Download Diagnostic protocol for

2007-TPDP-15 REV-01
Modified by the TPDP and edited by IPPC Secretariat
Agenda: 8
Xanthomonas axonopodis pv. citri
Annex to ISPM No. 27
INTERNATIONAL STANDARDS FOR
PHYTOSANITARY MEASURES
Annex to ISPM No. 27 (DIAGNOSTIC PROTOCOLS FOR REGULATED PESTS)
Xanthomonas axonopodis pv. citri
(200-)
CONTENTS
Endorsement ..................................................................................................................................................... 2
1.
Pest information ................................................................................................................................... 2
2.
Taxonomic information ....................................................................................................................... 2
3.
Detection ............................................................................................................................................... 2
3.1.
On symptomatic plant material .............................................................................................................. 2
3.2.
On asymptomatic plant material ............................................................................................................ 5
4.
Identification......................................................................................................................................... 6
5.
Records.................................................................................................................................................. 9
6.
Contact points for further information .............................................................................................. 9
7.
Acknowledgements............................................................................................................................... 9
8.
References ............................................................................................................................................. 9
Good protocol – still needs some work to be ready for country consultation. Hope it will be possible
to have this finalised next year. Timescale to be agreed with Enrique.
General comments:
1. Records section – the specific requirements for this organism are not listed (as decided yesterday). If
the first part is the same as ISPM 27, this should be indicated. Then there should be an indication of
the evidence that should be kept in cases where other NPPOs might be affected by the results
2. Information on specificity and reliability is not provided in the text.
3. Figure 1 could be improved. Should clearly reflect the options available. Are they all equally good?
Are the 4 techniques for symptomatic material options. (In Uruguay the reliability of the methods
varies according to the time of year).
4. What is the minimum requirement for diagnosis of the organism?
5. Refer to data sheets or databases EPPO PQR if they occur
6. Figure number 3 may not be necessary. Figure 6 is useful. Only include essential photographs in the
draft, other, useful photographs can be posted on the IPP.
7. Are bacteriophages used routinely for identification? If not, don’t include it or put a brief reference
to the technique for information.
1
8. Trade names – quote them only if the information is essential – for example if the
sensitivity/reliability etc has been demonstrated using that specific equipment etc.
Secretariat comments:
Put names of buffers and chemicals in full at first mention e.g. PBS, SDS, EDTA, PVP
Is there a need for a recipe for water agar? LUM??
Check the references to ensure they comply with instructions to authors
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2
Endorsement
This diagnostic protocol was adopted by the Commission on Phytosanitary Measures in ----.
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 sub-tropical conditions, being prevalent in many countries in
Asia and South America.
There are records from North America, Africa and Oceanea – if correct, these should be included
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 (ATCC 49118,
NCPPB 3234) causing (Asian canker) with a worldwide distribution in citrus-growing areas outside the
Mediterranean basin, group B strains (ATCC 51301, NCPPB 3237) (causing cancrosis B) found in South
America, group C strains (ATCC 51302, NCPPB 3233) (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,
Citrus aurantifolia (sour orange) and pummelo are also susceptible.CHECK WORDING - UNCLEAR
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 related to type A strains (Cubero and Graham, 2002, 2004) but affect only Mexican lime and
Citrus macrophila Webster (Alemow).
Suggest moving the information on reference strain numbers to the detection section
.
2.
Taxonomic information
Name:
Xanthomonas axonopodis pv. citri (Hasse) Vauterin et al., 1995
Synonyms:
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 (CBC)
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
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3
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 surrounded by a water-soaked and
a yellow halo margin.
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 and Fan, 1982; Civerolo and Helkie, 1981) and PCR are reliable methods for examining many
samples and can be completed within the same day
Isolation Include a description of the colony morphology in this section
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 g; peptone, 5 g; sodium phosphate anhydrous, 0.8 g; calcium
nitrate 7 H2O, 0.5 g; bacto agar, 20 g; distilled water, 1 L; pH: 7.2. Cycloheximide (100 mg/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 – 1st para – it is not clear that this paragraph refers to polyclonal antibodies.
Is there a need to refer to the IgG procedure or ELISA if there are kits – just refer to manufacturers
instructions.
2nd para – should be clearer that monoclonals are not advised for detection, should only be used for
identification
Kits containing the components for Xac detection are available commercially (detect between 104-105
cfu/ml). [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 overnight at 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 1/2000-1/4000 in PBS-Tween, are added and
incubated for 2 h 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 only 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 [Secretariat suggestion -Define at first use ? include
the recipe] at pH 7.2 and appropriate dilutions added to windows of each slide. Controls of normal (preimmune) 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-fading 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. This
procedure permitting detection in the range of 103 cells/ml.
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
[define?] 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. The PCR method allows
detection of 10 cfu/10ul or the equivalent of 103 cfu/ml.
Primers used in PCR.
Several sets of primers are available for diagnosis of Xac. Based on Hartung et al. (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
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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.]
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.
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 and 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
(J-RTpth3), 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. The RT-PCR method enable reliable detection less than 10 cfu
of Xac from diseased leaf lesions and 10 cfu from a dilution of cultured cells (Mavrodieva et al., 2004). This
method has not been compared with standard or nested PCRs
Inoculation test in leaf discs
This test uses 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 grapefruits 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., 2007).
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. The lower surface of each leaf is aseptically wounded by
puncturing with a needle or making small cuts with a scalpel and placed in 1% water agar plates with the
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lower surface up.[not sure if this is correct meaning?] 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
pustule development is 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 constitutes one sample.
The leaves and fruit samples are washed in peptone buffer(sodium chloride, 8.5 g; peptone, 1 g; Tween 20,
250 µl; distilled water, 1 l; pH: 7.2), incubated at room temperature and the sample is then concentrated
(OEPP/EPPO, 1998). Fruits samples are individually washed in sterile bags in peptone buffer, incubated at
room temperature (Verdier et al., 2007).
Preparation of leaf samples
Ten leaves are shaken for 20 minutes at room temperature into 50 ml peptone buffer (OEPP/EPPO, 1998).
For composite samples are used 100 leaves into 200 ml peptone buffer.
Supernatant is removed with a vacuum pump after centrifugation for 20 min at 6,000 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 g; peptone, 2 g; monosodium glutamate, 5 g; calcium nitrate, 0.3 g;
phosphate dipotassium anhydrous, 2 g; EDTA Fe, 1 mg; bacto agar, 17 g; 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
buffer. Aliquots (100 µl) of 1/10 and 1/100 dilution of each washing solution are streaked on XOS semiselective medium in triplicate. Growth is evaluated after incubation for 5-6 days at 28ºC (Verdier et al.,
2007).
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 obligatory 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.
Table 1. Biochemical characteristics of Xanthomonas axonopodis pv. citri
Catalase
Oxidase
Nitrate reduction
Hydrolysis of:
starch
casein
Tween 80
aesculin
Gelatin liquefaction
Pectate gel liquefied
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X.a. pv. citri
+
- or weak
+
+
+
+
+
+
7
Utilization of asparagine
Growth requires:
methionine
cysteine
0.02% TTC (w/v)
+
+
-
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 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 Are bacteriophages used routinely for diagnosis of citrus canker? If not, then just put a
reference, rather than details.
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.
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8
Automated techniques Not widely available, but used for bacterial identification. If it is available and if it is
a justified (are they reliable for Xac), they can be referred to, but not in detail.
Fatty acid analysis for identification of pure cultures is available from MIDI (Newark, 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′CAAGTTGCCTCGGAGCTATC-3′), based on the internally transcriber spacer (ITS) between the 16S and
23S genes, can then be used for universal identification of pure cultures of group A strains (Cubero and
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 and 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 and 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′-CTACGGCAAGGCGACGCTGCAG-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 phenolchloroform-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′-AAGTAAGTGACT-GGGGTGAGCG-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).
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
2007-TPDP-15 071003
9
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 high-contrast 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 and Civerolo, 1987).
5.
Records Refer to ISPM 27 and any additional records that might be required. Also include the
specific evidence that should be kept in cases where other NPPOs may be involved
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
- 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 non-compliance and emergency action) and where pests are found for the first time.
6.
Contact points for further information Are all these authors willing to be contacted? If so list
them and refer to authorship of the protocol in Section 7
[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 So. Riverbend Ave. Parlier, CA 93648, USA. Tel: (559) 596-2702, Fax: (559)
596-2701, E-mail: [email protected] .
2007-TPDP-15 071003
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7.
Acknowledgements suggest adding E Verdier here and the info on authorship – see Thrips palmi
protocol
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, 46113Moncada, Valencia, Spain, E-mail: [email protected].
[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).
8.
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