福州温室黄瓜枯萎病微生态研究
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摘要
本文以福州市超大现代农业集团黄山农场为试验地,调查了温室黄瓜的生态环境、病害种类及发病规律,较为系统地研究了温室黄瓜枯萎病的微生态环境,并初步探讨了温室黄瓜枯萎病的微生态控制技术。
1温室生态环境因子与黄瓜枯萎病关系分析
在温湿方面,2月温度达10℃左右,2月下旬及3月上旬,昼夜相对湿度最大值均可达90%以上,晚上甚至达到100%,这种低温高湿条件导致了温室黄瓜枯萎病早发。在土壤方面,温室黄瓜常采用砂质土壤栽培,枯萎病随土壤砂性增加而加剧。在病原菌方面,伴随温室连作栽培,尖孢镰刀菌逐年累积,同时逐年加重的温室根结线虫侵染为害形成的根部伤口易于枯萎病菌的侵染,导致黄瓜枯萎病常发和重发。
2温室黄瓜枯萎病胁迫下黄瓜微生态变化
染病黄瓜在不同生长期根际微生物数量结构性变化。在发芽期,染病黄瓜根际的细菌数量低于健康黄瓜;在结果期,染病黄瓜根际的真菌和放线菌的数量低于健康黄瓜。维持黄瓜根际微生态平衡中,前期细菌数量作用大,后期真菌和放线菌的作用大。
根据真菌形态特征,从温室黄瓜不同生态位初步鉴定出的丝状真菌35属,酵母菌6属,其中根际初步鉴定出25属,以镰刀菌属、青霉属、木霉属为优势菌。患病植株各生育期根际真菌种类均少于正常植株,在花期,黄瓜枯萎病发病处于高峰期时,患病植株的根际真菌多样性水平降到最低。病株的根、茎维管束真菌以镰刀菌为主,但种类较正常植株少;枯萎病株叶栖真菌以木霉为主;枯萎病植株的花表、果表的以酵母菌为主,真菌多样性较正常植株丰富。
根据放线菌形态特征,从温室黄瓜根际共分离并鉴定出放线菌15个属,以链霉菌属、链轮菌属为优势菌。正常根际全生育期都存在链轮菌属、链霉菌属,而病害根际在前期以链霉菌属为主,末期才分离到链轮菌属。
根据细菌形态特征、生理生化及16 srDNA序列,从温室黄瓜根际分离并鉴定出的细菌种类有9属。主要种类为芽孢杆菌,似杆菌类群,假单胞菌,短稳杆菌,类球红细菌,奇异变形菌。病菌降低的黄瓜根际细菌种类丰富度。T-RFLP及PCR-DGGE方法对黄瓜枯萎病、根结线虫病、黄瓜枯萎病与根结线虫病复合侵染的根际细菌的多样性进行分析。在PCR-DGGE分析中发现,除枯萎病和根结线虫复合侵染样品外,假单胞菌是优势菌。T-RFLP及PCR-DGGE方法则显示病菌提高了根际细菌种类丰富度,只是细菌种群的均度下降。
黄瓜患枯萎病后,亚硝化细菌、硝化细菌、纤维素分解细菌、氨化细菌、硫化细菌等功能菌群数量相应减少,反硝化细菌、反硫化细菌功能菌群数量相应增加,而自生固氮菌变化不稳定。秋茬患病植株功能菌群数量减少幅度比春茬减少幅度大。黄瓜感染枯萎病后,黄瓜根际过氧化氢酶活性、脲酶活性、纤维素酶活性、碱性磷酸酶活性减弱。
3温室黄瓜枯萎病的微生态控制技术探讨
通过菌株平板对峙检测实验,从黄瓜枯萎病根际筛选出真菌、细菌、放线菌拮抗菌株各2株,对枯萎病的抑制效果较好。经鉴定,这六株菌分别是:短稳杆菌,巨大芽胞杆菌,钦氏菌属,链轮菌属,木霉属,头孢霉属。拮抗真菌木霉菌、头孢霉菌与两株拮抗细菌的协同培养效果不好,两株放线菌链轮丝菌、钦氏菌对短稳杆菌有一定的拮抗效果,其他菌株两两间均未出现拮抗作用。对筛选出的木霉菌株、钦氏菌株、巨大芽孢杆菌株3株拮抗菌进行了发酵条件初步研究。同时对钦氏菌株进行发酵罐发酵条件摸索,获得该菌株大量产孢的条件,但还需进一优化发酵参数。盆栽接种试验表明,3株拮抗菌菌株防的效果均比治好。
Ecological environment, disease species and occurrence regularity of greenhouse cucumber were investigated Fujian Huangshan Chaoda Modern Agriculture (Holdings) Limited farm. Micro-ecological enviroment of greenhouse cucumber Fusarium wilt was systematically studied, and micro-ecological control technique was preliminarily investigated.
1. Relationship between greenhouse eco-environment and cucumber Fusarium wilt
Temperature and humidity: the temperature reached about 10℃in February and the maximum relative humidity of day and night could reach 90% or more, even at 100% in night. The low temperature and high humidity led to the early outbreak of Fusarium wilt of cucumber in greenhouse.
Soils: As sandy soil cultivation measures usually be used in cultivation of greenhouse cucumber, the Fusarium wilt of cucumber become more serious with increasing soil sand.
Pathogens: Fusarium oxysporum accumulated year by year accompanying cropping greenhouse cultivation measures. Root wound caused by incognita is more conducive to cucumber wilt infection.
2. Changes in micro-ecological of cucumber under the stress of cucumber wilt
The number of rhizosphere microbe was changed at different growth stages in infected cucumbers. In the germination stage, the number of rhizosphere bacteria of infected cucumber was less than that of normal cucumbers. In fruiting stage stage, the number of rhizosphere fungi and actinomycetes from infected cucumbers was less than that of healthy cucumbers. During the micro-ecological balance stage of cucumber rhizosphere, bacteria played a major role in early stage, but fungi and actinomycetes played majors roles in later stage.
According to fungi morphology, 35 species of filamentous fungi and 6 species of yeast were identified from different niches of cucumber. 25 species were identified from the rhizosphere of cucumber. Fusarium sp.., Penicillium sp.., and Trichoderma sp.. were dominant rhizosphere fungi. The infected cucumber had fewer rhizosphere fungi than that of healthly cucumbers. In the flower stage, when cucumber wilt outbroke at its peak, the diversity of rhizosphere fungi was declined to its lowest level. On the root vascular of infected cucumber, Fusarium sp. was the dominant strain and had few species. On the leaf surface of infected cucumber, Trichoderma sp. was the dominant strain. On the surface of flower and fruit of infected cucumber, yeast was the dominant strain and the infected cucumber had more species than normal cucumber.
According to actinomycetes morphology, 15 species were identified from the rhizosphere of cucumber. Streptomyces sp. .and Sprocket sp. .were dominant in normal. Contemporarily, Streptomyces sp.. was dominant in infected rhizosphere of cucumber in early stage and Sprocket sp. was isolated in late stage.
According to bacterium morphology, physiological and biochemical, and 16S rDNA sequence, 9 bacterial species were isolated and identified from cucumber rhizosphere in greenhouse. Bacillus、Pseudomonas、Bacteroidetes、Empedobacter、Proteus were dominant strains. The number of isolated bacterium species in infected rhizosphere was less than that of normal rhizosphere. Cultured-independent molecular technique was used to analyze the microbial diversity of cucumber rhizosphere soil. There were Fusarisum oxysporum infection rhizosphere soil, Meloidogyne incognita infection rhizosphere soil, complex infection by Meloidogyne incognita and Fusarisum oxysporum rhizosphere soil and normal soil inside and outside greenhouse. Based on denaturing gradient gel electrophoresis (DGGE), Pseudomonas was the dominant bacterium, except complex infected by Meloidogyne incognita and Fusarisum oxysporum. Compared with the normal, the infected had higher diversity index and smaller species eveness by T-RFLP and PCR-DGGE.
The quantity of Nitrite bacteria, Nitrobacteria, Cellulose-utilizing bacteria, Ammonifying bacteria and Sulphate reducer decreased in the rhizosphere of infected Fusarisum oxysporum cucumber respectively, but the quantity of Denitrifier, Desulphate reducer increased respectively. Change was unstable to erobic azotobacter in infected Fusarisum oxysporum. There was greater decrease in fall stubble than spring crop. Under Fusarium oxysporum stress, the activity of Catelase, Urease, Cellulase, Phosphatase were declined respectively.
3. Technology of micro-ecological control of cucumber Fusarium wilt in greenhouse
2 fungi, 2 bacterium, and 2 actinomycetes were selected from isolated microbe by confrontation to Fusarium oxysporum between the culture plate respectively. They were Empedobacter brevis sp., Bacillus megaterium sp., Chainia sp. , Sprocket sp. , Trichoderma sp., Cephalosporium sp. . The cooperative between antagonistic Trichoderma and antagonistic bacterium was ineffective, the same as that between antagonistic Cephalosporium sp. and antagonistic bacterium. Certain antagonistic effect was found between antagonistic Chainia sp. and Empedobacter brevis sp., the same antagonistic effect as between antagonistic Sprocket sp. and Empedobacter brevis sp. . Other antagonistic strains could be collaborative culture. Selected antagonistic strains Trichoderma sp., Bacillus megaterium sp. , Chainia sp. were studied on fermentation conditions by shaker respectively, accessing to a large number of conditions. The conditions for Chainia sp. fermentation were explored .But for fermentation bubbles, the fermentation parameters were needed to further optimize. By root injury treatment with Fusarium oxysporum, the result showed that prevention was more effective than remedy in pot inoculation test.
1温室生态环境因子与黄瓜枯萎病关系分析
在温湿方面,2月温度达10℃左右,2月下旬及3月上旬,昼夜相对湿度最大值均可达90%以上,晚上甚至达到100%,这种低温高湿条件导致了温室黄瓜枯萎病早发。在土壤方面,温室黄瓜常采用砂质土壤栽培,枯萎病随土壤砂性增加而加剧。在病原菌方面,伴随温室连作栽培,尖孢镰刀菌逐年累积,同时逐年加重的温室根结线虫侵染为害形成的根部伤口易于枯萎病菌的侵染,导致黄瓜枯萎病常发和重发。
2温室黄瓜枯萎病胁迫下黄瓜微生态变化
染病黄瓜在不同生长期根际微生物数量结构性变化。在发芽期,染病黄瓜根际的细菌数量低于健康黄瓜;在结果期,染病黄瓜根际的真菌和放线菌的数量低于健康黄瓜。维持黄瓜根际微生态平衡中,前期细菌数量作用大,后期真菌和放线菌的作用大。
根据真菌形态特征,从温室黄瓜不同生态位初步鉴定出的丝状真菌35属,酵母菌6属,其中根际初步鉴定出25属,以镰刀菌属、青霉属、木霉属为优势菌。患病植株各生育期根际真菌种类均少于正常植株,在花期,黄瓜枯萎病发病处于高峰期时,患病植株的根际真菌多样性水平降到最低。病株的根、茎维管束真菌以镰刀菌为主,但种类较正常植株少;枯萎病株叶栖真菌以木霉为主;枯萎病植株的花表、果表的以酵母菌为主,真菌多样性较正常植株丰富。
根据放线菌形态特征,从温室黄瓜根际共分离并鉴定出放线菌15个属,以链霉菌属、链轮菌属为优势菌。正常根际全生育期都存在链轮菌属、链霉菌属,而病害根际在前期以链霉菌属为主,末期才分离到链轮菌属。
根据细菌形态特征、生理生化及16 srDNA序列,从温室黄瓜根际分离并鉴定出的细菌种类有9属。主要种类为芽孢杆菌,似杆菌类群,假单胞菌,短稳杆菌,类球红细菌,奇异变形菌。病菌降低的黄瓜根际细菌种类丰富度。T-RFLP及PCR-DGGE方法对黄瓜枯萎病、根结线虫病、黄瓜枯萎病与根结线虫病复合侵染的根际细菌的多样性进行分析。在PCR-DGGE分析中发现,除枯萎病和根结线虫复合侵染样品外,假单胞菌是优势菌。T-RFLP及PCR-DGGE方法则显示病菌提高了根际细菌种类丰富度,只是细菌种群的均度下降。
黄瓜患枯萎病后,亚硝化细菌、硝化细菌、纤维素分解细菌、氨化细菌、硫化细菌等功能菌群数量相应减少,反硝化细菌、反硫化细菌功能菌群数量相应增加,而自生固氮菌变化不稳定。秋茬患病植株功能菌群数量减少幅度比春茬减少幅度大。黄瓜感染枯萎病后,黄瓜根际过氧化氢酶活性、脲酶活性、纤维素酶活性、碱性磷酸酶活性减弱。
3温室黄瓜枯萎病的微生态控制技术探讨
通过菌株平板对峙检测实验,从黄瓜枯萎病根际筛选出真菌、细菌、放线菌拮抗菌株各2株,对枯萎病的抑制效果较好。经鉴定,这六株菌分别是:短稳杆菌,巨大芽胞杆菌,钦氏菌属,链轮菌属,木霉属,头孢霉属。拮抗真菌木霉菌、头孢霉菌与两株拮抗细菌的协同培养效果不好,两株放线菌链轮丝菌、钦氏菌对短稳杆菌有一定的拮抗效果,其他菌株两两间均未出现拮抗作用。对筛选出的木霉菌株、钦氏菌株、巨大芽孢杆菌株3株拮抗菌进行了发酵条件初步研究。同时对钦氏菌株进行发酵罐发酵条件摸索,获得该菌株大量产孢的条件,但还需进一优化发酵参数。盆栽接种试验表明,3株拮抗菌菌株防的效果均比治好。
Ecological environment, disease species and occurrence regularity of greenhouse cucumber were investigated Fujian Huangshan Chaoda Modern Agriculture (Holdings) Limited farm. Micro-ecological enviroment of greenhouse cucumber Fusarium wilt was systematically studied, and micro-ecological control technique was preliminarily investigated.
1. Relationship between greenhouse eco-environment and cucumber Fusarium wilt
Temperature and humidity: the temperature reached about 10℃in February and the maximum relative humidity of day and night could reach 90% or more, even at 100% in night. The low temperature and high humidity led to the early outbreak of Fusarium wilt of cucumber in greenhouse.
Soils: As sandy soil cultivation measures usually be used in cultivation of greenhouse cucumber, the Fusarium wilt of cucumber become more serious with increasing soil sand.
Pathogens: Fusarium oxysporum accumulated year by year accompanying cropping greenhouse cultivation measures. Root wound caused by incognita is more conducive to cucumber wilt infection.
2. Changes in micro-ecological of cucumber under the stress of cucumber wilt
The number of rhizosphere microbe was changed at different growth stages in infected cucumbers. In the germination stage, the number of rhizosphere bacteria of infected cucumber was less than that of normal cucumbers. In fruiting stage stage, the number of rhizosphere fungi and actinomycetes from infected cucumbers was less than that of healthy cucumbers. During the micro-ecological balance stage of cucumber rhizosphere, bacteria played a major role in early stage, but fungi and actinomycetes played majors roles in later stage.
According to fungi morphology, 35 species of filamentous fungi and 6 species of yeast were identified from different niches of cucumber. 25 species were identified from the rhizosphere of cucumber. Fusarium sp.., Penicillium sp.., and Trichoderma sp.. were dominant rhizosphere fungi. The infected cucumber had fewer rhizosphere fungi than that of healthly cucumbers. In the flower stage, when cucumber wilt outbroke at its peak, the diversity of rhizosphere fungi was declined to its lowest level. On the root vascular of infected cucumber, Fusarium sp. was the dominant strain and had few species. On the leaf surface of infected cucumber, Trichoderma sp. was the dominant strain. On the surface of flower and fruit of infected cucumber, yeast was the dominant strain and the infected cucumber had more species than normal cucumber.
According to actinomycetes morphology, 15 species were identified from the rhizosphere of cucumber. Streptomyces sp. .and Sprocket sp. .were dominant in normal. Contemporarily, Streptomyces sp.. was dominant in infected rhizosphere of cucumber in early stage and Sprocket sp. was isolated in late stage.
According to bacterium morphology, physiological and biochemical, and 16S rDNA sequence, 9 bacterial species were isolated and identified from cucumber rhizosphere in greenhouse. Bacillus、Pseudomonas、Bacteroidetes、Empedobacter、Proteus were dominant strains. The number of isolated bacterium species in infected rhizosphere was less than that of normal rhizosphere. Cultured-independent molecular technique was used to analyze the microbial diversity of cucumber rhizosphere soil. There were Fusarisum oxysporum infection rhizosphere soil, Meloidogyne incognita infection rhizosphere soil, complex infection by Meloidogyne incognita and Fusarisum oxysporum rhizosphere soil and normal soil inside and outside greenhouse. Based on denaturing gradient gel electrophoresis (DGGE), Pseudomonas was the dominant bacterium, except complex infected by Meloidogyne incognita and Fusarisum oxysporum. Compared with the normal, the infected had higher diversity index and smaller species eveness by T-RFLP and PCR-DGGE.
The quantity of Nitrite bacteria, Nitrobacteria, Cellulose-utilizing bacteria, Ammonifying bacteria and Sulphate reducer decreased in the rhizosphere of infected Fusarisum oxysporum cucumber respectively, but the quantity of Denitrifier, Desulphate reducer increased respectively. Change was unstable to erobic azotobacter in infected Fusarisum oxysporum. There was greater decrease in fall stubble than spring crop. Under Fusarium oxysporum stress, the activity of Catelase, Urease, Cellulase, Phosphatase were declined respectively.
3. Technology of micro-ecological control of cucumber Fusarium wilt in greenhouse
2 fungi, 2 bacterium, and 2 actinomycetes were selected from isolated microbe by confrontation to Fusarium oxysporum between the culture plate respectively. They were Empedobacter brevis sp., Bacillus megaterium sp., Chainia sp. , Sprocket sp. , Trichoderma sp., Cephalosporium sp. . The cooperative between antagonistic Trichoderma and antagonistic bacterium was ineffective, the same as that between antagonistic Cephalosporium sp. and antagonistic bacterium. Certain antagonistic effect was found between antagonistic Chainia sp. and Empedobacter brevis sp., the same antagonistic effect as between antagonistic Sprocket sp. and Empedobacter brevis sp. . Other antagonistic strains could be collaborative culture. Selected antagonistic strains Trichoderma sp., Bacillus megaterium sp. , Chainia sp. were studied on fermentation conditions by shaker respectively, accessing to a large number of conditions. The conditions for Chainia sp. fermentation were explored .But for fermentation bubbles, the fermentation parameters were needed to further optimize. By root injury treatment with Fusarium oxysporum, the result showed that prevention was more effective than remedy in pot inoculation test.
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