Bacterial blight, caused by Xylophilus ampelinus (PANAGOPOULUS) WILLEMS et al. is a destructive disease of grapevine (Vitis vinifera). Incubation of cuttings at 15 °C for three days under moist conditions significantly increased the number of isolated X. ampelinus colonies. This treatment facilitates the isolation of the pathogen from infected material from which it could previously not be isolated

Botha, W. J.

Serfontein, J. J.

Serfontein, Suzel

Staphorst, J. L.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 36 (4), 209-210 (1997) Research Note The isolation and characterisation of Xylophilus ampelinus SuzEL SERFONTEIN, J. J. SERFONTEIN, W. J. BoTHA and J. L. STAPHORST S u m m a r y : Bacterial b1ight, caused by Xylophilus ampelinus (PANAGOPOULUS) WILLEMS et al. is a destructive disease of grapevine (Vitis vinifera). lncubation of cuttings at 15 ac for three days under moist conditions significant1y increased the number of iso1ated X. ampelinus co1onies. This treatment facili-tates the iso1ation of the pathogen from infected material from which it could previously not be isolated. K e y wo r d s : bacterial necrosis, Xylophilus ampelinus, Vitis vinifera, detection. Introduction: Bacterial blight of grapevine, caused by Xylophilus ampelinus (Panagopoulus) WILLEMS et al. is a destructive disease mostly of table grape cultivars in the Mediterranean region and in South Africa but may also oc-cur in Switzerland, Austria, Bulgaria, the Canary Islands, Sardinia, Argentina, Tunisia, Turkey and the former Yugo-slavia (BRADBURY 1973; GARAU et al. 1988). The pathogen survives in the vascular tissues of infected plants (PANAGOPOULUS 1987). The disease can be transmitted by propagation material, during grafting and by pruning knives. Bacteria are spread by moisture to plants where infection may take place through wounds, leaf scars and other sites. Irrfeetion may also occur without wounding (BRADBURY 1991). Panagopoulus (1987) found that 50% of apparently healthy canes from diseased vineyards in Crete were latently infected. No selective medium is available for the isolation of X. ampelinus and its slow growth rate on Nutrient Agar (NA) hampers its recovery because of interference by saprophytes. We found it difficult to isolate the bacterium from infected material with distinct symptoms collected dur-ing hot and dry periods. Material collected from the same plants during cool and wet periods yielded !arge numbers of the bacterium. Du PLESSIS (1940) was also unable to isolate X. ampelinus from diseased material (PANAGOPOULUS 1996) and GARAU et al. ( 1988) also failed to isolate the pathogen from material with bacterial blight symptoms that had immunofluorescent antigenic cells. To enable us to screen propagation material, it was necessary to develop a suitable enrichment technique for the isolation of X. ampelinus from infected and latently infected material. Materials and methods: B a c t e r i a l s t r a i n s : Twenty-eight South African isolates, 23 from the Paar! dis-trict and 5 from the De Doorns district were used in this Correspondence to: Dr. SuzEL SERFONTEIN, Agricultural Research Council, Plant Protection Research Institute, P/Bag X 134, Pretoria 0001, Republic of South Africa. Fax: +27-12-8081489. E-mail: nipbss@plant1.agric.za study. Three of the isolates from the Paar! district were iso-lated from symptomless plants. Reference cultures abtairred from the National Collection of Plant Pathogenic Bacteria (NCPPB) in England: X. ampelinus (NCPPB 2217; NCPPB 2218; NCPPB 2590) were included in the identification tests. Isolations were made in· September 1993 as described by P ANAGOPOULUS ( 1969) on Difco NA Cultures were maintained at room temperature on NA supplemented with 2% CaC03• Stock cultures were freeze dried. All the strains were tested for catalase and Kovacs oxidase activity, urease produc-tion, lipolysis ofTween 80, production of Hß from cysteine, production of acid from glucose in Hugh and Leifson's me-dium and the production of acid in Dyy's medium C from glucose, sucrose, Iactose, maltose, galactose and arabinose (BRADBURY 1991). Pathogenicity was determined according to PANA-GOPOULUS ( 1969) on Vitis vinifera cultivar Sultana grown in a greenhouse. Young shoots and leaves were inoculated. Metaboliefingerprints of strains using B i o 1 o g TM G N m i c r o p l a t e s : Six representative strains were subcultured on NA by picking a few colonies onto cotton swabs and streaking it on media covering the entire plate. NA was used instead of the recommended Su-crose Peptone Agar, as the isolates failed to grow on the latter medium. Plates were incubated for 48 h at 26 oc. Ten plates of NA were reinoculated by the same method and incubated for an additional 48 h. A cell suspension with a density of 0.30 ± 0.05 at 600 nm was made in sterile saline (0.83% NaCl) and Biolog™ GN microplates ( 150 f.ll per well) were inoculated as recommended. P1ates were read visually after62 h. E n ri c h m e n t o f b a c t e r i a i n p l a n t s a m p-1 e s : Suspensions of 4 X. ampelinus strains were made in sterile saline and counted by dilution plating. 1.5 ml of the bacterial suspension were sucked through 8 cm long cuttings of greenhouse-grown young grapevine shoots (cultivar Sultana) that included a node. The cuttings were placed in plastic bags containing wet cotton plugs, closed with rub-ber bands and incubated in the dark at 10, 12.5, 15, 17.5, 20 and 22.5 °C. After 3 d incubation, the basal ends of the shoots were placed in sterile saline and a vacuum was ap-plied at the distal end until 0.75 ml was extracted from each shoot. Extracts from two shoots which were not inoculated were also analysed for the presence of X. ampelinus before and after incubation. For the control the extracts of two in-oculated shoots were analysed before incubation. A number of cuttings with natural infections, showing bacterial blight symptoms from which the pathogen could not be isolated by conventional methods, were also included. Dilutions of the extracts were plated in triplicate on NA Colorries were counted after 7 d incubation. The experiment was repeated twice. Results: B a c t er i a l s t r a i n s : All the strains were catalase positive, Kovacs oxidase negative, strongly urease positive within 2-3 d, lipolysed Tween 80, produced H2S from cysteine, did not produce acid from glucose in Hugh and Leifson's medium, did not produce acid from glucose, sucrose, Iactose or maitose in Dye's medium C but produced acid from galactose and arabinose. The colonies were all 210 SuzEL SERFONTEIN, J. J. SERFONTEIN, W. J. BoTHA and J. L. STAPHORST light yellow on NA and approximately 1 mm in diameter after 7 d. All the strains were uniform in appearance and in reac-tions. Typical symptoms were produced by all the strains on shoots and leaves. The symptoms on the shoots took 3-4 weeks to develop and on the leaves l 0-14 d. Metaboliefingerprints of strains using B i o 1 o g ™ G N m i c r o p 1 a t e s : All the X. ampelinus strains tested in the Biolog™ GN microplates utilised only acetic acid, propionic acid, L-aspartic acid, L-glutamic acid and L-pyroglutamic acid. Enrichment of bacteria in plant s a m p 1 e s : Recovery of X. ampelinus from inoculated shoots was 100 to 1000 tim es greater than from the control (no incubation) after a 3-d incubation at temperatures below 17.5 oc (Table). Data were transformed by taking natural logarithms of the values. Differences between treatments and the control were subjected to Dunnett's T test. Treat-ments below 17.5 °C differed significantly from platings of shoots before incubation. At temperatures of 17.5 oc and above, saprophyte populations increased to Ievels that in-terfered with the detection of the pathogen on plates. From the naturally infected cuttings, the best recovery rate was obtained after incubation of the shoots for 3 d at 15 °C. No X. ampelinus colonies were cultured from shoots which were not inoculated. Tab I e Colony forming units recovered of X. ampelinus from inoculated cuttings on nutrient agar before and after 3 days of incubation at different temperatures Treatment Replicate I Replicate 2 Mean' temp., °C 10 l.Oxl()2 1.6x101 5.3xl0'1 no growth 9.1553822b 12.5 1.6x1(}1 5.3x101 6.6x10' 1.5x1Q6 10.8935479b 15 3.9x1Q2 6.0x104 1.7x104 1.3x101 9.6211262b 17.5 3.3x1Q6 saprophytes saprophytes saprophytes c -XJ saprophytes 4.6x104 l.lx1(Y saprophytes c -22.5 saprophytes saprophytes saprophytes saprophytes c -Controld 2 3 8 3.0x10' 2.9693921b Suspension 8.6x101 3.4x101 • Data were transformed by taking naturallogarithms of the values. b Treatments below 17.5 °C differed significantly from the control (no incubation). Differences between treatments and the control were subjected to Dunnett's T-test. ' No average could be determined as the plates were over-grown by saprophytes. d The control was colony forming units recovered from infiltrated cuttings before any incubation. Discussion: Difficulty was experienced in culturing X. ampelinus from diseased grapevine material onto medium. GERAU et al. (1988) also failed to isolate the organism from material in which the presence of the organism was con-firmed by immunofluo-rescent microscopy. The enrichment technique significantly improved the detection of X. ampe-linus from grapevine shoots by direct isolation. The organ-ism has also been detected in shoots from vine plantings without symptoms, previously thought tobe free of the dis-ease, using this enrichment method. Although the optimum incubation temperature of shoots at which the largest nurober of X. ampelinus colonies were recovered seemed to be 17.5 °C, problems were experienced with saprophyte inter-ference, especially from naturally infected cuttings. An in-cubation temperature of between 10 and 15 °C, lowered saprophyte interference. lt is important to note that other slow growing bacteria are also regularly isolated from grapevine material which can easily be confused with X. ampelinus. Confirmation of identity is therefore always necessary. A !arge proportion of the slow growing organisms were found tobe Gram-posi-tive. Confirmation tests are generallytime consuming andin our experience, Gram reaction, catalase test, oxidase test, urease production and the lypolisation ofTween 80 are some of the less time consuming tests that are helpful in prelimi-nary identification ofthe organism. Serological (GoRRIS et al. 1997) and polymerase chain reaction (BoTHA et al., unpub-lished data) techniques are available for the detection of X. ampelinus in grapevine material. If the nurober of bacteria in the plant extracts is increased, the sensitivity of these techniques will be improved. We thank the SouthAfrican Plant Improvement Organisation for financial assistance and RoELOF CoETZER from the Agricultural Research Council , Agrimetric Institute for statistical analysis. BRADBURY, J. F.; 1991 : Xylophilus ampelinus IMI descriptions of fungi and bacteria no. 1050. Mycopathologia 115, 63-64. BoTHA, W. J.; SERFONTEIN, S.; BERGER, D. K.; GREYLING, A. ; Detection of Xylophilus ampelinus in grapevine cuttings using the polymerase chain reaction . Submitted to Europ. J . Phytopathol. Du PLESsts, S. J.; 1940: Bacterial blight of vines (VIamsiekte) in South Africa caused by Erwinia vitivora (Bacc.) DuPI. Sei. Bull. Dep. Agricult. South Africa, No. 214. GARAU, R. ; Cuoust, M.; PROTA, U. ; 1988: Preliminary evidence of foci of "mal nero" of grapevine in Sardinia. (Abstract) . Rev. Plant Pathol. 67, 383 . GoRRts, M. T.; CAMBRA, M.; LoPEZ, M. M.; 1989: Production of monoclonal antibodies specific to Xylophilus ampelinus. In : KLEMENT, Z. (Ed .): Proc . 7th Intern. Conf. Pl;mt Pathogenic Bacteria, Budapest, Hungary, June 11-16, 1989, 913-921. Akademiai Kiado, Budapest. PANAOOPOULUS, C. G. ; 1969: The disease "tsilik marasi" of grapevine: Its description and identification of the cau sal agent (Xanthomonas ampelina sp. nov.). Ann . Inst. Phytopathol. Benaki 9, 59-81. - -; 1987: Recent research progress on Xanthomonas ampelina. Bull . OEPP 17, 225-230. 

The isolation and characterisation of Xylophilus ampelinus

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The isolation and characterisation of Xylophilus ampelinus

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