Selection Of Better Rhizobia Strains For Dolichos Lablab Infection And Their Inoculation Effects

Dolichos lablab L. is a legume forage or vegetable, belonging to genus Dolichos, Phaseolus, Papilionoideae of leguminous. It was widely distributed in tropical and subtropical regions because of its high-yield and good feeding quality. In current study we separated rhizobia from D. lablab and screened the excellent strains through biological characteristics of the rhizobia, investigated the influence of rhizobia on growth, development, nutritional characteristics, the yield and quality of D. lablab under the conditions of different soils, fertilization rates, and double inoculation of arbuscular mycorrhizal fungi and rhizobium. The results have laid a foundation for scientific cultivation and improving yield and quality of D. lablab. The main results are as follows:1Biological characteristic of the rhizobia from Dolichos lablab and better rhizobia strains selectedNine rhizobia strains were separated from nodules of D. lablab. The strains were salt-resistant, acid and alkali-resistant, dye-resistant and antibiotic-resistant. They could tolerate a salt condition of0to6%NaCl, and could live in a condition of pH4to11. However, the most suitable conditions for the rhizobia were:temperature27to29℃, pH6.0to8.0, concentration of NaCl0to0.12%. Among the nine strains, the nitrogenase activity, secretion of growth hormone and dissolving phosphorus of rhizobium bacteria R1and R2-2were significantly higher than those of other strains, indicating that R1and R2-2might have strong nitrogen-fixing effect and growth-promoting effects and could be developed as bio-fertilizer for D. lablab L. R2-3could dissolve inorganic and organic phosphorus, R3-2and R2-6could grow in pH11.0, R1,R2-4,R2-6could grow in6%NaCl, and they had the effects of phosphorus-dissolved and salt tolerance.2Inoculation effects of different rhizobia strainsNine rhizobia were separately inoculated to D. lablab (Rungao). The results showed that all nine rhizobia could form nodules with D. lablab and had an effect on its growth and nutrient uptake etc. Among the nine strains, nitrogen-fixing and growth-promoting effects of R1and R2-2were the best. Compare to control group (non-inoculated group), the biomass increased71.05%(R1) and67.62%(R2-2) and the average content of nitrogen, phosphorus, potassium, calcium, magnesium increased2.21%,0.28%,1.37%,1.33%,0.16%. The inoculation also improved nitrate reductase activity, chlorophyll content in leaves and root vigour of D. lablab at different degrees.3Response of Dolichos lablab's rhizobia to different soilsThe influence of rhizobia inoculation on nodulation, growth, photosynthesis, nutrient quality and absorption by Dolichos lablab as well as soil nutrients were conducted in four typical and representative soils widely distributed in Chongqing, two grey brown purple soils with pH4.06and6.14, a red brown purple soil with pH7.50and a yellow limestone soil with pH6.45. The results showed that the growth, yield and quality of D. lablab in the two grey brown purple soils with pH4.06and6.14were better than those in the yellow limestone soil and alkaline red brown purple soil. Rhizobia inoculation could form nodules in the slight acidic grey brown purple soil(pH6.14) and yellow limestone soil (pH6.45), could form fewer nodules or no nodules in acidic grey brown purple soil (pH4.09) and alkaline red brown purple soil (pH7.50), which indicated that higher or lower pH value was unfavorable for nodule formation. Nodule formation could improve D. lablab's nitrogen and phosphorus nutrition, net photosynthetic rate, promote the growth, increase biomass and quality to some extent. Positive correlations were obtained between biomass, nitrogen and phosphorus absorptions, and the soil nitrogen and phosphorus and nodule weight, indicating the nodule weight might reflect the nodule activity. The nodule number in the slight acidic grey brown purple soil was53.32%lower than that in the yellow limestone soil, while the size was lager and the nodule weight was1.66time to that in the yellow limestone soil, showing better inoculation. The selection of slight acidic and acidic grey brown purple soils and rhizobia inoculation could increase the yield and improve the quality of D. lablab in Chongqing,4The influence of nitrogen fertilizer on growth and nodulation effect of Dolichos lablabThe plants were seeded in pots with four nitrogen levels, No, N8, N40, N200(Ni=mg/kg dry soil). All pots were inoculated with Rl to investigate the influence of nitrogen fertilization on the growth, nodulation, nutrition absorption and rhizosphere microorganisms of D. lablab. The nodule number and weight were significantly higher for nitrogen fertilized plants than the control. The nodule number and weight increased first and then decreased with increasing nitrogen levels. This indicated that nodule formation of D. lablab required a certain amount of nitrogen. However, too much nitrogen supply might have an inhibitory effect. The biomass and NPK (nitrogen, phosphorus, potassium) uptake of D. lablab increased with increasing nitrogen levels. Compared with the control, total biomass for each nitrogen fertilizer level increased19.85%(N8),47.60%(N40) and65.55%(N200), respectively. The number of rhizosphere bacteria and azotobacter were the largest with high nitrogen level. Under low nitrogen level, the number of rhizosphere fungi and actinomycetes were the largest. Nodule weight positively correlated with nitrate reductase activity, chlorophyll content, root vigour, nutrient absorption and the number of rhizosphere bacteria and azotobacter. It was concluded that the growth of D. lablab demand more nitrogen. Under high nitrogen level, it could also maintain high nodule formation ability, improved nutrient absorption and biomass. Therefore, it is necessary to supply appropriate amount of nitrogen in D. lablab's cultivation practice. 5Effects of chemical fertilizer combined with organic manure and rhizobium inoculation on photosynthesis, nutrient absorption and soil enzymes of Dolichos lablabFertilization is one of the most important factors affecting the growth and feeding quality of forage. Chemical fertilizer combined with organic manure (CK, F, F0.75M0.25, F0.5M0.5, F0.25M0.75, M) were used to investigate the influence of fertilizer treatments on nodulation, growth, physiology, photosynthesis, feeding quality, nutrient absorption by D. lablab as well as soil enzyme activity and the quantity of rhizosphere microorganisms. The results showed D. lablab had better growth, yield and quality with F0.5M0.5and F0.75M0.25, followed by F and F0.5M0.5, and M, CK was the worst. Compared with F, the yield of F0.5M0.5and F0.75M0.25increased19.46%and16.49%, respectively. Plants with F and CK formed fewer nodules (19～26No./pot) and lower nodule weight (0.58-1.56g/pot). However, nodule number and nodule weight increased with increased manure proportion. When the proportion of organic fertilizer increased from25%to100%, nodule number increased from54to109nodule/pot, nodule weight from3.13to3.90g/pot. Fertilization could improve the nitrate reductase activity, chlorophyll content in leaves and root vigour. The diurnal variation of photosynthetic indexs under different fertilization were basically the same. There had no obvious lunch break. The diurnal variation of net photosynthetic rate, transpiration rate, stomatal conductance, CO2use efficiency, light use efficiency appeared typical "single peak" and the curve peak was around10:30PM. Different fertilization influenced the photosynthetic capacity of D. Lablab. The net photosynthetic rate, transpiration rate, stomatal conductance, CO2use efficiency, energy use efficiency in F0.5M0.5were the highest, CK and F were lower. The photosynthetic capacity was significantly positively correlated with growth, physiology and nodule formation. Organic fertilizer could improve D. lablab's contents of crude protein, crude fat, crude ash and nitrogen, phosphorus, potassium, and thus improve feeding quality. The ground biomass of F0.5M0.5and F0.75M0.25were the highest, of which the contents of protein, crude fat, crude ash, soluble sugar yield and nitrogen, phosphorus, potassium, calcium, magnesium absorption were also the highest. Compared with pure chemical fertilizer, organic manure could improve the soil enzyme (catalase, invertase, urease) activities. Correlation analysis showed shoot biomass (r2=0.6758), nitrogen (r2=0.7269), phosphorus (r2=0.9759) and potassium (r2=0.9488) absorption were positively correlated with nodule weight. Nodule formation could improve nitrogen, phosphorus and potassium nutrition, promote growth, increase biomass and quality to some extent. In cultivation practice in Chongqing, the selection of chemical fertilizer combined with organic manure and rhizobium inoculation could increase the yield and improve the quality of D. lablab.6Influence of AM fungi on Dolichos lablab's growth and nutrient absorptionFour Arbuscular mycorrhizal fungi (AMF)(three were Glomus intraradices, another was Glomus etunicatum) were inoculated to investigate the influence of AMF on growth, physiology and nutrient absorption of D. lablab. The results showed that four AMF could infect D. lablab and promote it's growth and nutrient absorption. The AMF BEG-193and BEG-167had the strongest mycorrhizal effect with highest mycorrhizal infection rate, growth rate and nutrient content. Compared with control, the biomass of D. lablab increased40.19%(BEG-193) and40.65%(BEG-167), respectively. Averagely, absorption of NPK (nitrogen, phosphorus, potassium) increased55.48%,86.94%and35.79%, respectively. Furthermore, nitrate reductase activity, chlorophyll content in leaves and root vigour of plants inoculated with AMF improved at different degrees.7Influence of co-inoculation of rhizobia and AM fungi on Dolichos lablab's growth and nodulation effectThe growth and nodulation effect of plants inoculated with both R1or BEG-193were better than those with single inoculation. The nitrate reductase activity, chlorophyll content in leaves, root vigour, biomass of double inoculated plants were significantly higher than those of single inoculation and uninoculated control. Compared with control, the biomass of R1,BEG-193and double inoculation increased21.30%,45.28%and64.65%, respectively; nitrogen accumulation increased40.66%,63.17%and96.70%, respectively; phosphorus accumulation increased38.11%,84.40%and142.17%, respectively; potassium accumulation increased20.24%,54.16%and69.14%, respectively. The number of bacteria, fungi, actinomycete and azotobacter in D. lablab's rhizosphere were larger with treatment of double inoculation than single inoculation and control.