Isolation&Pure Culture Of Tuber Indicum And Its Mycorrhiza Helper Bacteria(MHB) Selection

Tuber indicum have been exported to Europe for many years as the main commercial truffle from China. And then they were attracted attention because of highly similar of morphology and molecular sequence between T. indicum with the T. melanosporum Vittad.. The researches of classification, physiological ecology, mycorrhizal synthesis and so on for T. indicum were constantly specializing. The study was focused on Tuber indicum isolation and pure culture, and tried to select the Mycorrhiza Helper Bacteria (MHB) for mycorrhizal synthesis. A series of experiments were did and some available data for isolation, pure culture and MHBs were obtained, and that could be applied in mycorrhizal synthesis of truffle..The results from the study summarizing as following three aspects of isolation, pure culture of T. indicum:(1) The selection of medium. The common mediums, the successful mediums used for truffles culture, good reference value mediums used for other ectomycorrhizal fungi, as well as modified mediums;(2) The selection of isolation methods of truffles. The tissue separation method, spore separation method and mycorrhizal separation method were used in the experiment;(3) The selection of conditions of cultivation. Culture medias were kept in the incubator of25℃and no light after inoculating. Temperature gradient comparison were done after mycelium growing. The final results concluded as following as:(1) HM medium was suitable for mycelium growth. It was helpful for mycelium growth that to add pine needle and pine root extract substances in the medium;(2) Tissue isolation method was convenient, shortcut and this would be a preferred method. However, the success rate was lower. The steps of spore isolation method and mycorrhizal isolation method were more complex, and easily contaminated.In this study, Pinus armandii was experimental tree species, and eleven bacteria which were isolated from rhizosphere soil were experimental bacteria. T. indicum inocula and mycorrhizal media mixed with bacteria, and then were inoculated on Pinus armandii seedings. The Mycorrhiza Helper Bacteria were obtained by statistics and analysis of the quantity of mycorrhiza、plant height and ground diameter of the Pinus armandii mycorrhizal seedlings synthesized by T. indicum. There were three Mycorrhiza Helper Bacteria of2.4X109cfu/ml which were Pseudomonas sp. JCM5481(P143), Streptomyces sp. EN31(S191) and Variovorax paradoxus(V633) which had highly significant role in promoting (P<0.01) to the quantity of mycorrhizas plant height and ground diameter, as well as two Mycorrhiza Helper Bacteria of0.8X109cfu/ml which were Pseudomonas chlororaphis(P11)、Pseudomonas corrugate(P127) which had significant role in promoting (P<0.05) to the quantity of mycorrhiza、plant height and ground diameter. It seted up concentration gradient for four Pseudomonas in the experiment, and the results showed the different bacteria strains have their different suitable concentration respectively. It indicated that the concentration of bacteria was a key factor to get Mycorrhiza Helper Bacteria.