Genotypic Variation of Endophytic Nitrogen-Fixing Activity and Bacterial Flora in Rice Stem Based on Sugar Content

Enhancement of the nitrogen-fixing ability of endophytic bacteria in rice is expected to result in improved nitrogen use under low-nitrogen conditions. Endophytic nitrogen-fixing bacteria require a large amount of energy to fix atmospheric nitrogen. However, it is unknown which carbon source and bacteria would affect nitrogen-fixing activity in rice. Therefore, this study examined genotypic variations in the nitrogen-fixing ability of rice plant stem as affected by non-structural carbohydrates and endophytic bacterial flora in field-grown rice. In the field experiments, six varieties and 10 genotypes of rice were grown in 2017 and 2018 to compare the acetylene reduction activity (nitrogen-fixing activity) and non-structural carbohydrates (glucose, sucrose, and starch) concentration in their stems at the heading stage. For the bacterial flora analysis, two genes were amplified using a primer set of 16S rRNA and nitrogenase (NifH) gene-specific primers. Next, acetylene reduction activity was correlated with sugar concentration among genotypes in both years, suggesting that the levels of soluble sugars influenced stem nitrogen-fixing activity. Bacterial flora analysis also suggested the presence of common and genotype-specific bacterial flora in both 16S rRNA and nifH genes. Similarly, bacteria classified as rhizobia, such as Bradyrhizobium sp. (Alphaproteobacteria) and Paraburkholderia sp. (Betaproteobacteria), were highly abundant in all rice genotypes, suggesting that these bacteria make major contributions to the nitrogen fixation process in rice stems. Gammaproteobacteria were more abundant in CG14 as well, which showed the highest acetylene reduction activity and sugar concentration among genotypes and is also proposed to contribute to the higher amount of nitrogen-fixing activity.

Increasing the Legume–Rhizobia Symbiotic Efficiency Due to the Synergy between Commercial Strains and Strains Isolated from Relict Symbiotic Systems

The phenomenon of rhizobial synergy was investigated to increase the efficiency of nitrogen-fixing symbiosis of alfalfa (Medicago varia Martyn), common vetch (Vicia sativa L.) or red clover (Trifolium pratense L.). These plants were co-inoculated with the respective commercial strains Sinorhizobium meliloti RCAM1750, Rhizobium leguminosarum RCAM0626 or R. leguminosarum RCAM1365 and with the strains Mesorhizobium japonicum Opo-235, M. japonicum Opo-242, Bradyrhizobium sp. Opo-243 or M. kowhaii Ach-343 isolated from the relict legumes Oxytropis popoviana Peschkova and Astragalus chorinensis Bunge. The isolates mentioned above had additional symbiotic genes (fix, nif, nod, noe and nol) as well as the genes promoting plant growth and symbiosis formation (acdRS, genes associated with the biosynthesis of gibberellins and auxins, genes of T3SS, T4SS and T6SS secretion systems) compared to the commercial strains. Nodulation assays showed that in some variants of co-inoculation the symbiotic parameters of plants such as nodule number, plant biomass or acetylene reduction activity were increased. We assume that the study of microbial synergy using rhizobia of relict legumes will make it possible to carry out targeted selection of co-microsymbionts to increase the efficiency of agricultural legume–rhizobia systems.

The Abundance of the nifH Gene Became Higher and the nifH-Containing Diazotrophic Bacterial Communities Changed During Primary Succession in the Hailuogou Glacier Chronosequence, China

Primary successional ecosystems and the related soil development are often N limited. To date, N2-fixing communities during primary succession in alpine ecosystems have remained underexplored. In this study, we applied quantitative PCR (qPCR) quantitation and targeted amplicon sequencing of nifH in the Hailuogou Glacier foreland to investigate the succession of N2-fixing communities in five sites along a 62-year chronosequence. The abundance of the nifH gene increased along the primary succession in the chronosequence and correlated positively with pH, acetylene reduction activity, and water, organic C, total and available N, and available P contents. The increases in alpha diversity along the chronosequence may have been partly due to less competition for resources. In contrast to the clear separation based on soil properties, the changes in the diazotrophic community composition lacked a clear trend and were associated mostly with changes in soil available K and organic C contents. The changes among differentially abundant genera were possibly due to the changes in plant coverage and species composition. The whole primary succession of the diazotrophic communities was consistent with stochastic community assembly, which is indicative of low competitive pressure.

Promotion of biological nitrogen fixation activity of an anaerobic consortium using humin as an extracellular electron mediator

Nitrogen fertiliser is manufactured using the industrial Haber–Bosch process, although it is extremely energy-consuming. One sustainable alternative technology is the electrochemical promotion of biological nitrogen fixation (BNF). This study reports the promotion of BNF activity of anaerobic microbial consortia by humin, a solid-phase humic substance, at any pH, functioning as an extracellular electron mediator, to levels of 5.7–11.8 times under nitrogen-deficient conditions. This was evidenced by increased acetylene reduction activity and total nitrogen content of the consortia. Various humins from different origins promoted anaerobic BNF activity, although the degree of promotion differed. The promotion effected by humin differed from the effects of chemical reducing agents and the effects of supplemental micronutrients and vitamins. The promotion of anaerobic BNF activity by only reduced humin without any other electron donor suggested that humin did not serve as organic carbon source but as extracellular electron mediator, for electron donation to the nitrogen-fixing microorganisms. The next generation sequencing (NGS) of partial 16S rRNA genes showed the predominance of Clostridiales (Firmicutes) in the consortia. These findings suggest the effectiveness of humin as a solid-phase extracellular electron mediator for the promotion of anaerobic BNF activity, potentially to serve for the basis for a sustainable technology.

Microbial Consortium of PGPR, Rhizobia and Arbuscular Mycorrhizal Fungus Makes Pea Mutant SGECdt Comparable with Indian Mustard in Cadmium Tolerance and Accumulation

Cadmium (Cd) is one of the most widespread and toxic soil pollutants that inhibits plant growth and microbial activity. Polluted soils can be remediated using plants that either accumulate metals (phytoextraction) or convert them to biologically inaccessible forms (phytostabilization). The phytoremediation potential of a symbiotic system comprising the Cd-tolerant pea (Pisum sativum L.) mutant SGECdt and selected Cd-tolerant microorganisms, such as plant growth-promoting rhizobacterium Variovorax paradoxus 5C-2, nodule bacterium Rhizobium leguminosarum bv. viciae RCAM1066, and arbuscular mycorrhizal fungus Glomus sp. 1Fo, was evaluated in comparison with wild-type pea SGE and the Cd-accumulating plant Indian mustard (Brassica juncea L. Czern.) VIR263. Plants were grown in pots in sterilized uncontaminated or Cd-supplemented (15 mg Cd kg−1) soil and inoculated or not with the microbial consortium. Cadmium significantly inhibited growth of uninoculated and particularly inoculated SGE plants, but had no effect on SGECdt and decreased shoot biomass of B. juncea. Inoculation with the microbial consortium more than doubled pea biomass (both genotypes) irrespective of Cd contamination, but had little effect on B. juncea biomass. Cadmium decreased nodule number and acetylene reduction activity of SGE by 5.6 and 10.8 times, whereas this decrease in SGECdt was 2.1 and 2.8 times only, and the frequency of mycorrhizal structures decreased only in SGE roots. Inoculation decreased shoot Cd concentration and increased seed Cd concentration of both pea genotypes, but had little effect on Cd concentration of B. juncea. Inoculation also significantly increased concentration and/or accumulation of nutrients (Ca, Fe, K, Mg, Mn, N, P, S, and Zn) by Cd-treated pea plants, particularly by the SGECdt mutant. Shoot Cd concentration of SGECdt was twice that of SGE, and the inoculated SGECdt had approximately similar Cd accumulation capacity as compared with B. juncea. Thus, plant–microbe systems based on Cd-tolerant micro-symbionts and plant genotypes offer considerable opportunities to increase plant HM tolerance and accumulation.

Nitrogen fixation sensitivity related to water use efficiency at reproductive development in soybean

Soybean [Glycine max (L.) Merr.] nitrogen fixation is sensitive differentially to drought among different genotypes at different growth and development stages, which directly affects soybean yield. Acetylene reduction activity (ARA) response to a gradual drought and rewatering period at late podding (late R₃) and late seed fill (late R₅) were evaluated in two different water use efficiency (WUE) genotypes. Drought-stressed plants with high WUE (PI 372413) decreased ARA more insensitively than that of low WUE (PI 548534), and drought-stressed plants with low WUE (PI 548534) maintained low ARA level after stress alleviation at late R₅. The recovery ability of N₂ fixation was a genotypic difference with WUE at late reproductive development (late R₅), especially. Analysing relation between fraction of transpirable soil water (FTSW) and relative ARA, it was confirmed that PI 372413 with high WUE was more insensitive to water deficit and had drought tolerance by N₂ fixation and recovery ability with a threshold of 0.139–0.147 FTSW than PI 548534 with a threshold of 0.192–0.209 FTSW. The ability to recover N₂ fixation following drought during the reproductive developmental stage would be of an important value in the actual planting environment.

Nitrogen Fixation in Pozol, a Traditional Fermented Beverage

ABSTRACT Traditional fermentations have been widely studied from the microbiological point of view, but little is known from the functional perspective. In this work, nitrogen fixation by free-living nitrogen-fixing bacteria was conclusively demonstrated in pozol, a traditional Mayan beverage prepared with nixtamalized and fermented maize dough. Three aspects of nitrogen fixation were investigated to ensure that fixation actually happens in the dough: (i) the detection of acetylene reduction activity directly in the substrate, (ii) the presence of potential diazotrophs, and (iii) an in situ increase in acetylene reduction by inoculation with one of the microorganisms isolated from the dough. Three genera were identified by sequencing the 16S rRNA and nifH genes as Kosakonia, Klebsiella, and Enterobacter, and their ability to fix nitrogen was confirmed. IMPORTANCE Nitrogen-fixing bacteria are found in different niches, as symbionts in plants, in the intestinal microbiome of several insects, and as free-living microorganisms. Their use in agriculture for plant growth promotion via biological nitrogen fixation has been extensively reported. This work demonstrates the ecological and functional importance that these bacteria can have in food fermentations, reevaluating the presence of these genera as an element that enriches the nutritional value of the dough.

Diversity and Effectivity of Indigenous Mesorhizobium Strains for Chickpea (Cicer arietinum L.) in Myanmar

Chickpea (Cicer arietinum L.) is one of the world’s main leguminous crops that provide chief source of food for humans. In the present study, we characterized thirty isolates of indigenous chickpea rhizobia from Myanmar based on the sequence analysis of the bacterial 16S rRNA gene. The sequence analysis confirmed that all isolates were categorized and identified as the genus Mesorhizobium and they were conspecific with M. plurifarium, M. muliense, M. tianshanense, and M. sp. This is the first report describing M. muliense, M. tianshanense, and M. plurifurium from different geographical distribution of indigenous mesorhizobia of chickpea in Myanmar. In order to substitute the use of chemical fertilizers in legume production, there is a need for the production of Biofertilizers with rhizobial inoculants. The effectiveness of Myanmar Mesorhizobim strains isolated from soil samples of major chickpea growing areas of Myanmar for plant growth and nitrogen fixation were studied in pot experiments. The nodule dry weight and acetylene reduction activity of the plant inoculated with Mesorhizobium tianshanense SalCP19 was significantly higher than the other tested isolates in Yezin-4 chickpea variety. But, Mesorhizobium sp. SalCP17 was showed high level of acetylene reduction activity per plant in Yezin-6 chickpea variety.

Plant Tissue Localization and Morphological Conversion of Azospirillum brasilense upon Initial Interaction with Allium cepa L.

The genus Azospirillum is recognized as plant growth-promoting bacteria that exert beneficial effects on the host plant and is morphologically converted into cyst-like cells (i.e., c-form) in association with poly-β-hydroxybutyrate (PHB) accumulation in the cells under stress conditions. We constructed Azospirillum brasilense, labeled with reporter genes (gus/gfp, mCherry) and examined the plant tissue localization along with a morphological conversion into the c-form upon its initial interaction with onion seedlings (Allium cepa L.). The PHB granules in the A. brasilense cells were easily detected under fluorescence as “black holes”, rendering it possible to monitor the morphological conversion from vegetative to the c-form cells. The results showed that the A. brasilense cells on the surface of the roots and bulbs (underground stem) began converting at three days following inoculation and that the cell conversion was significantly advanced with time along with the cell population increase. The endophytic infection of A. brasilense into the bulb tissues was also confirmed, although these likely constituted vegetative cells. Moreover, the morphological conversion into the c-form was induced under nitrogen-restricted conditions. Analysis of the biochemical properties of the A. brasilense cells during cell conversion revealed that the acetylene reduction activity correlated positively with the PHB accumulation in the cells converting into the c-form under nitrogen-restricted conditions.

Changes in Acetylene Reduction Activities and nifH Genes Associated with Field-Grown Sweet Potatoes with Different Nursery Farmers and Cultivars

Sweet potato cultivars obtained from different nursery farmers were cultivated in an experimental field from seedling-stage to harvest, and the acetylene reduction activity (ARA) of different parts of the plant as well as the nifH genes associated with the sweet potatoes were examined. The relationship between these parameters and the plant weights, nitrogen contents, and natural abundance of 15N was also considered. The highest ARA was detected in the tubers and in September. Fragments of a single type of nitrogenase reductase gene (nifH) were amplified, and most of them had similarities with those of Enterobacteriaceae in γ-Proteobacteria. In sweet potatoes from one nursery farm, Dickeya nifH was predominantly detected in all of the cultivars throughout cultivation. In sweet potatoes from another farm, on the other hand, a transition to Klebsiella and Phytobacter nifH was observed after the seedling stage. The N2-fixing ability contributed to plant growth, and competition occurred between autochthonous and allochthonous bacterial communities in sweet potatoes.