Decreasing the utilization of nitrogen fertilizers through improvement the biological nitrogen fixation (BNF) in chickpea (Cicer arietinum) cropping area

Chickpea scientifically named Cicer arietinum is the worlds third ranking and Iran's first ranking legume in production tonnage. The area under production of this crop in Iran adds up to 59 thousand hectares with an annual yield of 24 thousand tons. The symbiotic Rhizobia on this plant are quite effective in fixing molecular nitrogen. Therefore the study of this symbiosis phenomenon is of great importance. The current study with a collection of 400 chickpea nodules followed by the isolation of some 130 isolates related to Mesorhizobium ciceri. One hundred of these isolated bacteria were found to be effective in plant infection for nitrogen fixing processes. An experiment with a completely randomized block design using 77 of the 100 selected isolates along with 4 imported strains were tested with three levels of nitrogen (N0, N35, mg/kg, and N70 mg/kg) for their potential in fixing the atmospheric nitrogen. Each treatment was replicated three times using a Leonard Jar containing sterile sand. The results showed significant differences (α = 0.01) in the N-content of the above ground plant portions caused by the plant showed significant differences (α= 0.05) for the different treatmens. Moreover the following specific primers were used in PCR amplification to insure the persence of nitrogen fixing gene and the evaluation of the strains to confirm as Rhizobium strains: 5ó- CGT TTT ACG GCA AGG GCG GTA TCG GCA- 3ó nifH1 5ó- TCC TCC AGC TCC TCC ATG GTG ATC GG- 3ó nifH2 5ó- AYT TCC TKG ARG ARA ACG GYG C-3ó nif1 5ó- ACG AGC AGA TGC TKG MKM AMT- 3ó nif2 For PCR amplification of samples the easiest method of preparing a target DNA was performed with excellent results in spite of its simplicity. Electrophoresis products of the PCR of M. ciceri isolates showed a clear band of amplified DNA at the region 780 nucleotide pair showing the products to confirm with the reported data. The PCR products were gels purified and sequenced. Amplification primers were used for sequencing both strands of the PCR products confirmed the PCR fragment containing 773 bp. The following primers were used for the determination of the polymorphism of the strains: 5ó- CTA CGG CAA GGC GAC GCT GAC G- 3ó BOXA1R 5ó- ATG TAA GCT CCT GGG GAT TCA C- 3ó ERIC1R 5ó- AAG TAA GTG ACT GGG GTG AGC G- 3ó ERIC2 5ó- AAT TTT CAA GCG TCG TGC CA- 3ó APO1 5ó- GGA AGT CGC C-3ó RAPD 5ó- GAC GAC GAC GAC _ 3ó SPH1 Experiment with the BOX and ERIC primers did not show enough polymorphism in M. ciceri, however SPH1, RAPD, and APO1 primers were suitable polymorphs for these bacteria, and thus could effectively be used as makers for variation studies of this species. An investigation on the cumulative cluster resulting from the matrix converted RAPD and APO1 primers indicated such a genetic variation. To study plasmid profiles of such samples using different variations of Eckhardt (in situ) technique, such as the one by Rosenberg (1981), or by Hynes (1984), or by Hashem (1994, 1996) and Simarov showed that non of the above techniques produced strong chemical bonds or reproducible results with M. ciceri under our laboratory conditions. More modifications of these methods would therefore be necessary for the prevailing conditions in Iran. Chickpea have been integrated into farming systems in arid and semiarid regions of Iran, However no information is available regarding rhizobium inoculation of this crop. so field studies conducted on 11 site in 2001-2004. The objectives of the research were determine the effects of rhizobium inoculation and micronutrient fertilization on nodule formation, N2-fixation, and their impacts on growth, yield and seed quality in chickpea and develop recommendations for optimizing rhizobium inoculation. The results of 11 sites with 16 effective that 10 strains with N treatment and controls in any of sites (2-3 native, 2 recommend strains form ICRICAT, 1 from ICARDA and 4-5 very effective strains from Iran) showed that use of 500-1000gr rhizobium inoculation increased yield and quality of seed as equal as optimal N fertilizer in all sites and in some sites effect of inoculation significantly is more then N fertilization. For the first time the information generated from this study will provide Iranian farmers with new and updated information in the area of seed inoculation with rhizobial inoculant and micronutrient fertilization of chickpea this study also inform the agricultural policy mekers about potential if Iran in organic production of chickpea in this country.