Cytospora paraplurivora sp. nov. isolated from orchards with fruit tree decline syndrome in Ontario, Canada

Abstract

A new species of Cytospora was isolated from cankered wood of Prunus spp. during a survey of orchards exhibiting symptoms of fruit tree decline syndrome in southern Ontario, Canada. We found isolates that are morphologically similar to species in the Cytosporaceae family, which is characterized by single or labyrinthine locules, filamentous conidiophores or clavate to elongate obovoid asci and allantoid, hyaline conidia. Multi-gene phylogenetic analysis of ITS, LSU, act and tef1- α showed that the isolates form a distinct clade, sister to Cytospora plurivora. Morphologically, our isolates showed differences in the length of conidia and culture characteristics compared to C. plurivora, suggesting the establishment of a new species. The species is described as Cytospora paraplurivora sp. nov. and placed in the family Cytosporaceae of Diaporthales. Additionally, we sequenced, assembled and characterized the genome of the representative isolate for this new species. The phylogenomic analysis confirms the species order and family level classification. C. paraplurivora sp. nov. has the potential to severely affect stone fruits production, causing cankers and dieback in stressed trees, and eventually leads to tree decline. Pathogenicity tests show that the species is pathogenic to Prunus persica var. persica.

Introduction

Stone fruit trees are economically important crops cultivated in Ontario, Canada. The Niagara Peninsula produces more than 90% of its peaches, nectarines and apricots [1]. Peach occupy the largest production areas, followed by nectarines and apricots. Fruit tree decline syndrome (FTDS) has been recently discovered in several orchards in this area of the province. The common symptoms include stem canker and dieback, wilting, increased suckering, leaf discolouration and eventual total collapse of the tree. Disease incidence ranged from 49 to 72% for apricots and nectarines (Fig 1). Similar disease symptoms were observed in stone fruits and apple trees in other regions of North America [2, 3]. A number of studies reported that Cytospora spp. was among the causative agents of canker and dieback diseases in fruit trees including Prunus spp. [4, 5]. Generally, members of the Cytospora spp. infect trees that are stressed by extreme weather conditions (drought or freezing), and by invading wounds in the bark caused by insect damage and improper pruning. These fungi commonly overwinter in a form of pycnidia embedded in the bark of cankered branches [6].

Fig 1. Nectarine trees showing fruit tree decline syndrome (FTDS) in southern Ontario, Canada.

The genus Cytospora (Cytosporacea, Diaporthales) was established by Ehrenberg in 1818. There are currently 676 records of Cytospora spp. registered in the IndexFungorum (www.indexfungorum.org; accessed Nov 2021). However, 163 species of the genus with available nucleotide sequences are listed in the NCBI taxonomy database (www.ncbi.nlm.nih.gov/taxonomy; accessed May 2022). Approximately, 150 species in the genus have been reported to cause diseases in more than 120 woody plants, which can result in significant commercial losses for growers [7–9]. The taxonomic species identification of Cytospora spp. is mainly based on morphological characteristics and molecular phylogenetic analysis. Recent studies described a number of new species of Cytospora on different hosts using multi-gene (ITS, LSU, act, tef1- α, tub2, and rpb2) phylogenetic analysis [5, 10–12]. This approach allows for the identification of cryptic and novel species within the genus. Obtaining high-quality, cost-effective genomic data is now possible through rapidly developing sequencing technologies. Whole-genome sequence data used in phylogenomics can contribute to resolving taxonomic uncertainties or support species reclassifications [13]. One such study found that a polyphagous plant pathogen, Corynespora olivacea was previously misclassified on family level [14].

The objective of this paper was to identify and characterize a novel Cytopora species associated with the decline of Prunus armeniaca, P. persica var. persica, P. persica var. nucipersica in Ontario, Canada. The characterization of the new species was performed following recently published guidelines [15, 16].

Materials and methods

Sample collection and isolation

Wood samples were collected in 2018–2021 from 30 apricot, 6 peach and 6 nectarine trees exhibiting extensive tree fruit decline symptoms from nine commercial orchards located in southern Ontario. Roots were symptom-free. The cankered and diseased wood sections from trunk and rootstock were cut into 1 cm pieces, and surface disinfested with 70% ethanol for 30 sec, followed by 1% NaClO for 20 min and three rinses in sterile distilled water. The samples were air-dried and placed on a 2% potato dextrose agar (PDA, Difco, USA) supplemented with kanamycin (50 mg L−1). The PDA plates were incubated at 22°C for 5 days in the dark. All fungal colony-forming units were hyphal-tip transferred to individual PDA plates and incubated at 22°C for 7 days in the dark. Purified mycelial isolates were classified into morphotypes prior to molecular identification. C. paraplurivora was characterized by fast-growing, white to cream with uneven lobate growth margin colonies.

Tree branches with fruiting structures (conidiomata) were checked separately. Single conidia isolations were performed using the protocol described by Chomnunti et al. [17]. For long-term preservation, fungal cultures were stored at −80°C in 30% glycerol. The holotype specimen was deposited in the Canadian National Mycological Herbarium (DAOM) and the living culture collection maintained by the Canadian Collection of Fungal Cultures (DAOMC).

Morphological examination

Conidiomata formed on the tree branches were described prior to sectioning with a sterile surgical scalpel. The macro-morphological structures were measured using a dissecting microscope (OMAX 2000X Infinity Compound Siedentopf Microscope with a Built-in Camera). In order to calculate the mean size of the structures, 10 conidiomata, 20 conidiogenous cells and 50 conidia were measured. The obtained measurements were recorded with minimum and maximum values and means were calculated. Radial growth of fungal colonies was estimated with two perpendicular measurements after 5 days of incubation. Morphology description [18] and color characterisation [19] were performed after 7 days.

DNA extraction, PCR amplification and Sanger sequencing

Genomic DNA was extracted from mycelium using the Plant/Fungi DNA Isolation Kit (Norgen Biotech Corp., Thorold, Canada) with the following modifications: fungal tissue was vortexed for 15 minutes with 1 mm glass beads and 500 μL lysis buffer and 1 μL RNase A prior to incubation at 65°C. After incubation on ice, the fungal mixture was centrifuged at 10,000 rpm to separate the lysate from the beads and biomass. During the column wash, the resin was dried by spinning for 10 minutes at 14,000 rpm. DNA was eluted at 10,000 rpm for 2 minutes. PCR amplifications were executed using C1000 Touch PCR thermal cycler (Bio-Rad, Hercules, USA) under conditions described in the references for each region. The internal transcribed spacer (ITS) region was amplified with the primer pair ITS1/ITS4 [20]. The primer pair LR0R/LR5 [21] was used to amplify the large subunit rRNA gene (LSU). The partial actin (ACT) region was amplified with the primer pair ACT512F/ACT783R [22], and the primer pair EF1-728F/ EF1-986R [22] was used to amplify partial translation elongation factor 1-alpha (tef1-α) gene sequences. The quality of the PCR products was examined using electrophoresis in 1% agarose gel. Sanger sequencing was carried out at Genome Quebec’s Sequencing Facility (Montreal, Canada).

Sequence alignment and phylogenetic analysis

The consensus sequences were built from Sanger chromatograms using BioEdit [23]. The ITS, LSU, act, tef1-α sequences from our study were uploaded on GenBank (http://www.ncbi.nlm.nih.gov/) on the Basic Local Alignment Search Tool (BLAST) [24] to find other similar sequences. The sequences of Cytospora spp. from the top BLAST hits were then extracted and added to recently published sequence datasets [5, 9]. Diaporthe vaccinii (CBS 160.32) of Diaporthaceae was used as the outgroup. All the sequences were initially aligned using CLUSTAL-X2 [25] and edited manually in MEGA-X [26]. Some characters were trimmed from both ends of the alignments. Randomized Accelerated Maximum Likelihood (RAxML) v. 8.0 [27] was used for Maximum Likelihood (ML) analysis [28] and MrBayes v. 3.2.7 for Bayesian Inference (BI) analysis [29].

ML analysis was performed using a general time reversible substitution (GTR) model with gamma-distributed rate of heterogeneity and proportion of invariant sites [30]. The model was selected with ModelTest-NG v. 0.1.7 [31] based on the Akaike Information Criterion [32]. Branch support was estimated with bootstrapping of 1000 replicates [33]. Bayesian probabilities (BP) values were defined by Markov Chain Monte Carlo (MCMC) sampling with the GTR model. Six simultaneous Markov Chains were run for 100,000 generations. The first 500 trees were discarded, and the remaining trees were used to calculate BP in the majority rule consensus tree. Phylograms were visualized using FigTree v. 1.4.4 [34]. The newly generated sequences were deposited in GenBank (Table 1). The alignments used in the analyses were submitted into TreeBase (www.treebase.org; ID: 29116).

Table 1. Strains used in the phylogenetic analysis with their culture accession and GenBank numbers.

Strains from this study are in bold and ex-types are marked with*. NA: not available.

Species	Strain	Host	Origin	GenBank accession numbers
				ITS	LSU	act	tef1-α
Cytospora ailanthicola	CFCC 89970*	Ailanthus altissima	China	MH933618	MH933653	MH933526	MH933494
C. ampulliformis	MFLUCC 16–0583*	Sorbus intermedia	Russia	KY417726	KY417760	KY417692	NA
	MFLUCC 16–0629	Acer platanoides	Russia	KY417727	KY417761	KY417693	NA
C. amygdali	LH357*	Prunus dulcis	USA	MG971853	NA	MG972002	MG971659
C. atrocirrhata	CFCC 89615	Juglans regia	China	KR045618	KR045700	KF498673	KP310858
	CFCC 89616	Juglans regia	China	KR045619	KR045701	KF498674	KP310859
C. beilinensis	CFCC 50493*	Pinus armandii	China	MH933619	MH933654	MH933527	MH933495
	CFCC 50494	Pinus armandii	China	MH933620	MH933655	MH933528	MH933496
C. berberidis	CFCC 89927*	Berberis dasystachya	China	KR045620	KR045702	KU710990	KU710913
	CFCC 89933	Berberis dasystachya	China	KR045621	KR045703	KU710991	KU710914
C. bungeanae	CFCC 50495*	Pinus bungeana	China	MH933621	MH933656	MH933529	MH933497
	CFCC 50496	Pinus bungeana	China	MH933622	MH933657	MH933530	MH933498
C. californica	9C-24*	Juglans regia	USA	MG971935	NA	MG972083	MG971645
C. carbonacea	CFCC 89947	Ulmus pumila	China	KR045622	KP310812	KP310842	KP310855
C. carpobroti	CMW 48981*	Carpobrotus edulis	S. Africa	MH382812	MH411216	NA	MH411212
C. celtidicola	CFCC 50497*	Celtis sinensis	China	MH933623	MH933658	MH933531	MH933499
	CFCC 50498	Celtis sinensis	China	MH933624	MH933659	MH933532	MH933500
C. ceratosperma	CFCC 89624	Juglans regia	China	KR045645	KR045724	NA	KP310860
	CFCC 89625	Juglans regia	China	KR045646	KR045725	NA	KP31086
C. ceratospermopsis	CFCC 89626*	Juglans regia	China	KR045647	KR045726	KU711011	KU710934
	CFCC 89627	Juglans regia	China	KR045648	KR045727	KU711012	KU710935
C. chrysosperma	CFCC 89629	Salix psammophila	China	KF765673	KF765689	NA	NA
	CFCC 89981	Populus alba	China	MH933625	MH933660	MH933533	MH933501
C. coryli	CFCC 53162*	Corylus mandshurica	China	MN854450	MN854661	NA	MN850758
C. davidiana	YW2014*	Populus davidiana	China	KM034870	NA	NA	NA
	CXY 1374	Populus davidiana	China	KM034869	NA	NA	NA
C. elaeagni	CFCC 89632	Elaeagnus angustifolia	China	KR045626	KR045706	KU710995	KU710918
	CFCC 89633	Elaeagnus angustifolia	China	KF765677	KF765693	KU710996	KU710919
C. elaeagnicola	CFCC 52883	Elaeagnus angustifolia	China	MK732342	NA	MK732345	NA
	CFCC 52884	Elaeagnus angustifolia	China	MK732343	NA	MK732346	NA
C. erumpens	CFCC 50022	Prunus padus	China	MH933627	MH933661	MH933534	MH933502
	MFLUCC 16–0580*	Salix x fragilis	Russia	KY417733	KY417767	KY417699	NA
C. eucalypti	KARE1585	Eucalyptus globulus	USA	MG971907	NA	MG972056	MG971617
C. euonymicola	CFCC 50499*	Euonymus kiautschovicus	China	MH933628	MH933662	MH933535	MH933503
	CFCC 50500	E. kiautschovicus	China	MH933629	MH933663	MH933536	MH933504
C. euonymina	CFCC 89993*	E.kiautschovicus	China	MH933630	MH933664	MH933537	MH933505
C. fraxinigena	MFLU 17–0880	Fraxinus ornus	Italy	MF190134	MF190079	NA	NA
C. fugax	CXY 1381	Populus ussuriensis	China	KM034853	NA	NA	NA
C. gigalocus	CFCC 89620*	Juglans regia	China	KR045628	KR045708	KU710997	KU710920
	CFCC 89621	Juglans regia	China	KR045629	KR045709	KU710998	KU710921
C. gigaspora	CFCC 50014	Juniperus procumbens	China	KR045630	KR045710	KU710999.	KU710922
	CFCC 89634*	Salix psammophila	China	KF765671	KF765687	KU711000	KU710923
C. granati	6F-45*	Punica granatum	USA	MG971799	NA	MG971949	MG971514
C. hippophaes	CFCC 89639	Hippophae rhamnoides	China	KR045632	KR045712	KU711001	KU710924
	CFCC 89640	Hippophae rhamnoides	China	KF765682	KF765698	KF765730	KP310865
C. japonica	CFCC 89956	Prunus cerasifera	China	KR045624	KR045704	KU710993	KU710916
C. joaquinensis	KARE975*	Populus deltoides	USA	MG971895	NA	MG972044	MG971605
C. junipericola	BBH 42444	Juniperus communis	Italy	MF190126	MF190071	NA	MF377579
	MFLU 17–0882*	Juniperus communis	Italy	MF190125	MF190072	NA	MF377580
C. juniperina	CFCC 50502	Juniperus przewalskii	China	MH933633	MH933667	MH933540	MH933508
	CFCC 50503	Juniperus przewalskii	China	MH933634	MH933668	MH933541	MH933509
C. leucosperma	CFCC 89622	Pyrus bretschneideri	China	KR045616	KR045698	KU710988	KU710911
	CFCC 89894	Pyrus bretschneideri	China	KR045617	KR045699	KU710989	KU710912
C. leucostoma	HigginsLake4	Alnus incana	USA	JX475137	NA	NA	JX438600
C. longiostiolata	MFLUCC 16–0628*	Salix fragilis	Russia	KY417734	KY417768	KY417700	NA
C. longispora	10F-57*	Prunus domestica	USA	MG971905	NA	MG972054	MG971615
C. mali	CFCC 50028	Malus pumila	China	MH933641	MH933675	MH933548	MH933513
	CFCC 50030	Malus pumila	China	MH933643	MH933677	MH933550	MH933524
C. melnikii	CFCC 89984	Rhus typhina	China	MH933644	MH933678	MH933551	NA
	MFLUCC 15–0851*	Malus domestica	Russia	KY417735	KY417769	KY417701	NA
C. nivea	MFLUCC 15–0860	Salix acutifolia	Russia	KY417737	KY417771	KY417703	NA
C. oleicola	KARE1021*	Olea europaea	USA	MG971944	NA	MG972098	MG971660
C. palm	CXY 1276	Cotinus coggygria	China	JN402990	NA	NA	KJ781296
C. parakantschavelii	MFLUCC 15–0857*	Populus sibirica	Russia	KY417738	KY417772	KY417704	NA
	MFLUCC 16–0575	Pyrus pyraster	Russia	KY417739	KY417773	KY417705	NA
C. parapistaciae	KARE270*	Pistacia vera	USA	MG971804	NA	MG971954	MG971519
C. paraplurivora	FDS-439	Prunus armeniaca	Canada	OL640182	OL640184	OL631586	OL631589
	FDS-564*	Prunus persica var. nucipersica	Canada	OL640183	OL640185	OL631587	OL631590
	FDS-623	Prunus persica var. persica	Canada	OL640181	OL640123	OL631588	OL631591
C. parasitica	MFLUCC 15–0507*	Malus domestica	Russia	KY417740	KY417774	KY417706	NA
	XJAU 2542–1	Malus sp.	China	MH798884	MH798897	NA	MH813452
C. paratranslucens	MFLUCC 15–0506*	Populus alba var. bolleana	Russia	KY417741	KY417775	KY417707	NA
	MFLUCC 16–0627	Populus alba	Russia	KY417742	KY417776	KY417708	NA
C. pistaciae	KARE443*	Pistacia vera	USA	MG971802	NA	MG971952	MG971517
C. platanicola	MFLU 17–0327*	Platanus hybrida	Italy	MH253451	MH253452	MH253449	NA
C. platyclade	CFCC 50504*	Platycladus orientalis	China	MH933645	MH933679	MH933552	MH933516
	CFCC 50505	Platycladus orientalis	China	MH933646	MH933680	MH933553	MH933517
C. platycladicola	CFCC 50039	Platycladus orientalis	China	KR045642	KR045721	KU711008	KU710931
C. plurivora	KARE1452*	Olea europaea	USA	MG971861	NA	MG972010	MG971572
	5L-29	Prunus persica	USA	MG971856	NA	MG972005	MG971567
C. populicola	KARE973*	Populus deltoides	USA	MG971891	NA	MG972040	MG971601
C. populina	CFCC 89644	Salix psammophila	China	KF765686	KF765702	KU711007	KU710930
C. populinopsis	CFCC 50032*	Sorbus aucuparia	China	MH933648	MH933683	MH933556	MH933520
C. pruinopsis	CFCC 50034*	Ulmus pumila	China	KP281259	KP310806	KP310836	KP310849
	CFCC 50035	Ulmus pumila	China	KP281260	KP310807	KP310837	KP310850
C. pruinose	CFCC 50036	Syringa oblata	China	KP310800	KP310802	KP310832	KP310845
C. prunicola	MFLU 17–0995*	Prunus sp.	Italy	MG742350	MG742351	MG742353	NA
C. quercicola	MFLU 17–0881	Quercus sp.	Italy	MF190128	MF190074	NA	NA
	MFLUCC 14–0868*	Quercus sp.	Italy	MF190129	MF190073	NA	NA
C. ribis	CFCC 50026	Ulmus pumila	China	KP281267	KP310813	KP310843	KP310856
	CFCC 50027	Ulmus pumila	China	KP281268	KP310814	KP310844	KP310857
C. rosae	MFLUCC 14–0845*	Rosa canina	Italy	MF190131	MF190075	NA	NA
C. rostrata	CFCC 89909*	Salix cupularis	China	KR045643	KR045722	KU711009	KU710932
C. rusanovii	MFLUCC 15–0854*	Salix babylonica	Russia	KY417744	KY417778	KY417710	NA
C. salicacearum	MFLUCC 15–0861	Salix x fragilis	Russia	KY417745	KY417779	KY417711	NA
	MFLUCC 15–0509*	Salix alba	Russia	KY417746	KY417780	KY417712	NA
C. salicicola	MFLUCC 15–0866	Salix alba	Russia	KY417749	KY417783	KY417715	NA
	MFLUCC 14–1052*	Salix alba	Russia	KU982636	KU982635	KU982637	NA
C. salicina	MFLUCC 15–0862*	Salix alba	Russia	KY417750	KY417784	KY417716	NA
	MFLUCC 16–0637	Salix x fragilis	Russia	KY417751	KY417785	KY417717	NA
C. schulzeri	CFCC 50040	Malus domestica	China	KR045649	KR045728	KU711013	KU710936
	CFCC 50042	Malus asiatica	China	KR045650	KR045729	KU711014	KU710937
C. sibiraeae	CFCC 50045*	Sibiraea angustata	China	KR045651	KR045730	KU711015	KU710938
	CFCC 50046	Sibiraea angustata	China	KR045652	KR045731	KU711015	KU710939
C. sophorae	CFCC 50048	Magnolia grandiflora	China	MH820401	MH820394	MH820409	MH820405
C. sophoricola	CFCC 89596	Styphnolobium japonicum var. pendula	China	KR045656	KR045735	KU711020	KU710943
	CFCC 89595*	Styphnolobium japonicum var. pendula	China	KR045655	KR045734	KU711019	KU710942
C. sophoriopsis	CFCC 89600*	Styphnolobium japonicum	China	KR045623	KP310804	KU710992	KU710915
C. sorbicola	MFLUCC 16–0584*	Acer pseudoplatanus	Russia	KY417755	KY417789	KY417721	NA
	MFLUCC 16–0633	Cotoneaster melanocarpus	Russia	KY417758	KY417792	KY417724	NA
C. spiraeae	CFCC 50049*	Spiraea salicifolia	China	MG707859	MG707643	MG708196	NA
	CFCC 50050	Spiraea salicifolia	China	MG707860	MG707644	MG708197	NA
C. spiraeicola	CFCC 53138*	Spiraea salicifolia	China	MN854448	MN854659	NA	MN850756
	CFCC 53139	Tilia nobilis	China	MN854449	MN854660	NA	MN850757
C. tamaricicola	CFCC 50507	Rosa multifolora	China	MH933651	MH933686	MH933559	MH933525
	CFCC 50508*	Tamarix chinensis	China	MH933652	MH933687	MH933560	MH933523
C. tanaitica	MFLUCC 14–1057*	Betula pubescens	Russia	KT459411	KT459412	KT459413	NA
C. translucens	CXY 1351	Populus davidiana	China	KM034874	NA	NA	NA
C. ulmi	MFLUCC 15–0863*	Ulmus minor	Russia	KY417759	NA	NA	NA
Diaporthe vaccinii	CBS 160.32	Vaccinium macrocarpon	USA	KC343228	NA	JQ807297	KC343954

DNA isolation, genome sequencing and characterization

Highly purified genomic DNA from the isolate FDS-564 was extracted from fungal mycelium grown in a liquid Potato Dextrose Broth for 5 days at room temperature, filtered and freeze-dried before following the modified DNA extraction protocol as previously described (Norgen Biotech Comp., Thorold, Canada). The library was prepared using a SMRT bell Express Template Prep Kit (PacBio, Menlo Park, USA). The library pool was sequenced on one SMRT cell using the PacBio Sequel II platform in the SickKids sequencing facility (Toronto, ON, Canada). The reads were assembled into contigs with the Canu assembler v. 2.1.1 [35]. The quality of genome assemblies was accessed using QUAST v. 5.0.2 [36]. The completeness of assembly was estimated with BUSCO v.4.0.5 employing the dataset Ascomycota_odb10 [37]. Repeat sequences were identified with RepeatMasker v. 4.0.9 [38] using the library of repeats for fungi obtained from Repbase [39]. Gene prediction was executed using Augustus v. 3.4.0 [40]. Augustus was trained with the gene structures from the representative genome of C. mali 03–8 (GCA_000818155) obtained using the MAKER2 pipeline v. 2.31.11 [41].

Phylogenomic analysis and genome alignment

The proteomes of 17 species of ascomycetes, including five species in the Cytosporaceae, were retrieved from the Mycocosm portal [42] (accessed May 2022) and the NCBI Genome database (accessed Dec 2021). Orthofinder v. 2.5.4 [43] was used to identify the single-copy orthogroups (SCO) in the species included in the analysis. Multiple sequence alignments were performed using MAFFT v. 7.489 [44]. The ML tree was produced with FastTree v. 2.1.10 [45]. All runs were performed using the default parameters. Mollisia scopiformis CBS 120377 (Leotiomycetes) was used as an outgroup (Table 2). The tree was visualized in FigTree v. 1.4.4 [34]. Genome alignment was performed with minimap2 v. 2.23 [46] and displayed as a dot-plot graph using D-Genies [47].

Table 2. Genomes used in phylogenomic analyses.

Strains from this study are in bold. NA: not available.

Species name	Strain	GenBank assembly accession	Host	Country	Reference
Botryosphaeria dothidea	sdau11-99	GCA_011503125	Malus sp.	China	[48]
Colletotrichum higginsianum	IMI 349063	GCA_001672515	Brassica rapa subsp. chinensis	Trinidad and Tobago	[49]
Cytospora chrysosperma	CFL2056	NA	NA	Canada	PI permission
Cytospora leucostoma	SXYLt	GCA_003795295	Prunus persica	China	[50]
Cytospora (Valsa) mali	03–8	GCA_000818155	Malus sp.	China	[51]
Cytospora (Valsa) mali var. pyri	SXYL134	GCA_000813385	Malus sp.	China	[51]
Cytospora paraplurivora	FDS-564	GCA_021272945	Prunus persica var. nucipersica	Canada	This study
Cytospora piceae	CFCC52841	GCA_016508685	Picea crassifolia	China	PI permission
Diaporthe ampelina	DA912	GCA_001006365	Vitis vinifera	USA	[52]
Diaporthe helianthi	7/96	GCA_001702395	Helianthus sp.	France	[53]
Macrophomina phaseolina	MS6	GCA_000302655	Corchorus olitorius	Bangladesh	[54]
Mollisia scopiformis	CBS 120377	GCA_001500285	Picea glauca	Canada	[55]
Neofusicoccum parvum	UCRNP2	GCA_000385595	Vitis vinifera	USA	[56]
Neurospora crassa	FGSC 73	GCA_000786625	NA	USA	[57]
Peltaster fructicola	LNHT1506	GCA_001592805	Malus sp.	China	[58]
Pyrenophora tritici-repentis	Ptr	GCA_000149985	Triticum sp.	USA	[59]
Pyricularia oryzae	70–15	GCA_000002495	Oryza sativa	USA	[60]
Zymoseptoria tritici	IPO323	GCA_000219625	Triticum sp.	Netherlands	[61]

Excised branch pathogenicity trials

Excised branches collected from non-symptomatic apricot, nectarine and peach trees were used to test the pathogenicity of C. paraplurivora isolates following the protocol by Arzanlou and Narmani [62]. Thirteen green lateral branches, with a mean diameter of 1 cm and 20 cm in length, were excised from each tree species. The leaves were removed, and the branches were surface disinfested with 70% ethanol for 10 min, rinsed 3 times with sterile water and air-dried. Ten branches were wounded and inoculated with 4 mm mycelium agar plug from a 5-day old cultures of each isolate and wrapped with Parafilm (Parafilm^® "M", MilliporeSigma, Canada). Three controls were inoculated with sterile PDA plugs. Excised branches were placed inside a clear plastic container with moist paper towels and incubated at room temperature in the dark. The branches were checked 12 days post-inoculation to measure the necrotic lesions. Longitudinal sections were made from the inoculation point for further lesion examination.

Living plants pathogenicity trials

The peach seedlings (cv. Loring) were grown from seeds and placed in a biosafety plant growth chamber under a temperature of 23°C and a photoperiod of 16 h. A completely randomized design was used for the experiment. Inoculations were performed on four-month-old seedlings. A total of five seedlings were wounded and inoculated with 4 mm mycelium agar plug from a 5-day old cultures of each isolate and wrapped with Parafilm (Fig 7). Five seedlings were inoculated with sterile PDA plugs as negative control. The developing lesions were measured 7, 10, 13 and 27 days post-inoculation (dpi).

Fig 7.

Symptoms of Cytospora paraplurivora infection on Prunus persica var. persica seedlings including A) 5-day old canker (ck) and gummosis (g); B) 7-day old canker; and C) 27-day old canker, conidiomata (cd) and gummosis.

Re-Isolation of the pathogen

C. paraplurivora was re-isolated from the excised branches and seedlings stem fragments and from fruiting structures (conidiomata). Three tissue fragments (0.5 cm long) were taken from the advanced necrosis and from the lesion edge of each branch and seedling. The tissue fragments were surface disinfested with 70% ethanol for 30 sec, followed by 1% NaClO for 20 min and three rinses in sterile distilled water. The samples were air-dried and placed on a 2% PDA supplemented with kanamycin (50 mg L⁻¹). The PDA plates were incubated at 22°C for 5 days in the dark. All fungal colony-forming units were hyphal-tip transferred to individual PDA plates and incubated at 22°C for 7 days in the dark. One conidiomata was taken from each infected seedling and the pathogen was re-isolated using the protocol described by Chomnunti et al. [17]. The identification of C. paraplurivora re-isolated from lesions and fruiting structures was made on the basis of morphological features using reference cultures and PCR with the primer pair EF1-728F/ EF1-986R [22], as described above, to fulfil Koch’s postulates.

Nomenclature

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In addition, new names contained in this work have been submitted to Index Fungorum and MycoBank from where they will be made available to the Global Names Index. The unique Index Fungorum number can be resolved and the associated information viewed through any standard web browser by appending the Index Fungorum number contained in this publication to the prefix www.indexfungorum.org/. The online version of this work is archived and available from the following digital repositories: PubMed Central, LOCKSS.

Results

The decline disease has affected up to 72% of the trees in the surveyed stone fruits orchards in southern Ontario. C. paraplurivora was the only Cytospora spp. isolated from symptomatic trees in all surveyed orchards. The incidence of C. paraplurivora on symptomatic trees was 50% in peaches, 57% in apricots and 100% in nectarines.

Phylogenetic analysis

The ML and BI analyses based on a concatenated alignment of ITS, LSU, act and tef1- α from 119 strains of Cytosporaceae produced phylogenetic trees with similar topology to those in recent taxonomic studies of Cytospora [5, 9, 12]. The best scoring RAxML tree is depicted in Fig 2. The strains of C. paraplurivora obtained in this study are clustered together and formed a distinct clade with strong bootstrap support values (100% ML, 1.00 BP). The closest species, C. plurivora is grouped with the new species in the well supported clade (100% ML, 1.00% BP). Broadly, the taxa with leucocytosporoid condiomata (C. amydgali, C. davidiana, C. erumpens, C. gigaspora, C. leucostoma, C. nivea, C. paratranslucens, C. rusanovii, C. sorbicola, C. translucens) also constitute a separate clade (88% ML, 1.00 BP).

Fig 2. Maximum likelihood (ML) phylogeny based on a concatenated ITS, LSU, act, tef1-α sequence alignment.

Bootstrap support values for ML ≥ 50% and BP ≥ 0.90 are defined as ML/BP above and below the nodes. Strains of the new species are in bold, and ex-type strains are marked with*. The tree is rooted to Diaporthe vaccinii (CBS 160.32).

Taxonomy

Cytospora paraplurivora Ilyukhin & Ellouze, sp. nov. (Fig 3).

Fig 3. C. paraplurivora on P. persica var. nucipersica (FDS-564).

A. Habit of conidiomata on branches. B. Longitudinal section through conidiomata. C. Transverse section of conidiomata. D. Conidiogenous cells. E. Conidia. F. Seven-day-old culture on PDA. Scale bars: A = 200 μm B-C = 100 μm, D-E = 5 μm.

Index Fungorum No: IF559353

Mycobank No: MB842135

Facesoffungi No: FoF19613

Etymology—the species epithet refers to Greek prefix “para-” meaning “close” and latin “plurivora” is the species name of Cytospora plurivora.

Holotype—DAOM 984922

Pathogenic on seedlings and branches of Prunus persica var. persica.

Sexual morph: not observed. Asexual morph: Conidiomata 440–720 × 220–310 μm diameter (n = 10), semi-immersed in host tissue, erumpent, discoid, solitary, circular to ovoid, scattered, multiloculate, with long ostiolar neck. Ostioles 125–160 μm diameter, at the same level as the disc or higher. Conidiophores unbranched, reduced to conidiogenous cells. Conidiogenous cells 7.5–10 μm (n = 20), blastic, phialidic, originate from inner layer of pycnidial wall, hyaline, smooth-walled. Conidia (4.8–)5.5–7.2(–7.4) × 1.2–1.5(–1.7) μm ($\overline{\mathrm{x}}$ = 6.4 × 1.3 μm, n = 50), hyaline, allantoid, somewhat elongate, aseptate.

Culture characteristics: colonies on PDA are fast-growing, reaching 6.2 cm in diameter after 5 days at 25^°C in the dark; initially white turning olivaceous after 5–7 days with thick texture at center, uneven lobate growth margin, lacking aerial mycelium; irregular, abundant pycnidia develop after 14 days. Hyphae hyaline, smooth, branched and septate.

Notes: based on morphological characteristics and phylogenetic analysis, C. paraplurivora is closest to C. plurivora isolated from different hosts in California (USA). This species is found to be associated with a canker disease of fruit (including Prunus spp.) and nut trees. Both species have common morphological characteristics but conidia of C. paraplurivora are notably longer than those of C. plurivora (6.4 × 1.3 μm versus 4.1 × 1.0 μm). Lack of olivaceous color of the C. plurivora cultures grown on PDA and incubated at the same conditions also differentiates these species [5].

Material examined: CANADA, southwestern Ontario, isolated from main stem of Prunus armeniaca, 2019, W. Ellouze, K.E. Schneider, FDS-439; southern Ontario, isolated from single conidium, pycnidia collected from branches of Prunus persica var. nucipersica, Nov. 2019, E. Ilyukhin, W. Ellouze FDS-564 (DAOM 984922 = holotype; DAOMC 252466 = ex-type living culture); southern Ontario, isolated from main stem of Prunus persica var. persica, Apr. 2021, W. Ellouze, K.E. Schneider, FDS-623 (DAOMC 252525 = ex-type living culture).

Genome assembly

A total of 6 854 783 single-end long reads (NCBI SRA accession no. SRR17267775) were generated from PacBio sequencing of FDS-564 and assembled into 211 contigs (> 1000 bp in size), with an N₅₀ of 658,741 bp, and a genome size of 39.7 Mb, which is within the size range of other species of Cytosporaceae (35.2 Mb for C. mali var. pyri SXYL134 to 41. 9 Mb for the representative genome of C. mali 03–8). Assessment of the completeness of the genome using BUSCO groups for fungi resulted in values of C:98.9% [D: 1.0%], F: 0.1%, n: 1 706 (C:complete [D:duplicated], F:fragmented, n: number of genes) which makes the genome sequence suitable for further analysis. The repeat content was 2.9% in the assembly of C. paraplurivora FDS-564. A total of 10,249 protein-coding genes were predicted, which is 9.2% fewer genes than predicted for the genome of C. mali 03–8 (11,284).

Phylogenomic analysis

A total of 3,482 orthogroups present in all species included in the analysis with 2,279 SCOs. The phylogenomic tree (Fig 4A) constructed from a concatenated amino acid alignment of SCOs showed that C. paraplurivora was closest to C. leucostoma SXYLt. This analysis placed C. paraplurivora within the Cytosporaceae family of the order Diaporthales with strong bootstrap support (100% ML). However, whole genome sequence alignment between C. paraplurivora and C. leucostoma indicated rather low similarity, where 84.31% of the syntenic blocks were less than 50% similar with a number of small gaps and some repeats (Fig 4B).

Fig 4.

A. ML tree indicating the placement of C. paraplurivora FDS-564 among 17 species of Ascomycetes. Bootstrap support for all nodes is 100. B. Dot plot graph showing syntenic blocks between genome sequences of C. leucostoma SXYLt and C. paraplurivora FDS-564.

Pathogenicity tests

Twelve days post-inoculation, the length of the necrotic lesions caused by C. paraplurivora on apricot, peach and nectarine excised branches were 22, 26 and 29 mm respectively. Lesions were significantly larger in nectarine cuttings compared to those in apricot (Fig 5A). No significant differences were observed between the mean lesion length caused by C. paraplurivora isolates FDS-564 and FDS-623 on peach seedlings 7, 10, 13 and 27 dpi (Fig 6). The canker lesions enlarged on both sides of inoculated incision. The infected peach seedlings start wilting 15 dpi and collapsed 30 dpi. The symptoms observed in the inoculated cuttings and seedlings, included flattening and discolouration of the bark, light brown wedge-shaped cankers, gummosis and abundant fruiting structures (conidiomata) development were similar to those observed on trees in the field (Figs 5B–5D and 7). C. paraplurivora was re-isolated from 90–100% of the inoculated cuttings and seedlings, fulfilling Koch’s postulates. Control cuttings and seedlings had no symptoms, and the fungus was not isolated from the wood.

Fig 5.

A. Mean lesion length caused by C. paraplurivora in Prunus spp. 12 days post-inoculation. B-D. Symptoms caused by C. paraplurivora (FDS-564) in excised branches of P. persica var. nucipercica 12 days post-inoculation.

Fig 6. Mean lesion length caused by C. paraplurivora isolates FDS-564 and FDS-623 in Prunus persica var. persica seedlings 7, 10, 13 and 27 days post-inoculation.

Discussion

Cytospora spp. are important plant pathogens with worldwide distribution causing canker and dieback diseases in woody plants. The genus has a broad host range that includes Castanea, Corylus. Eucaliptus, Juglans, Malus, Olea, Pistacia, Platanus, Prunus, and Salix. [5, 10, 12, 63, 64]. Pathogenic species of Cytospora were mainly isolated from wood cankers. The present study identifies C. paraplurivora sp. nov. isolated from both single conidia and cankered wood sampled from symptomatic apricot, nectarine, and peach trees in southern Ontario. Abundant conidiomata of C. paraplurivora sp. nov. were observed on trunk and branches of declining trees. C. paraplurivora sp. nov. spores dissemination mechanism might be similar to that of the sister species C. plurivora [65], other Cytospora spp. and fungal species causing Botryosphaeriaceae canker [66, 67]. The presence of conidiomata suggest that this species might spread across fruit tree orchards through spores dissemination by rain splash, wind and insect [65].

The symptoms caused by the new species resemble the leucostoma canker disease (perennial canker) of stone fruits, a widespread plant disease affecting crop yield in Canada and some regions of USA. C. leucostoma and C. cincta were found to be associated with this disease [68] and the species were identified solely by morphological characteristics. The studies on Cytospora spp. with the ITS sequence data refined the species identification of the leucocytosporoid species [69]. However, the later studies revealed that the protein-coding genes included in phylogenetic analysis allow for discrimination of closely related species of Cytospora [70]. In this study, using the sequence data of act and tef1-α genes suggested by Urbez-Torres et al. [39] improved the phylogenetic resolution of this species complex. This approach has also been extensively used to resolve phylogenies of other plant-associated fungal pathogens such as Diaporthe [71], Valsaria [72] and Diplodia [73].

Phylogenomic analysis using the genome sequence data of C. paraplurivora FDS-564 confirmed order and family level classification of the species. It is interesting to note, that the genome size of the new species is slightly larger than the average for Ascomycota (36.9Mb) [41] but significantly smaller compared to plant pathogens of Diaporthales such as Daiporthe citri 63.6Mb [74]. The high-quality genome assembly of C. paraplurivora will provide a valuable resource for the further study of host-pathogen interactions, pathogenicity-related genes, fungal biology, as well as comparative and population genomics analyses of Cytospora spp. and other fungal taxa.

The pathogenicity tests of C. paraplurivora on Prunus spp. performed in this study can be comparable to the trials of other pathogenic species of Cytospora on Malus spp. conducted in vitro under the same conditions with different timelines [75]. The phylogenetically close species C. paratranslucens caused necrotic lesions up to 10 cm on detached apple branches after 21 days [75]. The lesions up to 3.9 cm developed on twig segments of Malus sieversii inoculated with mycelium of C. parasitica after 8 days. This study showed that, highly virulent model species C. mali was able to cause the lesions up to 8.1 cm for the same time period [76]. Considering the time of post-inoculation with C. paraplurivora, it is assumed that the species can quickly colonize the plant tissue and cause the decline of infected trees. The pathogenicity tests on living peach seedlings confirmed the rapid spread of the canker lesions on the trunk of the inoculated plants, leading to the collapse of peach seedlings 30 dpi.

This paper is the first formal report for a new Cytospora species causing the canker of Prunus spp. associated with FTDS in Ontario. The findings suggest that C. paraplurivora has the potential to severely affect stone fruits production in Ontario. Accurate identification of pathogen(s) associated with stone fruit trees decline will support management of the disease. Further research needs to be conducted on emerging fungal pathogens and their associated infection mechanisms, to develop effective disease management strategies applicable to fruit tree orchards.

Data Availability

Alignment of DNA data are available in Treebase (submission ID: 29116) (https://www.treebase.org/treebase-web/home.html).Nucleotide sequences of Cytospora paraplurivora strains FDS-439, FDS-564 and FDS-623 were submitted to NCBI repository (https://www.ncbi.nlm.nih.gov) under accession numbers: OL640182, OL640183, OL640181 (ITS); OL640184, OL640185, OL640123 (LSU); OL631586, OL631587, OL631588 (act) and OL631589, OL631590, OL631591 (tef1-α). Cytospora paraplurivora strains FDS-564 Whole Genome Sequencing data was submitted to Genomes NCBI repository under accession numbers: JAJSPL000000000 (WGS), GCA_021272945 (Assembly) and SAMN24182651 (BioSample).

Funding Statement

This research was funded by Agriculture and Agri-Food Canada, grant number J-002199 awarded to Dr. Walid Ellouze. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.