猕猴桃细菌性溃疡病病原鉴定及室内防治药剂筛选

Pathogen identification of kiwifruit bacterial canker and screening of indoor control agents

  • 摘要: 【目的】明确广东省和平县猕猴桃细菌性溃疡病的病原菌,筛选出防治猕猴桃细菌性溃疡病的有效药剂,为猕猴桃细菌性溃疡病的诊断和防治提供理论依据。【方法】从广东省和平县采集猕猴桃细菌性溃疡病病枝,利用涂布平板法分离病原菌,依据柯赫氏法则验证其致病性,结合革兰氏染色、形态特征观察及16S rRNA和carA基因序列分析,明确病原菌的分类地位。分析不同温度对病原菌生长的影响,明确其与猕猴桃细菌性溃疡病常见病原菌丁香假单胞菌猕猴桃致病变种(Pseudomonas syringae pv. actinidiae)的区别。采用抑菌圈法和噻唑蓝(MTT)法,测定7种供试药剂1.5%四霉素AS、85%噻霉酮素TC、31.5%中生菌素TK、20%辛菌胺醋酸盐AS、98%喹啉铜TC、83.8%春雷霉素TC和98%硫酸链霉素TC的室内毒力。【结果】从猕猴桃细菌性溃疡病病枝中分离出1株菌株GD,通过柯赫氏法则验证确定其为猕猴桃细菌性溃疡病的病原菌。菌株GD的菌落呈乳白色至淡黄色,表面湿润、光滑,整体微微隆起。革兰氏染色结果表明菌株GD为革兰氏阴性菌,在显微镜下观察发现,菌体呈短杆状,两端钝圆,粗细较均匀,菌体大小为0.5~1.0 μm×2.0~3.0 μm。结合16S rRNA和carA基因序列分析,最终将其鉴定为胡萝卜软腐果胶杆菌猕猴桃亚种(Pectobacterium carotovorum subsp. actinidiae),不同于常见的猕猴桃细菌性溃疡病病原菌丁香假单胞菌猕猴桃致病变种。胡萝卜软腐果胶杆菌猕猴桃亚种最适生长温度为35 ℃,在40 ℃高温下仍能保持一定活力;而丁香假单胞菌猕猴桃致病变种最适生长温度为25 ℃,温度达35 ℃以上时其生长受抑制。室内毒力测定结果表明,1.5%四霉素AS对胡萝卜软腐果胶杆菌猕猴桃亚种和丁香假单胞菌猕猴桃致病变种的抑制效果最好,在6.25、3.125和1.5625 mg/mL浓度下对2种病原菌的抑菌圈直径均显著高于传统防治药剂98%硫酸链霉素TC(P<0.05),1.5%四霉素AS对胡萝卜软腐果胶杆菌猕猴桃亚种和丁香假单胞菌猕猴桃致病变种的半抑制浓度(IC50)分别为24.28和6.29 μg/mL。【结论】广东省和平县猕猴桃细菌性溃疡病病原菌为胡萝卜软腐果胶杆菌猕猴桃亚种,其最适生长温度为35 ℃,较常见的猕猴桃细菌性溃疡病病原菌丁香假单胞菌猕猴桃致病变种更耐高温。1.5%四霉素AS对胡萝卜软腐果胶杆菌猕猴桃亚种和丁香假单胞菌猕猴桃致病变种均有较好的抑制效果,可作为防治猕猴桃细菌性溃疡病的候选药剂。

     

    Abstract: 【Objective】 This study aimed to identify the pathogen of kiwifruit bacterial canker in Heping County, Guangdong Province, screen effective agents to control the disease, thereby providing a theoretical basis for the diagnosis and control of kiwifruit bacterial canker.【Method】 Diseased branches of kiwifruit bacterial canker were collected from Heping County, Guangdong Province. Pathogenic bacteria were isolated using the spread plate method, and their pathogenicity was verified according to Koch’s postulates. Based on Gram staining, morphological characteristics observation, and sequence analysis of 16S rRNA and carA genes, the taxonomic status of the pathogen was clarified. Effects of diffe-rent temperatures on pathogen growth were analyzed to clarify the difference between the pathogen and Pseudomonas syringae pv. actinidiae, a common pathogen of kiwifruit bacterial canker. Using the inhibition zone method and methyl thiazolyl tetrazolium (MTT) assay, indoor toxicity of seven tested agents (1.5% tetramycin AS, 85% thiadiazole copper TC, 31.5% zhongshengmycin TK, 20% cuaminosulfate acetate AS, 98% oxine-copper TC, 83.8% kasugamycin TC, and 98% streptomycin sulfate TC) was evaluated.【Result】 One bacterial strain, designated as GD, was isolated from the branches infected by kiwifruit bacterial canker. The strain GD was identified as the pathogen of kiwifruit bacterial canker based on Koch’s postulates, whose colonies were off-white to light yellow, with a smooth and humid surface and slight raised appearance. The strain GD was identified as a Gram-negative bacterium by Gram staining; microscopic observation revealed that the cells were short rod-shaped, with blunt and rounded ends, and they were relatively uniform in width, with their sizes of (0.5-1.0) μm×(2.0-3.0) μm. According to analysis of 16S rRNA and carA gene sequences, the strain was finally identified as Pectobacterium carotovorum subsp. actinidiae, which was different from the common kiwifruit bacterial canker pathogen Pseudomonas syringae pv. actinidiae. The optimal growth temperature of Pectobacterium carotovorum subsp. actinidiae was 35 ℃, and it still maintained certain viability in a high-temperature environment of 40 ℃; in contrast, the optimal growth temperature of Pseudomonas syringae pv. actinidiae was 25 ℃, and its growth was inhi-bited when the temperature exceeded 35 ℃. The results of indoor toxicity tests showed that 1.5% tetramycin AS demonstrated the strongest inhibitory effect against both Pectobacterium carotovorum subsp. actinidiae and Pseudomonas syringae pv. actinidiae. At the concentrations of 6.25, 3.125, and 1.5625 mg/mL, the diameters of inhibition zone against the two pathogens were significantly higher than those of the conventional control agent 98% streptomycin sulfate TC (P<0.05). The half maximal inhibitory concentration (IC50) values of 1.5% tetramycin AS against strain Pectobacterium carotovorum subsp. actinidiae and Pseudomonas syringae pv. actinidiae were 24.28 and 6.29 μg/mL, respectively.【Conclusion】 The kiwifruit bacterial canker pathogen in Heping County, Guangdong Province is Pectobacterium carotovorum subsp. actinidiae, whose optimal growth temperature is 35 °C, and it is more tolerant to high temperature than the common kiwifruit bacterial canker pathogen Pseudomonas syringae pv. actinidiae. 1.5% tetramycin AS shows excellent inhibitory effects on both Pectobacterium carotovorum subsp. actinidiae and Pseudomonas syringae pv. actinidiae, there by it can be taken as a candidate agent for controlling kiwifruit bacterial canker.

     

/

返回文章
返回