Tree inhabiting gnomoniaceous species from China, with Cryphogonomonia gen. nov. proposed

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 pinigen. et sp. nov., characterized by developed pseudostromata and ascospores with obvious hyaline sheath; Gnomoniopsis xunwuensissp. 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.

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 . 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. , 2011a(Mejía et al. , b, 2012Sogonov et al. 2008;Walker et al. 2010Walker et al. , 2012Walker et al. , 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. , 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;). 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.

Isolates
Fresh specimens of Gnomoniaceae-related fungi were collected from branches and leaves of hosts in Beijing, Jiangxi and Shaanxi provinces (Tables 1-3). 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 Se-qMan v.7.1.0 and reference sequences were retrieved from the National Center for Biotechnology   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  beck 2003). Two MCMC chains, started from random trees for 1,000,000 generations and trees, were sampled every 100 th 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 1-3).

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 parsimony-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. 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.
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.
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. 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.
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. 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).
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 Gnomo niaceae 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 . 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 Gnomo niaceae warrant the establishment of a new genus.
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.