Population Genetic Structure Of A New Anthracnose Of Camellia Oleifera And Endophytic Bactrria Colonization Pathway

Camellia oleifera anthracnose is one of the major diseases in China’s Camellia Produeing areas. It seriously damages Camellia oleifera quality and affect total yield.We found the major pathogen is not only Colletotrichum gloeosporioides. The new anthracnose pathogen is widely distributed and serious damaged.The study researched that new anthracnose pathogen of Camellia oleifera and its genetic structure of Colletotrichum fructicola.We used Green Fluorescent Protein Gene Tagged endophytic Bacillus subtilis Y13UV in the fields experiment which studied colonization pathway of Bacillus subtilis Y13UV.The main results were as followed:(1) Pathogen identification of a new anthracnose of Camellia oleifera based on multiple-gene phylogeny.We collected diseased C. fructicola leaves with the anthracnose syndrome from six provinces in China.A total of 23 isolates were obtained.Multiple-gene phylogenetic analyses indicated that these 23 isolates were grouped in the same clade as C. fructicola, with a bootstrap support of 100%.(2) Population genetic structure of C. fructicola.nrDNA ITS sequences were used to analyze the genetic structure and genetic diversity of C. fructicola populations consisting of 145 strains isolated from 10 sites. A total of 13 ITS haplotypes were identified among which the dominant haplotype (haplotype 5) included 127 specimens from the 10 sites. The genetic differentiation index, FST, revealed significant genetic differentiation between Suizhou populations and the others. AMOVA test showed that 13% of genetic differentiation occurred among population and 87% within population. The Mantel test showed no linear-correlation between genetic distance and geographical distance. Mismatch distribution analysis suggested there was population expansion for C. fructicola and gene flow was detected between populations.(3) Colonization pathway of Bacillus subtilis Y13UV.Plasmid was introduced into cells of Bacillus subtilis Y13UV by protoplasts conversion and GFP-tagged Y13 was acquired,inoculated of camellia seedlings with strains Y13UV-GFP by root drenching, leaf spraying and combination of root drenching and leaf spraying to investigate the colonization capacity of strains Y13UV-GFP in Camellia oleifera and its biocontrol mechanism.The results showed that strainsY13UV-GFP could invade Camellia oleifera tissues by root and leaves, reproduce and transfer in camellia oleifera tissues in Camellia oleifera.Over time, Y13UV-GFP shifted upward by root drenching and colonized stable in root and middle stem in 5 days after inoculation. The quantity of strain Y13UV-GFP stabled from 5 days to 15 days.Then the quantity of strain Y13UV-GFP reduced rapidly after 20 days.In 30 days the quantity of Y13UV-GFP was 0.53X103cfu/g.The quantity of Y13UV-GFP in high-positionin obviously more than low-position.Y13UV-GFP shifted downward by leaf spraying.The quantity of strain Y13UV-GFP stabled from 3 days to 10 days.Then the quantity reduced rapidly after 10 days.In 30 days,t 0.40×103cfu/g in leaves,0.27×103cfu/g in roots.The quantity of combination method showed a trend "fist decreasing then increasing".Stem colonization stabled from 3 days to 5 days and root colonization was continuously decreased.After 30 days,0.53 X 103cfu/g in roots,0.67 ×103cfu/g in leaves by combination inoculation. Combination inoculation quality in stem more than other two inoculation methods.The best method of inoculating with strains Y13UV-GFP was combination of root drenching and leaf spraying.Repeated inoculated was better for Y13 colonization.Control effect of combination of root drenching and leaf spraying is about 73.76%...