Progress 06/01/03 to 09/30/06
Outputs
Soybeans growing in the USA form nitrogen-fixing symbioses with Bradyrhizobium japonicum and Bradyrhizobium elkanii. It is possible for cowpea to nodulate with B. japonicum and B. elkanii but it is more common for cowpea to form symbioses with Bradyrhizobium spp. collectively referred to as "the cowpea miscellany group". The "cowpea miscellany" bradyrhizobia are not able to nodulate roots of soybean plants. Preliminary work on the effect of biosolid application on cowpea Bradyrhizobial had shown that two (73-3 and 73-28 from biosolid treatment of 73 tons/ha) cowpea bradyrhizobial isolates were able to effectively nodulate both cowpea and soybeans. The occurrence of this cross nodulation is a significant finding that warranted genetic analysis. Genomic DNA from these two isolates was amplified using random primers. The PCR was carried out with 40-60 ng of pure genomic DNA in a 25 ul reaction volume by mixing template DNA with 10X Gold Buffer, 1.5 mM MgCl, 20 mM dNTP
mix, 10 uM (each) primers and 1 U of AmpliTaq Gold polymerase. The PCR products from each 25 ul mixture were separated by horizontal gel electrophoresis with 2 % agarose gel for finger printing. The PCR-based fingerprint analysis showed that these two cowpea isolates are distinctly different from the Bradyrhizobium japonicum that nodulates only soybeans and the Bradyrhizobium sp. that nodulates cowpeas. The genetic fingerprinting results do not explain the nature of the common nod genes within these two isolates. Genetic Characterization of Two Cowpea Isolates - Results: Proper expression of the nodulation genes is critical for the establishment of a nitrogen-fixing symbiosis. The three evolutionary conserved genes, nodA, nod B and nod C, are required for legume nodulation in several Rhizobium and Bradyrhizobium species. The common nodulation genes (nodABC) are required for all modes of Rhizobium infection of plant roots. A detailed genetic examination of the isolates was carried out
to determine the genetic sequences of the nod ABC genes. Comparison of the nod ABC genes in the two cowpea isolates with strains of B. japonicum and B. sp. helps to explain the genetic factor that is responsible for the cross nodulation. The diversity among the strains was related to the sequence variations in the nod ABC genes. The genetic characterization of the two isolates helps to elucidate the exact nature of the symbiotic system resulting from these two cowpea isolates.
Impacts
The identification of specific sequence variations in the common nodulation gene will expand our knowledge on rhizobium-legume symbiosis. Isolates which possess genes that effectively nodulate both cowpeas and soybeans will mean that farmers can use one common inoculant when growing both crops. Commercial farmers use rhizobium inoculants to improve plant growth of legume crops like soybeans and cowpeas.
Publications
- Cousin C., Grant J., Dixon F., Beyene D., and van Berkum P. 2002. Influence of biosolids compost on the bradyrhizobial genotypes recovered from cowpea and soybean nodules. Arch. Microbiol. 177: 427-430.
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Progress 01/01/05 to 12/31/05
Outputs
DNA has been isolated from the following strains of Bradyhizobium: 73-3, 73-28, 146-5, 146-18, 0-3, 0-15, USDA 3656 (used as a control for cowpea) and USDA 6 (used as a control for soybean). Each sample of DNA was digested with EcoRI, HindIII, BamHI. The digested DNA was electrophoresed overnight and transferred to a nylon membrane for Southern Blot Analysis. The membrane was hydrolyzed with the nodA gene used as a probed that had been labeled with ECL direct nucleic acid labeling and detection kit. The Probe for nodA was prepared by following the manufacturers instructions for PCR-amplified gene products. The results of the Southern Blot Analysis indicated that samples from 73-3 and 73-28 share consecutive nucleotide sequences with USDA6 and USDA 3656 which was not the case with any of the DNA taken from the other sequences. In a second experiment, bacterial DNA samples obtained from mannitol agar plates containing the following strains of Bradyrhizobium: 73-3,
73-28, 146-5, 146-18, 0-3, 0-15, USDA 3656 (used as a control for cowpea) and USDA 6 (used as a control for soybean) was amplified using PCR: Twenty-five microliters of the selected DNA samples were suspended in water and boiled and the a cocktail containing the following was added-T 1X reaction buffer,2mM MgCl2,1U of Taq polymerase (all from Promega),0.05%Tween 20,0.05% Nonidet p-40, each deoxynucleoside triphosphate at a concentration of 0.2mM and 6.25 pmol of each primer (final concentration, 125mM). The following temperature cycle was used: 2min at 93,aC, 35 cycles consisting 0f 45s at 93,aC, 45s at 62,aC, and 2 min at 72,aC, and finally 5 min at 72,aC. The primer is currently being designed in that this experiment can be completed. After obtaining the PCR product, it will be digested with with endonucleases HhaI, HinfI, and RsaI, The restriction products were electrophoresed in 2.5% agarose gels at 80V for 2 hrs.Ethidium bromide-stained gels were photographed by using a UVP gel
documentation system, and the band patterns were scored manually. Direct sequencing of PCR product will be done after purification using Wizard PCR Preps Column, The purified products were electrophoresed in a 1% agarose gel to estimate the amount of DNA and then Sequenced by ABI PRISM Ready reaction dye terminator cycle sequencing kit with Amplitaq DNA polymerase FS (Perkin-Elmer). An estimated 100ng of DNA was used for each reaction together with 1.6pmol of primer, 4 microliters of ready reaction mixture, and enough water so that the final volume was 10 microliters. They use the same primers describe above. This process should give us a gene sequence that can be placed in the data base for a putative match.
Impacts
The citizens of the District of Columbia can benefit greatly from information obtained from this study in reference the increased yield of two very popular food sources. Glycine max, in particular, has been used to benefit the people of D.C. because of its documented assistance in reducing the threat of cardiovascular disease, which is the major cause of death in the United States. Both plants are high in fiber which is a major desirable nutrient in the fight against obesity.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
We identified two Bradyhizobium isolates obtained from biosolids amended soil at the University of the District of Columbia Muirkirk Research Farm in Beltsville, Maryland, not previously reported in the literature. In this study, the soil received either no biosolids application or 73 or 146 Mg/ha for three successive years. Thus, the isolates were named according to the concentration of biosolids applied to the soil and the isolate obtained from numbered cultures of all Bradyrhizobium nodules for the biosolids treatment. Seven genotypes were identified in this study with very different genetic fingerprints from those observed on the type strains Bradyrhizobium sp, which has been identified in the literature. Our focus was the two Bradyrhizobium isolates, 73-3 and 73-28 formed in symbioses. These isolates were significant because they nodulated both Vigna unguiculata and Glycine max. Although it is not uncommon to find bacterial strains that are standards for
nodulating G. max that will also noduate V. unguiculata, but the opposite has not been seen. Bacterial strains that routinely nodulate V. unguiculata will not nodulate G. max. Therefore our performing our initial study, it was a natural progression to to determine the gene/genes that relate to the shared nodulation phenotype. Our investigation, to date, indicates that biosolids treatment altered the genetic composition of the Bradyrhizobium population in the soil at Muirkirk Farm bring about this previously unreported phenomenon. The molecular aspects of this work was reported our laboratory in a refereed paper (Cousin et al., 2002) published in Arch Microbiol.177: 427-430; however, the current study proposes to continue the previous work and eventually isolate the gene or genes that relate to this shared nodulation phenotype. We initiated this study by growing the glycerol stocks of Bradyrhizobium isolates 73-3 and 73-28 (obtained from the previous study) in LB broth for 24 hours and
isolating the DNA (DNA kit from Stategene, La Jolla, Calif). The DNA was amplified using PCR reactions methods described by Martin et al.,1992. With a Taq DNA polymerase buffer combination (Promega, Madiaon, Wis) with Extender Strategene, La Jolla, Calif.). The PCR products were separated by horizontal gel electrophoresis with 1% (w/v) agarose gel amended with1% (w/v) Synergel and 0.5ug ethidium bromide using 0.5XTBE buffer. The DNA of these isolates were compared with sample DNA from isolates found in nodules from G. max/ USDA 6 and V. unguiculta /USDA 3954. These were used as positive controls in this project. The G. max and V. unguiculta nodules contained isolates that were compared with isolates found in nodules from G. max/ USDA 6 and V. unguiculta /USDA 3456. These two isolates were used as positive controls.
Impacts
While using biosolids as a fertilizer on cowpea and soybeans, we found two previously unknown strains of the bacterium Bradyrhizobium. This finding implies that biosolids compost treatment alter the genotypic composition of the Bradyrhizobium population in the soil at the Muirkirk Research Farm. Although it has been observed on many occasion that the bacterial species that normally nodulate soybean will also nodulate cowpea, the reverse has never been observed until our study. The reverse meaning that bacterial species that normally nodulate cowpea will not nodulate soybean. However, we found that 73-3 and 73-78, isolates nodulate cowpea and soybean. It is hypothesized that these two isolates of Bradyrhizobium share the same set of symbiotic genes. We will try to make this determination by comparing both with standard strains of Bradyrhizobium strain that normally nodulates cowpea (USDA 3456) and the strains the normally nodulates soybean (USDA 6) This information may
help to determine the mechanism that allows them to be effective in nodulation process of both cowpeas and soybeans . It will be valuable to future research to determine if biosolids cause DNA changes that effect nodulation and plant yield in cowpeas and soybeans.
Publications
- Cousin C, Grant J, Dixon F, Beyene D, and van Berkum P. 2002. Influence of biosolids compost on the bradyrhizobial genotypes recovered from cowpea and soybean nodules. Arch Microbiol.177: 427-430
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Progress 01/01/03 to 12/31/03
Outputs
The above grant was not awarded until September 2003. Currently, I am in the process of setting up my laboratory to begin this research.
Impacts
The project will examine DNA sequences and other characteristics of unique bacteria found in biosolids-amemded soils. By converting organic waste into composted biosolids may help increase nitrogen fixation and plant yield; and if verified, this can aid the disposal of biosolids, and benefit growers world-wide.
Publications
- No publications reported this period
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