Search results for: nodulation

Authors: Areej Javaid, Nishat Fatima, Mehwish Naseer

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There is a tremendous use of biofertilizers in agriculture to increase crop productivity. Pakistan spends a huge amount on the purchase of synthetic fertilizers every year. The use of natural compounds to harness crop productivity is the major area of interest nowadays due to being safe for human health and the environment as well. Legumes have the intrinsic quality to enrich the nutrient status of soil because of the presence of nitrogen fixation bacteria on nodules. This research determined the effect of biofertilizer on nodulation attributes and yield of the pea plant. Seeds of pea varieties were treated with a slurry of biofertilizer prepared in a 10% sugar solution just before seed sowing. The impact of biofertilizer on different parameters of growth, yield and nodulation was observed. Analysis of variance showed that plant height, days to flowering, number of nodes, days to first pod, root length and plant height exhibited significant genetic variation. All the yield parameters, including the number of pods per plant, number of seeds per pod, seed fresh and dry weight showed significant results under treatment. Among nodulation parameters, nodule number responded positively to biofertilizer treatment. Genotypes 2001-40 showed better performance followed by 2001-20 and LINA-PAK in all the parameters, whereas 2001-40 and 2001-20 performed well in nodulation and yield parameters. Consequently, seed treatment with biofertilizer before sowing is recommended to obtain higher crop yield.

Keywords: biological nitrogen fixation, correlation analysis, quantitative inheritance, varietal responses

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Authors: Hong Li, Tingxian Li, Xudong Wang, Qinghuo Lin

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Excess-iron (Fe) stresses involved in legume symbiotic nitrogen fixation are not understood. Our objectives were to investigate the tolerance of yardlong-bean plants to soil excess-Fe stress and antagonistic effects of organic amendments and rhizobial inoculants on plant root nodulation and stem ureide formation. The study was conducted in the tropical Hainan Island during 2012-2013. The soil was strongly acidic (pH 5.3±0.4) and highly variable in Fe concentrations(596±79 mg/kg). The treatments were arranged in a split-plot design with three blocks. The treatment effects were significant on root nodulation, stem ureide, amino acids, plant N/Fe accumulation and bean yields (P<0.05). The yardlong-bean stem allantoin, amino acids and nitrate concentrations and relative ureide % declined with high soil Fe concentrations (>300 mg/kg). It was concluded that the co-variance of excess Fe stress could inhibit legume symbiotic N fixation efficiency. Organic amendments and rhizobial inoculants could help improve crop tolerance to excess Fe stress.

Keywords: atmospheric N fixation, root nodulation, soil Fe co-variance, stem ureide, yardlong-bean plants

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Authors: Sarra Sobti, Baelhadj Hamdi-Aïssa

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The bacteria of the soil, usually called rhizobium, have a considerable importance in agriculture because of their capacity to fix the atmospheric nitrogen in symbiosis with the plants of the family of legumes. The present work was to study the effect of the salinity on growth and nodulation of alfalfa-rhizobia symbiosis at different agricultural experimental sites in Ouargla. The experiment was conducted in 3 steps. The first one was the isolation and characterization of the Rhizobia; next, the evolution of the isolates tolerance to salinity at three levels of NaCl (6, 8,12 and 16 g/L); and the last step was the evolution of the tolerance on symbiotic characteristics. The results showed that the phenotypic characterizations behave practically as Rhizobia spp, and the effects of salinity affect the symbiotic process. The tolerance to high levels of salinity and the survival and persistence in severe and harsh desert conditions make these rhizobia highly valuable inoculums to improve productivity of the leguminous plants cultivated under extreme environments.

Keywords: rhizobia, symbiosis, salinity, tolerance, nodulation, soil, Medicago sativa L.

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Authors: Yuda Purwana Roswanjaya, Wouter Kohlen, Rene Geurts

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The Trema genus represents a group of fast-growing tropical tree species within the Cannabaceae. Interestingly, five species nested in this lineage -known as Parasponia- can establish rhizobium nitrogen-fixing root nodules, similar to those found in legumes. Parasponia and legumes use a conserved genetic network to control root nodule formation, among which are genes also essential for mycorrhizal symbiosis (the so-called common symbiotic pathway). However, Trema species lost several genes that function exclusively in nodulation, suggesting a loss-of the nodulation trait in Trema. Strikingly, in a Trema orientalis population found in Malaysian Borneo we identified a truncated SYMBIOSIS RECEPTOR KINASE (SYMRK) mutant allele lacking a large portion of the c-terminal kinase domain. In legumes this gene is essential for nodulation and mycorrhization. This raises the question whether Trema orientalis can still be mycorrhized. To answer this question, we established quantitative mycorrhization assay for Parasponia andersonii and Trema orientalis. Plants were grown in closed pots on half strength Hoagland medium containing 20 µM potassium phosphate in sterilized sand and inoculated with 125 spores of Rhizopagus irregularis (Agronutrion-DAOM197198). Mycorrhization efficiency was determined by analyzing the frequency of mycorrhiza (%F), the intensity of the mycorrhizal colonization (%M) and the arbuscule abundance (%A) in the root system. Trema orientalis RG33 can be mycorrhized, though with lower efficiency compared to Parasponia andersonii. From this we conclude that a functional SYMRK kinase domain is not essential for Trema orientalis mycorrhization. In ongoing experiments, we aim to investigate the role of SYMRK in Parasponia andersonii mycorrhization and nodulation. For this two Parasponia andersonii symrk CRISPR-Cas9 mutant alleles were created. One mimicking the TorSYMRKRG33 allele by deletion of exon 13-15, and a full Parasponia andersonii SYMRK knockout.

Keywords: endomycorrhization, Parasponia andersonii, symbiosis receptor kinase (SYMRK), Trema orientalis

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Authors: Hong Li, Tingxian Li, Xudong Wang, Weibo Yang

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Co-limitation of iron (Fe) deficiency in legume nitrogen fixation process is not well understood. Our objectives were to examine how peanut plants cope with Fe deficiency with the rhizobial inoculants and N-nutrient treatments. The study was conducted in the tropical Hainan Island during 2012-2013. The soil was strongly acidic (pH 4.6±0.7) and deficient in Fe (9.2±2.3 mg/kg). Peanut plants were intercropped with cassava. The inoculants and N treatments were arranged in a split-plot design with three blocks. Peanut root nodulation, stem allantoin, amino acids and plant N derived from fixation (P) reduced with declining soil Fe concentrations. The treatment interactions were significant on relative ureide % and peanut yields (P<0.05). Residual fixed N from peanut plants was beneficial to cassava plants. It was concluded that co-variance of Fe deficiency could influence peanut N fixation efficiency and rhizobia and N inputs could help improving peanut tolerance to Fe deficiency stress.

Keywords: amino acids, plant N derived from N fixation, root nodulation, soil Fe co-variance, stem ureide, peanuts, cassava

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Authors: Sharon K. Mahlangu, Mustapha Mohammed, Felix D. Dakora

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Rural households in Africa depend largely on legumes as a source of high-protein food due to N₂-fixation by rhizobia when they infect plant roots. However, the legume/rhizobia symbiosis can exhibit some level of specificity such that some legumes may be selectively nodulated by only a particular group of rhizobia. In contrast, some legumes are highly promiscuous and are nodulated by a wide range of rhizobia. Little is known about the nodulation promiscuity of bacterial symbionts from wild legumes such as Aspalathus linearis, especially if they can nodulate cultivated grain legumes such as cowpea and Kersting’s groundnut. Determining the host range of the symbionts of wild legumes can potentially reveal novel rhizobial strains that can be used to increase nitrogen fixation in cultivated legumes. In this study, bacteria were isolated and tested for their ability to induce root nodules on their homologous hosts. Seeds were surface-sterilized with alcohol and sodium hypochlorite and planted in sterile sand contained in plastic pots. The pot surface was covered with sterile non-absorbent cotton wool to avoid contamination. The plants were watered with nitrogen-free nutrient solution and sterile water in alternation. Three replicate pots were used per isolate. The plants were grown for 90 days in a naturally-lit glasshouse and assessed for nodulation (nodule number and nodule biomass) and shoot biomass. Seven isolates from each of Kersting’s groundnut and cowpea and two from Rooibos tea plants were tested for their ability to nodulate soybean, mung bean, common bean and Bambara groundnut. The results showed that of the isolates from cowpea, where VUSA55 and VUSA42 could nodulate all test host plants, followed by VUSA48 which nodulated cowpea, Bambara groundnut and soybean. The two isolates from Rooibos tea plants nodulated Bambara groundnut, soybean and common bean. However, isolate L1R3.3.1 also nodulated mung bean. There was a greater accumulation of shoot biomass when cowpea isolate VUSA55 nodulated common bean. Isolate VUSA55 produced the highest shoot biomass, followed by VUSA42 and VUSA48. The two Kersting’s groundnut isolates, MGSA131 and MGSA110, accumulated average shoot biomass. In contrast, the two Rooibos tea isolates induced a higher accumulation of biomass in Bambara groundnut, followed by common bean. The results suggest that inoculating these agriculturally important grain legumes with cowpea isolates can contribute to improved soil fertility, especially soil nitrogen levels.

Keywords: legumes, nitrogen fixation, nodulation, rhizobia

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Authors: Mokgadi Miranda Hlongwane, Ntebogeng Sharon Mokgalaka, Felix Dapare Dakora

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Lessertia (L.) frutescens (syn. Sutherlandia frutescens) is a leguminous medicinal plant indigenous to South Africa. Traditionally, L. frutescens has been used to treat cancer, diabetes, epilepsy, fever, HIV, stomach problems, wounds and other ailments. This legume is endemic to the Cape fynbos, with large populations occurring wild and cultivated in the Cape Florist Region. Its widespread distribution in the Western Cape, Northern Cape, Eastern Cape and Kwazulu-Natal is linked to its increased use as a phytomedicine in the treatment of various diseases by traditional healers. The frequent harvesting of field plants for use as a medicine has made it necessary to undertake studies towards the conservation of Lessertia frutescens. As a legume, this species can form root nodules and fix atmospheric N₂ when in symbiosis with soil bacteria called rhizobia. So far, however, few studies (if any) have been done on the efficacy and diversity of native bacterial symbionts nodulating L. frutescens in South Africa. The aim of this project was to isolate and characterize L. frutescens-nodulating bacteria from five different locations in the Western Cape Province. This was done by trapping soil rhizobia using rhizosphere soil suspension to inoculate L. frutescens seedlings growing in sterilized sand and receiving sterile N-free Hoagland nutrient solution under glasshouse conditions. At 60 days after planting, root nodules were harvested from L. frutescens plants, surface-sterilized, macerated, and streaked on yeast mannitol agar (YMA) plates and incubated at 28 ˚C for observation of bacterial growth. The majority of isolates were slow-growers that took 6-14 days to appear on YMA plates. However, seven isolates were fast-growers, taking 2-4 days to appear on YMA plates. Single-colony cultures of the isolates were assessed for their ability to nodulate L. frutescens as a homologous host under glasshouse conditions. Of the 92 bacterial isolates tested, 63 elicited nodule formation on L. frutescens. Symbiotic effectiveness varied markedly between and among test isolates. There were also significant (p≤0.005) differences in nodulation, shoot biomass, photosynthetic rates, leaf transpiration and stomatal conductance of L. frutescens plants inoculated with the test isolates, which is an indication of their functional diversity.

Keywords: lessertia frutescens, nodulating, rhizobia, symbiotic effectiveness

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Authors: B. Makoudi, R. Ghanimi, M. Mouradi, A. Kabbadj, M. Farissi, J. J. Drevon, C. Ghoulam

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This work focuses on the responses of Vicia fabarhizobia symbiosis to phosphorus deficiency and their contribution to tolerate this constraint. The study was carried out on four faba bean varieties, Aguadulce, Alfia, Luz Otono, and Reina Mora submitted to two phosphorus treatments, deficient and sufficient and cultivated under field and greenhouse hydroaeroponic culture. Plants were harvested at flowering stage for growth, nodulation and phosphorus content assessment. Phosphatases in nodules and rhizospheric soil were analyzed. The impact of phosphorus deficiency on yield component was assessed at maturity stage. Under field conditions, phosphorus deficiency affected negatively nodule biomass and nodule phosphorus content with Alfia and Reina Mora showing the highest biomass reduction. The phosphatase activities in nodules and rhizospheric soil were increased under phosphorus deficiency. At maturity stage, under soil low available phosphorus, the pods number and 100 seeds weight were reduced. The genotypic variation was evident for almost all tested parameters.

Keywords: faba bean, phosphorus, rhizobia, yield

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Authors: Ayodele Adelusi Oyedeji

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The study was carried out under a controlled environment with the aim of examining remediation of crude oil polluted soil. The germination rate, heights and girths, number of leaves and nodulation was determined following standard procedures. Some physicochemical (organic matter, pH, nitrogen, phosphorous, potassium, calcium, magnesium and sodium) characteristics of soil used were determined using standard protocols. Results showed that at varying concentration of crude oil i.e 0 ml, 25 ml, 50 ml, 75 ml and 100 ml, Leuceana leucocephala had germination rate of 92%, 90%, 84%, 62% and 56% respectively, mean height of 73.70cm, 58.30cm, 49.50cm, 46.45cm and 41.80cm respectively after 16 weeks after planting (WAP), mean girth of 0.54mm, 0.34mm, 0.33mm, 0.21mm and 0.19mm respectively at 16 WAP, number of nodules 18, 10, 10, 6 and 2 respectively and number of leaves 24.00, 16.00, 13.00, 10.00 and 6.00 respectively. The organic matter, pH, nitrogen, phosphorous, potassium, calcium, magnesium, and sodium decreased with the increase in the concentration of crude oil. Furthermore, as the concentration of crude oil increased the germination rate, height, girth, and number of leaves and nodules decreased, suggesting the effect of crude oil on Leuceana leucocephala. The plant withstands the varying concentration of the crude oil means that it could be used for the remediation of crude oil contaminated soil in the Niger Delta region of Nigeria.

Keywords: ecosystem conservation, Leuceana leucocephala, phytoremediation, soil pollution

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Authors: Mouffok Ahlem, Belhamra Mohamed, Mouffok Sihem

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The use of nitrogen fertilizers inevitable consequence, the increase in the nitrate content of water, which may contribute to the production of nitrite and the formation of carcinogenic nitrosamines. The nitrogen fertilizer may also affect the structure and function of the microbial community. And the fight against eutrophication of aquatic environments represents a cost to the student statements. The agronomic, ecological and economic legumes such as faba beans and alfalfa are not demonstrated, especially in the case of semi-arid and arid areas. Osmotic stress due to drought and / or salinity deficit, nutritional deficiencies is the major factors limiting symbiotic nitrogen fixation and productivity of pulses. To study the symbiotic nitrogen fixation in faba bean (Vicia faba L.) and alfalfa (Medicago sativa L.) in the region of Biskra, we used soil samples collected from 30 locations. This work has identified several issues of ecological and agronomic interest. Evaluation of symbiotic potential of soils in the region of Biskra; by trapping technique, show different levels of susceptibility to rhizobial microflora. The effectiveness of the rhizobial symbiosis in both legumes indicates that air dry biomass and the amount of nitrogen accumulated in the aerial part, depends mainly on the rate of nodulation and regardless of the species and locality. The correlation between symbiotic nitrogen fixation and some physico-chemical properties of soils shows that symbiotic nitrogen fixation in both legumes is strongly related to soil conditions of the soil. Salinity disrupts the physiological process of growth, development and more particularly that of the symbiotic fixation of atmospheric nitrogen. Against by phosphorus promotes rhizobial symbiosis.

Keywords: rhizobia, faba bean, alfalfa, salinity

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Authors: Benselama Amel, Ounane S. Mohamed, Bekki Abdelkader

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A collection of 20 isolates from fresh Nodules of the legume plant Lablab purpureus was isolated. These isolates have been authenticated by seedling inoculation grown in jars containing sand. The results obtained after two months of culture have revealed that the 20 isolates (100% of the isolates) are able to nodulate their host plants. The results obtained were analyzed statistically by ANOVA using the software statistica and had shown that the effect of the inoculation has significantly improved all the growth parameters (the height of the plant and the dry weight of the aerial parts and roots, and the number of nodules). We have evaluated the tolerance of all strains of the collection to the major stress factors as the salinity, pH and extreme temperature. The osmotolerance reached a concentration up to 1710mm of NaCl. The strains were also able to grow on a wide range of pH, ranging from 4.5 to 9.5, and temperature, between 4°C and 40°C. Also, we tested the effect of the acidity, aluminum and ferric deficit on the Lablab-rhizobia symbiosis. Lablab purpureus has not been affected by the presence of high concentrations of aluminum. On the other hand, iron deficiency has caused a net decrease in the dry biomass of the aerial part. The results of all the phenotypic characters have been treated by the statistical Minitab software, the numerical analysis had shown that these bacterial strains are divided into two distinct groups at a level of similarity of 86 %. The SDS-PAGE was carried out to determine the profile of the total protein of the strains. The coefficients of similarity of polypeptide bands between the isolates and strains reference (Bradyrhizobium, Mesorizobium sp.) confirm that our strain belongs to the groups of rhizobia.

Keywords: SDS-PAGE, rhizobia, symbiosis, phenotypic characterization, Lablab purpureus

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Authors: Rahamdad Khan, Ijaz Ahmad Khan

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In modern agriculture the herbicides application are considered the most effective and fast in action against all types of weeds. But it’s a fact that the herbicide applicator cannot totally secure the crop plants from the possible herbicide injuries that further leads to several destructive changes in plant biochemistry. For the purpose pots studies were undertaken to test the tolerance order of chickpea against pre- emergence herbicides (Stomp 330 EC- Dual Gold 960 EC) and post- emergence herbicides (Topik 15 WP- Puma Super 75 EW- Isoproturon 500 EW) during 2012-13 and 2013-14. The experimental design was CRD with three replications. Plant height, number of branches plant-1, number of seeds plant-1, nodulation, seed protein contents and other growth related parameters in chickpea were examined during the investigations. The results indicate that all the enquire herbicides gave a significant variation to all recorded parameter of chick pea except nodule fresh and dray weight. Moreover the toxic effect of pre-emergence herbicide on chickpea was found higher as compared to post-emergence herbicides. Minimum chickpea plant height (50.50 cm), number of nodule plant-1 (17.83) and lowest seed protein (14.13 %) was recorded in Stomp 330 EC. Similarly the outmost seeds plant-1 (29.66) and number of nodule plant-1 (21) were found for Puma Super 75 EW. The results further showed that the highest seed protein content (21.75 and 21.15 %) was recorded for control/ untreated and Puma Super 75EW. Taking under concentration the possible negative impact of the herbicides the chemical application must be minimized up to certain extent at which the crop is mostly secure. However chemical weed control has many advantages so we should train our farmer regarding the proper use of agro chemical to minimize the loses in crops while using herbicides.

Keywords: chickpea, herbicides, protein, stomp 330 EC, weed

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Authors: Timea I. Hajnal-Jafari, Simonida S. Đurić, Dragana R. Stamenov

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Microbial inoculants from the group of symbiotic-nitrogen-fixing rhizobia are well known and widely used in production of legumes. On the other hand, nonsymbiotic plant growth promoting rhizobacteria (PGPR) are not commonly used in practice. The objective of this study was to examine the effects of soybean inoculation with symbiotic and nonsymbiotic bacteria on plant growth and seed yield of soybean. Microbiological activity in rhizospheric soil was also determined. The experiment was set up using a randomized block system in filed conditions with the following treatments: control-no inoculation; treatment 1-Bradyrhizobium japonicum; treatment 2-Azotobacter sp.; treatment 3-Bacillus sp..In the flowering stage of growth (FS) the number of nodules per plant (NPP), root length (RL), plant height (PH) and weight (PW) were measured. The number of pod per plant (PPP), number of seeds per pod (SPP) and seed weight per plant (SWP) were recorded at the end of vegetation period (EV). Microbiological analyses of soil included the determination of total number of bacteria (TNB), number of fungi (FNG), actinomycetes (ACT) and azotobacters (AZB) as well as the activity of the dehydrogenase enzyme (DHA). The results showed that bacterial inoculation led to the formation of root nodules regardless of the treatments with statistically no significant difference. Strong nodulation was also present in control treatment. RL and PH were positively influenced by inoculation with Azotobacter sp. and Bacillus sp., respectively. Statistical analyses of the number of PPP, SPP, and SWP showed no significant differences among investigated treatments. High average number of microorganisms were determined in all treatments. Most abundant were TNB (log No 8,010) and ACT (log No 6,055) than FNG and AZB with log No 4,867 and log No 4,025, respectively. The highest DHA activity was measured in the FS of soybean in treatment 3. The application of nonsymbiotic bacteria in soybean production can alleviate initial plant growth and help the plant to better overcome different stress conditions caused by abiotic and biotic factors.

Keywords: bacteria, inoculation, soybean, microbial activity

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Authors: Laura Villamizar, David Wright, Claudia Baena, Marie Foxwell, Maureen O'Callaghan

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Leguminous crops can be self-sufficient for their nitrogen requirements when their roots are nodulated with an effective Rhizobium strain and for this reason seed or soil inoculation is practiced worldwide to ensure nodulation and nitrogen fixation in grain and forage legumes. The most widely used method of applying commercially available inoculants is using peat cultures which are coated onto seeds prior to sowing. In general, rhizobia survive well in peat, but some species die rapidly after inoculation onto seeds. The development of improved formulation methodology is essential to achieve extended persistence of rhizobia on seeds, and improved efficacy. Formulations could be solid or liquid. Most popular solid formulations or delivery systems are: wettable powders (WP), water dispersible granules (WG), and granules (DG). Liquid formulation generally are: suspension concentrates (SC) or emulsifiable concentrates (EC). In New Zealand, R. leguminosarum bv. trifolii strain TA1 has been used as a commercial inoculant for white clover over wide areas for many years. Seeds inoculation is carried out by mixing the seeds with inoculated peat, some adherents and lime, but rhizobial populations on stored seeds decline over several weeks due to a number of factors including desiccation and antibacterial compounds produced by the seeds. In order to develop a more stable and suitable delivery system to incorporate rhizobia in pastures, two strains of R. leguminosarum (TA1 and CC275e) and several formulations and processes were explored (peat granules, self-sticky peat for seed coating, emulsions and a powder containing spray dried microcapsules). Emulsions prepared with fresh broth of strain TA1 were very unstable under storage and after seed inoculation. Formulations where inoculated peat was used as the active ingredient were significantly more stable than those prepared with fresh broth. The strain CC275e was more tolerant to stress conditions generated during formulation and seed storage. Peat granules and peat inoculated seeds using strain CC275e maintained an acceptable loading of 108 CFU/g of granules or 105 CFU/g of seeds respectively, during six months of storage at room temperature. Strain CC275e inoculated on peat was also microencapsulated with a natural biopolymer by spray drying and after optimizing operational conditions, microparticles containing 107 CFU/g and a mean particle size between 10 and 30 micrometers were obtained. Survival of rhizobia during storage of the microcapsules is being assessed. The development of a stable product depends on selecting an active ingredient (microorganism), robust enough to tolerate some adverse conditions generated during formulation, storage, and commercialization and after its use in the field. However, the design and development of an adequate formulation, using compatible ingredients, optimization of the formulation process and selecting the appropriate delivery system, is possibly the best tool to overcome the poor survival of rhizobia and provide farmers with better quality inoculants to use.

Keywords: formulation, Rhizobium leguminosarum, storage stability, white clover

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Authors: Zanele D. Ngwenya, Mustapha Mohammed, Felix D. Dakora

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Legumes are a major source of biological nitrogen, and therefore play a crucial role in maintaining soil productivity in smallholder agriculture in southern Africa. Through their ability to fix atmospheric nitrogen in root nodules, legumes are a better option for sustainable nitrogen supply in cropping systems than chemical fertilisers. For decades, farmers have been highly receptive to the use of rhizobial inoculants as a source of nitrogen due mainly to the availability of elite rhizobial strains at a much lower compared to chemical fertilisers. To improve the efficiency of the legume-rhizobia symbiosis in African soils would require the use of highly effective rhizobia capable of nodulating a wide range of host plants. This study assessed the morphogenetic diversity, photosynthetic functioning and relative symbiotic effectiveness (RSE) of groundnut, jack bean and soybean microsymbionts in Eswatini soils as a first step to identifying superior isolates for inoculant production. According to the manufacturer's instructions, rhizobial isolates were cultured in yeast-mannitol (YM) broth until the late log phase and the bacterial genomic DNA was extracted using GenElute bacterial genomic DNA kit. The extracted DNA was subjected to enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and a dendrogram constructed from the band patterns to assess rhizobial diversity. To assess the N2-fixing efficiency of the authenticated rhizobia, photosynthetic rates (A), stomatal conductance (gs), and transpiration rates (E) were measured at flowering for plants inoculated with the test isolates. The plants were then harvested for nodulation assessment and measurement of plant growth as shoot biomass. The results of ERIC-PCR fingerprinting revealed the presence of high genetic diversity among the microsymbionts nodulating each of the three test legumes, with many of them showing less than 70% ERIC-PCR relatedness. The dendrogram generated from ERIC-PCR profiles grouped the groundnut isolates into 5 major clusters, while the jack bean and soybean isolates were grouped into 6 and 7 major clusters, respectively. Furthermore, the isolates also elicited variable nodule number per plant, nodule dry matter, shoot biomass and photosynthetic rates in their respective host plants under glasshouse conditions. Of the groundnut isolates tested, 38% recorded high relative symbiotic effectiveness (RSE >80), while 55% of the jack bean isolates and 93% of the soybean isolates recorded high RSE (>80) compared to the commercial Bradyrhizobium strains. About 13%, 27% and 83% of the top N₂-fixing groundnut, jack bean and soybean isolates, respectively, elicited much higher relative symbiotic efficiency (RSE) than the commercial strain, suggesting their potential for use in inoculant production after field testing. There was a tendency for both low and high N₂-fixing isolates to group together in the dendrogram from ERIC-PCR profiles, which suggests that RSE can differ significantly among closely related microsymbionts.

Keywords: genetic diversity, relative symbiotic effectiveness, inoculant, N₂-fixing

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Authors: Jessica P. Fiorotti, Maria C. Freitas, Caio J. B. Coutinho-Rodrigues, Mariana G. Camargo, Emily S. Mesquita, Amanda R. C. Corval, Ricardo O. B. Bitencourt, Allan F. Marciano, Diva D. Spadacci-Morena, Patricia S. Golo, Isabele C. Angelo, Vania R. E. P. Bittencourt

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The bovine tick, Rhipicephalus microplus, is an arthropod of great importance in veterinary medicine leading to anemia, weight loss, animals' leather depreciation and also acting as a vector of many pathogens. In this way, the parasitism causes a loss of 3.24 billion dollars per year in Brazil. Knowingly, entomopathogenic fungi act as natural controller of some arthropods, acting mainly by active penetration through the cuticle. However, it can also act on the hemolymph and through the production of mycotoxins. Hemocytes are responsible for the cellular immune response and participate in the processes of phagocytosis, nodulation and encapsulation and may undergo changes when challenged by pathogens. The aim of the present study was to evaluate changes in R. microplus hemocytes after inoculation of Metarhizium robertsii using transmission electron microscopy. The isolate ARSEF 2575 and 200 engorged R. microplus females were used. The groups were divided into control, in which the females were inoculated with 5 μL of sterile distilled water solution and 0.1% Tween 80, and a group inoculated with 5 μL of fungal suspension at the concentration of 10⁷ conidia mL⁻¹. The experiment was performed in duplicate and each group contained 50 females. Twenty-four hours after fungal inoculation, hemolymph was collected through the cuticle dorsal surface perforation of the tick females. After collection, the hemolymph samples were centrifuged at 500 x g for 3 minutes at 4 °C, the plasma was discarded and the hemocyte pellet was resuspended in 50 μl PBS. The suspension material was fixed in 2% glutaraldehyde in Millonig buffer for three hours. After fixation, the material was centrifuged at 500 x g for 3 minutes, the supernatant was discarded and the cells were resuspended in a wash solution. Subsequently, the cells were post-fixed with 1% osmium tetroxide in phosphate buffer for one hour at room temperature and dehydrated in increasing concentrations of ethanol, and then embedded in Epon resin. The ultrathin sections were examined under the LEO EM 906E transmission electron microscopy at 80kV. The ultrastructural results revealed that.in control group, the cells were considered intact, in which the granulocytes were observed with granules of different electrodensities, intact mitochondria and cytoplasm without vacuolization. In addition, granulocytes showed plasma membrane projections similar to pseudopodia. Plasmatocytes presented as irregularly shaped cells, with the eccentric nucleus, agranular cytoplasm and some cells presented pseudopodia. Nevertheless, in the group exposed to the fungus, most of the cells presented in degeneration. The granulocytes found had fewer granules in the cytoplasm and more vacuoles. Plasmatocytes, after treatment, presented many vacuoles also in the cytoplasm and the lysosomes presented great amount of electrodense material in their interior. Thus, the results suggest that the fungus has a depressant action in the immune system of the tick, not only by the cell degranulation, but also suggesting that this leads to morphological changes in the hemocytes and may even trigger processes such as phagocytosis.

Keywords: bovine tick, cellular defense, entomopathogenic fungi, immune response

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