Identification Of Grape Anthracnose Resistance And Expression Analysis Of LysM RLK Gene Family In Grape

During the grape planting process,grape anthracnose is one of the most serious fungal diseases,mainly affecting the fruits during the coloring and ripening stages,causing them to become soft and rotten,seriously affecting the yield and quality of grapes.This study isolated a strain causing grape anthracnose from the grape fruit of ’White Banana’ in Fuzhou City,Fujian Province,and measured the indoor control effects of different fungicides on this anthracnose fungus.The resistance of six Chinese wild grape varieties(strains)and 46 common grape cultivar to anthracnose was evaluated by indoor inoculation in vitro.Finally,the LysM-RLK gene family of grape was identified and analyzed for expression.The main research results are as follows:1.A pathogen causing grape anthracnose disease was isolated from the ’White Banana’ grape fruit,named FN-1 and FN-2.Through morphological identification and combined with ITS,TUB,and ACT multi gene molecular identification,the pathogen was identified as C.fruticola in the composite species of Colletotrichum gloeosporioides.At the same time,the indoor toxicity of six fungicides to the strain was determined by the mycelial growth rate method.The results showed that carbendazim and prochloraz had the strongest inhibitory effect on the growth of the mycelium of fruit borne anthrax,followed by pyrazoxystrobin and thiophanate methyl,and thiram and mancozeb had the worst inhibitory effect on the growth of the mycelium.2.Through indoor leaf inoculation,52 different grape varieties(strains)were identified for their resistance to anthracnose.The results showed that different grape varieties had different resistance to anthracnose.Among them,V.davidii ‘Ziqiu’ and V.pesudoer were high resistance varieties to anthracnose,while V.amurensi ‘Zuoshan 1 hao’ and V.bryoniaefolia ‘Lan-2’ were high resistance varieties V.heyneana,‘Shang-24’,‘Cabernet Sauvignon’,‘Mars seedless’,‘Giant Rose’,‘Black Pearl’,and ‘Moldova’are disease resistant varieties.3.Twelve LysM-RLK genes were identified in the genome of Vitis vinifera L.They were divided into two subgroups: LysM-I and LysM-II,with LysM-I consisting of 4 members and LysM-II subgroup consisting of8 members.Bioinformatics analysis was conducted on them.Meanwhile,in the highly resistant anthracnose grape ‘Ziqiu’,it was found that Vd LysMRLK6,Vd LysM-RLK7,and Vd LysM-RLK8 were significantly upregulated by C.fructicola,SA,and JA.Among them,the expression level difference of Vd LysM-RLK6 gene was the most significant,8.52,3.73,and 3.91 times that before and after treatment,respectively.It is speculated that it may play an important role in the resistance of grapes to C.fructicola.4.The cDNA sequence of the Vd LysM-RLK6 gene was cloned from Vitis davidii using homologous cloning.The sequencing results showed that the sequence was 1866 bp long and encoded 621 amino acids.At the same time,the 1980 bp upstream promoter sequence of the gene was cloned and named p Vd LysM6.We constructed a full length and three GUS fusion expression vectors with 5’ terminal deletion promoter fragments:p Vd LysM6,p Vd LysM6-D1,p Vd LysM6-D2,and p Vd LysM6-D3.Instantaneous transformation of tobacco leaves for histochemical staining and GUS enzyme activity determination.The results showed that all four promoters exhibited promoter activity,and there were key elements of p Vd LysM6 promoter response to anthrax and SA induction between p Vd LysM6-D1 and p Vd LysM6-D2.