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Research Article
Multi-gene phylogeny and morphology of two new Phyllosticta (Phyllostictaceae, Botryosphaeriales) species from China
expand article infoCheng-Bin Wang‡, Jing Yang§, Yong Li‡, Han Xue‡, Chun-Gen Piao‡, Ning Jiang‡
‡ Key Laboratory of Biodiversity Conservation of National Forestry and Grassland Administration, Ecology and Nature Conservation Institute, Chinese Academy of Forestry, Beijing, China
§ Natural Resources and Planning Bureau of Rizhao City, Rizhao, China

Corresponding author: Ning Jiang ( n.jiang@caf.ac.cn )

Academic editor: Chitrabhanu Sharma Bhunjun
© 2023 Cheng-Bin Wang, Jing Yang, Yong Li, Han Xue, Chun-Gen Piao, Ning Jiang.
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: Wang C-B, Yang J, Li Y, Xue H, Piao C-G, Jiang N (2023) Multi-gene phylogeny and morphology of two new Phyllosticta (Phyllostictaceae, Botryosphaeriales) species from China. MycoKeys 95: 189-207. https://doi.org/10.3897/mycokeys.95.100414
Open Access

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.

Keywords

Ascomycota, morphology, new species, phylogeny, plant disease, taxonomy

Introduction

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 Garcinia oblongifolia, P. pterospermi was isolated from leaf spots of Pterospermum heterophyllum, 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 Quercus aliena in Anhui Province and Viburnum odoratissimum in Guangdong Province, respectively. This paper describes these species based on molecular evidence and morphological characteristics.

Materials and methods

Isolation and morphological observations

Samples of Quercus aliena and Viburnum odoratissimum 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.

DNA extraction, PCR amplification, and sequencing

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).

Phylogenetic analyses

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.

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

Species Strain no.* Host Location GenBank no.
ITS LSU tef1 act gapdh
Phyllosticta capitalensis species complex
P. acaciigena CPC 28295 T Acacia suaveolens Australia KY173433 KY173523 NA KY173570 NA
P. aloeicola CPC 21020 T Aloe ferox South Africa KF154280 KF206214 KF289193 KF289311 KF289124
CPC 21021 Aloe ferox South Africa KF154281 KF206213 KF289194 KF289312 KF289125
P. ardisiicola NBRC 102261 T Ardisia crenata Japan AB454274 NA NA AB704216 NA
P. aristolochiicola BRIP 53316 T Aristolochia acuminata Australia JX486129 NA NA NA NA
P. azevinhi MUCC0088 Ilex pedunculosa Japan AB454302 NA NA AB704226 NA
P. beaumarisii CBS 535.87 Muehlenbekia adpressa Australia NR_145235 NG_058040 KF766429 KF306232 KF289074
P. brazilianiae LGMF 330 T Mangifera indica Brazil JF343572 KF206217 JF343593 JF343656 JF343758
LGMF 334 Mangifera indica Brazil JF343566 KF206215 JF343587 JF343650 JF343752
P. capitalensis CBS 114751 Vaccinium sp. New Zealand EU167584 EU167584 FJ538407 FJ538465 KF289088
CBS 128856 T Stanhopea sp. Brazil JF261465 KF206304 JF261507 JF343647 JF343776
P. carochlae CGMCC 3.17317 T Caryota ochlandra China KJ847422 NA KF289178 KF289273 KF289092
P. cavendishii BRIP 57384 Musa cv. Lady finger Australia KC117644 KU697330 KF009695 KF014059 KU716085
BRIP 57383 Musa cv. Lady finger Australia KC117643 KU697329 KF009694 KF014058 KU716084
P. cordylinophila MFLUCC 10-0166 T Cordyline fruticosa Thailand KF170287 KF206242 KF289172 KF289295 KF289076
MFLUCC 12-0014 Cordyline fruticosa Thailand KF170288 KF206228 KF289171 KF289301 KF289075
P. doitungensis MFLU 21-0175 T Dasymaschalon obtusipetalum Thailand OK661033 OK661034 OL345581 NA NA
P. eugeniae CBS 445.82 T Eugenia aromatica Indonesia AY042926 KF206288 KF289208 KF289246 KF289139
P. fallopiae MUCC0113 T Fallopia japonica Japan AB454307 NA NA AB704228 NA
P. guangdongensis CFCC 58144 T Viburnum odoratissimum China OQ202160 OQ202170 OQ267758 OQ267764 OQ267770
CFCC 58766 Viburnum odoratissimum China OQ202161 OQ202171 OQ267759 OQ267765 OQ267771
CFCC 58772 Viburnum odoratissimum China OQ202162 OQ202172 OQ267760 OQ267766 OQ267772
P. ilicis-aquifolii CGMCC 3.14358 T Ilex aquifolium China JN692538 NA JN692526 JN692514 NA
CGMCC 3.14359 Ilex aquifolium China JN692539 NA JN692527 JN692515 NA
P. maculata CPC 18347 T Musa cv. Golygoly pot-pot Australia JQ743570 NA KF009700 KF014016 NA
BRIP 46622 Musa cv. Golygoly pot-pot Australia JQ743567 NA KF009692 KF014013 NA
P. mangiferae IMI 260576 T Mangifera indica India JF261459 KF206222 JF261501 JF343641 JF343748
P. mangifera-indicae MFLUCC 10-0029 T Mangifera indica Thailand KF170305 KF206240 KF289190 KF289296 KF289121
P. musaechinensis GZAAS 6.1247 Musa sp. China KF955294 NA KM816639 KM816627 KM816633
GZAAS 6.1384 Musa sp. China KF955295 NA KM816640 KM816628 KM816634
P. musarum BRIP 57803 Musa sp. Malaysia JX997138 NA KF009737 KF014055 NA
BRIP 58028 Musa sp. Australia KC988377 NA KF009738 KF014054 NA
P. oblongifoliae SAUCC210055 Garcinia oblongifolia China OM248442 OM232085 OM273890 OM273894 OM273898
SAUCC210052 T Garcinia oblongifolia China OM248445 OM232088 OM273893 OM273897 OM273901
P. paracapitalensis CPC 26517 T Citrus floridana Italy KY855622 KY855796 KY855951 KY855677 KY855735
CPC 26518 Citrus floridana Italy KY855623 KY855797 KY855952 KY855678 KY855736
P. parthenocissi CBS 111645 T Parthenocissus quinquefolia USA EU683672 NA JN692530 JN692518 NA
P. partricuspidatae NBRC 9466 T Parthenocissus tricuspidata Japan KJ847424 NA KJ847446 KJ847432 KJ847440
NBRC 9757 Parthenocissus tricuspidata Japan KJ847425 NA KJ847447 KJ847433 KJ847441
P. philoprina CBS 587.69 Ilex aquifolium Spain KF154278 KF206297 KF289206 KF289250 KF289137
P. phoenicis CBS 147091 Phoenix reclinata South Africa MW883442 MW883833 MW890098 MW890031 MW890050
P. pterospermi SAUCC210104 T Pterospermum heterophyllum China OM249954 OM249956 OM273902 OM273904 OM273906
SAUCC210106 Pterospermum heterophyllum China OM249955 OM249957 OM273903 OM273905 OM273907
P. rhizophorae NCYUCC 19-0352 T Rhizophora stylosa China MT360030 MT360039 NA MT363248 MT363250
NCYUCC 19-0358 Rhizophora stylosa China MT360031 MT360040 NA MT363249 MT363251
P. schimae CGMCC 3.14354 T Schima superba China JN692534 NA JN692522 JN692510 JN692506
P. schimicola CGMCC 3.17319 T Schima superba China KJ847426 NA KJ847448 KJ847434 KJ854895
CGMCC 3.17320 Schima superba China KJ847427 NA KJ847449 KJ847435 KJ854896
P. styracicola CGMCC3.14985 T Styrax grandiflorus China JX025040 NA JX025045 JX025035 JX025030
CGMCC3.14989 Styrax grandiflorus China JX025041 NA JX025046 JX025036 JX025031
P. vitis-rotundifoliae CGMCC 3.17322 T Vitis rotundifolia USA KJ847428 NA KJ847450 KJ847436 KJ847442
CGMCC 3.17321 Vitis rotundifolia USA KJ847429 NA KJ847451 KJ847437 KJ847443
Phyllosticta concentrica species complex
P. anhuiensis CFCC 54840T Quercus aliena China OQ202157 OQ202167 OQ267761 OQ267767 OQ267773
CFCC 55887 Quercus aliena China OQ202158 OQ202168 OQ267762 OQ267768 OQ267774
CFCC 58849 Quercus aliena China OQ202159 OQ202169 OQ267763 OQ267769 OQ267775
P. aspidistricola NBRC 102244 T Aspidistra elatior Japan AB454314 NA NA AB704204 NA
P. aucubae-japonicae MAFF 236703 T Aucuba japonica Japan KR233300 NA KR233310 KR233305 NA
P. bifrenariae CBS 128855 T Bifrenaria harrisoniae Brazil JF343565 KF206209 JF343586 JF343649 JF343744
CPC 17467 Bifrenaria harrisoniae Brazil KF170299 KF206260 KF289207 KF289283 KF289138
P. catimbauensis URM 7672 T Mandevilla catimbauensis Brazil MF466160 MF466163 MF466155 MF466157 NA
URM 7674 Mandevilla catimbauensis Brazil MF466161 MF466164 MF466153 MF466158 NA
P. citriasiana CBS 120486 T Citrus maxima Thailand FJ538360 KF206314 FJ538418 FJ538476 JF343686
P. citriasiana CBS 120487 Citrus maxima China FJ538361 KF206313 FJ538419 FJ538477 JF343687
P. citribraziliensis CBS 100098 T Citrus limon Brazil FJ538352 KF206221 FJ538410 FJ538468 JF343691
P. citricarpa CBS 127454 T Citrus limon Australia JF343583 KF206306 JF343604 JF343667 JF343771
P. citrichinensis ZJUCC 200956 T Citrus reticulata China JN791620 NA JN791459 JN791533 NA
ZJUCC 2010150 Citrus maxima China JN791662 NA JN791514 JN791582 NA
P. citrimaxima MFLUCC 10-0137 T Citrus maxima Thailand KF170304 KF206229 KF289222 KF289300 KF289157
P. concentrica CBS 937.70 Hedera helix Italy FJ538350 KF206291 FJ538408 KF289257 JF411745
CPC 18842 T Hedera sp. Italy KF170310 KF206256 KF289228 KF289288 KF289163
P. cussonia CPC 14873 T Cussonia sp. South Africa JF343578 KF206279 JF343599 JF343662 JF343764
CPC 14875 Cussonia sp. South Africa JF343579 KF206278 JF343600 JF343663 JF343765
P. elongata CBS 126.22 T Oxycoccus macrocarpos USA FJ538353 NA FJ538411 FJ538469 KF289164
P. ericarum CBS 132534 T Erica gracilis South Africa KF206170 KF206253 KF289227 KF289291 KF289162
P. gardeniicola MUCC0117 Gardenia jasminoides Japan AB454310 NA NA AB704230 NA
MUCC0089 Gardenia jasminoides Japan AB454303 NA NA NA NA
P. gwangjuensis CNUFC NJ1-12 T Torreya nucifera Korea OK285195 NA OM038511 OM001471 NA
CNUFC NJ1-12-1 Torreya nucifera Korea OK285196 NA OM038512 OM001472 NA
P. hostae CGMCC 3.14355 T Hosta plantaginea China JN692535 NA JN692523 JN692511 JN692503
CGMCC 3.14356 Hosta plantaginea China JN692536 NA JN692524 JN692512 JN692504
P. hymenocallidicola CBS 131309 T Hymenocallis littoralis Australia JQ044423 JQ044443 KF289211 KF289242 KF289142
CPC 19331 Hymenocallis littoralis Australia KF170303 KF206254 KF289212 KF289290 KF289143
P. hypoglossi CBS 101.72 Ruscus aculeatus Italy FJ538365 KF206326 FJ538423 FJ538481 JF343694
CBS 434.92 T Ruscus aculeatus Italy FJ538367 KF206299 FJ538425 FJ538483 JF343695
P. iridigena CBS 143410 T Iris sp. South Africa MG934459 NA MG934502 MG934466 NA
P. kerriae MAFF 240047 T Kerria japonica Japan AB454266 NA NA NA NA
P. kobus MUCC0049 Magnolia kobus Japan AB454286 NA NA AB704221 NA
P. ophiopogonis KACC 47754 Ophiopogon japonicus South Korea KP197057 NA NA NA NA
LrLF11 Lycoris radiata China MG543713 NA NA NA NA
P. paracitricarpa CPC 27169 T Citrus limon Greece KY855635 KY855809 KY855964 KY855690 KY855748
ZJUCC 200933 Citrus sinensis China JN791626 KY855813 JN791468 JN791544 KY855752
P. pilospora MUCC 2912a T Chamaecyparis pisifera var. plumose Japan LC542597 LC543423 LC543445 LC543465 NA
P. speewahensis BRIP 58044 T Orchids Australia KF017269 NA KF017268 NA NA
P. spinarum CBS 292.90 Chamaecyparis pisifera France JF343585 KF206301 JF343606 JF343669 JF343773
P. westeae BRIP 72390c T Clerodendrum inerme Australia OP599631 NA OP627090 NA NA
Phyllosticta cruenta species complex
P. abieticola CBS 112067 Abies concolor Canada KF170306 EU754193 NA KF289238 NA
P. cornicola CBS 111639 Cornus florida USA KF170307 NA NA KF289234 NA
P. cruenta CBS 858.71 Polygonatum odoratum Czech Republic MG934458 NA MG934501 MG934465 MG934474
P. cruenta MUCC0206 Polygonatum odoratum var. pluriflorum Japan AB454331 NA NA AB704237 NA
P. cryptomeriae KACC 48643 Juniperus chinensis var. sargentii Not given MK396559 NA NA NA NA
MUCC0028 Cryptomeria japonica Japan AB454271 NA NA AB704213 NA
P. foliorum CBS 447.68 T Taxus baccata Netherlands KF170309 KF206287 KF289201 KF289247 KF289132
P. gaultheriae CBS 447.70 T Gaultheria humifusa USA JN692543 KF206298 JN692531 KF289248 JN692508
P. hakeicola CBS 143492 T Hakea sp. Australia MH107907 MH107953 MH108025 MH107984 MH107999
P. hamamelidis MUCC149 Hamamelis japonica Japan KF170289 NA NA KF289309 NA
P. hubeiensis CGMCC 3.14986 T Viburnum odoratissimim China JX025037 NA JX025042 JX025032 JX025027
CGMCC 3.14987 Viburnum odoratissimim China JX025038 NA JX025043 JX025033 JX025028
P. illicii 24-1-1 T Illicium verum China MF198235 MF198240 MF198237 MF198243 NA
16-16-1 Illicium verum China MF198234 MF198239 MF198236 MF198242 NA
P. leucothoicola MUCC553 T Leucothoe catesbaei Japan AB454370 AB454370 NA KF289310 NA
P. ligustricola MUCC0024 T Ligustrum obtusifolium Japan AB454269 NA NA AB704212 NA
P. minima CBS 585.84 T Acer rubrum USA KF206176 KF206286 KF289204 KF289249 KF289135
P. neopyrolae CPC 21879 T Pyrola asarifolia Japan AB454318 AB454318 NA AB704233 NA
P. pachysandricola MUCC124 T Pachysandra terminalis Japan AB454317 AB454317 NA AB704232 NA
P. paxistimae CBS 112527 T Paxistima mysinites USA KF206172 KF206320 KF289209 KF289239 KF289140
P. podocarpicola CBS 728.79 T Podocarpus maki USA KF206173 KF206295 KF289203 KF289252 KF289134
P. pyrolae IFO 32652 Erica carnea Not given AB041242 NA NA NA NA
P. rubella CBS 111635 T Acer rubrum USA KF206171 EU754194 KF289198 KF289233 KF289129
P. sphaeropsoidea CBS 756.70 Aesculus hippocastanum Germany AY042934 KF206294 KF289202 KF289253 KF289133
P. telopeae CBS 777.97 T Telopea speciosissima Tasmania KF206205 KF206285 KF289210 KF289255 KF289141
P. yuccae CBS 112065 Yucca elephantipes USA KF206175 NA NA KF289237 NA
CBS 117136 Yucca elephantipes New Zealand JN692541 KF766385 JN692529 JN692517 JN692507
Phyllosticta owaniana species complex
P. austroafricana CBS 144593 T leaf spots of unidentified deciduous tree South Africa MK442613 MK442549 MK442704 MK442640 NA
P. carissicola CPC 25665 T Carissa macrocarpa South Africa KT950849 KT950863 KT950879 KT950872 KT950876
P. hagahagaensis CBS 144592 T Carissa bispinosa South Africa MK442614 MK442550 MK442705 MK442641 MK442657
P. owaniana CBS 776.97 T Brabejum stellatifolium South Africa FJ538368 KF206293 FJ538426 KF289254 JF343767
CPC 14901 Brabejum stellatifolium South Africa JF261462 KF206303 JF261504 KF289243 JF343766
P. podocarpi CBS 111646 Podocarpus falcatus South Africa AF312013 KF206323 KC357671 KC357670 KF289169
CBS 111647 Podocarpus lanceolata South Africa KF154276 KF206322 KF289232 KF289235 KF289168
P. pseudotsugae CBS 111649 Pseudotsuga menziesii USA KF154277 KF206321 KF289231 KF289236 KF289167
Phyllosticta rhodorae species complex
P. mimusopisicola CBS 138899 T Mimusops zeyheri South Africa KP004447 MH878626 NA NA NA
P. rhodorae CBS 901.69 Rhododendron sp. Netherlands KF206174 KF206292 KF289230 KF289256 KF289166
Phyllosticta vaccinii species complex
P. vaccinii ATCC 46255 T Vaccinium macrocarpon China KC193585 NA KC193582 KC193580 KC193583
LC 2795 Vitis macrocarpon USA KR233323 NA NA NA NA
P. vacciniicola CPC 18590 T Vaccinium macrocarpum USA KF170312 KF206257 KF289229 KF289287 KF289165
Outgroup
B. obtusa CMW 8232 T Conifers South Africa AY972105 NA DQ280419 AY972111 NA
B. stevensii CBS 112553 T culture from isotype of Diplodia mutila Not given AY259093 AY928049 AY573219 NA NA

Notes: *T = ex-type strains, NA = not available.

Results

Phylogenetic analyses

In this study, phylogenetic analyses contained sequences from 131 fungal samples representing 93 taxa, including two outgroup taxa, viz., Botryosphaeria obtusa (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).

Figure 1. 

Phylogram of Phyllosticta genus resulting from a maximum likelihood analysis based on a combined matrix of ITS, LSU, tef1, act and gapdh loci. The tree is artificially rooted to B. obtusa (CMW 8232) and B. stevensii (CBS 112553). ML bootstrap values (left, ML-BS ≥ 50%) and Bayesian posterior probabilities (right, BYPP ≥ 0.9) are given at the nodes. Ex-type strains are indicated in bold. Strains from the present study are marked in blue.

Taxonomy

Phyllosticta anhuiensis Ning Jiang & C.B. Wang, sp. nov.

MycoBank No: 847160
Fig. 2

Etymology

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

Description

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.

Figure 2. 

Morphology of Phyllosticta anhuiensis (CFCC 54840) A diseased leaf of Quercus aliena B conidiomata C conidiogenous cells D, E conidia F–H colonies on PDA, MEA and SNA after two weeks at 25 °C. Scale bars: 500 μm (B); 10 μm (C–E).

Culture characters

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.

Specimens examined

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

Notes

In the phylogeny analyses, P. anhuiensis groups sister to P. kerriae (MAFF 240047). P. kerriae was associated with Kerria japonica 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.

Phyllosticta guangdongensis Ning Jiang & C.B. Wang, sp. nov.

MycoBank No: 847161
Fig. 3

Etymology

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

Description

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.

Figure 3. 

Morphology of Phyllosticta guangdongensis (CFCC 58144) A diseased leaf of Viburnum odoratissimum B conidiomata C conidiogenous cells D, E conidia F–H colonies on PDA, MEA and SNA after two weeks at 25 °C. Scale bars: 500 μm (B); 10 μm (C–E).

Culture characters

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.

Specimens examined

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

Notes

Phylogeny indicates that P. anhuiensis groups sister to P. mangiferae (IMA 260576). P. mangiferae was associated with Mangifera indica 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.

Discussion

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).

Phyllosticta anhuiensis 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.

Acknowledgements

This research was funded by the National Microbial Resource Center of the Ministry of Science and Technology of the People’s Republic of China (NMRC-2022-7).

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