Query: Ralstonia solanacearum

title: A Streptomyces sp. NEAU HV9: Isolation, Identification, and Potential as a Biocontrol Agent against Ralstonia solanacearum of Tomato Plants
abstract: Ralstonia solanacearum is an important soil borne bacterial plant pathogen. In this study, an actinomycete strain named NEAU HV9 that showed strong antibacterial activity against Ralstonia solanacearum was isolated from soil using an in vitro screening technique. Based on physiological and morphological characteristics and 98.90% of 16S rRNA gene sequence similarity with Streptomyces panaciradicis 1MR 8T, the strain was identified as a member of the genus Streptomyces. Tomato seedling and pot culture experiments showed that after pre inoculation with the strain NEAU HV9, the disease occurrence of tomato seedlings was effectively prevented for R. solanacearum. Then, a bioactivity guided approach was employed to isolate and determine the chemical identity of bioactive constituents with antibacterial activity from strain NEAU HV9. The structure of the antibacterial metabolite was determined as actinomycin D on the basis of extensive spectroscopic analysis. To our knowledge, this is the first report that actinomycin D has strong antibacterial activity against R. solanacearum with a MIC (minimum inhibitory concentration) of 0.6 mg L 1 (0.48 mmol L 1) The in vivo antibacterial activity experiment showed that actinomycin D possessed significant preventive efficacy against R. solanacearum in tomato seedlings. Thus, strain NEAU HV9 could be used as BCA (biological control agent) against R. solanacearum, and actinomycin D might be a promising candidate for a new antibacterial agent against R. solanacearum.

title: Biological control of bacterial wilt of tomato caused by Ralstonia solanacearum using Pseudomonas species isolated from the rhizosphere of tomato plants
abstract: Tomato bacterial wilt, caused by Ralstonia solanacearum, is one of the devastating soil borne diseases of tomato plants in Nigeria. We investigated the potentiality of indigenous Pseudomonas species in controlling bacterial wilt of tomato. Seven Pseudomonas species were isolated and identified from the rhizosphere of tomato plants. The isolates were screened for enzyme production, and in vitro antimicrobial activity against pathogenic R. solanacearum using dual plate culture method. The biocontrol and growth promoting efficacy of the Pseudomonas species were studied in the screenhouse using a completely randomised design. P. aeruginosa, P. syringae and P. fluorescens produced lipase, protease and a amylase enzymes. All the Pseudomonas isolates except P. aureofaciens inhibited the growth of R. solanacearum. Since the tested species reduced the incidence of bacterial wilt and promoted the growth of the tested tomato genotypes, the indigenous Pseudomonas strain could be used to develop active biocontrol agents of tomato bacterial wilt in Nigeria.

title: Various antibacterial mechanisms of biosynthesized copper oxide nanoparticles against soilborne Ralstonia solanacearum
abstract: The substantial antimicrobial efficacy of nanoparticles against phytopathogens has been extensively investigated for advanced agricultural applications. However, few reports have focused on soilborne pathogenic bacteria. The aim of this study was to obtain sustainably synthesized copper oxide nanoparticles (CuONPs) using papaya leaf extracts and investigate the bactericidal activity of these CuONPs against Ralstonia solanacearum, the cause of bacterial wilt, under laboratory and greenhouse conditions. The results showed that CuONPs possessed strong antibacterial activity and that all R. solanacearum were killed after exposure to 250 mg mL 1 CuONPs. CuONPs could interact with bacterial cells to prevent biofilm formation, reduce swarming motility and disturb ATP production. Ultrastructural observations by transmission electron microscopy (TEM) revealed that after interactions with CuONPs, bacterial cells suffered significantly from nanomechanical damage to the cytomembrane, accompanied by the absorption of multiple nanoparticles. In addition, molecular studies identified the downregulation mechanism of a series of genes involving pathogenesis and motility. The control efficiency of CuONPs in tobacco bacterial wilt disease management under greenhouse conditions was verified by root irrigation application, demonstrating that as prepared CuONPs significantly reduced the disease occurrence and disease index. Our studies focused on developing biosynthesized nanoparticles as a biocompatible alternative for soilborne disease management.

title: Biogenic synthesis of iron oxide nanoparticles via Skimmia laureola and their antibacterial efficacy against bacterial wilt pathogen Ralstonia solanacearum.
abstract: Plant diseases are threat to global food security. The excessive use of agrochemicals is the leading cause of pesticides resistance and toxicity to beneficial life forms. The quest for innocuous and alternate antimicrobial agent is crucial in order to overcome the pathogen resistance and the birth of nanotech offers pledge to combat pathogenic organisms. In this study, a facile benign biogenic approach was adopted for the synthesis of biocompatible iron oxide nanoparticles (Fe2O3 NPs) via Skimmia laureola leaf extract and the synthesized nanoparticles were evaluated for their antibacterial efficacy against bacterial wilt pathogen Ralstonia solanacearum in vitro and in planta. Physico chemical characterization of the synthesized nanoparticles was performed through UV Visible spectroscopy, Fourier Transform Infrared Spectroscopy, X Ray Diffraction, Energy Dispersive X ray Spectroscopy and Scanning Electron Microscopy. The results revealed polydisperse nanoparticles in the size range of 56 nm to 350 nm. The culture media containing 6 mg/mL of Fe2O3 NPs dramatically inhibited the bacterial growth in vitro. Scanning electron microscopy revealed degenerative characteristics including degraded, shriveled and concentrated cell walls. Diseases severity was effectively reduced with 6% w/v of Fe2O3 NPs treated root zone in planta. Plant shoots, root length and fresh biomass were enhanced with Fe2O3 NPs treatments. The results indicated that the biosynthesized Fe2O3 NPs have the potential to control agriculturally important phytopathogen Ralstonia solanacearum in vitro and in planta.

title: Biocontrol of Bacterial Wilt Disease Through Complex Interaction Between Tomato Plant, Antagonists, the Indigenous Rhizosphere Microbiota, and Ralstonia solanacearum
abstract: Ralstonia solanacearum (biovar2, race3) is the causal agent of bacterial wilt and this quarantine phytopathogen is responsible for massive losses in several commercially important crops. Biological control of this pathogen might become a suitable plant protection measure in areas where R. solanacearum is endemic. Two bacterial strains, Bacillus velezensis (B63) and Pseudomonas fluorescens (P142) with in vitro antagonistic activity toward R. solanacearum (B3B) were tested for rhizosphere competence, efficient biological control of wilt symptoms on greenhouse grown tomato, and effects on the indigenous rhizosphere prokaryotic communities. The population densities of B3B and the antagonists were estimated in rhizosphere community DNA by selective plating, real time quantitative PCR, and R. solanacearum specific fliC PCR Southern blot hybridization. Moreover, we investigated how the pathogen and/or the antagonists altered the composition of the tomato rhizosphere prokaryotic community by 16S rRNA gene amplicon sequencing. B. velezensis (B63) and P. fluorescens (P142) inoculated plants showed drastically reduced wilt disease symptoms, accompanied by significantly lower abundance of the B3B population compared to the non inoculated pathogen control. Pronounced shifts in prokaryotic community compositions were observed in response to the inoculation of B63 or P142 in the presence or absence of the pathogen B3B and numerous dynamic taxa were identified. Confocal laser scanning microscopy (CLSM) visualization of the gfp tagged antagonist P142 revealed heterogeneous colonization patterns and P142 was detected in lateral roots, root hairs, epidermal cells, and within xylem vessels. Although competitive niche exclusion cannot be excluded, it is more likely that the inoculation of P142 or B63 and the corresponding microbiome shifts primed the plant defense against the pathogen B3B. Both inoculants are promising biological agents for efficient control of R. solanacearum under field conditions.

title: The type III effector RipB from Ralstonia solanacearum RS1000 acts as a major avirulence factor in Nicotiana benthamiana and other Nicotiana species
abstract: Ralstonia solanacearum is the causal agent of bacterial wilt in solanaceous crops. This pathogen injects approximately 70 effector proteins into plant cells via the Hrp type III secretion system in an early stage of infection. To identify an as yet unidentified avirulence factor possessed by the Japanese tobacco avirulent strain RS1000, we transiently expressed RS1000 effectors in Nicotiana benthamiana leaves and monitored their ability to induce effector triggered immunity (ETI) The expression of RipB strongly induced the production of reactive oxygen species and the expressions of defence related genes in N. benthamiana. The ripB mutant of RS1002, a nalixidic acid resistant derivative of RS1000, caused wilting symptoms in N. benthamiana. A pathogenicity test using R. solanacearum mutants revealed that the two already known avirulence factors RipP1 and RipAA contribute in part to the avirulence of RS1002 in N. benthamiana. The Japanese tobacco virulent strain BK1002 contains mutations in ripB and expresses a C terminal truncated RipB that lost the ability to induce ETI in N. benthamiana, indicating a fine tuning of the pathogen effector repertoire to evade plant recognition. RipB shares homology with Xanthomonas XopQ, which is recognized by the resistance protein Roq1. The RipB induced resistance against R. solanacearum was abolished in Roq1 silenced plants. These findings indicate that RipB acts as a major avirulence factor in N. benthamiana and that Roq1 is involved in the recognition of RipB.

title: Antibacterial activity of three essential oils and five monoterpenes against Ralstonia solanacearum phylotype II isolated from potato.
abstract: The present study describes the possibility of using some essential oils from three woody plants as bioagents against the growth of Ralstonia solanacearum isolates, a causal bacterium for Potato brown rot disease. From infected potato tubers showing typical symptoms of the brown rot disease, eight isolates of R. solanacearum were recovered, confirmed to belong to phylotype II according to the biochemical and physiological characteristics, and molecularly identified. Three essential oils extracted from Corymbia citriodora (leaves) Cupressus sempervirens (aerial parts) and Lantana camara (aerial parts) were evaluated for their antibacterial activity against eight isolates of R. solanacearum phylotype II. Results demonstrated that L. camara essential oil (concentration 5000 mg/mL) had the highest effects against the RsMo2, RsSc1 and Rs48, with inhibition zone (IZ) values of 17.33, 16.33, and 17.50 mm, respectively, also against Rs2 (IZ 14.33 mm) and RsIs2 (IZ 16 mm) C. citriodora oil showed the highest activity against RsBe2 (IZ 14 mm) RsFr4 (IZ 13.66 mm) and RsNe1 (IZ 13.66 mm) Gas Chromatography Mass Spectrometry (GC MS FID) analyzed the chemical composition of these essential oils. It was proved that L. camara leaves contains mainly trans caryophyllene (16.24% and a humulene (9.55% in C. citriodora oil were a citronellal (56.55% a citronellol (14.89% and citronellol acetate (13.04% and in Cup. sempervirens aerial parts were cedrol (22.17% and D3 carene (18.59% Five monoterpenes were evaluated against the most resistance Ralstonia isolate RsFr5 to the three studied essential oils and found that limonene had the highest effect against it compared with the lowest thymol. The results proved the strong bio effects of the essential oil from L. camara leaves as a natural product contained monoterpenes that can inhibit the growth of tested R. solanacearum phylotype II isolates.

title: Biocontrol of the Major Plant Pathogen Ralstonia solanacearum in Irrigation Water and Host Plants by Novel Waterborne Lytic Bacteriophages
abstract: Three new lytic bacteriophages were found to effectively control the pathogen Ralstonia solanacearum, a quarantine bacterium in many countries, and causative agent of bacterial wilt, one of the most important vascular plant diseases. Bacterial wilt management has been carried out with fluctuating effects, suggesting the need to find alternative treatments. In this work, three lytic phages were isolated from environmental water from geographically distant regions in Spain. They proved to specifically infect a collection of R. solanacearum strains, and some of the closely related pathogenic species Ralstonia pseudosolanacearum, without affecting non target environmental bacteria, and were able to lyze the pathogen populations within a wide range of conditions comprising environmental values of water temperatures, pH, salinity, and lack of aeration found in storage tanks. The three bacteriophages displayed high efficiency in controlling R. solanacearum, with reductions of the bacterial populations of several orders of magnitude in just a few hours, and proved to be able to survive in freshwater for months at environmental temperatures keeping activity on R. solanacearum, pointing out their suitability for field application through irrigation. Concerning their biocontrol potential, they were effective in reducing high populations of the pathogen in environmental water, and bacterial wilt incidence in planta by watering with either one phage or their combinations in assays with more than 300 plants. This is the first report on effective R. solanacearum biocontrol by applying single or combined bacteriophages through irrigation water in conditions mimicking those of the natural settings. The three phages belong to the Podoviridae family and are members of the T7likevirus genus. They are the first isolated phages from river water with activity against R. solanacearum, showing the longest persistence in natural water reported until now for phages with biocontrol potential, and consistently being able to control the disease in the host plant under environmental conditions. Consequently, the use of these bacteriophages for the prevention and/or biocontrol of the bacterial wilt disease caused by R. solanacearum has been patented. Evidence provided reveals the suitability of these waterborne phages to be effectively considered as a valuable strategy within the frame of sustainable integrated management programs.

title: Resistance against Ralstonia solanacearum in tomato depends on the methionine cycle and the &ggr; aminobutyric acid metabolic pathway
abstract: Bacterial wilt caused by Ralstonia solanacearum is a complex and destructive disease that affects over 200 plant species. To investigate the interaction of R. solanacearum and its tomato (Solanum lycopersicum) plant host, a comparative proteomic analysis was conducted in tomato stems inoculated with highly and mildly aggressive R. solanacearum isolates (RsH and RsM, respectively) The results indicated a significant alteration of the methionine cycle (MTC) and downregulation of g aminobutyric acid (GABA) biosynthesis. Furthermore, transcriptome profiling of two key tissues (stem and root) at three stages (0, 3 and 5 days post inoculation) with RsH in resistant and susceptible tomato plants is presented. Transcript profiles of MTC and GABA pathways were analyzed. Subsequently, the MTC associated genes SAMS2, SAHH1 and MS1 and the GABA biosynthesis related genes GAD2 and SSADH1 were knocked down by virus induced gene silencing and the plants' defense responses upon infection with R. solanacearum RsM and RsH were analyzed. These results showed that silencing of SAHH1, MS1 and GAD2 in tomato leads to decreased resistance against R. solanacearum. In summary, the infection assays, proteomic and transcriptomic data described in this study indicate that both MTC and GABA biosynthesis play an important role in pathogenic interaction between R. solanacearum and tomato plants.

title: Intra strain elicitation and suppression of plant immunity by Ralstonia solanacearum type III effectors in Nicotiana benthamiana
abstract: Effector proteins delivered inside plant cells are powerful weapons for bacterial pathogens, but this exposes the pathogen to potential recognition by the plant immune system. Therefore, effector acquisition must be balanced for a successful infection. Ralstonia solanacearum is an aggressive pathogen with a large repertoire of secreted effectors. One of these effectors, RipE1, is conserved in most R. solanacearum strains sequenced to date. In this work, we found that RipE1 triggers immunity in N. benthamiana, which requires the immune regulator SGT1, but not EDS1 or NRCs. Interestingly, RipE1 triggered immunity induces the accumulation of salicylic acid (SA) and the overexpression of several genes encoding phenylalanine ammonia lyases (PALs) suggesting that the unconventional PAL mediated pathway is responsible for the observed SA biosynthesis. Surprisingly, RipE1 recognition also induces the expression of jasmonic acid (JA) responsive genes and JA biosynthesis, suggesting that both SA and JA may act cooperatively in response to RipE1. Finally, we found that RipE1 expression leads to the accumulation of glutathione in plant cells, which precedes the activation of immune responses. R. solanacearum encodes another effector, RipAY, which is known to inhibit immune responses by degrading cellular glutathione. Accordingly, we show that RipAY inhibits RipE1 triggered immune responses. This work shows a strategy employed by R. solanacearum to counteract the perception of its effector proteins by the plant immune system.