Isolation and characterization of phosphate solubilizing fungi from soil treated with malic acid

Em face do aumento da demanda por alimentos resultante do crescimento populacional, a produção agrícola tem-se intensificado em todo o mundo. Para garantir a segurança alimentar futura, a disponibilidade de P para os cultivos torna-se central. O P desempenha papel importante no desenvolvimento das plantas, atuando como componente estrutural da célula ou desempenhando distintas funções nas reações metabólicas. Nos solos, apenas 0,1 % do P total está presente em formas solúveis disponíveis para as plantas. A aplicação de fertilizantes fosfatados é sempre fundamental para atender a demanda vegetal. No entanto, grande parte do P adicionado ao solo é fixada nas superfícies de argilas ou forma complexos pouco solúveis com Fe, Al e Ca, resultando em aumentos do custo de produção e diminuindo a sustentabilidade agrícola. O uso de microrganismos solubilizadores de fosfato (MSF) pode melhorar esse cenário, uma vez que eles têm a capacidade de disponibilizar P a partir de compostos minerais pouco reativos ou a partir da matéria orgânica do solo. Os MSF podem ser encontrados na rizosfera, região de intensa interação entre as raízes de plantas e a microbiota do solo. As raízes das plantas influenciam diretamente a atividade microbiana nesse local por meio de rizodeposições. As rizodeposições incluem açúcares, aminoácidos, ácidos orgânicos, dentre outros, que atraem, selecionam e sustentam populações microbianas específicas. Por exemplo, a liberação de ácido málico no solo aumenta as populações de MSF rizosféricos. Assim, o objetivo deste trabalho foi o de isolar fungos solubilizadores de fosfato de solo tratado com ácido málico e caracterizar a capacidade dos mesmos em solubilizar fosfatos de rocha e promover a liberação de P adsorvido ao solo, tanto na presença quanto na ausência desse composto. Amostras de solo foram incubadas durante 15 dias a 28 ºC, em meio NBRIP modificado, suplementado com 3,99 g L-1 de ácido málico. Após o período de incubação. diluições seriadas até 10-6 foram realizadas e plaqueadas em ágar NBRIP. Colônias que apresentaram halo de solubilização de fosfato, foram isoladas, purificadas e estocadas para avaliação do potencial de solubilização de diferentes fontes de P, dessorção de P e liberação de P, na ausência ou presencia do ácido málico. A suplementação do meio NBRIP com ácido málico favoreceu o aumento das populações de fungos solubilizadores de fosfato. Os maiores valores de P solúvel (mg L-1) obtidos para os isolados obtidos na presença de diferentes fontes de fósforo foram: 563,4 para o Ca3PO4; 26,2 para o FePO4; e 17, 8 para o AlPO4. Para os fosfatos de rocha, os maiores valores de P solúvel (mg L-1) foram 105,5 para Aipe; 76,3 para Patos; 49,1 para Catalão; e 26,1 para Araxá. Nos tratamentos com P fixado ao solo, o maior valor de P solúvel (mg L-1) obtido foi de 0,24, indicando baixo potencial de dessorção de P pelos fungos testados. Neste trabalho, a suplementação de meio NBRIP modificado com ácido málico não melhorou a capacidade de solubilização de fosfatos dos isolados fúngicos, mas contribuiu para a solubilização abiótica de todas as fontes de P avaliadas e para a dessorção de P do solo. Neste trabalho, o aumento das populações de fungos solubilizadores de fosfatos no solo e a maior solubilização/dessorção de P foram os principais efeitos do ácido málico na disponibilização de P, indicando o potencial desse metabólito em promover a mobilização dos reservatórios de P do solo de forma a contribuir com a nutrição fosfatada de plantas e microrganismos. PALAVRAS-CHAVE: Ácido málico. Fungos solubilizadores de fosfato. Rochas fosfóricas. P adsorvido.Considering the increased demand for food resulting from population growth, agricultural production has intensified around the world. To ensure future food security, the availability of P for crops becomes central. P plays an important role in plant development, acting as a structural component of the cell or playing different roles in metabolic reactions. In soils, only 0.1% of the total P is present in soluble forms available to plants. Thus, the application of phosphate fertilizers is essential to meet crop demand. However, much of the P added to the soil is fixed on clay surfaces or forms poorly soluble complexes with Fe, Al and Ca, resulting in production cost increases and decreasing agricultural sustainability. The use of phosphate solubilizing microorganisms (MSF) can improve this scenario, as they have the ability to make P available from poorly reactive mineral compounds or from soil organic matter. MSF can be found in the rhizosphere, a region of intense interaction between plant roots and soil microbiota. Plant roots directly influence microbial activity in this location through rhizodepositions. Rhizodepositions include sugars, amino acids, organic acids, among others, which attract, select and support specific microbial populations. For example, the release of malic acid in the soil increases rhizospheric MSF populations. Thus, the objective of this work was to isolate phosphate solubilizing fungi from soil treated with malic acid and characterize their ability to solubilize rock phosphates and promote the release of P adsorbed to the soil, both in the presence and absence of this compound. Soil samples were incubated for 15 days at 28 ºC, in modified NBRIP medium, supplemented with 3.99 g L-1 of malic acid. After the incubation period. serial dilutions up to 10-6 were made and plated on NBRIP agar. Colonies that showed halo of phosphate solubilization were isolated, purified and stored to evaluate the potential for solubilization of different P sources, P desorption and P release, in the absence or presence of malic acid. The supplementation of the NBRIP medium with malic acid favored the increase in the populations of phosphate solubilizing fungi. The highest values of soluble P (mg L-1) obtained for the isolates obtained in the presence of different phosphorus sources were: 563.4 for Ca3PO4; 26,2 for FePO4; and 17,8 for AlPO4. For rock phosphates, the highest values of soluble P (mg L-1) were 105,5 for Aipe; 76,3 for Ducks; 49,1 for Catalan; and 26,1 for Araxá. In treatments with P fixed to the soil, the highest value of soluble P (mgL-1) obtained was 0.24, indicating a low potential for P desorption by the tested fungi. In this work, the supplementation of NBRIP medium modified with malic acid did not improve the phosphate solubilization capacity of the fungal isolates, but it contributed to the abiotic solubilization of all evaluated P sources and to the desorption of P from the soil. In this work, the increased populations of phosphate-solubilizing fungi in the soil and the greater solubilization/desorption of P were the main effects of malic acid on P availability, indicating the potential of this metabolite to promote the mobilization of P reservoirs in the soil of to contribute to the phosphate nutrition of plants and microorganisms. KEYWORDS: Malic acid. Phosphate solubilizing fungi. Phosphoric rocks. Adsorbed P.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Guadua angustifolia Kunth leaves as a source for bioactive phenolic compounds: Optimization of ultrasound-assisted extraction using response surface methodology and antioxidant activities

This study evaluated the ultrasound-assisted extraction of phenolic compounds from Guadua angustifolia leaves, along with their optimization using response surface methodology. The effects of two sonication process conditions were determined using a central composite experimental design, with three levels (low, medium, and high) evaluated for time (10, 20, and 30 min) and temperature (20 °C, 35 °C, and 50 °C). A total of 12 experiments with four replicates were conducted at the central point, with the total phenol and flavonoid contents determined using the Folin-Ciocalteu colorimetric method and complexation with AlCl3, respectively. The optimized extract was analyzed using ultra-performance liquid chromatography (UPLC), and the antioxidant capacity of the optimized extract was determined by DPPH• (2,2-Diphenyl-1-Picrylhydrazyl) and ABTS•+ (2,2′-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) assays. Extraction at 50 °C for 20 min was found to favor the extraction of phenol and total flavonoids. The experimental validation of the total phenol and flavonoid content produced values of 7.39 mg gallic acid equivalents per gram of dry matter and 1.55 mg quercetin equivalents per gram of dry matter, respectively. These values suggest that the extraction process is reproducible, with a relative standard deviation of 22.9 % and 14.1 %, respectively. The chromatographic profile showed that optimization favored the visualization of phenolic compounds compared to the non-optimized extract. The optimized extract had higher antioxidant capacity than the non-optimized extract, with values of 209.23 and 144.76 μmol Trolox per gram extract for the DPPH• and ABTS•+ techniques, respectively. Thus, the conditions evaluated in the ultrasound-assisted extraction were an efficient technique capable of extracting the maximum amount of phenolic compounds with antioxidant activity from the leaves of G. angustifolia, showing its potential application in various industries

Dynamic of Phenolic Compounds in <i>Guadua angustifolia</i> Kunth under Chemical, Organic, and Biological Fertilization

Guadua angustifolia produces phenolic compounds, and this production may be influenced by the application of chemical, organic, and biological fertilizers. Currently, the effect of such fertilizers on the synthesis dynamics of this group of metabolites in bamboo is unknown. In this study, the total phenolic content (TPC) and total flavonoid content (TFC) in the leaves of plants fertilized with diammonium phosphate (DAP) and humus in combination with the biofertilizers Promofort®, Azospirillum brasilense, Pseudomonas fluorescens, and Stenotrophomonas sp. were determined using colorimetric techniques across three sampling events (four, five, and seven months after planting). Additionally, an approximation of the bacterial profile of G. angustifolia roots was performed using the DGGE-PCR fingerprint technique. Through repeated measures ANOVA (rmANOVA), it was determined that there is no statistically significant three-way interaction between humus or DAP application, biological fertilizers, and time for either TPC or TFC. However, there were interactions between the sampling event and the application of biological fertilizers for both TPC and TFC, with the latter being promoted by the application of Promofort®. Finally, NMDS analyses and heatmaps with hierarchical clustering showed that the composition and abundance of OTUs in the bacterial profile varied with fertilization type and increased over time