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Review Article
Morphological and molecular identification of Diaporthe species in south-western China, with description of eight new species
expand article infoWenxiu Sun, Shengting Huang, Jiwen Xia§, Xiuguo Zhang§, Zhuang Li§
‡ Yangtze University, Jingzhou, China
§ Shandong Agricultural University, Taian, China
Open Access

Abstract

Diaporthe species have often been reported as plant pathogens, endophytes and saprophytes, commonly isolated from a wide range of infected plant hosts. In the present study, twenty strains obtained from leaf spots of twelve host plants in Yunnan Province of China were isolated. Based on a combination of morphology, culture characteristics and multilocus sequence analysis of the rDNA internal transcribed spacer region (ITS), translation elongation factor 1-α (TEF), β-tubulin (TUB), calmodulin (CAL), and histone (HIS) genes, these strains were identified as eight new species: Diaporthe camelliae-sinensis, D. grandiflori, D. heliconiae, D. heterostemmatis, D. litchii, D. lutescens, D. melastomatis, D. pungensis and two previously described species, D. subclavata and D. tectonendophytica. This study showed high species diversity of Diaporthe in tropical rain forests and its hosts in south-western China.

Keywords

Diaporthaceae, Diaporthales, phylogeny, taxonomy, 8 new taxa

Introduction

Diaporthe is a genus in the Diaporthaceae family (Diaporthales), with the asexual morph previously known as Phomopsis and type species Diaporthe eres Nitschke collected from Ulmus sp. in Germany (Nitschke 1870). Nevertheless, with the implementation of “one fungus one name” nomenclature, the generic names Diaporthe and Phomopsis are no longer used for both morphs of this genus, and Rossman et al. (2015) gave priority to the older name Diaporthe Nitschke over Phomopsis (Sacc.) Bubák because it was published first, encountered commonly in literatures and represents the majority of species. The sexual morph of Diaporthe is characterized by: immersed perithecial ascomata and an erumpent pseudostroma with more or less elongated perithecial necks; unitunicate clavate to cylindrical asci; fusoid, ellipsoid to cylindrical, septate or aseptate, hyaline ascospores, biseriately to uniseriately arranged in the ascus, sometimes having appendages (Udayanga et al. 2011; Senanayake et al. 2017, 2018). The asexual morph is characterized by ostiolate conidiomata, with cylindrical phialides producing three types of hyaline, aseptate conidia (Udayanga et al. 2011; Gomes et al. 2013): type I: α-conidia, hyaline, fusiform, straight, guttulate or eguttulate, aseptate, smooth-walled; type II: β-conidia, hyaline, filiform, straight or hamate, aseptate, smooth-walled, eguttulate; type III: γ-conidia, rarely produced, hyaline, multiguttulate, fusiform to subcylindrical with an acute or rounded apex, while the base is sometimes truncate. The gamma conidia rarely produced and observed, those species described, having a third type of spores are D. ampelina (Berk. & M.A. Curtis) R.R. Gomes, Glienke & Crous, D. cinerascens Sacc., D. eres Nitschke, D. hongkongensis R.R. Gomes, C. Glienke & Crous, D. limonicola Guarnaccia & Crous, D. oncostoma (Duby) Fuckel, D. perseae (Zerova) R.R. Gomes, C. Glienke & Crous, D. raonikayaporum R.R. Gomes, C. Glienke & Crous (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 June 2020), but only one-fifth of these taxa have been studied with molecular data (Guo et al. 2020; Yang et al. 2020; Zapata et al. 2020). They are widely distributed and have a broad range of hosts from economically significant agricultural crops to ornamental plants including Camellia, Castanea, Citrus, Glycine, Helianthus, Juglans, Persea, Pyrus, Vaccinium and Vitis (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). Many Diaporthe species have been reported as destructive plant pathogens, innocuous endophytes and 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 them remain unclear (Vilka and Volkova 2015).

From previous studies, the methods of species identification and classification in genus Diaporthe were based on criteria such as morphological characters like the size and shape of ascomata (Udayanga et al. 2011) and conidiomata (Rehner and Uecker 1994). However, in recent studies, determining species boundaries only by morphological characters was demonstrated to be not always informative due to their variability under changing environmental conditions (Gomes et al. 2013). As for phylogenetic analysis for Diaporthe species, the use of a five-locus dataset (ITS-TUB-TEF-CAL-HIS) is the optimal combination for species delimitation as revealed by Santos et al. (2017). Thus, in recent years, many Diaporthe species have been described based on a polyphasic approach combined with morphological characterization and their host associations (Guarnaccia and Crous 2017; Gao et al. 2017; Yang et al. 2018, 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).

In this study, we propose eight novel species and two previously described species of Diaporthe, collected in Yunnan Province of China on twelve plant host genera, based on their morphological characters in culture, and molecular phylogenetic analysis.

Materials and methods

Isolation and morphological studies

The leaves of samples were collected from 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 consecutively immersing in 75% ethanol solution for 1 min, 5% sodium hypochlorite solution for 30 s, and finally rinsed in sterile distilled water for 1 min. The pieces were dried with sterilized paper towels and transferred on potato dextrose agar (PDA) in petri plates (Cai et al. 2009). All the PDA plates were incubated at biochemical 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 the fungal colonies were taken at 7 days and 15 days using a Powershot G7X mark II digital camera. Micromorphological characters were observed and documented in distilled water from microscope slides under Olympus SZX10 stereomicroscope and Olympus BX53 microscope, both supplied with Olympus DP80 HD color digital cameras to photo-document fungal structures. All fungal strains were stored in 10% sterilized glycerin at 4 °C for further studies. Voucher specimens were deposited in the Herbarium of Plant Pathology, Shandong Agricultural University (HSAUP). Living strain cultures were deposited in the Shandong Agricultural University Culture Collection (SAUCC). Taxonomic information on the new taxa was submitted to MycoBank (http://www.mycobank.org).

DNA extraction and amplification

Genomic DNA was extracted from fungal mycelia 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 was 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 visualized 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).

Phylogenetic analyses

Novel sequences generated from twenty strains in this study, and all reference available 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), the alignments and trees were deposited in TreeBASE (http://treebase.org/treebase-web/home.html).

Results

Phylogenetic analyses

Twenty fungal strains of Diaporthe isolates from 15 plant hosts were sequenced (Table 1). These were analyzed by using multilocus data (ITS, TUB, TEF, CAL and HIS) composed of 87 isolates of Diaporthe, with Diaporthella corylina (CBS 121124) as an outgroup taxon. A total of 2856 characters including gaps were obtained in the phylogenetic analysis, viz. ITS: 1–650, TUB: 651–1263, TEF: 1264–1705, CAL: 1706–2279, HIS: 2280–2856. Of these characters, 1395 were constant, 475 were variable and parsimony-uninformative, and 986 were parsimony-informative. For the BI and ML analyses, the substitution model GTR+I+G for ITS, HKY+I+G for TUB, TEF and CAL, GTR+G for HIS 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).

Table 1.

Species and GenBank accession numbers of DNA sequences used in this study with new sequences in bold.

Species Strain/Isolate Host/Substrate GenBank accession number
ITS TUB TEF CAL HIS
Diaporthe alnea CBS 146.46* Alnus sp. KC343008 KC343976 KC343734 KC343250 KC343492
D. anacardii CBS 720.97* Anacardium occidentale KC343024 KC343992 KC343750 KC343266 KC343508
D. baccae CBS 136972* Vaccinium corymbosum KJ160565 KJ160597
D. batatas CBS 122.21 Ipomoea batatas KC343040 KC344008 KC343766 KC343282 KC343524
D. camelliae-sinensis SAUCC194.92* Camellia sinensis MT822620 MT855817 MT855932 MT855699 MT855588
SAUCC194.103 Castanea mollissima MT822631 MT855828 MT855943 MT855710 MT855599
SAUCC194.104 Castanea mollissima MT822632 MT855829 MT855944 MT855711 MT855600
SAUCC194.108 Machilus pingii MT822636 MT855833 MT855948 MT855715 MT855603
D. canthii CBS 132533* Canthium inerme JX069864 KC843230 KC843120 KC843174
D. chamaeropis CBS 753.70 Spartium junceum KC343049 KC344017 KC343775 KC343291 KC343533
D. cinerascens CBS 719.96 Ficus carica KC343050 KC344018 KC343776 KC343292 KC343534
D. cissampeli CPC 27302 Cissampelos capensis KX228273 KX228384 KX228366
D. citri CBS 230.52 Citrus sinensis KC343052 KC344020 KC343778 KC343294 KC343536
D. collariana MFLUCC 17-2636* Magnolia champaca MG806115 MG783041 MG783040 MG783042
D. convolvuli CBS 124654 Convolvulus arvensis KC343054 KC344022 KC343780 KC343296 KC343538
D. cytosporella AR 5149 Citrus sinensis KC843309 KC843223 KC843118 KC843143
D. destruens SPPD-1 Solanum tuberosum JN848791 JX421691
D. dorycnii MFLU 17-1015* Dorycnium hirsutum KY964215 KY964099 KY964171
D. elaeagni CBS 504.72 Elaeagnus sp. KC343064 KC344032 KC343790 KC343306 KC343548
D. elaeagni-glabrae LC4802* Elaeagnus glabra KX986779 KX999212 KX999171 KX999281 KX999251
D. endophytica CBS 133811* Schinus terebinthifolius KC343065 KC344033 KC343791 KC343307 KC343549
D. eres AR5193* Ulmus laevis KJ210529 KJ420799 KJ210550 KJ434999 KJ420850
D. foeniculina CBS 123208 Foeniculum vulgare KC343104 KC344072 KC343830 KC343346 KC343588
D. fructicola MAFF 246408 Passiflora edulis LC342734 LC342736 LC342735 LC342738 LC342737
D. grandiflori SAUCC194.84* Heterostemma grandiflorum MT822612 MT855809 MT855924 MT855691 MT855580
D. heliconiae SAUCC194.75 Heliconia metallica MT822603 MT855800 MT855915 MT855682 MT855571
SAUCC194.77* Heliconia metallica MT822605 MT855802 MT855917 MT855684 MT855573
D. heterophyllae CPC 26215 Acacia heterophylla MG600222 MG600226 MG600224 MG600218 MG600220
D. heterostemmatis SAUCC194.85* Heterostemma grandiflorum MT822613 MT855810 MT855925 MT855692 MT855581
SAUCC194.102 Camellia sinensis MT822630 MT855827 MT855942 MT855709 MT855598
D. hickoriae CBS 145.26* Carya glabra KC343118 KC344086 KC343844 KC343360 KC343602
D. inconspicua CBS 133813* Maytenus ilicifolia KC343123 KC344091 KC343849 KC343365 KC343607
D. kongii T12509H* Helianthus annuus JF431301 KJ197272 JN645797
D. litchii SAUCC194.12 Elaeagnus conferta MT822540 MT855737 MT855854 MT855625 MT855509
SAUCC194.22* Litchi chinensis MT822550 MT855747 MT855863 MT855635 MT855519
D. longicolla FAU599 Glycine max KJ590728 KJ610883 KJ590767 KJ612124 KJ659188
D. lutescens SAUCC194.36* Chrysalidocarpus lutescens MT822564 MT855761 MT855877 MT855647 MT855533
D. macintoshii BRIP 55064a* Rapistrum rugostrum KJ197289 KJ197269 KJ197251
D. masirevicii BRIP 57330 Chrysanthemoides monilifera subsp. rotundata KJ197275 KJ197255 KJ197237
BRIP 57892a* Helianthus annuus KJ197276 KJ197257 KJ197239
D. melastomatis SAUCC194.55* Melastoma malabathricum MT822583 MT855780 MT855896 MT855664 MT855551
SAUCC194.80 Millettia reticulata MT822608 MT855805 MT855920 MT855687 MT855576
SAUCC194.88 Camellia sinensis MT822616 MT855813 MT855928 MT855695 MT855584
D. melonis CBS 507.78* Cucumis melo KC343142 KC344110 KC343868 KC343384 KC343626
D. miriciae BRIP 54736j* Helianthus annuus KJ197282 KJ197262 KJ197244
D. neilliae CBS 144.27 Spiraea sp. KC343144 KC344112 KC343870 KC343386 KC343628
D. nigra JZBH320170 Ballota nigra MN653009 MN887113 MN892277
D. nomurai CBS 157.29 Morus sp. KC343154 KC344122 KC343880 KC343396 KC343638
D. oncostoma CBS 100454 Robinia pseudoacacia KC343160 KC344128 KC343886 KC343402 KC343644
CBS 109741 Robinia pseudoacacia KC343161 KC344129 KC343887 KC343403 KC343645
D. ovalispora ZJUD93* Citrus limon KJ490628 KJ490449 KJ490507 KJ490570
D. parapterocarpi CPC 22729 Pterocarpus brenanii KJ869138 KJ869248
D. parvae PSCG 034* Pyrus bretschneideri MK626919 MK691248 MK654858 MK726210
D. passifloricola CPC 27480* Passiflora foetida KX228292 KX228387 KX228367
D. penetriteum LC3353* Camellia sinensis KP714505 KP714529 KP714517 KP714493
LC3394 Camellia sinensis KP267893 KP293473 KP267967 KP293544
D. phaseolorum CBS 116019 Caperonia palustris KC343175 KC344143 KC343901 KC343417 KC343659
CBS 116020 Aster exilis KC343176 KC344144 KC343902 KC343418 KC343660
D. phillipsii CAA 817* Dead twig MK792305 MN000351 MK828076 MK883831 MK871445
D. poincianellae URM 7932 Poincianella pyramidalis MH989509 MH989537 MH989538 MH989540 MH989539
D. pseudoinconspicua G26 Poincianella pyramidalis MH122538 MH122524 MH122533 MH122528 MH122517
D. psoraleae CPC 21634 Psoralea pinnata KF777158 KF777251 KF777245
D. pterocarpi MFLUCC 10-0571 Pterocarous indicus JQ619899 JX275460 JX275416 JX197451
MFLUCC 10-0575 Pterocarous indicus JQ619901 JX275462 JX275418 JX197453
D. pungensis SAUCC194.89 Camellia sinensis MT822617 MT855814 MT855929 MT855696 MT855585
SAUCC194.112* Elaeagnus pungens MT822640 MT855837 MT855952 MT855719 MT855607
D. ravennica MFLUCC 17-1029 Tamarix sp. KY964191 KY964075 KY964147
D. rosae MFLUCC 17-2658 Rosa sp. MG828894 MG843878 MG829273
D. rumicicola MFLUCC18-0739 Rumex sp. MH846233 MK049555 MK049554
D. saccarata CBS 116311* Protea repens KC343190 KC344158 KC343916 KC343432 KC343674
D. shennongjiaensis CNUCC 201905 Juglans regia MN216229 MN227012 MN224672 MN224551 MN224560
D. sojae CBS 100.87* Glycine soja KC343196 KC344164 KC343922 KC343438 KC343680
D. stictica CBS 370.54 Buxus sampervirens KC343212 KC344180 KC343938 KC343454 KC343696
D. subclavata ZJUD95* Citrus unshiu KJ490630 KJ490451 KJ490509 KJ490572
SAUCC194.66 Pometia pinnata MT822594 MT855791 MT855906 MT855674 MT855562
D. subellipicola KUMCC 17-0153 on dead wood MG746632 MG746634 MG746633
D. tectonendophytica MFLUCC 13-0471* Tectona grandis KU712439 KU743986 KU749367 KU749354
SAUCC194.11 Elaeagnus conferta MT822539 MT855736 MT855853 MT855624 MT855508
SAUCC194.63 Pometia pinnata MT822591 MT855788 MT855903 MT855672 MT855559
D. ueckerae FAU656* Cucumis melo KJ590726 KJ610881 KJ590747 KJ612122 KJ659215
D. unshiuensis CFCC 52595 Carya illinoensis MH121530 MH121607 MH121572 MH121448 MH121488
D. vangueriae CPC 22703 Vangueria infausta KJ869137 KJ869247
D. velutina LC4419* Neolitsea sp. KX986789 KX999222 KX999181 KX999286 KX999260
D. virgiliae CMW40755* Virgilia oroboides KP247573 KP247582
CMW40748 Virgilia oroboides KP247566 KP247575
D. zaobaisu PSCG 031* Pyrus bretschneideri MK626922 MK691245 MK654855 MK726207
Diaporthella corylina CBS 121124 Corylus sp. KC343004 KC343972 KC343730 KC343246 KC343488

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, 20 strains isolated in this study were assigned to 10 species, 8 of them are proposed and described here as new species (Fig. 1). Strains (SAUCC194.92, SAUCC194.103, SAUCC194.104 and SAUCC194.108) are D. camelliae-sinensis, strain (SAUCC194.84) – Diaporthe grandiflori, strains (SAUCC194.75 and SAUCC194.77) – D. heliconiae, strains (SAUCC194.85 and SAUCC194.102) – D. heterostemmatis, strains (SAUCC194.12 and SAUCC194.22) – D. litchii, strain (SAUCC194.36) – D. lutescens, strains (SAUCC194.55, SAUCC194.80 and SAUCC194.88) – D. melastomatis, strains (SAUCC194.89 and SAUCC194.112) – D. pungensis. One strain (SAUCC194.66) is of a previously described D. subclavata, and strains (SAUCC194.11 and SAUCC194.63) – of previously described D. tectonendophytica.

Figure 1. 

Phylogram of Diaporthe based on combined 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. Strains from this study are shown in red. Three branches were shortened to fit the page size – these are indicated by symbol (//) with indication number showing how many times they are shortened.

Taxonomy

Diaporthe camelliae-sinensis S.T. Huang, J.W. Xia, X.G. Zhang & Z. Li, sp. nov.

MycoBank No: 837600
Figure 2

Etymology

Named after the host Camellia sinensis on which it was collected.

Figure 2. 

Diaporthe camelliae-sinensis (SAUCC194.92) a leaf of host plant b, c surface (b) and reverse (c) sides of colony after incubation for 15 days on PDA d conidiomata e–h conidiophores and conidiogenous cells i beta conidia j–l alpha conidia and beta conidia m alpha conidia. Scale bars: 10 μm (e–m).

Diagnosis

Diaporthe camelliae-sinensis can be distinguished from the closely related species D. macintoshii R.G. Shivas et al. and D. vangueriae Crous based on ITS, TUB and TEF loci. Diaporthe camelliae-sinensis differs from D. macintoshii in smaller α-conidia and from D. vangueriae in shorter β-conidia.

Type

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on infected leaves of Camellia sinensis. 19 April 2019, S.T. Huang, HSAUP194.92, holotype, ex-holotype living culture SAUCC194.92.

Description

Asexual morph: Conidiomata pycnidial, multi-pycnidia grouped together, globose, black, erumpent, coated with white hyphae, thick-walled, exuding creamy to yellowish conidial droplets from central ostioles. Conidiophores hyaline, smooth, septate, branched, densely aggregated, cylindrical, straight to sinuous, swelling at the base, tapering towards the apex, 10–15 × 1.5–2 μm. Conidiogenous cells 8.5–12 × 2–2.8 μm, phialidic, cylindrical, terminal, slightly tapering towards the apex. Alpha conidia, hyaline, smooth, aseptate, ellipsoidal to fusoid, 2–4 guttulate, apex subobtuse, base subtruncate, 7.5–10 × 1.8–2.5 µm (mean = 8.5 × 2.2 μm, n = 20). Beta conidia hyaline, aseptate, filiform, sigmoid to lunate, mostly curved through 90–180°, tapering towards the apex, base truncate, 20–30 × 1.2–1.6 µm (mean = 25.6 × 1.3 μm, n = 20). Gamma conidia and sexual morph not observed.

Culture characteristics

Pure culture was isolated by subbing hyphal tips growing from surface sterilized diseased material. Colonies on PDA cover the Petri dish diameter after incubation for 15 days in dark conditions at 25 °C, cottony and radially with abundant aerial mycelium, sparse in the margin. With a tanned concentric ring of dense hyphae, white on surface side, white to pale yellow on reverse side.

Additional specimens examined

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, 19 April 2019, S.T. Huang. On infected leaves of Castanea mollissima, HSAUP194.103 and HSAUP194.104 paratype, living culture SAUCC194.103 and SAUCC194.104; on diseased leaves of Machilus pingii, HSAUP194.108 paratype, living culture SAUCC194.108.

Notes

Four isolates are clustered in a clade distinct from its closest phylogenetic neighbor, D. macintoshii and D. vangueriae. Diaporthe camelliae-sinensis can be distinguished from D. macintoshii in ITS, TUB and TEF loci (23/558 in ITS, 2/463 in TUB and 20/328 in TEF); from D. vangueriae in ITS and TUB loci (23/558 in ITS and 1/423 in TUB). Morphologically, Diaporthe camelliae-sinensis differs from D. macintoshii in having guttulate alpha conidia and smaller alpha conidia (7.5–10 × 1.8–2.5 vs. 8.0–11.0 × 2.0–3.0 μm) (Thompson et al. 2015). Furthermore, Diaporthe camelliae-sinensis differs from D. vangueriae in shorter beta conidia (20–30 × 1.2–1.6 vs. 28–35 × 1.5–2.0 μm) and D. camelliae-sinensis can produce alpha conidia, but D. vangueriae could not (Crous et al. 2014).

Diaporthe grandiflori S.T. Huang, J.W. Xia, X.G. Zhang & Z. Li, sp. nov.

MycoBank No: 837591
Figure 3

Etymology

Named after the host Heterostemma grandiflorum on which it was collected.

Diagnosis

Diaporthe grandiflori can be distinguished from the phylogenetically closely related species D. penetriteum Y.H. Gao & L. Cai in larger α-conidia and β-conidia.

Figure 3. 

Diaporthe grandiflori (SAUCC194.84) a leaf of Heterostemma grandiflorum b, c surface (b) and reverse (c) sides of colony after incubation for 15 days on PDA d conidiomata e conidiophores and conidiogenous cells f alpha conidia g, i alpha conidia and beta conidia h beta conidia. Scale bars: 10 μm (e–i).

Type

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on infected leaves of Heterostemma grandiflorum. 19 April 2019, S.T. Huang, HSAUP194.84, holotype, ex-holotype living culture SAUCC194.84.

Description

Asexual morph: Conidiomata pycnidial, subglobose to globose, solitary or aggregated in groups, black, erumpent, coated with white hyphae, thick-walled, exuding golden yellow spiral conidial cirrus from ostiole. Conidiophores hyaline, smooth, septate, branched, densely aggregated, cylindrical, straight to slightly sinuous, 9.5–16.5 × 1.9–2.8 μm. Conidiogenous cells 19.0–22.8 × 1.4–2.4 μm, cylindrical, multi-guttulate, terminal, tapering towards the apex. Alpha conidia abundant in culture, biguttulate, hyaline, smooth, aseptate, ellipsoidal, apex subobtuse, base subtruncate, 6.3–8.3 × 2.8–3.3 µm (mean = 7.5 × 2.9 μm, n = 20). Beta conidia, not numerous, hyaline, aseptate, filiform, slightly curved, tapering towards the apex, 21.5–30.5 × 1.5–2.1 µm (mean = 24.0 × 1.7 μm, n = 20). Gamma conidia not observed. Sexual morph not observed.

Culture characteristics

Pure culture was isolated by subbing hyphal tips growing from surface sterilized plant material. Colonies on PDA cover the Petri dish after 15 days kept in dark conditions at 25 °C, cottony with abundant aerial mycelium, white on surface side, white to grayish on reverse.

Notes

Phylogenetic analysis of a combined five loci showed that D. grandiflori (strain SAUCC194.84) formed an independent clade (Fig. 1) and is phylogenetically distinct from D. penetriteum. This species can be easily distinguished from D. penetriteum by 87 nucleotides difference concatenated alignment (24 in the ITS region, 1 TUB, 41 CAL and 21 HIS). Morphologically, D. grandiflori differs from D. penetriteum in larger α-conidia (6.3–8.3 × 2.8–3.3 vs. 4.5–5.5 × 1.5–2.5 μm) and longer β-conidia (21.5–30.5 × 1.5–2.1 vs. 16.5–27.5 × 1.0–2.0 μm) (Gao et al. 2016).

Diaporthe heliconiae S.T. Huang, J.W. Xia, X.G. Zhang & Z. Li, sp. nov.

MycoBank No: 837592
Figure 4

Etymology

Named after the host Heliconia metallica on which it was collected.

Diagnosis

Diaporthe heliconiae can be distinguished from the phylogenetically closely related species D. subclavata F. Huang, K.D. Hyde & Hong Y. Li in smaller α-conidia.

Figure 4. 

Diaporthe heliconiae (SAUCC194.77) a petiole of Heliconia metallica b, c surface (b) and reverse (c) sides of colony after incubation for 15 days on PDA d conidiomata on PDA e–g conidiophores and conidiogenous cells h beta conidia i alpha conidia and beta conidia j alpha conidia k alpha conidia and germinating conidia. All in water. Scale bars: 10 μm (e–k).

Type

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on the symptomatic petiole of Heliconia metallica. 19 April 2019, S.T. Huang, HSAUP194.77, holotype, ex-holotype living culture SAUCC194.77.

Description

Asexual morph: Conidiomata pycnidial, solitary or aggregated in groups, erumpent, thin-walled, superficial to embedded on PDA, dark brown to black, globose or subglobose, exuding creamy yellowish spiral conidial cirrus from the ostioles. Conidiophores hyaline, aseptate, cylindrical, straight to sinuous, branched, 16.5–25.0 × 1.3–1.8 µm. Alpha conidiogenous cells, cylindric-clavate, terminal, few guttulate, 11.5–18.0 × 1.0–1.5 µm. Beta conidiogenous cells, prismatic, terminal, few guttulate, 10.0–14.1 × 1.0–1.2 µm. Alpha conidia, hyaline, smooth, aseptate, ellipsoidal, 2–4 guttulate, apex subobtuse, base subtruncate, 5.0–6.5 × 2.0–2.5 µm (mean = 6.1 × 2.3 μm, n = 20). Beta conidia hyaline, aseptate, filiform, slightly curved, tapering towards the apex, 25.0–33.5 × 1.0–1.5 µm (mean = 29.4 × 1.3 μm, n = 20). Gamma conidia and sexual morph not observed.

Culture characteristics

Pure culture was isolated by subbing hyphal tips growing from surface sterilized infected plant material. Colonies on PDA cover the Petri dish diameter after incubation for 15 days in dark conditions at 25 °C. Aerial mycelium abundant, cottony, white, dense in the center, sparse near the margin. White on surface side, white to tanned on reverse side.

Additional specimen examined

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on the symptomatic petiole of Heliconia metallica. 19 April 2019, S.T. Huang, HSAUP194.75 paratype; living culture SAUCC194.75.

Notes

Diaporthe heliconiae clade comprises strains SAUCC194.75 and SAUCC194.77, closely related to D. subclavata in the combined phylogenetic tree (Fig. 1). Diaporthe heliconiae can be distinguished based on ITS, TUB and HIS loci from D. subclavata (16/489 in ITS, 8/357 in TUB and 3/470 in HIS). Morphologically, Diaporthe heliconiae differs from D. subclavata in its smaller α-conidia (5.0–6.5 × 2.0–2.5 vs. 5.5–7.2 × 2.2–2.9 μm). Furthermore, in Diaporthe heliconiae β-conidia were obtained size 25.0–33.5 × 1.0–1.5 µm, while in D. subclavata β-conidia were not obtained (Huang et al. 2015).

Diaporthe heterostemmatis S.T. Huang, J.W. Xia, X.G. Zhang & Z. Li, sp. nov.

MycoBank No: 837593
Figure 5

Etymology

Named after the host Heterostemma grandiflorum on which it was collected.

Diagnosis

Diaporthe heterostemmatis differs from its closest phylogenetic species D. subellipicola S.K. Huang & K.D. Hyde in ITS, TUB and TEF loci based on the alignments deposited in Tree-BASE.

Figure 5. 

Diaporthe heterostemmatis (SAUCC194.85) a leaf of host plant b, c surface (b) and reverse (c) sides of colony, after incubation for 15 days on PDA d conidiomata on PDA e, f conidiophores and conidiogenous cells g beta conidia h Alpha conidia i, j alpha conidia and beta conidia. Scale bars: 10 μm (e–j).

Type

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on infected leaves of Heterostemma grandiflorum. 19 April 2019, S.T. Huang, HSAUP194.85, holotype, ex-holotype living culture SAUCC194.85.

Description

Asexual morph: Conidiomata pycnidial, 3–5 pycnidia grouped together, globose, black, erumpent, exuding creamy to yellowish conidial droplets from ostioles. Conidiophores hyaline, septate, branched, elliptical or cylindrical, straight to sinuous, 6.5–10.5 × 2.5–4.5 μm. Conidiogenous cells 5.3–11.8 × 1.5–3.2 μm, phialidic, cylindrical, enlarged towards the base, tapering towards the apex, slightly curved, neck up to 5.5 μm long, 2.0 μm wide. Alpha conidia, hyaline, smooth, aseptate, ellipsoidal, biguttulate, apex subobtuse, base subtruncate, 5.8–7.5 × 2.5–3.3 µm (mean = 6.5 × 3.0 μm, n = 20). Beta conidia hyaline, aseptate, filiform, few guttulate, hooked and mostly curved through 90–180°, tapering towards both ends, 16.0–22.7 × 1.0–1.5 µm (mean = 20.4 × 1.2 μm, n = 20). Gamma conidia and sexual morph not observed.

Culture characteristics

Pure culture was isolated by subbing hyphal tips growing from surface sterilized plant material. Colonies on PDA cover the Petri dish diameter after incubation for 15 days in dark conditions at 25 °C. Aerial mycelium white, cottony, feathery, with concentric zonation, white on surface side, pale brown to black on reverse side.

Additional specimen examined

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on infected leaves of Camellia sinensis. 19 April 2019, S.T. Huang, HSAUP194.102 paratype; living culture SAUCC194.102.

Notes

This new species is proposed as the molecular data showed it forms a distinct clade with high support (ML/BI=98/1) and it appears most closely related to D. subellipicola. Diaporthe heterostemmatis can be distinguished from D. subellipicola by 57 nucleotides in concatenated alignment, in which 8 were distinct in the ITS region, 28 in the TUB region and 21 in the TEF region. Morphologically, D. subellipicola was observed only on the basis of the sexual morph and culture characteristics (Hyde et al. 2018).

Diaporthe litchii S.T. Huang, J.W. Xia, X.G. Zhang, Z. Li, sp. nov.

MycoBank No: 837595
Figure 6

Etymology

Named after the host Litchi chinensis on which it was collected.

Diagnosis

Diaporthe litchii differs from D. collariana R.H. Perrera & K.D. Hyde in smaller alpha conidia and shorter conidiophores.

Type

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on infected leaves of Litchi chinensis. 19 April 2019, S.T. Huang, HSAUP194.22, holotype, ex-holotype living culture SAUCC194.22.

Figure 6. 

Diaporthe litchii (SAUCC194.22) a leaf of host plant b, c surface (b) and reverse (c) sides of colony after incubation for 15 days on PDA d conidiomata e, f conidiophores and conidiogenous cells g, h beta conidia i alpha conidia and beta conidia j alpha conidia. Scale bars: 10 μm (e–j).

Description

Asexual morph: Conidiomata pycnidial, 3–5 pycnidia grouped together, globose, black, erumpent, coated with white hyphae, creamy to yellowish conidial droplets exuded from central ostioles. Conidiophores hyaline, branched, densely aggregated, cylindrical, 10.5–15.0 × 1.8–2.5 μm. Conidiogenous cells 7.5–9.5 × 1.5–2.0 μm, cylindrical, terminal, straight to sinuous. Alpha conidia, hyaline, smooth, aseptate, ellipsoidal to fusiform, biguttulate, 3.8–5.0 × 1.5–2.3 µm (mean = 4.7 × 2.0 μm, n = 20). Beta conidia hyaline, aseptate, filiform, few guttulate, slightly curved, tapering towards both ends, 20.0–28.0 × 1.2–1.8 µm (mean = 23.2 × 1.2 μm, n = 20). Gamma conidia and sexual morph not observed.

Culture characteristics

Pure culture was isolated by subbing hyphal tips growing from surface sterilized plant material. Colonies on PDA cover the Petri dish diameter after incubation for 15 days in dark conditions at 25 °C. Aerial mycelium abundant, white, cottony on surface, reverse white to pale brown with two concentric zonation.

Additional specimen examined

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on diseased leaves of Elaeagnus conferta. 19 April 2019, S.T. Huang, HSAUP194.12 paratype; living culture SAUCC194.12.

Notes

Diaporthe litchii comprises strains SAUCC194.12 and SAUCC194.22 can be distinguished from the closely related species D. collariana by 63 nucleotides difference in the concatenated alignment (9 in the ITS region, 34 TUB, 5 TEF and 15 CAL). Diaporthe litchii differs from D. collariana in smaller alpha conidia (3.8–5.0 × 1.5–2.3 vs. 4.7–5.6 × 1.7–2.2 μm) and shorter conidiophores (10.5–15.0 × 1.8–2.5 vs. 12–20 × 2.4–3.2 μm) (Perera et al. 2018).

Diaporthe lutescens S.T. Huang, J.W. Xia, X.G. Zhang & Z. Li, sp. nov.

MycoBank No: 837597
Figure 7

Etymology

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

Diagnosis

Diaporthe lutescens differs from D. pterocarpi (S. Hughes) D. Udayanga et al. and D. pseudoinconspicua T.G.L. Oliveira et al. in longer beta conidia and the types of conidia.

Figure 7. 

Diaporthe lutescens (SAUCC194.36) a leaves of host plant b, c surface (b) and reverse (c) sides of colony after incubation for 15 days on PDA d conidiomata e–g conidiophores and conidiogenous cells h, i beta conidia. Scale bars: 10 μm (e–i).

Type

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on leaves of Chrysalidocarpus lutescens. 19 April 2019, S.T. Huang, HSAUP194.36, holotype, ex-holotype living culture SAUCC194.36.

Description

Asexual morph: Conidiomata pycnidial, scattered or aggregated, black, erumpent, slightly raised above the surface of the culture medium, subglobose, exuding white creamy conidial droplets from central ostioles after 30 days incubation in light condition at 25 °C on PDA; pycnidial wall consists of black to dark brown, thin-walled cells. Conidiophores 10.2–17.0 × 1.8–3.0 μm, hyaline, unbranched, subcylindrical, septate, smooth, straight or slightly curved, obtuse at the apex, widened at base. Conidiogenous cells 5.7–9.1 × 1.4–2.6 μm, phialidic, cylindrical, terminal, straight to sinuous, tapering towards the apex. Beta conidia 20.8–28.8 × 1.2–2.0 μm (mean = 25.3 × 1.4 μm, n = 20), filiform, hyaline, straight or slightly curved, aseptate, base subtruncate, enlarged towards the apex. Alpha conidia and gamma conidia not observed.

Culture characteristics

Pure culture was isolated by subbing hyphal tips growing from surface sterilized infected plant material. Colonies on PDA cover the petri plate diameter after incubation for 15 days in dark conditions at 25 °C, initially white, becoming grayish, reverse pale brown, with concentric rings of dense and sparse hyphae, irregular margin, fluffy aerial mycelium. Pycnidia formed in 15 days.

Notes

From the phylotree, seen on Fig. 1, Diaporthe lutescens forms an independent clade and is phylogenetically distinct from D. pterocarpi and D. pseudoinconspicua. Diaporthe lutescens can be distinguished from D. pterocarpi in ITS, TUB, TEF and CAL loci by 77 nucleotide differences in concatenated alignment (43 in ITS, 2 in TUB, 29 in TEF and 17 in CAL), and from D. pseudoinconspicua in ITS, TUB, TEF, CAL and HIS loci by 65 nucleotide differences (18 in ITS, 3 in TUB, 23 in TEF, 8 in CAL and 13 in HIS). Moreover, D. lutescens differs from D. pterocarpi and D. pseudoinconspicua in having longer beta conidia (20.8–28.8 × 1.2–2.0 vs. 16.0–23.4 × 1.0–1.4 μm, and 20.8–28.8 × 1.2–2.0 vs. 18.0–21.0 × 1.0–1.5 μm). Furthermore, Diaporthe pterocarpi and D. pseudoinconspicua can produce α-conidia, but D. lutescens cannot (Crous et al. 2018a; Broge et al. 2020).

Diaporthe melastomatis S.T. Huang, J.W. Xia, X.G. Zhang & Z. Li, sp. nov.

MycoBank No: 837598
Figure 8

Etymology

Named after the host Melastoma malabathricum on which it was collected.

Diagnosis

Diaporthe melastomatis differs from D. parapterocarpi Crous in smaller α-conidia and the types of conidia.

Figure 8. 

Diaporthe melastomatis (SAUCC194.55) a branch with leaves of host plant b, c surface (b) and reverse (c) sides of colony after incubation for 15 days on PDA d conidiomata e, f conidiophores and conidiogenous cells g beta conidia h, i, k alpha conidia and beta conidia j alpha conidia. Scale bars: 10 μm (e–k).

Type

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on diseased leaves of Melastoma malabathricum. 19 April 2019, S.T. Huang, HSAUP194.55, holotype, ex-holotype living culture, SAUCC194.55.

Description

Asexual morph: Conidiomata pycnidial, subglobose to globose, black, erumpent, coated with white hyphae, thick-walled, yellowish spiral conidial cirrus exuded from ostioles. Conidiophores hyaline, smooth, septate, branched, densely aggregated, cylindric-clavate, straight to slightly sinuous, tapering towards the apex, 14.5–21.0 × 2.0–3.2 μm. Conidiogenous cells 9.5–13.0 × 1.5–2.5 μm, cylindrical, guttulate, terminal, tapering towards the base. Alpha conidia, hyaline, smooth, aseptate, oblong ellipsoidal, 2–4 guttulate, apex subobtuse, base subtruncate, 5.5–8.5 × 1.7–2.5 µm (mean = 6.8 × 2.1 μm, n = 20). Beta conidia abundant in the culture, hyaline, aseptate, filiform, multi-guttulate, sigmoid to lunate, mostly curved through 90–180°, tapering towards both ends, 25.0–33.5 × 1.1–2.0 µm (mean = 27.6 × 1.4 μm, n = 20). Gamma conidia and sexual morph not observed.

Culture characteristics

Pure culture was isolated by subbing hyphal tips growing from surface sterilized diseased material. Colonies on PDA cover the Petri diameter after incubation for 15 days in dark conditions at 25 °C, cottony and lobate with abundant aerial mycelium, hyphae white in the margin on surface side, with pale brown concentric ring of dense hyphae on reverse side.

Additional 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, HSAUP194.80 paratype, living culture SAUCC194.80; on infected leaves of Camellia sinensis, HSAUP194.88 paratype, living culture SAUCC194.88.

Notes

Diaporthe melastomatis is introduced based on the multi-locus phylogenetic analysis, with three isolates clustering separately in a well-supported clade (ML/BI = 100/1). Diaporthe melastomatis is most closely related to D. parapterocarpi, but distinguished based on ITS and TUB loci from D. parapterocarpi by 32 nucleotides difference in the concatenated alignment, in which 20 are distinct in the ITS region, 12 in the TUB region. Morphologically, Diaporthe melastomatis differs from D. parapterocarpi in its smaller alpha conidia (5.5–8.5 × 1.7–2.5 vs. 8.0–10.0 × 2.5–3.0 μm). Furthermore, Diaporthe melastomatis can produce beta conidia, but D. parapterocarpi cannot (Crous et al. 2014).

Diaporthe pungensis S.T. Huang, J.W. Xia, X.G. Zhang, Z. Li, sp. nov.

MycoBank No: 837599
Figure 9

Etymology

Named after the host Elaeagnus pungens on which it was collected.

Diagnosis

Diaporthe pungensis differs from its closest phylogenetic species D. inconspicua R.R. Gomes et al. and D. poincianellae T.G.L. Oloveira et al. in ITS, TUB, TEF, CAL and HIS loci based on the alignments deposited in Tree-BASE.

Figure 9. 

Diaporthe pungensis (SAUCC194.112) a leaf of host plant b, c surface (b) and reverse (c) sides of colony after incubation for 15 days on PDA d conidiomata on PDA e–h conidiophores and conidiogenous cells i, l beta conidia j, k alpha conidia and beta conidia. Scale bars: 10 μm (e–l).

Type

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on diseased leaves of Elaeagnus pungens. 19 April 2019, S.T. Huang, HSAUP194.112, holotype, ex-holotype living culture SAUCC194.112.

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 the ostioles. Conidiophores hyaline, aseptate, cylindrical, smooth, straight to sinuous, unbranched, 11.0–14.5 × 1.5–2.3 µm. Conidiogenous cells phialidic, cylindrical, terminal, 8.0–9.5 × 1.0–2.5 µm. Alpha conidia, hyaline, smooth, aseptate, ellipsoidal to fusoid, 2–3 guttulate, apex subobtuse, base subtruncate, 6.0–8.5 × 2.0–3.3 µm (mean = 6.6 × 2.5 μm, n = 20). Beta conidia hyaline, aseptate, eguttulate, filiform, slightly curved, tapering towards the apex, base truncate, some conidia are in the immature stage swollen in the middle, 24.0–28.9 × 1.0–2.0 µm (mean = 26.9 × 1.4 μm, n = 20). Gamma conidia not observed, sexual morph not observed.

Culture characteristics

Pure culture was isolated by subbing hyphal tips growing from surface sterilized plant material. Colonies on PDA cover the 3/4 of Petri dish diameter after incubation for 15 days in dark conditions at 25 °C, flat, cottony in the center with medium developed aerial mycelium, sparse in the outer region. With several concentric rings of dense and sparse hyphae, irregular margin, white on surface side, white to pale yellow and cinnamon speckle on reverse side.

Additional specimen examined

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on infected leaves of Camellia sinensis. 19 April 2019, S.T. Huang, HSAUP194.89 paratype, living culture SAUCC194.89.

Notes

Diaporthe pungensis forms a distinct clade with high support (ML/BI = 100/1), and differed with the closely related species (D. inconspicua and D. poincianellae) on ITS, TUB, CAL and HIS loci (94% in ITS, 92% in TUB, 70% in TEF, 92% in CAL and 92% in HIS; and 95% in ITS, 94% in TUB, 80% in TEF, 94% in CAL and 89% in HIS, respectively). Moreover, Diaporthe pungensis differs from D. inconspicua, in having guttulate of alpha conidia, and having larger alpha conidia (6.0–8.5 × 2.0–3.3 vs. 5.5–6.5 × 1.5–2 μm) (Bezerra et al. 2018). Furthermore, Diaporthe pungensis can produce two types of conidia (α-conidia and β-conidia), but D. poincianellae only produce a α-conidia(Crous et al. 2018b).

Diaporthe subclavata F. Huang, K.D. Hyde & H.Y. Li, Fung. Biol. 119: 343, 2015

Figure 10

Description

Asexual morph: Conidiomata pycnidial, multi-pycnidia grouped together, globose, black, erumpent, coated with white hyphae, creamy to yellowish conidial droplets exuded from central ostioles. Conidiophores hyaline, densely aggregated, cylindrical, straight to sinuous, tapering towards the apex, 13.5–23.0 × 2.0–3.0 μm. Alpha conidiogenous cells 7.0–10 × 1.8–2.5 μm, cylindrical, terminal, slightly curved. Beta conidiogenous cells 10.5–13.5 × 0.9–1.5 μm, cylindrical, hyaline, tapering towards the apex. Alpha conidia, hyaline, smooth, aseptate, ellipsoidal, multi-guttulate, apex subobtuse, base subtruncate, 4.7–5.8 × 2.4–2.9 µm (mean = 5.3 × 2.6 μm, n = 20). Beta conidia hyaline, aseptate, filiform, few guttulate, slightly curved, tapering towards the both ends, 25.5–32.0 × 1.0–1.6 µm (mean = 27.5 × 1.3 μm, n = 20). Gamma conidia and sexual morph not observed.

Figure 10. 

Diaporthe subclavata (SAUCC194.66) a leaf of Pometia pinnata b, c surface (b) and reverse (c) sides of colony after incubation for 15 days on PDA d conidiomata e–h conidiophores and conidiogenous cells i, j Beta conidia k, l Alpha conidia. Scale bars: 10 μm (e–l).

Culture characteristics

Pure culture was isolated by subbing hyphal tips growing from surface sterilized diseased material. Colonies on PDA cover the Petri dish diameter after incubation for 15 days in dark conditions at 25 °C. Aerial mycelium white, cottony, feathery, with concentric zonation, white on surface side, pale brown to black on reverse side.

Specimen examined

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, on infected leaves of Pometia pinnata. 19 April 2019, S.T. Huang, HSAUP194.66, living culture SAUCC194.66.

Notes

Diaporthe subclavata was originally described from the leaf with citrus scab of Citrus unshiu in Fujian Province, China (Huang et al. 2015). In the present study, isolated strain SAUCC194.66 from symptomatic leaves of Pometia pinnata was congruent with D. subclavata based on morphology and DNA sequences data (Fig. 1). We therefore present a description and illustration of D. subclavata as a known species for this clade, found on new host.

Diaporthe tectonendophytica M. Doilom, A. J. Dissanayake & K.D. Hyde, Fung. Div., 82: 163, 2016

Figure 11

Description

Asexual morph: Conidiomata pycnidial, aggregated, brownish to black, erumpent, subglobose, exuding white creamy conidial droplets from central ostioles after being kept for 30 days in light at 25 °C. Conidiophores 17.4–35.0 × 2.2–3.5 μm, hyaline, branched, subcylindrical, septate, straight or slightly curved, guttulate. Conidiogenous cells 11.3–15.0 × 1.7–2.5 μm (mean = 12.3 × 2.1 μm, n = 20), cylindric-clavate, hyaline, straight to slightly sinuous, tapering towards the apex. Beta conidia 25.0–31.8 × 0.9–1.8 μm (mean = 28.2 × 1.2 μm, n = 20), filiform, hyaline, guttulate, aseptate, hooked and mostly curved through 90–180°, swollen in the middle. Alpha conidia and Gamma conidia not observed.

Figure 11. 

Diaporthe tectonendophytica (SAUCC194.11) a leaf of host plant b, c surface (b) and reverse (c) side of colony after incubation for 15 days on PDA d conidiomata on PDA e, f conidiophores and conidiogenous cells g, h beta conidia. Scale bars: 10 μm (e–h).

Culture characteristics

Pure culture was isolated by subbing hyphal tips growing from surface sterilized diseased material. Colonies on PDA cover the Petri dish diameter after incubation for 15 days in dark conditions at 25 °C, aerial mycelium abundant, white to grayish on surface side, pale yellow on reverse with concentric zonation. Pycnidia are formed on 15th day or later.

Specimens examined

China, Yunnan Province: Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, 19 April 2019, S.T. Huang. On diseased leaves of Elaeagnus conferta HSAUP194.11, living culture SAUCC194.11; on diseased leaves of Pometia pinnata HSAUP194.63, living culture SAUCC194.63.

Notes

Diaporthe tectonendophytica was originally described from the asymptomatic branches of Tectona grandis in Thailand (Doilom et al. 2017). In the present study, two strains (SAUCC194.11 and SAUCC194.63) from symptomatic leaves of Elaeagnus conferta and Pometia pinnata were congruent with D. tectonendophytica based on morphology and DNA sequences data (Fig. 1). We therefore describe D. tectonendophytica as a known species for this clade.

Discussion

In the current study, 87 reference sequences (including an outgroup taxon) were selected based on BLAST searches of NCBIs GenBank nucleotide database and were included in the phylogenetic analyses (Table 1). Phylogenetic analyses based on five combined loci (ITS, TUB, TEF, CAL and HIS), as well as morphological characters of the non-sexual morph obtained in culture, contributed to knowledge of the diversity of Diaporthe species in Yunnan Province. Based on a large set of freshly collected specimens from Yunnan province, China, 20 strains of Diaporthe species were isolated from 12 host genera (Table 1). As a result, eight new species are proposed: Diaporthe camelliae-sinensis, D. grandiflori, D. heliconiae, D. heterostemmatis, D. litchii, D. lutescens, D. melastomatis, D. pungensis and two previously described species were described and illustrated, D. subclavata and D. tectonendophytica.

Previously, species identification of Diaporthe was largely referred to the assumption of host-specificity, leading to the proliferation of names (Gomes et al. 2013). However, based on a polyphasic approach and known morphology, more than one species of Diaporthe can colonize a single host, while one species can be associated with different hosts (Gomes et al. 2013; Gao et al. 2017; Guarnaccia and Crous 2017; Guarnaccia et al. 2018; Guo et al. 2020). Our study can well support this phenomenon. On the one hand, Diaporthe grandiflori (SAUCC194.84) and D. heterostemmatis (SAUCC194.85) were collected from Heterostemma grandiflorum; D. camelliae-sinensis (SAUCC194.92), D. heterostemmatis (SAUCC194.102), D. melastomatis (SAUCC194.88), and D. pungensis (SAUCC194.89) and were isolated from Camellia sinensis; D. litchii (SAUCC194.12) and D. tectonendophytica (SAUCC194.11) were known on Elaeagnus conferta. On the other hand, the species of D. camelliae-sinensis collected from three hosts (Camellia sinensis, Castanea mollissima, Machilus pingii) D. melastomatis sampled from three hosts (Camellia sinensis, Melastoma malabathricum, Millettia reticulata) and D. litchii sampled from two hosts (Elaeagnus conferta, Litchi chinensis). These studies revealed a high diversity of Diaporthe species from different hosts. The descriptions and molecular data 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. 31770016, 31750001, and 31900014) and the China Postdoctoral Science Foundation (no. 2018M632699).

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