拮抗青枯雷尔氏菌的放线菌筛选及其防病作用

doi:10.3864/j.issn.0578-1752.2024.07.009

Abstract

Abstract:

【Objective】Tobacco bacterial wilt caused by Ralstonia solanacearum is the main disease of tobacco in cash crops. In this paper, the actinomycetes with strong antagonism against R. solanacearum were screened from the soil of different habitats, and their antibacterial mechanism and biocontrol effect were determined, which provided a theoretical basis for the further development of anti-disease microbial agents.【Method】After screening the target actinomycetes by co-culture method and Oxford cup method, the target strain was identified by morphological studies, physiological and biochemical experiments and polygenic phylogenetic analysis. The minimum inhibitory concentration (MIC) of actinomycetes crude extract against R. solanacearum was determined by the 96-well plate method. After co-culture treatment of R. solanacearum with crude extract, the growth dynamics of R. solanacearum were detected and the morphological changes of R. solanacearum were observed. The effects on membrane permeability and membrane composition were observed by measuring β-galactosidase activity and propidium iodide (PI) fluorescence experiments and Fourier transform infrared spectroscopy. The antibacterial activity and mechanism of target actinomyces antagonistic R. solanacearum were preliminarily explored by measuring protein synthesis, extracellular polysaccharides (EPS), intracellular reactive oxygen species (ROS), and so on. The effect of the target actinomycetes on tobacco bacterial wilt control was determined by pot experiments.【Result】According to the morphological characteristics, physiological and biochemical experimental results and sequencing results, the target actinomyces strain Sa-21 was identified as Streptomyces rapamycinicus, and the diameter of the inhibition zone against R. solanacearum was 47.9 mm. The results of mechanism experiments showed that the minimum inhibitory concentration of Sa-21 strain crude extract inhibiting R. solanacearum was 0.5 μg·mL^-1, which also had a significant inhibitory effect on bacterial proliferation, and the inhibitory effect increased with the increase of crude extract concentration in the test range. Scanning electron microscopy showed that the structure of R. solanacearum changed after crude extract treatment, resulting in perforation and shrinkage of the bacteria, and with the increase of the concentration of crude extract in the test range, the number of ruptured bacteria increased, and the degree of shrinkage increased. After crude extract treatment, propidium iodide could pass through the cell membrane and bind to intracellular substances to fluoresce, and the activity of β-galactosidase was significantly increased, indicating that the permeability of the cell membrane of R. solanacearum was improved. Further studies showed that crude extract treatment could lead to the accumulation of intracellular reactive oxygen species and the decrease of extracellular polysaccharide production in R. solanacearium, indicating that the cell membrane was damaged to a certain extent. The results of potted plant disease control test showed that the relative control effect of 10-fold dilution of fermentation filtrate of treatment group was 67.61%, while the relative control effect of 10-fold dilution of fermentation filtrate of prevention group was 85.89%.【Conclusion】Streptomyces Sa-21 can inhibit the proliferation of R. solanacearum and destroy the cell membrane structure, which has a good control effect on tobacco bacterial wilt, and has certain development potential and application value.

Key words: Ralstonia solanacearum, Streptomyces rapamycinicus, actinomycete, biological control

LIAO XinLin, GUO Xin, YANG JiXue, SHAO JiaZhu, YUAN XinYu, HU JiaYan, CHEN XiaoXiao, JIANG DongHua. Screening of Actinomycetes Against Ralstonia solanacearum and Its Disease Prevention Function[J].Scientia Agricultura Sinica, 2024, 57(7): 1319-1334.

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References 43

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引物Primer	序列Sequence (5′-3′)
gyrB-F	GTGGCCGATTCCGGCAACCCCAACG
gyrB-R	TCAGATGTCGAGGA AGCGGACGTCC
recA-F	ATGGCAGGAACCGACCGCGAGAAGG
recA-R	TCAGCTCTTGGCCGCCGCGGCCTTG
rpoB-F	TTGGCCGCCTCGCGCAATGCCTCGA
rpoB-R	TCAGACCTCTTCGACGCTGCTCGGC

病情级别 Disease grade	症状 Symptom
0	全株无病The whole plant is disease-free
1	茎部部分出现褪绿斑，或发病一侧1/2以下叶片凋萎 Chlorotic spots appear on the stem part, or less than 1/2 of the leaves on the diseased side withers
3	茎部出现黑色条斑，但不超过茎高1/2，或发病一侧1/2至2/3叶片凋萎 Black stripe appears on the stem, but not more than 1/2 of the stem height, or 1/2 to 2/3 of the leaves on the diseased side withers
5	茎部黑色条斑超过茎高的1/2，但未达到茎顶部，或发病一侧2/3以上叶片凋萎 The black stripe on the stem exceeds 1/2 of the stem height, but does not reach the top of the stem, or more than 2/3 of the leaves on the diseased side withers
7	茎部黑色条斑到达茎顶部，或病株叶片全部凋萎 The black stripe on the stem reaches the top of the stem, or all the leaves of the diseased plant wither
9	病株基本枯死The diseased plant basically died

特征培养基 Characteristic medium	气生菌丝 Aerial mycelium	基内菌丝 Substrate mycelium	可溶性色素 Soluble pigment	菌落生长 Colony growth
高氏1号Gauze’s No. 1	白色White	黄绿色Yellow green	-	茂盛，快Lush, quick
察氏Cha’s	深灰色Dark grey	深灰色Dark grey	-	稀疏，慢Sparse, slow
葡萄糖天门冬素琼脂Glucose aspartin agar	白色White	淡黄色Pale yellow	-	稀疏，慢Sparse, slow
无机盐淀粉琼脂ISP4	深灰色Dark grey	黄绿色Yellow green	-	茂盛，快Lush, quick
甘油天门冬素琼脂ISP5	白色White	黄绿色Yellow green	-	稀疏，慢Sparse, slow

项目Item	结果Result	项目Item	结果Result
淀粉酶Amylase	+	硫化氢Hydrogen sulfide	-
纤维素酶Cellulase	+	黑色素Melanin	-
蛋白酶Protease	+	过氧化氢酶Catalase	+
甲基红Methyl red (MR)	-	脲酶Urease	+
V-P	-	脂肪酶Lipase	+

碳源种类Carbon source	结果Result	氮源种类Nitrogen source	结果Result
α-乳糖α-lactose	+++	蛋白胨Peptone	+++
D-麦芽糖D-maltose	+++	KNO₃	+++
D-木糖D-xylose	+	(NH4)₂SO₄	++
蔗糖Sucrose	+	谷氨酸Glutamate	-
L(+)-鼠李糖L(+)-rhamnose	++	甲硫氨酸Methionine	+
棉子糖Raffinose	+++	赖氨酸Lysine	+++
L(-)-阿拉伯糖L(-)-arabinose	+	亮氨酸Leucine	+++
肌醇Inositol	+++	组氨酸Histidine	+++
D-甘露醇D-mannitol	+++

Screening of Actinomycetes Against Ralstonia solanacearum and Its Disease Prevention Function

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振动峰分配 <BOLD>V</BOLD>ibration peak assignment	分类 Classification	振动峰<BOLD>V</BOLD>ariation peak (cm^-1)
振动峰分配 <BOLD>V</BOLD>ibration peak assignment	分类 Classification	CK	0.25 μg·mL^-1	10 μg·mL^-1
碳水化合物中的C-O, C-C str, 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 acid	1 060.175	1 061.139	1 062.585
磷酸二酯>PO₂中P=O的不对称伸缩振动峰 P=O stretching (antisymmetric) of>PO₂	主要是核酸Mainly nucleic acid	1 220.238	1 220.238	1 221.684
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-helix	1 540.363	1 541.327	1 541.327
酰胺I的β-折叠结构Amide I of β-pleated sheet structures	β-折叠β-pleated sheet	1 644.982	1 647.875	1 648.357
羟基O-H伸缩振动峰O-H str of hydroxyl groups	多糖和蛋白质Polysaccharide, protein	3 279.358	3 284.179	3 287.554

处理 Treatment	发病率 Incidence rate (%)	病情指数 Disease index	相对防治效果 Relative control effect (%)
阴性对照Negative control (CK1)	0	0	-
阳性对照Positive control (CK2)	86.68±2.54a	28.15±2.06a	-
治疗组Treatment group (T1)	60.02±1.93b	8.89±1.25b	67.61±3.72b
预防组Prevention group (T2)	26.64±1.78c	3.70±1.83c	85.89±4.28a

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[2]	GUO Ning, SUN Hua, MA HongXia, LIU ShuSen, ZHANG HaiJian, SHI Jie, ZHENG XiaoJuan, DONG YueGuang. Screening, Identification and Control Efficacy Analysis of Trichoderma Strains Against Maize Pythium Stalk Rot [J]. Scientia Agricultura Sinica, 2023, 56(22): 4453-4466.
[3]	WANG XuanDong, SONG Zhen, LAN HeTing, JIANG YingZi, QI WenJie, LIU XiaoYang, JIANG DongHua. Isolation of Dominant Actinomycetes from Soil of Waxberry Orchards and Its Disease Prevention and Growth-Promotion Function [J]. Scientia Agricultura Sinica, 2023, 56(2): 275-286.
[4]	SHA YueXia, HUANG ZeYang, MA Rui. Control Efficacy of Pseudomonas alcaliphila Strain Ej2 Against Rice Blast and Its Effect on Endogenous Hormones in Rice [J]. Scientia Agricultura Sinica, 2022, 55(2): 320-328.
[5]	CHEN Yang,ZHAO HongYi,YAN JunJie,HUANG Jian,GAO YuLin. Chemical Synthesis View on Sex Pheromones of Potato Tuberworm (Phthorimaea operculella) [J]. Scientia Agricultura Sinica, 2021, 54(3): 556-572.
[6]	CAO YuHan,LI ZiTeng,ZHANG JingYi,ZHANG JingNa,HU TongLe,WANG ShuTong,WANG YaNan,CAO KeQiang. Analysis of dsRNA Carried by Alternaria alternata f. sp. mali in China and Identification of a dsRNA Virus [J]. Scientia Agricultura Sinica, 2021, 54(22): 4787-4799.
[7]	HU ChangXiong,FAN Wei,ZHANG Qian,CHEN GuoHua,YIN HongHui,XU TianYang,YANG JinBo,YANG Hang,WU DaoHui,ZHANG XiaoMing. Control Effect of Orius similis on Frankliniella occidentalis Based on the Two-Sex Life Table and the Age-Stage-Specific Predation Rate [J]. Scientia Agricultura Sinica, 2021, 54(13): 2769-2780.
[8]	LI YangFan,SHAO MeiQi,LIU CHANG,GUO QingGang,WANG PeiPei,CHEN XiuYe,SU ZhenHe,MA Ping. Identification of the Antifungal Active Compounds from Bacillus amyloliquefaciens Strain HMB33604 and Its Control Efficacy Against Potato Black Scurf [J]. Scientia Agricultura Sinica, 2021, 54(12): 2559-2569.
[9]	LI Shu,WANG Jie,HUANG NingXing,JIN ZhenYu,WANG Su,ZHANG Fan. Research Progress and Prospect on Banker Plant Systems of Predators for Biological Control [J]. Scientia Agricultura Sinica, 2020, 53(19): 3975-3987.
[10]	ZHANG Lei,JIA Qi,WU Wei,ZHAO LuPing,XUE Bing,LIU HuanHuan,SHANG Jing,YONG TaiWen,LI Qing,YANG WenYu. Species Identification and Virulence Determination of Beauveria bassiana Strain BEdy1 from Ergania doriae yunnanus [J]. Scientia Agricultura Sinica, 2020, 53(14): 2974-2982.
[11]	WANG XiaoNing,LIANG Huan,WANG Shuai,FANG WenSheng,XU JingSheng,FENG Jie,XU Jin,CAO AoCheng. Function of Copper-Resistant Gene copA of Ralstonia solanacearum [J]. Scientia Agricultura Sinica, 2019, 52(5): 837-848.
[12]	SHA YueXia,SUI ShuTing,ZENG QingChao,SHEN RuiQing. Biocontrol Potential of Bacillus velezensis Strain E69 Against Rice Blast and Other Fungal Diseases [J]. Scientia Agricultura Sinica, 2019, 52(11): 1908-1917.
[13]	LI YuJia, LI Qian, ZHANG ZhiXiang, LI ShiFang. Screening and identification of peach endophytic bacteria with antagonism against Agrobacterium tumefaciens [J]. Scientia Agricultura Sinica, 2017, 50(20): 3918-3929.
[14]	ZHANG Bing-huo, LI Han-quan, LUO Juan-yan, YANG Jian-yuan, SHI Hong-qiu, SUN Feng-zhen. Influences of Actinomycete Strain JXJ-0136 on the Growth of Brassica chinensis and Vigna unguiculata and Its Phosphate Solubilization [J]. Scientia Agricultura Sinica, 2016, 49(16): 3152-3161.
[15]	LU Hui-hui, LIN Zhi-qiang, TAN Wan-zhong, LUO Hua-dong, XIAN Fei, BI Chao-wei, YU Yang, YANG Yu-heng. Insecticidal Protein Genes of Bacillus thuringiensis Strain CPB012 and Its Effects in Controlling Different Insect Pests [J]. Scientia Agricultura Sinica, 2015, 48(6): 1112-1121.