Transmission of mushroom virus X and the impact of virus infection on the transcriptomes and proteomes of different strains of Agaricus bisporus.

Cultivation of Agaricus bisporus is a large horticultural industry for many countries worldwide, where a single variety is almost grown exclusively. Mushroom virus X (MVX), a complex of multiple positive-sense single stranded RNA (ss(+)RNA) viruses, is a major pathogen of typical A. bisporus crops. MVX can manifest a variety of symptoms in crops and is highly infective and difficult to eradicate once established in host mycelium. Currently our knowledge regarding the molecular response of A. bisporus fruit bodies to MVX infection is limited. In order to study the response of different A. bisporus strains with different susceptibilities to MVX, we designed a model system to evaluate the in-vitro transmission of viruses in A. bisporus hyphae over a time-course, at two crucial phases in the crop cycle. The symptom expression of MVX in these varieties and the transcriptomic and proteomic response of fruit bodies to MVX-infection were examined. Transmission studies revealed the high potential of MVX to spread to uninfected mycelium yet not into the fruit bodies of certain strains in a crop. MVX affected colour and quality of multiple fruit bodies. Gene expression is significantly altered in all strains and between times of inoculation in the crop. Genes related to stress responses displayed differential expression. Proteomic responses revealed restriction of cellular signalling and vesicle transport in infected fruit bodies. This in-depth analysis examining many factors relevant to MVX infection in different A. bisporus strains, will provide key insights into host responses for this commercially important food crop.

Viral Interactions and Pathogenesis during Multiple Viral Infections in Agaricus bisporus.

Viral interactions during multiple viral infections were examined in Agaricus bisporus cultures harboring 9 viruses (comprising 18 distinct viral RNAs) by statistically analyzing their relative abundance in fruitbodies. Four clusters of viral RNA were identified that suggested synergism and coreplication. Pairwise correlations revealed negative and positive correlations between clusters, indicating further synergisms and an antagonism involving a group containing a putative hypovirus and four nonhost ORFan RNAs (RNAs with no similarity to known sequences) possibly acting as defective interfering RNAs. The disease phenotype was observed in 10 to 15% of the fruitbodies apparently randomly located among asymptomatic fruitbodies. The degree of symptom expression consistently correlated with the levels of the multipartite virus AbV16. Diseased fruitbodies contained very high levels of AbV16 and AbV6 RNA2; these levels were orders of magnitude higher than those in asymptomatic tissues and were shown statistically to be discretely higher populations of abundance, indicating an exponential shift in the replicative capacity of the virus. High levels of AbV16 replication were specific to the fruitbody and not found in the underlying mycelium. There appeared to be a stochastic element occurring in these viral interactions, as observed in the distribution of diseased symptoms across a culture, differences in variance between experiments, and a number of additional viruses undergoing the step-jump in levels between experiments. Possible mechanisms for these multiple and simultaneous viral interactions in single culture are discussed in relation to known virus-host regulatory mechanisms for viral replication and whether additional factors could be considered to account for the 1,000-fold increase in AbV16 and AbV6 RNA2 levels.IMPORTANCE How viruses interact in a multiple-virus infection was examined by quantifying the levels of 18 viral RNAs in fruiting cultures of the agriculturally cultivated fungus Agaricus bisporus and statistically analyzing and modeling their abundance. Synergistic, antagonistic, and neutral interactions occurred simultaneously in cultures. The viral RNAs were grouped into four clusters, each displaying similar relative abundance, and between clusters, further interactions were found with positive, negative, or no correlations. Mushroom fruitbodies showing disease symptoms were distributed apparently randomly across the culture. These symptoms were associated with the presence of viral RNAs from two different clusters at very high levels, 1,000-fold higher than asymptomatic fruitbodies. The role of viral interaction together with stochastic factors and the regulation of host antiviral defenses in pathogenesis are discussed.

Genomic Characterisation of Mushroom Pathogenic Pseudomonads and Their Interaction with Bacteriophages.

Bacterial diseases of the edible white button mushroom Agaricus bisporus caused by Pseudomonas species cause a reduction in crop yield, resulting in considerable economic loss. We examined bacterial pathogens of mushrooms and bacteriophages that target them to understand the disease and opportunities for control. The Pseudomonastolaasii genome encoded a single type III protein secretion system (T3SS), but contained the largest number of non-ribosomal peptide synthase (NRPS) genes, multimodular enzymes that can play a role in pathogenicity, including a putative tolaasin-producing gene cluster, a toxin causing blotch disease symptom. However, Pseudomonasagarici encoded the lowest number of NRPS and three putative T3SS while non-pathogenic Pseudomonas sp. NS1 had intermediate numbers. Potential bacteriophage resistance mechanisms were identified in all three strains, but only P. agarici NCPPB 2472 was observed to have a single Type I-F CRISPR/Cas system predicted to be involved in phage resistance. Three novel bacteriophages, NV1, ϕNV3, and NV6, were isolated from environmental samples. Bacteriophage NV1 and ϕNV3 had a narrow host range for specific mushroom pathogens, whereas phage NV6 was able to infect both mushroom pathogens. ϕNV3 and NV6 genomes were almost identical and differentiated within their T7-like tail fiber protein, indicating this is likely the major host specificity determinant. Our findings provide the foundations for future comparative analyses to study mushroom disease and phage resistance.

FISHing in fungi: Visualisation of mushroom virus X in the mycelium of Agaricus bisporus by fluorescence in situ hybridisation.

Agaricus bisporus is a commercial mushroom crop susceptible to a disease caused by a complex of viruses known collectively as mushroom virus X (MVX). Symptoms of MVX include bare patches and mushroom cap discolouration (browning) in the fruiting bodies, phenotypes associated with the viruses AbV6 and AbV16, respectively. Limited understanding exists of the localisation and mobilisation of these viruses within the mycelium of A. bisporus. To this end, a non-destructive fluorescence in situ hybridisation (FISH) method was developed for in situ targeting of AbV6 and AbV16 in A. bisporus mycelium. An MVX strain associated with the bare patch disease phenotype revealed predominantly high signal towards the growing edges of cultures when probed for AbV6, with a 'halo-effect' of high signal intensity around putative vacuoles. An MVX strain associated with the browning disease phenotype showed high signal intensities within reticulating networks of hyphae in a highly compartmentalised manner when probed for AbV16. Localisation of the two viruses in MVX-infected cultures appears independent, as both viruses were found in completely discrete areas of the mycelium in differential patterns. FISH detected low level presence of the two viruses, AbV6 and AbV16 in a number of cultures which had tested negative for the viruses by RT-PCR. This suggests that FISH may be more sensitive at detecting viruses at low levels than molecular methods. This study demonstrates that FISH is a powerful tool in the field of mycovirology.

Antimicrobial activity of Agaricus brasiliensis on Plasmopara viticola and its effect on the induction of resistance to the control of downy mildew on Isabel Precoce / Atividade antimicrobiana de Agaricus brasiliensis sobre Plasmopara viticola e seu efeito na indução de resistência para o controle de míldio em videiras Isabel Precoce

Agaricus brasiliensis include bioactive compounds that can act as antibiotics, bacteriostatic, fungistatic and nematostatic substances. In this sense, this study aimed to evaluate the effect of a single application of aqueous mycelial suspension (AMS) of A. brasiliensis in control of downy mildew (Plasmopara viticola) and resistance induction in 'Isabel Precoce' grapevines under greenhouse conditions. Treatments consisted of three doses of 1%, 5%, 10%, 15% and 20% AMS A. brasiliensis, as well as treatment with acibenzolar-S-methyl (ASM). The variables analyzed were: sporangiospore germination, disease severity, represented by the area under the disease progress curve (AUDPC), catalase enzyme activity, peroxidase and polyphenol. The 10%, 15% and 20% doses of AMS caused approximately 80% reduction in germination of P. viticola sporangiospores. The treatments did not show significant effects in reducing both the AUDPC of mildew and polyphenol oxidase enzyme activity. The A. brasiliensis aqueous mycelial suspension showed a fungitoxic effect on the germination of sporangiopores; however, it was not enough to reduce the severity of mildew in the 'Isabel Precoce' grapevines, even when acting on the catalase and peroxidase enzymes. Thus, experiments should be performed to verify the viability of the reproductive structures of the pathogen externalized in the vines when treated with A. brasiliensis AMS.(AU)

Agaricus brasiliensis possui compostos bioativos que apresentam atividade antimicrobiana e induz mecanismos de defesa em plantas contra patógenos. O objetivo deste trabalho foi avaliar a aplicação da suspensão miceliada aquosa de A. brasiliensis no controle do míldio (Plasmopara viticola) e indução de resistência em videiras Isabel Precoce. Os tratamentos foram: 0, 1, 5, 10, 15 e 20% da suspensão miceliada aquosa de A. brasiliensis, além do tratamento com acibenzolar-S-metil. As variáveis analisadas foram: germinação de esporangiósporos; severidade da doença, representada pela área abaixo da curva de progresso da doença; atividade da enzima catalase; peroxidase e polifenoloxidase. As doses 10, 15 e 20% de suspensão miceliada aquosa de A. brasiliensis proporcionaram redução de aproximadamente 80% na germinação dos esporangiósporos de P. viticola. Os tratamentos não apresentaram efeitos significativos na redução da área abaixo da curva de progresso da doença do míldio e na atividade da enzima polifenoloxidase. A dose de 10% da suspensão miceliada aquosa de A. brasiliensis reduziu a atividade de catalase e induziu a atividade da peroxidase. A suspensão miceliada aquosa de A. brasiliensis apresentou efeito fungitóxico na germinação de esporangióporos, entretanto não foi suficiente para reduzir a severidade do míldio da videira Isabel Precoce, mesmo atuando na atividade das enzimas catalase e peroxidase. Assim, experimentos deverão ser realizados para verificar a viabilidade das estruturas reprodutivas do patógeno exteriorizadas nas videiras quando tratadas com suspensão miceliada aquosa de A. brasiliensis.(AU)

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.

Viral Agents Causing Brown Cap Mushroom Disease of Agaricus bisporus.

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 noninfected mushrooms have been identified and sequenced. Ten of these RNA fragments have been found to be upregulated over 1,000-fold between diseased and nondiseased tissue but are absent from the Agaricus bisporus genome sequence and hybridize to double-stranded RNAs extracted from diseased tissue. We hypothesize that 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, nonsymptomatic, white mushrooms and at much greater levels (3,500 to 87,000 times greater) in infected mushrooms exhibiting brown coloration. In addition, differential screening revealed 9 upregulated and 32 downregulated host Agaricus bisporus transcripts. Chromametric analysis was able to distinguish color differences between noninfected 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.

Adaptabilidad de cepas brasileñas de Agaricus subrufescens Peck a la fructificación sobre diferentes capas de cobertura en cultivo comercial / Adaptability of Brazilian strains of Agaricus subrufescens Peck to fruiting on various casing materials in commercial crops

Antecedentes. Agaricus subrufescens Peck es un hongo cuyo cultivo ha despertado gran interés en todo el mundo en los últimos años, adquiriendo gran popularidad. Sus propiedades medicinales y culinarias hacen prever una rápida expansión del cultivo en todo el mundo. Objetivos. El trabajo plantea como objetivo la evaluación del efecto sobre los principales parámetros de producción de 3 cepas de Agaricus subrufescens que se han hecho fructificar sobre 5 capas de cobertura diferentes. Métodos. Se ha llevado a cabo un ciclo de cultivo de Agaricus subrufescens en condiciones controladas en el que se han evaluado los principales parámetros de producción. Resultados. Los mejores resultados han sido proporcionados por la cepa ABL 99/30. Las coberturas basadas en turba presentan mejor comportamiento que las basadas en suelo mineral. El mayor rendimiento (6,75 kg/m−2, eficiencia biológica 27,57 kg/dt) ha sido proporcionado por la combinación ABL 99/30-Euroveen. Conclusiones. Nuestros resultados evidencian que la combinación de la cepa ABL 99/30 utilizando una capa de cobertura basada en turba (Euroveen) ofrece un alto potencial para ser utilizada a escala comercial por el sector productor de hongos. La disponibilidad de alternativas a las especies de hongos comestibles cultivadas habitualmente puede suponer un mejor aprovechamiento de recursos y una mayor rentabilidad económica de la actividad (AU)

Background. Agaricus subrufescens Peck is a mushroom whose cultivation has aroused great interest worldwide in recent years, and is becoming increasingly popular. A rapid expansion of culture throughout the world is foreseen because of its medicinal and culinary properties. Aims. This work assesses the effect of 5 different casing layers on the production of 3 strains of Agaricus subrufescens. Methods. A growth cycle of Agaricus subrufescens under controlled conditions has been carried out. The main production parameters were evaluated. Results. The best results were provided by the ABL 99/30 strain. Peat-based casings have a better yield than those based on mineral soil. The highest yield (6.75 kg/m2, biological efficiency 27.57 kg/dt) was provided by the combination ABL 99/30-Euroveen. Conclusions. Our results suggest that the combination of the strain ABL 99/30 using a peat-based casing layer (Euroveen) offers a high potential for use on a commercial scale by the edible mushroom production sector. The availability of alternatives to the usually cultivated species can make better use of resources, and increase the profitability of this activity (AU)

[Morphology and structural peculiarities of Basidiomycetes pathogens].

The materials of studies of morphology and structural peculiarities of viruses, fungi and bacteria, which affect Basidiomycetes under biotechnology process and nature biocenosis conditions are given. The analysis of infection development in button mushroom (Agaricus bisporus) (J.Lge) Imbach and in oyster mushroom (Pleurotus ostreatus Kumm.), which served as model objects in the experiments of various levels of complexity has been carried out. Other kinds of edible and medicinal mushrooms, which were a source of biochemical fractions to form biologicals were investigated.

La France disease of the cultivated mushroom Agaricus bisporus in Poland.

Presence of the virus associated with La France disease was confirmed in the mushrooms collected from different farms located in Western Poland. Double-stranded RNA (dsRNA) was isolated from the mushrooms exhibiting a wide range of the disease symptoms including premature veil opening, brown-colored mushrooms, and loss of crop yield. The presence of dsRNA molecules (M1, M2, and L3) was confirmed by RT-PCR and sequencing. Furthermore, La France isometric virus (LFIV)-like particles were observed in the mushrooms extracts in electron microscopy. The LFIV infection was found in 120 of 200 mushroom samples tested. The amount of the infected samples indicated the high occurrence of La France disease that could be a threat to the mushroom industry in Poland.

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.

Identification of a subgenomic mRNA encoding the capsid protein of mushroom bacilliform virus, a single-stranded RNA mycovirus.

Mushroom bacilliform virus is unique among mycoviruses of the higher fungi in having a genome of positive-sense single-stranded RNA. We have identified a subgenomic mRNA molecule encoding the viral capsid protein in mushroom bacilliform virus-infected mycelium of Agaricus bisporus. Transcription of subgenomic RNA commences at the sequence ACAAAA, 47 nucleotides upstream of the initiating AUG.

Mushroom bacilliform virus RNA: the initiation of translation at the 5' end of the genome and identification of the VPg.

Mushroom bacilliform virus (MBV) is often found in cultivated mushrooms (Agaricus bisporus) with La France disease. MBV has a 4-kb ssRNA genome of positive-sense encoding four major open reading frames (ORFs). The arrangement of ORFs at the 5' end of the genome and the deduced amino-acid sequences of two of the putative gene products (protease and RNA-dependent RNA polymerase) show remarkable similarity to some plant viruses, particularly subgroup II luteoviruses. We show that this similarity extends to the translation strategy at the 5' end of the genome, the presence of a genome-linked protein (VPg), and the location of the VPg downstream of the protease motifs in the polypeptide encoded by ORF2.

RT-PCR detection of dsRNAs associated with La France disease of the cultivated mushroom Agaricus bisporus (Lange) Imbach.

A reverse transcription-polymerase chain reaction assay (RT-PCR) is described for the detection of double-stranded RNA (dsRNA) molecules (M1, M2, and L3) associated with La France disease of the cultivated mushroom Agaricus bisporus. RT-PCR was faster and more sensitive than current methods used to detect dsRNA, such as dsRNA extraction and analysis by electrophoresis. Another major advantage of RT-PCR was the detection of M1 dsRNA in rapidly prepared homogenates of sporophores and spawn, and in compost before sporophore production. The early detection of La France disease by RT-PCR will enable implementation of control measures by growers that may reduce losses in production time associated with a disease outbreak. Sequence analysis of dsRNA molecules in two Australian isolates showed that M1 was more conserved than M2 or L3 dsRNA.

Functional analysis of dsRNAs (L1, L3, L5, and M2) associated with isometric 34-nm virions of Agaricus bisporus (white button mushroom).

cDNA clones of dsRNAs associated with La France disease of Agaricus bisporus were isolated. Clones corresponding to L1 and L5 dsRNAs were sequenced. The deduced amino acid sequence of L1 dsRNA (1078 amino acids, Mr 121K) showed significant homology with RNA-dependent RNA polymerases of other dsRNA viruses. The deduced amino acid sequence of L5 dsRNA (724 amino acids, Mr 82K) showed no homology with known proteins. Amino acid sequences of tryptic digests of three virion-associated proteins were determined. The 34-nm virion-associated protein of Mr 115K was encoded by the L1 dsRNA, thus identifying this protein as the RNA-dependent RNA polymerase. The virion-associated protein of Mr 90K was encoded by the previously sequenced L3 dsRNA. A cDNA clone of the previously sequenced M2 dsRNA was expressed in Escherichia coli and antibodies raised against this protein reacted only with a protein present in the cytoplasm of diseased A. bisporus fruit bodies but not in the 34-nm virions.

PCR analysis of the viral complex associated with La France disease of Agaricus bisporus.

Reverse transcription PCR analysis was used to investigate the involvement of two RNA-genome viruses, La France isometric virus (LIV) and mushroom bacilliform virus (MBV), in the etiology of La France disease of the cultivated mushroom Agaricus bisporus. Reverse transcription PCR amplification of sequences targeted to the genomes of LIV and MBV, with a sensitivity of detection of < 10 fg of viral RNA, showed diseased mushrooms to be either singly infected by LIV or doubly infected by LIV and MBV. Of 70 geographically diverse diseased mushroom isolates, 100% were infected by LIV, whereas almost 60% of these isolates were coinfected by MBV. Of 58 mushroom isolates determined to be free of infection by LIV, 3 were found to be infected by MBV. This represents the first documented report of the independent replication of these two viruses. Our data support the hypothesis that La France disease is associated with infection by two autonomously replicating viruses in which LIV is the primary causal agent and MBV, although possibly pathogenic and capable of modulating symptoms, is not required for pathogenesis.