Molecular Inversion Probe: A New Tool for Highly Specific Detection of Plant Pathogens

<div><p>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 <i>Fusarium oxysporum</i> f.sp. <i>conglutinans</i> 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 <i>Fusarium oxysporum</i> f.sp. <i>conglutinans</i> from other fungal pathogens such as <i>Botrytis cinerea</i> and even pathogens of the same species such as <i>Fusarium oxysporum</i> f.sp. <i>lycopersici</i>. The MIP assay was able to detect the presence of the pathogen in infected <i>Arabidopsis thaliana</i> 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.</p></div

Disease symptom development and classification.

<p>The <i>A. thaliana</i> leaves were checked for visible symptoms from 1 day after inoculation (dai) until 25 dai and classified into 5 infection stages according to their severity. Stage 1∶0% symptom severity (3–6 dai); Stage 2: ∼25% symptom severity (7–10 dai); Stage 3: ∼50% symptom severity (11–15 dai); Stage 4: ∼75% symptom severity (16–20 dai); Stage 5∶100% symptom severity (21–25 dai).</p

MIP assay on infected <i>A. thaliana</i> plant tissue samples.

<p>Replicate MIP assays were performed on 2 µg of DNA extracted from the <i>A. thaliana</i> leaves belonging to each of the five infection stages as well as non-infected (healthy) leaves. Each reaction contained 2 µg of genomic DNA extracted from infected <i>A. thaliana</i> leaves. Non-infected <i>A. thaliana</i> was used as negative control. MIP assays were performed in triplicates and amplicon abundance was measured by quantifying the intensity of the DNA bands. Error bars represent standard deviation of the three replicates.</p

Effect of host DNA on the sensitivity of the MIP assay.

<p>Reactions containing 10 ng of Foc genomic DNA were mixed with either 1 µg or 2 µg of genomic DNA from <i>A. thaliana</i> in triplicates and analyzed in a MIP assay. A sample containing 2 µg of <i>A. thaliana</i> genomic DNA was used as negative control. Error bars represent the standard deviation of three replicates.</p

Oligonucleotide sequences.

<p>Oligonucleotide sequences.</p

Detection limit of the MIP assay.

<p>The MIP (MIPFOC01) was mixed with different amounts of Foc genomic DNA (160 ng, 80 ng, 40 ng, 20 ng, 10 ng, 5 ng, 2.5 ng and 1.25 ng) in triplicates and analyzed in a MIP assay. The PCR amplicon was electrophoresed through an agarose gel and the intensity of the DNA bands were measured. Error bars represent the standard deviation of three replicates.</p