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First Report of Didymella lethalis Associated with Roots of Pea, Subterranean Clover, and Winter Vetch in Germany, Switzerland, and Italy

Šišić, A., Baćanović-Šišić, J., Schmidt, H., Finckh, M. R.
Plant disease 2018 v.102 no.12 pp. 2642
DNA-directed RNA polymerase, Didymella, Pisum sativum, Trifolium subterraneum, Vicia villosa subsp. villosa, actin, agar, agroclimatology, air drying, autoclaving, biomass, cultivars, disease severity, endophytes, fungi, greenhouses, habitats, hosts, inoculum, internal transcribed spacers, leaves, malt extract, necrosis, oatmeal, pathogenicity, peas, phylogeny, pigmentation, planting, pycnidia, root rot, roots, sand, seeds, sodium hypochlorite, spores, sporulation, surveys, tubulin, wilting, winter, Germany, Italy, Switzerland
In the period between July 2013 and August 2017, a survey was conducted to examine populations of root-infecting fungi on pea (Pisum sativum), subterranean clover (Trifolium subterraneum), and winter vetch (Vicia villosa) grown in different European agroclimatic regions. Pea plants with black necrotic lesions on tap and lateral roots were observed across several fields in Germany, whereas subterranean clover roots collected in Switzerland and winter vetch roots collected in Italy lacked visible disease symptoms. Roots were washed, surface disinfected in 3% NaOCl for 10 s, rinsed in distilled water, air dried, cut into 1-cm pieces, and placed on Coon’s medium for incubation (Šišić et al. 2018). Didymella-like colonies were subcultured by single pycnidia transfers in Petri dishes containing malt extract (pigmentation and crystal formation) and oatmeal agar (production of pycnidia and sporulation) (Boerema et al. 2004). Of 801 Didymella isolates collected, 48 were identified as D. lethalis based on morphological characteristics. Species identity of the five representative isolates was confirmed by sequencing portions of the internal transcribed spacer region, β-tubulin, α-actin, and the RNA polymerase II second largest subunit (Chen et al. 2015). These included two German isolates from pea, two Swiss isolates from subterranean clover, and one Italian isolate from winter vetch. Using NCBI BLASTn analyses, including single and multigene phylogenies (RAxML), the sequences showed 100% identity to D. lethalis reference strain CBS 103.25. Newly generated sequences were submitted to GenBank under accession numbers MH325447 to MH325466. Each D. lethalis isolate was tested for pathogenicity in a greenhouse on spring pea cultivar Santana, winter pea cultivar EFB 33, subterranean clover cultivar Campeda, and winter vetch cultivar Hungvillosa. Seeds were germinated for 48 h prior to the planting of three seeds per 300-ml pot, which contained autoclaved sand. To prepare inoculum, isolates were cultured on PDA, and inoculations were performed after planting with 2 × 10⁴ spores/g of substrate. Four replicate pots arranged in a completely randomized design were used per treatment. Foot and root rot disease severity and biomass were assessed after 47 days. Each D. lethalis isolate inoculated on pea cultivar Santana caused severe foot and root necrosis, wilting of the lower leaves, and biomass reduction compared with the corresponding controls. In contrast, the infection response of pea cultivar EFB 33 was isolate specific. Brown-black lesions covering 20% of taproot were observed following inoculation with one Swiss isolate only, whereas the remaining D. lethalis isolates did not cause any visible disease symptoms or biomass reductions compared with the corresponding controls. Subterranean clover and winter vetch plants also showed no symptoms of the disease or biomass reduction when inoculated with any of the five isolates tested. However, all five isolates were successfully reisolated from roots of all hosts. No symptoms developed and no D. lethalis isolates were found in the roots of controls. To the best of our knowledge, this is the first report of D. lethalis infecting roots of pea and endophytic persistence of the species in roots of subterranean clover and winter vetch plants. Our results suggest that isolates of D. lethalis can be found in diverse habitats and that, in addition to a significant pathogenic potential of the species, the fungus is able to asymptomatically colonize various hosts that may serve as an inoculum reservoir in the absence of a susceptible crop.