两种木霉菌对黄瓜枯萎病菌生防作用及根际土壤微生物影响研究
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摘要
黄瓜枯萎病(Fusarium oxysporium f. sp. cucumerinum)是设施蔬菜最常见且危害严重的一种土传真菌病害,它的有效防治已成为蔬菜生产中急待解决的难题。长柄木霉(Trichoderma longbrachiatum)2号菌株和柠檬绿木霉(Trichoderma citrinoviride)4号菌株是由中国农业微生物菌种中心筛选的对多种蔬菜具有促生抗病作用的木霉菌。本论文从这两种木霉菌对黄瓜枯萎病菌的拮抗机制、长柄木霉施用后对黄瓜植株抗病性的诱导作用以及对根际土壤酶活性和土壤微生物区系变化的影响等方面,研究木霉菌对黄瓜枯萎病的生防作用和根际土壤微生物影响。旨在为揭示这两种木霉菌对蔬菜病害的生防作用机制和为长柄木霉的有效利用奠定基础。研究结果如下:
两种木霉菌与黄瓜枯萎病菌的对峙培养、显微观察和抑菌试验结果表明,这两种木霉菌对黄瓜枯萎病菌的拮抗机制中竞争、重寄生作用是重要的作用机制,同时也存在抗生作用。采用还原糖法对这两种木霉菌不同发酵时间几丁质酶和β-1, 3-葡聚糖酶活性进行测定,得出同一菌株在不同发酵时间两种酶活性大小的变化趋势大致相同。在相同发酵时间,木霉菌2号菌株的几丁质酶活性比木霉菌4号菌株的几丁质酶活性低,而木霉菌2号菌株的β-1, 3-葡聚糖酶活性比木霉菌4号菌株的β-1, 3-葡聚糖酶活性高。结合前期研究结果得知,菌株防治黄瓜枯萎病的能力与该菌株发酵产几丁质酶、β-1, 3-葡聚糖酶活性大小不存在简单的对应关系。
对长柄木霉菌施用后黄瓜不同生长时期与诱导抗病性相关的酶活变化进行了检测,结果为:根茎部及叶部苯丙氨酸解氨酶(PAL)活性都明显增强,其中以苗期根茎部和花期叶部PAL活性的增强最为显著,处理比对照的酶活分别高出1.33个酶活单位和1.11个酶活单位。根茎部和叶部超氧化物歧化酶(SOD)活性都明显增强,其中以末期根茎部和果期叶部SOD活性增强最为显著,处理比对照的酶活分别高出10.32个酶活单位和18.81个酶活单位。而苗期过氧化物酶(POD)活性降低、过氧化氢酶(CAT)活性增强。结果表明,长柄木霉菌施用后诱导黄瓜植株产生了抗病性,增强了植物的抗病能力。
对长柄木霉菌施用后黄瓜不同生长时期根际土壤中脲酶、过氧化氢酶、酸性磷酸酶等酶活性的变化进行了检测,结果为:苗期和末期处理的土壤脲酶活性高于对照,花期和果期处理的土壤脲酶活性低于对照。在黄瓜的不同生长时期,处理的土壤过氧化氢酶活性都高于对照;而处理的土壤酸性磷酸酶活性则低于对照,且在花期前后变化最为显著,最大降幅达20个酶活单位。
结合平板计数、DGGE、Biolog等方法对长柄木霉菌施用后黄瓜根际土壤微生物区系进行分析,结果表明:黄瓜根际土中可培养细菌、放线菌及真菌数量均明显减少;非根际土中可培养放线菌及真菌数量减少,而细菌数量增加。黄瓜根际土中细菌群落多样性降低,而放线菌和真菌群落多样性逐渐增加。土壤微生物碳源利用的能力在苗期增强,在其余时期均降低。
Cucumber Fusarium wilt is one of the most common and serious soilborne fungal diseases of vegetables in greenhouse. Its effective control had become the difficult problem to be solved eagerly in vegetable production. The strain No.2 of Trichoderma longbrachiatum and strain No.4 of T. citrinoviride screened by Agricultural Culture Collection of China (ACCC) and have growth-promoting and disease resistance-inducing effects on a variety of vegetable. In this paper, the interaction between the two Trichoderma species and Fusarium oxysporium f. sp. cucumerinum was studied. The enzymes related to inducing disease resistance in cucumber plant were studied at different growth stages of cucumber. At the same time, the effects on soil microbe were studied after Trichoderma longbrachiatum was applied through detecting the rhizosphere soil enzymes activities and analysing rhizosphere soil microbial communities at different growth stages of cucumber. The results will clarify the biocontrol mechanisms of the two Trichoderma species and establish basis for effective utilization of Trichoderma longbrachiatum. The main research results are as follows:
The results from dual culture, microscopic observation and inhibition tests showed that competition and mycoparasitism were the important mechanisms of the two Trichoderma species against pathogenic fungi, and antibiosis was also existed. The chitinase activity andβ-1, 3-glucanase activity of the two Trichoderma species were determined with the method of reducing sugar. The results indicated that the change tendency of the two enzymes activities of the same strain were roughly same at different fermentation time. At the same fermentation time, the chitinase activity of strain No.2 was lower than that of strain No.4, and theβ-1, 3-glucanase activity was reverse. Combining with the early research results, we could learn that there was no simple corresponding relation between the ability to biocontrol fungal diseases and the activities of chitinase andβ-1, 3-glucanase of the strains.
The activities of enzymes related to inducing plant disease resistance were determined at different cucumber growth stages after Trichoderma longbrachiatum was applied. The results were that the phenylalanine ammonia-lyase (PAL) activity significantly increased, and the PAL activity of rootstock at seedling stage and that of leaf at flowering stage increased most distinctly. The enzyme activities of the treatment were 1.33 units and 1.11 units higher than those of CK respectively. The superoxide dismutase (SOD) activity of rootstock and leaf significantly increased, and the SOD activity of rootstock at final stage and that of leaf at fruiting stage increased most distinctly. The enzyme activities of the treatment were 10.32 units and 18.81 units higher than those of CK respectively. The activities of peroxidase decreased and the activities of catalase increased at seedling stage. The results showed that the disease resistance was induced and the ability of disease resistance was enhanced after Trichoderma longbrachiatum was applied.
The activities of urease, catalase and acid phosphatase in rhizosphere soil were determined at different cucumber growth stages after Trichoderma longbrachiatum was applied. The results were that the urease activity of treatment was higher than that of CK at seedling stage and final stage. And the urease activity of treatment was lower than that of CK at flowering stage and fruiting stage. The catalase activity of treatment was higher than that of CK at all growth stages. The acid phosphatase activity of treatment was lower than that of CK at all growth stages. And the acid phosphatase activity changed significantly at flowering stage, the maximum decrease up to 20 enzyme activity units.
The rhizosphere soil microbial communities were analysed by plate count, DGGE and Biolog methods. The results showed that the numbers of bacteria, actinomyces and fungi were all lower than that of CK in rhizosphere soil; In non-rhizosphere soil, the numbers of actinomycetes and fungi were lower than that of CK while the number of bacteria was higher than that of CK; the diversity of bacteria decreased slightly, while that of actinomycetes and fungi increased gradually in the cucumber rhizosphere soil; the ability to utilize carbon sources of soil microorganisms increased at seedling stage and decreased at the other stages after Trichoderma longbrachiatum was applied.
两种木霉菌与黄瓜枯萎病菌的对峙培养、显微观察和抑菌试验结果表明,这两种木霉菌对黄瓜枯萎病菌的拮抗机制中竞争、重寄生作用是重要的作用机制,同时也存在抗生作用。采用还原糖法对这两种木霉菌不同发酵时间几丁质酶和β-1, 3-葡聚糖酶活性进行测定,得出同一菌株在不同发酵时间两种酶活性大小的变化趋势大致相同。在相同发酵时间,木霉菌2号菌株的几丁质酶活性比木霉菌4号菌株的几丁质酶活性低,而木霉菌2号菌株的β-1, 3-葡聚糖酶活性比木霉菌4号菌株的β-1, 3-葡聚糖酶活性高。结合前期研究结果得知,菌株防治黄瓜枯萎病的能力与该菌株发酵产几丁质酶、β-1, 3-葡聚糖酶活性大小不存在简单的对应关系。
对长柄木霉菌施用后黄瓜不同生长时期与诱导抗病性相关的酶活变化进行了检测,结果为:根茎部及叶部苯丙氨酸解氨酶(PAL)活性都明显增强,其中以苗期根茎部和花期叶部PAL活性的增强最为显著,处理比对照的酶活分别高出1.33个酶活单位和1.11个酶活单位。根茎部和叶部超氧化物歧化酶(SOD)活性都明显增强,其中以末期根茎部和果期叶部SOD活性增强最为显著,处理比对照的酶活分别高出10.32个酶活单位和18.81个酶活单位。而苗期过氧化物酶(POD)活性降低、过氧化氢酶(CAT)活性增强。结果表明,长柄木霉菌施用后诱导黄瓜植株产生了抗病性,增强了植物的抗病能力。
对长柄木霉菌施用后黄瓜不同生长时期根际土壤中脲酶、过氧化氢酶、酸性磷酸酶等酶活性的变化进行了检测,结果为:苗期和末期处理的土壤脲酶活性高于对照,花期和果期处理的土壤脲酶活性低于对照。在黄瓜的不同生长时期,处理的土壤过氧化氢酶活性都高于对照;而处理的土壤酸性磷酸酶活性则低于对照,且在花期前后变化最为显著,最大降幅达20个酶活单位。
结合平板计数、DGGE、Biolog等方法对长柄木霉菌施用后黄瓜根际土壤微生物区系进行分析,结果表明:黄瓜根际土中可培养细菌、放线菌及真菌数量均明显减少;非根际土中可培养放线菌及真菌数量减少,而细菌数量增加。黄瓜根际土中细菌群落多样性降低,而放线菌和真菌群落多样性逐渐增加。土壤微生物碳源利用的能力在苗期增强,在其余时期均降低。
Cucumber Fusarium wilt is one of the most common and serious soilborne fungal diseases of vegetables in greenhouse. Its effective control had become the difficult problem to be solved eagerly in vegetable production. The strain No.2 of Trichoderma longbrachiatum and strain No.4 of T. citrinoviride screened by Agricultural Culture Collection of China (ACCC) and have growth-promoting and disease resistance-inducing effects on a variety of vegetable. In this paper, the interaction between the two Trichoderma species and Fusarium oxysporium f. sp. cucumerinum was studied. The enzymes related to inducing disease resistance in cucumber plant were studied at different growth stages of cucumber. At the same time, the effects on soil microbe were studied after Trichoderma longbrachiatum was applied through detecting the rhizosphere soil enzymes activities and analysing rhizosphere soil microbial communities at different growth stages of cucumber. The results will clarify the biocontrol mechanisms of the two Trichoderma species and establish basis for effective utilization of Trichoderma longbrachiatum. The main research results are as follows:
The results from dual culture, microscopic observation and inhibition tests showed that competition and mycoparasitism were the important mechanisms of the two Trichoderma species against pathogenic fungi, and antibiosis was also existed. The chitinase activity andβ-1, 3-glucanase activity of the two Trichoderma species were determined with the method of reducing sugar. The results indicated that the change tendency of the two enzymes activities of the same strain were roughly same at different fermentation time. At the same fermentation time, the chitinase activity of strain No.2 was lower than that of strain No.4, and theβ-1, 3-glucanase activity was reverse. Combining with the early research results, we could learn that there was no simple corresponding relation between the ability to biocontrol fungal diseases and the activities of chitinase andβ-1, 3-glucanase of the strains.
The activities of enzymes related to inducing plant disease resistance were determined at different cucumber growth stages after Trichoderma longbrachiatum was applied. The results were that the phenylalanine ammonia-lyase (PAL) activity significantly increased, and the PAL activity of rootstock at seedling stage and that of leaf at flowering stage increased most distinctly. The enzyme activities of the treatment were 1.33 units and 1.11 units higher than those of CK respectively. The superoxide dismutase (SOD) activity of rootstock and leaf significantly increased, and the SOD activity of rootstock at final stage and that of leaf at fruiting stage increased most distinctly. The enzyme activities of the treatment were 10.32 units and 18.81 units higher than those of CK respectively. The activities of peroxidase decreased and the activities of catalase increased at seedling stage. The results showed that the disease resistance was induced and the ability of disease resistance was enhanced after Trichoderma longbrachiatum was applied.
The activities of urease, catalase and acid phosphatase in rhizosphere soil were determined at different cucumber growth stages after Trichoderma longbrachiatum was applied. The results were that the urease activity of treatment was higher than that of CK at seedling stage and final stage. And the urease activity of treatment was lower than that of CK at flowering stage and fruiting stage. The catalase activity of treatment was higher than that of CK at all growth stages. The acid phosphatase activity of treatment was lower than that of CK at all growth stages. And the acid phosphatase activity changed significantly at flowering stage, the maximum decrease up to 20 enzyme activity units.
The rhizosphere soil microbial communities were analysed by plate count, DGGE and Biolog methods. The results showed that the numbers of bacteria, actinomyces and fungi were all lower than that of CK in rhizosphere soil; In non-rhizosphere soil, the numbers of actinomycetes and fungi were lower than that of CK while the number of bacteria was higher than that of CK; the diversity of bacteria decreased slightly, while that of actinomycetes and fungi increased gradually in the cucumber rhizosphere soil; the ability to utilize carbon sources of soil microorganisms increased at seedling stage and decreased at the other stages after Trichoderma longbrachiatum was applied.
引文
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