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Multiplex PCR based detection method for Venturia species infecting pome and stone fruits

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Abstract

There are about 290 Venturia species listed in the mycobank database. Among them, a few are important and cause notable damage to pome and stone fruits. The most important Venturia spp. are V. inaequalis (apple scab), V. pyrina (pear scab), and V. carpophila (peach and almond scab). Species characterization in the genus Venturia is difficult because of overlapping morphological characteristics. It is necessary to develop sensitive and robust detection tools for the important Venturia species. Thirty-seven primers from V. inaequalis, V. pyrina, and V. carpophila genomes were designed. Many primers showed cross transferability and a few failed to generate any PCR amplicon. The pathogens V. inaequalis, V. pyrina, and V. carpophila are detected using Vi1, Pr3, and C4 primers, respectively. The Venturia inaequalis specific primer (Vi1) can also detected apple scab infection in the leaves. These species-specific primers were analyzed for the simultaneous detection of three Venturia spp. in a multiplex PCR. Real time PCR showed that these primers were able to detect the target DNA up to 0.001 ng concentration. The present study was conducted for the first time to develop species-specific primers for three important Venturia spp. Our primer set detected V. inaequalis, V. pyrina, and V. carpophila in a single multiplex PCR. The scientific community can now use these specific primers for the early detection of scab infection and species identification in pome and stone fruits.

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Acknowledgements

First author is thankful to Division of Plant Pathology, SKUAST-Kashmir, Shalimar for providing the necessary laboratory facility to carry out the research. The corresponding author is thankful to James D. Kelly (Distinguished Professor), Halima A. Awale and Nobel Amanda (Department of Plant Soil and Microbial Sciences, Michigan State University) for critical reading of the manuscript. This study was partially funded by Department of Biotechnology, Government of India New Delhi for providing financial assistance (Grant No:BT/PR17344/AGII/106/1005/2016).

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Authors and Affiliations

  1. Division of Plant Pathology, Faculty of Agriculture, Wadura Sopore, SKUAST-Kashmir, Srinagar, 193201, India

    Mohmmad Saleem Dar & Asha Nabi

  2. Directorate of Extension, SKUAST-Kashmir, Shalimar, Srinagar, 190025, India

    Mushtaq Ahmad

  3. Plant Virology and Molecular Plant Pathology Laboratory, Division of Plant Pathology, SKUAST-Kashmir, Shalimar, Srinagar, 190025, India

    Mahiya-Farooq, Mehraj D. Shah & Bilal A. Padder

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  1. Mohmmad Saleem Dar
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Correspondence to Bilal A. Padder.

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Supplementary file1 (DOCX 21 KB)

42360_2022_529_MOESM2_ESM.jpg

Supplementary Fig. 1. Designed primers showing cross amplification between different Venturia species. V. pyrina Pr4 primer showing cross amplification with C. humile, (1a); V. carpophila C2 and C3 primers show cross transferability with V. pyrina and C. humile (1b and1c); V. inaequalis Vi3 primer shows multiple banding pattern in other Venturia spp. (Fig. 1d) (JPG 311 KB)

42360_2022_529_MOESM3_ESM.jpg

Supplementary Fig. 2. PCR based validation of Venturia species-specific primer sets. V. carpophila primer C4 shows specificity in almond and peach scab isolates (2a, 2b); Species specificity of V. pyrina Pr3 primer (2c, 2d); V. inaequalis Vi1 primer shows specificity to Apple and Cotoneaster scab isolates (2e, 2f) (JPG 1984 KB)

42360_2022_529_MOESM4_ESM.tif

Supplementary Fig. 3. In planta PCR based specificity confirmation of V. inaequalis specific Vi1 primer using DNA extracted from infected and uninfected apple leaves as a template (TIF 2973 KB)

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Dar, M.S., Ahmad, M., Mahiya-Farooq et al. Multiplex PCR based detection method for Venturia species infecting pome and stone fruits. Indian Phytopathology 75, 941–950 (2022). https://doi.org/10.1007/s42360-022-00529-1

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