中国水稻科学 ›› 2023, Vol. 37 ›› Issue (2): 200-212.DOI: 10.16819/j.1001-7216.2023.220802
王炫栋, 余俊杰, 高润杰, 兰赫婷, 江樱姿, 齐文杰, 宋振, 蒋冬花()
收稿日期:
2022-08-04
修回日期:
2022-09-05
出版日期:
2023-03-10
发布日期:
2023-03-10
通讯作者:
蒋冬花
基金资助:
WANG Xuandong, YU Junjie, GAO Runjie, LAN Heting, JIANG Yingzi, QI Wenjie, SONG Zhen, JIANG Donghua()
Received:
2022-08-04
Revised:
2022-09-05
Online:
2023-03-10
Published:
2023-03-10
Contact:
JIANG Donghua
摘要:
【目的】评估沙阿霉素链霉菌Sz-11的促生能力和对水稻BLS的生防潜力,探究其初步的抑菌机制。【方法】用牛津杯法测定Sz-11对7种植物病原细菌和11种植物病原真菌的抗菌谱。通过浸种和浇灌试验评估Sz-11的促生潜力。SDS-PAGE电泳、琼脂糖凝胶电泳、CLSM、SEM、FTIR分析发酵液中的活性产物对Xooc蛋白质表达、基因组DNA合成、细胞膜通透性、细胞形态、细胞膜表面物质结构和组成的影响。4个水稻品种的盆栽防效试验,初步明确Sz-11对BLS的生防潜力。【结果】Sz-11对番茄叶斑病菌、苦瓜枯萎病菌、西瓜炭疽病菌、烟草赤星病菌等多种植物致病真菌都具有较好的拮抗活性,对水稻黄单胞菌(Xoo和Xooc)具有突出的拮抗作用。Sz-11能够促进水稻种子萌发生长,缩短萌发周期,加速营养生长期水稻有机质的积累和根系的分化。Sz-11能影响Xooc蛋白表达,改变细胞膜透性和膜表面物质组成,使细胞溶胀、变形、死亡,但不影响Xooc基因组DNA合成。盆栽试验显示,Sz-11对4个水稻品种BLS相对防效高达65.63%~84.38%,且提前预防效果优于发病后治理。【结论】Sz-11具有较广的抗菌谱,是一株有益的植物根际促生菌,通过作用于Xooc细胞膜和影响蛋白质合成来抑制病菌的生长繁殖,能有效防治水稻BLS的发生,具有较好的生防应用潜力。
王炫栋, 余俊杰, 高润杰, 兰赫婷, 江樱姿, 齐文杰, 宋振, 蒋冬花. 一株兼具防病促生功能的沙阿霉素链霉菌Sz-11[J]. 中国水稻科学, 2023, 37(2): 200-212.
WANG Xuandong, YU Junjie, GAO Runjie, LAN Heting, JIANG Yingzi, QI Wenjie, SONG Zhen, JIANG Donghua. Streptomyces zaomyceticus Sz-11, a Potential Biocontrol Agent with the Functions of Preventing Plant Diseases and Promoting Plant Growth[J]. Chinese Journal OF Rice Science, 2023, 37(2): 200-212.
菌株 Strain | 侵染植物 Infected plant | 抗性 Resistance |
---|---|---|
Xanthomonas oryzae pv. oryzicola, Xooc, RS105 | 水稻Oryza sativa L. | Rif |
Xanthomonas oryzae pv. oryzae, Xoo, P6 | 水稻Oryza sativa L. | / |
Pseudomonas syringae pv. tomato, Pst, DC3000 | 番茄Lycopersicon esculentum Miller | / |
Pseudomonas syringae pv. glycinea, Psg, NJAU | 大豆Glycine max (Linn.) Merr. | Cb |
Xanthomonas axonopodis pv. phaseoli, Xap, XS2 | 菜豆Phaseolus vulgaris Linn. | Cb |
Xanthomonas axonopodis pv. glycines, Xag, NEAU001 | 大豆Glycine max (Linn.) Merr. | Cb/Amp |
Ralstonia solanacearum, FR2 | 烟草Nicotiana tabacum L. | / |
Gaeumannomyces graminis, MG201 | 小麦Triticum aestivum L. | / |
Fusarium oxysporum f. sp. momordicae, Fom, FM04 | 苦瓜Momordica charantia L. | / |
Fusarium oxysporum f. sp. cucumerinum, Foc, FC27 | 黄瓜Cucumis sativus L. | / |
Fusarium pseudograminearum, FP68 | 小麦Triticum aestivum L. | / |
Penicillium expansum, MP001 | 苹果Malus pumila Mill. | / |
Alternaria solani, AS24 | 番茄Lycopersicon esculentum Miller | / |
Alternaria alternata, AL08 | 烟草Nicotiana tabacum L. | / |
Alternaria tomato, AT01 | 番茄Lycopersicon esculentum Miller | / |
Colletotrichum orbiculare, WT204 | 西瓜Citrullus lanatus (Thunb.) Matsum. et Nakai | / |
Gibberella zeae, GZ69 | 小麦Triticum aestivum L. | / |
Fusarium solani, FS023 | 杨树Populus L. | / |
表1 抗菌谱试验菌株
Table 1. Strains subjected to antibacterial spectrum test.
菌株 Strain | 侵染植物 Infected plant | 抗性 Resistance |
---|---|---|
Xanthomonas oryzae pv. oryzicola, Xooc, RS105 | 水稻Oryza sativa L. | Rif |
Xanthomonas oryzae pv. oryzae, Xoo, P6 | 水稻Oryza sativa L. | / |
Pseudomonas syringae pv. tomato, Pst, DC3000 | 番茄Lycopersicon esculentum Miller | / |
Pseudomonas syringae pv. glycinea, Psg, NJAU | 大豆Glycine max (Linn.) Merr. | Cb |
Xanthomonas axonopodis pv. phaseoli, Xap, XS2 | 菜豆Phaseolus vulgaris Linn. | Cb |
Xanthomonas axonopodis pv. glycines, Xag, NEAU001 | 大豆Glycine max (Linn.) Merr. | Cb/Amp |
Ralstonia solanacearum, FR2 | 烟草Nicotiana tabacum L. | / |
Gaeumannomyces graminis, MG201 | 小麦Triticum aestivum L. | / |
Fusarium oxysporum f. sp. momordicae, Fom, FM04 | 苦瓜Momordica charantia L. | / |
Fusarium oxysporum f. sp. cucumerinum, Foc, FC27 | 黄瓜Cucumis sativus L. | / |
Fusarium pseudograminearum, FP68 | 小麦Triticum aestivum L. | / |
Penicillium expansum, MP001 | 苹果Malus pumila Mill. | / |
Alternaria solani, AS24 | 番茄Lycopersicon esculentum Miller | / |
Alternaria alternata, AL08 | 烟草Nicotiana tabacum L. | / |
Alternaria tomato, AT01 | 番茄Lycopersicon esculentum Miller | / |
Colletotrichum orbiculare, WT204 | 西瓜Citrullus lanatus (Thunb.) Matsum. et Nakai | / |
Gibberella zeae, GZ69 | 小麦Triticum aestivum L. | / |
Fusarium solani, FS023 | 杨树Populus L. | / |
处理组 Treatment groups | 处理方法 Treatment methods |
---|---|
无菌水阴性对照组(CK1) Sterile water group, negative control (CK1) | 无菌剪刀45°剪叶后,喷雾无菌水,并套袋保持剪叶处湿润(24 h) Cut with sterile scissors at 45°followed by sterile water spraying and 20-h bagging to keep them moist by bagging |
Xooc菌液阳性对照组(CK2) Xooc positive control group (CK2) | 无菌剪刀45°剪叶后,喷雾接种Xooc水悬液,并套袋保持接种部位湿润(24 h) Cut leaves with sterile scissors at 45° followed by Xooc water suspension spraying and 24-h bagging to keep the inoculation site moist |
发酵液治疗处理组(T1) Fermentation liquid treatment group (T1) | 无菌剪刀45°剪叶后,喷雾接种Xooc水悬液,套袋保持接种部位湿润;接种3 h后,在叶片正反两面均匀喷洒目标菌株发酵液(剂量:100 µL/cm2),套袋保持接种部位湿润(24 h) Cut leaves with sterile scissors at 45° followed by Xooc water suspension spraying and bagging to keep the inoculation site moist. The fermentation liquid of the target strain was evenly sprayed on both sides of the leaves at a dosage of 100 µL/cm2 3hours after inoculation, and the inoculation site was hugged to keep it moist for 24 hours |
发酵液预防处理组(T2) Fermentation liquid prevention treatment group (T2) | 先用喷壶在叶片正反两面均匀喷洒目标菌株发酵液(剂量:100 µL/cm2),套袋保持接种部位湿润;3 h后,无菌剪刀45°剪叶,喷雾接种Xooc水悬液,套袋保持接种部位湿润(24 h) Firstly, the fermentation liquid of the target strain was evenly sprayed on both sides of the leaves with a watering can at the dosage of 100 µL/cm2 3 hours after inoculation, and the inoculation site was bagged to keep it moist. Sterile scissors were used to cut leaves at 45°3 h later, Xooc water suspension was sprayed, and the inoculation site was bagged to make it moist for 24 hours |
发酵滤液治疗处理组(T3) Fermentation filtrate treatment group (T3) | 处理方法同T1,但将目标菌株发酵液替换成过滤后不含菌体的发酵滤液 The treatment method was the same as T1, but the fermentation liquid of the target strain was replaced with a fermentation filtrate without bacteria after filtration |
发酵滤液预防处理组(T4) Fermentation filtrate prevention treatment group (T4) | 处理方法同T2,但将目标菌株发酵液替换成过滤后不含菌体的发酵滤液 The treatment method was the same as T2, but the fermentation liquid of the target strain was replaced with a fermentation filtrate without bacteria after filtration |
表2 水稻细菌性条斑病防效试验处理方法
Table 2. Treatment methods of rice bacterial leaf streak control effect test.
处理组 Treatment groups | 处理方法 Treatment methods |
---|---|
无菌水阴性对照组(CK1) Sterile water group, negative control (CK1) | 无菌剪刀45°剪叶后,喷雾无菌水,并套袋保持剪叶处湿润(24 h) Cut with sterile scissors at 45°followed by sterile water spraying and 20-h bagging to keep them moist by bagging |
Xooc菌液阳性对照组(CK2) Xooc positive control group (CK2) | 无菌剪刀45°剪叶后,喷雾接种Xooc水悬液,并套袋保持接种部位湿润(24 h) Cut leaves with sterile scissors at 45° followed by Xooc water suspension spraying and 24-h bagging to keep the inoculation site moist |
发酵液治疗处理组(T1) Fermentation liquid treatment group (T1) | 无菌剪刀45°剪叶后,喷雾接种Xooc水悬液,套袋保持接种部位湿润;接种3 h后,在叶片正反两面均匀喷洒目标菌株发酵液(剂量:100 µL/cm2),套袋保持接种部位湿润(24 h) Cut leaves with sterile scissors at 45° followed by Xooc water suspension spraying and bagging to keep the inoculation site moist. The fermentation liquid of the target strain was evenly sprayed on both sides of the leaves at a dosage of 100 µL/cm2 3hours after inoculation, and the inoculation site was hugged to keep it moist for 24 hours |
发酵液预防处理组(T2) Fermentation liquid prevention treatment group (T2) | 先用喷壶在叶片正反两面均匀喷洒目标菌株发酵液(剂量:100 µL/cm2),套袋保持接种部位湿润;3 h后,无菌剪刀45°剪叶,喷雾接种Xooc水悬液,套袋保持接种部位湿润(24 h) Firstly, the fermentation liquid of the target strain was evenly sprayed on both sides of the leaves with a watering can at the dosage of 100 µL/cm2 3 hours after inoculation, and the inoculation site was bagged to keep it moist. Sterile scissors were used to cut leaves at 45°3 h later, Xooc water suspension was sprayed, and the inoculation site was bagged to make it moist for 24 hours |
发酵滤液治疗处理组(T3) Fermentation filtrate treatment group (T3) | 处理方法同T1,但将目标菌株发酵液替换成过滤后不含菌体的发酵滤液 The treatment method was the same as T1, but the fermentation liquid of the target strain was replaced with a fermentation filtrate without bacteria after filtration |
发酵滤液预防处理组(T4) Fermentation filtrate prevention treatment group (T4) | 处理方法同T2,但将目标菌株发酵液替换成过滤后不含菌体的发酵滤液 The treatment method was the same as T2, but the fermentation liquid of the target strain was replaced with a fermentation filtrate without bacteria after filtration |
图1 Sz-11菌株抗菌谱 不同小写字母表示处理间差异显著,P<0.05。
Fig. 1. Antibacterial spectrum of strain Sz-11. Different lowercase letters represent significant differences between treatments(P<0.05). 1?Gaeumannomyces graminis, MG201; 2?Fusarium oxysporum f. sp. momordicae, Fom, FM04; 3?Penicillium expansum, MP001; 4?Alternaria solani, AS24; 5?Colletotrichum orbiculare, WT204; 6?Alternaria alternata, AL08; 7?Alternaria tomato, AT01; 8?Fusarium pseudograminearum, FP68; 9?Fusarium oxysporum f. sp. cucumerinum, Foc, FC27.
图2 Sz-11发酵液水稻浸种促生试验结果 不同小写字母表示处理间差异显著,P<0.05。
Fig. 2. Results of rice growth promoting experiment by soaking seed with Sz-11 fermentation liquid. Different lowercase letters represent significant differences between the treatments at P<0.05.
图4 Sz-11发酵滤液处理Xooc的SDS-PAGE蛋白电泳(A)和DNA琼脂糖凝胶电泳(B)结果 M?标记;N?高氏1号培养基对照;1,1/2,1/4,1/8?不同浓度发酵滤液处理。
Fig. 4. Results of SDS-PAGE protein electrophoresis(A) and DNA agarose gel electrophoresis (B) of Xooc treated with Sz-11 fermentation filtrate. M, Marker; N, Gause 1 medium treatment; 1, 1/2, 1/4, 1/8, Different concentrations of fermentation filtrate treatment.
图5 Sz-11发酵滤液处理对Xooc细胞膜通透性及表面疏水性的影响 A?CLSM图;B?Xooc细胞内β-半乳糖苷酶泄露;C?Xooc细胞表面疏水性变化。不同小写字母表示处理间差异显著,P<0.05。
Fig. 5. Effects of Sz-11 fermentation filtrate treatment on cell membrane permeability and surface hydrophobicity of Xooc. A, CLSM figure; B, Xooc intracellular β-galactosidase leakage; C, Changes of hydrophobicity on Xooc cell surface. Different lowercase letters represent significant differences between the treatments at P<0.05.
图7 不同浓度Sz-11发酵滤液处理后Xooc细胞的红外吸收光谱 对照?高氏1号培养基处理;T1?2倍稀释发酵滤液处理;T2?4倍稀释发酵滤液处理。
Fig. 7. Infrared absorption spectra of Xooc cell treated with different concentrations of Sz-11 fermentation filtrate. Control, Gause 1 medium treatment; T1, 1/2 fermentation filtrate; T2, 1/4 fermentation filtrate.
振动峰分配 Definition of the spectral assignment | 分类 Classification | 振动峰 Peak variation/(cm-1) | ||
---|---|---|---|---|
对照Control | T1 | T2 | ||
碳水化合物中的C-O, C-C str, C-O-H, C-O-C伸缩振动峰 C-O, C-C str, C-O-H, C-O-C def of carbohydrates | 糖原和核酸 Glycogen and nucleic acids | 1063.47 | 1063.62 | 1060.50 |
磷酸二酯>PO2中P=O的不对称伸缩振动峰 P=O str (asym) of>PO2 phosphodiesters | 主要是核酸 Mainly nucleic acids | 1244.96 | 1239.16 | 1236.03 |
COO中C=O对称伸缩振动峰 C=O str (sym) of COO | 氨基酸侧链,脂肪酸 Amino acid side chains, fatty acids | 1393.72 | 1393.87 | 1393.75 |
酰胺II中蛋白质N-H弯曲振动峰和C-N拉伸振动峰 Amide II (protein N-H bend, C-N stretch) | α螺旋 α helices | 1533.55 | 1539.65 | 1539.50 |
酰胺I的β-折叠结构Amide I of β-pleated sheet structures | β-折叠 β-pleated sheet | 1640.66 | 1634.86 | 1634.71 |
>CH2中C-H不对称伸缩振动峰 C-H str (asym) of>CH2 | 主要是脂类Mainly lipids | 2931.90 | 2926.08 | 2925.95 |
羟基O-H伸缩振动峰O-H str of hydroxyl groups | 多糖和蛋白质Polysaccharides, proteins | 3288.93 | 3283.10 | 3274.05 |
表3 不同浓度的Sz-11发酵滤液处理后Xooc细胞振动峰的变化
Table 3. Changes of vibrational peaks of Xooc cell treated with different concentrations of Sz-11 fermentation filtrate.
振动峰分配 Definition of the spectral assignment | 分类 Classification | 振动峰 Peak variation/(cm-1) | ||
---|---|---|---|---|
对照Control | T1 | T2 | ||
碳水化合物中的C-O, C-C str, C-O-H, C-O-C伸缩振动峰 C-O, C-C str, C-O-H, C-O-C def of carbohydrates | 糖原和核酸 Glycogen and nucleic acids | 1063.47 | 1063.62 | 1060.50 |
磷酸二酯>PO2中P=O的不对称伸缩振动峰 P=O str (asym) of>PO2 phosphodiesters | 主要是核酸 Mainly nucleic acids | 1244.96 | 1239.16 | 1236.03 |
COO中C=O对称伸缩振动峰 C=O str (sym) of COO | 氨基酸侧链,脂肪酸 Amino acid side chains, fatty acids | 1393.72 | 1393.87 | 1393.75 |
酰胺II中蛋白质N-H弯曲振动峰和C-N拉伸振动峰 Amide II (protein N-H bend, C-N stretch) | α螺旋 α helices | 1533.55 | 1539.65 | 1539.50 |
酰胺I的β-折叠结构Amide I of β-pleated sheet structures | β-折叠 β-pleated sheet | 1640.66 | 1634.86 | 1634.71 |
>CH2中C-H不对称伸缩振动峰 C-H str (asym) of>CH2 | 主要是脂类Mainly lipids | 2931.90 | 2926.08 | 2925.95 |
羟基O-H伸缩振动峰O-H str of hydroxyl groups | 多糖和蛋白质Polysaccharides, proteins | 3288.93 | 3283.10 | 3274.05 |
图8 4个品种水稻的病斑抑制率和防治效果 CK1?无菌水阴性对照;CK2?Xooc阳性对照;T1?发酵液治疗处理;T2?发酵液预防处理;T3?发酵滤液治疗处理;T4?发酵滤液预防处理。不同小写字母表示处理间差异显著,P<0.05。
Fig. 8. Inhibition rate and control effect of four rice varieties. CK1, Sterile water negative control; CK2, Xooc positive control; T1, Fermentation liquid cure treatment; T2, Fermentation liquid prevention treatment; T3, Fermentation filtrate cure treatment; T4, Fermentation filtrate prevention treatment. Different lowercase letters indicate significant difference at P<0.05.
处理 Treatment | 病情指数 Disease index | 相对防效 Relative control effect / % | ||||||
---|---|---|---|---|---|---|---|---|
湘两优900 Xiangliangyou 900 | 甬优15 Yongyou 15 | 嘉丰优2号 Jiafengyou 2 | 甬优1540 Yongyou 1540 | 湘两优900 Xiangliangyou 900 | 甬优15 Yongyou 15 | 嘉丰优2号 Jiafengyou 2 | 甬优1540 Yongyou 1540 | |
CK1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
CK2 | 60.00 e | 64.00 e | 48.67 d | 54.00 e | 0 | 0 | 0 | 0 |
T1 | 29.33 b | 39.33 c | 18.67 b | 24.00 b | 51.11 c | 38.54 b | 61.65 b | 55.56 b |
T2 | 19.33 a | 10.00 a | 10.00 a | 10.00 a | 67.78 d | 84.38 d | 79.45 c | 81.48 d |
T3 | 50.00 d | 46.00 d | 36.00 c | 32.67 d | 16.67 a | 28.13 a | 26.03 a | 39.51 a |
T4 | 40.00 c | 22.00 b | 18.67 b | 23.33 c | 33.33 b | 65.63 c | 61.65 b | 56.79 c |
表4 4个品种水稻的病情指数及相对防治效果
Table 4. Disease index and relative control effect of four rice varieties.
处理 Treatment | 病情指数 Disease index | 相对防效 Relative control effect / % | ||||||
---|---|---|---|---|---|---|---|---|
湘两优900 Xiangliangyou 900 | 甬优15 Yongyou 15 | 嘉丰优2号 Jiafengyou 2 | 甬优1540 Yongyou 1540 | 湘两优900 Xiangliangyou 900 | 甬优15 Yongyou 15 | 嘉丰优2号 Jiafengyou 2 | 甬优1540 Yongyou 1540 | |
CK1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
CK2 | 60.00 e | 64.00 e | 48.67 d | 54.00 e | 0 | 0 | 0 | 0 |
T1 | 29.33 b | 39.33 c | 18.67 b | 24.00 b | 51.11 c | 38.54 b | 61.65 b | 55.56 b |
T2 | 19.33 a | 10.00 a | 10.00 a | 10.00 a | 67.78 d | 84.38 d | 79.45 c | 81.48 d |
T3 | 50.00 d | 46.00 d | 36.00 c | 32.67 d | 16.67 a | 28.13 a | 26.03 a | 39.51 a |
T4 | 40.00 c | 22.00 b | 18.67 b | 23.33 c | 33.33 b | 65.63 c | 61.65 b | 56.79 c |
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