Screening Efficient Associative Nitrogen-fixing Bacteria On Three Gramineous Forages And Developing Microbe Inoculant

Some advanced instruments were applied in the research, such as gas chromatograph (GC), the fifteen nitrogen analyser with optical principle for determination of stable isotopes(NOI7), ultraviolet spectrophotometer, etc. The technique of MPN, the method of the fifteen nitrogen of isotope dilution and Salkowski colorimetry were also used. Associative nitrogen fixation bacterias (ANFB)were separated from soil away from roots(NRS), rhizoplan and surface of roots(RP)and soil adhering roots(RS) of Oat(avina satiav), smooth bromegrass( Bromus inermis. ) and highland barley(Hordeum vulgare L.). Ten associative nitrogen-fixing bacterias were isolated from turfgrass in Gansu province and Shanxi province by using special mediums(CCM, NFM). The abilities of bacteria, including fixing nitrogen, producing IAA, solubilizing phosphorus and working in tube, were determined. The strains separated from Oat and Wheat were reinoculated to Oat and smooth bromegrass,then the effect of promotion were determined. The amount of biological nitrogen was determined by isotope dilution as well. The results are as follows: 1.Associative nitrogen fixation bacterias(ANFB) were separated and enumerated from soil away from roots(NRS), rhizoplan or surface of roots(RP)and soil adhering roots(RS) of Oat, smooth bromegrass and highland barley.56 strains were from oat, 62 strains were from oat and 48 strains were from oat. Most strains are from rhizoplan or surface of roots, less from soil adhering roots and soil away from roots .The rate of RP from oat, highland barley and smooth bromegrass are 91.31%, 90.19%, 82.71%,respectively. RS are 6.80%, 7.41%, 11.48% and the rate of NRS are 1.89%, 2.40%, 5.81%.The total amount of oat and highland barley are larger than smooth bromegrass. 2. Associative nitrogen-fixing bacterias were isolated from turfgrass and forage (oat) in Gansu province by using special mediums(CCM, NFM). The abilities of bacteria, including nitrogen fixation, producing IAA, phosphorus solubilizing and working in tube, were determined. The results showed that nine in eleven ANFB strains could produce auxin (IAA), IAA concentration with L-Trp reached 2.16ug/ml～22.3ug/ml (nine strains over 10 ug/ml),IAA without L-Trp reached 0.81ug/ml～13.8ug/ml(six strains over 10ug/ml); six can dissolve organic phosphorus, three can dissolve inorganic phosphorus (phosphate solubilizing power reaching 58 ug/ml～82 ug/ml), one has double-function: two have no positive effect in big tube, others have positive effect. Shoot length increased 62.8%～142.8%, chlorophyll 2.78%～63.89%, Above dry weight 21.45%～150.48%, Under dry weight 62.8%～142.8%,Total length of root 2.27%～41.27%, Area of root 11.32%～84.9%; I017 and I012 were better in producing auxin, I016 was better in synthetic ability .They can be used as bio-fertilizer. 3. Eleven associative nitrogen-fixing bacterias were isolated from turfgrass in Gansu province and Shanxi province and alpine steppe in Gansu province. The ability and dynamic of producing aunix were determined in a week. The results showed that the amount was stable three days later and did not vary from then on. In order to validate the results, the dynamic auxin during14 days were determined as well. The concentrations of IAA in the two experiments were different, but the law of variety did not change. 4. The ANFB strains separated from oat, turfgrass and wheat were reinoculated to oat. The order of the strains which have better effect of promotion on oat is W5, W6, O5, C6, N4. All can germinate in one or two days ealier. 5. The ANFB strains separated from oat and wheat were reinoculated to smooth bromegrass. The order of the strains which have better effect of promotion on oat is W5, W6, C6, O5, N4. The results showed that chlorophyll content, leaf length, shoot length and yield of the reinoculated strains increased differently to some degree. 6. The ANFB strains separated from oat, turfgrass and wheat were also reinoculated to oat, and the quantity of biological fixing nitrogen were determined by ID. The high amount of biological nitrogen fixation strains were screened. The order of associative nitrogen fixation inoculant is N4>W6>W5>C6; The order of producing auxin inoculant isO6>I041>I033>O5>O3; The effect of mixture inoculant O5+I012+191 is better than N4+I012+191.