Dynamics of Aspergillus fumigatus in Azole Fungicide-Containing Plant Waste in the Netherlands (2016-2017).

The treatment of patients suffering from Aspergillus diseases is hampered due to infections with Aspergillus fumigatus that are already resistant to medical azoles. Previous work has suggested that A. fumigatus likely gains resistance through environmental azole exposure in so-called hot spots. Here, we investigated A. fumigatus resistance dynamics over time at three sites at which farmers used azole fungicides for crop protection. Over 16 months, 114 samples were taken from stockpiles of decaying plant waste. A. fumigatus and azole fungicide residues were ubiquitously present in the plant waste. On average, 105A. fumigatus CFU/g was recovered, of which roughly half were itraconazole and tebuconazole resistant. Similar tandem repeat-mediated resistance mechanisms were found in colonies cultured from plant waste as reported in clinical azole-resistant isolates. Our results show a consistent high burden of azole-resistant A. fumigatus in azole-containing plant waste and underscores the need to further investigate resistance-reducing interventions and transmission routes.IMPORTANCEAspergillus fumigatus is consistently present independently on season at a high abundance in plant waste material throughout the sampling period. Our study confirmed that long-term storage sites of azole-containing decaying plant material can indeed be considered hot spots, which can sustain resistance development and maintenance in A. fumigatus Roughly half of individual isolates were azole resistant and carried genetic mutations that are highly similar to those found in patients with azole-resistant invasive aspergillosis. Our work suggests that environmental sources of azole resistance in A. fumigatus may be important, underscoring the need for further studies on environment-to-patient transmission routes.

Cross-resistance to the new fungicide mefentrifluconazole in DMI-resistant fungal pathogens.

In the European Union (EU), regulation of sterol demethylation inhibiting (DMI) fungicides is tightened due to their suspected endocrine disrupting properties. However, the new DMI fungicide mefentrifluconazole was reported to have high fungicidal activity with minimal adverse side effects. In addition, some evidence suggests inconsistent cross resistance between mefentrifluconazole and other azoles. In this study, mefentrifluconazole and other triazoles were examined for activity to select pathogens sensitive or resistant to DMIs using mycelial growth tests on fungicide-treated culture medium or spray trials using cucumber plants. Cross-resistance was confirmed for all of the fungal species tested but activity levels varied. The sensitivity of Monilinia fructicola from peach to mefentrifluconazole was higher compared to other DMIs. In contrast, the inhibitory activity of mefentrifluconazole was equal or slightly inferior compared to difenoconazole, tebuconazole, propiconazole in Colletotrichum spp., Alternaria alternaria sp. complex and Cercospora beticola isolated from peach and sugar beet, respectively. Similar tendencies (i.e. equal or slightly inferior activity and cross-resistance) were observed for cucumber powdery mildew (Podosphaera xanthii) resistant to triflumizole, myclobutanil, and difenoconazole. Despite cross-resistance to other DMIs, mefentrifluconazole is a promising fungicide for fungal disease control on peach and other crops, with a reportedly more favorable toxicity profile.

Proteome-wide assessment of human interactome as a source of capturing domain-motif and domain-domain interactions.

Protein-protein interactions (PPIs) play a crucial role in various biological processes by establishing domain-motif (DMI) and domain-domain interactions (DDIs). While the existence of real DMIs/DDIs is generally assumed, it is rarely tested; therefore, this study extensively compared high-throughput methods and public PPI repositories as sources for DMI and DDI prediction based on the assumption that the human interactome provides sufficient data for the reliable identification of DMIs and DDIs. Different datasets from leading high-throughput methods (Yeast two-hybrid [Y2H], Affinity Purification coupled Mass Spectrometry [AP-MS], and Co-fractionation-coupled Mass Spectrometry) were assessed for their ability to capture DMIs and DDIs using known DMI/DDI information. High-throughput methods were not notably worse than PPI databases and, in some cases, appeared better. In conclusion, all PPI datasets demonstrated significant enrichment in DMIs and DDIs (p-value <0.001), establishing Y2H and AP-MS as reliable methods for predicting these interactions. This study provides valuable insights for biologists in selecting appropriate methods for predicting DMIs, ultimately aiding in SLiM discovery.

DMI-Fungicide Resistance in Venturia nashicola, the Causal Agent of Asian Pear Scab-How Reliable Are Mycelial Growth Tests in Culture?

Scab, caused by Venturia nashicola, is among the most serious diseases of Asian pears and control of this disease largely relies on sterol demethylation inhibitor (DMI) fungicides. However, pear growers have complained about field performance of DMIs since the mid-2000s. In this study, to evaluate pathogen sensitivity, mycelial growth tests and inoculation tests were conducted using DMI-amended culture medium and fungicide-sprayed potted pear trees, respectively. Results confirmed distribution of isolates resistant to fenarimol, hexaconazole, and difenoconazole in the field populations. Importantly, results from tests in culture did not fully correlate with those from tests in planta. Due to phenotypic instability of resistance and poor sporulation of this pathogen in culture, resistance is generally assessed by laborious and time-consuming inoculation with conidia collected from a field. To improve the result interpretation from in vitro tests, the isolates were genotyped: the CYP51 gene which encodes the target sterol 14α-demethylase was sequenced and various mutations have been detected in the coding sequence of DMI-resistant isolates. In addition to the detected single nucleotide polymorphisms, alternative mechanisms, not based on changes in the structure of the target protein, may also increase DMI resistance. Development of molecular methods for the diagnosis of DMI resistance seems to be challenging in V. nashicola.

De novo mutations in folate-related genes associated with common developmental disorders.

Folate deficiency is an environmental risk factor for several developmental disorders. De novo mutations (DNMs) also play important etiological roles in various developmental disorders. However, it remains unclear whether DNMs in folate-related genes (FRGs) contribute to developmental disorders. We obtained a list of 1,821 FRGs from folate metabolism pathways and the Comparative Toxicogenomics Database, along with data concerning DNMs in 15,404 cases and 3,391 controls from the Gene4Denovo database. We used a TADA-Denovo model to prioritize candidate disease-associated FRGs, and characterized these genes in terms of genic intolerance, functional networks, and expression patterns. Compared with the controls, FRGs were significantly enriched in likely damaging DNMs (ldDNMs) in patients with developmental disorders (1.54 ≤ odds ratio ≤ 3.39, P adj ≤ 0.0075). Furthermore, FRGs with ldDNMs rather than with likely non-damaging DNMs (lndDNMs) overlapped significantly among the five developmental disorders included in the datasets. The TADA-Denovo model prioritized 96 candidate disease-associated FRGs, which were intolerant to genetic variants. Their functional networks mainly involved pathways associated with chromatin modification, organ development, and signal transduction pathways. DNMT3A, KMT2B, KMT2C, and YY1 emerged as hub FRGs from the protein-protein interaction network. These candidate disease-associated FRGs are preferentially expressed in the excitatory neurones during embryonic development, and in the cortex, cerebellum, striatum, and amygdala during foetal development. Overall, these findings show that DNMs in FRGs are associated with the risk of developmental disorders. Further research on these DNMs may facilitate the discovery of developmental disorder biomarkers and therapeutic targets, enabling detailed, personalized, and precise folate treatment plan.

Point Mutations in the 14-α Sterol Demethylase Cyp51A or Cyp51C Could Contribute to Azole Resistance in Aspergillus flavus.

Infections caused by Aspergillus species are being increasingly reported. Aspergillus flavus is the second most common species within this genus causing invasive infections in humans, and isolates showing azole resistance have been recently described. A. flavus has three cyp51-related genes (cyp51A, cyp51B, and cyp51C) encoding 14-α sterol demethylase-like enzymes which are the target of azole drugs. In order to study triazole drug resistance in A. flavus, three strains showing reduced azole susceptibility and 17 azole susceptible isolates were compared. The three cyp51-related genes were amplified and sequenced. A comparison of the deduced Cyp51A, Cyp51B, and Cyp51C protein sequences with other protein sequences from orthologous genes in different filamentous fungi led to a protein identity that ranged from 50% to 80%. Cyp51A and Cyp51C presented several synonymous and non-synonymous point mutations among both susceptible and non-susceptible strains. However, two amino acid mutations were present only in two resistant isolates: one strain harbored a P214L substitution in Cyp51A, and another a H349R in Cyp51C that also showed an increase of cyp51A and cyp51C gene expression compared to the susceptible strain ATCC2004304. Isolates that showed reduced in vitro susceptibility to clinical azoles exhibited a different susceptibility profile to demethylation inhibitors (DMIs). Although P214L substitution might contribute to azole resistance, the role of H349R substitution together with changes in gene expression remains unclear.

Aspergillus fumigatus population dynamics and sensitivity to demethylation inhibitor fungicides in whole-crop corn, high moisture corn and wet grain corn silages.

BACKGROUND: Aspergillus fumigatus, the causal agent of aspergillosis in humans, is commonly present as a saprophyte in various organic substrates, such as spoiled silages. Aspergillosis is generally combated with demethylation inhibitor (DMI) fungicides, but the recent appearance of resistant medical and environmental strains made current treatment strategies less reliable. The goal of this study was to determine the evolution of A. fumigatus populations during the ensiling process of whole-crop corn, high moisture corn and wet grain corn, and to monitor the sensitivity of isolates from treated and untreated fields to one medical and one agricultural DMI fungicide. RESULTS: A. fumigatus was isolated from fresh forage at harvest at rather low concentrations (102 cfu g-1 ). The low frequency lingered during the silage process (at 60 and 160 days), whereas it significantly increased during air exposure (at 7 and 14 days of air exposure). Field treatment of corn with a mixture of prothioconazole and tebuconazole did not affect the sensitivity of A. fumigatus isolates. One of 29 isolates from the untreated plot was resistant to voriconazole. A unique amino acid substitution (E427K) was detected in the cyp51A gene of 10 of 12 sequenced isolates, but was not associated with DMI resistance. CONCLUSION: A. fumigatus significantly increased during aerobic deterioration of ensilaged corn after silo opening, compared with the low presence in fresh corn and during ensiling. Field treatment of corn with DMI fungicides did not affect the sensitivity of A. fumigatus isolates collected from fresh and ensiled corn. © 2019 Society of Chemical Industry.

Paralogous Cyp51s mediate the differential sensitivity of Fusarium oxysporum to sterol demethylation inhibitors.

BACKGROUND: As a soilborne fungus, Fusarium oxysporum can cause vascular wilt in numerous economically important crops. Application of antifungal drugs is the primary method for the control of F. oxysporum. Cyp51, a key enzyme of sterol biosynthesis is the main target of sterol demethylation inhibitors. RESULTS: The F. oxysporum genome contains three paralogous CYP51 genes (named FoCYP51A, FoCYP51B and FoCYP51C) that putatively encode sterol 14α-demethylase enzymes. Each of the three genes was able to partially complement the Saccharomyces cerevisiae ERG11 mutant. Growth assays demonstrated that deletion mutants of FoCYP51B, but not FoCYP51A and FoCYP51C were significantly retarded in hyphal growth. Deletion of FoCYP51A (ΔFoCyp51A and ΔFoCyp51AC) led to increased sensitivity to 11 sterol demethylation inhibitors (DMIs). Interestingly, FoCYP51B deletion mutants (ΔFoCyp51B and ΔFoCyp51BC) exhibited significantly increased sensitivity to only four DMIs (two of which are in common with the 11 DMIs mentioned earlier). Deletion of FoCYP51C did not change DMI sensitivity of F. oxysporum. None of the three FoCYP51s are involved in F. oxysporum virulence. The sensitivity of F. oxysporum isolates increased significantly when subjected to a mixture of different subgroups of DMIs classified based on the different sensitivities of FoCYP51 mutants to DMIs compared to the individual components. CONCLUSIONS: FoCYP51A and FoCYP51B are responsible for sensitivity to different azoles. These findings have direct implications for fungicide application strategies of plant and human diseases caused by F. oxysporum. © 2018 Society of Chemical Industry.

Spatiotemporal dynamics of fungicide resistance in the wheat pathogen Zymoseptoria tritici in France.

BACKGROUND: Management of pesticide resistance is a major issue in modern agricultural systems, particularly in the context of the broader challenge of reducing pesticide use. However, such management must be adapted to resistance dynamics, which remains challenging to predict due to its dependence on many biological traits of pests, interactions with the environment and pesticide use. We retrospectively studied the evolution of reported resistances to four modes of action (benzimidazoles, quinone outside inhibitors, sterol demethylation inhibitors and succinate dehydrogenase inhibitors), in French populations of the wheat pathogen Zymoseptoria tritici. RESULTS: We used statistical models to analyse the Performance trial network data set (2004-2017; ∼ 70 locations in France yearly). They highlighted contrasting behaviours between phenotypes, for example: (i) stable spatial distributions and colonization front structures over time, and (ii) different frequency growth rates at the national scale and between regions. CONCLUSION: We provide a quantitative description of the spatiotemporal patterns of resistance evolution for fungicides with several modes of action. Moreover, we highlight some unexpected resistance dynamics in France, with major differences between the north and south. This complex pattern of resistance evolution in French populations is consistent with previous descriptions of dynamics at the European scale. These results should make it easier to anticipate evolution locally and to improve the management of resistance. © 2019 Society of Chemical Industry.

Abundance, genetic diversity and sensitivity to demethylation inhibitor fungicides of Aspergillus fumigatus isolates from organic substrates with special emphasis on compost.

BACKGROUND: Aspergillus fumigatus is a widespread fungus that colonizes dead organic substrates but it can also cause fatal human diseases. Aspergilloses are treated with demethylation inhibitor (DMI) fungicides; however, resistant isolates appeared recently in the medical and also environmental area. The present study aims at molecular characterizing and quantifying A. fumigatus in major environmental habitats and determining its sensitivity to medical and agricultural DMI fungicides. RESULTS: A. fumigatus was isolated only rarely from soil and meadow/forest organic matter but high concentrations (103 to 107 cfu/g) were detected in substrates subjected to elevated temperatures, such as compost and silage. High genetic diversity of A. fumigatus from compost was found based on SSR markers, distinguishing among fungal isolates even when coming from the same substrate sample, while subclustering was observed based on mutations in cyp51A gene. Several cyp51A amino acid substitutions were found in 15 isolates, although all isolates were fully sensitive to the tested DMI fungicides, with exception of one isolate in combination with one fungicide. CONCLUSION: This study suggests that the tested A. fumigatus isolates collected in Italy, Spain and Hungary from the fungus' major living habitats (compost) and commercial growing substrates are not potential carriers for DMI resistance in the environment. © 2017 Society of Chemical Industry.

On the Coyne and Orr-igin of species: effects of intrinsic postzygotic isolation, ecological differentiation, x chromosome size, and sympatry on Drosophila speciation.

Coyne and Orr found that mating discrimination (premating isolation) evolves much faster between sympatric than allopatric Drosophila species pairs. Their meta-analyses established that this pattern, expected under reinforcement, is common and that Haldane's rule is ubiquitous in Drosophila species divergence. We examine three possible contributors to the reinforcement pattern: intrinsic postzygotic isolation, dichotomized as to whether hybrid males show complete inviability/sterility; host-plant divergence, as a surrogate for extrinsic postzygotic isolation; and X chromosome size, whether roughly 20% or 40% of the genome is X-linked. We focus on "young" species pairs with overlapping ranges, contrasted with allopatric pairs. Using alternative criteria for "sympatry" and tests that compare either level of prezygotic isolation in sympatry or frequency of sympatry, we find no statistically significant effects associated with X chromosome size or our coarse quantifications of intrinsic postzygotic isolation or ecological differentiation. Although sympatric speciation seems very rare in animals, the pervasiveness of the reinforcement pattern and the commonness of range overlap for close relatives indicate that speciation in Drosophila is often not purely allopatric. It remains to determine whether increased premating isolation with sympatry results from secondary contact versus parapatric speciation and what drives this pattern.

Explaining Darwin's corollary to Haldane's rule: the role of mitonuclear interactions in asymmetric postzygotic isolation among toads.

We examine the basis of Darwin's corollary to Haldane's rule, which describes viability and fertility differences between F1 produced from reciprocal crosses. We analyzed asymmetries in hybrid viability from >100 reciprocal crosses involving 36 toad species to test whether relatively high rates of mitochondrial vs. nuclear evolution produce dams with systematically less viable F1 hybrid progeny. We find no such effect, suggesting a predominant role for stochastic accumulation of asymmetric epistatic incompatibilities.

Critical practical aspects in the application of liquid chromatography-mass spectrometric studies for the characterization of impurities and degradation products.

Liquid chromatography-mass spectrometry (LC-MS) is considered today as a mainstay tool for the structure characterization of minor components like impurities (IMPs) and degradation products (DPs) in drug substances and products. A multi-step systematic strategy for the purpose involves high resolution mass and multi-stage mass studies on both the drug and IMPs/DPs, followed by comparison of their fragmentation profiles. Its successful application requires consideration of many practical aspects at each step. The same are critically discussed in this review.

Protective and curative efficacy of prothioconazole against isolates of Mycosphaerella graminicola differing in their in vitro sensitivity to DMI fungicides.

BACKGROUND: Septoria leaf blotch is the most important disease of wheat in Europe. To control this disease, fungicides of the 14α-demethylase inhibitor group (DMIs) have been widely used for more than 20 years. However, resistance towards DMIs has increased rather quickly in recent years. The objective of this study was to evaluate, on plants and under controlled conditions, the protective and curative efficacy of the DMI fungicide prothioconazole against three current isolates of M. graminicola, chosen to belong to different DMI-resistant phenotypes. Fungicide efficacy was assessed by visual symptoms and by quantitative real-time polymerase chain reaction (PCR). RESULTS: With a protective fungicide application, prothioconazole was always effective against each isolate. This was in accordance with the EC50 results. However, curative efficacy differed between the isolates. It remained at a good level, between 60 and 70% against one isolate, whereas it was strongly affected by late applications from 7 days post-inoculation with the two other isolates. CONCLUSION: A protective application of prothioconazole in wheat crops could be the best strategy to keep a high efficacy against Septoria leaf blotch.