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作物学报 ›› 2022, Vol. 48 ›› Issue (6): 1546-1557.doi: 10.3724/SP.J.1006.2022.14108

• 研究简报 • 上一篇    下一篇

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

王炫栋(), 杨孙玉悦, 高润杰, 余俊杰, 郑丹沛, 倪峰, 蒋冬花*()   

  1. 浙江师范大学化学与生命科学学院, 浙江金华 321004
  • 收稿日期:2021-06-22 接受日期:2021-09-10 出版日期:2022-06-12 网络出版日期:2021-10-15
  • 通讯作者: 蒋冬花
  • 作者简介:E-mail: xuandong189@163.com
  • 基金资助:
    国家自然科学基金项目(31570013);浙江省基础公益研究计划项目(LGN22C140004)

Screening Streptomyces against Xanthomonas axonopodis pv. glycines and study of growth-promoting and biocontrol effect

WANG Xuan-Dong(), YANG Sun-Yu-Yue, GAO Run-Jie, YU Jun-Jie, ZHENG Dan-Pei, NI Feng, JIANG Dong-Hua*()   

  1. College of Chemistry and Life Science, Zhejiang Normal University, Jinhua 321004, Zhejiang, China
  • Received:2021-06-22 Accepted:2021-09-10 Published:2022-06-12 Published online:2021-10-15
  • Contact: JIANG Dong-Hua
  • Supported by:
    National Natural Science Foundation of China(31570013);Zhejiang Province Basic Public Welfare Research Project(LGN22C140004)

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

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).

图1

Sl-3菌株发酵滤液拮抗Xag的抑菌圈(直径32.5 mm) A: 高氏1号液体培养基对照; B: Sl-3菌株发酵滤液。"

图2

Sl-3菌株菌落特征及显微结构 A、B、C: 菌落正背面; D、H: 气生菌丝; E、G: 基内菌丝; F、H: 孢子丝。"

图3

Sl-3菌株不同鉴别培养基生长情况"

表2

Sl-3菌株在不同鉴别培养基上的培养特征"

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

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

None
干燥、中部突起
Dry and central protrusion
++
ISP4 淡粉色
Light pink
粉白色
Pink-and-white

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

None
干燥、平整
Dry and flat
+++

图4

Sl-3菌株生理生化部分试验结果 “+++”表示生长旺盛; “++”表示生长良好; “+”表示生长。"

图5

16S rDNA PCR扩增产物(A)和基于16S rDNA序列构建的Sl-3菌株进化树(B)"

图6

Sl-3菌株粗提物和波尔多液对Xag的MIC CK1: 阴性对照; CK2: 阳性对照; *、**分别表示在0.05和0.01水平显著差异。"

图7

不同浓度Sl-3菌株粗提物处理下Xag生长曲线"

图8

不同浓度粗提物处理4 h扫描电子显微镜下Xag细胞形态 A: 对照; B: 1/2×MIC; C: 1×MIC; D: 2×MIC; E、F: 4×MIC。"

图9

不同浓度粗提物对Xag生物膜形成影响 *、**分别表示在0.05和0.01水平显著差异。"

图10

不同浓度粗提物对Xag胞外多糖产生的抑制率 *、**、***分别表示在0.05、0.01和0.001水平显著差异。"

图11

傅里叶变换红外光谱(FT-IR)不同浓度粗提物处理Xag胞外物质的变化"

表3

不同浓度粗提物处理Xag红外光谱振动峰分配"

振动峰分配
Definition of the spectral assignment
分类
Classification
振动峰Peak variation (cm-1)
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

图12

Sl-3菌株发酵滤液对大豆生长指标的影响 A: 株高; B: 茎粗; C: 根长; D: 鲜重; E: 干重。不同字母代表处理间差异显著(P<0.05)。"

表4

Sl-3菌株发酵滤液不同方法处理大豆的防治效果"

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

图13

Sl-3菌株发酵滤液不同方法处理大豆的防治效果 CK1: 无菌水对照; CK2: Xag阳性对照; M1: 喷洒发酵滤液后接种Xag; M2: 接种Xag后喷洒发酵滤液; M3: 喷洒波尔多液后接种Xag。"

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