Research Article
Print
Research Article
Two new species of Diaporthe (Diaporthaceae, Diaporthales) associated with Camellia oleifera leaf spot disease in Hainan Province, China
expand article infoHong Y. Liu, Dun Luo§, Han L. Huang§, Qin Yang
‡ Central South University of Forestry and Technology, Changsha, China
§ Guangxi State-owned Bobai Forest Farm, Yulin, China
Open Access

Abstract

Tea-oil tree (Camellia oleifera Abel.) is an important edible oil woody plant with a planting area over 3,800,000 hectares in southern China. Species of Diaporthe inhabit a wide range of plant hosts as plant pathogens, endophytes and saprobes. Here, we conducted an extensive field survey in Hainan Province to identify and characterise Diaporthe species associated with tea-oil leaf spots. As a result, eight isolates of Diaporthe were obtained from symptomatic C. oleifera leaves. These isolates were studied, based on morphological and phylogenetic analyses of partial ITS, cal, his3, tef1 and tub2 gene regions. Two new Diaporthe species (D. hainanensis and D. pseudofoliicola) were proposed and described herein.

Key words

DNA phylogeny, systematics, taxonomy, tea-oil tree, two new taxa

Introduction

Tea-oil tree, Camellia oleifera Abel., is a unique woody edible oil species in China, mainly distributed in the Qinling-Huaihe River area. It has a long history of cultivation and utilisation for more than 2300 years since ancient China (Zhuang 2008). Camellia oil, obtained from C. oleifera seeds, is rich in unsaturated fatty acids and unique flavours and has become a rising high-quality edible vegetable oil in China. The edible of tea-oil is also conducive to preventing cardiovascular sclerosis, anti-tumour, lowering blood lipid, protecting liver and enhancing human immunity (Wang et al. 2007). According to the Three-year Action Plan for Accelerating the Development of oil tea Industry, Hainan Province is listed as a key development area of oil tea and the total area of oil tea planting in the Province is planned to reach 16,667 hm2 by 2025. The development of C. oleifera industry is of great significance for the economic development of Hainan Province and the poverty alleviation of local farmers.

The expanding cultivation of C. oleifera over the last several decades has attracted increasing attention from plant pathologists to infectious diseases on this crop. Therein, diseases caused by Diaporthe species have become the emerging Camellia leaf diseases in southern China (Zhou and Hou 2019; Yang et al. 2021). During July and August of 2022, new leaf spots were detected on tea-oil tree with irregular, brownish-grey lesions, often associated with leaf margins. Infected leaves cultured on medium had dark pycnidia producing ellipsoid guttulate conidia, similar to that of Diaporthe species (Yang et al. 2021). The asexual morph is characterised by ostiolate conidiomata, with cylindrical phialides producing three types (alpha, beta and gamma conidia) of hyaline, aseptate conidia (Udayanga et al. 2011; Gomes et al. 2013).

Species identification criteria in Diaporthe has mainly relied on host association, morphology and culture characteristics (Mostert et al. 2001; Santos and Phillips 2009; Udayanga et al. 2011), which resulted in the description of over 200 species. 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; Udayanga et al. 2011). During the past decade, a polyphasic approach, based on multi-locus DNA data, morphological, phytopathological and phylogenetical analyses, has been employed for species boundaries in the genus Diaporthe (Huang et al. 2015; Gao et al. 2016, 2017; Guarnaccia and Crous 2017, 2018; Guarnaccia et al. 2018; Yang et al. 2018, 2020, 2021; Cao et al. 2022; Bai et al. 2023; Zhu et al. 2023).

The classification of Diaporthe has been on-going; however, little is known about species able to infect C. oleifera. Thus, the objective of the present study was to identify the prevalence of Diaporthe spp. associated with tea-oil tree leaf spot in the major plantations in Hainan Province, based on morphological and phylogenetic features.

Materials and methods

Fungal isolation

Leaves of C. oleifera with typical symptoms of leaf spots were collected from the main tea-oil camellia production fields in Hainan Province. Small sections (3 × 3 mm) were cut from the margins of infected tissues and surface-sterilised in 75% ethanol for 30 s, then sterilised in 5% sodium hypochlorite for 1 min, followed by three rinses with sterilised water and finally dried on sterilised filter paper (Yang et al. 2021). The sections were then plated on to PDA plates and incubated at 25 °C. Fungal growth was examined daily for up to 7 d. Isolates were then transferred aseptically to fresh PDA and purified by single-spore culturing (Fan et al. 2015). All fungal isolates were placed on PDA slants and stored at 4 °C. Specimens and axenic cultures are maintained in the Central South University of Forestry and Technology (CSUFT) in Changsha, Hunan Province.

Morphological and cultural characterisation

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 after 10 d. Colony colours were rated according to Rayner (1970). Cultures were examined periodically for the development of ascomata and conidiomata. The morphological characteristics were examined by mounting fungal structures in clear lactic acid and 30 measurements at 1000× magnification were determined for each isolate using a Leica compound microscope (DM 2500) with interference contrast (DIC) optics. Descriptions, nomenclature and illustrations of taxonomic novelties are deposited in MycoBank (Crous et al. 2004a).

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 quality was measured using the NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA), following the user manual (Desjardins et al. 2009). PCR amplifications were performed in a 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 primers CYLH4F (Crous et al. 2004b) 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 set T1 (O’Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995) was 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 undertaken using the same primer pairs and conditions as in Yang et al. (2018). PCR amplification products were assayed via electrophoresis in 2% agarose gels. DNA sequencing was performed using an ABI PRISM 3730XL DNA Analyzer with a BigDye Terminater Kit v.3.1 (Invitrogen, 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, 2021; Cao et al. 2022). 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 and Huelsenbeck 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. The nucleotide sequence data of the new taxa were deposited in GenBank (Table 1). The multilocus sequence alignments were deposited in TreeBASE (www.treebase.org) as accession S30780.

Table 1.

Isolates and GenBank accession numbers used in the phylogenetic analyses of Diaporthe.

Species Isolate GenBank accession numbers
ITS cal his3 tef1 tub2
Diaporthe acaciigena CBS 129521 KC343005 KC343247 KC343489 KC343731 KC343973
Diaporthe acericola MFLUCC 17-0956 KY964224 KY964137 NA KY964180 KY964074
Diaporthe acerigena CFCC 52554 MH121489 MH121413 MH121449 MH121531 NA
Diaporthe acuta PSCG 047 MK626957 MK691125 MK726161 MK654802 MK691225
Diaporthe acutispora LC6161 KX986764 KX999274 KX999235 KX999155 KX999195
Diaporthe aestuarium BRIP 59930a OM918686 NA NA OM960595 OM960613
Diaporthe alangii CFCC 52556 MH121491 MH121415 MH121451 MH121533 MH121573
Diaporthe albosinensis CFCC 53066 MK432659 MK442979 MK443004 MK578133 MK578059
Diaporthe alleghaniensis CBS 495.72 KC343007 KC343249 KC343491 KC343733 KC343975
Diaporthe ambigua CBS 114015 KC343010 KC343252 KC343494 KC343736 KC343978
Diaporthe ampelina STE-U 2660 AF230751 AY745026 NA AY745056 JX275452
Diaporthe amygdali CBS 126679 MH864208 KC343264 KC343506 KC343748 KC343990
Diaporthe amygdali syn. D. chongqingensis PSCG 435 MK626916 MK691209 MK726257 MK654866 MK691321
Diaporthe amygdali syn. D. fusicola CGMCC 3.17087 KF576281 KF576233 NA KF576256 KF576305
Diaporthe amygdali syn. D. garethjonesii MFLUCC 12-0542a KT459423 KT459470 NA KT459457 KT459441
Diaporthe amygdali syn. D. kadsurae CFCC 52586 MH121521 MH121439 MH121479 MH121563 MH121600
Diaporthe amygdali syn. D. mediterranea SAUCC194.111 MT822639 MT855718 MT855606 MT855836 MT855951
Diaporthe amygdali syn. D. ovoicicola CGMCC 3.17093 KF576265 KF576223 NA KF576240 KF576289
Diaporthe amygdali syn. D. sterilis CBS 136969 KJ160579 KJ160548 MF418350 KJ160611 KJ160528
Diaporthe amygdali syn. D. ternstroemiae CGMCC 3.15183 KC153098 NA NA KC153089 NA
Diaporthe anacardii CBS 720.97 KC343024 KC343266 KC343508 KC343750 KC343992
Diaporthe angelicae CBS 111592 KC343027 KC343269 KC343511 KC343753 KC343995
Diaporthe annellsiae BRIP 59731a OM918687 NA NA OM960596 OM960614
Diaporthe apiculata CFCC 53068 MK432651 MK442973 MK442998 MK578127 MK578054
Diaporthe aquatica IFRDCC 3051 JQ797437 NA NA NA NA
Diaporthe arctii DP0482 KJ590736 KJ612133 KJ659218 KJ590776 KJ610891
Diaporthe arecae CBS 161.64 KC343032 KC343274 KC343516 KC343758 KC344000
Diaporthe arengae CBS 114979 KC343034 KC343276 KC343518 KC343760 KC344002
Diaporthe aseana MFLUCC 12-0299a KT459414 KT459464 NA KT459448 KT459432
Diaporthe asheicola CBS 136967 KJ160562 KJ160542 NA KJ160594 KJ160518
Diaporthe aspalathi CBS 117169 KC343036 KC343278 KC343520 KC343762 KC344004
Diaporthe australafricana CBS 111886 KC343038 KC343280 KC343522 KC343764 KC344006
Diaporthe australiana CBS 146457 MN708222 NA NA MN696522 MN696530
Diaporthe australpacifica BRIP 60163d OM918688 NA NA OM960597 OM960615
Diaporthe baccae CBS 136972 KJ160565 MG281695 MF418264 KJ160597 MF418509
Diaporthe batatas CBS 122.21 KC343040 KC343282 KC343524 KC343766 KC344008
Diaporthe bauhiniae CFCC 53071 MK432648 MK442970 MK442995 MK578124 MK578051
Diaporthe beasleyi BRIP 59326a OM918689 NA NA OM960598 OM960616
Diaporthe beilharziae BRIP 54792 JX862529 NA NA JX862535 KF170921
Diaporthe benedicti SBen914 KM669929 KM669862 NA KM669785 NA
Diaporthe betulae CFCC 50469 KT732950 KT732997 KT732999 KT733016 KT733020
Diaporthe betulicola CFCC 51128 KX024653 KX024659 KX024661 KX024655 KX024657
Diaporthe betulina CFCC 52560 MH121495 MH121419 MH121455 MH121537 MH121577
Diaporthe biconispora ZJUD62 KJ490597 NA KJ490539 KJ490476 KJ490418
Diaporthe biguttulata ZJUD47 KJ490582 NA KJ490524 KJ490461 KJ490403
CFCC 52584 MH121519 MH121437 MH121477 MH121561 MH121598
Diaporthe bohemiae CBS 143347 MG281015 MG281710 MG281361 MG281536 MG281188
Diaporthe bounty BRIP 59361a OM918690 NA NA OM960599 OM960617
Diaporthe brasiliensis CBS 133183 KC343042 KC343284 KC343526 KC343768 KC344010
Diaporthe breyniae CBS 148910 ON400846 ON409189 ON409187 ON409188 ON409186
Diaporthe brumptoniae BRIP 59403a OM918702 NA NA OM960611 OM960629
Diaporthe caatingaensis URM7486 KY085927 KY115597 KY115605 KY115603 KY115600
Diaporthe camelliae-sinensis SAUCC194.92 MT822620 MT855699 MT855588 MT855932 MT855817
Diaporthe camelliae-oleiferae HNZZ027 MZ509555 MZ504685 MZ504696 MZ504707 MZ504718
Diaporthe canthii CPC 19740 JX069864 KC843174 NA KC843120 KC843230
Diaporthe carriae BRIP 59932a OM918691 NA NA OM960600 OM960618
Diaporthe caryae CFCC 52563 MH121498 MH121422 MH121458 MH121540 MH121580
Diaporthe cassines CPC 21916 KF777155 NA NA KF777244 NA
Diaporthe caulivora CBS 127268 MH864501 KC343287 KC343529 KC343771 KC344013
Diaporthe celticola CFCC 53074 MK573948 MK574587 MK574603 MK574623 MK574643
Diaporthe cercidis CFCC 52565 MH121500 MH121424 MH121460 MH121542 MH121582
Diaporthe chamaeropis CBS 454.81 KC343048 KC343290 KC343532 KC343774 KC344016
Diaporthe charlesworthii BRIP 54884m KJ197288 NA NA KJ197250 KJ197268
Diaporthe chiangmaiensis MFLU 18-1305 OK393702 NA NA OL439482 OK490918
Diaporthe chrysalidocarpi SAUCC194.35 MT822563 MT855646 MT855532 MT855760 MT855876
Diaporthe cichorii MFLUCC 17-1023 KY964220 KY964133 NA KY964176 KY964104
Diaporthe cinnamomi CFCC 52569 MH121504 NA MH121464 MH121546 MH121586
Diaporthe cissampeli CPC 27302 KX228273 NA KX228366 NA KX228384
Diaporthe citri AR3405 KC843311 KC843157 KJ420881 KC843071 KC843187
Diaporthe chensiensis CFCC 52567 MH121502 MH121426 MH121462 MH121544 MH121584
Diaporthe citriasiana CGMCC 3.15224 JQ954645 KC357491 KJ490515 JQ954663 KC357459
Diaporthe citrichinensis CGMCC 3.15225 JQ954648 KC357494 KJ420880 JQ954666 KJ490396
Diaporthe collariana MFLU 17-2770 MG806115 MG783042 NA MG783040 MG783041
Diaporthe compactum LC3083 KP267854 NA KP293508 KP267928 KP293434
Diaporthe conica CFCC 52571 MH121506 MH121428 MH121466 MH121548 MH121588
Diaporthe convolvuli CBS 124654 KC343054 KC343296 KC343538 KC343780 KC344022
Diaporthe coryli CFCC 53083 MK432661 MK442981 MK443006 MK578135 MK578061
Diaporthe crotalariae CBS 162.33 MH855395 JX197439 KC343540 GQ250307 KC344024
Diaporthe crousii CAA 823 MK792311 MK883835 MK871450 MK828081 MK837932
Diaporthe cucurbitae DAOM 42078 KM453210 NA KM453212 KM453211 KP118848
Diaporthe cuppatea CBS 117499 MH863021 KC343299 KC343541 KC343783 KC344025
Diaporthe cynaroidis CBS 122676 KC343058 KC343300 KC343542 KC343784 KC344026
Diaporthe cytosporella FAU461 KC843307 KC843141 MF418283 KC843116 KC843221
Diaporthe diospyricola CPC 21169 KF777156 NA NA NA NA
Diaporthe discoidispora ZJUD89 KJ490624 NA KJ490566 KJ490503 KJ490445
Diaporthe dorycnii MFLUCC 17-1015 KY964215 NA NA KY964171 KY964099
Diaporthe drenthii CBS 146453 MN708229 NA NA MN696526 MN696537
Diaporthe durionigena VTCC 930005 MN453530 NA NA MT276157 MT276159
Diaporthe elaeagni-glabrae LC4802 KX986779 KX999281 KX999251 KX999171 KX999212
Diaporthe endophytica CBS 133811 KC343065 KC343307 KC343549 KC343791 KC344033
Diaporthe eres AR5193 KJ210529 KJ434999 KJ420850 KJ210550 KJ420799
Diaporthe etinsideae BRIP 64096a OM918692 NA NA OM960601 OM960619
Diaporthe eucalyptorum CBS 132525 MH305525 NA NA NA NA
Diaporthe foeniculacea CBS 111553 KC343101 KC343343 KC343585 KC343827 KC344069
Diaporthe fraxini-angustifoliae BRIP 54781 JX862528 NA NA JX862534 KF170920
Diaporthe fraxinicola CFCC 52582 MH121517 MH121435 NA MH121559 NA
Diaporthe fructicola MAFF 246408 LC342734 LC342738 LC342737 LC342735 LC342736
Diaporthe fulvicolor PSCG 051 MK626859 MK691132 MK726163 MK654806 MK691236
Diaporthe ganjae CBS 180.91 KC343112 KC343354 KC343596 KC343838 KC344080
Diaporthe ganzhouensis CFCC 53087 MK432665 MK442985 MK443010 MK578139 MK578065
Diaporthe goulteri BRIP 55657a KJ197290 NA NA KJ197252 KJ197270
Diaporthe gossiae BRIP 59730a OM918693 NA NA OM960602 OM960620
Diaporthe grandiflori SAUCC194.84 MT822612 MT855691 MT855580 MT855809 MT855924
Diaporthe griceae BRIP 67014a OM918694 NA NA OM960603 OM960621
Diaporthe guangxiensis JZB320087 MK335765 MK736720 NA MK500161 MK523560
Diaporthe gulyae BRIP 54025 JF431299 NA NA JN645803 KJ197271
Diaporthe guttulata CGMCC 3.20100 MT385950 MW022470 MW022491 MT424685 MT424705
Diaporthe hainanenesis HNCM049 OR647684 NA OR671936 OR671944 OR671952
HNCM050 OR647685 NA OR671937 OR671945 OR671953
HNCM051 OR647686 NA OR671938 OR671946 OR671954
HNCM052 OR647687 NA OR671939 OR671947 OR671955
Diaporthe helianthi CBS 592.81 KC343115 KC343357 KC343599 KC343841 KC344083
Diaporthe heliconiae SAUCC194.77 MT822605 MT855684 MT855573 MT855802 MT855917
Diaporthe heterophyllae CPC 26215 MG600222 MG600218 MG600220 MG600224 MG600226
Diaporthe heterostemmatis SAUCC194.85 MT822613 MT855692 MT855581 MT855810 MT855925
Diaporthe hickoriae CBS 145.26 KC343118 KC343360 KC343620 KC343844 KC344086
Diaporthe hispaniae CBS 143351 MG281123 MG281820 MG281471 MG281644 MG281296
Diaporthe hongkongensis CBS 115448 KC343119 KC343361 KC343603 KC343845 KC344087
Diaporthe howardiae BRIP 59697a OM918695 NA NA OM960604 OM960622
Diaporthe hubeiensis JZB320123 MK335809 MK500235 NA MK523570 MK500148
Diaporthe hunanensis HNZZ023 MZ509550 MZ504680 MZ504691 MZ504702 MZ504713
Diaporthe incompleta LC6754 KX986794 KX999289 KX999265 KX999186 KX999226
Diaporthe inconspicua CBS 133813 KC343123 KC343365 KC343607 KC343849 KC344091
Diaporthe infecunda CBS 133812 KC343126 KC343368 KC343610 KC343852 KC344094
Diaporthe irregularis CGMCC 3.20092 MT385951 MT424721 NA MT424686 MT424706
Diaporthe isoberliniae CPC 22549 KJ869190 NA NA NA KJ869245
Diaporthe juglandicola CFCC 51134 KU985101 KX024616 KX024622 KX024628 KX024634
Diaporthe kochmanii BRIP 54033 JF431295 NA NA JN645809 NA
Diaporthe kongii BRIP 54031 JF431301 NA NA JN645797 KJ197272
Diaporthe krabiensis MFLUCC 17-2481 MN047100 NA NA MN433215 MN431495
Diaporthe lenispora CGMCC 3.20101 MT385952 MW022472 MW022493 MT424687 MT424707
Diaporthe litchicola BRIP 54900 JX862533 NA NA JX862539 KF170925
Diaporthe litchii SAUCC194.22 MT822550 MT855635 MT855519 MT855747 MT855863
Diaporthe lithocarpi CGMCC 3.15175 KC135104 KF576235 NA KC153095 KF576311
Diaporthe longicolla FAU599 KJ590728 KJ612124 KJ659188 KJ590767 KJ610883
Diaporthe longispora CBS 194.36 MH855769 KC343377 KC343619 KC343861 KC344103
Diaporthe lovelaceae BRIP 60163a OM918696 NA NA OM960605 OM960623
Diaporthe lusitanicae CBS 123212 MH863279 KC343378 KC343620 KC343862 KC344104
Diaporthe lutescens SAUCC194.36 MT822564 MT855647 MT855533 MT855761 MT855877
Diaporthe macadamiae CBS 146455 MN708230 NA NA MN696528 MN696539
Diaporthe macintoshii BRIP 55064a KJ197289 NA NA KJ197251 KJ197269
Diaporthe malorum CAA 734 KY435638 KY435658 KY435648 KY435627 KY435668
Diaporthe marina MFLU 17-2622 MN047102 NA NA NA NA
Diaporthe masirevicii BRIP 54256 KJ197276 NA NA KJ197238 KJ197256
Diaporthe mayteni CBS 133185 KC343139 KC343381 KC343623 KC343865 KC344107
Diaporthe maytenicola CPC 21896 KF777157 NA NA NA KF777250
Diaporthe mclennaniae BRIP 60072a OM918697 NA NA OM960606 OM960624
Diaporthe melastomatis SAUCC194.55 MT822583 MT855664 MT855551 MT855780 MT855896
Diaporthe melonis CBS 435.87 KC343141 KC343383 KC343625 KC343867 KC344109
Diaporthe middletonii BRIP 54884e KJ197286 NA NA KJ197248 KJ197266
Diaporthe minima CGMCC 3.20097 MT385953 MT424722 MW022496 MT424688 MT424708
Diaporthe minusculata CGMCC 3.20098 MT385957 MW022475 MW022499 MT424692 MT424712
Diaporthe miriciae BRIP 54736j KJ197282 NA NA KJ197244 KJ197262
Diaporthe monetii MF-Ha18-048 MW008493 MZ671938 MZ671964 MW008515 MW008504
Diaporthe moriniae BRIP 60190a OM918698 NA NA OM960607 OM960625
Diaporthe multigutullata CFCC 53095 MK432645 MK442967 MK442992 MK578121 MK578048
Diaporthe musigena CBS 129519 KC343143 KC343385 KC343267 KC343869 KC344111
Diaporthe myracrodruonis URM 7972 MK205289 MK205290 NA MK213408 MK205291
Diaporthe neoarctii CBS 109490 KC343145 KC343387 KC343629 KC343871 KC344113
Diaporthe neoraonikayaporum MFLUCC 14-1136 KU712449 KU749356 NA KU749369 KU743988
Diaporthe norfolkensis BRIP 59718a OM918699 NA NA OM960608 OM960626
Diaporthe nothofagi BRIP 54801 JX862530 NA NA JX862536 KF170922
Diaporthe novem CBS 127269 KC343155 KC343397 KC343639 KC343881 KC344123
Diaporthe ocoteae CPC 26217 KX228293 NA NA NA KX228388
Diaporthe oraccinii LC3166 KP267863 NA KP293517 KP267937 KP293443
Diaporthe ovalispora ZJUD93 KJ490628 NA KJ490570 KJ490507 KJ490449
Diaporthe oxe CBS 133186 KC343164 KC343406 KC343648 KC343890 KC344132
Diaporthe padina CFCC 52590 MH121525 MH121443 MH121483 MH121567 MH121604
Diaporthe pandanicola MFLUCC 17-0607 MG646974 NA NA NA MG646930
Diaporthe paranensis CBS 133184 KC343171 KC343413 KC343655 KC343897 KC344139
Diaporthe parapterocarpi CPC 22729 KJ869138 NA NA NA KJ869248
Diaporthe parvae PSCG 035 MK626920 MK691169 MK726211 MK654859 MK691249
Diaporthe pascoei BRIP 54847 JX862538 NA NA JX862538 KF170924
Diaporthe passiflorae CPC 19183 JX069860 KY435644 KY435654 KY435623 KY435674
Diaporthe passifloricola CPC 27480 KX228292 NA KX228367 NA KX228387
Diaporthe penetriteum LC3215 KP267879 NA KP293532 KP267953 NA
Diaporthe perjuncta CBS 109745 KC343172 KC343414 KC343656 KC343898 KC344140
Diaporthe perseae CBS 151.73 KC343173 KC343415 KC343657 KC343899 KC343141
Diaporthe pescicola MFLUCC 16-0105 KU557555 KU557603 NA KU557623 KU557579
Diaporthe phaseolorum AR4203 KJ590738 KJ612135 KJ659220 KJ590739 KJ610893
Diaporthe platzii BRIP 60353a OM918700 NA NA OM960609 OM960627
Diaporthe phillipsii CAA 817 MK792305 MK883831 MK871445 MK828076 MN000351
Diaporthe podocarpi-macrophylli LC6155 KX986774 KX999278 KX999246 KX999167 KX999207
Diaporthe pometiae SAUCC194.72 MT822600 MT855679 MT855568 MT855797 MT855912
Diaporthe pseudoalnea CFCC 54190 MZ727037 MZ753468 MZ781302 MZ816343 MZ753487
Diaporthe pseudofoliicola HNCM045 OR647680 NA OR671932 OR671940 OR671948
HNCM046 OR647681 NA OR671933 OR671941 OR671949
HNCM047 OR647682 NA OR671934 OR671942 OR671950
HNCM048 OR647683 NA OR671935 OR671943 OR671951
Diaporthe pseudomangiferae CBS 101339 KC343181 KC343423 KC343665 KC343907 KC344149
Diaporthe pseudophoenicicola CBS 176.77 KC343183 KC343425 KC343667 KC343909 KC344151
Diaporthe psoraleae CPC 21634 KF777158 NA NA KF777245 KF777251
Diaporthe psoraleae-pinnatae CPC 21638 KF777159 NA NA NA KF777252
Diaporthe pterocarpi CPC 22729 JQ619899 JX197451 NA JX275416 JX275460
Diaporthe pterocarpicola MFLUCC 10-0580a JQ619887 JX197433 NA JX275403 JX275441
Diaporthe pungensis SAUCC194.112 MT822640 MT855719 MT855607 MT855837 MT855952
Diaporthe pyracanthae CAA483 KY435635 KY435656 KY435645 KY435625 KY435666
Diaporthe racemosae CPC 26646 MG600223 MG600219 MG600221 MG600225 MG600227
Diaporthe raonikayaporum CBS 133182 KC343188 KC343430 KC343672 KC343914 KC344156
Diaporthe ravennica MFLUCC 16-0997 NA NA NA MT394670 NA
Diaporthe rhodomyrti CFCC 53101 MK432643 MK442965 MK442990 MK578119 MK578046
Diaporthe rhusicola CPC 18191 JF951146 KC843124 NA KC843100 KC843205
Diaporthe rosae MFLUCC 17-2658 MG828894 MG829273 NA NA MG843878
Diaporthe rosiphthora COAD 2914 MT311197 MT313691 NA MT313693 NA
Diaporthe rossmaniae CAA 762 MK792290 MK883822 MK871432 MK828063 MK837914
Diaporthe rostrata CFCC 50062 KP208847 KP208849 KP208851 KP208853 KP208855
Diaporthe rudis AR3422 KC843331 KC843146 NA KC843090 KC843177
Diaporthe saccarata CBS 116311 KC343190 KC343432 KC343674 KC343916 KC344158
Diaporthe sackstonii BRIP 54669b KJ197287 NA NA KJ197249 KJ197267
Diaporthe salicicola BRIP 54825 JX862531 NA NA JX862537 KF170923
Diaporthe sambucusii CFCC 51986 KY852495 KY852499 KY852503 KY852507 KY852511
Diaporthe schimae CFCC 53103 MK442640 MK442962 MK442987 MK578116 MK578043
Diaporthe schini CBS 133181 KC343191 KC343433 KC343675 KC343917 KC344159
Diaporthe schisandrae CFCC 51988 KY852497 KY852501 KY852505 KY852509 KY852513
Diaporthe schoeni MFLU 15-1279 KY964226 KY964139 NA KY964182 KY964109
Diaporthe sclerotioides CBS 296.67 MH858974 KC343435 KC343677 KC343919 KC344161
Diaporthe searlei BRIP 66528 MN708231 NA NA NA MN696540
Diaporthe sennae CFCC 51636 KY203724 KY228875 NA KY228885 KY228891
Diaporthe sennicola CFCC 51634 KY203722 KY228873 KY228879 KY228883 KY228889
Diaporthe serafiniae BRIP 55665a KJ197274 NA NA KJ197236 KJ197254
Diaporthe shaanxiensis CFCC 53106 MK432654 MK442976 MK443001 MK578130 NA
Diaporthe shawiae BRIP 64534a OM918701 NA NA OM960610 OM960628
Diaporthe siamensis MFLUCC 10-0573a JQ619879 JX197423 NA JX275393 JX275429
Diaporthe silvicola CFCC 54191 MZ727041 MZ753472 MZ753481 MZ816347 MZ753491
Diaporthe sojae FAU635 KJ590719 KJ612116 KJ659208 KJ590762 KJ610875
Diaporthe spinosa PSCG 383 MK626849 MK691129 MK726156 MK654811 MK691234
Diaporthe stictica CBS 370.54 KC343212 KC343454 KC343696 KC343938 KC344180
Diaporthe subclavata ZJUD95 KJ490630 NA KJ490572 KJ490509 KJ490451
Diaporthe subcylindrospora KUMCC 17-0151 MG746629 NA NA MG746630 MG746631
Diaporthe subellipicola KUMCC 17-0153 MG746632 NA NA MG746633 MG746634
Diaporthe subordinaria CBS 464.90 KC343214 KC343456 KC343698 KC343940 KC344182
Diaporthe taoicola MFLUCC 16-0117 KU557567 NA NA KU557635 KU557591
Diaporthe tectonae MFLUCC 12-0777 KU712430 KU749345 NA KU749359 KU743977
Diaporthe tectonendophytica MFLUCC 13-0471 KU712439 KU749354 NA KU749367 KU743986
Diaporthe tectonigena MFLUCC 12-0767 KX986782 KX999284 KX999254 KX999174 KX999214
Diaporthe terebinthifolii CBS 133180 KC343216 KC343458 KC343700 KC343942 KC344184
Diaporthe thunbergii MFLUCC 10-0576a JQ619893 JX197440 NA JX275409 JX275449
Diaporthe thunbergiicola MFLUCC 12-0033 KP715097 NA NA KP715098 NA
Diaporthe tibetensis CFCC 51999 MF279843 MF279888 MF279828 MF279858 MF279873
Diaporthe tulliensis BRIP 62248a KR936130 NA NA KR936133 KR936132
Diaporthe trevorrowii BRIP 70737a OM918703 NA NA OM960612 OM960630
Diaporthe ueckerae FAU656 KJ590726 KJ612122 KJ659215 KJ590747 KJ610881
Diaporthe ukurunduensis CFCC 52592 MH121527 MH121445 MH121485 MH121569 NA
Diaporthe undulata LC6624 KX986798 NA KX999269 KX999190 KX999230
Diaporthe unshiuensis CFCC 52594 MH121529 MH121447 MH121487 MH121571 MH121606
CFCC 52595 MH121530 MH121448 MH121488 MH121572 MH121607
Diaporthe vaccinii CBS 160.32 KC343228 KC343470 KC343712 KC343954 KC343196
Diaporthe vangoghii MF-Ha18-045 MW008491 MZ671936 MZ671962 MW008513 MW008502
Diaporthe vangueriae CBS 137985 KJ869137 NA NA NA KJ869247
Diaporthe vawdreyi BRIP 57887a KR936126 NA NA KR936129 KR936128
Diaporthe velutina LC4421 KX986790 NA KX999261 KX999182 KX999223
Diaporthe verniciicola CFCC 53109 MK573944 MK574583 MK574599 MK574619 MK574639
Diaporthe viniferae JZB320071 MK341551 MK500119 NA MK500107 MK500112
Diaporthe virgiliae CMW 40748 KP247556 NA NA NA KP247575
Diaporthe xishuangbanica LC6707 KX986783 NA KX999255 KX999175 KX999216
Diaporthe xunwuensis CFCC 53085 MK432663 MK442983 MK443008 MK578137 MK578063
Diaporthe yunnanensis LC6168 KX986796 KX999290 KX999267 KX999188 KX999228
Diaporthe zaobaisu PSCG 031 MK626922 NA MK726207 MK654855 MK691245
Diaporthella corylina CBS 121124 KC343004 KC343246 KC343488 KC343730 KC343972

Results

Phylogenetic analyses

The five-gene sequence dataset (ITS, cal, his3, tef1 and tub2) was analysed to infer the interspecific relationships within Diaporthe. The dataset consisted of 259 sequences including the outgroup taxon, Diaporthella corylina (CBS 121124). A total of 2909 characters including gaps (528 for ITS, 608 for cal, 563 for his3, 646 for tef1 and 564 for tub2) were included in the phylogenetic analysis. The best nucleotide substitution model for ITS, his3 and tub2 was TrN+I+G, while HKY+I+G was selected for both cal and tef1. The topologies resulting from ML and BI analyses of the concatenated dataset were congruent (Fig. 1). According to the phylogenetic tree, D. hainanensis and D. pseudofoliicola are new to science, based on the distinct and well-supported molecular phylogenetic placement with their closest described relatives. Phylogenetically, D. pseudofoliicola clustered together with D. longicolla and D. unshiuensis. Diaporthe hainanensis clustered together with D. cercidis and D. guangxiensis.

Figure 1. 

Phylogram of Diaporthe resulting from a Maximum Likelihood analysis, based on combined ITS, cal, his3, tef1 and tub2. Numbers above the branches indicate ML bootstraps (left, ML BS ≥ 50%) and Bayesian Posterior Probabilities (right, BPP ≥ 0.9). The tree is rooted with Diaporthella corylina. Isolates in the current study are in blue. “-” indicates ML BS < 50% or BI PP < 0.9.

Taxonomy

Diaporthe hainanensis Q. Yang, sp. nov.

MycoBank No: MycoBank No: 848328
Fig. 2

Diagnosis

Distinguished from D. cercidis in narrower alpha conidia; from D. guangxiensis in shorter beta conidia.

Etymology

In reference to the Hainan Province, from where the fungus was first collected.

Description

Asexual morph : Conidiomata on PNA pycnidial, globose or rostrated, black, erumpent in tissue, erumpent at maturity, 450–600 μm diam., often with pale yellowish conidial drops exuding from the ostioles. Conidiophores reduced to conidiogenous cells. Conidiogenous cells (10.5–)14.5–20(–21.5) × 1.4–1.8 μm (n = 30), aseptate, cylindrical, phialidic, straight or slightly curved. Alpha conidia (5.5–)7–8(–8.5) × 2.1–2.9 μm (n = 30), aseptate, hyaline, ellipsoidal, biguttulate. Beta conidia (21.5–)23–25 × 1.1 µm (n = 30), hyaline, aseptate, filiform, sinuous at one end, eguttulate.

Figure 2. 

Diaporthe hainanensis (HNCM049) A culture on PNA B, C conidiomata D conidiogenous cells E alpha conidia F beta conidia. Scale bars: 500 μm (B, C); 10 μm (C, F), 20 μm (E).

Culture characters

Culture incubated on PNA at 25 °C, originally white, fluffy aerial mycelium, becoming pale yellow with age, with visible solitary conidiomata pine needles after 15 days.

Specimens examined

China. Hainan Province: Chengmai County, on leaves of Camellia oleifera, 19°34'10"N, 110°18'09"E, 25 July 2022, Q. Yang (holotype CSUFT055; ex-type living culture: HNCM049; other living cultures: HNCM050, HNCM051 and HNCM052).

Notes

Four isolates representing D. hainanensis cluster in a well-supported clade (ML/BI = 100/1) and appear most closely related to D. cercidis on Cercis chinensis and D. guangxiensis on Macadamia sp. Diaporthe hainanensis can be distinguished from D. cercidis, based on ITS, his3, tef1and tub2 loci (13/458 in ITS, 5/455 in his3, 33/341 in tef1 and 5/401 in tub2); from D. guangxiensis, based on ITS, tef1 and tub2 loci (5/457 in ITS, 2/339 in tef1 and 16/403 in tub2). Morphologically, D. hainanensis differs from D. cercidis in narrower alpha conidia (2.1–2.9 μm vs. 3–3.5 μm) (Yang et al. 2018); from D. guangxiensis in shorter beta conidia (23–25 μm vs. 20–32 μm) (Manawasinghe et al. 2019).

Diaporthe pseudofoliicola Q. Yang, sp. nov.

MycoBank No: MycoBank No: 848327
Fig. 3

Diagnosis

Distinguished from D. longicolla in having smaller alpha conidia; from D. unshiuensis in having narrower conidiophores.

Etymology

The epithet “pseudofoliicola” refers to its habitat similar to Diaporthe foliicola.

Description

Asexual morph : Conidiomata on PDA pycnidial, 190–330 μm in diam., superficial, scattered on PDA, dark brown to black, globose, solitary or clustered in groups of 1–3 pycnidia. Pale yellow conidial drops exuding from ostioles. Conidiophores reduced to conidiogenous cells. Conidiogenous cells (10.5–)12.5–18(–22) × 1.3–1.5 μm (n = 30), phialidic, aseptate, cylindrical, straight, densely aggregated, terminal, slightly tapered towards the apex. Alpha conidia 5–6.5(–7) × 2.3–3.0 μm (n = 30), aseptate, hyaline, ellipsoidal to fusiform, biguttulate, both ends obtuse. Beta conidia (27.5–)30–33(–35.5) × 1.2–1.4 µm (n = 30), hyaline, aseptate, filiform, sinuous at one end, eguttulate.

Figure 3. 

Diaporthe pseudofoliicola (HNCM045) A culture on PDA B, C conidiomata D conidiogenous cells E alpha and beta conidia. Scale bars: 200 μm (B, C), 10 μm (D), 20 μm (E).

Culture characters

Culture incubated on PDA at 25 °C, originally flat with white fluffy aerial mycelium, becoming pale brown due to pigment formation, with yellowish-cream conidial drops exuding from the ostioles after 20 days.

Specimens examined

China. Hainan Province: Chengmai County, on leaves of Camellia oleifera, 110°15'16"E, 19°23'20"N, 25 July 2022, Q. Yang (holotype CSUFT050; ex-type living culture: HNCM045; other living cultures: HNCM046, HNCM047 and HNCM048).

Notes

Four isolates representing D. pseudofoliicola cluster in a well-supported clade (ML/BI = 100/1) and appear most closely related to D. longicolla on Glycine max and D. unshiuensis on Citrus unshiu. Diaporthe pseudofoliicola can be distinguished from D. longicolla, based on ITS, tef1 and tub2 loci (9/462 in ITS, 16/318 in tef1 and 4/444 in tub2); from D. unshiuensis, based on his3 and tef1 loci (51/457 in his3 and 17/318 in tef1). Morphologically, D. pseudofoliicola differs from D. longicolla in having smaller alpha conidia (5–6.5 × 2.3–3.0 μm vs. 6.9–7.2 × 1.6–2.8 μm) (Santos et al. 2011); from D. unshiuensis in having narrower conidiophores (1.3–1.5 μm vs. 1.4–2.6 μm) (Huang et al. 2015).

Discussion

In this study, an important oil-tea tree species, Camellia oleifera was investigated and Camellia leaf disease was found as a common disease in plantations in Hainan Province. Identification of our collections was conducted, based on isolates from symptomatic leaves of C. oleifera using five combined loci (ITS, cal, his3, tef1 and tub2), as well as morphological characters. Two new Diaporthe species were described, i.e. D. hainanensis and D. pseudofoliicola.

According to the USDA Fungal-host interaction database, there are six records of Diaporthe species associated with C. oleifera (https://nt.ars-grin.gov/fungaldatabases; accessed on 18 Sep 2023). These records are related to the following six Diaporthe species: D. eres, D. camelliae-oleiferae, D. hubeiensis, D. hunanensis, D. huangshanensis and D. sojae (Zhou and Hou 2019; Yang et al. 2021). Diaporthe eres, the type species of the genus, was described by Nitschke (1870) on Ulmus sp. collected in Germany, which has a widespread distribution and a broad host range as pathogens, endophytes or saprobes (Udayanga et al. 2014). Diaporthe eres differs from D. pseudofoliicola and D. hainanensis in having wider alpha conidia (3–4 μm in D. eres vs. 2.3–3.0 μm in D. pseudofoliicola vs. 2.1–2.9 μm in D. hainanensis) (Gomes et al. 2013); D. huangshanensis differs from D. pseudofoliicola in having shorter beta conidia (19.5–30 μm vs. 30–33 μm); from D. hainanensis in having wider alpha conidia (2.7–4.5 μm vs. 2.1–2.9 μm) (Zhou and Hou 2019). Yang et al. (2021) recorded four Diaporthe species, D. camelliae-oleiferae, D. hubeiensis, D. hunanensis and D. sojae, which were isolated from Camellia oleifera in Hunan Province and which can be distinguished from D. pseudofoliicola and D. hainanensis, based on DNA sequence data (Fig. 1).

As the species concept of Diaporthe has been greatly improved by using molecular data (Huang et al. 2015; Gao et al. 2016, 2017; Guarnaccia and Crous 2017; Guarnaccia et al. 2018; Yang et al. 2018, 2020, 2021; Manawasinghe et al. 2019; Guo et al. 2020; Jiang et al. 2021; Cao et al. 2022; Bai et al. 2023; Zhu et al. 2023), many new species have been discovered and reported in recent years. In this study, the Diaporthe isolates from C. oleifera were identified, based on sequence analysis and morphological characteristics. Future studies should focus on pathogenicity, epidemiology and fungicide sensitivity of the important plant fungal pathogen to develop effective management of C. oleifera disease and on the pathogenic molecular mechanism.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This study is financed by the Guilin Scientific Research [2022ZC] No. 27, the introduction of talent research start-up fund project of CSUFT (Project No.: 2019YJ025) and the Changsha Natural Science Foundation project (kq2202284).

Author contributions

Hong Y. Liu conceived and designed the study; Dun Luo ang Han L. Huang conducted the experiments; Qin Yang wrote the manuscript and revised.

Data availability

All of the data that support the findings of this study are available in the main text.

References

  • Bai Y, Lin L, Pan M, Fan XL (2023) Studies of Diaporthe (Diaporthaceae, Diaporthales) species associated with plant cankers in Beijing, China, with three new species described. MycoKeys 98: 59–86. https://doi.org/10.3897/mycokeys.98.104156
  • Crous PW, Gams W, Stalpers JA, Robert V, Stegehuis G (2004a) MycoBank: An online initiative to launch mycology into the 21st century. Studies in Mycology 50: 19–22.
  • Crous PW, Groenewald JZ, Risède JM, Simoneau P, Hywel-Jones NL (2004b) Calonectria species and their Cylindrocladium anamorphs: Species with sphaeropedunculate vesicles. Studies in Mycology 50: 415–430.
  • Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: More models, new heuristics and parallel computing. Nature Methods 9(8): 772. https://doi.org/10.1038/nmeth.2109
  • Desjardins P, Hansen JB, Allen M (2009) Microvolume protein concentration determination using the NanoDrop 2000c spectrophotometer. Journal of Visualized Experiments 33(33): 1–3. https://doi.org/10.3791/1610
  • Diogo E, Santos JM, Phillips AJ (2010) Phylogeny, morphology and pathogenicity of Diaporthe and Phomopsis species on almond in Portugal. Fungal Diversity 44(1): 107–115. https://doi.org/10.1007/s13225-010-0057-x
  • Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus (San Francisco, Calif. ) 12: 13–15.
  • Fan XL, Hyde KD, Udayanga D, Wu XY, Tian CM (2015) Diaporthe rostrata, a novel ascomycete from Juglans mandshurica associated with walnut dieback. Mycological Progress 14(10): 1–8. https://doi.org/10.1007/s11557-015-1104-5
  • Glass NL, Donaldson GC (1995) Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Applied and Environmental Microbiology 61(4): 1323–1330. https://doi.org/10.1128/aem.61.4.1323-1330.1995
  • Gomes RR, Glienke C, Videira SIR, Lombard L, Groenewald JZ, Crous PW (2013) Diaporthe: A genus of endophytic, saprobic and plant pathogenic fungi. Persoonia 31(1): 1–41. https://doi.org/10.3767/003158513X666844
  • Guarnaccia V, Groenewald JZ, Woodhall J, Armengol J, Cinelli T, Eichmeier A, Ezra D, Fontaine F, Gramaje D, Gutierrez-Aguirregabiria A, Kaliterna J, Kiss L, Larignon P, Luque J, Mugnai L, Naor V, Raposo R, Sándor E, Váczy KZ, Crous PW (2018) Diaporthe diversity and pathogenicity revealed from a broad survey of grapevine diseases in Europe. Persoonia 40(1): 135–153. https://doi.org/10.3767/persoonia.2018.40.06
  • Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0. Systematic Biology 59(3): 307–321. https://doi.org/10.1093/sysbio/syq010
  • Guo YS, Crous PW, Bai Q, Fu M, Yang MM, Wang XH, Du YM, Hong N, Xu WX, Wang GP (2020) High diversity of Diaporthe species associated with pear shoot canker in China. Persoonia 45(1): 132–162. https://doi.org/10.3767/persoonia.2020.45.05
  • Hall T (1999) BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98.
  • Huang F, Udayanga D, Wang X, Hou X, Mei X, Fu Y, Hyde KD, Li HY (2015) Endophytic Diaporthe associated with Citrus: A phylogenetic reassessment with seven new species from China. Fungal Biology 119(5): 331–347. https://doi.org/10.1016/j.funbio.2015.02.006
  • Jiang N, Voglmayr H, Piao CG, Li Y (2021) Two new species of Diaporthe (Diaporthaceae, Diaporthales) associated with tree cankers in the Netherlands. MycoKeys 85: 31–56. https://doi.org/10.3897/mycokeys.85.73107
  • Lombard L, Van Leeuwen GCM, Guarnaccia V, Polizzi G, Van Rijswick PC, Karin Rosendahl KC, Gabler J, Crous PW (2014) Diaporthe species associated with Vaccinium, with specific reference to Europe. Phytopathologia Mediterranea 53: 287–299. https://doi.org/10.14601/Phytopathol_Mediterr-14034
  • Manawasinghe IS, Dissanayake AJ, Li X, Liu M, Wanasinghe DN, Xu J, Zhao W, Zhang W, Zhou Y, Hyde KD, Brooks S, Yan J (2019) High genetic diversity and species complexity of Diaporthe associated with grapevine dieback in China. Frontiers in Microbiology 10: 1936. https://doi.org/10.3389/fmicb.2019.01936
  • Mostert L, Crous PW, Kang JC, Phillips AJ (2001) Species of Phomopsis and a Libertella sp. occurring on grapevines with specific reference to South Africa: Morphological, cultural, molecular and pathological characterization. Mycologia 93(1): 146–167. https://doi.org/10.1080/00275514.2001.12061286
  • Nitschke T (1870) Pyrenomycetes Germanici 2: 245. Breslau. Eduard Trewendt, Germany.
  • O’Donnell K, Cigelnik E (1997) Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Molecular Phylogenetics and Evolution 7(1): 103–116. https://doi.org/10.1006/mpev.1996.0376
  • Rambaut A, Drummond A (2010) FigTree v.1.3.1. Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, UK.
  • Rayner RW (1970) A mycological colour chart. Commonwealth Mycological Institute, Kew, UK.
  • Rehner SA, Uecker FA (1994) Nuclear ribosomal internal transcribed spacer phylogeny and host diversity in the coelomycete Phomopsis. Canadian Journal of Botany 72(11): 1666–1674. https://doi.org/10.1139/b94-204
  • Santos JM, Phillips AJL (2009) Resolving the complex of Diaporthe (Phomopsis) species occurring on Foeniculum vulgare in Portugal. Fungal Diversity 34: 111–125.
  • Santos JM, Vrandečić K, Ćosić J, Duvnjak T, Phillips AJL (2011) Resolving the Diaporthe species occurring on soybean in Croatia. Persoonia 27(1): 9–19. https://doi.org/10.3767/003158511X603719
  • Smith H, Wingfeld MJ, Coutinho TA, Crous PW (1996) Sphaeropsis sapinea and Botryosphaeria dothidea endophytic in Pinus spp. and Eucalyptus spp. in South Africa. South African Journal of Botany 62(2): 86–88. https://doi.org/10.1016/S0254-6299(15)30596-2
  • Udayanga D, Castlebury LA, Rossman AY, Chukeatirote E, Hyde KD (2014) Insights into the genus Diaporthe: Phylogenetic species delimitation in the D. eres species complex. Fungal Diversity 67(1): 203–229. https://doi.org/10.1007/s13225-014-0297-2
  • Udayanga D, Liu X, McKenzie EH, Chukeatirote E, Bahkali AH, Hyde KD (2011) The genus Phomopsis: Biology, applications, species concepts and names of common phytopathogens. Fungal Diversity 50(1): 189–225. https://doi.org/10.1007/s13225-011-0126-9
  • Yang Q, Fan XL, Guarnaccia V, Tian CM (2018) High diversity of Diaporthe species associated with dieback diseases in China, with twelve new species described. MycoKeys 39: 97–149. https://doi.org/10.3897/mycokeys.39.26914
  • Yang Q, Tang J, Zhou GY (2021) Characterization of Diaporthe species on Camellia oleifera in Hunan Province, with descriptions of two new species. MycoKeys 84: 15–33. https://doi.org/10.3897/mycokeys.84.71701
  • Zhuang RL (2008) Camellia oleifera. 2nd ed. China Forestry Press, Beijing.
login to comment