Innovative tools for detection of plant pathogenic viruses and bacteria

Abstract

Detection of harmful viruses and bacteria in plant material, vectors or natural reservoirs is essential to ensure safe and sustainable agriculture. The techniques available have evolved significantly in the last few years to achieve rapid and reliable detection of pathogens, extraction of the target from the sample being important for optimising detection. For viruses, sample preparation has been simplified by imprinting or squashing plant material or insect vectors onto membranes. To improve the sensitivity of techniques for bacterial detection, a prior enrichment step in liquid or solid medium is advised. Serological and molecular techniques are currently the most appropriate when high numbers of samples need to be analysed. Specific monoclonal and/or recombinant antibodies are available for many plant pathogens and have contributed to the specificity of serological detection. Molecular detection can be optimised through the automatic purification of nucleic acids from pathogens by columns or robotics. New variants of PCR, such as simple or multiplex nested PCR in a single closed tube, co-operative-PCR and real-time monitoring of amplicons or quantitative PCR, allow high sensitivity in the detection of one or several pathogens in a single assay. The latest development in the analysis of nucleic acids is micro-array technology, but it requires generic DNA/RNA extraction and pre-amplification methods to increase detection sensitivity. The advances in research that will result from the sequencing of many plant pathogen genomes, especially now in the era of proteomics, represent a new source of information for the future development of sensitive and specific detection techniques for these microorganisms.

Simultaneous detection of CTV, CEVd and HSVd using Arizona 861 S1 Citron and RT-PCR

“…In addition, these tests require specific indicator plants for each pathogen (Roistacher, 1991;Durán et al, 1993), which must be combined with the technique of sequential polyacrylamide gel electrophoresis (sPAGE). The most recent method for the detection of pathogens is PCR real time; however, this technique is still quite expensive, which is the principal problem for implementing its routine use in laboratories (López et al, 2003), though for detecting viral diseases in citrus, the results have been shown to be fast and reliable (Rizza et al, 2009;Loconsole et al, 2010).…”

Advancements in the diagnosis of bacterial plant pathogens: An overview

“…phaseolicola in bean seed extracts, Acidovorax avenae ssp. avenae in rice seeds (Schaad et al, 1995;López et al, 2003;Song et al, 2004).…”

Immunocapture and simple-direct-tube-reverse transcriptase polymerase chain reaction (RT-PCR) for detection of Rice yellow mottle virus

“…Polymerase chain reaction has been used successfully to detect a range of viruses including both DNA and RNA viruses (Chandler et al, 1998). However, detection of viral RNA by PCR requires reverse transcription (RT) of viral RNA prior to the reaction (Lopez et al, 2003). Rice viruses, except Rice tungro bacilliform virus (RTBV), are RNA viruses, and synthesis of cDNA of the viral genome by reverse transcription (RT) is necessary before the target RNA sequence is amplified (Uehara-Ichiki et al, 2013).…”