Aspergillus niger Mediated Biosynthesis of Bimetallic Copper-Selenium Nanoparticles and Their Roles in Enhancing Resistance to Ralstonia solanacearum.

The threats to the life and production of crops are exacerbated by climate change and the misuse of chemical pesticides. This study was designed to evaluate the effectiveness of biosynthesized silica nanoparticles (SiO2-NPs) as an alternative to pesticides against early blight disease of eggplant. Antifungal activity, disease index, photosynthetic pigments, osmolytes, oxidative stress, antioxidant enzymes activities were tested for potential tolerance of eggplant infected with Alternaria solani. Silica nanoparticles were successfully biosynthesized using Aspergillus niger through green and ecofriendly method. Results revealed that SiO2-NPs exhibited promising antifungal activity against A. solani where MIC was 62.5 µg/mL, and inhibition growth at concentration 1000 µg/mL recorded 87.8%. The disease Index (DI) as a result of infection with A. solani reached 82.5%, and as a result, a severe decrease in stem and root length and number of leaves occurred, which led to a sharp decrease in the photosynthetic pigments. However, contents of free proline, total phenol and antioxidant enzymes activity were increased in infected plants. On the other hand, the treatment with SiO2-NPs 100 ppm led to a great reduction in the disease Index (DI) by 25% and a high protection rate by 69.69%. A clear improvement in growth characteristics and a high content of chlorophyll and total carotenoids was also observed in the plants as a result of treatment with silica nanoparticles in (healthy and infected) plants. Interestingly, the noticeable rise in the content of infected and healthy plants of proline and phenols and an increase in the activity of super oxide dismutase (SOD) and polyphenol oxidase (PPO). It could be suggested that foliar application of SiO2-NPs especially 100 ppm could be commercially used as antifungal and strong inducer of plant physiological immunity against early blight disease.

Ralstonia solanacearum is a soil-borne pathogen that causes bacterial wilt and has a wide range of hosts in numerous plant species. This pathogen undergoes phenotypic conversion (PC) from a wild-type strain pathogenic form to a non-pathogenic form in broth culture, soil, plant, and water extract of the plant. Pre-inoculation of PC mutants to Solanum plants has been shown to have a protective effect against bacterial wilt disease. To select effective PC mutants as biocontrol agents against bacterial wilt disease in eggplant, control effect was investigated using five eggplant cultivars and 10 PC mutants. Control effects differed depending on PC mutants and eggplant cultivars. Two PC mutants (8224PC and 8103PC) showed highly suppressive effects in many eggplant cultivars and are anticipated to be practicable biocontrol agents. Furthermore, the antibacterial activities of PC mutants against the wild-type pathogen were tested to elucidate the relation between antibacterial activity of PC mutant and protective effect against bacterial wilt by PC mutant. Even the PC mutants that did not indicate antibacterial activity showed high suppression of bacterial wilt, suggesting that there are factors other than antibacterial activity involved in the suppression of bacterial wilt by PC mutant.

ABSTRACTRalstonia solanacearum, the causative agent of bacterial wilt, is a devastating bacterial plant pathogen with a wide range of hosts. We report here the first draft genome sequences for three strains of Ralstonia solanacearum isolated from infected potato, tomato, and pepper plants in Georgia.

Ralstonia solanacearum (Smith) is a soil-borne plant pathogen responsible for causing bacterial wilt and having wide host range which includes monocots, dicots, annual plants/trees and shrubs. It is a most destructive disease of solanceaous crops and ginger in north eastern region of India. The pathogen is primarily present in soils as saprophytic bacterium and it has ability to survive for long periods of time in various natural habitats. The bacterium causes sudden wilting in plants and difficult to detect at the initial level as similar symptom may also occur with many fungal organisms like Fusarium spp. and Verticillium spp. An attempt was made to develop a PCR-based rapid method for detection of this pathogen. This method requires only 3-5 hours against the conventional methods which generally require minimum 3 days to detect the pathogen. The PCR uses previously reported primer pairs for fliC gene (Rsol_fliC), which amplify 400bp region of fliC gene. The bacterial ooze from infected tissues was directly used as a source of DNA. The amplified product was cloned and sequenced for confirmation. The PCR based method developed in this report is very simple, robust and inexpensive and was successfully tested on four infected samples and further validated on over 50 samples of tomato which were infected by R. solanacearum.

Ralstonia solanacearum causes bacterial wilt on a wide range of plant hosts. Most strains of R. solanacearum are nonpathogenic below 20°C; however, Race 3 Biovar 2 (R3B2) strains are classified as quarantine pathogens because of their ability to infect crops, cause disease, and survive in temperate climates. We have identified race 1 biovar 1 Phylotype IIB Sequevar 4 strains present in Florida which were able to infect and produce wilt symptoms on potato and tomato at 18°C. Moreover they infected tomato plants at rates similar to strains belonging to R3B2. We determined that strains naturally nonpathogenic at 18°C were able to multiply, move in planta, and cause partial wilt when inoculated directly into the stem, suggesting that low temperature affects virulence of strains differently at early stages of infection. Bacterial growth in vitro was delayed at low temperatures, however it was not attenuated. Twitching motility observed on growing colonies was attenuated in nonpathogenic strains at 18°C, while not affected in the cool virulent ones. Using pilQ as a marker to evaluate the relative expression of the twitching activity of R. solanacearum strains, we confirmed that cool virulent strains maintained a similar level of pilQ expression at both temperatures, while in nonpathogenic strains pilQ was downregulated at 18°C.

Bacterial wilt caused by Ralstonia solanacearum (Rs) is one of the most important diseases found in ginger; however, the disease resistance mechanisms dependent on root bacteria and exudates are unclear. In the present study, we analyzed the changes in the composition of rhizobacteria, endobacteria, and root exudates during the pathogenesis of bacterial wilt using high-throughput sequencing and gas chromatography-mass spectrometry (GC-MS). Rs caused bacterial wilt in ginger with an incidence of 50.00% and changed the bacterial community composition in both endosphere and rhizosphere. It significantly reduced bacterial α-diversity but increased the abundance of beneficial and stress-tolerant bacteria, such as Lysobacter, Ramlibacter, Pseudomonas, and Azospirillum. Moreover, the change in rhizobacterial composition induced the changes in endobacterial and root exudate compositions. Moreover, the upregulated exudates inhibited ginger bacterial wilt, with the initial disease index (77.50%) being reduced to 40.00%, suggesting that ginger secretes antibacterial compounds for defense against bacterial pathogens.

The security of vegetable plants worldwide is threatened by bacterial wilts, one of the most infectious soil-borne bacterial plant diseases. This is caused by R. Solanacearum. Overuse of bactericides and antibiotics to combat bacterial wilt results in pesticide resistance and toxicity to beneficial living organisms. Consequently, nanoparticles are more beneficial, safe for the environment, and have strong antibacterial properties than conventional pesticides. In the present work, iron oxide nanoparticles (IONPs) and silver nanoparticles (AgNPs) were prepared by simple chemical, eco-friendly procedures, and characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), size distribution, zeta potential, ultraviolet-visible (UV-vis) absorption spectra, and Fourier transform infrared spectra (FTIR). In vitro and in vivo tests were also used to assess the nanoparticles’ antibacterial effectiveness against the phytopathogen R. solanacearum. The findings showed that NPs (nanoparticles) had strong antibacterial properties that changed according to concentration. The greenhouse toxicity study indicated that the NPs significantly impacted tomato bacterial wilt. The disease severity was successfully decreased by 27 and 67%, respectively, when IONPs and AgNPs were contrasted with the untreated infected plants that entirely wilted and died (100% disease severity). Therefore, as compared to infected plants, IONPs and AgNPs enhanced shoot and root length, fresh and dry weight, and chlorophyll content of tomato plants by two to five times. The findings show that the bacterial cell membranes were physically harmed by the direct attachment of NPs to their surfaces, as shown by transmission electron microscopy (TEM). In conclusion, this study provides evidence and strategies for preventing and controlling soil-borne bacterial wilt disease with an efficient and environmentally friendly metal oxide NPs. Furthermore, vegetable plant’s nutritional value is enhanced by iron, which is essential for all living things.

Tomato yield losses are mainly caused due to bacterial wilt (Ralstonia solanacearum) throughout the world. The disease management is mainly done through chemicals which lead to health risks. Among non-chemical strategies, development of resistant varieties can be a good alternative. The present study was conducted to screen the fifty seven different tomato genotypes against bacterial wilt using artificial inoculation technique under greenhouse conditions. The plants showing symptoms were examined using ooze test. Morphological and molecular characterization of the bacterial strains isolated from infected plants was carried out and bacterial phylotype I was determined using phylotype-specific multiplex PCR. The bacterial-infected tomato genotypes were categorized into highly resistant, resistant, moderately resistant, moderately susceptible, susceptible, highly susceptible and extremely susceptible lines. Seven tomato genotypes viz.RIL-118, Indam-1004, Arka Samrat, PKM-1, PED, EC-802390, and EC-816105 were found highly resistant to bacterial wilt. These genotypes were also evaluated for plant growth, yield and yield-related traits and fruit quality traits under field conditions. Maximum production (2533 g/plant) was observed for the genotype Arka Rakshak followed by EC816156 (2486 g/plant).while genotype EC815157 (200 g/plant) exhibited lowest production. In addition, fourteen bacterial wilt linked markers were validated for these genotypes where SCAR marker, SCU176-534 was found to be linked with the bacterial wilt resistance significantly. This study will be significant and useful in increasing tomato production and to develop new resistant tomato varieties through marker assisted breeding.

The current study aimed to determine the chemical compositions of ginger extract (GE) and to assess the antibacterial activities of GE against the ginger bacterial wilt pathogen Ralstonia solanacearum and to screen their mechanisms of action. A total of 393 compounds were identified by using ultra-performance liquid chromatography and tandem-mass spectrometry. The antibacterial test indicated that GE had strong antibacterial activity against R. solanacearum and that the bactericidal effect exhibited a dose-dependent manner. The minimum inhibitory concentration and minimum bactericidal concentration of R. solanacearum were 3.91 and 125 mg/ml, respectively. The cell membrane permeability and integrity of R. solanacearum were destroyed by GE, resulting in cell content leakage, such as electrolytes, nucleic acids, proteins, extracellular adenosine triphosphate and exopoly saccharides. In addition, the activity of cellular succinate dehydrogenase and alkaline phosphatase of R. solanacearum decreased gradually with an increase in the GE concentration. Scanning electron microscopy analysis revealed that GE treatment changed the morphology of the R. solanacearum cells. Further experiments demonstrated that GE delayed or slowed the occurrence of bacterial wilt on ginger. GE has a significant antibacterial effect on R. solanacearum, and the antibacterial effect is concentration dependent. The GE treatments changed the morphology, destroyed membrane permeability and integrity, reduced key enzyme activity and inhibit the synthesis of the virulence factor EPS of R. solanacearum. GE significantly controlled the bacterial wilt of ginger during infection. This research provides insight into the antimicrobial mechanism of GE against R. solanacearum, which will open a new application field for GE.

The destructive bacterial wilt disease caused by Ralstonia solanacearum leads to substantial losses in pepper production worldwide. Plant-derived pesticides exhibit advantages of high efficiency and broad spectrum when compared to traditional chemical pesticides. Artemisia annua and 'Tai Jiao' No. 1 were used as the experimental materials, and treated with 0.75 g·mL-1, 1.5 g·mL-1, and 3 g·mL-1 of A. annua extract and inoculated with R. solanacearum at a concentration of OD600 = 0.1 for 14 days. The inhibitory activity of A. annua extracts against R. solanacearum, as well as the disease index, defense enzyme activities, and defense-related substances contents of pepper seedlings were determined. The results showed that the Minimum Inhibitory Concentration (MIC) of the A. annua extract was 3 g·mL-1. As the concentration of A. annua extract increased, the extent of R. solanacearum cell crumpling intensified, accompanied by a gradual decline in its biofilm-forming ability. On the 14th day after treatment, the disease severity index and incidence rate were significantly reduced when the A. annua extract was applied at concentrations of 0.75 g·mL-1 and 3 g·mL-1. At both the 7th and 14th days after treatment, the application of A. annua extract at concentrations of 0.75 g·mL-1 and 3 g·mL-1 led to enhanced activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in peppers at different stages. Simultaneously, it reduced the levels of malondialdehyde (MDA) and hydrogen peroxide (H2O2), effectively scavenging reactive oxygen species and alleviating cellular lipid peroxidation. Furthermore, the extract increased the activities of polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL), as well as elevated the contents of soluble protein, flavonoids, and total phenols, ultimately enhancing the disease resistance of peppers. Considering the development costs, the application of A. annua extract at a concentration of 0.75 g·mL-1 demonstrates great potential for green control measures against bacterial wilt in peppers.

The First Complete Genome Resource of a Ralstonia solanacearum Phage UAM5 from Colombia.

In this study we investigated the ability of lansiumamide B to protect tobacco against bacterial wilt caused by Ralstonia solanacearum. The results of pot experiments indicated that lansiumamide B profoundly reduced the disease severity of bacterial wilt, with the control efficiency on tobacco bacterial wilt at 87.28% and 77.75% at 15 and 30 days, respectively. The activities of antioxidative enzymes, such as peroxidase (POD) and superoxide dismutase (SOD) were enhanced after treatment with lansiumamide B. The activity of phenylalanine ammonium lyase (PAL) and polyphenol oxidase (PPO) were also increased, which plays an important role in protecting the plant against pathogenic bacteria attack. The isozymes analysis results showed that the expressions of POD and PPO isozymes were enhanced after treatment with lansiumamide B. Detection of metabolites showed that the relative contents of resistance-related substances, such as Myo-inositol, Nicotine, Propanedioic acid, Lactic acid, Glycerol acid, Malic acid, L-threonic acid, D-lyxose, Ribitol, Tetradecanol, L-arabinose, Ribonic acid, Shikimic acid, Tartaric acid, 1, 2, 3-Propanetricarboxylic acid, L-arabitol and Octadecanoic acid in the groups of inoculation, BTH, and lansiumamide B were higher compared to solvent control. These results suggested that lansiumamide B might attenuate the harmful effects of Ralstonia solanacearum on tobacco plants.

Bacterial wilt pathogen Ralstonia solanacearum causes catastrophic loss in different plants across the genera and climatic conditions. It has a huge genetic diversity which affects tropical, subtropical, and warm temperate region. Apart from solanaceous plants, it affects a vast array of many other plant species. Wide host range and its survival capacity in various environments such as irrigation water and soil make it difficult to control R. solanacearum. Host resistance breakdown due to high genotype and environment interactions was frequently encountered. Therefore, integrated approach combining host plant resistance and cultural and biological control measures seems effective. Although excellent attempts have been made in management of R. solanacearum, still there is great opportunity to contribute to this problem for a stable solution. Varied chemical, cultural, agronomical, biological, biotechnological approaches, etc. have been used in addressing problem of Ralstonia with different levels of success. Biocontrol of R. solanacearum by different microorganisms has great potential. Microbes like Bacillus, Pseudomonas, Azotobacter, Streptomyces, etc. have been found suitable in suppressing bacterial wilt. This chapter focuses on different approaches of R. solanacearum biocontrol like the use of arbuscular mycorrhizal (AM) fungi, bacterial endophytes, bacteriophages, bacterial volatile compounds, chitosan, silicon, etc. in detail. It also briefs about present scenario of R. solanacearum control with future potential to be achieved.

Ralstonia solanacearum is a soil-borne bacterium causing the widespread disease known as bacterial wilt. Ralstonia solanacearum is also the causal agent of Moko disease of banana and brown rot of potato. Since the last R. solanacearum pathogen profile was published 10 years ago, studies concerning this plant pathogen have taken a genomic and post-genomic direction. This was pioneered by the first sequenced and annotated genome for a major plant bacterial pathogen and followed by many more genomes in subsequent years. All molecular features studied now have a genomic flavour. In the future, this will help in connecting the classical field of pathology and diversity studies with the gene content of specific strains. In this review, we summarize the recent research on this bacterial pathogen, including strain classification, host range, pathogenicity determinants, regulation of virulence genes, type III effector repertoire, effector-triggered immunity, plant signalling in response to R. solanacearum, as well as a review of different new pathosystems. Bacteria; Proteobacteria; β subdivision; Ralstonia group; genus Ralstonia. Ralstonia solanacearum is the agent of bacterial wilt of plants, characterized by a sudden wilt of the whole plant. Typically, stem cross-sections will ooze a slimy bacterial exudate. In the case of Moko disease of banana and brown rot of potato, there is also visible bacterial colonization of banana fruit and potato tuber. As a soil-borne pathogen, infected fields can rarely be reused, even after rotation with nonhost plants. The disease is controlled by the use of resistant and tolerant plant cultivars. The prevention of spread of the disease has been achieved, in some instances, by the application of strict prophylactic sanitation practices. Stock centre: International Centre for Microbial Resources-French Collection for Plant-associated Bacteria CIRM-CFBP, IRHS UMR 1345 INRA-ACO-UA, 42 rue Georges Morel, 49070 Beaucouzé Cedex, France, http://www.angers-nantes.inra.fr/cfbp/. Ralstonia Genome browser: https://iant.toulouse.inra.fr/R.solanacearum. GMI1000 insertion mutant library: https://iant.toulouse.inra.fr/R.solanacearumGMI1000/GenomicResources. MaGe Genome Browser: https://www.genoscope.cns.fr/agc/microscope/mage/viewer.php?

Bacterial wilt disease on banana is an important disease in Lumajang District and causes severe yield loss. Utilizing bacteriophage as natural enemy of pathogenic bacteria has been widely known as one of the control strategies. This research was aimed at determining the causing agent of bacterial wilt on banana isolated from Lumajang area, to obtain wide-host range bacteriophages against bacterial wilt pathogen and to know the basic characteristic of bacteriophages, particularly its nucleic acid type. Causative agent of bacterial wilt was isolated from symptomatic banana trees from seven districts in Lumajang area on determinative CPG plates followed by rapid detection by PCR technique using specific pair-primer. Bacteriophages were also isolated from soil of infected banana crop in Sukodono District. Morphological observation showed that all bacterial isolates have similar characteristic as common bacterial wilt pathogen,Ralstonia solanacearum. In addition, detection of FliC region in all isolates confirmed that all isolates wereR. solanacearumaccording to the presence of 400 bp of FliC DNA fragment. Moreover, two bacteriophages were obtained from this experiment (ϕRSSKD1 andϕRSSKD2), which were able to infect all nineR. solanacearumisolates. Nucleic acid analysis showed that the nucleic acid of bacteriophages was DNA (deoxyribonucleic acid).