Realization of protective and symbiotic properties of soybeans using fungicide seed treatment

Aim. The elaboration of efficient legume-rhizobial symbiosis systems, involving active strains of nodule bacteria, in the combination with fungicide seed treatment may be an alternative method of providing ecologically friendly nitrogen sources to plants and promoting their tolerance to the external factors, which is relevant for preservation and restoration of envi- ronmental quality. Therefore, the aim of our study was to determine the impact of pre-sowing seed treatment with fungi- cides, which differ in the action spectrum of active substances – Standak Top (fipronil, 250 g/l, thiophanate-methyl, 225 g/l, pyraclostrobin, 25 g/l) and Maxim XL (fludioxonyl, 25 g/l, metalaxyl, 10 g/l), on the intensity of the development of lipid peroxidation processes, the activity of antioxidant enzyme ascorbate peroxidase and nitrogen fixation activity in soybeans on the early stages of forming legume-rhizobial symbiosis. Methods. Microbiological (cultivation of a bacterial culture, seed inoculation), physiological (vegetative experiment), biochemical (spectrophotometric determination of the content of lipid peroxidation products and the activity of ascorbate peroxidase; measuring the nitrogen-fixation activity using a gas chro- matography). Results. It was found that pre-sowing fungicide treatment of soybean seeds and subsequent inoculation with active rhizobia of strain 634b did not result in the change in the content of TBA-active products in roots and root nodules (the values of indices were within the experiment deviation). At the same time, after seed inoculation using rhizobia and treat- ment with Maxim XL, there was an increase in the activity of ascorbate peroxidase in the roots from 20.3 to 30.8 %, and with Standak Top – from 20.0 to 29.8 % during the early stages of ontogenesis till the formation of the third ternate leaf. Here, the activity of the enzyme in root nodules increased by 24.7–40.3 % at the fungicidal effect. Our data demonstrate that the combination of fungicide seed treatment and inoculation with active rhizobia does not induce lipid peroxidation processes, but promotes the initiation of protective antioxidant properties in soybeans. It is accompanied with efficient functioning of the symbiotic apparatus, which is manifested in the increase in nitrogen-fixing activity of nodule bacteria, formed by active rhizobia of strain 634b after the seed treatment with Standak Top – by 98.3 and 78.1 % and after Maxim XL – by 78.6 and 196.2 % respectively, during the stages of the second and third ternate leaves. Conclusions. The pre-sowing soybean seed treatment with fungicides Standak Top and Maxim XL and the subsequent inoculation with active rhizobia of strain 634b does not induce the development of lipid peroxidation processes, but increases the activity of the antioxidant enzyme, ascor- bate peroxidase, in the roots and root nodules, which is accompanied with the efficient work of the symbiotic apparatus on the early stages of determining legume-rhizobial symbiosis. This method of seed treatment may be a novel measure, to use in the technologies of cultivating soybeans to enhance the realization of the symbiotic potential and meet the needs of plants in ecologically friendly nitrogen, and to promote the formation of their tolerance to the corresponding cultivation conditions.

Hydrogen Peroxide Accumulation Regulated by LjRbohD Modulates Nodulation of Lotus japonicus Under Blue Light

In many legumes, roots that are exposed to blue light do not form nodules, and blue light induces the biosynthesis of hydrogen peroxide (H2O2). The mechanism of blue light restraining nodulation is poorly understood. Whether H2O2 induced by blue light inhibits nodulation needs to be further studied. In this work, blue light could promote the production of H2O2, activate the expression of LjRbohD and LjRbohE, while inhibit the expression of LjRbohB. After applying exogenous H2O2 and diphenyleneiodonium chloride (DPI), the results show H2O2 induced by blue light represses the nodulation of MG20. The accumulated H2O2 may be generated by LjRbohD, which supported by Q-PCR. Cryptochrome 1A, a blue light photoreceptor, is high expression under blue light. However, there seems to be no direct relationship between LjRbohD and LjCry1A. On the contrary, LjRbohB, a positive governor in the regulation of nitrogen fixation activity in L. japonicus, may be negatively regulated by LjCry1A according to the hairy roots transformation results. Therefore, the mechanisms of regulating the nodulation in L. japonicus by LjRbohB and LjRbohD are quite different under blue light. Keywords: Louts japonicus, accumulated H2O2, blue light, nodulation, LjRbohs.

Transcriptomics differentiate two novel bioactive strains of Paenibacillus sp. isolated from the perennial ryegrass seed microbiome

Paenibacillus species are Gram-positive bacteria that have been isolated from a diverse array of plant species and soils, with some species exhibiting plant growth-promoting (PGP) activities. Here we report two strains (S02 and S25) of a novel Paenibacillus sp. that were isolated from perennial ryegrass (Lolium perenne) seeds. Comparative genomics analyses showed this novel species was closely related to P. polymyxa. Genomic analyses revealed that strains S02 and S25 possess PGP genes associated with biological nitrogen fixation, phosphate solubilisation and assimilation, as well as auxin production and transportation. Moreover, secondary metabolite gene cluster analyses identified 13 clusters that are shared by both strains and three clusters unique to S25. In vitro assays demonstrated strong bioprotection activity against phytopathogens (Colletotrichum graminicola and Fusarium verticillioides), particularly for strain S02. A transcriptomics analysis evaluating nitrogen fixation activity showed both strains carry an expressed nif operon, but strain S02 was more active than strain S25 in nitrogen-free media. Another transcriptomics analysis evaluating the interaction of strains with F. verticillioides showed strain S02 had increased expression of core genes of secondary metabolite clusters (fusaricidin, paenilan, tridecaptin and polymyxin) when F. verticillioides was present and absent, compared to S25. Such bioactivities make strain S02 a promising candidate to be developed as a combined biofertiliser/bioprotectant.

Rhamnolipid Enhances the Nitrogen Fixation Activity of Azotobacter chroococcum by Influencing Lysine Succinylation

The enhancement of nitrogen fixation activity of diazotrophs is essential for safe crop production. Lysine succinylation (KSuc) is widely present in eukaryotes and prokaryotes and regulates various biological process. However, knowledge of the extent of KSuc in nitrogen fixation of Azotobacter chroococcum is scarce. In this study, we found that 250 mg/l of rhamnolipid (RL) significantly increased the nitrogen fixation activity of A. chroococcum by 39%, as compared with the control. Real-time quantitative reverse transcription PCR (qRT-PCR) confirmed that RL could remarkably increase the transcript levels of nifA and nifHDK genes. In addition, a global KSuc of A. chroococcum was profiled using a 4D label-free quantitative proteomic approach. In total, 5,008 KSuc sites were identified on 1,376 succinylated proteins. Bioinformatics analysis showed that the addition of RL influence on the KSuc level, and the succinylated proteins were involved in various metabolic processes, particularly enriched in oxidative phosphorylation, tricarboxylic acid cycle (TCA) cycle, and nitrogen metabolism. Meanwhile, multiple succinylation sites on MoFe protein (NifDK) may influence nitrogenase activity. These results would provide an experimental basis for the regulation of biological nitrogen fixation with KSuc and shed new light on the mechanistic study of nitrogen fixation.

Microbially facilitated nitrogen cycling in tropical corals

Tropical scleractinian corals support a diverse assemblage of microbial symbionts. This ‘microbiome’ possesses the requisite functional diversity to conduct a range of nitrogen (N) transformations including denitrification, nitrification, nitrogen fixation and dissimilatory nitrate reduction to ammonium (DNRA). Very little direct evidence has been presented to date verifying that these processes are active within tropical corals. Here we use a combination of stable isotope techniques, nutrient uptake calculations and captured metagenomics to quantify rates of nitrogen cycling processes in a selection of tropical scleractinian corals. Denitrification activity was detected in all species, albeit with very low rates, signifying limited importance in holobiont N removal. Relatively greater nitrogen fixation activity confirms that corals are net N importers to reef systems. Low net nitrification activity suggests limited N regeneration capacity; however substantial gross nitrification activity may be concealed through nitrate consumption. Based on nrfA gene abundance and measured inorganic N fluxes, we calculated significant DNRA activity in the studied corals, which has important implications for coral reef N cycling and warrants more targeted investigation. Through the quantification and characterisation of all relevant N-cycling processes, this study provides clarity on the subject of tropical coral-associated biogeochemical N-cycling.

Nitrogen Fixation and Diazotrophic Community in Plastic-Eating Mealworms Tenebrio Molitor L.

Mealworms, the larvae of a coleopteran insect Tenebrio molitor L., are capable of eating, living on and degrading the non-hydrolyzable vinyl plastics as sole diet. However, vinyl plastics are carbon-rich but nitrogen-deficient. It remains puzzling how plastic-eating mealworms overcome the nutritional obstacle of nitrogen limitation. Here, we provide the evidence for nitrogen-fixation activity within plastic-eating mealworms. Acetylene reduction assays illustrate that the nitrogen-fixing activity ranges from 12.3 ± 0.7 to 32.9 ± 9.3 nmol ethylene·h− 1·gut− 1 and the corresponding fixed nitrogen equivalents of protein are estimated as 8.6 to 23.0 µg per day per mealworm. Nature nitrogen isotopic analyses of plastic-eating mealworms provide further evidence for the importance of nitrogen fixation as a new nitrogen source. Eliminating the gut microbial microbiota with antibiotics impairs the mealworm’s ability to fix nitrogen from atmosphere, indicating the contribution of gut microbiota to nitrogen fixation. By using the traditional culture-dependent technique, PCR and RT-PCR of nifH gene, nitrogen-fixing bacteria diversity within the gut was detected and the genus Klebsiella was demonstrated to be an important nitrogen-fixing symbiont. These findings first build the relationship between the plastic degradation (carbon metabolism) and nitrogen fixation (nitrogen metabolism) within mealworms. Combined with previously reported plastic-degrading capability and nitrogen-fixing activity, mealworms may be potential candidates for up-recycling of plastic waste to produce protein sources.

Nitrate Transport and Distribution in Soybean Plants With Dual-Root Systems

Nitrate absorbed by soybean (Glycine max L. Merr.) roots from the soil can promote plant growth, while nitrate transported to nodules inhibits nodulation and nodule nitrogen fixation activity. The aim of this study was to provide new insights into the inhibition of nodule nitrogen (N) fixation by characterizing the transport and distribution of nitrate in soybean plants. In this research, pot culture experiments were conducted using a dual root system of soybeans. In the first experiment, the distribution of 15N derived from nitrate was observed. In the second experiment, nitrate was supplied–withdrawal–resupplied to one side of dual-root system for nine consecutive days, and the other side was supplied with N-free solution. Nitrate contents in leaves, stems, petioles, the basal root of pealed skin and woody part at the grafting site were measured. Nitrate transport and distribution in soybean were analyzed combining the results of two experiments. The results showed that nitrate supplied to the N-supply side of the dual-root system was transported to the shoots immediately through the basal root pealed skin (the main transport route was via the phloem) and woody part (transport was chiefly related to the xylem). There was a transient storage of nitrate in the stems. After the distribution of nitrate, a proportion of the nitrate absorbed by the roots on the N-supply side was translocated to the roots and nodules on the N-free side with a combination of the basal root pealed skin and woody part. In conclusion, the basal root pealed skin and woody part are the main transport routes for nitrate up and down in soybean plants. Nitrate absorbed by roots can be transported to the shoots and then retranslocated to the roots again. The transport flux of nitrate to the N-free side was regulated by transient storage of nitrate in the stems.

Biological Nitrogen Fixation and Denitrification in Rhizosphere of Potato Plants in Response to the Fertilization and Inoculation

The study aim was to evaluate the potential nitrogen fixation and denitrification in the rhizosphere soil of potato plants, crop yield and output quality in response to the different fertilization systems and the inoculation with Azospirillum brasilense 410. Field stationary experiment was conducted between 2016 and 2019 with potato in a crop rotation system on leached chernozem soil. Farmyard manure, 40 t/ha, applied prior to potatoes planting promotes nitrogen fixation (0.8–2.0 times compared to control). However, it has also affected denitrification (in 1.4–2.2 times higher compared to control). The lowest rate of mineral fertilizers used in the experiment, N40P40K40, was shown as most environmentally feasible. Under its use the increase of soil nitrogenase activity and low denitrification levels were observed. Same trends were also noted for the medium fertilizer rate, N80P80K80. The highest doses of mineral fertilizers, N120P120K120, substantially affected the denitrification process and reduced the nitrogen fixation activity (in 1.9–2.2 times). The combination of manure with the medium fertilizers rate has also resulted in high denitrification levels, while the soil nitrogen fixation activity has restored only at flowering stage. Crop inoculation with A. brasilense combined with the manure application, has not affected studied processes. However, crop inoculation after the green manure intercropping has shown the growth of nitrogenase activity. Used on the mineral fertilizers background inoculation has activated nitrogen fixation and has ensured the decrease of denitrification levels, subject to the fertilization background. High fertilizer rates have hampered the inoculation efficiency. Inoculation has promoted crop yields on unfertilized and mineral backgrounds or following green manure. Crop inoculation following organic and the organo-mineral backgrounds had no significant effect, probably due to the competition for A. brasilense from microorganisms that have created a competitive environment for A. brasilense. Despite its environmental expediency, inoculation combined with the low fertilizer doses underperforms the action of inoculation combined with the medium fertilizer rates showing the latter as the compromise between the environmental requirements and crop productivity. The use of inoculation has promoted the accumulation of starch and ascorbic acid and has contributed to the reduction of nitrate contents in the tubers of inoculated plants.