|
|
|
|
|
| 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 |
|
|
|
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.
|
|
Received: 29 May 2015
Accepted: 09 March 2016
|
| Fund: This research was supported financially by the National Natural Science Foundation of China (31301610). |
|
Corresponding Authors:
ZHOU Zong-shan,Tel: +86-429-3598236, E-mail: zszhouqrj@163.com
E-mail: zszhouqrj@163.com
|
| 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.
|
| Abdollahzadeh J, Zolfaghari S. 2014. Efficiency of rep-PCRfingerprinting as a useful technique for molecular typingof plant pathogenic fungal species: Botryosphaeriaceaespecies as a case study. FEMS Microbiology Letters, 361,144-157Crous P W, Slippers B, Wingfield M J, Rheeder J, Marasas W F O, Philips A J L, Alves A, Burgess T, Barber P, Groenwald JZ. 2006. Phylogenetic lineages in the Botryosphaeriaceae.Studies in Mycology, 55, 235-253Denman S, Crous P W, Taylor J E, Kang J C, Pascoe I,Wingfield M J. 2000. An overview of the taxonomic historyof Botryosphaeria, and a re-evaluation of its anamorphsbased on morphology and ITS rDNA phylogeny. Studiesin Mycology, 45, 129-140Elfar K, Torres R, Díaz G A, Latorre B A. 2013. Characterizationof Diaporthe australafricana and Diaporthe spp. associatedwith stem canker of blueberry in Chile. Plant Disease, 97,1042-1050Espinoza J G, Briceño E X, Chávez E R, Úrbez-Torres J R,Latorre B A. 2009. Neofusicoccum spp. associated withstem canker and dieback of blueberry in Chile. PlantDisease, 93, 1187-1194Espinoza J G, Briceño E X, Keith L M, Latorre B A. 2008. Cankerand twig dieback of blueberry caused by Pestalotiopsisspp. and a Truncatella sp. in Chile. Plant Disease, 92,1407-1414Li L M, Wu L. 2011. The Research on Development of BlueberryIndustry in China. China Agriculture Press, China. (inChinese)Ma Z, Michailides T J. 2002. A PCR-based technique foridentification of Fusicoccum sp. from pistachio and variousother hosts in California. Plant Disease, 86, 515-520Martin R R, James D, Lévesque C A. 2000. Impacts of moleculardiagnostic technologies on plant disease management.Annual Review of Phytopathology, 38, 207-239Milholland R D. 1972. Histopathology and pathogenicityof Botryosphaeria dothidea on blueberry stems.Phytopathology, 62, 654-660Ni H F, Yang H R, Chen R S, Hung T H, Liou R F. 2012. Anested multiplex PCR for species-specific identificationand detection of Botryosphaeriaceae species on mango.European Journal of Plant Pathology, 133, 819-828Phillips A J L, Oudemans P V, Correia A, Alves A. 2006.Characterization and epitypification of Botryosphaeriacorticis the cause of blueberry cane canker. FungalDiversity, 21, 141-155Ridgway H J, Amponsah N T, Brown D S, Baskarathevan J, JonesE E, Jaspers M V. 2011. Detection of botryosphaeriaceousspecies in environmental samples using a multi-speciesprimer pair. Plant Pathology, 60, 1118-1127Sambrook J, Russell D W. 2001. Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, NY.Wang F. 2012. Identification, distribution, genetic diversity andPCR rapid detection of Botryosphaeria spp. causing peachtree gummosis in China. Ph D thesis, Huazhong AgriculturalUniversity, Wuhan, China. (in Chinese)Wang H, Qi M, Adrian J C. 1993. A simple method of preparingplant samples for PCR. Nucleic Acids Research, 21,4153-4154White T J, Bruns T, Lee S, Taylor J W. 1990. Amplificationand direct sequencing of fungal ribosomal RNA genesfor phylogenetic. In: Innis M A, Gelfand D H, Sninsky JJ, White T J, eds., PCR Protocols, a Guide to Methodsand Applications. Academics Press, San Diego. CA. pp.315-322Wright A F, Harmon P F. 2010. Identification of species in theBotryosphaeriaceae family causing stem blight on southernhighbush blueberry in Florida. Plant Disease, 94, 966-971Xu C N, Zhang H J, Zhou Z S, Hu T L, Wang S T, WangY N, Cao K Q. 2015. Identification and distribution ofBotryosphaeriaceae species associated with blueberry stemblight in China. European Journal of Plant Pathology, doi:10.1007/s10658-015-0724-6Xu C N, Zhou Z S, Chi F M, Wu Y X, Ji Z R, Zhang H J.2013. Research on the pathogen causing Botryosphaeriastem blight on blueberry. Acta Horticulturae Sinica, 40,231-236 (in Chinese)Xu C N, Zhou Z S, Zhang H J, Wu Y X, Chi F M. 2012.Identification of the pathogen causing stem canker ofblueberry. Acta Phytopathologica Sinica, 42, 532-535 (inChinese)Yu L, Impaprasert R, Xu S G, Wu X, Zhao J R. 2012. Firstreport of stem blight of blueberry caused by Botryosphaeriadothidea in China. Plant Disease, 96, 1697.Yu L, Impaprasert R, Zhao J R, Xu S G, Wu X. 2013. Stemdieback of high bush blueberries caused by Neofusicoccumparvum in China. New Disease Reports, 27, 3.Yue Q H, Zhao H H, Liang C, Li X D. 2013. The pathogencausing Phomopsis twig blight of blueberry. Mycosystema,32, 959-966 (in Chinese)Zhao H H, Yue Q H, Liang C. 2014. The pathogen causingPestalotiopsis twig dieback of blueberry. Mycosystema, 33,577–583. (in Chinese) |
| No Suggested Reading articles found! |
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
