Identification of Grapevine Crown Gall Bacteria

Transcription

1 Ann. Phypath. Soc. Japan 56: (1990) Identificati Grapevine Crown Gall Bacteria Isolated in Japan* Hiroyuki SAWADA**, Hiroyuki IEKI** Yuichi TAKIKAWA*** Abstract Recently, incidence crown gall disease has been gradually increasing in some varieties including ekyoho f in Japan. Twenty-five bacterial, which were obtained from galls s collected in Iwate, Yamanashi, Nagano Shimane Prefectures, were identified as Agrobacterium tumefaciens (Smith et Townsend 1907) Cn 1942 based ir ability induce galls, ma sunflower using needle prick inoculati method also based 90 cultural, physiological biochemical characteristics. Although differed from reference strains belging A. tumefaciens biovars 1 2 in characteristics, respectively, y were similar biovar 3 respect features presented in Bergey's Manual (1984) except for cleavage ethanol propiate. Based se results it suggested that belged A. tumefaciens biovar 3. (Received June 28, 1989) Key words:, crown gall, Agrobacterium tumefaciens, biovar 3. INTRODUCTION The incidence crown gall disease in Japan first reported in Presently, disease is widespread in several grape-growing prefectures is damaging mainly young trees some susceptible varieties such as ekyoho f in new plantatis nurseries27). Taxomic studies causal bacterium, Agrobacterium tumefaciens, have been very limited in Japan, its biovar remains be determined. Panagopoulos Psallidas18), who studied crown gall bacteria taxomically for first time, reported that ir cformed neir biovar 1 nor 2 which had been known hir18), proposed term ebiovar 3 f for ir isolated9,19). Afterwards, biovar 3 found be respsible for crown gall disease also in Hungary, South Africa Canada4,3,26). However, bacteriological properties biovar 3 have not been fully examined more detailed comparative studies or biovars are needed1,11). In this paper, we analyzed in detail taxomic characteristics crown gall bacteria isolated in Japan, cfirmed that y were identical A. tumefaciens biovar 3. Also problems relating differential characteristics between biovars were examined. MATERIALS AND METHODS Bacteria. In 1987, young, actively growing galls were sampled from affected trunks arms s in 9 orchards located in Iwate, Nagano Shimane Prefectures. Single

2 coly cultures were obtained from se materials using streak culture method King's B medium plates, were purified by restreaking. Their pathogenicity examined by procedure described below ly e isolate selected as a representative from pathogenic es derived from each diseased. The 22 obtained by se procedures in our laborary 3 supplied by Y. Terai (Yamanashi Fruit Tree Exp. Stn.) were used as (Table 1) in present experiment. Agrobacterium tumefaciens strains AtCl17) (biovar 1) (K. Ohta, Shizuoka Agr. Exp. Stn.), AtRll17) (biovar 2) (K. Ohta) Peach CG 8331 (biovar 2) (Y. Takikawa) were included as references (Table 2). All cultures were sred by freezing bacterial suspensis at -30C. The cultures for studies were grown pota-pepne-glucose agar medium (PPGA)16) kept at 4C during experiment. Media. The PPGA medium16) used ctained: pepne, 5g; glucose, 5g; Na2HPO4 E 12H2O, 3g; KH2PO4, 0.5g; NaCl, 3g; agar, 15g; pota decocti (200g/1,000ml), 1,000ml. The nutrient broth used in tests csisted : elab-lemco f Powder (Oxoid), 5g; pepne, 10g; NaCl, 2.5g; distilled water, 1,000ml, ph6.8. Proteose Pepne No.3 (Difco) used as pepne except for indole producti test where Bac-Trypne (Difco) employed. The or media used in physiological biochemical tests are referred literature cited. Pathogenicity. The ability bacterial induce galls tested stems ma (cv. Pterosa), sunflower (cv. Dairin Himawari) (cv. Rizamat) by needle prick inoculati method. Inoculum prepared by suspending a loopful material from a 24-hr-old PPGA slant culture in 2ml distilled water. The 4-8-week-old plants grown in a greenhouse were inoculated at 5-8 sites per internode by pricking through a drop inoculum a needle, were kept at 23C. The pathogenicity determined 30 days after inoculati based gall formati at site pricking. Table 1. Isolates crown gall bacteria used in present experiment a) Isolated by Mr. Y. Terai (Yamanashi Fruit Tree Exp. Stn.). Table 2. Strains Agrobacterium tumefaciens used as references a) Ohta, K. (1984)17). b) Culture Collecti Phypathogenic Bacteria, preserved in Laborary Plant Pathology, Shizuoka University, Shizuoka, Japan.

3 Ann. Phypath. Soc. Japan 56 (2). April, Bacteriological properties. Prior all tests, 24-hr-old PPGA slant sub-cultures were prepared from cultures maintained at 4C. A small amount material from subculture streaked over surface plate medium as a line a loop. When test medium placed in a tube, inoculum prepared by suspending whole slant subculture in 5ml sterile distilled water, transferred test medium a needle. Unless orwise stated, all cultures were incubated at 28C. The incubati periods are listed in Tables 3 4. Gram reacti performed according method Ryu21). The modified Yamanaka's method used for flagellar staining22). Morphology motility living cells were observed by phase-ctrast microscopy24). The methods Moore et al.15) were used test Kovacs' oxidase, producti 3-kelacse, formati a pellicle in ferric ammium citrate soluti, utilizati L-tyrosine citrate, growth D-1 agar, Schroth et al. medium New Kerr medium. For glucate oxidati, producti urease, amino acid decarboxylase phosphatase, hydrolysis starch Tween 80, method (1) described in Cowan Steel's Manual for Identificati Medical Bacteria, 2nd ed.2) used. Producti arginine dihydrolase, phenylalanine deaminase indole, liquefacti gelatin were tested by method (2), H2S formati tested by method (3) described in same manual2). The methods described in Cowan Steel's manual were also used analyze oxidative metabolism glucose, ONPG test, hydrolysis casein, litmus milk reacti, producti DNase2). Voges-Proskauer reacti methyl red test were performed in glucose-salt medium according method (1) Cowan Steel2). The methods Dye were used test nitrate reducti, nitrate respirati, growth facr requirement5). Producti lipase examined by method Lelliott et al.12), where cotn seed oil used as substrate in place margarine. The methods Lelliott et al. were also used examine levan formati, producti fluorescent pigment, pota st rot12). For Nile Blue test, producti tyrosinase ammia, methods Skinner23), Crosse Garrett3), Garrett et al.6) were used, respectively. Both methods Sneath25) Dye) were employed for hydrolysis esculin. Cleavage sugars organic acids tested by using Ayers, Rupp, Johns's syntic medium15) supplemented 0.02% yeast extract (Difco) as basal medium, which sugars salts organic acid were added give a 1% a 0.2% ccentrati, respectively. Temperature for growth lerance NaCl were tested in 1% pepne water. Producti pectinase examined using Pan's pectate gel medium at ph7.020). RESULTS Pathogenicity All 25 used were pathogenic ma, sunflower. There no difference amg se respect virulence sympms. The galls, which began appear 5-7 days after inoculati all plants used, grew actively during incubati period. Their surfaces became rough y were light-brown in color (Fig. 1). The diameter galls produced ma larger than 3mm, while that galls sunflower ranged from 1 2mm. The 3 reference strains also produced galls all three plants used. But diameter ir galls, which about 1mm, smaller than that galls produced by same host (Fig. 1). Bacteriological properties The reactis 25 were similar for 90 cultural, physiological biochemical characteristics tested in present experiment. And 65 characteristics coincided those reference strains belging biovars 1 2 (Table 3), while characteristics were different, respectively (Table 4). On or h, reactis

4 202 日本植 物 病 理 学 会 報 Fig. 1. in (biovar that 1) For because after In always 14 test for colies The which slants AtCl ible, it All period method. Upper: respect characteristics except for cleavage pre ethanol nutrient agar media In After PPGA16) medium. ctaining circular, litmus milk reducti, both media or cvex, culture, They glucose smooth, produced sucrose. former The translucent became white, where- began which Skinner Dye5), results period for cleavage gave but positive exhibited esculin no so after at all depended dihydrolase were reactis growth hydrolysis arginine by appear test slow that acids, 2 in turned medium kind extended media days 28 red test days, usually set organic or alkali. There. In that 7-10 days ir development, this reacti Since incubati a lag period for ability 1). scattered all time ir (biovar were a lag particular AtCl alg days. growth Their showed proved catabolism catabolize independent slant same be surfaces. color as reproduc- positive f. 23) after as edelayed acid compared gradually 17 slow blue sugars producti approximately scored by that remarkably described days. became after 3 inoculati (1984)11) npigmented, margin. propiate were test, biovar slime incubati reactis than propiate well litmus. indicated associated ethanol entire medium The more prick 2). brown. which used. those Bacteriology agar hydrolysis Sneath25), nutrient an latter in medium grew reduced esculin incubati glistening Systematic polysaccharide colies AtCl 平 成2年4月 4). extracellular surface as (Table abundant similar Manual propiate were Bergey's The 第2号 Galls stems induced by needle G-Ag-21 (biovar 3), lower: Peach CG 8331 (biovar sented 第56巻 gave at 5 days negative reactis prolged in 21 Nile Blue test, although incubati days. DISCUSSION The were aerobic, Gram negative, motile rods peritrichous flagella. They metabolized glucose oxidatively; produced catalase abundant extracellular polysaccharide slime glucose- or sucrose-ctaining media, but no gas nor pigment any media used; induced galls three plants examined. These features indicate that y belg genus Agrobacterium. In Bergey's Manual (1984)11), genus Agrobacterium is divided in 4 species mainly

5 Table 3. Bacteriological characteristics coinciding those reference strains a) Grapevine : see Table 1, reference strains: see Table 2. b) (): incubati period (day). basis pathogenicity types sympms induced plants. Namely genus csists crown-gall-forming A. tumefaciens, hairy-root-inducing A. rhizogenes, n-pathogenic A. radiobacter tumorigenic A. rubi which isolated from Rubus spp. In additi, re are 2-3 genetically phenotypically different groups in each species except for A. rubi7-10,26,28), which are assigned biovars 1, 2 311). Thus two steps are required identify agrobacteria: first are analyze pathogenicity which distinguishes species, secd study bacteriological properties that differentiate biovars1,11,15).

6 Table 4. Bacteriological characteristics differing from those reference strains a) Grapevine : see Table 1. b) Reference strains: see Table 2. c) Data from Bergey's Manual Systematic Bacteriology (1984)11). d) +: positive; (+): delayed positive; -: negative; d: varied by strains; NG: no growth. e) K: alkali producti; A: acid producti; R: reducti litmus. f) Different results were recorded by Sule (1978)26). The were found belg A. tumefaciens, since y were agrobacteria isolated from galls produced galls, ma sunflower. As for ir cultural, physiological biochemical characteristics, y were different from reference strains belging biovars 1 2 in characteristics, respectively, but gave similar reactis those biovar 3 described in Bergey's Manual (1984)11) except for cleavage ethanol propiate. For cleavage ethanol, Sule26) reported same results as those obtained by us. For cleavage propiate, variable results have been obtained amg biovar 3 strains19). These results show that se two characteristics are not uniform in biovar 3. Therefore, we assigned A. tumefaciens biovar 3. In previous reports crown gall diseases flower crops, A. tumefaciens biovars 2 have already been detected in Japan14,17). However, this is first report, our knowledge, identificati biovar 3 in Japan. Although ly biovar 3 could be isolated from s in present experiment, biovar 1 or 2 as well as 3 recovered from same host in Greece, Hungary, etc.18,19,26) Furr research is needed determine wher causal agents crown gall or than biovar 3 occur in Japan.

7 Ann. Phypath. Soc. Japan 56 (2). April, Since various bacteriological properties biovar 3 have not been fully examined, differential characteristics from or biovars remain be determined1,11). Furrmore, various cditis such as kind medium length incubati period were found affect results esculin hydrolysis, arginine dihydrolase cleavage sugars organic acids. Similar observatis have also been reported for oxidase7,18) H2S formati test7). Moreover, kind pepne used compositi nutrient broth were found affect growth agrobacteria significantly (data not shown). Therefore, furr detailed studies are needed clarify influence test cditis results determine differential characteristics biovars including biovar 3, by examining a sufficient number strains derived from various hosts localities. We wish thank Mr. Y. Terai Mr. K. Ohta for kindly providing reference strains, Dr. H. Tanaka Mr. T. Makino for ir helpful suggestis. Literature cited 1. Bradbury, J.F. (1986). In Guide Plant Pathogenic Bacteria. CAB Internatial Mycological Institute, Kew. pp Cowan, S.T. (1974). Cowan Steel's Manual for Identificati Medical Bacteria. 2nd ed. Cambridge Univ. Press, Cambridge. 3. Crosse, J.E. Garrett, M.E. (1963). J. appl. Bact. 26: Dhanvantari, B.N. (1983). Can. J. Bot. 61: Dye, D.W. (1968). N.Z. J. Sci. 11: Garrett, C.M.E., Panagopoulos, C.G. Crosse, J.E. (1966). J. appl. Bact. 29: Holmes, B. Roberts, P. (1981). J. appl. Bact. 50: Keane, P.J., Kerr, A. New, P.B. (1970). Aust. J. biol. Sci. 23: Kerr, A. Panagopoulos, C.G. (1977). Phypath. Z. 90: Kersters, K., De Ley, J., Sneath, P.H.A. Sackin, M. (1973). J. Gen. Microbiol. 78: Kersters, K. De Ley, J. (1984). In Bergey's Manual Systematic Bacteriology (Krieg, N.R. Holt, J.G. eds.). Vol.1. Williams & Wilkins, Baltimore, pp Lelliott, R.A., Billing, E. Hayward, A.C. (1966). J. appl. Bact. 29: Loubser, J.T. (1978). Plant Dis. Reptr. 62: Makino, T. Morita, H. (1985). Bull. Shizuoka Agr. Exp. Stn. 30: (in Japanese). 15. Moore, L.W., Kado, C.I. Bouzar, H. (1988). In Laborary Guide for Identificati Plant Pathogenic Bacteria (Schaad, N.W. ed.). 2nd ed. Amer. Phypath. Soc. Press, St. Paul. pp Nishiyama, K. Ezuka, A. (1977). Ann. Phypath. Soc. Japan 43: (in Japanese). 17. Ohta, K. Nishiyama, K. (1984). Ibid. 50: (in Japanese). 18. Panagopoulos, C.G. Psallidas, P.G. (1973). J. appl. Bact. 36: Panagopoulos, C.G., Psallidas, P.G. Alivizas, A.S. (1978). In Proc. 4th Int. Cf. Plant Path. Bact., Angers, pp Pan, A.M. (1959). Nature 183: Ryu, E. (1940). Kitasa Arch. Exp. Med. 17: Shirata, A. Go, M. (1981). Shokubutsu boeki 35: (in Japanese). 23. Skinner, F.A. (1977). J. appl. Bact. 43: Smibert, R.M. Krieg, N.R. (1981). In Manual Methods for General Bacteriology (Gerhart, P. et al. eds.). American Society for Microbiology, Washingn, D.C. pp Sneath, P.H.A. (1956). J. Gen. Microbiol. 15: Sule, S. (1978). J. appl. Bact. 44: Terai, Y., Asari, S., Ono, M. Ichikawa, K. (1987). Ann. Phypath. Soc. Japan 53: 405 (Abstr. in Japanese). 28. White, L.O. (1972). J. Gen. Microbiol. 72:

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