Mycology: Fungal Viruses

• Virus diseases are the most dangerous and difficult to control.
• Viruses have been increasingly found in association with fungi in recent years.
• This association means the fungus is either the vector or the host of the virus.
• Viruses or virus-like particles (VLPs) have been found in over 100 species from 73 genera of fungi, but only a few have been isolated and characterized.
• Terms for viruses of fungi include mycoviruses, fungal viruses, mycophages, double-stranded RNA (dsRNA) plasmids, and virus-like particles (VLPs).

History and Geographical Distribution

• In 1948, an infectious disease of white button mushrooms (Agaricus bisporus) was observed.
• The mushroom disease was at a Pennsylvania farm run by the La France brothers, and was named La France disease.
• A disease in England causing brown staining on the stipe was named 'brown disease', the most common symptom being water-logged patches on mushroom stipes, then named ‘watery stipe.’
• A similar disease was observed in the Pennsylvania mushroom industry, the cause was unknown so was named 'X-disease’.
• In India, viral and virus-like diseases have been found on button and oyster mushrooms.

Mycoviruses

• Mycoviruses are viruses that affect mushrooms.
• The majority of mycoviruses have double-stranded RNA.
• Approximately 30% of mycoviruses have positive sense, single-stranded RNA.
• Mycovirology is the study of viruses infecting fungi, also called Mycoviruses.

Host and Disease examples with Shape, Size and Country

• Agaricus bisporus, which can have La France, Watery stipe, X-disease, Die-back or mushroom disease is spherical, 25-35nm, and found in Australia, England and Holland.
• Agaricus bisporus can also be bacilliform club shaped, 18x50nm or 60-70 nm dia or 120-170 long with a spherical body of 40-50 nm & a cylindered tail 20-30 nm in dia, and found in the U.K, France, W.Germany and S.Africa.
• Agaricus bisporus can also be rods of varying length, 19x90nm, 19x35nm, 20x130nm, and found in Poland, GDR and China.
• Volvariella volvacea, which is spherical, 35nm, and found in China.
• Pleurotus spp. (P. Colombinus and P. Ostreatus) is spherical, 26+2nm, and found in France and India.
• P. Pulmonarius and P. Sapidus are which is spherical, 24nm, and found in China.
• P.Florida which have Flexousrods that are 40-600nm long.
• Ledodes is spherical, 20-45nm, and found in China and Japan.
• Ledodes which have stiff of 17x200x1200nm 15x700-900nm 18x1500nm 15x16x200-300nm, and found in Japan and China.

Detection Methods

• Diagnosing viral infections in mushrooms is difficult due to two reasons:
  • Mushrooms are anatomically simple organisms and respond to adverse stimuli in limited ways.
  • Symptoms like elongated stipe, water logging, yield loss, and bare patches can be induced by both viral infections and other factors.
  • Low virus concentration is another difficulty in diagnosis.
• Approaches to diagnose viral infection include:
  • Symptoms on the bed.
  • Comparative growth rates of mycelium on agar.
  • Direct electron-microscopic examination (EM).
  • Immunosorbent electron-microscopy (IEM or ISEM).
  • Polyacrylamide gel electrophoresis (PAGE).
  • Enzyme-linked immunosorbent assay (ELISA).
  • Reverse transcription-polymerase chain reaction assay (RT-PCR).

Symptoms

• Severe crop losses reported due to club-shaped virus in A. bisporus with difficulty to produce spawn from infected mushrooms.
• Virus diseases in L. edodes cause dwarfing, early maturity, hardened gills, and thickened, elongated or barrel-shaped stipes
• Additional symptoms include:
  • Mycelium does not permeate or disappears after normal spread, mushrooms appear in dense clusters with early maturity.
  • Mycelium from diseased sporophores on agar shows slow and degenerated growth compared to healthy mycelium.
  • Delayed appearance of pinheads, formation of fruiting primordia below the casing layer, and pilei are already opened when mushrooms appear.
  • Variable sporophore symptoms such as slow mycelial growth, abnormal mushroom development.
  • Slow pinhead development, dwarfing, delayed sporophore appearance, reduced yield.
  • Off-white cap color with early maturity, sporophores with elongated stems and small caps.
  • Elongated bent stipes, loosely attached mushrooms, accelerated post-harvest deterioration, and watery or streaking stipes.
  • A musty smell in the infested growing room.
  • Dwarfing or stipe elongation in Pleurotus due to virus infection.

Mycovirus Example

• The best known instance is with Cryphonectria parasitica hypo virus 1 (CHV1), a model for studying hypovirulence in fungi.
• The 9th ICTV report lists over 90 mycovirus species across 10 viral families, 20% unassigned to a genus or family.
• CHV1 can propagate in the genera Endothia and Valsa, which belong to Cryphonectriaceae and Diaporthaceae, respectively.
• A related virus was in Sclerotinia sclerotiorum conferring hypovirulence to its host.

Host Range and Incidence

• Mycoviruses are common in fungi and also found in all four phyla of the true fungi: Chytridiomycota, Zygomycota, Ascomycota, and Basidiomycota.
• Some viruses use fungi as vectors but cannot reproduce in the fungal cytoplasm so are different from mycoviruses.

Transmission

• Mycoviruses lack genes for 'cell-to-cell movement' proteins, moving intercellularly during cell division or via hyphal fusion.
• These may not need an external route due to many means of transmission and spread due to the fungal host’s life style.
  • Plasmogamy and cytoplasmic exchange over extended periods of time.
  • Production of vast amounts of asexual spores.
  • Overwintering via sclerotia.
  • More or less effective transmission into sexual spores.
• Transmission to sexually produced spores ranges from 0% to 100% depending on the virus-host combination.
• Research suggests transmission efficiencies are based on the hosts viral infection status and mycoviruses may regulate secondary mycoviral infection in Aspergillus species.

Disease Management

• Preventive measures include:
  • Steam the compost for 12 hours at 70°C and remove it quickly.
  • Spray wood with 2% sodium pentachlorophenate (with 0.5-1.0% soda) after drying, then spray with water.
  • Disinfect floors, doors, holes, shutters, racks, floors, and walls with formaldehyde.
  • Clean the manure yard and adjacent areas with formaldehyde.
  • Fit spore filters before filling and replace them according to the amount of dust in the air, use a fan for air extraction.
  • Apply pesticide against flies and cover the compost with moist paper immediately after spawning.
  • Wet the paper twice a week with a 2% solution of 40% commercial formaldehyde, repeat until a few days before casing, moisten the paper before carefully removing it.
  • Remove cuttings and litter rapidly and destroy.
  • Maintain cleanliness of the entire farm and surroundings, spray formaldehyde in the corridor.
  • Disinfect machines, refrigerator and other utilities with a formaldehyde solution.
  • Steam out the concerned room at the first sign of contamination.

Heat Therapy

• Infected cultures grown at 33C for 2 weeks, then sub-cultured and returned to 25C showed normal growth without virus.
• Increased sporophore yields were achieved with tissue and spore cultures from symptomatic sporophores of strains incubated at 32C for 2 weeks.
• More extensive spawn was observed on horse manure compost with symptomatic spawn incubated at 30C than at 23 or 27C.