半夏立枯病菌拮抗内生细菌的分离鉴定及功能分析

Isolation,identification and functional study of antagonistic endophytic bacteria against Pinellia ternata damping off (Rhizoctonia solani)

  • 摘要: 【目的】从药用植物半夏分离对半夏立枯病病原菌具有拮抗活性的内生细菌并初步探究其抑菌机制,为半夏立枯病的生物防治提供理论和实践依据。【方法】对湖北省潜江市南章莆半夏大棚种植基地的半夏主要病害进行调查和分离,通过柯赫氏法则进行致病性验证,筛选出致病性最强的菌株并进行种类鉴定。采用组织分离法对健康无病半夏进行内生细菌分离及鉴定,通过平板对峙试验筛选出对半夏立枯病病原菌拮抗活性最强的菌株并检测其抑菌谱,测定拮抗菌株无菌发酵液和挥发性物质的抑菌活性,检测拮抗菌株耐酸碱能力、胞外酶活性以及生物被膜的形成能力;通过平板试验检测温度和蛋白酶K对拮抗菌株无菌发酵液抑菌活性的影响;通过乙酸乙酯萃取法提取拮抗菌株的脂肽类活性物质并通过液相色谱—质谱联用仪(LC-MS)检测活性物质的主要成分。【结果】南章莆大棚半夏的主要病害是茎腐病、根腐病、叶斑病和病毒病,发病率分别为20.87%、7.03%、3.57%和4.08%,发病率最高的茎腐病主要是立枯丝核菌引起的半夏立枯病。从半夏种球组织中分离到5株细菌对半夏立枯病病原菌具有拮抗活性,其中菌株B2-7的抑菌效果最好,抑制率为79.60%,经鉴定为贝莱斯芽孢杆菌(Bacillus velezensis)。抑菌谱测定结果显示,菌株B2-7对12种植物病原真菌均具有拮抗活性,其抑菌率为61.11%~89.47%。菌株B2-7的挥发性物质和无菌发酵液对立枯丝核菌均具有拮抗作用,挥发性物质的抑菌率为41.18%,当无菌发酵液浓度为2.5%、5.0%、10.0%和20.0%时,其抑制率分别为39.40%、56.06%、73.89%和77.09%。菌株B2-7具有一定的耐酸性,但耐碱性较弱。菌株B2-7能产纤维素酶、淀粉酶、蛋白酶和分泌嗜铁素,形成生物被膜的能力较强,且随着菌液浓度的增加呈先增强后下降的变化趋势。菌株B2-7无菌发酵液对温度和蛋白酶K敏感,75 ℃高温处理拮抗活性下降至23.45%,蛋白酶K处理拮抗活性下降48.60%。菌株B2-7的脂肽类活性物质能抑制立枯丝核菌生长,抑菌带宽2.06 mm;LC-MS分析结果显示该菌株脂肽类活性物质中至少含有16种已知的有机化合物,其中包含surfactin、bacillaene、macrolactin和difficidin等多种已报道的拮抗物质。【结论】半夏内生细菌菌株B2-7对半夏立枯病病原菌具有较好的抑制作用,是防治半夏立枯病的潜力菌株,具有良好的应用前景。

     

    Abstract: 【Objective】An endophytic bacterium with antagonistic activity against Pinellia ternata damping off(Rhizoctonia solani)was isolated from the medicinal plant P. ternata,in order to provide theoretical and practical basis for biological control of P. ternata damping off.【Method】The main diseases of P. ternata in the greenhouse of Nanzhangpu in Qianjiangof Hubei were investigated and isolated. The pathogenicity test was conducted by Koch's rule to screen out the most pathogenic pathogens and identify their categories. Endophytic bacteria were isolated from healthy and disease-free P. ternata by tissue separation. The strain with the strongest antagonistic activity against R. solani was screened by plate confrontation,and the antifungal spectrum of the strain was determined by this method. The inhibitory activity against R. solani of aseptic fermentation broth and volatile substances from the strain were measured. The acid and alkali resistance, extracellular enzyme and the biofilm formation activity of the strain were detected. The effect of protease K and different temperature on the activity of the sterile fermentation fluid of this strain against R. solani was determined by plate test. The lipopeptide active substances were extracted by ethyl acetate extraction method and the main components of active substances were detected by liquid chromatography-mass spectrometry(LC-MS).【Result】The main diseases of P. ternata in the greenhouse of Nanzhangpu were tuber rot,root rot,leaf spot and viral disease,with the incidence of 20.87%, 7.03%,3.57% and 4.08%. The highest incidence of tuber rot was caused by R. solani. Five strains of bacteria isolated from P. ternata seed ball tissue had inhibitory activity to against P. ternata damping off pathogenic fungi. The strain named B2-7 had the highest inhibitory rate with 79.60%,which was identified as Bacillus velesiensis. The results of antifungal spectrum showed that the strain B2-7 had antagonistic activity against 12 different plant pathogenic fungi,and its inhibitory rate ranged from 61.11% to 89.47%. The volatile substances of strain B2-7 and the sterile fermentation solution had antagonistic effect on R. solani. The inhibitory activity against R. solani of volatile substances was 41.18%. When the concentrations of sterile fermentation fluid from B2-7 were 2.5%,5.0%,10.0% and 20.0%,the inhibitory rates for R. solani were 39.40%,56.06%,73.89% and 77.09%. Strain B2-7 had strong acid resistance,but alkaline resistance was weak. The strain B2-7 could produce cellulase,amylase,protease and secrete siderophores. The ability to form biofilm was strong,which increased first and then decreased with the increase of the concentration of bacterial solution. The sterile fermentation solution from B2-7 was sensitive to temperature and protease K,and the inhibitory activity decreased to 23.45% at 75℃ and decreased by 48.60% treated with protease K. The lipopeptide active substances of strain B2-7 could inhibit the growth of R. solani with an inhibition bandwidth of 2.06 mm. The results of LC-MS analysis showed that there were at least 16 known organic compounds in lipopeptide active substances,including surfactin,bacillaene,microlactin, difficidin and other antagonistic substances reported.【Conclusion】Endophytic bacterium B2-7 of P. ternata has good inhibitory activity against P. ternate damping off pathogenic fungi. It is a potential strain to control P. ternata damping off and has a good application prospect

     

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