Occurrence of Aspergillus fumigatus azole resistance in soils from Switzerland.

Aspergillus fumigatus is a fungal species causing diverse diseases in humans. The use of azoles for treatments of A. fumigatus diseases has resulted in azole resistance. Azoles are also widely used in the environment for crop protection, which resulted in azole resistance. Resistance is primarily due to mutations in cyp51A, which encodes the target protein for azoles. Here we addressed the occurrence of azole resistance in soils from a vast part of Switzerland. We aimed to associate the use of azoles in the environment with the occurrence of azole resistance. We targeted sample sites from different agricultural environments as well as sites with no agricultural practice (natural sites and urban sites). Starting from 327 sites, 113 A. fumigatus isolates were recovered (2019-2021), among which 19 were azole-resistant (15 with TR34/L98H and four with TR46/Y121F/T289A resistance mutations in cyp51A). Our results show that azole resistance was not associated with a specific agricultural practice. Azoles could be chemically detected in investigated soils, however, their presence was not associated with the occurrence of azole-resistant isolates. Interestingly, genetic markers of resistance to other fungicides were detected but only in azole-resistant isolates, thus reinforcing the notion that A. fumigatus cross-resistance to fungicides has an environmental origin. In conclusion, this study reveals the spreading of azole resistance in A. fumigatus from the environment in Switzerland. The proximity of agricultural areas to urban centers may facilitate the transmission of resistant strains to at-risk populations. Thus, vigilant surveillance is required to maintain effective treatment options for aspergillosis.

Aspergillus fumigatus is ubiquitous and causes diseases in humans. Antifungal drugs, and especially azoles, are used to combat A. fumigatus. Azoles are widely used in the environment, which exposes A. fumigatus and results in azole resistance. Azole resistance was investigated in Switzerland.

Environmental, bystander and resident exposure from orchard applications using an agricultural unmanned aerial spraying system.

Unmanned aerial spraying systems (UASS), i.e., unmanned aerial vehicles designed for pesticide applications, are widely used in East Asia and increasingly prevalent in other regions of the world, including North America and Europe. However, according to a recent report of the Organization for Economic Co-operation and Development, spray drift and exposure caused by these systems are not yet fully understood. In particular, there are at present no peer-reviewed reports on direct exposure of residents and bystanders to spray drift following UASS applications. This lack of data results in regulatory concerns with respect to the environment and human safety. The objective of this study was to quantify environmental, resident and bystander exposure following the application of a plant protection product to an orchard using a commercial UASS under field conditions. Using a fluorescent tracer, horizontal and vertical downwind drift data were collected and direct exposure of residents and bystanders located downwind the sprayed area to spray drift was quantified using display mannequins equipped with personal air sampling pumps. Spray drift and exposure inversely correlated with sampling height and downwind distance. Furthermore, drift and exposure were strongly influenced by wind speed and direction, albeit hardly affected by the growth stage of the trees. In addition, substantially less tracer was extracted from the filters of the air sampling pumps than from the coveralls worn by mannequins, suggesting that direct resident/bystander exposure to spray drift may predominantly occur via the dermal route. This report provides essential data on UASS spray drift potential that are relevant for environmental and health risk assessments related to these systems. The results are compared to predicted values of current regulatory models and previously reported field data on drift and exposure caused by different spraying equipment.

Ten Years of VINQUEST: First Insight for Breeding New Apple Cultivars With Durable Apple Scab Resistance.

Apple scab, caused by Venturia inaequalis, is a major fungal disease worldwide. Cultivation of scab-resistant cultivars would reduce the chemical footprint of apple production. However, new apple cultivars carrying durable resistances should be developed to prevent or at least slow the breakdown of resistance against races of V. inaequalis. One way to achieve durable resistance is to pyramid multiple scab resistance genes in a cultivar. The choice of the resistance genes to be combined in the pyramids should take into account the frequency of resistance breakdown and the geographical distribution of apple scab isolates able to cause such breakdowns. In order to acquire this information and to make it available to apple breeders, the VINQUEST project (www.vinquest.ch) was initiated in 2009. Ten years after launching this project, 24 partners from 14 countries regularly contribute data. From 2009 to 2018, nearly 9,000 data points have been collected. This information has been used to identify the most promising apple scab resistance genes for developing cultivars with durable resistance, which to date are: Rvi5, Rvi11, Rvi12, Rvi14, and Rvi15. As expected, Rvi1, together with Rvi3 and Rvi8, were often overcome, and have little value for scab resistance breeding. Rvi10 may also belong to this group. On the other hand, Rvi2, Rvi4, Rvi6, Rvi7, Rvi9, and Rvi13 are still useful for breeding, but their use is recommended only in extended pyramids of ≥3 resistance genes.

Vitis vinifera canes, a new source of antifungal compounds against Plasmopara viticola, Erysiphe necator, and Botrytis cinerea.

Methanolic and ethanolic crude extracts of Vitis vinifera canes exhibited significant antifungal activity against the three major fungal pathogens affecting grapevines, Plasmopara viticola, Erysiphe necator and Botrytis cinerea. The active extracts were analyzed by LC-PDA-ESI-MS, and selected compounds were identified. Efficient targeted isolation using medium-pressure liquid chromatography afforded six pure constituents in one step. The structures of the isolated compounds were elucidated by NMR and HRMS. Six identified compounds (ampelopsin A, hopeaphenol, trans-resveratrol, ampelopsin H, ε-viniferin, and E-vitisin B) presented antifungal activities against P. viticola. ε-Viniferin also exhibited a low antifungal activity against B. cinerea. None of the identified compounds inhibited the germination of E. necator. The potential to develop a novel natural fungicide against the three major fungal pathogens affecting V. vinifera from viticulture waste material is discussed.

Susceptibility to downy mildew in grape clusters: New microscopical and biochemical insights.

Grape clusters of different Vitis genotypes, including Vitis vinifera cvs Chasselas and Merlot, and two interspecific grape varieties, Solaris (cvs. Merzling* x (Saperavi severneyi x Muscat ottonel)) and 2091 (cvs. Gamaret x Bronner), are susceptible or resistant to downy mildew. These cultivars were inoculated with Plasmopara viticola at three developmental stages (BBCH stages 53, 69 and 75). Samples were examined by scanning electron microscopy and the synthesis of stilbenes was measured. Microscopical examinations of pedicels, rachis and calyptras showed important differences in stomatal structures within seasonal development. At BBCH 53, successful infections were observed on all tested cultivars and functional stomata were present, while no infections were observed after this stage. At BBCH 69 and 75, cracks were observed around the stomata and guard cells were unstructured or completely collapsed, leading to closed-like stomata. At BBCH 53, significant stilbene accumulation was quantified in 2091 and Solaris; pterostilbene and δ-viniferin were produced in large amounts. In the susceptible varieties, only piceid and resveratrol were induced. At the other two stages, the concentration of all measured stilbenes was undetectable. The critical roles of seasonal development and stilbenes in the resistance of grape clusters towards downy mildew are discussed.

Crosstalk and differential response to abiotic and biotic stressors reflected at the transcriptional level of effector genes from secondary metabolism.

Plant secondary metabolism significantly contributes to defensive measures against adverse abiotic and biotic cues. To investigate stress-induced, transcriptional alterations of underlying effector gene families, which encode enzymes acting consecutively in secondary metabolism and defense reactions, a DNA array (MetArray) harboring gene-specific probes was established. It comprised complete sets of genes encoding 109 secondary product glycosyltransferases and 63 glutathione-utilizing enzymes along with 62 cytochrome P450 monooxygenases and 26 ABC transporters. Their transcriptome was monitored in different organs of unstressed plants and in shoots in response to herbicides, UV-B radiation, endogenous stress hormones, and pathogen infection. A principal component analysis based on the transcription of these effector gene families defined distinct responses and crosstalk. Methyl jasmonate and ethylene treatments were separated from a group combining reactions towards two sulfonylurea herbicides, salicylate and an avirulent strain of Pseudomonas syringae pv. tomato . The responses to the herbicide bromoxynil and UV-B radiation were distinct from both groups. In addition, these analyses pinpointed individual effector genes indicating their role in these stress responses. A small group of genes was diagnostic in differentiating the response to two herbicide classes used. Interestingly, a subset of genes induced by P. syringae was not responsive to the applied stress hormones. Small groups of comprehensively induced effector genes indicate common defense strategies. Furthermore, homologous members within branches of these effector gene families displayed differential expression patterns either in both organs or during stress responses arguing for their non-redundant functions.

The rapid induction of glutathione S-transferases AtGSTF2 and AtGSTF6 by avirulent Pseudomonas syringae is the result of combined salicylic acid and ethylene signaling.

The expression of two members of the glutathione S-transferase (GST) multigene family was studied in Arabidopsis plants inoculated with an avirulent strain of Pseudomonas syringae pv. tomato (Pst). Accumulation of AtGSTF2 and AtGSTF6 transcripts started 4 and 2 h after inoculation, respectively, and clearly preceded the induction of the pathogenesis-related PR-1 gene. The aim of this work was to find the reason for the faster induction of the two GSTs compared with classical salicylic acid (SA)-regulated PR-proteins. Expression studies in Pst-inoculated SA-signaling mutants NahG and npr1 revealed that induction of both GSTs was SA-dependent and partially NPR1-independent. The induction of AtGSTF2 by Pst was also strongly repressed in the ethylene insensitive etr1 mutant. Both GSTs were induced by low amounts of SA (0.1 mM) and ethylene (0.1 ppm) while PR-1 gene expression was unaffected by ethylene. Interestingly, ethylene was about 50-fold less effective in NahG compared with wild-type plants thus suggesting a potentiation effect of SA on ethylene-induced accumulation of AtGST transcripts. Increased AtGST expression in plants inoculated with Pst correlated with increased production of SA and ethylene. However, the initial phase of AtGSTF6 induction was independent of SA- and ethylene-signaling. The jasmonate (JA)-insensitive mutant jar1 showed normal induction kinetics for both GSTs. Our data support the hypothesis that full expression of the pathogen-induced AtGSTF2 and, to a lesser extent AtGSTF6, is the result of combined SA- and ethylene-signaling and that early AtGSTF6 expression depends on additional unknown signaling mechanisms.