Tracking and assessment of Puccinia graminis f. sp. tritici colonization on rice phyllosphere by integrated fluorescence
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ORIGINAL ARTICLE
Tracking and assessment of Puccinia graminis f. sp. tritici colonization on rice phyllosphere by integrated fluorescence imaging and qPCR for nonhost resistance phenotyping Narayanasamy Prabhakaran1 · Aundy Kumar1 · Neelam Sheoran1 · Vaibhav Kumar Singh1 · Perumal Nallathambi2 Received: 18 April 2020 / Accepted: 10 November 2020 © Deutsche Phytomedizinische Gesellschaft 2020
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. Keywords Stem rust · Pathogen quantitation · qPCR · Confocal laser scanning microscopy · Scanning electron microscopy · Nonhost resistance · Rice · Wheat
Introduction The stem rust pathogen, Puccinia graminis f. sp. tritici, is a major threat of wheat cultivation in India and other countries. The occurrence of the three distinct rust diseases in different countries or regions of the world varies in terms of type, distribution, and degree of damage Electronic supplementary material The online version of this article (https://doi.org/10.1007/s41348-020-00405-y) contains supplementary material, which is available to authorized users. * Aundy Kumar [email protected] 1
Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi 110012, India
ICAR-Indian Agricultural Research Institute, Regional Station, Wellington‑643 231, The Nilgiris, Tamil Nadu, India
2
(Chen 2005; Chen et al. 2014; Kutschera and Hossfeld 2012). In order to mitigate
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