Abstract
Fluorescence microscopy and qPCR-based pathogen tracking tools were developed to elucidate the growth and development of Puccinia graminis f. sp. tritici on leaves of nonhost plant (rice: Oryza sativa L.). Stem rust race-Puccinia graminis f. sp. tritici 40A was identified using Internal Transcribed Spacer (ITS) sequences and gene sequences coding for hypothetical protein (PGTG_08233_hypothetical protein, HP). A reliable qPCR-based quantitation assay was developed for stem rust fungus exploiting HP primers that yielded 377-bp amplicon in conventional PCR and a forma specialis-specific 146 bp in qPCR with a detection sensitivity of 250-fg genomic DNA. The fungal cell wall N-acetyl-glucosamine was green fluorescence labelled and visualized on propidium iodide-stained nonhost leaf. This technique along with scanning electron microscopy allowed imaging of various developmental structures of Puccinia graminis f. sp. tritici 40A on plant epiphytic and endophytic niches. The microscopy coupled with qPCR-based pathogen load estimation revealed that the nonhost (rice) and host (wheat) phyllosphere surface supported uredospore germination, germ tube formation, hyphal elongation, epiphytic growth, stomatal entry and endophytic growth of fungus in an identical manner. However, the nonhost plant did not show any sign of rusting caused by uredospore production, which instead displayed induced H2O2 accumulation, on leaf. Development of qPCR-based Puccinia quantitation and green fluorescent tag-based qualitative assessment of Puccini proliferation will facilitate nonhost resistance phenotyping not only in rice but also in other nonhost plants of stem rust pathogen.
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The datasets during and/or analysed during the current study are available from the corresponding author on reasonable request (software application or custom code).
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Acknowledgements
The finding is an outcome of project (NFBSFARA/BS-4016/2013-14) funded by National Agricultural Science Fund, Indian Council of Agricultural Research, New Delhi. Various facilities provided by Directors, ICAR-IARI, New Delhi, are thankfully acknowledged.
Funding
The finding is an outcome of project (NFBSFARA/BS-4016/2013–14) funded by National Agricultural Science Fund, Indian Council of Agricultural Research, New Delhi.
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NP, NS, and AK conducted all experiments in particular qPCR and Fluorescence microscopy; PN, NP and VKS assisted plant inoculations; AK conceptualized the programme and supervised all experiments; NP and AK drafted and finalized the manuscript.
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Prabhakaran, N., Kumar, A., Sheoran, N. et al. Tracking and assessment of Puccinia graminis f. sp. tritici colonization on rice phyllosphere by integrated fluorescence imaging and qPCR for nonhost resistance phenotyping. J Plant Dis Prot 128, 457–469 (2021). https://doi.org/10.1007/s41348-020-00405-y
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DOI: https://doi.org/10.1007/s41348-020-00405-y