| Citrus brown spot causing by Alternaria alternata is one of the important diseases on young leaves, shoots and fruits. In recent years, it is reported to occur and expand constantly in wide citrus-growing region which has caused the great economic loss because of the production and quality declining.Resistance of citrus varieties against A. alternate was not known well in China any more than resistant genes. The fast and stable evaluation system about citrus varieties against citrus brown spot was not founded yet that has delayed disease control and research. Therefore, the stable pathogenicity evaluation system established and resistant varieties selected could support citrus production and control. Resistance genes would be necessary method for breeding.A fast and easy to operate pathogenicity evaluation system by indoor inoculation was to be established in the study. Resistance analysis of four main citrus varieties groups of Chongqing in which fifty-four different citrus varieties were evaluated by the system with three methods. Furthermore, based on the genetic map of "pear orange2X night honey2" and its molecular markers, the severity and incidence of A. alternata of68strains from F1was analysis by QTL mapping then verified by qPCR. The main results were as follows:(1) The citrus brown spot samples were collected from red tangerine at Wanzhou district. Then was separated, purified and identified indoors. Results indicate that the colonies, mycelium, conidia are similar to that of Alternaria sp.. Yellow halo is character of the disease. The PCR product from primers ITS1and ITS4was532bp. The sequencing results are compared with the similarities sequences in Gene Bank by BLAST. The highest similarity sequence was A. alternata which identity is up to99%. Isolated strain and A.alternata fall into one cluster with the PG2and PG3primer (endoPG)amplification sequences to construct evolutionary tree.(2) Leaves and fruits from satsuma mandarin caused by different wound with virulent HJX strain vaccination.At25℃in a moisture box regular surveys found that lesion was not found on non-inoculated and without wound samples, while occurred in other treatments by wound inoculation. The type of wound affected the disease development greatly. Lesions developed much faster on lower side than upper side of the leaves. No significant difference was observed on lower side among wounds of pricking one time, ten times and unwound. There were significant differences (P<0.01) among wounds of pricking one time, ten times and removing partial peel on fruits. Furthermore, six different citrus cultivars and two different virulent strains were used to test the reliability and stability of the method. The results showed that HJX strain made different on the pathogenicity of different cultivars and two strains made also different pathogenicity. Using detached leaves or fruits as plant material and inoculating on the lower side of leaf or removing partial peel on fruits, keeping at25℃for2days in moisture box to survey the necrosis as accurate, rapid evaluation of citrus brown spot virulence of methods indoor.(3) Fifty-four citrus varieties against A. alternata were evaluated the disease resistance and infection severity by disease system cluster analysis, average diameter and index method. These citrus varieties could be divided into the highly resistant, resistant, medium resistant, susceptible and highly susceptible varieties without immune. Citrus grandis Huyou, Citrus grandis Qiangdeleyou, Citrus sinensis Huashengdunqichen and Citrus grandis Liangpingyou are highly resistance. Most of tangerine (C. reticulata) cultivars are susceptible. This study aimed to definite the resistance difference in citrus varieties so as to provide a theoretical basis for citrus resistance breeding programs.(4) Based on the linkage map of contains334SSR markers, nine linkage group, the total length of844.2cM, the average figure from2.53cM molecular genetics Map, resistance analysis of "pear orange2X night honey2" and68recombinant inbred lines was done by leaf inoculation HJX strain in vitro with QTL5.0software. Eighteen QTL loci connecting with incidence distributed in LW2and LW3, including eleven QTL loci on LW2, named as qlt2.1, qlt2.2, qlt2.3, qlt2.4, qlt2.5, qlt2.6, qlt2.7, qlt2.8, qlt2.9, qlt2.10, qlt2.11, They explained23.9%,23.2%,21.2%,23.2%,20.4%,20.2%,17.8%,17.8%,17.4%,17.3%,17.4%of phenotypic variation respectively. Seven QTL loci on LW3, named as qlt3.1, qlt3.2, qlt3.2, qlt3.4, qlt3.5, qlt3.6, qlt3.7. They explained25%,18.4%,18.2%,17.9%,16.7%,16.6%and16.4%of phenotypic variation respectively. Eight QTL loci associated with citrus severity was detected including two loci on LW1, named qlty1.1, qlty1.2. They explained18.2%and15.8%of phenotypic variation respectively. Qlty2.1, qlty2.2and qlty2.3on LW2explained.17%,17%,16.3%. Qlty3.1and qlty3.2on LW3explained18.2%and17.4%. qlty6.1on LW6explained17.5%. These loci closely connected with the fixed markers which from EST-SSR of citrus genome such as cBSSR835E2, CMS30, Hcx4040, cSSR340, F13, cSSR202, moviemaker, cSSR309, cBDM83E1, cBMM74, cSSR128, cSSR27, cSSR32, HCX6020, CMS14, Hcx6026, F88and LcSSR115. The corresponding EST coding sequence could be research as new molecular marker assisted breeding.(5) The corresponding EST of LcSSR115, HCX6020, cSSR309, cSSR202, F88, cSSR340, cSSR32, Hcx4040was analysis by using the qPCR with moderate resistant C. reticulata Buzhihuo. The results show that six EST were up-regulated besides that of HCX602and cSSR32down-regulated. The study provided a theoretical basis for further resistant gene cloning and transformation of validation. |
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