The hidden treasures in endophytic fungi: a comprehensive review on the diversity of fungal bioactive metabolites, usual analytical methodologies, and applications.

This review provides a comprehensive overview of the key aspects of the natural metabolite production by endophytic fungi, which has attracted significant attention due to its diverse biological activities and wide range of applications. Synthesized by various fungal species, these metabolites encompass compounds with therapeutic, agricultural, and commercial significance. We delved into strategies and advancements aimed at optimizing fungal metabolite production. Fungal cultivation, especially by Aspergillus, Penicillium, and Fusarium, plays a pivotal role in metabolite biosynthesis, and researchers have explored both submerged and solid-state cultivation processes to harness the full potential of fungal species. Nutrient optimization, pH, and temperature control are critical factors in ensuring high yields of the targeted bioactive metabolites especially for scaling up processes. Analytical methods that includes High-Performance Liquid Chromatography (HPLC), Liquid Chromatography-Mass Spectrometry (LC-MS), Gas Chromatography-Mass Spectrometry (GC-MS), Nuclear Magnetic Resonance (NMR), and Mass Spectrometry (MS), are indispensable for the identification and quantification of the compounds. Moreover, genetic engineering and metabolic pathway manipulation have emerged as powerful tools to enhance metabolite production and develop novel fungal strains with increased yields. Regulation and control mechanisms at the genetic, epigenetic, and metabolic levels are explored to fine-tune the biosynthesis of fungal metabolites. Ongoing research aims to overcome the complexity of the steps involved to ensure the efficient production and utilization of fungal metabolites.

How Deep Can the Endophytic Mycobiome Go? A Case Study on Six Woody Species from the Brazilian Cerrado.

Elucidating the complex relationship between plants and endophytic fungi is very important in order to understand the maintenance of biodiversity, equity, stability, and ecosystem functioning. However, knowledge about the diversity of endophytic fungi from species of the native Brazilian Cerrado biome is poorly documented and remains largely unknown. These gaps led us to characterize the diversity of Cerrado endophytic foliar fungi associated with six woody species (Caryocar brasiliense, Dalbergia miscolobium, Leptolobium dasycarpum, Qualea parviflora, Ouratea hexasperma, and Styrax ferrugineus). Additionally, we investigated the influence of host plant identities on the structure of fungal communities. Culture-dependent methods coupled with DNA metabarcoding were employed. Irrespective of the approach, the phylum Ascomycota and the classes Dothideomycetes and Sordariomycetes were dominant. Using the cultivation-dependent method, 114 isolates were recovered from all the host species and classified into more than 20 genera and 50 species. Over 50 of the isolates belonged to the genus Diaporthe, and were distributed into more than 20 species. Metabarcoding revealed the phyla Chytridiomycota, Glomeromycota, Monoblepharomycota, Mortierellomycota, Olpidiomycota, Rozellomycota, and Zoopagomycota. These groups are reported for the first time as components of the endophytic mycobiome of Cerrado plant species. In total, 400 genera were found in all host species. A unique leaf endophytic mycobiome was identified in each host species, which differed not only by the distribution of fungal species, but also by the abundance of shared species. These findings highlight the importance of the Brazilian Cerrado as a reservoir of microbial species, and emphasize how endophytic fungal communities are diversified and adapted.

Genetic Diversity of Microcystin Producers (Cyanobacteria) and Microcystin Congeners in Aquatic Resources across Africa: A Review Paper.

Microcystins are produced by multifaceted organisms called cyanobacteria, which are integral to Africa's freshwater environments. The excessive proliferation of cyanobacteria caused by rising temperature and eutrophication leads to the production and release of copious amounts of microcystins, requiring critical management and control approaches to prevent the adverse environmental and public health problems associated with these bioactive metabolites. Despite hypotheses reported to explain the phylogeography and mechanisms responsible for cyanobacterial blooms in aquatic water bodies, many aspects are scarcely understood in Africa due to the paucity of investigations and lack of uniformity of experimental methods. Due to a lack of information and large-scale studies, cyanobacteria occurrence and genetic diversity are seldom reported in African aquatic ecosystems. This review covers the diversity and geographical distribution of potential microcystin-producing and non-microcystin-producing cyanobacterial taxa in Africa. Molecular analyses using housekeeping genes (e.g., 16S rRNA, ITS, rpoC1, etc.) revealed significant sequence divergence across several cyanobacterial strains from East, North, West, and South Africa, but the lack of uniformity in molecular markers employed made continent-wise phylogenetic comparisons impossible. Planktothrix agardhii, Microcystis aeruginosa, and Cylindrospermopsis raciborskii (presently known as Raphidiopsis raciborskii) were the most commonly reported genera. Potential microcystin (MCs)-producing cyanobacteria were detected using mcy genes, and several microcystin congeners were recorded. Studying cyanobacteria species from the African continent is urgent to effectively safeguard public and environmental health because more than 80% of the continent has no data on these important microorganisms and their bioactive secondary metabolites.

Methods used for the study of endophytic fungi: a review on methodologies and challenges, and associated tips.

Endophytic fungi are microorganisms that colonize the interior of plant tissues (e.g. leaves, seeds, stem, trunk, roots, fruits, flowers) in intracellular and/or extracellular spaces without causing symptoms of disease in host plants. These microorganisms have been isolated from plant species in a wide variety of habitats worldwide, and it is estimated that all terrestrial plants are colonized by one or more species of endophytic fungus. In addition, these microorganisms have been drawing the attention of researchers because of their ability to synthesize a wide range of bioactive molecules with potential for applications in agriculture, medicine and biotechnology. However, several obstacles come up when studying the diversity and chemical potential of endophytic fungi. For example, the usage of an inappropriate surface disinfection method for plant tissue may not eliminate the epiphytic microbiota or may end up interfering with the endophytic mycobiota, which consequently generates erroneous results. Moreover, the composition of the culture medium and the culture conditions can favor the growth of certain species and inhibit others, which generates underestimated results. Other inconsistencies can arise from the fungus misidentification and consequent exploration of its chemical potential. Based on the methodological biases that may occur at all stages of studies dealing with endophytic fungi, the objective of this review is to discuss the main methods employed in these studies as well as highlight the challenges derived from the different approaches. We also report associated tips to help future studies on endophytic fungi as a contribution.

Opposite Sides of Pantoea agglomerans and Its Associated Commercial Outlook.

Multifaceted microorganisms such as the bacterium Pantoea colonize a wide range of habitats and can exhibit both beneficial and harmful behaviors, which provide new insights into microbial ecology. In the agricultural context, several strains of Pantoea spp. can promote plant growth through direct or indirect mechanisms. Members of this genus contribute to plant growth mainly by increasing the supply of nitrogen, solubilizing ammonia and inorganic phosphate, and producing phytohormones (e.g., auxins). Several other studies have shown the potential of strains of Pantoea spp. to induce systemic resistance and protection against pests and pathogenic microorganisms in cultivated plants. Strains of the species Pantoea agglomerans deserve attention as a pest and phytopathogen control agent. Several of them also possess a biotechnological potential for therapeutic purposes (e.g., immunomodulators) and are implicated in human infections. Thus, the differentiation between the harmful and beneficial strains of P. agglomerans is mandatory to apply this bacterium safely as a biofertilizer or biocontroller. This review specifically evaluates the potential of the strain-associated features of P. agglomerans for bioprospecting and agricultural applications through its biological versatility as well as clarifying its potential animal and human health risks from a genomic point of view.

Insights into taxonomic diversity and bioprospecting potential of Cerrado endophytic fungi: a review exploring an unique Brazilian biome and methodological limitations.

Cerrado is the second largest biome in Brazil, and it is known for harboring a wide variety of endemic plant and microbial species, among which are endophytic fungi. Endophytic fungi are microorganisms capable of colonizing the interior of plant tissues without causing disease in host plants. Especially in the Cerrado biome, this group of microorganisms is still poorly studied and information on species estimation, ecological and evolutionary importance is not accurate and remains unknown. Also, it is extremely important to emphasize that great part of studies available on Cerrado endophytic fungi are national literature, including master's dissertations, course conclusion works or unpublished doctoral theses. The majority of these studies has highlighted that the endemic plant species are an important habitat for fungal endophytes, and new species have increasingly been described. Due to the lack of international literature on Cerrado endophytic fungi, the present review brings a bibliographic survey on taxonomic diversity and bioprospecting potential of fungal endophytes from a unique environment. This review also emphasizes the importance of studying Brazilian endophytic fungi from Cerrado as a source of new technologies (biofertilizer and biocontroller), since they are secondary metabolite-producing organisms with different biological activities for biotechnological, agricultural and pharmaceutical applications.

Draft genome sequence of the cyanobacterium Sphaerospermopsis aphanizomenoides BCCUSP55 from the Brazilian semiarid region reveals potential for anti-cancer applications.

Sphaerospermopsis aphanizomenoides is a filamentous nitrogen-fixing and bloom-forming cyanobacterium, which biomass can fertilize natural water with nutrients, especially through nitrogen fixation. The Sphaerospermopsis aphanizomenoides strain BCCUSP55 was previously isolated from a water supply reservoir in the Brazilian semiarid region, and its draft genome assembly coupled with the gene contents are reported here. The obtained BCCUSP55 draft genome comprised 254 scaffolds with a genome size estimated of 6,096,273 bp. In addition, it has 5250 predicted coding sequences (CDS) and the G + C content is 38.5%. Further, the BCCUSP55 draft genome presented the putative nocuolin A gene complete cluster, a natural oxadiazine that triggers apoptosis in human cancer cells. Thus, our results contribute to extend the knowledge on the genus Sphaerospermopsis and reveal its biotechnological potential.

Susceptibility of phytoplankton to the increasing presence of active pharmaceutical ingredients (APIs) in the aquatic environment: A review.

Human and veterinary pharmaceuticals either in the form of un-metabolized, incompletely metabolized, and metabolized drugs are increasingly present in aquatic ecosystems. These active pharmaceutical ingredients from pharmaceutical industries, hospitals, agricultural, and domestic discharges find their way into water systems - where they adversely affect non-target organisms like phytoplankton. Different aspects of phytoplankton life; ranging from growth, reproduction, morphology, physiology, biochemical composition, oxidative response, proteomics, and transcriptomics are altered by pharmaceuticals. This review discusses the currently available information on the susceptibility of phytoplankton to the ever-increasing presence of pharmaceutical products in the aquatic environment by focusing on the effect of APIs on the physiology, metabolome, and proteome profiles of phytoplankton. We also highlight gaps in literature concerning the salient underlining biochemical interactions between phytoplankton communities and pharmaceuticals that require an in-depth investigation. This is all in a bid to understand the imminent dangers of the contamination of water bodies with pharmaceutical products and how this process unfavorably affects aquatic food webs.

The presence of microcystins in the coastal waters of Nigeria, from the Bights of Bonny and Benin, Gulf of Guinea.

Microcystins (MCs) are the most studied toxins of cyanobacteria in freshwater bodies worldwide. However, they are poorly documented in coastal waters in several parts of the world. In this study, we investigated the composition of cyanobacteria and the presence of microcystins (MCs) in several coastal aquatic ecosystems of Nigeria. Direct morphological analysis revealed that members of the genus Oscillatoria were dominant with five species, followed by Trichodesmium with two species in Nigerian coastal waters. Oso Ibanilo had the highest cyanobacterial biomass (998 × 103 cells/L), followed by Rivers Ocean (156 × 103 cells/L). Except for the Cross River Ocean, cyanobacteria were present in all the investigated aquatic ecosystems. Ten (10) out of twenty water bodies examined had detectable levels of MCs. Furthermore, genomic DNA analysis for the mcyE gene of microcystin synthetase (mcy) cluster showed identities higher than 86% (query coverage > 96%) with toxic strains of cyanobacteria in all the samples analyzed. Also, the sequences of samples matched those of uncultured cyanobacteria from recreational lakes in Southern Germany. Our findings indicate that the presence of toxic cyanobacteria in coastal waters of Nigeria is of public and environmental health concern.

Cyanobacterial biodiversity of semiarid public drinking water supply reservoirs assessed via next-generation DNA sequencing technology.

Next-generation DNA sequencing technology was applied to generate molecular data from semiarid reservoirs during well-defined seasons. Target sequences of 16S-23S rRNA ITS and cpcBA-IGS were used to reveal the taxonomic groups of cyanobacteria present in the samples, and genes coding for cyanotoxins such as microcystins (mcyE), saxitoxins (sxtA), and cylindrospermopsins (cyrJ) were investigated. The presence of saxitoxins in the environmental samples was evaluated using ELISA kit. Taxonomic analyses of high-throughput DNA sequencing data showed the dominance of the genus Microcystis in Mundaú reservoir. Furthermore, it was the most abundant genus in the dry season in Ingazeira reservoir. In the rainy season, 16S-23S rRNA ITS analysis revealed that Cylindrospermopsis raciborskii comprised 46.8% of the cyanobacterial community in Ingazeira reservoir, while the cpcBAIGS region revealed that C. raciborskii (31.8%) was the most abundant taxon followed by Sphaerospermopsis aphanizomenoides (17.3%) and Planktothrix zahidii (16.6%). Despite the presence of other potential toxin-producing genera, the detected sxtA gene belonged to C. raciborskii, while the mcyE gene belonged to Microcystis in both reservoirs. The detected mcyE gene had good correlation with MC content, while the amplification of the sxtA gene was related to the presence of STX. The cyrJ gene was not detected in these samples. Using DNA analyses, our results showed that the cyanobacterial composition of Mundaú reservoir was similar in successive dry seasons, and it varied between seasons in Ingazeira reservoir. In addition, our data suggest that some biases of analysis influenced the cyanobacterial communities seen in the NGS output of Ingazeira reservoir.

Cylindrospermopsin induced changes in growth, toxin production and antioxidant response of Acutodesmus acuminatus and Microcystis aeruginosa under differing light and nitrogen conditions.

Growing evidence suggests that some bioactive metabolites (e.g. cyanotoxins) produced by cyanobacteria have allelopathic potential, due to their inhibitory or stimulatory effects on competing species. Although a number of studies have shown that the cyanotoxin cylindrospermopsin (CYN) has variable effects on phytoplankton species, the impact of changing physicochemical conditions on its allelopathic potential is yet to be investigated. We investigated the physiological response of Microcystis aeruginosa (Cyanobacteria) and Acutodesmus acuminatus (Chlorophyta) to CYN under varying nitrogen and light conditions. At 24h, higher microcystins content of M. aeruginosa was recorded under limited light in the presence of CYN, while at 120h the lower levels of the toxins were observed in the presence of CYN under optimum light. Total MCs concentration was significantly (p<0.05) lowered by CYN after 120h of exposure under limited and optimum nitrogen conditions. On the other hand, there were no significant (p>0.05) changes in total MCs concentrations after exposure to CYN under high nitrogen conditions. As expected, limited light and limited nitrogen conditions resulted in lower cell density of both species, while CYN only significantly (p<0.05) inhibited the growth of M. aeruginosa. Regardless of the light or nitrogen condition, the presence of CYN increased internal H2O2 content of both species, which resulted in significant (p<0.05) changes in antioxidant enzyme (catalase, peroxidase, superoxide dismutase and glutathione S-transferase) activities. The oxidative stress caused by CYN was higher under limited light and limited nitrogen. These results showed that M. aeruginosa and A. acuminatus have variable response to CYN under changing light and nitrogen conditions, and demonstrate that need to consider changes in physicochemical conditions during ecotoxicological and ecophysiological investigations.

Does anatoxin-a influence the physiology of Microcystis aeruginosa and Acutodesmus acuminatus under different light and nitrogen conditions?

Due to changing global climatic conditions, a lot of attention has been given to cyanobacteria and their bioactive secondary metabolites. These conditions are expected to increase the frequency of cyanobacterial blooms, and consequently, the concentrations of cyanotoxins in aquatic ecosystems. Unfortunately, there are very few studies that address the effects of cyanotoxins on the physiology of phytoplankton species under different environmental conditions. In the present study, we investigated the effect of the cyanotoxin anatoxin-a (ATX-A) on Microcystis aeruginosa (cyanobacteria) and Acutodesmus acuminatus (chlorophyta) under varying light and nitrogen conditions. Low light (LL) and nitrogen limitation (LN) resulted in significant cell density reduction of the two species, while the effect of ATX-A on M. aeruginosa was not significant. However, under normal (NN) and high nitrogen (HN) concentrations, exposure to ATX-A resulted in significantly (p < 0.05) lower cell density of A. acuminatus. Pigment content of M. aeruginosa significantly (p < 0.05) declined in the presence of ATX-A, regardless of the light condition. Under each light condition, exposure to ATX-A caused a reduction in total microcystin (MC) content of M. aeruginosa. The detected MC levels varied as a function of nitrogen and ATX-A concentrations. The production of reactive oxygen species (H2O2) and antioxidant enzyme activities of both species were significantly altered by ATX-A under different light and nitrogen conditions. Our results revealed that under different light and nitrogen conditions, the response of M. aeruginosa and A. acuminatus to ATX-A was variable, which demonstrated the need for different endpoints of environmental factors during ecotoxicological investigations.

Draft Genome Sequence of Cylindrospermopsis raciborskii (Cyanobacteria) Strain ITEP-A1 Isolated from a Brazilian Semiarid Freshwater Body: Evidence of Saxitoxin and Cylindrospermopsin Synthetase Genes.

Cylindrospermopsis raciborskii ITEP-A1 is a saxitoxin-producing cyanobacterium. We report the draft genome sequence of ITEP-A1, which comprised 195 contigs that were assembled with SPAdes and annotated with Rapid Annotation using Subsystem Technology. The identified genome sequence had 3,605,836 bp, 40.1% G+C, and predicted 3,553 coding sequences (including the synthetase genes).

Non-ribosomal peptides produced by Brazilian cyanobacterial isolates with antimicrobial activity.

Cyanobacterial strains isolated from terrestrial and freshwater habitats in Brazil were evaluated for their antimicrobial and siderophore activities. Metabolites of fifty isolates were extracted from the supernatant culture media and cells using ethyl acetate and methanol, respectively. The extracts of 24 isolates showed antimicrobial activity against several pathogenic bacteria and one yeast. These active extracts were characterized by Q-TOF/MS. The cyanobacterial strains Cylindrospermopsis raciborskii 339-T3, Synechococcus elongatus PCC7942, Microcystis aeruginosa NPCD-1, M. panniformis SCP702 and Fischerella sp. CENA19 provided the most active extracts. The 50 cyanobacterial strains were also screened for the presence of non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes and microcystin production. Putative fragment genes coding for NRPS adenylation domains and PKS keto-synthase domains were successfully PCR amplified from 92% and 80% of cyanobacterial strains, respectively. The potential therapeutical compounds siderophores were detected in five cyanobacterial isolates. Microcystin production was detected by ELISA test in 26% of the isolates. Further a protease inhibitor substance was detected by LC-MS/MS in the M. aeruginosa NPLJ-4 extract and the presence of aeruginosin and cyanopeptolin was confirmed by PCR amplification using specific primers, and sequenced. This screening study showed that Brazilian cyanobacterial isolates are a rich source of natural products with potential for pharmacological and biotechnological applications.

A NOVEL EPIPHYTIC CYANOBACTERIAL SPECIES FROM THE GENUS BRASILONEMA CAUSING DAMAGE TO EUCALYPTUS LEAVES(1).

A cyanobacterial mat colonizing the leaves of Eucalyptus grandis was determined to be responsible for serious damage affecting the growth and development of whole plants under the clonal hybrid nursery conditions. The dominant cyanobacterial species was isolated in BG-11 medium lacking a source of combined nitrogen and identified by cell morphology characters and molecular phylogenetic analysis (16S rRNA gene and cpcBA-IGS sequences). The isolated strain represents a novel species of the genus Brasilonema and is designated Brasilonema octagenarum strain UFV-E1. Thin sections of E. grandis leaves analyzed by light and electron microscopy showed that the B. octagenarum UFV-E1 filaments penetrate into the leaf mesophyll. The depth of infection and the mechanism by which the cyanobacterium invades leaf tissue were not determined. A major consequence of colonization by this cyanobacterium is a reduction in photosynthesis in the host since the cyanobacterial mats decrease the amount of light incident on leaf surfaces. Moreover, the cyanobacteria also interfere with stomatal gas exchange, decreasing CO2 assimilation. To our knowledge, this is the first report of an epiphytic cyanobacterial species causing damage to E. grandis leaves.