Effect of 2,4-Diacetylphloroglucinol Producing,Overproducing, and Nonproducing Pseudomonas fluorescens F113 in the Rhizosphere of Pea

Pseudomonas fluorescens F113lacZY and modified strains carrying different function modifications were assessed for their impact in the rhizosphere of pea. Strain F113lacZY naturally produces the anti-fungal metabolite 2,4-diacetylphloroglucinol (Phl) useful in plant disease control. The first modified strain of F113 was repressed in production of Phl, creating the Phl negative strain F113G22. The second was a plasmid based overproducer of Phl (F113Rif (pCUGP)). Both the F113lacZY and the F113Rif (pCUGP) strains increased the rhizoplane fungal populations, whereas the same strains reduced the rhizosphere soil fungal populations with respect to the control. Similar results were found with the rhizoplane and rhizosphere soil bacterial populations. The F113G22 treatment resulted in a significantly greater indigenous fluorescent Pseudomonas population than the F113lacZY and F113Rif (pCUGP) treatments and a greater total Pseudomonas population than the control, F113lacZY, and F113Rif (pCUGP) treatments. Overproduction of Phl did not affect the establishment of the introduced Pseudomonas population. None of the inocula displaced the indigenous populations, but the F113G22 inocula had an additive effect on the total Pseudomonas population. P (phosphatase), S (sulphatase), and N (urease) cycle enzyme activities were increased while C (glucosidase, NAGase) cycle activities were decreased by the F113lacZY and F113Rif (pCUGP) treatments, suggesting C leakage from the roots. Overall, most effects of inoculation compared to the wild type were found with the non-Phl-producing strain. Overproduction of Phl had little environmental effect in relation to wild-type inocula.     

Enhancing the biocontrol efficacy of Pseudomonas fluorescens F113 by altering the regulation and production of 2,4-diacetylphloroglucinol

Pseudomonas fluorescens F113 is an effective biocontrol agent against Pythium ultimum, the causative agent of damping-off of sugarbeet seedlings. Biocontrol is mediated via the production of the anti-fungal metabolite 2,4-diacetylphloroglucinol (Phl). A genetic approach was used to further enhance the biocontrol ability of F113. Two genetically modified (GM) strains, P. fluorescens F113Rif (pCU8.3) and P. fluorescens F113Rif (pCUP9), were developed for enhanced Phl production and assessed for biocontrol efficacy and impact on sugarbeet in microcosm experiments. The multicopy plasmid pCU8.3 contains the biosynthetic genes (phlA, C, B and D) and the putative permease gene (phlE) of F113 cloned into the rhizosphere stable plasmid pME6010, independent of external promoters. The plasmid pCUP9 consists of the Phl biosynthetic genes cloned downstream of the constitutive Plac promoter in pBBR1MCS. Introduction of pCU8.3 and pCUP9 into P. fluorescens F113 significantly altered the kinetics of Phl biosynthesis when grown in SA medium. A significant and substantial increase in Phl production by the GM strains was observed in the early logarithmic phase and stationary phase of growth compared with the wild-type strain. In microcosm, the two Phl overproducing strains proved to be as effective at controlling damping-off disease as the proprietary fungicide treatment, indicating the potential of genetic modification for plant disease control.     

Effect of inoculum preparation and formulation on survival and biocontrol efficacy of Pseudomonas fluorescens F113

Sugarbeet seeds used by farmers are often pelleted using an EB^TM-based mix. During the pelleting process, the seeds are dried immediately after application of the mix. In this work, the effects of inoculum preparation and formulation on survival and biocontrol efficacy of Pseudomonas fluorescens F113Rif were investigated using a 1:1 EB^TM/vermiculite mix and sugarbeet seeds pelleted with this material. Growing F113Rif for 3 d (28 °C) within the EB^TM/vermiculite mix amended with nutrients (sucrose asparagine broth), instead of adding the cells to the unamended mix immediately before drying the mix or the pelleted sugarbeet seeds, resulted in improved survival of the strain in the mix or on the seeds, respectively, during subsequent storage. A slower drying (20 h instead of 3 h) of the F113Rif-inoculated EB^TM/vermiculite mix to 11% w/w water content enhanced strain survival in the mix during storage, but the drying conditions studied had no effect on inoculant survival on the seed during storage when pelleted seeds were dried to 10% w/w water content. Biological control of damping-off disease of sugarbeet (caused by Pythium spp.) in soil microcosms was achieved when F113Rif was inoculated in the unamended mix 3 d before pelleting the seeds, but not when nutrient-amended mix was used. Inoculum preparation and drying of the formulation are key factors to consider when optimizing the use of a commercial EB^TM/vermiculite seed formulation for delivery of a biocontrol Pseudomonas inoculant.     

An integrated approach for the evaluation of biological control of the complex Polymyxa betae/Beet Necrotic Yellow Vein Virus,by means of seed inoculants

Rhizomania is an extremely severe sugarbeet disease caused by the complex Polymyxa betae/Beet Necrotic Yellow Vein Virus (BNYVV). A relatively small number of recently introduced sugarbeet cultivars characterized by a high tolerance to rhizomania are available on the market. An integrated approach was therefore developed using Pseudomonas fluorescens biological control agents (BCAs) in order to improve yield performance of cultivars characterized by a medium tolerance to the disease. A genetically modified biological control agent, Pseudomonas fluorescens F113Rif (pCUGP), was developed for enhanced production of the antimicrobial metabolite 2,4-diacetylphloroglucinol (Phl) and lacking an antibiotic resistance marker gene, making the strain suitable for field release. The ability of synthetic Phl and P. fluorescens F113Rif (pCUGP) to antagonize the fungal vector, P. betae, was assessed in microcosm trials. Results encouraged the preparation of multiple field trials in a soil naturally infested with P. betae/BNYVV, to determine the biocontrol efficacy of P. fluorescens F113Rif (pCUGP) and to assess its impact on sugarbeet yield and quality and on the indigenous microbial population. While the colonization ability of P. fluorescens F113Rif (pCUGP) was satisfactory at sugarbeet emergence (2.5×10⁶ CFU g^–1 root), control of rhizomania was not achieved. Inoculation of sugarbeet with Pseudomonas fluorescens F113Rif (pCUGP) did not affect crop yield and quality nor affect the numbers of selected microbial populations.     

Improvement of biological nitrogen fixation in Egyptian winter legumes through better management of Rhizobium

The diversity of rhizobia nodulating common bean ( Phaseolus vulgaris), berseem clover (Trifolium alexanderinum) and lentil (Lens culinaris) was assessed using several characterization techniques, including nitrogen fixation efficiency, intrinsic antibiotic-resistance patterns (IAR), plasmid profiles, serological markers and rep-PCR fingerprinting. Wide diversity among indigenous rhizobial populations of the isolates from lentil, bean and clover was found. Strikingly, a large percentage of the indigenous rhizobial population was extremely poor at fixing nitrogen. This emphasizes the need to increase the balance of highly efficient strains within the rhizobial population. Use of high-quality inocula strains that survive and compete with other less-desired and less-efficient N2-fixing rhizobia represents the best approach to increase biological nitrogen fixation of the target legume. In field-grown lentils, the inoculant strains were not able to outcompete the indigenous rhizobia and the native lentil rhizobia occupied 76–88% of the total nodules formed on inoculated plants. Nitrogen fixation by lentils, estimated using the ¹⁵N isotope dilution technique, ranged between 127 to 139 kg ha-1 in both inoculated and un-inoculated plants. With berseem clover, the inoculant strains were highly competitive against indigenous rhizobia and occupied 52–79% of all nodules. Inoculation with selected inocula improved N2 fixation by clover from 162 to 205 kg ha-1 in the three cuts as compared with 118 kg ha-1 in the un-inoculated treatment. The results also indicated the potential for improvement of N2 fixation by beans through the application of efficient N2-fixing rhizobia.     

Impact of 2,4-diacetylphloroglucinol-producing biocontrol strain Pseudomonas fluorescens F113 on intraspecific diversity of resident culturable fluorescent pseudomonads associated with the roots of field-grown sugar beet seedlings

The impact of the 2,4-diacetylphloroglucinol-producing biocontrol agent Pseudomonas fluorescens F113Rif on the diversity of the resident community of culturable fluorescent pseudomonads associated with the roots of field-grown sugar beet seedlings was evaluated. At 19 days after sowing, the seed inoculant F113Rif had replaced some of the resident culturable fluorescent pseudomonads at the rhizoplane but had no effect on the number of these bacteria in the rhizosphere. A total of 498 isolates of resident fluorescent pseudomonads were obtained and characterized by molecular means at the level of broad phylogenetic groups (by amplified ribosomal DNA restriction analysis) and at the strain level (with random amplified polymorphic DNA markers) as well as phenotypically (55 physiological tests). The introduced pseudomonad induced a major shift in the composition of the resident culturable fluorescent Pseudomonas community, as the percentage of rhizoplane isolates capable of growing on three carbon substrates (erythritol, adonitol, and L-tryptophan) not assimilated by the inoculant was increased from less than 10% to more than 40%. However, the pseudomonads selected did not display enhanced resistance to 2,4-diacetylphloroglucinol. The shift in the resident populations, which was spatially limited to the surface of the root (i.e., the rhizoplane), took place without affecting the relative proportions of phylogenetic groups or the high level of strain diversity of the resident culturable fluorescent Pseudomonas community. These results suggest that the root-associated Pseudomonas community of sugar beet seedlings is resilient to the perturbation that may be caused by a taxonomically related inoculant.     

Mutational Disruption of the Biosynthesis Genes Coding for the Antifungal Metabolite 2,4-Diacetylphloroglucinol Does Not Influence the Ecological Fitness of Pseudomonas fluorescens F113 in the Rhizosphere of Sugarbeets

The ability of Pseudomonas fluorescens F113 to produce the antibiotic 2,4-diacetylphloroglucinol (DAPG) is a key factor in the biocontrol of the phytopathogenic fungus Pythium ultimum by this strain. In this study, a DAPG-producing strain (rifampin-resistant mutant F113Rif) was compared with a nearly isogenic DAPG-negative biosynthesis mutant (Tn5::lacZY derivative F113G22) in terms of the ability to colonize and persist in the rhizosphere of sugarbeets in soil microcosms during 10 plant growth-harvest cycles totaling 270 days. Both strains persisted similarly in the rhizosphere for 27 days, regardless of whether they had been inoculated singly onto seeds or coinoculated in a 1:1 ratio. In order to simulate harvest and resowing, the roots were removed from the soil and the pots were resown with uninoculated sugarbeet seeds for nine successive 27-day growth-harvest cycles. Strains F113Rif and F113G22 performed similarly with respect to colonizing the rhizosphere of sugarbeet, even after nine cycles without reinoculation. The introduced strains had a transient effect on the size of the total culturable aerobic bacterial population. The results indicate that under these experimental conditions, the inability to produce DAPG did not reduce the ecological fitness of strain F113 in the rhizosphere of sugarbeets.     

Influence of the Size of Indigenous Rhizobial Populations on Establishment and Symbiotic Performance of Introduced Rhizobia on Field-Grown Legumes

Indigenous rhizobia in soil present a competition barrier to the establishment of inoculant strains, possibly leading to inoculation failure. In this study, we used the natural diversity of rhizobial species and numbers in our fields to define, in quantitative terms, the relationship between indigenous rhizobial populations and inoculation response. Eight standardized inoculation trials were conducted at five well-characterized field sites on the island of Maui, Hawaii. Soil rhizobial populations ranged from 0 to over 3.5 × 10⁴ g of soil^-1 for the different legumes used. At each site, no less than four but as many as seven legume species were planted from among the following: soybean (Glycine max), lima bean (Phaseolus lunatus), cowpea (Vigna unguiculata), bush bean (Phaseolus vulgaris), peanut (Arachis hypogaea), Leucaena leucocephala, tinga pea (Lathyrus tingeatus), alfalfa (Medicago sativa), and clover (Trifolium repens). Each legume was (i) inoculated with an equal mixture of three effective strains of homologous rhizobia, (ii) fertilized at high rates with urea, or (iii) left uninoculated. For soybeans, a nonnodulating isoline was used in all trials as the rhizobia-negative control. Inoculation increased economic yield for 22 of the 29 (76%) legume species-site combinations. While the yield increase was greater than 100 kg ha^-1 in all cases, in only 11 (38%) of the species-site combinations was the increase statistically significant (P ≤ 0.05). On average, inoculation increased yield by 62%. Soybean (G. max) responded to inoculation most frequently, while cowpea (V. unguiculata) failed to respond in all trials. Inoculation responses in the other legumes were site dependent. The response to inoculation and the competitive success of inoculant rhizobia were inversely related to numbers of indigenous rhizobia. As few as 50 rhizobia g of soil^-1 eliminated inoculation response. When fewer than 10 indigenous rhizobia g of soil^-1 were present, economic yield was significantly increased 85% of the time. Yield was significantly increased in only 6% of the observations when numbers of indigenous rhizobia were greater than 10 cells g of soil^-1. A significant response to N application, significant increases in nodule parameters, and greater than 50% nodule occupancy by inoculant rhizobia did not necessarily coincide with significant inoculation responses. No less than a doubling of nodule mass and 66% nodule occupancy by inoculant rhizobia were required to significantly increase the yield of inoculated crops over that of uninoculated crops. However, lack of an inoculation response was common even when inoculum strains occupied the majority of nodules. In these trials, the symbiotic yield of crops was, on average, only 88% of the maximum yield potential, as defined by the fertilizer N treatment. The difference between the yield of N-fertilized crops and that of N₂-fixing crops indicates a potential for improving inoculation technology, the N₂ fixation capacity of rhizobial strains, and the efficiency of symbiosis. In this study, we show that the probability of enhancing yield with existing inoculation technology decreases dramatically with increasing numbers of indigenous rhizobia.     

Role of 2,4-Diacetylphloroglucinol in the Interactions of the Biocontrol Pseudomonad Strain F113 with the Potato Cyst Nematode Globodera rostochiensis

The potato cyst nematode Globodera rostochiensis is an important pest of potato (Solanum tuberosum). Pseudomonas fluorescens F113, which produces 2,4-diacetylphloroglucinol (DAPG), was investigated as a potential biocontrol agent against G. rostochiensis. Exposure of nematode cysts to the pseudomonad, under in vitro conditions or in soil microcosms, almost doubled the ability of the eggs to hatch. The percentage of mobile juveniles was reduced threefold following their incubation in the presence of the pseudomonad, both in vitro and in soil. Results obtained with a transposon-induced DAPG-negative biosynthetic mutant of F113 and its complemented derivative with restored DAPG synthesis showed that the ability of strain F113 to produce DAPG was responsible for the increase in hatch ability and the reduction in juvenile mobility. Similar effects on egg hatch ability and juvenile mobility of G. rostochiensis were obtained in vitro by incubating nematode cysts and juveniles, respectively, in the presence of synthetic DAPG. DAPG-producing P. fluorescens F113 is proposed as a potential biocontrol inoculant for the protection of potato crops against the potato cyst nematode.     

Exploitation of gene(s) involved in 2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to a Pseudomonas strain.

Tn5 mutagenesis and complementation analysis were used to clone a 6-kb genomic fragment required for biosynthesis of 2,4-diacetylphloroglucinol (Phl) from fluorescent Pseudomonas sp. strain F113. A recombinant plasmid, pCU203, containing this region partially complemented a Phl production-negative mutant (F113G22) derived from strain F113. When sugar beet seeds were sown into an unsterilized soil, in which sugar beet was subject to damping-off by Pythium ultimum, the emergence of sugar beet seeds inoculated with strain F113 was significantly greater than that of seeds inoculated with F113G22. Transfer of pCU203 into eight other Pseudomonas strains conferred the ability to synthesize Phl in only one of these strains, Pseudomonas sp. strain M114. Strain M114(pCU203) showed enhanced antagonism towards P. ultimum in vitro and significantly increased the emergence of sugar beet seeds in the same soil compared with emergence induced by the parent strain M114.     

Impact of biocontrol Pseudomonas fluorescens CHA0 and a genetically modified derivative on the diversity of culturable fungi in the cucumber rhizosphere

Little is known about the effects of Pseudomonas biocontrol inoculants on nontarget rhizosphere fungi. This issue was addressed using the biocontrol agent Pseudomonas fluorescens CHA0-Rif, which produces the antimicrobial polyketides 2,4-diacetylphloroglucinol (Phl) and pyoluteorin (Plt) and protects cucumber from several fungal pathogens, including Pythium spp., as well as the genetically modified derivative CHA0-Rif(pME3424). Strain CHA0-Rif(pME3424) overproduces Phl and Plt and displays improved biocontrol efficacy compared with CHA0-Rif. Cucumber was grown repeatedly in the same soil, which was left uninoculated, was inoculated with CHA0-Rif or CHA0-Rif(pME3424), or was treated with the fungicide metalaxyl (Ridomil). Treatments were applied to soil at the start of each 32-day-long cucumber growth cycle, and their effects on the diversity of the rhizosphere populations of culturable fungi were assessed at the end of the first and fifth cycles. Over 11,000 colonies were studied and assigned to 105 fungal species (plus several sterile morphotypes). The most frequently isolated fungal species (mainly belonging to the genera Paecilomyces, Phialocephala, Fusarium, Gliocladium, Penicillium, Mortierella, Verticillium, Trichoderma, Staphylotrichum, Coniothyrium, Cylindrocarpon, Myrothecium, and Monocillium) were common in the four treatments, and no fungal species was totally suppressed or found exclusively following one particular treatment. However, in each of the two growth cycles studied, significant differences were found between treatments (e.g., between the control and the other treatments and/or between the two inoculation treatments) using discriminant analysis. Despite these differences in the composition and/or relative abundance of species in the fungal community, treatments had no effect on species diversity indices, and species abundance distributions fit the truncated lognormal function in most cases. In addition, the impact of treatments at the 32-day mark of either growth cycle was smaller than the effect of growing cucumber repeatedly in the same soil.     

Exploitation of gene(s) involved in 2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to a Pseudomonas strain.

Tn5 mutagenesis and complementation analysis were used to clone a 6-kb genomic fragment required for biosynthesis of 2,4-diacetylphloroglucinol (Phl) from fluorescent Pseudomonas sp. strain F113. A recombinant plasmid, pCU203, containing this region partially complemented a Phl production-negative mutant (F113G22) derived from strain F113. When sugar beet seeds were sown into an unsterilized soil, in which sugar beet was subject to damping-off by Pythium ultimum, the emergence of sugar beet seeds inoculated with strain F113 was significantly greater than that of seeds inoculated with F113G22. Transfer of pCU203 into eight other Pseudomonas strains conferred the ability to synthesize Phl in only one of these strains, Pseudomonas sp. strain M114. Strain M114(pCU203) showed enhanced antagonism towards P. ultimum in vitro and significantly increased the emergence of sugar beet seeds in the same soil compared with emergence induced by the parent strain M114.     

根瘤菌生物地理学的研究进展

根瘤菌是一类革兰氏阴性菌,能与特定的宿主植物共生形成根瘤,将空气中的分子态氮转变为植物可以利用的氨态氮。研究根瘤菌的生物地理分布格局及形成机制,不仅具有理论上的意义,还对根瘤菌接种剂的选择具有指导意义。目前,随着分子生物学技术的发展,以及根瘤菌多样性研究数据的积累,根瘤菌生物地理学取得了较大的进展。本文综述了根瘤菌生物地理学的研究方法及现状,并对今后的重点研究方向作了展望。     

Pseudomonas fluorescens DR54 Reduces Sclerotia Formation,Biomass Development,and Disease Incidence of Rhizoctonia solani Causing Damping-Off in Sugar Beet

Effects of the biocontrol strain, Pseudomonas fluorescens DR54, on growth and disease development by Rhizoctonia solani causing damping-off in sugar beet were studied in soil microcosms and in pot experiments with natural, clay-type soil. In pot experiments with P. fluorescens DR54-treated seeds, significantly fewer Rhizoctonia-challenged seedlings showed damping-off symptoms than when not inoculated with the biocontrol agent. In the rhizosphere of P. fluorescens DR54 inoculated seeds, the bacterial inoculant was present in high numbers as shown by dilution plating and immunoblotting. By the ELISA antibody technique and direct microscopy of the fungal pathogen grown in soil microcosms, it was shown that the presence of P. fluorescens DR54 on the inoculated seeds had a strong inhibitory effect on development of both mycelium biomass and sclerotia formation by R. solani. In the field experiment, plant emergence was increased by treatment with P. fluorescens DR54 and the inoculant was found to be the dominating rhizosphere colonizing pseudomonad immediately after seedling emergence.     

Interactive effects of synthetic nitrogen fertilizer and composted manure on ammonia volatilization from soils

The mutualism between legumes and nitrogen-fixing soil bacteria (rhizobia) is a key feature of many ecological and agricultural systems, yet little is known about how this relationship affects aboveground interactions between plants and herbivores. We investigated the effects of the rhizobia mutualism on the abundance of a specialized legume herbivore on soybean plants. In a field experiment, soybean aphid (Aphis glycines) abundances were measured on plants (Glycine max) that were either (1) treated with a commercial rhizobial inoculant, (2) associating solely with naturally occurring rhizobia, or (3) given nitrogen fertilizer. Plants associating with naturally occurring rhizobia strains exhibited lower aphid population densities compared to those inoculated with a commercial rhizobial preparation or given nitrogen fertilizer. Genetic analyses of rhizobia isolates cultured from field plants revealed that the commercial rhizobia strains were phylogenetically distinct from naturally occurring strains. Plant size, leaf nitrogen concentration, and nodulation density were similar among rhizobia-associated treatments and did not explain the observed differences in aphid abundance. Our results demonstrate that plant–rhizobia interactions influence plant resistance to insect herbivores and that some rhizobia strains confer greater resistance to their mutualist partners than do others.     

Competitiveness of a <Emphasis Type="Italic">Bradyrhizobium</Emphasis> sp. Strain in Soils Containing Indigenous Rhizobia

The success of rhizobial inoculation on plant roots is often limited by several factors, including environmental conditions, the number of infective cells applied, the presence of competing indigenous (native) rhizobia, and the inoculation method. Many approaches have been taken to solve the problem of inoculant competition by naturalized populations of compatible rhizobia present in soil, but so far without a satisfactory solution. We used antibiotic resistance and molecular profiles as tools to find a reliable and accurate method for competitiveness assay between introduced Bradyrhizobium sp. strains and indigenous rhizobia strains that nodulate peanut in Argentina. The positional advantage of rhizobia soil population for nodulation was assessed using a laboratory model in which a rhizobial population is established in sterile vermiculite. We observed an increase in nodule number per plant and nodule occupancy for strains established in vermiculite. In field experiments, only 9% of total nodules were formed by bacteria inoculated by direct coating of seed, whereas 78% of nodules were formed by bacteria inoculated in the furrow at seeding. In each case, the other nodules were formed by indigenous strains or by both strains (inoculated and indigenous). These findings indicate a positional advantage of native rhizobia or in-furrow inoculated rhizobia for nodulation in peanut.     

Effect of fungicides on survival of Rhizobium on seeds and the nodulation of bean (Phaseolus vulgaris L.)

The survival of Rhizobium leguminosarum biovar phaseoli on seeds of bean was tested, using the cultivar Carioca. The seeds were treated seven days before inoculation with Benlate, Vitavax, Banrot, Difolatan or Ridomil fungicides. The rhizobial strains used were: CIAT 899, CPAC 1135 and CIAT 652. Strain CIAT 899 showed greater survival on the seed with fungicide than the other strains. Two hours after the contact with fungicides strains CIAT 652 and CPAC 1135 had significantly lower numbers of rhizobia than the treatment without fungicide. The Benlate and Banrot fungicides had the greatest effect on survival of rhizobial strains. There was a drastic mortality of the two strains, CIAT 652 and CPAC 1135, on seeds treated with Benlate and Ridomil. Under field conditions, granular inoculation produced fewer nodules, but a similar total nodule weight as seed inoculation. Serological tests (ELISA) showed that seed treatment with Benlate in connection with seed inoculation reduced drastically the occurrence of inoculated strains in nodules, while the same fungicide treatment and inoculation applied in the seed furrow did not affect the survival of the inoculated strain.     

Rhizobial position as a main determinant in the problem of competition for nodulation in soybean

Selected Bradyrhizobium japonicum strains inoculated on soybean seeds often fail to occupy a significant proportion of nodules when a competitor rhizobial population is established in the soil. This competition problem could result from a genetic/ physiological advantage of the adapted soil population over the introduced inoculant or from a positional advantage, as the soil population already occupies the soil profile where the roots will penetrate, whereas the inoculant remains concentrated around the seeds. Here, we have assessed the contribution of these factors with a laboratory model in which a rhizobial population is established in sterile vermiculite. We observed that the wild-type strain B. japonicum LP 3004 was able to grow in pots with N-free plant nutrient solution-watered vermiculite for six or seven generations with a duplication rate of at least 0.7 day(-1). In addition, the rhizobial population persisted for 3 months with 10(6)-10(7) colony-forming units ml(-1) of the vermiculite-retained solution. N-starved, young rhizobial cultures are more efficient in performing several steps along their early association with soybean roots. However, N starvation during growth of rhizobia used for seed inoculation did not enhance their competitiveness against a 1 month vermiculite-established rhizobial population, which occupied more than 72% of the nodules. When a similarly established rhizobial population was recovered from the vermiculite and homogeneously suspended in plant nutrient solution, these cells were significantly less competitive (29% of nodules occupied) than rhizobia obtained from a fresh, logarithmic culture in a N-poor minimal medium, thus indicating that cell position rather than intrinsic competitiveness was the determinant for nodule occupation.     

Using amplicon sequencing of rpoB for identification of inoculant rhizobia from peanut nodules

To improve the nitrogen fixation, legume crops are often inoculated with selected effective rhizobia. However, there is large variation in how well the inoculant strains compete with the indigenous microflora in soil. To assess the success of the inoculant, it is necessary to distinguish it from other, closely related strains. Methods used until now have generally been based either on fingerprinting methods or on the use of reporter genes. Nevertheless, these methods have their shortcomings, either because they do not provide sufficiently specific information on the identity of the inoculant strain, or because they use genetically modified organisms that need prior authorization to be applied in the field or other uncontained environments. Another possibility is to target a gene that is naturally present in the bacterial genomes. Here we have developed a method that is based on amplicon sequencing of the bacterial housekeeping gene rpoB, encoding the beta-subunit of the RNA polymerase, which has been proposed as an alternative to the 16S rRNA gene to study the diversity of rhizobial populations in soils. We evaluated the method under laboratory and field conditions. Peanut seeds were inoculated with various Bradyrhizobium strains. After nodule development, DNA was extracted from selected nodules and the nodulating rhizobia were analysed by amplicon sequencing of the rpoB gene. The analyses of the sequence data showed that the method reliably identified bradyrhizobial strains in nodules, at least at the species level, and could be used to assess the competitiveness of the inoculant compared to other bradyrhizobia.