猕猴桃溃疡病菌拮抗菌的筛选、鉴定及其对植物病原真菌的抗性

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

摘要/Abstract

摘要：

从野生银杏中分离、筛选抗猕猴桃溃疡病内生细菌,并明确其对常见植物真菌病害的抑菌谱。采用“三步法”分离银杏内生细菌,并以猕猴桃溃疡病病原菌(Pseudomonas syringae pv. actinidiae,Psa)作为指标菌筛选拮抗菌;结合菌落形态特征、生理生化特征及16S rRNA基因序列对拮抗菌进行鉴定;开展盆栽防治实验研究其对猕猴桃溃疡病的防治效率,并应用平板对峙法研究其对多种植物病原真菌的拮抗作用。结果共得到7株对Psa具有拮抗活性的内生细菌,其中,以菌株MA23的拮抗活性最为显著,鉴定为蜡样芽孢杆菌。MA23发酵液上清对猕猴桃溃疡病的盆栽防治效率为93.6%,同时其对多种病原真菌也具有拮抗活性。从银杏中筛选到一株对Psa具有较好拮抗活性的内生蜡样芽孢杆菌MA23,其对多种植物病原真菌也具有拮抗活性。

关键词: 内生细菌, 猕猴桃溃疡病, 蜡样芽孢杆菌, 抗真菌活性

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

朱海云, 马瑜, 柯杨, 李勃. 猕猴桃溃疡病菌拮抗菌的筛选、鉴定及其对植物病原真菌的抗性[J]. 生物技术通报, 2021, 37(6): 66-72.

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.

图/表 7

图1 Psa拮抗内生菌的筛选 A-B:部分内生菌初筛抑菌圈;C:部分内生菌复筛抑菌圈(抑菌圈直径:17.86-26.46 mm);D:MA23复筛抑菌圈(平均抑菌圈直径32.74 mm)

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)

图2 MA23菌株的形态特征 A:菌落形态;B:革兰氏染色;C:芽孢染色

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

表1 MA23菌株生理生化鉴定结果

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	-

图3 MA23菌株的N-J系统发育树

Fig. 3 Neighbor-joining phylogenetic tree of MA23

图4 MA23对猕猴桃溃疡病的盆栽防治实验

Fig. 4 Pot experiment of MA23 controlling kiwifruit canker

图5 蜡样芽孢杆菌MA23对病原真菌的平板对峙实验 A,a:甘薯干腐病病原(Fusarium oxysporum);B,b:苹果干腐病病原(Botryosphaeria dothidea);C,c:苹果黑斑病病原(Alternaria alternata);D,d:番茄灰霉病病原(Botrytis cinerea);E,e:郁金香腐烂病病原(Trichoderma virens);F,f:棉花枯萎病病原(Fusarium oxysporum f.sp.Vasinfectum)。大写字母代表对照组,小写字母代表实验组

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

表2 MA23对不同植物病原真菌的抑制率

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

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