Four new species of Phyllosticta from China based on morphological and phylogenetic characterization

ABSTRACT Phyllosticta (Phyllostictaceae, Botryosphaeriales) species are widely distributed globally and constitute a diverse group of pathogenic and endophytic fungi associated with a broad range of plant hosts. In this study, four new species of Phyllosticta, i.e. P. endophytica, P. jiangxiensis, P. machili, and P. xinyuensis, were described using morphological characteristics and multi-locus phylogeny based on the internal transcribed spacer region (ITS) with intervening 5.8S rRNA gene, large subunit of rRNA gene (nrLSU), translation elongation factor 1-alpha gene (tef1), actin gene (act), and glyceraldehyde-3-phosphate dehydrogenase gene (gapdh). Phyllosticta machili is the first species of this genus reported to infect plants of the Machilus genus.


Introduction
The genus Phyllosticta (Phyllostictaceae, Botryosphaeriales, and Ascomycota) was established by Persoon (Persoon 1818). Presently, a total of 3,215 names are documented for Phyllosticta in the Index Fungorum (accessed on 29 March 2023). However, many of these names have been synonymised. Currently, 1,499 species are recognised in this genus (Bánki et al. 2022). Most members of Phyllosticta are important plant pathogens and endophytes with a wide host range and geographical distribution (Van der Aa and Vanev 2002). Some Phyllosticta species cause significant impacts on economic crops, such as citrus black spot caused by P. citricarpa (McAlpine) Aa, which is regarded as a quarantine pathogen in Europe and the USA (Baayen et al. 2002;Wulandari et al. 2009;Glienke et al. 2011), black rot disease on grapevines by P. ampelicida (Engelm.) Aa species complex (Wicht et al. 2012), and banana freckle disease by P. musarum (Cooke) Aa species complex (Pu et al. 2008;Wong et al. 2012). In addition, some species of Phyllosticta have also been isolated from a wide range of hosts as endophytic fungi. For example, P. capitalensis Henn. is an endophytic fungus distributed in more than 20 different families of plants in eight countries (Baayen et al. 2002;Wulandari et al. 2010;Glienke et al. 2011). However, endophytes can sometimes be transformed into pathogens, such as P. capitalensis, which can cause black freckle disease on Rubus chingii HU and leaf spots on Ligustrum japonicum Thunb. as a pathogen, thus affecting the visual value of ornamental plants (Sabahi et al. 2022;Zhang et al. 2022). Some species of Phyllosticta can survive by degrading organic material from dead plants as saprobes, such as P. carpogena (Shear) Aa and P. ericae Allesch (Trigiano et al. 2004) occurring on Rubus sp. (Rosaceae) and Erica carnea (Ericaceae), respectively (Van der Aa and Vanev 2002).
In China, some species of Phyllosticta can infect economic plants and cause economic losses. For example, P. theicola Curzi and P. capitalensis can both cause leaf spots on Camellia sinensis (L.) O. Ktze. (Cheng et al. 2019;Tian et al. 2019). Phyllosticta capitalensis can cause leaf spots on oil palms (Nasehi et al. 2020), and P. citricarpa can cause black spot disease on citrus leading to black spots on fruit and premature fruit drop (Wang et al. 2012;Tran et al. 2020).
The aim of this study was to identify Phyllosticta strains isolated from different host plants in China by morphological characterisation and multi-gene phylogenetic analysis.

Isolation and culture
Diseased leaf tissues and healthy tissues from different hosts were collected from China's Anhui, Hubei, Yunnan, and Jiangxi provinces. Fragments (5 mm × 5 mm) were taken from the margin of leaf lesions and healthy parts of leaves. All fragments were disinfected in 75% ethanol for 30 s, followed by 10% sodium hypochlorite for 3 min, washed in sterile water three times, then placed on potato dextrose agar (PDA), and cultured at room temperature (25 ± 3) °C. Cultures were dried after sporulation and sent as holotype to Chinese Academy of Forestry (CAF; http:// museum.caf.ac.cn), and ex-type living cultures of new species were deposited in the China Forestry Culture Collection Center (CFCC; http://cfcc.caf.ac. cn/) and Capital Normal University Culture Collection Center (CNUCC).

Morphological analysis
Isolates were cultured for 12-15 days and examined periodically until sporulation. The pycnidia and conidia on cultures were examined with a dissecting microscope (Nikon SMZ-1000) and an upright microscope (Olympus BX51). Mature pycnidia were immersed and crushed in water, releasing both conidia and conidiogenous cells on glass slides. Twenty of each structure, including pycnidia, conidiogenous cells, and conidia, were measured to calculate the average size as described. Colony colours were described based on the ColorHexa code (https:// www.colorhexa.com/).
Purification and sequencing were performed by Zhongkexilin Biotechnology Company (Beijing, China). DNA sequences using forward and reverse primers were aligned using Editseq v5.0 to obtain consensus sequences. The new sequences in this study were submitted to the GenBank. Other sequences of the ITS, nrLSU, tef1, act, and gapdh genes were downloaded from GenBank (Table 2). The segmentation homogeneity test was performed to determine the consistency of gene segments (Farris et al. 1994;Huelsenbeck et al. 2003). Aligned using the online MAFFT tool (https:// www.ebi.ac.uk/Tools/msa/mafft/) and edited using Gblocks (https://www.phylogeny.fr/one_task.cgi?task_ type = gblocks) by selecting DNA and all options for less stringent criteria.Strain names and sequences of new species in this study are indicated in bold. Isolates marked with "*" are ex-type or ex-epitype strains.
The phylogenetic analyses were based on a combined ITS, nrLSU, act, tef1, and gapdh matrix, including the type and representative species of Phyllosticta (Norphanphoun et al. 2020). A maximum parsimony (MP) tree was constructed with PAUP v4.0b10 (Swofford 2003). Trees were inferred using the heuristic search option with TBR branch swapping and 1,000 random sequence additions. Branches of zero length were collapsed and all equally most parsimonious trees were saved. Tree length (TL), consistency index (CI), retention index (RI), and rescaled consistency index (RC) were calculated for the generated parsimony trees. For the Bayesian inference (BI) analysis, MrModeltest v2.3 with the Akaike information criterion was used as a substitution model for the concatenated dataset (Nylander 2004). The GTR+I+G model was proposed for ITS, nrLSU, and gapdh, HKY+I +G for act, and GTR+G for tef1. 1,000,000 generations were launched with a random starting tree, and Markov chains were sampled every 100 generations. The analysis was stopped when the average standard deviation of the separation frequency was 0.01. During the burn-in phase of the analysis, the first 25% of trees were discarded, and the remaining trees were used to calculate the posterior probabilities (PPs).

Phylogenetic analysis
Single-gene phylogenetic trees were constructed before the multi-gene phylogenetic analysis to examine topology and clade support based on the ITS, nrLSU, act, tef1, and gapdh genes ( Supplementary  Figures 1, 2, 3, 4, and 5). The topology of the singlegene phylogenetic tree for ITS, act, and tef1 was similar to the multi-gene phylogenetic tree, but the support values of the deep nodes were lower. The topology of the single-gene phylogenetic tree of nrLSU and gapdh differed slightly from the multigene phylogenetic tree.
In the multi-locus phylogenetic analysis, 311 sequences of 81 isolates were used to construct a fivelocus phylogenetic tree; Botryosphaeria obtusa (Schwein.) Shoemaker (CMW 8232) was used as the outgroup. The dataset of five loci comprised 2,470 characters, including the alignment gaps, of which 623 characters were parsimony-informative, 305 parsimony-uninformative, and 1,542 constants. The analytical analysis yielded 1,000 similar trees, one of which (TL = 3,034, CI = 0.467, RI = 0.727, RC = 0.340, and HI = 0.533) is shown in Figure 1. The Bayesian tree confirmed the topology of the obtained tree using the maximum parsing method.
Culture characteristics: Colonies up to 4.3 cm in diameter at 7 days on PDA. Round with ciliate at the edge, dark lime green (#1f2820) to dark yellow (#8e7f00) on the obverse, greyish-white (#ececec) on the outer edge, and dark lime green (#171c1a) to light grey (#ececec) on the reverse side. Conidiomata are visible after 14 days with pale yellow (mostly white) (#ffffec) oozing on PDA.
Culture characteristics: Colonies up to 2.2 cm in diameter at 7 days on PDA. The entire edge is irregular, dark greyish tallow colour (#4a4a36) with aerial hyphae to greyish-white (#e0e0e0) on the obverse, dark greyish lime green (#2a392a) on the outer edge, and dark greyish lime green (#2a392a) to greyish-white (#e0e0e0) on the reverse side. Conidiomata are light grey (#c9c9c9) and visible after 14 days.
Culture characteristics: Colonies up to 6.9 cm in diameter at 7 days on PDA. Round with a scalloped margin, dark greyish-green centre (#5a6053) to greyishgreen edges (#808976) on the obverse and reverse sides. Formation of olive green to green lamellae as black masses. Conidiomata are visible after 12 days with light grey (mostly white) (#f8f8f8) tendrils on PDA.

Discussion
The initial identification of Phyllosticta species was mainly based on morphology, culture characteristics, and host associations. Van der Aa and Vanev (2002) revised more than 2,000 species in Phyllosticta and suggested more accurate and reliable morphological characteristics for its classification. However, the Phyllosticta species share many morphological similarities, making it difficult to classify them accurately using morphology alone. Hyde et al. (2010) proposed a connection between molecular data and the correct taxonomic unit using morphological characters in combination with molecular data to accurately identify fungi. Wikee et al. (2012) placed Phyllosticta within Phyllostictaceae using molecular phylogenetic analysis based on the ITS, nrLSU, act, tef1, and gapdh genes, combined with morphological characters. In this study, four new species were described based on morphology and phylogenetic analysis. Single-gene phylogenetic trees for each of the five genes were also constructed separately. However, the multi-locus phylogenetic tree provided better resolution for the Phyllosticta species. Including proteincoding genes in the analysis greatly facilitated species-level identification, also demonstrated by Wikee et al. (2011). Multi-locus phylogeny and three signal locus (ITS, tef1, and act) supported the four new species. The difference between the topology of the single-gene phylogenetic trees of nrLSU and gapdh with that of the multi-locus phylogenetic tree may be due to fewer available sequences (only 32 linked data for gapdh in 71 recognised species, and 34 linked data for gapdh in 71 recognised species). Among the phylogenetic trees for a single locus, the gapdh gene tree showed the greatest difference in topological structure compared with the multi-gene phylogenetic tree. This difference may be due to the few available genes for gapdh (only 37 linked data for gapdh in 70 recognised species).
Phyllosticta species can cause lesions on many plant species with a wide host range. The 10 strains in this study were collected from three host species, including Camellia oleifera, Machilus pauhoi, and Cunninghamia lanceolata. Among these strains, P. machili is the first reported isolation from M. pauhoi; whether the species is host-specific needs to be determined by pathogenicity testing. Until now, six species of Phyllosticta isolated from C. oleifera have been reported, i.e. P. camelliae, P. camelliaecola, P. capitalensis, P. erratica, P. theae, and P. theacearum. Among these species, P. capitalensis has a wide host range, with nearly 357 species, whereas the other species have been isolated only from C. oleifera (Norphanphoun et al. 2020). Phyllosticta cryptomeriae, P. concentrica, and P. cunninghamii have previously been isolated from Cu. lanceolata. In addition, Phyllosticta cunninghamii has also been isolated from Rhododendron cinnamomeum var. cunninghamii Paxton (Norphanphoun et al. 2020). P. cryptomeriae can be parasitic on Cryptomeria japonica (L.f.) D. Don. and P. concentrica has a wide range of hosts, with about 40 host species. Therefore, species of Phyllosticta may not have host specificity for C. oleifera and Cu. lanceolata. In addition, host specialisation in Phyllosticta may be related to lifestyle, and the endophytic fungus is less host-specific than the pathogenic fungus (Wikee et al. 2011); thus, P. capitalensis has a wider host range, which may be related to its endophytic lifestyle. A more indepth study of whether there is host specificity and how it relates to fungal lifestyle would require a comparative analysis of genomic data, such as comparing the number and type of carbohydrateactive enzymes.
According to Okane et al. (2003), Phyllosticta species, including pathogens, latent pathogens, and endophytes, are widely distributed and have a variety of lifestyles. Phyllosticta endophytica was isolated as an endophytic fungus from Cu. lanceolata in this study. Endophytes can transform into pathogenic organisms under certain circumstances (Rodriguez and Redman 2008;Rodriguez et al. 2009). Therefore, whether these species have specific hosts, their habitats, lifestyles, and the conditions under which they may transform need further investigation. This has important implications for the potential biological control of plant diseases.