Morphology and Phylogeny of Gnomoniopsis (Gnomoniaceae, Diaporthales) from Fagaceae Leaves in China

Gnomoniopsis (Gnomoniaceae, Diaporthales) is a well-classified genus inhabiting leaves, branches and fruits of the hosts in three plant families, namely Fagaceae, Onagraceae and Rosaceae. In the present study, eighteen Gnomoniopsis isolates were obtained from diseased leaves of Fagaceae hosts collected from Fujian, Guangdong, Hainan, Henan, Jiangxi and Shaanxi provinces in China. Morphology from the cultures and phylogeny based on the 5.8S nuclear ribosomal DNA gene with the two flanking internally transcribed spacer (ITS) regions, the translation elongation factor 1-alpha (tef1) and the beta-tubulin (tub2) genes were employed to identify these isolates. As a result, seven species were revealed, viz. Gnomoniopsis castanopsidis, G. fagacearum, G. guangdongensis, G. hainanensis, G. rossmaniae and G. silvicola spp. nov, as well as a known species G. daii. In addition, G. daii was firstly reported on the host Quercus aliena.

Gnomoniaceae is a large family of the Diaporthales, with currently 38 accepted genera, including Gnomoniopsis [16][17][18][19]. Gnomoniopsis, based on the type species G. chamaemori, is a well-studied genus in regard to morphology, phylogeny and host associations. This genus is characterized by having small, black perithecia immersed in the host tissue and one-septate, oval to fusiform ascospores, and is well-distinguished by phylogenies based on the 5.8S nuclear ribosomal DNA gene with the two flanking internally transcribed spacer (ITS) regions, the translation elongation factor 1-alpha (tef1) and the beta-tubulin (tub2) genes [20,21]. Species of Gnomoniopsis are currently known to inhabit only members of three plant families as hosts, viz. Fagaceae, Onagraceae and Rosaceae [20][21][22][23][24].
Until now, thirty species epithets of Gnomoniopsis have been recorded in Index Fungorum, six of them were reported from fagaceous trees [22]. Two species, Gnomoniopsis clavulata and G. paraclavulata, were firstly discovered on overwintered leaves of Quercus trees in the USA [20,21]. Subsequently, Gnomoniopsis smithogilvyi with its synonym G. castaneae were proposed from rotten fruits of Castanea in Australia and Europe by two independent studies [25,26]. However, these two names were proven to be a single species based on phylogeny and morphological characters [27]. Hence, G. castaneae becomes a synonym of G. smithogilvyi based on priority. In China, G. daii was described from rotten fruits and diseased leaves of Castanea mollissima [23,28]. Meanwhile, a different species named G. chinensis was reported to cause Chinese chestnut branch canker [29]. Later, Yang et al. described G. xunwuensis from leaf spots of Castanopsis fissa in China [24]. Since three Fagaceae-inhabiting species from China are now only known in the asexual morph, it is hard to separate them based on only morphological characters [23,24,29]. Hence, it is necessary to conduct phylogenetic analyses in order to recognize and identify the species [29].
Fagaceae is a common plant family widely distributed in the northern hemisphere, with seven genera namely Castanea, Castanopsis, Cyclobalanopsis, Fagus, Lithocarpus, Quercus and Trigonobalanus [30]. Previously, Gnomoniopsis has been reported from Castanea, Castanopsis and Quercus species [22]. The aims of present study are to investigate fagaceous hosts to collect Gnomoniopsis samples in China, and to identify them to species level based on combined morphology and phylogeny of ITS, tef1 and tub2 loci.

Field Sampling and Isolation
In the present study, we investigated leaf diseases of fagaceous trees in Fujian, Guangdong, Hainan, Henan, Jiangxi and Shaanxi provinces of China during 2018 and 2020. The diseased leaf samples were packed in paper bags and transferred to the laboratory for isolation. The infected leaves were firstly surface-sterilized for 1 min in 75% ethanol, 3 min in 1.25% sodium hypochlorite, and 1 min in 75% ethanol, then rinsed for 2 min in distilled water and blotted on dry sterile filter paper. Then samples were cut into 0.5 × 0.5 cm pieces using a double-edge blade, and transferred onto the surface of potato dextrose agar (PDA; 200 g potatoes, 20 g dextrose, 20 g agar per L) and malt extract agar (MEA; 30 g malt extract, 5 g mycological peptone, 15 g agar per L), and incubated at 25 • C to obtain the pure culture. The cultures were deposited in China Forestry Culture Collection Center (CFCC), and the specimens in the herbarium of the Chinese Academy of Forestry (CAF).

DNA Extraction, Sequencing and Phylogenetic Analyses
Genomic DNA was extracted from mycelia grown on cellophane-covered PDA using a cetyltrimethylammonium bromide (CTAB) method [31]. DNA was checked by electrophoresis in 1% agarose gel, and the quality and quantity were measured using a NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). Three partial loci, ITS region, tef1 and tub2 genes were amplified by the following primer pairs: ITS1 and ITS4 for ITS [32], EF1-688F and EF2 for tef1 [33], and T1/Bt2a and Bt2b for tub2 [34,35]. The polymerase chain reaction (PCR) conditions were as follows: an initial denaturation step of 5 min at 94 • C, followed by 35 cycles of 30 s at 94 • C, 50 s at 48 • C (ITS) or 54 • C (tub2) or 55 • C (tef1), and 1 min at 72 • C, and a final elongation step of 10 min at 72 • C. PCR products were assayed via electrophoresis in 2% agarose gels. DNA sequencing was performed using an ABI PRISM 3730XL DNA Analyser with a BigDye Terminator Kit v.3.1 (Invitrogen, Waltham, MA, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).
The sequences obtained in the present study were assembled using SeqMan v.7.1.0, and reference sequences were retrieved from the National Center for Biotechnology Information (NCBI), based on recent publications on the genus Gnomoniopsis [20][21][22][23][24]29]. Sequences of an accession of Apiognomonia errabunda (AR 2813) were added to represent the outgroup. The sequences were aligned using MAFFT v.6 and corrected manually using MEGA 7.0.21 [36].
The phylogenetic analyses of the ITS region and of a combined matrix of the three loci (ITS-tef1-tub2) were performed using Maximum Likelihood (ML) and Bayesian Inference (BI) methods. ML was implemented on the CIPRES Science Gateway portal (https:// www.phylo.org) using RAxML-HPC BlackBox 8.2.10 [37,38], employing a GTRGAMMA substitution model with 1000 bootstrap replicates. Bayesian inference was performed using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.0 [39]. Two MCMC chains, starting 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 > 0.9) were estimated in the remaining 7500 trees. Phylogenetic trees were viewed with FigTree v.1.3.1 and processed by Adobe Illustrator CS5. The nucleotide sequence data of the new taxa were deposited in GenBank, and the GenBank accession numbers of all accessions included in the phylogenetic analyses are listed in Table 1.

Morphological Identification and Characterization
The morphological data of the isolates collected in the present study were based on the cultures sporulating on PDA in the dark at 25 • C. The conidiomata were observed and photographed under a dissecting microscope (M205 C, Leica, Wetzlar, Germany). The conidiogenous cells and conidia were immersed in tap water, then the microscopic photographs were captured with an Axio Imager 2 microscope (Zeiss, Oberkochen, Germany) equipped with an Axiocam 506 color camera, using differential interference contrast (DIC) illumination. More than 50 conidia were randomly selected for measurement. Culture characteristics were recorded from PDA and MEA after 10 days incubation at 25 • C in the dark.

Phylogeny
The sequence dataset of the ITS gene matrix was analysed to infer the interspecific relationships within Gnomoniopsis. The dataset consisted of 56 sequences including one outgroup taxon, Apiognomonia errabunda (CBS 342.86). A total of 538 characters including gaps were included in the phylogenetic analysis. The topologies resulting from ML and BI analyses of the concatenated dataset were congruent ( Figure 1). Isolates from the present study formed seven individual clades representing seven species of Gnomoniopsis, including six new species and one known species.
The combined three-gene sequence dataset (ITS, tef1 and tub2) was further analysed to compare with results of the phylogenetic analyses of the ITS gene. The dataset consisted of 56 sequences including one outgroup taxon, Apiognomonia errabunda (CBS 342.86). A total of 1426 characters including gaps (538 for ITS, 348 for tef1 and 540 for tub2) were included in the phylogenetic analysis. The topologies resulting from ML and BI analyses of the concatenated combined dataset were congruent ( Figure 2). Isolates from the present study formed seven individual clades which were congruent with those in Figure 1
Culture characteristics-Colonies flat, spreading, with moderate aerial mycelium, folded surface and lobate margin, sienna to red-brown on MEA, dirty-white to slightly red-brown on PDA, occasionally forming red-brown conidiomata.
Material Notes-Five isolates from leaf spots of Castanopsis chunii, C. eryei, C. faberi, Lithocarpus glaber and Quercus variabilis clustered into a well-supported clade here newly described as Gnomoniopsis fagacearum, which is distinct from any known species phylogenetically (Figures 1 and 2). Morphologically, G. guangdongensis can be distinguished from the other Gnomoniopsis species by red-brown conidiogenous cells.
Culture characteristics-Colonies flat, spreading, with sparse to moderate aerial mycelium and diffuse margin, buff to fawn on MEA, dirty-white on PDA, with age forming narrow concentric zones, forming abundant dark brown conidiomata with creamy conidial masses.
Culture characteristics-Colonies flat, spreading, with sparse aerial mycelium and lobate to undulate margin, sienna to luteous on MEA, luteous on PDA, with age forming narrow concentric zones, forming abundant light brown conidiomata with creamy conidial masses.
Culture characteristics-Colonies flat, spreading, with sparse aerial mycelium and lobate to undulate margin, hazel on MEA, dirty-white on PDA, seldom forming dark brown conidiomata with brown conidial masses.
Notes-Two isolates from leaf spots of Castanopsis hainanensis clustered into a wellsupported clade here newly described as Gnomoniopsis rossmaniae, which is distinct from any known species phylogenetically (Figures 1 and 2). Morphologically, G. rossmaniae can be distinguished from the other Gnomoniopsis species by its aseptate to 1-septate, elongate-fusoid conidia.

Discussion
In the present study, six new Gnomoniopsis species (viz. G. castanopsidis, G. fagacearum, G. guangdongensis, G. hainanensis, G. rossmaniae and G. silvicola) are described and illustrated (Figures 3-10), and a new host, Quercus aliena, is reported for the known species G. daii. As noted in previous studies, the fungal genus Gnomoniopsis is so far only known from hosts of three plant families, Fagaceae, Onagraceae and Rosaceae [20,21,40], of which only one species, G. racemula was described from the family Onagraceae [20]. Hence, Fagaceae and Rosaceae are the main hosts for Gnomoniopsis species. Although several new Mycobank No.: 840974. Etymology-Name from "silva" = forest and "-cola" = inhabiting; with reference to its woody host.
Culture characteristics-Colonies flat, spreading, with moderate aerial mycelium and undulate margin, luteous to brown on MEA, dirty-white on PDA, forming abundant brown conidiomata with creamy conidial masses.
Notes-Two isolates from leaf spots of Castanopsis hystrix and Quercus serrata clustered into a well-supported clade here described as the new species Gnomoniopsis silvicola, which is distinct from any known species phylogenetically (Figures 1 and 2). Morphologically, G. silvicola has a bit smaller conidia than its phylogenetically close species G. daii (4.3-5.9 × 1.9-2.7 µm in G. silvicola vs. 5.1-6.3 × 2.3-3.6 µm in G. daii). In addition, G. silvicola is separated from G. daii in 34 bp differences in ITS.

Discussion
In the present study, six new Gnomoniopsis species (viz. G. castanopsidis, G. fagacearum, G. guangdongensis, G. hainanensis, G. rossmaniae and G. silvicola) are described and illustrated (Figures 3-10), and a new host, Quercus aliena, is reported for the known species G. daii. As noted in previous studies, the fungal genus Gnomoniopsis is so far only known from hosts of three plant families, Fagaceae, Onagraceae and Rosaceae [20,21,40], of which only one species, G. racemula was described from the family Onagraceae [20]. Hence, Fagaceae and Rosaceae are the main hosts for Gnomoniopsis species. Although several new species and host records are reported from Fagaceae in China herein, numerous additional hidden species might remain to be revealed from the widely spread fagaceous species in China.  So far, eleven Gnomoniopsis species were reported from fagaceous hosts, of which G. clavulata and G. paraclavulata were described from Quercus in the USA [20]. Gnomoniopsis smithogilvyi was reported as causal agent of sweet chestnut fruit rot in Australia, Europe and North America [25,26,[41][42][43][44][45]. The remaining eight species are only known from So far, eleven Gnomoniopsis species were reported from fagaceous hosts, of which G. clavulata and G. paraclavulata were described from Quercus in the USA [20]. Gnomoniopsis smithogilvyi was reported as causal agent of sweet chestnut fruit rot in Australia, Europe and North America [25,26,[41][42][43][44][45]. The remaining eight species are only known from China. They were well distinguished in phylogenetic analyses of the ITS gene and of combined matrices of ITS, tef1 and tub2 genes (Figures 1 and 2). The conidial characters as well as the hosts and distribution provide useful information for species delimitation (Table 2). G. smithogilvyi Castanea sativa (6.0-) 8 (-9.5) (2.0-) 2.5 (-4.0) 2.5-3.5 [25] Several Gnomoniopsis species are pathogens of leaves, branches or fruits [29,46]. For example, G. smithogilvyi causes sweet chestnut branch canker and fruit rot in in Australia, Europe and the USA [26,42,45], whereas in China G. daii is one of the main pathogens of Chinese chestnut causing fruit rot and leaf spot diseases [23,28]. In addition, G. chinensis causes branch canker of Chinese chestnut in China [29]. The newly described species of the present study were isolated from diseased leaves; however, additional studies are required to confirm their pathogenicity.
Institutional Review Board Statement: Not applicable for studies involving humans or animals.

Informed Consent Statement:
Not applicable for studies involving humans.

Data Availability Statement:
The sequences from the present study were submitted to the NCBI website (https://www.ncbi.nlm.nih.gov/) and the accession numbers were listed in Table 1.

Conflicts of Interest:
The authors declare no conflict of interest.