番茄灰霉病生防细菌TD-7的鉴定、发酵条件优化及其防治效果
|
摘要
通过平板稀释分离法,从多年生草本植物薄荷根际土壤中分离到一株对灰葡萄孢Botrytis cinerea抑菌活性良好的细菌TD-7。通过形态特征和16S rDNA、ITS序列分析,鉴定该菌为解淀粉芽胞杆菌Bacillus amyloliquefaciens,并对其发酵滤液抑菌机理、最优发酵条件、最优发酵培养液组分及防治效果进行了研究。研究发现,菌株TD-7发酵滤液对番茄灰霉菌孢子的萌发有强烈的抑制作用,对菌丝生长抑制率在95%以上。皿内酶分泌试验发现,该菌可分泌几丁质酶、纤维素酶和蛋白酶,未检测到β-1,3-葡聚糖酶活性。发酵滤液抑菌活性最强的条件为在250 mL三角瓶中,将种子液按3%的体积比接种到50～75 mL培养液中,初始pH 8.0,温度32～36℃,转速200 r/min,发酵5 d。通过正交优化,得到最佳发酵培养液配方为玉米粉3%、蛋白胨1.5%、KH_2PO_4 0.05%。经摇床发酵条件及发酵培养液组分优化,菌株TD-7发酵滤液抑菌活性提高了26.39%。该菌的发酵液对温室盆栽及田间大棚中的番茄灰霉病均具有良好的防治效果,分别为80.61%和87.70%。滤液稳定性试验结果表明,菌株TD-7发酵滤液中活性物质具有较广的pH适应范围(pH4.0～9.0),在高温和紫外光照射下较稳定,有制成生物制剂应用于番茄灰霉病生物防治的潜力。
The bacterial strain TD-7, which was isolated from the rhizoplane soil of the perennial plant peppermint by plate dilution method, had a good inhibitory effect on the tomato grey mould Botrytis cinerea. The bacterium was identified as Bacillus amyloliquefaciens based on morphological features and sequence analysis of 16S rDNA and ITS. We investigated the inhibition mechanism, optimal fermentation condition, optimal fermentation medium composition and control effect of the TD-7 fermentation filtrate. The results show that the fermentation filtrate had a strong inhibition effect on the spore germination of B. cinerea with the inhibition rate of the mycelium growth over 95%. In vitro, strain TD-7 can secrete chitinase, cellulase and protease, while the activity of beta-1,3-glucanase was undetectable. The optimal fermentation conditions for producing antimicrobial substance were 250 mL flask, volume ratio of seed liquid 3%, fermentation medium 50-75 mL, initial pH 8.0, temperature 32-36 ℃, rotation speed 200 r/min and fermentation time 5 d. Orthogonal test demonstrated that the optimal medium was composed of 3.0% corn meal, 1.5% peptone and 0.05% KH_2PO_4. By optimizing fermentation conditions of shake cultivation and components of fermentation medium, the inhibitory activity of TD-7 fermentation filtrate was increased by 26.39%. The TD-7 fermentation broth had remarkable control efficacies on the tomato grey mould in the pot and greenhouse experiment as 80.61% and 87.70%, respectively. The stability experiment of the TD-7 fermentation filtrate shows that the active substance had a broad adaption range to pH value(pH 4.0-9.0), and was stable under high temperatures and UV-treatment. This suggests that the strain TD-7 of B. amyloliquefaciens is promising to be developed into a biocontrol formulation for controlling the tomato grey mould.
The bacterial strain TD-7, which was isolated from the rhizoplane soil of the perennial plant peppermint by plate dilution method, had a good inhibitory effect on the tomato grey mould Botrytis cinerea. The bacterium was identified as Bacillus amyloliquefaciens based on morphological features and sequence analysis of 16S rDNA and ITS. We investigated the inhibition mechanism, optimal fermentation condition, optimal fermentation medium composition and control effect of the TD-7 fermentation filtrate. The results show that the fermentation filtrate had a strong inhibition effect on the spore germination of B. cinerea with the inhibition rate of the mycelium growth over 95%. In vitro, strain TD-7 can secrete chitinase, cellulase and protease, while the activity of beta-1,3-glucanase was undetectable. The optimal fermentation conditions for producing antimicrobial substance were 250 mL flask, volume ratio of seed liquid 3%, fermentation medium 50-75 mL, initial pH 8.0, temperature 32-36 ℃, rotation speed 200 r/min and fermentation time 5 d. Orthogonal test demonstrated that the optimal medium was composed of 3.0% corn meal, 1.5% peptone and 0.05% KH_2PO_4. By optimizing fermentation conditions of shake cultivation and components of fermentation medium, the inhibitory activity of TD-7 fermentation filtrate was increased by 26.39%. The TD-7 fermentation broth had remarkable control efficacies on the tomato grey mould in the pot and greenhouse experiment as 80.61% and 87.70%, respectively. The stability experiment of the TD-7 fermentation filtrate shows that the active substance had a broad adaption range to pH value(pH 4.0-9.0), and was stable under high temperatures and UV-treatment. This suggests that the strain TD-7 of B. amyloliquefaciens is promising to be developed into a biocontrol formulation for controlling the tomato grey mould.
引文
[1]康立娟,张小风,王文桥,等.灰霉菌的抗药性与适合度测定[J].农药学学报,2000,2(3):39-42.
[2]毛亚辉.设施番茄灰霉病防治现状及综合防治措施[J].天津农林科技,2012(1):16-17.
[3]薛德林,胡江春,李晓茹,等.宁康霉素对番茄灰霉病和晚疫病的防治效果[J].腐殖酸,2006(3):25-28.
[4]李桂霞,马汇泉,刘婧,等.番茄灰霉病高效拮抗菌株鉴定及抗菌活性研究[J].山东农业科学,2007,2:69-73.
[5]高苇,王勇,张春祥.番茄灰霉病拮抗细菌的筛选与鉴定[J].中国植保导刊,2016,36(8):16-25.
[6]安瑞平.内生细菌对黄瓜灰霉病的防治研究[J].安徽农业科学,2009,34:115.
[7]周阳薇,刘正坪,魏艳敏,等.植物根际土壤细菌的分离与鉴定[J].中国农学通报,2015,31(7):212-217.
[8]Vasanthakumari M M,Shivanna M B.Biological control of anthracnose of chilli with rhizosphere and rhizoplane fungal isolates from grasses[J].Archives of Phytopathology and Plant Protection,2013,46(14):1641-1666.
[9]Vasanthakumari M M,Shivanna M B.Biological control of sorghum anthracnose with rhizosphere and rhizoplane fungal isolates from perennial grasses of the Western Ghats of India[J].European Journal of Plant Pathology,2014,139(4):721-733.
[10]Roberts W K,Selitrennikoff C P.Plant and bacterial chitinases differ in antifungal activity[J].Microbiology,1988,134(1):169-176.
[11]Ghose T K.Measurement of cellulase activities[J].Pure and Applied Chemistry,1987,59:257-268.
[12]王明江,章如意,林多多,等.棉花黄萎病不同抗性品种内生菌数量调查与拮抗菌筛选[J].江苏农业科学,2010,38(2):102-104.
[13]韩立荣.一株土传病害生防菌的筛选及其功能开发[D].杨凌:西北农林科技大学,2010.
[14]赵新贝,上官妮妮,李月飞,等.番茄灰霉病拮抗细菌18BS-12发酵条件的优化及防治效果[J].西北农林科技大学学报(自然科学版),2016,44(6):116-124.
[15]石凤梅.80%腐霉利可湿性粉剂对番茄灰霉病的防效研究[J].黑龙江农业科学,2014,7:68-69.
[16]王芝凤.400 g/L嘧霉胺悬浮剂防治番茄灰霉病田间药效试验报告[J].农业科技与信息,2017(6):73-74.
[17]Ausubel F M,Brent R,Kingston R E,et al.Short protocols in molecular biology[M].New York:John Wiley and Sons,1995,11-13.
[18]权春善,王军华,徐洪涛,等.一株抗真菌解淀粉芽孢杆菌的分离鉴定及其发酵条件的初步研究[J].微生物学报,2006,46(1):7-12.
[19]Caldeira A T,Feio S S,Arteiro J M S,et al.Bacillus amyloliquefaciens CCMI 1051 in vitro activity against wood contaminant fungi[J].Annals of Microbiology,2007,57:29-33.
[20]Imen Z,Lobna J,Slim T,et al.Biocontrol activity of the endophytic Bacillus amyloliquefaciens strain CEIZ-11 against Pythium aphanidermatum and purification of its bioactive compounds[J].Biological Control,2016,100:54-62.
[21]Zhang S M,Wang Y X,Meng L Q,et al.Isolation and characterization of antifungal lipopeptides produced by endophytic Bacillus amyloliquefaciens TF28[J].African Journal of Microbiology Research,2012,6(8):1747-1755.
[22]张淑梅,姜威,孟利强,等.内生细菌TF28对番茄灰霉病的诱导抗性研究[J].安徽农业科学,2014,42(11):3253-3256.
[23]Mari M,Guizzardi M,Brunelli M,et al.Postharvest biological control of grey mould(Botrytis cinerea Pers.:Fr.)on fresh-market tomatoes with Bacillus amyloliquefaciens[J].Crop Protection,1996,15(8):699-705.
[24]杨桥,韩文菊,张文俊,等.产Macrolactin-A抗生素海洋解淀粉芽孢杆菌的鉴定及发酵条件优化[J].药物生物技术,2009,4:311-315,346.
[25]Chao S H,Cheng T H,Shaw C Y,et al.Characterization of a novel Pep F-like oligopeptidase secreted by Bacillus amyloliquefaciens 23-7A[J].Applied and Environmental Microbiology,2006,72(1):968-971.
[26]王英国,王军华,权春善,等.解淀粉芽孢杆菌抗菌活性物质的分离纯化及抑菌活性研究[J].中国生物工程杂志,2007,27(12):41-45.
[27]Arguelles-Arias A,Ongena M,Halimi B,et al.Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens[J].Microbial Cell Factories,2009,8(1):1-12.
[28]Kim P,Chung K C.Production of an antifungal protein for control of Colletotrichum lagenarium by Bacillus amyloliquefaciens MET0908[J].FEMS Microbiology Letters,2004,234(1):177-183.
[29]Koumoutsi A,Chen X H,Henne A,et al.Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42[J].Journal of Bacteriology,2004,186(4):1084-1096.
[30]Arrebola E,Jacobs R,Korsten L.Iturin A is the principal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens[J].Journal of Applied Microbiology,2010,108(2):386-395.
[31]刘俏,刘春善,范圣第.超滤解淀粉芽孢杆菌发酵液提取抗菌物质的研究[J].食品科技,2006,31(7):70-72.
[32]陈敏,郭旭文,李春远,等.解淀粉芽孢杆菌SC1150的抑菌活性及其液体发酵条件的优化[J].生态科学,2015,34(3):7-12.
[2]毛亚辉.设施番茄灰霉病防治现状及综合防治措施[J].天津农林科技,2012(1):16-17.
[3]薛德林,胡江春,李晓茹,等.宁康霉素对番茄灰霉病和晚疫病的防治效果[J].腐殖酸,2006(3):25-28.
[4]李桂霞,马汇泉,刘婧,等.番茄灰霉病高效拮抗菌株鉴定及抗菌活性研究[J].山东农业科学,2007,2:69-73.
[5]高苇,王勇,张春祥.番茄灰霉病拮抗细菌的筛选与鉴定[J].中国植保导刊,2016,36(8):16-25.
[6]安瑞平.内生细菌对黄瓜灰霉病的防治研究[J].安徽农业科学,2009,34:115.
[7]周阳薇,刘正坪,魏艳敏,等.植物根际土壤细菌的分离与鉴定[J].中国农学通报,2015,31(7):212-217.
[8]Vasanthakumari M M,Shivanna M B.Biological control of anthracnose of chilli with rhizosphere and rhizoplane fungal isolates from grasses[J].Archives of Phytopathology and Plant Protection,2013,46(14):1641-1666.
[9]Vasanthakumari M M,Shivanna M B.Biological control of sorghum anthracnose with rhizosphere and rhizoplane fungal isolates from perennial grasses of the Western Ghats of India[J].European Journal of Plant Pathology,2014,139(4):721-733.
[10]Roberts W K,Selitrennikoff C P.Plant and bacterial chitinases differ in antifungal activity[J].Microbiology,1988,134(1):169-176.
[11]Ghose T K.Measurement of cellulase activities[J].Pure and Applied Chemistry,1987,59:257-268.
[12]王明江,章如意,林多多,等.棉花黄萎病不同抗性品种内生菌数量调查与拮抗菌筛选[J].江苏农业科学,2010,38(2):102-104.
[13]韩立荣.一株土传病害生防菌的筛选及其功能开发[D].杨凌:西北农林科技大学,2010.
[14]赵新贝,上官妮妮,李月飞,等.番茄灰霉病拮抗细菌18BS-12发酵条件的优化及防治效果[J].西北农林科技大学学报(自然科学版),2016,44(6):116-124.
[15]石凤梅.80%腐霉利可湿性粉剂对番茄灰霉病的防效研究[J].黑龙江农业科学,2014,7:68-69.
[16]王芝凤.400 g/L嘧霉胺悬浮剂防治番茄灰霉病田间药效试验报告[J].农业科技与信息,2017(6):73-74.
[17]Ausubel F M,Brent R,Kingston R E,et al.Short protocols in molecular biology[M].New York:John Wiley and Sons,1995,11-13.
[18]权春善,王军华,徐洪涛,等.一株抗真菌解淀粉芽孢杆菌的分离鉴定及其发酵条件的初步研究[J].微生物学报,2006,46(1):7-12.
[19]Caldeira A T,Feio S S,Arteiro J M S,et al.Bacillus amyloliquefaciens CCMI 1051 in vitro activity against wood contaminant fungi[J].Annals of Microbiology,2007,57:29-33.
[20]Imen Z,Lobna J,Slim T,et al.Biocontrol activity of the endophytic Bacillus amyloliquefaciens strain CEIZ-11 against Pythium aphanidermatum and purification of its bioactive compounds[J].Biological Control,2016,100:54-62.
[21]Zhang S M,Wang Y X,Meng L Q,et al.Isolation and characterization of antifungal lipopeptides produced by endophytic Bacillus amyloliquefaciens TF28[J].African Journal of Microbiology Research,2012,6(8):1747-1755.
[22]张淑梅,姜威,孟利强,等.内生细菌TF28对番茄灰霉病的诱导抗性研究[J].安徽农业科学,2014,42(11):3253-3256.
[23]Mari M,Guizzardi M,Brunelli M,et al.Postharvest biological control of grey mould(Botrytis cinerea Pers.:Fr.)on fresh-market tomatoes with Bacillus amyloliquefaciens[J].Crop Protection,1996,15(8):699-705.
[24]杨桥,韩文菊,张文俊,等.产Macrolactin-A抗生素海洋解淀粉芽孢杆菌的鉴定及发酵条件优化[J].药物生物技术,2009,4:311-315,346.
[25]Chao S H,Cheng T H,Shaw C Y,et al.Characterization of a novel Pep F-like oligopeptidase secreted by Bacillus amyloliquefaciens 23-7A[J].Applied and Environmental Microbiology,2006,72(1):968-971.
[26]王英国,王军华,权春善,等.解淀粉芽孢杆菌抗菌活性物质的分离纯化及抑菌活性研究[J].中国生物工程杂志,2007,27(12):41-45.
[27]Arguelles-Arias A,Ongena M,Halimi B,et al.Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens[J].Microbial Cell Factories,2009,8(1):1-12.
[28]Kim P,Chung K C.Production of an antifungal protein for control of Colletotrichum lagenarium by Bacillus amyloliquefaciens MET0908[J].FEMS Microbiology Letters,2004,234(1):177-183.
[29]Koumoutsi A,Chen X H,Henne A,et al.Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42[J].Journal of Bacteriology,2004,186(4):1084-1096.
[30]Arrebola E,Jacobs R,Korsten L.Iturin A is the principal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens[J].Journal of Applied Microbiology,2010,108(2):386-395.
[31]刘俏,刘春善,范圣第.超滤解淀粉芽孢杆菌发酵液提取抗菌物质的研究[J].食品科技,2006,31(7):70-72.
[32]陈敏,郭旭文,李春远,等.解淀粉芽孢杆菌SC1150的抑菌活性及其液体发酵条件的优化[J].生态科学,2015,34(3):7-12.