João Sarkis Yunes

Widely used as inoculants in agriculture, nitrogen‐fixing bacteria can be associated with other plant growth‐promoting microorganisms (PGPMs) to increase crop grain yield. This study aimed to evaluate whether co‐inoculation with Rhizobium tropici and Azospirillum brasilense associated with microalgae or cyanobacteria enhances common bean (Phaseolus vulgaris) grain yield and its economic gains in contrasting sandy and clayey soils. Water‐soluble protein content and indole‐3‐acetic acid (IAA) production of two cyanobacteria (Anabaena cylindrica and Calotrix brevissima) and six Chlorophyta microalgae (Nannochloropsis oculata, Haematococcus pluvialis, Muriellopsis sphaerica, Chlorella protothecoides, Chlorella vulgaris, and Botryococcus braunii) were determined. Anabaena cylindrica and C. brevissima had the highest IAA production, 336.7 ± 44.7 and 94.1 ± 11.8 µg IAA mg⁻¹ of protein, respectively. Two field experiments were carried out to evaluate the triple inoculation of these Chlorophyta microalgae and cyanobacteria associated with R. tropici plus A. brasilense on the agronomic efficiency and profitability of common bean. Co‐inoculation of C. vulgaris plus R. tropici and A. brasilense in common bean had grain yield of 1277.5 kg ha⁻¹ in clayey soil and 2960.3 kg ha⁻¹ in sandy soil, increasing by 219.7 and 656.0 kg ha⁻¹, respectively, in relation to double inoculation of R. tropici + A. brasilense. In the sandy soil, common bean with triple co‐inoculation had the highest profit ($956 ha⁻¹), which was 25.6% higher than the N fertilized plants. Co‐inoculation with PGPMs generated the highest economic gains, and in addition, it is an eco‐friendly agronomic practice for sustainable food production.

Cyanobacterial blooms are a potential threat to human communities and ecosystems. Since the late 1980s, researchers have reported harmful cyanobacterial colonies in Patos Lagoon (PL), the largest coastal lagoon in South America. Most studies concerning harmful blooms in PL have focused on its biology and on its southernmost estuarine region, with little information about its displacement inside the lagoon and the influence of physical forces on its dynamics. This study uses satellite-derived information (normalized difference chlorophyll-a index – NDCI), river discharge data, and meteorological data (wind speed and direction, rainfall, and air temperature) to analyze two bloom episodes in PL, during the austral summer of 2019/2020, specifically in its larger, limnic portion. A 30-year meteorological time series was used to contrast the same summer period. Two remote sensing images from Sentinel-2 were taken of PL margins, near their central portion. The summer of 2019/2020 was drier when compared with the historical data, characterizing low river discharge. This environmental condition was coupled with high temperature, which implies thermal stratification in summer even at 2-m depth sites, which might have promoted cyanobacterial growth and accumulation inside PL. Moreover, weak winds (−1). The NDCI values represented the two days of blooms, with higher values occurring under higher water temperatures and low wind speeds.

In freshwater, saxitoxins (STX) are produced by different cyanobacteria genera, including Raphidiopsis. Data regarding cytogenotoxicity effects of STX on human cells are scarse, this merit further studies of its toxicology. This study assessed the cytotoxicity and the chromosome instability of STX on SHSY-5Y human cell line. The CBMN assay allows the detection of chromosome breaks and abnormal chromosomal segregation. Additionally, in silico systems biology approach, used to search for known and predicted interaction networks, was applied to study the interactions between STX and SHSY-5Y cellular components. The results of the CBMN assay demonstrated that STX concentrations of 2.5 - 10 µg/L induced cytostasis and chromosome instability in a dose-response relationship. Apoptosis was detected after exposure of SHSY-5Y cultured cells to STX concentration of 10 µg/L. The results of the systems biology analysis revealed the interaction of STX with proteins related with acetylcoline pathway, cell cycle regulation and apoptosis. Furthermore, combining the in vitro and in silico approachs, it was possible to suggest a mechanism of action of STX in SHSY-5Y cells. Overall, the data demonstrated the cytotoxicity and mutagenicity of environmentally relevant concentrations of STX. These results should be considered when setting up guidelines for monitoring STX in water supply.

The combined inoculation of Rhizobium (R. tropici+R. freirei), Azospirillum brasilense, and Anabaena cylindrica, a diazotrophic cyanobacterium, is a technology that has not yet been tested and established in the production of the common bean (Phaseolus vulgaris). The inoculation may be a promising strategy for increasing crop productivity by combining the benefits of biological nitrogen fixation with the production of plant growth phytohormones. Therefore, the objective of this study was to evaluate the co-inoculation of Rhizobium, Azospirillum brasilense, and Anabaena cylindrica as an alternative method for optimizing the symbiotic performance and development of the common bean at greenhouse conditions. The treatments were as follows: (T1) control without N and inoculation, (T2) N addition (100 kg N ha-1), (T3) Riz (addition of R. tropici+R. freirei), (T4) Azo (Azospirillum brasilense addition), (T5) Ana (Anabaena cylindrical addition), (T6) Riz+Azo, (T7) Riz+Ana, (T8) Azo+Ana, (T9) Riz+Azo+Ana. We used a completely randomized experimental design with four replications. The co-inoculation of Riz+Azo+Ana promoted plant height, root length and volume, shoot dry matter, accumulated shoot N, number and dry matter of nodules at flowering, number of grains per pod, hundred seed weight, and grain production of the common bean, contributing to increased yield per plant. We observed an increase in common bean grain yield ranging from 62 to 84% after double and triple co-inoculation of rhizobia with azospirilla and/or cyanobacteria, with the highest yield observed in the plants inoculated with Riz+Azo+Ana (84%), similar to those observed in plants after N addition. However, field experiments are necessary to elucidate the performances of the inoculated beneficial microorganisms.

Blooms of the saxitoxin-producing cyanobacterium Cylindrospermopsis raciborskii have been contaminating drinking water reservoirs in Brazil for many years. Although acute effects of saxitoxin intoxication are well known, chronic deleterious outcomes caused by repeated saxitoxin exposure still require further investigation. The aim of the present work is to investigate the effects of consumption of drinking water contaminated with C. raciborskii for 30 days on learning and memory processes in rats. The effects of saxitoxin (3 or 9 μg/L STX equivalents) or cyanobacteria on behavior was determined using the open field habituation task, elevated plus maze anxiety model task, inhibitory avoidance task, and referential Morris water maze task. No effects of saxitoxin consumption was observed on anxiety and motor exploratory parameters in the elevated plus maze and open field habituation tasks, respectively. However, groups treated with 9 μg/L STX equivalents displayed a decreased memory performance in the inhibitory avoidance and Morris water maze tasks. These results suggest an amnesic effect of saxitoxin on aversive and spatial memories.

The present work aimed to evaluate the antimycobacterial activity and cytotoxicity of Microcystis aeruginosa toxins, the MC-LR variant and purified extract of [D-Leu1] microcystin-LR. The antimicrobial activity of M. aeruginosa extract and microcystin was evaluated by resazurin microtiter assay against Mycobacterium tuberculosis, M. terrae, M. chelonae and M. kansasii. The cytotoxicity assay was performed by trypan blue exclusion against the HTC cell line. Antimicrobial activity was observed in the hexanic extract of M. aeruginosa (RST 9501 strain) against M. tuberculosis, including sensitive and resistant strains with minimal inhibitory concentrations (MIC) between 1.93 μM and 0.06 μM. The high activity of M. aeruginosa hexanic extract could be attributed to the major presence of the toxins MC-LR and [D-Leu1] MC-LR that showed activity at MIC between 53 and 0.42 μM against tested mycobacterial strains. Even at the highest concentration tested, no toxicity of M. aeruginosa extracts was identified against HTC cells. These preliminary results suggest that [D-Leu1] MC-LR is a promising candidate for the development of a new antimycobacterial agent.

Toxic cyanobacterial blooms are recurrent in Patos Lagoon, in southern Brazil. Among cyanotoxins, [D-Leu1] microcystin-LR is the predominant variant whose natural cycle involves water and sediment compartments. This study aimed to identify and isolate from sediment a bacterial strain capable of growing on [D-Leu1] microcystin-LR. Sediment and water samples were collected at two distinct aquatic spots: close to the Oceanographic Museum (P1), in Rio Grande City, and on São Lourenço Beach (P2), in São Lourenço do Sul City, southern Brazil.

Background: Many important toxins and antibiotics are produced by non-ribosomal biosynthetic pathways. Microcystins are a chemically diverse family of potent peptide toxins and the end-products of a hybrid NRPS and PKS secondary metabolic pathway. They are produced by a variety of cyanobacteria and are responsible for the poisoning of humans as well as the deaths of wild and domestic animals around the world. The chemical diversity of the microcystin family is attributed to a number of genetic events that have resulted in the diversification of the pathway for microcystin assembly. Results: Here, we show that independent evolutionary events affecting the substrate specificity of the microcystin biosynthetic pathway have resulted in convergence on a rare [D-Leu¹] microcystin-LR chemical variant. We detected this rare microcystin variant from strains of the distantly related genera Microcystis, Nostoc, and Phormidium. Phylogenetic analysis performed using sequences of the catalytic domains within the mcy gene cluster demonstrated a clear recombination pattern in the adenylation domain phylogenetic tree. We found evidence for conversion of the gene encoding the McyA₂ adenylation domain in strains of the genera Nostoc and Phormidium. However, point mutations affecting the substrate-binding sequence motifs of the McyA₂ adenylation domain were associated with the change in substrate specificity in two strains of Microcystis. In addition to the main [D-Leu¹] microcystin-LR variant, these two strains produced a new microcystin that was identified as [Met¹] microcystin-LR. Conclusions: Phylogenetic analysis demonstrated that both point mutations and gene conversion result in functional mcy gene clusters that produce the same rare [D-Leu¹] variant of microcystin in strains of the genera Microcystis, Nostoc, and Phormidium. Engineering pathways to produce recombinant non-ribosomal peptides could provide new natural products or increase the activity of known compounds. Our results suggest that the replacement of entire adenylation domains could be a more successful strategy to obtain higher specificity in the modification of the non-ribosomal peptides than point mutations.

Microcystins (MCs) are toxins produced by cyanobacteria during the blooms that could accumulate in aquatic animals and be relocated to higher trophic levels. Adenosine triphosphate (ATP) acts as an excitatory neurotransmitter and/or a neuromodulator in the extracellular space playing important roles in physiological and pathological conditions. The aim of this study was, therefore, to evaluate the acute effects of different concentrations of MC-LR on nucleoside triphosphate diphosphohydrolases and 5’-nucleotidade in adult zebrafish (Danio rerio) brain membranes. The results have shown no significant changes in ATP, adenosine diphosphate (ADP) and adenosine monophosphate (AMP) hydrolysis in zebrafish brain membranes. MC-LR in vitro also did not alter ATP, ADP and AMP hydrolysis in the concentrations tested. These findings show that acute exposure to MC-LR did not modulate ectonucleotidase activity in the conditions tested. However, additional studies including chronic exposure should be performed in order to achieve a better understanding about MC-LR toxicity mechanisms in the central nervous system.