枯草芽孢杆菌K-268的分离鉴定及对水稻稻瘟病的防病效果

doi:10.13560/j.cnki.biotech.bull.1985.2021-1124

摘要/Abstract

摘要：

生防细菌是绿色防控稻瘟病的有效措施。为了发掘高效拮抗稻瘟病菌的生防细菌,本研究采用平板对峙法从感病品种K020268的健康稻株叶片中筛选获得1株对稻瘟病菌抑制效果较好的拮抗细菌K-268,同时测定该菌的抑菌谱。通过形态学观察、生理生化鉴定16S rRNA 和gyrA序列分析对其进行菌种鉴定,并初步研究了该菌株的生物学特性和对稻叶瘟的防治效果。结果表明,菌株K-268对稻瘟病菌菌丝生长抑制率为86.30% ± 0.70%,同时对水稻纹枯病菌、玉米大斑病菌及柑橘沙皮病菌等供试的14株植物病原菌均有抑制作用;经鉴定菌株K-268为枯草芽孢杆菌（Bacillus subtilis）,该菌株对数生长期为14-32 h,生长最适温度为30℃,生长最适pH值为6.0-7.0,并且其具有良好的耐盐性。离体接种实验结果表明,稀释10倍即菌液浓度6×10⁸CFU/mL时,预防组和治疗组的叶瘟发病率分别为14.81%和23.46%,效果与稀释750倍的75%三环唑稀释液（WP）效果相当。活体喷雾接种结果表明,在接种稻瘟病菌分生孢子悬浮液（1×10⁶个/mL）前 24 h 喷施稀释10 倍（6×10⁸CFU/mL）的发酵液和接种稻瘟病菌分生孢子悬浮液后 24 h 喷施稀释10倍的发酵液对稻叶瘟的防效分别为59.00%和60.23%,效果接近500稀释液的40%稻瘟灵（EC）。因此K-268在水稻稻瘟病生物防治中具有一定的开发和应用价值。

关键词: 稻瘟病, 枯草芽孢杆菌, 分离鉴定, 生物防治

Abstract:

The use of biocontrol bacteria is an effective measure for green prevention and control of rice blast. In order to discover biocontrol bacteria with high efficiency against Magnaporthe oryzae,an antagonistic bacterium K-268 with the optimal inhibitory effect on M. oryzae from the healthy rice leaves of the susceptible variety K020268 was obtained by using the plate confrontation method and its antimicrobial spectrum was determined. Using morphological observation,physiological and biochemical identification 16S rRNA and gyrA sequence analysis,the identity of the strain was determined. In addition,the physical characteristics of the strain and its control effect against rice leaf blast were preliminarily studied. Results showed that strain K-268 had inhibitory rate of（86.30 ± 0.70）% on the mycelial growth of M. oryzae,and it demonstrated the inhibitory effects on 14 plant pathogenic fungi,such as Rhizoctonia solan,Exserohilum turcicum and Diaporthe citri,etc. Strain K-268 was identified to be Bacillus subtilis,its logarithmic growth period was 14-32 h,the optimal;growth temperature was 30℃,the optimal pH was 6.0-7.0,and had good salt tolerance. The results from in vitro inoculation experiments showed that the disease rate in the prevention group and the treatment group was14.81% and 23.46%,respectively,when the bacterial solution was diluted by 10 times to 6×10⁸ CFU;and the effect of which was not significantly different from that of the 750 times diluted 75% tricyclazole diluent（WP）. Results of in vivo spray inoculation showed that the control effects on the rice leaf blast were 59.00% and 60.23%,respectively,when the 10 times diluted fermentation liquid（6×10⁸CFU/mL）were sprayed 24 h before and after inoculation with Magnaporthe oryzae conidia suspension（1×10⁶ /mL）,respectively. These effects were close to the 40% rice blast spirit（EC）with 500 times dilution. Therefore,K-268 was confirmed to have certain development and application value in the biological control of rice blast.

Key words: rice blast, Bacillus subtilis, isolation and identification, biological control

祖雪, 周瑚, 朱华珺, 任佐华, 刘二明. 枯草芽孢杆菌K-268的分离鉴定及对水稻稻瘟病的防病效果[J]. 生物技术通报, 2022, 38(6): 136-146.

ZU Xue, ZHOU Hu, ZHU Hua-jun, REN Zuo-hua, LIU Er-ming. Isolation and Identification of Bacillus subtilis K-268 and Its Biological Control Effect on Rice Blast[J]. Biotechnology Bulletin, 2022, 38(6): 136-146.

图/表 11

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病原菌Pathogenic bacterium	抑菌率Bacteriostatic rate/%
水稻纹枯病菌R. solan	79.5±0.67
玉米大斑菌E.turcicum	47.6±0.33
柑橘沙皮病菌D. citri	21±5.7
草莓灰霉病菌 B. cinerea	49.6±1.8
茭白根腐病菌F. graminearum	43.5±10.3
辣椒枯萎病菌F. oxysporum	62±4.0
茄子根腐病菌F. solani	61.2±10.3
香樟炭疽病菌C. gloeosporioides	32.8±5
棉花黄萎病菌 V. dahliae	36.8±3.38
禾谷镰刀菌F.graminearum	56.7±0.33
烟草赤星病菌A. alternate	49.2±3.1
油菜菌核病菌S. sclerotiorum	56.3±2.33
黄瓜疫霉病菌P. melonis	56.8±0.0
水稻恶苗病菌F. moniliforme	69.2±0.88

病原菌Pathogenic bacterium	抑菌率Bacteriostatic rate/%
水稻纹枯病菌R. solan	79.5±0.67
玉米大斑菌E.turcicum	47.6±0.33
柑橘沙皮病菌D. citri	21±5.7
草莓灰霉病菌 B. cinerea	49.6±1.8
茭白根腐病菌F. graminearum	43.5±10.3
辣椒枯萎病菌F. oxysporum	62±4.0
茄子根腐病菌F. solani	61.2±10.3
香樟炭疽病菌C. gloeosporioides	32.8±5
棉花黄萎病菌 V. dahliae	36.8±3.38
禾谷镰刀菌F.graminearum	56.7±0.33
烟草赤星病菌A. alternate	49.2±3.1
油菜菌核病菌S. sclerotiorum	56.3±2.33
黄瓜疫霉病菌P. melonis	56.8±0.0
水稻恶苗病菌F. moniliforme	69.2±0.88

生理生化试验Physiological and biochemical tests	结果Result
接触酶试验Contact enzyme test	+
硝酸盐还原试验Nitrate reduction test	+
明胶液化试验Gelatin liquefaction test	+
硫化氢试验Hydrogen sulfide test	-
淀粉水解试验Starch hydrolysis test	-
吲哚试验Indole test	+
V.P试验V. P test	+
甲基红试验Methyl red test	-
石蕊牛奶试验Litmus milk test	胨化Peptonization
苯丙氨酸脱氨酶试验Phenylalanine deaminase test	-

生理生化试验Physiological and biochemical tests	结果Result
接触酶试验Contact enzyme test	+
硝酸盐还原试验Nitrate reduction test	+
明胶液化试验Gelatin liquefaction test	+
硫化氢试验Hydrogen sulfide test	-
淀粉水解试验Starch hydrolysis test	-
吲哚试验Indole test	+
V.P试验V. P test	+
甲基红试验Methyl red test	-
石蕊牛奶试验Litmus milk test	胨化Peptonization
苯丙氨酸脱氨酶试验Phenylalanine deaminase test	-

处理Treatment	发病率Disease incidence（-24 h）	发病率Disease incidence（+24 h）
清水对照CK Water control CK	98.77±1.23a	97±2.47a
NB	95.06±1.24ab	98.71±1.23a
75%三环唑（WP） 75% tricyclazole（WP）	12.34±1.23d	17.28±1.24d
6×10⁹CFU/mL	93.83±1.24ab	97.53±2.47a
6×10⁸CFU/mL	14.81±2.41d	23.46±1.24cd
6×10⁷CFU/mL	22.22±2.14c	27.16±2.47c