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Research Article
A new study of Nagrajomyces: with two new species proposed and taxonomic status inferred by phylogenetic methods
expand article infoLan Zhuo‡, Mei-Jun Guo‡, Qiu-Tong Wang‡, Hao Zhou‡, Meike Piepenbring§, Cheng-Lin Hou‡
‡ Capital Normal University, Beijing, China
§ Goethe University Frankfurt am Main, Frankfurt am Main, Germany

Corresponding author: Cheng-Lin Hou ( chenglin-hou@cnu.edu.cn )

Academic editor: Nattawut Boonyuen
© 2022 Lan Zhuo, Mei-Jun Guo, Qiu-Tong Wang, Hao Zhou, Meike Piepenbring, Cheng-Lin Hou.
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: Zhuo L, Guo M-J, Wang Q-T, Zhou H, Piepenbring M, Hou C-L (2022) A new study of Nagrajomyces: with two new species proposed and taxonomic status inferred by phylogenetic methods. MycoKeys 93: 131-148. https://doi.org/10.3897/mycokeys.93.93712
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Abstract

Nagrajomyces (incertae sedis, Ascomycota) is a monotypic genus with a previously unknown systematic position. In this report, two new species are proposed, Nagrajomyces fusiformis and Nagrajomyces laojunshanensis. These new taxa are proposed based on morphological characteristics evident via light microscopy and molecular data. Multi-locus phylogenetic analyses (ITS rDNA, nrLSU rDNA, RPB2, and TEF1-α) show that specimens recently collected in Yunnan Province, China are closely related to Gnomoniaceae. Both new species and known species were discovered repeatedly in their asexual developmental form exclusively on twigs of Rhododendron spp. (Ericaceae). This indicates a host specificity of Nagrajomyces spp. for species of Rhododendron.

Keywords

host specificity, Nagrajomyces, new taxa, phylogeny

Introduction

Gnomoniaceae is a distinct family of Diaporthales, established by Winter (1886). The traditional classification of species in Gnomoniaceae was mainly based on morphological features, such as the morphology of ascomata and ascospores as well as the position of necks (Barr 1978; Monod 1983). Sogonov et al. (2008) used phylogenetic analyses of molecular sequence data of several genes (TEF1-α, nrLSU, and RPB2) to revise the concepts of leaf-inhabiting genera, and discovered that several traditional genera in Gnomoniaceae are polyphyletic. Phylogenetic analyses indicate that host specificity can be used to circumscribe genera and species of Gnomoniaceae. Based on phylogenetic analyses and morphological characteristics, Senanayake et al. (2017) described new taxa and excluded some genera from Gnomoniaceae. Since then, additional genera have been introduced for species observed in sexual developmental stages, as well as those that have only been observed as pycnidial asexual morphs, and rarely for species known in both sexual and asexual forms (Senanayake et al. 2018; Crous et al. 2019; Jiang et al. 2019; Minoshima et al. 2019; Yang et al. 2020).

Many species of Gnomoniaceae are important plant pathogens, such as Apiognomonia errabunda (Roberge ex Desm.) Höhn, which causes oak anthracnose (Sogonov et al. 2007), Gnomoniopsis fructicola (G. Arnaud) Sogonov, which causes strawberry stem rot (Maas 1998), and Ophiognomonia leptostyla (Fr.) Sogonov, which causes walnut anthracnose (Neely and Black 1976). Species of Gnomoniaceae can also have wide host ranges, with species on Fagaceae, Onagraceae, and Rosaceae being frequently infected. Species of Rhododendron (Ericaceae), which is the largest genus of woody plants in the northern hemisphere, are also known hosts of species of Gnomoniaceae (Monod 1983).

Nagrajomyces (incertae sedis, Ascomycota) is a monotypic genus based on N. dictyosporus Mel’nik (Mel’nik 1984; Nag Raj 1993). It was discovered in Russia, where it grows on twigs of Rhododendron aureum and develops stalked, unilocular, or plurilocular conidiomata as well as muriform conidia, and each conidium bears an apical appendage that is single, unbranched, attenuated, flexuous and can be more than 100 μm long (Mel’nik 1984; Nag Raj 1993).

In the present study, two new species were discovered on twigs of Rhododendron spp. in Yunnan and assigned to the genus Nagrajomyces based on morphological characteristics, habitat, and host. Phylogenetic analysis revealed that the proposed Nagrajomyces species belong to Gnomoniaceae.

Materials and methods

Specimen collections and isolation

Fieldwork for the discovery of fungi was conducted during June 2021 in Yunnan Province, China. Fresh pycnidia were repeatedly discovered and collected on twigs of Rhododendron spp. Twigs with conidiomata were packed in paper bags and transported to the laboratory for morphological tests. Conidiomata were cut off in the laboratory using a razor blade, wrapped in paper packets, disinfected with 75% ethanol for 10 s, then 10% sodium hypochlorite for 2 min 30 s, and rinsed with distilled water three times. After absorbing the water with sterile filter paper, the conidiomata were transferred to potato dextrose agar (PDA) plates (Jiang et al. 2021) then incubated at 25 °C to obtain cultures. Dry specimens were deposited at the China Forest Biodiversity Museum of the Chinese Academy of Forestry (CAF; http://museum.caf.ac.cn) and the Herbarium of the College of Life Science, Capital Normal University (BJTC; http://smkxxy.cnu.edu.cn). Ex-type living cultures were deposited at the China Forestry Culture Collection Center (CFCC; http://cfcc.caf.ac.cn/).

Morphological analysis

Conidiomata were photographed and cut by hand using a razor blade under a Nikon SMZ-1000 stereomicroscope (Japan). Morphological characteristics of conidiomata, conidiophores, and conidia were photographed and measured with an Olympus EX-51 upright microscope (Japan), and for each structure at least 20 measurements were made. Color values were taken from ColorHexa (https://www.colorhexa.com/).

DNA extraction, polymerase chain reaction amplification, and phylogeny

Genomic DNA was extracted from specimens and cultures via the M5 Plant Genomic DNA Kit (Mei5 Biotechnology Co., Ltd., China) in accordance with the manufacturer’s instructions. Table 1 summarizes the primers used to obtain sequence data for ITS rDNA, nrLSU rDNA, RPB2, and TEF1-α, and the polymerase chain reaction (PCR) amplification protocols. PCR products were analyzed in 1% electrophoretic agarose gel with a 200-bp DNA ladder, purified, and sequenced by Beijing Zhongke Xilin Biotechnology Co., Ltd. (Beijing, China). SeqMan was used to align the sequences obtained by forward and reverse primers to obtain a consensus sequence. A partition homogeneity test was performed to determine the congruence of the four datasets (Farris et al. 1994). Sequences for phylogenetic analyses were selected based on Yang et al. (2020), supplemented by sequences of Apiosporopsis carpinea (Fr.) Mariani, Apiosporopsis sp., Juglanconis juglandina (Kunze) Voglmayr & Jaklitsch, Juglanconis oblonga (Berk.) Voglmayr & Jaklitsch, and Melanconis marginalis (Peck) Wehm. from Senanayake et al. (2018) used as outgroup taxa. All sequences used in this study are listed in Table 2. Subsequent alignments were generated with online MAFFT tools (https://www.ebi.ac.uk/Tools/msa/mafft/) and edited with Gblocks 0.91b (http://molevol.cmima.csic.es/castresana/Gblocks_server.html). The maximum likelihood (ML) tree was constructed using RAxML version 8.2.12 (Stamatakis et al. 2005; Stamatakis 2006; Stamatakis 2014) with GTRGAMMA model and 1000 bootstrap iterations. The multi-locus Bayesian Inference (BI) tree was built by MrBayes version 3.2.6 (Ronquist and Huelsenbeck 2003). Models of nucleotide substitution for each gene used in the Bayesian analysis were determined by MrModeltest v.2.3 (Nylander 2004). Analyses of four simultaneous Markov Chain Monte Carlo (MCMC) chains were run for 100,000,000 generations, and other operational methods were applied used as described by Guo et al. (2021). The maximum parsimony (MP) tree was constructed using PAUP version 4.0 beta 10 (Swofford 2003) with 1000 random sequence additions, 1000 maxtrees were obtained, and bootstrap analysis was conducted based on 1000 replicates, with 10 replicates of random stepwise additions of taxa. For further details see Guo et al. (2021). Trees were viewed via Treeview (Page 1996).

Table 1.
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Primer information and PCR amplification protocols.

Gene Primer pairs Reference Amplification conditions
ITS rDNA ITS1F/ITS4 White et al. (1990); Gardes and Bruns (1993) Phillips et al. (2008)
LSU rDNA LR0R/LR5 Vilgalys and Hester (1990); Rehner and Samuels (1994) Phillips et al. (2008)
TEF1-α EF1-728F/EF1-986R Carbone and Kohn (1999) Glass and Donaldson (1995)
RPB2 fRPB2-5F/fRPB2-7cR Liu et al. (1999) Liu et al. (1999)
Table 2.
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Sequences used in phylogenetic analyses. References to sequences generated in the present study are emphasized in bold.

Taxa Voucher ITS rDNA LSU rDNA RPB2 TEF1-α References
Alnecium auctum CBS 124263 KF570154 KF570154 KF570170 KF570200 Voglmayr and Jaklitsch (2014)
Ambarignomonia petiolorum CBS 116866 EU199193 AY818963 EU199151 – Mejía et al. (2008)
Ambarignomonia petiolorum CBS 121227 EU254748 EU255070 EU219307 EU221898 Mejía et al. (2008)
Amphiporthe tiliae CBS 119289 EU199178 EU199122 EU199137 – Mejía et al. (2008)
Apiognomonia errabunda AR 2813 DQ313525 – DQ862014 DQ313565 Sogonov et al. (2007)
Apiognomonia veneta MFLUCC 16-1193 MF190114 MF190056 – – Senanayake et al. (2017)
Apioplagiostoma populi 858501 KP637024 – – – Wijekoon et al. (2021)
Apiosporopsis carpinea CBS 771.79 – AF277130 – – Zhang and Blackwell (2001)
Apiosporopsis sp. Masuya 11Af2-1 – AB669034 – – Osono and Masuya (2012)
Asteroma alneum CBS 109840 EU167609 EU167609 – – Simon et al. (2009)
Asteroma sp. Masuya 8Ah9-1 – AB669035 – – Osono and Masuya (2012)
Cryptodiaporthe acerina AR 3822 EU254755 EU255075 EU219253 EU221879 Sogonov et al. (2008)
Cryptodiaporthe aubertii CBS 114196 KX929767 KX929803 KX929838 KX929732 Meyer et al. (2017)
Cryptosporella hypodermia CBS 116866 EU199181 AF408346 EU199140 – Mejía et al. (2008)
Ditopella biseptata MFLU 15-2661 MF190147 MF190091 MF377616 – Senanayake et al. (2017)
Ditopella ditopa CBS 109748 DQ323526 EU199126 EU199145 – Mejía et al. (2008)
Ditopellopsis sp. CBS 121471 EU254763 EU255088 EU219254 EU221936 Sogonov et al. (2008)
Flavignomonia rhoigena CFCC 53118 MK432674 MK429917 MK578102 – Jiang et al. (2019)
Flavignomonia rhoigena CFCC 53119 MK432675 MK429918 MK578103 – Jiang et al. (2019)
Gnomonia gnomon CBS 199.53 DQ491518 AF408361 EU219295 EU221885 Sogonov et al. (2008)
Gnomonia gnomon CBS 829.79 AY818957 AY818964 – EU221905 Sogonov et al. (2005)
Gnomoniella microspora BPI 877571 EU254765 – – – Sogonov et al. (2008)
Gnomoniopsis alderdunensis CBS 125680 GU320825 – – – Walker et al. (2010)
Gnomoniopsis chamaemori CBS 803.79 EU254808 EU255107 – – Sogonov et al. (2008)
Gnomoniopsis racemula AR 3892 EU254841 EU255122 EU219241 EU221889 Sogonov et al. (2008)
Juglanconis juglandina WU 35960 KY427145 KY427145 KY427195 KY427214 Voglmayr et al. (2017)
Juglanconis oblonga TFM FPH 2623 KY427153 KY427153 KY427203 KY427222 Voglmayr et al. (2017)
Mamianiella coryli BPI 877578 EU254862 – – – Sogonov et al. (2008)
Marsupiomyces epidermoidea MFLU 15-2921 – MF190058 – – Senanayake et al. (2017)
Marsupiomyces quercina MFLUCC 13-0664 MF190116 MF190061 – – Senanayake et al. (2017)
Melanconis marginalis BPI 748234 – – EU219299 EU221886 Sogonov et al. (2008)
Melanconis marginalis BPI 748446 EU199197 AF408373 EU219301 EU221991 Sogonov et al. (2008)
Neognomoniopsis quercina CBS 145575 MK876399 MK876440 – – Crous et al. (2019)
Nagrajomyces fusiformis CAF 800050 OP473599 OP473595 OP484756 OP484760 This study
Nagrajomyces fusiformis BJTC 1773 OP473602 OP473598 – OP484763 This study
Nagrajomyces laojunshanensis CFCC 58177 OP456161 OP473594 OP484755 OP484759 This study
Nagrajomyces laojunshanensis CAF 800049 OP473600 OP473596 OP484757 OP484761 This study
Nagrajomyces laojunshanensis BJTC 1849 OP473601 OP473597 OP484758 OP484762 This study
Occultocarpon ailaoshanense LCM 524.01 JF779849 JF779853 JF779856 – Mejía et al. (2011)
Occultocarpon ailaoshanense LCM 522.01 JF779848 JF779852 JF779857 JF779862 Mejía et al. (2011)
Ophiognomonia melanostyla LCM 389.01 JF779850 JF779854 JF779858 – Mejía et al. (2011)
Ophiognomonia vasiljevae AR 4298 EU254977 EU255162 EU219331 EU221999 Sogonov et al. (2008)
Phragmoporthe conformis AR 3632 – AF408377 – – Castlebury et al. (2002)
Plagiostoma aesculi AR 3640 EU254994 EU255164 EU219269 – Sogonov et al. (2008)
Plagiostoma rhododendri CBS 847.79 EU255044 EU255187 EU219272 – Sogonov et al. (2008)
Pleuroceras oregonense AR 4333 EU255060 EU255196 EU219313 EU221931 Sogonov et al. (2008)
Pleuroceras pleurostylum CBS 906.79 EU255061 EU255197 EU219311 EU221962 Sogonov et al. (2008)
Sirococcus conigenus BPI 871248 EU199201 EU199134 EU199157 – Mejía et al. (2008)
Sirococcus piceicola BPI 871166 EU199202 EU199135 EU199158 – Mejía et al. (2008)
Sirococcus tsugae BPI 871167 EU199203 EU199136 EU199159 – Mejía et al. (2008)
Sirococcus tsugae AR 4010 EF512478 EU255207 EU219289 EU221928 Sogonov et al. (2008)
Tenuignomonia styracis BPI 892786 – LC379289 LC379295 LC379283 Minoshima et al. (2019)
Tenuignomonia styracis BPI 892785 – LC379288 LC379294 LC379282 Minoshima et al. (2019)
Valsalnicola oxystoma AR 5137 JX519561 – – – Crous et al. (2012)
Valsalnicola oxystoma AR 4833 JX519559 JX519563 – – Crous et al. (2012)

Results

Phylogenetic analysis

Multi-locus phylogenetic analyses of species of Gnomoniaceae (Diaporthales) include sequences of 51 ingroup taxa and sequences of an outgroup formed by Apiosporopsis carpinea, Apiosporopsis sp., Juglanconis juglandina, J. oblonga, and Melanconis marginalis (Fig. 1). The multi-locus dataset (ITS rDNA, LSU rDNA, RPB2 and TEF1-α) comprises 2875 characters, of which 945 are parsimony-informative, 200 are parsimony-uninformative and 1730 are constant. Maximum parsimony analysis of sequences resulted in one most parsimonious tree with a length (TL) of 3730 steps, a consistency index (CI) of 0.463, a retention index (RI) of 0.690, and a homoplasy index (HI) of 0.537. Bayesian and maximum likelihood trees exhibited topologies similar to this parsimony tree.

Figure 1. 

Phylogenetic tree based on an ML analysis of combined ITS rDNA, nrLSU rDNA, RPB2, and TEF1-α sequences of species of Gnomoniaceae. Bootstrap support values for RAxML and maximum parsimony above 50% and Bayesian posterior probability values above 0.95 are shown at the nodes. The tree is rooted with sequences of Apiosporopsis carpinea, Apiosporopsis sp., Juglanconis juglandina, J. oblonga, and Melanconis marginalis. References to new sequences are in bold, and the names of the two new species are highlighted by colors.

The topology of the phylogenetic tree obtained in the current study was similar to the topology presented by Yang et al. (2020). Nineteen sequences of five specimens recently collected on Rhododendron spp. in China form a clade with high support values. This clade is sister to sequences of species of Siroccocus and Neognomoniopsis, but with poor support values. The newly discovered clade is divided into two small subclades labeled Nagrajomyces fusiformis and N. laojunshanensis.

Taxonomy

Nagrajomyces fusiformis C. L. Hou & L. Zhuo, sp. nov.

MycoBank No: 845666
Figs 2, 3

Etymology

The epithet fusiformis refers to fusoid conidia.

Type

China, Yunnan province, Lijiang, Yulong, 26°40'55"N, 99°54'01"E, alt. 2762 m, on dying twigs of Rhododendron vellereum Hutch. ex Tagg., 20 June 2021, coll. C.L. Hou, M.J. Guo, H. Zhou (holotype CAF 800050).

Diagnosis

This new species differs from N. dictyosporus and N. laojunshanensis by fusoid to elongate-fusoid conidia with pointed ends, usually 1-septate and smaller.

Figure 2. 

Micrographs of Nagrajomyces fusiformis (holotype CAF 800050) on twigs of Rhododendron vellereum A, B conidiomata on a dying twig C vertical section of a conidioma D conidiophores and conidia at diverse developmental stages E–H conidia with appendages. Scale bars: 2 mm (A); 200 µm (B); 100 µm (C); 10 µm (D–H).

Figure 3. 

Nagrajomyces fusiformis (holotype CAF 800050) A vertical section of a conidioma B conidiophores and conidia C conidia with appendages. Scale bars: 100 µm (A); 10 µm (B); 5 µm (C).

Description

Conidiomata solitary, pycnidial, irregularly plurilocular, subepidermal in origin, immersed at first, then becoming erumpent through the periderm of the host, 545–554 μm diameter, 520–546 μm high, peridium dark brown, 47.0–67.5 μm thick. Conidiophores ampulliform, smooth, hyaline, multiguttulate, 12–29 × 2.0–3.5 μm (x̄ = 19 × 3 μm, n = 20). Conidia fusoid to elongate-fusoid, 1-septate, cells equal, smooth, hyaline to pale brown, 13.5–19.0 × 3–4 μm (x̄ = 16.5 × 3.5 μm, n = 20), with a whip‑like appendage at the tip of each conidium, 30–77 μm (x̄ = 51 μm, n = 20) in length (Fig. 4). Sexual morph not observed.

Additional specimen examined

China, Yunnan Province, Lijiang, Laojunshan, 26°37'56"N, 99°43'30"E, alt. 3873 m, on dying twigs of Rhododendron vellereum, 20 June 2021, coll. C.L. Hou, M.J. Guo, H. Zhou (BJTC 1773).

Notes

Nagragomyces fusiformis differs from other species of Nagrajomyces by narrower and 1-septate conidia.

Nagrajomyces laojunshanensis C. L. Hou & L. Zhuo, sp. nov.

MycoBank No: 845665
Figs 4, 5

Etymology

The epithet laojunshanensis refers to the location where the type specimen was collected.

Type

China, Yunnan Province, Lijiang, Laojunshan, 26°39'44"N, 99°46'58"E, alt. 2910 m, on living twigs of Rhododendron cinnabarinum Hook. f., 20 June 2021, coll. C.L. Hou, M.J. Guo, H. Zhou (holotype CAF 800049). Ex-type culture CFCC 58177.

Diagnosis

This new species differs from N. fusiformis by conidia that are elongate-elliptical, blunter at both ends, and usually 3-septate and larger. Nagrajomyces laojunshanensis differs from N. dictyosporus by conidiomata that are unilocular and without stalks.

Description

Conidiomata solitary, pycnidial, unilocular, subglobose to ellipsoidal, subepidermal in origin, immersed at first, then becoming erumpent, 218–406 μm wide, 188–275 μm high, peridia black, 37–43 μm thick, opening irregularly in the upper part, with faint yellow content. Conidiophores ampulliform, smooth, hyaline, multiguttulate, 16.0–25.5 × 2–4 μm (x̄ = 21 × 3 μm, n = 20). Conidia elongate-elliptical, 1–3-septate, mostly 3-septate, smooth, hyaline, 18–23 × 5.5–7.0 μm (x̄ = 19.5 × 6.5 μm, n = 20), with a long, whip-like appendage at the tip of each conidium, 70–200 μm (x̄ = 143.5 μm, n = 20) in length. Sexual morph not observed.

Culture characteristics

Cultures (ex-type CFCC 58177) on PDA 8 cm diameter after 1 month, with irregular margins, sparse aerial mycelium, colonies with whitish margins, with center turning black olive (#3b3c36) with increasing age. On MEA, 5.7 cm diameter after 1 month, with irregular margins, colonies with beaver (#9f8170) -colored margins, with center turning black olive (#3b3c36) with increasing age. Conidia not observed.

Additional specimen examined

China, Yunnan province, Kunming, Luquan, Jiaozixueshan, 26°05'04"N, 102°50'54"E, alt. 3823 m, on living twigs of Rhododendron cinnabarinum Hook. f., 23 June 2021, coll. C.L. Hou, M.J. Guo, H. Zhou, (BJTC 1849).

Notes

Nagrajomyces laojunshanensis differs from N. dictyosporus by conidia that are colorless and conidiomata that are without stalks. Nagrajomyces laojunshanensis differs from N. fusiformis by elongate-elliptical conidia with blunter ends, which are longer (18–23 μm vs. 13–19 μm) and wider (5.7–7.0 μm vs. 2.8–3.7 μm). Conidia of N. laojunshanensis are mostly 3-septate, whereas those of N. fusiformis are 1-septate. Molecular sequence data confirm the presence of two distinct species.

Figure 4. 

Micrographs of Nagrajomyces laojunshanensis on Rhododendron cinnabarinum (holotype CAF 800049) A conidiomata on a living twig B ex-type culture (CFCC 58177) on PDA after 30 days, seen from above C ex-type culture (CFCC 58177) on MEA after 30 days, seen from above D vertical section of a conidioma E, F conidiophores and conidia G conidia forming a cirrus H conidium with appendage. Scale bars: 1 mm (A); 1 cm (B, C); 100 µm (D, E); 10 µm (F–H).

Figure 5. 

Nagrajomyces laojunshanensis (holotype CAF 800049) A vertical section of a conidioma B conidiophores and conidia C conidia with appendages. Scale bars: 50 µm (A); 10 µm (B); 5 µm (C).

Discussion

Morphologically, the most distinctive features of the new species of Nagrajomyces are septate conidia with long, single, apical appendages. The presence of this structure distinguishes them from all anamorphic genera known to belong to Gnomoniaceae. Both new species proposed in the present study and the known species N. dictyosporus inhabit twigs of Rhododendron. In spite of the absence of molecular data for the type species of Nagrajomyces, these two new species are accommodated in Nagrajomyces based on significant morphological features (distinctive conidia) and identical ecology.

Many coelomycetous genera have conidia with appendages (Nag Raj 1993), and some of them share morphological characteristics with the new species proposed in this study. For example, species of Uniseta and Urohendersonia have septate conidia and a long apical appendage attached to each conidium. Uniseta is a monotypic genus typified by U. flagellifera (Ellis & Everh.) Ciccar. (Ciccarone 1947). Nag Raj (1974, 1993) mentioned that U. flagellifera has a sexual morph called Cryptodiaporthe comptoniae (Schwein.) Barr (Barr 1991, syn. C. aubertii var. comptoniae (Schwein.) Wehm.) that is considered a synonym of Cryptodiaporthe aubertii (Westend.) Wehm. (Wehmeyer 1933). Conidia of this species are two-celled, hyaline, relatively inequilateral or curved, and bear a long flagellate appendage at one end (Wehmeyer 1933). Cryptodiaporthe acerina J. Reid & Cain and C. aubertii are included in the phylogenetic tree (Fig. 1) and located distant from the new species proposed herein. Furthermore, U. flagellifera differs from the new species proposed here by an asexual morph growing on branches of Comptonia asplenifolia damaged by fire (Ellis and Everhart 1889), while Nagrajomyces spp. develop on twigs of Rhododendron. Because of these differences, we consider the genus Uniseta to be separate from the genus Nagrajomyces.

Spegazzini (1902) introduced Urohendersonia Speg. with Ur. platensis Speg. as the type species. Nag Raj (1993) listed only five species in this genus, including the type species. Urohendersonia spp. differ from Nagrajomyces spp. by having globose to subglobose conidiomata immersed in host tissues, and yellowish brown to brown conidia each with an extracellular gelatinous appendage, and their host species (Nag Raj 1993). Urohendersonia spp. occur on diverse host species and various substrates of host, such as on leaves of Erythrina sp., Manihot carthagenensis, Pongamia pinnnata, Stipa spartea, or in the rhizospheres of Acerva persica and Dactyloctenium aegyptium (Nag Raj 1993; Wijayawardene et al. 2016). Unfortunately, there are no molecular sequence data available for any species within those genera.

In the phylogenetic analysis presented herein, the two new species, N. fusiformis and N. laojunshanensis form a clade with high support values, which is separate from other species of Gnomoniaceae represented by sequence data in GenBank. These two new species described in this study fill gaps in the molecular data of Nagrajomyces and also enable the taxonomic status of the new species to be determined.

A total of 38 genera are currently included in the family Gnomoniaceae based on morphological and molecular analyses (Senanayake et al. 2018; Crous et al. 2019; Jiang et al. 2019; Minoshima et al. 2019; Yang et al. 2020). Sexual morphs have been described for all but four; Asteroma, Flavignomonia, Millerburtonia, and Sirococcus. Sirococcus spp. are closely related to the new species described herein, whereas phylogenetic data indicate that the other three genera are distantly related to Nagrajomyces spp. Asteroma spp. have cylindrical to fusiform, acicular or broadly fusiform conidia (Senanayake et al. 2018). Conidia of Flavignomonia are cylindrical to oblong (Jiang et al. 2019), and conidia of Millerburtonia are filiform and aciculate (Ciferri 1951).

In addition to morphological characteristics and molecular sequence data, host ranges are often useful to delineate genera and species of Gnomoniaceae (Sogonov et al. 2008). Species of Gnomonia, for example, are generally associated with host plants in the Betulaceae family, mostly belonging to the subfamily Coryloideae (Sogonov et al. 2008). The two new species identified in the present study, and the known species, all develop on twigs of Rhododendron spp. indicating that they are specialized with respect to this host. The differences in conidiomatal structure could be explained by differences in host epidermal features or maturity. Two species of Gnomoniaceae are known to inhabit Rhododendron spp. Plagiostoma rhododendri (Auersw.) Sogonov was reported on dry twigs and inflorescences of Rhododendron hirsutum L., and occasionally on dead leaves of R. ferrugineum L. (Monod, 1983). Only the sexual form of this species has been described, and phylogenetic analysis places it somewhat distant to species of Nagrajomyces (Fig. 1). The second species is Gnomonia sp., reported on rotten leaves of R. ferrugineum (Rehm 1906). This species lacks a specific morphological description.

Rhododendron is the largest genus of woody plants in the northern hemisphere, and its species diversity is highest in the Himalaya-Hengduan Mountains and Southeast Asia (Chamberlain et al. 1996; Shrestha et al. 2018). Considering the host preference of Gnomoniaceae species and the biodiversity of Rhododendron worldwide, additional Gnomoniaceae species are expected to exist on these plants.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (grant number 31870629 and 32270012). We are grateful to the two anonymous reviewers whose comments and suggestions helped improve the manuscript.

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