Screening And Analysis Of Verticillium Dahliae-Related Mutants In Arabidopsis Thaliana And Biological Control Of Verticillium Wilt

Verticillium dahliae Klebahn is a soil-borne fungal pathogen causing vascular diseases. The pathogen penetrates the host through the roots, spreads through the xylem, and systemically colonizes both resistant and susceptible genotypes. Verticillium species are soil-borne fungi with worldwide distribution, causing vascular disease that results in severe yield and quality losses. Typical aboveground symptoms of Verticillium infection on Arabidopsis thaliana are leaf chloroses, vein clearing, and stunted growth. We choose the model plant Arabidopsis thaliana to explore cotton resistant genes. In addition, the test also studied microbial control Verticillium wilt on biological control.1?The screening of V. Dahliae-Related Mutants in Arabidopsis thalianawe use the Arabidopsis thaliana mutant screening system established at earlier stage in the research.Choose the seeds from EMS mutagenic seeds of Arabidopsis thaliana mutant library, growing vertically for9 days. Then droped 1?LVerticillium dahliae kleb Vd991 spore suspension at the root of seedlings(its concentration is 5×105 /m L). Some seedlings showed phenomenon of Verticillium wilt symptoms after inoculated 8 to 12 days, we should transfer the seeding to grow in the soil. More than 300 mutants were screened from the mutant library, which contains about 45200 seeds induced by EMS mutagenic the seeds of Arabidopsis thaliana mutant library. Two sensitive mutants se348, se579 and one resistant mutant se346,which could be steadily hereditary were obtained in the end by verifying the phenotype.After the mutant se348 inoculated Vd991 at the early stage on the culture medium, individual growth was stronger than the wild type Col-0. Inoculated for 8 days, cotyledons first faded green and turned yellow than Col-0, its cotyledon began to dry up with the increase of inoculation processing time, some euphyllas turned chlorosis. Put the inoculated seedlings into the soil to grow, we found that the mutant plant stunt, the life cycle was completed advance. After inoculated for 13 days, the pathogenic fungi content of se348 leaves was 4.2 times more than that of Col-0. The accumulation intensity of anthocyanin accumulation was lower than that of Col-0. The chlorophyll content declined more significantly than that of wild type. It shows that the mutant se348 is more sensitive to V.dahliae.After the mutant se579 inoculated Vd991 at the early stage on the culture medium, leaves first faded green, then were almost chlorosis, vein clearing, the growth was inhibited. The time of cotyledon became chlorosis is earlier than wild type, its cotyledon began to dry up with the increase of inoculation processing time, The number of chlorotic leaves increases. The life cycle was completed in advance by early bolting,eight days mutant plant stunt, tillering increased. The number of chlorotic leaves of mutant was more than wide type following a root-dip procedure. After inoculated for 12 days, the pathogenic fungi content of leaves was 4.75 times more than Col-0. The intensity of anthocyanin accumulation is increase but was lower than Col-0. The chlorophyll content declined more significantly than the wild type. It shows that the mutant se579 is more sensitive to V.dahliae.After the mutant se346 inoculated Vd991 at the early stage on the culture medium, se346 stunted growth, but its leaves were not chloroses. Anthocyanin accumulation increased form petiole, and increased more significantly. when inoculated for 20 days. Put the inoculated seedlings into the soil to grow, we found the mutant could grow normaly. The chlorophyll content change is not obvious. The pathogenic fungicontent of leaves was similar to Col-0. It shows that the mutant se346 is more resistance to V.dahliae.2?Genetic analysis and gene cloning of the mutant se346Phenotype of the first filial generation(F1) was similar to wild type Ler. And F2 generation represent represent segregation, its ratio of segregation of character approached to 3:1. Genetic analysis indicated that the mutant was a single recessive gene mutation. The lowest reorganization rate was located in the bottom of chromosome II by map-based cloning, near the maker T20P8(11500526-11599871bp) probably. Gene sequencing shows that the 52 th base cytosine was replaced by thymine, this gene mutation result in premature termination of protein(heme oxygenase) translation.3?Biological control of cotton Verticillium wiltBacillus pumilus Y106 is a kind of biocontrol bacterium which could be used for pathogenic fungi diseases controlling. The result of dural culture method showed that Y106 had quite strong fungistatic activities on V.dahliae, colony diameter of treatment group was 3.85 cm by co-culture for 10 days,decreased by 39.27% than the control group. When cultured V.dahliae called Vd-gfp77 which labelled with green fluorescent protein gene markers with Y106 in potato dextrose broth, we find that fluorescence absorbance value of Vd-gfp77 in the emission wavelength at 530 nm decreased from 217.43 to 97.49 withthe incresement of the Y106 concentration. Antagonistic experiment in the soil showed that the proliferation of V. dahliae were inhibited by Y106 in some extent, colony number of control group was 79,treatment group was 44, decreased by 44.3% than the control group with the incresement of the Y106 concentration. The control effect of Y106 on disease index deareased by 32.3%. Therefore, Y106 had strong fungistatic activities on V. dahliae and could prevent to control Cotton Verticillium wilt.