Sclerotinia sclerotiorum and Nectria radicicola: Understanding Mycoviruses

Cedric NDINGA MUNIANIA

Western Illinois University

My name is Cedric NDINGA MUNIANIA, a graduate student in the Biological Sciences Department at Western Illinois University.  This blog was developed to help inform people who are interested in fungal pathogenesis and myxovirus.  I will use Nectria radicicola and Sclerotinia sclerotiorum as model organisms to illustrate the topic, but these properties extend to other fungi as well. I hope this would be beneficial to you and will increase your interest in the subject.

Mycoviruses

            As the name explains, mycoviruses are viruses that affect fungi. They are widely spread throughout the different taxonomic groups of fungi including, pathogenic or symbiotic fungi (Ghabrial et al. 2009). They differ from other viruses because they lack an extracellular stage in their life cycle and remain in the infected fungus cytoplasm for their entire life (Cañizares et al. 2014). The effect of mycoviruses on fungi ranges from ineffective (asymptomatic) to very effective which includes phenotypic and physiological changes (Webster & Weber, 2007) (Fig 1).

History and Distribution : scientists came across mycoviruses for  the first time in 1962 following an aggressive fungal infection known as La France disease, which was affecting the commercial mushroom Agaricus bisporus  and causing a malformation of the fruiting bodies leading to low harvesting  (Ghabrial et al.  2009). Analysis of the mushroom showed dominance of three types of viruses specific to fungi which would later be referred as mycoviruses (Ghabrial et al. 2009). After this case, a large number of cases involving mycoviruses have been reported and mycoviruses have been found in every corner of the globe were fungi occur and they affect a large number of fungal taxonomic groups across all phyla.

Viral structure and mode of transmission:

            Most mycoviruses have a double stranded RNA (dsRNA) embedded in a membrane, but lack a capsid, a viral protein coat (Webster & Weber, 2007). However some specific mycoviruses have genome sequences for particular single-stranded RNA (ssRNA) that make dsRNA replicative intermediates (Cañizares et al. 2014). Due to their obligate intracellular life and the lack of a capsid, mycoviruses cannot efficiently be transmitted like other viruses but instead they are restricted to be transferred using intracellular mechanisms (Cañizares et al. 2014), which in most cases, include hyphae anastamosis or fusion, and asexual sporulation (Cortesi et al. 2001). Due to these reasons, along with the fact that some of the infected fungi could be asymptomatic,  it is difficult to study the prevalence and rate of infection of mycoviruses in fungal communities (Ghabrial et al. 2009).

Fungal immune system response and gene regulation

            The fungal immune system acts in different ways to fight against the virus and prevents it from spreading to the entire mycelium and other fungi occurring in the same area. In order to control the spread of virus, two molecular mechanisms are utilized, somatic incompatibility (SI) and RNA silencing (Nuss 2011). SI is the primary defense system used by the fungi to prevent spreading of the virus throughout the population. As previously described, mycoviruses mainly spread through the process of hyphal fusion or anastamosis. However, vegetative cell fusion is controlled by a set of loci referred as the Het Genes (HG). Therefore the interaction between two incompatible mycelia usually result in competition for substrate and cell death, preventing fusion of the two fungi (Glass & Dementhon 2006). This limits the transmission of the virus to only compatible mycelia, which in some cases have to come from the same fungus. The virus, however, has evolved as a response to this mechanism and behaves very differently when different alleles of the HG are expressed (Nuss 2011). For example, in C. parasitaca, the virus transmission rate is almost non-affected by the given allelic combination of the HG even though cell death is caused by SI (Nuss 2011). Also, it is observed that the virus transmission lowers when there is occurrence of rapid cell death (Nuss 2011). This tells us that not only the virus can be dependent to allelic variability of the HG but also to the rate of cell death for specific fungi (Biella et al. 2002).

            The second mechanism of fungal defense is through the route of RNA silencing. This well known process observed in animal and plant cells, is also done by fungi (Göhre et al. 2013).  In this process the host IS cleaves the dsRNA in to small fragments of siRNA and miRNA using enzymes known as dicer which has a RNAse-III domain. These fragments are then bound by the RNA Induced Silencing Complex (RISC) which blocks protein synthesis of viral RNA as well as its replication and spreads in the organism (Baulcombe 2004) (Fig 2). Fascinatingly, the virus has evolved some strategies to overcome RNA silencing. One efficient mode by which the virus performs these strategies is by the use of viral suppressors of RNA silencing (VSR), which requires a set of different mechanisms and proteins to suppress the RNA silencing (Nuss 2011). One major protein used to achieve this goal is protein P19, a viral protein that binds to ds siDNA with high affinity, preventing further binding with the RISC factor and thus preventing silencing of the viral RNA (Burgyan & Havelda 2011).  Interestingly, the P19 protein, in addition to stopping the RNA silencing, acts as a coat protein and helper components for viral transmission or transcriptional regulators (Burgyan & Havelda 2011). This means that they are enhancing virulence of the mycovirus. In addition to the VSR, a mutation or recombination in the fungi leading to synthesis of different dicer or argonaute proteins could result in failure of silencing by the host immune system (Nuss 2011).

Mycovirus Effects on Fungi and Benefits

            Depending on the fungus and stages in its life cycle, a virus can have alternating effects and some time a single virus or two closely related viruses can have different effects on two different fungi (Ahn & Lee 2000). In most cases mycovirus infections are asymptomatic and do not cause cell death (Asensio et al. 2011). However true, the virus could also display aggressive behaviors and cause major changes, such as change in hyphae shape or structure, low reproduction or infertility (Fig 3), lower mycotoxin production and hypovirulence or hypervirulence (Ghabrial et Suzuki 2009).  Although there are a plethora of distinguishable effects displayed by mycoviruses in which focus could be given, they could also be used as a biological control for hypovirulence, a type of infection where the virus lower virulence or pathogenicity of a pathogenic fungus; or hypervirulence, where the virus actually enhances virulence of a given fungus. The last is not well studied due to the fact that agricultural studies are majorly focusing on how to control pathogenic fungus affecting crops. 

Hypervirulence case: Nectria radicicola (Fungorum 2014)

Phylum: Ascomycota

Class: Sordariomycetes

Order: Hypocreales

Family: Nectriaceae

Genus: Nectria

            Nectria radicicola (anamorphic Cylindrocarpon destructans) is an ascomycetes homothallic fungus that causes seedling blight and roots rot in a variety of plants (Webster &Weber  2007). This fungus is particularly pathogenic to ginseng plants (Ahn & Lee 2000). Ginseng required about six years of cultivation before harvesting; therefore infection of the crops by this fungus causes major problems for agriculture in ginseng-growing regions(Ahn & Lee 2000). Genetics analysis of  N. radicicola isolated from ginseng have shown that mycoviruses with a specific sequence L1 dsRNA, enhances the virulence factor of  this fungus, causing higher degradation of the crop. Thus, selection of mycovirus to be used as biocontrol should exclude L1 dsRNA carrying mycoviruses. Interestingly these same mycoviruses have the opposite effect on Cryphonectria parasitica, a fungus that belongs to Cryphonectriaceae where infection with virus carrying this gene lead to hypovirulence (Ahn & Lee 2000).  

Hypovirulence: Sclerotinia sclerotiorum (Fungorum 2014)

 Phylum: Ascomycota

Class: Leotiomycetes

Order: Helotiales

Family: Sclerotiniaceae

Genus: Sclerotinia

            Sclerotinia sclerotiorum is a pathogenic ascomycetes placed with in the Sclerotinia genus. This fungus mainly reproduces asexually throughout the year and starts developing sexual fruiting bodies in the spring (Ghabrial et Suzuki 2009). This fungus represents a big problem for crop harvesting and infects mainly sunflower, soybean and oilseed rape. One major way to improve yield is to use mycoviruses as biocontrol which could make the fungus less virulent. In this specific case, Sclerotinia sclerotiorum debilitation-associated RNA-virus is used because this mycovirus infects the S. sclerotiorum with high affinity.  The strain Ep-1PN causes hypovirulence of the S. sclerotiorum (Ghabrial et Suzuki 2009). This is a very useful tool for biological control of agricultural crops (Fig 4).

Other interesting facts

            Mycoviruses can actually be involved in a three way symbiosis to allow plant growth in drought conditions. A good example is the mycovirus Curvularia thermal tolerance virus (CthTV) infection on the fungus Curvularia protuberata. CthTVcan activate specific genes on the fungus which now gives it the  ability to transfer heat resistance to plants (Marquez et al. 2006). Another interesting fact is that some mycovirus strains were classified and observed to belong to the same viral family or clade with animal and human pathogens. Mycoviruses such as SsDRV SSRV-L and SsRV-L are all included in the polyphyletic group of Rubi-Like Viruses, a groups which includes the human pathogen Hepatitis E Virus (Liu et al. 2009).  

Conclusion

            In retrospect, mycoviruses are very diverse and can affect a variety of fungi. These mycoviruses usually asymptotic can be aggressive and even lead to cell death and hypo/hypertolerance. For these reasons, mycoviruses are used as biological control in the agricultural and food industry to lower the effect of pathogenic fungi which can cause very dramatic consequences. In order to understand the full effect of mycoviruses in the environment, more in depth research needs to be performed, which will furthermore expand our knowledge on these organisms and their life cycle and biochemical processes.  

Reference

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