The Effects of Local Weed Species on Arbuscular Mycorrhizal Fungal Communities in an Organic Winter Wheat (Triticum durum L.) Field in Lebanon.

We examined the potential effects of weed species on the arbuscular mycorrhizal fungi (AMF) in an organic winter wheat (Triticum durum) field in Lebanon. In this agroecosystem, the field and its surroundings were covered with spontaneous vegetation corresponding to local weeds. The coexistence between wheat and weeds did not modify AM fungal community diversity and colonization in T. durum but changed their composition. We evidenced 22 operational taxonomic units (OTUs) specifically shared between wheat associated with weeds (Tdw) and weeds, regardless of their localization and 12 OTUs with an abundance of variation between wheat without neighboring weeds (Td) and Tdw. The number of AM propagules and total C and N contents were higher in soil covered with wheat associated with weeds (TdWsoil) vs. wheat without neighboring weeds (Tdsoil). In greenhouse experiments, the shoot biomass and root mycorrhizal intensity of Medicago sativa, used as a trap plant, were higher using TdWsoil vs. Tdsoil as the inoculum. Positive correlations were observed between soil AM propagule numbers and M. sativa shoot biomass, on the one hand and M. sativa mycorrhizal intensity, on the other hand. Weeds seemed to exert significant effects on root AM fungal composition in T. durum and these effects may contribute to enhanced AMF development in the field.

Molecular Characterization of Fungal Biodiversity in Long-Term Polychlorinated Biphenyl-Contaminated Soils.

Polychlorinated biphenyls (PCBs) belong to the organic pollutants that are toxic to humans and harmful to environments. Numerous studies dealing with the impact of PCBs on soil microorganisms have focused on bacterial communities. The effects of PCBs on fungal communities in three different PCB-polluted soils from former industrial sites were investigated using high-throughput sequencing of the internal transcribed spacer 1 region. Significant differences in fungal alpha diversity were observed mainly due to soil physico-chemical properties. PCBs only influenced the richness of the fungal communities by increasing it. Fungal composition was rather strongly influenced by both PCBs and soil properties, resulting in different communities associated with each soil. Sixteen Ascomycota species were present in all three soils, including Stachybotrys chartarum, Fusarium oxysporum, Penicillium canescens, Penicillium chrysogenum,Penicillium citrosulfuratum and Penicillium brevicompactum, which are usually found in PCB-polluted soils, and Fusarium solani, Penicillium canescens, Penicillium citrosulfuratum and Penicillium chrysogenum, which are known PCB degraders. This study demonstrated that PCBs influence the richness and the composition of fungal communities. Their influence, associated with that of soil physico-chemical properties, led to distinct fungal communities, but with sixteen species common to the three soils which could be considered as ubiquitous species in PCB-polluted soils.

Potentiality of Native Ascomycete Strains in Bioremediation of Highly Polychlorinated Biphenyl Contaminated Soils.

Polychlorinated biphenyls (PCBs) are organic pollutants that are harmful to environment and toxic to humans. Numerous studies, based on basidiomycete strains, have reported unsatisfactory results in the mycoremediation of PCB-contaminated soils mainly due to the non-telluric origin of these strains. The abilities of a five-Ascomycete-strain consortium in the mycoremediation of PCB-polluted soils and its performance to restore their sound functioning were investigated using mesocosm experiments associated with chromatography gas analysis and enzymatic activity assays. With the soil H containing 850 ppm PCB from which the strains had been isolated, a significant PCB depletion of 29% after three months of treatment was obtained. This led to an important decrease of PCBs from 850 to 604 ppm. With the soil L containing 36 ppm PCB, biodegradation did not occur. In both soils, the fungal biomass quantified by the ergosterol assay, did not increase at the end of the treatment. Biodegradation evidenced in the soil H resulted in a significantly improved stoichiometry of N and P acquiring enzymatic activities. This unprecedented study demonstrates that the native Ascomycetes display remarkable properties for remediation and restoration of functioning of the soil they originated from paving the way for greater consideration of these strains in mycoremediation.

Plant health status effects on arbuscular mycorrhizal fungi associated with Lavandula angustifolia and Lavandula intermedia infected by Phytoplasma in France.

We investigated root communities of arbuscular mycorrhizal fungi (AMF) in relation to lavender (Lavandula angustifolia) and lavandin (Lavandula intermedia) health status from organic and conventional fields affected by Phytoplasma infection. The intensity of root mycorrhizal colonization was significantly different between diseased and healthy plants and was higher in the latter regardless of agricultural practice. This difference was more pronounced in lavender. The root AMF diversity was influenced by the plant health status solely in lavender and only under the conventional practice resulting in an increase in the AMF abundance and richness. The plant health status did not influence the distribution of root AMF communities in lavandin unlike its strong impact in lavender in both agricultural practices. Finally, among the most abundant molecular operational taxonomic units (MOTUs), four different MOTUs for each plant species were significantly abundant in the roots of healthy lavender and lavandin in either agricultural practice. Our study demonstrated that the plant health status influences root colonization and can influence the diversity and distribution of root AMF communities. Its effects vary according to plant species, can be modified by agricultural practices and allow plants to establish symbiosis with specific AMF species.

Metabolic adaptation of fungal strains in response to contamination by polychlorinated biphenyls.

Polychlorinated biphenyls (PCBs) represent a large group of recalcitrant environmental pollutants. Up to now, many studies have focused on bioremediation of PCBs by fungal strains; however, the mechanisms of adaptation of these strains towards PCBs remain unknown despite their importance in developing effective bioremediation processes. We studied five species, each consisting of two strains isolated either from PCB-polluted or PCB-unpolluted substrates (control strains). We investigated their responses to PCB contamination by studying their tolerance to PCBs, their ability to reduce these pollutants, and their expression level of Laccase genes. In Thermothelomyces thermophila, Thermothelomyces heterothallica, Thermoascus crustaceus, and Fusarium solani, all the studied strains showed a similar tolerance and PCB degradation regardless of their origin. In Schizophyllum commune, while both strains showed similar resistance to PCBs, i.e., PCBs and their degradation products presented no toxicity for these strains, the rate of PCB degradation of the strain from a PCB-polluted environment was significantly slightly higher. The PCB degradation did not correlate with the expression level of genes encoding Laccases. These results demonstrate that the tolerance and PCB degradation by the fungal strains, which did not involve Laccase genes, required different adaptation systems which seem to be constitutive or rapidly inducible by PCB according to the fungal species.

Molecular screening of xerophilic Aspergillus strains producing mycophenolic acid.

Mycophenolic acid (MPA) is the fungal secondary metabolite displaying several biological properties. Up to now, screening of fungal strains producing MPA has mainly been the result of the search of this molecule in their culture medium by chemical methods. Here we developed a molecular approach by targeting the expression level of the MpaC gene encoding the polyketide synthase, one of the key enzymes involved in the MPA synthesis. Thirty xerophilic Aspergillus strains were identified using the RNA polymerase II subunit and the ß-tubulin genes. Seven Aspergillus species were evidenced. The expression level of the MpaC gene was quantified and compared to the MPA production rate. Only Aspergillus pseudoglaucus and all the eight strains of this species produced MPA. While the MpaC gene was not expressed or weakly expressed in the MPA non-producing strains, all the A. pseudoglaucus strains presented a high level of expression of this gene. The highest expression level of the MpaC gene among the MPA non-producing strains was significantly lower than the lowest expression level of this gene in the MPA producing strains. To our knowledge, this is the first study that demonstrates the effectiveness of molecular approach for the screening of MPA-producing species.

Autochthonous ascomycetes in depollution of polychlorinated biphenyls contaminated soil and sediment.

We investigated the capacity of a consortium of ascomycetous strains, Doratomyces nanus, Doratomyces purpureofuscus, Doratomyces verrucisporus, Myceliophthora thermophila, Phoma eupyrena and Thermoascus crustaceus in the mycoremediation of historically contaminated soil and sediment by polychlorinated biphenyls (PCBs). Analyses of 15 PCB concentrations in three mesocosms containing soil from which the fungal strains had previously been isolated, revealed significant PCB depletions of 16.9% for the 6 indicator PCBs (i-PCBs) and 18.7% for the total 15 PCBs analyzed after 6months treatment. The degradation rate did not statistically vary whether the soil had been treated with non-inoculated straw or colonized straw or without straw and inoculated with the consortium of the six strains. Concerning the sediment, we evidenced significant depletions of 31.8% for the 6 i-PCBs and 33.3% for the 15 PCB congeners. The PCB depletions affected most of the 15 PCBs analyzed without preference for lower chlorinated congeners. Bioaugmented strains were evidenced in different mesocosms, but their reintroduction, after six months treatment, did not improve the rate of PCB degradation, suggesting that the biodegradation could affect the bioavailable PCB fraction. Our results demonstrate that the ascomycetous strains potentially adapted to PCBs may be propitious to the remediation of PCB contaminated sites.

Potential of autochthonous fungal strains isolated from contaminated soils for degradation of polychlorinated biphenyls.

Up to now, most studies on polychlorinated biphenyl (PCB) bioremediation have examined the ability of model fungal strains to biodegrade PCBs. Yet, there is limited information concerning the potential of autochthonous filamentous fungal strains in the biodegradation of PCBs and their possible use in the environmental technologies. In this study, we investigated the capacity of autochthonous fungal strains in the biodegradation of PCBs by isolating 24 taxa from former industrial sites highly contaminated by PCBs. Microscopic and molecular analyses using the internal transcribed spacer (ITS) region revealed that the fungal strains belonged to the phyla Ascomycota (19 strains) and Zygomycota (five strains). The chromatography gas analysis revealed evidence of degradation of seven PCB congeners. With the exception of Circinella muscae which presented no degradation potential, the other fungal strains exhibited a rate of biodegradation ranging from 29 to 85 % after 7 d of incubation in liquid medium. Among these strains, Doratomyces nanus, Doratomyces purpureofuscus, Doratomyces verrucisporus, Myceliophthora thermophila, Phoma eupyrena, and Thermoascus crustaceus showed remarkable degradation ability (>70 %) regardless of the number of chlorine substituents on the biphenyl nucleus and a high tolerance towards PCBs. To our knowledge, this is the first study that demonstrates the ability of PCB degradation by these species and indicates the potential effectiveness of some autochthonous fungal strains in bioremediation systems.

Potentiality of the cox1 gene in the taxonomic resolution of soil fungi.

We explored the potential of the cox1 gene in the species resolution of soil fungi and compared it with the nuclear internal transcribed spacer (ITS) and small subunit (SSU)-rDNA. Conserved primers allowing the amplification of the fungal cox1 gene were designed, and a total of 47 isolates of Zygomycota and Ascomycota were investigated. The analysis revealed a lack of introns in >90% of the isolates. Comparison of the species of each of the six studied genera showed high interspecific sequence polymorphisms. Indeed, the average of nucleotide variations (4.2-11%) according to the genus, due mainly to the nucleotide substitutions, led to the taxonomic resolution of all the species studied regarding both ITS and SSU-rDNA, in which <88% were discriminated. The phylogenetic analysis performed after alignment of the cox1 gene across distant fungal species was in accordance with the well-known taxonomic position of the species studied and no overlap was observed between intra- and interspecific variations. These results clearly demonstrated that the cox1 sequences could provide good molecular markers for the determination of the species composition of environmental samples and constitute an important advance to study soil fungal biodiversity.

High sexual reproduction and limited contemporary dispersal in the ectomycorrhizal fungus Tricholoma scalpturatum: new insights from population genetics and spatial autocorrelation analysis.

Dispersal and establishment are fundamental processes influencing the response of species to environmental changes, and the long-term persistence of populations. A previous study on the symbiotic ectomycorrhizal fungus Tricholoma scalpturatum revealed strong genetic differentiations between populations in Western Europe, suggesting restricted dispersal for this wind-dispersed cosmopolitan fungus. Two distinct genetic groups (genetic groups 1 and 2), co-occurring in some locations, were also identified and could correspond to cryptic species. In the present work, we examine the reproductive strategy and dispersal biology of the two T. scalpturatum's genetic groups. Variable molecular markers (intersimple sequence repeats and intergenic spacer 2-restriction fragment length polymorphisms) and spatial autocorrelation analyses were used to examine fine-scale patterns (< 140 m) of genetic structure, in an effort to determine the physical scale at which genetic structure exists. A total of 473 fruit bodies were mapped and collected over 3 years from two plots located in the south of France, including 219 and 254 samples from group 1 and group 2, respectively. High genetic diversity and the presence of numerous small genets were observed in both groups. Autocorrelation analyses revealed significant positive spatial genetic structures of genets at close distances (up to few metres for both groups). Mantel tests confirmed this isolation-by-distance pattern. These results clearly demonstrate high sexual reproduction and spatial structuring of genets at very small geographical scales in this wind-dispersed ectomycorrhizal fungal species, a pattern consistent with restricted contemporary dispersal of spores.

Molecular evolution of mitochondrial ribosomal DNA in the fungal genus Tricholoma: barcoding implications.

The molecular evolution of the V6 and V9 domains of the mitochondrial SSU-rDNA was investigated to evaluate the use of these sequences for DNA barcodes in the Basidiomycota division. The PCR products from 27 isolates belonging to 11 Tricholoma species were sequenced. Both domains in the isolates belonging to the same species had identical sequences. All the species possess distinctive V9 sequences due to point mutations and insertion/deletion events. Secondary structures revealed that the insertion-deletion events occurred in regions not directly involved in the maintenance of the standard SSU-rRNA structure. The inserted sequences possess conserved motifs that enable their alignment among phylogenetically distant species. Hence, the V9 domain by displaying identical sequences within species, an adequate divergence level, easy amplification, and alignment represents an alternative molecular marker for the Basidiomycota division and opens the way for this sequence to be used as specific molecular markers of the fungal kingdom.

Population evidence of cryptic species and geographical structure in the cosmopolitan ectomycorrhizal fungus, Tricholoma scalpturatum.

Tricholoma scalpturatum is an ectomycorrhizal fungus that forms symbioses with roots of diverse trees and shrubs. It is commonly encountered in a wide range of habitats, across temperate ecosystems. A previous study has revealed a high genetic diversity at a local scale, and ruderal abilities. To examine genetic structure at a large geographical scale, a total of 164 basidiocarps were collected from 30 populations located in Western Europe, from Spain to Scandinavia. These samples were analyzed by three molecular methods with different levels of resolution: inter-simple sequence repeats (ISSRs), restriction fragment length polymorphisms (RFLPs) in the rDNA internal transcribed spacer (ITS), and ITS sequence analysis. Considerable genetic variation was found, and the morphospecies was separated into two genetic groups that were distinct from each other. The ISSR data and the relatively low percentage value (96%) of shared sequence polymorphisms in the ITS between isolates from the two groups, strongly suggest cryptic species and long-lasting separation. No geographical exclusion was detected for these two widely distributed taxa. However, high estimates of population differentiation were observed in each group, including between populations less than a few kilometers apart. This result provides evidence for limited gene flow and/or founding effects. It also indicates that T. scalpturatum does not constitute a random mating population, and the hypothesis of endemism cannot be excluded for this cosmopolitan wind-dispersed fungus.

Unusual accumulation of polymorphic microsatellite loci in a specific region of the mitochondrial genome of two mushroom-forming Agrocybe species.

The cob/tRNA(Tyr) mitochondrial regions of Agrocybe aegerita and of the related species Agrocybe chaxingu display an unusual clustering of four microsatellite loci constituted by motifs of one to six nucleotides whose number of repeats varied from three to 18. In A. chaxingu, these microsatellite loci are followed by a small region bearing one additional microsatelite and one minisatellite locus constituted by an octanucleotide motif repeated 13-18 times. In A. aegerita, this latter region is deleted. This is the first evidence of such an accumulation of microsatellites in mitochondrial genomes. The analyses of the microsatellite loci in 11 A. aegerita and in four A. chaxingu wild strains have shown extensive intraspecific and interspecific variations in the number of tandem repeats (VNTRs), suggesting that these loci could represent powerful molecular markers for strain fingerprinting. Up to 23 different alleles were present in the 15 Agrocybe studied strains, allowing the definition of 12 different haplotypes.

A CAPS test allowing a rapid distinction of Penicillium expansum among fungal species collected on grape berries, inferred from the sequence and secondary structure of the mitochondrial SSU-rRNA.

Penicillium expansum is a fungal species highly damageable for the postharvest conservation of numerous fruits. In vineyards, this fungus is sometimes isolated from grape berries where its presence may lead to the production of geosmin, a powerful earthy odorant, which can impair grapes and wines aromas. However, the discrimination of P. expansum from related fungi is difficult because it is based on ambiguous phenotypic characters and/or expensive and time-consuming molecular tests. In this context, the complete sequences and secondary structures of Penicillium expansum and Penicillium thomii mitochondrial SSU-rRNAs were achieved and compared with those of two other phylogenetically related Ascomycota: Penicillium chrysogenum and Emericella nidulans. The comparison has shown a high conservation in size and sequence of the core and of the variable domains (more than 80% of nt identity) of the four SSU-rRNAs, arguing for a close phylogenetic relationship between these four species of the Trichocomaceae family. Large (from 10 to 18 nt) inserted/deleted (indel) sequences were evidenced in the V1, V5 and V6 variable domains. The size variations (10 to 18 nt) of the V1 indel sequence allowed the distinction of the four species; the V5 indel (15 nt) was specifically recovered in E. nidulans; the V6 indel (16 nt), shared by the three Penicillium species, was lacking in E. nidulans. A couple of conserved primers (UI/R2) were defined to generate a PCR product containing the V1 to V5 variable domains. This product contained the two regions of the four SSU-rRNAs showing the highest rates of nt substitutions, namely the V2 variable domain and, surprisingly, a helix (H17) of the core. The H17 sequence was shown to specifically possess in P. expansum a recognition site for the ClaI restriction endonuclease. Hence, this enzyme generates a digestion pattern of the PCR product with two bands (350 bp+500 bp), specific to P. expansum and easily separable by agarose gel electrophoresis. This leads to a CAPS test usable for P. expansum discrimination among grape berries isolated filamentous fungi. The CAPS test was validated by a comparative analysis involving 29 strains belonging to 17 species currently isolated from grape berries in the Bordeaux vineyards.

The mitochondrial apocytochrome b genes of two Agrocybe species suggest lateral transfers of group I homing introns among phylogenetically distant fungi.

The Agrocybe chaxingu and Agrocybe aegerita mitochondrial apocytochrome b coding sequences are highly similar (97% of nt identity), but have highly different sizes (2312 and 4867nt, respectively), due to the presence of three large group IB introns: two (iAae1 and iAae2) in A. aegerita, one (iAch1) in A. chaxingu. All these introns encode a homing endonuclease (HE) similar to those described in introns of mitochondrial genes (cob, cox1, and nad5) from various organisms. Phylogenetic trees were built with these HE sequences. From these trees, the Agrocybe coding introns argue for recent lateral transfers, i.e., occurring after the separation of the two Agrocybe species, involving phylogenetically distant fungi such as members of the Ascomycota phylum (for iAch1 and iAae2) and, for the first time to our knowledge, a member of the Chytridiomycota phylum (for iAae1). The grouping of the HE gene (HEG) sequences according to the mitochondrial gene (cob, cox1, and nad5) where they are inserted, suggests modifications of the interactions between the HE and the recognized sequences, leading to new target genes. The largest distribution of the iAch1 HE, shared by several cob and cox1 mitochondrial genes from Ascomycota, Basidiomycota, and Chytridiomycota phyla, suggests a higher target flexibility of this HE, perhaps related to the presence of two different LAGLIDADG motifs in the catalytic site of the enzyme.

Secondary structure and molecular evolution of the mitochondrial small subunit ribosomal RNA in Agaricales (Euagarics clade, Homobasidiomycota).

The complete sequences and secondary structures of the mitochondrial small subunit (SSU) ribosomal RNAs of both mostly cultivated mushrooms Agaricus bisporus (1930 nt) and Lentinula edodes (2164 nt) were achieved. These secondary structures and that of Schizophyllum commune (1872 nt) were compared to that previously established for Agrocybe aegerita. The four structures are near the model established for Archae, Bacteria, plastids, and mitochondria; particularly the helices 23 and 37, described as specific to bacteria, are present. Within the four Agaricales (Homobasidiomycota), the SSU-rRNA "core" is conserved in size (966 to 1009 nt) with the exception of an unusual extension of 40 nt in the H17 helix of S. commune. The four core sequences possess 76% of conserved positions and a cluster of C in their 3' end, which could constitute a signal involved in the RNA maturation process. Among the nine putative variable domains, three (V3, V5, V7) do not show significant length variations and possess similar percentages of conserved positions (69%) than the core. The other six variable domains show important length variations, due to independent large size inserted/deleted sequences, and higher rates of nucleotide substitutions than the core (only 31% of conserved positions between the four species). Interestingly, the inserted/deleted sequences are located in few preferential sites (hot spots for insertion/deletion) where they seem to arise or disappear haphazardly during evolution. These sites are located on the surface of the tertiary structure of the 30S ribosomal subunit, at the beginning of hairpin loops; the insertions lead to a lengthening of existing hairpins or to branching loops bearing up to five additional helices.