解淀粉芽胞杆菌HR-2的分离鉴定及对水稻稻瘟病菌的拮抗效果

doi:10.13560/j.cnki.biotech.bull.1985.2020-0970

摘要/Abstract

摘要：

稻瘟病是一种严重威胁全球粮食安全的水稻真菌病害。本研究采用平板对峙法从湖南岳阳地区筛选出1株对水稻稻瘟病菌具有高效拮抗活性的菌株HR-2。通过形态特征验证、16S rRNA、gyrA和tuf基因序列比对分析,鉴定该菌株为解淀粉芽胞杆菌（Bacillus amyloliquefaciens）。抑菌广谱性测定结果表明菌株HR-2对水稻稻瘟病菌、油菜菌核病菌和番茄青枯病菌均有较强的拮抗效果,抑制率分别为82.34%、72.32%和72.64%。盆栽试验结果显示,菌株HR-2 10倍稀释液和100倍稀释液对水稻稻瘟病的防治效果分别达到63.81%和40.77%,其中HR-2 10倍稀释液的防效与50 mg/L嘧菌酯的防效相当。试验结果表明解淀粉芽胞杆菌菌株HR-2抑菌谱广,尤其对水稻稻瘟病具有显著的防治效果,可为抗稻瘟病生物防治药剂的开发奠定基础。

关键词: 水稻稻瘟病, 解淀粉芽胞杆菌, 分离鉴定, 生物防治

Abstract:

Magnaporthe oryzae is a rice fungal disease that seriously threatens global food security. A strain HR-2 with high antagonistic activity against M. oryzae was screened from Yueyang district,Hunan province by plate confrontation method in this study. The strain HR-2 was identified as Bacillus amyloliquefaciens by morphological verification,16S rRNA,gyrA and tuf gene sequence analysis. The results of broad-spectrum inhibition showed that the strain HR-2 had a fine inhibitory effect on M. oryzae,Sclerotinia sclerotiorum and Ralstonia solanacearum,and the inhibition rates were 82.34%,72.32% and 72.64%,respectively. The results of pot experiment demonstrated that the control effects of HR-2 10-times diluted solution and 100-times diluted solution on M. oryzae were 63.81% and 40.77% respectively,among which the control effects of HR-2 10-times diluted solution were similar to that of 50 mg/L azoxystrobin. The above results indicated that B. amyloliquefaciens HR-2 had a wide antibacterial spectrum and an obvious control effect on M. oryzae. Our research lays a foundation for the development of biological control agent against M. oryzae .

Key words: Magnaporthe oryzae, Bacillus amyloliquefaciens, isolated and identification, biological control

李瑾, 彭可为, 潘求一, 朱哲远, 彭迪. 解淀粉芽胞杆菌HR-2的分离鉴定及对水稻稻瘟病菌的拮抗效果[J]. 生物技术通报, 2021, 37(3): 27-34.

LI Jin, PENG Ke-wei, PAN Qiu-yi, ZHU Zhe-yuan, PENG Di. Isolation and Identification of Bacillus amyloliquefaciens HR-2 and Biological Control of Rice Blast[J]. Biotechnology Bulletin, 2021, 37(3): 27-34.

图/表 11

表1 16S rRNA、gyrA和tuf引物序列

引物名称	引物序列（5'-3'）
27 F	AGAGTTTGATCCTGGCTCAG
1492 R	GGTTACCTTGTTACGACTT
gyrA F	CAGTCAGGAAATGCGTACGTCCTT
gyrA R	CAAGGTAATGCTCCAGGCATTGCT
tuf GPF	ACGTTGACTGCCCAGGACAC
tuf GPR	GATACCAGTTACGTCAGTTGTACGGA

表2 7株菌株对5种供试病原菌的抑菌效果

供试病原菌	HR-2	G-1	G-2	G-3	G-4	G-10	HR-8
水稻稻瘟病菌	82.34±0.08a	82.54±0.04a	82.74±0.06a	83.93±0.06a	82.14±0.18a	81.55±0.16a	82.44±0.04a
油菜菌核病菌	72.32±0.04b	68.25±0.68a	68.06±0.68a	68.65±0.69a	/	/	/
番茄青枯病菌	72.62±0.02a	76.19±0.02a	73.81±0.01a	73.81±0.01a	/	/	/
小麦赤霉病菌	21.05±0.2a	/	13.16±0.05a	31.58±0.1a	/	/	/
西瓜枯萎病菌	26.32±0.16a	/	/	/	/	/	/

图1 菌株HR-2的抑菌活性图片显示菌株HR-2与水稻稻瘟病菌（A）、油菜菌核病菌（B）和番茄青枯病菌（C）对峙培养后病原菌生长情况;对照组（CK）用无菌水处理,实验组（处理）用HR-2菌悬液处理;对照组和实验组接种时间均相同

图2 菌株HR-2菌落形态

图3 菌株HR-2的16S rRNA扩增产物片段

图4 根据16S rRNA基因序列构建菌株HR-2系统发育树分支上的数值表示采用Neighbor-Joining方法构建系统树时,1000次计算时形成该节点的百分比;括号内为菌株的16S rRNA基因序列在GenBank中的登录号;标尺表示碱基置换频率

图5 根据gyrA基因序列构建菌株HR-2系统发育树分支上的数值表示采用Neighbor-Joining方法构建系统树时,1000次计算时形成该节点的百分比;括号内为菌株的gyrA基因序列在GenBank中的登录号;标尺表示碱基置换频率

图6 根据tuf基因序列构建菌株HR-2系统发育树分支上的数值表示采用Neighbor-Joining方法构建系统树时,1000次计算时形成该节点的百分比;括号内为菌株的tuf基因序列在GenBank中的登录号;标尺表示碱基置换频率

图7 解淀粉芽胞杆菌HR-2的生长曲线

表3 四组不同处理对稻瘟病的防治效果

处理	病情指数	防效/%
CK	46.75±0.35a	∕
100 times dilution	27.74±1.07b	40.77±1.91b
10 times dilution	16.96±0.92c	63.81±1.93a
50 mg/L Azoxystrobin	16.18±0.87c	65.48±1.84a

图8 四种不同处理的水稻叶片局部图图片显示水稻叶片处理7-8 d后叶瘟发病状况,根据叶片上的病斑数量评估不同处理对水稻稻瘟病的防治效果。对照组：将无菌水（abc）喷施到水稻叶面上,接种稻瘟孢子悬浮液。处理组：将HR-2-100（def）喷施到水稻叶面上,接种稻瘟孢子悬浮液;将HR-2-10（ghi）喷施到水稻叶面上,接种稻瘟孢子悬浮液;将50 mg/L嘧菌酯（jkl）喷施到水稻叶面上,接种稻瘟孢子悬浮液

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