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Detection of pea wilt pathogen Fusarium oxysporum f. sp. pisi using DNA-based markers

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Abstract

Identification of the fungus Fusarium oxysporum f. sp. pisi (Fop), the causal organism of wilt disease of pea, is a time consuming and arduous task. Diagnosis of Fop by traditional means requires more than 2 months and involves two steps, identification of species using morphological characters and formae specialis ‘pisi’ using pathogenicity assays. The ambiguous morphological differences between F. solani and F. oxysporum further complicate the diagnosis of F. oxysporum. A polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) based method was developed to detect Fop from India. A PCR–RFLP marker, HPACAPS1380, generated after restriction of 28S rDNA region with enzyme MvaI, detected accurately the Fop among several other fungi with detection sensitivity of 5 fg of Fop genomic DNA. In a mixture of Fop and pea DNA, the sensitivity was 500 pg of Fop DNA in 50 ng of pea DNA. The assay was further refined to detect the Fop from infected tissues and infested soil. The current assay can detect Fop from culture, plant tissues and soil in a considerably shorter period of time compared to traditional methods.

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Abbreviations

CTAB:

Cetyltrimethylammonium bromide

dNTP:

Deoxynucleotide triphosphate

EDTA:

Ethylenediamine tetraacetic acid

fg:

Fentagram

Fsp :

Fusarium solani f. sp. pisi

IGS:

Intergenic spacer

ITS:

Internal transcribed spacer

PCR–RFLP:

Polymerase chain reaction–restriction fragment length polymorphism

rDNA:

Ribosomal DNA

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Acknowledgements

The authors acknowledge financial grant received from the Department of Biotechnology, GOI, New Delhi to carry out the present study. We also thank Dr. S. Pande, International Crop Research Centre for Semi-Arid Tropics, Pattencheru, Hyderabad, for isolates of F. oxysporum f. sp. ciceris and Dr. Patil from University of Agricultural Sciences and Technology, Rahuri, Maharashtra for isolates of F. oxysporum f. sp. ciceris and F. oxysporum f. sp. udum and Dr. Anita Gotmore, Division of Crop Improvement, Central Institute for Cotton Research, Shankarnagar, Nagpur, Maharashtra for isolates of F. oxysporum f. sp. vasinfectum.

Funding

This study was funded partially by Grant No. BT/PR5599/AGR/02/282/2004 from Department of Biotechnology, Government of India, New Delhi.

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Authors and Affiliations

  1. Department of Agricultural Biotechnology, CSK Himachal Pradesh Agricultural University, Palampur, HP, 176 062, India

    Kamal Dev Sharma,  Hemlata, Rajeev Rathour & R. K. Kapila

  2. Department of Plant Pathology, CSK Himachal Pradesh Agricultural University, Palampur, HP, 176 062, India

    Y. S. Paul

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  1. Kamal Dev Sharma
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  2. Hemlata
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Correspondence to Kamal Dev Sharma.

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Sharma, K.D., Hemlata, Rathour, R. et al. Detection of pea wilt pathogen Fusarium oxysporum f. sp. pisi using DNA-based markers. J. Plant Biochem. Biotechnol. 27, 342–350 (2018). https://doi.org/10.1007/s13562-018-0443-0

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