Transcriptome and metabolome profiling in different stages of infestation of Eucalyptus urophylla clones by Ralstonia solanacearum.

Eucalyptus urophylla is an economically important tree species that widely planted in tropical and sub-tropical areas around the world, which suffers significant losses due to Ralstonia solanacearum. However, little is known about the molecular mechanism of pathogen-response of Eucalyptus. We collected the vascular tissues of a E. urophylla clone infected by R. solanacearum in the laboratory, and combined transcriptome and metabolome analysis to investigate the defense responses of Eucalyptus. A total of 11 flavonoids that differentially accumulated at the first stage or a later stage after infection. The phenylpropanoid of p-coumaraldehyde, the two alkaloids trigonelline and DL-ephedrine, two types of traditional Chinese medicine with patchouli alcohol and 3-dihydrocadambine, and the amino acid phenylalanine were differentially accumulated after infection, which could be biomarkers indicating a response to R. solanacearum. Differentially expressed genes involved in plant hormone signal transduction, phenylpropanoids, flavonoids, mitogen-activated protein kinase (MAPK) signaling, and amino acid metabolism were activated at the first stage of infection or a later stage, indicating that they may participate in the defense against infection. This study is expected to deliver several insights into the molecular mechanism in response to pathogens in E. urophylla, and the findings have far-reaching implications in the control of E. urophylla pathogens.

Ralstonia Strains from Potato-Growing Regions of Kenya Reveal Two Phylotypes and Epidemic Clonality of Phylotype II Sequevar 1 Strains.

Bacterial wilt, caused by the Ralstonia solanacearum species complex (RSSC), is the most destructive potato disease in Kenya. Studies were conducted to (i) determine the molecular diversity of RSSC strains associated with bacterial wilt of potato in Kenya, (ii) generate an RSSC distribution map for epidemiological inference, and (iii) determine whether phylotype II sequevar 1 strains exhibit epidemic clonality. Surveys were conducted in 2018 and 2019, in which tubers from wilting potato plants and stem samples of potential alternative hosts were collected for pathogen isolation. The pathogen was phylotyped by multiplex PCR and 536 RSSC strains typed at a sequevar level. Two RSSC phylotypes were identified, phylotype II (98.4%, n = 506 [sequevar 1 (n = 505) and sequevar 2 (n = 1)]) and phylotype I (1.6%, n = 30 [sequevar 13 (n = 9) and a new sequevar (n = 21)]). The phylotype II sequevar 1 strains were haplotyped using multilocus tandem repeat sequence typing (TRST) schemes. The TRST scheme identified 51 TRST profiles within the phylotype II sequevar 1 strains with a modest diversity index (HGDI = 0.87), confirming the epidemic clonality of RSSC phylotype II sequevar 1 strains in Kenya. A minimum spanning tree and mapping of the TRST profiles revealed that TRST27 '8-5-12-7-5' is the primary founder of the clonal complex of RSSC phylotype II sequevar 1 and is widely distributed via latently infected seed tubers. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Phylogenetic Distribution of Ralstonia solanacearum Species Complex Populations in Potato in Kenya.

Ralstonia solanacearum is a pathogen causing bacterial wilt disease of potato, resulting in 70% potato production losses in Kenya. A study was conducted to determine the diversity of R. solanacearum species complex strains within the main potato-growing regions of Kenya. Potato tubers were collected in different potato-growing regions of Kenya from visibly wilted potato plants as well as samples of tomato, irrigation water, and cultures for pathogen isolation. Genomic DNA was isolated from 135 purified cultures of RSSC isolates and PCR-amplified using multiplex and sequevar primers targeting the endoglucanase (egl) partial gene sequences. Pathogenicity tests using R. solanacearum strain (phylotype II sequevar I) were done on the cultivars Kenya Karibu, Shangi, Chulu, Wanjiku, and MoneyMaker. Phylogenetic analysis of the partial egl gene identified two genospecies, R. pseudosolanacearum sp. nov. (1.5%) and R. solanacearum (98.5%). All R. solanacearum strains clustered in sequevar I and were distributed in all the potato-growing regions surveyed. The cultivars were grown in a greenhouse for two cycles in a randomized complete block design and inoculated with R. solanacearum strain. The severity scores were assessed and the area under the disease progress curve (AUDPC) was determined. All the cultivars tested for pathogenicity exhibited wilting symptoms at varying intervals after infection, with none showing complete resistance to R. solanacearum. Cultivar Shangi exhibited minimum disease severity and progression of 41.14% and AUDPC of 1041.7, respectively, while 'Kenya Karibu' was the most susceptible with a high progression rate of 68.24% and AUDPC of 1897.5, respectively. 'MoneyMaker', 'Chulu', and 'Wanjiku' showed no significant difference in disease severity, depicting a simultaneous rate of infection among them. These findings provide valuable information to better understand the pathogen genetic diversity in Kenya and how it spreads.

Molecular Detection of Ralstonia solanacearum to Facilitate Breeding for Resistance to Bacterial Wilt in Potato.

Potato bacterial wilt is caused by the devastating bacterial pathogen Ralstonia solanacearum. Quantitative resistance to this disease has been and is currently introgressed from a number of wild relatives into cultivated varieties through laborious breeding programs. Here, we present two methods that we have developed to facilitate the screening for resistance to bacterial wilt in potato. The first one uses R. solanacearum reporter strains constitutively expressing the luxCDABE operon or the green fluorescent protein (gfp) to follow pathogen colonization in potato germplasm. Luminescent strains are used for nondestructive live imaging, while fluorescent ones enable precise pathogen visualization inside the plant tissues through confocal microscopy. The second method is a BIO-multiplex-PCR assay that is useful for sensitive and specific detection of viable R. solanacearum (IIB-1) cells in latently infected potato plants. This BIO-multiplex-PCR assay can specifically detect IIB-1 sequevar strains as well as strains belonging to all four R. solanacearum phylotypes and is sensitive enough to detect without DNA extraction ten bacterial cells per mL in complex samples.The described methods allow the detection of latent infections in roots and stems of asymptomatic plants and were shown to be efficient tools to assist potato breeding programs.

Rapid Loop-Mediated Isothermal Amplification for Detection of the Ralstonia solanacearum Species Complex Bacteria in Symptomatic Potato Tubers and Plants.

The Ralstonia solanacearum species complex (RSSC) is composed of several Ralstonia species and strains that are little related and show varied host range and distinct geographic distributions. The RSSC causes wilt disease, and can thus have severe economic consequences for many important crops and ornamental plants. One such is potato (Solanum tuberosum), where infection causes brown rot of the tubers. It is important that symptomatic tubers and plants can be rapidly and easily tested, as exclusion of infected material is a cornerstone of management of bacterial diseases. A suitable method is loop-mediated isothermal amplification, a rapid, DNA-based method that can be used for specific detection of plant pathogens in infected materials. The combination of this loop-mediated isothermal amplification assay for the RSSC with a simple sample preparation method is fit for purpose for identification of this devastating disease in symptomatic tubers and plants. This methodology is rapid and cost efficient, and can be carried out outside of conventional laboratory facilities.

Transboundary introduction of potato-infecting Ralstonia solanacearum in the Andaman Islands revealed by multilocus sequence typing: A potential threat to island agriculture.

Wilting of potato plants with an incidence of 20-30 % was observed for the first time in the agricultural farms of Andaman Islands, India. The infected plants showed wilting syndrome that included downward drooping of leaves, yellowing, and collapse of the entire plants. Characteristic milky-white exudate from the infected stem indicated bacterial etiology of the disease. Upon streaking onto 2, 3, 5 triphenyl-tetrazolium chloride amended nutrient medium, the bacterial exudate yielded characteristic creamy-white, fluidal, irregular colonies with the pink center. Upon inoculation, the randomly picked bacterial colonies, AN_PRSGr and AN_PRSCh, representing the two locations, incited wilt symptoms on one-month-old potato plants. The host range studies revealed that the isolates were pathogenic on tomato and eggplant but non-pathogenic to chili and Solanum torvum (wild eggplant). The 16S rRNA gene sequencing and the Ralstonia-specific PCR test confirmed the identity of AN_PRSGr and AN_PRSCh as Ralstonia solanacearum. Intra-species level classification revealed their identity as strains of race 1, biovar 3, and phylotype-I. Multilocus sequence typing (MLST)-based in-depth sequence alignment for phylogenetic analysis revealed the isolates AN_PRSGr and AN_PRSCh clustered with two mainland race 1/biovar 3/phylotype-I isolates of Kerala, India. However, the allelic profile-based goeBURST-analysis placed them as singletons in the global collection of Ralstonia solanacearum, conforming intra-racial/ intra-phylotype diversity within race 1/biovar3/phylotype-I strains. The molecular characterization.

Complete Genome Resources for Ralstonia Bacterial Wilt Strains UW763 (Phylotype I); Rs5 and UW700 (Phylotype II); and UW386, RUN2474, and RUN2279 (Phylotype III).

We share whole genome sequences of six strains from the Ralstonia solanacearum species complex, a diverse group of Betaproteobacteria that cause plant vascular wilt diseases. Using single-molecule real-time technology, we sequenced and assembled full genomes of Rs5 and UW700, two phylotype IA-sequevar 7 (IIA-7) strains from the southeastern United States that are closely related to the R. solanacearum species type strain, K60, but were isolated >50 years later. Four sequenced strains from Africa include a soil isolate from Nigeria (UW386, III-23), a tomato isolate from Senegal (UW763, I-14), and two potato isolates from the Madagascar highlands (RUN2474, III-19 and RUN2279, III-60). This resource will support studies of the genetic diversity, ecology, virulence, and microevolution of this globally distributed group of high-impact plant pathogens.[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.

Ralstonia solanacearum type III effector RipV2 encoding a novel E3 ubiquitin ligase (NEL) is required for full virulence by suppressing plant PAMP-triggered immunity.

Ralstonia solanacearum causes bacterial wilt disease in a broad range of plants, primarily through type Ⅲ secreted effectors. However, the R. solanacearum effectors promoting susceptibility in host plants remain limited. In this study, we determined that the R. solanacearum effector RipV2 functions as a novel E3 ubiquitin ligase (NEL). RipV2 was observed to be locali in the plasma membrane after translocatio into plant cells. Transient expression of RipV2 in Nicotiana benthamiana could induce cell death and suppress the flg22-induced pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) responses, mediating such effects as attenuation of the expression of several PTI-related genes and ROS bursts. Furthermore, we demonstrated that the conserved catalytic residue is highly important for RipV2. Transient expression of the E3 ubiquitin ligase catalytic mutant RipV2 C403A alleviated the PTI suppression ability and cell death induction, indicating that RipV2 requires its E3 ubiquitin ligase activity for its role in plant-microbe interactions. More importantly, mutation of RipV2 in R. solanacearum reduces the virulence of R. solanacearum on potato. In conclusion, we identified a NEL effector that is required for full virulence of R. solanacearum by suppressing plant PTI.

Identification of RipAZ1 as an avirulence determinant of Ralstonia solanacearum in Solanum americanum.

Ralstonia solanacearum causes bacterial wilt disease in many plant species. Type III-secreted effectors (T3Es) play crucial roles in bacterial pathogenesis. However, some T3Es are recognized by corresponding disease resistance proteins and activate plant immunity. In this study, we identified the R. solanacearum T3E protein RipAZ1 (Ralstonia injected protein AZ1) as an avirulence determinant in the black nightshade species Solanum americanum. Based on the S. americanum accession-specific avirulence phenotype of R. solanacearum strain Pe_26, 12 candidate avirulence T3Es were selected for further analysis. Among these candidates, only RipAZ1 induced a cell death response when transiently expressed in a bacterial wilt-resistant S. americanum accession. Furthermore, loss of ripAZ1 in the avirulent R. solanacearum strain Pe_26 resulted in acquired virulence. Our analysis of the natural sequence and functional variation of RipAZ1 demonstrated that the naturally occurring C-terminal truncation results in loss of RipAZ1-triggered cell death. We also show that the 213 amino acid central region of RipAZ1 is sufficient to induce cell death in S. americanum. Finally, we show that RipAZ1 may activate defence in host cell cytoplasm. Taken together, our data indicate that the nucleocytoplasmic T3E RipAZ1 confers R. solanacearum avirulence in S. americanum. Few avirulence genes are known in vascular bacterial phytopathogens and ripAZ1 is the first one in R. solanacearum that is recognized in black nightshades. This work thus opens the way for the identification of disease resistance genes responsible for the specific recognition of RipAZ1, which can be a source of resistance against the devastating bacterial wilt disease.

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.

Genome Resource: Ralstonia solanacearum Phylotype II Sequevar 1 (Race 3 Biovar 2) Strain UW848 From the 2020 U.S. Geranium Introduction.

Ralstonia solanacearum phylotype II sequevar 1 (RsII-1, formerly race 3 biovar 2) causes tomato bacterial wilt, potato brown rot, and Southern wilt of geranium. Strains in RsII-1 cause wilting in potato and tomato at cooler temperatures than tropical lowland R. solanacearum strains. Although periodically introduced, RsII-1 has not established in the United States. This pathogen is of quarantine concern and listed as a Federal Select Agent. We report a rapidly sequenced (<2 days) draft genome of UW848, a RsII-1 isolate introduced to the United States in geranium cuttings in spring 2020. UW848 belongs to the near-clonal cluster of RsII-1 global pandemic strains.

In Silico and In Vitro Analyses of Glucosamine and Indole Acetaldehyde Inhibit Pathogenic Regulator Gene phcA of Ralstonia solanacearum, a Causative Agent of Bacterial Wilt of Tomato.

In this study, medicinal plant (Solanum surattense)-associated bacteria were isolated and their extracellular secondary metabolites were extracted. Dual-plate application of crude secondary metabolites proved that SSL2I and SSL5 had a good inhibitory activity against Ralstonia solanacearum. These biocontrol bacteria were identified as Bacillus subtilis and Bacillus velezensis by 16S rRNA gene sequencing analysis. The crude extracts of secondary metabolites were identified based on high-resolution liquid chromatography/mass spectrometry (HR-LCMS) analysis. On the basis of HR-LCMS analysis, we selected the compounds such as glucosamine and indole acetaldehyde for in silico analysis and inhibition of pathogenic gene of phcA from R. solanacearum. The specificity of identified pathogenic gene of R. solanacearum and its cytoplasmic localization were identified by BLASTP and PSORTB bioinformatics tools. The protein-protein interaction between the identified secondary metabolites and pathogenic gene revealed that the compound had antagonistic potential against pathogenic gene of phcA. Furthermore, the synthetic forms of the above metabolites showed that both the compounds had the ability to inhibit R. solanacearum under in vitro condition. On the basis of in silico and in vitro analyses, it was concluded that medicinal plant-associated Bacillus spp. could be used as a biocontrol agent in managing wilt disease caused by R. solanacearum.

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.

Genome Resource of Two Potato Strains of Ralstonia solanacearum Biovar 2 (Phylotype IIB Sequevar 1) and Biovar 2T (Phylotype IIB Sequevar 25) Isolated from Lowlands in Iran.

Ralstonia solanacearum, the causal agent of bacterial wilt and brown rot disease, is one of the major pathogens of solanaceous crops, including potato, around the globe. Biovar 2T (phylotype II/sequevar 25) of R. solanacearum is adapted to tropical lowlands and is only reported in South America and Iran. Thus far, no genome resource of the biovar 2T of the pathogen has been available. Here, we present the near-complete genome sequences of the biovar 2T strain CFBP 8697 as well as strain CFBP 8695 belonging to biovar 2 race 3, both isolated from potato in Iran. The genomic data of biovar 2T will extend our understanding of the virulence features of R. solanacearum and pave the way for research on biovar 2T functional and interaction genetics.

Phage combination therapies for bacterial wilt disease in tomato.

Bacteriophages have been proposed as an alternative to pesticides to kill bacterial pathogens of crops. However, the efficacy of phage biocontrol is variable and poorly understood in natural rhizosphere microbiomes. We studied biocontrol efficacy of different phage combinations on Ralstonia solanacearum infection in tomato. Increasing the number of phages in combinations decreased the incidence of disease by up to 80% in greenhouse and field experiments during a single crop season. The decreased incidence of disease was explained by a reduction in pathogen density and the selection for phage-resistant but slow-growing pathogen strains, together with enrichment for bacterial species that were antagonistic toward R. solanacearum. Phage treatment did not affect the existing rhizosphere microbiota. Specific phage combinations have potential as precision tools to control plant pathogenic bacteria.

Molecular Epidemiology of Ralstonia solanacearum Species Complex Strains Causing Bacterial Wilt of Potato in Uganda.

Bacterial wilt (BW) caused by the Ralstonia solanacearum species complex (RSSC) is a serious threat to potato production in Uganda. However, little is known about the extent of the disease and the type of the pathogen strains involved. A nationwide survey was conducted to study BW prevalence and incidence in potato, and potato tuber and stem samples of potential alternative hosts were collected for pathogen isolation. DNA was extracted from pure cultures for genetic diversity studies. The pathogen was phylotyped by multiplex PCR; then, a subset of isolates was typed at sequevar level. Isolates of the same sequevar were then haplotyped using multilocus tandem repeat sequence typing (TRST) schemes. BW prevalence and incidence in potato farms were 81.4 and 1.7%, respectively. Three RSSC phylotypes were identified, with the majority of the strains belonging to Phylotype II (80%) followed by Phylotype I (18.5%) and III (1.5%). Phylotype I strains belonged to Sequevar 31, and Phylotype II strains belonged to Sequevar 1. Potato-associated Phylotype II Sequevar 1 strains were more diverse (27 TRST haplotypes) than nonpotato Phylotype I (5 TRST haplotypes). Mapping of TRST haplotypes revealed that three TRST haplotypes of Phylotype II Sequevar 1 strains play an important epidemiological role in BW of potato in Uganda being disseminated via latently infected seed.[Formula: see text]Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Comparative Evaluation of LAMP, qPCR, Conventional PCR, and ELISA to Detect Ralstonia solanacearum in Kenyan Potato Fields.

Bacterial wilt caused by Ralstonia solanacearum is considered among the most damaging diseases of potato in Sub-Saharan Africa and the most significant biotic constraint of potato production alongside late blight. Unlike late blight, which can be managed by chemical means, R. solanacearum can only be managed through cultural methods and clean seed. Laboratory testing to certify seed before planting is required to confirm the absence of the pathogen in Kenya. A loop-mediated isothermal amplification (LAMP) assay was developed using the UDP-(3-O-acyl)-N-acetylglucosamine deacetylase gene (IpxC) to screen seed potato for R. solanacearum strains. The assay was assessed using DNA extracted from R. solanacearum and other soil and potato pathogens to demonstrate specificity and sensitivity. The LAMP assay was validated using field samples from different potato growing regions of Kenya collected over two growing seasons and compared with established nucleic acid and protein-based assays. The IpxC LAMP assay was found to be specific and sensitive to R. solanacearum, detecting as low as 2.5 pg/µl of R. solanacearum DNA. Of the 47 potentially infected field samples collected, both IpxC LAMP and quantitative polymerase chain reaction (PCR) detected R. solanacearum DNA in 90% of the samples, followed by conventional PCR (86%) and ELISA (75%). This IpxC LAMP assay is a promising diagnostic tool to rapidly screen for R. solanacearum in seed potato with high sensitivity in Kenya. Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

[Construction of Tn5 transposon insertion mutants of Ralstonia solanacearum isolated from Pogostemon cablin].

Ralstonia solanacearum strain PRS-84 used in this study was isolated from diseased Pogostemon cablin plants in our previous study.The competent cells of R.solanacearum strain PRS-84 were transformed by electroporation with Tn5 transposon and then were plated on TTC agar plates containing kanamycin to select for kanamycin-resistant colonies.The detection of kanamycin-resistant gene in kanamycin-resistant colonies was performed by PCR.Further,the flanking fragments of Tn5 transposon insertion site in the mutants were amplified by inverse PCR,and the flanking fragments were sequenced and analyzed.The results indicated that the kanamycin-resistant colonies were obtained in the transformation experiment of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon.A specific band of approximately 700 bp was amplified by PCR from kanamycin-resistant colonies.The flanking sequences of Tn5 transposon insertion site in the transformants were obtained by inverse PCR.After sequencing and sequence analysis of Tn5 transposon insertion site in mutants,we preliminarily speculated that the Tn5 transposon inserted in the typ A gene,rec O gene and gid A gene in three mutants,respectively.A random mutagenesis system of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon has been established,and the Tn5 insertion mutants have been obtained.This study might facilitate the creation of mutant library and the discovery of the virulence gene of R.solanacearum isolated from P.cablin.

Type III secretion inhibitors for the management of bacterial plant diseases.

The identification of chemical compounds that prevent and combat bacterial diseases is fundamental for crop production. Bacterial virulence inhibitors are a promising alternative to classical control treatments, because they have a low environmental impact and are less likely to generate bacterial resistance. The major virulence determinant of most animal and plant bacterial pathogens is the type III secretion system (T3SS). In this work, we screened nine plant extracts and 12 isolated compounds-including molecules effective against human pathogens-for their capacity to inhibit the T3SS of plant pathogens and for their applicability as virulence inhibitors for crop protection. The screen was performed using a luminescent reporter system developed in the model pathogenic bacterium Ralstonia solanacearum. Five synthetic molecules, one natural product and two plant extracts were found to down-regulate T3SS transcription, most through the inhibition of the regulator hrpB. In addition, for three of the molecules, corresponding to salicylidene acylhydrazide derivatives, the inhibitory effect caused a dramatic decrease in the secretion capacity, which was translated into impaired plant responses. These candidate virulence inhibitors were then tested for their ability to protect plants. We demonstrated that salicylidene acylhydrazides can limit R. solanacearum multiplication in planta and protect tomato plants from bacterial speck caused by Pseudomonas syringae pv. tomato. Our work validates the efficiency of transcription reporters to discover compounds or natural product extracts that can be potentially applied to prevent bacterial plant diseases.

Identification of a DNA region associated with the cool virulence of Ralstonia solancearum strain UW551 and its utilization for specific detection of the bacterium's race 3 biovar 2 strains.

The cool virulent Ralstonia solanacearum race 3 biovar 2 (r3b2) strains cause destructive brown rot of potato. They are quarantined pathogens in Europe and Canada and select agent pathogens in the United States. We previously identified r3b2 (sequevars 1 and 2)-unique fragments that clustered into 32 regions in the genome of R. solanacearum. In this study, we targeted five of those regions for mutagenesis in order to determine whether they are involved in cool temperature-related biological functions for diagnostic purpose. Knockout mutants of four regions produced no changes to the biology of the r3b2 strain UW551. The mutation of region 13, which is 3,407 bp in size, resulted in significantly reduced twitching motility, attachment to the roots of tomato seedlings, and virulence under cool temperature conditions (18-24°C), although no significant difference was found under warm temperature conditions (24-30°C) as compared to the wild type strain. As a result, we designed primer pair Rs-CV-F and Rs-CV-R to target the region 13 for specific detection of r3b2 strains of R. solanacearum. Our assay specifically detected all the 34 r3b2 strains and none of the 56 non-r3b2 strains of R. solanacearum, nor any other five plant- or soil-associated bacteria including Enterobacter cloacae, Pseudomonas syringae pv. syringae, Xanthomonas campestris pv. campestris, X. citri, and R. pickettii. Unexpectedly, in silico analysis predicted that a recently deposited non-sequevar 1 or 2 Brazilian R. solanacearum strain RS489 would be recognized by our assay and by previously published r3b2-specific assays, although the cool-virulent status of this strain is unclear. Our PCR assay is the first to target a DNA region associated with cool-virulence that makes r3b2 strains highly regulated pathogens for specific detection of this important group of R. solanacearum.