新疆野苹果树内生细菌分离与鉴定及其对3种苹果病原菌的抑制作用

doi:10.11707/j.1001-7488.20200511

Abstract

Abstract:

Objective: In this study,endophytic bacteria were isolated and screened from a wild apple (Malus sieversii) in Xinjiang,which is an excellent germplasm resource in China,in order to obtain new bacterial resources for biological control of apple diseases. Method: Endophytic bacteria were isolated from the branches of M. sieversii by the methods of dilution plating and tissue isolation. Seven endophytic bacteria strains,with the strong inhibitory effect on three pathogenic fungi (Botryosphaeria dothidea,Colletotrichum gloeosporioides and Phomopsis mali),were selected by the method of plate confrontation. The 7 strains were identified based on morphological,physiological and biochemical characteristics,as well as 16S rDNA sequence analysis. Their pathogenicity was determined by using in vitro inoculation tests. Result: In this study,a total of 217 strains of endophytic bacteria were isolated and purified,among which seven strains (M-m7,M-m12,M-r12,P-m9,P-m11,L-m12 and P-r21) with strong inhibitory effects on mycelium growth of three pathogenic fungi of B. dothidea,C. gloeosporioides and P. mali were screened out. Their inhibition rates were 77.00%,72.60%,76.60%,60.64%,65.96%,65.81% and 71.79% on B. dothidea; 69.85%,64.95%,68.87%,68.37%,56.12%,53.54% and 65.66% on C. gloeosporioides; 77.63%,79.22%,72.60%,69.37%,70.27%,69.30% and 71.05% on P. mali,respectively. Strains M-m7,M-m12,P-m11 and L-m12 were identified as Bacillus subtilis,strains M-r12 and P-m9 were identified as B. elezensis and strain P-r21 was identified as B. amyloliquefaciens. Inoculation tests showed the 7 strains had no pathogenicity to apple tree itself. Conclusion: Seven strains of endophytic bacteria in M. sieversii can effectively suppress mycelium growth of B. dothidea,C. gloeosporioides and P. mali,and have no pathogenicity to apple tree.

Key words: Malus sieversii, endophytic bacteria, inhibition activity, Bacillus

CLC Number:

S763.13

Yongli Li,Yahong Wang,Le Chang,Hairu Yu,Zhou Zhou,Wenxia Zhao,Liangjian Qu. Isolation and Identification of Endophytic Bacteria from Xinjiang Wild Apple (Malus sieversii) and Their Antagonism against Three Pathogens of Apple Trees[J]. Scientia Silvae Sinicae, 2020, 56(5): 97-104.

Figures/Tables 5

Table 1

Fig.1

Fig.2

Table 2

Colony characteristics of seven endophytic bacterial strains"

菌株 Strain	NA上菌落特征 Colony characteristics in NA	PDA上菌落特征 Colony characteristics in PDA
M-m7	菌落白色；圆形；表面湿润；中央隆起，中心凹陷，有褶皱；不透明；有光泽；边缘光滑；生长速度慢White, round, wet surface, central bulge, depressed in the center, wrinkled, opaque, shiny, smooth edge, slow growth	菌落白色；近圆形；表面湿润；中央突起褶皱；半透明；有光泽；边缘光滑；生长速度快White, suborbicular, wet surface, central bulge, wrinkled, translucent, shiny, smooth edge, rapid growth
M-m12	菌落白色；圆形；表面湿润；中央隆起，中心凹陷，有褶皱；不透明；有光泽；边缘光滑；生长速度慢White, round, wet surface, central bulge, depressed in the center, wrinkled, opaque, shiny, smooth edge, slow growth	菌落白色；近圆形；表面湿润；中央突起褶皱；半透明；有光泽；边缘光滑、不整齐；生长速度快White, suborbicular, wet surface, central bulge, wrinkled, translucent, shiny, smooth edge, rapid growth
M-r12	菌落白色；圆形；表面微凸，中央凹陷，有褶皱；不透明；有光泽；边缘整齐；生长速度慢White, round, slightly convex surface, depressed in the center, wrinkled, opaque, shiny, neat edge, slow growth	菌落白色；近圆形；表面干燥；中央隆起，有褶皱；不透明；无光泽；边缘不整齐；生长速度快White, suborbicular, dry surface, central bulge, wrinkled, opaque, matted, ragged edge, rapid growth
P-m9	菌落白色；圆形；表面微凸，湿润；不透明；有光泽；边缘整齐；生长速度慢White, round, slightly convex surface, wet, opaque, shiny, neat edge, slow growth	菌落白色；近圆形；表面干燥；中央隆起，有褶皱；不透明；无光泽；边缘不整齐；生长速度快White, suborbicular, dry surface, central bulge, wrinkled, opaque, matted, ragged edge, rapid growth
P-m11	菌落白色；圆形；表面湿润；中央隆起，中心凹陷，有褶皱；不透明；有光泽；边缘光滑；生长速度慢White, round, wet surface, central bulge, depressed in the center, wrinkled, opaque, shiny, smooth edge, slow growth	菌落白色；近圆形；表面湿润；中央突起褶皱；不透明；有光泽；边缘光滑、不整齐或呈缺刻状；生长速度快White, suborbicular, wet surface, central bulge, wrinkled, opaque, shiny, smooth and ragged or notch edge, rapid growth
L-m12	菌落白色；圆形；表面干燥有褶皱；不透明；无光泽；边缘列齿；生长速度慢White, round, dry surface, wrinkled, opaque, matted, dentate edge, slow growth	菌落白色；菌落平展、近圆形；表面干燥；中央有褶皱；不透明；无光泽；边缘不整齐或呈缺刻状；生长速度快White, flat, suborbicular, dry surface, wrinkled in the center, opaque, matted, ragged or notch edge, rapid growth
P-r21	菌落白色；圆形；表面干燥有褶皱；不透明；无光泽；边缘列齿；生长速度慢White, round, dry surface, wrinkled, opaque, matted, dentate edge, slow growth	菌落白色；菌落平展、近圆形；表面干燥；中央有褶皱；不透明；无光泽；边缘不整齐或呈缺刻状；生长速度快White, flat, suborbicular, dry surface, wrinkled in the center, opaque, matted, ragged or notch edge, rapid growth

Table 2

Table 3

References 0

	陈学森,冯涛,张艳敏,等. 2006.新疆野苹果挥发性化合物组分的遗传多样性研究.全国野生果树资源研究与开发利用学术研讨会, 5-13
	Chen X S, Feng T, Zhang Y M, et al. 2006. Genetic diversity of volatile components in Xinjiang wild apple(Malus sieversii). National Symposium on Research, Development and Utilization of Wild Fruit Resource, 5-13.[in Chinese]
	东秀珠, 蔡妙英. 常见细菌系统鉴定手册. 北京: 科学出版社. 2001.
	Dong X Z , Cai M Y . Handbook of identification of common bacterial systems. Beijing: Science Press. 2001.
	方中达. 植病研究方法. 3版北京: 中国农业出版社. 1998.
	Fang Z D . Research methods of plant diseases. 3^rd Edition Beijing: China Agriculture Press. 1998.
	冯涛, 张红, 陈学森, 等. 新疆野苹果果实形态与矿质元素含量多样性以及特异性状单株. 植物遗传资源学报, 2006. 7 (3): 270- 276. doi: 10.3969/j.issn.1672-1810.2006.03.004
	Feng T , Zhang H , Chen X S , et al. Genetic diversity of fruit morphological traits and content of mineral element in Malus sieversii(Ldb.) Roem. and its elite seedlings. Journal of Plant Genetic Resources, 2006. 7 (3): 270- 276. doi: 10.3969/j.issn.1672-1810.2006.03.004
	李芳, 邹妍, 刘彬, 等. 新疆野苹果(Malus sieversii)内生菌的分离与鉴定. 天津农学院学报, 2015. 22 (4): 1- 5. doi: 10.3969/j.issn.1008-5394.2015.04.001
	Li F , Zou Y , Liu B , et al. Isolation and identification of endophytic fungi in Malus sieversii. Journal of Tianjin Agricultural College, 2015. 22 (4): 1- 5. doi: 10.3969/j.issn.1008-5394.2015.04.001
	李秀明. 2013.生防木霉菌T4和枯草芽孢杆菌B99-2制剂的研制及田间试验.上海:华东理工大学硕士学位论文.
	Li X M. 2013. Studies on the formulation of biocontrol agents Trichoderma harzianum T4 and Bacillus subtilis B99-2 and biocontrol in green house. Shanghai: MS thesis of East China University of Science and Technology.[in Chinese]
	马闯, 杨美玲, 张云秀, 等. 新疆野苹果(Malus sieversii)种群年龄结构及其动态特征. 干旱区研究, 2018. 35 (1): 156- 164.
	Ma C , Yang M L , Zhang Y X , et al. Age composition and dynamic characteristics of the main populations of endangered Malus sieversii. Arid Zone Research, 2018. 35 (1): 156- 164.
	彭兵, 张树斌, 贾宇, 等. 枯草芽胞杆菌菌株A抗菌蛋白的分离纯化及抗真菌机理. 中国农业科学, 2011. 44 (1): 67- 74. doi: 10.3864/j.issn.0578-1752.2011.01.008
	Peng B , Zhang S B , Jia Y , et al. Purification and antifungal mechanism of a kind of antifungal protein from strain A of Bacillus subtilis. Scientia Agricultura Sinica, 2011. 44 (1): 67- 74. doi: 10.3864/j.issn.0578-1752.2011.01.008
	秦伟, 耿文娟, 唐芳, 等. 新疆野苹果繁育特性研究. 新疆农业大学学报, 2010. 33 (4): 317- 321. doi: 10.3969/j.issn.1007-8614.2010.04.008
	Qin W , Geng W J , Tang F , et al. Study on reproduction characteristics for Malus sieversii. Journal of Xinjiang Agricultural University, 2010. 33 (4): 317- 321. doi: 10.3969/j.issn.1007-8614.2010.04.008
	秦伟, 刘立强, 廖康, 等. 新疆苹果种质资源亲缘关系的SSR分析. 新疆农业科学, 2011. 48 (1): 48- 52.
	Qin W , Liu L Q , Liao K , et al. SSR analysis on genetic relationships of Xinjiang Malus germplasm resources. Xinjiang Agricultural Sciences, 2011. 48 (1): 48- 52.
	苏静. 2007.苹果内生细菌的分离及其对苹果斑点落叶病的生物防治研究.西安:西北农林科技大学硕士学位论文.
	Su J. 2007. Isolation of endophytic bacteria from apple tree and biocontrol on apple spot leaf drop. Xi'an: MS thesis of Northwest A&F University.[in Chinese]
	田润炜, 蔡新斌, 刘丽燕, 等. 新疆野苹果种群年龄结构特征与动态分析. 西北植物学报, 2016. 36 (4): 811- 817.
	Tian R W , Cai X B , Liu L Y , et al. Characteristics of the age structure and dynamics of Malus sieversii population. Acta Botanica Boreali-Occidentalia Sinica, 2016. 36 (4): 811- 817.
	王勇, 王万立, 刘春艳, 等. 番茄红粉病致病病原的鉴定及其培养特性研究. 华北农学报, 2008. 23 (6): 97- 100.
	Wang Y , Wang W L , Liu C Y , et al. Studies on identification and cultural characterization of Trichothecium roseum. Acta Agriculturae Boreali-Sinica, 2008. 23 (6): 97- 100.
	文才艺, 吴元华, 田秀玲. 植物内生菌研究进展及其存在的问题. 生态学杂志, 2004. 23 (2): 86- 91. doi: 10.3321/j.issn:1000-4890.2004.02.019
	Wen C Y , Wu Y H , Tian X L . Recent advances and issues on the endophyte. Chinese Journal of Ecology, 2004. 23 (2): 86- 91. doi: 10.3321/j.issn:1000-4890.2004.02.019
	周婧. 2013.利用BIBAC文库建立胡杨转化基因组学平台及鉴定胡杨耐盐基因(簇)的研究.北京:中国林业科学研究院博士学位论文.
	Zhou J. 2013. Establishment of transgenomics for identifiying Populus euphratica Oliv. gene clusters by constructing BIBAC library. Beijing: PhD thesis of Chinese Academy of Forestry.[in Chinese]
	邹文欣, 谭仁祥. 植物内生菌研究新进展. 植物学报, 2001. 43 (9): 881- 892. doi: 10.3321/j.issn:1672-9072.2001.09.001
	Zou W X , Tan R X . Recent advances on endophyte research. Acta Botanica Sinica, 2001. 43 (9): 881- 892. doi: 10.3321/j.issn:1672-9072.2001.09.001
	吴传金, 陈学森, 曾继吾, 等. 新疆野苹果(Malus sieversii)超低温保存及其植株再生. 植物遗传资源学报, 2008. 9 (2): 243- 247.
	Wu C J , Chen X S , Zeng J W , et al. Cryopreservation of in vitro shoot tips of Malus sieversii by vitrification and its regeneration. Journal of Plant Genetic Resources, 2008. 9 (2): 243- 247.
	徐涛. 2012.苹果树皮内生真菌的分离及其对腐烂病的生防作用.保定:河北农业大学硕士学位论文.
	Xu T. 2012. Isolation of endophytic fungi from apple bark and their biological control effect on Valsa canker. Baoding: MS thesis of Hebei Agricultural University.[in Chinese]
	徐彦, 钟翡, 廖康, 等. 干旱胁迫下新源1号新疆野苹果抑制性差减文库的构建与初步分析. 农业生物技术学报, 2010. 18 (1): 149- 155. doi: 10.3969/j.issn.1674-7968.2010.01.023
	Xu Y , Zhong F , Liao K , et al. Construction and preliminary analysis of cDNA library of Malus sieversii under drought stress by suppression subtractive hybridization. Journal of Agricultural Biotechnology, 2010. 18 (1): 149- 155. doi: 10.3969/j.issn.1674-7968.2010.01.023
	闫鹏, 韩立群, 梅闯, 等. 新疆野苹果[Malus sieversii(Ledeb.)M. Roem.]植物学性状遗传多样性及相关性分析. 植物遗传资源学报, 2016. 17 (4): 683- 689.
	Yan P , Han L Q , Mei C , et al. Genetic diversity and correlation analysis of botanical characters in Xinjiang Wild Apple[Malus sieversii(Ledeb.)M. Roem.]. Journal of Plant Genetic Resources, 2016. 17 (4): 683- 689.
	闫秀娜, 李芳, 阎国荣, 等. 濒危植物新疆野苹果种子的萌发特性. 天津农学院学报, 2015. 22 (2): 33- 36. doi: 10.3969/j.issn.1008-5394.2015.02.009
	Yan X N , Li F , Yan G R , et al. Preliminary exploration on seed germination in endangered plant Malus sieversii. Journal of Tianjin Agricultural College, 2015. 22 (2): 33- 36. doi: 10.3969/j.issn.1008-5394.2015.02.009
	杨可, 郑柯斌, 黄晓慧, 等. 海洋生境贝莱斯芽孢杆菌TCS001的鉴定及抑真菌活性. 农药学学报, 2018. 20 (3): 333- 339.
	Yang K , Zheng K B , Huang X H , et al. Identification and antifungal activity of marine Bacillus velezensis strain TCS001. Chinese Journal of Pesticide Science, 2018. 20 (3): 333- 339.
	尹宝重, 刘博燕, 刘盼, 等. 苹果树皮内生菌对苹果树腐烂病的防治效果. 江苏农业科学, 2017. 45 (10): 84- 86.
	Yin B Z , Liu B Y , Liu P , et al. Control effect of apple bark endophytic bacteria on apple tree rot disease. Jiangsu Agricultural Sciences, 2017. 45 (10): 84- 86.
	于玮玮, 曹波, 龙鸿, 等. 新疆野苹果幼苗对盐胁迫的生理响应. 华北农学报, 2016. 31 (1): 170- 174.
	Yu W W , Cao B , Long H , et al. Physiological responses of Malus sieversii seedling under salinity stress. Acta Agriculturae Boreali-Sinica, 2016. 31 (1): 170- 174.
	Murashige T , Skoog F . A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiology Plant, 1962. 15 (3): 473- 497. doi: 10.1111/j.1399-3054.1962.tb08052.x

理化特征 Physiological and biochemical characteristics		菌株Strains
理化特征 Physiological and biochemical characteristics		M-m7	M-r12	M-m12	P-m9	P-m11	P-r21	L-m12
碳源利用 Utilization of carbon sources	甘露醇Mannitol	+	+	+	+	+	+	+
	蔗糖Sucrose	+	+	+	+	+	+	+
	麦芽糖Maltose	+	+	+	+	+	+	+
	葡萄糖Glucose	+	+	+	+	+	+	+
	乳糖Lactose	-	+	+	+	-	+	-
	L-阿拉伯糖L-Arabinose	ND	+	ND	+	ND	+	ND
	鼠李糖L-Rhamnose	+	+	+	+	+	+	+
	水杨苷Salicin	+	-	+	+	ND	-	+
	D-山梨醇D-Sorbitol	ND	+	ND	+	ND	+	ND
	甲醇Methanol	ND	+	ND	+	ND	+	ND
	乙醇Ethanol	ND	+	ND	+	ND	+	ND
	苹果酸Malic acid	ND	-	ND	-	ND	-	ND
	肌醇Inositol	ND	+	ND	+	+	+	ND
	果糖Fructose	+	ND	+	ND	+	ND	+
	木糖Xylose	+	ND	+	ND	+	ND	+
	棉子糖Raffinose	+	ND	+	ND	ND	ND	+
氮源利用 Utilization of nitrogen sources	KNO₃	+	+	+	+	+	+	+
氮源利用 Utilization of nitrogen sources	NH₄H₂PO₄	+	+	+	+	+	+	+
精氨酸双水解酶Arginine dihydrolase		ND	-	ND	-	ND	-	ND
苯丙氨酸脱氨酶Phenylalanine deaminase		-	-	-	-	-	-	-
硝酸盐还原Nitrate reductase		+	+	+	+	+	+	+
甲基红试验Methyl red test		-	-	-	-	-	+	+
V-P测定V-P test		-	+	+	+	+	+	-
厌氧性实验Anaerobic growth		-	-	-	-	-	-	-
淀粉水解Amylolysis		+	+	+	+	+	+	+
氧化酶Oxidase reaction		+	+	+	+	+	-	-
接触酶Catalase reaction	+	+	-	+	+	+	+
30 g·L^-1KOH溶解性试验30 g·L^-1 KOH solubility test		-	-	-	+	+	+	-
耐盐性 Growth in NaCl resolution	30 g·L^-1 NaCl	+	+	+	+	+	+	+
	50 g·L^-1 NaCl	+	-	+	-	+	+	+
	70 g·L^-1 NaCl	-	-	-	-	-	-	-
	100 g·L^-1 NaCl	-	-	-	-	-	-	-
牛奶胨化Milk coagulation		+	+	+	+	+	+	+
柠檬酸盐利用Citrate solution		+	+	-	+	+	+	+
运动性Motility		+	+	+	+	+	+	+
明胶液化Degradation of gelatin		+	+	+	+	+	+	+
革兰氏染色Gram staining		+	+	+	+	+	+	+

Isolation and Identification of Endophytic Bacteria from Xinjiang Wild Apple (Malus sieversii) and Their Antagonism against Three Pathogens of Apple Trees

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组织Tissues	NA	LB	PDA	RBA	MEA	MS	WA	AT	总数Total
韧皮部Phloem	14	10	15	0	23	7	4	0	73
木质部Xylem	28	20	20	14	28	23	11	0	144