【作者】 陈京元；

【导师】 嵇保中；

【作者基本信息】 南京林业大学 ， 森林保护学， 2006， 博士

【摘要】 湿地松(Pinus elliottii)是我国南方及湖北省重要的造林树种，其所产松脂是重要的化工原料。在湿地松育苗期间，往往因猝倒病的危害，严重影响了育苗和造林。国内外研究表明，松前猝倒病由丝核菌(Rhizoctonia)、腐霉菌(Pythium)、镰孢菌(Fusarium)、链格孢菌(Alternaria)、葡萄孢菌(Botrytis)、盘多毛孢菌(Pestalotia)等属的多种真菌侵染引起，且不同地域病原菌种类不尽相同。本研究以湿地松苗为研究对象，在详细研究松苗猝倒病主要病原菌和根际微生物分布与变化规律的基础上，以荧光假单胞菌和洋葱伯克霍尔德菌等引进与本土生防菌为研究材料，采用分子检测技术，对生防菌在松苗根际的定殖规律以及抑病效果进行了研究，主要内容及结论如下：(一)松苗根际微生物分布及数量变化规律湿地松根际土壤中存在着大量的微生物，每克土中真菌菌落数量一般在1×10~5～3.5×10~6CFU(colony forming units)左右，细菌每克土一般为10~7～10~8CFU；真菌数量变化的高峰期出现在9月份，细菌数量变化高峰期出现在苗木移栽的4月份。细菌的种群数量增长速度快于真菌，在根际表面定殖的时间也早于真菌。(二)松苗猝倒病主要病原真菌及致病性湖北省湿地松苗圃的松猝倒病主要病原菌为立枯丝核菌(Rhizoctonia solani Kühn.)、链格孢菌(Alternaria alternata(Fr.)Keissl.)和尖孢镰刀菌(Fusarium oxysporum Schl.)。立枯丝核菌的致病力最强，第7d致病率可达92.6％，尖孢镰刀菌致病力次之，第7d的致病率最高可达89.5％，链格孢菌致病力较弱，第7d的致病率为48％，但第10d可达80％。(三)生物防治细菌的筛选与鉴定选择镰刀菌属(Fusarium)中致病力最强的6株病原菌为供试菌株，对湿地松根际320株细菌分离物进行了拮抗性筛选，共获得27株具有拈抗性的细菌菌株，其中12株具有弱的拮抗性，5株具有中等程度的拮抗性，10株为强的拮抗性细菌菌株；用16S rDNA测序分析方法，对10株拮抗细菌分离物进行了鉴定，7株为洋葱伯克霍尔德菌(Burkholderia cepacia)，2株为枯草芽孢杆菌(Bacillus subtilis)，1株待定。(四)松苗根际生物防治细菌定殖规律研究用美国引进的8株荧光假单胞菌(Pseudomonas fluorescens Q2-87，1M1-96，HT5-1，Pf-5，MVP1-4，FFL1R8，2-79，Q37-87)菌株和本土筛选得到的1株洋葱伯克霍尔德菌(Burkholderia cepacia C23)菌株进行平皿对峙试验，供试病原真菌为立枯丝核菌A、B，尖孢镰刀菌和链格孢菌：结果表明9株生防菌对链格孢菌和丝核菌的拮抗能力强，对镰刀菌的拮抗能力都较弱；其中C23和Q2-87对丝核菌和链格孢菌都表现出较好的拮抗作用；室内外根际接菌后的分子检测表明：两个菌株在湿地松幼苗根际都具有较强的定殖能力。本研究从松苗根际筛选得到本土生防细菌，并获得了确定的室内外拮抗效果，初步明确了生防细菌定殖规律，是我国松苗猝倒病生物防治研究的重要进展。本文研究结果为应用生物防治解决松苗猝倒病防治难题奠定了基础，而松苗猝倒病的生物防治研究将有利于促进林木病害生物防治工作的深入和提高。更多还原

【Abstract】 Slash pine(Pinus elliottii) is an important afforesting tree species in Hubei Province and in the southern China. The turpentine is also the important raw chemical material. During slash pine seedling production, damping-off can often cause severe damages to seedlings production and afforesting quality because of its endangering. Studies from China and other countries indicated that pine seedling damping-off is caused by a number of fungal pathogens including genera Rhizoctonia, Pythium, Fusarium, Alternaria, Botrytis, and Pestalotia etc. The kinas of fungal pathogens vary in different areas and localities. The objectives of this study were to study dominant pathogens of pine seedling damping-off; to determine the distribution and variation of rhizobacteria; using Pseudomonas fluorescens and Burkholderia cepacia and other introduced and native biocontrol agents, to elucidate colonization of biocontrol bacteria in rhizosphere, and to evaluate the effectiveness of disease suppression of the biocontrol bacteria with molecular technology. Slash pine seedlings were used as host plants in this study. Results and conclusions of the research are as follows:(1) Distribution and population dynamics of microorganisms in pine seedling rhizospherc.The large quantities of microorganisms were present in the soil of slash pine rhizosphere. Populations of fungi were 1×10~5～3.5×10~6 CFU(colony forming units)/g of soil, but populations of bacteria were 10~7～10~8CFU/g of soil. The peak of fungal population was in September, but that of bacteria is in April after transplantation. Proliferation of bacteria is faster than fungi, and the time of bacterial colonization on rhizoplane was also earlier than fungi.(2) Dominating pathogenic fungi of slash pine seedling damping-off and their pathogenicities.The predominant pathogens in slash pine nurseries of Hubei Province were Rhizoctonia solani Kühn, Fusarium oxysporum(Fr.) Keissl. and Alternaria alternata Schl.. R. solani was the most virulent, and its infection rate reached 92.6% at 7th day. F. oxysporum is the second and the highest infection rate is 89.5% at 7th day. The pathogenicity of A. alternata was 48% at 7th day and weaker than other pathogens, but able to reach to 80% at 10th day.(3) Screening and identifying biocontrol bacteria. We selected 6 strains of the strongest virulent pathogens in genus Fusarium as target. Antagonistic test was conducted on plates, 320 bacteria isolates originated from slash pine rhizosphere were used to screen antagonists. We gained 27 antagonistic bacteria strains by selective media, wherein 12 strains were weak antagonist, 5 strains, intermediate antagonists and 10 strains, strong antagonists. Then 10 strong antagonistic strains were identified by 16S rDNA(ribosomal DNA) sequence analysis. The results were 7 strains of Burkholderia cepacia Yabuuchi et al., 2 strains of Bacillus subtilis(Ehrenberg) Cohn and one unknown strain.(4)Colonization patterns of bioeontrol bacteria in the pine seedling rhizosphere.We examined the antagonism on plates using 8 strains of Pseudomonas fluorescens Q2-87, 1M1-96, HT5-1, Pf-5, MVP1-4, FFL1R8, 2-79, Q37-87 introduced from the USA and one indigenous Burkholderia cepacia strain C23 from pine nurseries. The target pathogenic fungi are Rhizoctonia solani A, B, Fusarium oxysporum and Alternaria alternata. Results confirmed that all biocontrol bacteria suppressed Alternaria and Rhizoctonia well, but showed weak antagonism against Fusarium. C23 and Q2-87 were assured to be better antagonist against Rhizoctonia and Alternaria. After inoculation on root in the greenhouse and field, the results applying molecular assay(terminal dilution assay) showed that both Pseudomonas fluorescens Q2-87 and Burkholderia cepacia C23 colonized well.This study had screened and obtained the indigenous biocontrol bacteria from pine seedling rhizosphere, achieved the assured antagonistic effects in the greenhouse and field, primarily eluciated the colonizing patterns of biocontrol bacteria. It is an important progress about biocontrol researches of pine seedling damping-off in China. The results of this study established the foundation of exploiting biological control and solving the difficult problem of controlling pine seedling damping-off. Furthermore, biocontrol research of pine damping-off will be helpful to promote biocontrol of forest root disease into a higher level.更多还原