Synthesis and evaluation of pyridine-3-carboxamide analogs as effective agents against bacterial wilt in tomatoes.

This study focused on developing novel pyridine-3-carboxamide analogs to treat bacterial wilt in tomatoes caused by Ralstonia solanacearum. The analogs were synthesized through a multistep process and their structures confirmed using spectroscopy. Molecular docking studies identified the most potent analog from the series. A specific analog, compound 4a, was found to significantly enhance disease resistance in tomato plants infected with R. solanacearum. The structure-activity relationship analysis showed the positions and types of substituents on the aromatic rings of compounds 4a-i strongly influenced their biological activity. Compound 4a, with a chloro group at the para position on ring C and hydroxyl group at the ortho position on ring A, was exceptionally effective against R. solanacearum. When used to treat seeds, the analogs displayed remarkable efficacy, especially compound 4a which had specific activity against bacterial wilt pathogens. Compound 4a also promoted vegetative and reproductive growth of tomato plants, increasing seed germination and seedling vigor. In plants mechanically infected with bacteria, compound 4a substantially reduced the percentage of infection, pathogen quantity in young tissue, and disease progression. The analogs were highly potent due to their amide linkage. Molecular docking identified the best compounds with strong binding affinities. Overall, the strategic design and synthesis of these pyridine-3-carboxamide analogs offers an effective approach to targeting and controlling R. solanacearum and bacterial wilt in tomatoes.

Nonphytotoxic and pH-responsive ZnO-ZIF­8 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 IMPLICATION: The 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.

g-C3N4@CuO electrostatic self-assembly toward Ralstonia solanacearum: Insights from cytomembrane and motility disruption.

BACKGROUND: Ralstonia solanacearum, a notorious and refractory bacterial plant pathogen, threatens multiple vegetable crops and causes significant economic loss in agriculture. Long-term use of traditional medicines not only increases the problem of drug resistance, but also causes great environmental pollution. Therefore, there is an urgent need to develop new agents with high efficacy and low toxicity. RESULTS: In this study, we have synthesized and characterized graphitic carbon nitride incorporated copper oxide composite (g-C3N4@CuO), which showed higher antimicrobial effect than graphitic carbon nitride nanosheets (g-C3N4 nanosheets) and copper oxide nanoparticles (CuONPs). Ralstonia solanacearum exposed to g-C3N4@CuO exhibited higher levels of oxygen toxicity, cell membrane damage, DNA damage, motility disruption and even cell death compared to g-C3N4 nanosheets and CuONPs. In addition, g-C3N4@CuO was more effective in the control of tobacco bacterial wilt than g-C3N4 nanosheets and CuONPs. CONCLUSION: Thus, this study provides a new perspective on g-C3N4@CuO control of bacterial diseases in crops, and the mechanism is related to the destruction of cell membrane damage and motility disruption. © 2024 Society of Chemical Industry.

The regulation of BbLaeA on the production of beauvericin and bassiatin in Beauveria bassiana.

Beauvericin and bassiatin are two valuable compounds with various bioactivities biosynthesized by the supposedly same nonribosomal peptide synthetase BbBEAS in entomopathogenic fungus Beauveria bassiana. To evaluate the regulatory effect of global regulator LaeA on their production, we constructed BbLaeA gene deletion and overexpression mutants, respectively. Deletion of BbLaeA resulted in a decrease of the beauvericin titer, while overexpression of BbLaeA increased its production by 1-2.26 times. No bassiatin could be detected in ΔBbLaeA and wild type strain of B. bassiana, but 4.26-5.10 µg/mL bassiatin was produced in OE::BbLaeA. Furthermore, additional metabolites with increased production in OE::BbLaeA were isolated and identified as primary metabolites. Among them, 4-hydroxyphenylacetic acid showed antibacterial bioactivity against Ralstonia solanacearum. These results indicated that BbLaeA positively regulates the production of beauvericin, bassiatin and various bioactive primary metabolites.

Design, Synthesis and Antibacterial Activity of Novel Pyrimidine-Containing 4H-Chromen-4-One Derivatives*.

A series of pyrimidine-containing 4H-chromen-4-one derivatives were designed and synthesized by combining bioactive substructures. Preliminary biological activity results showed that most of the compounds displayed significant inhibitory activities in vitro against Xanthomonas axonopodis pv. Citri (X. axonopodis), Xanthomonas oryzae pv. oryzae (X. oryzae) and Ralstonia solanacearum (R. solanacearum). In particular, compound 2-[(3-{[5,7-dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-4H-1-benzopyran-3-yl]oxy}propyl)sulfanyl]-4-(4-methylphenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (4c) demonstrated a good inhibitory effect against X. axonopodis and X. oryzae, with the half-maximal effective concentration (EC50 ) values of 15.5 and 14.9 µg/mL, respectively, and compound 2-[(3-{[5,7-Dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-4H-1-benzopyran-3-yl]oxy}propyl)sulfanyl]-4-(3-fluorophenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (4h) showed the best antibacterial activity against R. solanacearum with an EC50 value of 14.7 µg/mL. These results were better than commercial reagents bismerthiazol (BT, 51.7, 70.1 and 52.7 µg/mL, respectively) and thiodiazole copper (TC, 77.9, 95.8 and 72.1 µg/mL, respectively). In vivo antibacterial activity results indicated that compound 4c displayed better curative (42.4 %) and protective (49.2 %) activities for rice bacterial leaf blight than BT (35.2, 39.1 %) and TC (30.8, 27.3 %). The mechanism of compound 4c against X. oryzae was analyzed through scanning electron microscopy (SEM). These results indicated that pyrimidine-containing 4H-chromen-4-one derivatives have important value in the research of new agrochemicals.

Diversity and antibacterial activity of fungal endophytes from Eucalyptus exserta.

BACKGROUND: Eucalyptus bacterial wilt caused by Ralstonia solanacearum is an important eucalyptus disease. Endophytic fungi, an important source of natural active substances, provide a new breakthrough for the control of plant diseases. RESULTS: In the present study, 80 endophytic fungal isolates were obtained from the healthy branches and fruits of Eucalyptus exserta. Fifteen distinct isolates (MK120854-MK120868) were selected for further taxonomic identification through morphological trait assessments and internal transcribed spacer (ITS) region-rRNA gene sequence analysis. Thirteen genera, namely, Phyllosticta, Penicillium, Eutypella, Purpureocillium, Talaromyces, Lophiostoma, Cladosporium, Pestalotiopsis, Chaetomium, Fusarium, Gongronella, Scedosporium and Pseudallescheria, were identified on the basis of their morphological characteristics. Members of the genus Phyllosticta were the primary isolates, with a colonization frequency (CF) of 27.5 %. Most of the fungal isolates displayed antibacterial activity. The crude extracts obtained from Lophiostoma sp. Eef-7, Pestalotiopsis sp. Eef-9 and Chaetomium sp. Eef-10 exhibited strong inhibition on the test bacteria, and Lophiostoma sp. Eef-7 was further cultured on a large scale. Three known compounds, scorpinone (1), 5-deoxybostrycoidin (2) and 4-methyl-5,6-dihydro-2 H-pyran-2-one (3), were isolated from the endophytic fungus Lophiostoma sp. Eef-7 associated with E. exserta. The structures of these compounds were elucidated by analysis of 1D and 2D NMR and HR-ESI-MS spectra and a comparison of their spectral data with published values. Compounds 1 and 2 showed weak antimicrobial activity against Ralstonia solanacearum. CONCLUSIONS: Endophytic fungi from Eucalyptus exserta may represent alternative sources of antimicrobial agents. Lophiostoma sp. Eef-7 can produce 2-azaanthraquinone derivatives and shows weak antibacterial activity against Ralstonia solanacearum.

Antibacterial and Antifungal Studies of Biosynthesized Silver Nanoparticles against Plant Parasitic Nematode Meloidogyne incognita, Plant Pathogens Ralstonia solanacearum and Fusarium oxysporum.

The possibility of using silver nanoparticles (AgNPs) to enhance the plants growth, crop production, and control of plant diseases is currently being researched. One of the most effective approaches for the production of AgNPs is green synthesis. Herein, we report a green and phytogenic synthesis of AgNPs by using aqueous extract of strawberry waste (solid waste after fruit juice extraction) as a novel bioresource, which is a non-hazardous and inexpensive that can act as a reducing, capping, and stabilizing agent. Successful biosynthesis of AgNPs was monitored by UV-visible spectroscopy showing a surface plasmon resonance (SPR) peak at ~415 nm. The X-ray diffraction studies confirm the face-centered cubic crystalline AgNPs. The scanning electron microscopy (SEM) and transmission electron microscopy (TEM) techniques confirm the rectangular shape with an average size of ~55 nm. The antibacterial and antifungal efficacy and inhibitory impact of the biosynthesized AgNPs were tested against nematode, Meloidogyne incognita, plant pathogenic bacterium, Ralstonia solanacearum and fungus, Fusarium oxysporum. These results confirm that biosynthesized AgNPs can significantly control these plant pathogens.

Design, synthesis, crystal structure and in vitro antimicrobial activity of novel 1,2,4-triazolo[1,5-a]pyrimidine-containing quinazolinone derivatives.

A series of novel 1,2,4-triazolo[1,5-a]pyrimidine-containing quinazolin-4(3H)-one derivatives (8a-8o) were designed, synthesized and assessed for their in vitro antibacterial and antifungal activities in agriculture. All the title compounds were completely characterized via 1H NMR, 13C NMR, HRMS and IR spectroscopic data. In particular, the molecular structure of compound 8f was further corroborated through a single-crystal X-ray diffraction measurement. The turbidimetric method revealed that some of the compounds displayed noticeable bactericidal potencies against the tested plant pathogenic bacteria. For example, compounds 8m, 8n and 8o possessed higher antibacterial efficacies in vitro against Xanthomonas oryzae pv. oryzae with EC50 values of 69.0, 53.3 and 58.9 µg/mL, respectively, as compared with commercialized agrobactericide bismerthiazol (EC50 = 91.4 µg/mL). Additionally, compound 8m displayed an EC50 value of 71.5 µg/mL toward Xanthomonas axonopodis pv. citri, comparable to control bismerthiazol (EC50 = 60.5 µg/mL). A preliminary structure-activity relationship (SAR) analysis was also conducted, based on the antibacterial results. Finally, some compounds were also found to have a certain antifungal efficacy in vitro at the concentration of 50 µg/mL.

Biosynthesis of Silver Chloride Nanoparticles by Rhizospheric Bacteria and Their Antibacterial Activity against Phytopathogenic Bacterium Ralstonia solanacearum.

Ralstonia solanacearum is the most destructive pathogen, causing bacterial wilt disease of eggplant. The present study aimed to develop green synthesis and characterization of silver chloride nanoparticles (AgCl-NPs) by using a native bacterial strain and subsequent evaluation of their antibacterial activity against R. solanacearum. Here, a total of 10 bacterial strains were selected for the biosynthesis of AgCl-NPs. Among them, the highest yield occurred in the synthesis of AgCl-NPs using a cell-free aqueous filtrate of strain IMA13. Ultrastructural observation revealed that the AgCl-NPs were spherical and oval with smooth surfaces and 5-35 nm sizes. XRD analysis studies revealed that these particles contained face-centered cubic crystallites of metallic Ag and AgCl. Moreover, FTIR analysis showed the presence of capping proteins, carbohydrates, lipids, and lipopeptide compounds and crystalline structure of AgCl-NPs. On the basis of phylogenetic analysis using a combination of six gene sequences (16S, gyrA, rpoB, purH, polC, and groEL), we identified strain IMA13 as Bacillus mojavensis. Three kinds of lipopeptide compounds, namely, bacillomycin D, iturin, and fengycin, forming cell-free supernatant produced by strain IAM13, were identified by MALDI-TOF mass spectrometry. Biogenic AgCl-NPs showed substantial antibacterial activity against R. solanacearum at a concentration of 20 µg/mL-1. Motility assays showed that the AgCl-NPs significantly inhibited the swarming and swimming motility (61.4 and 55.8%) against R. solanacearum. Moreover, SEM and TEM analysis showed that direct interaction of AgCl-NPs with bacterial cells caused rupture of cell wall and cytoplasmic membranes, as well as leakage of nucleic acid materials, which ultimately resulted in the death of R. solanacearum. Overall, these findings will help in developing a promising nanopesticide against phytopathogen plant disease management.

Antimicrobial evaluation of myricetin derivatives containing benzimidazole skeleton against plant pathogens.

A series of novel myricetin derivatives containing benzimidazole skeleton were constructed. The structure of compound 4g was further corroborated via X-ray single crystal diffractometer. The antimicrobial bioassays showed that all compounds exhibited potent inhibitory activities against Xanthomonas axonopodis pv. Citri (Xac), Ralstonia solanacearum (Rs) and Xanthomonas oryzae pv. Oryzae (Xoo) in vitro. Significantly, compound 4q showed the best inhibitory activities against Xoo, with the EC50 value of 8.2 µg/mL, which was better than thiodiazole copper (83.1 µg/mL) and bismerthiazol (60.1 µg/mL). In vivo experimental studies showed that compound 4q can treat rice bacterial leaf blight at 200 µg/mL, and the corresponding curative and protection efficiencies were 45.2 and 48.6%, respectively. Meanwhile, the antimicrobial mechanism of the compounds 4l and 4q were investigated through scanning electron microscopy (SEM). Studies showed that compounds 4l or 4q can cause deformation or rupture of Rs or Xoo cell membrane. These results indicated that novel benzimidazole-containing myricetin derivatives can be used as a potential antibacterial reagent.

Quorum Sensing Inhibition Attenuates the Virulence of the Plant Pathogen Ralstonia solanacearum Species Complex.

Strains of Ralstonia solanacearum species complex (RSSC) cause "bacterial wilt" on a wide range of plant species and thus lead to marked economic losses in agriculture. Quorum sensing (QS), a bacterial cell-cell communication mechanism, controls the virulence of RSSC strains by regulating the production of extracellular polysaccharide (EPS) and secondary metabolites, biofilm formation, and cellular motility. R. solanacearum strain OE1-1 employs (R)-methyl 3-hydroxymyristate (3-OH MAME) as a QS signal, which is synthesized by the PhcB methyltransferase and sensed by the PhcS/PhcRQ two-component system. We describe the design, synthesis, and biological evaluation of inhibitors of the phc QS system. Initial screening of a small set of QS signal analogues revealed that methyl 3-hydroxy-8-phenyloctanoate, named, PQI-1 (phc quorum sensing inhibitor-1), inhibited biofilm formation by strain OE1-1. To improve its inhibitory activity, the derivatives of PQI-1 were synthesized, and their QS inhibition activities were evaluated. PQIs-2-5 evolved from PQI-1 more strongly inhibited not only biofilm formation but also the production of ralfuranone and EPS. Furthermore, RNA-Seq analysis revealed that the PQIs effectively inhibited QS-dependent gene expression and repression in strain OE1-1. On the other hand, the PQIs did not affect the canonical QS systems of the representative reporter bacteria. These antagonists, especially PQI-5, reduced wilting symptoms of the tomato plants infected with strain OE1-1. Taken together, we suggest that targeting the phc QS system has potential for the development of chemicals that protect agricultural crops from bacterial wilt disease.

Antimicrobial Meroterpenoids and Erythritol Derivatives Isolated from the Marine-Algal-Derived Endophytic Fungus Penicillium chrysogenum XNM-12.

One new meroterpenoid-type alkaloid, oxalicine C (1), and two new erythritol derivatives, penicierythritols A (6) and B (7), together with four known meroterpenoids (2-5), were isolated from the marine algal-derived endophytic fungus Penicillium chrysogenum XNM-12. Their planar structures were determined by means of spectroscopic analyses, including UV, 1D and 2D NMR, and HRESIMS spectra. Their stereochemical configurations were established by comparing the experimental and calculated electronic circular dichroism (ECD) spectra for compound 1, as well as by comparison of the optical rotations with literature data for compounds 6 and 7. Notably, oxalicine C (1) represents the first example of an oxalicine alkaloid with a cleaved α-pyrone ring, whereas penicierythritols A (6) and B (7) are the first reported from the Penicillium species. The antimicrobial activities of compounds 1-7 were evaluated. Compounds 1 and 6 exhibited moderate antibacterial effects against the plant pathogen Ralstonia solanacearum with minimum inhibitory concentration (MIC) values of 8 and 4 µg/mL, respectively. Compound 6 also possesses moderate antifungal properties against the plant pathogen Alternaria alternata with a MIC value of 8 µg/mL.

Synthesis, In-Vitro and In-Silico Evaluation of Silver Nanoparticles with Root Extract of Withania somnifera for Antibacterial Activity via Binding of Penicillin-Binding Protein-4.

BACKGROUND: Metal Nanoparticles (NPs) have been widely used for various applications in biomedical sciences, including in drug delivery, and as therapeutic agents, but limited owing to their toxicity towards the healthy tissue. This warrants an alternative method, which can achieve the desired activity with much reduced or no toxicity. Being a biological product, Withania somnifera (W. somnifera) is environment friendly, besides being less toxic as compared to metal-based NPs. However, the exact mechanism of action of W. somnifera for its antibacterial activities has not been studied so far. OBJECTIVE: To develop "silver nanoparticles with root extract of W. somnifera (AgNPs-REWS)" for antimicrobial and anticancer activities. Furthermore, the analysis of their mechanism of action will be studied. METHODS: Using the in-silico approach, the molecular docking study was performed to evaluate the possible antibacterial mechanism of W. somnifera phytochemicals such as Anaferine, Somniferine, Stigmasterol, Withaferin A, Withanolide- A, G, M, and Withanone by the inhibition of Penicillin- Binding Protein 4 (PBP4). Next, we utilized a bottom-up approach for the green synthesis of AgNPs- REWS, performed an in-detail phytochemical analysis, confirmed the AgNPs-REWS by SEM, UVvisible spectroscopy, XRD, FT-IR, and HPLC. Eventually, we examined their antibacterial activity. RESULTS: The result of molecular docking suggests that WS phytochemicals (Somniferine, Withaferin A, Withanolide A, Withanolide G, Withanolide M, and Withanone) possess the higher binding affinity toward the active site of PBP4 as compared to the Ampicillin (-6.39 kcal/mol) reference molecule. These phytochemicals predicted as potent inhibitors of PBP4. Next, as a proof-of-concept, AgNPs- REWS showed significant antibacterial effect as compared to crude, and control; against Xanthomonas and Ralstonia species. CONCLUSION: The in-silico and molecular docking analysis showed that active constituents of W. somnifera such as Somniferine, Withaferin A, Withanolide A, Withanolide G, Withanolide M, and Withanone possess inhibition potential for PBP4 and are responsible for the anti-bacterial property of W. somnifera extract. This study also establishes that AgNPs via the green synthesis with REWS showed enhanced antibacterial activity towards pathogenic bacteria.

Biocontrol of tomato bacterial wilt by the new strain Bacillus velezensis FJAT-46737 and its lipopeptides.

BACKGROUND: There is an urgent need to discover biocontrol agents to control bacterial wilt. This study reports on a new lipopeptide-producing biocontrol strain FJAT-46737 and explores its lipopeptidic compounds, and this study investigates the antagonistic effects of these compounds. RESULTS: Based on a whole genome sequence analysis, the new strain FJAT-46737 was identified as Bacillus velezensis, and seven gene clusters responsible for the synthesis of bioactive secondary metabolites in FJAT-46737 were predicted. The antimicrobial results demonstrated that FJAT-46737 exhibited broad-spectrum antimicrobial activities in vitro against three bacteria and three fungi. Pot experiments showed that the control efficiencies for tomato bacterial wilt of the whole cultures, the 2-fold diluted supernatants and the crude lipopeptide of FJAT-46737 were 66.2%, 82.0%, and 96.2%, respectively. The above results suggested that one of the antagonistic mechanisms of FJAT-46737 was the secretion of lipopeptides consisting of iturins, fengycins and surfactins. The crude lipopeptides had significant antagonistic activities against several pathogens (including Ralstonia solanacearum, Escherichia coli and Fusarium oxysporum) and fengycins were the major antibacterial components of the lipopeptides against R. solanacearum in vitro. Furthermore, the rich organic nitrogen sources (especially yeast extracts) in the media promoted the production of fengycin and surfactin by FJAT-46737. The secretion of these two lipopeptides was related to temperature fluctuations, with the fengycin content decreasing by 96.6% and the surfactins content increasing by 59.9% from 20 °C to 40 °C. The optimal temperature for lipopeptide production by FJAT-46737 varied between 20 °C and 25 °C. CONCLUSIONS: The B. velezensis strain FJAT-46737 and its secreted lipopeptides could be used as new sources of potential biocontrol agents against several plant pathogens, and especially the bacterial wilt pathogen R. solanacearum.

Antibacterial Activities of Novel Dithiocarbamate-Containing 4H-Chromen-4-one Derivatives.

To aid the development of novel antibacterial agents that possess a innovative mechanism of action, we built a series of novel dithiocarbamate-containing 4H-chromen-4-one derivatives. We evaluated the activities of the derivatives against three plant pathogens Xanthomonas oryzae pv oryzae (X. oryzae pv o.), Ralstonia solanacearum (R. solanacearum), and Xanthomonas axonopodis pv citri (X. axonopodis pv c.). The results of the antibacterial bioassay showed that most of the target compounds displayed good inhibitory effects against X. oryzae pv o. and X. axonopodis pv c. Remarkably, compound E6 showed the best in vitro antibacterial activity against X. axonopodis pv c., with an EC50 value of 0.11 µg/mL, which was better than those of thiodiazole copper (59.97 µg/mL) and bismerthiazol (48.93 µg/mL). Compound E14 exhibited the best in vitro antibacterial activity against X. oryzae pv o., with an EC50 value of 1.58 µg/mL, which was better than those of thiodiazole copper (83.04 µg/mL) and bismerthiazol (56.05 µg/mL). Scanning electron microscopy analysis demonstrated that compounds E6 and E14 caused the rupture or deformation of the cell membranes for X. axonopodis pv c. and X. oryzae pv o., respectively. In vivo antibacterial activity test and the defensive enzymes activity test results indicated that the compound E14 could reduce X. oryzae pv o. more effectively than thiodiazole-copper or bismerthiazol.

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease.

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to agriculture. However, the Ralstonia model is considerably underexplored in comparison to other models involving bacterial plant pathogens, such as Pseudomonas syringae in Arabidopsis. Research targeted to understanding the interaction between Ralstonia and crop plants is essential to develop sustainable solutions to fight against bacterial wilt disease but is currently hindered by the lack of straightforward experimental assays to characterize the different components of the interaction in native host plants. In this scenario, we have developed a method to perform genetic analysis of Ralstonia infection of tomato, a natural host of Ralstonia. This method is based on Agrobacterium rhizogenes-mediated transformation of tomato roots, followed by Ralstonia soil-drenching inoculation of the resulting plants, containing transformed roots expressing the construct of interest. The versatility of the root transformation assay allows performing either gene overexpression or gene silencing mediated by RNAi. As a proof of concept, we used this method to show that RNAi-mediated silencing of SlCESA6 in tomato roots conferred resistance to Ralstonia. Here, we describe this method in detail, enabling genetic approaches to understand bacterial wilt disease in a relatively short time and with small requirements of equipment and plant growth space.

Target Discovery in Ralstonia solanacearum through an Activity-Based Protein Profiling Technique Based on Bioactive Oxadiazole Sulfones.

Ralstonia solanacearum is an extremely destructive and rebellious phytopathogen that can cause bacterial wilt diseases in more than 200 plant species. To explore and discover the potential targets in R. solanacearum for the purpose of developing new agrochemicals targeting this infection, here, we exploited a typical activity-based protein profiling technique for target discovery in R. solanacearum based on an activity-based probe 1 derived from bioactive oxadiazole sulfones. A total of 65 specific targets were identified with high confidence through a quantitative chemical proteomic approach. Three representative proteins (glycine cleavage system H protein, thiol peroxidase, and dihydrolipoamide S-succinyltransferase) were validated as the targets by using the immunoblotting analysis with their respective antibodies. Additionally, the in vitro interaction between the recombinant thiol peroxidase and probe 1 further confirmed that this protein was a target of oxadiazole sulfones. We anticipated that these discovered protein targets in R. solanacearum can stimulate the discovery and development of novel agrochemicals targeting bacterial infections caused by R. solanacearum.

Antibacterial activities against Ralstonia solanacearum and Xanthomonas oryzae pv. oryzae of 6-chloro-4-(4-substituted piperazinyl)quinazoline derivatives.

In this letter, a variety of simple 6-chloro-4-(4-substituted piperazinyl)quinazoline derivatives was prepared. Preliminary bioassays revealed that these compounds showed good antibacterial activities toward phytopathogens Ralstonia solanacearum and Xanthomonas oryzae pv. oryzae (Xoo). Among these derivatives, compounds 5a, 5d, 5e, 5f, 5p, 5q, 6b, and 6d exhibited potent inhibition effects against R. solanacearum with EC50 within 4.60-9.94 µg/mL, especially, compound 5g exerted the strongest activity with EC50 of 2.72 µg/mL; compound 6b possessed the best inhibitory activity toward Xoo with EC50 of 8.46 µg/mL. Subsequently, a good predictive three-dimensional quantitative structure-activity relationship (3D-QSAR) model was constructed via CoMFA to direct the future structural modification and optimization. Furthermore, the pathogens' topological studies were performed to explore the possible antibacterial mechanism. Given their simple frameworks and facile synthesis, title compounds can serve as the potential antibacterial leads.

Characterization of medicinal plant-associated biocontrol Bacillus subtilis (SSL2) by liquid chromatography-mass spectrometry and evaluation of compounds by in silico and in vitro methods.

This study explores the antimicrobial properties of bioactive secondary metabolites extracted from the medicinal plant (Solanum surattense)-associated Bacillus subtilis strain SSL2. The secondary metabolites were extracted from B. subtilis (SSL2) using ethyl acetate, acetone, butanol, chloroform and methanol solvents. The crude extract was tested against two wilt causing pathogens: Ralstonia solanacearum and Fusarium oxysporum. The results revealed that the ethyl acetate extract has maximum inhibition against both the pathogens tested in this study. Furthermore, liquid chromatography-mass spectrometry (LC-MS) analysis of ethyl acetate extract identified 80 different compounds based on mass-to-charge ratio, database difference, resolution of mass spectrum and so on. Among the 80 compounds, citrulline (m/z = 158.0917), chloramphenicol (m/z = 195.075) and carnitine (m/z 162.11) were further selected based on m/z ratio for in silico and in vitro analyses. The in silico analysis revealed that citrulline, chloramphenicol and carnitine inhibited the virulent genes phcA (R. solanacearum) and ste12 (F. oxysporum). Further, under in vitro condition, citrulline and chloramphenicol were found to inhibit the growth of R. solanacearum and F. oxysporum. On the basis of the biocontrol activity of B. subtilis (SSL2) in in silico and in vitro conditions, the bacteria could be used as a biocontrol agent against both bacterial and fungal wilt-causing pathogens. However, this needs to be tested in pot studies or field conditions before being used as biocontrol agents.Communicated by Ramaswamy H. Sarma.

Synthesis and antibacterial activity of solanum torvum mediated silver nanoparticle against Xxanthomonas axonopodis pv.punicae and Ralstonia solanacearum.

To meet the food demand of growing population, agricultural productivity needs to be increased by employing safe strategies without harming ecosystem. Silver nanoparticle (AgNP) using a green approach has become a promising substitute to the synthetic pesticides to overcome pest menace. In this study, AgNPs were synthesized from Solanum torvum fruit extract and their bactericidal property against phyto bacteria was shown. UV-vis spectroscopic observation revealed a surface resonance peak of 440 nm corresponding to the formation of AgNPs. Microscopic and particle-size analyses showed a nearly spherical size, with an average diameter of 27 nm. Surface charge and polydispersity index of the synthesized AgNPs were -11.8 mV and 0.29, respectively. Powder X-ray diffraction, energy-dispersive X-ray and Infrared spectroscopy techniques were used to explore phase formation, composition and possible biological molecules involved in AgNP formation. AgNPs exhibited minimum inhibitory concentrations of 6.25 µg mL-1 and 12.5 µg mL-1 against bacterial plant pathogens Xanthomonas axonopodis pv. punicae and Ralstonia solanacearum. In-vitro disk-diffusion assay showed inhibition zones of 11.4 ± 1 mm for R. solanacearum and 18.1 ± 1 mm for X. axonopodis pv. punicae treated with 50 µg mL-1 AgNPs. The AgNPs generated intracellular reactive oxygen species in the pathogens. DNA damage and DNA replication inhibition studies showed genotoxicity of AgNPs to the bacterial cells. A plant toxicity study demonstrated a nontoxic effect of the synthesized NPs. Overall; the results show that AgNPs can be used as an economically feasible, ecologically safe and effective approach to overcome bacterial diseases.