Study On Dual Culture And Spore Development Of AM Fungi Of Acaulospora

Arbuscular mycorrhiza(AM)fungi can be used as natural fertilizers and biocontrol agents,which have the functions of promoting the uptake of mineral nutrients in plant soil,enhancing plant resistance and disease resistance,improving soil structure and remediating heavy metal pollution in soil,and have a wide range of prospects in agricultural(forestry)production and applications.Since AM fungi have the characteristic of specialized symbiosis,they must rely on host plants to complete their life cycle,which makes AM fungal agents greatly limited in large-scale production.In this study,we established a dual culture system of five tobacco and belladonna plants with hairy roots and AM fungus(Acaulospora koskei)in vitro;constructed the genetic transformation of tobacco and belladonna plants by Agrobacterium rhizogenes to produce hairy roots and a surface disinfection method for AM fungal spores,and also studied Acaulospora koskei spore germination,mycelial growth conditions,structural changes and developmental processes,and obtained AM fungal propagules.The results of the study provide an effective method for the large-scale production of pure fungal agents of AM fungi,the utilization of AM fungal resources and the preservation of germplasm resources.The experimental results are as follows:1.Obtaining sterile seedlings: Tobacco(K326、K87、Va116、NC82、Bina No.1)and belladonna seeds were soaked in 75% alcohol for 1 min and 10% sodium hypochlorite for 15 min,inoculated on MS solid medium and incubated at 25°C,14 h/d light culture to obtain sterile seedlings.2.Induction of hairy roots: Agrobacterium rhizogenes C58C1 was used to infect the leaves of five species of tobacco(K326、K87、Va116、NC82、Bina No.1)and belladonna aseptic seedlings,and hairy roots were obtained by bacteriostatic and bactericidal culture,and the induction rates were NC82(83.33%)> Belladonna(63.33%)> K87(60.00%)> Bina 1 No.(56.67%)> Va116(53.33%)> K326(43.33%).Through morphological and molecular biological methods,it was proved that the six kinds of hairy roots induced by it were Ri T-DNA transformed roots,and the hairy roots were subcultured by both solid and liquid culture methods.Solid medium was more favorable for the growth of hairy roots.3.Spore identification and germination: AM fungal spores were isolated from soil by wet sieving and sucrose density centrifugation and were morphologically identified as Acaulospora koskei.The best disinfection time for Acaulospora koskei spores was10 min for solution A and 10 min for solution B;The best p H in water agar medium was6.0;The germination effect was best at the GR24 450pg/L and SPM 40mg/L,and the germination rate were 61.67% and 58.34% respectively;The germinated spores were identified by molecular biology,and the germinated spores were further proved to be Acaulospora koskei by establishing a phylogenetic tree.4.Establishment of a dual culture system: A dual culture system was established between the induced hairy roots of 6 species of plants and Acaulospora koskei,it was found that only tobacco Va116 hairy roots and Acaulospora koskei successfully established a dual culture system and produced new spores.The newly formed spores were milky white at first with a diameter of about 20-40μm,and when the spores matured,they were brown-black with a diameter of about 40-60μm.Through mycorrhizal infection,the invasion point,the distribution of extra-and intra-radicular mycelium,the formation of vesicle structures,and root endospores were observed,further confirming the successful establishment of the dual culture system.In this experiment,five stable Ri T-DNA hairy root induction systems of tobacco and belladonna were established,and a large number of hairy roots were obtained,which provided a good host material for the establishment of the dual culture system in the future;The optimal conditions for spore germination of Acaulospora koskei were explored,and a large number of aseptically germinated spores were obtained;a dual culture system of tobacco Va116 hairy roots and Acaulospora koskei was successfully established,new spores were produced,and the morphological characteristics of extraroot and intra-root hyphae,the formation of vesicle structures,and the development and growth of spores were recorded.Through mycorrhizal infection,the invasion point,the distribution of extra-and intra-radicular mycelium,the formation of vesicle structures,and root endospores were observed,further confirming the successful establishment of the dual culture system.