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Research Article
Four new Phragmidium (Phragmidiaceae, Pucciniomycetes) species from Rosaceae plants in Guizhou Province of China
expand article infoJing-E Sun‡, Qian Zhang‡, Wen-Mei Luo‡, Yuan-Qiao Yang‡, Hua-Ming An‡§, Yong Wang‡
‡ Guizhou University, Guiyang, China
§ Guizhou Engineering Research Center for Fruit Crops, Guiyang, China

Corresponding author: Yong Wang ( yongwangbis@aliyun.com )

Academic editor: Margarita Dueñas
© 2022 Jing-E Sun, Qian Zhang, Wen-Mei Luo, Yuan-Qiao Yang, Hua-Ming An, Yong Wang.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Sun J-E, Zhang Q, Luo W-M, Yang Y-Q, An H-M, Wang Y (2022) Four new Phragmidium (Phragmidiaceae, Pucciniomycetes) species from Rosaceae plants in Guizhou Province of China. MycoKeys 93: 193-213. https://doi.org/10.3897/mycokeys.93.90861
Open Access

Abstract

In this study, four new species of Phragmidium were proposed based on morphological and molecular characters. In morphology, Phragmidium rosae-roxburghii sp. nov. was distinguished to related taxa by its unique square to diamond-shaped urediniospores; Ph. rubi-coreani sp. nov. differed from Ph. barclayi and Ph. cibanum because of teliospores with fewer cells and shorter pedicels; urediniospores of Ph. potentillae-freynianae sp. nov. were bigger than Ph. duchesneae-indicae; and Ph. rosae-laevigatae sp. nov. produced bigger urediniospores than Ph. jiangxiense. The phylogenetic analyses based on the combination of two loci (ITS and LSU) also supported our morphological conclusion. In the meantime, three previously known species were also described herein.

Keywords

Basidiomycota, ITS, LSU, phylogeny, rust disease, taxonomy

Introduction

Phragmidium (Phragmidiaceae) was established by Link (1816) and characterized by laterally separated multicellular teliospores with pigmented bilaminar walls, and a thickened pedicel at the base (Wei 1988).

The genus was widely distributed around the world especially in the northern hemisphere, such as China, USA and Japan (Wei 1988; Zhuang 1989; Cummins and Hiratsuka 2003; Maier et al. 2003; Zhuang et al. 2012; Pscheidt and Rodriguez 2016; Liu et al. 2018, 2019, 2020; Zhao et al. 2021). Phragmidium species often caused severe rust diseases in Rosaceae plants (Rosa, Rubus, Potentilla, Sanguisorba, Duchesnea and Acaena). Species of Phragmidium have been reported growing on host plants of Rosa, Rubus, and Potentilla, with a few species on Sanguisorba (Cummins and Hiratsuka 2003; Maier et al. 2003; Yun et al. 2011; Pscheidt and Rodriguez 2016; Liu et al. 2018, 2019, 2020), Duchesnea (Zhao et al. 2021) and Acaena (McTaggart et al. 2016). Two species Ph. mucronatum (Pers.) Schltdl. and Ph. tuberculatum Jul. Müll., were common pathogens on ornamental roses worldwide (Wahyuno et al. 2001, 2002; Leen and Van Huylenbroeck 2007; Wilson and Aime 2014).

About 8000 species of rust fungi have been reported in the world (Zhao et al. 2021). Based on morphological features or host associations, 1200 species belonging to 71 genera of 15 families were previously reported in China. Over 70 Phragmidium species have been described (Cummins 1931; Arthur 1934; Zhuang et al. 1998, 2003, 2005, 2012; Wahyuno et al. 2001; Cummins and Hiratsuka 2003; Yang et al. 2015; Ali et al. 2017; Aime et al. 2018; Liu et al. 2018, 2019, 2020; Ono and Wahyuno 2019; Aime and McTaggart 2021; Zhao et al. 2021).

Traditionally, Phragmidium species are distinguished based on teliospores morphology (Wei 1988). According to Wahyuno et al. (2001) and Zhao et al. (2021) Phragmidium species are difficult to distinguish based only on morphology of asexual spore stages; thus, DNA data is essential for taxonomy and identification of Phragmidium species.

The combination of morphological and molecular characters has been applied in the taxonomy of rust fungi (Beenken et al. 2012; Beenken 2014; McTaggart et al. 2016, 2017; Liu et al. 2018, 2019, 2020; Ono and Wahyuno 2019; Zhao et al. 2021). Phragmidium includes more than 270 epithet records which are listed in MycoBank (https://www.mycobank.org) and Index Fungorum (http://www.indexfungorum.org) (accessed in October 2022). However, only 28 records were described and named by Chinese researchers, three Phragmidium taxa in Guizhou Province, Ph. duchesneae-indicae, Ph. nonapiculatum and Ph. kans were introduced by Zhao et al. (2021). In the present study, thirteen fresh rust specimens were collected on eight Rosaceae hosts, such as Duchesnea indica, Potentilla freyniana, P. kleiniana, Rosa roxbunghii, R. laevigata, Rosa sp., Rubus coreanus and Ru. parrifolius in Guizhou Province. This study aimed to determine the taxonomic status of the parasitic pecies of the Rosaceae in Guizhou Province through morphological and molecular characters. Meanwhile, we hope to contribute a significant amount of molecular data that may aid future studies and phylogenetic placement of Phragmidium in the Pucciniales.

Materials and methods

Sampling and microscopy observation

Thirteen fresh rust specimens were collected on branch and leaf from eight species of Rosaceae, Duchesnea indica, Potentilla freyniana, P. kleiniana, Rosa roxbunghii, R. laevigata, Rosa sp., Rubus coreanus and R. parrifolius in Guizhou Province, China. The spores from specimens were mounted in sterile water, on slides and observed using a Zeiss Scope 5 compound microscope (Axioscope 5, Jena, Germany), and photographed with an AxioCam 208 color (Jena, Germany) camera and saved as JPG files. Approximately 30 measurements were made of each feature using the ZEN 2.0 (blue edition) software. The Flora of China (http://www.efloras.org/flora_page.aspx?flora_id=4) was used to identify host plants (Liu et al. 2018). The rust specimens were deposited in the HGUP Herbarium of Department of Plant Pathology, Agricultural College, Guizhou University. Taxonomic details of our novel taxa were submitted to MycoBank (www.mycobank.org).

DNA extraction, PCR and sequencing

Rust spores were scraped from fresh plant tissues using a sterile scalpel. Total DNA of rust spores was extracted with a BIOMIGA Fungus Genomic DNA Extraction Kit (GD2416) according to the manufacturer’s protocol. Targeted sequences of internal transcribed spacer of rDNA (ITS) was amplified using primers ITS4rust (5’-CAGATTACAAATTTGGGCT-3’) (Beenken et al. 2012) and Rust2inv (5’-GATGAAGAACACAGTGAAA-3’) (Aime 2006), and the large subunit (LSU) of the ribosomal RNA gene was amplified using the primers No.4 (5’-ACCCGCTG AATTTAAGCATAT-3’)/No.11 (5’-CTCCTTGGTCCGTGTTTCAAGACGC-3’) (Van der Auwera et al. 1994), or LR6 (5’-CGCCAGTTCTGCTTACC-3’) (Vilgalys and Hester 1990), and LR0R (5’-ACCCGCTGAACTTAAGC-3’) (Hopple 1994). The PCR cycling conditions were as described by Liu et al (2018). The PCR amplicons from purification and sequencing were carried out at Sangon Biotech (Chengdu, China). Newly-generated sequences were deposited in GenBank (Table 1).

Table 1.
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Specimens and GenBank accession numbers of rust isolates included in this study.

Species Voucher specimens Host Locality ITS LSU
Phragmidium andersoni HMAS-53231 T Potentilla fruticosa Sinkiang, China N/A MG669120
Ph. altaicum BJFCR03247 Rosa albertii China MH285385 MH285381
BJFCR03246 Rosa albertii China MH285384 MH285380
BJFCR03217 T Rosa albertii China MH285383 MH285379
Ph. barclayi HMAS-67281 Rubus austrotibetanus Tibet, China N/A MG669117
Ph. barnardii BRIP 56945 Rubus parvifolius South Africa N/A KT199402
Ph. barnardii HGUP21035 Rubus parvifolius Guizhou, China OL684828 OL684839
Ph. biloculare BPI:881121 Potentilla flabellifolia USA N/A JF907670
Ph. butleri HMAS-67841 Rosa macrophylla Tibet, China N/A MG669118
Ph. chayuensis BJFC-R02532 T Rosa duplicata Tibet, China N/A MG669112
BJFC-R03014 T Rosa duplicata Tibet, China N/A MG669113
Ph. cibanum BJFCR02528 T Rubus niveus Tibet, China MH128370 MG669110
BJFCR03012 T Rubus niveus Tibet, China MH128371 MG669111
Ph. duchesneae-indicae HGUP21031 Duchesnea indica Guizhou, China OL684824 OL684835
HGUP21032 Duchesnea indica Guizhou, China OL684825 OL684836
Ph. fragariae WM 1317 Potentilla sterilis Europe N/A AF426217
Ph. fusiforme T-10 Rosa pendulina Switzerland N/A AJ715522
Ph. fructigenum HMUT100472 Rosa glomerata Guangdong, China N/A KU059168
Ph. griseum BJFCR03449 Rubus crataegifoliu Beijing, China MN264712 MN264730
BJFCR03451 Rubus crataegifoliu Beijing, China MN264713 MN264731
HMAS56906 Rubus crataegifoliu Beijing, China N/A MG669115
Ph. handelii BJFC-R01030 Rosa webbiana Gansu, China N/A KP407631
Ph. ivesiae BPI-877968 Potentilla gracilis USA N/A JF907673
BPI-863637 Potentilla gracilis USA N/A JF907672
BJFC-R01421 Rosa webbiana Gansu, China N/A KP407628
Ph. japonicum HMAS41585 Rosa laevigata Fujian, China MN264716 MN264734
IBAR8174 Rosa luciae Ibaraki, Japan MN882389 MN848143
Ph. jiangxiense BJFCR03452 Rosa laevigata Jiangxi, China MN264714 MN264732
BJFCR03453 T Rosa laevigata Jiangxi, China MN264715 MN264733
Ph. leucoaecium BJFCR02116 Rosa sp. Yunnan, China MN264718 MN264736
BJFCR02118 T Rosa sp. Yunnan, China MN264719 MN264737
Ph. longissima BJFC-R00338 Rosa lichiangensis Yunnan, China N/A KP407633
BJFC-R00360 Rosa lichiangensis Yunnan, China N/A KP407634
Ph. mexicanum BPI 843961 Potentilla indica Maryland, USA N/A JF907660
BPI 843829 Potentilla indica Virginia, USA N/A JF907659
Ph. mucronatum RUBO Rosa sp. Bochum, Germany N/A KU059171
TUB 012090 Rosa corymbifera Germany N/A AJ715520
Ph. montivagum HMAS67176 Rosa davurica China N/A KU059173
FO 47828 Rosa woodsii NA N/A AF426213
Ph. octoloculare HMAS-140416 Rubus biflorus Tibet, China N/A MG669119
Ph. potentillae HMAS53236 Potentilla virgata Sinkiang, China N/A MG669114
BJFCR00961 Potentilla chinensis Qinghai, China MN264720 MN264738
Ph. potentillae HGUP21034 Potentilla kleiniana Guizhou, China OL684827 OL684838
Ph. potentillae-canadensis BPI877886 Potentilla sp. North Carolina, USA N/A JF907667
BPI877885 Potentilla canadensis Maryland, USA N/A JF907668
Ph. potentillae-freynianae HGUP21033 T Potentilla freyniana Guizhou, China OL684826 OL684837
Ph. punjabense BA-65A T Rosa brunonii Pakistan N/A KX358854
BA-65B Rosa brunonii Pakistan N/A KX358855
Ph. rosae-laevigatae HGUP21036 T Rosa laevigata Guizhou, China OL684829 OL684840
HGUP21037 Rosa laevigata Guizhou, China OL684830 OL684841
Ph. rosae-multiflorae HMAS71053 Rosa multiflora Shanxi, China N/A KU059174
HMAS94924 Rosa multiflora Zhejiang, China N/A KU059175
BJFCR03454 Rosa multiflora Jiangxi, China MN264721 MN264739
Ph. rosae-roxburghii HGUP21025 T Rosa roxburghii Guizhou, China OL684818 OL684831
HGUP21026 Rosa roxburghii Guizhou, China OL684819 OL684832
HGUP21027 Rosa roxburghii Guizhou, China OL684820 N/A
HGUP21028 Rosa sp. Guizhou, China OL684821 OL678103
Ph. rosae-rugosae BJFCR03455 Rosa rugosa Jiangxi, China MN264722 MN264740
BJFCR03456 Rosa rugosa Beijing, China MN264723 MN264741
Ph. rubi-idaei WM 1024 Rubus idaeus Europe N/A AF426215
BRIP 59372 Rubus idaeus Australia N/A MW147044
Ph. rubi-oldhami HMAS-64306 Rubus pungens Sichuan, China N/A MG669116
Ph. rubi-corean HGUP21029 T Rubus coreanus Guizhou, China OL684822 OL684833
HGUP21030 Rubus coreanus Guizhou, China OL684823 OL684834
Phragmidium sp. HMAS41561 Rosa multiflora Fujian, China MN264717 MN264735
Ph. sanguisorbae BPI 872232 Sanguisorba minor USA N/A JF907674
ML 957 Sanguisorba minor Europe N/A AF426216
Ph. tormentillae BPI 843392 Potentilla sp. Maryland, USA DQ354553 DQ354553
BPI 877888 Potentilla simplex Tennessee, USA N/A JF907669
Ph. tuberculatum BJFCR00959 Rosa sp. Qinghai, China N/A KP407636
BPI 877978 Rosa sp. California, USA N/A KJ841919
BPI 843677 Rosa sp. Argentina N/A KJ841921
Ph. violaceum MCA2782 Rubus sp. France DQ142909 DQ142909
BPI 871510 Rubus sp. Oregon, USA DQ142910 DQ142910
BJFCR03457 Rubus sp. New Zealand MN264724 MN264742
Ph. warburgianum BJFCR03458 Rosa bracteata Japan MN264726 MN264744
BJFCR03459 Rosa bracteata Japan MN264727 MN264745
Ph. zangdongii BJFCR02447 T Rosa tibetica Tibet, China MH128372 MG669108
BJFCR03013 T Rosa tibetica Tibet, China MH128373 MG669109
Ph. zhouquensis BJFCR01516 T Rosa omeiensis Yunnan, China MN264728 MN264746
BJFCR01529 T Rosa omeiensis Yunnan, China MN264729 MN264747
Trachyspora intrusa BPI 843828 Alchemilla vulgaris Switzerland DQ354550

T = Type specimens. New specimens are in bold typeface.

Phylogenetic analyses

81 sequences, including originated from thirteen specimens and related sequences of Phragmidium spp. were aligned in the online version of MAFFT v. 7.307 (Katoh and Standley 2016). Trachyspora intrusa (BPI 843828) was selected as outgroup (Liu et al. 2020). The alignment document was edited using MEGA6 (Tamura et al. 2013) and manually adjusted when necessary.

All relevant sequences of ITS—LSU dataset were conducted using maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI) methods. ML analysis was performed using RAxML-HPC2 v.8.2.12 (Stamatakis 2014). Gaps were treated as “missing”. The MP analysis of the two loci (ITS and LSU) was implemented with PAUP v. 4.0b10 (Swofford 2002). The phylogenetic trees were generated using the heuristic search option with tree bisection reconnection (TBR) branch swapping and 1000 random sequence additions. The maxtrees was set to 5000. The tree length (TL), consistency index (CI), homoplasy index (HI), retention index (RI), and rescaled consistency index (RC) were calculated. Bayesian inference analysis was inferred by MrBayes 3.2.6 (Ronquist et al. 2012). The best model for two loci (ITS and LSU) was determined by MrModeltest v2 (Nylander 2004), ITS: HKY+G, LSU: GTR+I+G. BI were performed by six Markov chain Monte Carlo. These chains were run for 5 million generations, sampling tree every 100 generations. The first 25% of resulting trees were discarded as burn-in phase of each analysis, and trees were saved every 5000 generations. Alignment matrices have been uploaded as an attachment.

Results

Phylogenetic analyses

The phylogenetic trees accommodated 82 sequences listed in Table 1. The combined alignment including ITS (493 bp) and LSU (544 bp) regions consisted of 1067 characters, of which 585 were constant, 89 variable characters were parsimony uninformative, and 363 were parsimony informative. We built three phylogenetic trees, ML tree, MP tree and BI tree. The MP tree was selected to represent the phylogenetic relationship of different Phragmidium taxa (Fig. 1). MP analysis produced the following parameters: tree length (TL) = 1011; consistency index (CI) = 0.643; homoplasy index (HI) = 0.356; retention index (RI) = 0.898; and rescaled consistency index (RC) = 0.578. Phragmidium rubi-coreani on Rubus coreanus with telial, aecial and uredinial stages formed a small branch only. Phragmidium potentillae-freynianae and Ph. duchesneae-indicae constituted a distinct subclade with high statistical support (100 ML/99 MP/1.00 PP). Phragmidium rosae-laevigatae was phylogenetically sister to Ph. leucoaecium, Ph. japonicum, Ph. jiangxiense and Phragmidium sp. with high support (100 ML/100 MP/1.00 PP). The four aecial-uredinial fungi on Ro. roxburghii kept identical base composition on ITS and LSU gene regions and made up a distinct subclade to Ph. warburgianum with high support (100 ML/99 MP/1.00 PP). Our strains represented four novel taxa, which was also supported by comparison of the DNA base pair differences between our strains and related taxa on ITS and LSU gene region.

Figure 1. 

The maximum parsimony tree of 42 Phragmidium taxa based on ITS and LSU genes; host plants are also given.

The hosts of the Phragmidium species were mainly concentrated in Rosa, Rubus and Potentilla of Rosaceae (Fig. 1). Eighty-one Phragmidium strains clustered together as a clade, which was roughly divided into three subclades (Subclade I, Subclade II and Subclade III). For Subclade I with 16 species (Ph. rubi-coreani, Ph. barclayi, Ph. cibanum, Ph. violaceum, Ph. barnardii, Ph. griseum, Ph. rubi-idaei, Ph. altaicum, Ph. tuberculatum, Ph. octoloculare, Ph. sanguisorbae, Ph. punjabense, Ph. rubi-oldhami, Ph. butleri, Ph. zhouquensis and Ph. fragariae) (67 ML/59 MP), their hosts belonged to Rosa, Rubus, Potentilla, and Sanguisorba. Phragmidium rubi-coreani and Ph. rubi-ideai associated with host plants on the generic level had obvious genetic distance. Subclade II included 18 Phragmidium taxa (Ph. biloculare, Ph. potentillae, Ph. ivesiae, Ph. montivagum, Ph. fructigenum, Ph. zangdongii, Ph. fusiforme, Ph. handelii, Ph. rosae-rugosae, Ph. mucronatum, Ph. chayuensis, Ph. longissima, Ph. rosae-multiflorae, Ph. mexicanum, Ph. potentillae-canadensis, Ph. potentillae-freynianae, Ph. duchesneae-indicae and Ph. tormentillae) (95 ML), but their host plants only referred to Rosa, Potentilla and Duchesnea. Phragmidium potentillae-freynianae and Ph. duchesneae-indicae belonging to different generic host plants were accommodated to a branch (100 ML/99 MP/1.00 PP), but Ph. mexicanum and Ph. potentillae-canadensis formed a clade (99 ML/86 MP/1.00 PP) separated from Ph. potentillae-freynianae with the congeneric host plants. Phragmidium tormentillae associated with Potentilla canadensis (P. simplex) as its host formed an independent branch (97 ML/61 MP/0.94 PP). The Phragmidium host plants in Subclade III (Ph. rosae-roxburghii, Ph. warburgianum, Ph. japonicum, Ph. jiangxiense, Phragmidium sp., Ph. leucoaecium, Ph. rosae-laevigatae, Ph. andersoni) belonged to Rosa and Potentilla. Phragmidium rosae-laevigatae and Ph. rosae-roxburghii with the same generic host plants did not group together (97 ML /0.98 PP). Phragmidium japonicum, Ph. jiangxiense and Phragmidium sp. (HMAS51561) all from Rosa formed a branch (100 ML/100 MP/1.00 PP). Phragmidium andersoni collected from Potentilla fruticosa formed an independent branch.

RA×ML and MP bootstrap support values (MP ≥ 50%), and Bayesian posterior probability (PP ≥ 0.90) are marked on the nodes as (ML/MP/PP). Specimens from current study have put in bold and put an H in the selected holotypes. The outgroup was Trachyspora intrusa (BPI 843828). The scale bar indicates 30 expected changes per site.

Taxonomy

Phragmidium rosae-roxburghii J.E. Sun & Yong Wang bis, sp. nov.

MycoBank No: 845041
Figs 2, 3

Diagnosis

Phragmidium rosae-roxburghii easily to be distinguished by its unique square to diamond-shaped urediniospores.

Holotype

China. Guizhou Province, Panzhou city, 25°89'61"N, 104°56'07"W, 750 m, 21 Mar 2021, on Rosa roxburghii, coll. J.E. Sun & Y.Q. Yang, HGUP21025, ITS: OL684818, LSU: OL684831.

Etymology

Referring to the host, Rosa roxburghii, on which the fungus was first found.

Description

Spermogonia : unknown. Aecia formed on gold distinct, circular lesions on both sides of the stems, petioles and leaves, rarely produced on the abaxial leaf surface, scattered, flat oval to subglobose, powdery, 1.0–5.0 mm diam. Aeciospores formed in basipetal succession, oval o subglobose, 22–30 × 14–22 µm (mean 26 × 18 µm, n = 30), inclusions golden, to bright-yellow; wall 1.8–3.1 µm thick, colorless, mostly with irregularly elongated verrucae on the surface. Uredinia produced on the abaxial leaf surface, scattered to gregarious, hypophyllous, orange-colored or white, powdery, oval to rounded, 0.1–1.0 mm diam, paraphysis in the periphery of the uredinia, curved, 30–55 × 9–20 µm, colorless thin-walled. Urediniospores generally angular, square to diamond-shaped, yellowish to orange-colored, 20–30 × 16–21 µm (mean: 25 × 19 µm, n = 30), thick-walled, 0.5–2.0 µm thick, colorless, regularly echinulate with stout spines.

Rust diseases symptoms: In the early stage (March) of rust disease yellowish-orange powdery aecia formed on the stems and petioles on Rosa roxburghii and Rosa sp., the aecia were scattered, flat oval or nearly round and bordered (Fig. 2). In middle of June (Fig. 3), the upper surface of the lower leaves was turning yellow and orange spots gradually appeared on the under surface caused by uredinia, which are powdery, aggregated but without obvious boundaries.

Figure 2. 

Phragmidium rosae-roxburghii sp. nov. (HGUP21025, holotype) on Rosa roxburghii a–c aecia on stem and leaf pieces. d longitudinal section of aecium e–h aeciospores. Scale bars: 2 mm (b–c); 50 µm (d); 10 µm (e–h).

Figure 3. 

Phragmidium rosae-roxburghii sp. nov. (HGUP21026) on Rosa roxburghii a appearance of infected plants b uredinia on a leaf c longitudinal section of uredinium d paraphyses e–i urediniospores. Scale bars: 5 mm (b); 50 µm (c); 25 µm (d); 12.5 µm (e–i).

Habitat

Rosa roxburghii, Rosa sp.

Known distribution

China, Guizhou Province.

Additional material examined

China. Guizhou Province: Duyun city, 26°45'88"N, 106°98'42"W, 820 m, 22 Jun 2021, on Rosa roxburghii, coll. J.E. Sun, HGUP21026; Tongren city, 28°14'09"N, 108°34'03"W, 810 m, 04 Sep 2021, on Rosa roxburghii, coll. J.E. Sun, HGUP21027; Guiyang city, 26°44'74"N, 106°58'67"W, 960 m, 27 Mar, 2021, on Rosa sp., coll. J.E. Sun, HGUP21028.

Notes

Phragmidium rosae-roxburghii was the first species of Phragmidium described on Rosa roxburghii. It is easily to distinguish species by its unique square to diamond-shaped urediniospores, since in other Phragmidium species the urediniosporas are oval to nearly spherical (Yun et al. 2011; Ono 2012; Zhuang et al. 2012; Yang et al. 2015; Liu et al. 2018, 2019, 2020; Ono and Wahyuno 2019). In phylogeny, this species only kept a close relationship to Ph. warburgiana (Fig. 1) but its urediniospores are yellowish to orange-colored different to Ph. warburgiana with colorless urediniospores (Ono 2012). We proposed Ph. rosae-roxburghii as a new taxon.

Phragmidium rubi-coreani J.E. Sun & Yong Wang bis, sp. nov.

MycoBank No: 845042
Fig. 4

Diagnosis

Phragmidium rubi-coreani differs to Ph. barclayi by teliospores with fewer cells and shorter pedicels.

Holotype

China. Guizhou Province: Guiyang city, 26°45'86"N, 106°98'77"W, 970 m, 11 Apr, 2021, on Rubus coreanus, coll. J.E. Sun, HGUP21029, ITS: OL684822, LSU: OL684833.

Etymology

Referring to the host, Rubus coreanus, on which this species grows.

Description

Spermogonia : unknown. Aecia golden, produced on the abaxial leaf surface, hypophyllous, and 2.5–3.5 mm diam, subglobose to globose, powdery, 2.5–3.5 mm diam. Aeciospores produced in basipetal succession, subglobose, 14–24 × 10–23 µm (mean 19 × 16 μm, n = 30), bright yellow contents, thick-walled, 1.0–4.0 µm, colorless, echinulate; paraphyses clavate, not or weakly incurved, 38–61 μm long, thick-walled, wall 2.0–2.5 μm thick. Telia hypophyllous, scattered, 0.3–0.5 mm diam, chocolate-brown. Teliospores ellipsoid to cylindrical, 3–5 celled, constricted at the septa, bright orange, chocolate-brown to gray-brown, 29–74 × 14–37 µm (mean 50 × 25 μm, n = 30), thick-walled, wall 1.8–3.5 μm thick, colorless to chocolate-brown; pedicels not swollen at the base, 8–34 μm long, colorless. Uredinia formed on circular lesions on both sides of the leaves, powdery, yellow distinct, hypophyllous scattered, nearly oval, surrounded by host epidermis, 0.5–1.0 mm diam. Urediniospores: uredo-type, subglobose to oval, produced in basipetal succession, golden, or bright-yellow, 19–27 × 15–25 µm (mean 23 × 20 μm, n = 30), thick-walled, wall 0.8–1.5 µm thick, colorless, densely and minutely echinulate.

Rust diseases symptoms: The golden and powdery aecia were first produced on the underside of leaves. Then, scattered uredinia were formed, orange-colored and forming small round spots on the leaves. Chocolate-brown telia were produced on the leaf remnants (Fig. 4).

Figure 4. 

Phragmidium rubi-coreani sp. nov. (HGUP21029, holotype) on Rubus coreanus a gross features of infected leaves b uredinia on a leaf c–d longitudinal section of uredinium e paraphyses f urediniospores g aecia on a leaf h longitudinal section of aecium i–j aeciospores k telia on a leaf l longitudinal section of telium m–n Teliospores. Scale bars: 2 mm (b); 1 mm (g, k); 50 µm (c–e, h, l); 10 µm (f); 25 µm (i–j, m–n).

Habitat

Rubus coreanus.

Known distribution

China, Guizhou Province.

Additional material examined

China. Guizhou Province: Guiyang city, 27°10'30"N, 106°99'91"W, 830 m, 09 Apr 2021, on Rubus coreanus, coll. J.E. Sun, HGUP21030.

Notes

In the phylogenetic tree, Phragmidium rubi-coreani, Ph. barclayi and Ph. cibanum formed a branch (Fig. 1). However in morphology, teliospores of Phragmidium rubi-coreani have fewer septa and shorter pedicels (3–5-celled, 8–34 μm long) than Ph. barclayi (5–8-celled, 60–150 μm long) and Ph. cibanum (5–7-celled, 70–108 μm long) (Liu et al. 2018). Meanwhile, most reported Phragmidium taxa produce longer teliospores, such as Ph. zangdongii (29–74 × 14–37 µm vs. 82–110 × 23–31 μm); Ph. kanas (29–74 × 14–37 µm vs. 134–198 × 19–31 µm); Ph. potentillae-canadensis (29–74 × 14–37 µm vs. 48.1–86.8 × 30.1–33.3 µm) than the present species (Yun et al. 2011; Liu et al. 2018; Zhao et al. 2021). Thus, our fungus represented a novel taxon.

Phragmidium potentillae-freynianae J.E. Sun & Yong Wang bis, sp. nov.

MycoBank No: 845043
Fig. 5

Diagnosis

Different from the related taxa by its urediniospores catenulate, such as Ph. chayuensis, Ph. cibanum and Ph. tormentillae.

Holotype

China. Guizhou Province;, Guiyang city, 26°44'70"N, 106°59'65"W, 801 m, 27 Mar 2021, on Potentilla freyniana, coll. J.E. Sun, HGUP21033, ITS: OL684826, LSU: OL684837.

Etymology

Referring to the host, Potentilla freyniana, on which the fungus was first found.

Description

Spermogonia , aecia and telia not observed. Uredinia produced on the abaxial leaf surface, covering the entire lower surface of the leaves, hypophyllous, nearly oval, powdery, not surrounded by host epidermis, 0.1–1.0 mm diam, on densely orange spot, 0.1–1.0 mm diam. Urediniospores: uredo-type, subglobose to oval, produced in basipetal succession, 19–24 × 18–24 µm (mean 21.5 × 21 μm, n = 30), golden, or bright-yellow; thin-walled, wall 0.4–1.4 µm thick, colorless, densely and minutely echinulate.

Rust diseases symptoms: Large areas of orange powdery uredinia, covering almost the entire lower surface of the leaves, which are aggregated but without obvious boundaries (Fig. 5).

Figure 5. 

Phragmidium potentillae-freynianae sp. nov. (HGUP21033, holotype) on Potentilla freyniana. a–c uredinia on leaves d longitudinal section of uredinium e–i urediniospores. Scale bars: 2 mm (b–c); 50 µm (d–e); 25 µm (f–i).

Habitat

Potentilla freyniana.

Known distribution

China, Guizhou Province.

Notes

In the phylogenetic tree, Phragmidium potentillae-freynianae formed a well-supported clade allied to Ph. duchesneae-indicae (Fig. 1). Morphologically, its urediniospores are bigger than Ph. duchesneae-indicae (21.5 × 21 μm vs. 13–19 × 11–17 µm) (Zhao et al. 2021). The comparison of DNA base composition supports the morphological separation of this fungus as a new species.

Phragmidium rosae-laevigatae J.E. Sun & Yong Wang bis, sp. nov.

MycoBank No: 845044
Fig. 6

Diagnosis

Different from Ph. Jiangxiense mainly because of bigger urediniospores.

Holotype

China. Guizhou Province: Panzhou city, 25°64'56"N, 104°84'35"W, 1800 m, 19 Jul 2021, on Rosa laevigata, coll. J.E. Sun, HGUP21036, ITS: OL684829, LSU: OL684840.

Etymology

Referring to the host, Rosa laevigata, on which the fungus was first found.

Description

Spermogonia and aecia not observed. Uredinia produced on the abaxial leaf surface, hypophyllous, subglobose to globose, powdery, 0.1–0.5 mm diam, yellow, peripherally parphyses, hyaline, 20–31 × 10–17 µm. Urediniospores square to diamond-shaped, oval to nearly spherical, 23–35 × 16–30 µm (mean 29 × 23 µm, n = 30), orange-colored, thick-walled 0.5–2.0 µm thick, colorless, regularly echinulate with stout spines on the surface. Telia scattered compact, hypophyllous, golden, 0.1–0.5 mm diam. Teliospores (immature) oval, 24–60 × 8–20 µm (mean 50.5 × 25.5 μm, n = 30), with apical papillae (4.0–7.0 μm high, n = 10), too immature to know how many cells, orange-yellow; pedicels swollen at the base, 15–26 μm long, colorless, disconnected easily; wall 0.5–2.0 μm thick.

Rust diseases symptoms: As shown in Fig. 6, Uredinia and telia, which are bright-yellow and powdery are produced almost simultaneously on the lower surface of the yellowing and wilting leaves.

Figure 6. 

Phragmidium rosae-laevigatae sp. nov. (HGUP21036, holotype) on Rosa laevigata a gross features of infected leaves b uredinia and telia on a leaf c longitudinal section of telium d immature teliospores e longitudinal section of uredinium f–h urediniospores. Scale bars: 1 mm (b); 50 µm (c, e); 12.5 µm (d, f–h).

Habitat

Rosa laevigata.

Known distribution

China, Guizhou Province.

Additional material examined

China. Guizhou Province: Panzhou city, 25°61'81"N, 104°83'61"W, 1790 m, 19 Jul 2021, on Rosa laevigata, coll. J.E. Sun, HGUP21037.

Notes

Phylogenetically, Phragmidium rosae-laevigatae kept a close relationship to Ph. leucoaecium, Ph. japonicum and Ph. jiangxiense (Fig. 1). Morphologically, Phragmidium rosae-laevigatae has bigger urediniospores than Ph. jiangxiense (23–35 × 16–30 µm vs. 15–23 × 11–18 μm), but the uredinia and urediniospores of Ph. leucoaecium and Ph. japonicum were not observed (Liu et al. 2020). The comparison of DNA base composition also supported morphological conclusion. Thus, this fungus was also introduced as one novel taxon herein.

Phragmidium duchesneae-indicae P. Zhao & L. Cai, Fungal Diversity 5:1–58, 2021

MycoBank No: 557609
Fig. 7

Description

Spermogonia , aecia and telia not observed. Uredinia produced on the abaxial leaf surface, hypophyllous, nearly oval, golden, densely bright orange-yellow, powdery, not surrounding by host epidermis, 0.3–1.2 mm diam, without paraphyses. Urediniospores produced in basipetal succession, mostly globose, 17–22 × 15–20 µm (mean 19.5 × 17.5 μm, n = 30), inclusions yellowish, or bright-yellow; thick-walled, wall 0.7–1.8 µm thick, colorless, densely and minutely echinulate. Telia and teliospores see Zhao et al (2021).

Figure 7. 

Phragmidium duchesneae-indicae (HGUP21031) on Duchesnea indica a–c uredinia on leaves d longitudinal section of uredinium e–g urediniospores. Scale bars: 2 mm (b); 1 mm (c); 50 µm (d); 12.5 µm (e–g).

Habitat

Duchesnea indica

Known distribution

China, Guizhou Province.

Material examined

China. Guizhou Province: Guiyang city, 27°10'30"N, 106°99'91"W, 820 m, 09 Apr 2021, on Duchesnea indica, coll. J.E. Sun, HGUP21031; Guiyang city, 27°09'26"N, 106°98'90"W, 734 m, 04 Sep 2021, on Duchesnea indica, coll. J.E. Sun, HGUP21032.

Notes

Phragmidium duchesneae-indicae was first reported on D. indica by Zhao et al (2021). Our specimen had similar morphology to that described by Zhao et al (2021). GenBank accession numbers (ITS and LSU) of Ph. duchesneae-indicae have not been released, and our identification is based only on a morphological comparison.

Phragmidium potentillae (Pers.) P. Karst., Bidrag till Kännedom av Finlands Naturoch Folk, 31: 49, 1879

MycoBank No: 206190
Fig. 8

Description

Spermogonia and aecia not observed. Uredinia produced on the abaxial leaf surface, hypophyllous, nearly oval, powdery, densely bright orange, nearly oval, surrounding by host epidermis, 0.8–1.5 × 0.4–0.7 mm, and densely bright orange. Urediniospores angular to squarish, oval to nearly globose, produced in basipetal succession, 17–26 × 14–22 µm (mean 21.5 × 18 μm, n = 30), or bright–yellow to orange, immature urediniospores are colorless; thick-walled, wall 0.6–1.3 µm thick, colorless, densely and minutely echinulate. Telia and teliospores see Liu et al (2018).

Figure 8. 

Phragmidium potentillae (HGUP21034) on Potentilla kleiniana a–c uredinia on leaves d longitudinal section of uredinium e–j urediniospores. Scale bars: 1 mm (c); 50 µm (d); 12.5 µm (e–j).

Habitat

Potentilla kleiniana

Known distribution

China: Guizhou Province, Qinghai Province, Sinkiang Province; USA, the United Kingdom, Australia, Tasmania and Japan.

Material examined

China. Guizhou Province: Guiyang city, 27°09'26"N, 106°98'90"W, 730 m, 22 Jun 2021, on Potentilla kleiniana, coll. J.E. Sun, HGUP21034.

Notes

In the phylogenetic tree, HGUP21034 clustered with two sequences of specimens of Phragmidium potentillae (Fig. 1). The uredinia of P. potentillae described by Liu et al (2018), as 0.2–0.8 mm diam, smaller than in the specimen examined, 0.8–1.5 × 0.4–0.7 mm, the urediniospores mostly globose and echinulate, (18–25 × 15–21 μm vs. 17–26 × 14–22 µm).

Phragmidium barnardii Plowr. & G. Winter, Revue Mycologique Toulouse 8 (32): 208 (1886)

MycoBank No: 249450
Fig. 9

Description

Spermogonia , aecia and telia not observed. Uredinia produced on the abaxial leaf surface, hypophyllous, scattered to gregarious, oval to globose, orange, powdery, 0.1–1.0 mm diam, with hyaline and curved paraphyses, 26–39 × 10–13 µm. Urediniospores orange, 16–19 × 15–18 µm (mean: 17.5 × 16.5 µm, n = 30), nearly globose; thick-walled 1.3–2.2 µm, colorless, regularly echinulate with stout spines.

Figure 9. 

Phragmidium barnardii (HGUP21035) on Rubus sp. a–d uredinia on leaves e longitudinal section of uredinium f–h urediniospores. Scale bars: 1 mm (d); 50 µm (e); 12.5 µm (f–h).

Habitat

Rubus sp.

Known distribution

China, Guizhou Province; South Africa.

Material examined

China. Guizhou Province: Duyun city, 27°26'05"N, 107°38'91"W, 870 m, 26 Jun 2021, on Rubus sp., coll. J.E. Sun, HGUP21035.

Notes

Phragmidium barnardii was first reported on Rubus sp. by Winter (1886). Its DNA data was established by McTaggart et al (2016), although without description of morphological characteristics. We confirmed the specimens (HGUP21035) as Ph. barnardii, through phylogenetic analyse with DNA data from McTaggart et al. (2016).

Discussion

More than 70 Phragmidium species have been described in China, while many species without molecular data (Cummins 1931; Arthur 1934; Wahyuno et al. 2001; Cummins and Hiratsuka 2003; Zhuang et al. 2012; Yang et al. 2015; Ali et al. 2017). Recently, morphology and molecular data were gradually combined and used to describe the diversity of species in Phragmidium (Liu et al. 2018, 2019, 2020; Zhao et al. 2021). In the study, the four novel and three known species of Phragmidium were delineated based on phylogeny of the ITS and LSU gene regions and on morphological features.

The host plants of Ph. punjabense, Ph. warburgianum, Ph. rosae-rugosae, Ph. rosae-laevigatae and Ph. rosae-roxburghii all belong to Rosa, but Ph. potentillae-freynianae and Ph. potentilla occur on Potentilla sp. while Ph. rubi-coreani and Ph. barnardii occur on Rubus sp. However, the hosts of species with close phylogenetic relationships were not necessarily in the same genus. Phragmidium potentilla can be found on three plants (P. chinensia, P. kleiniana and P. virgata), and Ph. rosae-roxburghii can be parasitic on two Rosa plants (Rosa roxburghii and Rosa sp.). It might mean that host jumps also shaped the diversity of Phragmidium, like Pucciniales (McTaggart et al. 2016).

Phragmidium leucoaecium (BJFCR02118 and BJFCR02116), Ph. japonicum (HMAS41585), Ph. jiangxiense (BJFCR03452 and BJFCR03453) and Ph. rosae-laevigatae (HGUP21036 and HGUP21037) from Rosa formed a phylogenetic lineage, while three of the latter from the same host (Rosa laevigata) (Liu et al. 2020). This may be explained by geographical distribution, geography, climate, etc., but contradicts the concept of obligatory parasitism. We could guess that their hosts might not reflect taxonomic status of Phragmidium. Interestingly, Phragmidium tibeticum, Ph. sikangense and Ph. shensianum were named according to the collection locations (Dai 1979; Chen 2009). Their nomenclatures contradict the concept of obligatory parasitism for rust fungi, although might be easy to be understanding.

Acknowledgements

We would like to thank Dr Eric HC McKenzie for language editing. This research is supported by the following projects: National Natural Science Foundation of China (No. 31972222, 31660011), Program of Introducing Talents of Discipline to Universities of China (111 Program, D20023), Talent project of Guizhou Science and Technology Cooperation Platform ([2017]5788-5, [2019]5641, [2019]13), Guizhou Science, Technology Department of International Cooperation Base project ([2018]5806), the project of Guizhou Provincial Education Department ([2020]001), and Guizhou Science and Technology Innovation Talent Team Project ([2020]5001).

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