Frazier (1965) proposed that “the ability to transmit Pierce`s disease

Frazier (1965) proposed that
“the ability to transmit Pierce's disease is a group characteristic of the
Tettigellinae (currently Cicadellinae) and that each species of the group should
be a suspected vector until proven otherwise”
BGSS and GWSS
Glassy-winged sharpshooter
(GWSS)
Photo R. Coviello
Photo UC IPM
Homalodisca vitripennis
First detected in CA in 1989
High numbers on citrus (>6,000 per tree)
Polyphagous (estimated > 200 host plants)
Feeds on woody tissues of grapevines and
dormant plants
Highly dispersive
Nymphal transmissionof X. fastidiosa
GWSS is a less efficient vector than BGSS
BGSS
GWSS
Inoculation of two-year old wood tissue
Plant tissue
# plants
# infected plants
Transmission rate/group (%)
Green
shoot
44
Two-year old
wood
40
29
19
65.9
47.5
Percentage positive plants
50%
-----------------------Dormant Vine s ------------------------------------------------------------------------------------40% ----------Gree n Vines
30%
20%
10%
0%
8-Feb
Field
Lab
21-Feb
28-Feb
Date of Inoculation
Pooled dormant results
8-May
GWSS vs BGSS
GWSS lower transmission efficiency is
probably compensated by:
Transmission to two-year old wood (vine-to-vine
spread)
Transmission to dormant plants
Very high populations
Higher dispersal capacity than BGSS
- summary Vector transmission of X. fastidiosa
No vector species – pathogen strain specificity
Nymphs and adults transmit X. fastidiosa
No latent period
No transmission after molting
No transovarial transmission
Persistent in adults
P=NS
Xf
inoculum
Vector
number
Xf populations within vectors
Infection
level
• Infection level is determined
by the number of infective
vectors, not inoculum size
0
1
2
3
4
Number of infectious vectors
Higher vector ‘load’ = earlier symptoms
Are all X. fastidiosa isolates transmitted with similar efficiencies?
Significant differences observed among
strains (almond vs grape)
isolates within grape strain
No difference for test (recipient) plants
Do host plants determine transmission rates?
Source plant, but not test (recipient) plant, impacts
transmission rates for the same isolate of X. fastidiosa
What about pathogen/host plant combinations that may
function as reservoirs for vector acquisition?
Effect of strain and source plant on transmission
Significant differences
source plants*
isolate*
122 individuals
tested!
Vector behavior and transmission
Lastly, the host tissue preference hypothesis…
Sharpshooter plant tissue preference is a strong
component determining vector transmission efficiency
15”
apical
10”
medium
5”
basal
10 insects/plant
10 plants/sharpshooter species
Vector within-host feeding preference mediates transmission of a heterogeneously distributed pathogen
Where are the bugs?
Green sharpshooter - GSS
Daugherty et al. EE 2010
Blue-green sharpshooter - BGSS
No-choice X. fastidiosa acquisition by sharsphooters
from top and basal parts of the alfalfa plant
D
13”
E
A
2”
C
A
Impact on transmission efficiency…
Effect of plant tissue
Role of vector species
Transmission
Pathogen infection
rate/populations
GSS BGSS
1/10 0/10
12/37
10E5.5cfu/g
GSS BGSS
10/15 2/10
32/37
10E8.5cfu/g
15”
10”
5”
Choice - GSS much more efficient than BGSS from alfalfa to alfalfa
-infection levels and
symptoms accumulate
through time
-sharpshooters avoid
infected plants at later
dates
-spread peaks early,
then declines
Almeida Purcell 2006 AESA
Newman et al. 2003 AEM
Cell-cell signaling mutants are not transmissible because cells
do not colonize the foregut of leafhopper vectors
Newman et al. 2004 PNAS
Amido black (protein
specific stain)
Carbohydrate
binding proteins
NCM
Nitrocellulose
membrane coated
with polysaccharides
Xylella fastidiosa cells
bind to polysaccharides
in vitro through
membrane proteins
which act as lectins.
Killiny & Almeida 2009 AEM
Affinity
Binding to Copolymers (glucosylated polyacrylamide)
Binding to Foregut extracts in absence or presence of sugars
-Xf
+Xf
+Xf +D-glucose
Concentration
+Xf +D-galactose
glucosyl ligands
+Xf + N-acetylglucosamine
+Xf + chitobiose (N-acetylglucosamine)2
galactosyl ligands
+Xf + chitotriose (N-acetylglucosamine)3
+Xf +D-mannose
mannosyl ligands
Attachment of GFP- Bacteria to GWSS hindwings
No Bacteria
X. fastidiosa
X. campestris
pv. campestis
P. syringae
pv. syringae
E. herbicola
E. coli
no sugar
no Xf
0mM
50mM
100mM
200mM
500mM
Xylella fastidiosa cells OD 0.4
Killiny & Almeida 2009 AEM
1M
Transmission rates
Cell populations within insects
over time (qPCR)
Killiny & Almeida AEM 2009
Development of biofim on insect foregut
Time
0
Attachment Via CBPs
and HxfB
HxfA
Exopolysaccharides (gum)
protect the biofilm
Transition from attached cells
from laterally to polarly attached
Are exopolysaccharides (gum)
and fimbrial adhesins implicated
in biofilm formation?
Do type IV pili (long pili) play any
role in transmission?
Cell binary division allow double
layer biofilm formation
Almeida and Purcell
2006
Killiny unpublished Data
WT gumD
Meng et al 2005
Movement
within plant.
gumH
Killiny unpublished Data
Low
populations in
plant.
?
?
Insect transmission of fimbrial adhesins (left) and gum (right) mutants.
6E11: fimA-, 1A2: pilB-, DM12: fimA-/pilO-
Development of an artificial diet system is necessary to test the mutants that are
affected in multiplication or movement within the plant
Mechanical inoculation
Transmission
()
Xf in PWG
Insect feeding throw parafilm
(--)
Xf in PWG
Could host structural compounds, such as pectin or
glucan, regulate gene expression in X. fastidiosa and
control vector transmission?
Perez-Donoso PP 2010
Newman et al. AEM 2003
AH Purcell
jbei US DoE
Killiny & Almeida PNAS 2009
XFM-Pectin
XFM-Galacturonic acid
XFM-Rhamnose
Medium-dependent changes in the expression of genes
previously implicated in X. fastidiosa pathogenicity to plants
Fold changes in relation to
commonly used medium (PWG)
Fold changes in relation to
basal medium (XFM)
Immunological detection of Hxfs
Pectin and glucan induce regulons in X. fastidiosa
Transmission rate
*
100
12h
96h
75
*
50
25
0
TEM
TEM
pilB
fimA
hxfA
gumD
hxfB
gumH
rpfF
Attachment Via CBPs
and HxfB
HxfA
Exopolysaccharides protect
the biofilm
Transition from attached cells
from laterally to polarly attached
Cell binary division allow double
layer biofilm formation
Persistent in adults
Insect maintains Xf overwinter
Another environmental cue might exist in the insect foregut
to maintain the adhesive (transmissible) state of X. fastidiosa
Effect of chitin on X. fastidiosa’s growth
0,6
XFM
XFM-chitin
OD600
0,4
0,2
0
0
2
4
6
8
10
Log CFU/3ml
Days
*
7
Fold change
XFM
XFM-chitin
100
10
1
0,1
fimA hxfA hxfB pilY1 rpfF
y
6,5
Planktonic
cells
6
5,5
a
b
x
5
XFM
XFM-chitin
(GlcNac)3-MU overlay
assay
PD1826
chiA
PD1827
nahA
*
*
PD1828
bnmA
PD1829
bgl2
0,6
6
0,5
5
0,4
4
0,3
3
0,2
2
0,1
1
0
0
0
1
2
3
4
5 6
Days
7
8
Fold change
OD600
X. fastidiosa uses chitin as a carbon source
9 10
chitinase activity
XFM ∆C
XFM ∆C + Colloidal chitin
Attachment Via CBPs
and HxfB
HxfA
Exopolysaccharides protect
the biofilm
Transition from attached cells
from laterally to polarly attached
Cell binary division allow double
layer biofilm formation
Wheat germ agglutinin (WGA) Core of N-linked oligosaccharides
Concanavalin A (Con A) Branched mannose, carbohydrates with terminal mannose or glucose
Peanut agglutinin (PL) Terminal β-galactose
Lentil lectin (LL) Branched mannose with fucose linked α(1,6) to N-acetylglucosamine