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Novel specific primers for rapid identification of Macrophomina species

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Abstract

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.

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Acknowledgments

The first author thanks the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil) for a doctorate scholarship. A. R. Machado and K. C. Correia acknowledge the CNPq for financial support. S. J. Michereff also acknowledges the CNPq research fellowship.

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Authors and Affiliations

  1. Programa de Pós-Graduação em Fitopatologia, Universidade Federal Rural de Pernambuco, Recife, PE, Brazil

    Kledson M. Santos, Graziele S. Lima & Ana P. O. Barros

  2. Departamento de Micologia, Universidade Federal de Pernambuco, Recife, PE, Brazil

    Alexandre R. Machado & Cristina M. Souza-Motta

  3. Centro de Ciências Agrárias e da Biodiversidade, Universidade Federal do Cariri, Crato, CE, Brazil

    Kamila C. Correia & Sami Jorge Michereff

Authors
  1. Kledson M. Santos
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  2. Graziele S. Lima
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  3. Ana P. O. Barros
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  4. Alexandre R. Machado
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  5. Cristina M. Souza-Motta
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  6. Kamila C. Correia
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  7. Sami Jorge Michereff
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Corresponding author

Correspondence to Sami Jorge Michereff.

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All principles of ethical and professional conduct have been followed during this research and elaboration of this manuscript.

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The authors declare that they have no conflict of interest.

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Electronic supplementary material

Supplementary Table 1

(DOCX 15.4 kb)

Supplementary Table 2

(DOCX 17.2 kb)

Supplementary Table 3

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Supplementary Fig. 1

Detection of Macrophomina phaseolina, M. pseudophaseolina and M. euphorbiicola using the primer sets MpCalF/MpCalR, MsCalF/MsCalR and MeCalF/MeCalR, respectively. Lane M: DNA marker; lanes 2 to 6: M. phaseolina (MP) isolates CMM-2748, CMM-3540, CMM-3615, CMM-4048 and CMM-4149, respectively; lanes 7 to 11: M. pseudophaseolina (MS) isolates CMM-4029, CMM-4131, CMM-4155, CMM-4161, CMM-4231, respectively; lanes 12 to 16: M. euphorbiicola (ME) isolates CMM-2158, CMM-2718, CMM-4045, CMM-4134 and CMM-4145, respectively (PNG 938 kb)

High resolution image (TIF 796 kb)

Supplementary Fig. 2

Detection of Macrophomina phaseolina, M. pseudophaseolina and M. euphorbiicola using the primer sets MpTefF/MpTefR, MsTefF/MsTefR and MeTefF/MeTefR, respectively. Lane M: DNA marker; lanes 2 to 6: M. phaseolina (MP) isolates CMM-2748, CMM-3540, CMM-3615, CMM-4048 and CMM-4149, respectively; lanes 7 to 11: M. pseudophaseolina (MS) isolates CMM-4029, CMM-4131, CMM-4155, CMM-4161, CMM-4231, respectively; lanes 12 to 16: M. euphorbiicola (ME) isolates CMM-2158, CMM-2718, CMM-4045, CMM-4134 and CMM-4145, respectively (PNG 454 kb)

High resolution image (TIF 510 kb)

Supplementary Fig. 3

Evaluation of the specificity of the primer sets MpTefF/MpTefR, MsTefF/MsTefR and MeTefF/MeTefR in PCR assays using forty-two fungal isolates of the different genera (negative control). Lanes 1 to 42 Isolates in Table S3. Mp: M. phaseolina; Ms.: M. pseudophoseolina; Me: M. euphorbiicola (PNG 435 kb)

High resolution image (TIF 656 kb)

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Santos, K.M., Lima, G.S., Barros, A.P.O. et al. Novel specific primers for rapid identification of Macrophomina species. Eur J Plant Pathol 156, 1213–1218 (2020). https://doi.org/10.1007/s10658-020-01952-8

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