Exobasidium setsutaiense Nagao, 2025

Nagao, Hideyuki, Orihara, Takamichi & Kasuya, Taiga, 2025, Exobasidium setsutaiense sp. nov. (Exobasidiaceae) causing leaf blister on Rhododendron semibarbatum in Japan, Phytotaxa 684 (1), pp. 78-92 : 83-85

Exobasidium setsutaiense Nagao , sp. nov. FIGURE 3 .

Exobasidium butleri auct. non Sydow: Sawada, Tohoku Biological 1: 98.

MycoBank no.: 857431

Holotype:— JAPAN. Tochigi Prefecture, Nikko-shi, Hanaishi-cho, Botanical Gardens , Graduate School of Science , The University of

Tokyo, 544m alt. 5 Aug. 2021, Nagao KPM-NC-0029634, acc. nos. LC811668 / LC811679 , ex-holotype culture MAFF 247752. Paratype:— JAPAN. Iwate Prefecture, Setsutai-mura, 21 Jun. 1906, K. Sawada IUM-9112.

Additional specimens examined:— JAPAN, Tochigi Prefecture, Nikko-shi, Hanaishi-cho, Botanical Gardens , Graduate School of Science , The University of Tokyo, 544m alt. 6 July 2022, Nagao TI00267889 ; Tochigi Prefecture, Nikko-shi, Hanaishi-cho, Botanical Gardens , Graduate School of Science , The University of Tokyo, 641.0m alt. 14 July 2024, Nagao TI00272930 ; Gifu Prefecture, Nakatsugawa-shi, Kawaue , 20 May 2023, Nagao KPM-NC-0030309 and KPM-NC-0030310 ; Tokyo Metropolis, Nishitama-gun, Okutama-machi , Nokogiri-one , 688m alt. 27 May 2023, Nagao KPM-NC-0030311 ; Tokyo Metropolis, Nishitama-gun, Okutama-machi , Nokogiri-one , 541m alt. 27 May 2023 Nagao KPM-NC-0030312 .

Diagnosis:—It differs from E. butleri by septal numbers of basidiospores (3–4 vs 0–1 in E. butleri ).

Description:—The hymenium composed of basidia with 3–4 sterigmata and blastospores. Basidia clavate to cylindrical, 31–36 × 6.5–9.3 µm ( FIGURE 2F ), emerging directly from the host surface or through stomata. Sterigmata, 2.7–5.4 × 1.4–2.4 µm, developing outwardly and tapering toward the tip ( FIGURES 2E,2F ). Basidiospores ellipsoid, 10–19.7 × 2.9–6.3 µm, hyaline, smooth, one-celled when formed, becoming septate with 3–4 septa ( FIGURE 2D ).

Septate basidiospores germinating after 24 h when discharged onto an agar surface. Germ tubes of the basidiospores emerging from several cells and producing blastospores at the tips of germ tubes 24 h after discharge ( FIGURES 2G, 2H ). Germ tubes producing pseudohyphae and branching. Some basidiospores producing blastospores directly, i.e. budding ( FIGURES 2I, 2J ). Blastospores lacrimiform, subfusiform, and clavulate, 5–13 × 1 µm. Colonies composed of partially elongated pseudohyphae and blastospores on PDA. The surface of the colonies corrugate without a farinose appearance and 41. Salmon (5. 4YR/7. 9/5. 3) to 37. Flesh (8. 1R/7. 3/5. 8) according to A Mycological colour chart ( Rayner 1970), the same as the reverse of the colonies. Dark pigment not produced on PDA. Colonies glutinous and not fixing on the agar surface ( FIGURE 2K ).

Etymology:—Referring to Setsutai, the local village name in Iwate Prefecture, Japan where the first specimen of this fungus was collected by Dr. K. Sawada.

Symptom: — Blister on the leaves of R. semibarbatum was characterized by chlorotic leaf spots, 4.4–15.8 mm ( FIGURES 1D,1E ). Later, the center of infection became necrotic, whereas leaf blister on Rhododendron sect. Brachycalyx caused by Exobasidium yoshinagae Hennings (1902: 736) appears as a limited spot and is not developed outwardly with chlorotic area. On the leaves of lammas shoots grown in July 2024, blister also appeared. The appearance was circular ranging from 6 to 10 mm in diam ( FIGURE 1F ). Hymenium appeared densely in the center of lesions but the marginal chlorotic halo produced hymenium sparsely ( FIGURES 4A, 4B ). The lesion was slightly thickened ( FIGURES 4C, 4D ). The thickness of lesions ranged from 192.8–240.5 μm, while that of uninfected areas from 150.0–160.7 μm. The cell sizes of the upper epidermis of uninfected areas were 12.9–25.7 (32.1) × 15.4–30.3 (40.2) μm (mean: 20.9 × 24.7 μm), while those of infected areas were 12.9–25.7 × 21.4–38.6 (42.9) μm (mean: 19.8 × 30.5 μm). The cell sizes of palisade mesophyll of uninfected areas were 23.6–36.4 × 6.4–12.9 μm (mean: 30.3 × 9.5 μm), while those of infected areas were 23.6–34.3 × 8.6–10.7 (15) μm (mean: 29.6 × 10.1 μm). The cell sizes of spongy mesophyll of uninfected areas were 12.2–22.0 × 8.6–14.7 μm (mean: 17.0 × 12.6 μm), while those of infected areas were 10.7–27.9 × 8.6–19.3 μm (mean: 17.9 × 13.2 μm). No apparent hypertrophy cells were observed. These data suggested that the hyperplasia of spongy mesophyll caused a slight thickening of the infected area ( FIGURES 4C, 4D ).

Phylogenetic analysis:—The alignment included 1338 total characters of which 658 are constant (proportion = 0.491779), 271 variable characters are parsimony uninformative, while 409 characters are parsimony informative. The phylogenetic trees generated by NJ with high bootstrap value, 95 %, showed that E. setsutaiense is nested with E. dubium Raciborski (1909: 1172) , E. miyabei Nagao, Akimoto et Kishi (2003a: 5) , and E. yoshinagae from Rhododendron species in sect. Brachycalyx and Rhododendron dauricum Linnaeus (1753: 392) ( FIGURE 5 ), whereas the tree generated by MP and ML showed a sister clade with moderate bootstrap value 74% and 69%, respectively ( FIGURE 6 ). Rhododendron dauricum , a host of E. miyabei , belongs to sect. Rhododendron in subgen. Rhododendron L., whereas R. semibarbatum belongs to subgen. Mumeazalea .

Other members of the host plants belong to sect. Brachycalyx in subgen. Tsutsusi . This clade was highly supported with 96% and 87% by MP and ML, respectively, but the topology of E. yoshinagae clade, E. yoshinagae — E. dubium clade, and E. miyabei — E. setsutaiense clade were varied ( FIGURE 6 ). The phylogenetic position of Exobasidium species isolated from Ericaceae was highly supported with 100%, 95%, and 94% by NJ, MP, and ML, respectively.

The sequence similarity between E. setsutaiense and three closely related Exobasidium species:—The results from Needleman-Wunsch alignment of ITS and LSU sequences suggested that the similarities to sequences of E. setsutaiense LC 811668 and LC811679 were less than 96 % compared with E. miyabei OR 428370, E. yoshinagae AB 180340, and E. dubium AB 180346 for ITS and E. lushanense Zhen Ying Li & L. Guo EU 692789, E. dubium AB 178242, and E. yoshinagae AB 177551 for LSU, respectively ( TABLES 3, 4).

Figures

FIGURE 1. Symptom of blister on Rhododendron semibarbatum. A. The herbarium specimen of No. 9112 (IUM). Asterisk indicates discolored area, presumably chlorosis symptom. Arrow indicates a part of toothed leaf that is one of the specific characteristics of R. semibarbatum. B. Faintly farinose hymenium on the abaxial surface of R. semibarbatum (No. 1095). C. Chlorotic spot ranging from 8–10 mm in diam. on the adaxial surface of R. semibarbatum. Necrosis appeared dispersedly on the center of lesion (No. 5498). D. Field samples of blister on R. semibarbatum.Different hymenium appearance on the chlorosis spot. E. Farinose hymenium on the abaxial surface of R. semibarbatum (KPM-NC-0030311). F. Field samples of blister on the leaves of lammas shoot of R. semibarbatum (TI00272930). Photographed by Hideyuki Nagao.

FIGURE 2. Basidia and basidiospores of Exobasidium setsutaiense on blister of Rhododendron semibarbatum and colony appearance. Basidium (C) with sterigmata (arrowed) and basidiospores (A, B) with multiple septa (arrowed) from the herbarium specimen (No.9112). Basidium (F) and a part of basidium (E) with sterigmata (arrowed) and basidiospores (D) with multiple septa (arrowed) from the field sample (KPM-NC-0030312). Mode of germination of basidiospore is either by germ tube (G, H: KPM-NC-0030309) or by budding of blastospores (I, J: KPM-NC-0030312) after 48h on PDA. Arrows indicate blastospores. Arrowhead indicates branching point on germ tube. Colony on PDA after one month-old culture (K). Scale bars: A–F = 5 μm, G–J = 10 μm, K = 5 mm. Photographed by Hideyuki Nagao.

FIGURE 3. Basidium and basidiospores of Exobasidium setsutaiense from different samples. Basidium (B: KPM-NC-0030312)), basidiospores (A: KPM-NC-0029634, C: KPM-NC-0030309, D: KPM-NC-0030312)), and germinated basidiospores in the hymenium. Mode of germination of basidiospore is either by germ tube (E: KPM-NC-0030310, F: KPM-NC-0030309) or by budding of blastospores (D: KPM-NC-0030312)). Illustrated by Hideyuki Nagao.

FIGURE 4. Histological observation on blister on Rhododendoron semibarbatum infected by Exobasidium setsutaiense. A. An overview of cutting positions, B, C &D, for the histological observation. Blister (B & D) appeared slightly convex comparing with the non-infected area. B. Section at the middle of blister. Populated hymenial area was observed. C. Section at the non-infected area. Thickness of this area was about 150 μm (composition of cells). D. Section at the marginal part of infected area. Hymenium was sparsely observed. Thickness of this area was about 200 μm. ep: epidermis, hym: hymenium. Scale bar = 50 μm. Photographed by Hideyuki Nagao.

FIGURE 5. Neighbor joining tree of ITS-LSU concatenated data of Exobasidium. Bootstrap value is presented on the shoulders of branches. Values greater than 50% are indicated. The accession nos. for LSU and ITS are described in TABLE 2.

FIGURE 6. Quartet puzzling consensus Maximum likelihood (ML) and Maximum parsimony (MP) trees of ITS–LSU concatenated data of Exobasidium. Support value of ML (upper) and Bootstrap value of MP (lower) are presented on the shoulders of branches. Values greater than 50% are indicated. The accession nos. for LSU and ITS are described in TABLE 2.