Abstract
Highly specific detection methods, capable of reliably identifying plant pathogens are crucial in plant disease management strategies to reduce losses in agriculture by preventing the spread of diseases. We describe a novel molecular inversion probe (MIP) assay that can be potentially developed into a robust multiplex platform to detect and identify plant pathogens. A MIP has been designed for the plant pathogenic fungus Fusarium oxysporum f.sp. conglutinans and the proof of concept for the efficiency of this technology is provided. We demonstrate that this methodology can detect as little as 2.5 ng of pathogen DNA and is highly specific, being able to accurately differentiate Fusarium oxysporum f.sp. conglutinans from other fungal pathogens such as Botrytis cinerea and even pathogens of the same species such as Fusarium oxysporum f.sp. lycopersici. The MIP assay was able to detect the presence of the pathogen in infected Arabidopsis thaliana plants as soon as the tissues contained minimal amounts of pathogen. MIP methods are intrinsically highly multiplexable and future development of specific MIPs could lead to the establishment of a diagnostic method that could potentially screen infected plants for hundreds of pathogens in a single assay.
Highlights
Agriculture is a major economic activity with a total annual value of $1500 billion US dollars
The circularized molecular inversion probe (MIP), which is immune to exonuclease digestion, was amplified in a polymerase chain reaction (PCR) reaction to detectable levels using universal primers designed for two domains in the MIP backbone
The sensitivity, specificity and the dynamic range of the assay has been demonstrated in a singleplex MIP assay
Summary
Agriculture is a major economic activity with a total annual value of $1500 billion US dollars. Up to a third of the agricultural production is lost due to three major causes: disease outbreaks, insect attack and weed competition [1]. Losses caused by crop diseases are the most important issue globally, especially in agriculturally reliant countries. In the absence of resistance, the ideal method to control disease outbreaks is by early detection in the field before it spreads to neighboring farms. It is essential to develop new disease diagnostic technologies that are sensitive, reproducible, highly specific and able to detect multiple pathogens in a single assay
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