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

Abstract

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.

1. Introduction

Diaporthales is a species-rich fungal order usually associated with forest trees as endophytes, pathogens and saprophytes [1,2,3,4,5,6,7,8,9,10]. Amongst the numerous tree pathogens, the most notorious one is Cryphonectria parasitica (Cryphonectriaceae) causing chestnut (Castanea spp.) blight worldwide [11,12,13]. An example for endophytic lifestyle is Diaporthe biconispora (Diaporthaceae) and an additional six Diaporthe species that are endophytic in healthy Citrus tissues in China [14]. As an example of a saprophyte, Apiosporopsis carpinea (Apiosporopsidaceae) occurs on over-wintered leaves of Carpinus betulus [15].

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.

2. Materials and Methods

2.1. 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).

2.2. 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.

Table 1.

Strains and GenBank accession numbers used in this study.

Species	Country	Host	Host Family	Strain	GenBank Accession Number
					ITS	tef1	tub2
Apiognomonia errabunda	Switzerland	Fagus sylvatica	Fagaceae	AR 2813	DQ313525	DQ313565	DQ862014
Gnomoniopsis alderdunensis	USA	Rubus pedatus	Rosaeace	CBS 125679	GU320826	GU320813	GU320788
Gnomoniopsis alderdunensis	USA	Rubus parviflorus	Rosaeace	CBS 125680 *	GU320825	GU320801	GU320787
Gnomoniopsis alderdunensis	USA	Rubus parviflorus	Rosaeace	CBS 125681	GU320827	GU320802	GU320789
Gnomoniopsis chamaemori	Finland	Rubus chamaemorus	Rosaeace	CBS 804.79	GU320817	GU320809	GU320777
Gnomoniopsis chinensis	China	Castanea mollissima	Fagaceae	CFCC 52286 *	MG866032	MH545370	MH545366
Gnomoniopsis chinensis	China	Castanea mollissima	Fagaceae	CFCC 52287	MG866033	MH545371	MH545367
Gnomoniopsis chinensis	China	Castanea mollissima	Fagaceae	CFCC 52288	MG866034	MH545372	MH545368
Gnomoniopsis chinensis	China	Castanea mollissima	Fagaceae	CFCC 52289	MG866035	MH545373	MH545369
Gnomoniopsis clavulata	USA	Quercus falcata	Fagaceae	CBS 121255	EU254818	GU320807	EU219211
Gnomoniopsis castanopsidis	China	Castanopsis hystrix	Fagaceae	CFCC 54437 *	MZ902909	MZ936385	NA
Gnomoniopsis castanopsidis	China	Castanopsis hystrix	Fagaceae	CFCC 55878	MZ902910	MZ936386	NA
Gnomoniopsis comari	Finland	Comarum palustre	Rosaeace	CBS 806.79	EU254821	GU320810	EU219156
Gnomoniopsis comari	Finland	Comarum palustre	Rosaeace	CBS 807.79	EU254822	GU320814	GU320779
Gnomoniopsis comari	Switzerland	Comarum palustre	Rosaeace	CBS 809.79	EU254823	GU320794	GU320778
Gnomoniopsis daii	China	Castanea mollissima	Fagaceae	CFCC 54043 *	MN598671	MN605517	MN605519
Gnomoniopsis daii	China	Castanea mollissima	Fagaceae	CMF002B	MN598672	MN605518	MN605520
Gnomoniopsis daii	China	Quercus aliena	Fagaceae	CFCC 55517	MZ902911	MZ936387	MZ936403
Gnomoniopsis daii	China	Quercus aliena	Fagaceae	CFCC 55294B	MZ902912	MZ936388	MZ936404
Gnomoniopsis fagacearum	China	Castanopsis faberi	Fagaceae	CFCC 54288	MZ902913	MZ936389	MZ936405
Gnomoniopsis fagacearum	China	Quercus variabilis	Fagaceae	CFCC 54439	MZ902914	MZ936390	MZ936406
Gnomoniopsis fagacearum	China	Castanopsis eyrei	Fagaceae	CFCC 54414	MZ902915	MZ936391	MZ936407
Gnomoniopsis fagacearum	China	Lithocarpus glaber	Fagaceae	CFCC 54316 *	MZ902916	MZ936392	MZ936408
Gnomoniopsis fagacearum	China	Castanopsis chunii	Fagaceae	CFCC 54412	MZ902917	MZ936393	MZ936409
Gnomoniopsis fragariae = G. fructicola	USA	Fragaria vesca	Rosaeace	CBS 121226	EU254824	GU320792	EU219144
Gnomoniopsis fragariae = G. fructicola	France	Fragaria sp.	Rosaeace	CBS 208.34	EU254826	GU320808	EU219149
Gnomoniopsis fragariae = G. fructicola	USA	Fragaria sp.	Rosaeace	CBS 125671	GU320816	GU320793	GU320776
Gnomoniopsis guangdongensis	China	Castanopsis fargesii	Fagaceae	CFCC 54443 *	MZ902918	MZ936394	MZ936410
Gnomoniopsis guangdongensis	China	Castanopsis fargesii	Fagaceae	CFCC 54331	MZ902919	MZ936395	MZ936411
Gnomoniopsis guangdongensis	China	Castanopsis fargesii	Fagaceae	CFCC 54282	MZ902920	MZ936396	MZ936412
Gnomoniopsis guttulata	Bulgaria	Agrimonia eupatoria	Rosaeace	MS 0312	EU254812	NA	NA
Gnomoniopsis hainanensis	China	Castanopsis hainanensis	Fagaceae	CFCC 54376 *	MZ902921	MZ936397	MZ936413
Gnomoniopsis hainanensis	China	Castanopsis hainanensis	Fagaceae	CFCC 55877	MZ902922	MZ936398	MZ936414
Gnomoniopsis idaeicola	USA	Rubus sp.	Rosaeace	CBS 125672	GU320823	GU320797	GU320781
Gnomoniopsis idaeicola	USA	Rubus pedatus	Rosaeace	CBS 125673	GU320824	GU320798	GU320782
Gnomoniopsis idaeicola	France	Rubus sp.	Rosaeace	CBS 125674	GU320820	GU320796	GU320780
Gnomoniopsis idaeicola	USA	Rubus procerus	Rosaeace	CBS 125675	GU320822	GU320799	GU320783
Gnomoniopsis idaeicola	USA	Rubus procerus	Rosaeace	CBS 125676	GU320821	GU320811	GU320784
Gnomoniopsis macounii	USA	Spiraea sp.	Rosaeace	CBS 121468	EU254762	GU320804	EU219126
Gnomoniopsis occulta	USA	Potentilla sp.	Rosaeace	CBS 125677	GU320828	GU320812	GU320785
Gnomoniopsis occulta	USA	Potentilla sp.	Rosaeace	CBS 125678	GU320829	GU320800	GU320786
Gnomoniopsis paraclavulata	USA	Quercus alba	Fagaceae	CBS 123202	GU320830	GU320815	GU320775
Gnomoniopsis racemula	USA	Chamerion angustifolium	Onagraceae	CBS 121469 *	EU254841	GU320803	EU219125
Gnomoniopsis rossmaniae	China	Castanopsis hainanensis	Fagaceae	CFCC 54307 *	MZ902923	MZ936399	MZ936415
Gnomoniopsis rossmaniae	China	Castanopsis hainanensis	Fagaceae	CFCC 55876	MZ902924	MZ936400	MZ936416
Gnomoniopsis sanguisorbae	Switzerland	Sanguisorba minor	Rosaeace	CBS 858.79	GU320818	GU320805	GU320790
Gnomoniopsis silvicola	China	Castanopsis hystrix	Fagaceae	CFCC 54304	MZ902925	MZ936401	MZ936417
Gnomoniopsis silvicola	China	Quercus serrata	Fagaceae	CFCC 54418 *	MZ902926	MZ936402	MZ936418
Gnomoniopsis smithogilvyi	Australia	Castanea sp.	Fagaceae	CBS 130190 *	JQ910642	KR072534	JQ910639
Gnomoniopsis smithogilvyi	Australia	Castanea sp.	Fagaceae	CBS 130189	JQ910644	KR072535	JQ910641
Gnomoniopsis smithogilvyi	Australia	Castanea sp.	Fagaceae	CBS 130188	JQ910643	KR072536	JQ910640
Gnomoniopsis smithogilvyi	Italy	Castanea sativa	Fagaceae	MUT 401	HM142946	KR072537	KR072532
Gnomoniopsis smithogilvyi	New Zealand	Castanea sativa	Fagaceae	MUT 411	HM142948	KR072538	KR072533
Gnomoniopsis tormentillae	Switzerland	Potentilla sp.	Rosaeace	CBS 904.79	EU254856	GU320795	EU219165
Gnomoniopsis xunwuensis	China	Castanopsis fissa	Fagaceae	CFCC 53115 *	MK432667	MK578141	MK578067
Gnomoniopsis xunwuensis	China	Castanopsis fissa	Fagaceae	CFCC 53116	MK432668	MK578142	MK578068

Note: NA, not applicable. Ex-type strains are marked with *, and strains from present study are in black bold.

2.3. 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.

3. Results

3.1. 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.

Figure 1.

Phylogram of Gnomoniopsis resulting from a maximum likelihood analysis based on the ITS gene. Numbers above the branches indicate ML bootstrap values (left, ML BS ≥ 50%) and Bayesian Posterior Probabilities (right, BPP ≥ 0.9). The tree is rooted with Apiognomonia errabunda (CBS 342.86). Isolates from the present study are marked in blue, and taxa in bold face are studied in the present study.

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.

Figure 2.

Phylogram of Gnomoniopsis resulting from a maximum likelihood analysis based on a combined matrix of ITS, tef1 and tub2. Numbers above the branches indicate ML bootstrap values (left, ML BS ≥ 50%) and Bayesian Posterior Probabilities (right, BPP ≥ 0.9). The tree is rooted with Apiognomonia errabunda (CBS 342.86). Isolates from present study are marked in blue, and taxa in bold face are studied in the present study.

3.2. Taxonomy

Gnomoniopsis castanopsidis N. Jiang, sp. nov. Figure 3.

Figure 3.

Morphology of Gnomoniopsis castanopsidis (CFCC 54437). (A) Conidiomata formed on PDA; (B) Conidiogenous cells giving rise to conidia; (C–F) Conidia. Scale bars: A = 500 μm; (B–F) = 10 μm.

Mycobank No.: 840969.

Etymology—Named after the host genus, Castanopsis.

Description—Conidiomata pycnidial, aggregated or solitary, erumpent, globose to pulvinate, brown, 300–700 μm diam., exuding a creamy conidial mass. Conidiophores indistinct, often reduced to conidiogenous cells. Conidiogenous cells hyaline, smooth, multi-guttulate, cylindrical to ampulliform, attenuate towards apex, phialidic, 6.5–13 × 1.5–3 μm. Conidia aseptate, hyaline, smooth, multi-guttulate, oval to fusoid, straight or slightly curved, base truncate, (4.3–) 4.6–5.1 (–5.4) × (1.8–) 2.1–2.5 (–2.6) μm (n = 50), L/W = 1.8–2.6.

Culture characteristics—Colonies flat, spreading, with moderate aerial mycelium and undulate margin, fawn on MEA, dirty-white to fawn on PDA, forming abundant brown conidiomata with creamy conidial masses.

Material examined—CHINA, Hainan Province, Changjiang Li Autonomous County, on diseased leaves of Castanopsis hystrix, 16 November 2018, Yong Li (JNH0003 holotype; ex-type living culture, CFCC 54437); Ibid. (living culture CFCC 55878).

Notes—Two isolates from leaf spots of Castanopsis hystrix clustered into a well-supported clade named Gnomoniopsis castanopsidis, which is distinct from any known species phylogenetically (Figure 1 and Figure 2). Morphologically, G. castanopsidis is similar to G. silvicola in conidial size and shape. However, G. castanopsidis is separated from G. silvicola in 36 bp differences in ITS.

Gnomoniopsis daii C.M. Tian & N. Jiang, Forests 10(11/1016): 6 (2019). Figure 4.

Figure 4.

Morphology of Gnomoniopsis daii (CFCC 55517). (A) Conidiomata formed on PDA; (B) Conidiogenous cells giving rise to conidia; (C–F) Conidia. Scale bars: A = 500 μm; (B–F) = 10 μm.

Description—Conidiomata pycnidial, aggregated or solitary, erumpent, globose to pulvinate, brown, 200–600 μm diam., exuding a creamy conidial mass. Conidiophores indistinct, often reduced to conidiogenous cells. Conidiogenous cells hyaline, smooth, multi-guttulate, cylindrical, attenuate towards apex, phialidic, 7.5–19.5 × 2–3.5 μm. Conidia aseptate, hyaline, smooth, multi-guttulate, oval to fusoid, straight or slightly curved, base truncate, (5.1–) 5.6–6.1 (–6.3) × (2.3–) 2.8–3.2 (–3.6) μm (n = 50), L/W = 1.4–2.5.

Culture characteristics—Colonies flat, spreading, with moderate aerial mycelium and undulate margin, dirty-white to sienna on MEA, dirty-white to fawn on PDA, forming abundant brown conidiomata with creamy conidial masses.

Material examined—CHINA, Henan Province, Xinyang City, Shihe District, on diseased leaves of Quercus aliena, 7 August 2019, Yong Li (JNH0004; living culture, CFCC 55517); Ibid. (living culture CFCC 55294B).

Notes—Gnomoniopsis daii was initially described as the pathogen of Chinese chestnut (Castanea mollissima) fruit rot [23], and subsequently discovered to be the leaf spot pathogen of Chinese chestnut [28]. In the present study, two isolates from diseased leaves of Quercus aliena formed a well-supported clade with the ex-type strain of G. daii (Figure 1 and Figure 2). Hence, Gnomoniopsis daii is for the first time reported on the host genus Quercus.

Gnomoniopsis fagacearum N. Jiang, sp. nov. Figure 5.

Figure 5.

Morphology of Gnomoniopsis fagacearum (CFCC 54316). (A) Conidioma formed on PDA; (B) Conidiogenous cells giving rise to conidia; (C–F) Conidia. Scale bars: A = 300 μm; (B–F) = 10 μm.

Mycobank No.: 840970.

Etymology—Named after the host family, Fagaceae.

Description—Conidiomata acervular, solitary, erumpent, pulvinate, red-brown, 250–450 μm diam. Conidiophores indistinct, often reduced to conidiogenous cells. Conidiogenous cells red-brown, smooth, multi-guttulate, cylindrical, slightly curved, attenuate towards apex, phialidic, 16–33.5 × 2–5 μm. Conidia aseptate, hyaline or seldom red-brown, smooth, multi-guttulate, fusoid, straight or curved, base truncate, (9–) 9.6–11.4 (–12.6) × (2.8–) 3.1–4 (–4.5) μm (n = 50), L/W = 2.1–4.2.

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 examined—CHINA, Guangdong Province, Qingyuan City, Yangshan County, on diseased leaves of Lithocarpus glaber, 26 November 2019, Dan-Ran Bian (JNH0005 holotype; ex-type living culture, CFCC 54316); Jiangxi Province, Xinyu City, Fenyi County, on diseased leaves of Castanopsis faberi, 20 October 2019, Yong Li (living culture, CFCC 54288); Shaanxi Province, Hanzhong City, Foping County, on diseased leaves of Quercus variabilis, 13 August 2019, Yong Li (living culture, CFCC 54439); Fujian Province, Nanping City, Yanping County, on diseased leaves of Castanopsis eyrei, 13 July 2019, Dan-Ran Bian (living culture, CFCC 54414); Guangdong Province, Qingyuan City, Yangshan County, on diseased leaves of Castanopsis chunii, 26 November 2019, Dan-Ran Bian (living culture, CFCC 54412).

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 (Figure 1 and Figure 2). Morphologically, G. guangdongensis can be distinguished from the other Gnomoniopsis species by red-brown conidiogenous cells.

Gnomoniopsis guangdongensis N. Jiang, sp. nov. Figure 6.

Figure 6.

Morphology of Gnomoniopsis guangdongensis (CFCC 54443). (A) Conidioma formed on PDA; (B) Conidiogenous cells giving rise to conidia; (C–F) Conidia. Scale bars: A = 300 μm; (B–F) = 10 μm.

Mycobank No.: 840971.

Etymology—Named after the collection site, Guangdong Province.

Description—Conidiomata pycnidial, aggregated or solitary, erumpent, globose to pulvinate, dark brown, 150–600 μm diam., exuding a creamy conidial mass. Conidiophores indistinct, often reduced to conidiogenous cells. Conidiogenous cells hyaline, smooth, multi-guttulate, cylindrical to ampulliform, attenuate towards apex, phialidic, 12.5–24 × 1.5–3 μm. Conidia aseptate, hyaline, smooth, multi-guttulate, cylindrical, constricted at the middle, straight or slightly curved, base truncate, (4.3–) 4.6–5 (–5.2) × (1.4–) 1.6–1.8 (–2) μm (n = 50), L/W = 2.4–3.3.

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.

Material examined—CHINA, Guangdong Province, Qingyuan City, Yangshan County, on diseased leaves of Castanopsis fargesii, 26 November 2019, Dan-Ran Bian (JNH0006 holotype; ex-type living culture, CFCC 54443); Ibid. (living cultures CFCC 54331 and CFCC 54282).

Notes—Three isolates from leaf spots of Castanopsis fargesii clustered into a well-supported clade named Gnomoniopsis guangdongensis, which is distinct from any known species phylogenetically (Figure 1 and Figure 2). Morphologically, G. guangdongensis can be distinguished from the other Gnomoniopsis species by its conidia constricted at the middle.

Gnomoniopsis hainanensis N. Jiang, sp. nov. Figure 7.

Figure 7.

Morphology of Gnomoniopsis hainanensis (CFCC 54376). (A) Conidioma formed on PDA; (B) Conidiogenous cells giving rise to conidia; (C–F) Conidia. Scale bars: A = 300 μm; (B–F) = 10 μm.

Mycobank No.: 840972.

Etymology—Named after the collection site, Hainan Province.

Description—Conidiomata pycnidial, solitary, erumpent, globose to pulvinate, light brown, 100–300 μm diam., exuding a creamy conidial mass. Conidiophores indistinct, often reduced to conidiogenous cells. Conidiogenous cells hyaline, smooth, multi-guttulate, cylindrical, attenuate towards apex, phialidic, 16.5–26 × 2.5–4.5 μm. Conidia aseptate, hyaline, smooth, multi-guttulate, fusoid, straight, base truncate, (7.3–) 8–10 (–12.2) × (3.3–) 3.4–3.9 (–4.2) μm (n = 50), L/W = 1.9–3.3.

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.

Material examined—CHINA, Hainan Province, Changjiang Li Autonomous County, on diseased leaves of Castanopsis hainanensis, 16 November 2018, Yong Li (JNH0007 holotype; ex-type living culture, CFCC 54376); Ibid. (living culture CFCC 55877).

Notes—Two isolates from leaf spots of Castanopsis hainanensis clustered into a well-supported clade here newly described as Gnomoniopsis hainanensis, which is distinct from any known species phylogenetically (Figure 1 and Figure 2). G. guangdongensis is different from the phylogenetically close species G. fagacearum by its conidial size and length-width ratio (7.3–12.2 × 3.3–4.2 μm, L/W = 1.9–3.3 in G. guangdongensis vs. 9–12.6 × 2.8–4.5 μm, L/W = 2.1–4.2 in G. fagacearum).

Gnomoniopsis rossmaniae N. Jiang, sp. nov. Figure 8.

Figure 8.

Morphology of Gnomoniopsis rossmaniae (CFCC 54307). (A) Conidioma formed on PDA; (B) Conidiogenous cells giving rise to conidia; (C–F) Conidia. Scale bars: A = 300 μm; (B–F) = 10 μm.

Mycobank No.: 840973.

Etymology—In honor of Amy Y. Rossman for her contributions to the study of the fungal order Diaporthales.

Description—Conidiomata pycnidial, solitary, erumpent, pulvinate, dark brown, 250–650 μm diam., exuding a brown conidial mass. Conidiophores indistinct, often reduced to conidiogenous cells. Conidiogenous cells hyaline, smooth, multi-guttulate, cylindrical to ampulliform, attenuate towards apex, phialidic, 9–19 × 2–3 μm. Conidia aseptate to 1-septate, slightly constricted at septum, hyaline, smooth, multi-guttulate, elongate-fusoid, straight, base truncate, (10–) 11.6–14.6 (–16.1) × (3.1–) 3.3–3.9 (–4.1) μm (n = 50), L/W = 2.8–4.5.

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.

Material examined—CHINA, Hainan Province, Changjiang Li Autonomous County, on diseased leaves of Castanopsis hainanensis, 16 November 2018, Yong Li (JNH0008 holotype; ex-type living culture, CFCC 54307); Ibid. (living culture CFCC 55876).

Notes—Two isolates from leaf spots of Castanopsis hainanensis clustered into a well-supported clade here newly described as Gnomoniopsis rossmaniae, which is distinct from any known species phylogenetically (Figure 1 and Figure 2). Morphologically, G. rossmaniae can be distinguished from the other Gnomoniopsis species by its aseptate to 1-septate, elongate-fusoid conidia.

Gnomoniopsis silvicola N. Jiang, sp. nov. Figure 9.

Figure 9.

Morphology of Gnomoniopsis silvicola (CFCC 54418). (A) Conidiomata formed on PDA; (B) Conidiogenous cells giving rise to conidia; (C–F) Conidia. Scale bars: A = 500 μm; (B–F) = 5 μm.

Mycobank No.: 840974.

Etymology—Name from “silva” = forest and “-cola” = inhabiting; with reference to its woody host.

Description—Conidiomata pycnidial, aggregated or solitary, erumpent, globose to pulvinate, brown, 250–650 μm diam., exuding a creamy conidial mass. Conidiophores indistinct, often reduced to conidiogenous cells. Conidiogenous cells hyaline, smooth, multi-guttulate, cylindrical to ampulliform, attenuate towards apex, phialidic, 7–15 × 1.5–2.5 μm. Conidia aseptate, hyaline, smooth, multi-guttulate, oval to fusoid, straight or slightly curved, base truncate, (4.3–) 4.5–5.3 (–5.9) × (1.9–) 2.2–2.6 (–2.7) μm (n = 50), L/W = 1.7–2.5.

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.

Material examined—CHINA, Shaanxi Province, Hanzhong City, Foping County, on diseased leaves of Quercus serrata, 13 August 2019, Yong Li (JNH0009 holotype; ex-type living culture, CFCC 54418); Guangdong Province, Shaoguan City, Lechang County, on diseased leaves of Castanopsis hystrix, 4 December 2019, Dan-Ran Bian (living culture, CFCC 54304).

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 (Figure 1 and Figure 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.

4. 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 (Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9 and Figure 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.

Figure 10.

Gnomoniopsis cultures at 10 days. (A) G. silvicola (CFCC 54304) on MEA; (B) G. silvicola (CFCC 54304) on PDA; (C) G. rossmaniae (CFCC 54307) on MEA; (D) G. rossmaniae (CFCC 54307) on PDA; (E) G. guangdongensis (CFCC 54443) on MEA; (F) G. guangdongensis (CFCC 54443) on PDA; (G) G. hainanensis (CFCC 54376) on MEA; (H) G. hainanensis (CFCC 54376) on PDA; (I) G. fagaceaerum (CFCC 54316) on MEA; (J) G. fagaceaerum (CFCC 54316) on PDA; (K) G. silvicola (CFCC 54418) on MEA; (L) G. silvicola (CFCC 54418) on PDA; (M) G. castanopsidis (CFCC 54437) on MEA; (N) G. castanopsidis (CFCC 54437) on PDA; (O) G. daii (CFCC 55517) on MEA; (P) G. daii (CFCC 55517) on PDA.

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 (Figure 1 and Figure 2). The conidial characters as well as the hosts and distribution provide useful information for species delimitation (Table 2).

Table 2.

Comparison of Gnomoniopsis species on hosts belonging to Fagaceae.

Species	Host	Conidial Length (µm)	Conidial Width (µm)	L/W Ratio	Reference
G. castanopsidis	Castanopsis hystrix	(4.3–) 4.6–5.1 (–5.4)	(1.8–) 2.1–2.5 (–2.6)	1.8–2.6	This study
G. chinensis	Castanea mollissima	(6.0–) 6.5–8.5 (–9.0)	(2.2–) 2.7–3 (–3.5)	NA	[29]
G. clavulata	Quercus falcata	(5–) 6–6.5 (–8)	(2–) 2.5–3 (–4)	1.4–3.7	[20]
G. daii	Castanea mollissima	(5.0–) 5.5–7.0 (–8.0)	2.0–3.5	NA	[23,28]
G. daii	Quercus aliena	(5.1–) 5.6–6.1 (–6.3)	(2.3–) 2.8–3.2 (–3.6)	1.4–2.5	This study
G. fagacearum	Castanopsis chunii, C. eryei, C. faberi, Lithocarpus glaber and Quercus variabilis	(9–) 9.6–11.4 (–12.6)	(2.8–) 3.1–4 (–4.5)	2.1–4.2	This study
G. guangdongensis	Castanopsis fargesii	(4.3–) 4.6–5 (–5.2)	(1.4–) 1.6–1.8 (–2)	2.4–3.3	This study
G. hainanensis	Castanopsis hainanensis	(7.3–) 8–10 (–12.2)	(3.3–) 3.4–3.9 (–4.2)	1.9–3.3	This study
G. paraclavulata	Quercus alba	(6–) 7.5–8 (–9.5)	(2–) 3–3 (–3.5)	1.6–4.2	[20]
G. rossmaniae	Castanopsis hainanensis	(10–) 11.6–14.6 (–16.1)	(3.1–) 3.3–3.9 (–4.1)	2.8–4.5	This study
G. silvicola	Castanopsis hystrix and Quercus serrata	(4.3–) 4.5–5.3 (–5.9)	(1.9–) 2.2–2.6 (–2.7)	1.7–2.5	This study
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.

5. Conclusions

Eight Gnomoniopsis species are known from fagaceous hosts in China based on morphology and phylogeny, viz. G. chinensis on Castanea mollissima, G. castanopsidis on Castanopsis hystrix, G. daii on Castanea mollissima and Quercus aliena, G. fagacearum on Castanopsis chunii, Castanopsis eyrei, Castanopsis faberi, Lithocarpus glaber and Quercus variabilis, G. guangdongensis on Castanopsis fargesii, G. hainanensis on Castanopsis hainanensis, G. rossmaniae on Castanopsis hainanensis and G. silvicola on Castanopsis hystrix and Quercus serrata. They can be well distinguished by the combined approaches of morphology and phylogeny based on ITS, tef1 and tub2 genes.

Author Contributions

Conceptualization, Y.L. and N.J.; methodology, N.J. and D.-R.B.; software, N.J.; validation, N.J., S.-K.W., Y.L. and C.-G.P.; formal analysis, N.J.; investigation, Y.L.; resources, Y.L.; data curation, N.J.; writing—original draft preparation, N.J.; writing—review and editing, H.V.; visualization, N.J.; supervision, C.-G.P.; project administration, C.-G.P.; funding acquisition, C.-G.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Fundamental Research Funds for the Central Non-profit Research Institution of CAF (CAFYBB2018ZB001), and National Microbial Resource Center of the Ministry of Science and Technology of the People’s Republic of China (NMRC-2021-7).

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.