杨树内生细菌的分离、多样性分析及解淀粉芽孢杆菌PEBA20的抑菌活性研究
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摘要
杨树水泡型溃疡病是由葡萄座腔菌(Botryosphaeria dothidea)侵染引起的重大枝干病害,该病普遍发生,危害严重,防治困难,严重影响着杨树的经济效益及生态效益。因而,寻求对杨树病害合理有效的防治措施已势在必行。生物防治具有安全而又环境友好的特点,越来越受到人们的关注,并展现出重大的发展潜力。
有关杨树水泡溃疡病的生物防治研究,国内外报道较少。作为一类新型的生防因子,植物内生细菌是定植在健康植物的各种组织和器官中,与植物之间形成互惠共存、相互制约的一类微生物。从杨树体内分离筛选有益细菌并应用于杨树病害的生物防治实践中将具有重要的现实意义。
本研究旨在从杨树体中分离内生细菌并对其进行多样性分析;筛选出具有较高拮抗活性的内生细菌;并对内生细菌PEBA20进行系统的分类学鉴定,测定了该内生细菌的发酵条件及抑菌活性;而对其代谢产物—蛋白质进行初步纯化。主要研究结果如下:
1.于2007～2008年,3、6、9、12月4个月份,从泰安市城区选取多年生的三倍体毛白杨枝干共分离到126株内生细菌,并进行室内平板拮抗筛选测定,获得12株内生细菌对杨树水泡溃疡病病菌有较好的拮抗活性。通过4个月份的分离细菌,发现杨树体内含有大量的内生细菌,多样性较丰富。采用Shannon-Wiener分析其多样性,多样性指数(H)、丰度(D)、均匀度(J),发现在不同分离时间的杨树枝条中内生细菌的种类、数量和多样性指数明显不同。
2.通过培养性状、染色反应、生理生化特征分析和16SrDNA序列分析等方法对PEBA20进行鉴定。结果表明,该菌为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。对峙培养发现PEBA20对杨树水泡溃疡病病菌产生较宽的抑菌圈,表现较好的抑菌作用。抑菌谱测定结果表明,PEBA20菌具有广谱抑菌活性,对杨腐烂病菌、茄子绵疫病菌、杨树炭疽病菌、合欢枯萎病菌等12种病原真菌均有较好的抑制作用。PEBA20菌株的活体和发酵滤液均表现广泛的拮抗谱,对杨树腐烂病菌抑菌效果最好,抑制率分别为:89.37%、82.62%;对杨树水泡溃疡该菌也有较好抑菌作用,抑制率分别为:69.95%、63.2%。这说明PEBA20对杨树病害的防治有较好的效果。该菌株也表现出良好的抗细菌活性,对核桃黑斑病菌、烟草青枯病菌、马铃薯环腐病菌、巨大芽孢杆菌等细菌的抑菌圈均在12mm以上。
3.本研究初步探讨了不同培养条件下的发酵液对杨树水泡溃疡病菌的抑菌活性测定。测定不同培养条件的发酵液对杨树水泡溃疡病菌的抑菌效果,得出在NB培养基,培养120 h的PEBA20菌的发酵液,对杨树水泡溃疡病菌的抑菌效果最佳。通过二次正交设计优化发酵条件,计算得到该菌最适宜发酵条件为:T=28.5℃,rpm=140rpm,pH=5,通气量100mL,t=120h。
4. PEBA20菌发酵培养后能够产生抗菌蛋白,不同培养条件下的蛋白粗提液对杨树水泡溃疡病病菌有着较好的拮抗效果。PEBA20菌发酵液的蛋白组分和0-60%饱和度的硫酸铵盐析粗蛋白,经CM-Sepharose Fast Flow阳离子交换柱层析,发现PEBA20菌发酵后的蛋白质中含有阳离子蛋白质,并能通过CM-Sepharose Fast Flow获得初步分离;经DEAE-Sepharose Fast Flow阴离子交换柱层析,形成明显的蛋白质吸收峰,各峰相对独立,峰形较尖锐,且没有拖尾。PEBA20菌的初步分离,为进一步分离纯化奠定基础。
Poplar canker caused by Botryosphaeria dothidea, was a key stem disease which occurred widespreadly and was difficult to control.Biological control was an envirorment friendly method for the disease management with great potential development. There was little reports about biological control of Botryosphaeria dothidea. As a new type of biocontrol factors, plants endophytic bacteria were a class of microorganisms which colonied in a variety of tissues of healthy plants with mutually beneficial co-existence between plants. There was significance of controlling the diseases of poplars with endophytic bacteria isolated from the poplar body.
In this study, we isolated endophytic bacteria from the poplar trees and analysed the bacteria diversity, selected the endophytic bacteria with high antagonistic activity, identificated the endophytic bacteria strain of PEBA20 with systematic taxonomy identification, determined its optimization of fermentation conditions and antibacterial activity, prelimary purified the proteins. The main findings showed as follows:
1. 126 endophytic bacteria were isolated from branches of triploid Populus from Tai-an city in march, june, september and december, 2007 to 2008. We selected 12 poplar endophytic bacteria showed good antagonist activity to Botryosphaeria dothidea with flat antagonist screening method. We found a lot of poplar endophytic bacteria in poplars from these separated bacteria. Using Shannon-Wiener analysis with diversity index (H), abundance (D), evenness (J), the endophytic bacteria from poplar in different time showed significant difference.
2. We identificated PEBA20 as Bacillus amyloliquefaciens through cultivation, staining reaction, physiological or biochemical characteristic analysis and 16SrDNA sequence analysis. PEBA20 showed good antimicrobial effect on Botryosphaeria dothidea. The results showed that the antibacterial spectrum of PEBA20 was broad, the antibacterial activity were high against Valsa sordida、Phytophthora capsiciLeon、Glomerella cingulata、 Fusarium oxysporum and other 12 kinds of pathogenic fungi, the inhibitory rate of the bacteria and fermentation filtrate against Valsa sordidaI was 79.53% and 67.32% respectively, while the inhibitory rate against Botryosphaeria dothidea was 53.27% and 40.48% respectively. This indicated that PEBA20 has good effect to control the disease of poplar trees. PEBA20 also showed good antibateria activity against Xanthomonas jugandis、Ralstonia solanacearum、Clavibacter sepedonicum、Bacillus megaterium , the inhibition zones to bacteria were more than 12mm.
3. Antifungal activitives of different fermentation conditions of PEBA20 were determined. The results showed that culturing for 120h and cultured in NB medium were the favorable conditions for the antibiotic activity. We calculated the most suitable fermentation conditions for PEBA20 were T = 28.5℃, rpm = 140rpm, pH = 5, ventilation volume 100mL, t = 120h.
4. Crude protein of PEBA20 under different culture conditions had better effects to Botryosphaeria dothidea,indicating the existence of th antibiotic proteins. The column chromatography with CM-Sepharose Fast Flow and DEAE-Sepharose Fast Flow demonstreated that the protein with antibiotic activity could be purified.
有关杨树水泡溃疡病的生物防治研究,国内外报道较少。作为一类新型的生防因子,植物内生细菌是定植在健康植物的各种组织和器官中,与植物之间形成互惠共存、相互制约的一类微生物。从杨树体内分离筛选有益细菌并应用于杨树病害的生物防治实践中将具有重要的现实意义。
本研究旨在从杨树体中分离内生细菌并对其进行多样性分析;筛选出具有较高拮抗活性的内生细菌;并对内生细菌PEBA20进行系统的分类学鉴定,测定了该内生细菌的发酵条件及抑菌活性;而对其代谢产物—蛋白质进行初步纯化。主要研究结果如下:
1.于2007～2008年,3、6、9、12月4个月份,从泰安市城区选取多年生的三倍体毛白杨枝干共分离到126株内生细菌,并进行室内平板拮抗筛选测定,获得12株内生细菌对杨树水泡溃疡病病菌有较好的拮抗活性。通过4个月份的分离细菌,发现杨树体内含有大量的内生细菌,多样性较丰富。采用Shannon-Wiener分析其多样性,多样性指数(H)、丰度(D)、均匀度(J),发现在不同分离时间的杨树枝条中内生细菌的种类、数量和多样性指数明显不同。
2.通过培养性状、染色反应、生理生化特征分析和16SrDNA序列分析等方法对PEBA20进行鉴定。结果表明,该菌为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。对峙培养发现PEBA20对杨树水泡溃疡病病菌产生较宽的抑菌圈,表现较好的抑菌作用。抑菌谱测定结果表明,PEBA20菌具有广谱抑菌活性,对杨腐烂病菌、茄子绵疫病菌、杨树炭疽病菌、合欢枯萎病菌等12种病原真菌均有较好的抑制作用。PEBA20菌株的活体和发酵滤液均表现广泛的拮抗谱,对杨树腐烂病菌抑菌效果最好,抑制率分别为:89.37%、82.62%;对杨树水泡溃疡该菌也有较好抑菌作用,抑制率分别为:69.95%、63.2%。这说明PEBA20对杨树病害的防治有较好的效果。该菌株也表现出良好的抗细菌活性,对核桃黑斑病菌、烟草青枯病菌、马铃薯环腐病菌、巨大芽孢杆菌等细菌的抑菌圈均在12mm以上。
3.本研究初步探讨了不同培养条件下的发酵液对杨树水泡溃疡病菌的抑菌活性测定。测定不同培养条件的发酵液对杨树水泡溃疡病菌的抑菌效果,得出在NB培养基,培养120 h的PEBA20菌的发酵液,对杨树水泡溃疡病菌的抑菌效果最佳。通过二次正交设计优化发酵条件,计算得到该菌最适宜发酵条件为:T=28.5℃,rpm=140rpm,pH=5,通气量100mL,t=120h。
4. PEBA20菌发酵培养后能够产生抗菌蛋白,不同培养条件下的蛋白粗提液对杨树水泡溃疡病病菌有着较好的拮抗效果。PEBA20菌发酵液的蛋白组分和0-60%饱和度的硫酸铵盐析粗蛋白,经CM-Sepharose Fast Flow阳离子交换柱层析,发现PEBA20菌发酵后的蛋白质中含有阳离子蛋白质,并能通过CM-Sepharose Fast Flow获得初步分离;经DEAE-Sepharose Fast Flow阴离子交换柱层析,形成明显的蛋白质吸收峰,各峰相对独立,峰形较尖锐,且没有拖尾。PEBA20菌的初步分离,为进一步分离纯化奠定基础。
Poplar canker caused by Botryosphaeria dothidea, was a key stem disease which occurred widespreadly and was difficult to control.Biological control was an envirorment friendly method for the disease management with great potential development. There was little reports about biological control of Botryosphaeria dothidea. As a new type of biocontrol factors, plants endophytic bacteria were a class of microorganisms which colonied in a variety of tissues of healthy plants with mutually beneficial co-existence between plants. There was significance of controlling the diseases of poplars with endophytic bacteria isolated from the poplar body.
In this study, we isolated endophytic bacteria from the poplar trees and analysed the bacteria diversity, selected the endophytic bacteria with high antagonistic activity, identificated the endophytic bacteria strain of PEBA20 with systematic taxonomy identification, determined its optimization of fermentation conditions and antibacterial activity, prelimary purified the proteins. The main findings showed as follows:
1. 126 endophytic bacteria were isolated from branches of triploid Populus from Tai-an city in march, june, september and december, 2007 to 2008. We selected 12 poplar endophytic bacteria showed good antagonist activity to Botryosphaeria dothidea with flat antagonist screening method. We found a lot of poplar endophytic bacteria in poplars from these separated bacteria. Using Shannon-Wiener analysis with diversity index (H), abundance (D), evenness (J), the endophytic bacteria from poplar in different time showed significant difference.
2. We identificated PEBA20 as Bacillus amyloliquefaciens through cultivation, staining reaction, physiological or biochemical characteristic analysis and 16SrDNA sequence analysis. PEBA20 showed good antimicrobial effect on Botryosphaeria dothidea. The results showed that the antibacterial spectrum of PEBA20 was broad, the antibacterial activity were high against Valsa sordida、Phytophthora capsiciLeon、Glomerella cingulata、 Fusarium oxysporum and other 12 kinds of pathogenic fungi, the inhibitory rate of the bacteria and fermentation filtrate against Valsa sordidaI was 79.53% and 67.32% respectively, while the inhibitory rate against Botryosphaeria dothidea was 53.27% and 40.48% respectively. This indicated that PEBA20 has good effect to control the disease of poplar trees. PEBA20 also showed good antibateria activity against Xanthomonas jugandis、Ralstonia solanacearum、Clavibacter sepedonicum、Bacillus megaterium , the inhibition zones to bacteria were more than 12mm.
3. Antifungal activitives of different fermentation conditions of PEBA20 were determined. The results showed that culturing for 120h and cultured in NB medium were the favorable conditions for the antibiotic activity. We calculated the most suitable fermentation conditions for PEBA20 were T = 28.5℃, rpm = 140rpm, pH = 5, ventilation volume 100mL, t = 120h.
4. Crude protein of PEBA20 under different culture conditions had better effects to Botryosphaeria dothidea,indicating the existence of th antibiotic proteins. The column chromatography with CM-Sepharose Fast Flow and DEAE-Sepharose Fast Flow demonstreated that the protein with antibiotic activity could be purified.
引文
1. Buchanan RE,Gibbons NE(中国科学院微生物研究所《伯杰细菌鉴定手册》翻译组译).伯杰细菌鉴定手册(第八版)[M].北京:科学出版社,1984.
2.蔡妙英,刘津太,占立克译.芽孢杆菌属[M].戈登,海恩斯,帕格著,北京:农业出版社,1983.
3.陈志谊,陆凡,刘永锋.防治水稻病害微生物农药纹曲宁的研制及产业化[J].江苏农业学报,2003 ,19 (2):108.
4.程洪斌,刘晓桥,陈红漫.枯草芽孢杆菌防治植物真菌病害研究进展[J].上海农业学报,2006, 22(1) : 109- 112.
5.东秀珠,蔡妙英.常见细菌系统手册[M].北京,科学出版社,2001:62-65
6.方中达.植病研究方法(第三版)北京:中国农业出版社1998.12.
7.傅正擎,夏正俊,昊蔼民.内生菌防治棉花黄萎病机理[J].江苏农业学报,1999,15(4):211-215.
8.郭良栋.内生真菌研究进展[J].菌物系统,2001,20(1):148-152.
9.韩继刚,宋未.植物内生细菌研究进展及其应用潜力[J].自然科学进展,2004,14(4):374-379.
10.何红,蔡学清,陈玉森,等.辣椒内生芽孢杆菌(Bacillius subtilis)BS-2和BS-1防治香蕉炭疽病研究[J].福建农林大学学报,2002,31(4):441-443.
11.何红,蔡学清,关雄,胡方平.辣椒内生枯草芽孢杆菌(Bacillus subtilis) BS-1和B2防治辣椒炭疽病研究[J].植物病理学报, 2003,33 (2):170-173.
12.何红,蔡学清,洪永聪,等.辣椒内生细菌的分离及拮抗菌的筛选[J].中国生物防治,2002,18 (4):171-175.
13.何红,邱思鑫,蔡学清,等.辣椒内生细菌BS- I和BS-2在植物体内的定殖及鉴定[J].微生物学报,2004, 44( 1): 13-18.
14.黄海婵,裘娟萍.枯草芽孢杆菌防治植物病害的研究进展[J].浙江农业科学,2005,(3):213-219.
15.金静,王远路,刘建平等.三种杨树树皮真菌群落的研究[J].林业科学研究,2004,17(4):490-495.
16.金钧然,沈瑞祥.杨树林土壤微生物及优势菌对小穴壳菌抑制作用的研究.北京林业大学学报, 1989,11(3):79-84
17.金玲,巴峰.小麦内生有害细菌的发现和作用研究[J].植物病理学报,1996,26(2):123-126.
18.梁军,屈智巍,李忠宁,等.外生菌根菌和植物生长物质复合制剂对杨树扦插苗生长及抗逆性的效应.林业科学研究,2005a,18(6):717-721.
19.梁军,屈智巍,张颖,等.根际微生态保健剂对杨树生长及抗病性的效应.南京林业大学学报,2005b,29(5):45-49.
20.林东,徐庆,刘忆舟,等.枯草芽孢杆菌S0113分泌蛋白的抑菌作用及抗菌蛋白的分离纯化[J].农业生物技术学报,2001.9(1):77-80.
21.刘伟.芽孢杆菌(Bacillus spp.)拮抗菌株的筛选及TasA基因研究[D].[福建农林大学博士学位论文],福州:福建农林大学.2006,14-16.
22.刘晓光,高克祥,谷建才,等.毛白杨内生菌优势种毛壳ND35室内拮抗作用的研究.林业科学,1999,35(5):58-62
23.刘颖,徐庆,陈章良.抗真菌肽LP-I的分离纯化及特性分析[J].微生物学报,1999, 39( 5):441-447.
24.刘云霞,张青文,周明群. Bt杀虫基因向水稻内生细菌的转化研究[J].农业生物技术学报,1987,5(2):188-193.
25.龙良鲲,肖崇刚,窦彦霞.防治番茄青枯病内生细菌的分离与筛选[J].中国蔬菜,2003,2:19-21.
26.鲁素云.植物病害生物防治学[M].北京:北京农业大学出版社,1993:243-254.
27.乔宏萍,黄丽丽,康振生.小麦内生细菌及其对根茎主要病原真菌的抑制作用[J].应用生态学报,2006,17(4):690-694.
28.邱思鑫,何红,阮宏椿,等.内生芽孢杆菌TB2防治辣椒疫病效果及其机理初探[J].植物病理学报,2004,34(2):173-179.
29.邱思鑫.防病、促生植物内生芽孢杆菌[D].[福建农林大学博士学位论文,福州:福建农林大学.2004,139-141.
30.邱晓,裴炎,王喻宁,等.棉花体内的假单胞菌及其对棉苗根病的防效[J].植物保护学报,1990,17(4):303-306.
31.冉国华,张志元.植物内生细菌研究及其应用[J].海南大学学报,2004,22(4):366-373.
32.萨姆布鲁克J,弗里奇EF,曼尼阿蒂斯T(金冬雁等译).分子克隆实验指南(第二版)[M].北京.科学出版社,1998
33.宋子红,丁立孝,马伯军,等.花生内生菌的种群及动态分析[J].植物保护学报,1999,26(4):309-314.
34.汪家政,范明.蛋白质技术手册[M].北京,科学出版社,2000
35.王大耜.细菌分类基础北京[M].:科学出版社,1977.5
36.王金生主编.植物病原细菌学[M].北京:中国农业出版社,2000.12
37.王万能.烟草内生细菌118防治黑茎病的机理研究[J].西南农业大学学报,2003,25(1):28-31.
38.夏正俊,顾本康等.棉花内生及根际土壤细菌诱导棉花对大丽轮枝菌抗性的研究[J].中国生物防治,1996,12(1):7-10.
39.向玉英.杨树重要病害生物防治初探.林业科学研究,1989,2(1):93-98
40.杨安钢,毛积芳,药立波.生物化学与分子生物学实验技术[M].高等教育出版社,2001
41.杨海莲,孙晓璐,宋未.植物根际促生细菌和内生细菌的诱导抗病性的研究进展[J].植物病理学报,2000,30(2):106-110.
42.杨海莲,孙晓璐,宋未.植物内生细菌的研究[J].微生物学通报,1998,25(4):224-227.
43.杨海莲,孙晓璐,宋未等.水稻内生阴沟肠杆菌MR12的鉴定及其固氮和防病作用研究[J].植物病理学报,2001,31(1):92-93.
44.易图永,高必达,何昆.水稻纹枯病菌生防细菌的筛选[J].湖南农业大学学报,2000,26(2):116-118.
45.袁军,孙福在,田宏先.防治马铃薯环腐病有益内生细菌的分离和筛选[J].微生物学报,2002,42(3):270-274.
46.张铎.棉花黄萎病拮抗内生细菌的筛选鉴定及其抗菌物质研究[D].河南师范大学硕士学位论文,2006.
47.张星耀,骆有庆.中国森林重大生物灾害.北京:中国林业出版社,2003,93-119
48.赵达,傅俊范,裘季燕等.枯草芽孢杆菌在植病生防中的作用机制与应用[J].辽宁农业科学,2007(1): 46- 48.
49. Adhilcari T B, Joseph C M. Evaluation of bacteria isolated from rice for plant growth promotion and biological control of seedling diseaseof rice.[J].Can.J.Microbiol,2001,47:916-924.
50. Asaka O, Shoda M. Biocontrol of Rhizoctonia solani damping-off of tomato with Bacillus subtilis RB14 [J]. appl.Environ. Microbiol,1996, 62(11):4081-4085.
51. Baker K F. Evolving concepts of biological control of plant pathogens [J]. Ann. Rev. Phytopathol.,1987,25:67-85.
52. Benhamou N, Kloepper J W, Quadt-Hallmann A,et al. Induction of defense-related ultrastutural modifications in pea root tissues inoculated with endophytic bacteria. Plant Physio.,1996,112(3):919-929.
53. Benhamou N, Kloepper J W, 'Tuzum S. Induction of resistance against Fusarium wilt of tomato by combination of chitosan with an endophytic bacterial strain:ultrastructure and cytochemistry of the host response. Planta.,1998,204:153-168.
54. Boddey R M,Oliveira O C, Urquiaga S, et al. Biological nitrogen fixation associated with sugarcane and rice: contributions and prospects for improment[J].Plant Soil,1995,174:195-209.
55. Brooks D S, Gonzalez C F, Appel D N, et al. Evaluation of endophytic bacteria as potential biological control agents for oak wilt[J].Biol.control,1994,4:373-381.
56. CharlesW.Bacon, Ida E.Yates, Dorothy M.Hinton, et al.Biological Control of Fusarium moniliforme in maize [J].Eirvironmental Health Perspectives, 2001, 109(S2):325-332.
57. Chen C, Bauske E M, Musson G, et al. Biological control of Fusarium wilt on cotton by use of endophytic bacteria[J].Biol.Control,1995,5:83-91.
58. Doty SL, Dosher MR, Singleton GL, Moore AL, Van Aken B, Stettler RF, Strand SE, Gordon MP. Identification of an endophytic Rhizobium in stems of Populus. Symbiosis 2005,39(1): 27-35.
59. Hallmann J,Quadt-Hallmann A,Mahaffee W F,et al.Bacterial endophytes in agricultural crops[J].Canadian Journal of Microbiology,1997,43(10):895-914.
60. Hammerschmidt R, Kuc J. Induced resistance to disease in plants[J]. Kluwer Academic Publishers,DordrechtL, The Netherlands. 1995 , 182.
61. Hollis J P. Bacteria in the healthy potato tissue [J]. Phytopathol.,1951,41:197-209.
62. Jacobson C B, Pasternak J J,Glick B R. Partial purification and characterization of 1-amino-cyclopropane-1-carboxylate deamunase from the plant growth promoting rhizobacterium Pseudomonas putida GR12-2[J].Can.J.Microbiol.,1994,40:1019-1025.
63. Katumoto K. Two new species of Eudarluca hyperparasitic to Botryosphaeria[J]. Transactions of the Mycological Society of Japan., 1986,27(1):11-16
64. Kleopper J W,Schipper B,Bakker P A H M.Proposed elimination of the term endorhizosphere[J].Phytopathol.1992,82:726-727.
65. Lamb TG,Tonkyn DW , K Lluepfel DA.Movement of Pseudomonas aure of-aciensfrom the rhizosphere to aerial plant tissue [J].Can.J.Microbio,1996,1112-1120.
66. Lazarovits G,Nowak J. Rhizobacteria for improvement of plant growth and establishment[J]. Hortscience,1997,32:188-192.
67. Lodewyckx C,Vangronsveld J,Porteous Felal.Endophytic bacteria and their potential applicationsB[J].Critical Reviews in plant Sciences.2002,2(6):583-606.
68. Malik K A, Zafar Y, Mehnaz S, et al. Use of isotopes dilution for quantification of N2 fixation associated with the roots of kallar grass(Leptochloa fusca)[J].Biol.Fertil.Soil,1987,4:103-108.
69. Malik K A,Bilal R. Association of nitrogen-fixing, plant growth promoting rhizobacteria(PGPR)with kallar grass and rice[J].Plant Soil,1997,194:37-44.
70. Mpiga P, Belanger R R, Paulitz T C,et al. Increased resistance to Fusarium oxysporum f. sp. radicis-lycopersici in tomato plants treated with the endophytic bacterium Pseudomonas fluorescens stain 63-28.Physiol. Mol,Plant Pathol.1997,50:301-320.
71. Murty M G,Ladda J K. Influence of Azospirillum inoculation on the mineral uptake and growth of rice under hytroponic conditions[J].Plant Soil,1988,108:281-285.
72. Nowak J, Asiedn S K, Bensalim S, et al. From laboratory to applications: Challenges and progress with in vitro dual cultures of potato and beneficial bacteria[J].Plant Cell Tissue Organ. Cult,1998, 52:97-103.
73. Poon E S, Huang T C,Kuo T T. Possible mechanism of symptom inhibition of bacterial blight of rice by an endophytic bacterium isolated from rice[J].Bot.Bull.Academia Sinica,1977,18:61-70.
74. Quadt-Hallman A ,Benhamou N , Kloepper J W. Bacterial endophytes in cotton: mechanisms of entering the plant[J]. Can. J. Microbiol.1997b,43:557-582.
75. Quadt-Hallmann A , Kloepper J W.Immunological detection and localization of the cotton endophyte Enterobacter asburiae JM22 in differentplant species[J].Can.J.Microbiol,1996,42:1144-1154.
76. Ramamoorthy V, Viswanathan R, Raguchander T, et al. Induction of systemic resistance by plant growth promoting rhizobacteria in crop plants against pest and diseases. Crop Protection,2001,20:1-11.
77. Roger P A,Ladha J K. Biological N2 fixation in wetland rice fields: estimation and contribution to nitrogen balance[J].Plant Soil,1992,141:41-55.
78. Sharma V K,Nowak J. Enhancement of verticillium wilt resistance in tomato transplants by in vitro coculture of seedlings wilt a plantgrowth promoting rhizobacterium(Pseudomonas spp.strain PsJN) [J].Can.J.Microbiol,1998,44:528-536.
79. Sturz A V, Christie B R, Matheson B.G, et al. Endophytic bacterial communities in the periderm of potato tubers and their potential to improve resistance to soil-borne plant pathogens[J].Pant Pathol,1999,48:360-369.
80. Tien T M, Gaskins M H,Hubell D H. Plant growth substances produced by Azospirillum brasilense and their effect on the growth of pearl millet(Pennisetum americanum L.)[J].Appl.Environ.Microbiol,1979,37:1016-1024.
81. Ulrich K, Ulrich A, Ewald D.Diversity of endophytic bacterial communities in poplar grown under field conditions. FEMS Microbiol Ecol. 2008;63(2):169-80
82. Van Aken B, Peres CM, Doty SL, Yoon JM , Schnoor JL Methylobacterium populi sp. nov., a novel aerobic, pink-pigmented, facultatively methylotrophic, methane-utilizing bacterium isolated from poplar trees (Populus deltoides×pnigra DN34). Int J Syst Evol Microbiol 2004,54(4): 1191~1196.
83. Van buren A M, Andre C,Ishimaru C A. Biological control of the bacterial ring rot pathogen by endophytic bacteria isolated from potato[J].Phytopthol,1993,83:1406.
84. Van Peer R, Punte H L M, de Weger L A, et al. Characterization of root surface and endorhizosphere pseudomonads in relation to their colonization of roots[J]. App. Environ. Microbio1.1990 ,56:2462-2470.
85. Xu H, Griffith M, Patten C L, et al. Isolation of an antifreeze protein with ice nucleation activity from the plant growth promoting rhizobacterium Pseudomonas putida GR12-2[J].Can J.Microbiol,1998,44:64-73.
2.蔡妙英,刘津太,占立克译.芽孢杆菌属[M].戈登,海恩斯,帕格著,北京:农业出版社,1983.
3.陈志谊,陆凡,刘永锋.防治水稻病害微生物农药纹曲宁的研制及产业化[J].江苏农业学报,2003 ,19 (2):108.
4.程洪斌,刘晓桥,陈红漫.枯草芽孢杆菌防治植物真菌病害研究进展[J].上海农业学报,2006, 22(1) : 109- 112.
5.东秀珠,蔡妙英.常见细菌系统手册[M].北京,科学出版社,2001:62-65
6.方中达.植病研究方法(第三版)北京:中国农业出版社1998.12.
7.傅正擎,夏正俊,昊蔼民.内生菌防治棉花黄萎病机理[J].江苏农业学报,1999,15(4):211-215.
8.郭良栋.内生真菌研究进展[J].菌物系统,2001,20(1):148-152.
9.韩继刚,宋未.植物内生细菌研究进展及其应用潜力[J].自然科学进展,2004,14(4):374-379.
10.何红,蔡学清,陈玉森,等.辣椒内生芽孢杆菌(Bacillius subtilis)BS-2和BS-1防治香蕉炭疽病研究[J].福建农林大学学报,2002,31(4):441-443.
11.何红,蔡学清,关雄,胡方平.辣椒内生枯草芽孢杆菌(Bacillus subtilis) BS-1和B2防治辣椒炭疽病研究[J].植物病理学报, 2003,33 (2):170-173.
12.何红,蔡学清,洪永聪,等.辣椒内生细菌的分离及拮抗菌的筛选[J].中国生物防治,2002,18 (4):171-175.
13.何红,邱思鑫,蔡学清,等.辣椒内生细菌BS- I和BS-2在植物体内的定殖及鉴定[J].微生物学报,2004, 44( 1): 13-18.
14.黄海婵,裘娟萍.枯草芽孢杆菌防治植物病害的研究进展[J].浙江农业科学,2005,(3):213-219.
15.金静,王远路,刘建平等.三种杨树树皮真菌群落的研究[J].林业科学研究,2004,17(4):490-495.
16.金钧然,沈瑞祥.杨树林土壤微生物及优势菌对小穴壳菌抑制作用的研究.北京林业大学学报, 1989,11(3):79-84
17.金玲,巴峰.小麦内生有害细菌的发现和作用研究[J].植物病理学报,1996,26(2):123-126.
18.梁军,屈智巍,李忠宁,等.外生菌根菌和植物生长物质复合制剂对杨树扦插苗生长及抗逆性的效应.林业科学研究,2005a,18(6):717-721.
19.梁军,屈智巍,张颖,等.根际微生态保健剂对杨树生长及抗病性的效应.南京林业大学学报,2005b,29(5):45-49.
20.林东,徐庆,刘忆舟,等.枯草芽孢杆菌S0113分泌蛋白的抑菌作用及抗菌蛋白的分离纯化[J].农业生物技术学报,2001.9(1):77-80.
21.刘伟.芽孢杆菌(Bacillus spp.)拮抗菌株的筛选及TasA基因研究[D].[福建农林大学博士学位论文],福州:福建农林大学.2006,14-16.
22.刘晓光,高克祥,谷建才,等.毛白杨内生菌优势种毛壳ND35室内拮抗作用的研究.林业科学,1999,35(5):58-62
23.刘颖,徐庆,陈章良.抗真菌肽LP-I的分离纯化及特性分析[J].微生物学报,1999, 39( 5):441-447.
24.刘云霞,张青文,周明群. Bt杀虫基因向水稻内生细菌的转化研究[J].农业生物技术学报,1987,5(2):188-193.
25.龙良鲲,肖崇刚,窦彦霞.防治番茄青枯病内生细菌的分离与筛选[J].中国蔬菜,2003,2:19-21.
26.鲁素云.植物病害生物防治学[M].北京:北京农业大学出版社,1993:243-254.
27.乔宏萍,黄丽丽,康振生.小麦内生细菌及其对根茎主要病原真菌的抑制作用[J].应用生态学报,2006,17(4):690-694.
28.邱思鑫,何红,阮宏椿,等.内生芽孢杆菌TB2防治辣椒疫病效果及其机理初探[J].植物病理学报,2004,34(2):173-179.
29.邱思鑫.防病、促生植物内生芽孢杆菌[D].[福建农林大学博士学位论文,福州:福建农林大学.2004,139-141.
30.邱晓,裴炎,王喻宁,等.棉花体内的假单胞菌及其对棉苗根病的防效[J].植物保护学报,1990,17(4):303-306.
31.冉国华,张志元.植物内生细菌研究及其应用[J].海南大学学报,2004,22(4):366-373.
32.萨姆布鲁克J,弗里奇EF,曼尼阿蒂斯T(金冬雁等译).分子克隆实验指南(第二版)[M].北京.科学出版社,1998
33.宋子红,丁立孝,马伯军,等.花生内生菌的种群及动态分析[J].植物保护学报,1999,26(4):309-314.
34.汪家政,范明.蛋白质技术手册[M].北京,科学出版社,2000
35.王大耜.细菌分类基础北京[M].:科学出版社,1977.5
36.王金生主编.植物病原细菌学[M].北京:中国农业出版社,2000.12
37.王万能.烟草内生细菌118防治黑茎病的机理研究[J].西南农业大学学报,2003,25(1):28-31.
38.夏正俊,顾本康等.棉花内生及根际土壤细菌诱导棉花对大丽轮枝菌抗性的研究[J].中国生物防治,1996,12(1):7-10.
39.向玉英.杨树重要病害生物防治初探.林业科学研究,1989,2(1):93-98
40.杨安钢,毛积芳,药立波.生物化学与分子生物学实验技术[M].高等教育出版社,2001
41.杨海莲,孙晓璐,宋未.植物根际促生细菌和内生细菌的诱导抗病性的研究进展[J].植物病理学报,2000,30(2):106-110.
42.杨海莲,孙晓璐,宋未.植物内生细菌的研究[J].微生物学通报,1998,25(4):224-227.
43.杨海莲,孙晓璐,宋未等.水稻内生阴沟肠杆菌MR12的鉴定及其固氮和防病作用研究[J].植物病理学报,2001,31(1):92-93.
44.易图永,高必达,何昆.水稻纹枯病菌生防细菌的筛选[J].湖南农业大学学报,2000,26(2):116-118.
45.袁军,孙福在,田宏先.防治马铃薯环腐病有益内生细菌的分离和筛选[J].微生物学报,2002,42(3):270-274.
46.张铎.棉花黄萎病拮抗内生细菌的筛选鉴定及其抗菌物质研究[D].河南师范大学硕士学位论文,2006.
47.张星耀,骆有庆.中国森林重大生物灾害.北京:中国林业出版社,2003,93-119
48.赵达,傅俊范,裘季燕等.枯草芽孢杆菌在植病生防中的作用机制与应用[J].辽宁农业科学,2007(1): 46- 48.
49. Adhilcari T B, Joseph C M. Evaluation of bacteria isolated from rice for plant growth promotion and biological control of seedling diseaseof rice.[J].Can.J.Microbiol,2001,47:916-924.
50. Asaka O, Shoda M. Biocontrol of Rhizoctonia solani damping-off of tomato with Bacillus subtilis RB14 [J]. appl.Environ. Microbiol,1996, 62(11):4081-4085.
51. Baker K F. Evolving concepts of biological control of plant pathogens [J]. Ann. Rev. Phytopathol.,1987,25:67-85.
52. Benhamou N, Kloepper J W, Quadt-Hallmann A,et al. Induction of defense-related ultrastutural modifications in pea root tissues inoculated with endophytic bacteria. Plant Physio.,1996,112(3):919-929.
53. Benhamou N, Kloepper J W, 'Tuzum S. Induction of resistance against Fusarium wilt of tomato by combination of chitosan with an endophytic bacterial strain:ultrastructure and cytochemistry of the host response. Planta.,1998,204:153-168.
54. Boddey R M,Oliveira O C, Urquiaga S, et al. Biological nitrogen fixation associated with sugarcane and rice: contributions and prospects for improment[J].Plant Soil,1995,174:195-209.
55. Brooks D S, Gonzalez C F, Appel D N, et al. Evaluation of endophytic bacteria as potential biological control agents for oak wilt[J].Biol.control,1994,4:373-381.
56. CharlesW.Bacon, Ida E.Yates, Dorothy M.Hinton, et al.Biological Control of Fusarium moniliforme in maize [J].Eirvironmental Health Perspectives, 2001, 109(S2):325-332.
57. Chen C, Bauske E M, Musson G, et al. Biological control of Fusarium wilt on cotton by use of endophytic bacteria[J].Biol.Control,1995,5:83-91.
58. Doty SL, Dosher MR, Singleton GL, Moore AL, Van Aken B, Stettler RF, Strand SE, Gordon MP. Identification of an endophytic Rhizobium in stems of Populus. Symbiosis 2005,39(1): 27-35.
59. Hallmann J,Quadt-Hallmann A,Mahaffee W F,et al.Bacterial endophytes in agricultural crops[J].Canadian Journal of Microbiology,1997,43(10):895-914.
60. Hammerschmidt R, Kuc J. Induced resistance to disease in plants[J]. Kluwer Academic Publishers,DordrechtL, The Netherlands. 1995 , 182.
61. Hollis J P. Bacteria in the healthy potato tissue [J]. Phytopathol.,1951,41:197-209.
62. Jacobson C B, Pasternak J J,Glick B R. Partial purification and characterization of 1-amino-cyclopropane-1-carboxylate deamunase from the plant growth promoting rhizobacterium Pseudomonas putida GR12-2[J].Can.J.Microbiol.,1994,40:1019-1025.
63. Katumoto K. Two new species of Eudarluca hyperparasitic to Botryosphaeria[J]. Transactions of the Mycological Society of Japan., 1986,27(1):11-16
64. Kleopper J W,Schipper B,Bakker P A H M.Proposed elimination of the term endorhizosphere[J].Phytopathol.1992,82:726-727.
65. Lamb TG,Tonkyn DW , K Lluepfel DA.Movement of Pseudomonas aure of-aciensfrom the rhizosphere to aerial plant tissue [J].Can.J.Microbio,1996,1112-1120.
66. Lazarovits G,Nowak J. Rhizobacteria for improvement of plant growth and establishment[J]. Hortscience,1997,32:188-192.
67. Lodewyckx C,Vangronsveld J,Porteous Felal.Endophytic bacteria and their potential applicationsB[J].Critical Reviews in plant Sciences.2002,2(6):583-606.
68. Malik K A, Zafar Y, Mehnaz S, et al. Use of isotopes dilution for quantification of N2 fixation associated with the roots of kallar grass(Leptochloa fusca)[J].Biol.Fertil.Soil,1987,4:103-108.
69. Malik K A,Bilal R. Association of nitrogen-fixing, plant growth promoting rhizobacteria(PGPR)with kallar grass and rice[J].Plant Soil,1997,194:37-44.
70. Mpiga P, Belanger R R, Paulitz T C,et al. Increased resistance to Fusarium oxysporum f. sp. radicis-lycopersici in tomato plants treated with the endophytic bacterium Pseudomonas fluorescens stain 63-28.Physiol. Mol,Plant Pathol.1997,50:301-320.
71. Murty M G,Ladda J K. Influence of Azospirillum inoculation on the mineral uptake and growth of rice under hytroponic conditions[J].Plant Soil,1988,108:281-285.
72. Nowak J, Asiedn S K, Bensalim S, et al. From laboratory to applications: Challenges and progress with in vitro dual cultures of potato and beneficial bacteria[J].Plant Cell Tissue Organ. Cult,1998, 52:97-103.
73. Poon E S, Huang T C,Kuo T T. Possible mechanism of symptom inhibition of bacterial blight of rice by an endophytic bacterium isolated from rice[J].Bot.Bull.Academia Sinica,1977,18:61-70.
74. Quadt-Hallman A ,Benhamou N , Kloepper J W. Bacterial endophytes in cotton: mechanisms of entering the plant[J]. Can. J. Microbiol.1997b,43:557-582.
75. Quadt-Hallmann A , Kloepper J W.Immunological detection and localization of the cotton endophyte Enterobacter asburiae JM22 in differentplant species[J].Can.J.Microbiol,1996,42:1144-1154.
76. Ramamoorthy V, Viswanathan R, Raguchander T, et al. Induction of systemic resistance by plant growth promoting rhizobacteria in crop plants against pest and diseases. Crop Protection,2001,20:1-11.
77. Roger P A,Ladha J K. Biological N2 fixation in wetland rice fields: estimation and contribution to nitrogen balance[J].Plant Soil,1992,141:41-55.
78. Sharma V K,Nowak J. Enhancement of verticillium wilt resistance in tomato transplants by in vitro coculture of seedlings wilt a plantgrowth promoting rhizobacterium(Pseudomonas spp.strain PsJN) [J].Can.J.Microbiol,1998,44:528-536.
79. Sturz A V, Christie B R, Matheson B.G, et al. Endophytic bacterial communities in the periderm of potato tubers and their potential to improve resistance to soil-borne plant pathogens[J].Pant Pathol,1999,48:360-369.
80. Tien T M, Gaskins M H,Hubell D H. Plant growth substances produced by Azospirillum brasilense and their effect on the growth of pearl millet(Pennisetum americanum L.)[J].Appl.Environ.Microbiol,1979,37:1016-1024.
81. Ulrich K, Ulrich A, Ewald D.Diversity of endophytic bacterial communities in poplar grown under field conditions. FEMS Microbiol Ecol. 2008;63(2):169-80
82. Van Aken B, Peres CM, Doty SL, Yoon JM , Schnoor JL Methylobacterium populi sp. nov., a novel aerobic, pink-pigmented, facultatively methylotrophic, methane-utilizing bacterium isolated from poplar trees (Populus deltoides×pnigra DN34). Int J Syst Evol Microbiol 2004,54(4): 1191~1196.
83. Van buren A M, Andre C,Ishimaru C A. Biological control of the bacterial ring rot pathogen by endophytic bacteria isolated from potato[J].Phytopthol,1993,83:1406.
84. Van Peer R, Punte H L M, de Weger L A, et al. Characterization of root surface and endorhizosphere pseudomonads in relation to their colonization of roots[J]. App. Environ. Microbio1.1990 ,56:2462-2470.
85. Xu H, Griffith M, Patten C L, et al. Isolation of an antifreeze protein with ice nucleation activity from the plant growth promoting rhizobacterium Pseudomonas putida GR12-2[J].Can J.Microbiol,1998,44:64-73.