Molecular Identification And Culture Optimization Of Thelephora Ganbajun And Lactarius Deliciosus Isolates

Thelephora ganbajun, a species kind of ECM fungi in Yunnan Province, is a basidiomycete fungus of the Thelephora family. Deliciosus Lactarius is also ECM fungi (Lactarius). Both of them can symbiose with the pine Pinus yunnanensis, but the distribution range of Thelephora ganbajun is more narrower, and has be less studied. While the Lactarius deliciosus and Thelephora ganbajun growing environment similar and it has more research content which is applicable to Thelephora ganbajun Zang. Since there is no matured technology to control the generation of fruiting bodies, it is completely different from common saprophytic edible fungi, for example:Ganoderma lucidum, Flammulina velutipes, Auricularia auricular and so on, can not rely on accurate temperature, humidity, and nutritional conditions for the whole-year production, only rely entirely on wild collection. Especially the incidence of Thelephora ganbajun under natural conditions is lower than other mycorrhizal fungi, so the yield is lower. Due to the difficulty in separating the mycorrhizal fungi from the mycorrhizal organs or fruiting bodies, and a lot of mycorrhizal fungi on artificial medium growth slow or can not grow, therefore, it is an urgent task to improve the isolation and identification methods of mycorrhizal edible fungi.The aim of this study is to optimize the separation method and to isolate the mycelium of Deliciosus Lactarius and Thelephora ganbajun through single factor and orthogonal experiments investigating the optimal nutrition conditions and pH conditions of Yuxi and Xiaoshao isolate from Thelephora ganbajun. By Optimizating the culture medium and suitable culture conditions of L. deliciosus isolated through orthogonal experiments in comparison with the results of relevant studies, we shorten the culture time and get more mycelium to prepare inoculation; compare differences of two strain of Thelephora ganbajun on physiological characteristics and gene fragment sequences; then go on the test of synthetic mycorrhiza. The following is main results:1. Using the organization separation method. we isolated the pure culture of L. deliciosus, and established a new separation method--air conduction separation method, thus obtained Thelephora ganbajun mycelium.2. The optimum culture conditions of the strain of Thelephora ganbajun isolated from Shao Xiang is 20g glucose, peptone 2g, yeast powder 2g, magnesium sulfate 0.5g, KH2PO40.5g, initial pH5.5,1L water, and the strain of Thelephora ganbajun isolated from Yuxi is 20g glucose, ammonium sulfate 4g, yeast powder 2g, magnesium sulfate 0.5g, KH2PO40.5g, initial pH5.5,1L water; the optimum culture conditions of Deliciosus Lactarius strain is glucose 10g, peptone 4g, yeast powder 2g, magnesium sulfate 1g, 1gKH2PO4, PDA medium 1L.3. Three isolates of Deliciosus Lactarius and Thelephora ganbajun were identified as ITS sequence, two strains of Thelephora ganbajun proved to be identical in ITS sequence; the two strains are different from each other at a single nudeotide site in either the 12s RNA or the TUB sequences.and with two sites difference in 16s RNA sequences.4. Obtaining Yunnan pine seedlings with synthesized mycorrhizal of Deliciosus Lactarius and Thelephora ganbajun.