Wheat is the most economically important crop forming one quarter of Australian farm production. The wheat industry is severely affected by diseases, with fungal pathogens causing the most important economic losses in Australia. The application of fungicides and chemicals can control crop diseases to a certain extent, however, it is expensive and public concern for the environment has led to alternative methods of disease control to be sought, including the use of microorganisms as biological control agents. Microorganisms are abundant in the soil adjacent to plant roots (rhizosphere) and within healthy plant tissue (endophytic) and a proportion possess plant growth promotion and disease resistance properties. Actinobacteria are gram-positive, filamentous bacteria capable of secondary metabolite production such as antibiotics and antifungal compounds. A number of the biologically active endophytes belonging to the Actinobacteria phylum were isolated in our laboratory. A number of these isolates were capable of suppressing the wheat fungal pathogens Rhizoctonia solani, Pythium sp. and Gaeumannomyces graminis var. tritici, both in vitro and in planta indicating the potential for the actinobacteria to be used as biocontrol agents. The aim of this research was to investigate the molecular mechanisms underlying this plant-microbe interaction. The indigenous microbial populations present in the rhizosphere and endophytic environment are critical to plant health and disruptions of these populations are detrimental. The culture-independent technique Terminal Restriction Fragment Length Polymorphism (T-RFLP) was used to characterise the endophytic actinobacteria population of wheat roots under different conditions. Soils which support a higher number of indigenous microorganisms result in wheat roots with higher endophytic actinobacterial diversity and level of colonisation. Sequencing of 16S rRNA gene clones, obtained using the same actinobacteria-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity, and identified a number of Mycobacterium and Streptomyces species. It was found that the endophytic actinobacterial population of the wheat plants contained a higher diversity of endophytic actinobacteria than reported previously, and that this diversity varied significantly among different field soils. The endophytic actinobacteria have previously been shown to protect wheat from disease and enhance growth when coated onto the seed before sowing. As the endophytes isolated were recognised as potential biocontrol agents, the impact on the indigenous endophytic microbial population was investigated. Utilising the T-RFLP technique it was established that the use of a commercial microbial inoculant, containing a large number of soil bacterial and fungal strains applied to the soil, disrupts the indigenous endophyte population present in the wheat roots. The hypothesis is that non-indigenous microbes proliferate and dominate in the soil preventing a number of endophytic-competent actinobacterial genera from access to the seed and ultimately endophytic colonisation of the wheat roots. This dramatically reduces diversity of endophytes and level of colonisation. In contrast the use of a single endophytic actinobacteria endophyte inoculant results in a 3-fold increase in colonisation by the added inoculant, but does not significantly affect this indigenous population. Colonisation of healthy plant tissues with fungal endophytes has been shown to improve the competitive fitness with enhanced tolerance to abiotic and biotic stress and improved resistance to pathogens and herbivores. In this study the fungal endophyte population of wheat plants grown in four different soils was analysed using partial sequencing of 18S rRNA gene sequences. Sequence anlaysis of clones revealed a diverse range of fungal endophytes. In this diverse range of fungal endophytes a number sequences were highly similar to those of previously known fungal phytopathogens. A number of sequences detected were similar to fungal species previously identified in soil or plant material but not as endophytes. The remaining sequences were similar to fungal species without a known relationship with plants. Plants have developed an inducible mechanism of defence against pathogens. In addition to local responses plants have developed a mechanism to protect uninfected tissue through a signal that spreads systemically inducing changes in gene expression. In the model plant Arabidopsis thaliana activation of the Systemic Acquired Resistance (SAR) pathway and the Jasmonate (JA)/Ethylene (ET) pathway is characterised by the production of pathogenesis-related (PR) and antimicrobial proteins resulting in systemic pathogen resistance. Endophytic actinobacteria, isolated from healthy wheat roots in our laboratory, have been shown to enhance disease resistance to multiple pathogens in wheat when coated onto the seed before sowing. Real Time RT-PCR was used to determine if key genes in the SAR and JA/ET pathways were induced in response to inoculation with endophytic actinobacteria. Inoculation of wild-type Arabidopsis thaliana with selected strains of endophytic actinobacteria was able to �prime� the defence pathways by inducing low level expression of SAR and JA/ET genes. Upon pathogen infection the defence-genes are strongly up-regulated and the endophyte coated plants had significantly higher expression of these genes compared to un-inoculated plants. Resistance to the bacterial pathogen Erwinia carotovora subsp. carotovora was mediated by the JA/ET pathway whereas the fungal pathogen Fusarium oxysporum triggered primarily the SAR pathway. Further analysis of the endophytic actinobacteria-mediated resistance was performed using the Streptomyces sp. EN27 and Arabidopsis defence-compromised mutants. It was found that resistance to E. carotovora subsp. carotovora mediated by Streptomyces sp. EN27 occurred via a NPR1-independent pathway and required salicylic acid whereas the jasmonic acid and ethylene signalling molecules were not essential. In contrast resistance to F. oxysporum mediated by Streptomyces sp. EN27 occurred via a NPR1-dependent pathway but also required salicylic acid and was JA- and ET-independent. This research demonstrated that inoculating wheat with endophytic actinobacteria does not disrupt the indigenous endophytic population and may be inducing systemic resistance by activating defence pathways which lead to the expression of antimicrobial genes and resistance to a broad range of pathogens.

<p>Wood inhabiting fungi oposes a great problem for preservation of wooden surfaces everywhere, being the main problem of economic losses of wooden products.</p><p>A reference collection consisting of 9 different genus constituting of 21 different strains of wood-inhabiting fungi was used for identification of unknown species of mould and blue stain fungi on wood. The fungus DNA from the samples was isolated from malt extract agar. PCR (Polymerase Chain Reaction) was conducted on rDNA ITS1 and ITS2 regions for amplification of the DNA. The 21 samples were collected to a reference collection for identification of unknown species of fungi on wooden field samples using PCR and T-RFLP (Terminal Restriction Fragment Length Polymorphism).</p><p>PCR-based methods, sequencing and T-RFLP were proven to be simple and</p><p>accurate methods for detection and identification of fungi in their early stage.</p>

Wood inhabiting fungi oposes a great problem for preservation of wooden surfaces everywhere, being the main problem of economic losses of wooden products. A reference collection consisting of 9 different genus constituting of 21 different strains of wood-inhabiting fungi was used for identification of unknown species of mould and blue stain fungi on wood. The fungus DNA from the samples was isolated from malt extract agar. PCR (Polymerase Chain Reaction) was conducted on rDNA ITS1 and ITS2 regions for amplification of the DNA. The 21 samples were collected to a reference collection for identification of unknown species of fungi on wooden field samples using PCR and T-RFLP (Terminal Restriction Fragment Length Polymorphism). PCR-based methods, sequencing and T-RFLP were proven to be simple and accurate methods for detection and identification of fungi in their early stage.

Soil fungi play an important role in regulating plant communities as well as above and below ground ecosystem-level processes; conversely, plant communities may also affect the structure and functionality of these root-associating fungi. Alteration of these fungal communities due to non-native plant invasion has the potential to disrupt biogeochemical cycling, soil structure, and plant growth. Both beneficial symbionts such as arbuscular mycorrhizal fungi (AMF) as well as the total fungal community are potentially altered by aggressive invasive plant species in such a way as to disrupt existing native endophytic fungal communities in the soil post invasion. This disruption could provide a pathway for invasion and suggests the importance of investigating plant-fungal associations in invaded ranges. I used molecular techniques to characterize the fungal communities colonizing Vincetoxicum rossicum or Dog-strangling vine (DSV) and Alliaria petiolata or garlic mustard, both European natives that are currently well established in Eastern North America, as well as native plants that are commonly found persisting in the presence of dense colonies of DSV, as well as those same natives growing separately from DSV. Fungi colonizing different plant groups were analyzed using primers that target the internal transcribed spacer region of the ribosomal operon in order to amplify total fungal species (TF), as well as primers designed to exclusively amplify AMF using small subunit rRNA sequences. Significant differences were observed in the diversity of both the TF and the AMF communities colonizing native plants in the invaded sites relative to the uninvaded sites. Sequencing work indicated that DSV forms associations with a broad array of fungal partners relative to proximal native plants, suggesting the likelihood of it being a fungal generalist. As well, DSV was found to associate with described opportunistic AMF such as Glomus intraradices, G. caledonium, G. fasciculatum and G. mosseae, while natives growing within DSV patches were not. Finally, garlic mustard was found to have the dominant effect where DSV and garlic mustard were co-occurring. These findings support the ongoing investigations into plant invasion processes, and therefore contribute to the development of effective strategies for invasive species management as well as site restoration techniques.

Soil fungi play an important role in regulating plant communities as well as above and below ground ecosystem-level processes; conversely, plant communities may also affect the structure and functionality of these root-associating fungi. Alteration of these fungal communities due to non-native plant invasion has the potential to disrupt biogeochemical cycling, soil structure, and plant growth. Both beneficial symbionts such as arbuscular mycorrhizal fungi (AMF) as well as the total fungal community are potentially altered by aggressive invasive plant species in such a way as to disrupt existing native endophytic fungal communities in the soil post invasion. This disruption could provide a pathway for invasion and suggests the importance of investigating plant-fungal associations in invaded ranges. I used molecular techniques to characterize the fungal communities colonizing Vincetoxicum rossicum or Dog-strangling vine (DSV) and Alliaria petiolata or garlic mustard, both European natives that are currently well established in Eastern North America, as well as native plants that are commonly found persisting in the presence of dense colonies of DSV, as well as those same natives growing separately from DSV. Fungi colonizing different plant groups were analyzed using primers that target the internal transcribed spacer region of the ribosomal operon in order to amplify total fungal species (TF), as well as primers designed to exclusively amplify AMF using small subunit rRNA sequences. Significant differences were observed in the diversity of both the TF and the AMF communities colonizing native plants in the invaded sites relative to the uninvaded sites. Sequencing work indicated that DSV forms associations with a broad array of fungal partners relative to proximal native plants, suggesting the likelihood of it being a fungal generalist. As well, DSV was found to associate with described opportunistic AMF such as Glomus intraradices, G. caledonium, G. fasciculatum and G. mosseae, while natives growing within DSV patches were not. Finally, garlic mustard was found to have the dominant effect where DSV and garlic mustard were co-occurring. These findings support the ongoing investigations into plant invasion processes, and therefore contribute to the development of effective strategies for invasive species management as well as site restoration techniques.

Submitted by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-01-23T14:01:54Z No. of bitstreams: 5 22.pdf: 6729647 bytes, checksum: f44366fe5c167abbf5a70d5f3a327722 (MD5) license_url: 52 bytes, checksum: 3d480ae6c91e310daba2020f8787d6f9 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) license.txt: 2109 bytes, checksum: aa477231e840f304454a16eb85a9235f (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-02-10T12:46:43Z (GMT) No. of bitstreams: 5 22.pdf: 6729647 bytes, checksum: f44366fe5c167abbf5a70d5f3a327722 (MD5) license_url: 52 bytes, checksum: 3d480ae6c91e310daba2020f8787d6f9 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) license.txt: 2109 bytes, checksum: aa477231e840f304454a16eb85a9235f (MD5) / Made available in DSpace on 2015-02-10T12:46:43Z (GMT). No. of bitstreams: 5 22.pdf: 6729647 bytes, checksum: f44366fe5c167abbf5a70d5f3a327722 (MD5) license_url: 52 bytes, checksum: 3d480ae6c91e310daba2020f8787d6f9 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) license.txt: 2109 bytes, checksum: aa477231e840f304454a16eb85a9235f (MD5) Previous issue date: 2013 / O crescimento da popula??o mundial ocasiona alta na demanda por alimentos. O processo produtivo gera importante montante de res?duos que funcionam como fontes poluidoras de ?gua, solo e ar. Anualmente s?o usados no mundo aproximadamente 2,5 milh?es de toneladas de agrot?xicos. Dentre estes se encontra o clomazone, do grupo das isoxazolidinonas, um inibidor da s?ntese de caroten?ides e altamente lixivi?vel. De acordo com a problem?tica apresentada objetivou-se com este trabalho verificar a toler?ncia, por meio de avalia??es de crescimento, intoxica??o, an?lises anat?micas e de diversidade microbiana do solo, assim como a capacidade remediadora de doze esp?cies florestais nativas. Para montagem do experimento foi utilizado o delineamento em blocos ao acaso com quatro repeti??es. Foram feitas 3 aplica??es do herbicida clomazone com intervalos de 20 dias (aos 60, 80 e 100 dias ap?s o plantio), cada aplica??o foi correspondente ? metade da dose comercial de 2 L ha-1. Para as avalia??es de crescimento foram mensuradas a altura da planta, o di?metro do caule, o n?mero de folhas, a ?rea foliar e o ac?mulo de biomassa seca. Para as verifica??es anat?micas foram coletadas 2 folhas de cada planta sempre aos 7 dias ap?s a aplica??o do herbicida, nas duas primeiras aplica??es. Por meio de avalia??es micromorfom?tricas foram medidas na sec??o transversal das folhas das esp?cies florestais, a espessura e a ?rea ocupada pelos tecidos: epiderme adaxial e abaxial, par?nquima pali??dico e par?nquima lacunoso. Para estimativa de intoxica??o pelas plantas testadas, avaliou-se o efeito do produto por meio de notas de toxicidade. Para verifica??o de capacidade fitorremediadora das esp?cies arb?reas procedeu-se a semeadura da esp?cie indicadora sorgo (Sorghum bicolor (L.) Moench.) para indicativo do res?duo do herbicida no solo. Posteriormente em amostras de solo provenientes do experimento procedeu-se a an?lise de T-RFLP com intuito de caracterizar a diversidade microbiana presente. As esp?cies florestais sobreviveram ? aplica??o de clomazone, sendo que I. marginata, C. ferrea e S. brasiliensis apresentaram maior toler?ncia ao herbicida em rela??o ?s an?lise de crescimento, seguidas de S. parahyba, H. serratifolius repetindo-se I. marginata para avalia??es da integridade anat?mica. No entanto, n?o foi verificado, nas condi??es do experimento, remedia??o do solo para a maioria das esp?cies testadas. Contudo plantas de sorgo tiveram crescimento normal quando cultivadas em solo onde antes havia I. marginata. Os resultados de T-RFLP confirmaram a diversidade microbiana diferenciada associada a rizosfera de I. marginata. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Ci?ncia Florestal, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2012.

When paper is manufactured, deposits often form in the machines. These deposits are slimelike and can interfere with the papermaking process. The slimelike deposits are aggregates of micro-organisms, also known as biofilm. One single type of micro-organism can form a biofilm, but most biofilms consists of a mixture of several different kinds of micro-organisms and can form on about any conceivable surface. To control the aggregates of micro-organisms a slimecide is added, a so-called biocide. To examine what kind of bacteria that is included in the biofilm and also which bacteria that is killed or not killed by the biocide, Terminal Restriction Fragment Length Polymorphism analysis (T-RFLP) can be used.   In this report we examine biocides impact on biofilm produced in the laboratory.The biocides were first tested for possible interference with the PCR-step of the T-RFLP analysis. None of the tested ten biocides inhibited the PCR process the biofilm was formed on metal plates when these were lowered in a beaker with white water. Three different beakers were set up, one with addition of a biocide with active component 4,5-DICHLORO-1,2-DITHIOLONE from the start, one with the addition of the same biocide after three days and one with no addition at all of biocide. Samples were taken from the beakers and analyzed with T-RFLP.   In this report, we show that biocides affect planktonic and biofilm micro-organisms differently. There are however some micro-organisms in the biofilm that does not get affected by the biocide.   The experimental in this report is a good way of investigate the influence that biocides have on planktonic and biofilm micro-organisms, but to get even greater result the experiment should be done over a longer period of time and repeatedly.

>Magister Scientiae - MSc / The extreme conditions of Antarctic desert soils render this environment selective towards a diverse range of psychrotrophic microbial communities. Cracks and fissures in translucent quartz rocks permit an adequate amount of penetrating light, sufficient water and nutrients to support cryptic microbial development. Hypolithons colonizing the ventral surface of these quartz rocks have been classified into three types: cyanobacterial dominated (Type I),moss dominated (Type II) and lichenized (Type III) communities. Eukaryotic microbial communities were reported to represent only a minor fraction of Antarctic communities. In this study, culture independent techniques (DGGE, T-RFLP and clone library construction) were employed to determine the profile of the dominant eukaryotes, fungi and microalgae present in the three different hypolithic communities. The 18S rRNA gene (Euk for eukaryotes), internal transcribed spacer (ITS for fungi) and microalgal specific regions of the 18S rRNA gene, were the phylogenetic markers targeted for PCR amplification from hypolith metagenomic DNA. Results suggest that the three hypolith types are characterized by different eukaryotic, fungal and microalgal communities, as implied by nMDS analysis of the DGGE and T-RFLP profiles. Sequence analysis indicates close affiliation to members of Amoebozoa, Alveolata, Rhizaria (general eukaryote), Ascomycota (fungal) and Streptophyta (microalgal). Many of these clones may represent novel species. This study demonstrates that Dry Valley hypolithons harbour higher eukaryote diversity than previously recognised.Each hypolithon is colonized by specialized microbial communities with possible keystone species. The ecological role of the detected microorganisms in the hypolith environment is also theorized, and a trophic hierarchy postulated.

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