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Tree inhabiting gnomoniaceous species from China, with Cryphogonomonia gen. nov. proposed
expand article infoQin Yang§, Ning Jiang§, Cheng-Ming Tian§
‡ Central South University of Forestry and Technology, Changsha, China
§ Beijing Forestry University, Beijing, China
Open Access

Abstract

Species of Gnomoniaceae are commonly associated with leaf spot diseases of a wide range of plant hosts worldwide. During our investigation of fungi associated with tree diseases in China, several gnomoniaceous isolates were recovered from symptomatic branches and leaves on different woody plants in the Fagaceae, Pinaceae, and Salicaceae families. These isolates were studied by applying a polyphasic approach including morphological, cultural data, and phylogenetic analyses of partial ITS, LSU, tef1, rpb2 and tub2 gene sequences. As a result, three species were identified with characters fitting into the family Gnomoniaceae. One of these species is described herein as Cryphognomonia pini gen. et sp. nov., characterized by developed pseudostromata and ascospores with obvious hyaline sheath; Gnomoniopsis xunwuensis sp. nov. is illustrated showing sympodially branched conidiophore, oval or fusiform conidia; and one known species, Plagiostoma populinum. The current study improves the understanding of gnomoniaceous species causing diebacks and leaf spot on ecological and economic forest trees.

Keywords

forest trees, Gnomoniaceae, new genus, phylogeny, systematics

Introduction

The Gnomoniaceae (Diaporthales, Sordariomycetes, Ascomycota) is a family of perithecial ascomycetes that occur as endophytes, pathogens, or saprobes on growing and overwintered leaves of hardwood trees, shrubs, and herbaceous plants (Walker 2012). Many species in the Gnomoniaceae cause serious tree diseases such as cherry leaf scorch (Apiognomonia erythrostoma (Pers.) Höhn.), oak dieback (A. errabunda (Roberge) Höhn), sycamore canker (A. veneta (Sacc. & Speg.) Höhn), and chestnut dieback (Gnomoniopsis daii Tian & Jiang) (Sogonov et al. 2008; Walker et al. 2010; Jiang et al. 2019).

The sexual morph of Gnomoniaceae is characterized by ascomata that are generally immersed, solitary or aggregated in an undeveloped stroma (Rossman et al. 2007; Sogonov et al. 2008). The perithecia are dark brown to black and pseudoparenchymatous with central, eccentric, or lateral necks (Rossman et al. 2007; Sogonov et al. 2008). Asci usually have an inconspicuous or distinct apical ring. Ascospores are generally small, hyaline, uniseptate. The asexual morph is characterized by acervular or pycnidial, phialidic, with non-septate conidia (Monod 1983).

The generic concepts of Gnomoniaceae were recently revised based on a survey of leaf-inhabiting diaporthalean fungi (Sogonov et al. 2008). Phylogenetic analyses of molecular markers is the primary methodology for systematic studies of the Gnomoniaceae, however, host specificity and morphology can also be useful for species identification. Recent phylogenetic studies have shown that species of Gnomoniaceae often have a narrow host range associating with a single host genus or species (Mejía et al. 2008, 2011a, b, 2012; Sogonov et al. 2008; Walker et al. 2010, 2012, 2013). For example, Cryptosporella is a well-defined genus which was frequently limited to a single host species, especially in the host family Betulaceae, except for C. wehmeyeriana on Tilia spp. and type species C. hypodermia on Ulmus spp. (Mejía et al. 2008, 2011b).

Several fungal species of Gnomoniaceae, Cryptosporella platyphylla from Betula platyphylla, Flavignomonia rhoigena from Rhus chinensis, Gnomoniopsis daii and Ophiognomonia castaneae from Castanea mollissima, have been reported from China (Fan et al. 2016; Gong et al. 2017; Jiang and Tian 2019; Jiang et al. 2019). In the present study, tree inhabiting gnomoniaceous species, mainly on cankered branches and leaves, were surveyed in China. The aim of the present study was to identify these fungi via morphology and multi-locus phylogeny based on modern taxonomic concepts.

Materials and methods

Isolates

Fresh specimens of Gnomoniaceae-related fungi were collected from branches and leaves of hosts in Beijing, Jiangxi and Shaanxi provinces (Tables 13). Isolates from host material were obtained by removing a mucoid spores mass from perithecia and pycnidia-like conidiomata, spreading the suspension on the surface of 1.8% potato dextrose agar (PDA), and incubating at 25 °C for up to 24 h. Single germinating conidia/ascospore was removed and plated on to fresh PDA plates. Specimens are deposited in the Museum of the Beijing Forestry University (BJFC). Axenic cultures are maintained in the China Forestry Culture Collection Centre (CFCC).

Morphological analysis

Morphological observations of the asexual/sexual morph in the natural environment were based on features of the conidiomata or ascomata on infected plant tissues and micromorphology, supplemented by cultural characteristics. Ascomata and conidiomata from tree barks were sectioned by hand, using a double-edged blade and structures were observed under a dissecting microscope. The gross morphology of conidiomata or ascomata was recorded using a Leica stereomicroscope (M205 FA). Fungal structures were mounted in clear lactic acid and micromorphological characteristics were examined using a Leica compound microscope (DM 2500) with differential interference contrast (DIC) optics. Thirty measurements of each structure were determined for each collection. Colony characters and pigment production on PDA were noted after 10 d. Colony colors were described according to Rayner (1970).

DNA extraction, PCR amplification and sequencing

Total genomic DNA was extracted from fresh mycelium grown on PDA using a cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle 1990). PCR amplifications were performed in a DNA Engine Peltier Thermal Cycler (PTC-200; Bio-Rad Laboratories, Hercules, CA, USA). The primer sets ITS1 and ITS4 (White et al. 1990) were used to amplify the ITS region. The primer sets LR0R and LR7 (Vilgalys and Hester 1990; Vilgalys and Sun 1994) were used to amplify the nuclear ribosomal large subunit (LSU) region. The primer sets EF1-728F (Carbone and Kohn 1999) and EF1-1567R (Rehner 2001) were used to amplify a partial fragment of the translation elongation factor 1-α gene (tef1-α). The primer sets RPB2-5F and fRPB2-7cR (Liu et al. 1999) were used to amplify the partial RNA polymerase II subunit (rpb2) region. The primer sets T1 (O’Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995) were used to amplify the beta-tubulin gene (tub2). The PCR conditions were: an initial denaturation step of 5 min at 94 °C followed by 35 cycles of 30 sec at 94 °C, 50 sec at 48 °C (ITS, LSU) or 54 °C (tef-1α) or 55 °C (rpb2, tub2) and 1 min at 72 °C, and a final elongation step of 7 min at 72 °C. PCR amplification products were assayed via electrophoresis in 2% agarose gels. DNA sequencing was performed using an ABI PRISM 3730XL DNA Analyser with a BigDye Terminater Kit v.3.1 (Inv-itrogen, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

Phylogenetic analyses

The quality of our amplified nucleotide sequences was checked and combined by SeqMan v.7.1.0 and reference sequences were retrieved from the National Center for Biotechnology Information (NCBI), based on Mejía et al. (2011a), Senanayake et al. (2018), Jiang and Tian (2019), and Jiang et al. (2019), supplemented by sequences of Tenuignomonia styracis and Neognomoniopsis quercina from Crous et al. (2019) and Minoshima et al. (2019). Sequences were aligned using MAFFT v. 6 (Katoh and Toh 2010) and manually corrected using Bioedit 7.0.9.0 (Hall 1999).

The phylogenetical analyses were conducted using Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian inference (BI). MP was performed with PAUP v. 4.0b10 (Swofford 2003) using tree-bisection-reconnection (TBR) as the branch-swapping algorithm. Other calculated parsimony scores were tree length (TL), consistency index (CI), retention index (RI), and rescaled consistency (RC). ML was performed with RAxML (Stamatakis 2006) as implemented in raxmlGUI 1.3 (Silvestro and Michalak 2012), using the ML + rapid bootstrap setting and the GTRGAMMA substitution model with 1000 bootstrap replicates. BI was performed using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.0b4 (Ronquist and Huelsenbeck 2003). Two MCMC chains, started from random trees for 1,000,000 generations and trees, were sampled every 100th generation, resulting in a total of 10,000 trees. The first 25% of trees were discarded as burn-in of each analysis. Branches with significant Bayesian Posterior Probabilities (BPP) were estimated in the remaining 7500 trees. Phylogenetic trees were viewed with FigTree v.1.4.3 (Rambaut 2016) and processed by Adobe Illustrator CS5. Alignment and trees were deposited in TreeBASE (submission ID: S26271). The nucleotide sequence data of the new taxa have been deposited in GenBank (Tables 13).

Results

Phylogenetic analyses

The first sequences dataset for the ITS, LSU, tef1, and rpb2 was analyzed to focus on Gnomoniaceae. The alignment included 45 taxa, including the outgroup sequences of Melanconis marginalis (Table 1). The aligned four-locus datasets included 3388 characters. Of these, 2180 characters were constant, 198 variable characters were par-simony-uninformative and 1010 characters were parsimony informative. The heuristic search using maximum parsimony (MP) generated 4 parsimonious trees (TL = 3241, CI = 0.539, RI = 0.672, RC = 0.362), from which one was selected (Fig. 1). In the phylogenetic tree, two strains form a well-supported clade (MP/ML/BI=100/100/1) sister to the species Flavignomonia rhoigena from Rhus chinensis.

Figure 1. 

Maximum parsimony phylogram of Gnomoniaceae based on a combined matrix of ITS, LSU, tef1 and rpb2 genes. The MP and ML bootstrap support values above 50% are shown at the first and second position, respectively. Thickened branches represent posterior probabilities above 0.90 from BI. Scale bar: 80 nucleotide substitutions. Strains in this study are in blue and ex-type strains are in blod.

Table 1.

Strains and GenBank accession numbers used in the phylogenetic analyses of Gnomoniaceae

Species Strains Genbank accession number
ITS LSU tef1 rpb2
Alnecium auctum CBS 124263 KF570154 KF570154 KF570200 KF570170
Ambarignomonia petiolorum CBS 116866 EU199193 AY818963 NA EU199151
CBS 121227 EU254748 EU255070 EU221898 EU219307
Amphiporthe tiliae CBS 119289 EU199178 EU199122 NA EU199137
Anisogramma anomala 529478 EU683064 EU683066 NA NA
Anisogramma virgultorum 529479 EU683062 EU683065 NA NA
Apiognomonia errabunda AR 2813 DQ313525 NG027592 DQ313565 DQ862014
Apiognomonia veneta MFLUCC 16-1193 MF190114 MF190056 NA NA
Apioplagiostoma populi 858501 KP637024 NA NA NA
Asteroma alneum CBS 109840 EU167609 EU167609 NA NA
Asteroma sp. Masuya 8Ah9-1 NA AB669035 NA NA
Cryphognomonia pini CFCC 53020 MK432672 MK429915 MK578144 MK578100
CFCC 53021 MK432673 MK429916 MK578145 MK578101
Cryptosporella hypodermia CBS 116866 EU199181 AF408346 NA EU199140
Discula destructiva MD 254 AF429741 AF429721 AF429732 NA
Ditopella biseptata MFLU 15-2661 MF190147 MF190091 NA MF377616
Ditopella ditopa CBS 109748 DQ323526 EU199126 NA EU199145
Ditopellopsis sp. CBS 121471 EU254763 EU255088 EU221936 EU219254
Flavignomonia rhoigena CFCC 53118 MK432674 MK429917 NA MK578102
CFCC 53119 MK432675 MK429918 NA MK578103
CFCC 53120 MK432676 MK429919 NA MK578104
Gnomonia gnomon CBS 199.53 DQ491518 AF408361 EU221885 EU219295
CBS 829.79 AY818957 AY818964 EU221905 NA
Gnomoniopsis alderdunensis CBS 125680 GU320825 NA NA NA
Gnomoniopsis chamaemori CBS 803.79 EU254808 EU255107 NA NA
Gnomoniopsis racemula AR 3892 EU254841 EU255122 EU221889 EU219241
Mamianiella coryli BPI 877578 EU254862 NA NA NA
Marsupiomyces quercina MFLUCC 13-0664 MF190116 MF190061 NA NA
Marsupiomyces epidermoidea MFLU 15-2921 NA MF190058 NA NA
Melanconis marginalis CBS 109744 EU199197 AF408373 EU221991 EU219301
Neognomoniopsis quercina CBS 145575 MK876399 MK876440 NA NA
Occultocarpon ailaoshanense LCM 524.01 JF779849 JF779853 NA JF779856
LCM 522.01 JF779848 JF779852 JF779862 JF779857
Ophiognomonia melanostyla LCM 389.01 JF779850 JF779854 NA JF779858
Ophiognomonia vasiljevae AR 4298 EU254977 EU255162 EU221999 EU219331
Plagiostoma aesculi AR 3640 EU254994 EU255164 NA EU219269
Linospora capreae CBS 372.69 NA AF277143 NA NA
Pleuroceras oregonense AR 4333 EU255060 EU255196 EU221931 EU219313
Pleuroceras pleurostylum CBS 906.79 EU255061 EU255197 EU221962 EU219311
Phragmoporthe conformis AR 3632 NA AF408377 NA NA
Valsalnicola oxystoma AR 5137 JX519561 NA NA NA
AR 4833 JX519559 JX519563 NA NA
Sirococcus tsugae AR 4010 EF512478 EU255207 EU221928 EU219289
CBS 119626 EU199203 EU199136 EF512534 EU199159
Tenuignomonia styracis BPI 89278 NA LC379288 LC379282 LC379294

The second dataset with ITS, tef1 and tub2 sequences were analyzed in combination to infer the interspecific relationships within Gnomoniopsis. The alignment included 36 taxa, including the outgroup sequences of Apiognomonia veneta and Plagiostoma euphorbiae (Table 2). The aligned three-locus datasets included 2481 characters. Of these, 1443 characters were constant, 186 variable characters were par-simony-uninformative and 852 characters were parsimony informative. The heuristic search using maximum parsimony (MP) generated one parsimonious tree (TL = 2644, CI = 0.620, RI = 0.781, RC = 0.485), which is shown in Fig. 2. In the phylogenetic tree, three strains form a well-supported clade (MP/ML/BI=100/100/1) that does not include any previously described species.

Figure 2. 

Maximum parsimony phylogram of Gnomoniosis based on a combined matrix of ITS, tef1-α and tub2 genes. The MP and ML bootstrap support values above 50% are shown at the first and second position, respectively. Thickened branches represent posterior probabilities above 0.90 from BI. Scale bar: 80 nucleotide substitutions. Strains in this study are in blue and ex-type strains are in blod.

Table 2.

Strains and GenBank accession numbers used in the phylogenetic analyses of Gnomoniopsis

Species Strain Genbank accession number
ITS tef1 tub2
Apiognomonia veneta CBS 342.86 DQ313531 DQ318036 EU219235
Gnomoniopsis alderdunensis CBS 125679 GU320826 GU320813 GU320788
CBS 125680 GU320825 GU320801 GU320787
CBS 125681 GU320827 GU320802 GU320789
Gnomoniopsis chamaemori CBS 804.79 GU320817 GU320809 GU320777
Gnomoniopsis chinensis CFCC 52286 MG866032 MH545370 MH545366
CFCC 52287 MG866033 MH545371 MH545367
CFCC 52288 MG866034 MH545372 MH545368
CFCC 52289 MG866035 MH545373 MH545369
Gnomoniopsis clavulata CBS 121255 EU254818 GU320807 EU219211
Gnomoniopsis comari CBS 806.79 EU254821 GU320810 EU219156
CBS 807.79 EU254822 GU320814 GU320779
CBS 809.79 EU254823 GU320794 GU320778
Gnomoniopsis daii CFCC 54043 MN598671 MN605519 MN605517
CMF002B MN598672 MN605520 MN605518
Gnomoniopsis fructicola CBS 121226 EU254824 GU320792 EU219144
CBS 208.34 EU254826 GU320808 EU219149
CBS 125671 GU320816 GU320793 GU320776
Gnomoniopsis guttulata MS 0312 EU254812 NA NA
Gnomoniopsis idaeicola CBS 125672 GU320823 GU320797 GU320781
CBS 125673 GU320824 GU320798 GU320782
CBS 125674 GU320820 GU320796 GU320780
CBS 125675 GU320822 GU320799 GU320783
CBS 125676 GU320821 GU320811 GU320784
Gnomoniopsis macounii CBS 121468 EU254762 GU320804 EU219126
Gnomoniopsis occulta CBS 125677 GU320828 GU320812 GU320785
CBS 125678 GU320829 GU320800 GU320786
Gnomoniopsis paraclavulata CBS 123202 GU320830 GU320815 GU320775
Gnomoniopsis racemula CBS 121469 EU254841 GU320803 EU219125
Gnomoniopsis sanguisorbae CBS 858.79 GU320818 GU320805 GU320790
Gnomoniopsis smithogilvyi CBS 130190 JQ910642 KR072534 JQ910639
CBS 130189 JQ910644 KR072535 JQ910641
CBS 130188 JQ910643 KR072536 JQ910640
MUT 401 HM142946 KR072537 KR072532
MUT 411 HM142948 KR072538 KR072533
Gnomoniopsis tormentillae CBS 904.79 EU254856 GU320795 EU219165
Gnomoniopsis xunwuensis CFCC 53115 MK432667 MK578067 MK578141
CFCC 53116 MK432668 MK578068 MK578142
CFCC 53117 MK432669 MK578069 MK578143
Plagiostoma euphorbiae CBS 340.78 DQ323532 GU354016 GU367034

The third dataset with ITS, tef1 and tub2 sequences were analyzed in combination to infer the interspecific relationships within Plagiostoma. The alignment included 48 taxa, including the outgroup sequences of Apiognomonia errabunda (Table 3). The aligned three-locus datasets included 2311 characters. Of these, 1556 characters were constant, 204 variable characters were parsimony-uninformative and 551 characters were parsimony informative. The heuristic search using maximum parsimony (MP) generated 6 parsimonious trees (TL = 1462, CI = 0.685, RI = 0.779, RC = 0.534), from which one was selected (Fig. 3). In the phylogenetic tree, four strains from this study group in a well-supported clade with Plagiostoma populinum. The topologies resulting from MP, ML and BI analyses of the concatenated dataset were congruent.

Figure 3. 

Maximum parsimony phylogram of Plagiostoma based on a combined matrix of ITS, tef1-α and tub2 genes. The MP and ML bootstrap support values above 50% are shown at the first and second position, respectively. Thickened branches represent posterior probabilities above 0.90 from BI. Scale bar: 30 nucleotide substitutions. Strains in this study are in blue.

Table 3.

Strains and GenBank accession numbers used in the phylogenetic analyses of Gnomoniopsis.

Species Strain Genbank accession number
ITS tef1 tub2
Apiognomonia errabunda AR 4182 DQ313543 KJ509937 KJ509947
Plagiostoma aceris-palmati CBS 137265 KJ509959 KJ509938 KJ509949
Plagiostoma aesculi CBS 121905 EU254994 GU367022 GU354005
Plagiostoma amygdalinae CBS 791.79 EU254995 GU367030 GU354012
Plagiostoma apiculatum CBS 109775 DQ323529 GU367008 GU353990
CBS 126126 GU367066 GU367009 GU353991
Plagiostoma barriae LCM 601.01 GU367054 GU366997 GU353980
LCM 484.01 GU367053 GU366995 GU353979
Plagiostoma convexum CBS 123206 EU255047 EU219112 GU353994
Plagiostoma devexum CBS 123201 EU255001 GU367027 GU354010
Plagiostoma dilatatum LCM 403.02 GU367069 GU367012 GU353995
CBS 124976 GU367070 GU367014 GU353996
Plagiostoma euphorbiaceum CBS 816.79 EU255003 EU219158 GU354013
Plagiostoma euphorbiae CBS 340.78 DQ323532 GU367034 GU354016
CBS 817.79 KJ509960 GU367028 KJ509950
Plagiostoma exstocollum CBS 127662 GU367046 GU366988 GU353972
LCM 422.01 GU367043 GU366989 GU353969
Plagiostoma fraxini CBS 121258 EU255008 KJ509939 KJ509951
CBS 109498 AY455810 GU367033 GU354015
Plagiostoma geranii CBS 824.79 EU255009 GU367032 GU354014
Plagiostoma imperceptibile LCM 456.01 GU367059 GU367002 GU353984
Plagiostoma jonesii MFLUCC 16–1189 MF190159 NA MF377589
Plagiostoma mejianum CBS 137266 KJ509961 KJ509940 KJ509952
Plagiostoma oregonense CBS 126124 GU367073 GU367016 GU353999
Plagiostoma ovalisporum CBS 124977 GU367072 GU367015 GU353998
Plagiostoma petiolophilum AR 3821 EU255039 GU367025 GU354008
CBS 126123 GU367078 GU367023 GU354006
Plagiostoma populinum CFCC 53016 MK432677 MK578070 MK578146
CFCC 53017 MK432678 MK578071 MK578147
Plagiostoma populinum CBS 174.58 GU367074 GU367017 GU354000
CBS 144.57 GU367075 GU367018 GU354001
Plagiostoma pulchellum CBS 170.69 EU255043 KJ509941 GU353989
CBS 126653 GU367063 GU367006 GU353987
Plagiostoma rhododendri CBS 847.79 EU255044 GU367026 GU354009
Plagiostoma robergeanum CBS 121472 EU255046 GU367029 GU354011
Plagiostoma rubrosporum CBS 137267 KJ509962 KJ509942 KJ509953
Plagiostoma salicellum CBS 126121 GU367037 GU366977 GU353961
CBS 121466 EU254996 GU366978 GU353962
Plagiostoma salicicola MFLUCC 13–0656 MF190161 NA NA
Plagiostoma samuelsii CBS 125668 GU367051 GU366993 GU353977
LCM 596.01 GU367052 GU366994 GU353978
Plagiostoma triseptatum CBS 137268 KJ509963 KJ509943 KJ509954
Plagiostoma tsukubense CBS 137269 KJ509964 KJ509944 KJ509955
CBS 137270 KJ509965 KJ509945 KJ509956
Plagiostoma versatile CBS 124978 GU367038 GU366979 GU393963
LCM 598.01 GU367040 GU366981 GU393965
Plagiostoma yunnanense LCM 513.02 GU367036 GU366976 GU353960
CBS 124979 GU367035 GU366975 GU353959

Taxonomy

Cryphognomonia C.M. Tian & N. Jiang, gen. nov.

MycoBank No: 829509

Etymology

Crypho + gnomonia, referring to the cryptic stromata on hosts.

Type species

Cryphognomonia pini C.M. Tian & N. Jiang

Description

Pseudostromata erumpent, causing a pustulate bark surface. Central column yellowish to brownish. Stromatic zones lacking. Perithecia conspicuous, flask-shaped to spherical, umber to fuscous black, regularly scattered. Paraphyses deliquescent. Asci fusoid, 8-spored, biseriate, with an apical ring. Ascospores hyaline, clavate to cylindrical, smooth, multi-guttulate, symmetrical to asymmetrical, straight to slightly curved, bicellular, with a median septum distinctly constricted, with distinct hyaline sheath. Asexual morph: not observed.

Notes

Cryphognomonia was classified as a new genus in Gnomoniaceae throughout molecular data and the characteristics of sexual morph. Morphologically, Cryphognomonia can be distinguished from the other genera by pseudostromata and ascospores with obvious hyaline sheath.

Cryphognomonia pini C.M. Tian & N. Jiang, sp. nov.

MycoBank No: 829510
Figure 4

Diagnosis

Cryphognomonia pini differs from its closest phylogenetic neighbor, F. rhoigena, in ITS, LSU, tef1 and rpb2 loci based on the alignments deposited in TreeBASE.

Etymology

Named after the genus of the host plant from which the holotype was collected, Pinus.

Description

Pseudostromata erumpent, causing a pustulate bark surface, 650–1200 µm diam., containing up to 12 perithecia. Central column yellowish to brownish. Stromatic zones lacking. Perithecia conspicuous, flask-shaped to spherical, umber to fuscous black, regularly scattered, 350–600 µm diam. Paraphyses deliquescent. Asci fusoid, 8-spored, biseriate, with an apical ring, (60–)65–80(–90) × (21–)22–31(–35) µm. Ascospores hyaline, clavate to cylindrical, smooth, multi-guttulate, symmetrical to asymmetrical, straight to slightly curved, bicellular, with a median septum distinctly constricted, with distinct hyaline sheath, (15.5–)18–25(–27) × (8.5–)9.5–11.5(–12) µm. Asexual morph: not observed.

Culture characters

Cultures incubated on PDA at 25 °C in the dark, initially pale white, becoming olive-green after 3 wk. The colonies are flat, with regular margins; texture initially uniform, becoming compact after 1 month.

Specimens examined

China. Shaanxi Province: Ankang City, Huoditang forest farm, 33°26'7"N, 108°26'48"E, on branches of Pinus armandii, 8 June 2018, N. Jiang & C.M. Tian (holotype BJFC-S1725; ex-type living culture: CFCC 53020); 33°26'7"N, 108°26'48"E, on branches of Pinus armandii, 8 June 2018, N. Jiang & C.M. Tian (BJFC-S1726; living culture: CFCC 53021).

Notes

Cryphognomonia pini is the type species of Cryphognomonia, and occurs on Pinus armandii in China. Morphologically, Cryphognomonia pini is characterized based on bicellular ascospores with obvious hyaline sheath. In the phylogenetic tree, this species is most closely related to F. rhoigena (Fig. 1). However, Cryphognomonia pini can be distinguished from F. rhoigena based on ITS, LSU, tef1 and rpb2 loci (73/512 in ITS, 4/775 in LSU, 186/437 in tef1 and 90/1064 in rpb2).

Figure 4. 

Cryphognomonia pini on Pinus armandii (BJFC-S1725) A–C habit of ascomata on twigs D, E transverse section of ascomata F longitudinal section through ascomata G asci H, I ascospores. Scale bars: 2 mm (A); 500 μm (B–F); 10 μm (G–I).

Gnomoniopsis xunwuensis C.M. Tian & Q. Yang, sp. nov.

MycoBank No: 829529
Figure 5

Diagnosis

Gnomoniopsis xunwuensis differs from its closest phylogenetic neighbor, G. daii, in ITS, tef1 and tub2 loci based on the alignments deposited in TreeBASE.

Etymology

Named after the County (Xunwu), where the species was first collected.

Figure 5. 

Gnomoniopsis xunwuensis on Castanopsis fissa (BJFC-S1688) A symptoms on leaves of host plant B the colony on PDA C conidiomata on PDA D, E conidiophores attached with condia F conidia. Scale bars: 500 μm (C); 20 μm (D–F).

Description

On PDA: Conidiomata pycnidial, (115–)130–210(–250) μm diam., globose, solitary to gregarious, or occasionally coalescing, deeply embedded in the medium, erumpent, brown to dark black. White to cream conidial drops exuding from the ostioles. Conidiophores (40–)43–58(–60.5) × 2–2.5(–3) μm, cylindrical, hyaline, phiailidic, branched or sympodially branched, straight or slightly curved. Conidia oval or fusiform, straight to slightly curved, hyaline, multiguttules, (14–)16.5–20 × 4–5.5 µm.

Culture characters

Cultures incubated on PDA at 25 °C in the dark. Colony originally compact and flat with white aerial mycelium, then developing pale brown aerial mycelium at the center and blackish green mycelium at the marginal area, zonate with 2 well defined zones with regular edge; conidiomata dense, regularly distributed over agar surface.

Specimens examined

China. Jiangxi Province: Ganzhou City, Xunwu County, 24°40'50"N, 115°34'37"E, on leaves of Castanopsis fissa, 12 May 2018, Q. Yang, Y. Liu & Y.M. Liang (holotype BJFC-S1688; ex-type living culture: CFCC 53115); 24°52'20"N, 115°35'25"E, on leaves of Castanopsis fissa, 12 May 2018, Q. Yang, Y. Liu & Y.M. Liang (BJFC-S1689; living culture: CFCC 53116 and CFCC 53117).

Notes

Gnomoniopsis xunwuensis is associated with leaf spot of Castanopsis fissa, representing the first report from this host in China. It is characterized by sympodially branched conidiophore and oval or fusiform conidia. Morphologically, G. xunwuensis differs from G. daii in having bigger conidia (16.5–20 × 4–5.5 vs. 5.5–7 × 2–3.5 µm) (Jiang and Tian 2019). The phylogenetic inferences indicated this species as an individual well-supported clade (MP/ML/BI=100/100/1) in the genus Gnomoniopsis (Fig. 2).

Plagiostoma populinum (Fuckel) L.C. Mejía. Stud. Mycol. 68: 225. 2011.

Figure 6

Description

See Butin (1958)

Figure 6. 

Plagiostoma populinum on Populus tomentosa (BJFC-S1724) A–C habit of conidiomata on twigs D transverse section through conidiomata E longitudinal section through conidiomata F, G conidiogenous cells attached with conidia H, I condia. Scale bars: 2 mm (A); 1 mm (B, C); 500 μm (D, E); 10 μm (F–I).

Specimens examined

China. Beijing: Haidian district, 40°31'55"N, 116°20'24"E, on branches of Populus tomentosa, 12 November 2017, N. Jiang (BJFC-S1724; living culture: CFCC 53016 and CFCC 53017).

Notes

Plagiostoma populinum is a common plant pathogenic fungus causing poplar canker in China. The current identification follows previous descriptions and records (Butin 1958). In the present study, two isolates (CFCC 53016 and CFCC 53017) from symptomatic branches of Populus tomentosa were congruent with P. populinum based on morphology and DNA sequences data (Fig. 3). We therefore describe P. populinum as a known species for this clade.

Discussion

In this study, three gnomoniaceous species were identified based on morphological and molecular phylogenetic analyses. As a result, Cryphognomonia typified with C. pini is proposed as a new genus in Gnomoniaceae for its distinct phylogenic position and distinctive sexual morphs. Also, Gnomoniopsis xunwuensis strains were successfully isolated from leaf spot of Castanopsis fissa, and were identified as a new species in Gnomoniopsis, which was typified by Gnomoniopsis chamaemori having pycnidia with hyaline, oval, one-celled conidia (Walker et al. 2010).

The type species of Cryphognomonia, C. pini, is unique through its developed pseudostromata and ascospores with distinct hyaline sheath. In the molecular phylogeny, C. pini is closely related to species of F. rhoigena. Flavignomonia rhoigena is characterized by the formation of synnemata and no sexual morph is known for this species (Jiang et al. 2019). However, C. pini can be easily distinguished from F. rhoigena based on ITS, LSU, tef1 and rpb2 loci. Therefore, the unique morphology in combination with an isolated phylogenetic position within Gnomoniaceae warrant the establishment of a new genus.

Most species of Gnomoniopsis show host preference or potentially limited host specificity to genera in the Fagaceae, Onagraceae and Rosaceae (Sogonov et al. 2008). In the present study, isolates were collected from leaf spot of Castanopsis fissa, and described as a novel pathogen depending on its asexual state, G. xunwuensis. Four taxa, G. clavulata, G. daii, G. paraclavulata, and G. smithogilvyi, have been found on Fagaceae host plants. However, Gnomoniopsis xunwuensis can be easily distinguished from the four species in conidial size (16.5–20 × 4–5.5 µm in G. xunwuensis vs. 5.0–8.0 × 2.0–4.0 µm in G. clavulata vs. 5.0–8.0 × 2.0–3.5 µm in G. daii vs. 6.0–9.5 × 2.0–3.5 µm in G. paraclavulata vs. 4.9–9.8 × 2.9–4.9 µm in G. smithogilvyi), as well as supported by molecular data (Walker et al. 2010; Crous et al. 2012; Visentin et al. 2012).

Plagiostoma populinum is regarded as the pathogen responsible for poplar canker. Butin (1958) presented a full description with illustrations of this species as Cryptodiaporthe populea. Mejía et al. (2011a) treated C. populea as a synonym of P. populinum based on analyses of cultural and DNA sequence data. In this paper, P. populinum forms a highly supported monophyletic group (Fig. 3) characterized by having conidia with obvious hyaline sheath. It is the first time that we have been able to provide detailed morphological diagrams in China.

Acknowledgements

This study is financed by the Research Foundation of Education Bureau of Hunan Province, China (Project No.: 19B608), the introduction of talent research start-up fund project of CSUFT (Project No.: 2019YJ025) and National Natural Science Foundation of China (Project No.: 31670647). We are grateful to Chungen Piao, Minwei Guo (China Forestry Culture Collection Center (CFCC), Chinese Academy of Forestry, Beijing.

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