Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (10): 1885-1891.doi: 10.3864/j.issn.0578-1752.2016.10.005

• PLANT PROTECTION • Previous Articles     Next Articles

Development of the Agrobacterium tumefaciens Mediated Transformation System of Alternaria alternata Apple Pathotype

GU Xue-ying1, SHI Wen-xiao2, WANG Hong-kai 2, GUO Qing-yuan1   

  • Received:2015-12-29 Online:2016-05-16 Published:2016-05-16

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Abstract: 【Objective】 Alternaria alternata apple pathotype is one of the important pathogens on apple which can infect leaves and fruits seriously. The objective of this study is to establish the Agrobacterium tumefaciens mediated transformation system of Al. alternata apple pathotype, provide a technical basis for study molecular mechanisms of pathogenesis. 【Method】 Vector pKO1-HPH is a shuttle plasmid which is constructed on the backbone of pCAMBIA1300 containing hygromycin phosphotransferase gene (hph) and GFP gene. Fungal transformation was carried out by co-incubation of Ag. tumefaciens strain AGL1 containing plasmid pKO1-HPH with conidia of Al. alternata apple pathotype. Transformants were screened at PDA plates containing hygromycin B. Hph gene expression was detected by incubation of transformants on PDA plates containing 50 μg·mL-1 hygromycin B, GFP was detected using Green fluorescence microscope. Influences of fungal cultural time and pretreatment method of conidia on transformation were evaluated. The stability of transformants was determined by growing on PDA plates containing hygromycin B after five-generation successive growth. PCR assay was used to confirm the existence of the hph gene and GFP gene in transformants. The copy number of inserted T-DNA in transformants was determined by Southern blot. Pathogenicity test of some transformants was performed on apple fruits and leaves. 【Result】 Spores of Al. alternata apple pathotype were formed enough when 100 μL of spore suspension (about 105 spores) was spread on PCA plate and incubated for 36-48 h. The maximum of transformation efficiency was up to 200 transformants/107 conidia when pretreatment of conidia at 4℃ for 6 h before transformation. The resistance to hygromycin B in transformants was stable after five successive generations. Results of specific PCR showed the exogenous genes (GFP and hygromycin B) were integrated into genome of Al. alternata apple pathotype strain and single copy insertion was detected in genome of majority of the transformants according to southern blot analysis. Green fluorescence was detected in the mycelia and spores under the fluorescent microscopy, indicating GFP was expressed in spores and hyphae of transformants. Results of pathogenicity test on fruits and leaves showed that the pathogenicity of several transformants was reduced.【Conclusion】Ag. tumefaciens mediated transformation system of Al. alternata apple pathotype is developed in this research and useful for further studies on molecular interaction of apple and Al. alternata apple pathotype.

Key words: Alternaria alternate, apple pathotype, ATMT, pathogenicity

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