Existing position and extraction of antifungal substance produced by Chaetomium subaffine LB-1
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摘要:目的
对近缘毛壳Chaetomium subaffine菌株LB-1产生抑菌物质的部位及提取方法进行研究，为开发生防菌株LB-1防治植物病害奠定基础。
方法以番茄灰霉病菌Botrytis cinerea和玉米大斑病菌Exserohilum turcicum为指示菌，采用平板密封培养法检测菌株LB-1是否产生挥发性抑菌物质；采用菌体超声破碎和液态培养的方式检测菌株LB-1产生的非挥发性抑菌物质存在的部位；采用含毒培养基法和滤纸片法检测菌株LB-1培养液的硫酸铵沉淀、盐酸沉淀和有机溶剂萃取物的抑菌效果，以确定菌株LB-1培养液中抑菌物质的提取方法。
结果菌株LB-1与2种供试病原菌密封共培养时，对病原菌的生长没有明显影响，表明菌株LB-1不能产生挥发性抑菌物质。菌株LB-1菌体胞内提取物的抑菌活性与对照无差异，但其培养液对B. cinerea和E. turcicum的生长有较强的抑制作用，表明菌株LB-1产生的抑菌物质存在于菌体细胞外。菌株LB-1培养液的硫酸铵沉淀和盐酸沉淀对2种供试植物病原真菌的生长均无抑制效果，但有机溶剂萃取可获得菌株LB-1培养液中的抑菌物质，其中正丁醇萃取物抑菌效果最好，当其质量浓度为0.1 mg/mL时，对B. cinerea和E. turcicum的生长抑制率分别高达59.80%和58.37%。
结论菌株LB-1通过产生胞外非挥发性物质抑制植物病原真菌的生长，培养液中的抑菌物质可通过正丁醇萃取获得。
Abstract:ObjectiveThe existing position and extraction method of antifungal substance produced by Chaetomium subaffine strain LB-1 were studied in order to lay a foundation for the development of the strain LB-1 to control plant diseases.
MethodWith Botrytis cinerea and Exserohilum turcicum as test plant pathogens, the sealed plate assay was used to detect whether the strain LB-1 could produce volatile antifungal substance. The hyphae ultrasonic breaking and liquid culture methods were used to detect the existing position of nonvolatile antifungal substance produced by the strain LB-1. The extraction method of antifungal substance was determined by detecting the inhibitory effects of ammonium sulfate precipitation, hydrochloric acid precipitation and organic solvents extracts of strain LB-1 culture broth via poison plate assay and filter paper disc assay.
ResultThe strain LB-1 did not have obvious inhibitory effect on the growth of the two test plant pathogens when being co-cultured in a sealed plate with each plant pathogen, indicating that strain LB-1 could not produce volatile antifungal substance. The antifungal activity of the intracellular extract of the strain LB-1 was not different from that of the control, but its culture broth had a strong inhibitory effect on B. cinerea and E. turcicum, indicating that the antifungal substances produced by the strain LB-1 existed outside the mycelium. Neither ammonium sulfate precipitate nor hydrochloric acid precipitate of strain LB-1 culture broth showed inhibitory effect on B. cinerea and E. turcicum, but the organic solvent extract of strain LB-1 culture broth showed antifungal effect, and the inhibition rate of n-butanol extract was the highest. When the concentration was 0.1 mg/mL, the inhibition rates against B. cinerea and E. turcicum growth were 59.80% and 58.37% respectively.
ConclusionThe strain LB-1 inhibited the growth of plant pathogenic fungi by producing extracellular nonvolatile antifungal substances, and the antifungal substance in the culture broth can be extracted by n-butanol.
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图 1 滤纸片法检测的菌株 LB-1 培养液不同提取物对病原菌生长的抑制效果
每一培养皿中，左侧滤纸片为 DMSO 或缓冲液处理(CK)，右侧为有机溶剂萃取物或沉淀物处理
Figure 1. Antifungal effects of different extracts from culture broth of strain LB-1 on the growth of plant pathogens by filter paper disc assay
In each petri dish, the filter paper on the left side was treated with DMSO or buffer solution (CK), the right side was treated with organic extract or precipitation
表 1 菌株LB-1菌体胞内提取物质对病原菌生长的抑制效果
Table 1 Inhibitory effects of intracellular extract of strain LB-1 on the growth of plant pathogens
处理 Treatment 番茄灰霉病菌 Botrytis cinerea 玉米大斑病菌 Exserohilum turcicum 菌落直径/mm Diameter of colony 抑制率1)/% Inhibition rate 菌落直径/mm Diameter of colony 抑制率1)/% Inhibition rate 菌体胞内液 Intracellular extract 59.17±1.44 2.25a 61.17±1.89 1.47a 8.5 g/L NaCl 59.33±1.26 2.00a 60.83±1.26 1.96a 空白对照 Blank control 60.67±1.53 62.17±1.26 1) 同列数据后的不同小写字母表示差异显著(P < 0.05，LSD法) 1) Different lowercase letters in the same column indicate significant difference (P < 0.05，LSD method) 
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表 2 菌株LB-1不同培养天数的培养液对病原菌生长的抑制效果
Table 2 Inhibitory effects of culture broth of strain LB-1 on the growth of plant pathogens
培养时间/d Incubation time 番茄灰霉病菌 Botrytis cinerea 玉米大斑病菌 Exserohilum turcicum 菌落直径/mm Diameter of colony 抑制率1)/% Inhibition rate 菌落直径/mm Diameter of colony 抑制率1)/% Inhibition rate 3 54.00±0.10 10.00c 46.92±0.04 22.37c 7 48.17±0.06 18.75b 46.33±0.20 23.23c 10 45.37±0.16 22.95b 42.50±0.36 28.85b 15 35.33±0.15 38.00a 35.67±0.03 38.88a 20 34.67±0.08 39.00a 35.17±0.10 39.61a 空白对照 Blank control 60.67±1.53 62.17±0.13 1) 同列数据中的不同小写字母表示差异显著(P<0.05，LSD法) 1) Different lowercase letters in the same column indicate significant difference (P<0.05，LSD method)
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表 3 菌株LB-1培养液的硫酸铵沉淀物和盐酸沉淀物对病原菌生长的抑制效果
Table 3 Inhibitory effects of ammonium sulfate precipitation and hydrochloric acid precipitation of culture broth of strain LB-1 on the growth of plant pathogens
处理 Treatment 番茄灰霉病菌 Botrytis cinerea 玉米大斑病菌 Exserohilum turcicum 菌落直径/mm Diameter of colony 抑制率1)/% Inhibition rate 菌落直径/mm Diameter of colony 抑制率1)/% Inhibition rate 硫酸铵沉淀 Ammonium sulfate precipitate 60.33±1.53 1.24a 62.17±2.02 2.15a PBS缓冲液 PBS buffer solution 59.67±1.61 2.23a 62.83±1.15 1.20a 盐酸沉淀 Hydrochloric acid precipitate 60.50±1.50 0.99a 63.17±1.04 0.72a 磷酸缓冲液 Phosphate buffer solution 60.67±1.61 0.74a 62.83±1.15 1.20a 空白对照 Blank control 61.17±1.26 63.67±1.53 1)同列数据后的不同小写字母表示差异显著(P < 0.05，LSD法) 1)Different lowercase letters in the same column indicate significant difference (P < 0.05，LSD method)
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表 4 菌株LB-1培养液的有机溶剂萃取物及水相对病原菌生长的抑制效果
Table 4 Inhibitory effects of organic extracts and its aqueous phase of culture broth of strain LB-1 on the growth of plant pathogens
处理 Treatment 番茄灰霉病菌 Botrytis cinerea 玉米大斑病菌 Exserohilum turcicum 菌落直径/mm Diameter of colony 抑制率1)/% Inhibition rate 菌落直径/mm Diameter of colony 抑制率1)/% Inhibition rate 正丁醇粗提物 Crude extract of n-butanol 21.00±1.32 59.80a 23.00±1.80 58.37a 乙酸乙酯粗提物 Crude extract of ethyl acetate 47.67±1.26 20.10b 50.00±1.32 19.62b 石油醚粗提物 Crude extract of petroleum ether 51.17±1.89 14.89c 54.67±0.38 12.92c 三氯甲烷粗提物 Crude extract of chloroform 49.17±2.02 17.87bc 52.67±1.26 15.79c 正丁醇水相 Aqueous phase of n-butanol 60.00±1.00 1.74d 63.17±1.76 0.72d 乙酸乙酯水相 Aqueous phase of ethyl acetate 60.33±1.89 1.24d 63.00±1.80 0.96d 石油醚水相 Aqueous phase of petroleum ether 59.67±0.76 2.23d 62.83±2.02 1.20d 三氯甲烷水相 Aqueous phase of chloroform 59.17±2.25 2.98d 62.36±1.30a 1.88d 二甲基亚枫 Dimethylsulfoxide 59.83±1.44 1.99d 62.41±1.36 1.81d 空白对照 Blank control 61.17±1.26 63.67±1.53 1)同列数据后的不同小写字母表示差异显著(P < 0.05，LSD法) 1) Different lowercase letters in the same column indicate significant difference (P < 0.05，LSD method)
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