植物病原拮抗细菌的发酵条件作用机制及定殖规律的研究
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摘要
用稀释分离法从杭州近郊大棚蔬菜地根围土壤中共获得细菌菌株409枚,通过继代平皿对峙实验筛选出对Fusarium oxysporum f.sp.cucumerinum和Fusarium graminirum等多种植物病原菌有稳定拮抗作用的菌株ZJY-1、ZJY-13和ZJY-116。催芽实验和室内盆栽测定结果表明除ZJY-13外其余2个菌株均不会抑制种子的萌发且都具有明显的防病效果。引入田间小区后2个生防菌能有效防治黄瓜枯萎病和大麦赤霉病,显著降低病害的发生程度。此外,菌株ZJY-116有明显的促生作用,经ZJY-116种子处理的黄瓜种子播种后出苗早,幼根长而粗壮,子叶肥厚,黄瓜的平均单株产量以及果实均重都较健康对照高。
根据常规的形态学观察、生理生化测定和16Sr DNA序列的同源性分析,生防菌株ZJY-1和ZJY-116分别被鉴定为短短芽孢杆菌(Brevibacillus brevis)及枯草芽孢杆菌(Bacillus subtilis)。基因库中两菌株的登陆号分别为AY897210和AY897211。
摇瓶发酵实验结果表明玉米粉和麸皮是菌株ZJY-1和ZJY-116筛选所得的最佳碳源和氮源,且当玉米粉为30g L~(-1)、浮皮为10g L~(-1)时菌株的生物量达到最大。最适合ZJY-1发酵的优化条件为:温度30℃、pH 7左右、供氧充足;最适ZJY-116发酵的优化条件为:温度32℃、pH 7-7.5、氧气对发酵的影响不大。菌株ZJY-1主要通过产生能够抑制菌丝正常生长以及分生孢子萌发的抗菌代谢产物达到抑制病原菌的目的。最适合抗菌代谢物质产生的碳源为葡萄糖,氮源为明胶,向培养液中添加0.2%甘露醇可大大促进该物质的产生。
为研究两株拮抗细菌的定殖规律,将具有GFP荧光和氯霉素抗性的重组质粒pRP22-GFP,采用原生质体高频转化法,成功地导入生防菌Brevibacilluts brevis ZJY-1和Bacillus subtilis ZJY-116中,得到荧光标记菌株ZJY-1G和ZJY-116G。用这些标记菌株处理黄瓜种子及喷雾大麦穗部,通过平板稀释分离定期回收转化子,研究了GFP标记菌株在黄瓜根围和田间大麦穗部的定殖规律。结果表明,不论是在室内岔栽还是田间小区条件下,在黄瓜整个生育期两株生防菌株均能在根围稳定有效地定殖,并随着黄瓜盛花期和盛果期出现
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 × 10~4CFU g-1 root soil and 1.97× 10~4CFU 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 report
testified the feasibility of applying multifunctional microbial community in the environment.
根据常规的形态学观察、生理生化测定和16Sr DNA序列的同源性分析,生防菌株ZJY-1和ZJY-116分别被鉴定为短短芽孢杆菌(Brevibacillus brevis)及枯草芽孢杆菌(Bacillus subtilis)。基因库中两菌株的登陆号分别为AY897210和AY897211。
摇瓶发酵实验结果表明玉米粉和麸皮是菌株ZJY-1和ZJY-116筛选所得的最佳碳源和氮源,且当玉米粉为30g L~(-1)、浮皮为10g L~(-1)时菌株的生物量达到最大。最适合ZJY-1发酵的优化条件为:温度30℃、pH 7左右、供氧充足;最适ZJY-116发酵的优化条件为:温度32℃、pH 7-7.5、氧气对发酵的影响不大。菌株ZJY-1主要通过产生能够抑制菌丝正常生长以及分生孢子萌发的抗菌代谢产物达到抑制病原菌的目的。最适合抗菌代谢物质产生的碳源为葡萄糖,氮源为明胶,向培养液中添加0.2%甘露醇可大大促进该物质的产生。
为研究两株拮抗细菌的定殖规律,将具有GFP荧光和氯霉素抗性的重组质粒pRP22-GFP,采用原生质体高频转化法,成功地导入生防菌Brevibacilluts brevis ZJY-1和Bacillus subtilis ZJY-116中,得到荧光标记菌株ZJY-1G和ZJY-116G。用这些标记菌株处理黄瓜种子及喷雾大麦穗部,通过平板稀释分离定期回收转化子,研究了GFP标记菌株在黄瓜根围和田间大麦穗部的定殖规律。结果表明,不论是在室内岔栽还是田间小区条件下,在黄瓜整个生育期两株生防菌株均能在根围稳定有效地定殖,并随着黄瓜盛花期和盛果期出现
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 × 10~4CFU g-1 root soil and 1.97× 10~4CFU 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 report
testified the feasibility of applying multifunctional microbial community in the environment.
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