Effects of resveratrol, viniferins and pterostilbene on Plasmopara viticola zoospore mobility and disease development

The effects of stilbenes (resveratrol, δ- and ε-viniferins, and pterostilbene) on the mobility of zoospores of Plasmopara viticola and on subsequent disease development were studied in vitro. δ-viniferin and pterostilbene are the most toxic stilbenes concerning zoospore mobility (ED50 : 14.6 and 28.3 μM) and disease development (ED50 : 14.7 and 12.7 μM). The analysis of stilbenes in leaf cells of resistant (Solaris) and susceptible (Chasselas) grape cultivars artificially inoculated with P. viticola has shown that very high amounts of stilbenic phytoalexins accumulate at the site of infection of the resistant cultivar compared to the susceptible one

Development of a rapid and highly sensitive direct-PCR assay to detect a single conidium of Botrytis cinerea Pers.:Fr in vitro and quiescent forms in planta

“Direct-PCR” amplifications of Botrytis cinerea-specific genomic sequences, without any DNA purification step or time consuming sample preparation, were developed. A single copy sequence of 0.7 Kb in the Botrytis cinerea genome was amplified in reactions containing no more than 1 x 105 to 1 single conidium. As a demonstrative application, this assay was applied to detect B. cinerea in different parts of immature grape berries (at ‘pea size’), when previously inoculated with conidia at flowering. Using this method we showed the presence of quiescent Botrytis in the receptacle area only. Cloning and sequencing of the fragment confirmed the single sequence gene of B. cinerea. These results demonstrate that the method is easy to apply and of sufficiently high sensitivity to detect the presence of B. cinerea in immature grape berries. Its use for studies on the development of grey mould and improved control of the disease in vineyards is discussed

Downy mildew: is resistance linked to inoculum concentration?

Leaves of different Vitis vinifera cultivars, susceptible (‘Chasselas’ and ‘2185’), less susceptible (‘2142’) or resistant to downy mildew ('Solaris’ and ‘2091’), were inoculated with four different concentrations of an aqueous sporangia suspension of Plasmopara viticola (5 × 105, 2 × 105, 6 × 104 and 2 × 104 sporangia·ml-1). The infection rate of these samples was then examined by light microscopy and synthesis of stilbenes was analysed at infection sites. Infection rate increased parallel with inoculum concentration, but there was no correlation between the infection rate and resistance to P. viticola. Moreover, at the lowest inoculum concentration, the infection rate is similar for susceptible and resistant grapevine varieties. Quantification of stilbenes at 72 hpi showed that at the lowest inoculum concentration, the most susceptible grape variety synthesized the largest amount of stilbenes, whose level remained however below the ED50 values defined for each of them. Conversely, at the highest inoculum concentration, the most resistant varieties produced the highest amounts of the most toxic stilbenes against P. viticola. The critical role of the inoculum concentration used for artificial inoculation to evaluate grapevine resistance to downy mildew is discussed.

Leaf morphological characteristics and stilbene production differently affect downy mildew resistance of Vitis vinifera varieties grown in Italy

The degree of resistance to downy mildew of grape varieties belonging to the oenological tradition of Central Italy was evaluated by the analysis of plant responses to pathogen infections carried out in natural and controlled environments. Leaf morphological traits, such as hair and stomatal density, were determined for each variety, and the percentage of infected stomata and pathogen colonization of host mesophyll at 24, 48, and 72 hours post inoculation were assessed by epifluorescence microscopy. Furthermore, stilbene production at the site of Plasmopara viticola infection was analyzed at 72 hours post inoculation. Results indicate differences in resistance to downy mildew among selected varieties. Different significant values were detected among grapevines in the percentage of infected stomata and average number of successfully penetrated zoospores per stomata and per leaf surface unit. Differences also emerged in the rate of pathogen growth and stilbene production, signifying that defence mechanisms involved or induced during pathogen infection could be differentially effective among grapevine cultivars in limiting disease progression.

Histological and biochemical criteria for objective and early selection of grapevine cultivars resistant to Plasmopara viticola

Grapevine breeding is the most effective way to create cultivars resistant to downy mildew (Plasmopara viticola), and to reduce the number of fungicide applications. Four criteria, including histological and biochemical analyses, based on the level of different mechanisms of resistance to grapevine downy mildew, were tested on 42 different cultivars. Plantlets were artificially inoculated with downy mildew and the sporangia density was measured spectrophotometrically 6 d after infection. Callose synthesis in stomata and δ- and ε-viniferin levels at the site of infection were recorded 48 h after inoculation. These observations have allowed the 42 cultivars to be divided into 5 groups: very resistant (VR), resistant (R), less susceptible (LS), susceptible (S) and highly susceptible (HS). All 4 criteria have to be applied to assign the resistance level closer to field conditions. This method allows to rapidly evaluate the level of resistance of seedlings to downy mildew thereby leading to a reduction in duration  of the breeding program by several years.

Two New Primers Highly Specific for the Detection of Botrytis cinerea Pers. Fr.

Of thirty-nine Botrytis cinerea isolates originating in different host-plants and grown in pure cultures, twenty-six produced abundant grey aerial mycelium and sporulated intensely, whilst thirteen produced a thin mycelial layer, abundant sclerotia and secreted an unidentified yellow pigment in PDA culture media. The commonly used C729 +/– primers (5’-AGCTCGAGAGAGATCTCTGA-3’; 5’-CTGCAATGTTCTGCGTGGAA-3’) designed to detect B. cinerea did not amplify the DNA fragment of 0.73 kb in this smaller group of strains under standard conditions, whereas a shorter DNA fragment (0.60 kb) was amplified at a lower annealing temperature (50°C). This fragment was sequenced and two new internal primers were designed, BC108 + (5’-ACCCGCACCTAATTCGTCAAC-3’) and BC563 – (5’-GGGTCTTCGATACGGGAGAA-3’). These new primers were used to amplify a DNA fragment of 0.48 kb for the main group of 26 B. cinerea strains and a shorter fragment (0.36 kb) for the smaller group of 13 strains due to a deletion of 0.12 kb, which was not detected with the primers C729 +/–. All the strains were amplified to detect the presence or absence of Boty and Flipper transposable elements. No correlation was found between strains possessing the deletion and those belonging to either the vacuma or the transposa sibling species. Other closely related Botrytis species such as B. allii and B. fabae were not amplified with these primers, confirming their specificity for B. cinerea and enhancing the sensitivity of the molecular tools available to detect this fungus in host-plants

Fungal communities living in the wood of different cultivars of young Vitis vinifera plants

The fungal communities associated with fi ve Vitis vinifera cultivars grown in Switzerland (‘Humagne’, ‘Chasselas’, ‘Arvine’, ‘Gamaret’ and ‘Gamay’) were examined. Of the 703 fungal isolates obtained in pure culture, 66 operational taxonomic units (OTUs) were defi ned. The results show that: the great majority of the fungi isolated in this study were ascomycetes, with a high proportion of Sordariomycetes (mainly Hypocreales, Sordariales and Diaporthales); different fungal OTUs were associated with different cultivars; graft and rootstock contributed equally to the fungal community composition; Esca- or Petri-related species occurred sporadically in the different cultivars, with some of them occupying specifi c tissues or parts of the plant (e.g.: Botryosphaeriaceous species, Phaeomoniella chlamydospora and Phomopsis viticola); almost 25% of OTUs occurred in different plant parts in most cultivars, which suggests an easy spread outwards from the infected material (graft or rootstock), which might be explained by the fungal propagules being transported through the xylem vessels

Production of Fusaric Acid by Fusarium spp. in Pure Culture and in Solid Medium Co-Cultures.

The ability of fungi isolated from nails of patients suffering from onychomycosis to induce de novo production of bioactive compounds in co-culture was examined. Comparison between the metabolite profiles produced by Sarocladium strictum, by Fusarium oxysporum, and by these two species in co-culture revealed de novo induction of fusaric acid based on HRMS. Structure confirmation of this toxin, using sensitive microflow NMR, required only three 9-cm Petri dishes of fungal culture. A targeted metabolomics study based on UHPLC-HRMS confirmed that the production of fusaric acid was strain-dependent. Furthermore, the detected toxin levels suggested that onychomycosis-associated fungal strains of the F. oxysporum and F. fujikuroi species complexes are much more frequently producing fusaric acid, and in higher amount, than strains of the F. solani species complex. Fusarium strains producing no significant amounts of this compound in pure culture, were shown to de novo produce that compound when grown in co-culture. The role of fusaric acid in fungal virulence and defense is discussed

Azole Resistance of Environmental and Clinical Aspergillus fumigatus Isolates from Switzerland.

Aspergillus fumigatus is a ubiquitous opportunistic pathogen. This fungus can acquire resistance to azole antifungals due to mutations in the azole target (cyp51A). Recently, cyp51A mutations typical for environmental azole resistance acquisition (for example, TR <sub>34</sub> /L98H) have been reported. These mutations can also be found in isolates recovered from patients. Environmental azole resistance acquisition has been reported on several continents. Here we describe, for the first time, the occurrence of azole-resistant A. fumigatus isolates of environmental origin in Switzerland with cyp51A mutations, and we show that these isolates can also be recovered from a few patients. While the TR <sub>34</sub> /L98H mutation was dominant, a single azole-resistant isolate exhibited a cyp51A mutation (G54R) that was reported only for clinical isolates. In conclusion, our study demonstrates that azole resistance with an environmental signature is present in environments and patients of Swiss origin and that mutations believed to be unique to clinical settings are now also observed in the environment

Identification and Mode of Action of a Plant Natural Product Targeting Human Fungal Pathogens.

<i>Candida albicans</i> is a major cause of fungal diseases in humans, and its resistance to available drugs is of concern. In an attempt to identify novel antifungal agents, we initiated a small-scale screening of a library of 199 natural plant compounds (i.e., natural products [NPs]). <i>In vitro</i> susceptibility profiling experiments identified 33 NPs with activity against <i>C. albicans</i> (MIC <sub>50</sub> s ≤ 32 μg/ml). Among the selected NPs, the sterol alkaloid tomatidine was further investigated. Tomatidine originates from the tomato ( <i>Solanum lycopersicum</i> ) and exhibited high levels of fungistatic activity against <i>Candida</i> species (MIC <sub>50</sub> s ≤ 1 μg/ml) but no cytotoxicity against mammalian cells. Genome-wide transcriptional analysis of tomatidine-treated <i>C. albicans</i> cells revealed a major alteration (upregulation) in the expression of ergosterol genes, suggesting that the ergosterol pathway is targeted by this NP. Consistent with this transcriptional response, analysis of the sterol content of tomatidine-treated cells showed not only inhibition of Erg6 (C-24 sterol methyltransferase) activity but also of Erg4 (C-24 sterol reductase) activity. A forward genetic approach in <i>Saccharomyces cerevisiae</i> coupled with whole-genome sequencing identified 2 nonsynonymous mutations in <i>ERG6</i> (amino acids D249G and G132D) responsible for tomatidine resistance. Our results therefore unambiguously identified Erg6, a C-24 sterol methyltransferase absent in mammals, to be the main direct target of tomatidine. We tested the <i>in vivo</i> efficacy of tomatidine in a mouse model of <i>C. albicans</i> systemic infection. Treatment with a nanocrystal pharmacological formulation successfully decreased the fungal burden in infected kidneys compared to the fungal burden achieved by the use of placebo and thus confirmed the potential of tomatidine as a therapeutic agent