Dothiora eucalypti Lacerda, Gusmão, G.G. Barreto & A. Rodrigues, sp. nov.

Index Fungorum number: IF 902386; Mycobank number: MB 902386; Facesoffungi number: FoF 13860; Fig. 1

Etymology – In reference to the host Eucalyptus microcorys.

Holotype – HUEFS263169

Associated with leaves of Eucalyptus microcorys. Sexual morph: Not observed. Asexual morph: Coelomycetous. Colonies on PDA grow initially yeast-like, cream-colored, hyaline hyphae, presenting conidia with yeast-like budding. After seven days hyphae become melanized, giving rise to a hormonema-like synasexual morph, thick-walled, branched, smooth, septate, slightly constricted at septa, with integrated conidiogenous cells, producing hyaline, smooth, ellipsoidal to irregular conidia 5–10×1.5–3 μm (x̄=7.9×2.1 μm, n=30). Chlamydospores-like structures present. Conidiomata 100 µm diam., pycnidial, superficial to semi-immersed, solitary or aggregated, globose to irregular, unilocular, with central papillate ostiole, with or without protuberances at apex, dark brown to black, up to; wall 2–3 layers of brown textura angularis. Conidiophores 7–9×2–4 μm (x̄=8.1 × 2.9 μm, n=30), reduced to phialidic conidiogenous cells lining the inner cavity, obpyriform to obclavate, smooth, thin-walled hyaline. Conidia 4–12 × 1–2.8 μm (x̄=9.8×2.3 μm, n=30), aseptate, subcylindrical, oblong to irregular, smooth, sometimes with slightly truncate basis, hyaline.

Culture characteristics – Colonies on PDA at 28 °C after 7 days: initially yeast-like, cream-colored, but reaching 8.5 mm in diam., radial, olivaceous-black, black, filamentous, sometimes wrinkled with white borders; on PCA at 28 °C after 7 days: filamentous, black to brown, circular, lacking aerial mycelium, reaching 7 mm in diam.; on CMA at 28 °C after 7 days: flat, radial, olivaceous-black in the center, with sepia borders, reaching 8 mm diam.; on PDA at 15 °C: initially yeast-like, cream-colored, but after seven days filamentous, black to olivaceous-black, irregular to circular, reaching 4 mm diam.; on PCA at 15 °C after seven days: circular, filamentous, olivaceous-black, grey to black, olivaceous, with reddish borders, reddish soluble pigment release in the culture medium, reaching 4 mm diam.; on CMA at 15 °C after seven days: flat, circular to radial, black to olivaceous-black, with sepia borders, reaching 4 mm diam. No growth at 45 °C in any culture medium tested.

Material examined – Brazil. São Paulo, Rio Claro, leaf litter of E. microcorys F. Muell., 12 April 2015. L.T. Lacerda, LTL 15 (HUEFS263169, holotype as dry culture), ex-type CCMB 0728.

Additional material examined – Brazil. Botucatu, São Paulo, fungus garden of the leaf-cutting ant Atta sexdens (Linnaeus, 1758) on eucalyptus plantation, April 2012. J.S. Pereira and A. Rodrigues, (JSP06B5.2); id., Rio Claro, living leaves of E. microcorys, October 2015. L.T. Lacerda, (LTL 144; LTL 5_13); id., leaf litter of E. microcorys, April 2016. L.T. Lacerda, (LTL 6_8).

GenBank numbers – LTL 15 (ITS: MH777080; LSU MH777077); LTL 5_13 (ITS: MH777082; LSU: MH777075); LTL 6_8 (ITS: MH777081; LSU: MH777074); LTL 114 (ITS: MH777079; LSU: MH777076); JSP 06 B5.2 (ITS: KR093869; LSU: MH777078).

Notes – Based on a megablast search of NCBI GenBank nucleotide database, the closest hits using highly similar sequences for ITS are Kabatina thujae R. Schneid. & Arx (GenBank MH858857; Identities = 482/525 (92%), 18 gaps (3%)), K. juniperi (GenBank AY183367; Identities=482/526 (92%), 19 gaps (3%)) and Dothidea eucalypti Crous (GenBank NR156390; Identities = 479/524 (91%), 17 gaps (3%)). Closest hits using LSU sequences are Dothiora mahoniae (A.W. Ramaley) Crous (GenBank MH874022; Identities = 773/795 (97%), 4 gaps (0%))
Dothiora pistaciae (Quaedvl., Verkley & Crous) Crous (GenBank NG057996; Identities=771/793 (97%), 2 gaps (0%)) and Endoconidioma carpetanum (Bills, Peláes & Ruibal) Crous (GenBank MF611880; Identities=770/796 (97%), 5 gaps (0%)). Dothiora eucalypti can easily be distinguished from the closely related species by pycnidia with stronger protuberances at apex and the synasexual morph with smooth hormonema-like hyphae slightly constricted at septa. The phylogenetic analyses support that the five strains are distinct species (Fig. 2). Therefore, D. eucalypti is introduced as a novel species in the family Dothioraceae.

Dothiora eucalypti was found on leaf litter (strains LTL 6_8; LTL 15) and living leaves (strains LTL 144; LTL 5_13) of E. microcorys and in a leaf-cutting ant fungus garden on eucalyptus plantation (JSP06B5.2). Regarding the isolation of D. eucalypti from ant colonies, this event sheds some lights on the ecology of the fungus. Leaf-cutting ant species in the genus Atta have the habit of foraging on fresh leaves to nourish their mutualistic fungus, Leucoagaricus gongylophorus (Möller) Singer (De Fine Licht and Boomsma 2014). Thus, we assume that strain
JSP06B5.2 probably was foraged by ant workers and transported to the colony (Fig. 1).

Figure 1 – Dothiora eucalypti (HUEFS263169 holotype). a, b Aggregate and solitary pycnidia in culture with detail of the protuberances. c, d Longitudinal sections of pycnidia. e Detail of conidiogenous cells. f Conidia from pycnidia. g Hormonemalike hyphae with conidiogenous cells and conidia. Scale bars: a=100 μm, b=50 μm, c–d=20 μm, e–g=10 μm

Figure 2 – Phylogram generated based on combined ITS and LSU data of Dothiora eucalypti and allied taxa in Dothideales. The tree is rooted on Myriangium hispanicum CBS 247.33 and M. haraearum CBS 300.34. The newly generated sequences reindicated in blue bold. The alignment consisted of 69 sequences of Dothideomycetes, which had 1,424 characters, with 621 distinct patterns, 382 parsimony-informative, 109 singleton sites and 932 constant sites. The models for ML analysis were GTR+I+G for LSU and SYM+I+G for ITS. Bayesian inference was implemented with the same models as ML analysis. The best score tree with a final likelihood value of−10,496.107 is presented. The tree was inferred with Bayesian Inference (BI) and Maximum Likelihood (ML) algorithms. Only posterior probabilities≥0.90 and bootstrap values≥70% are shown