Molecular Mechanism Of Bacillus Spp.from Qinghai Tibet Plateau And Their VOCs In Alleviating Plant Biotic And Abiotic Stresses

Plants are sessile continuously affected by biotic and abiotic stresses that lead to a reduction in their productivity worldwide(Ansari et al.,2019).Biotic stresses are known to be very notorious involving different plant pathogens(Fungus,Bacteria,nematodes,insects,and viruses)that damage mostly all the important crops hence becoming a serious issue of the current time(Hashem et al.,2019).Moreover,among abiotic stresses,salinity ranks as the most severe environmental stressors highly limit wheat productivity and impose a major threat on the agricultural sector(Palaniyandi et al.,2014).The better management of these stresses ensures crop quality and productivity(Mahajan & Tuteja,2005;Tahir et al.,2017)The use of Plant Growth Promoting Rhizobacteria bacteria(PGPRs)has been reported to alleviate abiotic and biotic stresses in plants that provide a bright future for sustainable agriculture(Bharti et al.,2016).The current study aims to screen out Bacillus spp.strains obtained from Qinghai Tibet region China for their potential to alleviate different plant biological stresses and salt stress in wheat.Moreover,the study will focus to identify the potential broad-spectrum Bacillus volatiles against various phytopathogens to reduce the risk of different plant diseases.Biopesticides are considered safe and environmentally friendly as compared to the excessive chemical pesticides that pollute the environment with a negative effect on human health.Therefore,to identify Bacillus spp.strains or their volatiles with broad-spectrum antagonistic activity against different pathogens along with plant growth-promoting potential and stimulate induced system resistance in tomato is of the main objective in our research work.The significance of the current study is also highlighted by the fact that it will enable us to study the mechanism of high halophilic Bacillus strains involving the regulation of phytohormones,the key genes involved in growth promotion and salt resistance in wheat to reduce the risk of salinity.The results of the whole study are given below.1.Nematicidal Volatiles from Bacillus atrophaeus GBSC56 Promote Growth and Stimulate Induced Systemic Resistance in Tomato Against Meloidogyne incognitaBacillus 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 to control in partition plate experiment.Pure volatiles produced by GBSC56 were identified through Gas chromatography and mass spectrometry(GC-MS).Among 10 volatiles organic compounds(VOCs),three 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 that 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 infected roots that might reduce the adverse effect of oxidative stress-induced after infection.Moreover,genes responsible for plant growth promotion Sl CKX1,Sl IAA1 and exp18 were showed an upsurge in expression while AC0-1 was downregulated in infested plants.Furthermore,the defense-related genes(PR1,PR5,and Sl LOX1)in infected 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 of GBSC56 nematicidal volatiles by regulating antioxidant enzymes,the keys genes involved in plant growth promotion,and the defense mechanism of M.incognita-infested tomato plants to efficiently manage the rootknot disease.2.Nematicidal volatiles from Bacillus atrophaeus GBSC56 have broad-spectrum antagonistic potential to suppress Sclerotinia sclerotiorum with stimulation of induced systematic resistance in tomatoTo make biological control effective against multiple plant pathogens,research work is needed to find out Bacillus volatiles with broad-spectrum antagonistic potential for the purpose to alleviate the risk of complex diseases.In the current study,nematicidal VOCs produced by Bacillus atrophaeus GBSC56 obtain from the Qinghai Tibet region of China were evaluated for their broad-spectrum antagonistic potential against S.sclerotiorum that is known to cause complex diseases with other plant nematodes in a synergistic manner.The results showed that GBSC56-VOCs possessed a strong inhibitory effect against the S.sclerotiorum,induced high ROS production that leads to excessive oxidative stress and cell damage.The SEM and TEM analysis also revealed the morphological and ultra-structural deformities in terms of membrane damage and organelles disappearance in GBSC56-VOCs treated mycelial cells.Among 10 pure volatiles,Dimethyl Disulfide(DMDS),Methyl Isovalerate(MIV),2-Undecanaone(2-UD)and Ethyl Tiglate(ET)showed strong antagonistic potential against S.sclerotiorum,induced severe oxidative stress in mycelial cells and affect the virulence of the fungus by reducing the oxalic acid production.Moreover,downregulation of the expression of pathogenicity(B-GLU,OAH,CBH)and ROS scavenging genes(Ss CAT1,Ss SOD1 and Ss PO)was observed in pre-exposed S.sclerotiorum.Interestingly the biological control assay using host-plant leaves challenged with VOCs treated fungal mycelial plugs produced less lesion on the leaves.The volatiles(DMDS,MIV,and 2-UD)were found to upsurge the expression of defense-linked genes(PAL,GST,SOD,PPO,and HMGR)that stimulate induced systematic resistance against S.sclerotiorum disease progression on tomato stem in planta experiment.These findings provide the possibility of controlling complex disease of fungus and nematodes through volatiles with broad-spectrum antagonistic nature.3.Broad Spectrum Biocontrol Potential of Bacillus atrophaeus GBSC56 Volatiles Against Different Phytopathogenic BacteriaThe use of Bacillus broad-spectrum volatiles to control different bacterial plant pathogens is a topic of great interest among the researcher.In the current study,the volatiles produced by GBSC56 were studied for their broad-spectrum antagonistic potential against the most important notorious bacterial pathogens i.e.Xanthomonas oryzae pv.oryzae(Xoo),Pseudomonas syringae(Pst)and Ralstonia solanacearum(Rs).The result showed that among ten volatiles,four i.e.MIV,2-UD,and DMDS followed by ET have strong broad-spectrum biocontrol potential against the selected pathogens.The volatiles were found to induced oxidative stress inside the pathogen cells that leads to severe cell damage.Scanning and transmission electron microscopy reveal morphological and ultrastructural abnormalities in the pathogen’s cell exposed to volatiles.These abnormalities included loosening,and even at some places rupturing of the cell wall,movement of the cytoplasmic content towards these ruptured wall areas,in some cells a lack of differentiated materials in the cytoplasm.To the best of our knowledge,this is the first-time study to explore volatiles with broad-spectrum biocontrol potential against different bacterial plant pathogens.Moreover,the study provided detailed knowledge about volatiles with efficient broad-spectrum biocontrol potential which is suggested to be used in the agricultural sector to alleviate the risk of complex plant diseases and reduce the high cost of farmers for managing these diseases.4.Genetic screening and expression analysis of Bacillus spp.halophilic strains display their ability to reduce salt stress and regulate Phytohormones in wheatSoil salinity is one of the major factors negatively affecting the growth and productivity of important cereal crops including wheat resulting in significant losses worldwide.The current study was designed to alleviate salt stress on the wheat plant by inoculating halophilic Bacillus strains isolated from extreme rhizospheric environments of QinghaiTibetan plateau region China.The high halophilic strains NMCN1,LLCG23 and moderate halophilic FZB42 strain through genetic screening revealed the presence of genetic features corresponding to salt stress response,membrane transport,osmotic regulation,and signal transduction.Subsequently,the expression of genes involved in these pathways was also significantly higher in halophilic strains analyzed through q PCR under different salt concentrations as compared with control.The halophilic strains also help in inducing stress response in wheat plants by regulating abscisic acid,lipid peroxidation,and proline accumulation pathways in an efficient manner.Furthermore,through comparative analysis of growth promotion in wheat the high halophilic strains NMCN1 and LLCG23 showed significant results under high salt stress conditions(200 mmol)as compare with FZB42.Whereas,halophilic Bacillus strains NMCN1 and LLCG23 were able to upsurge the expression of phytohormones leading to significant improvement in plant growth under salt stress.Therefore,it is suggested that halophilic Bacillus strains can be further investigated to be used in different bioformulation for the purpose to obtain high production in saline conditions.