qPCR-based detection of Colletotrichum truncatum in soybean seeds
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qPCR-based detection of Colletotrichum truncatum in soybean seeds Manoel B. S. Júnior 1 & Mário L. V. Resende 1 & Edson A. Pozza 1 & Deila M. S. Botelho 1 & Acleide M. S. Cardoso 1 & Carolina S. Siqueira 1 & José C. Machado 1 & Alexandre R. M. Resende 1 & Gustavo C. D. Silveira 1 & Sarah S. C. Guimarães 1 Received: 6 February 2020 / Accepted: 8 June 2020 # Sociedade Brasileira de Fitopatologia 2020
Abstract Soybean seed infected with Colletotrichum truncatum is an important source of primary inoculum for anthracnose epidemics. Based on differences in the GAPDH gene sequences of Colletotrichum species, one pair of species-specific primers, CtruncF1/ CtruncR1, was designed to accurately detect C. truncatum in soybean seed samples. The primers amplified only a single PCR band of 211 bp from C. truncatum. SYBR Green qPCR using these primers enabled the detection of DNA of the target fungus in inoculated soybean seeds and in naturally infested seeds. The sensitivity of the method was 0.000253 ng/μL of C. truncatum DNA template, with an efficiency of 1.78 and a Ct of 30.09. These species-specific primers may be useful for certification of soybean seeds aimed at avoiding introduction of the pathogen into soybean-producing regions. Keywords Glycine max . Diagnostic . Molecular detection . Plant disease . Specific primers
Soybean (Glycine max L.) is the main grain crop in Brazil, with total planted area of 35 million hectares and production of 96 million bags (CONAB 2018). Anthracnose, caused by Colletotrichum truncatum, is considered one of the most frequent and harmful diseases that affect soybeans from seeds to adult plants, with infected soybean seeds being the main source of inoculum for the introduction and spread of the pathogen (Rogério et al. 2016). The disease reduces crop yield given the ability of the pathogen to affect seed germination and cause necrosis on petioles and veins, and spots on leaves, stems, and pods (Galli et al. 2007; Silva et al. 2012). Yield losses due to anthracnose have been estimated in up to 100% under conditions of temperature and humidity favorable for anthracnose epidemics to develop (Galli et al. 2007; Silva et al. 2012). Dias et al. (2016) found a significant association between soybean and incidence of soybean anthracnose in the pods in a 2-year study (r = − 0.85); on average, a reduction of 90 kg/h was estimated for every percent point increase in disease incidence.
* Sarah S. C. Guimarães [email protected] 1
Departamento de Fitopatologia, Universidade Federal de Lavras, Lavras, MG 37200-000, Brazil
The use of clean (pathogen-free) seeds is an important strategy in disease management in order to avoid or diminish the spread of pathogens. Therefore, accurate, sensitive, and fast diagnostic tools are needed to support seed phytosanitary certification. Species-specific primers in PCR assays can be used in seed certification program not only for presence/ absence result but also to provide a quantitative measure of the pathogens in seeds, other plant parts
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