Characterization Of The Mating Types In Natural Populations Of Botrytis Cinerea And Genetic Variation From Sexual Reproduction

Botrytis cinerea is an important pathogen with the broad host range over 1400 plant species,causing grey mould and heavy losses in crops during the pre-and post-harvest stages.B.cinerea is a heterothallic fungus with two mating types,and apothecia and ascospores are formed by sexual reproduction.However,B.cinerea mainly reproduces asexually,while sexual reproduction is not commonly seen in nature.Understanding the life cycles and modes of reproduction of the pathogen is essential for controlling the spread and prevalence of plant disease.Sexual reproduction is considered to benefit to the survival of species by generating genetic diversity and limiting the accumulation of deleterious mutations.The broad variability among fungal isolates and the large number of individual haplotypes and vegetative compatibility groups(VCGs)suggest that meiotic recombination occur in the populations of B.cinerea,while the direct experimental evidence is lacking.At present,the research on the variation of B.cinerea mostly focused on asexual reproduction variation,including mutation and horizontal gene transfer.The function of MAT gene of B.cinerea mostly focused on its influence on sexual reproduction and transcriptional regulation,but other functions(such as growth,pathogenicity and fungicide sensitivity)are poorly known.Crops(such as strawberries and tomatoes)cultivated in protected areas are in the optimum temperature and high humidity environment,which is suitable for the occurrence of B.cinerea.At present,the sexual reproduction potential of greenhouse or plastic-tunnel grown strawberry and tomato populations of B.cinerea is still unclear.Aiming at these problems above,the composition of mating type of B.cinerea populations from strawberry and tomato in plastic-tunnels was analyzed,the function of the main mating gene MAT1-1-1 and the influence of sexual reproduction on the genetic variation of B.cinerea were also studied.The results are summarized as follows:1.The composition of the mating types in the B.cinerea populations on strawberry and tomato in the protected areas in Hubei was determined.The mating types of 706 strains of B.cinerea collected from plastic tunnel-grown strawberry and tomato in Hubei were determined by multiplex PCR.The result showed that MAT1-1 and MAT1-2 strains were found to co-exist both in B.cinerea populations of protected areas.The overall ratio of the two mating types deviated from 1:1(?²=13.60,P=0.0002,df=1)with the main type of MAT1-2(56.9%)in Hubei population.In addition,further analysis in Hubei population showed that the isolates from strawberry and tomato differed in ratio of the two mating types,the strawberry isolates matched the one-to-one ratio(?²=0.01,P=0.9151,df=1),whereas the tomato isolates failed to match the one-to-one ratio(?²=26.03,P<0.0001,df=1);The frequencies of the two mating types differed significantly in the B.cinerea ioslates from different geographical counties or cities.In 7 out of 12 geographic subpopulations,the ratio of MAT1-1 to MAT1-2 matched 1:1,whereas in the remaining five geographic subpopulations(Jingmen,Jingzhou,Xiantao,Xiaogan and Yichang),the ratio of MAT1-1 to MAT1-2 did not match 1:1.These results suggested that sexual reproduction might be taking place in some B.cinerea populations on strawberry and tomato in the protected areas.2.The role of mating type gene BcMAT1-1-1 in the production of apothecia was clarified.BcMAT1-1-1deletion mutant was generated in MAT1-1 locus of wild type strain B05.10using the homologous recombibation method,and the function of BcMAT1-1-1 was analyzed by sexual development,biological characteristics,virulence and sensitivity to fludioxonil and fluazinam.The results showed that?BcMAT1-1-1 mutants had no difference from wild-type strain in colony morphology(mycelium,sclerotia,macroconidia and microconidia),mycelial growth rate,macroconidia and sclerotia production,macroconidia germination and pathogenicity on tobacco and Chinese cabbage.However,BcMAT1-1-1 is essential for sexual reproduction of B.cinerea.The results showed that all the cross combinations could not produce apothecium stipe and apothecium,whatever mutants were the female parent(?)or the male parent(?).3.The influence of sexual reproduction on genetic variation of B.cinerea was clarified.Apothecia and ascospores were induced under laboratory conditions using the cross combination(BY combination)of black sclerotia(B)strain HBtom544(MAT1-2,?)and Yellow sclerotia(Y)strain HBstr494(MAT1-1,?),and the cross combination(BB combination)B strain HBtom544(?)and B strain B05.10(MAT1-1,?).Fifty seven and thirty two monoascosporic progeny of B.cinerea were obtained from BY combination and BB combination,respectively.Variations among monoascospore progenies were evaluated by using the observation of colony morphology(hyphae,macroconidium and sclerotium),detection of mating type gene,measurement of mycelial compatibility group(MCG),as well as by determining the mycelium growth rate,virulence,and sensitivity against botryticides.(1)The sexual progenies of B.cinerea showed diversity in sclerotia production,color and colony morphology.All strains of BB progenies produced black sclerotia,while those of BY progenies produced two types of sclerotia,black and yellow.Eight different phenotypes(3 mycelial types,5 sclerotial types)were detected among these progenies.Four sclerotial types(S2?S5)were detected from BB progenies,with the majority subtype of S3(46.9%).While 3 mycelial types(M1,M3 and M4)with the majority subtype of M1(38.6%)and 5types of sclerotia types(S1?S5)with the main subtype of S2(26.3%)were found in BY progenies.(2)Both MAT1-1?MAT1-2 mating types are common in total sexual progenies of B.cinerea,and the overall ratio matched the 1:1 ratio.Thirty MAT1-1 isolates and twenty seven MAT1-2 isolates were observed in BY progenies(n=57),and the distribution ratio was 1:1(?²=0.16,P=0.6911,df=1).Similarly,15 MAT1-1 isolates and 17 MAT1-2 isolates were detected in BB progenies(n=32),and the distribution ratio also matched the 1:1 ratio(?²=0.13,P=0.7237,df=1).(3)The sexual progenies showed obvious diversity in mycelial compatibility group(MCG).Antagonistic interaction was obvious in mycelia contacts between most strains,dark pigmentation line or clear zone consisting of thin hyphae formed between the contacted colonies,indicating their mycelial incompatibility.The BB progenies were classified into 12MCGs(MCG 1^BB-12^BB)with the dominant group MCG 4^BBcontained 6 strains,while the other MCGs contained 1-5 strains,and 91.9%were incompatible.However,7 MCGs(MCG1^BY-7^BY)were identified in BY progenies,among which,MCG 1^BY,MCG 2^BY and MCG 4 ^BYcontained 16,15,and 13 strains,respectively,while the other MCGs contained 1 to 5 strains,and 79.8%were incompatible.Shannon index(H)and Simpson index(D)diversity index showed that BB progenies(H=2.6630,D=0.8945)had higher diversity than BY progenies(H=1.6709,D=0.7842).(4)Obvious diversity in mycelium growth rate,virulence and the sensitivity to botryticides were also detected in monoascospore isolates.Although the parental strains were all moderate virulence strains,the differentiation of high and weak virulence isolates was observed in monoascospore isolates,especially in the BY progenies(hypovirulence isolates accounted for 53.4%).All parental strains and progenies were sensitive to fludioxonil and fluazinam,but they differed significantly in sensitivity to both botryticides.Based on sequence alignment of the Bos1 which codes protein bound by fludioxonil of BY parents and progenies,two mutation sites of the parental strains HBstr494(?)and HBtom544(?)were observed respectively,i.e.,(P1186L+A1259T)and(I926T+A1259T).However,multiple amino acid mutations in Bos1 were found in BY progenies,among which A1259T mutation were found in both parents and progenies.These results indicated that sexual reproduction plays an important role in genetic variation of B.cinerea.All results of this study provided useful clues for understanding population structure,reproductive pattern and variation mechanism of B.cinerea on strawberry and tomato in protected areas.