Skip to main content
NCBI home page
MycoKeys logoLink to MycoKeys
. 2022 Jul 15;91:67–84. doi: 10.3897/mycokeys.91.86693

Pseudocryphonectriaelaeocarpicola gen. et sp. nov. (Cryphonectriaceae, Diaporthales) causing stem blight of Elaeocarpus spp. in China

Hua-Yi Huang 1, Huan-Hua Huang 1, Dan-Yang Zhao 1, Ti-Jiang Shan 2, Li-Li Hu 1,✉
PMCID: PMC9849075  PMID: 36760888

Abstract

Cryphonectriaceae is a diaporthalean family containing important plant pathogens of which Cryphonectriaparasitica is the most notorious one. An emerging stem blight disease on Elaeocarpusapiculatus (Elaeocarpaceae) and E.hainanensis was observed in Guangdong Province of China recently. Typical Cryphonectria blight-like symptoms including cankers on tree barks with obvious orange conidial tendrils were observed. Forty-eight isolates were obtained from diseased tissues and conidiomata formed on the hosts E.apiculatus and E.hainanensis. These isolates were further identified based on both morphology and molecular methods using the combined sequence data of the internal transcribed spacer (ITS) region, large subunit of the nrDNA (LSU), the translation elongation factor 1-alpha (tef1) and DNA-directed RNA polymerase II second largest subunit (rpb2) genes. As a result, the fungus represents an undescribed genus and species within the family Cryphonectriaceae. Hence, Pseudocryphonectriaelaeocarpicolagen. et sp. nov. is proposed herein to represent these isolates from diseased barks of E.apiculatus and E.hainanensis. Pseudocryphonectria differs from the other genera of Cryphonectriaceae in having dimorphic conidia. Further inoculation results showed that P.elaeocarpicola is the causal agent of this emerging blight disease in China, which can quickly infect and kill the hosts E.apiculatus and E.hainanensis.

Keywords: Ascomycota , phylogeny, plant disease, taxonomy

Introduction

Diaporthales represents a species-rich fungal order usually inhabiting plant tissues as pathogens, endophytes and saprophytes (Rossman et al. 2007; Senanayake et al. 2017; Voglmayr et al. 2017; Fan et al. 2018; Jaklitsch and Voglmayr 2020; Jiang et al. 2021a). Cryphonectriaceae is a pathogenic family within Diaporthales including several serious plant pathogens (Gryzenhout et al. 2006; Chen et al. 2016). For example, Cryphonectriaparasitica causes chestnut (Castanea spp.) blight disease worldwide (Rigling and Prospero 2017); Chrysoportheaustroafricana, Ch.cubensis and Ch.deuterocubensis result in eucalypt (Eucalyptus spp.) canker diseases in Africa, South America and Asia, respectively (Ferreira and Henfling 1976; Wingfield 1989; Old et al. 2003; Wang et al. 2020).

In a recent study, the family Cryphonectriaceae was re-evaluated based on morphological and molecular data of the ex-type strains, which accepted two subclades in the family with 21 genera and 55 species (Jiang et al. 2020). Subsequently, Capillaureum and Parvosmorbus were added to this family evidenced by both morphology and phylogeny (Ferreira et al. 2019; Wang et al. 2020). Currently, 23 genera were classified in Cryphonectriaceae based on morphological characters and combined sequence data of the internal transcribed spacer (ITS) region, large subunit of the nrDNA (LSU), and the translation elongation factor 1-alpha (tef1) and DNA-directed RNA polymerase II second largest subunit (rpb2) genes (Wijayawardene et al. 2018; Hyde et al. 2020; Jiang et al. 2020; Wang et al. 2020).

Cryphonectriaceae members are characterized by typical diaporthalean characters of perithecia with elongate beaks, often forming within stromatic tissues, deliquescent paraphyses, and asci that generally deliquesce, become detached from the perithecial wall when mature, and have a refractive apical annulus (Voglmayr et al. 2012; Senanayake et al. 2018; Jaklitsch and Voglmayr 2019; Jiang et al. 2019b; Fan et al. 2020; Udayanga et al. 2021). Species of Cryphonectriaceae except Aurantiosacculuscastaneae are different from the other diaporthalean taxa by owning orange stromatic tissues at some stage during their life cycle, which turn purple in 3% KOH and yellow in lactic acid (Gryzenhout et al. 2006; Jiang et al. 2019a).

Trees and shrubs of Elaeocarpus (Elaeocarpaceae) are evergreen plants, of which several species are planted along streets and in parks. E.apiculatus and E.hainanensis are commonly used as garden trees in Guangdong Province, however, suffering a serious stem blight disease currently. The present study aims to identify the causal agent based on modern taxonomic approaches and to confirm its pathogenicity.

Materials and methods

Sample survey, fungal isolation and morphology

In the present study, we investigated stem blight disease of Elaeocarpusapiculatus and E.hainanensis in Guangdong Province of China during 2020 and 2022. The disease symptoms on the Elaeocarpus trees generally occur on host stems and branches, with cankered barks and orange conidial tendrils (Fig. 1). Most diseased trees died within five months of infection during our investigations. Diseased barks with or without fruiting bodies were collected, packed in paper bags and transferred to the laboratory for isolation.

Figure 1.

Figure 1.

Open in a new tab

Symptoms caused by Pseudocryphonectriaelaeocarpicola on Elaeocarpus trees. A, B dead trees C–E cankered barks F, G orange conidial tendrils formed on the cankered barks.

The diseased barks without orange fungal fruiting bodies were firstly surface-sterilized for 2 min in 75% ethanol, 4 min in 1.25% sodium hypochlorite, and 1 min in 75% ethanol, then rinsed for 2 min in distilled water and blotted on dry sterile filter paper. Then diseased tissues were cut into 0.5 cm × 0.5 cm pieces using a double-edge blade, and transferred onto the surface of potato dextrose agar (PDA; 200 g potatoes, 20 g dextrose, 20 g agar per L), and incubated at 25 °C to obtain pure cultures. The diseased barks with fungal fruiting bodies were checked, and single conidial isolates were obtained from conidiomata by removing the mucoid conidial masses and spreading the suspension onto the surface of PDA. Agar plates were incubated at 25 °C to induce germination of the conidia. After inoculation for up to 48 h, single germinating conidium was then transferred to clean plates under a dissecting stereomicroscope with a sterile needle. The cultures were deposited in China Forestry Culture Collection Center (CFCC, http://cfcc.caf.ac.cn/), and the specimens in the herbarium of the Chinese Academy of Forestry (CAF, http://museum.caf.ac.cn/).

The morphological data of the new taxa in the present study were based on the conidiomata formed on the cankered barks, supplemented by cultural characters. The conidiomata were sectioned and photographed under a dissecting microscope (M205 C, Leica, Wetzlar, Germany). The conidiogenous cells and conidia were immersed in tap water, then the microscopic photographs were captured with an Axio Imager 2 microscope (Zeiss, Oberkochen, Germany) equipped with an Axiocam 506 color camera, using differential interference contrast (DIC) illumination. More than 50 conidia were randomly selected for measurement. Culture characters were recorded from PDA after 7 d incubation at 25 °C in the dark.

DNA extraction, PCR amplification and phylogenetic analyses

The fungal genomic DNA was extracted from mycelia grown on cellophane-covered PDA following the method in Doyle and Doyle (1990). DNA was checked by electrophoresis in 1% agarose gel, and the quality and quantity were measured using a NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). Four partial loci, ITS and LSU regions, tef1 and rpb2 genes were amplified by the following primer pairs: ITS1 and ITS4 for ITS (White et al. 1990), LR0R and LR5 for LSU (Vilgalys and Hester 1990), EF1-688F and EF2 for tef1 (Carbone and Kohn 1999), and RPB2-5F and RPB2-7cR for rpb2 (Liu et al. 1999). The polymerase chain reaction (PCR) conditions were as follows: an initial denaturation step of 5 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 50 s at 48 °C (ITS and LSU) or 54 °C (tub2) or 55 °C (rpb2), and 1 min at 72 °C, and a final elongation step of 10 min at 72 °C. PCR products were assayed via electrophoresis in 2% agarose gels. DNA sequencing was performed using an ABI PRISM 3730XL DNA Analyser with a BigDye Terminator Kit v.3.1 (Invitrogen, Waltham, MA, USA) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

The sequences obtained in the present study were assembled using SeqMan v. 7.1.0, and reference sequences were retrieved from the National Center for Biotechnology Information (NCBI), based on recent publications (Chen et al. 2018; Jiang et al. 2019a, 2020; Wang et al. 2020). The sequences were aligned using MAFFT v. 6 and corrected manually using MEGA v. 7.0.21 (Katoh and Toh 2010).

The phylogenetic analyses of combined matrixes of the ITS-LSU loci and four loci (ITS-LSU-tef1-rpb2) were performed using Maximum Likelihood (ML) and Bayesian Inference (BI) methods. ML was implemented on the CIPRES Science Gateway portal (https://www.phylo.org) using RAxML-HPC BlackBox 8.2.10 (Miller et al. 2010; Stamatakis 2014), employing a GTR-GAMMA substitution model with 1000 bootstrap replicates. Bayesian inference was performed using a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.0 (Ronquist and Huelsenbeck 2003). Two MCMC chains, starting from random trees for 1000000 generations and trees, were sampled every 100th generation, resulting in a total of 10000 trees. The first 25% of trees were discarded as burn-in of each analysis. Branches with significant Bayesian Posterior Probabilities (BPP > 0.9) were estimated in the remaining 7500 trees. Phylogenetic trees were viewed with FigTree v. 1.3.1 and processed by Adobe Illustrator CS5. The nucleotide sequence data of the new taxon were deposited in GenBank, and the GenBank accession numbers of all accessions included in the phylogenetic analyses are listed in Table 1.

Table 1.

Isolates and GenBank accession numbers used in the phylogenetic analyses.

Species Isolate GenBank Accession Number
ITS LSU tef1 rpb2
Amphilogiagyrosa CBS 112922* AF452111 AY194107 MN271818 MN271782
Amphilogiagyrosa CBS 112923 AF452112 AY194108 MN271819 MN271783
Aurantioporthecorni CMW 10526 DQ120762 AF408343 NA NA
Aurantioporthecorni CBS 245.90 MN172403 MN172371 MN271822 MN271784
Aurantiosacculusacutatus CBS 132181* JQ685514 JQ685520 MN271823 NA
Aurantiosacculuseucalyptorum CBS 130826* JQ685515 JQ685521 MN271824 MN271785
Aurantiosacculuscastaneae CFCC 52456* MH514025 MH514015 NA MN271786
Aurapexpenicillata CBS 115740* AY214311 AY194103 NA NA
Aurapexpenicillata CBS 115742 AY214313 MN172372 NA NA
Aurapexpenicillata CBS 115801 MN172404 MN172373 NA MN271787
Aurifilummarmelostoma CBS 124928* FJ890495 MH874934 MN271827 MN271788
Aurifilummarmelostoma CBS 124929 FJ882855 HQ171215 MN271828 MN271789
Capillaureumcaryovora CBL02* MG192094 MG192104 NA NA
Celoporthedispersa CBS 118782* DQ267130 HQ730853 HQ730840 NA
Celoportheeucalypti CBS 127190* HQ730837 HQ730863 HQ730850 MN271790
Celoportheguangdongensis CBS 128341* HQ730830 HQ730856 HQ730843 NA
Celoporthesyzygii CBS 127218* HQ730831 HQ730857 HQ730844 NA
Celoporthewoodiana CBS 118785* DQ267131 MN172375 JQ824071 MN271791
Chrysomorbuslagerstroemiae CBS 142594* KY929338 KY929328 MN271830 NA
Chrysomorbuslagerstroemiae CBS 142592 KY929330 KY929320 MN271831 NA
Chrysoportheaustroafricana CBS 112916* AF292041 AY194097 MN271832 NA
Chrysoportheaustroafricana CBS 115843 AF273473 MN172377 MN271833 NA
Chrysoporthecubensis CBS 118654* DQ368773 MN172378 MN271834 NA
Chrysoporthecubensis CBS 505.63 AY063476 MN172379 MN271835 MN271792
Chrysoporthehodgesiana CBS 115854* AY692322 MN172380 MN271836 MN271793
Chrysoporthehodgesiana CBS 115744 AY956970 MN172381 MN271837 NA
Chrysoportheinopina CBS 118659* DQ368777 MN172382 MN271838 NA
Chrysoporthesyzygiicola CBS 124488* FJ655005 MN172383 MN271839 NA
Chrysoporthezambiensis CBS 124503* FJ655002 MN172384 MN271840 NA
Corticimorbussinomyrti CBS 140205* KT167169 KT167179 MN271841 MN271794
Corticimorbussinomyrti CBS 140206 KT167170 KT167180 MN271842 MN271795
Cryphonectriacitrina CBS 109758* MN172407 EU255074 MN271843 EU219342
Cryphonectriadecipens CBS 129351 EU442657 MN172385 MN271844 MN271796
Cryphonectriadecipens CBS 129353 EU442655 MN172386 MN271845 MN271797
Cryphonectriajaponica CFCC 52148 MH514033 MH514023 MN271846 NA
Cryphonectriamacrospora CBS 109764 EU199182 AF408340 NA EU220029
Cryphonectrianeoparasitica CFCC 52146* MH514029 MH514019 MN271847 NA
Cryphonectriaparasitica ATCC 38755 MH843497 MH514021 NA DQ862017
Cryphonectriaparasitica CFCC 52150 AY141856 EU199123 MN271848 NA
Cryphonectriaquercus CFCC 52138* MG866024 NA MN271849 NA
Cryphonectriaquercicola CFCC 52141* MG866027 NA MN271850 NA
Cryphonectriaradicalis CBS 112917 AF452113 AY194101 NA NA
Cryptometrionaestuescens CBS 124007* GQ369457 MN172387 MN271851 MN271798
Cryptometrionaestuescens CBS 124008 GQ369458 HQ171211 MN271852 MN271799
Diaportheeres LC3198 KP267873 KY011845 KP267947 NA
Diversimorbusmetrosiderotis CBS 132866* JQ862871 JQ862828 MN271857 NA
Diversimorbusmetrosiderotis CBS 132865 JQ862870 JQ862827 MN271858 NA
Endothiachinensis CFCC 52144* MH514027 MH514017 MN271860 NA
Holocryphiaeucalypti CBS 115842* MN172411 MN172391 MN271882 MN271804
Holocryphiacapensis CBS 132870* JQ862854 JQ862811 MN271883 NA
Holocryphiagleniana CBS 132871* JQ862834 JQ862791 MN271884 NA
Holocryphiamzansi CBS 132874* JQ862841 JQ862798 MN271885 NA
Immersiportheknoxdaviesiana CBS 132862* JQ862765 JQ862755 MN271886 MN271805
Immersiportheknoxdaviesiana CBS 132863 JQ862766 JQ862756 MN271887 MN271806
Latruncellusaurorae CBS 125526* GU726947 HQ730872 MN271888 NA
Latruncellusaurorae CBS 124904 GU726946 HQ171213 MN271889 NA
Luteocirrhusshearii CBS 130776* KC197021 KC197019 MN271890 MN271807
Luteocirrhusshearii CBS 130775 KC197024 KC197018 MN271891 MN271808
Microthiahavanensis CBS 115855 DQ368735 MN172393 NA MN271811
Microthiahavanensis CBS 115841 DQ368736 MN172394 NA NA
Microthiahavanensis CBS 115758 DQ368737 MN172395 NA NA
Myrtonectriamyrtacearum CMW 46433* MG585736 MG585750 NA NA
Myrtonectriamyrtacearum CMW 46435 MG585737 MG585751 NA NA
Parvosmorbuseucalypti CSF2060 MN258787 MN258843 MN258829 NA
Parvosmorbusguangdongensis CSF10437 MN258795 MN258851 MN258837 NA
Pseudocryphonectriaelaeocarpicola CFCC 57515* ON489048 ON489050 ON456916 ON456918
Pseudocryphonectriaelaeocarpicola CFCC 57516 ON489049 ON489051 ON456917 ON456919
Rostraureumtropicale CBS 115725* AY167435 MN172399 MN271895 MN271814
Rostraureumtropicale CBS 115757 AY167438 MN172400 MN271896 MN271815
Ursicollumfallax CBS 118663* DQ368755 EF392860 MN271897 MN271816
Ursicollumfallax CBS 118662 DQ368756 MN172401 MN271898 MN271817
Open in a new tab

Note: NA, not applicable. Ex-type strains are marked with *, and strains from the present study are in black bold.

Pathogenicity tests

Three isolates of the new species Pseudocryphonectriaelaeocarpicola (ex-type strain: CFCC 57515, CFCC 57516 and CFCC 57517) were used for inoculations, and PDA plugs were used as the negative control. Three isolates were grown on PDA for four days at 25 °C before the tests. Inoculations were performed on 2-year-old seedlings of Elaeocarpusapiculatus and E.hainanensis, respectively. A total of 40 healthy seedlings were used for the pathogenicity tests. Five seedlings were inoculated with each isolate and the negative control. Inoculations were conducted following the method in Jiang et al. (2019a). The results were evaluated after ten days by measuring the lengths of the lesions on the cambium. The re-isolations were made from the resultant lesions from all tested seedlings by cutting small pieces of discolored xylem and placing them onto the PDA plates. Re-isolates were identified based on the ITS sequences. Differences among isolates in lesion length were analyzed by one-way analysis of variance (ANOVA) followed by least significant difference (LSD) tests. Statistical analysis was carried out by R software (v. 3.4.3) and considered as significant at p < 0.05.

Results

Incidence and isolates

Surveys of Elaeocarpusapiculatus and E.hainanensis stem blight were conducted in Guangdong Province during 2020 and 2022. Disease incidence was evaluated based on the percentage of the two hosts showing symptoms of all the investigated plants. As shown in Table 2, the disease incidences are all above 85% in seven locations, which indicates this disease poses a serious threat to these two tree hosts.

Table 2.

Occurrence and incidence of Elaeocarpusapiculatus and E.hainanensis stem blight in different locations in Guangzhou City.

District Location Host Diseased trees Dead trees Healthy Trees Total Disease incidence (%)
Tianhe Longdong Street E.apiculatus 9 10 0 19 100
Tianhe Guangdong tree Park E.apiculatus 14 9 2 25 92
Tianhe Shuanglin Street E.apiculatus 18 4 2 24 91.67
Tianhe Guangdong Eco-Engineering Polytechnic E.apiculatus 11 2 0 13 100
Tianhe South China Botanical Garden E.apiculatus 5 3 1 9 88.89
Liwan Meihua Middle School E.hainanensis 3 5 0 8 100
Yuexiu Luhu Park E.apiculatus 41 21 6 68 91.18
Open in a new tab

A total of 42 isolates were obtained from the symptomatic tissues of E.apiculatus and E.hainanensis, and six isolates from the conidiomata formed on the cankered barks. They are identical based on the sequence data, hence isolates CFCC 57515 from E.hainanensis and CFCC 57516 from E.apiculatus were selected for phylogenetic analyses.

Phylogenetic analyses

The sequence dataset of the ITS-LSU gene matrix was analysed to infer the genus and species relationships within Cryphonectriaceae. The dataset consisted of 71 sequences including one outgroup taxon, Diaportheeres (LC 3198). A total of 1580 characters including gaps were included in the phylogenetic analysis. The topologies resulting from ML and BI analyses of the concatenated dataset were congruent (Fig. 2). Isolates from the present study formed a distinct clade from the other genera of Cryphonectriaceae, which represents an undescribed genus.

Figure 2.

Figure 2.

Open in a new tab

Phylogram of Cryphonectriaceae resulting from a maximum likelihood analysis based on combined ITS and LSU loci. Numbers above the branches indicate ML bootstrap values (left, ML-BS ≥ 50%) and Bayesian Posterior Probabilities (right, BPP ≥ 0.9). The tree is rooted with Diaportheeres (LC 3198). Isolates from the present study are marked in blue, and ex-type strains are marked with *.

The combined four-loci sequence dataset (ITS, LSU, tef1 and rpb2) was further analysed to compare with results of the phylogenetic analyses of the ITS-LSU gene matrix. The dataset consisted of 50 sequences including one outgroup taxon, Diaportheeres (LC 3198). A total of 3226 characters including gaps (726 for ITS, 854 for LSU, 811 for tef1 and 835 for rpb2) were included in the phylogenetic analysis. The topologies resulting from ML and BI analyses of the concatenated combined dataset were congruent (Fig. 3). Isolates from the present study formed a distinct clade which was congruent with that shown in Fig. 2.

Figure 3.

Figure 3.

Open in a new tab

Phylogram of Cryphonectriaceae resulting from a maximum likelihood analysis based on combined ITS, LSU, tef1 and rpb2 loci. Numbers above the branches indicate ML bootstrap values (left, ML-BS ≥ 50%) and Bayesian Posterior Probabilities (right, BPP ≥ 0.9). The tree is rooted with Diaportheeres (LC 3198). Isolates from the present study are marked in blue, and ex-type strains are marked with *.

Taxonomy

. Pseudocryphonectria

Huayi Huang gen. nov.

794B2492-3F60-58B2-9645-A0B7C49D8EB5

844044

Etymology.

Named derived from pseudo- and the genus name Cryphonectria.

Type species.

Pseudocryphonectriaelaeocarpicola Huayi Huang

Description.

Sexual morph: Unknown. Asexual morph: Conidiomata pycnidial, aggregated or solitary, immersed under the host bark, subglobose to pulvinate, yellow to orange, multilocular, single ostiolate, forming long orange tendrils. Conidiophores cylindrical, aseptate, hyaline, sometimes reduced to conidiogenous cells. Conidiogenous cells lining inner cavity of conidiomata, phialidic, ampulliform, with attenuated or truncate apices, hyaline, smooth. Conidia dimorphic. Microconidia minute, aseptate, hyaline, smooth, cylindrical, straight. Macroconidia aseptate, hyaline, smooth, obclavate, straight or slightly curved.

Notes.

Pseudocryphonectria has typical orange cryphonectriaceous stromata, which turns purple the 3% KOH and yellow in lactic acid. This genus is characterized by its dimorphic conidia from the same conidioma, which is different from the other genera of Cryphonectriaceae (Chen et al. 2013, 2016, 2018; Beier et al. 2015; Jiang et al. 2020).

. Pseudocryphonectria elaeocarpicola

Huayi Huang sp. nov.

13EA0C08-31E7-579D-9475-B2CEF57EF13A

844045

Figs 4 , 5

Figure 4.

Figure 4.

Open in a new tab

Morphology of Pseudocryphonectriaelaeocarpicola from ElaeocarpushainanensisA, B habit of conidiomata on the host stem C transverse section through the conidioma D longitudinal section through the conidioma E conidiogenous cells giving rise to conidia F macroconidia and microconidia. Scale bars: 300 μm (C, D); 10 μm (E, F).

Figure 5.

Figure 5.

Open in a new tab

Morphology of Pseudocryphonectriaelaeocarpicola from PDAA, B colonies C, D orange conidiomata.

Etymology.

Named after the host genus, Elaeocarpus.

Description.

Sexual morph: Unknown. Asexual morph: Conidiomata pycnidial, aggregated or solitary, immersed under the host bark, subglobose to pulvinate, yellow to orange, 500–1200 μm wide, 150–450 μm high, multilocular, single ostiolate, forming long orange tendrils. Conidiophores cylindrical, aseptate, hyaline, sometimes reduced to conidiogenous cells. Conidiogenous cells lining inner cavity of conidiomata, phialidic, ampulliform, with attenuated or truncate apices, hyaline, smooth, 12.8–25.7 × 1.7–3.2 μm (n = 50). Conidia dimorphic. Microconidia minute, aseptate, hyaline, smooth, cylindrical, straight, (3.1–)3.3–4(–4.4) × (1.5–)1.6–2(–2.1) μm (n = 50), L/W = 1.6–2.7. Macroconidia aseptate, hyaline, smooth, obclavate, straight or slightly curved, (4.6–)5.1–6.1(–6.6) × (1.4–)1.6–2(–2.2) μm (n = 50), L/W = 2.5–3.9.

Culture characters.

Colonies on PDA flat, spreading, with aerial mycelium and entire margin, white to mouse grey, forming abundant orange conidiomata with orange conidial masses.

Specimens examined.

China, Guangdong Province, Guangzhou City, Meihua middle school, 23°8'37.94"N, 113°14'18.12"E, 24 m asl, on stems and branches of Elaeocarpushainanensis, 7 March 2022, Huayi Huang (CAF800051 holotype; ex-type living culture, CFCC 57515). Guangdong Province, Guangzhou City, Luhu Park, 23°9'11.15"N, 113°16'46.01"E, 92 m asl, on stems and branches of E.apiculatus, Huayi Huang, 15 March 2022 (CAF800055 paratype; ex-paratype living culture, CFCC 57516). Guangdong Province, Guangzhou City, Longdong straight street, 23°11'41.02"N, 113°22'8.33"E, 46 m asl, on stems and branches of E.apiculatus, Huayi Huang, 1 April 2022 (DY03, culture, CFCC 57517). Guangdong Province, Guangzhou City, South China botanical garden, 23°11'3.5"N, 113°21'41.53"E, 39 m asl, on stems and branches of E.apiculatus, Huayi Huang, 11 April 2022 (DY24, culture, DY24-2). Guangdong Province, Guangzhou City, Linke 1st street, 23°11'35.81"N, 113°22'46.69"E, 74 m asl, on stems and branches of E.apiculatus, Huayi Huang, 15 April 2022 (DY32; culture, DY32-1). Guangdong Province, Guangzhou City, Nonglin middle street, 23°11'23.84"N, 113°22'43.08"E, 46 m asl, on stems and branches of E.apiculatus, Huayi Huang, 15 April 2022 (DY42, culture, DY42-1).

Notes.

Pseudocryphonectriaelaeocarpicola is the sole species within the new genus, which causes serious stem blight of Elaeocarpus trees. Another notorious pathogen in Cryphonectriaceae, Cryphonectriaparasitica, causes serious chestnut worldwide. Morphologically, P.elaeocarpicola is similar to C.parasitica in the appearance of conidiomata with orange conidial tendrils formed on the host bark. However, P.elaeocarpicola can be distinguished from C.parasitica by its obvious dimorphic conidia (Jiang et al. 2019a). Phylogenetically, isolates of P.elaeocarpicola clustered into a distinct clade in the phylograms of Cryphonectriaceae (Figs 2, 3).

Pathogenicity tests

Ten days after inoculation on young seedlings of Elaeocarpusapiculatus and E.hainanensis, isolates CFCC 57515, CFCC 57516 and CFCC 57517 all caused death of the host, and formed orange conidiomata on the barks, and the negative control only produced minor lesions (Fig. 6). Statistical analyses of data showed no significant difference among three tested isolates on two hosts of E.apiculatus and E.hainanensis, however, significantly different from the negative control (Fig. 7). Isolates were obtained from lesions produced on tested seedlings, and were identical to Pseudocryphonectriaelaeocarpicola based on the sequence data and morphology of conidiomata formed on the barks. Hence, P.elaeocarpicola can quickly infect E.apiculatus and E.hainanensis, and kill the hosts.

Figure 6.

Figure 6.

Open in a new tab

Results of pathogenicity tests on Elaeocarpusapiculatus and E.hainanensis using isolates CFCC 57515, CFCC 57516 and CFCC 57517. Row 1: appearance of the hosts after incubation in 10 days; row 2: conidiomata formed on the barks.

Figure 7.

Figure 7.

Open in a new tab

Histogram of lesion lengths resulting from inoculation on Elaeocarpusapiculatus and E.hainanensis using isolates CFCC 57515, CFCC 57516 and CFCC 57517. Different letters above the error bars indicate treatments that were significantly different (p = 0.05).

Discussion

In the present study, the causal agent of stem blight on Elaeocarpusapiculatus and E.hainanensis was identified using both morphological and phylogenetical approaches, which revealed a new genus and species, namely Pseudocryphonectriaelaeocarpicola. Further pathogenicity test conducted on the two original hosts E.apiculatus and E.hainanensis confirmed the high virulence of the fungal pathogen. In ten days, the fungus can infect the host and kill both E.apiculatus and E.hainanensis. As shown in Table 2, the pathogen kills more than a half of the diseased adult trees during our investigations, which is similar to its relative fungus Cryphonectriaparasitica in pathogenicity (Rigling and Prospero 2017). Luckily, we timely discovered the fungus and report it herein, and the disease control studies have been in progress.

In the fungal order Diaporthales, many species were reported as forest pathogens causing leaf spots, cankers, fruit rot or blight diseases (Visentin et al. 2012; Pasche et al. 2016; Shuttleworth and Guest 2017; Jiang et al. 2021b; Pan et al. 2021; Lin et al. 2022), moreover, cryphonectriaceous members are known to be serious pathogens (Chen et al. 2013, 2016; Beier et al. 2015; Ferreira et al. 2019; Wang et al. 2020). This family is easily recognized based on the disease symptoms and their obvious orange conidioma formed on the cankered barks, together with their hyaline and small conidia (Gryzenhout et al. 2006). However, within this family, genera are similar in morphology which are usually distinguished by the molecular data (Jiang et al. 2020; Wang et al. 2020). Most genera in this family are known to own only one or two species; this may be caused by most samples on important trees like Fagaceae, Melastomataceae, and Myrtaceae and limited samples from the other hosts (Jiang et al. 2020; Wang et al. 2020). In the present study, Elaeocarpus (Elaeocarpaceae) usually being overlooked hosts, were found to be new hosts of Cryphonectriaceae pathogens.

There is still room for further exploration, such as the infection opportunity, sources of the primary infection and the alternative hosts of the pathogen. More importantly, the effective control methods to protect Elaeocarpus hosts are urgent to be studied due to the quick infection and high virulence.

Supplementary Material

XML Treatment for Pseudocryphonectria
XML Treatment for Pseudocryphonectria elaeocarpicola

Acknowledgements

This research was funded by Forestry Science and Technology Innovation Project of Guangdong (2020KJCX004) and the Guangdong Basic and Applied Basic Research Foundation (2019A1515011814). We thank Dr. Ning Jiang for his assistance with this paper.

Citation

Huang H-Y, Huang H-H, Zhao D-Y, Shan T-J, Hu L-L (2022) Pseudocryphonectriaelaeocarpicola gen. et sp. nov. (Cryphonectriaceae, Diaporthales) causing stem blight of Elaeocarpus spp. in China. MycoKeys 91: 67–84. https://doi.org/10.3897/mycokeys.91.86693

Funding Statement

This research was funded by Forestry Science and Technology Innovation Project of Guangdong (2020KJCX004).

References

  1. Beier GL, Hokanson SC, Bates ST, Blanchette RA. (2015) Aurantioporthecorni gen. et comb. nov., an endophyte and pathogen of Cornusalternifolia. Mycologia 107(1): 66–79. 10.3852/14-004 [DOI] [PubMed] [Google Scholar]
  2. Carbone I, Kohn LM. (1999) A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 3(3): 553–556. 10.1080/00275514.1999.12061051 [DOI] [Google Scholar]
  3. Chen SF, Wingfield MJ, Roux J. (2013) Diversimorbusmetrosiderotis gen. et sp. nov. and three new species of Holocryphia (Cryphonectriaceae) associated with cankers on native Metrosiderosangustifolia trees in South Africa. Fungal Biology 117(5): 289–310. 10.1016/j.funbio.2013.02.005 [DOI] [PubMed] [Google Scholar]
  4. Chen SF, Wingfield MJ, Li GQ, Liu FF. (2016) Corticimorbussinomyrti gen. et sp. nov. (Cryphonectriaceae) pathogenic to native Rhodomyrtustomentosa (Myrtaceae) in South China. Plant Pathology 65(8): 1254–1266. 10.1111/ppa.12507 [DOI] [Google Scholar]
  5. Chen SF, Liu QL, Li GQ, Wingfield MJ, Roux J. (2018) A new genus of Cryphonectriaceae isolated from Lagerstroemiaspeciosa in southern China. Plant Pathology 67(1): 107–123. 10.1111/ppa.12723 [DOI] [Google Scholar]
  6. Doyle JJ, Doyle JL. (1990) Isolation of plant DNA from fresh tissue. Focus 12: 13–15. 10.2307/2419362 [DOI] [Google Scholar]
  7. Fan XL, Bezerra JDP, Tian CM, Crous PW. (2018) Families and genera of diaporthalean fungi associated with canker and dieback of tree hosts. Persoonia 40(1): 119–134. 10.3767/persoonia.2018.40.05 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fan XL, Bezerra JDP, Tian CM, Crous PW. (2020) Cytospora (Diaporthales) in China. Persoonia 45(1): 1–45. 10.3767/persoonia.2020.45.01 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ferreira FA, Henfling JDM. (1976) O cancro do eucalipto causadopor Diaporthecubensis Bruner. Fitopatologia Brasileira 1: 129–169. [Google Scholar]
  10. Ferreira MA, de Oliveira MES, Silva GA, Mathioni SM, Mafia RG. (2019) Capillaureumcaryovora gen. sp. nov. (Cryphonectriaceae) pathogenic to pequi (Caryocarbrasiliense) in Brazil. Mycological Progress 18(3): 385–403. 10.1007/s11557-018-01461-3 [DOI] [Google Scholar]
  11. Gryzenhout M, Myburg H, Wingfield BD, Wingfield MJ. (2006) Cryphonectriaceae (Diaporthales), a new family including Cryphonectria, Chrysoporthe, Endothia and allied genera. Mycologia 98(2): 239–249. 10.1080/15572536.2006.11832696 [DOI] [PubMed] [Google Scholar]
  12. Hyde KD, Norphanphoun C, Maharachchikumbura SSN, et al. (2020) Refined families of Sordariomycetes. Mycosphere 11(1): 305–1059. 10.5943/mycosphere/11/1/7 [DOI] [Google Scholar]
  13. Jaklitsch WM, Voglmayr H. (2019) European species of Dendrostoma (Diaporthales). MycoKeys 59: 1–26. 10.3897/mycokeys.59.37966 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jaklitsch WM, Voglmayr H. (2020) The genus Melanconis (Diaporthales). MycoKeys 63: 69–117. 10.3897/mycokeys.63.49054 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jiang N, Fan XL, Tian CM. (2019a) Identification and pathogenicity of Cryphonectriaceae species associated with chestnut canker in China. Plant Pathology 68(6): 1132–1145. 10.1111/ppa.13033 [DOI] [Google Scholar]
  16. Jiang N, Fan XL, Crous PW, Tian CM. (2019b) Species of Dendrostoma (Erythrogloeaceae, Diaporthales) associated with chestnut and oak canker diseases in China. MycoKeys 48: 67–96. 10.3897/mycokeys.48.31715 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jiang N, Fan XL, Tian CM, Crous PW. (2020) Reevaluating Cryphonectriaceae and allied families in Diaporthales. Mycologia 112(2): 267–292. 10.1080/00275514.2019.1698925 [DOI] [PubMed]
  18. Jiang N, Voglmayr H, Bian DR, Piao CG, Wang SK, Li Y. (2021a) Morphology and phylogeny of Gnomoniopsis (Gnomoniaceae, Diaporthales) from Fagaceae leaves in China. Journal of Fungi 7(10): e792. 10.3390/jof7100792 [DOI] [PMC free article] [PubMed]
  19. Jiang N, Voglmayr H, Piao CG, Li Y. (2021b) Two new species of Diaporthe (Diaporthaceae, Diaporthales) associated with tree cankers in the Netherlands. MycoKeys 85: 31–56. 10.3897/mycokeys.85.73107 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Katoh K, Toh H. (2010) Parallelization of the MAFFT multiple sequence alignment program. Bioinformatics 26(15): 1899–1900. 10.1093/bioinformatics/btq224 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lin L, Pan M, Tian CM, Fan XL. (2022) Fungal richness of Cytospora species associated with willow canker disease in China. Journal of Fungi 8(4): 377. 10.3390/jof8040377 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Liu YJ, Whelen S, Hall BD. (1999) Phylogenetic relationships among ascomycetes: Evidence from an RNA polymerse II subunit. Molecular Biology and Evolution 16(12): 1799–1808. 10.1093/oxfordjournals.molbev.a026092 [DOI] [PubMed] [Google Scholar]
  23. Miller MA, Pfeiffer W, Schwartz T. (2010) Creating the CIPRES Science Gateway for Inference of Large Phylogenetic Trees; Institute of Electrical and Electronics Engineers: New Orleans, LA, USA, 1–7. 10.1109/GCE.2010.5676129 [DOI]
  24. Old KM, Wingfield MJ, Yuan ZQ. (2003) A manual of diseases of eucalypts in South-East Asia. Centre for International Forestry Research, Jakarta, Indonesia, 1–106.
  25. Pan M, Zhu HY, Liang LY, Tian CM, Fan XL. (2021) Studies of canker and dieback of oak tree in China, with two Cytospora species described. Plant Pathology 70(9): 2005–2015. 10.1111/ppa.13435 [DOI] [Google Scholar]
  26. Pasche S, Calmin G, Auderset G, Crovadore J, Pelleteret P, Mauch-Mani B, Barja F, Paul B, Jermini M, Lefort F. (2016) Gnomoniopsissmithogilvyi causes chestnut canker symptoms in Castaneasativa shoots in Switzerland. Fungal Genetics and Biology 87: 9–21. 10.1016/j.fgb.2016.01.002 [DOI] [PubMed] [Google Scholar]
  27. Rigling D, Prospero S. (2017) Cryphonectriaparasitica, the causal agent of chestnut blight: Invasion history, population biology and disease control. Molecular Plant Pathology 19(1): 7–20. 10.1111/mpp.12542 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ronquist F, Huelsenbeck JP. (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19(12): 1572–1574. 10.1093/bioinformatics/btg180 [DOI] [PubMed] [Google Scholar]
  29. Rossman AY, Farr DF, Castlebury LA. (2007) A review of the phylogeny and biology of the Diaporthales. Mycoscience 48(3): 135–144. 10.1007/S10267-007-0347-7 [DOI]
  30. Senanayake IC, Crous PW, Groenewald JZ, Maharachchikumbura SSN, Jeewon R, Phillips AJL, Bhat DJ, Perera RH, Li QR, Li WJ, Tangthirasunun N, Norphanphoun C, Karunarathna SC, Camporesi E, Manawasighe IS, Al-Sadi AM, Hyde KD. (2017) Families of Diaporthales based on morphological and phylogenetic evidence. Studies in Mycology 86(1): 217–296. 10.1016/j.simyco.2017.07.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Senanayake IC, Jeewon R, Chomnunti P, Wanasinghe DN, Norphanphoun C, Karunarathna A, Pem D, Perera RH, Camporesi E, McKenzie EHC, Hyde KD, Karunarathna SC. (2018) Taxonomic circumscription of Diaporthales based on multigene phylogeny and morphology. Fungal Diversity 93(1): 241–443. 10.1007/s13225-018-0410-z [DOI] [Google Scholar]
  32. Shuttleworth LA, Guest DI. (2017) The infection process of chestnut rot, an important disease caused by Gnomoniopsissmithogilvyi (Gnomoniaceae, Diaporthales) in Oceania and Europe. Australasian Plant Pathology 46(5): 397–405. 10.1007/s13313-017-0502-3 [DOI] [Google Scholar]
  33. Stamatakis A. (2014) RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30(9): 1312–1313. 10.1093/bioinformatics/btu033 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Udayanga D, Miriyagalla SD, Manamgoda DS, Lewers KS, Gardiennet A, Castlebury LA. (2021) Molecular reassessment of diaporthalean fungi associated with strawberry, including the leaf blight fungus, Paraphomopsisobscurans gen. et comb. nov. (Melanconiellaceae). IMA Fungus 12(1): 1–21. 10.1186/s43008-021-00069-9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Vilgalys R, Hester M. (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172(8): 4238–4246. 10.1128/jb.172.8.4238-4246.1990 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Visentin I, Gentile S, Valentino D, Gonthier P, Tamietti G, Cardinale F. (2012) Gnomoniopsiscastanea sp. nov.(Gnomoniaceae, Diaporthales) as the causal agent of nut rot in sweet chestnut. Journal of Plant Pathology 94: 411–419. [Google Scholar]
  37. Voglmayr H, Rossman AY, Castlebury LA, Jaklitsch WM. (2012) Multigene phylogeny and taxonomy of the genus Melanconiella (Diaporthales). Fungal Diversity 57(1): 1–44. 10.1007/s13225-012-0175-8 [DOI] [Google Scholar]
  38. Voglmayr H, Castlebury LA, Jaklitsch WM. (2017) Juglanconis gen. nov. on Juglandaceae, and the new family Juglanconidaceae (Diaporthales). Persoonia 38(1): 136–155. 10.3767/003158517X694768 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wang W, Li GQ, Liu QL, Chen SF. (2020) Cryphonectriaceae on Myrtales in China: Phylogeny, host range, and pathogenicity. Persoonia 45(1): 101–131. 10.3767/persoonia.2020.45.04 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. White TJ, Bruns T, Lee S, Taylor J. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protocols: A Guide to Methods and Applications 18: 315–322. 10.1016/B978-0-12-372180-8.50042-1 [DOI] [Google Scholar]
  41. Wijayawardene NN, Hyde KD, Lumbsch HT, Liu JK, Maharachchikumbura SSN, Ekanayaka AH, Tian Q, Phookamsak R. (2018) Outline of Ascomycota: 2017. Fungal Diversity 88(1): 167–263. 10.1007/s13225-018-0394-8 [DOI] [Google Scholar]
  42. Wingfield MJ. (1989) First record of Cryphonectria canker of Eucalyptus in South Africa. Phytophylactica 21: 311–313. [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

XML Treatment for Pseudocryphonectria
mycokeys.91.86693-treatment1.xml (11.4KB, xml)
XML Treatment for Pseudocryphonectria elaeocarpicola
mycokeys.91.86693-treatment2.xml (20.4KB, xml)

Articles from MycoKeys are provided here courtesy of Pensoft Publishers

RESOURCES