Influencing Factors On The Symbiosis Of Ammopiptanthus Mongolicus (Maxim.) With Rhizobia And Arbuscular Mycorrhizal Fungi(AMF)

The study of the cultivation and application of the precious and rare desert plant Ammopiptanthus mongolicus (Maxim.) in Beijing and the whole north area in China was less. Almost no one made the study from the symbiosis between the microorganism and the plant and influencing factors on the Symbiosis. So it is of great theoretical value and practical meaning to startup the study of symbiosis effect. In present work, based on the former study of the introduction, the paper got deep into branching out the direction of the item. Used the native rhizobia strains and the native AMF of A. mongolicus,combined with commercial AMF, the singal factors contain nod factors, flavonoid, Ca²⁺ and ABA on root hair deformation before nodules formation were analyzed. The optical electron microscope, scanning electron microscope (SEM), transmission microscope and energy-dispersive X-ray analysis(EDX) were used to analyze the changes of the structure and ultrastructure with nodules'growth. The qualitative and quantitative analysis of Ca²⁺ also were studied to make clear its function in root nodules'growth and nitrogen fixation. The main experimental results are as follows:1) 18 rhizobia strains of A. mongolicus were isolated and purificated from the different locations in northwest desert of China.3 nitrogen fixing effective strains contains ZW1,AZ1 and DK1-2 were selected out from these strains by re-nodulation. A native predominant AMF(Glomous.claroideum Schenk& Smith)was selected. The sand and 0.4 mmol·L^-1P level were selected for AMF spors growth from different cultivated media and different P level.2) The compatible N, P concentration were selected for the symbiosis of A. mongolicus and AM fungi and rhizobia. Under the lack of different nutrition element, we found the lack of N,P,K,Mg,Ca and Fe affect the shape and characters badly, the plant growth was restrained, especially lack of N,P,K, the growth almost stagnated. Under the level between 0-3.75 mmol·L^-1 of N, the nodules grew well; at the level of 0.4 mmol·L^-1 of P, AMF promoted the plant growth best.3) Combined with commercial fungi G.v, the study used the rhizobium strains ZW1,AZ1and AMF strains G.c,G.v, inoculated singlely or by combinations to A. mongolicus, and the best combination ZW1+G.v was sorted out for the plant growth. All the groups showed the better results on plant growth,nodulation and AMF infection to control,but the results had differences in those combinations,showed the importance of selection and cooperation between AMF and rhizobia. The plant biomass with group of ZW1+G.v was 171.0% to control, but didn't make the plant nearly have a strong dependence on the ZW1+G.v.4) The proper flavonoid was selected out for promotion on the interplay between the symbiosis and plant growth. Study showed that the biomass and nodule number of A. mongolicus, AMF infection rate, and hyphal enzyme activity of ALP and SDH were significantly promoted by flavonoids'action on Rhizobia & AMF. Flavonoid treatments had more obvious differences with Rhizobia &AMF treatments than control in these aspects,and obvious differences were found in different flavonoids (apigenin and hesperitin) or in different concentrations (0.15 umol L-1, 1.5 umol L-1and 15 umol L-1 )of the same flavonoid, and result with 1.5 umol L-1 is most significant in all of the groups. 15 umol L-1 of hesperitin had some inhabition on AMF infection, and hyphal enzyme activity of ALP and SDH on some groups,but didin't show inhabition or promotion on plant growth on the same group.The results showed that the promotion effects of nodulation of Rhizobia and AMF infection were more significant when an appropriate amount of flavonoid was added in the Rhizobia & AMF treatments.5) By the preliminary study on the root nodule formation of the A. mongolicus, various changes of A. mongolicus roots were observed. It was observed clearly that the position where rhizobia invaded the root of A. mongolicus was almost at the top of root hair and then entered the cortex. Signal factors contained nod factor(nf), flavonoids,Ca²⁺ and ABA on inducing the root hair deformation were studied. The former three factors demonstrated positive function. The higher the nod factors concentration and the activity, the more significantly it affected. 1.5μmol.L-1 of hesperitin was best in all flavonoid treaments, the percentage of seedlings with root hair deformation reached to 67.5% after 3 days.10 mmol.L-1 of Ca²⁺ with nf was most promotional in all treatments with or without nf, and the deformation rate fell by 20mmol.L-1 of Ca²⁺ even with nf. The results of them indicated that nf, flavonoid and Ca²⁺might play an important role in the recognition between rhizobium and its host plant.The function of ABA is opposite to nf. The higher the ABA concentration, the more significantly it inhibited in root root hair deformation,nodule number and the plant growth.The ABA inhibitor tungstate could conversed this situation.6) Root nodule development was studied with optics microscope and transmission electron microscopy. The nodule contains exodermis, cortex (contains vascular bundle), vagina matrix and vortex tissue,and it belongs to indefinite type. The infection threads were very abundant in the nodule cells and very wide in distribution. The rhizobia in young infected cells were small, few in number, spherical generally located near the cell walls or nucleus. The rhizobia in mature infected cells were larger,great in number, varied in shape,and distributed throughout the nodule cells.It also had much poly-hydroxy butanoic acid (PHB),the spherical rhizobia were seen to reproduce by fissiparism. The rhizobial morphology in senescent infected cells was irregular,their cytoplasm occured contraction and increased in electron—density, decomposed and had fibrilsome materials. it is not only so, their inside structures also became unclean,some rhizobia even became into a membrane—vesicular structures.7) The scanning electron microscope (SEM), transmission microscope and energy-dispersive X-ray analysis(EDAX) were used to make the qualitative and quantitative analysis of Ca²⁺ with the changes of the structure and ultrastructure by nodules'development to make clear the function of Ca²⁺ in root nodules'growth and nitrogen fixation. As a result,the content of Ca²⁺ was uplifted from the early forming stage to early senescent stage. In early development, the total distribution was much more comparatively. They distributed in cell walls, intercellular space, plasma membrane and bacteria premembrane, most presented in spots, lines or gobbet, illuminated the support and facilitation function of Ca in pushing the infection threads get into cells and promoted the rhizobia reproduction and growth,it also promoted the singal and materials communion between the rhizobia and plant cells. In mature cells, a normal of Ca²⁺ sediment could be seen,this was better for structural stability and exertion of function of bacteria in the nodule. In early senescent cells,too much Ca²⁺ localized in the cytoplasm near the bacteria,showed its promotion on bacteria and cells senility. Ca²⁺ scattered in disorder and droped a lot in senescent cells. As a result of SEM and EDAX, the tendency of Ca total content in different nodule development was the same as before. And the content from high to low in different structures was epidermis cells,vortex tissue and before nodule senility,but when nodule became senile seriously,the content from high to low was vortex tissue,cortex and epidermis cells.Rule of the total changes of intensity signal of EDAX and number of"calcium rich"zones were consistent with in SEM, calcium accumulated abundantly in the epidermis cells, which formed a calcium rich layer in four stages of nodule development.The study found that Ca²⁺ signal was related tightly with the nodule's development and its function, so their seedlings growth were also affected.