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Anti-Pythium activity of ZzAMP, a serine protease inhibitor (SPI) from Zingiber zerumbet rhizome, mediated by zoospore binding and oxidative stress

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Abstract

An abundance of protease inhibitors (PIs) in plant storage organs signifies their importance in plant defense. In below-ground storage tissues of resistant plants, PIs which are antimicrobial proteins (AMPs) stall host plant invasion, mediated by secretory proteases of necrotrophic soil-borne phytopathogens like Pythium spp., through competitive inhibition. A serine PI designated ZzAMP isolated from Zingiber zerumbet rhizomes was investigated for its inhibitory effect to zoospores of Pythium myriotylum, the etiological agent for soft rot disease, a major problem confronting ginger productivity. P. myriotylum zoospore inhibition by ZzAMP was observed to be optimal at pH 8.0 (79 ± 3.5%) with considerable stability at alkaline pH 12.0 (71 ± 1%). The optimal temperature yielding zoospore inhibition was determined as 45 °C (77.1 ± 0.2%) with protein stability observed over a wide range of temperatures from 25 to 65 °C. Peroxidase-dependent staining using 3, 3′-diaminobenzidine (DAB) to detect the generation of hydrogen peroxide (H2O2) by Pythium zoospores revealed browning of spores due to DAB stain uptake in ZzAMP treated spores compared to control, indicative of H2O2 burst. Localization experiments with fluorescein isothiocyanate (FITC)-labeled ZzAMP detected the labeled protein on the zoospore surface. Experiments thus revealed the mechanistic basis of zoospore inhibition by ZzAMP, which is of relevance considering the role of zoospores in mediating rapid disease spread and host invasion by Pythium species. The present study signifies the potential of ZzAMP in the development of formulations with applications in soft-rot disease control.

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Data availability

Data generated from this study are available from the corresponding author upon request.

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Acknowledgements

SR acknowledges the Research Fellowship received from Ministry of Human Resource and Development (MHRD), Government of India. Authors are grateful to NITC and CUK for the research facilities extended.

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Authors and Affiliations

  1. School of Biotechnology, National Institute of Technology Calicut, Calicut, 673 601, India

    Sharmila Raj

  2. Department of Biochemistry and Molecular Biology, Central University of Kerala (CUK), Kasaragod, Kerala, 671 320, India

    R. Aswati Nair

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  1. Sharmila Raj
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  2. R. Aswati Nair
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ARN conceived and designed the experiments. SR performed the experiments. SR and ARN analyzed the data. ARN wrote and revised the paper.

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Correspondence to R. Aswati Nair.

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Raj, S., Nair, R.A. Anti-Pythium activity of ZzAMP, a serine protease inhibitor (SPI) from Zingiber zerumbet rhizome, mediated by zoospore binding and oxidative stress. Eur J Plant Pathol 164, 429–437 (2022). https://doi.org/10.1007/s10658-022-02549-z

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