Download Diagnostic protocol for

2006-TPDP-12
DRAFT
Diagnostic protocol for regulated pests
Xanthomonas axonopodis pv. citri
General comments:
Perhaps include photos of symptoms?
Biochemical characteristics in a table
Bullet points would be useful for some of the sections
Information on sensitivity, specificity and reliability should be included
Include details on reference strains
Flow chart could be included, but not mandatory – would indicate minimum
requirements for detection and identification (but not a decision making scheme),
particularly if a combination of methods is required
Input from Asiatic experts would be useful
Index
Page
1. Pest information
2. Taxonomic information
3. Detection
3.1. Symptomatic Plant Material
3.2. Asymptomatic Plant Material
4. Identification
5. Records
6. Contact points for further information
7. Acknowledgements
8. References
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1. Pest information
Xanthomonas axonopodis pv. citri (Xac) (Hasse 1915) Vauterin et al., 1995, the causal
agent of citrus canker, causes severe damage of many cultivated species of Rutaceae,
primarily Citrus spp, Fortunella spp. and Poncirus spp., grown under tropical and subtropical conditions, being prevalent in many countries in Asia and South America.
There are distinct pathogenic groups among strains of Xac that correlated with
serological and genetic differences. The strains are generally divided into the following
groups: group A strains causing (Asian canker) with a worldwide distribution in citrusgrowing areas outside the Mediterranean basin, group B strains (causing cancrosis B)
found in South America, group C strains (causing Mexican lime cancrosis) in Brazil.
Vauterin et al. (1995) allocated the pathovar citri group A strains to Xac and used the
defective names pv. aurantifolii and pv. citrumelo without amendment. Group B strains
are restricted to lemon, although Mexican lime, sour orange and pummelo are also
susceptible. Group C is restricted to Mexican lime. These two canker types were
described in South America and were gradually supplanted by group A strains. Two
groups of strains, with restricted host range, have been identified within pathotype A
(Vernière et al., 1998; Sun et al., 2000), and designated as A* and Aw. These are closely
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related to type A strains (Cubero & Graham, 2002, 2004) but affect only Mexican lime
and Alemow.
2. Taxonomic information
Name:
Synonyms:
Xanthomonas axonopodis pv. citri (Hasse) Vauterin et al., 1995
Pseudomonas citri Hasse
Xanthomonas citri (Hasse) Dowson
Xanthomonas citri f.sp. aurantifoliae Namekata & Oliveira
Xanthomonas campestris pv. citri (Hasse) Dye
Xanthomonas citri (ex Hasse) nom. rev. Gabriel et al.
Xanthomonas campestris pv. aurantifolii Gabriel et al.
Common names:
Citrus bacterial canker
Citrus canker
Domain: Bacteria, Phylum: Proteobacteria, Class: Gammaproteobacteria, Order:
Xanthomonadales, Family: Xanthomonadaceae, Genus: Xanthomonas
3. Detection
3.1. On symptomatic plant material
Symptoms of citrus canker occur in any season on seedlings and young trees in which
appear a flush of abundant angular shoots from late summer through autumn. However,
the disease becomes sporadic as trees reach full fruiting development because when the
leaves are not young and the fruits reach their final size, they are not susceptible under
natural conditions and fewer angular shoots are produced. Disease severity also depends
on the susceptibility of the host plant species and cultivars (Goto, 1992). There is no
evidence that this pathogen is seedborne.
Xac can survive in diseased plant tissues, as an epiphyte on host and non-host plants,
and as a saprophyte on straw mulch or in soil. However, overwintering lesions,
particularly those formed on angular shoots, are the most important source of inoculum
for the following season. The bacteria are disseminated by rainwater running over the
surfaces of lesions and splashing onto healthy shoots.
Symptoms on branches
In dry conditions, the canker spot is corky or spongy, raised and has a ruptured surface,
while, in moist conditions the lesion enlarges rapidly, the surface remains unruptured
and oily at the margin. In the more resistant cultivars a callus layer may form between
the diseased and healthy tissue. The scar of a canker may be recognized by scraping the
rough surface with a knife to remove the outer corky layer, revealing light to dark
brown lesions in the healthy green bark tissues. The discoloured area can vary in shape
and in size from 5-10 mm, depending on the susceptibility of the host plant.
Symptoms on leaves
Bright yellow spots are first apparent on the underside, followed by erumpent brownish
lesions in both sides of the leaves which become rough, cracked and corky. The canker
may be sorrounded by a water-soaked and a yellow halo margin.
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Symptoms on fruits
As above, crater-like lesions develop in the surface of the fruit and may be scattered
singly over the fruit or several lesions may occur together with irregular contour.
Exudation of resinous substances may be observed in young infected fruits. The canker
never penetrates through the rind.
The samples can be tested by isolation on nutrient media, serological testing (ELISA,
IF), molecular testing (PCR) and bioassays (leaf discs or detached leaves). After
isolation, pathogenicity tests require several days to 2 weeks for symptoms to appear
and, thereafter, the cause of symptoms must be confirmed by more complex procedures.
Isolation of the causal organism can be performed by streaking lesion extracts onto
plates of suitable media. The appearance of the resulting colonies can be characteristic
for Xac but there are as yet no exclusively selective media available for pathovar
identification (e.g. pv. citri). Immunofluorescence (IF), ELISA (Civerolo & Fan, 1982;
Civerolo & Helkie, 1981) and PCR are reliable methods for examining many samples
and can be completed within several hours.
Isolation
Lesions are macerated in 0.5-1 ml saline (distilled sterile water with sodium chloride to
0, 85 %, pH: 7,0) sometimes they must be previously disinfected with 1%, sodium
hypochlorite during 1 minute, rinse 3 times with sterile distilled water and comminuted
in small pieces. Streak an aliquot of the extract on nutrient media. Suitable general
isolation media are nutrient agar supplemented with 0.1% glucose (NGA), YPGA (yeast
extract, 5 g; bactopeptone, 5g; glucose, 10 g; agar, 20 g; distilled water, 1L pH 7), or
Wakimoto medium: potato broth, 250 ml; saccharose, 15 gr; peptone, 5 gr; sodium
phosphate anhydrous, 0,8 gr; calcium nitrate 7 H2O, 0,5 gr; bacto agar, 20 gr; distilled
water, 1 L; pH: 7,2. Cycloheximide (100mg/l ) previously filter sterilized can be added
when necessary after autoclaving the media. Growth is evaluated after incubation at
28ºC for 3 days. In commercial fruit samples the bacteria can be stressed or having
difficulties for growing in the plates and more incubation days, or bioassays can be used
for recovering the bacteria from the samples.
DAS – ELISA:
A kits containing the components for Xac detection are available commercially.
Immunoglobulin (IgG) can be precipitated from polyclonal antibodies obtained against
Xac with ammonium sulphate, followed by two additional precipitations resuspensions
and dialysis in 0.15 mmol l-1 NaCl. Microtitre plates are coated with 200 µL carbonate
coating buffer (pH 9.6) containing 5-10 µg/ml IgG anti-Xac and incubated overnigth al
4ºC. After washing the plates successively three times with PBS-Tween, add 200 µl of
test samples, and negative controls (healthy plant material and another bacterial species)
and positive control (a reference strain of Xac). Incubate for 2 h at 37ºC. After washing
as before, 200 µl IgG anti-Xac conjugated with alkaline phosphatase diluted 1/20001/4000 in PBS-Tween, are added and incubated for 2 hs at 37°C. The washing is
repeated. Then 200 µl of p-nitrophenyl phosphate substrate buffer (1 mg/ml) are added
and plates are incubated for 30 to 60 min at room temperature. The absorbances are
quantified with a spectrophotometer equipped with a 405 nm filter. Criteria for the
determination of a positive sample is two times the OD value of healthy controls.
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Monoclonal antibodies are available for ELISA, but are mostly advised for
identification of pure cultures, due to low sensitivity of the detection in plant material
and the risk of non-specific reactions.
Immunofluorescence
Aliquots of 25 µl of each bacterial preparation or plant samples to be tested are pipetted
onto the windows of a plastic-coated multiwindow microscope slide, allowed to air-dry
and gently heat-fixed over a flame. Separate slides are set up for each test bacterium and
also, positive and negative controls, as for ELISA. Commercially available antiserum is
diluted with PBS at pH 7.2 and appropriate dilutions added to windows of each slide.
Controls of normal (pre-immune) serum at one dilution and of PBS are also added to the
slide. Slides are enclosed in a humid chamber and incubated at room temperature for 30
min. The droplets are shaken off the slides and they are rinsed with PBS and then
washed three times for 5 min in PBS. The slides are gently blotted dry. 25 µl of goat
anti-rabbit gamma globulin-fluorescein isothiocyanate conjugate (FITC) is pipetted into
each window at the appropriate a dilution. The slides are incubated, rinsed, washed and
blotted dry as before. 10 µl of 0.1 mmol l-1 phosphate-buffered glycerine (pH 7.6) with
an anti-fadding reagent, is added to each window and covered with a coverslip.
The slides are examined with a fluorescence microscope under immersion oil at x 600
or x 1000. The FITC will fluoresce bright green under the ultraviolet light of the
microscope. If the positive control with known bacterium fluoresces and the negative
controls of normal serum and PBS do not, examine the sample windows for bacterial
cell wall fluorescence, looking for the cells with the size and form of Xac.
Polymerase Chain Reaction (PCR)
DNA extraction from infected citrus tissue
For obtaining the more accurate PCR results, a DNA extraction protocol should be used
before amplification from plant material. The original DNA extraction by Hartung et al.
(1993) was performed with a CTAB protocol, but there are also commercial methods as
Quiagen DNA easy and an isopropanol protocol (that do not require phenol) that had
been extensively evaluated. For the isopropanol protocol (Llop et al., 1999) lesions or
other suspicious infected plant materials are cut into small pieces, covered with PBS
buffer and shaken in a rotary shaker for 20 min at room temperature. The supernatant is
filtered and centrifuged for 20 min at 10 000 g. The pellet is resuspended in 1 mL of
PBS. 500 µl is saved for further analysis or for direct isolation on agar plates. 500 µl of
the sample is centrifuged at 10 000 g for 10 min. The pellet is resuspended in 500 µL of
extraction buffer (200 mm Tris HCl pH 7.5, 250 mm NaCl, 25 mm EDTA, 0.5% SDS,
2% PVP) vortex and left for 1 h at room temperature with continuous shaking. The
suspension is centrifuged at 5000 g for 5 min, 450 µl of the supernatant is transferred
and 450 µl isopropanol is added. The suspension is mixed gently and left at room
temperature for 1 h. Precipitation can be improved by the use of Pellet Paint
Coprecipitant (Novagen, Darmstadt, DE) (Cubero et al., 2001). The suspension is
centrifuged at 13 000 g for 10 min, the supernatant is discarded and the pellet is dried.
The pellet is resuspended in 100 µl water. 5 µl of sample is used in a 50 µl PCR
reaction.
Primers used in PCR.
Several sets of primers are available for diagnosis of Xac. Based on Hartung et al.
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(1993), primers 2 (5′-CAC GGG TGC AAA AAA TCT-3′) and 3 (5′-TGG TGT CGT
CGC TTG TAT-3′) allow the amplification of a 222 bp DNA fragment only in A strains
and are the most frequently used in assays on plant material because of the sensitivity
reached. Primers J-pth1 (5′-CTTCAACTCAAAC-GCCGGAC-3′) and J-pth2 (5′CATCGCGCTGTTCGGGAG-3′) based on the nuclear localization signal in virulence
gene pthA allow the amplification of a 197 bp fragment in A, B and C strains (Cubero &
Graham, 2002). They are universal, but they showed lower sensitivity in plant material
detection.
For amplification with primers 2 and 3, the PCR reaction mix is prepared in a sterile vial
(per 50 µl reaction): PCR buffer (50 mm Tris HCl pH 9, 20 mm NaCl, 1% Triton ×100,
0.1% gelatin, 3 mm MgCl2), 1 µm each primer, 200 µm deoxynucleoside triphosphates,
and 1.25 units of Taq DNA polymerase. The components are mixed and 45 µL of the
mix is transferred into sterile PCR vials. The vials are kept with the PCR reaction mix
on ice. 5 µl of the extracted DNA, water control and positive control are added in the
specified order to the vials with the PCR reaction mix. The vials are placed in the
heating block of the thermal cycler and the following programme is run: 35 cycles of 70
s at 95°C (denaturation), 70 s at 58°C (annealing of primers), 60 s at 72°C (extension of
copy). The PCR product is analysed and the vials are stored at 4°C for use on the same
day, or at −20°C for longer. The PCR fragment of 222 bp should be detected in positive
samples in 2% agarose gel after electrophoresis and staining with ethidium bromide.
The water control should be negative in each case. If positive, the test should be
repeated. The gel is photographed if a permanent record is required. This protocol is
adviced as screening method for symptomatic material in the EPPO protocol
(OEPP/EPPO 2005).
Pair 4/7 [4-5′-TGT CGT CGT TTG TAT GGC-3′; 7-5′-GGG TGC GAC CGT TCA
GGA-3′] has proved useful for identification of A strains and shows variable results for
B and C strains (Vernière et al., 1998). Nested PCR, immunocapture and colorimetric
detection of nested PCR products for direct and sensitive detection of Xac in plants have
also been developed (Hartung et al.,1993).
Real-Time Polymerase Chain Reaction
After obtaining DNA from plant material by using protocol previously described (Llop
et al., 1999), the pellet is resuspended in 100μl in sterile ultrapure water, and stored at –
20°C until further use.
Oligo-nucleotide primers were designed using ABI PRISM Primer Express software
(version 2.0; Applied Biosystems, Foster City, CA). A set of primers J-pth3 (5'-ACC
GTC CCC TAC TTC AAC TCA A-3') and J-pth4 (5'-CGC ACC TCG AAC GAT
TGC-3') were designed based on sequences of the pth gene, a major virulence gene used
in other works specifically to detect CBC strains (Cubero & Graham, 2005). RT-PCR
was carried out by adding 2 µl of the template DNA to a reaction mixture containing
12.5 µl of QuantiMix Easy Kit (Biotools, B&M Labs, S.A.) which comprised
Quantimix Easy Mastermix and MgCl2 (50mM), 1 µl of 10 µM forward primer (JRTpth3), 1 µl of 10 µM reverse primer (J-RTpth4) in a final reaction volume of 25 µl.
RT-PCR reaction is completed in an ABI PRISM 7000 Sequence Detection System
(Applied Biosystems). Amplification conditions for all the primers and probes consisted
of an initial activation step of 15 min at 95°C followed by 40 cycles of 15 s at 95°C and
1 min at 60°C.
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Bioassay: in vitro inoculation tests in leaf discs
This test uses resident susceptible tissue to Xac inoculated with sample extracts in
appropriate conditions for the bacterial multiplication and development of incipient
pustules of the disease. Once inoculated with macerated lesions, it constitutes a very
sensitive (detect 102 cfu/ml) and specific diagnosis method.
The procedure for the application of this bioassay begin by sterilizing ELISA plates for
15 minutes in a microwave oven and the holes by 200 µl with sterile 1,5% agar-water
under laminate flow chamber at room temperature. Young grapefuits Duncan (Citrus
paradisi) leaves (light green) are disinfected for one minute with 1% sodium
hypochlorite. Rinse them 3 times with sterile distilled water and left them superficially
dry in the laminate flow chamber at room temperature. With a punch, previously
disinfected with 96º ethanol, obtain leaf discs. The leaf discs are placed back up in each
hole with agar-water. Fifty µl from macerated lesions, with 4 repetitions for each one,
are added.
A Xac suspension of 105 cfu/ml is used as a positive control, and saline sterile as a
negative one (4 repetitions for each one). Plates are incubated at a 28ºC for 12 days
under constant light and sealed with parafilm, achieving a relative dampness near to
100%. The formation of incipient whitish pustules in each of the leaf disks were
evaluated from the third day, using stereoscopic microscope and isolate Xac as under
isolation above. Even the symptomless discs can be analysed for detecting the presence
of living bacteria by isolation, after 12 days (Verdier et al., 2006).
Detached leaf enrichment
Xac can also be selectively enriched in wounded detached leaves of grapefruit cv.
Duncan, Mexican lime seedlings or citrumelo cv. Swingle seedlings. Young terminal
leaves from glasshouse-grown plants for 10 min in running tap water, surface-disinfect
in 1% sodium hypochlorite for 1 min, and aseptically rinse thoroughly with sterile
distilled water are washed. Each leaf by puncturing with a needle o making small cuts
with a scalped, through the lower surface is aseptically wounded and placed in 1%
water agar plates with the lower surface up. Droplets of 10-20 µl of the plant extracts
are added. Use positive and negative controls as for leaf discs bioassay. After 5-7 days
at 25ºC in a lighted incubator pustules development are evaluated and Xac is isolated as
above (OEPP/EPPO, 1998).
3.2. On asymptomatic plant material
In the absence of symptoms, leaf samples are taken from the trees; 10 leaves per tree
constitute one sample. The leaves and fruit samples are washed in peptone buffer,
incubated at room temperature and the sample is then concentrated (OEPP/EPPO,
1998). Fruits samples are individual washed in sterile bags in peptone buffer, incubated
at room temperature (Verdier et al., 2006).
Preparation of leaf samples
Ten leaves are shaken for 20 minutes at room temperature into 50 ml peptone buffer
(sodium chloride, 8,5 gr; peptone, 1 gr; Tween 20, 250 µl; distilled water, 1 l; pH: 7,2)
(OEPP/EPPO, 1998). For composite samples are used 100 leaves into 200 ml peptone
buffer.
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Supernatant is removed with a vacuum pump after centrifugation for 20 min at 6000 g
and the pellet is resuspended in 10 ml of 0,85% saline.
Aliquots (100 µl) of 1/100 and 1/1000 dilution of each washing solution are streaked on
XOS semi-selective medium in triplicate (saccharose, 20 gr; peptone, 2 gr; monosodium
glutamate, 5 gr; calcium nitrate, 0,3 gr; phosphate dipotassium anhydrous, 2 gr; EDTA
Fe, 1 mg; bacto agar, 17 gr; distilled water, 1 L; pH: 7,0; cycloheximide, 100 mg;
cephalexine, 20 mg; kasugamycine, 20 mg; methyl violet, 0,3 mg) (Monier, 1992).
Growth is evaluated after incubation at 28ºC for 5-6 days.
Preparation of fruit samples
Individual fruits are shaken for 20 minutes at room temperature in sterile bags,
containing 50 ml of peptone buffered water. Aliquots (100 µl) of 1/10 and 1/100
dilution of each washing solution are streaked on XOS semi-selective medium in
triplicate. Growth is evaluated after incubation for 5-6 days at 28ºC (Verdier et al.,
2006).
Bioassay: in vitro inoculation tests in leaf discs
The symptomless discs or detached leaves must be analysed for detecting the presence
of living bacteria by isolation, after 12 days, because the bacterial cells are multiplied in
the plant tissue and can be isolated in higher numbers (see above).
4. Identification
Description and biochemical characteristics
Xac is a Gram-negative, straight, rod-shaped bacterium measuring 1.5-2.0 x 0.5-0.75
µm. It is motile by means of a single polar flagellum. It shares many physiological and
biochemical properties with other members of the genus Xanthomonas. It is
chemoorganotrophic and obligately aerobic with the oxidative metabolism of glucose.
Colonies formed on medium with glucose are smooth, circular, creamy-yellow with
copious slime. The yellow pigment is xanthomonadin. Catalase is positive, but Kovacs'
oxidase is negative or weak; nitrate reduction is negative. Asparagine is not used as a
sole source of carbon and nitrogen simultaneously; various carbohydrates and organic
acids are used as a sole source of carbon. Hydrolysis of starch, casein, Tween 80 and
aesculin is positive. Gelatine and pectate gel are liquefied. Growth requires methionine
or cysteine and is inhibited by 0.02% triphenyltetrazolium chloride. Biovars may be
distinguished by utilization of mannitol (Bradbury, 1986). For further information on
the bacteriological properties of Xac see Goto (1992). Strains of groups B, C have many
properties in common with group A, but differences in the utilization of only a few
carbohydrates have been reported (Goto, 1980). Other techniques, such as ELISA, IF,
phage typing with citriphages, restriction fragment length polymorphism (RFLP) with
DNA probes and fatty acid composition analysis by gas chromatography can be utilised
for strain identification.
Pathogenicity tests
Xac and its pathotypes should be identified by pathogenicity on a panel of indicator
hosts such as Duncan grapefruit, Valencia sweet orange or Mexican lime, for confirmation of the diagnosis. Leaf assays by infiltration with a syringe with or without
needle on susceptible cultivars of Citrus hosts allow accurate identification of bacterial
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colonies. Lesions develop 7–14 days after inoculation of intact leaves or detached
leaves (Koizumi, 1971) after incubation at 25ºC at high humidity. With these assays the
eruptive callus-like reaction of Xac can readily be distinguished. Bacteria grown in
liquid media or colonies are scraped off from a freshly streaked agar plate and
suspended in sterile distilled water for inoculation into citrus. Concentration is adjusted
from 106 to 108cfu/ml. A negative and a positive control should always be included.
Plants inoculated with the positive control strain should be kept apart from other test
plants.
Indirect ELISA
ELISA kits containing all the necessary components for the identification of Xac are
available commercially. Positive control is also commercially available from the
manufacturers. The method used is indirect ELISA with monoclonal antibodies
described by Alvarez et al. (1991). In theory, all Xac strains can be identified, but it has
been reported that some phenotypically distinct A strains isolated in South-west Asia do
not react with the available monoclonal antibodies.
For identification of pure cultures, suspensions are centrifuged at about 10 000 rev min
for 2 min and the supernatant is discarded. 1 ml 1× PBS buffer is added and the cells are
resuspended by vortexing. The operation is repeated twice more. After the third wash,
the cells are resuspended in coating buffer. Bacterial concentration is adjusted
spectrophotometrically to OD6000.01 (about 2.5 × 107cfu/ml). 100 µl aliquots of the
samples are loaded onto ELISA microtiter plates (two wells per sample). Positive
control sample (from a reference culture or provided by manufacturer) and negative
buffer control wells with another bacteria should be included. The plates are incubated
overnight at 37°C until dry 200 µl blocking solution is added to each well (5% non-fat
dried milk in PBS buffer, 0.05 blocking component per mL buffer). The plates are
incubated for 30 min at room temperature and washed twice with 1× PBST. 100 µl of
prepared primary antibody is dispensed (prepare at the appropriate dilution in a solution
of 2.5% of dried milk in PBST). Plates are incubated up 1 h at room temperature, and
washed five times with 1× PBST. 100 µl of prepared enzyme conjugate per well is
dispensed (prepared at the appropriate dilution in a solution of 2.5% of dried milk in
PBST). Plates are incubated for 1 h at room temperature. After washing the plates, five
times with 1× PBST, 100 µl per well of freshly prepared substrate solution containing 1
mg ml−1 p-nitrophenyl phosphate in diethanolamine buffer, pH 9.8, is dispensed. The
plates are incubated for 30–60 min at room temperature. The O.D. is measured using a
spectrophotometer with a 405 nm filter at 405 nm. Positive samples are considered as
for DAS-ELISA.
Bacteriophages
Phage-typing is applicable to Xac with great reliability and many strains of Xac are
lysogenic (Okabe, 1961). Two virulent phages, Cp1 and Cp2, can infect 98% of the
strains isolated in Japan (Wakimoto, 1967). Similar results were also obtained in
Taiwan (Wu et al, 1993). The filamentous temperate phages and their molecular traits
have been studied in detail (Kuo et al, 1994; Wu et al, 1996). Phage Cp3 is specific to
the canker B strains (Goto et al, 1980). No phages specific to canker C strains have been
isolated.
Automated techniques
Fatty acid analysis for identification of pure cultures is available from MIDI (Newark,
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US) and from NCPPB (CSL,York, GB) among other. Biolog GN is an automated
method for identifying bacteria, based on the use of 95 substrates. It can be used for
identification at the species level and is commercially available from Biolog (Hayward,
US).
Molecular identification
The same sets of primers indicated for detection can be used for identification of
suspected strains. Another approach for producing universal primers for canker
producing strains utilized specific sequences in the intergenic spacer (ITS) regions of
16S and 23S ribosomal DNAs. Variation in the ITS sequences allows the design of
specific primers for A strains, to identify the Aw as an A strain, and differentiated the
Aw strain from the B and C pathotypes, even though these strains have a very similar
host
range
(Cubero
&
Graham,
2002).
Primers
J-Rxg
(5′GCGTTGAGGCTGAGACATG-3′) and J-RXc2 (5′-CAAGTTGCCTCGGAGCTATC3′), based on the internally transcriber spacer (ITS) between the 16S ansd 23S genes,
can then be used for universal identification of pure cultures of group A strains (Cubero
& Graham, 2002).
Molecular characterization
Features of citrus-attacking xanthomonads including Xac and the genus Xanthomonas
as a whole, have been characterized at the molecular level for the development of quick
and accurate methods for reclassification and identification. The procedures include
DNA-DNA hybridization (Vauterin et al, 1995), genomic fingerprinting (Lazo et al,
1987) and rep-PCR (Cubero & Graham, 2002).
Rep-PCR
BOX and ERIC-PCR (Louws et al., 1994) can be used for strain identification and
characterization under specific PCR conditions (Cubero & Graham, 2002). BOX PCR
reactions are carried out in 25-µL volumes containing 1× Taq buffer, 6 mm MgCl2, 2.4
µm concentration of primer BOX1R (5′-CTACG-GCAAGGCGACGCTGCAG-3′), 0.2
mm each deoxynucleoside thriphosphate and 2 U of Taq polymerase with a profile of
94°C (30 s), 48°C (30 s), and 72°C (1 min) for 40 cycles plus an initial step of 94°C for
5 min and a final step of 72°C for 10 min and using 5 µl of DNA extracted from
xanthomonad strains. DNA is extracted from bacterial supensions (absorbance at 600
nm from 0.2 to 0.5) following a single step of phenol-chloroform-isoamyl alcohol,
precipitated in ethanol, and re-suspended in ultrapure water. DNA is stored at −20°C
until further use. ERIC PCR reactions are carried out also in 25 volumes containing 1×
Taq buffer, 3 mm MgCl2, 1.2 µm concentration of primers ERIC1R (5′ATGTAAGCTCCT-GGGGATTCAC-3′)
and
ERIC2
(5′-AAGTAAGTGACTGGGGTGAGCG-3′) (Louws et al., 1994), 0.2 mm each deoxynucleoside triphosphate
and 2 U of Taq polymerase with the same profile as for BOX-PCR reactions. PCR
products are analysed in 3% agarose gel in 1X TAE buffer for 2 h at 110 V and stained
with ethidium bromide.
Genomic DNA fingerprinting
Extraction of DNA (Berman et al., 1981)
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10-ml liquid LB cultures of the test bacteria and of positive controls of Xac in 50-ml
flasks are grown for 18 h with gentle rotary shaking at 27°C. Genomic DNA is prepared
as follows. The pooled 20-ml culture is centrifuged (10 min at 10000 g) and the pellet is
resuspended in 10 ml of PBS (20 mmol l-1 potassium phosphate buffer, pH 6.9,
containing 150 mmol l-1 NaCl). After a second centrifugation the pellet is resuspended
in 5 ml of 50 mmol l-1 Tris, pH 8.0, containing 50 mmol l-1 EDTA. Eggwhite lysozyme
is added to a final concentration of 1 mg ml-1 and the tubes are put at 0°C for 30 min.
Then 1 ml of a freshly prepared lysing solution (0.5%) sodium dodecyl sulphate, 50
mmol l-1 Tris/HCl, pH 7.5, 400 mmol l-1 EDTA, and 1 mg ml-1 of pronase) is added to
each tube, and incubated at 50°C until the suspension clears. The lysate is extracted
with an equal volume of Tris buffer-saturated phenol (pH 7.8). After centrifugation
(9000 g for 10 min), the aqueous supernatant is transferred to a clean tube and sodium
acetate added to 0.3 mmol. After addition of two volumes of ethanol and mixing by
inversion, the nucleic acids are removed by spooling onto a glass pipette and dissolved
in 3 ml of TE (10 mmol l-1 Tris/HCl, pH 8.0, 1 mmol 1-1 EDTA) containing RNase A
(50 µg ml-1), After 30 min at 37°C, the solution is extracted with an equal volume of
chloroform and the DNA is spooled out of the solution by a second ethanol
precipitation. The DNA is dissolved in a minimal volume of TE and stored at 4°C until
used. The concentration of DNA in the sample can be estimated spectrophotometrically
(OEPP/EPPO, 1998).
DNA extracts (3-5 g) are digested with a restriction endonuclease. Reaction volumes
vary between 35 and 55 µl and buffer conditions are those recommended by the
supplier. Incubate at 37°C for 4 h. Load samples on a 1.5-mm-thick, 14-cm-long,
vertical 5% polyacrylamide gel, separate fragments by electrophoresis at 14 mA
constant current for 14 h in TBE (89 mmol l-1 Tris, 89 mmol l-1 boric acid, and 2 mmol
l-1 EDTA). During electrophoresis, the voltage increases from 50 V to 90 V. Gels are
stained with ethidium bromide (2 µg ml-1) for 60 min, then photographed on a
transilluminator using both an orange and a yellow filter and Polaroid type 55 highcontrast film. Genomic fingerprints of the test and reference extracts are compared
using the photograph, or with the negative and the aid of a photographic enlarger.
Furthermore for identification of pure cultures based on the pth gene, PCR reactions are
performed in volumes of 25 µl containing 1× Taq buffer, 3 mm MgCl2, 0.1 µm
concentration of primers J-pth1 and J-pth2, 0.2 mm each deoxynucleoside triphosphate,
1 U of Taq polymerase and 5 µl of extracted DNA. Amplification reaction conditions
consist of 93°C for 30 s, 58°C for 30 s, and 72°C for 45 s for 40 cycles plus an initial
step of 94°C for 5 min and a final step of 72°C for 10 min PCR products are visualized
and stained as above. (Hartung & Civerolo, 1987).
5. Records
The following records are to be kept:
- scientific name of the pest identified
- code or reference number of the sample (for traceability)
- nature of the infected/infested material including scientific name of host where
applicable origin of the infected/infested material
- description of signs or symptoms (including photographs where relevant)
- methods, including controls, used in the diagnosis and the results obtained with
each method for morphological methods, measurements, drawings or
photographs of the diagnostic features (where relevant), if applicable the
developmental stage
10
-
-
for biochemical and molecular methods, documentation of test results such as
photographs of diagnostic gels, ELISA printouts of results, on wich the
diagnosis was based
where appropriate, the magnitude of any infection/infestation (how many
individual pests found; how much damaged tissue)
the name of the laboratory and, where appropriate, the name of the person(s)
responsible for and/or who performed the diagnosis.
The conservation of culture(s) of the pest, preserved/mounted specimens, or test
materials (e.g. photograph of gels, ELISA plate printout results) is recommended in
cases of non-compliance (ISPM No. 13: Guidelines for the notification of noncompliance and emergency action) and where pests are found for the first time.
6. Contact points for further information
This protocol was originally drafted by E.F. Verdier, General Direction of Agricultural
Services, Biological Laboratories Department, Av. Millán 4703, CP: 12900,
Montevideo Uruguay. E-mail: [email protected].
Revised by R. Lanfrachi, Plant Pest and Diseases Laboratory, SENASA, Av. Ing.
Huergo 1001, CP: 1107, Buenos Aires, Argentina, E-mail: [email protected] and
M.M. López, Instituto Valenciano de Investigaciones Agrarias, IVIA, Carretera
Moncada a Náquera Km. 4,5, 46113-Moncada, Valencia, Spain, E-mail:
[email protected].
Further information on this organism can be obtained from: Dr. Edwin Civerolo. Office
of the Center Director, USDA-ARS 9611 S. Riverbend Avenue Parlier, CA, 93648.
USA.
Tel:
(559)
596-2702,
Fax:
(559)
596-2701,
E-mail:
[email protected] .
7. Acknowledgements
María M. López (Instituto Valenciano de Investigaciones Agrarias, IVIA, Moncada,
Valencia, Spain).
Rita Lanfrachi (Plant Pest and Diseases Laboratory, SENASA . Buenos Aires,
Argentina).
Jaime Cubero (Instituto Nacional de Investigación Agraria y Alimentaria, Madrid,
Spain).
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