: 1241 (Rhizoctonis solani) [1],,,,,,,,, [2 4],, [5,6], 1 材料与方法 1.1 材料玉米品种 : 13, 培养基 :1) (PDA) ; 2) (BPA) 病原菌 :(Rhizotonia solani)

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1 微生物学通报 AUG 20, 2008, 35(8): 1240~1245 Microbiology 研究报告 2008 by Institute of Microbiology, CAS 一株抗玉米纹枯病内生细菌的分离鉴定及其抗病促生作用 1 辜运富 2 张云飞 1* 张小平 ( ) ( ) 摘要 : 玉米纹枯病是玉米主要的真菌病害, 从优良玉米品种川单 13 号中分离到一株对玉米纹枯病菌具有明显抑制作用并能够促进玉米生长的内生细菌通过形态生理生化特性分析以及 16S rdna 序列同源性比较, 鉴定该菌株为枯草芽孢杆菌盆栽试验表明, 菌株能够抑制玉米纹枯病的发生, 抑菌率为 43.01%, 促生试验表明, 该菌能够显著促进玉米苗的生长, 促生作用与植物生长刺激素 (IAA) 的效果相似, 显示出良好的开发前景关键词 : 玉米纹枯病, 立枯丝核菌, 内生细菌, 16S rdna Isolation and Identification of One Anti-Rhizoctonis solani Endophytic Bacteria Strain from Corn and Its Antagonism and Promoting Research GU Yun-Fu 1 ZHANG Yun-Fei 2 ZHANG Xiao-Ping 1* (1. College of Environmental and Resource Science, Sichuan Agricultural University, Ya an ) (2. Institute of Biogas Science, Ministry of Agriculture, Chengdu ) Abstract: The corn sheath blight was the main fungus pathogen of the corn. One endophytic bacteria strain which could significantly inhibit corn sheath blight and promote corn growth was isolated from Chuandan 13 corn seeding. According to morphological, biophysiological and biochemical characteristics and 16S rdna sequence homology, the strain was identified as Bacillus subtilis, pot experiments indicated the strain could inhibit corn sheath blight from happening, the inhibit rate could reach 43.01%; promoting growth experiment revealed that it could significantly promote corn seed growth, the promoting effect was equal to that of IAA, which showed a good development prospect. Keywords: Corn sheath blight, Rhizoctonis solani, Endophytic bacteria, 16S rdna, 2500, 1.2, 90,, * 通讯作者 :Tel: ; : aumdwsb@sicau.edu.cn 收稿日期 : ; 接受日期 :

2 : 1241 (Rhizoctonis solani) [1],,,,,,,,, [2 4],, [5,6], 1 材料与方法 1.1 材料玉米品种 : 13, 培养基 :1) (PDA) ; 2) (BPA) 病原菌 :(Rhizotonia solani) (AG1-AI) 1.2 方法内生细菌的分离纯化 :1) ( 5 cm, 10 cm~15 cm, ) 5 g 2) 95 3 min~5 min min, 4, 0.5 ml,, 28 ~30 3 d~4 d,, 3 3) 2 cm~3 cm,,, 100 ml, 10 1, 10 6, 0.5 ml, 28 ~30 3 d~4 d, 3, 4), ) [7], 6) CNBH,, 抑菌作用测定 :1) PDA, 2.5 cm,,, 3,,, 1 ( CNBH) 2) (d=24 cm) 15 g, 6 cm ; ( CFU/mL), 5 h,, 10, d~20 d ( )=/ 100 ( )=/ 促生作用测定 : ( LB 72 h), CFU/mL,, IAA, 5 h,, 10, 10, 5 d 生理生化及形态鉴定 : (M.R) V-P (, 2001) [8] 细菌总 DNA 的提取 : BPA 28, 5000 r/min 15 min,, DNA [9], S rdna 的扩增纯化及克隆 :PCR 16S rdna, Primer 1(P1)5 - AGAGTTTGATCCTGGCTCAGAACGAACGCT-3 ;

3 1242 微生物学通报 2008, Vol.35, No.8 Primer 2 (P6)5 -TACGGCTACCTTGTTACGACTTC ACCCC-3 PCR 25 μl 2 PCR Master mix (TIANGen, Beijing), 0.5 μl (25 pmol/μl), 0.5 μl DNA, 23.5 μl 50 μl PCR 94 5 min; 94 1 min, s, 72 1 min 30 s, 35 ; 72 7 min PCR 1PCR UNIQ- PCR ( ) PCR pmd18-t (DH5α)( ), Amp + +X-gal+IPTG+LB S rdna 的测序及系统发育树的构建 :16S rdna, 1.5 kb BLAST GeneBank 16S rdna DNAMEGA 2 结果与分析 2.1 菌株 CNBH 的分离与生理生化鉴定 13 1, CNBH,,,,,,, 1,, 2.2 菌株 CNBH 的内生性检测 CNBH ( ), 1,,, 5 3, 3 ; 5,, 7, CNBH, Table 1 表 1 菌株 CNBH 的生理生化特性 Biophysiological and biochemical characteristics of strain CNBH Test item Results Gram staining + Nitrate + Starch hydrolysis + Amylase Catalase + L- Pectinose Spore staining + Shape Methyl red Gelatin liquefaction + V-P V-P test + Motility + Phenylalanine ammonia lyase Casein hydrolysis + Glucose oxidation + Mannitol + Litmus milk test +: ; : Note: +: Positive reaction; : Negative reaction 2.3 菌株 CNBH 的拮抗效果测定, CNBH, 26.3 mm, 1 1a CNBH, ( 1b ), ;, Strain code 表 2 Table 2 菌株 CNBH 突变株的回接分离 Isolate the mutant of strain CNBH Sampling time 3 d 5 d 7 d Root Stem Leaves Root Stem Leaves Root Stem Leaves CNBH CK +: ; : Note: +: Isolated; : Not isolated

4 : 1243 图 1 菌株 CNBH 对玉米纹枯病菌的影响 Fig. 1 Affection of strain CNBH to corn sheath blight, ( ) PDA,, CNBH 3 ( 3),, CNBH 32, 14.4 mm;, CNBH 14.88, 9.05 mm, CNBH, ; CNBH 2.4 菌株 CNBH 对玉米的促生作用测定 4 2,, CNBH,,,, BH, 48.0 mm, 17.5 mm, IAA CNBH,, 2.5 菌株 CNBH 稳定性检测 CNBH PDA 20,,,, CNBH, 5 5,, CNBH, 12,, Table 3 表 3 菌株 CNBH 对玉米苗期纹枯病的盆栽防治实验 The inhibition of strain CNBH to corn sheath blight in pots experiment Strain code Real plant numbers Plant numbers being infected (%) Infected rate Dead numbers (%) Dead rate Lesions diameters CK b CK a CNBH a (Duncan P=0.01) Note: The same letter after the numbers mean the treatments are not significantly different (P=0.01, LSR test) Treatments 表 4 菌株 CNBH 对玉米植株高度及主根长度的影响 ( 单位 :cm) Table 4 Affection of strain CNBH to corn stem and root(unit: cm) CK( ) CNBH CK(IAA) Length Root Stem Leaves Root Stem Leaves Average length 7.05 a 2.65 a c 4.43 b b 5.50 c (Duncan P=0.01) Note: The same letter after the numbers mean the treatments are not significantly different (P=0.01, LSR test)

5 1244 微生物学通报 2008, Vol.35, No.8 图 2 Fig. 2 菌株 CNBH 对玉米苗的促生作用 Promoting to corn seed of strain CNBH Passage numbers Passage numbers 表 5 菌株 CNBH 的稳定性检测 Table 5 The stability of strain CNBH Passage numbers Passage numbers mm, 2.6 菌株 CNBH 的分子生物学鉴定 CNBH 16S rdna PCR 1.5 kb GenBank, 8P119 WMB014 Blast CNBH 16S rdna GenBank 16S rdna, DNASTAR, (Bacillus subtilis) 16S rdna 95,, CNBH 3 图 3 菌株 CNBH 的系统发育树 Fig. 3 The phylogenetic tree of strain CNBH 3 讨论 3.1 内生细菌的研究意义 (endophytic bacteria) [10],,,,,,

6 : 1245,, [11,12] 3.2 关于内生枯草芽孢杆菌的研究 (B. subtilis), [13], [14] [15] [16] [17] ; [18],,,,,,, 13,, CNBH, CNBH,, CNBH 35,,,,, 参考文献 [1].. :, 1966, pp [2],,.., 2004, 23(2): [3],.. ( ), 2001, 32(2): [4],,,.., 2004, 15(4): [5],,,.., 2005, 22(4): [6],.., 1999, 29: [7],,,. BS-2 BS-1. ( ), 2003, 32(1): [8],.. :, 1999, pp [9],,,. DNA., 2002, 29 (6): [10] Di Fiore S. Entophytic bacteria and their possible role in the host plant Istven F. Azospirilum VI and related microorganisms: genetics, physiology, ecology. Heidelberg: Springer, 1995, pp [11] Kleopper JW, Beauehamp CJ. A review of issues related to measuring colonization of plant roots by bacteria. Canadian Journal of Microbiology, 1992, 38: [12],.., 1994, 20(5): [13] Dubnau, David A. Molecular biology an international series of monographs and text books. The molecular biology of the Bacilli.Volume1: Bacillus subtilis. New York: Academic Press, 1982, pp [14] Bai Y, Aoust FD, Smith DL, et al. Isolation of plant-growth-promoting Bacillus strains from soy-bean root nodules Can J Microbial, 2002, 48(3): [15] Choi EH, Lee SE, Kwon KS, et al. Isolation of nitrogen-fixing bacteria from gramineous crops and measurement of nitrogenase activity. Korean J of Microbial & Biotech, 2003, 31(1): [16] Wilhelm E, Arthofer W, Schafleitner R, et al. Bacillus subtilis an endophyte of chestnut (Castanea sativa) as antagonist against chestnut blight (Cryphonectria parasitica). Plant Cell, Tissue and Organ Culture, 1998, 52: [17],,,. BS-1 BS-2., 2004, 44(1): [18],,. B , 2006, 36(6):

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: 1241 (Rhizoctonis solani) [1],,,,,,,,, [2 4],, [5,6], 1 材料与方法 1.1 材料 玉米品种 : 13, 培养基 :1) (PDA) ; 2) (BPA) 病原菌 :(Rhizotonia solani)

: 1241 (Rhizoctonis solani) [1],,,,,,,,, [2 4],, [5,6], 1 材料与方法 1.1 材料玉米品种 : 13, 培养基 :1) (PDA) ; 2) (BPA) 病原菌 :(Rhizotonia solani)