Rizóbios isolados de fabáceas forrageiras do semiárido: biodiversidade e eficiência simbiótica

• Ano de defesa: 2018
• Autor(a) principal: Dias, Suelane de Melo
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal da Paraíba; Brasil; Solos e Engenharia Rural; Programa de Pós-Graduação em Zootecnia; UFPB
• Palavras-chave em Português: Caatinga; Diversidade microbiana; Leguminosas nativas; Nitrogênio; CNPQ::CIENCIAS AGRARIAS::ZOOTECNIA::PASTAGEM E FORRAGICULTURA
• Link de acesso: https://repositorio.ufpb.br/jspui/handle/123456789/14246

Biological nitrogen fixation is a natural process that occurs in associations of plants with diazotrophic bacteria. Most of the fodder fabaceae found in the Caatinga have high economic value and can be used to increase the nitrogen and organic matter input in the soil through biological nitrogen fixation and nutrient cycling. Thus the objective of this work was to characterize morphophysiologically isolated of rhizobia native to the forage species Desmanthus pernambucanus (L.) Thellung., Mimosa caesalpiniifolia Benth. and Mimosa tenuiflora (Wild) Poir. To evaluate the resistance to saline stress, acidity, aluminum toxicity and solubilization capacity of calcium phosphate, as well as to test the symbiotic affectivity of the isolates. Authenticate the native rhizobia isolates through the simultaneous amplification of fragments of the nifH and nodC genes by means of the duplex PCR technique. To determine the genetic diversity of the isolates by the amplified ribosomal ADN restriction analysis (PCR-RFLP) technique using the HhaI, MspI and HinfI endonucleases and quantify the production of in vitro indole compounds. The soil used as substrate for the cultivation of the fabaceae was collected at specific points in the municipalities of Pocinhos, São Sebastião de Lagoa de Roça and Boa Vista in the state of Paraíba. The nodules were obtained from the cultivation of a bait plant in a greenhouse. Bacteria from the nodules were isolated and purified in the laboratory and after the isolation protocol the bacteria were culturally evaluated and submitted to the tests of: Tolerance to low pH + Al3+, different saline media and solubilization of calcium phosphate in vitro. An experiment was then carried out to evaluate the symbiotic efficiency of the isolates inoculated in Vigna unguigulata (L.) Walp. under sterile conditions in a greenhouse. After isolation and purification of the isolates, ADN was extracted and subjected to the amplification of the fragments of symbiotic genes nifH and nodC for further evaluation of the genetic diversity accessed by PCR-RFLP. For this, the 16S rRNA gene was amplified using the 27F universal primers (AGAGTTTGATCMTGGCTCAG) and 1492R (TACGGYTACCTTGT TACGACTT). Restriction reactions were performed using the HhaI, MspI and HinfI endonucleases. The digestion product was subjected to horizontal electrophoresis in 3% agarose gel in 1% TAE buffer for 80 min at 100 Volts. For the quantification of Indolic Compounds, the colorimetric method was used to evaluate the colonies added with L-tryptophan (3.5 mg) and without L-tryptophan in YM culture medium. All isolates were evaluated as fast growing acidifying bacteria with the main morphological characteristics of irregular colonies with umbiculate elevation, smooth surface and moderate exopolysaccharide production. The DPBV41 and DPBV53 isolates did not show visible growth in the petri dish when submitted to pH 4.5 with and without aluminum, characterizing as non-tolerant isolates. The isolates most affected by the pH variation of the culture medium and aluminum toxicity were the bacteria isolated from the Desmanthus pernambucanus. The rhizobia colonies that were most likely to tolerate high salinity culture media were: MTBV77, MTP72, MTP37, DPBV42 and DPBV53 with maximum growth in all of the petri dishes. In the simultaneous amplification evaluations of symbiotic genes, among the 80 rhizobia isolates tested, 16 amplified only the nifH gene and five isolates amplified both nifH + nodC genes simultaneously. The genetic variability of the 21 rhizobia isolates xviii varied according to each restriction enzyme (HinfI, HhaI and MspI) and resulted in different restriction profiles transformed into binary data and evaluated through genetic dissimilarity matrix by the complement of the Jaccard coefficient. From the dissimilarity matrix a dendrogram was constructed by the Ward method, with the number of groups defined by the Mojena method, resulting in IV groups. On average, the distance between pairs of genotypes was 0.56. The most divergent pairs were the DPP1 and MTP44 isolates, with a distance of 0.89 and the most similar MTP72 and MTBV77 with a distance of 0.10. The production of Indole Compounds was higher in the samples where there was an increase of L-tryptophan, precursor of indole-acetic acid, by bacteria MTBV12, MTBV77, MTP78 and MTP37. The isolates of native rhizobia have high genetic diversity and are grouped based on the bait plant from which they are isolated. The production of Indole Compounds is variable among the selected isolates and is potentiated in the presence of the L-Tryptophan precursor. The native isolates of Mimosa tenuiflora are more tolerant to saline stress, acidity, and aluminum toxicity compared to other native bacteria isolated from Desmanthus pernambucanus and Mimosa caesalpinifollia. Most of the native rhizobia isolates, evaluated in this work, have low solubilization capacity of the calcium phosphate complex. All the bacteria isolated were able to nodulate the species Vigna unguigulata.