Pine Resin Canker Into The Risk Assessment And Molecular Biology Research

Pitch canker, caused by Fusarium circinatum Nirenberg & O'Donnell (=F. subglutinans(Wollenweb and Reinking)Nelson, Toussoun, and Marasas f. sp. pini(teleomorph: Gibberellacircinata Nirenberg and O'Donnell)), is a serious disease of conifers: A characteristicsymptom of infection on most hosts is a copious pitchy flow from cankers or necrotic tissue onlimbs, trunks, roots, and cones. Long-range spread of the disease may be connected to themovement of infected seed, seedlings, wood, vector, and soil. More recently, the disease hasbeen introduced into California, South Africa, Japan, and Chile.1) F. circinatum could grow on six different kinds of culture medium which were tested.On PDA plate, its growth was fastest, while on the Fusarium selective medium was slowest.The fungus grew well at 18～27℃, with optimum at 24℃. The treatment of whole darknesscould promote mycelium growth. The fungus also grew well at pH 5～9, with optimum at pH8.0. On the basic culture medium, galactose was the best carbon sources and sodium nitratewas the best nitrogen source. Vitamin did not distinctly affect hypha growth. Conidiosporescould germinate in all media including sterile water, while the optimal medium was 5% glucose.The conidia could germinate at 15～35℃, RH 85%～100% and pH 3～12, with the optimal at 25℃, RH 100% and pH 7.0. Darkness & enough oxygen were good for spores germination. Thelethal temperature for hypha was 55℃, and for spores was 52℃.2) Following the principle of systematicality, essentiality, practicality and specificity, anindex system for risk assessment of exotic forestry disease was established, consisting of 3different layers, i e. objective, criteria, and index layers, and 14 index parameters. In addition,the weight of index parameters was determined by analytic hierarchy process (AHP), andcomprehensive modeling and risk grading of exotic forestry disease were presented. Pest riskassessment (PRA) of F. circinatum was developed using above modeling. The PRA result was0.8237, and it belongs to very high devastating pest in China. It suggests that F. circinatumshould be listed the quarantine pest in China.3) AFLP fingerprinting analysis of F. circinatum and relative species was carried out. Tenprimer-paris that could generated abundant polymorphism fragments were screened out. Theyamplified 298 nucleotide acid fragments in the seventeen strains of Fusarium spp., 283polymorphism fragments among them. Percentage of polymorphism fragments produced byeach pair of AFLP primer was 94.97% in average, and varied from 89.29%-100%. All thesedata indicated that considerable genetic variation existed among F. circinatum and relativespecies at DNA level. Molecular genetic distances among Fusarium spp. were calculated, andthe relationship among them was described quantitatively. Compared with biological species,the result of cluster analysis were basically similar. Genetic diversity of E-AT/M-CAA AFLPfingerprinting of Fusarium spp. was analyzed, unique and difference bands for each specieswere determined, and all Fusarium section Liseola tested were identified based on the E-AT/M-CAA AFLP fingerprinting.4) The oligonucleotide primers G1/G2 (5'-GCGGTGTCGGTGTGCTTGTA-3'/5'-ACTCACGGCCACCAAACCAC-3'), derived from the differentiation of intergenic spacer (IGS)regions within Fungi, amplified a single 873 bp product from Fusarium spp.. The IGS DNAsequences of Fusarium spp. were gained from GenBank. Oligonucleotide primers S1/S2(5'-CTTACCTTGGCTCGAGAAGG-3'/5'-CCTACCCTACACCTCTCACT-3'), derived fromIGS DNA, amplified a product of 364 bp which was unique to F. circinatum. Thenested-PCR using primers G1/G2 and S1/S2 identified F. circinatum from other Fusarium spp..This PCR assay was proved to be highly sensitive with the detection limit of 5×10^-3 pg.μl^-1genomic DNA or 10 spores/100μl H₂O. The result indicates that the nested-PCR couldsuccessfully used to detect the presence of F. circinatum directly from infected plant tissue.