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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (6): 1546-1557.doi: 10.3724/SP.J.1006.2022.14108

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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 Online:2022-06-12 Published:2021-10-15
  • Contact: JIANG Dong-Hua E-mail:xuandong189@163.com;jdh@zjnu.cn
  • Supported by:
    National Natural Science Foundation of China(31570013);Zhejiang Province Basic Public Welfare Research Project(LGN22C140004)

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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

Table 1

Control effect test treatments in soybean"

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).

Fig. 1

Inhibitory zone of strain Sl-3 fermentation filtrate against Xag (diameter is 32.5 mm) A: Gause No.1 liquid medium control; B: fermentation filtrate of strain Sl-3."

Fig. 2

Colony characteristics and microstructure characteristics of strain Sl-3 A, B, C: colonies; D, H: aerial mycelium; E, G: substrate mycelium; F, H: chains of spores."

Fig. 3

Growth of strains Sl-3 in different differential media"

Table 2

Culture characteristics of strain Sl-3 on different differential media"

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		 +++

Fig. 4

Physiological and biochemical results of strain Sl-3 “+++”: abundant growth; “++”: moderate growth; “+”: growth."

Fig. 5

16S rDNA PCR amplification products (A) and strains Sl-3 evolutionary tree constructed based on 16S rDNA sequence (B)"

Fig. 6

Minimum inhibitory concentration of Xag by crude extract of strain Sl-3 and Bordeaux mixture CK1: negative control; CK2: positive control; *, ** mean significant differences at the 0.05 and 0.01 probability levels, respectively."

Fig. 7

Xag growth curve under different concentrations of crude extract treated of strain Sl-3"

Fig. 8

Xag cells were treated with different concentrations of crude extracts for four hours under scanning electron microscopy A: Control; B: 1/2×MIC; C: 1×MIC; D: 2×MIC; E, F: 4×MIC."

Fig. 9

Effect of crude extracts of different concentrations on Xag biofilm formation * and ** mean significant differences at the 0.05 and 0.01 probability levels, respectively."

Fig. 10

Inhibitory rate of Xag exopolysaccharides (EPS) production by crude extracts of different concentrations *, **, and *** mean significant differences at the 0.05, 0.01, and 0.001 probability levels, respectively."

Fig. 11

Fourier transform infrared spectroscopy (FT-IR) showing the chemical modifications in Xag with crude extracts of different concentrations"

Table 3

FT-IR vibrational peak assignment in response to Xag with crude extracts of different concentrations"

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

Fig. 12

Effects of fermentation filtrate of strain Sl-3 on the growth indices in soybean A: plant height; B: stem diameter; C: root length; D: fresh weight; E: dry weight. Different lowercase letters represent significant differences between the treatments at P<0.05."

Table 4

Control effect of different treatment methods on soybean with fermentation filtrate of strain Sl-3"

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

Fig. 13

Control effect of different treatment methods on soybean fermentation filtrate of strain Sl-3 CK1: sterile water negative control; CK2: Xag positive control; M1: Xag was inoculated after spraying fermentation filtrate; M2: spray fermentation filtrate after Xag inoculation; M3: Xag inoculated after spraying bordeaux liquid."

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