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Article

Diversity of RNA Viruses and Circular Viroid-like Elements in Heterobasidion spp. in Near-Natural Forests of Bosnia and Herzegovina

by
László Benedek Dálya
1,
Ondřej Hejna
2,
Marcos de la Peña
3,
Zoran Stanivuković
4,
Tomáš Kudláček
1,5 and
Leticia Botella
1,*
1
Department of Forest Protection and Wildlife Management, Faculty of Forestry and Wood Technology, Mendel University in Brno, Zemědělská 3, 613 00 Brno, Czech Republic
2
Department of Genetics and Biotechnologies, Faculty of Agriculture and Technology, University of South Bohemia in České Budějovice, Na Sádkách 1780, 370 05 České Budějovice, Czech Republic
3
Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-CSIC, 46022 Valencia, Spain
4
Department of Integrated Protection of Forest Ecosystems, Faculty of Forestry, University of Banja Luka, Vojvode Stepe Stepanovića Blvd. 75a, 78 000 Banja Luka, Bosnia and Herzegovina
5
Department of Forest Ecology, Faculty of Forestry and Wood Technology, Mendel University in Brno, Zemědělská 1, 613 00 Brno, Czech Republic
*
Author to whom correspondence should be addressed.
Viruses 2025, 17(8), 1144; https://doi.org/10.3390/v17081144
Submission received: 18 June 2025 / Revised: 25 July 2025 / Accepted: 12 August 2025 / Published: 20 August 2025
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Abstract

Heterobasidion root rot fungi represent a major threat to conifer forest stands, and virocontrol (biocontrol) has been proposed as an alternative strategy of disease management in recent years. Here, we investigated the occurrence of RNA viruses and viroid-like genomes in Heterobasidion annosum sensu lato in near-natural forests of Bosnia and Herzegovina (Dinaric Alps), a region previously unexplored in this regard. Seventeen H. annosum s.l. isolates were screened for virus presence by RNA Sequencing and bioinformatic analyses. In total, 32 distinct mycoviruses were discovered in the datasets, 26 of which were previously unknown. The detected viruses represent two dsRNA (Partitiviridae and Curvulaviridae), six linear ssRNA (Mitoviridae, Narnaviridae, Botourmiaviridae, Virgaviridae, Benyviridae, and Deltaflexiviridae) and three circular ssRNA (Dumbiviridae, Quambiviridae, and Trimbiviridae) virus families. In addition to the known circular ambiviruses with their hammerhead (HHRz) and hairpin (HPRz) ribozymes, two other smaller non-coding circular RNAs of ca. 910 bp each were identified encoding HHRz and deltavirus (DVRz) ribozymes in both polarities of their genomes. This study documents the first report of a putative viroid-like RNA agent in Heterobasidion, along with beny-like and deltaflexivirus-like viruses in Heterobasidion abietinum, and expands the known virosphere of Heterobasidion species in Southeastern European forests.

1. Introduction

Bosnia and Herzegovina has approximately 2.9 million ha of forests, which is about 57% of its total area [1]. The tree species composition is characterized by the dominance of oak (33%), pure beech (33%) and mixed coniferous–broadleaved stands (24%). In addition to providing significant commercial benefits [1], Bosnian forests are considered as a biodiversity hotspot giving shelter to numerous endemic and relict species [2]. The old-growth forests of the Dinaric Alps are especially important from naturalistic, cultural, and scientific perspectives not only regionally but also on the continental scale [3]. The importance of these ecosystems is increasing because of their role as a reference for the assessment of climate change impact in European forests [4]. Only small areas of valuable mixed stands of Norway spruce (Picea abies), silver fir (Abies alba), and European beech (Fagus sylvatica) remain intact to this day. Such stands are situated in the Janj, Lom, and Perućica old-growth forest reserves in the Republic of Srpska [5] and their vegetation belongs to the Piceo-Abieti-Fagetum dinaricum forest association [3].
Conifer tree species in Bosnia seem more vulnerable to climate change-induced environmental stress than broadleaves [6,7], and are therefore prone to higher degrees of biotic and abiotic damage [8]. The most important rotting fungal species contributing to the spruce declining process in Bosnia are Heterobasidion parviporum and Armillaria ostoyae [9,10]. Heterobasidion abietinum is also known to cause root and heart rot, which often leads to the drying out of fir trees in the territory of the Republic of Srpska [11]. The effectiveness of currently available forest protection methods to control Heterobasidion annosum sensu lato is insufficient. In recent years, the use of mycoviruses as biocontrol agents (BCAs) has been proposed as an alternative strategy against the pathogen, and a field trial in Finland [12] showed the hypovirulence-inducing potential of Heterobasidion partitivirus 13 from H. annosum (HetPV13-an1).
Comprehensive studies in Sweden [13], Finland [14], and Czechia [15] revealed the diverse virome of Heterobasidion spp. These investigations typically involved local, i.e., boreal or Central European Heterobasidion isolates, while some included specimens from other geographic regions. At present, little is known about viral communities hosted by Southeast European Heterobasidion strains. Greek H. annosum s.l. strains have been screened for dsRNA presence [16,17], but no further virus screenings were performed on material from the Balkans. The present work attempted to reduce this knowledge gap by exploring the occurrence and phylogenetic relationships of viruses associated with H. annosum s.l. in Bosnia.

2. Materials and Methods

2.1. Collection of Heterobasidion Isolates

Fruiting bodies of H. annosum s.l. were collected from coniferous forests of Bosnia, primarily from near-natural stands. Axenic cultures were isolated from basidiocarps and cultivated on 2% malt extract agar (MEA—HiMedia, Mumbai, Maharashtra, India) at 18 °C. In total, 17 Heterobasidion strains were included in the study (Figure 1 and Table S1), and were identified to the species level as described previously [15], following the dilution protocol of the Phire® Plant Direct PCR Kit (Thermo Fisher Scientific, Waltham, MA, USA).

2.2. RNA Extraction

Heterobasidion cultures were grown on cellophane-covered MEA plates at 22 °C in the dark for 2 weeks. After grinding the mycelia in liquid N2 using a sterile mortar and pestle, total RNA was extracted using RNAzol® RT (Sigma-Aldrich, Steinheim, Germany). Homogenization was performed by shaking for 1 min at 30 Hz in a Mixer Mill MM 400 (Retsch, Haan, Germany). RNA quality was visually assessed using a 1.2% agarose gel. RNA concentrations were measured by a Qubit® 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). The RNA solutions were stored at −80 °C.

2.3. Total RNA Sequencing

Three pools consisting of equal volumes with 0.5–1.5 µg of total RNA of six H. abietinum (NGS dataset ‘ABI’), three H. parviporum (‘PAR’), and seven H. annosum s.l. (six H. abietinum and one H. annosum; ‘BIH’) strains, as well as total RNA of a single H. annosum strain (‘2120’; Table S1), were sent for sequencing to the Institute of Applied Biotechnologies a.s. (Olomouc, Czechia). Ribosomal RNA was removed using the NEBNext® rRNA Depletion Kit v2 (New England Biolabs, Ipswich, MA, USA). The cDNA libraries were constructed using the NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, Ipswich, MA, US) and sequenced in paired-end (PE) (2 × 151 bp) on a NovaSeq 6000 (Illumina Inc., San Diego, CA, USA).

2.4. Bioinformatics

Each of the NGS datasets consisted of 588–955 million PE reads. Basecalling, adapter clipping, and quality filtering were performed using Bcl2fastq v2.20.0.422 Conversion Software (Illumina Inc., San Diego, CA, USA). The quality of raw reads was assessed using FastQC v0.11.9 [18]. Trimming of N bases, adapter sequences, and low-quality ends (<30) were processed with Cutadapt v4.7 [19]. Reads <50 bp were discarded from subsequent analyses. Post-trimming quality was reassessed with FastQC, and overrepresented ribosomal RNA sequences from Heterobasidion were removed using SortMeRNA v4.3.6 [20], with the SILVA database of rRNA serving as a reference (https://www.arb-silva.de/). STAR v2.7.9a was employed with default settings for read mapping to the host genome [21]. The NCBI GenBank assembly GCA_001457955.1 was used as the reference genome for H. annosum. After alignment, all mapped reads were discarded, and only unmapped reads were retained. To validate the presence of known viruses in the remaining reads, BWA v0.7.17-r1188 [22] was used to map the reads to NCBI viral RefSeq sequences at https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/ (1 February 2025). The coverage was calculated for each reference virus sequence by SAMtools v1.16.1 [23]. Coverage plots of every viral contigs were visualized in Geneious Prime® 2025.2.1 and they are available in 10.6084/m9.figshare.29634521.
Unmapped reads were then used for de novo assembly using SPAdes v3.15.5 with default settings [24]. Contigs shorter than 500 bp were discarded after this step. Final contigs were cross-referenced with several databases using BLAST v2.10.0 [25]. As a reference, we used a list of viral reference sequences from NCBI. BLASTn was applied for the nucleotide database accessible at https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/virus?SeqType_s=Nucleotide. Similarly, BLASTx was employed for the protein database available at https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/virus?SeqType_s=Protein, and also to search the UniProt database for a specified virus taxon at https://www.uniprot.org/uniprot/?query=taxonomy:10239. Contigs with an e-value higher than 1 × 10−3 were discarded. For BLASTn, the nt database from NCBI, available at https://ftp.ncbi.nlm.nih.gov/blast/db was used. For BLASTx, both the nr database from NCBI, accessible at https://ftp.ncbi.nlm.nih.gov/blast/db and UniProt Knowledgebase (UniProtKB), available at https://www.uniprot.org/uniprotkb/, were employed. The BLAST search was restricted to the best hit per contig. Within each RNA-Seq dataset, the virus candidate contigs (i.e., longer than 500 bp) were imported to Geneious Prime® 2024.0 (https://www.geneious.com) and subjected to further examination. After the manual correction of misassembles, the resulting viral genome sequences were saved and used in further analyses. The search for viral sequences encoding open reading frame (ORF) regions which lack significant similarity to known genes in databases (so-called ORFan analysis) was conducted as described in [26].
The screening of small self-cleaving ribozymes was carried out using the INFERNAL software v. 1.1.5. [27], which identifies RNA elements using secondary structure and nucleotide covariation modeling. Covariance models for the small ribozymes recently described from diverse ambiviruses, mitoviruses, and other viroid-like agents were used for the searches [28]. Secondary RNA structures of minimum free energy of the ambivirus genomes were calculated with the RNAfold program from the ViennaRNA Package 2.0 [29] and visualized by circular plots obtained with the jupiter software (https://github.com/rcedgar/jupiter).

2.5. Phylogenetic Analysis and Pairwise Comparison

Maximum Likelihood phylogenetic trees based on the amino acid (aa) sequences of either the RdRP or the replication protein were constructed using the bootstrapping algorithm implemented in MEGA11 [30]. The model with the lowest Bayesian Information Criterion score was selected for each tree.
Pairwise (pw) comparisons of viral sequences were performed in Geneious Prime® version 2025.2.1 using the MAFFT alignment algorithm.

2.6. DAPC Analysis of Ambiviruses

The dataset for DAPC analysis included 43 ambivirus RdRP nucleotide sequences from Finland (13 ambiviruses), Czechia (16 ambiviruses), and Bosnia (14 ambiviruses). DAPC analysis was performed using the package [31,32]. The number of axes retained in the Principal Component Analysis (PCA) step was set to 30. The number of axes retained in the Discriminant Analysis step was set to the number of populations minus one.

3. Results and Discussion

3.1. Virus and Viroid-like Agents Discovery

In total, 47 mycoviral and 2 mycoviroid-like sequence segments were reconstructed from contigs assembled using the RNA-Seq data covering 12 H. abietinum, 3 H. parviporum, and 2 H. annosum isolates (Table 1 and Table S1). Some of these segments were concatenated from two or three contigs. Interestingly, the negative-sense antigenome was assembled for 36% of the detected viral segments. No viral contigs were identified in H. annosum isolate 2120. One variant of Heterobasidion ourmia-like virus 1 (HetOlV1; Magoulivirus alphaheterobasidion) [33] was found in dataset PAR. The rest of the viruses were detected in datasets ABI and BIH (Table 1). The discovered segments belong to 3 distinct dsRNA viruses classified into two families and 29 ssRNA viruses representing nine families (Table 1). Six of these viruses were previously known. For eight viruses, two or three distinct variants were identified in either the same or different datasets (Table 1).
Additionally, seven ORFan contigs, 600–2300 nucleotides (nt) in length, were identified in datasets ABI and PAR (File S1). These may indicate the presence of putative viral segments with previously unknown genome organization.

3.2. Alphapartitiviruses

Four contigs with similarities to partitiviruses were found, either to an RdRP domain (50–70% identity with the first hit in BLASTx) or a coat protein (CP; 43–50%; Table 1). The contigs were deemed to represent two bisegmented partitiviruses named HetPV32 and HetPV33, which were paired based on 20 identical nt at the 5’ end of two of them (Figure 2). Conservation of the first 7–45 nt in the 5′ UTR of the sense RNA between the two genome segments is common in Partitiviridae. A stretch of identical nt at the beginning of the 5′ UTR was also observed in HetPV8 [34] and HetPV21 [33]. The length of the RdRP encoding dsRNA1 segment of HetPV32 and HetPV33 was ca. 2 kb, and the length of their coat or capsid protein (CP) encoding dsRNA2 segment was 1.8 kb (Figure 2), which falls within the typical range for alphapartitiviruses. The dsRNA1 segments of HetPV32 and HetPV33 had polyA tails of 18 and 32 bp, respectively. The two novel partitiviruses shared 40% pairwise (pw) aa identity in their RdRP and merely 22% pw aa identity in the CP. Both viruses were hosted by H. abietinum (Table 1), which was also the natural host of one HetPV1 variant from Greece [16]. Interestingly, the read coverage of HetPV32 dsRNA1 was an order of magnitude higher than that of dsRNA2, while for HetPV33, the difference was five-fold in the opposite direction (Table 1). This observation may reflect differences in the accumulation levels of the two genome segments in the host mycelium. Unique ratios of RdRP and CP genome segments of Heterobasidion partitiviruses were previously found [35]. Phylogenetic analysis of the Durnavirales order placed the novel partitiviruses in the Alphapartitivirus genus (Figure 3). HetPV33 is very closely related to HetPV4 and HetPV5, while HetPV32 fell into a branch separate from other alphapartitiviruses of Heterobasidion.
Table 1. Viruses and viroid-like elements detected in the present study.
Table 1. Viruses and viroid-like elements detected in the present study.
Mycovirus Acronym Segment GenBank Accession Host Species Length (nt) % GC Mapped Reads f Mean Depth BLASTX First g Identity Query Cover E Value
Heterobasidion partitivirus 32 HetPV32 dsRNA1 PP626369 H. abietinum 1934 d 47.7% d 260,018 d 18,931 d Sarcosphaera coronaria
partitivirus (QLC36816) 		50.1% 89% 0
dsRNA2 PP626370 H. abietinum 1816 54.7% 25,327 1939 Carrot cryptic virus (ACL93279) 43.0% 70% 3.0 × 10−92
Heterobasidion partitivirus 33 HetPV33 dsRNA1 PP626371 H. abietinum 1970 d 46.5%d 12,617 d 916 d Heterobasidion
partitivirus 4 (ADV15443) 		70.5% 94% 0
dsRNA2 PP626372 H. abietinum 1755 53.3% 59,967 4803 Tulasnella
partitivirus 3 (BDB07480) 		49.9% 83% 1.0 × 10−130
Heterobasidion RNA virus 6 HetRV6 dsRNA1 PP626373 H. abietinum OR H. annosum 1996 56.3% 1172 78 Heterobasidion
RNA virus 6 (ADW82833) 		93.1% 91% 0
dsRNA2 PP626374 H. abietinum OR H. annosum 1748 63.4% 915 69 Heterobasidion
RNA virus 6 (QED55787) 		93.7% 62% 0
Heterobasidion mitovirus 4 HetMV4 RNA1 PP626375 H. abietinum a 5292 42.1% 67,004 1685 Heterobasidion
mitovirus 3 (QED55406) h		 69.2% h 46% h 0 h
Heterobasidion mitovirus 5 HetMV5 RNA1 PP626376 H. abietinum 3715 40.9% 676,330 25,276 Heterobasidion
mitovirus 1 (AIF33766) h		 63.6% h 71% h 0 h
Heterobasidion mitovirus 6 HetMV6 RNA1 PP626377 H. abietinum 3521 41.4% 644,309 26,115 Heterobasidion
mitovirus 1 (AIF33766) h		 63.4% h 74%h 0 h
Heterobasidion mitovirus 7 HetMV7 RNA1 PP626378 H. abietinum OR H. annosum b 4314 41.1% 538,636 17,624 Lentinula edodes
mitovirus 1 (QOX06058)h		 38.8% h 51%h 2.0 × 10−160h
Heterobasidion narna-like virus 1 HetNlV1 RNA1 PP626379 H. abietinum a 3956 54.0% 34,130 1221 Heterobasidion
narna-like virus 1 (UHK02569) 		92.8% 93% 0
RNA2 PP626380 H. abietinum a 3998 55.8% 27,091 960 Heterobasidion
narna-like virus 1 (UHK02570) 		91.8% 92% 0
Heterobasidion narna-like virus 5 HetNlV5 RNA1 PP626381 H. abietinum OR H. annosum 3953 55.3% 77,698 2714 Heterobasidion
narna-like virus 4 (WBE16499) 		78.9% 96% 0
Heterobasidion narna-like virus 6 HetNlV6 RNA1 PP626382 H. abietinum OR H. annosum 1731 e 51.2 4745 379 Mbeech associated
narna-like virus 5 (WPR16644) 		53.0% 93% 0
RNA2 PP626383 H. abietinum OR H. annosum 2252 51.5 6832 394 Botrytis cinerea
binarnavirus 2 (QLF49184) 		45.9% 92% 0
Heterobasidion ourmia-like virus 1 HetOlV1 RNA1 PP626384 H. parviporum 2601 49.5 214,903 11,422 Heterobasidion
ourmia-like virus 1 (UHK02571) 		96.6% 73% 0
Heterobasidion ourmia-like virus 2 HetOlV2 RNA1 PP626385 H. abietinum a 2169 e 58.0 10,251 640 Heterobasidion
ourmia-like virus 2 (UOX39320) 		93.0% 67% 0
Heterobasidion ourmia-like virus 6 HetOlV6 RNA1 PP626386 H. abietinum 2516 51.2 12,337 658 Heterobasidion
ourmia-like virus 1 (WLV75612) 		76.9% 74% 0
Heterobasidion ourmia-like virus 7 HetOlV7 RNA1 PP626387 H. abietinum a 2366 50.5 11,364 566 Heterobasidion
ourmia-like virus 1 (WLV75612) 		79.4% 72% 0
Heterobasidion ourmia-like virus 8 HetOlV8 RNA1 PP626388 H. abietinum 2305 50.4 16,611 884 Heterobasidion
ourmia-like virus 1 (WLV75612) 		77.1% 76% 0
Heterobasidion ourmia-like virus 9 HetOlV9 RNA1 PP626389 H. abietinum OR H. annosum 2505 50.3 118,718 6411 Heterobasidion
ourmia-like virus 1 (WLV75612) 		81.4% 74% 0
Heterobasidion tobamo-like virus 2 HetTlV2 RNA1 PP626390 H. abietinum OR H. annosum 6675 e 49.3 1880 38 Heterobasidion
tobamo-like virus 1 (UOX39322) 		87.6% 64% 0
Heterobasidion beny-like virus 1 HetBlV1 RNA1 PP626391 H. abietinum OR H. annosum a 7563 d 57.2 d 268,134 d 4956 d Rhizoctonia cerealis
beny-like virus (WMI40034) 		31.1% 64% 2.0 × 10−162
Heterobasidion deltaflexivirus 1 HetDFV1 RNA1 PP626392 H. abietinum OR H. annosum a 6928 64.1 123,445 2322 Rhizoctonia solani
flexi-like virus 1 (QDW81317) 		33.1% 34% 2.0 × 10−107
Heterobasidion ambi-like virus 1 HetAlV1 RNA1 PP744446 H. abietinum 4754 49.2 822,963 24,597 Heterobasidion
ambi-like virus 1 (UHK02572) 		97.8% 38% 0
Heterobasidion ambi-like virus 2 HetAlV2 RNA1 PP744447 H. abietinum OR H. annosum 4286 49.2 938,195 30,286 Heterobasidion
ambi-like virus 2 (UHK02574) 		95.6% 41% 0
Heterobasidion ambi-like virus 30 HetAlV30 RNA1 PP744448 H. abietinum 5085 49.2 8663 241 Heterobasidion
ambi-like virus 6 (UOX39308) 		58.0% 42% 0
Heterobasidion ambi-like virus 31 HetAlV31 RNA1 PP744449 H. abietinum c 4876 49.9 32,263 922 Heterobasidion
ambi-like virus 3 (UHK02576) 		68.3% 42% 0
Heterobasidion ambi-like virus 32 HetAlV32 RNA1 PP744450 H. abietinum 4450 49.2 1,172,645 37,056 Heterobasidion
ambi-like virus 1 (UHK02572) 		83.3% 40% 0
Heterobasidion ambi-like virus 33 HetAlV33 RNA1 PP744451 H. abietinum 4368 49.2 505,254 16,380 Heterobasidion
ambi-like virus 2 (UHK02574) 		92.6% 40% 0
Heterobasidion ambi-like virus 34 HetAlV34 RNA1 PP744452 H. abietinum OR H. annosum 4956 50.2 110,813 3101 Heterobasidion
ambi-like virus 3 (UHK02576) 		67.3% 42% 0
Heterobasidion ambi-like virus 35 HetAlV35 RNA1 PP744453 H. abietinum OR H. annosum 4851 48.4 11,912 336 Heterobasidion
ambi-like virus 8 (UOX39312) 		61.6% 40% 0
Heterobasidion ambi-like virus 36 HetAlV36 RNA1 PP744454 H. abietinum OR H. annosum 4660 49.7 61,156 1779 Heterobasidion
ambi-like virus 12 (WOK44145) 		76.0% 44% 0
Heterobasidion ambi-like virus 37 HetAlV37 RNA1 PP744455 H. abietinum OR H. annosum 4767 48.8 54,368 1574 Heterobasidion
ambi-like virus 8 (UOX39312) 		65.9% 41% 0
Heterobasidion ambi-like virus 38 HetAlV38 RNA1 PP744456 H. abietinum OR H. annosum 4762 48.0 87,791 2543 Heterobasidion
ambi-like virus 8 (UOX39312) 		66.3% 41% 0
Heterobasidion ambi-like virus 39 HetAlV39 RNA1 PP744457 H. abietinum OR H. annosum 4632 49.0 21,324 602 Heterobasidion
ambi-like virus 10 (WOK44143) 		74.1% 44% 0
Heterobasidion ambi-like virus 40 HetAlV40 RNA1 PP744458 H. abietinum OR H. annosum 4287 48.4 53,587 1644 Heterobasidion
ambi-like virus 12 (WOK44145) 		87.7% 45% 0
Heterobasidion circular RNA 1 HetcRNA1 RNA1 PQ298351 H. abietinum 919 60.1 206,319 33,675 n.s.
Heterobasidion circular RNA 2 HetcRNA2 RNA1 PQ298352 H. abietinum 918 60.3 22,977 3754 n.s.
a Another virus variant was hosted by H. abietinum or H. annosum. b Two other virus variants were hosted by H. abietinum. c Another virus variant was hosted by H. abietinum. d Without polyA tail. e Partial genome sequence. f Using Geneious assembler with medium-low sensitivity. Corresponding BAM files with mapped reads for each viral contig are available at 10.6084/m9.figshare.29634521. g Search performed on 31.07.2024. h Using the Mold Protozoan Mitochondrial genetic code. n.s., no significant similarity found.

3.3. Orthocurvulavirus Annosi

A new variant of Heterobasidion RNA virus 6 (HetRV6; Orthocurvulavirus annosi) was detected, sharing 93–94% BLASTx identity with the variant HetRV6-ab6 ([17]; Table 1). The 2 kb long dsRNA1 segment corresponded to the RdRP, while dsRNA2, encoding a hypothetical protein (HP) with unknown function, was 1.7 kb long (Figure 2). A stretch of 39 identical bases was noticed at the 5′ termini of both segments (Figure 2). Comparable levels of conservation at the same region were documented in HetRV6-ab6 and -pa36 (GenBank OR644495, OR644496), as well as in orthocurvulaviruses of Curvularia protuberata [36], Sclerotium hydrophilum [37], Trichoderma harzianum [38], and Lactarius rufus [39]. Both segments of the new HetRV6 variant had a low depth of coverage compared to most other viruses reported here (Table 1), in agreement with [33]. Phylogenetically, HetRV6 formed a highly supported branch with Phlebiopsis gigantea curvulavirus 1 [40] within the Curvulaviridae family (Figure 3).
Viruses 17 01144 g003
Figure 3. Phylogenetic relationships within and between relevant families of the Durnavirales order. The Amalgaviridae family served as outgroup. The tree was built based on the alignment of RdRP aa sequences generated with MUSCLE in MEGA11 [30]. The evolutionary history was inferred by using the Maximum Likelihood method and the Le_Gascuel_2008 model [41]. A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories +G +I). All positions with less than 95% site coverage were eliminated; i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. Evolutionary analyses were conducted in MEGA11 with 1000 bootstrap repeats. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Branch lengths are proportional to the number of substitutions per site. Alphapartitiviruses and the orthocurvulavirus variant described in this study are denoted by a triangle.
Figure 3. Phylogenetic relationships within and between relevant families of the Durnavirales order. The Amalgaviridae family served as outgroup. The tree was built based on the alignment of RdRP aa sequences generated with MUSCLE in MEGA11 [30]. The evolutionary history was inferred by using the Maximum Likelihood method and the Le_Gascuel_2008 model [41]. A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories +G +I). All positions with less than 95% site coverage were eliminated; i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. Evolutionary analyses were conducted in MEGA11 with 1000 bootstrap repeats. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Branch lengths are proportional to the number of substitutions per site. Alphapartitiviruses and the orthocurvulavirus variant described in this study are denoted by a triangle.
Viruses 17 01144 g003

3.4. Mitoviruses

Four novel mitoviruses, named Heterobasidion mitovirus 4–7 (HetMV4–7), were derived from our data (Table 1). Their genomes were 3.5–5.3 kb in length and contained the mitoviral RdRP conserved domain (Figure 2). HetMV7 also possessed a short downstream ORF encoding an HP (Figure 2). HetMV4 earned the title of the longest mitovirus described to date ahead of HetMV3-an1 [42]. HetMV4 was represented by two virus variants, while HetMV7 by three (Table 1), with each variant sharing 98% aa pw identity over their RdRP. Heterobasidion abietinum was recorded as a host of each mitovirus described here (Table 1). The read coverage values of HetMV5–7 were among the highest observed in the present study (Table 1). The high accumulation levels of mitoviruses in Heterobasidion and other plant pathogenic fungi are a common phenomenon [15]. HetMV7 clustered with Lentinula edodes mitovirus 1 [43], while HetMV4–6 with HetMV1 [44] and HetMV3 [42] within the Duamitovirus genus of the Mitoviridae family (Figure 4).

3.5. Narna-like Viruses

Two new variants of Heterobasidion narna-like virus 1 (HetNlV1) [33] were detected, one of which surely inhabited H. abietinum. Each segment of both variants showed over 92% identity in BLASTx (Table 1). Another contig, almost 4 kb long and sharing 79% aa identity in its predicted RdRP with HetNlV4 [15], was designated as HetNlV5 RNA1 segment (Table 1; Figure 2). Two shorter sequences (~1.7 and 2.3 kb) were considered to constitute the genome of a novel bisegmented narna-like virus named HetNlV6 (Table 1; Figure 2). Their most significant BLASTx hits were Mbeech associated narna-like virus 5 and Botrytis cinerea binarnavirus 2 with 53% and 46% identity, respectively. Due to being incomplete at the 5’ end of its RNA1 segment and lacking the GDD motif within the RdRP, HetNlV6 was excluded from the phylogenetic analysis. All the other Heterobasidion narna-like viruses clustered tightly within Narnaviridae (Figure 4).

3.6. Ourmia-like Viruses

A new variant of HetOlV1 with 97% BLASTx identity to variant HetOlV1-RKU3.2.124 [33] was found in H. parviporum (Table 1), which remains the only host reported for this virus. Further, two variants of HetOlV2, each sharing 93% identity in BLASTx with the variant HetOlV2-CZ2072 [15], were revealed (Table 1). Both were incomplete at the 3’ end, and one variant infected H. abietinum (Table 1; Figure 2). Another four distinct ourmia-like viruses with high similarity (77–81% BLASTx identity) to HetOlV1 were discovered and named HetOlV6–9 (Table 1). Their monosegmented genomes of 2.3–2.5 kb contain a single predicted ORF encoding for a putative RdRP (Figure 2). HetOlV6–8 were present in H. abietinum (Table 1). HetOlV7 was represented by two different variants (Table 1). Pw aa identities of the RdRP between HetOlV2 and HetOlV7 variants were 98% and 97%, respectively. Phylogenetic analysis of the Lenarviricota phylum indicated that Heterobasidion ourmia-like viruses most likely belong to the Magoulivirus genus within the family Botourmiaviridae (Figure 4).

3.7. Tobamo-like Virus

A ~6.7 kb long sequence sharing 88% BLASTx identity with the recently described Heterobasidion tobamo-like virus 1 (HetTlV1) [15] was named HetTlV2 (Table 1). Only a partial genome of HetTlV2 could be reconstructed from our data. Based on pw alignments with the HetTlV1 genome sequence, there was a gap of ca. 773 bp between the truncated RdRP encoding ORF and the movement protein (MP) encoding ORF of the HetTlV2 genome (Figure 2). Furthermore, the HP at the 3’ end was incomplete. Most likely, HetTlV2 caused a low titer infection, manifested by the lowest read coverage in the present study, with a mean depth of 38 reads (Table 1). Oddly, the full genome of HetTlV1 was reconstructed at a mean depth of just 20 reads [15]. Therefore, RNA-Seq of a single host isolate may be a viable approach to obtain the complete genome sequence [15]. As opposed to HetTlV1, the first ORF of HetTlV2 encodes an HP. The largest ORF was predicted to encode a replicase comprising methyltransferase and helicase domains. The downstream ORF encodes the RdRP. Similarly to HetTlV1, −1 ribosomal frameshifting is presumably utilized to express these two proteins. The ORF encoding an MP contained the DEAD-like helicase superfamily and the helicase conserved C-terminal domains (Figure 2). Heterobasidion tobamo-like viruses occupied a separate phylogenetic branch and seem to be distantly related to other members of the family Virgaviridae (Figure 5).

3.8. Beny-like Virus

A 7.6 kb long contig showing the highest similarity (31% BLASTx identity) to Rhizoctonia cerealis beny-like virus was designated as Heterobasidion beny-like virus 1 (HetBlV1; Table 1). Its first ORF, containing the DEXS-box helicase and Benyviridae RdRP conserved domains, was projected to encode an RdRP (Figure 2). A shorter ORF encoding an HP was located downstream. The genomic sequence includes a polyA tail of 12 nt. HetBlV1 and another HetBlV1 variant detected in the same dataset (Table 1) shared 93% and 91% pw aa identities in their RdRP and HP, respectively. Classified members of the Benyviridae family infect plants, while the extension of this taxonomic group seems imminent as numerous viruses with similar properties have been recently discovered in fungal or insect metagenomic studies [46]. Phylogenetic analysis of the Alsuviricetes class placed HetBlV1 into a well-supported cluster consisting of mycoviruses and an entomovirus within Benyviridae (Figure 6).

3.9. Deltaflexivirus

As another novelty in the virome of Heterobasidion spp., we report the discovery of Heterobasidion deltaflexivirus 1 (HetDFV1), represented by a 6.9 kb long contig, with Rhizoctonia solani flexi-like virus 1 as its most significant BLASTx hit (33% identity; Table 1). The HetDFV1 genome encompassed a single predicted ORF encoding an RdRP, which included methyltransferase, helicase, and Deltaflexiviridae RdRP domains (Figure 2). Two HetDFV1 variants were present in the same dataset (Table 1) with 78% identity in their RdRP aa sequence. The Deltaflexiviridae family has traditionally accommodated plant and fungal viruses, although several affiliated viruses have been found in animals by metagenomics in recent years [47]. According to phylogenetic analysis, deltaflexiviruses could be of monophyletic origin, as no signs of cluster formation were apparent among putative members of the family (Figure 6).

3.10. Ambi-like Viruses

One contig detected in dataset ABI showed 98% BLASTx identity to the virus variant Heterobasidion ambi-like virus 1 (HetAlV1; Orthodumbivirus unoheterobasidii)-4R1 known from H. parviporum ([33]; Table 1). Another contig shared 96% BLASTx identity with the virus variant HetAlV2 (Orthodumbivirus duoheterobasidii)-RKU3.1.25 ([33]; Table 1). Both of these new variants had robust read support (Table 1). Eleven new viruses with moderate to high similarity to previously described ambi-like viruses of Heterobasidion were discovered and designated as HetAlV30–40 (Table 1). Each virus had a genome of 4.3–5.1 kb, containing two bidirectional ORFs. In the case of HetAlV30, an incomplete dimer of the genome sequence was assembled, which is common due to the circular genome organization of Ambiviricota [15,48,49,50]. The majority of the ambi-like viruses possessed hammerhead (HHRz) or hairpin (HPRz) ribozymes positioned at the C-termini of one or both ORFs (Table S2). The genome organization of HetAlV31, representative of the novel ambi-like viruses, is shown in Figure 2. HetAlV30–33 dwelled in H. abietinum and HetAlV31 had two variants (Table 1) with 90% nt pw identity in the RdRP. The ambi-like viruses of Heterobasidion were placed in three different families, where they grouped tightly together (Figure 7). Of the newly discovered ambiviruses, HetAlV30 belongs to the Quambiviridae family, HetAlV32 and HetAlV33 to Dumbiviridae, while the majority fell into Trimbiviridae.
The dataset BIH yielded 37 additional short contigs resembling ambi-like viruses of Heterobasidion (Table S3). These fragments have either zero or a single complete ORF, encoding either the putative RdRP or the HP. As we could not reliably compile them into coding complete genomes, we cannot confirm whether they belong to the viruses described above or to different ones.
The DAPC analysis of the ambiviruses’ genetic differentiation based on their host variant geographical origin (Finland, Czechia, and Bosnia) shows clear country-wise separated clusters (Figure 8, Table S4) and low overlap between them. Bosnia and Czechia are more separated along Discriminant Function 1 and Finland separated primarily along Discriminant Function 2. The tight clustering of Bosnian and Czech ambiviruses indicates low within-group variation, while the broader spread of Finnish samples suggests higher heterogeneity. These results suggest that the genetic variation of Heterobasidion ambiviruses is strongly influenced by geographic origin, potentially reflecting local evolutionary dynamics or host–virus associations. This pattern is particularly common among mycoviruses due to their intracellular transmission mode [51].

3.11. Viroid-like cRNAs

Two small circular RNAs (cRNAs) of 918 and 919 bp were identified in H. abietinum and named Heterobasidion circular RNA 1 and 2 (HetcRNA 1 and 2). Both lack ORFs in their genomes (supporting their non-coding nature) and contain self-cleaving ribozymes, specifically, a hammerhead ribozyme (HHRz) and a deltavirus-like ribozyme (DVRz), one in each polarity, enabling symmetric rolling-circle replication. Predicted secondary structures (Figure 9) reveal a hybrid architecture, combining rod-like structures typical of ambiviruses with branched features that are more characteristic of plant viroids. These cRNAs were detected using the INFERNAL pipeline in the library containing H. abietinum RNAs (ABI). The pw identity between them was low enough (40.1%) to consider them as two different putative viroid-like cRNAs.
Recent metatranscriptomic studies have greatly expanded the known diversity of viroid-like circular RNAs, highlighting major challenges in their classification and even in defining the boundaries of this class of replicators [28,52]. Our findings align with a growing body of evidence suggesting that viroid-like elements may also occur in fungi [28,52,53,54]. Previous work has demonstrated viroid replication or the processing of its RNAs in the yeast Saccharomyces cerevisiae [55] and in the cyanobacterium Nostoc (Nostocales) [56]. Tian et al. [57] demonstrated that apple viroids can naturally spread to fungi, suggesting the possible existence of mycoviroids. Prior to these findings, Wei et al. [58] showed that plant viroids can infect and induce symptoms in fungal pathogens like Cryphonectria parasitica and Fusarium graminearum. In 2022, Afanasenko et al. [59] reported that the potato spindle tuber viroid (PSTVd) can persist in pure cultures of Phytophthora infestans, providing evidence of bidirectional viroid transfer between plants and oomycetes. In 2023, Botryosphaeria dothidea circular RNAs (BdcRNAs) were discovered in Botryosphaeria dothidea, showing viroid-like genomic features and pathogenic effects on fungal hosts [60].
Although the biological function and replication mechanisms of HetcRNA1 and HetcRNA2 remain to be experimentally verified, their genomic architecture strongly supports their classification as putative mycoviroid-like agents, contributing to the expanding landscape of non-coding circular RNAs associated with fungi. Definitive classification as true mycoviroids would require demonstration of replication by the fungal host’s RNA polymerase [28]. Without such evidence, it remains possible that replication is mediated by a co-infecting viral polymerase, in which case these elements would be more accurately described as mycoviroid-like satellites [61].

4. Conclusions

Our small-scale survey consisting of 17 Bosnian H. annosum s.l. specimens revealed the existence of 32 mycoviruses representing overall 11 dsRNA or ssRNA families and two putative novel mycoviroids (Table 1), suggesting that the high biodiversity of Bosnian forests is coupled with a similarly diverse viral community. For comparison, the screening of 45 Czech H. annosum s.l. strains using a similar NGS methodology detected 25 viruses belonging to nine ssRNA families [15]. A possible cause of the observed difference in virus abundance between the two countries could be that the authors of [15] surveyed managed forests, while the present work focused on near-natural stands, known to accommodate higher viral diversity than disturbed habitats [62,63]. Among dsRNA viruses, the presence of O. annosi, the most common mycovirus species in Heterobasidion spp. [14], was confirmed. New members of Partitiviridae, the family with the largest number of Heterobasidion viruses described to date, were also discovered. However, ssRNA viruses of Heterobasidion, especially ambi-like viruses, seem to be more prevalent in Bosnia and display a much higher genetic variability. We report the first beny-like virus and deltaflexivirus associated with the root rot pathogen. Heterobasidion abietinum is recorded as a host of ssRNA viruses (i.e., narna-, ourmia-, ambi-like, and mitoviruses) and putative mycoviroid-like elements for the first time. The identification of HetcRNAs expands upon this growing field, raising new questions about the functional roles and evolutionary significance of viroid-like RNAs in fungi and, in particular, forest fungal pathogens.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/v17081144/s1; Table S1: Heterobasidion isolates studied; Table S2: Types and genomic position of the ribozymes detected in Heterobasidion ambiviruses; Table S3: Short contigs resembling ambi-like viruses detected in RNA-Seq dataset BIH; Table S4. Viruses included in the DAPC analysis. File S1. ORFan contigs detected. The name of the respective RNA-Seq dataset is indicated before the first underscore in each contig name. Coverage plot images and the corresponding BAM files with mapped reads for each viral contig are available at 10.6084/m9.figshare.29634521.

Author Contributions

L.B.D.: Conceptualization, formal analysis, investigation, data curation, writing—original draft, visualization, and funding acquisition. O.H.: Formal analysis, writing—review and editing, and resources. M.d.l.P.: Formal analysis and resources. Z.S.: Investigation, resources, and supervision. T.K.: Formal analysis. L.B.: Conceptualization, resources, writing—review and editing, supervision, and funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the European Regional Development Fund, Project “Phytophthora Research Centre”, Reg. No. CZ.02.1.01/0.0/0.0/15_003/0000453. M.dlP. was supported by the Generalitat Valenciana through the PROMETEO program, Project CIPROM/2022/21. The first author’s internship at the University of Banja Luka was supported by an Erasmus + grant (ERASMUS + 2020-1-CZ01-KA107-078115) funded by the EU.

Data Availability Statement

The RNA-Seq data have been deposited into the NCBI Sequence Read Archive database with the accession numbers SRR28491256, SRR28491257, SRR28491258, SRR28491259, SRR28496467, SRR28496590, and SRR28496591 under the BioProject PRJNA1093139. The mycoviral and mycoviroid-like genome sequences are available from the NCBI GenBank.

Acknowledgments

We are grateful to Mirjana Radović and her colleagues (The Public Institution National Park “Sutjeska”), Miloš Trifković (Department of Forest Protection and Wildlife Management, Mendel University in Brno), and Zoran Poljašević (NGO Ecological association “Ozrenski Studenac” Sočkovac) for guidance and technical assistance during the collection of Heterobasidion isolates.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Mataruga, M.; Ballian, D.; Terzić, R.; Daničić, V.; Cvjetković, B. State of forests in Bosnia and Herzegovina: Ecological and vegetation distribution, management and genetic variability. In Forests of Southeast Europe under a Changing Climate; Šijačić-Nikolić, M., Milovanović, J., Nonić, M., Eds.; Springer International Publishing: Cham, Switzerland, 2019; Volume 65, pp. 3–19. [Google Scholar] [CrossRef]
  2. Myers, N.; Mittermeier, R.A.; Mittermeier, C.; da Fonseca, G.A.B.; Kent, J. Biodiversity hotspots for conservation priorities. Nature 2000, 403, 853–858. [Google Scholar] [CrossRef] [PubMed]
  3. Govedar, Z.; Krstić, M.; Keren, S.; Babić, V.; Zlokapa, B.; Kanjevac, B. Actual and balanced stand structure: Examples from beech-fir-spruce old-growth forests in the area of the Dinarides in Bosnia and Herzegovina. Sustainability 2018, 10, 540. [Google Scholar] [CrossRef]
  4. Motta, R.; Alberti, G.; Ascoli, D.; Berretti, R.; Bilic, S.; Bono, A.; Milic, C.; Vojislav, D.; Finsinger, W.; Garbarino, M.; et al. Old-growth forests in the Dinaric Alps of Bosnia-Herzegovina and Montenegro: A continental hot-spot for research and biodiversity. Front. For. Glob. Change 2024, 7, 1371144. [Google Scholar] [CrossRef]
  5. Drinić, P. Taksacioni elementi sastojina jele, smrče i bukve prašumskog tipa u Bosni [Taxation elements of the fir-spruce-beech old-growth forests in Bosnia]. Rad. Šumarskog Fak. Univ. u Sarajev. 1956, 4, 107–160. [Google Scholar] [CrossRef]
  6. Drašković, B.; Gutalj, M.; Stjepanović, S.; Miletić, B. Estimating recent forest losses in Bosnia and Herzegovina by using the Copernicus and Corine land cover databases. Šumarski List 2021, 145, 581–589. [Google Scholar] [CrossRef]
  7. Vukmir, G.; Stanišljević, L.; Cero, M.; Cacan, M.; Marković, M.; Rudež, M.; Laganin, O.; Kostić, R.; Oprašić, S.; Ćatović, S.; et al. Initial National Communication (INC) of Bosnia and Herzegovina Under the United Nations Framework Convention on Climate Change; UNDP (United Nations Development Programme): Banja Luka, Bosnia and Herzegovina, 2019; Available online: https://www.undp.org/bosnia-herzegovina/publications/initial-national-communication-inc (accessed on 17 June 2025).
  8. Camarero, J.J.; Pizarro, M.; Gernandt, D.S.; Gazol, A. Smaller conifers are more resilient to drought. Agric. For. Meteorol. 2024, 350, 109993. [Google Scholar] [CrossRef]
  9. Stanivuković, Z.; Vasiljević, R. The most significant biotic harmful agents and their influence on the intensity of spruce (Picea abies Karst.) dieback on the Romanian plateau. Šumarstvo 2019, 3–4, 21–41. [Google Scholar]
  10. Zahirović, K.; Treštić, T.; Čabaravdić, A.; Dautbašić, M.; Mujezinović, O. Causitive agents of decay of Norway spruce/Picea abies (L.) Karst./on the mountain Zvijezda. Šumarski List 2019, 143, 155–160. [Google Scholar] [CrossRef]
  11. Karadžić, D.; Stanivuković, Z.; Milanović, S.; Milenković, I. Najznačajniji Prouzrokovači Infektivnih Bolesti u Šumama Republike Srpske [The Most Important Causes of Infectious Diseases in the Forests of the Republic of Srpska]; University of Banja Luka, Faculty of Forestry: Banja Luka, Bosnia and Herzegovina, 2019; 324p, /unibl/book/idKnjiga:2140, 978-99938-56-42-9; Available online: https://pub.unibl.org/s/lat/item/48960 (accessed on 11 August 2025).
  12. Piri, T.; Vainio, E.J.; Hantula, J. Preventing mycelial spread of Heterobasidion annosum in young Scots pine stands using fungal and viral biocontrol agents. Biol. Control 2023, 184, 105263. [Google Scholar] [CrossRef]
  13. Ihrmark, K.; Zheng, J.; Stenström, E.; Stenlid, J. Presence of double-stranded RNA in Heterobasidion annosum. For. Pathol. 2001, 31, 387–394. [Google Scholar] [CrossRef]
  14. Vainio, E.J.; Hantula, J. Taxonomy, biogeography and importance of Heterobasidion viruses. Virus Res. 2016, 219, 2–10. [Google Scholar] [CrossRef] [PubMed]
  15. Dálya, L.B.; Černý, M.; de la Peña, M.; Poimala, A.; Vainio, E.J.; Hantula, J.; Botella, L. Diversity and impact of single-stranded RNA viruses in Czech Heterobasidion populations. MSystems 2024, 9, e0050624. [Google Scholar] [CrossRef] [PubMed]
  16. Vainio, E.J.; Hakanpää, J.; Dai, Y.C.; Hansen, E.; Korhonen, K.; Hantula, J. Species of Heterobasidion host a diverse pool of partitiviruses with global distribution and interspecies transmission. Fungal Biol. 2011, 115, 1234–1243. [Google Scholar] [CrossRef] [PubMed]
  17. Vainio, E.J.; Hyder, R.; Aday Kaya, A.G.; Hansen, E.; Piri, T.; Doğmuş-Lehtijärvi, H.T.; Lehtijärvi, A.; Korhonen, K.; Hantula, J. Population structure of a novel putative mycovirus infecting the conifer root-rot fungus Heterobasidion annosum sensu lato. Virology 2012, 422, 366–376. [Google Scholar] [CrossRef]
  18. Andrews, S. FastQC: A Quality Control Tool for High Throughput Sequence Data. 2010. Available online: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (accessed on 10 January 2025).
  19. Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 2011, 17, 10–12. [Google Scholar] [CrossRef]
  20. Kopylova, E.; Noé, L.; Touzet, H. SortMeRNA: Fast and accurate filtering of ribosomal RNAs in metatranscriptomic data. Bioinformatics 2012, 28, 3211–3217. [Google Scholar] [CrossRef]
  21. Dobin, A.; Davis, C.A.; Schlesinger, F.; Drenkow, J.; Zaleski, C.; Jha, S.; Batut, P.; Chaisson, M.; Gingeras, T.R. STAR: Ultrafast universal RNA-seq aligner. Bioinformatics 2013, 29, 15–21. [Google Scholar] [CrossRef]
  22. Li, H.; Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 2009, 25, 1754–1760. [Google Scholar] [CrossRef]
  23. Li, H.; Handsaker, B.; Wysoker, A.; Fennell, T.; Ruan, J.; Homer, N.; Marth, G.; Abecasis, G.; Durbin, R. 1000 Genome project data processing subgroup. The sequence alignment/map format and SAMtools. Bioinformatics 2009, 25, 2078–2079. [Google Scholar] [CrossRef]
  24. Bankevich, A.; Nurk, S.; Antipov, D.; Gurevich, A.A.; Dvorkin, M.; Kulikov, A.S.; Lesin, V.M.; Nikolenko, S.L.; Pham, S.; Pevzner, P.A. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 2012, 19, 455–477. [Google Scholar] [CrossRef]
  25. Camacho, C.; Coulouris, G.; Avagyan, V.; Ma, N.; Papadopoulos, J.; Bealer, K.; Madden, T.L. BLAST+: Architecture and applications. BMC Bioinform. 2009, 10, 421. [Google Scholar] [CrossRef]
  26. Botella, L.; Hejna, O.; Kudláček, T.; Kovačiková, K.; Rost, M.; Forgia, M.; Raco, M.; Milenković, I.; Corcobado, T.; Maia, C.; et al. The virome of the panglobal, wide host-range plant pathogen Phytophthora cinnamomi: Phylogeography and evolutionary insights. Virus Evol. 2025, 11, veaf020. [Google Scholar] [CrossRef] [PubMed]
  27. Nawrocki, E.P.; Eddy, S.R. Infernal 1.1: 100-fold faster RNA homology searches. Bioinformatics 2013, 29, 2933–2935. [Google Scholar] [CrossRef] [PubMed]
  28. Forgia, M.; Navarro, B.; Daghino, S.; Cervera, A.; Gisel, A.; Perotto, S.; Aghayeva, D.N.; Akinyuwa, M.F.; Gobbi, E.; Zheludev, I.N.; et al. Hybrids of RNA viruses and viroid-like elements replicate in fungi. Nat. Commun. 2023, 14, 2591. [Google Scholar] [CrossRef] [PubMed]
  29. Lorenz, R.; Bernhart, S.H.; Höner zu Siederdissen, C.; Tafer, H.; Flamm, C.; Stadler, P.F.; Hofacker, I.L. ViennaRNA package 2.0. Algorit. Mol. Biol. 2011, 6, 26. [Google Scholar] [CrossRef]
  30. Tamura, K.; Stecher, G.; Kumar, S. MEGA11: Molecular evolutionary genetics analysis version 11. Mol. Biol. Evol. 2021, 38, 3022–3027. [Google Scholar] [CrossRef]
  31. Jombart, T. adegenet: A R package for the multivariate analysis of genetic markers. Bioinformatics 2008, 24, 1403–1405. [Google Scholar] [CrossRef]
  32. Jombart, T.; Ahmed, I. adegenet 1.3-1: New tools for the analysis of genome-wide SNP data. Bioinformatics 2011, 27, 3070–3071. [Google Scholar] [CrossRef]
  33. Sutela, S.; Piri, T.; Vainio, E.J. Discovery and community dynamics of novel ssRNA mycoviruses in the conifer pathogen Heterobasidion parviporum. Front. Microbiol. 2021, 12, 770787. [Google Scholar] [CrossRef]
  34. Vainio, E.J.; Capretti, P.; Motta, E.; Hantula, J. Molecular characterization of HetRV8-ir1, a partitivirus of the invasive conifer pathogenic fungus Heterobasidion irregulare. Arch. Virol. 2013, 158, 1613–1615. [Google Scholar] [CrossRef]
  35. Jurvansuu, J.; Kashif, M.; Vaario, L.; Vainio, E.J.; Hantula, J. Partitiviruses of a fungal forest pathogen have species-specific quantities of genome segments and transcripts. Virology 2014, 462–463, 25–33. [Google Scholar] [CrossRef]
  36. Márquez, L.M.; Redman, R.S.; Rodriguez, R.J.; Roossinck, M.J. A virus in a fungus in a plant: Three-way symbiosis required for thermal tolerance. Science 2007, 315, 513–515. [Google Scholar] [CrossRef] [PubMed]
  37. Wang, C.; Wu, J.; Zhu, X.; Chen, J. Complete nucleotide sequences of dsRNA2 and dsRNA7 detected in the phytopathogenic fungus Sclerotium hydrophilum and their close phylogenetic relationship to a group of unclassified viruses. Virus Genes 2016, 52, 823–827. [Google Scholar] [CrossRef] [PubMed]
  38. Liu, C.; Li, M.; Redda, E.T.; Mei, J.; Zhang, J.; Elena, S.F.; Wu, B.; Jiang, X. Complete nucleotide sequence of a novel mycovirus from Trichoderma harzianum in China. Arch. Virol. 2019, 164, 1213–1216. [Google Scholar] [CrossRef] [PubMed]
  39. Sutela, S.; Vainio, E.J. Virus population structure in the ectomycorrhizal fungi Lactarius rufus and L. tabidus at two forest sites in Southern Finland. Virus Res. 2020, 285, 197993. [Google Scholar] [CrossRef]
  40. Drenkhan, T.; Sutela, S.; Veeväli, V.; Vainio, E.J. Phlebiopsis gigantea strains from Estonia show potential as native biocontrol agents against Heterobasidion root rot and contain diverse dsRNA and ssRNA viruses. Biol. Control 2022, 167, 104837. [Google Scholar] [CrossRef]
  41. Le, S.Q.; Gascuel, O. An improved general amino acid replacement matrix. Mol. Biol. Evol. 2008, 25, 1307–1320. [Google Scholar] [CrossRef]
  42. Vainio, E.J. Mitoviruses in the conifer root rot pathogens Heterobasidion annosum and H. parviporum. Virus Res. 2019, 271, 197681. [Google Scholar] [CrossRef]
  43. Guo, M.; Shen, G.; Wang, J.; Liu, M.; Bian, Y.; Xu, Z. Mycoviral diversity and characteristics of a negative-stranded RNA virus LeNSRV1 in the edible mushroom Lentinula edodes. Virology 2021, 555, 89–101. [Google Scholar] [CrossRef]
  44. Vainio, E.J.; Jurvansuu, J.; Streng, J.; Rajamäki, M.-L.; Hantula, J.; Valkonen, J.P.T. Diagnosis and discovery of fungal viruses using deep sequencing of small RNAs. J. Gen. Virol. 2015, 96, 714–725. [Google Scholar] [CrossRef]
  45. Whelan, S.; Goldman, N. A general empirical model of protein evolution derived from multiple protein families using a Maximum-Likelihood approach. Mol. Biol. Evol. 2001, 18, 691–699. [Google Scholar] [CrossRef]
  46. Li, W.; Sun, H.; Cao, S.; Zhang, A.; Zhang, H.; Shu, Y.; Chen, H. Extreme diversity of mycoviruses present in single strains of Rhizoctonia cerealis, the pathogen of wheat sharp eyespot. Microbiol Spectr. 2023, 11, e0052223. [Google Scholar] [CrossRef]
  47. Canuti, M.; Rodrigues, B.; Lang, A.S.; Dufour, S.C.; Verhoeven, J.T.P. Novel divergent members of the Kitrinoviricota discovered through metagenomics in the intestinal contents of red-backed voles (Clethrionomys gapperi). Int. J. Mol. Sci. 2023, 24, 131. [Google Scholar] [CrossRef]
  48. Forgia, M.; Isgandarli, E.; Aghayeva, D.N.; Huseynova, I.; Turina, M. Virome characterization of Cryphonectria parasitica isolates from Azerbaijan unveiled a new mymonavirus and a putative new RNA virus unrelated to described viral sequences. Virology 2021, 553, 51–61. [Google Scholar] [CrossRef] [PubMed]
  49. Kuhn, J.H.; Botella, L.; de la Peña, M.; Vainio, E.J.; Krupovic, M.; Lee, B.D.; Navarro, B.; Sabanadzovic, S.; Simmonds, P.; Turina, M. Ambiviricota, a novel ribovirian phylum for viruses with viroid-like properties. J. Virol. 2024, 98, e00831-24. [Google Scholar] [CrossRef] [PubMed]
  50. Sutela, S.; Forgia, M.; Vainio, E.J.; Chiapello, M.; Daghino, S.; Vallino, M.; Martino, E.; Girlanda, M.; Perotto, S.; Turina, M. The virome from a collection of endomycorrhizal fungi reveals new viral taxa with unprecedented genome organization. Virus Evol. 2020, 6, veaa076. [Google Scholar] [CrossRef]
  51. Göker, M.; Scheuner, C.; Klenk, H.-P.; Stielow, J.B.; Menzel, W. Codivergence of mycoviruses with their hosts. PLoS ONE 2011, 6, e22252. [Google Scholar] [CrossRef]
  52. Koonin, E.V.; Lee, B.D. Diversity and evolution of viroids and viroid-like agents with circular RNA genomes revealed by metatranscriptome mining. Nucleic Acids Res. 2025, 53, 1278. [Google Scholar] [CrossRef]
  53. Sun, L.; Hadidi, A. Mycoviroids: Fungi as Hosts and Vectors of Viroids. Cells 2022, 11, 1335. [Google Scholar] [CrossRef]
  54. Lee, B.D.; Neri, U.; Roux, S.; Wolf, Y.I.; Camargo, A.P.; Krupovic, M.; Simmonds, P.; Kyrpides, N.; Gophna, U.; Dolja, V.V.; et al. Mining metatranscriptomes reveals a vast world of viroid-like circular RNAs. Cell 2023, 186, 646–661. [Google Scholar] [CrossRef]
  55. Friday, P.; Mukkara, R.A.; Owens, T.; Baumstark, M.F.; Bruist, M.F. Processing of potatospindle tuber viroid RNAs in yeast, a nonconventional host. J. Virol. 2017, 91, e01078-17. [Google Scholar] [CrossRef]
  56. Latifi, L.; Bernard, C.S. Replication of avocado sunblotch viroid in the cyanobacterium Nostocsp. PCC 7120. J. Plant Pathol. Microbiol. 2016, 7, 341. [Google Scholar] [CrossRef]
  57. Tian, M.; Wei, S.; Bian, R.; Luo, J.; Khan, H.A.; Tai, H.; Kondo, H.; Hadidi, A.; Andika, I.B.; Sun, L. Natural cross-kingdom spread of apple Scar Skin viroid from apple trees to fungi. Cells 2022, 11, 3686. [Google Scholar] [CrossRef] [PubMed]
  58. Wei, S.; Bian, R.; Andika, I.B.; Niu, E.; Liu, Q.; Kondo, H.; Yang, L.; Zhou, H.; Pang, T.; Lian, Z.; et al. Symptomatic plant viroid infections in phytopathogenic fungi. Proc. Natl. Acad. Sci. USA 2019, 116, 13042–13050. [Google Scholar] [CrossRef] [PubMed]
  59. Afanasenko, O.S.; Khiutti, A.V.; Mironenko, N.V.; Lashina, N.M. Transmission of potato spindle tuber viroid between Phytophthora infestans and host plants. Vavilovskii Zhurnal Genet. Sel. 2022, 26, 272–280. [Google Scholar] [CrossRef] [PubMed]
  60. Dong, K.; Xu, C.; Kotta-Loizou, I.; Jiang, J.; Lv, R.; Kong, L.; Li, S.; Hong, N.; Wang, G.; Coutts, R.H.A.; et al. Novel viroid-like RNAs naturally infect a filamentous fungus. Adv. Sci. 2023, 10, e2204308. [Google Scholar] [CrossRef]
  61. Navarro, B.; Turina, M. Viroid and viroid-like elements in plants and plant-associated microbiota: A new layer of biodiversity for plant holobionts. New Phytol. 2024, 244, 1216–1222. [Google Scholar] [CrossRef]
  62. Hermanns, K.; Marklewitz, M.; Zirkel, F.; Kopp, A.; Kramer-Schadt, S.; Junglen, S. Mosquito community composition shapes virus prevalence patterns along anthropogenic disturbance gradients. eLife 2023, 12, e66550. [Google Scholar] [CrossRef]
  63. Tirera, S.; de Thoisy, B.; Donato, D.; Bouchier, C.; Lacoste, V.; Franc, A.; Lavergne, A. The influence of habitat on viral diversity in neotropical rodent hosts. Viruses 2021, 13, 1690. [Google Scholar] [CrossRef]
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Figure 1. Map showing the sampling sites in Bosnia and Herzegovina together with the corresponding Heterobasidion spp. identified and the tree species from which they were collected. For complete information, see Table S1. The map was created in www.datawrapper.de.
Figure 1. Map showing the sampling sites in Bosnia and Herzegovina together with the corresponding Heterobasidion spp. identified and the tree species from which they were collected. For complete information, see Table S1. The map was created in www.datawrapper.de.
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Figure 2. Genome organization of mycoviruses detected in Heterobasidion, drawn to scale (indicating genome length in nucleotides). One representative of the novel ambi-like viruses was selected to illustrate the typical location of ribozymes within their circular genome. HetPV, Heterobasidion partitivirus; HetRV, Heterobasidion RNA virus; HetMV, Heterobasidion mitovirus; HetNlV, Heterobasidion narna-like virus; HetOlV, Heterobasidion ourmia-like virus; HetTlV, Heterobasidion tobamo-like virus; HetBlV, Heterobasidion beny-like virus; HetDFV, Heterobasidion deltaflexivirus; HetAlV, Heterobasidion ambi-like virus. HPRz, hairpin ribozyme; HHRz, hammerhead ribozyme; IB, identical base. Predicted open reading frames: RdRP, RNA-dependent RNA polymerase; CP, coat protein; HP, hypothetical protein; MP, movement protein. Conserved domains: pfam00680, RdRP (E values: 1.7 × 10−8, 1.21 × 10−12, 4.53 × 10−10); pfam05919, mitovirus RdRP (1.25 × 10−80, 9.81 × 10−88, 2.99 × 10−80, 1.71 × 10−88); cd23183, Botourmiaviridae RdRP (3.92 × 10−48, 7.2 × 10−47, 2.82 × 10−48, 1.44 × 10−52, 7.12 × 10−52, 1.75 × 10-51); pfam01660, methyltransferase (1.3 × 10−13, 2.6 × 10−24); pfam01443, helicase (2.61 × 10−29, 3.39 × 10−11); cd23251, Virgaviridae RdRP (3.11 × 10−54); smart00487, DEAD-like helicases superfamily (1.47 × 10−5); pfam00271, helicase conserved C-terminal domain (1.44 × 105); cd17933, DEXS-box helicase (6.52 × 10−5); cd23257, Benyviridae RdRP (4.0 × 10−22); cd23248, Deltaflexiviridae RdRP (2.22 × 10−70). The dashed line within the HetTlV2 genome symbolizes a gap, which could not be reconstructed from RNA-Seq data.
Figure 2. Genome organization of mycoviruses detected in Heterobasidion, drawn to scale (indicating genome length in nucleotides). One representative of the novel ambi-like viruses was selected to illustrate the typical location of ribozymes within their circular genome. HetPV, Heterobasidion partitivirus; HetRV, Heterobasidion RNA virus; HetMV, Heterobasidion mitovirus; HetNlV, Heterobasidion narna-like virus; HetOlV, Heterobasidion ourmia-like virus; HetTlV, Heterobasidion tobamo-like virus; HetBlV, Heterobasidion beny-like virus; HetDFV, Heterobasidion deltaflexivirus; HetAlV, Heterobasidion ambi-like virus. HPRz, hairpin ribozyme; HHRz, hammerhead ribozyme; IB, identical base. Predicted open reading frames: RdRP, RNA-dependent RNA polymerase; CP, coat protein; HP, hypothetical protein; MP, movement protein. Conserved domains: pfam00680, RdRP (E values: 1.7 × 10−8, 1.21 × 10−12, 4.53 × 10−10); pfam05919, mitovirus RdRP (1.25 × 10−80, 9.81 × 10−88, 2.99 × 10−80, 1.71 × 10−88); cd23183, Botourmiaviridae RdRP (3.92 × 10−48, 7.2 × 10−47, 2.82 × 10−48, 1.44 × 10−52, 7.12 × 10−52, 1.75 × 10-51); pfam01660, methyltransferase (1.3 × 10−13, 2.6 × 10−24); pfam01443, helicase (2.61 × 10−29, 3.39 × 10−11); cd23251, Virgaviridae RdRP (3.11 × 10−54); smart00487, DEAD-like helicases superfamily (1.47 × 10−5); pfam00271, helicase conserved C-terminal domain (1.44 × 105); cd17933, DEXS-box helicase (6.52 × 10−5); cd23257, Benyviridae RdRP (4.0 × 10−22); cd23248, Deltaflexiviridae RdRP (2.22 × 10−70). The dashed line within the HetTlV2 genome symbolizes a gap, which could not be reconstructed from RNA-Seq data.
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Figure 4. Phylogenetic relationships within and between relevant families of the Lenarviricota phylum. The Fiersviridae family served as outgroup. The tree was built based on the alignment of RdRP aa sequences generated with MUSCLE in MEGA11 [30]. The evolutionary history was inferred by using the Maximum Likelihood method and the Whelan And Goldman + Freq. model [45]. A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories +G +I). All positions with less than 95% site coverage were eliminated; i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. Evolutionary analyses were conducted in MEGA11 with 1000 bootstrap repeats. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Branch lengths are proportional to the number of substitutions per site. Ourmia-like, narna-like, and mitoviruses described in this study are denoted by a triangle.
Figure 4. Phylogenetic relationships within and between relevant families of the Lenarviricota phylum. The Fiersviridae family served as outgroup. The tree was built based on the alignment of RdRP aa sequences generated with MUSCLE in MEGA11 [30]. The evolutionary history was inferred by using the Maximum Likelihood method and the Whelan And Goldman + Freq. model [45]. A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories +G +I). All positions with less than 95% site coverage were eliminated; i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. Evolutionary analyses were conducted in MEGA11 with 1000 bootstrap repeats. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Branch lengths are proportional to the number of substitutions per site. Ourmia-like, narna-like, and mitoviruses described in this study are denoted by a triangle.
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Figure 5. Phylogenetic relationships of HetTlV2 (denoted by a triangle) with selected members of the Virgaviridae family. The Gammaflexiviridae family served as outgroup. The tree was built based on the alignment of replication protein aa sequences generated with MUSCLE in MEGA11 [30]. The evolutionary history was inferred by using the Maximum Likelihood method and the Le_Gascuel_2008 model [41]. A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories +G +I). All positions with less than 95% site coverage were eliminated; i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. Evolutionary analyses were conducted in MEGA11 with 1000 bootstrap repeats. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Branch lengths are proportional to the number of substitutions per site.
Figure 5. Phylogenetic relationships of HetTlV2 (denoted by a triangle) with selected members of the Virgaviridae family. The Gammaflexiviridae family served as outgroup. The tree was built based on the alignment of replication protein aa sequences generated with MUSCLE in MEGA11 [30]. The evolutionary history was inferred by using the Maximum Likelihood method and the Le_Gascuel_2008 model [41]. A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories +G +I). All positions with less than 95% site coverage were eliminated; i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. Evolutionary analyses were conducted in MEGA11 with 1000 bootstrap repeats. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Branch lengths are proportional to the number of substitutions per site.
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Figure 6. Phylogenetic relationships of HetBlV1 and HetDFV1 (both denoted by a triangle) with selected members of the Benyviridae and Deltaflexiviridae families. The Endornaviridae family served as outgroup. The tree was built based on the alignment of RdRP aa sequences generated with MUSCLE in MEGA11 [30]. The evolutionary history was inferred by using the Maximum Likelihood method and the Whelan And Goldman + Freq. model [45]. A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories +G +I). All positions with less than 95% site coverage were eliminated; i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. Evolutionary analyses were conducted in MEGA11 with 1000 bootstrap repeats. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Branch lengths are proportional to the number of substitutions per site.
Figure 6. Phylogenetic relationships of HetBlV1 and HetDFV1 (both denoted by a triangle) with selected members of the Benyviridae and Deltaflexiviridae families. The Endornaviridae family served as outgroup. The tree was built based on the alignment of RdRP aa sequences generated with MUSCLE in MEGA11 [30]. The evolutionary history was inferred by using the Maximum Likelihood method and the Whelan And Goldman + Freq. model [45]. A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories +G +I). All positions with less than 95% site coverage were eliminated; i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. Evolutionary analyses were conducted in MEGA11 with 1000 bootstrap repeats. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Branch lengths are proportional to the number of substitutions per site.
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Figure 7. Phylogenetic relationships of ambi-like viruses. The tree was built based on the alignment of RdRP aa sequences generated with MUSCLE in MEGA11 [30]. The evolutionary history was inferred by using the Maximum Likelihood method and the Le_Gascuel_2008 model [41]. A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories +G +I). All positions with less than 95% site coverage were eliminated; i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. Evolutionary analyses were conducted in MEGA11 with 1000 bootstrap repeats. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Branch lengths are proportional to the number of substitutions per site. Viruses described in this study are denoted by a triangle.
Figure 7. Phylogenetic relationships of ambi-like viruses. The tree was built based on the alignment of RdRP aa sequences generated with MUSCLE in MEGA11 [30]. The evolutionary history was inferred by using the Maximum Likelihood method and the Le_Gascuel_2008 model [41]. A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories +G +I). All positions with less than 95% site coverage were eliminated; i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. Evolutionary analyses were conducted in MEGA11 with 1000 bootstrap repeats. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Branch lengths are proportional to the number of substitutions per site. Viruses described in this study are denoted by a triangle.
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Figure 8. Country-wise DAPC plot of Heterobasidion ambiviruses. The two discriminant functions with the highest eigenvalues are displayed.
Figure 8. Country-wise DAPC plot of Heterobasidion ambiviruses. The two discriminant functions with the highest eigenvalues are displayed.
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Figure 9. Illustration of the discovered HetcRNAs in the RNA library ABI. Viroid ribozymes. Circular plots of the predicted RNA secondary structures of both HetcRNAs.
Figure 9. Illustration of the discovered HetcRNAs in the RNA library ABI. Viroid ribozymes. Circular plots of the predicted RNA secondary structures of both HetcRNAs.
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Dálya, L.B.; Hejna, O.; de la Peña, M.; Stanivuković, Z.; Kudláček, T.; Botella, L. Diversity of RNA Viruses and Circular Viroid-like Elements in Heterobasidion spp. in Near-Natural Forests of Bosnia and Herzegovina. Viruses 2025, 17, 1144. https://doi.org/10.3390/v17081144

AMA Style

Dálya LB, Hejna O, de la Peña M, Stanivuković Z, Kudláček T, Botella L. Diversity of RNA Viruses and Circular Viroid-like Elements in Heterobasidion spp. in Near-Natural Forests of Bosnia and Herzegovina. Viruses. 2025; 17(8):1144. https://doi.org/10.3390/v17081144

Chicago/Turabian Style

Dálya, László Benedek, Ondřej Hejna, Marcos de la Peña, Zoran Stanivuković, Tomáš Kudláček, and Leticia Botella. 2025. "Diversity of RNA Viruses and Circular Viroid-like Elements in Heterobasidion spp. in Near-Natural Forests of Bosnia and Herzegovina" Viruses 17, no. 8: 1144. https://doi.org/10.3390/v17081144

APA Style

Dálya, L. B., Hejna, O., de la Peña, M., Stanivuković, Z., Kudláček, T., & Botella, L. (2025). Diversity of RNA Viruses and Circular Viroid-like Elements in Heterobasidion spp. in Near-Natural Forests of Bosnia and Herzegovina. Viruses, 17(8), 1144. https://doi.org/10.3390/v17081144

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