Screening for potential mycoherbicides within the endophyte community of Phelipanche ramosa parasitizing tobacco.

Branched broomrape (Phelipanche ramosa (L.) Pomel) is an achlorophyllous root parasitic plant with a wide host range. Its complex management is leading to the abandonment of tobacco or oilseed rape cultivation in the most affected regions in France. Among broomrape regulation factors, soil microorganisms such as fungi seem to be a relevant biocontrol lever. The aim of this work was to detect potential mycoherbicides among fungal endophytic colonizers of P. ramosa parasitizing tobacco. Our hypothesis was that both the inhibitory of broomrape seed germination and the necrotic activities are characteristic of the fungal isolates whatever their taxonomic position. To test this hypothesis, we analysed the taxonomic and functional diversity of fungal isolates of symptomatic P. ramosa collected from infested tobacco-growing regions in France in order to identify one or more fungal strains for future biocontrol. The fungal isolates were characterized using morphological and molecular identification tools and tested for their ability to inhibit the germination of P. ramosa seeds, their necrotic activity on the stems of the pest and their non-pathogenicity to the host plant. We highlighted the specific richness of fungal colonizers associated with symptomatic P. ramosa. Among the 374 collected isolates, nearly 80% belonged to 19 Fusarium species. Eighty-seven isolates representative of this diversity also showed functional diversity by inhibiting seed germination of the parasite. The 20 best-performing isolates showed differences in germination inhibition of P. ramosa at the intraspecific level. Among these 20 fungal isolates, a set of 15 randomly selected isolates was tested for their necrotic activity on the parasite stems. Fusarium venenatum isolates showed dual competence, i.e. germination inhibition and necrotic activity, and were non-pathogenic to tobacco. This led us to discuss the potential mycoherbicidal effect of this fungal species on P. ramosa.

Weeds Harbor an Impressive Diversity of Fungi, Which Offers Possibilities for Biocontrol.

The use of herbicides for weed control is very common, but some of them represent a threat to human health, are environmentally detrimental, and stimulate herbicide resistance. Therefore, using microorganisms as natural herbicides appears as a promising alternative. The mycoflorae colonizing different species of symptomatic and asymptomatic weeds were compared to characterize the possible mycoherbicidal candidates associated with symptomatic weeds. A collection of 475 symptomatic and asymptomatic plants belonging to 23 weed species was established. A metabarcoding approach based on amplification of the internal transcribed spacer (ITS) region combined with high-throughput amplicon sequencing revealed the diversity of fungal communities hosted by these weeds: 542 fungal genera were identified. The variability of the composition of fungal communities revealed a dispersed distribution of taxa governed neither by geographical location nor by the botanical species, suggesting a common core displaying nonspecific interactions with host plants. Beyond this core, specific taxa were more particularly associated with symptomatic plants. Some of these, such as Alternaria, Blumeria, Cercospora, Puccinia, are known pathogens, while others such as Sphaerellopsis, Vishniacozyma, and Filobasidium are not, at least on crops, and constitute new tracks to be followed in the search for mycoherbicidal candidates. IMPORTANCE This approach is original because the diversity of weed-colonizing fungi has rarely been studied before. Furthermore, targeting both the ITS1 and ITS2 regions to characterize the fungal communities (i) highlighted the complementarity of these two regions, (ii) revealed a great diversity of weed-colonizing fungi, and (iii) allowed for the identification of potential mycoherbicides, among which were unexpected genera.

A Clonal Lineage of Fusarium oxysporum Circulates in the Tap Water of Different French Hospitals.

Fusarium oxysporum is typically a soilborne fungus but can also be found in aquatic environments. In hospitals, water distribution systems may be reservoirs for the fungi responsible for nosocomial infections. F. oxysporum was previously detected in the water distribution systems of five French hospitals. Sixty-eight isolates from water representative of all hospital units that were previously sampled and characterized by translation elongation factor 1α sequence typing were subjected to microsatellite analysis and full-length ribosomal intergenic spacer (IGS) sequence typing. All but three isolates shared common microsatellite loci and a common two-locus sequence type (ST). This ST has an international geographical distribution in both the water networks of hospitals and among clinical isolates. The ST dominant in water was not detected among 300 isolates of F. oxysporum that originated from surrounding soils. Further characterization of 15 isolates by vegetative compatibility testing allowed us to conclude that a clonal lineage of F. oxysporum circulates in the tap water of the different hospitals. IMPORTANCE: We demonstrated that a clonal lineage of Fusarium oxysporum inhabits the water distribution systems of several French hospitals. This clonal lineage, which appears to be particularly adapted to water networks, represents a potential risk for human infection and raises questions about its worldwide distribution.

Genus-Specific Primers for Study of Fusarium Communities in Field Samples.

Fusarium is a large and diverse genus of fungi of great agricultural and economic importance, containing many plant pathogens and mycotoxin producers. To date, high-throughput sequencing of Fusarium communities has been limited by the lack of genus-specific primers targeting regions with high discriminatory power at the species level. In the present study, we evaluated two Fusarium-specific primer pairs targeting translation elongation factor 1 (TEF1). We also present the new primer pair Fa+7/Ra+6. Mock Fusarium communities reflecting phylogenetic diversity were used to evaluate the accuracy of the primers in reflecting the relative abundance of the species. TEF1 amplicons were subjected to 454 high-throughput sequencing to characterize Fusarium communities. Field samples from soil and wheat kernels were included to test the method on more-complex material. For kernel samples, a single PCR was sufficient, while for soil samples, nested PCR was necessary. The newly developed primer pairs Fa+7/Ra+6 and Fa/Ra accurately reflected Fusarium species composition in mock DNA communities. In field samples, 47 Fusarium operational taxonomic units were identified, with the highest Fusarium diversity in soil. The Fusarium community in soil was dominated by members of the Fusarium incarnatum-Fusarium equiseti species complex, contradicting findings in previous studies. The method was successfully applied to analyze Fusarium communities in soil and plant material and can facilitate further studies of Fusarium ecology.

Fusarium diversity in soil using a specific molecular approach and a cultural approach.

Fusarium species are ubiquitous in soil. They cause plant and human diseases and can produce mycotoxins. Surveys of Fusarium species diversity in environmental samples usually rely on laborious culture-based methods. In the present study, we have developed a molecular method to analyze Fusarium diversity directly from soil DNA. We designed primers targeting the translation elongation factor 1-alpha (EF-1α) gene and demonstrated their specificity toward Fusarium using a large collection of fungi. We used the specific primers to construct a clone library from three contrasting soils. Sequence analysis confirmed the specificity of the assay, with 750 clones identified as Fusarium and distributed among eight species or species complexes. The Fusarium oxysporum species complex (FOSC) was the most abundant one in the three soils, followed by the Fusarium solani species complex (FSSC). We then compared our molecular approach results with those obtained by isolating Fusarium colonies on two culture media and identifying species by sequencing part of the EF-1α gene. The 750 isolates were distributed into eight species or species complexes, with the same dominant species as with the cloning method. Sequence diversity was much higher in the clone library than in the isolate collection. The molecular approach proved to be a valuable tool to assess Fusarium diversity in environmental samples. Combined with high throughput sequencing, it will allow for in-depth analysis of large numbers of samples.

Fungaemia caused by Fusarium proliferatum in a patient without definite immunodeficiency.

Recent literature has shown the growing importance of opportunistic fungal infections due to Fusarium spp. However, disseminated fusariosis remains rare in patients without neutropenia. We report a case of fungaemia in a 78-year-old French woman without definite immunodeficiency. Fusarium proliferatum grew from both central and peripheral blood cultures. Fever was the only clinical sign of the infection. An appropriate antifungal therapy with voriconazole led to the recovery of the patient. An environmental investigation was undertaken but failed to find a reservoir of Fusarium spores. A contaminated central venous catheter might have been the source of fungaemia.

Genetic diversity of Rhizoctonia solani associated with potato tubers in France.

The soilborne fungus Rhizoctonia solani is a pathogen of many plants and causes severe damage in crops around the world. Strains of R. solani from the anastomosis group (AG) 3 attack potatoes, leading to great yield losses and to the downgrading of production. The study of the genetic diversity of the strains of R. solani in France allows the structure of the populations to be determined and adapted control strategies against this pathogen to be established. The diversity of 73 French strains isolated from tubers grown in the main potato seed production areas and 31 strains isolated in nine other countries was assessed by phylogenetic analyses of (i) the internal transcribed spacer sequences (ITS1 and ITS2) of ribosomal RNA (rRNA), (ii) a part of the gene tef-1α and (iii) the total DNA fingerprints of each strain established by amplified fragment length polymorphism (AFLP). The determination of the AGs of R. solani based on the sequencing of the ITS region showed three different AGs among our collection (60 AG 3 PT, 8 AG 2-1 and 5 AG 5). Grouping of the strains belonging to the same AG was confirmed by sequencing of the gene tef-1α used for the first time to study the genetic diversity of R. solani. About 42% of ITS sequences and 72% of tef-1α sequences contained polymorphic sites, suggesting that the cells of R. solani strains contain several copies of ITS and the tef-1α gene within the same nucleus or between different nuclei. Phylogenetic trees showed a greater genetic diversity within AGs in tef-1α sequences than in ITS sequences. The AFLP analyses showed an even greater diversity among the strains demonstrating that the French strains of R. solani isolated from potatoes were not a clonal population. Moreover there was no relationship between the geographical origins of the strains or the variety from which they were isolated and their genetic diversity.

Ecological fitness of the biocontrol agent Fusarium oxysporum Fo47 in soil and its impact on the soil microbial communities.

Some nonpathogenic strains of Fusarium oxysporum can control Fusarium diseases responsible for severe damages in many crops. Success of biological control provided by protective strains requires their establishment in the soil. The strain Fo47 has proved its efficacy under experimental conditions, but its ecological fitness has not been carefully studied. In a series of microcosm studies, the ability of a benomyl-resistant mutant Fo47b10 to establish in two different soils was demonstrated. One year after its introduction at two concentrations in the disinfected soils, the biocontrol agent (BCA) established at similar high population densities, whereas in the nondisinfected soils it survived at lower densities, related to the initial concentrations at which it was introduced. The BCA behaved similarly in the two soils at temperatures ranging from 5 to 25 degrees C and soil water potentials between -0.01 and -1.5 MPa. In addition, terminal restriction fragment length polymorphism analysis of 16S and 18S rRNA showed that the structures of the bacterial and fungal communities evolved with time but were not significantly affected by the introduction of the BCA. Overall, the results showed that Fo47 is potentially a good BCA, able to establish in different soil environments without perturbing the investigated microbial structures.

High Genetic Diversity Among Strains of Fusarium oxysporum f. sp. vasinfectum from Cotton in Ivory Coast.

ABSTRACT Seventeen isolates of Fusarium oxysporum f. sp. vasinfectum from the Ivory Coast were characterized using vegetative compatibility group (VCG), restriction fragment length polymorphism of the ribosomal inter-genic spacer region (IGS), and mating type (MAT) idiomorph, and compared with a worldwide collection of the pathogen containing all available reference strains. Some of the isolates were identical to known reference strains for all three traits, whereas others had previously unknown varieties of IGS and (possibly) VCG. One or the other MAT idiomorph was present in each of the new isolates and the reference strains. The new isolates and reference strains were grouped based upon the three traits. Strains from the Ivory Coast were found in 7 of 11 groups detected, suggesting multiple sources for Fusarium wilt in the country. Despite the presence of both MAT idiomorphs among isolates, no evidence for recombination was found.

Defense responses of Fusarium oxysporum to 2,4-diacetylphloroglucinol, a broad-spectrum antibiotic produced by Pseudomonas fluorescens.

A collection of 76 plant-pathogenic and 41 saprophytic Fusarium oxysporum strains was screened for sensitivity to 2,4-diacetylphloroglucinol (2,4-DAPG), a broad-spectrum antibiotic produced by multiple strains of antagonistic Pseudomonas fluorescens. Approximately 17% of the F. oxysporum strains were relatively tolerant to high 2,4-DAPG concentrations. Tolerance to 2,4-DAPG did not correlate with the geographic origin of the strains, formae speciales, intergenic spacer (IGS) group, or fusaric acid production levels. Biochemical analysis showed that 18 of 20 tolerant F. oxysporum strains were capable of metabolizing 2,4-DAPG. For two tolerant strains, analysis by mass spectrometry indicated that deacetylation of 2,4-DAPG to the less fungitoxic derivatives monoacetylphloroglucinol and phloroglucinol is among the initial mechanisms of 2,4-DAPG degradation. Production of fusaric acid, a known inhibitor of 2,4-DAPG biosynthesis in P. fluorescens, differed considerably among both 2,4-DAPG-sensitive and -tolerant F. oxysporum strains, indicating that fusaric acid production may be as important for 2,4-DAPG-sensitive as for -tolerant F. oxysporum strains. Whether 2,4-DAPG triggers fusaric acid production was studied for six F. oxysporum strains; 2,4-DAPG had no significant effect on fusaric acid production in four strains. In two strains, however, sublethal concentrations of 2,4-DAPG either enhanced or significantly decreased fusaric acid production. The implications of 2,4-DAPG degradation, the distribution of this trait within F. oxysporum and other plant-pathogenic fungi, and the consequences for the efficacy of biological control are discussed.

Genetic Diversity of Pathogenic and Nonpathogenic Populations of Fusarium oxysporum Isolated from Carnation Fields in Argentina.

ABSTRACT In order to elucidate the origin of Fusarium oxysporum f. sp. dianthi in Argentina, the genetic diversity among pathogenic isolates together with co-occurring nonpathogenic isolates on carnation was investigated. In all, 151 isolates of F. oxysporum were obtained from soils and carnation plants from several horticultural farms in Argentina. The isolates were characterized using vegetative compatibility group (VCG), intergenic spacer (IGS) typing, and pathogenicity tests on carnation. Seven reference strains of F. oxysporum f. sp. dianthi also were analyzed and assigned to six different IGS types and six VCGs. Twenty-two Argentinean isolates were pathogenic on carnation, had the same IGS type (50), and belonged to a single VCG (0021). The 129 remaining isolates were nonpathogenic on carnation and sorted into 23 IGS types and 97 VCGs. The same VCG never occurred in different IGS types. Our results suggest that the pathogen did not originate in the local populations of F. oxysporum but, rather, that it was introduced into Argentina. Given the genetic homogeneity within Argentinean isolates of F. oxysporum f. sp. dianthi, either IGS type or VCG can be used for the identification of the forma specialis dianthi currently in Argentina.