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Biological Control 58 (2011) 160–166
Contents lists available at ScienceDirect
Biological Control
journal homepage: www.elsevier.com/locate/ybcon
Leaf blast (Magnaporthe oryzae) suppression and growth promotion by rhizobacteria
on aerobic rice in Brazil
Marta Cristina C. Filippi a,⇑, Gisele Barata da Silva b, Valácia L. Silva-Lobo a, Márcio Vinícius C.B. Côrtes a,
Alessandra Jackeline G. Moraes b, Anne S. Prabhu a
a
b
Embrapa Arroz e Feijão, Rodovia GO-462, km 12 Zona Rural C.P. 179, CP 179, Cep. 75375-000, Sto Antônio de Goiás, GO, Brazil
Laboratório de Proteção de Plantas, Universidade Federal Rural da Amazônia, Belem, PA, Brazil
a r t i c l e
i n f o
Article history:
Received 19 July 2010
Accepted 26 April 2011
Available online 1 May 2011
Keywords:
Oryza sativa
Pyricularia oryzae
Blast resistance
ISR
PR proteins
a b s t r a c t
Rice blast caused by Magnaporthe oryzae has the potential to cause 100% grain yield loss. The objective of
this investigation was to identify rhizobacteria showing potential for plant growth stimulation and resistance induction under greenhouse conditions. Bacterial isolates were collected from the rhizosphere of
rice plants from soils of Amazon, PA. Soil application with rhizobacteria was done by drenching with bacterial cell suspension before inoculating with virulent isolate of M. oryzae. Mass screening of 148 isolates
for growth promotion showed that 12.7% stimulated plant height, whereas 52.0% increased root length,
total biomass and root biomass. Based on these growth promotion results, 18 isolates were further tested
for in vitro inhibition of the pathogen and reduction of leaf blast severity (LBS) in greenhouse test. All
isolates inhibited pathogen growth and reduced disease severity from 16% to 95%. The two isolates showing the greatest suppression of leaf blast (Rizo-46 and Rizo-55) were further tested in a subsequent
greenhouse trial using three replications and three application methods (drenching the soil, 15 and
2 days before inoculation with rice pathogen, and spraying 2 days before inoculating with virulent
isolate). The soil drenching with isolate Rizo-55, 15 days prior to challenging with virulent isolate of
M. oryzae reduced LBS by 90%, whereas Rizo-46 applied 2 days before reduced LBS by 95%. The capacity
to suppress leaf blast by isolates Rizo-46 and Rizo-55 varied according to the mode of application. Also,
the enzymatic tests were conducted to quantitate the presence of proteins related to pathogenesis (PRPs)
during induction process of resistance by rhizobacteria. The enzyme activity of peroxidase, b-1,3-glucanase and chitinase greatly increased, and the results are in accord with greenhouse tests in relation to leaf
blast disease suppression.
Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction
Rice blast caused by Magnaporthe oryzae B.Couch [Pyricularia
grisea (Cooke) Sacc.] is the most destructive rice disease worldwide
causing grain yield losses of staggering dimension. Yield losses up
to 100% have been recorded in a recent outbreak of blast in Brazil,
in a newly released upland rice cultivar Colosso (Prabhu et al.,
2009). In an ecologically sustainable agriculture, blast disease control requires integrated management of genetic resistance, cultural
practices and chemical control. The durability of genetic resistance
in improved rice cultivars is limited due to high variability of the
pathogen (Ou, 1980). Consequently the application of fungicides
has been intensive in Brazil for reducing yield losses. The use of
resistance inducers, biotic as well as abiotic, are currently being
explored under commercial scale as major strategies for increasing
⇑ Corresponding author. Fax: +55 62 3533 2100.
E-mail address: [email protected] (M.C.C. Filippi).
1049-9644/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.biocontrol.2011.04.016
the durability of disease resistance and reducing toxic residues
resulting from indiscriminate use of chemicals.
Rhizobacteria are known to suppress plant diseases caused by
fungal pathogens and to promote plant growth. The application
of rhizobacteria in crop soils has direct and indirect benefits to
agricultural production and permit judicious use of agricultural
inputs (Lavie and Stotzky, 1986). In addition to improving plant
growth, rhizobacteria are directly involved in synthesis of phytohormones, solubilization of minerals such as phosphorus and
production of siderophores and iron chelates (Glick et al., 1995;
Bowen and Rovira, 1999). Besides mineralization, the rhizobacteria
may control plant pathogens and harmful microorganisms through
the production of antibiotics and degradation of cell wall by different enzymes (Antoun and Kloepper, 2001).
The reduction of disease severity in plants by plant growth
promoting rhizobacteria (PGPR) may occur by different mechanisms of suppression, such as competition for nutrients, antibiosis
through antimicrobial metabolite production (Ramamoorthy et al.,
2001) and inducted systemic resistance (ISR) (Van Loon and
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Marta Cristina C. Filippi et al. / Biological Control 58 (2011) 160–166
Pieterse, 2006; Van Loon, 2007). The systemic resistance induction
process increases enzymatic activity of peroxidase (PO) and phenol
oxidase (PPO) which are responsible for catalyzing lignin formation, and phenyl ammonia lyase (PAL) which is involved in the
biosynthesis of phytoalexins and phenols. ISR induction also
increases liposaccharides, a constituent of cell wall membranes
(LPS) (Radjacommare et al., 2004). The pathogenesis-related
proteins (PRPs) b-1,3-glucanase and chitinase, enzymes that
belong to PR-2 and PR-3 families, respectively (Van Loon and
Pieterse, 2006) have been related more often to SAR and sometimes to ISR. All these enzymes have been shown to be involved
in plant defense against pathogens in several pathosystems
(Kini et al., 2000).
The benefits of rhizobacterial associations have been observed
in various plant species such as chickpeas, eggplant (Kumar,
1998), beets (Thrane et al., 2000), radish (Leeman et al., 1995), sorghum (Chiarini et al., 1998), tomato (Hoffmann-Hergarten et al.,
1998), ornamentals (Yuen and Schroth, 1986) and cereals (Biswas
et al., 2000). The use of PGPR to control rice blast disease has been
observed by several other authors, however, these studies were
performed with isolates from rice grown under irrigated conditions
(Krishnamurthy and Gnanamanickam, 1998; Radjacommare et al.,
2004). Lucas et al. (2009) studied the use of two strains of PGPR in
the integrated management of rice blast in southern Spain and
observed that there is a direct relationship between inoculation
of seed with rhizobacteria and disease control, grain yield
improvement and head yield.
There is no information on the effect of PGPR in stimulating
plant growth and disease suppression in aerobic rice. In the present
study, attempts were made to select rhizobacteria associated with
upland rice roots which are capable of promoting growth stimulation as well as rice leaf blast disease suppression.
2. Material and methods
2.1. Rhizobacteria isolation
Forty-two samples of rhizosphere soil and roots were collected
from commercial rice fields of cultivars BRS Sertaneja, BRS Cambará and Primavera, after 40 days of seeding in the municipalities
of Paragominas and Dom Eliseu, PA, during 2008/09 rice growing
season. The soil where the samples were collected was Dark Red
Latosol according to the Brazilian classification system which is
equivalent to oxisol in the USA soil taxonomy. The soil showed
the following characteristics: pH range 5.1–5.9 and organic matter
48–55 g kg1. The organic content is considered high and favors
microbial activity. The sampling was done mainly in the following
two locations in Amazon basin: (1) Dom Eliseu, PA, latitude
04°170 3600 S and longitude 47°330 1500 W. (2) Paragominas, PA latitude 02°590 4500 S and longitude 47°210 100 0 W, altitude 90 m. The soil
samples from rhizosphere were collected from 40-day old rice
plants showing five fully opened leaves and tillering.
Rhizobacteria were isolated by the serial dilution method. Ten
grams of rhizosphere soil in 90 mL of sterile saline (0.85%) was
diluted to107 (Bharathi et al., 2004). An aliquot of 100 lL of each
dilution was distributed with the aid of the handle Drigalsky in
Petri dishes containing culture medium 523 (Kado and Heskett,
1970) and incubated at 28 °C for 24 h. Morphologically distinct
colonies were replicated and purified isolates totaling 210 were
stored in cryogenic tubes.
161
greenhouse conditions. An experiment was conducted for mass
screening of isolates with cultivar Primavera using a completely
randomized bloc design with 40 replications, one plant per replicate. The seeds, prior to seeding in plastic trays (15 30 10 cm) containing 3 kg of PlantmaxÒ substrate, were sterilized
with 90% alcohol and sodium hypochlorite solution. Each tray
was composed of eight lines and each line consisted of 10 plants,
totaling 80 plants per tray. The 149 treatments included 148
isolates of rhizobacteria and a control (trial drenched with water).
The bacterial suspension was adjusted to 550 nm (A550 = 0.1, corresponding to 108 CFU per mL) absorbance and applied by drenching the soil (100 ml/tray), 7 days after sowing or 15 days before
inoculation with the pathogen. Seven days after drenching the soil,
40 plants of each treatment were evaluated for plant height (PH),
root length (RL), total biomass (TB) and root biomass (RB).
The data were subjected to cluster analysis using average values
of each individual and the degree of similarity was obtained by the
standard Euclidean distance. The classes obtained were subjected
to analysis of variance (ANOVA) and means compared by Skott–
Knott test at 5% probability using the software SPSS version 16.0.
2.3. Antibiosis, enzymatic and phenotypic characterization of isolates
2.3.1. Antibiosis test
In order to identify antibiosis between rhizobacterial isolates
and M. oryzae, 18 isolates of rhizobacteria (19 treatments including
control) (Table 2) were selected based on the growth stimulation
results for an in vitro assay using a completely randomized design
with three replications. Five millimeter disc of M. oryzae mycelium
was transferred to the central part of a Petri dish containing potato
dextrose agar (PDA). Each isolate was streaked on four extremes of
the plate forming a square around the M. oryzae mycelium. The
evaluation was done by measuring the radial growth of M. oryzae.
The data were statistically analyzed and the means were compared
by Tukey test (P = 0.05) using the software SPSS, version 16.0.
2.3.2. Enzymatic characterization of isolates
The 18 most promising isolates of rhizobacteria identified in
previous bioassays (Section 2.2) were selected for the following
assays using a completely randomized bloc design with four
replications:
2.3.2.1. Phosphate solubilization (PS). Isolates of rhizobacteria were
grown in Petri plates containing GY medium (glucose–yeast
extract) and a phosphorus supplement according to SylvesterBradley et al. (1982). The plates were incubated at 28 °C for 3 days
and evaluated by identifying a translucent milky white growth
(positive observation) around the bacterial colony.
2.3.2.2. Production of indole acetic acid (IAA) or analogues. Isolates of
rhizobacteria were grown in Petri plates according to Cattelan et al.
(1999). The plates were evaluated by identifying a red halo formed
on the membrane (IAA positive).
2.3.2.3. Ferric siderophore production (Sid). Isolates of rhizobacteria
and one positive control (Pseudomonas sp.) were evaluated for its
ability to produce siderophore and to convert ferric ions (Fe III)
to soluble forms (Fe II) by chelation. Rhizobacterial isolates were
transferred to Petri plates containing agar and chrome azurol S
and incubated at 28 °C for 48 h according to Schwyn and Neilands
(1987). Colonies exhibiting a pink halo after incubation period
were considered positive.
2.2. Mass screening for plant growth stimulation
Out of 210 rhizobacteria isolates 148 were randomly selected
and evaluated for growth promotion of rice plants, under
2.3.2.4. b-1,3-glucanase in vitro production. Isolates of rhizobacteria
were evaluated for their ability to produce b-1,3-glucanase.
Glucanase activity was identified by adding b-1,3-glucan in a semi
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solid nitrogen free medium according to Renwick et al. (1991). The
plates were incubated for 3 days at 28 °C and evaluated by identifying as positive the formation of an orange halo.
2.4. Rhizobacteria for leaf blast suppression
2.4.1. Evaluation of isolates for leaf blast suppression
The 18 most promising isolates of rhizobacteria identified in
previous mass screening of 148 isolates (Section 2.2) were further
evaluated for their ability to suppress leaf blast under greenhouse
conditions. The test was conducted with the cultivar Primavera as
described in Section 2.2. The lay-out was a completely randomized
bloc design with three replications. The 19 treatments included 18
isolates of rhizobacteria and a non-treated control. The rhizobacteria isolates were applied to the rice plants in plastic trays by two
different methods: (1) drenching the soil (100 m mL/tray) 15 days
before challenging with the pathogen and; (2) drenching the soil
(100 mL/tray) 2 days before challenging with the pathogen.
Twenty-one day old rice plants were sprayed with a conidial suspension of isolate of M. oryzae (Py-162) which shows compatible
reaction on cv. Primavera, at a concentration of 3 105 conidia
mL1 following the method described by Filippi and Prabhu
(2001). The leaf blast reaction was observed at 2 days intervals
soon after the first lesion appearance, using the scale based on
percentage leaf area affected as described by Notteghem (1981).
The disease severity data were normalized using the arc sin transp
formation (arc sin x), for the analysis of variance and the averages were compared by Skott–Knott test (P = 0.05) using the
software SPSS version 16.0.
2.4.2. Rhizobacterial application method for leaf blast suppression
The two most promising isolates of rhizobacteria identified in
the bioassays described in Section 2.4.1 were used in further tests
to identify the most efficient application method. The lay-out and
cultivar were described in Section 2.2. The eight treatments
included two isolates, two non-treated controls (one for soil
drenching method and another for spray method) and three methods of application. The application methods were drenching the
soil with bacterial isolates: (1) 15 days before spray inoculation
of rice plants with the pathogen; (2) 2 days before inoculation of
rice plants with the pathogen; and (3) spraying the rice plants with
isolates of rhizobacteria, 2 days before inoculation with the pathogen. The challenge inoculation with the pathogen was conducted
as described in Section 2.4.1. Leaf blast severity assessed at 2-day
intervals was based on 24 rice plants per trail for AUPDC determination. The disease severity data were normalized using the arc sin
p
transformation (arc sin x), for the analysis of variance and the
averages were compared by Tukey test (P = 0.05) using the software SPSS version 16.0.
The dosage of proteins was executed according to methodology
of Bradford (1976).
2.5.2. b-1,3-Glucanase activity
Activity of b-1,3-glucanase in rice leaf extracts from different
treatments was assayed by measuring the rate of reducing sugar
production using laminarin as the substrate (Pan et al., 1991).
DNS reagent was used as the calorimetric agent. Activity was
expressed in units (U) mg1 protein. One unit was defined as the
enzyme activity catalyzing the formation of reducing sugar that
increases the absorbency of 1 unit of abs per hour. The experiment
was done in triplicate.
2.5.3. Peroxidase activity
Peroxidase activity was assayed by measuring the rate of
2,20 -azino-bis (3-rthylbenzthiazoline-6-sulfonic acid) oxidation,
using its own calorimetric property. One unit was defined as
the enzyme activity catalyzing the formation of 2,20 -azino-bis
(3-rthylbenzthiazoline-6-sulfonic acid) that increases the absorbency of 1 unit of abs per hour (Keesey, 1987).
2.5.4. Chitinase activity
Chitinase activity in rice leaf extracts from different treatments
was assayed by modified method of Pan et al. (1991). The rate of
N-actyl glucosamine production was measured using coloidal chitin as the substrate. DNS reagent was used as the calorimetric
agent. Activity was expressed in units (U) mg1 protein. One unit
was defined as the enzyme activity catalyzing the formation of
reducing sugar that increases the absorbency of 1 unit of abs per
hour.
3. Results
3.1. Screening rhizobacterial isolates for growth stimulation
The isolates were grouped into seven significantly different
classes based on the cluster analysis of 149 treatments (148 isolates and 1 control, n = 5.960) for plant height (PH), root length
(RL), total biomass (TB) and root biomass (RB) (Table 1). The class
7 was composed only by the isolate Rizo-235 which was superior
in PH, RL, and TB. The classes 7, 6, 5 and 4 were composed by 18
isolates and were statistically superior considering mean of all
parameters than class 3, formed by 70 isolates and the control.
The class 1 composed by 29 isolates showed reduced PH when
compared to the control (class 3). The others parameters RL and
Table 1
Mass screening of rhizobacteria for growth promotion.
2.5. Enzymatic activity on challenged plants with the pathogen
Isolatesa
The third leaf of 15 challenged rice plants described in Section
2.4.1 were collected at random to assay the enzymatic activity of
pathogenesis related proteins. The collected leaves were placed
in ice and frozen for posterior use. The quantitation of enzymes
was initially standardized by conducting exploratory trials in
which the best results were obtained with samples taken 72 h after
the inoculation with the challenger. The tests were performed in
triplicate.
2.5.1. Proteins extraction
A sample of five leaves was macerated in liquid nitrogen with
pistil until it became powder. The buffer solution was composed
of Tris–HCl 10 mM; NaCl [150 mM]; EDTA [2 mM]; DTT [2 mM];
PMSF [1 mM]; Leptin [10 lg mL1] and Aprotinin [10 lg mL1].
235
45, 49A, 49C, 66A,
97, 116B, 120, 246
107, 171, 193
46, 55, 66B, 82B,
91B, 100B, 126A
Control + 70 different
isolates
29 Different isolates
29 Different isolates
a
Class
Growth (cm)
Biomass (mg)
Plant
height
(PH)
Root
length
(RL)
Total
(TB)
Root
(RB)
7
6
45.77 A*
40.68 B
19.71 A
15.89 B
64.00 A
58.16 B
2.4 B
2.8 A
5
4
40.34 B
37.40 C
13.94 D
14.74 C
52.79 C
46.00 D
2.1 C
2.0 D
3
35.70 D
7.88 F
33.63 F
1.3F
2
1
34.09 D
31.75 E
12.56 E
11.19 E
38.33 E
38.80 E
2.01D
1.6 E
Isolates were clustered into groups according to Euclediana similarity matrix.
Means with different letters are significantly different at P = 0.05 according to
the Skott–Knott test, using the SPSS software, version 16.0.
*
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Marta Cristina C. Filippi et al. / Biological Control 58 (2011) 160–166
of each treatment (Table 2). However, 10 isolates (Rizo-55, Rizo171, Rizo-120, Rizo-246, Rizo-66A, Rizo-107, Rizo-49A, Rizo-100B,
Rizo-193 e Rizo-82B) suppressed LBS better when the soil was
drenched 15 days before than when drenched 2 days before pathogen inoculation. Six isolates (Rizo-46, Rizo-45, Rizo-235, Rizo-49C
and Rizo-97) showed statistically significant reduction in LBS when
the soil was drenched 2 days before inoculation with the pathogen
M. oryzae. Two isolates (Rizo-66B and Rizo-91B) showed comparable reduction of LBS regardless of time of soil drenching. The isolate
Rizo-55 applied15 days before reduced LBS by 90.7%, whereas the
Rizo-46 applied 2 days before reduced LBS by 95.03% (Table 2).
Table 2
Effect of soil drenching with cell suspension of rhizobacteria on rice blast severity
under greenhouse conditions.
Rice blast disease severity (%)b
Isolate
15
a
2
6.1 Aa*
6.6 Ab
6.6 Aa
7.5 Aa
8.7 Ba
10.9 Ba
11.3 Ba
12.3 Ba
12.4 Ba
14.7 Ca
15.3 Cb
15.4 Ca
19.8 Db
21.7 Db
24.1 Ea
25.8 Db
52.3 Fb
55.5 Fa
66.3 Ga
Rizo-55
Rizo-46
Rizo-171
Rizo-120
Rizo-246
Rizo-66A
Rizo-107
Rizo-49A
Rizo-100B
Rizo-193
Rizo-45
Rizo-82B
Rizo-235
Rizo-49C
Rizo-66B
Rizo-126A
Rizo-97
Rizo-91B
Control
14.3 Cb
3.3 Aa
23.4 Db
21.6 Db
15.0 Cb
24.1 Db
36.4 Eb
22.4 Db
28.8 Db
25.3 Db
5.1 Aa
43.6 Fb
16.1 Ca
11.1 Ba
30.9 Ca
11.9 Ba
17.6 Ca
45.1 Fa
66.3 Ga
3.3. Antibiosis, enzymatic and phenotypic characterization of
rhizobacterial isolates
In in vitro test all isolates showed reduction in mycelia growth
of M. oryzae. There was a significant difference between two groups
of isolates. The first one composed of 14 isolates (88.9%) (Rizo-45,
46, 49A, 49C, 55, 66A, 66B, 82B, 91B, 100B, 107, 116B, 171, 193 and
235) inhibited 85% of mycelia growth. The second group, composed of 4 isolates inhibited 67% of mycelia growth (Table 3).
Out of 18 isolates, eight were positive for PS, four for auxin
production or analogues and for fluorescence, 40% for b-glucanase
and none for Sid (hydroxyquinoline and CAS) (Table 3). No single
isolate was positive for all of the metabolites assessed.
a
Days before inoculation with Magnaporthe oryzae.
Percentage leaf area affected using a 10 grade scale (0–9) according to Notteghem (1981).
*
Means followed by different small letters in line and capital letters in column
are significantly different at P = 0.05 according to the Skott–Knott test, using
SPSS,16.1 software.
b
3.4. Rice leaf blast suppression by rhizobacterial isolates and
enzymatic activity
TB were statistically superior than class 3 (control) for the classes
7, 6, 5, 4, 2 and 1.
3.4.1. Disease progress: soil drenching vs. spraying rhizobacteria
The soil drenching treatments reduced significantly the area
under disease progress curve (AUDPC) compared with control
(Fig. 1). While drenching the soil with Rizo-55 and Rizo-46, 15 days
before inoculation with the pathogen M. oryzae decreased the
AUDPC to 51.36 (60%) and 44.54 (65%), the soil drenching 2 days
3.2. Screening rhizobacterial isolates for rice blast suppression
All 18 rhizobacterial isolates decreased leaf blast severity (LBS)
compared with the control, independent of the application method
Table 3
Antibiosis, enzymatic and phenotypic characterization of rhizobacterial isolates.
Isolate
Colony.
diameter
(mm)a
Reductionb
of colony
diameter (%)
Phosphatec
solubilization
Auxind
production
b-1,3-glucanasee
Siderophore
productionf
Fluorescenceg
Rizo-55
Rizo-91B
Rizo-49C
Rizo-45
Rizo-49A
Rizo-66A
Rizo-193A
Rizo-235
Rizo-100B
Rizo-171
Rizo-46
Rizo-82B
Rizo-107
Rizo-66B
Rizo-246
Rizo-120
Rizo-126A
Rizo-97
Control
9.01ah
9.76 a
10.06 a
10.48 a
10.76 a
10.76 a
10.95 a
11.73 a
13.67 a
15.16 a
15.33 a
15.48 a
15.98 a
29.45 a
33.46 b
39.80 b
40.25 b
41.50 b
80.00 c
90.99
90.24
89.94
89.50
80.36
89.24
89.05
88.27
86.33
84.84
84.67
84.52
84.02
85.73
66.69
60.20
59.75
58.50
80.00
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
a
b
c
d
e
f
g
h
Means represents 12 replications.
Colony growth reduction of Magnaporthe oryzae compared to the control.
Presence (+) or absence () of metabolite production by rhizobacterial isolates.
Presence (+) or absence () of auxin production by rhizobacterial isolates.
Presence (+) or absence () of b-1,3-glucanase activity by rhizobacterial isolates.
Presence (+) or absence () of siderophore activity by rhizobacterial isolates.
Presence (+) or absence () of fluorescence by rhizobacterial isolates.
Means followed by the same letters do not differ statistically by Scott–Knott test (5%).
Author's personal copy
164
Soil drenched 15 days before pathogen inoculation
40
30
20
3
10
2
0
0
72
96
120
144
Hours after challenge inoculation
Leaf blast severity (%)
Soil drenched 2 days before pathogen inoculation
1
3
2
Hours after challenge inoculation
Leaf blast severity (%)
C
3.00
2.50
2.00
1.50
1.00
0.50
0.00
CTR/water CTR/M.o
C
Plants sprayed 2 days before pathogen inoculation
40
1
2
20
10
3
0
0
72
96
120
R46/M.o
R55
R55/Mo
9
8
7
6
5
4
3
2
1
0
CTR/water CTR/M.o
50
30
R46
B
β-1.3 Glucanase activity (Abs. h-1.mg-1)
B
Peroxidase Activity (U.min-1.mg-1)
A
50
144
Hours aftert challenge inoculation
Fig. 1. Leaf blast disease progress (AUDPC) evaluated during 5 days after challenge
inoculation with M. oryzae under greenhouse conditions. Water control (1) Rizo-46
(2) and Rizo-55 (3), significant differences in AUDPC values were observed among
treatments and control according to Tukey, P = 0.05.
before inoculation with the pathogen decreased to 60.42 (52%) and
41.75 (67%), respectively. Spraying rice plants with Rizo-55 2 days
before inoculation with the pathogen significantly suppressed leaf
blast by 86% (AUDPC 17.8) compared to 20% by spraying with Rizo46 (AUDPC 101.56) and control (AUDPC 127.08).
3.4.2. Enzymatic activity
Drenching the soil with Rizo-46, 15 days before challenge inoculation increased the enzymatic activity of all three enzymes
peroxidase, b-1,3 glucanase and chitinase (Fig. 2A, B and C). A
significant increase was observed even when plants were not challenged with the pathogen. Soil drenched with Rizo 46 and plants
later challenged with the pathogen increased the peroxidase activity 2.5 times, b-1,3 glucanase 5.3 times and chitinase 18 times
compared with the activity in inoculated control. The isolate
Rizo-55 promoted lower enzymatic activity compared to Rizo-46
and controls. However, the peroxidase activity was higher in plants
not challenge with the pathogen (Fig. 2A).
Spraying Rizo-55 2 days before inoculation with the pathogen
promoted a statistically significant increase in enzymatic activity
compared to Rizo-46 and the inoculated control (Fig. 3A, B and
C). While peroxidase increased 4.8 times and b-1,3-glucanase
2.17 times, chitinase showed seven times increase.
Chitinase Activity (Abs.h-1.mg-1)
Leaf blast severity (%)
A
Marta Cristina C. Filippi et al. / Biological Control 58 (2011) 160–166
R46
R46/M.o
R55
R55Mo
0.12
0.10
0.08
0.06
0.04
0.02
0.00
CTR/water CTR/M.o
R46
R46/M.o
R55
R55/Mo
Fig. 2. Enzymatic activity of rice plants treated with rhizobacteria15 days before
challenge inoculation with M. oryzae. (A) Peroxidase, (B) b-1,3 glucanase and (C)
chitinase activities after soil drenching with R46 and R55 and challenge inoculation.
[CTR/water = plants treated with water; CTR/M.o = plants spray inoculated with
conidial suspension of M. oryzae; R46 = plants treated only with Rizo-46; R46/
Mo = plants treated with Rizo-46 and challenged with M. oryzae; R55 = plants
treated only with Rizo-55; R55/Mo = plants treated with Rizo-55 and challenged
with M. oryzae].
4. Discussion
4.1. Plant growth stimulation
In the present study, of 148 isolates of rhizobacteria evaluated,
18 isolates showed significant potential to stimulate growth and
suppress rice leaf blast in the early stages of development of rice
seedlings. The mass screening of isolates for growth promotion increased plant height by 22.0%, root length by 60.02%, total biomass
by 47.45% and total root biomass by 45.83%. Similar results were
obtained in irrigated rice, beans, tomato, radish and tea (Kamilova
et al., 2006; Mendonça, 2006; Ashrafuzzaman et al., 2009). This
percentage of growth promoting isolates is considered high compared to the published literature and may be attributed to the high
clay content and organic matter of the soil from where the isolates
of rhizobacteria were collected. These isolates may stimulate
growth by the production of auxins such as IAA or analogues which
alter plant metabolic pathways and make available nutrients. The
results showed that four of the 18 isolates were positive for IAA
Author's personal copy
Marta Cristina C. Filippi et al. / Biological Control 58 (2011) 160–166
Peroxidase Activity (U.min-1.mg-1)
A
3.00
2.50
2.00
1.50
1.00
0.50
0.00
CTR/water
CTR/M.o
R46
R46/Mo
R55
R55/Mo
β-1.3 Glucanase activity (Abs. h-1.mg-1)
B
Chitinase Activity (Abs.h-1.mg-1)
C
8
7
6
5
4
3
2
1
0
CTR/water
CTR/M.o
R46
CTR/water
CTR/M.o
R46
R46/Mo
R55
R55/Mo
0.12
0.1
0.08
0.06
0.04
0.02
0
R46/Mo
R55
R55/Mo
Fig. 3. Enzymatic activity of rice plants sprayed with rhizobacteria 2 days before
challenge inoculation with M. oryzae. (A) Peroxidase, (B) b-1,3 glucanase and (C)
chitinase activities spraying with R46 and R55 and challenge inoculation. [CTR/
water = plants treated with water; CTR/M.o = plants spray inoculated with conidial
suspension of M. oryzae; R46 = plants treated only with Rizo-46; R46/Mo = plants
treated with Rizo-46 and challenged with M. oryzae; R55 = plants treated only with
Rizo-55; R55/Mo = plants treated with Rizo-55 and challenged with M. oryzae].
and eight for phosphate solubilization. In studies on PGPR
conducted in vitro, Ashrafuzzaman et al. (2009) demonstrated that
only 22% of the tested isolates were positive for IAA production and
44% for phosphate solubilization. The phytostimulation can be
related to IAA or analogue production, gibberellic acid, pirroloquinolintera quinon (PQQ) compounds, alterations in the signaling
pathway, and a small portion by solubilization of P due to nonspecific enzyme activity (Rodriguez et al., 2006). All of these can
results in the increase of plant growth, probably as a result of photosynthetic efficiency and chlorophyll contents by the signaling of
glucose and abscisic acid (Van Loon, 2007; Ramos-Solano et al.,
2008). But the exact mechanisms are yet unclear.
165
compared to the control. Several other studies also showed positive
results utilizing PGPR for controlling leaf blast in irrigated rice
(Vleesschauwer et al., 2008; Lucas et al., 2009), and in other pathossystems (Silva et al., 2004). Studies on Arabidopsis indicated that
some bacterial components responsible for airborne chemical signaling triggers plant growth promotion and induced resistance
against plant pathogens (Ryu et al., 2003; Saravanakumar et al.,
2007). Although plant defense response and growth stimulation
are known to be interconnected, the antagonism and synergy
between these phytohormones and signaling networks is not known
(Herman et al., 2008b).
However, the disease progress evaluated during five consecutive days showed differences in their efficiency depending up on
the application method. Even though the AUDPC of both isolates
did not show statistical difference by soil drenching method
(Fig. 1 A and B), 86% of reduction was obtained by spraying
Rizo-55 2 days before challenge inoculation (Fig. 1 C). The differences in the efficiency of application method may be explained
due to the use of rhizobacterial inducers pertaining possibly to
different genera. The rhizobacteria Rizo-55 was fluorescent
whereas Rizo-46 was non fluorescent (Table 3) and further investigation is underway. The utilization of different bacteria to
increases protection against phytopathogens specifically in rice
with Pseudomonas fluorescens (Nandakumar et al., 2000; Radjacommare et al., 2004), and the Bacillus (Vasudevan et al., 2002) were
reported.
The two rhizobacteria exhibited distinct differences in their ability as inducers of ISR and enzymatic activity. The increase of peroxidase activity was detected in rice plants both by biotic as well as
abiotic inducers (Manandahar et al., 2000). In the present study,
the activity of peroxidase increased more in response to soil
drenching by both rhizobacterial isolates than in response to challenge inoculation after induction (Fig. 2). On the other hand, the
peroxidase activity was higher in response to challenge inoculation
by M. oryzae after induction by spraying Rizo-55 (Fig. 3 A). The
rhizobacterium Rizo-46 showed greater affinity to plant by soil
drenching, the habitat in which it was recognized as inducer. The
same was observed in spray inoculation method by Rizo-55, where
it was recognized as an inducer. These differences can be attributed
to specific interactions among the PGPR and plant (Bais et al., 2006).
In the present investigation, glucanase and chitinase activities
increased in rice plants inoculated by soil drenching with Rhiz46 and challenged with M. oryzae (R46/Mo, Fig. 2) and plants
sprayed with Rizo-55 (R-55, Fig. 3). These results further indicated
that rice plants were primed to respond more quickly and systematically to a determined challenger. Probably, some PGPR can
induce both SAR and ISR by first activating SA dependent pathway
(SAR) followed by stimulation of SA independent and ET/JA dependent pathways (ISR) (Herman et al., 2008a). Many researchers have
suggested that rice employs distinct mechanisms for its defense
against M. oryzae, (Park et al., 2006). Although antibiosis has been
shown to be one of the important mechanisms (Duffy and Defago,
1999), the effect of rhizobacteria inducing PR gene expression pattern against M. oryzae (Ahn et al., 2005; Park et al., 2006) should be
considered and further investigated. The results on enzymatic
activity are in accord with the observations on leaf blast disease
suppression obtained in the greenhouse study. Similar results were
observed in tea plants against Exobasidium vexans (Saravanakumar
et al., 2007) in tobacco plants, against Peronospora tabacina (Pan
et al., 1991).
4.2. Leaf blast disease suppression and enzymatic activity
While all 18 isolates selected for growth promotion were effective in suppressing the leaf blast severity, the isolates Rizo-46
applied 15 days before and Rizo-55 applied 2 days before spray inoculation with M. oryzae reduced LBS by 90% and 95%, respectively,
5. Conclusions
The multivariate analyses showed that two rhizobacterial isolates, Rizo-55 and Rizo-46 promoted simultaneously plant growth
Author's personal copy
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Marta Cristina C. Filippi et al. / Biological Control 58 (2011) 160–166
and reduced rice leaf blast severity. These isolates were able to induce plant defense by increasing enzymatic activity of pathogenesis related proteins.
Acknowledgments
We acknowledge with grateful thanks to FAPEG and Embrapa
for providing financial support for this research and CNPq for
awarding a research fellowship to the first author.
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