Studies Of Citrus Resistance To Colletotrichum Gloeosporioides

Colletotrichum Corda was a kind of plant pathogens, presenting global distributing, caused anthracnose of fruit trees, crops or other plants and resulted in great economic losses, especially under high temperature and high humidity conditions in tropics and subtropics. Anthracnose caused by Colletotrichum gloeosporioides was an important disease in citrus production, almost in each big citrus production areas all over the world, such as the United States, Brazil, Japan, China, etc. In China, citrus anthracnose was one of most common and serious diseases in citrus production, such as chongming in Shanghai, nan feng in Jiangxi, deqing in Guangdong and maguan in Yunnan, etc. When occurred seriously, citrus anthracnose could cause dry shoots, failure, and even the whole fruits falling in the orange gardens.At present, chemical control was the main method to control citrus anthracnose. But continuous using of fungicide could result in resistance of pathogenic bacteria to chemicals. Except for the effect of chemical control reduced, it was also easy to cause fruits with the pesticide residue that a potential threat to human health and the environment security. It was an effective, economic and security measure to prevent and control plant diseases for resistant varieties, and resistant evaluation was a key point in the process of resistance breeding. So far, the report about domestic research on resistance evaluation of citrus germplasm resources to anthracnose was less. Therefore, it became the priority of resistance breeding to citrus anthracnose for screening of citrus germplasm resources resistance to anthracnose and positioning related resistance genes.Our team studied 16 strains of citrus Colletotrichum spp. that saved in laboratory about species identification and genetic diversity analyses, found Colletotrichum gloeosporioides more virulent strain. To evaluate the disease resistance to anthracnose, fifty-two citrus varieties were analyzed with the artificial inoculation method by disease index、average diameter and system cluster analysis method. In addition, based on the citrus molecular marker genetic linkage map using F1 progenies of a cross between a local sweet orange cultivar Li 2(Citrus sinensis (L.) Osb.) and a tangor Wan 2(Citrus unshiu (Mark.) Marc.×Citrus sinensis (L.) Osb.), according to the severity and incidence of 68 strains citrus, we found some marker locis associated with resistance to anthracnose through QTL mapping. At last, some related genes of partial marker locis were validated using RT-PCR analysis. The main results in this study were as follows:(1) Through identification of morphology characters, such as the colonies, hypha, conidia, appressoriums, pathogenicity identification, identification of hyphal anastomosis; we found, fifteen strains were Colletotrichum gloeosporioides and one was Colletotrichum acutatum. Besides strain CQBB-QC that separated from navel orange in beibei Chongqing and strain SXHZ-WZMG-2 that separated from satsuma mandarin in hanzhong Shanxi were the same mycelium compatibility group, and they were similar to Colletotrichum gloeosporioides. Other than, two strains were citrus anthracnose pathogens and strongly virulent strains. Neither ITS or TUB2 gene sequences could distinguish the interspecific variation between Colletotrichum gloeosporioides and Colletotrichum acutatum, and not the intraspecific variation of Colletotrichum gloeosporioides. But GAPDH gene, GS gene, CHS gene and CAL gene, could distinguish the intraspecific variation of Colletotrichum gloeosporioides. What’s more, The results showed that citrus Colletotrichum spp. was rich in diversity under genetic polymorphism analysis with primer ERIC and BOX. And primer ERIC was superior to the BOX. Strain CQBB-QC and strain SXHZ-WZMG-2 gathered for a cluster when genetic distance was 8.(2) With the artificial inoculation method, fifty-two citrus varieties were analysed by disease index、average diameter and system cluster analysis methods. These citrus varieties could be divided into the highly resistant, resistant, medium resistant and susceptible varieties without immune and highly susceptible to anthracnose. Eight varieties were highly resistance, such as Citrus grandis Huyou, Citrus sinensis Wanlengqicheng and so on. Other species showed different degrees of resistance to anthracnose in the different evaluation methods. At the same time, F1 progenies of a cross between a tangor Wan 2(Citrus unshiu (Mark.) Marc.×Citrus sinensis (L.) Osb.) and a local sweet orange cultivar Li 2(Citrus sinensis (L.)Osb.) were analysed by three kinds of resistance evaluation methods, results showed that resistance difference was bigger.(3) Using 68 strains citrus in F1 progenies of a cross between a local sweet orange cultivar Li 2 (Citrus sinensis (L.) Osb.) and a tangor Wan 2 (Citrus unshiu (Mark.) Marc.×Citrus sinensis (L.) Osb.), with the artificial inoculation method, we analysed recognition resistance and defense resistance of citrus based on the incidence and severity of citrus leaves, and consequently performed QTL mapping of citrus resistance to anthracnose. Based on the linkage map of integrated 334 SSR markers with 9 linkage groups and covered 844.2 cM of citrus genome with an average genetic distance standing at 2.53 cM, applying Map QTL6.0, QTL related sites were detected and optimized by Interval Mapping (IM) and Multiple-QTL model (MQM) methods. Under the condition of the LOD≥2.5,10 QTL marker locis associated with citrus resistance to anthracnose were detected in 6 linkage group 1,2,3,4,8,9; respectively; such as cSSR250, cBMM88, LcSSR150, LcSSR135, cSSR340, cSSR36, cSSR197, ccSM146-100, cSSR230 and F21. They explained from 10.3% to 35% of phenotypic variation, respectively.(4) Reference to the resistance classification of 68 strains citrus in F1 progenies of a cross between a local sweet orange cultivar Li 2 (Citrus sinensis (L.) Osb.) and a tangor Wan 2 (Citrus unshiu (Mark.) Marc.×Citrus sinensis (L.) Osb.) to anthracnose,12 varieties were selected from highly resistant, resistant, medium resistant, susceptible and highly susceptible varieties to Colletotrichum gloeosporioides CQBB-QC. Then those genes with related resistance to anthracnose were verified by RT-PCR. The results showed that the expression quantitygene of gene LcSSR150 and gene ccSM146-110 increased. The expression pattern of gene cSSR250 was same on the first day and the tenth day after inoculation. Except for highly resistant varieties, the expression quantitygene of all varieties increased between the inoculation treatment and the contrast. There had no significantly difference for other genes between the inoculation treatment and the contrast on the first day and the tenth day after inoculation.