Plant-Associated Novel Didymellaceous Taxa in the South China Botanical Garden (Guangzhou, China)

The South China Botanical Garden (SCBG), one of the largest and oldest botanical gardens in China, conserves important plant germplasms of endangered species. Therefore, ensuring tree health and studying the associated mycobiome of the phyllosphere is essential to maintaining its visual aesthetics. During a survey of plant-associated microfungal species in SCBG, we collected several coelomycetous taxa. Phylogenetic relationships were evaluated based on the analyses of ITS, LSU, RPB2, and β-tubulin loci. The morphological features of the new collections were compared with those of existing species, emphasizing close phylogenetic affinities. Based on the morphological comparisons and multi-locus phylogeny, we introduce three new species. These are Ectophoma phoenicis sp. nov., Remotididymella fici-microcarpae sp. nov., and Stagonosporopsis pedicularis-striatae sp. nov. In addition, we describe a new host record for Allophoma tropica in the Didymellaceae. Detailed descriptions and illustrations are provided along with notes comparing allied species.


Introduction
The South China Botanical Garden (SCBG), Chinese Academy of Sciences, Guangzhou was established in 1929 [1]. This is the largest modern botanical garden in Guangzhou, Guangdong Province, and it extends over 2854 acres comprising around 2400 plant species including alpine, arctic, aquatic, Mediterranean, tropical, and desert examples [1]. SCBG is one of the top plant germplasm conservation institutions in China and it contains 710 of the verified Red List plant taxa [2]. There are more than 14,000 trees including Chinese endemic species in ex-situ conservation [3]. Tree health and disease management are rather important in SCBG.
In a survey of fungal species associated with plants in botanical gardens, we collected several saprobic, hyaline-spored, and didymellaceae-like coelomycetous taxa from the SCBG. The main objectives of this study are to analyze the taxonomic placement of our didymellaceous collections and then to describe the taxonomic novelties using morphology and the multi-locus phylogeny of ITS, LSU, RPB2, and β-tubulin sequences. Our study revealed that three of our isolates are new to science. Herein, we introduce these isolates as novel species namely Ectophoma phoenicis sp. nov., Remotididymella fici-microcarpae sp. nov., and Stagonosporopsis pedicularis-striatae sp. nov. A new collection of Allophoma tropica (R. Schneid. and Boerema) Qian Chen and L. Cai is described herein as a new host record. Detailed descriptions, illustrations, and notes are provided.

Sampling Sites, Specimens, and Isolates
Dead plant specimens were collected from the south China Botanical Garden, Guangzhou, Guangdong Province, China (Figure 1), between June to September 2021. Specimens were detached from the host using sterile blades and packed in sterilized paper bags. The collection site is characterized by a tropical climate with abundant sunshine and rainfall throughout the year. The average annual temperature is 22 °C with around 2125 mm of rainfall per year [23]. The collected dead samples (petioles, sepals, and stems) were brought to the laboratory in sterilized paper bags and examined with a stereomicroscope (Carl Zeiss Discovery V8). Conidia were cultured following the method described by Senanayake et al. [24]. The germinated conidia were aseptically transferred into fresh potato dextrose agar (PDA) plates, incubated at 25 °C in the dark to obtain pure cultures, and later transferred to PDA slants and stored at 4 °C for further study. Colony characters were recorded from PDA cultures. Fungarium specimens are deposited at the Herbarium of Zhongkai University of Agriculture and Engineering (ZHKU), and all the ex-type and living cultures are deposited at the Culture Collection of Zhongkai University of Agriculture and Engineering (ZHKUCC). Index Fungorum numbers (https://www.indexfungorum.org, accessed on 1 June 2022) and Facesoffungi numbers [25] were registered for the new species. The collected dead samples (petioles, sepals, and stems) were brought to the laboratory in sterilized paper bags and examined with a stereomicroscope (Carl Zeiss Discovery V8). Conidia were cultured following the method described by Senanayake et al. [24]. The germinated conidia were aseptically transferred into fresh potato dextrose agar (PDA) plates, incubated at 25 • C in the dark to obtain pure cultures, and later transferred to PDA slants and stored at 4 • C for further study. Colony characters were recorded from PDA cultures. Fungarium specimens are deposited at the Herbarium of Zhongkai University of Agriculture and Engineering (ZHKU), and all the ex-type and living cultures are deposited at the Culture Collection of Zhongkai University of Agriculture and Engineering (ZHKUCC). Index Fungorum numbers (https://www.indexfungorum.org, accessed on 1 June 2022) and Facesoffungi numbers [25] were registered for the new species.

Morphological Studies
Microscopic mounts of fruiting structures in sterilized tap water were examined and photographed using a stereomicroscope fitted with a camera (ZEISS Axiocam 208). The micromorphological characteristics such as the structure of the conidiomatal wall, conidiogenous cells, and conidia were studied and photographed using a compound microscope (Nikon Eclipse 80i) fitted with a digital camera (Canon 450D). All microscopic measurements were made with the Tarosoft image framework (v. 0.9.0.7).

DNA Extraction, PCR Amplification, and Sequencing
Fresh mycelia grown on PDA for two weeks at 25 • C in the dark were used for DNA extraction using a fungal genomic DNA extraction kit (Biospin DNA Extraction Kit, Bioer Technology, Co. Ltd., Hangzhou, China) following the manufacturer's protocols. Polymerase chain reactions (PCR) and sequencing were carried out for the complete ITS region, part of the LSU ribosomal DNA, the RNA polymerase II subunit (RPB2) gene, and the β-tubulin gene. The PCR amplification reactions were carried out with the following protocols in Table 1. The total volume of the PCR reaction was 25 µL containing 1 µL of DNA template, 1 µL of each forward and reverse primer, 12.5 µL of 2 × PCR Master Mix, and 9.5 µL of double-distilled sterilized water (ddH 2 O). All the PCR thermal cycles include a final hold at 4 • C. The PCR products were observed on a 1% agarose electrophoresis gel stained with ethidium bromide. Purification and sequencing of PCR products were carried out at Sunbiotech Company, Beijing, China. Sequence quality was checked, and sequences were condensed with DNASTAR Lasergene v. 7.1 [26]. Sequences derived in this study were deposited in GenBank and accession numbers were obtained (Table 2).   sampling for phylogenetic analyses. All the ex-type strains of species were included if available, and other authentic strains were selected when sequences from ex-type strains were unavailable. [27][28][29][30][31][32][33] were followed to obtain sequences from GenBank ( Table 2) [34] with default settings and manually adjusted using BioEdit 7.1.3 [35] when necessary. Maximum likelihood analysis was performed by RAxML [36] implemented in raxml-GUIv. 1.5 [37] using the ML + rapid bootstrap setting and the GTR + I + G model of nucleotide substitution with 1000 replicates. For the Bayesian inference (BI) analyses, the optimal substitution model for the combined datasets was determined to be GTR + I + G using the MrModeltest software v. 2.2 [38]. The BI analyses were computed in MrBayes v. 3.2.6 [39] with four simultaneous Markov chain Monte Carlo chains from random trees over 5 M generations (trees were sampled every 1000th generation). The distribution of log-likelihood scores was observed to check whether sampling was in the stationary phase and Tracer v1.5 was used to check if further runs were required to reach convergence [40]. The consensus tree and posterior probabilities were calculated after discarding the first 20% of the sampled trees as burn-in. The phylogram was visualized in FigTree v. 1.4 [41].

PHI Analyses
The PHI test was performed using SplitsTree4 v. 4.17.1 to determine the recombination level within phylogenetically closely related species. The concatenated four-locus dataset (ITS + LSU + RPB2 + β-tubulin) was used for the analyses. PHI test results (Φw) ≥0.05 indicated no significant recombination within the dataset. The relationships between closely related taxa were visualized in split graphs with both the Log-Det transformation and splits decomposition options.

Phylogenetic Analyses
The alignment comprised 2308 nucleotide characters (488 of ITS, 893 of LSU, 595 of RPB2, 332 of β-tubulin). Maximum likelihood analysis yielded the best ML tree ( After discarding the first 20% of generations in the Bayesian analyses, 4000 trees remained from which the 50% consensus tree and Bayesian Interference (BI) posterior probabilities were calculated ( Figure 2). All individual trees generated under different criteria from single gene datasets were similar in topology and not significantly different from the final trees generated from the concatenated datasets of the Didymellaceae. Topologies of the ML and Bayesian trees were similar to each other and there were no significant differences.
The phylogenetic analyses of this study ( Figure 2) showed that the Didymellaceae comprises a total of 27 well-supported genera. We included 32 sequences from four new collections representing eight isolates in this analysis. Newly generated sequences from two isolates (

Phylogenetic Analyses
The alignment comprised 2308 nucleotide characters (488 of ITS, 893 of LSU, 595 of RPB2, 332 of β-tubulin). Maximum likelihood analysis yielded the best ML tree (   After discarding the first 20% of generations in the Bayesian analyses, 4000 trees remained from which the 50% consensus tree and Bayesian Interference (BI) posterior probabilities were calculated ( Figure 2). All individual trees generated under different criteria from single gene datasets were similar in topology and not significantly different from the final trees generated from the concatenated datasets of the Didymellaceae. Topologies of the ML and Bayesian trees were similar to each other and there were no significant differences.
No species of Allophoma had been reported from Canna sp., and our collection is the first Allophoma species reported from this host genus. Two species, A. pterospermicola Qian Chen and L. Cai and A. thunbergiae Jun Yuan and Yong Wang, have been described from Guangxi and Guizhou Provinces in China [49,53], and A. tropica is the third species reported in the country. Notes: Ectophoma was established to accommodate two phoma-like taxa, Phoma multirostrata (P.N. Mathur, S.K. Menon and Thirum.) Dorenb. and Boerema and P. pereupyrena Gruyter, Noordel. and Boerema which formed a distinct lineage in the Didymellaceae [28]. Currently, there are four accepted species (Species Fungorum. http://www. speciesfungorum.org/Names/Names.asp, accessed on 14 August 2022). This genus is characterized by pycnidial conidiomata with one or more short necks, phialidic conidiogenous cells, and aseptate conidia [28]. Ectophoma species have been isolated from different woody and herbaceous plants as opportunistic plant pathogens or from soil inhabitants [51,54]. Ectophoma species have been reported from Greece, India, Iran, South Africa, and The Netherlands.
Culture characters: Colonies on PDA reaching 4 cm diam. after 7 days of incubation at 25 • C, circular, flat, floccose, filiform, pale brown to greyish brown, darker in the central area, then paler ring and darker ring around the center; reverse brown. Hyphae pale brown, branched, septate. No pigments or chlamydospores observed.
Culture characters: Colonies on PDA reaching 4 cm diam. after 7 days of incubation at 25 °C, circular, flat, floccose, filiform, pale brown to greyish brown, darker in the central area, then paler ring and darker ring around the center; reverse brown.  (Figure 2). A single gene comparison of ITS, RPB2, and β-tubulin locus of our isolates (ZHKUCC 22-0163, ZHKUCC 22-0164) with the type strain of E.iranica (CBS 144681) and E. multirostrata (CBS 274.60) revealed the base pair differences of 7/488, N/A, 3/332 and 6/488, 6/595, 2/332, respectively. The LSU sequences of all Ectophoma species are identical. The genetic distinctness and phylogenetic stability of our isolates were further confirmed by PHI analysis of the Ectophoma clade. The result showed that Φw = 0.99 and this means there was no significant genetic recombination (Φw ≥ 0.05) between these novel isolates with existing Ectophoma species ( Figure 5).  Morphologically, our collection differs from E. iranica by its floccose, grey colonies, apapillate pycnidia, cylindrical to doliiform, hyaline to grey conidiogenous cells, and ellipsoidal, straight conidia. In contrast, E. iranica has pale brown to greyish brown colonies, conidiomata with 1-2(3)-narrowed necks, and hyaline to pale brown, oblong to ellipsoidal, straight or sometimes very slightly curved conidia [29]. Ectophoma iranica is a phytopathogen that forms leaf spots on Dracaena compacta and Catharanthus roseus [29] while our collections are saprobes. Furthermore, our collections differ from E. multirostrata by its bi-guttulated conidia, and conidiomata with single ostiole while E. multirostrata is characterized by eguttulate conidia or sometimes with 2-3, polar guttules and necks with several ostioles. Ectophoma multirostrata is a plurivorous opportunistic plant pathogen isolated from soil and also from some plant samples [51] and our species is a saprobe.
Notes: Remotididymella is characterized by aseptate, hyaline, smooth-and thin-walled, allantoid or cylindrical, guttulate conidia. Currently, there are eight species listed under this genus (https://www.indexfungorum.org, accessed on 1 June 2022) and the sexual morph has been reported only for R. bauhiniae Jayasiri, E.B.G. Jones and K.D. Hyde [48]. Species of Remotididymella have been reported from Ageratina adenophora, Bauhinia sp., Capsicum annuum, Lycopersicon sp., and Solanum sp. as saprobes or pathogens. However, some species have been isolated from air, soil in tropical forests, and human respiratory tract [12,28,32,48]. Remotididymella species have been reported from China, Guadeloupe, Papua New Guinea, Thailand, The Republic of Fiji, and the United States.
Notes: Remotididymella is characterized by aseptate, hyaline, smooth-and thin-walled, allantoid or cylindrical, guttulate conidia. Currently, there are eight species listed under this genus (https://www.indexfungorum.org, accessed on 1 June 2022) and the sexual morph has been reported only for R. bauhiniae Jayasiri, E.B.G. Jones and K.D. Hyde [48]. Species of Remotididymella have been reported from Ageratina adenophora, Bauhinia sp., Capsicum annuum, Lycopersicon sp., and Solanum sp. as saprobes or pathogens. However, some species have been isolated from air, soil in tropical forests, and human respiratory tract [12,28,32,48]. Remotididymella species have been reported from China, Guadeloupe, Papua New Guinea, Thailand, The Republic of Fiji, and the United States.
Culture characters: Colonies on PDA reached 6 cm diam. after 7 days, circular, flat, filiform margin, center pale, margin darker, margin comprised of filiform hyphal tips, aerial mycelia less, brown, reverse pale brown. Cultures not sporulating and no pigments were produced.
can cause devastating diseases on a wide range of economically important plants, including those found in farmlands, forests, grasslands, and other natural ecosystems [5].
Culture characters: Colonies on PDA reached 7 cm diam. after 7 days, circular, flat, smooth, entire margin, aerial mycelia concentrated at the margin, brown, center dark, margin pale; reverse pale brown, center pale, margin darker. Cultures not sporulating and no pigments are produced.

Discussion and Conclusions
In a survey of the diversity and species richness of plant-associated fungi in the South China Botanical Garden, we collected several saprobic, hyaline-spored asexual species in the Didymellaceae. The Didymellaceae has recently undergone extensive revision based on its phylogenetic relationships and morphological characteristics [5,7,60]. In this study, we identified and introduced three new species in Didymellaceae (Ectophoma phoenicis, Remotididymella fici-microcarpae, and Stagonosporopsis pedicularis-striatae) along with a new host and locality record of Allophoma tropica based on polyphasic approaches according to the procedure of [61][62][63]. The guidelines for introducing novel species have been discussed in [64,65] were followed.
We described and illustrated a new locality report of Allophoma tropica. Allophoma tropica was recorded for the first time as a saprobe on leaves of Streptocarpus ionanthus and this study is the first record of Allophoma species on Canna in China. Furthermore, this study reported morphological characters of chlamydospores from cultures of Allophoma for the first time. The type collection of Allophoma tropica obtained from Streptocarpus ionanthus (H. Wendl.) Christenh. as a pathogen and morphological characters were obtained from pycnidia in culture while morphological characters of our collection were obtained from conidiomata on the substrate. Therefore, there are negligible differences in the morphological characteristics of fungi on a substrate and in media. There are four Ectophoma species listed in (Species Fungorum. http://www.speciesfungorum.org/Names/Names. asp, accessed on 14 August 2022). The type species of Ectophoma, E. multirostrata has been isolated from poultry farm soil and the live stem of Cucumis sativus in a greenhouse [28]. Ectophoma iranica and E. pomi are plant pathogens isolated from leaf spots of Dracaena compacta, Catharanthus roseus [29], and Coffea arabica [28]. Ectophoma insulana was isolated from the fruit of Olea europaea and from house dust [12]. Morphologically, our collections differ from E. iranica, and E. multirostrata and also our collection of Ectophoma phoenicis isolated from the dead petioles of Phoenix roebelenii are saprobic fungi. As such, this is the first record of saprobic behavior of Ectophoma species. Our collection of Ectophoma phoenicis was identified and introduced as a new species belonging to the genus Ectophoma.
Remotididymella fici-microcarpae is the first species in this genus reported from China and also from Ficus microcarpa. This was collected from dead stems as saprobes. However, Remotididymella species show a wide variation of life modes including plant and human pathogens and saprobes [12,32,48]. Morphologically, our collections differ from R. ageratinae by having immersed, brown, small conidiomata, large conidiogenous cells, and fusiform to allantoid conidia with indistinct guttules. Our collection of Remotididymella fici-microcarpae was identified and introduced as a new species belonging to the genus Remotididymella.
There are several Stagonosporopsis species that have been reported from China, and most of them are plant pathogens [31]. Stagonosporopsis vannaccii Baroncelli, Cafà, Castro, Boufleur and Massola was reported from leaf spots on Crassocephalum crepidioides in Guangxi [66] while Stagonosporopsis cucurbitacearum (Fr.) Aveskamp, Gruyter and Verkley are the cause of pumpkin gummy stem blight, which is one of the most devastating pumpkin crop diseases in North-East China [67]. Stagonosporopsis pogostemonis M. Luo, Y.H. Huang and Manawas. was reported from leaf spots and as a stem blight on Pogostemon cablin in South China [31]. However, Stagonosporopsis pedicularis-striatae was collected from dead stems as a saprobe. Morphologically, our collections differ from Stagonosporopsis astragali by their subglobose, slightly papillate, comparatively larger conidiomata (150-200 × 300-400 µm), and oblong to ellipsoid conidia. In terms of phylogenetic and morphological differences, we identified and introduced Stagonosporopsis pedicularisstriatae as a new species belonging to the genus Stagonosporopsis.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All sequence data are available in NCBI GenBank following the accession numbers in the manuscript.