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Amid growing environmental concerns and the imperative for sustainable agricultural practices, this study examines the potential of nitrogen-fixing cyanobacteria as biofertilizers, particularly in cotton cultivation. The reliance on synthetic nitrogen fertilizers (SNFs), prevalent in modern agriculture, poses significant environmental challenges, including greenhouse gas emissions and water system contamination. This research aims to shift this paradigm by exploring the capacity of cyanobacteria as a natural and sustainable alternative. Utilizing advanced metabarcoding methods to analyze the 16S rRNA gene, we conducted a comprehensive assessment of soil bacterial communities within cotton fields. This study focused on evaluating the diversity, structure, taxonomic composition, and potential functional characteristics of these communities. Emphasis was placed on the isolation of native N2-fixing cyanobacteria strains rom cotton soils, and their subsequent effects on cotton growth. Results from our study demonstrate significant plant growth-promoting (PGP) activities, measured as N2 fixation, production of Phytohormones, Fe solubilization and biofertilization potential of five isolated cyanobacterial strains, underscoring their efficacy in cotton. These findings suggest a viable pathway for replacing chemical-synthetic nitrogen fertilizers with natural, organic alternatives. The reintegration of these beneficial species into agricultural ecosystems can enhance crop growth while fostering a balanced microbial environment, thus contributing to the broader goals of global sustainable agriculture.

Scientific progress relies on clear and consistent definitions for effective communication and collaboration. The term "symbiosis" in the context of plant-microbe associations suffers from diverse interpretations, leading to ambiguity in classification of these associations. This review elaborates on the issue, proposing an inclusive definition as well as a keyword.

Nitrogen-fixing cyanobacteria from the order Nostocales are able to establish symbiotic relationships with diverse plant species. They are promiscuous symbionts, as the same strain of cyanobacterium is able to form symbiotic biological nitrogen-fixing relationships with different plants species. This review will focus on the different types of cyanobacterial-plant associations, both endophytic and epiphytic, and provide insights from a structural viewpoint, as well as our current understanding of the mechanisms involved in the symbiotic crosstalk. In all these symbioses, the benefit for the plant is clear; it obtains from the cyanobacterium fixed nitrogen and other bioactive compounds, such as phytohormones, polysaccharides, siderophores, or vitamins, leading to enhanced plant growth and productivity. Additionally, there is increasing use of different cyanobacterial species as bio-inoculants for biological nitrogen fixation to improve soil fertility and crop production, thus providing an eco-friendly, alternative, and sustainable approach to reduce the over-reliance on synthetic chemical fertilizers.

All known photosynthetic cyanobacteria carry a cytochrome c6 protein that acts transferring electrons from cytochrome b6f complex to photosystem I, in photosynthesis, or cytochrome c oxidase, in respiration. In most of the cyanobacteria, at least one homologue to cytochrome c6 is found, the so-called cytochrome c6B or cytochrome c6C. However, the function of these cytochrome c6-like proteins is still unknown. Recently, it has been proposed a common origin of these proteins as well as the reclassification of the cytochrome c6C group as c6B, renaming the new joint group as cytochrome c6BC. Another homologue to cytochrome c6 has not been classified yet, the formerly called cytochrome c6-3, which is present in the heterocyst-forming filamentous cyanobacteria Nostoc sp. PCC 7119. In this work, we propose the inclusion of this group as an independent group in the genealogy of cytochrome c6-like proteins with significant differences from cytochrome c6 and cytochrome c6BC, with the proposed name cytochrome c6D. To support this proposal, new data about phylogeny, genome localisation and functional properties of cytochrome c6-like proteins is provided. Also, we have analysed the interaction of cytochrome c6-like proteins with cytochrome f by isothermal titration calorimetry and by molecular docking, concluding that c6-like proteins could interact with cytochrome b6f complex in a similar fashion as cytochrome c6. Finally, we have analysed the reactivity of cytochrome c6-like proteins with membranes enriched in terminal oxidases of cyanobacteria by oxygen uptake experiments, concluding that cytochrome c6D is able to react with the specific copper-oxidase of the heterocysts, the cytochrome c oxidase 2.

Rice is one of the most important crops in the world and is considered a strategic crop for food security. Furthermore, the excessive use of chemical fertilizers to obtain high yields causes environmental problems. A sustainable alternative includes taking advantage of beneficial bacteria that promote plant growth. Here, we investigate the effect of five bacterial biofertilizers from halophytes on growth, and we investigate photosynthetic efficiency in rice plants grown under saline conditions (0 and 85 mmol L−1 NaCl) and future climate change scenarios, including increased CO2 concentrations and temperature (400/700 ppm and 25/+4 °C, respectively). Biofertilizers 1–4 increased growth by 9–64% in plants grown with and without salt in both CO2- temperature combinations, although there was no significant positive effect on the net photosynthetic rate of rice plants. In general, biofertilizer 1 was the most effective at 400 ppm CO2 and at 700 ppm CO2 +4 °C in the absence of salt. Inocula 1–5 also stimulated plant length at high CO2 levels without salt. Finally, the positive effect of biofertilization was attenuated in the plants grown under the interaction between salt and high CO2. This highlights the significance of studying biofertilization under stress interaction to establish the real potential of biofertilizers in the context of climate change conditions.

The Donana ˜ wetlands comprise an emblematic Mediterranean landscape protected as a UNESCO World Heritage Site. Some parts of these wetlands have been transformed into intensive rice cultivation areas, which are currently the most productive rice-growing areas in Europe. We examined the bacterial communities in these domesticated soils as they are key for plant health and productivity and have a strong influence on biochemical cycles. To identify the bacteria, we used metabarcoding analysis coupled with metabolic predictions and cooccurrence networks. This analysis was performed in the bulk and rhizosphere soils during different stages in the growing season. These soil compartments had a greater effect on the bacterial communities than the plant phenological stages. The diversity and richness of the bacterial population inhabiting the rhizosphere was much lower than that in the bulk soil, comprising taxa that were significantly more represented in this soil compartment, such as bacteria from the genus Hydrogenophaga, three genera from the order Rhizobiales, and unclassified genera from the families Desulfocapsaceae and Actinobacteria. Rhizosphere co-occurrence networks revealed a high number of negative connections, indicating unstable bacterial communities that may be highly influenced by biotic and abiotic factors. Rhizosphere networks mostly rely on two taxa belonging to the phyla Proteobacteria and Cyanobacteria, which are the predicted network hubs in this soil compartment. The bulk soil conserved high bacterial diversity and richness that was stable throughout the growth period of rice. Anaerobic bacteria from genera Marmoricola, the uncultured Gemmatimonadota bacteria SDR1034 terrestrial group, Anaerolinea, and the sulphur oxidizer, Thiobacillus were highly represented. This analysis provides valuable information for understanding bacterial diversity in the rhizosphere of rice cultivated in this region, which is critical for enhancing plant growth and product

Rice is one of the most important crops in the world and is considered a strategic crop for food security. Furthermore, the excessive use of chemical fertilizers to obtain high yields causes environmental problems. A sustainable alternative includes taking advantage of beneficial bacteria that promote plant growth. Here, we investigate the effect of five bacterial biofertilizers from halophytes on growth, and we investigate photosynthetic efficiency in rice plants grown under saline conditions (0 and 85 mmol L−1 NaCl) and future climate change scenarios, including increased CO2 concentrations and temperature (400/700 ppm and 25/+4 °C, respectively). Biofertilizers 1–4 increased growth by 9–64% in plants grown with and without salt in both CO2- temperature combinations, although there was no significant positive effect on the net photosynthetic rate of rice plants. In general, biofertilizer 1 was the most effective at 400 ppm CO2 and at 700 ppm CO2 +4 °C in the absence of salt. Inocula 1–5 also stimulated plant length at high CO2 levels without salt. Finally, the positive effect of biofertilization was attenuated in the plants grown under the interaction between salt and high CO2. This highlights the significance of studying biofertilization under stress interaction to establish the real potential of biofertilizers in the context of climate change conditions.

The Doñana wetlands comprise an emblematic Mediterranean landscape protected as a UNESCO World Heritage Site. Some parts of these wetlands have been transformed into intensive rice cultivation areas, which are currently the most productive rice-growing areas in Europe. We examined the bacterial communities in these domesticated soils as they are key for plant health and productivity and have a strong influence on biochemical cycles. To identify the bacteria, we used metabarcoding analysis coupled with metabolic predictions and co-occurrence networks. This analysis was performed in the bulk and rhizosphere soils during different stages in the growing season. These soil compartments had a greater effect on the bacterial communities than the plant phenological stages. The diversity and richness of the bacterial population inhabiting the rhizosphere was much lower than that in the bulk soil, comprising taxa that were significantly more represented in this soil compartment, such as bacteria from the genus Hydrogenophaga, three genera from the order Rhizobiales, and unclassified genera from the families Desulfocapsaceae and Actinobacteria. Rhizosphere co-occurrence networks revealed a high number of negative connections, indicating unstable bacterial communities that may be highly influenced by biotic and abiotic factors. Rhizosphere networks mostly rely on two taxa belonging to the phyla Proteobacteria and Cyanobacteria, which are the predicted network hubs in this soil compartment. The bulk soil conserved high bacterial diversity and richness that was stable throughout the growth period of rice. Anaerobic bacteria from genera Marmoricola, the uncultured Gemmatimonadota bacteria SDR1034 terrestrial group, Anaerolinea, and the sulphur oxidizer, Thiobacillus were highly represented. This analysis provides valuable information for understanding bacterial diversity in the rhizosphere of rice cultivated in this region, which is critical for enhancing plant growth and producti

All known photosynthetic cyanobacteria carry a cytochrome c 6 protein that acts transferring electrons from cytochrome b 6 f complex to photosystem I, in photosynthesis, or cytochrome c oxidase, in respiration. In most of the cyanobacteria, at least one homologue to cytochrome c 6 is found, the so-called cytochrome c 6B or cytochrome c 6C. However, the function of these cytochrome c 6-like proteins is still unknown. Recently, it has been proposed a common origin of these proteins as well as the reclassification of the cytochrome c 6C group as c 6B, renaming the new joint group as cytochrome c 6BC. Another homologue to cytochrome c 6 has not been classified yet, the formerly called cytochrome c 6-3, which is present in the heterocyst-forming filamentous cyanobacteria Nostoc sp. PCC 7119. In this work, we propose the inclusion of this group as an independent group in the genealogy of cytochrome c 6-like proteins with significant differences from cytochrome c 6 and cytochrome c 6BC, with the proposed name cytochrome c 6D. To support this proposal, new data about phylogeny, genome localisation and functional properties of cytochrome c 6-like proteins is provided. Also, we have analysed the interaction of cytochrome c 6-like proteins with cytochrome f by isothermal titration calorimetry and by molecular docking, concluding that c 6-like proteins could interact with cytochrome b 6 f complex in a similar fashion as cytochrome c 6. Finally, we have analysed the reactivity of cytochrome c 6-like proteins with membranes enriched in terminal oxidases of cyanobacteria by oxygen uptake experiments, concluding that cytochrome c 6D is able to react with the specific copper-oxidase of the heterocysts, the cytochrome c oxidase 2.

The exponential growth of the population, the trend towards organic food consumption and the increasingly restrictive regulations imposed by the European Union are forcing the agricultural sector to evolve towards more sustainable practices that are less harmful to the environment. In addition to these challenges, farmers need to cope with other biotic and abiotic factors affecting plant productivity, such as drought, diseases and pests. To that end, biotechnological approaches to obtain food in a sustainable way are being explored. The lower Guadalquivir region, at the South of Spain, contains the largest area devoted to intensive agriculture practices in Andalusia. This region faces two main problems, which are high salinity in irrigation water and N contamination by synthetic fertilizers (Paredes et al., 2020). In this work we provide two different biotechnological approaches to address these problems: 1) We have characterized salt resistance in nine rice varieties that are being cultivated in the Guadalquivir paddies. This analysis has been complemented through morphological, physiological and biochemical approaches, using analytical methods such as HPLC and mass chromatography, among others. We have identified metabolites that are overproduced in salt stress conditions and might be used for the early detection of salt stress in the plant. A comparative analysis of the different rice varieties analysed provided valued information about the different tolerance to salt. 2) In order to reduce the use of synthetic nitrogen fertilizers, we tested three bioinoculants that had been previously isolated from cotton soils. First, we characterized them biochemically for the PGPR activities, comprising N2 fixation and production of plant phytohormones. Effectiveness of these bioinoculants was assayed in microcosms experiments. We found a significant plant growth stimulation in two of the three bioinoculants evaluated.

Motivation: Cyanobacteria of the genus Nostoc are capable of establishing symbiosis relationships with many different types of plants. In these mutualistic relationships the cyanobacterium provides the plant with fixed nitrogen, while the plant provides the cyanobacterium with protection from hostile environments and carbon compounds as energy for N2 fixation. It has recently been described that Nostoc punctiforme performs a stable symbiosis with Oryza sativa (Álvarez et al., 2020). In order to know the molecular mechanisms involved in the recognition between the plant and the cyanobacterium, a proteomic study was carried out in the early stages of co-culture of both organisms. In this study, proteins with homology to the Nod factors of Rhizobium sp. were identified in Nostoc, which could be related to signaling in the plant. The aim of this work is to study the regulation of the expression of the genes encoding these Nod proteins by means of RT-qPCR. Methods: The expression of Nostoc punctiforme Nod genes was studied in response to the presence of the plant at 1, 2, 3, 5 and 7 days of co-culture. On the one hand, a Nostoc punctiforme culture grown at 25°C in Roux flasks with 1% CO2, continuous illumination and at 30°C was prepared. On the other hand, Oryza sativa seedlings were obtained germination of seeds under axenic conditions. At one week of growth, the seedlings were transplanted into flasks with hydroponic medium. Co-culture was performed by adding a fixed amount of Nostoc to the Oryza culture medium, and incubating the mixture in thermostated chambers at 25°C, 12h light/dark cycles and 75% relative humidity. RNA was extracted from Nostoc samples that had been in contact with the plant. As a control, Nostoc incubated without the plant was used. After RNA retrotranscription, the resulting cDNA was used to evaluate the expression of the genes of interest. Results: It was observed that the expression of certain Nod genes is activated in the presence of Oryza, a

Soil salinization is an environmental problem that adversely affects plant growth and crop productivity worldwide. As an alternative to the conventional approach of breeding salt-tolerant plant cultivars, we explored the use of plant-growth-promoting rhizobacteria (PGPR) from halophytic plants to enhance crop growth under saline conditions. Here, we report the effect of five PGPR consortia from halophytes on the growth of eight (alfalfa, flax, maize, millet, rice, strawberry, sunflower, and wheat) of the crops most commonly produced on salinized soils worldwide. To test the efficiency of halotolerant consortia, we designed a complex environmental matrix simulating future climate-change scenarios, including increased CO2 levels and temperature. Overall, biofertilizers enhanced growth of most crops with respect to non-inoculated control plants under different CO2 concentrations (400/700 ppm), temperatures (25/+4 °C), and salinity conditions (0 and 85 mM NaCl). Biofertilizers counteracted the detrimental effect of salinity on crop growth. Specifically, strawberry and rice showed the greatest positive additive response to inoculation in the presence of salt; above-ground biomasses were 35% and 3% greater, respectively, than their respective control grown without salt. Furthermore, depending on the interaction of environmental factors (salinity × CO2 × temperature) analyzed, the results varied—influencing the most effective biofertilizer determined for each crop now, or in the future. Our findings highlight the importance of conducting studies that consider stress interaction for realistic assessments of the potential of biofertilizers in a climate-changed world.

Soil salinization is an environmental problem that adversely affects plant growth and crop productivity worldwide. As an alternative to the conventional approach of breeding salt-tolerant plant cultivars, we explored the use of plant-growth-promoting rhizobacteria (PGPR) from halophytic plants to enhance crop growth under saline conditions. Here, we report the effect of five PGPR consortia from halophytes on the growth of eight (alfalfa, flax, maize, millet, rice, strawberry, sunflower, and wheat) of the crops most commonly produced on salinized soils worldwide. To test the efficiency of halotolerant consortia, we designed a complex environmental matrix simulating future climate-change scenarios, including increased CO2 levels and temperature. Overall, biofertilizers enhanced growth of most crops with respect to non-inoculated control plants under different CO2 concentrations (400/700 ppm), temperatures (25/+4 °C), and salinity conditions (0 and 85 mM NaCl). Biofertilizers counteracted the detrimental effect of salinity on crop growth. Specifically, strawberry and rice showed the greatest positive additive response to inoculation in the presence of salt; above-ground biomasses were 35% and 3% greater, respectively, than their respective control grown without salt. Furthermore, depending on the interaction of environmental factors (salinity × CO2 × temperature) analyzed, the results varied—influencing the most effective biofertilizer determined for each crop now, or in the future. Our findings highlight the importance of conducting studies that consider stress interaction for realistic assessments of the potential of biofertilizers in a climate-changed world.

Current agricultural productivity depends on an exogenous nutrient supply to crops. This is of special relevance in cereal production, a fundamental part of the trophic chain that plays a vital role in the human diet. However, our agricultural practices entail highly detrimental side-effects from an environmental point of view. Long-term nitrogen fertilization in croplands results in degradation of soil, water, and air quality, producing eutrophication and subsequently contributing to global warming. In accordance with this, there is a biotechnological interest in using nitrogen-fixing microorganisms to enhance crop growth without adding chemically synthesized nitrogen fertilizers. This is particularly beneficial in paddy fields, where about 60% of the synthetic fertilizer that has been applied is dissolved in the water and washed away. In these agricultural systems, N2-fixing cyanobacteria show a promising biotechnological potential as biofertilizers, improving soil fertility while reducing the environmental impact of the agricultural practice. In the current study, Andalusian paddy fields have been explored to isolate N2-fixing cyanobacteria. These endogenous microorganisms have been subsequently re-introduced in a field trial in order to enhance rice production. Our results provide valuable insights regarding the use of an alternative natural source of nitrogen for rice production.

Current agricultural productivity depends on an exogenous nutrient supply to crops. This is of special relevance in cereal production, a fundamental part of the trophic chain that plays a vital role in the human diet. However, our agricultural practices entail highly detrimental side-effects from an environmental point of view. Long-term nitrogen fertilization in croplands results in degradation of soil, water, and air quality, producing eutrophication and subsequently contributing to global warming. In accordance with this, there is a biotechnological interest in using nitrogen-fixing microorganisms to enhance crop growth without adding chemically synthesized nitrogen fertilizers. This is particularly beneficial in paddy fields, where about 60% of the synthetic fertilizer that has been applied is dissolved in the water and washed away. In these agricultural systems, N2-fixing cyanobacteria show a promising biotechnological potential as biofertilizers, improving soil fertility while reducing the environmental impact of the agricultural practice. In the current study, Andalusian paddy fields have been explored to isolate N2-fixing cyanobacteria. These endogenous microorganisms have been subsequently re-introduced in a field trial in order to enhance rice production. Our results provide valuable insights regarding the use of an alternative natural source of nitrogen for rice production.

Every year, large amounts of nitrogen fertilisers are used around the world to sustain the cultivation of rice. The wetland culture conditions required for rice cause nitrogen to leach and contaminate freshwater, triggering a number of adverse consequences for aquatic habitats. Dr Consolación Álvarez and Dr Vicente Mariscal Romero, from the Spanish National Research Council (CSIC), Spain, propose the development of new rice biofertilisers based on nitrogen-fixing cyanobacteria. The stable plant-cyanobacteria symbiosis can be exploited in rice crops to stimulate plant growth in the absence of synthetic nitrogen fertilisers.

Cyanobacteria are phototrophic microorganisms able to establish nitrogen-fixing symbiotic associations with representatives of all four of the major phylogenetic divisions of terrestrial plants. Despite increasing knowledge on the beneficial effects of cyanobacteria in rice fields, the information about the interaction between these microorganisms and rice at the molecular and structural levels is still limited. We have used the model nitrogen-fixing cyanobacterium Nostoc punctiforme to promote a long-term stable endophytic association with rice. Inoculation with this strain of hydroponic cultures of rice produces a fast adherence of the cyanobacterium to rice roots. At longer times, cyanobacterial growth in the proximity of the roots increased until reaching a plateau. This latter phase coincides with the intracellular colonization of the root epidermis and exodermis. Structural analysis of the roots revealed that the cyanobacterium use an apoplastic route to colonize the plant cells. Moreover, plant roots inoculated with N. punctiforme show both the presence of heterocysts and nitrogenase activity, resulting in the promotion of plant growth under nitrogen deficiency, thus providing benefits for the plant.