Microcystin influence on soil-plant microbiota: Unraveling microbiota modulations and assembly processes in the rhizosphere of Vicia faba.

Microcystins (MCs) are frequently detected in cyanobacterial bloom-impacted waterbodies and introduced into agroecosystems via irrigation water. They are widely known as phytotoxic cyanotoxins, which impair the growth and physiological functions of crop plants. However, their impact on the plant-associated microbiota is scarcely tackled and poorly understood. Therefore, we aimed to investigate the effect of MCs on microbiota-inhabiting bulk soil (BS), root adhering soil (RAS), and root tissue (RT) of Vicia faba when exposed to 100 µg L-1 MCs in a greenhouse pot experiment. Under MC exposure, the structure, co-occurrence network, and assembly processes of the bacterial microbiota were modulated with the greatest impact on RT-inhabiting bacteria, followed by BS and, to a lesser extent, RAS. The analyses revealed a significant decrease in the abundances of several Actinobacteriota-related taxa within the RT microbiota, including the most abundant and known genus of Streptomyces. Furthermore, MCs significantly increased the abundance of methylotrophic bacteria (Methylobacillus, Methylotenera) and other Proteobacteria-affiliated genera (e.g., Paucibacter), which are supposed to degrade MCs. The co-occurrence network of the bacterial community in the presence of MCs was less complex than the control network. In MC-exposed RT, the turnover in community composition was more strongly driven by deterministic processes, as proven by the beta-nearest taxon index. Whereas in MC-treated BS and RAS, both deterministic and stochastic processes can influence community assembly to some extent, with a relative dominance of deterministic processes. Altogether, these results suggest that MCs may reshape the structure of the microbiota in the soil-plant system by reducing bacterial taxa with potential phytobeneficial traits and increasing other taxa with the potential capacity to degrade MCs.

Health risk assessment of lake water contaminated with microcystins for fruit crop irrigation and farm animal drinking.

The health risks linked to the consumption of microcystin-accumulating crops have been increasing worldwide in toxic cyanobloom-occurring regions. The bioaccumulation of microcystins (MCs) in agricultural produce at environmentally realistic concentrations is poorly investigated. In this field study, we assessed the health risks of MCs in raw water used for irrigating fruit crops (bioaccumulation) and watering farm animals in the Lalla Takerkoust agricultural region (Marrakesh, Morocco). Thus, MCs were extracted from water and fruit samples and quantified by enzyme-linked immunosorbent assay in order to calculate the health risk indicators. MCs posed a high health-risk level to poultry and horses, with estimated daily intakes (EDI) being 14- and 19-fold higher than the recommended limits (3.1 and 2.3 µg MC-LR L-1), respectively. Furthermore, pomegranate posed the same level of risk, with EDI being 22- and 53-fold higher than the limit dose (0.04 µg MC-LR kg-1) for adults and children, respectively. There was an urgent need for guidelines regarding water use and management in MC-polluted areas, besides the setup of nature-based tools for toxin removal from raw water used in farming practices. Moreover, MCs could contaminate the human food chain, which implies further investigations of their potential accumulation in livestock- and poultry-based food.

Role of Rhizospheric Microbiota as a Bioremediation Tool for the Protection of Soil-Plant Systems from Microcystins Phytotoxicity and Mitigating Toxin-Related Health Risk.

Frequent toxic cyanoblooms in eutrophic freshwaters produce various cyanotoxins such as the monocyclic heptapeptides microcystins (MCs), known as deleterious compounds to plant growth and human health. Recently, MCs are a recurrent worldwide sanitary problem in irrigation waters and farmland soils due to their transfer and accumulation in the edible tissues of vegetable produce. In such cases, studies about the persistence and removal of MCs in soil are scarce and not fully investigated. In this study, we carried out a greenhouse trial on two crop species: faba bean (Vicia faba var. Alfia 321) and common wheat (Triticum aestivum var. Achtar) that were grown in sterile (microorganism-free soil) and non-sterile (microorganism-rich soil) soils and subjected to MC-induced stress at 100 µg equivalent MC-LR L-1. The experimentation aimed to assess the prominent role of native rhizospheric microbiota in mitigating the phytotoxic impact of MCs on plant growth and reducing their accumulation in both soils and plant tissues. Moreover, we attempted to evaluate the health risk related to the consumption of MC-polluted plants for humans and cattle by determining the estimated daily intake (EDI) and health risk quotient (RQ) of MCs in these plants. Biodegradation was liable to be the main removal pathway of the toxin in the soil; and therefore, bulk soil (unplanted soil), as well as rhizospheric soil (planted soil), were used in this experiment to evaluate the accumulation of MCs in the presence and absence of microorganisms (sterile and non-sterile soils). The data obtained in this study showed that MCs had no significant effects on growth indicators of faba bean and common wheat plants in non-sterile soil as compared to the control group. In contrast, plants grown in sterile soil showed a significant decrease in growth parameters as compared to the control. These results suggest that MCs were highly bioavailable to the plants, resulting in severe growth impairments in the absence of native rhizospheric microbiota. Likewise, MCs were more accumulated in sterile soil and more bioconcentrated in root and shoot tissues of plants grown within when compared to non-sterile soil. Thereby, the EDI of MCs in plants grown in sterile soil was more beyond the tolerable daily intake recommended for both humans and cattle. The risk level was more pronounced in plants from the sterile soil than those from the non-sterile one. These findings suggest that microbial activity, eventually MC-biodegradation, is a crucial bioremediation tool to remove and prevent MCs from entering the agricultural food chain.

Protective Role of Native Rhizospheric Soil Microbiota Against the Exposure to Microcystins Introduced into Soil-Plant System via Contaminated Irrigation Water and Health Risk Assessment.

Microcystins (MCs) produced in eutrophic waters may decrease crop yield, enter food chains and threaten human and animal health. The main objective of this research was to highlight the role of rhizospheric soil microbiota to protect faba bean plants from MCs toxicity after chronic exposure. Faba bean seedlings were grown in pots containing agricultural soil, during 1 month under natural environmental conditions of Marrakech city in Morocco (March-April 2018) and exposed to cyanobacterial extracts containing up to 2.5 mg·L-1 of total MCs. Three independent exposure experiments were performed (a) agricultural soil was maintained intact "exposure experiment 1"; (b) agricultural soil was sterilized "exposure experiment 2"; (c) agricultural soil was sterilized and inoculated with the rhizobia strain Rhizobium leguminosarum RhOF34 "exposure experiment 3". Overall, data showed evidence of an increased sensitivity of faba bean plants, grown in sterilized soil, to MCs in comparison to those grown in intact and inoculated soils. The study revealed the growth inhibition of plant shoots in both exposure experiments 2 and 3 when treated with 2.5 mg·L-1 of MCs. The results also showed that the estimated daily intake (EDI) of MCs, in sterilized soil, exceeded 2.18 and 1.16 times the reference concentrations (0.04 and 0.45 µg of microcysin-leucine arginine (MC-LR). Kg-1 DW) established for humans and cattle respectively, which raises concerns about human food chain contamination.

Mode of action and fate of microcystins in the complex soil-plant ecosystems.

Over the last decades, global warming has increasingly stimulated the expansion of cyanobacterial blooms in freshwater ecosystems worldwide, in which toxic cyanobacteria produce various congeners of cyanotoxins, mainly dominated by microcystins (MCs). MCs introduced into agricultural soils have deleterious effects on the germination, growth and development of plants and their associated microbiota, leading to remarkable yield losses. Phytotoxicity of MCs may refer to the inhibition of phosphatases activity, generating deleterious reactive oxygen species, altering gene functioning and phytohormones translocation within the plant. It is also known that MCs can pass through the root membrane barrier, translocate within plant tissues and accumulate into different organs, including edible ones. Also, MCs impact the microbial activity in soil via altering plant-bacterial symbioses and decreasing bacterial growth rate of rhizospheric microbiota. Moreover, MCs can persist in agricultural soils through adsorption to clay-humic acid particles and results in a long-term contact with the plant-microflora complex. However, their bioavailability to plants and half-life in soil seem to be influenced by biodegradation process and soil physicochemical properties. This review reports the latest and most relevant information regarding MCs-phytotoxicity and impact on soil microbiota, the persistence in soil, the degradation by native microflora and the bioaccumulation within plant tissues.

Phenotypic and genetic diversity of Moroccan rhizobia isolated from Vicia faba and study of genes that are likely to be involved in their osmotolerance.

Rhizobia are symbiotic nitrogen-fixing bacteria in root nodules of legumes. In Morocco, faba bean (Vicia faba L.), which is the main legume crop cultivated in the country, is often grown in marginal soils of arid and semi-arid regions. This study examines the phenotypic diversity of rhizobia nodulating V. faba isolated from different regions in Morocco for tolerance to some abiotic stresses. A total of 106 rhizobia strains isolated from nodules were identified at the species level by analysing 16S rDNA. Additionally, for selected strains recA, otsA, kup and nodA fragments were sequenced. 102 isolates are likely to belong to Rhizobium leguminosarum or R. laguerreae and 4 isolates to Ensifer meliloti. All strains tolerating salt concentrations of 428 or 342mM NaCl as well as 127 or 99mM Na2SO4 were highly resistant to alkaline conditions (pH 10) and high temperature (44°C). Three strains: RhOF4 and RhOF53 (both are salt-tolerant) and RhOF6 (salt-sensitive) were selected to compare the influence of different levels of salt stress induced by NaCl on growth and on trehalose and potassium accumulation. We find a direct correlation between the trehalose contents of the rhizobial strains and their osmotolerance.

Microcystin-tolerant Rhizobium protects plants and improves nitrogen assimilation in Vicia faba irrigated with microcystin-containing waters.

Irrigation of crops with microcystins (MCs)-containing waters-due to cyanobacterial blooms-affects plant productivity and could be a way for these potent toxins entering the food chain. This study was performed to establish whether MC-tolerant rhizobia could benefit growth, nodulation, and nitrogen metabolism of faba bean plants irrigated with MC-containing waters. For that, three different rhizobial strains-with different sensitivity toward MCs-were used: RhOF96 (most MC-sensitive strain), RhOF125 (most MC-tolerant strain), or Vicz1.1 (reference strain). As a control, plants grown without rhizobia and fertilized by NH4NO3 were included in the study. MC exposure decreased roots (30-37 %) and shoots (up to 15 %) dry weights in un-inoculated plants, whereas inoculation with rhizobia protects plants toward the toxic effects of MCs. Nodulation and nitrogen content were significantly impaired by MCs, with the exception of plants inoculated with the most tolerant strain RhOF125. In order to deep into the effect of inoculation on nitrogen metabolism, the nitrogen assimilatory enzymes (glutamine synthetase (GS) and glutamate synthase (GOGAT)) were investigated: Fertilized plants showed decreased levels (15-30 %) of these enzymes, both in shoots and roots. By contrast, inoculated plants retained the levels of these enzymes in shoots and roots, as well as the levels of NADH-GOGAT activity in nodules. We conclude that the microcystin-tolerant Rhizobium protects faba bean plants and improves nitrogen assimilation when grown in the presence of MCs.

Microbiote shift in the Medicago sativa rhizosphere in response to cyanotoxins extract exposure.

The bloom-containing water bodies may have an impact due to cyanotoxins production on other microorganisms and aquatic plants. Where such water is being used for crops irrigation, the presence of cyanotoxins may also have a toxic impact on terrestrial plants and their rhizosphere microbiota. For that purpose, PCR-based 454 pyrosequencing was applied to phylogenetically characterize the bacterial community of Medicago sativa rhizosphere in response to cyanotoxins extract. This analysis revealed a wide diversity at species level, which decreased from unplanted soil to root tissues indicating that only some populations were able to compete for nutrients and niches in this selective habitat. Gemmatimonas, Actinobacteria, Deltaproteobacteria and Opitutae mainly inhabited the bulk soil, whereas, the root-adhering soil and the root tissues were inhabited by Gammaproteobacteria and Alphaproteobacteria. The proportion of these populations fluctuated in response to cyanotoxins extract exposure. Betaproteobacteria proportion increased in the three studied compartments, whereas Gammaproteobacteria proportion decreased except in the bulk soil. This study revealed the potential toxicity of cyanotoxins extract towards Actinobacteria, Gemmatimonas, Deltaproteobacteria, and Gammaproteobacteria, however Clostridia, Opitutae and bacteria related with Betaproteobacteria, were stimulated denoting their tolerance. Altogether, these data indicate that crop irrigation using cyanotoxins containing water might alter the rhizosphere functioning.

Physiological and antioxidant responses of Medicago sativa-rhizobia symbiosis to cyanobacterial toxins (Microcystins) exposure.

Toxic cyanobacteria in freshwaters can induce potent harmful effects on growth and development of plants irrigated with contaminated water. In this study, the effect of cyanobacteria extract containing Microcystins (MC) on Medicago sativa-rhizobia symbiosis was investigated in order to explore plants response through biomass production, photosynthetic pigment and antioxidant enzymes analysis: Peroxidase (POD), Polyphenoloxidase (PPO) and Catalase (CAT). Alfalfa plants were inoculated with two endosymbiotic rhizobial strains: RhOL1 (MC less sensitive strain) and RhOL3 (MC more sensitive strain), to evaluate the rhizobial contribution on the plant response cultured under cyanobacterial toxins stress. The two rhizobia strains were identified as Ensifer meliloti by sequence analysis of their rrs and atpD genes. The chronic exposure to MC extract showed shoot, root and nodules dry weight decrease, in both symbiosis cultures. The rate of decline in plants inoculated with RhOL3 was higher than that in symbiosis with RhOL1 mainly at 20 µg L(-1) of MC. Cyanotoxins also reduced photosynthetic pigment content and generated an oxidative stress observed at cellular level. POD, PPO and CAT activities were significantly increased in leaves, roots and nodules of alfalfa plants exposed to MC. These enzyme activities were higher in plants inoculated with RhOL3 especially when alfalfa plants were exposed to 20 µg L(-1) of MC. The present paper reports new scientific finding related to the behavior of rhizobia-M. sativa associations to MC (Microcystins) for later recommendation concerning the possible use of these symbiosis face to crops exposure to MC contaminated water irrigation.

Physiological and biochemical defense reactions of Vicia faba L.-Rhizobium symbiosis face to chronic exposure to cyanobacterial bloom extract containing microcystins.

The presence of cyanotoxins, mainly microcystins (MCs), in surface freshwater represents a serious health risk to aquatic organisms living in the water body, as well as terrestrial animals and plants that are in contact with contaminated water. Consequently, the use of MCs contaminated water for irrigation represents a hazard for cultivated plants and could induce severe economical losses due to crops' yield reduction. The experimental approach undertaken in this work was exposing Vicia faba seedlings (inoculated with a Rhizobium strain resistant to MCs), to water supplemented with cyanobacterial crude extract containing total microcystins at a concentration of 50 and 100 µg/L (environmental relevant concentrations of MCs dissolved in the raw irrigation water from Lalla Takerkoust Lake-Marrakesh region). After chronic MCs exposure (2 months), biological and physiological parameters (plant growth, nitrogen uptake, mineral assimilation, and oxidative defense mechanisms) were evaluated. The results obtained showed evidence that chronic exposure to cyanobacterial bloom extract containing MCs strongly affected the physiological and biological plants activities; reduction of dry matter, photosynthetic activity, nodule number, and nitrogen assimilation. At the same time, an increase of oxidative stress was observed, as deduced from a significant increase of the activities of peroxidase, catalase, polyphenoloxidase, and phenylalanine ammonia lyase in leaves, roots, and nodules of faba bean plants exposed to cyanotoxins, especially at 100 µg/L of MCs. This experimentation constitutes a simulation of the situation related to cyanotoxins chronic exposure of seedlings-plants via the contaminated irrigation water. For this reason, once should take into consideration the possibility of contamination of agricultural crops and the quality of irrigation water should be by the way monitored for cyanotoxins biohazard.

Cyanobacterial extracts containing microcystins affect the growth, nodulation process and nitrogen uptake of faba bean (Vicia faba L., Fabaceae).

The use of irrigation water containing cyanobacterial toxins may generate a negative impact in both yield and quality of agricultural crops causing significant economic losses. We evaluated the effects of microcystins (MC) on the growth, nodulation process and nitrogen uptake of a Faba bean cultivar (Vicia faba L., Fabaceae), particularly the effect of MC on rhizobia-V. faba symbiosis. Three rhizobial strains (RhOF4, RhOF6 and RhOF21), isolated from nodules of local V. faba were tested. The exposure of rhizobia to MC showed that the toxins had a negative effect on the rhizobial growth especially at the highest concentrations of 50 and 100 µg/l. The germination of faba bean seeds was also affected by cyanotoxins. We registered germination rates of 75 and 68.75% at the toxin levels of 50 and 100 µg/l as compared to the control (100%). The obtained results also showed there was a negative effect of MC on plants shoot, root (dry weight) and total number of nodules per plant. Cyanotoxins exposure induced a significant effect on nitrogen assimilation by faba bean seedlings inoculated with selected rhizobial strains RhOF6 and RhOF21, while the effect was not significant on beans seedling inoculated with RhOF4. This behavior of tolerant rhizobia-legumes symbioses may constitute a very important pathway to increase soil fertility and quality and can represent a friendly biotechnological way to remediate cyanotoxins contamination in agriculture.

Allelopatic effects of cyanobacteria extracts containing microcystins on Medicago sativa-Rhizobia symbiosis.

The eutrophication of water leads to massive blooms of cyanobacteria potentially producers of highly toxic substances: cyanotoxins, especially microcystins (MC). The contamination of water used for irrigation by these toxins, can cause several adverse effects on plants and microorganisms. In this work, we report the phytotoxic effects of microcystins on the development of symbiosis between the leguminous plant Medicago sativa (Alfalfa) and rhizobia strains. The exposure of rhizobial strains to three different concentrations 0.01, 0.05 and 0.1 µg MC ml(-1) led to decrease on the bacteria growth. The strains of rhizobia Rh L1, Rh L2, Rh L3 and Rh L4 reduced their growth to, respectively, 20.85%, 20.80%, 33.19% and 25.65%. The chronic exposure of alfalfa seeds and seedlings to different MC concentrations affects the whole stages of plant development. The germination process has also been disrupted with an inhibition, which reaches 68.34% for a 22.24 µg MC ml(-1). Further, seedlings growth and photosynthetic process were also disrupted. The toxins reduced significantly the roots length and nodule formation and leads to an oxidative stress. Thus, the MCs contained in lake water and used for irrigation affect the development of symbiosis between M. sativa and Rhizobia.