PhoB-regulated phosphate assimilation of Ralstonia solanacearum is cross-activated by VsrB in Pi-abundant rich medium.

Ralstonia solanacearum is a devastating phytopathogen infecting a broad range of economically important crops. Phosphate (Pi) homeostasis and assimilation play a critical role in the environmental adaptation and pathogenicity of many bacteria. However, the Pi assimilation regulatory mechanism of R. solanacearum remains unknown. This study revealed that R. solanacearum pstSCAB-phoU-phoBR operon expression is sensitive to extracellular Pi concentration, with higher expression under Pi-limiting conditions. The PhoB-PhoR fine-tunes the Pi-responsive expression of the Pho regulon genes, demonstrating its pivotal role in Pi assimilation. By contrast, neither PhoB, PhoR, PhoU, nor PstS was found to be essential for virulence on tomato plants. Surprisingly, the PhoB regulon is activated in a Pi-abundant rich medium. Results showed that histidine kinase VsrB, which is known for the exopolysaccharide production regulation, partially mediates PhoB activation in the Pi-abundant rich medium. The 271 histidine of VsrB is vital for this activation. This cross-activation mechanism between the VsrB and PhoB-PhoR systems suggests the carbohydrate-Pi metabolism coordination in R. solanacearum. Overall, this research provides new insights into the complex regulatory interplay between Pi metabolism and growth in R. solanacearum.

Isolation and identification of NEAU-CP5: A seed-endophytic strain of B. velezensis that controls tomato bacterial wilt.

Bacterial wilt of tomato caused by Ralstonia solanacearum is a critical soilborne disease that drastically reduces yield. In the current study, an endophytic strain NEAU-CP5 with strong antagonistic activity against R. solanacearum was isolated from tomato seeds and characterized. The strain was identified as Bacillus velezensis based on 16S rRNA gene and whole genome sequence analysis. NEAU-CP5 can secrete amylase, protease, and cellulase, and also produce known antibacterial metabolites, including cyclo (leucylprolyl), cyclo (phenylalanyl-prolyl), cyclo (Pro-Gly), 3-benzyl-2,5-piperazinedione, pentadecanoic acid, eicosane, 2-methyoic acid, isovaleric acid, dibuty phthalate, and esters of fatty acids (HFDU), which may be responsible for its strong antibacterial activity. Fourteen gene clusters associated with antibacterial properties were also identified in the whole genome sequence of NEAU-CP5. Pot experiment demonstrated that the application of 108 CFU/mL NEAU-CP5 on tomato plants significantly reduced the incidence of tomato bacterial wilt by 68.36 ± 1.67 %. NEAU-CP5 also increased the activity of defense-related enzymes (CAT, POD, PPO, SOD, and PAL) in tomato plants. This is the first report of an effective control of bacterial wilt on tomato plants by B. velezensis and highlights the potential of NEAU-CP5 as a potential biocontrol agent for the management of tomato bacterial wilt.

Calcium modulation of bacterial wilt disease on potato.

Ralstonia solanacearum species complex (RSSC) is a phytopathogenic bacterial group that causes bacterial wilt in several crops, being potato (Solanum tuberosum) one of the most important hosts. The relationship between the potato plant ionome (mineral and trace elements composition) and the resistance levels to this pathogen has not been addressed until now. Mineral content of xylem sap, roots, stems and leaves of potato genotypes with different levels of resistance to bacterial wilt was assessed in this work, revealing a positive correlation between divalent calcium (Ca) cation concentrations and genotype resistance. The aim of this study was to investigate the effect of Ca on bacterial wilt resistance, and on the growth and virulence of RSSC. Ca supplementation significantly decreased the growth rate of Ralstonia pseudosolanacearum GMI1000 in minimal medium and affected several virulence traits such as biofilm formation and twitching motility. We also incorporate for the first time the use of microfluidic chambers to follow the pathogen growth and biofilm formation in conditions mimicking the plant vascular system. By using this approach, a reduction in biofilm formation was observed when both, rich and minimal media, were supplemented with Ca. Assessment of the effect of Ca amendments on bacterial wilt progress in potato genotypes revealed a significant delay in disease progress, or a complete absence of wilting symptoms in the case of partially resistant genotypes. This work contributes to the understanding of Ca effect on virulence of this important pathogen and provides new strategies for an integrated control of bacterial wilt on potato. IMPORTANCE: Ralstonia solanacearum species complex (RSSC) includes a diverse group of bacterial strains that cause bacterial wilt. This disease is difficult to control due to pathogen aggressiveness, persistence, wide range of hosts, and wide geographic distribution in tropical, subtropical, and temperate regions. RSSC causes considerable losses depending on the pathogen strain, host, soil type, environmental conditions, and cultural practices. In potato, losses of $19 billion per year have been estimated for this pathogen worldwide. In this study, we report for the first time the mineral composition found in xylem sap and plant tissues of potato germplasm with different levels of resistance to bacterial wilt. This study underscores the crucial role of calcium (Ca) concentration in the xylem sap and stem in relation to the resistance of different genotypes. Our in vitro experiments provide evidence of Ca's inhibitory effect on the growth, biofilm formation, and twitching movement of the model RSSC strain R. pseudosolanacearum GMI1000. This study introduces a novel element, the Ca concentration, which should be included into the integrated disease control management strategies for bacterial wilt in potatoes.

A robust high-throughput functional screening assay for plant pathogen effectors using the TMV-GFP vector.

Uncovering the function of phytopathogen effectors is crucial for understanding mechanisms of pathogen pathogenicity and for improving our ability to protect plants from diseases. An increasing number of effectors have been predicted in various plant pathogens. Functional characterization of these effectors has become a major focus in the study of plant-pathogen interactions. In this study, we designed a novel screening system that combines the TMV (tobacco mosaic virus)-GFP vector and Agrobacterium-mediated transient expression in the model plant Nicotiana benthamiana. This system enables the rapid identification of effectors that interfere with plant immunity. The biological function of these effectors can be easily evaluated by observing the GFP fluorescence signal using a UV lamp within just a few days. To evaluate the TMV-GFP system, we initially tested it with well-described virulence and avirulence type III effectors from the bacterial pathogen Ralstonia solanacearum. After proving the accuracy and efficiency of the TMV-GFP system, we successfully screened a novel virulence effector, RipS1, using this approach. Furthermore, using the TMV-GFP system, we reproduced consistent results with previously known cytoplasmic effectors from a diverse array of pathogens. Additionally, we demonstrated the effectiveness of the TMV-GFP system in identifying apoplastic effectors. The easy operation, time-saving nature, broad effectiveness, and low technical requirements of the TMV-GFP system make it a promising approach for high-throughput screening of effectors with immune interference activity from various pathogens.

Selection of the Dominant Endophytes Based on Illumina Sequencing Analysis for Controlling Bacterial Wilt of Patchouli Caused by Ralstonia solanacearum.

Bacterial wilt caused by Ralstonia solanacearum (RS) is one of the most devastating diseases in patchouli (Pogostemon cablin [Blanco] Benth.), which results in low yield and quality of patchouli. However, no stable and effective control methods have been developed yet. To evaluate the potential of dominant bacterial endophytes in biocontrol, the endophytic bacterial diversity of patchouli was investigated based on Illumina sequencing analysis, and the ability of isolates belonging to the dominant bacterial genera to control RS wilt of patchouli was explored in pot experiments. A total of 245 bacterial genera were detected in patchouli plants, with the highest relative abundance of operational taxonomic units belonging to the genus Pseudomonas detected in roots, leaves, and stems. The Pseudomonas isolates S02, S09, and S26 showed antagonistic activity against RS in vitro and displayed many plant growth-promoting characteristics, including production of indole-3-acetic acid, siderophores, and 1-aminocyclopropane-1-carboxylic acid deaminase and phosphate- and potassium-solubilizing capability. Inoculation of patchouli plants with the isolates S02, S09, and S26 significantly improved shoot growth and decreased the incidence of bacterial wilt caused by RS. The results suggest that screening of dominant bacterial endophytes for effective biocontrol agents based on Illumina sequencing analysis is more efficient than random isolation and screening procedures.

Genome-wide identification of the TIFY gene family in tobacco and expression analysis in response to Ralstonia solanacearum infection.

The TIFY gene family plays an essential role in plant development and abiotic and biotic stress responses. In this study, genome-wide identification of TIFY members in tobacco and their expression pattern analysis in response to Ralstonia solanacearum infection were performed. A total of 33 TIFY genes were identified, including the TIFY, PPD, ZIM&ZML and JAZ subfamilies. Promoter analysis results indicated that a quantity of light-response, drought-response, SA-response and JA-response cis-elements exist in promoter regions. The TIFY gene family exhibited expansion and possessed gene redundancy resulting from tobacco ploidy change. In addition, most NtTIFYs equivalently expressed in roots, stems and leaves, while NtTIFY1, NtTIFY4, NtTIFY18 and NtTIFY30 preferentially expressed in roots. The JAZ III clade showed significant expression changes after inoculation with R. solanacearum, and the expression of NtTIFY7 in resistant varieties, compared with susceptible varieties, was more stably induced. Furthermore, NtTIFY7-silenced plants, compared with the control plants, were more susceptible to bacterial wilt. These results lay a foundation for exploring the evolutionary history of TIFY gene family and revealing gene function of NtTIFYs in tobacco bacterial wilt resistance.

Comparative identification of WRKY transcription factors and transcriptional response to Ralstonia solanacearum in tomato.

In order to study the responses of tomato (Solanum lycopersicum) WRKY TFs to bacterial wilt caused by Ralstonia solanacearum, the most up-to-date genomes and transcriptional profiles were used to identify WRKY TFs in control and infected inbred lines. In total, 85 tomato WRKY TFs were identified and categorized into groups I, IIa + b, IIc, IId + e, and III. These WRKYs, especially those from group IIe, were mainly distributed at chromosome ends and in clusters. More than 45 % and 70 % of tomato WRKYs exhibited intraspecific and interspecific synteny, respectively. Nearly 60 % of tomato WRKYs (mainly in groups I and IIc) formed 73 pairs of orthologs with WRKYs in Arabidopsis and pepper, with Ka/Ks less than 1. Sixteen tomato WRKYs (mainly in groups IIa + b and IIc) responded strongly to biotic stress, and 12 differentially expressed WRKYs (mainly in groups III and IIb) were identified. RT-qPCR revealed that tomato WRKYs could respond to bacterial wilt through positive (predominant) or negative regulation. In particular, the interaction between Solyc03g095770.3 (group III) and Solyc09g014990.4 (group I) may play an important role. In brief, WRKY TFs were comprehensively identified in tomato and several bacterial wilt responsive genes were screened.

Pepper defense against Ralstonia solanacearum and High-temperature stress is positively regulated by CaMYB59.

We have functionally evaluated a transcription factor CaMYB59 for its role in pepper immune responses to Ralstonia solanacearum attack and high temperature-high humidity (HTHH). Exposure to R. solanacearum inoculation (RSI) and HTHH resulted in up-regulation of this nucleus-localized TF. Function of this TF was confirmed by performing loss of function assay of CaMYB59 by VIGS (virus-induced gene silencing). Plants with silenced CaMYB59 displayed not only compromised pepper immunity against RSI but also impaired tolerance to HTHH along with decreased hypersensitive response (HR). This impairment in defense function was fully linked with low induction of stress-linked genes like CaPO2, CaPR1, CaAcc and thermo-tolerance linked CaHSP24 as well as CaHsfB2a. Conversely, transient overexpression of CaMYB59 enhanced pepper immunity. This reveals that CaMYB59 positively regulated host defense against RSI and HTHH by means of HR like mimic cell death, H2O2 production and up-regulation of defense as well as thermo-tolerance associated genes. These changes in attributes collectively confirm the role of CaMYB59 as a positive regulator of pepper immunity against R. solanacearum. We recommend that such positive regulation of pepper defense is dynamically supported by phyto-hormone signaling and transcriptional web of defense genes. These integrated and interlinked events stabilize plant growth and survival under abiotic and biotic stresses.

Genome-wide analysis of the peanut CaM/CML gene family reveals that the AhCML69 gene is associated with resistance to Ralstonia solanacearum.

BACKGROUND: Calmodulins (CaMs)/CaM-like proteins (CMLs) are crucial Ca2+-binding sensors that can decode and transduce Ca2+ signals during plant development and in response to various stimuli. The CaM/CML gene family has been characterized in many plant species, but this family has not yet been characterized and analyzed in peanut, especially for its functions in response to Ralstonia solanacearum. In this study, we performed a genome-wide analysis to analyze the CaM/CML genes and their functions in resistance to R. solanacearum. RESULTS: Here, 67, 72, and 214 CaM/CML genes were identified from Arachis duranensis, Arachis ipaensis, and Arachis hypogaea, respectively. The genes were divided into nine subgroups (Groups I-IX) with relatively conserved exon‒intron structures and motif compositions. Gene duplication, which included whole-genome duplication, tandem repeats, scattered repeats, and unconnected repeats, produced approximately 81 pairs of homologous genes in the AhCaM/CML gene family. Allopolyploidization was the main reason for the greater number of AhCaM/CML members. The nonsynonymous (Ka) versus synonymous (Ks) substitution rates (less than 1.0) suggested that all homologous pairs underwent intensive purifying selection pressure during evolution. AhCML69 was constitutively expressed in different tissues of peanut plants and was involved in the response to R. solanacearum infection. The AhCML69 protein was localized in the cytoplasm and nucleus. Transient overexpression of AhCML69 in tobacco leaves increased resistance to R. solanacearum infection and induced the expression of defense-related genes, suggesting that AhCML69 is a positive regulator of disease resistance. CONCLUSIONS: This study provides the first comprehensive analysis of the AhCaM/CML gene family and potential genetic resources for the molecular design and breeding of peanut bacterial wilt resistance.

Comparative genomics and host range analysis of four Ralstonia pseudosolanacearum strains isolated from sunflower reveals genomic and phenotypic differences.

BACKGROUND: Bacterial wilt caused by Ralstonia solanacearum species complex (RSSC) is one of the devastating diseases in crop production, seriously reducing the yield of crops. R. pseudosolanacearum, is known for its broad infrasubspecific diversity and comprises 36 sequevars that are currently known. Previous studies found that R. pseudosolanacearum contained four sequevars (13, 14, 17 and 54) isolated from sunflowers sown in the same field. RESULTS: Here, we provided the complete genomes and the results of genome comparison of the four sequevars strains (RS639, RS642, RS647, and RS650). Four strains showed different pathogenicities to the same cultivars and different host ranges. Their genome sizes were about 5.84 ~ 5.94 Mb, encoding 5002 ~ 5079 genes and the average G + C content of 66.85% ~ 67%. Among the coding genes, 146 ~ 159 specific gene families (contained 150 ~ 160 genes) were found in the chromosomes and 34 ~ 77 specific gene families (contained 34 ~ 78 genes) in the megaplasmids from four strains. The average nucleotide identify (ANI) values between any two strains ranged from 99.05% ~ 99.71%, and the proportion of the total base length of collinear blocks accounts for the total gene length of corresponding genome was all more than 93.82%. Then, we performed a search for genomic islands, prophage sequences, the gene clusters macromolecular secretion systems, type III secreted effectors and other virulence factors in these strains, which provided detailed comparison results of their presence and distinctive features compared to the reference strain GMI1000. Among them, the number and types of T2SS gene clusters were different in the four strains, among which RS650 included all five types. T4SS gene cluster of RS639 and RS647 were missed. In the T6SS gene cluster, several genes were inserted in the RS639, RS647, and RS650, and gene deletion was also detected in the RS642. A total of 78 kinds of type III secreted effectors were found, which included 52 core and 9 specific effectors in four strains. CONCLUSION: This study not only provided the complete genomes of multiple R. pseudosolanacearum strains isolated from a new host, but also revealed the differences in their genomic levels through comparative genomics. Furthermore, these findings expand human knowledge about the range of hosts that Ralstonia can infect, and potentially contribute to exploring rules and factors of the genetic evolution and analyzing its pathogenic mechanism.

Effects of plant tissue permeability on invasion and population bottlenecks of a phytopathogen.

Pathogen genetic diversity varies in response to environmental changes. However, it remains unclear whether plant barriers to invasion could be considered a genetic bottleneck for phytopathogen populations. Here, we implement a barcoding approach to generate a pool of 90 isogenic and individually barcoded Ralstonia solanacearum strains. We used 90 of these strains to inoculate tomato plants with different degrees of physical permeability to invasion (intact roots, wounded roots and xylem inoculation) and quantify the phytopathogen population dynamics during invasion. Our results reveal that the permeability of plant roots impacts the degree of population bottleneck, genetic diversity, and composition of Ralstonia populations. We also find that selection is the main driver structuring pathogen populations when barriers to infection are less permeable, i.e., intact roots, the removal of root physical and immune barriers results in the predominance of stochasticity in population assembly. Taken together, our study suggests that plant root permeability constitutes a bottleneck for phytopathogen invasion and genetic diversity.

Transcriptomics and virus-induced gene silencing identify defence-related genes during Ralstonia solanacearum infection in resistant and susceptible tobacco.

Bacterial wilt (BW) caused by Ralstonia solanacearum is a globally prevalent bacterial soil-borne disease. In this study, transcriptome sequencing were subjected to roots after infection with the R. solanacearum in the resistant and susceptible tobacco variety. DEGs that responded to R. solanacearum infection in both resistant and susceptible tobacco contributed to pectinase and peroxidase development and were enriched in plant hormone signal transduction, signal transduction and MAPK signalling pathway KEGG terms. Core DEGs in the resistant tobacco response to R. solanacearum infection were enriched in cell wall, membrane, abscisic acid and ethylene terms. qRT-PCR indicated that Nitab4.5_0004899g0110, Nitab4.5_0004234g0080 and Nitab4.5_0001439g0050 contributed to the response to R. solanacearum infection in different resistant and susceptible tobacco. Silencing the p450 gene Nitab4.5_0001439g0050 reduced tobacco resistance to bacterial wilt. These results improve our understanding of the molecular mechanism of BW resistance in tobacco and solanaceous plants.

Ralstonia solanacearum pandemic lineage strain UW551 overcomes inhibitory xylem chemistry to break tomato bacterial wilt resistance.

Plant-pathogenic Ralstonia strains cause bacterial wilt disease by colonizing xylem vessels of many crops, including tomato. Host resistance is the best control for bacterial wilt, but resistance mechanisms of the widely used Hawaii 7996 tomato breeding line (H7996) are unknown. Using growth in ex vivo xylem sap as a proxy for host xylem, we found that Ralstonia strain GMI1000 grows in sap from both healthy plants and Ralstonia-infected susceptible plants. However, sap from Ralstonia-infected H7996 plants inhibited Ralstonia growth, suggesting that in response to Ralstonia infection, resistant plants increase inhibitors in their xylem sap. Consistent with this, reciprocal grafting and defence gene expression experiments indicated that H7996 wilt resistance acts in both above- and belowground plant parts. Concerningly, H7996 resistance is broken by Ralstonia strain UW551 of the pandemic lineage that threatens highland tropical agriculture. Unlike other Ralstonia, UW551 grew well in sap from Ralstonia-infected H7996 plants. Moreover, other Ralstonia strains could grow in sap from H7996 plants previously infected by UW551. Thus, UW551 overcomes H7996 resistance in part by detoxifying inhibitors in xylem sap. Testing a panel of xylem sap compounds identified by metabolomics revealed that no single chemical differentially inhibits Ralstonia strains that cannot infect H7996. However, sap from Ralstonia-infected H7996 contained more phenolic compounds, which are known to be involved in plant antimicrobial defence. Culturing UW551 in this sap reduced total phenolic levels, indicating that the resistance-breaking Ralstonia strain degrades these chemical defences. Together, these results suggest that H7996 tomato wilt resistance depends in part on inducible phenolic compounds in xylem sap.

Transcriptomic profiling reveals host-specific evolutionary pathways promoting enhanced fitness in the plant pathogen Ralstonia pseudosolanacearum.

The impact of host diversity on the genotypic and phenotypic evolution of broad-spectrum pathogens is an open issue. Here, we used populations of the plant pathogen Ralstonia pseudosolanacearum that were experimentally evolved on five types of host plants, either belonging to different botanical families or differing in their susceptibility or resistance to the pathogen. We investigated whether changes in transcriptomic profiles, associated with or independent of genetic changes, could occur during the process of host adaptation, and whether transcriptomic reprogramming was dependent on host type. Genomic and transcriptomic variations were established for 31 evolved clones that showed better fitness in their experimental host than the ancestral clone. Few genomic polymorphisms were detected in these clones, but significant transcriptomic variations were observed, with a large number of differentially expressed genes (DEGs). In a very clear way, a group of genes belonging to the network of regulation of the bacterial virulence such as efpR, efpH or hrpB, among others, were deregulated in several independent evolutionary lineages and appeared to play a key role in the transcriptomic rewiring observed in evolved clones. A double hierarchical clustering based on the 400 top DEGs for each clone revealed 2 major patterns of gene deregulation that depend on host genotype, but not on host susceptibility or resistance to the pathogen. This work therefore highlights the existence of two major evolutionary paths that result in a significant reorganization of gene expression during adaptive evolution and underscore clusters of co-regulated genes associated with bacterial adaptation on different host lines.

Gene expression changes throughout the life cycle allow a bacterial plant pathogen to persist in diverse environmental habitats.

Bacterial pathogens exhibit a remarkable ability to persist and thrive in diverse ecological niches. Understanding the mechanisms enabling their transition between habitats is crucial to control dissemination and potential disease outbreaks. Here, we use Ralstonia solanacearum, the causing agent of the bacterial wilt disease, as a model to investigate pathogen adaptation to water and soil, two environments that act as bacterial reservoirs, and compare this information with gene expression in planta. Gene expression in water resembled that observed during late xylem colonization, with an intriguing induction of the type 3 secretion system (T3SS). Alkaline pH and nutrient scarcity-conditions also encountered during late infection stages-were identified as the triggers for this T3SS induction. In the soil environment, R. solanacearum upregulated stress-responses and genes for the use of alternate carbon sources, such as phenylacetate catabolism and the glyoxylate cycle, and downregulated virulence-associated genes. We proved through gain- and loss-of-function experiments that genes associated with the oxidative stress response, such as the regulator OxyR and the catalase KatG, are key for bacterial survival in soil, as their deletion cause a decrease in culturability associated with a premature induction of the viable but non culturable state (VBNC). This work identifies essential factors necessary for R. solanacearum to complete its life cycle and is the first comprehensive gene expression analysis in all environments occupied by a bacterial plant pathogen, providing valuable insights into its biology and adaptation to unexplored habitats.

Quantitative detection of the Ralstonia solanacearum species complex in soil by qPCR combined with a recombinant internal control strain.

IMPORTANCE: DNA-based detection and quantification of soil-borne pathogens, such as the Ralstonia solanacearum species complex (RSSC), plays a vital role in risk assessment, but meanwhile, precise quantification is difficult due to the poor purity and yield of the soil DNA retrieved. The internal sample process control (ISPC) strain RsPC we developed solved this problem and significantly improved the accuracy of quantification of RSSC in different soils. ISPC-based quantitative PCR detection is a method especially suitable for the quantitative detection of microbes in complex matrices (such as soil and sludge) containing various PCR inhibitors and for those not easy to lyse (like Gram-positive bacteria, fungi, and thick-wall cells like resting spores). In addition, the use of ISPC strains removes additional workload on the preparation of high-quality template DNA and facilitates the development of high-throughput quantitative detection techniques for soil microbes.

Transcriptomic analysis of Ralstonia solanacearum in response to antibacterial volatiles of Bacillus velezensis FZB42.

Volatile organic compounds (VOCs), produced by a variety of microbial species and used as biological agents, have been demonstrated to play a significant role in controlling phytopathogens. In continuation of our previous studies, we aim to elucidate the underlying mechanisms and pathways involved in interactions between pathogens and microbial VOCs. In the current study, we tested how VOCs produced by Bacillus velezensis FZB42 affect the growth of Ralstonia solanacearum TBBS1 in vitro.Query The result showed that the colony growth of R. solanacearum was reduced with an inhibition rate of 0.83 ± 0.043 as compared to the control 1.7 ± 0.076, respectively. The number of viable cells of R. solanacearum was significantly decreased to 7.68 CFU/mL as compared to the control (9.02 CFU/mL). In addition, transcriptomic analysis of R. solanacearum in response to VOCs produced by FZB42 was performed to better understand the effect of VOCs on R. solanacearum. The transcriptional response of R. solanacearum to FZB42-VOCs was determined using an Illumina RNA-seq approach. The results revealed significant changes in the expression of 2094 R. solanacearum genes, including 593 upregulated and 1501 downregulated genes. To validate the RNA-seq results, the expression of 10 genes was quantified using RT-qPCR. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to functionally annotate differentially expressed genes. Significant changes were observed in genes directly or indirectly related to virulence, including those related to bacterial invasion, motility, chemotaxis, and secretion systems. Overall, RNA-seq profiling provides new insights into the possible fundamental molecular mechanisms that are responsible for the reduction in growth and virulence of R. solanacearum upon application of FZB42-VOC.

Integrative transcriptomic analysis unveils lncRNA-miRNA-mRNA interplay in tomato plants responding to Ralstonia solanacearum.

Ralstonia solanacearum, a bacterial plant pathogen, poses a significant threat to tomato (Solanum lycopersicum) production through destructive wilt disease. While noncoding RNA has emerged as a crucial regulator in plant disease, its specific involvement in tomato bacterial wilt remains limited. Here, we conducted a comprehensive analysis of the transcriptional landscape, encompassing both mRNAs and noncoding RNAs, in a tomato resistant line ('ZRS_7') and a susceptible line ('HTY_9') upon R. solanacearum inoculation using high-throughput RNA sequencing. Differential expression (DE) analysis revealed significant alterations in 7506 mRNAs, 997 lncRNAs, and 69 miRNAs between 'ZRS_7' and 'HTY_9' after pathogen exposure. Notably, 4548 mRNAs, 367 lncRNAs, and 26 miRNAs exhibited genotype-specific responses to R. solanacearum inoculation. GO and KEGG pathway analyses unveiled the potential involvement of noncoding RNAs in the response to bacterial wilt disease, targeting receptor-like kinases, cell wall-related genes, glutamate decarboxylases, and other key pathways. Furthermore, we constructed a comprehensive competing endogenous RNA (ceRNA) network incorporating 13 DE-miRNAs, 30 DE-lncRNAs, and 127 DEGs, providing insights into their potential contributions to the response against bacterial inoculation. Importantly, the characterization of possible endogenous target mimics (eTMs) of Sly-miR482e-3p via VIGS technology demonstrated the significant impact of eTM482e-3p-1 silencing on tomato's sensitivity to R. solanacearum. These findings support the existence of an eTM482e-3p-1-Sly-miR482e-3p-NBS-LRRs network in regulating tomato's response to the pathogen. Collectively, our findings shed light on the intricate interactions among lncRNAs, miRNAs, and mRNAs as underlying factors in conferring resistance to R. solanacearum in tomato.

Part II: Impedance-based DNA biosensor for detection of isolated strains of phytopathogen Ralstonia solanacearum.

In Part I, we demonstrated the complete development of a label-free, ultra-low sample volume requiring DNA-based biosensor to detect Ralstonia solanacearum, an aerobic non-spore-forming, Gram-negative, plant pathogenic bacterium, using non-faradaic electrochemical impedance spectroscopy (nf-EIS). We also presented the sensor's sensitivity, specificity, and electrochemical stability. In this article, we highlight the specificity study of the developed DNA-based impedimetric biosensor to detect various strains of R. solanacearum. We have collected seven isolates of R. solanacearum isolated from locally infected host plants (eggplant, potato, tomato, chilli, and ginger) from different parts of Goa, India. The pathogenicity of these isolates was tested on the eggplant, and the pathogen was confirmed by microbiological plating and polymerase chain reaction (PCR). We further report the insight into the DNA hybridization on the surface of Interdigitated Electrodes (IDEs) and the expansion of the Randles model for more accurate analysis. The interpretation of the sensor specificity is clearly demonstrated by the capacitance change observed at the electrode-electrolyte interface.