First Report of Sequence Type 1, Pathotype A Xanthomonas citri pv. citri from Lime and Lemon Fruit Originating from Bangladesh.

Abstract

Asiatic canker caused by Xanthomonas citri pv. citri, a quarantine pest in several countries (including the European Union), strongly impacts both national citrus markets in tropical and subtropical areas and international trade. This bacterium induces erumpent, callus-like lesions often with a water-soaked margin in a wide range of citrus species causing premature fruit drop and twig dieback. Long distance dispersal is mainly through infected propagative material and the role of fruit is still debated. During inspection of imported limes (C. aurantifolia) and lemons (C. limon) from Bangladesh from 2006 to 2009, canker-like infected fruits were intercepted by the UK plant health service. Typical corky lesions were surface sterilized and comminuted in 0.1% peptone solution. Suspensions were plated onto nutrient dextrose (ND) and yeast dextrose chalk (YDC) plates for bacterial isolation. After incubation for 3 to 7 days at 25°C, typical Xanthomonas-like yellow colonies were purified for identification. Identification of 18 isolates as Xanthomonas was carried out initially by fatty acid methyl ester (FAME) analysis. Identification at the species level (X. citri) was completed by sequencing of the gyrase B gene (4). PCR (3) was used to confirm the identity of these isolates using X. citri pv. citri CFBP 2525 as the positive control and distilled water as the negative control. The expected DNA fragment was only obtained from all of the bacterial isolates using primer pair 4/7 (3). Multilocus sequence analysis (MLSA) of four housekeeping genes (atpD, dnaK, efp, and gyrB) identified isolates from Bangladesh as two sequence types of X. citri pv. citri, ST1 (n = 5; GenBank Accession Nos. FJ376118, FJ376168, FJ376216, and FJ376251) and ST2 (n = 13; EU333904, EU333907, EU333910, and FJ376357), but not as any other xanthomonad pathogenic to citrus (2). Amplified fragment length polymorphism (AFLP) analysis of all X. citri pv. citri isolates from Bangladesh and additional reference isolates from pathotype A, A*, Aw and X. citri pv. aurantifolii (2) using Sac I/Msp I and four primer pairs (unlabelled MspI + 1 (A, C, T, or G) primers and 5'-labeled - SacI + C primer for the selective amplification step) confirmed identification as X. citri pv. citri. All five ST1 isolates grouped as a single cluster by AFLP, although not strongly supported by bootstrap analysis. Evolutionary genome divergences (EGD) computed from AFLP data ranged from 0.0000 to 0.0097 (median EGD 0.0055) suggested a relatively wide diversity within isolates originating from Bangladesh (median EGD from a worldwide pathotype A collection [n = 73] 0.0028) (2). When inoculated to Mexican lime SRA 140 and grapefruit cv. Duncan using a detached leaf assay (2), all the Bangladesh isolates produced typical extensive canker lesions on both species whereas the negative control (10 mM Tris buffer pH 7.2) did not, and Koch's postulates were fulfilled. To our knowledge, this is the first report of pathotype A assigned to ST1 by MLSA. All strains previously assigned to ST1 displayed a narrow host range (pathotype A*) (2). Our results further identify the Indian subcontinent as an area of relatively wide genetic diversity of X. citri pv. citri (1).