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摘要：解淀粉芽孢杆菌SQR9（Bacillus amyloliquefaciens SQR9）是本实验室从健康黄瓜植株根际分离的一株根际促生菌，研究发现它能显著抑制病原尖孢镰刀菌的生长，在盆栽和大田试验中表现出显著的促生和生防效果。在自然环境下特别是根际环境，混合菌群生物膜占主导地位，但实验室条件下的研究主要集中于研究单株菌的行为，关于菌株SQR9与其他微生物在生物膜形成过程中的互作鲜有报道。本研究采用绝对定量PCR对混合菌群生物膜中菌株SQR9进行定量，发现菌体数量比单独培养显著增加，说明可能存在某些微生物能够促进菌株SQR9生物膜的形成。分别提取混合生物膜和剩余培养液的DNA进行16S rDNA扩增并进行高通量测序，发现革兰氏阴性细菌寡养单胞菌（Stenotrophomonas sp.）在生物膜中的相对丰度显著高于培养液处理，其他微生物类群也存在于生物膜中，但在培养液中的相对丰度要高于生物膜中，我们推测土壤中寡养单胞菌能够与解淀粉芽孢杆菌SQR9互作，参与其生物膜和根际定殖过程。25798
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毕业论文关键词：解淀粉芽孢杆菌SQR9；生物膜；微生物互作
Preliminary identification of cucumber rhizospheric microorganisms that interact with Bacillus amyloliquefaciens SQR9
Abstract：Bacillus amyloliquefaciens SQR9, isolated and identified by our laboratory, was proved to be an outstanding PGPR (plant growth-promoting rhizobacteria) strain by its abilities to inhibit the growth of FOC (Fusarium oxysporum f. sp. cucumerinum J. H. Owen) and decrease the incidence of Fusarium wilt of cucumber. Although in most environments, especially in the rhizophere, multi-species biofilms are dominate, single-species biofilms are the focus of most current researches under laboratary condition. Researches on the interaction between B. amyloliquefaciens SQR9 and other rhizospheric microorganisms are limited. To explore such interaction, the number of B. amyloliquefaciens SQR9 in mixed-species biofilms were monitored by real-time PCR. Results showed that the biofilm formation abilities of SQR9 was improved by incubating with microorganisms from black soil, indicate that some unkown microorganisms may be involved in this process. The microbial communities in biofilm and liquid media were analyzed by high-throughput sequencing technology. Results indicate that the abundance of Stenotrophomonas in bioflim is significantly higher than that in liquid media. Other microflora also existed in biofilm, but played a dominant role in liquid media. We speculate that the interaction of Stenotrophomonas and SQR9 was involved in the biofilm formation and root colonization.
Key words: Bacillus amyloliquefaciens SQR9；biofilm；microbial interactions
目 录
摘要1
关键词1
Abstract1
Key words1
引言1
1材料与方法2
1.1材料 2
1.2方法 2
1.2.1黄瓜根际土的分离2
1.2.2黄瓜根际土菌体细胞的提取3
1.2.3生物膜共培养3
1.2.4提取生物膜和培养液中的DNA3
1.2.5定量PCR分析生物膜及游离细胞中菌株SQR9的数量3
1.2.6高通量测序5
2结果与分析5
2.1绝对定量PCR5
2.1.1引物特异性验证5
2.1.2转化子鉴定5
2.1.3标准曲线制作6
2.1.4生物膜及培养液中菌株SQR9的绝对定量6
2.2生物膜基因组高通量测序7
3讨论 8
致谢9
参考文献9
解淀粉芽孢杆菌SQR9根际互作微生物的初步鉴定\引言:
黄瓜土传枯萎病是黄瓜种植区主要的连作障害之一，由真菌尖孢镰刀菌黄瓜专化型FOC引起，严重影响黄瓜的产量和品质，研究以微生物防控为核心的综合防控具有重大意义。解淀粉芽孢杆菌SQR9是从黄瓜土传枯萎病发病区的健康植株根际分离的一株根际促生菌（PGPR），能强烈抑制尖孢镰刀菌，在盆栽试验和大田试验中表现出良好的防控黄瓜土传枯萎病和促进黄瓜生长的效果[1-2]。同时该菌株在实验室条件下表现了优异的生物膜形成、根际定殖、趋化性、可产生多种次生代谢产物（主要为抗生素类物质）等性状，是研究PGPR菌株在根际生态行为及植物-微生物互作的优良菌株[3-5]。