Advances in next-generation sequencing (NGS) allow for rapid development of genomics resources needed to generate molecular diagnostics assays for infectious agents. NGS approaches are particularly helpful for organisms that cannot be cultured, such as the downy mildew pathogens, a group of biotrophic obligate oomycetes that infect crops of economic importance. Unlike most downy mildew pathogens that are highly host-specific, Pseudoperonospora cubensis causes disease on a broad range of crops belonging to the family Cucurbitaceae. In this study, we identified candidate diagnostic markers for P. cubensis by comparing NGS data from a diverse panel of P. cubensis and P. humuli isolates, two very closely related oomycete species. P. cubensis isolates from diverse hosts and geographical regions in the United States were selected for sequencing to ensure that candidates were conserved in P. cubensis isolates infecting different cucurbit hosts. Genomic regions unique to and conserved in P. cubensis isolates were identified through bioinformatics. These candidate regions were then validated using PCR against a larger collection of isolates from P. cubensis, P. humuli, and other oomycetes. Overall seven diagnostic markers were found to be specific to P. cubensis. These markers could be used for pathogen diagnostics on infected tissue, or adapted for monitoring airborne inoculum with real-time PCR and spore traps.

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In August of 2013, garlic bulbs (Allium sativum) of the variety Chesnok Red grown and stored under dry conditions by a commercial producer in Buncombe County showed water-soaked, tan to salmon-pink lesions. Lesions on cloves became soft over time, slightly sunken, and had mycelium near the center of the bulb, which is characteristic of Fusarium rots on garlic (1,2). Approximately 10 to 20% of the bulbs inspected in the drying storage room were affected. Surface-sterilized tissue was excised from the margin of lesions on eight bulbs, plated onto acid potato dextrose agar (APDA), and incubated in the dark at room temperature (21°C). White to light pink colonies with abundant aerial mycelium and a purple pigment were obtained from all samples after 2 to 3 days of incubation. Inspection of colony morphology and reproductive structures under a microscope revealed that isolate characteristics were consistent with Fusarium proliferatum (Matsushima) Nirenberg. Microscopic morphological characteristics of the isolate included hyaline, septate hyphae; slender, slightly curved macroconidia with three to five septae produced in sporodochia; curved apical cell; and club-shaped, aseptate microconidia (measuring 3.3 to 8.3 × 1.1 to 1.3 μm) produced in chains by mono and polyphyalides. To further define the identity of the isolate, the beta-tubulin (Btub), elongation factor 1a (EF1a), and internal transcribed spacer (ITS) regions were amplified and sequenced (3). The resulting sequences were compared against the GenBank nucleotide database by using a BLAST alignment, which revealed that the isolate had 100% identity with F. proliferatum for the Btub, EF1a, and ITS regions (GenBank Accession Nos. AF291055.1, JX118976.1, and HF930594.1, respectively). Sequences for the isolate were deposited in GenBank under accessions KJ128963, KJ128964, and KJ128965. While there have been other reports of F. proliferatum causing bulb rot of garlic in the United States (1), to our knowledge, this is the first report in North Carolina. The finding is significant since F. proliferatum can produce a broad range of mycotoxins, including fumonisins, when infecting its host, which is a concern for food safety in Allium crops. References: (1) F. M. Dugan et al. Plant Pathol. 52:426, 2003. (2) L. J. du Toit and F. M. Dugan. Page 15 in: Compendium of Onion and Garlic Diseases and Pests. H. F. Schwartz and S. K. Mohan, eds. The American Phytopathological Society, St. Paul, MN, 2008. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.