No abstract

Antibiosis is assumed to be an essential mechanism exerted by potential biocontrol agents (BCAs) of Trichoderma spp. Therefore, in the present study, we report for the first time on the elucidation and production of viridiofungin A (VFA) from T. harzianum isolate T23 cultures and investigate the antifungal potential of VFA and some other secondary metabolites purified from T. harzianum cultures against Fusarium moniliforme. The bioautography assay revealed that T. harzianum isolates T16 and T23 excreted several secondary metabolites with antifungal activity. Following isolation and purification of the antifungal zones, three fractions (F223, F323 and F423) from extracts of isolate T23 and two fractions (F416 and F516) from extracts of isolate T16 exhibited pronounced fungitoxic activity in the bioautography and antibiotic disk assays against Cladosporium spp. and F. moniliforme, respectively. The structure of the antifungal metabolite in fraction F323 was identified as viridiofungin A (VFA), the first report of production of VFA by isolate T23 of T. harzianum. Following cultivation of isolate T23 in PDB medium for 9 days, 94.6 mg l −1 of VFA were determined. VFA and fraction F516 retarded the mycelial growth of F. moniliforme in the nonvolatile phase assay by >90% for each 250 μg ml −1 7 days post-inoculation (dpi). While VFA and fraction F416 showed both volatile and non-volatile effects, fraction F516 seemed to exhibit mainly non-volatile activity. Microscopic examination revealed that hyphae of F. moniliforme grown on VFA-amended medium were less branched and appeared thicker than untreated hyphae. Furthermore, in the presence of VFA, formation of chlamydospores by F. moniliforme was increased. Finally, the antifungal spectrum of VFA towards various important plant pathogens was evaluated. Germination of propagules of a variety of fungal pathogens in vitro was differentially inhibited by VFA. While in the presence of 100 μg ml −1 VFA conidial germination of V. dahliae was completely inhibited, a slightly higher concentration (150 μg ml −1 ) of the inhibitor was required to suppress germination of Phytophthora infestans sporangia or sclerotia of Sclerotinia sclerotiorum. Contrary to several reports in the literature, VFA proved to be fungistatic rather than fungicidal. However, neither VFA nor the other Trichoderma metabolites, such as 6PAP, F416 and F516, exhibited any antibacterial activity against Gram-positive and Gram-negative bacteria.

Fertilization management can affect plant performance and soil microbiota, involving still poorly understood rhizosphere interactions. We hypothesized that fertilization practice exerts specific effects on rhizodeposition with consequences for recruitment of rhizosphere microbiota and plant performance. To address this hypothesis, we conducted a minirhizotron experiment using lettuce as model plant and field soils with contrasting properties from two long-term field experiments (HUB-LTE: loamy sand, DOK-LTE: silty loam) with organic and mineral fertilization history. Increased relative abundance of plant-beneficial arbuscular mycorrhizal fungi and fungal pathotrophs were characteristic of the rhizospheres in the organically managed soils (HU-org; BIODYN2). Accordingly, defense-related genes were systemically expressed in shoot tissues of the respective plants. As a site-specific effect, high relative occurrence of the fungal lettuce pathogen Olpidium sp. (76–90%) was recorded in the rhizosphere, both under long-term organic and mineral fertilization at the DOK-LTE site, likely supporting Olpidium infection due to a lower water drainage potential compared to the sandy HUB-LTE soils. However, plant growth depressions and Olpidium infection were exclusively recorded in the BIODYN2 soil with organic fertilization history. This was associated with a drastic (87–97%) reduction in rhizosphere abundance of potentially plant-beneficial microbiota (Pseudomonadaceae, Mortierella elongata) and reduced concentrations of the antifungal root exudate benzoate, known to be increased in presence of Pseudomonas spp. In contrast, high relative abundance of Pseudomonadaceae (Gammaproteobacteria) in the rhizosphere of plants grown in soils with long-term mineral fertilization (61–74%) coincided with high rhizosphere concentrations of chemotactic dicarboxylates (succinate, malate) and a high C (sugar)/N (amino acid) ratio, known to support the growth of Gammaproteobacteria. This was related with generally lower systemic expression of plant defense genes as compared with organic fertilization history. Our results suggest a complex network of belowground interactions among root exudates, site-specific factors and rhizosphere microbiota, modulating the impact of fertilization management with consequences for plant health and performance.

Phytopathogenic fungal species of the Diaporthe/Phomopsis complex (DPC) are associated with three highly destructive diseases on soybean: seed decay, pod and stem blight, and stem canker. They are responsible for poor seed quality and significant yield reduction in most soybean-producing areas. Precise identification and classification of DPC species are important in understanding the epidemiology of disease and to develop effective control measures. Although cultural and morphological characteristics of DPC-associated pathogens have been described, establishing a more accurate taxonomic framework seems necessary for a revaluation of the taxonomy and phylogeny of DPC species. In this study, we focused on morphological and molecular analyses of species from DPC-damaged European soybean seeds obtained from several locations throughout Europe. Colony characteristics, conidia dimensions, existence of αand β-conidia, and formation of perithecia were evaluated in order to assign the isolates to a species morphologically. Phylogenetic relationships were determined based on sequences from betatubulin (TUB), translation elongation factor 1-alpha (TEF1), and nuclear ribosomal DNA internal transcribed spacers (ITS). All isolates were tested for pathogenicity on soybean with positive results. In this study, we present updated taxonomic data by combining morphological observations and molecular tools which placed 32 Diaporthe isolates into four DPC species: D. longicolla, D. caulivora, D. eres, and D. novem, which are well-known soybean pathogens.

The interaction of the pathogen Fusarium moniliforme and two antagonistic Trichoderma harzianum isolates was studied especially with respect to their secondary metabolites fusaric acid (FA) and 6-pentyl-alphapyrone (6PAP). Among 10 isolates of F. moniliforme screened for FA production on maize kernels, the isolate 8 accumulated the highest amount of FA (678 lg ⁄ g). Mycelial growth and production of FA by isolate 8, determined in different liquid media revealed that the highest biomass and FA were produced in Czapek Dox Broth (CDB) followed by RichardÕs solution. The amount of FA per gram mycelial dry weight reached its maximum in CDB and RichardÕs solution after 14 days of incubation. Mycelial growth and conidia production of both Trichoderma isolates (T16 and T23) were retarded by increasing concentrations of FA in agar medium. At FA concentration of 300 mg ⁄ ml the radial mycelial growth of the isolates T16 and T23 were retarded by 32.5% and 45%, respectively. Conidia production was diminished in a similar extent as mycelial growth. Both T. harzianum isolates were capable to degrade FA in potato dextrose broth medium, particularly when lower doses of FA were present. In the presence of 50 mg ⁄ ml FA in the culture medium, the isolates T23 and T16 reduced FA by 51.4% and 88.4%, respectively, 9 days post-inoculation. The antifungal metabolite 6PAP, isolated from T. harzianum T23 cultures, was introduced at different concentrations into 2-day-old cultures of F. moniliforme. After further 5 days of incubation of F. moniliforme in the presence of 6PAP, the FA contents per gram mycelial dry weight were significantly decreased compared to control cultures where 6PAP was absent. Dosages of 300 and 400 mg ⁄ l of 6PAP in the cultures retarded FA accumulations by 62.5% and 77.2%, respectively. The current results, however, provided the first evidence for activity of 6PAP, as a Trichoderma secondary metabolite, on degrading ⁄ synthesis suppression of the Fusarium toxin FA.