生防菌复配对烟草黑胫病的防治效果研究

2020-05-25 02:47李苗苗王晓强王东坤刘元德孙光军沈宏艾永峰崔志燕陈德鑫王凤龙
中国烟草科学 2020年2期
关键词:发酵

李苗苗 王晓强 王东坤 刘元德 孙光军 沈宏 艾永峰 崔志燕 陈德鑫 王凤龙

摘  要:为解决单一生防菌防效不稳定问题,通过平板对峙试验、相容试验、盆栽试验从6株生防菌中筛选对烟草疫霉菌(Phytophthora parasitica var. nicotianae)具有较高拮抗活性的复配菌株组合;利用分子生物学方法对各菌株的16S rRNA和gyrA基因进行系统进化分析,鉴定各菌株分类地位;并采用轮换因子法筛选各菌株的最适发酵培养基。结果表明,GY1、GY10和GY12发酵后混合施用对烟草黑胫病的防治效果最好,平板抑菌率达到86.9%,盆栽防效为74.53%,比GY1、GY10、GY12单独处理分别提高15.34%、27.42%和44.94%;分子鉴定表明,3株菌分别为贝莱斯芽孢杆菌(Bacillus velezensis)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)和枯草芽孢杆菌(Bacillus subtilis);并筛选得到了GY1、GY10、GY12的最适发酵培养基,发酵24 h后生物量相较于基础培养基分别增加了75.12%,92.31%和194.55%。

关键词:烟草黑胫病;芽孢杆菌;生防菌复配;发酵

Effect of Biocontrol Agents Mixture on Control of Tobacco Black Shank

LI Miaomiao1, WANG Xiaoqiang1, WANG Dongkun1, LIU Yuande2, SUN Guangjun3, SHEN Hong3,

AI Yongfeng3, CUI Zhiyan4, CHEN Dexin1,5*, WANG Fenglong1*

(1. Tobacco Research Institute of Chinese Academy of Agricultural Sciences, Qingdao 266000, China; 2. Linyi Tobacco Company of Shandong Province, Linyi, Shandong 276002, China; 3. Guizhou Tobacco Company, Guiyang 550004, China; 4. Shangluo Tobacco Company of Shaanxi Province, Shangluo, Shaanxi 726000, China; 5. Hainan Tobacco Company, Haikou 571199, China)

Abstract: In order to address the problem of unstable antibacterial activity of single biocontrol bacteria, a composite strains with high antagonistic activity against Phytophthora parasitica var. nicotianae was screened from six biocontrol bacteria by compatible test, antagonist test and pot test. The taxonomic status of each strain was identified with a phylogenetic analysis of the 16S rRNA and gyrA genes of the strains, and then the optimal fermentation medium for each strain was screened with a single factor test. The co-application of GY1, GY10 and GY12 after fermentation showed the best control effect on tobacco black shank, with the plate inhibition rate reached 86.9% and he potted control effect reached 84.21%, which increased by 17.58%, 26.74% and 35.9% compared with single strains of GY1, GY10 and GY12, respectively. Molecular identification showed that the three strains were Bacillus velezensis, Bacillus amyloliquefaciens and Bacillus subtilis, respectively. The optimal fermentation mediums of GY1, GY10, and GY12 were screened, where the biomass of GY1, GY10 and GY12 increased by 75.12%, 92.31%, and 194.55%, respectively, compared with the basic medium after 24 hours of fermentation.

Keywords: tobacco black shank; Bacillus; biocontrol agents mixture; fermentation

煙草黑胫病是由烟草疫霉菌(Phytophthora parasitica var. nicotianae)引起的根茎类病害[1],在我国各大烟区都有发生,每年造成的损失达数亿元[2],是制约我国烟草优质安全生产的重要因素[3]。烟草黑胫病的防治措施包括农业防治、抗病品种[4]、化学防治[5]和生物防治[6]等,但抗病品种培育难,地域性强[7];化学农药长期大量使用易造成农药残留、环境污染和抗药性[8]等问题。随着绿色防控理念的提出,环境友好的高效拮抗菌成为防治烟草黑胫病的一项重要措施。

2.1.4  盆栽防效试验  GY1、GY10、GY12的盆栽防效(表3)分别为64.62%、58.49%和51.42%;而GY5、GY8、GY9防效都在40%以下,与平板对峙结果一致。处理GY1-GY10-GY12发酵后混合的病情指数为20.00,盆栽防效为74.53%,相较于GY1、GY10、GY12单独处理分别提高15.34%、27.42%和44.94%。病情指数及发病率显著低于对照的78.52%和93.33%。综合平板对峙试验及盆栽试验结果选择GY1-GY10-GY12发酵后混合的复配方式进行后续试验。

2.2  菌株鉴定

利用16S rRNA基因构建的系统发育树显示,GY1与贝莱斯芽孢杆菌NRRL B-41580聚为一簇,序列同源性为89%;GY10与解淀粉芽孢杆菌NBRC 15535聚为一簇,序列同源性为91%;GY12与枯草芽孢杆菌DSM 10及JCM 1465聚为一簇,序列同源性为99%(图2 A、B、C)。同时gyrA基因系

统发育分析进一步表明,GY1与贝莱斯芽孢杆菌R1B序列同源性为100%;GY10与解淀粉芽孢杆菌TEB-31等序列同源性为100%;GY12与枯草芽孢杆菌NRRL BD-559等序列同源性为98%(图2 D)。因此认为GY1、GY10、GY12分别为贝莱斯芽孢杆菌、解淀粉芽孢杆菌及枯草芽孢杆菌。

2.3  生长曲线

GY1、GY12在20 h时到达平台期,OD600分别达到1.9和2.3,GY10在30 h时到达平台期,OD600为1.7(图3)。而GY1在28 h时OD600下降,推测可能与培养基中养分消耗过度,菌体沉淀等有关。

2.4  菌株最适生长培养基

GY1在以玉米粉为碳源时生物量最高,菌体量达8.37×108 cfu/mL,与其他处理差异显著(p≤0.05);GY10在以玉米粉做为碳源时生物量最高,菌体量达3.8×108 cfu/mL;GY12同样在以玉米粉做为碳源时生物量最高,菌体量达4.27×108 cfu/mL,与其他处理均差异显著(p≤0.05)(图4)。综合考虑发酵效率及成本后将玉米粉作为GY1、GY10、GY12的最佳生长碳源。

GY1在以酵母粉做为氮源时生物量最高,菌体量达6.6×108 cfu/mL,与其他处理差异显著(p≤0.05);GY10在以蛋白胨做为氮源时菌体量最高,

菌体量达5.8×108 cfu/mL;GY12在以酵母粉做为氮源时生物量最高,菌体量达6.4×108 cfu/mL(图5)。综合考虑发酵效率及成本后将酵母粉、蛋白胨、酵母粉分别作为GY1、GY10、GY12的最佳生长氮源。

GY1在KH2PO4做为无机盐时生物量最高,菌体量达7.53×108 cfu/mL;GY10在以CaCO3做为无机盐时生物量最高,菌体量达7.5×108 cfu/mL;GY12在以CaCO3做无机盐时生物量最高,菌体量达8.1×108 cfu/mL(图6)。综合考虑发酵效率及成本后将KH2PO4、CaCO3、CaCO3做为GY1、GY10、GY12的最佳生长无机盐。

因此确定3菌株的最适发酵培养基分别是GY1:玉米粉、酵母粉、KH2PO4;GY10:玉米粉、蛋白胨、CaCO3;GY12:玉米粉、酵母粉、CaCO3。

3  讨  论

芽孢杆菌在自然界分布广泛,由于其可以产生芽孢,具有较强的抗逆性,且很多菌株具有促进植物生长、防治植物病害的功能,因此在农业生产中

被广泛应用。芽孢杆菌促进植物生长主要是通过生物固氮、促进植物对营养元素的吸收、合成分泌植物生长调节物质、释放挥发性物质等方式进行[20-21]。除了对植物生长的促进作用,芽孢杆菌还被广泛用于植物病害的生物防治,其中枯草芽孢杆菌是生产上应用最为广泛的一类芽孢杆菌[22],此外解淀粉芽孢杆菌[23]、多粘类芽孢杆菌[24]、短小芽孢杆菌[25]、贝莱斯芽孢杆菌[26]等也被广泛应用。

芽孢杆菌在自然环境中具有抗逆性强、种群丰富、抗菌效果显著等明显优势[27],在植物病害的防治方面具有非常好的前景,但是目前对芽孢杆菌的研究主要集中在单个菌株,对多菌株共同开展的研究较少,在一定程度上限制了该类菌株的应用。尤其是在自然环境条件下单个菌株往往存在防效不稳定、防治靶标过于单一等问题,而多菌株复配可以有效的解决单一菌株存在的问题。张良等[28]发现两株生防菌复配对烟草黑胫病的防效达69.3%,明显高于单一菌株。喻会平等[29]将枯草芽孢杆菌B41和恶臭假单胞菌(Pseudomonas putida)B57复配,对烟草黑胫病的盆栽试验防效为75.72%,大田试验防效为67.99%,抑菌效果明显高于单一菌株。本研究采用平板对峙法测定6株生防菌对烟草黑胫病的拮抗作用,并将防效好的生防菌株复配,发现贝莱斯芽孢杆菌GY1、解淀粉芽孢杆菌GY10、枯草芽孢杆菌GY12对烟草疫霉菌的平板抑制率均达84%以上,且3菌株相容性好,无拮抗作用,适合复配。本研究中,复配菌GY1-GY10-GY12发酵后混合施用对烟草疫霉菌的平板抑菌率达到86.9%,比GY1、GY10、GY12单独处理略有提高(分别提高2.3%、2.7%和1%),但是盆栽防效显著高于单独处理,复配菌株的防效达到74.53%,比GY1、GY10、GY12单独处理分别提高15.34%、27.42%和44.94%。GY1-GY10-GY12发酵后混合对烟草疫霉菌的平板抑制效果影響不大,但明显提高了盆栽防效,原因可能与单一菌株土壤定殖能力差,三者共同施用增加了各菌株在烟草根际的定殖量和根际土壤微生物多样性有关;另外复配可以将三菌株优势互补,从而提高防效。

微生物发酵过程中菌体的生物量及抗菌产物的分泌受培养基成分影响较大[30],筛选菌株最适生长培养基对提高发酵效率有重要意义。张红艳等[31]优化地衣芽孢杆菌(Bacillus licheniformis)最佳发酵培养基组分,结果表明最佳配比的发酵培养基可提高发酵液的菌体浓度和芽孢率。张志焱等[32]通过单因子试验筛选出最适培养基,优化后抗菌肽的效价提高到了4.25×104 U/mL,是优化前的3.51倍。本试验首先通过绘制生长曲线得到了GY1、GY10、GY12的增殖趋势,再通过轮换因子法筛选3菌株的最适生长培养基,与直接筛选培养基相比提高了效率。采用筛选后的培养基发酵相较于基础培养基生物量(4.3×108、3.9×108、2.75×108 cfu/mL)分别提高75.12%,92.31%和194.55%。结果表明不同培养基成分对3种芽孢杆菌的生物量具有很大影响,筛选最适发酵培养基有助于提高发酵效率;同时本试验以玉米粉作为碳源,有效降低了发酵成本,该结果可以为菌株的规模化发酵提供借鉴。

4  结  论

结果表明,GY1-GY10-GY12发酵后混合是防治烟草黑胫病的高效复配组合,对烟草疫霉菌的平板抑菌率为86.9%;盆栽防效為74.53%;GY1、GY10、GY12分别为贝莱斯芽孢杆菌(B. velezensis)、解淀粉芽孢杆菌(B. amyloliquefaciens)和枯草芽孢杆菌(B. subtilis)。三菌株的最适生长培养基分别为GY1:玉米粉、酵母粉、KH2PO4;GY10:玉米粉、蛋白胨、CaCO3;GY12:玉米粉、酵母粉、CaCO3。

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基金項目:中华人民共和国科学技术部项目“烟草化学农药减施增效途径及技术研究”(2018YFD0201104-02);山东省自然科学基金“吡咯伯克霍尔德氏菌Lyc2抗细菌相关基因的鉴定与功能分析”(ZR2018BC037);中国烟草总公司贵州省公司科技项目“贵州烟草叶斑类病害成灾规律与绿色防控技术研究示范”(201920)

作者简介:李苗苗(1993-),女,在读硕士,主要从事植物病害防治研究。E-mail:1305047786@qq.com

*通信作者,E-mail:chendxtob@126.com;wangfenglong@caas.cn

收稿日期:2019-08-29                   修回日期:2019-12-23

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