无菌和带菌松材线虫对赤松的致病性
|
摘要
【目的】探讨无菌和带菌松材线虫对赤松的致病性,明确松材线虫在松树萎蔫病中的地位,为致病机制研究及病害防控提供依据。【方法】以赤松胚性愈伤组织为材料,每块组织接种无菌松材线虫250条,培养12天后,利用2,3,5-三苯基氯化四氮唑(TTC)染色法对愈伤组织进行染色,分析无菌松材线虫对赤松愈伤组织细胞活性的影响。以赤松组培苗和4年生盆栽实生苗为对象,每株接种无菌松材线虫和带菌松材线虫(组培苗250条,4年生苗3 000条),分别于18天和35天后统计2种类型线虫接种所致松苗萎蔫率,并对植株体内线虫进行再分离,分析2种类型松材线虫对赤松幼苗的致病能力。【结果】无菌松材线虫接种导致赤松胚性愈伤组织严重水渍化,TTC染色为米黄色或淡粉色,证明愈伤组织已失去活性。无菌松材线虫和带菌松材线虫接种均能导致赤松组培苗萎蔫,接种18天后,萎蔫率分别为70%和60%;萎蔫植株中均可再分离到松材线虫,其每株平均分别为599±567条和365+240条,二者差异显著(P<0.01)。剩余外观健康的赤松组培苗中也分离到线虫,但数量较少(仅10~20条)。2种类型松材线虫接种引起赤松盆栽苗同等程度的萎蔫,接种35天后的萎蔫率均达80%,无菌和带菌松材线虫致萎蔫植株中均可再分离到松材线虫,其每株平均分别为34 733±34 162条和25 057±21 410条,但差异不显著(P=0.508)。其余外观尚健康的松苗也进行线虫分离,2株接种无菌松材线虫的植株体内线虫分别为486条和22条;接种带菌松材线虫的2株松苗中,1株分离到646条线虫,另1株没有分离到线虫。【结论】无菌和带菌松材线虫均对赤松具有致病性,松材线虫是赤松萎蔫的直接原因,伴生细菌并非赤松萎蔫的必须因子。
【Objective】The aim of this study is to explore the pathogenicity of aseptic Bursaphelenchus xylophilus( pine wood nematode,PWN) and non-aseptic PWN on Pinus densiflora,to better understand the role of PWN in pine wilt disease development,and to provide useful information on pathogenicity mechanism and disease control. 【Method】The embryogenic calli of P. densiflora were inoculated with aseptic PWNs under aseptic conditions and cultured for 5 days.The effect of aseptic PWNs on the activity of embryogenic callus cells were evaluated by staining the embryogenic calli with the triphenyltetrazoliumchloride( TTC) solution. Tissue-cultured microshoots and 4-year-old seedlings cultured in green house were inoculated with aseptic PWNs and non-aseptic PWNs,respectively. In 18 and 35 days after inoculation,the pine wilt ratio was recorded and the PWNs were isolated from inoculated seedlings. The pathogenicity of aseptic PWN and non-aseptic PWN on P. densiflora was analyzed. 【Result】The inoculation by aseptic PWNs caused severe water stain in embryogenic calli of P. densiflora,and the TTC assay showed beige/light pink of embryogenic calli,revealing that the embryogenic cells lost viability. The embryogenic calli of control treatment remained healthy and showed bright red in TTC assay. Both the aseptic PWN and non-aseptic PWN wilted the tissue-cultured microshoots of P. densiflora. The wilting rates of the microshoots were 70% and 60% in 18 days after inoculation with aseptic PWNs and non-aseptic PWNs,respectively. PWNs were recovered from all wilted microshoots with average number of PWNs 599 + 567/microshoot for aseptic PWNs inoculation and 365 + 240/microshoot for non-aseptic PWNs. The number of nematodes recovered from wilted microshoots showed significant difference( P < 0. 01) between aseptic PWN and non-aseptic PWN treatments.Fewer PWNs( 10-20/microshoot) were also recovered from the remaining healthy-looking microshoots. The aseptic PWN could induce wilting of potted-seedlings of P. densiflora,as the same as non-aseptic PWN,with average of 80% wilting ratio in 35 days after inoculations. PWNs were recovered from wilted seedlings in both treatments. The number of recovered PWNs per seedling was 34 733 ± 34 162 and 25 057 ± 21 410 for aseptic PWNs and non-aseptic PWNs inoculations,respectively. No significant difference was found in number of recovered PWNs between the two treatments( P = 0. 508). The number of recovered PWNs in two healthy-looking seedlings of aseptic PWNs treatment was 486 and22,while only one healthy-looking seedlings of non-aseptic PWNs treatment contained 646 PWNs. 【Conclusion 】The aseptic and non-aseptic PWN could cause wilting of P. densiflora. The PWN is the main factor causing wilt of Japanese red pine,while the bacteria carried by the PWN are not necessary for the development of PWD.
【Objective】The aim of this study is to explore the pathogenicity of aseptic Bursaphelenchus xylophilus( pine wood nematode,PWN) and non-aseptic PWN on Pinus densiflora,to better understand the role of PWN in pine wilt disease development,and to provide useful information on pathogenicity mechanism and disease control. 【Method】The embryogenic calli of P. densiflora were inoculated with aseptic PWNs under aseptic conditions and cultured for 5 days.The effect of aseptic PWNs on the activity of embryogenic callus cells were evaluated by staining the embryogenic calli with the triphenyltetrazoliumchloride( TTC) solution. Tissue-cultured microshoots and 4-year-old seedlings cultured in green house were inoculated with aseptic PWNs and non-aseptic PWNs,respectively. In 18 and 35 days after inoculation,the pine wilt ratio was recorded and the PWNs were isolated from inoculated seedlings. The pathogenicity of aseptic PWN and non-aseptic PWN on P. densiflora was analyzed. 【Result】The inoculation by aseptic PWNs caused severe water stain in embryogenic calli of P. densiflora,and the TTC assay showed beige/light pink of embryogenic calli,revealing that the embryogenic cells lost viability. The embryogenic calli of control treatment remained healthy and showed bright red in TTC assay. Both the aseptic PWN and non-aseptic PWN wilted the tissue-cultured microshoots of P. densiflora. The wilting rates of the microshoots were 70% and 60% in 18 days after inoculation with aseptic PWNs and non-aseptic PWNs,respectively. PWNs were recovered from all wilted microshoots with average number of PWNs 599 + 567/microshoot for aseptic PWNs inoculation and 365 + 240/microshoot for non-aseptic PWNs. The number of nematodes recovered from wilted microshoots showed significant difference( P < 0. 01) between aseptic PWN and non-aseptic PWN treatments.Fewer PWNs( 10-20/microshoot) were also recovered from the remaining healthy-looking microshoots. The aseptic PWN could induce wilting of potted-seedlings of P. densiflora,as the same as non-aseptic PWN,with average of 80% wilting ratio in 35 days after inoculations. PWNs were recovered from wilted seedlings in both treatments. The number of recovered PWNs per seedling was 34 733 ± 34 162 and 25 057 ± 21 410 for aseptic PWNs and non-aseptic PWNs inoculations,respectively. No significant difference was found in number of recovered PWNs between the two treatments( P = 0. 508). The number of recovered PWNs in two healthy-looking seedlings of aseptic PWNs treatment was 486 and22,while only one healthy-looking seedlings of non-aseptic PWNs treatment contained 646 PWNs. 【Conclusion 】The aseptic and non-aseptic PWN could cause wilting of P. densiflora. The PWN is the main factor causing wilt of Japanese red pine,while the bacteria carried by the PWN are not necessary for the development of PWD.
引文
陈守常.2010.松材线虫病病原与致病机理研究进展.四川林业科技,31(1):18-25.(Chen S C.2010.Advance in researches on of pathogens and pathogenenic mechanism of pine wood nematode disease.Journal of Sichuan Forestry Science and Technology,31(1):18-25.[in Chinese])
池树友,韩正敏,何月秋.2006.无菌松材线虫对10年生黑松致病性的研究.林业科学,42(10):71-73.(Chi S Y,Han Z M,He Y Q.2006.Studies on the pathogenicity of 10years old black pine inoculated with aseptic pine wood nematode.Scientia Silvae Sinicae,42(10):71-73.[in Chinese])
池树友,何月秋,韩正敏,等.2008.松材线虫及携带细菌对黑松复合的侵染.福建林学院学报,28(1):92-96.(Chi S Y,He Y Q,Han Z M,et al.2008.Studies on complex infecting of pine wood nematodes and the bacterium carried by it.Journal of Fujian College of Forestry,28(1):92-96.[in Chinese])
郭道森,赵博光,高蓉.2001.利用愈伤组织验证细菌分离物B619与松材线虫病的关系.南京林业大学学报:自然科学版,25(5):71-74.(Guo D S,Zhao B G,Gao R.2001.Experiments on the relationship between the bacterium isolate B619 and the pine wilt disease by using calli of Pinus thunbergii.Journal of Nanjing Forestry University:Natural Science Edition,25(5):71-74.[in Chinese])
郭道森,赵博光,高蓉,等.2000.细菌分离物B619与松材线虫病关系的初步研究.南京林业大学学报:自然科学版,24(4):72-74.(Guo D S,Zhao B G,Gao R,et al.2000.A preliminary study on the relationship between the bacterium isolate B619 and pine wilt disease.Journal of Nanjing Forestry University:Natural Science Edition,24(4):72-74.[in Chinese])
洪英娣,赵博光,曹越.2003.松材线虫携带细菌的致病性.南京林业大学学报:自然科学版,27(2):45-48.(Hong Y D,Zhao B G,Cao Y.2003.Pathogenicity of bacteria carried by pine wood nematodes.Journal of Nanjing Forestry University:Natural Sciences Edition,27(2):45-48.[in Chinese])
刘华,梅兴国.2001.TTC法测定红豆杉细胞活力.植物生理学通讯,37(6):573-539.(Liu H,Mei X G.2001.Examining cell viability of Taxus chinensis with TTC(2,3,5-triphenyl-2H-tet-razolium chloride).Plant Physiology Communications,37(6):573-539.[in Chinese])
谈家金,向红琼,冯志新.2008.松材线虫伴生细菌的分离鉴定及其致病性.林业科技开发,22(2):23-26.(Tan J J,Xiang H Q,Feng Z X.2008.A preliminary study on isolation,identification and pathogenicity of the bacterium accompanying Bursaphelenchus xylophilus.China Forestry Science and Technology,22(2):23-26.[in Chinese])
谈家金,叶建仁.2003.松材线虫致病机理的研究进展.华中农业大学学报,22(6):613-617.(Tan J J,Ye J R.2003.Advance in research of pathogenetic mechanism of pine wood nematode.Journal of Huazhong Agricultural University,22(6):613-617.[in Chinese])
王敏敏,叶建仁,潘宏阳.2006.松材线虫病致病机理和防治技术研究进展.南京林业大学学报:自然科学版,30(2):103-107.(Wang M M,Ye J R,Pan H Y.2006.Research advances of pathogenic mechanism and controlling technology for pine wood nematode disease.Journal of Nanjing Forestry University:Natural Sciences Edition,30(2):103-107.[in Chinese])
吴静,朱丽华,许建秀,等.2015.抗松材线虫病赤松胚性愈伤组织的诱导及增殖.南京林业大学学报:自然科学版,39(1):17-21.(Wu J,Zhu L H,Xu J X,et al.2015.Inductionand proliferation of embryogenic callus in nematode-resistant Pinus densiflora.Journal of Nanjing Forestry University:Natural Sciences Edition,39(1):17-21.[in Chinese])
杨宝君.2002.松材线虫病致病机理的研究进展.中国森林病虫,21(1):27-31.(Yang B J.2002.Advance in research of pathogenetic mechanism of pine wood nematode.Forest Pest and Disease,21(1):27-31.[in Chinese])
叶建仁,黄麟.2012.松材线虫病病原学研究的几个问题.中国森林病虫,31(5):13-21.(Ye J R,Huang L.2012.Some aspects of the pathogen of the pine wilt disease.Forest Pest and Disease,31(5):13-21.[in Chinese])
张治宇,张克云,林茂松,等.2002.不同松材线虫群体对黑松的致病性测定.南京农业大学学报,25(2):43-46.(Zhang Z Y,Zhang K Y,Lin M S,et al.2002.Pathogenicity determination of Bursaphelenchus xylophilus isolates to Pinus thunbergii.Journal of Nanjing Agricultural University,25(2):43-46.[in Chinese])
赵博光,郭道森.2004.松材线虫携带的一株细菌分离及其致病性.北京林业大学学报,26(1):57-61.(Zhao B G,Guo D S.2004.Isolation and pathogenicity of a bacterium strain carried by pine wood nematode.Journal of Beijing Forestry University,26(1):57-61.[in Chinese])
朱丽华,季锦衣,吴小芹,等.2011.一种制备无菌线虫的方法.东北林业大学学报,39(6):65-67.(Zhu L H,Ji J Y,Wu X Q,et al.2011.A method for obtaining aseptic pine wood nematode.Journal of Northeast Forestry University,39(6):65-67.[in Chinese])
朱丽华,吴小芹,戴培培,等.2010.赤松离体培养植株再生体系的建立.南京林业大学学报:自然科学版,34(2):11-14.(Zhu L H,Wu X Q,Dai P P,et al.2010.In vitro plantlet regeneration from seedling explants of Pinus densiflora.Journal of Nanjing Forestry University:Natural Sciences Edition,34(2):11-14.[in Chinese])
Bolla I,Jordan W.1982.Cultivation of the pine wilt nematode,Bursaphelenchus xylophilus,in axenic culture media.Journal of Nematology,14(3):377-381.
Faria J M,Sena I,da Silva I V,et al.2015.In vitro co-cultures of Pinus pinaster with Bursaphelenchus xylophilus:a biotechnological approach to study pine wilt disease.Planta,241(6):1325-1336.
Han Z M,Hong Y D,Zhao B G.2003.A study on pathogenicity of bacteria carried by pine wood nematodes.J Phytopathol,151(11/12):683-689.
Kawazu K,Kaneko N.1997.Asepsis of the pine wood nematode isolate OKD-3 causes it to lose its pathogenicity.Jpn Nematology,27(2):76-80.
Kawazu K,Kaneko N,Hiranka K.1999.Reisolation of the pathogens from wilted red pine seedlings inoculated with the bacterium-carrying nematode isolates.Scientific Reports of Faculty of Agriculture,Okayama University,88:1-5.
Niu H T,Zhao L,Lu M,et al.2012.The ratio and concentration of two monoterpenes mediate fecundity of the pinewood nematode and growth of its associated fungi.PLo S ONE,7(2):e31716.doi:10.1371/journal.pone.0031716.
Oku H,Shiraishi T,Kurozumi S.1979.Participation of toxin in wilting of Japanese pines caused by a nematode.Naturwissenschaften,66(4):210.
Oku H.1980.Pine wilt toxin,the metabolite of a bacterium associated with a nematode.Naturwissenschaften,67(4):198-199.
Roriz M,Santos C,Vasconcelos M W.2011.Population dynamics of bacteria associated with different strains of the pine wood nematode Bursaphelenchus xylophilus after inoculation in maritime pine(Pinus pinaster).Experimental Parasitology,128(4):357-364.
Tamura H.1983.Pathogenicity of aseptic Bursaphelenchus xylophilus and associated bacteria to pine seedlings.Jpn Nematology,13:1-5.
Zhao B G,Wang H L,Han S F,et al.2003.Distribution and pathogenicity of bacteria species carried by Bursaphelenchus xylophilus in China.Nematology,5(6):899-906.
Zhao H,Chen C,Liu S,et al.2013.Aseptic Bursaphelenchus xylophilus does not reduce the mortality of young pine tree.Forest Pathology,43(6):444-454.
Zhu L H,Ye J,Negi S,et al.2012.Pathogenicity of aseptic Bursaphelenchus xylophilus.PLo S ONE,7(5):e38095.doi:10.1371/journal.pone.0038095.
池树友,韩正敏,何月秋.2006.无菌松材线虫对10年生黑松致病性的研究.林业科学,42(10):71-73.(Chi S Y,Han Z M,He Y Q.2006.Studies on the pathogenicity of 10years old black pine inoculated with aseptic pine wood nematode.Scientia Silvae Sinicae,42(10):71-73.[in Chinese])
池树友,何月秋,韩正敏,等.2008.松材线虫及携带细菌对黑松复合的侵染.福建林学院学报,28(1):92-96.(Chi S Y,He Y Q,Han Z M,et al.2008.Studies on complex infecting of pine wood nematodes and the bacterium carried by it.Journal of Fujian College of Forestry,28(1):92-96.[in Chinese])
郭道森,赵博光,高蓉.2001.利用愈伤组织验证细菌分离物B619与松材线虫病的关系.南京林业大学学报:自然科学版,25(5):71-74.(Guo D S,Zhao B G,Gao R.2001.Experiments on the relationship between the bacterium isolate B619 and the pine wilt disease by using calli of Pinus thunbergii.Journal of Nanjing Forestry University:Natural Science Edition,25(5):71-74.[in Chinese])
郭道森,赵博光,高蓉,等.2000.细菌分离物B619与松材线虫病关系的初步研究.南京林业大学学报:自然科学版,24(4):72-74.(Guo D S,Zhao B G,Gao R,et al.2000.A preliminary study on the relationship between the bacterium isolate B619 and pine wilt disease.Journal of Nanjing Forestry University:Natural Science Edition,24(4):72-74.[in Chinese])
洪英娣,赵博光,曹越.2003.松材线虫携带细菌的致病性.南京林业大学学报:自然科学版,27(2):45-48.(Hong Y D,Zhao B G,Cao Y.2003.Pathogenicity of bacteria carried by pine wood nematodes.Journal of Nanjing Forestry University:Natural Sciences Edition,27(2):45-48.[in Chinese])
刘华,梅兴国.2001.TTC法测定红豆杉细胞活力.植物生理学通讯,37(6):573-539.(Liu H,Mei X G.2001.Examining cell viability of Taxus chinensis with TTC(2,3,5-triphenyl-2H-tet-razolium chloride).Plant Physiology Communications,37(6):573-539.[in Chinese])
谈家金,向红琼,冯志新.2008.松材线虫伴生细菌的分离鉴定及其致病性.林业科技开发,22(2):23-26.(Tan J J,Xiang H Q,Feng Z X.2008.A preliminary study on isolation,identification and pathogenicity of the bacterium accompanying Bursaphelenchus xylophilus.China Forestry Science and Technology,22(2):23-26.[in Chinese])
谈家金,叶建仁.2003.松材线虫致病机理的研究进展.华中农业大学学报,22(6):613-617.(Tan J J,Ye J R.2003.Advance in research of pathogenetic mechanism of pine wood nematode.Journal of Huazhong Agricultural University,22(6):613-617.[in Chinese])
王敏敏,叶建仁,潘宏阳.2006.松材线虫病致病机理和防治技术研究进展.南京林业大学学报:自然科学版,30(2):103-107.(Wang M M,Ye J R,Pan H Y.2006.Research advances of pathogenic mechanism and controlling technology for pine wood nematode disease.Journal of Nanjing Forestry University:Natural Sciences Edition,30(2):103-107.[in Chinese])
吴静,朱丽华,许建秀,等.2015.抗松材线虫病赤松胚性愈伤组织的诱导及增殖.南京林业大学学报:自然科学版,39(1):17-21.(Wu J,Zhu L H,Xu J X,et al.2015.Inductionand proliferation of embryogenic callus in nematode-resistant Pinus densiflora.Journal of Nanjing Forestry University:Natural Sciences Edition,39(1):17-21.[in Chinese])
杨宝君.2002.松材线虫病致病机理的研究进展.中国森林病虫,21(1):27-31.(Yang B J.2002.Advance in research of pathogenetic mechanism of pine wood nematode.Forest Pest and Disease,21(1):27-31.[in Chinese])
叶建仁,黄麟.2012.松材线虫病病原学研究的几个问题.中国森林病虫,31(5):13-21.(Ye J R,Huang L.2012.Some aspects of the pathogen of the pine wilt disease.Forest Pest and Disease,31(5):13-21.[in Chinese])
张治宇,张克云,林茂松,等.2002.不同松材线虫群体对黑松的致病性测定.南京农业大学学报,25(2):43-46.(Zhang Z Y,Zhang K Y,Lin M S,et al.2002.Pathogenicity determination of Bursaphelenchus xylophilus isolates to Pinus thunbergii.Journal of Nanjing Agricultural University,25(2):43-46.[in Chinese])
赵博光,郭道森.2004.松材线虫携带的一株细菌分离及其致病性.北京林业大学学报,26(1):57-61.(Zhao B G,Guo D S.2004.Isolation and pathogenicity of a bacterium strain carried by pine wood nematode.Journal of Beijing Forestry University,26(1):57-61.[in Chinese])
朱丽华,季锦衣,吴小芹,等.2011.一种制备无菌线虫的方法.东北林业大学学报,39(6):65-67.(Zhu L H,Ji J Y,Wu X Q,et al.2011.A method for obtaining aseptic pine wood nematode.Journal of Northeast Forestry University,39(6):65-67.[in Chinese])
朱丽华,吴小芹,戴培培,等.2010.赤松离体培养植株再生体系的建立.南京林业大学学报:自然科学版,34(2):11-14.(Zhu L H,Wu X Q,Dai P P,et al.2010.In vitro plantlet regeneration from seedling explants of Pinus densiflora.Journal of Nanjing Forestry University:Natural Sciences Edition,34(2):11-14.[in Chinese])
Bolla I,Jordan W.1982.Cultivation of the pine wilt nematode,Bursaphelenchus xylophilus,in axenic culture media.Journal of Nematology,14(3):377-381.
Faria J M,Sena I,da Silva I V,et al.2015.In vitro co-cultures of Pinus pinaster with Bursaphelenchus xylophilus:a biotechnological approach to study pine wilt disease.Planta,241(6):1325-1336.
Han Z M,Hong Y D,Zhao B G.2003.A study on pathogenicity of bacteria carried by pine wood nematodes.J Phytopathol,151(11/12):683-689.
Kawazu K,Kaneko N.1997.Asepsis of the pine wood nematode isolate OKD-3 causes it to lose its pathogenicity.Jpn Nematology,27(2):76-80.
Kawazu K,Kaneko N,Hiranka K.1999.Reisolation of the pathogens from wilted red pine seedlings inoculated with the bacterium-carrying nematode isolates.Scientific Reports of Faculty of Agriculture,Okayama University,88:1-5.
Niu H T,Zhao L,Lu M,et al.2012.The ratio and concentration of two monoterpenes mediate fecundity of the pinewood nematode and growth of its associated fungi.PLo S ONE,7(2):e31716.doi:10.1371/journal.pone.0031716.
Oku H,Shiraishi T,Kurozumi S.1979.Participation of toxin in wilting of Japanese pines caused by a nematode.Naturwissenschaften,66(4):210.
Oku H.1980.Pine wilt toxin,the metabolite of a bacterium associated with a nematode.Naturwissenschaften,67(4):198-199.
Roriz M,Santos C,Vasconcelos M W.2011.Population dynamics of bacteria associated with different strains of the pine wood nematode Bursaphelenchus xylophilus after inoculation in maritime pine(Pinus pinaster).Experimental Parasitology,128(4):357-364.
Tamura H.1983.Pathogenicity of aseptic Bursaphelenchus xylophilus and associated bacteria to pine seedlings.Jpn Nematology,13:1-5.
Zhao B G,Wang H L,Han S F,et al.2003.Distribution and pathogenicity of bacteria species carried by Bursaphelenchus xylophilus in China.Nematology,5(6):899-906.
Zhao H,Chen C,Liu S,et al.2013.Aseptic Bursaphelenchus xylophilus does not reduce the mortality of young pine tree.Forest Pathology,43(6):444-454.
Zhu L H,Ye J,Negi S,et al.2012.Pathogenicity of aseptic Bursaphelenchus xylophilus.PLo S ONE,7(5):e38095.doi:10.1371/journal.pone.0038095.