福建猕猴桃细菌性枯萎病的病原菌鉴定

doi:10.13304/j.nykjdb.2022.1058

摘要/Abstract

摘要：

为明确近年来在福建省宁德市蕉城区石后乡猕猴桃苗圃新发生的一种细菌性病害病的原菌，并进一步探讨其发生流行规律，采用稀释分离法对采集病株进行病原菌的分离和纯化，对获得的细菌菌株进行致病性测定，通过柯赫氏法则验证、常规的细菌生物学特性、生理生化反应、Biolog 鉴定、16S rDNA和内切葡聚糖酶基因（egl）序列分析对病原菌进行分类鉴定。结果表明，从发病猕猴桃病株上获得9株细菌菌株，将其接种在健康的猕猴桃植株后，其发病症状与田间自然发病症状基本一致，且从接种后病株的茎秆和根部又重新分离到与原菌落形态相同的细菌。柯赫氏法则证实这9株细菌菌株为猕猴桃细菌性枯萎病的致病菌。这9株菌株在NA培养基上的菌落形态一致，均呈不规则型、扁平，并逐渐向四周扩散，不产生粘稠状物质，不会使培养基变色；在TTC培养基上，菌落颜色呈暗红色，产生可扩散至培养基中的红褐色色素，其流动性较差；在YDC培养基上，菌落呈乳白色。经综合分析，这9株细菌菌株均被鉴定为假茄科雷尔氏菌（Ralstonia pseudosolanacearum），生化型Ⅲ和演化型Ⅰ（亚洲组），序列变种14。这是首例青枯菌侵染猕猴桃的报道。以上研究结果为制定猕猴桃细菌性枯萎病精准有效的综合防控提供了理论依据。

关键词: 猕猴桃, 细菌性枯萎病, 病原菌鉴定, 生理生化测定, 假茄科雷尔氏菌, 16S rDNA

Abstract:

To identify the pathogenic agent of a newly bacterial wilt disease in kiwifruit， which found in Shihou village， Jiaocheng district， Ningde city， Fujian province in recent years， the bacterial strains were isolated and purified by the dilution separation method. The pathogenicity of the isolated strains was tested by inoculating on stems and roots of kiwifruit seedlings， and its taxonomic status were identified by Koch’s postulates， bacteria biological and biochemical characteristics， biolog test， PCR verification with the specific primer， evolutionary type was determined by multiplex PCR， analysis of 16S rDNA and endoglucanase gene （egl） sequences. The results showed that 9 strains were isolated and purified from the vascular tissues of the infected stems. Followed Kock’s postulates， the 9 strains could produce the similar symptoms on the inoculated kiwifruit seedlings， and the similar bacterial strains were also re-isolated. The colony morphology of the isolates was irregular， flat， spreading around， and could not produce viscous substances in NA medium， the colony color was dark red， producing reddish-brown pigment diffused into the medium and poor fluidity in TTC medium， while the colony was milky whitish in YDC medium. According to all kinds of analysis， the 9 isolates were identified as Ralstonia pseudosolanacearum， belonging to biovar Ⅲ， phylotype Ⅰ （Asia group）， sequevar 14. This was the first report of a disease caused by R.pseudosolanacearum on kiwifruit （Actinidia chinensis Planch）. Above results provided a theoretical basis for the accurate and effective prevention and control of kiwifruit bacterial wilt.

Key words: kiwi, bacterial blight wilt, pathogen identification, physiological and biochemical determination, Ralstonia pseudosolanacearum, 16S rDNA

中图分类号:

S663.4

季梦婷, 陈长江, 罗流河, 林志坚, 詹梦琳, 杨丙烨, 胡方平, 蔡学清. 福建猕猴桃细菌性枯萎病的病原菌鉴定[J]. 中国农业科技导报, 2024, 26(4): 144-152.

Mengting JI, Changjiang CHEN, Liuhe LUO, Zhijian LIN, Menglin ZHAN, Bingye YANG, Fangping HU, Xueqing CAI. Pathogen Identification of Kiwi Bacterial Wilt in Fujian[J]. Journal of Agricultural Science and Technology, 2024, 26(4): 144-152.

图/表 11

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引物名称 Primer name	引物序列 Primer sequence（5’-3’）	扩增片段分类归属 Classify remarks	大小 Size/bp
Nmult21：1F	CGTTGATGAGGCGCGCAATTT	演化型Ⅰ PhylotypeⅠ	144^［15］
Nmult21：2F	AAGTTATGGACGGTGGAAGTC	演化型Ⅱ Phylotype Ⅱ	372
Nmult23：AF	ATTACSAGAGCAATCGAAAGATT	演化型Ⅲ Phylotype Ⅲ	91
Nmult22：InF	ATTGCCAAGACGAGAGAAGTA	演化型Ⅳ Phylotype Ⅳ	213
Nmult22：RR	TCGCTTGACCCTATAACGAGTA		—
759F	GTCGCCGTCAACTCACTTTCC	青枯菌种特异性 Ralstonia specific-primer	280^［13］
760R	GTCGCCGTCAGCAATGCGGAATCG	青枯菌种特异性 Ralstonia specific-primer	280^［13］
27F	GTCGCCGTCAACTCACTTTCC	16S rDNA	1 500^［16］
1492R	GGTTACCTTGTTACGACTT	16S rDNA	1 500^［16］
Endo-F	ATGCATGCCGCTGGTCGCCGC	egl基因 egl gene	750^［17］
Endo-R	GCGTTGCCCGGCACGAACACC	egl基因 egl gene	750^［17］

引物名称 Primer name	引物序列 Primer sequence（5’-3’）	扩增片段分类归属 Classify remarks	大小 Size/bp
Nmult21：1F	CGTTGATGAGGCGCGCAATTT	演化型Ⅰ PhylotypeⅠ	144^［15］
Nmult21：2F	AAGTTATGGACGGTGGAAGTC	演化型Ⅱ Phylotype Ⅱ	372
Nmult23：AF	ATTACSAGAGCAATCGAAAGATT	演化型Ⅲ Phylotype Ⅲ	91
Nmult22：InF	ATTGCCAAGACGAGAGAAGTA	演化型Ⅳ Phylotype Ⅳ	213
Nmult22：RR	TCGCTTGACCCTATAACGAGTA		—
759F	GTCGCCGTCAACTCACTTTCC	青枯菌种特异性 Ralstonia specific-primer	280^［13］
760R	GTCGCCGTCAGCAATGCGGAATCG	青枯菌种特异性 Ralstonia specific-primer	280^［13］
27F	GTCGCCGTCAACTCACTTTCC	16S rDNA	1 500^［16］
1492R	GGTTACCTTGTTACGACTT	16S rDNA	1 500^［16］
Endo-F	ATGCATGCCGCTGGTCGCCGC	egl基因 egl gene	750^［17］
Endo-R	GCGTTGCCCGGCACGAACACC	egl基因 egl gene	750^［17］

测定项目 Measuring item	供试菌株 Test strain	GSRS1	FQRS1	测定项目 Measuring item		供试菌株 Test strain	GSRS1	FQRS1
淀粉水解 Starch hydrolysis	弱Weak -	+ +	弱Weak -	硝酸盐还原 Nitrate reduction	封管 Sealing tube	+/产气 +/Gas production	+/产气 +/Gas production	+/产气 +/Gas production
明胶液化 Gelatin liquefaction	-	+	+	硝酸盐还原 Nitrate reduction	开管 Open tube	+	+	+
果胶酶水解 Hydrolysis of pectinase	+	+	+	耐盐性 Salt tolerance	1%NaCl	+	+	+
果聚糖 Fructan	-	+	+		1.5%NaCl	+	+	+
H₂S试验 H₂S production	+	-	-		2%NaCl	-	-	-
精氨酸水解 Arginine hydrolysis	-	-	-	生长温度 Growth temperature	35 ℃	+	+	+
氧化酶试验 Oxidase test	+	-	-		37 ℃	+	+	+
PHB的积累 PHB accumulate	+	+	+		40 ℃	+	+	+
水解七叶灵 Esculin hydrolysis	-	-	-	石蕊牛乳 Litmus milk		+	+	+
KB 产荧光 Produce fluorescence on KB medium	-	-	-	吲哚产物 Indole production		-	-	-
YDC培养基 YDC medium	白色 White	白色White	白色 White	3% KOH		+	+	+

测定项目 Measuring item	供试菌株 Test strain	GSRS1	FQRS1	测定项目 Measuring item		供试菌株 Test strain	GSRS1	FQRS1
淀粉水解 Starch hydrolysis	弱Weak -	+ +	弱Weak -	硝酸盐还原 Nitrate reduction	封管 Sealing tube	+/产气 +/Gas production	+/产气 +/Gas production	+/产气 +/Gas production
明胶液化 Gelatin liquefaction	-	+	+	硝酸盐还原 Nitrate reduction	开管 Open tube	+	+	+
果胶酶水解 Hydrolysis of pectinase	+	+	+	耐盐性 Salt tolerance	1%NaCl	+	+	+
果聚糖 Fructan	-	+	+		1.5%NaCl	+	+	+
H₂S试验 H₂S production	+	-	-		2%NaCl	-	-	-
精氨酸水解 Arginine hydrolysis	-	-	-	生长温度 Growth temperature	35 ℃	+	+	+
氧化酶试验 Oxidase test	+	-	-		37 ℃	+	+	+
PHB的积累 PHB accumulate	+	+	+		40 ℃	+	+	+
水解七叶灵 Esculin hydrolysis	-	-	-	石蕊牛乳 Litmus milk		+	+	+
KB 产荧光 Produce fluorescence on KB medium	-	-	-	吲哚产物 Indole production		-	-	-
YDC培养基 YDC medium	白色 White	白色White	白色 White	3% KOH		+	+	+

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