Pathogen Identification Of Mango Malformation And Labeling Of Pathogen With GFP Gene

Mango malfortnation Disease is an important disease of mango (Mangifera indica L.) worldwide, Mango malformation is characterized by the abnormal development of vegetative shoots and inflorescences. Malformed vegetative buds are enlarged and crowd in the leaf axils or at the tips leading to profusely proliferated and bunched shoots. Flowers in a malformed inflorescence are producing no fruit or aborting early. In the serious cases, the disease also results in stunted trees with poor productivity. Although the disease has became more and more prevalent in mango producing areas and posed a great threat on mango production, efforts to developing control measures have not made too much progress in the last decades. Many species of Fusarium genus were reported to be associated with mango malformation in different countries or regions. Occurring of mango malformation has been detected in some mango orchards in Panzhihu, Sichuan Province, and Huaping County, Yunnan Province of China, for some time. Its etiology has not been determined yet. Therefore, the main purpose of this study is to establish the causal agent of mango malformation for these two locations. The results are as follows:1. The causal agent was identified as Fusarium proliferatum for the first time in China. The pathogen of mango malformation disease in Panzhi Hua and Huaping was identified as Fusarium proliferatum (Matsushima) Nirenberg on the basis of its colony characteristics, conidial morphology and analysis of DNA-sequences of ITS region of rDNA gene andβ-tubulin gene. This work also established F. proliferatum to be the fourth among mango malformation associated Fusarium spp verified through Koch's postulates.2. Biological characteristics of the pathogen were determined primarily. The best mycelial growth was recorded at 24℃and the optimum pH value was pH6. Among different C and N sources tested, fruit sugar and protein peptone were the best for the fungal mycelial growth. Light promoted mycelial growth. The optimum temperature for conidial germination was 24℃and the optimum pH was 5. The lethal temperature for the mycelium was 55℃for 20 min or 60℃for 5min.3. Green fluorescent protein gene (GFP gene) was transformed successfully to F. proliferatum and GFP expressed isolate was obtained for the first time. The protoplasts of a F. proliferatum isolate Mmdl have been prepared by digesting the mycelia with lywallzyme. Furthermore, the expression vector pPGT74-sGFP was transformed into the protoplasts with PEG mediated method. Transformants were regenerated and selected on hygromycin-containing medium. Single-spore isolates obtained and showed strong green fluorescence after several successive generations on the medium without hygromycin. The results showed that pPGT74-sGFP was successfully integrated into the isolate Mmdl. The presence of the green fluorescent protein DNA in the fungal cells was confirmed by PCR using a GFP-specific primer pair. The transformed isolates did not differ markedly from the wild type isolate in growth and morphological characteristics in vitro. Pathogenicity tests showed that the transformation process did not alter pathogenicity of the Mmdl.Establishment of the F. proliferatum as the causal agent of mango malformation in China and understanding of primary biological features of the fungus may help to the future control of the disease. The F. proliferatum isolate labeled with green fluorescent protein gene facilitates further study on its modes of penetration, colonization and extension in the mango tissue, as well as its spread.