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Sclerotinia sclerotiorum is a devastating necrotrophic pathogen that infects multiple crops, and its control is an unremitting challenge. In this work, we attempted to gain insights into the pivotal role of lipopeptides (LPs) in the antifungal activity of Bacillus amyloliquefaciens EZ1509. In a comparative study involving five Bacillus strains, B. amyloliquefaciens EZ1509 harboring four LPs biosynthetic genes (viz. surfactin, iturin, fengycin, and bacilysin) exhibited promising antifungal activity against S. sclerotiorum in a dual-culture assay. Our data demonstrated a remarkable upsurge in LPs biosynthetic gene expression through quantitative reverse transcription PCR during in vitro interaction assay with S. sclerotiorum. Maximum upregulation in LPs biosynthetic genes was observed on the second and third days of in vitro interaction, with iturin and fengycin being the highly expressed genes. Subsequently, Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry analysis confirmed the presence of LPs in the inhibition zone. Scanning electron microscope analysis showed disintegration, shrinkage, plasmolysis, and breakdown of fungal hyphae. During in planta evaluation, S. sclerotiorum previously challenged with EZ1509 showed significant suppression in pathogenicity on detached leaves of tobacco and rapeseed. The oxalic acid synthesis was also significantly reduced in S. sclerotiorum previously confronted with antagonistic bacterium. The expression of major virulence genes of S. sclerotiorum, including endopolygalacturonase-3, oxalic acid hydrolase, and endopolygalacturonase-6, was significantly downregulated during in vitro confrontation with EZ1509.

Fusarium graminearum and Fusarium verticillioides are fungal pathogens that cause diseases in cereal crops, such as Fusarium head blight (FHB), seedling blight, and stalk rot. They also produce a variety of mycotoxins that reduce crop yields and threaten human and animal health. Several strategies for controlling these diseases have been developed. However, due to a lack of resistant cultivars and the hazards of chemical fungicides, efforts are now focused on the biocontrol of plant diseases, which is a more sustainable and environmentally friendly approach. In the present study, the lipopeptide mycosubtilin purified from Bacillus subtilis ATCC6633 significantly suppressed the growth of F. graminearum PH-1 and F. verticillioides 7600 in vitro. Mycosubtilin caused the destruction and deformation of plasma membranes and cell walls in F. graminearum hyphae. Additionally, mycosubtilin inhibited conidial spore formation and germination of both fungi in a dose-dependent manner. In planta experiments demonstrated the ability of mycosubtilin to control the adverse effects caused by F. graminearum and F. verticillioides on wheat heads and maize kernels, respectively. Mycosubtilin significantly decreased the production of deoxynivalenol (DON) and B-series fumonisins (FB1, FB2 and FB3) in infected grains, with inhibition rates of 48.92, 48.48, 52.42, and 59.44%, respectively. The qRT-PCR analysis showed that mycosubtilin significantly downregulated genes involved in mycotoxin biosynthesis. In conclusion, mycosubtilin produced by B. subtilis ATCC6633 was shown to have potential as a biological agent to control plant diseases and Fusarium toxin contamination caused by F. graminearum and F. verticillioides.

This study elaborates inter-kingdom signaling mechanisms, presenting a sustainable and eco-friendly approach to combat biotic as well as abiotic stress in wheat. Fusarium graminearum is a devastating pathogen causing head and seedling blight in wheat, leading to huge yield and economic losses. Psychrophilic Bacillus atrophaeus strain TS1 was found as a potential biocontrol agent for suppression of F. graminearum under low temperature by carrying out extensive biochemical and molecular studies in comparison with a temperate biocontrol model strain Bacillus amyloliquefaciens FZB42 at 15 and 25 °C. TS1 was able to produce hydrolytic extracellular enzymes as well as antimicrobial lipopeptides, i.e., surfactin, bacillomycin, and fengycin, efficiently at low temperatures. The Bacillus strain-induced oxidative cellular damage, ultrastructural deformities, and novel genetic dysregulations in the fungal pathogen as the bacterial treatment at low temperature were able to downregulate the expression of newly predicted novel fungal genes potentially belonging to necrosis inducing protein families (fgHCE and fgNPP1). The wheat pot experiments conducted at 15 and 25 °C revealed the potential of TS1 to elicit sudden induction of plant defense, namely, H2O2 and callose enhanced activity of plant defense-related enzymes and induced over-expression of defense-related genes which accumulatively lead to the suppression of F. graminearum and decreased diseased leaf area.

Simple Summary: The Qinghai–Tibet Plateau is known as the “third pole of the world”. Due to the extreme geographical location, Qinghai–Tibet Plateau has unique ecosystems characterized by oxygen deficiency, low temperature, high salinity and alkalinity. We carried out the current study to explore the excellent extremophilic Bacillus strains via potential stress resistance as well as biocontrol properties in the Qinghai–Tibet Plateau. We found a Bacillus halotolerans strain with a promising ability to withstand harsh environments and which also exhibits an optimistic biocontrol activity against plant pathogens. We revealed the whole genome sequencing and its taxonomic position and elucidated its molecular functions that were responsible for enhancing stress tolerance as well as suppressing plant pathogens at the genetic level. Lastly, we identified this strain harbored the specific genes associated with stresses resistance, biocontrol function, and can be used as a biological agent in the agriculture field. Due to its topographical position and climatic conditions, the Qinghai–Tibet Plateau possesses abundant microorganism resources. The extremophilic strain KKD1 isolated from Hoh Xil possesses strong stress tolerance, enabling it to propagate under high salinity (13%) and alkalinity (pH 10.0) conditions. In addition, KKD1 exhibits promising biocontrol activity against plant pathogens. To further explore these traits at the genomic level, we performed whole-genome sequencing and analysis. The taxonomic identification according to the average nucleotide identity based on BLAST revealed that KKD1 belongs to Bacillus halotolerans. Genetic screening of KKD1 revealed that its stress resistance mechanism depends on osmotic equilibrium, membrane transportation, and the regulation of ion balance under salt and alkaline stress. The expression of genes involved in these pathways was analyzed using real-time quantitative PCR. The presence of different gene clusters encoding antimicrobial secondary metabolites indicated the various pathways by which KKD1 suppresses phytopathogenic growth. Moreover, the lipopeptides surfactin and fengycin were identified as being significant antifungal components of KKD1. Through comparative genomics analysis, we noticed that KKD1 harbored specific genes involved in stress resistance and biocontrol, thus providing a new perspective on the genomic features of the extremophilic Bacillus species.

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.

Plant growth-promoting rhizobacteria (PGPR) are beneficial soil microorganisms that colonize plant roots for nutritional purposes and benefit plants by increasing plant growth or reducing plant disease. To clarify the complex mechanisms underlying plant-PGPR interactions, transcriptomic analyses of the response of rice (Oryza sativa) 9311 to Bacillus amyloliquefaciens FZB42 were carried out using RNA-Seq technology. This resulted in the identification of 379 differentially expressed genes in rice roots and 719 in rice leaves following FZB42 interaction that are likely to be related to plant-PGPR interactions, plant stress-resistance, pathogen-resistance, growth and development and basic and energy metabolism. We focused on plant disease-resistance mechanisms induced by FZB42. Variations in the expression of crucial genes COI1, MEKK1, WRKY, and PR that are involved in signal transduction following plant-pathogen interaction at different interaction time points were examined. Significantly up-regulated expression of PR, LOX, and ERF genes involved in the salicylic acid, jasmonic acid, and ethylene signal transduction pathways, respectively, were detected, suggest that defense reactions in rice were induced following interaction with FZB42. Phytoalexin changes in rice induced by FZB42 were examined and momilactone A, phytocassane A, phytocassane D, phytocassane E levels were low at 12 h, rose between 24 and 48 h, suggesting a PGPR-mediated mechanism of plant pathogen resistance. The study indicated that plant responses to PGPR in multi-metabolism pathways, which includes improving plant pathogen-resistance by mediating multi-signal transduction that regulates the expression of PRs and other defense genes, as well as influencing fundamental metabolic pathways to enhance the plant’s physical condition.

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.

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.

Summary Oligosaccharide, a typical danger‐associated molecular pattern (DAMP), has been studied and applied as plant defence elicitor for several years. Here, we report a novel oligosaccharide, mannan oligosaccharide (MOS) with a degree of polymerization of 2–6, which was hydrolysed from locust bean gum by a newly reported enzyme, BpMan5. The MOS treatment can significantly enhance the generation of signalling molecules such as intracellular Ca2+ and reactive oxygen species. Subsequent defence events like stomata closure and cell death were also caused by MOS, eventually leading to the prevention of pathogen invasion or expansion. Transcriptional expression assay indicated that MOS activated mitogen‐activated protein kinase cascades in tobacco and rice via different cascading pathways. The expression levels of the defence‐related genes PR‐1a and LOX were both up‐regulated after MOS treatment, suggesting that MOS may simultaneously activate salicylic acid and jasmonic acid‐dependent signalling pathways. Furthermore, liquid chromatography‐mass spectrometry analysis showed that MOS led to the accumulation of four phytoalexins (momilactone A, phytocassane A, phytocassane D, and phytocassane E) in rice seedling leaves within 12–24 h. Finally, MOS conferred resistance in rice and tobacco against Xanthomonas oryzae and Phytophthora nicotianae, respectively. Taken together, our results indicated that MOS, a novel DAMP, could trigger multiple defence responses to prime plant resistance and has a great potential as plant defence elicitor for the management of plant disease.

Bacillus volatiles to control plant nematodes is a topic of great interest among researchers due to its safe and environmentally friendly nature. Bacillus strain GBSC56 isolated from the Tibet region of China showed high nematicidal activity against M. incognita, with 90% mortality as compared with control in a partition plate experiment. Pure volatiles produced by GBSC56 were identified through gas chromatography and mass spectrometry (GC-MS). Among 10 volatile organic compounds (VOCs), 3 volatiles, i.e., dimethyl disulfide (DMDS), methyl isovalerate (MIV), and 2-undecanone (2-UD) showed strong nematicidal activity with a mortality rate of 87%, 83%, and 80%, respectively, against M. incognita. The VOCs induced severe oxidative stress in nematodes, which caused rapid death. Moreover, in the presence of volatiles, the activity of antioxidant enzymes, i.e., SOD, CAT, POD, and APX, was observed to be enhanced in M. incognita-infested roots, which might reduce the adverse effect of oxidative stress-induced after infection. Moreover, genes responsible for plant growth promotion SlCKX1, SlIAA1, and Exp18 showed an upsurge in expression, while AC01 was downregulated in infested plants. Furthermore, the defense-related genes (PR1, PR5, and SlLOX1) in infested tomato plants were upregulated after treatment with MIV and 2-UD. These findings suggest that GBSC56 possesses excellent biocontrol potential against M. incognita. Furthermore, the study provides new insight into the mechanism by which GBSC56 nematicidal volatiles regulate antioxidant enzymes, the key genes involved in plant growth promotion, and the defense mechanism M. incognita-infested tomato plants use to efficiently manage root-knot disease.

Cyclodipeptides, formed from two amino acids by cyclodehydration, are produced naturally by many organisms, and are known to possess a large number of biological activities. In this study, we found that cyclo (l-Pro-l-Pro) and cyclo (d-Pro-d-Pro) (where Pro is proline) could induce defence responses and systemic resistance in Nicotiana benthamiana. Treatment with the two cyclodipeptides led to a reduction in disease severity by Phytophthora nicotianae and Tobacco mosaic virus (TMV) infections compared with controls. Both cyclopeptides triggered stomatal closure, induced reactive oxygen species production and stimulated cytosolic calcium ion and nitric oxide production in guard cells. In addition, the application of cyclodipeptides significantly up-regulated the expression of the plant defence gene PR-1a and the PR-1a protein, and increased cellular salicylic acid (SA) levels. These results suggest that the SA-dependent defence pathway is involved in cyclodipeptide-mediated pathogen resistance in N. benthamiana. We report the systemic resistance induced by cyclodipeptides, which sheds light on the potential of cyclodipeptides for the control of plant diseases.

In this study, the fully degradable soybean oil-based waterborne polyurethanes (SWPU) were prepared, and the mechanical properties, thermal properties and hydrophilicity of SWPU films were adjusted by changing the R value. First, soybean oil-based polyol was fabricated via epoxy soybean oil and ricinoleic acid in the absence of solvent and catalyst. Then, the resulting polyol was mixed with isophorone diisocyanate (IPDI), dimethylolpropionic acid (DMPA), trimethylamine (TEA) and ethylenediamine (EDA) to synthesize polyurethane emulsion with different R value (the molar ration of –NCO/–OH). The result showed that as the R value increased, the tensile strength of SWPU film was increased from 10.02 to 27.32 MPa, the initial decomposition temperature of polyurethane films was improved from 201 ℃ to 246 ℃, the Tg,h was increased from 68 to 130 ℃ and the water contact angle of SWPU films were enhanced from 82.4° to 95.5°. It can be explained that the degree of crosslinking of the polyurethane elastomer was improved and the interaction of the molecular chains became stronger with larger R value. Also, all SWPU films were placed in the 3 wt% sodium hydroxide solution at 45 ℃ to test the degradability, and the weight loss of samples at different degradation time were calculated, respectively. Finally, the morphology, FT-IR and tensile strength of samples was observed before and after degradation, and a possible mechanism of degradation was studied. The technique reported here demonstrated that it was possible to develop the degradable materials.

In this study, a graft modified Octa(aminopropyl) polyhedral oligomeric silsesquioxane (OPOSS)/ TEMPO-oxidized cellulose microfibrils (TOCMF) nanocomposite material (named PC) was prepared by a simple peptide-forming reaction to overcome the disadvantages of nanocellulose materials which have limited their application in environmentally friendly waterborne polyurethanes. The modified vegetable oil-based waterborne polyurethane (WPU) was prepared by reacting the excess amino group on the POSS with the -NCO group on the polyurethane matrix in the in-situ polymerization, in which the POSS particles function as a button-like bond between the oxidized cellulose and the polyurethane matrix. And the similar filamentous structure of TOCMF to the linear polyurethane matrix structure could greatly weaken the negative impact of the rigid structure of POSS particles on processability (Elongation at break). The performance test results showed that the mechanical properties, thermal stability and water resistance of the modified WPU composite film are greatly improved by the synergistic effect of the two nano materials. The initial decomposition temperature and water contact angle of the WPU composite film with a PC content of 0.7 wt.% were increased by 41.9 °C and 10°, respectively. Especially, the tensile strength of WPU/PC-0.7 wt.% was twice that of pure WPU.

Plants live in close association with large communities of microbes, some of which are foliar pathogens that invade tissues, primarily via stomata on the leaf surface. Stomata are considered part of an integral, innate immunity system capable of efficiently preventing pathogens from entering the host plant. Although Bacillus, a typical plant growth-promoting rhizobacterium, is known to induce stomatal closure, the substances participating in this closure and the mechanism involved in its regulation remain poorly understood. Here, we screened a mutant library and conducted site-specific mutagenesis experiments in order to identify such substances. We found that acetoin and 2,3-butanediol from B. amyloliquefaciens FZB42 induced stomatal closure in Arabidopsis thaliana and Nicotiana benthamiana. These two components could function either via root absorption or volatilization to restrict stomatal apertures, but root absorption was more efficient. Both substances invoked the salicylic acid and abscisic acid signaling pathways to close the stomata and stimulated accumulation of hydrogen peroxide and nitric oxide. The results present comprehensive evidence of how soil rhizobacteria may affect plant stomata, in a way that reinforces the evolved mutualism between the two groups of organisms, and provide potential alternative avenues of research towards reducing the incidence of disease in crops.

Bacillus amyloliquefaciens FZB42 is a plant growth-promoting rhizobacterium that induces resistance to a broad spectrum of pathogens. This study analyzed the mechanism by which FZB42 restricts leaf disease caused by Phytophthora nicotianae in Nicotiana benthamiana. The oomycete foliar pathogen P. nicotianae is able to reopen stomata which had been closed by the plant innate immune response to initiate penetration and infection. Here, we showed that root colonization by B. amyloliquefaciens FZB42 restricted pathogen-mediated stomatal reopening in N. benthamiana. Abscisic acid (ABA) and salicylic acid (SA)-regulated pathways mediated FZB42-induced stomatal closure after pathogen infection. Moreover, the defense-related genes PR-1a, LOX, and ERF1, involved in the SA and jasmonic acid (JA)/ethylene (ET) signaling pathways, respectively, were overexpressed, and levels of the hormones SA, JA, and ET increased in the leaves of B. amyloliquefaciens FZB42-treated wild type plants. Disruption of one of these three pathways in N. benthamiana plants increased susceptibility to the pathogen. These suggest that SA- and JA/ET-dependent signaling pathways were important in plant defenses against the pathogen. Our data thus explain a biocontrol mechanism of soil rhizobacteria in a plant.

A novel thermostable mannanase from a newly isolated Bacillus pumilus GBSW19 has been identified, expressed, purified and characterized. The enzyme shows a structure comprising a 28 amino acid signal peptide, a glycoside hydrolase family 5 (GH5) catalytic domain and no carbohydrate-binding module. The recombinant mannanase has molecular weight of 45 kDa with an optimal pH around 6.5 and is stable in the range from pH 5-11. Meanwhile, the optimal temperature is around 65 °C, and it retains 50% relative activity at 60 °C for 12h. In addition, the purified enzyme can be activated by several ions and organic solvents and is resistant to detergents. Bpman5 can efficiently convert locus bean gum to mainly M2, M3 and M5, and hydrolyze manno-oligosaccharides with a minimum DP of 3. Further exploration of the optimum condition using HPLC to prepare oligosaccharides from locust bean gum was obtained as 10mg/ml locust bean gum incubated with 10 U/mg enzyme at 50 °C for 24h. By using this enzyme, locust bean gum can be utilized to generate high value-added oligosaccharides with a DP of 2-6.

Fusarium graminearum (teleomorph: Ascomycota, Hypocreales, Gibberella , Gibberella zeae ) is a destructive fungal pathogen that threatens the production and quality of wheat and barley worldwide. Controlling this toxin-producing pathogen is a significant challenge. In the present study, the commercially available strain Bacillus amyloliquefaciens ( Bacteria , Firmicutes , Bacillales , Bacillus ) FZB42 showed strong activity against F. graminearum . The lipopeptide bacillomycin D, produced by FZB42, was shown to contribute to the antifungal activity. Purified bacillomycin D showed strong activity against F. graminearum , and its 50% effective concentration was determined to be approximately 30 μg/ml. Analyses using scanning and transmission electron microscopy revealed that bacillomycin D caused morphological changes in the plasma membranes and cell walls of F. graminearum hyphae and conidia. Fluorescence microscopy combined with different dyes showed that bacillomycin D induced the accumulation of reactive oxygen species and caused cell death in F. graminearum hyphae and conidia. F. graminearum secondary metabolism also responded to bacillomycin D challenge, by increasing the production of deoxynivalenol. Biological control experiments demonstrated that bacillomycin D exerted good control of F. graminearum on corn silks, wheat seedlings, and wheat heads. In response to bacillomycin D, F. graminearum genes involved in scavenging reactive oxygen species were downregulated, whereas genes involved in the synthesis of deoxynivalenol were upregulated. Phosphorylation of MGV1 and HOG1, the mitogen-activated protein kinases of F. graminearum , was increased in response to bacillomycin D. Taken together, these findings reveal the mechanism of the antifungal action of bacillomycin D. IMPORTANCE Biological control of plant disease caused by Fusarium graminearum is desirable. Bacillus amyloliquefaciens FZB42 is a representative of the biocontrol bacterial strains. In this work, the lipopeptide bacillomycin D, produced by FZB42, showed strong fungicidal activity against F. graminearum . Bacillomycin D caused morphological changes in the plasma membrane and cell wall of F. graminearum , induced accumulation of reactive oxygen species, and ultimately caused cell death in F. graminearum . Interestingly, when F. graminearum was challenged with bacillomycin D, the deoxynivalenol production, gene expression, mitogen-activated protein kinase phosphorylation, and pathogenicity of F. graminearum were significantly altered. These findings clarified the mechanisms of the activity of bacillomycin D against F. graminearum and highlighted the potential of B. amyloliquefaciens FZB42 as a biocontrol agent against F. graminearum .

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.

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.

An environmentally friendly waterborne polyurethane based on castor oil was prepared and modified by 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidized cellulose microfibers (TOCMF). The TOCMF with surface-attached active carboxyl groups was obtained by TEMPO/NaClO/NaBr system, and then the modified castor oil-based waterborne polyurethane was prepared by the synergistic action of covalent bond and hydrogen bond between TOCMF and prepolymer in the in-situ polymerization reaction. The introduction of carboxylate on the surface of TOCMF facilitated stabilization of the thickness of the double layer of the emulsion particles to form stable WPU/TOCMF dispersions. Benefiting from the filamentous structural effects of TOCMF and the covalent bond and hydrogen bond dual interactions in WPU matrix, WPU/TOCMF composite films showed improvement in tensile strength and thermal decomposition temperature. Tensile strength of composite films increased from 17.89 to 30.31 MPa with increasing TOCMF amount from 0 to 1.00 wt%. T5% of composite films increased from 202.6 to 239.3 °C with increasing TOCMF amount from 0 to 0.75 wt%. It was observed by SEM that the presence of agglomeration in the composite film with 1.25 and 1.50 wt% TOCMF, and the occurrence of agglomeration caused the performance of the composite film to deteriorate sharply.

UNLABELLED Both Bacillus subtilis and harpins stimulate plant growth and defence against various plant pathogens. In this study, B. subtilis 168 and two derivatives, surfactin producer OKB105 and combined surfactin and HpaGX ooc producer OKBHF, were applied to tomato plants to investigate the mechanisms underlying this effect. To evaluate colonization ability, strains were labelled with green fluorescent protein (GFP). Although biofilm distribution of the three strains was similar on root surfaces, Colonization populations of the two surfactin producers were approximately 2- to 3-fold higher than that of strain 168, and this was accompanied by significantly increased tomato growth. These results suggest that efficient colonization, possibly facilitated by surfactin production, enhanced the efficiency of plant growth promotion by B. subtilis. All three B. subtilis treatments caused plants to have less severe disease symptoms after inoculation with Ralstonia solanacearum, with plants treated with OKBHF being the most resistant, suggesting that hpaGX ooc improves biocontrol efficiency of B. subtilis. Analysis of defence-related genes showed a synergistic effect of HpaGX ooc on B. subtilis enhancement of the expression of the pathogenesis-related genes PR1b1 and PR-P2. In contrast, expression of the defence-related genes PINI and PINII was suppressed. SIGNIFICANCE AND IMPACT OF THE STUDY Bacillus subtilis and harpins are biological control agents with respective advantages. In this study, combinations of the both were applied to tomato in the form of hpaGX ooc -expressing B. subtilis, showed much better effects on resistance to wilt disease, and equivalent effects on plant growth promotion compared with the progenitor strain have a great potential in agricultural use.

Harpin proteins are well known as eliciters that induce multiple responses in plants, such as systemic acquired resistance, hypersensitive response, enhancement of growth, resistance to the green peach aphid, and tolerance to drought. Overexpression of Harpin-encoding genes enhances plant resistance to diseases in tobacco, rice, rape, and cotton; however, it is not yet known whether the expression of Harpin-encoding genes in vivo improves plant tolerance to abiotic stresses. The results of this study showed that overexpression of a Harpin-encoding gene hrf1 in rice increased drought tolerance through abscisic acid (ABA) signalling. hrf1- overexpression induces an increase in ABA content and promotes stomatal closure in rice. The hrf1 transgenic rice lines exhibited a significant increase in water retention ability, levels of free proline and soluble sugars, tolerance to oxidative stress, reactive oxygen species-scavenging ability, and expression levels of four stress-related genes, OsLEA3-1, OsP5CS, Mn-SOD, and NM_001074345, under drought stress. The study confirmed that hrf1 conferred enhanced tolerance to drought stress on transgenic crops. These results suggest that Harpins may offer new opportunities for generating drought resistance in other crops.

According to the change of environment, soil-dwelling Bacillus species differentiate into distinct subpopulations, such as spores and competent cells. Rap-Phr systems have been found to be involved in this differentiation circuit by interacting with major regulatory proteins, such as Spo0A, ComA, and DegU. In this study, we report that the plasmid-born RapQ-PhrQ system found in Bacillus amyloliquefaciens B3 affects three regulatory pathways in the heterologous host Bacillus subtilis. Expression of rapQ in B. subtilis OKB105 strongly suppressed its sporulation efficiency, transformation efficiency, and surfactin production. Co-expression of phrQ or addition of synthesized PhrQ pentapeptide in vitro could compensate for the suppressive effects caused by rapQ. We also found that expression of rapQ decreased the transcriptional level of the sporulation-related gene spoIIE and surfactin synthesis-related gene srfA; meanwhile, the transcriptional levels of these genes could be rescued by co-expression of phrQ and in vitro addition of PhrQ pentapeptide. Electrophoretic mobility shift (EMSA) result also showed that RapQ could bind to ComA without interacting with ComA binding to DNA, and PhrQ pentapeptide antagonized RapQ activity in vitro. These results indicate that this new plasmid-born RapQ-PhrQ system controls sporulation, competent cell formation, and surfactin production in B. subtilis OKB105.

The 3642 base pairs genomic region of Bacillus subtilis B3, which converts B. subtilis 168 into a producer of surfactin, includes srfDB3 encoding thioesterase, aspB3 encoding aspartate aminotransferase, lpaB3 encoding phosphopantetheinyl transferase and yczEB3 encoding YczE-like protein. Deletion analysis and functionally complement results indicated that truncation of srfDB3, aspB3 and yczEB3 did not affect biosynthesis of surfactin in B. subtilis 168. The lpaB3 gene with its promoter and ribosome-binding site is essential, whereas its transcription terminator is not necessary for biosynthesis of surfactin in B. subtilis 168.

Bacillus subtilis B3 was found to produce lipopeptides iturins and fengycin that have activity against several plant pathogens such as Fusarium graminearum, Rhizoctonia solani, Rhizoctonia cerealis, and Pyricularia grisea. A 3642-bp genomic region of B. subtilis B3 comprising srfDB3, aspB3, lpaB3, and yczEB3 genes that resulted in biosynthesis of surfactin in B. subtilis 168 was cloned, sequenced, and characterized. Among them, the srfDB3 gene encodes thioesterase, which is required for biosynthesis of surfactin in B. subtilis; the aspB3 gene encodes a putative aspartate aminotransferase-like protein; the lpaB3 encodes phosphopantetheinyl transferase, which shows high identity to the product of lpa-14 gene regulating the biosynthesis of iturin A and surfactin in B. subtilis RB14; the yczEB3 encodes a YczE-like protein with significant similarities in signal peptide and part of the ABC transport system. The genetic regions between the srfD gene and lpa gene from B. subtilis B3 and B. subtilis A13, which produces iturin A, contain an approximate 1-kb nucleotide fragment encoding an aspartate aminotransferase-like protein; however, the relevant regions from B. subtilis 168 and B. subtilis ATCC21332 producing surfactin comprise an approximately 4-kb nucleotide fragment encoding four unknown proteins. There is 73% identity between the Lpa family and the Sfp family, although both are highly conserved.

Bacillus amyloliquefaciens C06, an effective biological agent in controlling brown rot of stone fruit caused by Monilinia fructicola, was also found to produce extra-cellular mucilage and form mucoid colonies on semi-solid surfaces. This study aimed to characterize the extra-cellular mucilage produced by B. amyloliquefaciens C06 using transposon mutagenesis and biochemical and physical analyses. The mucilage production in B. amyloliquefaciens C06 was demonstrated to be associated with ywsC gene expression and characterized to be of high molecular weight, consisted of only glutamic acid and linked with non-peptide bonds, thus identified as gamma-polyglutamic acid (gamma-PGA). Compared with wild type B. amyloliquefaciens C06, its mutants deficient in producing gamma-PGA, e.g. M106 and C06DeltaywsC showed less efficiency in biofilm formation, surface adhesion and swarming ability. It was also demonstrated that gamma-PGA was not essential for C06 to form colony on semi-solid surfaces, but was able to improve its colony structure. In vivo evaluation showed that disruption of gamma-PGA production in C06DeltaywsC impaired its efficiency of colonizing apple surfaces.

Bacillus amyloliquefaciens C06, a potential agent in biological preservation of post-harvest fruit, was found to secrete extra-cellular γ-polyglutamic acid (γ-PGA) in liquid culture. In this work, M306, a transposon mutant of B. amyloliquefaciens C06, defective in forming structured colony and displaying enhanced ability of producing γ-PGA, was obtained. Inverse PCR and quantitative reverse transcription PCR (qRT-PCR) analysis demonstrated that the defective phenotype in M306 was associated with an ORF showing high similarity to RBAM_034550 from B. amyloliquefaciens FZB42. In this paper, the ORF was designated pbrA, standing for γ-PGA production and biofilm formation regulatory factor. qRT-PCR analysis also indicated that pbrA down-regulated mRNA expression of epsD and yqxM, the crucial genes involved in biofilm formation, but affected little on expression of ywtB, the gene directing γ-PGA synthesis. Evaluations in γ-PGA productivity of wild-type C06 and its mutants C06ΔepsA and C06ΔtasA, respectively, deficient in producing exopolysaccharides (EPS) and TasA, revealed that γ-PGA overproduction in M306 was probably due to the redistributed metabolic flux caused by defective production of EPS.

The early stage of plant-rhizobacteria interaction, affected by plant root exudates and plant-rhizobacteria surface contact, is considered to be critical for plant growth-promoting rhizobacteria colonizing plant roots and initiating the beneficial effects on plant growth. However, little is known about the mechanisms of plant-rhizobacteria surface contact involved in early stage of plant-rhizobacteria interaction. In order to reveal the molecular mechanisms of the surface contact, a rhizobacterium Bacillus amyloliquefaciens B55 was interacted with plant roots of rice R109 and used to perform a cDNA-based suppression-subtractive hybridization. Seven differentially expressed DNA fragments were identified. Except for the two fragments showing no matches to any known sequences in the Genbank, the other five fragments were found to have high homologies with the genes encoding 2-oxoglutarate dehydrogenase E1 component OdhA, aspartate ammonia-lyase AnsB, and hypothetical protein proposed to be involved in surface adhesion, acetolactate decarboxylase AlsD, and DNA mismatch repair protein MutL, respectively. The induced RNA expression levels of two putative genes ansB and odhA and an unmatched DNA fragment BD33 were verified by RT-PCR analysis.