Construction Of Genetic Transformation System Of Geotrichum Candidum

The litchi sour rot diesase, which is caused by the fungus, Geotrichum candidum, is one of litchi storage diseases. The disease is often happened in Fujian and Guangdong provinces. The influent factors on growth、sporulation and of G. candidum were mainly reported in many studies. But its molecular biology studies such as its pathogenicity and the interaction between the pathogen and host plant have not been found in details reporting. Understanding the molecular mechanisms of the pathogen and host paints are the basis for the development of new control strategies. Green fluorescent protein is a main marker genes for studying the fungal ecology and the interaction between pathogen and host plants. To further undersdand the interaction mechanism between G. candidum and host palnt, a genetic transformation system of G. candidum was constructed, and some preliminary reaserch results were obtained.This work further proved that the pathogen of lychee sour rot disease in Fujian was caused by the fungus Geotrichum candidum. The colonies of this fungus on PDA medium were white. The surface of the colony was powdery. The hyphae was colorless and had septum, dichotomouse or trichotomouse branching. The conidial stalks were short and not branched. The conidia were produced in chains.The growth temperature range was from18℃to32℃. At37℃and above the fungus would not grow on the solid medium. The optimum conditions for the mycelia growth were, growing on YEPD medium(pH6.0) at30℃, lighting for4-12h and shaking in the speed of180rpm.The optimum conditions for preparation and regeneration of protoplasts of G. candidum were as follows, the medium was YEPD, the driselase concentrations was Smg-ml-1driselase, and incubated at30℃for4h. The mycelia were2-day-old and regeneration medium was YEPD with20%sucrose as an osmotic stabillzer. At last the protoplasts were yielded of58.66×106cfu·ml-1and regeneration frequency was0.155%.The recombinant expless plasmids, pHAMT35G-gfp-Hmb and pHsp-gfp-Hmb were constructed. The PEG-mediated transformation system of G. candidum also was established. The different conversion rates by different plasmids and different molecular weight PEG were compared. The results showed that the conversion rate of plasmid pHAMT35G-gfp-Hmb was the highest, the conversion rate of a single plasmid was fairly higher than that of the total and the conversion rate of the PEG-MW3350was higher than that of the PEG-MW4400. The fluorescence was observed from transformants, which was further confermed by PCR that the green fluoresent protein gene was transferred into G. candidum genomic DNA. The transformed isolates were not different markedly from the wild type isolates on growing and morphological characteristics. Even after transferring for seven generations the green fluoresce also could been observed under a fluorescence microscopy. The colony and growth speed of the fungus kept the same as the wild type isolate. Pathogenicity tests showed that the transformation process did not alter the pathogenicity of G. candidum isolates.