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Development and evaluation of loop-mediated isothermal amplification assay for rapid and sensitive detection of potato cyst nematode, Globodera pallida from soil

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Abstract

Potato cyst nematodes, Globodera pallida and G. rostochiensis, are economically important and difficult to manage pests of the potato crop. The cyst of both the species looks similar and it is difficult to differentiate once it turns brown upon maturity. Early detection of the PCN at the species level is crucial to avoid its further spread and for adopting the appropriate management strategies. Therefore, in the present study, highly specific and sensitive loop-mediated isothermal amplification (LAMP) assay was developed to amplify mitochondrial-Sequence Characterized Amplified Region (SCAR) sequence of potato cyst nematode, G. pallida. The LAMP assay was completed within a shorter incubation period of 60 min at 60 °C followed by the reaction termination at 80 °C for 5 min. The developed LAMP assay exhibited high specificity for G. pallida and did not detect any other species including its sibling species, G. rostochiensis. In sensitivity tests, the assay detected G. pallida at 1000 times less DNA concentration (10 fg/µl) as compared to conventional PCR (10 pg/µl). In addition to this, the developed LAMP assay was tested for the detection of G. pallida directly from the soil samples, and even a single cyst mixed with soil was successfully detected by the developed assay. Moreover, the utility of low-cost instruments like hot water bath was also demonstrated for the detection of G. pallida from the soil. The developed LAMP is a rapid, highly specific, sensitive, and cost-effective technique for the species-specific detection of G. pallida. The developed assay will facilitate the rapid detection of G. pallida at quarantine stations as well as from the fields which will help to stop its further spread in new areas and also to devise effective management strategies for sustainable potato production.

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Funding

This research was funded by Indian Council of Agricultural Research-Central Potato Research Institute, Shimla under the institute code number “HORTCPRICIL201500600134” entitled “Biology, epidemiology, modeling and management of insect pests, vectors and cyst nematodes of potato”.

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Authors and Affiliations

  1. ICAR-Central Potato Research Institute, Shimla, 171 001, Himachal Pradesh, India

    Aarti Bairwa, Bhawna Dipta, Gaurav Verma, Kailash C. Naga, Sanjeev Sharma & Deepali Thakur

  2. ICAR-Central Potato Research Institute, Regional Station, The Nilgiris, Udhagamandalam, 643004, Tamil Nadu, India

    E. P. Venkatasalam & Priyank Hanuman Mhatre

  3. Department of Nematology, Tamil Nadu Agricultural University, Coimbatore, 641003, Tamil Nadu, India

    A. Shanthi

  4. ICAR-Indian Institute of Spices Research, Kozhikode, 673012, Kerala, India

    A. Jeevalatha

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  1. Aarti Bairwa
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  4. E. P. Venkatasalam
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Contributions

AB, GV, EPV, and PHM conceptualized the research. AB, BD, KCN, EPV, PHM, DT, and AS collected required experimental materials and performed the experiments. AB and EPV wrote the first draft of the manuscript. SS and AJ edited and reviewed the manuscript. PHM reviewed, revised, and redrafted the manuscript.

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Correspondence to Priyank Hanuman Mhatre.

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13205_2023_3542_MOESM1_ESM.tif

Supplementary file 1: Figure 1. Species-specific detection of Globodera pallida using newly designed forward (Gp-F3) and backward (Gp-B3) primers, Lane 1, agarose gel showing species-specific PCR amplification of approximately 187 bp sized amplicon for Globodera pallida; Lane 2, G. rostochiensis; Lane 3, no-template water control.

13205_2023_3542_MOESM2_ESM.tif

Supplementary file 2: Figure 2. Comparison of Globodera pallida mitochondrial-SCAR sequence with mitochondrial-CO I gene of different plant parasitic nematodes shows the specificity of designed primers. The position of LAMP primers designed for Globodera pallida mitochondrial-SCAR gene: Gp-F3 (forward outer primer); Gp-FIP (forward inner primer, a conjugated primer of F2 and a complementary sequence i.e., F1c); Gp-B3 (Backward outer primer), Gp-BIP (Backward inner primer, a conjugated primer of B2 and a complementary sequence i.e., B1c).

Supplementary file 3: (DOCX 15 KB)

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Bairwa, A., Dipta, B., Verma, G. et al. Development and evaluation of loop-mediated isothermal amplification assay for rapid and sensitive detection of potato cyst nematode, Globodera pallida from soil. 3 Biotech 13, 123 (2023). https://doi.org/10.1007/s13205-023-03542-x

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