Correlation Between Minichromosome And Pathogenicity Differntiation Of Colletotrichum Fructicola

Glomerella leaf spot(GLS)is a devastating disease on the Golden Delicious descendant and a new disease on apples in China.Colletotrichum fructicola is the main causal agent in China.Previous research from our lab indicated that there are two pathotypes within C.fructicola: the leaf-infecting GLS type and the non-leaf-infecting ABR type(causing bitter rot symptom on fruit).Omics analysis revealed that the GLS pathogenic strains specifically contain a gene cluster GLS-PGC1 that is highly expressed during GLS infection.We thus speculated that the emergence of GLS disease may be the outcome of horizontal transfer of a "leaf-pathogenic factors",leading to rapid pathogenicity differentiation.Minichromosome horizontal transfer might be an important mechanism.In this study,analyzing the correlation between GLS disease-related gene clusters and minichromosomes by karyotype analysis was performed with a combination of germ tube burst-based cytology,pulsed-field gel electrophoresis and southern hybridization.The results are as follows:1.The pathogenicity of C.fructicola strains from apple fruit,pepper,kiwi,nectarine,plum,tomato,Enamel plate,Passiflora,Rhododendron and so on was compared with the stains from apple leaves.Those strains(1104-7,PGYLP03)isolated from apple leaves can infect the leaves causing leaf spot symptom,and other strains(LJMX19,LC0146)isolated from other hosts can not infect apple leaves.Inoculation experiment confirmed the pathogenic differentiation between GLS isolates and non-GLS isolates of C.fructicola.2.Using the germ tube burst method,DAPI staining,it was found by fluorescence microscopy that the chromosome number of C.fructicola 1104-7 was more than 10,among which there were two short chromosomes,respectively are 0.37 ?m and 0.38 ?m.3.The system for karyotypes analysis of C.fructicola was stablished.The optimal conditions for karyotype pulsed-field gel electrophoresis are: STC dissolves Seakem Glod-embedded conidia overnight,sequentially lysed by NDS-SE(for embedded conidia and lysed conidia protease K is needed).0.7% agarose gel,0.5×TBE electrophoresis liquid,2 h at 4.8V/cm with a 60-60 s switch time ramp at an incouded angle of 120°,followed by32 h at 6 V/cm with a 60-20 s switch time ramp at an included angle of 120°,electrophoresis liquid temperature of 14 ?.Karyotype analysis confirmed that there are polymorphisms in their sizes and number of minichromosomes within C.fructicola strains,and one about 1.1Mb minichromosome was shared by all strains.4.PCR detection confirmed the GLS association of the GLS-PGC1 cluster.Southernhybridization with the SAL gene as a probe demonstrated that hybridization bands only be found in GLS strains,and the hybridization bands appeared on the 1.1 Mb minichromosome,the 2.2 Mb chromosome and on the DNA accumulation regions in loading pores.The results indicated that the GLS-PGC1 cluster was not only localized on minichromosome,but also on other chromsomes.It was showed that GLS pathogenicity was correlation with the minichromosome of C.fructicola.