Lepidosphaeria strobelii A.C. Lagashetti, D. Choudhary & S.K. Singh, sp. nov.
Index Fungorum number: IF 830723; MycoBank number: MB 830723; Facesoffungi number: FoF 06120, Fig. 1
Etymology – specific epithet refers to Prof. Gary Strobel, MSU, USA to commemorate his immense contribution in the field of biology of endophytic fungi.
Holotype – AMH-10126
Colour codes follow – Methuen Handbook of Colour (Kornerup and Wanscher 1978)
Isolated as an endophyte from Cinnemomum zeylanicum. Sexual morph Undetermined. Asexualmorph Hyphae septate, hyaline, thin-walled, 1–4 μm wide, simple to branched, smooth-walled, consisting of series of inflated cells (chlamydospores), globulated and hyphae showed frequent anastomosis and spirally twisting. Hyphal cells, cylindrical, 7–19.5 × 1.5–4 µm (x̅=12.5 × 2 µm, n=30). Chlamydospores hyaline as well as light to dark olivaceous brown, observed frequently, terminal to intercalary, solitary, in branched chains and in bunches, with thickened and darkened wall, variable in shape, globose to cylindrical, 4.5–11×5–9 µm (x̅=8.5×7.5 µm, n=30).
Culture characteristics – Colonies growing slowly on PDA reaching to 12–14 mm diam. after 2 weeks at 25 °C; colonies from above grey (4D1), circular, raised, rocky, with slightly cottony aerial mycelium, synnematous, entire with irregular margin; colony from below, violet brown (10F8), sulcate, with diffusible red pigment. Colonies on Sabouraud Dextrose agar (SDA) reaching to 12–13 mm diam. after 2 weeks at 25 °C; colonies from above greyish green (28C3), circular, raised, velvety with smooth, entire and irregular margin; colony from below violet brown (10F8), sulcate and with diffusible red pigment.
Material examined – INDIA, Karnataka, Belgaum (15.8497° N 74.4977° E), on living leaves of Cinnemomum zeylanicum, 29 November 2011, S.K. Singh (AMH 10126, holotype), ex-type living culture, NFCCI 4579.
GenBank numbers – ITS=MH790217, LSU=MK749433.
Notes – The proposed a new species, Lepidosphaeria strobelii is phylogenetically distinct from L. nicotiae with 99% MLBS support (Fig. 2) based on maximum likelihood analysis of a combined ITS and LSU sequence dataset. A megablast analysis, the ITS sequence of L. strobelii showed 95.23% (419/440) similarity and 6 gaps (1%) with L. nicotiae (CBS 559.71, Ex-type) and 96.08% (441/459) similarity and 2 gaps (0%) with L. nicotiae (1B0102). A comparison of the ITS sequence shows that L. strobelii differs from L. nicotiae, 21/441 base positions (4.7%) without gaps. Moreover, the source of isolation of these species are different. Type strain, L. nicotiae (CBS 559.71) was isolated from desert soil in Algeria and L. nicotiae (1B0102) was from totally suspended particles of air from Bangkok, Thailand. Whereas, L. strobelii (NFCCI 4579) was isolated as an endophyte from the leaves of Cinnemomum zeylanicum in India (Fig. 1). Morphologically, the new species could not be compared with L. nicotiae. Lepidosphaeria nicotiae is characterized by its sexual morph, forming cleistothecial, black, globose to subglobose ascomata, elongate-clavate asci, with a long pedicel, embedded in filiform, hyphae-like pseudoparaphyses, and pale brown to brown, oblong, tuberculate, 1-septate, finely echinulate ascospores (Doilom et al. 2018). While the sexual morph of L. strobelii is undetermined.
Figure 1 – Lepidosphaeria strobelii (AMH 10126, holotype; NFCCI 4579, ex-type culture). a, b Colonies morphology on potato dextrose agar (from surface and reverse views). c, d Colonies morphology on SDA, from surface and reverse views). e Stereoscopic view of colony growing on PDA. f, g Magnified view showing hyphal bundles. h Microscopic view of hyphal bundles. i Mycelia with septate hyphae (arrows). j Mycelia showing frequent anastomosis (arrows). k, l Mycelia with spiral twisting (arrows). m Young chlamydospores. n Mature chlamydospores in chain. o Mature chlamydospores in bunch. p Mature chlamydospore. Scale bars: i–p=20 μm
Figure 2 – Phylogram generated from maximum likelihood method for Lepidosphaeria strobelii (NFCCI 4579); using combined ITS and LSU sequence dataset. The evolutionary history was inferred based on the General Time Reversible model (Nei and Kumar 2000). The tree with the highest log likelihood (− 7009.2670) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. A discrete GAMMA distribution was used to model evolutionary rate differences among sites [5 categories (+G, parameter=0.5660)]. The rate variation model allowed for some sites to be evolutionarily invariable [(+I), 58.2766% sites]. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 35 nucleotide sequences. All positions with less than 85% site coverage were eliminated. That is fewer than 15% alignment gaps, missing data, and ambiguous bases were allowed at any position. There were a total of 1322 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Kumar et al. 2016). Sigarispora arundinis (formerly known as Lophiostoma arundinis) was used as an outgroup. Ex-type strains are in bold and newly generated sequence is in blue