硫藤黄链霉菌St-79对水稻白叶枯病的防效和促生作用

doi:10.16819/j.1001-7216.2022.211102

摘要/Abstract

摘要：

【目的】水稻白叶枯病是水稻的主要细菌性病害之一，严重影响水稻生产。本研究获得一株有效防治水稻白叶枯病的放线菌菌株，探索其对白叶枯病的防效和促进水稻生长的作用，为生防菌的开发利用提供科学依据。【方法】利用梯度稀释涂布法、共培养法和牛津杯法筛选拮抗水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae, Xoo)的放线菌菌株；通过形态特征、生理生化反应、16S rDNA序列和系统发育树分析对其进行分类鉴定；利用共培养法分析目标菌株的抗菌谱；通过96孔板微量稀释法、扫描电子显微镜、微生物粘附碳氢化合物法等探究目标菌株的抑菌作用；通过发酵滤液浸种与菌液喷洒土壤的方法探究其对水稻生长的促进作用；利用盆栽试验探索目标菌株发酵滤液对水稻白叶枯病的防治效果。【结果】从不同生境土壤中分离出140株放线菌菌株，最终筛选得到一株对Xoo具有强拮抗活性的放线菌菌株St-79，其发酵滤液抑菌圈直径为64 mm，并鉴定其为硫藤黄链霉菌(Streptomyces thioluteus)。菌株St-79对水稻细菌性条斑病菌(Xanthomonas oryzae pv. oryzicola)、大豆细菌性斑疹病菌(Xanthomonas axonopodis pv. glycines)、油菜黑腐病菌(Xanthomonas campestris pv. campestris)和番茄细菌性叶斑病菌(Pseudomonas syringae pv. tomato)均有拮抗作用。其乙酸乙酯粗提物具有良好的抑制Xoo生长的效果，粗提物对Xoo的最低抑菌浓度为8 μg/mL，最低杀灭浓度为32 μg/mL；扫描电镜观察结果显示，粗提物对Xoo细胞有很强的损伤作用；粗提物还能改变Xoo细胞内膜通透性，降低Xoo细胞的疏水性。促生试验结果显示，菌株St-79发酵滤液能够促进水稻幼苗的生长。盆栽试验结果显示，菌株St-79发酵滤液对水稻白叶枯病有良好的防治效果，在日本晴、甬优15、甬优1540上的相对防效为65.95%~87.23%，病斑抑制率为69.85%~95.8%，且预防效果优于治疗效果。【结论】获得一株有效防治水稻白叶枯病的放线菌，其对水稻白叶枯病菌有明显的抑制作用，并且可以促进水稻种子的萌发与幼苗的生长。

关键词: 放线菌, 硫藤黄链霉菌, 水稻白叶枯病菌, 发酵滤液, 生物防治, 促生作用

Abstract:

【Objective】 Rice bacterial blight (RBB) is one of the main bacterial diseases on rice, which seriously hinders rice production. The purpose of this study is to obtain an effective actinomycete against RBB and explore its biocontrol and growth-promoting effects, so as to lay a scientific basis for the development and utilization of biocontrol bacteria.【Method】 Actinomycetes against Xanthomonas oryzae pv. oryzae (Xoo) were screened by gradient dilution coating method, co-culture method and Oxford cup method. They were classified and identified by morphological traits, physiological and biochemical reactions, 16S rDNA sequences and phylogenetic-tree analysis. The antimicrobial spectrum of the target strain was analyzed by co-culture method. Antibacterial effect of the target strain was studied by 96-well microdilution method, scanning electron microscope and microbial adhesion hydrocarbon method; The promoting effect of fermentation liquid on rice growth was explored by soaking seeds and spraying soil. The control effect of fermentation liquid of the target strain on rice bacterial blight was studied in a pot experiment.【Result】 A total of 140 actinomycetes strains were isolated from soil in different habitats, and one actinomycete strain St-79 with the strongest antagonistic activity against Xoo was finally screened. The diameter of inhibition zone of fermentation liquid was 64 mm, and it was identified as Streptomyces thioluteus. The strain St-79 had antagonistic effects against Xanthomonas oryzae pv. oryzicola, Xanthomonas axonopodis pv. glycines, Xanthomonas campestris pv. campestris and Pseudomonas syringae pv. tomato. The ethyl acetate crude extract had good inhibitory effect on the growth of Xoo. The minimum inhibitory concentration of crude extract on Xoo was 8 μg/mL, and the minimum bactericidal concentration was 32 μg/mL. The observation results under a scanning electron microscopy showed that the crude extract had a strong damage effect on Xoo cells. Crude extracts can also change the permeability of Xoo cells and reduce the hydrophobicity of Xoo cells. The results of growth-promoting test showed that the fermentation liquid of St-79 could promote the growth of rice seedlings. The pot experiment results showed that the fermentation liquid of St-79 had good control effect on RBB. The relative control efficacies on Nipponbare, Yongyou 15 and Yongyou 1540 was 65.95%－87.23%, and the lesion inhibition rate were 69.85%－95.8%. The prevention effect was better than the post-disease treatment.【Conclusion】 An effective actinomycete against RBB was obtained, which had obvious inhibitory effect on RBB and could promote the germination and seedling growth of rice.

Key words: actinomycetes, Streptomyces thioluteus, Xanthomonas oryzae pv. oryzae, fermentation liquid, biocontrol, promoting growth

马静静, 潘妍妍, 杨孙玉悦, 王嘉琦, 蒋冬花. 硫藤黄链霉菌St-79对水稻白叶枯病的防效和促生作用[J]. 中国水稻科学, 2022, 36(6): 623-638.

MA Jingjing, PAN Yanyan, YANG Sunyuyue, WANG Jiaqi, JIANG Donghua. Control Effect of St-79 (Streptomyces thioluteus) on Rice Bacterial Blight and Its Growth-promoting Effect[J]. Chinese Journal OF Rice Science, 2022, 36(6): 623-638.

图/表 23

表1 防效试验设计

Table 1. Experimental design of control effect.

处理组 Treatment group	处理方法 Treatment method
空白对照组(CK₁) Blank control group(CK₁)	无菌剪刀45°剪叶，并套袋保持接种处湿润24 h。下同 Cut leaves with sterile scissors, and bag them to keep the inoculation site moist for for 24 h. The same below.
Xoo处理对照组(CK₂) Xoo treatment control group(CK₂)	无菌剪刀蘸取Xoo菌液后剪叶 Cut leaves with scissors following a dip into Xoo bacterial liquid.
处理组1(T₁) Treatment group 1(T₁)	按照CK₂的处理方法接种Xoo病菌；6 h后用喷壶将稀释4倍的目标菌株发酵滤液均匀地喷洒在伤口处(剂量：10 mL/株)，共喷洒3次(1 h喷1次)。下同 Inoculate Xoo bacteria according to the treatment method of CK₂; 6 h later, use a watering can to evenly spray the 4-fold dilution fermentation liquid of the target strain on the wound (dose: 10 mL/plant) for a total of 3 sprays (once every hour). The same below.
处理组2(T₂) Treatment group 2(T₂)	按照CK₂的处理方法接种Xoo病菌；6 h后用喷壶将稀释5倍的目标菌株发酵滤液均匀地喷洒在伤口处(剂量：10 mL/株) Inoculate Xoo bacteria according to the treatment method of CK₂; 6 h later, use a watering can to evenly spray the 5-fold dilution fermentation liquid of the target strain on the wound (dose: 10 mL/plant).
处理组3(T₃) Treatment group 3(T₃)	按照CK₂的处理方法接种Xoo病菌；6 h后用喷壶将稀释6倍的目标菌株发酵滤液均匀地喷洒在伤口处(剂量：10 mL/株) Inoculate Xoo bacteria according to the treatment method of CK₂; 6 h later, use a watering can to evenly spray the 6-fold dilution fermentation liquid of the target strain on the wound (dose: 10 mL/plant).
处理组4(T₄) Treatment group 4(T₄)	无菌剪刀剪叶后立即喷洒稀释4倍的目标菌株发酵滤液(剂量：10 mL/株)；6 h后按照CK₂的处理方法接种Xoo病菌 Cut leaves with sterile scissors and spray the 4-fold dilution fermentation liquid of the target strain on the wound (dose: 10 mL/plant); 6 h post-spraying, inoculate Xoo bacteria according to the CK₂ treatment method.

表1 防效试验设计

Table 1. Experimental design of control effect.

处理组 Treatment group	处理方法 Treatment method
空白对照组(CK₁) Blank control group(CK₁)	无菌剪刀45°剪叶，并套袋保持接种处湿润24 h。下同 Cut leaves with sterile scissors, and bag them to keep the inoculation site moist for for 24 h. The same below.
Xoo处理对照组(CK₂) Xoo treatment control group(CK₂)	无菌剪刀蘸取Xoo菌液后剪叶 Cut leaves with scissors following a dip into Xoo bacterial liquid.
处理组1(T₁) Treatment group 1(T₁)	按照CK₂的处理方法接种Xoo病菌；6 h后用喷壶将稀释4倍的目标菌株发酵滤液均匀地喷洒在伤口处(剂量：10 mL/株)，共喷洒3次(1 h喷1次)。下同 Inoculate Xoo bacteria according to the treatment method of CK₂; 6 h later, use a watering can to evenly spray the 4-fold dilution fermentation liquid of the target strain on the wound (dose: 10 mL/plant) for a total of 3 sprays (once every hour). The same below.
处理组2(T₂) Treatment group 2(T₂)	按照CK₂的处理方法接种Xoo病菌；6 h后用喷壶将稀释5倍的目标菌株发酵滤液均匀地喷洒在伤口处(剂量：10 mL/株) Inoculate Xoo bacteria according to the treatment method of CK₂; 6 h later, use a watering can to evenly spray the 5-fold dilution fermentation liquid of the target strain on the wound (dose: 10 mL/plant).
处理组3(T₃) Treatment group 3(T₃)	按照CK₂的处理方法接种Xoo病菌；6 h后用喷壶将稀释6倍的目标菌株发酵滤液均匀地喷洒在伤口处(剂量：10 mL/株) Inoculate Xoo bacteria according to the treatment method of CK₂; 6 h later, use a watering can to evenly spray the 6-fold dilution fermentation liquid of the target strain on the wound (dose: 10 mL/plant).
处理组4(T₄) Treatment group 4(T₄)	无菌剪刀剪叶后立即喷洒稀释4倍的目标菌株发酵滤液(剂量：10 mL/株)；6 h后按照CK₂的处理方法接种Xoo病菌 Cut leaves with sterile scissors and spray the 4-fold dilution fermentation liquid of the target strain on the wound (dose: 10 mL/plant); 6 h post-spraying, inoculate Xoo bacteria according to the CK₂ treatment method.

图1 5株放线菌菌株的菌落形态

Fig. 1. Colonies of 5 actinomyces strains.

图2 菌株St-79发酵滤液对Xoo的抑菌圈 A－高氏一号液体培养基对照；B－菌株St-79的发酵滤液处理。

Fig. 2. Inhibition zone of fermentation liquid of the strain St-79 against Xoo. A, Control with Kohl’s 1 liquid; B, Treatment with fermentation liquid of strain St-79.

图3 菌株St-79的菌落特征和显微特征 A、B－菌株St-79的菌落形态；C、D－光学显微镜下的气生菌丝体和孢子丝。

Fig. 3. Colony and microscopic observation of strain St-79. A and B, Colonies of strain St-79; C and D, Aerial mycelium and chains of spores under an optical microscope.

表2 菌株St-79生理生化试验结果

Table 2. Physiological and biochemical test results of strain St-79.

试验项目 Test item	试验结果 Reaction	试验项目 Test items	试验结果 Reaction
淀粉水解Hydrolysis of starch	++	硫化氢产生Hydrogen sulfide production	+++
纤维素水解Hydrolysis of cellulose	++	黑色素产生Melanin production	−
明胶液化Gelatin liquefaction test	+++	过氧化氢酶Catalase	+++
MR实验Methyl Red test	−	脲酶Urease	−
V-P实验Voges-Proskauer test	−	脂肪酶Lipase	+

表3 菌株St-79碳源、氮源利用试验结果

Table 3. Test results of utilization of carbon source and nitrogen source of strain St-79.

碳源种类 Type of carbon source	试验结果 Test result	氮源种类 Type of nitrogen source	试验结果 Test result
葡萄糖Glucose	+++	蛋白胨Peptone	+++
乳糖Lactose	++	KNO₃	++
麦芽糖Maltose	++	(NH₄)₂SO₄	+++
木糖Xylose	+	谷氨酸Glutamate	++
蔗糖Sucrose	++	甲硫氨酸Methionine	+
鼠李糖Rhamnose	++	赖氨酸Lysine	+++
肌醇Inositol	++	亮氨酸Leucine	+
阿拉伯糖L-arabinose	++	组氨酸Histidine	++
甘露醇mannitol	+++

图4 基于16S rDNA 序列构建的菌株St-79进化树

Fig. 4. Phylogenetic tree of strain St-79 based on the 16S rDNA sequence.

图5 菌株St-79对4种植物病原菌的抑制作用 A－水稻细菌性条斑病菌；B－油菜黑腐病菌；C－大豆细菌性斑疹病菌；D－番茄细菌性叶斑病菌。

Fig. 5. Antibacterial effect of fermentation liquid of strain St-79 against four kinds of plant pathogenic bacteria. A, X. oryzae pv. oryzicola; B, X. campestris pv. campestris; C, X. axonopodis pv. glycines; D, P. syringae pv. tomato.

表4 菌株St-79对4种植物病原细菌的抑制效果

Table 4. Inhibition zone of fermentation liquid from strain St-79 against four kinds of plant pathogenic bacteria.

植物病原细菌 Phytopathogenic bacteria	抑菌圈直径 Inhibition zone diameter /mm
水稻细菌性条斑病菌X. oryzae pv. oryzicola	34.85±0.49 b
油菜黑腐病菌X. campestris pv. campestris	33.95±0.35 b
大豆细菌性斑疹病菌X. axonopodis pv. glycines	32.25±0.70 b
番茄细菌性叶斑病菌P. syringae pv. tomato	19.95±0.21 a

表5 菌株St-79粗提物对Xoo的MIC和MBC

Table 5. MIC and MBC of crude extracts of target strain against Xoo.

菌株St-79乙酸乙酯粗提物浓度 Concentration of crude extracts of strain St-79/(μg·mL⁻¹)	粗提物作用24 h后培养液浊度 Turbidity of culture medium after treatment with crude extracts for 24 h	平板上菌体生长情况 Growth of bacteria on the plate
0	浑浊Muddy	有菌生长With Xoo
2	浑浊Muddy	有菌生长With Xoo
4	浑浊Muddy	有菌生长With Xoo
8	澄清Clarification	有菌生长With Xoo
16	澄清Clarification	有菌生长With Xoo
32	澄清Clarification	无菌生长Without Xoo
64	澄清Clarification	无菌生长Without Xoo
128	澄清Clarification	无菌生长Without Xoo
256	澄清Clarification	无菌生长Without Xoo

图6 不同浓度粗提物对Xoo生长的抑制作用

Fig. 6. Inhibitory effects of different concentrations of crude extracts on the growth of Xoo.

图7 不同浓度粗提物处理4 h后Xoo细胞扫描电子显微镜观察结果 A－对照；B－4 μg/mL；C－8 μg/mL；D－16 μg/mL；E－32 μg/mL。

Fig. 7. Xoo cells treated with crude extracts at different concentrations for 4 h under a scanning electron microscopy. A, Control; B, 4 μg/mL; C, 8 μg/mL; D, 16 μg/mL; E, 32 μg/mL.

图8 粗提物处理对Xoo细胞内膜通透性的影响不同小写字母表示在P < 0.05水平存在显著差异。

Fig. 8. Intimal permeability of Xoo cells treated with crude extracts. Different letters indicate significant difference at the P < 0.05 level.

图9 不同浓度粗提物处理4 h后对Xoo表面疏水性的影响不同小写字母表示在P < 0.05水平存在显著差异。

Fig. 9. Hydrophobicity assay of Xoo treated with different concentrations of crude extracts for 4 h. Different letters indicate significant difference at the P < 0.05 level.

表6 发酵滤液对水稻种子萌发率的影响

Table 6. Effect of fermentation liquid of St-79 on rice seed germination percentage. %

处理 Treatment	处理后天数Days after treatment/d
处理 Treatment	4	6	8
发酵滤液原液Crude fermentation liquid	0.00±0.00 a	0.00±0.00 a	0.00±0.00 a
高氏1号液体培养基原液 Crude Gauze’s medium No. 1	0.00±0.00 a	0.00±0.00 a	0.00±0.00 a
发酵滤液10倍稀释液1/10 fermentation liquid	25.33±3.06 b	63.33±1.15 b	72.67±1.15 c
高氏1号液体培养基10倍稀释液1/10 Gauze’s medium No. 1	23.00±3.00 b	59.33±1.15 b	66.00±2.00 b
发酵滤液50倍稀释液1/50 fermentation liquid	32.00±3.46 cd	72.00±2.00 c	78.00±2.00 d
高氏1号液体培养基50倍稀释液1/50 Gauze’s medium No. 1	28.67±3.06 bc	70.67±1.15 c	76.67±3.06 d
发酵滤液100倍稀释液1/100 fermentation liquid	53.33±3.06 e	91.33±2.31 f	96.67±1.15 g
高氏1号液体培养基100倍稀释液1/100 Gauze’s medium No. 1	29.33±1.15 bc	79.33±1.15 de	87.00±1.00 f
发酵滤液500倍稀释液1/500 fermentation liquid	39.33±1.15 d	82.67±2.31 e	84.67±1.15 ef
高氏1号液体培养基500倍稀释液1/500 Gauze’s medium No. 1	30.67±3.06 bc	76.00±2.00 cd	80.67±1.15 de
无菌水H₂O	30.00±2.00 bc	80.67±1.15 de	88.67±1.15 f

图10 发酵滤液浸种8 d后的水稻种子发芽情况不同小写字母表示在P < 0.05水平存在显著差异。

Fig. 10. Germination of rice seeds soaked in fermentation liquid for 8 days. Different letters indicate significant differences at the P < 0.05 level.

图11 土培条件下菌株St-79发酵滤液对水稻种子萌发率的影响 FL100、FL500分别表示菌株St-79发酵滤液稀释100倍和500倍；KN100、KN500分别表示高氏1号液体培养基稀释100倍和500倍，下同。不同小写字母表示在P < 0.05水平存在显著差异。

Fig. 11. Effect of fermentation liquid of strain St-79 on rice seed germination percentage in soil. FL100, FL500 indicate 1/100 and 1/500 fermentation liquid of strain St-79 respectively; KN100, KN500 indicate 1/100 and 1/500 Gauze’s medium No. 1 respectively. The same below. Different letters indicate significant differences at the P < 0.05 level.

图12 播种21 d后发酵滤液处理组与对照组盆栽长势比较

Fig. 12. Growth comparison of rice seedlings in fermentation liquid and control pots 21 days after sowing.

图13 菌株St-79发酵滤液对水稻幼苗株高、根长和茎粗的影响 A－株高；B－根长；C－茎粗。不同小写字母表示在P < 0.05水平存在显著差异。

Fig. 13. Effects of fermentation liquid of strain St-79 on plant height, root length and stem diameter of rice seedlings. A, Plant height; B, Root length; C, Stem diameter. Different letters indicate significant differences at the P < 0.05 level.

图14 菌株St-79发酵滤液对水稻幼苗鲜质量和干质量的影响 A－菌株St-79发酵滤液对水稻幼苗总鲜质量、地上部鲜质量和根鲜质量的影响；B－菌株St-79发酵滤液对水稻幼苗总干质量、地上部干质量、根干质量的影响。不同小写字母表示在P < 0.05水平存在显著差异。

Fig. 14. Effects of fermentation liquid of strain St-79 on wet weight and dry weight of rice seedlings. A, Effects of fermentation liquid of strain St-79 on fresh weight, shoot fresh weight and root fresh weight of rice seedlings; B, Effects of fermentation liquid of strain St-79 on dry weight, shoot dry weight and root dry weight of rice seedlings. Different letters indicate significant differences at the P < 0.05 level.

图15 不同处理下水稻品种病斑长度 A－甬优1540；B－甬优15；C－日本晴。CK1－空白对照；CK2－只接种Xoo；T1－接种Xoo 6 h后，喷洒稀释4倍的发酵滤液；T2－接种Xoo 6 h后，喷撒稀释5倍的发酵滤液；T3－接种Xoo 6 h后，喷撒稀释6倍的发酵滤液；T4－剪叶后喷洒稀释4倍的发酵滤液，6 h后接种Xoo。下同。

Fig. 15. Lesion length of rice varieties under different treatments. A, Yongyou 1540; B, Yongyou 15; C, Nipponbare. CK1, Blank control; CK2, Control with inoculating Xoo only; T1, Inoculation with Xoo before treatment with 4-fold dilution fermentation liquid; T2, Inoculation with Xoo before treatment with 5-fold dilution fermentation liquid; T3, Inoculation with Xoo before treatment with 6-fold dilution fermentation liquid; T4, Treatment with 4-fold dilution fermentation liquid before inoculation with Xoo, the same blow.

图16 不同处理下水稻品种病斑抑制率同一品种标相同小写字母者表示在P < 0.05水平存在显著差异。

Fig. 16. Lesion inhibition rate of rice varieties with different treatments For a variety, common letters indicate no significant difference at the P < 0.05 level.

表7 不同处理对3个水稻品种的病情指数及相对防效的影响

Table 7. Disease index and relative control effect on three rice varieties under different treatments.

处理 Treatment	病情指数Disease index			相对防效Relative control effect/%
处理 Treatment	甬优1540 Yongyou 1540	甬优15 Yongyou 15	日本晴 Nipponbare	甬优1540 Yongyou 1540	甬优15 Yongyou 15	日本晴 Nipponbare
CK₁	0	0	0	/	/	/
CK₂	95.66±0.29 e	95.93±0.52 e	97.41±0.52 e	0	0	0
T₁	16.93±0.42 b	22.02±0.55 b	18.16±1.06 b	82.31±0.29 c	77.05±0.70 c	81.35±1.19 c
T₂	23.13±1.05 c	27.40±1.59 c	23.19±0.84 c	75.84±0.86 b	71.23±1.53 b	76.20±1.00 b
T₃	30.93±0.26 d	32.76±0.24 d	27.98±0.24 d	67.67±0.55 a	65.85±0.07 a	71.28±0.09 a
T₄	12.23±1.58 a	15.00±0.79 a	14.07±0.52 a	87.23±1.53 d	84.38±0.74 d	85.56±0.62 d

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