Molecular Techniques for Detection of Microbial Pathogens

Abstract

Detection, identification and differentiation of microbial plant pathogens -oomycetes, fungi, bacteria, phytoplasmas, viruses and viroids -infecting various crops constitute the basic step for the development of effective crop disease management systems. The conventional cultural methods involving isolation and studying the morphological characteristics using microscope are labor intensive and cumbersome, yielding sometimes, inconclusive results. The molecular techniques, on the other hand, are able to provide precise, reliable and reproducible results rapidly, facilitating early disease management decisions. Biochemical, immunological and nucleic acid-based assays have been preferred, because of the distinct advantages over the conventional methods. The molecular techniques have been very useful in the identification of obligate pathogens causing diseases such as downy mildews, powdery mildews and rusts and also fungi that grow very slowly in the culture media, taking several weeks to produce spore forms that can be used for identification. The availability of antisera, primers and commercial kits has led to widespread application of the molecular techniques for on-site detection during field surveys for assessing the distribution of existing pathogens, occurrence of new/introduced pathogens or strains and for preventing introduction of new pathogens through seeds or planting materials. Furthermore, molecular techniques have been demonstrated to be useful in breeding programs to identify sources of resistance to disease(s). Several protocols for molecular techniques, useful for researchers and students are presented in the Appendix.

Diagnostic techniques for viroids

“…In order to replicate and move, they need the transcription machinery and the proteins of their hosts (Flores et al ., ). As a consequence, the use of serodiagnostic techniques for the detection of viroids is not possible (Hadidi et al ., ; Narayanasamy, ). Currently there are 32 viroid species approved by the International Committee on Taxonomy of Viruses (ICTV), which have been assigned to eight genera and two families, Avsunviroidae and Pospiviroidae (http://www.ictvonline.org/virustaxonomy.asp).…”

“…Symptom expression is influenced by the viroid strain and the host, which can result in latent (no symptoms) or mild to severe infection (Kovalskaya & Hammond, 2014). In addition, symptoms of viroid infection can also differ because host plants can be infected with one viroid or multiple pathogens simultaneously (Narayanasamy, 2008). Furthermore, expression of symptoms and viroid replication also depends on environmental conditions; viroids prefer high temperatures and light intensities (Flores, 2001;Hammond & Owens, 2006;G omez et al, 2008).…”

“…Viroids are the simplest plant pathogens in structure; they consist of naked, single‐stranded, circular, covalently closed, non‐encapsulated RNAs, between 246 and 401 nucleotides in size (Flores, ; Hammond & Owens, ). When introduced into their host plants, independent replication enables them to proliferate and to produce characteristic symptoms (Flores, ; Narayanasamy, ). They are essentially transcriptional parasites because their RNAs do not code for any proteins.…”

“…Because quarantine status includes only a few viroids, national implementation of certification schemes for the production of healthy planting material is an important measure for control of a broad range of viroids on many plants. All management strategies need the support of reliable and sensitive diagnostics, which demands the constant development of new detection methods (Narayanasamy, ; Jiang et al ., ). In this review, various methods used for the identification of viroids in their hosts are presented and discussed in terms of their advantages and disadvantages.…”