我国松材线虫携带细菌的种类和分布及与松材线虫病的关系
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摘要
松材线虫病(Bursaphelenchus xylophilus)是松属树种上最重要的病害之一，该病传播媒介复杂，寄主种类多，危害性大，尽管人们已对其研究了近半个世纪，但是对该病的真正病原还不清楚。目前很多学者认为，细菌在该病的病理过程中起着重要的作用。并且有人相继从松材线虫体上分离到了芽孢杆菌属和假单胞杆菌属的一些种，为弄清楚松材线虫携带的细菌种类，研究我国松材线虫所携带的细菌在区域和寄主上的分布，以及这些细菌对该病究竟有无关系等问题。本研究着重从我国松材线虫病发生的不同疫区、不同发病期、不同寄主松树病死木采样，从病木直接分离松材线虫携带细菌，经过鉴定，并筛选了有致萎活性的菌株，证实了它们与松材线虫病的关系，同时也对松材线虫与各类优势致病性菌株之间相互关系进行了初步探讨，主要研究结果如下：
1．根据松材线虫病在我国的发生、发展、分布和蔓延，选择我国松材线虫病发生的有代表陛的疫区：江苏、安徽、上海、湖北、浙江和广东等，在2001年春秋两次进行采样。采集了感染松材线虫病的黑松、马尾松病木，采用木块组织分离法，分离松材线虫所携带的细菌58株。利用传统的细菌鉴定方法和自动化细菌鉴定系统相结合的手段，对这些细菌进行了鉴定。发现在病木组织中松材线虫的体上存在着大量的细菌，其优势种群有三类，主要是假单胞菌、其次是泛菌和不解糖消化链球菌。荧光假单胞和泛菌1几乎在所有采样疫区、感病的黑松和马尾松组织内松材线虫体上均可检测到，其出现频率明显高于其他细菌菌株。并且分析了松材线虫携带的细菌在区域和寄主上的分布情况。
2．以培养60天的无菌黑松切根苗为实验材料，用细菌菌株的培养过滤原液和浓缩液，在无菌条件下，测定了58个菌株培养物的致萎活性。结果表明松材线虫携带的三大类细菌中，有致萎活性的菌株是假单胞菌属和泛菌属的一些种。其中，荧光假单胞菌和泛菌1的部分菌株的培养液致萎活性最强，大部分泛菌和少量假单胞菌的一些种致萎活性次之，不解糖消化链球菌对无菌黑松切根苗没有致萎活性。通过对过滤原液和浓缩液致萎能力的测定结果表明，细菌产生的某些活性物质对热不稳定，在高温条件下，部分活性成分有所改变，发生变性、分解、或转化成无毒成分，这对探讨细菌毒素的成分，明确细菌致萎的分子机理有重要的意义。
3．选择致萎活性不同的各类代表菌株，以培养4个月的无菌黑松苗和10年生黑松为试验材料，进行室内和野外人工接种。结果显示，用细菌单独接种不致病，无菌松材线虫单独接种不致病或发病轻且慢，二者混合接种、有菌线虫接种均能使黑松苗和野外多年生黑松发病严重，不致萎的不解糖消化链球菌和无菌线虫混合接种，寄主不发病或发病轻且慢，由此认为假单胞菌和泛菌的一些种与松材线虫在致病性上有协同作用。
4．接种后再分离结果显示，细菌单独接种的植株内都没有分离到细菌，无菌线虫单
独接种的没感病植株，分离到了线虫，但没有分离到细菌。无菌线虫单独接种发病的和细
菌与无菌线虫混合接种发病的植株都分离到了大量的线虫和细菌。由此说明，单独细菌不
能侵染成功，不能在寄主体内正常生长繁殖和传递扩散，必需有线虫的携带和传播，才能
发挥其毒害作用，无菌松村线虫单独接种也不能使松树表现症状，只有当与其伴随的某些
特异的细菌存在时，线虫在松树组织内才可大量繁殖并迅速扩展，由此可见松村线虫所携
带的细菌对松村线虫的致病性或其生长发育有影响。
5．松村线虫对其体上携带细菌的生长发育和繁殖有不同程度的促进作用，其中对荧
光假单胞菌促进作用最强，泛菌次之，对不解糖消化链球菌几乎没有作用。荧光假单胞菌
和泛菌1伴随在线虫体上，也能促进线虫的生长发育和繁殖，尤其是荧光假单胞能明显提
高线虫的产卵量和繁殖速度，促进线虫的个体发育，从而提高了线虫的致病活性。
以上研究都说明，松村线虫病的发生是松材线虫与其携带的某些特异的细菌共同作用
的结果，某些细菌对松村线虫病的发生有协同作用。
Pine wilt disease is one of the most important forest disease in the world .Although it has been researched more than half a century ,its pathogenicity is not clear .At the present time ,most people thought that the bacteria play an important role in the process of wilting ,and the bacteria Bacillus and Poseudomonas have been isolated from the surface of the pine wood nematode .In order to understand fully the species of the bacteria carried by pine wood nematodes and their distribution .We isolated bacteria from the disease wood and studied the identification and pathogenicity of them. The main result were as follows:
1. According to the emergence, development, distribution and extention of pine wilt disease in china , the following areas,which were Jiangsu Province , Shanghai city, Guangdong Province , Anhui province ,Hubei province and Zhejiang province etc, were choosed as the typical diseased areas. 58 bacterial strains were isolated from the diseased wood of pinus thunbergii or pinus massoniana which was infected by pine wood nematode in the spring and autumn of 2001, and identified by traditional methods and automatic bacteria identification system. Results showed that there were three dominant genus which belong to Pseudomonas, Pantoeu and Peptostreptococcus , Among those strains, some species of Pseudomonas and Pantoeu existed in different areas, different hosts ,and had wider distribution and higher frequent emergences than others. The results proved that the above-mentioned two species were the most dominant.
2. The toxicity of 58 bacterial strains were tested with the bacteria cultural filtered original liquid and concentrated liquid and aseptic black pine seedlings cultured for 60d under the aseptic condition. Results revealed that Pseudomonas, Pantoeu was mainly responsible for rapid wilting of pine trees, among which the activity of parts of Pseudomonas fluroscence, Pantoeu 1 were higher,. While that only a few of Pseudomonas strains and the most Pantoeu were the second, furthermore Peptostreptococcus asaccharolyticus has no wilt activity . the determination comparision between wilting activity of the filtered original and concentrated liquid showed that the active substances produced by bacteria was unstable in high temperature, and the active ingredients could be changed or decomposed to be inactive. The above results were significant in probing the elements of bacterial toxic substances and pathgenic mechanism of bacteria .
3. With aseptic black pine seedlings cultured for 4 months and 10 years black pine young trees , the artificial inoculation experiments in doors and outdoors were conducted
after choosing representative bacteria strains. The pathogenesis tests indicated that aseptic seedings and pinus thunberg wilting diseases did not occurred or occurred slowly and lightly as being inoculated with aseptic nematode ,and did not occurred too as being inoculated with bacteria separately,while inoculated with mixed suspension of them ,rapid wilting of pine trees can be caused.When we inoculate the combination of Peptostreptococcus asaccharolyticus and aseptic nematodes , black pine were still safe. Based on the results ,it was suggested that there was certain coorperation between nematode and some species of Pantoeu and Poseudomomas for the disease.
4. The reisolation results after inoculation showed that no bacteria was isolated in the healthy black pine inoculated with only bacteria or aseptic pine wood nematode, but a few of nematodes were isolated from the healthy black pine inoculated with only aseptic pine wood nematode. A lot of pine wood nematode and bacterium were isolated from the wilting black pine inoculated with the mixed suspension of aseptic pine wood nematode and bacteria. This suggested only bacteria can not invade successfully.grow and repropagate normally in host, transmit in pine tissues, if no nematode existence. While carried by nematode ,the bacteria can penetrate into pine tissues and develop toxic action normally. On the other hand , the aseptic ne
1．根据松材线虫病在我国的发生、发展、分布和蔓延，选择我国松材线虫病发生的有代表陛的疫区：江苏、安徽、上海、湖北、浙江和广东等，在2001年春秋两次进行采样。采集了感染松材线虫病的黑松、马尾松病木，采用木块组织分离法，分离松材线虫所携带的细菌58株。利用传统的细菌鉴定方法和自动化细菌鉴定系统相结合的手段，对这些细菌进行了鉴定。发现在病木组织中松材线虫的体上存在着大量的细菌，其优势种群有三类，主要是假单胞菌、其次是泛菌和不解糖消化链球菌。荧光假单胞和泛菌1几乎在所有采样疫区、感病的黑松和马尾松组织内松材线虫体上均可检测到，其出现频率明显高于其他细菌菌株。并且分析了松材线虫携带的细菌在区域和寄主上的分布情况。
2．以培养60天的无菌黑松切根苗为实验材料，用细菌菌株的培养过滤原液和浓缩液，在无菌条件下，测定了58个菌株培养物的致萎活性。结果表明松材线虫携带的三大类细菌中，有致萎活性的菌株是假单胞菌属和泛菌属的一些种。其中，荧光假单胞菌和泛菌1的部分菌株的培养液致萎活性最强，大部分泛菌和少量假单胞菌的一些种致萎活性次之，不解糖消化链球菌对无菌黑松切根苗没有致萎活性。通过对过滤原液和浓缩液致萎能力的测定结果表明，细菌产生的某些活性物质对热不稳定，在高温条件下，部分活性成分有所改变，发生变性、分解、或转化成无毒成分，这对探讨细菌毒素的成分，明确细菌致萎的分子机理有重要的意义。
3．选择致萎活性不同的各类代表菌株，以培养4个月的无菌黑松苗和10年生黑松为试验材料，进行室内和野外人工接种。结果显示，用细菌单独接种不致病，无菌松材线虫单独接种不致病或发病轻且慢，二者混合接种、有菌线虫接种均能使黑松苗和野外多年生黑松发病严重，不致萎的不解糖消化链球菌和无菌线虫混合接种，寄主不发病或发病轻且慢，由此认为假单胞菌和泛菌的一些种与松材线虫在致病性上有协同作用。
4．接种后再分离结果显示，细菌单独接种的植株内都没有分离到细菌，无菌线虫单
独接种的没感病植株，分离到了线虫，但没有分离到细菌。无菌线虫单独接种发病的和细
菌与无菌线虫混合接种发病的植株都分离到了大量的线虫和细菌。由此说明，单独细菌不
能侵染成功，不能在寄主体内正常生长繁殖和传递扩散，必需有线虫的携带和传播，才能
发挥其毒害作用，无菌松村线虫单独接种也不能使松树表现症状，只有当与其伴随的某些
特异的细菌存在时，线虫在松树组织内才可大量繁殖并迅速扩展，由此可见松村线虫所携
带的细菌对松村线虫的致病性或其生长发育有影响。
5．松村线虫对其体上携带细菌的生长发育和繁殖有不同程度的促进作用，其中对荧
光假单胞菌促进作用最强，泛菌次之，对不解糖消化链球菌几乎没有作用。荧光假单胞菌
和泛菌1伴随在线虫体上，也能促进线虫的生长发育和繁殖，尤其是荧光假单胞能明显提
高线虫的产卵量和繁殖速度，促进线虫的个体发育，从而提高了线虫的致病活性。
以上研究都说明，松村线虫病的发生是松材线虫与其携带的某些特异的细菌共同作用
的结果，某些细菌对松村线虫病的发生有协同作用。
Pine wilt disease is one of the most important forest disease in the world .Although it has been researched more than half a century ,its pathogenicity is not clear .At the present time ,most people thought that the bacteria play an important role in the process of wilting ,and the bacteria Bacillus and Poseudomonas have been isolated from the surface of the pine wood nematode .In order to understand fully the species of the bacteria carried by pine wood nematodes and their distribution .We isolated bacteria from the disease wood and studied the identification and pathogenicity of them. The main result were as follows:
1. According to the emergence, development, distribution and extention of pine wilt disease in china , the following areas,which were Jiangsu Province , Shanghai city, Guangdong Province , Anhui province ,Hubei province and Zhejiang province etc, were choosed as the typical diseased areas. 58 bacterial strains were isolated from the diseased wood of pinus thunbergii or pinus massoniana which was infected by pine wood nematode in the spring and autumn of 2001, and identified by traditional methods and automatic bacteria identification system. Results showed that there were three dominant genus which belong to Pseudomonas, Pantoeu and Peptostreptococcus , Among those strains, some species of Pseudomonas and Pantoeu existed in different areas, different hosts ,and had wider distribution and higher frequent emergences than others. The results proved that the above-mentioned two species were the most dominant.
2. The toxicity of 58 bacterial strains were tested with the bacteria cultural filtered original liquid and concentrated liquid and aseptic black pine seedlings cultured for 60d under the aseptic condition. Results revealed that Pseudomonas, Pantoeu was mainly responsible for rapid wilting of pine trees, among which the activity of parts of Pseudomonas fluroscence, Pantoeu 1 were higher,. While that only a few of Pseudomonas strains and the most Pantoeu were the second, furthermore Peptostreptococcus asaccharolyticus has no wilt activity . the determination comparision between wilting activity of the filtered original and concentrated liquid showed that the active substances produced by bacteria was unstable in high temperature, and the active ingredients could be changed or decomposed to be inactive. The above results were significant in probing the elements of bacterial toxic substances and pathgenic mechanism of bacteria .
3. With aseptic black pine seedlings cultured for 4 months and 10 years black pine young trees , the artificial inoculation experiments in doors and outdoors were conducted
after choosing representative bacteria strains. The pathogenesis tests indicated that aseptic seedings and pinus thunberg wilting diseases did not occurred or occurred slowly and lightly as being inoculated with aseptic nematode ,and did not occurred too as being inoculated with bacteria separately,while inoculated with mixed suspension of them ,rapid wilting of pine trees can be caused.When we inoculate the combination of Peptostreptococcus asaccharolyticus and aseptic nematodes , black pine were still safe. Based on the results ,it was suggested that there was certain coorperation between nematode and some species of Pantoeu and Poseudomomas for the disease.
4. The reisolation results after inoculation showed that no bacteria was isolated in the healthy black pine inoculated with only bacteria or aseptic pine wood nematode, but a few of nematodes were isolated from the healthy black pine inoculated with only aseptic pine wood nematode. A lot of pine wood nematode and bacterium were isolated from the wilting black pine inoculated with the mixed suspension of aseptic pine wood nematode and bacteria. This suggested only bacteria can not invade successfully.grow and repropagate normally in host, transmit in pine tissues, if no nematode existence. While carried by nematode ,the bacteria can penetrate into pine tissues and develop toxic action normally. On the other hand , the aseptic ne
引文
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