Ralstonia Pseudosolanacearum Research Articles

Draft genome sequence of Streptomyces noursei strain DS30.6, isolated from forest soil in Vietnam.

Streptomyces noursei strain DS30.6 was chosen to be resistant to Ralstonia pseudosolanacearum T2C-Rasto after being isolated from Vietnamese forest soil. The draft genome of this strain was reported to be 8,863,720 bp, with an average guanine-cytosine content of 72.67%.

Unraveling the in planta population dynamics of the plant pathogen Ralstonia pseudosolanacearum by mathematical modeling.

Ralstonia pseudosolanacearum, a plant pathogen responsible for bacterial wilt in numerous plant species, exhibits paradoxical growth in the host by achieving high bacterial densities in xylem sap, an environment traditionally considered nutrient-poor. This study combined in vitro experiments and mathematical modeling to elucidate the population dynamics of R. pseudosolanacearum within plants. To simulate the xylem environment, a tomato xylem-mimicking medium was developed. Then, a mathematical model was constructed using in vitro data and employed to simulate the dynamics of bacterial density and xylem sap composition during plant infection. The model accurately reproduced in planta experimental observations, including high bacterial densities and the depletion of glutamine and asparagine. Additionally, the model estimated the minimal number of bacteria required to initiate infection, the timing of infection post-inoculation, the bacterial mortality rate within the plant and the rate at which bacterial putrescine is assimilated by the plant. The findings demonstrate that xylem sap can sustain high bacterial densities, provides an explanatory framework for the presence of acetate, putrescine and 3-hydroxybutyrate in the sap of infected xylem and give clues as to the role of putrescine in the virulence of R. pseudosolanacearum.

Complete genome sequences of two novel Ralstonia jumbo phages isolated from leaf litter compost

Two Ralstonia phages, FLC1-1B and FLC4-3B, were isolated from leaf litter compost, using Ralstonia pseudosolanacearum, which is a causal agent of bacterial wilt disease, as a host. The genomic DNA sequences of FLC1-1B and FLC4-3B were determined and found to be 290,008 bp and 291,257 bp in length, respectively, and they were therefore classified as jumbo phages. However, they did not show high similarity to any jumbo phage genomic sequence in the NCBI nt database. The closest hit in a BLAST search was the jumbo phage ripduovirus RP12, with only 35% coverage and 77% sequence identity, whereas the FLC1-1B and FLC4-3B sequences were 99.0% identical. Based on these findings, FLC1-1B and FLC4-3B should be classified as members of a new genus in the order Caudoviricetes. FLC4-3B was found to suppress wilt disease in tomato plants, suggesting that it has potential as a biocontrol agent for managing R. pseudosolanacearum infections.

Commodity risk assessment of Petunia spp. and Calibrachoa spp. unrooted cuttings from Costa Rica.

The European Commission requested the EFSA Panel on Plant Health to evaluate the probability of entry of pests (likelihood of pest freedom at entry), including both regulated and non-regulated pests, associated with unrooted cuttings of the genera Petunia and Calibrachoa produced under physical isolation in Costa Rica. The relevance of any pest for this opinion was based on evidence collected according to specific criteria, following the methodology used for high-risk plants adapted for the specificity of this assessment. Twenty-two EU regulated pests (beet curly top virus, Bemisia tabaci, Chloridea virescens, Eotetranychus lewisi, Epitrix cucumeris, Epitrix tuberis, euphorbia mosaic virus, Helicoverpa zea, Liriomyza huidobrensis, Liriomyza sativae, Liriomyza trifolii, pepper golden mosaic virus, potato spindle tuber viroid, Ralstonia pseudosolanacearum, Ralstonia solanacearum, Spodoptera ornithogalli, squash leaf curl virus, Thrips palmi, tomato golden mosaic virus, tomato leaf curl Sinaloa virus, tomato spotted wilt virus, tomato yellow leaf curl virus) and one pest that is not regulated in the EU (Aleurodicus dispersus) fulfilled all relevant criteria and were selected for further evaluation. For these pests, the risk mitigation measures proposed in the technical dossier from Costa Rica were evaluated taking into account possible factors limiting their efficacies. Additionally, an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. The estimated degree of pest freedom varies among the pests evaluated, with tomato spotted wilt virus being the pest most frequently expected on the imported cuttings. The expert knowledge elicitation indicated, with 95% certainty that between 9927 and 10,000 bags containing unrooted cuttings of Petunia spp. and Calibrachoa spp. per 10,000 would be free of tomato spotted wilt virus.

Ralstonia solanacearum (phylotype II) isolated from Rosa spp. in the Netherlands is closely related to phylotype II isolates from other sources in the Netherlands and is virulent on potato

AbstractIn 2018, during an annual survey in the Netherlands, Ralstonia solanacearum phylotype II (phy II) was found in asymptomatic greenhouse rose plants at three geographic locations. These findings were remarkable, since previous findings of Ralstonia sp. in rose always concerned Ralstonia pseudosolanacearum phylotype I (phy I). Therefore, no information was available on the virulence of R. solanacearum phy II on rose. In this study, R. solanacearum phy II isolates PD 7421 and PD 7394, isolated in 2018 from asymptomatic ornamental rose (Rosa spp.), were assessed for their virulence in two rose cultivars (“Armando” and “Red Naomi”) at two temperatures. No typical symptoms were observed for PD 7421 and PD 7394 on the two rose cultivars, irrespective of the temperature. However, latent infections upon inoculation of these isolates on rose did occur. R. solanacearum phy II is known as a major potato pathogen, where it causes brown rot. Whole genome multilocus sequence typing analysis demonstrated that the isolates from rose were closely related to R. solanacearum phy II isolates previously found in seed potato and surface water in the Netherlands. Because of this close genetic relatedness, the virulence of PD 7421 and PD 7394 was also assessed in potato plants, where both isolates caused severe disease symptoms on the shoots as well as the daughter tubers. This implies that rose can act as a reservoir for R. solanacearum phy II and, in this way, can potentially be involved in spreading this bacterium.

Paenibacillus spp. may facilitate anaerobic soil disinfestation at low temperatures

We screened for bacteria that may facilitate the suppressive effect on Ralstonia pseudosolanacearum at temperatures < 30 °C after anaerobic soil disinfestation (ASD) with 1% v/v ethanol. Among 22 isolates grown anaerobically, we selected three that significantly suppressed R. pseudosolanacearum population after ASD at 25 °C. Based on the 16 S rRNA sequences, two isolates were inferred as Paenibacillus polymyxa and the other as Paenibacillus sp. Using qPCR primers designed for these strains, we found that all strains increased in the soil after ASD at 25 °C. After ASD with 1% v/v ethanol and with or without soil inoculation with these three strains in a concrete frame in the field at mean temperature of 26.3 °C, the R. pseudosolanacearum population was significantly lower in the inoculation treatment at a depth of 10 cm. These results suggest that these Paenibacillus strains have the potential to promote the ASD effect using 1% ethanol at low temperatures.

Changes in DNA methylation contribute to rapid adaptation in bacterial plant pathogen evolution.

Adaptation is usually explained by beneficial genetic mutations that are transmitted from parents to offspring and become fixed in the adapted population. However, genetic mutation analysis alone is not sufficient to fully explain the adaptive processes, and several studies report the existence of nongenetic (or epigenetic) inheritance that can enable adaptation to new environments. In the present work, we tested the hypothesis of the role of DNA methylation, a form of epigenetic modification, in adaptation of the plant pathogen Ralstonia pseudosolanacearum to the host during experimental evolution. Using SMRT-seq technology, we analyzed the methylomes of 31 experimentally evolved clones obtained after serial passages on 5 different plant species during 300 generations. Comparison with the methylome of the ancestral clone revealed a list of 50 differential methylated sites (DMSs) at the GTWWAC motif. Gene expression analysis of the 39 genes targeted by these DMSs revealed limited correlation between differential methylation and differential expression of the corresponding genes. Only 1 gene showed a correlation, the RSp0338 gene encoding the EpsR regulator protein. The MSRE-qPCR technology, used as an alternative approach for DNA methylation analysis, also found the 2 DMSs upstream RSp0338. Using site-directed mutagenesis, we demonstrated the contribution of these 2 DMSs in host adaptation. As these DMSs appeared very early in the experimental evolution, we hypothesize that such fast epigenetic changes can allow rapid adaptation to the plant stem environment. In addition, we found that the change in DNA methylation upstream RSp0338 remains stable at least for 100 generations outside the host and thus can contribute to long-term adaptation to the host plant. To our knowledge, this is the first study showing a direct link between bacterial epigenetic variation and adaptation to a new environment.

Contribution of the Sensor Histidine Kinases PhcS and VsrA to the Quorum Sensing of Ralstonia pseudosolanacearum Strain OE1-1.

The soilborne Gram-negative phytopathogenic beta-proteobacterium Ralstonia pseudosolanacearum strain OE1-1 produces methyl 3-hydroxymyristate (3-OH MAME) as the quorum sensing (QS) signal by the methyltransferase PhcB and senses the chemical, activating the LysR family transcriptional regulator PhcA, which regulates the QS-dependent genes responsible for QS-dependent phenotypes including virulence. The sensor histidine kinases PhcS and VsrA are reportedly involved in the regulation of QS-dependent genes. To elucidate the function of PhcS and VsrA in the active QS, we generated the phcS-deletion and vsrA-deletion mutants, which exhibited weak changes to their QS-dependent phenotypes including virulence. The phcS and vsrA-deletion mutant (ΔphcS/vsrA) had significant changes in its QS-dependent phenotypes and was nonvirulent, similar to the phcA-deletion mutant. The mutant (PhcS-H230Q) with a substitution of histidine to glutamine at amino acid position 230 in PhcS but not the mutant (VsrA-H256Q) with a substitution of histidine to glutamine at amino acid position 256 in VsrA exhibited significant changes in QS-dependent phenotypes and lost virulence. The transcriptome analysis with RNA-sequencing revealed significant alterations to the expression of QS-dependent genes in the ΔphcS/vsrA and PhcS-H230Q but not VsrA-H256Q, similar to the phcA-deletion mutant. The exogenous 3-OH MAME application led to a significantly enhanced QS-inducible major exopolysaccharide EPS I production of the strain OE1-1 and phcB-deletion mutant but not ΔphcS/vsrA and PhcS-H230Q. Collectively, results of the present genetic study suggested that PhcS contributes to QS along with VsrA and that histidine at amino acid position 230 of PhcS is required for 3-OH MAME sensing, thereby influencing QS-dependent phenotypes including virulence of the strain OE1-1. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

Plant-induced bacterial gene silencing: a novel control method for bacterial wilt disease.

Ralstonia pseudosolanacearum, a notorious phytopathogen, is responsible for causing bacterial wilt, leading to significant economic losses globally in many crops within the Solanaceae family. Despite various cultural and chemical control strategies, managing bacterial wilt remains a substantial challenge. This study demonstrates, for the first time, the effective use of plant-induced bacterial gene silencing against R. pseudosolanacearum, facilitated by Tobacco rattle virus-mediated gene silencing, to control bacterial wilt symptoms in Nicotiana benthamiana. The methodology described in this study could be utilized to identify novel phytobacterial virulence factors through both forward and reverse genetic approaches. To validate plant-induced gene silencing, small RNA fractions extracted from plant exudates were employed to silence bacterial gene expression, as indicated by the reduction in the expression of GFP and virulence genes in R. pseudosolanacearum. Furthermore, treatment of human and plant pathogenic Gram-negative and Gram-positive bacteria with plant-generated small RNAs resulted in the silencing of target genes within 48 hours. Taken together, the results suggest that this technology could be applied under field conditions, offering precise, gene-based control of target bacterial pathogens while preserving the indigenous microbiota.

Sigma S Involved in Bacterial Survival of Ralstonia pseudosolanacearum

Ralstonia pseudosolanacearum, a plant pathogenic bacterium that can survive for a long time in soil and water, causes lethal wilt in the Solanaceae family. Sigma S is a part of the RNA polymerase complex, which regulates gene expression during bacterial stress response or stationary phase. In this study, we investigated the role of sigma S in R. pseudosolanacearum under stress conditions using a rpoS-defective mutant strain of R. pseudosolanacearum and its wild-type strain. The phenotypes of rpoS-defective mutant were complemented by introducing the original rpoS gene. There were no differences observed in bacterial growth rate and exopolysaccharide production between the wild-type strain and the rpoS mutant. However, the wild-type strain responded more sensitively to nutrient deficiency compared to the mutant strain. Under the nutrient deficiency, the rpoS mutant maintained a high bacterial viability for a longer period, while the viability of the wild-type strain declined rapidly. Furthermore, a significant difference in pH was observed between the culture supernatant of the wild-type strain and the mutant strain. The pH of the culture supernatant for the wild-type strain decreased rapidly during bacterial growth, leading to medium acidification. The rapid decline in the wild-type strain’s viability may be associated with medium acidification and bacterial sensitivity to acidity during transition to the stationary phase. Interestingly, the rpoS mutant strain cannot utilize acetic acid, D-alanine, D-trehalose, and L-histidine. These results suggest that sigma S of R. pseudosolanacearum regulates the production or utilization of organic acids and controls cell death during stationary phase under nutrient deficiency.

The Micacocidin Production-Related RSc1806 Deletion Alters the Quorum Sensing-Dependent Gene Regulation of Ralstonia pseudosolanacearum Strain OE1-1.

The soil-borne phytopathogenic gram-negative bacterium Ralstonia solanacearum species complex (RSSC) produces staphyloferrin B and micacocidin as siderophores that scavenge for trivalent iron (Fe3+) in the environment, depending on the intracellular divalent iron (Fe2+) concentration. The staphyloferrin B-deficient mutant reportedly retains its virulence, but the relationship between micacocidin and virulence remains unconfirmed. To elucidate the effect of micacocidin on RSSC virulence, we generated the micacocidin productivity-deficient mutant (ΔRSc1806) that lacks RSc1806, which encodes a putative polyketide synthase/non-ribosomal peptide synthetase, using the RSSC phylotype I Ralstonia pseudosolanacearum strain OE1-1. When incubated in the condition without Fe2+, ΔRSc1806 showed significantly lower Fe3+-scavenging activity, compared with OE1-1. Until 8 days after inoculation on tomato plants, ΔRSc1806 was not virulent, similar to the mutant (ΔphcA) missing phcA, which encodes the LysR-type transcriptional regulator PhcA that regulates the expression of the genes responsible for quorum sensing (QS)-dependent phenotypes including virulence. The transcriptome analysis revealed that RSc1806 deletion significantly altered the expression of more than 80% of the PhcA-regulated genes in the mutant grown in medium with or without Fe2+. Among the PhcA-regulated genes, the transcript levels of the genes whose expression was affected by the deletion of RSc1806 were strongly and positively correlated between the ΔRSc1806 and the phcA-deletion mutant. Furthermore, the deletion of RSc1806 significantly modified QS-dependent phenotypes, similar to the effects of the deletion of phcA. Collectively, our findings suggest that the deletion of micacocidin production-related RSc1806 alters the regulation of PhcA-regulated genes responsible for QS-dependent phenotypes including virulence as well as Fe3+-scavenging activity. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Development of a tomato xylem-mimicking microfluidic system to study Ralstonia pseudosolanacearum biofilm formation.

The bacterial wilt pathogen Ralstonia pseudosolanacearum (Rps) colonizes plant xylem vessels and blocks the flow of xylem sap by its biofilm (comprising of bacterial cells and extracellular material), resulting in devastating wilt disease across many economically important host plants including tomatoes. The technical challenges of imaging the xylem environment, along with the use of artificial cell culture plates and media in existing in vitro systems, limit the understanding of Rps biofilm formation and its infection dynamics. In this study, we designed and built a microfluidic system that mimicked the physical and chemical conditions of the tomato xylem vessels, and allowed us to dissect Rps responses to different xylem-like conditions. The system, incorporating functional surface coatings of carboxymethyl cellulose-dopamine, provided a bioactive environment that significantly enhanced Rps attachment and biofilm formation in the presence of tomato xylem sap. Using computational approaches, we confirmed that Rps experienced linear increasing drag forces in xylem-mimicking channels at higher flow rates. Consistently, attachment and biofilm assays conducted in our microfluidic system revealed that both seeding time and flow rates were critical for bacterial adhesion to surface and biofilm formation inside the channels. These findings provided insights into the Rps attachment and biofilm formation processes, contributing to a better understanding of plant-pathogen interactions during wilt disease development.

Rapid detection of virulence-related genes by multiplex PCR in five pathogenic bacteria of mulberry bacterial wilt

Mulberry bacterial wilt is a devastating disease that is difficult to control and causes serious economic losses to the sericulture industry. This disease is mostly caused by a diverse group of pathogenic and opportunistic bacteria including, Ralstonia pseudosolanacearum, Pantoea ananatis, Enterobacter cloacae complex (ECC), Klebsiella pneumoniae species complex (KpSC), and K. oxytoca complex (KoC). Due to the lack of a rapid and reliable test to simultaneously detect these complex pathogens of mulberry wilt, we developed a multiplex PCR (mPCR) assay to detect five virulence-related genes carried by the pathogenic bacteria of mulberry bacterial wilt disease. The primers were designed for the virulence-related genes: pleD (GGDF structural domain-containing protein), yjfP (esterase), pelY (peripheral pectate lyase), ampD (N-acetyl-anhydromuranmyl-L-alanine amidase), and ripW (type III effector). Overall, the developed mPCR assay showed highly specific, sensitive and reproducible detection of target pathogens. Briefly, the results showed that the mPCR was highly specific in individual reactions, and the lowest detection concentration of the five pathogenic bacteria was 1.87 × 103 CFU/mL (DNA = 2.45 pg/μL). From 46 natural mulberry wilt samples, the mPCR detection rates of P. ananatis, ECC, KpSC, KoC and R. pseudosolanacearum were 8.69, 91.3, 34.7, 23.9 and 65.21%, respectively. The traditional culture media isolation methods showed comparable results. The pathogenicity test of 84 suspected pathogenic bacteria revealed that the morbidity (average morbidity level) caused by the pathogenic bacteria detected by mPCR was ≥ 65.5%, while the morbidity of the undetected pathogenic bacteria was ≤ 35.5%. Based on these results, we believe that the mPCR developed in the present study will be useful in rapid, reproducible, and sensitive detection of the pathogenic bacteria causing mulberry bacterial wilt including, R. pseudosolanacearum, P. ananatis, ECC, KpSC, and KoC.Graphical abstract

The biocontrol potentials of rhizospheric bacterium Bacillus velezensis K0T24 against mulberry bacterial wilt disease.

Mulberry bacterial wilt disease, caused by Ralstonia pseudosolanacearum, is a devastating soil-borne disease in the silk-mulberry-related industry. In this study, through high-throughput sequencing, we compared the rhizosphere bacterial composition of the mulberry-resistant cultivar (K10) and susceptible cultivar (G12), confirming Bacillus as a genus-level biomarker for K10. Next, twelve Bacillus spp. isolates, derived from the rhizosphere of K10, were screened for their antagonistic activity against R. pseudosolanacearum. The isolate showing strong antagonism was identified as B. velezensis K0T24 and selected for further analysis. The fermentation supernatant of B. velezensis K0T24 significantly inhibited the growth of R. pseudosolanacearum (82.47%) and the expression of its pathogenic genes. Using B. velezensis K0T24 in mulberry seedlings also increased defense enzyme activities and achieved a control efficacy of up to 55.17% against mulberry bacterial wilt disease. Collectively, our findings demonstrate the potential of B. velezensis K0T24 in suppressing mulberry bacterial wilt disease.

Bacillus amyloliquefaciens: a versatile antimicrobial Firmicute for the suppression of wilt caused by Ralstonia pseudosolanacearum in tomato

ABSTRACT Rhizosphere bacteria based biological control strategy is an eco-friendly approach to protect crops from various plant diseases. Among these, spore-forming Bacillus, abundant in the rhizosphere, provide unique advantages for plant health. This study focused on characterising two tomato rhizosphere-associated Bacillus amyloliquefaciens strains (Trb7 and Trb11), highly effective against the bacterial wilt pathogen Ralstonia pseudosolanacearum in vitro. Species identification was based on partial 16S rRNA gene sequencing, supported by phylogenetic analysis. Five antimicrobial peptide (AMP) genes (surfactin, fengycin, bacilysin, subtilin, and spore protein) were detected in both the isolates of B. amyloliquefqciens using specific molecular markers. Greenhouse trials showed that prophylactic application of these isolates led to an 83.4% reduction in tomato bacterial wilt, with only 16.6% wilt incidence compared to the 100% in the R. pseudosolanacearum-inoculated control. Moreover, these B. amyloliquefaciens isolates exhibited plant-growth-promoting traits in vitro. Interestingly, they also showed significant inhibition of mycelial growth against Sclerotium rolfsii and Alternaria alternata, major fungal pathogens in tomatoes. Overall, our findings highlight the potent bactericidal role of B. amyloliquefaciens isolates in suppressing R. pseudosolanacearum in tomatoes. Key highlights Two among the 100 Bacillus displayed antibacterial activity against Ralstonia pseudosolanacearum. Antibacterial firmicutes was identified as Bacillus amyloliquefaciens through the 16S rRNA gene sequence analysis. Five AMP genes (srfAA, fenD, bacA, spas, and spoVG) were identified in the isolates. Delivery of B. amyloliquefaciens drenching on rhizosphere significantly reduced wilt. Additionally, B. amyloliquefaciens showed the antifungal activity on Sclerotium rolfsii and Alternaria alternata.

Commodity risk assessment of Petunia spp. and Calibrachoa spp. unrooted cuttings from Kenya.

The European Commission requested the EFSA Panel on Plant Health to evaluate the probability of entry of pests (likelihood of pest freedom at entry), including both regulated and non-regulated pests, associated with unrooted cuttings of the genera Petunia and Calibrachoa produced under physical isolation in Kenya. The relevance of any pest for this opinion was based on evidence following defined criteria, based on the methodology used for High-Risk Plants adapted for the specificity of this assessment. Fourteen EU-regulated pests (Bemisia tabaci, cowpea mild mottle virus, Liriomyza huidobrensis, Liriomyza sativae, Liriomyza trifolii, potato leafroll virus, potato spindle tuber viroid, Ralstonia pseudosolanacearum, R. solanacearum, Scirtothrips dorsalis, tomato mild mottle virus, tomato spotted wilt virus, tomato yellow leaf curl virus and Xanthomonas vesicatoria) and six EU non-regulated pests (Aleurodicus dispersus, pepper veinal mottle virus, Nipaecoccus viridis, Phenacoccus solenopsis, Tetranychus neocaledonicus and tomato yellow ring virus) fulfilled all relevant criteria and were selected for further evaluation. For these pests, the risk mitigation measures proposed in the technical dossier from Kenya were evaluated, taking into account the possible limiting factors. Additionally, an expert judgement is given on the likelihood of pest freedom, taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. The estimated degree of pest freedom varies among the pests evaluated, with T. neocaledonicus being the pest most frequently expected on the imported cuttings. The Expert Knowledge Elicitation indicated, with 95% certainty, that between 9942 and 10,000 bags containing unrooted cuttings of Petunia spp. and Calibrachoa spp. per 10,000 would be free of T. neocaledonicus.

First report of Ralstonia pseudosolanacearum causing bacterial wilt on Rosa sp. in Greece.

In March 2021, a sample of nine-month-old, non-grafted, diseased rose (Rosa sp.) plants was sent by a grower to the Benaki Phytopathological Institute for examination. The plants exhibited symptoms of dieback with black necrosis of pruned shoots, brown discoloration of shoot and root vascular tissues, and whitish slime exudation on cutting wounds of the shoots. The symptoms resembled those caused by Ralstonia pseudosolanacearum (Tjou-Tam-Sin et al. 2016). According to the sample's information sheet, the sample had been collected in a commercial greenhouse rose crop for cut flowers with a 10% disease incidence in the area of Troizinia-Methana (Regional Unit of Islands, Greece). Microscopic examination of symptomatic shoot and root vascular tissues revealed masses of bacterial cells streaming out of them. Sections of symptomatic tissues were suspended in water and in the resulting suspension, bacteria of the R. solanacearum species complex (RSSC) were detected by an indirect immunofluorescence (IF) assay using polyclonal antibodies (Plant Research International, the Netherlands) and a qPCR assay (RS-I-F/RS-II-R primers, RSP-55T probe) (Vreeburg et al. 2016). Furthermore, colonies with typical characteristics of RSSC were isolated from vascular tissues of shoots and roots on non-selective (NA) and semi-selective (mSMSA) media (EPPO 2022), and their identification as RSSC was confirmed by the above-mentioned IF and qPCR assays. Also, the isolates were assigned to: i) biovar 3, based on their ability to metabolize three disaccharides (maltose, lactose, D(+) cellobiose) and three hexose alcohols (mannitol, sorbitol, dulcitol) producing acid (EU 2006) and ii) phylotype I, by multiplex conventional PCR (Opina et al. 1997; Fegan and Prior 2005). A representative isolate was selected for sequencing part of the genes: 16S rDNA (1464bp), mutS (729bp) and egl (795bp) with GenBank Accession Nos. OR102443, OR683617 and OR702781, respectively. Blast analysis of these sequences showed 100% identity with those of various RSSC strains (e.g. GenBank Ac. Nos. CP025741.1, CP021762.1, MF141029.1, respectively). The obtained egl sequence conforms with the characteristics of phylotype I based on the DNA barcoding tool (EPPO 2021) and is 100% identical to that of the Dutch strain PD7216 (MF141029.1) reported to be sequevar I-33 (Bergsma-Vlami et al. 2018). The pathogenicity of two isolates was tested by inoculating: i) tomato seedlings (cv. 'Belladona') at their stem between the cotyledons and the first true leaf (EU 2006) and b) rose plants (cv. 'Aqua' and 'Papa Meilland') at their shoot base (Tjou-Tam-Sin et al. 2016), with bacterial suspensions in water (108 cfu/ml). The inoculated plants were maintained at a day/night temperature about 28/20°C with tomato plants exhibiting leaf wilting (7-17 dpi) and rose plants exhibiting chlorosis and necrosis of leaves (17 dpi). The pathogen was re-isolated on mSMSA from both artificially infected plant species and identified by the IF assay described above, thus fulfilling Koch's postulates. This is the first diagnosis in Greece of: i) rose plants infected by a Ralstonia species and ii) a crop infected by R. solanacearum phylotype I that corresponds to the R. pseudosolanacearum species (EPPO 2022). Official phytosanitary measures imposed in the affected area include an annual survey of rose crops for the presence of this pathogen, aiming at an early detection and prevention of its spread in such a highly valued ornamental crop.

Characterization of Ralstonia pseudosolanacearum Diversity and Screening Tomato, Pepper, and Eggplant Resistance to Manage Bacterial Wilt in South Asia

In South Asia, bacterial wilt pathogens in the Ralstonia solanacearum species complex (RSSC) impose major constraints on eggplant, tomato, and pepper production. To improve the efficacy of bacterial wilt management, the goals of this study were to (i) conduct a survey of RSSC pathogens in Bangladesh and Nepal, (ii) characterize the genetic diversity of these isolates, and (iii) screen 37 tomato, eggplant, and pepper accessions for resistance to six representative isolates from South Asia. We isolated 99 isolates from Bangladesh and 20 isolates from Nepal and determined that all are phylotype I isolates of the Ralstonia pseudosolanacearum species. We sequenced and assembled draft genomes for 25 isolates. Phylogenomic analyses suggest that there is a wide diversity of endemic phylotype I isolates in South Asia and possible introductions of two clonal phylotype I lineages into Bangladesh and Nepal. We contextualize our newly described isolates based on prior reports of RSSC diversity in South Asia and global reports of RSSC pathogens on eggplant and pepper. Greenhouse trials revealed multiple tomato, eggplant, and pepper accessions that exhibit promising levels of resistance to six phylotype I isolates from South Asia. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Phage-Dependent Alteration of Rhizosphere Microbiota in Tomato Plants

Bacteriophages (phages) that infect bacterial pathogens are an alternative means of controlling bacterial diseases in humans, animals, and plants. However, the effects of targeted phage therapy on indigenous microbial communities are not fully understood. In this study, we hypothesized that phages infecting plant pathogenic bacteria play a role in modulating the microbial community in the plant rhizosphere. To explore this, we used the soil-borne plant bacterial pathogen Ralstonia pseudosolanacearum as the host bacterium and its phages as a model system in the tomato rhizosphere. The effect of phages on the microbiota was examined using a narrow host range phage RpY1 and a combination of two phages (RpY2 and RpT1, termed the phage combo) with a broad host range under the natural rhizosphere microbiota of tomato plants. Both RpY1 and the phage combo altered the tomato rhizosphere microbiota. The phage combo displayed phage effects mostly in the presence of R. pseudosolanacearum. However, RpY1 affected the rhizosphere microbiota even in the absence of the host bacterium. The effect of phage RpY1 on the microbiota was further investigated in the tomato rhizosphere using a synthetic community (SynCom) mimicking the natural tomato rhizosphere microbiota. Phage RpY1 affected the microbial community structure of the SynCom in the tomato rhizosphere in the absence of the host bacterium. Analyses of the natural microbiota and SynCom in the tomato rhizosphere indicated an indirect effect of phage RpY1 on the microbiota. This study suggests that phage application modulates the indigenous microbiota through unknown interactions with nonhost bacterial members in the plant rhizosphere. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Fermented botanical fertilizer controls bacterial wilt of tomatoes caused by Ralstonia pseudosolanacearum.

This study demonstrates the effect of fermented botanical product (FBP) on Ralstonia pseudosolanacearum-induced bacterial wilt disease and unravels its action mechanism. Soaking with diluted FBP solutions (0.1%-0.5%) significantly suppressed bacterial wilt in tomato plants, and FBP-treated tomato plants grew well against R. pseudosolanacearum infection. Growth assays showed that FBP had no antibacterial effect but promoted R. pseudosolanacearum growth. In contrast, few or no R. pseudosolanacearum cells were detected in aerial parts of tomato plants grown in FBP-soaked soil. Subsequent infection assays using the chemotaxis-deficient mutant (ΔcheA) or the root-dip inoculation method revealed that FBP does not affect pathogen migration to plant roots during infection. Moreover, FBP-pretreated tomato plants exhibited reduced bacterial wilt in the absence of FBP. These findings suggest that the plant, but not the pathogen, could be affected by FBP, resulting in an induced resistance against R. pseudosolanacearum, leading to a suppressive effect on bacterial wilt.