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松树枯萎病的流行与危害已导致世界森林生态系统产生了严重危机[1]。对松枯萎病的发生与流行机制的研究仍在开展与不断的深入。自从日本报道松材线虫(Bursaphelenchus xylophilus) 能引起松树枯萎后,松材线虫已被认为是松材线虫病的病原[2-3],我国把松树枯萎病称为松材线虫病。但是随着研究的深入,发现单纯地仅以松材线虫为病原还不能解释自然界复杂多样的松树枯死现象,可能是一种(或一类)松材线虫的伴生菌参与了致死松树或产生毒素导致松树枯萎[4-11]。进入本世纪后, 有关枯萎松树内松材线虫的伴生细菌种类和病理作用及与松材线虫的关系研究报道越来越多, 拓展和深化了松树枯萎病研究范围和层次[12-15]。但现在面临的问题是,随着分离鉴定的细菌种类越来越多[16-18],要确定究竟是哪种细菌与松材线虫最为密切,哪种细菌具有真正病原作用参与了松树枯死的过程等问题,需要深入研究。荧光假单胞杆菌(Pseudomonas fluorescens)是众多枯死松树内细菌中鉴定到种研究最多的菌种,但报道的只是在实验室得到的可引起黑松(Pinus thunbergii Parl.)苗木或细胞枯萎的一些结果[19-22],没有在林间进行调查来证实荧光假单胞杆菌协同松材线虫促使了松树枯萎。而且荧光假单胞杆菌是一种动物病原细菌,也是一种能抑制或拮抗植物病原细菌的生物防治细菌[23]。很少有动物病原细菌也能同时成为植物病原细菌的,这是因为动物病原细菌不能分泌产生纤维素酶,植物细胞的细胞壁对动物病原细菌具有天然的免疫性。松材线虫的耐久性幼虫(LⅣ幼虫)是侵入松树的第一虫态,也是离开松树的最终虫态[24]。LⅣ幼虫体上是否携带细菌,带的是何种细菌十分值得研究。因为LⅣ幼虫如携带细菌,表明这种细菌与松材线虫的关系密切,同时与松材线虫、松褐天牛一起参与了松树枯萎的全过程。在LⅣ侵入松树时,携带的细菌也直接进入了松树;在LⅣ离开松树时,这种细菌也被携带离开松树,该种细菌最有可能就是引起松树枯萎的病原细菌。按这个思路,作者开展了从松材线虫LⅣ幼虫体上分离鉴定细菌的研究,获得了香茅醇假单胞杆菌(Pseudomonas citronellolis Seubert),现将研究结果报道如下。
松材线虫(Bursaphelenchus xylophilus)耐久型幼虫(LⅣ)携带的香茅醇假单胞杆菌(Pseudomonas citronellolis)分离与鉴定
Isolated and Identified of the Bacteria Pseudomonas citronellolis from the Dauerlarva of Bursaphelenchus xylophilus (LⅣ)
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摘要:
目的 目前有关松材线虫与伴生细菌的关系及伴生细菌的病原作用是松树枯萎病研究的重点。为了揭示松材线虫与伴生细菌之间存在的密切关系,作者对松材线虫LⅣ幼虫携带的细菌进行了分离鉴定。 方法 根据培养性状和16S rDNA序列同源性以及系统发育学等方面进行分析鉴定。 结果 确定LⅣ幼虫携带的是香茅醇假单胞杆菌(Pseudomonas citronellolis),携带率为100%;每条LⅣ幼虫携带量在1.4×105~4.5×105。LⅣ幼虫生活在松褐天牛体内,是引起松材线虫病侵染流行的唯一虫态;新发现的香茅醇假单胞杆菌能分解纤维素及降解或合成萜烯和酚类化合物。 结论 LⅣ幼虫携带香茅醇假单胞杆菌的发现,揭示了松树、松褐天牛、松材线虫、细菌同为一体的紧密关系,并为揭示松树枯萎病机制提供了一种新病原和重要的研究思路。 Abstract:Objective This study is to reveal the relationship of pathogenic bacteria associated with the pine wood nematode. Method The bacteria carried by the dauerlarvae of Bursaphelenchus xylophilus (LⅣ) were isolated, and were identified with 16S rDNA sequence homology and phylogenetic analysis. Result It was showed that LⅣ could carry a large amount of bacteria and the carrying rate was as high as 100%; the amount of the bacteria carried was 1.4×105~4.5×105 on each LⅣ. The bacteria strain isolated from LⅣ was identified as Pseudomonas citronellolis. The 16S rDNA sequence of the strain No.14 was 1 448 bp in length and the similarity of P. citronellolis was 99% comparing with the data in the gene bank. Conclusion The preliminary conclusion is that the LⅣ with P. citronellolis formed a group of the compound pathogen; LⅣ is the only one linking pine tree with the longhorned beetle Monochamus alternatus; the bacteria of P. citronellolis could decompose cellulose and degrade or synthesis terpenes and phenolic compounds; The present discovery of LⅣ carrying P. citronellolis suggests that the pine tree, Monochamus alternatus, Bursaphelenchus xylophilus and P. citronellolis may be a compacted system, and perhaps it may reveal a part of the mechanisms that the bacteria is a main factor causing pine wilt disease and killing pine tree die finally. -
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[1] Paulo V, Manuel M. PWD: A Worldwide Threat to Forest Ecosystems[M]. Springer, 2008. [2] Kiyohara T, Tokushige Y. Inoculation experiments of nematode, Bursaphelenchus sp., onto pine trees[J]. Journal of Japanese Forest Society, 1971, 53(7):210-218. [3] Mamiya Y. Reproduction of pine lethal wilting disease by the inoculation of young trees with Bursaphelenchus lignicolus[J]. Japanese Journal of Nematology, 1972, 2:40-44. [4] Oku H, Shiraishi T, Kurozumi S. Pine wilt toxin, the metabolite of a bacteria associated with a nematode[J]. Naturwissenschaften, 1980, 67:198-199. [5] Tada M, Omizu A, Shiroishi M. Effect of phosphate on the growth and toxin-producing ability of a becterum isolated from pine wood nematode Bursaphelenchus lignicolus[J]. Science Report of Faculty Agriculture, Okayama University, Japan, 1981, 57:35-40. [6] Kawazu K, Zhang H, Kanazuki H. Accumulation of benzoic acid in suspension culture cells of Pinus thunbergii Parl. In response to phenyl acetic acid administration[J]. Bioscience Biotechnology and Biochemistry, 1996, 60:1410-1412. doi: 10.1271/bbb.60.1410 [7] Kawazu K, Zhang H, Yamashita H, et al. Relationship between the pathogenicity of the pine wood nematode, Bursaphelenchus xylophilus[J]. Bioscience Biotechnology and Biochemistry, 1996, 60:1413-1415. doi: 10.1271/bbb.60.1413 [8] Kawazu K, Kaneko N. Asepsis of the pine wood nematode isolate OKD-3 causes it to lose its pathogenicity[J]. Japanese Journal of Nematology, 1997, 27:76-80. doi: 10.3725/jjn1993.27.2_76 [9] Kawazu K, Kaneko N, Hiraoka K, et al. Reisolation of the pathogens from wilted red pine seedlings inoculated with the bacterium-carrying nematode, and the cause of difference in pathogenicity among pine wood nematode isolates[J]. Science Report of Faculty Agriculture, Okayama University, Japan, 1999, 88:1-5. [10] 曹越, 韩正敏, 李传道.松材线虫病感病松树中致萎毒性物质的研究[J].林业科学, 2001, 37(4):75-79. doi: 10.3321/j.issn:1001-7488.2001.04.012 [11] Han Z M, Hong Y D, Zhao B G. A study on pathogenicity of bacteria carried by pine wood Nematode[J]. Journal of Phytopathology, 2003, 151:683-689. doi: 10.1046/j.1439-0434.2003.00790.x [12] 赵博光, 郭道森, 高蓉, 等.细菌分离物b619与松材线虫病关系的初步研究[J].南京林业大学学报:自然科学版, 2000, 24(4):72-74. [13] 赵博光, 郭道森, 高蓉.松材线虫携带细菌部位的电镜观察[J].南京林业大学学报:自然科学版, 2000, 24(4):69-71. [14] Zhao B G, Wang H L, Han S F. Distribution and pathogenicity of bacteria species carried by Bursaphelenchus xylophilus in China[J]. Nematology, 2003, 5:899-906. doi: 10.1163/156854103773040817 [15] Zhao B G, Lin F. Mutualistic symbiosis between Bursaphelenchus xylophilus and bacteria of the genus Pseudomonas[J]. Forest Pathology, 2005, 35:339-345. doi: 10.1111/efp.2005.35.issue-5 [16] 谢立群, 巨为云, 赵博光.松材线虫病程中树体内线虫和细菌种群数量动态变化[J].林业科学, 2004, 40(4):124-129. doi: 10.3321/j.issn:1001-7488.2004.04.021 [17] 王慧利, 韩素芬, 赵博光.松材线虫携带细菌在疫区和寄主中分布及致病性研究[J].北京林业大学学报, 2004, 26(4):48-53. [18] 巨为云, 谢立群, 杨雪云, 等.不同来源松材线虫携带的细菌多样性[J].东北林业大学学报, 2008, 36(5):84-85. doi: 10.3969/j.issn.1000-5382.2008.05.032 [19] 池树友, 何月秋, 韩正敏, 等.松材线虫及携带细菌对黑松复合侵染[J].福建林学院学报, 2008, 28(1):92-96。 doi: 10.3969/j.issn.1001-389X.2008.01.022 [20] 李盛楠, 郭道森, 赵博光, 等.萤光假单孢杆菌鞭毛蛋白对黑松细胞的致死作用[J].西北植物学报, 2008, 28(11):2154-2158. doi: 10.3321/j.issn:1000-4025.2008.11.002 [21] 赵博光, 李良.萤光假单孢杆菌培养的无细胞滤液导致松萎蔫的病状观察[J].江西农业大学学报, 2008, 30(4):575-580. doi: 10.3969/j.issn.1000-2286.2008.04.001 [22] 孔令营, 郭道森, 赵博光, 等.荧光假单胞菌gcm5-1a胞外木质素过氧化物酶的初步纯化及性质研究[J].北京林业大学学报, 2010, 32(3):112-116. [23] 陈功友, 郑铁民, 毛庆裕.萤光假单胞杆菌和芽孢杆菌防治苹果叶果病害的研究[J].中国生物防治学报, 1993, 9(4):163-166. [24] Lai Y X. Distribution of nematode (Bursaphelenchus xylophilus) in the beetle Monochamus alternatus and its exiting transmission way. PWD: a worldwide threat to forest ecosystems by Paulo Vieira[C]. Manuel Moto, Springer, 2008, 243-254. [25] Kim O S, Cho Y J, Lee K, et al. Introducing EzTaxon-e:a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species[J]. International Journal of Systematic and Evolutionary Microbiology, 2012, 62:716-721. doi: 10.1099/ijs.0.038075-0 [26] Anzai Y, Kim H, Park J Y, et al. Phylogenetic affiliation of the Pseudomonas based on 16Sr RNA Sequence[J]. International Journal of Systematic and Evolutionary Microbiology, 2000, 50(4):1563-1589. doi: 10.1099/00207713-50-4-1563 [27] 平立岩.细菌泳动时的细胞取向:理论模拟与实验测定的研究进展[J].中国科学:生命科学, 2012, 42(4):259-268. [28] 来燕学.松树枯萎病枝条内的寄生虫和微生物群落[J].南京林业大学学报, 2006, 29(1):49-53. doi: 10.3969/j.issn.1000-2006.2006.01.012 [29] 来燕学.(拟)松材线虫耐久型四龄幼虫(LIV)的形态及其分化变异[J].林业科学研究, 2009, 22(2):294-298. doi: 10.3321/j.issn:1001-1498.2009.02.024 [30] Seubert W. Degradetion of isoprenoid compounds by micro-orgamisms isolation and characterization of an isoprenoid-degrading bacterium, Pseudomonas citrollolis, n. sp[J]. Journal of Bacteriology, 1960, 79:426-434. [31] 李平, 王焰新, 刘琨, 等.高效纤维素降解菌系的构建[J].地球科学-中国地质大学学报, 2009, 30(3):533-538. doi: 10.3321/j.issn:1000-2383.2009.03.018 [32] Norkraus B. Dedradation of cellulose[J]. Annual Review of Phytopathology, 1963, 1:325-350. doi: 10.1146/annurev.py.01.090163.001545 [33] Odani K, Sasaki S, Nishiyama Y, et al. Early symptom development of the pine wilt disease by hydrolytic enzymes produced by the pine wood nematode-cellulase as a possible candidate of the pathogen[J]. Journal of Japanese Forestry, 1985, 67(9):366-372. [34] Yamamoto N, Odani K, Sasaki S, et al. Cellulase exudation by the pine wood nematode -detection of activity in its crawling track[J]. Journal of the Japanese Forestry Society, 1986, 68(6):237-240. [35] Kusunoki M. Symptom development of pine wilt disease-histopathological observations with electionmicroscopes[J]. Ann Phytopath Soc Japan, 1987, 53(5):622-629. doi: 10.3186/jjphytopath.53.622 [36] Seubert W, Remberger U. Purification and mechanism of action of pyruvate carboxylase from Pseudomonas citrollolis[J]. Biochemistry, 1961, 334:401-414. [37] 史江红, 韩蕊, 宿凌燕, 等.某河水处理中17α-乙炔基雌二醇降解菌的分离鉴定及其降解特性[J].环境科学学报, 2010, 30(12):2411-2419. [38] Fall R R, Brown J L, Schaeffer T L. Enzyme recruitment allows the biodegradation of recalcitrant branched hydrocarbons by Pseudomonas citrollolis[J]. Applied and Environmental Microbiology, 1979, 38:715-722. [39] Choi Muv Huan, Sung Chul Yoon. Polyester Biosynthesis Characteristics of Pseudomonas citrollolis Grown on various carbon sources, including 3-Methy-Branched substrates[J]. Applied Environment Microbiologis, 1994, 60:3245-3254. [40] Scheffer T C, Cowling E B. Natural resistance of wood to microbial deterioration[J]. Annual Review of Phytopathology, 1966, 4:147-170. doi: 10.1146/annurev.py.04.090166.001051 [41] Goss J A, Cohen N D, Utter M F. Characterization of the subunit structure of Pyruvate carboxylase from Pseudomonas citronellolis[J]. The Journal of Biological Chemistry, 1981, 256(22):11819-11825. [42] Mina L, Hector R, Ray F. Multiple Acyl-Coenzyme a carboxylase in Pseudomonas citronellolis[J]. Biochemistry, 1976, 15(16):3465-3472. doi: 10.1021/bi00661a011