Simple sequence repeat markers useful for sorghum downy mildew (Peronosclerospora sorghi) and related species

<p>Abstract</p> <p>Background</p> <p>A recent outbreak of sorghum downy mildew in Texas has led to the discovery of both metalaxyl resistance and a new pathotype in the causal organism, <it>Peronosclerospora sorghi</it>. These observations and the difficulty in resolving among phylogenetically related downy mildew pathogens dramatically point out the need for simply scored markers in order to differentiate among isolates and species, and to study the population structure within these obligate oomycetes. Here we present the initial results from the use of a biotin capture method to discover, clone and develop PCR primers that permit the use of simple sequence repeats (microsatellites) to detect differences at the DNA level.</p> <p>Results</p> <p>Among the 55 primers pairs designed from clones from pathotype 3 of <it>P. sorghi</it>, 36 flanked microsatellite loci containing simple repeats, including 28 (55%) with dinucleotide repeats and 6 (11%) with trinucleotide repeats. A total of 22 microsatellites with CA/AC or GT/TG repeats were the most abundant (40%) and GA/AG or CT/TC types contribute 15% in our collection. When used to amplify DNA from 19 isolates from <it>P. sorghi</it>, as well as from 5 related species that cause downy mildew on other hosts, the number of different bands detected for each SSR primer pair using a LI-COR- DNA Analyzer ranged from two to eight. Successful cross-amplification for 12 primer pairs studied in detail using DNA from downy mildews that attack maize (<it>P. maydis & P. philippinensis</it>), sugar cane (<it>P. sacchari</it>), pearl millet (<it>Sclerospora graminicola</it>) and rose (<it>Peronospora sparsa</it>) indicate that the flanking regions are conserved in all these species. A total of 15 SSR amplicons unique to <it>P. philippinensis </it>(one of the potential threats to US maize production) were detected, and these have potential for development of diagnostic tests. A total of 260 alleles were obtained using 54 microsatellites primer combinations, with an average of 4.8 polymorphic markers per SSR across 34 <it>Peronosclerospora, Peronospora and Sclerospora </it>spp isolates studied. Cluster analysis by UPGMA as well as principal coordinate analysis (PCA) grouped the 34 isolates into three distinct groups (all 19 isolates of <it>Peronosclerospora sorghi </it>in cluster I, five isolates of <it>P. maydis </it>and three isolates of <it>P. sacchari </it>in cluster II and five isolates of <it>Sclerospora graminicola </it>in cluster III).</p> <p>Conclusion</p> <p>To our knowledge, this is the first attempt to extensively develop SSR markers from <it>Peronosclerospora </it>genomic DNA. The newly developed SSR markers can be readily used to distinguish isolates within several species of the oomycetes that cause downy mildew diseases. Also, microsatellite fragments likely include retrotransposon regions of DNA and these sequences can serve as useful genetic markers for strain identification, due to their degree of variability and their widespread occurrence among sorghum, maize, sugarcane, pearl millet and rose downy mildew isolates.</p

State of the Field of Plant Pathogen Diagnostic Assay Development and Validation

Diagnostic assays for plant pathogens are a critical component in the surveillance, detection, and management of disease in the production of food, feed, and fiber. Producers, crop consultants, and state and federal officials depend on rapid and accurate diagnostics for response, disease mitigation, crop management, and regulatory decisions affecting trade and export prices. Diagnostic assays undergoing development must be validated to understand how well the assay will perform on given sample matrices, in different user hands, and under varying laboratory conditions before deployment and application in diagnostic clinics and distribution as commercially available assays. The validation of diagnostic assays is a time-consuming, expensive, multitiered process, requiring pathogen and near neighbor reference materials, barcode and/or genome sequence data, and cross-laboratory partnerships. Regulatory agencies and diagnostic clinics do not often have fully validated assays to deploy during an outbreak of a novel pathogen. Our survey of recent publications describing the development of diagnostics for plant pathogens found that those detailing an appropriate and robust diagnostic assay validation process were few in number. The results point out the need for broad application of standards and rigor in the conception, development, and execution of diagnostic assay validation. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license