Extracellular Antimicrobial Substances Produced by Bacillus subtilis JN005 and Its Control Efficacy on Rice Leaf Blast

doi:10.16819/j.1001-7216.2020.9109

Abstract

Abstract:

【Objective】The purpose of this research is to assay stability, biological activity and efficacy of the extracellular antimicrobial substances produced by Bacillus subtilis JN005 against Magnaporthe oryzae. 【Method】The antimicrobial substances produced by B. subtilis JN005 was tested using different media. The activity of extracellular antimicrobial substances of JN005 strain against M. oryzae and its efficacy on rice leaf blast were measured by dural culture, optical microscope observation, indoor detached-leaf inoculation and live inoculation. 【Result】B. subtilis JN005 secreted protease, cellulase, amylase and β-1,3-glucanase and its extracellular antimicrobial substances showed inhibition activity against M. oryzae. The extracellular antimicrobial substances were stable in a wide range of pH values from 2 to 12 and at temperatures from 0℃ to 100℃. The antifungal activity was retained after treatment with proteinase K. The extracellular antimicrobial substance kept its activity even it was irradiated for 12 h under ultraviolet light or exposed to 4℃ for 3 months. The extracellular antimicrobial substances caused distortion of hyphae of M. oryzae, and hindered the germination of conidia. Indoor detached leaves inoculation of Xiangwanxian 12 showed that disease incidence was 34.07% as rice leaves were treated with 100-fold dilution of the extracellular antimicrobial substances, followed by inoculation with conidia suspension of M. oryzae (after 24 h). The control efficacy was close to that of 500 times-diluted 40% isoprothiolane EC. The disease incidence was 38.52% as rice leaves were inoculated with conidia suspension of M. oryzae and treated with 100-fold dilution of the extracellular antimicrobial substances after 24 h, significantly different from 1500 times-diluted 75% tricyclazole WP and 500 times-diluted 40% isoprothiolane EC. The potted plant experiment showed that efficacy was 75.32% as rice was inoculated with conidia suspension of M. oryzae followed by spraying 100-fold dilution of the extracellular antimicrobial substances before 24 h. The efficacy was 71.49% as rice was inoculated with conidia suspension of M. oryzae and followed by spraying of 100-fold dilution of the extracellular antimicrobial substances after 24 h. 【Conclusion】The extracellular antimicrobial substances produced by JN005 strain directly inhibited mycelial growth and conidia germination of M. oryzae and it has good environmental stability. The extracellular antimicrobial substances have better preventative efficacy and therapeutic efficacy on rice blast and should be further investigated and developed as such.

Key words: Bacillus subtilis, extracellular antimicrobial substance, rice blast, efficacy

摘要：

【目的】检测枯草芽孢杆菌JN005产生的胞外抗菌物质的稳定性和对稻瘟病菌的生物活性,及其对水稻叶瘟的防治效果。【方法】通过不同培养基检测枯草芽孢杆菌JN005产生的抑菌物质,结合平板对峙法、光学显微镜观察、室内离体和活体接种来检测JN005菌株胞外抗菌物质对稻瘟病菌拮抗活性和对水稻叶瘟防治效果。【结果】枯草芽孢杆菌JN005能分泌蛋白酶、纤维素酶、淀粉酶和β-1, 3-葡聚糖酶,其胞外抗菌物质对稻瘟病菌生长具有抑制作用。胞外抗稻瘟病菌物质在0℃~100℃和pH 2~12范围内活性较为稳定,且经蛋白酶K处理后不影响其活性,紫外照射12 h以及4℃保存3个月仍有活性。胞外抗菌物质能引起稻瘟病菌菌丝形态畸变,并抑制分生孢子的萌发。室内湘晚籼12号稻瘟病离体叶片接种表明,先用100倍胞外抗菌物质稀释液处理水稻叶片,24 h后接种稻瘟病菌分生孢子悬浮液(1×10⁵个/mL),其叶瘟发病率为34.07%,效果接近稀释500倍的40%稻瘟灵EC;而先接种稻瘟病菌分生孢子悬浮液,24 h后用100倍胞外抗菌物质稀释液处理,其发病率为38.52%,与稀释1500倍的75%三环唑(WP)和稀释500倍的40%稻瘟灵(EC)有显著性差异。活体喷雾接种试验表明,于湘晚籼12号秧苗长至3叶1心时,接种稻瘟病菌分生孢子悬浮液前24 h喷施稀释100倍的胞外抗菌物质稀释液,其叶瘟防效为75.32%;接种稻瘟病菌分生孢子悬浮液后24 h喷施稀释100倍的胞外抗菌物质稀释液,其防效为71.49%。【结论】JN005菌株胞外抗菌物质对稻瘟病菌的菌丝生长和孢子萌发有直接抑制作用,且对环境稳定性较好,对稻瘟病有较好的预防和治疗作用,有进一步的开发潜力。

关键词: 枯草芽孢杆菌, 胞外抗菌物质, 稻瘟病, 防治效果

CLC Number:

Huajun ZHU, Hu ZHOU, Zuohua REN, Erming LIU. Extracellular Antimicrobial Substances Produced by Bacillus subtilis JN005 and Its Control Efficacy on Rice Leaf Blast[J]. Chinese Journal OF Rice Science, 2020, 34(5): 470-478.

朱华珺, 周瑚, 任佐华, 刘二明. 枯草芽孢杆菌JN005胞外抗菌物质及对水稻叶瘟防治效果[J]. 中国水稻科学, 2020, 34(5): 470-478.

Figures/Tables 9

Fig. 1. Test of protease(A), cellulase(B), amylase(C) and β-1, 3-glucanase(D) produced by JN005 strain.

Fig. 2. Effects of volatile compounds(A) and extracellular antimicrobial substances(B) of JN005 strain on M. oryzae.

Fig. 3. Effect of different treatments on activity of extracellular antimicrobial substances of JN005 strain against M. oryzae.

Fig. 4. Effect of extracellular antimicrobial substances of JN005 strain on mycelia of M. oryzae. A, Treated with sterile water; B, Treated with PBS buffer; C-F, Treated with crude extract of extracellular antimicrobial substances.

Table 1 Control efficacy of crude extracts of JN005 extracellular antimicrobial substances on leaf blast (indoor detached-leaf inoculation).

处理 Treatment	稀释倍数 Dilution ratio	试验组A Test group A	试验组B Test group B
处理 Treatment	稀释倍数 Dilution ratio	发病率 Disease incidence/%	发病率 Disease incidence/%
清水 Water	0	98.5±0.7 a	97.8±1.3 a
PBS缓冲液 PBS buffer	0	97.0±2.0 a	99.3±0.7 a
40%稻瘟灵乳油 40% isoprothiolane (EC)	500	30.4±3.0 b	29.6±2.7 c
75%三环唑可湿性粉剂 75% tricyclazole (WP)	1500	20.0±1.3 c	23.0±1.5 d
胞外抗菌物质粗提液 Extracellular antimicrobial substances crude extract	100	34.1±3.2 b	38.5±2.7 b

Fig. 5. Effect of extracellular antimicrobial substances of JN005 strain on conidia of M. oryzae. A, Treated with sterile water; B, Treated with PBS buffer; C-F, Treated with crude extract of extracellular antimicrobial substances.

Fig. 6. Control efficacy of extracellular antimicrobial substances on rice leaf blast(indoor detached-leaf inoculation). A, Water treatment; B, Extracellular antimicrobial substances treatment.

Table 2 Control efficacy of crude extracts of JN005 extracellular antimicrobial substances on leaf blast(indoor live inoculation).

处理 Treatment	稀释倍数 Dilution ratio	试验组C Test group C			试验组D Test group D
处理 Treatment	稀释倍数 Dilution ratio	发病率 Disease incidence/%	病情指数 Disease index	防治效果 Control efficicy/%	发病率 Disease incidence/%	病情指数 Disease index	防治效果 Control efficicy/%
清水 Water	0	97.8±1.1 a	82.1±0.9 a	/	96.7±1.9 a	80.3±0.9 a	/
PBS缓冲液 PBS buffer	0	94.4±1.1 a	80.3±0.7 a	2.2±0.9 d	95.6±2.9 a	79.3±0.6 a	1.2±0.5 c
40%稻瘟灵乳油	500	35.6±2.9 b	15.1±0.4 c	81.7±0.5 b	37.8±2.9 b	13.7±0.7 c	83.0±0.9 a
40% isoprothiolane (EC)
75%三环唑可湿性粉剂	1500	25.6±2.9 c	11.3±0.7 d	86.3±0.7 a	27.8±1.1 c	12.5±0.8 c	84.4±1.1 a
75% tricyclazole (WP)
胞外抗菌物质粗提液	100	40.0±3.3 b	20.3±1.6 b	75.3±1.8 c	43.3±3.9 b	22.9±0.9 b	71.5±1.1 b
Crude extracts of extracellular antimicrobial substances

Fig. 7. Control efficacy of extracellular antimicrobial substances on leaf blast(indoor live inoculation). A, Water treatment; B, Extracellular antimicrobial substances treatment.

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