Cloning, Analysis And Detection Of The Mating-type Genes In Curvularia Lunata

Corn Curvularia leaf spot disease, caused by fungus Curvularia lunata, has been one of the major leaf spot diseases and constituted considerable threat to maize growing regions of China, In recent years, many studies mainly focused on pathogenic factors such as cell wall degradation enzymes, toxins, melanin and identification of pathogenic types. Sexual reproduction is a key factor on pathogenic differentiation of C.lunata.C.lunata is teleomorph:Cochliobolus lunata. The sexual state of C.lunata have never been found in nature at present. The single mating-type locus (MAT) controls sexual development, also controls mating, incompatibility, sexual-differentiation, sexual-development, the size of ascospore and other biological characteristics. At the same time, sexual reproduction produced the offspring would been beneficial to pathogenic and adaptability of the plant pathogenic fungi. Researching and exploring the mating-type of C.luMta in sexual reproduction have important practical applications and theoretical significance to understand the life cycle of the fungal pathogen and control and prevention disease. The purpose of this paper is to clone the mating type genes and bioinformatics analysis, find out the mating type of C.lunata to enrich machanism of molecular pathogenesis. Results were as follows:1. Cloning, bioinformatics analysis of mating type genes in C.lunataC.lunata ClMATl-1-1 and CIMAT1-2-1 were cloned (GenBank:KU749293.1, and KU749294.1) Full length of ClMAT1-1-1 and ClMAT1-2.1were 1190bp and 1087bp, containing an intron respectively.Homology comparison shows that ClMAT1-1-1/ClMAT1-2-1 gene flanking sequence homology is above 98%, and the MAT locus is highly conserved.ClMAT1-1-1/ClMAT1-2-1 gene encode 378 and 343 amino acids respectively. ClMAT1-1 protein containing a conservative alpha domain structure, belongs to the MAT-alphal family and is involved in sexual reproduction. ClMAT1-2 protein containing a conservative HMG domain box structure, and likely plays a signification role in DNA transcription, replication and protein interactions. Study found that the mating-type site on C.lunata is very similar to the site on C.heterostrophus. ClMAT1-1-1/ClMAT1-2-1 and mating type genes of C.heterostrophus, C.carbonum, C.ellisii, C.cymbopogonis show a close genetic relationship, and consistent with the morphological classification.2. Identification and classification of Curvularia lunata and its allied strainsA total of 208 strains were isolated from maize growing areas in eleven provinces of China such as Heilongjiang, Jilin, Liaoning, Hebei, Henan, Shandong, Sichuan, Anhui, Hubei, Guizhou and Yunnan during 2010-2016. Combined with the traditional morphological taxonomy and molecular biology method,189 strains of C.lunata,5 strains of C.geniculata,2 strains of B.spicifera,10 strains of B.zeicola and 2 strains of B.maydis were isolated and classificated, also found that the 5 fungal pathogens have a close genetic relationship. B.zeicola and B.maydisare in a branch and have close genetic relationship, other fungal pathogens(C.lunata, C.geniculata, B.spicifera) have a close genetic relationship and also in another branch.On the morphological classification, C.lunata and B.spicifera are very similar as morphologyical characteristics, such as three diaphragm (fake diaphragm)and also close genetic relationship.3. Distribution statistics of the mating-type in C.lunatain ChinaC.lunata is a heterothallic fungal pathogen, there are two kinds of mating type, Mating type 1-1(MAT1-1)and Mating type 1-2(MAT1-2). MAT1-1 was detected in all regions except the province of Hubei and Jilin. MAT1-2 was detected in all regions except the province of Shandong.MAT1-1strains (40.74%), MAT1-2 strains (42.33%) in 189 strains of C.lunata and the ratio of MAT1-1:MAT1-2 is close to 1:1. C.lunata in Yunnan accounted for 3.70% of all strains, the ratio of MAT1-1:MAT1-2 is close to 1:1. C.lunata in Liaoning accounted for 70.37% of all strains, the ratio of MAT1-1:MAT1-2 is also close to 1:1. We also found that MAT1-1 and MAT1-2 were both or not detected in the same strain, and in some areas the ratio of MAT1-1:MAT1-2 has a phenomenon which is called separation distortion.