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United States Department of Agriculture

Agricultural Research Service

Research Project: BIOLOGICALLY BASED MANAGEMENT STRATEGIES FOR CONTROL OF SOIL-BORNE PATHOGENS ....AN ALTERNATIVE TO METHYL BROMIDE PRE-PLANT SOIL FUMIGATION

Location: Floral and Nursery Plants Research Unit

Title: Genetic transformation of Fusarium oxysporum f.sp. gladioli with Agrobacterium to study pathogenesis in Gladiolus

Authors
item Lakshman, Dilip
item Pandey, Ruchi -
item Kamo, Kathryn
item Bauchan, Gary
item Amitava, Mitra -

Submitted to: European Journal of Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 20, 2012
Publication Date: December 9, 2012
Citation: Lakshman, D.K., Pandey, R., Kamo, K.K., Bauchan, G.R., Amitava, M. 2012. Genetic transformation of Fusarium oxysporum f.sp. gladioli with Agrobacterium to study pathogenesis in Gladiolus. European Journal of Plant Pathology. 133:729-738.

Interpretive Summary: Fusarium rot caused by Fusarium oxysporum f.sp. gladioli (Fog) is one of the most serious diseases of Gladiolus, both in the field and in stored bulbs. Traditionally, the pathogen had been controlled by fumigating soil with methyl bromide which is now banned, by hot water treatment, and to a limited extent with tolerant cultivars. However, very few chemical fungicides are available to control the disease, mostly due to insufficient knowledge on how the pathogen causes disease in gladiolus. In order to understand the pathogenic process at the cellular level, we have transformed the fungus with Agrobacterium tumefaciens to introduce green, yellow and cyan fluorescence genes. Transformed lines will also be useful to identify pathogenicity genes and screening of transgenically resistant gladiolus lines against the pathogen.

Technical Abstract: Fusarium rot caused by Fusarium oxysporum f.sp. gladioli (Fog) is one of the most serious diseases of Gladiolus, both in the field and in stored bulbs. In order to study the pathogenesis of this fungus, we have transformed Fog with Agrobacterium tumefaciens binary vectors containing the hygromycin B phosphotransferase (hph) gene and fluorescence reporter genes of EGFP (green), EYFP (yellow) or ECFP (cyan) using the AGL1 strain of A. tumefaciens. Hygromycin B (100 µg/ml) resistant colonies were observed only when acetosyringone was added to the co-cultivation medium. Transformed colonies are more clearly visible when co-cultivated on cellophane membrane than on Hybond -N+ membrane. Transformed lines were stably maintained through four serial passages on medium containing hygromycin B, and they expressed green, yellow or cyan fluorescence. PCR with hph-specific primers and Southern blotting with an hph-specific probe were positive from HygR lines but not from the untransformed isolate. The cyan fluorescence of the ECFP-transformed isolate was clearly distinguishable from the green autofluorescence of Gladiolus roots, signifying the potential of these lines for further histopathological investigations. Transformed lines will be useful for identifying pathogenicity related genes, screening transgenic resistance, and in studies of host-pathogen interactions.

Last Modified: 9/21/2014
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