Synthesis, characterization, and reusability of novel nanobiocomposite of endophytic fungus Aspergillus flavus and magnetic nanoparticles (Fe3O4) with dye bioremediation potential.

The incorrect disposal of textile dyes, such as Reactive Black 5 (RB5), causes several problems for living beings and the quality of the environment. Nanobiocomposites (NBC) produced from endophytic fungi (potentially remediation dyes-agents) and magnetic nanoparticles have high biotechnological potential due to their superparamagnetic behavior, which would allow their recovery through the magnetic field after the bioremediation process. This work aimed to obtain a new nanobiocomposite from the interaction of magnetite nanoparticles (Fe3O4) with the endophyte Aspergillus flavus (Af-CL-7) to evaluate its bioremediation capacity and to reduce the toxicity of RB5 and its reuse. Before obtaining the NBC, Af-CL-7 showed discoloration of RB5 and it was tolerant to all tested concentrations of this dye. The discovery of the nanobiocomposite textile dye bioremediator product presents a significant environmental advantage by addressing the issue of water pollution caused by textile dyes. The NBC called Af-Fe3O4 was successfully obtained with the magnetized endophyte, and their magnetic properties were verified by VSM analysis and by action of magnetic fields generated by Nd-Fe-B magnets SEM analyzes showed that the nanoparticles did not cause any damage to the hypha morphology, and TEM analyzes confirmed the presence of nanoparticles in the fungus wall and also inside the cell. The NBC Af-Fe3O4 and Af-CL-7 showed, respectively, 96.1% and 92.2% of RB5 discoloration in the first use, 91.1% e 86.2% of discoloration in the validation test, and 89.0% in NBC reuse. In the toxicological bioassay with Lactuca sativa seeds, NBC showed a positive reduction in the toxicity of RB5 after treatment, allowing the hypocotyl growth to be statistically similar to the control with water. Thus, we highlight the promising obtaining process of NBC that could be applied in bioremediation of contaminated waters, wherein the industrial economic cost will depend on the fermentation efficiency, biomass production and nanoparticle synthesis.

Meta-omics integration approach reveals the effect of soil native microbiome diversity in the performance of inoculant Azospirillum brasilense.

Plant growth promoting bacteria (PGPB) have been used as integrative inputs to minimize the use of chemical fertilizers. However, a holistic comprehension about PGPB-plant-microbiome interactions is still incipient. Furthermore, the interaction among PGPB and the holobiont (host-microbiome association) represent a new frontier to plant breeding programs. We aimed to characterize maize bulk soil and rhizosphere microbiomes in irradiated soil (IS) and a native soil (NS) microbial community gradient (dilution-to-extinction) with Azospirillum brasilense Ab-V5, a PGPB commercial inoculant. Our hypothesis was that plant growth promotion efficiency is a result of PGPB niche occupation and persistence according to the holobiont conditions. The effects of Ab-V5 and NS microbial communities were evaluated in microcosms by a combined approach of microbiomics (species-specific qPCR, 16S rRNA metataxonomics and metagenomics) and plant phenomics (conventional and high-throughput methods). Our results revealed a weak maize growth promoting effect of Ab-V5 inoculation in undiluted NS, contrasting the positive effects of NS dilutions 10-3, 10-6, 10-9 and IS with Ab-V5. Alpha diversity in NS + Ab-V5 soil samples was higher than in all other treatments in a time course of 25 days after sowing (DAS). At 15 DAS, alpha diversity indexes were different between NS and IS, but similar in all NS dilutions in rhizospheric samples. These differences were not persistent at 25 DAS, demonstrating a stabilization process in the rhizobiomes. In NS 10-3 +Ab-V5 and NS 10-6 Ab-V5, Ab-V5 persisted in the maize rhizosphere until 15 DAS in higher abundances compared to NS. In NS + Ab-V5, abundance of six taxa were positively correlated with response to (a)biotic stresses in plant-soil interface. Genes involved in bacterial metabolism of riboses and amino acids, and cresol degradation were abundant on NS 10-3 + Ab-V5, indicating that these pathways can contribute to plant growth promotion and might be a result of Ab-V5 performance as a microbial recruiter of beneficial functions to the plant. Our results demonstrated the effects of holobiont on Ab-V5 performance. The meta-omics integration supported by plant phenomics opens new perspectives to better understanding of inoculants-holobiont interaction and for developing better strategies for optimization in the use of microbial products.

Mechanisms of Mucor sp. CM3 isolated from the aquatic macrophyte Eichhornia crassipes (Mart.) Solms to increase cadmium bioremediation.

Bioremediation of toxic metals is a feasible and low-cost remediation tool to reduce metal contamination. Plant-fungus interactions can improve this technique. Eichhornia crassipes (Mart.) Solms is a macrophyte reported to bioremediate contaminated water. Thus, the present study aimed to isolate endophytic fungi from E. crassipes, select a highly cadmium (Cd) tolerant isolate and evaluate its bioremediation potential. This was evaluated by (1) the fungus tolerance and capacity to accumulate Cd; (2) Cd effects on cell morphology (using SEM and TEM) and on the fungal antioxidant defense system, as well as (3) the effect on model plant Solanum lycopersicum L. cultivar Calabash Rouge, inoculated with the endophyte fungus and exposed to Cd. Our results selected the endophyte Mucor sp. CM3, which was able to tolerate up to 1000 g/L of Cd and to accumulate 900 mg of Cd/g of biomass. Significant changes in Mucor sp. CM3 morphology were observed when exposed to high Cd concentrations, retaining this metal both in its cytoplasm and in its cell wall, which may be linked to detoxification and metal sequestration mechanisms related to the formation of Cd-GSH complexes. In addition, Cd stress induced the activation of all tested antioxidant enzymes - superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR) - in this endophytic fungus. Moreover, when inoculated in tomato plants, this fungus promoted plant growth (in treatments without Cd) and induced an increased metal translocation to plant shoot, showing its potential to increase metal bioremediation. Therefore, this study indicates that the isolated endophyte Mucor sp. CM3 can be applied as a tool in different plant conditions, improving plant bioremediation and reducing the environmental damage caused by Cd, while also promoting plant growth in the absence of contaminants.

The key role of indole-3-acetic acid biosynthesis by Bacillus thuringiensis RZ2MS9 in promoting maize growth revealed by the ipdC gene knockout mediated by the CRISPR-Cas9 system.

The bacterial biosynthesis of indole-3-acetic acid (IAA) is often related to the beneficial effects of plant growth-promoting rhizobacteria (PGPR) on plant development. In PGPR belonging to the Bacillus genus, the synthesis of IAA may occur through different metabolic pathways that are still poorly understood. B. thuringiensis (Bt) is well known for its insecticidal properties; however, its beneficial features are not limited to pest control. Our group has been studed the beneficial effects of Bt strain RZ2MS9 as growth promoter in a range of plant crops, including soybean, tomato, and maize. We recently demonstrated that bacterial IAA biosynthesis plays an important role in the ability of RZ2MS9 to benefit plant development. However, the molecular involved mechanisms in the IAA biosynthesis by this bacterium in the beneficial interaction with plants remain unclear. Here, we investigated the genetic basis of IAA biosynthesis by RZ2MS9. We knocked out the ipdC gene, involved in IAA biosynthesis via the tryptophan-dependent IPyA pathway, using the CRISPR-Cas9 system. Our results showed that, by disrupting the IPyA pathway, the amount of IAA synthesized by the mutant RZ2MS9 (ΔipdC) in the presence of tryptophan drops 57%. The gene knockout did not affect the bacterial growth, but it did affect its ability to colonize maize. Moreover, deactivating the ipdC gene in RZ2MS9 significantly reduces its ability to promote maize growth. ΔipdC performed worse than RZ2MS9 in almost all evaluated plant parameters, including total root length, projected root area, lateral roots, aerial part dry matter, and germination speed index. Therefore, we demonstrated that tryptophan-dependent IAA biosynthesis via the IPyA pathway by RZ2MS9 is strongly influenced by the ipdC gene. Furthermore, IAA biosynthesis by RZ2MS9 is a major mechanism used by this PGPR to promote maize growth.

Influence of plant growth-promoting endophytes Colletotrichum siamense and Diaporthe masirevici on tomato plants (Lycopersicon esculentum Mill.).

The protective and growth-promoting activities of Colletrotrichum and Diaporthe endophytes on tomato plants (Lycopersicon esculentum Mill.) are underexplored. We screened 40 endophytic fungi associated with Mexican shrimp plant (Justicia brandegeana) using an in vitro dual culture assay for Fusarium oxysporum, one of the most important phytopathogens of tomato plants. The three best antagonists, Colletotrichum siamense (JB224.g1), C. siamense (JB252.g1), and Diaporthe masirevicii (JB270), were identified based on multilocus sequence analysis. They were assessed in vitro for their inhibition of F. oxysporum and phosphate solubilisation capacity, and for the production of indole acetic acid. Greenhouse experiments verified the growth-promoting effects of these endophytes and the suppression of F. oxysporum symptoms in tomato plants.   Under greenhouse conditions, the JB252.g1 and JB270 isolates showed positive results for seedling emergence speed. The radicular system depth of plants inoculated with JB270 was greater than that in uninoculated plants (27.21 vs 21.95 cm). The soil plant analysis development chlorophyll metre (SPAD) index showed statistically significant results, especially for the endophyte JB224.g1 (36.99) compared to the control plants (30.90) and plants infected solely with F. oxysporum (33.64).

Differential Chemical Profile of Metabolite Extracts Produced by the Diaporthe citri (G-01) Endophyte Mediated by Varying the Fermented Broth pH.

Endophytic microorganisms show great potential for biotechnological exploitation because they are able to produce a wide range of secondary compounds involved in endophyte−plant adaptation, and their interactions with other living organisms that share the same microhabitat. Techniques used to chemically extract these compounds often neglect the intrinsic chemical characteristics of the molecules involved, such as the ability to form conjugate acids or bases and how they influence the solubilities of these molecules in organic solvents. Therefore, in this study, we aimed to evaluate how the pH of the fermented broth affects the process used to extract the secondary metabolites of the Diaporthe citri strain G-01 endophyte with ethyl acetate as the organic solvent. The analyzed samples, conducted by direct-infusion electrospray-ionization mass spectrometry, were grouped according to the pH of the fermented broth (i.e., <7 and ≥7). A more extreme pH (i.e., 2 or 12) was found to affect the chemical profile of the sample. Moreover, statistical analysis enabled us to determine the presence or absence of ions of high importance; for example, ions at 390.7 and 456.5 m/z were observed mainly at acidic pH, while 226.5, 298.3, and 430.1 m/z ions were observed at pH ≥ 7. Extraction at a pH between 4 and 9 may be of interest for exploring the differential secondary metabolites produced by endophytes. Furthermore, pH influences the chemical phenotype of the fungal metabolic extract.

Antimicrobial and antioxidant activities of secondary metabolites from endophytic fungus Botryosphaeria fabicerciana (MGN23-3) associated to Morus nigra L.

This study was to evaluate the biological activity of the extract of Botryosphaeria fabicerciana isolated from leaves of Morus nigra. The volatile compounds from the crude extract were analysed by GC-MS which demonstrate that mellein and ß-orcinaldehyde were are the major compounds. The best minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extract was observed against Gram-positive bacteria, with a MIC of 15.6 µg/mL towards B. cereus and MIC of 62.5 µg/mL towards S. aureus and B. subtilis. MBC values of 31.25 µg/mL, 62.5 µg/mL, and 250 µg/mL were observed towards B. cereus, B. subtilis, and S. aureus, respectively. The cytotoxicity analyses showed CC50 of 115 µg/mL. The crude extract showed antioxidant activity by the DPPH, ABTS, and FRAP assays. Therefore, the extract of the endophytic fungus presented biotechnological potential as an antibacterial and antioxidant agent.

Retrotransposons and multilocus sequence analysis reveals diversity and genetic variability in endophytic fungi-associated with Serjania laruotteana Cambess.

The composition of endophytic communities is dynamic and demonstrates host specificity; besides, they have great intra- and interspecific genetic variability. In this work, we isolated leaf endophytic fungi from Serjania laruotteana, identify them using multilocus analysis, and evaluate the genetic variability using IRAP (inter-retrotransposon amplified polymorphism) and REMAP (retrotransposon-microssatellite amplified polymorphism). A total of 261 fungi were isolated and 58 were identified. Multilocus phylogenetic analysis using the partial sequences from the ITS1-5.8S-ITS2 regions, elongation factor 1-alpha, ß-tubulin, actin, glyceraldehyde-3-phosphate dehydrogenase, and calmodulin genes identify that most strains belonged to the Colletotrichum and Diaporthe genera, other isolated genera were Xylaria, Phyllosticta, Muyocopron, Fusarium, Nemania, Plectosphaerella, Corynespora, Bipolaris, and Curvularia. The IRAP and REMAP analyzes were performed with Colletotrichum and Diaporthe genera and showed 100% of polymorphism and high intra- and interspecific variability. This is the first report of the diversity of endophytic fungi from S. laruotteana. In addition, it demonstrated that the IRAP and REMAP can be used to distinguish morphologically similar lineages, revealing differences even strains of the same species.

Bacillus thuringiensis RZ2MS9, a tropical plant growth-promoting rhizobacterium, colonizes maize endophytically and alters the plant's production of volatile organic compounds during co-inoculation with Azospirillum brasilense Ab-V5.

The beneficial features of Bacillus thuringiensis (Bt) are not limited to its role as an insecticide; it is also able to promote plant growth interacting with plants and other plant growth-promoting rhizobacterium (PGPR). The PGPR Bt strain RZ2MS9 is a multi-trait maize growth promoter. We obtained a stable mutant of RZ2MS9 labelled with green fluorescent protein (RZ2MS9-GFP). We demonstrated that the Bt RZ2MS9-GFP successfully colonizes maize's roots and leaves endophytically. We evaluated whether RZ2MS9 has an additive effect on plant growth promotion when co-inoculated with Azospirillum brasilense Ab-V5. The two strains combined enhanced maize's roots and shoots dry weight around 50% and 80%, respectively, when compared to the non-inoculated control. However, non-differences were observed comparing RZ2MS9 alone and when co-inoculated with Ab-V5, In addition, we used co-inoculation experiments in glass chambers to analyse the plant's volatile organic compounds (VOCs) production during the maize-RZ2MS9 and maize-RZ2MS9-Ab-V5 interaction. We found that the single and co-inoculation altered maize's VOCs emission profile, with an increase in the production of indoles in the co-inoculation. Collectively, these results increase our knowledge about the interaction between the Bt and maize, and provide a new possibility of combined application with the commercial inoculant A. brasilense Ab-V5.

The auxin-producing Bacillus thuringiensis RZ2MS9 promotes the growth and modifies the root architecture of tomato (Solanum lycopersicum cv. Micro-Tom).

Strains of Bacillus thuringiensis (Bt) are commonly commercialized as bioinoculants for insect pest control, but their benefits go beyond their insecticidal property: they can act as plant growth-promoters. Auxins play a major role in the plant growth promotion. However, the mechanism of auxin production by the Bacilli group, and more specifically by Bt strains, is unclear. In previous work, the plant growth-promoting rhizobacterium (PGPR) B. thuringiensis strain RZ2MS9 increased the corn roots. This drew our attention to the strain's auxin production trait, earlier detected in vitro. Here, we demonstrate that in its genome, RZ2MS9 harbours the complete set of genes required in two pathways that are used for Indole acetic acid (IAA) production. We also detected that the strain produces almost five times more IAA during the stationary phase. The bacterial application increased the shoot dry weight of the Micro-Tom (MT) tomato by 24%. The application also modified MT root architecture, with an increase of 26% in the average lateral root length and inhibition of the axial root. At the cellular level, RZ2MS9-treated MT plants presented elongated root cortical cells with intensified mitotic activity. Altogether, these are the best characterized auxin-associated phenotypes. Besides that, no growth alteration was detected in the auxin-insensitive diageotropic (dgt) plants either with or without the RZ2MS9 inoculation. Our results suggest that auxins play an important role in the ability of B. thuringiensis RZ2MS9 to promote MT growth and provide a better understanding of the auxin production mechanism by a Bt strain.

On the genetic architecture in a public tropical maize panel of the symbiosis between corn and plant growth-promoting bacteria aiming to improve plant resilience.

Exploring the symbiosis between plants and plant growth-promoting bacteria (PGPB) is a new challenge for sustainable agriculture. Even though many works have reported the beneficial effects of PGPB in increasing plant resilience for several stresses, its potential is not yet widely explored. One of the many reasons is the differential symbiosis performance depending on the host genotype. This opens doors to plant breeding programs to explore the genetic variability and develop new cultivars with higher responses to PGPB interaction and, therefore, have higher resilience to stress. Hence, we aimed to study the genetic architecture of the symbiosis between PGPB and tropical maize germplasm, using a public association panel and its impact on plant resilience. Our findings reveal that the synthetic PGPB population can modulate and impact root architecture traits and improve resilience to nitrogen stress, and 37 regions were significant for controlling the symbiosis between PGPB and tropical maize. In addition, we found two overlapping SNPs in the GWAS analysis indicating strong candidates for further investigations. Furthermore, genomic prediction analysis with genomic relationship matrix computed using only significant SNPs obtained from GWAS analysis substantially increased the predictive ability for several traits endorsing the importance of these genomic regions for the response of PGPB. Finally, the public tropical panel reveals a significant genetic variability to the symbiosis with the PGPB and can be a source of alleles to improve plant resilience. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01257-6.

Secondary metabolites of Curvularia sp. G6-32, an endophyte of Sapindus saponaria, with antioxidant and anticholinesterasic properties.

In the present study, the biological activity of an extract of the secondary metabolites (E-G6-32) produced by the Curvularia sp. G6-32 endophyte (isolated from the medicinal plant Sapindus saponaria L.) was investigated. The antioxidant potential was confirmed by the DPPH (22.5%) and ABTS (62.7%) assays, and the total phenolic compound content was 40 µg gallic acid equivalents/mg. The extract E-G6-32 displayed good inhibitory activity toward butyrylcholinesterase (BuChE; IC50 = 110 ± 0.05 µg mL-1). The extract E-G6-32 was subjected to spectroscopic and mass spectrometry analyses. Comparison with the literature data confirmed that (-)-asperpentyn (1) was a major component. Asperpentyn belongs to the epoxyquinone family, which has attractive structural complexity, diverse functional groups, and a broad range of biological activities, including specific enzyme inhibitory activity. Our results suggest that Curvularia sp. G6-32 is a promising source of bioactive secondary metabolites and contains (-)-asperpentyn, which has potential pharmaceutical interest.[Figure: see text].

Metabolic extract of the endophytic fungus Flavodon flavus isolated from Justicia brandegeana in the control of Alicyclobacillus acidoterrestris in commercial orange juice.

In this work, the antibacterial activity of a crude extract of the endophytic fungus Flavodon flavus (JB257), isolated from leaves of Justicia brandegeana, was evaluated against both the vegetative and sporulated forms of Alicyclobacillus acidoterrestris. The microdilution technique was performed in order to determine the antibacterial activity of the crude extract alone as well as in combination with the bacteriocin, nisin. The minimum inhibitory concentration (MIC) of the crude extract and nisin alone against A. acidoterrestris vegetative forms were 250 µg/mL and 31.5 µg/mL, respectively, while the minimum bactericidal concentrations (MBC) were 1000 µg/mL and 62.5 µg/mL,respectively. For A. acidoterrestris spores, treatment with the crude extract at a concentration of 500 µg/mL caused a 47% reduction in growth, while nisin at 62.5 µg/mL could reduce 100% of the growth. The in vitro evaluation of the crude extract combined with nisin against A. acidoterrestris by the Checkerboard method showed a synergistic interaction between the two compounds. In addition, greater selectivity towards bacterial cells over host cells, a human hepatocyte cell line, was achieved when the crude extract was combined with nisin, Using scanning electron microscopy, interferences in the cell membrane of A. acidoterrestris could be observed after treatment with the crude extract. The results presented in this study indicate that the crude extract of the endophyte F. flavus has biotechnological potential in the food industry, especially for the treatment of orange juices through the control of A. acidoterrestris.

Colletotrichum siamense, a Mycovirus-Carrying Endophyte, as a Biological Control Strategy for Anthracnose in Guarana Plants

Abstract Guarana, the fruit of Paullinia cupana, is known for its stimulating and medicinal properties by the Amazonian indigenous population and communities. However, it presents serious phytopathological problems, such as anthracnose disease caused by Colletotrichum spp. The objective of this study was to verify if C. siamense, a mycovirus-carrying endophytic fungus, could protect guarana seedlings, by reducing or by eliminating characteristic disease symptoms. Other physiological changes in the plant caused by the presence of this endophyte were also evaluated. The cuttings of the Cereçaporanga cultivar were dipped in a biological control suspension and planted in a specific substrate. After four months in the greenhouse, the seedlings were sprayed with a suspension of phytopathogen conidia, and a portion of these seedlings received the fungicide indicated for the crop to be compared with the control seedlings. After 28 days, the number of lesions, morphophysiological and macro characteristics, and leaf micronutrients were evaluated. The seedlings treated with C. siamense showed a lower percentage of lesions and an increased aerial part and root system compared to the other treatments. There were no significant differences between treatments regarding the percentage of macronutrients and micronutrients.

Toxicity of ß-(1→3, 1→6)-á´ -glucans produced by Diaporthe sp. endophytes on Metarhizium anisopliae (Metschnikoff) Sorokin assessed by conidia germination speed parameter / Toxicidade de ß- (1 → 3, 1 → 6) -á´ -glucanas produzidas por endófitos Diaporthe sp. sobre Metarhizium anisopliae (Metschnikoff) Sorokin avaliada pelo parâmetro de velocidade de germinação dos conídios

We have previously reported that ß-(1→3,1→6)-ᴅ-glucans produced by endophytes Diaporthe sp. G27-60 and G65-65 (GenBank accession codes JF766998 and JF767007, respectively) are promising anti-proliferation agents against human breast carcinoma (MCF-7) and hepatocellular carcinoma (HepG2-C3A) cells. However, the literature fails to describe the effects of Diaporthe exopolysaccharides (EPS) on eukaryotic healthy cells. The fungus Metarhiziumanisopliae has been employed as model-system to evaluate the toxicity of pharmaceutical and agricultural-interest substances, taking into account, among other parameters, the speed of conidia germination. Current study verified the effect of different concentrations of Diaporthe ß-glucans on the germination speed of M. anisopliae. Conidia were incubated with ß-glucans treatments (50, 200 and 400 µg/mL) at 28ºC, sampled during 24 h and analyzed by light microscopy. At the end of a 24-h incubation, the amount of germinated conidia reached ≈99% for controls and ranged between 97.7 and 98.6% for treatments. Bayesian analysis indicated that Diaporthe glucans had no toxicity on M. anisopliaeand the curve of germination occurred as expected for this fungal strain. Considering the validity of filamentous fungi as model-systems, results are important data on the toxicity of endophytic EPS on healthy cells and may be associated with our previous results obtained for these polymers against tumor cells.

Anteriormente, um estudo mostrou que ß-(1→3,1→6)-ᴅ-glucanas produzidas pelos endófitos Diaporthe sp. G27-60 e G65-65 (códigos de acesso no GenBank JF766998 e JF767007, respectivamente) são agentes promissores com ação antiproliferativa contra células HepG2-C3A (hepatoma humano) e MCF-7 (adenocarcinoma mamário humano). No entanto, os efeitos de exopolissacarídeos (EPS) produzidos por fungos do gênero Diaporthe em células eucarióticas sadias não estão descritos na literatura atual. O fungo Metarhiziumanisopliae tem sido utilizado como sistema-modelo para avaliar a toxicidade de substâncias de interesse farmacêutico e agronômico, considerando, entre outros parâmetros, a velocidade de germinação de conídios. O presente estudo teve como objetivo verificar os efeitos de diferentes concentrações de ß-glucanas produzidas por Diaporthe sp. sobre a velocidade de germinação de M. anisopliae. Os conídios foram incubados com os tratamentos de ß-glucanas (50, 200 e 400 µg/mL) a 28 ºC, com amostras coletadas ao longo de 24 h, e analisados por microscopia de luz. Ao final das 24 h de incubação, o total de conídios germinados nos controles foi de ≈99%, e variou entre 97,7 e 98,6% para os tratamentos. A análise bayesiana indicou que as glucanas de Diaporthe sp. não apresentaram toxicidade sobre M. anisopliae, e a curva de germinação atendeu ao esperado para essa linhagem fúngica. Considerando a validade dos fungos filamentosos como sistemas-modelo, esses resultados representam dados importantes sobre a toxicidade dos EPS de endófitos sobre células sadias e podem ser associados aos resultados anteriormente obtidos para esses polímeros em testes contra células tumorais.

Tibouchina granulosa (Vell.) Cogn (Melastomataceae) as source of endophytic fungi: isolation, identification, and antiprotozoal activity of metabolites from Phyllosticta capitalensis.

Endophytes are microorganisms that form symbiotic relationships with their own host. Included in this group are the species Phyllosticta capitalensis, a group of fungi that include saprobes that produce bioactive metabolites. The present study aimed to identify the cultivable endophytic fungal microbiota present in healthy leaves of Tibouchina granulosa (Desr.) Cogn. (Melastomataceae) and investigate secondary metabolites produced by a strain of P. capitalensis and their effects against both Leishmania species and Trypanossoma cruzi. Identification of the strains was accomplished through multilocus sequencing analysis (MLSA), followed by phylogenetic analysis. The frequency of colonization was 73.66% and identified fungi belonged to the genus Diaporthe, Colletotrichum, Phyllosticta, Xylaria, Hypoxylon, Fusarium, Nigrospora, and Cercospora. A total of 18 compounds were identified by high-resolution mass spectrum analysis (UHPLC-HRMS), including fatty acids based on linoleic acid and derivatives, from P. capitalensis. Crude extracts had activity against Leishmania amazonensis, L. infantum, and Trypanosoma cruzi, with inhibitory concentration (IC50) values of 17.2 µg/mL, 82.0 µg/mL, and 50.13 µg/mL, respectively. This is the first report of the production of these compounds by the endophytic P. capitalensis isolated from T. granulosa.

Bioprospection and molecular phylogeny of culturable endophytic fungi associated with yellow passion fruit

Endophytic microorganisms live inside the plants without causing any damage to their hosts. In the agricultural field, these endophytes might be a strategy of biological control for phytopathogens. We aimed to isolate endophytic fungifrom yellowpassion fruit (Passiflora edulis) leaves, evaluating its biocontrol capacity by in vitroantagonism against phytopathogen Colletotrichum sp. CNPU378. We also carried out greenhouse experiments in bean seedlings. A high colonization frequency was obtained (89%), and the molecular identification based on DNA sequencing attested Colletotrichumas the most frequent genus and minor occurrence of Curvulariaendophytes. The endophytes tested showed different types of competitive interactions in in vitro antagonism inhibition rate ranging from 28.8 to 48.8%. There were 10 promising antagonists tested for their antagonist activity of crude extracts of secondary metabolites, in which strain PE-36 (20.8%) stood out among the other strains evaluated. In the greenhouse assay, plants inoculated only with endophyte Colletotrichumsp. PE-36 was symptomless and suggest that the endophyte strengthened the growth promotion in common bean plants, especially in the root length and number of leaves when compared to control plantsand other treatments. Despite many fungiof Colletotrichumgenus being described as causative agents of anthracnose, in this study, the plant sampled was colonized predominantly by Colletotrichumendophytes living in asymptomatic relationship. By the way,we come across a Colletotrichumsp. endophyte able to antagonize a Colletotrichumsp. pathogen

Additive and heterozygous (dis)advantage GWAS models reveal candidate genes involved in the genotypic variation of maize hybrids to Azospirillum brasilense.

Maize genotypes can show different responsiveness to inoculation with Azospirillum brasilense and an intriguing issue is which genes of the plant are involved in the recognition and growth promotion by these Plant Growth-Promoting Bacteria (PGPB). We conducted Genome-Wide Association Studies (GWAS) using additive and heterozygous (dis)advantage models to find candidate genes for root and shoot traits under nitrogen (N) stress and N stress plus A. brasilense. A total of 52,215 Single Nucleotide Polymorphism (SNP) markers were used for GWAS analyses. For the six root traits with significant inoculation effect, the GWAS analyses revealed 25 significant SNPs for the N stress plus A. brasilense treatment, in which only two were overlapped with the 22 found for N stress only. Most were found by the heterozygous (dis)advantage model and were more related to exclusive gene ontology terms. Interestingly, the candidate genes around the significant SNPs found for the maize-A. brasilense association were involved in different functions previously described for PGPB in plants (e.g. signaling pathways of the plant's defense system and phytohormone biosynthesis). Our findings are a benchmark in the understanding of the genetic variation among maize hybrids for the association with A. brasilense and reveal the potential for further enhancement of maize through this association.

Gloeosporiocide, a new antifungal cyclic peptide from Streptomyces morookaense AM25 isolated from the Amazon bulk soil.

Actinobacteria are known by their ability to produce several antimicrobial compounds of biotechnological interest. Thus, in this study, we isolated and identified by partial 16S RNA sequencing ∼100 actinobacteria isolates from guarana (Paullinia cupana) bulk soil. Besides, we isolated from the actinobacteria Streptomyces morookaense AM25 a novel cyclic peptide, named gloeosporiocide, molecular formula C44H48N11O7S3 (calculated 938.2901), and characterized by the presence of cyclized cysteins to form three thiazols. The novel compound had activity against the plant pathogen Colletotrichum gloeosporioides, assayed by the paper disk diffusion method (42.7% inhibition, 0.1 mg disk-1) and by the microdilution assay (1.25 g L-1). Our results reveal the potential of the actinobacteria from the Amazon rhizospheric soils as biocontrol agents as well as producers of new compounds with antifungal activity. Thus, this work constitutes a step forward in the development of the biotechnology of actinobacteria in the production of compounds of agronomic interest.

ENZYMATIC AND ANTAGONIST ACTIVITY OF ENDOPHYTIC FUNGI FROM Sapindus saponaria L. (SAPINDACEAE) / Actividad enzimática y antagonista de los hongos endofíticos de Sapindus saponaria L. (Sapindaceae)

ABSTRACT Endophyte microorganisms have great biotechnological interest, with features applicable to different areas and are potentially useful in agriculture. The current study determines the biotechnological potential of endophytic fungi, isolated from leaves of Sapindus saponaria, to control phytopathogenic fungi and evaluate their enzyme production. Molecular taxonomy was performed by sequencing of the ITS1-5.8S-ITS2 ribosomal DNA region, identifying the genera Phomopsis, Sordariomycetes, Diaporthe, and Colletotrichum. In vitro antagonism against phytopathogens showed better results against Fusarium solani and provided inhibition indices between 41.8 % and 67.5 %. The endophytic strain SS81 (Diaporthe citri) presented the highest antagonism index against the pathogen. Against Glomerella sp. and Moniliophthora perniciosa, inhibition rates ranged between 18.7 % and 57.4 % and between 38.3 % and 64.8 %, respectively. Enzyme assays revealed that strain SS65 (Diaporthe sp.) produced 1.16 UI μmol/min of amylase; strain SS77 (Diaporthe sp.) produced 2.74 UI μmol/min of pectinase, and strain SS08 (Diaporthe sp.) produced 1.51 UI μmol/min of cellulase. Thus, the current study shows evidence the importance of isolated endophytes with phytoprotective properties of plants with medicinal properties as alternatives for biological control and natural sources of products with biotechnological interest.

RESUMEN Los microorganismos endofíticos tienen gran interés biotecnológico, con características aplicables a diferentes áreas y potencialmente útiles en la agricultura. El presente estudio determinó el potencial biotecnológico de los hongos endofíticos, aislados de las hojas de Sapindus saponaria, en el control de hongos fitopatógenos y evaluación de su producción de enzimática. La taxonomía molecular fue realizada por la secuencia de la región ITS1-5.8S-ITS2 del ADN ribosomal, identificando los géneros Phomopsis, Sordariomycetes, Diaporthe y Colletotrichum. El antagonismo in vitro contra fitopatógenos mostró mejores resultados contra Fusarium solani y proporcionó índices de inhibición de entre el 41,8 % y el 67,5 %. El linaje endofítico SS81 (Diaporthe citri) presentó el mayor índice de antagonismo contra los patógenos. Contra Glomerella sp. y Moniliophthora perniciosa, las tasas de inhibición variaron entre el 18,7 % y el 57,4 % y entre el 38,3 % y el 64,8 %, respectivamente. El ensayo enzimático reveló que el linaje SS65 (Diaporthe sp.) produjo 1,16 UI μmol / min de amilasa; el linaje SS77 (Diaporthe sp.) produjo 2,74 UI μmol / min de pectinasa; y el linaje SS08 (Diaporthe sp.) produjo 1,51 UI μmol / min de celulasa. Así, el presente estudio evidencia la importancia de los endófitos aislados con propiedades fitoprotectoras como alternativas para el control biológico y como fuentes naturales de productos con interés biotecnológico.