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Research Article
Two new species of Diaporthe (Diaporthaceae, Diaporthales) associated with tree cankers in the Netherlands
expand article infoNing Jiang§, Hermann Voglmayr|, Chun-Gen Piao, Yong Li
‡ Environment and Nature Conservation, Chinese Academy of Forestry, Beijing, China
§ Beijing Forestry University, Beijing, China
| University of Vienna, Vienna, Austria
Open Access

Abstract

Diaporthe (Diaporthaceae, Diaporthales) is a common fungal genus inhabiting plant tissues as endophytes, pathogens and saprobes. Some species are reported from tree branches associated with canker diseases. In the present study, Diaporthe samples were collected from Alnus glutinosa, Fraxinus excelsior and Quercus robur in Utrecht, the Netherlands. They were identified to species based on a polyphasic approach including morphology, pure culture characters, and phylogenetic analyses of a combined matrix of partial ITS, cal, his3, tef1 and tub2 gene regions. As a result, four species (viz. Diaporthe pseudoalnea sp. nov. from Alnus glutinosa, Diaporthe silvicola sp. nov. from Fraxinus excelsior, D. foeniculacea and D. rudis from Quercus robur) were revealed from tree branches in the Netherlands. Diaporthe pseudoalnea differs from D. eres (syn. D. alnea) by its longer conidiophores. Diaporthe silvicola is distinguished from D. fraxinicola and D. fraxini-angustifoliae by larger alpha conidia.

Keywords

Two new taxa, Diaporthe pseudoalnea, Diaporthe silvicola, taxonomy, two new taxa

Introduction

Diaporthe (syn. Phomopsis) is the type genus of Diaporthaceae in Diaporthales, commonly occurring as plant endophytes, pathogens and saprobes (Udayanga et al. 2014, 2015; Guarnaccia et al. 2017, 2018a, 2018b; Tibpromma et al. 2018; Yang et al. 2020; Dissanayake et al. 2020; Jiang et al. 2021). The sexual morph is characterized by immersed perithecial ascomata and an erumpent pseudostroma with more or less elongated perithecial necks, unitunicate clavate to cylindrical asci, and fusoid, ellipsoid to cylindrical, hyaline uni- to bicellular ascospores (Udayanga et al. 2011; Senanayake et al. 2017). The asexual morph is characterized by ostiolate conidiomata, with cylindrical phialides producing up to three types of hyaline, aseptate conidia (Udayanga et al. 2011; Gomes et al. 2013; Yang et al. 2018), and was previously classified as Phomopsis. Following the “one fungus one name” nomenclature, Rossman et al. (2015) recommended to use Diaporthe based on priority, necessitating the transfer of numerous Phomopsis species to Diaporthe.

Species of Diaporthe are known to cause plant diseases including dieback, canker, leaf spot, fruit rot, pod blights and seed decay. For example, D. citri, D. cytosporella and D. foeniculina caused melanose and stem end rot diseases of Citrus spp. (Udayanga et al. 2014), while Daporthe lithocarpi caused leaf spot disease of Castanea henryi in China (Jiang et al. 2021). Up to 19 Diaporthe species were confirmed to be associated with pear cankers in China (Guo et al. 2020), and eight species of Diaporthe were found to be the casual agents of Chinese grapevine dieback (Manawasinghe et al. 2019). Seven Diaporthe species were reported from blueberry twig blight and dieback diseases in Portugal (Hilário et al. 2020). Diaporthe biconispora and an additional six species were identified as endophytes from healthy Citrus tissues in China (Huang et al. 2015). Diaporthe constrictospora and an additional 11 species were isolated as saprobes from dead wood in karst formations in China (Dissanayake et al. 2020).

Diaporthe species were previously classified mainly based on host association and morphology (Rehner and Uecker 1994; Santos and Phillips 2009; Udayanga et al. 2011, 2014). However, several taxonomic studies of Diaporthales proved that phylogeny based on multiple genes is suitable to separate species (Voglmayr et al. 2012, 2017; Fan et al. 2018; Jiang et al. 2019, 2020; Jaklitsch and Voglmayr 2019, 2020). Species of Diaporthe are now characterised and circumscribed both by morphology and phylogeny of multi-locus DNA data, which revealed many cryptic species in recent years (Diogo et al. 2010; Lombard et al. 2014; Gao et al. 2016, 2017; Long et al. 2019; Yang et al. 2020, 2021; Zapata et al. 2020; Huang et al. 2021). To clarify the species boundaries of the Diaporthe eres complex, the Genealogical Phylogenetic Species Recognition principle (GCPSR) and the coalescent-based model Poisson Tree Processes (PTPs) were employed, which suggested that the Diaporthe eres species complex actually represents only a single species, D. eres (Hilário et al. 2021).

In the present study, Diaporthe samples from cankered branches of several tree species were collected in the Netherlands, and identified based on modern taxonomic approaches. As a result, two new species and two known species were identified, and the new species are described and illustrated herein.

Materials and Methods

Collection, examination and isolation

The fresh specimens of cankered branches were sampled from Alnus glutinosa, Fraxinus excelsior and Quercus robur in Utrecht, the Netherlands. Morphological characteristics of the conidiomata were determined under a Nikon AZ100 dissecting stereomicroscope. More than 20 conidiomata were sectioned, and 50 conidia were randomly selected for measurement using a Leica compound microscope (LM, DM 2500). Isolates were obtained by removing a mucoid conidial mass from conidiomata, spreading the suspension onto the surface of 1.8 % potato dextrose agar (PDA), and incubated at 25 °C for up to 24 h. Single germinating conidia were removed and plated onto fresh PDA plates. Cultural characteristics of isolates incubated on PDA in the dark at 25 °C were recorded, including the colony color and conidiomata structures. 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, PCR amplification and phylogenetic analyses

Genomic DNA was extracted from colonies grown on cellophane-covered PDA using a cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle 1990). DNA was checked by electrophoresis in 1 % agarose gel, and the quality and quantity were measured using a NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). Five partial loci, including the 5.8S nuclear ribosomal DNA gene with the two flanking internally transcribed spacer (ITS) regions, the calmodulin (cal), the histone H3 (his3), the translation elongation factor 1-alpha (tef1) and the beta-tubulin (tub2) genes were amplified by the primer pairs and polymerase chain reaction (PCR) process listed in Table 1. The PCR products were assayed via electrophoresis in 2 % agarose gels. DNA sequencing was performed using an ABI PRISM 3730XL DNA Analyser with a BigDye Terminator Kit v.3.1 (Invitrogen, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

Table 1.

Genes used in this study with PCR primers and process.

Locus 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 & Donaldson 1995;
O’Donnell and Cigelnik 1997

The quality of the amplified nucleotide sequences was checked and the sequences assembled using SeqMan v.7.1.0. Reference sequences were retrieved from the National Center for Biotechnology Information (NCBI), based on recent publications on the genus Diaporthe (Dissanayake et al. 2021; Gao et al. 2021; Huang et al. 2021; Sun et al. 2021, Wang et al. 2021; Yang et al. 2021). Sequences were aligned using MAFFT v. 6 (Katoh and Toh 2010) and corrected manually using MEGA 7.0.21. The best-fit nucleotide substitution models for each gene were selected using jModelTest v. 2.1.7 (Darriba et al. 2012) under the Akaike Information Criterion.

The phylogenetic analyses of the combined gene regions were performed using Maximum Likelihood (ML) and Bayesian Inference (BI) methods. ML was implemented on the CIPRES Science Gateway portal (https://www.phylo.org) using RAxML-HPC BlackBox 8.2.10 (Stamatakis 2014), employing a GTRGAMMA substitution model with 1000 bootstrap replicates. While BI was performed using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.0 (Ronquist et al. 2003). Two MCMC chains, started from random trees for 1000000 generations and trees, were sampled every 100th generation, resulting in a total of 10000 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 viewed with FigTree v.1.3.1 and processed by Adobe Illustrator CS5. The nucleotide sequence data of the new taxa were deposited in GenBank and are listed in Table 2.

Table 2.

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

Species Strain Host Origin GenBank accession numbers
ITS cal his3 tef1 tub2
Diaporthe acaciigena CBS 129521 Acacia retinodes Australia KC343005 KC343247 KC343489 KC343731 KC343973
D. acericola MFLUCC 17-0956 Acer negundo Italy KY964224 KY964137 NA KY964180 KY964074
D. acerigena CFCC 52554 Acer tataricum China MH121489 MH121413 MH121449 MH121531 NA
D. acerigena CFCC 52555 Acer tataricum China MH121490 MH121414 MH121450 MH121532 NA
D. acuta PSCG 047 Pyrus pyrifolia China MK626957 MK691125 MK726161 MK654802 MK691225
D. acutispora LC6161 Coffea China KX986764 KX999274 KX999235 KX999155 KX999195
D. alangii CFCC 52556 Alangium kurzii China MH121491 MH121415 MH121451 MH121533 MH121573
D. alangii CFCC 52557 Alangium kurzii China MH121492 MH121416 MH121452 MH121534 MH121574
D. albosinensis CFCC 53066 Betula albosinensis China MK432659 MK442979 MK443004 MK578133 MK578059
D. albosinensis CFCC 53067 Betula albosinensis China MK432660 MK442980 MK443005 MK578134 MK578060
D. alleghaniensis CBS 495.72 Betula alleghaniensis Canada MH121502 MH121426 MH121462 MH121544 MH121584
D. ambigua CBS 114015 Pyrus communis South Africa KC343010 KC343252 KC343494 KC343736 KC343978
D. ampelina STE-U 2660 Vitis vinifera France NA AY745026 NA AY745056 NA
D. amygdali CBS 126679 Prunus dulcis Portugal MH864208 KC343264 KC343506 KC343748 KC343990
D. anacardii CBS 720.97 Anacardium occidentale East Africa KC343024 KC343266 KC343508 KC343750 KC343992
D. angelicae CBS 111592 Heracleum sphondylium Austria KC343027 KC343269 KC343511 KC343753 KC343995
D. apiculatum CFCC 53068 Rhus chinensis China MK432651 MK442973 MK442998 MK578127 MK578054
D. apiculatum CFCC 53069 Rhus chinensis China MK432652 MK44297 MK442999 MK578128 MK578055
D. aquatica IFRDCC 3051 Aquatic habitat China JQ797437 NA NA NA NA
D. arctii DP0482 Arctium lappa Austria KJ590736 KJ612133 KJ659218 KJ590776 KJ610891
D. arecae CBS 161.64 Areca catechu India KC343032 KC343274 KC343516 KC343758 KC344000
D. arengae CBS 114979 Arenga engleri Hong Kong MF773664 KC343276 KC343518 KC343760 KC344002
D. aseana MFLUCC 12-0299a Unknown Thailand KT459414 KT459464 NA KT459448 KT459432
D. asheicola CBS 136967 Vaccinium ashei Chile KJ160562 KJ160542 NA KJ160594 KJ160518
D. aspalathi CBS 117169 Aspalathus linearis South Africa KC343036 KC343278 KC343520 KC343762 KC344004
D. australafricana CBS 111886 Vitis vinifera Australia KC343038 KC343280 KC343522 KC343764 KC344006
D. australiana CBS 146457 Macadamia Australia MN708222 NA NA MN696522 MN696530
D. baccae CBS 136972 Vaccinium corymbosum Italy MK370623 MG281695 MF418264 KJ160597 MF418509
D. batatas CBS 122.21 Ipomoea batatas USA KC343040 KC343282 KC343524 KC343766 KC344008
D. bauhiniae CFCC 53071 Bauhinia purpurea China MK432648 MK442970 MK442995 MK578124 MK578051
D. bauhiniae CFCC 53072 Bauhinia purpurea China MK432649 MK442971 MK442996 MK578125 MK578052
D. bauhiniae CFCC 53073 Bauhinia purpurea China MK432650 MK442972 MK442997 MK578126 MK578053
D. beilharziae BRIP 54792 Indigofera australis Australia JX862529 NA NA JX862535 KF170921
D. benedicti SBen914 Diaporthe benedicti USA KM669929 KM669862 NA KM669785 NA
D. betulae CFCC 50469 Betula platyphylla China KT732950 KT732997 KT732999 KT733016 KT733020
D. betulae CFCC 50470 Betula platyphylla China KT732951 KT732998 KT733000 KT733017 KT733021
D. betulicola CFCC 51128 Betula albo-sinensis China KX024653 KX024659 KX024661 KX024655 KX024657
D. betulicola CFCC 51129 Betula albo-sinensis China KX0246554 KX024660 KX024662 KX0246556 KX024658
D. betulina CFCC 52560 Betula albo-sinensis China MH121495 MH121419 MH121455 MH121537 MH121577
D. betulina CFCC 52561 Betula albo-sinensis China MH121496 MH121420 MH121456 MH121538 MH121578
D. biconispora ZJUD62 Citrus maxima China KJ490597 NA KJ490539 KJ490476 KJ490418
D. biguttulata ZJUD47 Citrus limon China KJ490582 NA KJ490524 KJ490461 KJ490403
D. bohemiae CBS 143347 Vitis vinifera Czech Republic MK300012 MG281710 MG281361 MG281536 MG281188
D. brasiliensis CBS 133183 Aspidosperma tomentosum Brazil KC343042 KC343284 KC343526 KC343768 KC344010
D. caatingaensis URM7485 Tacinga inamoena Brazil KY085927 KY115598 NA KY115604 KY115601
D. camelliae-sinensis SAUCC194.92 Camellia sinensis China MT822620 MT855699 MT855588 MT855932 MT855817
D. canthii CPC 19740 Canthium inerme South Africa JX069864 NA NA NA NA
D. caryae CFCC 52563 Carya illinoinensis China MH121498 MH121422 MH121458 MH121540 MH121580
D. caryae CFCC 52564 Carya illinoinensis China MH121499 MH121423 MH121459 MH121541 MH121581
D. cassines CPC 21916 Cassine peragua South Africa KF777155 NA NA KF777244 NA
D. caulivora CBS 127268 Glycine max Croatia MH864501 KC343287 KC343529 KC343771 KC344013
D. cercidis CFCC 52565 Cercis chinensis China MH121500 MH121424 MH121460 NA MH121582
D. cercidis CFCC 52566 Cercis chinensis China MH121501 MH121425 MH121461 NA MH121583
D. chamaeropis CBS 454.81 Chamaerops humilis Greece KC343048 KC343290 KC343532 KC343774 KC344016
D. charlesworthii BRIP 54884m Rapistrum rugostrum Australia KJ197288 NA NA KJ197250 KJ197268
D. chensiensis CFCC 52567 Abies chensiensis China MH121502 MH121426 MH121462 MH121544 MH121584
D. chensiensis CFCC 52568 Abies chensiensis China MH121503 MH121427 MH121463 MH121545 MH121585
D. chongqingensis PSCG 435 Pyrus pyrifolia China MK626916 MK691209 MK726257 MK654866 MK691321
D. chrysalidocarpi SAUCC194.35 Chrysalidocarpus lutescens China MT822563 MT855646 MT855532 MT855760 MT855876
D. cichorii MFLUCC 17-1023 Cichorium intybus Italy KY964220 KY964133 NA KY964176 KY964104
D. cinnamomi CFCC 52569 Cinnamomum China MH121504 NA MH121464 MH121546 MH121586
D. cinnamomi CFCC 52570 Cinnamomum China MH121505 NA MH121465 MH121547 MH121587
D. cissampeli CPC 27302 Cissampelos capensis South Africa KX228273 NA KX228366 NA KX228384
D. citri AR3405 Citrus USA KC843311 KC843157 KJ420881 KC843071 KC843187
D. citri CFCC 53079 Citrus sinensis China MK573940 MK574579 MK574595 MK574615 MK574635
D. citriasiana CGMCC 3.15224 Citrus unshiu China JQ954645 KC357491 KC490515 JQ954663 KC357459
D. citrichinensis CGMCC 3.15225 Citrus China JQ954648 KC357494 NA JQ954666 NA
D. collariana MFLU 17-2770 Magnolia champaca Thailand MG806115 MG783042 NA MG783040 MG783041
D. compactum LC3083 Camellia sinensis China KP267854 NA KP293508 KP267928 NA
D. conica CFCC 52571 Alangium chinense China MH121506 MH121428 MH121466 MH121548 MH121588
D. conica CFCC 52572 Alangium chinense China MH121507 MH121429 MH121467 MH121549 MH121589
D. constrictospora CGMCC 3.20096 Unknown China MT385947 MT424718 MW022487 MT424682 MT424702
D. convolvuli CBS 124654 Convolvulus arvensis Turkey KC343054 KC343296 KC343538 KC343780 KC344022
D. coryli CFCC 53083 Corylus mandshurica China MK432661 MK442981 MK443006 MK578135 MK578061
D. coryli CFCC 53084 Corylus mandshurica China MK432662 MK442982 MK443007 MK538176 MK578062
D. corylicola CFCC 53986 Corylus heterophylla China MW839880 MW836684 MW836717 MW815894 MW883977
D. corylicola CFCC 53987 Corylus heterophylla China MW839867 MW836685 MW836718 MW815895 MW883978
D. crotalariae CBS 162.33 Crotalaria spectabilis USA MH855395 JX197439 KC343540 GQ250307 KC344024
D. crousii CAA 823 Vaccinium corymbosum Portugal MK792311 MK883835 MK871450 MK828081 MK837932
D. cucurbitae DAOM 42078 Cucumis Canada KM453210 NA KM453212 KM453211 KP118848
D. cuppatea CBS 117499 Aspalathus linearis South Africa MH863021 KC343299 KC343541 KC343783 KC344025
D. cynaroidis CBS 122676 Protea cynaroides South Africa KC343058 KC343300 KC343542 KC343784 KC344026
D. cytosporella FAU461 Citrus limon Italy KC843307 KC843141 NA KC843116 KC843221
D. diospyricola CPC 21169 Diospyros whyteana South Africa KF777209 NA NA NA NA
D. discoidispora ZJUD89 Citrus unshiu China KJ490624 NA KJ490566 KJ490503 KJ490445
D. dorycnii MFLUCC 17-1015 Dorycnium hirsutum Italy KY964215 NA NA KY964171 KY964099
D. drenthii CBS 146453 Macadamia Australia MN708229 NA NA MN696526 MN696537
D. elaeagni-glabrae LC4802 Elaeagnus glabra China KX986779 KX999281 KX999251 KX999171 KX999212
D. ellipicola CGMCC 3.17084 Lithocarpus glaber China KF576270 NA NA KF576245 KF576294
D. endophytica CBS 133811 Schinus terebinthifolius Brazil KC343065 KC343307 KC343549 KC343791 KC344033
D. eres CBS 146.46 Alnus Netherlands KC343008 KC343250 KC343492 KC343734 KC343976
D. eres CBS 121004 Juglans USA KC343134 KC343376 KC343618 KC343860 KC344102
D. eres CGMCC 3.17081 Lithocarpus glabra China KF576282 NA NA KF576257 KF576306
D. eres CFCC 51632 Camptotheca acuminata China KY203726 KY228877 KY228881 KY228887 KY228893
D. eres CBS 139.27 Celastrus USA KC343047 KC343289 KC343531 KC343773 KC344015
D. eres CBS 143349 Vitis vinifera United Kingdom MG281017 MG281712 MG281363 MG281538 MG281190
D. eres AR5193 Ulmus Germany KJ210529 KJ434999 KJ420850 KJ210550 KJ420799
D. eres CFCC 52575 Castanea mollissima China MH121510 NA MH121470 MH121552 MH121592
D. eres CFCC 52576 Castanea mollissima China MH121511 MH121432 MH121471 MH121553 MH121593
D. eres CFCC 52577 Acanthopanax senticosus China MH121512 MH121433 MH121472 MH121554 MH121594
D. eres CFCC 52578 Sorbus China MH121513 MH121433 MH121473 MH121555 MH121595
D. eres CFCC 52579 Juglans regia China MH121514 NA MH121474 MH121556 NA
D. eres CFCC 52580 Melia azedarace China MH121515 NA MH121475 MH121557 MH121596
D. eres CFCC 52581 Rhododendron simsii China MH121516 NA MH121476 MH121558 MH121597
D. eres MAFF 625034 Pyrus pyrifolia Japan NA KJ435023 KJ420868 NA KJ420819
D. eres AR5211 Hedera helix France KJ210538 KJ435043 KJ420875 KJ210559 KJ420828
D. eres CGMCC 3.17089 Lithocarpus glabra China KF576267 NA NA KF576242 KF576291
D. eres MFLUCC 17-0963 Lonicera Italy KY964190 KY964116 NA KY964146 KY964073
D. eres DAOM 695742 Picea ruben Canada KU552025 NA NA KU552023 KU574615
D. eres MFLUCC 16-0113 Prunus persica China KU557563 NA KU557611 KU557631 KU55758
D. eres CBS 144.27 Spiraea USA KC343144 KC343386 KC343628 KC343870 KC344112
D. eres CBS 587.79 Pinus parviflora var Japan KC343153 KC343395 KC343637 KC343879 KC344121
D. eres CBS 338.89 Hedera helix Yugoslavia KC343152 KC343394 KC343636 KC343878 KC344120
D. eres MFLU 17-0646 Rosa United Kingdom MG828895 MG829274 NA MG829270 MG843877
D. eucalyptorum CBS 132525 Eucalyptus China MH305525 NA NA NA NA
D. foeniculacea CBS 111553 Foeniculum vulgare Spain MH854926 KC343343 KC343585 KC343827 KC344069
D. foeniculacea CFCC 54192 Quercus robur Netherlands MZ727033 NA MZ753474 MZ816339 MZ753483
D. foeniculacea M35 Quercus robur Netherlands MZ727034 NA MZ753475 MZ816340 MZ753484
D. foeniculacea M40-1 Quercus robur Netherlands MZ727035 NA MZ753476 MZ816341 MZ753485
D. foeniculacea M84 Quercus robur Netherlands MZ727036 NA MZ753477 MZ816342 MZ753486
D. fraxini-angustifoliae BRIP 54781 Fraxinus angustifolia Australia JX862528 KT459462 NA JX862534 NA
D. fraxinicola CFCC 52582 Fraxinus chinensis China MH121517 MH121435 NA MH121560 NA
D. fraxinicola CFCC 52583 Fraxinus chinensis China MH121518 MH121436 NA MH121559 NA
D. fulvicolor PSCG 051 Pyrus pyrifolia China MK626859 MK691132 MK726163 MK654806 MK691236
D. fusicola CGMCC 3.17087 Lithocarpus glabra China KF576281 KF576233 NA KF576256 KF576305
D. ganjae CBS 180.91 Cannabis sativa USA KC343112 KC343354 KC343596 KC343838 KC344080
D. ganzhouensis CFCC 53087 Unknown China MK432665 MK442985 MK443010 MK578139 MK578065
D. ganzhouensis CFCC 53088 Unknown China MK432666 MK442986 MK443011 MK578140 MK578066
D. garethjonesii MFLUCC 12-0542a Unknown Thailand KT459423 KT459470 NA KT459457 KT459441
D. goulteri BRIP 55657a Helianthus annuus Australia KJ197290 NA NA KJ197252 KJ197270
D. grandiflori SAUCC194.84 Heterostemma grandiflorum China MT822612 MT855691 MT855580 MT855809 MT855924
D. guangxiensis JZB320087 Vitis vinifera China MK335765 MK736720 NA MK500161 MK523560
D. gulyae BRIP 54025 Helianthus annuus Australia NA NA NA JN645803 KJ197271
D. guttulata CGMCC 3.20100 Unknown China MT385950 MW022470 MW022491 MT424685 MT424705
D. helianthi CBS 592.81 Helianthus annuus Serbia KC343115 KC343357 KC343599 KC343841 KC344083
D. heliconiae SAUCC194.77 Heliconia metallica China MT822605 MT855684 MT855573 MT855802 MT855917
D. heterophyllae CPC 26215 Acacia heterophylla France MG600222 MG600218 MG600220 MG600224 MG600226
D. heterostemmatis SAUCC194.85 Heterostemma grandiflorum China MT822613 MT855692 MT855581 MT855810 MT855925
D. hickoriae CBS 145.26 Carya glabra USA KC343118 KC343360 NA KC343844 KC344086
D. hispaniae CBS 143351 Vitis vinifera Spain MG281123 MG281820 MG281471 MG281644 MG281296
D. hongkongensis CBS 115448 Dichroa febrifuga China MK304388 KC343361 KC343603 KC343845 KC344087
D. hubeiensis JZB320123 Vitis vinifera China MK335809 MK500235 NA MK523570 MK500148
D. incompleta LC6754 Camellia sinensis China KX986794 KX999289 KX999265 KX999186 KX999226
D. inconspicua CBS 133813 Maytenus ilicifolia Brazil NA KC343365 KC343607 KC343849 KC344091
D. infecunda CBS 133812 Schinus terebinthifolius Brazil KC343126 KC343368 KC343610 KC343852 KC344094
D. irregularis CGMCC 3.20092 Unknown China MT385951 MT424721 NA MT424686 MT424706
D. isoberliniae CPC 22549 Isoberlinia angolensis Zambia KJ869190 NA NA NA KJ869245
D. juglandicola CFCC 51134 Juglans mandshurica China KU985101 KX024616 KX024622 KX024628 KX024634
D. kadsurae CFCC 52586 Kadsura longipedunculata China MH121521 MH121439 MH121479 MH121563 MH121600
D. kadsurae CFCC 52587 Kadsura longipedunculata China MH121522 MH121440 MH121480 MH121564 MH121601
D. kochmanii BRIP 54033 Helianthus annuus Australia NA NA NA JN645809 NA
D. kongii BRIP 54031 Helianthus annuus Australia NA NA NA NA KJ197272
D. lenispora CGMCC 3.20101 Unknown China MT385952 MW022472 MW022493 MT424687 MT424707
D. litchicola BRIP 54900 Litchi chinensis Australia LC041036 NA NA JX862539 NA
D. litchii SAUCC194.22 Litchi chinensis China MT822550 MT855635 MT855519 MT855747 MT855863
D. lithocarpus CGMCC 3.15175 Lithocarpus glabra China KC135104 KF576235 NA KC153095 KF576311
D. longicolla FAU599 Glycine max USA KJ590728 KJ612124 KJ659188 KJ590767 KJ610883
D. longispora CBS 194.36 Ribes Canada MH855769 KC343377 KC343619 KC343861 KC344103
D. lusitanicae CBS 123212 Foeniculum vulgare Portugal MH863279 KC343378 KC343620 KC343862 KC344104
D. lutescens SAUCC194.36 Chrysalidocarpus lutescens China MT822564 MT855647 MT855533 MT855761 MT855877
D. macadamiae CBS 146455 Macadamia Australia MN708230 NA NA MN696528 MN696539
D. macintoshii BRIP 55064a Rapistrum rugosum Australia KJ197289 NA NA KJ197251 KJ197269
D. mahothocarpus CGMCC 3.15181 Lithocarpus glabra China KC153096 NA NA KC153087 KF576312
D. malorum CAA 734 Malus domestica Portugal KY435638 KY435658 KY435648 KY435627 KY435668
D. masirevicii BRIP 54256 Glycine max Australia KJ197277 NA NA KJ197238 KJ197256
D. mayteni CBS 133185 Maytenus ilicifolia Brazil KC343139 KC343381 KC343623 KC343865 KC344107
D. maytenicola CPC 21896 Maytenus acuminata South Africa KF777157 NA NA NA KF777250
D. mediterranea SAUCC194.111 Machilus pingii China MT822639 MT855718 MT855606 MT855836 MT855951
D. melastomatis SAUCC194.55 Melastoma malabathricum China MT822583 MT855664 MT855551 MT855780 MT855896
D. melonis CBS 435.87 Glycine soja Indonesia KC343141 KC343383 KC343625 KC343867 KC344109
D. middletonii BRIP 54884e Rapistrum rugosum Australia KJ197286 NA NA KJ197248 KJ197266
D. minima CGMCC 3.20097 Unknown China MT385953 MT424722 MW022496 MT424688 MT424708
D. minusculata CGMCC 3.20098 Unknown China MT385957 MW022475 MW022499 MT424692 MT424712
D. miriciae BRIP 54736j Helianthus annuus Australia KJ197282 NA NA KJ197244 KJ197262
D. multigutullata CFCC 53095 Citrus maxima China MK432645 MK442967 MK442992 MK578121 MK578048
D. multigutullata CFCC 53096 Citrus maxima China MK432646 MK442968 MK442993 MK578122 MK578049
D. musigena CBS 129519 Musa Australia KC343143 KC343385 KC343267 KC343869 KC344111
D. neoarctii CBS 109490 Ambrosia trifida USA KC343145 KC343387 KC343629 KC343871 KC344113
D. neoraonikayaporum MFLUCC 14-1136 Tectona grandis Thailand KU712449 KU749356 NA KU749369 KU743988
D. nothofagi BRIP 54801 Nothofagus cunninghamii Australia JX862530 NA NA JX862536 KF170922
D. novem CBS 127269 Glycine max Croatia KC343155 KC343397 KC343639 KC343881 KC344123
D. ocoteae CPC 26217 Ocotea bullata France KX228293 NA NA NA KX228388
D. oraccinii LC3166 Camellia sinensis China KP267863 NA KP293517 KP267937 KP293443
D. ovalispora ZJUD93 Citrus limon China KJ490628 NA KJ490570 KJ490507 KJ490449
D. ovoicicola CGMCC 3.17093 Lithocarpus glabra China KF576265 KF576223 NA KF576240 KF576289
D. oxe CBS 133186 Maytenus ilicifolia Brazil KC343164 KC343406 KC343648 KC343890 KC344132
D. padina CFCC 52590 Padus racemosa China MH121525 MH121443 MH121483 MH121567 MH121604
D. padina CFCC 52591 Padus racemosa China MH121526 MH121444 MH121484 MH121568 MH121605
D. pandanicola MFLUCC 17-0607 Pandanaceae Thailand MG646974 NA NA NA MG646930
D. paranensis CBS 133184 Maytenus ilicifolia Brazil KC343171 KC343413 KC343655 KC343897 KC344139
D. parapterocarpi CPC 22729 Pterocarpus brenanii Zambia KJ869138 NA NA NA KJ869248
D. parvae PSCG 035 Pyrus bretschneideri China MK626920 MK691169 MK726211 MK654859 MK691249
D. pascoei BRIP 54847 Persea americana Australia MK111097 NA NA JX862538 KF170924
D. passiflorae CPC 19183 Passiflora edulis Netherlands JX069860 NA NA NA NA
D. passifloricola CPC 27480 Passiflora foetida Malaysia KX228292 NA KX228367 NA KX228387
D. penetriteum LC3215 Camellia sinensis China KP267879 NA NA KP293532 KP267953
D. perjuncta CBS 109745 Ulmus glabra Austria KC343172 KC343414 KC343656 KC343898 KC344140
D. perseae CBS 151.73 Persea gratissima Netherlands KC343173 KC343415 NA NA NA
D. pescicola MFLUCC 16-0105 Prunus persica China KU557555 KU557603 NA KY400831 KU557579
D. phaseolorum AR4203 Phaseolus vulgaris USA KJ590738 KJ612135 KJ659220 KJ590739 KJ610893
D. phillipsii CAA 817 Vaccinium corymbosum Portugal MK792305 MK883831 MK871445 MK828076 MN000351
D. podocarpi-macrophylli LC6155 Podocarpus macrophyllus Japan KX986774 KX999278 KX999246 KX999167 KX999207
D. pometiae SAUCC194.72 Pometia pinnata China MT822600 MT855679 MT855568 MT855797 MT855912
D. pseudoalnea CFCC 54190 Alnus glutinosa Netherlands MZ727037 MZ753468 MZ781302 MZ816343 MZ753487
D. pseudoalnea M2A Alnus glutinosa Netherlands MZ727038 MZ753469 MZ753478 MZ816344 MZ753488
D. pseudomangiferae CBS 101339 Mangifera indica Dominican Republic KC343181 KC343423 KC343665 KC343907 KC344149
D. pseudophoenicicola CBS 176.77 Mangifera indica Iraq KC343183 KC343425 KC343667 KC343909 KC344151
D. pseudotsugae MFLU 15-3228 Pseudotsuga menziesii Italy KY964225 KY964138 NA KY964181 KY964108
D. psoraleae CPC 21634 Psoralea pinnata South Africa KF777158 NA NA KF777245 KF777251
D. psoraleae-pinnatae CPC 21638 Psoralea pinnata South Africa KF777159 NA NA NA KF777252
D. pterocarpicola MFLUCC 10-0580a Pterocarpus indicus Thailand JQ619887 JX197433 NA JX275403 JX275441
D. pungensis SAUCC194.112 Elaeagnus pungens China MT822640 MT855719 MT855607 MT855837 MT855952
D. pyracanthae CAA483 Pyracantha coccinea Portugal KY435635 KY435645 KY435656 KY435625 KY435666
D. racemosae CPC 26646 Euclea racemosa South Africa MG600223 MG600219 MG600221 MG600225 MG600227
D. raonikayaporum CBS 133182 Spondias mombin Brazil KC343188 KC343430 KC343672 KC343914 KC344156
D. ravennica MFLUCC 16-0997 Clematis vitalba Italy NA NA NA MT394670 NA
D. rhusicola CPC 18191 Rhus pendulina South Africa JF951146 NA NA NA NA
D. rosae MFLUCC 17-2658 Rosa United Kingdom MG828894 MG829273 NA NA MG843878
D. rosiphthora COAD 2914 Rosa Brazil MT311197 MT313691 NA MT313693 NA
D. rossmaniae CAA 762 Vaccinium corymbosum Portugal MK792290 MK883822 MK871432 MK828063 MK837914
D. rostrata CFCC 50062 Juglans mandshurica China KP208847 KP208849 KP208851 KP208853 KP208855
D. rostrata CFCC 50063 Juglans mandshurica China KP208848 KP208850 KP208852 KP208854 KP208856
D. rudis AR3422 Laburnum anagyroides Austria KC843331 KC843146 NA KC843090 KC843177
D. rudis CFCC 54193 Quercus robur Netherlands MZ727039 MZ753470 MZ753479 MZ816345 MZ753489
D. rudis M86 Quercus robur Netherlands MZ727040 MZ753471 MZ753480 MZ816346 MZ753490
D. saccarata CBS 116311 Protea repens South Africa KC343190 KC343432 KC343674 KC343916 KC344158
D. sackstonii BRIP 54669b Helianthus annuus Australia KJ197287 NA NA KJ197249 KJ197267
D. salicicola BRIP 54825 Salix purpurea Australia JX862531 NA NA JX862537 KF170923
D. sambucusii CFCC 51986 Sambucus williamsii China KY852495 KY852499 KY852503 KY852507 KY852511
D. sambucusii CFCC 51987 Sambucus williamsii China KY852496 KY852500 KY852504 KY852508 KY852512
D. schimae CFCC 53103 Schima superba China MK442640 MK442962 MK442987 MK578116 MK578043
D. schimae CFCC 53104 Schima superba China MK442641 MK442963 MK442988 MK578117 MK578044
D. schimae CFCC 53105 Schima superba China MK442642 MK442964 MK442989 MK578118 MK578045
D. schini CBS 133181 Schinus terebinthifolius Brazil KC343191 KC343433 KC343675 KC343917 KC344159
D. schisandrae CFCC 51988 Schisandra chinensis China KY852497 KY852501 KY852505 KY852509 KY852513
D. schisandrae CFCC 51989 Schisandra chinensis China KY852498 KY852502 KY852506 KY852510 KY852514
D. schoeni MFLU 15-1279 Schoenus nigricans Italy KY964226 KY964139 NA KY964182 KY964109
D. sclerotioides CBS 296.67 Cucumis sativus Netherlands MH858974 KC343435 KC343677 KC343919 KC344161
D. searlei CBS 146456 Macadamia Australia MN708231 NA NA NA MN696540
D. sennae CFCC 51636 Senna bicapsularis China KY203724 KY228875 NA KY228885 KY228891
D. sennae CFCC 51637 Senna bicapsularis China KY203725 KY228876 NA KY228886 KY228892
D. sennicola CFCC 51634 Senna bicapsularis China KY203722 KY228873 KY228879 KY228883 KY228889
D. sennicola CFCC 51635 Senna bicapsularis China KY203723 KY228874 KY228880 KY228884 KY228890
D. serafiniae BRIP 55665a Helianthus annuus Australia KJ197274 NA NA KJ197236 KJ197254
D. shaanxiensis CFCC 53106 on branches of liana China MK432654 MK442976 MK443001 MK578130 NA
D. shaanxiensis CFCC 53107 on branches of liana China MK432655 MK432977 MK432002 MK578131 NA
D. siamensis MFLUCC 10-0573a Dasymaschalon Thailand NA JQ619897 NA JX275393 JX275429
D. silvicola CFCC 54191 Fraxinus excelsior Netherlands MZ727041 MZ753472 MZ753481 MZ816347 MZ753491
D. silvicola M79 Fraxinus excelsior Netherlands MZ727042 MZ753473 MZ753482 MZ816348 MZ753492
D. sojae FAU635 Glycine max USA KJ590719 KJ612116 KJ659208 KJ590762 KJ610875
D. spartinicola CPC 24951 Spartium junceμm Spain KR611879 NA KR857696 NA KR857695
D. spinosa PSCG 383 Pyrus pyrifolia China MK626849 MK691129 MK726156 MK654811 MK691234
D. sterilis CBS 136969 Vaccinium corymbosum Italy KJ160579 KJ160548 MF418350 KJ160611 KJ160528
D. stictica CBS 370.54 Buxus sampervirens Italy KC343212 KC343454 KC343696 KC343938 KC344180
D. subclavata ZJUD95 Citrus unshiu China KJ490630 NA KJ490572 KJ490509 KJ490451
D. subcylindrospora KUMCC 17-0151 Unknown China MG746629 NA NA MG746630 MG746631
D. subellipicola KUMCC 17-0153 Unknown China MG746632 NA NA MG746633 MG746634
D. subordinaria CBS 464.90 Plantago lanceolata South Africa KC343214 KC343456 KC343698 KC343940 KC344182
D. taoicola MFLUCC 16-0117 Prunus persica China KU557567 NA NA KU557636 KU557591
D. tectonae MFLUCC 12-0777 Tectona grandis Thailand KU712430 KU749345 NA KU749359 KU743977
D. tectonendophytica MFLUCC 13-0471 Tectona grandis Thailand KU712439 KU749354 NA KU749367 KU743986
D. tectonigena MFLUCC 12-0767 Camellia sinensis China KX986782 KX999284 KX999254 KX999174 KX999214
D. terebinthifolii CBS 133180 Schinus terebinthifolius Brazil KC343216 KC343458 KC343700 KC343942 KC344184
D. ternstroemia CGMCC 3.15183 Ternstroemia gymnanthera China KC153098 NA NA KC153089 NA
D. thunbergii MFLUCC 10-0576a Thunbergia laurifolia Thailand JQ619893 JX197440 NA JX275409 NA
D. thunbergiicola MFLUCC 12-0033 Thunbergia laurifolia Thailand KP715097 NA NA KP715098 NA
D. tibetensis CFCC 51999 Juglandis regia China MF279843 MF279888 MF279828 MF279858 MF279873
D. tibetensis CFCC 52000 Juglandis regia China MF279844 MF279889 MF279829 MF279859 MF279874
D. torilicola MFLUCC 17-1051 Torilis arvensis Italy KY964212 KY964127 NA KY964168 KY964096
D. toxica CBS 534.93 Lupinus angustifolius Australia KC343220 KC343462 KC343704 KC343946 KC344188
D. tulliensis BRIP 62248a Theobroma cacao Australia KR936130 NA NA KR936133 KR936132
D. ueckerae FAU656T Cucumis melo USA KJ590726 KJ612122 KJ659215 KJ590747 KJ610881
D. ukurunduensis CFCC 52592 Acer ukurunduense China MH121527 MH121445 MH121485 MH121569 NA
D. ukurunduensis CFCC 52593 Acer ukurunduense China MH121528 MH121446 MH121486 MH121570 NA
D. undulata LC6624 Unknown China KX986798 NA KX999269 KX999190 KX999230
D. unshiuensis ZJUD52 Citrus unshiu China KJ490587 NA KJ490529 KJ490466 KJ490408
D. unshiuensis CFCC 52594 Carya illinoensis China MH121529 MH121447 MH121487 MH121571 MH121606
D. unshiuensis CFCC 52595 Carya illinoensis China MH121530 MH121448 MH121488 MH121572 MH121607
D. vaccinii CBS 160.32 Oxycoccus macrocarpos USA MH121502 MH121426 MH121462 MH121544 MH121584
D. vangueriae CBS 137985 Vangueria infausta Zambia KJ869137 NA NA NA KJ869247
D. vawdreyi BRIP 57887a Psidium guajava Australia KR936126 NA NA KR936129 KR936128
D. velutina LC4421 Neolitsea China KX986790 NA KX999261 KX999182 KX999223
D. verniciicola CFCC 53109 Vernicia montana China MK573944 MK574583 MK574599 MK574619 MK574639
D. verniciicola CFCC 53110 Vernicia montana China MK573945 MK574584 MK574600 MK574620 MK574640
D. viniferae JZB320071 Vitis vinifera China MK341551 MK500119 NA MK500107 MK500112
D. virgiliae CMW 40748 Virgilia oroboides South Africa KP247556 NA NA NA KP247575
D. xishuangbanica LC6707 Camellia sinensis China KX986783 NA KX999255 KX999175 KX999216
D. xunwuensis CFCC 53085 Unknown China MK432663 MK442983 MK443008 MK578137 MK578063
D. xunwuensis CFCC 53086 Unknown China MK432664 MK442984 MK443009 MK578138 MK578064
D. yunnanensis LC6168 Unknown China KX986796 KX999290 KX999267 KX999188 KX999228
D. zaobaisu PSCG 031 Pyrus bretschneideri China MK626922 NA MK726207 MK654855 MK691245
Diaporthella corylina CBS 121124 Corylus NA KC343004 KC343246 KC343488 KC343730 KC343972

Results

Phylogenetic analyses

The five-gene sequence dataset (ITS, cal, his3, tef1 and tub2) was analysed to infer the interspecific relationships within Diaporthe. The dataset consisted of 307 sequences including one outgroup taxon, Diaporthella corylina (CBS 121124). A total of 2649 characters including gaps (516 for ITS, 576 for cal, 526 for his3, 507 for tef1 and 524 for tub2) were included in the phylogenetic analysis. Of these characters, 844 were constant, 318 were variable and parsimony-uninformative, and 1487 were parsimony-informative. The topologies resulting from ML and BI analyses of the concatenated dataset were congruent (Fig. 1). Isolates from the present study formed four individual clades representing four species of Diaporthe, of which isolates CFCC 54192, M35, M40-1 and M84 from Quercus robur represent D. foeniculacea, while CFCC 54193 and M86 from Q. robur represent D. rudis. CFCC 54191 and M79 from Fraxinus excelsior and CFCC 54190 and M2A from Alnus glutinosa represent two new species which are here described as D. silvicola and D. pseudoalnea, respectively.

Figure 1. 

Phylogram of Diaporthe resulting from a maximum likelihood analysis based on a combined matrix of ITS, cal, his3, tef1 and tub2. Numbers above the branches indicate ML bootstraps (left, ML BS ≥ 50 %) and Bayesian Posterior Probabilities (right, BPP ≥ 0.75). The tree is rooted with Diaporthella corylina. Isolates from present study are marked in blue.

Taxonomy

Diaporthe pseudoalnea N. Jiang, sp. nov.

MycoBank No: 840714
Fig. 2

Etymology

With reference to D. alnea, which was described from the same host genus, Alnus.

Description

Conidiomata pycnidial, discoid, immersed in bark, scattered, erumpent through the bark surface, with a solitary locule. Locule 800–1250 μm diam., undivided. Conidiophores 22–68.5 × 1.5–3 μm (av. = 39.8 × 2.2 μm, n = 50), cylindrical, attenuate towards the apex, hyaline, slightly brown at base, phialidic, unbranched, straight or slightly curved. Alpha conidia (5.8–)7.1–8.9(–11.2) × (1.5–)1.8–2.2(–2.7) μm (av. = 7.9 × 2.0 μm, n = 50), L/W = 3.2–4.7 (av. = 3.8, n = 50), hyaline, aseptate, subcylindrical with a nearly rounded apex, multi-guttulate, sometimes acute at both ends. Beta conidia not observed.

Figure 2. 

Diaporthe pseudoalnea from Alnus glutinosa A–C habit of conidiomata on branches D transverse section of conidiomata E longitudinal section through conidiomata F, G conidiophores and conidia H, I conidia. Scale bars: 2 mm (A), 500 μm (B, C, E), 200 μm (D), 10 μm (F–I).

Culture characters

Colonies are initially white with fluffy aerial mycelium, becoming dirty white after 2 weeks, and conidiomata are randomly distributed with orange conidial drops oozing out of the ostioles.

Specimens examined

NETHERLANDS. Utrecht City, on branches of Alnus glutinosa, 5°11’32” E, 52°05’22” N, 8 Apr. 2019, N. Jiang (holotype CAF800005 = JNH0001; ex-type living culture: CFCC 54190; other living culture: M2A).

Notes

Diaporthe nivosa and D. alnea were recorded from the host genus Alnus. Udayanga et al. (2014) investigated the lectotype of Diaporthe nivosa and revealed it as a Melanconis species based on a well-developed ectostromata and the ascospores characteristics, and Jaklitsch and Voglmayr (2020) treated it as a synonym of Melanconis marginalis ssp. marginalis. D. alnea has been reported from the Czech Republic, Germany, the Netherlands and the USA, and both sexual and asexual morphs have been described (Udayanga et al. 2014). However, applying the GCPSR principle, D. alnea has recently been considered to be a synonym of Diaporthe eres (Hilário et al. 2021), which has also been confirmed in our analyses where the ex-epitype isolate CBS 146.46 of D. alnea is placed within the D. eres clade (Fig. 1). Diaporthe pseudoalnea morphologically differs from D. alnea (now D. eres) by its longer conidiophores (22–68.5 × 1.5–3 μm in D. pseudoalnea vs. 9–16 × 1–2 μm in D. alnea; Udayanga et al. 2014). In our multi-gene analyses, D. pseudoalnea forms a distinct phylogenetic lineage which is placed remotely from the isolate CBS 146.46 of D. alnea (Fig. 1).

Diaporthe silvicola N. Jiang, sp. nov.

MycoBank No: 840715
Fig. 3

Etymology

Name from “silva” = forest and “-cola” = inhabiting; with reference to its woody host.

Description

Conidiomata pycnidial, conical, immersed in bark, scattered, erumpent through the bark surface, with a solitary locule. Locule 450–700 μm diam., undivided. Conidiophores 6.5–25 × 1.5–4 μm (av. = 15.4 × 2.4 μm, n = 50), cylindrical, attenuate towards the apex, hyaline, slightly brown, phialidic, unbranched, slightly curved. Alpha conidia (9.2–)10.1–12.3(–13.5) × (3.8–)4.2–4.9(–5.2) μm (av. = 11.5 × 4.5 μm, n = 50), L/W = 2.0–3.2 (av. = 2.5, n = 50), hyaline, aseptate, fusiform to oval, multi-guttulate, acute at both ends. Beta conidia not observed.

Figure 3. 

Diaporthe silvicola from Fraxinus excelsior A–C habit of conidiomata on branches D transverse section of conidiomata E longitudinal section through conidiomata F, I conidia G, H conidiophores and conidia. Scale bars: 2 mm (A), 1 mm (B), 500 μm (C), 200 μm (D, E), 10 μm (F–I).

Culture characters

Colonies are initially white, aerial mycelium turning grey at edges of plate, yellowish pigmentation developing in centre, conidiomata not produced until 2 weeks.

Specimens examined

NETHERLANDS. Utrecht City, on branches of Fraxinus excelsior in the forest ecosystem, 5°10’36” E, 52°05’32” N, 6 Jun. 2019, N. Jiang (holotype CAF800006 = JNH0002; ex-type living culture: CFCC 54191; other living culture: M79).

Notes

Diaporthe fraxini-angustifoliae was reported from Fraxinus angustifolia subsp. oxycarpa cv. Claret Ash in Australia (Tan et al. 2013). D. fraxinicola was described from Fraxinus chinensis in China (Yang et al. 2018). However, D. silvicola from Fraxinus excelsior in Netherlands differs from D. fraxini-angustifoliae and D. fraxinicola by obviously larger alpha conidia (9.2–13.5 × 3.8–5.2 μm in D. silvicola vs. 4–10 × 2–3 μm in D. fraxini-angustifoliae vs. 7–10 × 2.9–3.2 μm in D. fraxinicola; Tan et al. 2013; Yang et al. 2018).

Discussion

In this study, branch-inhabiting Diaporthe species were sampled from Alnus glutinosa, Fraxinus excelsior and Quercus robur in Utrecht, the Netherlands. Ten Diaporthe isolates were obtained and identified based on five combined loci (ITS, cal, his3, tef1 and tub2), as well as morphological characters from the natural substrates. The phylogenetic and morphological analyses revealed Diaporthe pseudoalnea sp. nov. from Alnus glutinosa, Diaporthe silvicola sp. nov. from Fraxinus excelsior, and D. foeniculacea and D. rudis from Quercus robur.

Phylogenetic analyses were conducted based on a combined DNA sequence matrix of five loci (ITS, cal, his3, tef1 and tub2) reported as useful markers to distinguish species of Diaporthe (Udayanga et al. 2014, 2015; Guarnaccia et al. 2017, 2018a, 2018b; Tibpromma et al. 2018; Yang et al. 2020; Dissanayake et al. 2020; Huang et al. 2021; Sun et al. 2021, Wang et al. 2021). The two novel species in this study can be distinguished from the other known species by all genes studied, but most effectively by cal, his3, tef1 and tub2. The multi-locus phylogenetic analysis grouped the isolates in two new clades, which support the introduction of the new species.

The utility of host association for Diaporthe species identification is limited because several species have wide host ranges (e.g., D. ere inhabits 282 different hosts; D. rudis inhabits 44 different hosts), and multiple Diaporthe species can infect a single host (e.g., nineteen Diaporthe species are associated with pear cankers in China) (Guo et al. 2020; Farr and Rossman 2021). Thus, a polyphasic approach of morphological, cultural, ecological and molecular data to identify Diaporthe samples or to introduce new species is essential.

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-2021-7).

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