Confirmation of Itersonilia perplexans Infecting Pyrethrum (Tanacetum cinerariifolium) in Australia

Abstract

Pyrethrum (Tanacetum cinerariifolium [Trevir.] Sch. Bip.) is grown to extract pyrethrins, which are active ingredients for insecticides. The Australian pyrethrum industry supplies over 50% of the world market. Surveys of Tasmanian crops in spring 2013 detected the presence of a fungus putatively identified as Itersonilia perplexans Derx. on foliage in 54 of 86 surveyed fields (Hay et al. 2015). This fungus was associated with necrotic leaf tips often spreading to encompass whole leaves. However, pathogenicity to pyrethrum was not confirmed. To isolate the fungus, tissue was excised from foliar lesions, surface sterilized using 0.4% NaClO, placed onto 2% water agar, and incubated at 20°C for 5 days. Colonies were pure-cultured by hyphal-tip transfer onto potato dextrose agar. Eleven isolates were cultured onto yeast mold agar (YMA) for 14 days at 15°C in the dark (Horita and Yasuoka 2002). Colonies were slow growing (1.9 to 2.3 mm/day), white to buff on both surfaces, with a darker center visible on lower surfaces. Mycelia were straight and hyaline with clamp connections at the septa. Squares transferred from the edge of YMA colonies onto microscope slides produced ballistoconidia that were aseptate, granular and lunate, kidney or lemon-shaped after 24 h. Ballistoconidia lengths and widths (n = 50/isolate) were 14.6 to 20.4 and 10.0 to 13.6 µm. Chlamydospores were not observed. These observations were consistent with descriptions of I. perplexans (Koike and Tjosvold 2001; Liu et al. 2015). All 11 isolates were sequenced across the internal transcribed spacer (ITS) region of rDNA (ITS; primers V9G/ITS4; de Hoog and van den Ende 1998; White et al. 1990), and large (LSU; primers LROR/LR7; Rehner and Samuels 1995), and small (SSU; NS1/NS4; White et al. 1990) subunits of rDNA (GenBank accession nos. KU563626 to KU563658). The ITS (673 bp), SSU (1,047 bp), and LSU (1,318 bp) differed by 3, 1, and 0 bp, respectively, across isolates. Maximum parsimony and maximum likelihood analyses of a concatenated three loci alignment with Cystofilobasidiales representatives (Liu et al. 2015) placed all isolates and the I. perplexans ex-neotype strain CBS 363.85 within a single monophyletic clade with 100% bootstrap support. Two representative isolates are stored at the Plant Pathology Herbarium (accession nos. BRIP 57986 and 57987). Leaves of 46-day-old pyrethrum plants (n = 45), generated from surface sterilized seed, were inoculated with a 1.5 × 105 ballistoconidia/ml suspension (equal mix of eight isolates) and maintained between 10 and 22°C under a 12-h photoperiod for 14 days. Brown necrotic leaf tips, consistent with reported field symptoms, were observed on 71% of plants and I. perplexans was recovered from 69% of symptomatic plants. For flower inoculations, pyrethrum plants were removed from fields as vegetative plants in spring and maintained in a greenhouse set at 20/14°C and 14/10 h day:night. Open flowers (10 per plant) were dipped into a 1.2 × 104 ballistoconidia/ml suspension mix of three isolates. Brown withered ray florets were observed on 10 of 12 plants 18 days postinoculation, matching those described in petal blight of chrysanthemum (McRitchie et al. 1973). I. perplexans was reisolated from 11 of 12 inoculated plants and one control plant (of 12) that exhibited the same symptoms. In both experiments, I. perplexans was identified based on its distinctive morphology. This confirms the pathogenicity of I. perplexans to both pyrethrum leaves and flowers.