Screening and Identification of an Antagonist Against the Pathogen of Kiwifruit Canker and Its Antifungal Activity to the Phytopathogenic Fungus

doi:10.13560/j.cnki.biotech.bull.1985.2020-0473

Abstract

Abstract:

The objective is to screen and identify the endophytic bacteria from wild Ginkgo biloba against the pathogen of kiwifruit canker,and to determine the bacteriostatic spectrum of common plant fungal diseases. Three-step method was used to isolate the endophytic bacteria from G. blioba. And then the Pseudomonas syringae pv.Actinidiae(Psa)was used as indicator bacterium for screening the antagonistic bacteria. Combination of morphological,physiological and biochemical characteristics and 16S rRNA sequence analysis was used to identify the antagonistic bacteria. Finally,the pot experiment was carried out to study its control efficacy against kiwifruit canker,and the confrontation method was used to evaluate the antifungal activity of the strain. Total 7 endophytic bacterial strains with antagonistic activities to Psa were obtained. The strain MA23 demonstrated the greatest antagonistic activity toward Psa and was identified as Bacillus cereus. The control efficacy of MA23 fermentation broth supernatant against kiwifruit canker was 93.6%. Meanwhile,MA23 had great capability of antagonism to some plant pathogenic fungi. In conclusion,endophytic bacterium B. cereus MA23 isolated from wild G. biloba not only has good antagonistic activity against the pathogen of kiwifruit canker,but also has significant inhibitory effect on a variety of plant pathogenic fungi.

Key words: endophytic bacteria, kiwifruit canker, Bacillus cereus, antifungal activity

ZHU Hai-yun, MA Yu, KE Yang, LI Bo. Screening and Identification of an Antagonist Against the Pathogen of Kiwifruit Canker and Its Antifungal Activity to the Phytopathogenic Fungus[J]. Biotechnology Bulletin, 2021, 37(6): 66-72.

Figures/Tables 7

Fig. 1 Screening of antagonistic endophytic bacteria against Psa A-B:Initial screening inhibition zones of partial endophytic bacteria. C:Re-screening inhibition zones of partial endophytic bacteria(The diameter of inhibition zone:17.86-26.46 mm). D:Re-screening inhibition zones of MA23(The average diameter of inhibition zone:32.74 mm)

Fig. 2 Morphological characteristics of MA23 A:Colony morphology. B:Result of Gram staining. C:Result of spore staining

Table 1 Physiological and biochemical identification results of MA23

鉴别特征 Characteristics	结果 Results
革兰氏染色 Gram staining	+
运动性 Motility	+
厌氧生长 Anaerobic growth	+
D-葡萄糖产酸 Acid production with D-glucose	+
D-葡萄糖产气 Gas production with D-glucose	-
接触酶 Catalase	+
V. P	+
硝酸盐还原 Nitrate deoxidization	+
淀粉水解 Amylolysis	+
酪蛋白水解 Casein hydrolyzation	+
明胶液化 Gelatin liquefaction	+
柠檬酸盐利用 Citrate utilization	+
卵黄反应 Egg yolk reaction	-

Fig. 3 Neighbor-joining phylogenetic tree of MA23

Fig. 4 Pot experiment of MA23 controlling kiwifruit canker

Fig. 5 Confrontation experiments of Bacillus cereus MA23 against pathogenic fungi A,a:Pathogen of sweet potato soft rot(Fusarium oxysporum). B,b:Pathogen of botryosphaeria canker on apple(Botryosphaeria dothidea). C,c:Pathogen of apple black spot(Alternaria alternata). D,d:Pathogen of tomato gray mold(Botrytis cinerea). E,e:Pathogen of rot in tulip(Trichoderma virens). F,f:Pathogen of fusarium wilt in cotton(Fusarium oxysporum f.sp.vasinfectum). Different capital letters refer to the control groups,and different lowercase letters refers to the experimental group

Table 2 Inhibition ratios of MA23 against different plant pathogenic fungi

供试病原菌 Pathogen for test	对照菌落半径 Semidiameter of control pathogen /mm	处理菌落半径 Semidiameter of treatment pathogen /mm	抑制率 Inhibition ratio/%
Botrytis cinerea	45.21	2.72	93.98
Alternaria alternata	43.6	3.34	92.34
Trichoderma virens	28.94	8.28	71.39
Fusarium oxysporum	28.89	8.54	70.44
Fusarium oxysporum f. sp. Vasinfectum	35.73	16.36	54.21
Botryosphaeria dothidea	28.56	20.02	29.90

References 29

[1]	朱海云, 李勃, 李燕, 等. 丁香假单胞菌猕猴桃致病变种的遗传多样性及进化关系[J]. 微生物学杂志, 2013, 33(4):66-71.
	Zhu HY, Li B, Li Y, et al. Relation of genetic diversity and evolution of kiwifruit pathogen Pseudomonas syringae pv. actinidiae[J]. J Microbiol, 2013, 33(4):66-71.
[2]	Monchiero M, Gullino ML, Pugliese M, et al. Efficacy of different chemical and biological products in the control of Pseudomonas syringae pv. actinidiae on kiwifruit[J]. Australas Plant Pathol, 2015, 44(1):13-23. doi: 10.1007/s13313-014-0328-1 URL
[3]	Han HS, Koh YJ, Hur JS, et al. Occurrence of the strA-strB streptomycin resistance genes in Pseudomonas species isolated from kiwifruit plants[J]. The Journal of Microbiology, 2004, 42(4):365-368.
[4]	Andersen GL. Occurrence and properties of copper-tolerant strains of Pseudomonas syringae Isolated from fruit trees in California[J]. Phytopathology, 1991, 81(6):648. doi: 10.1094/Phyto-81-648 URL
[5]	Wicaksono WA, Jones EE, Casonato S, et al. Biological control of Pseudomonas syringae pv. actinidiae(Psa), the causal agent of bacterial canker of kiwifruit, using endophytic bacteria recovered from a medicinal plant[J]. Biol Control, 2018, 116:103-112. doi: 10.1016/j.biocontrol.2017.03.003 URL
[6]	朱海云, 马瑜, 柯杨, 等. 猕猴桃细菌性溃疡病生防菌的筛选、鉴定及其防效初探[J]. 微生物学杂志, 2016, 36(5):90-96.
	Zhu HY, Ma Y, Ke Y, et al. Screening, identification and controlling effects of biocontrol strain against Chinese gooseberry or kiwi fruit bacterial canker[J]. J Microbiol, 2016, 36(5):90-96.
[7]	Hill R, Stark C, Cummings N, et al. Use of beneficial microorganisms and elicitors for control of Pseudomonas syringae pv. actinidiae in kiwifruit(Actinidia spp. )[J]. Acta Hortic, 2015(1095):137-146.
[8]	Reinhold-Hurek B, Hurek T. Living inside plants:bacterial endophytes[J]. Curr Opin Plant Biol, 2011, 14(4):435-443. doi: 10.1016/j.pbi.2011.04.004 pmid: 21536480
[9]	Ryan RP, Germaine K, Franks A, et al. Bacterial endophytes:recent developments and applications[J]. FEMS Microbiol Lett, 2008, 278(1):1-9. doi: 10.1111/fml.2008.278.issue-1 URL
[10]	Brader G, Compant S, Mitter B, et al. Metabolic potential of endophytic bacteria[J]. Curr Opin Biotechnol, 2014, 27:30-37. doi: 10.1016/j.copbio.2013.09.012 URL
[11]	Tontou R, Gaggia F, Baffoni L, et al. Molecular characterisation of an endophyte showing a strong antagonistic activity against Pseudomonas syringae pv. actinidiae[J]. Plant Soil, 2016, 405(1/2):97-106. doi: 10.1007/s11104-015-2624-0 URL
[12]	Fikri ASI, Rahman IA, Nor NSM, et al. Isolation and identification of local bacteria endophyte and screening of its antimicrobial property against pathogenic bacteria and fungi[C]// Selangor, Malaysia. Author(s), 2018.
[13]	李勃, 马瑜, 党永. 银杏中内生细菌的分离鉴定[J]. 果树学报, 2010, 27(4):566-569, 665.
	Li B, Ma Y, Dang Y. Isolation and identification of entophytic bacteria strain from Ginkgo biloba[J]. J Fruit Sci, 2010, 27(4):566-569, 665.
[14]	刘宁. 番茄灰霉病菌生防细菌BAB-1的鉴定及发酵工艺的优化[D]. 保定:河北农业大学, 2009.
	Liu N. Identification of biocontrol bacterial strain BAB-1 against tomato grey mold and optimization of its fermentation[D]. Baoding:Hebei Agricultural University, 2009.
[15]	倪志华, 张玉明, 周艳芬. 一株中性嗜盐菌Halobacillus dabanensis N522的分离鉴定及其抗菌活性研究[J]. 生物技术通报, 2016, 32(5):158-164.
	Ni ZH, Zhang YM, Zhou YF. Identification of a moderately halophilic bacterium Halobacillus dabanensis N522 and study of its antimicrobial activity[J]. Biotechnol Bull, 2016, 32(5):158-164.
[16]	Chikere CB, Ekwuabu CB. Molecular characterization of autochthonous hydrocarbon utilizing bacteria in oil-polluted sites at Bodo Community, Ogoni land, Niger Delta, Nigeria[J]. Nigerian Journal of Biotechnology, 2014, 27:28-33.
[17]	Altschul SF, Gish W, Miller W, et al. Basic local alignment search tool[J]. J Mol Biol, 1990, 215(3):403-410. pmid: 2231712
[18]	王莉衡, 柯杨, 强毅, 等. 芦荟内生菌内生哈茨木霉LH-7对植物病原菌的抗性[J]. 应用生态学报, 2014, 25(4):1130-1136.
	Wang LH, Ke Y, Qiang Y, et al. Inhibition effects and mechanisms of the entophytic fungus Trichoderma harzianum LH-7 from Aloe barbadensis[J]. Chin J Appl Ecol, 2014, 25(4):1130-1136.
[19]	王万清. 具有芘降解功能的植物内生细菌的分离筛选及其在小麦体内的定殖特性[D]. 南京:南京农业大学, 2015.
	Wang WQ. Isolation of Pyrene-degrading endophytic bacteria and inculation of them in wheat[D]. Nanjing:Nanjing Agricultural University, 2015.
[20]	路国兵, 张瑶, 冀宪领, 等. 植物内生细菌的侵染定殖规律研究进展[J]. 生物技术通报, 2007(3):88-92.
	Lu GB, Zhang Y, Ji XL, et al. Recent advance on the approaching and colonazition of endophytic bacteria[J]. Biotechnol Bull, 2007(3):88-92.
[21]	Rezaei M, Ghanbari M, Soltani M, et al. Production of bacteriocin by a novel Bacillus sp. strain RF 140, an intestinal bacterium of Caspian Frisian Roach(Rutillus frisii kutum)[J]. J Biotechnol, 2008, 136:S741.
[22]	Riley MA, Wertz JE. Bacteriocins:evolution, ecology, and application[J]. Annu Rev Microbiol, 2002, 56(1):117-137. doi: 10.1146/annurev.micro.56.012302.161024 URL
[23]	Yeaman MR, Yount NY. Mechanisms of antimicrobial peptide action and resistance[J]. Pharmacol Rev, 2003, 55(1):27-55. doi: 10.1124/pr.55.1.2 URL
[24]	郭霞, 郑雨焕. 美味牛肝菌菌塘中菌根促生菌的筛选与鉴定[J]. 菌物学报, 2018, 37(12):1802-1807.
	Guo X, Zheng YH. Isolation and identification of mycorrhiza helper bacteria from the colonized soil of Boletus edulis[J]. Mycosystema, 2018, 37(12):1802-1807.
[25]	孔令春, 魏松红, 宋鹏, 等. 抗稻瘟病的生防细菌筛选与鉴定[J]. 沈阳农业大学学报, 2018, 49(6):655-660.
	Kong LC, Wei SH, Song P, et al. Screening and identification of biocontrol bacteria against Pyricularia oryzae[J]. J Shenyang Agric Univ, 2018, 49(6):655-660.
[26]	张彦, 车建美, 刘波, 等. 蜡样芽孢杆菌ANTI-8098A在番茄内的定殖和对青枯病的防治研究[J]. 中国生物防治学报, 2011, 27(2):221-227.
	Zhang Y, Che JM, Liu B, et al. Colonization of Bacillus cereus ANTI-8098A in tomato plants and its biocontrol characteristics to bacterial wilt disease[J]. Chin J Biol Control, 2011, 27(2):221-227.
[27]	黄秋斌, 张颖, 刘凤英, 等. 蜡样芽孢杆菌B3-7在大田小麦根部的定殖动态及其对小麦纹枯病的防治效果[J]. 生态学报, 2014, 34(10):2559-2566.
	Huang QB, Zhang Y, Liu FY, et al. Colonization dynamics of Bacillus cereus B3-7 on wheat roots and control efficiency against sharp eyespot of wheat[J]. Acta Ecol Sin, 2014, 34(10):2559-2566.
[28]	王刚, 刘凤英, 王淼, 等. 内生细菌B3-7的运动性参与其在小麦根系的内生定殖和对小麦全蚀病的生物防治[J]. 植物病理学报, 2011, 41(5):526-533.
	Wang G, Liu FY, Wang M, et al. Motility of endophytic bacteria strain B3-7 involved in endophytic colonization of wheat roots and biological control of wheat take-all[J]. Acta Phytopathol Sin, 2011, 41(5):526-533.
[29]	李纪顺, 陈凯, 王贻莲, 等. 蜡样芽孢杆菌BCJB01在防治葡萄溃疡病方面的应用:中国, CN105767006A[P], 2016-07-20.
	Li JS, Chen K, Wang YL, et al. Application of Bacillus cereus BCJB01 in control of grapevine canker:China, CN201610141894. 5[P], 2016-07-20.