Study On Dual Culture And Spore Development Of Cetraspora Huaxica

The Arbuscular Mycorrhizal Fungi(AMF)belong to the subphylum Glomeromycotina and are endophytic mycorrhizal fungi that can establish a symbiotic relationship with 80% of the world’s terrestrial plants.Through its large hyphae network structure,AM fungi can expand the contact area between plant roots and soil,so as to promote the absorption of mineral elements such as nitrogen(N)and phosphorus(P)in soil,and play an important role in improving plant stress resistance and disease resistance,enhancing the host plant’s resistance to heavy metal stress,improving soil composition and repairing soil heavy metal pollution.Therefore,obtaining a large number of AM fungal agents is currently an important direction in AM fungal research.However,the characteristic of AM fungi as a specialized symbiosis severely limits the application of AM fungi in vitro culture and AM fungal agents in production practice.In this study,a new AM fungus species,Cetraspora huaxica,was used as the material to establish an in vitro double culture system with the hairy roots of four tobacco and Belladacea plants.The developmental processes of C.huaxica,including spore germination and hypha growth,were observed and recorded,and a large number of helper cells were obtained.In addition,the primary exploration of host-free culture of AM fungi was carried out,and the secondary spores of C.huaxica cysts were obtained,and the pure culture of C.huaxica cysts without host was initially realized.Specific test results are as follows:1.Tobacco(K87,Va116,NC82,Bina No.1)and Belladonna explants were infected with Agrobacterium rhizogenes C58C1 to induce hairy roots with a large number of white hairs and stable growth on hormone-free medium;Through morphological and molecular biological methods,it was proved that the five kinds of hairy roots induced by it were Ri T-DNA transformed roots.The induction rate of hairy roots: K87(91.67%)> Va116(86.11%)> Bina No.1(77.78%)> NC82(69.45%)>Belladonna(52.77%).2.AM fungi spores isolated from soil by wet sieving and sucrose density centrifugation were identified by morphological and molecular biological means as a new species of AM fungi and named Cetraspora huaxica.The best results were obtained using 75% alcohol disinfection for 1 min and liquid A and liquid B disinfection solutions for 12 min respectively for C.huaxica spores;in spore germination conditions:t the addition of GR24 at a concentration of 450 pg/L in water agar medium at pH 6.0effectively promoted spore germination.3.A dual culture system was established between four species of Tobacco(Va116,NC82,K87,Binar No.1)and the hairy roots of Belladonna with C.huaxica,and the hairy roots of Binar No.1 was selected as the optimal host for C.huaxica spores.In the course of the dual culture,the formation of auxiliary cells,the "D"-shaped ring structure,the "8"-shaped structure and the typical ALS structure formation of multilevel branches,hyphal healing and hyphal fusion were observed and recorded.The exogenous hormones methyl jasmonate MeJA and GR24 were effective in promoting the construction of the dual culture system;MeJA at 1 μmol/L effectively promoted hyphal branching,while GR24 had a significant effect on the formation of auxiliary cells,with the highest production of auxiliary cells at a concentration of 750 pg/L.4.The establishment of a host-free pure culture of C.huaxica was achieved by adding myristic acid to the MSR medium,which significantly increased the germination rate of spores,with a maximum spore germination rate of 82.22% at a fatty acid concentration of 500 μmol/L.Meanwhile,the addition of different concentrations of GR24 could effectively promote spore germination,hyphal growth and new spore production.The addition of GR24 at a concentration of 450 pg/L not only strongly stimulated spore germination,but also effectively accelerated the rate of spore germination,with a maximum spore germination rate of 95.56% by the 8th day;when the spores were cultured for the fourth month,the formation of new spores began to form,and the in vitro pure culture of C.huaxica was initially achieved.In this study,we successfully established the dual culture system of C.huaxica and plant hairy roots,and initially achieved the host-free pure culture of C.huaxica.The optimum pH,GR24 and MeJA concentrations for spore germination were screened.The growth of spore hyphae,mycelial healing,mycelial rings and auxiliary cells were recorded during the dual culture process.Meanwhile,the optimum concentrations of potassium myristate and exogenous hormone GR24 were screened in host-free pure culture,which promoted the germination of spores and mycelial growth of C.huaxica and the production of new spores,providing a theoretical method for the host-free culture of AM fungi.