| Glomerella leaf spot(GLS),mainly caused by Colletotrichum fructicola,is an emerging disease spreading rapidly in apple-producing areas in China.In the field,C.fructicola produces massive ascospores at late infection phase,the windborn spread of which plays important roles in disease epidemics.In most Ascomycota fungi,sexual fruiting body development and ascospore formation are regulated by mating type genes,requiring a recognition between MAT1-1-1 and MAT1-2-1.Individual heterothallic strain harbors either MAT1-1-1 or MAT1-2-1 whereas individual homothallic strain harbors both genes.In striking contrast,all known Colletotrichum genomes contain only MAT1-2-1 despite that some strains can reproduce homothallically.It is so far unclear whether Colletotrichum MAT1-2-1 regulates sexual reproduction.The C.fructicola strain 1104-6-B produces massive sterile and scattered perithecia on solid medium.When being co-cultivated with its sexually compatible strain 1104-7-W-S,a mating line,made up of aggregated fertile perithecia,forms at the contact zone.In this study,we have chosen 1104-6-B as the wild type strain,generated MAT1-2-1 gene deletion mutants,and analyzed the mutant phenotypes.Main results obtained are listed as follows:1.Based on polyethylene glycol(PEG)mediated protoplast transformation and homologous recombination,we generated MAT1-2-1 gene deletion mutants with hygromycin phosphate transferase as the selection marker.In total,12 knock-out mutants were obtained based on PCR screening of hygromycin-resistant transformants.2.We systematically compared the phenotypic differences between the wild type and MAT1-2-1 gene deletion mutants.There are no differences in two type strains’ s colony morphology,vegetative growth,conidial germination,appressorium differentiation and pathogenicity except that the mutants produced slightly reduced amount of conidia in potato dextrose broth(PDB)liquid medium.On oatmeal agar(OA),the mutants formed more conidial mass in the colony center.On OA,the mutants still produce perithecia with similar frequency,but with smaller diameter,which indicates that MAT1-2-1 affects perithecial developments.When co-cultivated with sexually compatible strain,the MAT1-2-1 gene deletion mutants still formed obvious mating line.However,perithecia on the mating line arecompletely sterile,lacking any ascus or ascospore.This suggests that MAT1-2-1 is a key regulator of ascospore and ascus developments in heterothallic mating.3.During this study,we have also observed that the laboratory C.fructicola strain is prone to mutate spontaneously.In total,four stable sectoring types were obtained from the progenies of a single-spore purified strain 1104.Type I(1104-7-W): produces dense aerial mycelia,the colony is light colored and contains no perithecia,produces much less conidia in liquid PDB medium,conidia are slender,and are nearly nonpathogenic;type II(1104-6-B):produces few aerial hyphae,colony dark-colored,most being sterile and scattered;type III(1104-7-W-S): produces few aerial hyphae,and a dark-colored colony containing a large number of aggregate and fertile perithecia;type IV(1104-6-B-S): produces scattered perithecia similar as 1104-6-B,yet the perithecia are completely sterile and the aerial hypha are much less and the colony is light-colored.When cocutivating strains of the four types in different pairwise combinations,only the combination between 1104-7-W-S and 1104-6-B forms mating line containing fertile perithecia.This study demonstrated that MAT1-2-1 gene affects perithecial development,and is a key regulator of ascospore and ascus developments.This study also obtained and clarified the biological trait variations among four sectoring mutants.These results would lay a foundation for further studies in the genetic regulation of C.fructicola sexual reproduction. |
PDF Full Text Request