拮抗大豆斑疹病菌放线菌菌株的筛选和促生作用及防效研究

doi:10.3724/SP.J.1006.2022.14108

摘要/Abstract

摘要：

大豆斑疹病在全球范围内发生, 严重影响大豆生产。为了避免化学杀菌剂的诸多缺点, 添加生防菌逐渐成为防治大豆斑疹病的一种替代策略。然而, 对大豆斑疹病生防菌剂开发和防效研究却鲜有报道。本试验采用梯度稀释涂布法从多种植物根际土壤样本中共分离出186株放线菌菌株。利用共培养法和牛津杯法筛选到8株拮抗大豆斑疹病菌(Xanthomonas axonopodis pv. glycines, Xag)的放线菌, 其中拮抗能力最强的是Sl-3菌株, 发酵滤液抑菌圈直径为(32.5±1.5) mm。根据形态特征、生理生化试验、16S rDNA序列和系统发育分析, 鉴定Sl-3菌株为丁香链霉菌(Streptromyces lilacinus)。丁香链霉菌Sl-3菌株乙酸乙酯粗提物具有良好的抑制Xag生长的效果。采用96孔板法测定粗提物最低抑菌浓度为64 μg mL^-1。扫描电镜观察结果显示Sl-3菌株的粗提物对Xag细胞有一定的损伤作用, 影响细胞分裂、细胞膜合成, 从而抑制细菌生长。粗提物还能抑制Xag胞外多糖的产生, 影响生物膜形成, 降低Xag的侵染毒力。傅里叶红外光谱法测定结果显示, 粗提物处理会对Xag细胞表面的脂质、蛋白质和核酸等物质的化学和生理指标造成一定的影响, 导致Xag细胞表面物质的结构及组成变化。促生试验结果显示Sl-3菌株发酵滤液能够促进大豆植株生长。盆栽试验结果显示, 丁香链霉菌Sl-3发酵滤液对大豆斑疹病有很好的防治效果, 相对防效达到93.17%, 且提前预防效果优于发病后治疗。

关键词: 放线菌, 丁香链霉菌, 大豆斑疹病菌, 抑菌作用, 生物防治

Abstract:

Soybean bacterial pustule disease occurs globally, seriously affecting soybean production. To avoid the many shortcomings of chemical fungicides, the addition of biocontrol bacteria has gradually become an alternative strategy for the prevention and treatment of soybean bacterial pustule disease. However, there are few reports on the development and prevention of soybean bacterial pustule disease biocontrol agents. In this experiment, a total of 186 strains of actinomycetes were isolated from a variety of plant rhizosphere soil samples using the gradient dilution coating method. Eight strains of actinomycete with antagonistic resistance to Xanthomonas axonopodis pv. glycines (Xag) were screened by coculture and Oxford cup methods. Among them, the strain Sl-3 performed the strongest antibacterial activity and the inhibition zone diameter was (32.5±1.5) mm. According to morphological characteristics, physiological and biochemical experiments, 16S rDNA sequencing and phylogenetic analysis, the strain Sl-3 was identified as Streptromyces lilacinus. The crude ethyl acetate extract of Streptromyces lilacinus Sl-3 has a good inhibitory effect on the growth of Xag. The lowest inhibitory concentration of the crude extract was determined to be 64 μg mL ^-1 by 96-well plate method. Scanning electron microscopy revealed that the crude extract of strain Sl-3 had a certain damage effect on Xag cells, which affected cell division and cell membrane synthesis, thus inhibiting the growth of bacteria. The crude extract also inhibited the production of Xag exopolysaccharide, affected the formation of biofilm, and reduced the toxicity of Xag infection. The results of fourier transform infrared spectroscopy showed that the chemical and physiological indexes of lipids, proteins and nucleic acids on the surface of Xag cells were affected by crude extract treatment, leading to changes in the structure and composition of Xag cell surface substances. The growth promotion test indicated that the fermentation filtrate of the strain Sl-3 can significantly promote the growth of soybean plants. The pot incubation test revealed that the fermentation filtrate of Streptomyces lilacinus Sl-3 had a good control effect on soybean bacterial pustule disease with a relative control effect of 93.17%, and the early prevention effect was better than the treatment after the onset.

Key words: actinomycete, Streptromyces lilacinus, Xanthomonas axonopodis pv. glycines (Xag), antibacterial effect, biocontrol

王炫栋, 杨孙玉悦, 高润杰, 余俊杰, 郑丹沛, 倪峰, 蒋冬花. 拮抗大豆斑疹病菌放线菌菌株的筛选和促生作用及防效研究[J]. 作物学报, 2022, 48(6): 1546-1557.

WANG Xuan-Dong, YANG Sun-Yu-Yue, GAO Run-Jie, YU Jun-Jie, ZHENG Dan-Pei, NI Feng, JIANG Dong-Hua. Screening Streptomyces against Xanthomonas axonopodis pv. glycines and study of growth-promoting and biocontrol effect[J]. Acta Agronomica Sinica, 2022, 48(6): 1546-1557.

图/表 17

表1

大豆防效试验处理"

处理组 Treatment group	处理方法 Treatment method
无菌水空白对照组(CK1) Sterile water blank control group (CK1)	刷子蘸取无菌水, 均匀刷涂在叶片正反两面, 并套袋保持接种处湿润(24 h)。 Dip the brush with sterile water, evenly brush on the front and back sides of the leaves, and cover the bag to keep the inoculation site moist (24 h).
Xag菌液处理对照组(CK2) Xag bacterial liquid treatment control group (CK2)	刷子蘸取Xag水悬液(OD₆₀₀=0.6), 均匀刷涂在叶片正反两面, 并套袋保持接种部位湿润(24 h)。 Dip the brush with Xag water suspension (OD₆₀₀=0.6), evenly brush it on the front and back sides of the leaves, and cover the bag to keep the inoculation site moist (24 h).
方法一处理组(M1) Method one treatment group (M1)	先用喷壶将目标菌株发酵滤液均匀的喷洒在叶片正反两面(剂量: 100 µL cm^-2), 共喷洒6次(0.5 h 次^-1); 喷洒12 h后, 按照CK2的处理方法接种Xag病菌, 并套袋保持接种部位湿润(24 h)。 First use a watering can to evenly spray the fermentation filtrate of the target strain on the front and back sides of the leaves (dose: 100 µL cm^-2) for a total of 6 sprays (0.5 h time^-1); after spraying for 12 h, inoculate Xag bacteria according to the CK2 treatment method, and bagging to keep the inoculation site moist (24 h).
方法二处理组(M2) Method two treatment group (M2)	按照CK2组的处理方法接种Xag病菌, 并套袋保持接种部位湿润(24 h); 接种12 h后按照M1中的方法喷洒目标菌株发酵滤液。 Inoculate Xag bacteria according to the treatment method of CK2 group, and keep the inoculation site moist (24 h) by bagging; spray the fermentation filtrate of the target strain according to the method in M1 after 12 hours of inoculation.
波尔多液处理组(M3) Bordeaux mixture treatment group (M3)	先用喷壶将波尔多液均匀的喷洒在叶片正反两面(剂量: 100 µL cm^-2), 共喷洒6次(0.5 h 次^-1); 喷洒12 h后, 按照CK2的处理方法接种Xag病菌, 并套袋保持接种部位湿润(24 h)。 First use a watering can to evenly spray Bordeaux mixture on the front and back sides of the leaves (dose: 100 µL cm^-2) for a total of 6 sprays (0.5 h time^-1); after spraying for 12 h, inoculate Xag bacteria according to the CK2 treatment method, and cover Keep the inoculation site moist with the bag (24 h).

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鉴别培养基 Differential medium	气生菌丝 Aerial mycelium	基内菌丝 Substrate mycelium	可溶性色素 Water-soluble pigment	菌落表面形态 Colony surface morphology	丰茂度 Abundance
察氏 Czapek dox	橘黄色 Croci	淡黄色 Faint yellow	无 None	干燥、略突起 Dry and slightly raised	++
葡萄糖天门冬素 Glucose asparagine	粉红色 Pink	淡粉色 Light pink	无 None	干燥、中部突起 Dry and central protrusion	++
ISP4	淡粉色 Light pink	粉白色 Pink-and-white	无 None	干燥、粉末状突起 Dry and powdery protrusions	+++
ISP5	深红色 Deepred	红黑色 Red and black	黄褐色 Tawny	干燥、略突起 Dry and slightly raised	++
高氏1号 Gause 1	紫红色 Amaranth	紫红色 Amaranth	无 None	干燥、平整 Dry and flat	+++

振动峰分配 Definition of the spectral assignment	分类 Classification	振动峰Peak variation (cm^-1)
振动峰分配 Definition of the spectral assignment	分类 Classification	Control	1×MIC	2×MIC
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	1067.76	1057.63	1062.89
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	1236.79	1233.52	1235.12
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	1387.89	1388.02	1388.25
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	1540.68	1527.58	1522.11
酰胺I的β-折叠结构 Amide I of β-pleated sheet structures	β-折叠 β-pleated sheet	1636.74	1636.49	1636.49
>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	2960.50	2929.79	2929.73
羟基O-H伸缩振动峰 O-H str of hydroxyl groups	多糖和蛋白质 Polysaccharides, proteins	3278.95	3274.56	3274.27

处理 Treatment	病情指数 Disease index	相对防效 Relative control effect (%)
CK1	0	0
CK2	89.56 ± 2.23 d	0
M1	6.12 ± 1.37 a	93.17 c
M2	12.33 ± 1.23 b	86.23 b
M3	25.87 ± 3.12 c	71.11 a