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| Species-specific PCR-based assays for identification and detection of Botryosphaeriaceae species causing stem blight on blueberry in China |
| XU Cheng-nan, ZHANG Hong-jun, CHI Fu-mei, JI Zhi-rui, DONG Qing-long, CAO Ke-qiang, ZHOU Zong-shan |
1、Research Institute of Pomology, Chinese Academy of Agricultural Sciences, Xingcheng 125199, P.R.China
2、College of Plant Protection, Agricultural University of Hebei, Baoding 071001, P.R.China |
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摘要 Botryosphaeriaceae species are important causal agents of blueberry stem blight worldwide. Blueberry stem blight has become an important disease, potentially affecting the quality and production of blueberries in China. It is difficult and time-consuming to identify at the species level using morphological methods. The aim of this study was to develop polymerase chain reaction (PCR) assays for the diagnosis and early detection of latent infections of blueberry stems by Botryosphaeria spp. Species-specific primers, based on the ribosomal DNA internal transcribed spacer region and β-tubulin gene, were designed and selected for use in PCR assays. Three primer pairs, Lt347-F/R for Lasiodiplodia theobromae, Np304-F/R for Neofusicoccum parvum and FaF/Bt2b for Botryosphaeria dothidea, successfully amplified specific PCR fragments of different sizes on pure cultures or from blueberry stems inoculated and naturally infected blueberry plants with three pathogens, respectively. These primers did not amplify any PCR fragments from other blueberry stem disease-associated pathogens, such as Phomopsis spp. and Pestalotiopsis spp. This PCR protocol could detect as low as 100 pg to 1 ng of purified fungal DNA. This PCR-based protocol could be used for the diagnosis and detection of these pathogens from pure cultures or from infected blueberry plants.
Abstract Botryosphaeriaceae species are important causal agents of blueberry stem blight worldwide. Blueberry stem blight has become an important disease, potentially affecting the quality and production of blueberries in China. It is difficult and time-consuming to identify at the species level using morphological methods. The aim of this study was to develop polymerase chain reaction (PCR) assays for the diagnosis and early detection of latent infections of blueberry stems by Botryosphaeria spp. Species-specific primers, based on the ribosomal DNA internal transcribed spacer region and β-tubulin gene, were designed and selected for use in PCR assays. Three primer pairs, Lt347-F/R for Lasiodiplodia theobromae, Np304-F/R for Neofusicoccum parvum and FaF/Bt2b for Botryosphaeria dothidea, successfully amplified specific PCR fragments of different sizes on pure cultures or from blueberry stems inoculated and naturally infected blueberry plants with three pathogens, respectively. These primers did not amplify any PCR fragments from other blueberry stem disease-associated pathogens, such as Phomopsis spp. and Pestalotiopsis spp. This PCR protocol could detect as low as 100 pg to 1 ng of purified fungal DNA. This PCR-based protocol could be used for the diagnosis and detection of these pathogens from pure cultures or from infected blueberry plants.
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Received: 29 May 2015
Accepted:
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| Fund: This research was supported financially by the National Natural Science Foundation of China (31301610). |
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Corresponding Authors:
ZHOU Zong-shan,Tel: +86-429-3598236, E-mail: zszhouqrj@163.com
E-mail: zszhouqrj@163.com
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| About author: XU Cheng-nan, Tel: +86-429-3598236, Mobile: +86-13998921065,E-mail: xuchengnan@caas.cn; |
Cite this article:
XU Cheng-nan, ZHANG Hong-jun, CHI Fu-mei, JI Zhi-rui, DONG Qing-long, CAO Ke-qiang, ZHOU Zong-shan.
2016.
Species-specific PCR-based assays for identification and detection of Botryosphaeriaceae species causing stem blight on blueberry in China. Journal of Integrative Agriculture, 15(3): 573-579.
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