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Mechanism of action of anti-fungals against mycotoxigenic species: from molecular to phenotypic efficacy

Final Report Summary - ANTIFUNGALVSMYCOTOX (Mechanism of action of antifungals against mycotoxigenic species: from molecular to phenotypic efficacy)

Mycotoxins have received considerable attention especially over the last three decades. Mycotoxicology is currently considered a subject of international importance. It has been estimated that roughly 25 % of the world's food crops are affected by mycotoxin contamination annually. The consumption of mycotoxin-contaminated commodities is related to several acute and chronic diseases in humans and animals.

There has been significant interest in finding effective natural compounds to substitute for the existing food grade preservatives to control mycotoxigenic food spoilage moulds. However, the modulation that the environmental conditions have on the effect of these compounds was not fully understood.

In this project, we identified a lack of knowledge with regard to how environmental factors affect natural antifungal compounds, and to the differential effects of different molecule types on the different mycotoxigenic fungi life cycle phases, i.e. germination, growth and mycotoxin production, in relation to mechanisms of inhibition.

The objectives of this project were to:

(a) examine and compare the efficacy of a range of concentrations of potassium sorbate, butylated hydroxyanisole and tebuconazole against different growth phases of key mycotoxigenic fungi under different environmental regimes;
(b) investigate efficacy on phenotypic mycotoxin production in relation to intermediate control treatments and different environmental conditions;
(c) examine toxin gene expression using reverse transcription polymerase chain reaction (RT-PCR) and a mycotoxin microarray in relation to intermediate control treatments under different environmental conditions;
(d) measure effects on viability, permeability and colony organisation using vital stains and confocal microscopy, flow cytometry and respiration measurements;
(e) compare inhibitory effects on intracellular oxidative species, endocytosis capabilities, mitochondria number and distribution by using fluorescent dyes and flow cytometry;
(f) work on data integration using bioinformatic approaches including projection to latent structure (PLS), principal component analysis (PCA) and neural networks (NNs).

On 31 August 2012, the project came to a completion, with all the objectives fulfilled. Indeed some additional data was obtained in some of the tasks proposed.

The ecology of several mycotoxigenic fungal species was studied using the traditional Petri plate method. The lethal doses of 50 and 90 % (LD50, LD90) for these compounds was calculated as described. However, a new technique to evaluate the efficacy of antifungal compounds was developed in parallel. This new technique used the Bioscreen C system to perform turbidimetric readings in an automated way with filamentous moulds for the first time. The proper settings and media compositions were developed during the project. Using this technique the behaviour of different fungal species to different antifungal compounds (tebuconazole, butilated hidroxyanisole, potassium sorbate and natamycin) was studied. An extra advantage of this technique is that it allowed the extraction and quantification of the mycotoxin produced and also the recovery of mycelium to perform gene expression experiments.

Significant developments were made with regard to mycotoxin chromatographic analyses and quantification in this project. For the first time, the suitability of solid-core stationary phase particles was tested for the analysis of aflatoxins. A method for the analysis of type A trichothecenes in oat-based medium was also developed. These results have been published in international leading peer-reviewed chromatography journals.

In the third phase of the project ('Multidisciplinary ecophysiology and functional molecular studies'), major scientific contributions were also made. The need for a rapid and robust method for the extraction and purification of fungal ribonucleic acid (RNA) was evident. In this period the first task was to develop a rapid method. This new method was recently published in the international Journal of Microbiological Methods. The method uses a bead beating system and a semi-automated purification approach.

With regard to the efficacy of antifungal compounds, interesting results showed that in vitro and in vivo results differ greatly with regard to toxin gene cluster expression. In some cases, intermediate concentrations of fungistats lead to an increase in regulatory gene expression that correlated positively with increased accumulation of the mycotoxin. In the fourth phase of the project, the flow cytometry technology was used to evaluate the mode of action of several antifungal compounds. FM-64 and SYTO-9 stain where used to identify alive / dead or damaged fungal spores treated with different antifungal compounds. Aspergillus flavus was used as model organism. The experiments were performed using different concentrations, different water activities, temperatures and different exposure times. The results showed that A. flavus spores were able to withstand most of the antifungal compound tested for at least 24 hours. Inactivation of spores was only achieved with butylated hidroxyanisole (BHA) at high concentrations and optimal germination environmental conditions.

In the last phase of the project, work is still ongoing as a result of the findings during the project. At the beginning of the project, a new method to evaluate the effect of environmental conditions on mycotoxigenic fungi was developed using the previously published Lambert-Pearson model. By using this model we have been able to study the dependence or independence of the observed effect from environmental conditions. Nominal logistic models have also been used to describe the effect of antifungal compounds under different environmental conditions. Ongoing analysis seeks to correlate growth data, toxin production and environment in order to predict the accumulation of mycotoxins using a mixed growth model modified in order to introduce extra factors.

The research developed in this project has proved to be of high impact. This research approach enhanced the understanding of the function of key biosynthetic genes in the toxin gene clusters involved in toxin production, and also integrated this with morphological / physiological data obtained with newly developed techniques. A number of research publications have been produced.