Research Article
Print
Research Article
Four new species of Diaporthe (Diaporthaceae, Diaporthales) from forest plants in China
expand article infoLingxue Cao, Dun Luo§, Wu Lin§, Qin Yang, Xiaojun Deng|
‡ Central South University of Forestry and Technology, Changsha, China
§ Guangxi State-owned Bobai Forest Farm, Yulin, China
| Guangxi Zhuang Autonomous Region Forestry Research Institute, Nanning, China
Open Access

Abstract

Species of Diaporthe inhabit a wide range of plant hosts as plant pathogens, endophytes and saprobes. During trips to collect forest pathogens in Beijing, Jiangxi, Shaanxi and Zhejiang Provinces in China, 16 isolates of Diaporthe were obtained from branch cankers and leaf spots. These isolates were studied by applying a polyphasic approach including morphological, cultural data, and phylogenetic analyses of the nuclear ribosomal internal transcribed spacer (ITS), calmodulin (cal), histone H3 (his3), partial translation elongation factor-1α (tef-1α) and β-tubulin (tub2) loci. Results revealed four new taxa, D. celticola, D. meliae, D. quercicola, D. rhodomyrti spp. nov. and two known species, D. eres and D. multiguttulata.

Keywords

Diaporthaceae, DNA phylogeny, four new taxa, systematics, taxonomy

Introduction

Diaporthe Nitschkes (syn. Phomopsis) is a large genus in the Diaporthaceae with plant pathogens, endophytes or saprobes (Muralli et al. 2006; Rossman et al. 2007; Santos and Phillips 2009; Santos et al. 2011; Udayanga et al. 2011, 2014a, b, 2015; Fan et al. 2015, 2018; Du et al. 2016; Dissanayake et al. 2017; Guarnaccia and Crous 2017, 2018; Guarnaccia et al. 2018; Yang et al. 2018, 2020, 2021a, b; Guo et al. 2020; Sun et al. 2021). Currently, more than 1100 epithets for Diaporthe and 950 for Phomopsis are listed in Index Fungorum (http://www.indexfungorum.org/; accessed 1 April 2022) with names often based on host association.

The family Diaporthaceae was established by von Höhnel (1917) and was accommodated in the order Diaporthales. Wehmeyer (1975) confined this family to include Diaporthe and Mazzantia. Later, Diaporthaceae was synonymised under Valsaceae (Barr 1978). However, analysis of LSU sequence data of diaporthalean taxa showed the distinct placement of Diaporthaceae in Diaporthales where it formed a well-supported clade (Castlebury et al. 2002). Diaporthe, the type genus of Diaporthaceae, is characterised by immersed ascomata and an erumpent pseudostroma with elongated perithecial necks (Gomes et al. 2013). Asci are unitunicate, clavate to cylindrical. Ascospores are fusoid, ellipsoid to cylindrical, hyaline, biseriate to uniseriate in the ascus, sometimes with appendages (Udayanga et al. 2011). The asexual morph is characterised by ostiolate conidiomata, with cylindrical phialides producing three types (alpha, beta, and gamma conidia) of hyaline, aseptate conidia (Udayanga et al. 2011; Gomes et al. 2013).

In China, the classification of Diaporthe has been progressing and the basis for the species identification is a combination of morphological, cultural and phylogenetical analyses (Huang et al. 2015; Gao et al. 2017; Guarnaccia and Crous 2017; Yang et al. 2017, 2018, 2020, 2021a, b; Manawasinghe et al. 2019; Jiang et al. 2021; Huang et al. 2021; Sun et al. 2021; Wang et al. 2021). The present study was conducted to identify Diaporthe species that cause dieback and leaf spot disease in Beijing, Jiangxi, Shaanxi and Zhejiang Provinces based on modern taxonomic concepts.

Materials and methods

Fungal isolation

From 2018 to 2020, sample collections have been ongoing in Beijing, Jiangxi, Shaanxi and Zhejiang Provinces, China (Table 1). Collected samples were taken to the laboratory for isolation and photographed, documented and then kept at 4 °C for further study.

Table 1.

Isolates and GenBank accession numbers of sequences used in this study.

Species Isolate GenBank accession numbers
ITS cal his3 tef-1α tub2
Diaporthe acaciigena CBS 129521* KC343005 KC343247 KC343489 KC343731 KC343973
Diaporthe acericola MFLUCC 17-0956* KY964224 KY964137 NA KY964180 KY964074
Diaporthe acerigena CFCC 52554* MH121489 MH121413 MH121449 MH121531 NA
Diaporthe acerigena CFCC 52555 MH121490 MH121414 MH121450 MH121532 NA
Diaporthe acuta PSCG 047* MK626957 MK691125 MK726161 MK654802 MK691225
Diaporthe acutispora LC6161* KX986764 KX999274 KX999235 KX999155 KX999195
Diaporthe alangii CFCC 52556* MH121491 MH121415 MH121451 MH121533 MH121573
Diaporthe alangii CFCC 52557 MH121492 MH121416 MH121452 MH121534 MH121574
Diaporthe albosinensis CFCC 53066* MK432659 MK442979 MK443004 MK578133 MK578059
Diaporthe albosinensis CFCC 53067 MK432660 MK442980 MK443005 MK578134 MK578060
Diaporthe alleghaniensis CBS 495.72* KC343007 KC343249 KC343491 KC343733 KC343975
Diaporthe ambigua CBS 114015* KC343010 KC343252 KC343494 KC343736 KC343978
Diaporthe ampelina STE-U 2660 AF230751 AY745026 NA AY745056 JX275452
Diaporthe amygdali CBS 126679* MH864208 KC343264 KC343506 KC343748 KC343990
Diaporthe amygdali syn. D. chongqingensis PSCG 435 MK626916 MK691209 MK726257 MK654866 MK691321
Diaporthe amygdali syn. D. fusicola CGMCC 3.17087 KF576281 KF576233 NA KF576256 KF576305
Diaporthe amygdali syn. D. garethjonesii MFLUCC 12-0542a KT459423 KT459470 NA KT459457 KT459441
Diaporthe amygdali syn. D. kadsurae CFCC 52586 MH121521 MH121439 MH121479 MH121563 MH121600
Diaporthe amygdali syn. D. kadsurae CFCC 52587 MH121522 MH121440 MH121480 MH121564 MH121601
Diaporthe amygdali syn. D. mediterranea SAUCC194.111 MT822639 MT855718 MT855606 MT855836 MT855951
Diaporthe amygdali syn. D. ovoicicola CGMCC 3.17093 KF576265 KF576223 NA KF576240 KF576289
Diaporthe amygdali syn. D. sterilis CBS 136969 KJ160579 KJ160548 MF418350 KJ160611 KJ160528
Diaporthe amygdali syn. D. ternstroemiae CGMCC 3.15183 KC153098 NA NA KC153089 NA
Diaporthe anacardii CBS 720.97* KC343024 KC343266 KC343508 KC343750 KC343992
Diaporthe angelicae CBS 111592* KC343027 KC343269 KC343511 KC343753 KC343995
Diaporthe apiculata CFCC 53068 MK432651 MK442973 MK442998 MK578127 MK578054
Diaporthe apiculata CFCC 53069 MK432652 MK442974 MK442999 MK578128 MK578055
Diaporthe aquatic IFRDCC 3051* JQ797437 NA NA NA NA
Diaporthe arctii DP0482* KJ590736 KJ612133 KJ659218 KJ590776 KJ610891
Diaporthe arecae CBS 161.64* KC343032 KC343274 KC343516 KC343758 KC344000
Diaporthe arengae CBS 114979* KC343034 KC343276 KC343518 KC343760 KC344002
Diaporthe arezzoensis MFLUCC 15-0127* MT185503 NA NA NA NA
Diaporthe aseana MFLUCC 12-0299a* KT459414 KT459464 NA KT459448 KT459432
Diaporthe asheicola CBS 136967* KJ160562 KJ160542 NA KJ160594 KJ160518
Diaporthe aspalathi CBS 117169* KC343036 KC343278 KC343520 KC343762 KC344004
Diaporthe australafricana CBS 111886* KC343038 KC343280 KC343522 KC343764 KC344006
Diaporthe australiana CBS 146457* MN708222 NA NA MN696522 MN696530
Diaporthe baccae CBS 136972* KJ160565 MG281695 MF418264 KJ160597 MF418509
Diaporthe batatas CBS 122.21 KC343040 KC343282 KC343524 KC343766 KC344008
Diaporthe bauhiniae CFCC 53071* MK432648 MK442970 MK442995 MK578124 MK578051
Diaporthe bauhiniae CFCC 53072 MK432649 MK442971 MK442996 MK578125 MK578052
Diaporthe beilharziae BRIP 54792* JX862529 NA NA JX862535 KF170921
Diaporthe benedicti SBen914* KM669929 KM669862 NA KM669785 NA
Diaporthe betulae CFCC 50469* KT732950 KT732997 KT732999 KT733016 KT733020
Diaporthe betulae CFCC 50470 KT732951 KT732998 KT733000 KT733017 KT733021
Diaporthe betulicola CFCC 51128* KX024653 KX024659 KX024661 KX024655 KX024657
Diaporthe betulicola CFCC 51129 KX024654 KX024660 KX024662 KX024656 KX024658
Diaporthe betulina CFCC 52560* MH121495 MH121419 MH121455 MH121537 MH121577
Diaporthe betulina CFCC 52561 MH121496 MH121420 MH121456 MH121538 MH121578
Diaporthe biconispora ZJUD62* KJ490597 NA KJ490539 KJ490476 KJ490418
Diaporthe biguttulata ZJUD47* KJ490582 NA KJ490524 KJ490461 KJ490403
Diaporthe bohemiae CBS 143347* MG281015 MG281710 MG281361 MG281536 MG281188
Diaporthe brasiliensis CBS 133183* KC343042 KC343284 KC343526 KC343768 KC344010
Diaporthe caatingaensis URM7486* KY085927 KY115597 KY115605 KY115603 KY115600
Diaporthe camelliae-sinensis SAUCC194.92* MT822620 MT855699 MT855588 MT855932 MT855817
Diaporthe canthi CPC 19740* JX069864 KC843174 NA KC843120 KC843230
Diaporthe caryae CFCC 52563* MH121498 MH121422 MH121458 MH121540 MH121580
Diaporthe caryae CFCC 52564 MH121499 MH121423 MH121459 MH121541 MH121581
Diaporthe cassines CPC 21916* KF777155 NA NA KF777244 NA
Diaporthe caulivora CBS 127268* MH864501 KC343287 KC343529 KC343771 KC344013
Diaporthe celticola CFCC 53074* MK573948 MK574587 MK574603 MK574623 MK574643
Diaporthe celticola CFCC 53075 MK573949 MK574588 MK574604 MK574624 MK574644
Diaporthe celticola CFCC 53076 MK573950 MK574589 MK574605 MK574625 MK574645
Diaporthe cercidis CFCC 52565* MH121500 MH121424 MH121460 MH121542 MH121582
Diaporthe cercidis CFCC 52566 MH121501 MH121425 MH121461 MH121543 MH121583
Diaporthe chamaeropis CBS 454.81* KC343048 KC343290 KC343532 KC343774 KC344016
Diaporthe charlesworthii BRIP 54884m* KJ197288 NA NA KJ197250 KJ197268
Diaporthe chensiensis CFCC 52567* MH121502 MH121426 MH121462 MH121544 MH121584
Diaporthe chensiensis CFCC 52568 MH121503 MH121427 MH121463 MH121545 MH121585
Diaporthe chrysalidocarpi SAUCC194.35* MT822563 MT855646 MT855532 MT855760 MT855876
Diaporthe cichorii MFLUCC 17-1023* KY964220 KY964133 NA KY964176 KY964104
Diaporthe cinnamomi CFCC 52569* MH121504 NA MH121464 MH121546 MH121586
Diaporthe cinnamomi CFCC 52570 MH121505 NA MH121465 MH121547 MH121587
Diaporthe cissampeli CPC 27302* KX228273 NA KX228366 NA KX228384
Diaporthe citri AR3405* KC843311 KC843157 KJ420881 KC843071 KC843187
Diaporthe citri CFCC 53079 MK573940 MK574579 MK574595 MK574615 MK574635
Diaporthe citriasiana CGMCC 3.15224* JQ954645 KC357491 KJ490515 JQ954663 KC357459
Diaporthe citrichinensis CGMCC 3.15225* JQ954648 KC357494 KJ420880 JQ954666 KJ490396
Diaporthe collariana MFLU 17-2770* MG806115 MG783042 NA MG783040 MG783041
Diaporthe compactum LC3083* KP267854 NA KP293508 KP267928 KP293434
Diaporthe conica CFCC 52571* MH121506 MH121428 MH121466 MH121548 MH121588
Diaporthe conica CFCC 52572 MH121507 MH121429 MH121467 MH121549 MH121589
Diaporthe constrictospora CGMCC 3.20096* MT385947 MT424718 MW022487 MT424682 MT424702
Diaporthe convolvuli CBS 124654 KC343054 KC343296 KC343538 KC343780 KC344022
Diaporthe coryli CFCC 53083* MK432661 MK442981 MK443006 MK578135 MK578061
Diaporthe coryli CFCC 53084 MK432662 MK442982 MK443007 MK538176 MK578062
Diaporthe corylicola CFCC 53986* MW839880 MW836684 MW836717 MW815894 MW883977
Diaporthe corylicola CFCC 53987 MW839867 MW836685 MW836718 MW815895 MW883978
Diaporthe crotalariae CBS 162.33* MH855395 JX197439 KC343540 GQ250307 KC344024
Diaporthe crousii CAA 823* MK792311 MK883835 MK871450 MK828081 MK837932
Diaporthe cucurbitae DAOM 42078* KM453210 NA KM453212 KM453211 KP118848
Diaporthe cuppatea CBS 117499 MH863021 KC343299 KC343541 KC343783 KC344025
Diaporthe cynaroidis CBS 122676* KC343058 KC343300 KC343542 KC343784 KC344026
Diaporthe cytosporella FAU461 KC843307 KC843141 MF418283 KC843116 KC843221
Diaporthe diospyricola CPC 21169* KF777156 NA NA NA NA
Diaporthe discoidispora ZJUD89* KJ490624 NA KJ490566 KJ490503 KJ490445
Diaporthe dorycnii MFLUCC 17-1015* KY964215 NA NA KY964171 KY964099
Diaporthe drenthii CBS 146453* MN708229 NA NA MN696526 MN696537
Diaporthe durionigena VTCC 930005* MN453530 NA NA MT276157 MT276159
Diaporthe elaeagni-glabrae LC4802* KX986779 KX999281 KX999251 KX999171 KX999212
Diaporthe endophytica CBS 133811* KC343065 KC343307 KC343549 KC343791 KC344033
Diaporthe eres AR5193* KJ210529 KJ434999 KJ420850 KJ210550 KJ420799
Diaporthe eres AR5211 KJ210538 KJ435043 KJ420875 KJ210559 KJ420828
Diaporthe eres CBS 587.79 KC343153 KC343395 KC343637 KC343879 KC344121
Diaporthe eres CFCC 52575 MH121510 NA MH121470 MH121552 MH121592
Diaporthe eres CFCC 52576 MH121511 MH121432 MH121471 MH121553 MH121593
Diaporthe eres CFCC 52577 MH121512 MH121433 MH121472 MH121554 MH121594
Diaporthe eres CFCC 52578 MH121513 MH121434 MH121473 MH121555 MH121595
Diaporthe eres CFCC 52579 MH121514 NA MH121474 MH121556 NA
Diaporthe eres CFCC 52580 MH121515 NA MH121475 MH121557 MH121596
Diaporthe eres CFCC 52581 MH121516 NA MH121476 MH121558 MH121597
Diaporthe eres CGMCC 3.15181 KC153096 NA NA KC153087 KF576312
Diaporthe eres CGMCC 3.17081 KF576282 NA NA KF576257 KF576306
Diaporthe eres CGMCC 3.17089 KF576267 NA NA KF576242 KF576291
Diaporthe eres DAOM 695742 KU552025 NA NA KU552023 KU574615
Diaporthe eres MAFF 625034 JQ807469 KJ435023 KJ420868 JQ807418 KJ420819
Diaporthe eres MFLU 17-0646 MG828895 MG829274 NA MG829270 MG843877
Diaporthe eres MFLUCC 16-0113 KU557563 KU557611 NA KU557631 KU557587
Diaporthe eres MFLUCC 17-0963 KY964190 KY964116 NA KY964146 KY964073
Diaporthe eres syn. D. alnea CBS 146.46 KC343008 KC343250 KC343492 KC343734 KC343976
Diaporthe eres syn. D. camptothecicola CFCC 51632 KY203726 KY228877 KY228881 KY228887 KY228893
Diaporthe eres syn. D. celastrina CBS 139.27 KC343047 KC343289 KC343531 KC343773 KC344015
Diaporthe eres syn. D. celeris CBS 143349 MG281017 MG281712 MG281363 MG281538 MG281190
Diaporthe eres syn.D. ellipicola CGMCC 3.17084 KF576270 NA NA KF576245 KF576294
Diaporthe eres syn. D. neilliae CBS 144.27 KC343144 KC343386 KC343628 KC343870 KC344112
Diaporthe eres syn. D. pulla CBS 338.89 KC343152 KC343394 KC343636 KC343878 KC344120
Diaporthe eres CSUFTCC101 ON076564 NA ON081664 ON081656 NA
Diaporthe eres CSUFTCC102 ON076565 NA ON081665 ON081657 NA
Diaporthe eres CSUFTCC103 ON076566 NA ON081666 ON081658 NA
Diaporthe eucalyptorum CBS 132525* MH305525 NA NA NA NA
Diaporthe foeniculacea CBS 111553* KC343101 KC343343 KC343585 KC343827 KC344069
Diaporthe fraxini-angustifoliae BRIP 54781* JX862528 NA NA JX862534 KF170920
Diaporthe fraxinicola CFCC 52582* MH121517 MH121435 NA MH121559 NA
Diaporthe fraxinicola CFCC 52583 MH121518 MH121436 NA MH121560 NA
Diaporthe fructicola MAFF 246408* LC342734 LC342738 LC342737 LC342735 LC342736
Diaporthe fulvicolor PSCG 051* MK626859 MK691132 MK726163 MK654806 MK691236
Diaporthe ganjae CBS 180.91* KC343112 KC343354 KC343596 KC343838 KC344080
Diaporthe ganzhouensis CFCC 53087* MK432665 MK442985 MK443010 MK578139 MK578065
Diaporthe ganzhouensis CFCC 53088 MK432666 MK442986 MK443011 MK578140 MK578066
Diaporthe goulteri BRIP 55657a* KJ197290 NA NA KJ197252 KJ197270
Diaporthe grandiflori SAUCC194.84* MT822612 MT855691 MT855580 MT855809 MT855924
Diaporthe guangxiensis JZB320087 MK335765 MK736720 NA MK500161 MK523560
Diaporthe gulyae BRIP 54025 JF431299 NA NA JN645803 KJ197271
Diaporthe guttulata CGMCC 3.20100* MT385950 MW022470 MW022491 MT424685 MT424705
Diaporthe helianthi CBS 592.81* KC343115 KC343357 KC343599 KC343841 KC344083
Diaporthe heliconiae SAUCC194.77* MT822605 MT855684 MT855573 MT855802 MT855917
Diaporthe heterophyllae CPC 26215* MG600222 MG600218 MG600220 MG600224 MG600226
Diaporthe heterostemmatis SAUCC194.85* MT822613 MT855692 MT855581 MT855810 MT855925
Diaporthe hickoriae CBS 145.26* KC343118 KC343360 KC343620 KC343844 KC344086
Diaporthe hispaniae CBS 143351* MG281123 MG281820 MG281471 MG281644 MG281296
Diaporthe hongkongensis CBS 115448* KC343119 KC343361 KC343603 KC343845 KC344087
Diaporthe hubeiensis JZB320123* MK335809 MK500235 NA MK523570 MK500148
Diaporthe incomplete LC6754* KX986794 KX999289 KX999265 KX999186 KX999226
Diaporthe inconspicua CBS 133813* KC343123 KC343365 KC343607 KC343849 KC344091
Diaporthe infecunda CBS 133812* KC343126 KC343368 KC343610 KC343852 KC344094
Diaporthe irregularis CGMCC 3.20092* MT385951 MT424721 NA MT424686 MT424706
Diaporthe isoberliniae CPC 22549* KJ869190 NA NA NA KJ869245
Diaporthe juglandicola CFCC 51134* KU985101 KX024616 KX024622 KX024628 KX024634
Diaporthe kochmanii BRIP 54033* JF431295 NA NA JN645809 NA
Diaporthe kongii BRIP 54031* JF431301 NA NA JN645797 KJ197272
Diaporthe krabiensis MFLUCC 17-2481* MN047100 NA NA MN433215 MN431495
Diaporthe lenispora CGMCC 3.20101* MT385952 MW022472 MW022493 MT424687 MT424707
Diaporthe litchicola BRIP 54900* JX862533 NA NA JX862539 KF170925
Diaporthe litchi SAUCC194.22* MT822550 MT855635 MT855519 MT855747 MT855863
Diaporthe lithocarpi CGMCC 3.15175* KC135104 KF576235 NA KC153095 KF576311
Diaporthe longicolla FAU599 KJ590728 KJ612124 KJ659188 KJ590767 KJ610883
Diaporthe longispora CBS 194.36* MH855769 KC343377 KC343619 KC343861 KC344103
Diaporthe lusitanicae CBS 123212* MH863279 KC343378 KC343620 KC343862 KC344104
Diaporthe lutescens SAUCC194.36* MT822564 MT855647 MT855533 MT855761 MT855877
Diaporthe macadamiae CBS 146455* MN708230 NA NA MN696528 MN696539
Diaporthe macintoshii BRIP 55064a* KJ197289 NA NA KJ197251 KJ197269
Diaporthe malorum CAA 734* KY435638 KY435658 KY435648 KY435627 KY435668
Diaporthe marina MFLU 17-2622* MN047102 NA NA NA NA
Diaporthe masirevicii BRIP 54256* KJ197276 NA NA KJ197238 KJ197256
Diaporthe mayteni CBS 133185* KC343139 KC343381 KC343623 KC343865 KC344107
Diaporthe maytenicola CPC 21896* KF777157 NA NA NA KF777250
Diaporthe melastomatis SAUCC194.55* MT822583 MT855664 MT855551 MT855780 MT855896
Diaporthe melonis CBS 435.87 KC343141 KC343383 KC343625 KC343867 KC344109
Diaporthe meliae CFCC 53089* MK432657 NA ON081662 ON081654 MK578057
Diaporthe meliae CFCC 53090 MK432658 NA ON081663 ON081655 MK578058
Diaporthe middletonii BRIP 54884e* KJ197286 NA NA KJ197248 KJ197266
Diaporthe minima CGMCC 3.20097* MT385953 MT424722 MW022496 MT424688 MT424708
Diaporthe minusculata CGMCC 3.20098* MT385957 MW022475 MW022499 MT424692 MT424712
Diaporthe miriciae BRIP 54736j* KJ197282 NA NA KJ197244 KJ197262
Diaporthe multigutullata CFCC 53095 MK432645 MK442967 MK442992 MK578121 MK578048
Diaporthe multigutullata CFCC 53096 MK432646 MK442968 MK442993 MK578122 MK578049
Diaporthe multigutullata CFCC 53098 MK573957 MK574592 MK574612 MK574632 MK574652
Diaporthe multigutullata CFCC 53099 MK573958 MK574593 MK574613 MK574633 MK574653
Diaporthe multigutullata CFCC 53100 MK573959 MK574594 MK574614 MK574634 MK574654
Diaporthe musigena CBS 129519* KC343143 KC343385 KC343267 KC343869 KC344111
Diaporthe myracrodruonis URM 7972 MK205289 MK205290 NA MK213408 MK205291
Diaporthe neoarctii CBS 109490* KC343145 KC343387 KC343629 KC343871 KC344113
Diaporthe neoraonikayaporum MFLUCC 14-1136* KU712449 KU749356 NA KU749369 KU743988
Diaporthe nothofagi BRIP 54801* JX862530 NA NA JX862536 KF170922
Diaporthe novem CBS 127269 KC343155 KC343397 KC343639 KC343881 KC344123
Diaporthe ocoteae CPC 26217* KX228293 NA NA NA KX228388
Diaporthe oraccinii LC3166* KP267863 NA KP293517 KP267937 KP293443
Diaporthe ovalispora ZJUD93* KJ490628 NA KJ490570 KJ490507 KJ490449
Diaporthe oxe CBS 133186* KC343164 KC343406 KC343648 KC343890 KC344132
Diaporthe padina CFCC 52590* MH121525 MH121443 MH121483 MH121567 MH121604
Diaporthe padina CFCC 52591 MH121526 MH121444 MH121484 MH121568 MH121605
Diaporthe pandanicola MFLUCC 17-0607* MG646974 NA NA NA MG646930
Diaporthe paranensis CBS 133184* KC343171 KC343413 KC343655 KC343897 KC344139
Diaporthe parapterocarpi CPC 22729 KJ869138 NA NA NA KJ869248
Diaporthe parvae PSCG 035 MK626920 MK691169 MK726211 MK654859 MK691249
Diaporthe pascoei BRIP 54847* JX862538 NA NA JX862538 KF170924
Diaporthe passiflorae CPC 19183* JX069860 KY435644 KY435654 KY435623 KY435674
Diaporthe passifloricola CPC 27480* KX228292 NA KX228367 NA KX228387
Diaporthe penetriteum LC3215 KP267879 NA KP293532 KP267953 NA
Diaporthe perjuncta CBS 109745* KC343172 KC343414 KC343656 KC343898 KC344140
Diaporthe perseae CBS 151.73 KC343173 KC343415 KC343657 KC343899 KC343141
Diaporthe pescicola MFLUCC 16-0105* KU557555 KU557603 NA KU557623 KU557579
Diaporthe phaseolorum AR4203* KJ590738 KJ612135 KJ659220 KJ590739 KJ610893
Diaporthe phillipsii CAA 817* MK792305 MK883831 MK871445 MK828076 MN000351
Diaporthe podocarpi-macrophylli LC6155* KX986774 KX999278 KX999246 KX999167 KX999207
Diaporthe pometiae SAUCC194.72* MT822600 MT855679 MT855568 MT855797 MT855912
Diaporthe pseudoalnea CFCC 54190* MZ727037 MZ753468 MZ781302 MZ816343 MZ753487
Diaporthe pseudomangiferae CBS 101339* KC343181 KC343423 KC343665 KC343907 KC344149
Diaporthe pseudophoenicicola CBS 176.77 KC343183 KC343425 KC343667 KC343909 KC344151
Diaporthe pseudotsugae MFLU 15-3228* KY964225 KY964138 NA KY964181 KY964108
Diaporthe psoraleae CPC 21634* KF777158 NA NA KF777245 KF777251
Diaporthe psoraleae-pinnatae CPC 21638* KF777159 NA NA NA KF777252
Diaporthe pterocarpi MFLUCC 10-0575* JQ619901 JX197453 NA JX275418 NA
Diaporthe pterocarpicola MFLUCC 10-0580a* JQ619887 JX197433 NA JX275403 JX275441
Diaporthe pungensis SAUCC194.112* MT822640 MT855719 MT855607 MT855837 MT855952
Diaporthe pyracanthae CAA483 KY435635 KY435656 KY435645 KY435625 KY435666
Diaporthe quercicola CSUFTCC104* ON076567 ON081670 ON081667 ON081659 NA
Diaporthe quercicola CSUFTCC105 ON076568 ON081671 ON081668 ON081660 NA
Diaporthe quercicola CSUFTCC106 ON076569 ON081672 ON081669 ON081661 NA
Diaporthe racemosae CPC 26646* MG600223 MG600219 MG600221 MG600225 MG600227
Diaporthe raonikayaporum CBS 133182* KC343188 KC343430 KC343672 KC343914 KC344156
Diaporthe ravennica MFLUCC 16-0997 NA NA NA MT394670 NA
Diaporthe rhodomyrti CFCC 53101* MK432643 MK442965 MK442990 MK578119 MK578046
Diaporthe rhodomyrti CFCC 53102 MK432644 MK442966 MK442991 MK578120 MK578047
Diaporthe rhusicola CPC 18191* JF951146 KC843124 NA KC843100 KC843205
Diaporthe rosae MFLUCC 17-2658* MG828894 MG829273 NA NA MG843878
Diaporthe rosiphthora COAD 2914 MT311197 MT313691 NA MT313693 NA
Diaporthe rossmaniae CAA 762* MK792290 MK883822 MK871432 MK828063 MK837914
Diaporthe rostrata CFCC 50062* KP208847 KP208849 KP208851 KP208853 KP208855
Diaporthe rostrata CFCC 50063 KP208848 KP208850 KP208852 KP208854 KP208856
Diaporthe rudis AR3422 KC843331 KC843146 NA KC843090 KC843177
Diaporthe saccarata CBS 116311* KC343190 KC343432 KC343674 KC343916 KC344158
Diaporthe sackstonii BRIP 54669b* KJ197287 NA NA KJ197249 KJ197267
Diaporthe salicicola BRIP 54825* JX862531 NA NA JX862537 KF170923
Diaporthe sambucusii CFCC 51986* KY852495 KY852499 KY852503 KY852507 KY852511
Diaporthe sambucusii CFCC 51987 KY852496 KY852500 KY852504 KY852508 KY852512
Diaporthe schimae CFCC 53103* MK442640 MK442962 MK442987 MK578116 MK578043
Diaporthe schimae CFCC 53104 MK442641 MK442963 MK442988 MK578117 MK578044
Diaporthe schini CBS 133181* KC343191 KC343433 KC343675 KC343917 KC344159
Diaporthe schisandrae CFCC 51988* KY852497 KY852501 KY852505 KY852509 KY852513
Diaporthe schisandrae CFCC 51989 KY852498 KY852502 KY852506 KY852510 KY852514
Diaporthe schoeni MFLU 15-1279* KY964226 KY964139 NA KY964182 KY964109
Diaporthe sclerotioides CBS 296.67* MH858974 KC343435 KC343677 KC343919 KC344161
Diaporthe searlei CBS 146456* MN708231 NA NA NA MN696540
Diaporthe sennae CFCC 51636* KY203724 KY228875 NA KY228885 KY228891
Diaporthe sennae CFCC 51637 KY203725 KY228876 NA KY228886 KY228892
Diaporthe sennicola CFCC 51634* KY203722 KY228873 KY228879 KY228883 KY228889
Diaporthe sennicola CFCC 51635 KY203723 KY228874 KY228880 KY228884 KY228890
Diaporthe serafiniae BRIP 55665a* KJ197274 NA NA KJ197236 KJ197254
Diaporthe shaanxiensis CFCC 53106* MK432654 MK442976 MK443001 MK578130 NA
Diaporthe shaanxiensis CFCC 53107 MK432655 MK432977 MK432002 MK578131 NA
Diaporthe siamensis MFLUCC 10-0573a* JQ619879 JX197423 NA JX275393 JX275429
Diaporthe silvicola CFCC 54191* MZ727041 MZ753472 MZ753481 MZ816347 MZ753491
Diaporthe sojae FAU635 KJ590719 KJ612116 KJ659208 KJ590762 KJ610875
Diaporthe spartinicola CPC 24951* KR611879 NA KR857696 NA KR857695
Diaporthe spinosa PSCG 383* MK626849 MK691129 MK726156 MK654811 MK691234
Diaporthe stictica CBS 370.54 KC343212 KC343454 KC343696 KC343938 KC344180
Diaporthe subclavata ZJUD95* KJ490630 NA KJ490572 KJ490509 KJ490451
Diaporthe subcylindrospora KUMCC 17-0151 MG746629 NA NA MG746630 MG746631
Diaporthe subellipicola KUMCC 17-0153* MG746632 NA NA MG746633 MG746634
Diaporthe subordinaria CBS 464.90 KC343214 KC343456 KC343698 KC343940 KC344182
Diaporthe taoicola MFLUCC 16-0117* KU557567 NA NA KU557635 KU557591
Diaporthe tectonae MFLUCC 12-0777* KU712430 KU749345 NA KU749359 KU743977
Diaporthe tectonendophytica MFLUCC 13-0471* KU712439 KU749354 NA KU749367 KU743986
Diaporthe tectonigena LC6512 KX986782 KX999284 KX999254 KX999174 KX999214
Diaporthe terebinthifolii CBS 133180* KC343216 KC343458 KC343700 KC343942 KC344184
Diaporthe thunbergii MFLUCC 10-0576a* JQ619893 JX197440 NA JX275409 JX275449
Diaporthe thunbergiicola MFLUCC 12-0033* KP715097 NA NA KP715098 NA
Diaporthe tibetensis CFCC 51999* MF279843 MF279888 MF279828 MF279858 MF279873
Diaporthe tibetensis CFCC 52000 MF279844 MF279889 MF279829 MF279859 MF279874
Diaporthe torilicola MFLUCC 17-1051* KY964212 KY964127 NA KY964168 KY964096
Diaporthe toxica CBS 534.93* KC343220 KC343462 KC343704 KC343946 KC344188
Diaporthe tulliensis BRIP 62248a* KR936130 NA NA KR936133 KR936132
Diaporthe ueckerae FAU656* KJ590726 KJ612122 KJ659215 KJ590747 KJ610881
Diaporthe ukurunduensis CFCC 52592* MH121527 MH121445 MH121485 MH121569 NA
Diaporthe ukurunduensis CFCC 52593 MH121528 MH121446 MH121486 MH121570 NA
Diaporthe undulate LC6624* KX986798 NA KX999269 KX999190 KX999230
Diaporthe unshiuensis CFCC 52594 MH121529 MH121447 MH121487 MH121571 MH121606
Diaporthe unshiuensis CFCC 52595 MH121530 MH121448 MH121488 MH121572 MH121607
Diaporthe vaccinii CBS 160.32* KC343228 KC343470 KC343712 KC343954 KC343196
Diaporthe vangueriae CBS 137985* KJ869137 NA NA NA KJ869247
Diaporthe vawdreyi BRIP 57887a* KR936126 NA NA KR936129 KR936128
Diaporthe velutina LC4421 KX986790 NA KX999261 KX999182 KX999223
Diaporthe verniciicola CFCC 53109* MK573944 MK574583 MK574599 MK574619 MK574639
Diaporthe verniciicola CFCC 53110 MK573945 MK574584 MK574600 MK574620 MK574640
Diaporthe viniferae JZB320071 MK341551 MK500119 NA MK500107 MK500112
Diaporthe virgiliae CMW 40748 KP247556 NA NA NA KP247575
Diaporthe xishuangbanica LC6707* KX986783 NA KX999255 KX999175 KX999216
Diaporthe xunwuensis CFCC 53085* MK432663 MK442983 MK443008 MK578137 MK578063
Diaporthe xunwuensis CFCC 53086 MK432664 MK442984 MK443009 MK578138 MK578064
Diaporthe yunnanensis LC6168* KX986796 KX999290 KX999267 KX999188 KX999228
Diaporthe zaobaisu PSCG 031* MK626922 NA MK726207 MK654855 MK691245
Diaporthella corylina CBS 121124* KC343004 KC343246 KC343488 KC343730 KC343972

A total of 16 isolates from host material were obtained by removing a mucoid conidia mass from conidiomata, spreading the suspension on the surface of 1.8% potato dextrose agar (PDA), and incubating at 25 °C for up to 24 h. Single germinating conidium was removed and plated onto fresh PDA plates. Specimens were deposited in the Museums of the Beijing Forestry University ( BJFC) and Central South University of Forestry and Technology ( CSUFT). Axenic cultures were maintained in the China Forestry Culture Collection Centre ( CFCC) and Central South University of Forestry and Technology Culture Collection ( CSUFTCC).

Morphological and cultural characterization

Agar plugs (6 mm diam) were taken from the edge of actively growing cultures on PDA and transferred onto 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 25 °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 in the 10-day culture. Colony features were rated according to the color charts of Rayner (1970). Cultures were examined periodically for the development of conidiomata. The microscopic examination was based on the morphological features of conidiomata obtained from the fungal growth mounted in clear lactic acid. At least 30 conidiomata and conidia were measured to calculate the mean size/length. Micro-morphological observations were done at ×1000 magnification using a Leica compound microscope (DM 2500) with interference contrast (DIC) optics. Descriptions, nomenclature, and illustrations of taxonomic novelties were deposited at MycoBank (www.MycoBank.org) (Crous et al. 2004).

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted from colonies grown on cellophane-covered PDA using a CTAB [cetyltrimethylammonium bromide] method (Doyle and Doyle 1990). For PCR amplifications of phylogenetic markers, five different primer pairs were used (Yang et al. 2018). The PCR conditions were: an initial denaturation step of 8 min at 95 °C followed by 35 cycles of 30 s at 95 °C, 30 s at 51 °C (ITS), 58 °C (his3) or 55 °C (cal, tef-1α, tub2), and 1 min at 72 °C, and a final elongation step of 5 min at 72 °C. PCR amplification products were assayed via electrophoresis in 2% agarose gels. DNA sequencing was performed using an ABI PRISM 3730XL DNA Analyzer with a BigDye Terminater Kit v.3.1 (Invitrogen, Waltham, MA, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

Phylogenetic analyses

The quality of our amplified nucleotide sequences was checked and combined by SeqMan v.7.1.0 and reference sequences (Table 1) were retrieved from the National Center for Biotechnology Information (NCBI), according to recent publications of the genus (Guo et al. 2020; Sun et al. 2021; Yang et al. 2021b). Sequences were aligned using MAFFT v. 6 (Katoh and Toh 2010) and manually corrected using Bioedit 7.0.9.0 (Hall 1999). Phylogenetic analyses were carried out with maximum likelihood analysis (ML), which was performed at the CIPRES web portal (Miller et al. 2010), 1000 rapid bootstrap replicates were run with GTRGAMMA model of nucleotide evolution. Bayesian inference analysis (BI) was performed in MrBayes v. 3.2.0 (Ronquist and Huelsenbeck 2003). 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 best nucleotide substitution model for ITS, his3 and tub2 was TrN+I+G, while HKY+I+G was selected for both cal and tef-1α. Phylogenetic trees were viewed in FigTree v1.4. The names of the isolates from the present study are marked in blue in the trees. Maximum likelihood bootstrap support values ≥ 75% (BT) are given at the nodes. Bayesian posterior probabilities ≥ 0.95 (PP) were thickened in the phylogenetic tree. Alignment and trees were deposited in TreeBASE (submission ID: S29621).

Results

Phylogenetic analyses

The sequence datasets for the ITS, cal, his3, tef-1α and tub2, were analysed in combination to infer the interspecific relationships within Diaporthe. The combined species phylogeny of the Diaporthe isolates consisted of 303 sequences, including the outgroup Diaporthella corylina (CBS 121124). A total of 2535 characters including gaps (512 for ITS, 524 for cal, 525 for his3, 463 for tef-1α, and 511 for tub2) were included in the phylogenetic analysis. Similar tree topologies were obtained by ML and BI methods, and the best scoring ML tree is shown in Fig. 1. The ML analysis yielded a tree with a likelihood value of ln: -76822.498401 and the following model parameters: alpha: 0.508079; Π(A): 0.214617, Π(C): 0.326518, Π(G): 0.235187 and Π(T): 0.223678. The phylogenetic tree inferred from the concatenated alignment resolved the sixteen Diaporthe isolates from branch cankers or leaf spots into six well-supported monophyletic clades that represent four novel species and two known species of Diaporthe (Fig. 1).

Figure 1. 

Phylogram of Diaporthe resulting from a maximum likelihood analysis based on combined ITS, cal, his3, tef-1α and tub2. The tree is rooted with Diaporthella corylina. Values above the branches indicate Maximum Likelihood bootstrap (left, ML BP ≥ 75%) and Bayesian probabilities (right, BI PP ≥ 0.95). Strains in current study are in blue and the ex-type cultures are in bold.

Taxonomy

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

MycoBank No: 832920
Fig. 2

Diagnosis

Distinguished from the other Diaporthe species based on DNA sequence data and characterised by conidiomata with single necks erumpent through the host bark.

Figure 2. 

Diaporthe celticola (BJFC-S1616) a, b habit of conidiomata on twig c transverse section through conidiomata d, e conidiogenous cells with alpha conidia f alpha conidia g, h conidiomata formed on PDA. Scale bars: 200 μm (b, c); 10 μm (d–f).

Etymology

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

Description

Conidiomata pycnidial, 535–605 × 210–225 μm diam, solitary and with single necks erumpent through host bark. Ectostromatic disc brown, one ostiole per disc, with yellowish cream conidial drops exuding from the ostioles. Tissue around the neck is cylindrical. Locule circular, undivided, 350–375 μm diam. Conidiophores reduced to conidiogenous cells. Conidiogenous cells unbranched, straight or sinuous, apical or base sometimes swelling, (8–)10.5–13(–14.5) × 1–1.5 μm (n = 30), L/W = 8.5–10.5. Alpha conidia hyaline, aseptate, ellipsoidal, biguttulate, (5–)6–7 × 3.5–4 μm (n = 30), L/W = 1.5–1.8. Beta conidia not observed.

Culture characters

Colony originally flat with white fluffy aerial mycelium, becoming light brown to olive-green mycelium with age, marginal area irregularly, with yellowish cream conidial drops exuding from the ostioles.

Specimens examined

China, Zhejiang Province: Hanzhou City, on branches of Celtis vandervoetiana, 12 May 2018, Q. Yang & Y.M. Liang (holotype BJFC-S1616; ex-type living culture: CFCC 53074; living cultures: CFCC 53075 and CFCC 53076).

Notes

Three strains representing Diaporthe celticola cluster in a well-supported clade (ML/BI = 100/1), and appear closely related to D. acaciigena. Diaporthe celticola can be distinguished based on ITS, cal, his3, tef-1α, and tub2 loci from D. acaciigena (29/473 in ITS, 68/442 in cal, 53/460 in his3, 79/330 in tef-1α, and 49/415 in tub2). Morphologically, D. celticola is characterised by conidiomata with single necks erumpent through the host bark and can be distinguished from D. acaciigena by smaller alpha conidia (6–7 × 3.5–4 vs. 10–11 × 6–6.5 μm) (Crous et al. 2011). This is the first occasion that Diaporthe species have been discovered from infected branches on Celtis vandervoetiana and demonstrates it to be a new species based on phylogeny and morphology.

Diaporthe eres Nitschke, Pyrenomyc. Germ. 2: 245, 1870.

Description

See Udayanga et al. (2014b).

Specimens examined

China. Beijing: Pinggu District, on branches of Populus × xiaohei, 10 July 2020, Q. Yang (CSUFT101; living cultures: CSUFTCC101, CSUFTCC102, and CSUFTCC103).

Notes

Diaporthe eres is the type species of the genus and was originally described by Nitschke (1870), from Ulmus sp. in Germany, which has a widespread distribution and a broad host range as an endophyte or saprobe, or pathogen causing leaf spots, stem cankers and diseases of woody plants (Udayanga et al. 2014b). In the present study, three isolates (CSUFTCC101, CSUFTCC102, and CSUFTCC103) are embedded into the D. eres species based on DNA sequence data (Fig. 1). We therefore describe D. eres as a known species for this clade.

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

MycoBank No: 829523
Fig. 3

Diagnosis

Distinguished from the phylogenetically closely-related species, D. podocarpi-macrophylli, in shorter alpha conidia.

Figure 3. 

Diaporthe meliae (BJFC-S1668) a, b habit of conidiomata on twig c transverse section through conidiomata d longitudinal section through conidiomata e, f conidiogenous cells g alpha conidia. Scale bars: 1 mm(b); 200 μm (c, d); 10 μm (e–g).

Etymology

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

Description

Conidiomata pycnidial, immersed in the host bark, scattered, erumpent through the bark surface, discoid, with a single locule. Ectostromatic disc dark brown, one ostiole per disc, (325–)330–375(–385) μm (n = 30) diam. Locule undivided, 420–640 × 385–515 μm (n = 30). Conidiophores reduced to conidiogenous cells. Conidiogenous cells (13.5–)15–26.5(–28) × 1.3–2.1(–2.3) μm (n = 30), L/W = 8.5–15.5, cylindrical, hyaline, branched, straight or slightly curved, tapering towards the apex. Alpha conidia hyaline, aseptate, fusiform, multi-guttulate, (6.7–)8–9.5(–10) × (2–)2.1–2.3 μm (n = 30), L/W = 3.4–4.5. Beta conidia not observed.

Culture characters

Colony originally flat with white felty aerial mycelium, becoming auburn furcate mycelium with age, with irregular margin, conidiomata absent.

Specimens examined

China, Shandong Province: Rizhao City, on branches of Melia azedarach, 20 April 2018, N. Jiang (holotype BJFC-S1668; ex-type living culture: CFCC 53089; living culture: CFCC 53090).

Notes

Two strains representing Diaporthe meliae cluster in a well-supported clade (ML/BI = 100/1), and appear closely related to D. podocarpi-macrophylli. Diaporthe meliae can be distinguished based on ITS, his3, tef-1α, and tub2 loci from D. podocarpi-macrophylli (4/459 in ITS, 15/455 in his3, 25/349 in tef-1α, and 14/401 in tub2). Morphologically, D. meliae can be distinguished from D. podocarpi-macrophylli by its longer conidiogenous cells (15–26.5 vs. 6–18 μm) and alpha conidia (8–9.5 vs. 3.5–8.5 μm) (Gao et al. 2017).

Diaporthe multiguttulata F. Huang, K.D. Hyde & Hong Y. Li, Fungal Biology 119(5): 343, 2015.

Fig. 4

Description

See Yang et al. (2021a).

Specimens examined

China, Jiangxi Province: Ganzhou City, on branches of Citrus maxima, 11 May 2018, Q. Yang, & Y.M. Liang (BJFC-S1615; living cultures: CFCC 53098, CFCC 53099, and CFCC 53100).

Figure 4. 

Diaporthe multiguttulata (BJFC-S1615) a, b habit of conidiomata on twig c transverse section through conidiomata d longitudinal section through conidiomata e–g conidiomata formed on PDA h conidiogenous cells i alpha conidia g beta conidia. Scale bars: 200 μm (b–d); 10 μm (h–j).

Notes

Diaporthe multiguttulata is characterised by ellipsoidal alpha conidia with one large guttulate, and was originally described as an endophyte from healthy branch of Citrus grandis in Fujian Province, China (Huang et al. 2015). Yang et al. (2021a) identified three isolates from Citrus maxima as D. multiguttulata based on DNA sequence data and confirmed from the morphological characters. In the present study, isolates (CFCC 53098, CFCC 53099, and CFCC 53100) from an additional specimen were observed and supplemented with beta conidia (Fig. 4j).

Diaporthe quercicola Q. Yang, sp. nov.

MycoBank No: 843494
Fig. 5

Diagnosis

Distinguished from the phylogenetically closely-related species, D. biguttulata, by its filiform, eguttulate alpha conidia.

Figure 5. 

Diaporthe quercicola (CSUFTCC104) a conidiomata formed on PDA b conidiogenous cells c alpha and beta conidia. Scale bars: 200 μm (a); 10 μm (b, c).

Etymology

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

Description

On PDA: Conidiomata pycnidial, 250–330 μm diam, globose, solitary or aggregated, deeply embedded in the medium, erumpent, single or clustered in groups of 3–5 pycnidia, coated with hyphae, cream to yellowish translucent conidial droplets exuded from the ostioles. Conidiophores reduced to conidiogenous cells. Conidiogenous cells hyaline, cylindrical, unbranched, straight, tapering towards the apex, (17–)20–26(–34.5) × 2.5–3.5 μm (n = 30), L/W = 6.5–9. Alpha conidia (6.5–)7–8.5(–9) × (1.5–)2–3 μm (n = 30), L/W = 3–4.5, aseptate, hyaline, fusiform, apex at both ends, eguttulate. Beta conidia hyaline, aseptate, filiform, straight or sinuous at one end, eguttulate, (21.5–)25.5–31(–33) × 1 µm (n = 30), L/W = 22.5–31.5.

Culture characters

Colony at first white, becoming dark brown with age. Aerial mycelium white, dense, fluffy, with yellowish conidial drops exuding from the ostioles after 20 days.

Specimens examined

China. Shaanxi Province: Xian City, on branches of Quercus aliena, 10 July 2020, Q. Yang (holotype CSUFTCC104; ex-type living culture: CSUFTCC104; living cultures: CSUFTCC105 and CSUFTCC106).

Notes

Three strains representing Diaporthe quercicola cluster in a well-supported clade (ML/BI = 100/1), and appear closely related to D. biguttulata. Diaporthe quercicola can be distinguished based on ITS, his3, and tef-1α loci from D. biguttulata (8/461 in ITS, 18/448 in his3, and 22/325 in tef-1α). Morphologically, D. quercicola can be distinguished from D. biguttulata by its fusiform, eguttulate alpha conidia and narrower beta conidia (1 vs. 0.9–1.6 μm) (Huang et al. 2015).

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

MycoBank No: 829525
Fig. 6

Diagnosis

Distinguished from the phylogenetically closely-related species, D. hongkongensis, in narrower beta conidia.

Figure 6. 

Diaporthe rhodomyrti (BJFC-S1660) a conidioma formed on PNA b conidiogenous cells c alpha and beta conidia. Scale bars: 500 μm (a); 10 μm (b, c).

Etymology

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

Description

On PNA: Conidiomata pycnidial, 500–850 μm diam, globose or rostrate, black, erumpent in tissue, erumpent at maturity, often with translucent conidial drops exuding from ostioles. Conidiophores reduced to conidiogenous cells. Conidiogenous cells (14.5–)15.5–23(–25.5) × 1.5–2 μm (n = 30), L/W = 8.5–13, cylindrical, hyaline, unbranched, septate, straight, tapering towards the apex. Alpha conidia abundant in culture, hyaline, aseptate, ellipsoidal, biguttulate, 6–7(–8.5) × 2–2.5(–3) μm (n = 30), L/W = 2.8–3.3. Beta conidia hyaline, aseptate, filiform, straight to sinuous, eguttulate, (15–)16.5–21.5(–23) × 1–1.5 µm (n = 30), L/W = 15.5–16.5.

Culture characters

Colony entirely white at surface, reverse with pale brown pigmentation, white, fluffy aerial mycelium.

Specimens examined

China. Jiangxi Province: Ganzhou City, on leaves of Rhodomyrtus tomentosa, 10 May 2018, Q. Yang & Y.M. Liang (holotype BJFC-S1660; ex-type living culture: CFCC 53101; living culture: CFCC 53102).

Notes

This new species is introduced as molecular data, and shows it to be a distinct clade with high support (ML/BI = 100/1) and appears closely related to Diaporthe hongkongensis. Diaporthe rhodomyrti can be distinguished based on ITS, cal, his3, tef-1α, and tub2 loci from D. hongkongensis (2/463 in ITS, 26/441 in cal, 11/434 in his3, 10/327 in tef-1α, and 2/420 in tub2). Morphologically, D. rhodomyrti can be distinguished from D. hongkongensis by its longer conidiogenous cells (15.5–23 vs. 5–12 μm) and narrower beta conidia (1–1.5 vs. 1.5–2 μm) (Gomes et al. 2013). This is the first time that Diaporthe species has been discovered from infected leaves on Rhodomyrtus tomentosa and demonstrate it as a new species based on phylogeny and morphology.

Discussion

In this study, investigations of forest pathogens in Beijing, Jiangxi, Shaanxi and Zhejiang Provinces was carried out. Identification of our collections was conducted, based on isolates from fruiting bodies using five combined loci (ITS, cal, his3, tef-1α, and tub2), as well as morphological characteristics. It includes Diaporthe eres and D. multiguttulata, as well as four new species named D. celticola, D. meliae, D. quercicola, and D. rhodomyrti.

Diaporthe (Diaporthaceae, Sordariomycetes) are species-rich asexual taxa, which are common pathogens that cause a variety of diseases, including dieback, stem cankers, leaf spots, leaf and pod blights, fruit rots and seed decay (Uecker 1988; Rehner and Uecker 1994; Mostert et al. 2001; Thompson et al. 2001; Santos et al. 2011). Because many Diaporthe species have overlapping morphological traits, sequence data is essential to resolve this genus and introduce new species (Udayanga et al. 2014a). Combined gene sequence of ITS, cal, his3, tef-1α, and tub2 is the optimal combination for species delimitation (Santos et al. 2017). However, removing the ITS locus has little effect on reconstructed phylogenies, identifying the cal-his3-tef-1α-tub2 four loci tree as almost equivalent to the five loci phylogenetic tree.

Many confusions occur in species separation of Diaporthe eres complex with 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). Fan et al. (2018) demonstrated the effectiveness of three loci, including cal, tef-1α and tub2, for the identification of the D. eres complex in walnut trees. Similarly, Yang et al. (2018) also used three-locus sequences to identify D. eres species associated with different hosts in China, and Chaisiri et al. (2021) revealed the phylogenetic analysis from the combined dataset of cal, his3, tef-1α and tub2 was highly effective, but the ITS region impeded species delimitation, which conforms with Yang et al. (2018).

Recently, several studies have been conducted associated with various hosts in China. For instance, the research conducted by Guo et al. (2020) revealed six novel Diaporthe species that infect pears and are responsible for pear shoot canker. Sun et al. (2021) showed high species diversity of Diaporthe in tropical rain forests, with description of eight new species. Wang et al. (2021) represented the first characterization of Diaporthe species associated with peach constriction canker in China, and contributed useful data for practicable disease management. Yang et al. (2021b) identified two new species from Camellia oleifera, which is an important edible oil woody plant in southern China. This study also characterises the taxonomic and morphological diversity of Diaporthe spp. associated with different hosts, which indicated there is a potential of Diaporthe species remains to be discovered in China.

Acknowledgements

This study is financed by National Natural Science Foundation of China (Project No.: 31670647) and the Research Foundation of Education Bureau of Hunan Province (Project No.: 19B608).

References

  • Barr ME (1978) The Diaporthales in North America with emphasis on Gnomonia and its segregates. Mycologia Memoir 7: 1–232.
  • Castlebury LA, Rossman AY, Jaklitsch WJ, Vasilyeva LN (2002) A preliminary overview of the Diaporthales based on large subunit nuclear ribosomal DNA sequences. Mycologia 94(6): 1017–1031. https://doi.org/10.1080/15572536.2003.11833157
  • Chaisiri C, Liu X, Lin Y, Fu Y, Zhu F, Luo C (2021) Phylogenetic and haplotype network analyses of Diaporthe eres species in China based on sequences of multiple loci. Biology 10(3): e179. https://doi.org/10.3390/biology10030179
  • Crous PW, Gams W, Stalpers JA, Robert V, Stegehuis G (2004) MycoBank: An online initiative to launch mycology into the 21st century. Studies in Mycology 50: 19–22.
  • Crous PW, Groenewald JZ, Shivas RG, Edwards J, Seifert KA, Alfenas AC, Alfenas RF, Burgess TI, Carnegie AJ, Hardy GESJ, Hiscock N, Hüberli D, Jung T, Louis-Seize G, Okada G, Pereira OL, Stukely MJC, Wang W, White GP, Young AJ, McTaggart AR, Pascoe IG, Porter IJ, Quaedvlieg W (2011) Fungal Planet description sheets: 69–91. Persoonia 26(1): 108–156. https://doi.org/10.3767/003158511X581723
  • Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: More models, new heuristics and parallel computing. Nature Methods 9(8): e772. https://doi.org/10.1038/nmeth.2109
  • Dissanayake AJ, Zhang W, Liu M, Hyde KD, Zhao WS, Li XH, Yan JY (2017) Diaporthe species associated with peach tree dieback in Hubei, China. Mycosphere 8(5): 533–549. https://doi.org/10.5943/mycosphere/8/5/2
  • Du Z, Fan XL, Hyde KD, Yang Q, Liang YM, Tian CM (2016) Phylogeny and morphology reveal two new species of Diaporthe from Betula spp. in China. Phytotaxa 269(2): 90–102. https://doi.org/10.11646/phytotaxa.269.2.2
  • Fan XL, Hyde KD, Udayanga D, Wu XY, Tian CM (2015) Diaporthe rostrata, a novel ascomycete from Juglans mandshurica associated with walnut dieback. Mycological Progress 14(10): 1–8. https://doi.org/10.1007/s11557-015-1104-5
  • Fan XL, Yang Q, Bezerra JDP, Alvarez LV, Tian CM (2018) Diaporthe from walnut tree (Juglans regia) in China, with insight of Diaporthe eres complex. Mycological Progress 17(7): 1–13. https://doi.org/10.1007/s11557-018-1395-4
  • Gomes RR, Glienke C, Videira SIR, Lombard L, Groenewald JZ, Crous PW (2013) Diaporthe: A genus of endophytic, saprobic and plant pathogenic fungi. Persoonia 31(1): 1–41. https://doi.org/10.3767/003158513X666844
  • Guarnaccia V, Groenewald JZ, Woodhall J, Armengol J, Cinelli T, Eichmeier A, Ezra D, Fontaine F, Gramaje D, Gutierrez-Aguirregabiria A, Kaliterna J, Kiss L, Larignon P, Luque J, Mugnai L, Naor V, Raposo R, Sándor E, Váczy KZ, Crous PW (2018) Diaporthe diversity and pathogenicity revealed from a broad survey of grapevine diseases in Europe. Persoonia 40(1): 135–153. https://doi.org/10.3767/persoonia.2018.40.06
  • Guo YS, Crous PW, Bai Q, Fu M, Yang MM, Wang XH, Du YM, Hong N, Xu WX, Wang GP (2020) High diversity of Diaporthe species associated with pear shoot canker in China. Persoonia 45(1): 132–162. https://doi.org/10.3767/persoonia.2020.45.05
  • Hall T (1999) BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98.
  • Huang F, Udayanga D, Wang X, Hou X, Mei X, Fu Y, Hyde KD, Li HY (2015) Endophytic Diaporthe associated with Citrus: A phylogenetic reassessment with seven new species from China. Fungal Biology 119(5): 331–347. https://doi.org/10.1016/j.funbio.2015.02.006
  • Huang ST, Xia JW, Zhang XG, Sun WX (2021) Morphological and phylogenetic analyses reveal three new species of Diaporthe from Yunnan, China. MycoKeys 78: 49–77. https://doi.org/10.3897/mycokeys.78.60878
  • Jiang N, Voglmayr H, Piao CG, Li Y (2021) Two new species of Diaporthe (Diaporthaceae, Diaporthales) associated with tree cankers in the Netherlands. MycoKeys 85: 31–56. https://doi.org/10.3897/mycokeys.85.73107
  • Lombard L, Van Leeuwen GCM, Guarnaccia V, Polizzi G, Van Rijswick PC, Karin Rosendahl KC, Gabler J, Crous PW (2014) Diaporthe species associated with Vaccinium, with specific reference to Europe. Phytopathologia Mediterranea 53: 287–299. https://doi.org/10.14601/Phytopathol_Mediterr-14034
  • Manawasinghe IS, Dissanayake AJ, Li X, Liu M, Wanasinghe DN, Xu J, Zhao W, Zhang W, Zhou Y, Hyde KD, Brooks S, Yan J (2019) High genetic diversity and species complexity of Diaporthe associated with grapevine dieback in China. Frontiers in Microbiology 10: e1936. https://doi.org/10.3389/fmicb.2019.01936
  • Miller MA, Pfeiffer W, Schwartz T (2010) Creating the CIPRES Science Gateway for Inference of Large Phylogenetic Trees. Institute of Electrical and Electronics Engineers: New Orleans, LA, USA. https://doi.org/10.1109/GCE.2010.5676129
  • Mostert L, Crous PW, Kang JC, Phillips AJ (2001) Species of Phomopsis and a Libertella sp. occurring on grapevines with specific reference to South Africa: Morphological, cultural, molecular and pathological characterization. Mycologia 93(1): 146–167. https://doi.org/10.1080/00275514.2001.12061286
  • Muralli TS, Suryanarayanan TS, Geeta R (2006) Endophytic Phomopsis species: Host range and implications for diversity estimates. Canadian Journal of Microbiology 52(7): 673–680. https://doi.org/10.1139/w06-020
  • Nitschke T (1870) Pyrenomycetes Germanici. Breslau. Eduard Trewendt, Germany. 2: e245.
  • Rayner RW (1970) A mycological colour chart. Commonwealth Mycological Institute, Kew, UK.
  • Rehner SA, Uecker FA (1994) Nuclear ribosomal internal transcribed spacer phylogeny and host diversity in the coelomycete Phomopsis. Canadian Journal of Botany 72(11): 1666–1674. https://doi.org/10.1139/b94-204
  • Santos JM, Phillips AJL (2009) Resolving the complex of Diaporthe (Phomopsis) species occurring on Foeniculum vulgare in Portugal. Fungal Diversity 34: 111–125.
  • Santos JM, Vrandečić K, Ćosić J, Duvnjak T, Phillips AJL (2011) Resolving the Diaporthe species occurring on soybean in Croatia. Persoonia 27(1): 9–19. https://doi.org/10.3767/003158511X603719
  • Santos L, Alves A, Alves R (2017) Evaluating multi-locus phylogenies for species boundaries determination in the genus Diaporthe. PeerJ 5: e3120. https://doi.org/10.7717/peerj.3120
  • Smith H, Wingfeld MJ, Coutinho TA, Crous PW (1996) Sphaeropsis sapinea and Botryosphaeria dothidea endophytic in Pinus spp. and Eucalyptus spp. in South Africa. South African Journal of Botany 62(2): 86–88. https://doi.org/10.1016/S0254-6299(15)30596-2
  • Sun W, Huang S, Xia J, Zhang X, Li Z (2021) Morphological and molecular identification of Diaporthe species in south-western China, with description of eight new species. MycoKeys 77: 65–95. https://doi.org/10.3897/mycokeys.77.59852
  • Thompson SM, Tan YP, Young AJ, Neate SM, Aitken EAB, Shivas RG (2001) Stem cankers on sunflower (Helianthus annuus) in Australia reveal a complex of pathogenic Diaporthe (Phomopsis) species. Persoonia 27(1): 80–89. https://doi.org/10.3767/003158511X617110
  • Udayanga D, Liu X, McKenzie EH, Chukeatirote E, Bahkali AH, Hyde KD (2011) The genus Phomopsis: Biology, applications, species concepts and names of common phytopathogens. Fungal Diversity 50(1): 189–225. https://doi.org/10.1007/s13225-011-0126-9
  • Udayanga D, Castlebury LA, Rossman AY, Hyde KD (2014a) Species limits in Diaporthe: Molecular re-assessment of D. citri, D. cytosporella, D. foeniculina and D. rudis. Persoonia 32(1): 83–101. https://doi.org/10.3767/003158514X679984
  • Udayanga D, Castlebury LA, Rossman AY, Chukeatirote E, Hyde KD (2014b) Insights into the genus Diaporthe: Phylogenetic species delimitation in the D. eres species complex. Fungal Diversity 67(1): 203–229. https://doi.org/10.1007/s13225-014-0297-2
  • Udayanga D, Castlebury LA, Rossman AY, Chukeatirote E, Hyde KD (2015) The Diaporthe sojae species complex: Phylogenetic re-assessment of pathogens associated with soybean, cucurbits and other field crops. Fungal Biology 119(5): 383–407. https://doi.org/10.1016/j.funbio.2014.10.009
  • Uecker FA (1988) A world list of Phomopsis names with notes on nomenclature, morphology and biology. Mycological Memoirs 13: 1–231.
  • von Höhnel FXR (1917) System der Diaportheen. Borntraeger.
  • Wang X, Guo Y, Du Y, Yang Z, Huang X, Hong N, Xu WX, Wang GP (2021) Characterization of Diaporthe species associated with peach constriction canker, with two novel species from China. MycoKeys 80: 77–90. https://doi.org/10.3897/mycokeys.80.63816
  • Wehmeyer LE (1975) The pyrenomycetous fungi. Mycologia Memoir 6: 1–250.
  • Yang Q, Fan XL, Du Z, Tian CM (2017) Diaporthe species occurring on Senna bicapsularis in southern China, with descriptions of two new species. Phytotaxa 302(2): 145–155. https://doi.org/10.11646/phytotaxa.302.2.4
  • Yang Q, Fan XL, Guarnaccia V, Tian CM (2018) High diversity of Diaporthe species associated with dieback diseases in China, with twelve new species described. MycoKeys 39: 97–149. https://doi.org/10.3897/mycokeys.39.26914
  • Yang Q, Tang J, Zhou GY (2021b) Characterization of Diaporthe species on Camellia oleifera in Hunan Province, with descriptions of two new species. MycoKeys 84: 15–33. https://doi.org/10.3897/mycokeys.84.71701