Establishment Of Genetic Transformation System For Lasiodiplodia Theobromae (Pat.) Griff. & Maubl

Several patogenic fungi cause post-harvest diseases of longan (Phyllanthus reticu latus Pior), in which the latent infection by Lasiodiplodia theobromae (Pat.) Griff. & Maubl is major one. It has the highest frequency (up to 46%) in fungal isolation from the post-harvest logan fruits. To further study the molecular mechanisms of disease, we tried to establish the transformation system of Lasiodiplodia theobromae in this study.First, we studied the capacity of conidiation of Lasiodiplodia theobromae (Pat.) Griff & Maubl under different culture conditions in order to obtain a large number of conidia for cultivating delicate mycelia to prepare for the protoplast. The results showed that oat and rice polish media are suitable for the fungus to produce stromata, and the quantity of stromata can reach the maximum when cultured at the pH 6.0 in media and under successive light for 15 days.Second, we optimized the conditions of the protoplast preparation and its regeneration. The fresh conidia were incubated in CM liquid medium for 30h and then the hyphae were collected, and digested by combination of enzyme (1%Nailase+1%Lyticase+1%Cellulase+1%Driselase) at 30℃by using sorbital (1M, pH 5.5) as buffer. The quantity and quality of the protoplast reached the maximum under this optimized condition.Last, we transformed plasmid pEGFP-H3 into the fungal protoplast with PEG-mediated transformation method. In total, 35 transformants were obtained, and 6 transformants were randomly selected to conduct further study. These clones were confirmed to be positive transformants by PCR verification and sequencing.In a summary, our results showed that we had primarily established a genetic transformation system of the fungus Lasiodiplodia theobromae.