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Research Article
Morphological and phylogenetic analyses reveal three new species of Diaporthe from Yunnan, China
expand article infoShengting Huang, Jiwen Xia§, Xiuguo Zhang§, WenXiu Sun
‡ Yangtze University, Jingzhou, China
§ Shandong Agricultural University, Taian, China
Open Access

Abstract

Species of Diaporthe have often been reported as plant pathogens, endophytes or saprobes, commonly isolated from a wide range of plant hosts. Sixteen strains isolated from species of ten host genera in Yunnan Province, China, represented three new species of Diaporthe, D. chrysalidocarpi, D. machili and D. pometiae as well as five known species D. arecae, D. hongkongensis, D. middletonii, D. osmanthi and D. pandanicola. Morphological comparisons with known species and DNA-based phylogenies based on the analysis of a multigene (ITS, TUB, TEF, CAL and HIS) dataset support the establishment of the new species. This study reveals that a high species diversity of Diaporthe with wide host ranges occur in tropical rainforest in Yunnan Province, China.

Keywords

Diaporthaceae, Diaporthales, phylogeny, taxonomy, three taxa new to science

Introduction

The genus Diaporthe (Diaporthaceae Diaporthales) with asexual morphs previously known as Phomopsis spp. is based on the type species Diaporthe eres Nitschke (1870) from Ulmus sp. in Germany. Rossman et al. (2015) proposed to use the name Diaporthe over Phomopsis in the context of the one fungus – one name initiative, because it was described first, is encountered commonly in literature and includes the majority of known species. The sexual morph of Diaporthe is characterised by immersed ascomata and an erumpent pseudostroma with elongated perithecial necks; asci are unitunicate, clavate to cylindrical; and ascospores are fusoid, ellipsoid to cylindrical, hyaline, biseriate to uniseriate in the ascus, sometimes with appendages (Udayanga et al. 2011; Senanayake et al. 2017, 2018). The asexual morph is characterised by ostiolate pycnidia with cylindrical phialides often producing three types of hyaline, aseptate conidia called α-conidia, β-conidia and γ-conidia (Udayanga et al. 2011; Gomes et al. 2013). The α-conidia and β-conidia are produced frequently, but the γ-conidia are rarely observed (Gomes et al. 2013; Guarnaccia and Crous 2017; Guo et al. 2020).

Currently, more than 1100 epithets of Diaporthe are listed in Index Fungorum (http://www.indexfungorum.org/; accessed 1 Nov. 2020), but only one-fifth of these taxa have been well-studied with ex-type cultures and supplementary DNA barcodes (Guo et al. 2020; Yang et al. 2020; Zapata et al. 2020). Species of Diaporthe are widely distributed and have a broad range of hosts including economically significant agricultural crops and ornamental plants such as species of Camellia, Castanea, Citrus, Glycine, Helianthus, Juglans, Persea, Pyrus, Vaccinium, Vitis and many more (van Rensburg et al. 2006; Santos and Phillips 2009; Crous et al. 2011a, b, 2016; Santos et al. 2011; Thompson et al. 2011; Grasso et al. 2012; Huang et al. 2013; Lombard et al. 2014; Gao et al. 2015, 2016, 2017; Udayanga et al. 2012, 2015; Guarnaccia et al. 2016; Dissanayake et al. 2017; Guarnaccia and Crous 2017; Fan et al. 2018; Senanayake et al. 2018; Guo et al. 2020). Diaporthe species have been reported as destructive plant pathogens, harmless endophytes or saprobes (Murali et al. 2006; Udayanga et al. 2012; Gomes et al. 2013; Ménard et al. 2014; Guarnaccia et al. 2016; Torres et al. 2016; Senanayake et al. 2018). However, the biology and lifestyle of some of these fungi remain unclear (Vilka and Volkova 2015).

In the past, methods of species identification of Diaporthe had previously been based only on host as well as morphological characters such as the size and shape of ascomata and conidiomata. Nowadays, molecular phylogenetic studies demonstrate that determining species boundaries only by morphological characters is not possible due to lack of host specificity and their variability under changing environmental conditions (Gomes et al. 2013). Phylogenetic analysis using a five-locus dataset (ITS-TUB-TEF-CAL-HIS) has been determined to be the optimal combination to identify species of Diaporthe species, as revealed by Santos et al. (2017). Many Diaporthe species are described based on a polyphasic approach together with morphological characterisation (Rehner and Uecker 1994; Udayanga et al. 2011; Gao et al. 2017; Guarnaccia and Crous 2017; Yang et al. 2018a, 2020; Crous et al. 2020; Dayarathne et al. 2020; Guo et al. 2020; Hyde et al. 2020; Li et al. 2020; Zapata et al. 2020).

The aim of this study was to explore the diversity of Diaporthe species from symptomatic leaves of plants in Yunnan Province. We present three novel species and five known species of Diaporthe, collected from species belonging to ten host genera, based on morphological characters and phylogenetic analysis.

Materials and methods

Isolation and morphological studies

Leaves of samples were collected in Yunnan Province, China. Isolations from surface sterilized leaf tissues were conducted following the protocol of Gao et al. (2014). Tissue fragments (5 × 5 mm) were taken from the margin of leaf lesions and surface-sterilized by immersing them in 75% ethanol solution for 1 min, 5% sodium hypochlorite solution for 30 s, and then rinsing in sterile distilled water for 1 min. The pieces were dried with sterilized paper towels and placed on potato dextrose agar (PDA) (Cai et al. 2009). PDA plates (90 mm) were incubated in an incubator at 25 °C for 2–4 days, and hyphae were picked out of the periphery of the colonies and inoculated onto new PDA plates.

Following 2–3 weeks of incubation, photographs of colonies were taken at 7 days and 15 days using a Powershot G7X mark II digital camera. Colour notations was done using the colour charts of Rayner (1970). Micromorphological characters were observed using an Olympus SZX10 stereomicroscope and Olympus BX53 microscope, both fitted with Olympus DP80 high definition colour digital cameras to document fungal structures. All fungal strains were stored in 10% sterilized glycerin at 4 °C for further studies. Voucher and type specimens were deposited in the Herbarium of Plant Pathology, Shandong Agricultural University (HSAUP). Living cultures were deposited in the Shandong Agricultural University Culture Collection (SAUCC). Taxonomic information of the new taxa was submitted to MycoBank (http://www.mycobank.org).

DNA extraction and amplification

Genomic DNA was extracted from fungal mycelium on PDA, using a modified cetyltrimethylammonium bromide (CTAB) protocol as described in Guo et al. (2000). The internal transcribed spacer regions with intervening 5.8S nrRNA gene (ITS), part of the beta-tubulin gene region (TUB), partial translation elongation factor 1-alpha (TEF), histone H3 (HIS) and calmodulin (CAL) genes were amplified and sequenced by using primers pairs ITS4/ITS5 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), EF1-728F/EF1-986R (Carbone and Kohn 1999), CAL-228F/CAL-737R (Carbone and Kohn 1999) and CYLH3F/H3-1b (Glass and Donaldson 1995; Crous et al. 2004), respectively.

PCR was performed using an Eppendorf Master Thermocycler (Hamburg, Germany). Amplification reactions were performed in a 25 μL reaction volume, which contained 12.5 μL Green Taq Mix (Vazyme, Nanjing, China), 1 μL of each forward and reverse primer (10 μM) (Biosune, Shanghai, China), and 1 μL template genomic DNA in amplifier, and were adjusted with distilled deionized water to a total volume of 25 μL.

PCR parameters were as follows: 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at a suitable temperature for 30 s, extension at 72 °C for 1 min and a final elongation step at 72 °C for 10 min. Annealing temperature for each gene were 55 °C for ITS, 60 °C for TUB, 52 °C for TEF, 54 °C for CAL and 57 °C for HIS. The PCR products were visualised on 1% agarose electrophoresis gel. Sequencing was done bi-directionally, conducted by the Biosune Company Limited (Shanghai, China). Consensus sequences were obtained using MEGA 7.0 (Kumar et al. 2016). All sequences generated in this study were deposited in GenBank (Table 1).

Table 1.

Species and Genbank accession numbers of DNA sequences used in this study. New sequences in bold.

Species Voucher Host/Substrare GeneBank accession number Reference
ITS TUB TEF CAL HIS
Diaporthe acuta PSCG 046 Pyrus pyrifolia MK626958 MK691224 MK654803 MK691124 MK726162 Guo et al. 2020
PSCG 047* Pyrus pyrifolia MK626957 MK691225 MK654802 MK691125 MK726161 Guo et al. 2020
D. acutispora LC6160 Camellia sasanqua KX986763 KX999194 KX999154 KX999273 KX999234 Gao et al. 2017
LC6161 Coffea sp. KX986764 KX999195 KX999155 KX999274 KX999235 Gao et al. 2017
D. amaranthophila MAFF 246900 Amaranthus tricolor LC459575 LC459579 LC459577 LC459583 LC459581 Rossman et al. 2015
MAFF 246901 Amaranthus tricolor LC459576 LC459580 LC459578 LC459584 LC459582 Rossman et al. 2015
D. angelicae CBS 111592* Heracleum sphondylium KC343027 KC343995 KC343753 KC343269 KC343511 Gomes et al. 2013
D. anhuiensis CNUCC 201901* Cunninghamia lanceolata MN219718 MN227008 MN224668 MN224549 MN224556 Zhou and Hou 2019
CNUCC 201902 Cunninghamia lanceolata MN219727 MN227009 MN224669 MN224550 MN224557 Zhou and Hou 2019
D. arctii DP0482 Arctium sp. KJ590736 KJ610891 KJ590776 KJ612133 KJ659218 Udayanga et al. 2015
D. arecae CBS 161.64* Areca catechu KC343032 KC344000 KC343758 KC343274 KC343516 Gomes et al. 2013
CBS 535.75 Citrus sp. KC343033 KC344001 KC343759 KC343275 KC343517 Gomes et al. 2013
SAUCC194.18 Persea americana MT822546 MT855743 MT855860 MT855631 MT855515 This study
D. arengae CBS 114979* Arenga engleri KC343034 KC344002 KC343760 KC343276 KC343518 Gomes et al. 2013
D. aseana MFLUCC 12-0299a* On dead leaves KT459414 KT459432 KT459448 KT459464 Dissanayake et al. 2017
D. beilharziae BRIP 54792* Indigofera australis JX862529 KF170921 JX862535 Tan et al. 2013
D. biconispora ZJUD 60 Citrus sinensis KJ490595 KJ490416 KJ490474 KJ490537 Huang et al. 2017
ZJUD 61 Fortunella margarita KJ490596 KJ490417 KJ490475 KJ490538 Huang et al. 2017
ZJUD 62 Citrus grandis KJ490597 KJ490418 KJ490476 KJ490539 Huang et al. 2017
D. brasiliensis CBS 133183* Aspidosperma tomentosus KC343042 KC344010 KC343768 KC343284 KC343526 Gomes et al. 2013
D. caatingaensis URM 7486* Tacinga inamoena KY085926 KY115600 KY115603 KY115597 KY115605 Crous et al. 2017
D. camporesii JZB320143 Urtica dioidca MN535309 MN561316 MN984254 Hyde et al. 2020
D. caricae-papayae NIBM-ABIJP Carica papaya MN335224 Rossman et al. 2015
D. caryae CFCC 52563 Carya illinoensis MH121498 MH121580 MH121540 MH121422 MH121458 Yang et al. 2018
CFCC 52564 Carya illinoensis MH121499 MH121581 MH121541 MH121423 MH121459 Yang et al. 2018
D. cercidis CFCC 52565 Cercis chinensis MH121500 MH121582 MH121542 MH121424 MH121460 Yang et al. 2018
D. chrysalidocarpi SAUCC194.33 Chrysalidocarpus lutescens MT822561 MT855758 MT855874 MT855645 MT855530 This study
SAUCC194.35* Chrysalidocarpus lutescens MT822563 MT855760 MT855876 MT855646 MT855532 This study
D. cichorii MFLUCC 17-1023* Cichorium intybus KY964220 KY964104 KY964176 KY964133 Dissanayake et al. 2017
D. compacta LC3083* Camellia sinensis KP267854 KP293434 KP267928 KP293508 Gao et al. 2016
D. cucurbitae CBS 136.25 Cucumis sativus KC343031 KC343999 KC343757 KC343273 KC343515 Udayanga et al. 2014
D. cuppatea CBS 117499 Aspalathus linearis KC343057 KC344025 KC343783 KC343299 KC343541 Udayanga et al. 2012
D. decedens CBS 109772 Corylus avellana KC343059 KC344027 KC343785 KC343301 KC343543 Gomes et al. 2013
D. eugeniae CBS 444.82 Eugenia aromatica KC343098 KC344066 KC343824 KC343340 KC343582 Gomes et al. 2013
D. fraxini-angustifoliae BRIP 54781* Fraxinus angustifolius JX862528 KF170920 JX862534 Tan et al. 2013
D. fulvicolor PSCG 051* Pyrus pyrifolia MK626859 MK691236 MK654806 MK691132 MK726163 Guo et al. 2020
PSCG 057 Pyrus pyrifolia MK626858 MK691233 MK654810 MK691131 MK726164 Guo et al. 2020
D. ganjae CBS 180.91* Cannabis sativa KC343112 KC344080 KC343838 KC343354 KC343596 Gomes et al. 2013
D. guangxiensis JZBH 320094* Vitis vinifera MK335772 MK500168 MK523566 MK736727 Manawasinghe et al. 2019
D. gulyae MF-Ha 17-042* Helianthus annuus MK024252 MK033488 MK039420 Thompson et al. 2011
D. hongkongensis CBS 115448* Dichroa febrifuga KC343119 KC344087 KC343845 KC343361 KC343603 Gomes et al. 2013
CGMCC 3.17102 Lithocarpus glaber KF576275 KF576299 KF576250 KF576227 Gao et al. 2015
LC 3478 Camellia sinensis KP267904 KP293484 KP267978 KP293553 Gao et al. 2017
SAUCC194.81 Millettia reticulata MT822609 MT855806 MT855921 MT855688 MT855577 This study
SAUCC194.87 Camellia sinensis MT822615 MT855812 MT855927 MT855694 MT855583 This study
D. huangshanensis CNUCC 201903 Camellia oleifera MN219729 MN227010 MN224670 MN224558 Zhou and Hou 2019
CNUCC 201904 Camellia oleifera MN219730 MN227011 MN224671 MN224559 Zhou and Hou 2019
D. infecunda CBS 133812* Schinus terebinthifolius KC343126 KC344094 KC343852 KC343368 KC343610 Gomes et al. 2013
D. krabiensis MFLUCC 17-2481* Bruguiera sp. MN047101 MN431495 MN433215 Dayarathne et al. 2020
D. litchiicola BRIP 54900* Litchi chinensis JX862533 KF170925 JX862539 Tan et al. 2013
D. limonicola CPC 28200* Citrus limon MF418422 MF418582 MF418501 MF418256 MF418342 Guarnaccia and Crous 2017
D. lusitanicae CBS 123212* Foeniculum vulgare KC343136 KC344104 KC343862 KC343378 KC343620 Phillips and Santos 2009
D. machili SAUCC194.69 Pometia pinnata MT822597 MT855794 MT855909 MT855677 MT855565 This study
SAUCC194.111* Machilus pingii MT822639 MT855836 MT855951 MT855718 MT855606 This study
D. malorum CAA752* Malus domestica KY435643 KY435671 KY435630 KY435661 KY435651 Santos et al. 2017
CAA740 Malus domestica KY435642 KY435670 KY435629 KY435660 KY435650 Santos et al. 2017
D. manihotia CBS 505.76 Manihot utilissima KC343138 KC344106 KC343864 KC343380 KC343622 Gomes et al. 2013
D. mayteni CBS 133185* Maytenus ilicicolia KC343139 KC344107 KC343865 KC343381 KC343623 Gomes et al. 2013
D. melitensis CPC 27873* Citrus limon MF418424 MF418584 MF418503 MF418258 MF418344 Guarnaccia and Crous 2017
D. middletonii BRIP 54884e* Rapistrum rugostrum KJ197286 KJ197266 KJ197248 Thompson et al. 2015
SAUCC194.27 Litchi chinensis MT822555 MT855752 MT855868 MT855639 MT855524 This study
SAUCC194.45 Lithocarpus glaber MT822573 MT855770 MT855886 MT855654 MT855542 This study
SAUCC194.46 Lithocarpus glaber MT822574 MT855771 MT855887 MT855655 MT855543 This study
SAUCC194.48 Lithocarpus craibianus MT822576 MT855773 MT855889 MT855657 MT855545 This study
D. millettiae GUCC9167* Millettia reticulata MK398674 MK502089 MK480609 MK502086 Long et al. 2019
D. multigutullata ZJUD 98* Citrus grandis KJ490633 KJ490454 KJ490512 KJ490575 Huang et al. 2015
D. musigena CBS 129519* Musa sp. KC343143 KC344111 KC343869 KC343385 KC343627 Crous et al. 2011
D. myracrodruonis URM7972 Myracrodruon urundeuva MK205289 MK205291 MK213408 MK205290 Silva et al. 2019
D. neoarctii CBS 109490* Ambrosia trifida KC343145 KC344113 KC343871 KC343387 KC343629 Gomes et al. 2013
D. novem CBS 127270* Glycine max KC343156 KC344124 KC343882 KC343398 KC343640 Santos et al. 2011
D. osmanthi GUCC9165* Osmanthus fragrans MK398675 MK502091 MK480610 MK502087 Long et al. 2019
SAUCC194.21 Litchi chinensis MT822549 MT855746 MT855862 MT855634 MT855518 This study
D. oxe CBS 133186* Maytenus ilicifolia KC343164 KC344132 KC343890 KC343406 KC343648 Gomes et al. 2013
CBS 133187 Maytenus ilicifolia KC343165 KC344133 KC343891 KC343407 KC343649 Gomes et al. 2013
D. pandanicola MFLUCC 17-0607 Pandanus sp. MG646974 MG646930 Tibpromma et al. 2018
SAUCC194.82 Millettia reticulata MT822610 MT855807 MT855922 MT855689 MT855578 This study
D. paranensis CBS 133184* Maytenus ilicifolia KC343171 KC344139 KC343897 KC343413 KC343655 Gomes et al. 2013
D. pascoei BRIP 54847* Persea americana JX862532 KF170924 JX862538 Tan et al. 2013
D. perseae CBS 151.73 Persea gratissima KC343173 KC344141 KC343899 KC343415 KC343657 Gomes et al. 2013
D. pescicola MFLU 16-0105* Prunus persica KU557555 KU557579 KU557623 KU557603 Dissanayake et al. 2017
D. podocarpi-macrophylli LC6155* Podocarpus macrophyllus KX986774 KX999207 KX999167 KX999278 KX999246 Gao et al. 2017
LC6200 Podocarpus macrophyllus KX986769 KX999201 KX999161 KX999276 KX999240 Gao et al. 2017
D. pometiae SAUCC194.19 Persea americana MT822547 MT855744 MT855861 MT855632 MT855516 This study
SAUCC194.72* Pometia pinnata MT822600 MT855797 MT855912 MT855679 MT855568 This study
SAUCC194.73 Heliconia metallica MT822601 MT855798 MT855913 MT855680 MT855569 This study
D. pseudomangiferae CBS 101339* Mangifera indica KC343181 KC344149 KC343907 KC343423 KC343665 Gomes et al. 2013
D. pseudophoenicicola CBS 462.69* Phoenix dactylifera KC343184 KC344152 KC343910 KC343426 KC343668 Gomes et al. 2013
D. pterocarpicola MFLUCC 10-0580a* Pterocarpus indicus JQ619887 JX275441 JX275403 JX197433 Udayanga et al. 2012
MFLUCC 10-0580b Pterocarpus indicus JQ619888 JX275442 JX275404 JX197434 Udayanga et al. 2012
D. pyracanthae CAA487* Pyracantha coccinea KY435636 KY435667 KY435626 KY435657 KY435647 Santos et al. 2017
D. racemosae CPC 26646* Euclea racemosa MG600223 MG600227 MG600225 MG600219 MG600221 Marin-Felix et al. 2018
D. raonikayaporum CBS 133182* Spondias mombin KC343188 KC344156 KC343914 KC343430 KC343672 Gomes et al. 2013
D. rossmaniae CAA 762* Vaccinium corymbosum MK792290 MK837914 MK828063 MK883822 MK871432 Hilario et al. 2020
D. sackstonii BRIP 54669b* Helianthus annuus KJ197287 KJ197267 KJ197249 Thompson et al. 2015
D. salinicola MFLU 18-0553* Xylocarpus sp. MN047098 MN077073 Dayarathne et al. 2020
MFLU 17-2592 Xylocarpus sp. MN047099 MN077074 Dayarathne et al. 2020
D. schini CBS 133181* Schinus terebinthifolius KC343191 KC344159 KC343917 KC343433 KC343675 Gomes et al. 2013
D. schoeni MFLU 15-2609 Schoenus nigricans KY964229 KY964112 KY964185 KY964141 Dissanayake et al. 2017
D. sennae CFCC 51636* Senna bicapsularis KY203724 KY228891 KY228885 KY228875 Yang et al. 2017
D. serafiniae BRIP 55665a* Helianthus annuus KJ197274 KJ197254 KJ197236 Thompson et al. 2015
D. spinosa PSCG 383* Pyrus pyrifolia MK626849 MK691234 MK654811 MK691129 MK726156 Guo et al. 2020
D. stewartii CBS 193.36* Cosmos bipinnatus FJ889448 JX275421 GQ250324 JX197415 Santos et al. 2010; Udayanga et al. 2012
D. subordinaria CBS 101711 Plantago lanceolata KC343213 KC344181 KC343939 KC343455 KC343697 Gomes et al. 2013
CBS 464.90 Plantago lanceolata KC343214 KC344182 KC343940 KC343456 KC343698 Gomes et al. 2013
D. taoicola PSGG485 Prunus persica MK626869 MK691227 MK654812 MK691120 MK726173 Dissanayake et al. 2017
D. tarchonanthi CPC 37479 Tarchonanthus littoralis MT223794 Crous et al. 2020
D. tectonigena MFLUCC 12-0767* Tectona grandis KU712429 KU743976 KU749371 KU749358 Doilom et al. 2016
D. terebinthifolii CBS 133180* Schinus terebinthifolius KC343216 KC344184 KC343942 KC343458 KC343700 Gomes et al. 2013
D. undulate LC6624* Unknown host KX986798 KX999230 KX999190 KX999269 Gao et al. 2017
LC8110 Unknown host KY491545 KY491565 KY491555 Gao et al. 2017
D. vawdreyi BRIP 57887a* Psidium guajava KR936126 KR936128 KR936129 Crous et al. 2015
D. viniferae JZBH 320071 Vitis vinifera MK341550 MK500112 MK500107 MK500119 Manawasinghe et al. 2019
JZBH 320072 Vitis vinifera MK341551 MK500113 MK500108 MK500120 Manawasinghe et al. 2019
D. xishuangbanica LC6707* Camellia sinensis KX986783 KX999216 KX999175 KX999255 Gao et al. 2017
Diaporthella corylina CBS 121124 Corylus sp. KC343004 KC343972 KC343730 KC343246 KC343488 Gomes et al. 2013

Phylogenetic analyses

Novel sequences generated from the sixteen strains in this study, and all reference sequences of Diaporthe species downloaded from GenBank, were used for phylogenetic analyses. Alignments of the individual locus were determined using MAFFT v. 7.110 by default settings (Katoh et al. 2017) and manually corrected where necessary. To establish the identity of the isolates at species level, phylogenetic analyses were conducted first individually for each locus and then as combined analyses of five loci (ITS, TUB, TEF, CAL and HIS regions). Phylogenetic analyses were based on maximum likelihood (ML) and Bayesian inference (BI) for the multi-locus analyses. For BI, the best evolutionary model for each partition was determined using MrModeltest v. 2.3 (Nylander 2004) and incorporated into the analyses. ML and BI were run on the CIPRES Science Gateway portal (https://www.phylo.org/) (Miller et al. 2012) using RaxML-HPC2 on XSEDE (8.2.12) (Stamatakis 2014) and MrBayes on XSEDE (3.2.7a) (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003; Ronquist et al. 2012), respectively. For ML analyses the default parameters were used and BI was carried out using the rapid bootstrapping algorithm with the automatic halt option. Bayesian analyses included five parallel runs of 5,000,000 generations, with the stop rule option and a sampling frequency of 500 generations. The burn-in fraction was set to 0.25 and posterior probabilities (PP) were determined from the remaining trees. The resulting trees were plotted using FigTree v. 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree) and edited with Adobe Illustrator CS5.1. New sequences generated in this study were deposited at GenBank (https://www.ncbi.nlm.nih.gov; Table 1) and the alignments and trees were deposited in TreeBASE: S27479 (http://treebase.org/treebase-web/home.html).

Results

Phylogenetic analyses

Sixteen strains of Diaporthe isolated from plant hosts from Yunnan, China, were grown in culture and used for analyses of molecular sequence data. Diaporthe spp. were analysed by using multilocus data (ITS, TUB, TEF, CAL and HIS) from 115 isolates of Diaporthe spp. and Diaporthella corylina (CBS 121124) as the outgroup taxon. A total of 3005 characters including gaps were obtained in the phylogenetic analysis, viz. ITS: 1–656, TUB: 657–1329, TEF: 1330–1860, CAL: 1861–2444, HIS: 2445–3005. Of these characters, 1349 were constant, 453 were variable and parsimony-uninformative, and 1203 were parsimony-informative. For the BI and ML analyses, the substitution model GTR+I+G for ITS, TUB, TEF and HIS, HKY+I+G for and CAL were selected and incorporated into the analyses. The ML tree topology confirmed the tree topologies obtained from the BI analyses, and therefore, only the ML tree is presented (Fig. 1).

Figure 1. 

Phylogram of Diaporthe spp. based on combined sequence data of ITS, TUB, TEF, CAL and HIS genes. The ML and BI bootstrap support values above 50% and 0.90 BYPP are shown at the first and second position, respectively. Strains marked with “*” are ex-type or ex-epitype. Codes referring to strains from the current study are written in red. Some branches were shortened to fit them to the page as indicated by two diagonal lines with the number of times a branch was shortened indicated.

Figure 1. 

Continued.

Figure 1. 

Continued.

ML bootstrap support values (≥ 50%) and Bayesian posterior probability (≥ 0.90) are shown as first and second position above nodes, respectively. Based on the five-locus phylogeny and morphology, nine isolates were assigned to five species, including Diaporthe arecae (1), D. hongkongensis (2), D. middletonii (4), D. osmanthi (1) and D. pandanicola (1), whereas seven isolates formed distinct well supported clades, which refer to novel species named D. chrysalidocarpi (2), D. machili (2) and D. pometiae (3), respectively.

Taxonomy

Diaporthe arecae (H.C. Srivast., Zakia & Govindar.) R.R. Gomes, Glienke & Crous, Persoonia 31: 16. (2013)

Figure 2

Subramanella arecae H.C. Srivast., Zakia & Govindar., in Srivastava, Banu and Govindarajan (1962). Basionym.

Description

Asexual morph: Conidiomata pycnidial, several pycnidia grouped together, globose, black, erumpent, exuding creamy to yellowish conidial droplets from ostioles. Conidiophores hyaline, septate, branched, cylindrical, straight to sinuous, 25.0–32.0 × 1.4–2.5 μm. Conidiogenous cells 10.5–20.7 × 1.4–2.0 μm, phialidic, cylindrical, swollen at base, tapering towards apex, slightly curved. Alpha conidia hyaline, smooth, aseptate, ellipsoidal, guttulate, apex subobtuse, base subtruncate, 7.5–10.0 × 1.8–3.0 µm (mean = 8.2 × 2.4 μm, n = 20). Beta conidia hyaline, aseptate, filiform, slightly curved, tapering towards base, 18.5–26.5 × 1.0–1.8 µm (mean = 24.3 × 1.4 μm, n = 20). Gamma conidia not observed. Sexual morph not observed.

Culture characteristics

Cultures incubated on PDA at 25 °C in darkness, growth rate 11.2–13.3 mm diam/day. Aerial mycelium white, cottony, feathery, abundant in center, sparse in margin, white on surface, reverse yellowish to tan.

Specimen examined

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on diseased leaves of Persea americana (Lauraceae). 19 April 2019, S.T. Huang, HSAUP194.18, living culture SAUCC194.18.

Figure 2. 

Diaporthe arecae (SAUCC194.18) a infected leaf of Persea americana b, c surface and reverse of a colony after 15 days on PDA d conidiomata e–g conidiophores and conidiogenous cells h beta conidia i alpha conidia j alpha conidia and beta conidia. Scale bars: 10 μm (e–j).

Notes

Diaporthe arecae (CBS 161.64) was originally described as Subramanella arecae on fruit of Areca catechu in India (Srivastava et al. 1962) and placed in Diaporthe by Gomes et al. (2013). The Diaporthe isolate from fruits of Citrus sp. (CBS 535.75) in Suriname was also placed in D. arecae by Gomes et al. (2013). In the present study, strain (SAUCC194.18) from symptomatic leaves of Persea americana was congruent with D. arecae based on morphology and DNA sequences data (Fig. 1). We therefore consider the isolated strain as D. arecae.

Diaporthe chrysalidocarpi S.T. Huang, J.W. Xia, W.X. Sun, & X.G. Zhang, sp. nov.

MycoBank No: 837812
Figure 3

Etymology

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

Diagnosis

Diaporthe chrysalidocarpi can be distinguished from the phylogenetically most closely related species D. spinosa by longer beta conidia (28.0–32.5 × 1.2–1.6 vs. 18.5–30.5 × 1.0–1.5 μm), and from other species D. fulvicolor by the types of conidia (D. chrysalidocarpi produces only beta conidia, while D. fulvicolor produces only alpha conidia) and several loci (25/491 in the ITS region, 18/471 TUB, 4/298 TEF, 28/458 CAL and 13/441 HIS).

Type

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on diseased leaves of Chrysalidocarpus lutescens (Palmae). 19 April 2019, S.T. Huang, HSAUP194.35 holotype, ex-type living culture SAUCC194.35.

Description

Asexual morph: Leaf spots irregular, pale brown in center, brown to tan at margin. Conidiomata pycnidial, scattered or aggregated, black, erumpent, raising above surface of culture medium, subglobose, exuding white or yellowish creamy conidial droplets from central ostioles after 30 days in light at 25 °C; pycnidial wall consists of black to dark brown, thin-walled cells. Conidiophores 27.5–35.0 × 1.4–2.0 μm, hyaline, slightly branched, swelling at base, subcylindrical, septate, smooth, straight or curved. Conidiogenous cells 10.5–23.0 × 1.4–1.8 μm, phialidic, cylindrical, terminal, straight to sinuous, tapering towards apex. Beta conidia 28.0–32.5 × 1.2–1.6 μm (mean = 30.3 × 1.3 μm, n = 20), filiform, hyaline, straight or slightly curved, aseptate, base subtruncate, tapering towards the base. Alpha conidia and gamma conidia not observed. Sexual morph not observed.

Culture characteristics

Cultures incubated on PDA at 25 °C in darkness, growth rate 13.3–15.2 mm diam/day, initially white, becoming greyish, reverse pale brown, with concentric rings of dense, sparse hyphae, irregular margin, fluffy aerial mycelium at center, pycnidia forming after 15 days.

Additional specimen examined

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on diseased leaves of Chrysalidocarpus lutescens (Palmae). 19 April 2019, S.T. Huang, HSAUP194.33 paratype; living culture SAUCC194.33.

Figure 3. 

Diaporthe chrysalidocarpi (SAUCC194.35) a diseased leaf of Chrysalidocarpus lutescens b, c surface and reverse of a colony after 15 days on PDA d, e conidiomata f, g conidiophores and conidiogenous cells h, i beta conidia. Scale bars: 10 μm (f–i).

Notes

Phylogenetic analysis of a combined five gene showed that D. chrysalidocarpi formed an independent clade (Fig. 1) and is phylogenetically distinct from D. spinosa and D. fulvicolor. This species can be distinguished from D. spinosa by 61 different nucleotides in the concatenated alignment (13/492 in the ITS region, 17/471 TUB, 4/298 TEF, 17/458 CAL and 10/441 HIS), and D. fulvicolor by 88 nucleotides (25/491 in the ITS region, 18/471 TUB, 4/298 TEF, 28/458 CAL and 13/441 HIS). Morphologically, D. chrysalidocarpi differs from D. spinosa in having longer beta conidia (28.0–32.5 × 1.2–1.6 vs. 18.5–30.5 × 1.0–1.5 μm) (Guo et al. 2020). Furthermore, Diaporthe chrysalidocarpi produces only beta conidia, while D. spinosa produces alpha conidia and beta conidia and D. fulvicolor produces only alpha conidia (Guo et al. 2020). Therefore, we establish this fungus as a novel species.

Diaporthe hongkongensis R.R. Gomes, Glienke, Crous, Persoonia 31: 23. (2013)

Figure 4

Description

Asexual morph: Conidiomata pycnidial, subglobose or globose, solitary, black, erumpent, coated with white hyphae, thick-walled, exuding creamy conidial droplets from central ostioles. Conidiophores hyaline, smooth, septate, unbranched, densely aggregated, cylindrical or clavate, straight to sinuous, swollen at base, tapering towards apex, 32.0–42.0 × 2.0–2.9 μm. Conidiogenous cells 20.0–24.2 × 1.3–2.3 μm, phialidic, cylindrical, terminal, slightly tapering towards apex. Alpha conidia, hyaline, smooth, aseptate, ellipsoidal or oval, 0–2 guttulate, apex subobtuse, base subtruncate, 5.5–7.0 × 2.0–2.5 µm (mean = 6.2 × 2.2 μm, n = 20). Beta conidia hyaline, aseptate, filiform, hamate, tapering towards both ends, mostly J-shaped, 21.5–27.0 × 1.4–1.8 µm (mean = 25.6 × 1.3 μm, n = 20). Gamma conidia not observed. Sexual morph not observed.

Culture characteristics

Cultures incubated on PDA at 25 °C in darkness, growth rate 19.0–21.5 mm diam/day, cottony, radial with abundant aerial mycelium, sparse at margin, with an obvious pale brown concentric ring of dense hyphae, white to grayish on surface with age, white to pale brown on the reverse side.

Figure 4. 

Diaporthe hongkongensis (SAUCC194.87) a diseased leaf of Camellia sinensis b, c surface and reverse of colony after 15 days on PDA d conidiomata e–g conidiophores and conidiogenous cells h beta conidia i alpha conidia. Scale bars: 10 μm (e–i).

Specimens examined

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, 19 April 2019, S.T. Huang. On diseased leaves of Millettia reticulata (Fabaceae) HSAUP194.81, living culture SAUCC194.81; on diseased leaves of Camellia sinensis (Theaceae) HSAUP194.87, living culture SAUCC194.87.

Notes

In the present study, two strains (SAUCC194.81 and SAUCC194.87) from symptomatic leaves of Millettia reticulata and Camellia sinensis were similar to Diaporthe hongkongensis (CGMCC 3.17102) (Gomes et al. 2013) and D. salinicola (MFLU 18-0553) (Dayarathne et al. 2020) based on DNA sequences data (Fig. 1). Morphologically, our strains were similar to Diaporthe hongkongensis, which was originally described with an asexual morph on fruits of Dichroa febrifuga in China, but the asexual morph of D. salinicola was undetermined. We therefore identify our strains as D. hongkongensis.

Diaporthe machili S.T. Huang, J.W. Xia, W.X. Sun, & X.G. Zhang, sp. nov.

MycoBank No: 837814
Figure 5

Etymology

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

Diagnosis

Diaporthe machili differs from D. caryae and D. sackstonii in the types of conidia (D. machili only produces beta conidia, while D. caryae produces alpha conidia and beta conidia, and D. sackstonii only produces alpha conidia), and from D. caryae in longer beta conidia (29.0–39.0 × 1.3–1.5 vs. 15.5–34.0 × 1.1–1.4 μm).

Type

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on diseased leaves of Machilus pingii (Lauraceae). 19 April 2019, S.T. Huang, HSAUP194.111 holotype, ex-holotype living culture SAUCC194.111.

Figure 5. 

Diaporthe machili (SAUCC194.111) a infected leaf of Machilus pingii b, c surface and reverse of colony after 15 days on PDA d, e conidiomata f–h conidiophores and conidiogenous cells i–k beta conidia. Scale bars: 10 μm (f–k).

Description

Asexual morph: Conidiomata pycnidial, aggregated, black, erumpent, subglobose to globose, exuding creamy conidial droplets from central ostioles after 30 days in light at 25 °C. Conidiophores 7.0–11.4 × 1.8–2.8 μm, hyaline, unbranched, densely aggregated, mostly ampulliform, cylindrical, guttulate, septate, straight or slightly curved, swelling at base, tapering towards apex. Beta conidia 29.0–39.0 × 1.3–1.5 μm (mean = 32.5 × 1.4 μm, n = 20), filiform, hyaline, aseptate, mostly curved, J-shaped, swelling in middle, tapering towards both ends. Alpha and gamma conidia not observed. Sexual morph not observed.

Culture characteristics

Cultures incubated on PDA at 25 °C in darkness, growth rate 16.3–17.5 mm diam/day, aerial mycelium abundant, white on surface, reverse white to pale yellow, with an obvious concentric zonation, pycnidia forming after 15 days.

Additional specimen examined

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on diseased leaves of Pometia pinnata (Sapindaceae). 19 April 2019, S.T. Huang, HSAUP194. 69 paratype; living culture SAUCC194. 69.

Notes

In the phylogenetic tree, Diaporthe machili forms an independent clade and is phylogenetically distinct from D. caryae and D. sackstonii (Fig. 1). Diaporthe machili can be distinguished from D. caryae in ITS, TUB, TEF, CAL and HIS loci by 67 nucleotide differences in concatenated alignment (5/459 in ITS, 10/416 in TUB, 15/334 in TEF, 7/454 in CAL and 30/455 in HIS), and from D. sackstonii in ITS, TUB and TEF loci by 58 nucleotide differences (12/559 in ITS, 23/486 in TUB and 23/348 in TEF). Moreover, Diaporthe machili differs from D. caryae in having longer beta conidia (29.0–39.0 × 1.3–1.5 vs. 15.5–34.0 × 1.1–1.4 μm). Diaporthe machili only produces beta conidia, while D. caryae produces alpha conidia and beta conidia, and D. sackstonii only produces alpha conidia (Thompson et al. 2015; Yang et al. 2018b).

Diaporthe middletonii R.G. Shivas, L. Morin, S.M. Thomps. & Y.P. Tan, Persoonia 35: 45. (2015)

Figure 6

Description

Asexual morph: Leaf spots discoid to irregular. Conidiomata pycnidial, scattered or aggregated in groups of 3–5 pycnidia, globose, black, erumpent, coated with white to greyish hyphae, thick-walled, exuding creamy translucent conidial droplets from central ostioles. Conidiophores hyaline, smooth, septate, unbranched, densely aggregated, cylindrical, straight to sinuous, tapering towards apex, 10.0–14.0 × 1.3–2.3 μm. Conidiogenous cells 5.0–9.5 × 1.3–1.7 μm, phialidic, cylindrical, terminal, slightly tapering towards apex. Alpha conidia hyaline, smooth, aseptate, biguttulate, ellipsoidal, oval, apex subobtuse, base subtruncate, 5.5–7.0 × 2.5–3.2 µm (mean = 6.3 × 2.8 μm, n = 20). Beta conidia hyaline, aseptate, filiform, mostly curved by 90–180°, tapering towards both ends, 26.0–36.5 × 1.0–1.6 µm (mean = 21.5 × 1.2 μm, n = 20). Gamma conidia not observed. Sexual morph not observed.

Figure 6. 

Diaporthe middletonii (SAUCC194.46) a infected leaf of Lithocarpus glaber b, c surface and reverse of colony after 15 days on PDA d, e conidiomata f–i conidiophores and conidiogenous cells j beta conidia k, l alpha conidia and beta conidia. Scale bars: 10 μm (f–l).

Culture characteristics

Cultures incubated on PDA at 25 °C in darkness, growth rate 22.5–24.0 mm diam/day, fluffy with abundant aerial mycelium, margin fimbriate, white on surface, white to pale yellow on reverse.

Specimens examined

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, 19 April 2019, S.T. Huang. On diseased leaves of Litchi chinensis (Sapindaceae), HSAUP194.27, living culture SAUCC194.27; on diseased leaves of Lithocarpus glaber (Fagaceae), HSAUP194.45, living culture SAUCC194.45; on diseased leaves of Lithocarpus glaber (Fagaceae), 19 April 2019, S.T. Huang, HSAUP194.46, living culture SAUCC194.46; on diseased leaves of Lithocarpus craibianus (Fagaceae), HSAUP194.48, living culture SAUCC194.48.

Notes

Diaporthe middletonii was originally described from the stem of Rapistrum rugosum (BRIP 54884e) (Brassicaceae) and Chrysanthemoides monilifera subsp. rotundata (BRIP 57329) (Asteraceae) in Australia (Thompson et al. 2015). In the present study, four strains (SAUCC194.27, SAUCC194.45, SAUCC194.46 and SAUCC194.48) are closely related to D. middletonii in the combined phylogenetic tree (Fig. 1). The differences between nucleotides in the concatenated alignment (17/565 in ITS, 9/494 in TUB and 10/340 in TEF) were minor. Morphologically, our strains were similar to D. middletonii by slightly shorter and wider alpha conidia (5.0–7.0 × 2.5–3.2 vs. 6.0–7.5 × 2.0–2.5 μm), and longer beta conidia (26.0–36.5 × 1.0–1.6 vs. 20.0–35.0 × 1.0–1.5 μm) (Thompson et al. 2015). We therefore identify our strains as Diaporthe middletonii.

Diaporthe osmanthi H. Long, K.D. Hyde, & Yong Wang bis, MycoKeys 57: 120. (2019)

Figure 7

Description

Conidiomata pycnidial, globose, 5–10 pycnidia grouped together, dark brown to black, exuding creamy to yellowish conidial droplets from central ostioles. Conidiophores hyaline, smooth, densely aggregated, branched, cylindric-clavate, 20.5–32.0 × 1.8–2.4 μm. Conidiogenous cells phialidic, hyaline, terminal, cylindrical, straight, 14.0–20.5 × 1.5–2.0 μm, tapered towards apex. Alpha conidia hyaline, aseptate, fusiform, tapering towards both ends, guttulate, 7.3–9.3 × 1.8–2.3 μm (mean = 8.5 × 2.0 μm, n = 20). Beta conidia hyaline, aseptate, filiform, curved, 22.0–28.5 × 1.0–2.0 μm (mean = 27.2 × 1.3 μm, n = 20). Gamma conidia not observed. Sexual morph not observed.

Culture characteristics

Cultures incubated on PDA at 25 °C in darkness, growth rate 12.0–13.5 mm diam/day, cottony with abundant aerial mycelium, sparse at margin. With several concentric rings of dense hyphae, white on surface, white to pale brown on reverse.

Specimen examined

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, 19 April 2019, S.T. Huang. On diseased leaves of Litchi chinensis (Sapindaceae) HSAUP194.21, living culture SAUCC194.21.

Figure 7. 

Diaporthe osmanthi (SAUCC194.21) a infected leaf of Litchi chinensis b, c surface and reverse of colony after 15 days on PDA d conidiomata e–g conidiophores and conidiogenous cells h, i beta conidia j, k alpha conidia. Scale bars: 10 μm (e–k).

Notes

Diaporthe osmanthi was originally described from the leaves of Osmanthus fragrans (Oleaceae) in Guangxi province, China (Long et al. 2019). In the present study, phylogenetic analyses (Fig. 1) indicated that the strain SAUCC194.21 is closely related to Diaporthe osmanthi and D. podocarpi-macrophylli (Gao et al. 2017). Morphological comparison indicated that this strain was most similar to D. osmanthi by the size of alpha conidia and beta conidia. We therefore identify this strain as belonging to D. osmanthi.

Diaporthe pandanicola Tibpromma & K.D. Hyde, MycoKeys 33: 44 (2018)

Figure 8

Description

Asexual morph: Conidiomata pycnidial, 3–5 pycnidia grouped together, superficial to embedded on PDA, erumpent, thin-walled, dark brown to black, globose or subglobose, exuding white creamy conidial mass from ostioles. Conidiophores hyaline, aseptate, cylindrical, smooth, straight to sinuous, unbranched, aggregated, 17.0–26.5 × 2.0–3.0 µm. Conidiogenous cells phialidic, cylindrical, terminal, 10.0–20.0 × 1.5–1.8 µm. Alpha conidia hyaline, smooth, aseptate, ellipsoidal, eguttulate, apex subobtuse, base subtruncate, 6.5–9.0 × 1.8–2.5 µm (mean = 7.5 × 2.0 μm, n = 20). Beta conidia hyaline, aseptate, filiform, curved, tapering towards apex, base truncate, 26.0–32.8 × 1.0–1.6 µm (mean = 29.0 × 1.3 μm, n = 20). Gamma conidia infrequent, aseptate, smooth, straight, hyaline, 12.5–14.5 × 1.3–1.8 µm (mean = 13.5 × 1.6 μm, n = 6). Sexual morph not observed.

Culture characteristics

Cultures incubated on PDA at 25 °C in darkness, growth rate 12.8–15.0 mm diam/day, flat, cottony in centre, with aerial mycelium sparse toward margin, white on surface, white to pale yellow on reverse.

Figure 8. 

Diaporthe pandanicola (SAUCC194.82) a infected leaf of Millettia reticulata b, c surface and reverse of colony after 15 days on PDA d conidiomata e–g conidiophores and conidiogenous cells h beta conidia i alpha conidia and gamma conidia j alpha conidia, beta conidia and gamma conidia. Scale bars: 10 μm (e–j).

Specimen examined

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on diseased leaves of Millettia reticulata (Fabaceae). 19 April 2019, S.T. Huang, HSAUP194.82, living culture SAUCC194.82.

Notes

Diaporthe pandanicola was originally described by Tibpromma et al. (2018) on healthy leaves of Pandanus sp. (Pandanaceae) as an endophytic fungus. Our strain (SAUCC194.82) is closely related to Diaporthe pandanicola based on phylogenetic analyses (Fig. 1). The differences of nucleotides in the concatenated alignment (19/533 in the ITS region and 11/351 in the TUB region) are less than 3%. Morphologically, our strain produces alpha conidia, beta conidia and gamma conidia, while Diaporthe pandanicola did not sporulate. We therefore identify our strains as Diaporthe pandanicola.

Diaporthe pometiae S.T. Huang, J.W. Xia, W.X. Sun, & X.G. Zhang, sp. nov.

MycoBank No: 837815
Figure 9

Etymology

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

Diagnosis

Diaporthe pometiae is similar to D. biconispora but differs in having smaller alpha conidia (5.7–8.3 × 2.2–3.0 vs. 6.0–10.5 × 2–3.5 μm) and types of conidia (D. pometiae produces beta conidia unlike D. biconispora).

Type

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on diseased leaves of Pometia pinnata (Sapindaceae). 19 April 2019, S.T. Huang, HSAUP194.72 holotype, ex-type living culture SAUCC194.72.

Description

Asexual morph: Leaf spots subcircular, fawn to dark brown. Conidiomata pycnidial, subglobose to globose, aggregated in groups, black, coated with white hyphae, thick-walled, exuding creamy droplets from ostioles. Conidiophores hyaline, smooth, slightly septate, branched, densely aggregated, cylindric-clavate, straight to slightly sinuous, 22.5–32.5 × 1.0–2.0 μm. Conidiogenous cells 15.0–22.5 × 1.0–1.5 μm, phialidic, cylindrical, multi-guttulate, terminal, tapering towards apex. Alpha conidia abundant in culture, 2–4 guttulate, hyaline, smooth, aseptate, ellipsoidal to oblong ellipsoidal, with both ends obtuse, 5.7–8.3 × 2.2–3.0 µm (mean = 6.7 × 3.1 μm, n = 20). Beta conidia, hyaline, aseptate, filiform, multi-guttulate, slightly curved, tapering towards to apex, 27.8–34.5 × 1.0–1.7 µm (mean = 21.7 × 1.4 μm, n = 20). Gamma conidia not observed. Sexual morph not observed.

Figure 9. 

Diaporthe pometiae (SAUCC194.72) a infected leaf of Pometia pinnata b, c surface and reverse of colony after 15 days on PDA d conidiomata e, f conidiophores and conidiogenous cells g beta conidia h alpha conidia and beta conidia. Scale bars: 10 μm (e–h).

Culture characteristics

Cultures incubated on PDA at 25 °C in darkness, growth rate 11.5–13.0 mm diam/day, cottony with abundant aerial mycelium, with a concentric zonation, white on surface, white to grayish on reverse.

Additional specimens examined

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, 19 April 2019, S.T. Huang. On diseased leaves of Persea americana (Lauraceae), HSAUP194.19 paratype, ex-paratype culture SAUCC194.19; on diseased leaves of Heliconia metallica (Musaceae), HSAUP194.73 paratype, ex-paratype culture SAUCC194.73.

Notes

Diaporthe pometiae is introduced based on the multi-locus phylogenetic analysis, with three isolates clustering separately in a well-supported clade (ML/BI = 100/1). Diaporthe pometiae is most closely related to D. biconispora, but distinguished based on ITS, TUB, TEF and HIS loci by 74 nucleotide differences in the concatenated alignment, in which 2/492 are distinct in the ITS region, 8/353 in the TUB region, 49/370 in the TEF region and 15/471 in the HIS region. Morphologically, Diaporthe pometiae differs from D. biconispora in its smaller alpha conidia (5.7–8.3 × 2.2–3.0 vs. 6.0–10.5 × 2–3.5 μm). Furthermore, Diaporthe pometiae produces beta conidia unlike D. biconispora (Huang et al. 2015).

Discussion

The Yunnan Province in southeastern China has a unique geography where three climatic regions meet: the eastern Asia monsoon region, the Tibetan plateau region, and the tropical monsoon region of southern Asia and Indo-China. The environment is conducive to growth of unusual microbial species. Species diversity in Yunnan Province is high compared to other parts of China.

Previously, species identification of Diaporthe relied on the assumption of host-specificity, leading to the proliferation of names. The morphological characters of Diaporthe could be changeable, as most taxa in culture do not produce all spore states of the asexual (alpha, beta and gamma conidia) or the sexual morph (Gomes et al. 2013). Based on a polyphasic approach and morphology, more than one species of Diaporthe can colonize a single host, while one species can be associated with several hosts (Gomes et al. 2013; Gao et al. 2017; Guarnaccia and Crous 2017; Guarnaccia et al. 2018; Guo et al. 2020). These studies revealed a high diversity of Diaporthe species from different hosts. Our study supports this phenomenon. For example, Diaporthe arecae (SAUCC194.18) and D. pometiae (SAUCC194.19) were collected from Persea americana; In addition, isolates of D. middletonii were obtained from three hosts (Litchi chinensis, Lithocarpus craibianus, L. glaber). As for host specificity, in our study, four species of Diaporthe, D. machili (SAUCC194.69), D. middletonii (SAUCC194.27), D. osmanthi (SAUCC194.21), and D. pometiae (SAUCC194.72) were isolated from Litchi chinensis and Pometia pinnata belong to the Sapindaceae, and D. litchiicola also was reported from Litchi chinensis in Queensland (Tan et al. 2013); however, D. machili (SAUCC194.111) also was isolated from Machilus pingii (Lauraceae), D. middletonii (SAUCC194.45) from Lithocarpus glaber (Fagaceae), D. osmanthi (GUCC 9165) from leaves of Osmanthus fragrans (Oleaceae) (Long et al. 2019), and D. pometiae (SAUCC194.19 and SAUCC194.73) from Persea americana (Lauraceae) and Heliconia metallica (Musaceae). These results provide evidence that many species are able to colonise diverse hosts and several different species could co-occur on the same host. It seems obvious that specificity does not occur at the family level.

For the current study, sixteen strains isolated from ten host genera represented three new species and five known species, based on morphological characters and phylogenetic analyses of the five combined loci (ITS, TUB, TEF, CAL and HIS). The descriptions and molecular data for species of Diaporthe represent an important resource for plant pathologists, plant quarantine officials and taxonomists.

Acknowledgements

This work was jointly supported by the National Natural Science Foundation of China (no. 31900014, 31770016, and 31750001) and the China Postdoctoral Science Foundation (no. 2018M632699).

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