Karine Vital

Variations in type III effector repertoires, pathological phenotypes and host range of Xanthomonas citri pv. citri pathotypes

The mechanisms determining the host range of Xanthomonas are still undeciphered, despite much interest in their potential roles in the evolution and emergence of plant pathogenic bacteria. Xanthomonas citri pv. citri (Xci) is an interesting model of host specialization because of its pathogenic variants: pathotype A strains infect a wide range of Rutaceous species, whereas pathotype A*/A(W) strains have a host range restricted to Mexican lime (Citrus aurantifolia) and alemow (Citrus macrophylla). Based on a collection of 55 strains representative of Xci worldwide diversity assessed by amplified fragment length polymorphism (AFLP), we investigated the distribution of type III effectors (T3Es) in relation to host range. We examined the presence of 66 T3Es from xanthomonads in Xci and identified a repertoire of 28 effectors, 26 of which were shared by all Xci strains, whereas two (xopAG and xopC1) were present only in some A*/A(W) strains. We found that xopAG (=avrGf1) was present in all A(W) strains, but also in three A* strains genetically distant from A(W) , and that all xopAG-containing strains induced the hypersensitive response (HR) on grapefruit and sweet orange. The analysis of xopAD and xopAG suggested horizontal transfer between X. citri pv. bilvae, another citrus pathogen, and some Xci strains. A strains were genetically less diverse, induced identical phenotypic responses and possessed indistinguishable T3E repertoires. Conversely, A*/A(W) strains exhibited a wider genetic diversity in which clades correlated with geographical origin and T3E repertoire, but not with pathogenicity, according to T3E deletion experiments. Our data outline the importance of taking into account the heterogeneity of Xci A*/A(W) strains when analysing the mechanisms of host specialization.

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.

Ligation-mediated PCR, a fast and reliable technique for insertion sequence-based typing of Xanthomonas citri pv. citri

Asiatic citrus canker, caused by Xanthomonas citri pv. citri, is a major disease threatening citrus crops throughout the world. The most common methods for strain differentiation of this pathogen are repetitive element sequence-based PCR (rep-PCR) and pulsed field gel electrophoresis (PFGE), using rare-cutting restriction enzyme analysis. We developed a ligation-mediated PCR targeting three insertion sequences (IS-LM-PCR) present as several copies in the genome of the fully sequenced strain 306 of X. citri pv. citri. This technique amplifies DNA fragments between an insertion sequence element and an MspI restriction site. The analysis of strains can be conducted within 24 h, starting from very small amounts of bacterial DNA, which makes IS-LM-PCR much less labor-intensive than PFGE. We used IS-LM-PCR to analyze a collection of 66 strains of X. citri pv. citri from around the world. The overall reproducibility of IS-LM-PCR reached 98% in this data set and its discriminatory power was markedly superior than rep-PCR. We suggest that IS-LM-PCR could be used for the global surveillance of non-epidemiologically related strains of X. citri pv. citri.

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 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-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 Kochs 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.