This work describes progress toward developing a model system to investigate <i>in plantae</i> suppression of human pathogens by <i>Stemphylium</i>-like fungal endophyte strains.

Pinus radiata is the most planted tree in the southern hemisphere. The planted trees are especially susceptible to pathogens, but even the native population, nowadays limited tomerely five locations, are threatened by diseases caused by arthropods, fungi and dehydration. Endophytes are bacteria or fungi that reside inside healthy plant tissue, and often have a beneficial effect on their hosts. Endophytes can help plants adapt to abiotic stress such as drought and protect them against pathogens and insect pests. Given the roles that endophytes play in host stress responses, it is possible that without studying endophytes we may not fully understand a plant’s response to increased temperatures and climate-induced disease.Using Illumina-sequencing of the 16S rRNA-gene the bacterial endophyte community in 15 trees from three of the remaining native populations were studied. By investigating trees from several sites geographical community differences were discovered. The three overall most dominating bacterial taxa can all be connected with genera known to contain members withanti-fungal properties.

>Magister Scientiae - MSc / Salinity is one of the major limiting factors to crop production, which consequently contributes to the risk of reduced food security. Among other factors, food security depends on availability of sufficient and nutritious food for humans. Livestock such as cattle and sheep are fed with various plant-based feeds; with Medicago sativa (commonly known as alfalfa or lucerne) being a very important forage/feed crop, so much that it is regarded as the queen of forage crops. However, alfalfa is severely affected by high soil salinity and thus its growth and yield are drastically reduced in soils with high NaCl content. Among the various alfalfa genotypes/varieties examined in this study, Agsalfa was identified as salt tolerant because it performed better under salt treatment compared to Magna601.

Bacterial endophytes, bacteria residing within plants, play an important role in the growth and development of plants and their ability to thrive under adverse conditions. The endophytes of Acer negundo, Ulmus pumila and Ulmus parvifolia trees sampled from a hydrocarbon-contaminated site were analyzed for variation between seasons and plant species. Branches from the same trees over a span of 3 seasons were collected and analyzed via culture dependent and culture independent methods. Numerous culture independent approaches were tested, culminating in the development of a new method for the amplification of endophytic bacterial ribosomal DNA that excludes plastid DNA. Community analyses using this new method in combination with T-RFLP showed significant differences between the endophytic communities of different plants species and of the same species growing in different seasons. The proposed technique can be used for the future study of endophytic communities of plants.

Bacterial endophytes, bacteria residing within plants, play an important role in the growth and development of plants and their ability to thrive under adverse conditions. The endophytes of Acer negundo, Ulmus pumila and Ulmus parvifolia trees sampled from a hydrocarbon-contaminated site were analyzed for variation between seasons and plant species. Branches from the same trees over a span of 3 seasons were collected and analyzed via culture dependent and culture independent methods. Numerous culture independent approaches were tested, culminating in the development of a new method for the amplification of endophytic bacterial ribosomal DNA that excludes plastid DNA. Community analyses using this new method in combination with T-RFLP showed significant differences between the endophytic communities of different plants species and of the same species growing in different seasons. The proposed technique can be used for the future study of endophytic communities of plants.

Masters of Science / Bacterial endophytes are able to improve the growth of their hosts through a number of different mechanisms such as nutrient uptake regulation, plant hormone production and regulation, siderophore production and phosphate solubilisation. They have also been shown to be able to provide protection to plants against various abiotic stressors, through various means such as oxidative stress protection. The purpose of this study was therefore to isolate endophytic bacteria from the stems of different weeds, to characterize their ability to use some of the most important growth promoting mechanisms including the ability to produce IAA, siderophores and ACC deaminase, what effect they had on the nutrient uptake in their hosts and to determine to what extent they could promote growth in the roots, stems and leaves of Brassica napus plants. In addition to this the endophytes were tested to see to what extent they could protect Brassica napus from the negative effects of vanadium stress and how this affected the plant physiologically in terms of morphology, overall biomass, the plants nutrient profile, lipid peroxidation and levels of cell death. The effect of vanadium stress on the oxidative state of Brassica napus was also monitored by determining the levels of stress induced reactive oxygen species (ROS) and the corresponding antioxidants that are responsible for regulating these reactive oxygen species. Six different endophytes (P1, P2, P3, P4, P5, P6) were isolated from different weed samples. Each endophyte was found to be able to significantly improve germination and growth in their host plant. Each isolate was able to improve the uptake of certain macronutrients and micronutrients in their respective hosts, while all of the isolates were shown to be capable of producing siderophores and ACC deaminase. One isolate had high levels of IAA production, with the remaining isolates producing small amounts of IAA. All isolates were also unable to solubilize phosphate. The five best performing endophytes (P1, P2, P3, P5, P6) in the preliminary growth trials were used in the follow up vanadium stressed growth trials, with endophyte P4 being left out of the remaining experiments. All of the endophytes showed improvements in growth promotion in comparison to the control, with endophyte treated plants showing both increased growth and biomass in both the non-stressed and vanadium stressed treatments of the vanadium stressed growth trial; however, the leaves of the vanadium stressed plants were significantly smaller than their non-stressed counterparts. When looking at the oxidative state it was found that vanadium stress caused a significant increase in the development of O2 -, H2O2 and •OH in the control and in addition to this it was shown that treatment with endophytes was able to cause a significant decrease in the levels of stress induced H2O2 and •OH in all of the treatments and O2 - for plants treated with endophyte P5. The noted change in the oxidative state of endophyte treated plants was attributed to an increase in the antioxidant activity of these plants, as it was found that endophyte treated plants showed a combination of increased activity for Superoxide dismutase, catalase and ascorbate peroxidase. This study has shown that endophytic bacteria from plant stems can be used to improve crop growth and yield, while simultaneously producing more nutrient dense crops from the same amount of land. It has also determined that endophytes P1, P2, P3, P5 and P6 are able to successfully provide protection to crop plants from the harmful effects of exposure to vanadium stress. This has great potential for improving food security locally and around the world, by allowing those who cannot gain access to large amounts of food to take in more nutrients from the same amount of food. Furthermore, it also presents the opportunity to use endophyte treatments to grow crops on land that has been previously contaminated with certain heavy metals. / 2023-12-01

Nos últimos anos, diversos protocolos e tecnologias têm sido propostos a fim de viabilizar ou otimizar a micropropagação de diversas culturas, bem como reduzir custos de produção. Dentre eles, tem ganhado destaque, o uso do meio de cultura líquido e do sistema de biorreator de imersão temporária (BIT). No entanto, observam-se diferenças de resposta entre espécies e metodologias, sendo necessários maiores estudos para um melhor conhecimento dos fatores que afetam os sistemas de micropropagação. Estudos recentes, baseados em técnicas moleculares, têm revelado que as culturas in vitro não são axênicas, como se acreditava, apresentando comunidades endofíticas onipresentes. Sabendo-se da importância desses microrganismos em plantas a campo, passou-se a questionar o papel destes no desenvolvimento e multiplicação de plantas in vitro. Diante deste cenário, este trabalho se propôs a comparar o desempenho de culturas in vitro em diferentes condições de cultivo: meio semissólido, meios líquido estático, sob agitação e, avaliando-se o crescimento/ multiplicação das plântulas e, naqueles onde houve diferença no desempenho, foram realizadas análises moleculares para a caracterização da comunidade microbiana presente na parte aérea das plantas. Foram utilizadas culturas de bromeliáceas e cana-de-açúcar, buscando sistemas que permitissem as avaliações pretendidas. Para isto foram instalados experimentos com Ananas comosus var. comosus (\'Imperial\' e \'Pérola\') e Aechmea nudicaulis, sob cultivo em meio líquido estático e sob agitação; e com Vriesea hieroglyphica E. Morren, sob cultivo em meio líquido estático, sob agitação e em BIT. Para a gramínea, cana-de-acúcar (Saccharum spp., variedade SP80-3280), foram avaliados o cultivo em meio líquido estático e em BIT. Foram também realizadas análises moleculares de plântulas de Dyckia distachya, que haviam sido cultivadas em meios de culturas semissólido, líquido sob agitação e estático. As culturas que apresentaram diferenças de desempenho entre os sistemas avaliados foram D. distachya, (sendo o melhor tratamento o meio líquido sob agitação) e cana-de-açúcar (melhor tratamento foi BIT) e estas foram consideradas como sistemas adequados para o estudo de como diferentes sistemas de cultivo in vitro podem influenciar na comunidade bacteriana das plantas. A caracterização da comunidade bacteriana de D. distachya foi realizada por T-RFLP (Terminal Restriction Fragment Length Polymorphism) e mostrou que o tratamento meio líquido sob agitação, o qual teve a maior produção de brotos em relação aos demais, diferiu quanto à abundância relativa das unidades taxonômicas operacionais (UTOs) encontradas. Para cana-de-açúcar foram realizadas a construção de bibliotecas de clones do gene 16S rRNA e PCR quantitativo em tempo real (qPCR). Estas análises mostraram não haver diferença significativa entre as bibliotecas dos tratamentos avaliados, no entanto, BIT apresentou 3,54 vezes mais cópias do gene 16S rRNA em relação ao tratamento meio líquido estático, nos permitindo inferir que também possui uma maior número de bactérias. Este estudo apresenta fortes indícios de que o sistema de cultivo in vitro utilizado influencia a comunidade microbiana presente nas plantas / In recent years, several protocols and technologies have been proposed for feasibility and optimization of micropropagation of different cultures as well as to reduce production costs. Among these, the use of liquid culture medium and the temporary immersion bioreactor system (TIB) have gained special attention. However, differences are observed among species and methodologies, being necessary more detailed studies for a better knowledge of the factors that affect the micropropagatiion systems. Recent studies, based on molecular techniques, have revealed that in vitro cultures are not axenic, as thought, presenting ubiquitous endophytic community. Knowing the importance of these microorganisms to field plants we would like to know more about their role in in vitro plants. In this scenario, this work proposes to compare the performance of in vitro cultures under different culture conditions: semisolid medium culture, liquid static and liquid medium under agitation, and where differences in in vitro performance were observed comparative molecular analysis of microbial community in the plantlets was performed. Bromeliads and sugarcane cultures were used seeking for model systems for these analyses. These experiments were conducted with Ananas comosus var. comosus (\'Imperial\' and \'Pérola\') and Aechmea nudicaulis cultured under liquid static medium and liquid under agitation, and with Vriesea hieroglyphica, we compared liquid static medium, liquid medium under agitation and TIB. For sugarcane (Saccharum spp. variety SP80-3280), liquid static medium and TIB was compared. Molecular analyses of Dyckia distachya plantlets, which had been grown in semisolid medium liquid static and liquid medium under agitation, were also carried out. Cultures that showed differences in performance among the systems evaluated were D. distachya, (with liquid medium under agitation as the best condition) and sugarcane (best treatment was BIT) and these were considered adequate to study the differences in the bacterial comunity of plants when grown in different in vitro conditions. The characterization of the microbial community of D. distachya was performed by T-RFLP (Terminal Restriction Fragment Length Polymorphism) and showed that the liquid medium under agitation, which had the highest number of shoots compare to the other culture conditions, also differed as to the relative abundance of Operational Taxonomic Units (OTUs). For sugarcane 16S rRNA gene clone libraries, as well as real-time PCR (qPCR) were performed. These analyses showed no significant differences between the libraries of the two treatments, however, BIT showed 3.54 times more copies of the 16S rRNA gene compared to cultures from static liquid medium, allowing us to infer a higher number of bacteria. This study provides strong evidence that the in vitro system used influences the microbial community present in plants

Nos últimos anos, diversos protocolos e tecnologias têm sido propostos a fim de viabilizar ou otimizar a micropropagação de diversas culturas, bem como reduzir custos de produção. Dentre eles, tem ganhado destaque, o uso do meio de cultura líquido e do sistema de biorreator de imersão temporária (BIT). No entanto, observam-se diferenças de resposta entre espécies e metodologias, sendo necessários maiores estudos para um melhor conhecimento dos fatores que afetam os sistemas de micropropagação. Estudos recentes, baseados em técnicas moleculares, têm revelado que as culturas in vitro não são axênicas, como se acreditava, apresentando comunidades endofíticas onipresentes. Sabendo-se da importância desses microrganismos em plantas a campo, passou-se a questionar o papel destes no desenvolvimento e multiplicação de plantas in vitro. Diante deste cenário, este trabalho se propôs a comparar o desempenho de culturas in vitro em diferentes condições de cultivo: meio semissólido, meios líquido estático, sob agitação e, avaliando-se o crescimento/ multiplicação das plântulas e, naqueles onde houve diferença no desempenho, foram realizadas análises moleculares para a caracterização da comunidade microbiana presente na parte aérea das plantas. Foram utilizadas culturas de bromeliáceas e cana-de-açúcar, buscando sistemas que permitissem as avaliações pretendidas. Para isto foram instalados experimentos com Ananas comosus var. comosus (\'Imperial\' e \'Pérola\') e Aechmea nudicaulis, sob cultivo em meio líquido estático e sob agitação; e com Vriesea hieroglyphica E. Morren, sob cultivo em meio líquido estático, sob agitação e em BIT. Para a gramínea, cana-de-acúcar (Saccharum spp., variedade SP80-3280), foram avaliados o cultivo em meio líquido estático e em BIT. Foram também realizadas análises moleculares de plântulas de Dyckia distachya, que haviam sido cultivadas em meios de culturas semissólido, líquido sob agitação e estático. As culturas que apresentaram diferenças de desempenho entre os sistemas avaliados foram D. distachya, (sendo o melhor tratamento o meio líquido sob agitação) e cana-de-açúcar (melhor tratamento foi BIT) e estas foram consideradas como sistemas adequados para o estudo de como diferentes sistemas de cultivo in vitro podem influenciar na comunidade bacteriana das plantas. A caracterização da comunidade bacteriana de D. distachya foi realizada por T-RFLP (Terminal Restriction Fragment Length Polymorphism) e mostrou que o tratamento meio líquido sob agitação, o qual teve a maior produção de brotos em relação aos demais, diferiu quanto à abundância relativa das unidades taxonômicas operacionais (UTOs) encontradas. Para cana-de-açúcar foram realizadas a construção de bibliotecas de clones do gene 16S rRNA e PCR quantitativo em tempo real (qPCR). Estas análises mostraram não haver diferença significativa entre as bibliotecas dos tratamentos avaliados, no entanto, BIT apresentou 3,54 vezes mais cópias do gene 16S rRNA em relação ao tratamento meio líquido estático, nos permitindo inferir que também possui uma maior número de bactérias. Este estudo apresenta fortes indícios de que o sistema de cultivo in vitro utilizado influencia a comunidade microbiana presente nas plantas / In recent years, several protocols and technologies have been proposed for feasibility and optimization of micropropagation of different cultures as well as to reduce production costs. Among these, the use of liquid culture medium and the temporary immersion bioreactor system (TIB) have gained special attention. However, differences are observed among species and methodologies, being necessary more detailed studies for a better knowledge of the factors that affect the micropropagatiion systems. Recent studies, based on molecular techniques, have revealed that in vitro cultures are not axenic, as thought, presenting ubiquitous endophytic community. Knowing the importance of these microorganisms to field plants we would like to know more about their role in in vitro plants. In this scenario, this work proposes to compare the performance of in vitro cultures under different culture conditions: semisolid medium culture, liquid static and liquid medium under agitation, and where differences in in vitro performance were observed comparative molecular analysis of microbial community in the plantlets was performed. Bromeliads and sugarcane cultures were used seeking for model systems for these analyses. These experiments were conducted with Ananas comosus var. comosus (\'Imperial\' and \'Pérola\') and Aechmea nudicaulis cultured under liquid static medium and liquid under agitation, and with Vriesea hieroglyphica, we compared liquid static medium, liquid medium under agitation and TIB. For sugarcane (Saccharum spp. variety SP80-3280), liquid static medium and TIB was compared. Molecular analyses of Dyckia distachya plantlets, which had been grown in semisolid medium liquid static and liquid medium under agitation, were also carried out. Cultures that showed differences in performance among the systems evaluated were D. distachya, (with liquid medium under agitation as the best condition) and sugarcane (best treatment was BIT) and these were considered adequate to study the differences in the bacterial comunity of plants when grown in different in vitro conditions. The characterization of the microbial community of D. distachya was performed by T-RFLP (Terminal Restriction Fragment Length Polymorphism) and showed that the liquid medium under agitation, which had the highest number of shoots compare to the other culture conditions, also differed as to the relative abundance of Operational Taxonomic Units (OTUs). For sugarcane 16S rRNA gene clone libraries, as well as real-time PCR (qPCR) were performed. These analyses showed no significant differences between the libraries of the two treatments, however, BIT showed 3.54 times more copies of the 16S rRNA gene compared to cultures from static liquid medium, allowing us to infer a higher number of bacteria. This study provides strong evidence that the in vitro system used influences the microbial community present in plants