Pathotype Identification of Xanthomonas citri pv. citri Strains Causing Citrus Canker in Vietnam.

Asiatic citrus canker caused by Xanthomonas citri pv. citri is a bacterial disease of major economic importance in tropical and subtropical citrus-producing areas. It probably originated in Asia (2). X. citri pv. citri induces erumpent, callus-like lesions with a water-soaked margin on aerial organs of the plants. Severe attacks cause premature fruit drop and twig dieback. This pathogen has consequently been subjected to international quarantine regulation and eradication efforts. Two pathogenic variants of X. citri pv. citri can be separated by their host range. X. citri pv. citri pathotype A strains cause severe infection worldwide in a wide range of citrus species; grapefruit (Citrus paradisi) is particularly susceptible. More recently, another group of strains from different areas of West Asia has been designated as X. citri pv. citri pathotype A* (4). These A* strains are genetically related to X. citri pv. citri, but their host range is primarily restricted to Mexican lime (C. aurantifolia) and they do not infect grapefruit. Strains similar in host range were later reported in Florida, Thailand, and Cambodia (2). In this study, we investigated the distribution of X. citri pv. citri pathotypes in Southeast Asia. A large survey on citrus was conducted in 14 provinces in the north (Ha Noi, Hung Yen, Nghe Han, Ha Ting, and Phu Tho) and south (Can Tho, Long An, Dong Nai, Tien Giang, Vinh Long, Ben Tre, Dong Thap, Vung Tau, and Lam Dong) of Vietnam. We collected 557 X. citri pv. citri isolates, after cultivation on KC semiselective medium (3), from citrus species, including 60 strains from Mexican lime in eight provinces. Ligation mediated (IS-LM)-PCR analysis using primers targeting three insertion sequences (1) was done on all Vietnamese strains and on additional reference strains of X. citri pv. citri-A, -A*, and X. citri pv. aurantifolii. IS-LM-PCR indicated that all Vietnamese isolates were pathotype A and did not include any with a restricted host range (X. citri pv. citri-A* and X. citri pv. aurantifolii). Amplified fragment length polymorphism (AFLP) analysis was carried out on a subset of 84 X. citri pv. citri strains, including 22 strains from Mexican lime from seven provinces. AFLP was carried out using SacI/MspI and four primer pairs (unlabeled MspI +1 [A, C, T or G] primers and 5'-labeled - SacI + C primer for the selective amplification step) (2) and the data confirmed that all Vietnamese X. citri pv. citri strains were genetically related to pathotype A strains. Mexican lime and Duncan grapefruit or pineapple sweet orange leaves were inoculated with 25 strains from lime (representative of the genetic diversity) using a detached leaf assay (3) and they produced typical canker lesions on both host species. In spite of the presence of pathotype A* strains in neighboring countries (2), no strains genetically or pathogenically related to this pathotype were identified in this collection. A survey of commercial Mexican lime orchards, especially in Vietnamese provinces bordering Cambodia, should be undertaken to detect and eradicate A* strains because these are known to strongly impact lime production in other parts of Asia (e.g., Thailand). References: (1) L. Bui Thi Ngoc et al. Appl. Environ. Microbiol. 75:1173, 2009. (2) L. Bui Thi Ngoc et al. FEMS Microbiol. Lett. 288:33, 2008. (3) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005. (4) C. Vernière et al. Eur. J. Plant Pathol. 104:477, 1998.

Development of 14 minisatellite markers for the citrus canker bacterium, Xanthomonas citri pv. citri.

We screened the genome of Xanthomonas citri pv. citri strain 306 for tandem repeats. A multiplex polymerase chain reaction protocol was used to assess the genetic diversity of 239 strains of X. citri pv. citri from Asia. The total number of alleles per locus ranged from three to 20. Using pooled data sets, 223 different haplotypes were identified. Successful amplifications were obtained at most loci for seven other X. citri pathovars. This typing scheme is expected to be useful at different spatial scales for population studies of pathovars of X. citri, several of which cause plant diseases of economic importance.

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 × 106 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.

First Report of Xanthomonas citri pv. citri Causing Citrus Canker in Mali.

Citrus canker, caused by Xanthomonas citri pv. citri, is a bacterial disease of economic importance in tropical and subtropical citrus-producing areas. X. citri pv. citri can cause severe infection in a wide range of citrus species and induces erumpent, callus-like lesions with water-soaked margins leading to premature fruit drop and twig dieback. It has consequently been subjected to eradication efforts and international regulations. Citrus canker occurs in Asia, South America, the United States, parts of Oceania, and some islands off the African continent (Comoros, Mauritius, Reunion, and Seychelles islands). It was described on the African continent, but in some cases, diagnosis errors might have occurred. The only well-documented outbreak occurred in South Africa where it was eradicated at the beginning of the twentieth century. In 2004, citrus canker symptoms on limes, sweet oranges, tangerines, and sour oranges were reported from different orchards around Bamako and in the Koulikoro Province of Mali. Isolations were performed on KC semiselective medium (2). PCR was used to check the identity of the pathogen, testing 21 Xanthomonas-like strains collected from citrus in the epidemic area. X. citri pv. citri strain CFBP 2525 from New Zealand was also used as the positive control, and the expected DNA fragment was obtained from all the isolates using primer pair 4/7 (1), whereas no fragment was observed for negative controls (distilled water as the template). Amplified fragment length polymorphism (AFLP) analysis of these X. citri pv. citri strains from Mali and other reference strains from X. citri pv. citri-A, -A*, and X. axonopodis pv. aurantifolii (3), using SacI/MspI and four primer pairs (unlabeled MspI + 1 [A, C, T, or G] primers and 5'-labeled-SacI + C primer for the selective amplification step), showed that the strains were closely related to X. citri pv. citri-A strains with a wide host range. On the basis of AFLP, the Mali strains were not closely related to X. axonopodis pv. aurantifolii. One week after inoculation, Duncan grapefruit and alemow citrus leaves inoculated with all strains from Mali by a detached leaf assay (3) developed erumpent, callus-like tissue at wound sites, similar to the reaction produced by X. citri pv. citri strain CFBP 2525 (positive control). A survey conducted in 2006 in nine orchards revealed disease incidences of 50, 15, 24, and 25% for lime, sweet orange, sour orange, and tangor groves, respectively. As of this report, citrus canker has spread to new citrus orchards and this might be due to the propagation and dissemination of infected material from small nurseries. To our knowledge, this represents the first outbreak of citrus canker in West Africa. Spread of the pathogen in Mali and neighboring countries should be monitored and a drastic surveillance of citrus nurseries in the region should be performed. References: (1) J. S. Hartung et al. Phytopathology 86:95, 1996. (2) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005. (3) C. Vernière et al. Eur. J. Plant Pathol. 104:477, 1998.

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

Asiatic citrus canker, caused by Xanthomonas axonopodis pv. citri (Xac-A), is a bacterial disease of economic importance in tropical and subtropical citrus-producing areas. Xac-A can cause severe infection in a wide range of citrus species and induces erumpent, callus-like lesions with a water-soaked margin. Severe attacks cause premature fruit drop and twig dieback. It has consequently been submitted to eradication efforts and international regulations. Recently, a group of strains with a host range restricted to Mexican lime (Citrus aurantifolia), but not infecting grapefruit (C. paradisi) known to be very susceptible to Xac-A, was described in different areas of southwest Asia, including Saudi Arabia, Oman, Iran, and India (3). Phenotypic and genetic similarities with Xac-A designated it as a variant called X. axonopodis pv. citri-A*. A variant with a similar restricted host range, designated X. axonopodis pv. citri-Aw, was also detected in Florida and likely originated from India (2). Amplified fragment length polymorphism (AFLP) analysis of 26 isolates from Thailand and additional reference isolates from Xac-A, -A*, -Aw, and X. axonopodis pv. aurantifolii (2,3) using SacI/MspI and four primer pairs (unlabeled MspI + 1 [A, C, T, or G] primers and 5'-labeled SacI + C primer for the selective amplification step), separated the Thai isolates into two distinct groups. A group composed of 20 isolates from different citrus species, including five isolates from lime, was closely related to strains with a large host range and previously identified as Xac-A. A second group, solely composed of six isolates from lime, was genetically related to Xac-A*. All Thai isolates were collected before 1991. Isolates genetically closed to Xac-A* originated from the central and northern provinces, whereas isolates related to Xac-A originated from all sampled provinces. On the basis of AFLP, no Thai isolate was related to X. axonopodis pv. aurantifolii. A specific X. axonopodis pv. citri nested-PCR assay (1) produced the expected fragments for all Thai isolates. Mexican lime leaves inoculated with Thai isolates of Xac-A and Xac-A* using a detached leaf assay (3) showed typical canker symptoms 1 week after inoculation. When inoculated to grapefruit or sweet orange, the Thai isolates genetically related to Xac-A* by AFLP analysis did not induce any canker symptoms, while isolates related to Xac-A produced canker symptoms on these two citrus species. In Thailand, Xac-A* induced severe symptoms on lime trees, including extensive defoliation and numerous twig cankers that often developed as diebacks. The Xac-A* variant appears epidemiologically important on lime, consistent with previous reports from southwest Asia. The detection of Xac-A* in Thailand makes it necessary to evaluate its geographic distribution in southeast Asia. References: (1) J. S. Hartung et al. Phytopathology 86:95, 1996. (2) X. A. Sun et al. Plant Dis. 88:1179, 2004. (3) C. Vernière et al. Eur. J. Plant Pathol. 104:477, 1998.