Multiple viral infections in Agaricus bisporus - Characterisation of 18 unique RNA viruses and 8 ORFans identified by deep sequencing.

Thirty unique non-host RNAs were sequenced in the cultivated fungus, Agaricus bisporus, comprising 18 viruses each encoding an RdRp domain with an additional 8 ORFans (non-host RNAs with no similarity to known sequences). Two viruses were multipartite with component RNAs showing correlative abundances and common 3' motifs. The viruses, all positive sense single-stranded, were classified into diverse orders/families. Multiple infections of Agaricus may represent a diverse, dynamic and interactive viral ecosystem with sequence variability ranging over 2 orders of magnitude and evidence of recombination, horizontal gene transfer and variable fragment numbers. Large numbers of viral RNAs were detected in multiple Agaricus samples; up to 24 in samples symptomatic for disease and 8-17 in asymptomatic samples, suggesting adaptive strategies for co-existence. The viral composition of growing cultures was dynamic, with evidence of gains and losses depending on the environment and included new hypothetical viruses when compared with the current transcriptome and EST databases. As the non-cellular transmission of mycoviruses is rare, the founding infections may be ancient, preserved in wild Agaricus populations, which act as reservoirs for subsequent cell-to-cell infection when host populations are expanded massively through fungiculture.

Brown mushroom symptom expression following infection of an Agaricus bisporus crop with MVX associated dsRNAs.

Mushroom Virus X (MVX) is associated with a range of symptoms observed in mushroom crops. The most prominent symptom in Ireland is the occurrence of 'brown' or 'off-white' mushrooms in white strain crops. The browning symptoms are associated with the presence of four low molecular weight dsRNAs: MVX(0.6), MVX(0.8), MVX(1.8) and MVX(2.0), however viral dsRNAs also occur in non-symptomatic mushrooms. Three virus-infected mushroom cultures containing MVX(1.8) and MVX(2.0) were used to infect experimental crops at different rates and at different times in the crop cycle to test the effect on symptom expression. Mushroom colour was measured by chromometer, and the ΔE value calculated. RT-PCR was used to test for the presence of MVX(1.8) dsRNA in harvested mushrooms. Results indicate that following infection, browning symptom expression is variable both within and between crops. Control mushrooms from 1st and 2nd flush had ΔE values of 7-12, with most being <10. In contrast, 1st flush mushrooms from virus infected treatments had ΔE values of 6-25, with most being >10 while 2nd flush mushrooms had ΔE values similar to controls. Only mushrooms with ΔE > 15 appeared visibly brown or off colour. The transient and inconsistent nature of MVX-associated browning symptoms is discussed.

Fungicide control of mushroom cobweb disease caused by Cladobotryum strains with different benzimidazole resistance profiles.

The benzimidazole fungicides thiabendazole and carbendazim, and the imidazole fungicide prochloraz-Mn, were tested for their efficacy in controlling cobweb disease of mushrooms caused by two Cladobotryum isolates. Isolate 202A was benzimidazole-sensitive in vitro and cobweb growth on the casing was well controlled by both benzimidazole fungicides in cropping experiments. Carbendazim also controlled the development of spotting symptoms much more effectively than thiabendazole. A second isolate (192B1) was benzimidazole-resistant and was highly resistant to thiabendazole in vitro but it showed some sensitivity to carbendazim in vitro at moderate to high concentrations. Despite this, carbendazim did not control disease symptoms in cropping experiments, confirming that isolate 192B1 is cross-resistant to other benzimidazole fungicides. Both isolates showed some sensitivity to prochloraz-Mn in vitro. This fungicide gave between 45% and 65% control of cobweb growth on the casing caused by either 202A or 192B1 but gave no control of spotting symptoms. Reducing the fungicide application volume did not give enhanced disease control. The emergence of benzimidazole resistance reduces the value of benzimidazoles in the control of mushroom pathogens. However, the lack of effective alternatives means they continue to have utility in cases where pathogens are still sensitive but this requires regular monitoring of pathogen populations for resistance.

Persistence of the fungicides thiabendazole, carbendazim and prochloraz-Mn in mushroom casing soil.

The persistence of the fungicides thiabendazole, carbendazim and prochloraz-Mn in mushroom casing soil was determined following their application at rates commonly used in the UK mushroom industry. Following drench applications, the concentration of all active ingredients was always higher in the top half of the casing soil layer than in with the bottom half. When carbendazim and prochloraz-Mn were applied using half the recommended volume of water per unit area, there was a tendency for carbendazim concentrations to be even higher in the top half of the casing soil, compared with the standard treatment, while concentrations of prochloraz-Mn were similar, irrespective of the volume of water used. Carbendazim and prochloraz-Mn concentrations in the top half of the casing layer decreased to < or = 13 mg kg(-1) by day 28/29, following different applications, whereas the thiabendazole concentration was consistently high during the course of the crop, being < or = 83 mg kg(-1) at day 31. Fungicides that do not persist at high concentrations in mushroom casing soil for the duration of the crop may not give good control of mushroom pathogens, particularly if the fungicide concentration falls to a level which is close to the EC50 value.

Double-stranded RNA elements associated with the MVX disease of Agaricus bisporus.

Double-stranded RNA (dsRNA) has been isolated from Agaricus bisporus fruit bodies exhibiting a wide range of disease symptoms. The symptoms which occurred singularly or in combination included; bare cropping areas on commercial beds (primordia disruption), crop delay, premature veil opening, off- or brown-coloured mushrooms, sporophore malformations and loss of crop yield. All symptoms were associated with loss of yield and/or product quality. Collectively, these symptoms are described as mushroom virus X (MVX) disease. The dsRNA titre was much lower than that previously encountered with the La France viral disease of mushrooms and a modified cellulose CF11 protocol was used for their detection. A broad survey of cultivated mushrooms from the British industry identified dsRNA elements ranging between 640 bp and 20.2 kbp; the majority have not previously been described in A. bisporus. 26 dsRNA elements were identified with a maximum of 17, apparently non-encapsidated dsRNA elements, in any one sample. Three dsRNAs (16.2, 9.4 and 2.4 kbp) were routinely found in mushrooms asymptomatic for MVX. Previously, La France disease was effectively contained and controlled by minimising the on-farm production and spread of basidiospores. Our on-farm observations suggest that MVX could be spread by infected spores and/or mycelial fragments.