Rhizobium Bacteria Research Articles

Rhizobium Inoculants Mitigate Corn Herbicide Residual Effects on Soybean Germination

Corn residual herbicides offer a practical approach to comprehensive weed management throughout the growing season. However, the use of residual pre-emergence herbicides can have a negative impact on crops grown in succession or within a rotation. A study was carried out to determine the effect of the residual activity of selected corn herbicides on soybeans. The objective of the study was to evaluate the impact of these herbicides on the germination of inoculated soybean seeds. Experiments were conducted in greenhouse conditions to check the carryover effect on soybean germination. Treatment combinations of two pre-herbicides and two inoculants were applied: atrazine (2241 g ai ha−1), mesotrione (105 g ai ha−1), and Bradyrhizobium japonicum, Bradyrhizobium japonicum + Bacillus subtilis, respectively. A randomized complete block design evaluated six treatment combinations, including the control. All treatments, except uninoculated treatments, presented efficacy in reducing the carryover effects of corn residual herbicides on the germination of soybeans. An increase in final germination percentage was observed with Bradyrhizobium japonicum + Bacillus subtilis co-inoculation plus atrazine (24% increase) and Bradyrhizobium japonicum plus mesotrione treatment combinations (19% increase). Inoculating soybean seeds with rhizobium bacteria can reduce the carryover effects on the germination of soybean seeds grown in soil applied with atrazine and mesotrione.

Promising protective treatment potential of endophytic bacterium Rhizobium aegyptiacum for ulcerative colitis in rats

Abstract Ulcerative colitis (UC) is an inflammatory condition of the intestine, resulting from an increase in oxidative stress and pro-inflammatory mediators. In this study, the extract of endophytic bacterium Rhizobium aegyptiacum was prepared for the first time using liquid chromatography-mass spectrometry (LC-MS). In addition, also for the first time, the protective potential of R. aegyptiacum was revealed using an in vivo rat model of UC. The animals were grouped into four categories: normal control (group I), R. aegyptiacum (group II), acetic acid (AA)-induced UC (group III), and R. aegyptiacum-treated AA-induced UC (group IV). In group IV, R. aegyptiacum was administered at 0.2 mg/kg daily for one week before and two weeks after the induction of UC. After sacrificing the rats on the last day of the experiment, colon tissues were collected and subjected to histological, immunohistochemical, and biochemical investigations. There was a remarkable improvement in the histological findings of the colon tissues in group IV, as revealed by hematoxylin and eosin (H&E) staining, Masson’s trichrome staining, and periodic acid-Schiff (PAS) staining. Normal mucosal surfaces covered with a straight, intact, and thin brush border were revealed. Goblet cells appeared magenta in color, and there was a significant decrease in the distribution of collagen fibers in the mucosa and submucosal connective tissues. All these findings were comparable to the respective characteristics of the control group. Regarding cyclooxygenase-2 (COX-2) immunostaining, a weak immune reaction was shown in most cells. Moreover, the colon tissues were examined using a scanning electron microscope, which confirmed the results of histological assessment. A regular polygonal unit pattern was seen with crypt orifices of different sizes and numerous goblet cells. Furthermore, the levels of catalase (CAT), myeloperoxidase (MPO), nitric oxide (NO), interleukin-6 (IL-6), and interlukin-1β (IL-1β) were determined in the colonic tissues of the different groups using colorimetric assay and enzyme-linked immunosorbent assay (ELISA). In comparison with group III, group IV exhibited a significant rise (P<0.05) in the CAT level but a substantial decline (P<0.05) in the NO, MPO, and inflammatory cytokine (IL-6 and IL-1 β) levels. Based on reverse transcription-quantitative polymerase chain reaction (RT-qPCR), the tumor necrosis factor-α (TNF-α) gene expression was upregulated in group III, which was significantly downregulated (P<0.05) by treatment with R. aegyptiacum in group IV. On the contrary, the heme oxygenase-1 (HO-1) gene was substantially upregulated in group IV. Our findings imply that the oral consumption of R. aegyptiacum ameliorates AA-induced UC in rats by restoring and reestablishing the mucosal integrity, in addition to its anti-oxidant and anti-inflammatory effects. Accordingly, R. aegyptiacum is potentially effective and beneficial in human UC therapy, which needs to be further investigated in future work.

Responsivity of Two Pea Genotypes to the Symbiosis with Rhizobia and Arbuscular Mycorrhiza Fungi-A Proteomics Aspect of the "Efficiency of Interactions with Beneficial Soil Microorganisms" Trait.

It is well known that individual pea (Pisum sativum L.) cultivars differ in their symbiotic responsivity. This trait is typically manifested with an increase in seed weights, due to inoculation with rhizobial bacteria and arbuscular mycorrhizal fungi. The aim of this study was to characterize alterations in the root proteome of highly responsive pea genotype k-8274 plants and low responsive genotype k-3358 ones grown in non-sterile soil, which were associated with root colonization with rhizobial bacteria and arbuscular mycorrhizal fungi (in comparison to proteome shifts caused by soil supplementation with mineral nitrogen salts). Our results clearly indicate that supplementation of the soil with mineral nitrogen-containing salts switched the root proteome of both genotypes to assimilation of the available nitrogen, whereas the processes associated with nitrogen fixation were suppressed. Surprisingly, inoculation with rhizobial bacteria had only a minor effect on the root proteomes of both genotypes. The most pronounced response was observed for the highly responsive k-8274 genotype inoculated simultaneously with rhizobial bacteria and arbuscular mycorrhizal fungi. This response involved activation of the proteins related to redox metabolism and suppression of excessive nodule formation. In turn, the low responsive genotype k-3358 demonstrated a pronounced inoculation-induced suppression of protein metabolism and enhanced diverse defense reactions in pea roots under the same soil conditions. The results of the study shed light on the molecular basis of differential symbiotic responsivity in different pea cultivars. The raw data are available in the PRIDE repository under the project accession number PXD058701 and project DOI 10.6019/PXD058701.

Zinc and Phosphate Solubilizing by Rhizobacteria Promotes Lettuce (Lactuca sativa L.) Growth in Salty Conditions

Due to climate change, the world is negatively affected by drought, temperature, salinity, and flood stress, leading to a significant decline in crop production. Lettuce is particularly significant when considering salt stress. To increase plant tolerance to salinity, various strategies are employed to support the development of agriculture. Rhizobacteria play a key role in regulating phosphorus (P) and zinc (Zn) homeostasis in plants. According to the study results, Rhizobium bacteria supported plant growth by improving the solubility of zinc and phosphate. These findings highlight the beneficial effects of plant growth-promoting rhizobacteria (PGPR) on the antioxidant system, which helps detoxify reactive oxygen species. The relationship between proline accumulation and antioxidant enzyme activities showed that PGPR inoculation enhanced the plant's defense mechanism against salt stress. In establishing this tolerance, increases in chlorophyll content, repair of membrane repair, and higher leaf relative humidity under salt stress were observed. PGPR also improved seedling height, diameter, and fresh and dry weight under stress by 70%, 51.4%, 55%, and 109%, respectively, due to the stress-mitigating effects of P and Zn. In conclusion, it is predicted that there will be a need to develop fertilization programs containing different rhizobacteria and Zn+P combinations. These programs would activate the antioxidant mechanism in saline soils, stabilize physiological processes, and positively impact plant growth.

Alleviation of Fe-induced chlorosis of soybean plants grown in calcareous soil by a freeze-dried iron fertilizer containing siderophores produced by Rhizobium radiobacter

The concerns about the unsustainability of traditional synthetic Fe chelates lead to the search for new environmentally friendly alternatives, such as siderophores-based fertilizers. In this work, the focus was on the evaluation of a bio-based Fe-chelate fertilizer obtained from the culture of the siderophore-producer bacterium Rhizobium radiobacter. The suitability of a freeze-dried fertilizer formulated from a R. radiobacter culture labeled with 57Fe to alleviate Fe chlorosis in soybean plants cultivated in calcareous soil was analyzed and the new potential Fe fertilizer was evaluated in comparison to the traditional synthetic chelate o,oEDDHA/57Fe3+. This natural chelate was able to maintain chlorophyll content stable during all the pot trials and presented greater Fe concentration in the remaining soil fractions serving as an Fe pool for a long time whereas o,oEDDHA/57Fe3+ could supply Fe quickly. The new bio-based Fe siderophore fertilizer, derived from R. radiobacter culture, could be a green substitute to conventional synthetic chelates to address Fe chlorosis in calcareous soil conditions.

Isolasi dan Karakterisasi Bakteri Rhizobium asal Bintil Akar Tanaman Kacang Tanah (Arachis hypogaea) dan Koro Rawe (Mucuna bracteata)

Nitrogen (N) is naturally available in the form of N2. Nitrogen fixation can be facilitated by microbes such as Rhizobium bacteria, which can establish symbiosis with the roots of legume plants to form root nodules that perform nitrogen fixation. This study aims to determine the characteristics of Rhizobium bacteria in the root nodules of peanuts (Arachis hypogaea) and koro rawe (Mucuna bracteata). The methods used in this research include collecting root nodules, isolating Rhizobium bacteria, and identifying and characterizing these bacteria for their nitrogen fixation and phosphate solubilization capabilities. Characterization results on YEMA + Congo red media indicated that 10 out of 16 isolates from peanut root nodules and 30 out of 55 isolates from koro rawe root nodules were positive for Rhizobium bacteria. The nitrogen fixation activity test revealed that only 4 isolates from peanut root nodules and 6 from koro rawe root nodules exhibited nitrogen fixation ability in Jensen's medium. The highest phosphate solubilization index was obtained from isolate KK.5.1.1 (0.5±0.08), while the lowest was from isolate KK.5.2.2 (0.09±0.06). This research underscores the significance of Rhizobium in enhancing nitrogen availability and phosphate solubilization for legume plants.

Cultural and Biochemical Characteristics of Rhizobium Present in Nodule of Pongamia Pinnata (l.) Pierre

The present investigation is an attempt to study the impact of rocky terrain on Rhizobium bacteria of root nodules isolated from legume trees. To study the morphological, cultural, and biochemical characteristics of bacterial strain the bacteria obtained from nodules of selected legume i.e. Pongamia pinnata. Rhizobia inhabited in root nodules of plant, grown in Kota university campus, rhizobia were isolated and inoculated on Yeast Extract Mannitol Agar (YEMA) medium and it’s morphological, cultural and biochemical characteristics were studied. It was observed that colonies were circular or irregular; light creamish, glistering, gelatinous, convex with entire margins. The bacteria were gram negative, rod shaped, aerobic, non-spore forming and slow-moving bacteria arranged single, in pairs and in clusters. It showed negative chemical reaction for indole, while showed positive reaction for citrate utilization, catalase, urease. By the help of bio chemical characteristics it was confirmed that isolated bacterial culture may be of Rhizobium pongamie and rocky terrain with high temperature of environment does not have any negative effect on the characters of Rhizobium, our findings was supported by many earlier investigations.

Seed inoculation with Rhizobium japonium bacteria improved fatty acid composition of different soybean (Glycine max L.) genotypes

BackgroundSoybean is an important crop for food security as it fulfills global oil requirements. Seed inoculation with bacteria is frequently used to increase its production; however, it could change the seed composition. Nevertheless, the genotypes respond differently to the bacteria. Therefore, it is necessary to assess the impact of Rhizobium bacteria on the seed composition. MethodsThis two-year (2018 and 2019) study investigated the effects of seed inoculation with Rhizobium japonicum on fatty acid composition of different soybean genotypes. Three frequently cultivated soybean genotypes, i.e., ‘Gapsoy16′, ‘Traksoy’, and ‘İlksoy’ were included in the study. The seeds were either inoculated with R. japonicum or sown without inoculation. The fatty acid profile, i.e., saturated fatty acids (palmitic and stearic acid) and unsaturated fatty acids (oleic, linoleic, linolenic, and arachidic acids) was determined, and the collected data were analyzed by single and multivariate analysis. ResultsSeed inoculation with R. japonicum significantly altered the fatty acid composition of different genotypes; however, varied effects were recorded for the genotype. Linoleic acid, oleic acid, and palmitic acid made up ∼ 33 % of total fatty acids in seeds. Linoleic acid contents varied between 30.78–34.02 %, whereas oleic acid contents ranged between 27.85–31.04 %. Similarly, palmitic acid contents differed between 15.53–16.93 %. The ‘İlksoy’ and ‘Gapsoy’ had the highest contents of palmitic and oleic acids, respectively. Overall, inoculation of bacteria increased the composition of unsaturated fatty acids and lowered saturated fatty acids. ConclusionSeed inoculation with R. japonicum increased the essential fatty acid composition in ‘Traksoy’ genotype. However, ‘İlksoy’ genotype recorded a decrease in unsaturated fatty acids. Therefore, ‘Traksoy’ can be inoculated with R. japonicum to improve fatty acid profile.

Response of Integrated Nutrient Management (INM) Practice on Growth, Yield and Quality Parameters of Fenugreek (Trigonella corniculate L.)

Integrated nutrient management methods assist improve fenugreek (Trigonella corniculate L.) development and yield by integrating the use of organic and inorganic fertilizers. These methods enhance soil fertility, increase nutrient availability, and improve plant health, all of which lead to increased crop yield, growth and quality. Future policies must place a high priority on the economical, sustainable, and efficient use of nutrient resources for the purpose to increase agricultural output. Thus, proper crop, water, soil, and land management along with integrated nutrient management are necessary for sustainable agriculture. Improved soil qualities and increased nutrient availability for agricultural plants come from the use of organic manures in conjunction with inorganic fertilizers. This enhances fenugreek growth, yield, and quality measures. The synthesis of carbohydrates, phytohormones, and even biofertilizers is enhanced by a nutritious diet. It also builds up the soil's organic status, which raises the availability of other nutrients and contributes to the maximum growth of crops. Vermicompost, farmyard manure, rhizobium, phosphorus- and potassium-soluble bacteria (PSB) are among the materials used in methi cultivation that support sustainable agriculture by enhancing soil fertility, nutrient availability, and general crop health, all of which lead to higher methi yields and quality. Organic matter is a storehouse of nutrients; applying both organic and inorganic fertilizer together can boost yields, improve soil fertility, raise crop input-use efficiency, and reduce the need for expensive fertilizers. This crop responds effectively to N provided through a combination of organic and inorganic sources. Majority of the nitrogen applied from different sources is not used by the first crop and is always reflected in the crop that follows. Therefore, it is necessary to assess how integrated nutrition management affected fenugreek's yield and nutrient uptake.

Rhizobial and passenger endophytes alleviates moisture stress in groundnut (Arachis hypogaea)

Moisture stress poses a significant threat to global agriculture, compromising crop yields and food security. In the quest for sustainable solutions, endophytic microorganisms have emerged as promising candidates for enhancing plant resilience to drought. The study's primary goal was to analyse the significance of bacterial endophytes, both rhizobial and passenger endophytes, in alleviating the effects of moisture stress. Here, PEG 6000 was used to test the drought endurance of the ten identified rhizobial and passenger endophytes. Rhizobium pusense S6R2, Enterobacter cloacae S23 and Bacillus tequilensis NBB13 were selected as best performing endophytes as they showed high tolerance of poly ethylene glycol (PEG) and maximum plant growth promoting traits like Indole Acetic Acid, exopolysaccharide production, biofilm formation, 1-aminocyclopropane1-carboxylate (ACC) deaminase activity, siderophore, zinc and phosphorous solubilisation even in PEG induced moisture stress condition. Metabolite analysis revealed that twenty-four significant compounds mostly belong to fatty acyls, amino acids, peptides, polyketides, and benzenoids were found in the root exudates of groundnut treated with endophytes. The best-performing endophytes were used in a pot culture experiment, with groundnut as the test crop. The current study found that co-inoculation of Rhizobium pusense S6R2 and Enterobacter cloacae S23 significantly increased nodule number, growth, photosynthetic pigment, anti-oxidant enzymes, and osmolyte under moisture stressed conditions when compared to other treatments. As a result, co-inoculation of Rhizobium and entophytic bacteria may be recommended as a bio-inoculant for groundnut for moisture stress alleviation after confirming the results in field evaluation.

Nghiên cứu đặc điểm hình thái, giải phẫu của loài Điên điển (Sesbania sesban (L.) Merr.) ở huyện Tân Hưng, tỉnh Long An

Fabaceae is the second largest flowering plant family (after the Asteraceae) with approximately 17,500 - 18,000 species belonging to 786 genera primarily distributed in tropical regions. In Vietnam, over 450 species from 90 genera have been reported, showcasing a rich diversity in habitat and distribution. Many species in this family are valuable for their timber such as Rosewood (Dalbergia cochinchinensis Pierre), and Sua wood (Dalbergia tonkinensis Prain); they also provide medicinal ingredients and food sources like Mung beans (Vigna radiata (L.) R.Wilczek) and Black gram (Vigna mungo (L.) Hepper). This research has been conducted to identify the morphological and anatomical characteristics of Sesbania sesban (L.) Merr., a species found in Tan Hung district, Long An province. Fresh samples of the species were collected and analyzed, describing the morphological features and anatomy of the reproductive and nutrient organs. The study revealed that the stems and leaves of S. sesban exhibit adaptations to strong light conditions, with stomata mainly concentrated on the lower leaf surface, stem hairs that aid in light reflection and moisture retention, and root nodules containing Rhizobium bacteria for nitrogen fixation. The anatomical structure of the nutrient organs showed strong development of pith and wood rings, indicating the adaptation of S. sesban to windy environments near river banks and canals.

Enhancing Bio-Fertilization Potential of Introduced Hedysarum Species Through Indirect Root Inoculation: Implications for Agro-Ecological Sustainability in Arid Lands

Saharan soils are devoid of Rhizobium bacteria specific to the Sulla plants which is indispensable for nitrogen fixation and soil fertility. The present study aims to test the possibility of formation nodules on the roots of some species of Hedysarum (Sulla) genus introduced in Ghardaïa region with indirect inoculation in the reason to optimize the use of nitrogen fertilizers. Three soil samples were chosen from three different districts of Algeria (Tizi-Ouzou, Sétif and El-Tarf) to be used as substrate and source of bacterial strains for three different species of Sulla (Hedysarum coronarium, Hedysarum carnosum, Hedysarum flexuosum). The results showed the formation of a considerable number of nodules in all treatments. The effect of species and samples of soils on the studied parameter was highly significant (p<0.000) at the 5% significance level. All cultivated species in the soil of Sétif could form significant more nodules compared to other soils. The disparity in the average number of nodules formed in the soil of Sétif represents a rate of 78.75% compared to the soil of Tizi-Ouzou. This study could contribute to improve fodder production of Sulla species cultivated in Ghardaïa region and also provides a forage rich in protein for local livestock.

The Effects of Frass and Vermicompost Fertilization on the Biometrical Parameters of Plant and Soil Quality, and the Rhizobiome, in Red Beet (Beta vulgaris L.) Cultivation

Insect frass and vermicompost hold potential applications as fertilizers, with their abilities to improve plant resilience against unfavorable environmental conditions and increase their resistance to pests and diseases. In this study, we explored the effects of vermicompost fertilization, mealworm frass, and superworm frass as potential plant fertilizers for red beet cultivation. We analyzed the connections among chemical parameters, rhizobiome structure and function, and the biometrics of fertilizer-treated plants. In general, soils enriched with vermicompost and superworm frass exhibited the highest macroelement contents. Dry superworm frass fertilization was characterized by the increased availabilities of total nitrogen, NH4-N, and NO3-N. The use of vermicompost and mealworm frass resulted in significantly higher red beet biomass values. The presence of the highest N-fixation potential and key hormonal substances involved in plant development, such as auxins and gibberellins, was demonstrated using wet superworm frass. The results indicated that wet superworm frass, similar to vermicompost and dry superworm frass, exhibits high chemoheterotrophic potential. This suggests an r-type strategy and high adaptive flexibility of rhizobial bacteria. As a consequence, both life in the root zone and the microbiome itself may be better adapted to sudden, unfavorable environmental changes or attacks by plant pathogens.

The microbial-driven nitrogen cycle and its relevance for plant nutrition.

Nitrogen (N) is a vital nutrient and an essential component of biological macromolecules such as nucleic acids and proteins. Microorganisms are major drivers of N-cycling processes in all ecosystems, including the soil and plant environment. The availability of N is a major growth-limiting factor for plants and it is significantly affected by the plant microbiome. Plants and microorganisms form complex interaction networks resulting in molecular signaling, nutrient exchange, and other distinct metabolic responses. In these networks, microbial partners influence growth and N use efficiency of plants either positively or negatively. Harnessing the beneficial effects of specific players within crop microbiomes is a promising strategy to counteract the emerging threats to human and planetary health due to the overuse of industrial N fertilizers. However, in addition to N-providing activities (e.g. the well-known symbiosis of legumes and Rhizobium spp.), other plant-microorganism interactions must be considered to obtain a complete picture of how microbial-driven N transformations might affect plant nutrition. For this, we review recent insights into the tight interplay between plants and N-cycling microorganisms, focusing on microbial N-transformation processes representing N sources and sinks that ultimately shape plant N acquisition.

Biological, physiological and economic characteristics of Onobrychis chorasanica Bunge ex Bois. (Sainfoin) under sowing conditions

Onobrychis chorasanica Bunge ex Bois. (Sainfoin) is a valuable pasture plant that is resistant to drought, produces high biomass and is not inferior to alfalfa in terms of nutrient content. It is urgent to research the possibilities of cultivation of this plant for the purpose of growing hay in hilly conditions. The presence of nitrogen-fixing Rhizobium bacteria in the roots of plants is important in increasing the fertility of desert soils and this feature increases its role in the creation of artificial grasslands in hilly conditions. In the experiments, it was shown that it is possible to reach 100 % germination by mechanical scarification of the seeds isolated from O. chorasanica pods using sandpaper. Germination of unscarred seeds in field conditions depends on the year and the depth of planting them in the soil. In a dry year, the highest fertility was observed to be 13.3-15.0 % in the options where the seeds were buried at a depth of 3-4 cm, and in years with abundant rainfall, this indicator was 35.3 % in the option where the seeds were buried at a depth of 3 cm. The viability of lawns in the 2nd year of life was 72-95 % in different options. Hay productivity of O. chorasanica was found to be 41 centners per ha in hilly conditions and seed yield was up to 250 kg. The results obtained in the research are promising for the intensification of feed production of O. chorasanica in local conditions. The obtained results serve to increase the biomass yield of pasture plants and the variety of used feeds in farms specializing in animal husbandry.

Development of approaches for genome editing of pea plants using CRISPR/Cas9 prime-editing technique

Mitogen-activated protein kinases (MAPKs) play an important role as intracellular regulators of signal pathways in plants. Some MAPKs have been shown to be induced in the roots of legume plants during symbiosis with nitrogen-fixing rhizobial bacteria. One of such signal regulators, MAPK6, was shown to be involved in the development of symbiosis between Pisum sativum L. pea plants and rhizobia. Using genetic engineering approaches, we overexpressed the MAPK6 gene in transgenic roots, that resulted in an increase in the number of nodules and the biomass of pea plants. New approaches for genome editing of pea plants have been designed using the CRISPR/Cas9 prime-editing technique, when the MAPK6 gene was used as a target. We have analyzed the transgenic roots of pea transformants and observed the presence of the gene encoding Cas9 the pegRNA sequence in the genome of transformants. Therefore, the possibility of using genetic engineering methods to obtain plants with increased efficiency of symbiosis will be investigated in future experiments.

Optimization of Rhizobium rhizogenes-mediated transformation, regeneration and characterization of Malus domesticaBorkh. Ri lines

This study explored the establishment and optimization of Ri (root inducing) technology for apple breeding, using the bacterium Rhizobium rhizogenes to obtain Ri lines with compact shoots and stronger root systems. The transformation and shoot regeneration for Malus domestica cultivars was studied in detail. Various R. rhizogenes strains, scion and rootstock genotypes, explant types, wounding methods and explant orientations were tested for hairy root induction. Most of the 16 tested strains, especially those with plasmid type III, induced hairy roots in the rootstock genotype ‘M26’. Although apple genotypes differed in response, in most of them roots were successfully induced using strains ATCC 15834, LMG 63 and LMG 150, with leaf blades outperforming petioles as explants. Wounding by scratching or sonication further improved transformation efficiency, as did placing leaf blades with their abaxial side upward on root induction medium. The majority (94%) of roots formed in one transformation experiment were tested PCR-positive for at least one T-DNA gene. Shoot regeneration experiments investigated salt concentrations, gelling agents, cytokinin types, concentrations, and a resting period on hormone-free medium. Shoot regeneration was highly genotype-dependent varying between 0 and 83%, whereas only minor, non-significant effects were observed for the treatments tested. Copy numbers of T-DNA genes were estimated using digital PCR for the first time in apple Ri lines. In the greenhouse, two Ri lines showed compact shoots and shorter leaves, but no enhanced root system. The improved protocol provides a valuable tool for breeders and scientists to obtain and further use Ri lines.

Probing the active site of Class 3 L-asparaginase by mutagenesis. I. Tinkering with the zinc coordination site of ReAV.

ReAV, the inducible Class-3 L-asparaginase from the nitrogen-fixing symbiotic bacterium Rhizobium etli, is an interesting candidate for optimizing its enzymatic potential for antileukemic applications. Since it has no structural similarity to known enzymes with this activity, it may offer completely new ways of approach. Also, as an unrelated protein, it would evade the immunological response elicited by other asparaginases. The crystal structure of ReAV revealed a uniquely assembled protein homodimer with a highly specific C135/K138/C189 zinc binding site in each subunit. It was also shown before that the Zn2+ cation at low and optimal concentration boosts the ReAV activity and improves substrate specificity, which indicates its role in substrate recognition. However, the detailed catalytic mechanism of ReAV is still unknown. In this work, we have applied site-directed mutagenesis coupled with enzymatic assays and X-ray structural analysis to elucidate the role of the residues in the zinc coordination sphere in catalysis. Almost all of the seven ReAV muteins created in this campaign lost the ability to hydrolyze L-asparagine, confirming our predictions about the significance of the selected residues in substrate hydrolysis. We were able to crystallize five of the ReAV mutants and solve their crystal structures, revealing some intriguing changes in the active site area as a result of the mutations. With alanine substitutions of Cys135 or Cys189, the zinc coordination site fell apart and the mutants were unable to bind the Zn2+ cation. Moreover, the absence of Lys138 induced atomic shifts and conformational changes of the neighboring residues from two active-site Ser-Lys tandems. Ser48 from one of the tandems, which is hypothesized to be the catalytic nucleophile, usually changes its hydration pattern in response to the mutations. Taken together, the results provide many useful clues about the catalytic mechanism of the enzyme, allowing one to cautiously postulate a possible enzymatic scenario.

Response of green gram [Vigna radiata (L) Wilczek] to application of biofertilizers and phosphorus

The present study was carried out at the Crop Research Centre, Carrier Point University, Kota (Rajasthan), during the kharif season of 2020 and 2021 to assess the effect of phosphorus, rhizobium and phosphate solubilizing bacteria on growth and yield attributes of green gram. Significantly increase in growth, yield attributes and yield of green gram were recorded with the application of phosphorus 50 kg P2 O5 /ha. In case of biofertilizers (PSB, Rhizobium and PSB + Rhizobium), all growth and yield attributes and yield were increased significantly. However, among treatment combinations, the maximum values of these parameters were observed with 50 kg P2 O5 /ha + biofertilizers (PSB + Rhizobium) in comparison to all other treatment combinations. The net returns and B: C ratio were also higher under 50 kg P2 O5 /ha + biofertilizers (PSB + Rhizobium).

Cattle dry manure fertilization increases forage yield of grass‐legume mixtures, while maintaining the legume proportion and root‐associated microbiota

AbstractThe aim of this study was to find ways to improve the forage yield of grass‐legume mixtures without compromising soil biodiversity. In Argentinean Patagonia, the effects of applying cattle dry manure (M) and urea (U) (0, 60, 120, or 240 kg N ha−1 year−1) were assessed on herbage production of irrigated fescue‐lotus mixtures, as well as on the activity/abundance of autochthonous arbuscular mycorrhizal fungi and N‐fixing rhizobium bacteria. We hypothesised that manure has advantages over urea in increasing forage yields while maintaining the proportion of legumes and root‐associated microbiota. The 120 U, 240 U, and 240 M resulted in the greatest forage production; however, yield varied depending on the source applied. The high productivity of the 120 U and 240 U was probably due to the fast grass growth immediately after fertilization, which resulted in a depressed growth of the legume. The high yield of the swards fertilized with 240 M was probably due to slight and delayed growth of grass without legume yield decline. The highest radiation interception was found in swards with a low legume proportion, suggesting a light competition from grass fertilized with the highest urea doses, which were also consistent with the highest N and P nutritional status. The microbial activity/abundance were not affected by fertilization, but the final number of nodules was positively associated with the legume proportion. In conclusion, manure fertilization increased forage yield of the mixtures, while preserving the legume proportion and the root‐associated microbiota. Our findings aid in reducing synthetic‐N fertilizers applied in pasture‐based livestock systems.