| This article mainly focused on the research of growth charactristics, host range, active metabolite composition, genetic transformation and green fluorescent protein lable of Phyllosticta citriasiana, a pathogen caused Citrus maxima cv. Guanximiyou black spot. The results were as follows:The mycelia growth of P. citriasiana was sensitive to the temperature. The mycelia growth was inhibited completely when the culture temperature was at≥40℃ or≤10℃ in the artificial culture condition. The mycelia growth rate increased initially and then decreased immediately at the culture temperature between 15℃ and 35℃. The mycelia grew fastest when cultureed at 25℃ and the colony diameter was 3.41cm for 7days, and followed by 30℃, the colony diameter was 3.25cm. The colony diameters were significant difference in 25℃ or 30℃ culture temperature from 15℃ (1.29cm×20℃(1.49cm) and 35℃(0.68cm) culture temperature. These results indicated that the mycelia grew fast when the culture temperature was at 25-30℃. The mycelia grew fastest when cultued on the SOA solid medium (9d) and there was a significant difference in the mycelia growth rate between SOA and OA medium. It indicated that it could promote mycelia growth by adding 2% sorbitol to the OA medium. The mycelia growth was sensitive to light intensity, with fastest growth in the dark, but the growth rate decreased significantly at 1980 Lux light intensity. The conidia germination rate of P. citriasiana increased initially and then decreased when the conidia germinated on the 1% glucose solution,10% glucose solution, and water from 0 to 72h. The germination rates were highest at 48h, with the rates at 61.4%,39.1% and 52.7%, respectively. It could significantly raise the conidia germination rate at 24h when the conidia germination solution(water) added 1% or 10% glucose(m/v). But there was no significant difference in the conidia germination rate by using water and 1% glucose solution as germination solution, when the germination time was 48h. It indicated that 1% or 10% glucose solution only could raise the the conidia germination rate of P. citriasiana within a short time period (in 24h). The conidia germination rate on the 0.7M NaCl solution was low, the max germination rate was only 16.4%(in 72h). The lethal temperature of mycelia and conidia was 55℃ for 15min at-water-bath.The pathogenicity tests of P. citriasiana on 28 speicess of Citrus plant showed that the pathogenicity range of this pathogen was narrow, except infection Citrus maxsima cv. Guanximyou, it could also infect pomelos such as C. maxima cv. Longyou 1, C. maxima cv. Longyou 2, C. maxima cv. Putaoyou, C. maxima cv. Pingshanyou, and infect lemons such as C. lemon cv. Feiminailaobaihua 2, C. lemon cv. Wufeng 2, C. lemon cv. Hainanyaoyong.The culture filtrate of the mycelia of P. citriasiana had biological activity to the leaves of Citurs maxia cv. Guanximiyou. It caused disease similar symptoms (yellow or tan spot) of P. citriasiana on the injured leaves. The active culture filtration was tolerant to high temperature, it still had biological activity to injured leaves of C.maxima cv. Guanximyou when it processed by water bath at 80℃ for 15min. The culture filtrate extraction from filtrate direct concentrated or ethyl acetate extracting firstly and then concentrated, were dissolved by methanol and then filtered. Both of resuspentions had a high UV absorption peak at 367nm. The concentrated extraciton dissolved by sterile water could make the injured leaves of Cynanchum chinense form the yellow black spot, but did not have biologicla activity to the injured leaves of Ageratum conyzoides.The fresh bright (or yellow) white peletons or mycilia pellets would be formed when the mycelia of P. citriasiana cultured on the CM, SCM, or 0.6M TB-Sucrose liquid media (at 25℃,120rpm,3d). Few protoplasts were gained when the mycelia were lysed by lysing enzyme or driselase alone. When 35mg/mL lysing enzyme and 10mg/mL driselase was used separately to digest mycelia for 3h, their protoplasts concentrations were only 8.167×105 numbers/mL and 1.0×105 numbers/mL, respectively. But when the mixture of 30mg/mL lysing enzyme+15mg/mL driselase was used, the protoplasts concentration could reach to 1.24×107numbers/mL. The regeneration rate of these protoplasts on the OCM solid media was 12.6%. The pCT74 plasmid (containing the GFP gene and hygromycin B resistance gene) was transferred into the protoplasts of P. citriasiana by PEG-CaCl2 mediated protoplast transformation and OCM solid media containing 150μg/mL-250μg/mL hygromycin B were used to selecte transformants. After fluorescence observasion and PCR verification, we had succeeded to obtain the transformant labled by green fluorescent protein (GFP). |
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