Note
Snow mold fungus Racodium therryanum is phylogenetically Herpotrichia juniperi
2021 Volume 62 Issue 6 Pages 406-409
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2021 Volume 62 Issue 6 Pages 406-409
Racodium therryanum is a snow mold causing snow blight and seed rot in conifers. The sexual stage of R. therryanum has not been found, however, it has been speculated that Herpotrichia juniperi, which causes brown felt blight, is the sexual stage of R. therryanum. In this study, we conducted phylogenetic analysis using 28S large subunit (LSU) rDNA, 18S small subunit (SSU) rDNA, translation elongation factor 1-alpha (tef1), and RNA polymerase II second largest subunit (rpb2). Four isolates were identified as R. therryanum based on their morphological characteristics; together with two strains of H. juniperi, they composed a clade supported by high bootstrap and Bayesian posterior probability values. Therefore, we conclude that the snow mold previously described as R. therryanum is phylogenetically H. juniperi.
Racodium therryanum is a snow mold fungus that causes snow blight (Racodium snow blight) and seed rot in conifers and some broadleaf tree species (Cheng & Igarashi, 1987; Sakamoto & Miyamoto, 2005; Sato, Shoji, & Ohta, 1960). Racodium snow blight has been reported from northern Japan in regions such as Hokkaido and Tohoku, which experience heavy snowfall in winter. Racodium therryanum is an asexual fungus that was originally isolated from Picea abies and identified by De Thumen (1880); it was recently isolated from seeds of Korean fir (Abies koreana) in Korea (Cho, Miyamoto, Takahashi, Hong, & Kim, 2007). Racodium therryanum (as‘Rhacodium’therryanum in Saccardo, 1899) is characterized by felty, dark brown mycelium and hyphae that are 6-7 µm in width (De Thumen, 1880; Saccardo, 1899). It produces oval or globose chlamydospores on mature cultures (Sakamoto & Miyamoto, 2005). In Japan, Sato et al. (1960) identified a snow mold fungus as R. therryanum based on morphological characteristics described by Saccardo (1899). The sexual stage of this fungus has not been found.
The taxonomic placement of R. therryanum is not clear. Recently, Abdollahzadeh, Groenewald, Coetzee, Wingfield, and Crous (2020) newly introduced the order Racodiales closely related to Capnodiales including the type species of the genus Racodium (Racodium rupestre), but they did not discuss the placement of R. therryanum. On the other hand, some researchers have pointed out the taxonomic relationship between R. therryanum and Herpotrichia juniperi. Berlese (1892) suggested that R. therryanum and H. juniperi appear to be the same species, but made no definitive conclusion. Saccardo (1899) also indicated that R. therryanum is probably the subiculum of H. juniperi. In addition, Bose (1961) referred R. therryanum as a synonym of H. juniperi. However, their accurate taxonomic relationship has remained unclear. Herpotrichia juniperi is a member of the family Melanommataceae within the order Pleosporales (Dothideomycetes, Ascomycota); it has brown hyphae that are 4-6 µm in width, and produces ascospores within globose to subglobose pseudothecia that are 200-450 µm in diam (Sivanesan, 1971). It is also known that H. juniperi has pycnidia which develop on the old culture (Bose, 1961), but their role in its life cycle is unclear. Herpotrichia juniperi is a causal agent of brown felt blight in conifers of midwestern Eurasia and North America (Kuz'michev, Sokolova, & Kulikova, 2001; Sinclair & Lyon, 2005; Oskay, Lehtijärvi, Dogmuş-Lehtijärvi, & Halmschlager, 2011). Sakamoto and Miyamoto (2005) indicated the similarity of snow blight symptoms caused by R. therryanum and H. juniperi, and recommended that their relationship be investigated through molecular analyses. In this study, we conducted phylogenetic analysis of R. therryanum and H. juniperi using four major gene regions: 28S large subunit (LSU) rDNA, 18S small subunit (SSU) rDNA, translation elongation factor 1-alpha (tef1), and RNA polymerase II second largest subunit (rpb2).
We used four isolates of R. therryanum; one was identified by Sato et al. (1960) (MAFF 410406, deposited at the National Agriculture and Food Research Organization; Table 1) and the other three were collected from a nursery or natural forest at the University of Tokyo Hokkaido Forest (Furano, Hokkaido, Japan; Iwakiri, Sakaue, Matsushita, & Fukuda, 2021) or the Hokkaido University Teshio Experimental Forest (Teshio, Hokkaido, Japan). The isolates were obtained either from seedlings of two conifer species (Picea jezoensis and Picea glehnii) showing snow blight symptoms or from decayed seeds in Apr or May in 2009, 2018, and 2020 (Table 1). All four isolates were identified as R. therryanum according to morphological characteristics described by Sato et al. (1960), namely, felty, dark green to dark gray mycelia.
| Isolates | Source | Locality | Latitude | Longitude | Collection date | GenBank accession numbers | |||
| LSU | SSU | tef1 | rpb2 | ||||||
| HFRN18-240 | Picea jezoensis seedling | Furano, Hokkaido | 43°17'03"N | 142°26'28"E | May, 2018 | LC618842 | LC618848 | LC618854 | LC618860 |
| HTSO20-756 | P. jezoensis seed | Teshio, Hokkaido | 45°02'34"N | 142°01'18"E | Apr, 2020 | LC618843 | LC618849 | LC618855 | LC618861 |
| MAFF 410406 | P. jezoensis seedling | Tomakomai, Hokkaido | N/A | N/A | Apr, 1952 | LC618844 | LC618850 | LC618856 | LC618862 |
| Rac09Pj11-1 | P. jezoensis seedling | Furano, Hokkaido | 43°13'11"N | 142°22'51"E | May, 2009 | LC618845 | LC618851 | LC618857 | LC618863 |
DNA was extracted from mycelia grown on potato dextrose agar plates at 5 °C for 1 mo using the PrepMan Ultra sample preparation reagent (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. The primers LR0R/LR5 (Rehner & Samuels, 1994), NS1/NS4 (White, Bruns, Lee, & Taylor, 1990), EF983F/EF2218R (Rehner & Buckley, 2005), and fRPB2-5f/fRPB2-7cR (Liu, Whelen, & Hall, 1999) were used for polymerase chain reaction (PCR) amplification of LSU, SSU, tef1, and rpb2, respectively, under the following thermal conditions: 95 °C for 2 min, followed by 35-40 cycles at 95 °C for 10 s, 55 °C for 10 s, 72 °C for 45 s, with a final extension at 72 °C for 10 min. The annealing temperatures for tef1 and rpb2 were 60 °C and 52 °C, respectively. The PCR products were purified and sequenced at Macrogen Japan (Kyoto, Japan). In addition to the four R. therryanum isolates, sequences of the strains H. juniperi CBS 467.64 and CBS 200.31 (for rpb2 only) and Herpotrichia macrotricha JCM 14419 were newly generated. The sequence data were edited using BioEdit 7.2.5 (Hall, 1999) and aligned using MUSCLE (Edgar, 2004) in MEGA7 (Kumar, Stecher, & Tamura, 2016). Concatenated sequences were aligned with 21 Melanommataceae species (Table 2). Melanomma pulvis-pyrius was selected as the outgroup. All aligned sequences were deposited in TreeBASE (ID: S27997). A phylogenetic tree was constructed using the maximum likelihood (ML) and Bayesian inference (BI) methods. ML estimation was conducted using IQ-TREE ver. 1.6.12 (Nguyen, Schmidt, von Haeseler, & Minh, 2015) with 1000 ultrafast bootstrap replicates. K2P+R3 was selected as the best evolution model for LSU via ModelFinder (Kalyaanamoorthy, Minh, Wong, von Haeseler, & Jermiin, 2017) in IQ-TREE. Similarly, F81+F+I, TIM2+F+G4, and TNe+G4 were selected as the best models for SSU, tef1, and rpb2, respectively. BI analysis was conducted using MrBayes 3.2.7 (Ronquist et al., 2012). The best model for each region was determined using MrModeltest v. 2.3 (Nylander, 2004) based on the hierarchical likelihood ratio test (hLRT). The best models were GTR+I+G for LSU, F81 for SSU, GTR+G for tef1, and SYM+I+G for rpb2. Four Marcov chains were run for 1,000,000 generations and trees were sampled every 100 generations. The first 25% of generations were discarded as burn-in. The phylogenetic tree was visualized using FigTree 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).
| Species | Strain | GenBank accession numbersa | |||
| LSU | SSU | tef1 | rpb2 | ||
| Alpinaria rhododendri | CBS 141994 | KY189973 | KY190004 | KY190009 | KY189989 |
| Aposphaeria corallinolutea | CBS 131287 | JF740330 | - | - | - |
| Bertiella macrospora | IL 5005 | GU385150 | - | - | - |
| Gemmamyces piceae | CBS 141555 | KY189976 | KY190006 | KY190011 | KY189992 |
| Herpotrichia juniperi | CBS 200.31 | DQ678080 | DQ678029 | DQ677925 | LC618864 |
| CBS 467.64 | LC618846 | LC618852 | LC618858 | LC618865 | |
| Herpotrichia macrotricha | GKM 196N | GU385176 | - | GU327755 | - |
| JCM 14419 | LC618847 | LC618853 | LC618859 | LC618866 | |
| Herpotrichia vaginatispora | MFLUCC 13-0865 | KT934252 | KT934256 | KT934260 | - |
| Melanocucurbitaria uzbekistanica | MFLUCC 17-0829 | MG829022 | MG829129 | MG829209 | - |
| Melanodiplodia tianschanica | MFLUCC 17-0805 | MG829023 | MG829130 | MG829210 | MG829256 |
| Melanomma pulvis-pyrius | CBS 124080 | GU456323 | GU456302 | GU456265 | GU456350 |
| Monotosporella tuberculata | CBS 256.84 | GU301851 | - | GU349006 | - |
| Muriformistrickeria rosae | MFLU 16-0227 | MG829028 | MG829135 | MG829215 | - |
| Muriformistrickeria rubi | MFLUCC 15-0681 | KT934253 | KT934257 | KT934261 | - |
| Phragmocephala atra | MFLUCC 15-0021 | KP698725 | KP698729 | - | - |
| Praetumpfia obducens | CBS 141474 | KY189984 | KY190008 | KY190019 | KY190000 |
| Pseudostrickeria muriformis | MFLUCC 13-0764 | KT934254 | KT934258 | KT934262 | - |
| Pseudotrichia mutabilis | PM 1 | KY189988 | - | KY190022 | KY190003 |
| Sarimanas pseudofluviatile | MAFF 239465 | LC001714 | LC001711 | - | - |
| Sarimanas shirakamiense | MAFF 244768 | LC001715 | LC001712 | - | - |
| Seifertia azaleae | DAOM 239136 | EU030276 | - | - | - |
| Seifertia shangrilaensis | MFLUCC 16-0238 | KU954100 | KU954101 | KU954102 | - |
a Sequences newly obtained in this study are indicated in bold
The sequences of the four DNA regions were 902 bp (LSU), 1094 bp (SSU), 1038 bp (tef1), and 1144 bp (rpb2) in length. The sequences of the four R. therryanum isolates showed high similarities with those of H. juniperi isolates CBS 200.31 (LSU, 99.7-99.9%; SSU 99.9-100%; tef1, 95.9-98.9%; rpb2, 95.9-97.6%) and CBS 467.64 (LSU, 99.3-99.6%; SSU, 99.9-100%; tef1, 95.4-98.4%; rpb2, 96.3-98.3%). Also, the combined LSU-SSU-tef1-rpb2 phylogenetic tree showed that the four R. therryanum isolates were grouped with the H. juniperi isolates, which were distinctively branched from other Melanommataceae species with high ML bootstrap (MB: 99%) and Bayesian posterior probability (BP: 0.99) values (Fig. 1). Therefore, all isolates tested in this study were identified as H. juniperi, and the snow mold fungus in Japan, which has been identified as R. therryanum, was revealed to phylogenetically be H. juniperi. Based on this result, we refer to the Japanese snow mold R. therryanum as H. juniperi in the following sections. Since H. juniperi was validly published as‘Sphaeria’juniperi in 1854 (Rabenhorst 1854), before the description of R. therryanum in 1880, Bose's description (1961) which synonymized R. therryanum with H. juniperi seems to be authentic. Type specimens and European isolates of R. therryanum should be re-examined to confirm the synonymization.
Although H. juniperi specimens isolated in Japan, Eurasia, and North America are phylogenetically the same species, their life cycles exhibit significant differences. In Japan, H. juniperi mycelia on infected twigs usually disappear soon after snowmelt (annual growth), whereas in Eurasia and North America, they grow perennially on the plant surface (Sinclair & Lyon, 2005). In midwestern Eurasia and North America, H. juniperi produces ascospores in summer or autumn, whereas in Japan it is always asexual and its sexual stage has not yet been found. Population genetic analyses have indicated that H. juniperi in Japan (discussed as R. therryanum) may undergo sexual reproduction (Iwakiri et al., 2021). Further studies of the life cycle of H. juniperi, especially in seasons when ascospores can be produced, are needed to elucidate differences among H. juniperi in Japan and in other regions.
The phylogenetic tree indicated that four Japanese H. juniperi isolates were divided into two well-supported subclades (MB > 90, BP > 0.95, Fig. 1). Iwakiri et al. (2021) found that H. juniperi in Japan (as R. therryanum) included several genetically differentiated groups. These results suggest the genetic diversity of H. juniperi in Japan. Schneider, Grünig, Holdenrieder, and Sieber (2009) reported that H. juniperi isolates from a mountainous region in Switzerland were divided into five genetically differentiated groups that may represent cryptic species. Further phylogenetic analyses of many samples from various regions are necessary to discuss the genetic diversity and species delimitations of H. juniperi.
The authors declare no conflicts of interest.
We appreciate Dr. Daisuke Sakaue for providing a Racodium therryanum culture. We also thank the staff of University of Tokyo Hokkaido Forest and Hokkaido University Teshio Experimental Forest for sample collection. This study was partly supported by Ministry of Education, Culture, Sports, Science and Technology-Japan project on“Joint usage / Education Center”in Forest Research Station, Field Science Center for Northern Biosphere, Hokkaido University. This work was financially supported by JSPS KAKENHI Grant Number JP18H02231.
