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This volume provides a collection of molecular protocols detailing the most common and modern techniques on fusarium wilt. Chapters guide readers through methods on initial isolation, molecular-based identification, genome characterization, generation of mutants, and characterization of interactions with other organisms including host plants. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.
Authoritative and cutting-edge, Fusarium wilt: Methods and Protocols aims to be a valuable resource for mycologists, plant pathologists, microbiologists, geneticists, and other scientists that have an interest in members of the Fusarium oxysporum species complex or closely related fungi.
List of contents
1. Pure culture and DNA sequence-based identification of Fusarium from symptomatic plants and diverse substrates.- Isolation of high-molecular weight (HMW) DNA from Fusarium oxysporum for long-read sequencing.- Karyotyping of Fusarium oxysporum by pulsed field gel electrophoresis and the germ tube burst method.- RNA-seq data analysis for differential expression.- Quantification and isolation of spontaneous colony growth variants.- Generation of a T-DNA insertion mutant library of Fusarium oxysporum using Agrobacterium tumefaciens.- Targeted gene disruption via CRISPR/Cas9 ribonucleoprotein complexes in Fusarium oxysporum.- CRISPR/Cas9 RNP-mediated gene fusion to assess protein quantification and subcellular localization in Fusarium oxysporum.- In vivo monitoring of cytosolic pH using the ratiometric pH sensor pHluorin.- Multi-pronged investigation of volatile compound-mediated interactions of Fusarium oxysporum with plants, fungi and bacteria.- Isolation of mitochondria from Fusarium species.- In planta gene expression analysis and colonization of Fusarium oxysporum.- Time-lapse imaging of root pathogenesis and fungal proliferation without physically disrupting roots.- High-throughput screening assays to identify plant natural products with antifungal properties against Fusarium oxysporum.- Screening and assessment of pisatin demethylase activity (PDA).-Detection and genotyping of Fusarium oxysporum f. sp. vasinfectum race 4 (VCG0114), the Fusarium wilt pathogen of cotton.
