The Sexual Reproduction And Genetic Diversity Of Phyllosticta Citricarpa And Phllosticta Citriasiana In China

Citrus Black Spot,caused by Phyllosticta citricarpa,and Pomelo Black Spot.caused by Phylloscticta citriasiana,are important fungal diseases on Citrus.Both of the pathogens are belong to Phyllosticta,Botryosphaeiaceae,Botryosphaeriales.Dothideomycetes.Pezizomycotina,Asomycota fungi.CBS and PBS can lead to premature fruit drop and cause spots on the leaf or fruit,which rendered such fruits unwanted on the fresh market and thus diminished or eliminated fruit’s commercial value.Moreover,P.citricarpa was listed pest by European Union and America though there were reports about occurrence of CBS in these places,which impairs the importation of the Citrus in China.And knowledge regarding the pathogens’biological characteristics like sexual reproduction,genetic mutation can fill the gap of the knowledge about two pathogen’s disease cycles and epidemiology,which further lay down the strong theoretical foundation for the control of these two diseases1.The research on sexual reproduction and population diversity of the P.citricarpa in ChinaAt first,191 P.citricarpa isolates were collected from Jiangxi,Sichuang(Chongqin)and Zhejiang province in China.And we determined each isoalte’s mating-type with mating type locus(MAT)specific primer pairs Gcmatl56f/Gcmatl 686r;Gcmat2686/Gmat21186rf.The result showed that the ratio of MAT1-1:MAT1-2 of P.citricarpa population from China after clone-correction is 69:69.which is not significantly deviatged from 1:1 by X2 test.With fine-scale(orchard.tree and Fruit or leaf),alternative mating type isolates can be found.The possibilities of founding alternative mating type isolates on single orchard,tree and fruit or leaf were 85.71%.33%and 82.35%respectively.This further suggested the possibility of sexual events.Using index of association(IA),the standard index of association(Rd)and proportion of compatibility to evaluate the sexual reproduction of P.citricarpa population from China.The results indicated IA,Rd and proportions of compatibility using 14 published SSR markers were 0.734(P<0.01).0.0582(P<0.01)and 0.229(P<0.01)in line.These results indicated the acceptance of mating.And the mixture distribution of two mating type isolates in UPGMA phylogenetic tree further support this assumption.Secondly,we genotyped 191 P.citricarpa isolates from China.From the results.we can see that 1)P.citricarpa population from China does not enjooyed polymorphism in PC 37 locus.which was different from all other populations.Besides.China population is the most diverse one based on the number of alleles,private alleles,gene and genotypic diversity.which may relate to its long co-evolutionary history with Citrus of in China,which further suggested that the origin of P.citricarpa may be in China.2)Moreover,we detected frequent gene flow in the basis of AMOVA,index of fixation and minimum spanning network.3)Finally,SRTUCTURE results gave clue to four genetic cluster of P.citricarpa population in China,and visualized the sexual,asexual reproduction and gene flow status.4)Given that Eureka lemon was introduced in China around a hundred years,we also focused on its divergence with other populations to figure out its origin.The minimum spanning network indicates that P.citricarpa from lemon was closely clustered with the ones from Nanfengmiju variety.2.The research on sexual reproduction and population diversity of the P.citriasiana in ChinaIn this study,we collected 94 P.citriasiana isolates from Guangdong,Guangxi and Fujian province in China.Using mating type genes of P.citricarpa,P.citriasiana as subject and our sequenced genome as query,we only find the homologous genes of MAT1-2 locus in our genome.Combined with chromosol walking method,we amplified MAT1-1 locus in other strains.Thus,the mating-type(MAT)loci of P.citriasiana were identified and characterized.Our analyses revealed that P.citriasiana has a heterothallic mating system with two alternative idiomorphs MAT1-1 and MAT1-2.Both idiomorphs are found in China and our surveys of natural isolates revealed that their ratio did not significantly deviate from 1:1.Additionally,we developed 14 microsatellite markers to genotype the P.citriasiana overall population.Based on the whole genome sequence of one strain,we developed 14 polymorphic microsatellite markers and analyzed 94 isolates from three provinces in South/Southeastern China.The results mirrored that the Chinese population of P.citriasiana has a high genotypic diversity,with evidence of recombination within individual subpopulations as well as in the whole Chinese sample.STRUCTURE analyses revealed that the Chinese samples belonged to seven genetic clusters with most of them broadly distributed among our analyzed geographic regions.Based on these results,we propose a model for the disease cycle of P.citriasiana and recommend a potential control strategy against the pomelo black spot disease in orchards.Based on the results,we clarified that both of conidiospre and ascospore are playing important role in epidemiology,both two diseases enjoyed a binary diease cycle.hence 1)the cleanness of the orchard of great importance to control PBS and CBS:2)The high genetic diversity of P.citriasiana and P.citriccarpa population in China and the existence of sexual reproduction of both pathogens equipped the pathogens abilities to overcome the natural or artificial barriers,which includes gaining the resistance to fungicides targeted on single gene spot on fungi;3)Given that CBS and PBS can be spread by the asexual reproductive materials of Citrus,it is vital to culture the pathogens-free seedling to restrict the expansion of pathogens.