Metabolomic approaches to explore chemodiversity in seeds of guaraná (Paullinia cupana) using UPLC-QTOF-MSE and NMR analysis.

The growing interest in health and well-being has spurred the evolution of functional foods, which provide enhanced health benefits beyond basic nutrition. Guaraná seeds (Paullinia cupana) have been widely studied and used as a functional food due to their richness in caffeine, phenolic compounds, amino acids, and other nutrients. This has established guaraná as a significant food supplement, with Brazil being the largest producer of the world. This study aims to propose a set of analytical methods to chemically evaluate fifty-six different guaraná clones, from the Guaraná Germplasm Active Bank, to accommodate the diverse requirements of the food industry. Metabolomic approaches were employed, in which a non-target metabolomic analysis via UPLC-QTOF-MSE led to the annotation of nineteen specialized metabolites. Furthermore, targeted metabolomics was also used, leading to the identification and quantification of metabolites by NMR. The extensive data generated were subjected to multivariate analysis, elucidating the similarities and differences between the evaluated guaraná seeds, particularly concerning the varying concentration levels of the metabolites. The metabolomics approach based on the combination of UPLC-QTOF-MSE, NMR and chemometric tools provided sensitivity, precision and accuracy to establish the chemical profiles of guaraná seeds. In conclusion, evaluating and determining the metabolic specificities of different guarana clones allow for their application in the development of products with different levels of specific metabolites, such as caffeine. This caters to various purposes within the food industry. Moreover, the recognized pharmacological properties of the annotated specialized metabolites affirm the use of guarana clones as an excellent nutritional source.

Isolation and Identification of Pigment-Producing Endophytic Fungi from the Amazonian Species Fridericia chica.

Pigments of fungal origin have aroused increasing interest in the food dye and cosmetic industries since the global demand for natural dyes has grown. Endophytic microorganisms are a source of bioactive compounds, and Amazonian plant species can harbor fungi with a wide range of biotechnological applications. Popularly known in Brazil as crajiru, Fridericia chica is a medicinal plant that produces a red pigment. In this study, a total of 121 fungi were isolated in potato dextrose agar from three plants. We identified nine pigment-producing endophytic fungi isolated from branches and leaves of F. chica. The isolates that showed pigment production in solid media were molecularly identified via multilocus analysis as Aspergillus welwitschiae, A. sydowii, Curvularia sp., Diaporthe cerradensis (two strains), Hypoxylon investiens, Neoscytalidium sp. (two strains) and Penicillium rubens. These isolates were subjected to submerged fermentation in two culture media to obtain metabolic extracts. The extracts obtained were analyzed in terms of their absorbance between 400 and 700 nm. The pigmented extract produced by H. investiens in medium containing yeast extract showed maximum absorbance in the red absorption range (UA700 = 0.550) and significant antioxidant and antimicrobial activity. This isolate can thus be considered a new source of extracellular pigment.

Trichoderma agriamazonicum sp. nov. (Hypocreaceae), a new ally in the control of phytopathogens.

The genus Trichoderma comprises more than 500 valid species and is commonly used in agriculture for the control of plant diseases. In the present study, a Trichoderma species isolated from Scleronema micranthum (Malvaceae) has been extensively characterized and the morphological and phylogenetic data support the proposition of a new fungal species herein named Trichoderma agriamazonicum. This species inhibited the mycelial growth of all the nine phytopathogens tested both by mycoparasitism and by the production of VOCs, with a highlight for the inhibition of Corynespora cassiicola and Colletotrichum spp. The VOCs produced by T. agriamazonicum were able to control Capsicum chinense fruit rot caused by Colletotrichum scovillei and no symptoms were observed after seven days of phytopathogen inoculation. GC-MS revealed the production of mainly 6-amyl-α-pyrone, 1-octen-3-ol and 3-octanone during interaction with C. scovillei in C. chinense fruit. The HLPC-MS/MS analysis allowed us to annotate trikoningin KBII, hypocrenone C, 5-hydroxy-de-O-methyllasiodiplodin and unprecedented 7-mer peptaibols and lipopeptaibols. Comparative genomic analysis of five related Trichoderma species reveals a high number of proteins shared only with T. koningiopsis, mainly the enzymes related to oxidative stress. Regarding the CAZyme composition, T. agriamazonicum is most closely related to T. atroviride. A high protein copy number related to lignin and chitin degradation is observed for all Trichoderma spp. analyzed, while the presence of licheninase GH12 was observed only in T. agriamazonicum. Genome mining analysis identified 33 biosynthetic gene clusters (BGCs) of which 27 are new or uncharacterized, and the main BGCs are related to the production of polyketides. These results demonstrate the potential of this newly described species for agriculture and biotechnology.

First report of Thielaviopsis ethacetica causing black rot in Bactris gasipaes fruit in Brazil.

The peach palm (Bactris gasipaes Kunth) is a palm of great importance to the population of the Brazilian Amazon region. Its fruits are an important food source for the local population (Alves and Flores, 1982). Between 2018 and 2021, peach palm fruits with black rot symptoms were collected in the state of Pará, in the municipalities of Juruti (020 09' 08'' S 560 05' 32W) and Santarém (20 26' 22''S 540 41' 55''W), Brazil. Symptomatic fruits detach easily from the bunch. When sectioned, tissues with black coloration and mycelia with white to black coloration were found (Fig. 1a-b). The incidence of the disease in orchards ranged from 5 to 50%. Then, direct isolation, was done by transferring fragments of mycelia and spores to a plate containing potato dextrose agar (PDA). Morphological markers of the asexual phase were evaluated by cultivating the isolates in malt extract agar (MEA) with fragments of Saccharum officinarum culm (Mbenoun et al., 2014). The colonies initially showed a white coloration but turned dark after eight days of cultivation. Colonies produced white mycelia with hyaline, unicellular, rectangular primary conidia (5.6-8.8 µm) (n=30) in length and 2.8-4.0 µm (n=30) in width (Fig. 1e). In the dark-colored mycelia, secondary conidia that formed exhibited three distinct shapes: cylindrical, light brown, and (6.6-11.6 µm x 3.0-3.7 µm) (n=30) (Fig 1. f-i); oblong to globose shape (5.0-10.0 x 3.0-5.3 µm) (n=30) (Fig 1. g); and ellipsoid-shaped (7.0-13.0 x 3.0-4.0 µm) (n=30) (Fig 1. h). Furthermore, unicellular aleuroconidia, produced in dark-colored colonies, exhibited cell walls (10.8-17.5 x 5.4-8.4 µm) (n=30) with a warty, dark-brown, ovoid-shaped appearance (Fig 1. j-k). Genomic DNA was isolated from 4-day-old cultures, and ITS-rDNA and TEF-1α were amplified from ITS1/ ITS2 (White et al., 1990) and EF1F/EF2R (Jacobs et al., 2004), respectively. Sequences were deposited in GenBank (ITS: OL623838, OL623839, OM643316, OL623840, and OL623841) (TEF1: OL631623, OM643318, OM643317, OL631624, and OL631625). Bayesian analysis concatenated were conducted with MrBayes v. 3.2.7 (Ronquist et al., 2011). Clustered the five isolates with the Thielaviopsis ethacetica reference isolate IMI 50560 with Bayesian posterior probability (Bpp = 0.99). (Fig. 2). The pathogenicity test, was conducted using the five isolates, that were were inoculated on six fruits early maturity. After the fruit were washed with neutral detergent and water, 0.5-cm-deep wounds were made with a sterile penknife. Next, 1 mL of primary and secondary conidia suspension at 1 x 105 spores/mL was added to each wound. Six control fruit were inoculated with distilled water. The fruits were kept in incubators at 25 °C with a 12 h photoperiod. The experiment was conducted twice (Alves and Flores, 1982). After five days of inoculation, all inoculated fruit showed characteristic symptoms (Fig 1. c), whereas the control fruit remained asymptomatic (Fig 1. d). The fungus reisolated from all inoculated fruit exhibited the same morphological markers as the inoculated fungus, thus completing Koch's postulates. Thielaviopsis ethacetica is an important pathogen in several palm species in sugarcane and pineapple crops in different parts of the world (Mbenoun et al., 2014; Borgens et al., 2019). This study is the first record of T. ethacetica causing black rot in B. gasipaes fruit in Brazil.

Larval gut microbiome of Pelidnota luridipes (Coleoptera: Scarabaeidae): high bacterial diversity, different metabolic profiles on gut chambers and species with probiotic potential.

Pelidnota luridipes Blanchard (1850) is a tropical beetle of the family Scarabaeidae, whose larvae live on wood without parental care. Microbiota of mid- and hindgut of larvae was evaluated by culture-dependent and independent methods, and the results show a diverse microbiota, with most species of bacteria and fungi shared between midgut and hindgut. We isolated 272 bacterial and 29 yeast isolates, identified in 57 and 7 species, respectively, while using metabarcoding, we accessed 1,481 and 267 OTUs of bacteria and fungi, respectively. The composition and abundance of bacteria and fungi differed between mid- and hindgut, with a tendency for higher richness and diversity of yeasts in the midgut, and bacteria on the hindgut. Some taxa are abundant in the intestine of P. luridipes larvae, such as Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria; as well as Saccharomycetales and Trichosporonales yeasts. Mid- and hindgut metabolic profiles differ (e.g. biosynthesis of amino acids, cofactors, and lipopolysaccharides) with higher functional diversity in the hindgut. Isolates have different functional traits such as secretion of hydrolytic enzymes and antibiosis against pathogens. Apiotrichum siamense L29A and Bacillus sp. BL17B protected larvae of the moth Galleria mellonella, against infection by the pathogens Listeria monocytogenes ATCC19111 and Pseudomonas aeruginosa ATCC 9027. This is the first work with the larval microbiome of a Rutelini beetle, demonstrating its diversity and potential in prospecting microbial products as probiotics. The functional role of microbiota for the nutrition and adaptability of P. luridipes larvae needs to be evaluated in the future.

Extracts of Amazonian Fungi With Larvicidal Activities Against Aedes aegypti.

The global increase in diseases transmitted by the vector Aedes aegypti, new and re-emerging, underscores the need for alternative and more effective methods of controlling mosquitoes. Our aim was to identify fungal strains from the Amazon rain forest that produce metabolites with larvicidal activity against Aedes aegypti. Thirty-six fungal strains belonging to 23 different genera of fungi, isolated from water samples collected in the state of Amazonas, Brazil were cultivated. The liquid medium was separated from the mycelium by filtration. Medium fractions were extracted with ethyl acetate and isopropanol 9:1 volume:volume, and the mycelia with ethyl acetate and methanol 1:1. The extracts were vacuum dried and the larvicidal activity was evaluated in selective bioassays containing 500 µg/ml of the dried fungal extracts. Larval mortality was evaluated up to 72 h. None of the mycelium extracts showed larvicidal activity greater than 50% at 72 h. In contrast, 15 culture medium extracts had larvicidal activity equal to or greater than 50% and eight killed more than 90% of the larvae within 72 h. These eight extracts from fungi belonging to seven different genera (Aspergillus, Cladosporium, Trichoderma, Diaporthe, Albifimbria, Emmia, and Sarocladium) were selected for the determination of LC50 and LC90. Albifimbria lateralis (1160) medium extracts presented the lowest LC50 value (0.268 µg/ml) after 24 h exposure. Diaporthe ueckerae (1203) medium extracts presented the lowest value of LC90 (2.928 µg/ml) at 24 h, the lowest values of LC50 (0.108 µg/ml) and LC90 (0.894 µg/ml) at 48 h and also at 72 h (LC50 = 0.062 µg/ml and LC90 = 0.476 µg/ml). Extracts from Al. lateralis (1160) and D. ueckerae (1203) showed potential for developing new, naturally derived products, to be applied in integrated vector management programs against Ae. aegypti.

Bacillus spp. metabolites are effective in eradicating Aedes aegypti (Diptera: Culicidae) larvae with low toxicity to non-target species.

The growing spread of dengue, chikungunya and Zika viruses demand the development of new and environmentally safe control methods for their vector, the mosquito Aedes aegypti. This study aims to find novel larvicidal agents from mutualistic (endophytic and rhizospheric) or edaphic bacteria that have no action against non-target organisms. Eleven out of the 254 bacterial strains tested were able to kill Ae. aegypti larvae. Larvicidal activity did not depend on presence of cells, since culture supernatants or crude lipopeptide extracts (CLEs) killed the larvae. Bacillus safensis BacI67 and Bacillus paranthracis C21 supernatants were the best performing supernatants, displaying the lowest lethal concentrations (LC50 = 31.11 µL/mL and 45.84 µL/mL, respectively). Bacillus velezensis B64a and Bacillus velezensis B15 produced the best performing CLEs (LC50 = 0.11 mg/mL and 0.12 mg/mL, respectively). Mass spectrometry analysis of CLEs detected a mixture of surfactins, iturins, and fengycins. The samples tested were weakly- or non-toxic to mammalian cells (RAW 264.7 macrophages and VERO cells) and non-target organisms (Caenorhabditis elegans, Galleria mellonella, Scenedesmus obliquus, and Tetrahymena pyriformis) - especially B. velezensis B15 CLE. The biosynthetic gene clusters related to secondary metabolism identified by whole genome sequencing of the four best performing bacteria strains revealed clusters for bacteriocin, beta-lactone, lanthipeptide, non-ribosomal peptide synthetases, polyketide synthases (PKS), siderophores, T3PKS, type 1 PKS-like, terpenes, thiopeptides, and trans-AT-PKS. Purification of lipopeptides may clarify the mechanisms by which these extracts kill Ae. aegypti larvae.

Mercury alters the rhizobacterial community in Brazilian wetlands and it can be bioremediated by the plant-bacteria association.

This study examined how soil mercury contamination affected the structure and functionality of rhizobacteria communities from Aeschynomene fluminensis and Polygonum acuminatum and how rhizobacteria mediate metal bioremediation. The strains were isolated using culture-dependent methods, identified through 16S rDNA gene sequencing, and characterized with respect to their functional traits related to plant growth promotion and resistance to metals and antibiotics. The bioremediation capacity of the rhizobacteria was determined in greenhouse using corn plants. The isolated bacteria belonged to the phyla Actinobacteria, Deinococcus-Thermus, Firmicutes, and Proteobacteria, with great abundance of the species Microbacterium trichothecenolyticum. The rhizobacteria abundance, richness, and diversity were greater in mercury-contaminated soils. Bacteria isolated from contaminated environments had higher minimum inhibitory concentration values, presented plasmids and the merA gene, and were multi-resistant to metals and antibiotics. Enterobacter sp._C35 and M. trichothecenolyticum_C34 significantly improved (Dunnett's test, p < 0.05) corn plant growth in mercury-contaminated soil. These bacteria helped to reduce up to 87% of the mercury content in the soil, and increased the mercury bioaccumulation factor by up to 94%. Mercury bioremediation mitigated toxicity of the contaminated substrate. Enterobacter sp._C35, Bacillus megaterium_C28, and Bacillus mycoides_C1 stimulated corn plant growth and could be added to biofertilizers produced in research and related industries.

Characterization and comprehensive analysis of the ecological interaction networks of bacterial communities in Paullinia cupana var. sorbilis by 16S rRNA gene metabarcoding.

Endophytes improve the host performance in areas of high plant endemicity. Paullinia cupana is an Amazonia plant species of economic and social importance due to the high caffeine concentration in its seeds. An interesting strategy to identify endophytic microorganisms with potential biotechnological application is to understand the factors that influence the endophytic community to rationalize the host management programs. We used the next-generation sequencing for bacterial 16S rRNA gene to examine how the P. cupana organ, genotype, and geographic location influenced its endophytic bacterial community. We obtained 1520 operational taxonomic units (OTUs) distributed in 19 phyla, 32 classes, 79 orders, 114 families and 174 genera. The P. cupana roots and leaves were specifically colonized by the bacterial genera Acidothermus and Porphyromonas, respectively, with high relative frequency. The plant organ type influenced the endophytic community's richness, diversity, OTUs composition, relative abundance of phyla and genera, and genera interaction network. However, the host plant genotype and geographic location influenced the composition and interaction among genera in the network analysis. Prevotella is a super-generalist genus in the interaction network of endophytic bacteria of P. cupana. This study revealed endophytic bacterial groups of importance to P. cupana and stressed that the host plant organ modulates the structure and interactions within this community. Our results indicated that the microbial community adapted to colonize P. cupana by adjusting to its composition and interaction network. The isolation of abundant and super-generalist bacterial genera shall help to examine their functionality to the composition and fitness of the endophytic community of P. cupana.

Diversity of cultivable bacterial endophytes in Paullinia cupana and their potential for plant growth promotion and phytopathogen control.

Endophytic bacteria occupy the same niche of phytopathogens and may produce metabolites that induce the host plant systemic resistance and growth. Host and environmental variables often determine the endophytic community's structure and composition. In this study, we addressed whether the plant genotype, organ, and geographic location influence the structure, composition, and functionality of endophytic bacterial communities in Paullinia cupana. To characterize the communities and identify strains with potential application in agriculture, we analyzed two P. cupana genotypes cultivated in two cities of the State of Amazonas, Brazil. Endophytic bacteria were isolated from surface-disinfested root, leaf, and seed tissues through the fragmentation and maceration techniques. The colonization rate, number of bacteria, richness, diversity, and functional traits were determined. The plant growth-promoting ability of selected bacterial strains was assessed in Sorghum bicolor. We identified 95 bacterial species distributed in 29 genera and 3 phyla (Proteobacteria, Actinobacteria, and Firmicutes). The colonization rate, richness, diversity, and species composition varied across the plant organs; the last parameter also varied across the plant genotype and location. Some strains exhibited relevant plant growth-promoting traits and antagonistic traits against the main phytopathogens of P. cupana, but they were not separated by functional traits. The main bacterial strains with plant growth-promoting traits induced S. bicolor growth. Altogether, our findings open opportunities to study the application of isolated endophytic bacterial strains in the bioprospection of processes and products.

Use of the IRAP marker to study genetic variability in Pseudocercospora fijiensis populations.

Pseudocercospora fijiensis is the etiological agent of black Sigatoka, which is currently considered as one of the most destructive banana diseases in all locations where it occurs. It is estimated that a large portion of the P. fijiensis genome consists of transposable elements, which allows researchers to use transposon-based molecular markers in the analysis of genetic variability in populations of this pathogen. In this context, the inter-retrotransposon-amplified polymorphism (IRAP) was used to study the genetic variability in P. fijiensis populations from different hosts and different geographical origins in Brazil. A total of 22 loci were amplified and 77.3 % showed a polymorphism. Cluster analysis revealed two major groups in Brazil. The observed genetic diversity (H E) was 0.22, and through molecular analysis of variance, it was determined that the greatest genetic variability occurs within populations. The discriminant analysis of principal components revealed no structuring related to the geographical origin of culture of the host. The IRAP-based marker system is a suitable tool for the study of genetic variability in P. fijiensis.