4.5 Article

Real-time PCR for detection and quantification of C. gloeosporioides s.l. growth in Stylosanthes and Arabidopsis

期刊

CROP PROTECTION
卷 159, 期 -, 页码 -

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ELSEVIER SCI LTD
DOI: 10.1016/j.cropro.2022.106021

关键词

Anthracnose; Colletotrichum gloeosporioides; Stylosanthes; Arabidopsis; Latent infection; qRT-PCR

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资金

  1. Key Research and Development Projects of Hainan Province [ZDYF2020211]
  2. Young Elite Scientists Sponsorship Program by CAST [2020QNRC001]
  3. Young Talents' Science and Technology Innovation Project of Hainan Association for Science and Technology [QCXM202001]
  4. National Natural Science Foundation of China [31872409]

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In this study, a real-time PCR assay based on ACT was developed for the detection and quantification of Colletotrichum gloeosporioides s.l. The assay displayed high specificity, allowing early detection at the symptomless phase of infection, and showed good correlation with microscopic observation and later disease symptoms. The method provides a rapid and effective detection tool for studying plant-Colletotrichum interactions, disease prediction, and control.
Colletotrichum gloeosporioides sensu lato (s.l.) is one of the most economically and scientifically important fungal pathogens, causing serious diseases in tropical and subtropical region. The detection of C. gloeosporioides s.l. is of importance for disease control. In this study, an ACT-based real-time PCR assay was developed for quantification of C. gloeosporioides s.l., which reliably detected as little as 100 fg genomic DNA, 100 copies of target DNA and 20 conidia. This method could recognize all four tested C. gloeosporioides s.l. isolates, while no amplification was observed in other Colletotrichum species and Botrytis cinerea, indicating the specificity of this assay. Detection and quantification of C. gloeosporioides s.l. was demonstrated in artificially and naturally infected host leaves. First, the real-time PCR analysis was performed using leaf samples collected at different time points post inoculation to monitor the growth of C. gloeosporioides s.l. over time. Secondly, the method was used to compare the resistance in two Stylosanthes cultivars and two Arabidopsis cultivars. Finally, naturally infected and symptomless leaves of Stylosanthes in the fields were tested by the real-time PCR method. Overall, the real-time PCR assay could allow the detection at early symptomless phase of infection; and the results was well correlated with microscopic observation and later disease symptoms. Therefore, our studies have provided a rapid and effective detection method for studying the plant-Colletotrichum interaction as well as disease prediction and control.

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