Dissertationen zum Thema „Plant growth promoting rhizobacterial (PGPR)“

Plant growth promoting rhizobacteria (PGPR) colonise plant roots and exert beneficial effects on plant growth and development. The mechanisms of action of these PGPR are not conclusively known, however, there is evidence for the role of indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase production by rhizobacteria in plant growth promotion. In this study, novel-PGPR were isolated from the rhizosphere of native species as well as agricultural crop species, as opposed to other work in this field in which potential PGPR are isolated from the rhizosphere of the target plant species. One hundred and sixty six bacteria were isolated from four rhizosphere soils in Western Australia and 72 isolates were assayed for the production of IAA. In the presence of the auxin precursor L-tryptophan (L-TRP) IAA production ranged from 0-37 1-Lg/ml. Five rhizosphere soils were screened for bacteria capable of utilizing ACC as a sole nitrogen source and 13 isolates were obtained. To ensure that the isolates were not potentially deleterious to host plants, 14 IAA producing (IAA-PGPR) and all rhizobacteria capable of using ACC as a sole nitrogen source (ACC-PGPR) were tested for their effects on germinating clover and wheat seedlings. Two IAA-PGPR isolates, NCH7 and PMK4, were inhibitory to wheat seedling germination and one ACC-PGPR isolate was inhibitory to clover seedlings. Based on these findings, 6 IAA-PGPR and 4 ACC-PGPR were screened for their effects on germinating wheat seedlings in gnotobiotic growth pouch assays. Prior to these tests, spontaneous rifampicin resistant mutants were generated for 6 isolates. The mutants, or the wild type isolates where rifampicin mutants were not generated, were (re)tested for their ability to produce IAA and utilize ACC. All 10 isolates produced IAA in the presence of L-TRP ranging from 0.11-2.97 1-Lg IAA/ 1-Lg cellular protein and 7 of the isolates grew on ACC amended medium. Bacterial growth was greatly increased in some isolates in the L-TRP amended media used in the auxin assay, suggesting some of the isolates have a requirement for tryptophan for optimal growth. The largest increases in root lengths in the gnotobiotic growth pouch assays were observed for seed treated with thhe ACC-PGPR, AWMK3 (81% increase). The IAA-PGPR treatments that increased root lengths were PMK4R (76%), WMK10R (66%) and NCH45 (33%). Increases in shoot lengths were recorded for seed treated with isolates WMK10R (42%), AWMK3 (11%), APMK2R (9%) and PMK9 (9%). A reduction in germination was observed in seed treated with some isolates, particularly PMK4R and WMK10R, which reduced germination by 34% and 20%, respectively. Five of the PGPR isolated in this study were tested in the field on 2 wheat cultivars at 3 locations in Konjonup and Wongan Hills and as a co-inoculant with a commercial rhizobial strain on peas at Kojonup. All the PGPR were delivered in the field using the clay based A1osca™ carrier technology. The increases in yields in response to the inoculation with the PGPR on peas and wheat were small and not significantly different from the controls. However, the yield of wheat was improved by four of the PGPR (NCH45, NCH54, PMK9, WMK10) at the Wongan Hills heavy soil site by 2 to 23% and by NCH54 and PMK9 at the Wongan Hills light soil site by 4% and 3%, respectively compared with the uninoculated controls. On the peas at Kojonup, nodulation was improved with the isolate PMK4 and these plots were visually more vigorous than the other treatments, however this growth was not significant. At harvest, four of the PGPR (NCH45, PMK4, PMK9 WMK1 0) improved pea yields compared to the Alosca™ control by 6-13%. These results suggest that further testing is warranted. Improvements to experimental design and sampling have been recommended to allow for the detection of statistically significant small percentage increases if they occur. The findings in this study demonstrate that novel PGPR can be isolated from non-target as well as target plant species and that the screening of rhizobacteria based on their in vitro auxin production and growth promoting effects in growth pouch assays is valid for the selection of effective PGPR.

Der Mangel an Nährstoffen im Boden, hauptsächlich an Phosphor (P) und Stickstoff (N), verbunden mit einem hohen Salzgehalt und der generellen Verarmung landwirtschaftlicher Böden , sind ein ernstes Problem für die landwirtschaftliche Produktion weltweit. Daher besteht ein dringender Bedarf an Forschung und Entwicklung geeigneter landwirtschaftlicher Praktiken, um ungünstige Bodenbedingungen zu verringern und wenn möglich die Fruchtbarkeit von Kulturland wiederherzustellen. Die Verwendung von Rhizobakterien, die das Pflanzenwachstum (PGPR) fördern, kann sich bei der Entwicklung von Strategien zur Erleichterung des Pflanzenwachstums unter normalen Wachstumsbedingungen sowie unter abiotischen Stress als nützlich erweisen. Diese Bakterien bieten ihren pflanzlichen Wirten Vorteile, indem sie die Aufnahme von Bodenmineralien fördern und Pflanzen vor schädlichen Umwelteinflüssen schützen. Die vorliegende Arbeit bewertet die Rolle von in Kamerun natürlich vorkommenden PGPR an Mais und untersucht deren Potenzial als Bioimpfstoffe zur Steigerung des Pflanzenwachstums in Kamerun. Wir prüfen die Hypothese, dass einheimische Bakteriengemeinschaften aus Kamerun einen hohen Anteil an Bakterien aufweisen, deren Eigenschaften Kulturpflanzen helfen, mit ungünstigen Bedingungen umzugehen. In der vorliegenden Arbeit wurden dazu Bakteriengemeinschaften der Rhizosphäre von in Kamerun angebautem Mais isoliert und untersucht. Zum ersten Mal erfolgte eine umfassende phylogenetische Zuordnung aller kultivierbaren Bakterien, auf Grundlage ihrer potenziellen Fähigkeiten zur Förderung des Pflanzenwachstums.
Nutrient deficiencies in soil, mainly in phosphorus (P) and nitrogen (N), coupled to salinity and the impoverishment of agricultural soils, are a severe problem for agricultural production worldwide. Therefore, there is an urgent need for research and development of more suitable agricultural practices in order to reduce unfavorable conditions, and if possible, to restore the fertility of cultivated lands. The use of rhizobacteria, which promote plant growth (PGPR), can prove useful in developing strategies to facilitate plant growth under normal as well as under abiotic stress conditions. These bacteria offer benefits to plant hosts by promoting the uptake of soil minerals and protecting plants from environmental stresses. The thesis evaluates the role of native PGPR associated with maize as potential bio-inoculants for plants growth in Cameroon. We hypothesized that native bacterial communities from Cameroon include a high potential of bacteria helping the plant cope with unfavorable conditions. Here, we provide for the first time a comprehensive phylogenetic affiliation of cultivable bacterial communities associated with maize rhizosphere grown in Cameroon in relationship to their potential plant growth-promoting abilities.

L'agricoltura moderna sta affrontando sfide come la perdita di fertilità del suolo, la variabilità climatica e gli attacchi di agenti patogeni in continuo aumento. Le pratiche agricole si stanno evolvendo verso sistemi sostenibili e rispettosi dell'ambiente. L'uso di biostimolanti (PBS, plant biostimulant) è una soluzione innovativa per affrontare le sfide di un’agricoltura sostenibile che garantisce un assorbimento ottimale dei nutrienti, una resa delle colture e tolleranza agli stress abiotici. In particolare, tra i diversi tipi di biostimolanti presenti sul mercato, i rizobatteri, classificati come Plant Growth Promoting Rhizobacteria (PGPR), offrono un nuovo approccio per promuovere la crescita delle piante, la mitigazione degli stress e l’aumento della resa colturale. Pertanto i PGPR sono considerati come una sorta di "probiotici" vegetali, poiché contribuiscono in modo efficiente alla nutrizione e all'immunità delle piante. L'obiettivo principale di questa tesi è isolare e identificare batteri presenti nella rizosfera di pomodoro (Solanum lycopersicum L.) che mostrano proprietà PBS, nonché valutare i meccanismi coinvolti nell'azione di promozione della crescita delle piante (Capitolo 2) e la genetica alla base di questi meccanismi (Capitolo 3 e 4). Infatti, una profonda comprensione dei meccanismi d’azione dei PGPR potrebbe colmare la mancanza di coerenza del dato di efficacia tra gli studi di laboratorio e gli studi in campo e stimolare la ricerca per la produzione e la commercializzazione di nuovi prodotti biostimolanti microbici.
Modern agriculture faces challenges such as loss of soil fertility, fluctuating climatic factors and increasing pathogen and pest attacks. Agricultural practices have been evolving towards organic, sustainable and environmentally friendly systems. The use of natural plant biostimulants (PBS) is an innovative solution to address the challenges in sustainable agriculture, to ensure optimal nutrient uptake, crop yield, quality and tolerance to abiotic stress. In particular, among different types of biostimulants present on the market, plant growth promoting rhizobacteria (PGPR) offer a novel approach for promoting plant growth, mitigate stress and increase crop yield. Hence, PGPR inoculants are now considered as a kind of plant ‘probiotics’, since they efficiently contribute to plant nutrition and immunity. The main goal of this thesis was to isolate and identify bacteria symbionts of tomato (Solanum lycopersicum L.) rhizosphere, which showed PBS properties and evaluate mechanism involved in the action of PGPR (Chapter 2), underlying genetics and physiological pathways (Chapter 3 and 4). Indeed, a deeply understanding of the mechanisms of plant growth promotion, could fulfill the lack of consistency between lab, greenhouse and field studies, and support commercialization of novel plant biostimulant products.

L'agricoltura moderna sta affrontando sfide come la perdita di fertilità del suolo, la variabilità climatica e gli attacchi di agenti patogeni in continuo aumento. Le pratiche agricole si stanno evolvendo verso sistemi sostenibili e rispettosi dell'ambiente. L'uso di biostimolanti (PBS, plant biostimulant) è una soluzione innovativa per affrontare le sfide di un’agricoltura sostenibile che garantisce un assorbimento ottimale dei nutrienti, una resa delle colture e tolleranza agli stress abiotici. In particolare, tra i diversi tipi di biostimolanti presenti sul mercato, i rizobatteri, classificati come Plant Growth Promoting Rhizobacteria (PGPR), offrono un nuovo approccio per promuovere la crescita delle piante, la mitigazione degli stress e l’aumento della resa colturale. Pertanto i PGPR sono considerati come una sorta di "probiotici" vegetali, poiché contribuiscono in modo efficiente alla nutrizione e all'immunità delle piante. L'obiettivo principale di questa tesi è isolare e identificare batteri presenti nella rizosfera di pomodoro (Solanum lycopersicum L.) che mostrano proprietà PBS, nonché valutare i meccanismi coinvolti nell'azione di promozione della crescita delle piante (Capitolo 2) e la genetica alla base di questi meccanismi (Capitolo 3 e 4). Infatti, una profonda comprensione dei meccanismi d’azione dei PGPR potrebbe colmare la mancanza di coerenza del dato di efficacia tra gli studi di laboratorio e gli studi in campo e stimolare la ricerca per la produzione e la commercializzazione di nuovi prodotti biostimolanti microbici.
Modern agriculture faces challenges such as loss of soil fertility, fluctuating climatic factors and increasing pathogen and pest attacks. Agricultural practices have been evolving towards organic, sustainable and environmentally friendly systems. The use of natural plant biostimulants (PBS) is an innovative solution to address the challenges in sustainable agriculture, to ensure optimal nutrient uptake, crop yield, quality and tolerance to abiotic stress. In particular, among different types of biostimulants present on the market, plant growth promoting rhizobacteria (PGPR) offer a novel approach for promoting plant growth, mitigate stress and increase crop yield. Hence, PGPR inoculants are now considered as a kind of plant ‘probiotics’, since they efficiently contribute to plant nutrition and immunity. The main goal of this thesis was to isolate and identify bacteria symbionts of tomato (Solanum lycopersicum L.) rhizosphere, which showed PBS properties and evaluate mechanism involved in the action of PGPR (Chapter 2), underlying genetics and physiological pathways (Chapter 3 and 4). Indeed, a deeply understanding of the mechanisms of plant growth promotion, could fulfill the lack of consistency between lab, greenhouse and field studies, and support commercialization of novel plant biostimulant products.

En las zonas semiáridas mediterráneas del sureste de España, las escasas e irregulares precipitaciones, y un largo y seco periodo de verano han contribuido drásticamente a la aceleración de los procesos de degradación del suelo. Los cambios ambientales como consecuencia de la pérdida de las comunidades naturales de plantas, vienen a menudo acompañados o precedidos por la degeneración de las propiedades físicas y químicas del suelo, además de por una pérdida o reducción de la actividad microbiana. Actualmente se acepta que la diversidad y actividad de la microbiota del suelo es la base de uno de los mecanismos que más contribuyen a la conservación del suelo, al desarrollo y mantenimiento de la cubierta vegetal y por ende, a la estabilidad y funcionamiento del ecosistema. Así pues, el objetivo principal de este trabajo fue evaluar, en áreas degradas, la eficacia de diversas cepas de rizobacterias promotoras del crecimiento vegetal (RPCV) junto a la aplicación de enmiendas orgánicas sobre el desarrollo de la cubierta vegetal y la calidad de las propiedades del suelo, así como verificar la efectividad como RPCV de varias cepas de actinobacterias, previamente aisladas de diferentes suelos de la Región de Murcia. Con este fin, se llevaron a cabo cinco ensayos diferentes: tres de ellos en condiciones de campo, utilizando diferentes enmiendas orgánicas y RPCV, un cuarto ensayo consistente en el aislamiento de cepas de actinobacterias de la rizosfera de un arbusto autóctono presente en dos localidades diferentes de la Región de Murcia, Rhamnus lycioides L. y un quinto y último ensayo focalizado en la verificación como RPCV de las cepas de actinobacterias previamente aisladas así como el estudio de la incidencia relativa del de origen de las cepas y el suelo sujeto a plantación en la efectividad de las mismas. En todos los experimentos desarrollados en condiciones de campo, se evaluaron tanto el crecimiento y la absorción de nutrientes por parte de la planta, así como las respuestas al estrés originado por la escasez de agua. Del mismo modo, se determinaron las propiedades físico-químicas, químicas y biológicas del suelo. Con respecto al ensayo de aislamiento de actinobacterias de suelo rizosférico, se llevaron a cabo diversas técnicas que permitieron aislar y purificar diferentes cepas, así como caracterizarlas e identificarlas. Como resultados principales del trabajo, se puede destacar que en los tres primeros ensayos, las rizobacterias empleadas promovieron, satisfactoriamente, el crecimiento de las plantas así como la absorción de nutrientes y su tolerancia al estrés. En el primer experimento, en el que se ensayó sobre Cistus albidus L. una mezcla de dos rizobacterias inmovilizadas en arcilla (Azospirillum brasilense y Pantoea dispersa) como inoculante microbiano y residuo de oliva como enmienda, se observó un efecto aditivo en el tratamiento combinado, consistente en la inoculación microbiana y la adición del residuo orgánico al mismo tiempo, que permitió acrecentar las propiedades bioquímicas y microbiológicas del suelo. En el segundo ensayo en campo, en el que se probaron las mismas rizobacterias y la misma enmienda sobre Pinus halepensis Mill., se determinó que la eficacia de la inoculación microbiana fue el tratamiento más efectivo sobre el desarrollo de la planta y sobre las propiedades del suelo. El tercer ensayo se desarrolló para verificar la eficacia de diferentes cepas libres de RPCV (Bacillus megaterium, Enterobacter sp., Bacillus thuringiensis y Bacillus sp.) y la adición de residuo de remolacha azucarera compostado como enmienda orgánica sobre Lavandula dentata L. En este caso, la selección de las rizobacterias efectivas y la combinación de su inoculación junto con la aplicación de la enmienda orgánica se consideró el punto crucial del que dependería la eficacia de esta técnica de revegetación. Con respecto al cuarto ensayo, desarrollado en condiciones de laboratorio, la metodologías utilizadas para el aislamiento caracterización e identificación de diferentes especies de actinobacterias se consideraron las adecuadas, obteniéndose cuatro cepas pertenecientes al género Streptomyces que reunían las condiciones necesarias para ser consideradas potenciales RPCV. En el quinto y último ensayo, en condiciones de campo, se determinó que las bacterias previamente aisladas preservaban las habilidades descritas en condiciones de laboratorio, verificándose su rol como RPCV. Sin embargo, deberían considerarse tanto el origen de la cepa como la fertilidad biológica del suelo sujeto a plantación como factores fundamentales para la selección de cepas de actinobacterias destinadas a uso en revegetación en ambientes semiáridos.
In Mediterranean semiarid zones of Southeast Spain, limited and irregular rainfalls and a long and dry summer periods have contributed drastically to the acceleration of soil degradation processes. Environmental changes as a consequence of loss of natural plant cover are often accompanied by the physical and chemical soil properties degeneration, and by a loss or reduction of microbial activity. It is a corroborated fact that the proper functioning and stability of terrestrial ecosystems depends, to a large extent, of the diversity and composition of their vegetal cover. However, the ecological mechanisms that adjust and maintain the peculiar diversity of plant species in an ecosystem throughout the time are only known in a fragmentary way. Nowadays, it is permissible to think that the soil microbiota diversity and activity constitute the basis of one of the mechanisms that influences on soil preservation, on the development and maintenance of the vegetal cover and, consequently, on the ecosystem stability and functioning. The main objective in this Thesis was to evaluate, in degraded areas, the effectiveness of diverse plant growth promoting rhizobacteria (PGPR) strains and the addition of an organic waste on plant performance and on the soil quality properties, as well as to verify the efficacy of some actinobacteria strains as PGPR, previously isolated from different soils of Murcia. So, five different assays were developed: three field experiments involved the use of different organic amendments and PGPR strains; a fourth assay based on the isolation of different actinobacterial strains from the rhizosphere of an autochthonous shrub, that occurs naturally in two distinct sites of Murcia, Rhamnus lycioides L. and a fifth and last experiment focused on the verification as PGPR of the previously isolated actinobacteria strains as well as the study of the relative incidence of both the strain origin and the characteristics of soil subjected to plantation. In the entire field assays it was evaluated the plants growth, nutrients uptake and the biochemical and/or physiological responses of the plants. The physical, physico-chemical and biological soil properties were also determined. With regard to the experiment focused to the actinobacteria isolation from rhizosphere soil, diverse techniques were carried out allowing isolating and purifying different strains as well as to characterise and identify them. The main results obtained in this Thesis can be summarised as follows: in the assays developed under field conditions, the assayed PGPR satisfactory promoted the plant growth, the nutrients uptake and the tolerance to water stress. In the first assay, it was tested the addition of a mixture of two immobilised PGPR in clay pellets (Azospirillum brasilense and Pantoea dispersa) as microbial inoculant and olive mill residue as organic amendment on the target plant Cistus albidus L., it was observed an additive effect in the combined treatment consisting of the microbial inoculation and the organic amendment applied jointly, allowing to enhance biochemical and microbiological soil properties. In the second field experiment, developed by using the same PGPR and organic residue than in the previous assay, it was determined that the most effective treatment to improve Pinus halepensis Mill. plant performance and soil conditions was the microbial inoculation. The third experiment was developed to verify the effectiveness of diverse PGPR free strains (Bacillus megaterium, Enterobacter sp., Bacillus thuringiensis and Bacillus sp.) and the application of sugar beet residue as organic amendment Lavandula dentata L. performance as target plant. The selection of the most efficient rhizobacteria strains and their combined effect with organic residue seems to be a critical point that drives the effectiveness of using these biotechnological tools in revegetation tasks. Regarding the fourth experiment, developed under laboratory conditions, the methodologies used to the actinobacteria isolation, characterisation and identification were successful. Four strains belonging to genus Streptomyces were obtained and they met the required abilities to consider them PGPR. The actinobacteria strains were tested in a fifth assay developed under field conditions being observed that the PGPR capacities were preserved. However, the strain origin and the biological fertility of plantation soil must be considered to an adequate actinobacteria strain selection to be used in restoration programs under semiarid conditions.

Wastewater or fish effluent (FE) from freshwater aquaculture can be a good and cheap liquid fertiliser for plants. However, while it represents a good source of nutrients to support plant growth in a system called aquaponics, it appears that its use needs to be optimised to take full advantage of the potential benefits. Apart from mineral amendments, the use of beneficial microorganisms that can have a direct impact on plant growth and nutrient utilisation could be a promising option. Plant growth promoting rhizobacteria (PGPR) are a group of rhizospheric bacteria, when introduced in association with the host plant at optimum density, can enhance plant growth and health. One well-known and versatile PGPR is Azospirillum brasilense that has numerous beneficial effects on plants. The production of phytohormones by the bacterium has been proposed as one of the major mechanisms responsible for the plant growth promoting effects observed in plants inoculated with Azospirillum. Hence, this PGPR could be a valuable input in vegetable production under an aquaponics system. In addition, despite the widespread studies conducted with this PGPR in various crops, there is no published report on vegetables fertilised with fish effluent or under an aquaponics system. This study focuses on evaluating the role of PGPR, particularly A. brasilense, on the growth and development of selected vegetable crops fertilised with fish effluent and using an aquaponics system. Strains of A. brasilense Sp7, Sp7-S and Sp245, Herbaspirillum seropedicea and Burkholderia phytofirmans PsJNT were used to inoculate seeds and/or seedlings by soaking and/or drenching. Inoculated and uninoculated seeds and seedlings were germinated and raised in controlled growing cabinets and a greenhouse, respectively. PGPR-inoculated vegetable seeds generally germinated faster and had better early seedling growth than uninoculated controls. Cucumber seeds inoculated with strains Sp7, Sp245 and H. seropedicea exhibited increase in germination percentage and shoot length by 9 and 20%, respectively, while all PGPR improved the germination vigour index, and enhanced length and weight of seedling roots by 25 and 23%, respectively. In tomato, Sp7-S enhanced the germination value, while most PGPR, except Sp7, significantly improved the germination vigour, root length (28%) and weight (37%) with superior vigor. In lettuce, Sp7-S, Sp245 and H. seropedicea inoculation resulted in longer roots (26%). Germination vigour was also improved by inoculation, except for B. phytofirmans. This improved germination and early seedling growth characteristics may influence future crop establishment and production. Of the two laboratory-based inoculation methods used, soaking appeared to be a better technique for enhanced early seedling growth by strains of A. brasilense. This effect could be related to their unique metabolic characteristics of the strains. The growth promoting effects of A. brasilense strains on the early seedling growth of vegetables varied between the bacterial strains and crop species, In particular, strains Sp7-S and Sp245 strongly enhanced root (85%) and shoot (75%) growth, germination value and vigour in tomato when inoculated by soaking. Sp245 increased endogenous plant IAA (indole-3-acetic acid) content of cucumber and lettuce by up to 100%, irrespective of inoculation method. This work demonstrates that the strains can be used for inoculation within the studied range of cell concentrations with or without plant growth promoting (PGP) effects. However, strain Sp7 appeared to be more influential at lower inoculum concentrations (about log10 6), while Sp7-S and Sp245 at log10 7 cfu mL-1 or higher. For instance, cucumber seeds inoculated with Sp7 log10 8 and 6, Sp7-S and Sp245 log10 8 and 7 cfu mL-1 increased seedling growth, vigour index and endogenous plant IAA by up to 55%. In lettuce, the inoculation with log10 6 of Sp7, log10 7 and 6 of Sp7-S, and log10 8 and 7 of Sp245 yielded superior seedling growth with improved seedling vigour, while log10 7 and 8 of Sp7 and Sp7-S, respectively, increased plant IAA concentration by more than 20%. In tomato, Sp7 at log10 6, Sp7-S and Sp245 at log10 7 enhanced the root biomass, while inoculation with all concentrations of Sp7 and Sp7-S, and log10 8 of Sp245 significantly increased plant IAA content by up to 300%. The inoculation with the bacterial cell suspension exerted more beneficial effects on the early seedling growth, vigor and endogenous plant IAA. In cucumber, seeds inoculated with bacterial cell and those treated with IAA solutions produced longer roots and shoots by 163 and 60%, respectively. Seedlings also exhibited superior vigor. These treatments, together with culture supernatant, and combined cell and supernatant, also increased endogenous plant IAA content, in which the combined cell and supernatant produced up to four-fold greater plant IAA concentrations. In lettuce, seeds inoculated with cell suspension produced longer roots (86%) with superior seedling vigour and elevated plant IAA. In tomato, inoculation with cell suspension and treatment with IAA solutions enhanced length of roots length by up to 52 and 188%, respectively, while all treatments significantly increased the plant IAA content by 70%. These results also demonstrate that bacterial cell suspension and combined cell and supernatant showed consistent effects on the expression of plant IAA. This work suggests that the endogenous IAA levels in the seeds during germination have been altered by the activity of live bacteria and phytohormones present in the supernatant. The altered root morphology of the seedlings due to A. brasilense inoculation might have enhanced the capacity of roots to absorb water and essential minerals leading to enhanced plant growth and metabolic activity. For instance, inoculated cucumber seedlings produced longer roots (23%), greater root biomass (19%), higher total phosphorus (15%), endogenous plant IAA (101%) and peroxidase activity (134%). In lettuce, inoculation increased root length (22%), peroxidase activity (53%) and plant IAA (38%). In addition, strain Sp7 enhanced the chlorophyll and protein contents by 25 and 42%, respectively. In tomato, inoculation resulted in longer roots (67%), larger leaves (22%), higher dry matter accumulation (33%), protein (15%) and endogenous plant IAA (94%) contents. Taller seedlings (12%) with larger stems (15%) and more developed leaves (9%) with greater fresh biomass (18%) were observed with Sp7 inoculation, while two-fold increase in peroxidase activity due to strain Sp245 was detected. On the other hand, inoculated basil seedlings grown in soil produced longer roots (90%), taller seedlings (19%) with more (25%) and larger (61%) leaves, which resulted in greater seedling biomass (61%) and phosphorus content (3%), and higher peroxidase activity (122%) particularly for those inoculated with Sp245 and Sp7, respectively. These plant growth promoting effects were also observed in basil grown in an aquaponics system. These include larger stems and leaves (25%), fresh weight yield (17%), peroxidase activity (73%), phosphorus (5%) and protein (23%) contents due to inoculation. The amount of endogenous plant IAA (27%) and chlorophyll (13%) contents were also increased by Sp7 and Sp7-S inoculation, respectively. This further suggests that A. brasilense could be a valuable agent to help maximize the usefulness of fish effluent or wastewater from freshwater aquaculture for vegetable seedling production. The 16S rDNA terminal restriction fragment length polymorphism (T-RFLP) analysis revealed that inoculation with A. brasilense has no adverse effect to the existing rhizobacterial communities (measured by the changes in the distribution of detectable operational taxonomic unit (OTU) (represented by TRF)) in the root rhizosphere of vegetables (i.e. lettuce, cucumber and basil) grown under different systems (i.e. sterile artificial substrate, soil and aquaponics). This highlights that this PGPR did not cause disturbance to the resident microbial communities or imbalance of the normal functioning of the system. In aquaponics, the presence of a substantial density of A. brasilense strains in the root rhizosphere of basil and the enhanced plant growth and physiological parameters of inoculated basil may imply that Azospirillum have successfully established a beneficial association with the existing bacterial populations. Moreover, this study demonstrates the potential of Azospirillum to be a practical agent for enhancing plant growth and development of vegetables fertilised with fish effluent and under aquaponics system. Directing future research endeavors to better understand the basic mechanisms occurring in the Azospirillum-plant interaction rather than exploring large scale application of this PGPR would support further development of the bioinoculant technology.

Nitrogen-fixing bacteria in soybean nodules convert atmospheric nitrogen to plant-available forms in exchange for carbon from the plant, but other non-nitrogen-fixing bacteria also reside in nodules, and their role in the nodule is not well understood. This study was conducted to determine the effect of three non-nitrogen-fixing Pseudomonas spp. strains isolated from nodules on soybean, and we hypothesized these strains benefit soybean. A greenhouse study in which two cultivars of soybean (Asgrow AG46X6 and Pioneer P48A60X) were treated with three fluorescent Pseudomonas spp. strains (referred to in this study as Bullseye, Pancake, and Starfish) and an uninoculated control. Soybeans were harvested at two time points: the R2/R3 growth stage and the R6 growth stage. Following each harvest, measures of growth, yield, and nitrogen fixation were taken, and data were analyzed using two non-parametric, multivariate analyses: multiple response permutation procedure (MRPP) and permutational multivariate analysis of variance (PERMANOVA). Both analyses showed soybeans of both cultivars treated with Pancake differed from controls following the first harvest but not the second. When analyzed individually, most metrics for growth, yield, and nitrogen fixation following the first harvest were not significantly different between Pancake and control treatments, but Pancake treatment means were still generally higher than controls. If metrics are considered collectively in conjunction with the results of the multivariate analyses, the results show Pancake generally increased soybean growth and nitrogen fixation. These findings support the hypothesis that non-nitrogen-fixing bacteria from nodules benefit plants, and such bacteria have the potential to serve as biofertilizers.
Master of Science in Life Sciences
Soybeans are one of the most commonly grown crops in the world, and nitrogen-fixing bacteria colonize the roots of soybeans and initiate the formation of spherical nodules attached to the roots. Inside the nodules, these bacteria convert atmospheric nitrogen to plant-available forms in exchange for sugar from the plant, and such bacteria reduce the need to add nitrogen fertilizer to agricultural fields. Other non-nitrogen-fixing bacteria also reside in nodules, but their role in the nodule is not well understood. If these bacteria benefit soybeans, they have the potential to serve as biofertilizers (microbial inoculants that promote plant growth). This study was conducted to determine whether non-nitrogen-fixing bacteria isolated from nodules benefit soybean. A greenhouse study in which two cultivars of soybean (Asgrow AG46X6 and Pioneer P48A60X) were grown in soil and were either left uninoculated or were inoculated with one of three strains of bacteria from the genus, Pseudomonas (referred to in this study as Bullseye, Pancake, and Starfish). Following harvest, measures of growth, yield, and nitrogen fixation were taken, and data showed the bacteria generally benefited the soybean plants. Although, these results showed the bacteria benefitted the plants, field trials and further testing in the greenhouse should be conducted before using these bacteria as commercial biofertilizers. Additionally, the effects of other non-nitrogen-fixing nodule bacteria on soybeans should also be tested to identify other beneficial strains, and the cost of production should be compared to the potential gains of using such bacteria before they are developed into biofertilizers.

Zur Aufklärung des Wirkungsmechanismus der toleranzerhöhenden Wirkung gegenüber Salinität des Pflanzenwurzeln besiedelnden PGPR Bacillus subtilis wurden bakterielle Stoffwechselprodukte der Stämme FZB24 und FZB41 bei der Testpflanze Tomate unter dem Einfluss von hohem Salzstress getestet. Das Kulturfiltrat mit der Gesamtheit der von B. subtilis produzierten Stoffwechselprodukte zeigte im axenischen Test zur Ermittlung des Wachstums nach 7-tägiger Behandlung der Sämlinge und nachfolgender Kultivierung unter Salzstress eine gewisse toleranzerhöhende Wirkung bei 0,1 %-Konzentration. Zur Produktaufschlüsselung wurde das Kulturfiltrat über Adsorberharz und HPLC fraktioniert. Diese Fraktionen, sowie die aus dem Kulturfiltrat nach 19 h Fermentation wurden ebenfalls bei Sämlingen axenisch getestet. Fraktionen mit verschiedenen Proteinen und Peptiden, die von B. subtilis produziert werden, zeigten teilweise eine konzentrationsabhängige Wirkung hinsichtlich der Wachstumsstimulierung und zugleich Toleranzerhöhung gegenüber Salzstress, weshalb nachfolgend ein Peptidextrakt aus B. subtilis einer Testung im axenischen System unterzogen wurde. Der Peptidextrakt zeigte gleichfalls eine erkennbare konzentrationsabhängige Wirkung. Mit gleichem Testsystem wurden Auxin-Präkursoren und Auxin selbst, die als Stoffwechselprodukte von B. subtilis nachgewiesen sind, sowohl als Wurzelbehandlung, wie auch als Blattbehandlung bei Sämlingen geprüft. Zusätzlich wurde die Wirkung der Auxine auf den Wassergehalt der Sämlinge unter Salzstress, sowie die Adventivwurzelbildung von Hypokotylsegmenten aus etiolierten Sämlingen in An- und Abwesenheit von Salinität getestet. Darüber hinaus wurde die Aufnahme und der Transport von Auxinen, ebenfalls bei Sprosssegmenten aus etiolierten Sämlingen in An- und Abwesenheit von Salinität geprüft. Schließlich wurde die Wirkung der Auxine auf das Wachstum und den Wassergehalt in einer Hydrokultur im Gewächshaus unter Salzstress ermittelt. Die Ergebnisse zeigen, dass namentlich Auxin-Präkursoren und z. T. Auxin als Stoffwechselprodukte von B. subtilis eine Erhöhung der Salzstresstoleranz bei der Testpflanze herbeiführen können, wenngleich die Wirkung auf die Salztoleranz sehr differenziert und unterschiedlich stark ausgeprägt war. Der vorhandene Effekt vor allem der Auxin-Präkursoren wird als offenbar bedeutendster Mechanismus für die wachstumsstimulierende und zugleich toleranzerhöhende Wirkung gegenüber Salinität des Rhizobakteriums bei Wurzelbesiedlung und Interaktion mit dem pflanzlichen Stoffwechsel diskutiert.
To find out the mode of tolerance increasing action against salinity of the plant root colonizing PGPR Bacillus subtilis, bacterial metabolites of the strains FZB24 and FZB41 were studied in the test plant tomato under the influence of high salinity. Because the culture filtrate with the whole range of produced metabolites by B. subtilis showed to a certain extent a tolerance increasing action at dilution of 0,1 % in axenic plant growth tests after 7 days treatment of seedlings and subsequent cultivation under salt stress, it has been fractionated with adsorber resin and HPLC. These fractions, as well as fractions from the culture filtrate after 19 h fermentation were tested also by seedlings in axenic culture. Fractions with different proteins and peptides, which were produced by B. subtilis, showed partly activities also depending of concentration with regard to the growth stimulation and at the same time tolerance increase against salt stress. Following also a peptide extract from B. subtilis was examined in the axenic plant test system, showing similarly a visible action depending of concentration. In the same test system there were tested further auxin precursors and auxin itself, which are known metabolites of B. subtilis, on seedlings both by root treatment and leaf treatment. Additionally was studied the action of auxins on the water content of the seedlings under salt stress, as well as on the adventitious root formation of hypokotyl segments from etiolated seedlings, in presence and absence of salinity. Finally it was studied the uptake and transport of auxins in segments of stems from etiolated seedlings in presence and absence of salinity. Lastly it was tested the action of auxins on plant growth and water content in a hydroponic cultivation under greenhouse conditions and salt stress. The results show that particularly auxin precursors and partly auxin as metabolites of B. subtilis can induce an increase in the salt stress tolerance of the test plant, although the action on the salt tolerance was differentiated and variable in its extent. The existing effect firstly of the auxin precursors is discussed as obviously main mechanism for the plant growth stimulating and at the same time tolerance increasing action of the rhizobacterium against salinity by root colonization and interaction with the plant metabolism.

Bacillus amyloliquefaciens FZB42 ist ein frei lebendes Bakterium, das Pflanzenwurzeln besiedelt und das Pflanzenwachstum durch viele verschiedene Wirkmechanismen anregt. In dieser Arbeit wurden die molekularen Grundlagen dieser positiven Wirkungen, die dieses „Pflanzenwachstum fördernde Rhizobakterium“ (PGPR) auf seine Wirte ausübt, untersucht. Um den gegenseitigen Austausch von B. amyloliquefaciens und seinen Wirtspflanzen zu entschlüsseln, wurden umfangreiche Proteomstudien durchgeführt. Es wurden Referenzkarten der extrazellulären und zytosolischen Proteinfraktionen erstellt. Die größte Anzahl an ausgeschiedenen Proteinen konnte während der stationären Phase beobachtet werden. Die identifizierten extrazellulären Proteine gehören verschiedenen Funktionsklassen an, wobei die prominentesten Klassen am Kohlenhydrat-Abbau und den Transport von Molekülen durch die Zellwand beteiligt sind. Die zytosolischen Extrakte von Kulturen, die in 1C-Medium bzw. Mineralmedium angezogen wurden, und in der zweidimensionalen Gelelektrophorese (2 DE) aufgetrennt wurden, ergaben 461 und 245 verschiedene Protein-Einträge. Die erstellten Referenz-Karten wurden anschließend verwendet, um Proteine und Prozesse, in an der Interaktion mit Pflanzen beteiligt sind, zu identifizieren. Dafür wurden die Bakterien Wurzelexudaten von Mais (Zea mays L.) ausgesetzt. Die Proteine aus zwei Stämmen, denen die globalen Transkriptionsregulatoren (Degu, AbrB) und vier Sigma-Faktoren (SigB, SigM, SigV, und SigX) fehlen, wurden ebenfalls untersucht, um ihre Beteiligung an den bakteriellen Reaktionen auf die Wurzelausscheidungen zu analysieren. Zusammenfassend ist dies die erste Studie, die umfangreiche Proteomdaten von Gram-positiven PGPR präsentiert, wobei gleichzeitig die Veränderung der Expression von extrazellulären und zytoplasmatischen Proteinen, nach Zugabe von Wurzelexudaten, ausgewertet wurde.
Bacillus amyloliquefaciens strain FZB42 is a free-living bacterium that competitively colonizes plant roots and stimulates plant growth by many different modes of action. The molecular basis of singular beneficial effects that this Plant Growth-Promoting Rhizobacteria (PGPR) exert on their hosts have been studied. To decipher the molecular cross-talk of B. amyloliquefaciens and its’ host plants as a whole system, an extensive proteomic approach was performed. Reference maps of the extracellular and cytosolic protein fractions were established. The highest number of secreted proteins was observed during stationary growth phase. Identified extracellular proteins belong to different functional classes, with the most prominent classes involved in carbohydrate degradation and transportation of molecules across the cell wall. Cytosolic extracts obtained from cultures grown in 1C and minimal media subjected to the 2 Dimensional Electrophoresis (2 DE), revealed 461 and 245 different protein entries, respectively. Created reference maps were subsequently used to identify proteins and processes involved in the interaction with plants, prior to exposure of bacteria to maize (Zea mays L.) root exudates. The proteomics of two strains lacking expression of genes coding for global transcriptional regulators (degU, abrB) and four sigma factors (sigB, sigM, sigV, and sigX) were also inves-tigated, in order to analyse their involvement in bacterial responses to root exudates. In summary, this is the first study presenting comprehensive proteomics of Gram-positive PGPR, evaluating at the same time changes in protein expression caused by addition of root exudates at the extracellular and cytosolic level.

Les bactéries promotrices de la croissance des plantes (PGPR) peuvent améliorer la performance et la tolérance des plantes lors de stress environnementaux. Arabidopsis thaliana est un modèle de choix pour étudier les mécanismes impliqués dans les interactions plante-bactéries. Nous avons analysé de multiples traits associés à la dynamique de croissance, au développement et la physiologie des végétaux afin d'évaluer les effets de l'inoculation par Phyllobacterium brassicacearum STM196, une PGPR isolée de la rhizosphère du colza, sur les réponses d'A. thaliana à des stress hydriques de différentes intensités. Grâce à des outils performants de phénotypage, nous avons développé une nouvelle approche d'analyse à haut-débit pour examiner l'implication de STM196 dans les stratégies de résistance des plantes au stress hydrique. Nos résultats montrent pour la première fois que les PGPR peuvent interférer dans les stratégies d'échappement des plantes grâce à des modifications de la croissance et du temps de floraison. De plus, STM196 induit une meilleure résistance au déficit hydrique modéré et une meilleure tolérance à la déshydratation sous une contrainte hydrique sévère. L'inoculation par STM196 peut ainsi représenter une valeur ajoutée aux stratégies de résistance intrinsèques aux plantes, ce qui est illustrée par sa remarquable capacité à promouvoir la survie et la production de biomasse végétale dans des environnements contrastés. Nos résultats soulignent l'importance des interactions plantes-bactéries dans les réponses des plantes à la sécheresse et offrent de nouvelles voies de recherches pour l'amélioration de la résistance à la sécheresse dans les cultures
Plant growth promoting rhizobacteria (PGPR) can enhance plant performance and plant tolerance to environmental stresses. Arabidopsis thaliana is a useful organism to study the mechanisms involved in plant-PGPR interactions. We analyzed multiple plant traits related to growth dynamics, development and physiology in order to assess the effects of Phyllobacterium brassicacearum STM196 strain, isolated from the rhizosphere of oilseed rape, on Arabidopsis responses to well-defined soil water availability. Using powerful tools for phenotyping, we developed a new high-throughput analysis to examine the implication of STM196 on plant strategies to cope with water stress. Our results show for the first time that PGPR can interfere in escape strategies of plants through modifications in plant growth and flowering time. Moreover, STM196 induced a better resistance to moderate water deficit and a better tolerance to dehydration under a severe stress. Inoculation by STM196 can represent an added value to plant resistance strategies, as illustrated by its remarkable ability to promote plant survival and biomass production under contrasted environments. Our results highlight the importance of plant-bacteria interactions in plant responses to drought and provide a new avenue of investigations to improve drought resistance in crops

Studies on plant growth-promoting rhizobacteria (PGPR) and fungi (PGPF) as biostimulants have shown significant positive effects on plant health, fruit yield, or pest management. However, very few published studies to date have been specific regarding their effects on strawberries (Fragaria × ananassa), particularly on soilborne disease prevalence in organically grown strawberries. Empirical data on the results of using these products in commercial growing applications under various conditions would be highly valuable, especially for organic growers who have limited synthetic chemical pesticides, herbicides and fertilizers registered for use. The objective of this study is to evaluate the efficacy of biostimulant supplementation on strawberries for improving fruit yield, fruit quality, and plant health in both high-tunnel, open-sided ‘hoophouse’ and field conditions. This study consisted of two research projects. The first project investigated the effects of commercially available PGPR-based biostimulant products on strawberry plant health. The three products contained differing proprietary combinations of PGPR, primarily from the Bacillus and Lactobacillus genera. Plants were grown in two different soil types: sandy and clay, in order to investigate the effects of biostimulant supplementation in different soil conditions. In fall of 2018, 160 ‘Monterey’ strawberry plants were grown in an outdoor hoophouse in 3-gallon pots. Plants were either treated monthly with a single bacterial biostimulant product (EM-1, Accomplish LM, or Armory), or left untreated as a control. Plants were grouped into 20 blocks, each block comprised of 8 plants (each of the four treatments replicated in both soil types). Fruit yield (g), fruit sugar content (Brix), and leaf SPAD absorbance levels were measured weekly from January 27 to June 26, 2019. The treatments tested had no significant effects on fruit yield, leaf SPAD absorbance or Brix; soil type, however, did significantly impact fruit yield, with higher yields in sandy soil. The second project was a field trial beginning in spring of 2020, in collaboration with Rutiz Farms in Arroyo Grande, CA, involving a total of 480 ‘Chandler’ strawberry plants. The farm is organically managed and has a history of soilborne diseases, including Verticillium dahliae. These plants were either treated monthly with one of three microbial biostimulant products: a product containing a proprietary strain of Trichoderma harzianum biocontrol fungus (TrichoSym), and two of the same PGPR-based products used the previous year (Accomplish LM and Armory); or left untreated as a control. The experiment was laid out in a randomized complete block design with four blocks, with each block consisting of 4 plots for each of the 4 treatments; each plot contained 30 plants. Fruit yield (g) per plot was measured weekly throughout the 2020 growing season and phenotypic disease incidence was measured biweekly. Soil samples were taken at three different points throughout the season, cultured on selective media, and analyzed to obtain estimates of V. dahliae colony-forming units (CFU) per gram soil. The treatments tested had no significant effect on fruit yield, phenotypic disease incidence, or V. dahliae CFU/g soil. The results are inconclusive as to whether this lack of effect is due to viability of the products themselves, ineffective application techniques resulting in lack of rhizosphere colonization, or some combination of these. Further research is needed to determine whether or not supplementation with microbial biostimulants can produce reliable, beneficial results in strawberries.

Thesis (M. Tech. (Agriculture)) -- Central University of Technology, Free State, 2013
ntroduction: Good quality Eucalyptus is of importance to South Africa’s pulp and paper industry. Limited land is available for forestry, therefore Eucalyptus with genotypes for good pulp and paper qualities, particularly hybrids, are bred and cloned via cuttings. Although these Eucalyptus clones keep the favourable genotypes in the population, many have difficulty with rooting. Research has shown that rhizobacteria can improve rooting. Thus, one strategy to enhance the rooting of cuttings is to use rhizobacterial preparations. The aim of this study was to characterise rhizobacterial communities of Eucalyptus hybrid and species and identify possible plant-growth promoting rhizobacteria (PGPR). Materials and methods: Rhizospheric samples were collected from Eucalyptus hybrids and species. The rhizobacterial communities were characterised using fatty acid methyl esters (FAME) analysis and denaturing gradient gel electrophoresis (DGGE). DGGE fragments were further sequenced to identify rhizobacteria. Results and discussion: FAME analysis successfully achieved a broad characterisation of the Eucalyptus hybrid and species rhizobacterial communities based on their fatty acid composition. Myristic acid (C14:0) was the most abundant fatty acid. DGGE profiles gave a molecular profile of the Eucalyptus hybrid and species rhizobacterial communities based on their DNA composition. Nitrosomona eutropha was present in all samples which illustrates a nitrogen-rich environment. Adhaenbacter aquaticus was unique to the better rooting Eucalyptus hybrid GU111. Conclusion: This study provided some insight into the diversity of rhizobacterial communities of Eucalyptus hybrids and species. Possible PGPR were identified and the observation made that the nature of the soil environment changes with the aging of the associated host. These findings allow further investigation into the formulation of potential rhizobacterial preparations for rooting enhancement of Eucalyptus cuttings.

碩士
國立嘉義大學
生物農業科技學系研究所
106
Plant growth-promoting rhizobacteria (PGPR) had been applied to plant growth widely. Previously, bacterial strains of genera of Bacillus and Pseudomonas have been isolated from soil based on their ability of secreting plant hormone IAA. Evidence of antimicrobial activity of these bacterial strains included detecting genes involved in synthesis of antimicrobial compounds, colony antagonistic analysis, antimicrobial activity of bacterial culture solution, and ability of secretion of microbial proteases. The antagonism assay of dual culture and volatile compounds of bacteria strains of Bacillus sp. and Pseudomonas sp. showed inhibition effect against Colletotrichum gloeosporioides and Fusarium oxysporum. In bacteria dual culture assay, the results showed that Bacillus sp. and Pseudomonas sp. inhibited the growth of Erwinia chrysanthemi. In soft rot inoculation test, the infected rate were decreased in Phalaenopsis orchid, Brassica rapa, variety chinensis and Brassica campestris, variety chinensis treated with bacteria and soybean fermentation product (SPH). In Fusarium wilt inoculation test, the infected ratio in banana seedlings treated with Pseudomonas sp. and SPH were decreased. In A. thaliana plant test, gene expression associated with Salicylic acid (SA) and Jasmonic acid (JA) pathways was increased after treated with Pseudomonas sp. and SPH. The western blot results showed the higher expression levels of antioxidant enzymes such as Catalase (CAT), and also defense relative protein such as Lipoxygenase (LOX)、Pathogenesis-related protein 2 (PR-2) and Pathogenesis-related protein 3 (PR-3). The results suggested that these PGPR strain and their protein hydrolysate products are potential products for the application of biocontrol.

Although the pesticide DDT has been banned from use in Canada for more than three decades, DDT still persists in Canadian farmlands at detectable levels. Much effort, such as incineration, thermal desorption, and bioremediation, has been used to remediate DDT contaminated soils, but so far it is either too expensive or impractically slow. In this study, a three-year period of field trials was performed to investigate phytoremediation of DDT contaminated soil. In the field trials, millet, fall rye, sugar beet, potato, and pumpkin, treated with plant growth-promoting rhizobacteria (PGPR) were planted on two sites. As well, untreated plants were planted as a control. Plant growth, and 4,4’-DDT plus 4,4’-DDE concentrations in plant tissues and soil were monitored regularly. Comparing the plant growth between PGPR treated and untreated, PGPR significantly promoted the plant growth. On site 1, the root length and root weight of fall rye treated with PGPR were 16% and 44% greater, respectively, compared to the untreated plants. The root and shoot dry weights of millet treated with PGPR were 38% and 47% greater than those untreated plants. Root dry weight of sugar beet treated with PGPR was increased by 74% compared to untreated sugar beet. A significant effect of growth promotion was also observed in pumpkin and potato treated with PGPR. Following plant growth, DDT detection in plants was performed. 4,4’-DDT and 4,4’-DDE were found in plant tissues of fall rye, millet, sugar beet, and pumpkin. The concentrations of 4,4’-DDT and 4,4’-DDE in fall rye roots were 0.61 and 0.59 μg/g, respectively. In pumpkin tissues at harvest, 4,4’-DDT and 4,4’-DDE concentrations were 0.67 and 1.64 μg/g in roots, 1.06 and 2.05 μg/g in the lower stems, and 0.2 and 0.32 μg/g in the upper stems. The data indicated that it is feasible to phytoremediate DDT from contaminated soil. In addition, 4,4’-DDT concentrations in soils with different plant species were determined. In millet plot on site 1, 4,4’-DDT concentration in rhizosphere soil dropped by 41% in 2006 compared to 4,4’-DDT concentration at t0. In sugar beet plot on site 1, 28% of 4,4’-DDT dropped in rhizosphere soil in 2007. In pumpkin plot on site 1, 4,4’-DDT in rhizosphere soil was decreased by 22% in 2007. The results show that 4,4’-DDT concentration in rhizosphere soil was significantly lower than the initial level of DDT. Based on the data of 4,4’-DDT in soils and plant tissues, a mass balance was constructed and calculated. The preliminary mass balance shows that the total amount that DDT decreased in rhizopshere soil approximately equals to the total amount of DDT accumulated in plant tissues. This indicates that phytoextraction is the mechanism of DDT phytoremediation. In addition, PGPR promoted plant growth and then enhanced the phytoremediation efficiency of DDT. Therefore, the research indicates that PGPR assisted phytoremediation has a great potential for remediation of DDT and other chlorinated aromatics from impacted soil.

Soil salinity is a widespread problem that limits crop yield throughout the world. The accumulation of soluble salts in the soil can inhibit plant growth by increasing the osmotic potential of interstitial water, inducing ion toxicity and nutrient imbalances in plants. Over the last decade, considerable effort has been put into developing economical and effective methods to reclaim these damaged soils. Phytoremediation is a technique that uses plants to extract, contain, immobilize and degrade contaminants in soil. The most common process for salt bioremediation is phytoextraction which uses plants to accumulate salt in the shoots, which is then removed by harvesting the foliage. As developing significant plant biomass in saline soils is an issue, a group of free-living rhizobacteria, called plant growth promoting rhizobacteria (PGPR), can be applied to plant seeds to aid plant growth by alleviating salt stress. The principle objective of this research was to test the efficacy of PGPR in improving the growth of plants on salt-impacted soils through greenhouse and field studies. In this research, previously isolated PGPR strains of Pseudomonas putida. UW3, Pseudomonas putida UW4, and Pseudomonas corrugata CMH3 were applied to barley (Hordeum valgare C.V. AC ranger), oats (Avena sativa C.V. CDC baler), tall wheatgrass (Agropyron elongatum), and tall fescue (festuca arundinacea C.V. Inferno). PGPR effects on plant growth, membrane stability, and photosynthetic activity under salt stress were examined. Greenhouse studies showed that plants treated with PGPR resulted in an increase in plant biomass by up to 500% in salt-impacted soils. Electrolyte leakage assay showed that plants treated with PGPR resulted in 50% less electrolyte leakage from membranes. Several chlorophyll a fluorescence parameters, Fv/Fm, effective quantum yield, Fs, qP, and qN obtained from pulse amplitude modulation (PAM) fluorometry showed that PGPR-treated plants resulted in improvement in photosynthesis under salt stress. Field studies showed that PGPR promoted shoot dry biomass production by 27% to 230%. The NaCl accumulation in plant shoots increased by 7% to 98% with PGPR treatment. The averaged soil salinity level at the CMS and CMN site decreased by 20% and 60%, respectively, during the 2008 field season. However, there was no evidence of a decrease in soil salinity at the AL site. Based on the improvements of plant biomass production and NaCl uptake by PGPR observed in the 2008 field studies, the phytoremediation efficiency on salt-impacted sites is expected to increase by 30-60% with PGPR treatments. Based on the average data of 2007 and 2008 field season, the time required to remove 25% of NaCl of the top 50 cm soil at the CMS, CMN and AL site is estimated to be six, twelve, and sixteen years, respectively, with PGPR treatments. The remediation efficiency is expected to accelerate during the remediation process as the soil properties and soil salinity levels improve over time.

A dissertation submitted in partial fulfilment of the requirements of the degree of Master of Science in Environmental science School of Animal, Plant and Environmental Sciences University of Witwatersrand, Johannesburg. March 2017.
Plant growth promoting rhizobacteria (PGPR) promotes plant growth in a variety of modes of action and also suppresses several phytopathogens causing plant diseases. There is evidence that Pseudomonas strains are able to induce systemic resistance, thereby enhancing the defensive capacity of many plant species, and they do so without any negative impact on the environment. Currently, many agricultural systems rely more on the use of chemical pesticides to combat plants diseases. The chemicals have several negative impacts on both human health and the environment. Therefore, there is need to investigate the ability to fight plant pathogens of alternatives like the Pseudomonas spp that do not harm the environment. Several strains of this genus are yet to be tested to see if they induce systemic resistance. Previous studies showed that bio surfactants produced by Pseudomonas koreensis exhibited strong effect against oomycetes P. ultimum in tomato plants. Induced systemic resistance (ISR) potential of P. koreensis following exposure to viruses has not been fully demonstrated to date. This study sought to investigate whether this strain has an effect on viruses and if it is able to induce systemic resistance against viral pathogens. The study started by growing the model plant N. benthamiana. The second stage involved carrying out assays of tobacco mosaic virus (TMV) after inoculating this virus in three bio treatments: (i) seed treatment of N. benthamiana with P. koreensis (referred to as the early treatment), (ii) root treatment at the transplanting stage (late treatment) and (iii) the control. In bio treatments (i) seeds were first sterilized by dipping them into 70% alcohol for 3 minutes and 0.1 % HgCl2 for 1 minute and washing them with distilled water. Each seed was then soaked into 20ml of bacteria suspension for 30 minutes and in (ii) a litre of P. koreensis culture was then poured onto the roots of 36 N. benthamiana plants. The bacteria suspension was added at 107 colony forming units per gram of soil to each tray. It was observed that disease severity was lower in the P. koreensis plant treatments than for the control. Results of this investigation have shown that P. koreensis can induce systemic resistance in foliar parts when plant seeds or roots are inoculated with this strain. This was demonstrated by separation of plant growth promoting rhizobacteria (PGPR) bacteria and TMV. Seeds and roots were inoculated with bacteria while the leaves were inoculated with TMV. The early bio treatment had the lowest mean number of necrotic lesions, and exhibited the mildest effects from TMV compared to the late bio treatment and control. Plants in the late bio treatment were moderately affected while the control was severely affected (P˂0.0001) ˂0.05. The early and the late bio treatment both had higher leaf surface area than the control; (P˂0.0001) ˂0.05. The early bio treatment lost the fewest leaves, and the late bio treatment lost a moderate number while the control lost the highest number (P˂0.0001)˂0.05.The reduced symptoms exhibited by plants inoculated with P. koreensis is an indication that P. koreensis has anti-viral activity against TMV. It was concluded that P. koreensis can reduce plant‟s viral susceptibility and result in ISR. It is hence proposed that P. koreensis can be used as a biological control (bio control) agent against viruses. Key words: Tobacco Mosaic Virus (TMV), Pseudomonas koreensis (P. koreensis), induced systemic resistance (ISR)
LG2018

Soil salinity affects an estimated one billion hectares worldwide. Excess salinity inhibits plant growth, limiting crop production. This is caused by osmotic stress in saline soil, nutrient imbalance and specific ion toxicity. There have been many methods of remediation investigated, including excavation, soil washing and phytoremediation. Phytoremediation involves the growth of plants on impacted soils to degrade or sequester contaminants. The remediation of salts relies on the uptake of ions into plant biomass where the salt is sequestered and the biomass can then be harvested. This method removes the salt from the site and leaves the top soil in place, which aids in revegetation after site remediation is completed. Plant-growth promoting rhizobacteria (PGPR) improves plant growth by lowering the levels of stress ethylene within the plant, thereby increasing the biomass available to sequester ions. The objectives of this research were to investigate the efficiency of phytoremediation of salt impacted soils in field remediation sites. Previously isolated strains of PGPR (UW3, Pseudomonas putida; UW4, Pseudomonas putida; and CMH3, Pseudomonas corrugata) were used in field trials involving the planting of oats (Avena sativa), annual ryegrass (Lolium multiflorum), tall wheatgrass (Agropyron elongatum) and tall fescue (Festuca arundinacea C.V. Inferno). The salt tolerance of various switchgrass (Panicum virgatum L.) cultivars (Cave-In-Rock, Southlow, Forestburg, and common) was compared to tall wheatgrass and Inferno tall fescue to investigate the potential of switchgrass for phytoremediation. Improvement of seed germination under salt stress by H2O2 pre-treatment was investigated both as an individual treatment and in combination with CMH3 treatment. The ion uptake into plant biomass was iii compared to the change in salinity, to determine how much of the decrease in site salinity is accounted for by uptake of salt by plants. H2O2 pretreatment resulted in a 50% increase in root and shoot emergence of tall wheatgrass under 75 mM NaCl stress compared to control treatments, which matched the germination improvement observed with PGPR treatment. The combination of H2O2 and CMH3 showed a similar improvement to root emergence under stress, but had no observable effect on shoot emergence when compared to the no-H2O2-no-PGPR control. Switchgrass cultivars showed a lower germination rate than tall wheatgrass at salt levels from 0 mM to 150 mM NaCl. The measured uptake of Na+, K+, Ca2+, Mg2+ and Cl- into plant biomass during a phytoremediation field trial was able to account for approximately 70% of the observed change in salinity in 2008. In 2009 the uptake of Na+ and Cl- into Kochia scoparia, a weed species that invaded the field site after a hard frost, was able to account for 36% of the observed change in salinity.

Upstream oil and gas production has caused soil salinity problems across western Canada. In this work we investigated the use of ACC (1-aminocyclopropane-1-carboxylate) deaminase-producing plant growth-promoting rhizobacteria (PGPR) and the arbuscular mycorrhizal fungus (AMF) Glomus intraradices to enhance the efficiency and feasibility of phytoremediation of saline soils. This work involved laboratory and field research for three sites in south east Saskatchewan, Canada. The three research sites were Cannington Manor South (CMS), Cannington Manor North (CMN) and Alameda (AL). CMS and AL were highly saline, while the CMN site had moderate salinity. Indigenous PGPR were isolated from these sites and tested in greenhouse experiments using authentic salt-contaminated soils taken from the research sites. Increased plant biomass by PGPR and/or AMF was observed. This growth promotion effect varied with plant species, soil salinity and soil fertility. The combination treatment of two previously isolated PGPR Pseudomonas putida UW3 and UW4 (noted as UW3+4) from farm soil in Ontario consistently promoted shoot growth of both barley and oats grown in saline soils by approximately 100%. The indigenous PGPR Pseudomonas corrugata (CMH3) and Acinetobacter haemolyticus (CMH2) also promoted plant growth on par with UW3+4. In addition, in one experiment where alfalfa was tested, UW3+4, CMH2 and CMH3 treatments not only enhanced shoot biomass but also increased root nodulation. For AMF effects, G. intraradices enhanced biomass of oats and barley. Furthermore, the AMF+CMH3 was effective in promoting growth of Topgun ryegrass, while AMF+CMH2 was beneficial for Inferno tall fescue growth in salt impacted soils. The concentration of NaCl in the plants grown in salt-impacted soils ranged from 24 – 83 g/kg. There was no evidence of an increase in NaCl concentrations of plant tissue by PGPR and/or AMF treatments. In addition, to determine the importance of nutrient addition to research sites, liquid fertilizer was applied to 2-week old plants. Results demonstrated that fertilizer effectively increased biomass, and more importantly the biomass of PGPR treated plants supplied with fertilizer was approximately 20% higher than that of plants treated with fertilizer alone. Therefore, research sites were then amended with compost before planting of the 2007 field trial. Plant growth promotion by UW3+4 and CMH3 was tested in the summer of 2007 in the field. Prior to planting, soils were sampled from each site for soil salinity analysis. Barley, oats, tall fescue and ryegrass treated with and without PGPR were sown in plots. The plant coverage condition, NaCl concentrations and biomass of plant shoots were assessed to evaluate the PGPR effect. The results showed that PGPR promoted shoot dry weight by 30% - 175%. The NaCl concentrations of barley, oats and tall fescue averaged 53 g/kg, 66 g/kg and 35 g/kg, respectively. There was no evidence of an increase in NaCl concentrations of plant tissue by PGPR in the field. The salt removal of the CMN site was the highest among three sites due to the large amount of shoot biomass produced. The amount of salt accumulated in the shoots on the CMN site is estimated to be 1580 kg per hectare per year when both barley and ryegrass are planted together as a mix and treated with PGPR. Based on the field data, the estimated time required to remove 50% salt in the top 50 cm soil is seven years with PGPR treatments, while it takes fifteen years to do so without PGPR. In conclusion, PGPR-promoted phytoremediation was proven to be a feasible and effective remediation technique for soils with moderate salinity.

Salt stress is a severe environmental challenge in agriculture, limiting the quality and productivity of the crops around the globe. Plant growth promoting rhizobacteria (PGPR) is proposed as a friendly solution to overcome those challenges. The desert plant endophytic bacterium, Enterobacter sp. SA187 has shown plant growth promotion and salt stress tolerance beneficial effect on the model plant Arabidopsis thaliana in vitro as well as under the field conditions on different crops. SA187 has a distinguished morphology of yellow colonies (SA187Y) that could be due to carotenoid biosynthesis. However, the bacteria tend to lose the yellow color upon incubation with the plants and the colonies turn to white (SA187W). In comparison to SA187Y, SA187W shows 50% reduction on the beneficial impact on A. thaliana fresh and dry weight of root and shoot system. By counting the CFU/plant, we showed that SA187Y and SA187W both have similar colonization rate in both shoots and roots. Under non-salt conditions, optimal bacterial colonization was observed on day 8 after inocubation, however, under the salt stress condition, the optimal colonization was observed at day 4. Moreover, during the time period of the incubation of the SA187Y with the plants, there was a consistent noticeable loss of the yellow color of the colonies. This change in color is only observed eight days after transfer and the number of white colonies increases with the increase of the incubation time. In addition, SA187W was GFP-tagged by Tn7 transposon system and visualized by confocal laser scanning microscopy. The SA187W-GFP colonies have shown a similar colonization pattern as SA187Y-GFP, bacteria were colonizing the differentiation zone and cell elongation zone in the roots. Finally, the gene expression of the carotenoid biosynthesis pathways genes in SA187Y showed an overall higher gene expression compared to SA187W. In conclusion, the color loss seems to affect the beneficial impact of the bacteria on plants. However, the reduced beneficial impact is not due to the colonization efficiency of bacteria on the plant roots but could be due to a regulation of gene expression of carotenoid biosynthesis.

Aflatoxin contamination is a qualitative problem in groundnut (Arachis hypogaea L.) occurring at both pre-and post-harvest stages. These aflatoxins are secondary metabolites produced by Aspergillus flavus and A. parasiticus and have carcinogenic, hepatotoxic, teratogenic and immuno-suppressive effects. Use of plant growth-promoting rhizobacteria (PGPR) is a viable and sustainable option in managing aflatoxin problem in groundnut. The objectives of our present study were to evaluate the prevalence of A. flavus infection and aflatoxin contamination in groundnut oil mills/traders of Telangana and Andhra Pradesh (AP); differentiate toxigenic and atoxigenic strains using cultural methods; identify a superior PGPR (Pseudomonas fluorescens) isolate and determine its mode of inhibition on A. flavus and aflatoxin contamination. Pod samples were collected from eight selected oil mills/traders in Mahaboobnagar, Rangareddy, Nizamabad, Karimnagar (Telangana); and Anantapur (AP) districts and A. flavus infection was enumerated. Further, aflatoxin contamination in kernels was estimated by indirect competitive ELISA. A total of 24 A. flavus isolates were obtained from the collected pod samples. These isolates were identified as toxigenic/atoxigenic using cultural detection methods on Yeast extract sucrose (YES) media and coconut agar medium (CAM). Ten native P. fluorescens isolates from groundnut rhizosphere were isolated and screened against A. flavus by dual culture and in vitro seed colonization (IVSC) assays. Superior PGPR isolate (Pf7) against mold infection and aflatoxin contamination was screened for determining its mode of inhibition using scanning electron microscopy (SEM). In Telangana, kernel infection by A. flavus ranged from 42 (Mahaboobnagar) to 90.7% (Nizamabad). After Nizamabad, samples from Rangareddy district recorded up to 90% kernel infection. In AP, Tadimarri mandal recorded kernel infection up to 29.3%, whereas Tadipatri recorded up to 59.3%. Aflatoxins in kernels from these mills in Telangana were highest in Rangareddy (1205.2 μg kg-1) followed by Karimnagar (365.5 μg kg-1). Oil mills of Nizamabad and Mahaboobnagar have recorded aflatoxins to a tune of 4.9 and 11.5 μg kg-1in Telangana

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].

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.

Due to climate changes, global temperature is projected to increase 1.5 °C or more. Consequentially, drought is expected to impact over 50% of the arable lands by 2050, affecting several crops, including maize, which is the third main food crop in the world. In fact, maize yield globally is expected to suffer a reduction of 15% because of drought stress. On the other hand, world population is predicted to reach 9 billion by 2050. Thus, there is the need to ensure food availability and security, to respond to an increased need to feed the growing population. In this context, rhizobacteria emerge as a more sustainable alternative or complement to chemical fertilizers. These bacteria exist naturally in the soil and are used to promote plant growth, and induce tolerance to abiotic factors, like drought, and are therefore called plant growth promoting rhizobacteria. Several strains have been applied in crops as biofertilizers, increasing productivity. Hence, this thesis aimed to explore the process of development of biofertilizers composed by plant growth promoting bacteria. To achieve that, maize plants were inoculated with rhizobacteria isolated from wild legumes, as well as with rhizobacteria isolated from maize plants and were grown in greenhouse under irrigated and drought conditions to screen for potential candidates to apply in the field. Posteriorly, in the field tests, maize yield was assessed to determine plant growth, and biochemical parameters were analyzed to understand how rhizobacteria inoculation improved maize development. Results evidenced maize growth and drought stress mitigation in the greenhouse, and biochemical parameters analyzed reveal the positive effect of bacterial inoculation, in the field. Additionally, rhizobacteria were isolated from maize plants growing under three levels of water deficit to understand if there would be any differences in their characteristics. Growth promotion abilities were evaluated as well as osmotolerance. In fact, microbial community associated with maize roots was affected by drought. Nevertheless, several strains were able to produce siderophores and bacteria isolated from conditions subject to water deficit had a lower osmotolerance. In general, the results of this thesis evidenced the potential of rhizobacteria to be applied in maize crops to mitigate drought stress and improve growth, ultimately increasing crop production.
Devido às alterações climáticas, prevê-se uma subida na temperatura mundial igual ou superior a 1.5 °C. Consequentemente, espera-se que a seca impacte mais de 50% da terra arável, afetando várias colheitas, incluindo o milho, que é a terceira principal colheita mundial. De facto, antecipa-se que a produtividade do milho a nível mundial diminua cerca de 15% devido ao stress hídrico. Por outro lado, a população mundial continua a aumentar, atingindo potencialmente 9 mil milhões até 2050. Logo, é fundamental garantir a disponibilidade de alimentos e segurança alimentar, de modo a responder às necessidades de uma população humana cada vez maior. Neste contexto, as rizobactérias surgem como uma alternativa mais sustentável ou um complemento ao uso de fertilizantes químicos. Estas bactérias existem naturalmente no solo e são usadas para promover o crescimento de plantas e para induzir tolerância a fatores abióticos, como a seca, e por isso são denominadas rizobactérias promotoras de crescimento de plantas. Várias espécies de bactérias têm sido aplicadas nas colheitas como biofertilizantes, aumentando a sua produtividade. Posto isto, esta tese tem como objetivo explorar o processo através do qual são desenvolvidos biofertilizantes compostos por rizobactérias promotoras de crescimento de plantas. Para tal, plantas de milho foram inoculadas com rizobactérias isoladas de raízes de leguminosas selvagens, bem como, rizobactérias isoladas de raízes de milho, e foram crescidas em estufa, sob condições normais, em que as plantas foram irrigadas e condições de seca, para encontrar potenciais candidatos para aplicar em testes de campo. Posteriormente, nos testes de campo, a produtividade do milho foi averiguada para determinar o crescimento do milho, e alguns parâmetros bioquímicos foram analisados de modo a entender-se se a inoculação com rizobactérias melhora o desenvolvimento desta colheita. Os resultados evidenciaram que a inoculação contribuiu para o aumento do crescimento do milho e a sua tolerância à seca em estufa e os parâmetros bioquímicos analisados revelam o efeito positivo da inoculação das bactérias nos estudos em campo. Adicionalmente, foram isoladas rizobactérias de raízes de milho que fora crescido em três níveis de défice hídrico para entender se haveria alguma diferença nas suas características. As capacidades de promoção de crescimento, bem como a osmotolerância foram avaliadas. De facto, a comunidade microbiana associada com as raízes do milho foi afetada pela seca. Ainda assim, várias estirpes foram capazes de produzir sideróforos e as bactérias isoladas de condições sujeitas a seca tiveram uma menor osmotolerância. No geral, os resultados desta tese evidenciam o potencial da aplicação de rizobactérias no milho para mitigar o stress causado pela seca e melhorar o seu crescimento, aumentando consequentemente a produtividade desta colheita.
Mestrado em Microbiologia

O estudo de técnicas agrícolas sustentáveis tornou-se fundamental face ao aumento da população e ao crescente desgaste dos recursos naturais. Assim, esta tese visa a integração de leguminosas num contexto de agricultura sustentável a nível europeu, utilizando para tal uma série de estudos de caso nos quais o papel da ervilha, da soja e da fava foi avaliado. O primeiro estudo de caso analisou culturas intercalares de cevada e ervilha e de que forma estas últimas podem afetar a nutrição da cevada, bem como que consequência traz a limitação de ferro (Fe) no caso da ervilha. Na cevada, destaca-se o aumento significativo (p<0.05) da concentração de Fe, magnésio (Mg), fósforo (P), cálcio (Ca) e zinco (Zn), quando intercalada com ervilha. Posteriormente, a limitação de Fe induzida durante o crescimento hidropónico da ervilha gerou o aumento (p<0.05) da concentração de Fe ao nível da parte aérea da planta enquanto que a concentração de Fe (p<0.0001), boro (B) (p<0.01) e Mg (p<0.05) diminuiu ao nível da raíz. Nas mesmas condições de limitação, a expressão da enzima Fe redutase foi de 0.022 μmol Fe/g FW-1 h-1, diminuindo 67% relativamente a uma situação de administração normal de Fe (100 μM Fe3+-EDDHA). Mais ainda, o gene nitrito redutase 1 (NIR1) aumentou (p<0.05) a sua expressão ao nível da raíz e da parte aérea da planta em situação de carência. O segundo estudo permitiu a análise do conteúdo mineral de favas previamente selecionadas pela sua maior percentagem de azoto (N), tendo em conta propriedades morfológicas do grão e parâmetros de rendimento. As variedades de fava com teor de N mais elevadas não foram indicativas de um perfil mineral melhorado e apresentaram-se heterogéneas dentro da mesma variedade. O terceiro incluiu a caraterização de estirpes de bactérias presentes em três inóculos comerciais destinados à produção de soja (Euralis, LegTech, RhizLiq), bem como avaliou o impacto da sua aplicação. A identificação molecular das estirpes Bradyrhizobium japonicum e Bradyrhizobium diazoefficiens nos inóculos testados mostrou que a sua dupla combinação com micorriza arbuscular é a mais eficaz para a maximização dos efeitos da inoculação na soja. Por último, o quarto estudo compreendeu a análise quantitativa da produção de hormona vegetal ácido 3-índole-acético (IAA) por parte de isolados de Rhizobium leguminosarum bv. viciae, relacionando a referida produção com a biomassa da ervilha de onde surgiram os isolados. Foi ainda efetuada uma análise qualitativa em placa da solubilização de fosfato nas mesmas amostras. As análises funcionais de rhizobia de ervilha demonstraram uma relação não significativa (R2=0,1324) entre a produção de hormona vegetal e a biomassa de cada uma das amostras, assim como também não ocorreu solubilização de fosfato na maioria dos casos. Globalmente, estes estudos contribuem para o desenvolvimento da literatura científica nesta área, confirmando o papel das leguminosas em técnicas agrícolas sustentáveis de melhoria da qualidade nutricional e agronómica das culturas.
The study of sustainable agricultural techniques has become essential with the emergence of population growth and the depletion of natural resources. In this way, this thesis aims to integrate legumes in a context of sustainable agriculture at a european level, using for that purpose a set of case studies in which the role of pea, soybean and faba bean was evaluated. The first case study examined the intercrop of barley and pea and how the latter can affect barley nutrition, as well as the consequence of limiting iron (Fe) in the case of pea. A significant increase (p<0.05) in the concentration of Fe, magnesium (Mg), phosphorus (P), calcium (Ca) and zinc (Zn) was observed in barley when intercropped with pea. The Fe limitation induced during hydroponic growth of pea increased Fe concentration (p<0.05) in the shoot, while the concentration of Fe (p<0.0001), boron (B) (p<0.01) and Mg (p<0.05) decreased at the root level. Under the same limiting conditions, the Fe reductase enzyme expression was of 0.022 μmol Fe/g FW-1 h-1, which was decreased 67% when compared to standard Fe administration (100 μM Fe3+-EDDHA). Moreover, the expression of the gene nitrite reductase 1 (NIR1) increased (p<0.05) in the root and shoot of the plants in Fe deficiency. The second study allowed the mineral content analysis of previously selected faba beans by their higher percentage of nitrogen (N), considering the grain morphological properties and yield parameters. The faba varieties with higher N content were not indicative of an improved mineral profile and were heterogeneous within the same variety. The third included the characterization of bacterial strains present in three commercial inoculums destined to soybean production (Euralis, LegTech, RhizLiq), as well as the impact of its application. Molecular identification of the strains Bradyrhizobium japonicum and Bradyrhizobium diazoefficiens in the inoculums tested showed that their double combination with arbuscular mycorrhiza is the most effective for maximizing the effects of soybean inoculation. Finally, the fourth study comprised the quantitative analysis of the production of 3-indole-acetic acid (IAA) hormone by Rhizobium leguminosarum bv. viciae isolates, relating this production to the pea biomass. A qualitative plate analysis of the phosphate solubilization of the previous isolates was also carried out. Functional analyses of pea rhizobia showed a non-significant relationship (R2=0.1324) between the hormone production and the samples biomass, as well as it did not occur phosphate solubilization in most cases. Globally, these studies contribute to the development of scientific literature in this area, confirming the role of legumes in sustainable agricultural techniques for improving the nutritional and agronomic quality of crops.