Fabien Guérin

Breakdown of the Scab Resistance Gene Vf in Apple Leads to a Founder Effect in Populations of the Fungal Pathogen Venturia inaequalis

ABSTRACT The recent breakdown of Vf, a major resistance gene to apple scab, provided an opportunity to analyze a population genetic process within the matching virulent subpopulation of the fungus Venturia inaequalis. We utilized the amplified fragment length polymorphism technique and allelic variation at four microsatellite loci to assess genetic structure of 133 isolates of V. inaequalis from a single commercial apple orchard sampled from one cultivar carrying the Vf gene (Judeline) and three cultivars devoid of the Vf gene. Both analyses indicated a strong decrease of the genetic diversity among isolates from the Vf cultivar compared with the high level of diversity among isolates from the three other cultivars. This leads to a high genetic differentiation between virVf and avrVf groups (F(ST) > 0.17). Analyses of the genetic distance between AFLP patterns based on the Jaccard index indicate that all virVf isolates could be assigned to a single clonal lineage. These results lead us to conclude that the clonal structure of the population isolated from the Vf cultivar is an example of a founder effect in response to a resistance gene breakdown and it is likely that this event occurred in the orchard during the sampling year.

A MLVA genotyping scheme for global surveillance of the citrus pathogen Xanthomonas citri pv. citri suggests a worldwide geographical expansion of a single genetic lineage.

MultiLocus Variable number of tandem repeat Analysis (MLVA) has been extensively used to examine epidemiological and evolutionary issues on monomorphic human pathogenic bacteria, but not on bacterial plant pathogens of agricultural importance albeit such tools would improve our understanding of their epidemiology, as well as of the history of epidemics on a global scale. Xanthomonas citri pv. citri is a quarantine organism in several countries and a major threat for the citrus industry worldwide. We screened the genomes of Xanthomonas citri pv. citri strain IAPAR 306 and of phylogenetically related xanthomonads for tandem repeats. From these in silico data, an optimized MLVA scheme was developed to assess the global diversity of this monomorphic bacterium. Thirty-one minisatellite loci (MLVA-31) were selected to assess the genetic structure of 129 strains representative of the worldwide pathological and genetic diversity of X. citri pv. citri. Based on Discriminant Analysis of Principal Components (DAPC), four pathotype-specific clusters were defined. DAPC cluster 1 comprised strains that were implicated in the major geographical expansion of X. citri pv. citri during the 20th century. A subset of 12 loci (MLVA-12) resolved 89% of the total diversity and matched the genetic structure revealed by MLVA-31. MLVA-12 is proposed for routine epidemiological identification of X. citri pv. citri, whereas MLVA-31 is proposed for phylogenetic and population genetics studies. MLVA-31 represents an opportunity for international X. citri pv. citri genotyping and data sharing. The MLVA-31 data generated in this study was deposited in the Xanthomonas citri genotyping database (http://www.biopred.net/MLVA/).

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.

New Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) Scheme for Fine-Scale Monitoring and Microevolution-Related Study of Ralstonia pseudosolanacearum Phylotype I Populations

ABSTRACT Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) is considered one of the most harmful plant diseases in the world. Special attention should be paid to R. pseudosolanacearum phylotype I due to its large host range, its worldwide distribution, and its high evolutionary potential. So far, the molecular epidemiology and population genetics of this bacterium are poorly understood. Until now, the genetic structure of the RSSC has been analyzed on the worldwide and regional scales. Emerging questions regarding evolutionary forces in RSSC adaptation to hosts now require genetic markers that are able to monitor RSSC field populations. In this study, we aimed to evaluate the multilocus variable-number tandem-repeat analysis (MLVA) approach for its ability to discriminate genetically close phylotype I strains and for population genetics studies. We developed a new MLVA scheme (MLVA-7) allowing us to genotype 580 R. pseudosolanacearum phylotype I strains extracted from susceptible and resistant hosts and from different habitats (stem, soil, and rhizosphere). Based on specificity, polymorphism, and the amplification success rate, we selected seven fast-evolving variable-number tandem-repeat (VNTR) markers. The newly developed MLVA-7 scheme showed higher discriminatory power than the previously published MLVA-13 scheme when applied to collections sampled from the same location on different dates and to collections from different locations on very small scales. Our study provides a valuable tool for fine-scale monitoring and microevolution-related study of R. pseudosolanacearum phylotype I populations. IMPORTANCE Understanding the evolutionary dynamics of adaptation of plant pathogens to new hosts or ecological niches has become a key point for the development of innovative disease management strategies, including durable resistance. Whereas the molecular mechanisms underlying virulence or pathogenicity changes have been studied thoroughly, the population genetics of plant pathogen adaptation remains an open, unexplored field, especially for plant-pathogenic bacteria. MLVA has become increasingly popular for epidemiosurveillance and molecular epidemiology studies of plant pathogens. However, this method has been used mostly for genotyping and identification on a regional or global scale. In this study, we developed a new MLVA scheme, targeting phylotype I of the soilborne Ralstonia solanacearum species complex (RSSC), specifically to address the bacterial population genetics on the field scale. Such a MLVA scheme, based on fast-evolving loci, may be a tool of choice for field experimental evolution and spatial genetics studies.

First Report of Ralstonia pseudosolanacearum Phylotype I Causing Bacterial Wilt on Rodrigues Island, Indian Ocean

The Ralstonia solanacearum species complex (Rssc) encompasses strains that are highly destructive on a wide host range, and some are listed as quarantine organisms in Europe, the United States, and many other countries. This soil-borne bacterial plant pathogen invades the roots and colonizes the xylem vessels, causing a bacterial wilt (BW) disease. Strains are distributed into four phylogenetic groups (phylotypes) linked to their geographical origin of evolution: phylotype I (Asia), phylotype II (America), phylotype III (Africa), and phylotype IV (Australia - Indonesia - Japan). Phylotypes are subdivided into sequevars, encompassing strains that share high homology on the endoglucanase gene partial sequence (750 bp) (Fegan and Prior 2005). Rodrigues is part of the Mascarene Islands in the Southwest Indian Ocean. In February 2016, a survey was conducted to identify the causal agent of BW, encountered for years on this island. Typical BW disease symptoms, including leaf epinasty and browning vessels, were observed on tomato (Solanum lycopersicum L.) in Terre Rouge and on chili pepper (Capsicum frutescens L.) in Nouvelle Decouverte and Montagne Malgache. Seventy-eight stem sections were collected, surface disinfected with 70% ethanol, and crushed into 2 ml of Tris-buffer, then processed for bacterial isolation by plating 50 µl on SM-1 medium (Granada and Sequeira 1983). Among all of the collected stems, typical Ralstonia pseudosolanacearum colonies grew on the semiselective medium from 51 of the 78 samples. Corresponding strains were subcultured and typed as R. pseudosolanacearum phylotype I (Safni et al. 2014), based on the PMX-PCR (Fegan and Prior 2005). A subset of six strains, two from each location, was selected for sequevar identification (Fegan et al. 1998). Strains CFBP8472 and CFBP8465 (Terre Rouge) were identified as sequevar I-31 (GenBank KX242311 and KX242312), but strains CFBP8467 and CFBP8468 (Montagne Malgache) along with CFBP8469 and CFBP8466 (Nouvelle Decouverte) were identified as sequevar I-33 (KX242313 to KX242316). This subset of strains was inoculated on five plants per strain by soil drenching on the susceptible tomato cultivar L390 (Lebeau et al. 2011) with 5 ml of a calibrated suspension (108 CFU/ml in Tris-buffer). Plants were grown in a fully controlled environment at day/night temperatures of 30/25°C ± 1°C under high relative humidity (∼90%). Sterile Tris-buffer inoculated negative controls were grown with the same parameters. Typical BW symptoms were observed within 6 days postinoculation and strains were reisolated and characterized using the same protocol as for the strains that were inoculated, fulfilling Kochs postulates. No symptoms were observed and no Rssc strains could be isolated from negative controls. To our knowledge, this is the first report of R. pseudosolanacearum phylotype I strains causing bacterial wilt on tomato and pepper on Rodrigues. Further studies need to be conducted on the prevalence and diversity of the Rssc strains causing the BW disease, in order to implement adapted control measures, such as the use of local and adapted resistant cultivars. Further surveys on other Southwest Indian Ocean islands such as Mauritius, Reunion, and Seychelles should be achieved to determine the potential origin of the R. pseudosolanacearum phylotype I strains isolated on Rodrigues island. (Texte integral)

Molecular Epidemiology of Bacterial Wilt in the Madagascar Highlands Caused by Andean (Phylotype IIB-1) and African (Phylotype III) Brown Rot Strains of the Ralstonia solanacearum Species Complex

The Ralstonia solanacearum species complex (RSSC) is a highly diverse cluster of bacterial strains found worldwide, many of which are destructive and cause bacterial wilt (BW) in a wide range of host plants. In 2009, potato production in Madagascar was dramatically affected by several BW epidemics. Controlling this disease is critical for Malagasy potato producers. The first important step toward control is the characterization of strains and their putative origins. The genetic diversity and population structure of the RSSC were investigated in the major potato production areas of the Highlands. A large collection of strains (n = 1224) was assigned to RSSC phylotypes based on multiplex polymerase chain reaction (PCR). Phylotypes I and III have been present in Madagascar for a long time but rarely associated with major potato BW outbreaks. The marked increase of BW prevalence was found associated with phylotype IIB sequevar 1 (IIB-1) strains (n = 879). This is the first report of phylotype IIB-1 strains in Madagascar. In addition to reference strains, epidemic IIB-1 strains (n = 255) were genotyped using the existing MultiLocus Variable-Number Tandem Repeat Analysis (MLVA) scheme RS2-MLVA9, producing 31 haplotypes separated into two related clonal complexes (CCs). One major CC included most of the worldwide haplotypes distributed across wide areas. A regional-scale investigation suggested that phylotype IIB-1 strains were introduced and massively spread via latently infected potato seed tubers. Additionally, the genetic structure of phylotype IIB-1 likely resulted from a bottleneck/founder effect. The population structure of phylotype III, described here for the first time in Madagascar, exhibited a different pattern. Phylotype III strains (n = 217) were genotyped using the highly discriminatory MLVA scheme RS3-MLVA16. High genetic diversity was uncovered, with 117 haplotypes grouped into 11 CCs. Malagasy phylotype III strains were highly differentiated from continental African strains, suggesting no recent migration from the continent. Overall, population structure of phylotype III involves individual small CCs that correlate to restricted geographic areas in Madagascar. The evidence suggests, if at all, that African phylotype III strains are not efficiently transmitted through latently infected potato seed tubers.