Cheng-Bin Wang,Jing Yang,Yong Li,Han Xue,Chun-Gen Piao,Ning Jiang
﻿Multi-gene phylogeny and morphology of two new Phyllosticta ( Phyllostictaceae , Botryosphaeriales ) species from China
Ascomycota,morphology,new species,phylogeny,plant disease,taxonomy,Fungi,Botryosphaeriales,Phyllostictaceae,Fungi,Botryosphaeriales,Phyllostictaceae
﻿Abstract Phyllosticta ( Phyllostictaceae , Botryosphaeriales ) includes plant pathogens, endophytes and saprobes, occurring on various hosts worldwide. During the present study, isolates associated with leaf spots were obtained from the hosts Quercus aliena and Viburnum odoratissimum , and identified based on morphological features and phylogenetic inference from the analyses of five loci (ITS, LSU, tef1, act and gapdh). Results supported the introduction of two novel species, namely Phyllosticta anhuiensis and P. guangdongensis . Phylogenetically, P. anhuiensis and P. guangdongensis formed two well-separated lineages in the P. concentrica and P. capitalensis species complexes, distinguishing from all presently accepted species in this genus by DNA sequence data. Morphologically, P. anhuiensis and P. guangdongensis have the typical structure of the genus Phyllosticta , and differed from their closely related species by the length of the conidial appendage.
﻿Multi-gene phylogeny and morphology of two new Phyllosticta ( Phyllostictaceae , Botryosphaeriales ) species from China

Phyllosticta (Phyllostictaceae, Botryosphaeriales) includes plant pathogens, endophytes and saprobes, occurring on various hosts worldwide. During the present study, isolates associated with leaf spots were obtained from the hosts Quercusaliena and Viburnumodoratissimum, and identified based on morphological features and phylogenetic inference from the analyses of five loci (ITS, LSU, tef1, act and gapdh). Results supported the introduction of two novel species, namely Phyllostictaanhuiensis and P.guangdongensis. Phylogenetically, P.anhuiensis and P.guangdongensis formed two well-separated lineages in the P.concentrica and P.capitalensis species complexes, distinguishing from all presently accepted species in this genus by DNA sequence data. Morphologically, P.anhuiensis and P.guangdongensis have the typical structure of the genus Phyllosticta, and differed from their closely related species by the length of the conidial appendage.

The genus Phyllosticta was established by Persoon (1818) and classified in Phyllostictaceae (Botryosphaeriales) (Phillips et al. 2019; Wijayawardene et al. 2020). Initially, Phyllosticta was placed in the Phyllostictaceae (Fries 1849). In a multi-locus phylogeny in the Dothideomycetes, Schoch et al. (2006) placed Phyllosticta into Botryosphaeriaceae (Botryosphaeriales), which was agreed upon by Crous et al. (2006) and Liu et al. (2012). Subsequently, Slippers et al. (2013) reinstated the Phyllostictaceae to accommodate Phyllosticta in terms of phylogenetic relationships. Recently, Pseudofusicoccum was added in this family based on the morphological characters of the conidia covered by a mucous sheath and molecular evidence (Phillips et al. 2019). The asexual morph of Phyllosticta is characterized by pycnidial conidiomata containing aseptate conidia surrounding with a mucoid layer and bearing a single apical appendage (van der Aa 1973; van der Aa and Vanev 2002; Wikee et al. 2011). The sexual morph of Phyllosticta is characterized by erumpent ascomata, 8-spored, clavate to broadly ellipsoid asci, ellipsoid to limoniform ascospores (van der Aa 1973; Wikee et al. 2011). Following the implementation of “one fungus one name” nomenclature rules, the name Phyllosticta (asexual state) was used over Guignardia (sexual state) and Leptodothiorella (spermatial state) (Glienke et al. 2011; Wikee et al. 2011). The Phyllosticta species identification solely delimited by morphology and host association may be difficult to assess (Wikee et al. 2011; Su and Cai 2012). Many species are difficult to distinguish due to slight morphological variation, and the mucoid layer or appendage will be absent or invisible in some species (van der Aa and Vanev 2002; Jin 2011; Wikee et al. 2011). Besides, the host range of Phyllosticta is unclear; some species exhibit the broadest host range while others do not (Wikee et al. 2011; Rashmi et al. 2019; Norphanphoun et al. 2020). To overcome the lack of morphological features and host range, phylogenetic approaches based on molecular loci were used to resolve the classification and identification of Phyllosticta species (Baayen et al. 2002; Wulandari et al. 2009; Wong et al. 2012; Wikee et al. 2013a). Based on the phylogenetic analyses of a combined ITS, LSU, tef1, act and gapdh sequence data, the current taxonomic classification of Phyllosticta comprises six species complexes i.e., P.capitalensis, P.concentrica, P.cruenta, P.owaniana, P.rhodorae and P.vaccinii species complexes (Norphanphoun et al. 2020). Currently, the polyphasic approach involving phylogenetic, morphological, and other analyses is used to clarify species boundaries (Norphanphoun et al. 2020; Zhang et al. 2022). Members of Phyllosticta species are known as pathogenic, endophytic, or rarely saprobic fungi associated with a variety of plants and have a worldwide distribution (van der Aa and Vanev 2002; Glienke et al. 2011; Wikee et al. 2011; Jiang et al. 2021; Wang et al. 2023). As pathogens, Phyllosticta species cause spots on the leaves or fruits of many economical plants (e.g., Musa spp., Citrus spp. and Vitis spp.), leading to substantial economic losses (Wang et al. 2012; Wong et al. 2012; Wikee et al. 2013b; Tran et al. 2017). As endophytes, some species were found associated with leaf spots but did not cause any symptom in pathogenicity tests, e.g., P.oblongifoliae was isolated from leaf spots of Garciniaoblongifolia, P.pterospermi was isolated from leaf spots of Pterospermumheterophyllum, and P.capitalensis was isolated from leaf spots of Citrus spp. (Wikee et al. 2013b; Tran et al. 2019; Zhang et al. 2022). In this study, two novel fungal species named P.anhuiensis and P.guangdongensis, were isolated from diseased leaves of Quercusaliena in Anhui Province and Viburnumodoratissimum in Guangdong Province, respectively. This paper describes these species based on molecular evidence and morphological characteristics.

Samples of Quercusaliena and Viburnumodoratissimum showing necrotic spots were obtained and collected from Anhui and Guangdong Provinces. Samples were surface-sterilized in 75% ethanol for 30 s, then sterilized in 1.5% sodium hypochlorite for 1 min, followed by three rinses with sterilized water and dried on sterilized filter paper, and cut into small sections (3 × 3 mm) from the margins of infected tissues. The sections were plated onto potato dextrose agar (PDA) plates and incubated at 25 °C. Hyphal tips from the edge of emerging colonies were transferred on fresh PDA plates and purified by single-spore culturing (Choi et al. 1999). The cultures and dried specimens of the new isolates have been deposited with the China Forestry Culture Collection Center (CFCC; http://cfcc.caf.ac.cn/) and the herbarium of the Chinese Academy of Forestry (CAF; http://museum.caf.ac.cn/). Colony features of cultures on PDA medium, synthetic low-nutrient agar (SNA), and malt extract agar (MEA) were recorded after 14 d incubation at 25 °C. After conidiomata appeared, fungal structures (including conidia, conidiogenous cells, and appendage) were measured and captured at least 50 measurements using a Nikon Eclipse 80i compound microscope with differential interference contrast optics.

Genomic DNA was extracted from fungal cultures grown on PDA medium using a CTAB method (Doyle and Doyle 1990). Polymerase chain reaction (PCR) amplification of the ITS, LSU, tef1, act, and gapdh loci were amplified using the primers: ITS1/ITS4 (White et al. 1990), EF1-728F/EF2 (O’Donnell et al. 1998; Carbone and Kohn 1999), ACT-512F/ACT-783R (Carbone and Kohn 1999) and Gpd1-LM/Gpd2-LM (Myllys et al. 2002), respectively. Amplification reactions were performed in a 20 μl reaction volume system containing 10 µl of 2× Taq Mix (Tiangen, China), 1 μl of each primer (10 μM), 1 μl template DNA (20 ng/μl) and 7 μL RNase-free water. PCR parameters were 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 55 °C for ITS, 51 °C for LSU, 48 °C for tef1 or 52 °C for act and gapdh, and 1 min at 72 °C, and a final elongation step of 10 min at 72 °C. The PCR products were purified and sequenced in Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

Newly generated in this study were combined using SeqMan v. 7.1.0, and reference sequences (Table 1) were downloaded from GenBank, according to the recent publication (Hattori et al. 2020; Norphanphoun et al. 2020; Crous et al. 2021; Bhunjun et al. 2022; Nguyen et al. 2022; Tan and Shivas 2022; Zhang et al. 2022). Alignments were done by MAFFT v. 7.036 (https://maft.cbrc.jp/alignment/server/) using default settings and manually improved using MEGA v.7.0 (Kumar et al. 2016). The phylogenetic analyses of the combined five loci (ITS, LSU, tef1, act and gapdh) were performed by maximum likelihood (ML) and Bayesian inference (BI). The ML research was conducted with the CIPRES web portal (Miller et al. 2017) using RAxML v. 8.2.12 (Stamatakis 2014) under the GTR+GAMMA model with 1000 bootstrap iterations. The BI analyses was performed by MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003). MrModelTest v. 2.3 (Nylander 2004) was used to determine the best-fit evolution model for each locus. Bayesian posterior probabilities (BYPP) were evaluated by Markov Chain Monte Carlo sampling (MCMC). Four Markov chains were performed for 2 million generations in two independent runs until the split deviation frequencies decreased below 0.01, and sampling every 100 generations. The first 25% of sampled trees were discarded as burn-in, and the remaining ones were used to calculate BYPP. Trees were visualized in FigTree 1.4 (http://tree.bio.ed.ac.uk/software/figtree), and the ML bootstraps (ML-BS) ≥ 50% and BYPP ≥ 0.9 were presented on nodes of the ML tree.

In this study, phylogenetic analyses contained sequences from 131 fungal samples representing 93 taxa, including two outgroup taxa, viz., Botryosphaeriaobtusa (CMW 8232) and B.stevensii (CBS 112553). The multi-locus datasets comprised 2460 characters including gaps, 521 for ITS, 764 for LSU, 297 for tef1, 248 for act and 630 for gapdh, with 1499/2460 conserved sites, 187/2460 variable sites, and 774/2460 parsimony informative. The best scoring RAxML tree with a final likelihood value of -22751.44. Estimated base frequencies were: A = 0.206387, C = 0.294301, G = 0.279093, T = 0.220219; substitution rates AC = 1.049607, AG = 3.135926, AT = 1.344881, CG = 1.068545, CT = 6.294467, GT = 1.00000; gamma distribution shape parameter α = 0.690585. In the phylogenetic tree (Fig. 1), Phyllosticta was divided into six distinct lineages as six species complexes, and our isolates formed two separate lineages represented two new species viz., P.anhuiensis (CFCC 54840, CFCC 55887 and CFCC 58849) and P.guangdongensis (CFCC 58144, CFCC 58766 and CFCC 58772).

Taxon classification Fungi Botryosphaeriales Phyllostictaceae Ning Jiang & C.B. Wang sp. nov. 81C08238-FAAB-5DCF-83D9-64BD9462A468 847160 Fig. 2

Referring to the Anhui Province, where the species was first collected.

Sexual morph: Unknown. Asexual morph: Conidiomata pycnidial, aggregated, black, erumpent, globose to pyriform, exuding gray to pale yellow conidial masses, 100–400 µm diam. Conidiophores subcylindrical to ampulliform, reduced to conidiogenous cells. Conidiogenous cells phialidic, hyaline, thin-walled, smooth, subcylindrical to ampulliform, 10–16 × 2.5–4.5 μm. Conidia 8.5–12 × 5.5–9 μm, (mean ± SD = 10 ± 1 × 7.2 ± 0.7 μm), solitary, hyaline, aseptate, thin and smooth-walled, coarsely guttulate, globose or ellipsoid to obvoid, enclosed in a thin persistent sheath, 1–1.5 μm thick, and bearing an apical mucoid appendage 4–6 × 1–2 μm, flexible, unbranched, tapering towards an acutely rounded tip.

Colonies on PDA flat, with irregular edge, slow growing, grayish-green to green, reaching a 90 mm diameter after two weeks. Colonies on MEA flat, undulate at the edge, slow growing, gray-white to gray, reaching a 70–80 mm diameter after two weeks. Colonies on SNA flat, slow growing, celandine green, reaching a 60–70 mm diameter after two weeks.

China, Anhui Province, Hefei City, leaf spots of Quercusaliena, Yong Li & Dan-ran Bian, 10 August 2019 (holotype CAF800072; ex-type culture: CFCC 54840). Ibid. (cultures: CFCC 55887 and CFCC 58849).

In the phylogeny analyses, P.anhuiensis groups sister to P.kerriae (MAFF 240047). P.kerriae was associated with Kerriajaponica in Japan (Motohashi et al. 2008). Comparison of DNA sequences of P.anhuiensis with P.kerriae (MAFF 240047), there is 99.4% (447/480 identities; 0/480 gaps) sequence similarity in ITS, 99.8% (554/555 identities, 0/480 gaps) in LSU, 98.6% (215/218 identities, 0/218 gaps) in tef1, and 97.7% (212/217 identities, 0/217 gaps) in act. Morphologically, P.anhuiensis can be distinguished from P.kerriae in having shorter appendage (4–6 µm in P.anhuiensis vs. 5–12.5 µm in P.kerriae) (Motohashi et al. 2008). Therefore, this species was regarded as a new species based on morphology and multi-locus phylogeny.

Taxon classification Fungi Botryosphaeriales Phyllostictaceae Ning Jiang & C.B. Wang sp. nov. A5308D23-E690-5AFA-B6FD-EE50F3BABB27 847161 Fig. 3

Referring to the Guangdong Province, where the species was first collected.

Sexual morph: Unknown. Asexual morph: Conidiomata pycnidial, aggregated, black, globose to pyriform, exuding opaque conidial masses, erumpent, 100–450 µm diam. Conidiophores subcylindrical to ampulliform, reduced to conidiogenous cells. Conidiogenous cells phialidic, subcylindrical to ampulliform, hyaline, smooth, 10–15 × 2.5–4 μm. Conidia 10–14 × 6–8 μm, (mean ± SD = 11.5 ± 1.3 × 7.5 ± 0.6 μm), solitary, hyaline, aseptate, thin and smooth-walled, ellipsoid to obovoid, coarsely guttulate, enclosed in a thin persistent mud sheath, 1–1.5 μm thick, with an apical mucoid appendage, 4.5–10 × 1–2 μm, flexible, unbranched, tapering towards an acutely rounded tip.

Colonies on PDA flat, slow growing, grayish-green in the center, and dark green at margin reaching 85 mm diameter after two weeks. Colonies on MEA slow growing, yellow in the center, white at undulate the margin, reaching a 20–25 mm diameter after two weeks. Colonies on SNA flat, slow growing, grayish-green, reaching a 25–30 mm diameter after two weeks.

China, Guangdong Province, Guangzhou City, leaf spot of Viburnumodoratissimum, Yong Li, 20 September 2022 (holotype CAF800073; ex-type culture: CFCC 58144). Ibid. (cultures: CFCC 58766 and CFCC 58772).

Phylogeny indicates that P.anhuiensis groups sister to P.mangiferae (IMA 260576). P.mangiferae was associated with Mangiferaindica leaves in Tanzania (Ebbels and Allen 1979; Glienke et al. 2011). Comparison of DNA sequences of P.anhuiensis with P.mangiferae (IMA 260576), there are 99.1% (471/475 identifies, 0/475 gaps) sequence similarity in ITS, 99.6% (760/763 identifies, 0/763 gaps) in LSU, 97.7% (211/216 identifies, 2/218 gaps) in tef1, 98.2% (221/225 identifies, 0/225 gaps) in act, and 98.4% (614/624 identifies, 6/624 gaps) in gapdh. Morphologically, P.guangdongensis can be distinguished from P.mangiferae in longer conidia (10–14 μm in P.guangdongensis vs. 8–12 µm in P.mangiferae) and shorter appendage (4.5–10 µm in P.guangdongensis vs. 7–13 µm in P.mangiferae) (Glienke et al. 2011). Therefore, this species was regarded as a new species based on morphology and multi-locus phylogenetic analyses.

Phyllosticta is a species-rich genus with more than 3211 records listed in the Index Fungorum (http://www.indexfungorum.org). For the Phyllosticta species identification, molecular data have proven useful in resolving species relationships (Okane et al. 2003; Su and Cai 2012; Guarnaccia et al. 2017; Norphanphoun et al. 2020; Zhang et al. 2022). ITS is a genetic marker for genus level, and combining it with additional loci (LSU, tef1, act and gapdh) is enough for species-level resolution (Jayawardena et al. 2019; Norphanphoun et al. 2020). In this study, based on the phylogenetic analyses of presently accepted species using five loci (ITS, LSU, tef1, act and gapdh), there are six species complexes and 93 species accepted in Phyllosticta (Table 1), viz., P.capitalensis species complex (including 33 species), P.concentrica species complex (including 28 species), P.cruenta species complex (including 22 species), P.owaniana species complex (including six species), P.rhodorae species complex (including two species), and P.vaccinii species complex species complex (including two species). P.anhuiensis and P.guangdongensis formed two well separated clades in the P.concentrica and P.capitalensis species complexes, distinguishing from all accepted species in this genus by DNA sequences data. Morphologically, our isolates have the typical structure of Phyllosticta (van der Aa and Vanev 2002). The asexual morph of species in the P.concentrica species complex is characterized by globose or ellipsoid to obvoid conidia enclosed in a thin persistent sheath with an apical mucoid appendage (Norphanphoun et al. 2020). The asexual morph of species in the P.capitalensis species complex are characterized by ellipsoid or ellipsoid to obovoid, ovoid, obpyriform conidia with a mucoid sheath with an apical mucoid appendage (Norphanphoun et al. 2020). Our isolates include the essential characteristics of their species complexes, and differ from their closest relatives by the size ranges of conidia and appendage (Motohashi et al. 2008; Glienke et al. 2011). Phyllostictaanhuiensis was isolated from Q.aliena in Anhui Province, and P.guangdongensis was isolated from V.odoratissimum in Guangdong Province. Among Phyllosticta species recorded from Quercus and Viburnum with sequence date and morphological features, P.capitalensis was isolated from Q.dentata and Q.variabilis in Japan; P.concentrica was isolated from Q.robur in Poland and Q.ilex in Ukraine; and P.hubeiensis was isolated from V.odoratissimum in China (Okane et al. 2003; Mulenko et al. 2008; Zhang et al. 2013; Farr and Rossman 2022). P.capitalensis and P.concentrica are common species reported from various plants, and P.hubeiensis was only recorded from V.odoratissimum (Wikee et al. 2013a, b; Zhang et al. 2013; Farr and Rossman 2022). Our isolates formed individual lineages as shown in Fig. 1, segregated from those three species. Morphologically, P.anhuiensis differs from P.capitalensis and P.concentrica by having longer conidiogenous cells (10–16 × 2.5–4.5 μm in P.anhuiensis vs. 7–10 × 3–5 in P.capitalensis vs. 7–10 × 3–6 μm in P.concentrica), shorter conidia (8.5–12 × 5.5–9 μm in P.anhuiensis vs. 10–14 × 5–7 μm in P.capitalensis vs. 10–14 × 6–9 μm in P.concentrica) and shorter appendage (4–6 × 1–2 μm in P.anhuiensis vs. 5–15 × 1–1.5 μm in P.concentrica) (Glienke et al. 2011; Wikee et al. 2013a); P.guangdongensis can be distinguished from P.hubeiensis in having shorter appendage (4.5–10 µm in P.guangdongensis vs. 7–12 µm in P.hubeiensis) (Zhang et al. 2013). In this study, we introduced two novel species from forestry trees. Previously, many Phyllosticta species were found in economic hosts, and with the investigation and study of Phyllosticta, many Phyllosticta will be found on forestry trees and this will improve our understanding of the species diversity.