Silages are key sources of forages
for over-wintering livestock in Belgium. Different crops are used for ensilage
such as whole maize plant, corncob-mix, grass, immature cereals, etc. Ensilaged
whole maize plant, which is produced on 160.000 ha/year in Belgium, takes the
major part of cattle forages. The grass silages also represent a big part of
the beef cattle and dairy heifers forages, with a yearly production of 250.000
ha (source: http://statbel.fgov.be/downloads/crp2004_fr.xls).
Under anaerobic conditions, these crops can be successfully preserved by bacterial
lactic acid fermentation. However, spoilage of silage material by the presence
and development of moulds is frequently observed and can result in numerous
problems for cattle feed and health. The development of these fungal contaminants
can be favored by numerous factors including soil contamination, insufficient
sugar content, failure to maintain anaerobiosis…
Project description
Objectives
The research entails to study the biological and
metabolic diversity of fungal contaminants in silage. The morphological characterization
of the isolated fungal strains will be related to the molecular characterization
and completed by phylogenic studies. The diversity of fungal strain isolated
form the various silages will be analyzed through RAPD and AFLP profiles and
DNA sequencing. The results of the morphological, taxonomic, phylogenic and
diversity will allow extending the BCCM/MUCL -collection through
the integration of subsets of well described and characterized fungal material
(silage contaminating fungi).
Within this project, new methods will be developed
for extraction and HPLC analysis of mycotoxins isolated for the different
silages. The analysis of mycotoxins will also be realized on samples issued
form the in vitro culture of fungal
contaminants and particular attention will be devoted to the less-known mycotoxins
and to other (potentially unknown) metabolites isolated from fungi. The information related to the mycotoxins
and production of metabolites of the fungal isolated from the silages will
be used to refine the phylogenic characterization of fungal strains. Toxicological
analysis will be conducted on silage samples et on pure fungal isolates extracts
in order to establish dangers linked to the presence of mycotoxines and other
fungal toxic compounds poorly or unknown. The capacity of pure mycotoxins
et contaminated silage extracts to induce genes will be evaluated on transgenic
cell cultures.
Five partners (BCCM/MUCL, CERVA-CODA-VAR, Hogeschool Gent,
CIPF, UA) will collaborate in the realization of this project.
Methodology
In a first step, different types of silages from
all regions of the country will be collected in order to have a broad view
of the situation of maize and herb silage in Belgium. Information will be
collected about the raw material of silage (plant, variety, phenologic state
of the plant at time of harvesting) and the ensilage system (type of silo,
design etc…). In addition, information will be collated on the symptoms developed by cattle (diseases, fertility
problems, allergic reactions etc...) fed with these silages to have some background
information on the possible effects of mycotoxins.
In a second step, the selected silages will be
analyzed through sampling in healthy as well as contaminated parts in order
to (1) isolate and identify molds and yeasts, (2) analyze mycotoxins from silage samples
and (3) evaluate the mycotoxin toxicity in silages.
(1) Isolation and identification
of molds and yeasts
Molds and yeasts will be isolated from fresh samples
and will be incubated on appropriate culture media. The fungal samples will
be maintained on mineral oil, by cryopreservation and lyophilisation.
For an accurate identification of species, regions
of taxonomic interest will be sequenced, based on the expertise of MUCL and
on the literature. The diversity within the different species isolated from
the different type of silages will be analyzed using RAPD and AFLP. This method
will allow us to obtain a picture of the possible populations within species.
(2) Analysis of mycotoxins
from silage samples
Preliminary mycotoxin markers analyses will be performed for
all collected samples of silages: deoxynivalenol (DON) and Patulin (PAT) on
Maize silage and PAT solely on grass silage. Depending on the preliminary
results, further analyzes will be made to find other mycotoxins.
(3) Mycotoxin toxicity in
silages: a gene expression and flow cytometric approach
The toxicity of mycotoxins will be evaluated on
living cell lines.This project introduces a new and versatile technology to
the assessment of mycotoxin detection and toxicity in the follow up of silages.
Starting from detection of mycotoxin toxicity, the technology allows for identification
of the mycotoxin family through determination of the molecular mode of action,
comparison and ranking of different contamination events in one or several
silages. The current assay consists of a battery of genetically transformed
bacterial strains and transgenic human liver cell lines. The working principle
of both systems is similar and is based on the induction of specific stress
gene promoters.
Interaction between the different partners
CIPF and HGent partners will collect about the
ensilages and all informations relative to these materials. HGent will isolate
fungal contaminants from silage samples, and will send the isolates to MUCL
for identification. MUCL will identify (morphologically and molecularly) and
cultivate the fungi in liquid medium. The culture supernatants will be sent
to CERVA for mycotoxin analyses. CERVA will extract and analyze the mycotoxin
from the silage samples and liquid medium cultures. UA will use the extracts
for the toxicological analyzes. MUCL, -as coordinator- will collect and integrate
the results.
Expected results and/or products
Fungal and mycotoxin contamination in maize and grass silage
feeds will be studied to gain an overview on
their effect on the forage quality in Belgium. The
study will let us to estimate the
toxicological effects of mycotoxins. In details,
the project will yield the following results:
Development of new methods for mycotoxin extraction and analysis from
maize silage matrix and grass silage matrix
An overview on the most important silage contaminating fungal species
Effect of inoculants and/or additives application on the quality of the
silage and on the fungal and mycotoxin contamination
The implementation of an on-line database on molds and mycotoxins of the
ensilages, being of use as support to the farmers for the management of
the problems of contamination of the ensilages by detailing the risks of
production and the toxicity of mycotoxins
Isolation of fungal species from moldy silage and characterization of those strains
is predisposed to valorize the biological material available in the BCCM collections by improving and expanding the existing knowledge
on the characteristics of the material. Characterization will
include morphological data, molecular
data (RAPD, AFLP, sequences), metabolite production
(mycotoxins) and toxicological data.
The private partners will gain a better insight in
the overall problematic of fungal contamination and consequent mycotoxin production
in silage. This will eventually
lead to improved quality of silage feed, reduction of contamination events
and hence reduction of costs.
Partners
Partner 1: Mycothèque
de l'Université catholique de Louvain (MUCL)
C3/00/22: Characterization of fungal species and mycotoxins contaminating silages in Belgium