In vivo testing of fungal inhibition

Introduction

Biological control of Fusarium oxysporum using other living organisms has been explored previously and identified Pseudomonas spp. as a possible control agent due to the production of growth inhibitory substances [1]. Pseudomonas putida is a root colonizing bacteria and is therefore expected to be found in the rhizosphere [2] where F. oxysporum attacks the plant. Engineering a bacterial platform for biological control of Fusarium oxysporum f. sp. cubense (Foc) based on P. putida therefore shows great potential. In our experiments, P. putida was genetically modified to overexpress four different fungal growth inhibitors (2,4-DAPG, DMDS & DMTS, pyoverdine and Chitinase) to prevent Foc from entering the roots of banana plants (see inhibition). Functionality was tested in vitro and in vivo by applying the control agent to the soil of pot grown banana plants (Figure 1). These plants were then infected with Foc. Adverse or beneficial effects of the active compounds produced by the genetically modified biological control agent on banana plants were investigated in vivo.

Application

Four different Pseudomonas putida strains are tested for their functionality as a biological control agents. The modified organisms were applied to the soil. Each organism produces a different active compound. Additional treatments involve a culture mix containing an equal share of each modified organism and a water control. Plants treated with the wild type Pseudomonas putida strain serve as control to confirm that the effects are based on the over expressed active compounds. Different treatments are listed below.

Treatments:

1. Wildtype P. putida KT2440
2. P. putida producing 2,4-DAPG
3. P. putida producing DMDS and DMTS
4. P. putida producing pyoverdine
5. P. putida producing chitinase
6. Culture mix of number 1,2,3, and 4
7. Water (control)

To give the control agent the possibility of colonizing the rhizosphere before confronting the plant with Foc the plants were inoculated with the different Pseudomonas putida constructs 48 hours prior to fungal inoculation. 5 ml of bacterial inoculum was applied by inserting it with a syringe close to the roots (at OD₆₀₀ of 0.25) (Figure 2). Two days later 5 ml of the fungal inoculum (spore-count of 1 million spores per ml) was applied likewise. Additionally, two maize kernels inoculated with Foc were applied to the soil of each plant to conserve the inoculum within the pot. Since the production of the fungal growth inhibitors in this test organisms is induced by IPTG the plants are watered with a solution containing IPTG.

Degree of wilt and fungal biomass of plants inoculated with Foc and the different biological control agents will be determined. Non-inoculated plants and plants solely inoculated with the biological control agent but not with Foc will serve as control. Possible positive or negative effects on growth will be tested by determining the above ground biomass of the banana plant after four weeks.

Results

Final results can unfortunately not be obtained before the Wiki-freeze so make sure to attend our presentation during the iGEM Jamboree in Boston to find out more.

Future work

References

1. Lemanceau P. Alabouvette C. (1991) Biological control of fusarium diseases by fluorescent Pseudomonas and non-pathogenic Fusarium. Crop Protection. Vol. 10 Issue 4: 279–286
2. Espinosa-Urgel M, Kolter R, Ramos JL.(2002) Root colonization by Pseudomonas putida: love at first sight. Microbiology. Feb;148(Pt 2):341-3.