| Alternaria leaf spot of chrysanthemum (Dendranthema×grandiflorum (Ramat.) Kitamura) caused by Alternaria species infecting is a significant and wide happened disease. This disease often happens heavily on condition of the high temperature and high humidity in the whole year production of chrysanthemum in greenhouse, when propagating rhizomes in summer and continuous cropping in field. Infections of Alternaria will debase the commercial value of the plants, even result in plant death. So, it is the most important target of chrysanthemum breeding to cultivate new cultivars resistant to the alternaria leaf spot. However, there was few reports of studies on the fungi caused alternaria leaf spot of chrysanthemum, screening materials that were resistant to this disease among related species of chrysanthemum, and improving resistance of chrysanthemum by transgene.To lay a solid foundation for utilization of chrysanthemum wild species'potential chacacters of disease resistance, and improving resistance of chrysanthemum cultivars by transgene, we carried out a series studies on isolation and identification of Alternaria sp. causing leaf spot in chrysanthemum, disease assessment to alternaria leaf spot in related species of chrysanthemum by seedling inoculation, the structural and biochemistry mechanisms of wild species with resistance to alternaria leaf spot, and transformation of chrysanthemum with hrfA gene. The main results are follows:1. Isolation, identification and inoculation of Alternaria sp. causing leaf spot in chrysanthemum (Dendranthema×grandiflorum). Six fungal isolates that can cause alternaria leaf spot were isolated from infected leaves of chrysanthemum. Morphological characters of conidia of six isolates on potato carrot agar medium and natural hosts was investigated, and found that in contrast with strain A02 and A09, the septums of the mid-body of conidia of strain A01,A03,A04 and A07 were more thicker, the abstriction of the main septums was more obvious.On natural hosts, strain A01,A02, A03,A04 and A09 usually formed single conidium or short conidia chain (3-7 conidia), and the short conidia chain of strain A02 and A09 branched. Strain A07 could form long conidia chain including more than 10 conidia and the conidia chain rarely branch. Results of observation of conidial morphology according to the method of Zhang (2003) showed that strain A01,A03,A04 and A07 could form long conidia chain including more than 10 conidia, the conidia chain did not branch or branch with a short side conidia chain (no more than 5 conidia), conidia chain of strain A02 and A09 contained no more than 5 conidia and abundant branches. According to the above results, A01,A03, A04 and A07 were identified as A. tenuissima strains, A02 and A09 were A. alternata strains.Analysis of 5.8S rDNA-ITS only can served as a assisting technique to differentiate difference between small-species and big-species of Alternaria. There was no significant difference in the pathogenicity between the six isolates. Results of artificial inoculation showed that the potential pathogenic minimum inoculum with six strains'condia mixture was 1×106/ml, and the optimal mycelia-concentration for inoculation was 1×107/ml.2.Evaluation of resistance of related species of Dendranthema and the allied genera seedling to alternaria leaf spot, difference in defense-related leaf morphological traits and response of enzymes to A. tenuissima. Disease assessment to measure severity of alternaria leaf spot were performed at 33 related species of Dendranthema and the allied genera. Results showed that most species have various degrees of susceptibility and infection indexes are ranged in 33.60～91.66. A. spathulifolius and A.marginatum are resistant and the infection index is ranged in 17.16-18.85, while A. pacificum, D. lavandulifolium, D. indicum (Shennongjia) and A. wilsoniana are moderate resistant and the infection index is ranged in 25.62-28.85.The leaf mophological traits of two resistant (A. spathulifolius and A. marginatum) and two highly susceptible wild species [D. indicum var. acutum and D. indicum (Guizhou)] have thick and high trichomes, high wax content, and high proportion of palisade tissue and sponge tissue. In resistant species, trichome density of upper leaves are 8.88 mm-2 and 17.84 mm-2, that of lower leaves are 11.36 mm-2 and 37.98 mm-2, trichome height are 1.01mm and 0.13mm, and wax content are 26.18 and 25.70.while in susceptible species, trichome density of upper leaves are 0.28 mm-2 and 2.48 mm-2, that of lower leaves are 0.32 mm-2 and 4.92 mm-2, trichome height are 0.08mm and 0.078mm, and wax content are 18.07 and 10.74.So, the leaf mophological traits is related with resistance of alternaria leaf spot. The activitiy of the enzymes, such as phenylalanine ammonia lyase (PAL),β-1,3-glucanase (GLU), and chitinase (CHT), is higher in the resistant species than that of in the highly susceptible ones. The activitiy of PAL and GLU are increasing and reach the peak value at 48h after inoculateion, while that of CHT at 72h. The activity changes of PAL, GLU and CHT before or after inoculation are associated with the resistance.PAL, GLU and CHT can be used as a biochemical marker for assessing resistance of seedlings of related species of chrysanthemum to alternaria leaf spot.3.Transgenic Chrysanthemum Plants Expressing a hrfA Gene. Results of regeneration analysis of chrysanthemum cultivars'Biyutai','Aoyunhuoju','Italy Red'and 'Shenma' on MS medium supplemented with 6-benzyladenine (6-BA) and naphthalene acetic acid (NAA) showed that genotype was the key factor that influenced the capability of regeneration,'Biyutai'had a high capability of regeneration and was a good transgene receptor. The optimal concentration of kanamycin (Km) supplemented to MS medium was 15 mg/L to select the transformed explants and regenerated shoots, and 7.5 mg/L to eliminate escape shoots.To inhibit growth of Agrobacterium tumefaciens after transformation and reduce toxicity to explants, the optimal concentration of carbenicillin was 400mg/L. Seventeen putative transformants of chrysanthemum derived from independent explants were obtained by Agrobacterium-mediated transformation. Polymerase chain reaction (PCR), Southern blot and Reverse transcription PCR (RT-PCR) analysis showed that the hpaGxoo gene was successfully integrated into the chrysanthemum genome and expressed in seven transgenic lines.Inoculation assays with the detached leaves and the whole plants both indicated that the presence of the transgene was associated with an improved level of resistance to alternaria leaf spot. Furthermore, transgenic chrysanthemums flowered significantly earlier than the wild-type plants did, this suggestted that the hpaGxoo gene probably accelerated chrysanthemum development. Setting rate of three transgenic lines decrease heavily and percent of growing seedling of two transgenic lines also decrease obviously, and these decrease probably ascribed to the 35S promoter. PCR and RT-PCR analysis of T1 transgenic plants of line 3,7 and 10 showed that the introduced hrfA gene could inherit and express stablely in the T1 progenies.
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