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Research Article
Characterization of Diaporthe species on Camellia oleifera in Hunan Province, with descriptions of two new species
expand article infoQin Yang‡, Jie Tang§, Guo Y. Zhou‡
‡ Central South University of Forestry and Technology, Changsha, China
§ Central South University of Forestry and Technology, Cahngsha, China

Corresponding author: Guo Y. Zhou ( zgyingqq@163.com )

Academic editor: Rungtiwa Phookamsak
© 2021 Qin Yang, Jie Tang, Guo Y. Zhou.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: 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
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. At present, relatively little is known about the taxonomy and genetic diversity of Diaporthe on C. oleifera. Here, we conducted an extensive field survey in Hunan Province in China to identify and characterise Diaporthe species associated with tea-oil leaf spots. As a result, eleven isolates of Diaporthe were obtained from symptomatic C. oleifera leaves. These isolates were studied by applying a polyphasic approach including morphological and phylogenetic analyses of partial ITS, cal, his3, tef1 and tub2 gene regions. Two new Diaporthe species (D. camelliae-oleiferae and D. hunanensis) were proposed and described herein, and C. oleifera was revealed to be new host records of D. hubeiensis and D. sojae. This study indicated there is a potential of more undiscovered Diaporthe species from C. oleifera in China.

Keywords

Camellia oleifera, DNA phylogeny, systematics, taxonomy, 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 utilization 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 flavors, 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-tumor, lowering blood lipid, protecting liver and enhancing human immunity (Wang et al. 2007). Hunan Province leads the country in C. oleifera production with the average of 3.3~40,000 hm2 to expand the cultivation area every year (Tan et al. 2018). By the end of 2017, the cultivation area of C. oleifera reached 1.4 million hm2, tea oil 290100 tons, and output value of 35 billion yuan (Tan et al. 2018). Thus, the development of C. oleifera industry is of great significance for the economic development of Hunan Province and the poverty alleviation of local farmers.

Diseases are a major constraint to C. oleifera production. Anthracnose disease caused by Colletotrichum species is one of the foremost diseases in southern China, which can infect leaves and fruits of C. oleifera, causing up to 40% fruit drop and up to 40% camellia seeds loss (Wang et al. 2020). During July and August of 2020, 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. 2020, 2021). Diaporthe species are responsible for diseases on a wide range of plant hosts, including agricultural crops, forest trees and ornamentals, some of which can cause substantial yield losses (Santos et al. 2011; Gomes et al. 2013; Udayanga et al. 2015; Gao et al. 2016; Guarnaccia and Crous 2017, 2018; Yang et al. 2018, 2020, 2021). For instance, D. ampelina, the causal agent of Phomopsis cane and leaf spot, is known as a severe pathogen of grapevines (Hewitt and Pearson 1988), infecting all green tissues and causing yield reductions of up to 30% in temperate regions (Erincik et al. 2001). Diaporthe citri is another well-known pathogen exclusively found on Citrus spp. causing melanose, stem-end rot and gummosis in all the citrus production area except Europe (Mondal et al. 2007; Udayanga et al. 2014a; Guarnaccia and Crous 2017, 2018).

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; Guarnaccia et al. 2018; Yang et al. 2018, 2020, 2021).

The classification of Diaporthe has been ongoing; 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 Hunan 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 Hunan 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. 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. 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).

Morphological and cultural characterization

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 done 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). Sequences were aligned using MAFFT v. 6 (Katoh and Toh 2010) and corrected manually using Bioedit 7.0.9.0 (Hall 1999). The best-fit nucleotide substitution models for each gene were selected using jModelTest v. 2.1.7 (Darriba et al. 2012) under the Akaike Information Criterion.

The phylogenetic analyses of the combined gene regions were performed using Maximum Likelihood (ML) and Bayesian Inference (BI) methods. ML was conducted using PhyML v. 3.0 (Guindon et al. 2010), with 1000 bootstrap replicates while BI was performed using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.0 (Ronquist et al. 2003). Two MCMC chains, started from random trees for 1,000,000 generations and trees, were sampled every 100th generation, resulting in a total of 10,000 trees. The first 25% of trees were discarded as burn-in of each analysis. Branches with significant Bayesian Posterior Probabilities (BPP) were estimated in the remaining 7500 trees. Phylogenetic trees were viewed with FigTree v.1.3.1 (Rambaut and Drummond 2010) and processed by Adobe Illustrator CS5. 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 S28703 and S22703.

Table 1.
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Isolates and GenBank accession numbers used in the phylogenetic analyses of Diaporthe.

Species Isolate Host Location GenBank accession numbers
ITS cal his3 tef1 tub2
D. acericola MFLUCC 17-0956 Acer negundo Italy KY964224 KY964137 NA KY964180 KY964074
D. acerigena CFCC 52554 Acer tataricum China MH121489 MH121413 MH121449 MH121531 NA
D. alangii CFCC 52556 Alangium kurzii China MH121491 MH121415 MH121451 MH121533 MH121573
D. alnea CBS 146.46 Alnus sp. Netherlands KC343008 KC343250 KC343492 KC343734 KC343976
D. amygdali CBS 126679 Prunus dulcis Portugal KC343022 KC343264 KC343506 AY343748 KC343990
D. angelicae CBS 111592 Heracleum sphondylium Austria KC343027 KC343269 KC343511 KC343753 KC343995
D. apiculatum CGMCC 3.17533 Camellia sinensis China KP267896 NA NA KP267970 KP293476
D. arecae CBS 161.64 Areca catechu India KC343032 KC343274 KC343516 KC343758 KC344000
D. arengae CBS 114979 Arenga enngleri Hong Kong KC343034 KC343276 KC343518 KC343760 KC344002
D. aseana MFLUCC 12-0299 Unknown dead leaf Thailand KT459414 KT459464 NA KT459448 KT459432
D. biguttulata CGMCC 3.17248 Citrus limon China KJ490582 NA KJ490524 KJ490461 KJ490403
CFCC 52584 Juglans regia China MH121519 MH121437 MH121477 MH121561 MH121598
D. camelliae-oleiferae HNZZ027 Camellia oleifera China MZ509555 MZ504685 MZ504696 MZ504702 MZ504718
HNZZ030 Camellia oleifera China MZ509556 MZ504686 MZ504697 MZ504708 MZ504719
HNZZ032 Camellia oleifera China MZ509557 MZ504687 MZ504698 MZ504709 MZ504720
D. celeris CPC 28262 Vitis vinifera Czech Republic MG281017 MG281712 MG281363 MG281538 MG281190
D. celastrina CBS 139.27 Celastrus sp. USA KC343047 KC343289 KC343531 KC343773 KC344015
D. cercidis CFCC 52565 Cercis chinensis China MH121500 MH121424 MH121460 MH121542 MH121582
D. charlesworthii BRIP 54884m Rapistrum rugostrum Australia KJ197288 NA NA KJ197250 KJ197268
D. chrysalidocarpi SAUCC194.35 Chrysalidocarpus lutescens China MT822563 MT855646 MT855532 MT855876 MT855760
D. cinnamomi CFCC 52569 Cinnamomum sp. China MH121504 NA MH121464 MH121546 MH121586
D. citriasiana CGMCC 3.15224 Citrus unshiu China JQ954645 KC357491 KJ490515 JQ954663 KC357459
D. citrichinensis CGMCC 3.15225 Citrus sp. China JQ954648 KC357494 NA JQ954666 NA
D. collariana MFLU 17-2770 Magnolia champaca Thailand MG806115 MG783042 NA MG783040 MG783041
D. conica CFCC 52571 Alangium chinense China MH121506 MH121428 MH121466 MH121548 MH121588
D. cucurbitae CBS 136.25 Arctium sp. Unknown KC343031 KC343273 KC343515 KC343757 KC343999
D. cuppatea CBS 117499 Aspalathus linearis South Africa KC343057 KC343299 KC343541 KC343783 KC344025
D. discoidispora ZJUD89 Citrus unshiu China KJ490624 NA KJ490566 KJ490503 KJ490445
D. drenthii BRIP 66524 Macadamia sp. South Africa MN708229 NA NA MN696526 MN696537
D. endophytica CBS 133811 Schinus terebinthifolius Brazil KC343065 KC343307 KC343549 KC343791 KC343065
D. eres AR5193 Ulmus sp. Germany KJ210529 KJ434999 KJ420850 KJ210550 KJ420799
D. fraxini-angustifoliae BRIP 54781 Fraxinus angustifolia Australia JX862528 NA NA JX862534 KF170920
D. fraxinicola CFCC 52582 Fraxinus chinensis China MH121517 MH121435 NA MH121559 NA
D. fructicola MAFF 246408 Passiflora edulis × P. edulis f. flavicarpa Japan LC342734 LC342738 LC342737 LC342735 LC342736
D. fusicola CGMCC 3.17087 Lithocarpus glabra China KF576281 KF576233 NA KF576256 KF576305
D. ganzhouensis CFCC 53087 Unknown China MK432665 MK442985 MK443010 MK578139 MK578065
D. garethjonesii MFLUCC 12-0542a Unknown dead leaf Thailand KT459423 KT459470 NA KT459457 KT459441
D. guangxiensis JZB320094 Vitis vinifera China MK335772 MK736727 NA MK523566 MK500168
D. helicis AR5211 Hedera helix France KJ210538 KJ435043 KJ420875 KJ210559 KJ420828
D. heterostemmatis SAUCC194.85 Heterostemma grandiflorum China MT822613 MT855692 MT855581 MT855925 MT855810
D. hubeiensis JZB320123 Vitis vinifera China MK335809 MK500235 NA MK523570 MK500148
HNZZ009 Camellia oleifera China MZ509553 MZ504683 MZ504694 MZ504705 MZ504716
HNZZ019 Camellia oleifera China MZ509554 MZ504684 MZ504695 MZ504706 MZ504717
D. hunanensis HNZZ023 Camellia oleifera China MZ509550 MZ504680 MZ504691 MZ504702 MZ504713
HNZZ025 Camellia oleifera China MZ509551 MZ504681 MZ504692 MZ504703 MZ504714
HNZZ033 Camellia oleifera China MZ509552 MZ5046802 MZ504693 MZ504704 MZ504715
D. kadsurae CFCC 52586 Kadsura longipedunculata China MH121521 MH121439 MH121479 MH121563 MH121600
D. litchicola BRIP 54900 Litchi chinensis Australia JX862533 NA NA JX862539 KF170925
D. lonicerae MFLUCC 17-0963 Lonicera sp. Italy KY964190 KY964116 NA KY964146 KY964073
D. masirevicii BRIP 57892a Helianthus annuus Australia KJ197277 NA NA KJ197239 KJ197257
D. miriciae BRIP 54736j Helianthus annuus Australia KJ197282 NA NA KJ197244 KJ197262
D. momicola MFLUCC 16-0113 Prunus persica China KU557563 KU557611 NA KU557631 KU55758
D. musigena CBS 129519 Musa sp. Australia KC343143 KC343385 KC343627 KC343869 KC344111
D. neilliae CBS 144.27 Spiraea sp. USA KC343144 KC343386 KC343628 KC343870 KC344112
D. nobilis CBS 113470 Castanea sativa Korea KC343146 KC343388 KC343630 KC343872 KC344114
D. oraccinii CGMCC 3.17531 Camellia sinensis China KP267863 NA KP293517 KP267937 KP293443
D. ovoicicola CGMCC 3.17093 Citrus sp. China KF576265 KF576223 NA KF576240 KF576289
D. pandanicola MFLU 18-0006 Pandanus sp. Thailand MG646974 NA NA NA MG646930
D. pascoei BRIP 54847 Persea americana Australia JX862532 NA NA JX862538 KF170924
D. passifloricola CBS 141329 Passiflora foetida Malaysia KX228292 NA KX228367 NA KX228387
D. penetriteum CGMCC 3.17532 Camellia sinensis China KP714505 NA KP714493 KP714517 KP714529
D. perseae CBS 151.73 Persea gratissima Netherlands KC343173 KC343415 KC343657 KC343899 KC344141
D. pescicola MFLUCC 16-0105 Prunus persica China KU557555 KU557603 NA KU557623 KU557579
D. pseudomangiferae CBS 101339 Mangifera indica Dominican Republic KC343181 KC343423 KC343665 KC343907 KC344149
D. pseudophoenicicola CBS 462.69 Phoenix dactylifera Spain KC343184 KC343426 KC343668 KC343910 KC344152
D. pulla CBS 338.89 Hedera helix Yugoslavia KC343152 KC343394 KC343636 KC343878 KC344120
D. racemosae CBS 143770 Euclea racemosa South Africa MG600223 MG600219 MG600221 MG600225 MG600227
D. schimae CFCC 53103 Schima superba China MK432640 MK442962 MK442987 MK578116 MK578043
D. schini CBS 133181 Schinus terebinthifolius Brazil KC343191 KC343433 KC343675 KC343917 KC344159
D. schoeni MFLU 15-1279 Schoenus nigricans Italy KY964226 KY964139 NA KY964182 KY964109
D. searlei BRIP 66528 Macadamia sp. South Africa MN708231 NA NA NA MN696540
D. sennicola CFCC 51634 Senna bicapsularis China KY203722 KY228873 KY228879 KY228883 KY228889
D. siamensis MFLUCC 10-573a Dasymaschalon sp. Thailand JQ619879 NA NA JX275393 JX275429
D. sojae FAU635 Glycine max USA KJ590719 KJ612116 KJ659208 KJ590762 KJ610875
HNZZ008 Camellia oleifera China MZ509547 MZ504677 MZ504688 MZ504699 MZ504710
HNZZ010 Camellia oleifera China MZ509548 MZ504678 MZ504689 MZ504700 MZ504711
HNZZ022 Camellia oleifera China MZ509549 MZ504679 MZ504690 MZ504701 MZ504712
D. spinosa PSCG Pyrus pyrifolia China MK626849 MK691129 MK726156 MK654811 MK691234
D. sterilis CBS 136969 Vaccinium corymbosum Italy KJ160579 KJ160548 MF418350 KJ160611 KJ160528
D. subclavata ICMP20663 Citrus unshiu China KJ490587 NA KJ490529 KJ490466 KJ490408
D. subellipicola MFLU 17-1197 on dead wood China MG746632 NA NA MG746633 MG746634
D. subordinaria CBS 464.90 Plantago lanceolata New Zealand KC343214 KC343456 KC343698 KC343940 KC344182
D. taoicola MFLUCC 16-0117 Prunus persica China KU557567 NA NA KU557635 KU557591
D. tectonae MFLUCC 12-0777 Tectona grandis Thailand KU712430 KU749345 NA KU749359 KU743977
D. tectonendophytica MFLUCC 13-0471 Tectona grandis Thailand KU712439 KU749354 NA KU749367 KU749354
D. tectonigena MFLUCC 12-0767 Tectona grandis Thailand KU712429 KU749358 NA KU749371 KU743976
D. terebinthifolii CBS 133180 Schinus terebinthifolius Brazil KC343216 KC343458 KC343700 KC343942 KC344184
D. tibetensis CFCC 51999 Juglandis regia China MF279843 MF279888 MF279828 MF279858 MF279873
D. tulliensis BRIP 62248a Theobroma cacao Australia KR936130 NA NA KR936133 KR936132
D. ukurunduensis CFCC 52592 Acer ukurunduense China MH121527 MH121445 MH121485 MH121569 NA
D. unshiuensis CGMCC 3.17569 Citrus unshiu China KJ490587 NA KJ490529 KJ490408 KJ490466
CFCC 52594 Carya illinoensis China MH121529 MH121447 MH121487 MH121571 MH121606
D. viniferae JZB320071 Vitis vinifera China MK341551 MK500107 NA MK500119 MK500112
D. xishuangbanica CGMCC 3.18282 Camellia sinensis China KX986783 NA KX999255 KX999175 KX999216
D. yunnanensis CGMCC 3.18289 Coffea sp. China KX986796 KX999290 KX999267 KX999188 KX999228
Diaporthella corylina CBS 121124 Corylus sp. China KC343004 KC343246 KC343488 KC343730 KC343972

Note: NA, not applicable. Strains in this study are marked in bold.

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 96 sequences including the outgroup taxon, Diaporthella corylina (CBS 121124). A total of 2520 characters including gaps (510 for ITS, 518 for cal, 533 for his3, 460 for tef1 and 499 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, two known species, D. hubeiensis and D. sojae, were part of Diaporthe.Diaporthe camelliae-oleiferae and D. hunanensis are new to science based on the distinct and well-supported molecular phylogenetic placement with their closest described relatives. Phylogenetically, D. camelliae-oleiferae clustered together with D. pandanicola and D. viniferae. Diaporthe hunanensis clustered together with D. chrysalidocarpi and other species, including D. drenthii, D. searlei and D. spinosa.

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 ≥ 0.75). The tree is rooted with Diaporthella corylina. Isolates in current study are in blue. “-” indicates ML BS < 50% or BI PP < 0.75.

Figure 1. 

Continued

Taxonomy

Diaporthe camelliae-oleiferae Q. Yang, sp. nov.

MycoBank No: 840451
Figure 2

Diagnosis

Distinguished from the phylogenetically closely-related species, D. pandanicola and D. viniferae based on DNA sequence data.

Etymology

Named after the host species, Camellia oleifera.

Description

Asexual morph: pycnidia on PDA 500–660 μm in diam., superficial, scattered on PDA, dark brown to black, globose, solitary, or clustered in groups of 3–5 pycnidia. Pale yellow conidial drops exuding from ostioles. Conidiophores reduced to conidiogenous cells. Conidiogenous cells (7.5–)10–14(–15.5) × 1.5–2.3 μm (n = 30), aseptate, cylindrical, straight, densely aggregated, terminal, slightly tapered toward the apex. Alpha conidia 5–6.5(–7.5) × 1.9–2.3 μm (n = 30), aseptate, hyaline, ellipsoidal to fusiform, biguttulate. Beta conidia (26.5–)28.5–31(–33) × 0.8–1.2 µm (n = 30), hyaline, aseptate, filiform, sinuous at one end, eguttulate.

Figure 2. 

Diaporthe camelliae-oleiferae (HNZZ027) A Culture on PDA B conidiomata C conidiogenous cells D–F alpha and beta conidia. Scale bars: 200 μm (B); 10 μm (C–D); 20 μm (E, F).

Culture characters

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

Specimens examined

China. Hunan Province: Zhuzhou City, on leaves of Camellia oleifera, 27°2'41"N, 113°19'17"E, 14 Aug. 2020, Q. Yang (holotype CSUFT027; ex-type living culture: HNZZ027; other living cultures: HNZZ030 and HNZZ032).

Notes

Three isolates representing D. camelliae-oleiferae cluster in a well-supported clade (ML/BI=100/1) and appear most closely related to D. pandanicola on Pandanus sp. and D. viniferae on Vitis vinifera. Diaporthe camelliae-oleiferae can be distinguished from D. pandanicola based on ITS and tub2 loci (24/462 in ITS and 11/401 in tub2); from D. viniferae based on ITS, cal, tef1 and tub2 loci (13/453 in ITS, 42/448 in cal, 7/339 in tef1 and 26/402 in tub2). Morphologically, D. camelliae-oleiferae differs from D. viniferae in having shorter alpha conidia (5–6.5 μm vs. 5–8.3 μm) (Manawasinghe et al. 2019); from D. pandanicola in having narrower alpha conidia (1.9–2.3 μm vs. 2.5–3.2 μm) (Huang et al. 2021).

Diaporthe hubeiensis Dissanayake, X.H. Li & K.D. Hyde

Figure 3

Manawasinghe, Dissanayake, Li, Liu, Wanasinghe, Xu, Zhao, Zhang, Zhou, Hyde, Brooks & Yan, Frontiers in Microbiology 10(no. 1936): 20 (2019)

Description

Asexual morph: pycnidia on PDA in culture, 700–885 μm in diam., superficial, scattered, dark brown to black, globose or subglobose. Conidiophores reduced to conidiogenous cells. Conidiogenous cells (6.5–)7–10(–11.5) × 2–3.5 μm (n = 30), aseptate, cylindrical, phiailidic, straight or slightly curved. Alpha conidia 5.8–8(–8.5) × 2.5–3.2 μm (n = 30), aseptate, hyaline, ellipsoidal to cylindrical, biguttulate, blunt at both ends. Beta conidia not observed.

Figure 3. 

Diaporthe hubeiensis (HNZZ019) A Culture on PDA B conidiomata C conidiogenous cells D alpha conidia. Scale bars: 500 μm (B); 10 μm (C–D).

Culture characters

Culture incubated on PDA at 25 °C, originally flat with white felted aerial mycelium, becoming dark brown mycelium due to pigment formation, conidiomata irregularly distributed over agar surface after 20 days.

Specimens examined

China. Hunan Province: Zhuzhou City, on leaves of Camellia oleifera, 27°2'35"N, 113°19'20"E, 14 Aug. 2020, Q. Yang (CSUFT019; living cultures: HNZZ019 and HNZZ009).

Notes

Diaporthe hubeiensis was originally described as pathogen of grapevines in Hubei Province, China (Manawasinghe et al. 2019). In the present study, two isolates (HNZZ019 and HNZZ009) are closely related to D. hubeiensis in the combined phylogenetic tree (Fig. 1). The differences of nucleotides in the concatenated alignment (1/460 in ITS, 3/458 in cal, 1/320 in his3 and 3/433 in tub2) are minor. Morphological comparison indicated that the isolates were similar to D. hubeiensis by the size of alpha conidia. We therefore identify the isolates as belonging to D. hubeiensis.

Diaporthe hunanensis Q. Yang, sp. nov.

MycoBank No: 840452
Figure 4

Diagnosis

Distinguished from its phylogenetically closely-related species, D. chrysalidocarpi, D. drenthii, D. searlei and D. spinosa based on DNA sequence data.

Etymology

In reference to the Hunan province, from where the fungus was first collected.

Description

Asexual morph: pycnidia on PDA 180–300 μm in diam., superficial, scattered, black, globose, solitary in most. Conidiophores reduced to conidiogenous cells. Conidiogenous cells (8–)9–15(–16.5) × 1.7–2.1 μm (n = 30), aseptate, cylindrical, phiailidic, straight or slightly curved. Alpha conidia 6.5–7.5(–8.5) × 2.4–2.9 μm (n = 30), aseptate, hyaline, ellipsoidal, biguttulate, both ends obtuse. Beta conidia not observed.

Figure 4. 

Diaporthe hunanensis (HNZZ023) A Culture on PDA B conidiomata C conidiogenous cells D alpha conidia. Scale bars: 500 μm (B); 10 μm (C–D).

Culture characters

Culture incubated on PDA at 25 °C, originally flat with white fluffy aerial mycelium, becoming pale brown with age, with visible solitary conidiomata at maturity after 18 days.

Specimens examined

China. Hunan Province: Zhuzhou City, on leaves of Camellia oleifera, 27°2'41"N, 113°19'17"E, 14 Aug. 2020, Q. Yang (holotype CSUFT 023; ex-type living culture: HNZZ023; living cultures: HNZZ025 and HNZZ033).

Notes

Three isolates representing D. hunanensis cluster in a well-supported clade (ML/BI=100/1) and appear most closely related to D. chrysalidocarpi on Chrysalidocarpus lutescens, D. drenthii and D. searlei on Macadamia sp., and D. spinosa on P. pyrifolia cv. Cuiguan. Diaporthe hunanensis can be distinguished from D. chrysalidocarpi based on ITS, cal, his3 and tub2 loci (7/457 in ITS, 28/448 in cal, 8/455 in his3 and 5/401 in tub2); from D. drenthii based on ITS, tef1 and tub2 loci (9/457 in ITS, 13/328 in tef1 and 23/401 in tub2); from D. searlei based on ITS and tub2 loci (10/457 in ITS and 12/401 in tub2); from D. spinosa based on ITS, cal, his3, tef1 and tub2 loci (8/458 in ITS, 31/448 in cal, 5/455 in his3, 8/328 in tef1 and 19/401 in tub2). Morphologically, D. chrysalidocarpi produces only beta conidia, while D. hunanensis produces alpha conidia (Huang et al. 2021); D. hunanensis differs from D. drenthii and D. searlei in wider alpha conidia (2.4–2.9 μm in D. hunanensis vs. 1.5–2.5 μm in D. drenthii vs. 1.5–2 μm in D. searlei) (Wrona et al. 2020); from D. spinosa in shorter alpha conidia (6.5–7.5 × 2.4–2.9 μm vs. 5.5–8 × 2–3.5 μm) (Guo et al. 2020). Therefore, we establish this fungus as a novel species.

Diaporthe sojae Lehman, Ann. Mo. bot. Gdn 10: 128 (1923)

Figure 5

Description

Sexual morph: perithecia on pine needles in culture, black, globose, 250–500 μm in diam., densely clustered in groups, deeply immersed with elongated, tapering perithecial necks protruding through substrata, 525–800 μm. Asci unitunicate, 8-spored, sessile, elongate to clavate, (35–)37–42(–44.5) × (8–)10–11.5 μm (n = 30). Ascospores hyaline, two-celled, often 4-guttulate, with larger guttules at centre and smaller one at ends, elongated to elliptical, slightly or not constricted at septum, (9–) 9.5–11.5 × 2.7–4 μm (n = 30). Asexual morph not observed.

Figure 5. 

Diaporthe sojae (HNZZ022) A Culture on PNA B ascomata C–E asci and ascospores. Scale bars: 500 μm (B); 10 μm (C–E).

Culture characters

Culture incubated on PNA at 25 °C, originally white, fluffy aerial mycelium, reverse yellowish pigmentation developing in centre, later becoming dark brown, with yellowish-cream drops exuding from the perithecia after 15 days.

Specimens examined

China. Hunan Province: Zhuzhou City, on leaves of Camellia oleifera, 27°2'41"N, 113°19'17"E, 14 Aug. 2020, Q. Yang (USUFT 022; living cultures: HNZZ022, HNZZ008 and HNZZ010).

Notes

Diaporthe sojae was first reported on pods and stems of soybean, and subsequently reported on a wide range of hosts (Dissanayake et al. 2015; Udayanga et al. 2015; Guo et al. 2020). It was also reported on some fruit trees in China, such as Vitis spp. (Dissanayake et al. 2015) and Citrus spp. (Huang et al. 2015). In the present, three isolates (HNZZ008, HNZZ010 and HNZZ022) are closely related to D. sojae in the combined phylogenetic tree (Fig. 1). The differences of nucleotides in the concatenated alignment (1/460 in ITS, 3/458 in cal, 1/320 in his3 and 3/433 in tub2) are minor. Compared with the description of the ex-type isolate FAU635, the isolate has wider asci (10–11.5 μm vs. 7–9 μm) (Udayanga et al. 2015). We therefore identify the isolates as belonging to D. sojae.

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 Hunan 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. It includes D. hubeiensis, D. sojae, as well as two new species named D. camelliae-oleiferae and D. hunanensis.

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 becoming the emerging Camellia leaf diseases in southern China (Gao et al. 2016; Guarnaccia et al. 2018; Yang et al. 2018; Zhou and Hou 2019). Understanding the diversity of Diaporthe species and the genetic variation within pathogen populations could help in developing sustainable disease management strategies.

According to the USDA Fungal–host interaction database, there are two records of Diaporthe species associated with C. oleifera (https://nt.ars-grin.gov/fungaldatabases/fungushost/fungushost.cfm) (accessed 9 September 2021). These records are related to the following two Diaporthe species: D. eres and D. huangshanensis (Zhou and Hou 2019). 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. 2014b). Diaporthe eres differs from D. camelliae-oleiferae and D. hunanensis in having wider alpha conidia (3–4 μm in D. eres vs. 1.9–2.3 μm in D. camelliae-oleiferae vs. 2.4–2.9 μm in D. hunanensis) (Gomes et al. 2003); D. huangshanensis differs from D. camelliae-oleiferae in having larger alpha conidia (5.7–8.4 × 2.7–4.5 μm vs. 5–6.5 × 1.9–2.3 μm); from D. hunanensis in having wider alpha conidia (2.7–4.5 μm vs. 2.4–2.9 μm) and longer conidiophores (12.1–23.5 μm vs. 9–15 μm) (Zhou and Hou 2019).

As the species concept of Diaporthe has been improved a lot 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), 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.

Acknowledgements

This study is financed by the Research Foundation of Education Bureau of Hunan Province, China (Project No.: 19B608) and the introduction of talent research start-up fund project of CSUFT (Project No.: 2019YJ025).

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