盐碱戈壁药用植物骆驼刺内生菌的分离、培养与鉴定
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摘要
骆驼刺(Alhagi sparsifolia Shap.)是新疆民间常用药用植物。为了解骆驼刺内生菌的多样性,获得骆驼刺的内生菌资源,本实验在新疆维吾尔克拉玛依盐碱戈壁(北纬45°16’,东经85°2’)采集骆驼刺。利用常规平板分离方法进行植物内生菌菌株的分离、培养,测定菌株16S r DNA基因序列,并结合系统发育分析进行鉴定。从骆驼刺中共分离到可培养内生菌50株,分属于葡萄球菌属(Staphylococcus)、芽胞杆菌属(Bacillus)、芽胞八叠球菌属(Sporosarcina)、微杆菌属(Exiguobacterium)、气球菌属(Aerococcus)、巨型球菌属(Macrococcus)、多米杆菌(Domibacillus)、巴尔加瓦菌属(Bhargavaea)、微球菌属(Micrococcus)、棒杆菌属(Corynebacterium)、考克菌属(Kocuria)、细杆菌属(Microbacterium)、副球菌属(Paracoccus)、马西利亚菌属(Massilia)和耐辐射球菌属(Deinococcus)15个菌属。其中葡萄球菌属占绝对优势,其次为芽胞杆菌属,为环境和植物当中广泛存在的菌属,分离获得的其他细菌与骆驼刺生长环境(盐渍化严重,高辐射)有关,内生菌多样性与骆驼刺在新疆干旱、寒冷、盐碱土壤环境中的适应性机制具有密切的联系。
Alhagi sparsifolia Shap. is a commonly used medicinal plant in Xinjiang. To understand the diversity of endophytic bacteria of Alhagi sparsifolia Shap.,and to get some microorganism resources and promote the development and utilization of microbial resources of plant endogenous bacteria and their important functional genes in the future,the samples from desert in Xinjiang(karamay north latitude 45° 16',longitude 85° 2')were collected to isolate the endophytes. Morphological observation and 16 S r DNA region sequence determination and phylogenetic tree were studied. 50 endophyte strains were obtained,and belonged to Staphylococcus,Bacillus,Sporosarcina,Exiguobacterium,Aerococcus,Macrococcus,Domibacillus,Bhargavaea,Micrococcus,Corynebacterium,Kocuria,Microbacterium,Paracoccus,Massilia,and Deinococcus. Among them,the genus Staphylococcus was the dominate group,followed by the genus Bacillus,which are widely existed in the environment and plant. The other isolated bacteria were related to the growth environment of camel spines(serious salinization and high radiation). It is found that the endophyte diversity is closely related to the adaptability mechanism of the plant in the arid,cold,saline alkali soil environment of Xinjiang.
Alhagi sparsifolia Shap. is a commonly used medicinal plant in Xinjiang. To understand the diversity of endophytic bacteria of Alhagi sparsifolia Shap.,and to get some microorganism resources and promote the development and utilization of microbial resources of plant endogenous bacteria and their important functional genes in the future,the samples from desert in Xinjiang(karamay north latitude 45° 16',longitude 85° 2')were collected to isolate the endophytes. Morphological observation and 16 S r DNA region sequence determination and phylogenetic tree were studied. 50 endophyte strains were obtained,and belonged to Staphylococcus,Bacillus,Sporosarcina,Exiguobacterium,Aerococcus,Macrococcus,Domibacillus,Bhargavaea,Micrococcus,Corynebacterium,Kocuria,Microbacterium,Paracoccus,Massilia,and Deinococcus. Among them,the genus Staphylococcus was the dominate group,followed by the genus Bacillus,which are widely existed in the environment and plant. The other isolated bacteria were related to the growth environment of camel spines(serious salinization and high radiation). It is found that the endophyte diversity is closely related to the adaptability mechanism of the plant in the arid,cold,saline alkali soil environment of Xinjiang.
引文
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[4]Lu Z H.Endophytic bacterial and fungi diversity of panax ginseng and their antimicrobial activity against pathogens[D].Shenyang:Shenyang Agricultural University,2016.卢占慧.人参内生菌群落多样性及拮抗菌株的抑菌作用研究[D].沈阳:沈阳农业大学,2016.
[5]Cao Y.Investigation on endophytes of euonymus japonicas and its bioactive metabolites[D].Yangling:Northwest Agricultural and Forestry University,2016.曹艳.冬青卫矛内生菌的分离及其活性代谢物研究[D].杨凌:西北农林科技大学,2016.
[6]Xu Y J.Research progress on pesources diversity of plant endophytes[J].Guangdong Agricultural Sciences,2011,38(24):149-152.徐亚军.植物内生菌资源多样性研究进展[J].广东农业科学,2011,38(24):149-152.
[7]Malfanova N,Lugtenberg B J J,Berg G.Bacterial endophytes:who and where,and what are they doing there?[M]//Malfanova N,Lugtenberg B J J,Berg G.eds.Molecular Microbial Ecology of the Rhizosphere.Hoboken,NJ,USA:John Wiley&Sons,Inc.,2013:391-403.
[8]Chen C Q,Xin K Y,Liu H,et al.Pantoea alhagi,a novel endophytic bacterium with ability to improve growth and drought tolerance in wheat[J].Sci Rep,2017,7:41564.
[9]Li J S,Liu Y M,Cai S Q.Study on resources,commodity circulation and folk application of uygur medical camel thorn[J].Journal of Medicine and Pharmacy of Chinese Minorities,1996,2(2):39-40.李君山,刘勇民,蔡少青.维吾尔医用骆驼刺类药材的资源、商品流通及民间应用研究[J].中国民族医药杂志,1996,2(2):39-40.
[10]Zhang G J.Study on chemical constituents of camel spine[D].Shenyang:Shenyang Pharmaceutical University,2009.张贵杰.骆驼刺的化学成分研究[D].沈阳:沈阳药科大学,2009.
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[12]Bao M,Kang M H.The summarize on research of endogenous bacteria[J].Qinghai Prataculture,2011,20(1):21-25.鲍敏,康明浩.植物内生菌研究发展现状[J].青海草业,2011,20(1):21-25.
[13]Xu G H.Preliminary analysis of variations of recent ecological environment of Xinjiang and countermeasures[J].Bimonthly of Xinjiang Meteorology,2004,27(4):5-6.徐羹慧.新疆生态环境近期变化与应对措施的初步分析[J].新疆气象,2004,27(4):5-6.
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[15]Cheng Y,Yuan L.Study on geographic distribution of endemic plant species in Xinjiang[J].Arid Zone Research,2011,28(5):854-859.程芸,袁磊.新疆植物特有种的地理分布规律[J].干旱区研究,2011,28(5):854-859.
[16]Feng Y,Pan B R.Study on floristic and ecology of species endemic to Xinjiang[J].Acta Botanica Yunnanica,2004,26(2):183-188.冯缨,潘伯荣.新疆特有种植物区系及生态学研究[J].云南植物研究,2004,26(2):183-188.
[17]Tohno M,Kitahara M,Matsuyama S,et al.Aerococcus vaginalis sp.nov.,isolated from the vaginal mucosa of a beef cow,and emended descriptions of Aerococcus suis,Aerococcus viridans,Aerococcusurinaeequi,Aerococcus urinaehominis,Aerococcus urinae,Aerococcus christensenii and Aerococcus sanguinicola[J].Int J Syst Evol Microbiol,2014,64(4):1229-1236.
[18]Pascual C,Lawson P A,Farrow J A E,et al.Phylogenetic analysis of the genus Corynebacterium based on16S rRNA gene sequences[J].Int J Syst Bacteriol,1995,45(4):724-728.
[19]Gobeli Brawand S,Cotting K,Gómez-Sanz E,et al.Macrococcus canis sp.nov.,a skin bacterium associated with infections in dogs[J].Int J Syst Evol Microbiol,2017,67(3):621-626.
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[21]Wang Z F.Effect of endophytic fungi on salt tolerance of wild barley(Hordeum brevisubulatum)[D].Lanzhou:Lanzhou University,2009.王正凤.内生真菌对野大麦耐盐性影响的研究[D].兰州:兰州大学,2009.
[22]Gou X Y.Effects of neotyphodium endophyte on salt tolerance to drunken horse grass(Achnatherum inebrians)[D].Lanzhou:Lanzhou University,2007.缑小媛.内生真菌对醉马草耐盐性的影响研究[D].兰州:兰州大学,2007.
[23]Zhao F G,He L F,Luo Q Y.Physiological ecology of plant stress[M].Beijing:Environmental Science and Engineering Publishing Center,Chemical Industry Press,2004.赵福庚,何龙飞,罗庆云.植物逆境生理生态学[M].北京:化学工业出版社环境科学与工程出版中心,2004.
[24]Wang W,Yu H F,Zhang Y H,et al.Effects of salt on the growth and physiological characteristics of sunflower seedlings[J].Acta Agriculturae Boreali-Sinica,2013,28(1):176-180.王伟,于海峰,张永虎,等.盐胁迫对向日葵幼苗生长和生理特性的影响[J].华北农学报,2013,28(1):176-180.
[25]Zhou G L,Luo X S,Tang Y L,et al.Kocuria flava sp.nov.and Kocuria turfanensis sp.nov.,airborne actinobacteria isolated from Xinjiang,China[J].Int J Syst Evol Microbiol,2008,58(6):1304-1307.
[26]Park E J,Kim M S,Roh S W,et al.Kocuria atrinae sp.nov.,isolated from traditional Korean fermented seafood[J].Int J Syst Evol Microbiol,2010,60(4):914-918.
[27]Zhang L M,Xi L J,Ruan J S,et al.Kocuria oceani sp.nov.,isolated from a deep-sea hydrothermal plume[J].Int J Syst Evol Microbiol,2017,67(1):164-169.
[28]Wang K B,Zhang L,Liu Y S,et al.Kocuria dechangensis sp.nov.,an actinobacterium isolated from saline and alkaline soils[J].Int J Syst Evol Microbiol,2015,65(9):3024-3030.
[29]Tang R,Yuan M L,Wu J,et al.Isolation and identification of an irradiation-resistant bacterium strain of Kocuria[J].Journal of Nuclear Agriculturae Sciences,2010,24(2):276-280.唐然,袁梦龙,吴菁,等.一株耐辐射考克氏菌的分离与鉴定[J].核农学报,2010,24(2):276-280.
[30]Kim S B,Nedashkovskaya O I,Mikhailov V V,et al.Kocuria marina sp.nov.a novel actinobacterium isolated from marine sediment[J].Int J Syst Evol Microbiol,2004,54(5):1617-1620.
[31]Kovács G,Burghardt J,Pradella S,et al.Kocuria palustris sp.nov.and Kocuria rhizophila sp.nov.,isolated from the rhizoplane of the narrow-leaved cattail(Typha angustifolia)[J].Int J Syst Bacteriol,1999,49(1):167-173
[32]Zhang D C,Li H R,Xin Y H,et al.Phaeobacter arcticus sp.nov.a psychrophilic bacterium isolated from the Arctic[J].Int J Syst Evol Microbiol,2008,58(6):1384-1387.
[33]Zhang S,Gan L Z,Qin Q M,et al.Paracoccus acridae sp.nov.,isolated from the insect Acrida cinerea living in deserted cropland[J].Int J Syst Evol Microbiol,2016,66(9):3492-3497.
[34]Sharma A,Dhar S K,Prakash O,et al.Description of Domibacillus indicus sp.nov.,isolated from ocean sediments and emended description of the genus Domibacillus[J].Int J Syst Evol Microbiol,2014,64(9):3010-3015.
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