油茶炭疽病检验检测技术研究
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摘要
油茶炭疽病是油茶树的主要病害之一,在我国长江流域以南各省的大面积油茶栽培区,特别是在我国油茶中心分布区的湖南、江西和广西等省发生普遍。油茶炭疽病害发生后,引起严重落果、落蕾、枝梢枯死,甚至整株衰亡,造成重大经济损失。本文系统分析了油茶炭疽病原菌胶孢炭疽菌的风险性并提出风险管理措施,研究了油茶炭疽病原菌形态学鉴定以及分子生物学快速检测方法,初步制备出油茶炭疽病原菌快速检测试剂盒。
(1)油茶炭疽病原菌风险性分析
我国有害生物的危险性综合评价标准分为4级,其中R值3.0～2.5为特别危险,2.4～2.0为高度危险,1.9～1.5为中度危险,1.4～1.0为低度危险,通过有害生物风险性分析,油茶炭疽菌胶孢炭疽菌的R值是1.55,这个结果说明其在我国南方属于中度危险的森林有害生物,我国正大力发展油茶产业,应将其列为油茶中心产区及周边地区的检疫对象。加强对油茶炭疽病菌的检疫力度,对苗木调运进行严格的检疫调运要求,阻止该菌扩散；同时加强检疫宣传,提高国民检疫意识,使茶农以及苗木商能自觉遵守检疫规程。在气候条件适宜该菌生存的地区,组织人员对苗木生产进行专门管理,管好源头。
(2)油茶炭疽病原菌形态学鉴定
通过繁殖器官的类型以及分生孢子梗的着生方式确定油茶炭疽病原菌所属黑盘孢目,油茶炭疽病菌的孢子为单细胞、无色,确定其为无色单孢族,根据刚毛着生位置,分生孢子形状,可确定其为刺盘孢菌属,根据孢子形态,分生孢子盘大小,以及刚毛的着生位置,结合油茶炭疽病引起病害的症状,最后确定油茶炭疽病菌为茶赤叶枯刺盘孢菌,即为胶孢炭疽菌。
(3)油茶炭疽病原菌分子生物学快速检测技术
根据ITS序列差异设计了油茶炭疽菌的特异性引物,对不同地理来源的13个菌株进行检测,均可扩增出约330bp的条带,对炭疽属胶孢炭疽菌以外21种炭疽菌均扩增不出条带。利用特异性引物常规PCR扩增可检测含量为lpg/25μL的病原菌,巢式PCR检测灵敏度为10 ag/25μL,检测灵敏度至少提高了100000倍,可以满足实际的需求。该方法可以在病害潜伏期,初发期进行病菌DNA的PCR扩增,迅速地鉴定出病原物的种类并可进行油茶炭疽病发病植株的病害诊断。以引物YT1和YT2作为检测油茶炭疽病原菌的特异性引物,使用巢式PCR技术,结合油茶组织总DNA的提取方法,初步制备出油茶无症状材料的炭疽病原菌快速检测试剂盒。
Camellia anthracnose mainly occurred in the China's Camellia producing areas in south of Changjiang river. Especially in the central producing areas, such as Hunan, Jiangxi and Guangxi provinces, it occurred previously. It causes serious drop in buds and fruits, sometimes resulting in the death of the branches and trunk, and caused the enormous economic losses. In this paper we systems analysis the risk of Colletotrichum gloeosporioides and brought forward the risk management measures. We also studied the morphological identification of pathogen and the rapid detection of molecular biology techniques, and then prepared rapid detection kit preliminarily.
(1) Risk analysis and risk management
Referring to the integrated evaluation of risk of other pest in China, criticality was grated to 4 as follow:R value (3.0-2.5) was regarded as most risk, R value (2.4-2.0) was regarded as high risk, R value (1.9-1.5) was regarded as middle risk, R value (1.4-1.0) was regarded as low risk. R value of Colletotrichum gloeosporioides was 1.55, it revealed that this pest had been graded to middle risk of forest pest. Especially as the pathogeny of Camellia anthracnose, Colletotrichum gloeosporioides should be as the quarantine objects in Hunan Province and surrounding areas. Strengthen the quarantine of Colletotrichum gloeosporioides in allocation and transportation of seedlings, prevent its distribution. At the same time strengthen propaganda, improve consciousness, let the famer and business abide by quarantine regulations. At the areas which climate is suit for Colletotrichum gloeosporioides, it should to manage the seedling produce.
(2) Morphologic identification
According the type of propagate organic and grow position of conidiophore, the pathogen of Camellia anthracnose have been identified as Melanconiales. Conidiophor of the pathogen were single cells, colorless. According the grow position of setae and the shape of conidiophor, it was sure as Colletotrichum. According the shape and size of conidiophor, the grow position of setae, and combined the disease symptom, it was finally identified the pathogen of Camellia anthracnose was Colletotrichum gloeosporioides.
(3) Rapid detection of molecular biology techniques
Based on differences in internal transcribed space (ITS) sequences of Camellia anthracnose, a pair of species-specific primers, YT1 and YT2, was synthesized. By using primers, we detected 13 strains come from different areas, all can be amplified a 300bp band. But the others 21 strains which were not belong to Colletotrichum gloeosporioides can't be amplified any bands. Using general PCR amplification it can detect pathogen which content was 1pg/25 u L, but by the nest-PCR the content of pathogen was only 10 ag/25 u L can be detected, the detection sensitivity increased 100000 times. The nest-PCR can amplify pathogen DNA at the latent period or incipient stage by PCR, and quickly identify the species of pathogen and diagnose disease of attacked plants by Camellia anthracnose. Using YT1 and YT2 specific primers and the nest-PCR technique, and combined with the DNA extraction methods of Oil Camellia tissue, we prepared the rapid detection kit preliminarily.
(1)油茶炭疽病原菌风险性分析
我国有害生物的危险性综合评价标准分为4级,其中R值3.0～2.5为特别危险,2.4～2.0为高度危险,1.9～1.5为中度危险,1.4～1.0为低度危险,通过有害生物风险性分析,油茶炭疽菌胶孢炭疽菌的R值是1.55,这个结果说明其在我国南方属于中度危险的森林有害生物,我国正大力发展油茶产业,应将其列为油茶中心产区及周边地区的检疫对象。加强对油茶炭疽病菌的检疫力度,对苗木调运进行严格的检疫调运要求,阻止该菌扩散；同时加强检疫宣传,提高国民检疫意识,使茶农以及苗木商能自觉遵守检疫规程。在气候条件适宜该菌生存的地区,组织人员对苗木生产进行专门管理,管好源头。
(2)油茶炭疽病原菌形态学鉴定
通过繁殖器官的类型以及分生孢子梗的着生方式确定油茶炭疽病原菌所属黑盘孢目,油茶炭疽病菌的孢子为单细胞、无色,确定其为无色单孢族,根据刚毛着生位置,分生孢子形状,可确定其为刺盘孢菌属,根据孢子形态,分生孢子盘大小,以及刚毛的着生位置,结合油茶炭疽病引起病害的症状,最后确定油茶炭疽病菌为茶赤叶枯刺盘孢菌,即为胶孢炭疽菌。
(3)油茶炭疽病原菌分子生物学快速检测技术
根据ITS序列差异设计了油茶炭疽菌的特异性引物,对不同地理来源的13个菌株进行检测,均可扩增出约330bp的条带,对炭疽属胶孢炭疽菌以外21种炭疽菌均扩增不出条带。利用特异性引物常规PCR扩增可检测含量为lpg/25μL的病原菌,巢式PCR检测灵敏度为10 ag/25μL,检测灵敏度至少提高了100000倍,可以满足实际的需求。该方法可以在病害潜伏期,初发期进行病菌DNA的PCR扩增,迅速地鉴定出病原物的种类并可进行油茶炭疽病发病植株的病害诊断。以引物YT1和YT2作为检测油茶炭疽病原菌的特异性引物,使用巢式PCR技术,结合油茶组织总DNA的提取方法,初步制备出油茶无症状材料的炭疽病原菌快速检测试剂盒。
Camellia anthracnose mainly occurred in the China's Camellia producing areas in south of Changjiang river. Especially in the central producing areas, such as Hunan, Jiangxi and Guangxi provinces, it occurred previously. It causes serious drop in buds and fruits, sometimes resulting in the death of the branches and trunk, and caused the enormous economic losses. In this paper we systems analysis the risk of Colletotrichum gloeosporioides and brought forward the risk management measures. We also studied the morphological identification of pathogen and the rapid detection of molecular biology techniques, and then prepared rapid detection kit preliminarily.
(1) Risk analysis and risk management
Referring to the integrated evaluation of risk of other pest in China, criticality was grated to 4 as follow:R value (3.0-2.5) was regarded as most risk, R value (2.4-2.0) was regarded as high risk, R value (1.9-1.5) was regarded as middle risk, R value (1.4-1.0) was regarded as low risk. R value of Colletotrichum gloeosporioides was 1.55, it revealed that this pest had been graded to middle risk of forest pest. Especially as the pathogeny of Camellia anthracnose, Colletotrichum gloeosporioides should be as the quarantine objects in Hunan Province and surrounding areas. Strengthen the quarantine of Colletotrichum gloeosporioides in allocation and transportation of seedlings, prevent its distribution. At the same time strengthen propaganda, improve consciousness, let the famer and business abide by quarantine regulations. At the areas which climate is suit for Colletotrichum gloeosporioides, it should to manage the seedling produce.
(2) Morphologic identification
According the type of propagate organic and grow position of conidiophore, the pathogen of Camellia anthracnose have been identified as Melanconiales. Conidiophor of the pathogen were single cells, colorless. According the grow position of setae and the shape of conidiophor, it was sure as Colletotrichum. According the shape and size of conidiophor, the grow position of setae, and combined the disease symptom, it was finally identified the pathogen of Camellia anthracnose was Colletotrichum gloeosporioides.
(3) Rapid detection of molecular biology techniques
Based on differences in internal transcribed space (ITS) sequences of Camellia anthracnose, a pair of species-specific primers, YT1 and YT2, was synthesized. By using primers, we detected 13 strains come from different areas, all can be amplified a 300bp band. But the others 21 strains which were not belong to Colletotrichum gloeosporioides can't be amplified any bands. Using general PCR amplification it can detect pathogen which content was 1pg/25 u L, but by the nest-PCR the content of pathogen was only 10 ag/25 u L can be detected, the detection sensitivity increased 100000 times. The nest-PCR can amplify pathogen DNA at the latent period or incipient stage by PCR, and quickly identify the species of pathogen and diagnose disease of attacked plants by Camellia anthracnose. Using YT1 and YT2 specific primers and the nest-PCR technique, and combined with the DNA extraction methods of Oil Camellia tissue, we prepared the rapid detection kit preliminarily.
引文
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