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. 2023 Mar 1;95:209–228. doi: 10.3897/mycokeys.95.98969

Two new species of Diaporthe (Diaporthaceae, Diaporthales) in China

Ya-Quan Zhu 1, Chun-Yan Ma 2, Han Xue 1, Chun-Gen Piao 1, Yong Li 1, Ning Jiang 1,✉
PMCID: PMC10210287  PMID: 37251990

Abstract

Species of Diaporthe have been reported as plant endophytes, pathogens and saprobes on a wide range of plant hosts. Strains of Diaporthe were isolated from leaf spots of Smilaxglabra and dead culms of Xanthiumstrumarium in China, and identified based on morphology and molecular phylogenetic analyses of combined internal transcribed spacer region (ITS), calmodulin (cal), histone H3 (his3), translation elongation factor 1-alpha (tef1) and β-tubulin (tub2) loci. As a result, two new species named Diaportherizhaoensis and D.smilacicola are identified, described and illustrated in the present study.

Keywords: Leaf spots, morphology, multi-gene phylogeny, taxonomy

Introduction

Diaporthe (Diaporthaceae,Diaporthales) is a species-rich genus with its asexual morph previously known as Phomopsis (Rossman et al. 2007; Udayanga et al. 2011, 2012a, 2014a, 2015; Dissanayake et al. 2017; Guarnaccia et al. 2018). The genus Diaporthe was established by Nitschke in 1870 and predates its sexual morph established in 1905, thus Diaporthe is recommended to be used for this genus following “one fungus one name” nomenclature (Nitschke 1870; Rossman et al. 2015).

The sexual morph of Diaporthe is characterized by immersed ascomata and an erumpent pseudostroma with single or multiple tapering perithecial necks. Asci are unitunicate, sessile and clavate to cylindrical. Ascospores are elliptical to fusiform, septate or aseptate, hyaline, biseriate to uniseriate in the ascus and sometimes have appendages (Udayanga et al. 2011; Senanayake et al. 2017, 2018). The asexual morph is characterized by black or dark brown conidiomata, with cylindrical phialides producing three types of aseptate and hyaline conidia (Type I: α-conidia, hyaline, fusiform, straight, guttulate or eguttulate, aseptate, smooth-walled; type II: β-conidia, hyaline, filiform, straight or hamate, aseptate, smooth-walled, eguttulate; type III: γ-conidia, rarely produced, hyaline, multiguttulate, fusiform to subcylindrical with an acute or rounded apex, while the base is sometimes truncate) (Udayanga et al. 2011; Gomes et al. 2013).

Species of Diaporthe are widely distributed, and infect a broad plant host range, e.g., agricultural crops, forest trees, vegetables, and fruits (Farr et al. 2002a, b; Crous 2005; Rossman et al. 2007; Udayanga et al. 2011, 2012a, b, 2014a, b, 2015; Gomes et al. 2013; Du et al. 2016; Dissanayake et al. 2017; Guarnaccia and Crous 2017; Fan et al. 2018). As plant pathogens, Diaporthe spp. cause severe diseases, e.g., blights, cankers, decay, dieback, leaf spots and wilt of many economically important plants in genera Castanea, Citrus, Helianthus, Macadamia, Rosa, Vaccinium and Vitis, resulting in major losses (Thompson et al. 2011; Huang et al. 2015; Guarnaccia et al. 2018, 2020; Hilário et al. 2020; Wrona et al. 2020; Caio et al. 2021; Jiang et al. 2021a).

The genus Diaporthe includes over 1000 epithets, mostly based on morphological characteristics and host associations (van der Aa et al. 1990; Santos et al. 2010; Guarnaccia et al. 2018). However, recent studies have shown that many species of Diaporthe are not host-specific, i.e., one species may infect more than one host species (Vrandecic et al. 2011; Bai et al. 2015; Zhang et al. 2018). And many Diaporthe species that are morphologically similar have proven to be genetically distinct (van Rensburg et al. 2006; Udayanga et al. 2011; Jiang et al. 2021b). Thus, polyphasic taxonomy is essential to identify and comprehensively characterize Diaporthe.

In the present study, we have analyzed five-locus dataset of combined nuclear ribosomal internal transcribed spacer (ITS), calmodulin (cal), histone (his3), translation elongation factor 1-alpha (tef1) and beta-tubulin (tub2). To aid the identification of two new species, we followed Norphanphoun et al. (2022) for the taxonomic treatments of Diaporthe. Norphanphoun et al. (2022) clustered Diaporthe into 13 workable species complexes namely D.arecae, D.biconispora, D.carpini, D.decedens, D.eres, D.oncostoma, D.pustulata, D.rudis, D.scobina, D.sojae, D.toxica, D.varians and D.vawdreyi species complexes. In addition, nine species were retained as singletons, viz., D.acerina, D.acutispora, D.crataegi, D.multiguttulata, D.ocoteae, D.perjuncta, D.pseudoalnea, D.spartinicola and D.undulata based on multilocus phylogeny.

In previous studies, Smilaxglabra and Xanthiumstrumarium have been reported as hosts of Diaporthe (Vrandecic et al. 2007, 2010; Gao et al. 2013; Thompson et al. 2018). D.eres (= D.mahothocarpi) and D.lithocarpi were identified as the cause agents of leaf spot disease based on morphology and phylogenetics on S.glabra in China (Gao et al. 2013). D.helianthi and D.longicolla, pathogens of X.strumarium, have been collected from blighted stems and branches in Croatia (Vrandecic et al. 2007, 2010). D.pseudolongicolla (= D.novem) has been reported as a branch dieback agent in X.strumarium in Australia (Thompson et al. 2018).

In this study, we introduce two new species namely Diaportherizhaoensis and D.smilacicola, collected from diseased plant tissues in China. We further provide descriptions, illustrations, and DNA sequence-based phylogeny to verify identification and placement.

Materials and methods

Isolation and morphological characterization

During 2021 and 2022, investigations were conducted to inspect for the presence of Diaporthe species associated with plant diseases in China. Leaves of Smilaxglabra and culms of Xanthiumstrumarium showing typical symptoms of Diaporthe were collected. Infected tissues were cut into 0.5 × 0.5 cm pieces using a double-edge blade, and surface sterilized as follows. These sections underwent initial immersion for 2 min in 0.5% sodium hypochlorite, followed by 1 min in sterile distilled water, 2 min in 75% ethanol, and, finally, 1 min in sterile distilled water. The disinfected fragments were then plated onto the surface of potato dextrose agar (PDA; 200 g potatoes, 20 g dextrose, 20 g agar per L) and malt extract agar (MEA; 30 g malt extract, 5 g mycological peptone, 15 g agar per L), and incubated at 25 °C to obtain the pure culture.

Species identification was based on morphological features of the new species produced on infected plant tissues and PDA plates. Conidiomata were sectioned by hand, using a double-edged blade and structures were observed under a dissecting microscope. Over 20 fruiting bodies were sectioned, and 50 conidia were selected randomly for measurement using Axio Imager 2 microscope (Zeiss, Oberkochen, Germany). Isolate characteristics incubated on PDA at 25 °C were observed and recorded at 7 days, including colony colour, texture and the arrangement of the conidiomata. The cultures were deposited in the China Forestry Culture Collection Center (CFCC; http://www.cfcc-caf.org.cn/), and the specimens in the herbarium of the Chinese Academy of Forestry (CAF; http://museum.caf.ac.cn/).

DNA extraction, amplification and sequencing

Genomic DNA was extracted from the fresh mycelium harvested from PDA plates after 7 days using a cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle 1990). For initial species confirmation, the internal transcribed spacer (ITS) region was sequenced for all isolates. The BLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to compare the resulting sequences with those in GenBank. After confirmation of Diaporthe species, four additional partial loci, including calmodulin (cal), histone H3 (his3), partial translation elongation factor 1-alpha (tef1) and part of the beta-tubulin gene region (tub2) genes were amplified. The primer pairs and amplification conditions for each of the above-mentioned gene regions are provided in Table 1. A PCR reaction was conducted in a 20 µL reaction volume, and the components were as follows: 1 µL DNA template (20 ng/μl), 1 µL forward 10 µM primer, 1 µL reverse 10 µM primer, 10 µL T5 Super PCR Mix (containing Taq polymerase, dNTP and Mg2+, Beijing TisingKe Biotech Co., Ltd., Beijing, China), and 7 µL sterile water. Amplifications were performed using a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA). Strands were sequenced in both directions using PCR primers. All amplified PCR products were estimated visually 1.4% agarose gels stained with ethidium bromide and then PCR positive products were sent to Sangon Biotech (Shanghai) Co., Ltd., (Beijing, China) for sequencing.

Table 1.

Loci used in this study with PCR primers and process.

Loci PCR primers PCR: thermal cycles: (Annealing temp. in bold) Reference
ITS ITS1/ITS4 (95 °C: 30 s, 48 °C: 30 s, 72 °C: 1 min) × 35 cycles White et al. 1990
cal CAL228F/CAL737R (95 °C: 15 s, 54 °C: 20 s, 72 °C: 1 min) × 35 cycles Carbone and Kohn 1999
his3 CYLH3F/H3-1b (95 °C: 30 s, 57 °C: 30 s, 72 °C: 1 min) × 35 cycles Crous et al. 2004
Glass and Donaldson 1995
tef1 EF1-728F/EF1-986R (95 °C: 15 s, 54 °C: 20 s, 72 °C: 1 min) × 35 cycles Carbone and Kohn 1999
tub2 T1(Bt2a)/Bt2b (95 °C: 30 s, 55 °C: 30 s, 72 °C: 1 min) × 35 cycles Glass and Donaldson 1995
O’Donnell and Cigelnik 1997
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Phylogenetic analyses

Sequences were edited and condensed with SeqMan v.7.1.0. The sequences generated in this study were supplemented with additional sequences obtained from GenBank (Table 2) based on blast searches and recent publications of the genus Diaporthe. The sequences were aligned with the MAFFT v.7 after which the alignments were manually corrected using MEGA v. 7.0. (Katoh and Toh 2010; Kumar et al. 2016). Phylogenetic analyses including Maximum Likelihood (ML) and Bayesian Inference (BI) methods were conducted for the single gene sequence data sets of the ITS, cal, his3, tef1 and tub2, and the combined data set of all five gene regions. ML analyses were conducted using RAxML-HPC BlackBox 8.2.10 on the CIPRES Science Gateway portal (https://www.phylo.org) (Miller et al. 2012), employing a GTRGAMMA substitution model with 1000 bootstrap replicates (Stamatakis 2014). BI analyses were conducted using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v.3.0 (Ronquist and Huelsenbeck 2003). Two Markov chain Monte Carlo (MCMC) chains were run from a random starting tree for 1,000,000 generations, resulting in a total of 10,000 trees. The first 25% of trees sampled were discarded as burn-in and the remaining trees were used to calculate the posterior probabilities. Branches with significant Bayesian Posterior Probabilities (BPP > 0.9) were estimated in the remaining 7,500 trees. Phylogenetic trees were viewed with FigTree v. 1.4 and processed by Adobe Illustrator CS5. The nucleotide sequence data of the new taxa were deposited in GenBank, and the GenBank accession numbers of all accessions included in the phylogenetic analyses are listed in Table 2.

Table 2.

Strains and GenBank accession numbers used in this study.

Species Location Host Strain GenBank Accession Number
ITS tef1 tub2 cal his3
Diaportheabsenteum China Camelliasinensis LC3429* KP267897 KP267971 KP293477 NA KP293547
D.absenteum China Camelliasinensis LC3564 KP267912 KP267986 KP293492 NA KP293559
D.acaciarum Tanzania Acaciatortilis CBS 138862* KP004460 NA KP004509 NA KP004504
D.acericola Italy Acernegundo MFLUCC 17-0956* KY964224 KY964180 KY964074 KY964137 NA
D.aceris Japan Acer sp. LC8112 KY491547 KY491557 KY491567 KY491575 NA
D.actinidiae New Zealand Actinidiadeliciosa ICMP 13683* KC145886 KC145941 NA NA NA
D.acuta China Pyruspyrifolia CGMCC 3.19600* MK626957 MK654802 MK691225 MK691124 MK726161
D.alangii China Alangiumkurzii CFCC 52556* MH121491 MH121533 MH121573 MH121415 MH121451
D.alangii China Alangiumkurzii CFCC 52557 MH121492 MH121534 MH121574 MH121416 MH121452
D.alnea Netherlands Alnus sp. CBS 146.46 KC343008 KC343734 KC343976 KC343250 KC343492
D.amaranthophila Japan Amaranthustricolor MAFF 246900 LC459575 LC459577 LC459579 LC459583 LC459581
D.ambigua South Africa Pyruscommunis CBS 114015* KC343010 KC343736 KC343978 KC343252 KC343494
D.angelicae Austria Heracleumsphondylium CBS 111592* KC343027 KC343753 KC343995 KC343269 KC343511
D.anhuiensis China Cunninghamialanceolata CNUCC 201901* MN219718 MN224668 MN227008 MN224549 MN224556
D.arctii Austria Arctiumlappa CBS 139280* KJ590736 KJ590776 KJ610891 KJ612133 KJ659218
D.arecae India Arecacatechu CBS 161.64* KC343032 KC343758 KC344000 KC343274 KC343516
D.arengae Hong Kong Arengaengleri CBS 114979* KC343034 KC343760 KC344002 KC343276 KC343518
D.arezzoensis Italy Cytisus sp. MFLUCC 15-0127 MT185503 NA NA NA NA
D.aseana Thailand Unidentified dead leaf MFLUCC 12-0299a* KT459414 KT459448 KT459432 KT459464 NA
D.australiana Australia Macadamia CBS 146457 MN708222 MN696522 MN696530 NA NA
D.batatas USA Ipomoeabatatas CBS 122.21* KC343040 KC343766 KC344008 KC343282 KC343524
D.beilharziae Australia Indigoferaaustralis BRIP 54792* JX862529 JX862535 KF170921 NA NA
D.biconispora China Citrusgrandis ZJUD62 KJ490597 KJ490476 KJ490418 MT227578 KJ490539
D.biguttulata China Citruslimon ZJUD47* KJ490582 KJ490461 KJ490403 NA KJ490524
D.brasiliensis Brazil Aspidosperma sp. CBS 133183* KC343042 KC343768 KC344010 KC343284 KC343526
D.caatingaensis Brazil Tacingainamoena CBS 141542* KY085927 KY115603 KY115600 NA KY115605
D.camelliae-oleiferae China Camelliaoleifera HNZZ027* MZ509555 MZ504707 MZ504718 MZ504685 MZ504696
D.caryae China Caryaillinoensis CFCC 52563* MH121498 MH121540 MH121580 MH121422 MH121458
D.caryae China Caryaillinoensis CFCC 52564 MH121499 MH121541 MH121581 MH121423 MH121459
D.cercidis China Cercischinensis CFCC 52565* MH121500 MH121542 MH121582 MH121424 MH121460
D.cercidis China Cercischinensis CFCC 52566 MH121501 MH121543 MH121583 MH121425 MH121461
D.chiangraiensis Thailand Bauhinia sp. MFLUCC 17-1669* MF190119 MF377598 NA NA NA
D.chrysalidocarpi China Chrysalidocarpuslutescens SAUCC194.35 MT822563 MT855760 MT855876 MT855646 MT855532
D.cichorii Italy Cichoriumintybus MFLUCC 17-1023* KY964220 KY964176 KY964104 KY964133 NA
D.cinmomi China Cinnamomum sp. CFCC 52569* MH121504 MH121546 MH121586 NA MH121464
D.cinmomi China Cinnamomum sp. CFCC 52570 MH121505 MH121547 MH121587 NA MH121465
D.citriasiana China Citrusunshiu CGMCC 3.15224* JQ954645 JQ954663 KC357459 KC357491 KJ490515
D.columnaris USA Vacciniumvitisidaea AR3612* AF439625 NA NA NA NA
D.compacta China Camelliasinensis CGMCC 3.17536* KP267854 KP267928 KP293434 NA KP293508
D.convolvuli Turkey Convolvulusarvensis CBS 124654* KC343054 KC343780 KC344022 KC343296 KC343538
D.cucurbitae Canada Cucumis sp. DAOM 42078* KM453210 KM453211 KP118848 NA KM453212
D.cuppatea South Africa Aspalathuslinearis CBS 117499* KC343057 KC343783 KC344025 KC343299 KC343541
D.cyatheae Taiwan Cyathealepifera YMJ 1364* JX570889 KC465406 KC465403 KC465410 NA
D.discoidispora China Citrusunshiu ZJUD89* KJ490624 KJ490503 KJ490445 NA KJ490566
D.drenthii Australia Macadamia CBS 146453 MN708229 MN696526 MN696537 NA NA
D.durionigena Vietnam Duriozibethinus VTCC 930005 MN453530 MT276157 MT276159 NA NA
D.endocitricola China Citrusmaxima ZHKUCC 20-0012* MT355682 MT409336 MT409290 MT409312 NA
D.endophytica Brazil Schinusterebinthifolius CBS 133811* KC343065 KC343791 KC344033 KC343307 KC343549
D.eucalyptorum China Eucalyptus CBS 132525* MH305525 NA NA NA NA
D.eugeniae Indonesia Eugeniaaromatica CBS 444.82* KC343098 KC343824 KC344066 KC343340 KC343582
D.fraxini-angustifoliae Australia Fraxinusangustifolia BRIP 54781* JX862528 JX862534 KF170920 NA NA
D.fructicola Japan Passifloraedulis × P.edulis f. MAFF 246408* LC342734 LC342735 LC342736 LC342738 LC342737
D.fulvicolor China Pyruspyrifolia CGMCC 3.19601* MK626859 MK654806 MK691236 MK691132 MK726163
D.ganjae USA Cannabissativa CBS 180.91* KC343112 KC343838 KC344080 KC343354 KC343596
D.goulteri Australia Helianthusannuus BRIP 55657a* KJ197290 KJ197252 KJ197270 NA NA
D.guangdongensis China Citrusmaxima ZHKUCC 20-0014* MT355684 MT409338 MT409292 MT409314 NA
D.guangxiensis China Vitisvinifera JZB320094* MK335772 MK523566 MK500168 MK736727 NA
D.gulyae Australia Helianthusannuus BRIP 54025* JF431299 JN645803 KJ197271 NA NA
D.guttulata China Unknown CGMCC 3.20100 MT385950 MT424685 MT424705 MW022470 MW022491
D.helianthi Serbia Helianthusannuus CBS 592.81* KC343115 KC343841 KC344083 KC343357 KC343599
D.heterostemmatis China Heterostemmagrandiflorum SAUCC194.85* MT822613 MT855925 MT855810 MT855692 MT855581
D.hongkongensis China Dichroafebrífuga CBS 115448* KC343119 KC343845 KC344087 KC343361 KC343603
D.hordei Norway Hordeumvulgare CBS 481.92* KC343120 KC343846 KC344088 KC343362 KC343604
D.huangshanensis China Camelliaoleifera CNUCC 201903* MN219729 MN224670 MN227010 NA MN224558
D.hubeiensis China Vitisvinifera JZB320123 MK335809 MK523570 MK500148 MK500235 NA
D.hunanensis China Camelliaoleifera HNZZ023* MZ509550 MZ504702 MZ504713 MZ504680 MZ504691
D.infecunda Brazil Schinus sp. CBS 133812* KC343126 KC343852 KC344094 KC343368 KC343610
D.infertilis Suriname Camelliasinensis CBS 230.52* KC343052 KC343778 KC344020 KC343294 KC343536
D.kochmanii Australia Helianthusannuus BRIP 54033* JF431295 JN645809 NA NA NA
D.kongii Australia Portulacagrandifla BRIP 54031* JF431301 JN645797 KJ197272 NA NA
D.krabiensis Thailand marine based habitats MFLUCC 17-2481* MN047101 MN433215 MN431495 NA NA
D.leucospermi Australia Leucospermum sp. CBS 111980* JN712460 KY435632 KY435673 KY435663 KY435653
D.limonicola Malta Citruslimon CPC 28200* NR_154980 MF418501 MF418582 MF418256 MF418342
D.litchiicola Australia Litchichinensis BRIP 54900* JX862533 JX862539 KF170925 NA NA
D.lithocarpi China Lithocarpusglabra CGMCC 3.15175* KC153104 KC153095 KF576311 KF576235 NA
D.longicolla USA Glycinemax FAU599 KJ590728 KJ590767 KJ610883 KJ612124 KJ659188
D.longispora Canada Ribes sp. CBS 194.36* KC343135 KC343861 KC344103 KC343377 KC343619
D.lusitanicae Portugal Foeniculumvulgare CBS 123212 KC343136 KC343862 KC344104 KC343378 KC343620
D.lusitanicae Portugal Foeniculumvulgare CBS 123213* MH863280 KC343863 KC344105 KC343379 KC343621
D.malorum Portugal Malusdomestica CAA 734* KY435638 KY435627 KY435668 KY435658 KY435648
D.manihotia Rwanda Manihotutilissima CBS 505.76 KC343138 KC343864 KC344106 KC343380 KC343622
D.masirevicii Australia Helianthusannuus BRIP 57892a* KJ197276 KJ197239 KJ197257 NA NA
D.mayteni Brazil Maytenusilicifolia CBS 133185 KC343139 KC343865 KC344107 KC343381 KC343623
D.megalospora Not stated Sambucuscanadensis CBS 143.27* KC343140 KC343866 KC344108 KC343382 KC343624
D.melitensis Malta Citruslimon CPC 27873* MF418424 MF418503 MF418584 MF418258 MF418344
D.melonis USA Cucumismelo CBS 507.78* KC343142 KC343868 KC344110 KC343384 KC343626
D.melonis Indonesia Glycinesoja CBS 435.87 KC343141 KC343867 KC344109 KC343383 KC343625
D.middletonii Australia Rapistrumrugostrum BRIP 54884e* KJ197286 KJ197248 KJ197266 NA NA
D.millettiae China Millettiareticulata GUCC9167* MK398674 MK480609 MK502089 MK502086 NA
D.minusculata China saprobic on decaying wood CGMCC 3.20098* MT385957 MT424692 MT424712 MW022475 MW022499
D.miriciae Australia Helianthusannuus BRIP 54736j* KJ197282 KJ197244 KJ197262 NA NA
D.musigena Australia Musa sp. CBS 129519* KC343143 KC343869 KC344111 KC343385 KC343267
D.myracrodruonis Brazil Astroniumurundeuva URM 7972* MK205289 MK213408 MK205291 MK205290 17
D.nelumbonis Taiwan Nelumbonucifera R. Kirschner 4114* KT821501 NA LC086652 NA NA
D.neoarctii USA Ambrosiatrifi CBS 109490* KC343145 KC343871 KC344113 KC343387 KC343629
D.neoraonikayaporum Thailand Tectonagrandis MFLUCC 14-1136* KU712449 KU749369 KU743988 KU749356 NA
D.oculi Japan Homosapiens HHUF 30565* LC373514 LC373516 LC373518 NA NA
D.osmanthi China Osmanthusfragrans GUCC9165* MK398675 MK480610 MK502091 MK502087 NA
D.ovalispora China Citruslimon CGMCC 3.17256* KJ490628 KJ490507 KJ490449 NA KJ490570
D.oxe Brazil Maytenusilicifolia CBS 133186* KC343164 KC343890 KC344132 KC343406 KC343648
D.pandanicola Thailand Pandanus sp. MFLUCC 17-0607* MG646974 NA MG646930 NA NA
D.paranensis Brazil Maytenusilicifolia CBS 133184* KC343171 KC343897 KC344139 KC343413 KC343655
D.pascoei Australia Perseaamericana BRIP 54847* JX862532 JX862538 KF170924 NA NA
D.passiflorae South America Passifloraedulis CBS 132527* JX069860 KY435633 KY435674 KY435664 KY435654
D.passifloricola Malaysia Passiflorafoetida CBS 141329* KX228292 NA KX228387 NA KX228367
D.perseae Netherlands Perseagratissima CBS 151.73* KC343173 KC343899 KC343141 KC343415 KC343657
D.pescicola China Prunuspersica MFLUCC 16-0105* KU557555 KU557623 KU557579 KU557603 NA
D.phaseolorum USA Phaseolusvulgaris AR4203* KJ590738 KJ590739 KJ610893 KJ612135 KJ659220
D.phoenicicola India Arecacatechu CBS 161.64* MH858400 GQ250349 JX275440 JX197432 NA
D.podocarpi-macrophylli China Podocarpusmacrophyllus CGMCC 3.18281* KX986774 KX999167 KX999207 KX999278 KX999246
D.pseudolongicolla Serbia Glycinemax PL42* JQ697843 JQ697856 NA NA NA
D.pseudolongicolla Croatia Glycinemax CBS 127269 KC343155 KC343881 KC344123 KC343397 KC343639
D.pseudomangiferae Dominican Republic Mangiferaindica CBS 101339* KC343181 KC343907 KC344149 KC343423 KC343665
D.pseudooculi Japan Homosapiens HHUF 30617* NR_161019 LC373517 LC373519 NA NA
D.pseudophoenicicola Spain Phoenixdactylifera CBS 462.69* KC343184 KC343910 KC344152 KC343426 KC343668
D.pseudophoenicicola Iraq Mangiferaindica CBS 176.77 KC343183 KC343909 KC344151 KC343425 KC343667
D.pterocarpicola Thailand Pterocarpusindicus MFLUCC 10-0580a* JQ619887 JX275403 JX275441 JX197433 NA
D.pyracanthae Portugal Pyracanthacoccinea CBS 142384* KY435635 KY435625 KY435666 KY435656 KY435646
D.racemosae South Africa Euclearacemosa CPC 26646* MG600223 MG600225 MG600227 MG600219 MG600221
D.raonikayaporum Brazil Spondiasmombin CBS 133182* KC343188 KC343914 KC344156 KC343430 KC343672
D.rhodomyrti China Rhodomyrtustomentosa CFCC 53101 MK432643 MK578119 MK578046 MK442965 MK442990
D.rhodomyrti China Rhodomyrtustomentosa CFCC 53102 MK432644 MK578120 MK578047 MK442966 MK442991
D.rizhaoensis China Xanthiumstrumarium CFCC 57562* OP955930 OP959767 OP959773 OP959782 OP959785
D.rizhaoensis China Xanthiumstrumarium CFCC 57563 OP955931 OP959766 OP959772 OP959781 OP959784
D.rizhaoensis China Xanthiumstrumarium CFCC 57564 OP955932 OP959765 OP959771 OP959780 OP959783
D.rosae Thailand Rosa sp. MFLUCC 17-2658* MG828894 NA MG843878 MG829273 NA
D.rosiphthora Brazil Rosa sp. COAD 2914* MT311197 MT313693 NA MT313691 NA
D.rossmaniae Portugal Vacciniumcorymbosum CAA762* MK792290 MK828063 MK837914 MK883822 MK871432
D.sackstonii Australia Helianthusannuus BRIP 54669b* KJ197287 KJ197249 KJ197267 NA NA
D.salinicola Thailand Xylocarpus sp. MFLU 18-0553* MN047098 MN077073 NA NA NA
D.sambucusii China Sambucuswilliamsii CFCC 51986* KY852495 KY852507 KY852511 KY852499 KY852503
D.sambucusii China Sambucuswilliamsii CFCC 51987 KY852496 KY852508 KY852512 KY852500 KY852504
D.schimae China Schimasuperba CFCC 53103* MK432640 MK578116 MK578043 MK442962 MK442987
D.schimae China Schimasuperba CFCC 53104 MK432641 MK578117 MK578044 MK442963 MK442988
D.schini Brazil Schinusterebinthifolius CBS 133181* KC343191 KC343917 KC344159 KC343433 KC343675
D.schoeni Italy Schoenusnigricans MFLU 15-1279* KY964226 KY964182 KY964109 KY964139
D.sclerotioides Netherlands Cucumissativus CBS 296.67* KC343193 KC343919 KC344161 KC343435 KC343677
D.searlei Australia Macadamia CBS 146456* MN708231 NA MN696540 NA NA
D.sennae China Sennabicapsularis CFCC 51636* KY203724 KY228885 KY228891 KY228875 NA
D.sennae China Sennabicapsularis CFCC 51637 KY203725 KY228886 KY228892 KY228876 NA
D.serafiniae Australia Helianthusannuus BRIP 55665a* KJ197274 KJ197236 KJ197254 NA NA
D.siamensis Thailand Dasymaschalon sp. MFLUCC 10-0573a* JQ619879 JX275393 JX275429 JX197423 NA
D.sinensis China Amaranthus sp. ZJUP0033-4* MK637451 MK660449 MK660447 NA MK660451
D.smilacicola China Smilaxglabra CFCC 54582* OP955933 OP959770 OP959776 OP959779 OP959788
D.smilacicola China Smilaxglabra CFCC 58764 OP955934 OP959769 OP959775 OP959778 OP959787
D.smilacicola China Smilaxglabra CFCC 58765 OP955935 OP959768 OP959774 OP959776 OP959786
D.sojae USA Glycinemax FAU635* KJ590719 KJ590762 KJ610875 KJ612116 KJ659208
D.spinosa China Pyruspyrifolia CGMCC 3.19602* MK626849 MK654811 MK691234 MK691129 MK726156
D.stewartii Not stated Cosmosbipinnatus CBS 193.36* MH867279 GQ250324 JX275421 JX197415 NA
D.subellipicola China on dead wood KUMCC 17-0153* MG746632 MG746633 MG746634 NA NA
D.subordinaria New Zealand Plantagolanceolata CBS 464.90* KC343214 KC343940 KC344182 KC343456 KC343698
D.taiwanensis Taiwan Ixorachinensis NTUCC 18-105-1* MT241257 MT251199 MT251202 MT251196 NA
D.taoicola China Prunuspersica MFLUCC 16-0117* KU557567 KU557635 KU557591 NA NA
D.tarchonanthi South Africa Tarchonanthuslittoralis CBS 146073* MT223794 NA MT223733 NA MT223759
D.tecomae Brazil Tabebuia sp. CBS 100547* KC343215 KC343941 KC344183 KC343457 KC343699
D.tectonae Thailand Tectonagrandis MFLUCC 12-0777* KU712430 KU749359 KU743977 KU749345 NA
D.tectonendophytica Thailand Tectonagrandis MFLUCC 13-0471* KU712439 KU749367 KU743986 KU749354 NA
D.tectonigena China Tectonagrandis MFLUCC 12-0767* KU712429 KU749371 KU743976 KU749358 NA
D.tectonigena China Camelliasinensis LC6512 KX986782 KX999174 KX999214 KX999284 KX999254
D.terebinthifolii Brazil Schinusterebinthifolius CBS 133180* KC343216 KC343942 KC344184 KC343458 KC343700
D.thunbergiicola Thailand Thunbergialaurifolia MFLUCC 12-0033* KP715097 KP715098 NA NA NA
D.tulliensis Australia Theobromacacao BRIP 62248a* KR936130 KR936133 KR936132 NA NA
D.ueckeri USA Cucumismelo FAU656* KJ590726 KJ590747 KJ610881 KJ612122 KJ659215
D.unshiuensis China Fortunellamargarita CGMCC 3.17566* KJ490584 KJ490463 KJ490405 NA KJ490526
D.unshiuensis China Caryaillinoensis CFCC 52594 MH121529 MH121571 MH121606 MH121447 MH121487
D.unshiuensis China Caryaillinoensis CFCC 52595 MH121530 MH121572 MH121607 MH121448 MH121488
D.vawdreyi Australia Psidiumguajava BRIP 57887a KR936126 KR936129 KR936128 NA NA
D.vexans USA Solanummelongena CBS 127.14 KC343229 KC343955 KC344197 KC343471 KC343713
D.viniferae China Vitisvinifera JZB320071* MK341550 MK500107 MK500112 MK500119 NA
D.vochysiae Brazil Vochysiadivergens LGMF1583* MG976391 MK007526 MK007527 MK007528 MK033323
D.xishuangbanica China Camelliasinensis CGMCC 3.18283* KX986784 KX999176 KX999217 NA NA
D.xishuangbanica China Camelliasinensis LC6707 KX986783 KX999175 KX999216 NA KX999255
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Notes: NA, not applicable. * ex-type strains.

Results

Phylogeny

In the present study, we followed Norphanphoun et al. (2022) for the species complexes treatments of Diaporthe. Firstly, we conducted a genus tree including all species belonging to this genus according to Norphanphoun et al. (2022). After that, the phylogenetic analysis revealed that three isolates (CFCC 57562, CFCC 57563 and CFCC 57564) clustered in a distinct clade in the D.sojae species complex, and three isolates (CFCC 54582, CFCC 58764 and CFCC 58765) clustered in a distinct clade in the D.arecae species complex (Figs 1, 2). The combined sequence alignments of D.arecae species complex comprised 62 strains, with D.vawdreyi (BRIP 57887a) and D.biconispora (ZJUD62) as the outgroup taxa. The dataset comprised 2791 characters including alignment gaps (634 for ITS, 381 for tef1, 791 for tub2, 499 for cal and 486 for his3). The combined sequence alignments of D.sojae species complex comprised 111 strains, with D.aceris (LC8112) and D.alnea (CBS 146.46) as the outgroup taxa. The dataset comprised 2799 characters including alignment gaps (671 for ITS, 483 for tef1, 483 for tub2, 593 for cal and 569 for his3). The final maximum likelihood tree topology was similar to Bayesian analysis.

Figure 1.

Figure 1.

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Phylogram of Diaporthesojae species complex resulting from a maximum likelihood analysis based on a combined matrix of ITS, cal, his3, tef1 and tub2 loci. Numbers above the branches indicate ML bootstrap values (left, ML BS ≥ 50%) and Bayesian posterior probabilities (right, BPP ≥ 0.9). Isolates from the present study are marked in bold blue.

Figure 2.

Figure 2.

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Phylogram of Diaporthearecae species complex resulting from a maximum likelihood analysis based on a combined matrix of ITS, cal, his3, tef1 and tub2 loci. Numbers above the branches indicate ML bootstrap values (left, ML BS ≥ 50%) and Bayesian posterior probabilities (right, BPP ≥ 0.9). Isolates from the present study are marked in bold blue.

Taxonomy

. Diaporthe rizhaoensis

Y.Q. Zhu & Ning Jiang sp. nov.

B0CFC039-E2A1-5035-8608-3273A2023E46

846816

Fig. 3

Figure 3.

Figure 3.

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Morphology of DiaportherizhaoensisA colonies on PDA, MEA and SNA at 25 °C after 2 weeks B habit of conidiomata on the host C transverse section of the conidioma D longitudinal section through the conidioma E–G conidiogenous cells with attached beta conidia H–J beta conidia. Scale bars: 500 µm (B); 100 µm (C, D); 10 µm (E–J).

Etymology.

Named after the collection site of the type specimen, Rizhao City.

Description.

Conidiomata pycnidial, small, scattered, slightly erumpent through bark surface, nearly flat, discoid, with a solitary undivided locule, 150–400 μm diam. Conidiogenous cells 6.7–11.4 × 1.6–3.0 μm, hyaline, unbranched, densely aggregated, mostly ampulliform, guttulate, aseptate, straight or slightly curved, swelling at base, tapering towards apex. Beta conidia 12.9–23.4 × 1.1–2.1 μm (mean = 18.7 × 1.4 μm, n = 50), hyaline, filiform, straight or slightly curved, aseptate, base subtruncate, tapering towards the base. Alpha conidia and gamma conidia not observed. Sexual morph not observed.

Culture characters.

Colonies on potato dextrose agar (PDA) flat, spreading, with flocculent aerial mycelium and entire edge, white, reaching a 90 mm diameter after 14 days at 25 °C; on malt extract agar (MEA) flat, spreading, with flocculent aerial mycelium and crenate edge, white, reaching a 90 mm diameter after 14 days at 25 °C, forming black conidiomata with black conidial masses; on synthetic low nutrient agar (SNA) flat, spreading, with flocculent aerial mycelium forming concentric rings and entire edge, white, reaching a 90 mm diameter after 14 days at 25 °C.

Materials examined.

China, Shandong Province, Rizhao City, Wulian County, Zhongzhi Town, on dead culms of Xanthiumstrumarium, 5 May 2022, Ning Jiang & Chengbin Wang (holotype CAF 800069; ex-holotype culture CFCC 57562). Shandong Province, Rizhao City, Wulian County, Xumeng Town, on dead culms of Xanthiumstrumarium, 5 May 2022, Ning Jiang & Chengbin Wang (cultures CFCC 57563 and CFCC 57564).

Notes.

Diaportherizhaoensis formed a distinct clade with high support (ML/BI = 100/1), and was close to D.guttulata and D.stewartia (Fig. 1). Diaportherizhaoensis is different from D.stewartia by host association (D.rizhaoensis on Xanthiumstrumarium vs. D.stewartia on Cosmosbipinnatus) (Harrison 1935; Dissanayake et al. 2020). In addition, D.guttulata and D.stewartia are only known in sexual morph. Moreover, Diaportherizhaoensis can be distinguished from D.guttulata (15/364 in cal, 5/428 in his3, 5/313 in tef1, and 1/408 in tub2) and D.stewartii (3/532 in ITS, 7/451 in cal, and 7/369 in tub2) by sequence data. Diaporthehelianthi, D.longicolla, D.pseudolongicolla (= D.novem) and D.rizhaoensis have been reported form the host Xanthiumstrumarium (Vrandecic et al. 2007, 2010; Petrović et al. 2018; Thompson et al. 2018). Morphologically, Diaporthehelianthi is a bit longer than D.rizhaoensis in the beta conidia, but not fully distinguished (Vrandecic et al. 2007, 2010). Morphology of D.longicolla and D.pseudolongicolla on Xanthiumstrumarium were not available. However, these four species are phylogenetically distinguished in the phylogram of D.sojae species complex (Fig. 1).

. Diaporthe smilacicola

Y.Q. Zhu & Ning Jiang sp. nov.

1BC54B14-2E2C-5952-8A5A-3DC760DCB39F

846818

Fig. 4

Figure 4.

Figure 4.

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Morphology of DiaporthesmilacicolaA colonies on PDA, MEA and SNA at 25 °C after 2 weeks B leaf spots on the host surface C conidiomata formed on the PDAD, E conidiogenous cells with attached alpha conidia F–G alpha conidia. Scale bars: 200 µm (C); 10 µm (D–G).

Etymology.

Named after the host genus, Smilax.

Description.

Leaf spots subcircular to irregular, pale brown to brown, with dark brown margin. Conidiomata pycnidial, scattered, subglobose to globose, black, erumpent, exuding faint yellow translucent conidial droplets from central ostioles, 150–350 μm diam. Conidiogenous cells 11–16.2 × 1.8–2.4 μm, hyaline, phialidic, cylindrical, terminal, slightly tapering towards the apex. Alpha conidia 5.7–9.7 × 2.0–3.5 μm (mean = 7.8 × 2.6 μm, n = 50), hyaline, aseptate, smooth, guttulate, ellipsoidal to oblong ellipsoidal, with both ends obtuse. Beta conidia and gamma conidia not observed. Sexual morph not observed.

Culture characters.

Colonies on PDA flat, with flocculent aerial mycelium and crenate edge, white to gray, reaching a 90 mm diameter after 14 days at 25 °C, forming black conidiomata with black conidial masses; on MEA flat, spreading, with flocculent aerial mycelium forming concentric rings, off-white to luteous, reaching a 90 mm diameter after 14 days at 25 °C; on SNA flat, spreading, with flocculent aerial mycelium forming concentric rings and entire edge, white, reaching a 90 mm diameter after 14 days at 25 °C.

Materials examined.

China, Hunan Province, Changsha City, Changsha County, Kaihui Town, on leaf spots of Smilaxglabra, 2 November 2020, Ning Jiang (holotype CAF 800070; ex-holotype culture CFCC 54582). Hunan Province, Shaoshan City, on leaf spots of Smilaxglabra, 2 November 2020, Ning Jiang (cultures CFCC 58764 and CFCC 58765).

Notes.

Three Diaporthe isolates representing D.smilacicola formed a well-supported clade (ML/BI = 100/1), and appear to be distinct from the other Diaporthe species phylogenetically (Fig. 2). Diaportheeres (= D.mahothocarpi), D.lithocarpi and D.smilacicola have been reported from the host S.glabra (Gao et al. 2013; Chaisiri et al. 2021). Morphologically, these three species are similar in conidial shape and size. However, Diaportheeres belongs to D.eres species complex, which is different from D.lithocarpi and D.smilacicola in D.arecae species complex. D.smilacicola is obviously different from D.lithocarpi based on sequence data (22/467 in ITS, 31/393 in cal, 52/317 in tef1, 19/420 in tub2) (Fig. 2).

Discussion

Based on the morphology and the multi-locus phylogeny, six isolates from the present study can be recognized as two new species of Diaporthe, viz. D.rizhaoensis from dead culms of Xanthiumstrumarium and D.smilacicola from leaf spots of Smilaxglabra.

Species identification in Diaporthe was primarily based on the assumption of host-specificity, which has largely impeded the progress of establishing a proper taxonomy of Diaporthe (Gomes et al. 2013). More than one species of Diaporthe can often be recovered from a single host and one species was found to be associated with different host plants (Gomes et al. 2013; Gao et al. 2017; Guarnaccia and Crous 2017; Guarnaccia et al. 2018; Guo et al. 2020). For example, D.eres can infect blackberry (Vrandecic et al. 2011), pear (Bai et al. 2015), and jujube (Zhang et al. 2018); D.pometiae was isolated from Heliconiametallica and Perseaamericana (Huang et al 2021); D.melastomatis was collected from three hosts namely Camelliasinensis, Melastomamalabathricum and Millettiareticulata (Sun et al. 2021); D.australiana, D.drenthii, D.macadamiae and D.searlei can cause diseases on macadamia in Australia and South Africa (Wrona et al. 2020) and seven endophytic Diaporthe species were discovered on Citrus trees (Huang et al. 2015). As was revealed in the present study, two additional species of Diaporthe were proposed from the host Smilaxglabra and Xanthiumstrumarium. This study further demonstrates that host association is not a robust character to distinguish members of Diaporthe.

Recently, the species classification of Diaporthe has become more dependent on DNA sequence-based methods rather than traditional morphological characterization. (Udayanga et al. 2014a, b, 2015; Fan et al. 2015; Gao et al. 2017; Guarnaccia and Crous 2017; Guarnaccia et al. 2018; Hyde et al. 2018, 2020; Yang et al. 2018, 2020, 2021; Long et al. 2019; Cao et al. 2022). The ITS sequence offers convincing proof for species demarcation and is recommended for identifying species boundaries in the genus Diaporthe (Santos and Phillips 2009, 2011; Thompson et al. 2011). However, the intraspecific variation is even greater than the interspecific variation, which makes it difficult to identify Diaporthe species using the ITS sequence alone (Crouch et al. 2009). Considering this, concatenation of a five-loci dataset (ITS-tef1-tub2-cal-his3) was recommended as the best combination for species identification within the genus (Udayanga et al. 2014; Fan et al. 2018; Yang et al. 2018; Guo et al. 2020). Two phylograms resulted from the present study also support the feasibility of the five loci data to separate species of Diaporthe.

The two newly introducing species could potentially be pathogens, because they were isolated from diseased plant tissues, and their pathogenicity should be evaluated in further studies. And, it is necessary to evaluate the effects of environmental conditions, such as temperature, pH, and carbon sources, on mycelium growth and pathogenicity.

Supplementary Material

XML Treatment for Diaporthe rizhaoensis
XML Treatment for Diaporthe smilacicola

Acknowledgements

This research was funded by Fundamental Research Funds for the Central Non-profit Research Institution of Chinese Academy of Forestry (grant CAFYBB2018ZB001), and National Microbial Resource Center of the Ministry of Science and Technology of the People’s Republic of China (grant NMRC-2021-7).

Citation

Zhu Y-Q, Ma C-Y, Xue H, Piao C-G, Li Y, Jiang N (2023) Two new species of Diaporthe (Diaporthaceae, Diaporthales) in China. MycoKeys 95: 209–228. https://doi.org/10.3897/mycokeys.95.98969

Funding Statement

This research was funded by Fundamental Research Funds for the Central Non-profit Research Institution of Chinese Academy of Forestry (grant CAFYBB2018ZB001), and National Microbial Resource Center of the Ministry of Science and Technology of the People’s Republic of China (grant NMRC-2021-7).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

XML Treatment for Diaporthe rizhaoensis
mycokeys.95.98969-treatment1.xml (26.4KB, xml)
XML Treatment for Diaporthe smilacicola
mycokeys.95.98969-treatment2.xml (17.2KB, xml)

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