油茶根腐病原菌及其PCR快速检测技术研究
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摘要
油茶根腐病Camellia oleifera root rot是普遍危害油茶树的一种根部病害。受害油茶根部腐烂,导致植株不能正常吸收养分和水分而干枯死亡,给油茶生产带来了严重的威胁,根腐病已经成为我国油茶可持续发展的限制性因子之一。本实验以油茶根腐病为研究对象,进行病原菌的分离鉴定、生物学特性、巢式PCR快速检测等研究。
(1)油茶根腐病病原菌的分离与鉴定。采集了14份发病的油茶根腐病的样本,用组织分离法进行分离,结果分离到同一种病原菌。将分离得到的菌株采用有伤接种和无伤接种两种方法,结果表明：采用的有伤将接种和无伤接种结果均显示此病原菌具有较强的致病性,接种后植株发病与田间自然发病症状相同,并且可再次分离到与接种体相同的病原菌。通过对其形态特征的观察及该菌的核糖体DNA(rDNA)内转录间隔区ITS区并测定其序列,并与Genbank中经Blast搜索获得的部分菌株构建分子系统发育树。基于形态特征和分子系统发育树,最后确定引起油茶根腐病的病原菌为镰刀属李瑟组层生镰刀菌Fusarium proliferatum。
(2)油茶根腐病原菌层生镰刀菌菌株G9生物学特性的研究。研究结果表明该病原菌在人工培养条件,生长的最适温度为25℃；最适宜生长的pH值为5-7；光照对该病原菌的生长速率无明显影响。分生孢子在相对湿度为90%以下不能生长,在相对湿度为100%(水滴)中才能很好的萌发；病菌生长的致死温度为60℃10 min；病菌生长较好的碳源为可溶性淀粉,氮源为氯化铵。
(3)油茶根腐病菌层生镰刀菌G9的巢式PCR快速检测体系建立。扩增层生镰刀菌核糖体DNA ITS区并测定其序列,比较该序列与GenBank中近似种的ITS序列差异,设计了特异性引物G1和G2,利用该对引物与ITS1和ITS4进行巢式PCR扩增检测层生镰刀菌。巢式PCR对层生镰刀菌的检测灵敏度可达100 ag基因组DNA,比常规扩增方法提高了10000倍。利用设计的G1/G2特异性引物与ITS区通用引物进行巢式PCR扩增,可灵敏的扩增出油茶根腐病菌层生镰刀菌DNA。
本研究分离鉴定了引起油茶根腐病的病原菌层生镰刀菌,为正确用药、合理防治油茶根腐病提供了理论依据；对该菌的生物学特性进行研究,明确病原菌的生活习性及其对环境条件的要求,有利于制定出有效防治油茶根腐病的方案。同时本研究建立的巢式PCR检测方法,为油茶根腐病的早期诊断与防治、病害的预测预报提供了一条行之有效的技术手段。
Camellia oleifera root rot was one kind of root rots which usually jeopardized Camellia oleifera.The root of Camellia oleifera harmed came to rot,which leaded to the death of the plant because of the unusual sorbation of nutiaton and water and brought serious danger to Camellia oleifera production. It has been one of limiting factors to Sustainable Development of Camellia oleifera.In this paper, Camellia oleifera root rot was identified and biological characteristics,nested PCR disease dianosis techolgoy were studied.
The pathogens of Camellia oleifera root rot had been separated and identified.Collected 14 species plant culivars infected Camellia oleifera root rot and only one pathogen was isolated.The isolate was inoculated the healthy plant in two means.The results showed that the plant had the same symptom with the speciemen which had gathered from filed.Then isolated from the plant agina,the fungi were were the same pathogen which had isolated from specimen, the pathogenic fungi had strong pathogenicity. Morphological characteristics were expatiated and sequenced the strain of the rDNA internal transcribed spacer (ITS). The homologous strain in the Genbank were download, molecular phylogenetic tree was constructed. Based on morphological characteristics and molecular phylogenetic tree, it can be concluded that the pathogens is Fusarium moniliform.
The pathogen's characterization had been studied.It showed that the optimum temperature and pH for mycelial growth of the pathogen were 25℃and pH 5-7.Different lighting time had no significant effect on mycelium growth of Fusarium moniliform.Condia could not germinate if the relative humidity was lower than 90%,the geimation rate was the higest in the water drop.The lethal temperature was 60℃,10minutes, Starch is the best carbon nutrient,NH4Cl is the best nitrogen nutrient.
Rapid and accurate detection of the pathogenic of Root Rot of Camellia oleifera diseased plant tissues was developed in the study. Based on differences in internal transcribed spacer(ITS) sequences of Fusarium., a pair of species-specific primers, G1/G2 was synthesized. The others species of Fusarium were used to test the specificity of the primers. G1/G2 amplified only a unique 400 bp band from CSUFT070109 strain. The detection sensitivity with G1/G2 was 1 pg of genomic DNA in 25μL recation solution. A nested PCR procedure using ITS1/ITS4 as the first-round primers and followed with G1/G2 increased detection sensitivity 10000-fold to 100 ag. The results suggested that the assay detected the pathogen more rapidly and accurately than the standard isolation methods.The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring, as well as guiding plant disease management.
In this study,we identified the pathogen causing Camellia oleifera root rot as Fusarium moniliform.providing information to protect and control Camellia oleifera correctly.this pathogen's characterization was studied to make clear its surrounding and make proper programme how to control root rot efficiently. Besides,through the nested PCR dianosis techololigy studied we could also established one efficient methods for early disease dianosis,pest control,monitoring and forecasting of Camellia oleifera root rot
(1)油茶根腐病病原菌的分离与鉴定。采集了14份发病的油茶根腐病的样本,用组织分离法进行分离,结果分离到同一种病原菌。将分离得到的菌株采用有伤接种和无伤接种两种方法,结果表明：采用的有伤将接种和无伤接种结果均显示此病原菌具有较强的致病性,接种后植株发病与田间自然发病症状相同,并且可再次分离到与接种体相同的病原菌。通过对其形态特征的观察及该菌的核糖体DNA(rDNA)内转录间隔区ITS区并测定其序列,并与Genbank中经Blast搜索获得的部分菌株构建分子系统发育树。基于形态特征和分子系统发育树,最后确定引起油茶根腐病的病原菌为镰刀属李瑟组层生镰刀菌Fusarium proliferatum。
(2)油茶根腐病原菌层生镰刀菌菌株G9生物学特性的研究。研究结果表明该病原菌在人工培养条件,生长的最适温度为25℃；最适宜生长的pH值为5-7；光照对该病原菌的生长速率无明显影响。分生孢子在相对湿度为90%以下不能生长,在相对湿度为100%(水滴)中才能很好的萌发；病菌生长的致死温度为60℃10 min；病菌生长较好的碳源为可溶性淀粉,氮源为氯化铵。
(3)油茶根腐病菌层生镰刀菌G9的巢式PCR快速检测体系建立。扩增层生镰刀菌核糖体DNA ITS区并测定其序列,比较该序列与GenBank中近似种的ITS序列差异,设计了特异性引物G1和G2,利用该对引物与ITS1和ITS4进行巢式PCR扩增检测层生镰刀菌。巢式PCR对层生镰刀菌的检测灵敏度可达100 ag基因组DNA,比常规扩增方法提高了10000倍。利用设计的G1/G2特异性引物与ITS区通用引物进行巢式PCR扩增,可灵敏的扩增出油茶根腐病菌层生镰刀菌DNA。
本研究分离鉴定了引起油茶根腐病的病原菌层生镰刀菌,为正确用药、合理防治油茶根腐病提供了理论依据；对该菌的生物学特性进行研究,明确病原菌的生活习性及其对环境条件的要求,有利于制定出有效防治油茶根腐病的方案。同时本研究建立的巢式PCR检测方法,为油茶根腐病的早期诊断与防治、病害的预测预报提供了一条行之有效的技术手段。
Camellia oleifera root rot was one kind of root rots which usually jeopardized Camellia oleifera.The root of Camellia oleifera harmed came to rot,which leaded to the death of the plant because of the unusual sorbation of nutiaton and water and brought serious danger to Camellia oleifera production. It has been one of limiting factors to Sustainable Development of Camellia oleifera.In this paper, Camellia oleifera root rot was identified and biological characteristics,nested PCR disease dianosis techolgoy were studied.
The pathogens of Camellia oleifera root rot had been separated and identified.Collected 14 species plant culivars infected Camellia oleifera root rot and only one pathogen was isolated.The isolate was inoculated the healthy plant in two means.The results showed that the plant had the same symptom with the speciemen which had gathered from filed.Then isolated from the plant agina,the fungi were were the same pathogen which had isolated from specimen, the pathogenic fungi had strong pathogenicity. Morphological characteristics were expatiated and sequenced the strain of the rDNA internal transcribed spacer (ITS). The homologous strain in the Genbank were download, molecular phylogenetic tree was constructed. Based on morphological characteristics and molecular phylogenetic tree, it can be concluded that the pathogens is Fusarium moniliform.
The pathogen's characterization had been studied.It showed that the optimum temperature and pH for mycelial growth of the pathogen were 25℃and pH 5-7.Different lighting time had no significant effect on mycelium growth of Fusarium moniliform.Condia could not germinate if the relative humidity was lower than 90%,the geimation rate was the higest in the water drop.The lethal temperature was 60℃,10minutes, Starch is the best carbon nutrient,NH4Cl is the best nitrogen nutrient.
Rapid and accurate detection of the pathogenic of Root Rot of Camellia oleifera diseased plant tissues was developed in the study. Based on differences in internal transcribed spacer(ITS) sequences of Fusarium., a pair of species-specific primers, G1/G2 was synthesized. The others species of Fusarium were used to test the specificity of the primers. G1/G2 amplified only a unique 400 bp band from CSUFT070109 strain. The detection sensitivity with G1/G2 was 1 pg of genomic DNA in 25μL recation solution. A nested PCR procedure using ITS1/ITS4 as the first-round primers and followed with G1/G2 increased detection sensitivity 10000-fold to 100 ag. The results suggested that the assay detected the pathogen more rapidly and accurately than the standard isolation methods.The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring, as well as guiding plant disease management.
In this study,we identified the pathogen causing Camellia oleifera root rot as Fusarium moniliform.providing information to protect and control Camellia oleifera correctly.this pathogen's characterization was studied to make clear its surrounding and make proper programme how to control root rot efficiently. Besides,through the nested PCR dianosis techololigy studied we could also established one efficient methods for early disease dianosis,pest control,monitoring and forecasting of Camellia oleifera root rot
引文
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