Novel specific primers for rapid identification of Macrophomina species

• Published in: European journal of plant pathology 2020-04, Vol.156 (4), p.1213-1218
• Main Authors: Santos, Kledson M., Lima, Graziele S., Barros, Ana P. O., Machado, Alexandre R., Souza-Motta, Cristina M., Correia, Kamila C., Michereff, Sami Jorge
• Format: Article
• Language: English
• Subjects: Actin; Agriculture; Amplification; Biomedical and Life Sciences; Calcium-binding protein; Calmodulin; Chemical synthesis; Ecology; Elongation; Geographical distribution; Host plants; Identification; Life Sciences; Phytopathogenic fungi; Plant Pathology; Plant Sciences; Polymerase chain reaction; Primers; Species; Translation elongation; Tubulin
• ISSN: 0929-1873; 1573-8469
• DOI: 10.1007/s10658-020-01952-8

Macrophomina is a widely distributed genus of phytopathogenic fungi with a wide range of plant hosts. The present study aimed to design specific primers for the rapid identification/detection of three Macrophomina species ( M. phaseolina , M. pseudophaseolina, and M. euphorbiicola ). The reference sequences of four nuclear genes actin (ACT), β-tubulin (βT), calmodulin (CAL) and translation elongation factor 1-alpha (TEF1-α) of each Macrophomina species were submitted for the generation of specific primers using automated software packages. The better specific primers set generated for detection of each species were selected and synthesized. Polymerase chain reaction (PCR)-based assays were conducted to verify the specificity with isolates of the three species of Macrophomina and 42 species of other genera. Three primer sets to amplify of regions CAL (MpCalF/MpCalR, MsCalF/MsCalR and MeCalF/MeCalR) and three primer sets to amplify of regions TEF-1α (MpTefF/MpTefR, MsTefF/MsTefR and MeTefF/MeTefR) were designed for M. phaseolina , M. pseudophaseolina, and M. euphorbiicola , respectively. The specific primers MpCalF/MpCalR from region CAL amplified only the isolates of M. phaseolina . However, the MsCalF/MsCalR and MeCalF/MeCalR amplified non-target isolates. The specific primers MpTefF/MpTefR, MsTefF/MsTefR, MeTefF/MeTefR from region TEF-1α, exhibited high specificity in amplifying only the target isolates. No fragment was detected from other fungal species tested, confirming high specificity of these primers. This is the first report to develop specific primers for the identification of M. phaseolina , M. pseudophaseolina, and M. euphorbiicola . The present study reveals that the primer sets can be used for molecular identification and will facilitate a large­scale survey of the distribution of species and monitoring the epidemics.