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Inglis, Peter Ward (1996) Internal Stipe Necrosis of Agaricus bisporus - Etiology and Molecular Genetic Studies. PhD thesis, University of Nottingham.

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Abstract

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.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Peberdy, John
Keywords:	Agaricus bisporus, button mushrooms, Internal Stipe Necrosis
Subjects:	S Agriculture > SB Plant culture
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID:	77102
Depositing User:	Hatton, Mrs Kirsty
Date Deposited:	03 Jan 2024 14:47
Last Modified:	03 Jan 2024 15:21
URI:	https://eprints.nottingham.ac.uk/id/eprint/77102

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