Plant Pathogen Ralstonia Research Articles

Bioprospecting of Selected Trichoderma spp. for Its Plant Growth Promoting and Biocontrol Properties

Bioprospecting for Trichoderma strains from peat soil collected at MARDI Sessang Station, Saratok, Sarawak, revealed promising candidates as plant growth-promoting microbes (PGPM) and producers of phytohormones such as indole-3-acetic acid (IAA) and gibberellic acid (GA3). The study aimed to identify Trichoderma strains with dual functions in biocontrol and plant growth enhancement. A total of seventeen strains were isolated and screened for their biocontrol activity against significant plant pathogens—Ralstonia solanacearum and Colletotrichum gloeosporioides and also their potential to produce IAA and GA3. Among the isolates, one strain exhibited strong antagonistic activity against R. solanacearum, a pathogen responsible for bacterial wilt, suggesting its potential for biocontrol applications. Additionally, two strains showed significant inhibitory effects against C. gloeosporioides, the causative agent of anthracnose. Four strains demonstrated the ability to produce IAA, an essential auxin for root development and overall plant growth promotion and two strains were identified as GA3 producers, contributing to plant growth by promoting cell elongation and germination. This study highlights the potential of Trichoderma strains from peat soils in Sarawak as valuable resources for sustainable agriculture. The dual functionality of these strains as biocontrol agents and phytohormone producers makes them promising candidates for integrated pest management (IPM) and biofertilizer development. Future research should focus on field trials and further molecular characterization to fully explore the potential of these Trichoderma isolates in enhancing crop productivity.

Isolation and identification of antifungal, antibacterial and nematocide agents from marine bacillus gottheilii MSB1

Pathogenic fungi employ numerous strategies to colonize plants, infect them, reduce crop yield and quality, and cause significant losses in agricultural production. The increasing use of chemical pesticides has led to various ecological and environmental issues, including the emergence of resistant weeds, soil compaction, and water pollution, all negatively impacting agricultural sustainability. Additionally, the extensive development of synthetic fungicides has adverse effects on animal and human health, prompting the exploration of alternative approaches and green strategies for phytopathogen control. Microorganisms living in sponges represent a promising source of novel bioactive secondary metabolites, potentially useful in developing new nematicidal and antimicrobial agents. This study focuses on extracting bioactive compounds from endosymbiotic bacteria associated with the marine sponge Hyrtios erect sp. (collected from NIOF Station, Hurghada, Red Sea, Egypt) using various organic solvents. Bacillus sp. was isolated and identified through 16 S rRNA gene sequencing. The biocidal activity of Bacillus gotheilii MSB1 extracts was screened against plant pathogenic bacteria, fungi, and nematodes. The n-butanol extract showed significant potential as a biological fungicide against Alternaria alternata and Fusarium oxysporum. Both n-hexane and ethyl acetate extracts exhibited negative impacts against the plant pathogenic bacteria Erwinia carotovora and Ralstonia solanacearum, whereas the n-butanol extract had a positive effect. Regarding nematicidal activity, ethyl acetate and n-butanol extracts demonstrated in-vitro activity against the root-knot nematode Meloidogyne incognita, which causes serious vegetable crop diseases, but the n-hexane extract showed no positive effects. The findings suggest that bioactive compounds from endosymbiotic bacteria associated with marine sponges, particularly B. gotheilii MSB1, hold significant potential as alternative biological control agents against plant pathogens. The n-butanol extract, in particular, displayed promising biocidal activities against various plant pathogenic fungi, bacteria, and nematodes. These results support further exploration and development of such bioactive compounds as sustainable, environmentally friendly alternatives to synthetic pesticides and fungicides in agricultural practices.

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.

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.

A pangenomic atlas reveals eco-evolutionary dynamics that shape type VI secretion systems in plant-pathogenic Ralstonia.

Soilborne Ralstonia solanacearum species complex (RSSC) pathogens disrupt microbial communities as they invade roots and fatally wilt plants. RSSC pathogens secrete antimicrobial toxins using a type VI secretion system (T6SS). To investigate how evolution and ecology have shaped the T6SS of these bacterial pathogens, we analyzed the T6SS gene content and architecture across the RSSC and their evolutionary relatives. Our analysis reveals that two ecologically similar Burkholderiaceae taxa, xylem-pathogenic RSSC and Paracidovorax, have convergently evolved to wield large arsenals of T6SS toxins. To understand the mechanisms underlying genomic enrichment of T6SS toxins, we compiled an atlas of 1,066 auxiliary T6SS toxin clusters ("aux" clusters) across 99 high-quality RSSC genomes. We classified 25 types of aux clusters with toxins that predominantly target lipids, nucleic acids, or unknown cellular substrates. The aux clusters were located in diverse genetic neighborhoods and had complex phylogenetic distributions, suggesting frequent horizontal gene flow. Phages and other mobile genetic elements account for most of the aux cluster acquisition on the chromosome but very little on the megaplasmid. Nevertheless, RSSC genomes were more enriched in aux clusters on the megaplasmid. Although the single, ancestral T6SS was broadly conserved in the RSSC, the T6SS has been convergently lost in atypical, non-soilborne lineages. Overall, our data suggest dynamic interplay between the lifestyle of RSSC lineages and the evolution of T6SSes with robust arsenals of toxins. This pangenomic atlas poises the RSSC as an emerging, tractable model to understand the role of the T6SS in shaping pathogen populations.IMPORTANCEWe explored the eco-evolutionary dynamics that shape the inter-microbial warfare mechanisms of a globally significant plant pathogen, the Ralstonia solanacearum species complex. We discovered that most Ralstonia wilt pathogens have evolved extensive and diverse repertoires of type VI secretion system-associated antimicrobial toxins. These expansive toxin arsenals potentially enhance the ability of Ralstonia pathogens to invade plant microbiomes, enabling them to rapidly colonize and kill their host plants. We devised a classification system to categorize the Ralstonia toxins. Interestingly, many of the toxin gene clusters are encoded on mobile genetic elements, including prophages, which may be mutualistic symbionts that enhance the inter-microbial competitiveness of Ralstonia wilt pathogens. Moreover, our findings suggest that the convergent loss of this multi-gene trait contributes to genome reduction in two vector-transmitted lineages of Ralstonia pathogens. Our findings demonstrate that the interplay between microbial ecology and pathogen lifestyle shapes the evolution of a genetically complex antimicrobial weapon.

Prevalence and diversity of TAL effector-like proteins in fungal endosymbiotic Mycetohabitans spp.

Endofungal Mycetohabitans (formerly Burkholderia) spp. rely on a type III secretion system to deliver mostly unidentified effector proteins when colonizing their host fungus, Rhizopus microsporus. The one known secreted effector family from Mycetohabitans consists of homologues of transcription activator-like (TAL) effectors, which are used by plant pathogenic Xanthomonas and Ralstonia spp. to activate host genes that promote disease. These 'Burkholderia TAL-like (Btl)' proteins bind corresponding specific DNA sequences in a predictable manner, but their genomic target(s) and impact on transcription in the fungus are unknown. Recent phenotyping of Btl mutants of two Mycetohabitans strains revealed that the single Btl in one Mycetohabitans endofungorum strain enhances fungal membrane stress tolerance, while others in a Mycetohabitans rhizoxinica strain promote bacterial colonization of the fungus. The phenotypic diversity underscores the need to assess the sequence diversity and, given that sequence diversity translates to DNA targeting specificity, the functional diversity of Btl proteins. Using a dual approach to maximize capture of Btl protein sequences for our analysis, we sequenced and assembled nine Mycetohabitans spp. genomes using long-read PacBio technology and also mined available short-read Illumina fungal-bacterial metagenomes. We show that btl genes are present across diverse Mycetohabitans strains from Mucoromycota fungal hosts yet vary in sequences and predicted DNA binding specificity. Phylogenetic analysis revealed distinct clades of Btl proteins and suggested that Mycetohabitans might contain more species than previously recognized. Within our data set, Btl proteins were more conserved across M. rhizoxinica strains than across M. endofungorum, but there was also evidence of greater overall strain diversity within the latter clade. Overall, the results suggest that Btl proteins contribute to bacterial-fungal symbioses in myriad ways.

RSSC-Lineage Multiplex PCR Assay Detects and Differentiates Ralstonia solanacearum, R. pseudosolanacearum, R. syzygii, and the R3bv2 Subgroup

Bacterial wilt strains in the Ralstonia solanacearum species complex (RSSC) pose serious threats to economically important crops worldwide. In 2014, Safni et al. proposed the reclassification of the RSSC into three genomospecies: R. solanacearum (Rsol), R. pseudosolanacearum (Rpseu), and R. syzygii (Rsyz). The revision requires the proper identification of strains for diagnostic and epidemiological studies. In response, we developed the inexpensive and user-friendly RSSC-Lineage Multiplex PCR, which effectively detects plant-pathogenic Ralstonia strains in general and also distinguishes between Rpseu, Rsol, Rsyz, and the high-security Select Agent “race 3 biovar 2” subgroup of Rsol, also known as the phylotype IIB-1 potato brown rot pandemic lineage. Genomes were retrieved from the NCBI GenBank database and screened for unique gene regions using OrthoMCL and other comparative genomic approaches. Specific primers were designed for each genomospecies, Ralstonia in general, and “race 3 biovar 2.” AT-rich flaps were added at the 5ʹ position of each primer to optimize the reaction thermodynamics. The specificity was tested in silico using the NCBI GenBank genome database and an in-house database. The in vitro specificity and accuracy of the tool was validated with 113 representative Ralstonia strains and 24 strains from other genera. The assay is highly specific, generating neither false positives nor false negatives. Primer set detection limits ranged from 10 to 100 pg. The assay also detected and differentiated strains from naturally and artificially inoculated plant hosts. This tool is highly specific, reliable, and economical for culture characterization, diagnostics, surveys, quarantine decisions, and epidemiological studies. [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 .

Effects of different dwarfing interstocks on the rhizosphere, endophytic bacteria, and drought resistance of apple trees

Rootstock is commonly used to enhance plant resistance to drought stress. However, it is necessary to investigate the effects of different rootstock, interstock, and scion combinations on rhizosphere and root endophytic bacteria under drought stress. We conducted a pot experiment to investigate how interstock [SH40, Jizhen 1 (J1), and Jizhen 2 (J2)] affects the drought tolerance and nitrogen (N) uptake and utilization of apple trees under drought stress. The results showed that the total dry weight, total chlorophyll content, carotenoid content, photosynthesis rate, and N absorption and utilization efficiency of apple trees decreased significantly, whereas relative electrolyte leakage increased significantly under drought stress. Membership function analysis showed that the apple plants with the J1 interstock had the greatest drought resistance. In addition, drought treatment significantly affected the diversity and composition of rhizosphere and root endophytic communities in all three rootstock/interstock/scion combinations. Further analysis revealed that the relative abundance of the plant pathogen Ralstonia was significantly increased in J2 drought-treated roots, compared to the other groups, whereas those of some potentially beneficial bacteria (0134_terrestrial_group, Phenylobacterium, Ellin6067, Kribbella, Chloronema, and Streptomyces) increased significantly in the J1 drought-treated sample. Co-occurrence network analysis showed that some potentially beneficial bacteria (Ellin6067, S0134_terrestrial_group, Pedomicrobium, and Subgroup_10) were significantly positively correlated with N content. These modifications of the rhizosphere and endophytic bacterial communities may influence the drought resilience and N uptake efficiency of different combinations of interstocks and scions. This study is a much-needed step towards understanding the stress response mechanism of scion–rootstock combinations.

Bacterial wilt affects the structure and assembly of microbial communities along the soil-root continuum

BackgroundBeneficial root-associated microbiomes play crucial roles in enhancing plant growth and suppressing pathogenic threats, and their application for defending against pathogens has garnered increasing attention. Nonetheless, the dynamics of microbiome assembly and defense mechanisms during pathogen invasion remain largely unknown. In this study, we aimed to investigate the diversity and assembly of microbial communities within four niches (bulk soils, rhizosphere, rhizoplane, and endosphere) under the influence of the bacterial plant pathogen Ralstonia solanacearum.ResultsOur results revealed that healthy tobacco plants exhibited more diverse community compositions and more robust co-occurrence networks in root-associated niches compared to diseased tobacco plants. Stochastic processes (dispersal limitation and drift), rather than determinism, dominated the assembly processes, with a higher impact of drift observed in diseased plants than in healthy ones. Furthermore, during the invasion of R. solanacearum, the abundance of Fusarium genera, a known potential pathogen of Fusarium wilt, significantly increased in diseased plants. Moreover, the response strategies of the microbiomes to pathogens in diseased and healthy plants diverged. Diseased microbiomes recruited beneficial microbial taxa, such as Streptomyces and Bacilli, to mount defenses against pathogens, with an increased presence of microbial taxa negatively correlated with the pathogen. Conversely, the potential defense strategies varied across niches in healthy plants, with significant enrichments of functional genes related to biofilm formation in the rhizoplane and antibiotic biosynthesis in the endosphere.ConclusionOur study revealed the varied community composition and assembly mechanism of microbial communities between healthy and diseased tobacco plants along the soil-root continuum, providing new insights into niche-specific defense mechanisms against pathogen invasions. These findings may underscore the potential utilization of different functional prebiotics to enhance plants’ ability to fend off pathogens.

HrpY protein of Ralstonia solanacearum exhibits spontaneous formation of pilus like assembly: analysis of its stability

Type 3 secretory system (T3SS), a complex protein machinery has a unique virulence mechanism that involves injecting effector proteins directly into host cells. The T3SS effector proteins are transported through an extracellular long hollow needle made up of multiple copies of a small protein. In T3SS of the plant pathogen Ralstonia solanacearum, the 8.6 kDa HrpY protein assembles into a large needle like apparatus (pilus) for transporting effector proteins. To study structural details of HrpY, we recombinantly expressed and purified HrpY in E. coli. The dynamic light scattering (DLS) analysis showed that rHrpY has spontaneously formed oligomers of large order (>100 nm). Transmission electron microscopy of rHrpY samples revealed that the large structures are tube like assembly having dimensions 86.3–166.6 nm and 5.8–6.8 nm in length and width respectively. Different molecular sizes of the purified rHrpY hindered the crystallization of the protein. The stability of oligomer assembly was studied with denaturants and surfactants. Denaturants like urea and guanidine HCl could not break them apart; however, detergents like SDS, sarkosyl, Octyl-β-Glucoside, CHAPS, Tween 20, Tween 80 and Triton X-100 showed disassembly of the oligomer. rHrpY assembly was found to withstand up to 50 °C and the circular dichroism analysis revealed that there is no significant change in the secondary structural composition with increase in temperature. However, change in the secondary structure was observed with the addition of SDS. Communicated by Ramaswamy H. Sarma

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.

The Ralstonia Research Community Rejects the Proposal to Classify Phylotype I Ralstonia into the New Species Ralstonia nicotianae

The Ralstonia solanacearum species complex is a group of globally important plant pathogens. Bacteria in this very large and genetically diverse group all colonize the xylem elements of angiosperm plants and cause high-impact wilting diseases of many crops. Because they threaten economic and food security, several R. solanacearum species complex subgroups are strictly regulated as quarantine pests. Biologically meaningful and consistent nomenclature is essential for organisms that have major economic and regulatory importance, such as plant-pathogenic Ralstonia. There are currently three species of Ralstonia wilt pathogens: R. pseudosolanacearum (corresponding to two phylogenetic groups that are described in the literature as phylotypes I and III), R. solanacearum (phylotypes IIA, IIB, and IIC), and R. syzygii (phylotype IV, containing three subspecies: subsp. syzygii, subsp. celebensis, and subsp. indonesiensis). A recent paper proposed reclassifying phylotype I as a new species named “ Ralstonia nicotianae.” The purpose of this commentary is to register our objection to the taxon “ Ralstonia nicotianae.” [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

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 exvivo 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.

QMRA of Ralstonia solanacearum in potato cultivation: Risks associated with irrigation water recycled through managed aquifer recharge

Agricultural aquifer storage recovery and transfer (ASTR) stores excess fresh water for later reuse in irrigation. Moreover, water quality improves because chemical pollutants and pathogens will be removed by degradation and attachment to the aquifer material. The source water may contain the bacterial plant pathogen Ralstonia solanacearum which causes plant infections and high yield losses. We used quantitative microbial risk assessment (QMRA) to investigate the removal of R. solanacearum during ASTR to predict infection risks of potato plants after irrigation with the recovered water. Laboratory experiments analyzed the ASTR treatment by investigating the bacterial die-off in the water phase and the removal by attachment to the aquifer sediment. Die-off in the water phase depends on the residence time and ranged between 1.3 and 2.7 log10 after 10 or 60 days water storage, respectively. A subpopulation of the bacteria persisted for a prolonged time at low concentrations which may pose a risk if the water is recovered too early. However, the natural aquifer sand filtration proofed to be highly effective in removing R. solanacearum by attachment which depends on the distance between injection and abstraction well. The high removal by attachment alone (18 log10 after 1 m) would reduce bacterial concentrations to negligible numbers. Upscaling to longer soil passages is discussed in the paper. Infection risks of potato plants were calculated using a dose-response model and ASTR treatment resulted in negligible infection risks of a single plant, but also when simulating the irrigation of a 5 ha potato field. This is the first QMRA that analyzed an agricultural ASTR and the fate of a plant pathogen focusing on plant health. QMRA is a useful (water) management tool to evaluate the treatment steps of water reclamation technologies with the aim to provide safe irrigation water and reduce risks disseminating plant diseases.

Biologically Synthesized Silver Nanoparticles Efficiently Control Plant Pathogenic Bacteria-Erwinia carotovora and Ralstonia solanacearum

Biogenic nanoparticles are considered effective alternatives to chemical pesticides for the management of pathogenic plant diseases. This study was focused on the synthesis of stable silver nanoparticles (AgNPs) to control challenging plant pathogenic bacteria in vitro and in planta. We synthesized AgNPs by reacting different proportions of silver nitrate and aqueous extract of Hedera nepalensis. The physicochemical properties of the synthesized AgNPs were determined by using various physical techniques. The TEM analysis revealed the AgNPs less than 50 nm in size and spherical shaped. For antibacterial assays, different concentrations (1000–15.62 µg/mL, 2-fold dilutions) of the extract-free AgNPs (Ef-AgNPs) or extract-mixed AgNPs (Em-AgNPs), and fruit extracts (FE) were used against plant pathogenic bacteria Erwinia carotovora subsp. carotovora, Erwinia carotovora subsp. atroseptica, and Ralstonia solanacearum. In the in vitro assays, we found significant inhibition of both bacterial species in response to maximum concentrations of AgNPs. Overall, Ef-AgNPs exhibited a higher percent inhibition of bacterial pathogens. In potato tubers assay, complete inhibition of Erwinia carotovora was observed, except for the lowest AgNPs concentration of 15.62 µg/mL. Similarly, exposure of tomato plants to Ralstonia solanacearum suspensions (OD600 = 0.2) in the soil-drenching experiment and post-exposure treatment with 1000 µg/mL and 125 µg/mL of AgNPs resulted in disease inhibition. This study provides the basis that biogenic nanoparticles prepared from Hedera nepalensis are one of the best substitutes to synthetic pesticide, having displayed better results to control the growth of phytopathogenic microbes. However, field studies need to be conducted in a controlled environment to scale up the current work and find out the efficacy of nanoparticles on a larger scale.

Analysis of the speciation process suggests a dual lifestyle in the plant pathogen Ralstonia solanacearum species complex

Analysis of the speciation process suggests a dual lifestyle in the plant pathogen Ralstonia solanacearum species complex

Chryseochelins-structural characterization of novel citrate-based siderophores produced by plant protecting Chryseobacterium spp.

Bacteria secrete siderophores whose function is to acquire iron. In recent years, the siderophores of several Chryseobacterium species were shown to promote the health and growth of various plants such as tomato or rice. However, the chemical nature of Chryseobacterium siderophores remained unexplored despite great interest. In this work, we present the purification and structure elucidation by nuclear magnetic resonance (NMR) spectroscopy and tandem mass spectrometry (MS/MS) of chryseochelin A, a novel citrate-based siderophore secreted by three Chryseobacterium strains involved in plant protection. It contains the unusual building blocks 3-hydroxycadaverine and fumaric acid. Furthermore, the unstable structural isomer chryseochelin B and its stable derivative containing fatty acid chains, named chryseochelin C, were identified by mass spectrometric methods. The latter two incorporate an unusual ester connectivity to the citrate moiety showing similarities to achromobactin from the plant pathogen Dickeya dadantii. Finally, we show that chryseochelin A acts in a concentration-dependent manner against the plant-pathogenic Ralstonia solanacearum strain by reducing its access to iron. Thus, our study provides valuable knowledge about the siderophores of Chryseobacterium strains, which have great potential in various applications.

Plant-Pathogenic Ralstonia Phylotypes Evolved Divergent Respiratory Strategies and Behaviors To Thrive in Xylem.

Bacterial pathogens in the Ralstonia solanacearum species complex (RSSC) infect the water-transporting xylem vessels of plants, causing bacterial wilt disease. Strains in RSSC phylotypes I and III can reduce nitrate to dinitrogen via complete denitrification. The four-step denitrification pathway enables bacteria to use inorganic nitrogen species as terminal electron acceptors, supporting their growth in oxygen-limited environments such as biofilms or plant xylem. Reduction of nitrate, nitrite, and nitric oxide all contribute to the virulence of a model phylotype I strain. However, little is known about the physiological role of the last denitrification step, the reduction of nitrous oxide to dinitrogen by NosZ. We found that phylotypes I and III need NosZ for full virulence. However, strains in phylotypes II and IV are highly virulent despite lacking NosZ. The ability to respire by reducing nitrate to nitrous oxide does not greatly enhance the growth of phylotype II and IV strains. These partial denitrifying strains reach high cell densities during plant infection and cause typical wilt disease. However, unlike phylotype I and III strains, partial denitrifiers cannot grow well under anaerobic conditions or form thick biofilms in culture or in tomato xylem vessels. Furthermore, aerotaxis assays show that strains from different phylotypes have different oxygen and nitrate preferences. Together, these results indicate that the RSSC contains two subgroups that occupy the same habitat but have evolved divergent energy metabolism strategies to exploit distinct metabolic niches in the xylem. IMPORTANCE Plant-pathogenic Ralstonia spp. are a heterogeneous globally distributed group of bacteria that colonize plant xylem vessels. Ralstonia cells multiply rapidly in plants and obstruct water transport, causing fatal wilting and serious economic losses of many key food security crops. The virulence of these pathogens depends on their ability to grow to high cell densities in the low-oxygen xylem environment. Plant-pathogenic Ralstonia can use denitrifying respiration to generate ATP. The last denitrification step, nitrous oxide reduction by NosZ, contributes to energy production and virulence for only one of the three phytopathogenic Ralstonia species. These complete denitrifiers form thicker biofilms in culture and in tomato xylem, suggesting they are better adapted to hypoxic niches. Strains with partial denitrification physiology form less biofilm and are more often planktonic. They are nonetheless highly virulent. Thus, these closely related bacteria have adapted their core metabolic functions to exploit distinct microniches in the same habitat.

Rhizosphere phage communities drive soil suppressiveness to bacterial wilt disease

BackgroundBacterial viruses, phages, play a key role in nutrient turnover and lysis of bacteria in terrestrial ecosystems. While phages are abundant in soils, their effects on plant pathogens and rhizosphere bacterial communities are poorly understood. Here, we used metagenomics and direct experiments to causally test if differences in rhizosphere phage communities could explain variation in soil suppressiveness and bacterial wilt plant disease outcomes by plant-pathogenic Ralstonia solanacearum bacterium. Specifically, we tested two hypotheses: (1) that healthy plants are associated with stronger top-down pathogen control by R. solanacearum-specific phages (i.e. ‘primary phages’) and (2) that ‘secondary phages’ that target pathogen-inhibiting bacteria play a stronger role in diseased plant rhizosphere microbiomes by indirectly ‘helping’ the pathogen.ResultsUsing a repeated sampling of tomato rhizosphere soil in the field, we show that healthy plants are associated with distinct phage communities that contain relatively higher abundances of R. solanacearum-specific phages that exert strong top-down pathogen density control. Moreover, ‘secondary phages’ that targeted pathogen-inhibiting bacteria were more abundant in the diseased plant microbiomes. The roles of R. solanacearum-specific and ‘secondary phages’ were directly validated in separate greenhouse experiments where we causally show that phages can reduce soil suppressiveness, both directly and indirectly, via top-down control of pathogen densities and by alleviating interference competition between pathogen-inhibiting bacteria and the pathogen.ConclusionsTogether, our findings demonstrate that soil suppressiveness, which is most often attributed to bacteria, could be driven by rhizosphere phage communities that regulate R. solanacearum densities and strength of interference competition with pathogen-suppressing bacteria. Rhizosphere phage communities are hence likely to be important in determining bacterial wilt disease outcomes and soil suppressiveness in agricultural fields.9CDhWVdEuw3PsngifdxC8SVideo

Bacterial volatile organic compounds attenuate pathogen virulence via evolutionary trade-offs.

Volatile organic compounds (VOCs) produced by soil bacteria have been shown to exert plant pathogen biocontrol potential owing to their strong antimicrobial activity. While the impact of VOCs on soil microbial ecology is well established, their effect on plant pathogen evolution is yet poorly understood. Here we experimentally investigated how plant-pathogenic Ralstonia solanacearum bacterium adapts to VOC-mixture produced by a biocontrol Bacillus amyloliquefaciens T-5 bacterium and how these adaptations might affect its virulence. We found that VOC selection led to a clear increase in VOC-tolerance, which was accompanied with cross-tolerance to several antibiotics commonly produced by soil bacteria. The increasing VOC-tolerance led to trade-offs with R. solanacearum virulence, resulting in almost complete loss of pathogenicity in planta. At the genetic level, these phenotypic changes were associated with parallel mutations in genes encoding lipopolysaccharide O-antigen (wecA) and type-4 pilus biosynthesis (pilM), which both have been linked with outer membrane permeability to antimicrobials and plant pathogen virulence. Reverse genetic engineering revealed that both mutations were important, with pilM having a relatively larger negative effect on the virulence, while wecA having a relatively larger effect on increased antimicrobial tolerance. Together, our results suggest that microbial VOCs are important drivers of bacterial evolution and could potentially be used in biocontrol to select for less virulent pathogens via evolutionary trade-offs.