松材线虫是重要的外来入侵生物，对我国的林业和生态环境造成了极大的威胁。松材线虫在入侵松树的过程中，会受到松树防御性化合物的胁迫。已有研究表明这些化合物可以抑制松材线虫的繁殖，目前尚不能明确松材线虫是否具有降解这些防御性化合物的能力。松材线虫体表和体内均存在着大量的共生细菌，本实验室前期在对松材线虫一个地理种群的共生细菌进行宏基因组分析时，发现松材线虫共生细菌的基因组中存在大量可以参与代谢这些化合物的基因。因此，我们推断松材线虫的共生细菌可能协助松材线虫降解寄主的防御性代谢化合物，从而提高松材线虫对寄主的适应性。 本研究旨在通过明确松树挥发性防御性化合物中对松材线虫行为影响最大的物质，筛选出松材线虫共生细菌中对这类化合物敏感的共生细菌菌株，并利用熏蒸法研究主要防御性化合物对线虫生长和繁殖的影响，以及对其共生细菌数量和多样性的影响。通过测试敏感菌株对化合物的耐受性和降解能力，明确松材线虫在受到防御性化合物胁迫条件下这些共生细菌是否可以起到协助作用。本研究的主要研究过程和结论如下： 1. 利用气质联用仪检测了美国、辽宁、浙江发生松材线虫病害的松木中的挥发性防御化合物的成分。发现α-蒎烯是3个样品中共有的主要成分。通过检测接种松材线虫的松苗挥发性防御化合物成分的变化情况，表明松苗在受到松材线虫的侵染后，化合物种类明显减少；但部分化合物相对含量增加，其中α-蒎烯增加最多。利用Pluronic F-127胶系统，测试松树挥发性化合物的提取液、α-蒎烯、β-蒎烯、月桂烯和石竹烯对松材线虫的吸引作用。结果显示，松树挥发性化合物提取液和α-蒎烯对线虫具有明显的吸引作用，其次是月桂烯和β-蒎烯，石竹烯对松材线虫几乎没有吸引作用。 2. 本研究采用接触法和熏蒸法，测试α-蒎烯对松材线虫的影响。通过接触法检测发现α-蒎烯对松材线虫的致死中浓度为92.93 mg/mL。利用不同水平的α-蒎烯熏蒸自然松材线虫和经过抗生素处理的少菌松材线虫，发现处理48h后，两线虫群体经蒎烯熏蒸的处理死亡率均高于空白对照，其中自然线虫群体比少菌线虫群体对α-蒎烯的影响更为敏感。以灰葡萄孢作为线虫的食源，蒎烯熏蒸培养5天后统计线虫数量。结果表明α-蒎烯可以抑制松材线虫繁殖，且自然线虫和少菌线虫在不同水平蒎烯熏蒸下繁殖力变化的趋势不同。 3. 采用熏蒸法测试α-蒎烯对松材线虫共生细菌的影响。细菌菌落计数结果表明，α-蒎烯熏蒸处理松材线虫后，自然松材线虫共生细菌的数量变化较为明显。中水平蒎烯中菌量最多；高水平中菌量最低，且低于空白对照。少菌线虫群体共生细菌的量在各水平蒎烯处理后均只有少量增加，差异不明显。实时荧光绝对定量检测结果与菌落计数结果中的变化趋势基本一致，两个群体中都是在中低浓度蒎烯条件下，细菌菌量较高。高通量测序结果显示，自然松材线虫群体OTU数量多于少菌线虫群体。经蒎烯处理自然线虫共生细菌的种群多样性明显降低。少菌线虫菌群的丰富度显升高。两组线虫的共生细菌中优势属均为Pseudomonas spp.和Stenotrophomonas spp.，自然线虫细菌中Pseudomonas spp.占细菌总量的70%以上，Stenotrophomonas spp.占20%左右。少菌线虫细菌中Stenotrophomonas spp.占比约70%，Pseudomonas spp.约占16%。经α-蒎烯处理后，Pseudomonas sp.相对含量增加，而Stenotrophomonas spp.、Delftia spp. 相对含量减少。 4．通过扫描电镜和荧光原位杂交观察发现，松材线虫体表和体内均存在着大量的共生细菌。本研究以蒎烯作为唯一碳源的固体培养基，对34个地理种群松材线虫进行耐受α-蒎烯菌株的筛选，分离得到了234个耐受菌株。通过16S测序鉴定出28个属种。其中优势种群为Achromobacter sp.、Pseudomonas sp.、Agrobacterium spp.、Novosphingobium sp.、Cupriavidus sp.、Rahnella sp.、Stenotrophomonas sp.等。利用不同筛选方法培养前期实验中的自然线虫和少菌线虫群体中的耐受蒎烯的菌株。结果表明两个线虫群体中共有的耐受菌株属种有Stenotrophomonas sp.、Pseudomonas sp.、Achromobacter sp.、Staphylococcus sp.和Ochrobactrum sp等。 5. 选取6个耐受α-蒎烯的优势菌株，即Achromobacter sp.、Pseudomonas sp.、Stenotrophomonas maltophilia、Cupriavidus sp.、Stenotrophomonas sp.和Pseudomonas putida，以蒎烯作为唯一碳源进行培养。通过监测细菌的生长情况和蒎烯的量，验证优势耐受菌株对蒎烯的降解能力。实验结果表明Stenotrophomonas maltophilia、Pseudomonas sp.、Cupriavidus sp.、Stenotrophomonas sp.可以在24h后菌量逐渐增加。但只有其中2个Pseudomonas sp.中蒎烯的量明显减少，说明其可以有效地降解蒎烯，并检测得到Pseudomonas sp.的代谢产物主要为松香芹醇、桃金娘醇和5-叔丁基间伞花烃等。通过与前人研究中蒎烯降解途径中的代谢产物进行对比，推测其可能具有3个代谢途径。 根据以上的研究结果，我们可以确定松材线虫共生细菌中存在可以降解蒎烯的功能菌株，在松材线虫受到蒎烯胁迫时，功能菌株的量增加明显。因此我们认为材线虫共生细菌可以协助松材线虫降解寄主防御性化合物α-蒎烯，提高松材线虫对松树防御性化合物的适应性，有利于松材线虫的入侵定殖。

Pinewood nematode, Bursaphelenchus xylophilus, is an important invasive species, which has a great threat to China's forestry and ecological environment. In the process of invading pine trees, pinewood nematodes are bound to be stressed by high-concentration pine defensive compounds. It is not yet clear whether pinewood nematodes have the ability to degrade these defensive compounds, although some studies have shown that these compounds can inhibit the growth of pinewood nematodes. There are a large number of symbiotic bacteria in the surface and the body of pinewood nematode. Through the metagenomic analysis of the symbiotic bacteria of a geographical population of pinewood nematodes in the early stage, it was found that the genes involved in detoxification metabolism, in the genome of the pinewood nematode symbiotic bacteria were very rich. Therefore, we conclude that the pinewood nematodes and commensal bacteria may complement each other in the detoxification metabolic pathway possibly, and the symbiotic bacteria assist the pinewood nematodes to degrade the defensive metabolic compounds of pine, and improve the adaptability of the pinewood nematode to the host. The aim of this study was to find out strains that they were sensitive to these compounds in pine nematode symbiotic bacteria by identifying the types of pine volatile defensive compounds which have the greatest impact on pinewood nematodes, and to test the tolerance and degradation of these compounds to the compounds. This research can clear the role that these pine worms can play together under the stress of compounds. The main research processes and conclusions of this study are as follows： 1. Volatile defense compounds in pine wood infected with pinewood nematode in the United States, Liaoning and Zhejiang, were detected by GC-MS. It was found that α-pinene was the main component in three samples. The change of volatile defense compounds in pine seedlings inoculated with pinewood nematode was analyzed. The results showed that the variety of volatile defense compounds in pine seedlings reduced significantly after being infected by pinewood nematode. The relative content of some compounds increased, and the most increased was detected for the α-pinene. Pluronic F-127 gum system was used to test the attraction of pinewood nematodes by extracts of pine volatile compounds, α-pinene, β-pinene, laurelene and caryophyllene. The extracts of volatile compounds from pine trees and α-pinene had obvious attraction to nematodes, followed by laurelene and β-pinene. Caryophyllene had little attraction to pine wood nematode. 2. The effects of α-pinene on pinewood nematode were tested by contact process and fumigation method. The median lethal concentration of α-pinene to pinewood nematodes was 92.93 mg/mL. Different levels of α-pinene were used to fumigate natural pinewood nematodes and antibiotic-treated pinewood nematodes. After 48 hours of treatment, the mortality of two nematodes fumigated with pinene was higher than that of control. Natural nematodes were more sensitive to the effects of α-pinene than those of the antibiotic-treated nematodes. The nematode fecundity was counted after 5 days of cultivation with Botrytis cinerea. The results showed that α-pinene could inhibit the reproduction of pinewood nematode, and the change of fecundity of the natural nematodes under three levels of pinene was different from that of the antibiotic-treated nematodes. 3. The effect of α-pinene on the symbiotic bacteria of pinewood nematodes was tested by fumigation. The results of bacterial colony counts showed that the number of symbiotic bacteria of natural pinewood nematode changed significantly after fumigation with α-pinene. The highest of the amount of bacteria was detected for the medium pinene. The lowest was the high pinene, which was lower than that in control. The amount of symbiotic bacteria increased slightly at all levels of pinene treatment in the antibiotic-treated pinewood nematodes, but the difference was not significant. The results of real-time absolute quantitative fluorescence detection and colony counting were similar. In both groups, the amount of bacteria was higher under the medium and low level of pinene. The results of high throughput sequencing showed that the number of OTU in natural pinewood nematodes was more than that in the antibiotic-treated pinewood nematodes. The diversity of natural nematode symbiotic bacteria treated with pinene decreased significantly. The richness of antibiotic-treated pinewood nematodes treated with pinene increased significantly. Pseudomonas spp. and Stenotrophomonas spp. were the dominant bacteria in both groups. Pseudomonas spp. accounted for more than 70% of the total bacteria in natural nematode bacteria and Stenotrophomonas spp. accounted for about 20%. Stenotrophomonas spp. accounted for 70% and Pseudomonas spp. accounted for 16%. The relative content of Pseudomonas sp. increased while that of Stenotrophomonas spp. and Delftia spp. decreased after treatment with α-pinene. 4. The photos of scanning electron microscopy and fluorescence in situ hybridization showed that a large number of symbiotic bacteria existed on the surface and in vivo of pinewood nematode. In the study, pinene was used as the sole carbon source in solid medium to screen strains resistant to α-pinene from 34 geographical populations of pinewood nematode, and 234 strains were isolated. Twenty-eight genera and species were identified by 16S sequencing. The dominant populations were Achromobacter sp., Pseudomonas sp., Agrobacterium spp., Novosphingobium sp., Cupriavidus sp., Rahnella sp., Stenotrophomonas sp. Different isolation methods were used to cultivate pinene-tolerant strains of natural nematodes and the antibiotic-treated nematodes which was tested in previous experiments. The results showed that the common tolerant strains in the two nematode populations were Stenotrophomonas sp., Pseudomonas sp., Achromobacter sp., Staphylococcus sp. and Ochrobactrum sp. 5. Six dominant strains, Achromobacter sp., Pseudomonas sp., Stenotrophomonas maltophilia, Cupriavidus sp., Stenotrophomonas sp. and Psudomonas putida, which were selected to culture with pinene as the sole carbon source. By monitoring the growth of bacteria and the amount of pinene, the degradation ability of pinene was verified. The results showed that Stenotrophomonas maltophilia, Pseudomonas sp., Cupriavidus sp., and Stenotrophomonas sp. could gradually increase after 24 hours. But only two of them, Pseudomonas sp., showed a significant degradation for the pinene. The metabolites of Pseudomonas sp. were mainly pinocarveol, Myrtenol and 5-tert-butyl cymene. Compared with the metab-olites in pinene degradation pathway in previous studies, we speculate that there may be three metabolic pathways. Based on our current results, we can confirm that there are functional strains which can degrade pinene in the symbiotic bacteria of pinewood nematode. When the reproduction of pinewood nematode was stressed by pinene, the amount of functional strains was increased obviously. Therefore, we believe that the symbiotic bacteria of pinewood nematode can help pinewood nematode to degrade the α-pinene, improve the adaptability of pinewood nematode to defensive compounds of pine trees, and facilitate the invasion and colonization of pinewood nematode.