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

Abstract 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 rhoigenagen. 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.

Gnomoniaceae was initially introduced with Gnomonia as the type (Winter 1886). Species in Gnomoniaceae 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 Diaporthales, 34 genera were accepted in the family Gnomoniaceae (Senanayake et al. 2018). Subsequently, Neognomoniopsis and Tenuignomonia were added based on both molecular and morphological evidence (Crous et al. 2019, Minoshima et al. 2019. Chinese gall (Rhus chinensis 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.

Sample collections and isolation
We conducted our fungal collection surveys from April to October in China, and found Rhus chinensis 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 (BJFC) and axenic cultures maintained in the China Forestry Culture Collection Centre (CFCC).

Morphological analysis
Recognition and identification of the fungal species on Rhus chinensis 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 Gnomoniaceae, and five isolates in the genus Synnemasporella. The phylogenetic analyses for the three gnomoniaceous isolates were conducted based on Senanayake et al. (2018), supplemented by sequences of Tenuignomonia styracis and Neognomoniopsis quercina from Crous et al. (2019) and Minoshima et al. (2019). Melanconis marginalis (CBS 109744) in Melanconidaceae 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 Tree-BASE (accession number: S25047).

Phylogenetic analyses
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 Gnomoniaceae. 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.

Flavignomonia rhoigena
Culture characters. On PDA at 25 °C in darkness, initially white, becoming olivegreen to black after 3 wk, zonate with 3-4 well defined zones. Conidiomata distributed concentrically over agar surface.  Notes. Flavignomonia rhoigena is the type species of Flavignomonia in the family Gnomoniaceae. 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.  Notes. Synnemasporella aculeans was proposed as a new combination in the new genus Synnemasporella based on the description of Cryptodiaporthe aculeans , 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 Rhus chinensis were congruent with S. aculeans based on morphology and DNA sequences data. This was the first time that the sexual morph of Synnemasporella aculeans in China had been collected.

Discussion
In this study, two diaporthalean species forming synnemata on Rhus chinensis were identified based on morphology and ITS, LSU, tef1, and rpb2 sequence datasets. As a result, Flavignomonia typified with F. rhoigena is proposed as a new genus in Gnomoniaceae for its distinct phylogenic position and distinctive asexual fruiting body, Also, Synnemasporella aculeans strains were successfully isolated from perithecia, pycnidia and synnemata, which was confirmed by molecular data.
Gnomoniaceae is a globally distributed fungal family on diverse plant hosts , 2011a, 2011b, Sogonov et al. 2008, Walker et al. 2012, Senanayake et al. 2017. Host specificity of this family has been confirmed to be important in the evolution (Walker et al. 2014). Our newly discovered genus Flavignomonia was only found on Rhus chinensis, and more Flavignomonia species might be collected from the plant family Anacardiaceae in the future.