Effect of probiotics on the maintenance of intestinal homeostasis after chemotherapy: systematic review and meta-analysis of pre-clinical studies.

Gastrointestinal mucositis (GIM) is an inflammation caused by antitumor therapy, especially after chemotherapy and radiotherapy. Currently in the clinical practice, only palliative measures are taken to treat GIM, representing the main clinical limitation in the management of this condition. Several studies have highlighted the potential benefits of probiotics for the management of GIM, but the actual role of these microorganisms in the maintenance of intestinal homeostasis remains elusive. In this context, here we aimed to realise a systematic review with meta-analysis to evaluate the effect of probiotics on experimental GIM. The meta-analysis showed that probiotics significantly suppressed the body weight loss related to GIM in rodents (95% confidence interval (CI): -2.67 to -0.70; I2=98%, P<0.00). Subgroup analysis showed that pre-treatment (≥7 days before chemotherapy) (95% CI: -8.84 to -0.17; I2=98%, P<0.04) with a high dose of probiotics (≥ 109 cfu/day) (95% CI: -2.58 to -0.28; I2=98%, P<0.00) comprising two or more microorganism species (95% CI: -6.49 to -0.28; I2=96%, P=0.03) remedied GIM more effectively. It was also revealed that fungi (specifically Saccharomyces boullardii) are more effective in remedying GIM than bacteria (P=0.03 vs P<0.00), and the mouse models are more receptive than rats to the enteroprotective effects of probiotics (95% CI: -4.76, -0.69; I2=97%, P=0.01). Qualitative analyses highlighted that probiotics suppress GIM through several mechanisms; they reduce the intestinal permeability, suppress the pro-inflammatory cytokine production while stimulating production and secretion of anti-inflammatory cytokines, inhibit the signalling pathways coupled to inflammation and apoptosis, accelerate the proliferation of enterocytes, reduce the levels of reactive oxygen species, and help maintain the protective mucus layer. In conclusion, this review highlights the therapeutic benefits of probiotics in experimental GIM.

First Report of Xanthomonas citri pv. citri-A Causing Asiatic Citrus Canker in Mayotte.

Asiatic citrus canker, caused by Xanthomonas citri pv. citri, is a bacterial disease of major economic importance in tropical and subtropical citrus-producing areas. X. citri pv. citri pathotype A can cause severe infection in a wide range of citrus species and induces erumpent, callus-like lesions with water-soaked margins evolving to corky cankers and leading to premature fruit and leaf drop and twig dieback on susceptible/very susceptible cultivars. A chlorotic halo is typically visible around canker lesions on leaves and young fruit, but not on mature fruit and twigs. This quarantine organism can strongly impact both national and international citrus markets. Long distance dispersal is mainly through infected propagative material. Asiatic citrus canker occurs on most islands in the Southwest Indian Ocean region (Comoros, Mauritius, Reunion, Rodrigues, and Seychelles islands), but was not yet reported in Mayotte (EPPO-PQR available at http://www.eppo.int ). In May 2012, typical canker-like symptoms were observed on sweet orange (Citrus sinensis) groves on Mtsamboro islet and soon after on the main island of Mayotte, mostly on sweet oranges, but also on Tahiti limes (C. latifolia) and mandarins (C. reticulata). Eighty-one Xanthomonas-like strains were isolated using KC semi-selective medium (4) from disease samples collected from both commercial groves and nurseries on different Citrus species located all over the island. Sixteen Xanthomonas-like isolates were tentatively identified as X. citri pv. citri based on a specific PCR assay with 4/7 primers (3). All strains but the negative control, sterile water, produced an amplicon of the expected size similar to X. citri pv. citri strain IAPAR 306 used as positive control. Multilocus sequence analysis targeting six housekeeping genes (atpD, dnaK, efp, gltA, gyrB, and lepA) (1,2) fully identified three strains from Mayotte (LJ225-3, LJ228-1, and LJ229-11) as X. citri pv. citri (and not other xanthomonad pathovars pathogenic to citrus or host range-restricted pathotypes of pathovar citri), and more specifically as sequence type ST2 composed of pathotype A strains of X. citri pv. citri (2) (including all strains from the Southwest Indian Ocean region). Eight strains were inoculated by a detached leaf assay (2) to Mexican lime SRA 140 (C. aurantifolia), Tahiti lime SRA 58, sweet orange cv. Washington Navel, alemow SRA 779 (C. macrophylla), and tangor cv. Ortanique (C. reticulata × C. sinensis) and developed typical erumpent, callus-like tissue at wound sites for all Citrus species, fulfilling Koch's postulates. Xanthomonas-like yellow colonies were reisolated from symptoms produced by the eight strains inoculated on Mexican lime. Boiled bacterial suspensions were assayed by PCR with 4/7 primers (3) and produced the expected 468-bp amplicon in contrast with the negative control (sterile water). No lesions developed on the negative control consisting of inoculations by 10 mM tris buffer (pH 7.2). Citrus canker-free nurseries and grove sanitation should be implemented for decreasing the prevalence of Asiatic canker in this island territory. References: (1) N. F. Almeida et al. Phytopathology 100:208, 2010. (2) L. Bui Thi Ngoc et al. Int. J. Syst. Evol. Microbiol. 60:515, 2010. (3) J. S. Hartung et al. Phytopathology 86:95, 1996. (4) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005.

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

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). References: (1) L. Bui Thi Ngoc et al. Appl. Environ. Microbiol. 75:1173, 2009. (2) L. Bui Thi Ngoc et al. Int. J. Syst. Evol. Microbiol. 60:515, 2010. (3) J. S. Hartung et al. Phytopathology 86:95, 1996. (4) N. Parkinson et al. Int. J. Syst. Evol. Microbiol. 57:2881, 2007.

First Report in Mali of Xanthomonas citri pv. mangiferaeindicae Causing Mango Bacterial Canker on Mangifera indica.

Bacterial canker (or black spot) of mango caused by Xanthomonas citri pv. mangiferaeindicae is an important disease in tropical and subtropical areas (1). X. citri pv. mangiferaeindicae can cause severe infection in a wide range of mango cultivars and induces raised, angular, black leaf lesions, sometimes with a chlorotic halo. Severe leaf infection may result in abscission. Fruit symptoms appear as small, water-soaked spots on the lenticels that later become star shaped, erumpent, and exude an infectious gum. Often, a "tear stain" infection pattern is observed on the fruit. Severe fruit infections cause premature drop. Twig cankers are potential sources of inoculum and weaken branch resistance to winds. Yield loss up to 85% has been reported at grove scale for susceptible cultivars (1). Suspected leaf lesions of bacterial canker were collected in July 2010 from mango trees in four, six, and three localities of the Koulikoro, Sikasso, and Bougouni provinces of Mali, respectively (i.e., the major mango-growing areas in this country). Nonpigmented Xanthomonas-like colonies were isolated on KC semiselective medium (3). Twenty-two strains from Mali were identified as X. citri pv. mangiferaeindicae based on IS1595-ligation-mediated PCR (4) and they produced fingerprints fully identical to that of strains isolated from Ghana and Burkina Faso. Five Malian strains (LH409, LH410, LH414, LH415-3, and LH418) were compared by multilocus sequence analysis (MLSA) to the type strain of X. citri and the pathotype strain of several X. citri pathovars, including pvs. anacardii and mangiferaeindicae. This assay targeted the atpD, dnaK, efp, and gyrB genes, as described previously (2). Nucleotide sequences were 100% identical to those of the pathotype strain of X. citri pv. mangiferaeindicae whatever the gene assayed, but differed from any other assayed X. citri pathovar. Leaves of mango cv. Maison Rouge from the youngest vegetative flush were infiltrated (10 inoculation sites per leaf for three replicate leaves on different plants per bacterial strain) with the same five strains from Mali. Bacterial suspensions (~1 × 105 CFU/ml) were prepared in 10 mM Tris buffer (pH 7.2) from 16-h-old cultures on YPGA (7 g of yeast, 7 g of peptone, 7 g of glucose, and 18 g of agar/liter, pH 7.2). The negative control treatment consisted of three leaves infiltrated with sterile Tris buffer (10 sites per leaf). Plants were incubated in a growth chamber at 30 ± 1°C by day and 26 ± 1°C by night (12-h/12-h day/night cycle) at 80 ± 5% relative humidity. All leaves inoculated with the Malian strains showed typical symptoms of bacterial canker a week after inoculation. No lesions were recorded from the negative controls. One month after inoculation, mean X. citri pv. mangiferaeindicae population sizes ranging from 5 × 106 to 1 × 107 CFU/lesion were recovered from leaf lesions, typical of a compatible interaction (1). To our knowledge, this is the first report of the disease in Mali. Investigations from local growers suggest that the disease may have been present for some years in Mali but likely less than a decade. A high disease incidence and severity were observed, suggesting the suitability of environmental conditions in this region for the development of mango bacterial canker. References: (1) N. Ah-You et al. Phytopathology 97:1568, 2007. (2) L. Bui Thi Ngoc et al. Int. J. Syst. Evol. Microbiol. 60:515, 2010. (3) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005. (4) O. Pruvost et al. Phytopathology 101:887, 2011.

Insertion sequence- and tandem repeat-based genotyping techniques for Xanthomonas citri pv. mangiferaeindicae.

Molecular fingerprinting techniques that have the potential to identify or subtype bacteria at the strain level are needed for improving diagnosis and understanding of the epidemiology of pathogens such as Xanthomonas citri pv. mangiferaeindicae, which causes mango bacterial canker disease. We developed a ligation-mediated polymerase chain reaction targeting the IS1595 insertion sequence as a means to differentiate pv. mangiferaeindicae from the closely related pv. anacardii (responsible for cashew bacterial spot), which has the potential to infect mango but not to cause significant disease. This technique produced weakly polymorphic fingerprints composed of ≈70 amplified fragments per strain for a worldwide collection of X. citri pv. mangiferaeindicae but produced no or very weak amplification for pv. anacardii strains. Together, 12 tandem repeat markers were able to subtype X. citri pv. mangiferaeindicae at the strain level, distinguishing 231 haplotypes from a worldwide collection of 299 strains. Multilocus variable number of tandem repeats analysis (MLVA), IS1595-ligation-mediated polymerase chain reaction, and amplified fragment length polymorphism showed differences in discriminatory power and were congruent in describing the diversity of this strain collection, suggesting low levels of recombination. The potential of the MLVA scheme for molecular epidemiology studies of X. citri pv. mangiferaeindicae is discussed.

First Report in Ghana of Xanthomonas citri pv. mangiferaeindicae Causing Mango Bacterial Canker on Mangifera indica.

Bacterial canker of mango (or bacterial black spot), caused by Xanthomonas citri pv. mangiferaeindicae, is an economically important disease in tropical and subtropical producing areas (1). X. citri pv. mangiferaeindicae can cause severe infection in a wide range of mango cultivars and induces raised, angular, black leaf lesions, sometimes with a chlorotic halo. Several months after infection, leaf lesions dry and turn light brown or ash gray. Severe leaf infection may result in abscission. Fruit symptoms appear as small water-soaked spots on the lenticels. These spots later become star shaped, erumpent, and exude an infectious gum. Often, a "tear stain" infection pattern is observed on the fruit. Severe fruit infections will cause premature fruit drop. Twig cankers are potential sources of inoculum and weaken resistance of branches to wind damage. Leaf lesions with suspected bacterial canker were collected in January 2010 from mango trees cv. Keitt in several blocks at the Integrated Tamale Fruit Company, Ghana. Non-pigmented Xanthomonas-like bacterial colonies were isolated on Kasugamycin-Cephalexin semiselective agar medium (3). On the basis of IS1595-Ligation Mediated-PCR data, 16 strains from Ghana produced identical fingerprints and were identified as X. citri pv. mangiferaeindicae (4). The haplotype corresponding to the Ghanaian strains had not been previously reported. On the basis of multidimensional scaling (4), this haplotype clustered together with a group of strains from multiple origins and the analysis was not informative as an aid for tracing back the outbreak. Five Ghanaian strains (LH2-3, LH2-6, LH2-8, LH2-11, and LH2-15) were compared by multilocus sequence analysis to the type strain of X. citri and the pathotype strain of several X. citri pathovars, including pvs. anacardii and mangiferaeindicae. This assay targeted the atpD, dnaK, efp, and gyrB genes as described previously (2). Nucleotide sequences were 100% identical to those of the pathotype strain of X. citri pv. mangiferaeindicae whatever the gene assayed, but differed from any other assayed X. citri pathovar. Mango cv. Maison Rouge leaves from the youngest vegetative flush were infiltrated (10 inoculation sites per leaf, three replicate plants) using inoculum of each of the same five Ghanaian strains made from suspensions in Tris buffer containing ~1 × 105 CFU/ml. Negative control treatments consisted of leaves infiltrated with sterile Tris buffer. Typical symptoms of bacterial canker were observed for all assayed strains a week after inoculation. No lesions were recorded from the negative control. One month after inoculation, mean X. citri pv. mangiferaeindicae population sizes ranging from 4 × 107 to 1 × 108 CFU/lesion were recovered from leaf lesions, typical of a compatible interaction (1). High disease prevalence was observed in Ghana, indicating the suitability of environmental conditions in this region for the development of mango bacterial canker. The budwood for these blocks was imported from Burkina Faso in 2002 and symptoms were observed in these blocks shortly after establishment. To our knowledge, this is the first report of mango bacterial canker in Western Africa. References: (1) N. Ah-You et al. Phytopathology 97:1568, 2007. (2) L. Bui Thi Ngoc et al. Int. J. Syst. Evol. Microbiol. 60:515, 2010. (3) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005. (4) O. Pruvost et al. Phytopathology. Online publication. DOI:10.1094/PHYTO-11-10-0304, 2011.

First Report in Burkina Faso of Xanthomonas citri pv. mangiferaeindicae Causing Bacterial Canker on Mangifera indica.

Bacterial canker of mango (or bacterial black spot) caused by Xanthomonas citri pv. mangiferaeindicae, is an economically important disease in tropical and subtropical areas (1). X. citri pv. mangiferaeindicae can cause severe infection on a wide range of mango cultivars and induces raised, angular, black leaf lesions, sometimes with a chlorotic halo. Fruit symptoms are black, star shaped, erumpent, and exude an infectious gum. A survey was conducted in Burkina Faso in May 2010 because budwood putatively associated with an outbreak of bacterial canker in Ghana had originated from Burkina Faso (3). Leaves and twigs with suspected bacterial canker lesions were collected from mango trees of the cvs. Amélie, Brooks, and Kent and from seedlings at five localities in Comoe and Houet provinces. Severe infections were observed on the sampled trees in Burkina Faso and leaf symptoms were typical of bacterial canker. Leaves were surface sterilized for 15 to 30 s with 70% ethanol, and nonpigmented, Xanthomonas-like bacterial colonies were isolated on KC semiselective agar medium (1). On the basis of an IS1595-ligation mediated PCR assay, 18 strains from Burkina Faso produced identical fingerprints and were identified as X. citri pv. mangiferaeindicae (4). The haplotype for strains from Burkina Faso was identical to that reported from Ghana (3). Three strains from Burkina Faso (LH127-2, LH130-1, and LH131-1) were compared by multilocus sequence analysis (MLSA) with the type strain of X. citri and the pathotype strain of several X. citri pathovars, including pvs. anacardii and mangiferaeindicae, targeting the atpD, dnaK, efp, and gyrB genes (2). Nucleotide sequences were 100% identical to those of the pathotype strain of X. citri pv. mangiferaeindicae, regardless of the gene assayed, but differed from any other X. citri pathovar assayed. Leaves of mango cv. Maison Rouge, taken from the youngest vegetative flush, were infiltrated (10 inoculation sites per leaf for three replicate leaves on different plants per bacterial strain) with the same three strains from Burkina Faso. Bacterial suspensions (approximately 1 × 105 CFU/ml) were prepared in 10 mM Tris buffer (pH 7.2) from 16-h-old solid cultures on YPG agar (7 g of yeast, 7 g of peptone, 7 g of glucose, and 18 g of agar per liter, pH 7.2). The negative control treatment consisted of three leaves infiltrated with sterile Tris buffer (10 sites per leaf). Plants were incubated in a growth chamber at 30 ± 1°C by day and 26 ± 1°C by night (12-h/12-h day/night cycle) at 80 ± 5% relative humidity. Typical symptoms of bacterial canker were observed for all assayed strains 1 week after inoculation; no symptoms were observed from negative control leaves. One month after inoculation, mean X. citri pv. mangiferaeindicae populations ranging from 2 × 107 to 8 × 107 CFU/leaf lesion were recovered, which was typical of a compatible interaction (1). The origin of inoculum associated with the bacterial canker outbreak in Burkina Faso is unknown. This report documents severe infections in Burkina Faso (including premature fruit drop due to severe fruit infections) and confirms the presence of bacterial canker in western Africa. A more extensive survey for the disease should be conducted in this region. References: (1) N. Ah-You et al. Phytopathology 97:1568, 2007. (2) L. Bui Thi Ngoc et al. Int. J. Syst. Evol. Microbiol. 60:515, 2010. (3) O. Pruvost et al. Plant Dis. 95:774, 2011. (4) O. Pruvost et al. Phytopathology 101:887, 2011.

First Report of Xanthomonas citri pv. citri Pathotype A Causing Asiatic Citrus Canker on Grapefruit and Mexican Lime in Senegal.

In February 2010, grapefruit (Citrus paradisi) and Mexican lime (C. aurantifolia) leaves with erumpent callus-like lesions were collected in Senegal in the Sebikotane area between Dakar and Thies. Similar symptoms have been observed by local farmers since 2008, and lesions were morphologically similar to those of citrus canker caused by Xanthomonas citri pv. citri (Asiatic canker) and X. citri pv. aurantifolii (South American canker). Lesions were primarily reported from grapefruit (cv. Shambar), which is the most frequent citrus species produced in this area, and Mexican lime, which is also commonly grown. Both species are very susceptible to X. citri pv. citri pathotype A, and Mexican lime is susceptible to X. citri pv. citri pathotype A* and X. citri pv. aurantifolii (4). Fifteen Xanthomonas-like strains were isolated from disease samples using KC semiselective medium (3). PCR with primer pair 4/7 (2) revealed that all the Senegalese strains and the X. citri pv. citri strain CFBP 2525 from New Zealand, used as a positive control, generated the expected DNA fragment, whereas no fragment was observed for negative controls (distilled water instead of the template). Insertion sequence ligation-mediated (IS-LM)-PCR analysis (1) of X. citri pv. citri strains from Senegal and reference strains of X. citri pv. citri pathotypes A and A* (1), with MspI and four primer pairs (unlabelled MspI primer and four 5'-labelled insertion sequence-specific primers targeting three IS elements), indicated that the strains from Senegal were related to X. citri pv. citri but not to pv. aurantifolii. They were closely related to X. citri pv. citri pathotype A strains, with a broad host range, present in the Indian subcontinent and Mali (C. Vernière, unpublished data). Multilocus sequence analysis of four partial housekeeping gene sequences (atpD, dnaK, efp, and gyrB) confirmed that four Senegalese strains were not related to X. citri pv. aurantifolii and showed a full sequence identity to X. citri pv. citri sequence type ST3 (2), fully consistent with IS-LM-PCR. Using a detached leaf assay (4), Duncan grapefruit, Pineapple sweet orange, and Mexican lime leaves inoculated with all strains from Senegal developed typical erumpent, callus-like tissue at wound sites 2 weeks after the inoculations. Xanthomonas-like colonies were reisolated and PCR amplification with the primer pair 4/7 produced the same 468-nt DNA fragment. This represents the fourth outbreak of citrus canker reported from Africa within the last 5 years, the other documented reports were from Ethiopia (2007) and Mali and Somalia (2008). High disease prevalence was observed in Senegal with incidence exceeding 90% in the orchards where lime and grapefruit were infected for 3 years, indicating the suitability of environmental conditions in this region for the development of Asiatic citrus canker. The origin of the inoculum associated with the reported canker outbreak in Senegal is currently unknown and the precise distribution of the pathogen needs to be thoroughly assessed. To our knowledge, this is the first documented report of the presence of Asiatic citrus canker in Senegal and this occurrence increases the threat to citriculture in West Africa. References: (1) L. Bui Thi Ngoc et al. FEMS Microbiol. Lett. 288:33, 2008. (2) L. Bui Thi Ngoc et al. Int. J. Syst. Evol. Microbiol. 60:515, 2010. (3) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005. (4) C. Vernière et al. Eur. J. Plant Pathol. 104:477, 1998.

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.