Promoting Effects Of Nitrogen Fixation Bacteria XD20 On Different Sugarcane Varieties

The use of large amounts of nitrogen fertilizer in crop production,although increasing the output,it also caused great pollution to the environment.Such as the greenhouse effects,diminishing stratospheric ozone and acid rain to changes in the global N cycle and nitrate pollution in surface and ground water.Sugarcane is an important sugar and energy crop in China.Guangxi is one of the major sugarcane planting province in China.Nitrogen fertilizer is one of the most expensive inputs in sugarcane production.If the application rate of nitrogen fertilizer in sugarcane production can be reduced,it is of great significance to sugarcane production in Guangxi.A large number of studies have shown that inoculation of endophytic nitrogen-fixing bacteria in sugarcane can promote the growth of sugarcane and increase the yield of sugarcane without applying or using less amount of nitrogen fertilizer.In this study,The strain XD20 isolated from the roots of the main sugarcane cultivar ROC22 in Guangxi,was identified by 16S rDNA and the growth charateristics were studied.After inoculation of the three micropropagated sugarcane varieties B8,ROC22 and GT40 with the strain,the effects of inoculated strain XD20 on the physiological characteristics of three different sugarcane varieties were compared,and the physiological basis of nitrogen fixing bacteria to promote sugarcane growth was discussed in order to provide reference basis for nitrogen reduction and synergism production of sugarcane.The results are shown as follows:1.The strain was identified by 16S rDNA and its growth characteristics were analyzed.16S rDNA was amplified by 27F/1492R primer,and a band about 1500 bp was obtained.The strain was identified as pantoea agglomerans by sequencina and phylogenetic tree analysis.Detection of nifH gene of strain XD20 using the universal primer PolyF/PolyR by PCR and amplification of 303 bp gene firagment was got.The nitrogenase activity of the strain XD20 was determined by gas chromatograph and the nitrogenase activity of the strain XD20 was 3187.8 nmolC2H4·mL-1·h-1.The Strain XD20 also has many other growth-promoting properties,such as producing indole acetic acid（IAA）,dissolving organic phosphorus and producing ferriophilic.2.Effect of culture conditions on the growth of the strains were studied.The effects of environmental factors such as different culture time,temperature,OSosmotic pressure and pH,on the growth of XD20 were measured using LB liquid medium as the basic medium.The results showed that the optimum growth temperature of XD20 was 30 ℃.And when the pH condition was neutral,the growth of strain XD20 was prosperous.The strain XD20 also has strong adaptability to osmotic pressure and high sucrose concentration and can utilized NO3-and NH4+to provide the required nitrogen for the growth of the strain3.The nifH gene in different parts of sugarcane were detected.Three sugarcane varieties,B8,ROC22 and GT40,were used as materials for tissue culture.The method of co-culture of bacteria suspension and tissue culture seedling was used.At 72 h after inoculation,the nifH gene could be amplified from the roots and aerial tissues of the seedlings,which indicated that strain XD20 had colonized in sugarcane plants.The relative expression of nifH gene was the highest in ROC22 plants at 3 days after inoculation.With the increase of inoculation time,the expression of nifH gene in B8 was higher than that in inoculation time,the expression of nifH gene in B8 was higher than that in ROC22 and GT40.4.After 60,90 and 120 days of inoculation,the plant height and chlorophyll content of sugarcane were determined,and the physiological activity indexes of+1 leaf were also measured.The results showed that inoculation treatment increased plant height and chlorophyll content,dry weight of above-ground and underground parts and the activity of nitrate reductase（NR）,glutamine synthetase（GS）,nitrate nitrogen content and soluble sugar and soluble protein content of sugarcane leaves were also increased.Comparing with the three sugarcane varieties,but the effects on B8 were most obvious.5.The effect of inoculation treatment on GS transcription in sugarcane was studied by RT-qPCR.The results indicated that XD20 increased the expression of scGS₁ and scGS₂ gene at mRNA level.