Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Deserts are the most abundant terrestrial biome on Earth, and microbial processes assume a major role since environmental stress severely limits higher plant and animal life. A major hurdle to developing an understanding of microbial ecology in deserts has been the lack of knowledge at the fundamental biodiversity level. This is due to lack of research focus and also the inherent bias in ‘traditional’ microbial diversity estimates based upon cultivation. In this thesis an evaluation of culture-independent approaches employing both DNA and RNA from environmental samples was made with comparison to more traditional cultivation techniques. These were then applied to soils from hot and cold deserts, and along stress gradients from semi-arid to hyper-arid. A literature review was first conducted to assess the extent of current knowledge and identify critical knowledge gaps. The scientific study was then carried out as follows. The second chapter presents an evaluation of fungal taxa using cultivation, DNA and RNA based techniques. The findings indicated major taxa are revealed in all approaches, yet differences in less abundant taxa occur. The third chapter describes fungal assemblages in the soils of the McMurdo Dry Valleys, a cold polar desert. In this study, RNA based approaches tracked active fungal assemblages, whilst DNA and cultivation revealed additional taxa. Chapter four analyzed microbial communities in the Thar Desert, a hot monsoon desert in India. This study revealed a diverse community that comprised known desiccation-tolerant taxa but also a phylogenetically broad range of bacteria, archaea and eukarya. Chapter 5 focuses on the delineation of total versus active microorganisms in environmental samples from the hot deserts. As with the initial experiments, this revealed that total and active assemblages track each other broadly in desert soils. A synthesis of the study revealed that certain common microbial phyla are likely well-adapted to xeric stress, although distinct hot and cold desert assemblages also develop. For such low-diversity systems it is likely that DNA-based approaches are reasonable tools for diversity analysis, and will be especially useful in arid systems when long periods of inactivity may confound attempts to estimate active populations. Broader significance of the study includes an increased appreciation of eukaryotic microbial presence in arid soils, and how latent soil microbiota may act as a reservoir for development of future microbial macro-structures (e.g. soil crust) that function in soil stabilization, and should therefore be included in conservation planning for deserts. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

The two main objectives in this study were investigating the microbial quality and microbial communities of 'use-by date' Mung bean sprouts by using conventional culture and 16S/18S rDNA PCR-DGGE methods, and evaluating the efficacy of natural antimicrobial substances, chemical disinfectants, and thermal treatments in reducing and inhibiting the growth of the pathogens on mung bean seeds. Retail samples of pre-packed mung bean sprouts were obtained from three retailers in the local area. The microbial quality and communities were evaluated on the 'use-by date'. The highest counts of total aerobic counts (7.86 log10 CFU/g), yeasts and moulds (7.0 log10 CFU/g), total lactic acid bacteria (6.24 log10 CFU/g) and total coliforms (6.63 log10 CFU/g) were found in samples from one shop and the DGGE band sequences also identified major populations of LAB from the same samples, These indicated poor quality and spoilage of the samples from this location and could be related to improper storage at temperatures above 5°C. The combination of conventional culture methods with the PCR-DGGE technique revealed a larger diversity of bacterial communities than eukaryotic ones based on the relative number of amplimers present on most of the OGGE gels. Identification based on band analysis revealed that the Enterobacteriaceae (29.6%), soil bacteria (20.4%), lactic acid bacteria (18.5%). yeast (14.8%). Pseudomonas spp. (13%), and Flavobacterium (3.7%) constituted the major populations in bean sprout samples. Cluster analysis of the OGGE patterns of both 16S and 18S rDNA amplimers found no strong relationship between sample sources and batches indicating the variability of natural populations. The use of natural antimicrobial products, such as a mixture of lime juice and vinegar (1: 1, pH 2.83) and bacteriocin-like substances produced by Pediococcus acidilactici, failed to reduce and inhibit the growth of Listeria monocytogenes on mung bean seeds. The former solution had higher antimicrobial efficiency in reducing the pathogen on seeds (1.93 log10 CFU/g) compared to the Pediococcus broth culture (1.22 log10 CFU/g), but both solutions failed to inhibit the re-growth of the pathogen during the sprouting process and also reduced seed germination percentage by 13-18%. The evaluation of efficacy of sequential washing using a combination of chemical treatments (two-step dipping) against the pathogens on seeds showed that a two-step dipping treatment in a solution containing 2% sodium hypochlorite for to min followed by 5% lactic acid solution for 5 min was the most effective treatment. This treatment achieved the highest reductions of L. monocytogenes (2.91 log10 CFU/g) and Salmonella Typhimurium (3.20 l0g10 CFU/g) after treatment and continued to reduce the pathogen during the sprouting process. This may be due to the chemical residues on treated seeds which lowered both pathogens on sprouted seeds to below the limit of detection <50 CFU/g) by direct plating without significantly affecting seed viability. The use of thermal treatments based on a hot and cold water dipping was found to be more effective in reducing the normal flora on seeds and less affecting of seed germination compared to microwave heating. The use of a hot and cold water dipping treatment at 92°c for 1 min followed by ice-cold water at 5°C for 30 sec achieved the highest reduction of L. monocytogenes on seeds (>5 log10 CFU/g) but had the lowest germination percentage (89%) compared to other hot and cold water dipping treatments. Microwave heating at 1-4 kW showed a poorer efficiency in reducing nonnal flora on seeds and severely affected seed viability. Overall, a two-step washing with 2% sodium hypochlorite followed by 5% lactic acid seems to be the most successful treatment in reducing and inhibiting the recovery of the pathogen during the sprouting process. However, the chemical residues on treated seed may become a negative image to apply this treatment in the sprout industry.

Blue Stilton is a blue-veined cheese manufactured in a restricted area of the UK, using lactic starter cultures plus a secondary culture of Penicillium roquefotti. The aim of this study was to determine the change in microflora during ripening of the cheese and to investigate potential microbial interactions. Additionally, the volatile compounds present in mature samples of cheeses exhibiting few blue veins were compared with those in good quality cheeses, showing ample blue veining. Experiments on cheeses from a single dairy, monitored during the ripening process, showed that the total Lactobacillus count increased from levels of around 103 cfu g-1 on day one to around 107 cfu g-1 after 8 weeks of ripening. This is comparable to values found in other cheeses including both mould-ripened and non mould-ripened varieties. Yeast counts were generally higher than those found in other cheeses and also increased to levels in the region of 107 g-1. The total viable count (TVC) decreased from around 109 g-1 initially, reflecting the presence of the starter bacteria, to 107 g.1, suggesting a decline in the starter bacteria similar to that found in other cheeses. Mature cheeses always exhibited similar numbers of microorganisms although the species varied between cheeses. High quality, mature, cheeses were compared with sub-standard cheeses from the same production site. The predominant species of lactobacilli in good quality cheeses were Lb. plantarum and Lb. curvatus, whereas in poor quality cheeses Lb. brevis predominated. This corresponded to the results of gas chromatography-olfactometry, which indicated the presence of fruity off flavours in poor quality cheeses. Several strains of these species were isolated, as indicated by differing capabilities in utilisation of a range of carbon sources. Yeast species also varied between good and poor quality cheeses with Candida sphaerlca and C. catenulata predominating in good cheeses and C. famata, C. lipolytica and C. catenulata also occurring in both good and poor quality samples. Strain differences were observed by the biochemical profiles and two strains of C. famata demonstrated inhibitory effects against P. roqueforti when incubated under anaerobiosis. It was concluded that these strains may affect the development of blue veins in Stilton cheese when maturation conditions encourage their proliferation. Comparisons were made between samples of cheeses from several Stilton producers and the results suggested that although the levels of the groups of microorganisms tested were similar, the species of lactobacilli and yeasts present were different. This suggests that the indigenous microflora may have a significant impact on the flavour of cheeses from individual production sites. It was concluded that the microflora of Blue Stilton cheese may have a significant impact on the quality of the product both in terms of flavour and the development of the blue veins.

An understanding of the movement of bacteria in soil is of importance in many areas of microbial ecology. The study of bacterial dispersal is of fundamental importance in understanding the dissemination of soil-borne plant pathogens and symbionts as well as human pathogens introduced into soil. In addition, the increasing interest in the use of bacterial inocula for improved plant nutrition, biological control of plant pathogens and bioremediation of contaminated soils, necessitates the study of movement of such inocula, both to optimise their function and determine their fate in the environment. The fate of inocula is of particular interest when such inocula are non-indigenous (genetically-modified or otherwise), where their impact on the environment outwith the target site is uncertain. Intact soil microcosms were used in the study of factors affecting the movement of bacterial inocula through soils in the presence of percolating water. Two ecophysiologically contrasting bacterial species (<i>Pseudomonas fluorescens</i> and <i>Bacillus subtilis</i>) were used as inocula. The strains were genetically marked with <i>lux</i> genes encoding bioluminescence and with antibiotic resistance markers (chromosomal integrations), these traits were used in the selective enumeration of the introduced bacteria against the indigenous soil populations. The effect of soil type on the leaching of <i>P. fluorescens</i> inocula was investigated using intact soil microcosms sampled from contrasting soils: Craibstone (loamy sand), Insch (sandy loam) and Cruden Bay (clay loam). It was found that cells of the inoculum were leached more rapidly, in greater numbers and over a longer period from the clay loam, than the two lighter textured soils. These differences were attributed to the interaction of the inocula with percolating water, as determined by the respective soil characteristics. Leaching of <i>B. subtilis</i> differed from that of <i>P. fluorescens</i>, in that the rate of movement through soil was slower and the number of colony forming units leached was less. It was found that the colony forming units of <i>B. subtilis</i> leached were predominantly in the form of spores. The differences in leaching of <i>B. subtilis</i> and <i>P. fluorescens</i> were attributed to the greater adsorption of vegetative cells of <i>B. subtilis</i> to the soils and the requirement for formation of spores prior to leaching.

This thesis describes research investigating the side-effects of pesticides on soil microbial biomass and microbial activity, with particular reference to two recently developed pesticides, a fungicide, epoxiconazole, and a herbicide, quinmerac. In a dose-responsee xperiment,a pplication of thesep esticidest o a sandy loam soil, at up to 10 and 20 times field rate, had no significant effect on soil microbial biomass C or ninhydrin-reactive N, over 84 days incubation. There was also no effect on soil respiration, except for the higher rate quinmerac-treated soil, which evolved 13% lessC02-Cthan the control. The rate of mineralisation of epoxiconazole and quinmerac, and their long-term effect on soil respiration, were measured in three contrasting soils: a sandy loam, a silty clay loam, and a clay soil, using 14C -labelled active ingredients. The kinetics of the pesticides' mineralisation were quite different, epoxiconazole being hyperbolic, while quinmerac was sigmoidal. The maximum amount of mineralisation of both pesticides occurred in the silty clay loam soil, which had the lowest microbial biomass content. The mineralisation of the pesticides was increased by the addition of ryegrass, with the greatest effect in the silty clay loam soil, probably because of the large ryegrass C: biomass C ratio. The mineralisation of epoxiconazole was affected by the ryegrass amendment much more than quinmerac. Further additions of the pesticides had no significant effect on soil respiration or pesticide mineralisation. The mineralisation of epoxiconazole and quimnerac was further investigated in the silty clay loam soil, using samples with different crop management histories, and the effects of ryegrass and glucose amendment. Pesticide mineralisation was shown to be related to the amount of soil microbial biomass, indicating that the difference in mineralisation rate between the three soil types above was not due to differences in their crop management, but innate differences in soil chemistry and microbiology. Ryegrass addition stimulated the mineralisation of epoxiconazole more than quinmerac, while the reverse was true for glucose, indicating that the pesticides were being degraded by two distinct fractions of the microbial biomass. The effects of long-term cumulative field application of the pesticides benomyl, chlorfenvinphos, aldicarb, triadimefon and glyphosate, on soil microbial biomass and mineralisation of soil organic matter were investigated. The addition of aldicarb consistently increased the microbial biomass, due to its beneficial effect on crop growth, but this effect was not reflected in the rate of organic matter mineralisation. However, in general, the continued application of these pesticides for up to 19 years, at slightly higher than the recommended rates, had very little effect on the soil microbial population. The effects of epoxiconazole and triadimefon on soil ergosterol content and microbial biomass C were compared in a sandy loam soil. Both pesticides temporarily reduced soil ergosterol by about 30%, while biomass C remained largely unaffected. However, when straw was added to the soils, the inhibition of ergosterol was still evident, as was an inhibitory effect on biomass C. The measurement of soil ergosterol was more sensitive to the pesticide effects than biomass C, and could be a useful test in determining changes in fungal populations.

Viruses have been shown to be responsible for considerable bacterial mortality and nutrient cycling in aquatic systems. As yet no detailed studies have been published on the role of viruses in natural soil bacterial communities despite common knowledge that viruses exist in the soil. This thesis sought to address some key questions on the ecology of soil bacterial viruses and their hosts. Disturbance through soil desiccation, nutrient inputs, rhizosphere effects and protozoan predation pressure were investigated. The first study of lysogeny in natural soil systems was also undertaken. The work presented here utilised tools and techniques commonplace in aquatic systems research and applied them to soil. A novel protocol was developed based on physical extraction of bacteria and viruses from soil and direct counting with epifluorescence microscopy. Physical extraction was achieved using shaking, ultrasound sonication and low speed centrifugation. The fluorochrome SYBR Gold was used to stain nucleic acid of extracted bacteria and viruses, and image analysis software used to determine bacterial cell volumes. Bacterial and viral abundances were in the region of 107-109 per gram of soil over a range of soil types. Significant fluctuations in viral and bacterial abundances were recorded at timescales of less than 24 h. Glucose and nitrogen addition led to substantial increases in bacterial and viral abundance. Loss of soil moisture resulted in peaks of viral abundance in sandy soils but not in a clay soil. A six-week microcosm study demonstrated that phage were not a significant regulator of bacterial abundance. Low levels of lysogeny were recorded over a range of soils when measured explicitly with Mitomycin C. The implication from that study was that viruses in soil behave differently to those in aquatic systems. Bacterial and viral abundances were highly coupled in most instances, irrespective of the potential activity of bacteria. Further fundamental studies are recommended.

No description available.

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1991. / Vita. Abstract. Includes bibliographical references. Also available via the Internet.