Transcription of genes in the biosynthetic pathway for fumonisin mycotoxins is epigenetically and differentially regulated in the fungal maize pathogen Fusarium verticillioides.

When the fungal pathogen Gibberella moniliformis (anamorph, Fusarium verticillioides) colonizes maize and maize-based products, it produces class B fumonisin (FB) mycotoxins, which are a significant threat to human and animal health. FB biosynthetic enzymes and accessory proteins are encoded by a set of clustered and cotranscribed genes collectively named FUM, whose molecular regulation is beginning to be unraveled by researchers. FB accumulation correlates with the amount of transcripts from the key FUM genes, FUM1, FUM21, and FUM8. In fungi in general, gene expression is often partially controlled at the chromatin level in secondary metabolism; when this is the case, the deacetylation and acetylation (and other posttranslational modifications) of histones are usually crucial in the regulation of transcription. To assess whether epigenetic factors regulate the FB pathway, we monitored FB production and FUM1, FUM21, and FUM8 expression in the presence of a histone deacetylase inhibitor and verified by chromatin immunoprecipitation the relative degree of histone acetylation in the promoter regions of FUM1, FUM21, and FUM8 under FB-inducing and noninducing conditions. Moreover, we generated transgenic F. verticillioides strains expressing GFP under the control of the FUM1 promoter to determine whether its strength under FB-inducing and noninducing conditions was influenced by its location in the genome. Our results indicate a clear and differential role for chromatin remodeling in the regulation of FUM genes. This epigenetic regulation can be attained through the modulation of histone acetylation at the level of the promoter regions of the key biosynthetic genes FUM1 and FUM21, but less so for FUM8.

The microbiology of Lascaux Cave.

Lascaux Cave (Montignac, France) contains paintings from the Upper Paleolithic period. Shortly after its discovery in 1940, the cave was seriously disturbed by major destructive interventions. In 1963, the cave was closed due to algal growth on the walls. In 2001, the ceiling, walls and sediments were colonized by the fungus Fusarium solani. Later, black stains, probably of fungal origin, appeared on the walls. Biocide treatments, including quaternary ammonium derivatives, were extensively applied for a few years, and have been in use again since January 2008. The microbial communities in Lascaux Cave were shown to be composed of human-pathogenic bacteria and entomopathogenic fungi, the former as a result of the biocide selection. The data show that fungi play an important role in the cave, and arthropods contribute to the dispersion of conidia. A careful study on the fungal ecology is needed in order to complete the cave food web and to control the black stains threatening the Paleolithic paintings.

The impact of arthropods on fungal community structure in Lascaux Cave.

AIMS: To determine the major components of the fungal population present in Lascaux Cave, France. The ceiling, walls, sediments and soil were colonized by Fusarium solani in 2001 and later, in 2006, black stains appeared. However, the origin of the successive fungal invasions is unknown as well as the ecology of the cave. METHODS AND RESULTS: The primers nu-SSU-0817F and nu-SSU-1536R were used for the direct amplification of fungal 18S-rDNA sequences from 11 samples. A total of 607 clones were retrieved. Eight out of the ten most abundant phylotypes corresponded to fungi associated with arthropods and represented about 50% of the clones. CONCLUSIONS: Entomophilous fungi play an important role in the cave and arthropods contribute to the dispersion of spores and fungal development. SIGNIFICANCE AND IMPACT OF THE STUDY: Choosing appropriate targets for control of fungal dispersal is dependent on knowing the causes of fungal colonization. A control of the arthropod populations seems to be a need in order to protect the rock art paintings in Lascaux Cave.

Fusarium oxysporum and its biocontrol.

Fusarium oxysporum is well represented among the rhizosphere microflora. While all strains exist saprophytically, some are well-known for inducing wilt or root rots on plants whereas others are considered as nonpathogenic. Several methods based on phenotypic and genetic traits have been developed to characterize F. oxysporum strains. Results showed the great diversity affecting the soil-borne populations of F. oxysporum. In suppressive soils, interactions between pathogenic and nonpathogenic strains result in the control of the disease. Therefore nonpathogenic strains are developed as biocontrol agents. The nonpathogenic F. oxysporum strains show several modes of action contributing to their biocontrol capacity. They are able to compete for nutrients in the soil, affecting the rate of chlamydospore germination of the pathogen. They can also compete for infection sites on the root, and can trigger plant defence reactions, inducing systemic resistance. These mechanisms are more or less important depending on the strain. The nonpathogenic F. oxysporum are easy to mass produce and formulate, but application conditions for biocontrol efficacy under field conditions have still to be determined.

Genetic diversity of Fusarium oxysporum populations isolated from different soils in France.

The genetic diversity of soil-borne populations of Fusarium oxysporum was assessed using 350 isolates collected from six different French soils. All isolates were characterised by restriction fragment analysis of the PCR-amplified ribosomal intergenic spacer (IGS). Twenty-six IGS types were identified among the 350 isolates analysed. Five to nine different IGS types were detected in each soil. None of the IGS types was common to all of the soils. An analysis of the molecular variance based on IGS type relationships and frequency revealed that the genetic structure of the populations of F. oxysporum varied widely among the soils. Some populations were both highly diverse within the soils and differentiated between the soils. A possible relationship between the intrapopulation or interpopulation level of diversity and some external factors such as the soil type or the crop history was evaluated. A subsample representative of the diversity of the six populations was further characterised by analysing the genomic distribution of two transposable elements, impala and Fot1. One to 10 copies of the impala element were present in most of the isolates, irrespective of their soil of origin. The Fot1 element was only detected in 40% of the isolates originating from the three populations less diverse in terms of IGS types, but in 82.6% of the isolates originating from the three more diverse populations.

Recovery of Mutants Impaired in Pathogenicity After Transposition of Impala in Fusarium oxysporum f. sp. melonis.

ABSTRACT The ability of transposon impala to inactivate genes involved in pathogenicity was tested in Fusarium oxysporum f. sp. melonis. Somatic excision of an impala copy inserted in the nitrate reductase-encoding niaD gene was positively selected through a phenotypic assay based on the restoration of nitrate reductase activity. Independent excision events were analyzed molecularly and shown to carry reinsertedimpala in more than 70% of the cases. Mapping of reinserted impala elements on large NotI-restriction fragments showed that impala transposes randomly. By screening 746 revertants on plants, a high proportion (3.5%) of mutants impaired in their pathogenic potential was recovered. According to the kinetics of wilt symptom development, the strains that were impaired in pathogenicity were clustered in three classes: class 1 grouped two strains that never induced Fusarium wilt symptoms on the host plant; class 2 and class 3 grouped 15 and 9 revertants which caused symptoms more than 50 and 30 days after inoculation, respectively. The first results demonstrate the efficiency of transposition in generating mutants affected in pathogenicity, which are usually difficult to obtain by classical mutagenesis, and open the possibility to clone the altered genes with impala as a tag.

Populations of Nonpathogenic Fusarium oxysporum Associated with Roots of Four Plant Species Compared to Soilborne Populations.

ABSTRACT The effect of the plant on the diversity of soilborne populations of Fusarium oxysporum was assessed after successive cultures of flax, melon, tomato, and wheat in separate samples of the same soil. Forty soil-borne isolates of F. oxysporum and forty root-colonizing isolates of each plant species were sampled during the first (T0) and fourth (T1) cultures. The population structures were assessed by a genotypic method based on restriction fragment analysis of polymerase chain reaction-amplified ri-bosomal intergenic spacer (IGS) DNA. Sixteen IGS types were defined among the four hundred isolates analyzed. The distributions of soil isolates among IGS types were similar at both sampling times. The structure of F. oxysporum populations associated with the roots of flax or melon did not differ from the structure of soilborne populations. In contrast, the structure of F. oxysporum populations associated with roots of wheat or tomato differed from the structure of soilborne populations. High frequencies were found for IGS type 4 among wheat isolates at both T0 and T1 and for IGS type 11 among tomato isolates at T1. Moreover, a high level of genetic divergence was obtained between IGS types 4 and 11. Our results suggest that tomato and wheat have a selective effect on soilborne populations of F. oxysporum and that this effect seems to be plant specific.

Effect of Two Plant Species, Flax (Linum usitatissinum L.) and Tomato (Lycopersicon esculentum Mill.), on the Diversity of Soilborne Populations of Fluorescent Pseudomonads.

Suppression of soilborne disease by fluorescent pseudomonads may be inconsistent. Inefficient root colonization by the introduced bacteria is often responsible for this inconsistency. To better understand the bacterial traits involved in root colonization, the effect of two plant species, flax (Linum usitatissinum L.) and tomato (Lycopersicon esculentum Mill.), on the diversity of soilborne populations was assessed. Fluorescent pseudomonads were isolated from an uncultivated soil and from rhizosphere, rhizoplane, and root tissue of flax and tomato cultivated in the same soil. Species and biovars were identified by classical biochemical and physiological tests. The ability of bacterial isolates to assimilate 147 different organic compounds and to show three different enzyme activities was assessed to determine their intraspecific phenotypic diversity. Numerical analysis of these characteristics allowed the clustering of isolates showing a high level (87.8%) of similarity. On the whole, the populations isolated from soil were different from those isolated from plants with respect to their phenotypic characteristics. The difference in bacteria isolated from uncultivated soil and from root tissue of flax was particularly marked. The intensity of plant selection was more strongly expressed with flax than with tomato plants. The selection was, at least partly, plant specific. The use of 10 different substrates allowed us to discriminate between flax and tomato isolates. Pseudomonas fluorescens biovars II, III, and V and Pseudomonas putida biovar A and intermediate type were well distributed among the isolates from soil, rhizosphere, and rhizoplane. Most isolates from root tissue of flax and tomato belonged to P. putida bv. A and to P. fluorescens bv. II, respectively. Phenotypic characterization of bacterial isolates was well correlated with genotypic characterization based on repetitive extragenic palindromic PCR fingerprinting.

Antagonistic Effect of Nonpathogenic Fusarium oxysporum Fo47 and Pseudobactin 358 upon Pathogenic Fusarium oxysporum f. sp. dianthi.

Pseudobactin production by Pseudomonas putida WCS358 significantly improves biological control of fusarium wilt caused by nonpathogenic Fusarium oxysporum Fo47b10 (P. Lemanceau, P. A. H. M. Bakker, W. J. de Kogel, C. Alabouvette, and B. Schippers, Appl. Environ. Microbiol. 58:2978-2982, 1992). The antagonistic effect of Fo47b10 and purified pseudobactin 358 was studied by using an in vitro bioassay. This bioassay allows studies on interactions among nonpathogenic F. oxysporum Fo47b10, pathogenic F. oxysporum f. sp. dianthi WCS816, and purified pseudobactin 358, the fluorescent siderophore produced by P. putida WCS358. Both nonpathogenic and pathogenic F. oxysporum reduced each other's growth when grown together. However, in these coinoculation experiments, pathogenic F. oxysporum WCS816 was relatively more inhibited in its growth than nonpathogenic F. oxysporum Fo47b10. The antagonism of nonpathogenic F. oxysporum against pathogenic F. oxysporum strongly depends on the ratio of nonpathogenic to pathogenic F. oxysporum densities: the higher this ratio, the stronger the antagonism. This fungal antagonism appears to be mainly associated with the competition for glucose. Pseudobactin 358 reduced the growth of both F. oxysporum strains, whereas ferric pseudobactin 358 did not; antagonism by pseudobactin 358 was then related to competition for iron. However, the pathogenic F. oxysporum strain was more sensitive to this antagonism than the nonpathogenic strain. Pseudobactin 358 reduced the efficiency of glucose metabolism by the fungi. These results suggest that pseudobactin 358 increases the intensity of the antagonism of nonpathogenic F. oxysporum Fo47b10 against pathogenic F. oxysporum WCS816 by making WCS816 more sensitive to the glucose competition by Fo47b10.

Effect of pseudobactin 358 production by Pseudomonas putida WCS358 on suppression of fusarium wilt of carnations by nonpathogenic Fusarium oxysporum Fo47.

Nonpathogenic Fusarium oxysporum Fo47b10 combined with Pseudomonas putida WCS358 efficiently suppressed fusarium wilt of carnations grown in soilless culture. This suppression was significantly higher than that obtained by inoculation of either antagonistic microorganism alone. The increased suppression obtained by Fo47b10 combined with WCS358 only occurred when Fo47b10 was introduced at a density high enough (at least 10 times higher than that of the pathogen) to be efficient on its own. P. putida WCS358 had no effect on disease severity when inoculated on its own but significantly improved the control achieved with nonpathogenic F. oxysporum Fo47b10. In contrast, a siderophore-negative mutant of WCS358 had no effect on disease severity even in the presence of Fo47b10. Since the densities of both bacterial strains at the root level were similar, the difference between the wild-type WCS358 and the siderophore-negative mutant with regard to the control of fusarium wilt was related to the production of pseudobactin 358. The production of pseudobactin 358 appeared to be responsible for the increased suppression by Fo47b10 combined with WCS358 relative to that with Fo47b10 alone.

Enhanced bacterial metabolism of a Pseudomonas strain in response to the addition of culture filtrate of a bacteriophagous amoeba.

In a previous work, Levrat et al. [21] showed an enhancement of the production of pyoverdin (siderophore) by Pseudomonas putida in the presence of amoeba. To explain the mechanism of stimulation, the hypothesis of production of stimulatory factors by amoeba was proposed. Filtrates of both mixed culture of bacteria and amoeba (Pseudomonas putida + Acanthamoeba castellanii) and of axenic culture of amoeba were added to the culture medium of Pseudomonas. The production of pyoverdin was increased in the presence of the filtrates. The maximum stimulation was observed with a 6 to 8 day old mixed culture filtrate at 2% final concentration. A higher amount of filtrate did not enhance the stimulation. General metabolisms like ammonium production or respiration were also enhanced in the presence of filtrate of mixed cultures. Filtrates of axenic culture of amoeba were also able to stimulate the production of pyoverdin by Pseudomonas. This stimulation of the bacterial metabolism was not correlated with a higher growth of the bacterial population. Then, the enhancement of the bacterial metabolic activity was not due to a rapid recycling of the bacterial biomass but rather to a production of stimulatory factors by amoeba.

Survival and inoculum potential of conidia and chlamydospores of Fusarium oxysporum f.sp. lini in soil.

The kinetics of survival and inoculum potential of Fusarium oxysporum f.sp. lini were studied in soil. Two types of inoculum were compared: microconidia freshly harvested from a laboratory-grown culture and microchlamydospores produced in sterilized soil. Introduced at the same inoculum densities into a natural soil, the two types of inoculum showed similar behavior; the inoculum densities changed little with time, at least during 100 days. However, the two types of inoculum did differ in disease potential. A higher percentage of microchlamydospores than microconidia germinated in the rhizosphere of flax seedlings, and the heterotrophic fluorescein diacetate hydrolysing activity of the microchlamydospores was 100 times higher than that of microconidia. Moreover, the microchlamydospores produced more disease on flax than the microconidia even at a much lower inoculum density.