Construction Of The Mutant Library Of Botryosphaeria Dothidea And Phenotype Analysis For The Mutants

Apple ring rot mainly caused by Botryosphaeria dothidea affects both trunks and fruitsand is one of the most serious apple diseases in China. B. dothidea is distributed world wideand is capable of infecting numerous plant species. The current study mainly focus onchemical control, infection process and pathogenicity differentiation. Nevertheless, on thefungal pathogenicity is still at an early stage, very little is currently known about thepathogenicity mechanisms of this fungus at the molecular level. Isolation of the genesinvolved in pathogenicity is crucial for fungal molecular biology, which may be approachedby genetic mutagenesis analysis. To reveal the molecular pathogenic mechanism ofBotryosphaeria dothidea as early as possible and provide theoretical basis and methods fordisease prevention and control, we established a mutant library of Botryosphaeria dothideathrough PEG and systematically analyze the phenotypes and pathogenicity of part of themutants in PEG mutant library of Botryosphaeria dothidea. Some mutants were acquiredassociated with the pathogen growth and pathogenicity by screening. The results are asfollows:1. Using the developed PEG mediated transformation of Botryosphaeria dothidea.Approximately5-10transformants per μg of vector DNA were obtained.860transformantswere got totally, and about37%of the transformants showed mitotic stability after alternatesubculturing onto media with and without selection for a minimum of four subcultures.Results of PCR amplification and Southern blot analysis proved that the transformationsucceeded and obtained three Monoclonal copy transformants.2. After systematically phenotypic comparation and analysis on the320PEGmutants andwild type strain0352, we found that, there were14mutants (about4.3%) changed obviouslyin the colony color. After7days’ culturing, compared with the wide type, one of them thefront color showed the stale grey and the back showed the black totally. Ten of them showedthe colonies’ color little green in the middle and milky white around. Three of them lost thepigment showed milk white totally. There were15mutants growing unevenly, colony shapeirregular and The aerial mycelium were very sparse.3.Through testing the growthrate,we observed that there were43(13.4%) mutants showed a increassing growth rate and three of them showed the declining growth rate Thegrowth rate of S69is the minimum which is only7mm/d, it decreased by43.3%comparedwith the wild type strain0352.4. We have made a detailed statistics and analysis on the pathogenicity of32strains andwild-type strain0352by inoculating detached Fuji apple tree branches and the leaves. Theresults showed that: most of the mutants did not show significant difference on pathogenicitycompared with wild-type strain0352. Only two mutants showed significant changes inpathogenicity. Three mutants showed a stronger pathogenicity than that of strain0352. Thepathogenicity of only one mutants was significantly decreased.