Search Results (23)

Understanding the dominant populations and biological functions of endophytic nitrogen-fixing bacteria in apple plants is of great significance for the healthy growth management and sustainable development of apple cultivation. In this study, we investigated the community diversity and potential plant growth-promoting abilities of endophytic nitrogen-fixing bacteria in different tissues of apple trees by combining high-throughput sequencing of the nifH gene with traditional isolation and cultivation techniques. Sequencing results revealed that the endophytic bacteria were affiliated with 10 phyla, 14 classes, 30 orders, 42 families, and 72 genera. Rhizobium was the dominant genus in the roots and twigs, while Desulfovibrio dominated the leaf tissues. The diversity and richness of endophytic bacteria in the roots were significantly higher than those in the leaves. Using four types of nitrogen-free media, a total of 138 presumptive endophytic nitrogen-fixing bacterial strains were isolated from roots, leaves, and twigs. These isolates belonged to 32 taxonomic groups spanning 5 phyla, 8 classes, 11 orders, 13 families, and 18 genera. The nifH gene was successfully amplified from the representative strains of all 32 groups using specific primers. Nitrogenase activity among the isolates ranged from 26.86 to 982.28 nmol/(h·mL). Some strains also exhibited the ability to secrete indole-3-acetic acid (IAA), solubilize phosphate and potassium, and produce siderophores. Six individual strains and three microbial consortia were tested for their plant growth-promoting effects on apple tissue culture seedlings. All treatments showed growth-promoting effects to varying degrees, with the RD01+RC16 consortium showing the most significant results: plant height, number of leaves, and chlorophyll content were 2.4, 3.3, and 4.2 times higher than those of the control, respectively. These findings demonstrate the rich diversity of endophytic nitrogen-fixing bacteria in apple plants and their promising potential for application in promoting host plant growth. Full article

Microbes such as bacteria and fungi play important roles in nutrient cycling in soils, often leading to the bioavailability of metabolically important mineral elements such as nitrogen (N), phosphorus (P), iron (Fe), and zinc (Zn). Examples of microbes with beneficial traits for plant growth promotion include mycorrhizal fungi, associative diazotrophs, and the N₂-fixing rhizobia belonging to the α, β and γ class of Proteobacteria. Mycorrhizal fungi generally contribute to increasing the surface area of soil-root interface for optimum nutrient uptake by plants. However, when transformed into bacteroids inside root nodules, rhizobia also convert N₂ gas in air into ammonia for use by the bacteria and their host plant. Thus, nodulated legumes can meet a high proportion of their N requirements from N₂ fixation. The percentage of legume N derived from atmospheric N₂ fixation varies with crop species and genotype, with reported values ranging from 50–97%, 24–67%, 66–86% 27–92%, 50–92%, and 40–75% for soybean (Gycine max), groundnut (Arachis hypogea), mung bean (Vigna radiata), pigeon pea (Cajanus cajan), cowpea (Vigna unguiculata), and Kersting’s groundnut (Macrotyloma geocarpum), respectively. This suggests that N₂-fixing legumes require little or no N fertilizer for growth and grain yield when grown under field conditions. Even cereals and other species obtain a substantial proportion of their N nutrition from associative and endophytic N₂-fixing bacteria. For example, about 12–33% of maize N requirement can be obtained from their association with Pseudomonas, Hebaspirillum, Azospirillum, and Brevundioronas, while cucumber can obtain 12.9–20.9% from its interaction with Paenebacillus beijingensis BJ-18. Exploiting the plant growth-promoting traits of soil microbes for increased crop productivity without any negative impact on the environment is the basis of green agriculture which is done through the use of biofertilizers. Either alone or in combination with other synergistic rhizobacteria, rhizobia and arbuscular mycorrhizal (AM) fungi have been widely used in agriculture, often increasing crop yields but with occasional failures due to the use of poor-quality inoculants, and wrong application techniques. This review explores the literature regarding the plant growth-promoting traits of soil microbes, and also highlights the bottle-necks in tapping this potential for sustainable agriculture. Full article

In rice, symbiotic N₂ fixation via nodule bacteroids does not take place naturally. Although N₂ fixation by endophytic and associative diazotrophs has been reported in rice, the main organs and seasonal regulation for the N₂ fixation have not been elucidated. In this study, seasonal changes in nitrogenase (acetylene reduction) activity and carbohydrate contents in elongated culm (EC), unelongated stem (US), and crown root (CR) were investigated in manure compost (MC)- and chemical fertilizer (CF)-applied rice. Nitrogenase activity increased after rooting (June) and reached the highest activity in US of MC-applied rice at panicle initiation (August). The sucrose content in EC continued to increase after rooting regardless of the applied materials, whereas the glucose content in US increased after rooting only in CF-applied rice, suggesting higher consumption of glucose in US of MC-applied rice. There were significant differences among bacterial microbiota in EC, US, and CR at panicle initiation. In addition, Clostridia class anaerobes were more abundant in US of MC-applied rice than in EC and CR of MC-applied rice. Such difference was not observed in US of CF-applied rice. These results suggest the suitability of US of MC-applied rice at panicle initiation as a site of N₂ fixation under anaerobic conditions. Full article

Gluconacetobacter diazotrophicus is a diazotrophic endophytic bacterium that promotes the growth and development of several plant species. However, the molecular mechanisms activated during plant response to this bacterium remain unclear. Here, we used the RNA-seq approach to understand better the effect of G. diazotrophicus PAL5 on the transcriptome of shoot and root tissues of Arabidopsis thaliana. G. diazotrophicus colonized A. thaliana roots and promoted growth, increasing leaf area and biomass. The transcriptomic analysis revealed several differentially expressed genes (DEGs) between inoculated and non-inoculated plants in the shoot and root tissues. A higher number of DEGs were up-regulated in roots compared to shoots. Genes up-regulated in both shoot and root tissues were associated with nitrogen metabolism, production of glucosinolates and flavonoids, receptor kinases, and transcription factors. In contrast, the main groups of down-regulated genes were associated with pathogenesis-related proteins and heat-shock proteins in both shoot and root tissues. Genes encoding enzymes involved in cell wall biogenesis and modification were down-regulated in shoots and up-regulated in roots. In contrast, genes associated with ROS detoxification were up-regulated in shoots and down-regulated in roots. These results highlight the fine-tuning of the transcriptional regulation of A. thaliana in response to colonization by G. diazotrophicus PAL5. Full article

Large quantities of cover crop residues in the soil, combined, or not, with the inoculation of seeds with diazotrophic bacteria, can increase organic matter (OM) and protect soil microorganisms, such as arbuscular mycorrhizal fungi (AMF) and dark septate endophytic (DSE) fungi. Thus, the use of these sustainable biotechnologies can benefit microbial interactions, soil fertility and rice production in the Brazilian Cerrado region. In this study, we evaluated the effects of maize and Urochloa ruziziensis, intercropped or individually, as cover crops and an inoculation of Azospirillum brasilense on the chemical (fertility) and biological (C–microbial biomass and C–CO₂ released) attributes of soil and the effects of root colonization by AMF and DSE on the yield of rice grown in succession in highlands. The experiment was conducted under field conditions, in a typical dystrophic Red Oxisol. The experimental design consisted of randomized blocks arranged in strips, incorporating a combination of eight residual cover crops: ((1) maize, (2) maize–I (I = inoculation of seeds with A. brasilense), (3) Urochloa (U. ruziziensis), (4) Urochloa–I, (5) maize + Urochloa–I, (6) maize + Urochloa–I, (7) maize–I + Urochloa and (8) maize–I + Urochloa–I). This was accompanied by two treatments of rice as a successor crop (inoculated or not with A. brasilense), with four replicates, totaling 64 experimental units. A cover crop and rice seed inoculation prompted increases in OM and AMF relative to DSE, while the inoculation of rice, regardless of the cover crop treatment, increased the soil’s P content. The combination of maize + Urochloa–I and inoculated rice as the next crop generated increases in its sum of bases (SBs) and cation exchange capacity (CEC). There was a 19% increase in rice grain yields when the seed was inoculated. Full article

Soil salinity has a negative impact on the biochemical properties of soil and on plant growth, particularly in arid and semi-arid regions. Using arbuscular mycorrhizal fungi (Glomus versiform) and foliar spray from compost tea as alleviating treatments, this study aimed to investigate the effects of alleviating salt stress on the growth and development of maize and wheat grown on a saline–sodic soil during the period of 2022/2023. Six treatments were used in the completely randomized factorial design experiment. The treatments included Arbuscular mycorrhizal fungus (AMF₀, AMF₁) and varied concentrations of compost tea (CT₀, CT₅₀, and CT₁₀₀). AMF colonization, the bacterial community and endosphere in the rhizosphere, respiration rate, growth parameters, and the productivity were all evaluated. The application of AMF and CT, either separately or in combination, effectively mitigated the detrimental effects caused by soil salinity. The combination of AMF and CT proved to be highly efficient in improving the infection rate of AMF, the bacterial community in the rhizosphere and endosphere, growth parameters, and grain yield of maize and wheat. Therefore, it can be proposed that the inoculation of mycorrhizal fungi with compost tea in saline soils is an important strategy for enhancing salt tolerance in maize and wheat plants through improving microbial activity, the infection rate of AMF, and overall maize and wheat productivity. Full article

Sub-Saharan African smallholder farmers face challenges due to limited access to commercial fertilizers, affecting food security. Exploring the benefits of intercropping is promising, but evaluating crop performance in specific agroecological contexts is crucial. This study in Vilankulo, Mozambique, conducted over two growth seasons (2018 and 2019), aimed to assess the benefits of intercropping maize (Zea mays L.) and cowpea (Vigna unguiculata L., Walp) (M+C) compared to maize (M) and cowpea (C) as sole crops. Key variables for comparison included dry matter yield (DMY), land equivalent ratio (LER), competitive ratio (CR), tissue nutrient concentration, nutrient recovery, and apparent N fixation (ANF). This study also examined the effects on cabbage (Brassica oleracea L.), cultivated as a succeeding crop, and soil properties. In 2018, maize plants were severely affected by drought and did not produce grain. This year, cowpea grain yields were 2.26 and 1.35 t ha⁻¹ when grown as sole crop or intercropped. In 2019, maize grain yield was 6.75 t ha⁻¹ when intercropped, compared to 5.52 t ha⁻¹ as a sole crop. Cowpea grain yield was lower when intercropped (1.51 vs. 2.25 t ha⁻¹). LER values exceeded 1 (1.91 and 1.53 for grain and straw in 2019), indicating improved performance in intercropping compared to sole crops. In 2019, CR was 1.96 for maize grain and 0.58 for cowpea grain, highlighting the higher competitiveness of maize over cowpea. Cowpea exhibited higher average leaf nitrogen (N) concentration (25.4 and 37.6 g kg⁻¹ in 2018 and 2019, respectively) than maize (13.0 and 23.7 g kg⁻¹), attributed to its leguminous nature with access to atmospheric N, benefiting the growth of maize in intercropping and cabbage cultivated as a succeeding crop. Cowpea also appears to have contributed to enhanced phosphorus (P) absorption, possibly due to access to sparingly soluble P forms. In 2019, ANF in M+C was 102.5 kg ha⁻¹, over 4-fold higher than in C (25.0 g kg⁻¹), suggesting maize accessed more N than could cowpea provide, possibly through association with endophytic diazotrophs commonly found in tropical grasses. Full article

Owing to the worldwide shortage of nitrogen (N) fertilizers, diazotrophic endophytes have received increasing attention as biofertilizers. In this study, we investigated the inoculation effects of a diazotrophic endophyte (Bradyrhizobium sp. AT1) on three different cultivars of sweet potato (cvs. Beniazuma, Ayamurasaki, and Kokei No. 14) under pot, container, and different field conditions. Following inoculation, the root length was increased in cvs. Beniazuma and Ayamurasaki but suppressed in cv. Kokei No. 14 in pots, filled with a mixture of vermiculite, potting soil, and pearlite. AT1 inoculation also increased shoot growth in cv. Beniazuma and tuber formation in cv. Ayamurasaki in containers filled with vermiculite, potting soil, and light-colored Andosol. In field experiments, carried out at two field sites with the three cultivars, AT1 inoculation increased the growth of cvs. Beniazuma and Ayamurasaki, but it had almost no effect on cv. Kokei No. 14. In addition to growth promotion, inoculation of micropropagated sweet potato cv. Beniazuma with AT1 led to N derived from air (Ndfa) and acetylene reduction activity (ARA) five months after inoculation. Our studies indicate that AT1 inoculation can enhance the growth of sweet potato and promote N₂ fixation. Full article

Traditional agriculture represents the most-extensive food-producing segment in the world. However, these agroecosystems are widely and closely associated with rural poverty, reflecting the dualism between the subsistence and the commodity-producing sector in the peripheric countries. Therefore, socially adapted technologies may be a reliable and helpful methodology to enhance subsistence crop production. Humic substances are natural organic biostimulants extractable as water suspensions from renewable sources such as agricultural biomass and farming residues. These easy-to-handle extracts may be mixed with plant-growth-promoting bacteria (PGPB) and used as biostimulants within a low-cost technological application in the circular economy strategy. Few investigations have been focused on the use of biostimulant practices on marginal or subsistence crops. Cassava (Manihot esculenta Crantz) and okra (Abelmoschus esculentus) are two essential foods for poor communities of rural territories in tropical and subtropical countries. The aim of this study was to evaluate the effect of the foliar application of a humic/PGPB mixed biostimulant on cassava and okra crops grown in an agricultural soil with very low natural fertility. In pot trials, the applied biostimulant improved the plant development with a 200% increase of the root weight in cassava, while the preservation of active diazotrophic bacteria was improved by 10- and 100-times in cassava and okra in the mixed formulation with humic acid. In real field systems, the plant treatment increased the yield of cassava and okra by 70% and 50%, respectively thereby allowing a simultaneous nitrogen savings with the best yield performance obtained at the lower N fertilization rate. The use of biostimulants can play a role in the transition process, helping the food security and the autonomy of impoverished farmers. Combining the elements of traditional knowledge and modern science is essential to create innovative technologies enabling the sustainable management of agroecosystems. Full article

Herbaspirillum seropedicae is an endophytic bacterium that can fix nitrogen and synthesize phytohormones, which can lead to a plant growth-promoting effect when used as a microbial inoculant. Studies focused on mechanisms of action are crucial for a better understanding of the bacteria-plant interaction and optimization of plant growth-promoting response. This work aims to understand the underlined mechanisms responsible for the early stimulatory growth effects of H. seropedicae inoculation in maize. To perform these studies, we combined transcriptomic and proteomic approaches with physiological analysis. The results obtained eight days after inoculation (d.a.i) showed increased root biomass (233 and 253%) and shoot biomass (249 and 264%), respectively, for the fresh and dry mass of maize-inoculated seedlings and increased green content and development. Omics data analysis, before a positive biostimulation phenotype (5 d.a.i.) revealed that inoculation increases N-uptake and N-assimilation machinery through differentially expressed nitrate transporters and amino acid pathways, as well carbon/nitrogen metabolism integration by the tricarboxylic acid cycle and the polyamine pathway. Additionally, phytohormone levels of root and shoot tissues increased in bacterium-inoculated-maize plants, leading to feedback regulation by the ubiquitin-proteasome system. The early biostimulatory effect of H. seropedicae partially results from hormonal modulation coupled with efficient nutrient uptake-assimilation and a boost in primary anabolic metabolism of carbon–nitrogen integrative pathways. Full article

The aim of the study was to select microbiological inoculants for a specific plant species, i.e., white lupine (Lupinus albus L.), to increase the efficiency of the diazotroph process. The research involved an in vitro assessment of interactions between the symbiotic bacteria (Bradyrhizobium sp. isolated from Nitragina and Nitroflora commercial preparations dedicated to white lupine) and selected endophytes (Pseudomonas fluorescens or Bacillus subtilis) used for seed coinoculation. In addition, selected morphological traits of plants (the weight and length of aboveground and belowground parts) were examined after the inoculation/coinoculation. The degree of root colonisation by selected endophytes used as individual inoculants and in combination with bacteria of the Bradyrhizobium genus was determined. The diazotrophic parameters were also investigated (nitrogenase activity, the number, and weight of nodules). The results showed no antagonistic interactions have been demonstrated between bacterial strains of the genus Bradyrhizobium sp. isolated from Nitragina and Nitroflora, and the endophytes Pseudomonas fluorescens or Bacillus subtilis used for the study. The applied coinoculation in vitro had a stimulating effect on the weight of the stems and roots of white lupine causing an average increase of 13% and 28%, respectively. The level of nitrogenase activity in the coinoculation variants increased from 3.5 nMC₂H₄ plant⁻¹ h⁻¹ to an average of 32.34 nMC₂H₄ plant⁻¹ h⁻¹. Full article

The sweet potato (Ipomoea batatas (L.) Lam.) is an important and widely grown crop, and the nitrogenase reductase (nifH) gene is the most widely sequenced marker gene used to identify nitrogen-fixing bacteria and archaea. There have been many examples of the isolation of the diazotrophic endophytes in sweet potatoes, and there has been no report on whether sweet potatoes and their wild ancestors harbored nifH genes. In this study, a comprehensive analysis of nifH genes has been conducted on these species by using bioinformatics and molecular biology methods. A total of 20, 19 and 17 nifH genes were identified for the first time in sweet potatoes, I. trifida and I. triloba, respectively. Based on a phylogenetic analysis, all of the nifH genes, except for g10233.t1, itf14g14040.t1 and itb14g15470.t1, were clustered into five independent clades: I, II, III, IV and V. The nifH genes clustered in the same phylogenetic branch showed a more similar distribution of conserved motifs and exons–introns than those of the other ones. All of the identified genes were further mapped on the 15 chromosomes of the sweet potato, I. trifida and I. triloba. No segmental duplication was detected in each genome of three Ipomoea species, and 0, 8 and 7 tandemly duplicated gene pairs were detected in the genome of the sweet potato, I. trifida and I. triloba, respectively. Synteny analysis between the three Ipomoea species revealed that there were 7, 7 and 8 syntenic gene pairs of nifH genes detected between the sweet potato and I. trifida, between the sweet potato and I. triloba and between I. trifida and I. triloba, respectively. All of the duplicated and syntenic nifH genes were subjected to purifying selection inside duplicated genomic elements during speciation, except for the tandemly duplicated gene pair itf11g07340.t2_itf11g07340.t3, which was subjected to positive selection. Different expression profiles were detected in the sweet potato, I. trifida and I. triloba. According to the above results, four nifH genes of the sweet potato (g950, g16683, g27094 and g33987) were selected for quantitative real-time polymerase chain reaction (qRT-PCR) analysis in two sweet potato cultivars (Eshu 15 and Long 9) under nitrogen deficiency (N0) and normal (N1) conditions. All of them were upregulated in the N1 treatment and were consistent with the analysis of the RNA-seq data. We hope that these results will provide new insights into the nifH genes in the sweet potato and its wild ancestors and will contribute to the molecular breeding of sweet potatoes in the future. Full article

Gluconacetobacter diazotrophicus has been the focus of several studies aiming to understand the mechanisms behind this endophytic diazotrophic bacterium. The present study is the first global analysis of the early transcriptional response of exponentially growing G. diazotrophicus to iron, an essential cofactor for many enzymes involved in various metabolic pathways. RNA-seq, targeted gene mutagenesis and computational motif discovery tools were used to define the G. diazotrophicusfur regulon. The data analysis showed that genes encoding functions related to iron homeostasis were significantly upregulated in response to iron limitations. Certain genes involved in secondary metabolism were overexpressed under iron-limited conditions. In contrast, it was observed that the expression of genes involved in Fe-S cluster biosynthesis, flagellar biosynthesis and type IV secretion systems were downregulated in an iron-depleted culture medium. Our results support a model that controls transcription in G. diazotrophicus by fur function. The G. diazotrophicusfur protein was able to complement an E. colifur mutant. These results provide new insights into the effects of iron on the metabolism of G. diazotrophicus, as well as demonstrate the essentiality of this micronutrient for the main characteristics of plant growth promotion by G. diazotrophicus. Full article

Sugarcane is an economically important crop that is used for the production of fuel ethanol. Diazotrophic bacteria have been isolated from sugarcane tissues, without causing visible plant anatomical changes or disease symptoms. These bacteria can be beneficial to the plant by promoting root growth and an increase in plant yield. Different rates of Biological Nitrogen Fixation (BNF) were observed in different genotypes. The aim of this work was to conduct a comprehensive molecular and physiological analysis of two model genotypes for contrasting BNF efficiency in order to unravel plant genes that are differentially regulated during a natural association with diazotrophic bacteria. A next-generation sequencing of RNA samples from the genotypes SP70-1143 (high-BNF) and Chunee (low-BNF) was performed. A differential transcriptome analysis showed that several pathways were differentially regulated among the two BNF-contrasting genotypes, including nitrogen metabolism, hormone regulation and bacteria recognition. Physiological analyses, such as nitrogenase and GS activity quantification, bacterial colonization, auxin response and root architecture evaluation, supported the transcriptome expression analyses. The differences observed between the genotypes may explain, at least in part, the differences in BNF contributions. Some of the identified genes might be involved in key regulatory processes for a beneficial association and could be further used as tools for obtaining more efficient BNF genotypes. Full article

Diazotrophic endophytic bacteria (DEB) are the key drivers of nitrogen fixation in rainfed soil ecosystems and, hence, can influence the growth and yield of crop plants. Therefore, the present work investigated the structure and composition of the DEB community at different growth stages of field-grown pearl millet plants, employing the cultivation-dependent method. Diazotrophy of the bacterial isolates was confirmed by acetylene reduction assay and amplification of the nifH gene. ERIC-PCR-based DNA fingerprinting, followed by 16S rRNA gene analysis of isolates recovered at different time intervals, demonstrated the highest bacterial diversity during early (up to 28 DAS (Days after sowing)) and late (63 DAS onwards) stages, as compared to the vegetative growth stage (28–56 DAS). Among all species, Pseudomonas aeruginosa was the most dominant endophyte. Assuming modulation of the immune response as one of the tactics for successful colonization of P. aeruginosa PM389, we studied the expression of the profile of defense genes of wheat, used as a host plant, in response to P. aeruginosa inoculation. Most of the pathogenesis-related PR genes were induced initially (at 6 h after infection (HAI)), followed by their downregulation at 12 HAI. The trend of bacterial colonization was quantified by qPCR of 16S rRNAs. The results obtained in the present study indicated an attenuated defense response in host plants towards endophytic bacteria, which is an important feature that helps endophytes establish themselves inside the endosphere of roots. Full article