Preparation On The Sustained-release Bacillus And Its Sustained Release Efficiency

Objective:In order to investigate the colonization dynamic of two have screend strains in this laboratory,Bacillus atrophaeus B44 and Bacillus subtilis S37,and to screen sustained-release coating material,then to prepare them as sustained-release microbial inoculum,a pot experiment was conducted to determine the control effect of cotton Rhizoctoniosis and its action mechanism.To provides a theoretical guidance for the more rational use of Bacillus inoculum to control the disease of cotton,and provides a feasible preparation method of Bacillus inoculum.Methods:The plasmid PGF4412 with GFP was transformed into two strains by modified Spizizen to label with fluorescence and Amp resistance.And then choose the tannic acid and ferric chloride,gelatin and poly glutamic acid,calcium carbonate and dopamine,three kinds of different material combinations of sustained-release to prepare of sustained-release Bacillus inoculum,and through the resistance medium counting method,to determine its storage stability,through pot experiment to determine its effect cycle in the rhizosphere soil of and control effect to cotton Rhizoctoniosis;choose three different chemical fungicides,fluorine Pyraoxystrobin 22%aniline?FS?,metalaxyl-myclobutanil?ME?and carbendazim?WP?,as well as mixed agent,water as CK,to compare the effect of sustained-release Bacillus inoculum in different temperature on cotton seed germination rate,plant height and other agronomic traits,to evaluate its protective effect on cotton seedlings under low temperature stress.By measuring Cotton Physiological and physiological indexes after three different temperatures in different days,the protective mechanism of sustained-release Bacillus inoculum on cotton seedlings under low temperature stress was evaluated.Results:1.The results showed that when the final concentration of ampicillin was 15?g/mL,B44strain have higher transformation rate and lower false positive rate,while S37 strain need20?g/m L.The two strains were only colonized on surface of cotton roots under fluorescence microscope.The more bacteria were used initially,the more bacteria will colonize in rhizosphere soil and surface of cotton roots.Besides B44 has higher control effect to cotton rhizoctoniosis when the initial dosage is higher,S37 also has the best control effect about 51.7%when the initial dosage is 1×10⁸CFU/g;2.After compare to three different materials,the calcium carbonate with dopamine are selected as optimal controlled release materials,as they have best embedding rate and releasing rate in PBS buffer solution with different pH in three materials combinations,which embedding rate is 87.4%.After storage 200 days at ordinary temperature,the bacillus survival rate is still as high as 91.33%.The pot experiment shows that the use of 2.5g/200 cotton seeds,the control effects of mixed Bacillus is 48.4%,while of controlled release preparation arrived54.6%,which have significant different.Besides,At the same concentration,the bacterial colony density of controlled release bacteria in soil declined to 4.33×10⁶ CFU/g,higher than that of mixed bacteria is 3.04×10⁶ CFU/g;3.All of three chemicals,mixed bacillus and sustained-release Bacillus inoculum could improve the germination rate of cotton under low temperature stress.At low temperature stress of 15^oC,the protective effect of sustained-release Bacillus treatment is more significant on cotton germination rate and seeding rate,which increased by 19.6%and 11.3%respectively,and after 5 days with low temperature treatment,the protein content of plant tissue increased 28.4%,H₂O₂ accumulation reduced 49.6%,the production of PAL increased28.3%.At 25^oC,sustained-release Bacillus could significantly promote the growth of cotton seedlings,but at lower temperature 5^oC,the effect of sustained-release Bacillus on Cotton Seedlings under low temperature stress was not significant.Conclusion:Two strains of B.atrophaeus B44 and B.subtilis S37 were labeled with plasmid PGF4412 by modified Spizizen method,which were found only colonize in the cotton seedling root surface and its rhizosphere soil,as well,the colonization on cotton roots and control effect to cotton rhizoctoniosis of two strains was positive correlation.After screened,poly-dopamine and calcium carbonate combination,was selected as the most ideal coating materials,which have better embedding effect on two strains.And storage stability of prepared sustained-release Bacillus at ordinary temperature after 200 d arrived 91.33%,and it's the colony density decline rate in the soil of was also lower than the mixed bacillus.Effects of different temperature on cotton seed germination,agronomic traits and physiological indexes,sustained-release Bacillus was found at 15^oC stress could improve the cotton seed germination rate,seeding rate and other physiological index significantly,but the effect is less than the chemical metalaxyl-myclobutanil and carbendazim,and similar with fluorine Pyraoxystrobin and mixed bacillus.This shows that two strains of Bacillus is more suitable for applicated on coot roots and its rhizosphere soil,the preparation of sustained-release Bacillus could increased the effect time in soil,and improved the control effect to cotton Rhizoctoniosis.