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    Isolation and assessment of attachment bacteria and yeasts for the biological control of Botrytis cinerea : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science at Massey University, Palmerston North, New Zealand
    (Massey University, 1997) Cook, Darryl W. M.; Cook, Darryl W. M.
    The biological control of Botrytis cinerea Pers. infection by microbial agents applied to the host surface has been based on a wide range of mechanisms of which resource competition, antibiosis and induced host resistance have been considered the most important. A 1995 review of antagonistic mechanisms concluded that biocontrol agent (BCA) colonisation of the plant host was critical for successful biocontrol but that few isolates appear to achieve this. Recent research has shown a reduced epiphytic growth prior to penetration of B. cinerea when conidia are applied as dry spores. Such pre-penetration infection morphology would provide little opportunity for antibiosis, resource competition or induced host resistance. Contemporary in vivo plant tissue assays and in vitro agar plate-based-assays have perpetuated the traditional biocontrol model based on such mechanisms hence an alternative approach was required. BCA selection based on microbial adhesion to the pathogen itself appeared to offer such an approach. An investigation of methods of B. cinerea conidial application showed that disease incidence was increased and development advanced from aerosol application of spores. Aerosol application was used as the standard technique for biocontrol experiments in the remainder of this study. A total of 12 bacterial and eight yeast candidates were obtained from the attachment assay. In vivo, 15 reduced disease by more than 90% in at least one combination of incubation temperature (1°C,7°C or 15°C) and BCA concentration (three-times to 60-times the B, cinerea population applied). When BCA application followed B. cinerea inoculation by up to 48 h, high biocontrol activity was observed. The five yeasts tested postharvest on kiwifruit conferred high biocontrol (>90%) when applied simultaneously or up to 48 h after B. cinerea inoculation. All eight bacterial and seven yeast BCA candidates also reduced disease incidence in stem wounds by more than 80% in glasshouse tomato plants. In vitro investigations into antagonistic mechanisms suggested that antibiosis was unlikely to be important in all but two of these bacterial BCAs. Production of endochitinase was common among the yeasts but there was no single presumptive mechanism for bacterial biocontrol. Variable levels of adhesion by BCA isolates were detected by light and electron microscopy and indicate that biocontrol may not be correlated quantitatively to the number of adhesion events. Adhesion of yeast and biocontrol activity were not affected by a monoclonal antibody to B. cinerea. However, bacterial adhesion and biocontrol activity were dramatically reduced indicating that the antibody blocked bacterial adhesion sites and that bacteria and yeast adhere to different sites on the pathogen. A monoclonal antibody-based ELISA immunoassay was developed to measure vegetative biomass of B. cinerea in infected tomato stem tissue with or without BCAs. The key to the successful application of this ELISA assay was the extraction of the pathogen antigen from the plant tissue using 0.1M copper sulphate and salts solution. Significant reductions in pathogen growth were detected in host tissue co-inoculated with B. cinerea and BCA. The attachment assay was an efficient isolation method that optimised use of laboratory resources and could be employed in future programmes as a presumptive test for biocontrol. With this determinative selection criterion, BCAs with desirable characteristics such as reduced importance of BCA application dose and timing were obtained. A comparison of these results with those in the literature led to the proposal for an alternative biocontrol model for B. cinerea that could supplement existing technologies.
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    Isolation, characterization and possible biocontrol application of Bdellovibrionaceae (BD) isolated from NZ sources : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) at Massey University
    (Massey University, 2008) Ahmed, Muftikhar
    Bdellovibrionaceae (BD) are unique, predatory, endoparasitic, Gram-negative bacteria. As the world's smallest living hunter they prey on other Gram-negative bacteria giving them potential as biological control agents. Prior to this study, however, there were no reports of BD in New Zealand. The overall aim of this research was to isolate BD from New Zealand sources, characterise them and investigate their potential role as a biological control agent. The history, characteristics, life cycle and mechanism of predation of this organism are reviewed and the possibility of the industrial applications of BD, are discussed. In this study, a halophilic species of BD was isolated from fourteen coastal sea water sites around New Zealand. Thirteen isolates were characterised using proven characterisation techniques including general, microscopic and molecular techniques. It was found that the isolates were taxonomically identical or very closely related to each other and belong to the genus Bacteriovorax. The predation pattern of BD isolates was examined against a group of Gram negative bacteria in solid and liquid media. The predation patterns and efficiencies of the different BD isolates were similar, which confirms that the BD isolates are closely related, are selective in their predation, and prey on some Gram-negative bacteria but not all. The rapid loss of culture viability of BD is well known, but no studies have been reported to date on the survival of pure cultures of BD at different temperatures. The survival rate of BD in dense suspensions at different temperatures without host bacteria was investigated and it was observed that pure BD cultures can be stored with minimal reduction in numbers at temperatures ranging from 4°C to 20°C. However, significant reductions in numbers were observed at -1 8"C, 30°C and 37°C after 13 to 16 days. The effects of the 13 New Zealand BD isolates on the growth of a population of Photobacterium phosphoreum were examined to select the best isolate for in vitro application. All of the isolates tested had considerable reduction effect against P. phosphoreum. Some isolates were more effective than others, despite their taxonomic similarity to each other. The isolate OT2 was selected for further studies based on these results. The in vitro efficacy of BD was assessed against late exponential cultures of a seafood spoilage bacterium, P. phosphoreum, originally isolated from Cod fillets from Denmark. Loglo reductions of P. phosphoreum and some other Gram-negative bacteria ranged from 4.5 to 4.8 after 9 h of incubation at 25OC. BD was effective in reducing the numbers of P. phosphoreum at pH 5.5 to 8.5 and salinity 0.9 to 4.5% (wlv). A significant interaction was observed between the prey and predator concentrations and nutrient concentration. Prey concentrations were observed to be the most vital factor in predation and the most favourable predation conditions were at a prey concentration of -8 loglo colony forming units (CFU)/mL, together with a predator concentration of 3 - 7 loglo plaque forming units (PFU)/mL and a prey : predator ratio of >5.0. The thresholds of the prey and predator concentrations for predation were observed to be 3.7 loglo CFUImL and 3.9 loglo PFUImL, respectively. The trials carried out in this study focused on the efficiency of BD on a pure culture of one organism, P. phosphoreum and not on mixed cultures of Gramnegative spoilage bacteria, the normal condition observed in saltwater fish. There has been very little research in this field and the results of these trials suggest further investigation into the effect of BD on mixed cultures of Gram-negative spoilage organisms is warranted. Since only one isolate of BD (OT2) was examined against only one spoilage bacterium (P. phosphoreum) in liquid medium, the evidence of these findings must be restricted to these particular conditions. Future studies, using a range of BD isolates against a mixture of spoilage and pathogenic organisms in solid medium are warranted. The biopreservation capability of BD in extending the shelf life of king salmon was evaluated. A significant effect was observed at 20°C but not at 10°C. At 20°C the shelf life was extended through extension of the lag phase of growth of the prey bacteria and a reduction in total numbers attained. Sensory evaluation of the salmon product being tested confirmed that the shelf life was extended. However, at 10°C there was no reduction in prey organisms, which suggested that the strain of BD used is ineffective at refrigeration temperatures.