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.

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.

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. 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. 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.