Studies On Zymosis Conditions, Functional Mechanism And Colonization Of Biocontrol Agents

409 strains of bacteria were obtained by isolating rhizosphere soil of protected vegetable fields near the HangZhou city. After repetitive co-culture on PDA plates, the strain ZJY-1 , ZJY-13 and ZJY-116 were screened for their stable antagonist toward Fusarium oxysporum f.sp. cucumerinum. Fusarium graminirum and other plant pathogens. The results of germination tests and pot trails showed that neither of the strain ZJY-1 and ZJY-116 could restrain germination of cucumber seed except ZJY-13. moreover, both strains could control plant diseases effectively. After introduced into the field, both biocontrol agents (BCA) gave significant protection against Fusarium wilt of cucumber and Fusarium head blight of barley (FHB), the diseases rate were reduced significantly, furthermore, ZJY-116 could also promote the growth of plant, the BCA-treated cucumber seeds germinated earlier, the young root is sturdier, and the cotyledon is more healthy than the check.The two strains were identified as Brevihacillus brevis and Bacillus suhtilis respectively by morphological, biochemical and physiological characteristics, as well as the 16 Sr DNA sequence analysis (The GenBank accession number of two BCA is AY897210 and AY897211 respectively).Fermentation experiments showed that cornmeal and wheat bran are the most suitable for strain ZJY-1 and ZJY-116 to grow, and the ultimate bioasis was achieved when the cornmeal is 30g L^-1 and the wheat bran is 10g L^-1. The optimized condition for ZJY-1 was the temperature at 30℃, pH 7 and the oxygen was abundantthe optimized condition for ZJY-116 was the temperature at 32℃. pH 7-7.5, the quantity of oxygen was not important.To monitor the colonization of two biocontrol agents, Brevibacillus brevis ZJY-1and Bacillus subtilis ZJY-116 in the environment, a green fluorescent protein (GFP)expression and chloramphenicol resistant plasmid pRP22-GFP was constructed andsuccessfully transformed into the BCA by high frequency transformation of their protoplastsand the stable transformants ZJY-1 G and ZJY-116G were obtained. Survival and colonizationof the Brevibacillus brevis ZJY-1 and Bacillus subtilis ZJY-116 strains in therhizosphere of cucumber and on spikes of barley were observed by periodically retrievethe GFP-tagged strains based on the selective markers above. The results in pot trials and plotexperiments showed that the both strains successfully colonized the rhizosphere ofcucumber during the whole growing season and the higher colonization level wasobserved during anthesis and fruition stage, even after the plant dead, the GFP-taggedstrain could maintained a high population, with 2.71 × 10⁴CFU g-1 root soil and 1.97× 10⁴CFU g-1 root soil of each strain in the pot 12 months later. Tracking the bacterialstrains introduced on the spikes of barley found that the two strains showed goodsurvival on both infected and healthy spikes in the field. When the BCA wereinoculated at 6.8 × 107CFU ml-1 during mid-anthesis, these bacterial strains stillmaintained a numerical level (×l05cfu grain-1 of barley) that could be detected untilthe spikes matured(37d later) and the bacteria population fluctuated relatively to therain events.By analyzing the population of major microorganisms, the activities of enzyme, as well as DGGE fingerprint of soil sample introduced with biocontrol agents, the effent of two strains on soil ecosystems have been studied in pot experiments, and the primary environmental security evaluation was made. Results showed that introduced BCA transiently changed the bacteria population, but the influence gradually vanished with the plant grow further. The fungi population in the BCA-introduced soil was almost always lower than that in the check, but the discrepancy was not remarkable. The reason for this maybe that introduced bacteria compete micro-niches and nutrition with the indigenous microorganisms. There was a little effect of introduced strains on the actinomyces, but the effect was less remarkable than that of shift of growing season.The influence of two BCA on the tested enzymes in the soil was diverse, except hydrogen peroxidase, the urease, fructosidase and dehydrogenase was affected to some extent. Study showed that the activities of three enzymes were generally higher than that of check during the plant growing season, the fertility level increased as a result, maybe that was the reason why the two BCA, especially ZJY-116 could promoting the growth of plant significantly Results also testified that the activities of soil enzymes would fluctuate closely related with the growing stage of plant. Generally, the activities increased when the plant grow well, the root exudates metabolites and the content of organic substances in soil was high. However, the activities decreased when the population of microflora were reduced after the plants were removed and the content of organic substances was low.The DGGE fingerprint analysis showed the effect of introduced BCA on microbial activity and population structure of resident microorganisms in soil during plant growing season were more pronounced than effects due to the inoculants. After introduced with BCA, there was no evident difference between the DGGE fingerprint of the soil sample obtained at different growing stage and that of check soil sample treated with sterilized distilled water, while the discrepancy among the soil sample at different growth stages was even more evident. The results were consistent with the analysis of parallel exponent.The feasibility of applying multifunctional micro-communities in the environment was studied by treating the cucumber seeds with the two strains ZJY-1( Brevibacillus brevis) and ZJY-116 (Bacillus mbtilis) singly or mixed with a chlorpyrifos degradation strain DSPS (Alcaligenes faecali) . The results showed that two agents kept their function of disease suppression and plant growth- promoting in the mixture with DSP3. Simultaneously, the results of chlorpyrifos content detected periodically in the rhizosphere soil showed that the degradation rate of DSP3 in the rhizosphere was not negatively affected even in the mixture with the two agents. Generally, this reporttestified the feasibility of applying multifunctional microbial community in the environment.