Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Centrolobium paraense Tul. conhecida popularmente como pau-rainha é uma leguminosa arbórea que ocorre em ilhas de mata, florestas de transição e matas de galerias das savanas de Roraima. Esta leguminosa se beneficia do processo de fixação biológica de nitrogênio (FBN) através da simbiose com bactérias do grupo rizóbio. No entanto, pouco se sabe sobre a diversidade e a eficiência dessas bactérias em simbiose com pau-rainha. Este estudo teve como objetivo a caracterização de rizóbios associados às raízes de pau-rainha em Roraima, bem como a avaliação da eficiência simbiótica na promoção do crescimento de mudas através da FBN. Para isso, plantas de pau-rainha foram cultivadas em solos coletados em ilhas de mata localizadas nos municípios de Mucajaí, Bonfim, Boa Vista e Normandia para obtenção dos nódulos, e posterior isolamento das bactérias em meio de cultura. As bactérias foram caracterizadas morfologicamente e agrupadas de acordo com os perfis gerados. Representantes de cada grupo foram avaliados quanto à capacidade de nodulação utilizando o feijão-caupi como planta hospedeira. Os que apresentaram resultados positivos foram caracterizados geneticamente através da técnica de BOX-PCR e sequenciamento parcial do gene 16S rRNA. Em seguida uma nova autenticação foi realizada, e os isolados que repetiram os resultados anteriores ou apresentaram similaridade com espécies reconhecidamente nodulíferas foram selecionados para teste de eficiência em pau-rainha. Para avaliar a eficiência da FBN, os rizóbios foram utilizados na inoculação de plântulas de pau-rainha sob condições estéreis de casa de vegetação durante 100 dias, procedendo à análise estatística para os parâmetros: nitrogênio total, massa seca da parte aérea, área foliar, massa seca de raiz, número de folíolos, altura da planta, diâmetro do caule, massa seca de nódulos e número de nódulos. Os identificados como Bradyrhizobium foram avaliados através do sequenciamento de cinco genesglnII, gyrB, rpoB, recA e dnaKpara análise de sequência multilocus (MLSA). De um total de 355 nódulos coletados, foram obtidos 178 isolados, sendo a maioria de crescimento lento com capacidade de alcalinizar o meio de cultura, compatíveis com o gênero Bradyrhizobium. A partir da caracterização morfológica foi gerado um dendrograma onde se constatou a formação de nove grupos com perfis distintos. Quarenta isolados foram selecionados para a autenticação e trinta e seis foram capazes de induzir a nodulação. A análise do BOX-PCR revelou-se que estes isolados apresentam pouca semelhança entre eles. No entanto a análise do sequenciamento do gene 16S rRNA, revelou que a maioria dos isolados pertenciam ao gênero Bradyrhizobium. Estes confirmaram a capacidade de nodulação e foram selecionados para ensaio de eficiência em pau-rainha juntamente com quatro isolados identificados comoRhizobium tropici e Pleomorphomonas oryzae. Os isolados pertencentes ao gênero Bradyrhizobium foram os mais eficientes, com destaque para os isolados ERR 326, ERR 399 e ERR 435 que proporcionaram os melhores resultados em todas as avaliações. A partir da MLSA ficou evidenciado que os isolados apresentaram grandes divergências com as estirpes de referência de Bradyrhizobium, indicando haver espécies novas colonizando o pau-rainha. / Centrolobium paraense Tul. popularly known as wood-queen is a leguminous tree that occurs in islands of forest, transition forest and gallery forests of Roraima savannas. This legume benefits from the process of biological nitrogen fixation (BNF) through symbiosis with rhizobia bacteria group. However, little is known about the diversity and efficiency of these bacteria in symbiosis with wood-queen. This study aimed to characterize rhizobia associated with the roots of wood-queen in Roraima, as well as evaluating the symbiotic effectiveness in promoting the growth of seedlings by FBN. For this, plants of wood-queen were grown in soils collected in forest islands located in the municipalities of Mucajaí, Bonfim, Boa Vista and Normandia to obtain the nodules, and subsequent isolation of bacteria in culture medium. The bacteria were morphologically characterized and grouped according to the profiles generated. Representatives of each group were evaluated for nodulation using cowpea as host plant, and those who tested positive were genetically characterized using the technique of BOX-PCR and partial sequencing of the 16S rRNA. Then a new authentication was performed, and that the isolated or repeated previous results showed similarity with known nodulating species were selected to test efficiency in wood-queen. To evaluate the efficiency of BNF, the strains were used to inoculate seedlings wood-queen under sterile conditions in a greenhouse for 100 days, carrying out statistical analysis for theparameters: total nitrogen, shoot dry weight, leaf area, root dry weight, number of leaves, plant height, stem diameter, dry mass and number of nodes. Those identified as Bradyrhizobium were evaluated by sequencing five genes glnII, gyrB, rpoB, recA and dnaK for multilocus sequence analysis (MLSA). From a total of 355 nodes collected were obtained 178 isolates, most slow growing with capacity to alkalinize the culture medium, compatible with the genus Bradyrhizobium. From the morphological characterization of a dendrogram was generated which demonstrated the formation of nine groups with distinct profiles. Forty isolates were selected for authentication and thirty six were able to induce nodulation. A BOX-PCR analysis revealed that these isolates show little similarity between them. However the analysis of 16S rRNA gene sequencing revealed that most isolates belonged to the genus Bradyrhizobium. These confirmed the nodulation and were selected for assay efficiency wood queen with four isolates identified as Rhizobium tropici and Pleomorphomonas oryzae. The isolates belonging to the genus Bradyrhizobium were the most efficient, especially for isolates ERR 326, ERR 399 and ERR 435 that provided the best results in all evaluations. From the MLSA was evident that the isolates showed large differences with the reference strains of Bradyrhizobium, indicating a new species colonizing the wood-queen.

Soil samples from different locations with varied soybean cultivation histories were taken from arable fields in 2018 in East Germany and Poland (Lower Silesia) to evaluate the specific microsymbionts of the soybean, Bradyrhizobium japonicum, one to seven years after inoculation. Soybeans were grown in the selected farms between 2011 and 2017. The aim of the experiment was to investigate whether there is a difference in rhizobia contents in soils in which soybeans have been recultivated after one to seven years break, and whether this could lead to differences in soybean plant growth. The obtained soil samples were directly transferred into containers, then sterilized soybean seeds were sown into pots in the greenhouse. After 94 days of growth, the plants were harvested and various parameters such as the nodular mass, number of nodules, and dry matter in the individual plant parts were determined. In addition, the relative abundances of Bradyrhizobium sp. in soil samples were identified by sequencing. No major decline in Bradyrhizobium sp. concentration could be observed due to a longer interruption of soybean cultivation. Soil properties such as pH, P, and Mg contents did not show a significant influence on the nodule mass or number, but seem to have an influence on the relative abundance of Bradyrhizobium sp. The investigations have shown that Bradyrhizobium japonicum persists in arable soils even under Central European site conditions and enters into an effective symbiosis with soybeans for up to seven years.:Introduction Materials and Methods Results Discussion Supplementary Materials Author Contributions Funding Acknowledgments Conflicts of Interest References

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.

Diazotrophic microbes reside in soybean nodules; however, other non-nitrogen fixing bacteria are a part of the interior nodule microbiome. Results from a previous greenhouse study show that a novel species of Pseudomonas associates with soybean nodules as a plant-growth promoting rhizobacteria (PGPR). This study observes the soybean growth promoting potential of Pseudomonas spp. in a field setting. Additionally, this study observed differences in soybean growth promotion based on amending the plant with isolated strains or a mixed culture of the species' strains. Two cultivars of soybean (Asgrow AG46X6 and Pioneer P48A60X) were either amended with isolated strains of the novel Pseudomonas spp. (referred to as PAMW1 and BUMW2 in this study), a mix of the two strains, or an uninoculated control. The study recorded measurements to observe growth, yield, and nitrogen fixation differences. The study uses two-way factorial ANOVAs and non-parametric, multivariate analyses to determine differences in growth promotion among samples. Soybean amended with PAMW1 has greater shoot mass, biomass, and height than other treatments. Through nonmetric multidimensional scaling (NMS), samples amended with a mixed culture or PAMW1 may be different regarding growth promotion relative to the non-amended samples. Univariate results support the hypothesis that the novel Pseudomonas spp. benefit soybean in a field setting. However, it is inconclusive whether a mixed culture amendment of multiple strains alters the overall growth promotion of soybean compared to samples amended with isolated strains. / Master of Science / Soil hosts a relatively abundant and diverse community of microorganisms. Moreover, the area of soil that interacts closely with plant roots and their associated exudates, called the rhizosphere, has a significantly greater microbial abundance than surrounding bulk soil. Interactions between microbes and the plant often promote plant growth because of secondary metabolites produced by these beneficial microbes. One particular bacterial species, belonging to the Pseudomonas genus, was discovered and extracted from the soybean nodule interior. Nitrogen-fixing bacteria predominantly reside in the soybean nodule, yet this microorganism cannot fix nitrogen. Although trace amounts of non-nitrogen-fixing bacteria reside in the soybean nodule, this novel species has a relatively high abundance. This study determines the benefits of this species in the soybean nodule. Following positive results in a greenhouse study, this field experiment observes variance in soybean growth and productivity based on their received bacterial amendment. For this study, two soybean cultivars were either amended with an isolated strain of this species, a mix of the two strains, or left uninoculated to serve as a control. Numerous recorded measurements serve as indices of soybean growth and productivity. The results suggest that this novel Pseudomonas species benefits the plant by significantly improving biomass. With further research, this species can potentially serve as an environmentally sensitive and sustainable alternative to fertilizers through its ability to promote soybean growth.

Na indução de resistência a planta possui mecanismos de defesa físicos e químicos para impedir a entrada e o desenvolvimento de patógenos e parasitas, incluindo fungos, bactérias, vírus, nematóides e até insetos. Estes mecanismos são ativados por infecções prévias ou pelo tratamento com agentes indutores (eliciadores) bióticos ou abióticos. Entre os agentes indutores bióticos, destaca-se a S. cerevisiae, que além da importância biotecnológica, tem demonstrado em estudos prévios potencial para o controle de doenças em várias plantas de importância econômica. Produtos à base de S. cerevisiae, como por exemplo o Agro-Mos® (carboidratos da parede celular da levedura) estão disponíveis no mercado, mas não como indutores de resistência. Já o indutor químico registrado como Bion® vem sendo comercializado e utilizado na indução de resistência em diversas espécies de plantas contra vários patógenos. Os objetivos deste trabalho foram purificar parcialmente frações de S. cerevisiae indutoras de resistência em pepineiro contra antracnose, causada por Colletotrichum lagenarium, e avaliar o efeito do extrato bruto autoclavado de S. cerevisiae, Agro-Mos® e Bion® na indução de resistência contra o inseto T. absoluta em tomateiro, o nematóide M. incognita em pepineiro, como também, verificar o efeito destes agentes na interação simbiótica entre soja e B. elkanii. Os resultados mostraram que o extrato bruto aquoso de S. cerevisiae autoclavado por 4 h foi o mais efetivo na redução da antracnose. Dessa maneira, o mesmo foi submetido à precipitação etanólica e o sobrenadante da precipitação foi fracionado utilizando-se Cromatografia de Troca Aniônica - CTA. Obtiveram-se quatro picos, sendo que os picos I (frações não ligada à resina DEAE-celulose) e II (frações ligadas à resina DEAE-celulose) foram os mais efetivos na proteção de plântulas de pepineiro reduzindo a severidade de antracnose em 80% e 72%, respectivamente. A aplicação foliar do extrato bruto aquoso de S. cerevisiae, Agro-Mos® e Bion® não afetou o desenvolvimento do inseto em tomateiro, como também, não interferiu significativamente na multiplicação do nematóide em raízes de pepineiro. Na interação simbiótica da soja com B. elkanii, os agentes testados não afetaram a nodulação por B. elkanii em raízes e o desenvolvimento vegetativo das plantas. Porém, a aplicação foliar do extrato bruto autoclavado de S. cerevisiae aumentou a quantidade de nitrogênio total da parte aérea das plantas. Finalmente, conclui-se que frações de S. cerevisiae induziram resistência em pepineiro contra C. lagenarium. Por sua vez, os agentes testados são foram eficientes no controle do inseto herbívoro e do nematóide e não demonstraram efeito negativo na interação soja - rizóbio. / In the resistance induction, the plant has physical and chemical defense mechanisms to avoid the entrance and the development of pathogens and parasites, including fungi, bacteria, virus, nematodes and even insects. These mechanisms are activated by previous infections or by the treatment with biotic and abiotic inducer agents. Among the biotic agents there is S. cerevisiae, that besides the biotechnological importance, was shown in previous studies to control diseases in several plants of economical importance. Products made of S. cerevisiae, as for exemple, the Agro-Mos® (formulated with carbohydrates from the cellular wall of the yeast) are available in the market, but not resistance inducers. The chemical inducer known as Bion® is already marketed and used to induced resistance in several plant species against several pathogens. The objectives of this work were to partially purify fractions of S. cerevisiae able to induce resistance in cucumber against anthracnose, caused by Colletotrichum lagenarium, and also evaluate the effect of the autoclaved crude aqueous extract from S. cerevisiae, Agro-Mos® and Bion® in the resistance induction against the insect T. absoluta in tomato plants, the nematode M. incognita in cucumber plants, as well as to verify the effect of the agents in the symbiotic interaction envolving soybean and B. elkanii. The results showed that the crude aqueous extract of S. cerevisiae autoclaved for 4 h was the most effective out in the reduction of cucumber anthracnose. Thus, the same extract was submitted to ethanolic precipitation and the obtained supernatant was fractioned by using Anion Exchange Chromatography - AEC. For peaks were obtained and peak I (non-adsorbed fraction to DEAE-Cellulose) and II (fraction adsorbed to DEAE-Cellulose) were the most effective out in the protection of the cucumber seedling by reducing anthracnose severity in 81% and 72% ,respectively. The application of the autoclaved extract of S. cerevisiae, Agro-Mos® and Bion® did not affect the development of the insect in tomato plants as well as did not interfere significantly in the multiplication of the nematode in cucumber roots. In the symbiotic interaction of soybean and B. elkanii, the tested agents did not affect the formation of nodules in soybean roots and the vegetative development of the plants. However, the foliar application of the autoclaved crude extract of S. cerevisiae significantly increased the amount of total nitrogen in the aerial part of the plants. Finally, it is concluded that the fractions (peaks I and II) of S. cerevisiae induced resistance of the cucumber plants. However the tested agents were not efficient in the control of the herbivore insect and the nematode and did not exhibit negative effects in the symbiotic interaction soybean and rhizobium.

Na indução de resistência a planta possui mecanismos de defesa físicos e químicos para impedir a entrada e o desenvolvimento de patógenos e parasitas, incluindo fungos, bactérias, vírus, nematóides e até insetos. Estes mecanismos são ativados por infecções prévias ou pelo tratamento com agentes indutores (eliciadores) bióticos ou abióticos. Entre os agentes indutores bióticos, destaca-se a S. cerevisiae, que além da importância biotecnológica, tem demonstrado em estudos prévios potencial para o controle de doenças em várias plantas de importância econômica. Produtos à base de S. cerevisiae, como por exemplo o Agro-Mos® (carboidratos da parede celular da levedura) estão disponíveis no mercado, mas não como indutores de resistência. Já o indutor químico registrado como Bion® vem sendo comercializado e utilizado na indução de resistência em diversas espécies de plantas contra vários patógenos. Os objetivos deste trabalho foram purificar parcialmente frações de S. cerevisiae indutoras de resistência em pepineiro contra antracnose, causada por Colletotrichum lagenarium, e avaliar o efeito do extrato bruto autoclavado de S. cerevisiae, Agro-Mos® e Bion® na indução de resistência contra o inseto T. absoluta em tomateiro, o nematóide M. incognita em pepineiro, como também, verificar o efeito destes agentes na interação simbiótica entre soja e B. elkanii. Os resultados mostraram que o extrato bruto aquoso de S. cerevisiae autoclavado por 4 h foi o mais efetivo na redução da antracnose. Dessa maneira, o mesmo foi submetido à precipitação etanólica e o sobrenadante da precipitação foi fracionado utilizando-se Cromatografia de Troca Aniônica - CTA. Obtiveram-se quatro picos, sendo que os picos I (frações não ligada à resina DEAE-celulose) e II (frações ligadas à resina DEAE-celulose) foram os mais efetivos na proteção de plântulas de pepineiro reduzindo a severidade de antracnose em 80% e 72%, respectivamente. A aplicação foliar do extrato bruto aquoso de S. cerevisiae, Agro-Mos® e Bion® não afetou o desenvolvimento do inseto em tomateiro, como também, não interferiu significativamente na multiplicação do nematóide em raízes de pepineiro. Na interação simbiótica da soja com B. elkanii, os agentes testados não afetaram a nodulação por B. elkanii em raízes e o desenvolvimento vegetativo das plantas. Porém, a aplicação foliar do extrato bruto autoclavado de S. cerevisiae aumentou a quantidade de nitrogênio total da parte aérea das plantas. Finalmente, conclui-se que frações de S. cerevisiae induziram resistência em pepineiro contra C. lagenarium. Por sua vez, os agentes testados são foram eficientes no controle do inseto herbívoro e do nematóide e não demonstraram efeito negativo na interação soja - rizóbio. / In the resistance induction, the plant has physical and chemical defense mechanisms to avoid the entrance and the development of pathogens and parasites, including fungi, bacteria, virus, nematodes and even insects. These mechanisms are activated by previous infections or by the treatment with biotic and abiotic inducer agents. Among the biotic agents there is S. cerevisiae, that besides the biotechnological importance, was shown in previous studies to control diseases in several plants of economical importance. Products made of S. cerevisiae, as for exemple, the Agro-Mos® (formulated with carbohydrates from the cellular wall of the yeast) are available in the market, but not resistance inducers. The chemical inducer known as Bion® is already marketed and used to induced resistance in several plant species against several pathogens. The objectives of this work were to partially purify fractions of S. cerevisiae able to induce resistance in cucumber against anthracnose, caused by Colletotrichum lagenarium, and also evaluate the effect of the autoclaved crude aqueous extract from S. cerevisiae, Agro-Mos® and Bion® in the resistance induction against the insect T. absoluta in tomato plants, the nematode M. incognita in cucumber plants, as well as to verify the effect of the agents in the symbiotic interaction envolving soybean and B. elkanii. The results showed that the crude aqueous extract of S. cerevisiae autoclaved for 4 h was the most effective out in the reduction of cucumber anthracnose. Thus, the same extract was submitted to ethanolic precipitation and the obtained supernatant was fractioned by using Anion Exchange Chromatography - AEC. For peaks were obtained and peak I (non-adsorbed fraction to DEAE-Cellulose) and II (fraction adsorbed to DEAE-Cellulose) were the most effective out in the protection of the cucumber seedling by reducing anthracnose severity in 81% and 72% ,respectively. The application of the autoclaved extract of S. cerevisiae, Agro-Mos® and Bion® did not affect the development of the insect in tomato plants as well as did not interfere significantly in the multiplication of the nematode in cucumber roots. In the symbiotic interaction of soybean and B. elkanii, the tested agents did not affect the formation of nodules in soybean roots and the vegetative development of the plants. However, the foliar application of the autoclaved crude extract of S. cerevisiae significantly increased the amount of total nitrogen in the aerial part of the plants. Finally, it is concluded that the fractions (peaks I and II) of S. cerevisiae induced resistance of the cucumber plants. However the tested agents were not efficient in the control of the herbivore insect and the nematode and did not exhibit negative effects in the symbiotic interaction soybean and rhizobium.

Pine rocklands are endangered ecosystems unique to south Florida, the Bahamas and Cuba. As a result of their karstic calcium carbonate­rich soil, these systems are limited in phosphorus and nitrogen, making symbiotic associations critical to plant growth. Four leguminous species (Cajanus cajan, Chamaecrista fasciculata, Tephrosia angustissima, and Abrus precatorious) were used to determine the relationship between rhizobial partners and plant performance, and the symbiosis related gene nifH was amplified to characterize the diversity of rhizobial symbionts. Plants were grown in soils from four different south Florida pine rocklands, and a salinity treatment was added to determine how storm surge and sea level rise could affect this symbiotic relationship. While plant performance and nodulation were highly impacted by soil type, salinity did not represent a significant effect. Phylogenetic analysis determined that all four plant species were found to associate with Bradyrhizobium spp. and no rhizobial shift between salinity treatment and soil type was found.