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Phomopsis Black Root Rot of Cucumber in Washington State Caused by Diaporthe sclerotioides

Fukada, H., Derie, M. L., Shishido, M., du Toit, L. J.
Plant disease 2018 v.102 no.8 pp. 1657
Cucumis sativus, Diaporthe, agar, black root rot, chloramphenicol, consensus sequence, cucumbers, death, direct seeding, disease transmission, genes, greenhouses, growth retardation, horticulture, hyphae, internal transcribed spacers, mycelium, organic foods, pathogenicity, pathogens, polymerase chain reaction, ribosomal DNA, roots, seedlings, sodium hypochlorite, solar radiation, tap roots, wilting, Austria, British Columbia, Denmark, France, Germany, Italy, Japan, Malaysia, Netherlands, Norway, United Kingdom, Washington (state)
Phomopsis black root rot of cucumber (Cucumis sativus L.), caused by Diaporthe sclerotioides (syn. Phomopsis sclerotioides), has been reported in Austria, Britain, Canada, Denmark, France, Germany, Italy, Japan, Malaysia, the Netherlands, and Norway (Shishido et al. 2016). In August 2017, wilted plants were observed in patches comprising 10% of a 5-ha organic cucumber crop in Skagit Co., WA, after fruit had formed. Crowns, tap roots, and secondary roots had pink to brown, dry, corky lesions, black pseudostromata, and rectangular pseudosclerotia within cortical cells. Sections of symptomatic roots and crowns (≤10 mm long) of eight plants selected randomly were surface-sterilized in 0.6% NaOCl for 3 to 5 min, triple-rinsed in sterilized water, dried on sterilized blotters, plated on water agar, and incubated at 24 ± 2°C with a natural day/night cycle. Hyphae were transferred to 1/2-strength PDA amended with chloramphenicol (100 ppm). After 2 weeks, each isolate covered the plate with gray-brown, dense mycelium. Flat, brown-black, irregular pseudostromata formed in the medium. Five isolates were grown in 1/2-strength potato dextrose broth on a shaker for 7 days, mycelia harvested, DNA extracted, and the internal transcribed spacer (ITS) 1-5.8S-ITS2 region of ribosomal DNA amplified using universal eukaryotic primers UN-UP18S42 and UN-LO28S576B (Schroeder et al. 2006). The sequences of isolates Dsl004, Dsl005, Dsl006, and Dsl008 (GenBank accession nos. MG434673.1, MG434674.1, MG434675.1, and MG434676.1, respectively) had 100% identity to that of a D. sclerotioides isolate (NR_111069.1); the sequence of Dsl002 (MG434672.1) had 99% identity because of a 7-nt insertion in the ITS1 region. All five isolates and DNA of D. sclerotioides ATCC18585 (ex-type culture) produced the expected 392-bp band of amplified DNA from the ITS rDNA using the highly specific primers CPs-1 and CPs-2 (Shishido et al. 2010). In addition, a 566-nt consensus sequence of the TEF 1-α gene of Dsl002, Dsl004 to Dsl006, and Dsl008 (MG868937) generated by PCR assay with primers EF-2F and EF-5AR (Taşkın et al. 2010) was 100% identical to that of D. sclerotioides ATCC18585 (MG868936). Pathogenicity of the five isolates on cucumber was verified in a greenhouse using two methods, direct-seeding and transplanting. Two-week-old colonized PDA plates of each isolate were cut into 3 mm³ pieces and mixed in Sunshine Potting Mix #1 (Sungro Horticulture, Canada) (half a plate/1.0 liter potting mix/15-cm-diameter pot). A nontreated seed (cv. Turbo) was then planted in the medium in each of five replicate pots/isolate, and a 10-day-old Turbo seedling was transplanted into each of five replicate pots/isolate. For the control treatment, potting medium was amended with noncolonized PDA (five direct-seeded and five transplanted replicates). Plants were maintained at 18 to 26°C with 10 h light/day. Dsl004, Dsl005, Dsl006, and Dsl008 caused wilting and death of plants in 3 weeks with the direct-seeding method, and in 4 to 5 weeks for the transplanting method. Dsl002 caused stunting and minor wilting. Control plants remained healthy. The roots of inoculated plants had corky lesions with pseudostromata and pseudosclerotia. D. sclerotioides was reisolated from roots of inoculated plants but not control plants. The species was confirmed morphologically and by ITS rDNA and TEF 1-α sequencing. This is the first report of Phomopsis black root rot of cucumber caused by D. sclerotioides in Washington State or anywhere in the U.S.A. The disease was reported in greenhouse cucumber in neighboring British Columbia, Canada, in 1972 (Ormrod and Christie 1972). Research is needed to assess prevalence of the pathogen and management strategies to limit spread of the disease.