Fusaric Acid Produced by Nitrogen Stress Fungal

The determination of the biochemical parameters that interfere in the production of a secondary metabolite is fundamental for the knowledge of the ideal conditions for this biosynthesis. Several studies have been carried out on the influence of different carbon and nitrogen sources on the production of fusaric acid in fungus cultures.

The fusaric acid acts in the metabolism of Zea mays, isolated mitochondria, inhibiting the electrons flow between the succinate-desidrogenase and the coenzyme Q, the activity of ATPase/ATP-sintase and probably also inhibiting, the alpha-cetoglutarato desidrogenase. Many cultures such as corn, rice, wheat, sorghum, banana, barley and passion fruit are affected by Fusarium spp.

In this work, we sought not only to determine the level of fusaric acid produced at the end of fungus growth, but also to monitor the production of this mycotoxin throughout the whole culture of the microorganism.

The objective of this work was to evaluate the relation between carbon and nitrogen ideal for producing the mycotoxin Fusaric acid by F. oxysporum f.sp. cubense.

Extraction of Fusaric acid: The F. oxysporum f. sp. cubense, was grown on oat liquid medium and homogenized with 100 mL of a methanol solution: K2HPO4 1%, pH 3.0, and centrifuged (10000 x g) for 20 minutes. The resulting supernatant was adjusted to pH 3.0 with HCl 2N. It was proceeded to the extraction by three times with 80 mL of methyl cloride. The resulting extract had its volume reduced to 40 mL in rota-vapour. Following 25 mL of an aqueous solution of NaHCO3 (5%) was added and a new extraction was performed twice. The fraction that contained NaHCO3 was adjusted to pH 3.0. The extraction with methyl chloride was proceeded two more times, when finally, the methyl chloride was removed under vacuum at 40 C in rotatory evaporator. HPLC determination: The obtained residue was resuspended in the mobile system and used in HPLC. The eluent system possessed the following composition: toluene, ethyl acetate, formic acid (50:40:20 vol, vol, vol). A Licrosorb-18 (ODS) column of 10 cm, with flow of 1.25 ml/min,c at temperature of 25 C was used. The UV detector at 210 nm, was used in the liquid chromatograph model CG-420. The standard fusaric acid was obtained from Sigma Co.

The Fusarium oxysporum f.sp. cubense, demonstrated to be a regular producer of fusaric acid. The results showed that the fusaric acid had its beginning of synthesis around 72 hours of fungus cultivation, and the beginning of the synthesis corresponded to the availability in the culture system of 1.6 g% of total carbohydrates and 0.3 g % protein. Therefore, the C:N ratio was of the order of 5:1. The exponential phase of production occurred between 72 and 120 hours of the growth process.

The low availability of nitrogen in the culture system was a decisive factor for this mycotoxin synthesis. The change in coloration of the culture medium, which changes from light beige to red-wine color over time, is indicative of the production of secondary metabolites. The decrease in the nitrogen level, which signals the onset of fusaric acid production, represents a deviation in the metabolism of the microorganism, and therefore the secondary metabolism.

Abstract published on http://www.spdiniz.com.br.