Morphological and Phylogenetic Analyses Reveal Three New Species of Phyllosticta (Botryosphaeriales, Phyllostictaceae) in China

The genus Phyllosticta has been reported worldwide and contains many pathogenic and endophytic species isolated from a wide range of plant hosts. A multipoint phylogeny based on gene coding combinatorial data sets for the internal transcribed spacer (ITS), large subunit of ribosomal RNA (LSU rDNA), translation elongation factor 1α (TEF1α), actin (ACT), and glycerol-3-phosphate dehydrogenase (GPDH), combined with morphological characteristics, was performed. We describe three new species, P. fujianensis sp. nov., P. saprophytica sp. nov., and P. turpiniae sp. nov., and annotate and discusse their similarities and differences in morphological relationships and phylogenetic phases with closely related species.


Introduction
Phyllosticta Pers.was initially established by Persoon in 1818 [1].At the outset, Phyllosticta was categorized within Botryosphaeriaceae [2,3].Later on, it was acknowledged as the anamorph of Guignardia, following the recommendations of Viala and Ravaz [4].The precedence of the earlier name is determined in accordance with the International Code for Nomenclature of Algae, Fungi, and Plants [5].Slippers et al. [6] placed Phyllosticta within Phyllostictaceae, Botryosphaeriales, relying on phylogenetic relationships.More recently, it has been reclassified in Botryosphaeriaceae based on compelling evidence from morphological characteristics and molecular data, particularly concerning conidia covered by mucus.The current classification of Phyllosticta poses challenges and warrants reevaluation.Phyllosticta is recognized as an endophytic fungi with the capability to induce leaf spot in plants, exhibiting a widespread distribution across the globe.Wang et al., Zhang et al., and van der Aa [7][8][9] have described 46 types of Phyllosticta, encompassing 12 sexual forms and 17 spermatial morphs.Van der Aa and Vanev have further modified all Phyllosticta species, accepting a total of 190 epithets [10].The intricate nature of the Phyllosticta classification system underscores the need for a thorough reassessment.
Phyllosticta represents a highly diverse group, with 3216 scientific names recorded under the genus according to the Index Fungorum search (www.indexfungorum.org,accessed on 25 October 2023) [8].Identifying Phyllosticta poses challenges based solely on morphological characteristics and host combinations, with the host range being broad and unclear.Some Phyllosticta species exhibit a wide range of hosts, while others do not.To overcome the limitations associated with morphological characteristics and host combinations, the integration of homologous gene DNA sequencing and comparative methods has significantly advanced our comprehension of the phylogeny of Phyllosticta species [5,8,[11][12][13][14][15]. Based on ITS, LSU, TEF1 alpha, ACT, and GPDH, five loci of phylogenetic analysis, Phyllosticta genetic system development can be divided into six species of composite groups: viz P. capitalensis species complex, P. concentrica species complex, P. cruenta species complex, P. owaniana species complex, P. rhodorae species complex, and P. vaccinii species complex [5,8].
Fujian province is situated in the north latitude range of 23 • 31 ′ -28 • 18 ′ N and the longitude range of 115 • 50 ′ -120 • 43 ′ E. This region experiences an average annual rainfall of 1500 mm, characterized by a subtropical maritime monsoon climate.Fungi were isolated from leaf spots and necrotic leaves of both Lonicera japonica and Turpinia montana samples.
For the molecular characterization, sequences from five gene loci were employed, including the internal transcribed spacer region of ribosomal DNA (ITS rDNA), the large subunit of ribosomal RNA (LSU rDNA), translation elongation factor 1α (TEF1α), actin (ACT), and glycerol 3-phosphate dehydrogenase (GPDH).Through a combination of phylogenetic and morphological analyses, the fungi were successfully identified as three new species.This discovery adds to the fungal diversity in the region and contributes to understanding of the local ecosystem.

Isolation and Morphology
The leaves of Lonicera japonica and Turpinia montana Vent, as well as saprophytic leaves collected in Mount Wuyi City, Fujian Province, China, were utilized for this study.Fragments (5 × 5 mm) were retrieved from damaged portions of the samples.Subsequently, the fragments were soaked in 75% alcohol for 1 min, followed by a single rinse with sterile water.Afterwards, they were immersed in a 5% sodium hypochlorite solution for 1 min and rinsed three times with sterile water.The samples were then dried on sterilized filter paper.The samples were inoculated on Potato dextrose agar plate (PDA: 200 g potatoes, 20 g of glucose, 20 g agar, 1000 mL of distilled water, pH 7.0), followed by 2-4 days of incubation at 25 • C. Subsequently, the AGAR portion with fungal mycelium from the periphery of the colony was transferred to a new PDA plate and photographed on days 7 and 15 using a digital camera (Canon Powershot G7X, Canon, Tokyo, Japan).After the appearance of conidia, the microscopic morphological characteristics of the fungi on PDA medium (including conidia, conidial cells, and appendage) were observed using an Olympus SZX10 (OLYMPUS, Tokyo, Japan) stereo microscope and an Olympus BX53 (OLYMPUS, Tokyo, Japan) microscope, and the colony characteristics of the fungi on the PDA medium were recorded.All the devices were equipped with an Olympus DP80 (OLYMPU, Tokyo, Japan) high-definition color digital camera to photograph the fungal structures.All the fungal strains were stored in 10% sterilized glycerine at 4 • C for followup research.The holotype specimens were deposited in the Herbarium of Plant Pathology, Shandong Agricultural University (HSAUP) and Herbarium Mycologicum Academiae Sinicae, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (HMAS).Ex-holotype living cultures were deposited in the Shandong Agricultural University Culture Collection (SAUCC).The taxonomic information of the new taxa was submitted to MycoBank (http://www.mycobank.org,accessed on 25 October 2023).

DNA Extraction and Amplification
The genomic DNA of the fungal mycelium growing on the PDA plate was extracted using a DNA extraction kit (GeneOn BioTech, Ludwigshafen am Rhein, Germany).The ITS, LSU, TEF1α, ACT, and GPDH regions were amplified using the primer pairs and polymerase chain reaction (PCR) programs specified in Table 1.The amplification reactions were conducted in a 20 µL reaction volume, comprising 12.5 µL of 2× Hieff Canace ® Plus PCR Master Mix (Yeasen Biotechnology, Shanghai, China, Cat No. 10154ES03), with 1 µL each (10 µM) of forward and reverse primers (TsingKe, Qingdao, China) and 1 µL of template genomic DNA.The volume was adjusted to a total of 25 µL with distilled deionized water.The PCR amplification products were observed on a 2% agarose electrophoresis gel.
DNA sequencing was carried out using an Eppendorf Master Thermocycler (Hamburg, Germany) at Tsingke Company Limited (Qingdao, China), bi-directionally.Consistent sequences were obtained using MEGA 7.0.All the sequences generated in this study were deposited in GenBank (Table S1).

Phylogenetic Analyses
The newly obtained sequencing data were processed using MEGA v.7.0 to ensure sequence consistency.Reference sequences were downloaded based on the GenBank numbers provided in the latest articles [8,13,[22][23][24].For phylogenetic inference, the sequence utilized by Zhang et al. [7] served as the foundation, considering the ITS-LSU-TEF1α-ACT-GAPDH sequence.MEGA v.7.0 software was employed to compare the new sequences from this study with those available in GenBank.
A phylogenetic analysis was conducted utilizing both the maximum likelihood (ML) and Bayesian inference (BI) algorithms.MrModelTest v.2.3 was employed to determine the optimal evolutionary model for each locus region [25], and this information was integrated into the BI analysis.The ML analysis was executed on the CIPRES Science Gateway portal (https://www.phylo.org,accessed on 25 October 2023) [26] using RAxML-HPC2 on XSEDE v. 8.2.12 [27][28][29][30].Bayesian inference was performed on a server with a Linux system.The ML analysis utilized default parameters, while the BI analysis was configured with a quick boot employing an automatic stop option.The Bayesian inference comprised four parallel runs of 50 million generations, employing stopping rules and a sampling frequency of 100 generations.The burn-in score was set to 0.25, and posterior probability (PP) was determined based on the remaining tree.The iTOL website (https://itol.embl.de,accessed on 25 October 2023) was utilized for plotting all the tree results and optimizing the tree construction and layout.The final results were presented using Adobe Illustrator CC 2019.

Taxonomy
MycoBank-No.MB850038.Etymology-The specific epithet "fujianensis" refers to Fujian City (China) where the type was collected.
Description-Phyllosticta fujianensis is leaf endogenic and associated with leaf spots.Its asexual morph exhibits pycnidial conidiomata, which are mostly aggregated in clusters, black, and erumpent.In PDA culture, it exudes write conidial masses within 10 days or longer.The conidial peduncle is not obvious and usually degenerated into a conidial cell.The conidiogenous cells terminal is 10.0-14.0 × 1.5-2.0µm and subcylindrical, ampulliform, hyaline, and smooth.The conidia hyaline is 10.0-11.0 × 3.5-5.0µm, mean ± SD = 10.7 ± 0.4 × 5.0 ± 0.2 µm, without a diaphragm, with thin, smooth walls.It is ovoid, ampoule-shaped, elliptic, or nearly spherical, wrapped in a thin mucous sheath of 1.0-2.0µm, and surrounded by hyalurons.The tip has a slimy appendage, and it is unbranched, soft, and tapering toward the tip.The sexual morphs are unknown; see Figure 2.
Culture characteristics-After 14 days at 25 • C in the dark, colonies of 65-76 mm diameter were found on the PDA, growing at 4.6-5.4mm/day.The colonies were greenishblack on the front and back sides, with moderate aerial mycelium on the surface and black social conidia.

Phyllosticta rhizophorae
Reduced to conidiogenous cells, subcylindrical to ampulliform     Description-The saprophytic leaf is endogenic and associated with saprophytic leaf spots.The asexual morph exhibits pycnidial conidiomata, mostly clustered and black, appearing erumpent.The PDA cultures oozed black conidial clumps, and pycnidia appeared after 10 days or more.The terminal ends of conidiogenous cells are oval to ampuleshaped, smooth, and clear, measuring 11.0-15.0× 2.5-3.5 µm.The conidia measure 13.0-15.0× 5.5-7.0 µm, mean ± SD = 13.4 ± 1.2 × 5.8 ± 0.3 µm.The conidia are solitary, transparent, without a diaphragm, with thin, smooth walls.They exhibit rough, oily spots or large central spots and are ovoid, ampulliform, oval or approximately spherical in shape.They are surrounded by a thin mucus sheath with a thickness of 1.3-2.7µmand are transparent at the top of the attachments, measuring 3.0-8.5 × 1.0-1.5 µm.The tip has a slimy appendage, is unbranched, soft, and tapers toward the tip.The sexual morphs unknown; see Figure 3. Culture characteristics-The colony on PDA grew on the whole 90 mm petri dish at 25 °C for 14 days, with a growth rate of 6.0-6.5 mm/day.The front and back sides of the colony were black-green, there was a medium number of air mycelia on the surface, and there were black conidia gathered together.
Additional specimen examined-China, Fujian Province: Wuyishan National Forest Culture characteristics-The colony on PDA grew on the whole 90 mm petri dish at 25 • C for 14 days, with a growth rate of 6.0-6.5 mm/day.The front and back sides of the colony were black-green, there was a medium number of air mycelia on the surface, and there were black conidia gathered together.
Culture characteristics-The colonies growing on PDA were cultured in darkness at 25 • C for 14 days, and the colonies grew in 90 mm petri dishes to 60-80 mm, with a growth rate of 4.2-5.7 mm/day.The front and back were black-green or black.There was air mycelium on the surface, and the black conidia were clustered together.

Discussion
The three isolates of Phyllotomycetes were obtained from Wuyishan National Park, Fujian Province, China, situated at coordinates 117°-118° E and 27°-28° N.This region experiences a typical subtropical monsoon climate, characterized by warm and humid conditions accompanied by ample rainfall.This climatic environment is conducive to the thriving growth of diverse microorganisms.Phyllosticta species identification traditionally relies on a combination of morphological characteristics and host association.However, due to the challenge of similar morphological features for taxonomic identification and homology analysis, the classification of Phyllosticta species has been complex, leading to a clu ered taxonomy.With advancements in molecular biology, the application of molecular data for species phylogeny has become increasingly sophisticated [5,33,43,52].Wang

Discussion
The three isolates of Phyllotomycetes were obtained from Wuyishan National Park, Fujian Province, China, situated at coordinates 117 • -118 • E and 27 • -28 • N.This region experiences a typical subtropical monsoon climate, characterized by warm and humid conditions accompanied by ample rainfall.This climatic environment is conducive to the thriving growth of diverse microorganisms.Phyllosticta species identification traditionally relies on a combination of morphological characteristics and host association.However, due to the challenge of similar morphological features for taxonomic identification and homology analysis, the classification of Phyllosticta species has been complex, leading to a cluttered taxonomy.With advancements in molecular biology, the application of molecular data for species phylogeny has become increasingly sophisticated [5,33,43,52].
Wang et al. [8] introduced six species complexes in Phyllosticta based on six gene loci, including the internal transcribed spacer of ribosomal RNA (ITS rDNA), large subunit of ribosomal RNA (LSU rDNA), translation elongation factor 1 alpha (TEF1α), actin (ACT), and glycerol-3-phosphate dehydrogenase (GPDH).The ITS, along with other loci such as LSU, TEF1α, ACT, and GPDH, allows for phylogenetic identification at the species level [5,53].In the present study, phylogenetic analyses of Phyllosticta species, as accepted in the latest paper, were conducted based on five loci.In this study, phylogenetic analyses of Phyllosticta species, as acknowledged in the most recent literature, were undertaken based on five genetic loci.The three newly discovered species were found within the P. capitalensis species complex and P. concentrica species complex.Consequently, the primary emphasis of the investigation was directed towards understanding the intricacies of the P. capitalensis and P. concentrica species complexes.
In this paper, we present a comprehensive examination of the multilocus phylogeny involving three Phyllosticta species isolates sourced from two host genera and saprophytic leaves.Additionally, we provide detailed illustrations of the morphological characteristics observed in culture.These isolates contribute novel insights into the species diversity of Phyllosticta within Fujian, China.We propose the recognition of three new species: Phyllosticta fujianensis, Phyllosticta saprophytica, and Phyllosticta turpiniae.Phyllosticta fujianensis was isolated from Lonicera japonica in Fujian Province, P. saprophytica was obtained from saprophytic leaves in Fujian Province, and P. turpiniae was isolated from Turpinia montana in Fujian Province.Phyllosticta exhibits typical morphological features, with asexual conidia being oval or oval to obovate, pearly-shaped, and featuring a mucous sheath and apical mucous appendage.The isolated strains conform to the morphological characteristics of Phyllosticta.
As of 25 October 2023, the Global Biodiversity Information Facility (GBIF) (https: //www.gbif.org/,accessed on 25 October 2023) encompasses 9678 geo-referenced records of Phyllosticta species reported globally.The main distribution locations for these species are in America, Asia, and Europe, with the United States having the most extensive distribution [54,55].Among the Phyllosticta species, P. carbitalensis, identified as a relatively weak plant pathogenic agent, is known to induce leaf spot diseases in various plants, including tea (Camellia sinensis), oil palm (Elaeis guineensis), Ricinus communis, and guava black spot [56,57] [8,14,33,44,45,[57][58][59][60] have notably enhanced the accuracy of Phyllosticta classification.This precision holds significant implications for the utilization and development of Phyllosticta species.In summary, Phyllosticta species showcase a wide distribution and manifest diverse lifestyles, encompassing pathogenicity, latency, and endophytic characteristics.Further research is warranted to ascertain whether Phyllosticta species exhibit host specificity and to elucidate the conditions governing the transition from endophytic to pathogenic behavior.

Figure 1 .Figure 1 .
Figure 1.Phylogram of the genus Phyllosticta based on a concatenated ITS, LSU, TEF1α, ACT, and GPDH sequence alignment, with Botryosphaeria obtusa (CMW 8232) and Botryosphaeria stevensii (CBS 112553) serving as outgroups.Maximum likelihood bootstrap support values and Bayesian inference posterior probabilities above 70% and 0.90 are shown at the first and second position, respectively.Ex-type cultures are indicated in bold face.Strains obtained in the current study are in red.Some branches are shortened for layout purposes-these are indicated by two diagonalFigure 1. Phylogram of the genus Phyllosticta based on a concatenated ITS, LSU, TEF1α, ACT, and GPDH sequence alignment, with Botryosphaeria obtusa (CMW 8232) and Botryosphaeria stevensii (CBS 112553) serving as outgroups.Maximum likelihood bootstrap support values and Bayesian inference posterior probabilities above 70% and 0.90 are shown at the first and second position, respectively.Ex-type cultures are indicated in bold face.Strains obtained in the current study are in red.Some branches are shortened for layout purposes-these are indicated by two diagonal lines with the number of times.The bar at the bottom-left represents the substitutions per site.Notes: Ex-type or ex-holotype strains are labeled with a star mark "*".

Table 1 .
Molecular markers and their PCR primers and programs used in this study.
. P. concentrica has been observed to cause leaf spot in Hedera helix and Boehmeria cylindrica.According to the current study, Phyllosticta is extensively distributed in China, and numerous researchers have reported new species of Phyllosticta and new records in the country.Contributions from Wang et al., Lin et al., Su et al., Liao et al., Tang et al., and Zhang et al.