Mating Characteristics Of Colletotrichum Fructicola Reveaeed By Fluorescent Labeled Nuclei

Glomerella leaf spot(GLS)is a devastating disease that occurrs in China's Golden Delicious apple in recent years,causing huge losses to the apple industry.In China,the main GLS pathogen is the Ascomycota fungus Colletotrichum fructicola.Its ascospore production is common in the field,which plays an important role in the long-range inoculum spread.Two types of mon-spore purified strains were obtained from an ascus in the laboratory: type1(1104-7-WS)is self-fertile,and produce well-developed asci and ascospores;type2(1104-6-B)develops abundant perithecia,but most were fertile,and the asci and ascospores were aberrant in case they were formed.In this study,we used 1104-7-WS and 1104-6-B as test strains.Based on gene knock-in technique,the fluorescent protein gene GFP and mCherry were integrated into the histone H1 locus of 1104-7-WS and 1104-6-B respectively,through which nuclear fluorescentlabeled strains were obtained.The melanin biosynthesis-related genes of the two strains were also knocked out to generate ‘albino' strains.The transformed strains were used to study the sexual developments of Colletotrichum fructicola.The following main results were obtained:1.When the strain 1104-7-WSGfp and the 1104-6-BmCherry strain were confronted on oatmeal plate,‘mating line' formed rapidly and contained asci and ascospores.Perithecium formation on the mating line was earlier than both strains,supporting that hyphal contact between the two strains speeds up sexual development.Further observation with perthecium on the mating line found that there were significant differences in the fluorescence labeling of ascospores in individual perthecium:(1).pure green type: all ascospores expressed GFP green fluorescence,speculated to be derived from 1104-7-WSGfp alone;(2).mixed type: containing both GFP-labeled spore and mCherry-labeled spore in the same ascus,speculated to be formed after hybridization between 1104-7-WSGfp and 1104-6-BmCherry;(3).pure red type: all ascospores contain red fluorescence speculated to be formed from 1104-6-BmCherry strain and to be stimulated by the "sex hormone" produced by 1104-7-WSGfp.2.Through confrontation culture assay with albino mutants expressing fluorescent nuclei,we concluded that the 1104-6-B strain is responsible for producing the perthecium and plays a “mother” role in the mating process of the two strains.The above studies showed that the sexual reproduction of Colletotrichum fructicola may occur in three different ways: the fertile homothallism system: strains contain intact sexual reproduction elements,and can finish sexual reproduction,producing perthecium,asci and ascospores all by itself,;semi-fertile homothallism systems: it is deficient in sex hormone production,and can not form or occasionally form aberrant perthecium and ascospores;such defect could be complemented by sexual hormone produed by the fertile homothallism strain;fertile-semi-fertile system: the self-fertile strain can hybridize with the hemi-fertile strain,efficiently produce perithecia,asci and ascospores,in which the semi-fertile strain acts as female parent responsible for producing perithecium during hybridization process.This study demonstrated the tetrad phenotype diversity of asci from different perithecia on the mating line,and laid a foundation for further study with the sexual reproduction mechanisms of C.fructicola.