Ralstonia Solanacearum Species Complex Research Articles

Contrasting genetic diversity and structure among Malagasy Ralstonia pseudosolanacearum phylotype I populations inferred from an optimized Multilocus Variable Number of Tandem Repeat Analysis scheme.

The Ralstonia solanacearum species complex (RSSC), composed of three species and four phylotypes, are globally distributed soil-borne bacteria with a very broad host range. In 2009, a devastating potato bacterial wilt outbreak was declared in the central highlands of Madagascar, which reduced the production of vegetable crops including potato, eggplant, tomato and pepper. A molecular epidemiology study of Malagasy RSSC strains carried out between 2013 and 2017 identified R. pseudosolanacearum (phylotypes I and III) and R. solanacearum (phylotype II). A previously published population biology analysis of phylotypes II and III using two MultiLocus Variable Number of Tandem Repeats Analysis (MLVA) schemes revealed an emergent epidemic phylotype II (sequevar 1) group and endemic phylotype III isolates. We developed an optimized MLVA scheme (RS1-MLVA14) to characterize phylotype I strains in Madagascar to understand their genetic diversity and structure. The collection included isolates from 16 fields of different Solanaceae species sampled in Analamanga and Itasy regions (highlands) in 2013 (123 strains) and in Atsinanana region (lowlands) in 2006 (25 strains). Thirty-one haplotypes were identified, two of them being particularly prevalent: MT007 (30.14%) and MT004 (16.44%) (sequevar 18). Genetic diversity analysis revealed a significant contrasting level of diversity according to elevation and sampling region. More diverse at low altitude than at high altitude, the Malagasy phylotype I isolates were structured in two clusters, probably resulting from different historical introductions. Interestingly, the most prevalent Malagasy phylotype I isolates were genetically distant from regional and worldwide isolates. In this work, we demonstrated that the RS1-MLVA14 scheme can resolve differences from regional to field scales and is thus suited for deciphering the epidemiology of phylotype I populations.

First phylotype analysis of Ralstonia solanacearum causing Eggplant bacterial wilt in the Republic of Guinea

Eggplant is one of the important cash crops in Guinea and it is cultivated in all the agroecological zones of the country with the areas and production are constantly growing. However, this crop is affected by bacterial wilt disease caused by bacteria of the Ralstonia solanacearum species complex (RSSC). in these production areas. In August 2018, a total of 81 strains were collected from stems of withered eggplant plant in the nine prefectures of the three administrative regions of Guinea. These strains were extracted and put on FTATM cards at the Foulayah laboratory in Guinea. for their molecular characterization the FTA cards were transported to the World vegetable center laboratory in Taiwan where all the 81 strains were confirmed as R. solanacearum . single 280 bp fragment resulted in all the isolates following polymerase chain reaction (PCR) amplification using the R. solanacearum specific universal primer pair 759/760. A phylotype specific multiplex PCR revealed that 55 of the 81 strains could be assigned to phylotypes. 85, 5% of this batch consisted of phylotype I; 3, 6% of phylotype II; 10, 9% of phylotype III. The objective of this study is to open the way to developing better eggplant BW management strategies for Guinea, including screening for eggplant lines resistant to phylotype I and phylotype III either together or separately for subsequent hybridization.

Quorum Sensing Inhibition Attenuates the Virulence of the Plant Pathogen Ralstonia solanacearum Species Complex.

Strains of Ralstonia solanacearum species complex (RSSC) cause "bacterial wilt" on a wide range of plant species and thus lead to marked economic losses in agriculture. Quorum sensing (QS), a bacterial cell-cell communication mechanism, controls the virulence of RSSC strains by regulating the production of extracellular polysaccharide (EPS) and secondary metabolites, biofilm formation, and cellular motility. R. solanacearum strain OE1-1 employs (R)-methyl 3-hydroxymyristate (3-OH MAME) as a QS signal, which is synthesized by the PhcB methyltransferase and sensed by the PhcS/PhcRQ two-component system. We describe the design, synthesis, and biological evaluation of inhibitors of the phc QS system. Initial screening of a small set of QS signal analogues revealed that methyl 3-hydroxy-8-phenyloctanoate, named, PQI-1 (phc quorum sensing inhibitor-1), inhibited biofilm formation by strain OE1-1. To improve its inhibitory activity, the derivatives of PQI-1 were synthesized, and their QS inhibition activities were evaluated. PQIs-2-5 evolved from PQI-1 more strongly inhibited not only biofilm formation but also the production of ralfuranone and EPS. Furthermore, RNA-Seq analysis revealed that the PQIs effectively inhibited QS-dependent gene expression and repression in strain OE1-1. On the other hand, the PQIs did not affect the canonical QS systems of the representative reporter bacteria. These antagonists, especially PQI-5, reduced wilting symptoms of the tomato plants infected with strain OE1-1. Taken together, we suggest that targeting the phc QS system has potential for the development of chemicals that protect agricultural crops from bacterial wilt disease.

Host adaptation and microbial competition drive Ralstonia solanacearum phylotype I evolution in the Republic of Korea.

Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) threatens the cultivation of important crops worldwide. We sequenced 30 RSSC phylotype I ( R. pseudosolanacearum ) strains isolated from pepper (Capsicum annuum) and tomato (Solanum lycopersicum) across the Republic of Korea. These isolates span the diversity of phylotype I, have extensive effector repertoires and are subject to frequent recombination. Recombination hotspots among South Korean phylotype I isolates include multiple predicted contact-dependent inhibition loci, suggesting that microbial competition plays a significant role in Ralstonia evolution. Rapid diversification of secreted effectors presents challenges for the development of disease-resistant plant varieties. We identified potential targets for disease resistance breeding by testing for allele-specific host recognition of T3Es present among South Korean phyloype I isolates. The integration of pathogen population genomics and molecular plant pathology contributes to the development of location-specific disease control and development of plant cultivars with durable resistance to relevant threats.

Genome- and Mass Spectrometry-Guided Discovery of Ralstoamides A and B from Ralstonia solanacearum Species Complex.

Strains of Ralstonia solanacearum species complex (RSSC) are devastating plant pathogens distributed globally with a wide host range and genetic diversity. Many RSSC strains harbor the polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) hybrid gene rmyA/rmyB for ralstonin production. We report that ralstoamides A (1) and B (2), which are ralstonin-like but shorter lipopeptides, were discovered from the Japanese strains using accumulated RSSC genome data and LC/MS-based metabolite analysis. Their structures, including absolute configurations, were elucidated by spectroscopic analysis and chemical techniques. ramA, a PKS-NRPS gene for ralstoamide production, was identified from the producer strains by genome sequencing and gene-deletion experiments. Based on the analysis of biosynthetic genes of ralstoamides and ralstonins, we suggest the occurrence of NRPS-module reduction of rmyA/rmyB genes in some RSSC strains. This possible molecular evolution changed not only the structures, but also the biological activity of RSSC lipopeptides.

First Report of Bacterial Wilt Disease of Tomato, Pepper and Gboma Caused by the Ralstonia solanacearum Species Complex in Togo.

Tomato (Solanum lycopersicum), pepper (Capsicum annum), and gboma (Solanum macrocarpon) are major vegetables in Togo, with many people depending on these crops for their livelihood. In December 2018, during the dry season with temperatures between 21°C to 35°C, tomato ('Petomech'), pepper ('Gboyebesse') and gboma (local landrace) showing wilt symptoms without foliar yellowing were collected from two locations, Tchouloum and CECO-AGRO sites in the Sotouboua Prefecture of Togo, ~300 km from the capital city of Lome. Disease incidence ranged between 10% to 50% in multiple fields. Cut stems of most wilting tomato, pepper and gboma plants produced bacterial ooze in water and vascular discoloration was visible in longitudinal stem sections. Ground cut stem tissue tested positive with Rs ImmunoStrips specific to the Ralstonia solanacearum species complex (RSSC) (Agdia Inc., Elkhart, IN, USA). Collected samples were stored at ambient temperature and cultured within 36 hr. Culturing sap from cut stems plated on modified SMSA medium (Engelbrecht 1994) yielded colonies with typical RSSC morphology: slow-growing, irregular, mucoid, and white with red centers. Genomic DNA was extracted from thirteen isolates: two from gboma, five from tomato and six from pepper. The expected 280-bp band was amplified from all 13 genomic DNAs following polymerase chain reaction (PCR) using the 759/760 RSSC-specific primer pair (Opina et al. 1997). PCR with the 630/631 primers, which identify the Race 3 biovar 2 RSSC subgroup, did not yield a product from any Togo isolate (Opina et al. 1997). The phylotype multiplex PCR identified all Togo isolates as belonging to the phylotype I subgroup, also called R. pseudosolanacearum (Prior et al. 2016; Fegan and Prior 2005). Phylotype control DNAs were from strains GMI1000 (phylotype I, Asia), K60 (phylotype II, Americas), CMR15 (phylotype III, Africa), and PSI07 (phylotype IV, Indondesia). Comparative genomic analysis of the partial endoglucanase (egl) gene, amplified with the Endo primer pairs (Poussier et al. 2000), revealed all Togo strains belonged to sequevar 17, a group known to cause bacterial wilt of peanut in China. (Xu et al. 2009). The egl sequences are in NCBI GenBank accessions MT572393 to MT572405. Koch's postulates were completed by inoculating 28-day-old bacterial wilt-susceptible 'Bonny Best' tomato plants by soil soak (Khokhani et al. 2018). Briefly, soil around each unwounded plant was drenched with 50 ml of a 108 CFU/mL suspension of bacteria grown from a single colony. Five plants were inoculated with each of four randomly selected Togo strains. RSSC phylotype I strain GMI1000 served as a positive control and water treated plants as negative controls. Plants were kept in a 28°C growth chamber with a 12 hr photoperiod. All RSSC inoculated plants were fully wilted within a week; symptoms resembled to those observed in the field. Water treated control plants did not wilt. Culturing sap from all inoculated plants on SMSA medium yielded colonies with typical RSSC morphology that tested positive with the Rs ImmunoStrips. This is the first identification of RSSC in Togo. These results will guide development of disease management strategies and regionally appropriate breeding of vegetable lines with resistance to the phylotype I RSSC strains present in Togo.

Potato-Infecting Ralstonia solanacearum Strains in Iran Expand Knowledge on the Global Diversity of Brown Rot Ecotype of the Pathogen.

Bacterial wilt and brown rot disease caused by Ralstonia solanacearum species complex (RSSC) is one of the major constraints of potato (Solanum tuberosum) production around the globe. During 2017 to 2018, an extensive field survey was conducted in six potato-growing provinces of Iran to monitor the status of bacterial wilt disease. Pathogenicity and host range assays using 59 bacterial strains isolated in Iran showed that they were pathogenic on eggplant, red nightshade, pepper, potato and tomato, while nonpathogenic on common bean, cowpea, cucumber, sunflower, zinnia and zucchini. PCR-based diagnosis revealed that the strains belong to the phylotype IIB/sequevar 1 (IIB/I) lineage of the RSSC. Furthermore, a five-gene multilocus sequence analysis and typing (egl, fliC, gyrB, mutS, and rplB) confirmed the phylogenetically near-homogeneous nature of the strains within IIB/I lineage. Four sequence types were identified among 58 IIB/1 strains isolated in Iran. Phylogenetically near-homogeneous nature of the strains in Iran raise questions about the mode of inoculum entry of the bacterial wilt pathogen into the country (one-time introduction versus multiple introductions), while the geographic origin of the Iranian R. solanacearum strains remains undetermined. Furthermore, sequence typing showed that there were shared alleles (haplotypes) and sequence types among the strains isolated in geographically distant areas in Iran, suggesting intranational transmission of the pathogen in the country.

Identification and characterization of Ralstonia spp. causing bacterial wilt disease of vegetables in Mali

Ralstonia solanacearum species complex is the most destructive and economically important bacterial pathogen of many plant species around the world. It is a particular constraint to the production of the solanaceous vegetables such as tomato, chilli pepper and African eggplant in West Africa. Its broad host range, ability to survive in the soil for long periods and ability to sustain latent infection, make it difficult to control. Identification, characterization and mapping the distribution of the causal Ralstonia spp. strains are necessary to help design effective control strategies. Wilted tomato, African eggplant and pepper plant samples were collected from fields in different regions of Mali. The causal bacterial strains were isolated from the samples and all were identified as Ralstonia pseudosolanacearum. Multiplex PCR using four phylotype specific primer pairs identified the presence of phylotype I strains distributed across all the vegetable production areas whereas phylotype III strains were limited to areas of Sikasso, Koulikoro and Segou to the south and east of Bamako city. Phylogenetic analysis of part of the conserved endoglucanase virulence gene sequences revealed four sequevars with most phylotype I strains identified as sequevars 46 and 31 with only a few as sequevar 14 and one sequevar 18. The phylotype III strains were all in the sequevar 23–48 group. Pathogenicity testing of a selected subset of the strains isolated in Mali showed them all to be pathogenic to the susceptible tomato cultivar Roma. To our knowledge, this is the first study on the molecular diversity within the Ralstonia spp. complex in Mali. This information is important for developing wilt management strategies including breeding for resistance and specifying quarantine restrictions.

Eco-friendly Management of Bacterial Wilt in Tomato Using Dried Powder of the Wild Arid-Land Medicinal Shrub Rhazya stricta.

Bacterial wilt (BW) disease caused by Ralstonia solanacearum species complex is a devastating plant disease that inflicts heavy losses to the large number of economic host plants it infects. In this study, the potential of dried powder of the arid-land medicinal shrub Rhazya stricta to control BW of tomato was explored. Both, in vitro and in planta studies were conducted, using different concentrations of dried powder of plant parts, and applied (surface mulched or mixed) to infested soil at 0, 10, and 20 days before transplanting (DBT). Aqueous extract of leaves (16% w/v) was found to be as effective as streptomycin (100 ppm) in inhibiting the in vitro growth of R. solanacearum. As evident from the scanning electron micrograph, 16% aqueous extract of leaves produced severe morphological changes, such as rupture of the bacterial cell walls. Results from the greenhouse experiments demonstrated that the higher powder dose (succulent shoot), namely, 30 g/kg of soil mixed with infested soil 20 DBT, was found to be the most effective in controlling BW. It increased root length (cm), shoot length (cm), and plant fresh biomass (g) by 55, 42, and 40%, respectively, over control plants. Mixing of plant powder with the artificially infested (35 ml of 108 CFU/ml per kilogram of soil) pot soil was better than surface mulching. The 30 g/kg of soil dose mixed with soil increased root length (cm), shoot length (cm), and plant fresh biomass (g) of treated plants by 67, 36, and 46%, respectively, over control plants. A 37% decrease in disease severity over the control was observed with drench application of 30 g of powder per kilogram of soil applied once at 20 DBT. Our results indicated that the dried powder (30 g/kg of soil) of leaves or succulent shoots of R. stricta, thoroughly mixed with soil, 20 DBT, could act as an effective control method against BW.

Super-Multiple Deletion Analysis of Type III Effectors in Ralstonia solanacearum OE1-1 for Full Virulence Toward Host Plants.

Ralstonia solanacearum species complex (RSSC) posses extremely abundant type III effectors (T3Es) that are translocated into plant cells via a syringe-like apparatus assembled by a type III secretion system (T3SS) to subvert host defense initiated by innate immunity. More than 100 T3Es are predicted among different RSSC strains, with an average of about 70 T3Es in each strain. Among them, 32 T3Es are found to be conserved among the RSSC and hence called the core T3Es. Here, we genetically characterized contribution of abundant T3Es to virulence of a Japanese RSSC strain OE1-1 toward host plants. While all the T3Es members of AWR family contributed slightly to virulence, those of the GALA, HLK, and SKWP families did not influence full virulence of OE1-1. Mutant OE1-1D21E (with deletion of all 21 T3Es members of four families) exhibited slightly impaired virulence, while mutant OE1-1D36E (deleting all 21 T3Es of 4 families and 15 core T3Es) exhibited substantially reduced virulence. Mutant OE1-1D42E (deleting all 21 T3Es of 4 families, 15 core T3Es and 6 extended core T3Es) failed to cause any disease on tobacco plants with leaf infiltration but retained faint virulence on tobacco plants with petiole inoculation. The proliferation of mutant OE1-1D42E in tobacco stems was substantially impaired with about three orders of magnitude less than that of OE1-1, while no impact in tobacco leaves if directly infiltrated into leaves. On the contrary, the OE1-1D42E mutant retained faint virulence on eggplants with leaf infiltration but completely lost virulence on eggplants with root-cutting inoculation. The proliferation of OE1-1D42E mutant both in eggplant leaves and stems was substantially impaired. Intriguingly, mutant OE1-1D42E still caused necrotic lesions in tobacco and eggplant leaves, indicating that some other than the 42 removed effectors are involved in expansion of necrotic lesions in host leaves. All taken together, we here genetically demonstrated that all the core and extended core T3Es are nearly crucial for virulence of OE1-1 toward host plants and provided currently a kind of T3Es-free strain that enables primary functional studies of individual T3Es in host cells.

Molecular Diversity and Pathogenicity of Ralstonia solanacearum Species Complex Associated With Bacterial Wilt of Potato in Rwanda

Bacterial wilt (BW), caused by Ralstonia solanacearum species complex (RSSC), leads to substantial potato yield losses in Rwanda. Studies were conducted to (i) determine the molecular diversity of RSSC strains associated with BW of potato, (ii) generate an RSSC distribution map for epidemiological inferences, and (iii) test the pathogenicity of predominant RSSC phylotypes on six commercial potato cultivars. In surveys conducted in 2018 and 2019, tubers from wilting potato plants were collected for pathogen isolation. DNA was extracted from 95 presumptive RSSC strain colonies. The pathogen was phylotyped by multiplex PCR and typed at sequevar level. Phylotype II sequevar 1 strains were then haplotyped using multilocus tandem repeat sequence typing (TRST) schemes. Pathogenicity of one phylotype II strain and two phylotype III strains were tested on cultivars Kinigi, Kirundo, Victoria, Kazeneza, Twihaze, and Cruza. Two RSSC phylotypes were identified, phylotype II (95.79%, n = 91) and phylotype III (4.21%, n = 4). This is the first report of phylotype III strains from Rwanda. Phylotype II strains were identified as sequevar 1 and distributed across potato growing regions in the country. The TRST scheme identified 14 TRST haplotypes within the phylotype II sequevar 1 strains with moderate diversity index (HGDI = 0.55). Mapping of TRST haplotypes revealed that a single TRST '8-5-12-7-5' haplotype plays an important epidemiological role in BW of potato in Rwanda. None of the cultivars had complete resistance to the tested phylotypes; the level of susceptibility varied among cultivars. Cultivar Cruza, which is less susceptible to phylotype II and III strains, is recommended when planting potatoes in the fields with history of BW.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Detection of Ralstonia pseudosolanacearum in drain water based on concentration, enrichment and the use of a duplex TaqMan PCR test

The Ralstonia solanacearum species complex (RSSC) can cause bacterial wilt in a wide variety of plant species, including a number of ornamental glasshouse crops. Recently in Europe, ornamental rose plants for the production of cut flowers and propagation materials have been strongly affected by Ralstonia pseudosolanacearum, phylotype I, biovar 3. To test for the presence of the pathogen in the glasshouse, sampling of water from a drainage gutter or well may be an efficient strategy since it is known that RSSC can be released from infected root systems in the water. A protocol was developed to detect low densities of R. pseudosolanacearum in drain water collected from rose growers. Drain water was filtered through a bacterial filter, the filtrate was collected and target bacteria enriched for 48 h in Semi‐selective Medium South Africa (SMSA) broth supplemented with sterilized tomato plant extracts. DNA extracted from the enrichment broth was analysed using a TaqMan test in a duplex format, based on specific egl sequences of RSSC and the use of an extraction and amplification control. The optimized protocol had a detection level of ≤1–10 colony forming units of R. pseudosolanacearum in drain water.

Potential evolutionary impact of integrative and conjugative elements (ICEs) and genomic islands in the Ralstonia solanacearum species complex

Ralstonia solanacearum, a soil-borne plant pathogen, encompasses a large number of strains known as R. solanacearum species complex (RSSC). Although it has been suggested that mobile genetic elements (MGEs) may play an important role in the RSSC genome, the evolutionary impact of these elements remains unknown. Here, we identified and analysed Integrative and Conjugative Elements (ICEs) and Genomic Islands (GIs) in the 121 genomes published for Ralstonia spp., including RSSC strains and three other non-plant pathogenic Ralstonia spp. Our results provided a dataset of 12 ICEs and 31 GIs distributed throughout Ralstonia spp. Four novel ICEs in RSSC were found. Some of these elements cover 5% of the host genome and carry accessory genes with a potential impact on the fitness and pathogenicity of RSSC. In addition, phylogenetic analysis revealed that these MGEs clustered to the same species, but there is evidence of strains from different countries that host the same element. Our results provide novel insight into the RSSC adaptation, opening new paths to a better understanding of how these elements affect this soil-borne plant pathogen.

Phylogenetic analysis and molecular signatures specific to the Ralstonia solanacearum species complex

Ralstonia solanacearum species complex is a highly destructive bacterial plant pathogen recognized as a group of genotypically-diverse strains with broad geographic distribution and wide host range. Strains are divided into four phylotypes (I, II, III and IV) relating to their geographical origins. Based on the polyphasic approach, phylotypes I and III have been reclassified as R. pseudosolanacearum, phylotype IV as R. syzygii species and phylotype II as R. solanacearum. The large data sets of whole genome sequences for the members of R. solanacearum allow us to discover novel molecular markers to evaluate the phylogenetic relationships within this species. In the phylogenetic tree based on concatenated amino acid sequences of 57 housekeeping genes, members of R. solanacearum species complex formed strongly supported clades that clearly distinguished as R. solanacearum, R. pseudosolanacearum and R. syzygii species. In this study, the conserved signature indels (CSIs) which are specifically present in protein sequences and can be used to differentiate various groups of Ralstonia species are reported. Comparative analysis of whole protein sequences from the three species has identified seven CSIs that are specific to R. solanacearum (phylotype II), 6 CSIs specific to R. pseudosolanacearum strains (phylotype I and III) and five CSIs that are specifically shared by members of R. syzygii (phylotype IV). These CSIs are flanked by conserved sequence regions, providing highly specific tools for identification of previously known or novel strains of these species.

Diversity and Evolutionary Dynamics of Antiphage Defense Systems in Ralstonia solanacearum Species Complex.

Over the years, many researchers have reported a great diversity of bacteriophages infecting members of the Ralstonia solanacearum species complex (RSSC). This diversity has driven bacterial evolution by leading the emergence and maintenance of bacterial defense systems to combat phage infection. In this work, we present an in silico study of the arsenal of defense systems that RSSC harbors and their evolutionary history. For this purpose, we used a combination of genomic, phylogenetic and associative methods. We found that in addition to the CRISPR-Cas system already reported, there are eight other antiphage defense systems including the well-known Restriction-Modification and Toxin-Antitoxin systems. Furthermore, we found a tenth defense system, which is dedicated to reducing the incidence of plasmid transformation in bacteria. We undertook an analysis of the gene gain and loss patterns of the defense systems in 15 genomes of RSSC. Results indicate that the dynamics are inclined toward the gain of defense genes as opposed to the rest of the genes that were preferably lost throughout evolution. This was confirmed by evidence on independent gene acquisition that has occurred by profuse horizontal transfer. The mutation and recombination rates were calculated as a proxy of evolutionary rates. Again, genes encoding the defense systems follow different rates of evolution respect to the rest of the genes. These results lead us to conclude that the evolution of RSSC defense systems is highly dynamic and responds to a different evolutionary regime than the rest of the genes in the genomes of RSSC.

Comparison of bacterial communities in soil samples with and without tomato bacterial wilt caused by Ralstonia solanacearum species complex

BackgroundRalstonia solanacearum is one of the most notorious soil-borne phytopathogens. It causes a severe wilt disease with deadly effects on many economically important crops. The microbita of disease-suppressive soils are thought that they can contribute to the disease resistance of crop plants, thus, evaluation of the microbial community and their interaction characteristics between suppressive soil (SS) and conducive soil (CS) will help to understand resistance mechanism. To do this, the bacterial community structure, correlation analysis with soil chemical properties, interaction network of SS (nearly no disease in three years), and CS (suffered heavy bacterial wilt disease) were analyzed.ResultsA higher bacterial community diversity index was found in SS, the relative abundance of Nocardioides, Gaiella and norank_f_Anaerolineaceae were significantly more than that of the CS. Moreover, the relative abundance of main genera Bacillus, norank_o_Gaiellales, Roseiflexus, and norank_o_Gemmatimonadaceae were significantly more than that of the CS. Redundancy analysis at the genus level indicated that the available phosphate played a key role in the bacterial community distribution, and its role was negatively correlated with soil pH, organic matter content, alkali-hydrolyzable nitrogen, and available potassium contents. Interaction network analysis further demonstrated that greater diversity at the genus level existed in the SS network and formed a stable network. Additionally, the species of Mycobacterium, Cyanobacteria, and Rhodobiaceae are the key components that sustain the network stability. Seven clusters of orthologous groups exhibited significant differences between SS and CS. Moreover, 55 bacterial strains with distinct antagonistic activities to R. solancearum were isolated and identified from the healthy tomato plant rhizosphere soil of the CS.ConclusionsOur findings indicate that the bacterial diversity and interaction network differed between the CS and SS samples, providing a good foundation in the study of bacterial wilt.

Construction of a single nucleotide polymorphism marker based QTL map and validation of resistance loci to bacterial wilt caused by Ralstonia solanacearum species complex in tomato

Bacterial wilt (BW), caused by Ralstonia solanacearum species complex is one of the major biotic factors limiting tomato production in the humid tropics. Pyramiding of resistance genes through marker-assisted selection is an efficient way to develop durable BW resistant cultivars. Tomato line ‘Hawaii 7996’ (H7996) is a stable and robust resistance source against various strains of the species complex. Major BW resistance quantitative trait loci (QTLs) Bwr-12 and Bwr-6, and several minor or strain specific QTLs have been coarse-mapped in this line, but none has been fine-mapped and validated. The objective of the current study was to construct a high density genetic map using single-nucleotide polymorphism (SNP) markers derived from genotyping-by-sequencing, fine-map Bwr-12 and Bwr-6 and determine the effects of these QTLs using a near isogenic line (NIL) population. A high density genetic map using 1604 SNP markers with an average distance of 0.82 cM was developed for 188 F9 recombinant inbred lines derived from the cross H7996 × WVa700. A total of seven QTLs associated with BW resistance to race 1-phylotype I (R. pseudosolanacearum) or/and race 3-phylotype II (R. solanacearum) strains were located on chromosomes 6 (Bwr-6.1, 6.2, 6.3 and 6.4) and 12 (Bwr-12.1, Bwr-12.2 and Bwr-12.3) with logarithm of odds (LOD) scores of 6.2–15.6 and 6.2–31.1, explaining 14.2–33.4% and 15.9–53.9% of the total phenotypic variation contributed from H7996, respectively. To validate the genetic effects of the two QTL regions, a set of 80 BC3F3 NILs containing different sections of Bwr-6 with or without Bwr-12 was phenotyped for disease severity after challenge with either race 1-phylotype I Pss4 or race 3-phylotype II Pss1632 BW strains over two seasons. Bwr-6.1 specific to Pss4 and Bwr-6.3 specific to Pss1632 were mapped to an interval of 5.0 cM (P < 0.05) between 6_33,444,000_SLM6-47 and 6_33,868,000_SLM6-124 SNP marker, and to 2.7 cM (P < 0.01) between positions 6_35,949,000 _SLM6-107 to 6_36,750,000_SLM6-82 marker, respectively. In addition, the specific effect of Bwr-12 for resistance to Pss4 (LOD score of 5.8-16.1, P < 0.01) was confirmed.

In silico cloning, expression and bioinformatics analysis of StZnT11 in Solanum tuberosum

Solanum tuberosum Zinc transporter 11 (StZnT11) is very important for maintaining zinc homeostasis in cells. The study on the expression of StZnT11 under abiotic stress and biotic stress laid a foundation for verifying the role of potato StZnT11 in the process of biotic stress of Ralstonia solanacearum species complex. According to the designated EST sequence, the homology of the original sequence was analyzed by using the Blast tool in NCBI, and a homologous object sequence with the highest similarity, coverage and e expectation value was selected. StZnT11 gene is obtained by Silico Cloning. The sequence and coding amino acid composition, physicochemical properties, molecular evolution, phosphorylation site and advanced structure of Solanum tuberosum StZnT11 gene were analyzed by bioinformatics method. The results showed that the cDNA gene is 1 300 bp in length, encoding a protein containing 348 amino acid residues, including 23 phosphorylation sites, one signal peptide and nine transmembrane regions, and is a hydrophobic protein located the plasma membrane. Through amino acid sequence alignment, StZnT11 protein has a high homology with zinc transporter from tobacco, tomato, pepper and other plants. The results of real-time fluorescence quantitative polymerase chain reaction showed that, StZnT11 is up-regulated by different concentrations of exogenous plant hormone abscisic acid (ABA). Tissue localization showed that StZnT11 was mainly expressed in specific tissues (phloem and leaf vascular bundles of stem vascular system). These results provide a theoretical basis for further experimental cloning and functional verification of the gene.

Ralstonia solanacearum causing potato bacterial wilt: host range and cultivars’ susceptibility in Rwanda

AbstractMembers of the Ralstonia solanacearum species complex (RSSC), causing potato bacterial wilt or brown rot, are highly contagious and there are no known cultivars with durable resistance to the pathogen. This study hypothesized (a) that crops intercropped or rotated with potato, plants in the same family, and plants grown in the neighbouring fields can host the pathogen and they can be potential sources of primary inoculum, and (b) that potato cultivars currently multiplied by the public tissue culture laboratory in Rwanda are less susceptible to the pathogen. Fourteen plant species and potato, and nine potato cultivars were tested for susceptibility to an RSSC phylotype II strain under greenhouse conditions. The bacteria induced symptoms on potato, tomato, tree tomato, sweet pepper, and eggplant only. Among the plant species with symptoms, potato, tomato, and tree tomato wilted completely. There was a significant difference in days to symptom expression and to complete wilting (p &lt; .0001). While all tested potato cultivars were found to be susceptible, the number of days to first symptom expression, days to complete wilting, area under the disease progress curve (AUDPC), and the number and weight of harvested tubers varied considerably. Cultivars Cruza, Kinigi, and CIP‐58 were less susceptible whereas the cultivars Gikungu, Kirundo, and Victoria were highly susceptible. There is a strong need to search for other sources of resistance. The results indicate that some plant species that might serve as a reservoir of the bacterium should be avoided in the vicinity of potato crops.

Characterization 3333 of tomato (Solanum lycopersicum) accessions for resistance to phylotype I and phylotype II strains of the Ralstonia solanacearum species complex under high temperatures

AbstractBacterial wilt of tomato caused by Ralstonia solanacearum species complex (RSSC) causes substantial yield losses in the tropics and subtropics. Disease management options by chemicals are limited, and host resistance is the cheapest and easiest means of control. However, sources of bacterial wilt resistance in tomato are limited. The disease often coincides with higher temperatures in the tropics, and resistance sources that are more heat stable are particularly valuable for breeding of tropically adapted tomato cultivars. The objectives of this study were to identify tomato accessions that demonstrate relatively high bacterial wilt resistance under high temperatures and to identify accessions that may possess QTLs other than Bwr‐6 and Bwr‐12 (two major disease resistance QTLs against bacterial wilt), which could be exploited in future breeding. Sixty‐seven tomato entries reported as bacterial wilt resistant were evaluated in a greenhouse against one strain each of phylotype I (Pss4) and phylotype IIB (Pss1632) of the RSSC (average temperature ≥29°C). Of those, five and 19 were homozygous for Bwr‐6 and Bwr‐12, respectively, and six were homozygous for both QTLs. Bwr‐12 contributed to resistance against phylotype I strain but not against the phylotype II strain. Bwr‐6 contributed to resistance against both phylotype strains. Entries with both QTLs as a group performed relatively better against the phylotype I strain. Entry “94T765‐24‐79”, which lacked Bwr‐6 and Bwr‐12, demonstrated relatively high resistance against the phylotype II strain and may carry new QTL/s. As new bacterial wilt resistance QTLs are mapped and markers designed, pyramiding multiple bacterial wilt resistance QTLs into new varieties should be straightforward, thereby increasing the chances of obtaining stable resistance.