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International Plant Protection Convention
Draft DP: Xanthomonas citri subsp. citri (text)
TPDP_2012_Nov_22
Agenda item: 7.3
DRAFT ANNEX to ISPM 27:2006 – Xanthomonas citri subsp. citri (2004-011)
Draft history
Date of this document
2012-10-03
Document category
Draft new annex to ISPM 27:2006 (Diagnostic protocols for regulated pests)
Current document stage
Meeting document, TPDP November 2012
Origin
Work programme topic: Bacteria, CPM-1 (2006)
Original subject: Xanthomonas axonopodis pv. citri (2004-011)
Major stages
-
Consultation on technical level
The first draft of this protocol was written by Enrique Verdier (General
Direction of Agricultural Services, Biological Laboratories Department,
Montevideo, Uruguay), Rita Lanfranchi (Plant Pests and Diseases
Laboratory, National Service of Agrifood Health and Quality (SENASA),
Capital Federal, Argentina), Maria M. López (Centro de Protección Vegetal y
Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA),
Spain).
The following experts also contributed to the preparation of the draft: Jaime
Cubero (Instituto Nacional de Investigación Agraria y Alimentaria (INIA),
Spain).
Main discussion points
during development of the
diagnostic protocol
To be added as necessary
Notes
-
CONTENTS
1.
Pest Information
2.
Taxonomic Information
3.
Detection
3.1
Detection in symptomatic plants
3.1.1 Symptoms
3.1.2 Sample preparation and isolation
3.2
Serological detection
3.2.1 Double antibody sandwich (DAS)-ELISA
3.2.2 Immunofluorescence (IF)
3.3
Molecular Detection
3.3.1 Polymerase Chain Reaction (PCR)
3.3.2 Real-time Polymerase Chain Reaction (Rt-PCR)
3.4
Pathogenicity tests
3.4.1 Inoculation test in leaf discs
3.4.2 Detached leaf enrichment
3.5
Detection in asymptomatic plants
4.
Identification
4.1
Description and biochemical characteristics
4.2
Pathogenicity tests
4.3
Indirect ELISA
4.4
Molecular identification
4.4.1 PCR detection
4.4.2 Rep-PCR fingerprinting
4.4.3 Genomic DNA fingerprinting
5.
Records
6.
Contact Points for Further Information
7.
Acknowledgements
8.
References
9.
Figures (see separate file)
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1.
Draft DP for Xanthomonas citri subsp. citri (text)
Pest Information
The nomenclature of Gabriel et al. (1989) has been reinstated and the accepted name for the citrus
bacterial canker pathogen is now Xanthomonas citri subsp. citri (Bull et al., 2010; Schaad et al.,
2006). Schaad et al. (2006) has published an emended classification of xanthomonad pathogens on
citrus and these names have now had formal uptake in the bacterial nomenclature (Bull et al., 2010).
X. citri subsp. citri (Xcc) (Hasse, 1915; Gabriel et al., 1989), the causal agent of citrus bacterial
canker, causes severe damage of many cultivated species of Rutaceae (EPPO, 1979), primarily Citrus
spp, Fortunella spp. and Poncirus spp., grown under tropical and sub-tropical conditions, being
prevalent in many countries in Asia, South America, Oceania, Africa and in the Florida State, USA
(CABI, 2006; EPPO, 2006).
There are distinct strains of citrus bacterial canker based on pathogenicity differences that also
correlate with serological and genetic differences. These strains are generally divided into the
following groups:
- Group A strains (Asian canker), caused by Xcc, infects most citrus hosts in the Rutaceae family.
Two groups of atypical Xcc strains with restricted host range have been identified within group A
(Vernière et al., 1998; Sun et al., 2000) and designated as A* and Aw. These are closely related to
Xcc type A strains (Cubero and Graham, 2002 and 2004) but affect only Citrus aurantiifolia
(Mexican lime) and Citrus macrophylla Webster (Alemow) in Florida (USA).
- Group B strains (causing cancrosis B), caused by X. fuscans subsp. aurantifolii, infect mainly
Citrus aurantiifolia (Mexican lime), Citrus limon (lemons), Citrus aurantium (sour orange) and
Citrus maxima (pummelo), and have only been found in South America.
- Group C strains (causing Mexican lime cancrosis); caused by X. fuscans subsp. aurantifolii, infect
Citrus aurantifolia (Mexican lime) in Brazil.
The last two canker types (Group A and B) were described in South America and were gradually
supplanted by group A strains. There is no evidence that this pathogen is seedborne.
2.
Taxonomic Information
Name:
Xanthomonas citri subsp. citri (Hasse) Vauterin et al.
Synonyms:
Xanthomonas axonopodis pv. citri (Hasse) Vauterin et al.
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.
Taxonomic position: Domain: Bacteria, Phylum: Proteobacteria, Class: Gammaproteobacteria,
Order: Xanthomonadales, Family: Xanthomonadaceae, Genus: Xanthomonas
Common names:
citrus bacterial canker (CBC), citrus canker (CC)
3.
Detection
3.1
Detection in symptomatic plants
3.1.1
Symptoms
Symptoms of citrus canker occur in any season on seedlings and young trees in which a flush of
abundant angular shoots appear from late summer through autumn (Figures 1-4). 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
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cultivars (Goto, 1992). Attacks of Phyllocnistis citrella, the citrus leaf miner increases the
susceptibility of leaves to citrus canker.
Xcc 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 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 on young infected fruits. The canker never penetrates through
the rind.
3.1.2
Sample preparation and isolation
Freshly prepared sample extracts are essential for successful isolation of Xcc from symptomatic
samples. However, when symptoms are very advanced or when the environmental conditions are not
favourable, the number of Xcc culturable cells can be very low and isolations can result in plates being
overcrowded with competing saprophytic or antagonistic bacteria. In particular, care should be taken
to not confuse Xcc colonies with Pantoea agglomerans, which is also commonly isolated from canker
lesions and produces yellow colonies on standard bacteriological media.
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 Xcc but there are as yet no
exclusively selective media available for pathovar identification (e.g. pv. citri).
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,
rinsed 3 times with sterile distilled water and comminuted in small pieces. An aliquot of the extract is
streaked on nutrient media. Suitable general isolation media are nutrient agar supplemented with 0.1%
glucose (NGA), yeast peptone glucose agar (YPGA) (yeast extract, 5 g; bactopeptone, 5g; glucose, 10
g; agar, 20 g; distilled water, 1l pH 7), or Wakimoto medium: potato broth (W) (250 ml; sucrose, 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. The colony morphology on media is round, mucoid, convex
and creamy-yellow, with smooth edges. Growth is evaluated after incubation at 28ºC for 3 to 5 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.
Identification of presumptive Xcc colonies can be made by morphological characteristics on nutrient
media, serological testing Enzyme-Linked Immunosorbent Assay (ELISA), Immunofluorescence (IF),
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Draft DP for Xanthomonas citri subsp. citri (text)
molecular testing Polymerase Chain Reaction (PCR), bioassays (leaf discs or detached leaves) and
pathogenicity tests.
Reference strains to be used as positive controls in all methods: group A strains (ATCC 49118, ICMP
24, NCPPB 3234, CFBP 2911, IBSBF 1594 = Xc 306), group B strains (ATCC 51301, NCPPB 3237)
and group C strains (ATCC 51302, NCPPB 3233, IBSBF 417) as examples.
3.2
Serological detection
3.2.1
Double antibody sandwich (DAS)-ELISA
Microtitre plate is coated with 200 µl/well carbonate coating buffer (Na2CO3, 1.59 g; NaHCO3, 2.93 g;
NaN3, 0.2 g; distilled water, 1 l; pH 9.6) containing immunoglobulins (IgG) anti-Xcc appropriately
diluted and incubated overnight at 4ºC. After washing the plates successively three times with
phosphate buffered saline with Tween (PBS-Tween) (NaCl, 8 g; KH2PO4, 0.2 g; Na2HPO4 12H2O, 2.9
g; KCl, 0.2 g; NaN3, 0.2 g.; Tween-20, 0.25 ml; distilled water, 1 l; pH 7.4), 200 µl/well of test
samples, and negative controls (healthy plant material) and positive control (a reference strain of Xcc)
are added. The plates are incubated for 2 h at 37ºC. After washing, 200 µl/well IgG anti-Xcc
conjugated with alkaline phosphatase at the appropriate dilution in PBS-Tween, are added and
incubated for 2 h at 37°C. The washing is repeated. Then 200 µl/well 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. The criterion
for the determination for a positive sample is two times the optical density (OD) value of healthy
controls. The sensitivity detection limit of DAS-ELISA is 104 to105 cfu/ml.
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. For specificity data, refer to the
technical information provided by the manufacturer. Some monoclonal antibodies have been reported
to cross-react with Xanthomonas axonopodis pv. phaseoli, Xanthomonas campestris pv. zinnea,
Xanthomonas citromelo and Xanthomonas hortorum pv. pelargonii. However, these pathovars are
unlikely to be present on citrus.
3.2.2
Immunofluorescence (IF)
Aliquots of 25 µl of each bacterial preparation or plant samples to be tested are pipetted onto the
windows of a plastic-coated multi-window 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 phosphate buffered saline (PBS) at
pH 7.2 and appropriate dilutions added to windows of each slide. Other 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.
Then 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 in darkness, rinsed, washed
and blotted dry as before. Finally 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 shows fluorescent rod shaped bacterial cells 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 Xcc. This procedure permits detection in the range of
103 cells/ml.
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Draft DP for Xanthomonas citri subsp. citri (text)
3.3
Molecular detection
3.3.1
Polymerase Chain Reaction (PCR)
TPDP_2012_Nov_22
- DNA extraction from infected citrus tissue
For obtaining 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 hexadecyltrimethylammonium bromide (CTAB) protocol, but there are also
commercial methods 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
ethylenediaminetetraacetic (EDTA), 0.5% sodium dodecyl sulphate (SDS), 2% polyvinylpyrrolidone
(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 (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 conventional PCR method allows
detection of 10 cfu/10ul or the equivalent of about 103 cfu/ml.
- Primers used in PCR
Several sets of primers are available for diagnosis of Xcc. 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 good specificity and sensitivity of about 102 cfu/ml reached. Primers
J-pth1 (5′-CTTCAACTCAAAC-GCCGGAC-3′) and J-pth2 (5′-CATCGCGCTGTTCGGGAG-3′)
based on the nuclear localization signal in the 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 (104 cfu/ml in plant material) than the previous ones. Comparative sensitivity of the
different protocols and primers in pure culture and fruit extracts has been reported (Golmohammadi et
al., 2007). The above primers have a reported high level of specificity to Xcc group A strains and have
been tested against a worldwide collection of Xanthomonas strains isolated from Citrus (Cubero and
Graham, 2002). However, the primers developed by Hartung et al. (1993) do not detect other citrus
canker pathotypes B and C or the atypical Xcc strains A* and Aw detected in Florida. The primers
developed by Cubero and Graham (2002) target the pth virulence gene present in all citrus canker
strains.
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 60 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.]
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Draft DP for Xanthomonas citri subsp. citri (text)
Pair 4/7 [4-5′-TGT CGT CGC 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 Xcc in plants have also been developed (Hartung et al. 1993).
3.3.2
Real-time Polymerase Chain Reaction (Rt-PCR)
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 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) and 0.5 µl of 10 µM Taqman probe (JTaq pth2) in a final reaction volume of 25 µl. Rt-PCR reaction is completed in an ABI PRISM 7000
Sequence Detection System. 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 provides similar specificity to the pth gene primers used in the conventional PCR
assay (Cubero and Graham 2002; 2005) and enable reliable detection less than 10 cfu of Xcc from
diseased leaf lesions and 10 cfu from a dilution of cultured cells (Mavrodieva et al. 2004). This
method has recently been compared with standard or nested PCRs (Golmohammadi et al. 2007) and
the sensitivity obtained is very good (10 cfu/ml).
3.4
Pathogenicity tests
3.4.1 Inoculation test in leaf discs
This test uses susceptible tissue to Xcc 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 this bioassay begins by sterilizing ELISA plates for 15 minutes in a microwave
oven and adding to the wells 200 µl of sterile 1.5% agar-water under laminate flow chamber at room
temperature. Young Citrus paradisi var. Duncan (grapefruits) leaves (light green) are disinfected for
one minute with 1% sodium hypochlorite. After rinsing them 3 times with sterile distilled water the
leaves are superficially dried in the laminate flow chamber at room temperature. The leaf disks,
obtained with a punch, previously disinfected with 96º ethanol, are placed back up in each hole with
agar-water. Fifty µl from macerated lesions, with 4 repetitions for each one, are added.
A Xcc 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 are evaluated from the third day, using stereoscopic microscope and
isolate Xcc 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).
3.4.2
Detached leaf enrichment
Xcc can also be selectively enriched in wounded detached leaves of Citrus paradisi var. Duncan
(grapefruit). Young terminal leaves from glasshouse-grown plants are wasted for 10 min in running
tap water, surface-disinfect in 1% sodium hypochlorite for 1 min, and aseptically rinse thoroughly
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with sterile distilled water. The lower surface of each leaf is aseptically wounded by puncturing with a
needle or by making small cuts with a scalpel and placed in 1% sterile 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 7 - 12 days at 25ºC in a lighted incubator, pustule development is
evaluated and Xcc is isolated as above (EPPO, 1998).
3.5
Detection in asymptomatic plants
In the absence of symptoms, leaf samples or fruits are taken from the trees and 10 leaves per tree
constitute 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 (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 (EPPO, 1998).
For composite samples 100 leaves into 200 ml peptone buffer are used.
The washing liquid is used for centrifugation and the 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 semiselective medium in triplicate (sucrose, 20 g; peptone, 2 g; monosodium glutamate, 5 g; calcium
nitrate, 0.3 g; phosphate dipotassium anhydrous, 2 g; EDTA Fe, 1 mg; cycloheximide, 100 mg;
cephalexine, 20 mg; kasugamycine, 20 mg; methyl violet 2B, 0.3 mg bacto agar, 17 g; distilled water,
1 l; pH: 7.0;) (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 semi-selective 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 multiply in the plant tissue and can be
isolated in higher numbers (see above).
4.
Identification
Detection of Xcc can be achieved by using cultural, biochemical and pathogenicity tests, serological or
molecular test. Identification requires isolation of Xcc-like colonies and a positive reaction from a
serological or molecular test. A positive reaction from the serological or molecular tests described
below is the minimum requirement to detect and identify Xcc during routine diagnosis of a pest widely
established in a country. In instances where the national plant protection organization (NPPO)
requires additional confidence in the identification of Xcc (e.g. detection in an area where Xcc is not
known to occur or detection in an imported consignment certified free of Xcc), further tests must be
done. Where the initial identification was done using a molecular method, subsequent tests should
isolate a pure Xcc culture and use biochemical, serological or molecular techniques, and pathogenicity
tests. Further tests may also be done to characterise the “pathotype” or “strain type present. In all
cases, positive and negative controls must be included in the tests. The recommended techniques are
described in the following sections.
4.1
Description and biochemical characteristics
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Xcc 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 an
oxidative metabolism of glucose. The yellow pigment is xanthomonadin.
Table 1. Key biochemical characteristics of Xanthomonas citri subsp. citri
Catalase
Oxidase
Nitrate reduction
Hydrolysis of:
starch
casein
Tween 80
aesculin
Gelatin liquefaction
Pectate gel liquefied
Utilization of asparagine
Growth requires:
methionine
cysteine
0.02% TTC (w/v)
X.c. subsp. citri
+
- or weak
+
+
+
+
+
+
+
+
-
Biovars may be distinguished by utilization of mannitol (Bradbury, 1986). For further information on
the bacteriological properties of Xcc see Goto (1992). Strains of groups B and C have many properties
in common with group A, but differences in the utilization of only a few carbohydrates have been
reported (Goto et al, 1980). Other techniques, such as ELISA, IF, phage typing with citriphages (Wu
et al, 1993 and 1996; Kuo et al, 1994; Goto et al, 1980) Restriction Fragment Length Polymorphism
(RFLP) with DNA probes can be utilised for strain identification.
4.2
Pathogenicity tests
Xcc and its pathotypes should be identified by pathogenicity on a panel of indicator hosts such as
Citrus paradisi var. Duncan (grapefruit), Citrus sinensis (Valencia sweet orange) or Citrus
aurantiifolia (Mexican lime), for confirmation of the diagnosis by young leaves inoculation.
Leaf assays by infiltration with a syringe with or without needle on susceptible cultivars of Citrus
hosts allow demonstration of pathogenicity 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 Xcc 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 10 6 to
108 cfu/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.
4.3
Indirect ELISA
The method generally use for culture identification is indirect ELISA with monoclonal antibodies
described by Alvarez et al. (1991). ELISA kits containing all the necessary components for the
identification of Xcc are available commercially. In theory, all Xcc 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.
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Pure culture suspensions are centrifuged at about 10,000 g 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× PBS-Tween. 100
µl of prepared primary antibody is dispensed (prepare at the appropriate dilution in a solution of 2.5%
of dried milk in PBS-Tween). Plates are incubated up 1 h at room temperature, and washed five times
with 1× PBS-Tween. 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 PBS-Tween). Plates are incubated for 1 h at
room temperature. After washing the plates, five times with 1× PBS-Tween, 100 µl per well of freshly
prepared substrate solution containing 1 mg/ml p-nitrophenyl phosphate in diethanolamine buffer, pH
9.8, is dispensed. The plates are incubated for 30–60 min at room temperature. The OD is measured
using a spectrophotometer with a 405 nm filter at 405 nm. Positive samples are considered as for
DAS-ELISA.
4.4
Molecular identification
Features of citrus-attacking xanthomonads including Xcc 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), Multilocus sequence analysis (Young et al. 2008),
and rep-PCR (Cubero and Graham, 2002; 2004).
4.4.1
PCR detection
Identification of pure cultures of suspect strains can be made 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). Other PCR
detection approaches have used universal primers that utilise specific sequences in the intergenic
transcribed spacer (ITS) regions of 16S and 23S ribosomal DNAs to identify citrus canker producing
strains. Variation in the ITS sequences has allowed 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 ITS region between the 16S and 23S genes, can
be used for universal identification of pure cultures of group A strains (Cubero and Graham, 2002).
4.4.2
Rep-PCR fingerprinting
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 25µl 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
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Draft DP for Xanthomonas citri subsp. citri (text)
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 MgCl 2, 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 (40 mmol l-1 Tris-acetate; pH 8.0, 1 mmol 1-1 EDTA) buffer for 2 h at 110 V
and stained with ethidium bromide.
4.4.3
Genomic DNA fingerprinting
- Extraction of DNA
10-ml liquid Luria Bertani (LB) cultures of the test bacteria and of positive controls of Xcc 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 10,000 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 (9,000 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) buffer 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 buffer and stored at 4°C until used. The concentration of DNA in the sample
can be estimated spectrophotometrically (EPPO, 1998).
- DNA digestion
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) buffer. 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.
5.
Records
Records and evidence should be retained as described in section 2.5 of ISPM 27:2006.
6.
Contact Points for Further Information
Further information on this organism can be obtained from:
General Direction of Agricultural Services, Biological Laboratories Department, Av. Millán 4703, CP
12900, Montevideo Uruguay (Enrique F. Verdier, E-mail: [email protected]).
Office of the Center Director, USDA-ARS. 9611 So. Riverbend Ave. Parlier, CA 93648, USA. (Ed
Civerolo, E-mail: [email protected]).
Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias
(IVIA), Carretera Moncada-Náquera km 4.5, 46113 Moncada (Valencia), Spain (María M.
López, [email protected]; tel.: +34 963424000; fax +34 963424001).
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Draft DP for Xanthomonas citri subsp. citri (text)
TPDP_2012_Nov_22
Instituto Nacional de Investigación Agraria y Alimentaria, INIA, Ctra de La Coruña km 6, Madrid,
Spain (Jaime Cubero, [details to be added])
7.
Acknowledgements
The first draft of this protocol was written by: E.F. Verdier, General Direction of Agricultural
Services, Biological Laboratories Department, Uruguay, and revised by R. Lanfranchi, SENASA,
Argentina and M.M. López, IVIA, Spain (see preceding section for addresses). In addition, J. Cubero,
INIA, Spain (see preceding section) was significantly involved in the development of this protocol.
8.
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9. Figures (see separate file)
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