Study On The Cultural And Genetic Characteristics Of The Sclerotia Producing By Morchella

The physiological and hereditary characters of genus Morchalle are complex and changeable, it is common that sclerotia and hyphal morphological changes and unstable sclerotia production happened frequently for culturing of different isolates, as well as relationship of sclerotia formation and morel fruiting is still in disputing.The groups of single spore isolates from fruiting bodies of Morchalle conica and Morchella crassipes were selected as experimental material in this study, which were collected from semi-artificial cultivation field in Yunnan provice.The microscopic and macroscopic morphology of mycelia and sclerotia of two group strains on different media were observated. The ability of substrate utilization of isolates was studied. AFLP(Amplified Fragment Length Polymorphism) technique was used to analysis the diversity of single spore isolates from M. conica.The expressing difference of gene related with sclerotial formation in single spore isolates was analyzed by mRNA differential display reverse transcription-PCR.The main results were described as follows:(1) All single spore strains isolated from same fruiting bodies of Morchalle conica and Morchella crassipes could be divided into nine types with stable properties under same cultural condition(YPD media) basing on cultural characteristics.The isolates were also divided into sclerotia-producing and non-sclerotia producing strains by sclerotial formation.(2) The media nutrition had impact on morel sclerotia formation and hyphac morphology. The sclerotia formation and hyphae morphology of the M.conica sclerotia-producing single spore strains are more stable than the M.conica sclerotia-producing multi spore strains and M.crasspies sclerotia-producing single spore strains.(3) The mating results showed that mating had a great effect on the morphological characteristics of two mating strains, for example, the shape and size of scletotia, the growth rate of mycelia and growth potential hade obvious difference compared with separate cultivation.especially on producing sclerotia traits, the producing ability of scletotia could exchange between two mating strains and even disappeared.(4) The cultural morphology of sclerotia-producing and non-sclerotia-produing strians under different liquid cultivation conditions (shakering and staticing) was observed. The macroscopic characteristics(thickness, density and colour and luster) of mycelia) of strains had significant difference, but no obvious difference could be observed in optical microscopy.lt was found that single spore isolates from same fruiting bodies of Morchalle conica and Morchella crassipes could produce sclerotia under static liquid cultivation but shaker cultivation.(5) A microscopic image library of sclerotia formation was obtained by observing the microstructure of mycelia in dfferent culture phases.(6) Forty-five single spore isolates and one tissue isolates from the same Morchalle conica fruiting body could be divided into four alignmental groups by using AFLP analyzsis.The495valid polymorphic bands range from l00bp to600bp were screened out by PCR amplifying with four pairs of primers, indicating that single spores from the same fruiting body had a very rich genetic diversity.50isolates cluster into11groups at the molecular level by Nei's Unbiased Measure of Genetic Identity and Genetic distance UPGAM cluster analysis of genetic similarity,especially the cluster result were correspond to four primitive morphological group between spot strains and non spot strains, the polymorphism index were consistcnt.Morphological polymorphisms were not closely related to genetic polymorphism especially sclerotia formation.The polymorphism genetic map obtained from this research may be a fingerprint of isolates in the furture.(7)Thc mRNA differences of two sclcrotia-producing strains and two non-sclcrotia-producing singel spore isolates from Morchella conica fruiring bodies named ZD-23and ZD-18were analyzed by mRNA differential display reverse transcription-PCR (DDRT-PCR) technique with72pairs of primers.13special represented differentive gene fragments related to sclerotia producting, were obtained and were validated by semiquantitive PCR. These gene fragment had certain homologies with Lipoprotein,cyclin-dependent kinase C-3, glycerophosphoryl diester phosphodiesterase, Rho GDP-dissociation inhibitor, GABA permease, ompA family protein, transcript antisense to ribosomal RNA protein, keratin-associated protein5-6, sodium calcium exchange protein and keratin-associated protein5-6. In addition, some positive fragments are hypothetical protein or gene fragments could not be found in NCBI database. These fragments are likely to be unique functional gene which are associated with producing morel sclerotia.The gene information relating with sclerotia may supplying important molecular foundation for morel sclerotia formation and the development of morel fruiting.In conclusion, although the morphological characteristics are diverse and susceptible to environment,but they are controled by genes under same cultivation condition.