【作者】 张立新；

【导师】 谢关林；

【作者基本信息】 浙江大学 ， 植物病理学， 2007， 博士

【摘要】 洋葱伯克氏菌（Burkholderia cepacia）是一种广泛存在于土壤、水和植物根围、与医院感染病人密切相关的革兰氏阴性细菌。它最初作为植物病原菌被认识，后来发现它也是医院中重要的人体条件致病菌，由该菌引起患者洋葱伯克氏菌综合症的致死事件在国内外均有报道。同时它在农业领域中具有生物防治、生物降解和促进植物生长等多种功能。近年来，该菌被认为不是一个种，而是一组基因型不同、表型相近的复合物，称为洋葱伯克氏菌群（Burkholderia cepacia complex，简称Bcc）。到目前为止已报道10个不同的基因型。现有研究认为自然环境可能是医院致病菌株的天然储存库，尤其在作物根围发现Bcc菌群丰富。但我国关于Bcc菌在自然环境中的研究极少，尤其在作物根围环境中的研究仍是空白。本研究选定了多年种植的水稻、玉米和大麦种植田块，对3种作物的根围进行Bcc基因型的多样性调查，了解基因型在不同作物根围的种类与分布，为评估其对人体的潜在致病性提供基础；结合医院来源的基因型Ⅰ和Ⅲ菌株，对不同基因型的菌株在苜蓿植物模型上进行毒力测定，探测根围来源的不同基因型菌株间的毒力程度；选取代表性的基因型菌株，对其在洋葱上进行致病性测定；同时对来源于根围的Bcc菌株进行拮抗植物病菌的离体筛选，以期挖掘具有生防效果的安全性菌株。取得了如下主要研究结果：（1）采用3种选择性培养基（PCAT、TB-T和BCSA）对水稻、玉米和大麦作物根围进行了Bcc菌的调查，共分离到628株Bcc疑似菌株。通过对这些细菌进行表型初步鉴定和PCR特异性扩增recA基因片段的分子鉴定，共确诊了149株洋葱伯克氏菌株，其中78株来自水稻根围，61株来自玉米根围，大麦根围仅分离到10株。结果表明，PCAT和TB-T两种选择性培养基对Bcc菌株的选择性优于BCSA培养基。水稻和玉米根围存在丰富的Bcc菌株，在PCAT选择性平板上，能分离到55％～72％比例的Bcc菌，在根围的种群密度达到10⁵～10⁶cfu·g^-1。而对于大麦根围，在PCAT选择平板上分离到的Bcc菌株比例很小，仅8％左右，表明Bcc菌在大麦根围种群密度低。（2）采用限制性内切酶HaeⅢ对来源于水稻和玉米根围的139株Bcc菌的recA基因片段进行了限制性酶切（RFLP）分析，以探测Bcc菌的遗传多样性。结果表明，在水稻根围的78株Bcc菌中，发现4种不同的限制性酶切图谱（A、B、H和K）；在玉米根围的61株Bcc菌中，也存在4种不同的限制性酶切图谱（A、E、I和M），这表明Bcc分离物具有明显的遗传多态性。（3）利用recA-HaeⅢ限制性酶切分析、基因型特异性PCR扩增方法和recA基因的序列分析对来自水稻和玉米根围的139株Bcc菌，以及来自医院的14株Bcc菌进行了基因型的鉴定和划分。在水稻根围存在基因型Ⅰ、ⅢB和Ⅴ，其中以基因型Ⅴ的菌株数量最多，占分离于水稻根围Bcc菌株数的76.9％。基因型Ⅴ为水稻根围Bcc菌的优势基因型，这一结果是国内外的首次报道；在玉米根围存在基因型Ⅰ、ⅢB、Ⅴ和Ⅸ，其中以基因型ⅢB为玉米根围的优势基因型，占分离Bcc菌株数的42.6％。这表明在水稻和玉米两种作物根围中，优势基因型存在明显的差异。在医院来源的菌株中，存在基因型Ⅰ和ⅢA，其中ⅢA型菌株数较多，占菌株总数的64.3％。在本研究的Bcc菌分离物中，其中20个Bcc分离物具有相同的recA-HaeⅢRFLP图谱Ⅰ，采用基因型ⅢA的特异性引物表现为PCR阳性扩增。然而，其两个代表菌株（M229和M279）的recA基因序列在聚类树中与基因型ⅢD参考菌株以很低的自举置信水平值聚类在一起（BCL≤28％），低自举置信水平值的存在表明进一步种的多样性可能存在，因此这些Bcc菌株没有被鉴定为基因型。菌株R456在分离的Bcc菌株中具有唯一的RFLP图谱H，通过对其recA基因序列的分析，它在聚类树中单独形成一个分支，但该菌株的recA基因序列与其它基因型（包括基因型ⅢB、Ⅳ和Ⅸ）具有97％的序列相似性。这表明该分离物与其它基因型是不同的。然而，该菌株的recA序列与以前报道的菌株BC14具有99％的序列相似性，这表明这些菌株可能为新的基因型。（4）采用苜蓿植物模型对来源于自然环境和医院中的67株Bcc菌进行了毒力测定，同时也对毒力基因BCESM和cblA的存在与否进行了特异性PCR检测。结果表明，来源于医院的基因型Ⅰ和Ⅲ菌株均对苜蓿幼苗有较强的毒力，幼苗子叶黄化或白化，根短小、畸形，对苜蓿幼苗的平均发病率分别达到69％和68％。来源于自然环境的Bcc菌株中，基因型ⅢB也对苜蓿幼苗表现出强毒力，幼苗症状类似于医院致病菌的致病效果，幼苗平均发病率为55％；基因型Ⅰ菌株对苜蓿幼苗的毒力程度较轻，有的菌株没有致幼苗发病；基因型Ⅴ和Ⅸ的多数菌株对苜蓿幼苗发病率很低，部分菌株不致幼苗发病。这说明来源于自然环境的基因型ⅢB菌株与医院致病菌的基因型ⅢA和Ⅰ菌株具有相同的毒力，表明自然环境中的基因型ⅢB菌可能为潜在的人体条件致病菌。同时研究表明，在这些Bcc菌株中，未检测到BCESM和cblA这两个毒力基因。（5）对分离于医院和自然环境的20株代表性Bcc菌进行（?）烟草过敏反应和洋葱致病性试验。在测试的20株Bcc菌中，基因型Ⅰ、Ⅲ和Ⅴ的菌株都导致了烟草的过敏性坏死反应，仅有1株基因型Ⅸ菌株呈阴性反应，表明多数洋葱伯克氏菌可能为植物致病菌。来源于医院的基因型Ⅰ和Ⅲ菌株均能引起洋葱鳞茎发病，与洋葱致病菌株LMG1222表现出相似的致病效果，说明这些菌株既是人体条件致病菌，也是洋葱的致病菌。在来源自然环境的Bcc菌中，基因型Ⅰ和Ⅲ的菌株也能引起洋葱发病；基因型Ⅴ菌株引起洋葱鳞茎产生坏死小斑点，但继续培养后，斑点并没有扩大；基因型Ⅸ的菌株未能使洋葱发病。（6）对经鉴定后的77株Bcc菌进行了拮抗植物病原真菌的离体筛选。结果表明，34株细菌对黄瓜枯萎病菌和灰霉病菌具有不同程度的拮抗活性，占总测试菌株的54.5％。其中以基因型Ⅴ内拮抗细菌占的比例最高，基因型Ⅲ内的拮抗菌株占的比例最少，仅占5.7％。23株基因型Ⅴ菌株能够明显抑制病原生长，形成较大的抑菌圈（直径15～20mm），且对立枯丝核病菌、疫霉病菌和其它枯萎病菌也表现了较强的拮抗活性。选取了4株平板拮抗活性强的菌株进行了代谢产物活性、致病性及毒力测定。结果表明，拮抗细菌的培养滤液对灰霉病菌和立枯丝核病菌有较好的拮抗活性，但对枯萎病菌无拮抗活性。它们对苜蓿幼苗的毒力很低，在洋葱上不致病，未检测到BCESM和cblA两个毒力基因，初步表明它们可能是安全性菌株。更多还原

【Abstract】 Burkholderia cepacia is a gram-negative bacterium commonly found in soil, Water, plant rhizosphere, patients and hospital environment. It was first described as a phytobacterium. Subsequently, it emerged as an opportunistic human pathogen in hospital, and caused "cepacia sydrome" resulting in death of the patients in China and other countries. However, B. cepacia strains have attracted considerable interest as plant pest antagonists, plant growth-promoting rhizobacteria and bioremediation agents of toxic substances. Recently, B. cepacia has been classified into ten genotypically distinct but phenotypically similar species (genomovars) referring to the B. cepacia complex (Bcc). The current findings showed that the natural environmental may serve as a "reservoir" for pathogenic strains, and the rhizospheres of crop plants were regarded as abundant sources of Bcc strains. However, little information is available for Bcc populations from natural environment, especially in the rhizosphere of major cereal crops in China.The distribution and diversity of Bcc population were surveyed in the rhizospheres of rice, maize and barley with monoculture for several years for understanding Bcc genomovars in different crop rhizosphere and providing key information in risk analysis of Bcc. Based on Bcc isolates from the rhizospheres and clinical sources, the virulence study of the Bcc strains in alfalfa model was performed to detect the virulence degree of different genomovar strains. The pathogenicity of different Bcc genomovar strains was tested on onion bulbls, and antagonistic screening of Bcc isolates against phytopathogenic fungi was also conducted in this study for more promising and safe strains in biocontrol.The RFLP assay with enzyme HaeIII was performed to detect genetic variability among 139 Bcc isolates from the rhizosphere of rice and maize. The results showed that four different RFLP patterns (A, B, H and K) were found among 78 Bcc isolates from rice rhizosphere, and four other different patterns (A, E, I and M) were also detected among 61 Bcc isolates from maize rhizosphere, revealing considerable variability among the Bcc isolates.The identification of Bcc genomovars was performed among 139 Bcc isolates from the rhizosphere of rice and maize, and 14 Bcc isolates from clinical samples by a combination of recA-HaeIII restriction fragment length polymorphism assays, species-specific PCR tests and recA gene sequence analysis. Three Bcc species including B. cepacia, B. cenocepacia recA lineage IIIB and B. vietnamiensis; were recovered from rice rhizosphere. The B. vietnamiensis isolates showed highest numbers with76.9% of the total. To our knowledge, this is the first report of B. vietnamiensis dominating in the rice rhizosphere. B. cenocepacia dominated the maize rhizosphere, followed by B. cepacia, B. pyrrocinia and B. vietnamiensis. It indicated that predominant Bcc species vary dramatically in the rhizosphere of maize and rice. B. cepacia and B. cenocepacia recA lineage IIIA were found among the Bcc isolates from clinical sources and the later was higher with 64.3% of the total.Twenty Bcc isolates sharing the recA RFLP pattern were PCR positive with B. cenocepacia IIIA-specific primers. Two representatives of these isolates, M229 and M279, clustered with B. cenocepacia recA lineage IIID strains with the least robust group within the phylogenetic analysis (BCL≤28%). The existence of a low bootstrap value suggests that further species diversity may be present. These Bcc isolates have therefore not been assigned to a genomovar. One isolate (R456) that gave a unique RFLP profile could not be assigned to a genomovar by recA gene sequence analysis. The recA gene sequence of this isolate had 97% identity to those from several genomovars including B. cenocepacia recA lineage IIIB, B. stabilis and B. pyrrocinia, and it formed a single discrete cluster within the phylogenetic tree. This indicates that the isolate is equally divergent from several of the other genomovars. However, its recA sequence had 99% identity to that of the previously described strain BC14 with which they probably belong to a novel Bcc group.Sixty-seven Bcc isolates from natural environment and clinical samples were tested for virulence in the alfalfa model, and PCR tests were also performed to detect the genes with BCESM and cblA. The results showed that the strains of B. cepacia and B. cenocepacia caused symptoms of yellow leaves, stunted roots, and brown necrotic lesions on alfalfa seedlings with mean percentages of the infected seedlings by B. cepacia and B. cenocepacia from clinical sources being 69% and 68%, respectively. Among the Bcc strains from natural environment, B. cenocepacia recA lineage IIIB strains caused symptoms in 55% of the alfalfa seedlings; Some strains of B. cepacia did not cause symptoms of seedlings; Most strains of B. vietnamiensis and B. pyrronicia did not cause symptoms of seedlings. It indicated that B. cenocepacia recA lineage IIIB strains from natural environment were similar to B. cenocepacia recA lineage IIIA and B. cepacia strains from clinical sources in symptoms of the seedlings, and they may be potential human opportunistic pathogen. The genes with BCESM and cblA did not been detected among these Bcc strains.Twenty representative Bcc strains from’natural environment and clinical sources were tested for hypersensitive reaction (HR) on tobacco and pathogenicity tests on onion. All strains including B. cepacia, B. cenocepacia and B. vietnamiensis, except for one B. pyrrocinia strain, had HR on tobacco, revealing that most Bcc strains may be pathogenic. B. cepacia and B. cenocepacia strains from clinical sources induced symptoms on onion which were similar to those caused by strain LMG1222. It indicated these strains were both human opportunistic pathogen and onion phytopathogen. Among the Bcc strains from natural environment, B. cepacia and B. cenocepacia strains caused symptoms on onion; B. vietnamiensis produced small dot lesions on onion without extending after inoculation for 14 d; Strains of 5. pyrrocinia did not cause symptoms on onion.Seventy-seven Bcc strains were selected to screen for their antagonism against pathogenic fungi of crops in vitro. Thirty-four Bcc strains, 54.5% of the taotal showed antagonistic effect on Fusarium oxysporum f. sp. cucumerinum Owen and Botrytis cinerea. The percentage of antagonistic bacteria was highest in B. vietnamiensis, and lowest in B. cenocepacia; 23 B. vietnamiensis strains significantly inhibited growth of the two pathogens, showing 15-20 mm of inhibition zone. And they also had higher antagonistic activity against Rhizoctonia solania, Phytophthara capsici Leonian and other Fusarium oxysporum. Four antagonistic bacteria were selected to investigate for antagonistic activities of their culture filtrates. It showed that the culture filtrates of the bacteria had certain inhibitory effect on Rhizoctonia solania and Botrytis cinerea, and no effect on Fusarium oxysporium. The four antagonistic bacteria could be considered as safe strains since they were almost aviruience on alfalfa seedlings, not pathogenic on onion and the virulence genes with BCESM and cblA were not detected.更多还原