Molecular investigation of pseudomonads causative of Agaricus bisporus blotch disease in New Zealand mushroom farms
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Presented within this thesis is the molecular investigation of selected pseudomonad species inducing blotch disease of Agaricus bisporus in New Zealand mushroom farms. This work represents part of the ongoing research that is aimed at gaining a comprehensive understanding of bacterial pathogenicity toward fungi. Essentially, this thesis can be loosely defined as having two major sections, (i) population characterisation of pseudomonads able to induce blotch disease in New Zealand and (ii) the molecular investigations undertaken to understand bacteria/fungal interactions, with particular focus on elucidating induction of blotch disease of A. bisporus. This thesis originally set out with the objective of investigating 'Pseudomonas gingeri'. Prior to commencing this study, P. ginger; had been described as the causative organism of 'ginger blotch' disease of the commercially produced mushroom, A. bisporus. However, continuing observations of varying disease symptoms and varying phenotypes of bacteria isolated from 'ginger blotch' disease lesions suggested that more than orie organism was causing 'ginger blotch'. A New Zealand national survey of mushrooms exhibiting disease symptoms of 'ginger blotch' was undertaken and from this, 33 pseudomonads capable of causing blotch were characterised to determine the degree of species diversity. Results identified three major findings: 1) the diversity of pseudomonads capable of causing blotch discolourations of A. bisporus is considerably more extensive than previously thought. 2) The organism previously described as P. ginger; is not solely responsible for ginger discolourations of A. bispoms ('ginger blotch'); and 3) a particular blotch discolouration may be caused by more than a single pseudomonad species. The result of these findings affecting the classification of previously identified P. gingeri is discussed. Furthermore, the isolation and characterisation of a novel Pseudomonas isolate, NZI7, is reported that exhibited all phenotypic criteria described for the identification of P. tolaasii, yet showed no genetic similarity. The impact of NZI7 identification is that it demonstrates limitations to the previously accepted phenotypic techniques for P. Tolaasii identification. The observation of many different blotch discolourations being caused by several different Pseudomonas species, termed blotch causing organisms (BCOs), altered the subsequent research direction to a holistic approach to determine genetic determinants involved in colonisation and blotch formation amongst these diverse BCOs. Phenotypic analyses of putative pathogenicity determinants (PPDs) described in other virulent bacteria were performed on all 33 BCOs including; auxotrophy, biofilm formation, protease, lipase and chitinase production. Abiotic biofilm formation under varying conditions was the only consistent PPD phenotype shown by all BCOs. As it was not feasible to carry out detailed investigations on all 33 BCOs, a single BCO P. putida NZ103 was selected for Tn5 mutagenesis to identify the role of NZ103 PPDs in blotch discolouration From a total of 5000 mini-Tn5kmlacZ2 transposon generated NZ103 mutants, 45 were selected based on deficiencies in one or a combination of the PPDs analysed. Furthermore, these 45 NZ103 mutants were assessed using in vitro A. bisporus tissue bioassays and from these, 13/45 had substantial blotch reduction. Ten isolates were chosen from these NZ103 blotch reduced mutants and the genetic regions flanking the mini-Tn5kmlacZ2 insertion point were characterized by restriction mapping, subcloning, nucleotide sequencing and comparison to entries in the BLAST database. Genetic similarities of genes interrupted in these 10 NZ103 mutants included: genes encoding two-component regulatory systems such as gacS/lemA/rtpA; proteins involved in transcriptional regulation (such as the LysR family); probable 2-hydroxyacid dehydrogenase involved in energy metabolism; a possible integron gene; outer membrane protein OprF involved in osmoregulation and cell shape; a metalloprotease secretion gene aprE; and several extracellular binding receptors which may serve as chemoreceptors. These putative identifications and their relationship to current literature is discussed with a focus on interpreting how such genes may be involved in either bacterial/fungal interactions and/or blotch formation of A. bisporus. A summary of all results and discussions within the scope of this thesis is presented within a number of 'models' that have been created to encompass critical factors involved in blotch disease and pose questions for future research to build upon. Finally, Chapter 8 emphasises areas for future research that would further the understanding of bacterial/fungal interactions as well as benefit management of mushroom blotch diseases.