Rozprawy doktorskie na temat „Growth of Microbe”

Abandoned agricultural lands in the Intermountain West are plagued by dense, persistent non-native vegetation. Targeted restoration tools are required to remove the competitive advantage of these non-natives while also removing the soil legacies they leave behind. Activated carbon (AC) is one such tool, with the ability to disrupt the mechanisms of allelopathy, positive plant-soil feedbacks, and altered nutrient cycling commonly used by non-native species. Previous studies have shown the success of high concentrations of AC in native plant community restoration on a small scale. Here, our goals are twofold: first, to test AC effectiveness in restoring desirable plant communities on a larger scale, and secondly, to identify the primary mechanism, allelopathy versus microbial changes, through which AC impacts native and non-native species. A large scale AC treatment in Methow Valley, Washington tested the effectiveness of AC restoration at a large scale and tested five concentrations and two types of AC to determine lowest effective application. Following treatment, sites were monitored for vegetation cover for three years. The large-scale application produced similar results to the previous study at a 1000 g/m2 application rate, with a 28% increase in the ratio of desirable:undesirable species cover and a decrease to 25% undesirable species cover. However, the effectiveness of AC concentrations below 1000 g/m2 cannot yet be determined and may require a longer time scale and additional monitoring to assess restoration success. A greenhouse experiment was performed, which used native and non-native species common to the study site, grown in pairs in sterilized and live AC-treated soils to separate AC effects on allelopathy from that of microbial interactions. Both native and non-native species experienced a 25% decreased biomass in AC-treated live soils, with a minimal decrease in A- treated sterile soils for native species and no effect in AC-treated sterile soils for non-native species. Overall, AC live soils produced a positive effect on relative abundance; the ratio of native to non-native biomass was highest in AC-treated live soils. From these results, it is concluded that the primary pathway through which AC works is changes in the plant-microbial interactions of both native and non-native species.

Many different bacteria have essential roles in the process ofrecycling organic waste, making them useful tools when it comes toestablishing artificial ecosystems, a key technology to master inthe expansion of human space travel.In order to further investigate bacteria growth conditions duringspace travel, the MOREBAC experiment was formulated. The objectivewas to design an experimental setup and develop measurementequipment with the capability of confirming successfulresuscitation of freeze-dried bacteria in space by measuringbacteria growth, on-board the student-built MIST-satellite.The experimental setup prototype consisted of an acrylic chipwherein the bacteria would be placed during experiments and anoptical measurements configuration using a photosensor with thepurpose of detecting bacteria cell growth. For experimentalenvironment monitoring, a temperature sensor and a pressure sensorwere calibrated.An Arduino Nano microcontroller was programmed to control allelectrical components during measurements. During the opticaldensity measurements blue dyed water and E.coli bacteria innutrition media were used as test samples.Provided varying blue dye or bacteria cell concentrations, in theform of dilution series and growth-over-time-series, the equipmentproved capable of producing measurements that indicate the opticaldensity of the test sample.Furthermore, a prototype experiment protocol simulating eventsthat will occur in the final experiment design, was implementedand was able to produce real-time monitoring graphs of optical,temperature and pressure measurements, as well as documentation ofall events and measurement data.
MOREBAC
MIST

The microorganisms colonizing plants can have a significant effect on host phenotype, mediating such processes as pathogen resistance, stress tolerance, nutrient acquisition, growth, and reproduction. Research regarding plant-microbe interactions has focused almost exclusively on vascular plants, and we know comparatively little about how bryophytes -- including mosses, liverworts, and hornworts -- are influenced by their microbiomes. Ceratodon purpureus is a dioecious, cosmopolitan moss species that exhibits sex-specific fungal communities, yet we do not know whether these microbes have a differential effect on the growth and physiology of male and female genotypes. Using a common-garden design, we reared ten axenic genotypes of C. purpureus in a controlled environmental chamber. Clonal C. purpureus replicates, with and without the addition of a microbial inoculation, were used to test the effect of a mixed microbial community on vegetative growth, sex expression, photosynthetic efficiency (Fv/Fm and ETR), and chlorophyll content (CFR) for male and female mosses. We found that microbes had a negative impact on the growth and photosynthesis efficiency of C. purpureus, and this effect varied among genotypes of C. purpureus for ETR and growth. Microbes also had a positive, sex-specific effect on chlorophyll content in C. purpureus, with males exhibiting lower CFR values in the absence of microbes. C. purpureus sex expression was marginally negatively affected by microbe addition, but gametangia production was low overall in our experiment. We also conducted preliminary surveys using direct counts from moss ramets to assess the community composition of epiphytic algae associated with our microbe addition and control C. purpureus. These surveys identified three algal morphospecies in association with the microbe addition C. purpureus genotypes, as well as cyanobacteria, nematodes, rotifers, and testate amoeba. No algae, cyanobacteria, or micro-fauna were observed in the control plants. Transplantation of a mixed microbial community from field-to-laboratory conditions may be applied to other bryophyte species under varying environmental conditions to provide insight into how these diminutive yet important ecosystems will respond to environmental perturbation.

Recent preliminary data have suggested that microbe-to-plant signals, and plant internal signals elicited by microbial signals, affect aspects of plant physiology, development and growth. The reported research investigated the responses of plants to signal compounds of microbial and plant origin, such as lipo-chitooligosaccharides (LCOs - signal molecules in rhizobia-legume associations), chitin and chitosan (present in fungal cell walls), and phenolic compounds (salicylic acid, acetylsalicylic acid and gentisic acid - internal signals in plants, often affected by signals from microbes). Phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) are key enzymes of the phenylpropanoid pathway. Oligomers of chitin and chitosan increased the activities of both PAL and TAL in soybean leaves. The degree of increase was dependent on oligomer chain length and time after treatment. LCO [Nod Bj V (C18:1 , MeFuc)] was isolated from Bradyrhizobium japonicum strain 532C. When Arabidopsis thaliana plants were grown for two weeks on agar containing this LCO (10-8M) or chitin pentamer (10-4 M), they had greater root length, root diameter, root surface area and number of root tips than control plants. Chitosan (tetramer and pentamer) did not have this effect. Chitin and chitosan were also tested for effects on corn and soybean photosynthetic rates and growth. High molecular weight chitosan generally reduced photosynthetic rates, but did not reduce the growth of corn or soybean. However, foliar application of 10-6 M LCO to corn leaves increased photosynthetic rates (up to 36%). Foliar application of lumichrome (10-5 and 10-6 M), a breakdown product of riboflavin produced by some rhizosphere bacteria, to corn (C4 plant) and soybean (C3 plant) increased photosynthetic rates (up to 6%). Foliar application of lumichrome (10-5 M) increased soybean leaf area and shoot dry weight. Foliar application of SA, acetyl salicylic acid (ASA) and gentisic acid (GT

Les plantes vivent en étroite relation avec des populations complexes de microorganismes, y compris des espèces de rhizobactéries communément appelées rhizobactéries promotrices de la croissance des plantes (PGPR). Les PGPR conférèrent aux plantes une meilleure croissance et tolérance aux stress biotiques et abiotiques mais les mécanismes moléculaires impliqués dans ce processus restent largement inconnus. En utilisant un système expérimental in vitro, la plante modèle Arabidopsis thaliana et la souche PGPR bien caractérisée Pseudomonas simiae WCS417r, nous avons réalisé un ensemble complet d'analyses phénotypiques, d’expressions géniques et biochimiques. Nos résultats montrent que PsWCS417r induit des modifications transcriptionnelles majeures du transport du sucre et d'autres processus biologiques clés liés à la croissance, au développement et à la défense des plantes. En utilisant une approche de génétique inverse, nous avons également démontré que AtSWEET11 et AtSWEET12, deux gènes transporteurs de sucre dont l'expression est réprimée par les souches bactériennes étudiées chez Arabidopsis thaliana, sont fonctionnellement impliqués dans les effets favorisant la croissance et le développement des plantules. Nos résultats révèlent que la régulation du transport de sucres joue un rôle important dans les effets bénéfiques des interactions plantes-rhizobactéries. Nous avons étendu notre étude à deux autres souches de PGPR (Pseudomonas fluorescens PICF7, Burkholderia phytofirmans PsJN) et à une autre souche non-PGPR (Escherichia coli DH5α). Ces trois souches bactériennes sont capables de modifier elles aussi l’expression de plusieurs gènes codant des transporteurs de sucre (essentiellement des gènes des familles AtSWEET et AtERD6-like), soit dans les racines, soit dans les parties aériennes des plantules d’Arabidopsis. Globalement, nos résultats révèlent une régulation transcriptionnelle conservée ou spécifique de certains gènes codants pour des transporteurs de sucres lors des interactions plante-PGPR. Enfin, nous avons effectué l'identification et la caractérisation d'une souche Bacillus megaterium, RmBm31, isolée de nodules racinaires de la légumineuse Retama monosperma. Notre étude révèle que RmBm31 est une bactérie endophyte produisant de l'IAA et possédant un grand nombre de gènes associés à des caractères favorisant la croissance des plantes. En utilisant la plante modèle Arabidopsis, nous avons démontré que cette souche présente des effets bénéfiques sur la croissance et le développement des plantules via la production de composés volatils. Ces effets semblent impliquer des mécanismes de signalisation indépendants de l'auxine
Plants live in close relationships with complex populations of microorganisms, including rhizobacteria species commonly referred to as Plant Growth Promoting Rhizobacteria (PGPR). PGPR able to confer to plants an improved productivity but the molecular mechanisms involved in this process remain largely unknown. Using an in vitro experimental system, the model plant Arabidopsis thaliana, and the well characterized PGPR strain Pseudomonas simiae WCS417r, we have carried out a comprehensive set of phenotypic, gene expression, and biochemical analyses. Our results show PsWCS417r induces major transcriptional changes in sugar transport and in other key biological processes linked to plant growth, development and defense. Using a reverse genetic approach, we also demonstrate that AtSWEET11 and AtSWEET12, two sugar transporter genes whose expression is down-regulated by the PGPR, are functionally involved in its plant-growth promoting effects. Altogether, our findings reveal regulation of plant sugar transport plays a crucial role in determining the fate of plant-rhizobacteria interactions. We extended our study to two other PGPR and a non PGPR strain. Overall, our results show that all three bacterial strains tested are able to alter the expression of several plant sugar transporter genes (essentially genes of the AtSWEET and AtERD6-like families), either in roots or in shoot, and either in physical contact with the seedling roots or via the production of volatile compounds only. Altogether, our findings reveal conserved and strain-specific trancriptional regulation of sugar transport during plant-PGPR interactions. Lastly, we report the identification and characterization of a Bacillus megaterium endophytic strain, RmBm31, isolated from root nodules of the legume species Retama monosperma. Our study reveals RmBm31 is an IAA-producing endophytic bacterium that possess a large set of genes associated with plant growth promoting traits. Using the model plant species Arabidopsis, we demonstrate this strain display beneficial effects on plant growth and root development via the production of volatile compounds. These effects seem to involve auxin-independent signaling mechanisms

Research was conducted to observe bacterial growth on the surface of metals in a static bioreactor. Metal and non-metal samples were subjected to bacterial exposure (1 day and 9 days). The metal samples were surface treated prior to bacterial exposure. The microstructures of the surface treated samples were analyzed by optical microscopy. After exposure, the microstructures of the samples were analyzed by scanning electron microscopy (SEM). The analysis suggested that microbial attachment on the surface was related to the underlying microstructure of steel. The preferential attachment of microbes could potentially be influenced by cathodic and anodic regions created by the electrolytic cells.

Abstract Periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA) is a syndrome characterized by regular, high-fever episodes with healthy periods in between. In a classic phenotype of the syndrome, the fevers begin in childhood before the age of five, and fever flares are accompanied by aphthous stomatitis, pharyngitis, and/or cervical adenitis. The etiology of the syndrome is unknown, but tonsillectomy (TE) has been shown to be an effective treatment for the disease. The purposes of this study were as follows: (1) to assess the long-term outcome of PFAPA patients treated by TE with either the classic or incomplete phenotype (later onset of the disease and/or missing oropharyngeal symptoms), (2) to compare the health and growth of PFAPA patients with healthy controls, and (3) to compare the histological and microbiological findings of the tonsils of PFAPA patients with controls via conventional and modern sequencing technologies. In this approximately 9-year follow up, 97% (n = 56) of patients with the classic phenotype and all patients (n = 50) with the incomplete phenotype achieved a prompt and constant response after TE. There were no differences in either the length of fever episodes or flares between patients with both the classic and incomplete phenotypes. The health and growth of 119 PFAPA patients was compared to that of sex- and age-matched controls (n = 230), and no differences in prevalence of chronic diseases or growth were found between the groups. Infections, oral thrush, and pollen allergy were more common in the history of the PFAPA patients than in the controls. Microbiological and histological findings of the tonsils of PFAPA patients (n = 31) were compared with the findings of the controls (n = 24) who had undergone TE for other reasons. Biofilm formation and Candida albicans were more frequently found among PFAPA patients than the controls, but Staphylococcus aureus, varicella zoster, and herpes simplex viruses were more common in the controls. While comparing the bacterial microbiota between the groups, we found significant differences in the presence and relative abundance of many bacteria. For example, Cyanobacteria were more common and abundant in the case samples than in the controls. Because the long-term outcome after TE was excellent, both in classic and incomplete PFAPA patients; a new diagnostic criteria for the syndrome is proposed. The microbes of the tonsils in PFAPA patients differ from that of the controls, which may play an important role in triggering the inflammatory processes that lead to symptoms of PFAPA
Tiivistelmä Periodic fever, aphthous stomatitis, pharyngitis, adenitis (PFAPA) syndrooma, on oireyhtymä, jossa potilaat kärsivät hyvin säännöllisesti ilmaantuvista, toistuvista kuumejaksoista, joiden välillä potilaat ovat terveitä. Klassisessa tautimuodossa kuumeilut alkavat lapsuudessa ennen viiden vuoden ikää ja kuumevaiheeseen liittyy liitännäisoireita: suun limakalvojen rakkuloita, nielutulehdusta ja/tai kaulan imusolmukkeiden suurentumista. Oireyhtymän syytä ei tiedetä, mutta nielurisaleikkaus (TE) on osoittautunut tehokkaaksi hoidoksi. Tutkimuksen tarkoituksena oli (1) arvioida PFAPA potilaiden vointia pitkäaikaisseurannassa TE:n jälkeen ja vertailla taudinkuvaa niiden PFAPA potilaiden välillä, joilla oli klassinen PFAPA tai epätyypillinen PFAPA. (2) Lisäksi tutkimme myös TE:lla hoidettujen PFAPA potilaiden sairastuvuutta, yleistä terveydentilaa ja kasvua vertaamalla näitä sukupuoli- ja ikävakioituihin kontrolleihin ja (3) selvitimme mikrobiologisia ja histologisia löydöksiä PFAPA potilaiden nielurisoissa verrattuna muista syistä TE:ssa käyneiden lasten nielurisoihin. Tässä noin yhdeksän vuoden seurannassa TE:n jälkeen oli täysin parantunut 97% (n = 56) potilaista, joilla oli klassinen PFAPA, ja kaikki (n = 50) potilaat, joilla oli epätyypillinen PFAPA (tauti oli alkanut viiden ikävuoden jälkeen ja/tai klassiset liitännäisoireet puuttuivat). Kuumeprofiilit eivät muilta osin eronneet ennen nielurisaleikkausta näissä ryhmissä. PFAPA potilaiden (n = 119) kasvu ja yleinen terveydentila eivät eronneet väestökontrolleista (n = 230). Krooniset ja autoimmuunisairaudet olivat yhtä harvinaisia molemmissa ryhmissä. Potilaat raportoivat sairastaneensa enemmän infektioita ja sammasta lapsuudessa ja heillä oli enemmän siitepölyallergioita. PFAPA potilaiden (n = 31) ja muista syistä TE:ssa käyneiden lasten (n = 24) nielurisojen mikrobiologiaa ja histologiaa tutkittiin ja vertailtiin. Biofilmimuodostusta nielurisan pinnalla ja Candida albicansia löytyi enemmän tapauksilta kuin kontrolleilta, kun taas Staphylococcus aureusta, varicella zoster- ja herpes simplex -viruksia tavattiin enemmän kontrolleilla. Myös mikrobiomi erosi ryhmien välillä, esimerkiksi syanobakteerit olivat yleisempiä PFAPA risoissa kuin kontrolleilla. Klassisten ja epätyypillisten PFAPA potilaiden terveydentila TE:n jälkeen oli pitkäaikaisseurannassamme erinomainen ja siksi ehdotamme, että PFAPA –syndrooman diagnostisia kriteereitä tulisi muuttaa. Nielurisojen mikrobisto on erilainen kontrolleihin verrattuna ja tällä voi olla merkitystä PFAPA syndrooman inflammatorisessa prosessissa

La levure et la microalgue sont des microorganismes très étudiés pour la production de composés à haute valeur ajoutée pour des secteurs tels que l’agroalimentaire et l’énergie. Ce travail de thèse propose un procédé de culture mixte entre la levure Saccharomyces cerevisiae et la microalgue Chlorella vulgaris pour la croissance des deux espèces tout en limitant le rejet en CO2. Le procédé repose sur la symbiose mutuelle entre les deux organismes autour des échanges de gaz, qui est rendu possible en imposant une co-dominance en termes de population. Les populations doivent être équilibrées pour que les microalgues puissent gérer la production de CO2. Le procédé est réalisé en photo-bioréacteur de 5 litres non-aéré et fermé, afin d’éviter les échanges gazeux avec l’environnement externe. Dans cette configuration, le CO2 est produit sous forme dissoute et directement accessible aux microalgues, évitant les phénomènes de dégazage et de dissolution. Les populations de levures et de microalgues atteignent une concentration égale (20 millions de cellules par ml) au bout de 24 heures de culture, restent stables jusqu’à la fin de la culture (168 heures) et les microalgues recyclent 12% du CO2 produit par les levures. Un modèle cinétique de la levure et de la microalgue en culture mixte est développé en combinant les modèles individuels de la levure et de la microalgue. Le modèle prédictif de la levure prend en compte les possibles voies métaboliques impliquées dans la fermentation et la respiration de ces voies est prédite en y intégrant des facteurs de limitation. Le modèle de la microalgue est basé sur l’activité photosynthétique. Les résultats de ce travail montrent la faisabilité du procédé de culture mixte entre hétérotrophe et autotrophe et pourrait apporter les bases pour le développement d’un procédé écologique à faible impact environnemental
Yeast and microalgae are microorganisms widely studied for the production of high-value compounds used in food and energy area. This work proposes a process of mixed culture of Saccharomyces cerevisiae and Chlorella vulgaris for both growth and CO2 mitigation. The process relies on mutual symbiosis between the two organisms through gas exchange, which is possible by engineering the co-dominance of populations. The two populations must be balanced in such a way so that microalgae can cope with the rate of CO2 production by the yeast activity. The process is performed in non-aerated 5l-photo-bioreactor fitted with a fermentation lock to prevent gas exchange with the outside atmosphere. With this set-up, the CO2 is produced in dissolved form and is available to the microalgae avoiding degassing and dissolution phenomena. The two organism populations are balanced at approximately 20 millions cells per ml, 12% CO2 produced by yeast was reutilized by microalgae within 168 hours of culture. A yeast and microalgae growth model in mixed culture is developed by combining each individual growth model. The predictive yeast model considers the possible metabolic pathways involved in fermentation and respiration and imposes limitation factors on these pathways, in this manner, the model can predict the partition of these pathways. The microalgae individual model is based on the photosynthetic activity. The results of this work show the feasibility of such process and could provide a basis for the development of a green process of low environmental impact

This thesis is concerned with the characterisation, fabrication and testing of devices capable of generating and detecting terahertz (THz) radiation. Such devices are based on semiconductor photoconductors grown under low temperature (LT) conditions using the technique of Molecular Beam Epitaxy (MBE). The absorption of a pulsed or continuous wave (CW) signal by these structures in conjunction with the presence of an electric field generates photocurrent, which is fed into an antenna structure fabricated on the surface of the active layers. As a result of such a sequence, a THz signal is generated and radiated from the substrate side into free space. Therefore, the efficiency of the devices is determined by the characteristics of the photoconductors and the geometry of the designed antenna structures. The desired material characteristics are high absorption at the corresponding illumination wavelength, high dark resistivity, high electron mobility and sub picosecond carrier lifetime. The determination of these characteristics for all the structures grown in this work composes the characterisation part of the thesis. The fabrication part comprises of the design of several antenna structures with various geometrical characteristics, while the testing part consists of their evaluation as THz sources and detectors in a time-domain spectroscopy (TDS) system under pulsed excitation. To date, THz devices based on low temperature grown GaAs (LT-GaAs) photoconductors have been reported to be the most efficient. However, their operational wavelength, at 800 nm, requires very expensive and complex components spurring interests in solutions consisting of devices operating at longer wavelengths, where cheaper and simpler components exist. The most desirable and practical operational wavelength is the telecommunication one at 1.55 μm. Thus, the biggest challenge is the development of efficient devices operating at this illumination wavelength. In this work, devices operating at the very important wavelength of 1.55 μm as well as at the wavelengths of 1 μm and 800 nm are presented. The key findings for the long wavelength devices (1.55 μm) demonstrate photoconductors with ultrafast carrier lifetimes (~ 120 fs), high resistivity (> 105 Ω / sq), high mobility (> 1000 cm2 / Vs) and system responses with spectral range up to 3 THz and power-to-noise ratio of 60 dB. These characteristics are among the best ever reported for such material systems, making them efficient THz devices for various optoelectronic applications, especially for telecommunication laser-driven CW THz systems.

One of the major challenges for agricultural research in the 21st century is to increase crop productivity to meet the growing demand for food and feed. Biotic (e.g. plant pathogens) and abiotic stresses (e.g. soil salinity) have detrimental effects on agricultural productivity, with yield losses being as high as 60% for major crops such as barley, corn, potatoes, sorghum, soybean and wheat, especially in semi-arid regions such as Saudi Arabia. Plant growth promoting bacteria isolated from pioneer desert plants could serve as an eco-friendly, sustainable solution for improving plant growth, stress tolerance and health. In this dissertation, culture-independent amplicon sequencing of bacterial communities revealed how native desert plants influence their surrounding bacterial communities in a phylogeny-dependent manner. By culture-dependent isolation of the plant endosphere compartments and a number of bioassays, more than a hundred bacterial isolates with various biochemical properties, such as nutrient acquisition, hormone production and growth under stress conditions were obtained. From this collection, five phylogenetically diverse bacterial strains were able to promote the growth of the model plant Arabidopsis thaliana under salinity stress conditions in a common mechanism of inducing transcriptional changes of tissue-specific ion transporters and lowering Na+/K+ ratios in the shoots. By combining a number of in vitro bioassays, plant phenotyping and volatile-mediated inhibition assays with next-generation sequencing technology, gas chromatography–mass spectrometry and bioinformatics tools, a candidate strain was presented as a multi-stress tolerance promoting bacterium with potential use in agriculture. Since recent research showed the importance of microbial partners for enhancing the growth and health of plants, a review of the different factors influencing plant-associated microbial communities is presented and a framework for the successful application of microbial inoculants in agriculture is proposed. The presented work demonstrates a holistic approach for tackling agricultural challenges using microbial inoculants from desert plants by combining culturomics, phenomics, genomics and transcriptomics. Microbial inoculants are promising tools for studying abiotic stress tolerance mechanisms in plants, and they provide an eco-friendly solution for increasing crop yield in arid and semi-arid regions, especially in light of a dramatically growing human population and detrimental effects of global warming and climate change.

Bacterial population during 3.2 billion years of early earth was responsible for modifying the greenhouse gas concentration in the early earth. Here we studied the primitive facultative anaerobe E.coli K12, known to contain pathways of mixed acid fermentation in stressed oxygen deficient environment. We carried out chamber experiments where bacteria were grown in M9 minimal media containing 0.4% glucose in crimp sealed chambers for a period of 7 days-12 days. Growth rate for the bacteria were monitored using optical density measurements and CFU values on LB agar. δ13C of CO2 was analysed using Gas Bench peripheral connected with IRMS MAT 253. We observed an approximate enrichment of 3‰ in the 13C data of respired CO2 from the 1st day of incubation till 12 days of incubation under stressed conditions. We suggest that enrichment in δ13C captured a shift in the carbon source from glucose to acetate.

Cyclodipeptides (CDPs) are the smallest, most stable cyclic peptides that are synthesized as secondary metabolites by bacteria. The aim of this study was to investigate the effect of the Pseudomonas argentinensis (SA190) and four (CDPs), named as cis-cyclo-(Pro-Phe) (Cyclo2), cis-cyclo-(Pro-Leu) (Cyclo3), cis-cyclo-(Pro-Tyr) (Cyclo4) and cis-cyclo-(Pro-Val) (Cyclo5), with three concentrations (1µM, 100nM, and 10nM), on the growth of Arabidopsis thaliana under normal plant growth conditions [1/2MS media], salt conditions [125 mM NaCl] and drought conditions [25% PEG]. Moreover, we determined the most effective CDPs with optimal concentration. It was found that cis-cyclo-(Pro-Tyr) (Cyclo4) at a concentration of 100nM had an effect on the plant growth and can mimic the effect of SA190 under normal [1/2MS media] conditions. Also, cis-cyclo-(Pro-Tyr) (Cyclo4) at a concentration of 1µM can mimic the effect of SA190 under salt conditions [125mM NaCl]. Finally, cis-cyclo-(Pro-Val) (Cyclo5) at a concentration of 1µM can mimic the effect of SA190 under drought conditions [25% PEG].

The effect of Ascophyllum nodosum extracts on the nodulation and growth of alfalfa was investigated. Plant growth assay revealed that alfalfa treated with 2 g L-1 ANE exhibited a significant increase in leaf area. Under salt stress, alfalfa treated with 0.5 g L-1 ANE exhibited a significant increase in total length compared to controls. A root hair deformation assay indicated that ANE 0.5 g L-1 stimulated the synthesis of Nod factors secreted by rhizobia thus accelerate root hair deformation of alfalfa. Similarly, ANE 0.5 g L-1 caused an increase in nodC gene expression suggesting that ANE may act similarly to flavonoids in the rhizobium-legume symbiosis. Under field conditions, ANE increased the total number of functional nodules, total root length and total leaf area. Taken together, the results suggest that ANE may contain compound(s) that promote specific metabolic pathway both in alfalfa and bacterium thus enhance the symbiotic relationship.

Symbioses between microbes and multicellular eukaryotes are found in all biomes, and encompass a spectrum of symbiotic lifestyles that includes parasitism and disease, commensalism, and mutually beneficial interdependent host-microbe relationships. Regardless of outcome, these symbiotic lifestyles are governed by a complex molecular "courtship" between microbe and potential host. This courtship is the primary determinant of the host range of a given microsymbiont. Host immunity poses a formidable barrier to the establishment of host-microbe relationships, and the majority of microbial suitors will be thwarted by it. Only by successfully "wooing" the host cell's immune defenses with the appropriate molecular signals can a microsymbiont successfully colonize its host. A strategy common to microsymbionts across the spectrum of symbiotic lifestyles and host organisms is the delivery of microbial-encoded effector proteins into the cytoplasm of host cells to manipulate the host cell's molecular machinery for the purposes of subverting host immunity. Bacteria, in particular, have adapted a number of secretion systems for this purpose. The most well-characterized of these is the type III secretion system (T3SS), a molecular apparatus that specializes in injecting type III effector (T3Es) proteins directly into host cells. The work in this thesis focuses on T3Es of plant-associated bacteria, with particular emphasis on mutualistic bacteria. We present evidence that collections of T3Es from Sinorhizobium fredii and Bradyrhizobium japonicum are, in stark contrast to those of phytopathogenic bacteria, in a co-evolutionary equilibrium with their hosts. This equilibrium is characterized by highly conserved T3E collections consisting of many "core" T3Es with little variation in nucleotide sequence. The T3Es of Mesorhizobium loti MAFF303099 suggest a completely different picture of the evolution of T3Es. MAFF303099 recently acquired its T3SS locus, and the work in this thesis provides an evolutionary snapshot of a mutualist that is innovating a T3E collection primarily through horizontal gene transfer. Collectively, this work represents the first comprehensive catalog of T3Es of rhizobia and, in the case of Sinorhizobium and Bradyrhizobium, the first evidence of purifying selection for T3Es.
Graduation date: 2012

碩士
國立中興大學
土壤環境科學系所
103
Cultivated soil of Taiwan have low organic matter, low soil fertility and weak preserving nutrients due to high temperature and rainfall.The application of organic matter can increase soil fertility and improve soil activity. Recently, biological fertilizer can be mixed into organic fertilizer and apply soils, and that can increase beneficial microbes and improve soil fertility. The application of biological fertilizer not only promote crop growth, reduce disease, and increase the income amount to achieve improving the quality of crops. Palygorskite have large surface area and dispersive characteristic, and that also have channel pore of high surface area and surface activity. High adsorptive activity of palygorskite can increase crop yields, reduce the heavy metal content and improve soil physical and chemical properties. The objectives of experiment studies were to add different microorganisms into palygorskite that were mixed and applied to leafy vegetables (B. r. chinensis and Brassica chinensis Linn) of pot experiment. We counted crop harvest in the yield survey and root bacteria situation, and to explore the interaction between clay minerals and microbes. The results showed the application of palygorskite could increase crops yields in only palygorskite treatment, and the vegetable yields (B. r. chinensis and Brassica chinensis Linn) of acidified palygorskite 20 g treatment were the highest. B. r. chinensis was 68.42 g / pot and Brassica chinensis Linn was 63.87 g / pot. Two vegetable yields of mixed palygorskite and microbes treatment were more than that of palygorskite treatment, that showed microbes could promate vegetable growth. 10 g acidified palygorskite added Bacillus licheniformis treatment could cause the highest yields of vegetable (B. r. chinensis 76.73 g / pot and Brassica chinensis Linn 69.51 g / pot). In the addition of Saccharomyces cerevisiae, B. r. chinensis yields of 20 g acidified palygorskite treatment were hightest (80.07 g / pot), while Brassica chinensis Linn yields of 20 g palygorskite treatment were hightest (77.49 g / pot). Furthermore, the CPU of two microbes in rhizosphere was increasing with applied amounts of palygorskite due to microbes absorbed to nutrients of decomposed organic matter, and that increased the growth of vegetable root and shoot. The experiment results also indicated the mixed application of palygorskite and microbes apparently improved vegetable growth, and that could be applied on agricultural development with lower cost. In future, we expect the application of natural minerals coupling with microbe can make good agricultural product in Taiwan''s cultural soils.

Tese de mestrado, Biologia Evolutiva e do Desenvolvimento, Universidade de Lisboa, Faculdade de Ciências, 2020
Interactions between microorganisms and plants have occurred for millions of years. Combining the capacity of light and CO2 usage by the plants with the capacity of efficient substrate usage by their microbiota, the water-to-land-transition was possible. Since then, plants and plant microbiota have coevolved, and today, the microbiome is considered as an extension of the plant’s genetic assembly. However, the agricultural revolution led to progressive alterations in habitat, crop managing practices, and breeding to promote crop production changing their evolutionary trajectory. Moreover, the trajectory of the co-evolution between crops and their microbiome is also changed. The soil, the plant, and the microorganisms are connected and impact each other. The rhizosphere is considered to be the most dynamic interface on Earth, and the microorganisms that exist there might promote plant growth and resilience. These microorganisms are referred to as Plant Growth-Promoting Microbes (PGPM), including Plant Growth-Promoting Rhizobacteria (PGPR) and Arbuscular Mycorrhizal Fungi (AMF). The experimental work is divided into three chapters according to the plant-model. In the first one, microorganisms (AMF and bacteria) were collected from a wild relative of chrysanthemum (Dendranthema grandiflora). Plant Growth Promoting Rhizobacteria (PGPR) was submitted for molecular analysis. Three of them (previously selected by plant growth-promoting traits) were inoculated on five chrysanthemum commercial cultivars to test their impact on plant performance and root microbiome assembly. AMF was also inoculated with the same propose. PGPR impacted the number of nodes and root biomass of commercial chrysanthemum cultivars. AMF affected the root biomass of cultivars of chrysanthemum cultivated in autoclaved and non-autoclaved soil. AMF root colonization was not found. All the treatments impacted the microbiome assembly in the tested commercial cultivars. Concluding, PGPR and AMF obtained from wild chrysanthemum impacted growth performance and microbiome assembly in five commercial cultivars. In the second one, AMF collected from wild chrysanthemum were proliferated using millet (Panicum miliaceum) as a host in order to have an inoculum of two morphotypes. The two morphotypes of AMF spores were successfully multiplied in millet roots and sorrowing soil. Millet appeared as a good host for the propagation of AMF spores. Lastly, in the third chapter, the same AMF were inoculated in sorghum (Sorghum bicolor) to test their impact on plant performance and resilience against Striga hermonthica, a parasitic weed. Sorghum growth performance and resilience were impacted by the presence of S. hermonthica and/or AMF in the soil. AMF root colonization on sorghum was observed and AMF treatments reduced S. hermonthica germination by 59%. However, the reduction of the germination of S. hermonthica seeds did not increase the sorghum growth performance, so more studies are needed to understand these mechanisms. Promising results were found, but additional work is needed to understand how these inocula are best applied in the field and the mechanisms behind it. It is also necessary to comprehend how the entire microbiome is affected by the inocula and how these changes impact growth performance and resilience in both, chrysanthemum, and sorghum species.

Anaerobic digestion (AD) is becoming a widely adopted technology for conversion of organic waste and nutrient-rich fertiliser production due to its cost-effectiveness and sustainability. In this study, a batch experiment was conducted using five different types of food waste and cow dung (CD). No significant difference was observed among the four substrates that produced the highest methane (P<0.05). Based on the batch experiment results, two substrates were selected for semi-continuous digestion and the highest methane yield (67%) was obtained from co-digestion (CO). PCR-DGGE results revealed higher bacterial and archaeal diversity indices in CO as compared to mono-digestion of CD and mixed food waste. The high-throughput sequence analyses revealed that the Operational Taxonomic Units (OTUs) belonging to the phyla Bacteroidetes, followed by Firmicutes, Actinobacteria and Proteobacteria, were dominant in all treatments. The enhanced methane production in CO could be attributed to the neutral pH and partial shift of archaea from Methanosaeta to Methanosarcina. The digestate and fresh CD were screened for plant growth promoting bacteria (PGPB), nutrient and heavy metal content. The dung contained higher concentrations of heavy metals (P<0.05) and potential pathogens in comparison to the digestate. The use of digestate may, therefore, enhance soil fertility with minimal negative environmental effects.
School of Agriculture and Life Sciences
M. Sc. (Life Sciences)

Public tendency, of late, is to reduce liberal use of harmful synthesized chemicals for promoting plant health. Today, biological control is becoming a commonly cited disease control option. Biological control agents (BCAs) not only control disease , but also promote plant growth. Application of biological control is based largely on knowledge of control mechanisms employed by antagonists, as well as the means of application that will ensure that an antagonistic population is established. Knowing the advantages is not the only factor that should be considered before application commences as, the disadvantages must be clearly outlined and explored further before a constructive decision as on implementation of biological control. A literature review was undertaken to provide the necessary technical information about biological control, its potential uses, methods of application, mechanisms of action employed, advantages and disadvantages associated with biological control application, public perceptions and the potential future of biological control. Diseases encountered within the KwaZulu-Natal Midlands on pasture and turf grasses were determined by a once-off survey conducted over 1999/2000. The aim of the survey was to determine broadly the management practices of farmers and groundsmen in KwaZulu-Natal and the potential impact of these on the occurrence of weeds, insects and diseases. The survey also addressed the level of existing knowledge about biological control and willingness to apply such measures. In the pasture survey, farmers were questioned about: soil type, grass species common used, irrigation , fertilization and liming, grazing programs and weed, insect and disease occurrences and control measures implemented. The same aspects were addressed in a survey to a representative sample of groundsmen (turfgrass production) , including also: topdressing, greens base used, drainage systems, mowing practices and decompaction principles. The survey showed correlation between pest incidence and management practices implemented. In terms of pest control, both farmers and groundsmen indicated a stronger preference to the use of herbicides , insecticides and fungicides. Use of fungicides for disease control by farmers is considered an often unfeasible expense, rather more emphasis was placed on implementing cultural control methods. At present farmers do not apply biological control strategies, but they did indicate much interest in the topic. Alternatives to current, or lack of current, disease management strategies are important considerations, with two new diseases identified in the KwaZulu-Natal Midlands just within the period of this thesis. Biological control strategies are implemented by 8% of the groundsmen surveyed, with emphasis being placed on augmenting the already present natural predators rather than the introduction of microbial antagonists. Although often mis-diagnosed by farmers Helminthosporium leaf spot is a common disease in the KwaZulu-Natal Midlands on Pennisetum clandestinum (kikuyu), This disease reduces pasture quality and detracts from the aesthetic appearance and wearability of turfgrasses. Helminthosporium leaf spot is incited by a complex of causal agents , Bipolaris was confirmed as the casual agent of Helminthosporium leaf spot on kikuyu at Cedara. Disease control by two BCAs, Bacillus (B. subtilis Ehrenberg & Cohn.) and Trichoderma (T. harzianum Rifai), as commercial formulations was tested against the fungicide, PUNCH EXTRA®. In vitro, Trichoderma was shown to be aggressive in controlling Bipolaris sp. In vivo, disease control achieved with Trichoderma kd was comparative with PUNCH XTRA® but not statistically different (P>=0.05). Trichoderma and Bacillus provided better disease control in comparison to an untreated control. Improved growth of Lolium sp. was determined in vitro, with Trichoderma kd and Bacillus B69 treatments. The microbe-based treatments accounted for growth stimulation, with significant (P<=O.05) growth differences noted. A microbial activator, MICROBOOST®was added to the treatments to improve microbial efficiency. Improved plant growth with MICROBOOST® applications was shown. Improved growth associated with microbial treatments, Trichoderma harzianum kd; Bacillus subtilis B69 and Gliocladium virens Miller, Gibens, Foster and con Arx. ,was also determined in vivo at Cedara, on L.perenne L., Festuca rubra L. and Agrostis stolonifera L. Establishment of a suppressive soil with antagonistic microbes resulted in significant (P<=O.05) effects on final grass coverage (except G. virens), as well increased root and shoot lengths (P<=O.05). Increased germination rates, as expressed in vitro, were not shown in vivo. Microbial activity with the application of MICROBOOST® showed little effect on germination but increased root and shoot lengths significantly (P<=O.05). Increased weed growth associated with the treatments (except G. virens) was considered a drawback of the microbial-treatments. Microbial treatments were also applied to pasture grasses. An in vitro grazing trial was established at Cedara, using L. multiflorum L. to evaluate the microbe-based treatments Trichoderma kd, Bacillus B69 and G. virens for improved pasture establishment and for increased grazing preference by Dohne Merino sheep. Trichoderma kd was associated with increased dry and wet biomass , but lower dry matter yields in comparison to the control. Only G. virens accounted for a higher dry matter percentage than the control. However, differences between the control and the microbial treatments was very small and not significant (P>=0.05). Of the three grazing observations made, sheep showed no grazing preference to plots with or without microbial treatments In general, the body of this research has shown that microbial treatments have the potential for increased disease control and growth stimulation of grasses. However, lack of significant differences between microbial treatments and controls has raised the question as to effect of external factors on microbial activity and survival, especially in vivo. This raises the question as to the validity of the use of microbial treatments where growth conditions cannot be controlled , remembering that the cost of establishment must be covered by the economic returns from utilization.
Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

The plant root system is the connection between the aboveground biomass and the soil system, and it requires a healthy soil environment to support optimum root growth. A healthy and extensive root system allows the plant to explore a greater soil volume for water and nutrients, and thereby increase the likelihood of more productive crop.Intensive vegetable production systems with little or no organic materials incorporated into soil combined with heavy farm traffic and use of chemical fertilizers often lead to detrimental impacts on soil health characteristics, reducing capacity to support extensive root system development. Crops in these systems may also be more susceptible to high incidence and severity of soil-borne root diseases, which further reduces the root system and yield capacity of the crop. Pathogen damage to crop root systems is a major threat to crop production, and while current management systems can mitigate against this threat, alternative strategies based on building soil health through building soil organic carbon content and strong plant root systems is more sustainable. This research investigated the effects of different organic materials to soil microbial communities, plant root system development and crop performance in cropping soils.

Associations between leguminous plants and symbiotic nitrogen-fixing bacteria (rhizobia) are a classic example of mutualism between a eukaryotic host and a specific group of prokaryotic microbes. Rhizobia improve plant yield furnishing fixed nitrogen; therefore, they are highly used as inoculants, especially in sustainable agriculture. Though this association is species-specific, legume roots are exposed to heterogeneous rhizobial populations where different compatible strains are present and, could be infected by more than one strain. It is known that within the same rhizobial species different strains may have different competition capabilities, but detailed analyses able to predict the rhizobial competitive phenotype based exclusively on their genome are still lacking. In this thesis, we performed a bacterial genome-wide association analysis (GWAS) to define which genetic traits are responsible for an improved competitive phenotype in the model species Sinorhizobium meliloti. A panel of thirteen S. meliloti strains, whose genome is completely sequenced, has been selected and tested against three S. meliloti reference competitor strains (Rm1021, AK83 and BL225C) in a Medicago sativa nodule occupancy test. The measure of competition phenotypes previously obtained in the competition tests, in combination with strains genomic sequences of strains tested, were used to build-up a k-mer-based statistical models for each set of competition experiments. The obtained models were then applied to evaluate the accuracy in predicting the competition abilities of strains in the three competition patterns (vsRm1021, vsAK83 and vsBL225C). The competitive abilities of S. meliloti strains against two partners, BL225C and Rm1021, were well predict by the predictive models, as shown by the coefficient of determination R2 (equal to 0.96 and 0.84, respectively). Four strains showing the highest competition phenotypes (> 60% single strain nodule occupancy; GR4, KH35c, KH46 and SM11) versus BL225C were used to identify k-mers associated with the competition phenotype. The most significantly associated k-mers (p <0.05) were mapped on the genomic sequences of the S. meliloti strains used. Most of the k-mers were located on the symbiosis-related megaplasmid pSymA and on genes coding for transporters, proteins involved in the biosynthesis of cofactors and proteins related to metabolism (i.e. glycerol, fatty acids) suggesting that competition abilities reside in multiple genetic determinants comprising several cellular components. The identification of the best rhizobial inoculants in two new effective breeding lines of pea (Pisum sativum) used in Lithuania (DS 3637-2 and DS 3795-3) was also investigated. Six rhizobial strains, isolated from pea plants, which could be used as potential inoculants, were phylogenetically identified and extensive phenotypically characterized by Phenotype Microarray. All the strains belonged to the Rhizobium leguminosarum group, and were subdivided into three groups related to Rhizobium anhuiense, Rhizobium leguminosarum bv. viciae and R. sophorae/R. laguerreae. Differences observed with Phenotype microarray were linked to different phylogeny of the strains. In terms of symbiotic efficiency, six strains showed different symbiotic performances depending on the breeding line used. In particular, Rhizobium anhuiense strain Z1 (the reference strain) and Rhizobium leguminosarum bv. viciae 14ZE were the best symbiotic inoculants with breeding lines DS 3637-2 and DS 3795-3, respectively.

Tese no âmbito do Doutoramento em Biociências, ramo de especialização em Biotecnologia, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e tecnologia da Universidade de Coimbra.
The interest in plant growth promoting rhizobacteria (PGPR) and arbuscular mycorrhizal (AM) fungi for agricultural purposes (e.g. enhancement of crop yield and nutrition, promotion of plants resilience to abiotic stress) is rising. Yet, large-scale applications of these microbes have been hampered by the lack of data on their field performance and the feasibility of the inoculation methods, especially in the case of AM fungi. Seed coating, a technique in which seeds are covered with minor amounts of exogenous materials (including microbial inoculants), is a potential tool to deliver microbes at large-scale. This technique has been gaining attention in the agricultural sector. A literature review revealed that seed coating has been applied to more than 50 plant species (e.g. wheat, tomato, maize, melon, bean, clover), including seeds with different characteristics (dimensions, forms, textures). Mostly studied for the application of various species of PGPR (especially from the genera Pseudomonas and Bacillus), seed coating is not so frequently explored for inoculation of AM fungi or microbial consortia. The improvement of crop productivity and protection of plants against pathogens have been the main focus of research on microbial seed coating, while a smaller portion has been aimed at enhancing crops resistance to abiotic stresses. One of the main goals of this PhD thesis was to evaluate seed coating as a deliver system for the inocula of AM fungi and PGPR. Overall, seed coating allowed the application of minor amounts of AM fungi and PGPR to the seeds of three selected agricultural crops: maize, cowpea and chickpea. Further, by comparing inoculation of AM fungi through direct soil inoculation (conventional) with seed coating, similar AM root colonization was obtained despite a reduction in the amount of applied inocula. Contrary to AM fungi, the presence of PGPR coated on the seeds could not be confirmed in the rhizosphere and roots of the inoculated crops. It is well known that both AM fungi and PGPR have the ability to improve soil fertility and enhance plant nutrition, which can bring benefits for plant growth and development. By increasing nutrient availability and nutrient use efficiency, these plant beneficial microbes (PBM) can assist farmers to reduce their dependence on chemical fertilizers. The results obtained in this thesis showed that coating seeds with PGPR and AM fungi had a significant impact on plant shoot nutrient concentrations under different fertilization regimes. For instance, maize seeds coated with AM fungi (Rhizophagus irregularis) increased shoot nutrient concentration (nitrogen, phosphorus, potassium, magnesium and zinc), comparing with non-inoculated plants. Nutrient contents on maize shoot were boosted by R. irregularis inoculation, particularly in the treatments where fertilization was reduced or absent. On the contrary, maize coated with PGPR (Pseudomonas fluorescens) presented most of the nutritional increments when full fertilization was applied. Nevertheless, in both inoculation treatments, despite the nutrient enhancements, no improvement in plant biomass was obtained. These results confirm that PBM can increase plant nutrient uptake. PGPR and AM fungi are known to confer drought resistance to plants. Coating seeds with PGPR (Pseudomonas putida) showed a general positive influence in the plant productivity, especially under moderated water deficit. Seed coating with AM fungi (R. irregularis singly or in consortia with P. putida) promoted nutrient uptake, leaf pigment contents and gas exchange parameters of cowpea, yet mostly when plants where under no water deficit. Mainly, these results emphasized the importance of selecting the PBM that better potentiate plant resilience to abiotic stresses, in order to obtain the best benefits from the inoculation. Field experiments are essential to validate the benefits of microbial seed coating and its feasibility for large-scale applications. A comparison between chickpea coated with a single AM fungal isolate of R. irregularis and multiple isolates of the same fungal species under greenhouse and field conditions showed that plants inoculated with multiple AM fungal isolates performed better (e.g. higher biomass, increased grain yield) than those inoculated with a single AM isolate. Seed coating proved to be an appropriate tool to deliver AM fungi with benefits for chickpea plants at both experimental scales, but particularly relevant under field conditions. The mixture of multiple R. irregularis isolates was also used in consortium with Pseudomonas libanensis for coating cowpea seeds. This treatment significantly improved crop productivity in comparison with non-inoculated plants and plants inoculated with R. irregularis single-isolate + P. libanensis. The results showed improvements in grain lipid content, soil physicochemical properties (pH and soil organic matter), and crop yield under low-input agricultural systems. AM fungi and PGPR should be selected for microbial seed coating formulation according to their affinity with the host crop, growing conditions (e.g. soil properties) and farming practice (e.g. irrigation and fertilization), in order to obtain economical profits. This thesis is a contribution to the knowledge on microbial seed coating and highlights the potential of seed coating as a microbial delivery tool and the benefits of its use in different agricultural conditions. Microbial seed coating can be of great use for sustainable agricultural systems. Yet, in order to allow its large-scale application as a cost-effective technique for PGPR and AM fungi inoculation, further development is necessary.
O interesse em rizobactérias promotoras de crescimento de plantas (RPCP) e fungos arbusculares micorrízicos (FAM) para fins agrícolas (e.g. melhoramento do valor nutricional e rendimento de culturas, promoção de resiliência de plantas a factores abióticos) tem vindo a aumentar. Contudo, a aplicação em larga escala destes microrganismos tem sido dificultada pela escassez de dados sobre a performance destes em campo e viabilidade dos métodos de inoculação, em particular, no caso dos FAM. O revestimento de sementes, uma técnica que consiste em cobrir sementes com pequenas quantidades de materiais exógenos (incluindo inoculantes microbianos), representa uma potencial ferramenta para inocular microrganismos em grande escala. O revestimento de sementes tem vindo a ganhar importância no sector agrícola. Segundo a revisão da literatura realizada nesta tese, o revestimento de sementes foi usado em mais de 50 espécies de plantas (e.g. trigo, tomate, milho, melão, feijão, trevo), abrangendo sementes com diferentes características (dimensões, formas, texturas). Principalmente estudado para aplicação de várias espécies de RPCP (em particular do género Pseudomonas e Bacillus), o revestimento de sementes não tem sido tão frequentemente usado para inoculação dos FAM ou consórcios microbianos. O aumento da produtividade e proteção de culturas agrícolas contra agentes patogénicos têm sido o principal foco da investigação sobre revestimento de sementes com microrganismos benéficos. Por outro lado, os estudos referentes ao melhoramento da resistência de plantas a stresses abióticos através do revestimento de sementes têm sido consideravelmente menores. Um dos principais objectivos desta tese foi avaliar a técnica de revestimento de sementes como método de inoculação para FAM e RPCP. De uma forma geral, o revestimento de sementes permitiu a aplicação de pequenas quantidades de FAM e RPCP em sementes de três culturas: milho, feijão-frade e grão-de-bico. Uma comparação entre inoculação directa no solo (convencional) e revestimento de sementes com FAM, mostrou que, apesar da redução na quantidade de inoculo aplicado, a eficiência do fungo na colonização nas raízes da planta alvo foi similar. Contrariamente aos FAM, a presença de RPCP na rizosfera e raízes das culturas selecionadas não foi confirmada. É de conhecimento geral que tanto os FAM como RPCP têm a capacidade de melhorar a fertilidade do solo e o estado nutricional das plantas, o que pode ser de grande proveito para o desenvolvimento e crescimento destas. Através do aumento da disponibilidade ou eficiência de utilização dos nutrientes, estes microrganismos promotores de crescimento de plantas (MPCP) podem ajudar os agricultores a reduzir a dependência de fertilizantes sintéticos. De acordo com os resultados obtidos nesta tese, sementes revestidas com RPCP e FAM tiveram um impacto significativo no estado nutricional das plantas quando submetidas a diferentes regimes de fertilização. Por exemplo, sementes de milho revestidas com FAM (Rhizophagus irregularis) apresentaram um aumento significativo na concentração de nutrientes na parte área (azoto, fósforo, potássio, magnésio e zinco), quando comparado com plantas não inoculadas. A concentração de nutrientes, na parte área do milho, foi estimulada pela inoculação de R. irregularis, em particular, em tratamentos com fertilização reduzida ou ausente. Pelo contrário, o aumento na concentração de nutrientes em milho revestido com RPCP (Pseudomonas fluorescens) foi superior quando plena fertilização foi aplicada. Contudo, apesar do incremento nutricional, em geral, não se verificaram aumentos a nível da biomassa da planta em ambas as inoculações. Estes resultados confirmam que os inoculantes podem influenciar de forma positiva a absorção de nutrientes pelas plantas. RPCP e FAM são conhecidos por conferir resistência a plantas sobre stress hídrico. O revestimento de sementes com RPCP (Pseudomonas putida) mostrou, em geral, um efeito benéfico na productividade do feijão-frade, em especial, quando submetido a deficit hídrico moderado. Por sua vez, o revestimento de sementes com FAM (R. irregularis) individual ou em consórcio com P. putida promoveu a absorção de nutrientes pela planta, o conteúdo de pigmentos nas folhas e parâmetros de troca gasosa, contudo, na sua maioria na ausência de stress hidríco. Estes resultados realçam a importância de selecionar os MPCP que melhor fomentem a resiliência das plantas a stresses abióticos, a fim de tirar melhor partido da inoculação. Experiências de campo são indispensáveis para corroborar os benefícios do revestimento de sementes com microrganismos e a viabilidade para aplicações em grande escala. Uma comparação entre grão-de-bico revestido com um único isolado de R. irregularis e com uma mistura de vários isolados de R. irregularis, em estufa e em campo, mostrou que as plantas revestidas com múltiplos isolados tiveram um melhor desempenho (e.g. incremento na biomassa e na produção de grão). O revestimento de sementes mostrou ser uma ferramenta adequada para a inoculação de FAM com vantagens para a produção de grão-de-bico em ambas as escalas experimentais, em particular, para as condições de campo. A mesma mistura de isolados de R. irregularis foi usada em consórcio com Pseudomonas libanensis para revestir sementes de feijão-frade. Este tratamento aumentou significativamente a produtividade da cultura em comparação com plantas não inoculadas e plantas inoculadas com único tipo de isolados de R. irregularis + P. libanensis. Os resultados revelaram melhoramentos no conteúdo lipídico das sementes, nas propriedades físico-químicas do solo (pH e matéria orgânica do solo) e no rendimento da cultura, num sistema agrícola de baixo input. FAM e RPCP devem ser selecionadas de acordo com a sua afinidade com a planta alvo, condições de cultivo (e.g. propriedades do solo) e práticas agrícolas (e.g. irrigação, fertilização), de forma a obter lucros. Esta tese contribui para aumentar o conhecimento sobre revestimento de sementes com microrganismos, e realça o potencial da técnica como ferramenta de inoculação de FAM e RPCP e os seus benefícios em diferentes condições agrícolas. O revestimento de sementes com microrganismos pode ser de grande interesse para sistemas agrícolas sustentáveis. Contudo, de forma a permitir o uso em larga escala, como um método eficiente para a inoculação de RPCP e FAM, é necessário apostar no seu melhoramento e desenvolvimento.