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Research Article
Pseudocryphonectria elaeocarpicola gen. et sp. nov. (Cryphonectriaceae, Diaporthales) causing stem blight of Elaeocarpus spp. in China
expand article infoHua-Yi Huang, Huan-Hua Huang, Dan-Yang Zhao, Ti-Jiang Shan§, Li-Li Hu
‡ Protection and Utilization, Guangdong Academy of Forestry, Guangzhou, China
§ South China Agricultural University, Guangzhou, China
Open Access

Abstract

Cryphonectriaceae is a diaporthalean family containing important plant pathogens of which Cryphonectria parasitica is the most notorious one. An emerging stem blight disease on Elaeocarpus apiculatus (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, Pseudocryphonectria elaeocarpicola gen. 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, Cryphonectria parasitica causes chestnut (Castanea spp.) blight disease worldwide (Rigling and Prospero 2017); Chrysoporthe austroafricana, 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 Aurantiosacculus castaneae 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 Elaeocarpus apiculatus 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. 

Symptoms caused by Pseudocryphonectria elaeocarpicola 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
Amphilogia gyrosa CBS 112922* AF452111 AY194107 MN271818 MN271782
Amphilogia gyrosa CBS 112923 AF452112 AY194108 MN271819 MN271783
Aurantioporthe corni CMW 10526 DQ120762 AF408343 NA NA
Aurantioporthe corni CBS 245.90 MN172403 MN172371 MN271822 MN271784
Aurantiosacculus acutatus CBS 132181* JQ685514 JQ685520 MN271823 NA
Aurantiosacculus eucalyptorum CBS 130826* JQ685515 JQ685521 MN271824 MN271785
Aurantiosacculus castaneae CFCC 52456* MH514025 MH514015 NA MN271786
Aurapex penicillata CBS 115740* AY214311 AY194103 NA NA
Aurapex penicillata CBS 115742 AY214313 MN172372 NA NA
Aurapex penicillata CBS 115801 MN172404 MN172373 NA MN271787
Aurifilum marmelostoma CBS 124928* FJ890495 MH874934 MN271827 MN271788
Aurifilum marmelostoma CBS 124929 FJ882855 HQ171215 MN271828 MN271789
Capillaureum caryovora CBL02* MG192094 MG192104 NA NA
Celoporthe dispersa CBS 118782* DQ267130 HQ730853 HQ730840 NA
Celoporthe eucalypti CBS 127190* HQ730837 HQ730863 HQ730850 MN271790
Celoporthe guangdongensis CBS 128341* HQ730830 HQ730856 HQ730843 NA
Celoporthe syzygii CBS 127218* HQ730831 HQ730857 HQ730844 NA
Celoporthe woodiana CBS 118785* DQ267131 MN172375 JQ824071 MN271791
Chrysomorbus lagerstroemiae CBS 142594* KY929338 KY929328 MN271830 NA
Chrysomorbus lagerstroemiae CBS 142592 KY929330 KY929320 MN271831 NA
Chrysoporthe austroafricana CBS 112916* AF292041 AY194097 MN271832 NA
Chrysoporthe austroafricana CBS 115843 AF273473 MN172377 MN271833 NA
Chrysoporthe cubensis CBS 118654* DQ368773 MN172378 MN271834 NA
Chrysoporthe cubensis CBS 505.63 AY063476 MN172379 MN271835 MN271792
Chrysoporthe hodgesiana CBS 115854* AY692322 MN172380 MN271836 MN271793
Chrysoporthe hodgesiana CBS 115744 AY956970 MN172381 MN271837 NA
Chrysoporthe inopina CBS 118659* DQ368777 MN172382 MN271838 NA
Chrysoporthe syzygiicola CBS 124488* FJ655005 MN172383 MN271839 NA
Chrysoporthe zambiensis CBS 124503* FJ655002 MN172384 MN271840 NA
Corticimorbus sinomyrti CBS 140205* KT167169 KT167179 MN271841 MN271794
Corticimorbus sinomyrti CBS 140206 KT167170 KT167180 MN271842 MN271795
Cryphonectria citrina CBS 109758* MN172407 EU255074 MN271843 EU219342
Cryphonectria decipens CBS 129351 EU442657 MN172385 MN271844 MN271796
Cryphonectria decipens CBS 129353 EU442655 MN172386 MN271845 MN271797
Cryphonectria japonica CFCC 52148 MH514033 MH514023 MN271846 NA
Cryphonectria macrospora CBS 109764 EU199182 AF408340 NA EU220029
Cryphonectria neoparasitica CFCC 52146* MH514029 MH514019 MN271847 NA
Cryphonectria parasitica ATCC 38755 MH843497 MH514021 NA DQ862017
Cryphonectria parasitica CFCC 52150 AY141856 EU199123 MN271848 NA
Cryphonectria quercus CFCC 52138* MG866024 NA MN271849 NA
Cryphonectria quercicola CFCC 52141* MG866027 NA MN271850 NA
Cryphonectria radicalis CBS 112917 AF452113 AY194101 NA NA
Cryptometrion aestuescens CBS 124007* GQ369457 MN172387 MN271851 MN271798
Cryptometrion aestuescens CBS 124008 GQ369458 HQ171211 MN271852 MN271799
Diaporthe eres LC3198 KP267873 KY011845 KP267947 NA
Diversimorbus metrosiderotis CBS 132866* JQ862871 JQ862828 MN271857 NA
Diversimorbus metrosiderotis CBS 132865 JQ862870 JQ862827 MN271858 NA
Endothia chinensis CFCC 52144* MH514027 MH514017 MN271860 NA
Holocryphia eucalypti CBS 115842* MN172411 MN172391 MN271882 MN271804
Holocryphia capensis CBS 132870* JQ862854 JQ862811 MN271883 NA
Holocryphia gleniana CBS 132871* JQ862834 JQ862791 MN271884 NA
Holocryphia mzansi CBS 132874* JQ862841 JQ862798 MN271885 NA
Immersiporthe knoxdaviesiana CBS 132862* JQ862765 JQ862755 MN271886 MN271805
Immersiporthe knoxdaviesiana CBS 132863 JQ862766 JQ862756 MN271887 MN271806
Latruncellus aurorae CBS 125526* GU726947 HQ730872 MN271888 NA
Latruncellus aurorae CBS 124904 GU726946 HQ171213 MN271889 NA
Luteocirrhus shearii CBS 130776* KC197021 KC197019 MN271890 MN271807
Luteocirrhus shearii CBS 130775 KC197024 KC197018 MN271891 MN271808
Microthia havanensis CBS 115855 DQ368735 MN172393 NA MN271811
Microthia havanensis CBS 115841 DQ368736 MN172394 NA NA
Microthia havanensis CBS 115758 DQ368737 MN172395 NA NA
Myrtonectria myrtacearum CMW 46433* MG585736 MG585750 NA NA
Myrtonectria myrtacearum CMW 46435 MG585737 MG585751 NA NA
Parvosmorbus eucalypti CSF2060 MN258787 MN258843 MN258829 NA
Parvosmorbus guangdongensis CSF10437 MN258795 MN258851 MN258837 NA
Pseudocryphonectria elaeocarpicola CFCC 57515* ON489048 ON489050 ON456916 ON456918
Pseudocryphonectria elaeocarpicola CFCC 57516 ON489049 ON489051 ON456917 ON456919
Rostraureum tropicale CBS 115725* AY167435 MN172399 MN271895 MN271814
Rostraureum tropicale CBS 115757 AY167438 MN172400 MN271896 MN271815
Ursicollum fallax CBS 118663* DQ368755 EF392860 MN271897 MN271816
Ursicollum fallax CBS 118662 DQ368756 MN172401 MN271898 MN271817

Pathogenicity tests

Three isolates of the new species Pseudocryphonectria elaeocarpicola (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 Elaeocarpus apiculatus 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 Elaeocarpus apiculatus 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 Elaeocarpus apiculatus 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

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, Diaporthe eres (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. 

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 Diaporthe eres (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, Diaporthe eres (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. 

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 Diaporthe eres (LC 3198). Isolates from the present study are marked in blue, and ex-type strains are marked with *.

Taxonomy

Pseudocryphonectria Huayi Huang, gen. nov.

MycoBank No: 844044

Etymology

Named derived from pseudo- and the genus name Cryphonectria.

Type species

Pseudocryphonectria elaeocarpicola 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.

MycoBank No: 844045
Figs 4, 5

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.

Figure 4. 

Morphology of Pseudocryphonectria elaeocarpicola from Elaeocarpus hainanensis A, 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).

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.

Figure 5. 

Morphology of Pseudocryphonectria elaeocarpicola from PDA A, B colonies C, D orange conidiomata.

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 Elaeocarpus hainanensis, 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

Pseudocryphonectria elaeocarpicola is the sole species within the new genus, which causes serious stem blight of Elaeocarpus trees. Another notorious pathogen in Cryphonectriaceae, Cryphonectria parasitica, 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 Elaeocarpus apiculatus 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 Pseudocryphonectria elaeocarpicola 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. 

Results of pathogenicity tests on Elaeocarpus apiculatus 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. 

Histogram of lesion lengths resulting from inoculation on Elaeocarpus apiculatus 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 Elaeocarpus apiculatus and E. hainanensis was identified using both morphological and phylogenetical approaches, which revealed a new genus and species, namely Pseudocryphonectria elaeocarpicola. 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 Cryphonectria parasitica 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.

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.

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