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Morphological and phylogenetic analyses reveal two new species of Sporocadaceae from Hainan, China

Species of Sporocadaceae have often been reported as plant pathogens, endophytes or saprophytes and are commonly isolated from a wide range of plant hosts. The isolated fungi were studied through a complete examination, based on multilocus phylogenies from combined datasets of ITS/ tub2 / tef1 , in conjunction with morphological characteristics. Nine strains were isolated from Ficus microcarpa , Ilex chinensis and Schima superba in China which represented four species, viz ., Monochaetia schimae sp. nov. , Neopestalotiopsis haikouensis sp. nov. , Neopestalotiopsis piceana and Pestalotiopsis licualicola . Neopestalotiopsis piceana was a new country record for China and first host record from Ficus macrocarpa. Pestalotiopsis licualicola was first report from Ilex chinensis in China.

To date, most phylogenetic studies addressing genera of Sporocadaceae have been based solely on ITS and LSU sequences (Barber et al. 2011;Tanaka et al. 2011;Jaklitsch et al. 2016), or on concatenated datasets of more genes but with incomplete datasets (Senanayake et al. 2015;Wijayawardene et al. 2016). In this study, we made a collection of the established genera Monochaetia, Neopestalotiopsis and Pestalotiopsis species from leaves of Ficus microcarpa, Ilex chinensis and Schima superba in Hainan Province, China. The inventories allowed establishing two new species that are described here.

Isolation and morphological studies
The samples were collected from Hainan Province, China. The strains were isolated from diseased leaves of Ficus microcarpa, Ilex chinensis and Schima superba using surface disinfected tissue fragments (0.5 × 0.5 cm) taken from the margin of leaf lesions (Gao et al. 2014;Jiang et al. 2021a). Surface disinfection consisted of steps including immersion in 75% ethanol for 30 s, 5% sodium hypochlorite (Aladdin, Shanghai, China) for 1 min, and sterile distilled water for 30 s. The pieces were dried with sterilized paper towels and placed on potato dextrose agar (PDA). All plates were incubated at 25 °C for 3-4 days. Then, hyphae were picked out of the periphery of the colonies and inoculated onto new PDA plates. Photographs of the colonies were taken at 7 and 15 days using a Powershot G7X mark II digital camera. Micromorphological characters were observed using an Olympus SZX10 stereomicroscope and Olympus BX53 microscope, all fitted with Olympus DP80 high definition colour digital cameras to photodocument fungal structures. The size of conidia was measured by software Digimizer (https://www.digimizer.com/), and thirty individual measurements were obtained for each character. All fungal strains were stored in 10% sterilised glycerin at 4 °C for further studies. The holotype specimens were deposited in the Herbarium of Plant Pathology, Shandong Agricultural University (HSAUP). Ex-type cultures were deposited in the Shandong Agricultural University Culture Collection (SAUCC). Taxonomic information on the new taxa was submitted to MycoBank (http://www.mycobank.org).
PCR was performed using an Eppendorf Master Thermocycler (Hamburg, Germany). Amplification reactions were performed in a 50 μL reaction volume, which contained 25 μL Green Taq Mix (Vazyme, Nanjing, China), 2 μL of each forward and reverse primer (10 μM) (Tsingke, Beijing, China), and 2 μL template genomic DNA, to which distilled deionized water was added. PCR parameters were as follows: 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at a suitable temperature for 30 s, extension at 72 °C for 1 min and a final elongation step at 72 °C for 7 min. Annealing temperature was 55 °C for ITS, 54 °C for tub2, 52 °C for tef1. The PCR products were visualised on 1% agarose electrophoresis gel. Sequencing was done bi-directionally, conducted by the Tsingke Biotechnology Company Limited (Qingdao, China). Consensus sequences were obtained using MEGA 7.0 or MEGA-X (Kumar et al. 2016). All sequences generated in this study were deposited in GenBank (Table 1).

Phylogeny
Newly generated sequences in this study were aligned with additional related sequences downloaded from GenBank (Table 1) using MAFFT 7 online service with the Auto strategy (Katoh et al. 2019, http://mafft.cbrc.jp/alignment/server/). To establish the identity of the isolates at the species level, phylogenetic analyses were conducted first individually for each locus and then as combined analyses of three loci (ITS, tub2 and tef1). Phylogenetic analyses were based on maximum likelihood (ML) and Bayesian inference (BI) for the multi-locus analyses. For BI, the best evolutionary model for each partition was determined using MrModeltest v. 2.3 (Nylander 2004) and incorporated into the analyses. ML and BI were run on the CIPRES Science Gateway portal (https://www.phylo.org/) (Miller et al. 2012) using RaxML-HPC2 on XSEDE v. 8.2.12 (Stamatakis 2014) and MrBayes on XSEDE v. 3.2.7a (Huelsenbeck and Ronquist 2001;Ronquist and Huelsenbeck 2003;Ronquist et al. 2012), respectively. Four Markov chains were run for two runs from random starting trees for 10,000,000 generations (ITS + tub2 + tef1) until the split deviation frequency value < 0.01, and trees were sampled every 1000 generation. The first quarter generations were discarded as burn-in. A majority rule consensus tree of all remaining trees was calculated. The resulting trees were plotted using FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree) and edited with Adobe Illustrator CC 2019. New sequences generated in this study were deposited at GenBank (https://www.ncbi.nlm.nih.gov; Table 1). The final concatenated sequence alignments were deposited in TreeBase (http://purl.org/phylo/treebase/phylows/study/TB2:S29480). Isolates marked with "T" are ex-type or ex-epitype strains.
Culture characteristics. Colonies on PDA occupying an entire 90 mm petri dish in 7 days at 25 °C in darkness, growth rate of 7.0-14.0 mm/day, edge undulate, white to grey white, with moderate aerial mycelium on the surface, with black, gregarious conidiomata; reverse similar in colour.

Neopestalotiopsis piceana
Culture characteristics. Colonies on PDA incubated at 25 °C in the dark with an average radial growth rate of 9.0-14.0 mm/day and occupying an entire 90 mm petri dish in 7 d, with edge undulate, whitish, aerial mycelium on surface, fruiting bodies black, concentric; reverse of culture yellow to pale brown.
Culture characteristics. Colonies on PDA reaching 70.0-80.0 mm diam after 7 d at 25 °C, growth rate 9.0-12.0 mm/day, edge entire, whitish to pale honey coloured, with sparse aerial mycelium on the surface, with black, gregarious conidiomata; reverse similar in colour.
Notes. In the present study, two strains (SAUCC210087 and SAUCC210088) from symptomatic leaves of Ilex chinensis were clustered to Pestalotiopsis licualicola clade ) based on phylogeny (Fig. 1). Morphologically, our strains were the same as P. licualicola, which was originally described with an asexual morph on leaves of Licuala grandis in China. The sexual morph of P. licualicola was undetermined yet. This is the first time this species has been reported in Ilex chinensis (Aquifoliaceae) in China.

Discussion
Based on phylogeny and morphology, nine strains from three host species (Ficus microcarpa, Ilex chinensis and Schima superba) were described as well as two new species (Monochaetia schimae sp. nov. and Neopestalotiopsis haikouensis sp. nov.) and two known species (Neopestalotiopsis piceana and Pestalotiopsis licualicola). In the genus Monochaetia, most species were found on Fagaceae hosts, including Castanea pubinervis (Monochaetia dimorphospora), Castanea mollissima (Monochaetia castaneae), Quercus pubescens (Monochaetia monochaeta) and etc. In our study, the species of Monochaetia (M. schimae) was first reported from Schima superba (Theaceae). Ilex was widely grown as an evergreen tree all over the world and isolated many pathogens, endophytes or saprophytes (Alfieri et al. 1984;Maharachch. et al. 2014;de Silva et al. 2017;Solarte et al. 2018). More than 100 strains (Xylariales) have been isolated from the genus Ilex. Among these, there was 13 pestalotia-like fungi, and we compare morphology with my new collection. In morphology, the conidia size of Pestalotiopsis humicola is similar to Neopestalotiopsis haikouensis. Phylogenetic analyses of Maharachch. et al. (2014) and the current study show Neopestalotiopsis and Pestalotiopsis are different genus. The known species Neopestalotiopsis piceana was described from Picea sp. (Pinaceae) in United Kingdom (Maharachch. et al. 2014) and Pestalotiopsis licualicola was described from Licuala grandis (Palmaceae) in China . In this study, Neopestalotiopsis piceana was a new record for China and first reported from Ficus macrocarpa (Moraceae), Pestalotiopsis licualicola was first reported from Ilex chinensis (Aquifoliaceae) in China, so we described and illustrated N. piceana and P. licualicola again. Species in genera have multi-septate and more or less fusiform conidia with a single apical and basal appendage (Monochaetia, Seiridium); other genera do not form appendages (Nonappendiculata) or have 2-4 appendages (Pestalotiopsis, Ciliochorella, Neopestalotiopsis, Pseudopestalotiopsis) (Maharachch. et al. 2014;Bonthond et al. 2018;Liu et al. 2019). Our study supported this phenomenon.
As many pestalotioid species have overlapping morphological traits, sequence data is essential to resolve these three genera and introduce new species (Jeewon et al. 2002;de Silva et al. 2017;Norphanphoun et al. 2019). Combined gene sequences of ITS, tub2 and tef1 can provide a better resolution for Monochaetia. However, more genes are needed to provide better resolution and support in Neopestalotiopsis. In the previous studies, members of Sporocadaceae are of particular interest with regard to the production of secondary metabolites, e.g. Bartalinia, Morinia and Pestalotiopsis (Collado et al. 2006;Gangadevi and Muthumary 2008;Liu et al. 2009). Pestalotiopsis fici was shown to possess a very high number of gene clusters involved in bioactive compound synthesis . Owing to Pestalotiopsis and other genus in this family sharing the same evolutionary history, it is important to report novel species and screen for novel metabolites in future studies.