Diversidade e mecanismos de promoção do crescimento de plantas utilizando abordagem dependente e independente de cultivo por microrganismos obtidos de milho cultivado em diferentes fontes de fósforo

Abstract

The use of phosphate fertilizers in agriculture is important due to the high requirement of this nutrient for the plants. However, much of the phosphorus (P) added to soil as soluble P is rapidly adsorbed to iron, aluminum or calcium oxides, which can lead to a complexation of 80% of the P applied to the soil. Triple superphosphate (TSP) is an excellent source of soluble P. However, due to its high cost of production, the use of, cheaper P sources, such as rock phosphate (RP) has been an attractive alternative in developing countries. A drawback is the low reactivity of RP, but the use of P solubilizing microorganisms to improve the availability of this nutrient is investigated. These microorganisms, in addition to improving P solubilization, may also play a role in promoting the growth of plants. They can be used for the development of microbial inoculants aimed at increasing the productivity and sustainability of important agricultural crops such as maize. In this work, we compared the effects of three years of fertilization with RP and TSP on the diversity of the maize rhizosphere microbial community using independent cultivation method. In addition, we obtained bacterial isolates from the rhizosphere of maize cultivated with RP and without P addition and evaluated the potential of these and of endophytic bacteria also isolated from maize for the solubilization of rock phosphate (Araxá) and iron phosphate in vitro. Pearl millet growth-promotion under RP fertilization was also evaluated in greenhouse tests. Furthermore, we investigated the presence of genes related to plant growth-promotion in 9 sequenced genomes. We also analyzed the draft genome of Serratia marcescens SmCNPMS2112 and S. marcescens SmUFMG85 isolated from maize that stood out in the in vitro and greenhouse trials. These genomes were compared among themselves and to other strains of Serratia isolated from different environments, aiming at the identification of genes related to the endophytic lifestyle, promotion of plant growth and pathogenicity. Proteobacteria phylum was dominant in all phosphate fertilization conditions, while Oxalobacteraceae (mainly Massilia sp. and Herbaspirillum sp.) were enriched in the soil fertilized with RP. Klebsiella sp. was the second most abundant taxon in soil treated with RP. Burkholderia sp. and Bacillus sp. were enriched in the soil fertilized with RP when compared to soil treated with TSP. As for fungi, Glomeromycota presented higher abundance in RP-treated soils, and the most abundant genera were Scutellospora sp. and Racocetra. These taxa are already described as important for P solubilization / acquisition in soil fertilized with RP. Maize cultivated in the TSP and RP treated soil presented similar productivity, and a positive correlation was detected for the Pcontent and the microbial community of these soils. Bacteria belonging to the Enterobacteriaceae family were predominant in the cultures obtained from the rhizosphere soil of maize cultivated with and without RP, and the main genera were Klebsiella and Enterobacter. Among the sources of P we evaluated, Araxá phosphate was solubilized to a higher extent, when compared to iron phosphate and independent of the P source, the endophytic bacteria solubilized P more efficiently than the rhizospheric ones. In addition, eight in nine bacteria evaluated for the capacity to promote the growth of millet contributed to the improvement of at least one of the growth parameters evaluated,and the most efficient strains were Bacillus megaterium UFMG50, Ochrobactrum sp. CNPMS2088 and Serratia marcescens CNPMS2112. These bacteria contain genes related to P metabolism, production of organic acids and phytohormones. Whole genome phylogenetic analysis together with the analysis of conserved phylogenetic markers of Serratia sp., it was observed that the conserved part of the genome contributed more to the separation, whereas the accessory portion of the total genome did not affect the phylogenetic distribution. Numerous predicted functions were shared between Serratia sp. of different lifestyles, such as solubilization, hydrolysis and phosphate acquisition, iron acquisition, indole acetic acid (IAA) production, motility, adhesion and biofilm formation, bacteriocin production, antibiotics, and genes that confer drug resistance and oxidizing agents, as well as many virulence factors. Therefore, the differences that define a particular lifestyle among the Serratia sp. evaluated are probably more related to variations in gene expression than to genomic content per se. In addition, 3 prophages were detected in the S. marcescens SmUFMG85 genome and in the S. marcescens SmCNPMS2112 genome. Comparative analysis, along with the search for genomic islands, showed that the unique and accessory regions are located in genomic islands. In the genome of S. marcescens SmCNPMS2112, they contained, type III secretory system, protease, chitinase, fimbriae, siderophore and β-lactamase. While in S. marcescens SmUFMG85 the type IV secretion system was obtained, genes related to conjugation and transposition, chemotaxis, iron acquisition and production of rhamnolipids. Thus, it can be concluded that the repertoire of detected genes confirms the endophytic style of the isolates (SmUFMG85 and SmCNPMS2112) and the potential to promote plant growth. In addition, the role of the S. marcescens SMUFMG85 and SmCNPMS2112 mobilomes, especially from genomic islands, was evident for the acquisition of various functions that may confer adaptability to the host plant in addition to other environments.Differences in the composition of microbial communities associated to the type of P fertilization were observed in this study. Even though it is not possible to establish causal relationships, our in vitro and greenhouse tests allowed us to identify taxa involved in the solubilization of RP and in plant growth-promotion. Likewise, the tests done in vitro and in greenhouse show several bacteria as candidates for the development of bioinoculants for agriculture.