甜瓜枯萎病菌拮抗细菌在不同耕作土壤中的分布及其功能研究
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摘要
从杭州菜园地、嘉兴大棚甜瓜田采集不同来源根际土壤和非根际土壤共14批(每批3份)，利用平板稀释法和对峙法测定细菌、枯萎病菌数量及拮抗细菌的分布，结果显示，轮作后细菌总量明显增加，枯萎病菌数量降低，细菌和真菌比值(B／F值)增加，同时土壤中细菌数量越多，拮抗细菌的比例越高，尤其以大蒜与甜瓜轮作细菌数量最多，拮抗细菌比例也最高；细菌数量以菜园土壤中最高，甜瓜根际土壤次之，非根际土壤中数量最少。从14批土壤中共分离获得840个细菌分离物，在离体条件下测定他们对甜瓜枯萎病菌(Fusarium oxysporum f.sp melonis)的拮抗能力，结果显示43个菌株具有拮抗性，占总检测数的5.11％，其中分离自甜瓜根际土壤和非根际土壤的拮抗菌株为17个和10个，分别占5.67％和3.33％；分离自菜园蔬菜根际的拮抗菌株16个，占6.67％。经复筛后从中获得15个拮抗性较好的菌株，平板对峙法测定拮抗指数大小，结果表明，拮抗指数在2.0以上的拮抗细菌有9个，其中大多数来自根际土壤，1.0～2.0之间的有4个，1.0以下的有2株。数据显示，土壤中细菌数量的多少与拮抗细菌的分布有一定的关系，细菌总量与拮抗细菌数成正比；菜园土中细菌数量与拮抗细菌比例高于大棚甜瓜土壤；甜瓜大棚根际土壤细菌数量与拮抗细菌比例都明显高于非根际土壤，而且根际土壤中筛选出的拮抗细菌普遍拮抗指数高，离体拮抗作用强。
选取离体拮抗作用较好的9个拮抗菌株进行拮抗稳定性和抑菌谱测定，筛选出拮抗作用较稳定且抑菌谱宽的拮抗菌株7个，各菌株的培养滤液对菌丝生长和分生孢子萌发的抑制作用测定表明，它们均表现不同程度的抑菌活性，其中菌株Hc2和Hc3的抑制作用最强，培养滤液稀释50％时能完全抑制菌丝生长；凹玻片法测定拮抗细菌培养滤液与分生孢子悬浮液混合后抑制孢子萌发率的结果表明，Ec50值越低，抑制力越强，Hc3菌株的培养滤液在5％时抑制率也达50％。
对7株拮抗细菌处理盆栽甜瓜种子后发现，Hc1，Hc2，Hc3和Jt6四个拮抗细菌表现较好的促生作用，其中菌株Hc2对芽和胚根的促生效果最好；Hc1、Hc3和Jt6则对幼苗的生长有一定的促进作用。在对甜瓜幼苗的防效试验中，4株拮抗细菌在处理后10天有很好的防效，相对防效最高可达65.4％，但是在处理后20天，防效显著降低。
16个桔抗菌株的细菌学、Biolog和分子生物学（RenPCR）鉴定表明，阴性粘抗菌
株大多数属于Pseudomonas spp，是土壤中的优势菌株，部分属于Burkholderta SPP．；
Biolog 鉴定相 《 性在 0．50 以上的种有 尸ae峪lviosa；Wu订d；Presinovorans；
叭o删censC P v个法广人。州和 Bu啪oWM c叩acm。
Fourteen batches (42 samples) of different soils were collected from vegetable garden and muskmelon field in Hangzhou and Jiaxin of Zhejiang province and the proportion of antagonistic bacteria to the total in the soils was measured. The result showed that the ratio of total number of bacteria and Fusarium oxysporum improved in the soils of crop rotation, the number of Fusarium oxysporum greatly decreased and the antagonistic bacteria increased. Especially in the soil of muskmelon and garlic rotation the number of the antagonistic bacteria was the highest. Eight hundreds and forty bacterial isolates were isolated from the soil samples and screened in vitro for antagonists against Fusarium oxysporum f.sp.melonis. It showed that 43 strains, 5.11% of total, were antagonistic to the pathogen. Among them 17 strains from the rhizosphere of muskmelon soil, 5.67% of total, were antagonists; 10 strains from the non-rhizosphere soil of muskmelon, 3.33% antagonists and 16 strains from rhizosphere soil of the vegetable garden, 6.67% of total antagonists. Fifteen strains were further tested for the antagonistic index, of which 9 strains were above 2.0 and 4 strains were between 1.0 to 2.0. The distribution of antagonistic bacteria was correlated to the total number of bacteria in the soils. In general the more the number of bacteria were in the soil, the higher the proportion of antagonistic bacterium. The proportion of the antagonistic bacteria to total number in the vegetable garden soil was higher than those of the melon soil; the proportion in the soil of rhizosphere was also higher than those of in non-rhizosphere soil. It was noted that the antagonistic bacteria from the rhizosphere soil were stronger against the pathogen than those from non-rhizosphere in vitro.
Nine stronger antagonistic strains were further tested for their antagonistic stability and anti-fungal spectra and 7 of them selected. Their culture filtrates were tested for their effect on inhibiting mycelial growth and conidial germination. It showed that strains Hc2 and Hc3 were strongest among the 7 strains. At the concentration of 50% they completely suppressed mycelial growth. Even if at the concentration of 5% they still inhibited 50% of conidia germination. The treatments of the melon seed dressing in pots showed that 4 strains, Hcl, Hc2, Hc3 and Jt6, were not only antagonistic to the pathogens but also had effect on
growth-promoting of the melon seedlings. They performed better control effect 10 days after treatment and the highest relatively control effect reached 65.4%. However, the control effect decreased obviously 20 days after the treatment. Identification of the antagonistic bacterial strains by bacteriological, Biolog and molecular biological test (RAPD-PCR) showed that among 16 Gram negative strains most of them belong to Pseudomonas spp. and they were predominant in the isolation. The species with over 0.50 of Biolog similarity were P.aeruginosa; P.putida; P.resinovorans; P.fluorescens C; P.viridilivida A Burkholderia cepacia.
选取离体拮抗作用较好的9个拮抗菌株进行拮抗稳定性和抑菌谱测定，筛选出拮抗作用较稳定且抑菌谱宽的拮抗菌株7个，各菌株的培养滤液对菌丝生长和分生孢子萌发的抑制作用测定表明，它们均表现不同程度的抑菌活性，其中菌株Hc2和Hc3的抑制作用最强，培养滤液稀释50％时能完全抑制菌丝生长；凹玻片法测定拮抗细菌培养滤液与分生孢子悬浮液混合后抑制孢子萌发率的结果表明，Ec50值越低，抑制力越强，Hc3菌株的培养滤液在5％时抑制率也达50％。
对7株拮抗细菌处理盆栽甜瓜种子后发现，Hc1，Hc2，Hc3和Jt6四个拮抗细菌表现较好的促生作用，其中菌株Hc2对芽和胚根的促生效果最好；Hc1、Hc3和Jt6则对幼苗的生长有一定的促进作用。在对甜瓜幼苗的防效试验中，4株拮抗细菌在处理后10天有很好的防效，相对防效最高可达65.4％，但是在处理后20天，防效显著降低。
16个桔抗菌株的细菌学、Biolog和分子生物学（RenPCR）鉴定表明，阴性粘抗菌
株大多数属于Pseudomonas spp，是土壤中的优势菌株，部分属于Burkholderta SPP．；
Biolog 鉴定相 《 性在 0．50 以上的种有 尸ae峪lviosa；Wu订d；Presinovorans；
叭o删censC P v个法广人。州和 Bu啪oWM c叩acm。
Fourteen batches (42 samples) of different soils were collected from vegetable garden and muskmelon field in Hangzhou and Jiaxin of Zhejiang province and the proportion of antagonistic bacteria to the total in the soils was measured. The result showed that the ratio of total number of bacteria and Fusarium oxysporum improved in the soils of crop rotation, the number of Fusarium oxysporum greatly decreased and the antagonistic bacteria increased. Especially in the soil of muskmelon and garlic rotation the number of the antagonistic bacteria was the highest. Eight hundreds and forty bacterial isolates were isolated from the soil samples and screened in vitro for antagonists against Fusarium oxysporum f.sp.melonis. It showed that 43 strains, 5.11% of total, were antagonistic to the pathogen. Among them 17 strains from the rhizosphere of muskmelon soil, 5.67% of total, were antagonists; 10 strains from the non-rhizosphere soil of muskmelon, 3.33% antagonists and 16 strains from rhizosphere soil of the vegetable garden, 6.67% of total antagonists. Fifteen strains were further tested for the antagonistic index, of which 9 strains were above 2.0 and 4 strains were between 1.0 to 2.0. The distribution of antagonistic bacteria was correlated to the total number of bacteria in the soils. In general the more the number of bacteria were in the soil, the higher the proportion of antagonistic bacterium. The proportion of the antagonistic bacteria to total number in the vegetable garden soil was higher than those of the melon soil; the proportion in the soil of rhizosphere was also higher than those of in non-rhizosphere soil. It was noted that the antagonistic bacteria from the rhizosphere soil were stronger against the pathogen than those from non-rhizosphere in vitro.
Nine stronger antagonistic strains were further tested for their antagonistic stability and anti-fungal spectra and 7 of them selected. Their culture filtrates were tested for their effect on inhibiting mycelial growth and conidial germination. It showed that strains Hc2 and Hc3 were strongest among the 7 strains. At the concentration of 50% they completely suppressed mycelial growth. Even if at the concentration of 5% they still inhibited 50% of conidia germination. The treatments of the melon seed dressing in pots showed that 4 strains, Hcl, Hc2, Hc3 and Jt6, were not only antagonistic to the pathogens but also had effect on
growth-promoting of the melon seedlings. They performed better control effect 10 days after treatment and the highest relatively control effect reached 65.4%. However, the control effect decreased obviously 20 days after the treatment. Identification of the antagonistic bacterial strains by bacteriological, Biolog and molecular biological test (RAPD-PCR) showed that among 16 Gram negative strains most of them belong to Pseudomonas spp. and they were predominant in the isolation. The species with over 0.50 of Biolog similarity were P.aeruginosa; P.putida; P.resinovorans; P.fluorescens C; P.viridilivida A Burkholderia cepacia.
引文
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