Hypovirulence-associated Mycoviruses Research Articles

A novel hypovirulence-associated Hadaka virus 1 (HadV1-LA6) in Fusarium oxysporum f. sp. cubense.

Fusarium oxysporum f. sp. cubense (Foc) poses a significant threat to banana crops as a lethal fungal pathogen. The global spread of Foc underscores the formidable challenges associated with traditional management methods in combating this pathogen. This study delves into the hypovirulence-associated mycovirus in Foc. From Foc strain LA6, we isolated and characterized a novel member of the Hadakaviridae family, named Hadaka virus 1 strain LA6 (HadV1-LA6). HadV1-LA6 comprises 10 genomic RNA segments, with RNA1 to RNA7 sharing 80.9%-95.0% amino acid sequence identity with known HadV1-7n, while RNA8 to RNA10 display significantly lower identity. HadV1-LA6 demonstrates horizontal transmission capabilities in an all-or-none fashion between different Foc strains via coculturing. Phenotypic comparisons highlight that HadV1-LA6 significantly reduces the growth rates of its host fungus under cell wall stress and oxidative stress conditions. Importantly, HadV1-LA6 attenuates Foc's virulence in detached leaves and banana plants. This study represents the first introduction of a novel hypovirulence-associated Hadaka virus 1 in Foc.IMPORTANCEFusarium wilt of banana (FWB) is a severe fungal disease caused by soil-borne Fusarium oxysporum f. sp. cubense (Foc). Among various strategies, biocontrol emerges as a safe, ecologically friendly, and cost-effective approach to managing FWB. In this study, we focus on exploring the potential of a novel hypovirulent member of hadakavirid, HadV1-LA6. Previous reports suggest that HadV1 shows no apparent effect on the host. However, through phenotypic assessments, we demonstrate that HadV1-LA6 significantly impedes the growth rates of its host fungus under stress conditions. More importantly, HadV1-LA6 exhibits a remarkable capacity to attenuate Foc's virulence in detached leaves and banana plants. Furthermore, HadV1-LA6 could be horizontally transmitted between different Foc strains, presenting a promising resource for revealing the molecular mechanism of the interaction between Hadaka virus 1 and its host.

Plants interfere with non-self recognition of a phytopathogenic fungus via proline accumulation to facilitate mycovirus transmission

Non-self recognition is a fundamental aspect of life, serving as a crucial mechanism for mitigating proliferation of molecular parasites within fungal populations. However, studies investigating the potential interference of plants with fungal non-self recognition mechanisms are limited. Here, we demonstrate a pronounced increase in the efficiency of horizontal mycovirus transmission between vegetatively incompatible Sclerotinia sclerotiorum strains in planta as compared to in vitro. This increased efficiency is associated with elevated proline concentration in plants following S. sclerotiorum infection. This surge in proline levels attenuates the non-self recognition reaction among fungi by inhibition of cell death, thereby facilitating mycovirus transmission. Furthermore, our field experiments reveal that the combined deployment of hypovirulent S. sclerotiorum strains harboring hypovirulence-associated mycoviruses (HAVs) together with exogenous proline confers substantial protection to oilseed rape plants against virulent S. sclerotiorum. This unprecedented discovery illuminates a novel pathway by which plants can counteract S. sclerotiorum infection, leveraging the weakening of fungal non-self recognition and promotion of HAVs spread. These promising insights provide an avenue to explore for developing innovative biological control strategies aimed at mitigating fungal diseases in plants by enhancing the efficacy of horizontal HAV transmission.

Characterization of two novel fusariviruses co-infecting a single isolate of phytopathogenic fungus Botrytis cinerea.

A wide diversity of mycoviruses has been reported from Botrytis species, some with the potential to suppress the pathogenic abilities of this fungus. Considering their importance, this study was devised to find potential hypovirulence-associated mycoviruses found in Botrytis cinerea strains isolated from Pakistani strawberry fields. Here we report the complete genome characterization of two fusariviruses co-infecting a single isolate of phytopathogenic fungus B. cinerea (Kst14a). The viral genomes were sequenced by deep sequencing using total RNA fractions of the Kst14a isolate. The identified viruses were tentatively namedBotrytis cinerea fusarivirus 9 (BcFV9) and Botrytis cinerea fusarivirus 3a (BcFV3a). Both viruses had a single-segmented (ssRNA) genome having a size of 6424 and 8370 nucleotides encoding two discontinuous open reading frames (ORFs). ORF-1 of both mycoviruses encodes for a polyprotein having a conserved domain of RNA-dependent RNA polymerase (RdRP) and a helicase domain (Hel) which function in RNA replication, while ORF2 encodes a hypothetical protein with an unknown function, respectively. Phylogenetic analysis indicated that BcFV9 made a clade with the genus Alphafusarivirus and BcFV3a fall in the genus Betafusarivirus in the family Fusariviridae. To our knowledge, this is the first report of two fusariviruses identified in isolates of B. cinerea from Pakistan. Both mycoviruses successfully transfected to a compatible strain of B. cinerea (Mst11). A comparison of virus-free (VF) and virus-infected (VI) isogenic lines showed the presence of these viruses was causing hypovirulence in infected strains. Virus-infected strains also had a small lesion size while testing the pathogenicity via apple assay.

Mycovirus: Biocontrol agent against S. sclerotiorum of Rapeseed

Plant disease control is one of the essential aspects of food production Use of pesticide causes severe damage to our environment and have terrible effects on human health. It has been observed that the abundance use of pesticides also disturbs natural populations of various beneficial insects. Now environmental pollution has become a global problem, and due to the negative effect of chemical control, we must focus on new methods to control plant diseases, and the best of them is to use biological control method; this method includes the use of various beneficial microorganism having the potential to control plant diseases. Viruses that infect fungi are called as mycoviruses, and some of them could confer hypovirulence and thus have the capacity to control fungal diseases. Sclerotinia sclerotiorum, a widespread ascomycete fungus, can attack more than 450 plant species and subspecies. It causes stem rot on rapeseed and leads to substantial economic loss each year. Previously, several hypovirulence-associated mycoviruses have been identified from S. sclerotiorum, suggesting that this fungus could host various mycoviruses. Keywords: Biological control, Hypovirulence, Mycovirus, Sclerotinia sclerotiorum, RNA-seq, virus transmission

Hypovirulence-associated mycovirus epidemics cause pathogenicity degeneration of Beauveria bassiana in the field

BackgroundThe entomogenous fungus Beauveria bassiana is used as a biological insecticide worldwide, wild B. bassiana strains with high pathogenicity in the field play an important role in controlling insect pests via not only screening of highly virulent strains but also natural infection, but the pathogenicity degeneration of wild strains severely affected aforementioned effects. Previous studies have showed that multiple factors contributed to this phenomenon. It has been extensively proved that the mycovirus infection caused hypovirulence of phytopathogenic fungi, which has been used for plant disease biocontrol. However, it remains unknown whether the mycovirus epidemics is a key factor causing hypovirulence of B. bassiana naturally in the field.MethodsWild strains of B. bassiana were collected from different geographic locations in Jilin Province, China, to clarify the epidemic and diversity of the mycoviruses. A mycovirus Beauveria bassiana chrysovirus 2 (BbCV2) we have previously identified was employed to clarify its impact on the pathogenicity of host fungi B. bassiana against the larvae of insect pest Ostrinia furnacalis. The serological analysis was conducted by preparing polyclonal antibody against a BbCV2 coat protein, to determine whether it can dissociate outside the host fungal cells and subsequently infect new hosts. Transcriptome analysis was used to reveal the interactions between viruses and hosts.ResultsWe surprisingly found that the mycovirus BbCV2 was prevalent in the field as a core virus in wild B. bassiana strains, without obvious genetic differentiation, this virus possessed efficient and stable horizontal and vertical transmission capabilities. The serological results showed that the virus could not only replicate within but also dissociate outside the host cells, and the purified virions could infect B. bassiana by co-incubation. The virus infection causes B. bassiana hypovirulence. Transcriptome analysis revealed decreased expression of genes related to insect epidermis penetration, hypha growth and toxin metabolism in B. bassiana caused by mycovirus infection.ConclusionBeauveria bassiana infected by hypovirulence-associated mycovirus can spread the virus to new host strains after infecting insects, and cause the virus epidemics in the field. The findings confirmed that mycovirus infection may be an important factor affecting the pathogenicity degradation of B. bassiana in the field.

Mycoviruses: Antagonistic Potential, Fungal Pathogenesis, and Their Interaction with Rhizoctonia solani.

Mycoviruses, or fungal viruses, are prevalent in all significant fungal kingdoms and genera. These low-virulence viruses can be used as biocontrol agents to manage fungal diseases. These viruses are divided into 19 officially recognized families and 1 unclassified genus. Mycoviruses alter sexual reproduction, pigmentation, and development. Spores and fungal hypha spread mycoviruses. Isometric particles mostly encapsulate dsRNA mycoviruses. The widespread plant-pathogenic fungus Rhizoctonia solani, which has caused a rice sheath blight, has hosted many viruses with different morphologies. It causes significant crop diseases that adversely affect agriculture and the economy. Rice sheath blight threatens the 40% of the global population that relies on rice for food and nutrition. This article reviews mycovirology research on Rhizoctonia solani to demonstrate scientific advances. Mycoviruses control rice sheath blight. Hypovirulence-associated mycoviruses are needed to control R. solani since no cultivars are resistant. Mycoviruses are usually cryptic, but they can benefit the host fungus. Phytopathologists may use hypovirulent viruses as biological control agents. New tools are being developed based on host genome studies to overcome the intellectual challenge of comprehending the interactions between viruses and fungi and the practical challenge of influencing these interactions to develop biocontrol agents against significant plant pathogens.

Hypovirulence of Colletotrichum gloesporioides Associated with dsRNA Mycovirus Isolated from a Mango Orchard in Thailand.

The pathogenic fungus Colletotrichum gloeosporioides causes anthracnose disease, which is an important fungal disease affecting the production of numerous crops around the world. The presence of mycoviruses, however, may have an impact on the pathogenicity of the fungal host. Here, we describe a double-stranded RNA (dsRNA) mycovirus, which was isolated from a field strain of C. gloeosporioides, Ssa-44.1. The 2939 bp genome sequence comprises two open reading frames (ORFs) that encode for a putative protein and RNA-dependent RNA polymerase (RdRp). The Ssa-44.1 mycovirus is a member of the unclassified mycovirus family named Colletotrichum gloeosporioides RNA virus 1 strain Ssa-44.1 (CgRV1-Ssa-44.1), which has a phylogenetic similarity to Colletotrichum gleosporioides RNA virus 1 (CgRV1), which was isolated from citrus leaves in China. In C. gloeosporioides, CgRV1-Ssa-44.1 was shown to be linked to hypovirulence. CgRV1-Ssa-44.1 has a low spore transfer efficiency but can successfully spread horizontally to isogenic virus-free isolates. Furthermore, CgRV1-Ssa-44.1 had a strong biological control impact on C. gloeosporioides on mango plants. This study is the first to describe a hypovirulence-associated mycovirus infecting C. gloeosporioides, which has the potential to assist with anthracnose disease biological management.

Hypovirulence-associated mycovirus in Fusarium sp. isolated from apple orchards of Himachal Pradesh, India

Infection of fungi with mycoviruses can lead to hypovirulence and control the pathogenicity of fungus causing diseases in crops. In the present study, ten Fusarium isolates with reduced mycelia growth were obtained from mulberry twigs buried in the rhizosphere of apple trees in Himachal Pradesh. All isolates except isolate 2 of Fusarium led to complete fruit rotting after 12 days of spore inoculation. After curing of Fusarium isolate 2, culture exhibited normal morphology, pigmentation, and growth rate. Fusarium strain cured for mycovirus was found to be more pathogenic as compared to uncured strain. The growth and pathogenicity phenotype were not fully restored in comparison to wild type. This study thus documents the presence of hypovirulence associated mycoviruses in Fusarium sp. isolated from apple rhizosphere of Himachal Pradesh. Recently, mycovirus induced hypovirulence has gained a lot of attention worldwide.

A Novel Ourmia-Like Mycovirus Confers Hypovirulence-Associated Traits on Fusarium oxysporum.

Fusarium wilt caused by Fusarium oxysporum f. sp. momordicae (FoM) is an important fungal disease that affects the production of bitter gourd. Hypovirulence-associated mycoviruses have great potential and application prospects for controlling the fungal disease. In this study, a novel ourmia-like virus, named Fusarium oxysporum ourmia-like virus 1 (FoOuLV1), was isolated from FoM strain HuN8. The viral genomic RNA is 2,712 nucleotides (nt) in length and contains an open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) using either standard or mitochondrial codes. In strain HuN8, there was also a FoOuLV1-associated RNA segment with 1,173 nt in length with no sequence homology. Phylogenetic analysis showed that FoOuLV1 is a member of the genus Magoulivirus of the family Botourmiaviridae. FoOuLV1 was found to be associated with hypovirulence in FoM. Moreover, FoOuLV1 and its hypovirulence trait can be transmitted horizontally to other FoM strains and also to other formae speciale strains of F. oxysporum. In addition, FoOuLV1 showed significant biological control effect against the bitter gourd Fusarium wilt. To our knowledge, this study reveals the first description of a hypovirulence-associated ourmia-like mycovirus, which has the potential to the biological control of Fusarium wilt.

A 2-kb Mycovirus Converts a Pathogenic Fungus into a Beneficial Endophyte for Brassica Protection and Yield Enhancement

Mycoviruses are viruses that infect fungi, and hypovirulence-associated mycoviruses have the potential to control fungal diseases. However, it is unclear how mycovirus-mediated hypovirulent strains live and survive in the field, and no mycovirus has been applied for field crop protection. In this study, we found that a previously identified small DNA mycovirus (SsHADV-1) can convert its host, Sclerotinia sclerotiorum, from a typical necrotrophic pathogen to a beneficial endophytic fungus. SsHADV-1 downregulates the expression of key pathogenicity factor genes in S. sclerotiorum during infection. When growing in rapeseed, the SsHADV-1-infected strain DT-8 significantly regulates the expression of rapeseed genes involved in defense, hormone signaling, and circadian rhythm pathways. As a result, plant growth is promoted and disease resistance is enhanced. Field experiments showed that spraying DT-8 at the early flowering stage can reduce the disease severity of rapeseed stem rot by 67.6% and improve yield by 14.9%. Moreover, we discovered that SsHADV-1 could also infect other S. sclerotiorum strains on DT-8-inoculated plants and that DT-8 could be recovered from dead plants. These findings suggest that the mycoviruses may have the ability to shape the origin of endophytism. Our discoveries suggest that mycoviruses may influence the origin of endophytism and may also offer a novel strategy for disease control in which mycovirus-infected strains are used to improve crop health and release mycoviruses into the field.

Sclerotinia minor Endornavirus 1, a Novel Pathogenicity Debilitation-Associated Mycovirus with a Wide Spectrum of Horizontal Transmissibility.

Sclerotinia minor is a phytopathogenic fungus causing sclerotinia blight on many economically important crops. Here, we have characterized the biological and molecular properties of a novel endornavirus, Sclerotinia minor endornavirus 1 (SmEV1), isolated from the hypovirulent strain LC22 of S. minor. The genome of SmEV1 is 12,626 bp long with a single, large open reading frame (ORF), coding for a putative protein of 4020 amino acids. The putative protein contains cysteine-rich region (CRR), viral methyltransferase (MTR), putative DEXDc, viral helicase (Hel), and RNA-dependent RNA polymerase (RdRp) domains. The putative protein and the conserved domains are phylogenetically related to endornaviruses. SmEV1 does not contain a site-specific nick characteristic of most previously described endornaviruses. Hypovirulence and associated traits of strain LC22 and SmEV1 were readily cotransmitted horizontally via hyphal contact to isolates of different vegetative compatibility groups of S. minor. Additionally, SmEV1 in strain LC22 was found capable of being transmitted vertically through sclerotia. Furthermore, mycelium fragments of hypovirulent strain LC22 have a protective activity against attack by S. minor. Taken together, we concluded that SmEV1 is a novel hypovirulence-associated mycovirus with a wide spectrum of transmissibility, and has potential for biological control (virocontrol) of diseases caused by S. minor.

Horizontal and vertical transmission of the hypovirulence-associated mycovirus Fusarium oxysporum f. sp. dianthi virus 1

Mycovirus Fusarium oxysporum f. sp. dianthi virus 1 (FodV1) has been recently described infecting isolate Fod 116 of Fusarium oxysporum f. sp. dianthi (Fod). FodV1 is a new member of the family Chrysoviridae, and its genome consists of four dsRNA segments ranging from 2.6 to 3.5 kb. Presence of high levels of FodV1 in its original fungal host correlated with alteration of some phenotypic traits, including virulence. In this work we have analysed if FodV1 can be transferred horizontally to another Fod isolate by hyphal anastomosis, and vertically through conidiation, and if the mycovirus accumulates in the recipient isolate at similar levels that those found in the donor one. Moreover, we have investigated if the new virus-infected isolate reproduces the same phenotypic alterations that the original virus-infected does. Results indicated that FodV1 transfers horizontally between compatible isolates by hyphal anastomosis, reaching a high level of accumulation in the recipient isolate, and vertically during sporogenesis. Presence of FodV1 in the new fungal host reduced the growth rate and altered the morphology of the colony on solid medium, and diminished the conidiation rate in liquid medium. More interestingly, FodV1 induced hypovirulence in its new fungal host. Results contained in this work constitute the basis for further research on the application of mycovirus FodV1 to the control of Fusarium wilt diseases.

Two Novel Hypovirulence-Associated Mycoviruses in the Phytopathogenic Fungus Botrytis cinerea: Molecular Characterization and Suppression of Infection Cushion Formation.

Botrytis cinerea is a necrotrophic fungus causing disease on many important agricultural crops. Two novel mycoviruses, namely Botrytis cinerea hypovirus 1 (BcHV1) and Botrytis cinerea fusarivirus 1 (BcFV1), were fully sequenced. The genome of BcHV1 is 10,214 nt long excluding a poly-A tail and possesses one large open reading frame (ORF) encoding a polyprotein possessing several conserved domains including RNA-dependent RNA polymerase (RdRp), showing homology to hypovirus-encoded polyproteins. Phylogenetic analysis indicated that BcHV1 may belong to the proposed genus Betahypovirus in the viral family Hypoviridae. The genome of BcFV1 is 8411 nt in length excluding the poly A tail and theoretically processes two major ORFs, namely ORF1 and ORF2. The larger ORF1 encoded polypeptide contains protein domains of an RdRp and a viral helicase, whereas the function of smaller ORF2 remains unknown. The BcFV1 was phylogenetically clustered with other fusariviruses forming an independent branch, indicating BcFV1 was a member in Fusariviridae. Both BcHV1 and BcFV1 were capable of being transmitted horizontally through hyphal anastomosis. Infection by BcHV1 alone caused attenuated virulence without affecting mycelial growth, significantly inhibited infection cushion (IC) formation, and altered expression of several IC-formation-associated genes. However, wound inoculation could fully rescue the virulence phenotype of the BcHV1 infected isolate. These results indicate the BcHV1-associated hypovirulence is caused by the viral influence on IC-formation-associated pathways.

Virus-mediated suppression of host non-self recognition facilitates horizontal transmission of heterologous viruses.

Non-self recognition is a common phenomenon among organisms; it often leads to innate immunity to prevent the invasion of parasites and maintain the genetic polymorphism of organisms. Fungal vegetative incompatibility is a type of non-self recognition which often induces programmed cell death (PCD) and restricts the spread of molecular parasites. It is not clearly known whether virus infection could attenuate non-self recognition among host individuals to facilitate its spread. Here, we report that a hypovirulence-associated mycoreovirus, named Sclerotinia sclerotiorum mycoreovirus 4 (SsMYRV4), could suppress host non-self recognition and facilitate horizontal transmission of heterologous viruses. We found that cell death in intermingled colony regions between SsMYRV4-infected Sclerotinia sclerotiorum strain and other tested vegetatively incompatible strains was markedly reduced and inhibition barrage lines were not clearly observed. Vegetative incompatibility, which involves Heterotrimeric guanine nucleotide-binding proteins (G proteins) signaling pathway, is controlled by specific loci termed het (heterokaryon incompatibility) loci. Reactive oxygen species (ROS) plays a key role in vegetative incompatibility-mediated PCD. The expression of G protein subunit genes, het genes, and ROS-related genes were significantly down-regulated, and cellular production of ROS was suppressed in the presence of SsMYRV4. Furthermore, SsMYRV4-infected strain could easily accept other viruses through hyphal contact and these viruses could be efficiently transmitted from SsMYRV4-infected strain to other vegetatively incompatible individuals. Thus, we concluded that SsMYRV4 is capable of suppressing host non-self recognition and facilitating heterologous viruses transmission among host individuals. These findings may enhance our understanding of virus ecology, and provide a potential strategy to utilize hypovirulence-associated mycoviruses to control fungal diseases.

New insights into mycoviruses and exploration for the biological control of crop fungal diseases.

Mycoviruses are viruses that infect fungi. A growing number of novel mycoviruses have expanded our knowledge of virology, particularly in taxonomy, ecology, and evolution. Recent progress in the study of mycoviruses has comprehensively improved our understanding of the properties of mycoviruses and has strengthened our confidence to explore hypovirulence-associated mycoviruses that control crop diseases. In this review, the advantages of using hypovirulence-associated mycoviruses to control crop diseases are discussed, and, as an example, the potential for Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) to control the stem rot of rapeseed (Brassica napus) is also introduced. Fungal vegetative incompatibility is likely to be the key factor that limits the wide utilization of mycoviruses to control crop diseases; however, there are suggested strategies for resolving this problem.

Extracellular transmission of a DNA mycovirus and its use as a natural fungicide

Mycoviruses are thought not to be infectious as free particles and to lack an extracellular phase in their life cycles, limiting the broad use of hypovirulence-associated mycoviruses in controlling fungal disease. Here, we demonstrate that purified particles of a DNA mycovirus, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), are infectious when applied extracellularly to its host Sclerotinia sclerotiorum. Virus particles isolated from an infected host can infect the hyphae of virus-free S. sclerotiorum directly when applied to hyphae grown on potato dextrose agar or sprayed on leaves of Arabidopsis thaliana and Brassica napus, regardless of vegetative compatibility affiliation. When applied to leaves, the virus can suppress the development of lesions. SsHADV-1 can also reduce disease severity and enhance rapeseed yield significantly under field conditions. SsHADV-1 has a narrow host range; it can infect Sclerotinia minor and Sclerotinia nivalis, sister species of S. sclerotiorum, and cause debilitation of these two fungi, but cannot infect or transfect other tested fungi, such as Botrytis cinerea, which shares the same family with S. sclerotiorum. Virus particles are likely to be very stable on the leaves of A. thaliana plants because viral DNA could be detected at 15 d postinoculation on unwounded leaves and at 10 d postinoculation on wounded leaves, respectively; however, this virus could not infect and move in plant cells. Our findings may prompt a reconsideration of the generalization that mycoviruses lack an extracellular phase in their life cycles and stimulate the search for other DNA mycoviruses with potential use as natural fungicides.