Real-time monitoring of Ralstonia solanacearum infection progress in tomato and Arabidopsis using bioluminescence imaging technology

Background Ralstonia solanacearum, one of the most devastating bacterial plant pathogens, is the causal agent of bacterial wilt. Recently, several studies on resistance to bacterial wilt have been conducted using the Arabidopsis-R. solanacearum system. However, the progress of R. solanacearum infection in Arabidopsis is still unclear. Results We generated a bioluminescent R. solanacearum by expressing plasmid-based luxCDABE. Expression of luxCDABE did not alter the bacterial growth and pathogenicity. The light intensity of bioluminescent R. solanacearum was linearly related to bacterial concentrations from 104 to 108 CFU·mL−1. After root inoculation with bioluminescent R. solanacearum strain, light signals in tomato and Arabidopsis were found to be transported from roots to stems via the vasculature. Quantification of light intensity from the bioluminescent strain accurately reported the difference in disease resistance between Arabidopsis wild type and resistant mutants. Conclusions Bioluminescent R. solanacearum strain spatially and quantitatively measured bacterial growth in tomato and Arabidopsis, and offered a tool for the high-throughput study of R. solanacearum-Arabidopsis interaction in the future.

Epidemic networks and potential sources of bacterial wilt infection in a potato seed network in Kenya.

Potato seed systems in Kenya are largely informal, characterized by high seed degeneration due to the buildup of seed- and soil-borne diseases, including bacterial wilt caused by Ralstonia solanacearum. Informal sources of seed include neighbors, local markets and farmer-saved seed, and present a risk for spread and establishment of disease. To understand the larger context of potato disease risk in Kenya, we used network analysis to evaluate (1) epidemic risk through potato trade networks centered around East Africa, and (2) locations in East Africa likely to be particularly important for epidemic management because of their high potato cropland connectivity. We evaluated the interactions of the key stakeholders in a potato seed system and used network analysis to identify locations that are likely to be important for the spread of infection of R. solanacearum in a potato seed distribution network in Meru, Kenya. Household details, seed sources, quantities sold, pest incidence and management practices, knowledge about seed degeneration and farmers' sources of information on potato production were obtained and analyzed. The survey revealed that self-saved, neighbors, seed companies, friends, exchange, and markets are the main seed sources. Only 43% of total seed transacted was certified. Users of uncertified seeds have high disease risk, and this is an especially important risk if their roles in the network give them the potential to be 'superspreaders' of disease.

Isolation and Complete Genome Sequence Analysis of Kosakonia cowanii Pa82, a Novel Pathogen Causing Bacterial Wilt on Patchouli

Pogostemon cablin (patchouli), an important medicinal and aromatic plant, is widely used in traditional Chinese medicine as well as in perfume industry. Patchouli plants are susceptible to bacterial wilt disease, which causes significant economic losses by reduction in yield and quality of the plant products. However, few studies focus on the pathogens causing bacterial wilt on patchouli. In this study, strain Pa82 was isolated from diseased patchouli plants with typical bacterial wilt symptoms in Guangdong province, China, and was confirmed to be a highly virulent pathogen of patchouli bacterial wilt. Comparative sequence analysis of 16S rRNA gene showed that the strain was closely related to Kosakonia sp. CCTCC M2018092 (99.9% similarity) and Kosakonia cowanii Esp_Z (99.8% similarity). Moreover, phylogenetic tree based on 16S rRNA gene sequences showed that the strain was affiliated with genus Kosakonia. Further, the whole genome of strain Pa82 was sequenced, and the sequences were assembled and annotated. The complete genome of the strain consists of one chromosome and three plasmids. Average nucleotide identity (ANI) and phylogenetic analysis revealed that the strain belongs to Kosakonia cowanii (designated Kosakonia cowanii Pa82). Virulence-related genes of the strain involved in adherence, biofilm formation, endotoxin and other virulence factors were predicted. Among them, vgrG gene that encodes one of the type VI secretion system components was functionally validated as a virulence factor in Kosakonia cowanii Pa82 through construction of Tn5 insertion mutants and identification of mutant defective in virulence.

Biocontrol Potential of Chitin and Chitosan Extracted from Black Soldier Fly Pupal Exuviae against Bacterial Wilt of Tomato

Globally, Ralstonia solanacearum (Smith) is ranked one of the most destructive bacterial pathogens inducing rapid and fatal wilting symptoms on tomatoes. Yield losses on tomatoes vary from 0 to 91% and most control measures are unaffordable to resource-poor farmers. This study investigated the antimicrobial activities of chitin and chitosan extracted from black soldier fly (BSF) pupal exuviae against R. solanacearum. Morphological, biochemical, and molecular techniques were used to isolate and characterize R. solanacearum for in vitro pathogenicity test using disc diffusion technique. Our results revealed that BSF chitosan significantly inhibited the growth of R. solanacearum when compared to treatments without chitosan. However, there was no significant difference in the antibacterial activities between BSF and commercial chitosan against R. solanacearum. Soil amended with BSF-chitin and chitosan demonstrated a reduction in bacterial wilt disease incidence by 30.31% and 34.95%, respectively. Whereas, disease severity was reduced by 22.57% and 23.66%, when inoculated tomato plants were subjected to soil amended with BSF chitin and chitosan, respectively. These findings have demonstrated that BSF pupal shells are an attractive renewable raw material for the recovery of valuable products (chitin and chitosan) with promising ability as a new type of eco-friendly control measure against bacterial wilt caused by R. solanacearum. Further studies should explore integrated pest management options that integrate multiple components including insect-based chitin and chitosan to manage bacterial wilt diseases, contributing significantly to increased tomato production worldwide.

Metabolomic Evaluation of Ralstonia solanacearum Cold Shock Protein Peptide (csp22)-Induced Responses in Solanum lycopersicum

Ralstonia solanacearum, the causal agent of bacterial wilt, is one of the most destructive bacterial plant pathogens. This is linked to its evolutionary adaptation to evade host surveillance during the infection process since many of the pathogen’s associated molecular patterns escape recognition. However, a 22-amino acid sequence of R. solanacearum-derived cold shock protein (csp22) was discovered to elicit an immune response in the Solanaceae. Using untargeted metabolomics, the effects of csp22-elicitation on the metabolome of Solanum lycopersicum leaves were investigated. Additionally, the study set out to discover trends that may suggest that csp22 inoculation bestows enhanced resistance on tomato against bacterial wilt. Results revealed the redirection of metabolism toward the phenylpropanoid pathway and sub-branches thereof. Compared to the host response with live bacteria, csp22 induced a subset of the discriminant metabolites, but also metabolites not induced in response to R. solanacearum. Here, a spectrum of hydroxycinnamic acids (especially ferulic acid), their conjugates and derivatives predominated as signatory biomarkers. From a metabolomics perspective, the results support claims that csp22 pre-treatment of tomato plants elicits increased resistance to R. solanacearum infection and contribute to knowledge on plant immune systems operation at an integrative level. The functional significance of these specialized compounds may thus support a heightened state of defense that can be applied to ward off attacking pathogens or toward priming of defense against future infections.

Metabolic Profiling of Resistant and Susceptible Tobaccos Response Incited by Ralstonia pseudosolanacearum Causing Bacterial Wilt

The causal agent of bacterial wilt, Ralstonia pseudosolanacearum, can cause significant economic losses during tobacco production. Metabolic analyses are a useful tool for the comprehensive identification of plant defense response metabolites. In this study, a gas chromatography-mass spectrometry (GC-MS) approach was used to identify metabolites differences in tobacco xylem sap in response to R. pseudosolanacearum CQPS-1 in two tobacco cultivars: Yunyan87 (susceptible to R. pseudosolanacearum) and K326 (quantitatively resistant). Metabolite profiling 7 days post inoculation with R. pseudosolanacearum identified 88 known compounds, 42 of them enriched and 6 depleted in the susceptible cultivar Yunyan87, while almost no changes occurred in quantitatively resistant cultivar K326. Putrescine was the most enriched compound (12-fold) in infected susceptible tobacco xylem, followed by methyl-alpha-d-glucopyranoside (9-fold) and arabinitol (6-fold). Other sugars, amino acids, and organic acids were also enriched upon infection. Collectively, these metabolites can promote R. pseudosolanacearum growth, as shown by the increased growth of bacterial cultures supplemented with xylem sap from infected tobacco plants. Comparison with previous metabolic data showed that beta-alanine, phenylalanine, and leucine were enriched during bacterial wilt in both tobacco and tomato xylem.

Identification of candidate type 3 effectors that determine host specificity associated with emerging Ralstonia pseudosolanacearum strains

Ralstonia pseudosolanacearum (Rps), previously known as R. solanacearum phylotypes I and III is one of the causal agents of bacterial wilt, a devastating disease that affects more than 250 plant species. Emerging Rps strains were identified infecting new hosts. P824 Rps strain was isolated from blueberry in Florida. Rps strains including PD7123 were isolated from hybrid tea roses in several countries through Europe. P781 is a representative strain of Rps commonly found on mandevilla in Florida. UW757 is a strain isolated from osteospermum plants originating in Guatemala. These strains are phylogenetically closely related and of economic importance on their respective hosts. The objective of this study is to associate the Type 3 Effectors (T3Es) repertoire of these four strains with host specificity. Candidate T3E associated with host specificity to blueberry, tea rose, osteospermum, and mandevilla were identified by sequence homology. Pathogenicity assays on 8 hosts including, blueberry, mandevilla, osteospermum and tea rose with the 4 strains showed that both P824 and PD7123 are pathogenic to blueberry and tea rose. P781 is the only strain pathogenic to mandevilla and P824 is the only strain non-pathogenic to osteospermum. Hypotheses based on correlation of T3E presence/absence and pathogenicity profiles identified 3 candidate virulence and 3 avirulence T3E for host specificity to blueberry and tea rose. Two candidate avirulence T3E were identified for mandevilla, and one candidate virulence for osteospermum. The strategy applied here can be used to reduce the number of host specificity candidate genes in closely related strains with different hosts.