A further outbreak of citrus canker near Darwin

Transcription

1 Australasian Plant Pathology (I 995) 24: A further outbreak of citrus canker near Darwin Patricia BroadbentA, R N. PitkethleyB, D. BarnesA, J. BradleyA, C. DephoffA, E.L. Civeroloc, M.R Gillings* and P.C. FahyA ANSW Agriculture, Biological and Chemical Research Institute,PMB 10, Rydalmere, New South Wales *Northern Temtory Department of Primary Industry and Fisheries, Berrimah Agricultural Research Centre, Bemmah,Northern Territory 0828 CUSDA, ARS, Agricultural Research Center, Beltsville,Maryland, MD 20705, United States of America Abstract Citrus canker (caused by Xanthomonas campestris pv. citri Group A) was detected in a pummelo orchard at Lambell's Lagoon near Darwin in All citrus trees in the orchard were eradicated. Surveys of horticultural holdings at Lambell's Lagoon in 1992 failed to detect symptoms of citrus canker. Leaf washings from trees in each orchard failed to detect X. c. citri using leaf enrichment or immunofluorescence techniques. Other xanthomonad isolates were obtained and these produced a slight callusing with watersoaking around wounds made in detached leaves of Duncan grapefruit. In 1993, canker symptoms were observed on lime and grapefruit twigs and fruit in a mixed citrus orchard 500 m from the 1991 outbreak site. This outbreak was confirmed as Group A of X. c. citri on the basis of pathogenicity tests, fatty acid analyses of recovered bacteria, irnmunofluorescence microscopy and by genomic DNA fingerprinting. Introduction Australia has had five previous outbreaks of citrus canker: in the Northern Territory in 1912 (Hill 1918), on Home Island in the Cocos (Keeling) Islands in 1981, on ChristmasIsland in 1985 (Shivas 1987), on Thursday Island in the Torres Strait in 1984 (Jones et al. 1984) and near Darwinin 1991 (Broadbent et al. 1992). The 1912 outbreak of canker in the Northern Territory most likely originated from Japan or China, since citrus trees were imported into Darwin from these sources, and nearly every ship trading between these countries and Australia landed small consignments of citrus fruits in Darwin. As far as possible, every citrus tree in the Territory was carefully examined for canker in All citrus trees in proximity to cankeraffected trees were destroyed. Land in the vicinity of areas in which canker had been detected was placed under quarantine for a period of 5 years. By 1922 it was obvious that the initial eradication campaign had not been successful. Hill then recommended that all citrus trees in the Northern Territory be destroyed (Mertin 1952). The destruction campaign was modified in that only trees north of the 19th parallel were dug out andburned. It was not until 1925 that permission was granted to grow citrus trees again in the Northern Territory. Plantings were stringently inspected, and reinfection of trees was not found. No symptoms of canker were found during a survey of 4400 trees in 1950 in regions that were featured in the original surveys (Mertin 1952). In April 1991, during a survey of citrus in northern Australia as part of the Northern Australia Quarantine Strategy, the senior author observed symptoms consistent with citrus canker in a young pummelo (Citrus grandis &] Osbeck) orchard of 150 trees at Lambell's Lagoon near Darwin. The causal agent was identified as Xanthomonas campestris pv, citri (Hasse) Dye (Asiatic group or Group A), based on pathogenicity on a series of citrus hosts, fatty acid profiles and genomic DNA fingerprinting (Broadbent et al. 1992). Eradication of trees in the pummelo orchard was undertaken by burning in situ. A thorough survey of citrus trees in orchards and home gardens in the Lambell's Lagoon area

2 conducted in May 1991 (Pitkethley, personal communication) failed to detect further infected trees. Movement of citrus fruits and propagating material from the Northern Territory to other states was prohibited by the Northern Territory Department of Primary Industry and Fisheries. In this paper we report the results of surveys for citrus canker at Lambell's Lagoon in 1992 and 1993, and the detection of a further outbreak of the disease in Methods Surveys for citrus canker 1992 A total of 466 citrus trees in 26 orchards and home gardens at Lambell's Lagoon, a horticultural subdivision 57 km SE of Darwin, was inspected for canker symptoms from May A commercial nursery at Bees Creek and an orchard at Howard Springs were also surveyed. Leaves were collected randomly from trees at each site on 28 and 29 May and forwarded to the laboratory at the Quarantine Station, Rydalmere, to test for epiphytic X. c. citri The survey of citrus trees at Lambell's Lagoon was repeated in late May The following leaf and fruit samples were forwarded in sealed plastic bags to the quarantine laboratory at the Quarantine Station, Rydalmere: 1. Leaves of grapefruit (C. paradisi Macf.) and West Indian lime (C. aurantifolia [Christm.] Swing), with symptoms indicative of citrus canker (samples 39-42, 44-47), from a mixed orchard of 34 citrus trees at Lambell's Lagoon. A lime fruit with canker lesions (sample 43) was forwarded from the same property. 2. Further symptomatic and asymptomatic leaves from affected grapefruit, lime and Washington navel orange trees (C. sinensis [L.]) Osb.) (samples 55-6 l), in the same orchard, symptomatic fruit (samples 55, 57-59) and leaves from asymptomatic trees (samples 62-66). 3. Asymptomatic leaves (sample 73) of young trees bought from the affected orchard and planted elsewhere at Lambell's Lagoon; asymptomatic leaves (sample 72) of Ruby red grapefruit planted adjacent to the suspect trees. 4. Asymptomatic leaves of (a) lemon trees (C. limon (L.) Burm. f.) (sample 74) bought from the affected orchard and planted at Humpty Doo ; (b) mixed citrus (sample 75) planted adjacent to these trees in the same orchard. 5. Asymptomatic leaves of pummelo, lemon, mandarin (C. reticulata Blanco) and cumquat (Fortunella margarita [Lour.] Swing.) (samples 67-71) from trees in a nursery in Darwin, with possible connections to the 1991 canker outbreak. Detached leaf enrichments X. c. citri was selectively enriched in wounded, detached leaves or excised leaf tissue discs of Duncan grapefruit, West Indian lime, sour orange (C. aurantium L.), Eureka lemon, sweet orange, Swingle citrumelo (Poncirus trifoliata (L.) Raf. x CC. paradisi) and Troyer citrange (P. trifoliata x C. sinensis). Young terminal leaves from glasshouse-grown plants were washed for 10 min in running tap water, surface disinfested in 1% sodium hypochlorite for 2 min and rinsed thoroughly with sterile distilled water and placed with the lower surface up on 1% water agar in a Petri dish. Lesion extracts were produced by finely chopping a suspect canker lesion in a drop of sterile water. Leaf washings were obtained by pummelling 20 leaves in 100 ml sterile distilled water for 30 sec using a 'stomacher', the liquid was spun at rpm for 10 min and the pellet resuspended in 200 pl sterile water. Each leaf was wounded by making five punctures with a 27 gauge needle through the lower surface. Droplets (10-20 pl) of aqueous lesion extract or leaf washings were placed on the puncture wounds on the lower surface of the detached leaf. Reactions were observed in the detached leaves 5-7 days later and isolations of X. c. citri attempted. An alternative to the above method, used in 1992, was to float surface sterilised discs of citrus leaves in ELISA wells containing the leaf washings or lesion extracts. Bacterial isolations These were carried out under quarantine conditions at the Quarantine Station at Rydalmere from (a) canker lesions on field samples of leaves and fruit, (b) lesions which developed in detached leaf assays and (c) leaf washings from field samples. Individual lesions were dissected from leaves, finely chopped in separate drops of sterile water in sterile Petri dishes, and left for 15 min before

3 streaking the liquid from the macerate onto sucrose-peptone agar (SPA) (sucrose 20 g/l, peptone 5 g/l, K2mo4 0.5 g/l, MgS04.7H g/l, and agar 15 g/l, ph ). Leaf washings were serially diluted and plated on the same medium. Single yellow, domed, mucoid colonies, indicative of Xanthomonas, were selected after three days at 26OC and were restreaked twice to obtain pure strains. Pathogenicity tests The aggressiveness of each Xanthomonas strain was evaluated with detached-leaf assays. The bacteria were grown on SPA for 2 days at 26OC and a dilution of approximately 108 cwml was made in sterile distilled water. Drops (10 pl) of bacterial suspension were placed on each wound site on the lower surface of detached leaves and reactions observed 5-7 days later. Antiserum production Isolates of,y. c. citri (DAR and DAR from canker lesions on pummel0 leaves from the 1991 Darwin outbreak and DAR from West Indian lime from the 1986 outbreak on Thursday Island), were forwarded to the USDA-ARS Beltsville for antiserum production and comparison with groups A,B,C andd of citrus canker (Table 1). Immunofluorescence Immunofluorescence microscopy has been used to detect epiphytic X. c. citri from leaves (Gottwald et al. 1988). Antisera to two group A strains of X. c. citri (Xc 62 from Japan or Xc 362 from the 1991 Darwin outbreak) were prepared at USDA Beltsville. Immunoglobulin G (IgG) was fractionated from whole serum using the protein A column procedure (Miller and Stone 1978). Antisera were conjugated to fluorescein isothiocyanate for indirect immunofluorescence, according to the method of Brlansky and Lee (1990). Leaf washings were made from asymptomatic leaves as described above. Samples from symptomatic leaves were prepared by chopping the lesions in a few drops of sterile distilled water. The resulting supernatant was transferred to a 10 ml syringe, then pushed through a Nucleopore 25 mm Swin-lok filter assembly with a multiple holder adaptor (Nucleopore) that contained a 5.0 pm polycarbonate membrane for trapping cellular debris in one holder and a 0.2 pm black polycarbonate membrane for trapping bacteria in the second holder. The 0.2 pm membrane was removed, placed sample side upward in a 35 x 10 mm plastic Petri dish, and cut in half with a scalpel. Three drops of a 1:20 dilution of FITC-labelled IgG specific for X. c. citri (Xc62 or Xc362) were spread on the surface of one half of the membrane. On the other half, TRITClabelled normal serum IgG was spread. All samples of IgG were diluted in a Tris-BSA-gelatin antibody buffer (20 mm Tris, 0.9% NaCl, ph 8.2 containing 0.1% bovine serum albumin, 1.O% gelatin, and 2.0 mm NaNO,). Incubations in the IgG were for min at room temperature and were followed by rinsing in Tris-BSAgelatin buffer for 5 min. Indirect antibody labelling was carried out according to the method of Brlansky and Lee (1990), using a primary unlabelled IgG followed by a fluorescent-labelled goat antirabbit IgG. Incubation times in primary IgG (diluted 1:20 in Tris-BSA-gelatin antibody buffer) were min, followed by washing in Tris-BSAgelatin antibody buffer for 5 min, incubation in the secondary labelling IgG (diluted 1:20) for min, and washing in Tris-BSA-gelatin antibody buffer for 5 min. The membranes were Table 1 Strains of Xanthomonas campestris pv. citri used in serological assays and for antiserum production Laboratory strain Xc 362 (DAR 65862) Xc 363 (DAR 65860) Xc 366 (DAR 65870) Xc 62 Xc 64 Xc 69 Xc 70 Xc 90 - Group A A A A B B C D Host and country of origin C. grandis, Darwin, Australia C. grandis, Darwin, Australia C, aurantifolia, Thursday Island, Australia C. reticulata, Japan C. limon, Argentina C. limon, Argentina C. aurantifolia, Brazil C. aurantifolia, Mexico 92

4 then mounted on glass microscope slides and viewed with a Leitz epifluorescence microscope using a 50 watt mercury lamp. They were viewed in the blue excitation range (excitation filter BP490, beam splitter FT 520, and suppression filter LP 520) for FITC. Reactions were rated as follows: ++ = strong fluorescence, cells highly visible; + = weak fluorescence, cell shapes visible; - = no visible fluorescence. survey and the 1993 outbreak at Lambell's Lagoon, as well as the 1984 and 1986 outbreaks on Thursday Island, were compared in dendrograms using the method described in the MIS Library Generation Software Users Manual (Anon. 1988). Results Bacterial identification Fatty acid analyses Each strain was grown on SPA for 2-3 days at 28OC to obtain actively growing colonies, then subcultured onto BBL trypticase-soy broth with 1.5% Difco Bacto Agar (TSBA). The TSBA plate cultures were grown at 28 rt 1 C for 24 h 1 h under aerobic conditions. Approximately 40 mg of bacterial culture was harvested from each plate using a platinum loop. Fatty acids were extracted from each culture, converted to fatty acid methyl esters, and assayed by the method of Sasser (1990~). The fatty acid methyl ester extracts were injected into a Hewlett-Packard 5898A gas-liquid chromatograph, where component fatty acids were separated by a 25 m x 0.2 mm high resolution capillary column with 0.33 pm cross-linked 5% phenyl methyl silicone coating prior to flame ionisation detection. Separated peaks were identified by the MIDI Microbial Identification system (MIS) standards and software (Microbial ID Inc, Newark, Delaware) (Anon. 1987). Each extract was named by comparing the combined calculated percentage area of each of the fatty acid peaks for each strain with profiles of known bacteria held in the computer systems library database, linked to the gas-liquid chromatograph, using the MIDI MIS software. A similarity index was produced for each strain by matching to the closest species in the MIDI MIS TSBA aerobe library version 3.7. The similarity index is based on statistical similarity, which expresses how closely the composition of an unknown isolate compares with the fatty acid composition of the library matches. The library of fatty acid profiles is computer-searched using a pattern recognition program. A similarity index of is reported to be an excellent match (Anon. 1987). Data from fatty acid analyses of strains of bacteria isolated in the 1991 outbreak, survey The 1992 field survey of 466 trees in 26 orchards and home gardens in the Lambell's Lagoon horticultural subdivision was carried out 36 days after the end of the wet season. No symptoms of citrus canker were found. Injury by leaf miners (Phyllocnistis citrella Stainton) and greasy spot lesions (Stenella sp.) were common. Rainfall at Lambell's Lagoon in the wet season was 87.0 cm compared to cm in (Figure 1). Leaf enrichments Washings of citrus leaves sampled in 1992 from each horticultural holding at Lambell's Lagoon (a total of 38 samples) were tested in leaf enrichment assays for the presence of Xanthomonas spp. pathogenic to citrus. Floating leaf pieces in washings in ELISA wells was an unsatisfactory method as the leaf discs soon became overgrown with fungi and bacteria. Leaf enrichment tests using needle pricks in intact leaves inoculated with the leaf washings were more successful. No symptoms typical of the eruptive lesions of canker A were produced. However, some leaf washings caused a small amount of callus-like tissue at the wound site on leaves of Duncan grapefruit (Figure 2), but not sweet orange. Water inoculations produced no response. Bacterial isolations were carried out on SPA from the lesions produced on detached leaves. All yellow colonies were streaked and subcultured for subsequent identification by fatty acid analyses and for pathogenicity testing. Fatty acid analyses The fatty acid profiles of the bacteria isolated were compared to entries in the MIDI MIS TSBA aerobe library version 3.7. Four were identified as Xanthomonas spp. (Table 2), but none matched the library entry for X. c. citri in the database. By comparison, a xanthomonad OAR 65859) isolated from the 1991 Darwin outbreak of canker gave a similarity index of to X. c. cltri in the TSBA library 93

5 O N D J F M A M J J A S Month Figure 1 Rainfall data for l99oi9 1 ( H ), 91/92 ( A ) and 92/93 ( + ) for Lambell's Lagoon. Figure 2 Leaf enrichment on detached Duncan grapefruit leaf using leaf washings from sample 2B (Musk lime) in the 1992 survey. database. The results for the unweighted pair matchings for the ten bacterial isolates from the 1992 Northern Territory survey, the Torres Strait control strain DAR and the mean TSBA library entries for X. c. citri (group A) and the Florida strain of X. c. citrumelo, are displayed in the dendrogram (Figure 3). Researchers at Microbial ID Inc. have found from experience that 'the relatedness of entries at the genus, species and subspecies levels is approximately 25, 10, and 6 Euclidean distances respectively' (Sasser 1990b). The dendrogram analysis of the 1992 survey isolates shows that the control strain DAR closely matched X, c. citri (Group A) (Euclidian Distance (E.D.) = 2.2), indicating a relationship at the subspecies level. Isolates (E) andh92/83 (J) had an E.D. of approximately 16 from the X. c. citri (Group A) strain indicating a relationship at the genus, but not the species or subspecies level. Isolates (L) and (W) had an E.D. of approximately 27 from the X. c. citri (Group A) strain, indicating a possible relationship at the genus level only. The remaining isolates (B), (M),

6 Table 2 Similarity indices for fatty acid methyl ester profiles generated by comparing bacteria isolated from the 1992 Northern Territory citrus canker survey with a reference strain (DAR 65859) from the 1991 Northern Territory citrus canker survey Accession Identification by fatty acid analyses Similarity Index* (MIDI MIS number TSBA aerobe library version 3.7) Q92172 (B) Pseudomonaspaucimobilis Holmes, Owen, Evans, Malnick and Willcox 1977 Flavobacterium meningosepticum King (F) Xanthomonasmaltophilia (Hugh 1981) Swings, de Vos, van den Mooter, de Ley 1983 Q92183 (J) Xanthomonas campestris pv. pruni (Smith 1903) Dye 1978 Q92183 (L) Xanthomonas campestris pv. vasculorum (Cobb 1893) Dye (M) Pseudomonas saccharophila Doudoroff Q (BB) Curtobacterium pussilum (Iizuka and Komagata ) Yamada and Komagata 1972 Q (T) Flavobacterium meningosepticurn Q (W) Xanthomonas campestris pv. vasculovum Q (X) Pseudomonas saccharophila DAR65859 Reference strain Xanthomonas cam~estris vv. citri *The similarity index is a numerical value which express how closely the comparison of an unknown isolate compares with the fatty acid composition of the library matches. A similarity index of 0.6 to 1.0 is an excellent match (1.0 is the highest possible). A MIS identification giving only one match with a similarity index greater than has a strong likelihood of being correct. Single-match identifications with similarities less than may indicate an organism not in the library, but related to the MIS match. (Anon. 1987). Q (BB), Q (T) and (X) were all related to the known strain of X. c. citri (Group A) with an E.D. greater than 3 8. Therefore, it can be concluded that these six isolates were not xanthomonads. Pathogenicity testing Of the isolates identified as Xanthomonas sp., Q92/78(F), Q92/83(J), Q92/83(L) and Q92/107(W) caused slight callusing with water-soaked areas around the wound (Figure 4) in detached, wounded leaves of Duncan grapefruit, giving a very minor reaction compared to the eruptive callusing produced by X. c, citri. The response in detached wounded leaves of sweet orange and sour orange was less than for Duncan grapefruit. Immunofluorescence X. c. citri was not detected in leaf washings from samples in the 1992 survey by immunofluorescence microscopy of bacteria that were trapped on 0.2 pm black polycarbonate membranes and incubated in FITC labelled immunoglobulin (IgG) prepared against X. c. citri (isolate Xc 62). Similarly none of the purified Xanthomonas isolates reacted with Xc 62 antiserum, indicating that the bacteria were unrelated to X. c. citri survey In the survey conducted in late May 1993 (2 weeks after completion of the wet season), lime and grapefruit trees were observed with leaf, twig and fruit symptoms consistent with canker (Figure 5). The affected orchard of 34 trees of various citrus species and cultivars was in close proximity (500 m) to the 1991

7 Culture No. GI (BB) Q92/107(X) Q92/85(M) Q92/72(B) Q92/73(D) Q92/107(T) Q (W) Q92/83(L) X.c.citrumelo TSBA library mean DAR65859 X.c.citri (control) X.c.citriGroup A TSBA library mean Euclidian distance Figure 3 Dendrogram based on fatty acid compositions of bacterial isolates from the 1992 citrus canker survey, compared to a control strain of Xanthomonas campestris pv. citri (DAR 65859) from the 1991 survey and to the TSBA library version 3.7 mean data entries for X. c. citri and X. c, citrumelo. (See Table 2 for identifications of isolates). canker outbreak. Symptoms were on the spring flush and consisted of raised, roughly circular, corky scabs 4-5 mm in diameter. Most canker lesions were not active and had been overrun with fungal mycelium (principally Fusarium spp.). Lesions that were still active had a dark green, oily ring at the margin and a prominent yellow halo. Frequently the infection of leaves followed the course taken by citrus leaf miners (Phyllocnistis citrella) and other injuries. Detached leaf enrichments Callus-like lesions developed on wounded detached leaves of four citrus hosts inoculated with leaf washings from (a) all symptomatic leaves (samples 39-43, 44-47,55-59) (b) 216 symptomless leaf samples (55-6 1) from affected trees, (c) 116 fruit (55-6 1) with canker lesions, (d) a budstick (59) with a canker lesion. None of the washings of samples from (a) symptomless trees in the affected orchard at Lambell's Lagoon (samples 62-66) (b) young trees bought from the affected orchard

8 and planted elsewhere (sample 74) or (c) trees in a nursery (samples 67-71) with connections to the canker outbreak produced symptoms in wounded detached citrus leaves (Table 3). Bacterial isolations Few macerates, obtained by finely chopping canker lesions in drops of sterile saline, yielded yellow, domed, mucoid colonies, indicative of Xanthomonas, as the SPA plates were overrun by other bacteria. This problem was partially overcome by plating on SPA to which sodium benzyl penicillin (100 ppm, unitsl) and crystal violet (140 mg/l) had been added. The polymerase chain reaction was used to screen mixed culture suspensions for the presence of X. c. citri (Gillings et al. 1995). Isolations from callus-like tissue in the detached leaf enrichments were more successful. All yellow colonies were streaked and subcultured for subsequent identification by fatty acid analyses and for pathogenicity testing. Immunofluorescence Results of the immunofluorescence microscopy of bacteria that were trapped from leaf washings or lesion extracts on 0.2 pm black polycarbonate membranes and incubated in FITC-labelled IgG prepared against X. c. citri (isolate Xc 362) showed good correlation with the results of leaf enrichments (Table 3). Fatty acid analyses The fatty acid profiles of bacteria isolated from the 1993 outbreak were compared to entries in the MIDI MIS TSBA aerobe library version 3.7. Five isolates were identified asx c. citri (Table 4). The dendrogram of the 1993 survey strains (Figure 6) indicates that they are all related at the species level and that the strains labelled X. c. citri were related at the subspecies level (ED. = 5.3). Strain DAR lay outside the subspecies level (E.D. = 8.6 from the strains of X, c. citri), and the closest match was to X. c. zinniae. The standard strain DAR was represented across the spread of the strains within the X. c. citri group indicating the variability that can be obtained by multiple GC runs and/or multiple fatty acid extractions of the one strain. The two reference strains X. c. campestris and X. c. phaseoli were included to give a comparison to other related pathovars of X. campestris. Both groups lay outside the subspecies level of relatedness to the strains of X. c. citri. Pathogenicity testing of bacterial isolates Bacterial strains DAR 65973(i), DAR 65973(ii) and DAR 65977, identified by fatty acid analyses as X. c. citri were tested for their pathogenicity in wounded, detached leaves of Duncan grapefruit, Eureka lemon, Ruby Blood sweet orange, Troyer citrange and West Indian lime. All gave callus-like lesions, typical of canker (Figure 7). Reisolations from inoculated detached leaves (DAR and 65984) were compared with direct isolations (DAR and 65977) by genomic fingerprinting (Gillings et al. 1995) and no differences were detected. Serological relationship of Darwin strain to pathotypes of X. c. citri Strong positive reactions occurred in indirect ELISA tests using antiserum to strains 366, 362 or 363 of X c. citri with pure culture cells of Xc 62 (pathotype A) from Japan. Lesser reactions occurred with Xc 362 and 363. Reactions to Xc 70 (group C) were greater than for group B (Xc 64 and Xc 69) or D (Xc 90). Discussion Figure 4 Pathogenicity testing of xanthomonad isolate Q92/83(J) in a wounded detached leaf (lower surface)of Duncan grapefruit. The identification of X. c. citri as the causal agent of the 1993 canker outbreak at Lambell's Lagoon is based on pathogenicity tests, fatty acid analyses of recovered bacteria, immunofluorescence microscopy (loc. cit.) and by genomic DNA fingerprinting and positive PCR assays (Gillings et al. 1995). Such a multiple

9 Figure 5 Symptoms of citrus canker on (a) a leaf of Ruby grapefruit, (b) fruit and (c) twig of West Indianlime collected during 1993 survey. 98

10 Table 3 Comparison of detection methods for Xanthomonas campestris pv. citri in leaf washings from field samples collected in the 1993 survey Sample Leaf enrichments Direct isolations Immunofluorescence ofx c. citri Symptomatic leaves Asymptomatic leaves Negative control Symptomatic leaves Asymptomatic leaves Symptomatic fruit + (DAR 65982) - + (DAR 65984) - - Stem lesion Asymptomatic leaves

11 Euclidian distance Culture no D AM6922 Xc. campestr~s I I I I I I DAR30537 xc. campestrlis I D AR30546 DAR65859 (A) Xc. campesrr~s x c. cirri..... DAR65977 x c. cirri... DAR65973(i) x c. cirri.... DAR65869 x c cirri..... DAR65859(B) X c. cirri..... DAR65973(ii) x c. cirri..... TSBA v3.7 (mean) DAR65870 DAR65859(C) DAR65859fD) DAR65859(E) DAR65984 DAR65982 DAR65983 X c. cirri... X c. cifri.... X c. cirri... Xc cii//:... x c. cirri..... x c. citri... xc. c/f/i... Xcundet... DAR65944 (G C run 2) DAR65945 (G C run 2) D AR65944 (GC runl) DAR65945 (G C run 1) X cphaseok Xc.phaseo/i Xc.phaseo/i Xc.phaseo/i ,t'?' 'r Strain Subspecies Species Figure 6 Dendrogram of the fatty acid compositions of citrus strains from the 1993 citrus canker survey with comparisons to: 1. the TSBA library version 3.7 mean entry for Xanthomonas campestris pv. citri, 2. strainsdar anddar from the 1984 citrus canker outbreak on Thursday Island, 3. several extractions of a control strain of X. c. citri PAR 65859) from the 1991 survey to show the variability in fatty acid compositions for one strain, (A) separate extraction, run on ; (B) separate extraction, run on ; (C) concentrated rerun of DAR 65859(A), run on ; (D) average of three runs:- DAR 65859@), DAR 65859(C) and DAR 65859(E); (E) separate extraction, runon , 4. known comparison strains of X. c. citri and X. c. phaseoli, from the NSW Agriculture Plant Pathology Herbarium DAR culture collection. 100

12 Table 4 Similarity indices for fatty acid methyl ester profiles generated from strains of Xanthomonas campestris pv. citri from the 1993 canker survey against MIDI MIS TSBA aerobe library version 3.7 DAR No. Identification Similarity index (i)* X. c. citri (ii)a X. c. citri X. c. citri X. c. citri X. c. citri ADAR 65973(i) and DAR 65973(ii) were isolated from the same leaf lesion. Figure 7 Pathogenicity testing of Xanthomonas campestris pv. citri (DAR 65977) in a wounded, detached leaf (lower surface) of Duncan grapefruit. approach to formally confirming the identity of the causal agent is necessary because at least three distinct groups of strains of Xanthomonas are involved in the canker diseases of citrus (Stall and Civerolo 1991). Other xanthomonads can cause disease in citrus or citrus relatives e.g. X. c. citrumelo causes bacterial spot of Citrus spp. while other xanthomonads have been isolated from Feronia elephantatum and Clausena lansium (Stall and Civerolo 1991). In the present study, unrelated xanthomonads caused a minor reaction of slight callusing with a water soaked margin around wounded areas in detached Duncan grapefruit leaves. The outbreak of citrus canker in 1993 occurred in an orchard 500 m from the site of the 1991 canker outbreak. Genomic DNA fingerprints of cultures of X. c. citri from each outbreak were indistinguishable, providing strong evidence that the same clonal line of group A was involved in both outbreaks (Gillings et al. 1995). The question that arises is why canker was not detected in 1992 if the 1993 outbreak was the result of spread from the 1991 infection site. The assumption is that infection took place during or prior to 1991 from infected pummelo trees associated with the 1991 canker outbreak and that the bacteria survived epiphytically or in undetected lesions until disease expression in Leaf washings of three composite samples of leaves (grapefruit, lemon and lime; and orange, mandarin and tangelo) from the orchard involved in the 1993 canker outbreak were negative for X. c. citri by leaf enrichment tests and immunofluorescence microscopy in Either the epiphytic populations of X. c. citri were too low for detection, or they were not uniformly distributed on all leaves. Short distance dissemination of the pathogen may have occurred via infected plant material (not known to have taken place), wind-driven rain, insects (e.g. leaf miner), contaminated pruning and harvesting tools, or equipment and personnel. No sharing of equipment or personnel has been documented. The possibility that both Darwin outbreaks arose from an as yet unidentified source, possibly outside the Lambell's Lagoon area, cannot be discounted. X. c. citri may be 'resident' for a long time on citrus trees in a symptomless association (Goto 1972). In Florida, trees sometimes did not develop canker lesions until 2 years after they were removed from contaminated nurseries (Berger et al. 1914). Survival of the bacterium on non-citrus weed and grass hosts has also been reported (Civerolo 198 1). An analysis of rainfall data at Lambell9s Lagoon for the wet seasons , 1991/2, (Figure 1) shows that higher rainfall occurred during the years when the canker outbreaks occurred: cm in 90/91 and cm in 92/93 compared with 87.0 cm in 91/92. In the Darwin area, conditions essential for serious canker outbreaks prevail during the rainy season from October to March. Mean maximum temperatures during this period are consistently above 30 C with mean minimum temperatures about 25 C. A major growth flush occurs in October with some new leaves forming throughout the wet season (Broadbent 1992).

13 Dissemination of X. c. citri and subsequent incidence of canker are directly related to windblown rain especially at wind speeds in excess of 6 mlsec with light rain (Serizawa 1981). While total rainfall during the wet season was considerably less in , the incidence of light rain and strong wind should have been satisfactory for bacterial dissemination and disease development. In 1993, canker lesions were observed on West Indian lime and grapefruit trees, which are more susceptible than the mandarin and tangelo cultivars also present in the affected orchard. Difficulty was experienced in isolating X. c. citri directly from canker lesions in both West Indian lime and grapefruit, due to age of the lesions, and contamination by other bacteria. Populations of cells of X. c. citri decline in lesions as they stop expanding and fewer bacteria are released from older lesions due to suberisation (Timmer et al ) The failure in May 1992 to detect canker bacteria which may have been present epiphytically and the difficulty in culturing X. c. citri from lesions at the end of the wet season in 1993 casts doubt on the timing of the canker surveys. The canker lesions were present on the growth flush formed at the beginning of the wet season (October-November) and were 6 months old at the time the survey was conducted. The decision to survey at the end of the wet season had been made to avoid possible spread of X. c. citri by survey personnel on hands, clothing, shoes, equipment or vehicle wheels. The infected orchard found in 1993 has been eradicated. However, young trees were removed from this orchard to other sites, prior to detection of canker, and it is possible that the outbreak has not been eradicated. Further surveys in , commencing at the start of the wet season did not detect canker symptoms. In the United States of America, delimiting surveys for citrus canker are repeated four times a year for two years following any new detection. Surveys follow the flushes of new growth as closely as possible. During the third year, at least two additional surveys are conducted. Following eradication of infected trees, no new host plant can be planted for two years (Anon. 1986). The recent occurrences of canker in more remote areas of Australia and controversies over survey techniques, fruit treatments and claims for compensation for the removal of asymptomatic trees, point to the need for an action plan to be formulated to deal with future outbreaks of canker, whether on remote islands (e.g. in the Torres Strait) or in major citrus growing areas. References Anon. (1986) - Action Plan. Citrus Canker Disease. USDA, APHIS. Anon. (1987) - HP 5898A Microbial identification system operating manual, Hewlett-Packard Company, Avondale, PA Anon. (1988) - MIS Library Generation Software Users Manual. Microbial ID, In., Newark, Delaware. Berger, E.W., Stevens, H.E. and Sterling, F. (1914) - Citrus canker II. Florida Agricultural Experiment Station Bulletin pp. Brlansky, R.H. and Lee, R.F. (1990) - Detection of Xanthomonas campestris pv. citrumelo and X. citri from citrus using membrane entrapment immunofluorescence. PlantDisease 74: Broadbent, P., Fahy, P.C., Gillings, M.R., Bradley, J.K., and Barnes, D. (1992) -Asiatic citrus canker detected in a pummel0 orchard in northern Australia. PlantDisease 76: Broadbent, P. (1992) - Perceived vulnerability of citrus to canker in the major citrus growing areas of Australia. Australasian Plant Pathology 21: Civerolo, E.L. (1981) - Citrus bacterial canker disease: an overview. Proceedings of the InternationalSociety of Citnculture I: Gillings, M.R., Fahy, P.C., Broadbent, P. and Barnes, D. (1995) - A second outbreak of Asiatic citrus canker in the Northern Temtory rapidly identified using the polymerase chain reaction. Australasian PlantPathology24: Goto, M. (1 972) - Survival of Xanthomonas citri in the bark tissue of citrus trees. Canadian Journal of Botany 50: Gottwald, T.R., McGuire, R.G. and Garrow, S. (1988) -Asiatic citrus canker: spatial and temporal spread in simulated new planting situations in Argentina. Phytopathology 78: Hill, G.F. (1 91 8) - History of citrus canker in the Northern Territory. Bulletin of the Northern Territory 1 8.

14 Jones, D.R., Moffett, M.L. and Navaratnam, S.J. (1984) - Citrus canker on Thursday Island. Australasian PlantPathology. 13: Mertin, J.V. (1952) - Plant quarantine survey in Northern Territory. Journal of the Australian InstituteofAgriculturalScience 18: Miller, T. J. and Stone, H.O. (1 978) - The rapid isolation of ribonuclease-free immunoglobulin G by protein-a-sepharose affinity chromatography. Journal of ImmunologicalMethods 24: Pruvost, O., Harting, J.S., Civerolo, E.L., Dubois C. and Perrier, X. (1 992) - Plasmid DNA fmgerprints distinguish pathotypes ofxanthomonas campestris pv, citn; the causal agent of citrus bacterial canker disease. Phytopathologv82: Sasser, M. (1990a) - Identification of bacteria through fatty acid analysis. In Methods of Phytobacteriologv (Eds Z. Klement, K. Rudolph and D.C. Sands), pp Akademiai Kiadio, Budapest. Sasser, M. (1990b) - Tracking a strain using the Microbial Identification System. Technical Note # 102, Newark, Delaware. Serizawa, S. (1 981) - Recent studies on the behaviour of the causal bacterium of the citrus canker. Proceedings of the Internationalsociety of Citriculture I: Shivas, R.G. (1987) - Citrus canker (Xanthomonas campestris pv, citn] and banana leaf rust (Uredo musae) at Christmas Island, Indian Ocean. AustralasianPlantPathologv 16: Stall, R.E. and Civerolo, E.L. (1991) - Research relating to the recent outbreak of citrus canker in Florida. Annual Review ofphytopathology29: Timmer, L.W., Gottwald, T.R. Zitko, S.E. (1991) - Bacterial exudation from lesions of Asiatic citrus canker and citrus bacterial spot. Plant Disease 75: Manuscript received 5 September 1994, accepted 27 October 1994.