Article

Major cereal crops benefit from biological nitrogen fixation when inoculated with the nitrogen-fixing bacterium Pseudomonas protegens Pf-5 X940

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

A main goal of biological nitrogen fixation (BNF) research has been to expand the nitrogenfixing ability to major cereal crops. In this work, we demonstrate the use of the efficient nitrogen-fixing rhizobacterium Pseudomonas protegens Pf-5 X940 as a chassis to engineer the transfer of nitrogen fixed by BNF to maize and wheat under non-gnotobiotic conditions. Inoculation of maize and wheat with Pf-5 X940 largely improved nitrogen content and biomass accumulation in both vegetative and reproductive tissues, and this beneficial effect was positively associated with high nitrogen fixation rates in roots. 15N isotope dilution analysis showed that maize and wheat plants obtained substantial amounts of fixed nitrogen from the atmosphere. Pf-5 X940-GFP-tagged cells were always reisolated from the maize and wheat root surface but never from the inner root tissues. Confocal laser scanning microscopy confirmed root surface colonization of Pf-5 X940-GFP in wheat plants, and microcolonies were mostly visualized at the junctions between epidermal root cells. Genetic analysis using biofilm formation-related Pseudomonas mutants confirmed the relevance of bacterial root adhesion in the increase in nitrogen content, biomass accumulation and nitrogen fixation rates in wheat roots. To our knowledge, this is the first report of robust BNF in major cereal crops.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... Also, other P. protegens strains synthesize metabolites with antifungal activity, such as 2,4-DAPG (DAPG) [23,[55][56][57], pyocyanin [56,58], pyrrolnitrin (PR) [18,56], pyoluteorin (PL) [56,57], orfamide A [41,56,59], rhizoxin A [56,60], cyanide acid (HCN) [56,61], and chitinases [41,42,62]. For example, in addition to its plant-growth-promoting abilities, P. protegens CHA0 isolated from roots of the tobacco plant can inhibit the oomycete Pythium ultimatum, Fusarium oxysporum basidiomycete, and even some herbivorous insects [26,[63][64][65][66]. Also, P. protegens Pf-5 is a strain that is closely phylogenomically related with P. protegens E1BL2 and also inhibits the growth of phytopathogenic fungi through the production of DAPG, PR, PL, HCN, and rhizoxin [67]. Although no antifungal activities have been reported with P. protegens EMM-1, this strain isolated from the maize rhizosphere of Rojo Criollo landrace inhibits the growth of Pseudomonas putida, Pseudomonas syringae, and Ralstonia solanacearum phytopathogenic bacteria and Klebsiella pneumoniae, Burkholderia cepacia complex, and Streptococcus beta-hemolytic clinical pathogenic bacteria [68,69]. ...
... A bioinoculant of Azospirillum brasiliense achieved grain yields between 6 and 9 tons/ha [72,73], while another bioinoculant containing Bacillus subtilis 160 reached 12.8 tons/ha [74]. Although it is not appropriate to compare field trials performed in different conditions, the maize productivity with a bioinoculant prepared with P. protegens Pf-5 X940 led to a substantial 115% increase in productive biomass [67]. Our field trial in this study reached an average yield of 16.6 tons/ha and 115.27% of productive biomass, which further validated the efficacy of P. protegens E1BL2 as a bioinoculant. ...
... Our field trial in this study reached an average yield of 16.6 tons/ha and 115.27% of productive biomass, which further validated the efficacy of P. protegens E1BL2 as a bioinoculant. performed in different conditions, the maize productivity with a bioinoculant prepared with P. protegens Pf-5 X940 led to a substantial 115% increase in productive biomass [67]. ...
Article
Full-text available
The relationships between plants and bacteria are essential in agroecosystems and bioinoc-ulant development. The leaf endophytic Pseudomonas protegens E1BL2 was previously isolated from giant Jala maize, which is a native Zea mays landrace of Nayarit, Mexico. Using different Mexican maize landraces, this work evaluated the strain's plant growth promotion and biocontrol against eight phytopathogenic fungi in vitro and greenhouse conditions. Also, a plant field trial was conducted on irrigated fields using the hybrid maize Supremo. The grain productivity in this assay increased compared with the control treatment. The genome analysis of P. protegens E1BL2 showed putative genes involved in metabolite synthesis that facilitated its beneficial roles in plant health and environmental adaptation (bdhA, acoR, trpE, speE, potA); siderophores (ptaA, pchC); and extracellular enzymes relevant for PGPB mechanisms (cel3, chi14), protection against oxidative stress (hscA, htpG), nitrogen metabolism (nirD, nit1, hmpA), inductors of plant-induced systemic resistance (ISR) (flaA, flaG, rffA, rfaP), fungal biocontrol (phlD, prtD, prnD, hcnA-1), pest control (vgrG-1, higB-2, aprE, pslA, ppkA), and the establishment of plant-bacteria symbiosis (pgaA, pgaB, pgaC, exbD). Our findings suggest that P. protegens E1BL2 significantly promotes maize growth and offers biocontrol benefits, which highlights its potential as a bioinoculant.
... As our understanding of the mechanisms and regulation of nitrogenase N2 fixation and the nature of how and why these diazotrophs form these symbiotic relationships with the host plants open potential opportunities to enable non-leguminous crops to benefit from BNF. As cereals are the most widely grown food crops and are the source of the largest proportion of calories consumed by the human population, there has been considerable synthetic biology research investigating and developing the tools to transfer nodule-based symbiotic nitrogen fixation to cereal crops (Fox et al. 2016). A second approach to enhancing crop N nutrition via N fixing soil microbes involves research focusing on free-living N fixing bacteria. ...
... A second approach to enhancing crop N nutrition via N fixing soil microbes involves research focusing on free-living N fixing bacteria. There are different approaches that are being explored for this and one of them is to transfer nitrogen fixation ability from non-native diazotrophs to plant host-colonizing rhizobacteria by introducing genomic islands that can encode the nitrogenase activity (Fox et al. 2016) into free-living bacteria that readily colonize plant roots. Fox et al. (2016) demonstrated that by transfer of X940 genomic island from Pseudomonas A1501 to the aerobic root-associated beneficial bacterium, Pseudomonas protegens Pf-5, followed by the inoculation of maize and wheat plants with this genetically modified bacterium, enabled the host plant's root surface (rhizoplane) and rhizosphere to be colonized by Pf-5, providing enough radiolabeled fixed nitrogen to the roots to confer higher grain and biomass yields. ...
... There are different approaches that are being explored for this and one of them is to transfer nitrogen fixation ability from non-native diazotrophs to plant host-colonizing rhizobacteria by introducing genomic islands that can encode the nitrogenase activity (Fox et al. 2016) into free-living bacteria that readily colonize plant roots. Fox et al. (2016) demonstrated that by transfer of X940 genomic island from Pseudomonas A1501 to the aerobic root-associated beneficial bacterium, Pseudomonas protegens Pf-5, followed by the inoculation of maize and wheat plants with this genetically modified bacterium, enabled the host plant's root surface (rhizoplane) and rhizosphere to be colonized by Pf-5, providing enough radiolabeled fixed nitrogen to the roots to confer higher grain and biomass yields. ...
Chapter
Full-text available
It is now clear that the root microbiome, which consists of bacteria, archaea, and fungi that colonize both the rhizosphere and the internal space of the root, is one of the most complex ecosystems in nature and is very important for root and plant health and function.In this chapter we have focused on the role of the root microbiome functional traits in improvement of nutrient acquisition and abiotic stress tolerance, with a focus on drought stress, the biocontrol of root and shoot plant diseases, and the role of root-associated microbes in both producing plant growth-promoting hormones and impacting the plant hormone metabolism and signaling pathways to alter root growth. Additionally, we have also endeavored to give the readers an introduction into the rapid advances in this field, from the metagenomic analyses that now have become relatively routine for the study of “what is there” in the root microbiome, regarding microbial composition, diversity, and abundance, to nascent studies beginning to study the plant and microbial molecular and physiological mechanisms and processes that underlie how the microbiome is assembled, and how the microbiome confers improved functional crop traits. Furthermore, given the incredible complexity of this ecosystem, we discuss the recent research involving systems biology analysis of the root microbiome, which will be critical in deciphering the trait–function links and interactions between roots and soil microbes. Finally, we also discuss the agricultural and genetic interventions that are being employed to modify the root microbiome via inoculation of the seed and plant with potentially beneficial soil microbes, as well as the studies looking at the role of plant genetic and molecular variation in impacting the composition and function of the microbiome.KeywordsRoot microbiomeRhizobacteriaFunctional traitsMetagenomicsPlant growth
... Diazotrophic bacteria, such as Azospirillum, Herbaspirillum, and Pseudomonas. sp, belong to the plant growth-promoting rhizobacteria (PGPR) due to their ability to convert N2 into ammonia, and they have been used as inoculants in practice to improve plant biomass and nitrogen (N) content [6][7][8][9]. Nitrogen fixation is quantified using the 15 N-dilution method, which compares the performance of nitrogen-fixing plants and non-fixing reference plants. This method has been used to obtain quantitative estimates of the proportion of plant N obtained from biological nitrogen fixation (BNF) [10]. ...
... This is attributed to associative, or free-living diazotrophs that fix dinitrogen sufficient for their own needs, and do not excrete N into the environment [19]. Nevertheless, the BNF activity of diazotrophic bacteria can potentially provide more N nutrition for the host plant, particularly if the diazotrophic bacteria are engineered to excrete ammonium [8,13,14,20,21]. It has been well documented that ammonium-excreting nitrogen-fixing bacteria can be obtained by means of two main strategies, including the inhibition of ammonium assimilation and interference with the mechanisms by which ammonium inhibits either nitrogenase synthesis or activity [22]. ...
... In addition, genetic modifications on P. stutzeri such as gene deletion and recombination, can be simply obtained. For example, a recent study by Fox et al. [8] described the beneficial rhizobacterium Pseudomonas protegens Pf-5, containing the nitrogen fixation island from P. stutzeri A1501 via the recombinant cosmid X940, which showed high nitrogenase activity and ammonium production [20]. ...
Article
Full-text available
Diazotroph mutants designed using metabolic engineering to excrete surplus ammonium were used to enhance nitrogen fixation and plant growth, as the levels of nitrogen fixation attained with diazotrophs are insufficient for the plant’s needs. In this study, wild-type (A1501) and engineered ammonium-excreting (1568/pVA3) strains of nitrogen-fixing Pseudomonas stutzeri strains were tested in vitro based on plant growth-promoting traits, such as phosphate solubilization ability, indole acetic acid (IAA) production and nitrogenase activities, as well as ammonium excretion as affected by mannitol-mediated osmotic stress. The maize plant growth-promoting effect of the A1501 and 1568/pVA3 strains was evaluated in pots and in the field, and the 15N-dilution technique was employed to assess the proportion of plant nitrogen derived from nitrogen fixation. The results demonstrate that the 1568/pVA3 strain displayed higher IAA production and nitrogenase activity than A1501 and released significant quantities of ammonium. After 50 days, in all of the conditions assayed, maize inoculated with 1568/pVA3 accumulated more plant biomass (3.3% on average) and fixed N (39.4% on average) than plants inoculated with A1501. In the field experiment, the grain yield of maize was enhanced by 5.6% or 5.9% due to the inoculation of seeds with 1568/pVA3 in the absence or presence of exogenous N fertilizer, respectively. Therefore, the engineered P. stutzeri strain tested in the greenhouse and field was shown to perform better than the wild-type strain with respect to maize growth parameters and biologically fixed nitrogen.
... Most studies isolate bacteria from the rhizosphere of plants subjected to a specific stress and test the ability of those bacteria to improve plant resistance to such stress [5]. Bacteria capable of growing in suboptimal environments have also been found in soils not affected by harsh conditions [14,15], and few studies have focused on the positive interaction between plants and bacteria isolated from the rhizosphere of such soils [16,17]. ...
... However, plants can also benefit from the interaction with bacteria isolated from the rhizosphere of other plant species [16,[19][20][21]. The most commonly reported mechanisms of plant growth promotion are the improvement of plant nutritional status by P solubilization [16,20] and N-fixation [19] and the bacterial synthesis of PGP compounds, such as indole acetic acid (IAA) [19,20] and aminocyclopropane-1-carboxylic acid (ACC) deaminase [19][20][21]. ...
... However, plants can also benefit from the interaction with bacteria isolated from the rhizosphere of other plant species [16,[19][20][21]. The most commonly reported mechanisms of plant growth promotion are the improvement of plant nutritional status by P solubilization [16,20] and N-fixation [19] and the bacterial synthesis of PGP compounds, such as indole acetic acid (IAA) [19,20] and aminocyclopropane-1-carboxylic acid (ACC) deaminase [19][20][21]. ...
Article
Full-text available
Plant growth promoting (PGP) bacteria are known to enhance plant growth and protect them from environmental stresses through different pathways. The rhizosphere of perennial plants, including olive, may represent a relevant reservoir of PGP bacteria. Here, seven bacterial strains isolated from olive rhizosphere have been characterized taxonomically by 16S sequencing and biochemically, to evaluate their PGP potential. Most strains were identified as Pseudomonas or Bacillus spp., while the most promising ones belonged to genera Pseudomonas and Curtobacterium. Those strains have been tested for their capacity to grow under osmotic or salinity stress and to improve the germination and early development of Triticum durum subjected or not to those stresses. The selected strains had the ability to grow under severe stress, and a positive effect has been observed in non-stressed seedlings inoculated with one of the Pseudomonas strains, which showed promising characteristics that should be further evaluated. The biochemical and taxonomical characterization of bacterial strains isolated from different niches and the evaluation of their interaction with plants under varying conditions will help to increase our knowledge on PGP microorganisms and their use in agriculture.
... 18 Several recent studies exploited genetically engineered Pseudomonas as inoculants to enhance plant productivity; for example, N-fixing P. protegens pf-5 mutants were employed for plant growth promotion in some pot experiments. [19][20][21] Pseudomonas protegens CHA0 is a well-characterized biocontrol agent capable of colonizing the rhizosphere, promoting plant growth, and controlling plant pathogens. 22,23 Complete genomic data of CHA0 revealed that it has a genome of 6.87 Mbp but no Nfixation genes, and furthermore, no nitrogenase activity was detected in this strain. ...
... 39,40 Several recent studies exploited genetically modified Pseudomonas as inoculants to enhance plant biomass production for example, N-fixing P. protegens pf-5 mutants were applied for plant growth promotion in pot experiments. [19][20][21] Results in this study showed that N-fixing CHA0-ΔretS-nif inoculation could significantly enhance the growth and production of ginger plants when compared to the uninoculated control and the wild-type strain-treated plants, which suggested that inoculating with CHA0-ΔretS-nif was more effective than inoculating with wild-type CHA0 for plant growth promotion. Moreover, these enhancements were maintained under low N-fertilizer input conditions (approximately 85% of full N supply), indicating that the capacity of the mutant strain to fix biological N can partially compensate for the need for N-fertilizers. ...
... This finding is in agreement with earlier investigations showing that reasonable use of captured N 2 -fixation ability can largely improve N uptake in maize, wheat, and tomato plants. 19,41 The novelty of our study is that proposed an improved N-fertilizer management strategy that can be applied to enhancing glasshouse vegetable production in northern China. Most previous studies applied microbial inoculants to benefit major cereal crop yields and to reduce N fertilization. ...
Article
Full-text available
BACKGROUND Excessive nitrogen (N) fertilization in glasshouse fields greatly increases N loss and fossil‐fuel energy consumption resulting in serious environmental risks. Microbial inoculants are strongly emerging as potential alternatives to agrochemicals and offer an eco‐friendly fertilization strategy to reduce our dependence on synthetic chemical fertilizers. Effects of a N‐fixing strain Pseudomonas protegens CHA0‐ΔretS‐nif on ginger plant growth, yield, and nutrient uptake, and on earthworm biomass and the microbial community were investigated in glasshouse fields in Shandong Province, northern China. RESULTS Application of CHA0‐ΔretS‐nif could promote ginger plant development, and significantly increased rhizome yields, by 12.93% and 7.09%, respectively, when compared to uninoculated plants and plants treated with the wild‐type bacterial strain. Inoculation of CHA0‐ΔretS‐nif had little impact on plant phosphorus (P) acquisition, whereas it was associated with enhanced N and potassium (K) acquisition by ginger plants. Moreover, inoculation of CHA0‐ΔretS‐nif had positive effects on the bacteria population size and the number of earthworms in the rhizosphere. Similar enhanced performances were also found in CHA0‐ΔretS‐nif‐inoculated ginger plants even when the N‐fertilizer application rate was reduced by 15%. A chemical N input of 573.8 kg ha⁻¹ with a ginger rhizome yield of 1.31 × 10⁵ kg ha⁻¹ was feasible. CONCLUSIONS The combined application of CHA0‐ΔretS‐nif and a reduced level of N‐fertilizers can be employed in glasshouse ginger production for the purpose of achieving high yields while at the same time reducing the inorganic‐N pollution from traditional farming practices. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
... Inoculation of plants with some of these genetically modified A. vinelandii strains substituted for at least some synthetic N-fertilizer for plant growth (Ambrosio et al. 2017;Bageshwar et al. 2017;Mus et al. 2022). Similar results were obtained using other bacterial species similarly modified (Fox et al. 2016;Santos et al. 2017;Schnabel and Sattely 2021a). Under laboratory growth conditions, NH 4 + excretion is considerably more pronounced in mutant strains and/ or under conditions when cell growth and biomass production slow down (Bali et al. 1992;Ortiz-Marquez et al. 2012;Ortiz-Marquez et al. 2014;Ambrosio et al. 2017;Ambrosio and Curatti 2021). ...
... Unregulated expression of the nif genes in recombinant P. protegens Pf-5 resulted in NH 4 + release and N-fertilization of plants. However, it also resulted in a fitness burden on the cells, which led to a rapid decline of the bacterial population in the soil (Setten et al. 2013;Fox et al. 2016). ...
Article
Full-text available
Non-symbiotic N2-fixation would greatly increase the versatility of N-biofertilizers for sustainable agriculture. Genetic modification of diazotrophic bacteria has successfully enhanced NH4⁺ release. In this study, we compared the competitive fitness of A. vinelandii mutant strains, which allowed us to analyze the burden of NH4⁺ release under a broad dynamic range. Long-term competition assays under regular culture conditions confirmed a large burden for NH4⁺ release, exclusion by the wt strain, phenotypic instability, and loss of the ability to release NH4⁺. In contrast, co-inoculation in mild autoclaved soil showed a much longer co-existence with the wt strain and a stable NH4⁺ release phenotype. All genetically modified strains increased the N content and changed its chemical speciation in the soil. This study contributes one step forward towards bridging a knowledge gap between molecular biology laboratory research and the incorporation of N from the air into the soil in a molecular species suitable for plant nutrition, a crucial requirement for developing improved bacterial inoculants for economic and environmentally sustainable agriculture. Key points • Genetic engineering for NH4⁺ excretion imposes a fitness burden on the culture medium • Large phenotypic instability for NH4⁺-excreting bacteria in culture medium • Lower fitness burden and phenotypic instability for NH4⁺-excreting bacteria in soil
... Being a leguminous crop, chickpea xes the atmospheric nitrogen by association with Rhizobium species 30 which differentiates it from other cereal crops 79 . It stores the xed nitrogen in the nodules present in its root system and converts it into ammonia that can be used by the plant 22,84 . It was estimated that about 70 kg of nitrogen per hectare is xed annually by chickpea 78 , which helps in providing nitrogen not only to the host but also to the subsequent crops grown 45 thereby, helping the farmers in reducing the cost of production. ...
Preprint
Full-text available
Chickpeas, a widely cultivated legume, actively fix atmospheric nitrogen in root nodules through a symbiotic relationship with rhizobia bacteria. A recombinant inbred line (RIL) population, progressing from F2 to F7 generations, was developed in a short-period of 18 months using the Rapid Generation Advancement (RGA) protocol. The F 7 RILs were evaluated during the 2020-21 and 2021-22 crop seasons under typical field conditions to quantify the effects of nodulation on seed yield (SY) and its associated traits. The analysis of variance revealed a highly significant difference (P < 0.01) among genotypes for seed yield and other agronomic traits, with no significant seasonal effect. In the pooled analysis, nodulating genotypes (NG) exhibited a substantial increase (P < 0.01) in SY (62.55%), 100-seed weight (SW100; 12.21%), harvest index (HI; 6.40%), number of pods per plant (NPPP; 39.55%), and number of seeds per plant (NSPP; 44.37%) compared to non-nodulating genotypes (NNG). Both NG and NNG exhibited a significant (P < 0.01) positive correlation between SY and NPPP (r=0.64 and 0.63), NSPP (r=0.66 and 0.61), HI (r=0.27), and number of primary branches per plant (PBr) (r=0.31), respectively. The top-performing genotypes for yield and related traits were predominantly nodulating. Genotype-trait bi-plot analysis identified nine nodulating genotypes as the most adaptable across the two seasons—six for SY, plant height, SW100, and three for days to first flowering and maturity. These findings underscore the critical role of nodulation in maximizing chickpea yields and the significant yield penalties associated with non-nodulation. To boost chickpea production, future breeding efforts should focus on developing genotypes with high compatibility with rhizobium strains.
... Furthermore, members of Pseudomonas have been related to detoxification activities in other curculionide beetles and to insecticide resistance in other insects, such as S. frugiperda larvae, due to their high efficacy for the biodegradation of toxic compounds (Ceja-Navarro et al. 2015;Almeida et al. 2017). Many Pseudomonas species also are nitrogen fixers under microaerophilic conditions (Desnoues et al. 2003;Fox et al. 2016;Sanow et al. 2023), supporting the idea that S. acupunctatus' gut bacterial communities provides nitrogen to its host. However, all these ideas require functional testing. ...
Article
Full-text available
The agave weevil, Scyphophorus acupunctatus, is a pest of agave. Its larvae cause damage to agaves by boring holes in the plant. Boring requires that the insect consume the constituents of its host plant, which contains sugars and many recalcitrant polymers. It has been hypothesized for many years that the gut bacterial communities of S. acupunctatus play a role in its ability to metabolize agave components. However, studies exploring this insect's gut bacterial communities have yet to be performed. In this work, we used a 16S rRNA gene-based metabarcoding approach to characterize the gut bacterial communities of field-collected agave weevils from different localities in Mexico. We found that external factors, including host plants, have important effects on the structure of the gut bacterial communities of S. acupunctatus. Despite this variability, we found a discrete core bacterial community mainly composed of the genera Prevotella, Pectinatus, Liquorilactobacillus, Secundilactobacillus, Paucilactobacillus, and Pseudomonas. These genera may be necessary for S. acupunctatus as metabolic helpers and/or gatekeepers. Additional studies are needed to fully assess the functionality of the gut bacterial community of this species in terms of its metabolic contribution, which may help to decipher their potential ecological implications. The information we provided here is the first step for guiding further questions.
... Despite its abundance in the atmosphere as nitrogen gas (N 2 ), it cannot be directly assimilated by plants [43]. Previous studies have confirmed that certain strains belonging to the Pseudomonas genus, such as P. koreensis CY4, P. entomophila CN11, P. protegens Pf-5 X940, and P. stutzeri A1501, possess nitrogen-fixing capabilities, which can enhance plant nutrient uptake and growth [44][45][46]. The growth of Pseudomonas sp. ...
Article
Full-text available
Growth-promoting endophytic bacteria possess substantial potential for sustainable agriculture. Here, we isolated an endophytic bacterium, Pseudomonas sp. En3, from the leaf endosphere of Populus tomentosa and demonstrated its significant growth-promoting effects on both poplar and tomato seedlings. The phosphorus solubilization and nitrogen fixation abilities of strain En3 were confirmed via growth experiments on NBRIP and Ashby media, respectively. Salkowski staining and HPLC-MS/MS confirmed that En3 generated indole-3-acetic acid (IAA). The infiltration of En3 into leaf tissues of multiple plants did not induce discernible disease symptoms, and a successful replication of En3 was observed in both poplar and tobacco leaves. Combining Illumina and Nanopore sequencing data, we elucidated that En3 possesses a circular chromosome of 5.35 Mb, exhibiting an average G + C content of 60.45%. The multi-locus sequence analysis (MLSA) and genome average nucleotide identity (ANI) supported that En3 is a novel species of Pseudomonas and constitutes a distinct phylogenetic branch with P. rhizosphaerae and P. coleopterorum. En3 genome annotation analysis revealed the presence of genes associated with nitrogen fixation, phosphate solubilization, sulfur metabolism, siderophore biosynthesis, synthesis of IAA, and ethylene and salicylic acid modulation. The findings suggest that Pseudomonas sp. En3 exhibits significant potential as a biofertilizer for crop and tree cultivation.
... Furthermore, there is strong evidence that the naturally occurring plant microbiome has a significant role in developing and controlling plant diseases (Bulgarelli et al., 2013). Fox et al. (2016) expanded the nitrogen-fixing ability to important cereal crops using rhizobacteria. The nitrogen-fixing rhizobacterium Pseudomonas protegens Pf-5 X940 was successfully used as a chassis to design the transfer of nitrogen fixed by BNF to maize and wheat. ...
... For example, by ingesting Pseudomonas protegens Pf-5 X940, 15 nitrogen isotope dilution analyses showed that corn and wheat produced significant amounts of fixed nitrogen by this organism. In addition, the colony in wheat plants was formed on the root surface of Pf-5 X940 expressing GFP [88]. ...
Article
Full-text available
Ammonia is an important chemical that is widely used in fertilizer applications as well as in the steel, chemical, textile, and pharmaceutical industries, which has attracted attention as a potential fuel. Thus, approaches to achieve sustainable ammonia production have attracted considerable attention. In particular, biological approaches are important for achieving a sustainable society because they can produce ammonia under mild conditions with minimal environmental impact compared with chemical methods. For example, nitrogen fixation by nitrogenase in heterogeneous hosts and ammonia production from food waste using microorganisms have been developed. In addition, crop production using nitrogen-fixing bacteria has been considered as a potential approach to achieving a sustainable ammonia economy. This review describes previous research on biological ammonia production and provides insights into achieving a sustainable society.
... However, the traditional rhizobium nitrogen fixation can only be applied in legume plants. Currently, in order to apply BNF technology to more non-leguminous plants, associated nitrogen-fixing bacteria (NFB) have been isolated and developed as an efficient biofertilizer agent in agricultural production [6,7]. The associated NFB strain Kosakonia radicincitans GXGL-4A was isolated from maize roots. ...
Article
Full-text available
Nitrogen is an important factor affecting crop yield, but excessive use of chemical nitrogen fertilizer has caused decline in nitrogen utilization and soil and water pollution. Reducing the utilization of chemical nitrogen fertilizers by biological nitrogen fixation (BNF) is feasible for green production of crops. However, there are few reports on how to have more ammonium produced by nitrogen-fixing bacteria (NFB) flow outside the cell. In the present study, the amtB gene encoding an ammonium transporter (AmtB) in the genome of NFB strain Kosakonia radicincitans GXGL-4A was deleted and the △amtB mutant was characterized. The results showed that deletion of the amtB gene had no influence on the growth of bacterial cells. The extracellular ammonium nitrogen (NH4⁺) content of the △amtB mutant under nitrogen-free culture conditions was significantly higher than that of the wild-type strain GXGL-4A (WT-GXGL-4A), suggesting disruption of NH4⁺ transport. Meanwhile, the plant growth-promoting effect in cucumber seedlings was visualized after fertilization using cells of the △amtB mutant. NFB fertilization continuously increased the cucumber rhizosphere soil pH. The nitrate nitrogen (NO3⁻) content in soil in the △amtB treatment group was significantly higher than that in the WT-GXGL-4A treatment group in the short term but there was no difference in soil NH4⁺ contents between groups. Soil enzymatic activities varied during a 45-day assessment period, indicating that △amtB fertilization influenced soil nitrogen cycling in the cucumber rhizosphere. The results will provide a solid foundation for developing the NFB GXGL-4A into an efficient biofertilizer agent.
... Nitrogen transformation genes-nitrogen regulation response regulator GlnG, nitrogen PTS system EIIA component, nitrogen regulatory protein PII, GlnK, and nitrogen regulation protein NtrB, that were detected in our resolved MAG-Pseudomonas (Table 2, Supplementary Table S2), has also been previously reported in other Pseudomonas spp [57][58][59]. All the nitrogen transformation gene functions that were detected in our MAG-Pseudomonas can be essential in helping to fulfill the plant host's need for nitrogen, especially in N-depleted soils [60][61][62][63]. NtrB also plays a role in nitrogen metabolism and can regulate the nitrogen dynamics under nitrogen-deprived and enriched environments [64]. ...
Article
Full-text available
Background Climate change will result in more frequent droughts that can impact soil-inhabiting microbiomes (rhizobiomes) in the agriculturally vital North American perennial grasslands. Rhizobiomes have contributed to enhancing drought resilience and stress resistance properties in plant hosts. In the predicted events of more future droughts, how the changing rhizobiome under environmental stress can impact the plant host resilience needs to be deciphered. There is also an urgent need to identify and recover candidate microorganisms along with their functions, involved in enhancing plant resilience, enabling the successful development of synthetic communities. Results In this study, we used the combination of cultivation and high-resolution genomic sequencing of bacterial communities recovered from the rhizosphere of a tallgrass prairie foundation grass, Andropogon gerardii . We cultivated the plant host-associated microbes under artificial drought-induced conditions and identified the microbe(s) that might play a significant role in the rhizobiome of Andropogon gerardii under drought conditions. Phylogenetic analysis of the non-redundant metagenome-assembled genomes (MAGs) identified a bacterial genome of interest – MAG- Pseudomonas . Further metabolic pathway and pangenome analyses recovered genes and pathways related to stress responses including ACC deaminase; nitrogen transformation including assimilatory nitrate reductase in MAG- Pseudomonas, which might be associated with enhanced drought tolerance and growth for Andropogon gerardii. Conclusions Our data indicated that the metagenome-assembled MAG -Pseudomonas has the functional potential to contribute to the plant host’s growth during stressful conditions. Our study also suggested the nitrogen transformation potential of MAG-Pseudomonas that could impact Andropogon gerardii growth in a positive way. The cultivation of MAG- Pseudomonas sets the foundation to construct a successful synthetic community for Andropogon gerardii . To conclude, stress resilience mediated through genes ACC deaminase, nitrogen transformation potential through assimilatory nitrate reductase in MAG- Pseudomonas could place this microorganism as an important candidate of the rhizobiome aiding the plant host resilience under environmental stress. This study, therefore, provided insights into the MAG- Pseudomonas and its potential to optimize plant productivity under ever-changing climatic patterns, especially in frequent drought conditions.
... Free-living nitrogen-fixing prokaryotes contribute to nitrogen requirement of wheat (Dellagi et al., 2020), up to 76 and 32% for shoots and roots, respectively (Majeed et al., 2015). In addition, higher yields were observed for T. aestivum inoculated with engineered strains able to fix nitrogen constitutively (Fox et al., 2016). The diazotroph community varies in size and activity with plant species (Perin et al., 2006;Mao et al., 2013;Bouffaud et al., 2016) and cultivars of T. aestivum, with a higher number of rhizosphere diazotrophs for Xiaoyan than for other T. aestivum cultivars (Mahoney et al., 2017). ...
... Engineering BNF in plants is a major longstanding goal of plant biotechnology. Earlier strategies to reduce global dependence on N fertilizers included the use of diazotrophic bacteria to colonize the rhizosphere [31][32][33][34][35][36] . The introduction of bacterial nif genes into cereals to increase crop productivity offers a more direct approach in which the plants fix their own N. Multigene transfer technology allows the optimization of different combinations of heterologous genes from diverse origins, as well regulatory elements such as promoters and targeting peptides. ...
Article
Full-text available
Engineering cereals to express functional nitrogenase is a long-term goal of plant biotechnology and would permit partial or total replacement of synthetic N fertilizers by metabolization of atmospheric N 2 . Developing this technology is hindered by the genetic and biochemical complexity of nitrogenase biosynthesis. Nitrogenase and many of the accessory proteins involved in its assembly and function are O 2 sensitive and only sparingly soluble in non-native hosts. We generated transgenic rice plants expressing the nitrogenase structural component, Fe protein (NifH), which carries a [4Fe-4S] cluster in its active form. NifH from Hydrogenobacter thermophilus was targeted to mitochondria together with the putative peptidyl prolyl cis‐trans isomerase NifM from Azotobacter vinelandii to assist in NifH polypeptide folding. The isolated NifH was partially active in electron transfer to the MoFe protein nitrogenase component (NifDK) and in the biosynthesis of the nitrogenase iron-molybdenum cofactor (FeMo-co), two fundamental roles for NifH in N 2 fixation. NifH functionality was, however, limited by poor [4Fe-4S] cluster occupancy, highlighting the importance of in vivo [Fe-S] cluster insertion and stability to achieve biological N 2 fixation in planta . Nevertheless, the expression and activity of a nitrogenase component in rice plants represents the first major step to engineer functional nitrogenase in cereal crops.
... Genus Pseudomonas is an important member of PGPRs with phosphorus-and silicate-solubilizing capacity, auxin production and nitrogen fixation functions [45][46][47][48]. Among the strains isolated in this study, the strains in this genus were P8 with good phosphorous solubilizing capability, P32 with good auxin production capability, P10, P11, P21 and P26 with silicate solubilizing capability, and P22 with good nitrogen fixation capability. ...
Article
Full-text available
Microbial metabolites in rhizosphere soil are important to plant growth. In this study, microbial diversity in blueberry plant rhizosphere soil was characterized using high-throughput amplicon sequencing technology. There were 11 bacterial phyla and three fungal phyla dominating in the soil. In addition, inorganic-phosphate-solubilizing bacteria (iPSB) in the rhizosphere soil were isolated and evaluated by molybdenum-antimony anti-coloration method. Their silicate solubilizing, auxin production, and nitrogen fixation capabilities were also determined. Eighteen iPSB in the rhizosphere soil strains were isolated and identified as Buttiauxella, Paraburkholderia and Pseudomonas. The higher phosphorus-solubilizing capacity and auxin production in blueberry rhizosphere belonged to genus Buttiauxella sp. The strains belong to genus Paraburkholderia had the same function of dissolving both phosphorus and producing auxin, as well as silicate and nitrogen fixation. The blueberry seeds incubated with the strains had higher germination rates. The results of this study could be helpful in developing the plant growth-promoting rhizobacteria (PGPR) method for enhancing soil nutrients to blueberry plant.
... Among these activities, those which assist bacteria in the uptake of the macro and micronutrients N, P and Fe are not unexpected as the mangrove habitat is relatively nutrient poor. Nitrogen is equally important to plants, as a vital component of chlorophyll as well as amino and nucleic acids and it has been previously reported that the content of nitrogen in nonleguminous plants can be increased by the association with species of Bacillus (Thatoi et al., 2020) and Pseudomonas (Fox et al., 2016). Hydrolytic enzymes produced by the nitrogen fixing Bacillus sp. and Pseudomonas sp. ...
Article
Saline soils resulting from anthropogenic activity and climate change present a challenge to future food security. Towards addressing this, we isolated and characterized halotolerant bacteria from a Malaysian mangrove forest, and explored their effect on morpho-physiological and biochemical parameters of banana plantlets under salt stress. A total of 88 rhizobacterial and 16 endophytic bacterial isolates collected from the roots and rhizosphere of Rhizophora apiculata, Avicennia alba and Sonneratia alba, were found to tolerate up to 400 mM of sea salt. Based on best performance in multiple plant growth traits, three rhizobacterial strains RB1, RB3 and RB4 and three endophytic bacterial strains EB1, EB2 and EB3 were used for further analysis. The rhizobacterial strains were identified as Bacillus sp. and endophytic bacteria as Pseudomonas sp. based on 16 S rRNA gene sequence. SEM observation confirmed colonization of each strain on banana plantlet roots. When colonized plantlets were subjected to 90 mM salt and compared to uninoculated (control) and mock inoculated plants, improved plant growth was observed with each of the strains, especially with bacterial strains EB3 and RB3. Biochemical analysis of plantlets revealed that root colonization with EB3 and RB3 enhanced levels of plant chlorophyll (> 5-fold), carotenoid (> 2.85-fold) and proline (2.6-fold and 2.3-fold), while plantlets also showed reduced MDA content (0.45-fold and 0.51-fold), significantly reduced generation of ROS (0.23-fold and 0.47-fold) and lower levels of electrolyte leakage (0.77 and 0.51-fold). Antioxidant enzymes also showed enhanced activity with EB3 and RB3. Our results indicate that these halotolerant Bacillus and Pseudomonas strains from the mangrove have multifunctional plant growth promoting activity and can reduce salt stress in bananas. This data provides a reference for exploring halotolerant microbes from hypersaline environments to overcome salt stress in plants.
... It is noteworthy to mention the genetic modification of the Pf-5 strain of P. protegans with a genomic island (X940) encoding nitrogen fixation. Inoculation of maize and wheat with Pf-5 X940 largely improved nitrogen content and biomass accumulation in vegetative and reproductive tissues, and this beneficial effect was positively associated with high nitrogen fixation rates in roots (Fox et al. 2016). ...
Preprint
Full-text available
The results of our previous studies showed that 8 examined bacterial strains originating from the apple phyllosphere or soil environment showed both antagonistic activity towards E. amylovora and the protective ability of apple flowers and terminal shoots against fire blight. Five of the strains represented bacterial species in which this activity was observed for the first time (L16 Pseudomonas vancouverensis , 3M Pseudomonas chlororaphis subsp. aureofaciens , 43M Enterobacter ludwigii , 59M Pseudomonas protegens and 35M Pseudomonas congelans ) (Mikiciński et al. 2020). We now present an attempt to explain the potential mechanisms related to the biocontrol capacity of the first four of the above mentioned strains. The studied strains were very effective in protecting pear fruitlet slices against fire blight. The disease severity after preventive treatment of the slices with the tested strains ranged from 0.0 to 0.6, while that of the untreated slices was 4.0 (rating scale: 0.0–4.0). Among the mechanisms studied, the L16 strain, characterized by the highest antagonistic activity, showed the ability to produce siderophores, biosurfactant, hydrogen cyanide (HCN), salicylic acid (SA) and indole-3-acetic acid (IAA). The L16 strain also degraded nicotinic acid. The 43M strain showed the lowest activity, producing only IAA and degrading nicotinic acid. A study of the detection of genes encoding antibiotics characteristic of pseudomonads showed the presence of prnD and gacA in the 3M strain and phlD , pltB , pltC and gacA in 59M. However, none of the genes sought were detected in the L16 strain.
... The greenness index of wild-type and mutant plants was analyzed using CCM-200-Opti-Sciences by measuring the light transmittance between red (650 nm) and near-infrared (940 nm) chlorophyll absorption ranges (Torres-Dorante et al., 2016). Total nitrogen content in leaves was measured by the Kjeldahl method, as previously (Fox et al., 2016). ...
Article
Full-text available
The multifunctional channel NOD26, identified and extensively studied (both biochemically and biophysically) in soybean, is a major protein component of the symbiosome membrane. The water and ammonia transport activities of NOD26 are thought to be important for nodule development, osmotic balance, and ammonia efflux from the symbiosome. However, the widely accepted relevance of NOD26 in nitrogen-fixing symbiosis has never been explored in planta. Recently, we have reported the emergence of NOD26 in the nitrogen-fixing clade of angiosperms via tandem duplication. Here, we characterized the two copies of NOD26 from Medicago truncatula (Medtr8g087710 and Medtr8g087720) in their transport abilities, and at gene expression and genetic levels. Similar to their homologous soybean gene, MtNOD26 genes encode water and ammonia transport activities in heterologous expression systems. By using multiple transcriptional studies (RT-qPCR, transcriptional fusion and RNA-Seq analyses), we found that the expression of MtNOD26 copies is restricted to the nodule and gradually increases from the bacteria-free meristematic region to the nitrogen-fixation zone. Under nitrogen-limiting soil conditions, the homozygous insertional mutant lines of these two MtNOD26 genes had the same aberrant nodulation phenotype and chlorosis. Similar to uninoculated wild-type plants, inoculated mutants were unable to grow in minimal medium without a nitrogen source. Using the CRISPR/Cas9 system, we have edited the orthologous NOD26 genes in Medicago sativa (alfalfa), generating plants with aberrant nodules, chlorosis and impaired grow under nitrogen-limiting conditions. Collectively, our findings suggest functional equivalence between NOD26 copies and underline a crucial role of NOD26 in symbiotic nitrogen fixation.
... This strain also secreted NH 4 + into the adjoining medium. Subsequent greenhouse experiments showed improved yields in maize and wheat inoculated with this engineered strain, and using 15 N isotope dilution analysis, demonstrated N 2 fixation in roots (Fox et al., 2016). A further refinement of P. protegens Pf-5 achieved high levels of inducible nitrogenase activity with reduced O 2 and NH 4 sensitivity using the nif clusters regulated by mutated nifA from P. stutzeri and A. vinelandii (Ryu et al., 2020). ...
Article
Full-text available
The demand for nitrogen (N) for crop production increased rapidly from the middle of the twentieth century and is predicted to at least double by 2050 to satisfy the on-going improvements in productivity of major food crops such as wheat, rice and maize that underpin the staple diet of most of the world’s population. The increased demand will need to be fulfilled by the two main sources of N supply – biological nitrogen (gas) (N2) fixation (BNF) and fertilizer N supplied through the Haber-Bosch processes. BNF provides many functional benefits for agroecosystems. It is a vital mechanism for replenishing the reservoirs of soil organic N and improving the availability of soil N to support crop growth while also assisting in efforts to lower negative environmental externalities than fertilizer N. In cereal-based cropping systems, legumes in symbiosis with rhizobia contribute the largest BNF input; however, diazotrophs involved in non-symbiotic associations with plants or present as free-living N2-fixers are ubiquitous and also provide an additional source of fixed N. This review presents the current knowledge of BNF by free-living, non-symbiotic and symbiotic diazotrophs in the global N cycle, examines global and regional estimates of contributions of BNF, and discusses possible strategies to enhance BNF for the prospective benefit of cereal N nutrition. We conclude by considering the challenges of introducing in planta BNF into cereals and reflect on the potential for BNF in both conventional and alternative crop management systems to encourage the ecological intensification of cereal and legume production.
... Free-living nitrogen-fixing prokaryotes contribute to nitrogen requirement of wheat (Dellagi et al., 2020), up to 76 and 32% for shoots and roots, respectively (Majeed et al., 2015). In addition, higher yields were observed for T. aestivum inoculated with engineered strains able to fix nitrogen constitutively (Fox et al., 2016). The diazotroph community varies in size and activity with plant species (Perin et al., 2006;Mao et al., 2013;Bouffaud et al., 2016) and cultivars of T. aestivum, with a higher number of rhizosphere diazotrophs for Xiaoyan than for other T. aestivum cultivars (Mahoney et al., 2017). ...
Article
Full-text available
Wheat, one of the major crops in the world, has had a complex history that includes genomic hybridizations between Triticum and Aegilops species and several domestication events, which resulted in various wild and domesticated species (especially Triticum aestivum and Triticum durum ), many of them still existing today. The large body of information available on wheat-microbe interactions, however, was mostly obtained without considering the importance of wheat evolutionary history and its consequences for wheat microbial ecology. This review addresses our current understanding of the microbiome of wheat root and rhizosphere in light of the information available on pre- and post-domestication wheat history, including differences between wild and domesticated wheats, ancient and modern types of cultivars as well as individual cultivars within a given wheat species. This analysis highlighted two major trends. First, most data deal with the taxonomic diversity rather than the microbial functioning of root-associated wheat microbiota, with so far a bias toward bacteria and mycorrhizal fungi that will progressively attenuate thanks to the inclusion of markers encompassing other micro-eukaryotes and archaea. Second, the comparison of wheat genotypes has mostly focused on the comparison of T. aestivum cultivars, sometimes with little consideration for their particular genetic and physiological traits. It is expected that the development of current sequencing technologies will enable to revisit the diversity of the wheat microbiome. This will provide a renewed opportunity to better understand the significance of wheat evolutionary history, and also to obtain the baseline information needed to develop microbiome-based breeding strategies for sustainable wheat farming.
... PGPB promote plant growth directly usually by facilitating resource acquisition or modulating plant hormone levels, or indirectly by decreasing the inhibitory effects of various biotic and abiotic stresses (Glick, 1995). For instance, the diazotrophic bacteria are able to x nitrogen and provide it to plants (Fox et al., 2016;Geddes et al., 2015;Ren et al., 2019). Some soil bacteria were described to promote solubilization and bioavailability of inorganic or organic phosphorus by synthesizing low molecular weight organic acids or various phosphatases (Zeng et al., 2017;Rodrıǵuez&Fraga, 1999;Rodriguez et al., 2004). ...
Preprint
Full-text available
A novel diazotrophic bacterium, designated CCTCC AB 2021101 T , was isolated from fresh roots of kiwifruit. Cells of strain CCTCC AB 2021101 T were Gram-negative, aerobic and rod-shaped, with motility provided by peritrichous flagella. The 16S rRNA analysis showed that strain CCTCC AB 2021101 T belongs to the genus Azospirillum and is closely related to Azospirillum melinis (98.32%), Azospirillum oryzae (97.73%), Azospirillum lipoferum (96.98%), Azospirillum humicireducens (96.49%) and Azospirillum largimobile (96.01%) and lower sequence similarity (<96.0 %) to all other species of the genus Azospirillum . Strain CCTCC AB 2021101 T was able to grow well at 35–40℃ and pH 6.0–7.0, and tolerated up to 3.0 % (w/v) NaCl. The major saturated fatty acids are C 14:0 , C 16:0 and C 18:0 . C 18:1 ω 7c and C 16:0 3-OH were the major unsaturated and hydroxylated fatty acid. The G+C content was 67.8 mol%. Strain CCTCC AB 2021101 T gave positive amplification for dinitrogen reductase ( nifH gene). Highest nifH gene sequence similarities were obtained with Azospirillum brasilense AWB14 T (95.9%), Azospirillum zeae Gr24 T (95.56%), Azospirillum picis DSM 19922 T (96.79%), Azospirillum lipoferum B22 T (94.88%) and Azospirillum oryzae COC8 T (94.88%). The activity of the nitrogenase of the strain was further confirmed by acetylene-reduction assay, which was recorded as 81 nmol ethylene h ⁻¹ . Based on these data, strain CCTCC AB 2021101 T is considered to represent a novel endophytic diazotrophs species in the genus Azospirillum , for which the name Azospirillum actinidiae sp. nov. is proposed. The type strain is CCTCC AB 2021101 T .
... There is evidence that nif clusters transfer laterally between species (Kechris, Lin, Bickel, & Glazer, 2006;Yan et al., 2008); still, the transfer of the nif cluster possesses many confront. It has also been reported that the nif gene can be acquired by the nonnitrogen-fixing heterologous host through horizontal gene transfer and confers the ability to fix nitrogen (Dixon & Postgate, 1972;Fox et al., 2016;Setten et al., 2013). The first successful transfer of nif genes was reported in gram-negative bacterium K. pneumoniae strain M5a1 by both transduction (Streicher, Gurney, & Valentine, 1971) as well as conjugation approaches (Dixon & Postgate, 1972). ...
Article
Biofertilizers are the substances containing variety of microbes having the capacity to enhance plant nutrient uptake by colonizing the rhizosphere and make the nutrients easily accessible to plant root hairs. Biofertilizers are well known for their cost effectiveness, environment-friendly nature, and composition. These are effective alternatives to the hazardous synthetic fertilizers. This chapter covers various types of microbial biofertilizers pronouncing symbiotic and free-living nitrogen-fixers, phosphorus-solubilizer and mobilizers, their formulations, applications of few commercially available biofertilizers toward sustainable agriculture, and recent approaches to develop next-generation biofertilizers.
... There is evidence that nif clusters transfer laterally between species (Kechris, Lin, Bickel, & Glazer, 2006;Yan et al., 2008); still, the transfer of the nif cluster possesses many confront. It has also been reported that the nif gene can be acquired by the nonnitrogen-fixing heterologous host through horizontal gene transfer and confers the ability to fix nitrogen (Dixon & Postgate, 1972;Fox et al., 2016;Setten et al., 2013). The first successful transfer of nif genes was reported in gram-negative bacterium K. pneumoniae strain M5a1 by both transduction (Streicher, Gurney, & Valentine, 1971) as well as conjugation approaches (Dixon & Postgate, 1972). ...
Chapter
Full-text available
Purple nonsulfur bacteria (PNSB), a diverse group of photosynthetic microorganisms, inhabit in a wide variety of aquatic habitats where sunlight penetrates. These microorganisms show pigmentation ranging from deep red to brown and propagate under anoxic conditions. Depending on the presence of nutrients, oxygen concentration, and light intensity, they can shift their modes of metabolism between photoautotrophy, photoheterotrophy, and chemoheterotrophy. Most promisingly, many of the PNSB are known to thrive in the presence of various toxicants such as heavy metals and thus play an important role in the remediation of contaminated sites. PNSB are one of the most potential candidates for the production of biohydrogen, taking us a step further towards the era of “green technology.” They also serve as a potential source of single-cell proteins, enzymes, biofertilizers, carotenoids, plant growth-producing hormones, and several precursor molecules. This chapter discusses the versatile and important role of PNSB in biotechnology.
... Diazotrophic Paenibacillus beijingensis BJ-18 provided nitrogen for wheat, maize and cucumber plants and promoted plant growth, nitrogen uptake and metabolism . A recombinant nitrogen-xing Pseudomonas protegens Pf-5 X940 was constructed by introducing the nif genes of Pseudomonas stutzeri A1501 via the X940 cosmid to the bene cial rhizobacterium Pseudomonas protegens Pf-5, and inoculation of Arabidopsis, alfalfa, tall fescue and maize with Pf-5 X940 increased the ammonium concentration in soil and plant productivity under nitrogen-de cient conditions (Fox et al. 2016;Setten et al. 2013). Inoculation with Azospirillum brasilense Ab-V5 cells enriched with exopolysaccharides and polyhydroxybutyrate enhances the productivity of maize under low N fertilizer input (Oliveira et al. 2017) Paenibacillus polymyxa WLY78 is a nitrogen-xing bacterium containing a compact nif gene cluster consisting of 9 genes (nifBHDKENXhesAnifV) (Wang et al. 2013Xie et al. 2014). ...
Preprint
Full-text available
Aims To study nitrogen contribution to cucumber derived from nitrogen fixation of Paenibacillus polymyxa WLY78. Methods The nif gene cluster deletion mutant (ΔnifB-V) of P. polymyxa WLY78 is constructed by a homologous recombination method. The GFP-labeled ΔnifB-V mutant was used to inoculate cucumber and to study colonization by confocal laser scanning microscope. The effects of plant-growth promotion were investigated by greenhouse experiments. The nitrogen fixation contribution was estimated by ¹⁵N isotope dilution experiments. Results Deletion of nif gene cluster of P. polymyxa WLY78 resulted in complete loss of nitrogenase activity. Observation by laser confocal microscopy revealed ΔnifB-V mutant can effectively colonize cucumber root, stem and leaf tissues, like wild-type P. polymyxa WLY78. Greenhouse experiments showed that inoculation with P. polymyxa WLY78 can significantly enhance the lengths and dry weights of cucumber roots and shoots, but inoculation with ΔnifB-V mutant can not. ¹⁵N isotope dilution experiments showed that cucumber plants derive 25.93% nitrogen from nitrogen fixation performed by P. polymyxa WLY78, but the ΔnifB-V mutant nearly can not provide nitrogen for plant growth. Conclusions This present study demonstrates that nitrogen fixation performed by P. polymyxa WLY78 is responsible for cucumber growth promotion.
... There is growing interest in the development of effective associative N fixation for cereal crops, especially maize, rice, and wheat (Mus et al., 2016;Bloch et al., 2020;Mahmud et al., 2020), and as well for perennial forage and bioenergy grasses (Roley et al., 2019;Bahulikar et al., 2020). These range from simply isolating, testing, and deploying the most effective natural plant-associated diazotrophs of target plant species, based primarily on plant growth promotion (Fox et al., 2016), to current attempts to edit the genomes of such bacteria to remove genetic controls that prevent N fixation and NH 3 release in agricultural soils containing potentially high levels of mineral and organic-N (Barney et al., 2017;Bueno Batista and Dixon, 2019). Decades of genetic, genomic, and biochemical research and technology development provide a basis for attempts to edit or engineer diazotrophs for optimal association with non-legumes (Bueno Batista and Dixon, 2019). ...
Article
Full-text available
Nitrogen (N) is an essential but generally limiting nutrient for biological systems. Development of the Haber-Bosch industrial process for ammonia synthesis helped to relieve N limitation of agricultural production, fueling the Green Revolution and reducing hunger. However, the massive use of industrial N fertilizer has doubled the N moving through the global N cycle with dramatic environmental consequences that threaten planetary health. Thus, there is an urgent need to reduce losses of reactive N from agriculture, while ensuring sufficient N inputs for food security. Here we review current knowledge related to N use efficiency (NUE) in agriculture and identify research opportunities in the areas of agronomy, plant breeding, biological N fixation (BNF), soil N cycling, and modeling to achieve responsible, sustainable use of N in agriculture. Amongst these opportunities, improved agricultural practices that synchronize crop N demand with soil N availability are low-hanging fruit. Crop breeding that targets root and shoot physiological processes will likely increase N uptake and utilization of soil N, while breeding for BNF effectiveness in legumes will enhance overall system NUE. Likewise, engineering of novel N-fixing symbioses in non-legumes could reduce the need for chemical fertilizers in agroecosystems but is a much longer-term goal. The use of simulation modeling to conceptualize the complex, interwoven processes that affect agroecosystem NUE, along with multi-objective optimization, will also accelerate NUE gains.
... Since the microorganism has enormous potential related to enhancing soil and crop productivity, plant microbiome engineering offers us a great scope to enhance crop yields and quality. Transfer of bacterial N 2 fixing genes to cereals has offered a great possibility to meet the plant nitrogen requirements (Bageshwar et al. 2017;Fox et al. 2016;Geddes et al. 2015Geddes et al. , 2019Lugtenberg and Kamilova 2009;Mondy et al. 2014;Pankievicz et al. 2015). Transgenic plants that produced opine molecules, through gene transfer from Agrobacterium, were shown to enrich their rhizosphere with bacteria able to catabolize opines (Mondy et al. 2014;Oger et al. 1997;Savka et al. 2013). ...
Chapter
The persistence, survival, and availability of the applied agri-inputs (water, fertilizers, and other soil/plant amendments) including microbial inoculants in the rhizosphere of crop plants have become a major problem in agriculture. Rhizosphere engineering is an innovative approach through which the soil biophysical properties are modified to influence plant–microbiome–soil interactions to enhance soil and crop productivity through higher input use efficiency. The basic components of rhizosphere engineering include soil, plant, and microbes, which could be modified to optimize water and nutrient transport as well as microbial activity at the root–soil interface. Though genetic modification of crop plants and microbial engineering has taken back seat because of consumer awareness on human and environmental health, rhizosphere modification through agronomic approaches is the only hope at present to improve soil and crop productivity in an eco-friendly and sustainable manner. Natural way of modification of crop rhizosphere is expected to make soil healthy by avoiding indiscriminate use of plant protection chemicals and fertilizers. Hence, rhizoengineering approach should be advocated to farming community through eco-friendly farm amendments, instead of engineering crops or microorganisms.
... Diazotrophic Paenibacillus beijingensis BJ-18 provides nitrogen for wheat, maize and cucumber plants and promotes plant growth, nitrogen uptake and metabolism . A recombinant nitrogen-xing Pseudomonas protegens Pf-5 X940 was constructed by introducing the nif genes of Pseudomonas stutzeri A1501 via the X940 cosmid to the bene cial rhizobacterium Pseudomonas protegens Pf-5, and inoculation of Arabidopsis, alfalfa, tall fescue and maize with Pf-5 X940 increased the ammonium concentration in soil and plant productivity under nitrogen-de cient conditions (Fox et al. 2016;Setten et al. 2013). Inoculation with Azospirillum brasilense Ab-V5 cells enriched with exopolysaccharides and polyhydroxybutyrate enhances the productivity of maize under low N fertilizer input (Oliveira et al. 2017;) Paenibacillus polymyxa WLY78 is a nitrogen-xing bacterium containing a compact nif gene cluster consisting of 9 genes (nifBHDKENXhesAnifV) (Wang et al. 2013;Xie et al. 2014). ...
Preprint
Full-text available
Aims This study aimed to compare the effect on colonization, plant-growth promotion and nitrogen fixation contribution by inoculation with Paenibacillus polymyxa wild-type and Nif⁻mutant. Methods Paenibacillus polymyxa wild-type and Nif⁻ mutant was labeled with GFP and then the GFP-labeled bacteria were used to inoculate cucumber. The colonization patterns of P. polymyxa WLY78 in these plants were observed under the confocal laser scanning microscope. The effects of plant-growth promotion were investigated by greenhouse experiments. The nitrogen fixation contribution was estimated by ¹⁵N isotope dilution experiments. Results Observation by laser confocal microscopy revealed that both P. polymyxa WLY78 and ΔnifB-V mutant can effectively colonize cucumber root, stem and leaf tissues. Greenhouse experiments showed that inoculation with P. polymyxa WLY78 can significantly enhance the lengths and fresh wights of cucumber roots and shoots, but inoculation with ΔnifB-V mutant can not. ¹⁵N isotope dilution experiments showed that cucumber plants derive 25.93% nitrogen from nitrogen fixation performed by P. polymyxa WLY78, but the ΔnifB-V mutant nearly can not provide nitrogen for plant. Conclusions This present study demonstrates that nitrogen fixation plays an import role in promoting plant growth.
... Inoculation of plants with ammonium-excreting genetically modified bacteria proved to substitute for at least some synthetic N-fertilizer for plant growth (Ambrosio et al. 2017;Bageshwar et al. 2017;Fox et al. 2016;Rosenblueth et al. 2018;Santos et al. 2017). ...
Article
Full-text available
There is an increasing interest in the use of N2-fixing bacteria for the sustainable biofertilization of crops. Genetically-optimized bacteria for ammonium release have an improved biofertilization capacity. Some of these strains also cross-feed ammonium into microalgae raising additional concerns on their sustainable use in agriculture due to the potential risk of producing a higher and longer-lasting eutrophication problem than synthetic N-fertilizers. Here we studied the dynamic algal cross-feeding properties of a genetically-modified Azotobacter vinelandii strain which can be tuned to over-accumulate different levels of glutamine synthetase (GS, EC 6.3.1.20) under the control of an exogenous inducer. After switching cells overaccumulating GS into a noninducing medium, they proliferated for several generations at the expense of the previously accumulated GS. Further dilution of GS by cell division slowed-down growth, promoted ammonium-excretion and cross-fed algae. The final bacterial population, and timing and magnitude of algal N-biofertlization was finely tuned in a deferred manner. This tuning was in accordance with the intensity of the previous induction of GS accumulation in the cells. This bacterial population behavior could be maintained up to about 15 bacterial cell generations, until faster-growing and nonammonium excreting cells arose at an apparent high frequency. Further improvements of this genetic engineering strategy might help to align efficiency of N-biofertilizers and safe use in an open environment. Key points • Ammonium-excreting bacteria are potential eutrophication agents • GS-dependent deferred control of bacterial growth and ammonium release • Strong but transient ammonium cross-feeding of microalgae
Article
The earth’s surface constitutes a layer of soil around it which is termed as pedosphere. Soil holds millions of microbes that are involved in improving soil fertility. The increasing use of chemical fertilizers has become a major factor which is deteriorating soil microflora. It has resulted in decreased soil fertility. Soil organisms are involved in a number of processes like cycling of soil nutrients and providing them to plants. They are also involved in volatilization that may lead to nutrient loss. Microorganisms have an important role in carbon, nitrogen, and sulfur transformations, as well as organic matter degradation. They have an impact on the global nutrient and carbon cycle. The soil microflora is also involved in modulating the various physico-chemical properties of soil like pH, moisture, temperature etc. Soil properties and soil microorganisms are highly correlated with each other. The huge diversity of microorganisms in soil also plays a central role in regulating and supporting various ecosystem services. This review highlights the crucial role of different microbes in various nutrient cycling which is one of the major concerns to address the decreasing status of soil nutrients. It also covers various physico-chemical properties which affects soil microbial community and various ecosystem services provided by microbial activity.
Article
None declared.Conflicts of interestMicrobial interactions impact the functioning of microbial communities. However, microbial interactions within host-associated communities remains poorly understood. Here, we report that the beneficiary rhizobacterium Niallia sp. RD1 requires the helper Pseudomonas putida H3 for bacterial growth and beneficial interactions with the plant host. In the absence of the helper H3 strain, the Niallia sp. RD1 strain exhibited weak respiration and elongated cell morphology without forming bacterial colonies. A transposon mutant of H3 in a gene encoding succinate-semialdehyde dehydrogenase displayed much attenuated support of RD1 colony formation. Through subsequent addition of succinate to the media, we found that succinate serves as a public good that supports RD1 growth. Comparative genome analysis highlighted that RD1 lacked the gene for sufficient succinate, suggesting its evolution as a beneficiary of succinate biosynthesis. The syntrophic interaction between RD1 and H3 efficiently protected tomato plants from bacterial wilt and promoted the tomato growth. The addition of succinate to the medium restored complex II-dependent respiration in RD1 and facilitated the cultivation of various bacterial isolates from the rhizosphere. Taken together, we delineate energy auxotrophic beneficiaries ubiquitous in the microbial community, and these beneficiaries could benefit host plants with the aid of helpers in the rhizosphere.
Article
Full-text available
Biological nitrogen fixation (BNF) by diazotrophic bacteria is one of the oldest and most crucial processes in nature. In this process, bacteria form symbiotic associations with plants, capturing atmospheric nitrogen and making it readily available to them. The diversity of nitrogen-fixing bacteria is vast. Recent advancements in molecular biology techniques have enabled the identification of new genera and species capable of fixing nitrogen and providing other types of nutrients for plants. From an agronomic perspective, this process is fundamental in increasing crop productivity sustainably and-cost-effectively. This review aims to categorize the most recent updates on the diversity of nitrogen-fixing bacteria and showcase the main advances in the genetic improvement of legumes for this characteristic. Recent research has revealed a wide diversity of species applicable to various crops of agronomic interest, and many of these bacteria have been used either alone or in consortium with other microorganisms. This study demonstrates the agricultural potential of these new discoveries and the vast possibilities for expanding research into the diversity of microorganisms responsible for BNF in agriculture.
Article
Uncontrolled usage of chemical fertilizers, climate change due to global warming, and the ever-increasing demand for food have necessitated sustainable agricultural practices. Removal of ever-increasing environmental pollutants, treatment of life-threatening diseases, and control of drug-resistant pathogens are also the need of the present time to maintain the health and hygiene of nature, as well as human beings. Research on plant–microbe interactions is paving the way to ameliorate all these sustainably. Diverse bacterial endophytes inhabiting the internal tissues of different parts of the plants promote the growth and development of their hosts by different mechanisms, such as through nutrient acquisition, phytohormone production and modulation, protection from biotic or abiotic challenges, assisting in flowering and root development, etc. Notwithstanding, efficient exploitation of endophytes in human welfare is hindered due to scarce knowledge of the molecular aspects of their interactions, community dynamics, in-planta activities, and their actual functional potential. Modern “-omics-based” technologies and genetic manipulation tools have empowered scientists to explore the diversity, dynamics, roles, and functional potential of endophytes, ultimately empowering humans to better use them in sustainable agricultural practices, especially in future harsh environmental conditions. In this review, we have discussed the diversity of bacterial endophytes, factors (biotic as well as abiotic) affecting their diversity, and their various plant growth-promoting activities. Recent developments and technological advancements for future research, such as “-omics-based” technologies, genetic engineering, genome editing, and genome engineering tools, targeting optimal utilization of the endophytes in sustainable agricultural practices, or other purposes, have also been discussed.
Article
Full-text available
Agricultural products such as tea, chocolate, coffee and wine are valued for their sensorial and nutritional qualities. Variation in the growing conditions of a crop can influence the plant's phenotype, thus it behooves agriculturalists to optimize the conditions on their farms to grow the highest quality product. The set of growing conditions associated with a certain geographic location and its influence on the product's chemistry is known as terroir. Although terroir plays a significant role in marketing and consumer appreciation as well as product identity and valorization, rarely are the biochemical differences or the factors creating them very well understood. The word derives from the Latin for "land", suggesting terroir is simply a function of the geographical location where a plant grew, while in its modern usage, terroir is understood to be the result of soil type, climate, landscape, topography, biotic interactions and agricultural practice. Except for fermented food products like wine and chocolate, plant associated microbiomes have been little studied for their contribution to a crop's terroir; however, modern metagenomics and metabolomics technologies have given scientists the tools to better observe how microbial diversity can impact the chemical variation in plant products. Differences in the microbiomes inhabiting plant organs can change phytochemistry by altering host metabolism, for example increasing the nutrients absorbed by roots that then are deposited in leaves, seeds and fruits. Plant associated microbes can consume plant molecules, removing them from the metabolome, or they can contribute smells and flavors of their own. This review aims to synthesize research into rhizosphere, endosphere, phyllosphere, spermosphere, carposphere, and anthosphere microbiome influences on plant biochemistry and crop derived products, while helping to increase the appreciation that beneficial microbes are able to contribute to agriculture by improving phytochemical quality.
Article
The endophytic nitrogen-fixing bacterium A02 belongs to the genus Curtobacterium (Curtobacterium sp.) and is crucial for the nitrogen (N) metabolism of cassava(Manihot esculenta Crantz). We isolated the A02 strain from cassava cultivar SC205 and used the 15N isotope dilution method to study the impacts of A02 on growth and accumulation of N in cassava seedlings. Furthermore, the whole genome was sequenced to determine the N-fixation mechanism of A02. Compared with low N control (T1), inoculation with the A02 strain (T2) showed the highest increase in leaf and root dry weight of cassava seedlings, and 120.3 nmol·(mL·h) was the highest nitrogenase activity recorded in leaves, which were considered the main site for colonization and N-fixation. The genome of A02 was 3,555,568 bp in size and contained a circular chromosome and a plasmid. Comparison with the genomes of other short bacilli revealed that strain A02 showed evolutionary proximity to the endophytic bacterium NS330 (Curtobacterium citreum) isolated from rice (Oryza sativa) in India. The genome of A02 contained 13 nitrogen fixation (nif) genes, including 4 nifB, 1 nifR3, 2 nifH, 1 nifU, 1 nifD, 1 nifK, 1 nifE, 1 nifN, and 1 nifC, and formed a relatively complete N fixation gene cluster 8-kb long that accounted for 0.22% of the whole genome length. The nifHDK of strain A02(Curtobacterium sp.) is identical to the Frankia alignment. Function prediction showed high copy number of the nifB gene was related to the oxygen protection mechanism. Our findings provide exciting information about the bacterial genome in relation to N support for transcriptomic and functional studies for increasing N use efficiency in cassava.
Chapter
The area of soil exposed to root activity is called the rhizosphere which harbors diverse microbes that can aid in plant growth and resistance against biotic and abiotic stresses. Rhizosphere microbiome is defined as all the microbial species found in the rhizosphere, which have one of the most complex and diverse ecosystems on the planet. These rhizosphere microbial communities interact with the plants as beneficial or detrimental interactions. Beneficial rhizosphere microbes promote plant growth through abiotic stress tolerance, absorption of nutrients in plants and antagonism against several phytopathogens, while parasitic interaction causes diseases of plants which are economically important, leading to challenges in food security and reduction in productivity. In this chapter, we have discussed in detail the various interactions on microbe-plant and microbe-microbe interaction and also the role of rhizosphere microbiome in plant health and resistance.KeywordsBeneficialDetrimentalPlant healthMicrobesRhizosphere
Article
Full-text available
Background: Molecular identification of a wide range of organisms capable of carrying out biological nitrogen fixation (BNF) are diverse in nature and significantly improves plant growth. Biological N2 fixation reflects the activity of a phylogenetically diverse list of microorganisms. Molecular characterization provides efficient means to identify organisms with the potential of N2 fixation. Applying these techniques in an array of environments has considerably broadened our understanding of the suite of organisms that can carry out BNF. Methods: Thirty-four strains of free living N2 fixing bacterial strains were isolated from diverse plants cultivated in North Gujarat, including wheat, cotton, castor and pearl millet, using a nitrogen-free selective medium. Acetylene reduction assay was used to check the ability of all bacteria to fix nitrogen. Hybridization with nifH probe derived from Azotobacter vinelandii with isolated free-living nitrogen-fixing bacteria showed a positive result. The selected strains were characterized by molecular analysis like; ARDRA and 16S rDNA sequencing. Result: Based on molecular characterization 17 strains to known groups of nitrogen-fixing bacteria, including organisms from the genus Azotobacter, Pseudomonas, Enterobacter, Arthrobacter, Bacillus, Variovorax, Nocardiodies, Rhodococcus, Mycobacterium, Planococcus, Microbacterium have been identified. One of the strains was identified as unknown bacteria. The potential strains were identified by 16srDNA analysis and also corroborated by morphological and biochemical characterization.
Article
Full-text available
BNF is a fascinating phenomenon which contributes to protect the nature from environmental pollution that can be happened as a result of heavy nitrogen applications. The importance of BNF is due to its supply of the agricultural lands with about 200 million tons of N annually. In this biological process, a specific group of bacteria collectively called rhizobia fix the atmospheric N in symbiosis with legumes called symbiotic nitrogen fixation and others (free living) fix nitrogen gas from the atmosphere termed asymbiotic. Several trials were done by scientists around the world to make cereals more benefited from nitrogen gas through different approaches. The first approach is to engineer cereals to form nodulated roots. Secondly is to transfer nif genes directly to cereals and fix N without Rhizobium partner. The other two approaches are maximizing the inoculation of cereals with both of diazotrophs or endophytes. Recently, scientists solved some challenges that entangle engineering cereals with nif genes directly and they confirmed the suitability of mitochondria and plastids as a suitable place for better biological function of nif genes expression in cereals. Fortunately, this article is confirming the success of scientists not only to transfer synthetic nitrogenase enzyme to Escherichia coli that gave 50% of its activity of expression, but also move it to plants as Nicotiana benthamiana. This mini review aims at explaining the future outlook of BNF and the challenges limiting its transfer to cereals and levels of success to make cereals self nitrogen fixing.
Chapter
Today, alternative control methods less harmful than chemical control methods are sought against diseases that cause serious yield losses in crops. Biological disease control agents are in the spotlight of many researchers as a promising approach in this regard. These agents, which are increasingly used as biological fertilizers, are widely used in the field of vegetable cultivation. The biological control activity of these microorganisms depends on the functioning of their complex physiological and molecular mechanisms in harmony. This harmony between plants and microorganisms appears as the ultimate result of millions of years of improvement and has started an invisible but endless belowground warfare. The purpose of this chapter is to reveal the details of the symbiotic agreements between the microorganisms involved in this warfare and the plants and to bring together in detail the combat tools and equipment used by the microorganisms against the pathogens. In this chapter, information about current microorganisms applied to vegetables was presented by bringing together detailed physiological and molecular explanations about the action mechanism of their symbiosis and pathogen biocontrol activity. It is a recent update that starts with explaining communication between microorganisms, plants, and pathogens, continues explaining the regulation of related gene expression, includes explaining physiological responses, and also includes current practices on vegetables based on families.
Article
Full-text available
Aims To study nitrogen contribution to cucumber derived from nitrogen fixation of Paenibacillus polymyxa WLY78. Methods The nif gene cluster deletion mutant (ΔnifB-V) of Paenibacillus polymyxa WLY78 was constructed by a homologous recombination method. The effects of plant-growth promotion were investigated by greenhouse experiments. The nitrogen fixation contribution was estimated by ¹⁵N isotope dilution method (also being called the ¹⁵N natural abundance technique). Results Deletion of nif gene cluster of P. polymyxa WLY78 resulted in complete loss of nitrogenase activity. Greenhouse experiments showed that inoculation with P. polymyxa WLY78 could significantly enhance the lengths and dry weights of cucumber roots and shoots, but inoculation with ΔnifB-V mutant could not. ¹⁵N isotope dilution experiments showed that cucumber plants derive 25.93% nitrogen from nitrogen fixation performed by P. polymyxa WLY78, but the ΔnifB-V mutant nearly could not provide nitrogen for plant growth. Conclusions This present study demonstrated that nitrogen fixation performed by P. polymyxa WLY78 contributes to plant growth.
Article
Full-text available
Agricultural productivity relies on synthetic nitrogen fertilizers, yet half of that reactive nitrogen is lost to the environment. There is an urgent need for alternative nitrogen solutions to reduce the water pollution, ozone depletion, atmospheric particulate formation, and global greenhouse gas emissions associated with synthetic nitrogen fertilizer use. One such solution is biological nitrogen fixation (BNF), a component of the complex natural nitrogen cycle. BNF application to commercial agriculture is currently limited by fertilizer use and plant type. This paper describes the identification, development, and deployment of the first microbial product optimized using synthetic biology tools to enable BNF for corn (Zea mays) in fertilized fields, demonstrating the successful, safe commercialization of root-associated diazotrophs and realizing the potential of BNF to replace and reduce synthetic nitrogen fertilizer use in production agriculture. Derived from a wild nitrogen-fixing microbe isolated from agricultural soils, Klebsiella variicola 137-1036 (“Kv137-1036”) retains the capacity of the parent strain to colonize corn roots while increasing nitrogen fixation activity 122-fold in nitrogen-rich environments. This technical milestone was then commercialized in less than half of the time of a traditional biological product, with robust biosafety evaluations and product formulations contributing to consumer confidence and ease of use. Tested in multi-year, multi-site field trial experiments throughout the U.S. Corn Belt, fields grown with Kv137-1036 exhibited both higher yields (0.35 ± 0.092 t/ha ± SE or 5.2 ± 1.4 bushels/acre ± SE) and reduced within-field yield variance by 25% in 2018 and 8% in 2019 compared to fields fertilized with synthetic nitrogen fertilizers alone. These results demonstrate the capacity of a broad-acre BNF product to fix nitrogen for corn in field conditions with reliable agronomic benefits.
Article
Full-text available
Plants contain diverse microbial communities. The associated microorganisms confer advantages to the host plant, which include growth promotion, nutrient absorption, stress tolerance, and pathogen and disease resistance. In this review, we explore how agriculture is implementing the use of microbial inoculants (single species or consortia) to improve crop yields, and discuss current strategies to study plant-associated microorganisms and how their diversity varies under unconventional agriculture. It is predicted that microbial inoculation will continue to be used in agriculture.
Chapter
The rise in the global population and the growing demand for agricultural products have become a matter of grave concern for the present generation. To address this current challenge, it is vital to provide adequate amounts of nitrogen in a form that is easily accessible to the crop. The roles of the nitrogen-fixing (nif) genes in microorganisms are very important because commercially prepared nitrogen is not sufficient enough to meet the nitrogen demand. The present chapter provides an insight into the world of nif genes, their associated roles, and how they are regulated. The evolution of nif genes, structural and functional role of each nif gene, the gradual development in research of various genes, and its related clusters identified in different crops and microbes are discussed. The chapter also sheds light on the current research and developments on nif genes across the globe and areas where more research must be encouraged. The present chapter intends to provide researchers understanding of nif genes and their evolution and regulation mechanisms, which could be helpful in further studies.
Chapter
The concept of functional diversity assists in comprehension of natural intricacy during the widespread cooperation that microorganisms depict with other populations and ecological systems as how they communicate. Principally, microorganisms consist of several traits that describe their function inside ecological systems. Plant root associated bacteria that inhabit the plant root as ectophytes or endophytes and be capable of precisely boosting plant development through enhanced nourishment, production of plant hormones, mitigation of deleterious effect of different plant associated pathogens, and fighting for colonization sites on plants are referred as plant growth-promoting rhizobacteria (PGPR). Therefore, the application of specific nitrogen-fixing plant growth-promoting rhizobacteria in the form of microbial inoculants (single or mixed) can minimize the addiction of chemically synthesized fertilizers without compromising the crop yield. Despite their potential to enhance crop productivity and improved crop protection, nitrogen-fixing PGPR still have to cover a long distance to compete as effective bioinoculants. Therefore, there is urgent need to learn the functional diversity of nitrogen-fixing PGPR for sustainable crop production. Keeping in view the author make effort to evaluate the current development related with functional diversity about nitrogen-fixing PGPR along with their mode of action.
Article
Full-text available
ABSTRACT Background: Agriculture is a major contributor to environmental and soil degradation. Soil microorganisms are essential to improve plant growth, crop yields and stress-tolerance. Objective: To characterize maize early plant-response in a seedbed setting to native consortia of isolated microorganisms from arid zones. Methods: Sixteen fungal and 16 bacterial isolates from arid soils were identified by MALDI-TOF MS and confirmed using morphological characteristics. Ten biofertilizers were tested in replicates (n=100) in maize under seedbed conditions. Consortia were formulated based on growth promoting traits, including mainly Penicillium and Pseudomonas species. After 45 days, biofertilizers were evaluated according to plant height, and shoot and root fresh weight. Results and Conclusions: Penicillium and Pseudomonas were the predominant genera identified. Most strains are potential candidates for biofertilizer formulation based on their growth promoting traits. Bacterial consortia mainly promoted plant caulinar development, while the combination of fungal and bacterial species markedly increased root development. Eight biofertilizer consortia from arid zones had positive effects at early developmental stage of maize under seedbed conditions compared to uninoculated plants.
Chapter
Abiotic stresses are supposed to negate crop productivity leading to food insecurity. Plant growth-promoting rhizobacteria (PGPR) can significantly facilitate plant growth directly or indirectly by suppressing various plant pathogens, producing different phytohormones, mineralization and decomposition of organic matter, triggering antioxidant system, producing siderophores, and improving the bioavailability of different mineral nutrients. Among the PGPRs, Pseudomonas are ubiquitous and their occurrence in stressed environment has also been reported. Pseudomonas can produce different enzymes and metabolites that help plants withstand varied biotic and abiotic stresses. Pseudomonas-induced salt or drought tolerance has extensively studied at the physiological and biochemical levels in plants. The potential of Pseudomonas has also been explored under some other abiotic stress factors like water, temperature, nutrients, and heavy metals. Furthermore, in this chapter, we will elaborate the interactions among plant, Pseudomonas spp., and various abiotic environmental stresses with an objective to explore the underlying mechanisms and stress tolerance in plants as promises of Pseudomonas.
Chapter
Most contemporary agricultural practices involve the use of synthetic fertilizers which have been linked to numerous deleterious consequences such as eutrophication of water bodies and emission of greenhouse gases. Biofertilizers offer viable and environmentally friendly alternatives. The positive effects of plant growth-promoting rhizobacteria have extensively been demonstrated several agronomically important crops under both controlled and field conditions. Despite the large volume of literature documenting the potential of these microbial inoculants as biofertilizers, their practical application has largely been hampered by several factors. This chapter presents the current knowledge of biofertilizer research, commercialization, and practical applications from the global perspective. The constraints facing their research and global application are also articulated. Finally, some prospects regarding their future research, commercialization and practical application for sustainable cropping systems are critically elucidated. It is anticipated that this will enable the full evaluation of the potential prospects of biofertilizers for sustainable agriculture and ecosystems globally.
Article
Full-text available
Using cereal crops as examples, we review the breeding for tolerance to the abiotic stresses of low nitrogen, drought, salinity and aluminium toxicity. All are already important abiotic stress factors that cause large and widespread yield reductions. Drought will increase in importance with climate change, the area of irrigated land that is salinized continues to increase, and the cost of inorganic N is set to rise. There is good potential for directly breeding for adaptation to low N while retaining an ability to respond to high N conditions. Breeding for drought and salinity tolerance have proven to be difficult, and the complex mechanisms of tolerance are reviewed. Marker-assisted selection for component traits of drought in rice and pearl millet and salinity tolerance in wheat has produced some positive results and the pyramiding of stable quantitative trait locuses controlling component traits may provide a solution. New genomic technologies promise to make progress for breeding tolerance to these two stresses through a more fundamental understanding of underlying processes and identification of the genes responsible. In wheat, there is a great potential of breeding genetic resistance for salinity and aluminium tolerance through the contributions of wild relatives.
Chapter
Full-text available
Among the land plants, legumes are unique as they establish symbiotic relationship with soil-borne, nitrogen-fixing bacteria known as rhizobia to meet their nitrogen demand. This symbiotic interaction is considered a promising component of sustainable agriculture due to its economic and ecological benefits. However, the scope of this symbiosis, which is currently limited to legumes, needs to be extended to non-legumes, in particular to economically important cereal crops, to achieve sustainable production of staple crops. This chapter explores the feasibility of transferring the symbiotic nitrogen fixing machinery to non-legume crops.
Article
Full-text available
Plant production systems globally must be optimized to produce stable high yields from limited land under changing and variable climates. Demands for food, animal feed, and feedstocks for bioenergy and biorefining applications, are increasing with population growth, urbanization and affluence. Low-input, sustainable, alternatives to petrochemical-derived fertilizers and pesticides are required to reduce input costs and maintain or increase yields, with potential biological solutions having an important role to play. In contrast to crops that have been bred for food, many bioenergy crops are largely undomesticated, and so there is an opportunity to harness beneficial plant-microbe relationships which may have been inadvertently lost through intensive crop breeding. Plant-microbe interactions span a wide range of relationships in which one or both of the organisms may have a beneficial, neutral or negative effect on the other partner. A relatively small number of beneficial plant-microbe interactions are well understood and already exploited; however, others remain understudied and represent an untapped reservoir for optimizing plant production. There may be near-term applications for bacterial strains as microbial biopesticides and biofertilizers to increase biomass yield from energy crops grown on land unsuitable for food production. Longer term aims involve the design of synthetic genetic circuits within and between the host and microbes to optimize plant production. A highly exciting prospect is that endosymbionts comprise a unique resource of reduced complexity microbial genomes with adaptive traits of great interest for a wide variety of applications. © 2014 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.
Article
Full-text available
Significance To date, three different nitrogenase systems [molybdenum (MoFe), vanadium (VFe), and iron-only (FeFe)] have been found in nature. The MoFe nitrogenase has been studied extensively, but the alternative vanadium-dependent (Vnf) and iron-only (Anf) systems are less well characterized, particularly with respect to components required for their biosynthesis and activity. We have engineered an artificial FeFe nitrogenase system in Escherichia coli that combines anf structural genes with accessory nitrogen fixation genes ( nif ) to provide a minimal 10-gene cluster that supports the biosynthesis and activity of the FeFe nitrogenase. Our findings underscore the potential for the future engineering of nitrogen fixation in eukaryotes because the Anf system can bypass limitations in molybdenum availability in plant organelles.
Article
Full-text available
The availability of nitrogen is one of the major limiting factors to crop growth. In the developed world, farmers use unsustainable levels of inorganic fertilisers to promote crop production. In contrast, in the developing world inorganic fertilisers are often not available and small-holder farmers suffer the resultant poor yields. Finding alternatives to inorganic fertilisers is critical for sustainable and secure food production. Bacteria and Archaea have evolved the capability to fix atmospheric nitrogen to ammonia, a form readily usable in biological processes. This capability presents an opportunity to improve the nutrition of crop plants, through the introduction into cereal crops of either the nitrogen fixing bacteria or the nitrogenase enzyme responsible for nitrogen fixation. While both approaches are challenging, recent advances have laid the groundwork to initiate these biotechnological solutions to the nitrogen problem.
Article
Full-text available
Most biological nitrogen fixation is catalyzed by molybdenum-dependent nitrogenase, an enzyme complex comprising two component proteins that contains three different metalloclusters. Diazotrophs contain a common core of nitrogen fixation nif genes that encode the structural subunits of the enzyme and components required to synthesize the metalloclusters. However, the complement of nif genes required to enable diazotrophic growth varies significantly amongst nitrogen fixing bacteria and archaea. In this study, we identified a minimal nif gene cluster consisting of nine nif genes in the genome of Paenibacillus sp. WLY78, a gram-positive, facultative anaerobe isolated from the rhizosphere of bamboo. We demonstrate that the nif genes in this organism are organized as an operon comprising nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV and that the nif cluster is under the control of a σ(70) (σ(A))-dependent promoter located upstream of nifB. To investigate genetic requirements for diazotrophy, we transferred the Paenibacillus nif cluster to Escherichia coli. The minimal nif gene cluster enables synthesis of catalytically active nitrogenase in this host, when expressed either from the native nifB promoter or from the T7 promoter. Deletion analysis indicates that in addition to the core nif genes, hesA plays an important role in nitrogen fixation and is responsive to the availability of molybdenum. Whereas nif transcription in Paenibacillus is regulated in response to nitrogen availability and by the external oxygen concentration, transcription from the nifB promoter is constitutive in E. coli, indicating that negative regulation of nif transcription is bypassed in the heterologous host. This study demonstrates the potential for engineering nitrogen fixation in a non-nitrogen fixing organism with a minimum set of nine nif genes.
Article
Full-text available
Biological nitrogen fixation is a complex process requiring multiple genes working in concert. To date, the Klebsiella pneumoniae nif gene cluster, divided into seven operons, is one of the most studied systems. Its nitrogen fixation capacity is subject to complex cascade regulation and physiological limitations. In this report, the entire K. pneumoniae nif gene cluster was reassembled as operon-based BioBrick parts in Escherichia coli. It provided ∼100% activity of native K. pneumoniae system. Based on the expression levels of these BioBrick parts, a T7 RNA polymerase-LacI expression system was used to replace the σ(54)-dependent promoters located upstream of nif operons. Expression patterns of nif operons were critical for the maximum activity of the recombinant system. By mimicking these expression levels with variable-strength T7-dependent promoters, ∼42% of the nitrogenase activity of the σ(54)-dependent nif system was achieved in E. coli. When the newly constructed T7-dependent nif system was challenged with different genetic and physiological conditions, it bypassed the original complex regulatory circuits, with minor physiological limitations. Therefore, we have successfully replaced the nif regulatory elements with a simple expression system that may provide the first step for further research of introducing nif genes into eukaryotic organelles, which has considerable potentials in agro-biotechnology.
Article
Full-text available
To study colonization of the tomato root system, we previously have described a gnotobiotic quartz sand system, in which seedlings inoculated with one or two bacterial strains were allowed to grow. Here we present a scanning electron microscope description of the colonization of the tomato root system by Pseudomonas fluorescens biocontrol strain WCS365, with emphasis on spatial-temporal colonization patterns, based on an improved scanning electron microscopy procedure. Upon inoculation of the germinated seed, proliferation on the seed coat was observed for 2 to 3 days. Within 1 to 3 days, micro-colonies developed, mainly at the root base. Most micro- colonies were localized in junctions between epidermal root cells, whereas others were found in indented parts of the epidermal surface. Downward to the root tip, only single bacterial cells were found. Colonization progressed down the root, initially as single cells. A semi-transparent film appeared to enclose the root surface and micro-colonies present on the root. After 7 days, micro-colonies had developed at positions where only single cells were observed previously and distribution of the bacteria along the root varied from ≃106 CFU per cm of root near the root base to ≃102 to 103 CFU per cm of root near the root tip. Similar colonization patterns were found for the P. fluorescens biocontrol strains CHA0 and F113, and P. putida strain WCS358, as well as for four species that have repeatedly been isolated from tomato roots from a commercial tomato field near Granada, Spain. In contrast, four Rhizobium strains and one Acinetobacter radioresistens strain showed poor colonization and micro-colonies were not observed. Based on the described data, we present a model for colonization of the deeper root parts after seed inoculation by P. fluorescens biocontrol strains, in which single cells occasionally establish on a deeper root section where they sometimes develop into micro-colonies. We hypothesize that microcolonies are the sites where the intracellular N-acyl-L-homoserine lactone concentration is sufficiently high to cause maximal production of biocontrol factors such as antibiotics and exoenzymes and that micro-colonies explain the relatively high conjugation frequency observed between pseudomonads in the rhizosphere.
Article
Full-text available
Nitrogen is the second most critical factor for crop production after water. In this study, the beneficial rhizobacterium Pseudomonas protegens Pf-5 was genetically modified to fix nitrogen using the genes encoding the nitrogenase of Pseudomonas stutzeri A1501 via the X940 cosmid. Pf-5 X940 was able to grow in L medium without nitrogen, displayed high nitrogenase activity and released significant quantities of ammonium to the medium. Pf-5 X940 also showed constitutive expression and enzymatic activity of nitrogenase in ammonium medium or in nitrogen-free medium, suggesting a constitutive nitrogen fixation. Similar to Pseudomonas protegens Pf-5, Pseudomonas putida, Pseudomonas veronii and Pseudomonas taetrolens but not Pseudomonas balearica and Pseudomonas stutzeri transformed with cosmid X940 showed constitutive nitrogenase activity and high ammonium production, suggesting that this phenotype depends on the genome context and that this technology to obtain nitrogen-fixing bacteria is not restricted to Pf-5. Interestingly, inoculation of Arabidopsis, alfalfa, tall fescue and maize with Pf-5 X940 increased the ammonium concentration in soil and plant productivity under nitrogen-deficient conditions. In conclusion, these results open the way to the production of effective recombinant inoculants for nitrogen fixation on a wide range of crops.
Article
Full-text available
Background Nitrogen is an essential nutrient in plant growth. The ability of a plant to supply all or part of its requirements from biological nitrogen fixation (BNF) thanks to interactions with endosymbiotic, associative and endophytic symbionts, confers a great competitive advantage over non-nitrogen-fixing plants.ScopeBecause BNF in legumes is well documented, this review focuses on BNF in non-legume plants. Despite the phylogenic and ecological diversity among diazotrophic bacteria and their hosts, tightly regulated communication is always necessary between the microorganisms and the host plant to achieve a successful interaction. Ongoing research efforts to improve knowledge of the molecular mechanisms underlying these original relationships and some common strategies leading to a successful relationship between the nitrogen-fixing microorganisms and their hosts are presented.Conclusions Understanding the molecular mechanism of BNF outside the legume-rhizobium symbiosis could have important agronomic implications and enable the use of N-fertilizers to be reduced or even avoided. Indeed, in the short term, improved understanding could lead to more sustainable exploitation of the biodiversity of nitrogen-fixing organisms and, in the longer term, to the transfer of endosymbiotic nitrogen-fixation capacities to major non-legume crops.
Article
Full-text available
The rise in the world demand for food poses a challenge to our ability to sustain soil fertility and sustainability. The increasing use of no-till agriculture, adopted in many areas of the world as an alternative to conventional farming, may contribute to reduce the erosion of soils and the increase in the soil carbon pool. However, the advantages of no-till agriculture are jeopardized when its use is linked to the expansion of crop monoculture. The aim of this study was to survey bacterial communities to find indicators of soil quality related to contrasting agriculture management in soils under no-till farming. Four sites in production agriculture, with different soil properties, situated across a west-east transect in the most productive region in the Argentinean pampas, were taken as the basis for replication. Working definitions of Good no-till Agricultural Practices (GAP) and Poor no-till Agricultural Practices (PAP) were adopted for two distinct scenarios in terms of crop rotation, fertilization, agrochemicals use and pest control. Non-cultivated soils nearby the agricultural sites were taken as additional control treatments. Tag-encoded pyrosequencing was used to deeply sample the 16S rRNA gene from bacteria residing in soils corresponding to the three treatments at the four locations. Although bacterial communities as a whole appeared to be structured chiefly by a marked biogeographic provincialism, the distribution of a few taxa was shaped as well by environmental conditions related to agricultural management practices. A statistically supported approach was used to define candidates for management-indicator organisms, subsequently validated using quantitative PCR. We suggest that the ratio between the normalized abundance of a selected group of bacteria within the GP1 group of the phylum Acidobacteria and the genus Rubellimicrobium of the Alphaproteobacteria may serve as a potential management-indicator to discriminate between sustainable vs. non-sustainable agricultural practices in the Pampa region.
Article
Full-text available
AimsBecause of its high dry matter (DM) productivity, elephant grass (Pennisetum purpureum) is an ideal candidate for biomass production for biofuel production if low N fertilizer rates are used to avoid high fossil fuel inputs. The objective of this study was to investigate the potential of different elephant grass genotypes to obtain contributions of plant-associated biological N2 fixation (BNF). MethodsThree field experiments with 4 or 5 different genotypes were conducted on low-fertility Acrisols, two in Rio de Janeiro State and one in Espirito Santo for the evaluation of DM and N accumulation and 15N abundance. ResultsDM and N accumulation rates of four genotypes in the two experiments in Rio State stabilized at high levels after 2years of growth. In all experiments the spontaneously-occurring weeds in the plots were significantly higher in 15N abundance than the elephant grass genotypes. The lower 15N abundance of the elephant grass was shown not to be due to lower δ15N abundance at depth in the soil. ConclusionsFour of the grass genotypes obtained between 18 and 70% of their N from BNF amounting to inputs of between 36 and 132kg N ha−1yr−1. KeywordsN natural abundance–Bioenergy crop–Elephant grass genotypes–N2 fixation–N accumulation– Pennisetum purpureum
Article
Full-text available
The metabolic capacity for nitrogen fixation is known to be present in several prokaryotic species scattered across taxonomic groups. Experimental detection of nitrogen fixation in microbes requires species-specific conditions, making it difficult to obtain a comprehensive census of this trait. The recent and rapid increase in the availability of microbial genome sequences affords novel opportunities to re-examine the occurrence and distribution of nitrogen fixation genes. The current practice for computational prediction of nitrogen fixation is to use the presence of the nifH and/or nifD genes. Based on a careful comparison of the repertoire of nitrogen fixation genes in known diazotroph species we propose a new criterion for computational prediction of nitrogen fixation: the presence of a minimum set of six genes coding for structural and biosynthetic components, namely NifHDK and NifENB. Using this criterion, we conducted a comprehensive search in fully sequenced genomes and identified 149 diazotrophic species, including 82 known diazotrophs and 67 species not known to fix nitrogen. The taxonomic distribution of nitrogen fixation in Archaea was limited to the Euryarchaeota phylum; within the Bacteria domain we predict that nitrogen fixation occurs in 13 different phyla. Of these, seven phyla had not hitherto been known to contain species capable of nitrogen fixation. Our analyses also identified protein sequences that are similar to nitrogenase in organisms that do not meet the minimum-gene-set criteria. The existence of nitrogenase-like proteins lacking conserved co-factor ligands in both diazotrophs and non-diazotrophs suggests their potential for performing other, as yet unidentified, metabolic functions. Our predictions expand the known phylogenetic diversity of nitrogen fixation, and suggest that this trait may be much more common in nature than it is currently thought. The diverse phylogenetic distribution of nitrogenase-like proteins indicates potential new roles for anciently duplicated and divergent members of this group of enzymes.
Article
Full-text available
Bacterial genes associated with a single trait are often grouped in a contiguous unit of the genome known as a gene cluster. It is difficult to genetically manipulate many gene clusters because of complex, redundant, and integrated host regulation. We have developed a systematic approach to completely specify the genetics of a gene cluster by rebuilding it from the bottom up using only synthetic, well-characterized parts. This process removes all native regulation, including that which is undiscovered. First, all noncoding DNA, regulatory proteins, and nonessential genes are removed. The codons of essential genes are changed to create a DNA sequence as divergent as possible from the wild-type (WT) gene. Recoded genes are computationally scanned to eliminate internal regulation. They are organized into operons and placed under the control of synthetic parts (promoters, ribosome binding sites, and terminators) that are functionally separated by spacer parts. Finally, a controller consisting of genetic sensors and circuits regulates the conditions and dynamics of gene expression. We applied this approach to an agriculturally relevant gene cluster from Klebsiella oxytoca encoding the nitrogen fixation pathway for converting atmospheric N(2) to ammonia. The native gene cluster consists of 20 genes in seven operons and is encoded in 23.5 kb of DNA. We constructed a "refactored" gene cluster that shares little DNA sequence identity with WT and for which the function of every genetic part is defined. This work demonstrates the potential for synthetic biology tools to rewrite the genetics encoding complex biological functions to facilitate access, engineering, and transferability.
Article
Full-text available
There are increasing applications of diazotrophic rhizobacteria in the sustainable agriculture system. A field experiment on young immature oil palm was conducted to quantify the uptake of N derived from N(2) fixation by the diazotroph Bacillus sphaericus strain UPMB-10, using the (15)N isotope dilution method. Eight months after (15)N application, young immature oil palms that received 67% of standard N fertilizer application together with B. sphaericus inoculation had significantly lower (15)N enrichment than uninoculated palms that received similar N fertilizers. The dilution of labeled N served as a marker for the occurrence of biological N(2) fixation. The proportion of N uptake that was derived from the atmosphere was estimated as 63% on the whole plant basis. The inoculation process increased the N and dry matter yields of the palm leaflets and rachis significantly. Field planting of young, immature oil palm in soil inoculated with B. sphaericus UPMB-10 might mitigate inorganic fertilizer-N application through supplementation by biological nitrogen fixation. This could be a new and important source of nitrogen biofertilizer in the early phase of oil palm cultivation in the field.
Article
Full-text available
Rhizobial bacteria enter a symbiotic association with leguminous plants, resulting in differentiated bacteria enclosed in intracellular compartments called symbiosomes within nodules on the root. The nodules and associated symbiosomes are structured for efficient nitrogen fixation. Although the interaction is beneficial to both partners, it comes with rigid rules that are strictly enforced by the plant. Entry into root cells requires appropriate recognition of the rhizobial Nod factor signaling molecule, and this recognition activates a series of events, including polarized root-hair tip growth, invagination associated with bacterial infection, and the promotion of cell division in the cortex leading to the nodule meristem. The plant's command of the infection process has been highlighted by its enforcement of terminal differentiation upon the bacteria within nodules of some legumes, and this can result in a loss of bacterial viability while permitting effective nitrogen fixation. Here, we review the mechanisms by which the plant allows bacterial infection and promotes the formation of the nodule, as well as the details of how this intimate association plays out inside the cells of the nodule where a complex interchange of metabolites and regulatory peptides force the bacteria into a nitrogen-fixing organelle-like state.
Article
Full-text available
The toxR gene of Vibrio cholerae encodes a transmembrane, DNA-binding protein that activates transcription of the cholera toxin operon and a gene (tcpA) for the major subunit of a pilus colonization factor. We constructed site-directed insertion mutations in the toxR gene by a novel method employing the chromosomal integration of a mobilizable suicide plasmid containing a portion of the toxR coding sequence. Mutants containing these new toxR alleles had an altered outer membrane protein profile, suggesting that two major outer membrane proteins (OmpT and OmpU) might be under the control of toxR. Physiological studies indicated that varying the concentration of the amino acids asparagine, arginine, glutamate, and serine caused coordinate changes in the expression of cholera toxin, TcpA, OmpT, and OmpU. Changes in the osmolarity of a tryptone-based medium also produced coordinate changes in the expression of these proteins. Other environmental signals (temperature and pH) had a more pronounced effect on the expression of cholera toxin and TcpA than they did on the outer membrane proteins. These results suggest that certain environmental signals (i.e., osmolarity and the presence of amino acids) are tightly coupled to the expression of toxR-regulated proteins and therefore may be signals that are directly sensed by the ToxR protein.
Article
Full-text available
Using physical and genetic data, we have demonstrated that Rhizobium meliloti SU47 has a symbiotic megaplasmid, pRmeSU47b, in addition to the previously described nod-nif megaplasmid pRmeSU47a. This plasmid includes four loci involved in exopolysaccharide (exo) synthesis as well as two loci involved in thiamine biosynthesis. Mutations at the exo loci have previously been shown to result in the formation of nodules which lack infection threads (Inf-) and fail to fix nitrogen (Fix-). Thus, both megaplasmids contain genes involved in the formation of nitrogen-fixing root nodules. Mutations at two other exo loci were not located on either megaplasmid. To mobilize the megaplasmids, the oriT of plasmid RK2 was inserted into them. On alfalfa, Agrobacterium tumefaciens strains containing pRmeSU47a induced marked root hair curling with no infection threads and Fix- nodules, as reported by others. This plant phenotype was not observed to change with A. tumefaciens strains containing both pRmeSU47a and pRmeSU47b megaplasmids, and strains containing pRmeSU47b alone failed to curl root hairs or form nodules.
Article
Full-text available
To visualize simultaneously different populations of pseudomonads in the rhizosphere at the single cell level in a noninvasive way, a set of four rhizosphere-stable plasmids was constructed expressing three different derivatives of the green fluorescent protein (GFP), namely enhanced cyan (ECFP), enhanced green (EGFP), enhanced yellow (EYFP), and the recently published red fluorescent protein (RFP; DsRed). Upon tomato seedling inoculation with Pseudomonas fluorescens WCS365 populations, each expressing a different autofluorescent protein followed by plant growth for 5 days, the rhizosphere was inspected using confocal laser scanning microscopy. We were able to visualize simultaneously and clearly distinguish from each other up to three different bacterial populations. Microcolonies consisting of mixed populations were frequently observed at the base of the root system, whereas microcolonies further toward the root tip predominantly consisted of a single population, suggesting a dynamic behavior of microcolonies over time. Since the cloning vector pME6010 has a broad host range for gram-negative bacteria, the constructed plasmids can be used for many purposes. In particular, they will be of great value for the analysis of microbial communities, for example in processes such as biocontrol, biofertilization, biostimulation, competition for niches, colonization, and biofilm formation.
Article
Full-text available
The use of Tn7-based systems for site-specific insertion of DNA into the chromosome of Gram-negative bacteria has been limited due to the lack of appropriate vectors. We therefore developed a flexible panel of Tn7 delivery vectors. In one group of vectors, the miniTn7 element, which is inserted into the chromosome, contains a multiple cloning site (MCS) and the kanamycin, streptomycin or gentamicin resistance markers. Another group of vectors intended for tagging with green fluorescent protein (GFP) carries the gfpmut3* gene controlled by the modified lac promoter PA1/04/03, several transcriptional terminators, and various resistance markers. These vectors insert Tn7 into a specific, neutral intergenic region immediately downstream of the gene encoding glucosamine-6-phosphate synthetase (GlmS) in the tested fluorescent Pseudomonas strains. The gfp-tagging vector containing a gentamicin-resistance marker is useful for tagging strains carrying a Tn5 transposon. Tn5 transposons often carry kanamycin-resistance-encoding genes and are frequently used to generate bacterial mutants and to deliver reporter constructions in gene expression studies. To demonstrate the utility of a dual marker/reporter system, the Tn7-gfp marker system was combined with a Tn5-delivered luxAB reporter system in Pseudomonas fluorescens. The system allowed detection of gfp-tagged cells in the barley rhizosphere, while expression of the Tn5-tagged locus could be determined by measuring bioluminescence.
Article
Full-text available
Confocal microscopy combined with three-dimensional olive root tissue sectioning was used to provide evidence of the endophytic behaviour of Pseudomonas fluorescens PICF7, an effective biocontrol strain against Verticillium wilt of olive. Two derivatives of the green fluorescent protein (GFP), the enhanced green and the red fluorescent proteins, have been used to visualize simultaneously two differently fluorescently tagged populations of P. fluorescens PICF7 within olive root tissues at the single cell level. The time-course of colonization events of olive roots cv. Arbequina by strain PICF7 and the localization of tagged bacteria within olive root tissues are described. First, bacteria rapidly colonized root surfaces and were predominantly found in the differentiation zone. Thereafter, microscopy observations showed that PICF7-tagged populations eventually disappeared from the root surface, and increasingly colonized inner root tissues. Localized and limited endophytic colonization by the introduced bacteria was observed over time. Fluorescent-tagged bacteria were always visualized in the intercellular spaces of the cortex region, and no colonization of the root xylem vessels was detected at any time. To the best of our knowledge, this is the first time this approach has been used to demonstrate endophytism of a biocontrol Pseudomonas spp. strain in a woody host such as olive using a nongnotobiotic system.
Article
Full-text available
Humans continue to transform the global nitrogen cycle at a record pace, reflecting an increased combustion of fossil fuels, growing demand for nitrogen in agriculture and industry, and pervasive inefficiencies in its use. Much anthropogenic nitrogen is lost to air, water, and land to cause a cascade of environmental and human health problems. Simultaneously, food production in some parts of the world is nitrogen-deficient, highlighting inequities in the distribution of nitrogen-containing fertilizers. Optimizing the need for a key human resource while minimizing its negative consequences requires an integrated interdisciplinary approach and the development of strategies to decrease nitrogen-containing waste.
Article
Using physical and genetic data, we have demonstrated that Rhizobium meliloti SU47 has a symbiotic megaplasmid, pRmeSU47b, in addition to the previously described nod-nif megaplasmid pRmeSU47a. This plasmid includes four loci involved in exopolysaccharide (exo) synthesis as well as two loci involved in thiamine biosynthesis. Mutations at the exo loci have previously been shown to result in the formation of nodules which lack infection threads (Inf-) and fail to fix nitrogen (Fix-). Thus, both megaplasmids contain genes involved in the formation of nitrogen-fixing root nodules. Mutations at two other exo loci were not located on either megaplasmid. To mobilize the megaplasmids, the oriT of plasmid RK2 was inserted into them. On alfalfa, Agrobacterium tumefaciens strains containing pRmeSU47a induced marked root hair curling with no infection threads and Fix- nodules, as reported by others. This plant phenotype was not observed to change with A. tumefaciens strains containing both pRmeSU47a and pRmeSU47b megaplasmids, and strains containing pRmeSU47b alone failed to curl root hairs or form nodules.
Article
Pseudomonas fluorescens PICF7, an indigenous olive roots inhabitant, displays endophytic lifestyle in this woody crop and exerts biocontrol against the fungal phytopathogen Verticillium dahliae Here we report microscopy evidence that the strain PICF7 is also able to colonize and persist on/in wheat and barley root tissues. Root colonization of both cereal species followed a similar pattern to that previously reported in olive, including inner colonization of the root hairs. This demonstrates that strain PICF7 can colonize root systems of distant botanical species. Barley plants germinated from PICF7-treated seeds showed enhanced vegetative growth. Moreover, significant increases in the number of grains (up to 19.5%) and grain weight (up to 20.5%) per plant were scored in this species. In contrast, growth and yield were not significantly affected in wheat plants by the presence of PICF7. Proteomics analysis of the root systems revealed that different proteins were exclusively found depending on the presence or absence of PICF7 and only one protein with hydrogen ion transmembrane transporter activity was exclusively found in both PICF7-inoculated barley and wheat plants but not in the controls.
Article
The vast majority of Pseudomonas species are unable to fix atmospheric nitrogen. Although several studies have demonstrated that some strains belonging to the genus Pseudomonas sensu stricto do have the ability to fix nitrogen by the expression of horizontally acquired nitrogenase, little is known about the mechanisms of nitrogenase adaptation to the new bacterial host. Recently, we transferred the nitrogen fixation island from Pseu-domonas stutzeri A1501 to the non-nitrogen-fixing bacterium Pseudomonas protegens Pf-5, and interestingly, the resulting recombinant strain Pf-5 X940 showed an uncommon phenotype of constitutive nitrogenase activity. Here, we integrated evolutionary and functional approaches to elucidate this unusual phenotype. Phylogenetic analysis showed that polyhydroxybutyrate (PHB) biosyn-thesis genes from natural nitrogen-fixing Pseudomonas strains have been acquired by horizontal transfer. Contrary to Pf-5 X940, its derived PHB-producing strain Pf-5 X940-PHB exhibited the inhibition of nitrogenase activity under nitrogen-excess conditions, and displayed the typical switch-on phenotype observed in natural nitrogen-fixing strains after nitrogen deficiency. This indicates a competition between PHB production and nitrogen fixation. Therefore, we propose that horizontal transfer of PHB biosynthesis genes could be an ancestral mechanism of regulation of horizontally acquired nitrogenases in the genus Pseudomonas.
Article
Nitrogen is an essential nutrient for all organisms that must have been available since the origin of life. Abiotic processes including hydrothermal reduction, photochemical reactions, or lightning discharge could have converted atmospheric N2 into assimilable NH4(+), HCN, or NOx species, collectively termed fixed nitrogen. But these sources may have been small on the early Earth, severely limiting the size of the primordial biosphere. The evolution of the nitrogen-fixing enzyme nitrogenase, which reduces atmospheric N2 to organic NH4(+), thus represented a major breakthrough in the radiation of life, but its timing is uncertain. Here we present nitrogen isotope ratios with a mean of 0.0 ± 1.2‰ from marine and fluvial sedimentary rocks of prehnite-pumpellyite to greenschist metamorphic grade between 3.2 and 2.75 billion years ago. These data cannot readily be explained by abiotic processes and therefore suggest biological nitrogen fixation, most probably using molybdenum-based nitrogenase as opposed to other variants that impart significant negative fractionations. Our data place a minimum age constraint of 3.2 billion years on the origin of biological nitrogen fixation and suggest that molybdenum was bioavailable in the mid-Archaean ocean long before the Great Oxidation Event.
Article
Snakin-1, a peptide produced by higher plants, has broad-spectrum antibiotic activity, inhibiting organisms ranging from Bacteria to Eukaryotes. However, the mode of action against target organisms is poorly understood. As a first step to elucidate the mechanism, we screened a mutation library of Pseudomonas fluorescens Pf-5 in LB and agar medium supplemented with alfalfa snakin-1 (MsSN1). We identified three biofilm formation-related Pseudomonas mutants that showed increased resistance to MsSN1. Genetic, physiological and bioinformatics analysis validated the results of the mutant screens, indicating that bacterial adhesion protein lapA is probably the target of MsSN1. Collectively, these findings suggest that snakin-1 acts on microbial adhesion properties.