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Bacillus strains are well studied for antagonistic effect against fungal pathogens, but the selection of potential antifungal strains is laborious and time-consuming process. Newly developed genetic markers and LC-MS based detection was undertaken simultaneously to detect eight antimicrobial compounds viz., surfactin, bacillomycin, iturin, fengycin/plipastatin, bacilysin, bacillaene, bacillibactin and plantazolicin in forty-seven Bacillus strains. Out of these strains, 19 were positive for the presence of marker genes encoding antimicrobial compounds. Bacillus strains FZB42, EZ1509, EZ1507, VM10, GBAC46, VM49 and LLCG43 possessed genes for maximum number of antimicrobial compounds. LC-MS analysis of antimicrobial compounds showed corresponding results except OKB105 and 168. Contrary to marker-based detection of genes, LC-MS analysis revealed that OKB105 can produce surfactin but unable to synthesize fengycin, while 168 was deficit in both compounds. To assay antifungal potential, 19 Bacillus strains and their methanolic extracts were tested in vitro to inhibit mycelial growth of S. sclerotiorum. Results revealed that EZ1509, VM10, GBAC46, VM49 and FZB42 showed highest inhibitory activity. A bioassay on detached rapeseed leaves demonstrated that strains VM10, EZ1509, FZB42 and GBAC46 were excellent in reducing lesion diameter, while, OKB105 and 168 were completely ineffective to control S. sclerotiorum. Interestingly, antifungal activity of Bacillus strains was positively co-related to the number of antimicrobial genes, indicating their role in antifungal activity of Bacillus strains. Our findings suggest that combining genetic markers and LC-MS analysis can rapidly screen Potential Bacillus strains with antifungal attributes and this screening method can serve as foundation for the development of new biopesticides.

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.

Many species of plant-pathogenic gram-negative bacteria deploy the type III (T3) secretion system to secrete virulence components, which are mostly characteristic of protein effectors targeting the cytosol of the plant cell following secretion. Xanthomonas oryzae pv. oryzae (Xoo), a rice pathogen causing bacterial blight disease, uses the T3 accessory protein HrpE to assemble the pilus pathway, which in turn secretes transcription activator-like (TAL) effectors. The hrpE gene can execute extensive physiological and pathological functions beyond effector secretion. As evidenced in this study, when the hrpE gene was deleted from the Xoo genome, the bacteria incur seriouimpairments in multiplication, motility, and virulence. The virulence nullification is attributed to reduced secretion and translocation of PthXo1, which is a TAL effector that determines the bacterial virulence in the susceptible rice varieties. When the HrpE protein produced by prokaryotic expression is applied to plants, the recombinant protein is highly effective at inducing the defense response. Moreover, leaf photosynthesis efficiency is enhanced in HrpE-treated plants. These results provide experimental avenues to modulate the plant defense and growth tradeoff by manipulating a bacterial T3 accessory protein.

Abiotic stress in plants pose a major threat to cereal crop production worldwide and cold stress is also notorious for causing a decrease in plant growth and yield in wheat. The present study was designed to alleviate cold stress on plants by inoculating psychrophilic PGPR bacteria belonging to Bacillus genera isolated from extreme rhizospheric environments of Qinghai-Tibetan plateau. The genetic screening of psychrophilic Bacillus spp. CJCL2, RJGP41 and temperate B. velezensis FZB42 revealed presence of genetic features corresponding to cold stress response, membrane transport, signal transduction and osmotic regulation. Subsequently, the time frame study for the expression of genes involved in these pathways was also significantly higher in psychrophilic strains as analyzed through qPCR analysis at 4 ℃. The inoculated cold tolerant Bacillus strains also aided in inducing stress response in wheat by regulating abscisic acid, lipid peroxidation and proline accumulation pathways in a beneficial manner. Moreover, during comparative analysis of growth promotion in wheat all three Bacillus strains showed significant results at 25 ℃. Whereas, psychrophilic Bacillus strains CJCL2 and RJGP41 were able to positively regulate the expression of phytohormones leading to significant improvement in plant growth under cold stress.

Background: The bacterium Pantoea ananatis is associated with devastating diseases in many crops that cause serious economic losses. We previously isolated strain DZ-12 from maize brown rot leaves and, genome sequencing revealed that it belongs to P. ananatis and contains a large, endogenous plasmid, pDZ-12. Virulence plasmids are essential for pathogenesis in many bacterial pathogens. However, nothing was known regarding the role of this plasmid in P. ananatis pathogenicity in maize. Results: Here, we eliminated the endogenous plasmid from P. ananatis by substituting its native replicon with a temperature-sensitive replicon. The resulting temperature-sensitive plasmid could be cured by growing cells at high temperature (37 °C). Loss of pDZ-12 from P. ananatis DZ-12 led to decreased disease severity in maize plants, suggesting the endogenous plasmid was important for pathogenesis. Meanwhile, loss of pDZ-12 also affected the ability of the bacterium to form biofilms. The method described here, which is efficient and needs only two steps to cure the endogenous plasmid without antibiotic resistance, was also shown to work in Bacillus subtilis, and may be generally applicable in bacteria. Conclusions: This study provides the first evidence that the endogenous plasmid of P. ananatis DZ-12 is important for pathogenesis in maize plants and in the ability of this species to form biofilms. It also presents the first report on curing plasmid DNA from P. ananatis.

Background: Pantoea ananatis, a Gram-negative bacterium and species of the family Enterobacteriaceae, is associated with diseases in a wide range of crops. Diseases caused by P. ananatis are devastating and can result in serious economic losses. We previously isolated strain DZ-12 from maize brown rot leaves and, genome sequencing revealed that it belongs to P. ananatis and contains a large, endogenous plasmid, pDZ-12. Virulence plasmids are essential for pathogenesis in many bacterial pathogens. However, nothing was known regarding the role of this plasmid in the pathogenicity of P. ananatis in maize plants. Results: Here, we developed a simple and efficient strategy to eliminate the endogenous plasmid by substituting its native replicon with a temperature-sensitive replicon. The resulting temperature-sensitive plasmid could be cured by growing cells at high temperature (37 ℃). Loss of pDZ-12 from P. ananatis DZ-12 led to decreased disease severity in maize plants, suggesting the endogenous plasmid was important for pathogenesis. Meanwhile, loss of pDZ-12 also affected the ability of the bacterium to form biofilms. The method described here, which is efficient and needs only two steps to cure the endogenous plasmid without antibiotic resistance, was also shown to work in the Gram-positive bacterium Bacillus subtilis, and may be generally applicable in bacteria. Conclusions: This study we provide the first evidence that the endogenous plasmid of P. ananatis DZ-12 is important for pathogenesis in maize plants and in the ability of this species to form biofilms, which further our understanding of the pathogenicity of P. ananatis. It also presents the first report on curing plasmid DNA from P. ananatis. Keywords: Pantoea ananatis, endogenous plasmid, replicon, temperature-sensitive

Fusarium graminearum is a notorious pathogen that causes Fusarium head blight (FHB) in cereal crops. It produces secondary metabolites, such as deoxynivalenol, diminishing grain quality and leading to lesser crop yield. Many strategies have been developed to combat this pathogenic fungus, however, considering the lack of resistant cultivars and likelihood of environmental hazards upon using chemical pesticides, efforts have shifted toward the biocontrol of plant diseases, which is a sustainable and eco-friendly approach. Fengycin, derived from Bacillus amyloliquefaciens FZB42, was purified from the crude extract by HPLC and further analyzed by MALDI-TOF-MS. Its application resulted in structural deformations in fungal hyphae, as observed via scanning electron microscopy. In planta experiment revealed the ability of fengycin to suppress F. graminearum growth and highlighted its capacity to combat disease incidence. Fengycin significantly suppressed F. graminearum, and also reduced the deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), and zearalenone (ZEN) production in infected grains. To conclude, we report that fengycin produced by B. amyloliquefaciens FZB42 has potential as a biocontrol agent against F. graminearum and can also inhibit the mycotoxins produced by this fungus.

Fusarium graminearum is a notorious pathogen that causes Fusarium head blight (FHB) in cereal crops. It produces secondary metabolites, such as deoxynivalenol, diminishing grain quality and leading to lesser crop yield. Many strategies have been developed to combat this pathogenic fungus; however, considering the lack of resistant cultivars and likelihood of environmental hazards upon using chemical pesticides, efforts have shifted toward the biocontrol of plant diseases, which is a sustainable and eco-friendly approach. Fengycin, derived from Bacillus amyloliquefaciens FZB42, was purified from the crude extract by HPLC and further analyzed by MALDI-TOF-MS. Its application resulted in structural deformations in fungal hyphae, as observed via scanning electron microscopy. In planta experiment revealed the ability of fengycin to suppress F. graminearum growth and highlighted its capacity to combat disease incidence. Fengycin significantly suppressed F. graminearum, and also reduced the deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), and zearalenone (ZEN) production in infected grains. To conclude, we report that fengycin produced by B. amyloliquefaciens FZB42 has potential as a biocontrol agent against F. graminearum and can also inhibit the mycotoxins produced by this fungus.

The bacterium Pantoea ananatis is associated with devastating plant diseases that cause serious economic losses. Strain DZ-12 was previously isolated from maize brown rot leaves in Hebei Province, China and its genome sequencing revealed that it belongs to P. ananatis. It contains a large, endogenous plasmid, pDZ-12. Different studies have shown that virulence determinants are frequently carried on plasmids. To determine whether pDZ-12 from P. ananatis has any effect on pathogenicity, the plasmid was eliminated by substituting its native replication genes with temperature-sensitive replication genes. The resulting temperature-sensitive plasmid could be cured by growing cells at high temperature (37℃). Loss of pDZ-12 from P. ananatis DZ-12 led to a decreased disease severity in maize plants suggesting that the endogenous plasmid is important for pathogenesis. Loss of pDZ-12 also affected the ability of the bacterium to form biofilms. The study provides the first evidence that the endogenous plasmid of P. ananatis DZ-12 is important for pathogenesis in maize plants and carries genes involved in biofilm formation. This study also presents the first report on curing a plasmid from P. ananatis.