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High diversity of Diaporthe species associated with dieback diseases in China, with twelve new species described
expand article infoQin Yang, Xin-Lei Fan, Vladimiro Guarnaccia§|, Cheng-Ming Tian
‡ Beijing Forestry University, Beijing, China
§ Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands
| University of Stellenbosch, Matieland, South Africa
Open Access

Abstract

Diaporthe species have often been reported as important plant pathogens, saprobes and endophytes on a wide range of plant hosts. Although several Diaporthe species have been recorded in China, little is known about species able to infect forest trees. Therefore, extensive surveys were recently conducted in Beijing, Heilongjiang, Jiangsu, Jiangxi, Shaanxi and Zhejiang Provinces. The current results emphasised on 15 species from 42 representative isolates involving 16 host genera using comparisons of DNA sequence data for the nuclear ribosomal internal transcribed spacer (ITS), calmodulin (cal), histone H3 (his3), partial translation elongation factor-1α (tef1) and β-tubulin (tub2) gene regions, as well as their morphological features. Three known species, D. biguttulata, D. eres and D. unshiuensis, were identified. In addition, twelve novel taxa were collected and are described as D. acerigena, D. alangii, D. betulina, D. caryae, D. cercidis, D. chensiensis, D. cinnamomi, D. conica, D. fraxinicola, D. kadsurae, D. padina and D. ukurunduensis. The current study improves the understanding of species causing diebacks on ecological and economic forest trees and provides useful information for the effective disease management of these hosts in China.

Keywords

Dieback, DNA phylogeny, Systematics, Taxonomy

Introduction

The genus Diaporthe Nitschke represents a cosmopolitan group of fungi occupying diverse ecological behaviour as plant pathogens, endophytes and saprobes (Muralli et al. 2006, Rossman et al. 2007, Garcia-Reyne et al. 2011, Udayanga et al. 2011, 2012a, b, 2014a, b, 2015, Gomes et al. 2013, Fan et al. 2015, Du et al. 2016, Dissanayake et al. 2017b, Guarnaccia and Crous 2017, Yang et al. 2017a, b, 2018, Guarnaccia et al. 2018, Marin-Felix et al. 2018). Diaporthe species are responsible for diseases on a wide range of plant hosts, including agricultural crops, forest trees and ornamentals, some of which are economically important. Several symptoms such as root and fruit rots, dieback, stem cankers, leaf spots, leaf and pod blights and seed decay are caused by Diaporthe spp. (Uecker 1988, Rehner and Uecker 1994, Mostert et al. 2001, Santos et al. 2011, Thompson et al. 2011, Udayanga et al. 2011). For example, D. ampelina, the causal agent of Phomopsis cane and leaf spot, is known as a severe pathogen of grapevines (Hewitt and Pearson 1988), infecting all green tissues and causing yield reductions of up to 30% in temperate regions (Erincik et al. 2001). Diaporthe citri is another well-known pathogen exclusively found on Citrus spp. causing melanose, stem-end rot and gummosis in all the citrus production areas except Europe (Mondal et al. 2007, Udayanga et al. 2014a, Guarnaccia and Crous 2017, 2018). Similarly, stem canker, attributed to several Diaporthe spp., is one of the most important diseases of sunflower (Helianthus annuus) worldwide (Muntañola-Cvetković et al. 1981, Thompson et al. 2011).

Several species of Diaporthe include a broad number of endophytes associated with hosts present in temperate and tropical regions (Udayanga et al. 2011). Gomes et al. (2013) considered that D. endophytica is a sterile endophyte on Schinus terebinthifolius and Maytenus ilicifolia based on molecular phylogeny. Huang et al. (2015) distinguished seven undescribed Diaporthe species associated with citrus in China. Moreover, some endophytes have been shown to act as opportunistic plant pathogens. For instance, D. foeniculina has been found as both endophyte and opportunistic pathogen on various herbaceous weeds, ornamentals and fruit trees (Udayanga et al. 2014a, Guarnaccia et al. 2016).

The genus Diaporthe (syn. Phomopsis) was established by Nitschke (1870). Species identification criteria in Diaporthe were originally based on host association, morphology and culture characteristics (Mostert et al. 2001, Santos and Phillips 2009, Udayanga et al. 2012). As a consequence, a broad increase in the number of proposed Diaporthe species occurred. More than 1000 epithets for Diaporthe and 950 for Phomopsis were listed in Index Fungorum (2018) (http://www.indexfungorum.org/) (accessed 1 March 2018). The abolishment of the dual nomenclature system for pleomorphic fungi raised the question about which generic name to use. Given that both names are well known amongst plant pathologists and have been equally used, Rossman et al. (2015) proposed that the name Diaporthe (Nitschke 1870) has priority over Phomopsis (Saccardo and Roumeguère 1884) and has been adopted as the generic name in recent major studies (Gomes et al. 2013, Udayanga et al. 2014a, b, 2015, Fan et al. 2015, Huang et al. 2015, Du et al. 2016, Gao et al. 2017, Yang et al. 2017a, b, c, 2018).

The sexual morph of Diaporthe is characterised by immersed ascomata and an erumpent pseudostroma with elongated perithecial necks. Asci are unitunicate, clavate to cylindrical. Ascospores are fusoid, ellipsoid to cylindrical, hyaline, biseriate to uniseriate in the ascus and sometimes with appendages (Udayanga et al. 2011). The asexual morph is characterised by ostiolate conidiomata, with cylindrical phialides producing three types of hyaline, aseptate conidia (Udayanga et al. 2011). Previously, species identification of Diaporthe was largely referred to the assumption of host-specificity, leading to the proliferation of names (Gomes et al. 2013). More than one species of Diaporthe can colonise a single host, while one species can be associated with different hosts (Santos and Phillips 2009, Diogo et al. 2010, Santos et al. 2011, Gomes et al. 2013). In addition, considerable variability of the phenotype characters is present within a species (Rehner and Uecker 1994, Mostert et al. 2001, Santos et al. 2010, Udayanga et al. 2011, 2012a). Species identification is essential for understanding the epidemiology and plant diseases management and to guide the implementation of phytosanitary measures (Santos and Phillips 2009, Udayanga et al. 2011, Santos et al. 2017). Thus, molecular data are necessary to resolve Diaporthe taxonomy and, during the recent years, many species have been described through a polyphasic approach together with morphology (Gomes et al. 2013, Udayanga et al. 2014a, b, 2015, Huang et al. 2015, Gao et al. 2017, Guarnaccia and Crous 2017, Yang et al. 2018). Santos et al. (2017) revealed that the use of a five-loci dataset (ITS-cal-his3-tef1-tub2) is the optimal combination for species delimitation, showing the ribosomal ITS locus as the least informative, which is contrary to the result of Santos et al. (2010).

Although the classification of Diaporthe has been on-going, species are currently being identified based on a combination of morphological, cultural, phytopathological and phylogenetical analyses (Gomes et al. 2013, Huang et al. 2013, 2015, Udayanga et al. 2014a, b, 2015, Fan et al. 2015, Du et al. 2016, Gao et al. 2016, 2017, Guarnaccia and Crous 2017, Hyde et al. 2017, 2018, Guarnaccia et al. 2018, Jayawardena et al. 2018, Perera et al. 2018a, b, Tibpromma et al. 2018, Wanasinghe et al. 2018). However, fungi isolated from forest trees in China were recorded in old fungal literature without any living culture and molecular data (Teng 1963, Tai 1979, Wei 1979). The current study aimed to investigate the major ecological or economic trees in China by large-scale sampling and to identify isolates via morphology and multi-locus phylogeny based on modern taxonomic concepts. From 2015 to 2017, several surveys were conducted in six Provinces representing 16 host genera. The objectives of the present study were (i) to provide a multi-gene phylogeny for the genus Diaporthe based on a large set of freshly collected specimens in China; (ii) to identify Diaporthe taxa associated with disease symptoms or non-symptomatic tissues of various host genera distributed over six Provinces in China; (iii) to define the species limits of D. eres and closely related species based on multi-gene genealogies.

Materials and methods

Isolates

From 2015 to 2017, fresh specimens of Diaporthe were collected from symptomatic or non-symptomatic twigs or branches from Beijing, Heilongjiang, Jiangsu, Jiangxi, Shaanxi and Zhejiang Provinces in China (Table 1). A total of 105 isolates were obtained by removing a mucoid spore mass from conidiomata and spreading the suspension on the surface of 1.8% potato dextrose agar (PDA) in a Petri dish and incubating at 25 °C for up to 24 h. Single germinating conidia were transferred on to fresh PDA plates. Forty-two representative Diaporthe strains were selected based on cultural characteristics on PDA, conidia morphology and ITS sequence data. Specimens were deposited in the Museum of the Beijing Forestry University (BJFC). Axenic cultures are maintained in the China Forestry Culture Collection Centre (CFCC).

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

Species Isolate Host Location GenBank accession numbers
ITS cal his3 tef1 tub2
D. acaciarum CBS 138862 Acacia tortilis Tanzania KP004460 N/Aa N/Aa N/Aa KP004509
D. acaciigena CBS 129521 Acacia retinodes Australia KC343005 KC343247 KC343489 KC343731 KC343973
D. acericola MFLUCC 17-0956 Acer negundo Italy KY964224 KY964137 N/Aa KY964180 KY964074
D. acerigena CFCC 52554 Acer tataricum China MH121489 MH121413 MH121449 MH121531 N/Aa
CFCC 52555 Acer tataricum China MH121490 MH121414 MH121450 MH121532 N/Aa
D. acutispora CGMCC 3.18285 Coffea sp. China KX986764 KX999274 N/Aa KX999155 KX999195
D. alangii CFCC 52556 Alangium kurzii China MH121491 MH121415 MH121451 MH121533 MH121573
CFCC 52557 Alangium kurzii China MH121492 MH121416 MH121452 MH121534 MH121574
CFCC 52558 Alangium kurzii China MH121493 MH121417 MH121453 MH121535 MH121575
CFCC 52559 Alangium kurzii China MH121494 MH121418 MH121454 MH121536 MH121576
D. alleghaniensis CBS 495.72 Betula alleghaniensis Canada KC343007 KC343249 KC343491 KC343733 KC343975
D. alnea CBS 146.46 Alnus sp. Netherlands KC343008 KC343250 KC343492 KC343734 KC343976
D. ambigua CBS 114015 Pyrus communis South Africa KC343010 KC343252 KC343494 KC343736 KC343978
D. ampelina STEU2660 Vitis vinifera France AF230751 AY745026 N/Aa AY745056 JX275452
D. amygdali CBS 126679 Prunus dulcis Portugal KC343022 KC343264 KC343506 AY343748 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 CGMCC 3.17533 Camellia sinensis China KP267896 N/Aa N/Aa KP267970 KP293476
D. aquatica IFRDCC 3051 Aquatic habitat China JQ797437 N/Aa N/Aa N/Aa N/Aa
D. arctii CBS 139280 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 enngleri Hong Kong KC343034 KC343276 KC343518 KC343760 KC344002
D. aseana MFLUCC 12-0299a Unknown dead leaf Thailand KT459414 KT459464 N/Aa KT459448 KT459432
D. asheicola CBS 136967 Vaccinium ashei Chile KJ160562 KJ160542 N/Aa 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. baccae CBS 136972 Vaccinium corymbosum Italy KJ160565 N/Aa MF418264 KJ160597 N/Aa
D. batatas CBS 122.21 Ipomoea batatas USA KC343040 KC343282 N/Aa KC343766 KC344008
D. beilharziae BRIP 54792 Indigofera australis Australia JX862529 N/Aa N/Aa JX862535 KF170921
D. benedicti BPI 893190 Salix sp. USA KM669929 KM669862 N/Aa KM669785 N/Aa
D. betulae CFCC 50469 Betula platyphylla China KT732950 KT732997 KT732999 KT733016 KT733020
CFCC 50470 Betula platyphylla China KT732951 KT732998 KT733000 KT733017 KT733021
D. betulicola CFCC 51128 Betula albo-sinensis China KX024653 KX024659 KX024661 KX024655 KX024657
CFCC 51129 Betula albo-sinensis China KX024654 KX024660 KX024662 KX024656 KX024658
D. betulina CFCC 52560 Betula albo-sinensis China MH121495 MH121419 MH121455 MH121537 MH121577
CFCC 52561 Betula costata China MH121496 MH121420 MH121456 MH121538 MH121578
CFCC 52562 Betula platyphylla China MH121497 MH121421 MH121457 MH121539 MH121579
D. bicincta CBS 121004 Juglans sp. USA KC343134 KC343376 KC343618 KC343860 KC344102
D. biconispora CGMCC 3.17252 Citrus grandis China KJ490597 KJ490539 KJ490539 KJ490476 KJ490418
D. biguttulata CGMCC 3.17248 Citrus limon China KJ490582 N/Aa KJ490524 KJ490461 KJ490403
CFCC 52584 Juglans regia China MH121519 MH121437 MH121477 MH121561 MH121598
CFCC 52585 Juglans regia China MH121520 MH121438 MH121478 MH121562 MH121599
D. biguttusis CGMCC 3.17081 Lithocarpus glabra China KF576282 N/Aa N/Aa KF576257 KF576306
D. bohemiae CPC 28222 Vitis vinifera Czech Republic MG281015 MG281710 MG281361 MG281536 MG281188
D. brasiliensis CBS 133183 Aspidosperma tomentosum Brazil KC343042 KC343284 KC343526 KC343768 KC344010
D. caatingaensis CBS 141542 Tacinga inamoena Brazil KY085927 N/Aa N/Aa KY115603 KY115600
D. camptothecicola CFCC 51632 Camptotheca acuminata China KY203726 KY228877 KY228881 KY228887 KY228893
D. canthii CBS 132533 Canthium inerme South Africa JX069864 KC843174 N/Aa KC843120 KC843230
D. caryae CFCC 52563 Carya illinoensis China MH121498 MH121422 MH121458 MH121540 MH121580
CFCC 52564 Carya illinoensis China MH121499 MH121423 MH121459 MH121541 MH121581
D. cassines CPC 21916 Cassine peragua South Africa KF777155 N/Aa N/Aa KF777244 N/Aa
D. caulivora CBS 127268 Glycine max Croatia KC343045 KC343287 N/Aa KC343771 KC344013
D. celeris CPC 28262 Vitis vinifera Czech Republic MG281017 MG281712 MG281363 MG281538 MG281190
D. celastrina CBS 139.27 Celastrus sp. USA KC343047 KC343289 KC343531 KC343773 KC344015
D. cercidis CFCC 52565 Cercis chinensis China MH121500 MH121424 MH121460 MH121542 MH121582
CFCC 52566 Cercis chinensis China MH121501 MH121425 MH121461 MH121543 MH121583
D. chamaeropis CBS 454.81 Chamaerops humilis Greece KC343048 KC343290 KC343532 KC343774 KC344016
D. charlesworthii BRIP 54884m Rapistrum rugostrum Australia KJ197288 N/Aa N/Aa KJ197250 KJ197268
D. chensiensis CFCC 52567 Abies chensiensis China MH121502 MH121426 MH121462 MH121544 MH121584
CFCC 52568 Abies chensiensis China MH121503 MH121427 MH121463 MH121545 MH121585
D. cichorii MFLUCC 17-1023 Cichorium intybus Italy KY964220 KY964133 N/Aa KY964176 KY964104
D. cinnamomi CFCC 52569 Cinnamomum sp. China MH121504 N/Aa MH121464 MH121546 MH121586
CFCC 52570 Cinnamomum sp. China MH121505 N/Aa MH121465 MH121547 MH121587
D. cissampeli CBS 141331 Cissampelos capensis South Africa KX228273 N/Aa KX228366 N/Aa KX228384
D. citri AR 3405 Citrus sp. USA KC843311 KC843157 N/Aa KC843071 KC843187
D. citriasiana CGMCC 3.15224 Citrus unshiu China JQ954645 KC357491 KJ490515 JQ954663 KC357459
D. citrichinensis CGMCC 3.15225 Citrus sp. China JQ954648 KC357494 N/Aa JQ954666 N/Aa
D. collariana MFLU 17-2770 Magnolia champaca Thailand MG806115 MG783042 N/Aa MG783040 MG783041
D. compacta CGMCC 3.17536 Camellia sinensis China KP267854 N/Aa KP293508 KP267928 KP293434
D. conica CFCC 52571 Alangium chinense China MH121506 MH121428 MH121466 MH121548 MH121588
CFCC 52572 Alangium chinense China MH121507 MH121429 MH121467 MH121549 MH121589
CFCC 52573 Alangium chinense China MH121508 MH121430 MH121468 MH121550 MH121590
CFCC 52574 Alangium chinense China MH121509 MH121431 MH121469 MH121551 MH121591
D. convolvuli CBS 124654 Convolvulus arvensis Turkey KC343054 KC343296 KC343538 KC343780 KC344022
D. crotalariae CBS 162.33 Crotalaria spectabilis USA KC343056 KC343298 KC343540 KC343782 KC344024
D. cucurbitae CBS 136.25 Arctium sp. Unknown KC343031 KC343273 KC343515 KC343757 KC343999
D. cuppatea CBS 117499 Aspalathus linearis South Africa KC343057 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 N/Aa KC843116 KC843221
D. diospyricola CPC 21169 Diospyros whyteana South Africa KF777156 N/Aa N/Aa N/Aa N/Aa
D. discoidispora ZJUD89 Citrus unshiu China KJ490624 N/Aa KJ490566 KJ490503 KJ490445
D. dorycnii MFLUCC 17-1015 Dorycnium hirsutum Italy KY964215 N/Aa N/Aa KY964171 KY964099
D. elaeagni-glabrae CGMCC 3.18287 Elaeagnus glabra China KX986779 KX999281 KX999251 KX999171 KX999212
D. ellipicola CGMCC 3.17084 Lithocarpus glabra China KF576270 N/Aa N/Aa KF576245 KF576291
D. endophytica CBS 133811 Schinus terebinthifolius Brazil KC343065 KC343307 KC343549 KC343791 KC343065
D. eres AR5193 Ulmus sp. Germany KJ210529 KJ434999 KJ420850 KJ210550 KJ420799
CFCC 52575 Castanea mollissima China MH121510 N/Aa MH121470 MH121552 MH121592
CFCC 52576 Castanea mollissima China MH121511 MH121432 MH121471 MH121553 MH121593
CFCC 52577 Acanthopanax senticosus China MH121512 MH121433 MH121472 MH121554 MH121594
CFCC 52578 Sorbus sp. China MH121513 MH121434 MH121473 MH121555 MH121595
CFCC 52579 Juglans regia China MH121514 N/Aa MH121474 MH121556 N/Aa
CFCC 52580 Melia azedarace China MH121515 N/Aa MH121475 MH121557 MH121596
CFCC 52581 Rhododendron simsii China MH121516 N/Aa MH121476 MH121558 MH121597
D. eucalyptorum CBS 132525 Eucalyptus sp. Australia NR120157 N/Aa N/Aa N/Aa N/Aa
D. foeniculacea CBS 123208 Foeniculum vulgare Portugal KC343104 KC343346 KC343588 KC343830 KC344072
D. fraxini-angustifoliae BRIP 54781 Fraxinus angustifolia Australia JX862528 N/Aa N/Aa JX862534 KF170920
D. fraxinicola CFCC 52582 Fraxinus chinensis China MH121517 MH121435 N/Aa MH121559 N/Aa
CFCC 52583 Fraxinus chinensis China MH121518 MH121436 N/Aa MH121560 N/Aa
D. fukushii MAFF 625034 Pyrus pyrifolia Japan JQ807469 N/Aa N/Aa JQ807418 N/Aa
D. fusicola CGMCC 3.17087 Lithocarpus glabra China KF576281 KF576233 N/Aa KF576256 KF576305
D. ganjae CBS 180.91 Cannabis sativa USA KC343112 KC343354 KC343596 KC343838 KC344080
D. garethjonesii MFLUCC 12-0542a Unknown dead leaf Thailand KT459423 KT459470 N/Aa KT459457 KT459441
D. goulteri BRIP 55657a Helianthus annuus Australia KJ197290 N/Aa N/Aa KJ197252 KJ197270
D. gulyae BRIP 54025 Helianthus annuus Australia JF431299 N/Aa N/Aa KJ197271 JN645803
D. helianthi CBS 592.81 Helianthus annuus Serbia KC343115 KC343357 KC343599 KC343841 KC344083
D. helicis AR5211 Hedera helix France KJ210538 KJ435043 KJ420875 KJ210559 KJ420828
D. heterophyllae CBS 143769 Acacia heterohpylla France MG600222 MG600218 MG600220 MG600224 MG600226
D. hickoriae CBS 145.26 Carya glabra USA KC343118 KC343360 KC343602 KC343844 KC344086
D. hispaniae CPC 30321 Vitis vinifera Spain MG281123 MG281820 MG281471 MG281644 MG281296
D. hongkongensis CBS 115448 Dichroa febrífuga China KC343119 KC343361 KC343603 KC343845 KC344087
D. incompleta CGMCC 3.18288 Camellia sinensis China KX986794 KX999289 KX999265 KX999186 KX999226
D. inconspicua CBS 133813 Maytenus ilicifolia Brazil KC343123 KC343365 KC343607 KC343849 KC344091
D. infecunda CBS 133812 Schinus terebinthifolius Brazil KC343126 KC343368 KC343610 KC343852 KC344094
D. isoberliniae CPC 22549 Isoberlinia angolensis Zambia KJ869133 N/Aa N/Aa N/Aa KJ869245
D. juglandicola CFCC 51134 Juglans mandshurica China KU985101 KX024616 KX024622 KX024628 KX024634
CFCC 51135 Juglans mandshurica China KU985102 KX024617 KX024623 KX024629 KX024635
D. kadsurae CFCC 52586 Kadsura longipedunculata China MH121521 MH121439 MH121479 MH121563 MH121600
CFCC 52587 Kadsura longipedunculata China MH121522 MH121440 MH121480 MH121564 MH121601
CFCC 52588 Acer sp. China MH121523 MH121441 MH121481 MH121565 MH121602
CFCC 52589 Acer sp. China MH121524 MH121442 MH121482 MH121566 MH121603
D. kochmanii BRIP 54033 Helianthus annuus Australia JF431295 N/Aa N/Aa JN645809 N/Aa
D. kongii BRIP 54031 Portulaca grandiflora Australia JF431301 N/Aa N/Aa JN645797 KJ197272
D. litchicola BRIP 54900 Litchi chinensis Australia JX862533 N/Aa N/Aa JX862539 KF170925
D. lithocarpus CGMCC 3.15175 Lithocarpus glabra China KC153104 KF576235 N/Aa KC153095 KF576311
D. longicicola CGMCC 3.17089 Lithocarpus glabra China KF576267 N/Aa N/Aa KF576242 KF576291
D. longicolla ATCC 60325 Glycine max USA KJ590728 N/Aa KJ659188 KJ590767 KJ610883
D. longispora CBS 194.36 Ribes sp. Canada KC343135 KC343377 KC343619 KC343861 KC344103
D. lonicerae MFLUCC 17-0963 Lonicera sp. Italy KY964190 KY964116 N/Aa KY964146 KY964073
D. lusitanicae CBS 123212 Foeniculum vulgare Portugal KC343136 KC343378 KC343620 KC343862 KC344104
D. macinthoshii BRIP 55064a Rapistrum rugostrum Australia KJ197289 N/Aa N/Aa KJ197251 KJ197269
D. mahothocarpus CGMCC 3.15181 Lithocarpus glabra China KC153096 N/Aa N/Aa KC153087 KF576312
D. malorum CAA734 Malus domestica Portugal KY435638 KY435658 KY435648 KY435627 KY435668
D. maritima DAOMC 250563 Picea rubens Canada N/Aa N/Aa N/Aa N/Aa KU574616
D. masirevicii BRIP 57892a Helianthus annuus Australia KJ197277 N/Aa N/Aa KJ197239 KJ197257
D. mayteni CBS 133185 Maytenus ilicifolia Brazil KC343139 KC343381 KC343623 KC343865 KC344107
D. maytenicola CPC 21896* Maytenus acuminata South Africa KF777157 N/Aa N/Aa N/Aa KF777250
D. melonis CBS 507.78 Cucumis melo USA KC343142 KC343384 KC343626 KC343868 KC344110
D. middletonii BRIP 54884e Rapistrum rugostrum Australia KJ197286 N/Aa N/Aa KJ197248 KJ197266
D. miriciae BRIP 54736j Helianthus annuus Australia KJ197282 N/Aa N/Aa KJ197244 KJ197262
D. momicola MFLUCC 16-0113 Prunus persica China KU557563 KU557611 N/Aa KU557631 KU55758
D. multigutullata ZJUD98 Citrus grandis China KJ490633 N/Aa KJ490575 KJ490512 KJ490454
D. musigena CBS 129519 Musa sp. Australia KC343143 KC343385 KC343627 KC343869 KC344111
D. neilliae CBS 144.27 Spiraea sp. USA KC343144 KC343386 KC343628 KC343870 KC344112
D. neoarctii CBS 109490 Ambrosia trifida USA KC343145 KC343387 KC343629 KC343871 KC344113
D. neoraonikayaporum MFLUCC 14-1136 Tectona grandis Thailand KU712449 KU749356 N/Aa KU749369 KU743988
D. nobilis CBS 113470 Castanea sativa Korea KC343146 KC343388 KC343630 KC343872 KC344114
D. nothofagi BRIP 54801 Nothofagus cunninghamii Australia JX862530 N/Aa N/Aa JX862536 KF170922
D. novem CBS 127270 Glycine max Croatia KC343155 KC343397 KC343640 KC343881 KC344123
D. ocoteae CBS 141330 Ocotea obtusata France KX228293 N/Aa N/Aa N/Aa KX228388
D. oraccinii CGMCC 3.17531 Camellia sinensis China KP267863 N/Aa KP293517 KP267937 KP293443
D. ovalispora ICMP20659 Citrus limon China KJ490628 N/Aa KJ490570 KJ490507 KJ490449
D. ovoicicola CGMCC 3.17093 Citrus sp. China KF576265 KF576223 N/Aa 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
CFCC 52591 Padus racemosa China MH121526 MH121444 MH121484 MH121568 MH121605
D. pandanicola MFLU 18-0006 Pandanus sp. Thailand MG646974 N/Aa N/Aa N/Aa MG646930
D. paranensis CBS 133184 Maytenus ilicifolia Brazil KC343171 KC343413 KC343655 KC343897 KC344139
D. parapterocarpi CPC 22729 Pterocarpus brenanii Zambia KJ869138 N/Aa N/Aa N/Aa KJ869248
D. pascoei BRIP 54847 Persea americana Australia JX862532 N/Aa N/Aa JX862538 KF170924
D. passiflorae CBS 132527 Passiflora edulis South America JX069860 N/Aa KY435654 N/Aa N/Aa
D. passifloricola CBS 141329 Passiflora foetida Malaysia KX228292 N/Aa KX228367 N/Aa KX228387
D. penetriteum CGMCC 3.17532 Camellia sinensis China KP714505 N/Aa KP714493 KP714517 KP714529
D. perjuncta CBS 109745 Ulmus glabra Austria KC343172 KC343414 KC343656 KC343898 KC344140
D. perseae CBS 151.73 Persea gratissima Netherlands KC343173 KC343415 KC343657 KC343899 KC344141
D. pescicola MFLUCC 16-0105 Prunus persica China KU557555 KU557603 N/Aa KU557623 KU557579
D. phaseolorum AR4203 Phaseolus vulgaris USA KJ590738 N/Aa KJ659220 N/Aa KP004507
D. podocarpi-macrophylli CGMCC 3.18281 Podocarpus macrophyllus China KX986774 KX999278 KX999246 KX999167 KX999207
D. pseudomangiferae CBS 101339 Mangifera indica Dominican Republic KC343181 KC343423 KC343665 KC343907 KC344149
D. pseudophoenicicola CBS 462.69 Phoenix dactylifera Spain KC343184 KC343426 KC343668 KC343910 KC344152
D. pseudotsugae MFLU 15-3228 Pseudotsuga menziesii Italy KY964225 KY964138 N/Aa KY964181 KY964108
D. psoraleae CBS 136412 Psoralea pinnata South Africa KF777158 N/Aa N/Aa KF777245 KF777251
D. psoraleae-pinnatae CBS 136413 Psoralea pinnata South Africa KF777159 N/Aa N/Aa N/Aa KF777252
D. pterocarpi MFLUCC 10-0571 Pterocarpus indicus Thailand JQ619899 JX197451 N/Aa JX275416 JX275460
D. pterocarpicola MFLUCC 10-0580a Pterocarpus indicus Thailand JQ619887 JX197433 N/Aa JX275403 JX275441
D. pulla CBS 338.89 Hedera helix Yugoslavia KC343152 KC343394 KC343636 KC343878 KC344120
D. pyracanthae CAA483 Pyracantha coccinea Portugal KY435635 KY435656 KY435645 KY435625 KY435666
D. racemosae CBS 143770 Euclea racemosa South Africa MG600223 MG600219 MG600221 MG600225 MG600227
D. raonikayaporum CBS 133182 Spondias mombin Brazil KC343188 KC343430 KC343672 KC343914 KC344156
D. ravennica MFLUCC 15-0479 Tamarix sp. Italy KU900335 N/Aa N/Aa KX365197 KX432254
D. rhusicola CBS 129528 Rhus pendulina South Africa JF951146 KC843124 N/Aa KC843100 KC843205
D. rosae MFLU 17-1550 Rosa sp. Thailand MG828894 N/Aa N/Aa N/Aa MG843878
D. rosicola MFLU 17-0646 Rosa sp. UK MG828895 N/Aa N/Aa MG829270 MG843877
D. rostrata CFCC 50062 Juglans mandshurica China KP208847 KP208849 KP208851 KP208853 KP208855
CFCC 50063 Juglans mandshurica China KP208848 KP208850 KP208852 KP208854 KP208856
D. rudis AR3422 Laburnum anagyroides Austria KC843331 KC843146 N/Aa KC843090 KC843177
D. saccarata CBS 116311 Protea repens South Africa KC343190 KC343432 KC343674 KC343916 KC344158
D. sackstonii BRIP 54669b Helianthus annuus Australia KJ197287 N/Aa N/Aa KJ197249 KJ197267
D. salicicola BRIP 54825 Salix purpurea Australia JX862531 N/Aa N/Aa JX862537 JX862531
D. sambucusii CFCC 51986 Sambucus williamsii China KY852495 KY852499 KY852503 KY852507 KY852511
CFCC 51987 Sambucus williamsii China KY852496 KY852500 KY852504 KY852508 KY852512
D. schini CBS 133181 Schinus terebinthifolius Brazil KC343191 KC343433 KC343675 KC343917 KC344159
D. schisandrae CFCC 51988 Schisandra chinensis China KY852497 KY852501 KY852505 KY852509 KY852513
CFCC 51989 Schisandra chinensis China KY852498 KY852502 KY852506 KY852510 KY852514
D. schoeni MFLU 15-1279 Schoenus nigricans Italy KY964226 KY964139 N/Aa KY964182 KY964109
D. sclerotioides CBS 296.67 Cucumis sativus Netherlands KC343193 KC343435 KC343677 KC343919 KC344161
D. sennae CFCC 51636 Senna bicapsularis China KY203724 KY228875 N/Aa KY228885 KY228891
CFCC 51637 Senna bicapsularis China KY203725 KY228876 N/Aa KY228886 KY228892
D. sennicola CFCC 51634 Senna bicapsularis China KY203722 KY228873 KY228879 KY228883 KY228889
CFCC 51635 Senna bicapsularis China KY203723 KY228874 KY228880 KY228884 KY228890
D. serafiniae BRIP 55665a Helianthus annuus Australia KJ197274 N/Aa N/Aa KJ197236 KJ197254
D. siamensis MFLUCC 10-573a Dasymaschalon sp. Thailand JQ619879 N/Aa N/Aa JX275393 JX275429
D. sojae FAU635 Glycine max USA KJ590719 KJ612116 KJ659208 KJ590762 KJ610875
D. spartinicola CBS 140003 Spartium junceum Spain KR611879 N/Aa KR857696 N/Aa KR857695
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 ICMP20663 Citrus unshiu China KJ490587 N/Aa KJ490529 KJ490466 KJ490408
D. subcylindrospora MFLU 17-1195 Salix sp. China MG746629 N/Aa N/Aa MG746630 MG746631
D. subellipicola MFLU 17-1197 on dead wood China MG746632 N/Aa N/Aa MG746633 MG746634
D. subordinaria CBS 464.90 Plantago lanceolata New Zealand KC343214 KC343456 KC343698 KC343940 KC344182
D. taoicola MFLUCC 16-0117 Prunus persica China KU557567 N/Aa N/Aa KU557635 KU557591
D. tectonae MFLUCC 12-0777 Tectona grandis China KU712430 KU749345 N/Aa KU749359 KU743977
D. tectonendophytica MFLUCC 13-0471 Tectona grandis China KU712439 KU749354 N/Aa KU749367 KU749354
D. tectonigena MFLUCC 12-0767 Tectona grandis China KU712429 KU749358 N/Aa KU749371 KU743976
D. terebinthifolii CBS 133180 Schinus terebinthifolius Brazil KC343216 KC343458 KC343700 KC343942 KC344184
D. thunbergii MFLUCC 10-576a Thunbergia laurifolia Thailand JQ619893 JX197440 N/Aa JX275409 JX275449
D. thunbergiicola MFLUCC 12-0033 Thunbergia laurifolia Thailand KP715097 N/Aa N/Aa KP715098 N/Aa
D. tibetensis CFCC 51999 Juglandis regia China MF279843 MF279888 MF279828 MF279858 MF279873
CFCC 52000 Juglandis regia China MF279844 MF279889 MF279829 MF279859 MF279874
D. torilicola MFLUCC 17-1051 Torilis arvensis Italy KY964212 KY964127 N/Aa KY964168 KY964096
D. toxica CBS 534.93 Lupinus angustifolius Australia KC343220 KC343462 C343704 KC343946 KC344188
D. tulliensis BRIP 62248a Theobroma cacao fruit Australia KR936130 N/Aa N/Aa KR936133 KR936132
D. ueckerae FAU656 Cucumis melo USA KJ590726 KJ612122 KJ659215 KJ590747 KJ610881
D. ukurunduensis CFCC 52592 Acer ukurunduense China MH121527 MH121445 MH121485 MH121569 N/Aa
CFCC 52593 Acer ukurunduense China MH121528 MH121446 MH121486 MH121570 N/Aa
D. undulata CGMCC 3.18293 Leaf of unknown host China-Laos border KX986798 N/Aa KX999269 KX999190 KX999230
D. unshiuensis CGMCC 3.17569 Citrus unshiu China KJ490587 N/Aa KJ490529 KJ490408 KJ490466
CFCC 52594 Carya illinoensis China MH121529 MH121447 MH121487 MH121571 MH121606
CFCC 52595 Carya illinoensis China MH121530 MH121448 MH121488 MH121572 MH121607
D. vaccinii CBS 160.32 Oxycoccus macrocarpos USA KC343228 KC343470 KC343712 KC343954 KC344196
D. vangueriae CPC 22703 Vangueria infausta Zambia KJ869137 N/Aa N/Aa N/Aa KJ869247
D. vawdreyi BRIP 57887a Psidium guajava Australia KR936126 N/Aa N/Aa KR936129 KR936128
D. velutina CGMCC 3.18286 Neolitsea sp. China KX986790 N/Aa KX999261 KX999182 KX999223
D. virgiliae CMW40748 Virgilia oroboides South Africa KP247566 N/Aa N/Aa N/Aa KP247575
D. xishuangbanica CGMCC 3.18282 Camellia sinensis China KX986783 N/Aa KX999255 KX999175 KX999216
D. yunnanensis CGMCC 3.18289 Coffea sp. China KX986796 KX999290 KX999267 KX999188 KX999228
Diaporthella corylina CBS 121124 Corylus sp. China KC343004 KC343246 KC343488 KC343730 KC343972

Morphological analysis

Agar plugs (6 mm diam.) were taken from the edge of actively growing cultures on PDA and transferred on to the centre of 9 cm diam Petri dishes containing 2% tap water agar supplemented with sterile pine needles (PNA; Smith et al. 1996) and potato dextrose agar (PDA) and incubated at 20–21 °C under a 12 h near-ultraviolet light/12 h dark cycle to induce sporulation as described in recent studies (Gomes et al. 2013, Lombard et al. 2014). Colony characters and pigment production on PNA and PDA were noted after 10 d. Colony colours were rated according to Rayner (1970). Cultures were examined periodically for the development of ascomata and conidiomata. The morphological characteristics were examined by mounting fungal structures in clear lactic acid and 30 measurements at 1000× magnification were determined for each isolate using a Leica compound microscope (DM 2500) with interference contrast (DIC) optics. Descriptions, nomenclature and illustrations of taxonomic novelties are deposited in MycoBank (www.MycoBank.org; Crous et al. 2004b).

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted from colonies grown on cellophane-covered PDA using a modified CTAB [cetyltrimethylammonium bromide] method (Doyle and Doyle 1990). DNA was estimated by electrophoresis in 1% agarose gel and the quality was measured using the NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA), following the user manual (Desjardins et al. 2009). PCR amplifications were performed in a DNA Engine Peltier Thermal Cycler (PTC-200; Bio-Rad Laboratories, Hercules, CA, USA). The primer sets ITS1/ITS4 (White et al. 1990) were used to amplify the ITS region. The primer pair CAL228F/CAL737R (Carbone and Kohn 1999) were used to amplify the calmodulin gene (cal) and the primer pair CYLH4F (Crous et al. 2004a) and H3-1b (Glass and Donaldson 1995) were used to amplify part of the histone H3 (his3) gene. The primer pair EF1-728F/EF1-986R (Carbone and Kohn 1999) were used to amplify a partial fragment of the translation elongation factor 1-α gene (tef1). The primer sets T1 (O’Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995) were used to amplify the beta-tubulin gene (tub2); the additional combination of Bt2a/Bt2b (Glass and Donaldson 1995) was used in case of amplification failure of the T1/Bt2b primer pair. Amplifications of different loci were performed under different conditions (Table 2). PCR amplification products were assayed via electrophoresis in 2% agarose gels. DNA sequencing was performed using an ABI PRISM® 3730XL DNA Analyser with a BigDye Terminater Kit v.3.1 (Invitrogen, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

Genes used in this study with PCR primers, process and references.

Gene PCR primers (forward/reverse) PCR: thermal cycles: (Annealing temp. in bold) References of primers used
ITS ITS1/ITS4 (95 °C: 30 s, 51 °C: 30 s, 72 °C: 1 min) × 35 cycles White et al. 1990
cal CAL228F/CAL737R (95 °C: 15 s, 55 °C: 20 s, 72 °C: 1 min) × 35 cycles Carbone and Kohn 1999
his3 CYLH4F/H3-1b (95 °C: 30 s, 58 °C: 30 s, 72 °C: 1 min) × 35 cycles Glass and Donaldson 1995, Crous et al. 2004a
tef1 EF1-728F/EF1-986R (9 °C: 15 s, 55 °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, Glass and Donaldson 1995

Phylogenetic analyses

DNA generated sequences were used to obtain consensus sequences using SeqMan v.7.1.0 DNASTAR Lasergene Core Suite software programme (DNASTAR Inc., Madison, WI, USA). Sequences were aligned using MAFFT v.6 (Katoh and Toh 2010) and edited manually using MEGA6 (Tamura et al. 2013). Two different datasets were employed to estimate two phylogenetic analyses: one for Diaporthe species and one for Diaporthe eres complex. The first analysis was undertaken to infer the interspecific relationships in Diaporthe. All the Diaporthe isolates recovered from samples collected during this study and additional reference sequences of Diaporthe species were included in the dataset of combined ITS, cal, his3, tef1, and tub2 regions (Table 1), with Diaporthella corylina (CBS 121124) as outgroup. The second analysis focused on the Diaporthe eres complex based on cal, tef1 and tub2 loci (Table 3) according to recent publications (Gao et al. 2014, 2015, 2016, Udayanga et al. 2014b, Tanney et al. 2016, Fan et al. 2018), with Diaporthe citri (AR3405) as outgroup. Maximum Parsimony analysis was performed by a heuristic search option of 1000 random-addition sequences with a tree bisection and reconnection (TBR) algorithm. Maxtrees were set to 5000, branches of zero length were collapsed and all equally parsimonious trees were saved. Other calculated parsimony scores were tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency (RC). Maximum Likelihood analysis was performed with a GTR site substitution model (Guindon et al. 2010). Branch support was evaluated with a bootstrapping (BS) method of 1000 replicates (Hillis and Bull 1993).

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

Species Isolate/culture collection Host Location GenBank accession numbers
CAL TEF1-α TUB
D. alleghaniensis CBS 495.72 Betula alleghaniensis Canada KC343249 GQ250298 KC843228
D. alnea CBS 146.46 Alnus sp. Netherlands KC343250 KC343734 KC343976
CBS 159.47 Alnus sp. Netherlands KC343251 KC343735 KC343977
LCM22b.02a Alnus sp. USA KJ435020 KJ210557 KJ420825
LCM22b.02b Alnus sp. USA KJ435021 KJ210558 KJ420826
D. betulina CFCC 52560 Betula albo-sinensis China MH121419 MH121537 MH121577
CFCC 52561 Betula costata China MH121420 MH121538 MH121578
CFCC 52562 Betula platyphylla China MH121421 MH121539 MH121579
D. bicincta CBS 121004 Juglans sp. USA KC343376 KC343860 KC344102
D. biguttusis CGMCC 3.17081 Lithocarpus glabra China N/Aa KF576257 KF576306
D. camptothecicola CFCC 51632 Camptotheca acuminata China KY228881 KY228887 KY228893
D. celastrina CBS 139.27 Celastrus sp. USA KC343289 KC343773 KC344015
D. chensiensis CFCC 52567 Abies chensiensis China MH121426 MH121544 MH121584
CFCC 52568 Abies chensiensis China MH121427 MH121545 MH121585
D. citri AR3405 Citrus sp. USA KC843157 KC843071 KC843187
D. citrichinensis ZJUD034 Citrus sp. China KC843234 KC843071 KC843187
ZJUD034B Citrus sp. China KJ435042 KJ210562 KJ420829
D. ellipicola CGMCC 3.17084 Lithocarpus glabra China N/Aa KF576245 KF576291
D. eres AR5193 Ulmus laevis Germany KJ434999 KJ210550 KJ420799
AR5196 Ulmus laevis Germany KJ435006 KJ210554 KJ420817
DP0438 Ulmus minor Austria KJ435016 KJ210553 KJ420816
LCM114.01a Ulmus sp. USA KJ435027 KJ210545 KJ420787
LCM114.01b Ulmus sp. USA KJ435026 KJ210544 KJ420786
FAU483 Malus sp. Netherlands KJ435022 JQ807422 KJ420827
DAN001A Daphne laureola France KJ434994 KJ210540 KJ420781
DAN001B Daphne laureola France KJ434995 KJ210541 KJ420782
AR5197 Rhododendron sp. Germany KJ435014 KJ210552 KJ420812
CBS 439.82 Cotoneaster sp. UK JX197429 GQ250341 JX275437
AR3519 Corylus avellana Austria KJ435008 KJ210547 KJ420789
FAU506 Cornus florida USA KJ435012 JQ807403 KJ420792
FAU570 Oxydendrum arboreum USA KJ435025 JQ807410 KJ420794
AR3723 Rubus fruticosus Austria KJ435024 JQ807354 KJ420793
FAU522 Sassafras albida USA KJ435010 JQ807406 KJ420791
DP0666 Juglans cinerea USA KJ435007 KJ210546 KJ420788
DP0667 Juglans cinerea USA KC843155 KC843121 KC843229
AR3560 Viburnum sp. Austria KJ435011 JQ807351 KJ420795
AR5224 Hedera helix Germany KJ435036 KJ210551 KJ420802
AR5231 Hedera helix Germany KJ435038 KJ210555 KJ420818
AR5223 Acer nugundo Germany KJ435000 KJ210549 KJ420830
CBS 109767 Acer sp. Austria KC343317 KC343801 KC344043
DLR12a Vitis vinifera France KJ434996 KJ210542 KJ420783
DLR12b Vitis vinifera France KJ434997 KJ210543 KJ420784
AR4347 Vitis vinifera Korea KJ435030 JQ807356 KJ420805
AR4355 Prunus sp. Korea KJ435035 JQ807359 KJ420797
AR4367 Prunus sp. Korea KJ435019 JQ807364 KJ420824
AR4346 Prunus mume Korea KJ435003 JQ807355 KJ420823
AR4348 Prunus persici Korea KJ435004 JQ807357 JQ807357
AR3669 Pyrus pyrifolia Japan KJ435002 JQ807415 KJ420808
D. eres AR3670 Pyrus pyrifolia Japan KJ435001 JQ807416 KJ420807
AR3671 Pyrus pyrifolia Japan KJ435017 JQ807417 KJ420814
AR3672 Pyrus pyrifolia Japan KJ435023 JQ807418 KJ420819
DP0591 Pyrus pyrifolia New Zealand KJ435018 JQ807395 KJ420821
AR4369 Pyrus pyrifolia Korea KJ435005 JQ807366 KJ420813
DP0180 Pyrus pyrifolia New Zealand KJ435029 JQ807384 KJ420804
DP0179 Pyrus pyrifolia New Zealand KJ435028 JQ807383 KJ420803
DP0590 Pyrus pyrifolia New Zealand KJ435037 JQ807394 KJ420810
AR4373 Ziziphus jujuba Korea KJ435013 JQ807368 KJ420798
AR4374 Ziziphus jujuba Korea KJ434998 JQ807369 KJ420785
AR4357 Ziziphus jujuba Korea KJ435031 JQ807360 KJ420806
AR4371 Malus pumila Korea KJ435034 JQ807367 KJ420796
FAU532 Chamaecyparis thyoides USA KJ435015 JQ807408 KJ435015
CBS 113470 Castanea sativa Australia KC343388 KC343872 KC344114
AR4349 Vitis vinifera Korea KJ435032 JQ807358 KJ420822
AR4363 Malus sp. Korea KJ435033 JQ807362 KJ420809
CFCC 52575 Castanea mollissima China N/Aa MH121552 MH121592
CFCC 52576 Castanea mollissima China MH121432 MH121553 MH121593
CFCC 52577 Acanthopanax senticosus China MH121433 MH121554 MH121594
CFCC 52578 Sorbus sp. China MH121434 MH121555 MH121595
CFCC 52579 Juglans regia China N/Aa MH121556 N/Aa
CFCC 52580 Melia azedarace China N/Aa MH121557 MH121596
CFCC 52581 Rhododendron simsii China N/Aa MH121558 MH121597
D. helicis AR5211 Hedera helix France KJ435043 KJ210559 KJ420828
D. longicicola CGMCC 3.17089 Lithocarpus glabra China N/Aa KF576242 KF576291
D. mahothocarpus CGMCC 3.15181 Lithocarpus glabra China N/Aa KC153087 KF576312
D. maritima DAOMC 250563 Picea rubens Canada N/Aa N/Aa KU574616
D. momicola MFLUCC 16-0113 Prunus persica China N/Aa KU557631 KU55758
D. neilliae CBS 144. 27 Spiraea sp. USA KC343386 KC343870 KC344112
D. padina CFCC 52590 Padus racemosa China MH121443 MH121567 MH121604
CFCC 52591 Padus racemosa China MH121444 MH121568 MH121605
D. phragmitis CBS 138897 Phragmites australis China N/Aa N/Aa KP004507
D. pulla CBS 338.89 Hedera helix Yugoslavia KC343394 KC343878 KC344120
D. vaccinii DF5032 Vaccinium corymbosum USA KC849457 JQ807380 KC843225
FAU633 Vaccinium macrocarpon USA KC849456 JQ807413 KC843226
FAU446 Vaccinium macrocarpon USA KC849455 JQ807398 KC843224
CBS 160.32 Vaccinium macrocarpon USA KC343470 GQ250326 JX270436
FAU 468 Vaccinium macrocarpon USA KC849458 JQ807399 KC843227

Bayesian inference (BI) analysis, employing a Markov chain Monte Carlo (MCMC) algorithm, was performed (Rannala and Yang 1996). MrModeltest v. 2.3 was used to estimate the best-fit model of nucleotide substitution model settings for each gene (Posada and Crandall 1998). 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 the burn-in phase of each analysis. Branches with significant Bayesian posterior probabilities (BPP) were estimated in the remaining 7500 trees.

Sequences data were deposited in GenBank (Table 1). The multilocus sequence alignments were deposited in TreeBASE (www.treebase.org) as accession S22702 and S22703. The taxonomic novelties were deposited in MycoBank (Crous et al. 2004b).

Results

Collection of Diaporthe strains

Forty-two representative Diaporthe strains were isolated from 16 different host genera (Table 1) collected from six Provinces (Beijing, Heilongjiang, Jiangsu, Jiangxi, Shaanxi and Zhejiang) in China. All of these strains were isolated from symptomatic or non-symptomatic branches or twigs and preserved in the China Forestry Culture Collection Centre (CFCC).

Phylogenetic analyses

The first sequences dataset for the ITS, cal, his3, tef1, and tub2 was analysed in combination to infer the interspecific relationships within Diaporthe. The combined species phylogeny of the Diaporthe isolates consisted of 236 sequences, including the outgroup sequences of Diaporthella corylina (culture CBS 121124). A total of 2948 characters including gaps (516 for ITS, 568 for cal, 520 for his3, 486 for tef1 and 858 for tub2) were included in the phylogenetic analysis. The maximum likelihood tree, conducted by the GTR model, confirmed the tree topology and posterior probabilities of the Bayesian consensus tree. For the Bayesian analyses, MrModeltest suggested that all partitions should be analysed with dirichlet state frequency distributions. The following models were recommended by MrModeltest and used: GTR+I+G for ITS, cal and his3, HKY+I+G for tef1 and tub2. The topology and branching order of ML were similar to BI analyses (Fig. 1). Based on the multi-locus phylogeny and morphology, 42 strains were assigned to 15 species, including 12 taxa which we describe here as new (Fig. 1).

Figure 1. 

Phylogram of Diaporthe from a maximum likelihood analysis based on combined ITS, cal, his3, tef1 and tub2. Values above the branches indicate maximum likelihood bootstrap (left, ML BP ≥ 50%) and bayesian probabilities (right, BI PP ≥ 0.70). The tree is rooted with Diaporthella corylina. Strains in the current study are in blue.

The second dataset with cal, tef1 and tub2 sequences were analysed to focus on the Diaporthe eres complex. The alignment included 86 taxa, including the outgroup sequences of Diaporthe citri (Table 3). The aligned three-locus datasets included 1148 characters. Of these, 881 characters were constant, 105 variable characters were parsimony-uninformative and 162 characters were parsimony informative. The heuristic search using maximum parsimony (MP) generated 105 parsimonious trees (TL = 438, CI = 0.669, RI = 0.883, RC = 0.591), from which one was selected (Fig. 2). Based on the multi-locus phylogeny and morphology, seven strains were identified as D. eres, seven strains formed three distinct clades embedded in the D. eres complex, i.e. D. betulina, D. chensiensis and D. padina. MP and ML bootstrap support values above 50% are shown as first and second position, respectively. The branches with significant Bayesian posterior probability (≥ 0.70) in Bayesian analyses were thickened in the phylogenetic tree. The current results, based on the three genes (cal, tef1 and tub2), suggest that D. eres clade could be separated from other species in this complex (Fig. 2). However, D. biguttusis (CGMCC 3.17081), D. camptothecicola (CFCC 51632), D. ellipicola (CGMCC 3.17084), D. longicicola (CGMCC 3.17089), D. mahothocarpus (CGMCC 3.15181) and D. momicola (MFLUCC 16-0113) were clustered in D. eres clade and thus treated as the synonyms of D. eres in the current study.

Figure 2. 

Phylogram of Diaporthe eres complex based on combined cal, tef1 and tub2. Values above the branches indicate maximum parsimony bootstrap (left, MP BP ≥ 50%) and maximum likelihood bootstrap (right, ML BP ≥ 50%). Values below branches represent posterior probabilities (BI PP ≥ 0.70) from Bayesian inference. The tree is rooted with Diaporthe citri. Strains in the current study are in blue. The ex-type/ex-epitype culture is in bold.

Taxonomy

Diaporthe acerigena C.M. Tian & Q. Yang, sp. nov.

MycoBank No: MB824703
Figure 3

Diagnosis

Diaporthe acerigena can be distinguished from the phylogenetically closely related species D. oraccinii in larger alpha conidia.

Holotype

CHINA. Shaanxi Province: Qinling Mountain, on symptomatic twigs of Acer tataricum, 27 June 2017, N. Jiang (holotype: BJFC-S1466; ex-type culture: CFCC 52554).

Etymology

Named after the host genus on which it was collected, Acer.

Description

On PDA: Conidiomata pycnidial, globose, solitary or aggregated, deeply embedded in the medium, erumpent, dark brown to black, 185–270 μm diam, whitish translucent to cream conidial drops exuding from the ostioles. Conidiophores 14.5–17 × 1.4–2.9 μm, cylindrical, hyaline, phiailidic, branched, straight to sinuous. Alpha conidia 7–10 × 2.1–2.9 μm (av. = 8.6 × 2.5 μm, n = 30), aseptate, hyaline, ellipsoidal, rounded at one end, slightly apex at the other end, usually with two-guttulate. Beta conidia not observed.

Figure 3. 

Diaporthe acerigena (CFCC 52554) A Alpha conidia B–C Conidiophores D Culture on PDA and conidiomata. Scale bars: 20 μm (A–C), 200 μm (D).

Culture characters

Cultures incubated on PDA at 25 °C in darkness. Colony at first white, becoming dark brown in the centre with age. Aerial mycelium white, dense, fluffy, with cream conidial drops exuding from the ostioles.

Additional specimens examined

CHINA. Shaanxi Province: Qinling Mountain, on symptomatic twigs of Acer tataricum, 27 June 2017, N. Jiang, living culture CFCC 52555 (BJFC-S1467).

Notes

Two strains representing D. acerigena cluster in a well-supported clade and appear most closely related to D. oraccinii. Diaporthe acerigena can be distinguished from D. oraccinii based on ITS, his3, tef1 and tub2 loci (5/469 in ITS, 8/429 in his3, 8/326 in tef1 and 5/358 in tub2). Morphologically, D. acerigena differs from D. oraccinii in the longer and larger alpha conidia (8.6 × 2.5 vs. 6.6 × 1.9 μm) (Gao et al. 2016).

Diaporthe alangii C.M. Tian & Q. Yang, sp. nov.

MycoBank No: MB824704
Figure 4

Diagnosis

Diaporthe alangii can be distinguished from the phylogenetically closely related species D. tectonae and D. tulliensis by the size of conidiophores and alpha conidia.

Holotype

CHINA. Zhejiang Province: Tianmu Mountain, on symptomatic branches of Alangium kurzii, 19 Apr. 2017, Q. Yang (holotype: BJFC-S1468; ex-type culture: CFCC 52556).

Etymology

Named after the host genus on which it was collected, Alangium.

Description

Conidiomata pycnidial, immersed in bark, scattered, erumpent through the bark surface, discoid, with a solitary undivided locule. Ectostromatic disc black, one ostiole per disc, 135–330 μm diam. Locule circular, undivided, 290–445 μm diam. Conidiophores 6–12 × 1.4–2 μm, cylindrical, hyaline, phiailidic, unbranched, straight. Alpha conidia 6.5–8 × 2 μm (av. = 7 × 2 μm, n = 30), aseptate, hyaline, ellipsoidal, biguttulate, mostly with one end obtuse and the other acute, occasionally submedian constriction. Beta conidia not observed.

Figure 4. 

Diaporthe alangii (CFCC 52556) A Habit of conidiomata on branches B Transverse section of conidioma C Longitudinal section of conidioma D Alpha conidia E Conidiophores F Culture on PDA. Scale bars: 200 μm (B–C), 10 μm (D–E).

Culture characters

Cultures incubated on PDA at 25 °C in darkness. Colony initially white, producing beige pigment after 7–10 d. The colony is flat, felty with a thick texture at the centre and marginal area, with thin texture in the middle, lacking aerial mycelium, conidiomata absent.

Additional specimens examined

CHINA. Zhejiang Province: Tianmu Mountain, on symptomatic branches of Alangium kurzii, 19 Apr. 2017, Q. Yang, living culture CFCC 52557 (BJFC-S1469); ibid. living culture CFCC 52558 (BJFC-S1470); ibid. living culture CFCC 52559 (BJFC-S1471).

Notes

Four isolates clustered in a clade distinct from its closest phylogenetic neighbour, D. tectonae and D. tulliensis. Diaporthe alangii can be distinguished from D. tectonae in cal, tef1 and tub2 loci (6/458 in cal, 4/308 in tef1 and 11/407 in tub2); from D. tulliensis in ITS, tef1 and tub2 loci (6/462 in ITS, 8/308 in tef1 and 10/701 in tub2). Morphologically, D. alangii differs from D. tectonae in shorter conidiophores (6–12 vs. 11–18 μm) and longer alpha conidia (6.5–8 vs. 5.5–6 μm); from D. tulliensis in shorter conidiophores (6–12 vs. 15–20 μm) (Crous et al. 2015, Doilom et al. 2017).

Diaporthe betulina C.M. Tian & Q. Yang, sp. nov.

MycoBank No: MB824705
Figure 5

Diagnosis

Diaporthe betulina can be distinguished from the phylogenetically closely related species D. betulae in smaller locule and wider alpha conidia.

Holotype

CHINA. Heilongjiang Province: Yichun city, on symptomatic branches of Betula platyphylla, 27 July 2016, Q. Yang (holotype: BJFC-S1472; ex-type culture: CFCC 52562).

Etymology

Named after the host genus on which it was collected, Betula.

Description

Conidiomata pycnidial, conical, immersed in bark, scattered, erumpent through the bark surface, with a solitary undivided locule. Ectostromatic disc brown to black, one ostiole per disc, 290–645 μm diam. Ostiole medium black, up to the level of disc. Locule undivided, 670–905 μm diam. Conidiophores 12.5–17.5 × 1.5–2 μm, cylindrical, hyaline, phiailidic, branched, straight or slightly curved. Alpha conidia hyaline, aseptate, ellipsoidal to fusiform, 0–2-guttulate, sometimes acute at both ends, 8–10 × 2.5–3 μm (av. = 9 × 2.6 μm, n = 30). Beta conidia hyaline, aseptate, filiform, straight or hamate, eguttulate, base subtruncate, tapering towards one apex, 26–32.5 × 1 µm (av. = 30 × 1 µm, n = 30).

Figure 5. 

Diaporthe betulina (CFCC 52562) A Habit of conidiomata on branches B Transverse section of conidioma C Longitudinal section of conidioma D Conidiophores E Alpha conidia F Beta conidia G Culture on PDA and conidiomata. Scale bars: 500 μm (A–C), 10 μm (D–F).

Culture characters

Cultures incubated on PDA at 25 °C in darkness. Colony flat with white felty aerial mycelium, turning white to dark brown aerial mycelium, conidiomata irregularly distributed on the agar surface.

Additional specimens examined

CHINA. Heilongjiang Province: Yichun city, on symptomatic branches of Betula albo-sinensis, 27 July 2016, Q. Yang, living culture CFCC 52560 (BJFC-S1473); on symptomatic branches of Betula costata, 27 July 2016, Q. Yang, living culture CFCC 52561 (BJFC-S1474).

Notes

Diaporthe betulina was isolated from Betula spp. cankers in Heilongjiang Province. Three strains representing D. betulina cluster in a well-supported clade and appear most closely related to D. betulae, which was also isolated from Betula platyphylla in Sichuang Province (Du et al. 2016). Diaporthe betulina can be distinguished based on ITS, his3, tef1 and tub2 loci from D. betulae (11/461 in ITS, 9/453 in his3, 12/336 in tef1 and 7/695 in tub2). Morphologically, D. betulina differs from D. betulae in smaller locule (470–945 vs. 600–1250 μm) and wider alpha conidia (3–4 vs. 2.5–3 μm) (Du et al. 2016).

Diaporthe biguttulata F. Huang, K.D. Hyde & H.Y. Li, 2015

Figure 6

Description

Conidiomata pycnidial, immersed in bark, scattered, erumpent through the bark surface, discoid, with a single locule. Ectostromatic disc dark brown, one ostiole per disc, 160–320 μm diam. Locule undivided, 235–350 μm diam. Conidiophores 8.5–11 × 1.5 μm, cylindrical, hyaline, branched, straight or slightly curved, tapering towards the apex. Alpha conidia hyaline, aseptate, ellipsoidal to oval, 2-guttulate, usually rounded at both ends, occasionally with one end acute, 7–8.5 × 1.5–2 μm (av. = 6.5 × 2.6 μm, n = 30). Beta conidia not observed.

Figure 6. 

Diaporthe biguttulata (CFCC 52584) A Habit of conidiomata on branches B Transverse section of conidioma C Longitudinal section of conidioma D Alpha conidia E Conidiophores F Culture on PDA. Scale bars: 200 μm (B–C), 10 μm (D–E).

Culture characters

Cultures incubated on PDA at 25 °C in darkness. Colony originally flat with white aerial mycelium, becoming pale grey, with dense aerial mycelium in the centre and sparse aerial mycelium at the marginal area, conidiomata absent.

Specimens examined

CHINA. Zhejiang Province: Tianmu Mountain, on symptomatic branches of Juglans regia, 20 Apr. 2017, Q. Yang, living culture CFCC 52584 and CFCC 52585 (BJFC-S1504).

Notes

Diaporthe biguttulata was originally described from a healthy branch of Citrus limon in Yunnan Province, China (Huang et al. 2015). In the present study, two isolates (CFCC 52584 and CFCC 52585) from symptomatic branches of Juglans regia were congruent with D. biguttulata based on morphology and DNA sequences data (Fig. 1). We therefore describe D. biguttulata as a known species for this clade.

Diaporthe caryae C.M. Tian & Q. Yang, sp. nov.

MycoBank No: MB824706
Figure 7

Diagnosis

Diaporthe caryae differs from its closest phylogenetic neighbour, D. charlesworthii and D. sackstonii, in ITS, tef1 and tub2 loci based on the alignments deposited in TreeBASE.

Holotype

CHINA. Jiangsu Province: Nanjing city, on symptomatic twigs of Carya illinoensis, 10 Nov. 2015, Q. Yang (holotype: BJFC-S1476; ex-type culture: CFCC 52563).

Etymology

Named after the host genus on which it was collected, Carya.

Description

Conidiomata pycnidial, immersed in bark, scattered, slightly erumpent through the bark surface, nearly flat, discoid, with a solitary undivided locule. Ectostromatic disc brown to black, one ostiole per disc. Locule undivided, 310–325 μm diam. Conidiophores 7–11 × 1.4–2.2 μm, cylindrical, phialidic, unbranched, sometimes inflated. Alpha conidia hyaline, aseptate, ellipsoidal or fusiform, eguttulate, obtuse at both ends, 7–8.5 × 2.1–2.5 μm (av. = 8 × 2.3 μm, n = 30). Beta conidia hyaline, aseptate, filiform, straight or hamate, eguttulate, base subtruncate, tapering towards one apex, 15.5–34 × 1.1–1.4 µm (av. = 27.5 × 1.2 µm, n = 30).

Figure 7. 

Diaporthe caryae (CFCC 52563) A Transverse section of conidioma B Longitudinal section of conidioma C Culture on PDA D Alpha conidia E Conidiophores F Beta conidia. Scale bars: 200 μm (A), 100 μm (B), 10 μm (D, F), 20 μm (E).

Culture characters

Cultures incubated on PDA at 25 °C in darkness. Colony at first flat with white felty mycelium, becoming black in the centre and black at the marginal area with age, conidiomata not observed.

Additional specimens examined

CHINA. Jiangsu Province: Nanjing city, on symptomatic twigs of Carya illinoensis, 10 Nov. 2015, Q. Yang, living culture CFCC 52564 (BJFC-S1477).

Notes

Two strains representing D. caryae cluster in a well-supported clade and appear closely related to D. charlesworthii and D. sackstonii. Diaporthe caryae can be distinguished based on ITS, tef1 and tub2 loci from D. charlesworthii (50/468 in ITS, 107/338 in tef1 and 90/707 in tub2); from D. sackstonii (4/440 in ITS, 13/340 in tef1 and 23/701 in tub2). Morphologically, D. caryae can be distinguished from D. charlesworthii by its shorter conidiophores (7–11 vs. 15–35 μm); from D. sackstonii by its longer alpha conidia (7–8.5 vs. 6–7 μm) (Thompson et al. 2015).

Diaporthe cercidis C.M. Tian & Q. Yang, sp. nov.

MycoBank No: MB824707
Figure 8

Diagnosis

Diaporthe cercidis can be distinguished from the phylogenetically closely related species D. pescicola in larger alpha conidia.

Holotype

CHINA. Jiangsu Province: Nanjing city, on twigs and branches of Cercis chinensis, 11 Nov. 2015, Q. Yang (holotype: BJFC-S1478; ex-type culture: CFCC 52565).

Etymology

Named after the host genus on which it was collected, Cercis.

Description

Conidiomata pycnidial, immersed in bark, scattered, slightly erumpent through the bark surface, nearly flat, discoid, with a solitary undivided locule. Ectostromatic disc grey to brown, one ostiole per disc. Locule circular, undivided, 135–200 μm diam. Conidiophores 7–17 × 1.4–2.1 μm, phialidic, unbranched, straight or slightly curved, tapering towards the apex. Alpha conidia hyaline, aseptate, fusiform to oval, biguttulate, 6.5–10 × 3–3.5 μm (av. = 8.6 × 3.3 μm, n = 30). Beta conidia hyaline, aseptate, filiform, straight or hamate, eguttulate, 20–28.5 × 1–1.3 µm (av. = 25.5 × 1.2 µm, n = 30).

Figure 8. 

Diaporthe cercidis (CFCC 52565) A Habit of conidiomata on branches B Transverse section of conidioma C Longitudinal section of conidioma D Alpha conidia E Beta conidia F Conidiophores G Culture on PDA and conidiomata. Scale bars: 100 μm (B–C), 10 μm (D–F).

Culture characters

Cultures incubated on PDA at 25 °C in darkness showed colony at first white, becoming pale brown with yellowish dots with age, flat, with dense and felted mycelium, with visible solitary or aggregated conidiomata at maturity.

Additional specimens examined

CHINA. Jiangsu Province: Yangzhou city, on twigs and branches of Ginkgo biloba, 11 Nov. 2015, N. Jiang, living culture CFCC 52566 (BJFC-S1479).

Notes

Diaporthe cercidis is distinguished from D. pescicola in the ITS, cal and tef1 loci (13/458 in ITS, 47/442 in cal and 6/328 in tef1). Morphologically, D. cercidis differs from D. pescicola in shorter conidiophores (7–17 vs. 21–35 μm) and larger alpha conidia (6.5–10 × 3–3.5 vs. 6–8.5 × 2–3 μm) (Dissanayake et al. 2017a).

Diaporthe chensiensis C.M. Tian & Q. Yang, sp. nov.

MycoBank No: MB824708
Figure 9

Diagnosis

Diaporthe chensiensis differs from its closest phylogenetic neighbour, D. vaccinii, in ITS, cal, his3 and tef1 loci based on the alignments deposited in TreeBASE.

Holotype

CHINA. Shaanxi Province: Ningshan County, Huoditang forest farm, on symptomatic twigs of Abies chensiensis, 5 July 2017, Q. Yang (holotype: BJFC-S1480; ex-type culture: CFCC 52567).

Etymology

Named after the host species on which it was collected, chensiensis.

Description

Conidiomata pycnidial, immersed in bark, scattered, slightly erumpent through the bark surface, discoid, with a single locule. Ectostromatic disc white to brown, one ostiole per disc, 200–325 μm diam. Locule undivided, 385–540 μm diam. Conidiophores 8.5–13 × 2–3 μm, cylindrical, hyaline, phiailidic, unbranched, straight or slightly curved, tapering towards the apex. Alpha conidia hyaline, aseptate, smooth, ellipsoidal, biguttulate, rounded at both ends, 6.5–11 × 2–2.2 μm (av. = 8.5 × 2.1 μm, n = 30). Beta conidia present on the host, hyaline, eguttulate, smooth, filiform, hamate, 21–28.5 × 0.8–1.1 μm (av. = 25 × 1 μm, n = 30).

Figure 9. 

Diaporthe chensiensis (CFCC 52567) A–B Habit of conidiomata on branches C Transverse section of conidioma D Longitudinal section of conidioma E Alpha conidia F Beta conidia G Conidiophores H Culture on PDA and conidiomata. Scale bars: 500 μm (B), 200 μm (C–D), 10 μm (E), 20 μm (F).

Culture characters

Cultures incubated on PDA at 25 °C in darkness. Colony originally flat with white felted aerial mycelium, becoming light brown mycelium due to pigment formation, conidiomata irregularly distributed over agar surface, with yellowish conidial drops exuding from the ostioles.

Additional specimens examined

CHINA. Shaanxi Province: Ningshan County, Huoditang forest farm, on symptomatic twigs of Abies chensiensis, 5 July 2017, Q. Yang, living culture CFCC 52568 (BJFC-S1481).

Notes

Diaporthe chensiensis occurs in an independent clade (Fig. 1) and is phylogenetically distinct from D. vaccinii. Diaporhe chensiensis can be distinguished from D. vaccinii by 57 nucleotides in concatenated alignment, in which 14 were distinct in the ITS region, 13 in the cal region, 10 in the his3 region, 15 in the tef1 region and 15 in the tub2 region. Although this species belongs to the D. eres complex, it is, however, distinct from the known species within the complex (Fig. 2).

Diaporthe cinnamomi C.M. Tian & Q. Yang, sp. nov.

MycoBank No: MB824709
Figure 10

Diagnosis

Diaporthe cinnamomi differs from its closest phylogenetic species D. discoidispora in ITS, his3 and tef1 loci based on the alignments deposited in TreeBASE.

Holotype

CHINA. Zhejiang Province: Linan city, on symptomatic twigs of Cinnamomum sp., 22 Apr. 2017, Q. Yang (holotype: BJFC-S1482; ex-type culture: CFCC 52569).

Etymology

Named after the host genus on which it was collected, Cinnamomum.

Description

On PDA: Conidiomata pycnidial, globose, solitary or aggregated, deeply embedded in the substrate, erumpent, dark brown to black, 170–235 μm diam., whitish translucent to cream conidial drops exuding from the ostioles. Conidiophores 11–25 × 1.5–2 μm, cylindrical, hyaline, branched, straight or curved, tapering towards the apex. Alpha conidia hyaline, aseptate, ellipsoidal to oval, biguttulate, rounded at both ends, 5–7 × 2.5–3 μm (av. = 6 × 2.9 μm, n = 30). Beta conidia not observed.

Figure 10. 

Diaporthe cinnamomi (CFCC 52569) A Culture on PDA B Conidiomata C Alpha conidia D Conidiophores. Scale bars: 200 μm (B), 10 μm (C–D).

Culture characters

Cultures incubated on PDA at 25 °C in darkness showed colony originally flat with white felty mycelium, developing petaloid mycelium after 7–10 d and turning yellowish at the centre and brownish at the marginal area after 15 d. Conidiomata erumpent at maturity.

Additional material examined

CHINA. Zhejiang Province: Linan city, on symptomatic twigs of Cinnamomum sp., 22 Apr. 2017, Q. Yang, living culture CFCC 52570 (BJFC-S1483).

Notes

Diaporthe cinnamomi comprises strains CFCC 52569 and CFCC 52570 closely related to D. discoidispora in the combined phylogenetic tree (Fig. 1). Diaporthe cinnamomi can be distinguished based on ITS, his3 and tef1 loci from D. discoidispora (4/460 in ITS, 17/448 in his3 and 38/339 in tef1).

Diaporthe conica C.M. Tian & Q. Yang, sp. nov.

MycoBank No: MB824710
Figure 11

Diagnosis

Diaporthe conica is phylogenetically and morphologically distinct from D. rostrata, in smaller locule and alpha conidia.

Holotype

CHINA. Zhejiang Province: Tianmu Mountain, on symptomatic branches of Alangium chinense, 20 Apr. 2017, Q. Yang (holotype: BJFC-S1484; ex-type culture: CFCC 52571).

Etymology

Named after the conical conidiomata.

Description

Conidiomata pycnidial, 420–580 μm diam., solitary and with single necks erumpent through the host bark. Tissue around the neck is conical. Locule oval, undivided, 385–435 μm diam. Conidiophores reduced to conidiogenous cells. Conidiogenous cells unbranched, straight or sinuous, apical or base sometimes swelling, 19–23.5 × 2.8 μm. Alpha conidia hyaline, aseptate, ellipsoidal, biguttulate, 5.5–7 × 2.3–3 μm (av. = 6.5 × 2.6 μm, n = 30). Beta conidia not observed.

Figure 11. 

Diaporthe conica (CFCC 52571) A–B Habit of conidiomata on branches C Longitudinal section of conidioma D Alpha conidia E–F Conidiophores G Culture on PDA and conidiomata. Scale bars: 300 μm (B–C), 10 μm (D–F).

Culture characters

Cultures incubated on PDA at 25 °C in darkness. Colony white to yellowish, with dense and felted mycelium, lacking aerial mycelium, with maize-coloured conidial drops exuding from the ostioles.

Additional material examined

CHINA. Zhejiang Province: Tianmu Mountain, on symptomatic branches of Alangium chinense, 20 Apr. 2017, Q. Yang, living culture CFCC 52572 (BJFC-S1485); ibid. living culture CFCC 52573 (BJFC-S1486); ibid. living culture CFCC 52574 (BJFC-S1487).

Notes

Four isolates clustered in a clade distinct from further Diaporthe species based on DNA sequence data. Morphologically, this species is characterised by conical conidiomata, which is similar with D. rostrata from Juglans mandshurica. However, D. conica differs from D. rostrata by having smaller locule and alpha conidia (310–385 vs. 620–1100 μm in locule; 5.5–7 × 2.3–3 vs. 8.5–11.5 × 4–5 μm in alpha conidia) (Fan et al. 2015).

Diaporthe eres Nitschke, 1870

Figure 12

= Diaporthe biguttusis Y.H. Gao & L. Cai, 2015.

= Diaporthe camptothecicola C.M. Tian & Qin Yang, 2017.

= Diaporthe ellipicola Y.H. Gao & L. Cai, 2015.

= Diaporthe longicicola Y.H. Gao & L. Cai, 2015

= Diaporthe mahothocarpus (Y.H. Gao, W. Sun & L. Cai) Y.H. Gao & L. Cai, 2015.

= Diaporthe momicola Dissan., J.Y. Yan, Xing H. Li & K.D. Hyde, 2017.

Description

Conidiomata pycnidial, immersed in bark, erumpent through the bark surface, serried, with a single locule. Ectostromatic disc obviously, brown to black, with one ostiole per disc, 245–572 μm diam. Ostiole medium black, up to the level of disc. Locule circular, undivided, 335–450 μm diam. Conidiophores 10.5–19 × 1–1.5 μm, cylindrical, hyaline, unbranched, straight or slightly sinuous. Conidiogenous cells phialidic, cylindrical, terminal. Alpha conidia hyaline, aseptate, ellipsoidal to lanceolate, one guttulate at each end, 6–7.5 × 1.5–2.5 μm (av. = 6.5 × 2 μm, n = 30). Beta conidia not observed.

Figure 12. 

Diaporthe eres (CFCC 52575) A–B Habit of conidiomata on branches C Transverse section of conidioma D Longitudinal section of conidioma E Alpha conidia F Conidiophores G Culture on PDA and conidiomata. Scale bars: 500 μm (B), 200 μm (C–D), 10 μm (E–F).

Culture characters

Cultures on PDA incubated at 25 °C in darkness. Colony with white felty aerial mycelium, becoming white felted aerial mycelium in the centre and grey-brown mycelium at the marginal area, conidiomata irregularly distributed over agar surface.

Specimens examined

CHINA. Beijing: Pinggu district, on symptomatic branches of Castanea mollissima, 1 Nov. 2016, N. Jiang, living culture CFCC 52576 (BJFC-S1489); ibid. living culture CFCC 52577 (BJFC-S1490). Heilongjiang Province: Liangshui Nature Reserve, on symptomatic twigs of Acanthopanax senticosus, 29 July 2016, Q. Yang, living culture CFCC 52580 (BJFC-S1493). Heilongjiang Province: Harbin city, Botanical garden, on symptomatic twigs of Sorbus sp., 2 Aug. 2016, Q. Yang, living culture CFCC 52575 (BJFC-S1488). Shaanxi Province: Zhashui County, on symptomatic branches of Juglans regia, 29 July 2016, Q. Yang, living culture CFCC 52579 (BJFC-S1492). Zhejiang Province: Yangzhou city, on symptomatic twigs of Melia azedarace, 8 July 2017, N. Jiang, living culture CFCC 52578 (BJFC-S1491). Zhejiang Province: Tianmu Mountain, on symptomatic twigs of Rhododendron simsii, 20 Apr. 2017, Q. Yang, living culture CFCC 52581 (BJFC-S1494).

Notes

Diaporthe eres, the type species of the genus, was described by Nitschke (1870) on Ulmus sp. collected in Germany, which has a widespread distribution and a broad host range as a pathogen, endophyte or saprobe causing leaf spots, stem cankers and diseases of woody plants (Udayanga et al. 2014b). Fan et al. (2018) indicated that D. biguttusis, D. ellipicola, D. longicicola and D. mahothocarpus should be treated as synonyms of D. eres using cal, tef1 and tub2 gene regions. In this study, we extended the work presented in Fan et al. (2018) and found seven additional strains belonging to D. eres. Additionally, the phylogenetic tree demonstrated that D. camptothecicola and D. momicola should also be treated as synonyms of D. eres (Fig. 2). Diaporthe camptothecicola from Camptotheca acuminate and D. momicola from Prunus persica are described and illustrated based on the combined ITS, cal, his3, tef1 and tub2 regions (Dissanayake et al. 2017a, Yang et al. 2017c). Both of the two species are embedded in the D. eres complex. However, ITS analysis resulted in an unresolved phylogenetic tree without definitive bootstrap at the internodes, highly discordant to the trees resulting from the other four genes (Udayanga et al. 2014b). Therefore, the ITS region was not used in the combined analysis in the current study. To further investigate this complex, a second set of four (cal, his3, tef1 and tub2), three (cal, tef1 and tub2), two (tef1 and tub2) and one (tef1) data matrices were performed following Santos et al. (2017) and Fan et al. (2018). The results showed that the three genes analyses (cal, tef1 and tub2) appeared to be a better species recognition (Fig. 2). When it comes to this species complex, sequences supported by Udayanga et al. (2014b) are necessary to perform a more robust phylogenetic tree, clarifying the real species boundaries in this group in the future work.

Diaporthe fraxinicola C.M. Tian & Q. Yang, sp. nov.

MycoBank No: MB824711
Figure 13

Diagnosis

Diaporthe fraxinicola can be distinguished from the closely related species D. oraccinii and D. acerigena (described above) based on ITS, tef1 and tub2 loci. Diaporthe fraxinicola differs from D. oraccinii in larger alpha conidia and from D. acerigena in wider alpha conidia.

Holotype

CHINA. Shaanxi Province: Zhashui city, Niubeiliang Reserve, on symptomatic twigs of Fraxinus chinensis, 7 July 2017, Q. Yang (holotype: BJFC-S1495; ex-type culture: CFCC 52582).

Etymology

Named after the host genus on which it was collected, Fraxinus.

Description

Conidiomata pycnidial, immersed in bark, scattered, slightly erumpent through the bark surface, nearly flat, discoid, with a single locule. Ectostromatic disc grey to dark brown, circular to ovoid, one ostiole per disc, 150–325 μm diam. Locule circular, undivided, 275–480 μm diam. Conidiophores 10.5–17.5 × 2.1–3.2 μm, hyaline, branched, cylindrical to clavate, straight, tapering towards the apex. Alpha conidia hyaline, aseptate, ellipsoidal to oval, 2–3-guttulate, rounded at both ends, 7–10 × 2.9–3.2 μm (av. = 8.5 × 3 μm, n = 30). Beta conidia hyaline, filiform, straight or hamate, eguttulate, aseptate, base subtruncate, tapering towards one apex, 19–29.5 × 1.4 µm (av. = 24.5 × 1.4 µm, n = 30).

Figure 13. 

Diaporthe fraxinicola (CFCC 52582) A–B Habit of conidiomata on branches C Transverse section of conidioma D Longitudinal section of conidioma E Alpha conidia F Beta conidia G Culture on PDA and conidiomata. Scale bars: 500 μm (B), 200 μm (C), 100 μm (D), 10 μm (E–F).

Culture characters

Cultures incubated on PDA at 25 °C in darkness. Colony originally flat with white aerial mycelium, becoming yellowish, dense and felted aerial mycelium with age, with visible solitary or aggregated conidiomata at maturity.

Additional material examined

CHINA. Shaanxi Province: Zhashui city, Niubeiliang Reserve, on symptomatic twigs of Fraxinus chinensis, 7 July 2017, Q. Yang, living culture CFCC 52583 (BJFC-S1496).

Notes

This new species is introduced as molecular data, shows it to be a distinct clade with high support (ML/BI=100/1) and it appears most closely related to D. oraccinii and D. acerigena. Diaporthe fraxinicola can be distinguished from D. oraccinii by 22 nucleotides in concatenated alignment, in which 6 were distinct in the ITS region, 8 in the tef1 region and 8 in the tub2 region; from D. acerigena by 27 nucleotides in concatenated alignment, in which 11 were distinct in the ITS region, 3 in the tef1 region and 13 in the tub2 region. Morphologically, D. fraxinicola differs from D. oraccinii in longer and larger alpha conidia (7–10 × 2.9–3.2 vs. 5.5–7.5 × 0.5–2 μm); differs from D. acerigena in larger alpha conidia (2.9–3.2 vs. 2.1–2.9 μm) (Gao et al. 2016).

Diaporthe kadsurae C.M. Tian & Q. Yang, sp. nov.

MycoBank No: MB824713
Figure 14

Diagnosis

Diaporthe kadsurae differs from its closest phylogenetic species D. fusicola and D. ovoicicola in ITS, cal and tef1 loci based on the alignments deposited in TreeBASE.

Holotype

CHINA. Jiangxi Province: Shangrao city, Sanqing Mountain, on symptomatic branches of Kadsura longipedunculata, 1 Apr. 2017, B. Cao, Y.M. Liang & C.M. Tian (holotype: BJFC-S1497; ex-type culture: CFCC 52586).

Etymology

Named after the host genus on which it was collected, Kadsura.

Description

Conidiomata pycnidial, immersed in bark, scattered, slightly erumpent through the bark surface, nearly flat, discoid, with a single locule. Ectostromatic disc obviously, brown to black, one ostiole per disc. Locule undivided, 475–525 μm diam. Conidiophores 7–11 × 1.8–2.9 μm, cylindrical, hyaline, unbranched, straight or slightly curved, tapering towards the apex. Alpha conidia hyaline, aseptate, oval or fusoid, biguttulate, 5.5–7.5 × 2.1–2.9 μm (av. = 6.5 × 2.5 μm, n = 30). Beta conidia not observed.

Figure 14. 

Diaporthe kadsurae (CFCC 52586) A Habit of conidiomata on branches B Transverse section of conidioma C Longitudinal section of conidioma D Alpha conidia E Conidiophores F Culture on PDA. Scale bars: 200 μm (B–C), 10 μm (D–E).

Culture characters

Cultures incubated on PDA at 25 °C in darkness. Colony originally flat with white aerial mycelium, becoming dense and felted aerial mycelium in the centre and grey to black mycelium at the marginal area with solitary conidiomata at maturity.

Additional specimens examined

CHINA. Jiangxi Province: Shangrao city, Sanqing Mountain, on symptomatic branches of Kadsura longipedunculata, 1 Apr. 2017, B. Cao, Y.M. Liang & C.M. Tian, living culture CFCC 52587 (BJFC-S1498); Yunbifeng National Forest Park, on symptomatic twigs of Acer sp., 31 Mar. 2017, B. Cao, Y.M. Liang & C.M. Tian, living culture CFCC 52588 (BJFC-S1499); ibid. living culture CFCC 52589 (BJFC-S1500).

Notes

This new species is introduced as molecular data show it to be a distinct clade with high support (ML/BI=100/1) and it appears most closely related to D. fusicola and D. ovoicicola. Diaporthe kadsurae can be distinguished from D. fusicola by 11 nucleotides in concatenated alignment, in which 4 were distinct in the ITS region and 7 in the cal region; from D. ovoicicola by 25 nucleotides in concatenated alignment, in which 12 were distinct in the ITS region, 6 in the cal region and 7 in the tef1 region. Morphologically, D. kadsurae differs from D. fusicola and D. ovoicicola in shorter conidiophores (7–11 μm in D. kadsurae vs. 11–24.1 μm in D. fusicola; 7–11 μm in D. kadsurae vs. 14.2–23.6 μm in D. ovoicicola) (Gao et al. 2014).

Diaporthe padina C.M. Tian & Q. Yang, sp. nov.

MycoBank No: MB824714
Figure 15

Diagnosis

Diaporthe padina can be distinguished from the phylogenetically closely related species D. betulae in smaller conidiomata and alpha conidia.

Holotype

CHINA. Heilongjiang Province: Liangshui Nature Reserve, on symptomatic twigs of Padus racemosa, 31 July 2016, Q. Yang (holotype: BJFC-S1501; ex-type culture: CFCC 52590).

Etymology

Named after the host genus on which it was collected, Padus.

Description

Conidiomata pycnidial, immersed in bark, scattered, slightly erumpent through the bark surface, discoid, with a single locule. Ectostromatic disc light brown, one ostiole per disc, 330–520 μm diam. Locule circular, undivided, 250–550 μm diam. Conidiophores 5.5–12.5 × 1–1.5 μm, hyaline, unbranched, cylindrical, straight or slightly curved. Alpha conidia hyaline, aseptate, ellipsoidal to fusiform, eguttulate, 7–8 × 1.5–2 μm (av. = 7.5 × 1.8 μm, n = 30). Beta conidia hyaline, filiform, straight or hamate, eguttulate, aseptate, base truncate, 21–24 × 1 µm (av. = 22 × 1 µm, n = 30).

Figure 15. 

Diaporthe padina (CFCC 52590) A–B Habit of conidiomata on branches C Transverse section of conidioma D Longitudinal section of conidioma E Alpha and beta conidia F, I Beta conidia G–H Conidiophores J Culture on PDA and conidiomata. Scale bars: 500 μm (B), 200 μm (C–D), 10 μm (E–I).

Culture characters

Cultures incubated on PDA at 25 °C in darkness. Colony originally flat with white aerial mycelium, becoming grey to brown in the centre, with pale grey, felted, valviform mycelium at the marginal area and aggregated conidiomata at maturity.

Additional material examined

CHINA. Heilongjiang Province: Liangshui Nature Reserve, on symptomatic twigs of Padus racemosa, 31 July 2016, Q. Yang, living culture CFCC 52591 (BJFC-S1502).

Notes

Four strains representing D. padina cluster in a well-supported clade and appear closely related to D. betulae. This species is phylogenetically closely related to, but clearly differentiated from, D. betulae by 40 different unique fixed alleles in ITS, cal, his3, tef1 and tub2 loci (4, 7, 10, 13 and 6 respectively) based on the alignments deposited in TreeBASE. Morphologically, D. padina differs from D. betulae in smaller conidiomata and alpha conidia (250–550 vs. 600–1250 μm in conidiomata; 7–8 × 1.5–2 vs. 8.5–11 × 3–4 μm in alpha conidia) (Du et al. 2016).

Diaporthe ukurunduensis C.M. Tian & Q. Yang, sp. nov.

MycoBank No: MB824715
Figure 16

Diagnosis

Diaporthe ukurunduensis can be distinguished from the phylogenetically closely related species D. citrichinensis in longer conidiophores and shorter alpha conidia.

Holotype

CHINA. Shaanxi Province: Qinling Mountain, on symptomatic twigs of Acer ukurunduense, 27 June 2017, Q. Yang (holotype: BJFC-S1503; ex-type culture: CFCC 52592).

Etymology

Named after the host species on which it was collected, Acer ukurunduense.

Description

Conidiomata pycnidial, immersed in bark, serried, slightly erumpent through the bark surface, nearly flat, discoid, with a single locule. Ectostromatic disc dark brown to black, one ostiole per disc. Locule circular, undivided, 165–215 μm diam. Conidiophores 11.5–18 × 1.5 μm, hyaline, branched, cylindrical, straight or curved. Alpha conidia hyaline, aseptate, ellipsoidal to oval, biguttulate, 5–6 × 2.1–2.9 μm (av. = 5.5 × 2.5 μm, n = 30). Beta conidia not observed.

Figure 16. 

Diaporthe ukurunduensis (CFCC 52592) A Habit of conidiomata on branches B Transverse section of conidioma C–D Alpha conidia E Conidiophores F Culture on PDA. Scale bars: 200 μm (B), 10 μm (C–E).

Culture characters

Cultures incubated on PDA at 25 °C in darkness. Colony originally flat with white aerial mycelium, becoming brown to pale black in the centre, dense, felted, conidiomata not observed.

Additional specimens examined

CHINA. Shaanxi Province: Qinling Mountain, on symptomatic twigs of Acer ukurunduense, 27 June 2017, Q. Yang, living culture CFCC 52593 (BJFC-S1503).

Notes

Diaporthe ukurunduensis comprises strains CFCC 52592 and CFCC 52593 closely related to D. citrichinensis in the combined phylogenetic tree (Fig. 1). Diaporthe ukurunduensis can be distinguished from D. citrichinensis based on ITS and tef1 loci (10/470 in ITS and 4/336 in tef1).

Diaporthe unshiuensis F. Huang, K.D. Hyde & H.Y. Li, 2015

Figure 17

Description

On PNA: Conidiomata pycnidial, globose or rostrated, black, erumpent in tissue, erumpent at maturity, 260–500 μm diam, often with translucent conidial drops exuding from the ostioles. Conidiophores 18–28.5 × 1.4–2.1 μm, cylindrical, hyaline, branched, septate, straight or curved, tapering towards the apex. Alpha conidia abundant in culture, hyaline, aseptate, ellipsoidal to fusiform, biguttulate, sometimes with one end obtuse and the other acute, 6.5–8.5 × 2.1–2.5 μm (av. = 7.8 × 2.3 μm, n = 30). Beta conidia not observed.

Figure 17. 

Diaporthe unshiuensis (CFCC 52594) A Culture on PNA B Conidiomata C Alpha conidia D Conidiophores. Scale bars: 500 μm (B), 10 μm (C–D).

Culture characters

Cultures incubated on PNA at 25 °C in darkness. Colony entirely white at surface, reverse with pale brown pigmentation, white, fluffy aerial mycelium.

Specimens examined

CHINA. Jiangsu Province: Nanjing city, on non-symptomatic twigs of Carya illinoensis, 10 Nov. 2015, Q. Yang, living culture CFCC 52594 and CFCC 52595 (BJFC-S1476).

Notes

Diaporthe unshiuensis was originally described from twigs of non-symptomatic Fortunella margarita in Zhejiang Province, China (Huang et al. 2015). In the present study, two isolates from twigs of asymptomatic Carya illinoensis were congruent with D. unshiuensis based on morphology and DNA sequences data (Fig. 1). We therefore describe D. unshiuensis as a known species for this clade.

Discussion

The current study described 15 Diaporthe species from 42 strains based on a large set of freshly collected specimens. It includes 12 new species and 3 known species, which were sampled from 16 host genera distributed over six Provinces of China (Table 1). In this study, 194 reference sequences (including outgroup) were selected based on BLAST searches of NCBIs GenBank nucleotide database and included in the phylogenetic analyses (Table 1). Phylogenetic analyses based on five combined loci (ITS, cal, his3, tef1 and tub2), as well as morphological characters, revealed the diversity of Diaporthe species in China, mainly focusing on diebacks from major ecological or economic forest trees.

Several studies have been conducted associated with various hosts in China. For instance, the research conducted by Huang et al. (2015) revealed seven apparently undescribed endophytic Diaporthe species on Citrus. Gao et al. (2016) demonstrated that Diaporthe isolates, associated with Camellia spp., could be assigned to seven species and two species complexes. Recently, Diaporthe has been revealed as paraphyletic by Gao et al. (2017), showing that Ophiodiaporthe, Pustulomyces, Phaeocytostroma and Stenocarpella embed in Diaporthe s. lat. and eight new species of Diaporthe were introduced from leaves of several hosts. However, the identification of Diaporthe species associated with dieback of forest trees has rarely been studied, thus a large-scale investigation of Diaporthe spp. was conducted from 2015 to 2017. This study provides the first molecular phylogenetic frame of Diaporthe diversity associated with dieback in China, combined with morphological descriptions.

Diaporthe eres, the type species of the genus, was initially described by Nitschke (1870), from Ulmus sp. collected in Germany. The major problem with this generic type was the lack of an ex-type culture or ex-epitype culture, although a broad species concept has historically been associated with D. eres (Udayanga et al. 2014b). Udayanga et al. (2014b) designed strain AR5193 as the epitype of D. eres and provided the phylogram of this complex using seven loci (ITS, act, Apn2, cal, his3, FG1093, tef1 and tub2), amongst which the tef1, Apn2 and his3 genes were recognised as the best markers for defining species in the D. eres complex. Moreover, they showed that poorly supported non-monophyletic grouping was observed when ITS sequences were included in the combined analysis. In this study, although we conducted phylogenetic analysis as performed in previous studies on Diaporthe species (Santos et al. 2017), much confusion has, however, occurred in species separation of the D. eres complex (Fig. 1). Especially, the ITS region could lead to a confused taxonomic situation within this species complex. We found the three-gene analysis, excluding the ITS and his3 regions, resulted in a more robust tree congruent with Udayanga et al. (2014b) and resolved the species boundaries within the D. eres species complex. The isolates, clustering with D. eres in this study, occur on multiple hosts from many different geographic locations. This study revealed three new species belonging to the D. eres complex, i.e. D. betulina, D. chensiensis and D. padina. It also shows D. biguttusis, D. camptothecicola, D. ellipicola, D. longicicola, D. mahothocarpus and D. momicola were clustered in D. eres and should be treated as synonyms of D. eres, which is in conformity with Fan et al. (2018).

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/). Thus, during the past decade, a polyphasic approach, employing multi-locus DNA data together with morphology and ecology, has been employed for species boundaries in the genus (Crous et al. 2012, Udayanga et al. 2014a, b, Huang et al. 2015, Gao et al. 2016, 2017, Guarnaccia and Crous 2017, 2018, Hyde et al. 2017, 2018, Yang et al. 2017a, b, 2018, Guarnaccia et al. 2018, Jayawardena et al. 2018, Perera et al. 2018a, b, Tibpromma et al. 2018, Wanasinghe et al. 2018).

Further studies are required in order to conduct an extensive collection of Diaporthe isolates, to resolve taxonomic questions and to redefine species boundaries. Multiple strains from different locations should also be subjected to multi-gene phylogenetic analysis to determine intraspecific variation. The descriptions and molecular data of Diaporthe species provided in this study represent a resource for plant pathologists, plant quarantine officials and taxonomists for identification of Diaporthe.

Acknowledgements

This study is financed by National Natural Science Foundation of China (Project No.: 31670647). We are grateful to Chungen Piao, Minwei Guo (China Forestry Culture Collection Center (CFCC), Chinese Academy of Forestry, Beijing.

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