瓜类细菌性果斑病菌拮抗细菌的筛选及其抑菌作用

doi:10.13560/j.cnki.biotech.bull.1985.2022-0449

摘要/Abstract

摘要：

从土壤中筛选出抑制哈密瓜细菌性果斑病的拮抗菌株，明确其分类地位，测定其生长条件及防病效果。采用梯度稀释法及平板对峙法进行菌株的分离及筛选，综合形态学观察、生理生化测定以及分子鉴定等多方面对菌株进行鉴定，确定生防菌种属，并测定其最适生长条件，利用平板对峙法测定其抑菌作用，并分别采用针刺接种法、叶面喷施法对生防菌的离体叶片和盆栽防病试验进行测定。本研究从土样中共分离出196株细菌，经对峙筛选发现有20株细菌具有抑菌效果，其中编号为131、791的两株细菌对哈密瓜细菌性果斑病病原菌具有较好的平板拮抗效果，抑菌圈直径分别为（19.03±0.13）mm，（17.55±0.29）mm。经鉴定菌株131为沙福芽孢杆菌（Bacillus safensis），菌株791为贝莱斯芽孢杆菌（B. velezensis）。菌株131的最适生长条件为NB培养基，培养18 h，温度37℃，pH 7，接种量为2%。菌株791的最适生长条件为KB培养基，培养18 h，温度37℃，pH 6，接种量为1%。菌株131和791对哈密瓜细菌性果斑病的离体叶片防效分别为83.33%和87.53%，室内盆栽防效分别为69.86%和77.99%，同时也对多种病原真菌具有抑制作用。菌株131和791均为对哈密瓜细菌性果斑病具有较好防效的芽孢杆菌（Bacillus），对瓜类作物细菌性果斑病的防治提供了理论补充，奠定了果斑病害生物防治的基础。

关键词: 哈密瓜细菌性果斑病, 拮抗, 鉴定, 盆栽防效, 抑菌作用, 最适生长条件

Abstract:

The antagonistic strains against bacterial fruit blotch（BFB）of cantaloupe were screened from soil, their taxonomic status was clarified, and their growth conditions and disease prevention effects were determined. Separation and screening were carried out by plate dilution method and confrontation method, and the species was identified by comprehensive morphological, physiological, biochemical and molecular identification. After determining the species of biocontrol bacteria, the optimal growth conditions were determined, and the bacteriostatic effect was determined by the plate confrontation method. The leaves and potted plants of biocontrol bacteria were measured by acupuncture inoculation method and foliar spraying method respectively. In this study, 196 strains of bacteria were isolated from soil samples, 20 strains of them were found to have bacteriostatic effect via confrontation method. Among them, two strains numbered 131 and 791 had better plate antagonism effect on the pathogen of BFB, and the diameters of the inhibition zones were（19.03±0.13）mm and（17.55±0.29）mm, respectively. The strain 131 was identified as Bacillus safensis and strain 791 as B. velezensis. The optimal growth condition of strain 131 was NB medium, cultured for 18 h, temperature was 37℃, pH 7 and the inoculum was 2%. The optimal growth condition of strain 791 was KB medium, cultured for 18 h, temperature was 37℃, pH 6 and the inoculation amount was 1%. Strain 131 and 791 were 83.33% and 87.53% in vitro, 69.86% and 77.99% in indoor pots, respectively; meanwhile they had inhibitory effects on various pathogens. In summary, strain 131 and 791 are Bacillus with good control effect on BFB, which provides theoretical supplements for the prevention and control of BFB and lays a solid foundation for BFB.

Key words: bacterial fruit blotch of cantaloupe, antagonism, identification, control effect on potted plant, bacteriostatic effect, optimal growth conditions

张玲, 张荣意, 刘盛科, 谭志琼. 瓜类细菌性果斑病菌拮抗细菌的筛选及其抑菌作用[J]. 生物技术通报, 2023, 39(1): 253-263.

ZHANG Ling, ZHANG Rong-yi, LIU Sheng-ke, TAN Zhi-qiong. Screening of Antagonistic Bacteria for Bacterial Fruit Blotch of Cucurbits and Its Antibacterial Effects[J]. Biotechnology Bulletin, 2023, 39(1): 253-263.

图/表 14

表1 PCR条件

Table 1 PCR conditions

类型Type	引物Primer（5'-3'）	反应体系Reaction system	循环条件Cyclic condition
16S rRNA	27F: AGAGTTTGATCCTGGCTCAG 1492R: TACCTTGTTACGACTT	2×Es Taq MasterMix 25 μL，ddH₂O 19 μL，引物各2 μL，DNA模板2 μL	95℃预变性5 min；94℃变性60 s，55℃退火90 s，72℃延伸1 min，共30个循环；72℃延伸10 min
gyrB	UP-1S: GAAGTCATCATGACCGTTCTGC- AYGCNGGNGGNAARTTYGA UP-2Sr: AGCAGGGTACGGATGTGCGA- GCCRTCNACRTCNGCRTCNGTCAT	2×Es Taq MasterMix 25 μL，ddH₂O 21 μL，引物各1 μL，DNA模板2 μL	94℃预变性10 min；94℃变性30 s，52℃退火30 s，72℃延伸1 min，共35个循环；72℃延伸10 min

表2 病情等级分级标准

Table 2 Classification criteria of disease grade

病情等级 Disease grade	分类标准 Classification criterion
0级Level 0	病斑数量为0
1级Level 1	总的叶片面积中病斑占比< 5%
2级Level 2	5% ≤总的叶片面积中病斑占比< 15%
3级Level 3	15% ≤总的叶片面积中病斑占比< 25%
4级Level 4	25% ≤总的叶片面积中病斑占比< 35%
5级Level 5	35% ≤总的叶片面积中病斑占比< 45%
6级Level 6	45% ≤总的叶片面积中病斑占比< 55%
7级Level 7	55% ≤总的叶片面积中病斑占比< 65%
8级Level 8	65% ≤总的叶片面积中病斑占比< 75%
9级Level 9	总的叶片面积中病斑占比≥75%

图1 两株拮抗菌对西瓜噬酸菌的抑制效果

Fig. 1 Inhibitory effects of two strains on A.citrulli

图2 菌株131、791的菌落图及革兰氏染色图 A、B分别为菌株131的菌落图和革兰氏染色图；C、D分别为菌株791的菌落图和和革兰氏染色图

Fig. 2 Colony and Gram stain of strain 131 and 791 A and B are the colony map and Gram-stained map of strain 131，respectively；C and D are the colony map and Gram-stained map of strain 791，respectively

表3 菌株131、791的生理生化鉴定结果

Table 3 Physiological and biochemical identification resu-lts of strain 131 and 791

指标Index	结果Result
指标Index	131	791
革兰氏染色Gram stain	+	+
接触酶试验Catalase test	+	+
氧化酶试验Oxidase test	+	+
厌氧生长Anaerobic growth	-	-
2% NaCl	+	+
10% NaCl	+	+
明胶液化Gelatin liquefaction	+	+
淀粉水解Amylohydrolysis	+	+
甲基红试验MR	+	+
V-P	+	+
碳源利用Carbon source utilization
棉子糖Raffinose	+	+
半乳糖Galactose	+	+
乳糖Lactose	+	+
葡萄糖Glucose	+	+
蔗糖Sucrose	+	+
甘露醇Mannitol	+	+
氮源利用Nitrogen source utilization
硝酸铵Ammonium nitrate	+	+
氯化铵Ammonium chloride	+	+
尿素Urea	+	+
硫酸铵Ammonium sulfate	+	+
硝酸钾Potassium nitrate	+	+
蛋白胨Peptone	+	+

图3 菌株131、791的16S rRNA基因电泳图（A）和gyrB基因电泳图（B）

Fig. 3 16S rRNA gene electropherogram (A) and gyrB gene electropherogram (B) of strain 131 and 791

图4 菌株131基于16S rRNA基因和gyrB基因的系统发育树 Bacillus safensis：沙福芽孢杆菌；Bacillus pumilus：短小芽孢杆菌；Bacillus invictae：无敌芽孢杆菌；Bacillus australimaris：南海芽胞杆菌；Bacillus stratosphericus：同温层芽孢杆菌；Bacillus xiamenensis：厦门芽孢杆菌

Fig. 4 Phylogenetic tree of strain 131 based on 16S rRNA genes and gyrB genes

图5 菌株791基于16S rRNA基因和gyrB基因的系统发育树 Bacillus velezensis：贝莱斯芽孢杆菌；Bacillus atrophaeus：萎缩芽孢杆菌；Bacillus vallismortis：死谷芽胞杆菌；Bacillus halotolerans：耐盐芽胞杆菌；Bacillus axarquiensis：阿萨尔基亚芽孢杆菌

Fig. 5 Phylogenetic tree of strain 791 based on 16S rRNA genes and gyrB genes

图6 菌株131、791的生长曲线

Fig. 6 Growth curves of strain 131 and 791

图7 菌株131、791在不同条件下的生长状况小写字母表示在0.05水平的差异显著性

Fig. 7 Growth status of strain 131 and 791 under different conditions Lower letters indicates the significance of the difference at the 0.05 level

图8 两株生防菌对哈密瓜细菌性果斑病的离体叶片效果 A：只接种水的叶片；B：只接种BFB菌液的叶片；C、D：分别接种菌株131、791菌液和BFB菌液的叶片。图中结果均为处理后第7天统计

Fig. 8 Effects of two biocontrol bacteria on the isolated leaves of bacterial fruit blotch of cantaloupe A：Leaf only inoculated with water. B：Leaf only inoculated with the BFB liquid. C and D：Leaves inoculated with the bacterial liquid of strain 131，791 and the BFB liquid，respectively. The results in the figure were counted on the seventh day after treatment

表4 菌株131和791的盆栽防病效果

Table 4 Control efficacy of strain 131 and 791 in potting

试验处理 Treatment	病情指数 Disease index	防治效果 Control efficacy/%
只喷水Spray water only	-	-
只喷病菌菌液Spray BFB bacterial solution only	77.41 ± 0.64a	-
喷131菌液和病菌菌液 Spray 131 bacterial solution and BFB bacterial solution	23.33 ± 1.11b	69.86
喷791菌液和病菌菌液 Spray 791 bacterial solution and BFB bacterial solution	17.04 ± 1.28c	77.99

图9 两株菌株对细菌性果斑病的盆栽防治效果 A：只喷水；B：只喷BFB菌液；C：同时喷131菌液和BFB菌液；D：同时喷791菌液和BFB菌液。图中结果均为处理后第7天统计

Fig. 9 Control efficacy of two strains against BFB in potting A：Spray water only. B：Spray BFB bacterial solution only. C：Simultaneously spray 131 bacterial solution and BFB bacterial solution. D：Simultaneously spray 791 bacterial solution and BFB bacterial solution. The results in the figure were counted on the seventh day after treatment

表5 菌株131、791对其它病原菌的抑菌作用

Table 5 Inhibition percentage of strain 131，791 against other pathogen

病原真菌 Pathogenic fungus	抑菌直径 Inhibition diameter/mm		抑菌率 Inhibition rate/%
病原真菌 Pathogenic fungus	131	791	131	791
小麦赤霉病病菌 F. graminearum	0	10.50±0.50c	0	12.35
火龙果炭疽病病菌 C. gloeosporioides	7.10±0.10b	11.00±0.41c	8.35	12.94
黄瓜叶斑病病菌 P. cichorii	8.40±0.63b	21.25±0.75a	9.88	25.00
甜瓜蔓枯病病菌 D. bryoniae	0	16.25±0.63b	0	19.12
荔枝霜疫霉 P. litchii	12.00±1.15a	14.00±1.63b	14.12	16.47

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