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Tracking and assessment of Puccinia graminis f. sp. tritici colonization on rice phyllosphere by integrated fluorescence imaging and qPCR for nonhost resistance phenotyping
Journal of Plant Diseases and Protection ( IF 2 ) Pub Date : 2020-11-25 , DOI: 10.1007/s41348-020-00405-y
Narayanasamy Prabhakaran , Aundy Kumar , Neelam Sheoran , Vaibhav Kumar Singh , Perumal Nallathambi

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.



中文翻译:

跟踪和评估小麦条锈菌f。sp。整合荧光成像和qPCR技术在水稻叶缘小麦的定殖中进行非寄主抗性表型分析

开发了荧光显微镜和基于qPCR的病原体跟踪工具来阐明Puccinia graminis f的生长和发育。sp。非寄主植物的叶片上的小麦(大米:水稻)。茎锈锈病-Puccinia graminis f。sp 使用内部转录间隔区(ITS)序列和编码假设蛋白(PGTG_08233_hypothetical protein,HP)的基因序列鉴定了tritici 40A。利用HP引物开发了一种可靠的基于qPCR的定量分析方法,用于干锈菌,该引物在常规PCR中产生377 bp的扩增子,在qPCR中产生特殊形式的特异性146 bp,检测灵敏度为250 fg基因组DNA。真菌细胞壁N-乙酰基-氨基葡萄糖经过绿色荧光标记,并在碘化丙啶染色的非寄主叶片上可视化。这项技术与扫描电子显微镜一起,可以对小麦条锈菌的各种发育结构进行成像。sp 。Tritici 40A对植物的附生和内生生态位。显微镜结合基于qPCR的病原体负荷估算显示,非寄主(大米)和寄主(小麦)的叶球表面以相同的方式支持真菌的孢子萌发,胚管形成,菌丝伸长,附生生长,气孔进入和内生生长。然而,非寄主植物没有显示出由孢子孢子产生引起的任何生锈迹象,而是表现出诱导的H 2 O 2。积累,在叶子上。基于qPCR的Puccinia定量分析和基于绿色荧光标记的Puccini增殖定性评估的发展将不仅促进水稻,而且还促进茎锈病病原体的其他非寄主植物的非寄主抗性表型。

更新日期:2020-11-25
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