Detection of Bacterial and Phytoplasmal Pathogens

Abstract

Bacterial pathogens classified as prokaryotes are much simpler in structure and smaller in size and they have less complex structural features compared to fungal plant pathogens. As the morphological characteristics of bacterial cells are less variable, biological, biochemical, physiological, immunological and genomic characteristics have to be determined for reliable identification and meaningful classification of bacterial pathogens. Detection and identification of bacterial plant pathogens present in whole plants and in propagative plant materials have been possible by employing isolation on cultural media and metabolic fingerprinting methods. But they are labor-intensive and require long time. Results often are inconclusive. Isozyme analysis, direct colony thin layer chromatography and gel electrophoresis techniques have been successfully applied for the detection of some bacterial pathogens. Immunoassays and nucleic acid-based assays have become widely accepted techniques, providing more sensitive and specific detection and quantification of bacterial pathogens affecting a wide range of host plant species. However, the major limitation of the molecular techniques is their inability to discriminate living cells from the dead ones. Attempts have been made to overcome this limiting factor by using DNA binding dyes and estimating pathogen RNAs as an indicator of cell viability. The diagnostic methods have both merits and demerits and hence, appropriate method has to be selected based on cost-effectiveness and possibility of obtaining reliable results rapidly to suit the requirements of the investigation concerned.Phytoplasmas are cell wall-less, nonculturable bacteria belonging to Mollicutes. Lack of cultural characteristics has made obligatory to study the morphological characteristics of phytoplasmas present in the phloem cells of infected plant hosts by observing under electron microscopes. As most of the phytoplasmas look alike in the ultrathin sections, the morphological characters have no diagnostic value. Histochemical methods using DNA binding dyes have been employed to localize the phytoplasmas in the host cells. Application of immunoassays and nucleic acidbased techniques has been effective in providing information for the identification and differentiation of phytoplasams. Polyclonal and monoclonal antibodies have been generated for detection of phytoplasmas infecting several plant species. Universal and species-specific primers and probes have been designed based on

Immunodominant membrane proteins of phytoplasmas

“…These antibodies can serve as specific tools for investigating the expression patterns of IDPs within the infected host plant. In addition, these antibodies have been used to localize phytoplasma via in situ immunofluorescence, immunosorbent electron microscopy and goldlabelled antibody decoration (Narayanasamy, 2011). The recombinant IDPs are also important tools for studying protein-protein interactions by pull-down assays and farwestern blot analysis (Galetto et al, 2011;Suzuki et al, 2006).…”

Tube-Wise Diagnostic Microarray for the Multiplex Characterization of the Complex Plant Pathogen Ralstonia solanacearum

“…Diagnostic protocols mainly rely on techniques that offer compatible routine application procedures and provide for user-friendly environments, good industrial production capacities, and a high level of reliability. Techniques that primarily employ immunoassays and immune-strip tests (ImmunoStrip®; Agdia, Elkhart, IN) are mainly used for the detection of Rssc strains at the species level (Danks and Barker, 2000 ), and although these assays are simple and affordable, they are known to produce false positives (Narayanasamy, 2010 ). DNA-based approaches using conventional PCR (Table 1 ) can identify the Rssc at the species level (Huang and Schell, 1990 ; Seal et al, 1993 ; Opina et al, 1997 ; Lee and Wang, 2000 ; Glick et al, 2002 ; Schonfeld et al, 2003 ), the phylotype level (Fegan and Prior, 2005 ); and when these approaches are coupled with sequencing capabilities, they can be used to produce phylogenetic trees (Lane, 1991 ; Taghavi et al, 1996 ; Fegan and Prior, 2005 ; Prior and Fegan, 2005b ).…”