Biological activity of lipids extracted from two isolates of Fusarium oxysporum (environmental and clinical) in Galleria mellonella


Objective
To evaluate the virulence of conidia and the biological activity of lipids extracted from two Fusarium oxysporum (F. oxysporum) isolates: environmental and clinical in the Galleria mellonella (G. mellonella) model.

METHODS:
Fungi:
Fusarium oxysporum environmental isolate was obtained from decomposing plant material with code 15H07 for the Grupo Interdisciplinario de Estudios Molecualres (GIEM), Natural and Exact Sciences Faculty, Universidad de Antioquia. 
Fusarium oxysporum clinical isolate was obtained from a patient with onychomycosis with code 09155 for the Grupo Micología Médica, School of Medicine, Universidad de Antioquia.
 Lipids:
The lipids extracted were obtained by the soxhlet method with three solvents of low, intermedium and high polarity (hexane, dichloromethane and methanol). The lipids were characterized for high performance liquid chromatography mass spectrometry (HPLC-MS) and mono and bidimensional thin layer chromatography (TLC).
G. mellonella model:
G. mellonella caterpillar in the final larval instar stage were injected into the hemocoel via the last proleg with 104, 105, 106 and 107 conidia/mL from both F. oxysporum isolates using Tween 80 as surfactant. To evaluate the biological activity: 1) the lipids extracted were injected at 102, 103 and 104 µg/mL using Tween 20 for their emulsification and 2) co-stimulus with conidia and lipids were injected to the G. mellonella caterpillars.

RESULTS:

The confirmation that both isolates of Fusarium spp corresponded to Fusarium oxysporum was done by the ITS1, ITS4 and calmodulin gene sequencing by the Centro Nacional de Secuenciación, SIU, UdeA.
The average mortality of caterpillar at 48 hours post-injection with 104, 105, 106 and 107 conidias/mL of the environmental isolate was 30%, 53.3%, 93.3% and 100%, respectively. The average mortality of the caterpillar at 48 hours post-injection with 104, 105, 106 and 107 conidias/mL of the clinical isolate was 6.7%, 16.7%, 23.3% and 76.7%, respectively and also with the infection with this isolate was observed external cuticle growth on the dead larvae. The evaluation of the biological activity of the lipids extracted with methanol from the environmental isolate show early melanization since first hour of injection and lasted until 48 hours. The average mortality of the caterpillar injected with 102, 103 and 104 µg/mL of methanolic lipid extracted from the environmental isolate was 0%, 30% and 96.7%, respectively. The chemical characterization of the methanol extracted lipids from the environmental isolate with chromatographic techniques showed the presence of higher amount of compounds on this isolate compared with the clinical isolate. The caterpillar mortality with the co-stimulus of conidia and methanol lipid extracted from the environmental isolate was approximately 100% compared with individual treatments.

CONCLUSION:
There are significative differences on the conidia virulence, chemical composition and biological activity of the lipid extracted from the environmental and clinical F. oxysporum isolates evaluated.

3 / Biological activity of lipids extracted from two isolates of Fusarium oxysporum (environmental and clinical) in Galleria mellonella