生物技术通报 ›› 2021, Vol. 37 ›› Issue (6): 66-72.doi: 10.13560/j.cnki.biotech.bull.1985.2020-0473
收稿日期:
2020-04-22
出版日期:
2021-06-26
发布日期:
2021-07-08
作者简介:
朱海云,女,硕士,研究方向:应用生物技术;E-mail: 基金资助:
ZHU Hai-yun(), MA Yu(), KE Yang, LI Bo
Received:
2020-04-22
Published:
2021-06-26
Online:
2021-07-08
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。
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
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 | - |
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
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 |
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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