Transcapsidation and Polysomal Encapsulation as Putative Strategies for the Genome Protection of the Novel Diplodia fraxini Fusagravirus 1 (DfFV1)

Abstract

Two novel dsRNA mycoviruses were found in different isolates of Diplodia fraxini, NW FVA 1581 and NW FVA 1706, which were isolated from a root, associated with stem collar necrosis of Fraxinus excelsior L. Both mycelia are infected by a novel fusagravirus, which was named Diplodia fraxini fusagravirus 1 (DfFV1), and isolate NW FVA 1706 is additionally infected by a novel partitivirus, which was denominated as Diplodia fraxini partitivirus 1 (DfPV1). The one-segmented, bicistronic genome of DfFV1 is composed of about 8,500 bp. Their ORFs are connected by a 1 slippery heptamer sequence and the 3’ terminal ORF is coding for the viral RdRp. The genome of DfPV1 is composed of three, monocistronic dsRNA segments ranging from 1,755 bp (dsRNA 1) over 1,588 bp (dsRNA 2) to 1,233 bp (dsRNA 3). Based on genome organization and phylogenetic positions, DfFV1 was assigned to the proposed family of “Fusagraviridae” and DfPV1 to the genus Gammapartitivirus within the family of Partitiviridae. Ultra-structural analysis showed that polysomal structures were stabilized in the single infection and none of these structures could be isolated in the double infection. It is assumed that DfFV1 has an opportunistic lifestyle, being either protected by ribosomes or by transcapsidation from particles of DfPV1.

Full text

Article Not peer-reviewed version
Transcapsidation and Polysomal
Encapsulation as Putative
Strategies for the Genome
Protection of the Novel
Diplodia
fraxini
Fusagravirus 1 (DfFV1)
Tobias Lutz , Steffen Bien , Gitta Jutta Langer , Cornelia Heinze *
Posted Date: 13 July 2023
doi: 10.20944/preprints202307.0901.v1
Keywords: Fusagravirus; Partitivirus; Co-infection; Transcapsidation; Polysomal encapsulation; Diplodia
fraxini
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Article
Transcapsidation and Polysomal Encapsulation as
Putative Strategies for the Genome Protection of the
Novel Diplodia Fraxini Fusagravirus 1 (DfFV1)
Tobias Lutz 1, Steffen Bien 2, Gitta Jutta Langer 2 and Cornelia Heinze 1,*
1 University of Hamburg, Institute of Plant Science and Microbiology, Molecular Phytopathology,
Ohnhorststr. 18, 22609 Hamburg, Germany; tobias.lutz@uni-hamburg.de, cornelia.heinze@uni-hamburg.de
2 Nordwestdeutsche Forstliche Versuchsanstalt, Grätzelstr. 2, 37079 Göttingen, Germany;
steffen.bien@nw-fva.de, gitta.langer@nw-fva.de
* Correspondence: cornelia.heinze@uni-hamburg.de; Tel.: +49-40-42816-227
Abstract: Two novel dsRNA mycoviruses were found in different isolates of Diplodia fraxini, NW-FVA 1581
and NW-FVA 1706, which were isolated from a root, associated with stem collar necrosis of Fraxinus excelsior
L. Both mycelia are infected by a novel fusagravirus, which was named Diplodia fraxini fusagravirus 1 (DfFV1),
and isolate NW-FVA 1706 is additionally infected by a novel partitivirus, which was denominated as Diplodia
fraxini partitivirus 1 (DfPV1). The one-segmented, bicistronic genome of DfFV1 is composed of about 8,500 bp.
Their ORFs are connected by a -1 slippery heptamer sequence and the 3’-terminal ORF is coding for the viral
RdRp. The genome of DfPV1 is composed of three, monocistronic dsRNA segments ranging from 1,755 bp
(dsRNA 1) over 1,588 bp (dsRNA 2) to 1,233 bp (dsRNA 3). Based on genome organization and phylogenetic
positions, DfFV1 was assigned to the proposed family of “Fusagraviridae” and DfPV1 to the genus
Gammapartitivirus within the family of Partitiviridae Ultra-structural analysis showed that polysomal structures
were stabilized in the single infection and none of these structures could be isolated in the double infection. It
is assumed that DfFV1 has an opportunistic lifestyle, being either protected by ribosomes or by
transcapsidation from particles of DfPV1.
Keywords: Fusagravirus; Partitivirus; Co-infection; Transcapsidation; Polysomal encapsulation;
Diplodia fraxini
1. Introduction
The rapid spread of the invasive Ascomycete Hymenoscyphus fraxineus (T. Kowalski) Baral,
Queloz & Hosoya affects the natural ash species in Europe and their decline increased in the last three
decades. Especially in Northern Europe, ash trees already have been eradicated [1]. Different fungi
are associated with infected trees and among them, members of the Botryosphaeriaceae are the main
species involved in ash dieback etiology [2]. Within this family, Diplodia mutila (Fr.) Mont.
(teleomorph: Botryosphaeria stevensii Shoemaker) was one of the most reported species [3,4]. Diplodia
fraxini (Fr.) Fr. (Botryodiplodia fraxini (Fr.) Sacc.) has been referred to Diplodia mutila (D. mutila) in
previous studies [5], as both species are very similar and closely related. Based on the work of Alves
et al. [6], D. mutila and Diplodia fraxini (D. fraxini) can be differentiated by morphological and
phylogenetic markers. Diplodia fraxini causes dark brown inner bark lesions on F. excelsior that spread
up and down from the site of infection.
In all main taxa of fungi and oomycetes, mycoviruses are widespread [reviewed in 7]. Since the
first discovery of a mycovirus which causes morphological alterations in cultivated mushrooms by
Hollings [8], the knowledge about mycoviruses has expanded rapidly in the last decade. Mycoviruses
can encode their proteasome on positive sense single-stranded RNA (+ssRNA), which was latest
discovered to be the most widespread strategy among mycoviruses, on negative sense
single-stranded RNA (-ssRNA) or on positive sense single-stranded DNA (+ssDNA). Besides these,
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2
many mycoviruses possess a genome which consists of double-stranded RNA (dsRNA) [reviewed in
7].
Within the order of Durnavirales, currently six families are recognized by the International
Committee on Taxonomy of Viruses (ICTV): Amalgaviridae, Curvulaviridae, Fusariviridae, Hypoviridae,
Picobirnaviridae and Partitiviridae (https://ictv.global/taxonomy). Members of the Family Partitividae
encode a RdRp and a capsid protein on two dsRNA segments which range in total from 3,000–4,800
bp. However, additional defective or satellite dsRNA segments may also be present. The segments
are separately encapsidated by identical capsid subunits in isometric particles with sizes ranging
from 25 to 43 nm [9]. According to the ICTV, members of the family are separated in five genera
(Alphapartitivirus, Betapartitivirus, Cryspovirus, Deltapartitivirus, Gammapartitivirus) [9]. Recently, two
novel genera, “Epsilonpartitivirus” and “Zetapatitivirus” were proposed by Jiang et al. [10] and Nerva
et al. [11]. While partitiviruses found in fungi, plants and insects are accommodated in the genera
Alpha-, Beta- and Deltapartitivirus or in the proposed genus “Epsilonpartitivirus”, members of the genus
Gammapartitivirus and of the proposed genus “Zetapatitivirus” are exclusively described from fungal
hosts [reviewed in 7,9]. Beside their host range, the different genera are distinguished by features of
the dsRNA, the size of the segments and the molecular weight (MW) of the capsid protein subunits
[9]. While the 3’-terminus of members of the Alpha- and Betapartitiviruses genus is polyadenylated, no
poly(A) tail is found on gammapartitiviruses. Several members of the Partitiviridae family are not
bipartite but tripartite [12,13]. The function of the third segment is still unknown and it may not be
always detectable [12].
Viruses in the proposed family of “Fusagraviridae” [14] contain a bicistronic dsRNA genome
which ranges from 8,500 bp for Trichoderma atroviride mycovirus 1 [15] to 10,200 bp for
Cryphonectria naterciae fusagravirus 1 [16]. Viruses of this family were detected in fungi, plants [17]
and insects [18]; however, they are, according to Ayllón and Vainio [7] mostly found in ascomycetes.
The two ORFs are believed to be separated by a -1 ribosomal frameshifting, which is mediated by a
heptameric slippery sequence with the consensus nucleotides (nts) XXXX (any nucleotide) YY (either
A or U) and Z (not G) upstream of the 5’-proximal (ORF 1) stop codon and a Recoding Stimulatory
Element (RSE) immediately downstream from the slippery site [14,19–22]. While ORF 1 encodes a
hypothetical protein, the 3´-proximal ORF (ORF 2) encodes a protein with RdRp motifs and in some
species a Phytoreo_S7 domain was detected [14]. Although for several fusagra-like viruses, the
expression of a capsid protein was verified [17] and particles were obtained by sucrose density
centrifugation [15], others suggested a capsidless nature of fusagraviruses [18,23,24].
In many publications, species within the genus Botryosphaeria were described to be viral hosts
[25–27], the first report of a virus in Diplodia sp. was described from Diplodia seriata (De Not) with a
multiinfection [28]. Until now, no viruses were described from D. fraxini. In here, we describe the
novel partitivirus Diplodia fraxini partitivirus 1 (DfPV1) and two strains of the novel Diplodia fraxini
fusagravirus 1 (DfFV1) which we isolated from two independent isolates of D. fraxini. We showed
that the single infection of DfFV1 and the double infection of DfFV1 and DfPV1 differ in the formation
of their ultrastructural patterns.
2. Materials and Methods
2.1. Fungal Isolates, Propagation And Species Determination
The Diplodia fraxini strains NW-FVA 1581 and NW-FVA 1706 were isolated from trunk tissues
of Fraxinus excelsior L. The sampled European ash trees were affected by ash dieback caused by H.
fraxineus and exhibited stem collar necrosis and rots. Isolation of both D. fraxini strains and the sample
sites were described in Langer [5], in which these strains were referred to Botryosphaeria stevensii
(Anamorph: Diplodia mutila). Briefly, NW-FVA 1581 was isolated from wood tissue sampled in
Schleswig-Holstein, Germany (Tree 8, N54° 40.264' E9°41.202'). NW-FVA 1706 was sampled from
necrotic tissue in Schleswig-Holstein, Germany, (Tree 26, N54° 05.238' E10° 23.159').
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Cultivation of mycelium was performed in the dark at room temperature on solid complete
medium (CMS) as stated in Leach et al. [29]. For harvesting, the mycelium was separated from the
medium by a cellophane sheet.
For the phylogenetic analysis of the fungal isolates, genomic DNA was extracted using the
method of Damm et al. [30]. The 5.8S nuclear ribosomal gene with the two flanking internal
transcribed spacers (ITS-1 and ITS-2) was amplified using the primer pairs ITS-1F [31] and ITS-4 [32].
Additionally, partial sequences of the translation elongation factor 1α (TEF1-α) and of the β-tubulin
gene (TUB) were generated using the primer pairs EF1-728F [33], EF1-1567R [34] and Bt2a+Bt2b [35].
Obtained sequences of PCR products were submitted to GenBank and are displayed in Table 1.
Table 1. GenBank accession IDs of the sequenced isolates NW-FVA 1581 and NW-FVA 1706.
Taxon Isolate Acc. ID ITS Acc. ID TEF1-
α
Acc. ID TUB
D. fraxini NW-FVA 1581
OR050980
OR079892
OR079888
D. fraxini NW-FVA 1706
OR050981
OR079893
OR079889
For phylogenetic analysis, three single locus datasets (ITS, EF1-α, TUB), including appropriate
reference sequences retrieved from GenBank, were aligned automatically using MAFFT v. 7.308
[36,37] and manually adjusted where necessary. The concatenated ITS-EF1-α-TUB sequence-dataset
was analyzed using Bayesian Inference (BI) and Maximum Likelihood (ML). For BI analysis, the best
fit model of evolution for each partition was estimated by MEGA7 [38]. Posterior probabilities were
determined by Markov Chain Monte Carlo sampling (MCMC) in MrBayes v. 3.2.6 [39,40] as
implemented in Geneious R11 [41], using the estimated models of evolution. Four simultaneous
Markov chains were run for 1 million generations and trees were sampled every 100th generation.
The first 2000 trees, which represent the burn-in phase of the analysis, were discarded and the
remaining 8000 trees were used to calculate posterior probabilities in the majority rule consensus tree.
The ML analysis was performed by RAxML v. 8.2.11 [42,43] as implemented in Geneious R11 [41],
using the GTRGAMMA model with the rapid bootstrapping and search for best scoring ML tree
algorithm, including 1000 bootstrap replicates.
2.2. DsRNA Extraction, Virus-Like Particle Purification, Protein Analysis and Electron Microscopy
Double stranded RNA was extracted from mycelium using the dsRNA Extraction kit (iNtRON
Biotechnology, Seongnam-Si, South Korea) and was analyzed by 1 % (w/v) agarose gel
electrophoresis. Virus like particles (VLPs) and polysomes (PSs) were extracted as described for the
betachrysovirus Fusarium graminearum virus-China 9 (FgV-ch9) in Lutz et al. [44]. The dsRNA of
VLPs and PSs was extracted by peqGOLD TriFastTM (VWR life sciences, Radnor, Pennsylvania, USA)
according to the manufacturer’s protocol. The protein patterns of VLPs and PSs were analyzed by a
12.5 % (w/v) SDS-PAGE visualized by Coomassie-Brilliant Blue staining. Bands were cut from the gel
and sequenced with LC-MS/MS by a nano-liquid chromatography system (Dionex UltiMate 3000
RSLCnano, ThermoFisher Scientific, Waltham, Massachusetts, USA) and analyzed by means of the
Proteome Discoverer 2.0 (ThermoFisher Scientific) by the Universitätsklinikum Hamburg-Eppendorf
(UKE, Hamburg, Germany). VLPs and PSs were examined by electron microscopy (LEO 906E, Zeiss,
Germany) with 2 % (w/v) uranyl acetate contrasting.
2.3. Virus Sequence Determination
From VLPs isolated dsRNA was submitted to Next-Generation Sequencing. The libraries were
prepared according to Nextera XT DNA Library Preparation Kit (Illumina Inc., San Diego, CA, USA)
and run on a NextSeq 2000 (Illumina Inc., San Diego, CA, USA) instrument at the Leibniz Institute
DSMZ (Braunschweig, Germany) as pair-end reads (2 × 151). De novo assembly of contigs was
performed by using Geneious Prime software (Biomatters, Auckland, New Zealand, version
2021.2.2). The extreme 5′- and 3′-termini were determined by single-primer amplification technique
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(SPAT) using an oligonucleotide with a phosphorylated 5′-terminus and a 2′,3′-dideoxyC-group
(23ddC) at the 3′-terminus as a blocker to prevent self-ligation
(5′-PO4-TCTCTTCGTGGGCTCTTGCG-23ddC-3′) according to Zhong et al. [45], RT and PCR.
Sequences of further primers used for sequencing are displayed in Table S1. Amplicons were cloned
into pGEM®-T Vector (Promega Corporation, Fitchburg, Wisconsin, USA) and sequenced. Nucleic
acid sequences and ORFs were analyzed by SnapGene (GSL Biotech, San Diego, California, USA) and
BLAST on the NCBI website [46]. Alignments of protein sequences and phylogenetic analysis were
performed using MEGA X (version 10.2.4; 38,47) with the respective algorithm. The 5’- and 3’-termini
of the dsRNA segments of DfPV1 were aligned using the Muscle algorithm [48–51] in default settings.
Alignments for the ML analysis were prepared with the Clustal Omega algorithm [48,52–54] in
default settings. A bootstrap test was conducted with 1000 replicates for the construction of a ML
tree. For DfFV1a and DfFV1b, the model by Le and Gascuel [55] with frequencies and gamma
distribution of 5 (LG+G+F) was used. The ML tree of DfPV1, was constructed using the model by Le
and Gascuel [55] and a gamma distribution of 5 (LG+G). Phylogenetic analysis was carried out after
sequence alignment of the RdRp of virus sequences found by BLASTp with an E-value of 0.0 and
were adjusted by hand where necessary. The ML tree of DfFV1a and DfFV1b was rooted with
sequences of RdRps of the Magnaporthe oryzae Chrysovirus 1 D/B (MoCV1-D/B) of the Chrysoviridae
family [56,57]. The ML tree of DfPV1 was rooted by the use of RdRps of Heterobasidion partitivirus
3 and 12 from the genus Alphapartitivirus [58,59]. Figures were generated and edited by Unipro
UGENE (ugene.net, version 1.32.0), INKSCAPE (inkscape.org, version 1.1) and SnapGene. Conserved
protein domains were identified by conserved domain database (CDD) search on the NCBI website
[60–63].
2.4. Verification of Virus Presence by RT-PCR
To screen both isolates, NW-FVA 1581 and NW-FVA 1706, for the presence of DfPV1 and DfFV1
by reverse transcriptase PCR (RT-PCR), cDNA was synthesized by using 100 U Maxima H Minus
Reverse Transcriptase (ThermoFisher Scientific) with random primers according to the
manufacturer’s instructions from dsRNA extracted from mycelium and from VLPs. Presence of
DfPV1 was verified by amplification of a 274 bp fragment of the RdRp gene encoded on segment 1
by using primer pair number 1 and 7 (Table S1) and by amplification of a 498 bp fragment from the
ORF 1 gene of DfFV1 with primer pair number 8 and 9 (Table S1).
3. Results
3.1. NW-FVA 1581 and NW-FVA 1706 were Determined as D. fraxini and Harbor dsRNAs
The isolates NW-FVA 1581 and NW-FVA 1706 were determined as D. fraxini by the evaluation
of the ITS, the TEF1-α and TUB genes. The phylogenetic analysis is displayed in Figure S1. On CMS,
both isolates grew at the same speed covering the medium of a 6.5 mm petri dish within 21 days.
While the mycelium of isolate NW-FVA 1581 developed aerial and colored hyphae, the mycelium of
NW-FVA 1706 produced fewer aerial hyphae without extended pigmentation (Figure 1).
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Figure 1. Diplodia fraxini NW-FVA 1581 and NW-FVA 1706 growing on CM
s
for 21 days in the dark
from the front and the back.
3.2. NW-FVA 1581 is Infected by a Novel Fusagravirus and NW-FVA 1706 Additionally by a Novel
Partitivirus
Extraction of dsRNA from mycelium of NW-FVA 1581 and NW-FVA 1706 suggested the
presence of a virus in both isolates (Figure S2). Next Generation Sequencing and completion by SPAT
showed the presence of three segments of 1,755 bp (dsRNA 1), 1,588 bp (dsRNA 2) and 1,233 bp
(dsRNA 3) in isolate NW-FVA 1706 and in both isolates bands of 8,720 bp in NW-FVA 1706 and 8,644
bp in NW-FVA 1581.
The ORF of dsRNA 1 (1755 bp) is flanked by 66 nts at the 5’-NTR and 69 nts at the 3’-NTR. It
encodes the protein P1 which consists of 539 amino acids (aa) and a calculated MW of 62.06 kDa
(Figure 2C). The viral RdRp of Botryospheria dothidea partitivirus 2 (BdPV2, acc. ID: WFJ08489.1)
showed the highest similarity to P1 (80.45 % identical aa, E-value 0.0). Further in silico analysis by
CDD search showed RdRp motifs (acc. ID: pfam00680) in the RT_like superfamily (acc. ID: cl02808)
being present from position 46 to 492 (E-value 1.41e-85). The ML analysis showed that P1 clusters in
the genus Gammapartitivirus together with BdPV2 and BdPV3 and with Aspergillus ochraceous virus
(AoV).
The ORF of dsRNA 2 encodes for protein P2, consisting of 435 aa with a calculated MW of 47.18
kDa flanked by 84 nts at the 5’-NTR and 199 nts at the 3’-NTR (Figure 2C). It showed highest similarity
to the capsid protein of Penicillium brevicompactum partitivirus 1 (PbPV1, 65.97 % identical aa,
E-value 0.0, acc. ID: AYP71817.1) found in sea cucumber Holothuria poli [64]. Due to the BLASTp
search, ORF 2 is hypothetically coding for the capsid subunits of the virus particle.
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Figure 2. Genome organization and phylogenetic analysis of DfFV1a/b and DfPV1. The dsRNA
segments are displayed as horizontal lines with their respective UTRs at each terminus. ORFs are
represented as boxes with start and stop codon positions indicated above and underneath the boxes.
Note that the genome organization is not drawn to scale. Maximum-likelihood tree of DfFV1a/b and
DfPV1 and selected viruses. 1000 bootstrap replicates were performed, their values are displayed at
the nodes. The scale bar corresponds to the genetic distance. The colored dots indicate the novel
viruses. A: Genome organization of DfFV1a. B Maximum-likelihood tree of DfFV1a/b. Additionally,
the genetic distance is indicated above the interrupted lines. The names of viruses are abbreviated as
follows: BcV1: Botrytis cinerea RNA virus 1; DfFV1a/b: Diplodia fraxini fusagravirus 1a/b; FgMV3:
Fusarium graminearum dsRNA mycovirus-3; FpFV1: Fusarium poae fusagravirus 1; FpMV1:
Fusarium poae mycovirus 1; FpV2/3: Fusarium poae dsRNA virus 2/3; MoCV1 B/D: Magnaporthe
oryzae chrysovirus 1; MpFV1-5: Macrophomina phaseolina fusagravirus 1-5;MpV: Macrophomina
phaseolina double-stranded RNA; MV-D: Monilinia virus D; PvMV961/962: Phomopsis viticola
mycovirus 961/962; RnFV1: Rosellinia necatrix fusagravirus 1; SsFV1/2: Sclerotinia sclerotiorum
fusagravirus 1/2; SsMV-L: Sclerotinia sclerotiorum dsRNA mycovirus-L; SsMV-L-WX1/2: Sclerotinia
sclerotiorum dsRNA mycovirus-L-WX1/2; TaV1: Trichoderma asperellum dsRNA virus 1; ThV1:
Trichoderma hamatum dsRNA virus 1; ThV2: Trichoderma harzianum dsRNA virus 2. C: Genome
organization of DfPV1. D: Maximum-likelihood tree of DfPV1. Alpha(α)- and
Gamma(γ)partitiviruses are indicated on the right. The names of viruses are abbreviated as follows:
BdV2/3: Botryosphaeria dothidea partitivirus 2/3; DfPV1: Diplodia fraxini partitivirus 1; AoV:
Aspergillus ochraceous virus; PdPV1: Penicillium digitatum partitivirus 1; PsV-S/F: Penicillium
stoloniferum virus S/F; GaRV-MS: Gremmeniella abietina RNA virus MS1; CchPV1: Cordyceps
chanhua partitivirus 1; OPV1: Ophiostoma partitivirus 1; DdV1/2: Discula destructiva virus 1/2; FsV1:
Fusarium solani virus 1; HPV3/12: Heterobasidion partitivirus 3/12. E: Clipping of the alignments of
NTRs of dsRNA segments of DfPV1. The 5’-terminus is displayed at the top and the 3’-terminus at
the bottom.
Segment 3 (dsRNA 3) codes for protein P3, consisting of 272 aa and with a calculated size of
30.75 kDa. The ORF is flanked by a 181 nts 5’-NTR and a 233 nts 3’-NTR (Figure 2C). Highest identity
was found with the corresponding protein deduced from the unverified sequence of segment 3 of
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BdPV2 (acc. ID: MZ044012.1) with 70.97 % identical aa. Analysis by BLASTp revealed additional five
sequences which produced significant alignments. The highest similarity of P3 was shared with an
unknown protein from Aspergillus fumigatus partitivirus 1 (AfPV1, 34.86 % identical aa, E-value
1e-25, acc. ID: CAA7351346.1) and the lowest similarity with a hypothetical protein of Trichoderma
harzianum partitivirus 3 (ThPV3, 25.86 % identical aa, E-value 4e-17, acc. ID: WGH72996.1). Even
though AoV clusters in the same branch when comparing the aa sequences of the RdRps, it only
shows 32.6 % identical aa when comparing P3 (acc. ID: AYP71820.1). For the other virus, BdPV3,
which also clusters in the same branch, no third segment was reported.
The heptamer 5´-CGCAAAA-3´ of the extreme 5´-termini and the trimer of the extreme
3´-termini (5´-TCC-3´) are identical in all segments. In segment 3, a 36 nts insertion starts at position
14 which separates a conserved stretch (Figure 2E).
In total, the genome of DfPV1 consists of 4,576 bp. Due to the high similarity to
gammapartitiviruses, we denominate the tri-segmented virus as Diplodia fraxini partitivirus 1
(DfPV1). The complete sequences were deposited in GenBank (acc. ID: OR199886 - OR199888).
The complete sequences of the 8,720 bp isolated from NW-FVA 1706 and the 8644 bp isolated
from NW-FVA 1581 share 90.36 % identical nts and have a GC content of 51 %. Due to their high
similarity, a detailed description will be given to the 8720 bp dsRNA. The dsRNA harbors two
discontinuous open reading frames, which may be connected by a -1 slippery heptamer sequence
5’-4645GGAAAAC4651-3’.
The 5´ proximal ORF (ORF 1) starts at position 538, is terminated at position 4,599 and codes for
a hypothetical protein (P1) consisting of 1,354 aa with a calculated MW of 150.98 kDa (Figure 2A). A
BLASTp search revealed highest similarity (41.96 % identical aa, E-value 0.0) to the hypothetical
protein of Macrophomia phaseolina fusagravirus 2 (MpFV2, acc. ID: QK002083.1).
The 3´ proximal ORF (ORF 2) starts at position 5109 and terminates at position 8621. It encodes
a protein (P2) of 1,345 aa with a calculated MW of 151.59 kDa (Figure 2A) and showed highest
similarity (40.58 % identical aa, E-value 0.0) to the RdRp of Macrophomina phaseolina fusagravirus
3 (MpFV3, acc. ID: QKO02086.1). Conserved motifs of the RT-like superfamily (acc. ID: cl02808) were
detected by CDD search between position 513 and 791 (E-value 2.02e-12) suggesting that ORF 2 is
coding for the viral RdRp. No Phytoreo_S7 domain was detected by CDD search.
To further analyze the taxonomic position of the two strains of DfFV1, a ML tree was constructed
based on the aa sequences of both putative viral RdRps. Both viruses cluster together and build a
clade with fusagraviruses found in Macrophomina phaseolina. Therefore, we denominated the viruses
as Diplodia fraxini fusagravirus 1a and as Diplodia fraxini fusagravirus 1b. Both sequences were
deposited at GenBank (acc. ID: OR224544 and OR228587).
3.3. Single and Double Virus Infections Result in Different Ultra-Structures and dsRNA Patterns
To further analyze the viral structure of DfFV1a/b, VLPs and PSs were purified from 21 d old
mycelium from both fungal isolates and subjected to electron microscopy. In isolate NW-FVA 1706,
isometric particles with an average size of around 25 nm were detected (Figure 3B). In fungal isolate
NW-FVA 1581, exclusively cauliflower-like structures were detected (Figure 3A, left). The same
cauliflower-like structures were observed when a protocol for polysome enrichment was applied for
mycelium of NW-FVA 1581 (Figure 3A, right), and no structures were detected for NW-FVA 1706
when the same protocol was applied (not shown).
The protein pattern of VLPs and is shown in Figure 3C. In extractions of VLPs of NW FVA 1581,
a band in the size of about 90 kDa is present (lane 2). Note, that polysomal structures were purified
when the protocol for VLP purification was applied. Protein sequencing of the band observed in the
particle preparation resulted in peptides corresponding to the P1 sequence (Figure S3). Polysome
extraction revealed a slightly smaller band with the MW of about 80 kDa (lane 4). In addition, a typical
band pattern for polysomes is visible below 40 kDa. When extracting VLPs from NW-FVA 1706, two
bands with sizes between 55 and 40 kDa are visible, but no band in the range of 80 to 90 kDa (lane 1).
Peptides corresponding to P2 of DfPV1 were obtained by protein sequencing (Figure S4). After
polysome extraction, no proteins were detected (lane 3).
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Figure 3. Electron micrographs of VLPs and PSs examined by transmission electron microscopy
contrasted with 2 % /w/v) uranyl acetate and SDS-PAGE (12.5 % w/v) stained with Coomassie Brilliant
Blue. The black bar within the electron micrographs corresponds to 100 nm. A: Ultra-structures
obtained from mycelium of NW-FVA 1581 using a protocol for VLPs-isolation (left) and protocol for
PSs-isolation (right). B: Isometric particles obtained from mycelium of NW-FVA 1706 with a protocol
for VLPs-isolation. C: Protein patterns of VLPs and PSs isolations. M, PageRuler Prestained Protein
Ladder (Thermo Fisher Scientific). 1, VLPs isolated from NW-FVA 1706. 2, VLPs isolated from NW-
FVA 1581. 3, PSs isolated from NW-FVA 1706. 4, PSs isolated from NW-FVA 1581.
RNA extraction from VLPs purified from NW-FVA 1581 showed exclusively a band of about
9,000 bp (Figure 4A, lane 2) which corresponds with the segment size of 8,644 bp, four bands
corresponding to the sizes of the segments of DfFV1 and DfPV1 were visible at around 9,000 bp and
at 1,500 to 2,000 bp, when RNA was extracted from VLPs, purified from NW-FVA 1706 (Fig 4A, lane
1).
Figure 4. Agarose gel electrophoresis (1 % w/v) of dsRNA extracted from VLPs and PSs isolated from
mycelium of NW-FVA 1581 and NW-FVA-1706. M, GeneRuler 1 kb plus DNA ladder (Thermo Fisher
Scientific). The sizes of the marker are given on the left. 1, RNA pattern isolated from NW-FVA 1706.
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2, RNA pattern isolated from NW-FVA 1581 A: Viral dsRNA extracted from VLPs. B: Viral RNA
extracted from PSs.
A band of about 9,000 bp according to the size of DfFV1b was detected when RNA was extracted
from NW-FVA 1581 PSs (Figure 4B, lane 2). In addition, bands in the range from 1,000 to 2,000 bp
were detected which we addressed to host rRNA and mRNA. Neither a viral band nor host RNA was
present when RNA was derived from PSs from NW-FVA 1706 (Figure 4B; lane 1).
3.4. Single and Double Infections were Verified by RT-PCR from Mycelium and VLPs
To verify the presence of the viruses DfPV1 and DfFV1 in the two fungal isolates, NW-FVA 1581
and NW-NVA 1706, RT-PCR with species specific primers was conducted using dsRNA extracted
from fungal tissue and RNA extracted from VLPs. In isolate NW-FVA 1706, both viruses were
detected irrespective of the template used, while in isolate NW-FVA 1581 only DfFV1b was detected
(Figure 5).
Figure 5. Agarose gel electrophoresis (1% w/v) of RT-PCR products to detect DfPV1 and DfFV1 in
NW-FVA 1581 and NW-FVA 1706 from RNA isolated from VLPs and from dsRNA extraction from
mycelium. M, GeneRuler 1 kb plus DNA ladder (Thermo Fisher Scientific). The sizes of the marker
are given on the left. When DfFV1 was detected, band sizes of 498 bp, and when DfPV1 was detected,
band sizes of 274 bp were expected. The fungal isolates are indicated at the bottom. RNA was isolated
from VLPs and used as template in lane 1,2,5,6. RNA was isolated from mycelium and used as
template in lane 3,4,7,8. Lane 1: Detection of DfPV1. Lane 2: Detection of DfFV1. Lane 3: Detection of
DfPV1. Lane 4: Detection of DfFV1. Lane 5: Detection of DfPV1. Lane 6: Detection of DfFV1. Lane 7:
Detection of DfPV1. Lane 8: Detection of DfFV1. Lane 9: Water control RT-PCR for DfPV1. Lane 10:
Water control RT-PCR for DfFV1.
4. Discussion
The two strains, NW-FVA 1581 and NW-FVA 1706, originated from different forest stands of
Northern Germany. Although they were preliminary classified as Botryosphaeria stevensii/Diplodia
mutila sl. [5], we could unequivocally address them to D. fraxini in our study. In NW-FVA 1581, we
detected a single virus infection, in NW-FVA 1706 a double infection and both isolates showed a
different phenotype. The single infected NW-FVA 1581 produced numerous aerial hyphae which
turned brownish after several days of incubation, while the mycelium of NW-FVA 1706, which
harbors the double infection, stayed whitish with reduced aerial hyphae production. Similarly, Alves
et al. [65] reported several phenotypes from strains of D. fraxini. However, no data about the virome
of these isolates are available. Therefore, the morphotype-development cannot be addressed to the
genotype or virome of the two strains.
In NW-FVA 1706, we detected four dsRNAs and addressed the three smaller bands after
sequencing to monocistronic segments with sizes of 1,755 bp (segment 1), 1,588 bp (segment 2) and
1,233 bp (segment 3). Protein 1, which is encoded on segment 1, shows RdRp motifs and is closest
related to the respective protein of BdPV2. The putative capsid protein, which is encoded on segment
2, is closest related to the respective segment of PbPV1. The rules of the ICTV for the establishment
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of new species of partitiviruses claim 90 % or less sequence identity for the RdRp and 80 % or less for
the capsid protein. According to the rules, the three dsRNAs found in NW-FVA 1706 are segments of a
new tri-partite member of the genus Gammapartitivirus within the Partitiviridae family and therefore
we denominated it as Diplodia fraxini partitivirus 1 (DfPV1). The ascomycete host D. fraxini and the
lack of a 3’ poly(A) additionally correspond with the ICTV demarcation criteria for
gammmapartitiviruses. Members of this genus are usually described to be bipartite, however species
with a tripartite genome are listed as definite species of the Partitiviridae family and the genome of
the putative member Ustilaginoidea virens gammapartitivirus 1 (UvPV-1) is even divided into four
segments. According to the 9th report on subviral agents by the ICTV [66], satellite-like nucleic acids
are defined to be distinct from their helper virus and are either coding for no or for a non-structural
protein. As it was shown for an additional segment (dsRNA 4) for the Fusarium solani alternavirus
1 (FsAV1) [67], the third segment of DfPV1 also has extended NTRs and is therefore distinct from
segment 1 and 2. The criterium of encoding either no protein or a non-structural protein is fulfilled
since protein patterns of VLPs did not show any band with the expected size of about 30 kDa. Several
related proteins to P3 were found by BLASTp which are the corresponding proteins of other tripartite
gammapartitiviruses. There is no obvious pattern identifiable which can be linked to the RdRp
similarity and the pre- or absence of the third segment regarding to their taxonomical relationship.
Within the clade, BdPV2 and BdPV3 are more closely related to DfPV1 than AoV, even though no
third segment was described for BdPV3. Due to their close relationship regarding the RdRp and their
similar P3, BdPV2 and DfFV1 may have a common ancestor. Additionally, both fungal hosts are
members of the Botryosphaeria genus which supports this hypothesis. In contrast, the genus
Aspergillus belongs to the class Eurotiomyces and is not related to the genus Botryosphaeria which is
accommodated in the class Dothideomycetes. Therefore, the third segment of AoV may have been
acquired independently by horizontal gene transfer (HGT) as it was speculated by Wang et al. [68]
for a papain-like protease domain on dsRNA 2 of Sclerotinia sclerotiorum megabirnavirus 1 (SsMBV1),
for a Phytoreo_S7 domain in non-phytoreoviruses by Liu et al. [24] and from Lutz et al. [67] for
segment 4 of FsAV1.
The sequences of the two bands of about 9,000 bp which were detected in both isolates, NW-FVA
1581 and NW-FVA 1706, are putative strains of the same virus since they share 90.36 % identical nts.
In the 5´-NTR of NW-FVA 1581, a deletion of 79 nts was detected and verified by RT-PCR. No
Phytoreo_S7 domain was detected by CDD search for neither of them.
The putative RdRp showed highest similarity to the MpFV3 (40.58 % identical aa) and clusters
within a distinct clade. Due to sequence characteristics as genome size, the putative coding strategy
by a -1 frameshift and lengths of the 5´- and 3´-NTRs, we classify the two viruses found in the fungal
isolates, NW-FVA 1706 and NW-FVA 1581, as strains of a new member of the proposed family
“Fusagraviridae” which was suggested by Wang et al. [14] and denominate them as Diplodia fraxini
fusagravirus 1a (NW-FVA 1706) and as Diplodia fraxini fusagravirus 1b (NW-FVA 1581).
A capsidless [18,23] as well as encapsidated [15,17] nature was discussed for fusagraviruses. Our
results suggest that the viral RNA of DfFV1 is protected by P1 of DfFV1 together with ribosomal
proteins in a single infection. The protection of a viral genome by polysomes was also suggested for
the capsidless narnaviruses which encode a RdRp on their (+)ssRNA genome [69]. In an
ambigrammatic way, a function-less protein on the complementary (-)ssRNA is translated and is
involved to encapsulate and protect both strands [70]. Wilkinson et al. [70] discussed this process to
be performed by “frozen polysomes” which are unable to detach from the 3´-terminus, a mechanism
which has to be reversible and may be performed by virus encoded proteins. The structures obtained
by VLP and PS enrichments from DfFV1 infected NW-FVA 1581 may be based on those “frozen
polysomes”. The protein encoded from ORF1 with its unknown function may serve as the factor to
support the freezing process and the conversion of the fungal metabolism to prevent stalled
polysomes from degradation by no-go decay [71]. A large ORF on the (+)ssRNA and (-)ssRNA is
discussed to be necessary for an extended coverage of the complete viral genome [72]. Due to the
absence of a large ORF at the complementary strand of DfFV1, we only could give an explanation for
the viral (+)ssRNA encapsulation. Since we isolated dsRNA from PS enrichments, this mechanism
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seems unlikely to be involved. Other unknown factors must be responsible for the stabilization of
these polysomal structures in case the encapsulation of the genome by ribosomes is based on the
proposed mechanism. Whether the protein encoded on ORF 1 is involved in the switch of translation
to freezing and back or whether a completely different mechanism is involved has to be investigated.
The ultra-structures recovered from the double infection of NW-FVA 1706 differ from that of
NW-FVA 1581, although both cultures were grown under identical conditions. From mycelium of
NW-FVA 1706, no polysomal structures could be isolated, neither in a VLP nor in a PS enrichment.
When using the protocol for VLP purification, isometric particles were recovered containing dsRNA
from both viruses, DfPV1 and DfFV1. Since no ORF 1-related putative capsid protein was detected,
it is unlikely that the protein encoded on ORF 1 builds the viral shell for the fusagravirus genome as
it was shown for Cryphonectria carpinicola fusagravirus 1 (CcFGV1) by Das et al. [15]. However,
transcapsidation of the replicative from was described as a common feature of members of the
Yadokaviridae family with a given distantly related partner [73]. We hypothesize, that the dsRNA of
the novel fusagravirus DfFV1 was transcapsidated by partitiviral capsids since exclusively particles,
with the typical size for partitivirueses of around 25 nm were detected.
Another co-infection of a fusagravirus and a partitivirus was described from Rosellinia necatrix
[74], but no data of the ultra-structure and their RNA content are available yet. It will be interesting
whether the fusagravirus genome is transcapsidated by the partitiviral capsid in this infection and
whether the fusagraviruses are generally promiscuous and use for the protection of their genome
either their own capsid, a capsid from a co-infecting virus or ribosomes together with P1 of DfFV1.
Supplementary Materials: The following supporting information can be downloaded at the website of this
paper posted on Preprints.org.
Author Contributions: Conceptualization, Cornelia Heinze; Funding acquisition, Gitta Jutta Langer and
Cornelia Heinze; Investigation, Steffen Bien and Cornelia Heinze; Methodology, Steffen Bien; Project
administration, Gitta Jutta Langer and Cornelia Heinze; Supervision, Gitta Jutta Langer and Cornelia Heinze;
Writing—original draft, Steffen Bien, Gitta Jutta Langer and Cornelia Heinze; Writing—review & editing,
Cornelia Heinze.
Funding: This project is financed by the Agency for Renewable Resources (FNR) in the program
“Waldklimafonds“ [Forest and Climate Fund] (2219WK22A4 and 2219WK22G4) funded by the German Federal
Ministry of Food and Agriculture and the GermanFederalMinistry for Environment, Nature Conservation and
Nuclear Safety.
Institutional Review Board Statement: Not applicable
Informed Consent Statement: Not applicable
Data Availability Statement: The data presented in this study are openly available in this work and its
supplementary material
Conflicts of Interest: The authors declare no conflict of interest.
Acknowledgments: We thank Elke Woelken for electron microscopy and Birgit Hadeler for technical assistance.
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