Internal Stipe Necrosis of Agaricus bisporus - Etiology and Molecular Genetic Studies

The button mushroom, Agaricus bisporus is the most popular mushroom in cultivation worldwide, and is the most valuable protected crop in the UK, with an estimated wholesale value exceeding £250 million. In 1991 a new disease emerged in mushroom crops in the UK, called Internal Stipe Necrosis (ISN). Crop losses due to this disease may reach 10 %, since affected mushrooms must be downgraded or discarded. Symptoms take the form of a variable browning reaction in the central region of the mushroom stipe, which may also demonstrate varying degrees of internal collapse. During an exhaustive study of ISN over the past 3 years, it was found that an unusual enteric bacterium was consistently associated with the disease, along with diverse members of the Pseudomonas fluorescens complex, which probably represent secondary colonisers. Several strains of the enteric bacterium reproduced ISN symptoms in trials in which mushrooms were injected with bacteria and in trials where bacteria were sprayed onto otherwise normal mushroom beds. Isolates collected from deliberate infection experiments were shown to be identical to the applied strains by the use of restriction fragment length polymorphism (RFLP) studies, using a cloned 16s rRNA gene isolated from a representative strain of the enteric bacteria. These bacteria therefore appear to satisfy Koch's Postulates as the causative agent of ISN. Conventional biochemical profiles identified the ISN causative agent as Ewingella americana, an unusual species previously unknown in mushrooms or their growing environment. This identification was confirmed by genomic DNA hybridisation using a range of reference strains taxonomically related to and including E. americana. Evidence presented suggests that E. americana produces a single endo-acting chitinase. The significance of this enzyme in ISN pathogenesis is discussed. This 33 kDa enzyme has been purified by hydrophobic interaction chromatography and the encoding gene cloned and expressed in E. coli. Sequence analysis of this gene (designated chiA) revealed an open reading frame of 921 bp, with a deduced peptide size corresponding closely to the size of the purified enzyme. The deduced amino acid sequence was most similar to the chitinase II of Aeromonas sp. No. 10S-24 and, to a lesser extent, the chitinase of Saccharopolyspora erythraeus. Alignment with other chitinases, however, revealed very low homology with the exception of two conserved motifs in the catalytic domain of these enzymes. The E. americana sequence also lacks the chitin binding and Type III fibronectin homology units common to many bacterial chitinases. Deletion of a conserved motif, which has previously been implicated as forming the active site of chitinases, produced a product retaining significant chitinolytic activity. Such evidence may lead to a reappraisal of the significance of this motif in catalysis

Internal Stipe Necrosis of Agaricus bisporus - Etiology and Molecular Genetic Studies

The button mushroom, Agaricus bisporus is the most popular mushroom in cultivation worldwide, and is the most valuable protected crop in the UK, with an estimated wholesale value exceeding £250 million. In 1991 a new disease emerged in mushroom crops in the UK, called Internal Stipe Necrosis (ISN). Crop losses due to this disease may reach 10 %, since affected mushrooms must be downgraded or discarded. Symptoms take the form of a variable browning reaction in the central region of the mushroom stipe, which may also demonstrate varying degrees of internal collapse. During an exhaustive study of ISN over the past 3 years, it was found that an unusual enteric bacterium was consistently associated with the disease, along with diverse members of the Pseudomonas fluorescens complex, which probably represent secondary colonisers. Several strains of the enteric bacterium reproduced ISN symptoms in trials in which mushrooms were injected with bacteria and in trials where bacteria were sprayed onto otherwise normal mushroom beds. Isolates collected from deliberate infection experiments were shown to be identical to the applied strains by the use of restriction fragment length polymorphism (RFLP) studies, using a cloned 16s rRNA gene isolated from a representative strain of the enteric bacteria. These bacteria therefore appear to satisfy Koch's Postulates as the causative agent of ISN. Conventional biochemical profiles identified the ISN causative agent as Ewingella americana, an unusual species previously unknown in mushrooms or their growing environment. This identification was confirmed by genomic DNA hybridisation using a range of reference strains taxonomically related to and including E. americana. Evidence presented suggests that E. americana produces a single endo-acting chitinase. The significance of this enzyme in ISN pathogenesis is discussed. This 33 kDa enzyme has been purified by hydrophobic interaction chromatography and the encoding gene cloned and expressed in E. coli. Sequence analysis of this gene (designated chiA) revealed an open reading frame of 921 bp, with a deduced peptide size corresponding closely to the size of the purified enzyme. The deduced amino acid sequence was most similar to the chitinase II of Aeromonas sp. No. 10S-24 and, to a lesser extent, the chitinase of Saccharopolyspora erythraeus. Alignment with other chitinases, however, revealed very low homology with the exception of two conserved motifs in the catalytic domain of these enzymes. The E. americana sequence also lacks the chitin binding and Type III fibronectin homology units common to many bacterial chitinases. Deletion of a conserved motif, which has previously been implicated as forming the active site of chitinases, produced a product retaining significant chitinolytic activity. Such evidence may lead to a reappraisal of the significance of this motif in catalysis

Sobrevivência em solo e detecção de co-transformantes de Trichoderma harzianum por PCR "nested"

The objective of this work was to evaluate the survival of two Trichoderma harzianum cotransformants, TE 10 and TE 41, carrying genes for green fluorescent protein (egfp) and for resistance to benomyl, during four weeks in a contained soil microcosm. Selective culture media were used to detect viable fungal material, whose identity was confirmed by the observation of the fluorescent phenotype by direct epifluorence microscopy. PCR using two nested primer pairs specific to the egfp gene was also used to detect the transformed fungi. Although it was not possible to reliably detect the egfp gene directly from soil extracts, an enrichment step involving selective culture of soil samples in liquid medium prior to DNA extraction enabled the consistent detection of the T. harzianum co-transformants by nested PCR for the duration of the incubation period.O objetivo deste trabalho foi avaliar a sobrevivência de dois co-transformantes de Trichoderma harzianum, TE 10 e TE 41, expressando o gene da proteína de fluorescência verde (egfp) e resistência a benomil, por um período de quatro semanas em microcosmo de solo, sob condições controladas. Foi utilizado um meio seletivo para detecção de material fúngico viável, o qual foi confirmado por observação quanto ao fenótipo de fluorescência em microscópio de epifluorescência direta. O fungo transformado foi detectado por PCR "nested", utilizando-se dois pares de primers específicos para o gene egfp. Foram utilizados meios líquidos enriquecidos no cultivo de amostras de solo, permitindo uma detecção consistente de co-transformantes de T. harzianum, uma vez que não foi possível a detecção do gene egfp por PCR de amostras de DNA extraídas diretamente de solo

Novos isolados de Trichoderma antagônicos a Sclerotinia sclerotiorum

Quarenta e nove isolados de Trichoderma obtidos no centro-oeste do Brasil foram avaliados quanto a sua atividade antagônica in vitro contra Sclerotinia sclerotiorum (agente causal do mofo branco) e identificados com base nas sequências ITS do DNA ribossômico nuclear. Os testes de cultivo pareado mostram que todos os isolados exibiram algum antagonismo, com um máximo de 77% de inibiação micelial e inibição total da produção de escleródios. Dois isolados se destacaram como os mais promissores, considerando ambos os parâmetros avaliados (CEN1253 - T. koningiopsis e CEN1265 - T. brevicompactum). Cinco espécies diferentes foram identificadas: T. harzianum (23), T. spirale (9), T. koningiopsis (8), T. brevicompactum (7) and T. asperellum (2). Estes isolados estão armazenados na Coleção de Fungos para Controle Biológico da Embrapa e as informações obtidas nos experimentos serão incorporadas na base de dados de ativos biológicos, no sistema de informações de recursos genéticos, e disponibilizados para estudos futuros.Forty-nine isolates of Trichoderma from the Brazilian Midwest were evaluated for their antagonistic activity in vitro against Sclerotinia sclerotiorum (causal agent of white mold), which were then identified based on their nuclear ribosomal ITS sequences. Paired culture tests showed that all isolates exhibited some antagonism, with a maximum of 77% mycelial inhibition and complete inhibition of sclerotia production. Two isolates were found to be the most promising biocontrol agents, considering both antagonistic parameters (CEN1253 - T. koningiopsis and CEN1265 - T. brevicompactum). Five different species were identified: T. harzianum (23), T. spirale (9), T. koningiopsis (8), T. brevicompactum (7) and T. asperellum (2). These isolates are stored in the Embrapa Fungi Collection for Biological Control and the information obtained in the experiments will be incorporated into the database of biological assets within the genetic resources information system (Allele) and be made available for further studies

Tourist Photographers and the Promotion of Travel: the Polytechnic Touring Association, 1888–1939

The Polytechnic Touring Association (PTA) was a London-based, originally philanthropic turned commercial travel firm whose historical origins coincided with the arrival of the Kodak camera in 1888 – thus, of popular (tourist) photography. This article examines the PTA’s changing relationship with tourist photographers, and how this influenced the company’s understanding of what role photography could play in promoting the tours, in the late nineteenth and early twenty century. This inquiry is advanced on the basis of the observation that, during this time, the PTA’s passage from viewing tourists as citizens to educate, to customers to please, paralleled the move from using photography-based images to mixed media. Such a development was certainly a response to unprecedented market demands; this article argues that it should also be considered in relation to the widening of photographic perceptions engendered by the democratization of the medium, to which the PTA responded, first as educator, then as service provider. In doing so, the article raises several questions about the shifting relationship between “high”, or established, and “low”, or emerging, forms of culture, as mass photography and the mass marketing of tourism developed

Evaluating the population impact of hepatitis C direct acting antiviral treatment as prevention for people who inject drugs (EPIToPe) – a natural experiment (protocol)

Hepatitis C virus (HCV) is the second largest contributor to liver disease in the UK, with injecting drug use as the main risk factor among the estimated 200 000 people currently infected. Despite effective prevention interventions, chronic HCV prevalence remains around 40% among people who inject drugs (PWID). New direct-acting antiviral (DAA) HCV therapies comine high cure rates (>90%) and short treatment duration (8 to 12 weeks). Theoretical mathematical modelling evidence suggests HCV treatment scale-up can prevent transmission and substantially reduce HCV prevalence/incidence among PWID. Our primary aim is to generate empirical evidence on the effectiveness of HCV ‘Treatment as Prevention’ (TasP) in PWID

Survival in soil and detection of co-transformed Trichoderma harzianum by nested PCR Sobrevivência em solo e detecção de co-transformantes de Trichoderma harzianum por PCR "nested"

The objective of this work was to evaluate the survival of two Trichoderma harzianum co-transformants, TE 10 and TE 41, carrying genes for green fluorescent protein (egfp) and for resistance to benomyl, during four weeks in a contained soil microcosm. Selective culture media were used to detect viable fungal material, whose identity was confirmed by the observation of the fluorescent phenotype by direct epifluorence microscopy. PCR using two nested primer pairs specific to the egfp gene was also used to detect the transformed fungi. Although it was not possible to reliably detect the egfp gene directly from soil extracts, an enrichment step involving selective culture of soil samples in liquid medium prior to DNA extraction enabled the consistent detection of the T. harzianum co-transformants by nested PCR for the duration of the incubation period.<br>O objetivo deste trabalho foi avaliar a sobrevivência de dois co-transformantes de Trichoderma harzianum, TE 10 e TE 41, expressando o gene da proteína de fluorescência verde (egfp) e resistência a benomil, por um período de quatro semanas em microcosmo de solo, sob condições controladas. Foi utilizado um meio seletivo para detecção de material fúngico viável, o qual foi confirmado por observação quanto ao fenótipo de fluorescência em microscópio de epifluorescência direta. O fungo transformado foi detectado por PCR "nested", utilizando-se dois pares de primers específicos para o gene egfp. Foram utilizados meios líquidos enriquecidos no cultivo de amostras de solo, permitindo uma detecção consistente de co-transformantes de T. harzianum, uma vez que não foi possível a detecção do gene egfp por PCR de amostras de DNA extraídas diretamente de solo