The Application of Molecular Methods in Diagnostics of Phytopathogenic Viruses, Fungi and Fungus-Like Organisms
Chapter from the book: Vucelić-Radović, B et al. 2019. Application of Molecular Methods and Raman Microscopy/Spectroscopy in Agricultural Sciences and Food Technology.
The aim of this chapter is to describe some of the protocols which are used in the diagnostics of plant viruses, fungi and fungus-like organisms. Special adaptation of certain protocols is required for each of this group of pathogens, and even more every type of virus, fungus or fungus-like organism, each type of raw material in terms of plant organs, plant host species, time of year and the conditions in the laboratory. The protocols of molecular methods for detection, identification and characterization of phytopathogenic organisms cited here in include detailed instructions for its application. This chapter contains specific recommendations for selection of plant material, extraction of total RNAs or DNAs, detailed procedure for RT-PCR or PCR depending on phytopathogenic organisms and instruction for interpretation of obtained results. The chapter also focuses on application of DNA cloning in plant virology. The text on DNA cloning contains instruction for basic steps including amplification of DNA fragment that we want to clone, ligation of PCR product with vector DNA, insertation of recombinant DNA into bacteria cells, the reproduction of bacteria together with the recombinant DNA, screening clones with recombinant DNA, and plasmid extraction and purification from the bacterial cells.