Draft DP for Xanthomonas citri subsp. citri (text) TPDP_2012_Nov_22
DRAFT ANNEX to ISPM27:2006 – Xanthomonas citri subsp. citri (2004-011)
Draft history
Date of this document / 2012-10-03Document category / Draft new annex to ISPM27:2006 (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)
1. 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 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), 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 (pre-immune) serum at one dilution and of PBS are also added to the slide. Slides are enclosed in a humid chamber and incubated at room temperature for 30 min. The droplets are shaken off the slides and they are rinsed with PBS and then washed three times for 5 min in PBS. The slides are gently blotted dry. 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.
3.3 Molecular detection
3.3.1 Polymerase Chain Reaction (PCR)
- 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.