Several mycoviruses have been reported and characterized in R. necatrix since 2004, through detection of double-stranded (ds) RNA that may be the genome of dsRNA viruses or a replicative form of single-stranded (ss) RNA viruses. The overall incidence of mycoviruses in R. necatrix was determined in a study performed in 2015. A large-scale screening of over 1000 Japanese isolates revealed an overall dsRNA incidence of approximately 20% (3). However, the incidence of dsRNA in R. necatrix was found to be only 14% in a 2018 study, where out of 79 Mediterranean isolates (62 from Spain, 16 from Israel, and 1 from Italy), 11 tested positive for dsRNA (22). Therefore, mycovirus prevalence in R. necatrix differs depending on the sampling location, being approximately 20% in Japan and approximately 14% in the Mediterranean region. Nevertheless, it should be noted that these infection rates are based on investigating potentially the same or duplicate isolates. In an older 2004 study (23), R. necatrix isolates were assigned into mycelial compatibility groups (MCGs) and the detection frequency of dsRNA in each group was determined to prevent duplication. Out of the total of 186 MCGs investigated, 45 were tested positive for dsRNA illustrating a 24.2% infection rate. The frequencies of dsRNA prevalence in MCGs from cultivated and uncultivated lands were significantly different, respectively 24.2% and 11.8%. Other similar studies reported an incidence of mycoviruses in R. necatrix between 14 to 21% (23–25). Most of these viruses are latent but some cause visible phenotypic alterations.

Viruses from the family Spinareoviridae are not only found in fungi but are reported to infect mammals, birds, reptiles, fish, arthropods including crustaceans, molluscs and plants. Spinareoviruses form non-enveloped icosahedral particles 60–85 nm in diameter, composed of 1–3 concentric protein layers. Their segmented linear dsRNA genome is between 23 and 29 kbp in size, with each of their 9–12 segments ranging from 0.5 to 4.8 kbp in length (26, 27). The presence of reoviruses in a fungal host, namely C. parasitica, was reported for the first time in 1994 (28). The reoviruses were named mycoreovirus 1 (MyRV1) and 2 (MyRV2), but their genome sequences were not fully determined until 2004 (29). Later, another reovirus designated as Rosellinia necatrix mycoreovirus 3 (RnMRV3) was detected in the R. necatrix hypovirulent strain W370, obtained from a root of Japanese pear in Yamato, Hiroshima, and placed in the genus Mycoreovirus in the family Spinareoviridae (24). The 12 genomic dsRNA segments (S1–S12) of RnMYRV3 range from 943 bp to 4,143 bp in length and have a 3-3-6 electrophoretic profile on 5% (^w/_v) polyacrylamide gel electrophoresis (PAGE). Figure 1A shows the genome organization of RnMyRV3. All RnMyRV3 segments were genetically unique, each having one long open reading frame (ORF), flanked by 5’ and 3’ untranslated regions (UTRs). The largest segment encodes an RNA-dependent RNA polymerase (RdRP) and the second largest a putative capsid protein (CP). Two more segments encode a guanylyl transferase and an NTP-binding protein, while the rest encode proteins of unknown function. Interestingly, one of the RnMyRV3 segments, namely segment 8 that is absent in C. parasitica MyRV1, was occasionally lost during subculturing of infected fungal strains (30). Segment-specific panhandle structures were found in all segments, which may act as a guiding site for the RdRP. RnMyRV3 particles are 80 nm in diameter, non-enveloped and double-layered, with an inner and an outer capsid layer (31–33). The internal capsid of RnMyRV3 is 50 nm in diameter and has surface projections (turrets or spikes).

The virus-infected R. necatrix hypovirulent strain W370 was cured by hyphal tipping and the resulting virus-free strain recovered its virulence, suggesting that RnMyRV3 attenuates fungal virulence and may be used to control white root rot (30). In another study, RnMyRV3 was also found to reduce mycelial growth rate and induce hypovirulence to the host fungus. When transmitted to R. necatrix strain RT37-1, RnMyRV3 infection resulted in hypovirulence on apple seedlings, with mortality ranging between 0 to 17% for seedlings inoculated with virus-infected strains and 50 to 100% for seedlings inoculated with virus-free strains.

RnMyRV3 led to upregulation of RNA silencing related genes and the abundance of vsRNAs derived from RnMyRV3 was found to be 13.0% (14). The counter-defense strategy of RnMyRV3 against RNA silencing was also investigated in a different study. As indicated by the RnMyRV3 suppressed RNA silencing of green fluorescent protein (GFP), reduced accumulation of GFP-small (s) RNAs and increased accumulation of GFP-dsRNA, observations suggesting that it interferes with dsRNA fragmentation and has RNA silencing suppressor activity (34).

In 2004, a bisegmented dsRNA mycovirus was isolated from R. necatrix strain W779, originally collected from a bait twig buried in a Japanese pear orchard in Ibaraki prefecture (23). The mycovirus was designated as Rosellinia necatrix megabirnavirus 1 (RnMBV1) and a distinct virus family, Megabirnaviridae, was proposed for to accommodate it based on its biological and molecular attributes. Megabirnaviruses form non-enveloped, icosahedral particles, 50-55 nm in diameter. Their bisegmented linear dsRNA genome is approximately 16.1 kbp in size, with two segments ranging from 7.2 to 8.9 kbp in length (35). The word ‘mega’ (‘large’ in Greek, mégas) indicates the relative large genome size, while the word ‘bi’ (‘two’ in Latin) refers to bisegmented nature of the genome, similar to the families Birnaviridae and Picobirnaviridae (6). However, it is noteworthy that these three families, Megabirnaviridae, Birnaviridae and Picobirnaviridae, show different genomic organization with no considerable sequence similarity (6, 36). RnMBV1 has two genomic dsRNA segments 8,931 bp and 7,180 bp in length, as shown in Figure 1B . Both segments are bicistronic, possessing two ORFs each. ORF1 and ORF2 of the largest segment encode for CP and RdRP, respectively. Both segments exhibit substantial levels of sequence similarity in the lengthy 5′ untranslated region of roughly 1.6 kbp, aside from the rigorously conserved 5′ (24 nt) and 3′ (8 nt) terminal sequences. RnMBV1 particles are rigid, spherical and approximately 50 nm in diameter (37).

RnMBV1 is transmitted horizontally via hyphal anastomosis and is homogenously distributed within R. necatrix colonies. RnMBV1 infection results in severe reduction of both R. necatrix growth and virulence against plant hosts, and therefore has strong potential as a biocontrol agent of white root rot (37, 38). The factor(s) responsible for R. necatrix attenuation are unknown but RnMBV1 mutants lacking dsRNA2 result in milder impact on growth and virulence demonstrating the important role of RNA2. An RnMBV1 strain with genome rearrangement was isolated and named RnMBV1-RS1. In addition to dsRNA1 with two open reading frames (ORFs) encoding the CP and the RdRP, RnMBV1-RS1 had a new segment dsRNAS1, which emerged as a result of an ORF1 deletion and an ORF2 partial tandem duplication, retaining a much shorter 5’ untranslated region (UTR). R. necatrix transfected with RnMBV-RS1 virions maintained its virulence on host plants, in contrast to RnMBV1-infected R. necatrix. This suggests that dsRNAS1 is transcribed and packaged, and that dsRNA2, while dispensable for virus replication, is required for virulence reduction in R. necatrix (39).

Transcriptome profiling was performed to investigate gene expression alterations in the Japanese R. necatrix strain W97, which exhibited hypovirulence following transfection with RnMBV1 from strain W779 (37). In total, 545 and 615 genes were found to be up- and down-regulated, respectively, in virus-infected as compared to virus-free R. necatrix. Differential gene expression analysis suggested that primary and secondary metabolism is deregulated in virus-infected R. necatrix. Genes encoding transcriptional regulators, plant cell wall-degrading enzymes, and factors involved in toxin production, such as cytochalasin E, were also differentially expressed (40).

Similar to RnMyRV1, RnMBV1 infection led to upregulation of RNA silencing genes. The abundance of vsRNAs derived from RnMBV1 was found to be 24.9%, higher as compared to RnMyRV1 (section 1.2) and other mycoviruses infecting R. necatrix, including a partitivirus (section 1.4), a quadrivirus (section 1.5), and a victorivirus (section 1.6). Notably, RnMBV1 and RnMyRV1 were the only two mycoviruses examined that upregulated RNA silencing genes in R. necatrix, leading to vsRNA abundance over 10% (14).

Members of the family Partitiviridae infect fungi and plants. Partitiviruses form non-enveloped, icosahedral particles, 25-43 nm in diameter. Their bisegmented, linear dsRNA genome with two segments ranging from 3.0 to 4.8 kb in length (41, 42). In addition to the families Megabirnaviridae and Reoviridae, members of the family Partitiviridae are also widespread in R. necatrix (6, 25, 43). Many R. necatrix strains tested were co-infected by multiple partitiviruses, but only a few of them have been molecularly and biologically characterized so far.

Rosellinia necatrix partitivirus 1 (RnPV1) was discovered infecting isolate W8, collected from grapevine in Okayama, Japan. Genome organization of RnPV1 is shown in Figure 1C , with the largest segment encoding RdRP and the smallest encoding CP. The UTRs of the two segments were similar with approximately 70% nt identity at the 5’ termini, but different at the 3’ termini, which contained adenosine-uracil rich elements (AREs) (44). Electron microscopy of purified RnPV1 particles indicated that they were approximately 25 nm in diameter. RnPV1 infection did not lead to upregulation of RNA silencing genes and the abundance of vsRNAs derived from RnPV1 was found to be 6.8% (41).

In a separate study, RnPV7 was discovered infecting isolate 7-11/W1142 following a controlled environment experiment (section 1.7) and was assigned to genus Alphapartitivirus. RnPV7 infection had no effect on host growth or virulence.

According to one report (25), a total of 20 partitiviruses from 16 R. necatrix strains, belonging to 5 previously reported species and 15 new species, for which the names Rosellinia necatrix partitivirus 11 to 25 were proposed and which were assigned to genera Alphapartitivirus and Betapartitivirus. For 13 of these novel partitiviruses, transfection experiments were performed to introduce purified virions into reference strains of the natural host R. necatrix and a heterologous host C. parasitica. The effects of partitivirus infection were assessed in R. necatrix W97, together with C. parasitica wild-type EP155 and its Δdcl2 KO mutant, an RNA silencing deficient strain. Initially, the quintuply infected (RnPV1, RnPV14-RnPV17) W97 transfectant displayed considerably reduced growth; its mycelium was fluffy and white in color, while the mycelium of the virus-free strain was off-white and more mat-like in appearance. Subsequent hyphal fusion with virus-free W97 led to the manifestation of a milder growth defect. Even after repeated coculturing, EP155 stably maintained single infection with RnPV11 from W98 or RnPV20 from W1134, and double infection with RnPV14 and RnPV16 from W744. All infected EP155 strains showed reduction pigmentation, growth rate, and aerial hyphae. Δdcl2, when singly infected by RnPV11 or RnPV20 and doubly infected by RnPV14 and RnPV16, also exhibited reduced growth with irregular margins. Conversely, EP155 infected by RnPV18 and RnPV19 was either asymptomatic or showed mild reduction in growth rate (25).

In a recent study of isolates from South Korea, R. necatrix KACC 40168 was reported to be infected with two viruses, one of which was a new member of the genus Alphapartitivirus in the family Partitiviridae and hereafter designated as RnPV26 (44).

A tetrasegmented dsRNA virus was reported in R. necatrix strain W1075 from a Japanese pear orchard in the Saga prefecture in 2012 (45). Similar to RnMBV1, a new family Quadriviridae was established to accommodate Rosellinia necatrix quadrivirus 1 (RnQV1). Quadriviruses form non-enveloped, icosahedral particles, 45 nm in diameter. Their tetrasegmented linear dsRNA genome is between 16.8 and 17.1 kbp in size, with four segments ranging from 3.5 to 5.0 kbp in length (46). An agarose gel electrophoretic profile of the four genomic dsRNAs of RnQV1 is very much similar to that of a member of the family Chrysoviridae, Helminthosporium victoriae virus 145S (HvV145S), although their size ranges are different: 3.9–4.9 kbp for RnQV1 and 2.8–3.6 kbp for HvV145S (6). All 4 dsRNA segments, i.e., dsRNAs 1 to 4, possess a single large ORF flanked by 5’ and 3’ UTRs. RnQV1 dsRNAs 2 and 4 encode for CPs while dsRNA3 encodes for RdRP as shown in Figure 1D . The infected mycelia were found to contain rigid spherical particles, approximately 45 nm in diameter (45). RnQV1 infection did not lead to upregulation of RNA silencing genes and the abundance of vsRNAs derived from RnQV1 was found to be 1.2% (41).

Later in the same year, the biological and molecular characterization of a second quadrivirus strain termed Rosellinia necatrix quadrivirus 1 strain W1118 (RnQV1-W1118) was reported. Commonalities with the first quadrivirus (RnQV1-W1075) included its spherical particle morphology, quadripartite genome structure, 72–82% sequence similarity between homologous proteins, terminal sequence heterogeneity, and ability to cause a latent infection. Conversely, distinguishing features included different conserved terminal sequences and the degree of susceptibility to proteolytic degradation of the two major capsid proteins, which is thought to occur during virion purification. In particular, the numbers of the strictly conserved sequences are different between the two viruses: 9 vs 7 nt and 12 vs 14 nt for the 5’- and 3’-terminal sequences of RnQV1-W1118 and RnQV1-W1075, respectively. In RnQV1-W1118, the 5’-terminal sequences are more conserved than the 3’-terminal sequences, while the opposite is observed for Rn W1075. Several highly conserved sequence stretches are detected at both ends in addition to the strictly shared sequences (47).

Members of the family Pseudototiviridae infect fungi and protozoa. Pseudototiviruses form non-enveloped, icosahedral particles, 40 nm in diameter, and have monosegmented, linear dsRNA genomes, ranging from 4.6 to 7.0 kbp in length (2023 Release, MSL #39). R. necatrix strains W1028 to W1030 from the experimental orchard of apple trees (Nagano Fruit Tree Experiment Station) at Suzaka, Nagano prefecture, Japan, were confirmed to be coinfected by members of the genus Victorivirus, family Pseudototiviridae, together with partitiviruses (48, 49). Figure 1E shows the genome organization of the monosegmented Rosellinia necatrix victorivirus 1 (RnVV1) with two large overlapping ORFs, encoding CP and RdRP. Similarities and differences to other reported victoriviruses include respectively the presence of a UAAUG pentamer sequence element, facilitating translation termination/reinitiation at the junction between two ORFs and sequence divergence with 41% RdRP amino acid sequence identity to its closest relative, Botryotinia fuckeliana totivirus 1. The RnVV1 particles were spherical, approximately 40 nm in diameter.

Molecular and biological characterization of RnVV1-W1029 has been reported (49). The effects of RnVV1 on the phenotype of the natural host R. necatrix were negligible. The colony morphology of four strains with the same host background was compared: R. necatrix W563 (virus-free field isolate believed to be isogenic to W1029), W1029 (virus-infected), W1029-T20 and W1029-T31 (partially cured via hyphal tipping; RnPV3-free). No effects on appearance or growth rate were observed regardless of whether the host fungus was uninfected (in case of W563) or infected with RnVV1 alone (T20 and T31) or in combination with RnPV3 (W1029). In another experiment, RnVV1 particles were purified from W1029 and used to transfect strain W97, which belongs to a different vegetative compatibility group. Three transfectants (Tf1, Tf2 and Tf3) were compared to parental virus-free W97 strain, concluding that RnVV1 infected R. necatrix asymptomatically under lab conditions.

Further experiments were performed to expand the RnVV1 (and RnPV3) host range and study its phenotype in C. parasitica. Viruses purified from W1029 were used to transfect C. parasitica Δdcl-2 mutant. Three types of transfectants were obtained: A26 doubly infected with RnVV1 and RnPV3; A12, singly infected with RnVV1; and A6, singly infected with RnPV3. In C. parasitica, phenotypic changes were observed in the Δdcl-2 strain singly infected with RnVV1 which showed altered phenotype and reduced growth. Interestingly, comparison of the RNA silencing-competent (wild-type EP155) and -defective (Δdcl-2) strains of C. parasitica infected with RnVV1 showed that RNA silencing acted against the virus to repress its replication, which was restored by coinfection with Cryphonectria parasitica hypovirus 1 (CHV1) or transgenic expression of an RNA silencing suppressor, CHV1 p29. This was the first study reporting host range expansion and RNA silencing of a member of the family Pseudototiviridae (49). Interestingly, RnVV1 infection did not lead to upregulation of RNA silencing genes and the abundance of vsRNAs derived from RnQV1 was found to be 0.3% in R. necatrix (41).

Members of the family Endornaviridae infect plants, fungi and oomycetes. Endornaviruses are capsidless and have monosegmented, linear, positive-sense (+) ssRNA genomes, ranging from 9.7 to17.6 kb in length. This family has two genera, Alphaendornavirus and Betaendornavirus, which accommodate viruses on the basis of genome size, respectively greater than 11.9 kb and less than 10.7 kb, and fungal host (50). Three R. necatrix isolates, designated as 6-31, 6-33 and 6-35, were reported to be infected with an endornavirus for the first time. Rosellinia necatrix endornavirus 1 (RnEV1) was placed in genus Betaendornavirus because of its relatively short genome of 9,369 nt. A large ORF encoded a protein with a methyltransferase domain, a cysteine rich region, an RNA helicase domain and an RdRP domain, as shown in Figure 2A . RnEV1 infection had no effect on host growth or virulence (50, 51).

Interestingly, natural RnEV1 infection occurred when the virus-free R. necatrix strain W97 was incubated in a soil sample from an apple tree in Suzaka under glasshouse conditions, giving rise isolates 6-31, 6-33 and 6-35. A similar phenomenon was observed with the virus-free R. necatrix strain W370T1, which was found to be infected with RnPV7 (section 1.4) and gave rise to isolate 7-11/W1142, following incubation in a soil sample from a forest in Morioka under glasshouse conditions. This observation raises an important question: how did these mycoviruses get transmitted to R. necatrix? No R. necatrix genetically different from the original strains W97 and W370T1 were isolated from soil after incubation, suggesting that these mycoviruses could not have been transmitted from infected R. necatrix already present in the soil samples. Therefore, it was hypothesized that the spread of these mycoviruses may have been facilitated by another fungus or through interactions with soil organisms such as nematodes or micro-arthropods. No horizontal transmission of RnEV1 and RnPV7 between incompatible R. necatrix strains was observed on agar plates during in vitro experiments (50, 51). However, the experiment was performed in soil samples that may have contained plant exudates such as proline. Proline was recently shown to weaken incompatibility between fungi and improve mycovirus transmission (52). This may also help explain mycovirus infection of R. necatrix, potentially through cross-species transmission of mycoviruses.

Members of the family Fusariviridae infect fungi and oomycetes. Fusariviruses are capsidless and have monosegmented, linear +ssRNA genomes, which range from 5.9 to 10.7 kb in length and may be monocistronic, bicistronic, tricistronic or quadricistronic (53). R. necatrix strain NW10 from an apple tree in Nagano, Japan, was found to be infected Rosellinia necatrix fusarivirus 1 (RnFV1), related to Fusarium graminearum virus 1 (FgV1) and hence placed in the newly established family Fusariviridae. The 6,286 nt genome of RnFV1 has two ORFs, as shown in Figure 2B . The largest encoding a protein with the RdRP and RNA helicase domains typical of fusariviruses. Both of these domains showed moderate level of sequence identity to each other. The smallest ORF encoded a conserved protein with unknown function, which contained a motif from a structural maintenance of chromosomes (SMC) protein. RnFV1 was successfully transferred between incompatible fungal strains using a zinc ion-based technique. RnFV1 caused a latent infection, and therefore has no potential to be used as a biocontrol agent (54).

The family Yadokariviridae, which includes the genera Alphayadokarivirus and Betayadokarivirus, accommodates capsidless, non-segmented, linear +ssRNA viruses, 3.3 to 6.3 kb in length that hijack capsids from phylogenetically distant dsRNA viruses, resulting in spherical and non-enveloped particles 33–50 nm in diameter. Yadokariviruses likely replicate within the hijacked heterocapsids using their own RdRP, imitating the replication process of dsRNA viruses. Through their interaction with the capsid-donating dsRNA viruses, yadokariviruses can influence their fungal hosts in both positive and negative ways (55).

Co-infection with the 6.3 kb long alphayadokarivirus Yado-kari virus 1 shown in Figure 2C and its unclassified capsid donor, Yado-nushi virus 1, led to a growth defect in strain Rn-1032 from Japan but also promoted the accumulation of the donor virus (48, 56). Conversely, the betayadokarivirus Yado-kari virus 4a from strain Rn-454, reduced the levels of its capsid donor dsRNA virus and alleviated the growth defect caused by the associated dsRNA virus. Another betayadokarivirus, Yado-kari virus 3 from strain Rn-454, has no impact on either its capsid donor or the host fungus (57).

These findings contribute to our understanding of viral ecology and virus-host interactions in fungal systems. The effects shown by yadokariviruses on their capsid donors and host fungi illustrate how mycoviruses can exhibit a wide range of behaviors, from promoting viral replication to mitigating harmful effects on the host fungus. This variability could be driven by genetic differences between viruses, their ability to take over the host machinery, or the ecological context in which the viruses exist. Furthermore, these results raise interesting questions about the potential for viral interactions to influence fungal disease dynamics, especially in phytopathogenic fungi like R. necatrix.

A study was performed to screen R. necatrix strains for viral infections and it was reported that different strains of R. necatrix were infected with at least six different viral families. The viral sequences represent families such as Megabirna-, Partiti-, Fusari-, Yado-kariviridae, described in previous sections, but also Hypo-, Fusagra-, and Megatotiviridae. Hypoviruses are capsidless viruses with monosegmented, linear, +ssRNA genomes ranging from 7.3 to 18.3 kb in length and may be monocistronic or bicistronic (58). Conversely, fusagraviruses form non-enveloped, icosahedral capsids approximately 30 nm in diameter, have monosegmented, linear, dsRNA genomes ranging from 8 to 11 kb in length and are bicistronic (59). Finally, megatotiviruses form non-enveloped, spherical particles 40-50 nm in diameter and have monosegmented, linear, dsRNA genomes ranging from 10-12.5 kb in length (Taxon Details | ICTV) (22). The hypovirulent effect of mycovirus infection in the Mediterranean R. necatrix strain Rn459, causing root rot in avocado, was reported (22, 60). Rn459 was found to be infected with RnPV10 (section 1.4) and at least three other viruses: Rosellinia necatrix hypovirus 2 (RnHV2), Rosellinia necatrix fusagravirus 1 (RnFGV1), and Rosellinia necatrix virga-like virus (RnVLV). The latter resembles members of the family Virgaviridae, plant viruses with non-enveloped rod-shaped capsids 17-24 nm in diameter and up to 300 nm in length, and monosegmented, linear, +ssRNA genomes ranging from 6.3 to 13.0 kb in length (61).

Following spontaneous loss of RnFGV1 from the original strain, hyphal tipping was used to cure the cultures of virus infection in order to examine the effects on growth in vitro and virulence on avocado plants. The fungal strain obtained, Rn459_PV10F/VLVF, was confirmed to be cured of RnPV10 and RnVLV infection but still retained RnHV2. Rn459_PV10F/VLVF manifested a phenotype different from the original Rn459 strain, growing faster in vitro and being more virulent on avocado plants. These collective results suggest that RnPV10 and RnVLV, alone or in combination, contribute to confer hypovirulence to R. necatrix.

A mixed dsRNA mycovirus infection in a Korean R. necatrix isolate, including a fusagravirus and a partitivirus was reported (44). The two mycoviruses were detected by next-generation sequencing analysis of purified dsRNAs samples. The first dsRNA virus had a complete genome sequence of 8,868 bp in size and contained two large ORFs 1 and 2, overlapped by 22 bp containing a canonical (−1) slippery heptanucelotide sequence of UUUAAAC. Phylogenetic analysis showed that it clustered with RnFGV3 and other fusagraviruses and showed similarity with the RnFGV3 hypothetical protein and RdRP. The virus was named Rosellinia necatrix fusagravirus 4 due to its genomic organization, sequence similarity, and phylogenetic analysis, which indicate that it belongs to a novel species of the family Fusagraviridae.