First Report of Xanthomonas citri pv. citri-A* Causing Citrus Canker on Lime in Cambodia.

Asiatic citrus canker caused by Xanthomonas citri pv. citri (X. citri pv. citri-A) is detrimental to citrus production in tropical and subtropical areas. The bacterium can cause severe infection on many citrus species, initially causing water-soaked leaf lesions that become erumpent and necrotic, often with a chlorotic halo. Severe infection causes premature fruit drop and twig dieback. X. citri pv. citri-A has consequently been subject to eradication and international quarantine regulations. In the 1990s, strains with a host range restricted to Mexican lime (Citrus aurantifolia), but not infecting grapefruit (C. paradisi), were described in different areas of Southwest Asia (4). This variant was designated X. citri pv. citri-A* because of its phenotypic and genetic similarities with X. citri pv. citri. Lime leaves with canker lesions were collected in 2007 from a citrus nursery in Kandal Province, Cambodia and isolations were performed with KC semiselective medium (3). Four Xanthomonas-like strains were further characterized by PCR alongside positive control strain CFBP 2525 from New Zealand. The expected DNA fragment was obtained using primer pair 4/7 (2) from the bacterial strains but not when distilled water was used as a template. Amplified fragment length polymorphism (AFLP) analysis of the four X. citri pv. citri strains from Cambodia and reference strains X. citri pv. citri-A (CFBP 2525, CFBP 2900, LMG 9322), -A* (CFBP 2911, JF90-2, JK2-10, JK143-1, JM47-2), and X. citri pv. aurantifolii (CFBP 2866, CFBP 2868, CFBP 2901) using SacI/MspI and four primer pairs (1) separated the Cambodian strains into two distinct haplotypes (i.e., AFLP fingerprint patterns). One haplotype was closely related (evolutionary genome divergences [EGD] ≤0.006 [1]) to X. citri pv. citri-A strains with a wide host range and the other was most genetically related to a strain of X. citri pv. citri-A* from Thailand (EGD of 0.003). On the basis of AFLP, the Cambodian isolates were not related to X. citri pv. aurantifolii (EGD values of >0.060). When inoculated to Mexican lime and Duncan grapefruit using a detached leaf assay in which inoculum droplets containing ∼1 × 10 CFU were deposited on wounds (4), the strains genetically related to X. citri pv. citri-A produced typical canker lesions on both citrus species a week after inoculation, whereas the Cambodian strains related to X. citri pv. citri-A* by AFLP analysis only produced canker lesions on lime. Our finding extended the geographical distribution of pathotype A*. Identification of both pathotypes from a few samples collected in a nursery suggests a potential for large-scale distribution of these strains within the citrus orchards in Cambodia, where the most important citrus crop is sweet orange, suggesting that the occurrence of X. citri pv. citri-A* is of moderate economic significance, in contrast with X. citri pv. citri-A strains with a wide host range. Diseased citrus nursery plants are a major source of primary inoculum in developing countries. Sanitation of citrus nurseries against citrus canker in Cambodia is a prerequisite for improved management of the disease. References: (1) N. Ah-You et al. Phytopathology 97:1568, 2007. (2) J. S. Hartung et al. Phytopathology 86:95, 1996. (3) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005. (4) C. Vernière et al. Eur. J. Plant Pathol. 104:477, 1998.