Nectria-related fungi causing dieback and canker diseases in China, with Neothyronectriacitri sp. nov. described

Abstract To clarify phylogenetic relationships amongst Nectria, Neothyronectria and Thyronectria in Nectriaceae, we examined detailed morphological characters and performed phylogenetic analyses of a concatenated dataset, based on the ITS, LSU, tef1 and tub2 DNA sequences of fungal specimens in China. Four species of nectria-related fungi were identified, i.e. Nectriadematiosa, N.pseudotrichia, Neothyronectriacitri and Thyronectriapinicola. The newly described species, Neothyronectriacitri, is characterised by its ascomatal wall with bright yellow scurf, unitunicate asci, each with 4-spored and ascospores allantoid to short-cylindrical, uniseriate, muriform, hyaline to slightly yellowish-brown. This species has affinities with other one known species of Neothyronectria and can be distinguished by molecular data.


Introduction
Nectriaceae Tul. & C. Tul., typified by the genus Nectria (Fr.) Fr., was established by Tulasne and Tulasne (1865) to include nectria-related fungi having brightly pigmented ascomata with fusiform to allantoid ascospores and globose to fusiform phialidic conidia (Rossman et al. 1999, 2013, Rossman 2000, Lombard et al. 2015, Maharachchikumbura et al. 2015, Huang et al. 2018, Yang et al. 2018. Members of the family are unified by phenotypic characters such as uniloculate ascomata that are yellow, orange-red to purple and phialidic asexual morphs. Lombard et al. (2015) defined the generic concepts in Nectriaceae, based on a multi-gene phylogenetic analysis and resolved 47 genera supported by morphological observations. Since then, Neothyronectria was proposed as a new genus to accommodate the species, Neothyronectria sophorae, which is known only from the pycnidial asexual morph (Crous et al. 2016) and Cosmosporella was proposed as a new genus (Huang et al. 2018), thus 49 genera are now accepted in the Nectriaceae. Nectria, typified by N. cinnabarina (Tode: Fr.) Fr., was initially established by Fries (1849). Some species of Nectria are weak parasites of woody plants (Samuels et al. 2009, Hirooka et al. 2011. Hirooka et al. (2012) reviewed the genus, based on the type and additional herbarium specimens, and accepted 29 species. They also monographed the genus Thyronectria as Pleonectria but because Thyronectria (1875) is older, it has priority over Pleonectria (1876) as explained by Jaklitsch and Voglmayr (2014). Many members of Nectria and Thyronectria occur on dead corticated twigs or branches of woody plants worldwide mainly in temperate and subtropical regions (Hirooka et al. 2012, Jaklitsch and Voglmayr 2014, Zeng and Zhuang 2016. To date, 42 species of Thyronectria have been accepted (Jaklitsch and Voglmayr 2014, Voglmayr et al. 2016, Zeng and Zhuang 2016, Lechat et al. 2018. During trips to collect forest pathogens in China, several nectria-related fungi associated with canker or dieback diseases were collected. Based on a multi-locus phylogeny (ITS, LSU, tef1 and tub2), we identified four nectria-related species in three genera of Nectriaceae and propose one new species in Neothyronectria.

Isolates
Fresh specimens were collected from infected branches or twigs of diverse hosts from Beijing, Heilongjiang, Jiangxi, Shaanxi and Xinjiang provinces, China. Strains were isolated from fresh diseased branches and grown from ascospores or conidia by spreading the suspension on the surface of 1.8% potato dextrose agar (PDA), incubated at 25 °C for up to 24 h. Single germinating conidia were removed and transferred to fresh potato dextrose agar (PDA) plate. Specimens and isolates of the new species have been deposited in the Museum of Beijing Forestry University (BJFC). Axenic cultures are maintained in the China Forestry Culture Collection Center (CFCC).

Morphological analysis
Morphological observations of the sexual and asexual morph in the natural environment were based on features of the fruiting bodies produced on infected plant tissues and micromorphology, supplemented by cultural characteristics. Gross morphology of fruiting bodies was recorded using a Leica stereomicroscope (M205 FA). Perithecia, pycnidia, synnemata and stromata were observed and described. To test ascomatal wall reactions, 3% KOH and 100% lactic acid (LA) were used. The micromorphological characteristics were examined by mounting fungal structures in clear lactic acid and 30 measurements at 1000× magnification were determined for each isolate using a Leica compound microscope (DM 2500) with differential interference contrast (DIC) optics. Colony characters and pigment production on PDA were noted after 10 d. Colony colours were described according to Rayner (1970). Longitudinal descriptions, nomenclature and illustrations of taxonomic novelties are deposited in MycoBank (http://www.MycoBank.org; Crous et al. 2004).

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from colonies grown on cellophane-covered PDA, using a modified CTAB [cetyltrimethylammonium bromide] method (Doyle andDoyle 1990, Zhang et al. 2010). For PCR amplifications of phylogenetic markers, four different primer pairs were used (Table 1). 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 of the combined gene regions were performed using Maximum Parsimony (MP), Maximum-Likelihood (ML) and Bayesian Inference (BI) methods. The data were edited in AliView version: 1.19-beta1k and the evolutionary model obtained using MrModeltest v. 2.3 (Nylander et al. 2008) under the Akaike Information Criterion (AIC) performed in PAUP v. 4.0b10. The MP analysis was performed by a heuristic search option of 1000 random-addition sequences with a tree bisection and reconnection (TBR) algorithm. Maxtrees were set to 5000, branches of zero length were collapsed and all equally parsimonious trees were saved. Other calculated parsimony scores were tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency (RC). ML was performed using RAxML-HPC v.8 on XSEDE in CIPRES Science Gateway (Miller et al. 2010, Stamatakis 2014 with 1000 rapid bootstrap replicates using the GTR+I+G model of nucleotide substitution. BI was implemented by MrBayes v. 3.0b4 (Ronquist and Huelsenbeck 2003) with GTR+I+G as the best-fit model. Posterior Probabilities (PP) were estimated by Markov Chain Monte Carlo sampling (MCMC) in MrBayes v. 3.0b4 (Huelsenbeck and Ronquist 2001). 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 the burn-in phase of each analysis. Branches with significant Bayesian Posterior Probabilities (BPP) were estimated in the remaining 7500 trees. Phylogenetic trees were viewed with FigTree v.1.3.1 (Rambaut and Drummond 2010) and processed by Adobe Illustrator CS5. Alignment and trees were deposited in TreeBASE (submission ID: 24366). The nucleotide sequence data of the new taxon have been deposited in GenBank (Table 1).
The phylogeny, resulting from the MP analysis of combined gene sequence data, is shown in Fig. 1. Overall, the topologies obtained from the different phylogenetic analyses were mostly similar and the best scoring MP tree is illustrated here. The MP and ML bootstrap support values above 50% are shown at the first and second position, respectively. Branches with significant BPP (≥ 0.95) in Bayesian analyses were thickened in the phylogenetic tree. Note. Members of Nectria are typically weak parasites of woody plants and occur on hardwood trees and shrubs throughout the temperate zone of the northern hemisphere (Samuels et al. 2009, Hirooka et al. 2011. The genus Nectria is characterised by well-developed stromata, subglobose to globose, red to dark red, fleshy, soft-textured, uniloculate, warted perithecia that become cupulate when dry and are associated with coelomycetous asexual morphs. Asci are unitunicate and clavate to cylindrical in shape. Ascospores are variable and usually broadly ellipsoid to long-fusiform, hyaline to yellow brown, smooth to striate and non-to multi-septate or muriform (Rossman et al. 1999, Hirooka et al. 2009, Maharachchikumbura et al. 2015. Note. Nectria dematiosa has a broad host range and is widely distributed in China, occurring as the most commonly Nectria species (Yang et al. 2018). This study is the first report of N. dematiosa from Betula platyphylla and Tilia mandshurica. Note. Nectria pseudotrichia is one of the common tropical fungi in the genus Nectria and is distinguished in the genus by having muriform ascospores and a synnematous asexual morph. Type species. Neothyronectria sophorae Crous & Thangavel, Persoonia 37: 329, 2016. Note. The genus Neothyronectria was described by Crous & Thangavel (2016) based on the only species, N. sophorae, which is known from a pycnidial asexual morph. Neothyronectria is characterised by pycnidial conidiomata that exude a creamy mucoid conidial mass and hyaline, ampulliform to subcylindrical conidia. In this study, we collected and illustrated here one additional taxon in Neothyronectria. Etymology. Named after the host genus on which it was collected, Citrus. Description. Mycelium not visible around ascomata or on the host. Stromata erumpent through epidermis, up to 0.6 mm high and 1 mm diam., pseudoparenchymatous, cells forming textura angularis to t. globulosa, intergrading with ascomatal wall. Ascomata superficial on well-developed stromata, scattered to aggregated in groups of 3-10, subglobose to globose, 200-270 μm diam., rarely slightly cupulate upon drying, sometimes with only a depressed apical region, yellowish-brown to grey, apical region slightly darker, no colour change in KOH or LA, sometimes surface scurfy or scaly, bright yellow to greenish-yellow. Ascomatal surface cells forming textura globulosa or t. angularis, sometimes including bright yellow scurf, 9-15 μm diam., walls pigmented, uniformly about 1.5 μm thick. Ascomatal wall 27-46 μm thick, of two regions: outer region 22-35 μm thick, intergrading with stroma, cells forming textura globulosa or t. angularis, walls pigmented, about 1.5 μm thick; inner region 9-15 μm thick, of elongate, thin-walled, hyaline cells, forming textura prismatica. Asci clavate, unitunicate, 53.5-65 × 8.5-11 μm, with inconspicuous ring at apex, 4-spored. Ascospores allantoid to short-cylindrical, uniseriate, rounded at both Note. Neothyronectria citri, as described here, is known from an ascomatal sexual morph phylogenetically allied to species of Allantonectria and Thyronectria (Fig. 1). In this study, two strains representing Neothyronectria citri cluster in a well-supported clade and appear most closely related to Neothyronectria sophorae, which was isolated from Sophora microphylla in New Zealand (Crous et al. 2016). Neothyronectria citri can be distinguished, based on ITS, LSU and tub2 loci from Neothyronectria sophorae (16/464 in ITS,9/772 in LSU and 60/494 in tub2).  Note. Thyronectria Sacc. was established by Saccardo (1875) to include nectria-like fungi with immersed ascomata and muriform ascospores and characterised by well-developed erumpent stromata which are often covered with yellow-green amorphous scurf and ascospores that sometimes bud in the ascus to produce ascoconidia Voglmayr 2014, Lombard et al. 2015). Members of the genus occur on dead corticated twigs or branches of woody plants worldwide mainly in temperate and subtropical regions (Hirooka et al. 2012, Jaklitsch andVoglmayr 2014).
Culture characters. Cultures incubated on PDA at 25 °C in darkness. Colony surface cottony with aerial mycelium, becoming yellowish-brown due to pigment formation, small reddish-brown sporodochial conidial masses produced after 3-4 wk.
Note. The hosts of Thyronectria pinicola, synonymised with Pleonectria pinicola, are restricted to Pinus. Members of the genus distributed in Asia (China, Japan, Pakistan), Australia, Europe (Germany, Russia), North America (USA) and South America (Chile) (Jaklitsch and Voglmayr 2014). The asexual morph of T. pinicola in the natural environment has long, sterile hyphae extending from the hymenium and abundant conidiophores (Figs 4E-G). In the present study, two isolates from twigs of Pinus sylvestris var. mongolica were congruent with T. pinicola, based on morphology and DNA sequences data (Fig. 1). We therefore describe T. pinicola as a known species for this clade.

Discussion
In this investigation of nectria-related fungi in China, we identified four species in three genera (Nectria, Neothyronectria and Thyronectria) of Nectriaceae, based on four combined loci (ITS, LSU, tef1 and tub2), as well as morphological characters. It includes Nectria dematiosa, N. pseudotrichia, and Thyronectria pinicola as well as one new species named Neothyronectria citri. The new species is characterised by well-developed erumpent stromata that are often covered with yellow-green amorphous scurf; asci unitunicate, clavate, with inconspicuous ring at apex, each with 4-spored; ascospores allantoid to short-cylindrical, uniseriate, muriform, hyaline to slightly yellowish.
Species revised by Rossman et al. (1999) in Nectria were monographed by Hirooka et al. (2012), who recognised three genera, i.e. Allantonectria, Nectria and Pleonectria. Allantonectria, based on Allantonectria miltina, was recognised as a monotypic genus with small, aseptate ascospores, trichoderma-like conidiophores and occurring on monocotyledonous plants. The genus Thyronectria (as Pleonectria) is characterised by having ascomata with bright yellow scurf, ascospores that often bud to produce ascoconidia inside or outside of the asci and/or a pycnidial anamorph (Hirooka et al. 2012). Based on the lack of bright yellowish scurf on the ascomata, the genus Nectria is easily distinguished from Allantonectria and Thyronectria. In this study, Neothyronectria citri was identified as a new species in Neothyronectria, which was typified by Neothyronectria sophorae having ampulliform to subcylindrical conidia (Crous et al. 2016). Unlike species of Thyronectria, Neothyronectria did not produce ascoconidia but they have bright yellow scurf on the ascomatal wall.
In the taxonomy of hypocrealean fungi, the reaction of the perithecial wall to KOH is considered as an important character (Rossman et al. 1999, Zeng andZhuang 2016). Most species of Allantonectria and Thyronectria have perithecial colour turning darker to blood-red or purple in KOH. However, some species in Thyronectria display a weak or negative reaction to KOH, which might be influenced by the presence of scurf covering the perithecia or their dark-coloured ascomata (Hirooka et al. 2012, Jaklitsch and Voglmayr 2014, Zeng and Zhuang 2016. In our study, the dark perithecial walls of Neothyronectria citri do not change colour in KOH but the major features, such well-developed stromata and ascomata with bright yellow scurf, as well as the molecular data, also provide strong evidence that it belongs to Neothyronectria.