In silico analysis of the biodiversity of endophytic fungi isolated from the Brazilian Cerrado / Análise in silico da biodiversidade de fungos endofíticos isolados do Cerrado brasileiro

Abstract Endophytic microorganisms are those that inhabit the interior of plant tissues and organs without causing damage to the plant, triggering a mutualistic interaction. These endophytes are known to produce compounds with various applications in the areas of biotechnology, pharmaceutical industry and agriculture; therefore, molecular methods are used to investigate the phylogeny of these organisms. The Brazilian Cerrado is the second largest biome in Brazil and is considered a hotspot, with a high diversity of endemic fauna and flora. The objective of this work was to analyze, through in silico analysis, the phylogeny of cultivable endophytic fungi isolated from plants found in the Brazilian Cerrado biome. A search was carried out for articles that worked with endophytes in the period between 2012 and 2022. The dendrogram was generated using the Neighbor-joining method based on the ITS1-5.8S-ITS2 conserved region obtained by GenBank codes of isolated endophytes. The genera Xylaria, Diaporthe, and Colletotrichum were isolated in more than three plants in the seven articles found. Most of the articles found related to the biodiversity of endophytic fungi from the Brazilian Cerrado aim at the bioprospecting of bioactive compounds, through culture-dependent methods; as such, a part of endophytic diversity may be lost due to the inability of certain endophytes to grow in artificial media. In silico analysis can assist in the investigation of phylogenetic relationships between endophytic fungi and has the potential to guide future work aimed at prospecting for bioactive compounds, phylogenetic identification, or biodiversity of this group of endophytes.

Resumo Microrganismos endofíticos são aqueles que vivem no interior de tecidos e órgãos vegetais sem causar dano à planta, desencadeando uma interação mutualística. Esses endófitos são conhecidos por produzir compostos com diversas aplicações nas áreas de biotecnologia, indústria farmacêutica e agricultura; logo, métodos moleculares são utilizados para investigar a filogenia destes organismos. O Cerrado brasileiro é o segundo maior bioma do Brasil e é considerado um hotspot, com uma alta diversidade de fauna e flora endêmica. O objetivo deste trabalho foi analisar, por meio da análise in silico, a filogenia de fungos endofíticos cultiváveis isolados de plantas encontradas no bioma Cerrado brasileiro. Foi realizada uma busca por artigos que trabalharam com endófitos no período entre 2012 e 2022. O dendrograma foi gerado usando o método Neighbor-joining baseado na região conservada ITS1-5.8S-ITS2 obtida pelos códigos GenBank de endófitos isolados. Os gêneros Xylaria, Diaporthe e Colletotrichum foram isolados em mais de três plantas nos sete artigos encontrados. A maior parte dos artigos encontrados relacionados à biodiversidade de fungos endofíticos do Cerrado brasileiro visam à bioprospecção de compostos bioativos, através de métodos dependentes de cultura; como tal, uma parte da diversidade endofítica pode ser perdida devido à incapacidade de certos endófitos crescerem em meios artificiais. A análise in silico pode auxiliar na investigação das relações filogenéticas entre fungos endofíticos e tem potencial para orientar trabalhos futuros voltados à prospecção de compostos bioativos, identificação filogenética ou biodiversidade deste grupo de endófitos.

Effect of Monocerin, a Fungal Secondary Metabolite, on Endothelial Cells.

This study reports the isolation and identification of the endophytic fungus Exserohilum rostratum through molecular and morphological analysis using optical and transmission electron microscopy (TEM), as well as the procurement of its secondary metabolite monocerin, an isocoumarin derivative. Considering the previously observed biological activities of monocerin, this study was performed on human umbilical vein endothelial cells (HUVECs) that are widely used as an in vitro model for several different purposes. Important parameters, such as cell viability, senescence-associated ß-galactosidase, cellular proliferation by using 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE), apoptosis analysis with annexin, cellular morphology through scanning electron microscopy (SEM), and laser confocal analysis were evaluated after exposing the cells to monocerin. After 24 h of exposure to monocerin at 1.25 mM, there was more than 80% of cell viability and a low percentage of cells in the early and late apoptosis and necrosis. Monocerin increased cell proliferation and did not induce cell senescence. Morphological analysis showed cellular integrity. The study demonstrates aspects of the mechanism of action of monocerin on endothelial cell proliferation, suggesting the possibility of its pharmaceutical application, such as in regenerative medicine.

Effect of monocerin, a fungal secondary metabolite, on endothelial cells

This study reports the isolation and identification of the endophytic fungus Exserohilum rostratum through molecular and morphological analysis using optical and transmission electron microscopy (TEM), as well as the procurement of its secondary metabolite monocerin, an isocoumarin derivative. Considering the previously observed biological activities of monocerin, this study was performed on human umbilical vein endothelial cells (HUVECs) that are widely used as an in vitro model for several different purposes. Important parameters, such as cell viability, senescence-associated β-galactosidase, cellular proliferation by using 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE), apoptosis analysis with annexin, cellular morphology through scanning electron microscopy (SEM), and laser confocal analysis were evaluated after exposing the cells to monocerin. After 24 h of exposure to monocerin at 1.25 mM, there was more than 80% of cell viability and a low percentage of cells in the early and late apoptosis and necrosis. Monocerin increased cell proliferation and did not induce cell senescence. Morphological analysis showed cellular integrity. The study demonstrates aspects of the mechanism of action of monocerin on endothelial cell proliferation, suggesting the possibility of its pharmaceutical application, such as in regenerative medicine.

Short-Term Effect in Soil Microbial Community of Two Strategies of Recovering Degraded Area in Brazilian Savanna: A Pilot Case Study.

The Brazilian Cerrado is a highland tropical savanna considered a biodiversity hotspot with many endemic species of plants and animals. Over the years, most of the native areas of this biome became arable areas, and with inadequate management, some are nowadays at varying levels of degradation stage. Crop-livestock integrated systems (CLIS) are one option for the recovery of areas in degradation, improving the physicochemical and biological characteristics of the soil while increasing income and mitigating risks due to product diversification. Little is known about the effect of CLIS on the soil microbial community. Therefore, we perform this pilot case study to support further research on recovering degraded areas. The bacterial and fungal soil communities in the area with CLIS were compared to an area under moderate recovery (low-input recovering - LI) and native savanna (NS) area. Bacterial and fungal communities were investigated by 16S and ITS rRNA gene sequencing (deep rRNA sequencing). Ktedonobacteraceae and AD3 families were found predominantly in LI, confirming the relationship of the members of the Chloroflexi phylum in challenging environmental conditions, which can be evidenced in LI. The CLIS soil presented 63 exclusive bacterial families that were not found in LI or NS and presented a higher bacterial richness, which can be related to good land management. The NS area shared 21 and 6 families with CLIS and LI, respectively, suggesting that the intervention method used in the analyzed period brings microbial diversity closer to the conditions of the native area, demonstrating a trend of approximation between NS and CLIS even in the short term. The most abundant fungal phylum in NS treatment was Basidiomycota and Mucoromycota, whereas Ascomycota predominated in CLIS and LI. The fungal community needs more time to recover and to approximate from the native area than the bacterial community. However, according to the analysis of bacteria, the CLIS area behaved differently from the LI area, showing that this treatment induces a faster response to the increase in species richness, tending to more accelerated recovery. Results obtained herein encourage CLIS as a sustainable alternative for recovery and production in degraded areas.

Multifunctional potential of endophytic bacteria from Anacardium othonianum Rizzini in promoting in vitro and ex vitro plant growth.

Anacardium othonianum Rizzini, a cashew tree native to the Brazilian Cerrado, is economically important due to its applications in the food, chemical and pharmaceutical industries. However, A. othonianum yields a crop with low productivity due to a number of factors, such as nutritionally poor soils, drought and losses due to pests and diseases. Brazil is one of the nine largest cashew nut producers worldwide, and sustainable technologies are needed to increase the productivity of this crop. In this context, the use of endophytic microorganisms could promote plant growth and provide protection against phytopathogens. In this study, the isolation of the root endophytic community of A. othonianum led to the characterization of 22 distinct bacterial strains with multifunctional traits for plant growth promotion. The results of in vitro assays to assess auxin synthesis, phosphate solubilization, phosphatase and siderophore production and biocontrol against Fusarium oxysporum led to the selection of Acinetobacter lwoffii Bac109 and Pantoea agglomerans Bac131 as the most promising strains. The reinoculation of the Bac109 and Bac131 strains onto A. othonianum seeds showed that the treatment containing a mixture of these strains was the most effective in promoting increases in the biometric parameters of early plant growth. Thus, this study highlights the biotechnological potential of a consortium of A. lwoffii Bac109 and P. agglomerans Bac131 for future applications in sustainable cashew cultivation.