Efficacy of Peat and Liquid Inoculant Formulations of Bradyrhizobium japonicum Strain WB74 on Growth, Yield and Nitrogen Concentration of Soybean (Glycine max L.)

South African soils generally lack native Bradyrhizobium strains that nodulate and fix atmospheric nitrogen (N2) in soybeans (Glycine max L.). It is therefore very important to inoculate soybeans with products that contain effective Bradyrhizobium strains as active ingredients. In this study, a field experiment was conducted on two bioclimatic zones in South Africa during the 2019/2020 season to assess the effect of Bradyrhizobium japonicum strain WB74 inoculant formulation on nitrogen fixation, growth and yield improvement in soybeans. The first bioclimatic zone was characterized by a sandy clay loam soil, whereas the second bioclimatic zone has a sandy loam soil. The results showed that inoculation of soybeans with both peat and liquid formulations of Bradyrhizobium japonicum WB74 increased nitrogen uptake, which resulted in yield increase. The amount of N fixed was measured as 15N isotopes and increased with all treatments compared to the uninoculated control in both liquid and peat inoculant formulations. In bioclimatic zone A, slightly better results were obtained using the liquid formulation (1.79 t ha−1 for liquid compared to 1.75 t ha−1 for peat treatments), while peat formulations performed better in bioclimatic zone B (1.75 t ha−1 for peat compared to 1.71 t ha−1 for liquid treatments). In both areas higher yields were obtained with the formulations used in this study compared to the registered standards (treatment T3). The findings in this study provide vital information in the development and application of formulated microbial inoculants for sustainable agriculture in South Africa.

Polyvinylpyrrolidone (PVP) and Na-Alginate Addition Enhances the Survival and Agronomic Performances of a Liquid Inoculant of Bradyrhizobium japonicum for Soybean (Glycine max (L.) Merr.)

Nontoxic polymers PVP and Na-alginate may provide a favorable environment for the survival of bacteria. Therefore, PVP and Na-alginate were added to a growth medium to develop a liquid inoculant of Bradyrhizobium japonicum strain. The strain was identified by 16S rDNA sequencing. The addition of PVP (1.8%) and Na-alginate (0.2%) in the medium promoted a better survival (1.93 × 109 cells mL−1) of B. japonicum strain compared to the control (3.50 × 102 cells mL−1) after 6 months of storage. The combination of PVP and Na-alginate ensured 1.53 × 107 cells mL−1 up to 12 months of storage under ambient temperature (28 ± 2 °C), whereas PVP (1.8%) or Na-alginate (0.2%) alone produced similar cell counts only up to 8 months and 6 months, respectively. Consecutive field experiments proved the efficacy of the liquid inoculant on nodulation and yield of soybean. The combination of PVP and Na-alginate-based inoculation of B. japonicum strain significantly increased the nodule number per plant, number of pods per plant, number of seeds per pod, seed yield, and yield per hectare (p ≤ 0.05). Thus, the combination of PVP- and Na-alginate-based inoculation of B. japonicum has great potential to popularize the organic cultivation of soybean.

Role of nitrogen deficiency on growth and development near isogenic by E genes lines of soybean co-inoculated with nitrogen-fixing bacteria

Nitrogen deficiency is a limiting factor in increasing efficiency of crop production in terrestrial ecosystems, and the transformation of inert nitrogen to forms that can be assimilated by plants is mediated by soil microorganisms. Symbiotic nitrogen-fixing bacteria and roots depend on each other and have developed various mechanisms for symbiotic coexistence. The aim of this work was to investigate the role of nitrogen deficiency on growth and development near isogenic by E genes lines of soybean (Glycine max (L.) Merr.): short-day (SD) line with genotype Е1е2е3(Е4е5Е7), and photoperiodic insensitive (PPI) line with genotype е1е2е3(Е4е5Е7) grown from seeds inoculated with active strains of Bradyrhizobium japonicum against the background of local populations of diazotrophs of the genus Azotobacter spp. and establish how the soybean – Bradyrhizobium symbiosis will develop as the genes of both microsymbionts and macrosymbionts are responsible for the formation of the symbiotic complex. Plants were grown in a vegetation chamber, in sand culture. To assess the quantitative composition of microorganisms in the rhizosphere and rhizoplanes, 6 plants were selected from each soybean line, then separation of the zones of the rhizosphere and rhizoplanes was performed using the method of washing and the resulting suspension was used for inoculation on dense nutrient media (mannitol-yeast agar medium and Ashby medium). The results of study showed that seed inoculation and co-inoculation provides faster formation of the symbiotic soybean – Bradyrhizobium complex. Differences in nodulation rates between the short-day line with genotype Е1е2е3(Е4е5Е7), and a photoperiodic insensitive line with genotype е1е2е3(Е4е5Е7) were identified. Determination of the amount of B. japonicum on the medium of mannitol-yeast agar in the rhizosphere and rhizoplane showed that inoculation by B. japonicum strain 634b caused a significant increase in the amount B. japonicum in the rhizosphere and rhizoplane in both soybean lines, comparison with non-inoculated seeds. Then, co-inoculation by B. japonicum strain 634b + Azotobacter chroococcum significantly increased the amount of B. japonicum only in the rhizoplane and decreased their number in the rhizosphere. Determination of the amount of A. chroococcum on the Ashby elective medium in the rhizosphere and rhizoplane showed that the inoculation by B. japonicum strain 634b caused a significant decrease in the amount of A. chroococcum both in the rhizosphere and in the rhizoplane of the PPI line of soybean, and in the rhizosphere the SD line, in comparison with non-inoculated seeds. That can testify to the competitive interaction of these microorganisms. However, the co-inoculation by B. japonicum strain 634b + A. chroococcum in the SD line significantly increased the number of A. chroococcum in the rhizoplane and decreased their number in the rhizosphere, in the PPI line their number decreased in the rhizoplane and increased in the rhizosphere, in comparison with non-inoculated seeds. Probably, the E genes (their dominant or recessive state) of soybean isogenic lines affect the regulation of the content and distribution of sugars. It was established that the nitrogen deficiency stimulated development of the root system of plants and the synthesized sugars were distributed predominantly to the root system growth. We suppose that the seeds’ inoculation had extended sugar consumption to the symbiont, due to which it compensates the lack of nitrogen, but leads to a slower growth of the root system.

Latitudinal Characteristic Nodule Composition of Soybean-Nodulating Bradyrhizobia: Temperature-Dependent Proliferation in Soil or Infection?

A species-specific latitudinal distribution of soybean rhizobia has been reported; Bradyrhizobium japonicum and B. elkanii dominate in nodules in northern and southern areas, respectively. The aim of this study was to elucidate whether temperature-dependent proliferation in soil or infection is more reliable for determining the latitudinal characteristic distribution of soybean-nodulating rhizobia under local climate conditions. Three study locations, Fukagawa (temperate continental climate), Matsue and Miyazaki (humid sub-tropical climate), were selected in Japan. Each soil sample was transported to the other study locations, and soybean cv. Orihime (non-Rj) was pot-cultivated using three soils at three study locations for two successive years. Species composition of Bradyrhizobium in the nodules was analyzed based on the partial 16S rRNA and 16S–23S rRNA ITS gene sequences. Two Bradyrhizobium japonicum (Bj11 and BjS10J) clusters and one B. elkanii (BeL7) cluster were phylogenetically sub-grouped into two (Bj11-1-2) and four clusters (BjS10J-1-4) based on the ITS sequence. In the Fukagawa soil, Bj11-1 dominated (80–87%) in all study locations. In the Matsue soil, the composition was similar in the Matsue and Miyazaki locations, in which BeL7 dominated (70–73%), while in the Fukagawa location, BeL7 decreased to 53% and Bj11-1 and BjS10J-3 increased. In the Miyazaki soil, BeL7 dominated at 77%, and BeL7 decreased to 13% and 33% in the Fukagawa and Matsue locations, respectively, while BjS10J-2 and BjS10J-4 increased. It was supposed that the B. japonicum strain preferably proliferated in the Fukagawa location, leading to its nodule dominancy, while in the Miyazaki location, temperature-dependent infection would lead to the nodule dominancy of B. elkanii, and both factors would be involved in the Matsue location.

EFFECTS OF COMBINED INOCULATION OF BAMBARA GROUNDNUT (Vigna subterranean L. Verdc.) WITH GLOMUS MOSSEA AND BRADYHRHIZOBIUM JAPONICUM ON NITROGEN AND PHOSPHOROUS UPTAKE IN SHOOT, PLANT BIOMASS, LEAF CHLOROPHYLL AND MYCORRHIZAL INOCULATION EFFICIENCY (MIE).

The response of Bambara groundnut to co-inoculation with Abuscular mychorrhizal fungi (G. mossea) and Bradhyrhizobium japonicum (strain USDA110) with regard to leaf chlorophyll, percentage shoot nitrogen and phosphorus, nodule weight and plant biomass was studied. Bambara plants were grown under screen house conditions in pots. Plants were inoculated with 1ml of B. japonicum USDA 110 strain (109 cfu /ml), Mychorrizal was applied to the plants 10g, and 20g (90 spores/g) and water was applied at 10ml, 20 ml and 50ml every other day. The obtained results showed that dual inoculation activity was able to improve both nitrogen and phosphorus in plant shoot, MIE, but did not improve biomass and leaf chlorophyll when compared with plants subjected to single inoculation with only G. mossea and only B. japonicum. More Nitrogen and Phosphorus was retained in the shoot of plants co-inoculated with B. japonicum and 20g G. mossea when given 50ml of water and also had higher biomass. Leaf chlorophyll reduced in plants as flowering approached. B. japonicum was able to positively influence and establish symbiosis with G.mossea and synergistically effectively act as “mycorrhiza helper bacteria” (MHB) when both were co-inoculated in Bambara plant.

The Storage Efficiency of Immobilized Bradyrhizobium japonicum Strain Using Encapsulation Method

Summary There is a growing need for new formulations of carriers with better protection for bacterial inoculum. One of the newer techniques in inoculum making is encapsulation method. With this method, the whole bacterial cells are immobilized in defined space – matrix, where the cells are protected from environmental activities before use. Encapsulation of the inoculum was performed with ionic gelation method. The alginate-based microparticles (500-600 µm) containing viable B. japonicum strain were solidified in CaCl2. The initial number of viable bacteria in every sample was 9.0 log CFU/ml. Chitosan coated particles had a higher mortality rate than non-coated particles, with 1.3 log CFU/ml in lyophilized and wet microparticles stored at room temperature. High viability of B. japonicum was registered in wet particles stored at constant −20°C for thirty days with a viability rate of 8.84 log CFU/ml.