Taxonomy of two synnematal fungal species from Rhus chinensis, with Flavignomonia gen. nov. described.

Rhus chinensis represents a commercially and ecologically important tree species in China, but suffers from canker diseases in Jiangxi Province. Synnemata, pycnidia and ascomata were discovered on cankered tissues. Strains were obtained from single ascospore or conidium within the fruiting bodies and identified based on morphological comparison and the phylogenetic analyses of partial ITS, LSU, tef1 and rpb2 gene sequences. As a result, two species were confirmed to represent two kinds of synnemata. One of these species is described herein as Flavignomonia rhoigena gen. et sp. nov.; and Synnemasporella aculeans is illustrated showing ascomata, pycnidia and synnemata. Flavignomonia is distinguished from Synnemasporella by the colour of the synnematal tips. Additionally, Flavignomonia can be distinguished from the other gnomoniaceous genera by the formation of synnemata. Ning Jiang, Qin Yang, Ying-Mei Liang, Chengming Tian.

Introduction

Many species are important branch canker pathogens, forming acervuli or pycnidia on diseased tissues (Rossman et al. 2007, Senanayake et al. 2017, Jiang et al. 2018, 2019, Wijayawardene et al. 2018, Yang et al. 2018, Voglmayr et al. 2019). However, two diaporthalean species with synnemata were reported to cause cankers, namely (syn. ) and (Fan et al. 2018). These two species differ from the other diaporthalean taxa in conidiomata and form a distinct clade phylogenetically, which was named and distinguished by Fan et al. (2018).

was initially introduced with as the type (Winter 1886). Species in formed upright perithecia, with or without long or short necks and presence or absence of stromatic tissues (Barr 1978, Sogonov et al. 2008, Walker et al. 2012). In the recent monograph of , 34 genera were accepted in the family (Senanayake et al. 2018). Subsequently, and were added based on both molecular and morphological evidence (Crous et al. 2019, Minoshima et al. 2019).

Chinese gall ( Mill.) has a range of uses as source of medicine, dye and oil, and has a wide distribution in China (Wang et al. 2014). However, cankers were found to be associated with different ascomata during our fungal collection trips in Jiangxi Province, China. The objectives of the present study were to identify these fungi based on morphological and phylogenetic evidence.

Materials and methods

Sample collections and isolation

We conducted our fungal collection surveys from April to October in China, and found to be one of the major tree species in Jiangxi Province. Twigs, branches and stems were carefully checked, and diseased tissues were cut into small pieces and packed in paper bags. Isolates were obtained by transferring the ascospores or conidial masses from ascomata to sterile PDA plates, incubating at 25 °C until spores germinated. Single germinating spores were transferred onto new PDA plates, which were kept at 25 °C in darkness. Specimens were deposited in the Museum of the Beijing Forestry University () and axenic cultures maintained in the China Forestry Culture Collection Centre ().

Morphological analysis

Recognition and identification of the fungal species on was based on fruiting bodies formed on the bark. Ascomata and conidiomata were sectioned by hand using a double-edged blade, and microscopic structures were observed under a dissecting microscope. At least 10 conidiomata/ascomata, 10 asci, and 50 conidia/ascospores were measured to calculate mean and standard deviation. Measurements are reported as maxima and minima in parentheses and the range representing the mean plus and minus the standard deviation of the number of measurements given in parentheses (Voglmayr et al. 2017). Microscopy photographs were captured with a Nikon Eclipse 80i compound microscope equipped with a Nikon digital sight DS-Ri2 high definition colour camera, using differential interference contrast illumination. Nomenclatural novelties and descriptions were deposited in MycoBank (Crous et al. 2004).

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted from colonies grown on cellophane-covered PDA plates using a modified 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/ITS4 (White et al. 1990) were used to amplify the ITS region. The primer pair LR0R/LR5 (Vilgalys and Hester 1990) was used to amplify the LSU region. The primer pairs EF1-688F/EF1-986R or EF1-728F/TEF1-LLErev (Carbone and Kohn 1999; Jaklitsch et al. 2005; Alves et al. 2008) were used to amplify tef1 gene. The primer pair dRPB2-5f/dRPB2-7r (Voglmayr et al. 2016) was used to amplify the rpb2 gene. The polymerase chain reaction (PCR) assay was conducted as described by Fan et al. (2018). PCR amplification products were assayed via electrophoresis in 2 % agarose gels. DNA sequencing was performed using an ABI PRISM 3730XL DNA Analyzer with a BigDye Terminater Kit v.3.1 (Invitrogen, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

Phylogenetic analyses

The preliminary identities of the isolates sequenced in this study were obtained by conducting a standard nucleotide BLAST search using the sequences generated from the above primers of the different genomic regions (ITS, LSU, tef1 and rpb2). The BLAST results showed that three isolates were grouped in the family , and five isolates in the genus . The phylogenetic analyses for the three gnomoniaceous isolates were conducted based on Senanayake et al. (2018), supplemented by sequences of and from Crous et al. (2019) and Minoshima et al. (2019). (CBS 109744) in was selected as the out-group taxon. All sequences were aligned using MAFFT v. 6 (Katoh and Toh 2010) and edited manually using MEGA v. 6 (Tamura et al. 2013). Phylogenetic analyses were performed using PAUP v. 4.0b10 for maximum parsimony (MP) analysis (Swofford 2003), and PhyML v. 3.0 for Maximum Likelihood (ML) analysis (Guindon et al. 2010).

MP analysis was run using a heuristic search option of 1000 search replicates with random additions of sequences with a tree bisection and reconnection algorithm. Other calculated parsimony scores were tree length (TL), consistency index (CI), retention index (RI), and rescaled consistency (RC). ML analysis was performed using a GTR site substitution model including a gamma-distributed rate heterogeneity and a proportion of invariant sites (Guindon et al. 2010). The branch support was evaluated using a bootstrapping method of 1000 replicates (Hillis and Bull 1993). The matrix was partitioned for the different gene regions. Phylograms were shown using FigTree v. 1.4.3 (Rambaut 2016). Novel sequences generated in the current study were deposited in GenBank (Table 1) and the aligned matrices used for phylogenetic analyses in TreeBASE (accession number: S25047).

Table 1.

Strains used in the phylogenetic tree and their culture accession and GenBank numbers. Strains from this study are in bold.

Species	Strains	GenBank numbers
		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
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
Synnemasporella aculeans	CFCC 52094	MG682086	MG682026	MG682066	MG682046
Synnemasporella aculeans	CFCC 53123	MK432679	MK429920	MK578148	MK578105
	CFCC 53124	MK432680	MK429921	MK578149	MK578106
	CFCC 53125	MK432681	MK429922	MK578150	MK578107
	CFCC 53126	MK432682	MK429923	MK578151	MK578108
	CFCC 53127	MK432683	MK429924	MK578152	MK578109
Synnemasporella toxicodendri	CFCC 52097	MG682089	MG682029	MG682069	MG682049
Tenuignomonia styracis	BPI 89278	NA	LC379288	LC379282	LC379294

Results

The alignment based on the combined sequence dataset (ITS, LSU, tef1, and rpb2) included 42 in-group taxa and one out-group taxon, comprising 3368 characters in the aligned matrix. Of these, 2201 characters were constant, 224 variable characters were parsimony-uninformative and 943 characters were parsimony informative (282 from the ITS-LSU, 280 from tef1, 381 from rpb2). The MP analysis resulted in nine equally most parsimonious trees with identical tree backbone. The best ML tree (lnL = −20604.0384) was compatible with the MP strict consensus tree, except for unsupported clades in Fig. 1. As the trees obtained from different analytical methods were similar, only the ML tree was present in Fig. 1. The phylogram based on the four gene sequence matrix indicated that the three strains from the present study represent a novel genus in .

Figure 1.

Phylogenetic tree based on an ML analysis of a combined DNA dataset of ITS, LSU, tef1 and rpb2 gene sequences for all genera with DNA data and some species of . Bootstrap values ≥ 50 % for MP and ML analyses are presented at the branches. The scale bar represents the number of changes per site.

Taxonomy

C.M. Tian, Q. Yang & N. Jiang gen. nov.

DAD63D30-357D-53BE-8FEA-5EBACB4FB8AA

829530

Diagnosis.

is distinguished from by the orange tips of its synnemata.

Type species.

C.M. Tian & Q. Yang

Etymology.

The generic name is derived from the colour of synnemata (flavus = yellow) and the genus name .

Description.

Sexual morph: not observed. Asexual morph: Conidiomata synnematal. Synnemata long and determinate, growing from host tissue, with brown base and orange tip, straight to curved, parallel, with flat to slightly concave and dark zone of conidiogenous cells and host tissue at their bases. Conidiophores reduced to conidiogenous cells. Conidiogenous cells phialidic, aggregated, hyaline, straight to curved, cylindrical, arranged adjacent to one another at the end of the synnema, producing a single conidium. Conidia cylindrical to oblong, smooth, multiguttulate, hyaline.

Notes.

is included in based on DNA sequences data. is morphologically similar to in forming synnemata (Wehmeyer 1933, Fan et al. 2018). However, , typified with , is distinguished from species by its orange synnematal tips and hyaline conidia (Fan et al. 2018).

C.M. Tian & Q. Yang sp. nov.

7F7D0EAD-2C04-5A91-B125-2DF50F71C843

829531

Figure 2

Figure 2.

on (BJFC-S1766, holotype) A–C habit of conidiomata on twigs D transverse section through synnema E longitudinal section through synnema F, I conidiogenous cells attached with conidia G conidiomata on PDA H condia J the colony on PDA. Scale bars: 1 mm (B); 500 μm (C); 100 μm (D); 10 μm (F, H–I); 200 μm (G).

can be distinguished from other gnomoniaceous species by the formation of synnemata.

Named after the host genus, .

Sexual morph: not observed. Asexual morph: Conidiomata synnematal. Synnemata (650–)750–1100 µm high, 150–300 µm diam, determinate, growing from host tissue, with brown base and orange tip, straight to curved, parallel, with flat to slightly concave and dark zone of conidiogenous cells and host tissue at their bases. Conidiophores reduced to conidiogenous cells. Conidiogenous cells (12.5–)16–22(–25) × 2 μm, phialidic, aggregated, hyaline, straight to curved, cylindrical, arranged adjacent to one another at the end of the synnema, producing a single conidium. Conidia cylindrical to oblong, smooth, multiguttulate, hyaline, (5–)5.5–7(–8) × 1.5–2 µm.

Culture characters.

On PDA at 25 °C in darkness, initially white, becoming olive-green to black after 3 wk, zonate with 3–4 well defined zones. Conidiomata distributed concentrically over agar surface.

Specimen examined.

CHINA, Jiangxi Province, Ganzhou City, Xunwu County, , on branches of , 14 May 2018, Q. Yang, Y. Liu & Y.M. Liang (holotype BJFC-S1766, ex-type living cultures CFCC 53118, CFCC 53119 and CFCC 53120).

is the type species of in the family . It can be easily distinguished from the other gnomoniaceous genera by its unique conidiomata (Walker et al. 2004, Senanayake et al. 2018, Crous et al. 2019, Minoshima et al. 2019).

(Schwein.) X.L. Fan & J.D.P. Bezerra, Persoonia 40: 130. 2018.

C8503EB3-6F7E-529D-BB7A-97E1F130DB8E

Figure 3 , 4

Figure 3.

Asexual morphology of on (BJFC-S1740) A, B habit of pycnidia on twigs C transverse section of pycnidium D longitudinal section through pycnidium E conidia F, G conidiogenous cells and conidia H, I habit of synnemata on twigs J longitudinal section through synnema K the colony on PDA L, N conidiogenous cells bearing conidia M conidia. Scale bars: 500 μm (B–D, I, J); 10 μm (E–G, L–N).

Figure 4.

Sexual morphology of on (BJFC-S1745) A, B habit of ascomata on twigs C transverse section of ascomata D longitudinal section through ascomata E, F asci G ascospores. Scale bars: 500 μm (B–D); 10 μm (E–G).

Sexual morph: See Wehmeyer (1933) and Fan et al. (2018). Asexual morph: See Fan et al. (2018).

Specimens examined.

CHINA, Jiangxi Province, Ganzhou City, Xunwu County, , on branches of , 14 May 2018, Q. Yang, Y. Liu & Y.M. Liang (BJFC-S1740, living culture CFCC 53123); Ganzhou City, Fengshan forest park, , on branches of , 15 May 2018, Q. Yang, Y. Liu & Y.M. Liang (BJFC-S1753, living culture CFCC 53124 and CFCC 53125). , on branches of , 16 May 2018, Q. Yang, Y. Liu & Y.M. Liang (BJFC-S1745, living culture CFCC 53126 and CFCC 53127).

was proposed as a new combination in the new genus based on the description of (Fan et al. 2018), which was introduced producing perithecial ascomata, and an asexual morph producing sporodochial and/or pycnidial conidiomata (Wehmeyer 1933). In the present study, five isolates from canker tissues on were congruent with based on morphology and DNA sequences data. This was the first time that the sexual morph of in China had been collected.

Discussion

In this study, two diaporthalean species forming synnemata on were identified based on morphology and ITS, LSU, tef1, and rpb2 sequence datasets. As a result, typified with is proposed as a new genus in for its distinct phylogenic position and distinctive asexual fruiting body, Also, strains were successfully isolated from perithecia, pycnidia and synnemata, which was confirmed by molecular data.

Nineteen fungal species have been recorded from the commercially and ecologically important tree species in China, including , , , , , , , , , , , , , , sp., , , and two synnematal species from branch cankers in this study (Farr and Rossman 2019). and , described and illustrated in the present study can be easily recognized by the asexual fruiting bodies, and they differ from each other in the colour of the synnematal tips.

is a globally distributed fungal family on diverse plant hosts (Mejía et al. 2008, 2011a, 2011b, 2012, Sogonov et al. 2008, Walker et al. 2012, Senanayake et al. 2017, 2018). Host specificity of this family has been confirmed to be important in the evolution (Walker et al. 2014). Our newly discovered genus was only found on , and more species might be collected from the plant family in the future.