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Research Article
New species and records of Diaporthe from Jiangxi Province, China
expand article infoQin Yang§, Ning Jiang, Cheng-Ming Tian
‡ Beijing Forestry University, Beijing, China
§ Central South University of Forestry and Technology, Changsha, China
Open Access

Abstract

Diaporthe species have often been reported as important plant pathogens, saprobes and endophytes on a wide range of plant hosts. Although several Diaporthe species have been recorded, little is known about species able to infect forest trees in Jiangxi Province. Hence, extensive surveys were recently conducted in Jiangxi Province, China. A total of 24 isolates were identified and analysed using comparisons of DNA sequence data for the nuclear ribosomal internal transcribed spacer (ITS), calmodulin (cal), histone H3 (his3), partial translation elongation factor-1α (tef1) and β-tubulin (tub2) gene regions, as well as their morphological features. Results revealed five novel taxa, D. bauhiniae, D. ganzhouensis, D. schimae, D. verniciicola, D. xunwuensis spp. nov. and three known species, D. apiculatum, D. citri and D. multigutullata.

Keywords

DNA phylogeny, five new taxa, forest trees, systematics, taxonomy

Introduction

The genus Diaporthe Nitschke (Sordariomycetes, Diaporthales) represents a cosmopolitan group of fungi occupying diverse ecological behaviour as plant pathogens, endophytes and saprobes (Muralli et al. 2006; Rossman et al. 2007; Udayanga et al. 2014, 2015; Fan et al. 2015, 2018; Guarnaccia and Crous 2017; Guarnaccia et al. 2018; Yang et al. 2018, 2020; Manawasinghe et al. 2019; Marin-Felix et al. 2019). Diaporthe species are responsible for diseases on a wide range of plant hosts, including agricultural crops, forest trees and ornamentals, some of which are economically important. Several symptoms, such as root and fruit rots, dieback, stem cankers, leaf spots, leaf and pod blights and seed decay are caused by Diaporthe spp. (Uecker 1988; Rehner and Uecker 1994; Mostert et al. 2001; Santos et al. 2011; Thompson et al. 2011; Udayanga et al. 2011).

Diaporthe was historically considered as monophyletic, based on its typical sexual morph and Phomopsis asexual morph (Gomes et al. 2013). However, Gao et al. (2017) recently revealed its paraphyletic nature, showing that Mazzantia (Wehmeyer 1926), Ophiodiaporthe (Fu et al. 2013), Pustulomyces (Dai et al. 2014), Phaeocytostroma and Stenocarpella (Lamprecht et al. 2011) are embedded in Diaporthe s. lat. Furthermore, Senanayake et al. (2017) recently included additional two genera in Diaporthe s. lat., namely Paradiaporthe and Chiangraiomyces.

Species identification criteria in Diaporthe were originally based on host association, morphology and culture characteristics (Mostert et al. 2001; Santos and Phillips 2009; Udayanga et al. 2011), which led to the description of over 200 species (Hyde et al. 2020). Some species of Diaporthe were reported to colonise a single host plant, while other species were found to be associated with different host plants (Santos and Phillips 2009; Diogo et al. 2010; Santos et al. 2011; Gomes et al. 2013). In addition, considerable variability of the phenotypic characters was found to be present within a species (Rehner and Uecker 1994; Mostert et al. 2001; Santos et al. 2010; Udayanga et al. 2011). During the past decade, a polyphasic approach, based on multi-locus DNA data, morphology and ecology, has been employed for species boundaries in the genus Diaporthe (Crous et al. 2012; Huang et al. 2015; Gao et al. 2016, 2017; Guarnaccia and Crous 2017; Guarnaccia et al. 2018; Yang et al. 2018, 2020). The classification of Diaporthe has been progressing and the basis for the species identification is a combination of morphological, cultural, phytopathological and phylogenetical analyses (Gomes et al. 2013; Udayanga et al. 2014, 2015; Fan et al. 2015; Huang et al. 2015; Gao et al. 2016, 2017; Guarnaccia and Crous 2017; Guarnaccia et al. 2018; Yang et al. 2018, 2020; Manawasinghe et al. 2019).

In Jiangxi Province, China, some forest trees were observed to be infected with fungal pathogens that cause dieback and leaf spots. Cankered branches and leaves with typical Diaporthe fruiting bodies were also found in the area. However, we found that only limited research had been undertaken regarding the fungal pathogens isolated from forest trees in Jiangxi Province. Hence, the present study was conducted to identify Diaporthe species that cause dieback and leaf spots disease in the forest trees in Jiangxi Province through morphological and multi-locus phylogenetic analyses, based on modern taxonomic concepts.

Materials and methods

Isolates

Fresh specimens of Diaporthe were isolated from the collected branches and leaves of six host plants during the collection trips conducted in Jiangxi Province (Table 1). A total of 24 isolates were established by removing a mucoid conidia mass from conidiomata, spreading the suspension on the surface of 1.8% potato dextrose agar (PDA) and incubating at 25 °C for up to 24 h. A single germinating conidium was plated on to fresh PDA plates. Specimens were deposited at the Museum of the Beijing Forestry University (BJFC). Axenic cultures were maintained at the China Forestry Culture Collection Centre (CFCC).

Table 1.

Reference sequences included in molecular phylogenetic analyses of Diaporthe.

Species Isolate Host Location GenBank accession numbers
ITS cal his3 tef1 tub2
D. acericola MFLUCC 17-0956 Acer negundo Italy KY964224 KY964137 NA KY964180 KY964074
D. acerigena CFCC 52554 Acer tataricum China MH121489 MH121413 MH121449 MH121531 NA
D. acutispora CGMCC 3.18285 Coffea sp. China KX986764 KX999274 NA KX999155 KX999195
D. alangii CFCC 52556 Alangium kurzii China MH121491 MH121415 MH121451 MH121533 MH121573
D. alnea CBS 146.46 Alnus sp. Netherlands KC343008 KC343250 KC343492 KC343734 KC343976
D. ampelina STEU2660 Vitis vinifera France AF230751 AY745026 NA AY745056 JX275452
D. amygdali CBS 126679 Prunus dulcis Portugal KC343022 KC343264 KC343506 AY343748 KC343990
D. angelicae CBS 111592 Heracleum sphondylium Austria KC343027 KC343269 KC343511 KC343753 KC343995
D. apiculatum CGMCC 3.17533 Camellia sinensis China KP267896 NA NA KP267970 KP293476
CFCC 53068 Rhus chinensis China MK432651 MK442973 MK442998 MK578127 MK578054
CFCC 53069 Rhus chinensis China MK432652 MK442974 MK442999 MK578128 MK578055
CFCC 53070 Rhus chinensis China MK432653 MK442975 MK443000 MK578129 MK578056
D. arctii CBS 139280 Arctium lappa Austria KJ590736 KJ612133 KJ659218 KJ590776 KJ610891
D. arecae CBS 161.64 Areca catechu India KC343032 KC343274 KC343516 KC343758 KC344000
D. arengae CBS 114979 Arenga enngleri Hong Kong KC343034 KC343276 KC343518 KC343760 KC344002
D. aseana MFLUCC 12-0299a Unknown dead leaf Thailand KT459414 KT459464 NA KT459448 KT459432
D. bauhiniae CFCC 53071 Bauhinia purpurea China MK432648 MK442970 MK442995 MK578124 MK578051
CFCC 53072 China MK432649 MK442971 MK442996 MK578125 MK578052
CFCC 53073 China MK432650 MK442972 MK442997 MK578126 MK578053
D. beilharziae BRIP 54792 Indigofera australis Australia JX862529 NA NA JX862535 KF170921
D. betulicola CFCC 51128 Betula albo-sinensis China KX024653 KX024659 KX024661 KX024655 KX024657
D. biconispora CGMCC 3.17252 Citrus grandis China KJ490597 KJ490539 KJ490539 KJ490476 KJ490418
D. biguttulata CGMCC 3.17248 Citrus limon China KJ490582 NA KJ490524 KJ490461 KJ490403
CFCC 52584 Juglans regia China MH121519 MH121437 MH121477 MH121561 MH121598
D. bohemiae CPC 28222 Vitis vinifera Czech Republic MG281015 MG281710 MG281361 MG281536 MG281188
D. brasiliensis CBS 133183 Aspidosperma tomentosum Brazil KC343042 KC343284 KC343526 KC343768 KC344010
D. caatingaensis CBS 141542 Tacinga inamoena Brazil KY085927 NA NA KY115603 KY115600
D. caryae CFCC 52563 Carya illinoensis China MH121498 MH121422 MH121458 MH121540 MH121580
D. celeris CPC 28262 Vitis vinifera Czech Republic MG281017 MG281712 MG281363 MG281538 MG281190
D. celastrina CBS 139.27 Celastrus sp. USA KC343047 KC343289 KC343531 KC343773 KC344015
D. cercidis CFCC 52565 Cercis chinensis China MH121500 MH121424 MH121460 MH121542 MH121582
D. charlesworthii BRIP 54884m Rapistrum rugostrum Australia KJ197288 NA NA KJ197250 KJ197268
D. cinnamomi CFCC 52569 Cinnamomum sp. China MH121504 NA MH121464 MH121546 MH121586
D. citri AR 3405 Citrus sp. USA KC843311 KC843157 NA KC843071 KC843187
CFCC 53079 Citrus sinensis China MK573940 MK574579 MK574595 MK574615 MK574635
CFCC 53080 Citrus sinensis China MK573941 MK574580 MK574596 MK574616 MK574636
CFCC 53081 Citrus sinensis China MK573942 MK574581 MK574597 MK574617 MK574637
CFCC 53082 Citrus sinensis China MK573943 MK574582 MK574598 MK574618 MK574638
D. citriasiana CGMCC 3.15224 Citrus unshiu China JQ954645 KC357491 KJ490515 JQ954663 KC357459
D. citrichinensis CGMCC 3.15225 Citrus sp. China JQ954648 KC357494 NA JQ954666 NA
D. collariana MFLU 17-2770 Magnolia champaca Thailand MG806115 MG783042 NA MG783040 MG783041
D. conica CFCC 52571 Alangium chinense China MH121506 MH121428 MH121466 MH121548 MH121588
D. cucurbitae CBS 136.25 Arctium sp. Unknown KC343031 KC343273 KC343515 KC343757 KC343999
D. cuppatea CBS 117499 Aspalathus linearis South Africa KC343057 KC343299 KC343541 KC343783 KC344025
D. discoidispora ZJUD89 Citrus unshiu China KJ490624 NA KJ490566 KJ490503 KJ490445
D. endophytica CBS 133811 Schinus terebinthifolius Brazil KC343065 KC343307 KC343549 KC343791 KC343065
D. eres AR5193 Ulmus sp. Germany KJ210529 KJ434999 KJ420850 KJ210550 KJ420799
D. fraxini-angustifoliae BRIP 54781 Fraxinus angustifolia Australia JX862528 NA NA JX862534 KF170920
D. fraxinicola CFCC 52582 Fraxinus chinensis China MH121517 MH121435 NA MH121559 NA
D. fructicola MAFF 246408 Passiflora edulis × P. edulis f. flavicarpa Japan LC342734 LC342738 LC342737 LC342735 LC342736
D. fukushii MAFF 625034 Pyrus pyrifolia Japan JQ807469 NA NA JQ807418 NA
D. fusicola CGMCC 3.17087 Lithocarpus glabra China KF576281 KF576233 NA KF576256 KF576305
D. ganjae CBS 180.91 Cannabis sativa USA KC343112 KC343354 KC343596 KC343838 KC344080
D. ganzhouensis CFCC 53087 Unknown dead wood China MK432665 MK442985 MK443010 MK578139 MK578065
CFCC 53088 Unknown dead wood China MK432666 MK442986 MK443011 MK578140 MK578066
D. garethjonesii MFLUCC 12-0542a Unknown dead leaf Thailand KT459423 KT459470 NA KT459457 KT459441
D. guangxiensis JZB320094 Vitis vinifera China MK335772 MK736727 NA MK523566 MK500168
D. gulyae BRIP 54025 Helianthus annuus Australia JF431299 NA NA KJ197271 JN645803
D. helicis AR5211 Hedera helix France KJ210538 KJ435043 KJ420875 KJ210559 KJ420828
D. heterophyllae CBS 143769 Acacia heterohpylla France MG600222 MG600218 MG600220 MG600224 MG600226
D. hispaniae CPC 30321 Vitis vinifera Spain MG281123 MG281820 MG281471 MG281644 MG281296
D. hubeiensis JZB320123 Vitis vinifera China MK335809 MK500235 NA MK523570 MK500148
D. incompleta CGMCC 3.18288 Camellia sinensis China KX986794 KX999289 KX999265 KX999186 KX999226
D. infecunda CBS 133812 Schinus terebinthifolius Brazil KC343126 KC343368 KC343610 KC343852 KC344094
D. juglandicola CFCC 51134 Juglans mandshurica China KU985101 KX024616 KX024622 KX024628 KX024634
D. kadsurae CFCC 52586 Kadsura longipedunculata China MH121521 MH121439 MH121479 MH121563 MH121600
D. kochmanii BRIP 54033 Helianthus annuus Australia JF431295 NA NA JN645809 NA
D. kongii BRIP 54031 Portulaca grandiflora Australia JF431301 NA NA JN645797 KJ197272
D. litchicola BRIP 54900 Litchi chinensis Australia JX862533 NA NA JX862539 KF170925
D. lithocarpus CGMCC 3.15175 Lithocarpus glabra China KC153104 KF576235 NA KC153095 KF576311
D. lonicerae MFLUCC 17-0963 Lonicera sp. Italy KY964190 KY964116 NA KY964146 KY964073
D. lusitanicae CBS 123212 Foeniculum vulgare Portugal KC343136 KC343378 KC343620 KC343862 KC344104
D. masirevicii BRIP 57892a Helianthus annuus Australia KJ197277 NA NA KJ197239 KJ197257
D. middletonii BRIP 54884e Rapistrum rugostrum Australia KJ197286 NA NA KJ197248 KJ197266
D. miriciae BRIP 54736j Helianthus annuus Australia KJ197282 NA NA KJ197244 KJ197262
D. momicola MFLUCC 16-0113 Prunus persica China KU557563 KU557611 NA KU557631 KU55758
D. multigutullata ZJUD98 Citrus grandis China KJ490633 NA KJ490575 KJ490512 KJ490454
D. multigutullata CFCC 53095 Citrus maxima China MK432645 MK442967 MK442992 MK578121 MK578048
CFCC 53096 Citrus maxima China MK432646 MK442968 MK442993 MK578122 MK578049
CFCC 53097 Citrus maxima China MK432647 MK442969 MK442994 MK578123 MK578050
D. musigena CBS 129519 Musa sp. Australia KC343143 KC343385 KC343627 KC343869 KC344111
D. neilliae CBS 144.27 Spiraea sp. USA KC343144 KC343386 KC343628 KC343870 KC344112
D. neoarctii CBS 109490 Ambrosia trifida USA KC343145 KC343387 KC343629 KC343871 KC344113
D. oraccinii CGMCC 3.17531 Camellia sinensis China KP267863 NA KP293517 KP267937 KP293443
D. ovoicicola CGMCC 3.17093 Citrus sp. China KF576265 KF576223 NA KF576240 KF576289
D. pandanicola MFLU 18-0006 Pandanus sp. Thailand MG646974 NA NA NA MG646930
D. pascoei BRIP 54847 Persea americana Australia JX862532 NA NA JX862538 KF170924
D. passifloricola CBS 141329 Passiflora foetida Malaysia KX228292 NA KX228367 NA KX228387
D. penetriteum CGMCC 3.17532 Camellia sinensis China KP714505 NA KP714493 KP714517 KP714529
D. perjuncta CBS 109745 Ulmus glabra Austria KC343172 KC343414 KC343656 KC343898 KC344140
D. perseae CBS 151.73 Persea gratissima Netherlands KC343173 KC343415 KC343657 KC343899 KC344141
D. pescicola MFLUCC 16-0105 Prunus persica China KU557555 KU557603 NA KU557623 KU557579
D. podocarpi-macrophylli CGMCC 3.18281 Podocarpus macrophyllus China KX986774 KX999278 KX999246 KX999167 KX999207
D. pseudomangiferae CBS 101339 Mangifera indica Dominican Republic KC343181 KC343423 KC343665 KC343907 KC344149
D. pseudophoenicicola CBS 462.69 Phoenix dactylifera Spain KC343184 KC343426 KC343668 KC343910 KC344152
D. psoraleae-pinnatae CBS 136413 Psoralea pinnata South Africa KF777159 NA NA NA KF777252
D. pterocarpicola MFLUCC 10-0580a Pterocarpus indicus Thailand JQ619887 JX197433 NA JX275403 JX275441
D. pulla CBS 338.89 Hedera helix Yugoslavia KC343152 KC343394 KC343636 KC343878 KC344120
D. pyracanthae CAA483 Pyracantha coccinea Portugal KY435635 KY435656 KY435645 KY435625 KY435666
D. racemosae CBS 143770 Euclea racemosa South Africa MG600223 MG600219 MG600221 MG600225 MG600227
D. rostrata CFCC 50062 Juglans mandshurica China KP208847 KP208849 KP208851 KP208853 KP208855
D. sackstonii BRIP 54669b Helianthus annuus Australia KJ197287 NA NA KJ197249 KJ197267
D. sambucusii CFCC 51986 Sambucus williamsii China KY852495 KY852499 KY852503 KY852507 KY852511
D. schimae CFCC 53103 Schima superba China MK432640 MK442962 MK442987 MK578116 MK578043
CFCC 53104 Schima superba China MK432641 MK442963 MK442988 MK578117 MK578044
CFCC 53105 Schima superba China MK432642 MK442964 MK442989 MK578118 MK578045
D. schini CBS 133181 Schinus terebinthifolius Brazil KC343191 KC343433 KC343675 KC343917 KC344159
D. schisandrae CFCC 51988 Schisandra chinensis China KY852497 KY852501 KY852505 KY852509 KY852513
D. schoeni MFLU 15-1279 Schoenus nigricans Italy KY964226 KY964139 NA KY964182 KY964109
D. sennae CFCC 51636 Senna bicapsularis China KY203724 KY228875 NA KY228885 KY228891
D. serafiniae BRIP 55665a Helianthus annuus Australia KJ197274 NA NA KJ197236 KJ197254
D. siamensis MFLUCC 10-573a Dasymaschalon sp. Thailand JQ619879 NA NA JX275393 JX275429
D. sojae FAU635 Glycine max USA KJ590719 KJ612116 KJ659208 KJ590762 KJ610875
D. sterilis CBS 136969 Vaccinium corymbosum Italy KJ160579 KJ160548 MF418350 KJ160611 KJ160528
D. subclavata ICMP20663 Citrus unshiu China KJ490587 NA KJ490529 KJ490466 KJ490408
D. subellipicola MFLU 17-1197 Dead wood China MG746632 NA NA MG746633 MG746634
D. subordinaria CBS 464.90 Plantago lanceolata New Zealand KC343214 KC343456 KC343698 KC343940 KC344182
D. taoicola MFLUCC 16-0117 Prunus persica China KU557567 NA NA KU557635 KU557591
D. tectonae MFLUCC 12-0777 Tectona grandis China KU712430 KU749345 NA KU749359 KU743977
D. tectonendophytica MFLUCC 13-0471 Tectona grandis China KU712439 KU749354 NA KU749367 KU749354
D. tectonigena MFLUCC 12-0767 Tectona grandis China KU712429 KU749358 NA KU749371 KU743976
D. terebinthifolii CBS 133180 Schinus terebinthifolius Brazil KC343216 KC343458 KC343700 KC343942 KC344184
D. ternstroemia CGMCC 3.15183 Ternstroemia gymnanthera China KC153098 NA NA KC153089 NA
D. thunbergii MFLUCC 10-576a Thunbergia laurifolia Thailand JQ619893 JX197440 NA JX275409 JX275449
D. tibetensis CFCC 51999 Juglandis regia China MF279843 MF279888 MF279828 MF279858 MF279873
D. tulliensis BRIP 62248a Theobroma cacao Australia KR936130 NA NA KR936133 KR936132
D. ukurunduensis CFCC 52592 Acer ukurunduense China MH121527 MH121445 MH121485 MH121569 NA
D. unshiuensis CGMCC 3.17569 Citrus unshiu China KJ490587 NA KJ490529 KJ490408 KJ490466
CFCC 52594 Carya illinoensis China MH121529 MH121447 MH121487 MH121571 MH121606
D. undulata CGMCC 3.18293 Leaf of unknown host China-Laos border KX986798 NA KX999269 KX999190 KX999230
D. vawdreyi BRIP 57887a Psidium guajava Australia KR936126 NA NA KR936129 KR936128
D. verniciicola CFCC 53109 Vernicia montana China MK573944 MK574583 MK574599 MK574619 MK574639
CFCC 53110 Vernicia montana China MK573945 MK574584 MK574600 MK574620 MK574640
CFCC 53111 Vernicia montana China MK573946 MK574585 MK574601 MK574621 MK574641
CFCC 53112 Vernicia montana China MK573947 MK574586 MK574602 MK574622 MK574642
D. viniferae JZB320071 Vitis vinifera China MK341551 MK500107 MK500119 MK500112
D. virgiliae CMW40748 Virgilia oroboides South Africa KP247566 NA NA NA KP247575
D. xishuangbanica CGMCC 3.18282 Camellia sinensis China KX986783 NA KX999255 KX999175 KX999216
D. xunwuensis CFCC 53085 Unknown dead wood China MK432663 MK442983 MK443008 MK578137 MK578063
CFCC 53086 Unknown dead wood China MK432664 MK442984 MK443009 MK578138 MK578064
D. yunnanensis CGMCC 3.18289 Coffea sp. China KX986796 KX999290 KX999267 KX999188 KX999228
Diaporthella corylina CBS 121124 Corylus sp. China KC343004 KC343246 KC343488 KC343730 KC343972

Morphological observation

Agar plugs (6 mm diam.) were taken from the edge of actively-growing cultures on PDA and transferred on to the centre of 9 cm diam. Petri dishes containing 2% tap water agar, supplemented with sterile pine needles (PNA; Smith et al. 1996) and potato dextrose agar (PDA) and incubated at 25 °C under a 12 h near-ultraviolet light/12 h dark cycle to induce sporulation, as described in recent studies (Gomes et al. 2013; Lombard et al. 2014). Colony characters and pigment production on PNA and PDA were noted in the 10-day culture. Colony features were rated according to the colour charts of Rayner (1970). Cultures were examined periodically for the development of conidiomata. The microscopic examination was based on the morphological features of conidiomata obtained from the fungal growth, mounted in clear lactic acid. At least 30 conidia were measured to calculate the mean size/length. Micro-morphological observations were done at 1000× magnification using a Leica compound microscope (DM 2500) with interference contrast (DIC) optics. Descriptions, nomenclature and illustrations of taxonomic novelties were deposited at MycoBank (www.MycoBank.org).

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted from colonies grown on cellophane-covered PDA, using a CTAB (cetyltrimethylammonium bromide) method (Doyle and Doyle 1990). DNA was estimated by electrophoresis in 1% agarose gel and the yield was measured using the NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA), following the user manual (Desjardins et al. 2009). The PCR amplifications were performed in the DNA Engine Peltier Thermal Cycler (PTC-200; Bio-Rad Laboratories, Hercules, CA, USA). The primer set ITS1/ITS4 (White et al. 1990) was used to amplify the ITS region. The primer pair CAL228F/CAL737R (Carbone and Kohn 1999) was used to amplify the calmodulin gene (cal) and the primer pair CYLH4F (Crous et al. 2004) and H3-1b (Glass and Donaldson 1995) were used to amplify part of the histone H3 (his3) gene. The primer pair EF1-728F/EF1-986R (Carbone and Kohn 1999) was used to amplify a partial fragment of the translation elongation factor 1-α gene (tef1). The primer sets T1 (O’Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995) were used to amplify the beta-tubulin gene (tub2); the additional combination of Bt2a/Bt2b (Glass and Donaldson 1995) was used in case of amplification failure of the T1/Bt2b primer pair. The PCR amplifications of the genomic DNA with the phylogenetic markers were done using the same primer pairs and conditions as in Yang et al. (2018). The PCR products were assayed via electrophoresis in 2% agarose gels, while the DNA sequencing was performed using an ABI PRISM 3730XL DNA Analyser with a BigDye Terminater Kit v.3.1 (Inv-itrogen, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

Phylogenetic analyses

The quality of the amplified nucleotide sequences was checked and combined using SeqMan v.7.1.0 and reference sequences were retrieved from the National Center for Biotechnology Information (NCBI), based on recent publications on the genus Diaporthe (Guarnaccia et al. 2018; Yang et al. 2018, 2020). Sequences were aligned using MAFFT v. 6 (Katoh and Toh 2010) and corrected manually using Bioedit 7.0.9.0 (Hall 1999). The best-fit nucleotide substitution models for each gene were selected using jModelTest v. 2.1.7 (Darriba et al. 2012) under the Akaike Information Criterion.

The phylogenetic analyses of the combined gene regions were performed using Maximum Likelihood (ML) and Bayesian Inference (BI) methods. ML was conducted using PhyML v. 3.0 (Guindon et al. 2010), with 1000 bootstrap replicates while BI was performed using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.0 (Ronquist et al. 2003). Two MCMC chains, started from random trees for 1,000,000 generations and trees, were sampled every 100th generation, resulting in a total of 10,000 trees. The first 25% of trees were discarded as burn-in of each analysis. Branches with significant Bayesian Posterior Probabilities (BPP) were estimated in the remaining 7500 trees. Phylogenetic trees were viewed with FigTree v.1.3.1 (Rambaut and Drummond 2010) and processed by Adobe Illustrator CS5. Sequence alignment and phylogenetic trees were deposited in TreeBASE (submission ID: S25213). The nucleotide sequence data of the new taxa were deposited in GenBank (Table 1).

Results

The phylogenetic position of the 24 isolates of Diaporthe was determined by the phylogenetic analysis of the combined ITS, cal, his3, tef1 and tub2 sequences data. Reference sequences of the representative species used in the analysis were selected from Yang et al. (2018) and supplemented with sequences from GenBank. The ITS, cal, his3, tef1 tub2 and combined data matrices contained 522, 541, 529, 520, 535 and 2 659 characters with gaps, respectively. The alignment comprised of 142 strains together with Diaporthella corylina (culture CBS 121124) which was selected as the outgroup. The best nucleotide substitution model used for the analysis of ITS, his3 and tub2 was TrN+I+G, while HKY+I+G was used for cal and tef1. The topologies resulting from ML and BI analyses of the concatenated dataset were congruent (Fig. 1) and the sequences from the 24 Diaporthe isolates formed eight distinct clades as shown in Fig. 1, representing five undescribed species and three known species.

Figure 1. 

Phylogram of Diaporthe from a Maximum Likelihood analysis based on combined ITS, cal, his3, tef1 and tub2. Values above the branches indicate Maximum Likelihood bootstrap (left, ML BP ≥ 50%) and Bayesian probabilities (right, BI PP ≥ 0.90). The tree is rooted with Diaporthella corylina. Strains in current study are in blue font and the ex-type cultures are in bold font.

Figure 11. 

Continued.

Taxonomy

Diaporthe apiculatum Y.H. Gao & L. Cai, in Gao, Liu & Cai, Syst. Biodiv. 14: 106. 2016.

Figure 2

Description

Conidiomata pycnidial, discoid, immersed in bark, scattered, slightly erumpent through bark surface, with a solitary undivided locule. Ectostromatic disc yellowish to grey, one ostiole per disc, (300–)305–357(–368) μm diam. Ostiole medium black, up to level of disc. Locule undivided, (338–)357–450(–464) μm diam. Conidiophores reduced to conidiogenous cells. Conidiogenous cells cylindrical, hyaline, densely aggregated, phiailidic, unbranched, straight or slightly curved. Beta conidia hyaline, aseptate, filiform, hamate, eguttulate, base subtruncate, tapering towards one apex, (26.5–)30–39.5(–43) × 1.5–2 µm. Alpha conidia not observed.

Figure 2. 

Diaporthe apiculatum on Rhus chinensis (BJFC-S1680) a, b habit of conidiomata in wood c transverse section of conidiomata d longitudinal section through conidiomata e conidiogenous cells attached with beta conidia f the colony on PDA. Scale bars: 200 μm (b–d); 10 μm (e).

Culture characters

Colony originally flat with white fluffy aerial mycelium, becoming yellowish to pale green mycelium with age, marginal area irregular, conidiomata absent.

Specimens examined

China. Jiangxi Province: Ganzhou City, Fengshan Forest Park, on branches of Rhus chinensis, 25°45'12"N, 115°00'41"E, 23 Jul 2018, Q. Yang, Y. Liu, Y.M. Liang & C.M. Tian (BJFC-S1680; living culture: CFCC 53068, CFCC 53069 and CFCC 53070).

Notes

Diaporthe apiculatum was originally described as an endophyte from healthy leaves of Camellia sinensis in Jiangxi Province, China (Gao et al. 2015). In the present study, three isolates (CFCC 53068, CFCC 53069 and CFCC 53070) from symptomatic branches of Rhus chinensis were found congruent with D. apiculatum, based on DNA sequence and morphological data (Fig. 1). The clade was, therefore, confirmed to be D. apiculatum and was found to be both an endophyte and a pathogen.

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

MycoBank No: 829519
Figure 3

Diagnosis

Distinguished from the phylogenetically closely-related species D. psoraleae-pinnatae in alpha and beta conidia.

Etymology

Named after Bauhinia, the host genus where the fungus was isolated.

Description

Conidiomata pycnidial, immersed in bark, scattered, slightly erumpent through bark surface, nearly flat, discoid, with a solitary undivided locule. Ectostromatic disc grey to brown, one ostiole per disc. Locule circular, undivided, (180–)200–290(–300) μm diam. Conidiophores reduced to conidiogenous cells. Conidiogenous cells hyaline, cylindrical, unbranched, straight, tapering towards the apex. Alpha conidia hyaline, aseptate, ellipsoidal to fusiform, biguttulate to multi-guttulate, (7.5–)9–13(–14) × (1.5–)2–2.5(–3) μm. Beta conidia hyaline, aseptate, filiform, straight to sinuous, eguttulate, (25–)28.5–40(–43) × 1 µm.

Figure 3. 

Diaporthe bauhiniae on Bauhinia purpurea (BJFC-S1621) a habit of conidiomata in wood b transverse section of conidiomata c longitudinal section through conidiomata d the colony on PDA e conidiogenous cells attached with alpha conidia f Alpha conidia g Beta conidia. Scale bars: 100 μm (b, c); 10 μm (e–h).

Culture characters

Colony at first white, becoming wine-red in the centre with age. Aerial mycelium white, dense, fluffy, conidiomata absent.

Specimens examined

China. Jiangxi Province: Ganzhou City, on branches of Bauhinia purpurea, 25°52'21"N, 114°56'44"E, 11 May 2018, Q. Yang, Y. Liu & Y.M. Liang (holotype BJFC-S1621; ex-type living culture: CFCC 53071; living culture: CFCC 53072 and CFCC 53073).

Notes

Three isolates representing D. bauhiniae cluster in a well-supported clade and appear most closely related to D. psoraleae-pinnatae. Diaporthe bauhiniae can be distinguished from D. psoraleae-pinnatae, based on ITS and tub2 (38/458 in ITS and 11/418 in tub2). Morphologically, D. bauhiniae differs from D. psoraleae-pinnatae in having narrower alpha conidia (2–2.5 vs. 2.5–3 μm) and the beta conidia of D. psoraleae-pinnatae were not observed (Crous et al. 2013).

Diaporthe citri (H.S. Fawc.) F.A. Wolf, J. Agric. Res., Washington 33(7): 625, 1926.

Figure 4

Description

Leaf spots subcircular to irregular, pale brown, with dark brown at margin. Pycnidia solitary, scattered on the leaf surface. Pycnidial conidiomata in culture, globose, erumpent, single or clustered in groups of 3–5 pycnidia, coated with hyphae, cream to yellowish translucent conidial droplets exuded from ostioles. Conidiophores reduced to conidiogenous cells. Conidiogenous cells hyaline, unbranched, septate, straight, slightly tapering towards the apex, 14.5–25 × 2–3 μm. Alpha conidia hyaline, aseptate, rounded at one end, apex at the other end, usually with two large guttulate, (9.5–)10.5–12 × 3.5–4.5 μm. Beta conidia not observed.

Figure 4. 

Diaporthe citri on Citrus sinensis (BJFC-S1658) a, b symptoms on leaves of host plant c culture on PDA (30d) d conidiomata e alpha conidia f conidiophores and alpha conidia. Scale bars: 10 μm (e, f).

Culture characters

Colony originally flat with white fluffy aerial mycelium, becoming greyish mycelium with age, with yellowish-cream conidial drops exuding from the ostioles.

Specimens examined

China. Jiangxi Province: Ganzhou City, on leaves of Citrus sinensis, 24°59'44"N, 115°31'01"E, 13 May 2018, Q. Yang, Y. Liu & Y.M. Liang (BJFC-S1658; living culture: CFCC 53079 and CFCC 53080); 24°59'45"N, 115°31'02"E, 13 May 2018, Q. Yang, Y. Liu & Y.M. Liang (BJFC-S1659; living culture: CFCC 53081 and CFCC 53082).

Notes

Diaporthe citri is a widely distributed species in citrus-growing regions. In the present study, four isolates (CFCC 53079, CFCC 53080, CFCC 53081 and CFCC 53082) from symptomatic leaves of Citrus sinensis were congruent with D. citri, based on DNA sequence and morphological data (Fig. 1). The clade was, therefore, confirmed to be D. citri.

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

MycoBank No: 829522
Figure 5

Diagnosis

Distinguished from the phylogenetically closely-related species D. vawdreyi in having longer conidiophores and wider alpha conidia.

Etymology

Named after Ganzhou City where the species was first collected.

Description

On PDA: Conidiomata pycnidial, subglobose, solitary, deeply embedded in the medium, erumpent, dark brown to black. Pale yellow conidial drops exuding from ostioles. Conidiophores (12–)15.5–21 × 1.5–2 μm, cylindrical, hyaline, phiailidic, branched, straight or slightly curved. Alpha conidia 6.5–8.5(–9) × 2–2.5(–3) μm, aseptate, hyaline, ellipsoidal to fusiform, rounded at one end, slightly apex at the other end, biguttulate. Beta conidia hyaline, aseptate, filiform, sinuous at one end, eguttulate, (21.5–)25.5–31(–33) × 1 µm.

Figure 5. 

Diaporthe ganzhouensis on unknown host (BJFC-S1678) a the colony on PDA and conidiomata b alpha and beta conidia c conidiogenous cells and alpha conidia. Scale bars: 10 μm (b, c).

Culture characters

Colony at first white, becoming yellowish with age. Aerial mycelium white, dense, fluffy, with visible solitary conidiomata at maturity.

Specimens examined

China. Jiangxi Province: Ganzhou City, unknown dead wood, 25°45'17"N, 115°00'41"E, 23 Jul 2018, Q. Yang, Y. Liu, Y.M. Liang & C.M. Tian (holotype BJFC-C004; ex-type culture: CFCC 53087; living culture: CFCC 53088).

Notes

Diaporthe ganzhouensis comprises the isolates CFCC 53087 and CFCC 53088, revealed to be closely related to D. vawdreyi in the combined phylogenetic tree (Fig. 1). Diaporthe ganzhouensis can be distinguished, based on ITS, tef1 and tub2 loci from D. vawdreyi (6/456 in ITS, 63/357 in tef1 and 40/469 in tub2). Diaporthe ganzhouensis differs morphologically from D. vawdreyi in having longer conidiopores (15.5–21 vs. 6–15 μm) and wider alpha conidia (2–2.5 vs. 1.5–2 μm) (Crous et al. 2015).

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

Figure 6

Description

Conidiomata pycnidial, 692–750(–800) μm diam., solitary and with single necks erumpent through host bark. Tissue around neck is cylindrical. Locule circular, undivided, 450–565(–600) μm diam. Conidiophores reduced to conidiogenous cells. Conidiogenous cells unbranched, straight or slightly curved, apical or base sometimes swelling, (8.5–)9–10.5(–11) × 1.5–2 μm. Alpha conidia hyaline, aseptate, ellipsoidal, biguttulate or with one large guttulate, rounded at one end, slightly apex at the other end, occasionally submedian constriction, (7.5–)8–9(–10.5) × 4–5(–5.5) μm. Beta conidia not observed.

Figure 6. 

Diaporthe multiguttulata on Citrus maxima (BJFC-S1614) a, b habit of conidiomata on twig c conidiomata on PDA d transverse section through conidiomata e longitudinal section through conidiomata f conidiogenous cells attached with alpha conidia g alpha conidia h the colony on PDA. Scale bars: 200 μm (b, d, e); 10 μm (f, g).

Culture characters

Colony originally flat with white felty aerial mycelium, becoming pale green mycelium with age, margin area irregularly, with visible solitary conidiomata at maturity.

Specimens examined

China. Jiangxi Province: Ganzhou City, on branches of Citrus maxima, 25°51'28"N, 114°55'19"E, 11 May 2018, Q. Yang, Y. Liu & Y.M. Liang (BJFC-S1614; living culture: CFCC 53095, CFCC 53096 and CFCC 53097).

Notes

Diaporthe multiguttulata was originally described as an endophyte from a healthy branch of Citrus grandis in Fujian Province, China (Huang et al. 2015). In the present study, three isolates (CFCC 53095, CFCC 53096 and CFCC 53097) from symptomatic branches of Citrus maxima were congruent with D. multigutullata, based on DNA sequence data and confirmed from the morphological analysis (Fig. 1). The clade, therefore, was verified as D. multigutullata which could exist both as an endophyte and a pathogen.

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

MycoBank No: 829526
Figure 7

Diagnosis

Distinguished from the phylogenetically closely-related species D. sennae in having larger alpha conidia and longer beta conidia.

Etymology

Named after the host genus Schima on which the fungus was isolated.

Description

Leaf spots subcircular to irregular, pale brown, with dark brown at margin. Pycnidia solitary, scattered on the leaf surface. Pycnidial conidiomata in culture, globose, (150–)173–357(–373) µm in its widest diam., erumpent, single or clustered in groups of 3–5 pycnidia, coated with hyphae, cream to yellowish translucent conidial droplets exuded from ostioles. Conidiophores reduced to conidiogenous cells. Conidiogenous cells hyaline, unbranched, septate, straight, slightly tapering towards the apex. Alpha conidia scarce, hyaline, aseptate, ellipsoidal to spindle-shaped, four small guttulate, (7.5–)8–8.5(–9) × 2.5–3 μm. Beta conidia abundant, hyaline, aseptate, filiform, straight to sinuous at one end, eguttulate, (25–)27.5–38.5(–40.5) × 1–1.5 µm.

Figure 7. 

Diaporthe schimae on Schima superba (BJFC-S1661) a symptoms on leaves of host plant b the colony on PDA c conidiomata on PDA d conidiophores cells attached with beta conidia e Alpha conidia. Scale bars: 10 μm (d, e).

Culture characters

Colony entirely white, with fluffy aerial mycelium, concentric zonation, margin fimbricate, reverse slightly yellowish.

Specimens examined

China. Jiangxi Province: Ganzhou City, Fengshan Forest Park, on leaves of Schima superba, 25°44'22"N, 114°59'40"E, 15 May 2018, Q. Yang, Y. Liu & Y.M. Liang (holotype BJFC-S1661; ex-type culture: CFCC 53103); 24°40'51"N, 115°34'36"E, 15 May 2018, Q. Yang, Y. Liu & Y.M. Liang (BJFC-S1662; living culture: CFCC 53104); 24°40'52"N, 115°34'54"E, 15 May 2018, Q. Yang, Y. Liu & Y.M. Liang (BJFC-S1663; living culture: CFCC 53105).

Notes

Diaporthe schimae occurs in an independent clade (Fig. 1) and was revealed to be phylogenetically distinct from D. sennae. Diaporhe schimae can be distinguished with D. sennae by 41 nucleotides in concatenated alignment, in which three were distinct in the ITS region, 20 in the tef1 region and 18 in the tub2 region. Diaporthe schimae differs morphologically from D. sennae in having larger alpha conidia and longer beta conidia (8–8.5 × 2.5–3 vs. 5.5–6.3 × 1.5–1.7 µm in alpha conidia; 27.5–38.5 vs. 18.4–20 µm in beta conidia) (Yang et al. 2017a).

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

MycoBank No: 832921
Figure 8

Diagnosis

Distinguished from the phylogenetically closely-related species D. rostrata in having smaller alpha conidia; and from D. juglandicola in having wider alpha conidia.

Etymology

Named after the host genus Vernicia on which the fungus was isolated.

Description

Conidiomata pycnidial, 825–1050 × 445–500 μm diam., solitary and with single necks erumpent through host bark. Tissue around neck is conical. Locule circular, undivided, 400–665 μm diam. Conidiophores reduced to conidiogenous cells. Conidiogenous cells unbranched, straight or sinuous, 14.5–21.5 × 1–1.5 μm. Alpha conidia hyaline, aseptate, ellipsoidal to fusiform, with 1–2-guttulate, 7–8.5 × 3–3.5 μm. Beta conidia not observed.

Figure 8. 

Diaporthe verniciicola on Vernicia montana (BJFC-S1622) a, b habit of conidiomata on twig c transverse section through conidiomata d longitudinal section through conidiomata e alpha conidia f conidiophores g culture on PDA (30d). Scale bars: 500 μm (b); 200 μm (c); 10 μm (e, f).

Culture characters

Colony white to yellowish, with dense and felted mycelium in the centre, lacking aerial mycelium, conidiomata absent.

Specimens examined

China. Jiangxi Province: Ganzhou City, on branches of Vernicia montana, 24°40'51"N, 115°34'52"E, 12 May 2018, Q. Yang, Y. Liu & Y.M. Liang (holotype BJFC-S1622; ex-type culture: CFCC 53109); 24°40'52"N, 115°34'50"E, 12 May 2018, Q. Yang, Y. Liu & Y.M. Liang (BJFC-S1623; living culture: CFCC 53110); 24°45'14"N, 115°34'00"E, 12 May 2018, Q. Yang, Y. Liu & Y.M. Liang (BJFC-S1624; living culture: CFCC 53111); 25°44'15"N, 114°59'32"E, 15 May 2018, Q. Yang, Y. Liu & Y.M. Liang (BJFC-S1624; living culture: CFCC 53112).

Notes

Two isolates of D. verniciicola clustered in a well-supported clade (ML/BI = 100/1) and appeared closely related to D. rostrata and D. juglandicola (Fig. 1). Morphologically, D. verniciicola is similar to D. rostrata characterised by conidiomata with single necks erumpent through the host bark. However, the new taxon can be distinguished from D. rostrata in having smaller alpha conidia (7–8.5 × 3–3.5 vs. 8.5–11.5 × 4–5 μm) (Fan et al. 2015) and D. verniciicola differs from D. juglandicola in having wider alpha conidia (3–3.5 vs. 2.5–3 μm) (Yang et al. 2017b). This is the first discovery of a Diaporthe species isolated from infected branches or twigs on Vernicia montana and was confirmed as a new species, based on phylogeny and morphology.

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

MycoBank No: 829521
Figure 9

Diagnosis

Distinguished from the phylogenetically closely-related species D. oraccinii in having longer conidiophores and larger alpha conidia.

Etymology

Named after the county (Xunwu) where the species was first collected.

Description

On PDA: Conidiomata pycnidial, globose, solitary or aggregated, deeply embedded in the medium, erumpent, dark brown to black. Hyaline conidial drops exuding from ostioles. Conidiophores (18.5–)21.5–30(–32.5) × 1–1.5(–2) μm, cylindrical, hyaline, phiailidic, unbranched, straight to sinuous. Alpha conidia (6.5–)7–8.5 × 2–3 μm, aseptate, hyaline, ellipsoidal to fusiform, rounded at one end, slightly apex at the other end, usually with 2-guttulate. Beta conidia not observed.

Figure 9. 

Diaporthe xunwuensis on unknown host (BJFC-S1679) a the colony on PDA and conidiomata b alpha conidia c conidiogenous cells attached with alpha conidia. Scale bars: 10 μm (a–c).

Culture characters

Colony at first white, becoming dark brown in the centre with age. Aerial mycelium white, dense, fluffy, with black conidial drops exuding from the ostioles.

Specimens examined

China. Jiangxi Province: Ganzhou City, unknown dead wood, 25°45'17"N, 115°00'41"E, 23 Jul 2018, Q. Yang, Y. Liu, Y.M. Liang & C.M. Tian (holotype BJFC-C003; ex-type culture: CFCC 53085; living culture: CFCC 53086).

Notes

Two isolates representing D. xunwuensis clustered in a well-supported clade and appear most closely related to D. oraccinii. Diaporthe xunwuensis can be distinguished from D. oraccinii, based on ITS, his3 and tef1 loci (5/471 in ITS, 5/432 in his3 and 5/325 in tef1). Morphologically, D. xunwuensis differs from D. oraccinii in having longer conidiopores (21.5–30 vs. 10.5–22.5 μm) and larger alpha conidia (7–8.5 × 2–3 vs. 5.5–7.5 × 0.5–2 μm) (Gao et al. 2016).

Discussion

The current study described eight Diaporthe species from 24 strains, based on a large set of freshly-collected specimens. It includes five new species and three known species, which were sampled from six host genera distributed in Jiangxi Province of China (Table 1). In this study, 142 reference sequences (including outgroup) were selected, based on BLAST searches of NCBIs GenBank nucleotide database and included in the phylogenetic analyses (Table 1). Phylogenetic analyses, based on five combined loci (ITS, cal, his3, tef1 and tub2), as well as morphological characters, revealed the diversity of Diaporthe species in Jiangxi Province, mainly focusing on diebacks from major ecological or economic forest trees.

The identification and characterisation of novel taxa and new host records indicate the high potential of Diaporthe to evolve rapidly. In the present study, five species were first reported in China as pathogens. Amongst these species, D. bauhiniae was characterised by having longer alpha conidia (9–13 × 2–2.5 μm). Diaporthe ganzhouensis and D. xunwuensis were isolated from unknown dead wood, but D. ganzhouensis can be distinguish from D. xunwuenesis in having beta conidia and was supported by analysis of the sequence data. Diaporthe schimae was identified as the most widespread species from isolates collected in Jiangxi Province. Diaporthe verniciicola have conidiomata with single necks erumpent through the host bark. Furthermore, two new host records were described, D. apiculatum from Rhus chinensis and D. multiguttulata from Citrus maxima.

Recent plant pathological studies have revealed that several Diaporthe species cause disease, particularly to important plant hosts on a wide range of economically-significant agricultural crops, such as blueberries, citrus, grapes, oaks, sunflowers, soybeans, tea plants, tropical fruits, vegetables and various trees (van Rensburg et al. 2006; Santos and Phillips 2009; Santos et al. 2011; Thompson et al. 2011; Grasso et al. 2012; Lombard et al. 2014; Huang et al. 2015; Udayanga et al. 2015; Gao et al. 2016; Guarnaccia et al. 2018; Yang et al. 2020). For example, research conducted by Huang et al. (2015) revealed seven endophytic Diaporthe species on Citrus; Gao et al. (2016) demonstrated that Diaporthe isolates associated with Camellia spp. could be assigned to seven species and two species complexes; Guarnaccia et al. (2018) explored the occurrence, diversity and pathogenicity of Diaporthe species associated with Vitis vinifera and revealed four new Diaporthe species; Yang et al. (2018) provided the first molecular phylogenetic framework of Diaporthe diversity associated with dieback diseases in China. Following the adoption of DNA sequence-based methods, Diaporthe taxonomy is actively changing, with numerous species being described each year.

The present study is the first evaluation of Diaporthe species, associated with dieback diseases in Jiangxi Province using the combined morphology and molecular data and provided useful information for evaluating the pathogenicity of various species. Multiple strains from different locations should also be subjected to multi-locus phylogenetic analysis to determine intraspecific variation and redefine species boundaries. The descriptions and molecular data of Diaporthe species, provided in this study, represent a resource for plant pathologists, plant quarantine officials and taxonomists for identification of Diaporthe.

Acknowledgements

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

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