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Xanthomonas Oryzae EuropeanBlackwell Publishing Ltd and Mediterranean Plant Protection Organization PM 7/80 (1) Organisation Européenne et Méditerranéenne pour la Protection des Plantes Diagnostics Diagnostic Xanthomonas oryzae Specific scope Specific approval and amendment This standard describes a diagnostic protocol for Xanthomonas Approved in 2007-09. oryzae pathovars oryzae and oryzicola. in the United States and India (Jones et al., 1989; Gnanamanickam Introduction et al., 1993). The species Xanthomonas oryzae includes two pathovars, Bacterial leaf streak of rice (BLS) is caused by X. oryzae pv. namely, oryzae and oryzicola (Swings et al., 1990). These oryzicola. The disease is present in Tropical Asia, West Africa bacterial pathogens are closely related organisms and were and Australia (OEPP/EPPO, 2006b). X. oryzae pv. oryzicola earlier named as pathovars of Xanthomonas campestris. Rice is has reached epidemic proportions in recent years in China. the main host for both pathogens, which are seed-borne and Other hosts affected by the pathogen are: Oryza spp., Leersia seed-transmitted. spp., Leptochloa filiformis, Paspalum orbiculare, Zizania Bacterial leaf blight of rice (BLB) was first reported in aquatica, Z. palustris and Zoysia japonica. No races have been Fukuoka Prefecture, Japan, during 1884 in rice affected by X. recorded, however, differences in virulence of strains have been oryzae pv. oryzae. This disease is considered one of the most observed in many countries (Vera Cruz et al., 1984; Adhikari & serious rice diseases worldwide although it has declined in Mew, 1985; Saddler, 2002b). incidence in Japan since the mid 1970s. Nevertheless it is still Further information on the biology and ecology of the prevalent worldwide. The disease was reported in South East species can be found in the EPPO data sheet on X. oryzae Asia in the early 1960s, where it is currently widespread, and it (OEPP/EPPO, 1997) still affects the rice crop in its severe form (Goto, 1992). It has also been reported in several African countries, in Australia, Identity North America (Lousiana and Texas, US), Central and South America (OEPP/EPPO, 2006a) but it is only of economic Taxonomic position: Kingdom: Bacteria; Phylum: Proteobacteria; importance in Asia. Other reported hosts include wild or minor Class: Gammaproteobacteria; Order: Xanthomonodales; Family: cultivated Poaceae: Brachiaria mutica, Cenchrus ciliaris, Xanthomonodaceae; Genus: Xanthomonas (Dowson, 1939) Cyperus difformis, C. rotundus, Cynodon dactylon, Echinochloa Name: Xanthomonas oryzae pv. oryzae (Ishiyama 1922) crus-galli, Leersia spp. (Leersia hexandra, L. oryzoides), Swings et al. (1990) Leptochloa chinensis, Oryza spp., Panicum maximum, Paspalum Synonyms: Xanthomonas campestris pv. oryzae (Ishiyama scrobiculatum, Zizania aquatica, Z. latifolia, Z. palustris, 1922) Dye 1978; Xanthomonas oryzae (Ishiyama, 1921) and Zoysia japonica (Li et al., 1985; Bradbury, 1986; Valluva- Dowson 1943; Other synonyms are Xanthomonas itoana, paridasan & Mariappan, 1989; EPPO/CABI, 1997; Saddler, Xanthomonas kresek, Xanthomonas translucens f. sp. oryzae 2002a). Cyperaceae (sedges) that are naturally infected include EPPO computer code: XANTOR Cyperus difformis and C. rotundus. Variation in virulence Phytosanitary categorization: EPPO A1 list no. 2, EU annex has been observed between isolates, and many races have been II/A1 as Xanthomonas campestris pv. oryzae. described; however, the different races have not been clearly defined with specific reactions being assigned to each variety Xanthomonas oryzae pv. oryzicola (Mew, 1987). The diversity of the pathogen has been analysed based on virulence and PCR-based DNA fingerprinting Name: Xanthomonas oryzae pv. oryzicola (Fang et al., 1956) (George et al., 1994). Low virulence strains have been reported Swings et al. (1990) © 2007 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 37, 543–553 543 544 Diagnostics Fig. 1 Symptoms of X. oryzae pv. oryzae (bacterial leaf blight). (a) Rice field plants with X. oryzae pv. oryzae symptoms; (b) X. oryzae pv. oryzae symptoms in rice nursery beds; (c) Rice seedlings showing X. oryzae pv. oryzae symptoms; (d) Wilting of transplanted seedlings; (e) Kresek symptoms in field plants; (f) Progressive development of bacterial leaf blight in rice plants grown from infected seed. Synonyms: Xanthomonas campestris pv. oryzicola (Fang They start as small water-soaked stripes from the tips where et al., 1956) Dye 1978; Xanthomonas oryzicola (Fang et al., water pores are found and rapidly enlarge in length and width, 1956) Dowson 1943; Xanthomonas translucens f. sp. oryzicola forming a yellow lesion with a wavy margin along the lead (Fang et al., 1956) Bradbury 1971 edges. Later on, diseased areas turn white to grey. These lesions EPPO computer code: XANTTO can develop on one or both sides of the leaf and occasionally Phytosanitary categorization: EPPO A1 list no. 3, EU annex along the midribs, and leaf blight symptoms generally occur II/A1 as Xanthomonas campestris pv. oryzicola. from maximum tillering stage and onwards. In young lesions, drops of bacterial ooze can be observed early in the morning. On panicles the disease causes grey to light brown lesions on Detection glumes that result in infertility and low quality of the grains. Kresek is the result of systemic infection that is common in the Disease symptoms tropics in young plants and during the tillering stage of susceptible X. oryzae pv. oryzae and X. oryzae pv. oryzicola can be clearly cultivars. Leaves of infected plants wilt, roll up, turn grey-green distinguished by symptoms, which reflect the differences in and whither, and entire plants finally die. Surviving plants look their modes of infection. stunted and yellowish. Yellow or pale yellow leaves are due to systemic infections that appear at tillering stage; the youngest Bacterial leaf blight of rice (BLB) leaves become uniformly pale yellow or show a broad yellow X. oryzae pv. oryzae enters either through wounds or hydathodes, stripe, and bacteria are found in the internodes and crowns of multiplies in the epitheme and moves to the xylem vessels affected stems, but not in the leaf itself (Ou, 1985; Goto, 1992). where active multiplication results in blight on the leaves. The symptoms of the disease include leaf blight, wilting (kresek), Bacterial leaf streak of rice (BLS) (Fig. 1) and pale yellow leaves. Leaf blight is characterized by X. oryzae pv. oryzicola is a foliar disease that appears at any wavy elongated lesions, which develop along the leaf margins. growth stage of the host. Cells of X. oryzae pv. oryzicola enter © 2007 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 37, 543–553 Xanthomonas oryzae 545 Fig. 1 Continued be observed as tiny yellow beads. The narrow, long, translucent lesions may coalesce, forming large patches, and severely affected fields appear burnt. It is at this stage that leaves wither, turn brown and eventually die, and the disease can be difficult to distinguish from bacterial leaf blight (Ou, 1985). Isolation Symptomatic samples are processed individually or in small groups. Precautions are made to avoid cross contaminations when collecting the samples and during the extraction process. The samples should be processed as soon as possible after collection and conserved at 4–8°C until use. Freshly prepared sample extracts are necessary for a successful isolation. Several Fig. 2 Symptoms of bacterial leaf streak in rice plants. procedures have been used for the isolation of X. oryzae pv. oryzae from symptomatic and asymptomatic plant parts, including seeds. Most of the procedures described for the through the stomata and multiply in the parenchyma tissues of isolation of X. oryzae pv. oryzae from rice plants, can also be the leaves. X. oryzae pv. oryzicola infects mainly the parenchyma applied for the isolation of X. oryzae pv. oryzicola. However, it of the cells of the leaves, but is not systemic. Initial symptoms is known that both bacteria grow slowly on the isolating media are small water-soaked, transparent interveinal streaks (Fig. 2), and can be overgrown by fast growing contaminants. Often these which may elongate and darken. The transparent streaks contaminants are of yellow colour (e.g. Pantoea agglomerans differentiate leaf streak lesions from those of X. oryzae pv. and Xanthomonas-like saprophytic bacteria) thus making oryzae that are opaque against the light. Bacterial exudates can difficult the observations of colonies of the target organisms and © 2007 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 37, 543–553 546 Diagnostics are often visually undistinguishable in colony morphology, growth, and colour from strains of both pathogens. Culturing may fail from advanced stages of infection due to competition or being overgrown by saprophytic organisms and also from seeds with low levels of inoculum or under bad conditions of storage. If disease symptoms are typical but isolation is negative, the isolation step should be repeated. Direct plating from symptomatic and asymptomatic leaves collected from the field Bacterial leaf blight in temperate regions can usually be observed during the latter part of the seed bed stage (Ou, 1985). The bacteria can be detected from the upper part of infected leaves before symptoms appear (Goto, 1965; Misukami & Wakimoto, 1969; Sakthivel et al., 2001). Tabei (1967) reported that symptomless rice seedlings may carry the pest. The isolation of Xanthomonas from symptomatic material is preferable and can be performed using Peptone sucrose agar (PSA), Nutrient Broth Yeast Extract agar medium
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