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Title: LAMP detection assays for boxwood blight pathogens: a comparative genomics approach

item Wight-Malapi, Martha
item Demers, Jill
item VELTRI, DANIEL - Oak Ridge Institute For Science And Education (ORISE)
item MARRA, ROBERT - Connecticut Agricultural Experiment Station
item Crouch, Jo Anne

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/27/2016
Publication Date: 5/20/2016
Citation: Wight-Malapi, M., Demers, J.E., Veltri, D.P., Marra, R.E., Crouch, J. 2016. LAMP detection assays for boxwood blight pathogens: a comparative genomics approach. Scientific Reports. doi: 10.1038/srep26140.

Interpretive Summary: Fungi cause many serious diseases of ornamental and crop plants around the world. One of these, Calonectria pseudonviculata, causes a severe disease called boxwood blight. Sometimes the blight fungus may be present in seemingly healthy boxwood leaves, allowing it to go undetected by plant health inspectors. This makes it very easy for the fungus to travel into disease-free areas on “clean” plants and start new infections. In this study, a set of diagnostic assays based on DNA sequences were developed that can detect the boxwood blight fungus in small amounts, even when it cannot be seen by the naked eye. Because DNA sequences most commonly used for diagnostic assays are not specific to the boxwood blight pathogen, we used three novel approaches to identify unique DNA target sequences. The complete genome sequences of the boxwood blight pathogens and related fungi were mined to identify DNA regions that would be 100% diagnostic, and not cross-react with other fungi living in and around boxwood plants. Several good target DNA candidates were identified, and three were fully developed and tested in the lab as diagnostic assays. The diagnostic assays were tested on ~500 different fungi, and were shown to be fully specific to the boxwood blight pathogen. Plant pathologists, quarantine officials and other scientists will be able to use these tools to detect the presence of this fungus and help prevent the spread of boxwood blight disease.

Technical Abstract: Rapid and accurate molecular diagnostic tools are critical to efforts to minimize the impact and spread of emergent pathogens. The identification of diagnostic markers for novel pathogens presents several challenges, especially in the absence of information about population diversity, and where genetic resources are limited. The objective of this study was to use comparative genomics datasets to develop unique target regions suitable for the diagnosis of two fungal species causing a newly emergent blight disease of boxwood. Candidate marker regions for loop-mediated isothermal amplification (LAMP) assays were identified from draft genomes of Calonectria henricotiae and Calonectria pseudonaviculata, as well as three related species not associated with this disease. To increase the probability of identifying unique targets, we used three approaches to mine genome datasets, based on (i) unique regions, (ii) polymorphisms, and (iii) presence/absence of regions across datasets. From a pool of candidate markers, we demonstrated LAMP assay specificity by testing related fungal species, common boxwood pathogens, and environmental samples containing 445 diverse fungal taxa. This comparative-genomics-based approach to the development of LAMP diagnostic assays for fungi is the first of its kind, and could be easily applied to diagnostic marker development for other newly emergent plant pathogens.