Phylotype and sequevar variability of Ralstonia solanacearum in Brazil,an ancient centre of diversity of the pathogen

The β‐proteobacterium Ralstonia solanacearum causes bacterial wilt of many plant species. Knowledge of phylotype and sequevar variability in populations of this microorganism is useful for implementing control measures, particularly host resistance. To this end, 301 isolates of R. solanacearum were collected from different geographic regions and hosts in Brazil. Their phylotype and sequevar characterization was used to determine the amount and distribution of phenetic and phylogenetic variability. Isolates were classified into phylotypes I (n = 48), clade 1; and phylotype II, clades 2–5. Phylotype II was divided into subclusters IIA (n = 112) and IIB (n = 141). Phylotype II was widely distributed, whereas phylotype I isolates were found in Central, Northern, and Northeastern regions of Brazil. There were 108 haplotypes identified among endoglucanase (egl) gene sequences from 301 isolates and 32 haplotypes among DNA repair (mutS) gene regions from 176 isolates. The egl and mutS sequence analyses identified eight known (1, 4, 7, 18, 27, 28, 41 and 50) and four new (54, 55, 56 and 57) sequevars. Phylotype IIB showed high diversity in sequevars and host range. Multiplex PCR, using primers specific to the Moko ecotype, characterized banana and long pepper isolates as sequevar 4 and 4/NPB, respectively. This constitutes the first report of the emergent ecotype IIB/4NPB in a new host, long pepper. The majority of sequevars were associated with geographic regions. This high variability of R. solanacearum in Brazil suggests use of host resistance to control bacterial wilt should be mainly focused by region.     

Wide genetic diversity of Ralstonia solanacearum strains affecting tomato in Trinidad,West Indies

The genetic diversity of Ralstonia solanacearum causing bacterial wilt of tomato in Trinidad was assessed using the hierarchical phylotyping scheme and rep‐PCR DNA fingerprinting. Seventy‐one isolates were collected in 2003 on infected tomato crops in the four main vegetable cropping areas of Trinidad (North, Central, South‐East and South). Two phylotypes were present, with phylotype II being much more prevalent (66%) than phylotype I (34%). Phylotype II strains consisted mainly of sequevar 7 in Central and South‐East, and sequevar 35 in North, South‐East and South. This is the first report of sequevar 7 outside south‐eastern USA. In contrast, no ‘brown rot’ (phylotype IIB/1, race 3 biovar 2) or emerging strains of phylotype IIB/4NPB were identified. Rep‐PCR data were used to assess population genetic structure. No significant clustering by geographical distance was found, suggesting regular gene flow among cropping areas (via waterways, plant or soil). However, the population from Central was significantly differentiated from the others, containing only phylotype II/seq 7 strains, with a high degree of clonality, suggesting a possible recent introduction from abroad. The South population was less aggressive and more genetically diverse, suggesting horizontal gene transfers within the population, even among isolates of different phylotypes. Phylotype I and phylotype II populations differed slightly in clonality levels, with indications of more frequent recombination events within phylotype I populations. Possible factors influencing genetic diversity and distribution within the island are discussed.     

Phylogenetic relationships and population structure of Ralstonia solanacearum isolated from diverse origins in Taiwan

Bacterial wilt caused by Ralstonia solanacearum is a destructive disease for many crops. The aim of this study was to investigate the phylogenetic relationships and genetic structure of an R. solanacearum population from diverse origins in Taiwan. All 58 tested isolates belonged to phylotype I, except the two biovar 2 isolates from potato. These belonged to phylotype IIB sequevar 1 and were identical to known potato brown rot strains, which were probably introduced. Phylotype I isolates were grouped into 10 sequevars. Sequevar 15 was predominant (34 out of 56 isolates). Its distribution covered the whole island and it was largely associated with solanaceous crops such as tomato, and with tomato field soil. A total of 14 haplotypes were identified based on a partial endoglucanase gene sequence. Parsimony network analysis revealed that haplotype A was the oldest haplotype in the local population. It encompassed all but one of the sequevar 15 isolates. Large variation in virulence on tomato was observed among the 58 isolates, and seven pathotypes were identified. Significant genetic differentiation was detected among pathotypes. Moreover, genetic differentiation was detected between biovar 3 and biovar 4 subgroups and between the strains associated with solanaceous and non‐solanaceous species, but none was detected between strains from different geographic origins. The results suggest that the phylotype I population in Taiwan is homogeneous, while mutation and local adaptation to specific ecological niches keep shaping the population.     

Resistance to Taiwanese race 1 strains of Ralstonia solanacearum in wild tomato germplasm

A total of 252 wild Solanum accessions and one population of 49 introgression lines of LA716 were screened for resistance to a race 1/biovar 4/phylotype I strain Pss186 of Ralstonia solanacearum. Most wild tomato accessions were highly susceptible. However, five accessions of S. pennellii, i.e. LA1943, LA716, LA1656, LA1732 and TL01845 were resistant to strain Pss186. These accessions were then challenged against two other race 1/phylotpye I strains Pss4 and Pss190, which were more aggressive. All the five S. pennellii accessions were susceptible to Pss4, but displayed high to moderate resistance to Pss190 with a percentage of wilted plants ranging from 0% to 60%. Pss190 is an aggressive strain that made a resistant tomato line Hawaii 7996 susceptible. Thus, the results found in this study provide evidence of the presence of strain-specific resistance. LA3501, which has an introgression segment on chromosome 6, was found to be resistant to Pss186 among the screened introgression lines. This confirms the importance of resistance trait loci on chromosome 6 that have been identified by other studies. This is the first report of S. pennellii being resistant to bacterial wilt. These new resistant sources will provide breeders with more resources to breed for stable resistance to bacterial wilt of tomato.     

Diversity of Ralstonia solanacearum causing potato bacterial wilt in Iran and the first record of phylotype II/biovar 2T strains outside South America

The diversity of 40 strains of Ralstonia solanacearum causing bacterial wilt of potato in the major potato-growing areas of Iran was assessed. Based on rep-PCR genomic fingerprinting, strains fell into two distinct groups. The first group contained 37 of the 40 strains and the second consisted of three strains from a narrow tropical region in Iran. The three strains from the narrow tropical region were found to be phenotypically and genotypically most similar to R. solanacearum biovar 2T strains, whereas all other strains were phenotypically and genotypically identified as being R. solanacearum biovar 2/race 3. Phylogenetic analysis of endoglucanase gene sequence information of two of the strains from the tropical region revealed that they belonged to phylotype II of the R. solanacearum species complex and had 100% sequence similarity to a biovar 2T strain from potato in Peru. This is the first report of the presence of R. solanacearum phylotype II/biovar 2T in Iran and the first report of the existence of this group of R. solanacearum outside South America.     

Diversity and distribution of Ralstonia solanacearum strains in Guatemala and rare occurrence of tomato fruit infection

Fifty-nine Ralstonia solanacearum isolates from diverse crops and regions were collected and characterized to determine the distribution and diversity of this soilborne pathogen in Guatemala. Three distinct types were present: a phylotype I, sequevar 14 strain, probably originating from Asia, infecting tomatoes and aubergines at moderate elevations; a phylotype II, sequevar 6 strain of American origin causing Moko disease in lowland banana plantations; and a phylotype II, sequevar 1 (race 3 biovar 2) strain causing brown rot on potatoes, Southern wilt of Pelargonium spp. and bacterial wilt of greenhouse tomatoes at high elevations. These data on strain diversity will inform effective regional efforts to breed for wilt resistance. A sensitive enrichment method did not detect the pathogen in fruits from naturally infected commercial tomato plants in Guatemalan fields and greenhouses, although it was detected in 6% of fruits from a wilt-resistant hybrid. Low numbers of R. solanacearum cells were also infrequently detected in fruits from plants artificially inoculated in the growth chamber with either race 3 biovar 2 or a phylotype II tomato strain.     

Genetic,biochemical and pathogenic diversity of Pseudomonas syringae pv. pisi strains

Pseudomonas syringae pv. pisi is a seedborne pathogen distributed worldwide that causes pea bacterial blight. Previous characterization of this pathogen has been carried out with relatively small and/or geographically limited samples. Here, a collection of 91 strains are examined that include strains from recent outbreaks in Spain (53 strains) and from 14 other countries, and that represent all races and the new race 8, including the type race strains. This collection was characterized on the basis of 55 nutritional tests, genetic analysis (rep‐PCR, amplification of AN3 and AN7 specific markers, and multilocus sequence typing (MLST)) and pathogenicity on the differential pea cultivars to identify races. Principal component analysis and distance dendrograms confirm the existence of two genetic lineages within this pathovar, which are clearly discriminated by the AN3/AN7 markers, rep‐PCR and MLST. Strains from races 1 and 7 amplified the AN3 marker; those from races 2, 6 and 8 amplified AN7, while strains of races 3, 4 and 5 amplified either AN3 or AN7. Nevertheless, strains were not grouped by race type by any of the genetic or biochemical tests. Likewise, there was no significant association between metabolic and/or genetic profiling and the geographical origin of the strains. The Spanish collection diversity reflects the variability found in the worldwide collection, suggesting multiple introductions of the bacteria into Spain by contaminated seed lots.     

Current classification of Ralstonia solanacearum and genetic diversity of the strains in Japan

Ralstonia solanacearum is the causal organism of bacterial wilt of more than 200 species representing 50 families of plants in tropical, subtropical, and warm temperate regions in the world. Traditionally classified into five races based on differences in host range, R. solanacearum has also been grouped into six biovars on the basis of biochemical properties. With recent developments in molecular biology, various DNA-based analyses have been introduced and used to confirm that this binary system does not completely represent the diversity within R. solanacearum strains. Therefore, a new hierarchical classification scheme has been suggested, which defines R. solanacearum as a species complex and reorganized the concept of the species as a monophyletic cluster according to a phylogenetic analysis based on genomic sequence data. Here we discuss the current bacterial wilt situation and genetic relationships based on the recent classification system of Japanese R. solanacearum strains as well as worldwide strains. We also review the genetic, biochemical, and pathological characteristics of R. solanacearum strains, in particular, those affecting potato and Zingiberaceae plants as distinctly important pathogens in relation to continuously problematic and recent emergent diseases in Japan.     

PCR-based specific detection of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> race 4 strains

Two primer sets were designed based on the sequence of polymorphic bands that were derived from repetitive sequence-based polymerase chain reaction (rep-PCR) fingerprinting and specifically detected in Ralstonia solanacearum race 4 strains (ginger, mioga, and curcuma isolates). One primer set (AKIF-AKIR) amplified a single band (165bp) from genomic DNA obtained from all mioga and curcuma and some ginger isolates; another set (21F-21R) amplified one band (125bp) from the other ginger isolates. These primer sets did not amplify the bands from genomic DNA of other R. solanacearum strains or of other related bacteria. PCR detection limit for the pathogen was 2 × 10²cfu.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB118756 and AB118757     

Detection and Visualization of the Major Acidic Exopolysaccharide of Ralstonia solanacearum and its Role in Tomato Root Infection and Vascular Colonization

Exopolysaccharides play an important role in the pathogenicity of Ralstonia solanacearum. We compared in vitro and in planta exopolysaccharide production of the pathogenic strain AW1 with that of three related mutant strains impaired in both their exopolysaccharide production and aggressiveness on tomato. The distinction between the two hexosamine-rich exopolysaccharides, namely the N-acetyl-glucosaminorhamnan and the major N-acetyl-galactosamine-containing acidic polymer was emphasized. The major acidic polymer was identified specifically by electron microscopy using glutaraldehyde/ruthenium red/uranyl acetate staining, by immunofluorescence using specific monoclonal antibodies and correlated to an appropriate biochemical analysis. The two mutant strains AW1-1 and AW-19A were totally devoid of any production of the major exopolysaccharide in vitro or in planta whatever the technique used. Infection and vascular colonization of tomato roots by the pathogenic strain were also compared to those of the mutant strains by light microscopy. Pathogenicity on tomato was assessed by root infection without any artificial injury. Light microscopy showed that the two mutant strains AW1-1 and AW-19A were poorly infective and unable to invade xylem vessels, while they induced defence mechanisms in root tissues and appeared aggregated or degenerated within cortical infection pockets. These two mutant strains were non-pathogenic or weakly aggressive, respectively. In contrast, the pathogenic strain AW1 and the hypoaggressive AW1-41 strains, which produce large amounts of the major acidic exopolysaccharide in planta, were both infective and invasive, and tomato root tissues exhibited only limited defence reactions. Thus, the major acidic exopolysaccharide produced by Ralstonia solanacearum is involved in root infection and vascular colonization, though its precise role is still unknown.     

Molecular identification of some African strains of Ralstonia solanacearum from eucalypt and potato

Ralstonia solanacearum is a known bacterial pathogen of eucalypt and potato plants in Africa. A survey was undertaken to detect this pathogen in eucalypt plantations in South Africa, the Democratic Republic of Congo, and Uganda. Numerous bacterial strains were isolated from trees with symptoms typical of bacterial wilt, but only seven were positively identified as R. solanacearum. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, based on the hrp (hypersensitive response and pathogenicity) gene region was used to determine and group the biovars of these R. solanacearum strains. The eucalypt isolates and one potato isolate formed a biovar 3 cluster, whereas the two other potato isolates formed a cluster that corresponded to biovar 2. Amplified fragment length polymorphism (AFLP) analysis confirmed these clusters. Therefore, PCR-RFLP can be used as a reliable diagnostic technique to enable researchers to rapidly identify the pathogen.     

Molecular characterization of Ralstonia solanacearum strains from Ethiopia and tracing potential source of bacterial wilt disease outbreak in seed potatoes

Bacterial wilt, caused by Ralstonia solanacearum, is emerging as a major threat to potato production in Ethiopia, reaching epidemic proportions in the Chencha district recently, with a prevalence of 97% of potato fields in 2015. The recent disease outbreak in the district coincided with a significant introduction of seed potatoes. This research was therefore initiated to genetically characterize the pathogen so as to trace its source, identify its relationship with outbreaks in the rest of the country, and make intervention recommendations. Ralstonia solanacearum isolates were sampled both from seed and ware potato fields in Chencha and from seed potato fields in production regions suspected of being potential sources of the pathogen. Multiplex PCR and phylogenetic analysis of partial endoglucanase gene sequences identified all of the isolates as phylotype IIB sequevar 1. VNTR sequence analysis distinguished 11 different haplotypes, nine of which were unique to the Chencha district. However, one of the haplotypes was common to all seed potato producer regions of Ethiopia except for the Shashemene area. The unique and diverse VNTR haplotypes of the pathogen in Chencha indicates that it is well established in the district. When a geographical map of the VNTR haplotypes was superimposed with the main cross‐regional seed potato distribution pattern of the country, it became evident that the pathogen was being disseminated via latently infected seed from the Holeta‐Jeldu area in the Central Highlands of Ethiopia. Identification of largely uninfected highland districts and multiplication of high‐grade seed potato exclusively in those districts should be given priority.     

防治青枯病工程菌Hrp-菌株的发酵培养基配方优化

在摇瓶发酵条件下,采用全因子实验设计法和正交实验设计法对工程菌Hrp-菌株的培养基碳、氮源配方进行优化,确定了最适碳源为葡萄糖、细玉米面,最适氮源为硫酸铵、细豆饼粉;对这四个碳氮源因子的浓度进行L9（34）的正交试验,最终确定出该菌的最适发酵培养基碳、氮源配方为（g/L）：葡萄糖8g、细玉米面5g、细豆饼粉35g、硫酸铵2g。优化后的培养基配方使摇瓶发酵液菌量从1.34×10^10cfu/ml提高至3.30×10^10cfu/ml。     

An improved enrichment broth for the sensitive detection of Ralstonia solanacearum (biovars 1 and 2A) in soil using DAS–ELISA

A reliable, sensitive, low-cost and easy-to-use technique is described for the detection of Ralstonia solanacearum (the causal organism of bacterial wilt, BW) in soil. A total of 273 potato isolates belonging to five different biovars (Bv), originating from 33 countries worldwide, were tested and successfully detected by antibodies produced at the International Potato Center (CIP). Isolates of R. solanacearum belonging to Bv1 and Bv2A were successfully detected by double antibody sandwich–enzyme-linked immunosorbent assay (DAS–ELISA) at low population levels after incubation of soil suspensions for 48 h at 30°C in a new semiselective broth containing a potato tuber infusion. Detection thresholds of 20 and 200 CFU g⁻¹ inoculated soil were obtained for Bv1 and Bv2A, respectively. Sensitivity of detection of Bv2A was similar or even higher in five different inoculated soil types. No cross-reactions were obtained in DAS–ELISA after enrichment of soil suspensions (i) prepared from 23 different soils sampled in BW-free areas in six departments of Peru; and (ii) inoculated with 10 identified bacteria and 136 unknown isolates of soil microbiota isolated from eight different locations. Only the blood disease bacterium gave a low-level reaction after enrichment. In naturally infested soils, average sensitivities of 97·6 (SE 14·8) and 100·9 (SE 22·6) CFU g⁻¹ were obtained for biovars 1 and 2A, respectively. By making serial dilutions of the soil suspension before enrichment, densities of R. solanacearum could be determined in a semiquantitative way. Results also showed that composite samples of five soils could be analysed to assess field soil populations without reducing detection sensitivity.     

Phylogenetic relationships of Ralstonia solanacearum species complex strains from Asia and other continents based on 16S rDNA, endoglucanase, and hrpB gene sequences

The 16S rDNA, endoglucanase, and hrpB genes were partially sequenced for Asian strains of Ralstonia solanacearum spp. complex, including 31 strains of R. solanacearum and two strains each of the blood disease bacterium (BDB) and Pseudomonas syzygii. Additional sequences homologous to these DNA regions, deposited at DDBJ/EMBL/GenBank databases were included in the analysis. Various levels of polymorphisms were observed in each of these DNA regions. The highest polymorphism (approximately 25%) was found in the endoglucanase gene sequence. The hrpB sequence had about 22% poly-morphism. The phylogenetic analysis consistently divided the strains into four clusters, as distinctly shown on the phylogenetic trees of 16S rDNA, hrpB gene, and endo-glucanase gene sequences. Cluster 1 contained all strains from Asia, which belong to biovars 3, 4, 5, and N2. Cluster 2 comprised the Asian strains of R. solanacearum (as biovars N2 and 1) isolated from potato and clove, as well as BDB and P. syzygii. Cluster 3 contained race 3 biovar 2 strains from potato, race 2 biovar 1 strains from banana, and race 1 biovar 1 strains isolated from America, Asia, and other parts of the world. Cluster 4 was exclusively composed of African strains. The results of the study showed the distribution and diversity of the Asian strains, which are present in three of the four clusters. The similarity of Asian strains to those in the other regions was also observed.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AY464950 to AY465050     

Characterization of Pseudomonas syringae pv. actinidiae (Psa) isolated from France and assignment of Psa biovar 4 to a de novo pathovar: Pseudomonas syringae pv. actinidifoliorum pv. nov.

Since 2008, bacterial canker of kiwifruit (Actinidia deliciosa and A. chinensis) caused by Pseudomonas syringae pv. actinidiae (Psa) has resulted in severe economic losses worldwide. Four biovars of Psa can be distinguished based on their biochemical, pathogenicity and molecular characteristics. Using a range of biochemical, molecular and pathogenicity assays, strains collected in France since the beginning of the outbreak in 2010 were found to be genotypically and phenotypically diverse, and to belong to biovar 3 or biovar 4. This is the first time that strains of biovar 4 have been isolated outside New Zealand or Australia. A multilocus sequence analysis based on four housekeeping genes (gapA, gltA, gyrB and rpoD) was performed on 72 strains representative of the French outbreak. All the strains fell into two phylogenetic groups: one clonal corresponding to biovar 3, and the other corresponding to biovar 4. This second phylogenetic group was polymorphic and could be divided into four lineages. A clonal genealogy performed with a coalescent approach did not reveal any common ancestor for the 72 Psa strains. Strains of biovar 4 are substantially different from those of the other biovars: they are less aggressive and cause only leaf spots whereas Psa biovars 1, 2 and 3 also cause canker and shoot die‐back. Because of these pathogenic differences, which were supported by phenotypic, genetic and phylogenetic differences, it is proposed that Psa biovar 4 be renamed Pseudomonas syringae pv. actinidifoliorum pv. nov. Strain CFBP 8039 is designated as the pathotype strain.