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Morphological and phylogenetic analyses reveal two new species and a new record of Phyllosticta (Botryosphaeriales, Phyllostictaceae) from Hainan, China
expand article infoZhaoxue Zhang‡§, Xiaoyong Liu‡, Xiuguo Zhang‡, Zhe Meng‡
‡ Shandong Normal University, Jinan, China
§ Shandong Agricultural University, Taian, China

Corresponding author: Zhe Meng ( zmeng@sdnu.edu.cn )

Academic editor: Huzefa Raja
© 2022 Zhaoxue Zhang, Xiaoyong Liu, Xiuguo Zhang, Zhe Meng.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Zhang Z, Liu X, Zhang X, Meng Z (2022) Morphological and phylogenetic analyses reveal two new species and a new record of Phyllosticta (Botryosphaeriales, Phyllostictaceae) from Hainan, China. MycoKeys 91: 1-23. https://doi.org/10.3897/mycokeys.91.84803
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Abstract

The fungal genus Phyllosticta has been reported from all around the world and accommodates numerous pathogenic and endophytic species isolated from a wide range of plant hosts. Based on multilocus phylogenies from a combined dataset of genes encoding internal transcribed spacer (ITS), large subunit of ribosomal RNA (LSU rDNA), translation elongation factor 1 alpha (TEF1α), actin (ACT) and glycerol-3-phosphate dehydrogenase (GPDH), in conjunction with morphological characteristics, we describe two new species P. oblongifoliae sp. nov. and P. pterospermi sp. nov., as well as a new Chinese record P. capitalensis. Their similarity and dissimilarity to morphologically-allied and phylogenetically-related species are also annotated and discussed.

Keywords

multigene phylogeny, new species, taxonomy

Introduction

Phyllosticta Pers. was introduced by Persoon (1818) and P. convallariae Pers. was designated as the type species (Donk 1968). Since Phyllosticta is distinct from other genera in that family, Seaver (1922) treated it in the family Phyllostictaceae Fr. of the order Phyllostictites. Nevertheless, Phyllosticta was accommodated in the family Botryosphaeriaceae Theiss. & Syd. (in Botryosphaeriales C.L. Schoch et al.) in several major studies (e.g. Crous et al. 2006; Schoch et al. 2006; Liu et al. 2012). However, the phylogenetic analyses by Wikee et al. (2013a) allocated Phyllosticta in a clade sister to Botryosphaeriaceae. As a result, the genus is currently accepted in the family Phyllostictaceae, in the order Botryosphaeriales.

A total of 3,213 names are documented for Phyllosticta in the Index Fungorum (accessed on 31 March 2022) (Hongsanan et al. 2020; Wijayawardene et al. 2020). However, many of these names have been synonymised (van der Aa and Vanev 2002). Currently, 1499 species are accepted in the genus (Bánki et al. 2022). The majority of the Phyllosticta species are known to infect a broad range of hosts and cause plant diseases, such as leaf and fruit spots (Wikee et al. 2013a; Zhou et al. 2015; Lin et al. 2017). Van der Aa (1973) revised this genus and established his own morphological criteria, i.e. aseptate pycnidia and hyaline conidia that are usually covered by a mucoid layer and bear a single apical appendage. According to these criteria, van der Aa and Vanev (2002) re-classified Phyllosticta and accepted 190 species. Other species were recombined into Asteromella Pass. & Thüm., Diaporthe Fuckel, Guignardia Viala & Ravaz, Leptodothiorella Höhn. and Phoma Sacc. A rare tropical species from the Brazilian Cerrado, P. xylopiae-sericeae Furlan. & Dianese, although morphologically well documented (Furlanetto and Dianese 1998), remains to be molecularly characterised. Recently, DNA sequencing of orthologous genes has greatly improved our knowledge of fungal phylogeny. Since van der Aa and Vanev (2002), several studies have shown that phylogenetic analyses can help delineate species in Phyllosticta (Baayen et al. 2002; Wulandari et al. 2009; Glienke et al. 2011; Wikee et al. 2011). More recently, new species of Phyllosticta have been increasingly described, based on a combination of molecular data and morphological features (Su and Cai 2012; Wang et al. 2012, 2013; Wong et al. 2012; Zhang et al. 2012, 2013; Wikee et al. 2013a; Wulandari et al. 2013; Crous et al. 2014, 2015, 2016, 2017, 2018, 2019, 2021; Zhou et al. 2015; Guarnaccia et al. 2017; Lin et al. 2017; Hattori et al. 2020; Norphanphoun et al. 2020). Norphanphoun et al. (2020) assembled all species denoted as Phyllosticta in GenBank, analysing a comprehensive dataset of five loci and consequently proposing six species complexes, 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.

Hainan Province (18°10'–20°10'N, 108°37'–111°05'E) is an island in southern China, with an annual mean temperature of 22–27 °C and an annual precipitation of 1000–2600 mm. Bawangling National Forest Park is located in the southwest of Hainan, with a typical tropical rainforest climate. Fungi associated with leaf spots were collected from Rhapis excelsa, Garcinia oblongifolia and Pterospermum heterophyllum. Using sequences of five gene loci, which include 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). We also incorporated their morphology and then identified these fungi as three species of the P. capitalensis species complex, including two new species, as well as a species new to China, based on morphology and phylogenetic analyses.

Materials and methods

Isolation and morphological studies

Leaves of Rhapis excelsa, Garcinia oblongifolia and Pterospermum heterophyllum showing necrotic spots were collected at the Bawangling National Forest Park, Hainan Province, China. Isolates were obtained using a tissue isolation method (Jiang et al. 2021). Fragments (5 × 5 mm) were taken from the margin of leaf lesions, surface-sterilised by immersing consecutively in 75% ethanol solution for 1 min, 5% sodium hypochlorite solution for 30 s and then rinsed in sterile distilled water for 1 min (Jiang et al. 2021). The sterilised fragments were dried with sterilised paper towels and placed on potato dextrose agar (PDA: 200 g potato, 20 g dextrose, 20 g agar, 1000 ml distilled water, pH 7.0) and incubated at 25 °C for 2–4 days. Subsequently, portions of agar with fungal mycelium from the periphery of the colonies were transferred into new PDA plates and photographed on the 7th and 15th days by a digital camera (Canon Powershot G7X). An inoculum of the purified colonies was placed on 2% malt extract agar (MEA:20 g malt extract, 20 g soy peptone, 15 g agar, 1000 ml distilled water, pH 5.6) and incubated under continuous near-UV light at room temperature to promote sporulation (Braun et al. 2018). Micromorphological characters were observed using an Olympus SZX10 stereomicroscope and Olympus BX53 microscope, all fitted with an Olympus DP80 high-definition colour digital camera to photo-document fungal structures. All fungal strains were stored in 10% sterilised glycerine at 4 °C for further studies. Structural measurements were taken using the Digimizer software (https://www.digimizer.com/), with thirty measurements taken for each character. The holotype specimens were deposited in the Herbarium of Plant Pathology, Shandong Agricultural University (HSAUP). Ex-holotype living cultures were deposited in the Shandong Agricultural University Culture Collection (SAUCC). Taxonomic information of the new taxa was submitted to MycoBank (http://www.mycobank.org).

DNA extraction and sequencing

Genomic DNA was extracted from fungal mycelia grown on PDA, using a modified cetyltrimethylammonium bromide (CTAB) protocol as described in Guo et al. (2000). The internal transcribed spacer region (ITS) with intervening 5.8S rRNA gene, large subunit of rRNA gene (LSU), translation elongation factor 1-alpha gene (tef1), actin gene (ACT) and glyceraldehyde-3-phosphate dehydrogenase gene (GPDH) were amplified and sequenced by using the primer pairs ITS5/ITS4 (White et al. 1990), LROR/LR5 (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.

PCR was performed using an Eppendorf Master Thermocycler (Hamburg, Germany). Amplification reactions were carried out in a 25 μl reaction volume, which contained 12.5 μl 2×Green Taq Mix (Vazyme, Nanjing, China), 1 μl of each forward and reverse primer (10 μM stock; Biosune, Shanghai, China), 1 μl template genomic DNA (approximately 10 ng/μl) and 9.5 μl distilled deionised water. PCR parameters were as follows: 94 °C for 5 min; 35 cycles of denaturation at 94 °C for 30 s, annealing at a suitable temperature for 50 s and extension at 72 °C for 1 min; and a final elongation step at 72 °C for 10 min. The suitable annealing temperatures for the genes were 55 °C for ITS, 51 °C for LSU, 52 °C for ACT, 48 °C for tef1 and 52 °C for GPDH, respectively. PCR products were checked through a 1% agarose gel electrophoresis, stained with GelRed and visualised by a UV light. Sequencing was performed bi-directionally by Biosune Company Limited (Shanghai, China). Consensus sequences were obtained using MEGA v. 7.0 (Kumar et al. 2016). All sequences generated in this study were deposited in GenBank (Table 1).

Table 1.
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Species and GenBank accession numbers of DNA sequences used in this study.

Species1 Voucher2 Host/Substrate Country GenBank accession number
ITS LSU tef1 ACT GPDH
Phyllosticta acaciigena CPC 28295 * Acacia suaveolens Australia KY173433 KY173523 ‒ KY173570 ‒
P. aloeicola CPC 21020 * Aloe ferox South Africa KF154280 KF206214 KF289193 KF289311 KF289124
CPC 21021 Aloe ferox South Africa KF154281 KF206213 KF289194 KF289312 KF289125
P. ardisiicola NBRC 102261 * Ardisia crenata Japan AB454274 AB454274 ‒ AB704216 ‒
P. aristolochiicola BRIP 53316 * Aristolochia acuminata Australia JX486129 ‒ ‒ ‒ ‒
P. azevinhi MUCC0088 Ilex pedunculosa Japan AB454302 AB454302 ‒ AB704226 ‒
P. beaumarisii CBS 535.87 Muehlenbekia adpressa Australia AY042927 KF306229 KF289170 KF306232 KF289074
P. brazillianiae LGMF 330 * Mangifera indica Brazil JF343572 KF206217 JF343593 JF343656 JF343758
LGMF 333 Mangifera indica Brazil JF343574 KF206216 JF343595 JF343658 JF343760
P. camelliae MUCC0059 Camellia japonica Japan AB454290 AB454290 AB704223
P. capitalensis CBS 128856 * Stanhopea graveolens Brazil JF261465 KF206255 JF261507 KF289289 JF343776
CBS 226.77 Baccaurea ramiflora Brazil FJ538336 KF206289 FJ538394 FJ538452 JF343718
CBS 356.52 Paphiopedilum callosum Germany FJ538342 KF206300 FJ538400 FJ538458 KF289087
CBS 100175 Ilex sp. Not given FJ538320 KF206327 FJ538378 FJ538436 JF343699
CBS 101228 Citrus sp. Brazil FJ538319 KF206325 FJ538377 FJ538435 KF289086
CBS 114751 Nephelium lappaceum Hawaii EU167584 EU167584 FJ538407 FJ538465 KF289088
CBS 115047 Vaccinium sp. New Zealand FJ538323 KF206318 FJ538381 FJ538439 KF289077
CBS 115049 Aspidosperma polyneuron Brazil FJ538324 KF206317 FJ538382 FJ538440 KF289084
CBS 117118 Bowdichia nitida Brazil FJ538339 JQ743603 FJ538397 FJ538455 KF289090
CBS 120428 Musa acuminata Indonesia JN692544 KF206315 JN692532 JN692520 JN692509
CBS 123373 Sansevieria sp. Netherlands FJ538341 JQ743604 FJ538399 FJ538457 JF343703
CPC 13987 Protea repens Portugal KF206183 KF206281 KF289176 KF289263 KF289083
CPC 16592 Citrus limon Argentina KF206187 KF206270 KF289273 KF289178 KF289092
CPC 17468 Cymbidium sp. Brazil KF206188 KF206259 KF289189 KF289284 KF289120
CPC 20256 Ophiopogon japonicus Thailand KC291337 KF206247 KC342557 KC342534 KF289089
CPC 20257 Ficus benjamina Thailand KC291338 KF206246 KC342558 KC342535 KF289099
LGMF219 Citrus sinensis Brazil KF206202 KF206220 JF261490 KF289306 JF343737
LGMF220 Citrus sinensis Brazil KF206203 KF206219 JF261488 KF289307 JF343735
LGMF222 Citrus sinensis Brazil KF206204 KF206218 JF261492 KF289308 JF343739
SAUCC210144 Rhapis excelsa China OM571175 OM571179 OM640045 OM640047 OM640049
SAUCC210148 Rhapis excelsa China OM571176 OM571180 OM640046 OM640048 OM640050
P. carochlae CGMCC 3.17317 * Caryota ochlandra China KJ847422 ‒ KJ847444 KJ847430 KJ847438
CGMCC 3.17318 Caryota ochlandra China KJ847423 ‒ KJ847445 KJ847431 KJ847439
P. cavendishii BRIP 554196 * Musa cv. Formosana Taiwan JQ743562 ‒ KF009743 KF014080 ‒
BRIP 58008 Banana Australia KC988365 ‒ KF009742 KF014071 ‒
P. cordylinophila CPC 20261 * Cordyline fruticosa Thailand KF170287 KF206242 KF289172 KF289295 KF289076
CPC 20277 Cordyline fruticosa Thailand KF170288 KF206228 KF289171 KF289301 KF289075
P. eugeniae CBS 445.82 Eugenia aromatica Indonesia AY042926 KF206288 KF289208 KF289246 KF289139
P. fallopiae MUCC0113 * Fallopia japonica Japan AB454307 AB454307 ‒ ‒ ‒
P. harai MUCC0043 Aucuba japonica Japan AB454281 AB454281 ‒ AB704219 ‒
P. hubeiensis CGMCC 3.14986 * Viburnum odoratissimim China JX025037 ‒ JX025042 JX025032 JX025027
CGMCC 3.14987 Viburnum odoratissimim China JX025038 ‒ JX025043 JX025033 JX025028
P. ilicis-aquifolii CGMCC 3.14358 * Ilex aquifolium China JN692538 ‒ JN692526 JN692514 ‒
CGMCC 3.14359 Ilex aquifolium China JN692539 ‒ JN692527 JN692515 ‒
P. maculata CPC 18347 * Musa cv. Goly-goly pot-pot Australia JQ743570 ‒ KF009700 KF014016 ‒
BRIP 46622 Musa cv. Goly-goly pot-pot Australia JQ743567 ‒ KF009692 KF014013 ‒
P. mangiferae IMI 260.576 * Mangifera indica India JF261459 KF206222 JF261501 JF343641 JF343748
CPC 20260 Arecaceae Thailand KF206193 KF206243 KF289187 KF289294 KF289114
P. mangifera-indica MFLUCC 10–0029 * Mangifera indica Thailand KF170305 KF206240 KF289190 KF289296 KF289121
P. miurae MUCC0065 Lindera praecox Japan AB454291 AB454291 ‒ AB704224 ‒
P. musaechinensis GZAAS6.1247 Musa. sp. China KF955294 ‒ KM816639 KM816627 KM816633
GZAAS6.1384 Musa. sp. China KF955295 ‒ KM816640 KM816628 KM816634
P. musarum BRIP57803 Musa. sp. Malaysia JX997138 ‒ KF009737 KF014055 ‒
BRIP58028 Musa. sp. Australia KC988377 ‒ KF009738 KF014054 ‒
P. oblongifolae SAUCC210055 Garcinia oblongifolia China OM248442 OM232085 OM273890 OM273894 OM273898
SAUCC210054 Garcinia oblongifolia China OM248443 OM232086 OM273891 OM273895 OM273899
SAUCC210053 Garcinia oblongifolia China OM248444 OM232087 OM273892 OM273896 OM273900
SAUCC210052 * Garcinia oblongifolia China OM248445 OM232088 OM273893 OM273897 OM273901
P. paracapitalensis CPC 26517 * Citrus floridana Italy KY855622 KY855796 KY855951 KY855677 KY855735
CPC 26518 Citrus floridana Italy KY855623 KY855797 KY855952 KY855678 KY855736
CPC 26700 Citrus floridana Italy KY855624 KY855798 KY855953 KY855679 KY855737
CPC 26701 Citrus floridana Italy KY855625 KY855799 KY855954 KY855680 KY855738
CPC 26805 Citrus floridana Italy KY855626 KY855800 KY855955 KY855681 KY855739
CPC 26806 Citrus floridana Italy KY855627 KY855801 KY855956 KY855682 KY855740
CPC 28120 Citrus limon Spain KY855628 KY855802 KY855957 KY855683 KY855741
P. paracapitalensis CPC 28121 Citrus limon Spain KY855629 KY855803 KY855958 KY855684 KY855742
CPC 28122 Citrus limon Spain KY855630 KY855804 KY855959 KY855685 KY855743
CPC 28123 Citrus limon Spain KY855631 KY855805 KY855960 KY855686 KY855744
CPC 28127 Citrus limon Spain KY855632 KY855806 KY855961 KY855687 KY855745
CPC 28128 Citrus limon Spain KY855633 KY855807 KY855962 KY855688 KY855746
CPC 28129 Citrus limon Spain KY855634 KY855808 KY855963 KY855689 KY855747
P. parthenocissi CBS 111645 * Parthenocissus quinquefolia USA EU683672 ‒ JN692530 JN692518 ‒
P. partricuspidatae NBRC 9466 * Parthenocissus tricuspidata Japan KJ847424 ‒ KJ847446 KJ847432 KJ847440
NBRC 9757 Parthenocissus tricuspidata Japan KJ847425 ‒ KJ847447 KJ847433 KJ847441
P. philoprina CBS 587.69 Ilex aquifolium Spain KF154278 KF206297 KF289206 KF289250 KF289137
CBS 616.72 Ilex aquifolium Germany KF154279 KF206296 KF289205 KF289251 KF289136
P. pterospermi SAUCC210104 * Pterospermum heterophyllum China OM249954 OM249956 OM273902 OM273904 OM273906
SAUCC210406 Pterospermum heterophyllum China OM249955 OM249957 OM273903 OM273905 OM273907
P. rhizophorae NCYUCC 19–0352 * Rhizophora stylosa Taiwan MT360030 MT360039 ‒ MT363248 MT363250
NCYUCC 19–0358 Rhizophora stylosa Taiwan MT360031 MT360040 ‒ MT363249 MT363251
P. schimae CGMCC 3.14354 * Schima superba China JN692534 ‒ JN692522 JN692510 JN692506
P. schimicola CGMCC 3.17319 * Schima superba China KJ847426 ‒ KJ847448 KJ847434 KJ854895
CGMCC 3.17320 Schima superba China KJ847427 ‒ KJ847449 KJ847435 KJ854896
P. styracicola LC1642 * Styrax gradiflorus China JX025040 ‒ JX025045 JX025035 JX025030
P. vitis-rotundifoliae CGMCC 3.17321 Vitis rotundifolia USA KJ847429 ‒ KJ847451 KJ847437 KJ847443
CGMCC 3.17322 * Vitis rotundifolia USA KJ847428 ‒ KJ847450 KJ847436 KJ847442

1Newly generated sequences in this study are in bold. 2Isolates marked with “*” are ex-type or ex-epitype strains.

Phylogenetic analyses

The generated consensus sequences were subjected to BLAST searches to identify closely-related sequences in the NCBI’s GenBank nucleotide database (Zhang et al. 2000). For phylogenetic inferences, based on ITS-LSU-tef1-ACT-GPDH sequences, a subset of sequences from the alignments of Norphanphoun et al. (2020) was used as the backbone. Newly-generated sequences in this study were aligned with related sequences retrieved from GenBank (Table 1) using MAFFT 7 online tool with the Auto strategy (Katoh et al. 2019; http://mafft.cbrc.jp/alignment/server/). To establish the identity of the isolates at species level, phylogenetic analyses were first performed for each locus individually and then all loci were concatenated together for a unified analysis (ITS-LSU-tef1-ACT-GPDH).

Phylogenetic analyses were carried out with Maximum Likelihood (ML) and Bayesian Inference (BI) algorithms. The best evolutionary model for each partition was determined using MrModelTest v. 2.3 (Nylander 2004) and incorporated into the BI analyses. ML and BI run on the CIPRES Science Gateway portal (https://www.phylo.org/; Miller et al. 2012) using RAxML-HPC2 on XSEDE v. 8.2.12 (Stamatakis 2014) and MrBayes on XSEDE v. 3.2.7a (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003; Ronquist et al. 2012), respectively. Default parameters were used for the ML analyses and the rapid bootstrapping with the automatic halt option was set for the BI analyses. Bayesian Inference included four parallel runs of 10,000,000 generations, with the stop rule option and a sampling frequency of 1,000 generations. Burn-in fraction was set to 0.25 and posterior probabilities (PP) were determined from the remaining trees. All resultant trees were plotted using FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree) and the layout of the trees was edited in Adobe Illustrator CC 2019.

Results

Phylogenetic analyses

A total of 86 isolates representing the Phyllosticta species were phylogenetically analysed, of which 84 isolates in the P. capitalensis species complex were considered as ingroup and two strains of Phyllosticta hubeiensis (CGMCC 3.14986, CGMCC 3.14987) in the P. cruenta species complex were used as outgroup. The final alignment contained 2665 concatenated characters, viz. 1–733 (ITS), 734–1499 (LSU), 1500–1790 (tef1), 1791–2042 (ACT), 2043–2665 (GPDH). Of these characters, 1964 were constant, 126 were variable and parsimony-uninformative and 575 were parsimony-informative. MrModelTest recommended that the Bayesian Inference should use Dirichlet base frequencies for the ITS, LSU, tef1, ACT and GPDH data partitions. The GTR+I+G model was proposed for ITS, LSU and GPDH, while HKY+G for tef1 and ACT. The MCMC analysis of the five concatenated genes was run for 1,520,000 generations, resulting in 30,402 trees. The initial 7,600 trees generated in the burn-in phase were discarded, while the remaining trees were used to calculate posterior probabilities in the majority rule consensus trees. The alignment contained a total of 876 unique site patterns (ITS: 358, LSU: 69, tef1: 170, ACT: 137, GPDH: 142). The topology of the ML tree confirmed the tree topology obtained from the Bayesian Inference and, therefore, only the ML tree is presented (Fig. 1). The 86 strains were assigned to 34 species, based on the five-gene phylogeny (Fig. 1). The present study revealed three species, viz. Phyllosticta oblongifolae sp. nov., P. pterospermi sp. nov. and P. capitalensis. The P. oblongifolae sp. nov. was a sister group to P. eugeniae (0.98/81) and the P. pterospermi sp. nov. was closely related to P. mangiferae (0.99/92).

Figure 1. 

Phylogram of the Phyllosticta capitalensis species complex, based on a concatenated ITS, LSU, tef1, ACT and GPDH sequence alignment, with Phyllosticta hubeiensis (CGMCC 3.14986, CGMCC 3.14987) of the P. cruenta species complex serving as outgroup. Bayesian Inference posterior probabilities and Maximum Likelihood bootstrap support values above 0.70 and 70% 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 left-bottom represents substitutions per site.

Taxonomy

The taxa described belong in family Phyllostictaceae.

Phyllosticta oblongifoliae Z.X. Zhang, X.Y. Liu, Z. Meng & X.G. Zhang, sp. nov.

MycoBank No: 843232
Fig. 2

Etymology

The specific epithet “oblongifoliae” refers to the host plant Garcinia oblongifolia.

Type

China, Hainan Province: Bawangling National Forest Park, on diseased leaves of Garcinia oblongifolia, 19 May 2021, Z.X. Zhang (holotype, HSAUP210052; ex-type SAUCC210052).

Description

Leaf endogenic and associated with leaf spots. Asexual morph: Conidiomata pycnidial, mostly aggregated in clusters, black, erumpent. In MEA culture exuding colourless to opaque conidial masses within 10 days or longer. Pycnidial wall multilayered, textura angularis, brown to dark brown, up to 30 μm thick; inner walls hyaline. Conidiophores indistinct, often reduced to conidiogenous cells. Conidiogenous cells terminal, subcylindrical, ampulliform, hyaline, smooth, 9.0–14.0 × 2.5–4.5 μm. Conidia 8.0–13.0 × 6.0–8.0 μm, mean ± SD = 10.0 ± 1.3 × 7.2 ± 0.5 μm, hyaline, aseptate, thin and smooth walled, coarsely guttulate or with a single large central guttule, ovoid, ampulliform, ellipsoidal to subglobose, enclosed in a thin mucoid sheath, 1.0–2.0 μm thick and bearing a hyaline, apical mucoid appendage, 3.0–8.5 × 1.0–1.5 μm, flexible, unbranched, tapering towards an acutely rounded tip.

Figure 2. 

Phyllosticta oblongifoliae (SAUCC210052) a diseased leaf of Garcinia oblongifolia b, c colonies (left-above, right-reverse) after 15 days on PDA (b) and MEA (c) d conidiomata e–h conidiogenous cells with conidia i–j conidia. Scale bars: 10 μm (e–j).

Culture characteristics

. Colonies on PDA occupying an entire 90 mm Petri dish in 14 days at 25 °C in darkness, with a growth rate of 6.0–6.5 mm/day, greenish-black in obverse and reverse. Colonies on MEA 82–86 mm in diameter after 14 days at 25 °C in darkness, with a growth rate of 5.7–6.2 mm/day, undulate at edge, white to grey white in obverse and reverse, with moderate aerial mycelia on the surface, with black, gregarious conidiomata.

Additional specimens examined

China, Hainan Province: Bawangling National Forest Park, on diseased leaves of Garcinia oblongifolia, 19 May 2021, Z.X. Zhang, HSAUP210053, living culture SAUCC210053; on diseased leaves of Garcinia oblongifolia, 19 May 2021, Z.X. Zhang, paratype HSAUP210054, ex-paratype living culture SAUCC210054; on diseased leaves of Garcinia oblongifolia, 19 May 2021, Z.X. Zhang, paratype HSAUP210055, ex-paratype living culture SAUCC210055.

Notes

Phyllosticta oblongifoliae is introduced, based on the multi-locus phylogenetic analysis as the strain clustered into a well-supported clade (Fig. 1; 1.00/100), which is closely related to Phyllosticta ugeniae (0.98/81), but distinguished, based on molecular data, ITS, LSU, tef1, ACT and GPDH loci by 57 nucleotide differences in the concatenated alignment. Morphologically, P. oblongifoliae (SAUCC210052) differs from P. ugeniae (CBS 445.82) in its shorter and wider conidia (8.0–13.0 × 6.0–8.0 vs. 9.6–16.8 × 4.8–6.0 μm) (Wikee et al. 2013a). Therefore, we establish this fungus as a novel species (Jeewon and Hyde 2016).

Phyllosticta pterospermi Z.X. Zhang, X.Y. Liu, Z. Meng & X.G. Zhang, sp. nov.

MycoBank No: 843233
Fig. 3

Type

China, Hainan Province: Bawangling National Forest Park, on diseased leaves of Pterospermum heterophyllum, 19 May 2021, Z.X. Zhang (holotype, HSAUP210104; ex-holotype living culture SAUCC210104).

Etymology

The specific epithet “pterospermi” refers to the genus name of the host plant Pterospermum heterophyllum.

Description

Leaf endogenic and associated with leaf spots. Asexual morph: Conidiomata pycnidial, mostly aggregated in clusters, black, erumpent. On MEA, pycnidia exudes yellow conidial masses, within 15 days or longer. Pycnidial walls multilayered, textura angularis, brown, up to 30 μm thick; inner walls of hyaline. Conidiophores indistinct, often reduced to conidiogenous cells. Conidiogenous cells, cylindrical, hyaline, smooth, 7.5–11.0 × 2.5–4.5 μm. Conidia 8.0–12.0 × 4.5–8.5 μm, mean ± SD = 9.8 ± 0.9 × 7.3 ± 0.7 μm, hyaline, aseptate, thin and smooth-walled, coarsely guttulate or with a single large central guttule, obovoid, ellipsoidal to subglobose, enclosed in a thin mucoid sheath, 1.0–2.0 μm thick and bearing a hyaline, apical mucoid appendage, 4.0–6.8 × 1.5–3.0 μm, flexible, unbranched, tapering towards an acutely rounded tip.

Figure 3. 

Phyllosticta pterospermi (holotype SAUCC210104) a diseased leaf of Pterospermum heterophyllum b, c colonies (left-above, right-reverse) after 15 days on PDA (b) and MEA (c) d conidiomata e–h conidiogenous cells with conidia i–j conidia. Scale bars: 10 μm (e–j).

Culture characteristics

Colonies on PDA 80–90 mm in diameter after 14 days at 25 °C in darkness, with a growth rate of 5.7–6.5 mm/day, undulate at edge, grey white to greyish-green in obverse and reverse. Colonies on MEA 82–86 mm in diameter after 14 days at 25 °C in darkness, with a growth rate of 5.8–6.2 mm/day, undulate at edge, grey white to yellow in obverse and reverse, with moderate aerial mycelia on the surface, with black, gregarious conidiomata.

Additional specimen examined

China, Hainan Province: Bawangling National Forest Park, on diseased leaves of Pterospermum heterophyllum. 19 May 2021, Z.X. Zhang, paratype HSAUP210106, ex-paratype living culture SAUCC210106.

Notes

Two isolates from leaf spots of Pterospermum heterophyllum phylogenetically clustered into a well-supported clade (1.00/100), which is closely related to P. ardisiicola (0.90/62) and P. mangiferae (0.99/91; Fig. 1). However, P. pterospermi differs from P. ardisiicola by 30 nucleotides (13/603 in ITS, 3/553 in LSU and 14/248 ACT) and from P. mangiferae by 29 nucleotides (7/567 in ITS, 2/763 in LSU, 3/215 in tef1, 3/226 in ACT and 14/643 in GPDH). In morphology, they are distinguished by hosts and conidial size (8.0–12.0 × 4.5–8.5 μm in P. pterospermi vs. 7.0–11.0 × 5.0–7.5 μm in P. ardisiicola vs. 10.0–12.0 × 6.0–7.0 μm in P. mangiferae). Furthermore, P. pterospermi differs from P. ardisiicola and P. mangiferae by wider conidiogenous cells (7.5–11.0 × 2.5–4.5 μm vs. 5.0–12.5 × 1.2–2.5 μm) and from P. mangiferae in having longer conidiogenous cells (7.5–11.0 × 2.5–4.5 μm vs. 6.0–10.0 × 3.0–4.0 μm) (Motohashi et al. 2008; Glienke et al. 2011). Therefore, we establish this strain as P. pterospermi sp. nov. (Jeewon and Hyde 2016).

Phyllosticta capitalensis Henn., Hedwigia 48: 13. 1908

Fig. 4

Description

Leaf endogenic and associated with leaf spots. Asexual morph: Conidiomata pycnidial, mostly aggregated in clusters, black, erumpent. In MEA, cultures exuded colourless to opaque conidial masses, appeared on pycnidia after 10 days or longer. Pycnidial walls of multilayered, textura angularis, brown to dark brown, up to 35 μm thick; inner walls hyaline. Conidiophores subcylindrical to ampulliform, frequently reduced to conidiogenous cells or branching from a basal supporting cell, coated in mucoid layer, 8.0–14.0 × 3.0–5.0 μm. Conidiogenous cells terminal, subcylindrical to ampulliform, hyaline, smooth, 8.0–11.0 × 3.0–4.5 μm. Conidia 9.0–12.5 × 5.0–7.0 μm, mean ± SD = 10.6 ± 0.9 × 6.2 ± 0.5 μm, solitary, hyaline, aseptate, thin and smooth walled, coarsely guttulate or with a single large central guttule, ovoid, ampulliform, ellipsoidal to subglobose, enclosed in a thin mucoid sheath, 1.3–2.7 μm thick and bearing a hyaline, apical mucoid appendage, 3.0–8.5 × 1.0–1.5 μm, flexible, unbranched, tapering towards an acutely rounded tip. Spermatia hyaline, smooth, guttulate to granular, bacilliform, 6.0–8.2 × 1.3–2.0 μm, occurring in conidioma with conidia. Sexual morph: Ascomata shape and wall like those of the conidiomata. Asci bitunicate, hyaline, clavate to broadly fusoid-ellipsoid, with visible apical chamber, 2 μm diam., 45–85 × 9–13 μm. Ascospores bi- to multiseriate, hyaline, smooth, granular to guttulate, aseptate, straight, rarely curved, widest in the middle, limoniform with obtuse ends, 15–18 × 6–7 μm.

Figure 4. 

Phyllosticta capitalensis (holotype SAUCC210144) a diseased leaf of Rhapis excelsa b, c colonies (left-above, right-reverse) after 15 days on PDA (b) and MEA (c) d conidiomata e asci and ascospores f asci, ascospores and conidia g conidiogenous cells with conidia h conidia i spermatia. Scale bars: 10 μm (e–i).

Culture characteristics

Colonies on PDA occupying an entire 90 mm Petri dish in 14 days at 25 °C in darkness, with a growth rate of 6.0–6.5 mm/day, greenish-black in obverse and reverse. Colonies on MEA 82–86 mm in diameter after 14 days at 25 °C in darkness, with a growth rate of 5.7–6.2 mm/day, undulate at edge, white to grey white in obverse and reverse, with moderate aerial mycelia on the surface, with black, gregarious conidiomata.

Specimens examined

China, Hainan Province: Bawangling National Forest Park, on diseased leaves of Rhapis excelsa (Thunb.) Henry ex Rehd, 19 May 2021, Z.X. Zhang, HSAUP210144, living culture SAUCC210144; on diseased leaves of Rhapis excelsa. 19 May 2021, Z.X. Zhang, HSAUP210148, living culture SAUCC210148.

Notes

Based on morphological features, Hennings (1908) described Phyllosticta capitalensis and Glienke et al. (2011) added molecular data. The holotype (CBS 128856) of P. capitalensis was collected from Stanhopea graveolens (Glienke et al. 2011). In our current study, two isolates (SAUCC210144, SAUCC210148), collected from diseased leaves of Rhapis excelsa, cluster in the P. capitalensis clade (Fig. 1). Although four other species are also in this clade, we consider these two isolates as P. capitalensis, based on their morphological characters, such as granular to guttulate ascospores (15–18 × 6–7 vs. 15–17 × 5–6 μm), subcylindrical to ampullate conidiogenous cells (8.0–11.0 × 3.0–4.5 vs. 7–10 × 3–5 μm), ellipsoidal to subglobose conidia (9–12.5 × 5–7 vs. 11–12 × 6–7 μm) and hyaline, apical mucoid appendages (3–8.5 × 1–1.5 vs. 6–8 × 1–1.5 μm).

Discussion

Compared to other parts of China, species richness is highly diverse in Hainan Province, especially in Bawangling National Forest Park, which has a typical tropical rainforest climate. The environment favours growth of unusual microbial species. Historically, Phyllosticta species have been identified by morphology and host association. However, overlapping morphology makes it difficult to pinpoint homologous characters and, consequently, traditional identification of Phyllosticta species has long been a complicated endeavour (Norphanphoun et al. 2020). This issue has led to confusion in the taxonomy of Phyllosticta. Molecular phylogenetics has promoted species delimitation and species complex determination (Baayen et al. 2002; Okane et al. 2003; Motohashi et al. 2009; Wulandari et al. 2009; Glienke et al. 2011; Wikee et al. 2012). Norphanphoun et al. (2020) introduced six species complexes in Phyllosticta, based on five gene loci encoding 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). Amongst these, the P. capitalensis species complex consisted of 28 cryptic species, P. acaciigena, P. aloeicola, P. ardisiicola, P. aristolochiicola, P. azevinhi, P. beaumarisii, P. brazilianiae, P. capitalensis, P. carochlae, P. cavendishii, P. cordylinophila, P. eugeniae, P. fallopiae, P. ilicis-aquifolii, P. maculata, P. mangiferae, P. mangifera-indicae, P. musaechinensis, P. musarum, P. paracapitalensis, P. parthenocissi, P. partricuspidatae, P. philoprina, P. rhizophorae, P. schimae, P. schimicola, P. styracicola and P. vitis-rotundifoliae. In this study, we focus our analyses on the P. capitalensis species complex and report two new species and one new Chinese record.

Multilocus phylogeny, as well as morphological characters observed in culture, described and illustrated herein eight isolates of Phyllosticta species from three host genera, which contributed knowledge to the diversity of Phyllosticta species in Hainan, China. Two new species are proposed: P. oblongifoliae sp. nov. and P. pterospermi sp. nov. This is the first time we report Phyllosticta species from Pterospermum heterophyllum (Sterculiaceae). In a recent study, Allophoma pterospermicola was reported as pathogenic to Pterospermum (Marin-Felix et al. 2019). In reality, the number of phytopathogenic fungi from the Pterospermum host is inherently small. The known species Phyllosticta capitalensis (synonym Guignardia mangiferae; Baayen et al. 2002) was described multiple times from Stanhopea graveolens (Orchidaceae) in Brazil (Glienke et al. 2011). In this study, we describe and illustrate Phyllosticta capitalensis again. Each of these species show typical morphological characteristics of Phyllosticta, i.e. conidia with mucilaginous sheaths and an apical appendage (van der Aa 1973).

Phyllosticta capitalensis is a cosmopolitan endophytic species reported in more than 300 host records in Fungal Databases (https://nt.ars-grin.gov/fungaldatabases/index.cfm) (Okane et al. 2001, 2003; Baayen et al. 2002; Glienke et al. 2011; Wikee et al. 2013b; Wu et al. 2014; Zhang et al. 2015; Tran et al. 2019; Hattori et al. 2020). As a weak pathogen, P. capitalensis causes leaf spots on tea (Camellia sinensis), oil palm (Elaeis guineensis), Ricinus communis and black spot disease on Psidium guajava (Cheng et al. 2019; Nasehi et al. 2019; Liao et al. 2020; Tang et al. 2020).

Acknowledgements

This work was jointly supported by the National Natural Science Foundation of China (nos. 31900014, U2002203, 31750001)

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Supplementary material

Supplementary material 1 

The combined ITS, LSU, tef1, ACT and GAPDH sequences

Zhaoxue Zhang, Xiaoyong Liu, Xiuguo Zhang, Zhe Meng

Data type: Phylogenetic.

Explanation note: The combined ITS, LSU, tef1, ACT and GAPDH sequences.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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