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.

Revisiting the source of wilt symptoms: X-ray microcomputed tomography provides direct evidence that Ralstonia biomass clogs xylem vessels

Ralstonia cause wilt diseases by colonizing xylem vessels and disrupting water transport. The dogma is that bacterial biomass clogs vessels and reduces the flow of xylem sap due to Ralstonia abundance. However, the physiological mechanism of xylem disruption during bacterial wilt is untested. Using a tomato and Ralstonia pseudosolanacearum GMI1000 model, we visualized and quantified spatiotemporal dynamics of xylem disruption during bacterial wilt. First, we measured stomatal conductance of leaflets on mock-inoculated and wilt-symptomatic plants. Wilted leaflets had reduced stomatal conductance, as did turgid leaflets on the same petiole as wilted leaflets. Next, we used X-ray microcomputed tomography (X-ray microCT) and light microscopy to differentiate between mechanisms of xylem disruption: blockage by bacterial biomass, blockage by vascular tyloses, or sap displacement by gas embolisms. We imaged intact plant stems to quantify embolized vessels. Embolized vessels were rare, but infected plants with low bacterial populations had a non-significant trend of more vessel embolisms. To test that vessels are clogged during bacterial wilt, we imaged excised stems after brief dehydration. Most vessels in mock-infected plants emptied their contents after excision, but non-conductive clogged vessels were abundant in infected plants by 2 days post infection. At wilt onset when bacterial populations exceeded 5x108 cfu/g stem tissue, approximately half of the vessels were clogged with electron-dense bacterial biomass. We found no evidence of tyloses in X-ray microCT reconstructions or from light microscopy of preserved stems. Therefore, bacterial blockage of vessels appears to be the principal cause of xylem disruption during Ralstonia wilt.

Light modulates important physiological features of Ralstonia pseudosolanacearum during the colonization of tomato plants

Ralstonia pseudosolanacearum GMI1000 (Rpso GMI1000) is a soil-borne vascular phytopathogen that infects host plants through the root system causing wilting disease in a wide range of agro-economic interest crops, producing economical losses. Several features contribute to the full bacterial virulence. In this work we study the participation of light, an important environmental factor, in the regulation of the physiological attributes and infectivity of Rpso GMI1000. In silico analysis of the Rpso genome revealed the presence of a Rsp0254 gene, which encodes a putative blue light LOV-type photoreceptor. We constructed a mutant strain of Rpso lacking the LOV protein and found that the loss of this protein and light, influenced characteristics involved in the pathogenicity process such as motility, adhesion and the biofilms development, which allows the successful host plant colonization, rendering bacterial wilt. This protein could be involved in the adaptive responses to environmental changes. We demonstrated that light sensing and the LOV protein, would be used as a location signal in the host plant, to regulate the expression of several virulence factors, in a time and tissue dependent way. Consequently, bacteria could use an external signal and Rpsolov gene to know their location within plant tissue during the colonization process.

Diversity of Ralstonia pseudosolanacearum, the causal agent of bacterial wilt on Eucalyptus pellita in Indonesia

Siregar BA, Giyanto, Hidayat SH, Siregar IZ, Tjahjono B. 2021. Diversity of Ralstonia pseudosolanacearum, the causal agent of bacterial wilt on Eucalyptus pellita in Indonesia. Biodiversitas 22: 2538-2545. The Ralstonia species complex was initially classified into five races and five biovars but the classification could not accommodate the isolates' phylogenetic history or geographic origins. A phylotype and sequevar system is based on the geographic distribution and characteristics of endoglucanase (egl) and hypersensitive response and pathogenicity (hrp) gene sequences. This study aims to describe pathogen diversity of the causal agent of bacterial wilt on Eucalyptus pellita F. Muell. Pathogens were isolated from wilting seedlings and trees in several Eucalyptus plantations. The phenotypic diversity analysis included biovar, exopolysaccharide quantification and virulence tests, while genotypic diversity included phylotypes and sequevar determination based on egl gene sequences. A total of 35 strains were isolated from the field and nurseries of Eucalyptus in various locations. All isolates were confirmed as Ralstonia species complex based on morphological characteristics and molecular studies using species-specific primers. These isolates were dominantly classified as biovar 3 and 4 and had a high variation on virulence and EPS production. Based on the egl sequence's alignment, 29 strains of Phylotype I are grouped into four sequevar references (sequevars 14, 17, 18, 30) and new a sequevar 58. This study shows that strains of R. pseudosolanacearum causing bacterial wilt on E. pellita in Indonesia have high phenotypic and genotypic diversities.

Low Temperature Induces Infectious Nonresuscitatable VBNC Cells in Ralstonia Pseudosolanacearum

Ralstonia pseudosolanacearum and other members of Ralstonia solanacearum species complex (RSSC) causes the disease bacterial wilt in many crops of economic importance. The organism is known to form Viable But Non Culturable cells (VBNC). VBNCs resuscitate invitro during the “resuscitation window” period and are infectious Previous studies have identified nonresuscitatable VBNCs in various bacterial genus including RSSC, however their infectivity was not elucidated and described. In this work, VBNCs of two Ralstonia pseudosolanacearum strains were generated by exposing the microcosms to psychrophilic stress, UV-C radiation and 70% isopropanol. Both resuscitatable and nonresuscitatable VBNCs were observed in psychrophilic and UV-C stressed microcosms. The nonresuscitatable VBNCs generated at psychrophilic temperature were found infective. Based on resuscitation properties, nonresuscitatable VBNCs can be considered as a different VBNC type from resuscitatable VBNCs.