Transcriptomic analysis of Ralstonia solanacearum in response to antibacterial volatiles of Bacillus velezensis FZB42.

Volatile organic compounds (VOCs), produced by a variety of microbial species and used as biological agents, have been demonstrated to play a significant role in controlling phytopathogens. In continuation of our previous studies, we aim to elucidate the underlying mechanisms and pathways involved in interactions between pathogens and microbial VOCs. In the current study, we tested how VOCs produced by Bacillus velezensis FZB42 affect the growth of Ralstonia solanacearum TBBS1 in vitro.Query The result showed that the colony growth of R. solanacearum was reduced with an inhibition rate of 0.83 ± 0.043 as compared to the control 1.7 ± 0.076, respectively. The number of viable cells of R. solanacearum was significantly decreased to 7.68 CFU/mL as compared to the control (9.02 CFU/mL). In addition, transcriptomic analysis of R. solanacearum in response to VOCs produced by FZB42 was performed to better understand the effect of VOCs on R. solanacearum. The transcriptional response of R. solanacearum to FZB42-VOCs was determined using an Illumina RNA-seq approach. The results revealed significant changes in the expression of 2094 R. solanacearum genes, including 593 upregulated and 1501 downregulated genes. To validate the RNA-seq results, the expression of 10 genes was quantified using RT-qPCR. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to functionally annotate differentially expressed genes. Significant changes were observed in genes directly or indirectly related to virulence, including those related to bacterial invasion, motility, chemotaxis, and secretion systems. Overall, RNA-seq profiling provides new insights into the possible fundamental molecular mechanisms that are responsible for the reduction in growth and virulence of R. solanacearum upon application of FZB42-VOC.

Bacillus spp.-Mediated Growth Promotion of Rice Seedlings and Suppression of Bacterial Blight Disease under Greenhouse Conditions.

Rice (Oryza sativa L.) is a major cereal and staple food crop worldwide, and its growth and production are affected by several fungal and bacterial phytopathogens. Bacterial blight (BB) is one of the world's most devastating rice diseases, caused by Xanthomonas oryzae pv. oryzae (Xoo). In the current study, Bacillus atrophaeus FA12 and B. cabrialesii FA26 were isolated from the rice rhizosphere and characterized as having broad-range antifungal and antibacterial activities against various phytopathogens, including Xoo. In addition, the selected strains were further evaluated for their potent rice growth promotion and suppression efficacy against BB under greenhouse conditions. The result shows that FA12 and FA26, applied as seed inoculants, significantly enhanced the vigor index of rice seedlings by 78.89% and 108.70%, respectively. Suppression efficacy against BB disease by FA12 and FA26 reached up to 59.74% and 54.70%, respectively, in pot experiments. Furthermore, MALDI-TOF MS analysis of selected strains revealed the masses ranged from m/z 1040 to 1540, representing that iturins and fengycin are the major antimicrobial compounds in the crude extracts, which might have beneficial roles in rice defence responses against BB. In conclusion, FA12 and FA26 possess broad-range antagonistic activity and have the capability to promote plant growth traits. More importantly, applying these strains has a high potential for implementing eco-friendly, cost-effective, and sustainable management practices for BB disease.

Suppression of Sclerotinia sclerotiorum by the Induction of Systemic Resistance and Regulation of Antioxidant Pathways in Tomato Using Fengycin Produced by Bacillus amyloliquefaciens FZB42.

Lipopeptides from Bacillus species exhibit promising biological control activity against plant pathogens. This study aimed to explore the potential of purified fengycin to induce systemic resistance in tomato against Sclerotinia sclerotiorum. Bacillus amyloliquefaciens FZB42, its mutant AK1S, and their corresponding metabolites showed in vitro inhibition of S. sclerotiorum mycelium. Fengycin derived from an AK1S mutant was purified and identified through HPLC and MALDI-TOF-MS, respectively. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed structural deformities in the fungal mycelium. Moreover, fengycin induced the accumulation of reactive oxygen species (ROS) in S. sclerotiorum mycelium and downregulated the expression of ROS-scavenging genes viz., superoxide dismutase (SsSOD1), peroxidase (SsPO), and catalase (SsCAT1) compared to the untreated control. Furthermore, the lesion size was dramatically reduced in fengycin-treated tomato plants compared to plants infected with S. sclerotiorum only in a greenhouse experiment. Additionally, the transcriptional regulation of defense-related genes GST, SOD, PAL, HMGR, and MPK3 showed the highest upsurge in expression at 48 h post-inoculation (hpi). However, their expression was subsequently decreased at 96 hpi in fengycin + S. sclerotiorum treatment compared to the plants treated with fengycin only. Conversely, the expression of PPO increased in a linear manner up to 96 hpi.

Volatile Compounds of Endophytic Bacillus spp. have Biocontrol Activity Against Sclerotinia sclerotiorum.

To develop an effective biological agent to control Sclerotinia sclerotiorum, three endophytic Bacillus spp. strains with high antagonistic activity were isolated from maize seed and characterized. In vitro assays revealed that the Bacillus endophytes could produce volatile organic compounds (VOC) that reduced sclerotial production and inhibited mycelial growth of S. sclerotiorum. Gas chromatography-mass spectrometry revealed that the selected strains produced 16 detectable VOC. Eight of the produced VOC exhibited negative effects on S. sclerotiorum, while a further four induced accumulation of reactive oxygen species in mycelial cells. A mixture of VOC produced by Bacillus velezensis VM11 caused morphological changes in the ultrastructure and organelle membranes of S. sclerotiorum mycelial cells. The bromophenol blue assay revealed a yellow color of untreated fungal mycelium, which grew faster and deeper from 24 to 72 h postinoculation, as an indication of reduced pH. The potassium permanganate (KMnO4) titration assay showed that the rate of oxalic acid accumulation was higher in minimal salt liquid medium cultures inoculated with untreated fungal plugs compared with the Bacillus VOC-treated ones. Interestingly, biological control assays using host-plant leaves challenged with treated fungal mycelial plugs produced reduced lesions compared with the control. These findings provide new viable possibilities of controlling diseases caused by S. sclerotiorum using VOC produced by Bacillus endophytes.

Effect of volatile compounds produced by Ralstonia solanacearum on plant growth promoting and systemic resistance inducing potential of Bacillus volatiles.

BACKGROUND: Microbial volatiles play an expedient role in the agricultural ecological system by enhancing plant growth and inducing systemic resistance against plant pathogens, without causing hazardous effects on the environment. To explore the effects of VOCs of Ralstonia solanacearum TBBS1 (Rs) on tobacco plant growth and on plant growth promoting efficiency of VOCs produced by Bacillus subtilis SYST2, experiments were conducted both in vitro and in planta. RESULTS: The VOCs produced by SYST2 significantly enhanced the plant growth and induced the systemic resistance (ISR) against wilt pathogen Rs in all experiments. The SYST2-VOCs significantly increased PPO and PAL activity and over-expressed the genes relating to expansin, wilt resistance, and plant defense while repressed the genes relating to ethylene production. More interestingly, VOCs produced by pathogen, Rs had no significant effect on plant growth; however, Rs-VOCs decreased the growth promoting potential of SYST2-VOCs when plants were exposed to VOCs produced by both SYST2 and Rs. The co-culture of SYST2 and Rs revealed that they inhibited the growth of each other; however, the inhibition of Rs by SYST2-VOCs appeared to be greater than that of SYST2 by Rs-VOCs. CONCLUSION: Our findings provide new insights regarding the interaction among SYST2-VOCs, Rs-VOCs and plant, resulting in growth promotion and induced systemic resistance against the bacterial wilt pathogen Rs. This is the first report of the effect of VOCs produced by pathogenic microorganism on plant growth and on plant growth-promoting and systemic resistance-inducing potential of PGPR strain SYST2.

FvSet2 regulates fungal growth, pathogenicity, and secondary metabolism in Fusarium verticillioides.

Histone H3 lysine 36 methylation (H3K36me) is generally associated with activation of gene expression in most eukaryotic cells. However, the function of H3K36me in filamentous fungi is largely unknown. Set2 is the sole lysine histone methyltransferase (KHMTase) enzyme responsible for the methylation of H3K36 in Saccharomyces cerevisiae. In the current study, we identified a single ortholog of S. cerevisiae Set2 in Fusarium verticillioides. We report that FvSet2 is responsible for the trimethylation of H3K36 (H3K36me3). The FvSET2 deletion mutant (ΔFvSet2) showed significant defects in vegetative growth, FB1 biosynthesis, pigmentation, and fungal virulence. Furthermore, trimethylation of H3K36 was found to be important for active transcription of genes involved in FB1 and bikaverin biosyntheses. These data indicate that FvSet2 plays an important role in the regulation of secondary metabolism, vegetative growth and fungal virulence in F. verticillioides.

Volatile organic compounds produced by a soil-isolate, Bacillus subtilis FA26 induce adverse ultra-structural changes to the cells of Clavibacter michiganensis ssp. sepedonicus, the causal agent of bacterial ring rot of potato.

Rhizobacterial volatile organic compounds (VOCs) play an important role in the suppression of soil-borne phytopathogens. In this study, the VOCs produced by a soil-isolate, Bacillus subtilis FA26, were evaluated in vitro for their antibacterial activity against Clavibacter michiganensis ssp. sepedonicus (Cms), the causal agent of bacterial ring rot of potato. The VOCs emitted by FA26 inhibited the growth of Cms significantly compared with the control. Scanning and transmission electron microscopy analyses revealed distorted colony morphology and a wide range of abnormalities in Cms cells exposed to the VOCs of FA26. Varying the inoculation strategy and inoculum size showed that the production and activity of the antibacterial VOCs of FA26 were dependent on the culture conditions. Headspace solid-phase microextraction/gas chromatography-mass spectrometry analyses revealed that FA26 produced 11 VOCs. Four VOCs (benzaldehyde, nonanal, benzothiazole and acetophenone) were associated with the antibacterial activity against Cms. The results suggested that the VOCs produced by FA26 could control the causal agent of bacterial ring rot of potato. This information will increase our understanding of the microbial interactions mediated by VOCs in nature and aid the development of safer strategies for controlling plant disease.

Involvement of FvSet1 in Fumonisin B1 Biosynthesis, Vegetative Growth, Fungal Virulence, and Environmental Stress Responses in Fusarium verticillioides.

Fusarium verticillioides (teleomorph, Gibberella moniliformis) is an important plant pathogen that causes seedling blight, stalk rot, and ear rot in maize (Zea mays). During infection, F. verticillioides produce fumonsins B1 (FB1) that pose a serious threat to human and animal health. Recent studies showed that Set1, a methyltransferase of H3K4, was responsible for toxin biosynthesis in filamentous fungi. However, to date, the regulation of FvSet1 on FB1 biosynthesis remains unclear. In the current study, we identified only one Set1 ortholog in F. verticillioides (FvSet1) and found that the deletion of FvSET1 led to various defects in fungal growth and pathogenicity. More interestingly, the FvSET1 deletion mutant (ΔFvSet1) showed a significant defect in FB1 biosynthesis and lower expression levels of FUM genes. FvSet1 was also found to play an important role in the responses of F. verticillioides to multiple environmental stresses via regulating the phosphorylation of FvMgv1 and FvHog1. Taken together, these results indicate that FvSet1 plays essential roles in the regulation of FB1 biosynthesis, fungal growth and virulence, as well as various stress responses in F. verticillioides.

Bacillus volatiles adversely affect the physiology and ultra-structure of Ralstonia solanacearum and induce systemic resistance in tobacco against bacterial wilt.

Volatile organic compounds (VOCs) produced by various bacteria have significant potential to enhance plant growth and to control phytopathogens. Six of the most effective antagonistic Bacillus spp. were used in this study against Ralstonia solanacearum (Rsc) TBBS1, the causal agent of bacterial wilt disease in tobacco. Bacillus amyloliquefaciens FZB42 and Bacillus artrophaeus LSSC22 had the strongest inhibitory effect against Rsc. Thirteen VOCs produced by FZB42 and 10 by LSSC22 were identified using gas chromatography-mass spectrometry analysis. Benzaldehyde, 1,2-benzisothiazol-3(2 H)-one and 1,3-butadiene significantly inhibited the colony size, cell viability, and motility of pathogens and negatively influenced chemotaxis. Transmission and scanning electron microscopy revealed severe morphological and ultra-structural changes in cells of Rsc. Furthermore, VOCs altered the transcriptional expression level of PhcA (a global virulence regulator), type III secretion system (T3SS), type IV secretion system (T4SS), extracellular polysaccharides and chemotaxis-related genes, which are major contributors to pathogenicity, resulting in decreased wilt disease. The VOCs significantly up-regulated the expression of genes related to wilt resistance and pathogen defense. Over-expression of EDS1 and NPR1 suggest the involvement of SA pathway in induction of systemic resistance. Our findings provide new insights regarding the potential of antibacterial VOCs as a biocontrol tool against bacterial wilt diseases.