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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) International Plant Protection Convention Page 1 of 12 TPDP_2012_Nov_22 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 Page 2 of 12 International Plant Protection Convention Draft DP for Xanthomonas citri subsp. citri (text) TPDP_2012_Nov_22 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), International Plant Protection Convention Page 3 of 12 TPDP_2012_Nov_22 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. Page 4 of 12 International Plant Protection Convention 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.] International Plant Protection Convention Page 5 of 12 TPDP_2012_Nov_22 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 Page 6 of 12 International Plant Protection Convention Draft DP for Xanthomonas citri subsp. citri (text) TPDP_2012_Nov_22 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 International Plant Protection Convention Page 7 of 12 TPDP_2012_Nov_22 Draft DP for Xanthomonas citri subsp. citri (text) 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. Page 8 of 12 International Plant Protection Convention Draft DP for Xanthomonas citri subsp. citri (text) TPDP_2012_Nov_22 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 International Plant Protection Convention Page 9 of 12 TPDP_2012_Nov_22 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). Page 10 of 12 International Plant Protection Convention 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. References Álvarez, A.M., Benedict, A.A., Mizumoto, C.Y., Pollard, L.W. & Civerolo, E.L. 1991. Analysis of Xanthomonas campestris pv. citri and X.c. pv. citrumelo with monoclonal antibodies. Phytopathology, 81: 857- 865. Bradbury, J.F. 1986. Guide to Plant Pathogenic Bacteria. Wallingford, UK, CAB International. pp.1-332. Bull, C.T., De Boer, S.H., Denny, T.P., Firrao, G., Fischer-Le Saux, M., Saddler, G.S., Scortichini, M., Stead, D.E. & Takikawa, Y. 2010 Comprehensive list of names of plant pathogenic bacteria, 1980 – 2007. Journal of Plant Pathology 92(3): 551-592. CABI. 2006. Crop Protection Compendium. CAB International 2006 Edition. Civerolo, E.L. & Fan, F. 1982. Xanthomonas campestris pv. citri detection and identification by enzyme-linked immunosorbent assay. Plant Disease, 66: 231-236. Civerolo, E.L. & Helkie, C. 1981. Indirect enzyme-linked immunosorbent assay of Xanthomonas campestris pv. citri. In Proceedings of the Fifth International Conference on Plant Pathogenic Bacteria, pp. 105-112. Cali (Colombia). Cubero, J. & Graham, J.H. 2002. Genetic relationship among worldwide strains of Xanthomonas causing canker in citrus species and design of new primers for their identification by PCR. Applied and Environmental Microbiology, 68: 1257–1264. Cubero, J. & Graham, J.H. 2004. The leucine-responsive regulatory protein (lrp) gene for characterization of the relationship among Xanthomonas species. International Journal of Systematic and Evolutionary Microbiology, 54: 429–437. Cubero, J. & Graham, J.H. 2005. Quantitative real time Polymerase Chain reaction for bacterial enumeration and allelic discrimination to differentiate Xanthomonas strains on citrus. Phytopathology, 95: 1333-1340. Cubero, J., Graham, J.H. & Gottwald, T.R. 2001. Quantitative PCR meted for diagnosis of citrus bacterial canker. Applied and Environmental Microbiology, 67: 2849–2852. EPPO. 1979. Xanthomonas axonopodis pv. citri. Data Sheets on Quarantine Pests. EPPO A1 list No1. EPPO. 1998. Phytosanitary procedure Xanthomonas axonopodis pv. citri. Inspection, test and survey methods. EPPO Standard PM 3/27(1). EPPO. 2006. PQR database (version 4.5). Paris, France: European and Mediterranean Plant Protection Golmohammadi M, Cubero J, Peñalver J, Quesada JM, López MM, Llop P. 2007. Diagnosis of Xanthomonas axonopodis pv. citri, causal agent of citrus canker in commercial fruits by isolation and PCR based methods. Journal of Applied Microbiology (in press). Goto, M. 1992. Citrus canker. In J. Kumer, H.S. Chaube, U.S. Singh & A.N. Mukhopadhyay, eds. Plant Diseases of International Importance, Vol. III, Diseases of Fruit Crops. New Jersey, USA: Prentice Hall. Goto, M., Takahashi, T. & Messina, M.A. 1980. A comparative study of the strains of Xanthomonas campestris pv. citri isolated from citrus canker in Japan and cancrosis B in Argentina. Annals of the Phytopathological Society of Japan, 46: 329-338. Hartung, J.S. & Civerolo, E.L. 1987. Genomic fingerprinting of Xanthomonas campestris pv. citri strains from Asia, South America and Florida. Phytopathology, 77: 282-285. Hartung, J.S., Daniel, J.F., Pruvost, O.P. & Civerolo, E.L. 1993. Detection of Xanthomonas campestris pv. citri by the polymerase chain reaction method. Applied and Environmental Microbiology, 59 (4): 1143-1148. International Plant Protection Convention Page 11 of 12 TPDP_2012_Nov_22 Draft DP for Xanthomonas citri subsp. citri (text) Koizumi, M. 1971. A quantitative determination method for Xanthomonas citri by inoculation into detached citrus leaves. Bull. Hort. Res. Sta., Japan, SER. B, No 11: 167-182. Kuo, T.T., Chiang, C.C., Chen, S.Y., Lin, J.H. & Kuo, J.L. 1994. A long lytic cycle in filamentous phage Cf1tv infecting Xanthomonas campestris pv. citri. Archives of Virology, 135: 253-264. Lazo, G.R., Roffey, R. & Gabriel, D.W. 1987. Pathovars of Xanthomonas campestris are distinguishable by restriction fragment-length polymorphism. International Journal of Systematic Bacteriology, 37: 214-221. Louws, F.J., Fulbright, D.W., Taylor Stephens, C. & Bruijn, F.J. 1994. Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR. Applied and Environmental Microbiology, 60: 2286–2295. Llop, P., Caruso, P., Cubero, J., Morente, C. & López, M.M. 1999. A simple extraction procedure for efficient routine detection of pathogenic bacteria in plant material by polymerase chain reaction. Journal of Microbiology Methods, 37: 23–31. Mavrodieva, V., Levy, L. & Gabriel, D.W. 2004. Improved Sampling Methods for Real-Time Polymerase Chain Reaction Diagnosis of Citrus Canker from Field Samples. Phytopathology, 94: 61-68. Monier, L. 1992. Contribution à la mise au point d´un milieu de culture semi-sélectif pour la détection de Xanthomonas campestris pv. citri, agent du chancre bactérien des agrumes. École Nationale d´Ingénieurs des Travaux de l´Horticulture et du Paysage d´Angers. Institut de Recherches sur les Fruits et Agrumes IRFA. pp. 62. Schaad NW, Postnikova E, Lacy GH, Sechler A, Agarkova I, Stromberg PE, Stromberg VK, Vidaver AK. 2005. Reclassification of Xanthomonas campestris pv. citri (ex Hasse 1915) Dye 1978 forms A, B/C/D, and E as X. smithii subsp. citri (ex Hasse) sp. nov. nom. rev. comb. nov., X. fuscans subsp. aurantifolii (ex Gabriel 1989) sp. nov. nom. rev. comb. nov., and X. alfalfae subsp. citrumelo (ex Riker and Jones) Gabriel et al., 1989 sp. nov. nom. rev. comb. nov.; X. campestris pv malvacearum (ex smith 1901) Dye 1978 as X. smithii subsp. smithii nov. comb. nov. nom. nov.; X. campestris pv. alfalfae (ex Riker and Jones, 1935) dye 1978 as X. alfalfae subsp. alfalfae (ex Riker et al., 1935) sp. nov. nom. rev.; and "var. fuscans" of X. campestris pv. phaseoli (ex Smith, 1987) Dye 1978 as X. fuscans subsp. fuscans sp. nov. Systematic and Applied Microbiology, 28: 494-518. Schaad NW, Postnikova E, Lacy GH, Sechler A, Agarkova I, Stromberg PE, Stromberg VK, Vidaver AK. 2006. Emended classification of xanthomonad pathogens on citrus. Systematic and Applied Microbiology, 29: 690 -695. Sun, X., Stall, R.E., Cubero, J., Gottwald, T.R., Graham, J.H., Dixon, W.D., Schubert, T.S., Peacock, M.E., Dickestein, E.R. & Chaloux, P.H. 2000. Detection of a unique isolate of citrus canker bacterium from Key lime in Wellington and Lake Worth, Florida. Proceedings of the International Citrus Canker Research Workshop. Fort Pierce (US). http://doacs.state.fl.us/canker. Vauterin, L., Hoste, B., Kersters, K. & Swings, J. 1995. Reclassification of Xanthomonas. International Journal of Systematic Bacteriology, 45: 472-489. Verdier, E., Zefferino, E. & Méndez, S. 2007. Survival of Xanthomonas axonopodis pv. citri on the surface of citrus fruit after postharvest treatment (submitted for publication). Vernière, C., Hartung, J.S., Pruvost, O.P., Civerolo, E.L., Álvarez, A.M., Maestri, P. & Luisetti, J. 1998. Characterization of phenotypically distinct strains of Xanthomonas axonopodis pv. citri from Southwest Asia. European Journal of Plant Pathology, 104: 477-487. Wu, W.C., Lee, S.T., Kuo, H.F. & Wang, L.Y. 1993. Use of phages for identifying the citrus canker bacterium Xanthomonas campestris pv. citri in Taiwan. Plant Pathology, 42: 389-395. Wu, W.C., Chen, T.T. & Wang, Y.R. 1996. Characterization of five filamentous phages from Xanthomonas campestris pv. citri. Plant Pathology Bulletin, 5: 1-14. Young, J.M., Park, D.C., Shearman, H.M., & Fargier, E. 2008. A multilocus sequence analysis of the genus Xanthomonas. Systematic and Applied Microbiology 31(5): 366-377. 9. Figures (see separate file) Page 12 of 12 International Plant Protection Convention