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2 - Grifa.org

International Congress
BIOLOGICAL PRODUCTS: WHICH GUARANTEES FOR
THE CONSUMERS
Milan
October 15-16th 2002
Congress Centre CARIPLO
Via Romagnosi 6
Milan
Co-sponsors
Università di Milano
Assometab, Cesena
Shimadzu Italia, Milano
Main Sponsors
SIPCAM SpA, Milano
BioTecnologie B.T., Todi PG
Fondazione CARIPLO, Milano
Acknowledgement
We are grateful to the CARIPLO Foundation for
providing their prestigious Congress Centre
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Programme
Tuesday October 15th
9:30- 9:45
Registration
9:45-10:15
Opening:
M. Trevisan (GRIFA president, University of Piacenza),
L. Villa (Chairman of the Faculty of Pharmacy, University of Milan),
On. L. Bellotti (Responsible of Biological Committee, MiPAF),
M. Cocucci (Chairman of the Faculty of Agriculture, University of Milan),
P. Cabras (University of Cagliari)
SESSION 1 – Chairmen: M. Trevisan, E. Conti
10:15-10:55
V. Rotondo
FIAO, Italy
The situation of organic farming in Italy: legislation and need for research
10:55-11:15
Coffee break
11:15-12:00
J. Casida
University of California, Berkeley, USA
Botanical insecticides: reflections and perspectives
12:00-12:20
Y. Siderer, E. Anklam
European Commission JRC, Geel, Belgium
Need for research and appropriate analytical tools for organic food control
12:20-12:40
C. Tuberoso
Università di Cagliari
Multiresidual methods for the determination of
products from organic farming
vegetable extract residues in
12:40 -13:00 R. Lo Curto, F. Vilasi, T. Pellicanò, P. Munafò, G. Dugo
Università degli Studi di Messina
Presence of ochratoxin in experimental wines related to pesticides treatments
employed on grapes
13:00-14:30
Lunch
14:30-15:00
Poster session
SESSION 2 – Chairmen: P. Cabras, M. Solfrizzo
15:00-15:40
C. Regnault-Roger
Université de Pau et des Pays de l'Adour, France
Prospects for new botanical insecticides: the example of Mediterranean
aromatic plants.
2
15:40-16:20
G. Lozzia
Università degli Studi di Milano
Reliability and perspectives for the biological control of phytophagous insects
16:20-16:40
M. Kelderer, E. Elias
Centro Sperimentale Laimburg, Bolzano
Efficacy of Ranya speciosa as natural insecticide
16:40-17:00
Coffee break
17:00-17:40
S.K. Reilly
U.S. Environmental Protection Agency, USA
Biopesticides and their registration in the United States
17:40-18:00
M. Rubbiani
Istituto Superiore di Sanità, Roma
Biopesticides: evaluation process following the application of the directive
91/414/CE
18:00-18:20
A. Ragni
BioTecnologie BT (Italy)
Entomopathogenic nematodes in biological control: reality and prospectives
20:00
Social dinner (SOCREA, via Cino del Duca 8)
Wednesday October 16th
SESSION 3 – Chairmen: M. Taccheo Barbina, G. Lozzia
9:00-9:40
L. Gullino, A. Garibaldi
Università di Torino
Crop defence from fungal pathogens in organic farming: problems, effective tools
and perspectives
9:40-10:20
M. Solfrizzo
CNR, Bari
Toxicity and analysis of micotoxins and their presence in some organic foodstuffs
10:20-10:40
B. Beretta, C. Ballabio, F. Tacchini, A. Cattaneo, C.L. Galli, C. Gigliotti, P. Restani
Determination of the content of mycotoxins in commercial products from organic
and traditional farming
10:40-11:00
Coffee break
3
11:00-11:40
A. Evidente
Università di Napoli Federico II, Portici
The fungal phytotoxins and their potential role as herbicides in control methods
against weeds
11:40-12:00
A. Santomauro, G. Tauro, M. Sorrenti, F. Faretra
Università di Bari
Protection of grapevine from powdery mildew by using natural substances and the
microbial antagonist Ampelomyces quisqualis
12:00-12:20
A. Di Muccio
Istituto Superiore di Sanità
Experiences in the controls of pesticide residues in Italian products from organic
farming
12:20-12:30
M. Benuzzi
ASSOMETAB
BioControl agents for organic agriculture: a devoloping field without
appriopriate rules
12:30-13:00
G. Imbroglini
MiPAF
The commitment of MiPAF for organic farming
13:00-13:10
Closing remarks: M. Trevisan, P. Cabras
4
MAIN SPEAKERS
· V. Rotondo
FIAO, Italy
6
· J. Casida
University of California, Berkeley, USA
8
· C. Regnault-Roger
Université de Pau et des Pays de l'Adour, France
10
· G. Lozzia
11
· S.K. Reilly
U.S. Environmental Protection Agency, USA
18
· L. Gullino
Università di Torino
19
· M. Solfrizzo
CNR, Bari
20
· A. Evidente
Università di Napoli Federico II, Portici
21
5
LA SITUAZIONE DEL BIOLOGICO IN ITALIA: ASPETTI NORMATIVI E FABBISOGNO DI RICERCA
Vincenzo Rotondo
In rappresentanza FIAO
C/o Suolo e Salute S.r.l., P.zza Mazzini, 42 Nettuno di Roma
Ad oggi il territorio italiano offre la maggiore produzione da agricoltura biologica dell’Unione Europea. Il trend di
crescita è sempre in aumento sia per le richieste del consumo che in offerta da parte della produzione. Dall’elaborazione
dei dati forniti dagli Organismi di controllo operanti in Italia risulta che gli operatori del settore sono passati dai 54.004
del 2000 ai 60.509 del 2001, suddivisi in 56.440 produttori agricoli ( di cui 1.600 produttori/trasformatori), 3.947
trasformatori e 122 importatori.
La distribuzione degli operatori sul territorio nazionale vede il 65% nel sud del paese, il 23% nel centro, il 22 nel nord.
Per quanto riguarda la loro presenza nelle diverse aree geografiche delle attività produttive, si rileva la prevalenza al sud
dei produttori agricoli (68%, contro il 20% al nord e il 12% al centro), ed al nord dei trasfo rmatori(47% contro il 19% al
centro ed il 34 al sud), e degli importatori (82% contro l’11% al centro ed il 7% al sud).
La superficie interessata, in conversione o interamente convertita ad agricoltura biologica risulta pari a 1.237.640 ettari,
pari all’8% circa della SAU. Le principali colture riguardano i foraggi ed i cereali (221.436 ettari), i prati e pascoli
(241.157 ettari), che nel loro insieme rappresentano il 70% circa degli investimenti. Seguono in ordine di importanza le
coltivazioni arboree (olivo, vite, agrumi e frutta) con il 20% e le colture orticole ed industriali (leguminose da granella,
prodotti orticoli, colture industriali) 4%.
Per le produzioni animali, distinte sulla base delle principali tipologie produttive, al 31.12.2001, si segnala la seguente
situazione: bovini 330.701 (latte e carne), ovi-caprini 327.891, pollame 648.693, conigli 1.682, api, in arnie, 48.228.
L’attività di controllo, esercitata dagli organismi autorizzati dal Ministero delle Politiche Forestali e dalla Provincia
Autonoma di Bolzano, si è concretizzata in 72.896 visite ispettive, prelevamento ed analisi di 7.332 campioni. L’attività
di controllo ha portato al rilevamento di 2.074 irregolarità ed all’applicazione, in via definitiva, di 1.367 sanzioni.
Numero operatori suddivisi per regione ed attività (dati provvisori)
N. OPERATORI CONTROLLATI
REGIONE
ABRUZZO
BASILICATA
CALABRIA
CAMPANIA
EMILIA ROMAGNA
FRIULI VENEZIA
GIULIA
LAZIO
LIGURIA
LOMBARDIA
MARCHE
MOLISE
PIEMONTE
PUGLIA
SARDEGNA
SICILIA
TOSCANA
TRENTINO ALTO
ADIGE
UMBRIA
VALLE D’AOSTA
VENETO
TOTALI
TOTALE GENERALE
P
T
I
942
656
7.087
1.782
4.535
243
113
33
131
174
531
58
2
4
39
1
1.057
689
7.938
1.960
5.105
302
2.415
314
1.023
1.807
476
3.250
6.470
7.798
12.225
1.923
551
225
65
379
129
34
312
361
88
424
318
97
4
23
2
12
3
7
2
2.640
383
1.425
1.938
510
3.574
6.834
7.886
12.649
2.248
650
948
18
1.257
81
2
392
4
19
1.033
20
1.668
56.440
3.947
122
60.509
6
Il settore oggi è regolamentato in ambito comunitario con i Regolamenti 2092/91 con tutte le integrazioni e
modificazioni successive ed il Regolamento 1804/99. In ambito nazionale il D.L. 220/95 è il riferimento del
recepimento nazionale alla normativa comunitaria.
A causa dello sviluppo consistente e della complessità nell’offrire la più ampia garanzia al consumo risulta necessario
revisionare la normativa al fine di modificare parte dei contenuti, sia a livello nazionale attraverso una legge delega che
in considerazione della grande opportunità in prospettiva alla potenzialità del suo territorio, valorizzi ed incentivi le
produzioni biologiche, sia a livello comunitario in modo tale da razionalizzare e contemplare in un’unica disciplina di
settore la materia del biologico, determinando anche maggiore chiarezza, armonizzazione tra i vari comparti
dell’agroalimentare , creare i giusti riferimenti per l’informazione, la promozione, la progettazione ed in modo
particolare la ricerca.
Grande assente ingiustificata è la ricerca nel settore dell’agricoltura biologica.
Nel nostro paese la sperimentazione a supporto del metodo biologico è piuttosto limitata e trascurata dagli enti preposti,
salvo rare eccezioni dovute a volontà individuali. Si registra altresì, uno scollegamento fra le realtà produttive e le unità
di ricerca. Molti problemi di entità apparentemente complessa possono essere risolti attraverso lo sviluppo di attività
sperimentali specifiche , da osservazioni e prove accurate che abbisognano di studio e tecnologie appropriate.
Mancano fondamentalmente alternative valide per sostituire i prodotti rameici, conferme sull’utilizzazione di
determinati principi attivi e corretto impiego, soluzioni ai problemi tecnici delle nostre aree come il risanamento di
terreni inquinati dalle sostanze chimiche utilizzate decenni fa (clororganici), individuazione degli antagonisti dei
parassiti e loro corretto impiego nella lotta biologica, incremento dello studio sulle foraggere proteiche sia per il
fabbisogno zootecnico che come miglioratrici dei suoli, proteggere la biodiversità, aumentare l’attenzione sull’uso
indiscriminato di sementi OGM, ecc. Il settore resta in attesa di interventi a favore di queste attività che porterebbero
indiscutibilmente giovamento a chi produce e stimolo per chi vuole iniziare.
7
BOTANICAL INSECTICIDES:
REFLECTIONS AND PERSPECTIVES
John E. Casida
Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management,
University of California, Berkeley, California 94720-3112, USA
Importance and History. Insects are our main competitors for a limited supply of food and fiber. Pests destroy
about one-third of the world food supply during growth, harvesting and storage, even with the use of pesticides and
other control measures. The insecticides have evolved from inorganics to botanicals to synthetic organics. The inorganic
insecticides have almost disappeared from use on crops for reasons of inadequate effectiveness and safety. The
botanicals and synthetic organics have many similarities yet important differences and limitations. The author presents
some reflections and perspectives on these relationships from a half century of personal experience in insecticide
research.
Five Major Botanicals. There are five major botanical insecticides, i.e. pyrethrum, rotenone, nicotine, neem
and ryanodine. Pyrethrum extract from the flowers of the white daisy Chrysanthemum cinerariaefolium is the most
important. The countries of production include Kenya, Tanzania and Australia. The effectiveness is enhanced and the
economics improved by adding piperonyl butoxide for stabilization from metabolic detoxification in insects. Pyrethrum
is also important because its active ingredients, the pyrethrins, provided the model for the synthetic pyrethroids which
now constitute 20% of the market value of all insecticides. Rotenone from the roots of Derris elliptica and
Lonchocarpus utilis has for 150 years been used to control chewing insects and as a piscicide to kill unwanted fish.
Rotenone has been the prototype for Complex I respiratory inhibitors but not for economically successful structural
modifications. Home gardeners recommend rotenone use. Nicotine, the alkaloid of tobacco plants (and cigarettes), has
declined in use for decades but serendipitously served as the mode of action but not structural prototype of the new
synthetic neonicotinoids of high effectiveness and apparent safety. Neem has important local uses in India and China
and the extract with azadirachtin active ingredient is an effective crop protectant acting as an antifeedant. Ryania is the
ground stemwood of Ryania speciosa. The supply is limited but the effectiveness is high for lepidopterous larvae.
Supply and Composition. One of the limitations of botanical insecticides is consistent supply with adequate
standards of effectiveness and safety. This is a constant challenge even with the best producer organizations. Pyrethrum
once had an allergen now removed by a defined cleanup procedure. We analyzed cubé resin (the commercial rotenone
supply) and found 11 stilbenes and flavonoids and 29 rotenoids (including one that incorporated chlorine from the
solvent extraction conditions). Nicotine has been extensively studied except perhaps its environmental degradation
products. Neem contains many active ingredients; we described nimbolide and epoxyazadiradione as cytotoxic agents.
The principal active ingredient of Ryania was long described as ryanodine until we found that 9,21-dehydroryanodine
was of even greater importance.
Toxicology. Botanicals are more difficult to evaluate toxicologically than synthetic insecticides. They usually
have one or two major active ingredients (e.g. pyrethrins I and II in pyrethrum extract) but there may also be many
related bioactive compounds (e.g. in neem). Unless highly purified they also have hundreds to thousands of other
extractives or natural products which can vary with the plant source and processing. Critical evaluation of toxicology
requires production and use samples of consistent composition. Toxicology studies on pyrethrum extract meet current
standards but those for other botanicals are lacking in scope, although this does not necessarily mean hazards are
involved.
Residues of botanical insecticides are rarely a known problem. The pyrethrins photodecompose rapidly to
compounds of greatly reduced neurotoxicity. The same applies to the rotenoids and ryania components. Natural
products are generally considered to be easily metabolized. They have few or no halogens or refractory substituents.
Quite the contrary they have phenol, alcohol and ketone substituents, and alkene moieties that are amenable to ready
degradation. The analyses are more difficult than with most synthetics: the lack of halogens limits the analytical
detector sensitivity; multiple components and related but inactive analogs complicate the process.
Mode of Action and Resistance. Mode of action is an important aspect of insecticide use, safety and
toxicology. The same targets are involved in the most part with the botanicals and synthetics. Major insecticides act on
four principal targets in the nervous system.
8
target
botanical
synthetic
sodium channel –
voltage dependent
pyrethrins
DDT, pyrethroids
chloride channel GABA-gated
picrotoxinin*
cyclodienes, lindane,
fiproles
acetylcholinesterase
physostigmine*
organophosphates
methylcarbamates
nicotinic receptor
nicotine
neonicotinoids
*never a major commercial insecticide
The synthetics indicated above constitute about 90% of the insecticide market. This has major implications in the
selection for resistance leading to higher required doses and less effective control. Selection with the synthetics can lead
to target site resistance to the botanicals, e.g. the use of synthetic pyrethroids and even DDT results in selection of
strains resistant to pyrethrins.
Prototypes. Botanicals and other natural products are primary sources and prototypes in the search for new
insecticides. Screening of plant extracts enables the search of thousands of compounds as mixtures. This can be done at
random or with knowledge of potential usefulness from practice or folklore. Bioassay-directed isolation and modern
characterization techniques can often lead to a defined structure in weeks. This practice for a half century has turned up
potent compounds usually with problems of inadequate selectivity for insects versus mammals. The variety and source
of organisms are not infinite and limitations in this approach are already evident in the number of new isolates that
turned out to be previously known components with insecticidal activity. Medicinal plants and herbs are frequently used
or proposed to control insect pests. A literature search will usually reveal the active ingredient and often the mode of
action. Isolation of the effective component will usually verify that one is using a new source of an old control
chemical. The approach is still good but with increasing limitations.
Botanicals are more likely than synthetics to give leads for completely new types of insecticides. With
synthetics, the problem is to generate a new lead compound and then prepare massive numbers of analogs and rapidly
optimize their structure. Combinatorial procedures help here. Fast throughput screening allows rapid sifting for
effectiveness and mode of action.
Organic Agriculture. Botanical insecticides and natural foods fit well together. Rapidly increasing knowledge
helps choose the best materials and insures their effectiveness and safety. The natural chemical defenses of plants used
as botanical insecticides offer continued benefits in organic agriculture with confidence that the consumer and
environment will be adequately protected.
9
PROSPECT FOR NEW BOTANICAL INSECTICIDES :
THE EXAMPLE OF MEDITERRANEAN AROMATIC PLANTS
Catherine Regnault-Roger
Laboratoire d’Ecologie Moléculaire
Université de Pau et des Pays de l’Adour-France
BP 11 55 – F-64013 PAU Cedex
[email protected]
Fifty years of sustained struggles against harmful insect using chemical synthetic molecules has produced perverse
secondary effects like mammalian toxicity, insect resistance ans ecological hazards. Now, IPM has to face up to the
economic and ecological consequences of the use of pest control measures. The diversification of the approaches
inherent to IPM is necessary for friendly environnemental management. Among the alternative strategies, the use of
plants with insecticidal allelochemicals appaer to be promissing. The developpement of this ecochemical approache
requires the identification of efficient extracts or compounds from new sources of tropical but also temperate plants.
Aromatic plants are abundant on the Mediterranean periphery and they have been used as spices, pot-herb or medicinal
plants since Antiquity. Numerous industrial applications have then been implemented in perfumery, cosmetics and
detergents, pharmacology and fine chemistry as well as aromatics for the food industry. A recently-appeared field, IPM,
may also be prospected as an application field for aromatic Mediterranean plants: they contain a large range of
allelochemicals, which develop insecticidal properties. These insecticidal activities could be considered as
complementary or renewal strategies in IPM.
In Southwestern of France, the use of aromatic plants as traditional protectors of stored products is an old custom. We
evaluated in our laboratory the efficiency of these traditional customs: we observed that some plants were insecticidal,
especially those belonging to Lamiaceae. Thymus spp, Origanum spp, Rosmarinus sp were among the most active.
Our first insect model was Acanthoscelides obtectus Say (Bruchidae, Coleoptera) and its host plant Phaseolus vulgaris,
Leguminosae. HPLC analyses were conducted to identify main components and bioassays to determine their biological
activities on the beetle. The toxicity on adults and the inhibition of insect reproduction were evaluated. Essential oils
were active, however not exclusively as some hydrodistillated residues also exerted a toxic activity. The main active
compounds were identified as monoterpens and polyphenols.
Then, their effects on insects of different kinds like Ceratitis capitata (Diptera), Rhopalosiphum padi and
Metopolophium dirrhodum (Aphids) or Lepidoptera were tested. These evaluations showed that a large variability could
be observed in the response of different insect species to a same compound. Variability of plant chemical patterns
induced by climatic, pedological and genetic variations was also noticed.
According to these results, structure-activity relationships are discussed here in regard to their efficiency to control pest
insects and to their suitability to be considered as potential biopesticides.
References :
C.REGNAULT-ROGER, B.J.R. PHILOGENE, C.VINCENT, 2002 : Biopesticides d’origine
végétale, Lavoisier (Paris), pp 337.
C.REGNAULT-ROGER : The potential of botanical essential oils for insect pest control, Integrated Pest Managements
Reviews, 2, 25-34 (1997).
10
RELIABILITY AND PROSPECTIVES FOR THE BIOLOGICAL CONTROL OF PHYTOPHAGOUS
INSECTS
Giuseppe Carlo Lozzia, Ivo E. Rigamonti
Istituto di Entomologia agraria, Università degli Studi di Milano
In the last years there has been an explosion in the widespread of foodstuffs and other “biological” products that are
associated with an image of naturalness and hygienic safety by the public, even if these concepts are wrongly thought to
be closely linked. In reality, notwithstanding the great emphasis on the use of “natural” techniques and of the exclusion
of artificial products, biological agriculture is not so different from the most modern “conventional” strategies, and is
largely based on the use of active ingredients, which are natural but still toxic, that have all the problems of “pesticides”
(residues on foodstuffs, side effects on non-target organisms, etcetera) but on a different scale. This situation, both for
biological and traditional agriculture, is not due to a “strategic” trust in pesticides but to a lack of total efficacy in low
impact management techniques, and in particular to biological control.
Up to now, as a matter of fact, there is no culture of a certain importance for which the pest management can be given
solely to, or even mainly to, biological control. The situation is decidedly better if we only refer to animal organisms. In
this case there are consolidated strategies, at least theoretically; which refer to the reconstitution of balances and to the
improvement of the action against natural enemies, which are carried out in practice through the use of four
“techniques”: the diffusion of antagonists, the elimination of worrying sources and the adoption of environmental and
cultural management measures. Furthermore, the use of pheromones and other “modern” methods gives the possibility
to enlarge the possibilities of success. Modern strategies are based on this framework especially with the understanding
that a culture is a real ecosystem where limiting natural factors have a considerably important role. The most recent
research is analysing the influence of different cultural practices on the coenosys settled in the agro-ecosystem. The aim
of all this is, on the one hand to favour the diffusion of techniques with beneficial effects, and on the other to limit the
repercussions of harmful interventions which cannot be eliminated, as some phytosanitary treatments (Bassino, 1987).
One of the areas where the use of biological control practices is widely used, is in glasshouses cultures. The quick
succession of cultures one different from the other, with brief periods when there are no cultures, create a very
particular environment where techniques of permanent biological control of phytophagous insects have no possibility of
success. For this reason, there are usually routine “inundative” releases of antagonists in glasshouses and in tunnels.
Using this strategy the biological tool is almost like a chemical treatment. Large quantities of predators or parasitoids
are introduced into the structure in order to quickly bring down the population of the target pest and to keep it thereafter
under the damage level until the end of the cultivation cycle. Often the release has to be repeated over and over again
and more than one species of antagonists are introduced. In these environments phytophagous insects with determinate
characteristics tend to become affirmed (polyphagous, high number of generations, very prolific) which are able to
damage the cultures that follow, that, as already mentioned, can be very different one from another, and in any case the
species always tend to be the same ones. Numerous Rhynchota are particularly abundant (whiteflies, aphids, scale
insects), but Agromyzidae Diptera can also be easily found, as well as some Thysanoptera (Frankliniella occidentalis
(Pergande), Heliothrips haemorrhoidalis (Bouché), Thrips tabaci Lindemann) and spider mites. The low number of pest
species makes it possible for key antagonists to be restricted to a few units. All this has led to the birth and the diffusion
of structures, the commercial insectary, that have become similar in organisation to an industry, whereby the most
important natural enemies of every phytophagous insect is bred in large quantities and after put on sale on a large scale.
The breeding of auxiliaries is very complex (figure 1) requiring the development of refined and very reliable
techniques.
11
Preparation of the medium
(plant or diet)
breeding of the victim
Multiplication of the victim
Multiplication of the auxiliary
Collection
Packaging
Stock
Product quality control
Delivery
Figure 1 - Breeding process in a commercial insectary.
The high cost involved, together with the need to have a sure market has until now limited considerably the field of use
to protected environments which, on the one hand, usually host highly precious and expensive cultures and, on the
other, do not allow the dispersion of the antagonists, optimising their efficacy. Among the species to be found in
breeding there are numerous Hymenoptera parasitoids as the Aphelinidae Encarsia formosa Gahan, active against
whiteflies, or the Eulophidae Diglyphus isaea (Walker) which attacks the Agromyzidae Lyriomiza trifolii (Burgess),
and among the predators the Neuroptera Crysopidae Chrysoperla carnea (Stephens) an enemy of many aphids and the
Phytoseiid mite Phytoseiulus persimilis Athias-Henriot an antagonist of spider mites. To be fair, it has to be said that
many micro-organisms, in a broad sense, are produced commercially for the same purpose as Bacteria (Bacillus
thuringiensis Berliner), fungi (Beauveria bassiana (Bals.)) or Nematodes (Steinernema feltiae (Filipjev) and S.
carpocapsae (Weiser)).
For cultures to be found exclusively in the open field, an example of a nearly optimal situation can be supplied by
viticulture, which in the last decades has led to the affirmation of different practices of biological control which cover
all the kinds of strategies that have been mentioned above. This evolution has been made easier by the pre-existence of
a remarkably complex arthropod coenosys and therefore highly stable (Boller and Remund, 1986; Boller and Basler,
1987). The brief description of the different measures in the following paragraphs allows us to understand what are the
lines of research and what is the path that makes the practical enactment possible.
The first measure, the elimination of a worrying source, has been the rationalisation of the use of pesticides which, since
the 80’s has helped to rebuild the populations of Phytoseiids, predator mites responsible for the biological control of
Tetranychidae and Eriophyidae. It has been proved that the choice of products, in particular of fungicides are
fundamental for the maintenance of Phytoseiids and since the effects tend to accumulate, even substances which are
relatively little toxic can be extremely harmful if they are used repeatedly or in particular moments of the season.
However, in answer to specific conditions it may be possible to use active toxic substances as dithiocarbamates, dinocap
or powdery sulphur. In this case it is necessary to know the characteristics and the critical moments for the dominating
species in the concerned area, since they are different in the various Italian regions. For example, Kampimodromus
aberrans (Oudemans) is really sensitive and requires the use of the least harmful active ingredient possible from those
on offer. Typhlodromus pyri Scheuten is more tolerant instead but has moments of high sensitivity when it has to be
protected fully. In the more northern areas this period coincides with the regrowth of the vegetation, when there are a
few females and in addition they are very “stressed” due to the adverse winter conditions. In the more southern areas
this normally takes place in the summertime, as T. pyri does not like high temperatures very much. If done correctly, the
effects can be limited to a selection pressure which favours the affirmation of the more resistant Phytoseiids without
compromising their efficacy. The protection of these predators is furthermore enhanced by the support offered by the
practices of habitat management aimed at the diffusion of tree areas. Here, populations of Phytoseiids settle undisturbed
which interact with the culture thanks to the passive diffusion of individuals carried over by the air currents and which
give the possibility to overcome critical moments through the repopulating of populations decimated by erroneous
cultural practices or even by the recolonisation of the culture (figure 2).
12
Trees
Vineyard
Spontaneous
flora
Weeds
Passive diffusion
Figure 2 - Phenology of the movements of Phytoseiid populations in the grapevine agroecosystem in Northern Italy.
A general presence of trees and shrubs is not the only requirement but well defined elements able to host the interesting
species for the culture is. Therefore K. aberrans is abundant in our regions on many hosts (Celtis australis, Corylus
avellana, Ficus carica, Fraxinus excelsior) (Duso et al., 1993; Coiutti, 1993) while T. pyri is important only on Rubus
fruticosus and Sambucus nigra (Lozzia and Rigamonti, 1990) to be added to Acer spp., Cornus sanguinea, Corylus
avellana, Lonicera xylosteum and Ulmus spp. in Switzerland (Boller et al., 1988).
At a later stage, measures of environmental management have been proposed, that involve, in different periods of the
season, both the natural vegetation and cultivation, able to obtain that Empoasca vitis Goethe be under the danger
threshold. The evolution of the process has been long and hard. It has been known for a long time that E. vitis carries
out its cycle on different hosts and hibernates on conifers and other evergreen plants different from those that it attacks
(Vidano, 1958) and that, starting from the beginning of May, there is a mass migration from the plants where it
overwinters towards the cultures as soon as they blossom. The plants that are involved in rapid succession are the
following: firstly the apple tree, roses and some stone fruits then the grapevine, which seems to be the favourite plant on
which most of the population concentrates (figure 3).
13
rose, bramble, dogrose
birch
hazelnut
apple tree
vine
Picea sp.
March
April
May
June
and eggs
, and of Empoasca vitis, adults
Figure 3 – Phenology of Anagrus atomus, adults
eggs
, on different host plants (from Cerutti et al., 1989, modified).
and
The species stays here until summer when, from the beginning of August, the hibernating females which leave the
vineyards start to appear. The first step has been to identify the key antagonist, identified as Anagrus atomus Haliday,
an Hymenoptera Mymaridae, oophagous parasitoid that controlled “naturally” in some vineyards this leafhopper, being
able to hit a part between 20 and 82% of the germs (Arzone et al., 1988; Vidano et al., 1988; Cerutti et al., 1990). Later
on Cerutti et al. (1989) studied the cycles of E. vitis and of A. atomus highlighting the fundamental importance of the
maintenance of plants on which the parasitoid spends the last stages of the season and passes the winter and above all,
on which it starts again its activity in April just before the sprouting of the grapevine (figure 3). Therefore it is the adults
of the first generation of the year that migrate on the culture starting from the end of May. The success of the
colonisation of the vineyard by the parasite therefore depends on the presence of suitable host plants. They include
different Rosaceae (Rubus spp., Rosa spp., the apple tree), Lonicera sp., Corylus avellana and also Betula pendula. In
such structured areas the action of A. atomus on the leafhopper is decisive. Very similar results had been obtained in
North America too, where the species involved were Anagrus epos Gir. and Erythroneura elegantula Osb.. In this
way in numerous cases the permanent control of these harmful leafhopper in different European countries and in North
America has been obtained. At present, though, the diffusion of these strategies is obstructed by too low threshold
levels which over-estimate the symptoms, such as the leaf reddening, which probably are of little importance. As a
matter of fact the threshold level is 2 or even 1 specimen per leaf while it has been noted that there are no effective
damages even if there are more than 3 specimens per leaf (Lozzia and Rigamonti, 1994). Also considering the different
sensitivity of the cultivars it would be a good idea to increase these threshold levels in order to avoid that useless
treatments reduce parasitoid populations, obviously where the environmental conditions described above exist or where
it is thought they will start. In this general framework Jacobiasca lybica (Bergevin & Zanon) is not included. In Italy it
is mainly to be found in Sardinia, it is harmful at lower densities than with respect to the preceeding species and does
not seem, at present as far as is known, to be sufficiently controlled by natural antagonists.
In the last years a programme of inoculative releases has been started in many regions, whose aim is to rebuild the
balance between an exotic phytophagous insect, the Rhynchota Flatidae Metcalfa pruinosa (Say) and one of its
parasitoids, the Hymenoptera Dryinidae Neodryinus typhlocybae (Ashmead). In this case, too, the introduction of the
auxiliary in the environment is the final result of a long research process. The leafhopper arrived in Italy from Northern
America at the end of the 70’s (Zangheri and Donadini, 1980) and since then it has spread in different European
countries. Thanks to the fact that it is polyphagous it has colonised the most diverse environments, including many
cultures to which it is harmful, especially due to its abundant production of honeydew and to the problems linked to
this. In the first years after its arrival, the research for its natural enemies in the area of origin was carried out and the
key antagonist N. typhlocybae was identified. After this a breeding on a small scale of this Dryinidae was started in
order to evaluate its capabilities in the laboratory and with the specimens obtained the first experimental tests were
carried out in the field. After having obtained a mass production, the launch on a large scale was started at the end of the
90’s (Girolami et al., 1996). The technique is the classic one adopted in these cases. Taking advantage of the polyphagy
14
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Oophagous parasitoids of
leafhoppers
X
X
Pollen for predatory mites
X
X
X
X
X
Predatory mites
Oophagous parasitoids
Pupal parasitoids
X
X
X
Larval parasitoids
Urtica dioica
Lamium maculatum
Daucus carota
Aegopodium podagraria
Galium mollugo
Origanum vulgare
Rosa canina
Rubus fruticosus
Lonicera xylosteum
Nectar for parasitoids
Nectar for Lepidoptera
of the host, a relatively limited number of parasitoids is introduced on natural plants surrounding a plot. In this way the
auxiliaries can multiply without the risk of being killed by phytosanitary treatments normally carried out on the
grapevines. Once the population has reached a sufficient density level a flow of individuals begins and reaches the
culture and settles there; if there are no harmful interferences the colonisation is permanent but even if this is not so the
“reservoir” of the population on natural plants can guarantee a remarkable level of protection. The critical moments in
these cases are usually the capability of the antagonist to adapt to the new environment, in particular the overcoming of
the bad season, and its diffusion potential on a large radius starting from the introduction points. At the present moment
there are no definitive results as yet as to the success of this project.
A last example, which includes the combination of more measures, is the control of the grape berry moths Lobesia
botrana (Den & Schiff.) and Eupoecilia ambiguella (Hb.). The first solution is the progressive substitution of normal
treatments carried out with pesticides with techniques with a progressively lower impact on the environment, as the use
of growth regulators, of microbiological insecticides and lately with mating disruption. This last strategy uses, as is
known, large quantities of sexual pheromones whose aim is to “confuse” the males making them incapable of
distinguishing the traces secreted by the females, thus not allowing the coupling and as a consequence the reproduction.
This solution, optimal from the environmental point of view, has different “technical” limitations though, thus obtaining
the best results only if it is used on large and homogenous areas and in the presence of low populations. This last
constraint has led in the past different Authors to suggest to bring down the moths populations with ad hoc treatments
and this has not allowed the use of mating disruption in many areas in Italy. Together with this, and to eliminate these
limitations in part, habitat management measures are being introduced, whose aim is to improve the action of the
natural antagonists of the moths, in particular of the parasitoids. Since the majority of these species in the adult stage is
glyciphagous, it has been very important to guarantee them the presence of a suitable source of food, which is made up
of nectar plants in flower within or near the vineyard. The critical points are therefore two: on the one hand the presence
of species that produce nectar and are attractive for the parasitoids and, on the other hand, the continuous presence of
plants in flower. This can be obtained by sowing suitable species and with the alternate mowing of the rows.
These are clear examples of the holistic approach, which is typical of the most recent evolutions in cultural management
strategies. The control of a phytophagous insect is obtained by putting together different techniques both preventive and
curative, which involve different aspects of its biology and of its relations with the rest of the agroecosystem.
Furthermore, practices such as weeding and alternate mowing have a more complex meaning. They allow to maintain a
continuous presence of flowers, which are a fundamental source of food for many useful or indifferent arthropods
(figure 4), and they allow not only the increase of the number of their species but also the presence of denser and more
stable populations (Remund et al., 1989; Remund et al., 1992).
X
X
X
X
X
X
X
Figure 4 - Ecological meaning of the flora between the lines and in the adjoining areas of the vineyard with respect to
the antagonists of grape berry moths, their alternative hosts and other auxiliary species.
This data makes us suppose that in these environments a regulation of the harmful insects and mites at low population
levels and a stability of the whole agroecosystem is possible. Numerous species of parasitoids and predators which find
a great number of alternative hosts or preys among the insects and mites to be found on flowers and grass are
favourably hit by this presence. The pollen of anemophile species, instead, when it reaches the grapevine, is an
15
important alternative foodstuff for the Phytoseiids (Wiedmer and Boller, 1990; Remund and Boller, 1992; Engel and
Ohnesorge,1994) that are able to colonise even the grass flora, in particular many dicotyledons that then become
another reservoir of useful species available for the grapevine (Lozzia and Rigamonti, 1998).
Biological control, as can be seen from the above examples and from numerous other cases, has often been an
efficacious and reliable instrument for a permanent solution linked to infestations of single phytophagous insects.
However, it still cannot be considered suitable for the management of pests on the whole, not even limiting itself to
animal organisms. Also in the case of the wine grapevine, which is perhaps the culture that today has the best situation,
there have been problems following the expansion of flavescence dorée, a phytoplasma disease carried by Scaphoideus
titanus Ball, whose control requires the carrying out of treatments against the vector, interventions that risk to frustrate
also the results obtained with biological control against other phytophagous insects of the grapevine. This situation can
be generalised and applied to all viral, bacterial or phytoplasma diseases transmitted by vector organisms, whether they
be insects, mites or nematodes. In these cases, in fact, it is necessary to maintain the populations of the parasite at
extremely low density levels that cannot be obtained through the action of natural antagonists which for their own
existence, need the presence of a suitable quantity of victims. Only the availability of resistant plants will make
treatments against vectors superfluous in the future.
The management of vector species, the one of key phytophagous insects of the various cultures for which usually
“natural” control is not sufficient, the side effects of fungicide treatments are at present and in the near future the main
obstacles to the creation of a phytosanitary defence against phytophagous insects only using biological control
strategies. Another factor that must be considered and that today limits the reliability of biological control is the
dynamic nature of agroecosystems. The appearance of new phytophagous insects, the pullulations of secondary or
occasional species, the expansion of the area of numerous pests and so on, are highly probable events but they cannot be
forecasted, and this makes it necessary to have continuous adjustments to the methods adopted even if they are widely
experimented and to study new strategies. The same variability that exists between different areas whereby a culture is
diffused implies the need not to generalise solutions but to find those more suitable to the single realities through a
continuous process of experimentation. To sum up, we can say that biological control is without a doubt the main
weapon on hand in modern agriculture for the management of phytophagous insects but it still requires many studies
before all its potential can be used to the full and for a certain period of time it will be necessary to use more invasive
techniques.
BIBLIOGRAPHY
ARZONE A., VIDANO C., ARNÒ C., 1988 - Predators and parasitoids of Empoasca vitis and Zygina rhamni
(Rhynchota Auchenorrhyncha). In Vidano C. & Arzone A. (ed.), Proc. 6th Auchenorrhyncha Meeting. CNRIPRA, Torino: 623-629.
BASSINO J.P., 1987 - Principles of integrated protection in vine growing. A.A. Balkema Ed., Rotterdam, Proc.
Meeting E.C. Experts’ Group “integrated Pest Control in Viticulture”, Portoferraio, Italy, 26-28/9/1 985: 329336.
BOLLER E., REMUND U., 1986 - Der Rebberg als vielfältiges Agro-Oekosystem. Schweiz. Z. Obst-Weinbau, 122 (2):
45-50
BOLLER E., BASLER P., 1987 - Pflanzenschutzmassnahmen in Weinbau im Rahmen der integrierte Produktion.
Schweiz. Z. Obst-Weinbau, 123 (2): 61-63.
BOLLER E.F, REMUND U., CANDOLFI M.P., 1988 - Hedges as potential sources of Typhlodromus pyri, the most
important predatory mite in vineyards of northern Switzerland. Entomophaga, 33: 249-255.
CERUTTI F., DELUCCHI V., BAUMGÄRTNER J., RUBLI D., 1989 - Ricerche sull’ecosistema “vigneto” nel Ticino:
II. La colonizzazione dei vigneti da parte della cicalina Empoasca vitis Goethe (Hom., Cicadellidae,
Typhlocybinae) e del suo parassitoide Anagrus atomus Haliday (Hym., Mymaridae), e importanza della flora
circostante. Mitt. Schweiz. Ent. Ges., 62: 253-267.
CERUTTI F., BAUMGÄRTNER J., DELUCCHI V., 1990 - Ricerche sull’ecosistema “vigneto” nel Ticino: III.
Biologia e fattori di mortalità di Empoasca vitis Goethe (Homoptera, Cicadellidae, Typhlocybinae). Mitt.
Schweiz. Ent. Ges., 63: 43-54.
COIUTTI C., 1993 - Acari Fitoseidi su piante arboree spontanee e coltivate in Friuli Venezia Giulia. Frustula entomol.,
n.s. 16: 65-77.
DUSO C., TORRESAN L., VETTORAZZO E., 1993 - La vegetazione spontanea come riserva di ausiliari:
considerazioni sulla diffusione degli Acari Fitoseidi (Acari Phytoseiidae) in un vigneto e sulle piante spontanee
contigue. Boll. Zool. agr. Bachic., Ser. II, 25: 183 -203.
ENGEL R. VON, OHNESORGE B., 1994 – Die Rolle von Ersatznahrung und Mikroklima im System Typhlodromus
pyri Scheuten (Acari, Phytoseiidae) – Panonychus ulmi Koch (Acari, Tetranychidae) auf Weinreben. 1.
Untersuchungen im Labor. J. Appl. Ent., 118: 129-150.
GIROLAMI V., CONTE L., CAMPORESE P., BENUZZI M., MARTIR G. R., DRADI D., 1996 - Possibilità di
controllo biologico della Metcalfa pruinosa. Inf.re Agrario. 52 (25): 61-65.
LOZZIA G.C., RIGAMONTI I.E., 1990 - Influenza dell'ambiente e delle tecniche agrocolturali sulla presenza dei
fitoseidi (Acarina: Phytoseiidae) in alcuni vigneti del Nord Italia. Atti Giornate Fitopatologiche 1990, 1: 449458.
16
LOZZIA G.C., RIGAMONTI I.E., 1994 - Modello di gestione integrata dell'agroecosistema vigneto. Atti Convegno
"La difesa integrata dell'uva da tavola e da vino per gli aiuti comunitari del regolamento CEE 2078/92", Latina,
26 XI 1994: 75-98.
LOZZIA G.C., RIGAMONTI I.E., 1998 – Effects of weed management on phytoseiid populations in vineyards of
Lombardy (Italy). Boll. Zool. Agr. Bachic., Ser II, 30: 69-78.
REMUND U., NIGGLI U., BOLLER E.F., 1989 - Faunistische und botanische Erhebungen in einem Rebberg der
Ostschweiz. Einfluss der Unterwichsbewirtschaftung auf das Ökosystem Rebberg. Landwirtschaft. Schweiz.
Band. 2: 393-408.
REMUND U., GUT D., BOLLER E.F., 1992 – Beziehungen zwischen Begleitflora und Arthropodenfauna in
ostschweizer Rebbergen. Einfluss der botanischen Vielfalt auf okologische Gleichgewichte. Schweiz. Zeit.
Obst- Weinbau, 128 (20): 528-540.
REMUND U., BOLLER E.F., 1992 - Blühende Rebberge in der Ostschweiz: 3. Ergänzende Pollenfrassversuche mit
Raubmilben. Schweiz. Zeit. Obst- Weinbau, 128: 237-240.
VIDANO C., 1958 - Le cicaline italiane della vite (Hemiptera Typhlocybidae). Boll. Zool. agr. Bachic., Ser. 11, 1: 61115.
VIDANO C., ARNÒ C., ALMA A., 1988 - On the Empoasca vitis intervention threshold on vine (Rhynchota
Auchenorrhyncha). In Vidano C. & Arzone A. (ed.), Proc. 6th Auchenorrhyncha Meeting. CNR-IPRA, Torino:
517-524.
WIEDMER U., BOLLER E.F., 1990 – Blühende Rebberge in der Ostschweiz: 2. Zum Pollenangebot auf den
Rebenblättern. Schweiz. Zeit. Obst- Weinbau, 126: 426-431.
ZANGHERI S., DONADINI P., 1980 - Comparsa nel Veneto di un Omottero neartico: Metcalfa pruinosa Say
(Homoptera, Flatidae). Redia, 63: 301-305
17
BIOPESTICIDES AND THEIR REGISTRATION IN THE UNITED STATE
Sheryl K. Reilly
Chief, Biochemical Pesticides Branch
Biopesticides and Pollution Prevention Division
Office of Pesticides Programs
U.S. Environmental Protection Agency
Biopesticides play an important role in organic crop production. Their proper use can significantly enhance the quality
and production of crops targeted to address specific “niche market” needs. Biopesticides are regulated by the U.S.
Environmental Protection Agency (USEPA) as pesticides and the cost of commercial development can be significant.
Of the two broad classes of pesticides currently recognized by the USEPA, namely conventional chemicals and
biological pesticides (or biopesticides), a substantial number belong to the biopesticide class, specifically the
biochemical category. By definition, a biochemical pesticide is classified as such if it can be shown to possess a nontoxic mode of action on the target pest, and if it is naturally occurring or structurally similar and functionally identical to
a naturally occurring substance. Biochemical pesticides are generally active at low concentrations and fairly specific in
terms of their effects on a pest. USEPA is required by law to assure that any pesticide use in commerce will not result
in unreasonable adverse effects to humans or the environment. In order to make this assessment, data requirements
(e.g., mammalian toxicology, product identification and analytical methods, non-target plant and animal toxicology,
fate, etc.) have been established for pesticide product registration. Registrants must address each data requirement,
either by submitting a valid study or a request for a waiver of those requirements, prior to receiving formal product
registration by the USEPA. Waivers of required data are based on scientific rationale or information that is known in
the publically available, peer-reviewed scientific literature. For biochemical pesticides, the acceptability of data and/or
waivers, as well as any proposed label uses, are reviewed by the Biopesticides and Pollution Prevention Division
(BPPD) in the Office of Pesticide Programs of the USEPA. This division of the USEPA was formed in 1994 to facilitate
the development of biopesticide products which are important tools in an integrated pest management program and
offer alternatives to conventional chemical pesticides. Given the time and expense associated with product
development, commercialization, and registration, registrants should work closely with the USEPA, grower groups,
university and extension personnel, trade group(s) and others to ensure a successful product development process.
18
DIFESA DELLE COLTURE DAI PATOGENI FUNGINI IN AGRICOLTURA BIOLOGICA: PROBLEMI,
MEZZI DISPONIBILI E PROSPETTIVE
Maria Lodovica Gullino, Angelo Garibaldi
DI.VA.P.R.A. – Patologia vegetale, Università di Torino
La difesa delle colture dall’attacco dei numerosi patogeni fungini da sempre rappresenta un problema di non facile ed
immediata soluzione in molti sistemi colturali. Ciò è tanto più vero nel caso delle produzioni biologiche.
La situazione può essere più o meno complessa nel caso di sistemi colturali differenti: ad esempio è attualmente
possibilmente (ma non lo sarà forse più domani) proteggere la vite dagli attacchi di patogeni fungini mentre più
complessa è la situazione per il riso o per le colture ornamentali.
In questa relazione vengono pertanto presi in considerazione i principali problemi causati da patogeni fungini in alcuni
sistemi colturali, evidenziando le difficoltà che si incontrano o che si possono incontrare nel loro contenimento. Si
esaminano i mezzi di difesa disponibili, valutando, ove possibile, criticamente le reali possibilità di impiego,
sottolineando l’esigenza, anche per il comparto dell’agricoltura biologica, che tutti i prodotti ammessi passino il vaglio
di una idonea valutazione, a tutela del produttore e del consumatore.
Viene ribadito il concetto che, in modo particolare nel caso delle produzioni biologiche, la difesa delle colture ini zia con
l’adozione di tecniche colturali idonee e con l’impiego di varietà resistenti o scarsamente suscettibili nei confronti dei
più importanti parassiti.
Vengono forniti alcuni esempi relativi a colture di importanza economica in Italia e si discutono criticamente le
prospettive future.
19
TOXICITY AND ANALYSIS OF MYCOTOXINS AND THEIR OCCURRENCE IN SOME ORGANIC
PRODUCTS
M. Solfrizzo, M. Pascale, A. Visconti, G. Avantaggiato
CNR, Institute of Science of Food Production, Viale Einaudi 51, 70125 Bari, I taly
Fumonisins, ochratoxin A, and deoxynivalenol are among the most important mycotoxins due to their toxicity and
frequent occurrence in foodstuffs, including cereals. Fumonisins are mainly produced by Fusarium verticillioides e F.
proliferatum on maize before and soon after harvest. These toxins can cause equine leukoencephalomalacia, porcine
pulmonary oedema, liver and kidney cancer in rodents, nephrotoxicity and immunosuppression. The occurrence of
ochratoxin A in cereals depends on geographic areas and is related to the presence of Penicillium verrucosum and
Aspergillus ochraceus. Ochratoxin A has a strong nephrotoxicity and, at higher dosage, is carcinogenic on renal
proximal tubule. Deoxynivalenol occurs in wheat, barley, oats, rye, spelt and maize and less frequently in rice,
sorghum, and triticale. It is produced by F. graminearum e F. culmorum two important plant pathogens. Recent
toxicological evaluation by Joint FAO/WHO Expert Committee on Food Additives have established provisional
maximum tolerable daily intakes of 2 e 1 µg/kg of body weight for fumonisins and deoxynivalenol, respectively. The
provisional tolerable weekly intake of ochratoxin A of 0.1 µg/kg of body weight has been confirmed.
For the analysis of cereal based food aimed to assess mycotoxin intake the use of validated analytical methods is
essential. At this regard the European Committee for Standardization (CEN) has established method acceptability
criteria for each mycotoxin. Recently, we have developed and validated by a collaborative study a new HPLC method
for the determination of fumonisins in maize and maize based foods which has been adopted by CEN as European
Standard Method. Official standard methods based on HPLC are available for the analysis of ochratoxin A in cereals,
whereas a suitable method that respond to CEN criteria needs to be produced for deoxynivalenol.
The consumption of organic products in Europe has increased in the last years but few information are available on their
mycotoxin contamination as compared to conventional products. It is well known that agricultural practices play a role
on mycotoxin formation in cereals.
A total of 461 cereal samples collected in Italy, including 186 samples originating from organic production (136 soft
wheat and 50 spelt) harvested in 1998-2000 and 275 samples from conventional production (138 soft wheat and 137
durum wheat) harvested in 1999-2000 have been analysed for deoxynivalenol in our laboratory. A higher incidence of
positive samples was found in organic wheat (65-100% of positive samples) and organic spelt (100% of positive
samples) as compared to conventional wheat (38-80% of positive samples), whereas the highest levels of
deoxynivalenol were found in conventional wheat (up to 6465 µg/kg). In particular, mean levels of deoxynivalenol in
positive samples of organic soft wheat, organic spelt, conventional soft wheat and conventional durum wheat were 70180 µg/kg, 148-192 µg/kg, 219-604 µg/kg e 251-1097 µg/kg, respectively.
20
THE FUNGAL PHYTOTOXINS AND THEIR POTENTIAL ROLE AS HERBICIDES IN INTEGRATED
CONTROL METHODS AGAINST WEEDS.
Antonio Evidente
Dipartimento di Scienze del Suolo, della Pianta e dell’Ambiente, Università di Napoli Federico II, Via Università 100,
80055 Portici
The man weed battle started long time ago due to the great hazard resulted from their wide diffusion in
agricultural crops and pastures. Weeds have created heavy problems in reforestation activities and in important
economical infrastructures. In the last 50 years, beside the traditional agrarian practises, the most diffused methods in
controlling weeds are the use of chemical herbicides which are responsible for major environmental pollution with
human and animal health risks.
Therefore, numerous studies have been carried out to develop biological control using natural weed enemies as
insects, viruses, bacteria and fungi but a particular interest has been directed towards phytopathogenic fungi which are
the most common plant pathogens that in respect to other microorganisms may be applied with safety and simplicity.
Phytopathogenic fungi are responsible, together with other microbial agents, of severe damages to important
agrarian forestall and ornamental plants Frequently, these fungal species produce phytotoxins, bioactive metabolites
with different chemical structure, mode of action, host specificity, biological activity and environmental impact. Toxins
are generally substances which interfere with plant metabolism of which many are directly responsible of pathogenesis
while others are not considered toxins at all1,2.
Studies conducted in the last years have concentrated on controlling weed diffusion in important agrarian crops.
Fungal pathogens isolated from some infected tissue of weeds are generally belonging to important taxonomic genera
(Alternaria, Ascochyta, Drechslera, Fusarium, Melanconis, Phoma, Pyrenophora ). Some phytotoxins were isolated and
characterised to determine their role in developing new methods for weed control, Their direct application or that of
their derivatives or/and analogues with the pathogen may increase the herbicidal activity so improving the efficacy in
terms of pathogenesis, virulence and herbicidal selectivity
Structure elucidation and stereochemistry of phytotoxins may allow to plan their total enantioselective
synthesis in order to obtain amounts suitable to carry out experiments in green houses and in fields to develop strategies
for the biological integrated management of weeds. Some toxins may be derivatised to modulate their biological activity
in terms of phytotoxicity and selectivity and to obtain new natural safe herbicides.
Furthermore, phytotoxins can be used to develop a method for their analysis in complex samples. Therefore, if
the phytotoxins are virulence features they may be used as biomarkers for in vitro selection of the best fungal strain to
be used in the integrated management methods. In this communication, a brief overview will be presented on the results
obtained on developing strategies to biologically control of Chenopodium album, Striga hermonthica and Orobanche
ramosa, infesting many important agrarian crops like maize, beet root, millet, sorghum, sunflower, tomato, legumes,
etc2,3.
References
1. Ballio, A.; Graniti, A. Experientia, 1991, 47, 751-826.
2. Evidente, A.; Motta, A Phytotoxins from fungi, pathogenic for agrarian, forestal and weedy plants. In Bioactive
Compounds from Natural Sources, Tringali, C. (Ed.), Taylor & Francis, London, 2001. Chapter 12, pp. 473-525.
3. Evidente, A.; Abouzeid, M.A. Characterization of Phytotoxins from Phytopathogenic Fungi and their Potential Use
as Herbicides in Integrated Crop Management. In Handbook of Sustainable Weed Management, Harminder, P.S.
(Ed.), The Haworth Press Inc., Binghamton, NY, USA (2002) in press.
21
ORAL COMMUNICATIONS
· E. Anklam
European Commission JRC, Geel, Belgium
23
· C. Tuberoso
Università di Cagliari
24
· T. Pellicanò
Università degli Studi di Messina
25
· M. Kelderer
Centro Sperimentale Laimburg, Bolzano
30
· M. Rubbiani
Istituto Superiore di Sanità, Roma
31
· A. Ragni
BioTecnologie BT (Italy)
32
· P. Restani
38
· A. Santomauro
Università di Bari
39
· A. Di Muccio
· M. Benuzzi
ASSOMETAB
41
· G. Imbroglini
MiPAF
42
22
NEED FOR RESEARCH AND APPROPRIATE ANALYTICAL TOOLS FOR ORGANIC FOOD CONTROL
Yona Siderera, and Elke Anklamb
a
Interdisciplinary Centre for Technological Analysis and Forecasting, at Tel Aviv University, Ramat -Aviv, Israel
b
European Commission, Joint Research Centre, Food Products Unit, B -2440 Geel, Belgium
Organic farming and the organic food market are growing fast. Due to health concerns, environmental consciousness,
social status considerations and other reasons, consumers are interested in the products of organic farming. At the same
time, they want more information about these products.
Inspection procedures for confirming the origin of products seem to be insufficient and need to be supported by
analytical tools. Not only should the products purchased on the market be checked for their authenticity but also
products used during the various production steps (e.g. fertilisers, soil, animal feed, pesticides). Analytical research
work should accompany the expansion of organic farming and organic food production. This should take into
consideration different climate conditions, the variety of soils and soil constituents, the variety of plants and plants
strains, and husbandry of livestock related to animal welfare. At the same time, the products (vegetables, fruits, milk,
meat, and processed food) should be studied for their own qualities.
Research into agricultural crops with various cultivation methods and the resulting natural or processed food is complex
and lengthy. It is very difficult to compare the contents of nutrients and vitamins, as the constituents of such substances
depend heavily on storage time, conditions and technology. Fast and general answers cannot be given on methods for
verifying authenticity and safety. Attention should be focused on the planning of experiments, taking into consideration
many variable parameters like soil quality, climatic and geographical conditions and crop variety. It is important to
study a large number of samples, to be able to deduce meaningful conclusions from the results of the research. The
research protocol should involve a large enough number of research participants as well. The results of such a
combined study could then be used as analytical tools for assessing organic products. Later on, a farmer or a retail outlet
would be able to add to the label that the product was checked and, e.g., found “clean” of pesticides, or that mycotoxins
were found to be below legal levels after harvesting. These tests would be beneficial for interested customers and for
the organic market sustainability. It should be emphasised that this approach of collaborative structured studies is
preferable to the work of a single researcher wanting to study a particular question of her or his interest (or the
stakeholder’s interest).
Once analytical tools have been developed, firstly for a small number of products, but in higher numbers along the
years, then legislation on organic food products might be changed to include a call for analytical tests to support the
inspection system.
23
METODO MULTIRESIDUO PER LA DETERMINAZIONE DI RESIDUI DI ESTRATTI VEGETALI IN
ALIMENTI BIOLOGICI
C.I.G. Tuberoso
Dip. di Tossicologia Università di Cagliari
Via Ospedale, 72 Cagliari
Nell'agricoltura biologica si effettua il controllo dei pesticidi di sintesi, in quanto la normativa 2092/91 non ne consente
l'uso, per cui la sola presenza di residui a livelli > di 0,01 mg/kg rende gli alimenti non conformi. I pesticidi naturali per
i quali è consentito l'uso invece, di norma, non vengono controllati, principalmente per carenza di metodiche analitiche
rapide, semplici e con buona sensibilità. Anche a livello di ricerca gli studi su questi pesticidi naturali sono molto
limitati, per cui la conoscenza del loro livello di residui negli alimenti è quasi completamente sconosciuta. Gli insetticidi
estratti da piante più utilizzati sono quelli ottenuti dalla Azadirachta indica, Derris elliptica, Ryania speciosa, e
Chrysanthemum cinerariaefolium i cui principi attivi sono rispettivamente: azadirachtin, rotenone, rianodina con
deidrorianodina e piretrina I e II.
Il presente lavoro propone una metodica che consente la determinazione di questi antiparassitari naturali in maniera
semplice e sensibile. La tecnica utilizzata è la cromatografia liquida accoppiata alla massa (HPLC-MS)
Inizialmente si è ottenuta la separazione cromatografica dei principi attivi utilizzando una colonna C18, un gradiente di
H2O e CH3CN al flusso di 0,2 ml/min ed un rivelatore a serie di diodi settato alle lunghezze d’onda ottimali per la
determinazione dei singoli principi attivi. Nella determinazione col detector di massa è stata impiegata una sorgente
APCI e tutti gli antiparassitari sono stati analizzati in SIM con due metodi (differenti per le temperature del probe, CDL
e Block) che permettono di ottimizzare la risposta per la deidrorianodina, la rianodina e l’azadiractina (metodo 1) e per
il rotenone e le piretrine (metodo 2).
L’estrazione dei principi attivi da matrici vegetali è stata effettuata con acetato di etile, solvente che si è dimostrato
ottimale per il recupero quantitativo di tutti i principi attivi. L'estratto non subiva alcuna purificazione in quanto non
presentava alcun interferente. Nelle determinazioni quantitative la soluzione madre veniva preparata in estratto di
matrice non trattata per eliminare l'effetto matrice.
I limiti di determinazione ottenuti sono compresi tra 2 e 10 ppb. Il rotenone è l’antiparassitario che fornisce la risposta
migliore permettendo di rilevare fino a 2 ppb.
24
PRESENCE OF OCHRATOXIN IN EXPERIMENTAL WINES RELATED TO PESTICIDES
TREATMENTS EMPLOYED ON GRAPES
R. Lo Curto*, F. Vilasi*, T. Pellicanò*, P. Munafò**, G.mo Dugo*,
* Università degli Studi di Messina, Dip.to Chimica Organica e Biologica.
** Centro Analisi & Servizi S. r. l. via U. La Malfa, 18 - 98051 Barcellona P. G., Messina, Italia.
Introduction
Mycotoxins are a class of highly toxic chemical compounds produced, under particularly environmental conditions, by
several moulds developing in many foodstuffs. Their presence is depending upon several factors such as: fungal strains,
climate and geographical conditions, cultivation techniques and foodstuffs conservation (1). Mycotoxins may occur in
various vegetal products as cereals, dried fruits, coffee beans, cocoa and beverages as beer and wine (2-4). Among
mycotoxins very important are the ochratoxins. This term indicates a group of metabolites, having showing similar
chemical structure, produced by strains of the genus Aspergillus (A. ochraceus) and Penicillium (P. ferrucosum) (1,5,6).
The most studied, both for its diffusion and toxicological importance is Ochratoxin A (OTA) R-N-[(5-chloro-3,4dihydro-8-hydroxy-3-methyl-1-oxo-1H-2-benzopyran-7-yl)carbonyl]-phenylalanine. It has an oral DL50 level of 20
mg/kg in rats and pig (7). OTA is highly toxic and causes severe animal and human intoxications, for example “Porcine
nephropathy” and “Balcan Endemic nephropathy” (8-10). OTA exhibits nephrotoxic and teratogen activites and
moreover suppressive actions on immune system, causing a diminution of immune globulin level and of other humoral
factors in mice and chicken and a reduction of cell immune response (10,11).
Recently, more attention was focused on Ochratoxin A levels in commonly consumed foods, especially fruits and
cereals (12,13) and in their fermentation products like beer (14-16) and wine (17-25).
As far as wine is concerned, available data regarding the presence of OTA are very discordant, some Authors reported a
high toxin concentration (up to 7.0 ng\ml) with considerable level of contamination (incidence up to 92%) in red wine
produced in southern regions of Europe and in North Africa. Other Authors reported OTA contamination levels ranging
3.9% for white wines to 16.6% for red wines, even if these data were obtained by different analytical methods.
Generally red wines contain a greater amount of OTA than white or rosè ones. These differences can be attributed to
climatic factors, grape cultivation and storage conditions apart from wine-making techniques. The content of OTA in 23
samples of red and white wine, produced in the year 2000 by three experimental vite, situated in three different Italian
regions treated with different pesticides see tables 1,2,3), is reported in this work (26).
Materials and methods
Chemicals and Reagents
Standard of OTA, sodium chloride, polyethylene glycol (Peg 8000), sodium hydrogencarbonate, glacial acetic acid and
toluene were obtained from Fluka (Milano-Italy). Immunoaffinity columns were purchased from Vicam (Waterton,
MA, USA).Acetonitrile, methanol and water (HPLC grade) were supplied from Carlo Erba Reagents (Milano-Italy).
Wine samples
A total of 23 wine samples (16 white 7 red samples), produced in the year 2000 by three experimental vite, situated in
three countries “Avellino”-Campania, “Catania”-Sicily and “Grosseto”-Tuscany, were analysed.
Wine characteristics and pesticide treatments
White wines came from Sicily and Campania in the crop year 2000. Sicilian wines were produced from 15 -20 years old
plants, grown up on Etna (300 m asl, S. Venerina, Catania, Italy), in a vulcanic soil. Vines were grafted with Inzolia and
Carricante varieties in 1:1 ratio. Wines from Campania were produced from 25 years old plants grown up on
Montefredane hills (700 m asl, Avellino, Italy), in a clayey soil. Vines were grafted with Fiano d’Avellino variety. Red
wines from Tuscany were produced from 25 years old plants, cultivated in Maremma Toscana coast (100 m asl,
Grosseto, Italy), on a calcareous soil. Vines were grafted with Sangiovese variety, Morellino clone. Vinification process
was run within 24 hours after grape harvesting and it was run using the following scheme:
White Vinification
Newly – cropped Vitis vinifera grapes from Sicily and Campania were crushed destemmed and then soft pressed by a
pneumatic press. Must was treated with SO2 (30 g/hl), pectolitic enzymes (1.5 g/hl) and vitamin C (5 g/hl) to favour
clarification before fermentation; temperature was maintained at 8°C for 24 hours. Clear must was spiked with 20 g/hl
of thiamine and ammonium phosphate as fermentation coadjuvans and 30 g/hl of selected yeasts and fermentation was
run at 15°C. In order to remove lees, after fermentation wine was decanted into a tank and spiked with SO2 (5 g/hl). Ten
days later wine was decanted again and treated with SO2 (5 g/hl). Wine was finally filtered twice trough 1 m and 0.45m
cardboards filters, spiked with SO2 (2-3 g/hl), bottled in dark bottles and maintained at 4°C for the duration of the
experimentation. Each grape sample was separately vinificated, following the above mentioned protocol.
Red vinification
Newly – cropped Vitis vinifera grapes from Tuscany were crushed and destemmed, then spiked with SO2 (5
g/hl), added with selected yeasts (30 g/hl) and let to ferment for 10 days at 28°C, making three fullings a day. Wine was
drawn from the vat and the vinasses pressed by a hydraulic press. After 4 weeks, lees were removed and the wine
25
spiked with SO2 (2-3 g/hl). Wine was spiked again with SO2 (2-3 g/hl) and bottled in dark bottles at 4°C for the duration
of the experimentation.
Each grape sample was separately vinificated following the above mentioned protocol. All wines were stored in
the dark at 4°C, and each sample was open immediately prior to analysis.
Pesticide treatments.
Pesticides were used at the doses recommended by the manufacturer and were sprayed with a manual sprayer in
the three Italian vines. Each Sicilian samples but four, was subjected to a Sulfur treatment, followed by organic
pesticide treatments, applied during grape ripening and repeated for six times every 15 days. Two samples were treated
only with Sulfur (dry and wettable powder), one sample was treated only with organic pesticides (Dinocap –
Penconazole) and one sample only with water in order to have a comparison with treated samples (table 1). Each
sample from Campania but four, was subjected twice to Sulfur treatments every 10 days, followed by organic pesticide
treatments applied during the vines maturation phase and repeated 6 times every 15 days. Two samples were treated
only with Sulfur (dry and wettable powder), one sample was treated only with organic pesticides (Dinocap –
Penconazole) and one sample only with water (table 2). Each Tuscan sample but four, was subjected twice to Sulfur
treatments every 8 days, followed by organic pesticides tratments, applied during the vines maturation phase and
repeated 9 times every 12 days. Two samples were treated only with Sulfur (dry and wettable powder), one sample was
treated only with organic pesticides (Dinocap – Penconazole) and one sample only with water (table 3).
Apparatus
The analytical method proposed by Visconti et al. for OTA determination in wine was utilized. Utilizing commercial
immunoaffinity columns and HPLC equipped whit a RF detector carried out Ochratoxin A determination.The
chromatographic analysis was run by using a Shimadzu HPLC system equipped with a System Controller SCL-10 A, an
RF-1AXL detector (lex=330 nm, lex=460nm), a LC 10A pump, a Rheodyne injector with a loop of 20 ml and a
reversed-phase Supelco column C18 (15 cm x 4.6 mm, 5 mm particles) equipped with a guard filter (0.5 mm). Analyses
were run at room temperature in isocratic conditions with a mobile phase composed of water \ acetonitrile \ acetic acid
99:99:2 v\v\v at a flow rate of 1 ml\min. OTA quantification was made by measuring peak areas at OA retention time
and by comparing them with calibration curve. Using a wine sample added of OTA made confirmation of OTA
identification standard. HPLC chromatogram of a standard solution of Ocratoxin A is shows in Fig.1. The sensibility
test of analytical method was of 0.01 ng\ml.
Results and discussion
Values of OTA found in wines are shown in tables 4 for Sicily and Tuscan respectively. OTA was not found in white
wines produced in Campania while, of eight samples from Sicily, only six were found to be contaminated; the higher
value found was 0.03 ng\ml in the sample treated with dinocap. The sample treated with quinoxifen and the treated with
water were found not contaminated with OTA. Because of OTA concentration in this wine is very low, it is not possible
to correlate its presence with pesticide treatments.Values of OTA found in red wines show that all samples are
contaminated. The sample produced with grapes treated only with sulfur 80 PBWG, showed a Ochratoxin A content
(2.00 ng\ml) higher than the other samples, followed by sample obtained from grapes treated with powdered sulfur
(0.71 ng\ml). The concentration of OTA in the other samples spanned from 0.07 ng\mg in sample treated with
“azoxystrobin” to 0.24 ng\mg in sample treated with “dinocap and penconazole”. The values of OTA found, are
comparable with those reported in literature on micotoxins presence in red wines.
The experimental viticulture located in Campania is not subject to pollution from OTA producing moulds. The
experimental Sicilian cultivar seems is more easily contaminated with OTA since, of eight samples, six were found
contaminated.
Values of OTA found in red wines show that experimental vite located in Tuscany is strongly subject to infection from
OTA producing fungi. Synthetic pesticides can reduce the OTA concentration from 96.5% in the sample treated with
azoxystrobin, to 88% in the sample treated with dinocap and penconazole. Since OTA is strictly related to the growth of
some toxigenic fungi on grapes, the different Ochratoxin A content in detected wine samples can be considered an
efficiency test of the pesticides used.
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Castegnaro, M., Plestina, R., Dirheimer, G., Chernozemsky, I. N., Bartsch, H. (1991). Mycotoxins. Endemic
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Pfohl-Leszkowicz, A., Pinelli, E., Bartsch, H., Mohr, U., Castegnaro, M. (1998). Sex- and strain-specific
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MacDonald, S., Wilson, P., Barnes, K., Damant, A., Massey, R., Mortby, E., Shepherd, M. J. (1999).
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Nakajima, M., Tsubouchi, H., Miyabe, M. (1999). A survey of ochratoxin A and aflatoxins in domestic and
imported beers in Japan by immunoaffinity and liquid chromatography. J. AOAC Int., 82, 897-902.
Visconti, A., Pascale, M., Centonze, G. (2000). Detrmination of ochratoxin A in domestic and imported beers
in Italy by immunoaffinity clean-up liquid chromatography. J. Chromatogr. A, 888, 321-326.
Visconti, A., Pascale, M., Centonze, G. (1999). Determination of ochratoxin A in wine by means of
immunoaffinity column clean-up and high-performance liquid chromatography. J. Chromatogr. A, 864, 89101.
Leitner, A., Zollner, P., Paolillo, A., Stroka, J., Papadopoulu-Bouraoui, A., Jaborek, S., Anklam, E., Lindner,
W. (2002). Comparison of methods for the determination of ochratoxin A in wine. Analytica Chimica Acta,
453, 33-41.
Soleas, G. J., Yan, J., Goldberg, D.M. (2001). Assay ochratoxin A in wine and beer by high-pressure liquid
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Majerus, P., Otteneder, H. (1996). Detection and occurrence of ochratoxin A in wines and grape juice. Dtsch.
Lebensm.-Rundsch, 92,388-390.
Burdaspal, P. A., Legarda, T. M. (1999). Ochratoxin A in wines and grape musts and juices produced in Spain
and other European countries. Alimentaria, 299, 107-113.
Otteneder, H., Majerus, P. (2000). Occurrence of Ochratoxin A (OTA) in wines: influence of the type wine and
its geographical origin. Food Addit. Contam., 17, 793-798.
Ospital, M., Cazabeil, J. M., Betbeder, A. M., Tricard, C., Creppy, E.E., Medina, B. (1998). Ochratoxin A in
wines. Rev. Fr. Oenol., 169, 16-18.
Festas, I., Herbert, P., Santos, L., Cabral, M., Barros, P. (2000). Alves, Ochratoxin A in some Portuguese
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R. Lo Curto, T. Pellicanò, F. Vilasi, P. Munafò, G.mo Dugo Food. Chem.(2003) (in press)
27
Figure 1: HPLC chromatogram of standard OTA
Table 1: Pesticide treatments on “Fiano di Avellino”(Campania) variety grapes
Wine samples
Sicily 1
Sicily 3
Sicily 4
Sicily 5
Sicily 8
Sicily 9
Sicily 10
Sicily 11
n° of pesticide treatments during grape ripening
1 Sulfur 80 Pb
6 Quinoxyfen250 SC
1 Sulfur 80 Pb
6 Fenarimol 12 SC
1 Sulfur 80 Pb
6 Azoxystrobin 250 SC
1 Dinocap 350 EC 6 Penconazole 100 EC
1 Sulfur 80 Pb
6 Sulfur 80 Pb
1 Sulfur 80 Pb
6 Dinocap350EC
1 Sulfur Powder
6 Sulfur Powder
1 Water
6 Water
Table 2: Pesticide treatments on “Inzolia” and “Carricante”(Sicily) varieties grapes
Wine samples
Campania 1
Campania 2
Campania 3
Campania 4
Campania 5
Campania 6
Campania 7
Campania 8
2 Sulfur 80 Pb
6 Quinoxyfen 250 SC
2 Sulfur 80 Pb
6 Fenarimol 12 SC
2 Sulfur 80 Pb
2 Sulfur 50 PS
6 Sulfur 50 PS
2 Dinocap350EC
6 Penconazole 100 EC
2 Sulfur 80 Pb
6 Dinocap 350 EC
2 Water
6 Water
2 Sulfur 80 Pb
6 Sulfur 80 Pb
28
Table 3: Pesticide treatments on “Sangiovese” (Tuscany) variety grapes
Wine Samples
Tuscan 1
Tuscan 2
Tuscan 3
Tuscan 4
Tuscan 5
Tuscan 6
Tuscan 7
2
2
2
2
2
2
2
Sulfur 80 Pb
9 Quinoxyfen 250 SC
Sulfur 80 Pb
9 Fenarimol 12 SC
Sulfur 80 Pb
Dinocap 350 EC
9 Penconazole 100 EC
Sulfur Powder
9 Sulfur Polvere
Sulfur 80 PBWG
9 Sulfur 80 PBWG
Dinocap 350 EC
6+3 Sulfur + Quinoxyfen 250 SC
Table 4: Concentration of OTA in wine samples
Wine Samples
Sicily 1
Sicily 3
Sicily 4
Sicily 5
Sicily 8
Sicily 9
Sicily 10
Sicily 11
Tuscan 1
Tuscan 2
Tuscan 3
Tuscan 4
Tuscan 5
Tuscan 6
Tuscan 7
nd: not detected
Ochratoxin A (mg L-1)
n. d.
0.02
0.01
0.02
0.02
0.03
0.01
traces
0.22
0.11
0.07
0.24
0.71
2.00
0.10
29
EFFICACIA DELLA RYANIA SPECIOSA, COME INSETTICIDA NATURALE
Markus Kelderer, Ellen Elias
Centro Sperimentale Laimburg (BZ)
Ryania speciosa (Vahl) è un arbusto appartenente alla famiglia delle Flacourtiacee presente nel clima tropicale del
Centro- e Sudamerica. Le prime notizie di questa pianta risalgono al 1897 quando Cortes segnaló di aver trovato una
pianta con un alcaloide tossico chiamato dagli indigeni ‚Matacucaracha‘.
Negli anni 40 in agricoltura il legno macinato di Ryania speciosa venne usata contro diversi insetti nocivi. (es. in
frutticoltura contro Cydia pomonella). Gli allegati 2b del Regolamento sull’agricoltura biologica 2092/91 inizialmente
citavano preparati a base di Ryania speciosa come compatibili con i principi dell’agricoltura biologica. Con il Reg.
1488/97 la Ryania speciosa venne tolta da tale lista poiché non era registrata come fitofarmaco in nessun paese della
Comunitá Europea. In mancanza di alternative nel contenimento di alcuni tortricidi cardinali in frutticoltura biologica su
richiesta delle associazioni dei produttori, l’argomento venne inserito tra i temi prioritari da affrontare nell’ambito dei
progetti finalizzati ‚Difesa in agricoltura biologica‘. I lavori svolti nell’ambito dei 3 anni di ricerca sono:
1) Raccolta bibliografica delle conoscenze finora presenti
2) Sviluppo di un metodo di estrazione capace di estrarre il quantitativo maggiore di Ryanodina componente
principale degli alcaloidi presenti nel legno di Ryania speciosa. A tale proposito si saggiarono diversi solventi
(acqua, metanolo, etanolo, diclormetano, acetone, ecc.), diversi metodi di estrazione (Soxhlet, bollitura a riflusso,
Ultraturrax, Estrazione con SFE, ) e diverse metodologie per determinare la Ryanodina (cromatografia su strato
sottile TLC e HPLC).
3) Biotest in laboratorio su diversi lepidotteri carpofagi (Cydia pomonella, Lobesia botrana, Pandenmis heperana,
Adoxophyes orana, Mamestra brassicae) confrontando legno macinato di Ryania speciosa, Ryanodina pura ed
estratti standardizzati. Vennero fatte sia prove contro le uova, le larve e gli adulti dei lepidotteri citati
determinandone la DL50.
4) Ricerca di sinergisti (Piperonilbutossido, ecc. ) per esaltare l’efficacia della Ryanodina come principio attivo.
5) L’efficacia in pieno campo di diverse formulazioni a base di Ryania contro diversi fitofagi.
6) Determinazione della persistenza del principio attivo in condizioni di pieno campo.
7) Determinazione dei metodi analitici per il riscontro dei residui.
Risultati:
Tra i metodi di estrazione si è dimostrato particolarmente efficacie e di facile esecuzione il metodo Soxhlet usando
come solvente il metanolo; la determinazone della Ryanodina si è effettuata con HPLC/UV-VIS con colonna RP-18.
In laboratorio gli estratti di Ryania Speciosa non hanno dimostrato di avere un efficacia contro adulti ne contro uova dei
citati fitofagi. L’efficacia contro le larve si è dimostrata variabile in funzione della specie. L’aggiunta di sinergisti ha
migliorato l’efficacia degli estratti. Tale aumento dell‘efficacia varia a seconda della specie in oggetto. Le prove di
campo confermano i risultati riscontrati in laboratorio.
30
BIOPESTICIDES: EVALUATION PROCESS FOLLOWING THE APPLICATION
OF THE DIRECTIVE 91/414/CE
M. Rubbiani
Laboratorio di Tossicologia Applicata
Viale Regina Elena 299 - 00161 Roma - ITALIA
E mail: [email protected]
Directive 91/414/CE for new and existing (revision) biopesticides requests the same kind of full evaluation, in terms of
risk assessment, provided for chemicals.
The first application of the guidelines immediately showed the obvious differences between the two types of products
(chemical and micro-organism based pesticides), and the need to distinguish within the critical end points addressed to
both of them.
This yielded to revise the data requirements for risk assessment (Annex II part B and Annex III part B of the Directive),
because of the foreseeable problems due to the specific nature of the micro-organisms based pesticides.
The different aspects regarding the critical end points of the evaluation process, such as mode of action,
characterisation, toxicological and microbiological features, as well as the possible risk for human and environment are
fairly highlighted.
Moreover, the problems raised in relation to the use and handling of these products, as well as the possibility to develop
new strategies for a more suitable toxicological and ecotoxicological evaluation (correctly hazard- and risk- based) are
reported.
The activity within Directive 91/414/CE evaluation procedures at EU level and the activity of the National Committee
for the registration of biopesticides are also shortly described.
31
ENTOMOPATHOGENIC NEMATODES IN BIOLOGICAL CONTROL: REALITY AND PROSPECTIVES
M. Ricci and A. Ragni
BioTecnologie BT srl Pantalla di Todi 06050 Perugia Italy
Tel: ++39 – 075 - 895091 Fax: ++39 – 075 – 888 776 E-mail: [email protected], [email protected]
General description
Entomopathogenic nematodes (EPNs) are small (about 1 mm in length) round worms and are obliged parasite of
insects. In the last few decades, they have been studied to develop new bio control agents (BCAs).
EPNs belong to the phylum Nematoda, class Secernentea, order Rhabditida, sub-order Rhabditina, super-family
Rhabditoidea, families Steinernematidae and Heterorhabditidae. So far, about 35 species have been described, but every
year new ones are added.
Bio-insecticide products based on EPNs consist of the infective juveniles (IJs) which represents a stage of their life
cycle that can survive out of the insect bodies without feeding, relying on their fat reserves. IJs can survive in the soil
for long periods (if the soil is wet they can survive for months) until locate a suitable insect host (Gaugler, 1988).
Mode of Action and Life Cycle
In the soil, the IJ searches for and infects a suitable insect host penetrating into its haemocoel via natural body openings
(mouth, anus, spiracles). Once in the haemocoel, the IJs release a symbiotic bacterium (Photorhabdus or Xenorhabdus
spp.) into the haemolymph. The bacteria rapidly multiply, killing the insect by septicaemia within 24-48 hours. The
nematodes feed upon the bacteria and degraded insect tissue and develop to first generation adult males and females (in
the Steinernematidae family) or hermaphrodites (in the Heterorhabditidae family). In both cases, after the eggs have
been fertilized, they are laid in the insect body or remain in the mother body. The hatched larvae complete their cycle
passing thorough 5 stages. EPNs can complete 1 to 3 generations, depending on the host insect size. As the insect
resource becomes exhausted, most of the juveniles differentiate into a particular third stage that becomes the survival
form of the life cycle (Infective Juvenile). The insect cuticle then ruptures and the IJs escape into the surrounding
environment (Wouts 1980; Poinar 1990).
Behaviour
Because of their potential as biological control agents for insect pests, a great deal of research has been conducted on
their behaviour and ecology, in particular the behavioural ecology of host-finding. Host-finding typically involves five
steps; (I) host habitat selection, (II) localized search, (III) host orientation, (IV) host acceptance (or attachment), and (V)
host suitability (Vinson, 1975). Individual species adopt different approaches to each of these steps. Host searching
strategies can be divided into two approaches. Ambushing, where the environment moves past a waiting animal, is most
efficient against high densities of highly mobile hosts. Cruising, where the animal moves through the environment, is
most efficient against sedentary and widely distributed hosts (Huey and Pianka, 1981). The entomopathogenic
nematodes Steinernema carpocapsae and Heterorhabditis bacteriophora appear to occupy similar habitats and use
similar hosts, yet they exhibit different searching behaviours. S. carpocapsae appears to be an ambusher and H.
bacteriophora appears to be a cruiser. For these two different strategies energy reserves are potentially the most
important endogenous regulatory factor. Nematode infective stages are non-feeding and rely on stored lipids as the
principal energy reserve. S. carpocapsae is on average 30% lipid by dry weight (Womersley, 1990). The rate of decline
in lipid levels may be correlated with their foraging activity. Thus, many parasite infective stages remain inactive unless
stimulated. S. carpocapsae becomes inactive without stimulation while H. bacteriophora remains active (Gaugler and
Campbell, 1991). Increased age, and the subsequent depletion of lipids, decreased the pathogenicity of S. carpocapsae
and H. bacteriophora when the host was farther away (Vanninen, 1990).
Symbiotic bacteria
Symbiosis
Entomopathogenic nematodes are symbiotically associated with specific bacteria. Steinernematidae have their
mutualistic relationship with bacteria of the genus Xenorhabdus and Heterorhabiditae are associated with bacteria of the
genus Photorhabdus. The two bacteria genera are gram-negative, belonging to the family of Enterobacteriaceae.
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Xenorhabdus spp. and Photorhabdus spp. are carried in the intestine of the infective stages of nematodes; Steinernema
spp. have a special intestinal vesicle where the bacteria are kept while Heterorhabditis spp. have their symbionts in the
lumen of the anterior part of the gut.
Once the nematodes have penetrated a host, they release the bacteria symbiont in the haemolymph causing a
septicaemia and the host dies. Inside the larval cadavers the bacteria growth and nematodes develop to adult stages,
multiply and sexually reproduce. Although the nematodes can grow axenically (without any micro-organism) or on
other bacteria, their reproduction is optimal only in presence of their natural symbiont. Thus, Xenorhabdus spp. and
Photorhabdus spp. have the status of obligate symbiont because they provide essential nutrients for the correct
multiplication of the nematodes (Boemare et al., 1997).
The nematode-bacterium complex represents an exciting example of co-evolution that involves the interaction between
the host and the nematode-bacterium complex, the relationship within the two symbiotic partners and the interaction
between the bacterium and its bacteriocines. To investigate these three connections, it is necessary to study the insect
defence reactions, the pathogenicity of the nematodes and the bacterium pathogenicity, symbiotic properties and
lysogeny.
Insects have an immune system based on phagocytosis and encapsulation of exogenous agents. The recognition of
foreign bodies is mediated by the production of some humoral factors (Boemare et al., 1997). Entomopahtogenic
nematodes are able to depress or to escape the immune defence of insect larvae. This is obtained both with nonrecognition by insect humoral factors and by escaping the phagocytosis and encapsulation. The nematodes secrete an
immune-depressive factor against insect immuno-proteins directed against the symbiotic bacteria. As been reported also
that axenic nematodes produce toxin(s) able to cause paralysis and death of the host (Simoes, 1994). Symbiotic bacteria
display also pathogenic action them-self: they produce toxins and secrete enzymatic complex (proteases, lipases and
phospholipases) that help the establishment of septicaemia in the host. Thus both partners of the symbiotic complex
collaborate to kill the host. It is also been reported that axenic S. glaseri or its sole symbiont X. poinarii, have no
entomopathogenic action, but this was restored after re-association of both partners (Akhurst and Boemare, 1990). The
observed lysogenity of the bacterial symbiont is also useful for the symbiosis relationship because it helps the
symbionts to compete with closely related bacteria.
Production of useful molecules
Both Xenorhabdus and Photorhabdus spp. can be grown independently from they nematodes partners under standard
laboratory conditions. In vitro, bacteria secrete several extracelluar products: proteases, chitinases, lipases,
phospholipases, antibacterial and antimycotic substances. Those compounds are produced also in vivo and the enzymes
digest the tissues of dead larvae in order to provide nutrients for both bacteria and nematodes. The antifungal and
antibacterial products are used to preserve the cadavers from the colonisation of other microorganisms. Those active
substances have also been tested for their use in biolgical control of phytopatogenic fungi (Ng and Webster, 1997).
In vitro, X. nematophilus and X. bovienii produce aliphatic amides (Park and Paik, 2001) and dithiolpyrrolones tested
for antineoplastic activity (Webster and Chen, 1999).
Recently, strains of Photorhabdus capable to secrete exotoxins (Bowen et al., 1998), or to produce endotoxins (Ragni et
al., 1997), with oral insecticidal activity, have been described. This new source of natural toxins could help both the
fight against the development of insect resistance as well as novel sources of specific activities in combination with
other known bio-pesticides.
Target organisms
Due to their poor resistance to dry conditions, EPNs are mainly applied against soil dwelling insects. Otiorrhynchus
sulcatus F. (Coleoptera, Curculionidae) is considered one of the major worldwide insect pests. The larvae feed on the
root system of several plant species (mainly ornamentals and nursery stock) producing its consequent decay and very
significant economic losses. A part other Curculionids such as Conorhychus mendicus, Balaninus elephas, Diaprepes
abbreviatus and Cylas formicarius on ornamentals, berries, citrus, sugar beet and chestnuts, other pests are: Scarabeids:
Popilia japonica, Maladera matrida, Phyllopertha horticola , Aphodius spp. on lawns, orchards, nurseries and sweet
potatoes; Diptera: Sciarids, Phorids, Scatopsids, Cecidomids on mushrooms, greenhouses; Lepidoptera: Noctuids on
vegetable crops. Several studies have been performed to find out if EPNs can be used on the aerial part of the plants
against chewing and sucking insect, but, yet, without any important success.
Application Technology
Particular strategies must be developed in order to insure the successful delivery of the nematodes to the target site and
target insect. Many parameters must be investigated to improve EPNs performance. One of these is the determination of
target insect life-stage susceptibility, since different life stages of different species are not equally susceptible. It has
been shown that pest population levels and behaviour have a great influence on nematode performance and must be
considered carefully. Often, larval stages of insects such as borers are not accessible to nematodes. Selecting the most
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appropriate nematode species and/or strain is important for efficacy and commercial development. Abiotic factors such
as soil type, soil temperature and moisture, and biotic factors, including pathogens and predators, can greatly influence
the nematodes ability to effectively kill the target pest.
Application strategies, including field dosage, volume, irrigation and appropriate application methods, are very
important, especially if nematodes are to be integrated with other control strategies. Compatibility with other
agrochemicals has been demonstrated allowing the use of EPNs in existing Integrated Pest Management programs. Also
crop morphology and plant phenology must be considered. Additional researches have shown the potential for
entomopathogenic nematodes to be used in other habitats (e.g. aquatic, foliar, and cryptic), and in manure.
EPNs are applied to the crops by common sprayers (also by drip irrigation); attentions should be taken in order to avoid
sedimentation of nematodes by continuous agitation and to avoid exposure of nematodes to direct sunlight, preparing
the suspension under a shade and applying the product at late afternoon. It is important to pre-irrigate the soil with at
least 6 mm of water and an irrigation post application of about 6-12 mm of water. The treated area should be irrigated
frequently (at least every 1-3 days with a minimum of 6 mm of water) to maintain appropriate soil moisture for EPNs.
Production
EPNs can be produced in vivo using insects (in general, the last instar larvae of the Lepidoptera Galleria mellonella).
This method requires low technology but, in the other hand, requires a constant source of healthy insects and a large
amount of manpower and thus costs are enormous, in particular, in western countries. EPNs can be produced in vitro,
both in solid and in liquid media. The latter method is the most sophisticated because requires innovative know how and
modern facilities and equipment; this method allows a strong reduction in the production process costs.
Formulation
Entomopathogenic nematode based-product requires a reliable and stable formulation. This has been a difficult task,
because the larger markets are demanding a product with a minimum shelf life of six months when stored at room
temperatures (20°-25°C). Nematode products contain living animals that have certain temperature, oxygen and moisture
requirements necessary for their survival and effectiveness as control agents, cannot reach this market requirements.
While no nematode formulation has been completely successful in reaching these goals, some have come very close
(Georgis, 1992) such as a product based on the nematode Steinernema carpocapasae formulated in granules. A part this
exception, due to the particular physiology of this species, the most common available formulations are based on clays
with a limited shelf life (1-2 months) at 5-10°C.
Perspectives
To broaden the use of EPNs in Biological Control or IPM Programs, it is necessary to achieve positive results in the
following fields:
New Agricultural Targets; for instance: Grillotalpa grillotalpa, Melolontha melolontha, Leaf miners, Fruit flies
(Bactrocera sp., Ceratitis sp., Ragoletis sp.), Aleurodids, Aphids.
Market Needs; following the market needs, several aspects need to be improved. The reduction of production costs may
be achieved by new technology and/or new strains with new activity, higher infectivity and higher production. The
product shelf life can be extended by new technology and new strains capable to live in dry conditions.
Extended applications; the following applications may extend the use of EPNs: foliar application, stock farm use (ticks,
fleas, flies), domestic use (flies, cockroaches, fleas).
Particular attention is dedicated to new entomopatogenic nematodes able to survive and parasitize hosts at extreme
environmental conditions such as cold, warm and dry environments and to the exploitation of the biological active
molecules produced by the symbiotic bacteria. Related to these topics, BioTecnologie B.T., a small Biotech company,
based in Umbria Region (Italy), is developing two innovative products: a new strain of EPN active at very low
temperatures and an innovative bio-insecticide based on endotoxins produced by a particular strain of the nematode
symbiotic bacterium Photorhabdus luminescens:
- Entomopathogenic nematode active at low temperature
Many soil dwelling insect pests can or should be controlled when the temperatures are below 12°C (late autumn or early
spring). The most important representative is Otiorhynchus sulcatus (Coleoptera, Curculionidae), a serious pest of soft
fruits and ornamental plants. The inadequacies of chemical pesticides, the banning of the most persistent ones (e.g.
aldrin) and the increasing dispersal of the insect, has led to an urgent need of a new and safer pest control method
(Zimmermann, 1996). The available EPN based products, lack on effectiveness at temperatures below 12 -15°C.
A nematode strain, belonging to the species Steinernema kraussei, has been discovered and it is very active against O.
sulcatus larvae even at 3°C (Ricci and Fridlender, 1999). The strain is also effective against larvae of B. elephas at 8°C.
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It can be reproduced in vivo at 15°C and in liquid culture at 20°C without loosing its cold activity. A product based on
this nematode is actually under development.
-
Bio-insecticide based on endotoxins produced by a particular strain of the nematode symbiotic bacterium
Photorhabdus luminescens
It has been discovered (Ragni et al., 1997) a newly characterize strain of Photorhabdus luminescens, denominated
XP01, which is highly toxic when directly administered by food contamination assays to neonates larvae of the
Lepidopetran pests Mamestra brassicae and Cydia molesta. Further characterization made with Mamestra brassicae, as
target insect, showed that the activity of this strain is caused by an intracellular compound that is not secreted into the
culture media. In fact, the surnatant of fermented broth of XP01 had no toxic activity and this discovery was different
from what was reported for the W-14 strain of P. luminescens (Bowen et al., 1998). It was demonstrated that the toxic
effect of XP01 is maintained even when the bacterial cells are killed. The discovery of Photorhabdus luminescens strain
XP01 indicates the existence of a presumably new family of entomotoxic proteins present in Photorhabdus and
probably in Xenorhabdus spp. These insecticidal toxins could be used as an active bio-pesticide ingredient in a similar
manner to the delta-endotoxins of B. thuringiensis. This new source of natural toxins could help both the fight against
the development of insects resistance as well as serve as sources of novel specific activities in combination with other
known biopesticides.
Further studies could lead to understanding of the genetic control involved in the expression of XP01 insecticidal
toxins.
Conclusions: An Analysis of the Commercial Entomopathogenic Nematode Produc t
In this type of analysis, commercial and scientific factors should be sorted into four categories either as ‘Strengths,
‘Weakness’, ‘Opportunity’, or ‘Threat’ (source: Pest Management Resource Centre with the following information as
available at the site: http://www.pestmanagement.co.uk/special/microb/com_prod.html).
-
-
-
-
Product Strengths
Consistent supply available; supply will be consistent when sales forecasts is accurate and production levels are
predictable. Most EPN species can be produced on a large scale, up to a capacity of 80,000 litre fermenters, at a
concentration of 150-300,000 infective juveniles per millilitre.
The product is safe and ‘natural’; comprehensive data packages have been developed to support the safety claims
made about EPN products. Workers benefit from not having to wear protective clothing during product application.
Studies have also shown that EPN applications do not have a significant impact on non-target insects (Georgis et al.,
1991).
Products do not require registration; registration regulations vary from country to country and have not
been standardized in Europe. In most countries EPN’s are regarded as animals and are exempt from pesticide
regulations. This means that once efficacy data has been generated, products can be introduced. The commercial
advantage of this is that product sales can begin at least three years faster than conventional products.
Products are easy to use; no additional equipment is required to apply EPN products, instructions have been
designed to be similar to conventional pesticides making their use familiar to growers.
Product Weaknesses
-
-
-
The insect life cycle; the susceptible stage of the insect life cycle may be in an inaccessible niche, or in a niche
unsuitable for EPN survival. For example, excessively wet-dry soil or foliage.
Insect behaviour; insect behaviour or physiology may reduce EPN product efficacy. Gaugler (1988) suggested
that the tendency of quiescent soil insects, especially pupae, to release carbon dioxide in bursts rather than
continuously reduced the ability of infective juveniles to follow a chemical gradient to the host. It has been reported
that when exposed to infective juveniles of Steinernema carpocapsae Weiser (All strain) American cockroaches,
Peripaneta americana (L), actively groomed nematodes from legs and antennae to prevent infection.
Efficacy may be limited at hot and cold temperature; for example, control of black vine weevil Otiorhynchus
sulcatus (F.) larvae in blackcurrant crops by Steinernema carpocapsae was limited at temperatures below 15 °C.
Product formulation; products should have a shelf life at room temperature. This can provide more flexibility in the
distribution system. For example, a product shelf life at room temperature makes it easier to deliver and store the
product in a warm climate, or the distributor could reduce costs by taking more products at one delivery and risk a
temporary reduction in sales. Conversely, if the growers have cold storage facilities, as in the mushroom market, a
sophisticated formulation may not be required, reducing development costs.
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- EPN products must be applied to moist soil; EPN’s can survive dry conditions (Womersley, 1990) but cannot search
for and locate the insect host.
Product Opportunities
-
-
-
The market value; it is important that the market supports growing sales of the newly introduced product at a
price that immediately provides a return on the investment of the initial product development.
No chemical available / phytotoxic effects/ chemical not effective; For example, in American cranberry crops
no conventional chemical is available to control the Cranberry Girdler Chrysoteuchia topiaria, or strawberry root
weevil Otiorhynchus ovatus (L). Growers were limited to using cultural methods to prevent pest damage until an
EPN product was developed. In mushroom crops application of insecticides reduced yields.
EPN searching behaviour; in field grown crops it is particularly difficult to apply insecticides at the point
where the pest is causing damage. In strawberry plants grown under plastic access to the roots where black vine
weevil larvae cause damage can be difficult. However, by applying EPN’s through T-Tape irrigation systems
infective juveniles introduced below the soil surface can search for and infect larvae developing in the strawberry
roots.
Knowledge of the pest life cycle; expert knowledge obtained from universities and extension services can be
used to determine the correct time for product application. It is important that the insect biology is understood to
prevent ‘mysterious’ product failures.
Product Threats
Grower confidence; any change in the product can cause problems with grower confidence. Even a change in the
colour of the product container can cause suspicions that something is not quite right with the production system.
Product education; in the initial stages of product introduction distributor and grower education is vital. If the
growers misuse the product and observe damaged crops it is unlikely that they will continue to take risks on a ‘new’
idea. It is easy for a poor reputation to build up and almost impossible to recover confidence in the market.
-
Competitive conventional insecticides; the development of new conventional chemicals may address
application problems, for example slow release formulations.
References
Akhurst, RJ and N. Boemare. 1990. Biology and taxonomy of Xenorhabus. In: Entomopathogenic nematodes in
biological control R. Gaugler and H.K. Kaya, eds. CRC Press; Boca Raton FL. pp. 75 -90.
Boemare N., A. Givaudan, M. Brehélin and C. Laumond. 1997. Review article. Symbiosis and pathogenicity of
nematode-bacterium complex. Symbiosis Vol. 22: 21-45
Gaugler, R. (1988). Ecological considerations in the biological control of soil-inhabiting insects with entomopathogenic
nematodes. Agriculture, Ecosystems and Environment 24 351-360.
Bowen D.J., T.A. Rocheleau, M. Blackburn, O. Andreev, E. Golubeva, R. Bhartia, and R.H. ffrench-Constant. 1998.
Insecticidal toxin from the bacterium Photorhabdus luminescens. Science. 280. 2129-2132
Gaugler, R. and J. F. Campbell, 1991, Entomopathogenic nematode behavioural response to oxamyl, Ann. appl. Biol.
119, p. 131-138
Georgis, R., Kaya, H.K., and Gaugler, R. (1991). Effect of steinernematid and heterorhabditid nematodes (Rhabditida:
Steinernematidae and Heterorhabditidae) on non-target arthropods. Environ. Entomol. 20:815-822.
Georgis, R. 1992. Present and future prospects for entomopathogenic nematode products. Biocontrol Science and
Technology 2: 83-99.
Huey, R. B., and E. R. Pianka, 1981, Ecological consequences of foraging mode, Ecology 62, 991 -999.
Ng K.K. and Webster J.M.. 1997. Antimycotic activity of Xenorhabdus bovienii (Enterobacteriaceae) metabolites
against Phyotophthora infestans on potato plants. Can. J. Plant Pathology 19 (2): 125-136.
Park S.H. & Paik S.U. 2001. Novel aliphatic amide having anticancer property. PCT Application WO01/49656
Poinar, G. O., Jr., 1990, Biology and Taxonomy of Steinernematidae and Heterorhabditidae, In Entomopathogenic
Nematodes in Biological Control, R. Gaugler and H. K. Kaya Eds, CRC Press, Boca Raton. pp. 23 -58
Ragni A., Valentini F and Fridlender B.1997. “Insecticidal Bacteria”. PCTIL97/00246
Ricci M. and Fridlender B., New entomopathogenic nematodes active at low temperatures. PCT/IL99/00218
Simoes, N. 1994. Virulence factors produced by the entomopathogenic nematode Steinernema carpocapsae during
parasitism. Proce. of the Vith Intern. Colloquium of Invertebrate Pathology Aug.28-Sept. 2. 1994. Montpellier, France
p. 116-119
Vanninen, I., 1990, Depletion of endogenous lipid reserves in Steinernema feltiae and Heterorhabditis bacteriophora
and effect on infectivity, Proceedings and Abstracts of the 5th International Collogium on Invertebrate Pathology and
Microbial Control, Adelaide.
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Vinson, S. B., 1975, Biochemical coevolution between parasitoids and their hosts, In Evolutionary Strategies of
Parasitic Insects and Mites, P. W. Price Ed., Academic Press, New York.
Webster J.M & G. Chen. 1999. Dithiolpyrrolones and their corresponding and dioxides as antineoplastic agents. PCT
Application WO99/12543.
Womersley, C. Z., Dehydration Survival and Anhdrobiotic Potential, 1980. In Entomopathogenic Nematodes in
Biological Control, R. Gaugler and H. K. Kaya Eds, CRC Press, Boca Raton.
Wouts, W.M., 1980. Biology, life cycle and redescription of Neoaplectana bibionis Bovien, 1937 (Nematoda:
Steinernematidae). J. Nematology 12 1 62-72.
Zimmermann, G., 1996. Microbial control of vine weevil. Proceedings of the second international workshop on vine
weevil (Otiorhynchus sulcatus Fabr.) (Coleoptera, Curculionidae). Braunschweig, Germany, May 21-23, 1996.
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DETERMINAZIONE DEL CONTENUTO DI MICOTOSSINE IN PRODOTTI COMMERCIALI
PROVENIENTI DA AGRICOLTURA TRADIZIONALE E BIOLOGICA
B.Beretta, C.Ballabio, F.Tacchini, A.Cattaneo, C.L.Galli, C.Gigliotti*, P.Restani
Dipartimento di Scienze Farmacologiche - Università degli Studi di Milano, via Balzaretti, 9 - 20133 Milano
*Dipartimento di Produzione Vegetale - Università di Milano, via Celoria, 2 - 20133 Milano
Le micotossine sono metaboliti secondari prodotti da alcuni funghi filamentosi che possono infestare le derrate
alimentari. Questi composti sono stabili e permangono nell'alimento dopo la morte del micete produttore; sono resistenti
ai lunghi periodi di conservazione ed ai comuni trattamenti industriali e casalinghi di preparazione dei cibi.
Tra le micotossine più importanti dal punto di vista agro-economico e della salute pubblica si ritrovano le ocratossine e
la patulina.
La patulina è prodotta da alcune specie di Aspergillus e Penicillium che possono contaminare alcuni frutti e cereali ed in
particolare la mela. E' una molecola relativamente instabile, ma è termoresistente se posta in ambienti acquosi e acidi.
Diversi studi hanno portato a considerare tale composto come potenziale cancerogeno per l’uomo, anche se ulteriori
approfondimenti sono necessari per comprendere se l’effetto cancerogeno possa manifestarsi alle concentrazioni
normalmente riscontrate negli alimenti.
Le ocratossine sono prodotte da alcune specie di Aspergillus e Fusarium e sono note per la loro nefrotosicità. Il
composto più tossico presente in questo gruppo è l'Ocratossina A (OTA), una molecola abbastanza stabile che può
passare inalterata attraverso la catena alimentare e ritrovarsi intatta in carne, cereali e derivati,
L’oggetto di questo studio si articola nella valutazione:
· della presenza di patulina in omogeneizzati e succhi di mela provenienti da diverse strategie agricole (coltivazione
tradizionale, lotta integrata e agricoltura biologica);
· della presenza di OTA in farine di cereali destinate alla prima infanzia, provenienti da diverse strategie agricole
(coltivazione tradizionale, lotta integrata e agricoltura biologica);
· della qualità tossicologica dei prodotti analizzati in funzione della diversa provenienza agricola.
Per la ricerca della patulina sono stati analizzati 23 omogeneizzati di mela provenienti da agricoltura tradizionale,
biologica e da lotta integrata e 21 succhi di mela provenienti da agricoltura tradizionale e biologica.
Per la ricerca dell'OTA sono stati analizzati 30 lotti di semolino, 33 lotti di crema di riso, 26 lotti di crema di mais e
tapioca e 30 lotti di crema multicereali. Anche questi prodotti derivavano da agricoltura tradizionale, biologica e da lotta
integrata.
Il protocollo di analisi prevede una prima fase di estrazione-purificazione del campione ed una seconda fase di analisi in
HPLC.
Tutti i campioni di omogeneizzati analizzati presentavano concentrazioni di patulina al di sotto del limite di legge
stabilito in 50 mg/kg; la concentrazione maggiore riscontrata era di 6,39 mg/kg e 5,97 mg/kg per i prodotti da coltura
tradizionale e da lotta integrata, rispettivamente. L'analisi statistica non ha evidenziato differenze significative tra valori
determinati nei campioni provenienti dalle due strategie agricole considerate.
Il più alto valore di patulina riscontrato nei succhi di mele provenienti da agricoltura tradizionale era 3,03 mg/kg, mentre
i risultati relativi ai prodotti biologici erano estremamente variabili con un valore massimo di 28,24 mg/kg. Dal
confronto dei dati, dal punto di vista statistico, si è evidenziata una differenza significativa tra le due tipologie di
prodotti inclusi nello studio.
Per quanto riguarda la ricerca dell'OTA, nessun campione di crema di mais e tapioca e nessun lotto dei prodotti
provenienti da lotta integrata conteneva la micotossina in quantità rilevabili. Tra i lotti di semolino analizzati
provenienti da coltivazione tradizionale, 2 dei 6 campioni risultati positivi superavano il valore limite fissato per legge
in 0,5 mg/kg (0,58 e 0,65 mg/kg); mentre un solo lotto biologico presentava O,18 mg/kg di OTA. Tra le creme di riso
incluse nello studio solo nei campioni biologici è stata rilevata la OTA ed in particolare due lotti superavano il limite di
legge, con valori di 0,7 e 0,74 mg/kg. Le analisi delle creme multicereali hanno evidenziato la presenza di OTA solo nei
prodotti da agricoltura tradizionale e nessun campione superava il limite di legge.
Dai risultati ottenuti, il contenuto di OTA non sembra direttamente correlabile alla pratica agricola seguita; infatti nei
semolini i valori più alti si sono riscontrati nei campioni provenienti da agricoltura tradizionale, mentre per le creme di
riso si è verificato l’opposto.
Le conclusioni di questo studio suggeriscono che la presenza di micotossine è un problema quanto mai attuale; i
prodotti in commercio da noi analizzati, pur essendo nella quasi totalità dei casi nei limiti di legge, indicano la assoluta
necessità di controllare le derrate alimentari e non trascurare i prodotti biologici che per le modalità di coltivazione e
conservazione possono essere a maggior rischio di contaminazione.
Beretta B., Gaiaschi A., Galli C.L., Restani P., 2000, Patulin in apple-based foods: occurrence and safety evaluation.
Food Add. Contam., 17, 399-406.
Beretta B., De Domenico R., Gaiaschi A., Ballabio C., Galli C.L., Restani P., 2002, Ochratoxin A in cereal-based baby
food: occurrence and safety evaluation. Food Add. Contam., 19, 70-75.
38
PROTECTION OF GRAPEVINE FROM POWDERY MILDEW BY USING NATURAL SUBSTANCES AND
THE MICROBIAL ANTAGONIST AMPELOMYCES QUISQUALIS
Agostino Santomauro (1), Giuseppe Tauro(2), Maurizio Sorrenti, Francesco Faretra (1)
(1)
(2)
Dipartimento Protezione delle Piante e Microbiologia Applicata, University of Bari
Centro di Ricerca e Sperimentazione in Agricoltura "Basile Caramia", Locorotondo (Bari)
Powdery mildew, caused by Uncinula necator (Schw.) Burr., is one of the most common and severe diseases of
grapevine, causing heavy yield losses wherever the crop is intensively grown. Many fungicides are available for its
control. Recently, however, the rapid diffusion of organic agriculture has stressed out the lack of solid experimental
data on the effectiveness of natural substances and microbial antagonists that are potential alternatives to the use of
chemicals.
The present paper reports the results of field trials carried out in 1998, 2000 and 2001 in order to evaluate the
effectiveness of natural substances and the mycoparasite Ampelomyces quisqualis Ces. for the control of grapevine
powdery mildew.
The statistical scheme of randomized blocks with four replications and plots of 8-10 plants was adopted in all
trials. The first two sprays were always carried out at the beginning and at the end of blossoming. Afterwards,
treatments were carried out at one-week intervals until véraison. The tested spray schedules are listed below.
Tested compounds
Ampelomyces quisqualis +
Mineral oil
Ampelomyces quisqualis +
Pinolene
Sodium bicarbonate +
Mineral oil
Sodium bicarbonate +
Pinolene
Sodium bicarbonate
Foliar fertilizer
Powder milk
Mineral oil
Pinolene
Pinolene
Potassium salts of fatty acids
Sulphur
Sulphur
Formulates
AQ 10 (Bio Intrachem)
Ultra Fine Oil (Bio Intrachem)
AQ 10 (Bio Intrachem)
Vapor-Gard (Bio Intrachem)
RPH (Carlo Erba)
RPH (Carlo Erba)
RPH (Carlo Erba)
Trym (Italpollina)
Vigor Latte (Mignini)
Nu - Film 17 (Chimiberg)
MYX 403.2-050 (Mycogen)
Zolfo WG (Bayer)
Kumulus Tecno (Syngenta)
Rates
(g or ml/ha)
50 +
2000
50 +
2000
5000 +
2000
5000 +
2000
5000
150
10000
10000
10000
10000
10000
2500
5000
Year of employment
1998 2000
2001
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Symptom severity was assessed two to four times for each trial by observing 250-300 bunches per plot and
counting infected berries. An empirical scale with 8 class of infection was used to calculate the following parameters:
the percentage of infected organs (Tehon’s “prevalence”), the disease severity (Tehon’s “destructiveness”) and its
weighted average value (according to the McKinney Index). All data were submitted to variance analysis and mean
values were separated by Duncan’s Multiple Range Test.
During 1998 and 2000, powdery mildew infections interested 77% and 65% of bunches in untreated plots,
respectively. The values of McKinney Index, however, were not very high, reaching 17% and 13%, respectively. Under
these conditions of medium disease pressure, all of the tested compounds showed to be effective against powdery
mildew, decreasing significantly symptom severity on both berries and rachis, as compared to the untreated check. In
particular, sodium bicarbonate (used either alone or in mixture with mineral oil) and pinolene showed the highest
effectiveness, allowing reductions up to 90% of symptom severity. These results were substantially confirmed even
under the higher disease pressure occurred in the trial carried out in 2001. In that year, powdery mildew symptoms were
observed on 88% of bunches in untreated plots, with a value of McKinney Index as high as 34%. Even under such
conditions, pinolene and sodium bicarbonate, used either alone or in mixture, allowed a significant reduction of the
disease. Moreover, very interesting results were obtained with powder milk that yielded the best control of the disease.
Repeated sprays with pinolene or potassium salts of fatty acids had some negative side effects on bunches, by
removing waxes from the surface of berries and causing persistent bad smell. Repeated sprays with milk left visible
residues, thus getting dirty bunches. Such effects represent a serious limitation for their usage on table grape, but should
be negligible for wine grape.
In conclusion, the tested natural substances and A. quisqualis showed an interesting, although not complete,
effectiveness against grapevine powdery mildew. Yet, their exclusive usage in the protection of table grape seems can
39
not be recommended due to their partial effectiveness and negative side effects. On the other hand, they might found a
larger application in the protection of wine grape from powdery mildew in organic agriculture as well as in integrated
disease management. However, further investigations are needed in order improve knowledge and rule out any
detrimental effects on taste, flavor and other qualitative characteristics of wine.
Acknowledgements: Work supported by the Italian Ministry of Agricultural and Forestry Policies in the frame of the
research project “Technical tools for crop protection in organic agriculture” – Sub-project “Natural substances in crop
protection from fungal diseases”.
40
I MEZZI TECNICI PER L'AGRICOLTURA BIOLOGICA: UN SETTORE IN SVILUPPO SENZA LA CERTEZZA
DELLA NORMATIVE CHE LO REGOLANO
Massimo Benuzzi
Presidente ASSOMETAB (Associazione Italiana dei produttori e distributori di mezzi tecnici per l’agricoltura biologica
e ecocompatibile)
L’Agricoltura biologica è uscita da una fase pionieristica nella quale le cosiddette “scelte di vita” avevano maggior
importanza nella gestione dell’azienda rispetto a decisioni di tipo imprenditoriale; l’incremento delle superfici
interessate e l’esigenza di soddisfare la sempre più crescente domanda di “cibi puliti” pone il settore di fronte a precise
scelte. Anche la Grande Distribuzione Organizzata ha recentemente dimostrato che il prodotto biologico può entrare
nella sua gamma, promuovendo anche marchi propri che contraddistinguono una specifica linea di agricoltura
biologica. Nello stesso tempo diventa sempre più chiaro che i prodotti da agricoltura biologica potranno ulteriormente
crescere in quantità e qualità, con le adeguate remunerazioni per tutti i soggetti della filiera, solo se verranno garantite in
modo sicuro e trasparente la certificazione delle produzioni biologiche.
Senza entrare nel merito del “controllo dei controllori” o dell’esistenza o meno di problemi nella catena di
certificazione, ASSOMETAB vuole mettere in evidenza quanto al momento attuale sia stato poco considerato il tema
dei mezzi tecnici per l’agricoltura biologica.
Prima di tutto è necessario sottolineare che Istituti di ricerca e Università stanno, attraverso le aziende interessate,
sempre di più proponendo nuove soluzioni ai problemi tecnici che scaturiscono dalle esigenze degli operatori del
biologico. Infatti negli ultimi anni sono stati risolti numerosi problemi fitoiatrici, non solo legati all’agricoltura
biologica, con l’avvento di nuovi insetticidi e fungicidi di origine naturale, soluzioni tecniche alternative alla chimica.
Fondamentale è il ruolo delle aziende legate a questo settore che hanno investito e stanno investendo nella ricerca, nella
messa a punto di nuovi biopesticidi (ma anche di fertilizzanti) e anche nelle relative registrazioni. Si riesce così a
coniugare economia e ecologia, ovvero due cose che, fino a poco tempo fa, sembravano inconciliabili. Se non ci fossero
aziende indirizzate a questo settore dell’agricoltura probabilmente i produttori biologici dovrebbero attendere che anche
le multinazionali agrochimiche decidano di muoversi per promuovere lo sviluppo del settore. Forse, in quel caso,
dovrebbero armarsi di tanta pazienza……
Il fatto che in Italia sia presente una Associazione come ASSOMETAB con aziende che investono nella ricerca di
fertilizzanti e fitofarmaci per il biologico proprio nel paese europeo con le superficie più ampia a biologico, forse, non è
un caso.
Anche gli operatori del biologico (agricoltori e tecnici) devono avere presente che scegliere aziende che investono nel
biologico (come quelle di ASSOMETAB) significa dare una mano a soggetti che sicuramente reinvestiranno buona
parte dei ricavi in nuovi prodotti per il biologico, innescando così un circolo virtuoso; magari un concime o un Bacillus
thuringiensis di una multinazionale sono più economici, ma in un’ottica più lungimirante (quella che fa crescere un
settore), privilegiare una azienda rispetto ad un altra equivale ad effettuare una scelta che potrebbe rivelarsi importante
per lo sviluppo dl settore.
Questo sviluppo è però attualmente minacciato dal Decreto 290, più specificamente dai punti 1 e 2 dall’articolo 38 che
liberalizzando in modo assolutamente inadeguato il settore ha permesso ad aziende con poche scrupoli di mettere sul
mercato mezzi tecnici non testati che possono screditare tutto il segmento.
Riportiamo alcuni esempi di quali problemi tale DPR ha scatenato:
- Come potranno gli enti certificatori garantire che i prodotti biologici siano sicuri quando i fitofarmaci per
produrli non sono adeguatamente controllati ?
- Come potrà la GDO garantire che i propri marchi siano realmente aderenti a quanto vantato nelle promozioni
pubblicitarie ?
- Perché aziende che hanno investito ingenti somme in registrazioni si devono trovare fronteggiare una
concorrenza “sleale” che mette sul mercato fitofarmaci prodotti e controllati da “apprendisti stregoni” ?
- Perché quando un insetticida biologico non è registrato su una coltura, quello simile (o che per lo meno vanta
di esserlo con il nuovo DPR) può essere invece impiegato dovunque ? In pratica per i fitofarmaci registrati
esiste la necessità di registrare i prodotti su ciascuna coltura indicando anche i residui ammessi; nel caso di
prodotti “regolarizzati” con il DPR 290 invece non si pone alcun limite su nessuna coltura, creando di fatto una
disparità assolutamente assurda.
- Perche il DPR 290 non specifica assolutamente niente circa i tempi di carenza; forse il rotenone regolarmente
registrato deve avere 10 giorni di tempo di carenza e quello ammesso dal DPR 290 nessun giorno? I Bacillus
thuringiensis registrati 3 e i Bacillus thuringiensis non passati al vaglio del Ministero della Salute nessuno ?
Significa che è meglio non farsi controllare ?
Questi sono alcuni punti che vorremmo far sapere a chi si interessa veramente del settore.
ASSOMETAB ha avanzato al MIPAF sin dall’estate scorsa le sue proposte per una normativa chiara ed efficace sui
mezzi tecnici per l’agricoltura biologica. In sintesi una registrazione semplificata che abbrevi significativamente i
lunghi tempi (e i relativi costi) prima dell’immissione sul mercato, ma che mantenga elevate garanzie per il
consumatore, l’agricoltore e l’ambiente.
41
IMPEGNO DEL MIPAF NEL BIOLOGICO
Giancarlo Imbroglini
In questi ultimi tempi, si è osservata una notevole crescita dell’agricoltura biologica in Italia che ha portato il nostro
Paese a detenere il primato in Europa per numero di aziende e per superficie coltivata a biologico ed a co llocarsi al 3°
posto nel mondo. Chi coltiva, alleva, prepara e importa, secondo il metodo di produzione biologico, deve sottostare alle
disposizioni contenute nel Regolamento della Comunità Europea n. 2092/91 e successive modifiche e integrazioni.
Nell’allegato II B del suddetto Regolamento sono elencati i prodotti fitosanitari che possono essere impiegati per la
difesa delle colture. I prodotti ammessi sono, però, in numero esiguo, dotati, generalmente, di scarsa efficacia e inoltre,
per alcuni di essi, quali i sali di rame, sono stati fissati limiti massimi di impiego. Questa situazione concorre fortemente
a frenare la crescita delle produzioni biologiche. Gli approfondimenti scientifici che si rendono, pertanto, necessari
devono tendere a fornire risposte esaustive alle numerose problematiche che gli operatori del biologico si trovano a
dover affrontare ed a vagliare con cura le innovazioni strategiche per la difesa non chimica delle colture. La ricerca in
agricoltura biologica è caratterizzata da una maggiore complessità rispetto all’agricoltura convenzionale in quanto la
gestione della produzione si fonda su sistemi olistici che rendono necessaria l’armonizzazione delle pratiche
agronomiche nel loro complesso. Anche la difesa fitosanitaria va inquadrata in un’ottica di sistema complesso e non
come verifica del miglior prodotto per risolvere un problema specifico. Nell’ambito delle attività di tutela e sviluppo
dell’agricoltura biologica, il Ministero delle Politiche Agricole e Forestali (MiPAF), autorità di controllo del settore a
livello nazionale, ha promosso e sostiene un programma per la trattazione delle diverse tematiche che riguardano la
difesa delle piante coltivate secondo il metodo biologico con riferimento sia agli aspetti normativi che agli aspetti
sperimentali. Ha cercato, in tal modo, di supportare l’agricoltura biologica con finanziamenti specifici. Con il progetto
“Difesa delle Produzioni in agricoltura biologica”, di durata triennale, ci si è posti l’obiettivo di investigare sui mezzi
tecnici utilizzabili per la difesa in agricoltura biologica valutandone l’efficacia, cercando di mettere a punto i dosaggi
ottimali, le epoche di trattamento più convenienti, il numero di trattamenti, nonché gli eventuali effetti secondari a loro
ascrivibili. L’obiettivo è anche quello di individuare altri mezzi di difesa, compatibili con il metodo di produzione
biologico, in grado di contrastare le numerose avversità di pre e post-raccolta.
42
POSTER
1. Agosteo G.E., Ambrosio M.: Prove di lotta all'oidio dello zucchino con prodotti alternativi in
pieno campo
2. Arnoldi A., D'Agostina A., Boschin G.: Lupinus Albus, an ancient European legume
particulary suitable for organic farming, may become an useful source of functional ingredients
3. Barrese E., Benincasa C., Lombardo N., Mazzalupo I., Pellegrino M., Perri E., Sindona
G.: Benzo(a)pyrene in organic virgin olive oils
4. Cabizza M., Melis M., Cabras P.: Effetto delle cere epicuticulari di frutta e vegetali sulla
fotodegradazione del rotenone
5. Castagnoli M., Liguori M., Nannelli R., Simoni S.: Insetticidi d'origine vegetale e acari
6. Casucci C., Monaci E., Perucci P.: Changes of biochemical parameters in soil amended with
moist olive husks
7. Casulli F., Santamauro A., Tauro G., Gatto M.A., Faretra F.: Natural compounds in the
control of powdery mildew on cucurbits in organic agriculture
8. Citarrei F., Scribano M., Coranelli S., Cellerino C., Quattrocchi L., Concezzi L., Ragni A.:
Methods for evaluation, in controlled conditions, of the growth of oilseed rape promoted by
bacteria
9. Convertini G., Donato F., Sasanelli N., D'Addabbo T.: Effetto dello spandimento di residui
dell'industria olearia sulla fertilità del suolo e su nematodi fitoparassiti
10. D'Addabbo T., Sasanelli N.: Nematicidal activity of acqueous extracts from rue (Ruta
Graveolens L.)
11. Gargani E., Del Bene G.: Valutazione dell'efficacia insetticida di Ryania, Rotenone,
Azadiractina e Beauveria Bassiana
12. Dugo G.mo, Giuffrida D., Drogo A., La Pera L.: Determination of Cd (II), Cu (II), Pb (II)
and Zn (II) in biological and not biological citrus essential oils by derivative potentiometric
stripping analysis (dPSA)
13. Floris I., Satta A., Cabras P., Angioni A.: Impiego del timolo nel controllo della varroosi delle
api. Efficacia, persistenza e residui
14. Pertot I., Delaiti M., Forti D.: Assesment of phytotoxicity to Grapevine of traditional and new
copper compounds used in copper reduction strategies in organic viticulture and its relationship
with environmental conditions and number of treatments
43
15. Fusari F., Petrini A.: Organic wheat quality and production: the results of three years of trials
16. Iannotta N.: Il controllo della "mosca delle olive" (Bactrocera oleae Gmel.) con metodi
consentiti in coltivazione biologica
17. Kelderer M., Casera C., Lardschneider E.: Il contenimento della ticchiolatura in frutticoltura
biologica
18. La Pera L., Lo Turco V., Lo Curto S., Mavrogeni E., Dugo G.mo: Influence of different
treatments on Cd (II), Cu (II), Pb (II) and Zn (II) content in Sicilian olive oils
19. La Torre G.L., Pellicanò T.M., Pollicino D., Alfa M., Dugo G.mo: Determination of phenolic
compounds in experimental wines subjected to different pesticides treatments
20. La Torre A., Donnarumma L., Lolletti D., Imbroglini G.: Control of apple scab in organic
farming
21. Maietti A., Mazzotta L., Saletti C., Mirolo G., Berveglieri M., Tedeschi P., Brandolini V.:
Contaminazione da micotossine in alimenti di produzione biologica
22. Maini P.: Peptidati di rame: prodotti innovativi a basso dosaggio a base di rame, chelato ad
amino acidi e peptidi
23. Perri E., Lombardo N., Muzzalupo I., Urso E., Pellegrino M., Sindona G., Benincasa C.,
Cavallo C.: Characterization of organic virgin olive oils from coratina cultivar
24. Perri E., Mazzalupo I., Rizzuti B., Pellegrino M., Sindona G., Benincasa C., Cavallo C.:
Characterization of organic virgin olive oils from ogliarola salentina cultivar
25. Pertot I., De Luca F., Vecchione A., Zulini L.: Efficacy evaluation of biological control
agents against Plasmopara viticola
26. Pulga A., Valmori I.: Il controllo delle poerazioni di campo come base della rintracciabilità a
garanzia del consumatore e dell'ambiente
27. Ricci M., Colli M., Barcarotti R., Ragni A.: Eco-toxicity of the entomopathogenic bacteriumnematode symbiotic complex toward non-target organisms
28. Ragni A., Valentini F.: Photorhabdus e Xenorhabdus: a new source of useful compounds for
biological control
29. Ricci M., Flek G., Colli M., Barcarotti R., Quattrocchi L., Coranelli S., Concezzi L., Fifi
A.P., De Nicola R., Scribano M., De Berardinis M., Citarrei F., Ragni A.: Bioassays for
screening and quality control of products with insecticidal, fungicidal and nematocidal activity,
based on plant extracts, microorganisms and chemical molecules
30. Scribano M., Ragni A.: Isolation of new biological control agents from Umbria region
terrotory
44
31. Pietri A., Bertuzzi T., Barbieri G., Rossi F.: Protein content and mycotoxins contamination in
organic and conventional wheat
45
32. Tedeschi P., Maietti A., Mazzotta D., Vecchiati G., Romano P., Brandolini V.:
Determinazione di residui metallici nelle pere e influenza sull'impatto ambientale
33. Varvaro L., Antonelli M., Balestra G.M., Fabi A., Scermino D., Vuono G.: Investigations
on the bactericidal activity of some natural products
34. Cravedi P., Molinari F. :Ricerca su insetticidi utilizzabili secondo il metodo di produzione
biologico di prodotti agricoli
35. Salvo F., Saitta M., Di Bella G., La Pera L., Dugo G.mo: Fungicides and heavy metals in
wine samples produced from experimental grapes subjected to different pesticides treatments
36. Capasso R., De Martino A., Cristinzio G., Di Maro A., Parente A.: New a-elicitin isoforms
from Phytophthora hybernalis as protein elicitors for a potential employment in the biological
pest mangement
46
1
PROVE DI LOTTA ALL’OIDIO DELLO ZUCCHINO CON PRODOTTI
ALTERNATIVI IN PIENO CAMPO
G.E. Agosteo, M. Ambrosio
Dipartimento di Agrochimica ed Agrobiologia - Università Mediterranea di Reggio Calabria
Premessa
La coltura dello zucchino in Calabria è ampiamente diffusa sia pieno campo che in ambiente protetto. L’intensità delle
infezioni di oidio (Sphaerotheca fusca Blumer, Erysiphe cichoracearum D.C.) su questa coltura è particolarmente
elevata. A differenza del melone che dispone di un’ampia gamma di varietà resistenti o tolleranti alla malattia, per lo
zucchino è necessario ricorrere a ripetuti interventi di lotta chimica, pena il precoce decadimento vegetativo delle
piante. La gestione della lotta all’oidio, per il rispetto degli intervalli di sicurezza dei fungicidi, è complicata dalla
scalarità della raccolta. Lo zolfo, unico fungicida antioidico autorizzato in agricoltura biologica, può dare problemi di
selettività su alcune varietà di cucurbitacee, soprattutto in presenza di temperature elevate, quali quelle del periodo
primaverile-estivo nel meridione d’Italia.
Con la presente prova si è inteso saggiare l’efficacia antioidica su zucchino in pieno campo, in un ambiente di
produzione meridionale caratterizzato da forte pressione di malattia, di alcuni prodotti di origine minerale, quali
bicarbonato di sodio e fosfato monopotassico, il cui impiego è stato sperimentato con successo in altri ambienti di
coltivazione (Minuto et al., 1998).
Materiali e metodi
La prova è stata realizzata nell’anno 2001, nella Piana di Lamezia Terme (CZ), su zucchino, cv. Clarita, allevato in
pieno campo, con un ciclo colturale di circa 70 giorni. Il trapianto è stato realizzato il 25 maggio, le piante hanno
occupato il terreno in un periodo stagionale (giugno – luglio) caratterizzato da temperature particolarmente favorevoli al
patogeno. E’ stato utilizzato uno schema sperimentale a blocchi randomizzati con 7 tesi (Tab.1) e 4 ripetizioni.
Tab.1 - Tesi a confronto, dosi, intervalli tra le applicazioni
Nome commerciale e/o p.a. (%) e Dosi (ml o g/hl) Intervallo tra
Tesi
Principio attivo
società produttrice formulazione p.c.
p.a. le applicazioni
1 Zolfo bagnabile
90 Pb
500
450
7 giorni
Zolfo bagnabile - Isagro
2 Bicarbonato di sodio
Solvay
100 Pb
750
750
7 giorni
3 Bicarbonato di sodio
Solvay
100 Pb
1000
1000
7 giorni
1000
7 giorni
4 KH2PO4 (0-52-34)
Haifa Chemicals Ltd 100 Cris.sol. 1000
5 Trifloxystrobin
Flint - Bayer
50 WG
25
12,5
14 giorni
Zolfo bagnabile,
Zolfo bagnabile - Isagro
90 Pb
500
450
7 giorni
6
penconazole
Topas 10 EC - Syngenta 10,52 Le
35
3,5
14 giorni
7 Testimone
\
\
\
\
\
Ciascuna parcella era costituita da 12 piante contigue sulla fila. I trattamenti hanno avuto inizio l’11 giugno, prima della
comparsa dei sintomi, e sono stati effettuati con le cadenze riportate nella tab.1. Nella tesi 6 (trattamento aziendale),
sono stati realizzati due interventi con zolfo seguiti, alla comparsa dei sintomi, dal penconazole. La lettura dei sintomi è
stata realizzata a 42 e 60 giorni dall’inizio del ciclo, su 4 foglie per pianta (1 dei palchi basali, 2 dei palchi medi ed 1
apicale), da 5 piante centrali a parcella, sulla base di una scala di malattia con 6 classi d’infezione (0=0; 1=0-1, 2=1-5,
3=5-20, 4=20-40, 5= >40 % di area fogliare infetta) (EPPO, 1997).
Sono stati determinati gli indici di diffusione (percentuale di foglie infette), gravità G = Σ (f . v) n-1 ed intensità (indice
di McKinney) (I = [Σf . v (N.X)-1] .100), dove f = frequenza dei casi per ciascuna classe d’infezione, v= valore di classe
corrispondente, n = numero di casi infetti, N = numero totale delle osservazioni, X = valore massimo della classe nella
scala d’infezione. I dati sono stati sottoposti ad analisi della varianza, le medie sono state separate con il test di Duncan
per P=0,05 e P=0,01. I valori percentuali sono stati trasformati nei corrispondenti valori angolari.
47
Risultati
I risultati conseguiti sono riportati nella tab.2.
Tab.2 – Indici di malattia a 42 e 60 giorni dall’inizio del ciclo colturale
Rilievo a 42 gg
Rilievo a 60 gg
Tesi
Diffusione
Gravità
Intensità Diffusione
Gravità
1. Zolfo bagnabile
86,2 b AB 3,52ab A
60,7 b B
98,7 a AB 4,61ab AB
2. Bicarb. di sodio 750 g/hl
83,7 b BC 3,45 ab A
57,2 bc BC 98,7 a AB 4,53 bc BC
3. Bicarb. di sodio 1000 g/hl
78,7 bc BC 3,52 ab A
57,6 bc BC 96,2 ab AB 4,47 bcde BC
4. KH2PO4 1000 g/hl
83,7 b BC 3,20 b A
52,7 cd BC 100 a A
4,51 bcd BC
5. Trifloxystrobin
26,2 d D
1,35 c B
7,5 eD
92,5 bc BC 4,32 de C
6 .Zolfo, Penconazole
75,0 c C
3,40 ab A
50,7 d C
97,5 ab AB 4,39 cde BC
7. Testimone
96,2 a A
3,72 a A
69,7 a A
100 a A
4,77 a A
Le medie sono state separate secondo il test di Duncan. Valori seguiti da stesse lettere non
significativamente fra loro per P= 0,05 (lettere minuscole) e P= 0,01 (lettere maiuscole)
Intensità
91,5 b AB
89,5 bc B
86,25 c B
89,75 bc B
80 d C
86,75 c B
95,5 a A
differiscono
La pressione di malattia è stata molto elevata (96% e 100% di foglie infette nel testimone, rispettivamente, nei due
rilievi). Alla data del 1° rilievo il trifloxystrobin, strobilurina di sintesi, ha fatto rilevare indici di malattia molto
contenuti, con infezioni limitate alle sole foglie basali. Tutte le altre tesi si sono differenziate statisticamente dal
testimone ma con indici di malattia elevati. Il fosfato monopotassico ha fatto rilevare una migliore attività antioidica
rispetto allo zolfo ed un indice complessivo di malattia (intensità) non differente statisticamente dalla tesi di riferimento
aziendale (zolfo, penconazole). Il bicarbonato di sodio (sia allo 0,75 che all’1%) ha mostrato un’attività analoga a
quella dello zolfo.
In prossimità della fine del ciclo colturale (2° rilievo) le differenze fra le tesi si sono notevolmente ridotte ed il
contenimento della malattia è stato insoddisfacente. L’intensità di malattia è stata statisticamente differente dal
testimone in tutte le tesi, grazie soprattutto ad un minore peso della gravità. Il trifloxystrobin ha mostrato i valori di
intensità più contenuti e, unico fra le tesi saggiate, una diffusione statisticamente differente dal testimone. Il bicarbonato
di sodio all’1% ha fatto rilevare un’intensità minore rispetto allo zolfo, in linea con la tesi aziendale (zolfo,
penconazole). Alle dosi utilizzate non sono stati osservati effetti fitotossici sulle piante.
Conclusioni
Un soddisfacente contenimento delle infezioni di oidio su zucchino, negli ambienti di coltivazione meridionali
caratterizzati da forte pressione di malattia, richiede l’impostazione di programmi di lotta che facciano ricorso a
fungicidi con azione curativa. Fosfato monopotassico e bicarbonato di sodio hanno evidenziato un’attività significativa,
migliore od analoga a quella dello zolfo, di cui costituiscono pertanto una valida alternativa in applicazioni di tipo
preventivo.
Lavori citati
EPPO, 1997. Powdery mildew of cucurbits and other vegetables, PP 1/57(3). EPPO Standards. Guidelines for the
efficacy evaluation of plant protection products. Vol. 2, EPPO Parigi, 81-85.
MINUTO A., MINUTO G., GULLINO M.L., GARIBALDI A., 1998. Prove di lotta al mal bianco dello zucchino. In:
Atti Giornate Fitopatologiche, 655-660.
48
2
LUPINUS ALBUS, AN ANCIENT EUROPEAN LEGUME PARTICULARLY SUITABLE FOR ORGANIC
FARMING, MAY BECOME AN USEFUL SOURCE OF FUNCTIONAL INGREDIENTS
Anna Arnoldi, Alessandra D’Agostina, Giovanna Boschin
DISMA, Section of Chemistry – University of Milan ([email protected])
Lupin is a protein rich grain legume typical of the Mediterranean region that produces seeds with a protein content, both
qualitatively and quantitatively similar to that of soybean. A very interesting advantage of lupin in respect to soybean is
that there are no commercially available genetically modified varieties, which makes it an ideal crop for organic
farming. In addition it is particularly valuable in Mediterranean areas, owing to its drought resistant properties, and,
being a legume, it may be used in rotation with other cultures to reduce the use of fertilisers. For these reasons the
European Commission has included it in the list of plants that merit specific research efforts (1).
The protein content of lupin, varying from species to species, is around 35 to 40% in Lupinus albus. About 10% of the
total protein content consists of albumins, while the rest is represented by globulins, showing a 1:1 legumin
(11S)/vicilin (7S) ratio. Ultracentrifugation of the protein solutions leads to 4 fractions with sedimentation coefficients
of 15S, 11S, 7S and 2S. The major fractions are the 11S and the 7S fractions, which account for about 33 to 37% of
total proteins (2). In addition, lupin contains a specific protein fraction, conglutin g, which accounts for about 5% of
total proteins and has the exceptional characteristic of being a sulphur-rich protein, thus containing amino acids that are
scarce in other grain legumes.
In respect to soybean, lupin kernels contain lower amounts of antinutrients, potentially of consumer concern, when
using protein ingredients in food preparations. Trypsin inhibitor concentration is, in fact, 0.18 mg/g in lupin vs. 17.90
mg/g in soybean, phytate concentration is 0.44 mg/g vs. 1.59 mg/g, saponin concentration is 1.4 mg/g vs. 1.9 mg/g, and
the concentration of oligosaccharides of the raffinose family is 4.6 mg/g vs. 5.7 mg/g (3). Lupin alkaloids, which, also
for their bitter taste, represented a strong limitation for the human consumption of some old lupin varieties, have now
been reduced to very low levels in the “sweet lupin” varieties (4). Sweet lupin flour has been suggested for use in bread,
cookies and milk substitutes. Manufacturers advertise that these products have an increased dietary fibre content and
high protein value. Additionally lupin seeds do not contain phyto-hormones, in particular isoflavones that, according to
recent literature data, have serious toxicological potentials.
In the last decade, the increasing awareness of the health consequences of an incorrect diet and some scandals in the
meat market (BSE, epizootic aphtha, contamination with polychloro-dibenzodioxines and/or furans residues) have
encouraged the consumers to go back to legumes as a source of valuable proteins. In this situation, lupin seeds have all
the characteristics to become one of the main source of vegetable food ingredients in the Mediterranean area.
1.
2.
3.
4.
European Economic and Social Committee, Section for Agriculture, Rural Development and the Environment,
‘New impetus for a plan for plant protein crops in the Community’, Brussels, 4 December 2001.
Guegen J, Cerletti P. In New and Developing Sources of Food Proteins. Hudson, BJF ed., Chapman and Hall,
London 1994, 145-193.
Champ M. Proceedings 4th Europ. Conference on Grain Legumes, 2001, 109-113.
Muzquiz M, Pedrosa MM, Cuadrado C, Ayet G, Burbano C, Brenes A In Recent Advances of Research in
Antinutritional Factors in Legume Seeds and Rape Seeds . Jasman AJM et al eds, EAAP Publication N. 93,
Wageningen Press, 1998, 387-390.
49
3
BENZO(a)PYRENE IN ORGANIC VIRGIN OLIVE OILS
a
Elena Barrese, bCinzia Benincasa, aNicola Lombardo, bFabio Mazzotti, aInnocenzo Muzzalupo, aMassimiliano
Pellegrino, aEnzo Perri, bGiovanni Sindona
a)
b)
Istituto Sperimentale per l'Olivicoltura, 87030 Rende (CS)-Italy
Dipartimento di Chimica, Università della Calabria, via P. Bucci, cubo 15/c I -87030 Arcavacata di Rende (CS)-Italy
Introduction
Polycyclic aromatic hydrocarbons (PAHs) are environmentally hazardous organic compounds because of their known
or suspected carcinogenicity. PAHs are mostly formed during an incomplete combustion of organic material and they
are known as highly stable contaminants present in many foods. This contamination can be a result of sorption from the
environment or from food preparation methods. Air pollution is the main source of PAHs in the edible parts of plants.
Hence, the determination of PAHs in foods such as olives and olive oil is important for human health. Benzo(a)pyrene
(BaP) and Benzo(e)pyrene (BeP) are the best known PAHs and they have been often used as indicators of the presence
of PAHs in foods. Several authors have reported the presence of Benzopyrenes (BP) in edible oils and fats. However,
there are few reports on the BP content in olive oils (Mariani et al., 1984; Menichini et al., 1991; Fiume et al.2002)
while there has been no reports on the BP content in organic virgin olive oils. In general, the detected levels of BP in the
olive oils analyzed were in the range of a few mg/kg of olive oil (Pupin et al., 1996; Vazquez Troche et al., 2000).
However, olive oil refining involving deodorization, bleaching and charchoal treatment reduces the content of PAHs.
Therefore, paradoxically, higher levels of BP may be found in virgin olive oils, irrespective of farming system adopted,
since they are, by definition, unrefined and untreated.
Currently no legislation exists regarding BP levels in vegetable oils in European Union. However, the International
Olive Council (IOC), the German food industries and some European nations (Italy, Spain and Portugal) recommend
for olive-pomace oil and refined olive-pomace oil a BaP maximum level of 2 mg/kg. Therefore, there is a need to verify
the level of BP in European olive oils before a regulation occur. The goal of this preliminary work was to assess the
presence and determine the level of BP in organic virgin olive oils from the most important olive growing region of
Italy: Apulia.
Plant material. In 2000/2001 harvest, samples of organically cultivated olive drupes of Coratina cv from a farm at
Andria (Bari, Italy) were collected 10 days before the traditional start of harvest, in the middle of the harvest period and
10 days after the end of harvest time. The olives were handpicked each time from 5 olive tree of the same cultivar.
Work-up of plant material. Olive drupes (10 Kg) were crushed with a hammer mill and the oil was extracted by
centrifugation after 20 minutes of malaxation at RT.
Analytical procedures.Extraction and clean-up procedure were conducted by slightly modifying Vazquez Troche et al.
(2000) protocol. The final extract was analyzed by a Hewlett Packard HP 5973 Mass Spectrometer interfaced with a HP
6890A GC (Agilent Technologies, Waldronn, GmbH) and BaP detected as molecular ion (m/z 252) by Selected Ion
Monitoring (SIM) mode. Quantification was obtained by the external standard method.
Detection limit for BP in the acetonitrile eluate was 0.3 mg/kg of olive oil. The standard calibration curves obtained with
a linear coefficient of 0,999 generated from triplicate 1 ml injections at different concentration show a linear range from
0.3 mg/kg to 5.4 mg/kg with good-to-excellent R2 values. For reproducibility test three concentrations were analyzed and
the average coefficient of variation (CV%) was 5%. The recovery obtained for spiked samples with 4 mg/Kg of BaP was
66%. As expected, also the organic olive oils are affected with a PAHs. In fact, the levels of BaP and BeP in organic
virgin olive oils from Coratina cv. at Andria (Apulia) ranged from 0.39 and 0.85 mg/kg, and 0.38 and 0.60 mg/kg ,
respectively, while the average content was 0.56 mg/Kg and 0.52 mg/Kg, respectively (table 1). Although the BP values
were low, the determination of the other “heavy” PAHs is still required to assess the real intakes of PAHs from the total
diet of apulian olive oil consumer. Therefore, the determination of the most important “heavy” PAHs in organic virgin
olive oil by GC-MS is in progress.
50
Table 1. Benzo(a)pyrene and benzo(e)pyrene content in organic virgin olive oils from Coratina cv. at Andria
(Apulia).
Cultivar
Origin
Harvest time
BaP (mg/Kg of olive oil)
BeP (mg/Kg of olive oil)
Coratina
Andria (Apulia)
11/07/00
0.85
0,38
Coratina
Andria (Apulia)
12/11/00
0.45
0,60
Coratina
Andria (Apulia)
01/02/01
0.39
0,58
Mean + Std. dev.
0.56+ 0.20
0.52+ 0.10
Acknowledgements
We thank Dr. Cosimo Cavallo (Regione Puglia), Dr. Nino Paparella (C.I.Bi., Bari), Dr. Nicola Panaro (C.I.Bi.,
Bari) and Drs. Edoardo and Giancarlo Ceci Ginistrelli for olive sampling, data collection on farming systems and olive
grove management. We thank also Prof. Angelo Putignano and Prof. Francesco Prudentino (I.T.A.S. of Ostuni-BR) for
olive oil extraction.
References
F. Fiume, F. Ferrieri, G. Froio, S. Spinello, O Lattarulo, G. Fanuzzi, 2002, Determinazione di idrocarburi policiclici
aromatici in oli alimentari, Riv. Ital. Sostanze grasse, LXXIV, 151-155.
C. Mariani, E. Fedeli,1984, Idrocarburi policiclici aromatici negli oli vegetali, Riv. Ital. Sostanze Grasse, 61, 305 -315.
E. Menichini, A. Bocca, F. Merli F., Ianni, F. Monfredini, 1991, Polycyclic aromatic hydocarbons in olive oils on the
italian market, Food Addit. Contam., 8(3), 363-369.
A. M. Pupin and Maria Cecilia Figueiredo Toledo, Benzo(a)pyrene in olive oils on the brazilian market, Food
Chemistry, 1996, 55(2), 185-188.
S. Vazquez Troche, M.S. Garcìa Falcòn, S. Gonzales Amigo, M.A. Lage Yusty, J. Simal Lozano, Enrichment of
benzo(a)pyrene in vegetable oils and determination by HPLC-FL, Talanta 2000, 51, 1069-1076.
51
4
EFFETTO DELLE CERE EPICUTICULARI DI FRUTTA E VEGETALI SULLA FOTODEGRADAZIONE
DEL ROTENONE
M. Cabizza, M. Melis, P.Cabras
Dip. di Tossicologia Università di Cagliari
Via Ospedale, 72 Cagliari
E' stato studiato l'effetto delle cere epicuticulari di frutta e ortaggi sulla fotodegradazione del rotenone alla luce solare
La fotodegradazione è stata condotta utilizzando capsule di petri all'interno delle quali veniva posto il rotenone con (o
senza) la cera di susine, nettarine, mele, pere, melanzane e pomodoro. Le capsule sono state esposte alla luce solare e i
campioni prelevati a intervalli di tempo prestabiliti Dopo l'esposizione al sole i campioni sono stati ripresi con
acetonitrile:acqua 50:50 (v/v), iniettati per l'analisi HPLC e sono stati determinati il rotenone e i suoi principali
metaboliti: 6',7'-epoxy-rotenone a e b, rotenolone 6ab,12aa, rotenolone 6ab,12ab, deidrorotenone.
Il tempo di semivita del rotenone irradiato alla luce solare senza cere era 1,0 ore. In presenza di cere di pomodoro non
c'erano sensibili variazioni mentre con cera di nettarine e susine il tempo di semivita risultava maggiore. Le cere di
mele, pere e melanzane aumentavano la cinetica di fotodegradazione del p.a.
Il rotenone irradiato al sole in assenza di cere formava 5 metaboliti più importanti (6',7'epoxy-rotenone a e b,
rotenolone 6ab,12aa, rotenolone 6ab,12ab, deidrorotenone e un metabolita con tr 5,37) mentre irradiando il rotenone
in presenza di cere si avevano differenze nella formazione di tali metaboliti sia dal punto di vista qualitativo che
quantitativo. Solo irradiando il rotenone in presenza di cere di pomodoro tali metaboliti si formavano
contemporaneamente mentre con tutte le altre cere impiegate esistono differenze nella loro formazione a seconda del
tipo di cera utilizzato. La cera di pere inibisce la formazione di tutti i metaboliti ad eccezione del deidrorot enone e del
rotenolone 6ab,12ab.
Il metabolita rotenolone 6ab,12ab si forma sempre nel rotenone irradiato sia in assenza che in presenza di cere ed è il
metabolita quantitativamente più abbondante.
52
5
INSETTICIDI D’ORIGINE VEGETALE E ACARI
Castagnoli M., Liguori M., Nannelli R., Simoni S.
Sezione di Acarologia, Istituto Sperimentale per la Zoologia Agraria. Firenze
Different formulations of extracts of neem, derris, equisetum, ryania, wormwood, garlic, chrysanthemum, and
bitter wood were tested on the phytophagous mite Tetranychus urticae, the phytoseiid Neoseiulus californicus, the
stored food mite Lepidoglyphus destructor to evaluate their different toxicity on eggs and females and the effects on the
female fecundity.
On the whole, these botanical pesticides were more effective on eggs than on motile stages. The highest
toxicity was registered on N. californicus.
The extracts based on low concentrations of azadirachtina and neem oil were the most toxic for the females and
the eggs of the tetranychid and did not significantly affect the survival and fecundity of the phytoseiid. Derris and
wormwood extracts were the most effective on the stored food mite.
KEY WORDS: Tetranychus urticae, Neoseiulus californicus, Lepidoglyphus destructor, neem, derris, ryania, wormwood,
chrysanthemum, bitter wood.
INTRODUZIONE
Negli ultimi anni una maggiore sensibilità verso le conseguenze negative per l’ambiente determinate dall’uso
di molti pesticidi di sintesi ha determinato anche nel nostro Paese una crescita di interesse nei confronti dei prodotti
d’origine vegetale ritenuti più facilmente degradabili. L'uso di questi prodotti commercializzati sia come insetticidi o
più genericamente come biostimolanti e coadiuvanti ad azione limitatrice nei confronti di artropodi nocivi si è
particolarmente affermato in agricoltura biologica. Nell'ottica di una valutazione complessiva dei loro effetti, si rende
quindi sempre più necessaria la verifica anche sulle specie non target.
L’efficacia di sostanze naturali come rotenone, piretro e quassia è nota da tempo, più recente è l’interesse per i
derivati di Azadiracta indica (A. Juss) ritenuti in grado di esplicare azione tossica, repellente, fagodeterrente e inibitrice
della crescita su moltissime specie di artropodi (SCHMUTTERER, 1990). Per quanto riguarda gli acari le conoscenze sono
molto limitate e, se poche sono le ricerche dedicate all'attività biologica di estratti di neem (MANSOUR et al.,1987,1997;
SUNDARAN & SLOANE, 1995; SPOLLEN & ISMAN, 1996; MOMEN et al., 1997; PAPAIOANNOU-SOULIOTIS et al., 1997;
TSOLAKIS et al., 1997), ancora meno sono quelle che prendono in esame altri principi di origine naturale (PERRUCCI,
1995; GULATI & MATHUR, 1995; CASTAGNOLI et al., 2002).
Nell' ambito di un più vasto programma di indagini, l’efficacia di alcuni principi attivi di origine vegetale è
stata saggiata su tre delle più comuni specie di acari: Tetranychus urticae Koch, Neoseiulus californicus (McGregor) e
Lepidoglyphus destructor (Schrank) (CASTAGNOLI et al., 2000; NANNELLI & SIMONI, 2001). Il primo è un fitofago
estremamente polifago e in grado di causare seri danni su un’ampia gamma di colture, il secondo è un fitoseide
predatore frequentemente associato al tetranichide e in grado di contrastarne le pullulazioni, mentre il terzo è un acaro
astigmata che colonizza le più disparate derrate alimentari immagazzinate che vanno dai cereali ai salumi e ai formaggi.
MATERIALI E METODI
I prodotti saggiati e le relative diluizioni, che corrispondono alle dosi massime consigliate dai produttori, sono
elencati nelle tabelle 1-3. In laboratorio sono stati valutati gli effetti sulla schiusura delle uova e sulla sopravvivenza
delle femmine su un totale di 80 esemplari o uova per prodotto. Per il fitofago e il predatore è stata presa in
considerazione anche l’incidenza sulla fecondità.
I metodi usati erano diversificati a seconda dello stadio e delle caratteristiche della specie: “dip method” (HELLE &
OVERMEER, 1985) e “microimmersion bioassay” (modificato da DENNEHY et al., 1993) sono stati usati rispettivamente
per tutte le uova e per le femmine del fitofago e del predatore, mentre per le femmine dell’astigmata è stato usato il
metodo "impregnated filter paper bioassay" secondo THIND & MUGGLETON (1998).
I risultati sono stati espressi come percentuale di tossicità globale (E), combinando mortalità e fecondità delle
femmine del trattato e del non trattato (OVERMEER & VAN ZOON, 1982) o come mortalità di ABBOTT (1925), tenendo
conto dell’eventuale mortalità naturale registrata nel testimone.
53
RISULTATI
Derivati del neem (Azadirachta indica)
In generale i prodotti saggiati sono risultati più efficaci sulle uova che sulle forme mobili delle tre specie. Per
quanto concerne il fitofago, la maggiore efficacia sulle uova si è avuta con il prodotto in cui l'azadiractina era
addizionata all'olio di neem e con quello in cui questo principio attivo era miscelato ad oli vegetali e lecitina di soia
(tab.1, prodotti 1 e 2). Questi stessi formulati non determinano un incremento della fecondità di T. urticae come invece
è stato osservato per gli altri prodotti. Sul predatore N. californicus l'azadiractina addizionata agli oli vegetali e lecitina
di soia (tab.1, prodotto 2) oltre ad incidere pesantemente sulla schiusura delle uova determina la più alta mortalità delle
femmine. Su L. destructor non si osserva una rilevante attività tossica di questo gruppo di prodotti; l'unico che
determina un'apprezzabile riduzione della schiusura delle uova è quello con la più bassa percentuale di azadiractina
(tab.1, prodotto 5).
Piretrine
Mostrano azione tossica sulle uova del fitofago e soprattutto su quelle dell'astigmata, ma non sulle femmine di
queste due specie. Per quanto concerne il fitoseide l'azione varia dall'effetto positivo della formulazione con piretro al
20% (tab.2, prodotto 1) a quello negativo del formulato con piretro al 25% (tab.2, prodotto 3). Ciò è imputabile quasi
certamente alla diversa formulazione: in quest'ultimo le piretrine naturali sono miscelate ad oli vegetali, mentre il primo
è una soluzione idroalcolica.
Tabella 1. Estratti da neem (A = Azadiractina A): composizione e dose dei prodotti usati e loro effetto (E = tossicità
globale, Abb. = mortalità secondo Abbott) su femmine e uova di due specie di acari di interesse agrario ( T. urticae e N.
californicus) e di una specie di acaro delle derrate (L. destructor).
T. urticae
Prodotti
N. californicus
L. destructor
composizione
dose
femmine
E%
uova
Abb. %
femmine
E%
uova
Abb. %
femmine
Abb. %
uova
Abb. %
1
0,4% A + 0,09%
olio di neem
4g/l
12,98
100
15,00
9,08
-2,86
-4,35
2
n. 1 + oli veget.
e lecit. di soia
4g+2g/l
0,00
70,68
90,12
72,73
-3,90
17,39
3
1% A + olio di
sesamo
2,5g/l
-50,86
20,05
4,88
33,76
-4,76
-26,09
4
n. 3 + oli veg. e
lecit. soia
2,5g+2g/l
-86,01
20,05
66,40
-2,59
-2,66
4,35
5
0,15% A
3cc/l
-17,56
20,05
-4,98
63,33
6,49
30,43
6
3% A (Oikos)
1,5cc/l
-16,65
15,94
8,94
0,00
12,50
21,74
7
10% A
(Neemazal)
3g/l
-17,87
28,99
35,50
-1,27
----
----
54
Tabella 2. Piretrine: composizione e dose dei prodotti usati e loro ef fetto (E = tossicità globale, Abb. = mortalità
secondo Abbott) su femmine e uova di due specie di acari di interesse agrario (T. urticae e N. californicus) e di una
specie di acaro delle derrate (L. destructor).
T. urticae
Prodotti
N. californicus
L. destructor
composizione
dose
femmine
E%
uova
Abb. %
femmine
E%
uova
Abb. %
femmine
Abb. %
uova
Abb. %
1
piretro 20%
4,5cc/l
-65,55
48,03
-23,37
-3,89
-8,42
91,30
2
n. 1 + acidi grassi
30%
4,5cc+4g/l
-18,04
10,72
--
--
4,00
86,96
3
piretro 25%
(Biopiren Plus)
1,6g/l
-25,54
2,53
91,98
-1,27
----
----
Derivati da altre sostanze vegetali
I derivati del derris manifestano tossicità soprattutto nei confronti delle femmine e delle uova del predatore e
dell’astigmata, sul fitoseide la mortalità delle femmine supera con due prodotti il 90%. Sul fitofago il rotenone 5% (tab.
3, prodotto 1) ha efficacia nei confronti delle femmine e il rotenone 5% addizionato di oli vegetali e lecitina di soia
(tab.3, prodotto2) risulta più tossico per le uova.
Variabili sono gli effetti degli altri formulati: solo l'assenzio agisce contro le uova del fitoseide e del fitofago,
mentre la quassia riesce a controllare moderatamente le popolazioni dell'astigmata (tab. 3).
55
Tabella 3. Estratti da vegetali vari: composizione e dose dei prodotti usati e loro effetto (E = tossicità globale, Abb. =
mortalità secondo Abbott) su femmine e uova di due specie di acari di interesse agrario ( T. urticae e N. californicus) e
di una specie di acaro delle derrate (L. destructor).
T. urticae
Prodotti
N. californicus
L. destructor
composizione
dose
femmine
E%
uova
Abb. %
femmine
E%
uova
Abb. %
femmine
Abb. %
uova
Abb. %
1
rotenone 5%
4g/l
46,92
32,04
57,69
42,85
78,30
26,09
2
n. 1 + oli veg. e
lecit. soia
4g+2g/l
-3,85
93,33
99,66
62,33
44,32
34,78
3
rotenone 6%
(Rotena)
3cc/l
----
----
94,95
-1,27
----
----
4
riania 70% + oli
veg. e lecit. soia
4g+2g/l
6,47
40,03
-35,75
-3,89
24,85
-30,43
5
assenzio 80%
8g/l
26,74
37,37
-3,84
70,13
2,04
-26,09
6
n. 5 + oli veg. e
lecit. soia
8g2g/l
27,65
18,71
13,57
25,97
9,86
-30,43
7
aglio 85%
1g/l
-1,80
16,05
33,56
0,00
-7,05
-30,43
8
n. 7+ oli veg. e
lecit. soia
1g+2g/l
29,08
33,38
-10,13
0,00
37,82
-26,09
9
quassia 50%
5cc/l
-35,41
-6,66
----
----
45,34
47,83
10
n. 9 +
acidi grassi 30%
5cc+4g/l
20,00
-2,66
----
----
52,76
34,78
CONSIDERAZIONI CONCLUSIVE
I prodotti con lo stesso principio attivo miscelati o addizionati con differenti bagnanti ed emulsionanti
determinano una diversa mortalità sulle specie considerate da cui si deduce che anche gli additivi possono condizionare
l’entità delle risposte.
Nell’ambito dei prodotti saggiati, in generale, si può osservare che quelli che esplicano azione acaricida sono
più tossici per il predatore che per il fitofago del quale aumentano spesso la fecondità. Per gli acari delle piante, i
risultati più promettenti si hanno essenzialmente con il prodotto contenente azadirachtina A a bassa concentrazione e
olio di neem: questo formulato pur non essendo efficace sugli adulti del tetranichide, determina alta mortalità delle loro
uova, e non incide invece sulla vitalità delle uova e delle femmine del fitoseide predatore, né sulla loro fecondità.
Per l'acaro delle derrate, gli estratti di derris e quassia sono risultati i prodotti più tossici sugli stadi mobili
mentre, sulle uova, gli estratti a base di piretro hanno determinato la più alta mortalità.
RIASSUNTO
I derivati da neem, derris, equiseto, riania, assenzio, aglio, crisantemo e quassia somministrati puri o con
l’aggiunta di bagnanti od emulsionanti sono stati saggiati su Tetranychus urticae (specie fitofaga), Neoseiulus
56
californicus (fitoseide predatore) e Lepidoglyphus destructor (astigmata comunemente conosciuto come acaro delle
derrate alimentari) per valutare la loro tossicità sulle uova e le femmine e gli effetti sulla fecondità.
Su tutte le specie i prodotti saggiati sono stati più efficaci sulle uova che sulle forme mobili. La tossicità più
alta sulle femmine è stata evidenziata sul predatore.
Solo i prodotti a base di azadiractina A a bassa concentrazione e olio di neem risultano efficaci sugli adulti e
sulle uova del tetranichide incidendo poco sulla vitalità delle uova e delle femmine del fitoseide. Per l’acaro delle
derrate gli estratti di derris e quassia sono risultati i più tossici.
BIBLIOGRAFIA
ABBOTT W.S., 1925 - A method of computing the effectiveness of an insecticide .- J. Econ. Entomol., 18: 265-267.
CASTAGNOLI M., SIMONI S., GOGGIOLI D., 2000 - Attività biologica di sostanze vegetali nei confronti di Tetranychus
urticae Koch (Acari Tetranichidae) e del suo predatore Neoseiulus californicus (McGregor)(Acari
Phytoseiidae).- Redia, 83: 141-150.
CASTAGNOLI M., ANGELI G., LIGUORI M., FORTI D., SIMONI S., 2002 - Side effects of botanical insecticides on
predatory mite Amblyseius andersoni (Chant).- J. Pest Science 75: 122-127
DENNEHY T.J., FURNHANNM A.W., DENHOLM I., 1993 - The microimmersion bioassay: a novel method for the topical
application of pesticides to spider mites.- Pesticide Sci., 93: 47-54.
HELLE W., OVERMEER W.P.J., 1985 - Toxicological test methods.- In: Spider mites, their biology, natural enemies and
control. Vol 1A, Elsevier, Amsterdam, New York, Oxford, Tokyo: 391 -395.
GULATI R., MATHUR S., 1995 - Effect of Eucalyptus and Mentha leaves and Curcuma rhizomes on Tyrophagus
putrescentiae (Scrank) (Acarina Acaridae) in wheat.- Experimental & Applied Acarology, 19: 511-518.
MANSOUR F.A., ASCHER K.R.S., OMARI N.,1987 - Effects of neem (Azadirachta indica) seed kernel extracts from
different solvents on the predacious mite Phytoseiulus persimilis and the phytophagous mite Tetranychus
cinnabarinus. - Phytoparasitica, 15: 125-130.
MANSOUR F.A., ASCHER K.R.S., ABO-MOCH F., 1997 - Effects of Neemgard on phytophagous and predacious mites
and spiders. - Phytoparasitica, 25: 333-336.
MOMEN F.M., REDA A.S., AMER S.A.A., 1997 - Effect of neem Azal-F on Tetranychus urticae and three predacious
mites of the family Phytoseiidae.- Acta Phytopathol. Hungarica, 32: 355-362.
NANNELLI R., SIMONI S., 2001 - Valutazione della tossicità di sostanze vegetali su femmine e uova di Lepidoglyphus
destructor (Schrank) (Acari Glycyphagidae).- Redia, 84: 129-140.
OVERMEER W.P.J., VAN ZOON A.Q., 1982 - A standardized method for testing the side effects of pesticides on the
predacious mite Amblyseius potentillae (Acarina Phytoseiidae). - Entomophaga, 27: 357-364.
PAPAIOANNOU-SOULIOTIS P., MARKOYANNAKI-PRINTZIOU D., ZOAKI-MALIOSSOVA D., 1997 - Side effects of Neemark
(Azadirachta indica A. Juss ) and two vegetable oils formulations on Tetranychus urticae Koch and its predator
Phytoseiulus persimilis Athias-Henriot.- Boll Zool. agr. Bachic. (ser II), 32: 25 -33.
PERRUCCI S., 1995 - Acaricidal activity of some essential oils and their constituents against Tyrophagus longior a mite
of stored food.- Journal of Food Protection, 58 (5): 560 -563.
SCHMUTTERER H.,1990 - Properties and potential of natural pesticides from the neem tree, Azadirachta indica.- Annu.
Rev. Entomol., 35: 271-297.
SPOLLEN K.M., ISMAN M.B., 1996 - Acute and sublethal effects of a neem insecticide on the commercial biological
control agents Phytoseiulus persimilis and Amblyseius cucumeris (Acari Phytoseiidae) and Aphidoletes
aphidimyza (Diptera Cecidomyiidae).- J. Econ. Entomol., 89: 1379-1386.
SUNDARAN K.M.S., SLOANE L., 1995 - Effects of pure and formulated azadirachtin, a neem -based biopesticides, on the
phytophagous spider mite Tetranychus urticae Koch. - J. Environ. Sci. Health, B, Pesticides Food Contam.
Agric. Wastes., 30: 811-814
THIND B.B., MUGGLETON J., 1998 - A new bioassay for the detection of resistance to pesticides in the stored product
mite Acarus siro (Acari Acaridae).- Experimental & Applied Acarology, 22: 543-552.
TSOLAKIS H., LETO G., RAGUSA S., 1997 - Effects of some plant materials on Tetranychus urticae Koch (Acariformes
Tetranychidae ) and Typhlodromus exhilaratus Ragusa (Parasitiformes Phytoseiidae). - ANPP-Fourth Int.
Conf. on pests in Agriculture, Montpellier 6-8 January, 1997, pp. 239-245.
57
6
CHANGES OF BIOCHEMICAL PARAMETERS IN SOIL AMENDED WITH MOIST OLIVE HUSKS
Cristiano Casucci, Elga Monaci and Piero Perucci
DIBIAGA, Ancona University, Via Brecce Bianche, 60131 Ancona, ITALY
Key words: amendment, enzyme activity, soil fertility
·
·
·
·
·
·
On the last twenty years the disposal of organic residues coming from agricultural crop and food industry is a very
important problem relating to ecological risk, in particular in the middle Italy where the production of olive husks from
oil-mill is very diffuse. Therefore, the use of olive husks like amendment can represent both a solution for problems
connected to their disposal than a contribution to give an answer to the impoverishment of soils organic matter content
by actual agricultural practices. The aim of this research was to evaluate the influence, both in laboratory and in field
conditions, on some biochemical parameters correlated with soil fertility of the amendment with moist olive husks at
different dose.
Field experiments were carried out for a short-time (three years) in two soils amended with legal dose (D, 20 ton ha-1
for year, equivalent to 1.47 g of organic C/kg of soil) and double dose (2D). Laboratory experiments in the same soils
were carried out for one year and an additional dose of ten-fold field dose (10D) was employed.
In field trials the biochemical parameters didn’t show significant changes in comparison with no-amended soils and,
when present, their generally were positive.
Concerning laboratory experiment the results were:
microbial biomass-C content: no-significant increases at the beginning of experiment for D and 2D but significant
and constant increases during the entire period for 10D were found;
soil oxidation capacity: an inhibiting effect due to amendment was observed at the beginning of the trial which
disappeared after the first month of incubation;
FDA-hydrolase capacity: an initial significant decrease at all doses, which disappeared after two month from
amendment;
o-DPO activity: amendment at D and 2D didn’t cause significant changes, while a strong inhibition at 10D was
observed. This tendency persisted during the early drawings;
b-glucosidase activity: the initial inhibiting effect (two months) was followed by an increase correlated with the
organic amendment dose;
alkaline phosphatase activity: any influence was observed at D and 2D; 10D determined a significant decrease
during the first two months, at the third month the activity value returned to the original values of controls.
58
7
NATURAL COMPOUNDS IN THE CONTROL OF POWDERY MILDEW ON CUCURBITS IN ORGANIC
AGRICULTURE
Fedele Casulli(1), Agostino Santomauro (1), Giuseppe Tauro (2), Maria Antonia Gatto (1), Franco Faretra (1)
(1)
(2)
Dipartimento Protezione delle Piante e Microbiologia Applicata, University of Bari, Italy
Centro di Ricerca e Sperimentazione in Agricoltura "B asile Caramia", Locorotondo (Bari), Italy
In Italy, Cucurbitaceae are important vegetable crops grown especially in Central and Southern Italy (Sicily, Latium
and Apulia). Melon (Cucumis melo L.), watermelon [Citrullus lanatus (Thunb) Matsum et Nakai], courgette (Cucurbita
pepo L.) and cucumber (Cucumis sativus L.) are the prevalent species being cultivated on a surface of 21,400, 14,200,
12,200 and 1,500 hectares, respectively (Istat, 2001). Powdery mildew, caused by Sphaerotheca fusca (Fr.) S. Blumer
[syn. S. fuliginea (Schlecht. ex Fr). Poll.] is the most common and severe disease on the crops. Many fungicides are
available for an effective control of the pathogen, but in view of their negative side effects, people look to more
consumer- and environment-friendly alternatives, including natural compounds. The present paper reports the results of
trials carried out under glasshouse and field conditions in order to evaluate the effectiveness of natural compounds in
the control of powdery mildew in organic agriculture.
Zucchini squash (Cucurbita pepo L., cv. Striato pugliese) and cucumber plants (C. sativus, cv. Mezzo lungo di
Polignano) were grown individually in plastic pots (10 cm in diameter) in a glasshouse at 20-24°C and RH 70-80%,
under daylight. Four-replicated plants, maintained at the two-three leaf stage by pruning were used for each thesis. The
plants were artificially inoculated with S. fusca by spraying a suspension containing about 5×105 fresh conidia per ml.
The tested compounds, including several natural substances allowed in organic agriculture (Table 1), were applied
either 3 days before or 3 days after the artificial inoculation with the pathogen. Untreated check plants were sprayed
only with water. Numbers and sizes of fungal colonies were assessed 11 days after inoculation on 10-cm2 of the surface
of each leaf.
Natural substances were tested in field trials on melon (C. melo, cv. Barattiere) carried out during the summer. The
statistical design was the complete randomised blocks with four replications and plots were made up of 8-10 plants in
all trials. Spray schedules always were started before the appearance of first disease symptoms; treatments were
repeated at one-week intervals. The severity of powdery mildew symptoms was periodically assessed evaluating the
percentage of infected leaf surface. All data were submitted to variance analysis and mean values were separated by
Duncan’s Multiple Range Test.
According to the reduction of symptoms severity on treated plants as compared to the untreated check, the tested
compounds were grouped in effective (higher than 70%), moderately effective (from 40 to 70%) or poorly effective
(less than 40%) (Table 1).
A good control of powdery mildew fungi was achieved in glasshouse as well as in field by fresh and dried milk (10%),
which reduced infections over 90%. Among the other tested compounds, calcium and magnesium chloride, potassium,
sodium or ammonium phosphate dibasic, pinolene, sodium bicarbonate and the mixtures of mineral oil with sodium
bicarbonate or sodium silicate, significantly reduced the severity of S. fusca on leaves. These compounds showed the
highest effectiveness when applied 3-4 days after the artificial inoculation of S. fusca. In the field, the same compounds
yielded a satisfactory control of powdery mildew on the upper leaf surface of cucurbits, but they showed poor
effectiveness against infections on stems and lower leaf surface, especially under high disease pressure. Mineral oil was
effective when applied 3-4 days before artificial inoculation of S. fusca; this corroborate the finding that the compound
acts also as inducer of defence mechanisms in plants. Ammonium phosphate monobasic, sodium silicate, sodium
hydroxide, trisodium and tripotassium phosphate proved poorly effective at non-phytotoxic concentration while calcium
carbonate, calcium sulphate, calcium silicate, sodium and potassium chloride, sodium and potassium phosphate
monobasic were poorly effective against S. fusca.
59
Table 1. Effectiveness against Sphaerotheca fusca of natural compounds tested in greenhouse and in field (a).
Compounds
Rates
Trials
(%)
Glasshouse
Field
Pinolene
1
+++
+++
Fresh milk
100
+++
Dried milk
10
+++
+++
Mineral oil
1
+++
+++
Sodium bicarbonate (NaHCO 3)
0.5
+++
+++
Mineral oil + sodium bicarbonate
1+0.5
+++
+++
Mineral oil + sodium silicate
1+0.5
+++
+++
Potassium phosphate dibasic (K2HPO4)
0.5
+++
++
Sodium phosphate dibasic (Na 2HPO4)
0.5
+++
Ammonium phosphate dibasic [(NH4)2HPO4)]
1
+++
Calcium chloride (CaCl2)
0.5-1
+++
Magnesium chloride (MgCl2)
1
+++
Sodium silicate (Na2O.2SiO2)
0.5
++
Sodium hydroxide (NaOH)
0.25
++
Ammonium phosphate monobasic (NH4H2PO4)
1
++
Tripotassium phosphate (K3PO4)
0.5
++
Trisodium phosphate (Na3PO4)
0.5
++
Potassium phosphate monobasic (KH2PO4)
1
+
Sodium phosphate monobasic (NaH 2PO4)
0.5
+
Potassium chloride (KCl)
1
+
Sodium chloride (NaCl)
0.3-0.5
+
Calcium carbonate (CaCO3)
1
+
Calcium sulphate (CaSO4)
1
+
Calcium silicate (CaO.2SiO2)
1.5-2
+
(a)
+++ effective; ++ moderately effective; + poorly effective.
The results herein discussed show that several compounds are effective in the early stages of infection or under low
disease pressure, when applied repeatedly at short time intervals. Their effectiveness against powdery mildew proved to
be influenced by temperature, time of application and especially by the uniformity of distribution on plants. In fact, field
trials showed that one of their most important limits is the lack of control of powdery mildew on the lower surface of
leaves. Cucurbits and other crops requiring sprays during a long harvesting time would benefit greatly from the usage
of natural substances in crop protection.
Acknowledgements: Work supported by the Italian Ministry of Agricultural and Forestry Policies in the frame of the
research project “Technical tools for crop protection in organic agriculture” – Sub-project “Natural substances in crop
protection from fungal diseases”.
60
8
METHODS FOR EVALUATION, IN CONTROLLED CONDITIONS, OF THE GROWTH OF OILSEED
RAPE PROMOTED BY BACTERIA
F. Citarrei, M. Scribano, S. Coranelli, C. Cellerino, L. Quattrocchi, L. Concezzi, and A. Ragni
Tel: ++39 – 075 - 895091 Fax: ++39 – 075 – 888 776
E-mail: [email protected]
Abstract
Growth promoters are microorganisms able to increase growth and productivity of plants by: atmospheric nitrogen
fixation, mineral salts solubilization and siderophore synthesis (ferrous natural chelants), production of substances
similar to plants hormones and with antagonistic effect towards phytopathogenic microorganisms. Growth promoters
can influence the availability of nutritive elements or they can improve the phytosanitary conditions of the plants. In the
present work we have tested the ability of some bacteria strain to promote the growth of oilseed rape [Brassica napus
(DC) Metzger var. oleifera, Brassiceae] by using two types of experimentations: in vitro, in laboratory and in vivo, in
greenhouse. The in vitro test was conducted with sterile seedlings monoxenically associated with bacteria isolates. In
the in vivo trials, the seeds were treated with bacteria prior to sawing. Both methods are able to select and evaluate the
promoting action of the bacteria towards the oilseed rape although the in vitro is faster and discriminate better the
promoting effect of the bacteria. In vitro test
Objective
To test the ability of some bacteria strains to promote the rapeseed growth in vitro
Materials and Methods
Sterile rape seeds were sown on MS/2 culture media; sterile seedlings were transplanted in MS culture media inoculated
with Pseudomonas sp. strain MK280 (Fig.1). This strain is able to produce siderophores that, as natural chelants, make
iron available for plants. The treatments in the Pseudomonas test are reported in Table 1.The following growth
parameters were checked at 15 and 30 days: plant height, plant fresh weight and plant dry weight. Table 1.
Experimental conditions of rape seed in vitro test with Pseudomonas sp. MK280
Codes
FeCl3+MK280
FeCl3
Fe FeEDTA+MK280
FeEDTA
Treatment description
Ferric chloride + bacteria MK280
Ferric chloride without bacteria MK280
Without iron, without bacteria MK280
Chelated iron + bacteria
Chelated iron without bacteria MK280
Figure 1. Sterile seedlings of oilseed rape rapes transplanted
in MS culture media in a plant cell culture vessel.
61
Figure 2. Oilseed rape plants grown in vitro with different treatments.
For explication of codes, see Table 1.
Fe -
FeEDTA
FeEDTA
+ MK280
FeCl3
FeCl3+MK280
62
Figure 3. Effect of Pseudomonas sp. MK280 on rapeseed in vitro growth: plant height
plant height 15 days
25
cm
20
15
10
5
0
FeCl3 + MK280
FeCl3
FeEDTA + MK280
FeEDTA
Fe -
Treatments
Figure 4. Effect of Pseudomonas sp. MK280 on rapeseed in vitro growth: plant fresh and dry weight
fresh w eight 15 days
g
1,2
fresh w eight 30 days
1
dry w eight 15 days
0,8
dry w eight 30 days
0,6
0,4
0,2
0
FeCl3 +
MK280
FeCl3
FeEDTA +
MK280
FeEDTA
Fe -
Treatm ents
Greenhouse trial
Objective
To test the ability of some bacteria strains to promote the rapeseed growth in controlled conditions.
The trial was conducted using the following bacteria: Azospirillum brasilense (strain LMD78.36) or Pseudomonas sp.
(strains MK280, Hv37a and AGS195B). The seeds were treated with bacteria strain cell suspensions; then the treated
seeds were sown in a pot (one seed/pot) containing natural soil (Nitrogen= 0.08%; Organic matter =1.2%; Iron = 10.77
mg kg-1 )The strains were tested on 20 plants, 10 plants were fertilized with Nitrogen (60 kg/ha) after 40 days from
sowing; 10 were not fertilized. The following growth parameters were checked at 15 and 30 days: plant height, plant
fresh weight and plant dry weight.
Table 2. Colony Forming Unit from rape seeds treated with bacteria before sowing in greenhouse.
Bacteria
C.F.U/seed
A. brasiliense LMD78.36
6 x 103
Pseudomonas sp. MK280
1.2 x 105
Pseudomonas sp. HV37a
6 x 105
63
3 x 106
Pseudomonas sp. AGS195B
Figure 5. Bacterial seed treatment effect on rapseed: plant height
100
plant height 80 days + N
cm
80
60
40
20
0
Control
Hv37aR2
LMD78.36
AGS195B
MK280
Treatments
g
Figure 6. Bacterial seed treatment effect on rapseed plant: dry weight
dry weight 40 days
dry weight 80 days
dry weight 80 days + N
2,5
2
1,5
1
0,5
0
Control
Hv37aR2
LMD78.36
AGS195B
MK280
Treatments
Results and Conclusions
Results of the Pseudomonas sp. in vitro test were reported in Figures 2, 3 and 4. The strain MK280 promoted the
rapeseed growth when a little available iron form (FeCl3) is present in the culture media. Results of the greenhouse trial
were reported in Figures 5 and 6. In the treatment without Nitrogen, after 80 days from sowing, the plants treated with
Pseudomonas sp. strain MK280 showed a higher height and dry weight then control and plants treated by others
bacteria.
The present study showed that both methods were able to select and evaluate the promoting action of the bacteria
towards the oilseed rape although the in vitro was faster and discriminated better the promoting effect of the bacteria.
Acknowledgements
The study was funded by GAL Alto Tevere Valle delle Genti, Citta di Castello (Leader II project).
Research work of F. Citarei, M. Scribano and C. Cellerino was supported by grant of 3A Parco Tecnologico
Agroalimentare dell’Umbria, in the frame of Umbria Region Training programm (European Social Forum found).
64
9
IMPROVEMENT OF SOIL PROPERTIES AND PHYTOPARASITIC NEMATODES SUPPRESSION BY
COMPOSTED OLIVE MILL WASTE
Convertini Grazia (1), Ferri Donato (1), Sasanelli Nicola (2), D’Addabbo Trifone (2)
(1) Istituto Sperimentale Agronomico (MIPAF) – Via Celso Ulpiani, 5 - 70125 Bari, Italy. E-mail:
[email protected]
(2) Istituto per la Protezione delle Piante - Sez. di Bari - C.N.R. - Via Amendola 165/A, 70126 Bari, Italy. E -mail:
[email protected]
Summary.
The effect of composted olive mill wastes (composted - OMW) soil amendments on phytoparasitic nematodes and on
soil fertility was investigated in a sandy soil heavely infested by Meloidogyne incognita in southern Italy (Apulia
region). Composted olive pomace obtained by mixing fresh solid cake coming from the three-phase decanter (for olive
oil production) with farmyard manure (10, 20 and 40 t ha -1), another compost obtained by mixing exhausted solid
cake (chemical extraction of residue olive oil) with wheat straw and poultry manure (10, 20 and 40 t ha -1) and raw
sewage supplied at 40 and 80 m3 ha-1 , were compared with two controls : i) untreated soil; ii) treatment with
fenamiphos (traditional nematicide) at 0.3 t ha-1. Tomato crop yield, soil nematode population and root gall index were
recorded in all the plots. Soil fertility parameters, as total N, nitrates, ammonium, total, extracted and humified organic
C, NaHCO3-P, exchangeable bases, heavy metals were determined for the most significant rate of each amendment.
Crop yield was enhanced in all amended plots and pomace based composts were also suppressive on M. incognita.
Moreover, compost amendments increased soil organic matter content, improved N availability and protected soil
organic fractions from the fast decomposition occurring in the semi-arid conditions of southern Italy.
Keywords: Control, Meloidogyne incognita, nematodes, olive mill wastes, soil fertility.
INTRODUCTION
The disposal of olive mill wastes (OMW), fresh pomace and raw sewage, represents a serious environmental
problem in the areas of cultivation of olive, as large amounts of these materials are produced in a short period every
year (Ranalli and De Mattia, 1996).
The use of these materials as soil amendment could represent a possible alternative solution to this problem. Recent
studies showed that in the soil characterized by poor fertility, OMW incorporation into the soil improves some chemical
soil properties at different texture in southern Italy (Ferri et al., 2001). Also soil porosity, aggregates stability,
hydrological properties were improved by the application of OMW (Pagliai, 1996), although contrasting results derived
from previous experiments (Bonari and Ceccarini, 1993 ).
Moreover, a suppressive action on soil phytoparasitic nematodes has been also reported among the effects of olive
mill wastes incorporation into the soil (Rodriguez-Kabana et al., 1995; D’Addabbo and Sasanelli, 1996, Sasanelli et al.,
2002).
Objective of the field experiment described in this paper was a comparative evaluation of the effect of fresh and
exhausted composted olive pomace and raw sewage application on a population of the root-knot nematode
Meloidogyne incognita and on the chemical properties of the soil.
MATERIAL AND METHODS
A sandy soil at Monteroni (province of Lecce, southern Italy), heavily infested by Meloidogyne incognita (Kofoid
et White) Chitw. (Pi = 9 eggs and juveniles/cm3 soil), was subdivided in 12 m2 plots, spaced 1 m each other, according
to a randomized block design with four replicates for each treatment. Composted olive pomace obtained by adding
fresh solid cake (93%) from a three-phase decanter (for olive oil production) with farmyard manure (7 %) and a
compost obtained by mixing exhausted pomace solid cake (91%) (after the chemical extraction of residue olive oil) with
wheat straw (2%) and poultry manure (7 %) were distributed on the plot surface at 10, 20 and 40 t ha -1 rates and then
incorporated into the soil at 25 – 30 cm depth by rotavation. Raw sewage was added at the dosages of 40 and 80 m 3 ha-1.
Untreated soil and 300 kg ha-1 fenamiphos applied before transplanting were used as controls. One month old seedlings
of tomato (Lycopersicum esculentum L.), cv. Tondino di Zagaria, were transplanted into the plots three months after the
treatments. During the growing season the field received the usual cultural practices.
At the end of the crop cycle in each plot the tomato yield was recorded, the nematode gall infestation index on the
roots was estimated on a 0 – 5 scale (0 for not galled roots and 5 for roots completely deformed by many large galls)
(Di Vito et al., 1979) and the nematode population density was determined in the soil (Coolen, 1979).
65
Soil samples were collected in the plots treated with : i) no input (CONTR); ii) fenamiphos (300 kg ha -1)
(F.MPHOS); iii) raw sewage (80 mc ha-1) (R.S.); iv) exhausted pomace solid cake compost ( 20 t ha -1) (EOPC); v) fresh
pomace solid cake compost (20 t ha -1) (FOPC), after the treatments and harvesting to evaluate soil N-NO3, N-NH4
exchangeable, NaHCO3 - P (Olsen method), exchangeable bases, CEC, heavy metals concentration , total organic
carbon (TOC), total extracted (TEC) and humified carbon (humic and fulvic acids, CHA + CFA) (Sequi et al., 1986;
Italian Ministry of Agricultural Resources, 1994). The degree of humification, DH% = (CHA + CFA)/TEC*100, the
humification rate, HR% = (CHA + CFA)/TOC*100, and the humification index, HI= NH / (CHA + CFA), were also
calculated.
Data were statistically analysed by the analysis of variance and means compared by Duncan’s Multiple Range Test
(P=0.05).
RESULTS AND DISCUSSION
In the field experiment all the composts significantly increased the tomato yield compared to the untreated control,
but were statistically lower than chemical treatment (Table 1).
All the treatments significantly reduced the root infestation index and, with the exception of the lowest rates of raw
sewage and exhausted pomace compost, the soil nematode population (Table 1).
Results from this experiment and their comparison with those from the previous field experiments evidenciated that
incorporation of OMW into the soil could result in a suppression, but not in a complete eradication of phytoparasitic
nematode populations and, at low initial soil infestation, also in a crop yield increase. Therefore, the aim of soil
amendments with these materials should be a progressive reduction of infestation level under the tolerance limit of the
target nematode species (Sasanelli, 1994).
Table 1 - Effects of different olive mill wastes amendments on Meloidogyne incognita on tomato (cv. Tondino di
Zagaria).
Treatment
Untreated control
Fenamiphos
Dose
(t x
ha-1)
0,3
Tomato
yield
(t x ha-1)
25.4
a
40.5
e
Root gall index
(0 – 5)
4,5
2,7
b
a
Eggs and
juveniles/cm3
soil
19.8
d
10.6
abc
Raw sewage
4
32.3
bc
2,4
a
15.1
cd
8
29.0
b
2,5
a
5.2
a
Exhausted olive pomace compost
10
36.8
d
2,1
a
13.7
bcd
20
36.6
d
2,2
a
9.2
abc
40
36.3
d
2,2
a
11.2
abc
Fresh olive pomace compost
10
32.1
bc
2,8
a
5.3
a
20
33.7
cd
2,2
a
7.1
ab
40
35.8
cd
2,6
a
9.3
abc
Data are means of four replications; data followed by the same letter on the same column are not significantly
different according to Duncan’s Multiple Range Test (P = 0.01).
In the Figure 1 A can be observed a poor increasing trend of soil N-NO3 contents respectively for “R.S.”,
“EOPC”, “FOPC” treatments and lowest N-NO3 content in “fenamiphos-treated” plots.
Soil NaHCO3-P content was unaffected from experimental treatments, but soil exchangeable K showed higher
value in "fenamiphos-treated" plots (Figure 1A). On the contrary, in “FOPC-treated” plots higher “exchangeable-Ca”
content was recorded , while similar trends were observed in exchangeable Ca in “fenamiphos-”, “R.S.-”, “EOPCtreated” plots. No differences were observed on exchangeable Na and Mg among treated plots compared with the
“control” (Figure 1 B ).
Among the heavy metals of the soil (Figure 1 C ), only Zn content was higher in “EOPC-“ and “FOPC-treated”
plots probably because during the composting processes of solid residual cake of olive milling ( fresh or exhausted ) a
Zn concentration could be verified.
Soil organic C (Figure 1 D) contents (total and extracted) seem lower in the plots treated with OMW (raw or
composted) in comparison to other treatments, because the incorporation into the soil of fresh organic matter improves
biological activity (Ocio et al., 1991). No variations were recorded on humified organic C, probably for the short period
of trial.
66
A
140
a
2000
100
ab b
b
CONTR.
F.MPHOS
R.S.
60
EOPC
b
40
n.s.
1500
F.MPHOS
R.S.
1000
EOPC
FOPC
n.s.
n.s.
500
0
0
N-NO3
NaHCO3-P
Ca
Exch.-K
C
120
7
n.s.
100
6
a
b
b
F.MPHOS
a
( g kg-1 )
b
R.S.
EOPC
40
FOPC
D
a
a
b
b
b
CONTR.
F.MPHOS
4
a
3
R.S.
a
b
b
EOPC
b
FOPC
2
n.s.
20
Na
5
CONTR.
80
Mg
Soil exchangeable bases
Soil available nutrients
(mg kg-1)
FOPC
n.s.
20
60
B
ab
b
CONTR.
b b
80
(mg kg-1)
(mg kg-1)
2500
a
120
n.s.
n.s.
1
0
0
Zn
Cu
Pb
Total
Soil heavy metals
Extract.
Humified
Soil organic Carbon
Figure 1 – Soil fertility variations as affected by experimental treatments.
(For each parameter columns followed by different letters are significanty different at P<0.05 according to
Duncan’s Multiple Range test)
Soil properties reported in the Table 2 show that experimental treatments play a similar role on pH and
CEC. The degree of humification (DH) results significantly higher in “R.S.-treated” plots than in
“control” plots; on the other hand in “fenamiphos-” and “FOPC-treated” plots this parameter appears very lower
than in “control” plots. Humification rate
(HR) in all treated plots is lower than control. Finally humification index (HI) of “fenamiphos-“ and “FOPC-“ treated
plots is higher than other plots similarly to the findings of degree of humification (DH).
Table 2 - Variations of some soil properties as affected by experimental t reatments.
Humification parameters
CEC
(meq/100 g)
DH (%)
HR (%)
CONTR
8.30
9.55
20.4 b (1)
9.1 a
F.MPHOS
8.32
10.13
16.0 c
7.4 c
R.S.
8.20
9.81
22.7 a
8.1 b
EOPC
8.29
9.06
21.3 b
8.1 b
FOPC
8.23
9.29
16.3 c
6.1 c
(1 ) – Values followed by the same letter are not significantly different at P < 0.05
according to Duncan’s Multiple Range Test.
Treatments
pH
HI
3.9 b
5.2 a
3.4 b
3.7 b
5.1 a
In conclusion OMW soil amendments could be suggested for integrated nematode management strategies, as it
provides a good pest suppressivity.
After the first year of trial is very hard to record soil fertility variations as affected by experimental treatments. The
experimental data show that both OMW soil amendments and traditional nematicide (fenamiphos) don’t determine
decrease of soil available nutrients, soil exchangeable bases. On the other hand the effects on soil heavy metals (Zn,
Cu, Pb) are unsignificant, when were compared all the treatments with “control”. Very interesting trends were observed
on soil organic C and humification parameters. The apparent increase observed on soil organic C (TOC, TEC) in the
plots untreated (CONTR) and treated with fenamiphos (F.MPHOS) seems in contrast with a light increase of
67
humification parameters recorded in the other plots (R.S.-, EOPC-, FOPC-treated). Probably, the incorporation into the
soil of OMW (raw or composted) determines an improvement of soil o.m. transformations.
Moreover it is necessary to continue experimental research to verify when OMW and traditional nematicide effects
are evident and eventually different on soil fertility dynamics.
Work carried out in the frame-work of the O.P. CNR-MURST, Law 95/95, National Research Program: “Reflui Agro Industriali – Sottoprogetto Reflui Oleari”.
REFERENCES
Bonari E. e L. Ceccarini, 1993. Sugli effetti dello spargimento delle acque di vegetazione sul terreno agrario: risultati
di una ricerca sperimentale. Genio Rurale, 5: pp.60-67
Coolen W. A., 1979. Methods for the extraction of Meloidogyne spp. and other nematodes from roots and soil. In:
Root-knot nematodes (Meloidogyne species) Systematics, Biology and Control. (F. Lamberti and C.E. Taylor Eds),
Academic Press, London, UK, pp. 317-329.
D’Addabbo T. and N. Sasanelli, 1996. Effect of olive pomace soil amendment on Meloidogyne incognita.
Nematologia mediterranea, 24, pp. 91-94.
Di Vito M., F. Lamberti e A. Carella. 1979. La resistenza del pomodoro nei confronti dei nematodi galligeni:
prospettive e possibilità. Rivista di Agronomia 13: pp. 313-322.
Ferri, D., G. Convertini, F. Montemurro e C. Vitti, 2001. Evoluzione di alcune proprietà chimiche di terreni pugliesi
ammendati con reflui oleari. Atti Giornata di Studio “ Prospettive di utilizzzione agronomica dei reflui oleari” ( Bari, 14
febbraio).
MIRAAF, 1994. Metodi ufficiali di analisi chimica del suolo.
Ocio J.A., P.C. Brookes and D.S. Jenkinson, 1991. Field incorporation of straw and its effects on soil microbial
biomass and soil inorganic N. Soil Biol. Biochem. 23: pp.171-176.
Pagliai M., 1996. Effetti della somministrazione di acque reflue di frantoi oleari sulle caratteristiche fisiche del suolo.
Atti Convegno Int. su “Trattamento e riciclaggio in agricoltura dei sottoprodotti dell’industria olearia”. Lecce, 8-9
marzo.
Ranalli A. and G. De Mattia. 1996. Role of piloted biological processes in the purification of oil mill waste water.
Riv. Ital. Sost. Grasse, LXXIII: pp.61-66.
Rodriguez-Kabana R., V. Estaun, R.J. Pinochet and O. Marfa’, 1995. Mixtures of olive pomace with different
nitrogen sources for the control of Meloidogyne spp. on tomato. Journal of Nematology, 27 (4S), pp. 575 -584.
Sasanelli N. 1994. Tables of Nematode-Pathogenicity. Nematologia mediterranea, 22: p.153-157.
Sasanelli N., T. D’Addabbo, G. Convertini and D. Ferri, 2002. Soil Phytoparasitic Nematodes Suppression and
Changes of Chemical Properties Determined by Waste Residues from Olive Oil Extraction. Proceedings of 12th ISCO
Conference, May 26-31, 2002 Beijing China. Vol. III: pp. 588-592.
Sequi, P., M. De Nobili, L. Leita and G. Cercignani, 1986. A new index of humification. Agrochimica, 30: pp.175 –
179
68
10
NEMATICIDAL ACTIVITY OF AQUEOUS EXTRACTS FROM RUE (RUTA GRAVEOLENS L.)
T. D’Addabbo and N. Sasanelli
Istituto per la Protezione delle Piante- Cnr, Sezione di Bari
Via G. Amendola 165/A – 70126 Bari. Italy
Summary. The effect of leaf and root aqueous crude extracts and of root leachates of R. graveolens on the root-knot
nematodes Meloidogine arenaria, M. hapla, M. incognita and M. javanica, and on the beet cyst nematode Heterodera
schachtii was investigated in a in vitro experiment. Aqueous extracts were prepared by soaking green leaves or roots in
distilled water and root leachates were collected by drenching the rue cultivated soil with excess tap water. Egg masses
of each Meloidogyne species and cysts of H. schachtii were incubated in the test solutions over a four weeks period.
Immersion in rue extracts significantly suppressed juvenile emergence from the egg masses of all the four
Meloidogyne species and from cysts of H. schachtii. Leaf extract was always more effective than root extract for
Meloidogyne species, whereas no difference resulted in H. schachtii. No synergic effect derived from the combination
of the two extracts. No significant emergence reduction resulted from the immersion in rue root leachates for any of the
tested nematode species.
INTRODUCTION
Several plants are reported to minimize nematode damage in vegetable and field crops by producing nematicidal
(killing) and nematistatic (suppressive) organic compounds. These natural compounds can be released from the roots of
living plants or by plant tissues incorporated into the soil as a green manure (Grainge and Ahmed, 1988).
The biocidal effect of extracts from rue (Ruta graveolens L.) was previously reported on insects, fungi and weeds
(Aliotta et al., 1994; 1999; Oliva et al., 1999), but few information is available on the effect on phytoparasitic
nematodes. Therefore an experiment was undertaken to investigate the in vitro effect of leaf and root aqueous extracts
and of root leachates of R. graveolens on four different species of root-knot nematodes, Meloidogine arenaria (Neal)
Chitw., M. hapla Chitw., M. incognita (Kofoid et White) Chitw.and M. javanica (Treub) Chitw. and on the beet cyst
nematode Heterodera schachtii Schmidt.
MATERIALS AND METHODS
Aqueous extracts of rue were prepared by soaking 100 g green leaves or roots, or 50 g when in combination, in 400 ml
distilled water for 24 hrs. Tissues were then macerated in a blender and the suspension filtered through filter paper.
Root leachates were collected from three month old plants, cultivated in fifteen 2,500 cm3 clay pots, by drenching the
soil with excess tap water. The leachates were then centrifuged at 1,300 g for 30 min, stored in plastic bottles, and kept
in a freezer until required. In the experiment on H. schachtii each plant extract or root leachate was adjusted to 3 mM
adding an equal volume of 6 mM of zinc sulphate (Clarke and Shepherd, 1966).
The four Meloidogyne populations were reared on tomato cv. Roma in a glasshouse at 20-25 °C, whereas H. schachtii
was collected from an infested field at Avezzano (L’Aquila). Batches of twenty egg masses (averaging 20,000 eggs per
mass) of each Meloidogyne species or 100 cysts of H. schachtii (123 egg/cyst) were placed on 2 cm diam sieves (215
mm aperture) and each sieve in a 3.5 cm diam Petri dish. Three ml of each test solution, were then added to four batches
of egg masses or cysts. Distilled water (for Meloidogyne species) or 3 mM zinc sulphate solution (for H. schachtii) and
a 5 mg/ml aqueous solution of fenamiphos were used as controls.
The dishes were arranged in a complete randomized block design with four replicates of each treatment: egg masses of
Meloidogyne species were incubated at 25 °C, whereas the cysts of H. schachtii were mantained at 20 °C. Emerging
juveniles were removed and counted at weekly intervals, renewing the hatching solutions at the same time, over a nine
or ten weeks period, respectively for Meloidogyne species and H. schachtii. Egg masses and cysts were removed from
the test solutions after the first four weeks and the incubation continued in distilled water or, for cysts of H. schachtii, in
the zinc sulphate aqueous solution.
At the end of the experiment the egg masses were immersed in a 1% sodium hypochlorite aqueous solution (Hussey and
Barker, 1973), whereas the cysts were crushed, according to Seinhorst and Den Ouden (1966). Unhatched eggs from
egg masses and cysts were then counted. Numbers of juveniles emerging weekly were expressed as cumulative percent
of the total initial population. Data were statistically analysed and compared by LSD’s test.
69
Emergence of juveniles from the egg masses immersed in leaf and root extracts of Ruta graveolens, either alone or
combined, was significantly suppressed compared to the water control in all the four Meloidogyne species (Table 1).
Leaf extract was always more effective than root extract and resulted statistically more suppressive also than
fenamiphos solution. No synergic effect was derived from the combination of the two extracts, as final hatch was higher
than in leaf extract alone. Egg masses immersed in root leachates gave a final hatch similar to control.
The ultimate egg hatch of H. schachtii cysts incubated in single root and leaf extracts was significantly less than in 3
mM zinc sulphate and fenamiphos (Fig. 1). Suppressivity of the extracts decreased when in combination, as no
difference there was in final hatch compared to the control. There was no difference between leaf and root extracts,
whereas their combination resulted in a significantly higher egg hatch. As found in Meloidogyne species, no significant
emergence reduction was found for the root leachates.
In conclusion, the experiment evidenciated the nematicidal properties of rue extracts. The nematicidal compounds
seemed to be present in all the plant tissues, but especially in leaves. A possible antagonistic effect of leaf and root
extracts emerged from the results, that could be attributable to a negative interaction of the active principles responsible
for the suppressive effect. The absence of a nematicidal effect in root leachates indicated that nematicidal compounds
are present only in plant tissues, but not released into the soil.
LITERATURE CITED
Aliotta, G., and G. Cafiero, (1994). Potential allelochemicals from Ruta graveolens L. and their action on radish seeds.
Journal of Chemical Ecology 20 (11): 2761-2775.
Clarke A.J. and A.M. Shepherd, (1966). Inorganic ions and the hatching of Heterodera spp. Annals of Applied
Biology 58: 49–508.
Grainge M. and S. Ahmed, (1988). Handbook of Plants with Pest-Control Properties. (J. Wiley and Sons eds.) New
York, pp. 238-248.
Hussey R.S. and K.R. Barker, (1973). A comparison of methods of collecting inocula of Meloidogyne spp. including a
new technique. Plant Disease Reporter 57: 1025-1028.
Landolt, P. J.,R. W. Hofstetter and L. L. Biddick , (1999). Plant essential oils as arrestants and repellents for
neonate larvae of the codling moth (Lepidoptera: Tortricidae). Environmental Entomology 28 (6): 954-960.
Oliva, A. and E. Lahoz, (1999). Fungistatic activity of Ruta graveolens extract and its allelochemicals. Journal of
Chemical Ecology 25 (3): 519-526.
Seinhorst J.W. and H. Den Ouden (1966). An improvement of Bijloo’s method for determining the egg content of
Heterodera cysts. Nematologica (12): 170-171.
Table 1. Effect of root and leaf extracts and root leachates of rue (Ruta graveolens L.) on the total percent cumulative
hatch of four different Meloidogyne species.
Meloidogyne species
Treatments
M. arenaria M. hapla M. incognita M. javanica
Leaf extract
12
6
12
3
Root extract
59
43
61
23
Root and leaf extract
20
21
49
21
Root leachates
83
91
80
89
Fenamiphos
38
47
29
31
Distilled water
81
87
75
92
L.S.D. 0.05
11
12
15
15
L.S.D. 0.01
15
15
20
20
70
40
% cumulative hatch
35
30
25
20
15
10
5
0
LE
RE
LRE
LCH
FEN
CTRL
0,05
0,01
Fig. 1. Effect of rue (Ruta graveolens L.) extracts on the percent cumulative hatch of Heterodera schachtii (LE = leaf
extract; RE = root extract; LRE = leaf + root extract; LCH = root leachates; FEN = 5 mg/ml fenamiphos aqueous
solution; CTRL = 3 mM zinc sulphate solution).
71
11
VALUTAZIONE DELL’EFFICACIA INSETTICIDA DI RYANIA, ROTENONE, AZADIRACTINA E
BEAUVERIA BASSIANA
Elisabetta Gargani, Giovanna Del Bene
Istituto Sperimentale per la Zoologia Agraria – Via Lanciola, 12 A - 50125 Firenze.
e-mail: [email protected]
Nell’ambito del P.F. MiPAF “Difesa delle produzioni in agricoltura biologica”, l’ISZA, sezione di Entomologia
Agraria, ha effettuato prove sperimentali sull’efficacia insetticida di prodotti di origine naturale (Del Bene et al., 2000):
ryania, rotenone, azadiractina e Beauveria bassiana. Le prove sono state condotte in laboratorio, in serra e in campo su
varie specie vegetali di interesse ortoflorovivaistico, infestate sia naturalmente sia artificialmente da insetti appartenenti
agli ordini dei Tisanotteri (Tripidi Heliothrips haemorrhoidalis e Frankliniella occidentalis), Rincoti (Triozide
Lauritrioza alacris e Aleurodide Trialeurodes vaporariorum), Lepidotteri (Gracillariide Phyllocnistis citrella) e
Coleotteri (Crisomelide Pyrrhalta viburni). Sono stati inoltre studiati gli effetti collaterali nei confronti del parassitoide
Imenottero Afelinide Encarsia formosa.
I prodotti sono stati sperimentati in formulazioni e dosaggi diversi con una sola applicazione o 2-3 applicazioni a
distanza di 7 giorni. I controlli sono stati effettuati di norma a cadenza settimanale, conteggiando gli individui vivi e
morti, distinti per stadio di sviluppo. Sono state calcolate le percentuali di sopravvivenza e di mortalità (M) secondo
Abbott.
RYANIA
Il principio attivo, costituito dagli alcaloidi ryanodine, è estratto da Ryania speciosa, arbusto della famiglia
Flacourtiaceae, nativo di Trinidad e del Bacino dell’Amazzonia. Le ryanodine agiscono soprattutto per ingestione e
hanno una persistenza di due settimane; sono tossiche per i mammiferi (Tremblay, 1985; Casida et al., 1987).
Sono stati impiegati 2 formulati sperimentali, Tigosan liquido (3-6 ml/l) e Tigosan polvere (3-5 g/l), di cui solo quello
in polvere ha dimostrato una certa efficacia (tab. 1).
Tab. 1 – Percentuali di mortalità (sec. Abbott) ottenute con Ryania.
Formulato
Dose ml/l Insetto
Stadio
Tigosan L
3
H. haemorrhoidalis Nean. - adulti
6
Tigosan WP
3
F. occidentalis
Tigosan WP
5
Nean. - adulti
5
L. alacris
Tigosan WP
5
Ninfe I-II
Ninfe III-V
T. vaporariorum *
Tigosan WP
5 – 2tr.
Neanidi I
5 – 2tr.
Nean. II-IV
P. citrella
Tigosan WP
5
Larve I-III
P. viburni
Tigosan WP
5
Larve I
Larve II-III
Larve I-II
Adulti
Adulti
* Nessun effetto su E. formosa (100% sfarfallamento)
Pianta
% controllo
viburno - labor.
7,5
"
"
22
mirto - campo
22
pomodoro - serra
55,6
fagiolino - labor.
40
alloro - labor.
45,2
"
"
10,5
pomodoro - serra
11,5
"
"
10,4
agrumi - vivaio
0
viburno - labor.
100
"
"
53,3
viburno - serra
82,5
viburno - labor.
66,7
viburno - campo
0
Ryania ha dato esiti variabili contro P. viburni: in laboratorio la mortalità delle larve di I età ha raggiunto il 100 % con
la dose di 5 g/l, mentre quella delle larve di età superiore è stata del 55,3% e quella degli adulti del 66,7%; in serra è
stata confermata l’efficacia sulle larve I-II (82,5 % M) mentre in campo non è stato registrato alcun controllo degli
adulti. E’ risultata mediamente efficace nei confronti di F. occidentalis su pomodoro in serra (55,6%M) e selettiva nei
confronti di E. formosa.
72
ROTENONE
Il principio attivo è estratto dalle radici di Derris elliptica e di altre Papillionaceae appartenenti ai generi Lonchocarpus
e Tephrosia; agisce per contatto e ingestione (La Torre et al., 2002). Ha bassa persistenza e si decompone rapidamente
con l’esposizione all’aria e alla luce; non è selettivo nei confronti degli insetti utili né dei fitoseidi (Tsolakis et al., 1997)
ed è tossico per i vertebrati (Tremblay, 1985). In Italia il prodotto è stato registrato come liquido emulsionabile ad
ampio spettro di azione, alla concentrazione del 6% di p.a. (“Rotena” Serbios, Tossico) e al la concentrazione del 4%
(“Bioroten” Intrachem, Nocivo).
Sono stati impiegati 3 formulati: Derisan (non registrato, 3-6 ml/l); Rotena (2,5 ml/l); Bioroten (3 ml/l) (tab. 2).
Tab. 2 – Percentuali di mortalità (sec. Abbott) ottenute con Rotenone.
Formulato Dose ml/l Insetto
Stadio
Pianta
% controllo
Derisan
6
H . haemorrhoidalis Nean. - adulti
viburno - labor.
50
Rotena
2,5 – 2tr.
viburno - campo
0
F. occidentalis
Derisan
3
Nean. - adulti
pomodoro - serra
0
fagiolino - labor.
13,4
Bioroten
3 – 3tr.
limonium - serra
49,3
E. phyllireae
Derisan
6
NinfeIV-V
fillirea - labor.
0
L. alacris
Rotena
2,5
Ninfe I-II
alloro - labor.
93,1
Ninfe III-V
"
"
44,6
Rotena
2,5 – 3tr. T. vaporariorum*
Nean. I
pomodoro -serra
55,7
Nean. II-IV
"
"
60
P. citrella
Rotena
2,5
Larve I-II
agrumi - vivaio
0
P. viburni
Derisan
3
Larve I
viburno - labor.
7,9
Bioroten
3
Larve I-II
viburno - serra
100
Rotena
2,5
Adulti
viburno - labor.
93,3
*Effetti negativi su E. formosa (sfarfallamento da 8, 75% a 32,5%)
Rotenone è risultato mediamente efficace nei confronti di H. haemorrhoidalis con mortalità del 50% (Derisan) e
verso le neanidi di T. vaporariorum con 55,7 – 60% M (Rotena); assai efficace verso le giovani ninfe di L. alacris con
mortalità del 93,1% (Rotena). Nei confronti di P. viburni, la resa del prodotto è stata assai diversa a seconda dello
stadio trattato e del formulato usato. Infatti mentre le larve di I età, stadio più vulnerabile, sono sopravvissute in
laboratorio al trattamento con Derisan, la mortalità di larve I-II trattate con Bioroten è stata del 100% e quella degli
adulti trattati con Rotena è stata del 93,3%. Rotena ha avuto comunque effetti collaterali negativi molto pesanti su E.
formosa, le cui percentuali di sfarfallamento sono state ridotte significativamente.
AZADIRACTINA
Dall’albero Azadirachta indica (neem), della famiglia Meliaceae, originario dell’India, viene
estratto un olio contenente numerosi principi attivi appartenenti alla classe dei limonoidi (azadiractine), con proprietà
antiparassitarie (Schmutterer, 1990; La Torre et al., 2002). L’estratto ha azione come regolatore di crescita,
fagodeterrente e repellente e riduce la fecondità degli adulti e la vitalità delle uova (Rovesti e Deseo, 1990; Bezzi e
Caden, 1991; Capella et al., 2000); agisce per ingestione e contatto e presenta un elevato potere di penetrazione (Isman
et al., 1991). In Italia è commerciato come biostimolante vegetale (“Stardoor” Intrachem, EC 4,5%); inoltre sono stati
registrati come insetticidi due prodotti a base di azadiractina A, che rappresenta la componente con le più elevate
proprietà insetticide (“Oikos” Sipcam e “Diractin” Serbios, EC 3%, non classificati tossicologicamente).
I formulati impiegati sono stati 4: Olnisan (non registrato, 2,5 ml/l); Stardoor (0,7 ml/l); Oikos (1-1,5 ml/l); Diractin
(1,5 ml/l) (tab. 3).
73
Tab. 3 – Percentuali di mortalità (sec. Abbott) ottenute con Azadiractina.
Formulato Dose ml/l Insetto
Stadio
Pianta
% controllo
Oikos
1,5 – 2tr.
H. haemorrhoidalis Nean. - adulti viburno - campo
90,7
F. occidentalis
Olnisan
2,5
Nean. - adulti pomodoro - serra
9,4
fagiolino - labor.
34
Oikos
1,5 – 3tr.
limonium - serra
89,9
Diractin
1,5 – 3tr.
"
"
86,6
L. alacris
Stardoor
0.7
Ninfe I-II
alloro - labor.
89,4
Ninfe III-V
"
"
28,7
Olnisan
2,5
Ninfe I-V
alloro - vivaio
29,5
T. vaporariorum*
Stardoor
0,7
Nean. I
pomodoro- serra
18,7
P. citrella
Olnisan
2,5
Larve I-II
Oikos
1,5 – 2tr.
Diractin
1,5 – 2tr.
P. viburni
Olnisan
2,5
Larve I
Diractin
1,5 – 3tr.
Larve I-II
Stardoor
0,7
Adulti
Oikos
1 – 2tr.
Adulti
*Effetti negativi su E. formosa (sfarfallamento 17,5%)
agrumi - vivaio
"
"
"
"
viburno - labor.
viburno - serra
viburno - labor.
viburno - campo
18
48,1
55,6
10,5
90,6
13,3
0
I diversi formulati, escluso Olnisan, hanno dato risultati positivi nei confronti delle ninfe di I e II età di L. alacris
(Stardoor 89,4 % M), nei confronti di F. occidentalis (Oikos 89,9% M, Diractin 86,6% M) e H. haemorrhoidalis (Oikos
90,7% M); hanno mostrato un ottimo contenimento di larve I-II di P. viburni (Diractin 90,6% M) e una certa efficacia
contro larve I-II di P. citrella (Oikos 48,15% M, Diractin 55,6% M). Gli effetti collaterali nei confronti di E. formosa
sono stati negativi con Stardoor .
BEAUVERIA BASSIANA
E’ un Deuteromicete agente del “calcinaccio” del baco da seta, scoperto nel 1835 da Agostino Bassi, che ne ipotizzò
per primo l’uso nella lotta agli insetti dannosi. Le spore del fungo, a contatto con l’insetto, germinano penetrando
attraverso la cuticola, mediante una combinazione di azioni meccaniche ed enzimatiche: lo sviluppo successivo del
fungo, accompagnato da produzione di tossine, determina la morte dell’insetto solitamente in 3-5 giorni (Magnano di
San Lio e Vacante, 1989; Benuzzi e Santopolo, 2001; La Torre et al., 2002). B. bassiana ha ampio spettro di attività e
agisce per contatto in ambiente umido. Come bioinsetticida è stato prodotto a partire da un ceppo isolato su
Anthonomus grandis: il formulato registrato in Italia è “Naturalis” (Intrachem, Irritante), sospensione concentrata al
7,16% di B. bassiana, contenente 2,3 x 107 conidiospore/ml (tab. 4).
Tab 4 – Percentuali di mortalità (sec. Abbott) ottenute con B. bassiana.
Formulato Dose ml/l
Insetto
Stadio
Naturalis
1 – 2tr.
1,5 – 3tr.
H. haemorrhoidalis
F. occidentalis
Nean. - adulti
Nean. - adulti
1
1,5 – 3tr.
1– 3tr.
1 – 2tr.
P. citrella
P. viburni
Larve I-II
Larve I-II
Adulti
Pianta
viburno - campo
rosa - serra
limonium - serra
agrumi - vivaio
viburno - serra
viburno - labor.
viburno - campo
% controllo
41,3
81
85
0
81,1
55,3
3,4
Naturalis (1-1,5 ml/l), a seguito di trattamenti ripetuti, ha assicurato un controllo soddisfacente in serra di F.
occidentalis (81 –85 % M) e di larve I-II di P. viburni (81,1% M), ma non di H. haemorrhoidalis in campo (41,35 %
M), né di adulti di P. viburni in laboratorio (55,3% M) e in campo (3,4% M).
Riassumendo, i prodotti biologici testati hanno dato risultati variabili a seconda dell’impiego in laboratorio o campo,
in base alla diversa formulazione e dose, alla specie di insetto e alla sua fase di sviluppo al momento del trattamento.
Risultati chiaramente positivi (controllo > 90%) sono stati ottenuti con:
- ryania contro larve I di P. viburni (Tigosan WP: 100% M.);
- rotenone nei confronti di P. viburni (adulti: Rotena: 93,3% M , larve I-II: Bioroten: 100% M) e di L. alacris (ninfe III: Rotena: 93,1% M);
- azadiractina contro H. haemorrhoidalis (Oikos: 90,7% M.) e larve I-II di P. viburni (Diractin: 90,6% M).
Hanno comunque garantito un controllo superiore all’ 80%:
- ryania nei confronti di larve I-II di P. viburni (Tigosan WP: 82,5% M);
74
- azadiractina nei confronti di F. occidentalis ( Oikos: 89,9% M, Diractin: 86,6% M), ninfe I-II di L. alacris (Stardoor:
89,4% M);
- B. bassiana nei confronti di F. occidentalis (81% M su rosa, 85,05 % M su limonium) e di P. viburni (larve I-II 81,1
% M).
Nei confronti di E. formosa si sono avuti effetti collaterali negativi con Rotena e Stardoor, mentre ryania è risultata
selettiva.
Bibliografia
BENUZZI M., SANTOPOLO F., 2001 – Naturalis: bioinsetticida a base di Beauveria bassiana.- Informatore
Fitopatologico, 4: 61-64.
BEZZI A., CADEN S., 1991 – Piante insetticide e pesticide.- Erboristeria domani. Ottobre: 65-79.
CAPELLA A., GUARNONE A., DOMENICHINI P., 2001 – Azadiractina: caratteristiche, attività biologica e strategie di
impiego su melo e ortive in coltura protetta.- Notiziario sulla protezione delle piante, 13: 59-63.
CASIDA J.E., P ESSAH I.N., SEIFERT J., WATERHOUSE A.L., 1987 – Ryania insecticide: chemistry, biochemistry and
toxicology.- In Pesticide science and biotechnology. Proceedings of the 6th international congress of pesticide
chemistry., Ottawa, Canada, 10-15 August 1986: 177-182.
DEL BENE G., GARGANI E., LANDI S., 2000 – Evaluation of plant extracts for insect control.- Journal of Agriculture and
Environment for International Development, 94(1): 43-61.
ISMAN M.B., KOUL O., ARASON J.T., STEWART J., SALLOUM G.S., 1991 – Developing a neem-based insecticide for
Canada.- Memoirs of the Entomological Society of Canada, 159: 39-47.
LA TORRE A., ALEGI S., IMBROGLINI G., 2002 – Mezzi di difesa in agricoltura biologica.- Informatore Agrario, 16
Suppl.1: 4-42.
MAGNANO DI SAN LIO G., VACANTE V., 1989 – I funghi entomopatogeni nella lotta biologica contro i fitofagi.Informatore Fitopatologico, 11: 17-25.
ROVESTI L., DESEO K.V., 1990 – Azadirachta indica A Juss (Neem) e sue potenzialità nella lotta contro gli insetti.Informatore Fitopatologico, 11: 27-32.
SCHMUTTERER H., 1990 – Properties and potential of natural pesticide from the neem tree, Azadirachta indica.- Ann.
Revue Entomology: 271-297.
TREMBLAY E., 1985 – Entomologia Applicata. Vol. 1. Generalità e mezzi di controllo. Liguori Ed., Napoli: 203 pp.
TSOLAKIS H., LETO G., RAGUSA S., 1997 – Effects of some materials on Tetranichus urticae Koch (Acariformes,
Tetranichidae) and Typhlodromus exhilaratus Ragusa (Parasitiformes, Phytoseiidae).- 4th International
Conference on pest in agriculture, 6-8 January, Le Corum, vol. I: 239-245.
75
12
DETERMINATION OF Cd (II), Cu (II), Pb (II), AND Zn (II) IN BIOLOGICAL AND NOT BIOLOGICAL
CITRUS ESSENTIAL OILS BY DERIVATIVE POTENTIOMETRIC STRIPPING ANALYSIS (dPSA)
Dugo Giacomo, Giuffrida Daniele, Drogo Alessandra, La Pera Lara
Dipartimento di Chmica Organica e Biologica Università di Messina Salita Sperone, 31, 90166 S. Agata -Messina
Citrus essential oils are complex mixtures of many classes of volatile (85-98%) and not volatile (15-2%)
compounds (1); they are used in food and cosmetic farm as aromatizer and in pharmaceutical industry as additives of
some drugs (2). Their wide use implies a strict control of the presence of organic and inorganic contaminants as heavy
metals. There is a lack of available data regarding to the presence of heavy metals: some reports concerning to the
micro-elements composition of citrus peel extracts were found (3,4). Metals levels in citrus essential oils mostly
depend on the type of soil, agrochemical treatments, but also on extraction procedures as scraping or pressing, since
the fruits inevitably come in contact with metallic surfaces (5). In this work potentiometric stripping analysis is used to
determine the content of Cd (II), Cu (II), Pb (II) and Zn (II) in lemon, mandarin, orange and bergamot non biological
essential oils produced in Sicily and Calabria in the crop year 1999 (6). Moreover 13 samples of bergamot biological
essential oils and 13 samples of non-biological essential oils produced in Calabria in the crop year 2000, were studied.
Reagents
All oils are sampled in dark glassy bottles, with blind nipples, and stored at 4°C until the analyses. Ultra pure
hydrochloric acid (34-37%), Hg (II) (1000 mgmL-1, 1M in hydrochloric acid) and Cd (II), Cu (II), Pd (II), Pb (II), Zn (II)
(1000 mgmL-1, 0.5 N in HNO3) standard solutions were purchased by Panreac (Barcelona, Spain); Ga (NO 3)3×3H2O (5 g,
99.9%) was purchased by Aldrich Chem. Co. (Milwakee, WI, USA). The extracts were filtrated on a carbon column
Supelclean ENVI-Carb SPE (0.5 g, 6 mL), purchased by Supelco (Bellefonte, PA, USA).
Apparatus
Metals analysis are carried out by a PSA ION 3 potentiometric stripping analyzer (Steroglass, S. Martino in Campo,
Perugia, Italy), connected to an IBM-compatible personal computer. The analyzer operates under the control of the
NEOTES 2.0.1 software package (Steroglass). The determination is carried out in a conventional three-electrode cell.
The working electrode was a glassy carbon one coated with a thin mercury film; reference electrode was an Ag/AgCl
electrode (KCl 3M), and a platinum wire auxiliary electrode was used.
Procedures
Hydrochloric acid extraction
A 8.60 g (10.00 mL) sample aliquot and a 10 mL volume of 36% ultrapure-hydrochloric acid (Panreac, Barcellona), are
introduced in a teflon beaker. The extraction was carried out for about 30 min under magnetic stirring at the temperature
of 90 °C. The acid sample, was transferred in a separating funnel and let to cool for about 5 minutes. In order to favour
the separation of the two phases, the mixture is spiked with 1.0 mL ultrapure methanol, then the acid layer was collected
in a 20.00 mL flask. The organic layer is extracted again with 4.50 mL of concentrated hydrochloric acid for 5 min,
under the same conditions described, and then washed with 4.50 mL of boiling water; washings were added to the acid
extracts and made up to the mark with water. The obtained solution was passed through a carbon column. The filtered
solution was used for the simultaneous determination of Cd (II), Cu (II), Pb (II), and Zn (II).
Potentiometric determination
All analysis were executed in a conventional three electrodes cell; working electrode was a glassy carbon electrode
coated with a Hg film, the reference one was an Ag/AgCl electrode, the counter was a Pt electrode. The concentrations
of Cd (II), Cu (II), Pb (II), and Zn (II) were simultaneously determined. 1.00 mL of acid extract, 17.50 mL of ultrapure water, 1.00 mL of 1000 mg mL-1 Hg (II) as oxidant agent and 0.50 mL of 10 mg mL-1 Ga (III) - in order to prevent
Cu-Zn complexes formation on the mercury film (6)- were put into the electrochemical cell. The quantitative analysis
was executed by the multiple points standard additions method. The potentiometric parameters are reported on table 1.
Method performances
76
Recovery and repeatabilty tests were menaged by emploing the above described method to lemon, mandarin, orange
and bergamot essential oils. Cd (II), Cu (II), Pb (II), and Zn (II) recoveries always spanned from 95.00 -100.50%
evidencing that metals quantification remained unaffected by the clean-up steps. The repeatability was > 95.00% for
Cd (II), Cu (II), Pb (II), and Zn (II) in all types of oils. Detection limits obtained for the four analytes, ranged from
0.10 to 0.98 ng g -1 in lemon, mandarin, sweet orange and bergamot essential oils.
Application
Figure 1 show the concentration of Cd, Cu, Pb and Zn in lemon, sweet orange, mandarin in Sicilian and Calabrian
bergamot essential oils. It’s evident that Cd is the metal present in the lowest amount and Zn in the highest, in all types
of essential oils. Cd concentration spanned from 2 to 23.3 ng g-1; Cu from 17.8 to 380.0 ng g-1; Pb ranged from 75.9 to
180.3 ng g-1 and Zn from 804.5 to 1647.5 ng g -1.
Moreover, the concentration of Cd, Cu, Pb and Zn was determined in 13 samples of biological bergamot essential oils,
and 13 non biological ones. Figure 2 evidences that there was any significant difference between biological and non
biological oils metals level.
LITERATURE
1. Di Giacomo A., Mincione B.Gli oli essenziali agrumari in Italia. 1994 Ed. Laruffa, p.196.
2. Gorinstein S., Martin-Belloso O., Park Y. S., Haruenkit R., Lojek A., Ciz, M., Caspi A., Libman, I., Trakhtenberg
S.Comparison of some biochemical characteristics of different citrus fruit. Food Chem. 2001, 74, 309 -315.
3. Simpkins W.A., Honway, L., Wu M., Harrison M., Goldberg D. Trace elements in Australian orange juice and
other products. Food Chem, 2000, 71, 423-433.
4. Di Giacomo, A. Tecnologia dei prodotti agrumari. Parte II. 1988, duplicated lecture notes, Univrsità di Reggio
5.
6.
Calabria, 95-120.
La Pera L., Saitta M., Di Bella G., Dugo G.mo. Simultaneous determination of Cd (II), Cu (II) Pb (II) and Zn (II) in
citrus essential oils by derivative potentiometric stripping analysis. J. Agric. Food Chem. 2003 in press
La Pera L., Lo Curto S., Visco A., Dugo G.mo. Derivative Potentiometric Stripping Analysis (dPSA) used for
determination of cadmium, copper, lead and zinc in in Sicilian olive oils. J. Agric. Food Chem. 2002, 50, 30903094.
77
1600,00
1400,00
1200,00
ng/g
1000,00
Cd
Cu
Pb
Zn
800,00
600,00
400,00
200,00
0,00
Lemon
Mandarin
Sweet orange
Bergamot
Figure 1.Metals content of lemon, mandarin, sweet orange and bergamot essential oil from 1999.
343,2
350
303,6
300
250
ng/g
200
Nonbio
Bio
200,4
166,1
142,0
128,6
Cu
Pb
150
100
50
0
Zn
Fig 2: mean metals content in biological and non biological bergamot essential oils produced in Calabria in the
crop year 2000.
78
13
IMPIEGO DEL TIMOLO NEL CONTROLLO DELLA VARROOSI DELLE API.
EFFICACIA, PERSISTENZA E RESIDUI.
I. Floris1, A. Satta 2, P. Cabras3, A. Angioni 3
1
Dipartimento di Protezione delle Piante - Sezione di Entomologia agraria, Università di Sassari, Via De Nicola, 07100
Sassari.
2
Istituto per lo Studio degli Ecosistemi – Sezione Ecologia applicata e Controllo biologico - CNR Sassari, Via De
Nicola, 07100 Sassari.
3
Dipartimento di Tossicologia, Università di Cagliari, Via Ospedale 72, 09124 Cagliari.
SUMMARY
During the last years, after Varroa destructor resistance have been detected to some important synthetic acaricides,
many alternative ways of varroa control based on natural products were tested. According to the literature, thymol
seems to deserve a strong interest for the treatment of varroa infestation. In this paper, the results of an apiary trial
conducted during the summer of 2001 in a Mediterranean environment (Sardinia, Italy) were reported. The
effectiveness of thymol, the persistence in two acaricide formulations (gel - Apiguard and vermiculite – Api Life
VAR®) and the residues in honey and wax were evaluated. Both the thymol formulations, after the treatments, reduced
significantly the levels o mite infestations of both adult bees and sealed brood (average of about 90%). However, a
considerable colony-to-colony variability in effectiveness was recorded. A moderate negative effect of the thymol
treatments on the colony development was also observed. During two weeks of treatments, the bees removed about all
the applied product (gel or vermiculite). Residues found in honey varied from 0.40 and 8.80 mg/Kg for Apiguard and
from 0.12 and 4.03 mg/Kg for Api Life VAR®, but according to EU regulation No. 2377/90 thymol is a non-toxic
veterinary drug, which do not need a MRL (maximal residue limit). The wax residues reached relatives higher levels
than honey since thymol is a fat-soluble ingredient (average of 21.6±13.0 and 147.7±188.9 for Api Life VAR and
Apiguard, respectively). However, after the treatment, thymol rapidly evaporates from wax.
INTRODUZIONE
L’acaro Varroa destructor Anderson & Trueman, agente della Varroosi delle api, rappresenta il più temibile parassita di
Apis mellifera L. ed il suo controllo in apiario impone il ricorso a trattamenti acaricidi, spesso mediante l’impiego di
sostanze di sintesi (Acrinathrine, Clorbenzialate, Chlorfenvinphos, Chlordimerform, Fenotiazine, Bromopropilate ,
Fluvalinate, Amitraz, Coumaphos, Cymiazole, Flumethrin, Tetradifon), con conseguenti problemi di farmaco-resistenza
e di residui nei prodotti dell’alveare, già segnalati in letteratura (Milani, 1999; Wallner, 1999).
Negli ultimi anni, si sta affermando, a livello mondiale, l’impiego di sostanze di origine naturale, ed in particolare di
alcuni acidi organici (acido formico, acido ossalico e acido lattico) e del timolo (Imdorf et al., 1999; Calderone, 1999;
Whittington et al., 2000).
Nel presente lavoro, è stata studiata l’efficacia del timolo nel controllo della Varroosi in un ambiente mediterraneo,
valutandone altresì i residui nel miele e nella cera e verificandone la persistenza nelle due diverse formulazioni
commerciali impiegate.
Materiali e Metodi
Prova in apiario
La prova è stata condotta in un apiario della Sardegna centro-meridionale (Oristano) nel periodo giugno-luglio del 2001.
Una postazione di 15 alveari con arnie Dadant-Blatt standard da 10 favi e colonie derivate da Apis mellifera ligustica
Spin., è stata ripartita in 3 gruppi di 5 alveari ciascuno, omogenei per consistenza ed infestazione, valutate,
rispettivamente, mediante rilievi preliminare sulla superficie di covata e tramite campionamento di api adulte (300 api
per alveare) e di covata opercolata (300 cellette per alveare ) (Floris et al., 2001).
Per il trattamento sono state utilizzate due formulazioni a base di timolo: Api Life VARâ e Apiguard. La prima,
confezionata in tavolette di vermiculite da circa 12 g contenenti circa il 74% di timolo (pari a circa 9 g di timolo per
tavoletta), il 3,7% di mentolo, il 3,7% di canfora e il 16% di eucaliptolo; mentre nella seconda formulazione il timolo
era incluso in gelatina nella percentuale del 25% e confezionato in vaschette contenenti 50 g di gel (12,5 g di timolo).
Un gruppo di alveari è stato trattato con Api Life VAR, uno con Apiguard ed il terzo è stato tenuto come testimone. Il
trattamento è stato eseguito il 21 Giugno 2001 impiegando rispettivamente due mezze tavolette di Api Life VAR ed una
vaschetta di timolo in gel per alveare. Un secondo trattamento è stato effettuato il 6 Luglio 2001.
79
Durante l’esperimento, per due volte la settimana, è stato eseguito il conteggio delle femmine di Varroa cadute sul
fondo dell’arnia. Alla fine della prova sono stati nuovamente eseguiti i rilievi, per valutare consistenza e livello di
infestazione degli alveari trattati e testimone.
Prima, durante e dopo il trattamento, sono stati inoltre eseguiti i prelievi di miele e di cera da ciascun alveare trattato e
testimone, nonché dei formulati dai soli alveari trattati, per valutare la persistenza e i residui del timolo.
I campioni di miele (~ 5 g per campione) sono stati prelevati da 100 cellette disopercolate scelte a caso da tre favi del
nido di ciascun alveare, aspirandolo con una siringa. Il campione di cera (~ 10 g) è stato ottenuto tagliando 3 porzioni di
circa 2 cm2 prelevati da diversi favi del nido di ciascun alveare. Infine, i prelievi delle formulazioni sono stati effettuati
asportando un frammento di gelatina o di vermiculite. I campioni sono stati conservati in freezer (-18°C) fino al
momento dell’analisi.
L’efficacia dei trattamenti è stata valutata impiegando come parametro la percentuale di mortalità M (Henderson e
Tilton, 1955) calcolata come:
é æ Bc × At öù
M = 100 × ê1 - ç
÷ú
ë è Bt × Ac øû
dove Bc e Bt rappresentano la percentuale di infestazione (degli adulti o della covata opercolata) prima del trattamento,
rispettivamente negli alveari testimone (Bc) e in quelli trattati (Bt); Ac e At rappresentano la percentuale di infestazione
dopo il trattamento, rispettivamente negli alveari testimone e in quelli trattati.
Reattivi
Gli standard analitici del Timolo (98%), Cineolo (99%), Mentolo (96%) e Canfora (96%) sono stati ottenuti dalla
Aldrich Chemical, Janssen (Geel, Belgium), Carlo Erba (Milano Italia), rispettivamente. Le soluzioni madre (1000
mg/Kg) sono state preparate in acetone (SupraSolv, Merck, Darmstadt, Germany). Le soluzioni di lavoro sono state
preparate giornalmente, per diluizione con estratti in dietil etere di miele e cera non trattati (controlli). Il dietil etere era
solvente per analisi (Merck, Darmstadt, Germany).
Procedure di estrazione
Formulati - Un g di ciascun formulato è stato pesato in un provettone da 50 ml con tappo a vite, venivano poi aggiunti
20 ml of metanolo (Chromanorm per HPLC, Prolabo) ed il protettone agitato (15 min) in un agitatore rotante e poi
sonificato per 5 min in un bagno ad ultrasuoni (Transsonic T460). Un’aliquota di metanolo (100 ml) veniva diluita a 5
ml con acetone ed iniettata in GC.
Miele - Un g di miele veniva pesato in un provettone da 15 ml con tappo a vite e veniva solubilizzato con 2 g d’acqua,
venivano poi aggiunti 2 ml di etere dietilico, ed il protettone agitato (10 min) in un agitatore rotante. Dopo separazione
delle fasi, un’aliquota dell’estratto era iniettata in GC.
Cera - Si pesavano 0,5 g di cera in un provettone da 25 ml con tappo a vite, si aggiungevano 5 ml di miscela
metanolo/acqua (1:1) ed il provettone veniva immerso in un bagnomaria a 70 °C fino a dissoluzione della cera, e quindi
agitato in vortex per 1 min. Dopo raffreddamento a temperatura ambiente, venivano aggiunti 5 ml di etere dietilico, ed il
provettone sottoposto ad agitazione per 15 min. in agitatore rotante. Dopo sepa-razione delle fasi, l’estratto etereo
veniva centrifugato ed un’aliquota iniettata in GC.
Recuperi
Campioni non trattati di miele e cera venivano fortificati con 0,1, 0,5, 2,0 e 5,0 mg/Kg of timolo, mentolo, cineolo e
canfora, ed analizzati secondo le metodiche descritte precedentemente. Le analisi sono state replicate quattro volte per
ogni livello di fortificazione. La media dei recuperi ottenuti per il miele era 97% (range 84-111%) con un coefficiente di
variazione massimo (CV) del 5,6%; e per la cera 89% (range 82-98%) con un coefficiente di variazione massimo (CV)
di 8,5%.
Gascromatografo
I campioni sono stati analizzati usando un gascromatografo HRGC 5160 Serie Mega equipaggiato con un detector FID
80 , un’autocampionatore AS 800, un’iniettore split-splitless (Carlo Erba, Milano, Italia), e collegato ad un integratore
HP 3396 A (Hewlett Packard, Avondale, PA, USA). La colonna era una capillare in silice fusa DB-5 MS (5% PhenylMethyl-Polysiloxane) 30 m x 0.25 mm id e 0.1mm di spessore del film (J&W Scientific, Folsom, CA). L’iniettore ed il
detector erano tenuti ad una temperatura di 100 °C e 200 °C, rispettivamente. Le analisi dei formulati venivano eseguite
iniettando 2 ml in modalità split, con rapporto di splitaggio di 1:10, mentre tutti gli altri campioni venivano analizzati
iniettando 2 ml in modalità splitless (30 s). La temperatura del forno era programmata come segue: 60 °C (5 min), salita
fino a 130 °C (2 °C/min) ed isoterma per 4 min, salita fino a 180 °C (10 °C/min). L’elio era il gas di trasporto e di
makeup a 1,8 e 30 ml/min, rispettivamente. La fiamma del FID era alimentata da aria ed idrogeno a 250 e 30 ml/min,
rispettivamente. Le rette di taratura venivano calcolate fra le altezze dei picchi e le concentrazioni utilizzando il metodo
80
dello standard esterno. E’ stata trovata una buona linearità nel range 0,1-5,0 mg/Kg con un coefficiente di correlazione
di 0,9994. Il cleanup non era necessario in quanto non vi erano picchi interferenti. Il limite di determinazione (Their and
Zeumer, 1987) era di 0,1 mg/Kg.
Analisi Statistica
I dati acquisiti durante la sperimentazione sono stati sottoposti ad analisi statistica (ANOVA) previa trasformazione
angolare (arcsin Öy/100), nel caso di valori percentuali, al fine di ridurre l’eterogeneità della varianza. Nelle tabelle e
nelle figure sono riportati I valori non trasformati. Quando I test F risultavano significativi, le medie sono state separate
applicando il test della Differenza Minima Significativa (LSD con a = 0,05).
RISULTATI E DISCUSSIONE
Efficacia dei trattamenti
La tabella I evidenzia come, prima dell’ inizio degli esperimenti, nessuna differenza statisticamente significativa fosse
riscontrabile fra i tre gruppi sperimentali, relativamente alla superficie di covata opercolata ed ai livelli di infestazione.
La tabella II riporta i valori per gli stessi parametri registrati al termine del trattamento.
Tab. I – Infestazione e consistenza degli alveari prima del trattamento.
Gruppo di Alveari
Infestazione covata
Infestazione api adulte
Superficie di covata
opercolata (%)
(%)
(cm2)
Api Life Var®
2,6 ± 1,2
3,1 ± 1,7
2.387 ± 1.071
Apiguard
2,6 ± 1,0
2,8 ± 1,0
2.425 ± 1.128
Testimone
2,8 ± 2,2
2,1 ± 0,9
2.368 ± 977
Le medie nella stessa colonna non sono significativamente differenti (ANOVA, P > 0,05)
Tab. II – Infestazione e consistenza degli alveari dopo il trattamento.
Gruppo di Alveari
Infestazione covata
Infestazione api adulte
Superficie di covata
opercolata (%)
(%)
(cm2)
Api Life Var®
0,7 ± 0,5 a
0,4 ± 0,5 a
902 ± 319 a
Apiguard
1,7 ± 0,6 a
1,5 ± 1,0 a
1.090 ± 263 a
Testimone
8,4 ± 4,8 b
8,0 ± 6,1 b
1.598 ± 282 b
Le medie nella stessa colonna seguite dalla stessa lettera non sono significativamente differenti (ANOVA, P > 0,05 LSD
test)
I livelli di infestazione, sia della covata sia degli adulti, sono diminuiti nei trattati ed aumentati nel testimone, mentre la
superficie di covata opercolata è diminuita in tutti e tre i gruppi anche se in misura maggiore nei trattati. Le differenze
riscontrate sono risultate statisticamente significative solo tra i trattati ed il testimone. Tali differenze, evidenziano un
effetto del timolo sulla consistenza delle colonie, con una flessione più marcata negli alveari trattati rispetto ai
testimoni.
L’efficacia, espressa in termini di percentuale di mortalità, nel gruppo trattato con Apiguard, è risultata pari a 90,4 ±
8,3% ed a 95,5 ± 8,7%, considerando, rispettivamente, il livello di infestazione della covata e degli adulti. Nel gruppo
trattato con Api Life VAR®, considerando i medesimi parametri nello stesso ordine, sono stati riscontrati valori pari a
74,8 ± 13,1% e di 81,3 ± 15,5%. Tali differenze non sono risultate statisticamente significative per effetto dell’elevata
variabilità rilevata all’interno di ogni gruppo, che evidenzia un’azione di tale composto volatile presumibilmente
condizionata da fattori biologici e microclimatici interni all’alveare.
L’andamento della caduta degli acari durante l’esperimento, è riportato nella Figura 1. La mortalità più elevata é stata
riscontrata nel gruppo trattato con Apiguard durante la prima settimana di trattamento e, in seguito alla sostituzione
delle vaschette, nella terza settimana.
81
700
600
Apiguard
Api Life Var
Controllo
N° di varroe
500
400
300
200
100
16
-1
9/
V
II
12
-1
6/
V
II
912
/V
II
58/
V
II
25/
V
II
28
/V
I2/
V
II
25
-2
8/
V
I
21
-2
5/
V
I
17
-2
1/
V
I
0
Fig. 1- Andamento della caduta degli acari
Persistenza e residui del timolo
a) Metodo analitico
La metodica messa a punto nel presente lavoro per l’estrazione del timolo è risultata estremamente rapida e non ha
richiesto alcun clean-up rispetto ai metodi riportati in precedenti lavori (Bogdanov et al. 1998, Martel e Zeggane, 2002;
Nozal et al. 2002), in quanto gli estratti non hanno evidenziato picchi interferenti. The average recovery for honey was
97% (range 84-111%) with a maximum coefficient of variation (CV) of 5.6%; and for wax it was 89% (range 82-98%)
with a maximum coefficient of variation (CV) of 8.5%. A good linearity was achieved in the 0.1-5.0 mg/Kg range with
a correlation coefficient of 0.9994. Cleanup was unnecessary because there were no interference peaks. The limit of
determination (Their and Zeumer, 1987) was 0,1 mg/Kg.
b) Residui
Inizialmente si è proceduto ad un controllo del titolo del timolo in entrambi i formulati. Secondo quanto riportato in
etichetta una tavoletta di Api Life Var® dovrebbe contenere il 2,52% di vermiculite ed il resto di oli essenziali; un
controllo da noi effettuato ha accertato la presenza di vermiculite in quantità superiore all’11% (11,4±0,64). A tale
risultato si è pervenuti pesando dopo essiccamento in stufa, quantità note di Api Life Var®, precedentemente sottoposte
all’estrazione degli oli essenziali secondo la metodica descritta. I titoli in timolo dell’Api Life Var e dell’Apiguard,
determinati gas cromatograficamente, sono risultati essere del 56% contro il 74% e del 18% contro il 25% dichiarati.
I campioni di Api Life Var ed Apiguard, prelevati durante l’esperimento, si presentavano “Propolizzati” (disgregati
dall’azione delle api).
I residui di timolo nel miele prelevato dalle arnie trattate con Api Life Var® variano notevolmente da alveare ad alveare
(Tab. III). Alla fine della prima settimana dal trattamento, la media dei residui di timolo nel miele, prelevato dalle 5
arnie prese in considerazione, è stata di 1,97 ± 1,54 mg/Kg; tale valore è diminuito nel corso della seconda settimana
fino a 0,75 ± 0,44 mg/Kg. A questo punto, sostituite le vecchie tavolette, i residui aumentano nuovamente nel corso
della terza settimana (1,05 ± 1,01 mg/Kg) per decadere nel corso dei sette giorni successivi fino al valore di 0,62 ± 0,57
mg/Kg. Da tale andamento si evince che il rilascio più abbondante del timolo nelle nuove tavolette viene in parte
trattenuto dal miele, dal quale tuttavia evapora in parte nell’arco della seconda settimana.
Nei campioni di miele prelevati dagli alveari trattati con Apiguard, possiamo notare la stessa grande variabilità di
concentrazione del residuo di timolo, con un’analoga decadenza della concentrazione tra prima e seconda e tra terza e
quarta settimana di trattamento (Tab. IV). I valori alla fine della prima e della terza settimana sono significativamente
più elevati rispetto a quelli riscontrati con l’uso di Api Life Var®. Ciò può essere messo in correlazione con la maggior
concentrazione del timolo nell’Apiguard rispetto all’ Api Life Var® e con le differenti modalità di erogazione di tale
composto.
Nel miele prelevato dagli alveari testimone, la concentrazione di timolo, quando presente, è molto modesta (0,11 ± 0,21
mg/Kg) (Tab. III).
82
Tab. III - Residui di timolo(mg/Kg) nel miele (Media±Dev.St.).
Formulazione
Prelievo a 7gg
Prelievo a 14gg
dal 1° tratt.
dal 1° tratt.
Api Life Var®
1,97±1,54
0,75±0,44
Apiguard
3,07±1,80
0,89±0,70
Prelievo a 7gg
dal 2° tratt.
1,05±1,01
2,55±3,50
Prelievo a 14gg
dal 2° tratt.
0,62±0,57
0,96±0,61
In generale, i residui nel miele si presentano altamente variabili come già riscontrato da Bogdanov et al. (1998). Tenuto
conto che si tratta di un composto molto volatile, la variabilità potrebbe essere in funzione delle differenti condizioni
microclimatiche che possono verificarsi negli alveari in rapporto alle diverse condizioni biologiche delle colonie.
I residui di timolo nei campioni di cera, prelevati prima e al termine della quarta settimana di trattamento, sono riportati
nelle tabella IV.
Tab.IV - Residui di timolo (mg/Kg) nella cera (Media±Dev.St.).
Formulazione
Prima del trattamento
Api Life Var®
n.d.
Apiguard
1,0±0,7
Alla fine del trattamento
21,6±13,0
147,7±188,9
Nei campioni di cera prelevati prima del trattamento dagli alveari testimone, la concentrazione del timolo, quando
presente, ha valori pari a 2,9 ± 4,6 mg/Kg, alla fine del trattamento i valori sono di 1,5 ± 0,8 mg/Kg. La medesima
natura apolare di timolo e cera comporta evidentemente una maggiore presenza del composto nella cera stessa piuttosto
che nel miele.
In definitiva, sulla base dei risultati acquisiti nel presente studio, si conferma la buona efficacia del timolo, in
riferimento alle due differenti formulazioni testate, nel controllo della Varroosi in apiario, con una riduzione percentuale
media dell’infestazione del 90 % e del 95% circa se riferita alla covata o alle api adulte. Questi risultati, comparati con
quelli ottenuti con altri acaricidi nello stesso ambiente (Floris et al., 2001; Floris et al., 2001), dimostrano la validità del
timolo, seppure con una modesta incidenza negativa del trattamento sullo sviluppo delle colonie, come mezzo
alternativo agli acaricidi di sintesi per il controllo della Varroa, con il vantaggio che, trattandosi di un composto di
origine naturale, non presenta alcun limite massimo di residuo nel miele (Imdorf et al., 1999; Wallner, 1999).
BIBLIOGRAFIA
Bogdanov S., Imdorf A., Kilchenmann V., 1998 - Residues in wax and honey after Apilife Var treatment. Apidologie
1998, 29, 513-524.
Calderone N.W., 1999 – Evaluation of formic acid and Thymol-based blend of natural products for fall control of
Varroa jacobsoni (Acari: Varroidae) in colonies of Apis mellifera (Hymenoptera: Apidae). J. Econ. Entomol.,
92(2): 253-260.
Floris I., Cabras P., Garau V.L., Minelli E. V., Satta A., Troullier J., 2001 - Persistence and Effectiveness of Pyretroids
in Plastic Strips Against Varroa Jacobsoni (Acari: Varroidae) and Mite Resistance in a Mediterranean Area. J.
Econ. Entomol. 2001, 94(4): 806 -810.
Floris I., Satta A., Garau V.L., Melis M., Cabras P., Aloul N., 2001 - Effectivness, persistence, and residue of amitraz
plastic strips in the apiary control of Varroa destructor. Apidologie 2001, 32, 557-585.
Henderson C. F., Tilton E. W., 1955 - Tests with acaricides against brown wheat mite. J. Econ. Entomol., 1995, 48,
157-161.
Imdorf A., Bogdanov S., Ibáñez Ochoa R., Calderone N.W., 2001 – Use of essential oils for the control of Varroa
jacobsoni Oud. In honey bee colonies. Apidologie, 30 (2-3): 209-228.
Martel A.C., Zeggane S. – Determination of acaricides in honey by high-performance liquid chromatography with
photodiode array detection. Journal of Chromatography A. 954(1-2):173-180, 2002 Apr 19.
Milani N., 1999 – The resistance of Varroa jacobsoni Oud. to acaricides. Apidologie, 30 (2-3): 229-234.
Nozal M.J., Bernal J.L., Jimenez J.J., Gonzales M.J. Higes M., 2002 – Extraction of thymol, eucalyptol, menthol, and
camphor residues from honey and beeswax – Determination by gas chromatography with flame ionization
detection. Journal of Chromatography A. 954(1-2):207-215, 2002 Apr 19.
Their H.P., Zeumer H., - 1987 – Manual of Pesticides Residues Analysis: VCH: Weinheim, Germany, Vol.1, pp. 37-74.
Wallner K., 1999 - Varroacides and their residues in bee products. Apidology, 1999, 30, 235 -248.
Whittington R., Winston M.L., Melathopoulos A.P., Higo H.A., 2000 – Evaluation of the botanical oils neem, thymol,
and canola sprayed to control Varroa jacobsoni Oud. (Acari: Varroidae) and Acarapis woodi (Acari:
Tarsonemidae) in colonies of honey bees (Apis mellifera L., Hymenoptera: Apidae). American Bee Journal, 140
(7): 567-572.
83
14
ASSESMENT OF PHYTOTOXICITY TO GRAPEVINE OF TRADITIONAL AND NEW COPPER
COMPOUNDS USED IN COPPER REDUCTION STRATEGIES IN ORGANIC VITICULTURE AND ITS
RELATIONSHIP WITH ENVIRONMENTAL CONDITIONS AND NUMBER OF TREATMENTS
Ilaria Pertot, Marco Delaiti, Diego Forti
Istituto Agrario di S. Michele all’Adige, via Mach 1, S. Michele all’Adige, (TN), Italy
[email protected]
Introduction
In organic viticulture Plasmopara viticola control is based almost exclusively on copper. The expected restrictions on
copper used in organic agriculture in the European Union have stimulated the research of alternatives and the
optimisation of copper allowed quantity. To compare efficacy against downy mildew of different copper compounds
several researches have been done, but phythotoxicity has received scant attention, particularly in relation to copper
reduction strategies in organic viticulture. Phytotoxicity risk must be taken into consideration during grapevine
phenological cycle, because copper compounds could affect the quality of grape and the vegetative balance of the plant.
Researches have shown that, using the same Cu2+ dosage, efficacy and rainfastness of different copper compounds and
formulations are the same (Pertot et al., 2002). Reduction in annual copper quantity will be possible with a rationale
use of dosages, that can vary between 30 to 70 g Cu2+/hl during the vegetative stage of the plant, depending on weather
conditions and infection risk evaluation. The rationale dosages used must be associated with a careful protection against
primary infections, the execution of treatments before forecasted rain and the increasing of frequency application in the
period of maximum plant growth.
Among new copper formulations, compounds and adjuvants only copper peptidate could allow a consistent reduction in
copper annual quantity, giving at the same time a adequate efficacy level against the disease (Pertot et al., l.c.).
The aim of this research was: i) the evaluation, with low dosages, of phytotoxicity to grapevine in field of new copper
formulations compared to the traditional ones; ii) the identification in controlled conditions of the factors that induce
phytotoxicity.
Materials and metods
The trials were done in the experimental organic vineyard of Istituto Agrario di S. Michele all’Adige, located in
Rovereto (TN, Italy). Chardonnay was the used cv. Test products, reference products and untreated control were
arranged in a suitable statistical design (fully randomised design, with treatments replicated four times). Products were
applied using a normal volume of 12 hl/ha, with a four times concentrated sprayer (tab.1). Phytotoxicity assessment was
done following EPPO standars, guideline PP1/135(2). For each repetition twenty five leaves were randomly collected
and classified using a scale depending on the symptoms and the percentage of leaf surface affected.
2001
active ingredient commercial name
bordeaux mixture P. Bordolese Dispers
copper hydroxide Kocide 2000
copper sulphate Cutril
cuprous oxide
Cobre Nordox
copper peptidate Peptiram 5
copper peptidate Peptiram 7
Cu2+ g/ha
360, 600, 840
300
150
2002
active ingredient
commercial name
bordeaux mixture P. Bordolese Dispers
copper hydroxide Heliocuivre
copper peptidate
Peptiram 7 (2 times)
copper peptidate
Peptiram 7 (3 times)
cuprous oxide
Cobre Nordox
copper oxychloride ICC0087
Cu2+ g/ha
360, 600
150
360, 600
Tab.1 Field treatments in 2001 and 2002
In greenhouse controlled conditions the influence of temperature (5, 15, 25, 35° C), leaf wetness and number of
treatments (1, 3, 5, 7) on phytotoxicity of copper peptidate (Peptiram 5) was evaluated. Potted plants, cv. Pinot gris,
were used. For each treatment were used five plants with two shoots each. Phytotoxicity evaluation was done using
three classes of intensity: low (from 0 to 0,5 % of leaf surface damaged), middle (from 0,6 to 5%) and high (from 5,1 to
10 %) and indicated as phytotoxicity index.
84
Results
In field trials, phytotoxicity (fig. 3) was related to particular environmental conditions (which were identified both in
2001 and 2002 in prolonged leaf wetness), but the degree of damage depended on the copper compound used. Copper
peptidates, followed by cuprous oxide, induced the highest phytotoxicity level (fig.1).
Fig. 1 field results in 2001 (left) and 2002 (right). Same letter means that results are not significantly different
(P=0.05).
2.5
2
d
c
1.5
b
1
0.5
a
a
a
phytotoxicity index (%)
2.5
2
d
1.5
bc
1
0.5
cd
b
a
a
0
0
e
ure
ide
ate
ate
ide
xid
ixt
tid
tid
lor
rox
so
p
p
h
m
d
u
e
e
c
p
p
x
hy
pro
xy
au
er
er
er
cu
ro
pp
pp
e
rde
pp
o
o
p
o
o
c
c
p
b
c
co
e
ate
ure
ide
ate
ate
xid
ixt
tid
tid
lph
rox
so
p
p
u
m
d
u
e
e
s
y
x
er
rp
rp
pro
rh
au
pe
pe
pp
cu
pe
p
p
o
rde
p
c
o
o
o
c
c
b
co
100
In controlled conditions, low temperature, but also high ones, if associated with leaf wetness, induced phytotoxic effects
(fig. 4), which were evident at cytological level before inducing macroscopic alterations on leaves. At cellular level
copper precipitate was visible. Chemical composition of precipitates was determined by X ray microanalysis.
Phytotoxicity increased with the increasing of the number of treatments.
80
60
40
20
0
5°C
15°C
25°C
35°C
100
0
80
1
60
3
40
5
20
7
0
5°C
temperature
15°C
25°C
35°C
temperature
Fig. 2 Phytotoxicity index (percentage of leaf infected) on plants treated with an increasing number of application at
different temperature. Effect on dry plants (left) and on wet plants (rightside).
85
Fig 3 – 4 Symptoms of phytotoxicity in field and in greenhouse.
Discussion and conclusion
When long leaf wetness periods combined with low or very high temperatures are frequent it is necessary to be very
careful in using copper compounds, as copper peptidate. Copper peptidate, which could be useful to reduce the annual
allowed copper amount per hectar, must be used only when grapevine is less sensible to copper phytotoxicity, avoiding
application until the end of flowering. Copper peptidate repeated treatments whithout periods of rains increase the risk
of phytotoxicity. In this case could be useful to reduce dosages in the following treatments.
Acknowledgements
This work was funded by the Italian Ministry of Agriculture (MiPAF), research project “La difesa delle colture in
agricoltura biologica”.
References
I. Pertot, M. Delaiti, E. Mescalchin, M. Zini, D. Forti (2002) Attività antiperonosporica di nuove formulazioni di
composti rameici utilizzati a dosi ridotte e prodotti alternativi al rame impiegabili in viticoltura biologica, Atti giornate
fitopatologiche 2002.
AA.VV. (1997) Guidelines for the efficacy evaluation of plant protection products, vol. 1-2 OEPP/EPPO, France.
86
15
ORGANIC WHEAT QUALITY AND PRODUCTION: THE RESULTS OF THREE YEARS OF TRIALS
Fabio Fusari, Antonella Petrini
The relevant increasing of farms and areas interested in organic productions in Italy draws attention to the need of an
experimentation which can help this sector for an equilibrate growth. CERMIS (Research and Experimental Centre for
Plant Improvement “N. Strampelli”), in collaboration with ASSAM (Agency for Food and Agriculture Services in
Marche region), has begun in 1998 an experimental project in the sector of autumn-vernal cereals in which the main
objectives are identifying the varieties to employ and optimizing the cultivation techniques. A correct varietal choice is
essential, in organic agriculture, in order to obtain acceptable productive and qualitative results. The morphological and
physiological characteristics which make cultivars suitable for this kind of cultivation are: rusticity, resistance or
tolerance to the main phytopathies, ability of competition with weeds and greater efficiency in the use of nitrogen. An
other aspect to consider, especially in the case of bread wheat, is the qualitative one, because the milling industry
requires distinct technological characteristics of the stocks according to the different kinds of product (bread, biscuits,
‘panettone’, etc.). Qualitative characteristics, intrinsic in a specific variety, are also affected by pedological, climatic
and agronomic factors; so the evaluation of each single variety within a cultivation system is fundamental, especially
when the external inputs are reduced as in the case of organic agriculture.
The activity within the project
Since from 1998 a parcel trial for the varietal comparison of bread wheat has been carried out by an organic farm
situated along the valley of Potenza river (Macerata, Italy) on a medially fertile, tendentially clayey, soil. The
cultivation techniques which have been adopted are the ones usually employed by that farm, admitted by the EC Reg.
2092/91. The main agronomic and qualitative attributes have been taken and statistically analysed in accordance with
the methodologies adopted by the wheat national network in Italy. The twenty bread wheat varieties that have been
evaluated have been chosen, between the ones registered to the national catalogue, on the basis of their agronomic,
merchandise (qualitative classes) and commercial characteristics. Also the old cultivar Abbondanza has been inserted in
the trial, in order to verify its behaviour in the case of an organic method of cultivation.
Table 1 - Production indexes (*) of the tested varieties
Graph 1 - Average yield (t/ha) of the 13 varieties
common to the three years of trials
YEAR
Variety name
1999
2000
2001
average
4,0
ABBONDANZA
108
101
134
114
EUREKA
101
109
135
115
3,5
SERIO
113
104
108
108
3,0
CENTAURO
109
111
97
105
2,5
BOLERO
93
110
94
99
2,0
COLFIORITO
107
92
90
96
SAGITTARIO
86
99
101
95
ENESCO
91
101
87
93
1,0
MIETI
109
95
63
89
0,5
PANDAS
103
113
36
84
0,0
SIBILLA
87
98
73
86
VALLEROSA
95
86
67
83
SANGIACOMO
98
81
61
80
ETECHO
157
157
GUADALUPE
140
140
TREMIE
128
128
TIBET
124
124
GENIO
118
118
BILANCIA
109
109
SIRMIONE
Field average (t/ha)
78
3,39
2,75
2,11
1,5
1999
2000
2001
Graph 2 - Mean alveographic parameters (W
and P/L) during the three years of trials
w
180
P/L
1,0
0,9
160
0,8
140
0,7
120
0,6
100
0,5
80
78
0,4
60
2,65
0,3
40
(*) 100= field average
0,2
20
0,1
0
0,0
1999
2000
2001
Results
From the analysis of mean results of the thirteen varieties common to the three years of trials, it’s evident that a
progressive loss of yields over the years (graph 1), due mainly to the anomalous meteorological trend during the two
last grain seasons, has taken place.
87
In particular the farming year 2000/2001 has been characterized by a mild and humid winter and by low temperatures in
April, which have damaged the early varieties and the cultivars that are susceptible to some fungal diseases, mainly
yellow and brown rust.
In the table 1 the production indexes of the varieties tried out over the three-year period 1998-2001 are summarized.
The cultivars which highlight the best performances are Abbondanza, Eureka and Serio which, in that period, have
always given indexes above average. Also the mean index of the variety Centauro is above 100, while the production
index of the cultivar Pandas decreases dramatically because of the chilling damages suffered during the grain season
2000/2001.
Among the varieties tested only during the farming year 2000/2001 the index of the cultivar Sirmione is below average,
while Etecho and Guadalupe are in evidence for their very high indexes, even if for these varieties it is necessary to wait
for further experimental evidences.
Qualitative characteristics
On a grain sample, mixture of the three repetitions, technological analyses (alveogram, falling number, protein and
gluten content) have been performed in order to establish the qualitative characteristics of the harvested product.
Examining the graph 2, where the mean data of the thirteen varieties common to the three years of trials are reported, it
can be observed that W values are basically low, whereas the ratio P/L indicates a tendential imbalance because of an
excess of tenacity. Probably this qualitative decay is due to an unfavourable seasonal trend both in 1998/1999 and in
2000/2001, and to a reduced nitrogen availability during the main stages of the vegetative cycle. Only in the grain
season 1999/2000 the quality appears medially sufficient, so that in this case most of the varieties can be set in the
respective qualitative classes according to the Synthetic Quality Index (ISQ) calculated on the basis of the protein
content and of the alveographic and farinographic characteristics.
Conclusions
In general the results which have been obtained demonstrate that organic agriculture gives the chance for satisfying
yields combined to a middle-low technological quality, anyhow sufficient for the production of biscuits and
breadmaking flours. The improvement of technological quality for the wheat cultivated with the organic methods
requires a careful varietal choice, and the use of techniques able to guarantee an adequate availability of assimilable
nitrogen in the soil.
*CERMIS – Centro Ricerche e Sperimentazione per il Miglioramento Vegetale “N. Strampelli”, Tolentino (MC) Email: [email protected]
88
16
IL CONTROLLO DELLA “ MOSCA DELLE OLIVE” (BACTROCERA OLEAE Gmel.) CON METODI
CONSENTITI IN COLTIVAZIONE BIOLOGICA
N. Iannotta
Istituto Sperimentale per l’Olivicoltura, Rende (CS)
Introduzione
Una cultura ecologista ed igienico-salutista recentemente instauratasi in Italia ed in altri Paesi europei e lo stimolo degli
incentivi comunitari, ha portato un notevole flusso di olivicoltori in direzione della coltivazione biologica. Le superfici
olivicole investite in questa forma di coltivazione si sono enormemente dilatate in questi ultimi anni, per effetto della
accresciuta domanda di prodotti “certificati” e per l’aumentato interesse economico verso un tipo di produzione in
grado di avvantaggiarsi dell’ulteriore valore aggiunto del “biologico”. Tale incremento di superfici investite nel
comparto olivicolo, già di per sé economicamente importante in Italia (soprattutto nel Mezzogiorno), ha interessato
nell’ultimo decennio diverse decine di migliaia di ettari e recenti indagini riferiscono che ben il 10% dell’olivicoltura
convenzionale ha convertito la propria produzione ai sistemi biologici.
La difesa fitosanitaria in coltura biologica, specialmente negli ambienti meridionali, si pone come un oggettivo limite
alla competitività economica dell’olivicoltura, soprattutto in considerazione della lotta alla “mosca delle olive”, il più
dannoso parassita dell’olivo, della quale non si può prescindere.
Come è ben noto, i prodotti fitosanitari impiegabili in biologico sono contenuti nell’allegato II B del Reg. CEE 2092/91,
ma le notizie reperibili in letteratura circa la loro efficacia, il loro impatto ambientale ed il loro impatto tossicologico
sulla qualità del prodotto sono alquanto scarse ed incomplete.
Da diversi anni l’Istituto Sperimentale per l’Olivicoltura ha avviato specifiche ricerche tendenti a verificare la reale
efficacia di alcuni prodotti compresi nella citata tabella, ed in qualche caso anche la presenza di residui sul prodotto. In
questa sede si illustrano le prove effettuate nell’ultimo biennio, concernenti il trappolaggio massale e la lotta al dittero
con principi attivi ammessi in coltivazione biologica.
Materiali e metodo
Le prove di mass-trapping si sono svolte negli anni 2000-2001 nel campo sperimentale dell’Istituto Sperimentale per
l’Olivicoltura in Rende (CS), dove la “mosca” è presente in maniera massiccia causando notevoli danni, costituito da
piante giovani (10-15 anni) di cultivar diverse. In questa ricerca è stato sperimentato il dispositivo bersaglio “Attract
and Kill” (Suneco s.r.l). Esso è stato applicato manualmente alle piante di olivo, in numero di 100 x Ha (33 con
bicarbonato d’ammonio e feromone; 67 con solo bicarbonato d’ammonio), in luglio dei due anni, e mantenuto fino alla
raccolta (fine ottobre). Il campo così predisposto (tesi A) è stato confrontato con un’altra parcella (tesi B) trattata con
metodo antidacico convenzionale (dimetoato al superamento della soglia di intervento), e con una parcella testimone
(tesi T), trattata solo con acqua. Nella parcella trattata convenzionalmente sono stati effettuati due trattamenti, uno ai
primi di settembre e l’altro ai primi giorni di ottobre, con dimetoato alla concentrazione di 60gr/hl di p.a.
In un’altra parcella della stessa azienda è stato effettuato un trattamento con rame (ossicloruro di rame allo 0,5%).
Le prove di lotta con rotenone e azadiractina sono state effettuate negli anni 2000-2001 nel campo sperimentale
dell’A.R.S.S.A. in Mirto-Crosia (CS). Ai fini della difesa fitosanitaria, il campo è da diversi anni trattato con metodi
compatibili con la coltivazione biologica. Esso è stato suddiviso in tre parcelle corrispondenti ad altrettante tesi: A,
trattata con rotenone (p.c. ROTENA) alla dose di 300 cc/hl, addizionato da 300 g/hl di olio bi anco e 50g/hl di bagnante;
B, trattata con azadiractina A (p.c. DIRACTIN) alla dose di 150 cc/hl, addizionata con 50g/hl di bagnante; C, trattata
con sola acqua. I trattamenti sono stati effettuati, alle prime ore del mattino, al superamento della soglia del 20% di
infestazione attiva. Essi sono stati effettuati il 25 settembre del 2000, unico trattamento dell’anno, ed il 7 settembre e 4
ottobre del 2001 (due trattamenti).
I rilievi, per tutte le tesi, sono stati effettuati a scadenza decadica (da luglio a fine ottobre), ed hanno riguardato: a)
andamento della popolazione adulta presente in campo, per ogni tesi, rilevato mediante monitoraggio con cartelle
cromotropiche; b) andamento dell’infestazione attiva, per ogni tesi, rilevato mediante campionamento a random ed
esame di 100 drupe.
89
Risultati
Nelle figg.1-2 sono riportati i dati (media nel biennio) riferiti all’andamento dei voli e dell’infestazione attiva nelle
diverse tesi della prova di mass-trapping.
25
Tesi A
20
Tesi A
Tesi B
%
Tesi T
Tesi B
15
Tesi T
10
5
Fig.1: Andamento della popolazione adulta (catture) nelle diverse
tesi.
25/10
15/10
4/10
24/9
13/9
3/9
23/8
13/8
2/8
25/10
4/10
15/10
24/9
3/9
13/9
23/8
2/8
13/8
22/7
12/7
0
2/7
n° catture
200
180
160
140
120
100
80
60
40
20
0
Fig.2: Andamento dell'infestazione attiva (uova, larve,pupe) nelle
diverse tesi.
Dalla fig. 1 si desume che la maggior presenza del fitofago si registra nella tesi testimone, dove ai rilievi del 15 e del 23
ottobre raggiunge rispettivamente il numero di 81 e 192 esemplari catturati in media per trappola. Nello stesso periodo
più contenute appaiono le catture delle rimanenti tesi, con valori fra loro non significativamente differenti. La fig. 2
mostra i risultati relativi all’infestazione attiva rilevata nelle diverse tesi fino al 25 ottobre, epoca ritenuta ottimale per la
raccolta nell’azienda dove si è svolta la prova. I dati relativi alle tesi trattate (A e B) evidenziano come l’andamento
dell’infestazione attiva, da settembre in poi, sia in queste parcelle inferiore rispetto al testimone. Nel confronto fra esse
si nota un migliore andamento della tesi A fino alla prima decade di ottobre; successivamente, a seguito del trattamento,
la curva della tesi B subisce una flessione riportando l’andamento dell’infestazione attiva inferiormente a quella della
tesi A.
Le figg. 3-4 mostrano i dati relativi al trattamento con il rame (tesi A), posti in confronto al trattamento convenzionale
con dimetoato (tesi B) e con il testimone (tesi T).
n°catture x trappola
20
200
180
160
140
120
100
80
60
40
20
0
Te si A
TesiA
Te si B
15
TesiB
T r a t t a me n t o
Te si T
Trattamento
TesiT
10
5
0
2/7
12/7
22/7
2/8
13/8
23/8
3/9
13/9
24/9
2/ 8
4/10 15/10 25/10
Fig.3: Andamentodellapopolazioneadulta(catture) nellediverse tesi
9/ 8
16/ 8
23/ 8
30/ 8
6/ 9
13/ 9
20/ 9
27/ 9
4/ 10
11/ 10 18/ 10 25/ 10
Fig.4. Andamento dell'infestazione attiva
(uova, larve, pupe) nelle diverse tesi.
La fig.3 evidenzia una efficacia dei trattamenti contro gli adulti, con effetti simili tra loro (rame e dimetoato), come
dimostrano gli andamenti delle catture inferiori nelle tesi trattate rispetto al testimone. Dall’esame della fig. 4 si desume
che la tesi trattata con ossicloruro di rame mostra un andamento inferiore al testimone e, per oltre un mese, anche
rispetto alla tesi trattata con dimetoato.
Nella fig.5 si rappresentano gli andamenti della popolazione adulta nel campo della prova con
impiego di rotenone e azadiractina.
Anno 2000
trattamento
100
Anno2001
80
120
70
trattamento
trattamento
60
Catture
60
40
50
40
30
20
20
10
0
Tesi A (Rotenone)
Tesi B (Azadiractina)
9/10
2/10
25/9
18/9
11/9
4/9
28/8
21/8
14/8
7/8
31/7
24/7
3/7
Tesi C (Controllo)
23/10
Tesi B (Azadiractina)
Fig.5: Andamento della popolazione adulta negli anni in osservazione.
16/10
Tesi A (Rotenone)
17/7
0
8/7 18/7 7/8 17/8 28/8 7/9 18/9 28/9 9/10 20/10 30/10 9/11 20/11 30/11
10/7
Catture
80
Tesi C (Controllo)
90
Nel primo anno si può notare un andamento caratterizzato da picchi di presenza di adulti notevoli, registrati nel mese di
agosto, probabilmente a causa delle favorevoli condizioni ambientali, ma dopo il trattamento è visibile l’effetto del
rotenone che ha ridotto la presenza in campo del dittero sia rispetto alla tesi controllo che rispetto alla tesi trattata con
azadiractina.
Nel secondo anno, con le diverse condizioni climatiche, i picchi di presenza si registrano in settembre ed ottobre,
epoche in cui sono stati effettuati i trattamenti. Questi ultimi hanno prodotto, per entrambi i prodotti saggiati, una
notevole riduzione delle catture nelle parcelle trattate.
La fig.6 mostra i risultati ottenuti nei due anni nelle diverse tesi, in relazione all’infestazione attiva. L’esame
complessivo dei dati indica l’efficacia dei trattamenti, sia relativamente al rotenone che all’azadiractina, nel
contenimento delle infestazioni daciche. Infatti, in entrambi gli anni in osservazione, i trattamenti hanno prodotto
sempre una riduzione dell’infestazione attiva contenendone i valori intorno al 20%, fino al momento della raccolta.
Nella tesi controllo, nelle stesse circostanze, i valori registrati sono risultati più alti, ben oltre il limite del 20%.
C arole a (2000)
100
80
60
% 40
20
0
C arole a (2001)
80
T rat t am en t o
40
T rat t am en t o
T rat t am en t o
22/9
%
23/8
60
20
0
T esi B (A zadiract in a)
T esi C (Co n t ro llo )
T esi C (Co n t ro llo )
22/10
T esi A (Ro t en o n e)
T esi B (A zadiract in a)
12/10
2/10
12/9
2/9
13/8
3/8
29/11
19/11
9/11
30/10
20/10
10/10
30/9
20/9
10/9
31/8
21/8
11/8
T esi A (Ro t en o n e)
Fig. 6: Andamento dell’infestazione attiva nel biennio 2000-2001
La tab.1 riporta i risultati delle analisi relativamente ai residui di rotenone riscontrati su olive e olio (comunicazione
personale del professor P. Cabras - Dipartimento di tossicologia dell’Università di Cagliari).
Tab.1: Residui di rotenone (mg/Kg±SD) riscontrati in olive e olio a diversi giorni dal trattamento.
Giorni dopo il trattamento
Olive
Olio
0
0.99 ± 0.04
—
2
0.52 ± 0.13
1.89 ± 0.18
5
0.44 ± 012
1.05 ± 0.12
9
0.19 ± 0.04
0.51 ± 0.05
12
0.11 ± 0.02
0.53 ± 0.18
Ricordando che il limite legale ammesso dalle normative vigenti è di 0.04 mg/Kg di residuo su olive a 10 giorni dal
trattamento, risulta evidente come tale limite sia stato sempre superato, anche dopo 12 giorni dal trattamento. Inoltre i
valori ottenuti su olio, ancorché privi di riferimenti legislativi, appaiono particolarmente elevati, indice di una tendenza
alla liposolubilità del rotenone.
Conclusioni
Relativamente al mass-trapping, dai risultati ottenuti nei rilievi effettuati (catture, infestazione attiva), appare evidente
l’azione di contenimento esercitato dal dispositivo bersaglio “Attract and Kill” contro la “mosca delle olive”. Inoltre,
ove si considerino anche i benefici effetti conseguibili con questo innovativo metodo (assenza di rischi tossicologici per
il prodotto, alta selettività di azione contro l’insetto e perciò con bassissimo impatto ambientale, economicità del
metodo per il ridotto numero di dispositivi impiegati rispetto al mass-trapping tradizionale) risulta ancor più evidente la
validità del suo impiego. Tuttavia la validità assoluta del metodo va verificata ed ulteriormente sperimentata anche in
altre realtà olivicole in diverse condizioni ambientali, e, inoltre, va considerata quale utile mezzo di integrazione con
altri sistemi di lotta “alternativa”, come ad esempio la raccolta effettuata nell’epoca ottimale.
Circa i principi attivi saggiati (rame, rotenone ed azadiractina), ammessi all’impiego nella coltivazione biologica, essi
hanno mostrato buona efficacia nel contenimento di B. oleae . Tale efficacia è apparsa evidente contro la popolazione
adulta, specialmente nel secondo anno di prove con due trattamenti, ma soprattutto contro la popolazione
91
preimmaginale. L’infestazione attiva, infatti, nelle tesi trattate e sulle diverse cultivar, si è sempre attestata su valori
compresi entro il 20%, limite ritenuto valido per l’ottenimento di un prodotto (olio) di qualità. Nel confronto tra
rotenone e azadiractina, il primo ha mostrato avere una maggiore, sia pur lieve, efficacia rispetto alla azadiractina.
Quest’ultimo prodotto, indicato come “fagoinibitore” e “regolatore di crescita”, ha in realtà funzionato come
“insetticida” , visto l’effetto sull’abbattimento della popolazione adulta e la mortalità delle larve riscontrata nell’esame
dei campioni di olive. Tuttavia nell’ipotesi di un loro più largo impiego in olivicoltura, destano notevole perplessità i
risultati delle analisi sui residui del prodotto, che per il rotenone figurano in misura eccessiva, tanto nelle olive quanto
nell’olio. Inoltre, non sono nemmeno noti gli effetti che questi prodotti provocano nell’impatto con l’ecosistema, per i
quali, vista la loro attività insetticida non selettiva, non è difficile immaginarne una incidenza negativa nei confronti
dell’artropodofauna utile, soprattutto per l’azadiractina che all’effetto insetticida unisce anche quello di fagoinibitore
(alterazione delle abitudini alimentari) e di bioregolatore (alterazione ormonale della crescita e della metamorfosi).
Occorrono ulteriori specifiche prove sperimentali per approfondire le conoscenze al riguardo e, ove si accertassero
negativi impatti ambientali e si confermassero rischiose presenze di residui nel prodotto, per questi principi attivi si
imporrebbe una revisione della tabella IIB del REG.CEE 2092/91.
Bibliografia
Bagnoli B., Petacchi R., 2001- Problematiche relative all’utilizzo di prodotti di origine vegetale per il controllo della
mosca delle olive. Pisa, aprile (in press).
Belcari A., Bobbi E., 1999 – L’impiego del rame nel controllo della mosca delle olive, Bactrocera oleae. Informatore
fitopatologico, 12: 52-53
Delrio G., Lentini A., 1993 – Applicazione della tecnica delle catture massali contro il Dacus oleae in due comprensori
olivicoli dalla Sardegna. Atti del Convegno “Olivicoltura”, Firenze 1991:41-45.
Haniotakis G., Kozyrakis M., Fitsakis T., Antonidaki A., 1991 – An effective mass trapping method for the control
of Dacus oleae. J. Econ. Entom. , 84
Iannotta N., Perri L., Rinaldi R., 1994 - Control of the olive-fly by mass-trapping in Calabria. Acta Horticulturae, n.
356: 411-413.
Iannotta N., Monardo D., Perri L., Tocci C., Zaffina F., 2000 - Esperienze di lotta alla Bactrocera oleae (Gmel.) con
sistemi conformi al Reg. CEE 2092/91. Atti Sem. “Metodi e sistemi innovativi dell’olivicoltura biologica e sostenibile”
, Rende (Cs) :123-126.
Iannotta N., 2001- La lotta naturale ai parassiti dell’olivo. Olivo e olio n° 5: 16-26.
Iannotta N., 2001- Esperienze di lotta contro Bactrocera oleae (Gmel) con metodi conformi al Reg. CEE 2092/91.
Relazione al convegno “L’olivicoltura biologica e la lotta contro la mosca delle olive”. Pisa, aprile (in press).
Iannotta N., Lombardo N., Maiolo B., Parlati M.V., Scazziota B., 2002 - Controllo di Bactrocera oleae (Gmel.) con
un prodotto fitosanitario naturale compatibile con la produzione biologica. Atti Conv. “Produzioni alimentari e qualità
della vita”, 4-8 Sett.2000-Sassari: 333-338.
Petacchi R., Rizzi I., Guidotti D., Toma M., 2001 – Informatizzazione della raccolta e gestione dei dati nei
programmi finalizzati al controllo della mosca dell’olivo: l’esperienza della Regione Toscana nella tecnica delle
“catture massali”, Pisa, aprile (in press).
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17
Il contenimento della ticchiolatura in frutticoltura biologica
Markus Kelderer1, Claudio Casera, Ewald Lardschneider
Centro Sperimentale Agrario Forestale Laimburg (BZ)
Spesso i produttori biologici sono obbligati dall’andamento del mercato a scegliere le stesse cultivar coltivate in
frutticoltura convenzionale ed integrata. La maggior parte di queste sono abbastanza sensibili alla ticchiolatura e
non è facile arrivare a fine stagione senza gravi danni. La ricerca condotta presso il Centro Sperimentale
Laimburg dimostra che è possibile controllare la ticchiolatura delle pomacee usando bassi dosaggi di rame,
polisulfuro di calcio ed argille acide in combinazione con lo zolfo. Il controllo è efficace se questi prodotti
vengono usati nel giusto modo.
I punti principali di questo lavoro sperimentale sono:
a)
b)
c)
d)
Confronto e riduzione del dosaggio di diverse sostanze attive alternative al rame
Tempi più appropriati di applicazione delle sostanze attive (preventivo, tempestivo, curativo)
Tecniche di applicazione (atomizzatore, sistemi d’irrigazione per aspersione)
Una particolare attenzione viene posta attualmente agli interventi sanitari per ridurre l’inoculo nel
frutteto
a) In cultivar molto sensibili alla ticchiolatura (es. Golden Delicious) l’efficacia delle miscele argille
acide/zolfo a volte non sono sufficienti, mentre il rame dimostra una eccellente efficacia anche a
dosaggi molto bassi (150g di rame metallico per trattamento ed ettaro), provocando però su certe varietà
problemi di rugginosità. Tra le formulazioni a base di zolfo, è da segnalare in particolar modo il
polisolfuro di calcio. Questo è abbastanza efficace e viene tollerato abbastanza bene anche dalla pianta.
Da quando questo prodotto è stato introdotto negli allegati 2b del regolamento comunitario 2092/91 la
maggior parte dei produttori usa questa sostanza attiva in dosaggi che variano da 10 a 30kg per
trattamento ed ettaro in funzione dell’andamento climatico durante la stagione.
b) Gli ultimi anni i produttori biologici hanno usato questi fungicidi soprattutto in modo preventivo. Per
ridurre il numero di trattamenti ed aumentarne l’efficacia, a partire del 1997 al Centro Sperimentale
Laimburg, sono stati sperimentati sia in laboratorio che in pieno campo, trattamenti tempestivi (scab
stop) su foglia bagnata e trattamenti curativi. I risultati dei trattamenti tempestivi su foglia bagnata sono
incoraggianti e i produttori stanno introducendo queste tecniche nei loro frutteti. Il tempo ideale per
effettuare questi trattamenti è abbastanza breve, perciò è necessario poter disporre di una tecnica di
applicazione veloce ed efficace.
c) Una possibile opzione nell’ambito delle tecniche di applicazione e l’uso del sistema di irrigazione per
aspersione. In diverse prove vennero confrontati metodi di applicazione con atomizzatori e sistemi di
irrigazione per aspersione. I due metodi di applicazione a parità di dosaggio e principio attivo ad ettaro
hanno fornito risultati confrontabili.
d) La quantità di inoculo presente è determinante per il grado di pressione della malattia nel frutteto.
Ridurre l’inoculo può diventare determinante per riuscire a controllare certe patologie. A partire dal
2001 sono in atto prove per determinare come possa essere ridotto l’inoculo nei frutteti e qual è l’effetto
di queste misure preventive sull’incidenza della malattia.
93
18
INFLUENCE OF DIFFERENT TREATMENTS ON Cd (II), Cu (II), Pb (II) AND Zn (II) CONTENT IN
SICILIAN OLIVE OILS
La Pera Lara, Lo Turco Vincenzo, Lo Curto Simona, Mavrogeni Ekaterini, Dugo Giacomo
Dipartimento di Chmica Organica e Biologica Università di Messina Salita Sperone, 31, 90166 S. AgataMessina
Metals can be found in fatty food as edible oils that are often subjected to refining, bleaching, and
deodorization, during which they inevitably come in contact to metallic surfaces (1). Trace levels of some metal
ions like Cu (II) could catalyse the oxidation reaction of fatty acid chains, with conseguent a deleterious effect on
oil flavor and shelf-life of oils (2).
Metals contamination of olive oil could be due to their presence in the soil, water and, particularly for
lead, in the air (3). Many studies have been carried out in which the presence of trace metals in olive oils was
correlated to agronomical techniques, harvesting methods, oil extraction and packaging procedures (4, 5). The
purpose of this paper was to explore the correlation between the pesticide treatments occurred in olive growing
and the content of metals found in oils.
The analytical techniques frequently used for the subsequent determinations are emission and absorption
spectrophotometric techniques as well as electroanalytical techniques (2,6).
In this paper derivative potentiometric stripping analysis (dPSA) was used to determine copper (II), lead
(II), cadmium (II) and zinc (II), in samples of virgin olive oils from Sicily (7-9).
Samples
Seven olive oils samples belonging to Nocellara del Belice variety, produced in the crop year 2000 -2001
from plants grown up on sandy soils in the zone of Valle del Belice (Trapani), were studied. Each of these
samples was originated from olive grove subjected to different pesticides treatments as dimethoate/fenthion,
dimethoate, copper oxy-chlorine/dimethoate, copper oxy-chlorine/fenthion; biological treatments as copper oxychlorine and biological fight and no treatment.
Oil samples were stored in dark glassy bottles at 4°C for the time of analysis.
Chemicals
Ultra pure hydrochloric acid (34-37%), Hg (II) (1000 mgmL-1, 1M in hydrochloric acid) and Cd (II), Cu
(II), Pb (II), Zn (II), Ga (III) and Pd (II) (1000 mg mL-1, 0.5 N in nitric acid) standard solutions were purchased
by Panreac Quimica (Barcelona, Spain). The extracts are filtrated on a carbon column Supelclean ENVI -Carb
SPE (0.5 g, 6 mL), purchased by Supelco (Bellefonte, PA, USA).
Apparatus
Determinations were carried out using a PSA ION3 potentiometric stripping analyzer (Steroglass, S.
Martino in Campo, Perugia, Italy), connected to an IBM-compatible personal computer. The analyzer operated
under the control of the NEOTES software package (Steroglass).
Electrode Preparation (plating) and potentiometric determination
Working electrode was a glassy carbon electrode coated with a Hg film, the reference one was an
Ag/AgCl electrode; the counter was a Pt electrode. The plating of the working electrode was effected putting in
the electrochemical cell a 20 mL volume of a 1000 mgmL-1 Hg standard solution and carrying out the
electrolysis, at –950 mV against the reference electrode, for 1 min.
The determination of Cd, Cu and Pb in oily extract was executed at pH 0.5, putting in the electrochemical
cell a 3 mL volume of elute, a 17 mL volume of ultra pure H2O and a 1 mL volume of Hg (II) standard solution
as oxidant agent. The determination of Zn was executed at pH 1.8, putting in the electrochemical cell a 1 mL
volume of elute, 18.5 mL of ultra pure H2O and 0.5 mL of Hg (II) standard solution as oxidant agent. The
quantitatve analysis was carried out by the multiple standard addition method. Metals determination was
managed under analytical conditions described in table 1.
RESULTS AND DISUSSION
Method
The repeatability of the method was attested at 86.4 % for cadmium, at 94.9% for copper, at 99.0% for
lead and at 98.9% for zinc. Recoveries were carried out by spiking a sample of olive oil at different levels.
94
Obtained recoveries percent were: 84.5±9.9% for Cd, 97.3±2.7% for Cu, 1 00.7±0.7 % for Pb and 83.4±1.7 % for
Zn. Instrumental detection limits were: 1.2 ngg-1 for Cd, 3.6 ngg-1 for Cu, 5.9 ngg-1 for Pb and 14.3 ngg-1 for Zn.
Application
Obtained results are reported on Table 1. Cd (II) was not found in any olive oil sample. The highest
content of Cu (II) was found in the sample originated from biological strategy (96.1±5.3 ng g-1). A lower
content of the metal was presented by the samples produced from plants treated with organic pesticides
associated or not to copper oxy-chlorine, such as copper oxy-chlorine/dimethoate (91.4±5.1 ng g -1) and copper
oxy-chlorine/fenthion (60.0±3.1 ng g-1). A smaller level of Cu was found in samples originated from
dimethoate/fenthion (56.8±3.3 ng g-1), dimethoate (38.3±0.4 ng g -1) and no treated sample (44.8±1.5 ng g -1).
The smallest Cu concentration was found in the oil originated from copper oxy-chlorine treatment (26.0±2.3 ng
g-1).
Oil samples produced from olive grove treated with organic pesticides - copper oxychlorine/fenthion
(222.6±2.3 ng g -1), dimethoate (179.2 ±1.9 ng g -1), dimethoate/fenthion (165.0±1.5 ng g-1) - showed a higher
lead amount respect to samples treated only with copper oxy-chlorine (47.5±0.5 ng g-1), no-treated sample
(34.6±0.4ng g-1) and the sample from biological strategy (17.3±0.2 ng g -1), which presented the lowest Pb
content (fig.1). An exception was represented by the sample treated with copper oxy-chlorine/dimethoate
(32.8±0.3 ng g -1).
Samples produced from olive groves treated with copper oxy-chlorine/fenthion (576.0±6.3 ng g-1)
presented the highest content of Zn, followed by biological strategy (550.0±5.8 ng g -1) and copper oxychlorine/dimethoato (433.2±4.7 ng g -1) samples; the oils treated with dimethoate (176.0 ±1.9 ng g -1), copper
oxy-chlorine (161.5±1.7 ng g -1), dimethoate/fenthion (140.8±1.5
ng g -1), showed a Zn level remarkably
lower. The smallest concentration of the metal was found in no-treated sample (59.8±0.7 ng g -1).
CONCLUSION
Samples produced from no treated olive grove and by plants treated only with copper oxy-chlorine,
always showed a lower content of Cu (II), Pb (II) and Zn (II) respect to samples originated from olive grove
treated with the organophosphorated pesticides dimethoate and fenthion. These molecules, which contain S, P
and O atoms, may influence the mechanism of metals uptake of the plants by complexing the cations (10).
Samples originated from biological fight always present a high amount of the micronutrient Cu (II) and Zn (II)
and a very low concentration of Pb (II).
LITERATURE
1.
Martín-Polvillo M., Albi T. and Guinda A. Determination of trace elements in edible vegetable
oils by atomic absorption spectrophotometry. J. Am. Oil Chem. Soc. 1994, 71, 347 -350.
2.
Calapaj R., Chiricosta S., Saija G. and Bruno E. Method for the determination of heavy metals
in vegetable oils by graphite furnace atomic absorption. At. Spectrosc. 1988, 9, 107-110.
3.
Paoletti R., Nicosia S., Clementi F., Fumagalli G. 1st ed. “Tossicologia degli alimenti”, 1999,
Utet, Torino.
4.
De Leonardis A., Macciola V. and De Felice M. 1997. La determinazione del ferro e del rame
negli oli vergini d'oliva mediante spettrofotometria ad assorbimento atomico. Riv. Ital. Sostanze Grasse 74:
149.
5.
Garrido M.D., Frías I., Díaz C. and Hardisson A. Concentrations of metals in vegetable edible
oils. Food Chem. 1994, 50, 237-240.
6.
Wahdat F., Hinkel S. and Neeb R. Direct inverse voltammetric determination of Pb, Cu and Cd
in some edible oils after solubilization. Fresenius' Z. Anal. Chem. 1995, 352, 393-396.
7.
La Pera L., Lo Curto S., Dugo G.mo, Lo Coco F. and Liberatori A. Determination of Copper
(II), lead (II), cadmium (II) and zinc (II) in olive oils from Sicily by derivative potentiometric stripping
analysis (dPSA). Presented at “European Conference of Advanced Technology for Safe and High Quality of
Food”, Berlin, 5-7 December, 2001.
8.
La Pera L., Lo Curto S., Visco A., La Torre L. and Dugo G.mo Derivative potentiometric
stripping analysis (dPSA) used for the determination of cadmium, copper, lead and zinc in Sicilian olive
oils. J. Agric. Food Chem. 2002, 95, 3090 -3093.
9.
La Pera, L.; Lo Coco, F.; Mavrogeni, E; Giuffrida, D.; Dugo, G.mo. Determination of Copper
(II), Lead (II), Cadmium (II) and Zinc (II) in virgin olive oils produced in Sicily and Apulia by Derivative
Potentiometric Stripping Analysis. Ital. J. Food Science 2002 in press.
10. Kaim W.; Schwedersky B. Bioinorganic Chemistry: inorganic elements in the chemistry of
life. Vth ed. Wiley & Sons Ltd, West Sussex, England, 1996.
95
Table 1. Analytical conditions for determination of Pb, Cu, Cd and Zn.
Pb
Cu
Integration range
mV
-580; -380
-380; -140
mV
-1200; -100
-1200; -100
Potential range
mV
50 x 5 s
50 x 5 s
Conditioning potential
mV
-1200
-1200
Accumulation potential
min
2
2
Accumulation time
s
10
10
Stripping time
mV
0
0
Acquisition final potential
300
300
Sampling time
ms
mV
-430
-260
Discharge potential
turn/s 2
2
Agitation speed
n.
4
4
Cycles
n.
2
2
Standard additions
Cd
-760; -590
-1200; -100
50 x 5 s
-1200
2
10
0
300
-640
2
4
2
Zn
-1100; -750
-1200; -100
50 x 5 s
-1200
2
10
0
300
-950
2
4
2
Table 2. concentration of Cd, Cu, Pb and Zn in olive oils from different pesticides treatments
Treatments
Biological fight
Cd
(ng g-1)
<1.2
Cu
(ng g-1)
96.1±5.3
Pb
(ng g-1)
17.3±0.2
Zn
(ng g-1)
550.0±5.8
no-treatments
<1.2
44.8±1.5
34.6±0.4
59.8±0.7
Copper oxychlorine
<1.2
26.0±2.3
47.5±0.5
161.5±1.7
Copper oxychlorine fenthion
Copper oxychlorine dimetoate
Dimethoate
<1.2
<1.2
<1.2
60.0±3.1
91.4±5.1
38.3±0.4
222.6±2.3
32.8±0.3
179.2±1.9
576.0±6.3
433.2±4.7
176.0±1.9
Dimethoate
fenthion
<1.2
56.8±3.3
165.0±1.5
140.8±1.5
96
250,00
200,00
[Pb] (ng/g)
Copper-Fenthion
Dimethoate-Fenthion
150,00
Dimethoate
No treatments
100,00
Copper-Dimethoate
Copper
50,00
Biological fight
0,00
Figure 1.Pb content found in olive oils from different treatments
97
19
DETERMINATION OF PHENOLIC COMPOUNDS IN EXPERIMENTAL WINES SUBJECTED TO
DIFFERENT PESTICIDES TREATMENTS
G. L .La Torre, T. M. Pellicanò, D.Pollicino, M. Alfa, G.mo Dugo
Università degli Studi di Messina; Dip.to di Chimica Organica e Biolog ica
INTRODUCTION
Wine is a complex fluid; it contains water, sugars, acids, alcohols and a wide range of phenolic compounds. The
phenolics can be derived from grapes and wood, or they can be metabolites from yeasts. Phenolics occur
naturally in various families of plants, but grapes and related products are considered one of the most important
dietary sources of these substances (1, 2). Phenolic compounds are an important group of substances that
contribute to several sensorial characteristics such as colour, flavour, astringency and hardness of wine.
Furthermore, these compounds are important in food hygiene because of their bactericidal effects and,
consequently, they constitute an interesting index for evaluating the quality of a wine (3 - 5). Some researchers
suggest that a regular consumption of a moderate quantity of wine, especially red (6-8), is therapeutic for human
health. This protective effect has been associated with an increase in the plasma of the level of high-density
lipoprotein (HDL)-cholesterol and with a decrease of platelet activity, because of the effect of ethanol and
polyphenolic components (9). Epidemiological studies have shown that a moderate wine consumption helps to
prevent the development of coronary heart disease (10,11).
The major molecules responsibles for this physiologic protection are phenolic compounds such as resveratrol
and flavonoids. Moreover, it has been reported that resveratrol, quercetin and other polyphenolics are cancer
chemo-preventive agents, since through the inhibition of cyclooxygenase they restrain cellular events associated
with tumour initiation, promotion and progression, and stimulate quinone reductase – an enzyme that detoxifies
carcinogens (12,13). In recent years, interest has been focused on resveratrol (3,5,4’-trihydroxystilbene), a
phytoalexin produced by the plant as a defence response to some exogenous stimuli, such as ultraviolet radiation,
chemical stressor and, particularly, microbial infections (15). It is well-known that resveratrol is present in the
chemical form of cis- and trans-resveratrol and of cis- and trans-piceid ( b-glucosides of resveratrol) (16). The
presence of resveratrol has been detected in numerous types of wines, originating from various countries (USA,
France, Italy, Spain, Japan), and large variations in its content are reported. They depend on grape cultivar,
geographic origin, wine type, Botrytis infection and enological practices. Generally, many authors found that
higher contents of trans-resveratrol (0.2 - 13 mg/L) are usually present in red wines that have had a prolonged
contact between the must and skins, whereas lower concentrations (0.1 - 0.8 mg/L) are usually present in white
wine (17, 18). In rosé wines the levels of resveratrol monomers were between white and red wines (18). About
b-glucosides of resveratrol, no proportionality between the levels of cis- and trans-resveratrol and their
glucosides were reported (19), whereas piceid concentrations from 0.3 mg/L to 9 mg/L in red wines and from 0.1
mg/L to 2.2 mg/L in white wines have been detected (14). Studies on resveratrol content in wines are numerous
(20-24); they are often focussed on the different grape cultivars and associated with the identification of
polyphenolic compounds, of potential biological activities, such as quercetin, myricetin, etc. The phenolic
compounds are secondary plant metabolites that are contained within the skin, seed, and flesh of grapes and are
extracted into wines (especially red) during the process of vinification. The types and concentrations of the
phenolic compounds in wine may depend on a number of factors: grape variety and ripening stage, soil and
climatic conditions. Moreover, the processes of viticulture and vinification, which vary between countries,
regions and wine-makers, determine the content and profile of phenolic compounds in wine (14). The
concentration of phenolic compounds is marked by temperature, so places with high temperatures and drier
conditions disfavour the presence of these compounds in wines.(16)
The purpose of this study was to determine the polyphenolic compounds (resveratrol, piceid, and flavonoids) in
wines from three different Italian regions, obtained from grapes (Sangiovese, Fiano d’Avellino, Inzolia Caricante (50/50)) treated with different pesticides and to investigate the probable influence of the different
treatments on the quality of the wines.
Chemicals and materials
trans-Resveratrol was purchased from Sigma Chemical Co. Stock solution of cis-resveratrol was produced by UV
irradiation of trans-resveratrol in methanol for 30 min at 254 nm. Solid-phase extraction (SPE) cartridges were
98
Supelco Supelclean LC-18 SPE tubes (3ml). The other phenolic compoundswere quercetin, rutin (quercetin-3-Orutinoside), kaempferol, kaempferol-3-glucoside, kaempferol-3-rutinoside, rhamnetin, isorhamnetin, and were
also supplied by Sigma.
Wine Analysed
The analyses were carried out on 23 Italian wine samples (red and white), coming from three different Italian
vineyards: one located in Tuscany, one in Campania and one in Sicily. In particular, wines from Tuscany were
produced from 30 years old plants (vines were grafted with Sangiovese variety, Morellino clone) and each sample
was vinified red. Wines from Campania were produced from 20 years old plants (vines were grafted with Fiano
d’Avellino variety) and each sample was vinified white. Sicilian wines were produced from 15-20 years old
plants (vines were grafted with Inzolia and Carricante variety in ratio 1:1) and each sample was vinified white.
The vineyard was subdivided in thesis; each thesis consisted of four rows of vines and the remotest was located
with the function to prevent drift effect. The fungicides treatments were carried out at the end of blossoming and
maturation. The scheme of experimental work provided the treatment of each thesis with different antioidics and
antiperonosporics: sulphur, azoxystrobin, dinocap, penconazolo, fenarimol, quinoxyfen (tab.1).
Analysis of cis- and trans-resveratrolo
cis- and trans-resveratrol were extracted from wines using the SPE cartridges prior to the HPLC analysis. For the
analysis of total wine resveratrol was used an enzymatic hydrolysis method, improved by our working group (25).
The wine resveratrol O-glycosides (trans- and cis-piceid) were totally hydrolysed in ~ 9 hours after incubation
with b-glucosidase at 50°C; then the trans- and cis-aglycones were measured by HPLC after solid phase
extraction (SPE). An ODS Hypersil 5mm (250x4,6 mm i.d.) column was used as the stationary phase and was
preceded by a precolumn of the same material. The mobile phase consisted of phase (A) water/acetic acid (pH =
3) and phase (B) acetonitrile/acetic acid (pH = 3). cis- and trans-resveratrol were eluted with a gradient time
program and the eluted was monitored at 285 and 307 nm, where cis- and transisomers have absorbance
maxima, respectively. All chromatographic experiments were performed at room temperature with a flow rate
1ml/min. The chromatograms were recorded according to the retention time. Before the HPLC analysis the
extract were filtered through a 0,45mm glass-microfiber GMF Whatman chromatographic filter.
HPLC analysis methodology for flavonoids
The analysis were carried out on aliquot of wine filtered through a 0,45 mm nylon Supelco chromatographic filter,
using an HPLC system operating at room temperature with a flow–rate of 0,8 ml/min.. The spectrophotometric
detector was a photodiode array operating at wavelengths of 265 nm for aglycon-flavonoids and 254 for
flavonoid-glucoside. The analytical column was a Alltech Alltime C-18, 5 mm ( 250 x 4,6 mm i.d.). A SUPELCO
guard column packed with the same stationary phase was also used. The mobile phase consisted of
H2O/CH3COOH at pH= 3 (phase A) and CH3CN/CH3COOH at pH = 3 (phase B). Elution was carried out in
gradient.
Analytical data related to the free isomeric forms of resveratrol (cis- and trans-) and to the total resveratrol (free
and glucosides isomers) are reported in fig. 1,2 and 3. This investigation, once again, clearly shows that red wines
have a greater resveratrol content than white wines, regardless of the pesticide treatments of the vines. Analysing
the data related to wines from Campania, it can be noticed that in the samples Thesis 3, 4, 5 and 6 the amount of
cis-resveratrol is between 0.04 and 0.05 mg/L, while the samples Thesis 1, Thesis 2 and Thesis 7 have an amount
of cis-resveratrol impossible to verify with the adopted analytic method. The concentration of trans-resveratrol
results highest in the sample Thesis 5 (0.13 mg/L), and a lowest in the sample Thesis 3 (0.07 mg/L). The total
concentration of cis- and trans-resveratrol deserve a particular attention because, generally, it is similar in every
sample while, this balance does not occur in free form of cis- and trans-resveratrol. In fact, the concentration of
free trans-resveratrol is a little lower than that of total trans-resveratrol, but in sample Thesis 2 it is even almost
identical; the same situation does not occur in cis-resveratrol. Examining the concentration of free cis-resveratrol
to that of total, it results that the amount of cis-piceid is much higher than that of trans-piceid, regardless of the
pesticides treatment. A completely different situation is shown for wines produced and vinified in Sicily. In this
case, the concentration of total trans-resveratrol (between 0.17 and 0.34 mg/L) is always higher than that of total
cis-resveratrol (between 0.09 and 0.20 mg/L). Moreover, differently from what was noticed in wines produced
and vinified in Campania, the cis- and trans-piceid concentration in each sample is comparable, and is between a
minimum value of 0.03 mg/L and a maximum of 0.18 mg/L. The samples Thesis 4 and Thesis 5 differ from this
general trend, as they are characterized by an amount of cis-piceid clearly higher than that of trans-isomer. As
shown in fig.3, the wines obtained from cv Sangiovese have very different quantities of free trans-resveratrol and
they range from 0.22 to 1.76 mg/L. The same trend can be noticed with free cis-resveratrol, but in this case the
concentrations are more homogeneous. Comparing total cis- and trans-resveratrol concentrations to those of the
equivalent free isomers, it emerges that the levels of trans-piceid are approximately twice as high as those of the
corresponding cis-piceid, except for samples Thesis 4 and Thesis 5 that present comparable levels. The
99
investigation of the flavonoids regarded the identification and quantification of seven components (quercetin,
rutin, kaempferol, kaempferol-3-glucoside, kaempferol-3-rutinoside, rhamnetin, isorhamnetin) through a fast
method that involves the use of wine as it is, as suggested by Viñas et al. (14).
The wine samples from Campania are characterized by a higher content of flavonoids and flavonoid-glucosides
and show a composition, both qualitatively and quantitatively, similar. Of the seven compounds analysed in the
flavonoids group, quercetin (<0.52-7.51 mg/L), rutin (2.72-2.90 mg/L), kaempferol (<0.25-4.29 mg/L),
kaempferol-3-glucoside (2.38-2.56 mg/L), kaempferol-3-rutinoside (<0.64 mg/L), rhamnetin (9.98-10.06 mg/L),
isorhamnetin (<0.24-4.20 mg/L) rhamnetin presented the highest average content in all the samples. The sample
Thesis 4, however, differ for the quercetin content, as shown in Table 3; moreover, the kaempferol is not
detectable in samples Thesis 1 and Thesis 2, so as isorhamnetin in sample Thesis 1. A similar trend, both
qualitative and quantitative, can be seen in samples of Sicilian wines, as shown in Table 4. In comparison with the
wines from Campania, the Sicilian wines have a higher content in quercetin. The wines from Tuscany deserve
particular attention. They present only rutin, quercetin, kaempferol-3-glucoside, kaempferol-3-rutinoside and each
one of them shows a great distribution of the above-mentioned components, as evidenced in Table 5, where the
samples Thesis 1, 4, 6 and 7 are particularly interesting because they present the content in
kaempferol-3-glucoside which is not detectable with this analytic method. Furthermore, the sample Thesis 2 has a
high content in quercetin together with a very low content in kaempferol-3-rutinoside. Analysing the red and
white wines it appears that the phenolic content in these samples varies with vinification approach. The results
show that the red wines contain high concentration of phenolics with a variation from 1608 to 2111 mg/L. A
comparison between wines from Sicily and Campania indicates that the total phenol content in the first samples
has an averaged value of 351.6 mg/L, while for the second one this value is 304.3 mg/L.
CONCLUSIONS
The present study clearly attests that the white wines, regardless of the pesticide treatment originally supplied to
the vine, show levels of resveratrol lower than the red wines. Moreover, these values are comparable with data
reported in literature for wines obtained with white vinification. The studied samples show different
concentrations of resveratrol content, which are due not only to vinification process (red or white), but also to
pesticide treatments. Resveratrol concentrations evaluated for these three experimentations present a slightly
homogeneous distribution and values are not easily correlated to different pesticide treatments. Obviously
different factors such as climatic conditions, different exposure of vineyards to atmospheric agents and different
agronomical management can produce variations in wine composition. The wines produced from grapes treated
with Quinoxyfen (Thesis 1) show, however, a total phenol content and an antioxidant activity higher than all the
samples examined. Moreover, the studied wines sometimes present a cis-/trans- resveratrol ratio inverted to the
one of correspondent piceid, that is cis-piceid higher than trans-isomer. Nonetheless, as experimentally verified,
this result is not due to the preparation techniques for analysing samples. This is quite peculiar, as the transisomers are usually more stable than the correspondent cis-isomers.
LITERATURE CITED
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Shahidi, F.; Naczk, M. In Phenolic – Sources, Chemistry, Effects, Applications; Technomics,
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Stacchini, A.; Draisci, R.; Lucentini, L. Il vino rosso diminuisce il rischio d’infarto? Commento ai dati
recenti della letteratura. L’Enotecnico, 1994, 3, 69-72.
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Ruf, J. C. Wine and polyphenols related to platelet aggregation and atherothrombosis. Drugs Experiments
and Clinical Research 1999, 25, 125-131.
10. Renaud, S.; De Lorgelil, M. Wine, alcohol, platelets and French paradox for coronary heart disease. Lancet
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11. Gronbaek, M.; Deis, A.; Sorensen, T.; Becker, U.; Schnohr, P.; Jensen, G. Mortality associated with
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20.
21.
22.
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24.
25.
Agullo, G.; Gamet, L.; Besson, C.; Domigne, C.; Remesy, C. Quercetin exerts a preferential cytotoxic
effect on active dividing colon carcinoma HT29 and Caco-2-cells. Cancer Letter 1994, 87, 55-63.
Jang, M.; Pezzuto, J. M. Cancer chemopreventive activity of resveratrol. Drugs Experiments and Clinical
Research 1999, 213, 65-77.
Viñas, P.; Lòpez-erroz, C.; Marin-Hernàndez, J. J.; Hernàndez-Cordoba, M. Determination of phenols in
wines by liquid chromatography with photodiode array and fluorescence detections. J. Chrom. A 2000,
871, 85-93.
Langcake, P.; Pryce, R. J. The production of resveratrol by “Vitis Vinifera” and other members of the
“Vitacee” as a response to infection or injury. Physiology and Plant Pathology 1976, 9, 77-86.
Goldberg, D. M.; Yan, J.; Ng, E.; Diamandis, E. P.; Karumanchiri, A.; Soleas, G.; Waterhouse, A. L. A
global survey of trans-resveratrol concentrations in commercial wines. Am J. Enol. Vitic. 1995, 46 (2),
159-165.
Dugo, G.mo; Bambara, G.; Salvo, F.; Saitta, M.; Lo Curto, S. Contenuto di resveratrolo in vini rossi da
uve alloctone e autoctone prodotte in Sicilia. L’enologo 2000, 12, 79-84.
Romero-Pérez, A. I.; Lamuela-Raventòs, R.; Waterhouse, A. L. ; De La Torre-Boronat, M. C. Level of cisand trans-Resveratrol and their glucosides in white and rosè Vitis Vinifera Wines from Spain. J. Agric.
Food Chem. 1996, 44, 2124-2128.
Waterhouse, A. L.; Lamuela-Raventòs, R. The occurrence of piceid, a stilbene glucoside in grape berries.
Phytochemistry. 1994, 12, 571-573.
Vrhovsek, U.; Wendelin, S.; Eder, R. Effectes of various vinification techniques on the concentration of
cis- and trans-resveratrol and resveratrol glucoside isomers in wine. Am.J.Enol.Vitic. 1997, 48 (2), 214219.
Nevado, J. J. B.; Salcedo, A. M. C.; Penavo, G. C. Simultaneous determination of cis-and trans-resveratrol
in wines by capillary zone electrophoresis. Analyst 1999, 124, 61-66.
Sato, M.; Suzuki, Y.; Okutsuka, K. Contents of resveratrol, piceid, and their isomers in commercially
available wines made from grapes cultivated in Japan. Biosci., Biotecnol., Biochem. 1997, 61 (11), 18001805.
Chu, Q.; O’Dwyer, M.; Zeele M. G. Directed analysis of resveratrol in wine by micellar electrokinetic
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Merillon, J. M. Determination of stilbenes (trans-Astringin, cis- and trans-Piceid, and cis- and transResveratrol) in Portuguese Wines. J. Agric. Food Chem. 1999, 47, 2666-2670.
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analysis of resveratrol glucosides in wine. Anal.Chim.Acta (Submitted).
101
Table 1 Scheme of experimental treatment of each wine sample with antioidium and antiperonospora
Wine Sample
Campania
Thesis 1
Thesis 2
Thesis 3
Thesis 4
Thesis 5
Thesis 6
Thesis 7
Sicily
Thesis 1
Thesis 2
Thesis 3
Thesis 4
Thesis 5
Thesis 6
Thesis 7
Tuscany
Thesis 1
Thesis 2
Thesis 3
Thesis 4
Thesis 5
Thesis 6
Thesis 7
Pesticides
n° treatments
Pesticides
n° treatments
Powdered sulphur
Powdered sulphur
Powdered sulphur
Dinocap
Wettable sulphur
Wettable sulphur
Powdered sulphur
2
2
2
2
2
2
2
Quinoxyfen
Fenarimol
Azoxystrobin
Penconazole
Wettable sulphur
Dinocap
Powdered sulphur
6
6
6
6
6
6
6
Powdered sulphur
Powdered sulphur
Powdered sulphur
Dinocap
Wettable sulphur
Wettable sulphur
Powdered sulphur
2
2
2
2
2
2
2
Quinoxyfen
Fenarimol
Azoxystrobin
Penconazole
Wettable sulphur
Dinocap
Powdered sulphur
6
6
6
6
6
6
6
Powdered sulphur
Powdered sulphur
Powdered sulphur
Dinocap
Wettable sulphur
Dinocap
Powdered sulphur
2
2
2
2
2
2
2
Quinoxyfen
Fenarimol
Azoxystrobin
Penconazole
Wettable sulphur
Sulphur + Quinoxyfen
Powdered sulphur
6
6
6
6
6
6+3
6
Table 2 - Detection limits for the analyses of flavonoids
Wine
Sample
Rutin
Kaempferol- KaempferolKaempferol Quercetin Isorhamnetin
3-Rutinoside 3-Glucoside
Thesis 1
2.72
< 0.64
2.54
< 0.25
7.52
4.16
10.02
Thesis 2
2.79
< 0.64
2.58
< 0.25
7.52
4.16
10.02
Thesis 3
2.72
< 0.64
2.57
< 0.25
7.58
4.17
10.03
Thesis 4
2.72
< 0.64
2.56
< 0.25
7.59
4.17
10.02
Thesis 5
2.72
< 0.64
2.57
< 0.25
7.53
4.17
10.02
Thesis 6
2.72
< 0.64
2.56
< 0.25
7.52
4.16
10.02
Thesis 7
2.72
< 0.64
2.46
< 0.25
7.51
4.16
10.03
Rhamnetin
102
Tab. 3 Flavonol content (mg/ml) in white wines from Campania
Rutin
mg/ml
0.03
Kaempferol
0.25
Kaempferol-3-glucoside
0.51
Kaempferol-3-rutinoside
0.64
Quercetin
0.52
Rhamnetin
0.55
Isorhamnetin
0.24
Flavonoid
Table 4 Flavonol content (mg/ml) in white wines from Sicily
Wine Sample
Rutin
Kaempferol3-Rutinoside
Kaempferol3-Glucoside
Kaempferol
Quercetin
Isorhamnetin
Rhamnetin
Thesis 1
2.72
< 0.64
2.41
< 0.25
< 0.52
< 0.24
10.06
Thesis 2
2.90
< 0.64
2.39
< 0.25
< 0.52
4.20
10.02
Thesis 3
2.85
< 0.64
2.39
4.29
< 0.52
4.17
10.03
Thesis 4
2.84
< 0.64
2.56
4.29
7.51
4.17
10.02
Thesis 5
2.88
< 0.64
2.39
4.29
< 0.52
4.17
9.98
Thesis 6
2.74
< 0.64
2.38
4.29
< 0.52
4.16
10.02
Thesis 7
2.85
< 0.64
2.38
4.29
< 0.52
4.17
10.02
Table 5 Flavonol content (mg/ml) in red wines from Tuscany
Wine
Sample
Rutin
Kaempferol3-Rutinoside
KaempferolKaempferol Quercetin Isorhamnetin Rhamnetin
3-Glucoside
Thesis 1
13.04
48.95
< 0.51
< 0.25
24.67
< 0.24
< 0.55
Thesis 2
13.67
3.58
5.68
< 0.25
60.15
5.93
< 0.55
Thesis 3
19.45
26.48
6.37
< 0.25
21.36
< 0.24
< 0.55
Thesis 4
22.08
29.55
< 0.51
< 0.25
27.47
< 0.24
< 0.55
Thesis 5
13.91
38.55
4.35
< 0.25
29.61
< 0.24
< 0.55
Thesis 6
14.83
44.32
< 0.51
< 0.25
40.15
< 0.24
< 0.55
Thesis 7
12.87
39.02
< 0.51
< 0.25
20.11
< 0.24
< 0.55
103
Fig.1
CONCENTRATION OF RESVERATROL ISOMERS
IN WHITE WINES FROM SICILY
trans-resveratrol
0.36
trans-piceid
0.28
total transresveratrol
cis-resveratrol
mg/L
0.21
cis-piceid
total cisresveratrol
0.14
0.07
0.00
Thesis 1
Thesis 2
Thesis 3
Thesis 4
Thesis 5
Thesis 6
Thesis 7
Samples
IN WHITE WINES FROM CAMPANIA
0.17
trans- resveratrol
trans-piceid
total trans- resveratrol
cis-resveratrol
cis-piceid
total cis-resveratrol
0.14
mg/L
0.11
0.08
0.05
0.02
0.00
Thesis 1
Thesis 2
Thesis 3
Thesis 4
Thesis 5
Thesis 6
Thesis 7
Samples
Fig. 2
104
IN RED WINES FROM TUSCANY
3,50
trans-resveratrol
3,00
trans-piceid
total trans-resveratrol
mg/L
2,50
cis-resveratrol
cis-piceid
2,00
total cis-resveratrol
1,50
1,00
0,50
0,00
Thesis 1
Thesis 2
Thesis 3
Thesis 4
Thesis 5
Thesis 6
Thesis 7
samples
Fig.3
105
20
CONTROL OF APPLE SCAB IN ORGANIC FARMING
Anna La Torre, Lucia Donnarumma, Daniele Lolletti, Giancarlo Imbrogli ni
Istituto Sperimentale per la Patologia Vegetale – Via C. G. Bartero, 22 - Roma
Introduction
Apple scab, which is caused by Venturia inequalis (Cke.) Wint., is considered the mayor disease of apple. In
organic agriculture, formulations of synthetic origin for defence are not admitted and plant protection is
essentially based on the use of mineral products as copper and sulphur. Copper is more effective than sulphur
(sulphur is ineffective at below 10°C, phytotoxic over 28°C and it has greater persistence). Use of copper
involves several problems: it accumulates in soil, causes phytotoxicity phenomena, when employed on full
vegetation, it gives rust phenomena on some varieties of apples. It is necessary to find protection alternatives
strategies, limiting the negative effects of use formulations of this element and assuring, at the same time,
sufficient protection against the diseases.
Fig. 1: - Spilocea pomi conidia
Fig. 2: - Experimental field
The trial has been done in a farm near Latina (Latium, central Italy), which as been converted, in 1993, to the
method of organic farming (cv Royal Gala grafted on M9). Treatments and doses are indicated in table 1. Some
compounds, for their formulation, release low amounts of copper. They are: Cuivrol, commercialized like
fertiliser (18% of copper metal); Cuprobenton, composed by copper oxychloride and copper sulfate hydrate
(15% of copper metal) coformulated with benthic clay (70%); Blue Copper Formula 2, made up with copper
hydroxide (24% in shape of crystals of the dimension of 0,35 mµ). As alternative products have been tested
Myco-sin (sulphureous clay, yeasts, equisetum extracts) and calcium polysulphide. The trials have been
conducted as recommended in EPPO/OEPP PP 1/5(3). The esteem of the infectious risk has been assessed on
the Mills criteria, monitoring instrumentally the climatic parameters by a thermoigrograph and a pluviometer.
The assessments to estimate therapeutic efficacy have been carried out on 4 central plants of every parcel
estimating the number of leaves for every treatment and percentage of leaves surface interested by apple scab,
using a scale with 5 classes of attack. Results obtained, after arc sin trasformation, have been evaluated by
Duncan test.
106
Table 1 – Fungicides used during the trial
Active ingredient
Formula
Commercial
formula
Copper Pro 50
WDG
Water-dispersible
granules
Cuprobenton DC
(WDG)
Wettable powder
Copper metal with B, Mo, Zn
Rame Azzurro
Formula 2
Cuivrol
Suspension
concentrate
Wettable powder
Calcium polysulphide
Polisenio
Liquid
Sulphureous clay, leavening
substances,equisetum extracts
Myco-sin*
Wettable powder
Copper oxycloride
Copper oxycloride,
Copper sulfate hydrate
Copper hydroxide
a.s. % o Dose f. c.
g/l
ml - g/hl
50
700; 600; 400;
340; 288; 200;
100
25
5
350
1100; 500
18
700; 300; 270
80
220; 160
Dose a.s.
g/hl
350; 300;
200; 170;
144; 100;
50
275; 125
55; 25
77, 56
126; 54;
48,6
18000; 1800;1500 14400;
1440;
1200
1500; 1000;
700; 500
Dose Cu++
g/hl
350; 300;
200; 170;
144; 100,
50
165;75
52,8; 38,4
126;54;
48,6
117; 60,8
*The treatments have been done in accordance with Biagra company:
at phenologycal phase “leaf fall” one treatment has been done with Bordeaux mixture (9kg/ha); at “beginning of bud swelling”, “bud
burst” and “flowering” treatment with Myco-sin and wettable sulphur; at “bud swelling” “pre-flowering” and “fruit swelling”
treatment with Myco-sin and sea-weed; at “fruit setting” one treatment has been done with copper sulfate (4 kg/ha).
Results
Table 2 shows the development of the infections during the trial and the activity of the various formulations over time.
Table 2 – Percentage of infected leaves
Treatments
Copper oxycloride
Copper oxycloride,
Copper sulfate
hydrate
Copper hydroxide
Copper metal with B,
Mo, Zn
Calcium
polysulphide
Sulphureous
clay, leavening
substances,equisetum
extracts
Control
Assessment
12/04/00
3.5 aA
4.3 aA
Assessment
18/05/00
15.3 aA
26.7 abAB
% infect leaves
Assessment
Assessment
20/06/00
20/07/00
25.8 aAB
30.4 aA
33.1 abABC 52.2 bB
Assessment
16/08/00
33.9 aA
53.3 bcBC
4.5 aA
10.2 aAB
17.1 aA
41.7 cBC
22.9 aA
46.9 bcABC
38.3 aA
61.9 cB
43.1 abAB
72.5 dD
1.0 aA
34.7 bcABC
45.1 bcABC
56.3 bcB
63.8 cdCD
7.5 aA
46.9 cCD
48.6 bcBC
61.3 bcB
65.6 cdCD
23.1 bB
61.0 dD
51.9 cC
63.4 cB
68.5 dCD
Different letters indicate significant different values by Duncan test (for P=0,05, lower-case letters, and for
P=0,01 capital letters)
107
Fig. 3 reports the assessment related to the degree of infection found on the leaves. On the fruits the first
symptoms appeared on 11 May; results on the degree of protection offered by different products are reported in
table 3. During the trial, the climatic conditions have turned out favourable for the development of pathogen
(Fig. 4). Elevate infection degrees, found on the leaves and the fruits, caused low yields.
Fig. 3 - Percentage of leaf damage at different date for various products
LEGEND
d
c
45
INFECT LEAF INDEX (%)
d d
d
50
40
a
a
30
a
15
c
b
c
e
a
c
d
a
b
a
10
c
c
a
b
25
20
b
a
b
a
35
d
a
CC
a
CU
16/08/00
b
c
a
5
0
20/07/00
20/06/00
a
a
a
IR
c
d
c
b b
CC = Copper
oxicloride
CU = Copper
oxicloride, Copper
sulphate hydrate
IR = Copper hydroxide
CV = Copper metal
with B, Mo, Zn
PS = Calcium
polysulphide
M+S = Sulphureous
clay, leavening
substances,
equisetum extracts
TEST = Control
a
CV
THESES
b
18/05/00
DATE
OF ASSESSMENT
12/04/00
PS
M+S
TEST
Different letters indicate significant different value by Duncan test for P=0,05
Fig. 4 -Climate during the trial: directions about phenologycal phases and about infections of apple scab
100
30
90
70
20
60
15
50
Rain (mm)
Temperature (°C) Relative Umidity (%)
25
80
40
10
30
20
5
10
0
0
Infections:
March
April
May
June
JuLy Phenologic
phases:
RG
OT
MA
BR
PF
CP
AL
FN
LEGENDA:
= infections of apple scab
RG= Bud burst
OT= Mouse-ear stage
MA= Appearance of flower buds BR= Pink bud
CP= Petal fall
AL=Fruit setting
FN= Walnut fruit
IF= Fruit swelling
108
Rain
IF
Mean temperature
PF= Full flowering
Mean relative humidity
Table 3 – Efficacy of different treatment against apple scab on fruits (Scale OEPP/EPPO)
Treatments
%
no attack
Assessment
20/07/00
% Fruits with
1-3 spots
% Fruits with
%
more than 3
no attack
spots
9.6 aA
70.1 cD
27.8 bB
55.3 bC
Copper oxycloride
61.7 eD
26.2 aAB
39.0 cB
Copper oxycloride, 38.0 dC
Copper sulfate
hydrate
Copper hydroxide
58.5 eD
28.7 abAB
11.7 aA
Copper metal with B, 27.6 cBC
38.2 bcB
38.8 cB
Mo, Zn
Calcium polysulphide 24.7 bcB
37.8 bcB
41.7 cBC
18.1 bB
27.1 aAB
56.3 dCD
Sulphureous
clay, leavening
substances,equisetum
extracts
Control
6.1 aA
20.2 aA
68.2 eE
Different letters indicate significant different values by Duncan test
P=0,01 capital letters)
Assessment
18/08/00
% Fruits with
% Fruits with
1-3 spots
more than 3 spots
18.9 aA
28.6 bAB
4.9 aA
17.6 abABC
69.0 cD
48.8 bBC
19.6 aA
33.0 bB
6.9 aAB
21.0 bcABC
46.5 bBC
36.2 aAB
32.5 bB
35.6 bB
24.3 bcBC
32.6 cCD
29.2 aA
28.3 bAB
46.1 dD
(for P=0,05, lower-case letters, and for
Conclusions
By the results obtained in the operating conditions of the trial, we can deduce that the various formulates have
against apple scab an acceptable activity related to the degree of attack found in the control parcels. Best results
have been obtained with copper oxicloride, reference product in our trials, and also copper hydroxicloride;
second best results were obtained with the use of Cuprobenton (copper compounds and active bentonites).
Calcium polysulphide has given partial protection, however, it is a positive result because its employment, also
after grown resumption, is permitted by Reg. CE n. 1073/2000. Cuivrol, constituted from oligoelements B, Cu,
Mo, Zn and Myco-sin, made up of sulphureous clay were less effective. Activity against apple scab, not always
satisfactory, manifested by several products, could in part be related to the strong pressure of the pathogen that
has found favourable climatic conditions for the insurgence and spread of the disease.
References
- Boschieri S., Mantinger H.,1991. Produzione biologico-organica del melo presso il centro per la
sperimentazione di Laimburg. Otto anni di esperienze. L’informatore Agrario, 44: 97-101.
- C.M.I. Descriptions of Pathogenic Fungi and Bacteria No.401,1974, Kew.
109
-
-
Direttiva del Consiglio 91/414/CEE relativa all’immissione in commercio dei prodotti fitosanitari (G.U.
19.8.1991, n. L 230).
Donnarumma L., La Torre A., 2000. Sali di rame in agricoltura biologica e possibili alternative.
Informatore Fitopatologico, 4: 27-31.
Gentili G., Ravagli S., 1994. Applicazione di tecniche di lotta guidata alle malattie crittogamiche dei
fruttiferi e della vite in Emilia- Romagna: risultati delle sperimentazioni effettuate nel periodo 19861993. Atti Giornate Fitopatologiche,3: 79-86.
Govi G., 1955. Risultati di prove di lotta contro la ticchiolatura del melo. Frutticoltura VI: 517-531.
McKinney H.H.,1923. Influence of soil temperature and moisture on infection of wheat seedlings by
Helminthosporium sativum. Journal of Agricultural Research, 5: 195-217.
Mills W. D., 1944. Efficient use of sulfur dust and sprays during rain to control apple scab. Ext. Bull.
Cornell Agric. Exp. Sta., 630.
OEPP/EPPO, 1997. Directives pour l’evaluation biologique des produits phytosanitaires, vol. 2
Fongicides & Bactericides, PP 1/5(3): 15-18.
110
21
CONTAMINAZIONE DA MICOTOSSINE IN ALIMENTI DI PRODUZIONE BIOLOGICA
A. Maietti1, L. Mazzotta1, C. Saletti2, G. Mirolo3, M. Berveglieri2,
P. Tedeschi1, V. Brandolini 1
1
Dipartimento di Scienze Farmaceutiche – Università di Ferrara; 2Dipartimento di Sanità Pubblica – Azienda
USL di Ferrara; 3A.R.P.A. – Ferrara
INTRODUZIONE
Le micotossine sono metaboliti secondari prodotti da muffe, tossici per gli animali superiori. La formazione di
queste sostanze è strettamente connessa alla crescita fungina anche se la presenza di funghi tossigeni in un
prodotto non indica automaticamente la presenza di micotossine. Le tossine tuttavia possono persistere per lungo
tempo dopo la crescita vegetativa e la morte del fungo. L’analisi micologica basata sulla numerazione delle unità
vitali e l'identificazione delle specie fungine non permette di quantificare il rischio tossico proprio di un prodotto
alimentare; questo rischio non può che essere determinato con un'analisi chimico-fisica.
Le aflatossine sono un gruppo di micotossine prodotte da ceppi di Aspergillus flavus, A. parasiticus e A. nomius,
con simile struttura chimica. Si ritiene che l'A. flavus produca le aflatossine B1 e B2, mentre l'A. parasiticus
produca le aflatossine B1, B2, G1, G2. Le contaminazioni degli alimenti con aflatossine si verificano
principalmente nelle zone a climi caldi e umidi come le zone tropicali e subtropicali, ma poiché i Paesi delle
zone climatiche più fredde importano prodotti da queste aree, le aflatossine sono un problema mondialmente
riconosciuto. L'aflatossina B1 è il tipo più frequentemente riscontrato, mentre la presenza di aflatossine B2, G1,
G2 è generalmente associata alla presenza dell'aflatossina B1 e di solito sono presenti in quantità minore.
Le muffe capaci di produrre micotossine sono contaminanti assai diffusi negli alimenti e nei prodotti agricoli;
crescita e produzione di tossine possono avvenire sia in campo che in magazzino.
Le derrate alimentari possono subire una contaminazione diretta che avviene normalmente su matrici vegetali
quali cereali, frutta secca, semi oleaginosi, spezie; oppure indiretta attraverso l’alimentazione degli animali da
allevamento con foraggi e mangimi contaminati.
La qualità delle materie prime, il controllo dell'ambiente di conservazione, i trattamenti fisici e chimici, la pulizia
dei sili e dei trasporti, sono la chiave del controllo dello sviluppo fungino. La prevenzione della contaminazione
fungina potrebbe essere pertanto più difficoltosa nei prodotti biologici che per definizione possono essere
sottoposti a trattamenti solo con prodotti naturali consentiti. Le micotossine possono causare vari effetti tossici di
tipo acuto, subacuto, teratogeno, mutageno, cancerogeno; esse evidenziano diversi tipi di tossicità in dipendenza
della dose, dell'organo interessato, del sesso, dell'età e della specie.
Per l'Italia, le micotossine rappresentano un problema connesso con l'importazione di derrate da Paesi a clima
caldo e umido, mentre la contaminazione dei prodotti locali è poco frequente ed è a livelli piuttosto contenuti sia
per motivi climatici che per le migliori tecniche agronomiche, di raccolta e di conservazione dei prodotti stessi.
La Comunità Economica Europea con il Regolamento CEE n.1525/98 ha fissato dei limiti massimi accettabili
sia per il contenuto globale delle aflatossine (4.0 mg/Kg) che per quello della sola aflatossina B1 (2.0 mg/Kg) e
del suo metabolita aflatossina M1 nel latte (0.05 mg/Kg).
Il consumo di prodotti da agricoltura biologica è in espansione ed è pertanto maggiore la quota di popolazione a
rischio con particolare esposizione verso il settore ortofrutticolo (40%) seguito da pasta e riso (8%). Per tale
motivo si è ritenuto interessante svolgere un'indagine mirata alla valutazione della eventuale presenza di
micotossine (Aflatossina B1 e totali), nei prodotti "biologici" commercializzati nella provincia di Ferrara al fine
di incrementare le necessarie informazioni sotto il profilo della sicurezza alimentare.
Le principali tipologie produttive dell'agricoltura biologica sulle quali si è incentrata la ricerca di micotossine
sono state le seguenti: cereali e paste alimentari, Muesli biologico, pane integrale biologico; riso integrale
biologico e riso soffiato biologico, farine biologiche, fette biscottate, crackers, gallette ed affini, e biscotti,
composta di frutta biologica.
MATERIALI E METODI
I campionamenti sono stati effettuati presso la grande distribuzione nel rispetto della normativa vigente ed i
campioni raccolti sono stati analizzati per verificare la presenza di Aflatossine B1 e totali.
Procedimento di estrazione: 25 g di campione macinato ed omogeneizzato sono mescolati a 10 g di celite e
dispersi in una miscela acetonitrile/acido acetico. Dopo 30 minuti di agitazione si filtra. Il filtrato viene trattato
inizialmente con acqua, piombo acetato neutro e ammonio solfato, e successivamente estratto con toluene. La
111
fase organica è portata a secco ed il residuo ripreso con esano e acido trifluoroacetico, quindi riportato
nuovamente a secco e ripreso con acetonitrile e acqua.
La determinazione analitica è effettuata con un HPLC Perkin Elmer dotato di detector fluorimetrico SFM 25
Kontron, iniettore Rehodyne con loop da 20 ml e colonna C18 (5 mm x 25 cm). La fase eluente è una soluzione
costituita da acqua 79 %, acido acetico 7 %, alcol isopropilico 7 % e acetonitrile 7 %.
I prodotti esaminati in questa ricerca sono tra quelli maggiormente ricercati dal consumatore nell’ambito dei
prodotti derivati da agricoltura biologica. In questo lavoro sono presentati i dati riguardanti frutta secca, cereali e
legumi, semi oleaginosi, spezie ed erbe infusionali realtivi a campionamenti effettuati negli anni 1999 e 2000.
Nell’anno 1999 sono stati esaminati 82 campioni di frutta secca, cereali e legumi; 413 campioni di semi
oleaginosi; 65 campioni di spezie ed erbe infusionali. Nell’anno 2000 sono stati esaminati 137 campioni di frutta
secca, cereali e legumi; 430 campioni di semi oleaginosi; 29 campioni di spezie ed erbe infusionali.
FRUTTA SECCA, CEREALI E LEGUMI
Anno 1999
Ricerca:
Aflatossine
Tot.
camp
Camp
pos.
B1
82
3
B1+B2+G1+G2
82
3
Numero di campioni
< LR*
0-2
2-4
75
4
1
74
4
1
4-10
1
10-50
> 50
1
1
1
1
Standard di
riferimento
(Circolare n.10
del 9/6/99)
2 mg/kg
4 mg/kg
LR* = limite di rilevabilità
Campioni non conformi
Campioni
non
Conformi
3,7 %
Campioni
Conformi
96,3 %
Campioni
non
Conformi
3,7 %
Campioni
Conformi
96,3 %
(B1 + B2 + G1 + G2)
B1
Anno 2000
Ricerca:
Aflatossine
Tot.
camp
Camp
pos.
Numero di campioni
< LR*
0-2
B1
137
1
135
1
B1+B2+G1+G2
121
1
119
1
2-4
4-10
10-50
1
> 50
Standard di
riferimento
(Circolare n.10
del 9/6/99)
2 mg/kg
4 mg/kg
112
Campioni
non
Conformi
0,7 %
Campioni
non
Conformi
0,8%
Campioni
Conformi
99,3 %
B1
Campioni
Conformi
99,2 %
(B1 + B2 + G1 + G2)
SEMI OLEAGINOSI
Anno 1999
Ricerca:
Aflatossine
Tot
camp
B1
413
B1+B2+G1+G2
413
Camp
pos
Numero di campioni
< LR*
0-2
2-4
4-10
10-50
> 50
23
387
3
5
7
6
5
20
383
6
4
7
7
6
Standard di
riferimento
(Circolare n.10
del 9/6/99)
2 mg/kg
4 mg/kg
Campioni
non
Conformi
5,6 %
Campioni
Conformi
94,4 %
Campioni
Conformi
95,2 %
Campioni
non
Conformi
4,8 %
(B1 + B2 + G1 + G2)
B1
Anno 2000
Ricerca:
Aflatossine
Tot
camp
Camp
pos
B1
430
18
408
4
5
2
5
6
Standard di
riferimento
(Circolare n.10
del 9/6/99)
2 mg/kg
B1+B2+G1+G2
430
15
408
4
3
4
5
6
4 mg/kg
Numero di campioni
< LR*
0-2
2-4
4-10
10-50
> 50
113
Campioni
non
Conformi
4,2 %
Campioni
non
Conformi
3,5 %
Campioni
Conformi
95,8 %
Campioni
Conformi
96.5 %
B1
(B1 + B2 + G1 + G2)
SPEZIE ED ERBE INFUSIONALI
Anno 1999
Ricerca:
Aflatossine
Tot
camp
B1
65
B1+B2+G1+G2
65
Camp
pos
Numero totale di campioni
< LR*
0-10
10-20
20-50
2
53
10
2
2
53
10
1
Standard di
riferimento
(Circolare n.10
del 9/6/99)
10 mg/kg
> 50
20 mg/kg
1
Campioni
Conformi
96,9 %
Campioni
non
Conformi
3,1 %
Campioni
non
Conformi
3,1 %
Campioni
Conformi
96,9 %
(B1 + B2 + G1 + G2)
B1
Anno 2000
Ricerca:
Aflatossine
Tot
camp
B1
29
B1+B2+G1+G2
29
Camp
pos
Numero di campioni
< LR*
0-4
4-8
8-10
1
26
2
1
1
26
2
1
10- 0
> 50
Standard di
riferimento
(Circolare
n.10
del 9/6/99)
10 mg/kg
20 mg/kg
114
Campioni
non
Conformi
3,4 %
Campioni
non
Conformi
3,4 %
Campioni
Conformi
96,6 %
B1
Campioni
Conformi
96,6 %
(B1 + B2 + G1 + G2)
Relativamente all’anno 1999 a seconda della matrice considerata il grado di contaminazione da aflatossina B1 e
aflatossine totali è risultato variare dal 3 % nelle erbe infusionali e spezie, al 3,7 % nella frutta secca, al 6 % nei
semi oleaginosi, mentre nei cereali e legumi analizzati non sono state reperite aflatossine.
Il trend si è mantenuto nell’anno 2000 per quanto riguarda spezie ed erbe infusionali; per i semi oleaginosi è
stata riscontrata una maggior percentuale di campioni conformi ai limiti indicati dalla circolare n. 10 del 9/6/99.
Per la categoria frutta secca cereale e legumi i campioni contaminati da aflatossine sono risultati al di sotto del 1
%. Tale risultato è ancor più significativo se si considera che per questa categoria di prodotti il numero di
campioni analizzati è stato circa il doppio.
All'interno delle diverse classi alimentari sono state individuate alcune matrici più a rischio: tra i semi oleaginosi
i pistacchi e le arachidi; tra le spezie i peperoncini e le polveri derivate ed infine tra la frutta secca i fichi.
I dati ottenuti confermano l'alto rischio alimentare per prodotti provenienti da paesi terzi (Turchia, India, Iran, e
altri) dove non sempre le condizioni di stoccaggio e trasporto delle derrate alimentari possono garantire la
corretta conservabilità e salubrità dei prodotti.
CONCLUSIONI
Il consumo di prodotti da agricoltura biologica è in espansione ed è maggiore da parte di persone che hanno
scelto un’alimentazione prevalentemente vegetariana. Il consumo maggiore di prodotti biologici viene registrato
nel settore ortofrutticolo, con il 40% delle preferenze sul totale dei consumatori, seguito da pasta e riso con l’8 ed
il 2-3% dei prodotti confezionati. I prodotti considerati nel presente lavoro hanno una larga diffusione tra i
consumatori di prodotti biologici.
I risultati ottenuti dalla indagine effettuata in questo biennio sono stati soddisfacenti e ci hanno permesso di
ricavare le seguenti informazioni :
·
Sono necessari controlli mirati sia allo stoccaggio presso i centri di produzione (per cereali di
produzione locale) sia nella grossa distribuzione (ipermercati);
·
Un più attento controllo deve essere effettuata su matrici a particolare rischio quali semi oleaginosi
(mandorle, pistacchi, arachidi e noci), spezie (peperoncino e polveri derivate), caffè; e in particolare quelle
provenienti da paesi terzi dove non sempre le condizioni di trasporto e stoccaggio garantiscono la salubrità
dei prodotti;
·
Devono essere in oltre monitorati i prodotti locali insilati quali grano, riso, sfarinati.
Infine va ricordato che gli alimenti che sono risultati maggiormente a rischio di aflatossine sono arachidi e
derivati, mais e derivati, noci brasiliane, mandorle, fichi secchi, alcune spezie (peperoncino); ma che una cattiva
conservazione può far comparire le aflatossine anche in prodotti non considerati a rischio.
Bibliografia
- Nasir M.S., Jolley M.E., “Development of a fluorescence polarization assay for the determination of
aflatoxins in grains”. J. Agric. Food Chem. 50(11):3116-21 (2002).
- Schatzki T.F., Haddon W.F., “Rapid, non-destructive selection of peanuts for high aflatoxin content by
soaking and tandem mass spectrometry”. J. Agric. Food Chem. 50(10):3062-9 (2002).
115
-
Sobolev V.S., Dorner J.W., “Cleanup procedure for determination of aflatoxins in major agricultural
commodities by liquid chromatography”. J. AOAC Int. 85(3):642 -5 (2002).
Regolamento CE 1525/98
Circolare n°10 del 09/06/99 Gazzetta Ufficiale della Repubblica Italiana
Barbieri G., Bergamini C., Ori E, Resca P. Journal of Food Science 1313 -1331(1994)
Finoli C., Ferrari M. Industrie Alimentari, 732-736 (1994)
Brera C., Miraglia M., Colatosti M.: Microchemical Journal, 59 (1), 45 -49 (1998)
Brera C., Miraglia M. Microchemical Journal, 54 (4), 465 -471 (1996)
R.C. Garden, M.M. Wattman, P.J.L. Taylor and M.W Stow Journal of Chromatography, 648, 485 -490
(1993)
Brera C. Rapporti ISTISAN 34, 203-214 (1996)
116
22
PEPTIDATI DI RAME: PRODOTTI INNOVATIVI
A BASSO DOSAGGIO A BASE DI RAME, CHELATO AD AMINO ACIDI E PEPTIDI
Paolo Maini
U.R. GRIFA no. 42 - c/o SICIT 2000 SpA, Chiampo VI
Il rame, insieme allo zolfo, è forse il più indispensabile dei prodotti utilizzati per la difesa delle colture
nell'Agricoltura Biologica (AB). Vite, patata, pomodoro, olivo e molte frutticole non si potrebbero assolutamente
produrre senza rame in AB.
A livello europeo, i Paesi del Nord vorrebbero eliminare del tutto il rame dalla lista dei prodotti ammessi, a causa
dell'inquinamento delle falde acquifere in quei suoli, principalmente acidi. Nel Sud Europa la prevalenza di suoli
alcalini evita la percolazione e quindi il problema è meno sentito. E’ vero però che i Paesi del Nord sono i
consumatori di una buona parte delle nostre produzioni biologiche e quindi le loro richieste devono essere tenute
in debita considerazione. A livello normativo (Reg. CEE 2092/91) l’utilizzo del rame era stato concesso fino al
31 marzo 2002, e ciò avrebbe significato la morte dell'AB, se non fossero intervenuti gli organismi dei
produttori biologici con proposte di riduzione dell’uso al minimo. Fortunatamente, è stata accettata la proposta di
"…una riduzione graduale dell’uso del rame, con un limite massimo di 8 kg di rame metallico per ettaro e per
anno. Dopo 4 anni, il rame dovrà essere ulteriormente ridotto e successive riduzioni verranno decise in base al
progresso della ricerca sulle possibili alternative. La quantità di rame ad ettaro e per anno andrà calcolata come
media quinquennale, in modo da poter fronteggiare annate climaticamente avverse con quantità leggermente
maggiori di rame, per poi recuperare nelle annate meno difficili."
Ed è proprio sulla linea delle suddette normative che si posizionano i nuovi ed innovativi formulati a base di
rame complessato ad amino acidi e peptidi (PEPTIDATI o Polipeptidati di rame), capaci di ridurre fortemente i
dosaggi del rame ad ettaro.
Peptiram 5 e Peptiram 7, peptidati dim rame chelato ad amino acidi e peptidi, rispettivamente a base di solfato e
di idrossido di rame e registrati dalla SICIT 200 SpA nell'aprile del 2002, si differenziano nettamente da tutti i
prodotti fungicidi convenzionali a base di rame. Essi possono rappresentare, pertanto, una nuova e valida
alternativa nel controllo delle malattie fungine e batteriche delle colture agrarie, particolarmente nell'AB,
tenendo in conto anche il fatto che il rame, pur essendo utilizzato da più di cento anni, non ha mai prodotto
fenomeni di resistenza.
I due nuovi Peptidati di rame sono caratterizzati da una speciale struttura basata su complessi del rame con
amino acidi e peptidi (Figura 1). Il metallo, così legato, non segue più la normale via di penetrazione nelle
cellule vegetali con meccanismo “cationico”, ma penetra con gli stessi meccanismi delle sostanze organiche. La
forte riduzione dei dosaggi/ha di rame che ne deriva, può arrivare fino ad un quinto o ad un decimo,
rispettivamente con Peptiram 5 e Peptiram 7.
Figura 1: Esempi di chelati tra Rame ed amino acidi. A sinistra: 1 atomo di Cu, 2 moli di Glicina e 1 mole di
H2O; a destra:1 atomo di Cu , 1 mole di Istidina e 1 mole di Asparagina .
Forse proprio per questo meccanismo di facile penetrazione è da notare l'efficacia mostrata verso malattie
fungine solitamente non controllate dal Cu, quali certe ruggini. Mentre in altre colture (ad es:. olivo,
cucurbitacee) prevale l'azione stimolante degli amino acidi con notevoli rigogli vegetativi e produttivi, in alcune
colture (ad es.: vite), a causa della rapida assimilazione, si possono verificare fenomeni fitotossici, in funzione
della varietà, dello stadio fisiologico o delle condizioni ambientali. L'importanza dell'impiego del rame nella
vite, ha portato a saggiare la sensibilità varietale su 16 cv., con buoni risultati, escluso il moscato.
117
La necessità di dover ridurre i dosaggi di rame per ettaro, ha spinto vari Organismi regionali o affini, che si
occupano di AB, a fare numerose prove con formulazioni o tecniche nuove. Negli ultimi due-tre anni anche i
Peptiram sono stati inseriti in tali programmi e generalmente i risultati sono stati tra i più soddisfacenti.
Nel corso di questa ultima annata, dopo la registrazione dei due prodotti, sono state impostate anche numerose
prove dimostrative su varie colture.
L'impiego dei due Peptiram, inoltre, è del tutto soddisfacente anche sul piano igienico-tossicolgico, in quanto
analisi dei residui di rame su vite e pomodoro hanno evidenziato valori non diversi dai testimoni (Figura 2). Ciò
potrebbe essere particolarmente interessante per i produttori biologici di vini passiti, ove, in qualche caso, si
sfiorano i livelli residuali massimi.
3
UVA
POMODORO
Residui Cu , mg/kg
2,16
1,42
1,5
0,91
0,72
0,82
0,84
0
Controllo Peptiram 5 Peptiram 7 Controllo Peptiram 5 Peptiram 7
6 kg/ha
1,5 kg/ha
300 g/100L 100 g/100L
Figura 2: Residui di Cu (mgKg-1) da prove ed analisi secondo le BPL.
118
23
CHARACTERIZATION OF ORGANIC VIRGIN OLIVE OILS FROM CORATINA CULTIVAR
E. Perri, N. Lombardo, I. Muzzalupo, E. Urso, M.
Pellegrino
Istituto Sperimentale per l’Olivicoltura
C.da Li Rocchi, 87036 Rende (CS), Italy
[email protected]
G. Sindona, C. Benincasa
Dipartimento di Chimica
Università della Calabria
I-87030 Arcavacata di Rende (CS), Italy
C. Cavallo
Regione Puglia
I.P.A., Brindisi, Italy
Introduction
In spite of the growing interest in organically cultivated olive grove and organic olive oil in European and
international markets, there are very few reports on the qualitative, nutritional and organoleptic characteristics of
these products (Gutierrez et al., 1999; Perri et al., 1999; Perri et al., 2001; Perri et al., 2002). Recently, a
research project dealing with the “Characterisation of olive oils from the South of Italy obtained by organic
farming systems” has been co-funded by the EU and the Italian Ministry of Agriculture (B07 Project,
Multiregional Operational Programme, 2 Measure). The principal aim of this research project was to classify and
characterise the nutritional and organoleptic quality of organic olive oils and to compare the quality of organic
olive oil of important Italian cultivars from selected producing areas to conventional and/or integrated olive oils
from the same cultivars and areas. This paper presents some results of the second year of activity in Apulia of the
above research project.
Plant material. In 2000, triplicate samples of organically and integrated cultivated olive drupes of Coratina cv
from two adjacent farms at Andria (Bari, Italy) were collected 10 days before the traditional start of harvest, in
the middle of the harvest period and 10 days after the end of harvest time. To compare organically produced
olive oils versus integrated olive oils the olives were handpicked from the same cultivars under comparable
pedo-climatic, harvesting and productive conditions and the analytical and sensory data compared on the basis of
the same stage of olive maturity monitored by Jaen index (Gutierrez et al., 1999).
Analytical procedures. The measurements of free acidity, fatty acid methyl esters, peroxide index, specific
extinction coefficients K232 and K270, Dk, and panel test have been determined according to the European Official
Methods of Analysis (EC Regulation N. 2568/91 July, 1991). Total phenols were determined by colorimetry
using the Folin-Ciocalteu reagent using caffeic acid as external standard. Oleuropein (olp) was determined in
virgin olive oil extracts by tandem mass spectrometry with Atmospheric Pressure Chemical Ionization (APCIMS/MS) under Selected Reaction Monitoring (SRM) condition (Perri et al., 1999b).
The analytical data arising from the second year of sampling confirm the preliminary results published on the
first paper (Perri et al., 2002): i) olive oil from Coratina cv. is characterised by high level of total phenols and
high percentages of oleic acid in a wide range of stages of maturity of the drupes; ii) all the examined olive oil
samples were ascribable to the extra virgin olive oil grade on the basis of analytical data relating to free acidity,
peroxide number, specific extinction coefficients K232 and K270 , Dk, fatty acid methyl esters, sterol and
organoleptic score (panel test); iii) ecologically grown olives can produce high-quality oil thanks to the low
Bactrocera oleae infestations; iv) as shown by the variance analysis (table 1), analytical and sensory data
relating to organic and integrated olive oils are very similar. In fact, the only statistical significant difference
observed between organic and integrated olive oils corresponding to the means of the sensory score of integrated
olive oil (table 1) may be considered negligible from the quality point of view.
Acknowledgements
The Italian Ministry of Agriculture and European Union (Multiregional Operational Programme, Measure 2, B07
Project) supported this research work. We thank Dr. Nino Paparella, Dr. Nicola Panaro (C.I.Bi., Bari) and Drs.
Edoardo and Giancarlo Ceci Ginistrelli for olive sampling, data collection on farming systems and olive grove
management. We thank also Prof. Angelo Putignano and Prof. Francesco Prudentino (I.T.A.S. of Ostuni-BR) for
olive oil extraction.
Literature cited
Gutierrez R. F., Arnaud T., Albi M., Influence of ecological cultivation on virgin olive oil quality, JAOCS, 1999,
76:617-621.
119
Perri E., Palopoli A., Pellegrino M., Sirianni R., Miele D., 1999a, La caratterizzazione degli oli di oliva calabresi
ottenuti da agricoltura biologica, Proceeding of Seminario-Laboratorio “Metodi e sistemi innovativi
dell’olivicoltura biologica e sostenibile: stato della ricerca e della sperimentazione”, Rende, april 14-16, pp147153.
Perri E, Raffaelli A., Sindona, 1999b, G., Quantitation of oleuropein in virgin olive oil by ionspray mass
spectrometry-selected reaction monitoring, J. Agric. Food Chem., 47(10), 4156-4160.
Perri E., Rizzuti B., Pellegrino M., Salvo F., Spartà G., 2001, Valutazione organolettica degli oli di oliva siciliani
campionati nell’ambito del progetto POM B07 ”Caratterizzazione degli oli di oliva meridionali da agricoltura
biologica” nell’annata 1999-2000, in Corso per tecnici degustatori d’olio extravergine e vergine d’ oliva,
Regione Siciliana, Alcamo.
Perri E., Rizzuti B., Pellegrino M., Paparella N., Panaro N., Cavallo C., 2002, Characterisation of italian virgin
olive oils from organic farming systems, Atti “4th Intenational Symposium on olive growing”, Valenzano (BA),
25-30 settembre - Acta Horticulturae (in corso di stampa).
Perri E., Lombardo N., Parlati M.V., Fodale A., Mulè R., Pellegrino M., Salvo F., Spartà G., 2002,
Caratterizzazione di oli di oliva da agricoltura biologica provenienti dalla Sicilia, Atti del Convegno
Iternazionale di Olivicoltura, Spoleto, 22-23 aprile 2002.
Table 1. Mean values of the main quality parameters of olive oil of Coratina cv. from Andria (BA) and
significant differences in the means of the same treatment and between treatments.
Ripeness index
Parameter
Culture
2.2
3.2
Mean(a)
Organic
Acidity
0.28 n.s.
0.23 n.s.
0.26
Integrated
0.28 n.s.
0.28 n.s.
0.28
n.s.
n.s.
n.s.
(a)
Peroxide
Organic
Integrated
2.93 n.s.
2.60 b
n.s.
3.20 n.s.
3.27 a
n.s.
3.07
2.93
n.s.
Total phenols
Organic
Integrated
485.80 n.s.
432.13 n.s.
n.s.
418.13 n.s.
382.20 n.s.
n.s.
451.97
407.16
n.s.
Organoleptic score
Organic
Integrated
Oleic acid
Organic
Integrated
7.00 n.s.
7.37 n.s.
*
76.73 n.s.
76.02 n.s.
n.s.
7.53 n.s.
7.53 n.s.
n.s.
74.86 n.s.
76.31 n.s.
n.s.
7.27
7.45
n.s.
75.79
76.16
n.s.
Organic
Oleuropein (mg/kg)
0.126 A
0.090 B
0.108
Overall means. Differences within the same row (Tukey test): n.s.=not significant; different letters
indicate a significant differences at P 0.05 (Tukey test). Differences within the same column (Tukey test):
n.s.=not significant; * P<0.05.
120
24
CHARACTERIZATION OF ORGANIC VIRGIN OLIVE OILS FROM OGLIAROLA SALENTINA
CULTIVAR
E. Perri, N. Lombardo, I. Muzzalupo, B.
Rizzuti, M. Pellegrino
Istituto Sperimentale per l’Olivicoltura
C.da Li Rocchi, 87036 Rende (CS), Italy
[email protected]
G. Sindona, C. Benincasa
Dipartimento di Chimica
Università della Calabria
I-87030 Arcavacata di Rende (CS),
Italy
C. Cavallo
Regione Puglia
I.P.A., Brindisi, Italy
Introduction
The organic extra-virgin olive oil would be able to have high nutritional and sensory quality as necessary
requirement to become part of those Italian products that are able to survive in the national and international
market. However, there are very few reports on the qualitative, nutritional and organoleptic characteristics of
these products (Gutierrez et al., 1999; Perri et al., 1999a; Perri et al., 2001; Perri et al., 2002). Recently, a
research project dealing with the “Characterisation of olive oils from the South of Italy obtained by organic
farming systems” has been co-funded by the EU and the Italian Ministry of Agriculture (B07 Project,
Multiregional Operational Programme, 2 Measure). The principal aim of this research project was to classify and
characterise the nutritional and organoleptic quality of organic olive oils and to compare the quality of organic
olive oil of important Italian cultivars from selected producing areas to conventional and/or integrated olive oils
from the same cultivars and areas. This paper presents some results of the second year of activity in Apulia of the
above research project.
Plant material. In 2000/2001 harvest, triplicate samples of organically and conventional cultivated olive drupes
of Ogliarola salentina cv. from two adjacent farms at Villa castelli (Brindisi, Italy) were collected 10 days before
the traditional start of harvest, in the middle of the harvest period and 10 days after the end of harvest time. To
compare organically produced olive oils versus conventional olive oils the olives were handpicked from the
same cultivars under comparable pedo-climatic, harvesting and productive conditions and the analytical and
sensory data compared on the basis of the same stage of olive maturity monitored by Jaen index (Gutierrez et al.,
1999).
Analytical procedures. The measurements of free acidity, fatty acid methyl esters, peroxide index, specific
extinction coefficients K232 and K270, Dk, and panel test have been determined according to the European Official
Methods of Analysis (EC Regulation N. 2568/91 July, 1991). Total phenols were determined by colorimetry
using the Folin-Ciocalteu reagent using caffeic acid as external standard. Oleuropein (olp) was determined in
virgin olive oil extracts by tandem mass spectrometry with Atmospheric Pressure Chemical Ionization (APCIMS/MS) under Selected Reaction Monitoring (SRM) condition (Perri et al., 1999b).
According with the results of the first year of observation (Perri et al., 2002), from the data of quality parameters
of table 1, it turns out that the olive oil from Ogliarola salentina cv. was characterised by medium to low level of
total phenols and low percentages of oleic acid. Moreover, only the olive oils produced in the first period of
harvest were ascribable without any tolerance to the extra virgin olive oil grade on the basis of organoleptic
score (panel test). This is probably due to an increase of the percentages of Bactrocera oleae infestations
observed in the second harvest period. As shown by the variance analysis (table 1), analytical and sensory data
relating to organic and conventional olive oils are very similar probably because of similar percentages of olive
fly infestation due to ineffective pesticide treatments. Finally, an increase of olp content in olive oil versus Jaen
index was also observed.
Acknowledgements
The Italian Ministry of Agriculture and European Union (Multiregional Operational Programme, Measure 2, B07
Project) supported this research work. We thank Mr. Lorenzo Elia and Amalio Cassese for olive sampling, data
collection on farming systems and olive grove management. We thank also Prof. Angelo Putignano and Prof.
Francesco Prudentino (I.T.A.S. of Ostuni-BR) for olive oil extraction.
121
Literature cited
Gutierrez R. F., Arnaud T., Albi M., Influence of ecological cultivation on virgin olive oil quality, JAOCS, 1999,
76:617-621.
Perri E., Palopoli A., Pellegrino M., Sirianni R., Miele D., 1999a, La caratterizzazione degli oli di oliva calabresi
ottenuti da agricoltura biologica, Proceedings of Seminario-Laboratorio “Metodi e sistemi innovativi
dell’olivicoltura biologica e sostenibile: stato della ricerca e della sperimentazione”, Rende, april 14-16, pp147153.
Perri E, Raffaelli A., Sindona, 1999b, G., Quantitation of oleuropein in virgin olive oil by ionspray mass
spectrometry-selected reaction monitoring, J. Agric. Food Chem., 47(10), 4156 -4160.
Perri E., Rizzuti B., Pellegrino M., Salvo F., Spartà G., 2001, Valutazione organolettica degli oli di oliva siciliani
campionati nell’ambito del progetto POM B07 ”Caratterizzazione degli oli di oliva meridionali da agricoltura
biologica” nell’annata 1999-2000, in Corso per tecnici degustatori d’olio extravergine e vergine d’oliva,
Regione Siciliana, Alcamo.
Perri E., Rizzuti B., Pellegrino M., Paparella N., Panaro N., Cavallo C., 2002, Characterisation of italian virgin
olive oils from organic farming systems, Atti “4th Intenational Symposium on olive growing”, Valenzano (BA),
25-30 settembre - Acta Horticulturae (in corso di stampa).
Perri E., Lombardo N., Parlati M.V., Fodale A., Mulè R., Pellegrino M., Salvo F., Spartà G., 2002,
Caratterizzazione di oli di oliva da agricoltura biologica provenienti dalla Sicilia, Atti del Convegno
Iternazionale di Olivicoltura, Spoleto, 22-23 aprile 2002.
Table 1. Mean values of the main quality parameters of olive oil of Ogliarola salentina cv. from Villa Castelli
(Brindisi, Italy) and significant differences in the means of the same treatment and between treatments.
Ripeness index
Parameter
Culture
2.0
4.1
Mean(a)
Organic
Acidity
0.42 n.s.
0.37 n.s.
0.40
Conventional
(as % of oleic acid)
0.28 n.s
0.33 n.s.
0.30
n.s.
n.s.
n.s.
Peroxide (mg O2/kg)
Organic
Conventional
5.40 n.s.
5.07 n.s.
n.s.
5.80 n.s.
5.40 n.s.
n.s.
5.60
5.23
n.s.
Total phenols
(mg/kg)
Organic
Conventional
Organoleptic score
Organic
Conventional
Oleic acid (%)
Organic
Conventional
225.63 n.s.
266.23 A
n.s.
7.00 A
6.66 a
n.s.
66.22 n.s.
65.15 n.s.
n.s.
180.13 n.s.
177.10 B
n.s.
5.53 B
5.53 b
n.s.
66.21 n.s.
68.74 n.s.
n.s.
202.88
221.67
n.s.
6.27
6.00
n.s.
66.22
66.94
n.s.
Organic
Oleuropein (mg/kg)
0.073 A
0.120 B
0.096
(a) Overall means.
Differences within the same row (Tukey test): n.s.=not significant; means in row followed by the same letter are
not significantly different at P 0.01 (capital letters) or P 0.05. Differences within the same column (Tukey test):
n.s.=not significant, * P<0.05.
122
25
EFFICACY EVALUATION OF BIOLOGICAL CONTROL AGENTS AGAINST
PLASMOPARA VITICOLA
Ilaria Pertot, Federica De Luca, Antonella Vecchione, Luca Zulini
Istituto Agrario di San Michele all’Adige, via E. Mach 1, S. Michele all’Adige (TN)
[email protected]
Keywords
downy mildew, Plasmopara viticola, biological control agents, organic agriculture.
Introduction
Downy mildew causal agent, Plasmopara viticola (Berk. et Curt.) Berl. et De Toni, is one of the most important
grapevine pathogens, causing great losses if no protective treatments are applied (Kortekamp, 1997).
Nowadays consumer and farmer are much more concerned about food, health and environment safety (Butt,
2001). The conditions for using copper will be restricted by European Union by fixing a ceiling on use expressed
in terms of kilograms of copper per hectare per year in organic agriculture. Antagonist microorganisms could be
a possible alternative to copper in downy mildew controlling. Epidemiological characteristics of downy mildew
probably would not allow a complete field control based on a single biocontrol agent (BCA). A possible solution
for improving efficacy of BCA could be the integration of several micro-organisms with different action
mechanisms on specific stages of disease: i) during oospore overwintering and germination, for primary
inoculum reduction ii) and during sporangia germination for protection against secondary infections.
Micro-organisms were isolated from grapevine leaf material and rhizosphere of 18 different vineyards located in
the north –east of Italy. Vineyards were abandoned and chemically untreated for at least three years. Microorganisms were isolated on potato dextrose agar, malt agar and nutrient sucrose agar and grown at 20°C. Several
isolation methods were used: inocula were based on leaf washing water, necrotic leaf portions grounded in sterile
water with mortar and pestle, necrotic leaf pieces, infected leaf dish maintained in contact with naturally
degraded leaf material and root surface material. For each kind of material used, all morphologically different
colonies of fungi and bacteria were collected. A sample of 46 isolates was evaluated for sporangia germination
and infection inhibition activity. A sample of 33 isolates was evaluated for overwintering oospores inhibition
activity.
The evaluation methods used were:
i)
oospore germination inhibition, based on a modified floating leaf disk method (Hill);
ii)
inhibition of sporangia germination, based on reduction percentage of sporangia germination in a 1:1
water solution of the isolate culture broth and P. viticola sporangia;
iii)
infection inhibition, based on the reduction of the number of sporangiophore developed on isolate
culture broth treated leaves.
Several micro-organisms have induced a partial inhibition of sporangia germination (tab. 1), but the complete
control of infection on leaf can be achieved with only few of them.
123
Tab. 1 - Biological Control Agents (BCA) efficacy on sporangia germination and
infection process inhibition.
BCA
% germinated
sporangia
Inhibition of infection
process
Level of
inhibition
1
3
yes
C
2
2
no
A
3
0
no
B
4
3
no
A
5
4
no
A
6
0
no
B
7
3
no
A
8
0
partial
B
9
2
yes
B
10
4
yes
A
11
0
no
B
12
0
no
B
13
untreated
no
-
14
1
no
A
15
0
no
B
16
0
no
B
17
1
yes
D
18
1
no
A
A) No sporangia germination inhibition as well as infection process.
B) Partial sporangia germination inhibition or infection process.
C) Inhibition of infection process, but no inhibition of sporangia germination.
D) Sporangia germination inhibition as well as infection process.
Twenty micro-organisms have given a good control on conidiophore development (fig.1) with less than thirty
conidiophore/cm2 developed. Best results have been found on oospore germination: 42% of the tested
microorganisms completely inhibited of oospore germination (fig. 2), while the remaining 58% showed some
degrees of efficacy on the oospore germination inhibition and on the time necessary for oospore germination in
optimal conditions.
124
Fig. 1 – Effects of Biological Control Agents (BCA) on sporangiophore development of P. viticola (in vitro leaf
infections).
g
18
29
fg
22
efg
13
15
def
28
ef
cde
20
Untreated
bcd
27
bc
1
24
5
abc
16
Isolated
19
8
14
21
ab
25
10
3
6
26
23
17
a
12
11
9
7
4
2
0
10
20
30
40
50
60
70
80
90
N° Sporangiophore/cm2
125
100
Fig. 2 – Efficacy evaluation of BCA on overwintering oospore germination.
BCA Efficacy on overwintering
oospore germination.
N° of sporulated disk
BCA which have partially inhibited the
overwintering oospore germination.
16
14
12
10
8
6
4
2
0
Not sporulated
42%
A
A
98 119
35
A
22
2
11
86
2A
34
99
44
Isolate
F
F
77 120
13
37
A
49
A
59
69
93
A
d
10 ate
e
tr
un
Time for oospore germination in samples treated with
BCA which have which have partially inhibited the
overwintering oospore germination.
Sporulated
58%
N° days for oospore
germination
7
6
5
4
3
2
1
0
d
te
ea
tr
un
2
11
2A
69
13
37
93
A
10
A
A
98 119
Isolate
A
22
99
44
F
F
77 120
A
59
35
86
34
A
49
A strong reduction of the overwintering inoculum could be useful in downy mildew control, in fact new
epidemiological studies have demonstrated that primary infections play an important role in the disease
development (Pertot et al., 2002). If biocontrol activity of the studied micro-organisms is confirmed in field
trials, the combined effect of several BCAs on the different biological stages of the pathogen will be evaluated.
In such way BCAs could help in reducing copper quantity in organic viticulture.
Acknowledgements
This work was funded by the Fund for Research of the Autonomous Province of Trento, Italy, Research project
AGRIBIO.
References
Kortekamp A. (1997) Epicoccum nigrum Link: A biological control agent of Plasmopara viticola (Berk. et Curt.)
Berl. et De Toni. Vitis 36 (4), 215-216.
Butt T. M., Jackson C., Magan N. (2001) Introduction- Fungal Biological Control Agents: Progress, Problems
and Potential. In: Fungi as biocontrol Agents. Progress, Problems and Potential. CABI Publishing, Wallingford,
UK, 1-8.
Hill G. () IOBC Bulletin.
Pertot I., Gobbin D., Gessler C. (2002) Epidemiology of Plasmopara viticola: field distribution of primary and
secondary infections in the early stage of the season and microclimate impact on infection severity. In:
Proceedings of the 4th International Workshop on Powdery and Downy Mildew in Grapevine. Napa, California,
U.S.A., September 30-October 04, 2002.
126
26
IL CONTROLLO DELLE OPERAZIONI DI CAMPO COME BASE DELLA RINTRACCIABILITÀ A
GARANZIA DEL CONSUMATORE E DELL’AMBIENTE
Alessandro Pulga (AIAB – ICEA – Bologna)
Ivano Valmori (Image Line – Banca dati Biolgest – Faenza)
Dall’aprile del 2002 è stato creato un servizio su internet destinato alla gestione dei dati relativi alle operazioni
colturali svolte presso le aziende agricole in coltivazione biologica.
L’iniziativa ha avuto come obiettivo quello di raccogliere le informazioni relativamente a:
- registrazione di tutti gli interventi di difesa fitosanitaria e controllo delle avversità (svolta con prodotti
fitosanitari, biologici e naturali, ausiliari e sistemi basati su trappole e feromoni);
- registrazione delle fertilizzazioni minerali ed organiche;
- registrazione di tutte le operazioni di campo (lavorazioni, sfalci, etc.);
- registrazione delle operazioni colturali (potature, diradamenti, raccolta, etc.);
- registrazione degli eventi atmosferici;
- registrazione dei conferimenti;
- gestione dei magazzini aziendali;
- tenuta del quaderno di campagna in base alla normativa 290/01 (registro dei trattamenti effettuati).
Il tutto viene reso fruibile direttamente su Internet, sul sito www.quadernodicampagna.it, permettendo a tecnici
ed agricoltori di interagire con il sistema. Gli agricoltori, di fatto unici depositari delle informazioni reali sul
ciclo produttivo della merce, sono incentivati ad utilizzare il sistema in quanto ricevono dallo stesso una serie di
agevolazioni operative quali:
- controllo incrociato delle registrazioni dei prodotti utilizzati in base alle banche dati dei fitofarmaci;
- verifica del rispetto degli intervalli di sicurezza;
- verifica delle giacenze e delle scorte di magazzino;
- simulazione delle operazioni colturali al fine di verificarne la correttezza.
I vantaggi del progetto.
Per i tecnici:
- erogare un servizio tecnico direttamente dalla propria sede (ricetta on-line);
- tenere traccia dei consigli forniti per ogni appezzamento di ogni agricoltore;
- verificare la correttezza delle operazioni svolte.
Per gli agricoltori:
- massima semplicità di utilizzo del sistema;
- possibilità di assolvere agli obblighi imposti dalla normativa vigente in materia di agricoltura biologica
(registrazioni obbligatorie previste dal Reg. CEE 2092/91 e D.lgs. 220/95);
- fornire tutti i dati utili richiesti dai clienti (in particolare dalla GDO);
- nessun software da installare e aggiornare, ma collegamento al servizio tramite area riservata attraverso
internet, anche con un semplice modem standard;
- garanzia del salvataggio dei dati (backup);
- disponibilità della versione sempre più efficiente e con banche dati aggiornate settimanalmente.
Per gli Organismi di Controllo e le autorità pubbliche di sorveglianza:
- possibilità di acquisire tutte le informazioni – utili ai fini del controllo e della certificazione – in formato
elettronico;
- possibilità di dialogo e di scarico dei dati nei propri sistemi informatici;
- i dati in formato elettronico sono più facilmente fruibili e permettono controlli più veloci ed efficaci.
Per i consumatori ed il mercato:
- massima trasparenza nel ciclo produttivo e garanzia di poter risalire all’origine del prodotto ed entrare
nel dettaglio di tutti gli interventi effettuati dal campo alla tavola;
- possibilità di sapere cosa si consuma.
Questo sistema costituisce sicuramente un primo importante strumento per creare la base dati fondamentale per
sviluppare sistemi di rintracciabilità, dalle fasi a monte della filiera produttiva fino al consumatore finale.
Questo sistema, opportunamente implementato e validato da un Ente di certificazione di parte terza, può
diventare strumento utile a tutte le organizzazioni interessate, al fine dell’ottenimento di certificazioni volontarie
di prodotto e della certificazione di rintracciabilità di filiera in conformità alla norma UNI 10939.
127
27
ECO-TOXICITY OF THE ENTOMOPATHOGENIC BACTERIUM-NEMATODE SYMBIOTIC
COMPLEX TOWARD NON-TARGET ORGANISMS
M. Ricci, M. Colli, R. Barcarotti and A. Ragni
Tel: ++39 – 075 - 895091 Fax: ++39 – 075 – 888 776 E-mail: [email protected]
Introduction
In agriculture, the use of bio-control agents is growing; this is due, in particular, to their ability to avoid pest’s
resistance and to their friendly environmental impact.
A mini-review of the entomopathogenic bacterium-nematode symbiotic complex toward non-target organisms is
presented; bibliographic data and original data coming from research are shown. In particular it is mentioned a
work which first demonstrated specificity of the entomopathogenic bacterium-nematode symbiotic complex
toward target organisms (insects).
The present work concerns three sections related to the eco-toxicity of entomopatogenic nematodes: a) Short
review; b) Demonstration of entomopathogenic nematodes host specificity; c) Laboratory results toward nontarget hosts: Earthwoms, Scorpions and Millipeds.
a) Short review
Poinar (1990) reported that although Steinernematids and Heterorhabditids are considered entomopathogenic
nematodes because all natural infections have involved insects, they do have the ability to enter and kill noninsect invertebrates, at least in the laboratory (Wayne et al., 1987). Steps of their infection process are normally
completed in most insects but various obstacles to their completion occur with many non-insect invertebrates
(Wayne et al., 1987). The effect of these nematodes on vertebrates is of a great concern. Off all the vertebrates
(among the following classes: Pisces, Reptilia, Amphibia, Aves and Mammalia) thus far challenged with
infective stage of Steinernematids and Heterorhabditids, only young tadpoles of frogs and toads were vulnerable.
But the reason was given to the fact that when penetrating into the host the nematodes were carrying foreign
bacteria.
b) Demonstration of entomopathogenic nematodes host specificity ( Ricci et al., 1994)
Introduction
Entomopathogenic nematode infective juveniles (IJs) locate and parasitise potential hosts. The success of this
process depends with suitability of the host. Ricci et al. (1994) and Lewis et al. (1996), demonstrated that a
nematode's subsequent degree of attraction to CO2, stimulated by contact with the cuticle of an arthropod,
should signify that the exposed IJs recognize it as a potential host. The level of stimulation caused to S.
carpocapsae, a nematode that forages primarily by ambushing passing hosts, by 12 potential hosts (3
Lepidoptera, 4 Coleoptera, 1 Orthoptera, 1 Blattodea, 1 Diptera and 2 non-insect arthropods) was compared. To
determine whether arthropods that stimulate a high level of attraction of IJs are suitable hosts, three parameters
were tested: nematode induced mortality, nematode invasion rate and reproductive potential.
Behavioral Recognition Assay: nematodes were exposed for a certain time to the cuticle of different insects or
non-insects or inert materials, then exposed to chemical volatile cues emitted by the insects Galleria mellonella
in an apparatus called Agar Plate Arena (see Figure 1). After a certain time the nematodes that accumulated in
the proximity of the cue emissions, were collected and counted.
Nematode Infectivity Assay: host mortality was recorded after 2 days in Petri dishes at the concentrations of 200
and 500 nematodes per host.
Nematode Invasion Assay: Nematodes that penetrated the host during the Infectivity assay were counted.
Reproductive Potential Assay: pre-weighed hosts were exposed to 500 IJs. After 2 days the cadavers were
transferred on a nematode collecting trap. Every 2 days, the emerged IJs were collected until the exploitation of
all the host tissues was completed.
128
Results and Discussion
S. carpocapsae was differentially stimulated to be attracted to G. mellonella volatiles by the contact of the
cuticle of different arthropods. Agrotis ipsilon pupa, Leptinotarsa decemlineata, Isopoda sp., Blattella
germanica, Musca domestica and Diplopoda sp. were not significantly different from the controls. A. ipsilon
larva, Tenebrio molitor, Acheta domesticus, G. mellonella, Diabrotica virgifera virgifera and Popilia japonica
induced a significantly higher "activation" to S. carpocapsae IJs compare to the controls. It seems that S.
carpocapsae can discriminate even among different stage of the same species in fact the value of A. ipsilon larva
is more than twice that of the pupa.
The same hosts that induced the higher attraction to the volatiles were more susceptible to the nematodes.
The nematodes, established (penetration rate and reproduction index) better in the group of arthropods that
where better stimulated by G. mellonella volatiles.
It has been demonstrated that, at least one entomopathogenic nematode, S. carpocapsae, could discriminate
among suitable and not suitable hosts.
Figure 1: Agar Plate Arena
G. mellonella larvae
Pipette
tip
Application point of nematodes
Chamber with gradient
Plexiglas lid
N ematodes
Glass plate
Agar substrate
Rubber
c) Laboratory results toward non-target hosts:
The non-insect organisms, earthworms, scorpions and millipedes have been challenged with different strains of
entomopathogenic nematodes.
Material and methods
Hosts: the non-insect organisms, earthworms, scorpions and millipedes have been captured under stones, and
stored at 12°C for 24 hours prior the experiment.
Nematodes: were reared in Galleria mellonella last instar larvae by standard rearing procedures.
For earthworms the following nematode strains were tested: Heterorhabditis bacteriophora NJ, Steinernema
kraussei N0093 and S. feltiae UK.
For Scorpions and Millipedes: H. bacteriophora NJ, S. kraussei N0093, S. sp. N0166, S. sp. N0167, S. sp.
N0168, H. sp. N0169, H. sp. N0170. The infectivity of nematodes was tested in parallel on 100 mg larvae of
Tenebrio molitor (10 larvae per box with 1500 nematodes). There were 3 repetitions per treatment. Incubation
was done at 23°C for 5 days. Controls (only water) were always included.
Tests with Earthworms: in 10x7x5 cm plastic boxes, 50 g of peat with 75% humidity, 5 earthworm were
challenged with 3000 infective nematodes. There were 3 repetitions per treatment. Incubation was done at 23°C
for 7 and 14 days.
Tests with Millipedes: in 10x7x5 cm plastic boxes, 50 g of peat with 75% humidity, 3 millipedes were
challenged with 1500 infective nematodes. There were 3 repetitions per treatment. Incubation was done at 23°C
for 5 days.
Tests with Scorpions: they were tested in Petri dishes with 3000 nematodes per individual for 5 days at 23°C.
Results
Mortality of Earthworms challenged with entomopathogenic nematodes (see Tab. 1) did not differed among
treatments both at 7 (P=05957) and 14 days (P=0,7520).
129
Tab. 1
Average Earthworm
mortality at 7 days
Treatments
Steinernema feltiae UK
S. kraussei N0093
Heterorhabditis bacteriophora NJ
Control
6,67 %
20,00 %
0,00 %
33,33 %
Average Earthworm
mortality at 14 days
6,67 %
20,00 %
0,00 %
33,33 %
Mortality of the Insect Tenebrio molitor and the non-insects Millipedes and Scorpions challenged with
entomopathogenic nematodes (see Tab. 2) revealed as follows: the effect of nematodes is not different from the
control in Millipedes (P=0,66) and in Scorpions (P=1)
Tab. 2
Treatments
S. kraussei N0093
H. bacteriophora NJ
S. sp. N0166
S. sp. N0167
S. sp. N0168
H. sp. N0169
H. sp. N0170
Control
Tenebrio molitor
larvae (insects)
mortality
Millipedes
(non-insects)
mortality
73,33 %
86,67 %
73,33 %
93,33 %
40,00 %
6,67 %
96,67 %
0,00 %
0,00 %
0,00 %
0,00%
0,00 %
0,00 %
11,11 %
11,11 %
11,11 %
Scorpions
(non-insects)
mortality
0,00 %
0,00 %
0,00 %
0,00 %
0,00 %
0,00 %
0,00 %
0,00 %
Conclusions and Discussion
Unlike other microbial pesticides, such as Bacillus thuringiensis, viruses and fungi, entomopathogenic
nematodes are exempt from registration in the USA and other countries.Currently, all available evidence
(bibliography) indicates that beneficial nematodes and their associated bacteria have no negative impact on nontarget organisms in the agrarian environment (Akhurst 1990; Georgis et al., 1991).
Entomopathogenic nematodes have the ability to discriminate among suitable and not suitable different potential
hosts.
Recent experiments (see above), demonstrated that while different strains of entomopathogenic nematodes were
lethal to insect larvae (Tenebrio molitor), did not infect non-target organisms such as Heartworms, Scorpions and
Millipedes.
In conclusion entomopathogenic nematodes are safe to humans and to non-target organisms, non-polluting and
thus environmentally safe and acceptable.
Acknowledgment
This research was partially founded by MURST grant Cluster C06-07 L. 488-92
References:
Akhurst, R.J. 1990. Safety to nontarget invertebrates of nematodes of econ omically important pests. In: Laird et
al., pp. 233-240.
Georgis, R., Kaya, H.K. and Gaugler, R. 1991. Effect of steinernematid and heterorhabditid nematodes
(Rhabditida: Steinernematidae: Heterorhabditidae) on nontarget arthropods. Environ. Entomol. 20(3) : 815-822.
Lewis E.E., Ricci M. and Gaugler R., 1996. Host recognition behaviour predicts host suitability in the
entomopathogenic nematode Steinernema carpocapsae (Rhabditida: Steinernematidae) Parasitology (1996) 1136, 573-581
Poinar Jr, G.O., 1990. Taxonomy and Biology of Steinernematidae and Heterorhabditidae . In: (Eds. R. Gaugler
and H.K. Kaya) Entomopathogenic nematodes in biological control, pp. 23 -60. CRC Press, Boca Raton, U.S.A.
Ricci M., Lewis E.E. and Gaugler R., 1994. Host discrimination by Steinernema carpocapsae (Weiser) All strain;
at Debrecen, Hungary: Cost 819 Symposium & Workshop on Entomopathogenic Nematodes. In: C.T. Griffin,
R.L. Gwynn & J.P. Masson (Eds.) COST 819, Biotechnology, Ecology and transmission strategies of
130
entompathogenic nematodes. CSC-EC EAEC, Brussels, Luxembourg. p. 105 Smart Jr., G.C., 1995.
Entomopathogenic Nematodes for the Biological control of Insects . Supplement to the J. of Nematology (27),
529-534.
Wayne, L.G., Brenner, D.J., Colwell, R.R., Grimont, P.A.D., Kandler, O., Krichevsky, M.I., Moore, L.H.,
Moore, W.E.C., Murray, R.G.E., Stackebrandt, E., Starr, M.P. and Truper, H.G., 1987. Report on the ad hoc
committee on reconciliation of approaches to bacterial systematics . Int. J. Syst. Bacteriol., 37, 463.
131
28
PHOTORHABDUS AND XENORHABDUS: A NEW SOURCE OF USEFUL COMPOUNDS FOR
BIOLOGICAL CONTROL
A. Ragni and F. Valentini
IntroductionThe genera Photorhabdus and Xenorhabdus are symbiotic Enterobacteriaceae bacteria
associated with entomopathogenic nematodes of the genera Heterorhabditis and Steinernema, respectively. The
characteristics of both Photorhabdus and Xenorhabdus genera have been reviewed by Forst & Nealson (1996),
and by Forst et al. (1997).
Recently Bowen et al. (1998a; 1998b) and Blackburn et al. (1998) discovered and characterized an insecticidal
toxin complex that it is secreted into the culture medium by Photorhabdus luminescens strain W-14. Except for
this specific strain, the insecticidal capability of entomopathogenic soil nematode symbiotic bacteria has been
reported only when carried into the insect by the nematodes or artificially injected into their haemocel.
This work reports the isolation of a unique strain Photorhabdus sp., named XP01, selected among 12
Photorhabdus and Xenorhabdus isolates, that displays an insecticidal activity when fed to the neonate larvae of
several Lepidoptera pests.Materials and Methods.
Bacteria isolation
The bacteria strains tested were isolated as described by Akhurst (1980), from cadavers of Galleria mellonella
infected with Heterorhabditis spp. or with Steinernema spp. strains belonging to the collection of
entomopathogenic nematodes held at BioTecnologie S.r.l (BT) laboratories. A list of the isolated strains is
shown on Table 1. Cultures were maintained by weekly subculturing on Nutrient Agar plates and incubated in
the dark at 25°C. Inocula for liquid cultures were prepared as fermented broth aliquots containing glycerol at a
final concentration of 15% (v/v) and stored at -80°C.
Bacterial preparations
Whole cultures were obtained by growing the strains in a Casein peptone plus yeast extract liquid medium in the
dark at 25°C and shaking at 180 rpm , for a period of either 19 or 24 hours. Technical powder of XP01 was
prepared by freeze-drying the pellet obtained by centrifugation the bacterial culture at 6,238 x g for 40 min. The
pellet was washed three times in a neutral Ringer’s solution prior freezing. Freeze-drying resulted in cells dead
since plating the resuspended technical powder gave no colony growing on Nutrient Agar.
Insects rearing and bioassay techniques
Colonies of Mamestra brassicae, Cydia molesta, Spodoptera littoralis, Helicoverpa armigera, Scotia segetum
and Agrotis ipsilon are continuously reared at BT insectarium under controlled environmental conditions and fed
with semi-synthetic diets.
Insecticidal activity of bacterial suspensions was measured by using an overlay technique bioassay. Bacterial
preparations at different serial dilutions were applied in 50 µl aliquots in the wells of a diet bioassay trays
(surface area 175 mm2) containing 1 g of an insect meridic diet. As control, the insect diet was treated with
deionized sterile water. After drying, one unfed 1st instar larva (neonate) was placed in each well. Each
treatment was applied to 32 insects. Mortality was scored after 5 days of incubation at 27°C.
Results.
Preliminary Screenings
A total of 12 Photorhabdus and Xenorhabdus bacterial isolates were assayed for insecticidal activity against
neonates of Mamestra brassicae and of Cydia molesta in a surface contamination assay. As shown in Figure 2,
neonates of Cydia molesta were more susceptible than those of Mamestra brassicae when exposed to fermented
broths of Photorhabdus and Xenorhabdus isolates. Treatments with XP01 and XP04 fermented broths resulted in
100 % mortality of C. molesta neonates. In this species the mortality rate ranged from 25 to 41% for treatments
with XP136, XP1007 and XP127. Low activity (from 0% to 19% mortality) against C. molesta neonates was
recorded when treatments were done with the remaining bacterial strains. A 100% mortality of Mamestra
brassicae neonates was observed when the insects were treated with XP01. A lower mortality equal to 74% was
observed when XP04 was used. A residual activity (5% larval mortality) was recorded when treatment was done
with strain XP10.
132
After this preliminary screening and due to the high activity of XP01 against both larvae insect tested, this strain
was selected for further characterization of its insecticidal activity.
The XP01 activity was further tested on several other lepidopteran species. For convenience purposes only
macrolepidopteran species were used in these assays. Results of this second screening are shown in Figure 3.
The whole culture of XP01 showed the capability to kill all the five species treated, being most effective against
M. brassicae, S. segetum and A. ipsilon.
The insecticidal activity of freezed dried technical powder, made from the cell pellet of fermented broth, was
tested against neonates of M. brassicae. As seen before with other bacterial preparations, the observed mortality
of insects was proportional to the applied dose (Figure 4).
Discussion.
Photorhabdus and Xenorhabdus species are bacteria known to live in symbiotic association with
entomopathogenic soil nematodes. Mechanisms involved in the pathogenicity of these bacteria resulting from
their release by entomopathogenic nematodes in insects, have been extensively studied. A great deal of
variability in the bacterial dose needed to kill different insects has been reported. The effect both depending on
the bacterial strain used as well as the target insect studied.
The present study describes the discovery of a newly characterize strain of Photorhabdus luminescens,
denominated XP01, which is highly toxic when directly administered by food contamination assays to neonates
larvae of several Lepidopteran pests including Mamestra brassicae and Cydia molesta.
It was demonstrated that the toxic effect of XP01 is maintained even when a technical powder is used. Since
technical powders do not contain alive cells, this result indicates that once the toxin has been synthesized alive
bacterial cells are not further required for activity. This characteristic of XP01 insecticidal capability is similar to
what has been described for Bacillus thuringiensis toxins (Schnepf et al., 1998).
The discovery of the insecticidal activity of Photorhabdus luminescens strain XP01 indicates the existence of a
presumably new family of entomotoxic proteins present in Photorhabdus and probably Xenorhabdus spp. These
insecticidal toxins could be used as an active bio-pesticide ingredient in a similar manner to the delta-endotoxins
of B. thuringiensis. This new source of natural toxins could help both the fight against the development of
insects resistance as well as serve as sources of novel specific activities in combination with other known
biopesticides.Acknowledgements
We thank G. Flek for rearing insects for tests, L. Quattrocchi and R. Lorenzini for their assistance in
microbiology and bioassay works.
References
Akhurst, R.J. 1980. Morphological and Functional Dimorphism in Xenorhabdus spp., of bacteria symbiotically
associated with insect pathogenic nematodes Neoplectana and Heterorhabditis. J. Gen. Microbiol. 121: 303309.
Blackburn, M., E. Golubeva, D. Bowen, and. R.H. Ffrench-Constant. 1998. A novel insecticidal toxin from
Photorhabdus luminescens, toxin complex a (Tca), and its histopathological effect on the midgut of Manduca
sexta. Appl. Environ. Microbiol. 64: 3036-3041.
Bowen, D.J., T.A. Rocheleau, M. Blackburn, O. Andreev, E. Golubeva, R. Bhartia, and R.H. Ffrench-Constant.
1998a. Insecticidal toxin from the bacterium Photorhabdus luminescens. Science. 280: 2129-2132.
Bowen, D.J., and J.C. Ensign. 1998b. Purification and characterization of a high-molecular-weight insecticidal
protein complex produced by the entomopathogenic bacterium Photorhabdus luminescens. Appl. Environ.
Microbiol. 64: 3029-3035.
Forst, S. and K. Nealson. 1996. Molecular biology of the symbiotic-pathogenic bacteria Xenorhabdus and
Photorhabdus spp. Microbiol. Rev. 60: 21-43.
Forst, S., B. Dowds, N.E. Boemare, and E. Stackebrandt. 1997. Xenorhabdus and Photorhabdus spp.: bugs that
kill bugs. Annu. Rev. Microbiol. 51: 47-72.
Schnepf, E., N. Crickmore, J. Van Rie, D. Lerecluse, J. Baum, J. Feitelson, D.R. Zeigler, and D.H. Dean. 1998.
Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62: 775 -806.
133
Photorhabdus strain XP01cells showing inclusion bodies.
TABLE 1. Bacterial isolates tested
Bacterial isolate
Bacterial genus or species
XP01
Photorhabdus luminescens
XP1007
XP127
XP81
XPHDO
XPLon
XPMib
XP04
XP05
Xenorhabdus sp.
XP10
XP136
XP98
FIG. 2. Mortality of neonates of two Lepidopteran pests treated with non-diluted fermented broth of several
bacteria strains. Bacterial broths were normalized in terms of number of cells per ml before the use. Each bar
represents the percent mortality obtained treating a total of 32 insects.
Larval mortality (%)
120
100
80
60
40
20
0
XP01
XP04
XP05
XP10
XP1007
XP127
XP136
XP81
XP98
XPHDO
XPLon
XPMib
Strain
Mamestra brassicae
Cydia molesta
134
FIGURE 3. Insecticidal activity of fermented broth of XP01 strain against neonates of Mamestra brassicae,
100
80
60
40
20
0
1:1
1:4
1:8
1:16
untreated
Dilution
Spodoptera littoralis
Helicoverpa armigera
Scotia segetum
Mamestra brassicae
Agrotis ipsilon
Spodoptera littoralis, Helicoverpa armigera, Scotia segetum and Agrotis ipsilon. n= 32 larvae for each treatment
FIGURE 4. Insecticidal activity of fermented broth XP01 strain and of XP01 technical powder (resuspended to
the original broth concentration of suspended solids) against neonates of Mamestra brassicae. Each point
represents the percent mortality obtained treating a total of 96 insects in 3 replicate bioassays with 32 insect
each.
100
80
60
40
20
0
1:1
1:2
1:4
1:8
1:16
Dilution rate
XP01broth
XP01 F.D. Technical powder
135
29
BIOASSAYS FOR SCREENINGS AND QUALITY CONTROL OF PRODUCTS WITH
INSECTICIDAL, FUNGICIDAL AND NEMATOCIDAL ACTIVITY, BASED ON PLANT EXTRACTS,
MICROORGANISMS AND CHEMICAL MOLECULES
M. Ricci, G. Flek, M. Colli, R. Barcarotti, L. Quattrocchi, S. Coranelli, L. Concezzi, A.P. Fifi, R. De Nicola, M.
Scribano, M. De Berardinis, F. Citarrei and A. Ragni
Introduction
A bioassay is an experiment where a living organism (insect, plant, phytopathogenic nematode and others) is
used as test (Robertson J.L. & Preisler H.K.). It is a fundamental tool in the R & D of agricultural products.
Bioassays are used to discover new molecules and/or toxins, to compare different pesticides, to evaluate the
feasibility of excipients for formulations, attractants and/or repellents, and for the quality control of products
ready for the market. Does not exist a universal bioassay: for each purposes a specific bioassy should be used or,
if not available, should be developed (Ricci et al., 1996). A good bioassay should be cheap, fast, should allow the
repeatability of the results in different dates and different sites, should discriminate two products with little
differences, and should predict, as far as possible, the further performance of the product in field conditions.
Here, are presented a series of bioassays that are used, adapted and developed by the company BioTecnologie
B.T. S.r.l., for its R&D programs or for services for third parties.
Bioassays
Commercial companies that produce and sell bio-products for agricultural use, based on Entomopathogenic
Nematodes, Fungi, Bacteria and Viruses, have to set up a bioassay unit in order to perform screening programs
and to carry out quality control of the production and quality control of final products.
The bioassay should be a compromise between the need to assure scientific results and the need to save time and
money.
To support a rational bioassay unit, other sections are needed: Insectary, Plant Pathogen Maintenance and
Greenhouses.
Screenings
Screenings are performed in order to discover new active ingredients or to support the development of products.
According to the different needs, different bioassays are designed.
At BioTecnologie B.T., since 1992, several procedures for bioassays have been developed.
Toxicity bioassays and feeding behavior tests are performed daily for inside and outside (external service)
research purposes.
Quality Control of Bio-products for agricultural pest control
The first duty of the quality control system is to assess what is declared on the label of the product in terms of
product weight and titer of active ingredient.
Even if bio-products are based on non-pathogenic (to human beings) microorganisms, the producer has to assure
that his product is free of pathogenic contaminants (Ricci and Fridlender, 1998).
Stability is an important parameter in terms of maintenance of the product characteristics during storage and
transportation.
The activity is the parameter that shows the ability of the product to control the target insects in real conditions.
This is the harder parameter to assess because a simple, cheap, fast and reliable laboratory bioassay has to be
found in order to predict the activity of the product under field conditions
Insectary
A multiple-species insect rearing facility for research support on bio-insecticide development is operating at
BioTecnologie B.T. S.r.l., in Umbria – Italy, since 1992.
136
Several colonies of Lepidopteran, Coleopteran and Dipteran species have been established and are maintained on
a regular basis whereas other species are reared on a temporary basis when needed. Standardized rearing
procedures have been developed that ensure consistent quality of the test insects.
The following species are currently reared:
Lepidopteran:
Agrotis ipsilonMamestra brassicae, Spodoptera littoralis, S. exigua, Helicoverpa armigera, Lobesia botrana,
Cydia molestaColeopteran:
Otiorhynchus sp., Tenebrio molitorDipteran:
Culex pipiens pipiens
Plant Pathogen Colony Maintainance
Several pathogens are maintained regularly in vitro to support the research and development of bio-phytosanitary
products.
The following fungi species are currently maintained:
Fusarium culmorum, F. crookwellense, F. oxysprum dianthii, F. sambucinum, F. solani, F. graminearum, F.
akuminatum, F. oxysporum, Rhizoctonia solani, Alternaria solani, Botrytis cynerea, Penicillum italicum,
Cercospora beticola, Phytophthora cinnamoni, Pyranochaeta lycopersici, Phoma spp., Microdochium nivale
The following phytopathogenic nematode species is currently reared:
Meloidogyne incognita
Greenhouses
Modern and well-equipped greenhouses support the growth of different plant species for insect and
phytopathogenic nematode rearing, pathogen maintenance and for glasshouse assays.
The following plants have been grown:
Corn, Melon, Tomato, Pepper, Cyclamen, Impatient, Tobacco, Celery, Rapeseed .
Conclusions:
Due to its facility and its experience, BioTecnologie B.T., is able to design, develop and perform several
bioassays for the development and the quality control of pesticides and biopesticides.
References
Ricci M., Glazer I. and Gaugler R., 1996. Comparison of Bioassays to Measure Virulence of Different
Entomopathogenic Nematodes. Biocontrol Science and Technology (1996) 6, 235 -245
Ricci M. and Fridlender B., 1998. Quality Control of a Biological Insecticide Based on Entomopathogenic
Nematodes: NEMA-BIT. At Gent, B: Cost 819 Workshop on Quality Control of Entomopathogenic Nematodes.
p. 16 and 34-35
Robertson J.L. &. Priesler H.K, 1992. Pesticide bioassays with arthropods. CRC Press, Boca Raton.
Pictures
Bioassay Tray: most of the screenings for micro-organisms
and molecules against target insects are carried out in this
particular tray where individual insect is tested on an artificial
diet
137
Insectary: the Lepidopteran room, where different
lepidopteran species are reared in standard conditions
Tenebrio-Assay: researchers are carrying
on a bioassay
Greenhouse: a trial is the greenhouse
Phytopathogenic fungi: a bioassay to
screen micro-organisms and molecules
active against phytopathogenic fungi
138
139
30
ISOLATION OF NEW BIOLOGICAL CONTROL AGENTS FROM UMBRIA REGION
M. Scribano and A. Ragni
Summary
Samples of soil were collected in two areas of the Umbria region, Valnerina and Oasi di Alviano; the samples
were used for the isolation of entomopathogenic agents through a selective technique based on the usage of
bait. There were isolated 13 different samples of entomopathogenic nematodes, 25 entomopathogenic fungi, 27
sporogenous bacteria and 44 non-sporogenous bacteria. The activities of these microbiological agents were
verified through in vitro and in vivo tests.
1. Sampling: 80 soil samples (1 Kg each) were collected till the depth of 20/25 cm from two natural areas of
Umbria
region:
Valnerina
and
Oasi
di
Alviano.
2. Baiting with Galleria mellonella1: the soil samples were put in containers together with a bait, larvae of the
Galleria mellonella insect; they were incubated in the dark at 25°C. Periodically the boxes were controlled; the
dead insects were collected and used later for the isolation of microbiological agents.
3. Isolation of nematodes: the collection of nematodes was carried out through White-trap1: the gummyconsistence Galleria cadavers were placed on a filter paper imbibed with Ringer solution in order to allow the
nematodes to emerge (fig1). The entomopathogenic power of collected nematodes was tested through infections
of Galleria mellonella larvae2: 7 last-instar larvae of Galleria mellonella were treated with 100 nematodes per
larva.
4. Isolation of fungi3: the hyphae, developed on the surface of the turgid cadavers, were streaked on PDA (potato
dextrose agar) Petri dishes containing 150 mg/ml of streptomycin. Than the dishes were incubated at 25°C.
5. Isolation of bacteria:
•Non sporogenous bacteria: the flabby cadavers of Galleria were homogenized in a physiological solution; the
suspension was plated on NA (nutrient agar) Petri dishes containing 150 mg/ml of nystatin. Than the dishes were
incubated at 25°C.
•Sporogenous bacteria: an aliquot of the insect cadaver suspension was treated for 12 minutes at 78°C and then
was streaked on NA.
The entomopathogenic power of the isolated bacteria towards Mamestra brassicae (Lepidoptera) was verified
though in vivo tests using an overlay technique bioassay4: 32 neonates of M. brassicae were treated with the
fermented broths of the isolated bacteria. Identification of bacteria was performed with gallery API 50 CHB/E
Medium.
Results:
Only 3 out of 27 of the sporogenous bacteria caused 100% mortality towards Mamestra brassicae. API 50
CHB/E test showed that two of three active bacteria strains were Bacillus thuringiensis species as confirmed by
microscopical observation of the sporulating cells (fig.2). B. thuringiensis is the most important microbiological
agent for control of pest insects5. The third strain is being identified.The entomopathogenic power of the 13
isolated nematode strains were tested through infections of Galleria mellonella; the obtained mortality was high
although variable, as illustrated in Table1.
Conclusion
The study has shown the possibility of isolating samples of micro-organisms useful for the biological control
from natural habitats. The study will need more screenings in order to evaluate the entomopathogenic power,
the conditions of use and the possibility of production of the discovered micro-organisms for using them in the
integrated pest management.
Acknowledgements
140
M. Scribano research work was supported by grant of 3A Parco Tecnologico Agroalimentare dell’Umbria, in the
frame of Umbria Region Training programm (European Social Forum found).
References
.K.V. Deseo Kovacs, L. Rovesti. 1992. Lotta biologica contro i fitofagi; Edagricole
2.
Bedding, R.A. & Akhurst, R. J. 1975. A simple technique for the detection of insect parasitic rhabditid
nematodes in soil;
Poinar, G. O. 1979. In: Nematodes for biological control of insects. CRC Press, Boca Raton, Florida; pp.227.
3
Burge, M. N. 1988. Fungi in biological contol systems. Manchester Univ. Press, Manchester & New York;
pp.296;
4
Deseo, K. V. 1991. Batteri entomopatogeni come mezzi di lotta microbiolo gica. Inf. Fitopat. 10, 7-13.
5
Li, R. S., Jarrett, P. & Burges, H. D. 1987.– Importance of spores, crystal and delta endotoxin in the
pathogenicity of different varieties of Bacillus thuringiensis in Galleria Mellonella. J. Invert. Pathol. 50; 277284.
1
Figure 1. White-trap with entomopathogenic nematode parasitized insects
141
Figure 2. Vegative (a) and sporulating cells (b) of B. thuringiensis isolate
a
b
Table1. Mortality of last-instar larvae of G. mellonella
treated with nematodes isolates at the dose of
100 nematodes per larva. n=7
142
Nematode isolate
3
4
9
12
13
14
27
29
38
39
53
58
73
% Mortality
100
40
60
100
100
100
40
55
70
100
100
100
70
143
31
PROTEIN CONTENT AND MYCOTOXINS CONTAMINATION IN ORGANIC AND
CONVENTIONAL WHEAT
Pietri A., Bertuzzi T., Barbieri G. and Rossi F.
Istituto di Scienze degli Alimenti e della Nutrizione, Università Cattolica del Sacro Cuore, Facoltà di Agraria,
Piacenza.
Introduction
The importance of organic farming in terms of foods production and consumer confidence is increasing.
Numerous reports show as a growing number of people believe that organic foods are healthier than
conventional ones, particularly for the absence of pesticide residue in organic foods. However cereals from
organic production were sometimes found to have higher mycotoxins content than conventionally produced
foods, probably because if synthetic pesticide are not used there is a lower crop protection towards parasite
fungi. However due to some inconsistent results, a clear trend could not be identified. In respect of the proteins
content, a trend to lower values in organically produced cereals was observed.
Aim of this work was to verify the content of ocratoxin A (OTA), trichothecenes, ergosterol and crude protein in
organic and conventional wheat produced in the area of Piacenza (North Italy).
Thirty-five samples of wheat (20 organic and 15 conventional) were collected during storage and analyzed for
theirs mycotoxins contamination and crude protein content. OTA was extracted with a mixture of CH3OH:3%
NaHCO3 water solution(50:50) and purified using an immunoaffinity column (Ochraprep). OTA was determined
by reversed-phase HPLC with fluorescence detection (lecc=333 nm e lem=470 nm); a Select-B RP-8 column,
125x4mm (Merck), was employed at room temperature with a mobile phase of acetonitrile:acidified water (2%
CH3COOH)(41:59) at 1.2 ml/min. The detection limit was 0.010 mg/kg.
Deoxynivalenol (DON), 3 and 15-acetyl-deoxynivalenol (3-AcDON and 15-AcDON), nivalenol (NIV), HT-2
and T-2 were determined. These trichothecenes were extracted with acetonitrile:water (84:16) for 90 minutes and
an aliquot (6 ml) of the filtered extract was purified by Mycosep 227 column (Romer Labs). The final extract
was derivatised with N-trimethylsilylimidazole and analysed by GC-MS using an ion trap detector.
Trichothecenes were separated with a RTX-5 capillary column, 30mx0.25mm i.d. (Restek), and quantified by the
selected ion monitoring technique; the detection limit was 2 mg/kg.
Ergosterol analysis was performed according to AFNOR (Norme Francaise, 1991). After extraction with KOH
alcoholic solution, an aliquot (20 ml) of the filtered extract was purified by Extrelut column (Merck). Ergosterol
was determined by HPLC with UV detection (l=280 nm); a Superspher Si 60 125x4 mm column (Merck) was
employed with a mobile phase of n-hexane:isoamyl alcohol=98:2 at 1.0 ml/min. Detection limit was 0.4 mg/kg.
The N content was determined using the Kjeldahl technique. Statistical analysis of data was performed with the
statistical package SAS, using the PROC UNIVARIATE to verify the normality of data distribution and PROC
GLM and PROC NPAR1WAY for analysis of variance.
Results
The analyzed samples had a low contamination of mycotoxins, OTA levels were always lower than legal
threshold of 3 mg/kg and no significant differences were observed between the two types of wheat. The 45% of
organic samples were contaminated by OTA against a 40% in conventional wheat, however the most
contaminated sample (0.53 mg/kg) was found in conventional cereals.
The only detected trichothecene was DON, confirming the hypothesis that it is the most widespread in cereals.
Only 25% of organic wheat showed a DON contamination (max value = 17.1 mg/kg) while all conventional
samples were contaminated (max value =219.1 mg/kg). The DON content of organically produced wheat was
significant lower than conventionally grown one (2.85 mg/kg vs 99.67 mg/kg; P<0.01). According to ergosterol
content, all samples were of good quality since they had a contamination lower than 8 mg/kg, limit usually
accepted as index of wheat quality.
A higher crude protein level was observed in conventional when compared to organic wheat (12.96% vs 10.51;
P<0.01), this difference is probably due to a lower N fertilization.
144
In our trial organic wheat showed a lower mycotoxins contamination than conventional wheat, on the other hand
the adoption of organic production system leads to a reduction in crude protein content. Further studies are
required in order to clarify the effect of organic cultivation system on nutritive value and content of
antinutritional factor in wheat.
145
32
DETERMINAZIONE DI RESIDUI METALLICI NELLE PERE
E INFLUENZA SULL’IMPATTO AMBIENTALE
P. Tedeschi1, A. Maietti1, D. Mazzotta1, G. Vecchiati 1, P. Romano 2, V. Brandolini 1
1
Dipartimento di Scienze Farmaceutiche – Università di Ferrara
Dipartimento di Biologia, Difesa a Biotecnologie Agroforestali -Università della Basilicata
2
INTRODUZIONE
La presenza di residui di antiparassitari nelle derrate alimentari sia di origine animale (carni, latte, uova,
burro, eccetera) sia di origine vegetale (ortofrutticoli freschi, conservati o lavorati industrialmente, vino, olio),
determina, collateralmente alle preoccupazioni di ordine igienico-sanitario, anche ripercussioni negli scambi
commerciali, specialmente a livello internazionale. Al fine di limitare il più possibile la presenza dei residui di
fitofarmaci nelle derrate agricole e ridurre conseguentemente i rischi di intossicazioni acute e croniche per i
consumatori, si stanno studiando e valutando sistemi di coltivazione che consentano l’applicazione delle più
moderne tecniche di "produzione integrata" e di “produzione biologica”.
Per quanto riguarda la difesa dalle crittogame i formulati a base di rame sono prodotti chimici ammessi dai
disciplinari di produzione integrata e biologica. E’ stato dimostrato che il rame svolge un ruolo chiave in
numerose funzioni biochimiche e metaboliche degli organismi ma un suo accumulo può provocare fenomeni
tossici gravi. La dieta occidentale contiene abitualmente una quantità di rame più che sufficiente a ricoprire il
fabbisogno giornaliero che per un adulto sano, di età compresa tra i 23 e i 50 anni, è di circa 2 mg (1,5-3 mg).
Ne sono particolarmente ricchi i legumi, il pesce, i crostacei, la carne, i cereali e le noci.. Da studi epidemiologici
condotti precedentemente, risulta che per pazienti con elevati livelli di rame (> 1.43 mg/l) il rischio relativo di
morte per malattie cardiovascolari e tumori è quattro volte più elevato che nei soggetti con livelli normali
(<1.05mg/l). Ancora oggi le accurate ricerche che vengono condotte sia in campo medico, sia in campo
alimentare, mettono in evidenza l’elevata probabilità di inquinamento dei cibi con i residui dei trattamenti
chimici. Sarebbe quindi necessario approfondire ulteriormente le conoscenze sul tipo di impatto ambientale e
sulla contaminazione degli alimenti da parte dei residui di rame e di altri metalli presenti nelle formulazioni
commerciali.
MATERIALI E METODI
PROVE DI CAMPO
La parte sperimentale di campo è stata realizzata presso un frutteto di pere “Conference” suddiviso secondo lo
schema del “blocco randomizzato”. Le tesi a confronto erano le seguenti:
· Prova di difesa biologica per cui è stato previsto solo l’uso di ossicloruro di rame;
· Prova di difesa integrata: per cui è stato previsto l’uso di poltiglia bordolese integrata con altri principi
attivi di sintesi;
· Prova di controllo senza trattamenti anticrittogamici.
PREPARAZIONE DEI CAMPIONI.
Le foglie, il terreno e la parte edule delle pere (polpa e buccia) opportunamente sminuzzati e resi il più possibile
omogenei dopo essere stati preventivamente asciugati in stufa a 90°C per 4 h sono stati essiccati in stufa a
120°C per 1 h in capsule Petri e poi lasciati raffreddare in essiccatore.
SPETTROSCOPIA DI ASSORBIMENTO ATOMICO
I campioni costituiti sono stati sottoposti a mineralizzazione pesando circa 1 grammo di foglie e terreno e 2
grammi di frutti e trattandoli con 5 mL di HNO3 "supra-pur" al 65% in contenitori di teflon (tipo PTFE/PFA)
comunemente chiamati “bombe” con applicato ad ognuno un sistema refrigerante a ricaduta. Si inseriscono i
campioni in un forno a microonde (Millestone MLS 1200, FKV) opportunamente modificato, e si trattano
secondo uno schema "Tempi di irraggiamento/ Potenza di irraggiamento" appositamente messo a punto. Si
lasciano raffreddare i campioni nei provettoni, quindi si aggiunge, ad ognuno di essi, 1 mL di H2O2 a 130 volumi
146
per "strippare" i vapori nitrosi residui mediante saturazione dell'ambiente con O 2. Si scalda nuovamente ed
infine si lascia definitivamente raffreddare: si ottengono così soluzioni limpide, più o meno incolori.
Le analisi sono state effettuate in triplo e per la lettura dei campioni è stato utilizzato uno spettrofotometro per
assorbimento atomico Perkin Elmer 1100B dotato di una lampada a catodo cavo, a singolo o multielemento, di
tipo Intensitron TM Perkin Elmer e di un bruciatore a premiscelazione al Titanio, a fenditura singola di 10 cm,
per fiamma ad aria /acetilene (2145/2400°C SIO S.p.A. Milano).
Come prova di controllo (bianco) è stata eseguita una mineralizzazione con i soli reagenti al fine di osservare
loro eventuali interferenze nella determinazione quantitativa dei metalli presenti.
La prima parte del lavoro è stata incentrata sulla messa a punto del metodo di mineralizzazione ed analisi dei
diversi campioni oggetto della ricerca. Per stimare gli eventuali effetti dovuti ad interferenze da matrice sono
state eseguite numerose prove di recupero basate sul metodo delle aggiunte. In particolare ogni matrice
considerata (terreno, foglie e pere) è stata addizionata (fortificata) con soluzioni standard di elemento in esame e
tali da determinare sui campioni incrementi noti e pari a 0,1 e 1 mg/g.
Dall’esame complessivo dei risultati ottenuti riportati in tabella 1, si può evidenziare come tutti i valori di
recupero siano compresi fra il 97,8% e il 102% per cui si può affermare che la metodologia in esame rientra
pienamente nei criteri di attendibilità previsti dalle raccomandazioni IUPAC.
Nella tabella 2 sono riportate le concentrazioni medie dei diversi metalli presenti nei campioni di foglie e terreno
raccolti in tempi diversi all’interno delle parcelle sperimentali trattate secondo i criteri di “Difesa Biologica” e di
“Difesa Integrata”.
Tabella 1. Valori medi di recupero (% ± SD) determinati per i diversi metalli presenti nelle matrici analizzate e
ottenuti con il metodo delle aggiunte a diversi livelli di concentrazione ( mg/g).
==================================================================
Recupero (% ±SD)
--- ---------------------------------------------------------------------------------------Terreno
Foglie
Pere
Metallo
--------------------------------------------------------------------------------------------Concentrazioni addizionate ( mg/g)
-- ------------------------------------------------------------------------------------------0.1
1.0
0.1
1.0
0.1
1.0
___________________________________________________________________________
Rame
100.5±1.2 100.3±1.7
98.2±2.1 99.6±1.5
98.2±1.9 100.1±1.6
Ferro
99.8±2.3
97.9±3.1
101.0±1.9
98.0±2.7
Zinco
100.3±2.1
98.2±2.8
96.4±3.8
99.1±2.7
101.2±2.8 100.0±1.4
98.3±1.4
99.2±2.2
100.2±3.1
98.3±2.8
102.0±2.5
99.9±2.9
Nichel
102.0±2.6
98.4±3.0
100.4±2.8
98.2±3.1
98.6±3.2
98.1±3.4
Cromo
100.5±3.0
102.1±2.8
99.2±3.1
98.7±2.4
97.8±2.7 100.4±3.3
Magnesio
99.2±2.8 101.1±2.1
==================================================================
L’ipotesi nulla H0 sull’intercetta è stata valutata per p=0,01. Il limite di rivelabilità è stato calcolato secondo le
raccomandazioni IUPAC per p=0,01.
Il principale interesse della ricerca era incentrato sulla rilevazione delle concentrazioni di rame che si sarebbero
determinate in relazione al numero e al tipo di trattamenti eseguiti, e da un primo gruppo di metalli considerati
indispensabili: ferro, zinco e magnesio. Da ciò e sulla base dei risultati ottenuti, si può osservare che il contenuto
tali metalli nelle foglie e terreno aumenta considerevolmente dalla prima alla seconda raccolta nei campioni
trattati e rispetto al testimone, mentre nella terza raccolta l’aumento è molto contenuto e non si differenzia
significativamente per le due modalità di difesa. Il testimone, costituito da piante che non hanno subito
trattamenti chimici, presenta ridotti contenuti di rame.
Le differenze riscontrate non sono significative nell’ambito di ciascuna raccolta, tuttavia, per il ferro, si nota che
i campioni del testimone presentano costantemente concentrazioni superiori a quelle dei campioni trattati. Nichel
e cromo sono rilevabili in concentrazioni limitate in tutte le tesi e permangono pressoché costanti per tutto l’arco
della sperimentazione.
147
Tabella 2. Concentrazioni medie (µg/g) dei residui di metalli presenti nelle foglie e nei terreni provenienti dai
campi sperimentali di difesa biologica ed integrata. F = Foglie; T = Terreno.
Difesa
Prelievo
Rame
Ferro
Zinco
Magnesio
Nichel
Cromo
F
T
F
T
F
T
F
T
F
T
F
T
Biologica
1° P
31,5
90,2
53,6
10983
n.d.
70,0
3505
5815
2,7
41,6
0,6
10,3
Integrata
1° P
45,1
93,6
58,5
9076
n.d.
104,0
3471
6591
3,1
46,7
0,4
13,5
Testimone
1° P
18,9
77,0
63,4
11329
n.d.
57,1
3741
6214
4,0
37,9
0,8
10,0
Biologica
2° P
424,2
131,2
73,4
23010
45,7
127,0
3481
6289
3,9
42,5
1,1
10,3
Integrata
2° P
401,0
125,7
69,4
22795
48,5
121,4
3192
6646
3,5
43,5
1,1
9,6
Testimone
2° P
9,7
103,6
87,3
23779
46,8
131,4
3373
6507
5,1
43,3
1,0
11,3
Biologica
3° P
462,3
97,7
75,7
22367
60,6
118,8
3811
6736
1,4
44,0
1,2
12,0
Integrata
3° P
460,0
93,3
99,6
24114
55,2
109,4
3305
7078
2,2
45,5
1,4
13,8
Testimone
3° P
18,6
91,4
100,1
24300
46,8
124,9
3732
7282
1,0
46,0
2,3
12,2
148
L’ultima parte della ricerca è stata dedicata alle determinazioni sui frutti. I risultati ottenuti sono riportati in
tabella 3. Per quanto riguarda gli elementi indispensabili ferro, zinco e magnesio si può notare che sono presenti
a valori pressoché costanti e senza notevoli differenze tra i due sistemi di difesa. Unica eccezione un leggero
aumento delle concentrazioni del magnesio nel secondo prelievo specie utilizzando il sistema di difesa integrata.
Per il cromo va sottolineato come esso sia presente in concentrazioni molto ridotte in tutte le tesi e in tutte e tre
le raccolte mentre per il nichel i valori sono risultati sempre al di sotto del limite di rivelabilità dello strumento.
Tabella 3. Concentrazioni medie ( mg/g) dei residui di metalli presenti nei
frutti provenienti dai campi sperimentali di difesa biologica ed integrata.
Difesa
Prelievo
Pere
Rame
Biologica
Integrata
1°P
1°P
2,64
3,41
Ferro
3,04
2,57
Zinco
Magnesio
Nichel
Cromo
70,85
<0,5
0,26
1,40
0,08
80,73
<0,5
0,17
Testimone
1°P
1,08
2, 72
1,02
75,03
<0,5
0,26
Biologica
2°P
3,20
1,00
1,08
91,95
<0,5
0,13
Integrata
2°P
3,80
1,25
1,18
101,83
<0,5
0,16
Testimone
2°P
1,28
1,25
1,50
94,90
<0,5
0,12
Per quanto concerne il rame, essendo l’elemento sottoposto ad attenzione particolare in quanto principio attivo
antiparassitario, l’analisi complessiva evidenzia che le tesi trattate contengono concentrazioni di metallo sempre
superiori a quelle del testimone; inoltre, va sottolineato l’aumento generale delle concentrazioni nella seconda
raccolta rispetto alla prima.
149
CONCLUSIONI
La ricerca intrapresa, anche se limitata ad una sola annata agraria per cui sono auspicabili ulteriori conferme, ha potuto
mettere in evidenza alcuni punti di sicuro interesse. Innanzitutto vanno evidenziati i risultati relativi alle determinazioni
eseguite sui frutti che, rappresentando la parte edule e commerciale, interessano principalmente i produttori e i
consumatori.
I risultati ottenuti evidenziano un quadro positivo; in particolare, si è dimostrata valida la difesa biologica per quanto
riguarda gli aspetti igienico-sanitari e di conservazione. Confrontando infatti le percentuali di frutti colpiti alla raccolta
si può osservare che il valore più basso è quello relativo alla difesa biologica (46,75%) contro il 54,87% della difesa
integrata e l’81% del testimone. Per ciò che concerne invece i residui di rame presenti nel terreno, nelle foglie e frutti
trattati secondo le modalità di difesa biologica e integrata, le relative concentrazioni risultano sempre inferiori al limite
di tolleranza previsto dalla vigente legislazione e che per la frutta è fissato in 20 mg/g.
Da queste indicazioni si può quindi dedurre che le preparazioni a base di rame possono costituire un valido metodo di
difesa anticrittogamica essendo selettive ed efficaci anche a basse concentrazioni oltre ad essere di sicuro interesse data
la loro limitata pericolosità per ciò che concerne gli effetti antropotossici.
La prosecuzione della ricerca potrà fornire ulteriori elucidazioni su altre colture ma soprattutto mettere in evidenza in
un programma colturale completo quali siano i rischi/benefici dovuti all’applicazione delle più attuali strategie di difesa
al fine di migliorare le produzioni sia dal punto di vista quantitativo che qualitativo andando quindi maggiormente
incontro alle pressanti richieste dei consumatori.
Bibliografia
- Aceto M., Abollino O., Bruzzoniti MC., Mantasti E., Sarzanini C., Malandrino M. (2002). Determination of metals in
wine with atomic spectroscopy (flame-Aas, GF-AAS and ICP-AES); a review. Food Addit Contam. 19: 126-33.
- Anderson R.A., Bryden N.A., Polansky M.N., Deuster P.A. (1995). Acute exercise effects on urinary losses and
serum concentration of copper and zinc of moderately trained and untrained men consuming a controlled diet. Analyst
120: 867-870.
- Brandolini V., Tedeschi P., Capece A., Maietti A., Mazzotta D., Salzano G., Paparella A., Romano P. (2002).
Saccharomyces cerevisiae wine strains differing in copper resistance exhibit different capability to reduce copper
content in wine. World Journal of Microbiology & Biotechnology . 18: 499-503.
- Chow P.Y.T., Chua T.H., Tang K.F., Ow B.Y. (1995). Dilute acid digestion procedure for the determination of lead,
copper and mercury in traditional chinese medicine by atomic absorption spectrometry. Analyst 120, 1221-1223.
- Epsein L., Bassein S. (2001). Pesticide applications of copper on perennial crops in California, 1993 to 1998. J.
Environ Qual. 30(5) : 1844-1847.
- Hamey PY., Harris CA. (1999). The variation of pesticide residues in fruits and vegetables and the associated
assessment of risk. Regul. Toxicol. Pharmacol. 30: 34-41.
- Tahan J.E., Sànchez J.M., Granadillo V.A., Cubillàn H.S., Romero R.A. (1995). Concentration of total Al, Fe, Hg,
Na, Pb and Zn in commercial canned seafood determined by spectrometric means after mineralization by microwave
heating. J. Agric. Food. Chem. 43: 910-915.
150
33
INVESTIGATIONS ON THE BACTERICIDAL ACTIVITY OF SOME NATURAL PRODUCTS
Varvaro L., M. Antonelli, G. M. Balestra, A. Fabi, D. Scermino e G. Vuono
Dipartimento di Protezione delle Piante, Università degli Studi della Tuscia,
Via S. Camillo de Lellis, 01100, Viterbo, Italy
The Council Regulation (EEC) no. 2092/91 shows the rules on organic production in agriculture to ensure both
consumers and producers. The products for plant protection (copper compounds, propolis a nd sodium silicate) are listed
in the Annex II part B of this regulation. The main chemicals used in organic agriculture to control bacterial plant
diseases are cupric compounds. The last EEC Regulation (Reg. no. 473/2002) limits the use of copper and force the
researchers to study new way in controlling bacterial diseases. The utilization of natural substances, such as propolis
and sodium silicate, is one way susceptible to be investigated in order to better know their bactericidal or bacteriostatic
effect on some host pathogen interactions.
In particular, the activity of propolis and sodium silicate, alone or in association with different copper
compounds, were in vitro studied toward a large number of phytopathogenic bacterial strains, both Gram positive and
Gram negative. Furthermore, some in vivo tests were performed against some bacterial diseases of tomato, by spraying
commercial products containing propolis and sodium silicate and on hazelnut using copper compounds. In vitro results
show that propolis is active against the growth of the most of Gram positive bacterial strains, even at relatively low
concentrations, while it is very poorly active on almost all essayed Gram negative ones. Antagonistic action of sodium
silicate resulted evident only at high concentrations; in particular a certain in vitro inhibition was detected on
Pseudomonas syringae pv. tomato the agent of bacterial speck of tomato, but it was not confirmed by in vivo tests. An
inhibitory effect on epiphytic populations of Xanthomonas vesicatoria, the agent of bacterial spot of tomato, was
observed till to 7 days after the treatment. Sodium silicate was able to control also epiphytic populations of Clavibacter
michiganensis subsp. michiganensis, the agent of bacterial canker of tomato. The in vitro and in vivo studies on the
effect of different copper compounds in controlling phytopathogenic bacteria showed some interesting results. Copper
oxychloride and new chemicals, like tri-basic copper sulphate or pentahydrate copper sulphate, were sprayed on
hazelnut plants showing dieback symptoms. The obtained results confirmed the preventive bactericidal effect of copper
oxychloride against the pathogens and, moreover, pointed out the good effectiveness of the pentahydrate copper
sulphate. In fact, even if this last compound is characterized by a copper concentration five time lower than the copper
oxychloride, and although it has been sprayed at reduced field-doses, it gave the same results in controlling the hazelnut
disease.
All results showed that propolis and sodium silicate could be useful in controlling epiphytic bacterial
populations of some phytopathogenic bacteria on tomato, especially in organic agriculture. On the other hand, copper is
confirmed to be used to diminish the populations of phytopathogenic bacteria and to reduce in such a way the disease
diffusion. The opportunity to successfully use chemical products characterized by low copper concentration is important
to safeguard the environment and the human health.
151
34
RICERCA SU INSETTICIDI UTILIZZABILI SECONDO IL METODO DI PRODUZIONE BIOLOGICO DI
PRODOTTI AGRICOLI
Fabio Molinari – Piero Cravedi
Istituto di Entomologia e Patologia vegetale – Facoltà di Agraria U.C.S.C. – Piacenza – e-mail [email protected]
Introduzione
La protezione del pesco con il metodo biologico di produzione è attuata da alcuni anni con vari prodotti ad azione
antiparassitaria di cui non è ben nota l’efficacia.
Varia attività sperimentale è stata condotta negli anni con finalità non sempre aderenti alle esigenze del metodo.
La valutazione dell’efficacia dei prodotti antiparassitari non può essere fatta prescindendo dal metodo complessivo di
coltivazione che è determinante nell’influenzare la sensibilità delle piante agli attacchi parassitari. La carenza di
informazioni sull’argomento non consente di avere un quadro preciso del ruolo che certi prodotti possono svolgere nella
difesa delle colture (pesco e di melone nella presente ricerca). L’obiettivo che ci si propone di ottenere dati che, tenendo
conto della filosofia del metodo della produzione biologico, risultino valutabili secondo criteri scientifici.
l’Istituto di Entomologia di Piacenza svolge dal 1998 una Ricerca su insetticidi utilizzabili secondo il metodo di
produzione biologico di prodotti agricoli nell’ambito del Progetto: Difesa delle produzioni in agricoltura biologica
promosso dal MINISTERO PER LE POLITICHE AGRICOLE.
La ricerca si inserisce sulla “produzione integrata” nei pescheti che da anni l’Istituto di Entomologia di Piacenza sta
conducendo in varie regioni italiane in collegamento con le principali istituzioni di ricerca europee.
Materiali e Metodi
Sono state effettuate prove parcellari per determinare l’efficacia nel controllo di insetti di alcuni prodotti per
l’agricoltura biologica.
Inizialmente i prodotti da sperimentare erano quassine ed oli vegetali su afide verde del pesco e su Aphis gossypii su
cucurbitacee, ryania su Cydia molesta su pesco.
In corso di svolgimento, anche in relazione all’interesse mostrato da parte dei produttori interpellati per l’esecuzione
della sperimentazione, sono state inserite prove con Azadiractina in sostituzione di quelle con oli vegetali, che non è
stato possibile allestire per mancanza di interesse da parte di tecnici ed agricoltori; in fase avanzata sono state inserite
prove per la valutazione di ryanodine contro Cydia funebrana.
Pertanto le prove effettuate nel progetto sono state:
Quassio contro M. persicae su pesco (su reinfestazioni dopo la fioritura)
Quassio contro A. gossypii su melone (anguria)
Ryania contro C. molesta su pesco
Ryania contro Cydia funebrana su susino
Azadiractina3 contro C. molesta su pesco
Azadiractina3 contro M. persicae su pesco
Azadiractina3 contro A. gossypii su melone (anguria)1 - Polvere micronizzata (400 g/hl)
2 - polvere setacciata 180 mm, con contenuto di ryanodine 0,1%. (600 g/hl)
3 - Oikos, Sipcam (100 g/hl)
Il progetto prevede una prima fase dedicata alla determinazione dell’efficacia, mentre nelle fasi successive verranno
ricercati i criteri per un’utilizzazione ottimale dei prodotti.
Lo schema di base è stato il seguente:
Aphis gossypii. Prove parcellari secondo lo schema proposto dall’EPPO su cotone. Trattamento effettuato all’inizio
dell’arrivo degli insetti alati e ripetuto dopo 7 giorni.
Myzus persicae. Prove parcellari. 1998-1999: trattamenti effettuati sulle reinfestazioni in post-fioritura quando l’azione
degli antagonisti inizia a manifestarsi e potrebbe essere influenzata negativamente da trattamenti a base di piretro o
rotenone.
2000-2001: concentrando maggiormente l’attenzione sull’azadiractina, sono stati valutati gli effetti di trattamenti pre e
postfiorali.
Cydia molesta. Prove parcellari. Trattamenti effettuati sulle larve della seconda generazione per meglio evidenziare il
livello di efficacia.
Cydia funebrana. Prova a parcelloni sulle larve di seconda generazione. I trattamenti sono stati ripetuti 3 volte (2 per
ryania) a distanza di 8-10 giorni.
Risultati
152
Vengono qui riportati i risultati di alcune prove rappresentative. Aphis gossypii: le scarse infestazioni nei campi
sperimentali non hanno consentito di trarre indicazioni chiare. Nella prova di Castellazzo Bormida (a)
Myzus persicae: le prime prove hanno evidenziato una limitata attività del quassio sulle reinfestazioni, mentre più
interessante è risultata l’azione di azadiractina; buona l’efficacia prefiorale, chiaramente migliorata dalla ripetizione del
trattamento in postfioritura (b).
Cydia molesta. Sia azadiractina sia rianodine hanno mostrato una attività variabile, ma complessivamente tale da
suggerire ulteriori approfondimenti, sia sulle modalità applicative, sia sulle formulazioni; in particolare le rianodine
hanno presentato alcune difficoltà in fase di preparazione e di distribuzione, e nonostante ciò hanno fornito risultati
quasi sempre in linea, e talvolta superiori a quelli dei preparati a base di Bacillus thuringiensis.. Un esempio è riportato
in figura c.
Cydia funebrana. E’ stata effettuata una sola prova nel 2002 su parcelloni con 2 repliche. Dal grafico (d) si può
osservare che nelle parcelle trattate con ryania e azadiractina si è registrato un calo nell’infestazione, mentre in quelle
trattate con Bacillus thuringiensis una scarsa azione può essere evidenziata solo dalla riduzione dell’incremento del
fitofago, con un risultato finale comunque non differente rispetto al testimone.
153
35
FUNGICIDES AND HEAVY METALS IN WINE SAMPLES PRODUCED FROM EXPERIMENTAL
GRAPES SUBJECTED TO DIFFERENT PESTICIDES TREATMENTS
Salvo Francesco, Saitta Marcello, Di Bella Giuseppa, La Pera Lara, Dugo Giacomo
Dipartimento di Chmica Organica e Biologica Università di Messina Salita Sperone, 3190166 S. Agata -Messina
Wine is a wildely consumed beverage with thousand years traditions, so an accurate quantitative knowledge of
the presence of toxic elements as pesticides and heavy metals, is very important from both toxicological and enological
point of view. The use of pesticides is closely associated with viticulture (1). Many factors influence the presence of
heavy metals in wine: type of soil, wine-processing equipment, vinification methods (2). The purposes of this work are
both to quantify pesticides residues and trace metals in samples of Italian wines, and to study the possible correlation
between the presence these contaminants (3).
Wine farms that comply with the Rule Cee 2078/92 for wine grape defense, have to respect all the provided
restrictions regarding to the use of pesticides.
To control the growth of powdery mildew (Uncinula necator), the use of sulfur, azoxystrobin, dinocap,
fenarimol, penconazole, quinoxyfen, is allowed. Mildew epidemic weakens the vines, causing huge production losses
and negatively influencing the quality of wines. Also an incorrect use of fungicide and frequency of their applications,
maight alter the quality of the final product.
The determination of fungicide residues in wine is well documented, even if the reported extraction procedures
are complex and expensive; meanwhile there is a lack of available data regarding to the extraction of dinocap residue.
In this work a quick chromatographic method for the simultaneous extraction of azoxystrobin, dinocap, fenarimol,
penconazole, quinoxyfen residues in wine samples is described (4).
The same samples of wine were subjected to trace metal determination as Cd (II), Cu (II), Pb (II) and Zn (II).
Heavy metals dosage was carried out by derivative potentiometric stripping analysis (3), which allowed to execute the
determinations in a short time directly on the wine sample appropriately acidified. The presence of Cd (II), Cu (II), Pb
(II) and Zn (II) in white and red wine sample, was correlated to the treatments applied on grapes.
Samples description
All the samples of white and red wine were produced in the crop year 2000-2001.White wines came from Sicily
and Campania red wine from Tuscany. Sicilian wines were produced from Inzolia and Carricante grape variety; wines
from Campania were produced Fiano d’Avellino grape variety. Red wines from Tuscany were vinified from Sangiovese
grape variety, Morellino clone.
The fungicide treatments were carried out on the end of blossoming and maturation; they were used at the doses
recommended by the manufacturers and were sprayed with a manual sprayer every 7-12 days (9 treatments) The last
treatments was executed about 28 days before the grape harvest. Only dinocap, which is generally used to start the
defence strategy, was applied 3 times. A random-block scheme was used with four replications for each test, and each
block contained 60 plants.
HRGC analysis
A HRGC 5300 mega Series Carlo Erba Instruments gas chromatograph was used for determination of five
fungicides. It was fitted with an electron-capture detection (ECD) system and split-splitless injector, connected to a
Hewlett Packard 3394 A reporting integrator.
A SPB-5 fused – silica column (30 m x 0.25 mm i.d.) was employed, with 5% diphenyl 95% dimethyl siloxane
as liquid phase (film thickness 0.25 mm) (Supelco). The injector and detector were operated at 250° C and 280°C,
respectively.
The sample (1 mL) was injected in the split mode (1:3), and the oven temperature was programmed as follows:
200°C (1 min) raised to 270°C (5°C/min), held for 20 min. Helium was the carrier gas and nitrogen the make-up gas
both at 150 Kpa.
Ten grams of wine was collected in an Erlenmeyer flask with a teflon-lined screw-cap. A 20 mL volume of
hexane-ethyl acetate (9:1, v/v) was added, and the mixture was put in a sonicator (Transsonic T420, Elma) for 30 min.
A 2 mL volume of supernatant was evaporated under nitrogen flow; 0.2 mL of the internal standard solution
(bromophos-methyl 1.00 mg/L) was added, and the mixture was directly analyzed by gas chromatography. No clean-up
was necessary because there were no interference in the chromatogram.
Previously, a blank assay was employed to check for the absence of residuals in wine. Untreated samples of wine
were fortified with 0.01, 0.1 and 1.0 mg/Kg of fungicides. The samples were allowed to equilibrate for 60 min prior to
extraction, and were processed according to the above procedure. The recovery assays were replicated 3 times:
recoveries ranged fro 75.9 to 121.5 %. The detection limits were evaluated as the peak height that is two times respect
to the bottom noise. For azoxystrobin, fenarimol, penconazole and quinoxyfen the calculated detection limit is 5 pg/mL;
154
while for dinocap is 10 pg/mL. For the determination of sulfur residue, a HRGC Dani 86.10, fitted with a FPD Dani
86/70 detector and a Mega 68 fused Silica column, were used; the detection limit was 2 ng/mL.
Derivative potentiometric stropping analysis
For metals determination an ION 3 Steroglass potentiometric stripping analyzer equipped with a conventional
three electrodes cell, was used. The working electrode was a glassy carbon one covered with a thin mercury film.
Before each analysis an appropriate volume of wine was treated with HCl 2M lowering the pH to 2, in order to ensure
that all metals present remain in unbound –free forms. Cd (II), Cu (II) and Pb (II) were determined simultaniously, their
oxidation potential were respectively –590 mV, -185 mV and –400mV. Zn was separately determined, its oxidation
potential was –950 mV. Quantitative analysis was always carried out by the method of standard additions.
RESULTS and CONCLUSION
The residues found in all wines were below the limits that Italian law (D.M. 19 may 2000) provides for grapes
and wines. Particularly no quinoxyfen residues were found in the wines; also in wines originated from treated grapes,
no dinocap residues were found probabily because of an inferior number of tratments applied on vines (table 1).
Maximum accetable levels of Cd (II), Cu (II), Pb (II), and Zn (II) in wines, has been estabilished by the Italian
Republic (5) and by the European Commission (6) respectively at 100
ng L-1, 1000 ng L-1, 300 ng L-1, and 500 ng L-1. As tables 2 a-c evidence that the metals content of the studied wines was
always under the acceptable levels, particularly Cd was not found in any sample. The content of Cu, determined in
wines coming from the three regions ranged from 10.5 to 415.7 ng mL-1 and was not influenced by fungicides
treatments applied on grapes. The concentration of Pb spanned from 3.6 to 17.4 ng mL -1 and was regularly influenced
by pesticides treatments (fig. 1): the highest amounts were extimated for samples treated only with sulfur and with
azoxystrobin while the lowest were found in wines trated with quinoxyfen and dinocap. The level of Zn was in the range
21.6-263.0 ng mL-1 and showed a regular behaviour respect to the treatments (fig.2) only in white wines: the highest
amounts were extimated for samples treated with fenarimol and azoxystrobin, and as already observed, the lowest in
samples treated with dinocap. For red wines the highest Zn contents were evaluated in samples treated with azoxstrobin
and in the one trated with sulfur, the lowest in the sample treated with fenarimol.
LITERATURE
1.
Fernadez-Cornejo, J. Environmental and economic consequences of technology adoption: IPM in
viticulture. Agricultural Economics 1998, 18, 145-155.
2.
Golimowsky, J.; Valenta, P.; Wolfgang-Nurnberg, H. Toxic Trace Metals in Food II. A Comparative
Study of The Levels of Toxic Trace Metals in Wine by Differential Pulse Anodic Stripping Voltammetry and
Electrothermal Atomic Absorption Spectrometry. Z.Lebensm. Unters. Forsh. 1979 b, 168, 439-443.
3.
Salvo, F.; La Pera, L.; Di Bella, G.; Nicotina, M.; Dugo, G.mo. Influence of mineral and organic
pesticides treatments on Cd (II), Cu (II) Pb (II) and Zn (II) content determined by Derivative Potentiometric Stripping
Analysis in Italian Red and white wines. J. Agric. Food Chem. 2003 in press
4. Dugo, G.mo; Visco A.; Saitta M.; Vinci, V.; Di Bella G.Dosaggio di quinoxyfen su prodotti vitivinicoli
siciliani. Vigne vini, 2001, 91, 7-8.
5. Repubblica Italiana; Caratteristiche e Limiti di alcune Sostanze nel Vino. 1986, december D.M. 29.
6. European Commission; 1997 Doc III/5125/95/ Rev.3.
Table 1: Fungicide residues (mg /Kg + s.d.) in wine.
Sicily
Campania
Tuscany
azoxystrobin
21±6
5±1
12±2
dinocap
n.d.
n.d.
n.d.
fenarimol
13±2
6±2
70±6
penconazole
10±1
8±2
12±1
quinoxyfen
n.d.*
n.d.*
n.d.*
n.d. < 10 mg /Kg
n.d.* < 5 mg /Kg
155
Table 2 a, b, c: Cd (ng mL -1), Cu (ng mL-1), Pb (ng mL-1) and Zn (ng mL-1) concentrations in 8 samples of wines
from Sicily (a), 8 from Campania (b), and 8 from Tuscany (c) : one of each group was treated with water only (blank),
the others were subjacted to different organic and inorganic pesticides treatment. Each value was the mean of three
determinations.
(a)
Samples
Treatments
Cd
Cu
Pb
Zn
Blank
Water
ORGANIC
TREATMENTS
1
Quinoxifen
2
Fenarimol
3
Azoxystrobin
4
Dinocap-Penconazole
5
Dinocap
mean ± sd
INORGANIC
TREATMENTS
6
S WP
7
S DP
mean ± sd
total mean ± sd
n.d.= not detectable < 1.5 ng mL -1
S WP= sulfur wettable powder
S DP=sulfur dry powder
n.d
20.6±0.7
9.0±0.9
21.6±1.1
n.d
n.d
n.d
n.d
n.d
63.3±1.8
27.8±1
26.8±0.8
23.2±0.7
24.4±1.2
33.1±17.0
10.3±1.1
11.4±0.9
16.7±1.4
9.6±0.8
13.0±1.3
12.2±2.8
23.7±0.8
53.1±0.3
52.6±1.3
26.7±0.6
21.6±1.0
35.5±15.9
n.d
n.d
36.1±1.2
29.4±1.3
32.8±4.7
31.5±13.7
13.8±1.0
26.0±1.3
19.9±8.6
13.7±5.6
47.2±1.6
53.7±2.0
50.5±4.6
37.4±15.3
(b)
Samples
Treatments
Cd
Cu
Pb
Zn
Blank
ORGANIC
1
2
3
4
5
mean ± sd
INORGANIC
6
7
mean ± sd
total mean ± sd
Water
TREATMENTS
Quinoxifen
Fenarimol
Azoxystrobin
Dinocap-Penconazole
Dinocap
n.d
31.0±0.8
3.6±0.3
77.6±2.0
n.d
n.d
n.d
n.d
n.d
185.3±5.9
184.2±2.7
203.0±1.9
247.1±6.5
72.4±1.0
178.8±64.5
4.2±0.2
5.1±0.3
13.1±1.2
4.7±0.8
4.1±0.4
6.2±2.9
111.9±1.7
205.5±4.3
200.8±4.7
109.1±3.0
142.1±2.6
133.9±79.3
TREATMENTS
S WP
S DP
n.d
n.d
110.8±4.0
174.6±4.0
142.7±45.1
151.1±72.7
12.6±0.2
15.4± 0.8
14.0±2.0
7.8±4.9
178.8±2.5
176.6±2.9
177.7±1.6
150.3±47.2
Samples
Treatments
Cd
Cu
Pb
Zn
Blank
ORGANIC
1
2
3
4
5
mean ± sd
INORGANIC
6
7
mean ± sd
total mean ± sd
Water
TREATMENTS
Quinoxifen
Fenarimol
Azoxystrobin
Dinocap-Penconazole
Dinocap
n.d
10.5±0.5
5.0±0.4
105.0±2.9
n.d
n.d
n.d
n.d
n.d
415.7±12.5
103.3±2.3
397.0±14.9
253.9±10.0
325.2±7.3
304.4±128.7
12.6±0.9
14.4±1.0
17.4±1.1
9.0±0.8
9.5±0.7
12.6±3.5
263.0±8.0
166.7±6.3
103.3±5.2
179.3±7.0
152.2±5.4
172.9±58.0
n.d
n.d
20.1±2.3
61.4±3.3
40.8±28.2
198.4±169.3
17.2±0.2
16.6±1.0
16.9±0.5
12.7±4.5
266.9±9.1
257.8±8.9
262.4±6.4
186.8±68.2
(c)
TREATMENTS
S WP
S DP
156
30,0
Sicily
Campania
Tuscany
20,0
Sicily
Campania
15,0
Tuscany
[Pb](ng/ml)
[Pb](ng/ml)
25,0
10,0
5,0
rD
fe
ui
Su
lfu
xy
no
lfu
Su
en
D
D
in
in
oc
oc
ap
ap
(Q
-P
P
n)
P
rW
zo
na
co
xi
str
zo
A
Q
le
in
ob
ol
rim
na
Fe
ui
no
W
xi
ph
at
er
en
0,0
Figure 1: Pb content of wines samples from different treatments
300,0
250,0
[Zn] (ng/ml)
200,0
Sicily
Campania
Tuscany
150,0
100,0
50,0
rD
P
n)
lfu
Su
xy
ap
(Q
ui
no
lfu
Su
oc
oc
D
in
in
D
fe
rW
P
le
zo
na
co
ap
-P
en
zo
xi
st r
ob
in
ol
Fe
na
en
xi
ph
rim
A
Q
ui
no
W
at
er
0,0
Figure 2: Zn content of wines samples from different treatm ents
157
36
NEW a-ELICITIN ISOFORMS FROM Phytophthora hybernalis AS PROTEIN ELICITORS FOR A
POTENTIAL EMPLOYMENT IN THE BIOLOGICAL PEST MANAGEMENT
R. Capasso*, A. De Martino*, G. Cristinzio**, A. Di Maro*** and A. Parente***
*Dipartimento di Scienze della Pianta, del Suolo e dell’Ambiente, Università degli Studi di Napoli “Federico II”,
Portici, Italy. E-mail: [email protected]
** Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università degli Studi di
Napoli “Federico II”, Portici, Italy.
*** Dipartimento di Scienze della Vita, Seconda Università degli Studi di Napoli, Caserta, Italy.
The microbial naturally occurring compounds which are able to induce plant defence mechanisms against
phytopathogenic fungi and bacteria are named elicitors and could be consequently employed in strategies for the
biological control of plant diseases.
The genus Phytophthora produces protein elicitors, named a- and b-elicitins, which show a relative molecular weight
of 10 KDa and 98 amino acids. Their characteristic biological activities are the induction of a hypersensitive response
(HR) and systemic acquired resistance (SAR) in tobacco with the formation of phytoalexins, PR proteins and salycilic
acid; moreover, they induce protection against phytopathogenic microorganisms in tobacco and in some species of
Brassicaceae1.
In the present communication we describe the isolation and the preliminary data on the chemical characterization and
elicitor activity on tobacco (Nicotiana tabacum L., cv Rustica) of three new elicitin isoforms H-a1, H-a2 e H-a3,
produced in the culture filtrates of an isolate of Phytophthora hybernalis, the causal agent of rot of citrus fruits, isolated
from diseased plants in the sorrentina peninsula.
The three isoforms have been isolated by four purification steps, of which the final step was represented by a
chromatographic purification on a reverse-phase column (RP-18) eluted at low pressure with a gradient of wateracetonitrile.
It was determined their absolute molecular weight by MALDI Mass Spectrometry, obtaining values of 10239.24 for Ha1, 10235.94 for H-a2 and 10244.28 uma for H-a3.
In studies of elicitor activity carried out testing the hypersensitive response on tobacco, the three isoforms showed
different intensity responses, that is H-a1 proved to be the most active, followed by H-a2, whereas H-a3 proved to be
inactive.
Studies will be carried out on the determination of their primary structure and elicitor effects on other plants of
agricultural interest. The very low dose required for their positive biological effects on tobacco, their protein nature
and the no toxicity of the elicitins for adults2, render these new compounds and those previously described 1,3,4 very
interesting for investigations on their possible use in biological pest management programs.
REFERENCES
1. Ponchet, M., Panabieres, F., Milat, M.-L., Mikes, V., Montillet, J.-L., Suty, L., Triantaphylides, C.,
Tirilly, Y., Blein, J.-P., 1999. Are elicitins cryptograms in plant-Oomycete communications? Cell Mol.
Life Sci. 56, 1020-1047.
2. Mikes, V., Blein, J.-P., Milat, M.-L., Ponchet, M., Ricci, P., 1998. Use of elicitins as lipid carriers
and their medical use. Patent PCT Int. Appl. 31 pp Coden; PIXXD2.
3. Capasso, R., Cristinzio, G., Evidente, A.,Visca, C., Ferranti, P., Del Vecchio Blanco, F., Parente, A.,
Elicitin 172 from an isolate of Phytophthora nicotianae, pathogenic to tomato. Phytochem. 50,
703-709.
4. Capasso, R., Cristinzio, G., Di Maro, A. Ferranti, P., Parente, A., 2001. Syringicin a new a-elicitin from
an isolate of Phytophthora syringae, pathogenic to citrus fruit. Phytochem. 58, 257-262.
158

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