General Xaj diagnostic/identification protocols

New procedures for the
detection of
Xanthomonas arboricola
pv. juglandis in plant
samples
Sophie Gironde and Charles Manceau
Steps in the project
! Characterization of X. a. pv. juglandis
! Antibiotic resistance and selective
medium.
! BIO-PCR and BIO RT-PCR.
Development of a selective
medium.
Objective:
Set up a culture medium allowing an elective
growth to Xanthomonas arboricola pv.
juglandis from plant extracts.
I.
Sensitivity of X. a. pv. juglandis to a large range of
antibiotics.
II.
Tests of medium selectivity.
III. Tests for isolation from plant samples.
Sensitivity of X. a. pv. juglandis to a
large range of antibiotics.
• 28 antibiotics belonging to 12 families were
tested versus 5 strains of X. a. pv. juglandis
– 2 strains isolated from fruit and leaf spots
• CFBP1022 and 12581
– 3 strains isolated from oozing cankers
• 12582, 12767 and 12785
Materials and methods
Determination of the Minimum Inhibitory
Concentrations (MIC) of antibiotics
Sterile steal sticks were dipped in
bacterial
suspensions
(106
UFC/mL).
Each dipped stick lied 4 µL of
bacterial suspension onto agar
surface.
Two replicates were
performed.
The plates were incubated for 3
to 6 days at 28°C.
YPGA + antibiotic [0,5-128 mg/L]
Lecture
Positive results:
- Growth identical to this
and YPGA
- separated colonies
(more than 4 colonies per
repetition)
Negative results:
- No growth
- Velum 6 days after
inoculation
- less than 4 colonies per
repetition
Results
MIC (mg/L) of antibiotic vs. Xanthomonas arboricola pv. juglandis.
Bacitracine
Colistine
Xanthomonas arboricola
bacterial spots
CFBP 1022
12581
12582
128
64
128
<=0,5
<=0,5
<=0,5
pv. juglandis
O.V.C.
12767
12785
>128
>128
<=0,5
<=0,5
Oxytétracycline
Tétracycline
4
1
2
<=0,5
2
<=0,5
2
<=0,5
2
<=0,5
Céphalexine
Céfalotine
Céfuroxime
128
64
32
32
8
8
>128
128
32
>128
128
64
>128
128
64
Kasugamycine
Gentamicine
Spectinomycine
Kanamycine
Streptomycine
Neomycine
>128
8
32
4
4
32
>128
8
8
2
1
16
>128
4
32
2
2
16
64
8
8
2
1
16
64
8
8
1
1
16
Lincomycine
>128
32
>128
>128
>128
Pénicilline G
Carbénicilline
Ampicilline
Xanthomonas arboricola pv. Juglandis
Non C.V.S.
C.V.S.
CFBP 1022
12581
12582
12767
12785
8
1
8
16
8
8
1
4
8
8
16
2
4
32
8
Triméthoprime
16
>128
>128
128
128
A. nalidixique
A. oxolinique
4
<=0,5
8
1
8
2
8
2
16
2
Vancomycine
8
4
16
32
16
Chloramphénicol
4
2
4
2
2
Novobiocine
2
4
8
8
8
Tellurite
16
16
32
32
32
Azide Na
16
16
16
16
16
Rifampicine
Rifamycine SV
<=0,5
4
<=0,5
8
<=0,5
8
<=0,5
8
<=0,5
8
No difference between strains isolated from fruit, leaf and oozing canker (excepted
cefalotine)
Cephalexine has been chosen to be incorporated into the medium.
Isolation from plant extracts
Printing F2
YPGA
•
•
Printing F2
YPGA+C30
All assays (10 leaves) display a significative reduction of the epiphytic microbial
growth onto Y.P.G.A. medium supplemented with Cephalexine (30 mg/mL).
Almost only yellow colonies grew on the medium. These bacteria were identifed as
X. a. pv. juglandis
Isolation fromplant extract
Grinding F3
Témoin
Y.P.G.A.
Grinding F3
Y.P.G.A. + C30
Y.P.G.A.+C30 : Separated colonies of Xanthomonas in diluted suspension
drops
Search for specific genomic
markers.
Objective:
Find genomic sequences specific to X. a. pv.
juglandis and to the lineage that causes Oozing
Vertical Cankers
Strategies:
Suppresive Substractive Hybridization (SSH)
Cloning of AFLP DNA bands
Cloning of AFLP DNA fragments
Polyacrylamide gel (7%) after electrophoris of
A.F.L.P. products.
left : amplification of X. arboricola strains D8,
D14, D33, et D44 that cause OVC; and strains
D3, D15, D22 et D59 that do not cause OVC with
the selective primer MspI-TA .
4
8
1
5
Right: Idem with MspI-TG primer.
2
6
7
3
Cloned bands:
OVC:
1 : 380 pb
2 : 350 pb
3 : 185 pb
6 : 190 pb
7 : 160 pb
X. a. pv. juglandis:
4 : 500 pb
5 : 360 pb
8 : 420 pb
5 primer sets were
designed and tested
for speficity
Bacterial
strains
Xaj 2528
Xaj 2568
Xaj 12581
Xaj 12588
Xaj 12783
Xaj 12574
Xaj 12580
Xaj 12711
Xaj 12768
Xaj 12785
1caF/1CaR
selected for
detection reagents
Xa celebensis
3523
Xa populi 3123
Xa pruni 2535
Xa corylina 1159
Xa fragariae
6771
X axonopodis
citri 2525
X axonopodis
vesicatoria 6864
X campestris
campestris 5241
X oryzae oryzae
2532
X translucens
translucens
2054
Pseudomonas
syringae 12836
1CaF/1CaR 1CbF/1CbR 3AaF/3AaR 4AaF/4AaR 4BaF/4BaR
+
+
+
+
+
+
+
+
+
+
/
-
+
+
+
+
+
+
+
+
+
+
/
-
+
+
+
+
+
+
+
+
+
+
/
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
/
+
-
-
-
+
-
/
+
(218 pb)
2aF/2aR
+
+
+
+
+
+
+
+
+
+
+
+
+
-
Set up a BIO-PCR procedure for the sensitive
detection of X. a. pv. juglandis in plant samples
• Spread 0.1mL of plant extract (grinding or washing
material) on the surface of Y.P.G.A. supplemented with
cycloheximide (50 mg/L), Cephalexine (30mg/L)
• Incubate at 27°C for 20 - 24 hours
• Wash the plate surface with 2 mL of sterile distilled water
• Boil the suspension for 5 min
• Put on ice
• Centrifuge 1mL at 13 000 g for 2 min.
• Use 3 !L for PCR
BIO-PCR on plant samples
Result obtained with
the 1CaF/1CaR
primer set
X. a. pv. juglandis
Contaminated trees
Healthy trees
MW
H2O
100 bp
As soon as one cfu is
expected a clear
signal was obtained
on the gel
Uncontaminated trees are rare, almost
all walnut trees were contaminated in
June 2008.
MW
Real time PCR
A Taqman probe was designed between 1CaF and
1CaR complementing sequences and labelled with
the FAM dye and the corresponding quencher
The PCR reactions were performed on DNA extracts
in the same manner that for BIO-PCR
BIO-RT-PCR
" Clear separation of positive and negative samples
" Save time and manipulation
assays
Positive controls
(dilutions of
extracts of pure
culture of X. a. pv.
juglandis
healthy samples
and negative
control
Extraction of nucleic acid (7/7)
! FTA cards Whatman: An alternative extraction tool
for collection, shipment, archiving
and purification of nucleic acid from
a wide variety of biological samples
- grind the plant sample onto the paper
- let dry
- take a sample disk
- wash the disk 3 times in 200 !l of FTA
purification reagent
- rinse the disk twice in 200 !l of TE8 buffer
-Put the disk in the PCR mix
and run the PCR
Conclusion
• An early detection procedure is available for X.
a. pv. juglandis
–
–
–
–
Specific
Easy to use
Rapid (less than 48 hours)
Detect viable bacteria
• Need more research for the specific detection of
strains that cause OVC
– Possible with AFLP
• But still difficult to handle
– Potential interest of VNTR (microsatellite)
Perspectives
• Use MLVA to analysis the genetic structure of
populations that cause:
–
–
–
–
Vertical oozing canker
Brown apical necrosis (Univ. Gerona)
Geographical populations
…
• Collaboration with Brion Duffy
– The genomic sequence of X. a pv. pruni use as
genomic ressource to find VNTR useful for MLVA in
X. arboricola pathovars.