一株分离自黑翅土白蚁的粘质沙雷氏菌的鉴定及特性
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摘要
【目的】为了对白蚁进行有效防治,对获得的1株对白蚁具有毒杀作用的菌株进行鉴定与分析。【方法】从感染死亡的黑翅土白蚁上筛选出1株产红色素的菌株。划线分离、纯化后得到较纯的菌株,命名为SM1,将其接种于健康的黑翅土白蚁上,可以使健康的白蚁身体变红死亡。根据菌株SM1形态学特性、生理生化性质、红色色素及16S r DNA的同源性,对其进行了鉴定。【结果】在基础培养基上,菌落呈圆形,易挑起,有轻微臭味,边缘整齐光滑黏稠; SM1直径为1～3 mm,中间朱红色不透明,呈隆起状。菌株SM1经10项生理生化指标测定,确定此菌为粘质沙雷氏菌。此外,克隆得到此菌的16S r DNA片段大小为1 418 bp,与粘质沙雷氏菌的同源性为99%,因此进一步判定该菌为粘质沙雷氏菌。通过超声波辅助丙酮浸提发酵液,可以得到较高纯度的红色色素。将色素溶于乙醇溶液后,用紫外全波长扫描分析。色素在535 nm左右处存在一个明显吸收峰,确定该红色色素是灵菌红素,从而进一步判定该菌为粘质沙雷氏菌。【结论】从黑翅土白蚁上筛选的SM1菌株为粘质沙雷氏菌,研究结果为白蚁的生物防治奠定了理论基础。
【Objective】With increasing attention on the ecological environment,biological control of termites needs to be developed.【Method】A strain of a bacteria producing red pigment was screened from Odontotermes formosanus. After being separated and purified,a purer strain was obtained and named SM1. Then,SM1 was inoculated into O. formosanus,which caused the healthy termites to turn red and die. The bacteria was identified based on its morphological ch-aracteristics,physiological and biochemical characteristics,red pigment,and 16 S r DNA. 【Result】The bacterial colony was round,easy to pick up,had slight odor,was smooth and sticky,and had a diameter of 1-3 mm and a middle vermilion that was not transparent. In addition,ten physiological and biochemical characteristics of the SM1 strain were measured. These results showed that the bacterium was Serratia marcescens. Furthermore,the size of its cloned16 S r DNA fragment was 1 418 bp,and the homology with Serratia marcescens was 99%. Therefore,the bacteria was determined to be Serratia marcescens. Further,a higher purity of red pigment could be obtained by ultrasonic-assisted acetone extraction,and the pigment was dissolved in ethanol solution and then analyzed by ultraviolet full wavelength scanning. The results showed that the pigment had an obvious absorption peak at 535 nm,which suggested that the red pigment was a prodigiosin. 【Conclusion】The bacterium screened from Odontotermes formosanus was Serratia marcescens,which created a theoretical foundation for the biological control of termites.
【Objective】With increasing attention on the ecological environment,biological control of termites needs to be developed.【Method】A strain of a bacteria producing red pigment was screened from Odontotermes formosanus. After being separated and purified,a purer strain was obtained and named SM1. Then,SM1 was inoculated into O. formosanus,which caused the healthy termites to turn red and die. The bacteria was identified based on its morphological ch-aracteristics,physiological and biochemical characteristics,red pigment,and 16 S r DNA. 【Result】The bacterial colony was round,easy to pick up,had slight odor,was smooth and sticky,and had a diameter of 1-3 mm and a middle vermilion that was not transparent. In addition,ten physiological and biochemical characteristics of the SM1 strain were measured. These results showed that the bacterium was Serratia marcescens. Furthermore,the size of its cloned16 S r DNA fragment was 1 418 bp,and the homology with Serratia marcescens was 99%. Therefore,the bacteria was determined to be Serratia marcescens. Further,a higher purity of red pigment could be obtained by ultrasonic-assisted acetone extraction,and the pigment was dissolved in ethanol solution and then analyzed by ultraviolet full wavelength scanning. The results showed that the pigment had an obvious absorption peak at 535 nm,which suggested that the red pigment was a prodigiosin. 【Conclusion】The bacterium screened from Odontotermes formosanus was Serratia marcescens,which created a theoretical foundation for the biological control of termites.
引文
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[11]GIRI A V,ANANDKUMAR N,M UTHUKUMARAN G,et al. A novel medium for the enhanced cell growth and production of prodigiosin from Serratia marcescens isolated from soil[J]. BMC Microbiology,2004,4:11-18.DOI:10.1186/1471-2180-4-11.
[12]TANIKAWA T,NAKAGAWA Y,MATSUYAMA T. Transcriptional downregulator hex S controlling prodigiosin and serrawettin W1 biosynthesis in Serratia marcescens[J]. Microbiology and Immunology,2006,50(8):587-596. DOI:10. 1111/j. 1348-0421.2006.tb03833.x.
[13]LEWIS S M,CORPE W A. Prodigiosin-producing bacteria from marine sources[J]. Applied Microbiology,1964,12(1):13-17.DOI:10.1007/BF02046758.
[14]JONG S L,YONG S K,SOOYEON P. Exceptional production of both prodigiosin and cycloprodigiosin as major metabolic constituents by a novel marine bacterium,Zooshikella rubidus S1-1[J].Applied and Environmental Microbiology,2011,77(14):4967-4973.DOI:10.1128/AEM.01986-10.
[15]黄麟,杨济云,李秋成,等.龙爪槐枝干溃疡病的病原研究[J].南京林业大学学报(自然科学版),2017,41(5):175-179.DOI:10.3969/j.issn.1000-2006.201701004.HUANG L,YANG J Y,LI Q C,et al. Identification of the pathogen causing stem canker on Sophora japonica Linn. var. japonica f. pendula Hort[J]. Journal of Nanjing Forestry University(Natural Sciences Edition),2017,41(5):175-179.
[16]赵银娟,薛斌,李桂娥,等.一株耐高温抑菌黏质沙雷氏菌的鉴定及其红色素的初步分离[J].安徽农业科学,2013,41(7):2829-2831,DOI:10.3969/j.issn.0517-6611.2013.07.004.ZHAO Y J,XUE B,LI G E,et al. Identification of thermo-stability Serratia marcescens strain inhibiting bacterial and preliminary isolation of red pigment[J].Journal of Anhui Agricultural Sciences,2013,41(7):2829-2831.
[17]汤方,崔荔,唐进根,等.黑翅土白蚁和黑胸散白蚁对药剂传毒效率的差异[J].南京林业大学学报(自然科学版),2007,31(6):69-72.DOI:10.3969/j.issn.1000-2006.2007.06.016.TANG F,CUI L,TANG J G,et al. Difference in transmission efficiency to pesticides between Odontotermes formosanus(Shiraki)and Reticulitermes chinensis(Snyder)[J]. Journal of Nanjing Forestry University(Natural Sciences Edition),2007,31(6):69-72.
[18]邓彩萍,刘红霞,闫喜中,等.罹病光肩星天牛幼虫分离菌株的16Sr DNA系统发育分析[J].安徽农业科学,2008,36(18):7672-7673,7682.DOI:10.3969/j.issn.0517-6611.2008.18.045.DENG C P,LIU H X,YAN X Z,et al. Phylogenetic analysis of the 16S r DNA of a strain isolated from diseased larva of Anoplophora glabripennis(Motsch.)[J]. Journal of Anhui Agricultural Sciences,2008,36(18):7672-7673,7682.
[19]张晶,覃伟权,阎伟,等.一株对红棕象甲幼虫和卵有致病力的病原菌的分离鉴定[J].热带作物学报,2011,32(12):2331-2335.DOI:10.3969/j.issn.1000-2561.2011.12.027.ZHANG J,QIN W Q,YAN W,et al. Islation and identification of a pathogenic strain of Rhynchophorus ferrugineus Oliver[J]. Chinese Journal of Tropical Crops,2011,32(12):2331-2335.
[20]杨建云,纪春艳,凌冰,等.黄曲条跳甲幼虫致病菌的鉴定及其对黄曲条跳甲杀虫活性的研究[J].中国生物防治学报,2014,30(3):434-440. DOI:10. 3969/j. issn. 2095-039X. 2014.03.025.YANG J Y,JI C Y,LING B,et al. Isolation and identification of bacteria from Phyllotreta striolata(Fabricius)and determination of its insecticidal bioactivity[J]. Chinese Journal of Biological Control,2014,30(3):434-440.
[2]MONTANER B,PEREZ-TOMAS R. The prodigiosins:a new family of anticancer drugs[J]. Current Cancer Drug Targets,2003,3(1):57-65.DOI:10.2174/1568009033333772.
[3]BABASHPOUR S,AMINZADEH S,FARROKHI N,et al. Characterization of a Chitinase(Chit62)from Serratia marcescens B4A and its efficacy as a bioshield against plant fungal pathogens[J].Biochemical Genetics,2012,50(9/10):722-735.DOI:10.1007/s10528-012-9515-3.
[4]WANG K,YAN P S,CAO L X,et al. Potential of chitinolytic Serratia marcescens strain JPP1 for biological control of Aspergillus parasiticus and Aflatoxin[J]. BioMed Research International,2013:397142.DOI:10.1155/2013/397142.
[5]NOBUTAKA S,MASAMI N,KEN W,et al. Synergistic antifungal activity of the cul ture filtrates of Serratia marcescens strain B2 and chemical fungicides against the sclerotia viability of the rice sheath blight pathogen,Rhizoctonia solani[J]. Biocontrol Science,2005,10(3):97-100.DOI:10.4265/bio.10.97.
[6]LAVANIA M,CHAUHAN P S,CHAUHAN S V,et al. Induction of plant defense enzymes and phenolics by treatment with plant growth-promoting rhizobacteria Serratia marcescens NBRI1213[J].Current Microbiology,2006,52(2):363-368. DOI:10. 1007/s00284-005-5578-2.
[7]CHAKRABORTY U,CHAKRABORTY B N,CHAKRABORTY A P. Influence of Serratia marcescens TRS-1 on growth promotion and induction of resistance in Camellia sinensis against Fomes la maoensis[J]. Journal of Plant Interactions,2010,5(4):261-272.DOI:10.1080/17429140903551738.
[8]TING A S,MEON S,KADIR J,et al. Induction of host defence enzymes by the endophytic bacterium Serratia marcescens,in banana plantlets[J]. International Journal of Pest Management,2010,56(2):183-188.DOI:10.1080/09670870903324198.
[9]FRSTNER A. Chemistry and biology of roseophilin and the prodigiosin alkaloids:a survey of the last 2500 years[J]. Angewand echemie International Edtion in English,2003,42(31):3582-3603.DOI:10.1002/anie.200300582.
[10]SLATER H,CROW M,EVERSON L,et al. Phosphate availability regulates biosynthesis of two antibiotics,prodigiosin and carbapenem,in Serratia via both quorum-sensing-dependent and independent pathways[J]. Molecular Microbiology,2003,47(2):303-320.DOI:10.1046/j.1365-2958.2003.03295.x.
[11]GIRI A V,ANANDKUMAR N,M UTHUKUMARAN G,et al. A novel medium for the enhanced cell growth and production of prodigiosin from Serratia marcescens isolated from soil[J]. BMC Microbiology,2004,4:11-18.DOI:10.1186/1471-2180-4-11.
[12]TANIKAWA T,NAKAGAWA Y,MATSUYAMA T. Transcriptional downregulator hex S controlling prodigiosin and serrawettin W1 biosynthesis in Serratia marcescens[J]. Microbiology and Immunology,2006,50(8):587-596. DOI:10. 1111/j. 1348-0421.2006.tb03833.x.
[13]LEWIS S M,CORPE W A. Prodigiosin-producing bacteria from marine sources[J]. Applied Microbiology,1964,12(1):13-17.DOI:10.1007/BF02046758.
[14]JONG S L,YONG S K,SOOYEON P. Exceptional production of both prodigiosin and cycloprodigiosin as major metabolic constituents by a novel marine bacterium,Zooshikella rubidus S1-1[J].Applied and Environmental Microbiology,2011,77(14):4967-4973.DOI:10.1128/AEM.01986-10.
[15]黄麟,杨济云,李秋成,等.龙爪槐枝干溃疡病的病原研究[J].南京林业大学学报(自然科学版),2017,41(5):175-179.DOI:10.3969/j.issn.1000-2006.201701004.HUANG L,YANG J Y,LI Q C,et al. Identification of the pathogen causing stem canker on Sophora japonica Linn. var. japonica f. pendula Hort[J]. Journal of Nanjing Forestry University(Natural Sciences Edition),2017,41(5):175-179.
[16]赵银娟,薛斌,李桂娥,等.一株耐高温抑菌黏质沙雷氏菌的鉴定及其红色素的初步分离[J].安徽农业科学,2013,41(7):2829-2831,DOI:10.3969/j.issn.0517-6611.2013.07.004.ZHAO Y J,XUE B,LI G E,et al. Identification of thermo-stability Serratia marcescens strain inhibiting bacterial and preliminary isolation of red pigment[J].Journal of Anhui Agricultural Sciences,2013,41(7):2829-2831.
[17]汤方,崔荔,唐进根,等.黑翅土白蚁和黑胸散白蚁对药剂传毒效率的差异[J].南京林业大学学报(自然科学版),2007,31(6):69-72.DOI:10.3969/j.issn.1000-2006.2007.06.016.TANG F,CUI L,TANG J G,et al. Difference in transmission efficiency to pesticides between Odontotermes formosanus(Shiraki)and Reticulitermes chinensis(Snyder)[J]. Journal of Nanjing Forestry University(Natural Sciences Edition),2007,31(6):69-72.
[18]邓彩萍,刘红霞,闫喜中,等.罹病光肩星天牛幼虫分离菌株的16Sr DNA系统发育分析[J].安徽农业科学,2008,36(18):7672-7673,7682.DOI:10.3969/j.issn.0517-6611.2008.18.045.DENG C P,LIU H X,YAN X Z,et al. Phylogenetic analysis of the 16S r DNA of a strain isolated from diseased larva of Anoplophora glabripennis(Motsch.)[J]. Journal of Anhui Agricultural Sciences,2008,36(18):7672-7673,7682.
[19]张晶,覃伟权,阎伟,等.一株对红棕象甲幼虫和卵有致病力的病原菌的分离鉴定[J].热带作物学报,2011,32(12):2331-2335.DOI:10.3969/j.issn.1000-2561.2011.12.027.ZHANG J,QIN W Q,YAN W,et al. Islation and identification of a pathogenic strain of Rhynchophorus ferrugineus Oliver[J]. Chinese Journal of Tropical Crops,2011,32(12):2331-2335.
[20]杨建云,纪春艳,凌冰,等.黄曲条跳甲幼虫致病菌的鉴定及其对黄曲条跳甲杀虫活性的研究[J].中国生物防治学报,2014,30(3):434-440. DOI:10. 3969/j. issn. 2095-039X. 2014.03.025.YANG J Y,JI C Y,LING B,et al. Isolation and identification of bacteria from Phyllotreta striolata(Fabricius)and determination of its insecticidal bioactivity[J]. Chinese Journal of Biological Control,2014,30(3):434-440.