Transformation of Agrobacterium tumefaciens-Mediated Colletotrichum gloeosporioides and Identification of Transformants

【Objective】The objective of this study is to optimize the Agrobacterium tumefaciens-mediated transformation(ATMT) technology system of Colletotrichum gloeosporioides,screen and identify the transformants and compare the difference of biological characteristics and pathogenicity between wild-type strain and transformants.【Method】Using the conidia of C.gloeosporioides strain LXS010101 as transformation recipients,A.tumefaciens strain EHA105 carrying plasmid pBIG3C harboring the hygromycin B phosphotransferase gene(hph) was transformed into C.gloeosporioides.The obtained transformants were screened and identified by hygromycin B resistance,PCR and Southern blot analysis.Partial transformants were selected randomly and analyzed on the colony morphology,mycelia growth rate,conidia development and pathogenicity.【Result】Successful transformation of C.gloeosporioides was performed and the highest efficiency reached on 439 transformants per 1×105 spores.The optimal transformation conditions were that 1×105 spores per milliliter of C.gloeosporioides spore suspension were co-cultured with Agrobacterium cells at 22℃ for 24 h,in the presence of co-culture medium containing acetosyringone(AS) at 200 μmol.mL-1.The transformants were stable when grown on PDA medium without hygromycin B for five times and were verified by PCR amplification with the hph primers and by Southern blot analysis with the hph probe.The results showed that all the detected transformants could be amplified the target bands and all the T-DNA were single-copy inserted into the genome of C.gloeosporioides.Compared with the wild-type strain LXS010101,most of the transformants did not change on the colony morphologies,however,in which 23.33% of the transformants decreased the mycelia growth rate significantly and two strains ATJ-3 and ATJ-15 could not produce conidia.In the other 28 transformants,39.29% of the strains reduced the spore germination rate and 25.00% of the strains changed appressorium formation.In addition,11 transformants with decreased their pathogenicity were characterized and ATJ-19 even completely lost its ability to infect apple.【Conclusion】The successful optimization of ATMT system of C.gloeosporioides and partial transformants were analyzed primarily indicated the usefulness of this approach for functional genetic analysis and revealing the pathogenesis mechanism in this important pathogenic fungus.