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. 2023 May 24;9(6):603. doi: 10.3390/jof9060603

Integrative Taxonomy of Novel Diaporthe Species Associated with Medicinal Plants in Thailand

Jutamart Monkai 1,2, Sinang Hongsanan 1,2, Darbhe J Bhat 3,4, Turki M Dawoud 3, Saisamorn Lumyong 1,2,5,*
Editor: Lei Cai
PMCID: PMC10301816  PMID: 37367539

Abstract

During our investigations of the microfungi on medicinal plants in Thailand, five isolates of Diaporthe were obtained. These isolates were identified and described using a multiproxy approach, viz. morphology, cultural characteristics, host association, the multiloci phylogeny of ITS, tef1-α, tub2, cal, and his3, and DNA comparisons. Five new species, Diaporthe afzeliae, D. bombacis, D. careyae, D. globoostiolata, and D. samaneae, are introduced as saprobes from the plant hosts, viz. Afzelia xylocarpa, Bombax ceiba, Careya sphaerica, a member of Fagaceae, and Samanea saman. Interestingly, this is the first report of Diaporthe species on these plants, except on the Fagaceae member. The morphological comparison, updated molecular phylogeny, and pairwise homoplasy index (PHI) analysis strongly support the establishment of novel species. Our phylogeny also revealed the close relationship between D. zhaoqingensis and D. chiangmaiensis; however, the evidence from the PHI test and DNA comparison indicated that they are distinct species. These findings improve the existing knowledge of taxonomy and host diversity of Diaporthe species as well as highlight the untapped potential of these medicinal plants for searching for new fungi.

Keywords: Asexual morph, classification, Diaporthaceae, new host records, saprobes

1. Introduction

Medicinal plants are essential for sustaining human health and livelihoods according to their ethnobotanical uses and therapeutic purposes [1,2]. They have also contributed to maintaining biodiversity in forest ecosystems and supporting natural recreation in urban ecosystems [1,2]. Fungi are usually encountered in medicinal plants, where they can affect their hosts in both beneficial and harmful manners [2,3,4]. As pathogens, they impair plant health and productivity [4]; whereas, as endophytes, they promote plant growth and produce a diverse array of secondary metabolites, which have been exploited for the development of new drugs and pharmaceutical products [2,3]. Thus, studies of fungi associated with medicinal plants represent a significant repository for the estimation of fungal diversity, the discovery of novel fungi and fungal–plant interactions, as well as the bioprospecting of new bioactive compounds and their biotechnological applications [5,6,7,8,9,10,11,12].

Diaporthe species are a large and diverse group of fungi known as endophytes, saprobes, and plant pathogens, with worldwide distribution and a broad range of host associations [13,14,15,16,17,18]. Pathogenic Diaporthe species cause various plant diseases, such as blight, cankers, diebacks, fruit rots, leaf spots, and wilts, on forest trees [19,20,21,22] and many agricultural crops such as citrus, grapevine, peach, soybean, sunflower, and tea [23,24,25,26,27,28]. Morphologically, Diaporthe is characterized by pseudostromatic ascomata that usually have black lines in the host substrate, along with elongated perithecial necks for the sexual morph [29], and asexual morph consisting of ostiolate conidiomata, aseptate, and polymorphic (alpha, gamma, and beta), and hyaline conidia [14]. However, identifying Diaporthe species based solely on morphological data is challenging due to their polyphyletic nature and the presence of numerous cryptic species [30,31,32]. Recent studies have used multilocus phylogeny, including internal transcribed spacers (ITS), the translation elongation factor 1-α (tef1-α), β-tubulin (tub2), calmodulin (cal), and histone H3 (his3), along with morphological characteristics, to accurately identify and classify Diaporthe species [15,19,23,26,31,33,34,35,36]. Norphanphoun et al. [32] classified Diaporthe into 13 species complexes based on a comprehensive sequence dataset of five loci (ITS, tef1-α, tub2, cal, and his3) to assist species delineation. The integrative approach based on cultural, ecological, morphological, and molecular characteristics is advantageous for accurately identifying Diaporthe species [22,27,28,35,36,37].

Taxonomic studies of Diaporthe revealed a variety of medicinal plants as their hosts [38]. However, most of these studies have been conducted in temperate zones (i.e., [15,16,17,21,24,26,28]). Knowledge of Diaporthe associated with medicinal plants in the tropics is still limited [31,32]. Therefore, this study aims to identify and describe isolates of Diaporthe associated with several medicinal plants in Thailand using both morphological and molecular analyses. To better illustrate the placements of the five new species, their morphological descriptions, micrographs, and updated phylogenetic trees are presented and discussed.

2. Materials and Methods

2.1. Sample Collection and Morphological Examination

Fresh fungal specimens were collected from the dead leaves and woody twigs of various medicinal plants in urban parks and forest areas in the Chiang Mai and Tak provinces of Thailand in 2019 and 2022. Collected samples were investigated for macro- and micro-morphological structures using a Nikon SMZ800N stereo microscope (Nikon Instruments Inc., Melville, NY, USA) and photomicrographed with a Nikon Eclipse Ni compound microscope attached to a Nikon DS-Ri2 camera system (Nikon Instruments Inc., Melville, NY, USA). The measurement of each structure (i.e., conidiomata, conidiomatal walls, conidiophores, conidiogenous cells, and conidia) was taken using the Tarosoft (R) Image Frame Work program. All figures were modified using Adobe Photoshop CS6 Extended version 10.0 software (Adobe Systems, San Jose, CA, USA).

2.2. Fungal Isolation and Preservation

Pure cultures were obtained from single spore isolation on 2% water agar (WA), and germinated conidia were aseptically transferred to potato dextrose agar (PDA) [39]. Fungal cultures were incubated at 25 °C for four to six weeks and then examined for colony morphology and spore production. Herbarium material and pure culture of Diaporthe globoostiolata were deposited in the herbarium of Mae Fah Luang University (MFLU) and the Mae Fah Luang University Culture Collection (MFLUCC), Chiang Rai Province, Thailand. Herbarium materials and pure cultures of D. afzeliae, D. bombacis, D. careyae, and D. samaneae were deposited in the Herbarium of the Department of Biology (CMUB) and the Culture Collection of Sustainable Development of Biological Resources Laboratory, Faculty of Science, Chiang Mai University (SDBR-CMU), Chiang Mai Province, Thailand. The numbers of Index Fungorum and Faces of Fungi were acquired as outlined in the Index Fungorum [40] and Jayasiri et al. [41].

2.3. DNA Extraction, PCR Amplification, and Sequencing

A DNA Extraction Mini Kit (FAVORGEN, Ping-Tung, Taiwan) was used to extract genomic DNA from fungal colonies grown on PDA for two weeks. Five phylogenetic markers including internal transcribed spacers (ITS), translation elongation factor 1-α (tef1-α), β-tubulin (tub2), calmodulin (cal), and histone H3 (his3) were amplified using the primer pairs ITS5/ITS4 [42], EF1-728F/EF1-986R [43], Bt2a/Bt2b [44], CAL228F/CAL737R [43], and CYLH3F/H3-1b [44,45], respectively. The PCR conditions for each gene region were carried out as described by Jiang et al. [21]. The purification of PCR products was processed using a PCR Clean-up Gel Extraction NucleoSpin® Gel and PCR Clean-up Kit (Macherey-Nagel, Düren, Germany). The sequence analysis was operated by the genetic analyzer at 1ST Base Company (Kembangan, Malaysia).

2.4. Phylogenetic Analyses

The sequences obtained in this study were submitted through a BLASTn search in GenBank (www.ncbi.nlm.nih.gov/blast/, assessed on 1 March 2023) to determine the most similar taxa. The initial phylogenetic analysis was conducted based on the ITS sequence dataset from Norphanphoun et al. [32] to identify the placement of our isolates within species complexes. The newly generated sequences and their related sequences were then selected for the concatenated ITS, tef1-α, tub2, cal, and his3 sequence dataset based on the BLASTn search results and updated literature [18,22,32,46,47,48] (Table 1). The alignment of a single locus dataset was performed using MAFFT v.7 (http://mafft.cbrc.jp/alignment/server/index.html, assessed on 1 March 2023) [49] and the ambiguous sites were manually adjusted using BioEdit 7.1.3.0 [50]. The phylogenetic trees of single locus and combined datasets were analyzed using maximum likelihood (ML) and Bayesian inference (BI) criteria. Tree topologies from single locus analyses were also compared and no conflicts were found.

Table 1.

List of taxa and their GenBank accession numbers included in the phylogenetic analyses.

Taxa Names Culture Accession
No. 		GenBank Accession No.
ITS tef1-α tub2 cal his3
Cytospora disciformis CBS 116827 T KY051801 KX965072 KX964907 N/A N/A
C. leucostoma SXYLt * LKEB00000000 N/A N/A N/A N/A
Diaporthe acuta CGMCC 3.19600 T MK626957 MK654802 MK691225 MK691124 MK726161
D. acuta PSCG045 MK626956 MK654809 MK691223 MK691123 MK726160
D. afzeliae SDBR-CMU467 T OQ600199 OQ603502 OQ678279 OQ646882 OQ646886
D. ampelina CBS 114016 T AF230751 AY745056 JX275452 AY745026 N/A
D. anhuiensis CNUCC 201901 T MN219718 MN224668 MN227008 MN224549 MN224556
D. anhuiensis CNUCC 201902 MN219727 MN224669 MN227009 MN224550 MN224557
D. annellsiae BRIP 59731a T OM918687 OM960596 OM960614 N/A N/A
D. arecae CBS 161.64 T KC343032 KC343758 KC344000 KC343274 KC343516
D. arecae CBS 535.75 KC343033 KC343759 KC344001 KC343275 KC343517
D. arengae CBS 114979 T KC343034 KC343760 KC344002 KC343276 KC343518
D. aseana MFLUCC 12-0299a T KT459414 KT459448 KT459432 KT459464 N/A
D. baccae CPC 20585 KJ160564 KJ160596 N/A N/A N/A
D. betulicola CFCC 51128 T KX024653 KX024655 KX024657 KX024659 KX024661
D. betulicola CFCC 51129 KX024654 KX024656 KX024658 KX024660 KX024662
D. bohemiae CBS 143347 T MG281015 MG281536 MG281188 MG281710 MG281361
D. bohemiae CBS 143348 MG281016 MG281537 MG281189 MG281711 MG281362
D. bombacis SDBR-CMU468 T OQ600198 OQ603501 OQ678278 OQ646881 OQ646885
D. bounty BRIP 59361a T OM918690 OM960599 OM960617 N/A N/A
D. camelliae-oleiferae HNZZ027 T MZ509555 MZ504707 MZ504718 MZ504685 MZ504696
D. camelliae-oleiferae HNZZ030 MZ509556 MZ504708 MZ504719 MZ504686 MZ504697
D. camelliae-sinensis SAUCC194.103 MT822631 MT855943 MT855828 MT855710 MT855599
D. camelliae-sinensis SAUCC194.92 T MT822620 MT855932 MT855817 MT855699 MT855588
D. canthii CBS 132533 T JX069864 KC843120 KC843230 KC843174 N/A
D. careyae SDBR-CMU469 T OQ600196 OQ603449 OQ678276 OQ646879 OQ646883
D. carpini CBS 114437 KC343044 KC343770 KC344012 KC343286 KC343528
D. cercidis CFCC 52565 T MH121500 MH121542 MH121582 MH121424 MH121460
D. chamaeropis CBS 454.81 T KC343048 KC343774 KC344016 KC343290 KC343532
D. chamaeropis CBS 753.70 KC343049 KC343775 KC344017 KC343291 KC343533
D. chiangmaiensis MFLUCC 18-0544 T OK393703 OL439483 N/A N/A N/A
D. chiangmaiensis MFLUCC 18-0935 OK393704 OL439484 N/A N/A N/A
D. chiangmaiensis MFLUCC 21-0212 OK393702 OL439482 OK490918 N/A N/A
D. cinerascens CBS 719.96 KC343050 KC343776 KC344018 KC343292 KC343534
D. cissampeli CBS 141331 T KX228273 N/A KX228384 N/A KX228366
D. corylicola CFCC 53986 T MW839880 MW815894 MW883977 MW836684 MW836717
D. cytosporella FAU461 T KC843307 KC843116 KC843221 KC843141 MF418283
D. decedens CBS 109772 KC343059 KC343785 KC344027 KC343301 KC343543
D. decedens CBS 114281 KC343060 KC343786 KC344028 KC343302 KC343544
Taxa Names Culture Accession
No. 		GenBank Accession No.
ITS tef1-α tub2 cal his3
D. decorticans CBS 114200 KC343169 KC343895 KC344137 KC343411 KC343653
D. decorticans CBS 114649 KC343170 KC343896 KC344138 KC343412 KC343654
D. delonicis MFLU 16-1059 T MT215490 N/A MT212209 N/A N/A
D. detrusa CBS 109770 KC343061 KC343787 KC344029 KC343303 KC343545
D. detrusa CBS 114652 KC343062 KC343788 KC344030 KC343304 KC343546
D. elaeagni-glabrae CGMCC 3.18287 T KX986779 KX999171 KX999212 KX999281 KX999251
D. elaeagni-glabrae LC4806 KX986780 KX999172 KX999213 KX999282 KX999252
D. endocitricola ZHKUCC 20-0012 T MT355682 MT409336 MT409290 MT409312 N/A
D. endocitricola ZHKUCC 20-0013 MT355683 MT409337 MT409291 MT409313 N/A
D. eugeniae CBS 444.82 T KC343098 KC343824 KC344066 KC343340 KC343582
D. eugeniae DPFT23 MK110366 MK117267 MK122799 N/A N/A
D. fibrosa CBS 109751 KC343099 KC343825 KC344067 KC343341 KC343583
D. fibrosa CBS 113830 KC343100 KC343826 KC344068 KC343342 KC343584
D. foeniculina CBS 111553 T KC843295 KC843104 KC843209 KC843129 N/A
D. foeniculina (=D. foeniculacea) CBS 123208 T KC343104 KC343830 KC344072 KC343346 KC343588
D. foeniculina (=D. rhoicola) CBS 129528 T JF951146 KC843100 KC843205 KC843124 N/A
D. forlicesenica MFLUCC 17-1015 T KY964215 KY964171 KY964099 N/A N/A
D. fraxini-angustifoliae BRIP 54781 T JX862528 JX862534 KF170920 N/A N/A
D. fraxini-angustifoliae MFLUCC 15-0748 KT459428 KT459446 KT459430 KT459462 N/A
D. fulvicolor CGMCC 3.19601 T MK626859 MK654806 MK691236 MK691132 MK726163
D. fulvicolor PSCG 057 MK626858 MK654810 MK691233 MK691131 MK726164
D. globoostiolata MFLUCC 23-0025 T OQ600200 OQ603503 OQ678280 N/A N/A
D. gossiae BRIP 59730a T OM918693 OM960602 OM960620 N/A N/A
D. guangxiensis JZB320091 MK335769 MK523564 MK500165 MK736724 N/A
D. guangxiensis JZB320094 T MK335772 MK523566 MK500168 MK736727 N/A
D. hickoriae CBS 145.26 T KC343118 KC343844 KC344086 KC343360 KC343602
D. hispaniae CBS 143351 T MG281123 MG281644 MG281296 MG281820 MG281471
D. hispaniae CBS 143352 MG281124 MG281645 MG281297 MG281821 MG281472
D. hongkongensis CBS 115448 T KC343119 KC343845 KC344087 KC343361 KC343603
D. howardiae BRIP 59697a T OM918695 OM960604 OM960622 N/A N/A
D. huangshanensis CNUCC 201903 T MN219729 MN224670 MN227010 N/A MN224558
D. huangshanensis CNUCC 201904 MN219730 MN224671 MN227011 N/A MN224559
D. hunanensis HNZZ023 T MZ509550 MZ504702 MZ504713 MZ504680 MZ504691
D. hunanensis HNZZ025 MZ509551 MZ504703 MZ504714 MZ504681 MZ504692
D. hungariae CBS 143353 T MG281126 MG281647 MG281299 MG281823 MG281474
D. hungariae CBS 143354 MG281127 MG281648 MG281300 MG281824 MG281475
Taxa Names Culture Accession
No. 		GenBank Accession No.
ITS tef1-α tub2 cal his3
D. impulsa CBS 114434 KC343121 KC343847 KC344089 KC343363 KC343605
D. impulsa CBS 141.27 KC343122 KC343848 KC344090 KC343364 KC343606
D. inconspicua CBS 133813 T KC343123 KC343849 KC344091 KC343365 KC343607
D. inconspicua URM7776 MG696772 MG710414 MG710395 MG710391 MG710410
D. isoberliniae CPC 22549 T KJ869133 N/A KJ869245 N/A N/A
D. juglandicola CFCC 51134 T KU985101 KX024628 KX024634 KX024616 KX024622
D. juglandicola CFCC 51135 KU985102 KX024629 KX024635 KX024617 KX024623
D. krabiensis MFLUCC 17-2481 T MN047101 MN433215 MN431495 N/A N/A
D. limonicola CPC 27869 MF418419 MF418498 MF418579 MF418253 MF418339
D. imonicola CPC 28200 T NR_154980 MF418501 MF418582 MF418256 MF418342
D. litchiicola BRIP 54900 T JX862533 JX862539 KF170925 N/A N/A
D. lithocarpi CGMCC 3.15175 T KC153104 KC153095 KF576311 KF576236 N/A
D. ithocarpi CGMCC 3.17098 KF576276 KF576251 KF576300 KF576228 N/A
D. lithocarpi LC3079 KP267851 KP267925 KP293431 N/A KP293505
D. lutescens SAUCC 194.36 T MT822564 MT855877 MT855761 MT855647 MT855533
D. macintoshii BRIP 55064a T KJ197289 KJ197251 KJ197269 N/A N/A
D. maytenicola CPC 21896 T KF777157 N/A KF777250 N/A N/A
D. melastomatis SAUCC194.55 T MT822583 MT855896 MT855780 MT855664 MT855551
D. melastomatis SAUCC194.80 MT822608 MT855920 MT855805 MT855687 MT855576
D. meliae CFCC 53089 T MK432657 ON081654 MK578057 N/A ON081662
D. meliae CFCC 53090 MK432658 ON081655 MK578058 N/A ON081663
D. melitensis CPC 27873 T MF418424 MF418503 MF418584 MF418258 MF418344
D. melitensis CPC 27875 MF418425 MF418504 MF418585 MF418259 MF418345
D. millettiae GUCC9167 T MK398674 MK480609 MK502089 MK502086 N/A
D. musigena CBS 129519 T KC343143 KC343869 KC344111 KC343385 KC343627
D. musigena HKFZL006 MK050110 MK054238 MK079660 N/A N/A
D. nebulae Phom240 KY511315 MH708543 KY511346 N/A N/A
D. nebulae PMM1681 T KY511337 MH708552 KY511369 N/A N/A
D. nelumbonis R. Kirschner 4114 T KT821501 N/A LC086652 N/A N/A
D. nigra JZB320170 T MN653009 MN892277 MN887113 N/A N/A
D. norfolkensis BRIP 59718a T OM918699 OM960608 OM960626 N/A N/A
D. oculi HHUF 30565 T LC373514 LC373516 LC373518 N/A N/A
D. oncostoma CBS 100454 KC343160 KC343886 KC344128 KC343402 KC343644
D. oncostoma CBS 589.78 KC343162 KC343888 KC344130 KC343404 KC343646
D. osmanthi GUCC9165 T MK398675 MK480610 MK502091 MK502087 N/A
D. pandanicola MFLUCC 17-0607 T MG646974 N/A MG646930 N/A N/A
D. parapterocarpi CPC 22729 T KJ869138 N/A KJ869248 N/A N/A
D. parvae CGMCC 3.19599 T MK626919 MK654858 MK691248 N/A MK726210
D. parvae PSCG035 MK626920 MK654859 MK691249 MK691169 MK726211
D. pascoei BPPCA147 MK111091 MK117255 MK122790 N/A N/A
Taxa Names Culture Accession
No. 		GenBank Accession No.
ITS tef1-α tub2 cal his3
D. pascoei BRIP 54847 T JX862532 JX862538 KF170924 N/A N/A
D. perseae BPPCA257 MK111098 MK117256 MK122791 N/A N/A
D. perseae CBS 151.73 T KC343173 KC343899 KC344141 KC343415 KC343657
D. pescicola MFLUCC 16-0105 T KU557555 KU557623 KU557579 KU557603 N/A
D. pescicola PSCG036 MK626855 MK654796 MK691226 MK691116 MK726159
D. phillipsii CAA817 T MK792305 MK828076 MN000351 MK883831 MK871445
D. phillipsii CAA818 MK792307 MK828078 MN000352 MK883833 MK871447
D. phoenicicola CBS 161.64 T MH858400 GQ250349 JX275440 JX197432 N/A
D. phoenicicola KUC21217 KT207733 N/A KT207633 N/A N/A
D. phoenicicola KUC21243 KT207761 N/A KT207659 N/A N/A
D. phoenicicola PBMR340 MK111086 MK117271 MK122805 N/A N/A
D. phoenicicola PBMR345 MK111088 MK117275 MK122810 N/A N/A
D. podocarpi-macrophylli CGMCC 3.18281 T KX986774 KX999167 KX999207 KX999278 KX999246
D. podocarpi-macrophylli LC6229 KX986771 KX999164 KX999204 KX999277 KX999243
D. poincianellae URM 7932 T MH989509 MH989538 MH989537 MH989540 MH989539
D. portugallica CPC 34247 T MH063905 MH063911 MH063917 MH063893 MH063899
D. portugallica CPC 34248 MH063906 MH063912 MH063918 MH063894 MH063900
D. pseudoinconspicua URM 7873 MH122535 MH122530 MH122521 MH122525 MH122518
D. pseudoinconspicua URM 7874 T MH122538 MH122533 MH122524 MH122528 MH122517
D. pseudomangiferae CBS 101339 T KC343181 KC343907 KC344149 KC343423 KC343665
D. pseudomangiferae CBS 388.89 KC343182 KC343908 KC344150 KC343424 KC343666
D. pseudooculi HHUF 30617 T NR_161019 LC373517 LC373519 N/A N/A
D. pseudophoenicicola CBS 462 69 T KC343184 KC343910 KC344152 KC343426 KC343668
D. pseudophoenicicola HNQZ01 MN424520 MN424562 MN424534 MN424576 MN424548
D. psoraleae CBS 136412 T KF777158 KF777245 KF777251 N/A N/A
D. psoraleae pinnatae CBS 136413 T KF777159 N/A KF777252 N/A N/A
D. pterocarpi MFLUCC 10-0571 T JQ619899 JX275416 JX275460 JX197451 N/A
D. pterocarpi MFLUCC 10-0588 JQ619900 JX275417 JX275461 JX197452 N/A
D. pterocarpicola MFLUCC 10-0580 T JQ619887 JX275403 JX275441 JX197433 N/A
D. pungensis SAUCC 194.112 T MT822640 MT855952 MT855837 MT855719 MT855607
D. pungensis SAUCC 194.89 MT822617 MT855929 MT855814 MT855696 MT855585
D. ravennica MFLUCC 15-0479 T KU900335 N/A KX432254 N/A N/A
D. ravennica MFLUCC 17-1029 KY964191 KY964147 KY964075 N/A N/A
D. rhodomyrti CFCC 53101 T MK432643 MK578119 MK578046 MK442965 MK442990
Taxa Names Culture Accession
No. 		GenBank Accession No.
ITS tef1-α tub2 cal his3
D. rhodomyrti CFCC 53102 MK432644 MK578120 MK578047 MK442966 MK442991
D. rostrata CFCC 50062 T KP208847 KP208853 KP208855 KP208849 KP208851
D. rostrata CFCC 50063 KP208848 KP208854 KP208856 KP208850 KP208852
D. rumicicola JZB320006 T MK066126 MK078545 MK078546 N/A N/A
D. rumicicola MFLUCC 18-0739 MH846233 MK049554 MK049555 N/A N/A
D. saccarata CBS 116311 T KC343190 KC343916 KC344158 KC343432 KC343674
D. salinicola MFLU 17-2592 MN047099 MN077074 N/A N/A N/A
D. salinicola MFLU 18-0553 T MN047098 MN077073 N/A N/A N/A
D. samaneae SDBR-CMU470 T OQ600197 OQ603500 OQ678277 OQ646880 OQ646884
D. schimae CFCC 53103 T MK432640 MK578116 MK578043 MK442962 MK442987
D. schimae CFCC 53104 MK432641 MK578117 MK578044 MK442963 MK442988
D. schisandrae CFCC 51988 T KY852497 KY852509 KY852513 KY852501 KY852505
D. schisandrae CFCC 51989 KY852498 KY852510 KY852514 KY852502 KY852506
D. scobina CBS 251.38 KC343195 KC343921 KC344163 KC343437 KC343679
D. searlei CBS 146456 T MN708231 N/A MN696540 N/A N/A
D. sennae CFCC 51636 T KY203724 KY228885 KY228891 KY228875 N/A
D. sennae CFCC 51637 KY203725 KY228886 KY228892 KY228876 N/A
D. spinosa CGMCC 3.19602 T MK626849 MK654811 MK691234 MK691129 MK726156
D. spinosa PSCG388 MK626860 MK654798 MK691229 MK691128 MK726171
D. stictica CBS 370.54 T KC343212 KC343938 KC344180 KC343454 KC343696
D. taiwanensis NTUCC 18-105-1 T MT241257 MT251199 MT251202 MT251196 N/A
D. taiwanensis NTUCC 18-105-2 MT241258 MT251200 MT251203 MT251197 N/A
D. taoicola MFLUCC 16-0117 T KU557567 KU557635 KU557591 N/A N/A
D. taoicola PSCG485 MK626869 MK654812 MK691227 MK691120 MK726173
D. tectonigena LC6512 KX986782 KX999174 KX999215 KX999284 KX999254
D. tectonigena MFLUCC 12-0767 T KU712429 KU749371 KU743976 KU749358 N/A
D. thunbergiae MFLUCC 10-0576a T JQ619893 JX275409 JX275449 JX197440 N/A
D. thunbergiae MFLUCC 10-0576b JQ619894 JX275410 JX275450 JX197441 N/A
D. toxicodendri FFPRI420990 LC275193 LC275217 LC275225 LC275201 LC275209
D. vangueriae CPC 22703 T KJ869137 N/A KJ869247 N/A N/A
D. velutina CGMCC 3.18286 T KX986790 KX999182 KX999223 N/A KX999261
D. velutina LC4419 KX986789 KX999181 KX999222 KX999286 KX999260
D. verniciicola CFCC 53109 T MK573944 MK574619 MK574639 MK574583 MK574599
D. verniciicola CFCC 53110 MK573945 MK574620 MK574640 MK574584 MK574600
D. viniferae JZB320071 T MK341550 MK500107 MK500112 MK500119 N/A
D. viniferae JZB320072 MK341551 MK500108 MK500113 MK500120 N/A
D. woolworthii CBS 148.27 KC343245 KC343971 KC344213 KC343487 KC343729
D. xishuangbanica CGMCC 3.18282 KX986783 KX999175 KX999216 N/A KX999255
D. xishuangbanica CGMCC 3.18283 T KX986784 KX999176 KX999217 N/A N/A
D. zaobaisu CGMCC 3.19598 T MK626922 MK654855 MK691245 N/A MK726207
Taxa Names Culture Accession
No. 		GenBank Accession No.
ITS tef1-α tub2 cal his3
D. zaobaisu PSCG032 MK626923 MK654856 MK691246 N/A MK726208
D. zhaoqingensis ZHKUCC 22-0056 T ON322885 N/A ON315074 ON315000 ON315015
D. zhaoqingensis ZHKUCC 22-0057 ON322886 ON315043 ON315075 N/A ON315016
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The ex-type cultures are indicated with the superscript “T” and the newly generated sequences are indicated in bold. “N/A” indicates the sequence is unavailable. “*” indicates a whole genomic DNA strain.

ML and BI analyses were performed using RAxML-HPC2 on XSEDE (v.8.2.12) [51] and MrBayes on XSEDE v.3.2.7a [52,53,54] in the CIPRES Science Platform V3.3 (https://www.phylo.org/portal2/home.action, assessed on 1 March 2023) [55]. The GTRGAMMA model of the bootstrapping phase with 1000 bootstrap iterations was set as the parameter for ML analysis [51]. The best nucleotide substitution model was determined using MrModeltest v.2.3 [56], and GTR + I + G was selected as the best-fitting model for the ITS, tef1-α, tub2, cal, and his3 datasets. For BI analysis, six simultaneous Markov chains were set to run 10,000,000 generations with a sampling frequency of 100 generations. The burn-in phase was set as 0.25, and the posterior probabilities (PP) were evaluated from the remaining trees. The phylogenetic trees resulting from the ML and BI analyses were visualized by the FigTree v1.4.0 program [57] and adjusted using Adobe Photoshop CS6 software (Adobe Systems, San Jose, CA, the USA). Novel obtained sequences were registered for GenBank accession numbers.

2.5. Genealogical Concordance Phylogenetic Species Recognition Analysis

The recombination level between new species and their most closely related taxa was examined using the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) model [58,59]. A pairwise homoplasy index (PHI) test was implemented by SplitsTree4 using the LogDet transformation and split decomposition options [60,61]. A PHI test result (Φw) above 0.05 indicated no significant recombination in the dataset. In addition, split graphs were generated for visualization of the relationship between closely related species.

3. Results

3.1. Molecular Phylogeny

The combination of the ITS, tef1-α, tub2, cal, and his3 sequence datasets comprised 191 Diaporthe strains, with Cytospora disciformis CBS 116,827 and C. leucostoma SXYLt as the outgroups. The aligned sequence dataset contained a total of 3020 characters with gaps in the order of ITS (1–588), tef1-α (589–992), tub2 (993–1800), cal (1801–2522), and his3 (2523–3020). The final RAxML analysis resulted in the best scoring tree with a final optimization likelihood value of -42610.603037. The matrix comprised 2000 distinct alignment patterns, with 33.63% undetermined characters or gaps. The estimated base frequencies were as follows: A = 0.218896, C = 0.324616, G = 0.235283, and T = 0.221205; substitution rates AC = 1.207023, AG = 3.073601, AT = 1.095752, CG = 0.816932, CT = 4.008593, and GT = 1.000000; and gamma distribution shape parameter of 0.398901. The phylogenetic trees generated from the ML and BI analyses revealed similar topologies. The newly recovered isolates formed five monophyletic lineages within three species complexes as follows: D. afzeliae, D. bombacis, and D. globoostiolata were clustered within the D. arecae species complex; D. samaneae was grouped in the D. oncostoma species complex; and D. careyae was associated with the D. carpini species complex (Figure 1).

Figure 1.

Figure 1

Figure 1

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Phylogenetic tree obtained from the RAxML analysis of the combined ITS, tef1-α, tub2, cal, and his3 sequence data. Bootstrap support values for ML equal to or greater than 60% and Bayesian posterior probabilities equal to or greater than 0.90 PP are indicated at the nodes as ML/PP. The ex-type strains are in black, and the new isolates obtained in this study are in red. The tree is rooted in Cytospora disciformis (CBS 116827) and C. leucostoma (SXYLt).

3.2. Genealogical Concordance Phylogenetic Species Recognition Analysis

In the PHI analysis, there was no evidence of significant recombination (Φw > 0.05) between each new species (Diaporthe afzeliae, D. bombacis, D. globoostiolata, and D. samaneae) and their closely related taxa in the combined ITS, tef1-α, tub2, cal, and his3 sequence dataset (Figure 2a–d). The results of PHI analysis also revealed no significant recombination (Φw > 0.05) between D. zhaoqingensis and D. chiangmaiensis (Figure 2e). This evidence confirms that they are distinct species.

Figure 2.

Figure 2

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The split graphs of a PHI test result of (a) Diaporthe afzeliae, (b) D. bombacis, (c) D. globoostiolata, and (d) D. samaneae with their closely related taxa, and (e) D. zhaoqingensis and D. chiangmaiensis using the LogDet transformation and split decomposition options. New species in each graph are indicated in red.

3.3. Taxonomy

Diaporthe afzeliae Monkai and S. Lumyong, sp. nov.

Index Fungorum number: IF900377; Faces of fungi number: FoF 14091; Figure 3

Etymology: Refers to the host genus, Afzelia, from which the holotype was collected.

Holotype: CMUB39998

Saprobic on dead wood of Afzelia xylocarpa. Sexual morph: undetermined. Asexual morph: Coelomycetous. Conidiomata: 200–300 high × 450–850 μm diam., pycnidial, stromatic, subepidermal, immersed, clustered, ovoid to subconical, elongated, dark brown to black, ostiolate, and multi-loculate. Ostioles: up to 120 μm wide, subglobose or conical, dark brown, and papillate. Conidiomatal wall: up to 40 μm wide, comprising several layers of thin-walled cells, arranged in textura angularis, with dark brown outer layers and hyaline to pale brown inner layers. Conidiophores: 9–26.7 × 1.7–3 μm (x = 15.8 × 2.3 μm, n = 30), tightly aggregated, subcylindrical, hyaline, septate, branched, and straight to sinuous. Conidiogenous cells: 8.2–18 × 1.4–2.7 μm (x = 12 × 2 μm, n = 30), subcylindrical to ampulliform, tapering towards apex, hyaline, phialidic, and terminal, with visible periclinal thickening and a prominent collarette. Alpha conidi:a 5.6–10.4 × 2.3–2.8 μm (x = 8.5 × 2.3 μm, n = 30), ellipsoid to elongate fusiform, obtuse at apex, subtruncate at base, sometimes with a denticle attached to the base, aseptate, hyaline, smooth-walled, and eguttulate. Beta conidia: not observed.

Culture characteristics: Colonies on PDA reached 5 cm diam. after 10 days at 25 °C, effuse, fluffy, lobate margin, originally white, becoming grey and yellow grey mycelium with age, yellowish to pale brown in reverse, with numerous black dots developing as the fruiting bodies (conidial production not seen).

Material examined: Thailand, Chiang Mai Province, Kanjanapisak Park, on dead wood of Afzelia xylocarpa (Kurz) Craib (Fabaceae), 4 April 2022, J. Monkai, KJ32 (CMUB39998, holotype), ex-type living culture, SDBR-CMU467.

Notes: Diaporthe afzeliae formed a sister clade to D. searlei and D. pterocarpicola (Figure 1). Diaporthe afzeliae can be distinguished from D. searlei CBS 146,456 by 0.84% and 2.22% base pair differences in ITS (5/598 bp) and tef1-α (11/495 bp) and D. pterocarpicola MFLUCC 10-0580 in 3.5%, 0.8%, 1.84%, and 3.79% base pair differences in ITS (18/515 bp), tef1-α (3/373 bp), tub2 (8/435 bp), and cal (17/448 bp). Diaporthe afzeliae is different from D. searlei by its wider conidia {5.6–10.4 × 2.3–2.8 vs. 5–9 × 1.5–2 μm} [62] and D. pterocarpicola by its narrower conidia {5.6–10.4 × 2.3–2.8 vs. (5–)6–7(–8) × (2–)2.5(–3.5) μm} [33]. Moreover, D. afzeliae was isolated as a saprobe from Afzelia xylocarpa, while D. searlei was associated with the husk rot of Macadamia sp. [62] and D. pterocarpicola infected leaves of Pterocarpus indicus [33].

Diaporthe bombacis Monkai and S. Lumyong, sp. nov.

Index Fungorum number: IF900378; Faces of fungi number: FoF 14092; Figure 4

Etymology: Refers to the host genus, Bombax, from which the holotype was collected.

Holotype: CMUB39995

Saprobic on dead wood of Bombax ceiba. Sexual morph: undetermined. Asexual morph: Coelomycetous. Conidiomata: 220–330 high × 270–430 μm diam., pycnidial, stromatic, subepidermal, immersed, clustered, subglobose to ovoid, dark brown to brown, ostiolate, and uni-to multi-loculate. Ostioles: up to 90 μm wide, central, subglobose, dark brown. Conidiomatal wall: up to 40 μm wide, comprising a few layers of thin-walled cells, arranged in textura angularis, with dark brown outer layers and hyaline to pale brown inner layers. Conidiophores: 6.2–24 × 1.5–2.8 μm (x = 15.8 × 2 μm, n = 30), tightly aggregated, subcylindrical, hyaline, septate, branched, and straight to sinuous. Conidiogenous cells: 4.5–12 × 1.4–2.4 μm (x = 8 × 2 μm, n = 30), subcylindrical to ampulliform, tapering towards the apex, hyaline, phialidic, and terminal, with visible periclinal thickening; collarette not observed. Alpha conidia: 6–9.4 × 1.7–3 μm (x = 7.6 × 2.4 μm, n = 30), ellipsoid to elongate fusiform, obtuse at apex, subtruncate at base, aseptate, hyaline, smooth-walled, and eguttulate. Beta conidia: not observed.

Culture characteristics: Colonies on PDA reached 5 cm diam. after 10 days at 25 °C, effuse, fluffy, lobate at the margin, originally white, becoming yellowish to pale brown mycelium with age, yellowish to pale brown in reverse, with numerous black dots developing as the fruiting bodies (conidial production not seen).

Material examined: Thailand, Chiang Mai Province, Kanjanapisak Park, on dead wood of Bombax ceiba L. (Bombacaceae), 4 April 2022, J. Monkai, KJ12 (CMUB39995, holotype), ex-type living culture, SDBR-CMU468.

Notes: Diaporthe bombacis formed a distinct clade adjacent to D. eugeniae (Figure 1). Diaporthe bombacis can be distinguished from D. eugeniae CBS 444.82 in 0.7%, 0.85%, 4.98%, 2.68%, and 1.92% base pair differences in ITS (4/571 bp), tef1-α (6/352 bp), tub2 (20/402 bp), cal (13/485 bp), and his3 (9/469 bp). Diaporthe bombacis resembles D. eugeniae in having stromatic and uni-to multi-loculate conidiomata with ostioles [63]. However, D. bombacis differs from D. eugeniae in having longer alpha conidia {6–9.4 × 1.7–3 vs. 6 × 2–3 μm} and the absence of beta conidia [63]. Diaporthe eugeniae was reported from Eugenia aromatica [63], while D. bombacis was found on Bombax ceiba.

Figure 3.

Figure 3

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Diaporthe afzeliae (CMUB39998, holotype). (a) Conidiomata on host substrate (indicated with the red arrow). (b–d) Section through conidiomata (indicated with the red arrow). (e) Ostiole. (f) Conidiomatal walls. (g–i) Conidiogenous cells giving rise to conidia. (j–m) Alpha conidia. (n,o) Colonies on PDA, (n) from above and (o) from reverse. Scale bars: (a) = 500 μm, (b–d) = 200 μm, (e,f) = 20 μm, (g,h) = 10 μm, and (i–m) = 5 μm.

Figure 4.

Figure 4

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Diaporthe bombacis (CMUB39995, holotype). (a,b) Conidiomata on host substrate (indicated with the red arrow). (c,d) Section through conidioma. (e) Ostiole. (f) Conidiomatal walls. (g,h) Conidiogenous cells giving rise to conidia. (i–k) Alpha conidia. (l,m) Colonies on PDA, (l) from above and (m) from reverse. Scale bars: (a,b) = 500 μm, (c,d) = 100 μm, (e,f) = 20 μm, (g,h) = 10 μm, and (i–k) = 5 μm.

Diaporthe careyae Monkai and S. Lumyong, sp. nov.

Index Fungorum number: IF900379; Faces of fungi number: FoF 14093; Figure 5

Etymology: Refers to the host genus, Careya, from which the holotype was collected.

Holotype: CMUB39996

Saprobic on dead wood of Careya sphaerica. Sexual morph: undetermined. Asexual morph: Coelomycetous. Conidiomata: 100–180 high × 150–320 μm diam., pycnidial, immersed to semi-immersed, erumpent, solitary to gregarious, subglobose to ovoid, dark brown to reddish-brown, uni-to bi-loculate, ostiolate, and lacking necks. Conidiomatal wall: up to 20 μm wide, comprising a few layers of thick-walled cells, arranged in textura angularis, with reddish-brown outer layers and hyaline to brown inner layers. Conidiophores: reduced to conidiogenous cells. Conidiogenous cells: 4.8–10.7 × 1.4–2.5 μm (x = 8 × 2 μm, n = 30), subcylindrical, tapering towards apex, producing 1–2 conidia, hyaline, phialidic, terminal, with visible periclinal thickening and a prominent collarette. Alpha conidia: 7–12 × 1.8–3 μm (x = 9.4 × 2.6 μm, n = 30), oblong to ellipsoid, obtuse at apex, subtruncate at base, straight to slightly curved or asymmetrical, 0–1(–2) septate, hyaline, smooth-walled, and bi- to multi-guttulate. Beta conidia: not observed.

Culture characteristics: Colonies on PDA reached 9 cm diam. after 10 days at 25 °C, effuse, sparse hyphae, filiform margin, originally white, becoming grey with age, yellowish to light brown in reverse.

Material examined: Thailand, Chiang Mai Province, Chiang Mai University, near Angkaew Reservoir, on dead wood of Careya sphaerica Roxb. (Lecythidaceae), 16 March 2022, J. Monkai, AK02 (CMUB39996, holotype), ex-type living culture, SDBR-CMU469.

Notes: Diaporthe careyae formed a well-supported monophyletic lineage basal to species in the D. carpini species complex (100% ML, 1.00 PP, Figure 1). Phylogenetically, this species was not clustered with any Diaporthe species, and the base pair difference between closely related species was not possible to compare. The morphological characteristics of D. careyae are distinct from other Diaporthe species in having septate and oblong alpha conidia. Thus, D. careyae was proposed as a new species based on its distinctive morphology and phylogenetic placement.

Figure 5.

Figure 5

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Diaporthe careyae (CMUB39996, holotype). (a) Conidiomata on host substrate (indicated with the red arrow). (b,c) Section through conidioma. (d–f) Conidiogenous cells giving rise to conidia. (g–l) Alpha conidia. (m,n) Colonies on PDA, (m) from above and (n) from reverse. Scale bars: (a) = 500 μm, (b,c) = 50 μm, (d–f) = 10 μm, and (g–l) = 5 μm.

Diaporthe globoostiolata Monkai and S. Lumyong, sp. nov.

Index Fungorum number: IF900380; Faces of fungi number: FoF 14094; Figure 6

Etymology: Refers to the globular shape of the ostiole.

Holotype: MFLU 23-0063

Saprobic on dead leaves of a member of Fagaceae. Sexual morph: undetermined. Asexual morph: Coelomycetous. Conidiomata: 90–120 high × 110–180 μm diam., pycnidial, semi-immersed, partly erumpent, solitary, subconical to subglobose, dark brown to black, uni-loculate, with ostiolar necks protruding through host surface. Ostioles: up to 80 μm wide, central, globose, black, papillate. Conidiomatal wall: up to 20 μm wide, comprising a few layers of thin-walled cells, arranged in textura angularis, with dark brown outer layers and hyaline to pale brown inner layers. Conidiophores: reduced to conidiogenous cells. Conidiogenous cells: 3.5–11.4 × 1.4–3.7 μm (x = 6.5 × 2.2 μm, n = 30), subcylindrical to ampulliform, slightly tapering towards the apex, hyaline, monophialidic, terminal, with visible periclinal thickening and a prominent collarette. Alpha conidia: 6–9.6 × 1.8–2.8 μm (x = 7.6 × 2.2 μm, n = 30), fusiform to ellipsoid, obtuse at both ends, aseptate, hyaline, smooth-walled, and mono- to bi-guttulate. Beta conidia: 13.2–22 × 1–1.8 μm (x = 16.8 ×1.4 μm, n = 30), filiform, tapering towards apex, truncate at base, straight to slightly curved, hyaline, smooth-walled, and eguttulate.

Culture characteristics: Colonies on PDA reached 9 cm diam. after 10 days at 25 °C, effuse, fluffy, lobate margin, originally white, becoming pale yellowish mycelium with age, yellowish to pale brown in reverse.

Material examined: Thailand, Tak Province, Tambon Chiang Tong, Wang Chao District, on dead leaves of a member of Fagaceae, 22 August 2019, P. Sysouphanthong, TS1-5 (MFLU 23-0063, holotype), ex-type living culture, MFLUCC 23-0025.

Notes: Diaporthe globoostiolata formed a well-supported clade basal to D. hongkongensis (99% ML, 1.00 PP, Figure 1). Diaporthe globoostiolata can be distinguished from D. hongkongensis CBS 115,448 in 1.23%, 3.55%, and 3.67% base pair differences in ITS (7/571 bp), tef1-α (12/338 bp), and tub2 (16/436 bp). Diaporthe globoostiolata and D. hongkongensis have overlapping sizes of alpha conidia {6–9.6 × 1.8–2.8 vs. (5–)6–7(–8) × (2–)2.5(–3) μm} [15]. However, the beta conidia of D. globoostiolata are slightly shorter than those of D. hongkongensis {13.2–22 × 1–1.8 vs. 18–22 × 1.5–2 μm} [15]. Our isolate and its closely related taxa, which are D. hongkongensis and D. lithocarpi, were found on the same host (member of the family, Fagaceae) [30,64,65].

Diaporthe samaneae Monkai and S. Lumyong, sp. nov.

Index Fungorum number: IF900381; Faces of fungi number: FoF 14095; Figure 7

Etymology: Refers to the host genus, Samanea, from which the holotype was collected.

Holotype: CMUB39997

Saprobic on dead wood of Samanea saman. Sexual morph: undetermined. Asexual morph: Coelomycetous. Conidiomata: 300–480 high × 290–740 μm diam., pycnidial, stromatic, superficial to semi-immersed, erumpent, clustered, subglobose to ovoid, elongate, dark brown to brown, multi-loculate, and ostiolate. Conidiomatal wall: up to 50 μm wide, comprising a few layers of thin-walled cells, arranged in textura angularis, with brown outer layers and hyaline to pale brown inner layers. Conidiophores: 7.5–31.7 × 1.5–2.7 μm (x = 19 × 2 μm, n = 30), tightly aggregated, subcylindrical, hyaline to pale brown, septate, branched, straight to sinuous, and smooth. Conidiogenous cells: 5.2–14.3 × 1.5–2.7 μm (x = 9.7 × 2 μm, n = 30), subcylindrical to ampulliform, tapering towards apex, hyaline, phialidic, terminal, with visible periclinal thickening and a prominent collarette. Alpha conidia: 7–11 × 1.8–2.8 μm (x = 8.4 × 2.4 μm, n = 30), ellipsoid to elongate fusiform, obtuse at apex, subtruncate at base, aseptate, hyaline, smooth-walled, eguttulate, forming basipetal chains of two or more conidia on phialidic neck. Beta conidia: not observed.

Culture characteristics: Colonies on PDA reached 9 cm diam. after 10 days at 25 °C, effuse, sparse hyphae, filiform margin, originally white, becoming pale yellowish mycelium with age, yellowish to pale brown in reverse, with numerous black dots developing as the fruiting bodies (conidial production not seen).

Material examined: Thailand, Chiang Mai Province, Charoen Prathet Public Park, on dead wood of Samanea saman (Jacq.) Merr. (Fabaceae), 27 March 2022, J. Monkai, JS01 (CMUB39997, holotype), ex-type living culture, SDBR-CMU470.

Notes: Diaporthe samaneae formed an independent lineage and are closely related to D. inconspicua and D. pseudoinconspicua (97% ML, 1.00 PP, Figure 1). Diaporthe samaneae can be distinguished from D. inconspicua CBS 133,813 in 3%, 1.68%, 0%, 2.71%, and 0.84% base pair differences in ITS (17/567 bp), tef1-α (5/298 bp), tub2 (0/423 bp), cal (11/406 bp), and his3 (4/479 bp) and D. pseudoinconspicua URM 7874 in 3.93%, 1.08%, 0.64%, 2.47%, and 1.01% base pair differences in ITS (18/458 bp), tef1-α (3/278 bp), tub2 (3/467 bp), cal (10/405 bp), and his3 (5/496 bp). Diaporthe samaneae differs from D. inconspicua and D. pseudoinconspicua in having longer alpha conidia {7–11 × 1.8–2.8 vs. 5.5–6.5 × 1.5–2 μm and 5–7.5(–8.5) ×2–2.5(–3.5) μm} [66,67]. The host preference of D. inconspicua is the species of Maytenus, Poincianella, and Spondias [15,66], while D. pseudoinconspicua was associated with Poincianella [67]. Both species, D. inconspicua and D. pseudoinconspicua, were reported as endophytes, while D. samaneae was reported as a saprobe from Samanea.

Figure 6.

Figure 6

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Diaporthe globoostiolata (MFLU 23-0063, holotype). (a) Conidioma on host substrate (indicated with the red arrow). (b) Section through conidioma. (c) Ostiole. (d–g) Conidiogenous cells giving rise to conidia. (h,i) Alpha and beta conidia. (j,k) Colonies on PDA, (j) from above and (k) from reverse. Scale bars: (a) = 200 μm, (b,c) = 20 μm, and (d–i) = 5 μm.

Figure 7.

Figure 7

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Diaporthe samaneae (CMUB39997, holotype). (a,b) Conidiomata on host substrate (indicated with the red arrow). (c,d) Section through conidiomata. (e) Conidiomatal walls. (f–i) Conidiogenous cells giving rise to conidia. (j–n) Alpha conidia. (o,p) Colonies on PDA, (o) from above and (p) from reverse. Scale bars: (a,b) = 500 μm, (c,d) = 200 μm, (e) = 20 μm, (f–i) = 10 μm, and (j–n) = 5 μm.

4. Discussion

This study describes five novel species of Diaporthe in Thailand. Aside from the phenotypic traits, phylogenetic and PHI analyses based on the combined sequence datasets of ITS, tef1-α, tub2, cal, and his3 were successfully applied to justify the novel species. In particular, tub2, cal, and his3 have a high discrimination power for distinguishing species in Diaporthe, and this is consistent with the results from other studies [15,18,22,35,36,37].

Our study also gains better insight into the phylogenetic relationships within Diaporthe, especially in the D. arecae species complex. Diaporthe zhaoqingensis and D. chiangmaiensis were clustered together in the same clade (98% ML, 1.00 PP) and not so well separated (Figure 1). Therefore, we compared the base pair differences between the type strains of D. zhaoqingensis ZHKUCC 22-0056 and D. chiangmaiensis MFLUCC 18-0544. There are 1.38% base pair differences in ITS (7/508 bp) between the ex-type of both strains. In the tef1-α gene region, there are 0.33% base pair differences (1/300 bp) between the type strains of D. chiangmaiensis MFLUCC 18-0544 and D. zhaoqingensis ZHKUCC 22-0057. There are 4.94% base pair differences (19/385 bp) in the tub2 gene region, between D. chiangmaiensis MFLUCC 21-0212 and the type strain of D. zhaoqingensis ZHKUCC 22-0056. However, some genes from the type strains were not available to compare. The PHI test result also showed that D. zhaoqingensis and D. chiangmaiensis were not conspecific, indicating that they are different species (Figure 2e). Diaporthe zhaoqingensis was isolated as an endophyte on Morinda officinalis [18], and D. chiangmaiensis was isolated from Magnolia lilifera as an endophyte and saprobe [47]. However, the morphological characteristics of these two species could not be compared as only gamma conidia were observed in D. zhaoqingensis while alpha conidia were observed in D. chiangmaiensis [18,47]. Therefore, more sequence data such as the tub2, cal, and his3 of the type strain of D. chiangmaiensis are needed to resolve their taxonomic placements and confirm whether they are distinct species.

Furthermore, the new species, D. careyae, was shown to be distinct from other Diaporthe species based on its morphology and phylogeny. The conidia of D. careyae were 0–1(–2) septate, whereas aseptate conidia were a typical characteristic of Diaporthe. The septation of conidia has been reported in some Diaporthe species (e.g., D. foeniculina and D. saccarata) [17,68], however, their phylogenetic placements were not closely related to D. careyae. It is noteworthy that there are some singleton species that were not grouped into any species complex, and their taxonomic positions remain unclear [32]. In addition, most species of Diaporthe lack sequence data and have incomplete morphological descriptions [31,32]; therefore, further extensive sampling is needed in order to unravel the taxonomic circumscription of this genus.

The newly introduced species of Diaporthe were associated with different medicinal plants, comprising D. afzeliae on Afzelia xylocarpa, D. bombacis on Bombax ceiba, D. careyae on Careya sphaerica, and D. samaneae on Samanea saman. These plant species have been used as traditional medicines in tropical countries, including Thailand, and have been reported on concerning their various phytochemicals and pharmacological activities [69,70,71,72,73,74,75]. To the best of our knowledge, none of the Diaporthe species have been isolated from these host genera, making this the first report of such a host association [38]. Moreover, a new species, D. globoostiolata, was found on a member of Fagaceae. Some plant genera in Fagaceae, such as Castanopsis, Quercus, and Lithocarpus, have also been reported on regarding their medicinal usage and pharmacological properties [76,77,78,79]. Furthermore, more than 30 Diaporthe species have been recorded from the host family Fagaceae [38]. This study reflects the high genetic diversity and phenotypic variation within Diaporthe and expands our understanding of the diversity and host relationships of the Diaporthe species associated with medicinal plants in tropical regions. However, future studies are necessary to investigate the disease symptoms and evaluate the pathogenicity of these Diaporthe isolates as they are important for tree health assessments and management.

Acknowledgments

J.M., S.H. and S.L. are thankful for the partial support of Chiang Mai University, Thailand. J.M. is grateful to Phongeun Sysouphanthong and Areerat Manowong for their assistance during this research. D.J.B. and T.M.D. gratefully acknowledge the financial support under the Distinguished Scientist Fellowship Programme (DSFP), at King Saud University, Riyadh, Saudi Arabia. Shaun Pennycook from Landcare Research, Auckland, New Zealand, is thanked for advising on the taxon name.

Author Contributions

Conceptualization, J.M. and S.H.; methodology, J.M. and S.H.; software, J.M. and S.H.; validation, S.H., D.J.B. and S.L.; formal analysis, J.M. and S.H.; investigation, J.M.; resources, J.M.; data curation, J.M.; writing—original draft preparation, J.M.; writing—review and editing, J.M., S.H., D.J.B., T.M.D. and S.L.; visualization, J.M.; supervision, S.L.; project administration, S.L.; funding acquisition, S.L. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All sequences generated in this study were submitted to GenBank (https://www.ncbi.nlm.nih.gov, accessed on 1 April 2023).

Conflicts of Interest

The authors declare no conflict of interest.

Funding Statement

This research was funded by the National Research Center of Thailand (No. 42A650198).

Footnotes

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Associated Data

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

Data Availability Statement

All sequences generated in this study were submitted to GenBank (https://www.ncbi.nlm.nih.gov, accessed on 1 April 2023).

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