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. 2024 Sep 13;108:317–335. doi: 10.3897/mycokeys.108.128983

New species of Diaporthe (Diaporthaceae, Diaporthales) from Bauhiniavariegata in China

Yaquan Zhu 1, Lei Ma 2, Han Xue 1, Yong Li 1, Ning Jiang 1,✉
PMCID: PMC11415621  PMID: 39310741

Abstract

Diaporthe species are known as endophytes, saprobes and pathogens infecting a wide range of plants and resulting in important crop diseases. In the present study, four strains of Diaporthe were obtained from diseased leaves of Bauhiniavariegata in Guangdong Province, China. Phylogenetic analyses were conducted to identify these strains using five gene regions: internal transcribed spacer (ITS), calmodulin (cal), histone H3 (his3), translation elongation factor 1-α (tef1) and β-tubulin (tub2). The results combined with morphology revealed two new species of Diaporthe named D.bauhiniicola in D.arecae species complex and D.guangzhouensis in D.sojae species complex.

Key words: Diaporthales, morphology, multi-gene phylogeny, taxonomy, two new taxa

Introduction

Diaporthe (syn. Phomopsis) is the type genus of Diaporthaceae in Diaporthales (Hyde et al. 2014; Maharachchikumbura et al. 2016). Before the implementation of “one fungus, one name”, it has been a common practice to use two names for the fungal species with pleomorphic life cycles (Taylor 2011). The genus Diaporthe established in 1870 predates Phomopsis established in 1905, thus Diaporthe is recommended for use (Rossman et al. 2015). More than 1200 epithets for Diaporthe have been listed in Index Fungorum with names often based on host association (http://www.indexfungorum.org/, accessed June 2024).

The teleomorph of Diaporthe is characterized by aggregated spherical ascomata with tapering necks, unitunicate, 8-spored, elongate to clavate asci, and septate or aseptate, elongated to elliptical, hyaline ascospores with larger guttules at center and smaller ones at the ends (Senanayake et al. 2018; Yang et al. 2020). The anamorph is characterized by black, ostiolate pycnidia containing cylindrical phialides often producing three types of hyaline, aseptate conidia called α-conidia, β-conidia and γ-conidia (Udayanga et al. 2012a; Dissanayake et al. 2017; Fan et al. 2018). The α-conidia and β-conidia are produced frequently, but the γ-conidia are rarely observed (Gomes et al. 2013).

Diaporthe species are associated with a wide range of plant hosts as pathogens, endophytes and saprobes of crops, forest trees and ornamentals (Farr et al. 2002a; Crous 2005; Udayanga et al. 2012b, 2014a, 2014b, 2015; Jiang et al. 2021; Zhu et al. 2023). As plant pathogens, Diaporthe species cause severe diseases, e.g., leaf spots, blights, dieback, scab, decay, stem end rots and wilt of many economically important plants including species of Citrus (Guarnaccia and Crous 2017), Macadamia (Wrona et al. 2020), Rosa (Caio et al. 2021), Vaccinium (Farr et al. 2002b), Vitis (Manawasinghe et al. 2019) and many more (Yang et al. 2018, 2021; Guarnaccia et al. 2020; Guo et al. 2020; Ariyawansa et al. 2021). In addition, Diaporthe species can live inside the healthy host tissues as endophytes (Huang et al. 2015; Dong et al. 2021). In addition, species of Diaporthe have been also reported as saprobes from different woody hosts (Dissanayake et al. 2020).

Species identification of Diaporthe has traditionally been based on host as well as morphological characters such as the size and shape of fruiting bodies and spores (Mostert et al. 2001; Santos and Phillips 2009). 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; Huang et al. 2021; Sun et al. 2021; Cao et al. 2022). Moreover, many Diaporthe species that are morphologically similar have proven to be genetically distinct (van Rensburg et al. 2006; Yang et al. 2018). Phylogenetic analysis using a five-locus dataset (ITS-tef1-tub2-cal-his3) has been widely used to identify species of Diaporthe species (Santos et al. 2017; Marin-Felix et al. 2019; Hilário et al. 2021b; Norphanphoun et al. 2022). Diaporthe was clustered into 13 groups, 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 and nine singletons as D.acerina, D.acutispora, D.crataegi, D.multiguttulata, D.ocoteae, D.perjuncta, D.pseudoalnea, D.spartinicola and D.undulata based on multilocus phylogeny (Norphanphoun et al. 2022; Hongsanan et al. 2023).

Bauhiniavariegata is a flowering plant species belonging to Fabaceae. It is native to China and cultivated as an ornamental tree in subtropical and tropical climate for its scented flowers. The aim of the present study was to identify new isolates collected from diseased leaves of Bauhiniavariegata in China following the combined approaches of morphology and phylogeny in the genus Diaporthe.

Materials and methods

Isolation and morphological characterization

In 2022, a plant disease investigation was conducted in Guangdong Province, China. Small and irregular leaf spots were observed on the leaves of Bauhiniavariegata, and 14 leaves were collected for isolation. The leaves were firstly surface-sterilized for 1 min in 75% ethanol, 3 min in 1.25% sodium hypochlorite and 1 min in 75% ethanol, rinsed for 2 min in distilled water and blotted on dry sterile filter paper. Then, the discolored areas were cut into 0.5 × 0.5 cm pieces and transferred to the surface of potato dextrose agar plates (PDA; 200 g potatoes, 20 g dextrose, 20 g agar per litre), incubated at 25 °C to obtain pure cultures. The cultures were deposited in the China Forestry Culture Collection Center (CFCC; http://cfcc.caf.ac.cn/) and the specimen was deposited in the Herbarium of the Chinese Academy of Forestry (CAF; http://museum.caf.ac.cn/).

The isolates were grown on PDA, MEA and SNA plates, incubated at 25 °C under a 12 h near-ultraviolet light/12 h dark cycle to induce sporulation. Colony characters and pigment production on PDA, MEA and SNA were noted for the 10-day culture. Microscopic structures of the fungi growing on medium were mounted in water and examined under an Axio Imager 2 microscope (Zeiss, Oberkochen, Germany). At least 30 measurements were made for each structure examined.

DNA extraction, amplification and sequencing

The genomic DNA was extracted from the fresh mycelium harvested from PDA plates after seven days using a cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle 1990). For initial genus confirmation, the internal transcribed spacer (ITS) region was sequenced. After confirmation of Diaporthe species, four additional gene regions coding for translation elongation factor 1-alpha (tef1), beta-tubulin (tub2), calmodulin (cal) and his-tone H3 (his3) were sequenced. The primer pairs and amplification conditions for each of the above-mentioned gene regions are provided in Table 1.

Table 1.

Loci assessed in this study with used PCR primers and program.

Loci Primers PCR: Thermal Cycles: (Annealing Temp. in Bold) Reference
ITS ITS1f/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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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). All amplified PCR products were evaluated visually with 1.4% agarose gels stained with ethidium bromide and PCR positive products sent to Sangon Biotech (Shanghai) Co., Ltd., (Beijing, China) for sequencing. Strands were sequenced in both directions using PCR primers. The new sequences generated in this study, as well as the reference sequences of all isolates used in the present study, are listed in Table 2.

Table 2.

Isolates and GenBank accession numbers used in the phylogenetic analyses of Diaporthe.

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.bauhiniicola China Bauhiniavariegata CFCC 58154* PP864723 PP938599 PP938603 PP938607 PP938611
D.bauhiniicola China Bauhiniavariegata GZ13B PP864724 PP938600 PP938604 PP938608 PP938612
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 ZHKUCC20-0012* 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 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 ZHKUCC20-0014* MT355684 MT409338 MT409292 MT409314 NA
D.guangxiensis China Vitisvinifera JZB320094* MK335772 MK523566 MK500168 MK736727 NA
D.guangzhouensis China Bauhiniavariegataa CFCC 58151* PP864725 PP938601 PP938605 PP938609 PP938613
D.guangzhouensis China Bauhiniavariegata GZ13E PP864726 PP938602 PP938606 PP938610 PP938614
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 Passiflaedulis 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.pseudobauhiniae Thailand Bauhinia sp. MFLU 17-1670 MF190118 MF377599 NA NA NA
D.pseudobauhiniae Thailand Bauhinia sp. MFLUCC 17-1669* MF190119 MF377598 NA NA NA
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.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.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.

Phylogeny

For the phylogenetic analysis, sequences of reference Diaporthe species and related taxa were downloaded from NCBI GenBank based on recent publications on the genus Diaporthe (Norphanphoun et al. 2022) (Table 2). Downloaded sequences were aligned together with the sequences obtained in the present study using MAFFT version 7.526 (Katoh and Standley 2013) and manually corrected using Bioedit 7.0.9.0 (Hall 1999). The phylogenetic analyses of the combined gene regions were performed using Maximum Likelihood (ML) and Bayesian Inference (BI) methods. ML was conducted using PhyML v. 3.0 (Guindon et al. 2010), with 1000 bootstrap replicates while BI was performed using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.0 (Ronquist and Huelsenbeck 2003). Two MCMC chains, started from random trees for 1,000,000 generations and trees, were sampled every 100th generation, resulting in a total of 10,000 trees. The first 25% of trees were discarded as burn-in of each analysis. Branches with significant Bayesian Posterior Probabilities (BPP) were estimated in the remaining 7500 trees. Phylogenetic trees were visualized with FigTree v.1.3.1 (Rambaut and Drummond 2010) and processed by Adobe Illustrator CS5. The nucleotide sequence data of the new taxa were deposited in GenBank (Table 2)

Results

Phylogenetic analyses

In the present study, we inferred a genus tree of Diaporthe covering a large proportion of sequence data available as last summarized by Norphanphoun et al. (2022). Two strains CFCC 58154 and GZ13B formed a clade in the D.arecae species complex, and the other strains CFCC 58151 and GZ13E in the D.sojae species complex.

In the D.arecae species complex, the combined sequence alignments comprised 61 strains, with D.eucalyptorum (CBS 13252), D.biconispora (ZJUD62) and D.vawdreyi (BRIP 57887a) as the outgroup taxa. The dataset comprised 2662 characters including alignment gaps (590 for ITS, 499 for cal, 485 for his3, 375 for tef1 and 713 for tub2). CFCC 58154 and GZ13B from Bauhiniavariegata formed a distinct clade close to D.sennae (Fig. 1). In the D.sojae species complex, the combined sequence alignments comprised 166 strains (Fig. 2), with D.aceris (LC8112) and D.alnea (CBS 146.46) as the outgroup taxa. The dataset comprised 3025 characters including alignment gaps (602 for ITS, 592 for cal, 521 for his3, 483 for tef1 and 827 for tub2). CFCC 58151 and GZ13E from B.variegata clustered in a distinct clade close to D.tulliensis (Fig. 2).

Figure 1.

Figure 1.

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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 in bold and ex-type strains are marked with *.

Figure 2.

Figure 2.

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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 in bold and ex-type strains are marked with *.

Taxonomy

. Diaporthe bauhiniicola

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

2D6AD0F1-0758-54ED-B0EB-B5BDDB93E656

854183

Fig. 3

Figure 3.

Figure 3.

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Morphology of DiaporthebauhiniicolaA colonies on PDA, MEA and SNA at 25 °C after 2 weeks B a diseased leaf of BauhiniavariegataC conidioma formed on PDA after 30 days D conidiogenous cells with attached alpha conidia E–G alpha conidia. Scale bars: 200 µm (C); 10 µm (D–J).

Holotype.

China • Guangdong Province, Guangzhou City, Luhu Park, 23°9'11.15"N, 113°16'46.01"E, 92 m asl, on diseased leaves of Bauhiniavariegata, 8 Aug 2022, Yong Li, Chengbin Wang & Yaquan Zhu, (holotype: CAF800094; ex-type culture: CFCC 58154).

Etymology.

Named after the host genus, Bauhinia.

Description.

Conidiomata formed on PDA pycnidial, scattered to aggregated, black, erumpent, raising above surface of culture medium, subglobose, 150–300 μm diam., exuding white or yellowish creamy conidial droplets from central ostioles after 30 days at 25 °C. Conidiophores reduced to conidiogenous cells. Conidiogenous cells hyaline, unbranched, septate, straight, slightly tapering towards the apex, 6.0–15.0 × 1.5–4.0 μm. Alpha conidia hyaline, aseptate, ellipsoidal to spindle-shaped, biguttulate or with one guttulate, 4.5–7.0 × 2.0–3.0 μm. Beta conidia and gamma conidia not observed. Teleomorph not observed.

Culture characteristics.

Colonies covering entire plate after 2 weeks. On PDA with profuse aerial mycelium, white surface, reverse fulvous. On MEA with fluffy aerial mycelium, dirty white surface, reverse ochreous. On SNA white sparse aerial mycelium, surface and reverse white.

Additional material examined.

China • Guangdong Province, Guangzhou City, Luhu Park, 23°9'11.15"N, 113°16'46.01"E, 92 m asl, on diseased leaves of Bauhiniavariegata, 8 Aug 2022, Yong Li, Chengbin Wang & Yaquan Zhu, living culture GZ13B.

Notes.

Two strains representing Diaporthebauhiniicola clustered in a clade distinct from its closest phylogenetic neighbour, D.sennae (Fig. 1). D.sennae has been reported from the host Sennabicapsularis in China (Yang et al. 2017). D.bauhiniicola differs from D.sennae by wider alpha conidia (4.5–7.0 × 2.0–3.0 μm in D.bauhiniicola vs. 5.0–6.5 × 1.5–1.8 μm in D.sennae) (Yang et al. 2017). Diaporthebauhiniicola differs from D.sennae in nucleotide sequence data (18/529 in ITS, 5/490 in cal, 15/351 in tef1, 14/677 in tub2) (Yang et al. 2017).

. Diaporthe guangzhouensis

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

2A0959D8-088A-5FA5-A7A2-765C956730CF

854184

Fig. 4

Figure 4.

Figure 4.

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Morphology of DiaportheguangzhouensisA colonies on PDA, MEA and SNA at 25 °C after 2 weeks B the leaf of BauhiniavariegataC conidiomata D conidiogenous cells with attached beta conidia E–G beta conidia. Scale bars: 200 µm (C); 10 µm (D–J).

Etymology.

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

Holotype.

China • Guangdong Province, Guangzhou City, Longdong straight street, 23°11'41.02"N, 113°22'8.33"E, 46 m asl, on diseased leaves of Bauhiniavariegata, 8 Aug 2022, Yong Li, Chengbin Wang & Yaquan Zhu, (holotype: CAF800095; ex-type culture: CFCC 58151).

Description.

Conidiomata pycnidial, scattered to aggregated, black, erumpent, raising above surface of culture medium, subglobose, 150–450 µm diam, exuding white or yellowish creamy conidial droplets from central ostioles after 30 days at 25 °C. Conidiophores 12.5–24.5 × 1–2.5 μm, cylindrical, hyaline, unbranched, straight to sinuous. Conidiogenous cells densely aggregated, phiailidic, unbranched, straight or slightly curved, 5.5–10 × 2.0–7.5 μm. Beta conidia filiform, hyaline, straight or slightly curved, aseptate, 17.0–29.5 × 1.0–2.0 μm. Alpha conidia and gamma conidia not observed. Teleomorph not observed.

Culture characteristics.

Colonies covering entire plate after 2 weeks. On PDA with profuse aerial mycelium, white surface, reverse amber. On MEA with fluffy aerial mycelium, dirty white surface, reverse ochreous. On SNA white sparse aerial mycelium, surface and reverse white.

Additional material examined.

China • Guangdong Province, Guangzhou City, Longdong straight street, 23°11'41.02"N, 113°22'8.33"E, 46 m asl, on diseased leaves of Bauhiniavariegata, 8 Aug 2022, Yong Li, Chengbin Wang & Yaquan Zhu, living culture GZ13E.

Notes.

Diaportheguangzhouensis from the present study is phylogenetically close to D.tulliensis (Fig. 2). Diaportheguangzhouensis differs from D.tulliensis in nucleotide sequence data (5/526 in ITS, 9/347 in tef1, 13/711 in tub2) (Crous et al. 2015). In addition, host and distribution data are vital for species identification (D.guangzhouensis inhabiting Bauhiniavariegata in China vs. D.tulliensis inhabiting Theobromacacao in Australia) (Crous et al. 2015).

Discussion

In the current study, phylogenetic analyses based on five combined loci (ITS, cal, his3, tef1 and tub2), as well as morphological characters of the anamorph obtained in culture, revealed D.bauhiniicola and D.guangzhouensis spp. nov. from Bauhiniavariegata, which contributed to our knowledge of the diversity of Diaporthe species in China.

Diaporthepseudobauhiniae (syn. D.chiangraiensis, Chiangraiomycesbauhiniae) was described as a saprobic fungus on branches of Bauhinia sp. in Thailand (Senanayake et al. 2017). D.bauhiniae was introduced from branches of B.purpurea in China (Yang et al. 2021). Hence, a total of four species of Diaporthe have been recorded from the host genus Bauhinia. Phylogenetically, D.bauhiniae belongs to D.varians species complex; D.bauhiniicola belongs to D.arecae species complex; D.guangzhouensis and D.pseudobauhiniae belong to D.sojae species complex (Figs 1, 2) (Norphanphoun et al. 2022). Furthermore, D.guangzhouensis and D.pseudobauhiniae formed different clades in D.sojae species complex (Fig. 2). Morphologically, D.bauhiniicola has larger alpha conidia than D.pseudobauhiniae, but longer alpha conidia than D.bauhiniae (4.5–7.0 × 2.0–3.0 μm in D.bauhiniicola vs. 3–5 × 2–4 μm in D.pseudobauhiniae vs. 7.5–14 × 1.5–3 μm in D.bauhiniae) (Senanayake et al. 2017; Yang et al. 2021). D.guangzhouensis shares similar beta conidia size with D.pseudobauhiniae that are shorter and wider than D.bauhiniae (17.0–29.5 × 1.0–2.0 μm in D.guangzhouensis vs. 18–38 × 1.5–2 μm in D.pseudobauhiniae vs. 25–43 × 1 µm in D.bauhiniae) (Senanayake et al. 2017; Yang et al. 2021). Another species named Phomopsisbauhiniae was recorded on the branches of Bauhiniavariegata in Spain, however, this species was only studied in morphology and has not been combined in Diaporthe (Uecker 1988). Diaporthebauhiniicola has shorter but wider alpha conidia than P.bauhiniae morphologically (Uecker 1988). The molecular analyses are necessary for the species P.bauhiniae based on the ex-type culture in the future.

The initial species concept of Diaporthe based on the assumption of host-specificity, resulted in the introduction of more than 1000 taxa (http://www.indexfungorum.org/). However, more than one species of Diaporthe have been often discovered from the same host (Gomes et al. 2013; Guarnaccia and Crous 2017; Guarnaccia et al. 2020; Guo et al. 2020). For example, D.caryae and an additional 18 Diaporthe species are associated with pear shoot canker in China (Guo et al. 2020); D.sennae and D.sennicola inhabit branches of Sennabicapsularis causing canker diseases (Yang et al. 2017). The current study further supports this phenomenon.

Diaporthe is considered as a species-rich genus. Nevertheless, an emerging perspective posits that the quantity of recognized Diaporthe species may have been substantially overestimated. The D.amygdali species complex has been proven a single species evidenced from the genealogical concordance phylogenetic species recognition principle (GCPSR) and coalescence-based models: general mixed yule-coalescent (GMYC) and poisson tree processes (PTP), with several species becoming synonyms (Hilário et al. 2021b). Similarly, several species in the D.eres species complex such as D.betulae and D.padina were treated as synonyms (Hilário et al. 2021a). A comprehensive study is necessary to clarify species boundaries of Diaporthe in the future. This will help improve our understanding of the species concept within this genus.

Supplementary Material

XML Treatment for Diaporthe bauhiniicola
XML Treatment for Diaporthe guangzhouensis

Citation

Zhu Y, Ma L, Xue H, Li Y, Jiang N (2024) New species of Diaporthe (Diaporthaceae, Diaporthales) from Bauhinia variegata in China. MycoKeys 108: 317–335. https://doi.org/10.3897/mycokeys.108.128983

Funding Statement

This study was supported by Fundamental Research Funds of CAF (CAFYBB2023PA002), and the National Microbial Resource Center of the Ministry of Science and Technology of the People’s Republic of China (NMRC-2023-7).

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This study was supported by Fundamental Research Funds of CAF (CAFYBB2023PA002).

Author contributions

Conceptualization: LM, YL, NJ. Methodology: YZ. Formal analysis: HX. Investigation: YL. Data Curation: LM, HX. Writing - Original draft: YZ. Writing - Review and Editing: NJ. Visualization: NJ.

Author ORCIDs

Yaquan Zhu https://orcid.org/0000-0002-3296-239X

Han Xue https://orcid.org/0000-0003-0414-6237

Yong Li https://orcid.org/0000-0002-4406-1329

Ning Jiang https://orcid.org/0000-0002-9656-8500

Data availability

All of the data that support the findings of this study are available in the main text.

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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 bauhiniicola
mycokeys.108.128983-treatment1.xml (15.2KB, xml)
XML Treatment for Diaporthe guangzhouensis
mycokeys.108.128983-treatment2.xml (19.1KB, xml)

Data Availability Statement

All of the data that support the findings of this study are available in the main text.

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