Resistance to Fusarium oxysporum f. sp. gladioli in transgenic Gladiolus plants expressing either a bacterial chloroperoxidase or fungal chitinase genes

Authors: Kamo, Kathryn; Lakshman, Dilip; Pandey, Ruchi; Guaragna, Mary; Okubara, Patricia; Rajasekaran, Kanniah - Rajah; Cary, Jeffrey; Jordan, Ramon

Submitted to: Plant Cell Tissue and Organ Culture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/29/2015
Publication Date: 11/30/2015
Publication URL: http://handle.nal.usda.gov/10113/61738
Citation: Kamo, K.K., Lakshman, D.K., Pandey, R., Guaragna, M.A., Okubara, P.A., Rajasekaran, K., Cary, J.W., Jordan, R.L. 2015. Resistance to Fusarium oxysporum f. sp. gladioli in transgenic Gladiolus plants expressing either a bacterial chloroperoxidase or fungal chitinase genes. Plant Cell Tissue And Organ Culture. 124:541.

Interpretive Summary: Gladiolus is a popular horticultural crop throughout the world. A major pathogen of Gladiolus is the soilborne fungus Fusarium oxysporum that can persist in the soil for long periods. In the past, Fusarium was controlled using methyl bromide, but this broad-spectrum fumigant has been phased out for application to soil due to health and environmental concerns, and effective alternatives other than planting in a new location where Fusarium is not well established are not currently available. This study reports the development of transgenic Gladiolus plants containing either an exochitinase, endochitinase, or chloroperoxidase gene, and the antifungal effect of these transgenes on Fusarium oxysporum sp. Gladioli (Fog). The three transgenes in this study resulted in several Gladiolus lines that were able to inhibit the growth of Fog for a period of time. Cell extract from one of the Gladiolus plant lines containing the endochitinase gene was effective in inhibiting colony formation from germinated spores in vitro. The shoots and roots of one plant line containing the endochitinase gene and one plant line with the chloroperoxidase gene appeared to be more resistant to Fog than the non-transformed, regenerated plants. Plant pathogens are a significant concern when growing many crops, but only 10% of the transgenic plants registered for field trials in North America during the last 15 years were engineered for disease resistance. This low percentage of transgenic plants engineered for disease resistance indicates the difficulty in engineering for pathogen immunity. The results from this study show the possibility of engineering for Fusarium resistance in Gladiolus. Genetic resistance would offer a sustainable alternative to the nursery and bulb industry for control of Fusarium oxysporum f. sp. gladioli.

Technical Abstract: Three antifungal genes, a non-heme chloroperoxidase from Pseudomonas pyrrocinia, and an exochitinase and endochitinase from Fusarium venetanum under regulation by the CaMV 35S promoter, were used to transform Gladiolus for resistance to Fusarium oxysporum f. sp. gladioli. Gladiolus plants were confirmed to be transgenic by Southern hybridization. Semi-quantitative RT-PCR of RNA isolated from leaves and roots demonstrated expression of the Fusarium exochitinase and endochitinase genes in transgenic plants compared to controls. All transgenic plants expressing the Fusarium exochitinase or endochitinase gene had chitinase activity higher than that of the control plants. Semi-quantitative RT-PCR verified that three of the four plant lines with the chloroperoxidase gene expressed the transgene in leaves and roots while no expression was detected in control plants. Western hybridization confirmed the presence of the chloroperoxidase protein in both leaves and roots of transgenic plants. Cell extracts from one endochitinase plant line inhibited growth of germinated F. oxysporum spores more consistently than extracts from the four chloroperoxidase and three endochitinase plant lines. Three chloroperoxidase, two exochitinase, and three endochitinase transgenic plant lines sustained a significantly (P<0.05) lower density of hyphae on roots compared to roots of non-transformed Gladiolus plants three to four days following exposure of the roots to Fusarium. Shoots from two plant lines, one containing a chloroperoxidase and the other an endochitinase gene, had less necrosis when rated on a scale of 1-3 and appeared visually to be healthier and without obvious Fusarium infection than non-transformed, regenerated Gladiolus plants 17-21 days following exposure to Fusarium oxysporum.