A Comparative Study of the Strains of Xanthomonas campestris pv. citri Isolated from Citrus Canker in Japan and Cancrosis B in Argentina

Strains of Xanthomonas campestris pv. citri (Hasse 1915) Dye 1978 isolated from citrus canker in Japan (canker A) and those from cancrosis B in Argentina (canker B) were compared by pathological, physiological, serological and phage susceptibility properties. Strains of canker B organism were characterized by being significantly less virulent than those of canker A organism on various citrus plants such as Unshu, Natsudaidai, lemon and navel orange. Strains of canker B organism also differed from those of canker A organism in small size of colonies on agar plates, delayed lag period in liquid media, inability to utilize lactose and maltose, susceptibility to a new phage Cp 3, and lack of an antigenic component of the latter. Ten out of twenty-one strains of canker B organism were unable to utilize malonate which was consistently utilized by strains of canker A organism. All strains of canker B organism utilized mannitol, while one-half of the strains of canker A organism did not utilize this substrate. It was concluded that such differences supported the differenti ation of canker B organism as a distinct group within the pathovar of X. campestris pv. citri. (Received March 15, 1980) Introduction A bacterial disease of citrus called cancrosis B, false citrus canker or type B canker has long been known in Argentina, Paraguay and Urguay1,2,9,10,11,15). Although the disease produces symptoms closely resembling common citrus canker caused by Xanthomonas campestris pv. citri (Hasse 1915) Dye 1978 on susceptible citrus plants, it has been said that the disease is distinguishable in pathological characteristics such as host range or disease severity from common citrus canker which has been called cancrosis A, asian canker or true canker in these countries. Cancrosis B which will be referred to hereafter as canker B, has been acknowledged as a mild disease compared with common citrus canker which is subsequently referred to as canker A2,4,15). Alcaraz1) found some differences in biochemical, cultural and pathological properties between the causal organisms of canker B from Argentina and canker A from Brazil. In Brazil, Namekata 12, 11) reported that a strain of X. citri isolated from Mexican lime (Citrus aurantifolia (Christm.) Swing. ) was different from the strains from other citrus in pathological, physiological and serological characteristics. The Mexican * This study was supported in part by research grant No . 2480470f Ministry of Education, Science and Culture, Japan ** Faculty of Agriculture, Shizuoka University , 836 Ohya, Shizuoka 422, Japan静 岡 大 学 農 学 部 *** Instituto Nacional de Tecnologia Agropecuaria , Concordia, Entre Rios, Argentina 330 日本植物病理学会報 第46巻 第3号 昭和55年7月 lime strain was strongly virulent on Mexican lime, weakly virulent on Tahiti lime (C. latifolia Tanaka) and avirulent on other citrus, so he named this strain X. citri f. sp. aurantifolia. These information indicate the necessity for the extensive studies on the strains of X. campestris pv. citri from different parts of the world. The objective of this study was to compare the pathological, physiological, biochemical and serological properties as well as phage susceptibility of the strains of canker B organism from Argentina with those of the strains of canker A organism from Japan. Materials and Methods Bacteria. The cultures used in the study are shown in Table 1. These bacteria were grown on potato-sucrose agar stab (potato 200g, sucrose 10g, agar 15g, distilled water 1,000ml, pH 6.8), and maintained at 4C after being layered with liquid paraffin. Table 1. History of the cultures used in the study a) Isolated by M. A. Messina in Argentina b) Culture No. 22 was isolated by M. A. Messina in Argentina. The rest were isolated by M. Goto in Japan c) Code numbers in INTA. Concordia, Argentina d) Code numbers in the Laboratory of Plant Pathology. Shizuoka University. Japan e) Citrus sp. f) C. reticulata Blanc g) Poncirus trifoliata Raf. h) Goto et al. (8) Ann. Phytopath. Soc. Japan 46 (3). July, 1980 331 noculation tests. Inoculations were made in a greenhouse on leaves of two-year-old seedlings of Natsudaidai (Citrus natsudaidai Hayata), three-year-old trees of lemon (C. limon Burm. cv. Lisbon), Unshu (C. unshu Marc. cv. Sugiyama), Yuzu (C. junos Sieb. et Zucc.), and navel orange (C. sinensis Osbeck). Leaves were inoculated by spray and pin-prick methods with bacteria suspended in water at a concentration of approximately 108 cells/ml; the suspensions were prepared from 24-hr-old potato-sucrose agar slant cultures. Inoculated leaves were maintained humid overnight in polyethylene bags. Physiologicai and biochemical characteristics. Growth rates and temperature relations were analysed with a temperature-gradient biophotorecorder (Toyo Kagaku Sangyo Co. Ltd., Tokyo). For determination of minimum and maximum growth temperatures, turbidity of culture medium was continuously recorded for 5 days. The basal medium used for carbohydrate utilization tests consisted of: (NH4)2•@ HPO4 1.0g, KCl 0.2g, MgSO4•E7H2O 0.2g, carbon source 10g, bromthymol blue (0.4% solution) 4ml, agar 15g, distilled water 1,000ml, pH 6.8. The method described by Young19) was used for production of reducing substances from sucrose and Dye's method5) for utilization of asparagine. Paton's pectate gel medium17) was used for pectolytic activities. NaCl tolerance was tested in peptone sucrose broth (peptone 10g, sucrose 10g, distilled water 1,000ml, pH 6.8). The methods described by Cowan and Steel3) were followed in testing for the following characters: oxidation-fermentation tests, gelatin liquefaction (method 1), hydrolysis of arginine (method 2), aesculin, Tween 80, starch and casein, gluconate test (method 1), reduction of nitrate (method 1), production of urease (method 1), tyrosinase and lecithinase, production of hydrogen sulphide (method 1), methyl red test, acetoin production (Barritt method), litmus milk reaction and decarboxylases (MƒÓller's method). All tests were conducted at 28 C. Bacteriophage. Phage Cp 1 and Cp 2 were obtained from the culture collection of phytopathogenic bacteria in the Laboratory of Plant Pathology, Shizuoka University. Phage Cp 3 was isolated from the soil under Natsudaidai trees in the experimental plot of Shizu oka University with isolate No. 21 as the host bacterium. The Natsudaidai trees were moderately infected only with the canker A organism. The phages were propagated in peptone-sucrose broth, treated with chloroform and lyophilized or preserved at -30C. Antisera. Antisera were prepared by injecting rabbits with whole living cells of the strains No. 42 and No. 43, both of which were canker A organism isolated in Japan. Titres of these antisera were 2,560 to 5,120 in homologous combinations. Agglutination and gel diffusion tests were used to compare the serological relatedness of strains of canker A and B organisms. Whole living cells were used for agglutination tests, whereas sonicated cell suspensions were used for gel diffusion tests.