油茶炭疽病拮抗内生细菌的筛选、发酵及抑菌机理研究
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摘要
油茶炭疽病(Camellia oleifera anthracnose)是我国油茶产区的主要病害之一,由胶孢炭疽菌(Colletotrichum gloeosporioides)引起。该病常引起落果、落蕾,落果率一般在20%～40%,严重时达60%以上,降低了油茶产量。当前对油茶炭疽病的防治主要通过种植抗性品种和化学防治来控制,但易引发病原菌抗药性、环境污染等,效果不理想。安全、高效的无公害生物防治受到人们的广泛关注。本研究从油茶健康叶子中分离筛选出对油茶炭疽病病菌有抑制作用的拮抗菌,然后对防治效果好的生防菌株进行发酵条件优化及抑菌机理研究,研究结果如下：
(1)油茶炭疽病拮抗内生细菌的筛选与鉴定。从油茶健康植株分离到内生细菌156株。通过平板对峙和液体发酵离体初筛试验,得到对油茶炭疽病菌有抑制作用的菌株9株,抑制率均达到75%以上。进一步通过盆栽活体复筛,筛到一株强烈抑制油茶炭疽病的拮抗菌株Y13,中南林业科技大学菌种保藏号为CSUFT B0013.结合菌落形态、生理生化特征及16S rDNA序列分析,内生拮抗菌Y13鉴定是枯草芽孢杆菌Bacillus subtilis。
(2)油茶炭疽病拮抗内生细菌Y13的发酵条件优化。单因子试验表明：拮抗菌Y13最佳培养基为NYBD培养基,最佳碳源是葡萄糖,最佳氮源是牛肉膏和酵母膏；在温度30℃,初始pH 7.0,摇瓶装液量为50mL,接种量7%,摇床转速140 r/min,发酵周期72h的条件下可获得最大抑菌活性。正交试验表明：Y13菌株获得最大抑菌活性的培养基为葡萄糖25g,酵母膏10g,牛肉膏5g,NaCl浓度为1.2%。菌株获得最大生长量的培养基为葡萄糖15g,酵母膏10g,牛肉膏3g,NaCl浓度为1.5%。
(3)油茶炭疽病拮抗内生细菌Y13的抑菌机理研究。拮抗菌Y13连续转接10次,均具有明显的抗菌作用,拮抗作用比较稳定。Y13能够抑制油茶炭疽病菌菌丝体的生长和分生孢子的萌发。同时,Y13对植物病原真菌有较广的抑菌谱,对油茶叶枯病菌、番茄早疫病菌、黄瓜枯萎病菌、水稻纹枯病菌的抑制效果较好,抑制率分别为88.5%、84.3%、81.8%、80.0%和80.1%,拮抗活性较强,具有较好的生防应用潜力。拮抗菌Y13定殖力测定表明,拮抗菌最终定殖菌量为7.65×103CFU/g。接种炭疽病菌对拮抗细菌的定殖有一定影响,特别是在接种炭疽病菌1d后接种拮抗细菌和同时接种炭疽病菌与拮抗细菌的情况下,拮抗细菌的定殖能力显著下降,表明拮抗细菌Y13的防病效果要好于治病效果。
Camellia oleifera anthracnose caused by Colletotrichum gloeosporioides is one of the major diseases in China's Camellia producing areas.It occurs broadly and causes serious drop in buds and leaves, sometimes resulting in the death of the tree.It occurs up to 20%～40% of the growing regions are infected with this disease, reducing up to 80% of the total yield. Now, the most way of control the disease is combine the breeding variety to chemical control, but the effect is uncomfortable. Biological control has become an important way to plant disease for its safe, efficient and pollution-free features, which was attentioned by people.The most aim of the experiment was isolated and screened the strong antagonistic bacteria against C. gloeosporioides from the healthy leaves of Camellia oleifera, identified the strain, optimised the aimed strain fermented condition and its inhibiting Mechanisms against C. gloeosporioides. The results showed that:
(1)Isolating and screening the strong antagonistic bacteria against C. gloeosporioides from the healthy leaves of Camellia oleifera and identifing the strain.156 bacterial strains isolated from the healthy leaves of Camellia oleifera collected from heyang city, Hunan province.9 strains inhibiting Colletotrichum gloeosporioides were screened out by inhibitory zone method and the aseptic filtrate treatment. Through pot experiment test in vivo,it was found that isolated Y13 was the most effective antagonist against Colletotrichum gloeosporioides,which preservation number is CSUFT B0013.The identification of Y13 was performed with traditional bacterial systemic identification method. The result showed that the physiological and biochemical character of Y13 is similar to that of Bacillus subtilis. Then, the molecular identification of Y13 was performed. Through the comparative 16s rDNA sequences analysis, the 16s rDNA sequence of Y13 had high homology with typical Bacillus subtilis (99%).
(2)Optimising the aimed strain fermented condition. Through single factor and orthogonal test, the optimal fermentation conditions of producing antibiotic products of Bacillus subtilis Y13 were given out. The optimal culture conditions of the bacteria for the antibacterial protein producing are pH7.0,30℃,7%,140r/min, NYBD medium with shaking for 72h. Orthogonal test show that when Y13 got highest inhibiting activity, composition of the best media were glucose 25g, beef extract 10g, yeast extract 5g, NaCl 1.2%;when Y13 grow best,glucose 15g, beef extract 10g, yeast extract 3g, NaCl 1.5%.
(3)Study on its inhibiting Mechanisms against C. gloeosporioides. The strain Y13 always has the function of antifungal after undergo 10 transfer of culture. The effect of antifungal is stabile. Y13 could inhibit the growth of mycelia and conidia germination. Meanwhile,Y13 strain showed better repressing activity on Colletotrichum gloeosporioides. Wide antifungal spectrum was found through the leavening trials, which showed Y13 also had better repressing impact onFusarium oxysporium、Thanatephorus cucumeris、Alternaria solani、PestalotiopsisStey.Their inhibition rates were 88.5%、84.3 %、81.8%、80.0%和80.1%, respectively. Bacillus subtilis Y13 antagonistic against C.gloeosporioides was induced to be rifampicin-resistant and used for monitoring its colonization on leaves of Camellia oleifera. Experiments showed that the introduced antagonistic bacteria could colonize on Camellia oleifera concentration 7.65×103CFU/g. In comparison with inoculation of antagonistic bacteria only, pre-inoculation of C.gloeosporioides had a negative effect on colonization of the antagonists,which indicated that the antagonistic bacteria was better in prevention than curation for grey mould.
(1)油茶炭疽病拮抗内生细菌的筛选与鉴定。从油茶健康植株分离到内生细菌156株。通过平板对峙和液体发酵离体初筛试验,得到对油茶炭疽病菌有抑制作用的菌株9株,抑制率均达到75%以上。进一步通过盆栽活体复筛,筛到一株强烈抑制油茶炭疽病的拮抗菌株Y13,中南林业科技大学菌种保藏号为CSUFT B0013.结合菌落形态、生理生化特征及16S rDNA序列分析,内生拮抗菌Y13鉴定是枯草芽孢杆菌Bacillus subtilis。
(2)油茶炭疽病拮抗内生细菌Y13的发酵条件优化。单因子试验表明：拮抗菌Y13最佳培养基为NYBD培养基,最佳碳源是葡萄糖,最佳氮源是牛肉膏和酵母膏；在温度30℃,初始pH 7.0,摇瓶装液量为50mL,接种量7%,摇床转速140 r/min,发酵周期72h的条件下可获得最大抑菌活性。正交试验表明：Y13菌株获得最大抑菌活性的培养基为葡萄糖25g,酵母膏10g,牛肉膏5g,NaCl浓度为1.2%。菌株获得最大生长量的培养基为葡萄糖15g,酵母膏10g,牛肉膏3g,NaCl浓度为1.5%。
(3)油茶炭疽病拮抗内生细菌Y13的抑菌机理研究。拮抗菌Y13连续转接10次,均具有明显的抗菌作用,拮抗作用比较稳定。Y13能够抑制油茶炭疽病菌菌丝体的生长和分生孢子的萌发。同时,Y13对植物病原真菌有较广的抑菌谱,对油茶叶枯病菌、番茄早疫病菌、黄瓜枯萎病菌、水稻纹枯病菌的抑制效果较好,抑制率分别为88.5%、84.3%、81.8%、80.0%和80.1%,拮抗活性较强,具有较好的生防应用潜力。拮抗菌Y13定殖力测定表明,拮抗菌最终定殖菌量为7.65×103CFU/g。接种炭疽病菌对拮抗细菌的定殖有一定影响,特别是在接种炭疽病菌1d后接种拮抗细菌和同时接种炭疽病菌与拮抗细菌的情况下,拮抗细菌的定殖能力显著下降,表明拮抗细菌Y13的防病效果要好于治病效果。
Camellia oleifera anthracnose caused by Colletotrichum gloeosporioides is one of the major diseases in China's Camellia producing areas.It occurs broadly and causes serious drop in buds and leaves, sometimes resulting in the death of the tree.It occurs up to 20%～40% of the growing regions are infected with this disease, reducing up to 80% of the total yield. Now, the most way of control the disease is combine the breeding variety to chemical control, but the effect is uncomfortable. Biological control has become an important way to plant disease for its safe, efficient and pollution-free features, which was attentioned by people.The most aim of the experiment was isolated and screened the strong antagonistic bacteria against C. gloeosporioides from the healthy leaves of Camellia oleifera, identified the strain, optimised the aimed strain fermented condition and its inhibiting Mechanisms against C. gloeosporioides. The results showed that:
(1)Isolating and screening the strong antagonistic bacteria against C. gloeosporioides from the healthy leaves of Camellia oleifera and identifing the strain.156 bacterial strains isolated from the healthy leaves of Camellia oleifera collected from heyang city, Hunan province.9 strains inhibiting Colletotrichum gloeosporioides were screened out by inhibitory zone method and the aseptic filtrate treatment. Through pot experiment test in vivo,it was found that isolated Y13 was the most effective antagonist against Colletotrichum gloeosporioides,which preservation number is CSUFT B0013.The identification of Y13 was performed with traditional bacterial systemic identification method. The result showed that the physiological and biochemical character of Y13 is similar to that of Bacillus subtilis. Then, the molecular identification of Y13 was performed. Through the comparative 16s rDNA sequences analysis, the 16s rDNA sequence of Y13 had high homology with typical Bacillus subtilis (99%).
(2)Optimising the aimed strain fermented condition. Through single factor and orthogonal test, the optimal fermentation conditions of producing antibiotic products of Bacillus subtilis Y13 were given out. The optimal culture conditions of the bacteria for the antibacterial protein producing are pH7.0,30℃,7%,140r/min, NYBD medium with shaking for 72h. Orthogonal test show that when Y13 got highest inhibiting activity, composition of the best media were glucose 25g, beef extract 10g, yeast extract 5g, NaCl 1.2%;when Y13 grow best,glucose 15g, beef extract 10g, yeast extract 3g, NaCl 1.5%.
(3)Study on its inhibiting Mechanisms against C. gloeosporioides. The strain Y13 always has the function of antifungal after undergo 10 transfer of culture. The effect of antifungal is stabile. Y13 could inhibit the growth of mycelia and conidia germination. Meanwhile,Y13 strain showed better repressing activity on Colletotrichum gloeosporioides. Wide antifungal spectrum was found through the leavening trials, which showed Y13 also had better repressing impact onFusarium oxysporium、Thanatephorus cucumeris、Alternaria solani、PestalotiopsisStey.Their inhibition rates were 88.5%、84.3 %、81.8%、80.0%和80.1%, respectively. Bacillus subtilis Y13 antagonistic against C.gloeosporioides was induced to be rifampicin-resistant and used for monitoring its colonization on leaves of Camellia oleifera. Experiments showed that the introduced antagonistic bacteria could colonize on Camellia oleifera concentration 7.65×103CFU/g. In comparison with inoculation of antagonistic bacteria only, pre-inoculation of C.gloeosporioides had a negative effect on colonization of the antagonists,which indicated that the antagonistic bacteria was better in prevention than curation for grey mould.
引文
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