| 可可毛色二孢拮抗菌的鉴定及发酵条件优化 |
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| 引用本文： | 刘双龙,杨德洁,牛晓庆,杨福孙,覃伟权.可可毛色二孢拮抗菌的鉴定及发酵条件优化[J].南方农业学报,2022,53(8):2186-2195. |
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| 作者姓名： | 刘双龙 杨德洁 牛晓庆 杨福孙 覃伟权 |
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| 作者单位： | 1. 中国热带农业科学院椰子研究所/海南省院士创新平台/海南省槟榔产业工程研究中心, 海南文昌 571339;2. 海南大学热带作物学院, 海南海口 570228 |
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| 基金项目： | 海南省院士创新平台资金项目（琼科〔2019〕 251号） |
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| 摘 要： | 【目的】筛选对可可毛色二孢菌（Lasiodiplodia theobromae）具有拮抗作用的生防菌株，为由可可毛色二孢菌引起的槟榔根腐病的生物防治提供菌种资源及指导。【方法】以海南发病槟榔园健康槟榔根组织及根际土壤为试验材料，采用组织匀浆法和平板稀释梯度培养法分别分离槟榔根内生细菌及根际土壤中的细菌，应用平板对峙法筛选出对可可毛色二孢菌具有拮抗作用的菌株；通过平板对峙法测定拮抗菌株菌液对3种槟榔根部病害病原真菌尖孢镰刀菌 （Fusarium oxysporum）、奇异根串珠霉菌 （Thielaviopsis paradoxa）和可可毛色二孢菌的抑制作用；通过形态学观察、生理生化测定和16S rDNA序列分析对拮抗菌株进行鉴定；采用盆栽试验初步验证拮抗菌株对由可可毛色二孢菌引起的槟榔根腐病的防治效果；采用单因素变化试验等筛选拮抗菌株的最佳发酵条件。【结果】经平板对峙法筛选出5株拮抗作用较强的菌株，其中菌株wrj-2-5的抑菌率最高，为74.07%；菌株wrj-2-5菌液对可可毛色二孢菌、尖刀镰刀菌和奇异根串珠霉菌菌丝生长均有较强的抑制作用，抑制率均在70.00%以上。综合菌株形态学观察、生理生化测定和16S rDNA分析结果，将菌株wrj-2-5鉴定为铜绿假单胞菌（Pseudomonas aeruginosa）。室内盆栽试验结果表明，菌株wrj-2-5对可可毛色二孢菌引起的槟榔根腐病具有较好的防病作用，对照组发病率为60.00%，试验组长势正常未发病。拮抗菌wrj-2-5最佳培养基配方为酵母浸粉1.5 g、葡萄糖1.5 g、 MgSO4·7H2O 0.2 g、水100 mL；最佳发酵条件为转速240 r/min、培养温度28 ℃、培养时间36 h、初始培养基pH 6、初始接种量8%。【结论】菌株wrj-2-5为铜绿假单胞菌，其对可可毛色二孢菌、尖孢镰刀菌和奇异根串珠霉菌均有较好的抑制作用，对由可可毛色二孢菌引起的槟榔根腐病有较好的防治效果，可作为研制槟榔根腐病生防菌剂的候选菌株资源。
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| 关 键 词： | 可可毛色二孢菌 拮抗细菌 鉴定 生物防治 |
| 收稿时间： | 2021-11-03 |
Identification and fermentation condition optimization of antagonistic bacteria against Lasiodiplodia theobromae |
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| LIU Shuang-long,YANG De-jie,NIU Xiao-qing,YANG Fu-sun,QIN Wei-quan.Identification and fermentation condition optimization of antagonistic bacteria against Lasiodiplodia theobromae[J].Journal of Southern Agriculture,2022,53(8):2186-2195. |
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| Authors: | LIU Shuang-long YANG De-jie NIU Xiao-qing YANG Fu-sun QIN Wei-quan |
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| Affiliation: | 1. Coconut Research Institute of Chinese Academy of Tropical Agricultural Sciences/Innovation Platform for Academicians of Hainan Province/Hainan Engineering Research Center of Arecanut Industry, Wenchang, Hainan 571339, China;2. College of Tropical Crops, Hainan University, Haikou, Hainan 570228, China |
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| Abstract: | 【Objective】To screen antagonistic biocontrol strains against Lasiodiplodia theobromae，so as to provide bacterial resources and guidance for areca root rot caused by L.theobromae.【Method】 Healthy areca root and rhizosphere soil in the areca orchard where root rot disease was detected in Hainan were taken as test materials.Tissue homogenization method and plate dilution gradient culture method were used to isolate root endophytic bacteria and rhizosphere soil bacteria，and flat-stand method was used to screen antagonistic bacterial strains.Inhibitory effect of the antagonistic strains on 3 kinds of pathogenic fungi of areca root diseases，Fusarium oxysporum，Thielaviopsis paradoxa and Lasiodiplodia theobromae，was determined through the flat-stand method.The strains were identified through morphological observation，physiological and biochemical determination and 16S rDNA sequence analysis.The pot experiment was conducted to verify the efficacy of the antagonistic strains against by areca root rot caused by L.theobromae.Single factor change experiment was used to screen the best fermentation conditions for antagonistic strains.【Result】 Five strains with strong antagonistic effect were screened through flat-stand method，in which strain wrj-2-5 had the highest average inhibitory rate of 74.07%.Strain wrj-2-5 had a strong inhibitory effect on the mycelial growth of F.oxysporum，T.paradoxa and L.theobromae，and the inhibition rate was above 70.00%.Taking results of morphological observation，physiological and biochemical determination and 16S rDNA analysis into account，strain wrj-2-5 was identified as Pseudomonas aeruginosa.The indoor pot test showed that the strain wrj-2-5 had a good preventive effect on areca root rot caused by L.theobromae.Disease incidence rate of the control group was 60.00%，and in the experimental group，plants grew well and no disease was detected.It was found that the optimized medium formula of antagonistic strain wrj-2-5 was as follows:1.5 g yeast extract，1.5 g glucose，0.2 g MgSO4·7H2O and 100 mL water；the optimized fermentation condition was as follows: rotating speed 240 r/min，temperature 28℃，culture time 36 h，initial pH 6 and inoculation amount of 8%.【Conclusion】 Strain wrj-2-5 is P.aeruginosa，which has a good inhibitory effect on the F.oxysporum，T.paradoxa and L.theobromae，and it has a good control effect on areca root rot caused by L.theobromae，which can be taken as resources for making areca root rot disease biocontrol bacterial agent. |
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