Identification and Characterization of Leaf-Inhabiting Fungi from Castanea Plantations in China

Two Castanea plant species, C. henryi and C. mollissima, are cultivated in China to produce chestnut crops. Leaf spot diseases commonly occur in Castanea plantations, however, little is known about the fungal species associated with chestnut leaf spots. In this study, leaf samples of C. henryi and C. mollissima were collected from Beijing, Guizhou, Hunan, Sichuan and Yunnan Provinces, and leaf-inhabiting fungi were identified based on morphology and phylogeny. As a result, twenty-six fungal species were confirmed, including one new family, one new genus, and five new species. The new taxa are Pyrisporaceae fam. nov., Pyrispora gen. nov., Aureobasidium castaneae sp. nov., Discosia castaneae sp. nov., Monochaetia castaneae sp. nov., Neopestalotiopsis sichuanensis sp. nov. and Pyrispora castaneae sp. nov.


Introduction
Castanea, a plant genus well-known for edible chestnuts and hard timber, is distributed worldwide. The most four famous species are American chestnut (C. dentata), Chinese chestnut (C. mollissima), European chestnut (C. sativa) and Japanese chestnut (C. crenata). C. mollissima is widely cultivated as the crop in most provinces of China. In recent years, another Castanea species, C. henryi, was planted in Hunan Province of China to replace C. mollissima for its higher economic benefits.
In the long cultivation history of chestnut trees, fungal diseases have caused serious economic and ecological problems. Chestnut blight caused by Cryphonectria parasitica is the most notorious one, which devasted Castanea dentata forests in North America and weakened the other chestnut species in plantations [1][2][3]. Several emerging pathogens were recently reported in Castanea sativa from Australia and Europe, viz. Gnomoniopsis smithogilvyi (syn. G. castaneae) [4,5], Phytophthora cinnamomi [6], Sirococcus castanea [7].
In China, previous studies have revealed a high fungal diversity associated with chestnut branch cankers. For example, three Coryneum species [8], two Cryphonectria species [2], six Cytospora species and seven Dendrostoma species were described from the cankered branches [9,10]. In addition, some cryptic species were discovered, such as Aurantiosacculus castaneae [2], Endothia chinensis [2], Melanops castaneicola and Neopseudomelanconis castaneae [11,12]. Due to the high fungal diversity on chestnut branches, we collected leaf samples from two chestnut species, Castanea henryi and C. mollissima in China. In the present study, fungal taxa associated with the symptomatic leaves were identified based on morphological and molecular approaches, which is a fundamental task for the subsequent research on chestnut leaf diseases.

Field Sampling and Isolation
From 2017 to 2020, we investigated Castanea plantations of C. henryi and C. mollissima in the Beijing, Guizhou, Hunan, Sichuan and Yunnan Provinces of China. The disease symptoms were recorded (Figure 1), and 176 fresh leaf samples were collected and packed in sealed plastic bags. These leaf samples were transported to the laboratory for fungal isolation within ten days. Fresh isolates were acquired by removing spore masses in fruiting bodies or surfacesterilized leaf tissues onto the surfaces of potato dextrose agar (PDA; 200 g potatoes, 20 g dextrose, 20 g agar per L) using axenic syringe needles. Then plates were incubated in the dark at 25 • C until germination. Hyphal tips were cut and transported to new PDA plates, and incubated in the dark at 25 • C.

Morphological Identification and Characterization
Fungal species on Chinese chestnut leaves were initially observed based on ascomata and conidiomata formed on the leaf surface or PDA, under a dissecting stereomicroscope (AZ100, Nikon, Tokyo, Japan), then asci, ascospores, conidiogenous cells and conidia were photographed using a Leica compound microscope (DM 2500, Leica, Wetzlar, Germany). Cultural characteristics of isolates incubated on PDA in the dark at 25 • C were recorded.

DNA Extraction, Sequencing and Phylogenetic Analysis
Genomic DNA was extracted from mycelium grown on PDA using a CTAB (cetyltrime thylammonium bromide) method [13]. Then PCR (polymerase chain reaction) was conducted for each genus using selected genes and primers (Table 1). The PCR conditions were set 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 52 • C (ITS, LSU) or 54 • C (act, cal, chs-1, gapdh, his3, rpb2, tub2, tef1) and 1 min at 72 • C, and a final elongation step of 7 min at 72 • C. The PCR amplification products were sequenced using an ABI PRISM ® 3730XL DNA Analyzer with BigDye ® Terminater Kit v. 3.1 (Invitrogen) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).
The sequences obtained in this study (Table 2) were supplemented with the additional sequences retrieved from GenBank. The sequences were aligned and checked manually using MEGA6. Ambiguous regions were excluded from the analyses and gaps were treated as missing data. Phylogenetic analyses were carried out with maximum likelihood analysis (ML), which was performed at the CIPRES web portal [14]. Bayesian inference analysis (BI) was performed in MrBayes v. 3.2.0 [15]. Phylogenetic trees were viewed in FigTree v1. 4. The names of the isolates from present study are marked in blue in the trees. Maximum likelihood bootstrap support values ≥50% (BT) and Bayesian posterior probabilities ≥0.90 (PP) are given at the nodes respectively.
Culture characteristics-Colonies on PDA reaching up to 40 mm in 10 days, flat, initially white-grey mycelium, gradually becoming greenish to dark brown, with white hyphae at the margin; reverse black.
Culture characteristics-Colonies on PDA reaching up to 40 mm in 7 days, spreading, smooth, flat, rapidly turning to olivaceous black, with dark green, irregular margins, covered with slimy masses of conidia.
Culture characteristics-Colonies on PDA reaching up to 50 mm in 10 days, flat with entire edge, forming concentric circles, olivaceous black, sterile. Notes-Discosia was recorded on leaf spots of Chinese chestnut leaves in the monograph of chestnut diseases [44]. However, the fungus was not identified to the species level. In present study, we collected Discosia samples from Beijing (North China), Hunan and Yunnan (South China), and found it different from any known species [43,45,46]. From the phylogram (Figure 9), Discosia brasiliensis was the closest species to D. castaneae, but they could be distinguished by their conidial width (3.5-4.5 µm in D. castaneae vs. 2-3 µm in D. brasiliensis) [46].  Notes-Monochaetia concentrica and M. kansensis were recorded to inhabit Castanea leaves, but Monochaetia castaneae from present study is narrower than them (4.7-6.6 µm in M. castaneae vs. 6.5-8.5 µm in M. concentrica vs. 6.0-8.0 µm in M. kansensis) [47]. Monochaetia castaneae is phylogenetically close to M. junipericola from twigs of Juniperus communis (Figure 11), but they are distinguished by hosts and conidial sizes (18.8-27.3    Notes-Neopestalotiopsis asiatica was described based on the endophytic isolate from unknown trees in China under the name of Pestalotiopsis asiatica [49], but subsequently transferred to the genus Neopestalotiopsis [50]. In the present study, several isolates of N. asiatica were obtained from Chinese chestnut diseased leaves ( Figure 13).  Notes-Neopestalotiopsis brasiliensis was described from rotted fruits of Psidium guajava in Brazil [51]. In present study, strains from diseased chestnut leaves formed a supported clade with the ex-type strain COAD 2166 (Figure 13), and shared similar morphology. Hence, we identified our strains as N. brasiliensis, which represented a new host and geographical record. , fusoid, ellipsoid to subcylindrical, straight to slightly curved, 4-septate; basal cell conic to obconic with a truncate base, hyaline, minutely verruculose and thin-walled, 3.5-5 µm long; three median cells doliiform, versicolourous, second cell from base pale brown to olivaceous, 3.5-6 µm long; third cell honey brown, 4.5-6.5 µm long; fourth cell brown, 4.5-6 µm long; apical cell 3.5-6 µm long, hyaline, subcylindrical, rugose and thin-walled; with 2 or 3 tubular apical appendages, arising from the apical crest, unbranched, filiform, 8-15 µm long; basal appendage present, 1.5-4 µm long.
Culture characteristics-Colonies on PDA reaching up to 60 mm in 7 days, dense aerial mycelium on the surface with undulate edge, white. Fruiting bodies were observed after 15 days.
Culture characteristics-Colonies on PDA reaching up to 60 mm in 7 days, flat, initially white mycelium, gradually becoming pale brownish, with cottony aerial mycelium and fringed margin; reverse pale yellowish.
Notes-The fungal order Diaporthales was well-classified based on both morphology and phylogeny in recent years ( Figure 23) [56][57][58][59]. In this study, the sexual morph was observed on Castanea leaves, showing the typical characters of Diaporthales, the asci with distinct apical ring. Additionally, the asexual morph is distinctive based on the conidiogenous cells with pyriform base and long neck. Hence, we proposed a new family to accommodate this species.      Notes-The genus Coniella was well classified recently [60]. However, Coniella castaneicola was not studied for lacking of fresh collections and DNA data. In present study, we obtained fresh Coniella isolates from Castanea mollissima, and found it distinct from others in the phylogram ( Figure A11). However, specimen in present study shared similar conidial morphology with the original description of Coniella castaneicola (20 × 2-2.5 µm) [61], hence we temporarily assign it to C. castaneicola.
Culture characteristics-Colonies on PDA reaching up to 60 mm in 14 days, flat, creamy white, aerial mycelium forming concentric rings. Conidiomata were formed after 15 days.
Notes-Tubakia dryinoides was described from Castanea crenata and Quercus phillyraeoides in Japan [62]. In present study, strains from diseased chestnut leaves formed a supported clade with the ex-type strain NBRC 9267 ( Figure A12), and shared similar morphology. Hence, we identified our strains as T. dryinoides, which represented a new host and geographical record. Culture characteristics-Colonies on PDA reaching up to 60 mm in 5 days, flat with entire edge, aerial mycelium dense, cottony, grey to dark grey in the centre, white at the margin; reverse greyish green.
Material Notes-Colletotrichum fructicola was described from Coffea arabica in Thailand [63], and subsequently found to infect several economic plants in China, such as Camellia sinensis, Citrus sinensis, Morus alba, Pyrus pyrifolia and Vitis vinifera [64]. In present study, strains from diseased chestnut leaves formed a supported clade with Colletotrichum fructicola ( Figure A13), and shared similar morphology. Hence, we identified our strains as C. fructicola, and Castanea henryi and C. mollissima represented two new host records. Notes-Colletotrichum nymphaeae was recorded to be associated with several hosts, including Camellia oleifera, Citrus aurantifolia, Juglans regia, Malus domestica, Prunus salicina and Vitis vinifera [69]. In the present study, strains from diseased chestnut leaves formed a supported clade with Colletotrichum nymphaeae ( Figure A15). Hence, we identified our strains as C. nymphaeae, and Castanea mollissima represented a new host record.

Discussion
Castanea henryi and C. mollissima are two crops currently cultivated in plantations of China, and suffering from cankers, leaf spots and fruit rot diseases commonly. During our investigations in the past years, Cryphonectria parasitica and Dendrostoma spp. were commonly occurring in most plantations, causing mild to serious cankers depending on the management [2,10]. Gnomoniopsis chinensis caused fatal stem and branch canker disease in only Hebei Province [60]. Compared to the cankers, leaf spots are usually neglected. In present study, we focused on the leaf-inhabiting fungi, identified them to 26 fungal species using phenotypic characters and the multi-locus phylogeny.
From Table 3, most fungi (92.3%) belong to Sordariomycetes and the rest two species belong to Dothideomycetes. This result is nearly congruent as we expected, because Sordariomycetes is a species-rich class and contains many plant pathogens [69]. Within Sordariomycetes, Amphisphaeriales (45.8%), Botryosphaeriales (12.5%), Diaporthales (33.3%) and Glomerellales (20.8%) are identified. They contain famous plant pathogens such as pestalotioid taxa, Botryosphaeria-like taxa, diaporthalean fungi and Colletotrichum species. These fungi were documented in the monograph of chestnut disease by Xie in 1998 [44]. However, genus and species concepts have changed a lot in recent years. For example, the old name Colletotrichum gloeosporioides has been expanded to a group of species named Colletotrichum gloeosporioides species complex [70], hence the chestnut-inhabiting Colletotrichum needs to be re-identified to particular one or several species. The genus Pestalotiopsis s. l. was separated into three genera, namely Pestalotiopsis s. s., Neopestalotiopsis and Pseudopestalotiopsis based on phylogeny [50]. In addition, species with similar morphology from the same host, especially resulting into same symptoms, are not easy to be distinguished without molecular approach in previous studies. Gnomoniopsis daii from Chinese chestnut and Gnomoniopsis smithogilvyi (G. castaneae) from European chestnut were likely identified to Phomopsis (now Diaporthe) species for extremely similar morphology.
According to the filed investigation and sample observation, Colletotrichum spp., Neopestalotiopsis spp., Pestalotiopsis kenyana and Phyllosticta capitalensis are now common pathogens in plantations of Castanea henryi and C. mollissima. Pathogenicity tests and disease control methods are required to be conducted in the future.        .  Table 137994. The scale bar indicates 0.004 nucleotide changes per site. Isolate from this study is marked in blue, ex-type strains are marked with *, and the identified species is marked in yellow. Figure A7. Phylogram generated from RAxML analysis based on combined ITS, tef1 and tub2 sequence data of Pestalotiopsis isolates. The tree was rooted to Neopestalotiopsis magna (MFLUCC 12-652). The scale bar indicates 0.04 nucleotide changes per site. Isolates from this study are marked in blue, ex-type strains are marked with *, and the identified species is marked in yellow. Figure A8. Phylogram generated from RAxML analysis based on combined ITS, LSU, rpb2, tef1 and tub2 sequence data of Robillarda isolates. The tree was rooted to Strickeria kochii (CBS 140411). The scale bar indicates 0.04 nucleotide changes per site. Isolates from this study are marked in blue, ex-type strains are marked with *, and the identified species is marked in yellow.        Figure A13. Phylogram generated from RAxML analysis based on combined ITS, act, chs-1, gapdh and tub2 sequence data of Collectrichum gloeosporiodes species complex isolates. The tree was rooted to Collectrichum catinaense (CBS 142417) and C. boninense (CBS 123755). The scale bar indicates 0.03 nucleotide changes per site. Isolates from this study are marked in blue, ex-type strains are marked with *, and the identified species are marked in yellow. Figure A14. Phylogram generated from RAxML analysis based on combined ITS, act, chs-1, gapdh and tub2 sequence data of Collectrichum boninense species complex isolates. The tree was rooted to Collectrichum gloeosporiodes (CBS 112999). The scale bar indicates 0.03 nucleotide changes per site. Isolates from this study are marked in blue, ex-type strains are marked with *, and the identified species is marked in yellow. Figure A15. Phylogram generated from RAxML analysis based on combined ITS, act, chs-1, gapdh and tub2 sequence data of Collectrichum acutatum species complex isolates. The tree was rooted to Collectrichum anthrisci (CBS 125334). The scale bar indicates 0.04 nucleotide changes per site. Isolates from this study are marked in blue, ex-type strains are marked with *, and the identified species is marked in yellow.