Study On Biocontrol Mechanism Of Bacillus Licheniformis TG116

Plant diseases caused by filamentous fungi are the main problems in agricultural production.There are many criticisms in conventional chemical control methods,such as pesticide residues,environmental pollution and so on.Therefore,the biological control represented by environment-friendly biocontrol agents has been gradually favored by people,and has made great progress.At present,many biocontrol agents with good control effect on plant diseases have been isolated and put into production practice.Bacillus spp.is the most studied biocontrol resource among bacteria.It has been found that Bacillus can prevent and cure diseases via niche competition,direct antagonism,inducing plant production system defense and promoting plant growth.Biocontrol of Bacillus is one of the important biocontrol mechanisms by directly producing protease,cellulase,chitinase and other hydrolysis enzymes to hydrolyze the cell wall of pathogenic fungi to kill pathogenic fungi.TG116 is an endophytic Bacillus licheniformis isolated from the tuber of Typhonium giganteum Engl.in our laboratory.Previous studies have shown that TG116has broad-spectrum antagonism to plant pathogenic fungi and has potential development value,howerer its bacteriostatic mechanism is not clear.This study takes TG116 as the research object,and some antibacterial protein components of TG116were isolated,purified and identified by chromatography and mass spectrometry.The biocontrol mechanism was discussed at the physiological,biochemical,morphological and molecular biological levels.The main findings are as follows:1.TG116 has broad-spectrum antibacterial activity to plant pathogenic fungi,strong growth ability and environmental adaptability.2.TG116 fermentation broth had antibacterial activity against plant pathogenic fungi,the optimum fermentation conditions were as follows:30?,pH 7.5,120 h.Increasing oxygen content could improve the bacteriostatic activity of fermentation broth.3.70%saturation?NH₄?₂SO₄ is the best precipitation condition.The crude protein has stable bacteriostatic activity and biological activities such as protease and cellulase,and it has good temperature stability and UV irradiation stability.4.The optimum single factor fermentation conditions for protease production of TG116 were as follows:40?,pH 8.0,48 h,the higher the dissolved oxygen content,the higher the protease activity.After optimization by response surface methodology,the optimum fermentation conditions were as follows:40.8?,pH 8.0,55 h,and the protease activity was increased by 4 times.The enzymatic characteristics showed that the optimum reaction was 50?,pH 8.5.The protease has good stability,more than 80%activity could be retained after 3 h water bath at 40?.It retains more than 60%activity after water bath at 50?for 2 h,and inactivates at 60?.When pH is higher than 7,the protease has good stability and the activity remains basically unchanged,but it is rapidly inactivated in acidic environment.Therefore,it is alkaline protease.Metal ions Mn²⁺,Co²⁺have a certain activation effect on protease activity,other metal ions such as Mg²⁺,Ca²⁺,Na⁺,Zn²⁺,K⁺have a certain inhibitory effect.Denaturants have a strong inhibitory effect,the higher the concentration,the stronger the inhibition.EDTA has the strongest inhibition among them.5.The optimum single factor fermentation conditions for cellulase production from TG116 were as follows:40.0?,pH 7.0,51 h,fermentation volume 75 mL.After optimization by response surface methodology,the optimum fermentation conditions were as follows:39.0?,pH 7.0 and 51.0 h,which could increase the cellulase activity by 2 times.The enzymatic characteristics showed that the optimum reaction was 50?,pH 3.5.The cellulase has good stability,more than 80%activity could be retained after3 h water bath at 40?.It retains more than 60%activity after 2 h water bath at 50?,and inactivates at 60?.When pH was lower than 6,the cellulase had good stability and the activity remained basically unchanged,but in the alkaline environment,the enzyme was basically inactivated.Indicating that the cellulase was acid cellulose.Metal ions like Mg²⁺,Fe²⁺,Zn²⁺,Ca²⁺,NH₄⁺and Li⁺have a certain activation effect on cellulase activity,other metal ions like K⁺,Mn²⁺have a certain inhibitory effect.Denaturant has a strong inhibitory effect,and the higher the concentration,the stronger the inhibition.Tris has the strongest inhibitory effect among them.6.The crude protein precipitated by 70%saturation?NH₄?₂SO₄ was purified by DEAE Sepharose Fast Flow column chromatography,and 12 different components were obtained,of which 5 had obvious bacteriostatic activity.One of the components was analyzed by Sephadex G-50 molecular sieve chromatography and SDS-PAGE polyacrylamide gel electrophoresis.An obvious band was obtained and identified as extracellular serine protease.The molecular weight of the protease was about86.17 kDa.Microscopic observation showed that the enzyme could lead to abnormal phenomena such as mycelial expansion and folding of pathogenic fungi.The protease can also affect the germination and development of spores,such as the decrease of germination rate,the deformity or expansion of germinated spore bud tube cells,the formation of vesicles and so on.7.Using the reported serine protease coding gene as a reference,a fragment of1617 bp in length was cloned by designing specialized primers.It was proved by informatics that encoded extracellular serine protease,named Pase gene,and submitted to GenBank?MK659579?.The relative molecular weight of the protease was 53.84kDa,theoretical isoelectric point 5.28,molecular formula C₂₃₂₁H₃₇₁₅N₆₂₃O₇₅₇S₁₂.The N-terminal of the protein contains obvious hydrophobicity and three transmembrane structures.The secondary structures of the protein are?-helix,?-rotation angle,extension chain,irregular crimp and so on.There are three conserved sequences,peptidase inhibitor I9,peptidase S8 family and cell surface serine protease?PA_C5a_like?.Analysis of its possible location in the cell,may exist in the cytoplasm,mitochondrial matrix,peroxisome and lysosome.The N-terminal of the protein contains a signal peptide,which may be related to the secretion of the protein into the extracellular process.Finally,the position of protein motif was analyzed,and it was found that there might be a N-glycosylation site,a cAMP and cGMP dependent protein kinase phosphorylation site,a protein kinase C phosphorylation site,a casein kinase II phosphorylation site,a N-nutmeg acylation site,a serine protease,subtilis enzyme family,histidine active site and serine protease,subtilis enzyme family,serine active site.The existence of these sites may be closely related to the function of the protein.