Project description:Agaricus bisporus is a soil-inhabiting fungus which is cultivated for production of white button mushrooms. A disease of A. bisporus has been previously described with a range of disease symptoms (yield loss, pinning delay, cap distortions and cap browning) which has been given collective name of “Mushroom Virus X” (MVX). The causes of this disease are not clear however prior to this research an association was found between the disease and double-stranded RNA molecules in the mushroom fruitbodies. The experiment was designed to examine causes and host responses of the disease causing the Brown Cap symptom in the cultivated mushroom A. bisporus. This microarray experiment was performed before the Agaricus bisporus genome was sequenced. The gene sequences used to design probes were from known and novel A. bisporus sequences and sequences of transcript fragments identified by Suppression Subtractive Hybridization of non-symptomatic and virus-diseased A. bisporus mushroom fruitbodies. The A. bisporus mushroom fruitbodies were grown on composted wheat straw using commercial cultivation procedures. The gene expression comparison was made of RNA isolated from 32 mushroom fruitbodies (Agaricus bisporus) samples: 20 samples from 5 separate virus-infected commercial mushroom farms with crops displaying the brown symptom (4 replicate samples per farm) and 12 samples from a non-infected crop grown at the University of Warwick. The precise composition of the viral load was the subject of this and future research/papers. Abstract of Manuscript submitted to Applied and Environmental Microbiology: Characterizing the viral agents causing brown cap mushroom disease of Agaricus bisporus by Daniel Eastwood, Julian Green, Helen Grogan, and Kerry Burton (Paper #AEM01093-15). The symptoms of viral infections of fungi range from cryptic to severe but there is little knowledge of the factors involved in this transition of fungal/viral interactions. Brown Cap Mushroom Disease of the cultivated Agaricus bisporus is economically important and represents a model system to describe this transition. Differentially expressed transcript fragments between mushrooms showing the symptoms of Brown Cap Mushroom Disease and control white non-infected mushrooms have been identified and sequenced. Ten of these RNA fragments have been found to be up-regulated over a thousand-fold between diseased and non-diseased tissue but are absent from the Agaricus bisporus genome sequence and hybridise to double-stranded RNA’s extracted from diseased tissue. We hypothesize these transcript fragments are viral and represent components of the disease-causing agent, a bipartite virus with similarities to the family Partitiviridae. The virus fragments were found at two distinct levels within infected mushrooms, at raised levels in infected, non-symptomatic, white coloured mushrooms and much greater levels (3,500-87,000 times greater) in infected mushrooms exhibiting brown colouration. In addition, differential screening revealed 9 up-regulated and 32 down-regulated host Agaricus bisporus transcripts. Chromametric analysis was able to distinguish colour differences between non-infected white mushrooms and white infected mushrooms at an early stage of mushroom growth. This method may be the basis for an ‘on-farm’ disease detection assay.

Project description:Agaricus bisporus is a soil-inhabiting fungus which is cultivated for production of white button mushrooms. A disease of A. bisporus has been previously described with a range of disease symptoms (yield loss, pinning delay, cap distortions and cap browning) which has been given collective name of “Mushroom Virus X” (MVX). The causes of this disease are not clear however prior to this research an association was found between the disease and double-stranded RNA molecules in the mushroom fruitbodies. The experiment was designed to examine causes and host responses of the disease causing the Brown Cap symptom in the cultivated mushroom A. bisporus. This microarray experiment was performed before the Agaricus bisporus genome was sequenced. The gene sequences used to design probes were from known and novel A. bisporus sequences and sequences of transcript fragments identified by Suppression Subtractive Hybridization of non-symptomatic and virus-diseased A. bisporus mushroom fruitbodies. The A. bisporus mushroom fruitbodies were grown on composted wheat straw using commercial cultivation procedures. The gene expression comparison was made of RNA isolated from 32 mushroom fruitbodies (Agaricus bisporus) samples: 20 samples from 5 separate virus-infected commercial mushroom farms with crops displaying the brown symptom (4 replicate samples per farm) and 12 samples from a non-infected crop grown at the University of Warwick. The precise composition of the viral load was the subject of this and future research/papers. Abstract of Manuscript submitted to Applied and Environmental Microbiology: Characterizing the viral agents causing brown cap mushroom disease of Agaricus bisporus by Daniel Eastwood, Julian Green, Helen Grogan, and Kerry Burton (Paper #AEM01093-15). The symptoms of viral infections of fungi range from cryptic to severe but there is little knowledge of the factors involved in this transition of fungal/viral interactions. Brown Cap Mushroom Disease of the cultivated Agaricus bisporus is economically important and represents a model system to describe this transition. Differentially expressed transcript fragments between mushrooms showing the symptoms of Brown Cap Mushroom Disease and control white non-infected mushrooms have been identified and sequenced. Ten of these RNA fragments have been found to be up-regulated over a thousand-fold between diseased and non-diseased tissue but are absent from the Agaricus bisporus genome sequence and hybridise to double-stranded RNA’s extracted from diseased tissue. We hypothesize these transcript fragments are viral and represent components of the disease-causing agent, a bipartite virus with similarities to the family Partitiviridae. The virus fragments were found at two distinct levels within infected mushrooms, at raised levels in infected, non-symptomatic, white coloured mushrooms and much greater levels (3,500-87,000 times greater) in infected mushrooms exhibiting brown colouration. In addition, differential screening revealed 9 up-regulated and 32 down-regulated host Agaricus bisporus transcripts. Chromametric analysis was able to distinguish colour differences between non-infected white mushrooms and white infected mushrooms at an early stage of mushroom growth. This method may be the basis for an ‘on-farm’ disease detection assay. A gene expression comparison was made between diseased mushroom displaying the brown cap symptom with characteristic double-strand RNA profiles (banding pattern on gels) and non-symptomatic virus-free mushrooms. In total RNA was isolated from 32 mushroom fruitbody (Agaricus bisporus) samples: 20 samples from 5 separate virus-infected commercial mushroom farms with crops displaying the brown symptom (4 replicate samples per farm) and 12 samples from a non-infected crop grown at the University of Warwick. Commercially-grown mushrooms are produced in “flushes” at approximately weekly intervals. The samples were collected from commercial farms when symptoms were reported to us but these were from different flushes: Farm1 from the 2nd flush; Farm 2 from the 1st flush; Farm 3 from the 3rd flush; Farm 4 from the 1st flush; and Farm 9 from the 1st flush. To allow for comparisons on the basis of Flush Number, the non-infected mushrooms grown at the University of Warwick were sampled from the first, second and third flushes, 4 mushrooms sampled from each flush.

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Double stranded RNA elements from the mushroom Agaricus bisporus

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