Study On The Role Of Sexual Reproduction In Population Genetic Variation Of Cochliobolus Heterostrophus Causing Southern Corn Leaf Blight In Sichuan Province

Southern corn leaf blight caused by Cochliobolus heterostrophus（anamorph:Bipolaris maydis）,is one of the most serious diseases in corn production worldwide.The pathogenic fungus is characterized by complicated physiological differentiation and high genetic variation,and therefore brings considerable difficulties in corn resistance breeding and disease control.Sexual recombination plays important role in fungal variation;however,it is still unclear whether the genetic variation of C.heterostrophus population is attributed to the presence of cryptic sexual cycle.In this study,firstly,on the basis of identifying the genetic diversity of natural population,mating strategies and the probability of sexual recombination of C.heterostrophus isolates from the maize-growing regions in Sichuan Province will be evaluated through analyzing the mating type and fertility.Then,construct phylogenetic trees based on mating type gene sequences to analyze whether mating type genes can be used in the phylogenetic study of helminthosporioid fungi.Thirdly,in order to better detect the structure of sexual generation,the asci and ascospores of C.heterostrophus were stained with Fluorescent Brightener 28,Nile Red and DAPI and examined using a fluorescence microscope.Additionally,the environmental factors affecting the development of pseudothecia will be screened by optimizing the induced conditions.At last,the F₁ and F₂ populations of sexual recombination will be constructed using high compatibly parental cross combinations.And the culture characters,virulence and ISSR molecular markers between sexual progenies populations and parental generations will be analyzed to reveal the role of sexual reproduction in genetic diversity of C.heterostrophus.This research will lay an important foundation on the variation mechanism and population evolution of the pathogenic fungi.Results are shown below.1.A total of 697 B.maydis isolates were collected from 133 sampling sites from Sichuan and Yunnan provinces from 2012-2018.Isolates possessed rich variation in colony characteristics,mycelial growth rates and conidiation.Among them,84.07%isolates had average growth rates of more than 6.00mm/d on PDA medium,and 52.22%isolates had conidiation of more than 6×10⁴ conidia per cm².Based on morphological characteristics,pathogenicity testing,and phylogenetic analysis of the r DNA internal transcribed spacer（r DNA-ITS）and glyceraldehyde-3-phosphate dehydrogenase（GAPDH）genes,all these isolates were identified as B.maydis.Virulence differentiation was observed among B.maydis,and isolates with high virulence caused larger fusiform,elliptical or subrotund lesions 6-8 mm in length and accounted for 47.2%of the total tested isolates.ISSR-PCR system was built in this study.A total of 93 loci were detected in 219 isolates of B.maydis and B.zeicola using 9 screened ISSR primers and as high as 93.55%of these loci were polymorphic loci.According to the geographical origin,211 B.maydis isolates were divided into 6 populations.Total genetic diversity of the six populations was 0.1929;genetic diversity within the six populations was 0.1528;genetic differentiation coefficient among populations was0.1799 and gene flow was 2.2798.Clustering analysis indicated that genetic diversity of different populations had highly correlations with the geographical origin.2.The mating type（MAT）gene,r DNA-ITS,GAPDH,1,3,8-trihydroxynaph-thalene reductase（Brn1）and translation elongation factor（EF）gene of 46 Bipolaris and Exserohilum isolates from corn leaf blight were selected and sequenced for phylogenetic analysis.Then reference nucleotide sequences were downloaded from NCBI and the phylogenetic constructions of concatenated and individual gene trees were performed.The results showed that individual and multi-genes combined construction trees could accurate cluster the species of these Bipolaris,Curvularia,Exserohilum and Drechslera which belong to helminthosporioid fungi.MAT gene can be used in the phylogenetic study of helminthosporioid fungi.3.Under the fluorescence microscope,the cell walls of ascospores were stained bright blue by Fluorescent Brightener 28,which greatly improved the visibility of ascospores.Mature ascospore contained 59-91 nuclei,and each cell contained 4-11nuclei.Meanwhile,the ascospore could be stained bright orange-red by Nile Red,indicating they were rich in lipids.Ascospore suspension was inoculated on maize leaf segments to test the virulence of ascospore.Germ tubes formed from one or more cell at 2-6h after inoculation.At 12-24h,numerous appressoria differentiated from germ tubes.After 24h,the appressoria penetrated the epidermal cell walls directly and numerous invading hyphae formed.At 48h after inoculation,more water-soaked spots appeared and obvious symptoms were observed.It proved that ascospore were virulent to maize.When Sach’s medium was used as basal medium,mature corn leaves were used as inducer,biotin concentration was 0.02 g/L,Fe Cl₃ concentration was 0.06 g/L at dark,p H was 5-7,temperature was 24℃,it was best condition for the formation and maturation of pseudothecia.4.The mating type of 544 C.heterostrophus isolates were detected by polymerase chain reaction technology.Results showed that two mating types,MAT1-1（52.57%）and MAT1-2（47.43%）,were widespread in nature.The ratio of each mating type was consistent with a 1:1 ratio（χ²=1.441,p=0.230）.Mating type distribution was irrespective of the geographical location or years of collection of isolates.Of the 544 isolates,483 isolates（88.79%）were fertile and produced ascospores,and 61 isolates（11.21%）were sterile and did not produce ascospores when mated with standard tested isolates.The fertility levels were different among the tested isolates.High fertile,medium fertile and low fertile isolates accounted for12.32%,27.39%and 49.08%,respectively.Population fertility structure varied among different geographical locations,and fertility of isolates may be related to the geographic location.5.According to the results of fertility analysis,6 pairs of parent hybridization combinations with higher combining ability were selected for constructing the sexual progeny populations.A total of 264 isolates of F₁ and 180 isolates of F₂ were obtained.214 of the 444 isolates were MAT1-1,and 230 were MAT1-2,which accounted for48.20%and 51.80%,respectively.The ratio of each mating type was consistent with a1:1 ratio（χ²=0.071,p=0.789）.Mating type distribution was consistent in F₁ and F₂populations.Fertility structure varied among sexual progenies.The high fertile,medium fertile,low fertile and sterile isolates accounted for 1.13%,25.23%,52.25%and 21.40%,respectively.There was no significant difference between F₁ and F₂populations in fertility structure,and showed a decreasing tendency of fertility compared to natural population.Sexual progenies produced obvious differentiation from their parental isolates in culture characters,virulence and DNA polymorphism.Some sexual progenies showed enhanced mycelium growth and conidiation capacity,and more virulent to maize.It indicated that sexual reproduction was one of the factors leading to the variation of C.heterostrophus.In conclusion,C.heterostrophus natural population is complicated and has rich genetic diversity.The MAT1-1 and MAT1-2 isolates are commonly found to be balanced overall in the natural population.And the cross combinations between MAT1-1 and MAT1-2 isolates are generally fertile.These indicate that cryptic sexual cycle of C.heterostrophus may be under way and plays important roles in fungal genetic variation and disease cycle.