Morpho-Molecular Characterization of Microfungi Associated with Phyllostachys (Poaceae) in Sichuan, China

In the present study, we surveyed the ascomycetes from bamboo of Phyllostachys across Sichuan Province, China. A biphasic approach based on morphological characteristics and multigene phylogeny confirmed seven species, including one new genus, two new species, and five new host record species. A novel genus Paralloneottiosporina is introduced to accommodate Pa. sichuanensis that was collected from leaves of Phyllostachys violascens. Moreover, the newly introduced species Bifusisporella sichuanensis was isolated from leaves of P. edulis, and five species were newly recorded on bamboos, four species belonging to Apiospora, viz. Ap. yunnana, Ap. neosubglobosa, Ap. jiangxiensis, and Ap. hydei, and the last species, Seriascoma yunnanense, isolated from dead culms of P. heterocycla. Morphologically similar and phylogenetically related taxa were compared. Comprehensive descriptions, color photo plates of micromorphology are provided.


Introduction
Bamboo is currently classified in the subfamily Bambusoideae of the extensive grass family Poaceae, and distributed worldwide. It comprises circa 1000 to 1500 species in up to 90 genera [1] and more than 70 species in Phyllostachys (Bambusoideae, Poaceae) [2,3]. Most bamboos are distributed in Southeast Asia, with China as the distribution center [4]. There are about 21 species of Phyllostachys in Sichuan, including Phyllostachys edulis (Carriere) J. Houzea, P. heteroclada Oliver, and P. violascens 'Prevernalis' S.Y. Chen et C.Y. Yao. Bamboos of Phyllostachys play an important role in native economy and ecology. They are used in furniture, and construction (e.g., fishing rods, flutes, flooring materials, chairs.) [5,6]. Bamboo shoots are used as food for humans and animals such as pandas [7,8]. In addition, it is an important ornamental plant for the landscape in China because of its evergreen and graceful appearance [9].
A review of the literature on bamboo-associated fungi reveals that nearly 1500 species have been described or recorded worldwide [10], including economically important pathogenic fungi, and a large number of saprobic and endophytic fungi [1,[11][12][13]. Most bambusicolous fungi have been reported from Asia, especially Japan and Thailand, a few known from India and South America [1,12,[14][15][16][17][18]. However, few studies have investigated the diversity and phylogeny on bamboo in China. The taxonomic studies on bambusicolous fungi are of great significance [19][20][21]. According to the literature review, about 85 species associated with Phyllostachys have been recorded. Teng [22] first reported the fungus Oedocephalum glomerulosum (Bull.) Sacc. on Phyllostachys in 1932. Tai listed 36 species of Phyllostachys from bamboo based on the reports on Chinese fungal resource until 1973 [23]. Chen investigated the phytogeography of forest fungi in China, North America, and Siberia, from which 33 species were found associated with Phyllostachys [24]. However, most of those identifications were conducted lacking molecular data and detailed micromorphology, and as most bamboos are unidentified, the relationship of bambusicolous fungi with bamboo species is not clear.
Due to the high fungal diversity on Phyllostachys, an ongoing investigation was conducted in several main producing or planting areas of bamboo Phyllostachys in Sichuan Province, China, including Ya'an City, Qionglai City, Chengdu City, and Yibin City. In this study, we provide detailed taxonomic features combining morphology and phylogeny on the fungi associated with Phyllostachys from Sichuan Province, China, which is a fundamental task for the bioresource collection on bambusicolous fungi.

Specimen Collection and Morphological Study
From 2020 to 2021, the specimens were collected from leaves, branches, and culms. The samples were kept in plastic bags and taken back to the laboratory after being photographed with a Sony DSC-HX3 digital camera. The fungi were isolated into pure culture based on single spore isolation [25]. Glass slide specimens were prepared by free-hand slicing with double-sided blades for morphologic observation. Morphological characteristics of ascomata and sporodochia were observed using a dissecting microscope, the NVT-GG (Shanghai Advanced Photoelectric Technology Co. Ltd., Shanghai, China), and photographed with a VS-800C micro-digital camera (Shenzhen Weishen Times Technology Co. Ltd., Shenzhen, China). An Olympus BX43 compound microscope with an Olympus DP22 digital camera was used to observe and photograph the microstructure of asci, ascospores, conidiophores, and conidia. Measurements were performed using Tarosoft ® Image Frame Work v.0.9.7 (Tarosoft (R), Nontha Buri, Thailand). Specimens were deposited at the Herbarium of Sichuan Agricultural University, Chengdu, China (SICAU), and pure cultures were deposited at the Culture Collection in Sichuan Agricultural University (SICAUCC).
Amplification reactions were performed in 25 µL of total reaction that contained 22 µL Master Mix (Beijing TsingKe Biotech Co., Ltd., Beijing, China), 1 µL each of forward and reverse (10 µM) primers and 1 µL of DNA template. The amplification reactions were performed as described by Dai et al. [16] and Wang et al. [36]. PCR products were purified and sequenced at TsingKe Biological Technology Co., Ltd. (Chengdu, China). The resulting sequences were submitted to GenBank.

Sequence Alignment and Phylogenetic Analyses
Based on blast searches in GenBank, using ITS, LSU, SSU, tef1-α, tub2, rpb1, or rpb2 sequence data, separate phylogenetic analyses were carried out to determine the placements of each fungal group (Table 1). Sequences for phylogenetic analyses were selected mainly from recently published literature and phylogenetic related sequences based on BLAST searches in GenBank (Table A1). Datasets were aligned using MAFFT v.7.407 [37], and ambiguous regions were excluded with BioEdit version 7.0.5.3 [38]. Maximum likelihood (ML) and Bayesian inference (BI) were constructed as described in Xu et al. [39]. The phylogram was visualized with FigureTree v. 1.4.3 and edited using Adobe Illustrator CS6 (Adobe Systems Inc., San Jose, CA, USA). Table 1. Selected genes for polymerase chain reaction of each genus.
The concatenated aligned dataset of ITS, LSU, SSU, tef1-α sequences, including 124 ingroup taxa within Phaeosphaeriaceae and two outgroup taxa in Leptosphaeriaceae, were used for the phylogenetic analyses of Paralloneottiosporina. The alignment contained 5851 characters (ITS = 1469, LSU = 1433, SSU = 1548, tef1-α = 1401), including gaps. The best scoring RAxML tree with a final likelihood value of −46908.078740 is presented. The matrix had 2382 distinct alignment patterns, with 55.68% of undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.246158, C = 0.236637, G = 0.264322, T = 0.252883, with substitution rates AC = 1.087661, AG = 2.657942, AT = 2.045792, CG = 0.863381, CT = 6.106747, GT = 1.000000. The gamma distribution shape parameter α = 0.263651, and the tree length = 7.503091. In the phylogenetic tree generated from ML and BI analyses, the novel species Paralloneottiosporina sichuanensis (SICAUCC 22-0074, SICAUCC 22-0075) constitutes a moderately supported independent lineage (63% ML/0.99 BYPP statistical support) with the species Alloneottiosporina thailandica (MFLUCC 15-0576) ( Figure 3).  Phylogram generated from RAxML analysis based on combined ITS, LSU, tub2, and tef1α sequence data of Apiosporaceae. Bootstrap support values for maximum likelihood (ML, left) higher than 50% and Bayesian posterior probabilities (BYPP, right) equal to or greater than 0.90 are indicated at the nodes, respectively. The sequences from ex-type strains are marked by a superscript symbol T. The newly generated sequences are written in red. Arthrinium species with yellow background were temporarily not combined to Apiospora.
Phylogenetic analyses of a concatenated aligned dataset (ITS, LSU, rpb1, tef1-α), including 70   Phylogram generated from RAxML analysis based on combined ITS, LSU, tub2, and tef1-α sequence data of Apiosporaceae. Bootstrap support values for maximum likelihood (ML, left) higher than 50% and Bayesian posterior probabilities (BYPP, right) equal to or greater than 0.90 are indicated at the nodes, respectively. The sequences from ex-type strains are marked by a superscript symbol T. The newly generated sequences are written in red. Arthrinium species with yellow background were temporarily not combined to Apiospora.

Figure 2.
Phylogram generated from RAxML analysis based on combined ITS, LSU, rpb1, and tef1-α sequence data of Magnaporthaceae and Pyriculariaceae. Bootstrap support values for maximum likelihood (ML, left) higher than 50% and Bayesian posterior probabilities (BYPP, right) equal to or greater than 0.90 are indicated at the nodes, respectively. The sequences from ex-type strains are marked by a superscript symbol T. The newly generated sequence is written in red.
Notes: Bifusisporella was introduced as a new genus to accommodate B. sorghi based on morphology and phylogeny. At present, Bifusisporella comprises only the ex-type species B. sorghi, and no records on its sexual morph. The new species B. sichuanensis is well-supported within Bifusisporella, which suggests that there is a need to amend the morphological circumscriptions of the genus.
Notes: Paralloneottiosporina resembles Alloneottiosporina in asexual status having semiimmersed, unilocular, gregarious, glabrous conidiomata, but Paralloneottiosporina differs in absent of microconidia, conidia without mucoid appendages, bigger conidia, fewer layers of conidiomatal wall. The macroconidia of Alloneottiosporina species are usually accompanied with mucoid appendages at both ends, and microconidia are produced near the ostiolar channel. Moreover, colonies are whitish to bright orange-pink on PDA in Paralloneottiosporina, but olivaceous-black in Alloneottiosporina [43]. Based on morphological characteristics and molecular phylogeny, the new genus is introduced in Phaeosphaeriaceae.

Discussion
In this study, we confirmed seven species of saprophyte or parasitism from leaves and culms of Phyllostachys, corresponding to four genera. Microfungi are abundant on culms and leaves of bamboo as pointed out by Dai et al. [45]. Ascomycetes are the most abundant species on bamboo, with about 1150 taxa having been recorded [45]. Furthermore, the number of saprophytic fungi is more than that of pathogenic fungi [16,36].
The genus Apiospora Sacc. was recognized and described by Saccardo considering Ap. montagnei designated as the type species [46]. Apiospora has been widely accepted as a synonym for Arthrinium after Ellis [47]. Crous and Groenewald combined Apiospora species to be sexual morphs of Arthrinium species and synonymized under Arthrinium [40]. However, Pintos and Alvarado found that the morphological and ecological differences between Apiospora and Arthrinium are sufficient to support the taxonomic separation of the two genera. As a result, fifty-five species of Arthrinium were combined to Apiospora [48]. In this study, given the phylogenetic analysis with species of Apiospora and Arthrinium, in which 10 species of Arthrinium (Ar. agari, Ar. arctoscopi, Ar. fermenti, Ar. koreanum, Ar. mori, Ar. phaeospermum, Ar. pusillispermum, Ar. sargassi, Ar. taeanense, Ar. marinum) are clustered in a well-supported clade within Apiospora, future studies are needed to better understand the combination of previous Arthrinium species with Apiospora. Apiospora species have a worldwide distribution and can be found on various hosts. Most species occurred on the plants in Poaceae, although some were known from Amaranthaceae, Juncaceae, Euphorbiaceae, Cyperaceae, Restionaceae, Fagaeaeand, even seaweeds [48,49]. To date, more than 25 species have been found on bamboo, most species were saprobic on dead bamboo culms, and a few species have been reported as pathogens. For example, Ap. arundinis causes brown culm streak of Phyllostachys praecox, and Ap. kogelbergensis causes blight disease of Bambusa intermedia [16,41,50,51]. Apiospora. hydei, Ap. neosubglobosa, and Ap. jiangxiensis were saprophytic on unidentified bamboo culms and leaves [41,52]. Apiospora yunnansis has been reported on bamboo culms of Phyllostachys nigra and P. heteroclada, which can cause bamboo blight disease of P. heteroclada [53,54]. In this study, four known species, Apiospora hydei, Ap. neosubglobosa, Ap. jiangxiensis, and Ap. yunnansis, were newly recorded on Phyllostachys nigra, P. heteroclada, P. bissetii, and P. aurea respectively.
At present, Bifusisporella only comprises the ex-type species B. sorghi. In this study, we provide taxonomic details for another new species, B. sichuanensis, that was collected from living leaves of Phyllostachys edulis. B. sorghi was isolated as an endophyte from healthy sorghum leaves in Brazil by Silva et al. [42]. However, B. sichuanensis is pathogenic, causing tar spot on bamboo leaves. In addition, the sexual stage in this genus is supplemented.
Phaeosphaeriaceae is one of the most important and species-rich families in Pleosporales with diverse lifestyles [55,56], and may be found on herbaceous stems or monocotyledonous culms, branches, leaves, flowers, and woody substrates [57,58]. Currently, more than 70 genera are accommodated in Phaeosphaeriaceae [59]. Most genera in this family were introduced as monotypic genera, such as Acericola, Banksiophoma, Bhagirathimyces, Bhatiellae, Brunneomurispora, Camarosporioides, Elongaticollum, Equiseticola, Hydeopsis, Jeremyomyces, Mauginiella, Melnikia, Neoophiobolus, Neosphaerellopsis, Neostagonosporella, Ophiobolopsis, and Parastagonosporella, among others. Due to these genera being represented by a single species, resulting in few samples that could be used for taxon, the phylogenetic relationships with the related genera are sometimes not well-resolved. Based on morphological characteristics and multigene phylogeny, a novel genus, Paralloneottiosporina, is introduced to accommodate Pa. sichuanensis sp. nov. According to the field investigation, Pa. sichuanensis can cause leaf blight that eventually leads to leaf necrosis and plant decline in severe cases. Besides Ph. violascens, leaf blight caused by Pa. sichuanensis has also been observed on P. heterocycla and P. tianmuensis. This indicates that Pa. sichuanensis may be a common parasitic fungus on bamboos.
As only three species are accommodated within Seriascoma, more research is also needed for better understanding this genus [60]. Seriascoma is presently known as saprobic on decaying wood and dead bamboo in the terrestrial or freshwater habitats distributed in China and Thailand [16,44,61,62]. Seriascoma. yunnanense is found on dead branches of bamboo in Yunnan. In this study, S. yunnanense was saprophytic on Phyllostachys edulis.
The previous studies have revealed a high fungal diversity associated with bamboo Phyllostachys. In recent years, 10 species belonging to seven genera have been described from bamboo of Phyllostachys, including two new genera, Neostagonosporella and Parakarstenia, established by Yang et al. on P. heteroclada in Sichuan Province [54,58,[63][64][65][66][67][68][69]. However, the knowledge about bambusicolous fungi is incomplete and mainly remains at cataloguing stage [14]. The previous studies of identification were mostly based on morphological characteristics, and lacked molecular data. Moreover, their hosts were poorly documented or unknown [70], and specimens were absent for further re-examination. Therefore, these species need to be recollected, epitypified, and sequenced [10], and new species need to be discovered and described.

Conflicts of Interest:
The authors declare no conflict of interest.