Determination Of Phylogenetic Species Of Fusarium Graminearum Clade And Characterization Of Transgenic Wheat Containing An Antibody Fusion And Transgenic Rice Carrying Antifungal Peptides

Wheat and maize scab or Fusarium head blight(FHB) caused by Fusarium graminearum clade not only reduces yields,but more importantly produces various mycotoxins during the infection of crops.These Fusarium mycotoxins directly accumulate in cereal grains and are detrimental to human and animal health.FHB epidemics occur in different areas in China where agro-environments vary greatly. Furthermore,natural resistance is inadequate,and there is no immune or highly resistance germplasm available in wheat or closely related crops.This has slowed down the progress in breeding resistant cultivars in wheat.Thus,investigation of phylogenetic species and mycotoxin chemotypes of F.graminearum clade from different regions throughout China, identification of resistant transgenic wheat mediated by Fusarium-specific antibody fusions are required.This will certainly be very important for the identification of resistant crop varieties,reduction of mycotoxins,food safety and breeding cultivars resistant to scab in wheat.In rice a huge annual loss of yields is due to fungal diseases such as rice blast caused by Magnaporthe grisea and sheath blight caused by Rizoctonia solani and the selection of transgenic rice expressing multiple foreign genes that are resistant to rice blast and sheath blight is required.This will certainly be very important for breeding cultivars resistant to blast and sheath blight in rice.In this dissertation there are three main focuses dealing with F.graminearum and improvement of resistance to scab and rice fungal diseases.These include:1) study of phylogenetic species and mycotoxins of F.graminearum clade to reveal how many types of species and mycotoxin chemotypes from F.graminearum clade throughout China,2) resistant evaluation of transgenic wheat expressing Fusarium-specific antibody fusions after inoculation with F.graminearum to identify highly resistant transgenic wheat lines, and 3) resistant assays of transgenic rice expressing multiple genes after inoculation with Magnaporthe grisea and Rizoctonia solani to screen transgenic rice lines resistant to the two fungal diseases.The main results are presented below:1.A total of 460 Fusarium strains isolated from diseased wheat and maize from 13 provinces in China were assayed with primers Fg16F/R specific for F.graminearum clade and two SCAR types,ⅠandⅤ,were revealed.Thirty strains were randomly selected for sequencing of their three genes coding for Tri101,reductase-like gene and Histone 3. According to the fixed nucleotides used by others recently for identification of phylogenetic species of F.graminearum,comparison and blast analysis of three gene sequences derived from thirty strains indicated that there are two phylogenetic species,F. asiaticum and F.graminearum,of F.graminearum clade throughout China.Phylogenetic species are completely congruent with SCAR typing.The strains of SCAR typeⅠbelong to F.graminearum while SCAR typeⅤconsists of the strains from F.asiaticum.Among 460 strains assayed,342 strains are F.asiaticum and 118 strains are F.graminearum.Thus, F.asiaticum is the predominant species of F.graminearum clade in China.Based on the climate data derived from 30 years from the period 1970 to 1999,F.asiaticum is distributed mostly in the areas where the annual average temperatures were above 15℃, whereas F.graminearum is predominantly from the areas where the annual average temperatures were 15℃or lower.2.Primers specific for mycotoxin chemotypes were designed according to gene sequences of mycotoxin metabolic pathways in F.graminearum clade and used for a series of PCR to identify mycotoxin chemotypes of Fusarium 342 strains.The results indicated there are three mycotoxin chemotypes in F.graminearum clade in China, 3-AcDON(176 strains,51.46%),15-AcDON(113 strains,33.04%) and NIV(53 strains, 15.5%).In addition,there are two different groups of 15-AcDON-producing strains in F. graminearum clade in China.Strains with 11 bp repeats within their Tri7 gene belong to F. graminearum while strains without the 11 bp repeats reside in F.asiaticum.The later is not reported group of 15-AcDON-producing strains,which were designed as a new 15-AcDON group.The 11 bp repeat could be used as molecular marker for differentiation of F.asiaticum and F.graminearum that produce 15-AcDON mycotoxin.3.Fusarium-specifie antibody fusions were introduced into immature embryos for the first time by.biolistic bombardment,resulting in fifteen regenerated transgenic wheat plants after selection on herbicide media.T1 plants were then inoculated with F.asiaticum by single floret inoculation and disease symptom was evaluated 7,14 and 21 days post inoculation(dpi).The results showed that seven plants displayed a significantly enhanced resistance 21 dpi.The integration and expression of the transgenes in theses transgenic plants were demonstrated by PCR and RT-PCR.Resistance evaluation of all T2 plants derived from these seven transgenic wheat plants after inoculation with the same strain resulted in selection of six plants with significantly improved resistance comparable to that of the best resistant wheat cultivar Sumai3.Therefore,Fusarium-specific antibody fusion-mediated resistance to FHB is stably inherited and thus could be used as a valuable scab resistance gene for molecular breeding of new wheat cultivars with improved scab resistance.4.Three antifungal peptides,a chitinase gene from wheat(Chi),a peptide from Aspergillus giganteus(AG) and a peptide from maize(MBP),together with hygromicin gene were used for co-transformation of calli derived from mature rice embryos.After screening on hygromycin media thirty-six positive transgenic rice lines were selected, from which seventeen plants containing three genes were identified by PCR.Southern blot and Western blot analyses showed the integration and expression of the transgenes. Inoculation of T2 plants with Magnaporthe grisea and Rizoctonia solani identified six resistant transgenic rice lines that displayed an intermediate resistant level for the both fungal pathogens.These results indicated that the expression of multiple genes in transgenic plants could improve resistcance characters.In addition,after segregation in T2 generation,resistant transgenic rice plants without hygromycin gene but with transgenes were selected.This result indicated that after co-bombardment with multiple genes, transgenic lines without any antibiotic genes could be generated to produce environment-safe transgenic crops.