Soil-borne Bacterial Pathogen Research Articles

Nonphytotoxic and pH-responsive ZnO-ZIF‑8 loaded with honokiol as a “nanoweapon” effectively controls the soil-borne bacterial pathogen Ralstonia solanacearum

The development of intelligently released and environmentally safe nanocarriers not only aligns with the sustainable agricultural strategy but also offers a potential solution for controlling severe soil-borne bacterial diseases. Herein, the core-shell structured nanocarrier loaded with honokiol bactericide (honokiol@ZnO-ZIF-8) was synthesized via a one-pot method for the targeted control of Ralstonia solanacearum, the causative agent of tobacco bacterial wilt disease. Results indicated that honokiol@ZnO-ZIF-8 nanoparticles induced bacterial cell membrane and DNA damage through the production of excessive reactive oxygen species (ROS), thereby reducing bacterial cell viability and ultimately leading to bacterial death. Additionally, the dissociation mechanism of the nanocarriers was elucidated for the first time through thermodynamic computational simulation. The nanocarriers dissociate primarily due to H+ attacking the N atom on imidazole, causing the rupture of the Zn-N bond under acidic conditions and at room temperature. Furthermore, honokiol@ZnO-ZIF-8 exhibited potent inhibitory effects against other prominent Solanaceae pathogenic bacteria (Pseudomonas syringae pv. tabaci), demonstrating its broad-spectrum antibacterial activity. Biosafety assessment results indicated that honokiol@ZnO-ZIF-8 exhibited non-phytotoxicity towards tobacco and tomato plants, with its predominant accumulation in the roots and no translocation to aboveground tissues within a short period. This study provides potential application value for the intelligent release of green pesticides. Environment implicationThe indiscriminate use of agrochemicals poses a significant threat to environmental, ecological security, and sustainable development. Slow-release pesticides offer a green and durable strategy for crop disease control. In this study, we developed a non-phytotoxic and pH-responsive honokiol@ZnO-ZIF-8 nano-bactericide based on the pathogenesis of Ralstonia solanacearum. Thermodynamic simulation revealed the dissociation mechanism of ZIF-8, with different acidity controlling the dissociation rate. This provides a theoretical basis for on-demand pesticide release while reducing residue in the. Our findings provide strong evidence for effective soil-borne bacterial disease control and on-demand pesticide release.

Cell wall-mediated root development is targeted by a soil-borne bacterial pathogen to promote infection

Plant pathogens manipulate host development, facilitating colonization and proliferation. Ralstonia solanacearum is a soil-borne bacterial pathogen that penetrates roots and colonizes plants through the vascular system, causing wilting and death. Here, we find that RipAC, an effector protein from R.solanacearum, alters root development in Arabidopsis, promoting the formation of lateral roots and root hairs. RipAC interacts with CELLULOSE SYNTHASE (CESA)-INTERACTIVE PROTEIN 1 (CSI1), which regulates the activity of CESA complexes at the plasma membrane. RipAC disrupts CESA-CSI1 interaction, leading to a reduction in cellulose content, root developmental alterations, and a promotion of bacterial pathogenicity. We find that CSI1 also associates with the receptor kinase FERONIA, forming a complex that negatively regulates immunity in roots; this interaction, however, is not affected by RipAC. Our work reveals a bacterial virulence strategy that selectively affects the activities of a host target, promoting anatomical alterations that facilitate infection without causing activation of immunity.

Inhibition of Ralstonia solanacearum by Warburgia ugandensis stem bark and leaf crude extracts obtained using organic solvents

Ralstonia solanacearum is a soil-borne bacterial pathogen that poses significant threat to the Solanaceae family and other crops. It causes widespread bacterial wilt, a devastating disease that affects the plant's water transport system, leading to wilting and death. Numerous chemical agents and treatment methods have been employed in attempts to control R. solanacearum, but are ineffective. The study aimed to determine the in vitro efficacy of W. ugandensis stem bark and leaf crude extracts against R. solanacearum. W. ugandensis stem bark and leaf crude extracts were obtained using organic solvents viz. methanol, ethanol, dichloromethane and hexane. In vitro, antagonistic activities against R. solanacearum of all organic crude extracts of W. ugandensis were determined by standard agar well diffusion assay on Kelman’s 2, 3, 5- triphenyl tetrazolium chloride medium in triplicates. Two-way analysis of variance (ANOVA) was used in the statistical analysis of the mean diameter inhibition zones. All the organic solvents crude extracts of W. ugandensis were inhibitive against R. solanacearum. However, the stem bark crude extracts exhibited significantly higher efficacy against R. solanacearum compared to the leaf crude extracts. The crude extracts were subjected to a serial dilution to determine the minimum inhibitory concentration (MIC). W. ugandensis stem bark dichloromethane crude extracts had the lowest MIC of 1 mg/ml. W. ugandensis stem bark dichloromethane crude extracts were most effective against R. solanacearum. Further research is important to determine the bioactive compounds against R. solanacearum in W. ugandensis stem bark dichloromethane crude extracts.

A bacterial type III effector targets plant vesicle-associated membrane proteins.

The soilborne bacterial pathogen Ralstonia solanacearum is one of the most destructive plant pathogens worldwide, and its infection process involves the manipulation of numerous plant cellular functions. In this work, we found that the R. solanacearum effector protein RipD partially suppressed different levels of plant immunity triggered by R. solanacearum elicitors, including specific responses triggered by pathogen-associated molecular patterns and secreted effectors. RipD localized in different subcellular compartments in plant cells, including vesicles, and its vesicular localization was enriched in cells undergoing R. solanacearum infection, suggesting that this specific localization may be particularly relevant during infection. Among RipD-interacting proteins, we identified plant vesicle-associated membrane proteins (VAMPs). We also found that overexpression of Arabidopsis thaliana VAMP721 and VAMP722 in Nicotiana benthamiana leaves promoted resistance to R. solanacearum, and this was abolished by the simultaneous expression of RipD, suggesting that RipD targets VAMPs to contribute to R. solanacearum virulence. Among proteins secreted in VAMP721/722-containing vesicles, CCOAOMT1 is an enzyme required for lignin biosynthesis, and mutation of CCOAOMT1 enhanced plant susceptibility to R. solanacearum. Altogether our results reveal the contribution of VAMPs to plant resistance against R. solanacearum and their targeting by a bacterial effector as a pathogen virulence strategy.

Duality of immune recognition by tomato and virulence activity of the Ralstonia solanacearum exo-polygalacturonase PehC.

Ralstonia solanacearum is a devastating soil-borne bacterial pathogen capable of infecting many plant species, including tomato (Solanum lycopersicum). However, the perception of Ralstonia by the tomato immune system and the pathogen's counter-defense strategy remain largely unknown. Here, we show that PehC, a specific exo-polygalacturonase secreted by Ralstonia, acts as an elicitor that triggers typical immune responses in tomato and other Solanaceous plants. The elicitor activity of PehC depends on its N-terminal epitope, and not on its polygalacturonase activity. The recognition of PehC specifically occurs in tomato roots and relies on unknown receptor-like kinase(s). Moreover, PehC hydrolyzes plant pectin-derived oligogalacturonic acids (OGs), a type of damage-associated molecular pattern (DAMP), which leads to the release of galacturonic acid (GalA), thereby dampening DAMP-triggered immunity (DTI). Ralstonia depends on PehC for its growth and early infection and can utilize GalA as a carbon source in the xylem. Our findings demonstrate the specialized and dual functions of Ralstonia PehC, which enhance virulence by degrading DAMPs to evade DTI and produce nutrients, a strategy used by pathogens to attenuate plant immunity. Solanaceous plants have evolved to recognize PehC and induce immune responses, which highlights the significance of PehC. Overall, this study provides insight into the arms race between plants and pathogens.

Legionella pneumophila and Free-Living Nematodes: Environmental Co-Occurrence and Trophic Link.

Free-living nematodes harbor and disseminate various soil-borne bacterial pathogens. Whether they function as vectors or environmental reservoirs for the aquatic L. pneumophila, the causative agent of Legionnaires' disease, is unknown. A survey screening of biofilms of natural (swimming lakes) and technical (cooling towers) water habitats in Germany revealed that nematodes can act as potential reservoirs, vectors or grazers of L. pneumophila in cooling towers. Consequently, the nematode species Plectus similis and L. pneumophila were isolated from the same cooling tower biofilm and taken into a monoxenic culture. Using pharyngeal pumping assays, potential feeding relationships between P. similis and different L. pneumophila strains and mutants were examined and compared with Plectus sp., a species isolated from a L. pneumophila-positive thermal source biofilm. The assays showed that bacterial suspensions and supernatants of the L. pneumophila cooling tower isolate KV02 decreased pumping rate and feeding activity in nematodes. However, assays investigating the hypothesized negative impact of Legionella's major secretory protein ProA on pumping rate revealed opposite effects on nematodes, which points to a species-specific response to ProA. To extend the food chain by a further trophic level, Acanthamoebae castellanii infected with L. pneumphila KV02 were offered to nematodes. The pumping rates of P. similis increased when fed with L. pneumophila-infected A. castellanii, while Plectus sp. pumping rates were similar when fed either infected or non-infected A. castellanii. This study revealed that cooling towers are the main water bodies where L. pneumophila and free-living nematodes coexist and is the first step in elucidating the trophic links between coexisting taxa from that habitat. Investigating the Legionella-nematode-amoebae interactions underlined the importance of amoebae as reservoirs and transmission vehicles of the pathogen for nematode predators.

Soil properties, rhizosphere bacterial community, and plant performance respond differently to fumigation and bioagent treatment in continuous cropping fields.

Continuous cropping barriers lead to huge agriculture production losses, and fumigation and biological agents are developed to alleviate the barriers. However, there is a lack of literature on the differences between strong chemical fumigant treatment and moderate biological agent treatment. In this study, we investigated those differences and attempted to establish the links between soil properties, rhizosphere microbial community, and plant performance in both fumigation- and bioagent-treated fields. The results showed that the fumigation had a stronger effect on both soil functional microbes, i.e., ammonia oxidizers and soil-borne bacterial pathogens, and therefore, led to a significant change in soil properties, higher fertilizer efficiency, lower disease infections, and improved plant growth, compared with untreated control fields. Biological treatment caused less changes to soil properties, rhizosphere bacterial community, and plant physiology. Correlation and modeling analyses revealed that the bioagent effect was mainly direct, whereas fumigation resulted in indirect effects on alleviating cropping barriers. A possible explanation would be the reconstruction of the soil microbial community by the fumigation process, which would subsequently lead to changes in soil characteristics and plant performance, resulting in the effective alleviation of continuous cropping barriers.

Small changes in rhizosphere microbiome composition predict disease outcomes earlier than pathogen density variations

Even in homogeneous conditions, plants facing a soilborne pathogen tend to show a binary outcome with individuals either remaining fully healthy or developing severe to lethal disease symptoms. As the rhizosphere microbiome is a major determinant of plant health, we postulated that such a binary outcome may result from an early divergence in the rhizosphere microbiome assembly that may further cascade into varying disease suppression abilities. We tested this hypothesis by setting up a longitudinal study of tomato plants growing in a natural but homogenized soil infested with the soilborne bacterial pathogen Ralstonia solanacearum. Starting from an originally identical species pool, individual rhizosphere microbiome compositions rapidly diverged into multiple configurations during the plant vegetative growth. This variation in community composition was strongly associated with later disease development during the later fruiting state. Most interestingly, these patterns also significantly predicted disease outcomes 2 weeks before any difference in pathogen density became apparent between the healthy and diseased groups. In this system, a total of 135 bacterial OTUs were associated with persistent healthy plants. Five of these enriched OTUs (Lysinibacillus, Pseudarthrobacter, Bordetella, Bacillus, and Chryseobacterium) were isolated and shown to reduce disease severity by 30.4–100% when co-introduced with the pathogen. Overall, our results demonstrated that an initially homogenized soil can rapidly diverge into rhizosphere microbiomes varying in their ability to promote plant protection. This suggests that early life interventions may have significant effects on later microbiome states, and highlights an exciting opportunity for microbiome diagnostics and plant disease prevention.

A Fast and Easy Method to Study Ralstonia solanacearum Virulence upon Transient Gene Expression or Gene Silencing in Nicotiana benthamiana Leaves.

Ralstonia solanacearum is a devastating soil-borne bacterial pathogen that causes disease in multiple host plants worldwide. Typical assays to measure virulence of R. solanacearum in laboratory conditions rely on soil-drenching inoculation followed by observation and scoring of disease symptoms. Here, we describe a novel inoculation protocol to analyze the replication of R. solanacearum upon infiltration into the leaves of Nicotiana benthamiana, in which gene expression has been altered using Agrobacterium tumefaciens. The protocol includes five major steps: 1) growth of N. benthamiana plants; 2) infiltration of A. tumefaciens; 3) R. solanacearum inoculation; 4) sample collection and bacterial quantitation; 5) data analysis and representation. The transient gene expression or gene silencing prior to R. solanacearum inoculation provides a straightforward way to perform genetic analysis of plant functions involved in the interaction between pathogen and host, using the appropriate combination of A. tumefaciens and R. solanacearum strains, with high sensitivity and accuracy provided by the quantitation of bacterial numbers in plant tissues.

Management of bacterial wilt (Ralstonia solanacearum) of potato: focus on natural bioactive compounds

The bacterial wilt disease caused by Ralstonia solanacearum is an extremely destructive soil borne bacterial pathogen to potato. It appeared as rapid and fatal wilting symptoms in the host. The pathogen entered through different wounds and easily disseminated via infected biological material, soil, contaminated irrigation water, surface water, farm equipment etc. and could survive for many years in association with alternate hosts. It is a widely distributed and very much diversified soil borne pathogen having an unusually broad host range with long-term survivable ability. Direct yield losses caused by the pathogen varied from 30 to 90% depending on different factors such as cultivar, weather factors, soil type, cropping pattern and strain etc. Bacterial wilt continued to be an economically serious problem for field-grown potatoes in many tropical, subtropical and warmer areas of the world including Bangladesh. But the effectiveness of conventional management is limited because of some special biological features of the bacteria. Mostly protective methods and chemical control remain ineffective, antibiotics show hardly any effect, and efficacious biocontrol method has yet to be developed against the organism. However, during the recent decades, some natural bioactive compounds, viz. propolis, honey, turmeric, magnesium chloride, cow dung, aromatic rice extract, iodine, sodium bicarbonate etc. have got attention for their effectiveness in inhibiting a range of serious bacterial pathogens from both Gram positive and Gram negative types. As no conventional method has been found effective alone, such compounds could be tested for their effectiveness against the very successful soil borne bacteria to overcome the traditional management limitations.J. Biodivers. Conserv. Bioresour. Manag. 2018, 4(1): 73-92

Hydroxycoumarins: New, effective plant-derived compounds reduce Ralstonia pseudosolanacearum populations and control tobacco bacterial wilt

Plant wilt disease caused by the soilborne bacterial pathogen Ralstonia pseudosolanacearum is one of the most devastating plant diseases; however, no effective protection against this disease has been developed. Coumarins are important natural plant-derived compounds with a wide range of bioactivities and extensive applications in medicine and agriculture. In the present study, three hydroxycoumarins (Hycs), umbelliferone (UM), esculetin (ES) and daphnetin (DA) significantly inhibited the growth of R. pseudosolanacearum on solid medium in a concentration-dependent manner, and the minimum inhibitory concentration (MICs) of these compounds was 325 mg L−1, 125 mg L−1 and 75 mg L−1, respectively. The percentage of live cells of R. pseudosolanacearum when supplemented with UM, ES, and DA was 63.61%, 17.81% and 7.23%, respectively, which were significantly lower than the DMSO treatment with 92%. Furthermore, irrigating roots with hydroxycoumarins (Hycs) 24 h before inoculation with R. pseudosolanacearum significantly delayed the occurrence of tobacco bacterial wilt, with the control efficiency of the DA treatment (the most efficient of Hycs treatment) 80.03%, 69.83%, 59.19%, 45.49%, 44.12%, 38.27% at 6, 8, 10, 12, 14, and 16 days after inoculation, respectively. Compared with the DMSO treatment, the pathogen populations of tobacco stems supplemented with 100 mg L−1 DA were the lowest, with population significantly reduced by 22.46%, 27.34%, and 18.06% at 4, 7, and 10 days after inoculation, respectively. Based on this study, these Hycs could be applied as potential protective agents in the management of tobacco bacterial wilt.

L-Histidine Induces Resistance in Plants to the Bacterial Pathogen Ralstonia solanacearum Partially Through the Activation of Ethylene Signaling.

Wilt disease in plants, which is caused by the soil-borne bacterial pathogen Ralstonia solanacearum, is one of the most devastating plant diseases. We previously detected bacterial wilt disease-inhibiting activity in an extract from yeast cells. In the present study, we purified this activity and identified one of the substances responsible for the activity as the amino acid histidine. The exogenous application of l-histidine, but not d-histidine, inhibited wilt disease in tomato and Arabidopsis plants without exhibiting any antibacterial activity. l-Histidine induced the expression of genes related to ethylene (ET) biosynthesis and signaling as well as the production of ET in tomato and Arabidopsis plants. l-Histidine-induced resistance to R. solanacearum was partially abolished in ein3-1, an ET-insensitive Arabidopsis mutant line. Resistance to the fungal pathogen Botrytis cinerea, which is known to require ET biosynthesis or signaling, was also induced by exogenously applied l-histidine. These results suggest that l-histidine induces resistance to R. solanacearum and B. cinerea partially through the activation of ET signaling in plants.

Screening of suitable carriers for Bacillus amyloliquefaciens strain QL-18 to enhance the biocontrol of tomato bacterial wilt

Soilborne plant diseases seriously limit agricultural production. Due to environmental concerns, sustainable agricultural practices must involve the use of environmentally friendly alternatives, such as biocontrol microbes (BMs), to suppress various plant diseases. However, the success of BM application largely depends on the carrier formulation. An ideal carrier that can support the survival of a BM while discouraging the growth of the target pathogen is expected to enhance the performance of the BM in plant disease control. In this study, various commercial composts and peats were evaluated as carriers for Bacillus amyloliquefaciens QL-18, a BM used to control tomato bacterial wilt (TBW) caused by the soilborne bacterial pathogen Ralstonia solanacearum. Among the evaluated carriers, rapeseed cake compost (RCC) was found to be the best carrier, acting as both a substrate and food source for B. amyloliquefaciens QL-18. RCC facilitated the growth and survival of strain QL-18 by supplying adequate levels of dissolved carbon and nitrogen. Moreover, aqueous extracts of RCC alone inhibited R. solanacearum growth, suggesting that certain RCC components are toxic to the pathogen. Greenhouse and field experiments demonstrated that RCC improved the ability of B. amyloliquefaciens QL-18 to reduce the incidence of TBW and the population of the pathogen in the tomato rhizosphere. Although other carriers maintained large populations of B. amyloliquefaciens QL-18, similar to those obtained with RCC, they were not deemed suitable carriers because they constituted a poor nutrient supply for B. amyloliquefaciens QL-18 growth; thus, they had less of an impact on disease control. The principle of BM carrier selection should be adopted in the future when developing new bio-products for the effective control of soilborne plant diseases.

Impact of Bacillus amyloliquefaciens S13-3 on control of bacterial wilt and powdery mildew in tomato.

Biological control is a non-hazardous technique to control plant diseases. Researchers have explored microorganisms that show high plant-disease control efficiency for use as biological control agents. A single soil application of Bacillus amyloliquefaciens strain S13-3 suppressed tomato bacterial wilt caused by Ralstonia solanacearum, a soilborne bacterial pathogen, through production of antibiotics augmented possibly by induction of systemic acquired resistance. Soil application also controlled tomato powdery mildew disease through induction of systemic acquired resistance. S13-3 showing bifunctional activity with a single application to soil may be an innovative biological control agent against bacterial wilt and powdery mildew in tomato.

Isolation and identification of a virulent Ralstonia solanacearum by fliC gene amplification and induction of chitinase by 2-amino butyric acid for control of bacterial wilt in tomato plants

Ralstonia solanacearum is a devastating, soil borne bacterial pathogen of tomato. The pathogen is nonmotile in planta but highly motile in culture. On the basis of physiological and biochemical characteristics 26 isolates have been purified and identified as Ralstonia solanacearum. The flic gene is responsible for the movement of bacteria. Ralstonia specific fliC gene amplification is the indication of virulence of the pathogen. In the present study one R. solanacearum isolate has been identified by PCR amplification of the fliC gene using fliC gene specific primer. Following isolation and identification of the virulent isolate, fresh tomato plants were induced by application of 2- amino butyric acid (ABA). The defense enzyme, chitinase was estimated in treated plants. Treated inoculated plants did not show any visible symptoms of wilt even after 14 days of inoculation. Significantly it was observed that chitinase was increased in the 2-ABA-treated plants and also in the treated-inoculated plants. The increased chitinase activity in the treated plants showed that 2-ABA has the resistance inducing capacity in tomato plants against Ralstonia solanacearum.

Identification of Natural Diterpenes that Inhibit Bacterial Wilt Disease in Tobacco, Tomato and Arabidopsis

The soil-borne bacterial pathogen Ralstonia solanacearum invades a broad range of plants through their roots, resulting in wilting of the plant, but no effective protection against this disease has been developed. Two bacterial wilt disease-inhibiting compounds were biochemically isolated from tobacco and identified as sclareol and cis-abienol, labdane-type diterpenes. When exogenously applied to their roots, sclareol and cis-abienol inhibited wilt disease in tobacco, tomato and Arabidopsis plants without exhibiting any antibacterial activity. Microarray analysis identified many sclareol-responsive genes in Arabidopsis roots, including genes encoding or with a role in ATP-binding cassette (ABC) transporters, and biosynthesis and signaling of defense-related molecules and mitogen-activated protein kinase (MAPK) cascade components. Inhibition of wilt disease by sclareol was attenuated in Arabidopsis mutants defective in the ABC transporter AtPDR12, the MAPK MPK3, and ethylene and abscisic acid signaling pathways, and also in transgenic tobacco plants with reduced expression of NtPDR1, a tobacco homolog of AtPDR12. These results suggest that multiple host factors are involved in the inhibition of bacterial wilt disease by sclareol-related compounds.

Finally, proof of weapons of mass destruction.

Allelopathy (one species' use of chemicals to harm other species) may be a key ingredient in successful invasions of alien plants into established communities. Bais et al. show that in response to elicitation by common soil fungi, spotted knapweed (Centaurea maculosa) launches an ineffective defense against the fungi that results in extensive collateral damage to neighboring plants. Specifically, the flavonoid (-)-catechin, released from the roots of knapweed, produces a massive reactive oxygen species (ROS)-mediated oxidative burst, Ca2+ signaling, and rapid cell death in those unadapted native species whose habitats the plant regularly invades. The roots release both (+) and (-) enantiomers, but only the (-) enantiomer functions as a weapon of mass destruction; the (+) enantiomer inhibits the growth of numerous common soil-borne bacterial pathogens. Eliciting apoptotic response for a competitive advantage is an example of signal cross-talk between the genomes of interacting organisms and highlights how the internal signaling of one organism can be used by others to adjust their phenotypes in an adaptive manner. The study provides strong circumstantial evidence for an allelopathic interaction, but the genetic manipulation of (-)-catechin release would allow researchers to determine if these responses occur in nature. Precise genetic control over the release of secondary metabolites from plants would benefit ecological research.

Finally, Proof of Weapons of Mass Destruction

Allelopathy (one species' use of chemicals to harm other species) may be a key ingredient in successful invasions of alien plants into established communities. Bais et al. show that in response to elicitation by common soil fungi, spotted knapweed (Centaurea maculosa) launches an ineffective defense against the fungi that results in extensive collateral damage to neighboring plants. Specifically, the flavonoid (–)-catechin, released from the roots of knapweed, produces a massive reactive oxygen species (ROS)-mediated oxidative burst, Ca2+ signaling, and rapid cell death in those unadapted native species whose habitats the plant regularly invades. The roots release both (+) and (–) enantiomers, but only the (–) enantiomer functions as a weapon of mass destruction; the (+) enantiomer inhibits the growth of numerous common soil-borne bacterial pathogens. Eliciting apoptotic response for a competitive advantage is an example of signal cross-talk between the genomes of interacting organisms and highlights how the internal signaling of one organism can be used by others to adjust their phenotypes in an adaptive manner. The study provides strong circumstantial evidence for an allelopathic interaction, but the genetic manipulation of (–)-catechin release would allow researchers to determine if these responses occur in nature. Precise genetic control over the release of secondary metabolites from plants would benefit ecological research.