Use Of Fungicides In Agriculture Research Articles

Essential oils in post-harvest disease management: Metabolic impact on Narince (Vitis vinifera L. cv) grapes against Botrytis cinerea

There is an urgent need for alternative, environmentally friendly post-harvest disease management solutions because of the growing concern over the use of chemical fungicides in agriculture and their detrimental impact on human health and the environment. This study examines the effects of thymol, eugenol, and 1,8-cineole essential oils on the sugar, organic acid, and amino acid profiles of 'Narince' grape berries as well as their effectiveness against gray mold (Botrytis cinerea). Our study has noted that the application of EOs, particularly eugenol, significantly alters the metabolic profile of 'Narince' grapes, enhancing sugar content and modifying the levels of organic acids and amino acids, which suggests a potential for improving grape quality and resistance to Botrytis cinerea. The combined use of eugenol with other EOs and fungicides, as indicated by the treatments OF and OSF, not only displayed a synergistic effect in elevating the concentration of key metabolites but also appeared to be more effective in combating fungal infection compared to individual EO applications. The PCA analysis and subsequent heatmap visualization revealed distinct metabolic clustering among the treatments, with combined EO treatments, especially those including eugenol, markedly influencing the amino acid profile of the berries. Our investigation's results suggest that EOs may offer a viable, eco-friendly alternative to traditional fungicides. Our study also adds to the body of knowledge supporting the advancement of integrated pest management strategies that are efficacious yet environmentally considerate, aligning with the global shift towards more sustainable practices in agriculture and food security.

Detection of Strobilurin Fungicides in Trout Streams within an Agricultural Watershed

The use of strobilurin fungicides in agriculture has increased steadily during the past 25 years, and although strobilurins have minimal water solubility, they regularly appear in surface waters, at times in concentrations approaching toxic levels for aquatic life. The present study examined concentrations of strobilurin fungicides in designated trout streams draining an agricultural watershed in southeastern Minnesota, USA, where fungicides may have contributed to a recent fish kill. Water samples (n = 131) were analyzed for the presence of five different strobilurin fungicides (azoxystrobin, fluoxastrobin, picoxystrobin, pyraclostrobin, trifloxystrobin). Samples were collected via grab and automated sampling during baseflow and stormflow events throughout an entire crop-growing season from sites on each of the three forks of the Whitewater River. Detection frequencies for the five strobilurins ranged from 44 to 82%. Fluoxastrobin and pyraclostrobin concentrations were above known toxic levels in 3% and 15% of total samples analyzed, respectively. The highest concentrations were detected in mid-summer (mid-June to mid-August) samples, coincident with likely strobilurin applications. Lower concentrations were present in water samples collected during the nonapplication periods in spring and fall, suggesting groundwater–stream interactions or steady leaching of fungicides from watershed soils or stream sediments. Further study is required to determine strobilurin concentrations in sediments, soils, and groundwater. Better tracking and guidance regarding strobilurin use is necessary to adequately protect aquatic life as fungicide use continues to increase.

Systemic biochemical changes in pepper (Capsicum annuum L.) against Rhizoctonia solani by kale (Brassica oleracea var. acephala L.) green manure application

BackgroundIn the search for new alternatives to avoid the problems associated with the use of synthetic chemical fungicides in agriculture, the use of green manure (GrM) could help combat fungal diseases of crops, such as those produced by the necrotrophic pathogen Rhizoctonia solani. In the case of the use of Brassica tissues as GrM, it could have an elicitor capacity for systemic plant resistance.ResultsWe used kale leaves as a GrM and applied it to pepper plants infected with R. solani. The application of freeze-dried kale tissues to the roots of pepper plants produced a systemic activation of foliar defences via the salicylic acid (SA) and ethylene (ET) pathways, significantly reducing pathogen damage. In addition, this systemic response led to the accumulation of secondary defence metabolites, such as pipecolic acid, hydroxycoumarin and gluconic acid, in leaves. Remarkably, pepper plants treated with lyophilised kale GrM accumulated glucosinolates when infected with R. solani. We also confirmed that autoclaving removed part of the glucobrassicin (85%) and sinigrin (19%) content of the kale tissues.ConclusionsGrM kale tissues can activate systemic defences in bell pepper against foliar pathogens through SA/ET hormonal pathways, accumulating secondary defence metabolites.

Detrimental consequences of tebuconazole on redox homeostasis and fatty acid profile of honeybee brain

Excessive use of azole fungicides in agriculture poses a potential threat to honeybees and other pollinator insects; however, the detailed effects of these molecules remain largely unclear. Hence, in the present study it was aimed to investigate the acute sublethal effects of tebuconazole on the redox homeostasis and fatty acid composition in the brain of honeybees. Our findings demonstrate that tebuconazole decreased total antioxidant capacity, the ratio of reduced to oxidized glutathione and disturbed the function of key antioxidant defense enzymes along with the induction of lipid peroxidation indicated by increased malondialdehyde levels, while it also altered the fatty acid profile of the brain. The present study highlights the negative impact of tebuconazole on honeybees and contributes to the understanding of potential consequences related to azole exposure on pollinator insects’ health, such as the occurrence of colony collapse disorder.

Transcriptomic Profile of Penicillium digitatum Reveals Novel Aspects of the Mode of Action of the Antifungal Protein AfpB.

Antifungal proteins (AFPs) from filamentous fungi are promising biomolecules to control fungal pathogens. Understanding their biological role and mode of action is essential for their future application. AfpB from the citrus fruit pathogen Penicillium digitatum is highly active against fungal phytopathogens, including its native fungus. Our previous data showed that AfpB acts through a multitargeted three-stage process: interaction with the outer mannosylated cell wall, energy-dependent cell internalization, and intracellular actions that result in cell death. Here, we extend these findings by characterizing the functional role of AfpB and its interaction with P. digitatum through transcriptomic studies. For this, we compared the transcriptomic response of AfpB-treated P. digitatum wild type, a ΔafpB mutant, and an AfpB-overproducing strain. Transcriptomic data suggest a multifaceted role for AfpB. Data from the ΔafpB mutant suggested that the afpB gene contributes to the overall homeostasis of the cell. Additionally, these data showed that AfpB represses toxin-encoding genes, and they suggest a link to apoptotic processes. Gene expression and knockout mutants confirmed that genes coding for acetolactate synthase (ALS) and acetolactate decarboxylase (ALD), which belong to the acetoin biosynthetic pathway, contribute to the inhibitory activity of AfpB. Moreover, a gene encoding a previously uncharacterized extracellular tandem repeat peptide (TRP) protein showed high induction in the presence of AfpB, whereas its TRP monomer enhanced AfpB activity. Overall, our study offers a rich source of information to further advance in the characterization of the multifaceted mode of action of AFPs. IMPORTANCE Fungal infections threaten human health worldwide and have a negative impact on food security, damaging crop production and causing animal diseases. At present, only a few classes of fungicides are available due to the complexity of targeting fungi without affecting plant, animal, or human hosts. Moreover, the intensive use of fungicides in agriculture has led to the development of resistance. Therefore, there is an urgent need to develop antifungal biomolecules with new modes of action to fight human-, animal-, and plant-pathogenic fungi. Fungal antifungal proteins (AFPs) offer great potential as new biofungicides to control deleterious fungi. However, current knowledge about their killing mechanism is still limited, which hampers their potential applicability. AfpB from P. digitatum is a promising molecule with potent and specific fungicidal activity. This study further characterizes its mode of action, opening avenues for the development of new antifungals.

Natural extracts against agricultural pathogens: A case study of Celtis australis L.

Plant extracts and other plant products have been used as an alternative to synthetic fungicides or an additional way to reduce their use. The choice of plant extracts and their application depends on their functional characteristics, availability, cost-effectiveness, and their impact on phytopathogens, and also on the environment. Therefore, the present study aims to assess the potential of Celtis australis methanolic extracts as source of compounds with antifungal activity. Methanolic extracts prepared from leaves and unripe mesocarps of C.australis collected from different localities of Montenegro (Podgorica-PG, Donja Gorica-DG, and Bar-BR) were evaluated for their phenolic compounds' composition as well as antifungal and cytotoxic properties. Obtained results revealed that extracts contain various bioactive constituents including phenolic acids, flavonoids, and their derivatives. The predominant phenolic acid was ferulic acid, identified in leaf samples from DG (187.97 mg/100 g dw), while isoorientin was the most abundant phenolic compound found in all examined samples. Regarding antifungal potential of the tested samples, all but one (prepared from mesocarp BR) possessed higher activity than Previcur, a commercial systemic fungicide intended to control seedlings. In vitro studies on HaCaT cell line showed that the extracts had no toxic effect toward the tested cell line. These results lead to the conclusion that methanolic extracts of C.australis can become an alternative to the use of synthetic fungicides in agriculture. Those extracts represent natural biodegradable fungicides and enable more efficient control of pathogenic fungi.

Ternary Mixture of Azoxystrobin, Boscalid and Pyraclostrobin Disrupts the Gut Microbiota and Metabolic Balance of Honeybees (Apis cerana cerana)

There is a growing risk of pollinators being exposed to multiple fungicides due to the widespread use of fungicides for plant protection. A safety assessment of honeybees exposed to multiple commonly used fungicides is urgently required. Therefore, the acute oral toxicity of the ternary mixed fungicide of ABP (azoxystrobin: boscalid: pyraclostrobin = 1:1:1, m/m/m) was tested on honeybees (Apis cerana cerana), and its sublethal effect on foragers' guts was evaluated. The results showed that the acute oral median lethal concentration (LD50) of ABP for foragers was 12.6 μg a.i./bee. ABP caused disorder of the morphological structure of midgut tissue and affected the intestinal metabolism; the composition and structure of the intestinal microbial community was perturbed, which altered its function. Moreover, the transcripts of genes involved in detoxification and immunity were strongly upregulated with ABP treatment. The study implies that exposure to a fungicide mixture of ABP can cause a series of negative effects on the health of foragers. This work provides a comprehensive understanding of the comprehensive effects of common fungicides on non-target pollinators in the context of ecological risk assessment and the future use of fungicides in agriculture.

Recommended rates of azoxystrobin and tebuconazole seem to be environmentally safe but ineffective against target fungi

The use of fungicides in agriculture has been playing a role in the enhancement of agricultural yields through the control of pathogens causing serious diseases in crops. Still, adverse environmental and human health effects resulting from its application have been reported. In this study, the possibility of readjusting the formulation of a commercial product combining azoxystrobin and tebuconazole (active ingredients – AIs; Custodia®) towards environmentally safer alternative(s) was investigated. Specifically, the sensitivity of non-target aquatic communities to each AI was first evaluated by applying the Species Sensitivity Distributions (SSDs) approach. Then, mixtures of these AIs were tested in a non-target organism (Raphidocelis subcapitata) denoting sensitivity to both AIs as assessed from SSDs. The resulting data supported the design of the last stage of this study, where mixtures of those AIs at equivalent vs. alternative ratios and rates as in the commercial formulation were tested against two target fungal species: Pyrenophora teres CBS 123929 and Rhynchosporium secalis CBS 110524. The comparison between the sensitivity of non-target aquatic species and the corresponding efficacy towards target fungi revealed that currently applied mixture and rates of these AIs are generally environmentally safe (antagonistic interaction; concentrations below the EC1 for R. subcapitata and generally below the HC5 for aquatic non-target communities), but ineffective against target organisms (maximum levels of inhibition of 70 and 50% in P. teres CBS 123929 and R. secalis CBS 110524, respectively). Results additionally suggest a potentiation of the effects of the AIs by the other formulants added to the commercial product at tested rates. Overall, this study corroborates that commercial products can be optimized during design stages based on a systematic ecotoxicological testing for ingredient interactions and actual efficacy against targets. This could be a valuable pathway to reduce environmental contamination during transition to a more sustainable agricultural production.

Fatty acid profile of the liver lipids under acute fungicide action and intake of a biologically active preparation in rats

The widespread use of fungicides in agriculture leads to a negative consequences for both humans and the environment. The acute effect of fungicides containing tebuconazole alone or in combination with triadimefon on fatty acid (FAs) composition of the total lipids in the liver of rats was studied. FAs content was analyzed by gas chromatography with the use of flame ionization detector. The results obtained indicate that the acute effect of triazole fungicides one-time oral administration to rats is characterized by toxic reactions and is accompanied by the total liver lipids FAs profile redistribution, similar for the studied fungicides. In particular, the reduction in the total content of monounsaturated FAs (by 26% – 37%) and in the ω3/ω6 ratio was observed. It was demonstrated that the subsequent two-week introduction of the preparation based on Milk thistle seeds oil extract had a positive effect and improved the FAs profile of lipids in dynamics after fungicide intake. Keywords: fatty acids, fungicides, Milk thistle seeds extract, rat liver lipids, triazoles

Fatal Fungicide-Associated Triazole-Resistant Aspergillus fumigatus Infection, Pennsylvania, USA.

We report a fatal infection in a 65-year-old immunocompromised male patient caused by pan-triazole–resistant Aspergillus fumigatus containing a TR34/L98H genetic mutation linked to agricultural fungicide use. Clinical and environmental surveillance of triazole-resistant A. fumigatus is needed in the United States to prevent spread and guide healthcare and agricultural practices.

Reduced efficacy of biocontrol agents and plant resistance inducers against potato early blight from greenhouse to field

Early blight in potato, caused by Alternaria solani, is mainly controlled by frequent applications of synthetic fungicides. Reducing the use of synthetic fungicides in agriculture is desired to reach an overall sustainable development since the active components can be harmful for humans and for the ecosystem. In integrated pest management, IPM, the idea is to combine various measures, including optimized crop management, crop rotation, use of resistant cultivars, biological control agents (BCAs), plant resistance inducers, and fertilizers, to decrease the dependence on traditional chemical fungicides. In this paper, we present the results from greenhouse and field trials where we evaluated the effect of strategies aimed at reducing our reliance on synthetic fungicides including treatments with biological control agents (BCAs) (Pythium oligandrum, Polygandron®, and Bacillus subtilis, Serenade®) and plant resistance inducers (silicon products HortiStar® and Actisil®) for early blight in potato. The agents were applied separately or in combination with each other or with synthetic fungicides. In the greenhouse, trials application of these agents resulted in 50–95% reduction of infection by A. solani, but their combination did not generally improve the outcome. However, the effects were much smaller in the hand-sprayed field trials, 20–25% disease reduction and almost disappeared in full-scale field trials where application was done with tractor sprayers. In this article, we discuss possible reasons behind the drop in efficacy from greenhouse trials to full-size field evaluation.

Control of Fungal Diseases and Fruit Yield Improvement of Strawberry Using Bacillus velezensis CE 100.

Due to the increasing health and environmental risks associated with the use of fungicides in agriculture, alternatives—such as using plant growth-promoting bacteria (PGPB) to suppress phytopathogens—that simultaneously improve plant yield, are important. This study evaluated the biocontrol efficiency of Bacillus velezensis CE100 against Macrophomina phaseolina and Fusarium oxysporum f. sp. fragariae, the respective causal agents for charcoal rot and fusarium wilt diseases in strawberry, and its potential to enhance strawberry growth and fruit production. B. velezensis CE 100 produced fungal cell wall-degrading enzymes, chitinases, and β-1,3-glucanases; and inhibited the mycelial growth of M. phaseolina and F. oxysporum f. sp. fragariae by 64.7% and 55.2%, respectively. The mycelia of both phytopathogenic fungi showed severe swelling and rupturing of the hyphae compared to the smooth, normal growth in the control group. Moreover, B. velezensis CE100 produced up to 2.8 units/mL of indole-3-acetic acid (IAA) during incubation and enhanced root biomass in strawberries. Consequently, B. velezensis CE 100 not only increased the fruit yield of strawberries by controlling the fungal diseases but also through enhancing plant growth. The findings of this study indicate that B. velezensis CE100 could be a safe, ecofriendly biocontrol alternative to chemical fungicides in strawberry production.

Phylogenetic Distribution of csp1 Types in Aspergillus fumigatus and Their Correlates to Azole Antifungal Drug Resistance.

ABSTRACTIn Aspergillus fumigatus, the repetitive region of the csp1 gene is one of the most frequently used loci for intraspecies typing of this human pathogenic mold. Using PCR amplification and Sanger sequencing of only a single marker, csp1 typing is readily available to most laboratories and highly reproducible. Here, I evaluate the usefulness of the csp1 marker for resistance detection and epidemiologic stratification among A. fumigatus isolates. After resolving nomenclature conflicts from published studies and adding novel csp1 types, the number of known types now adds up to 38. Their distribution mostly correlates with A. fumigatus population structure, and they are also meaningful for narrowly defined cases of azole resistance phenotypes. Isolates carrying the pandemic resistance allele TR34/L98H show signs of interclade crossing of strains with t02 or t04A, into the t11 clade. Furthermore, absolute differences in voriconazole MIC values between t02/t04B versus t11 TR34/L98H isolates indicate that the genetic background of resistance mutations may have a pivotal role in cross-resistance phenotypes and, thus, clinical outcome and environmental selection. Despite the general genetic similarity of isolates with identical csp1 types, outcrossing into other clades is also observed. The csp1 type alone, therefore, does not sufficiently discriminate genetic clades to be used as the sole marker in epidemiologic studies.IMPORTANCE Aspergillus fumigatus is a ubiquitously distributed saprophytic mold and a leading cause of invasive aspergillosis in human hosts. Pandemic azole-resistant strains have emerged on a global scale, which are thought to be propagated through use of azole-based fungicides in agriculture. To perform epidemiologic studies, genetic typing of large cohorts is key. Here, I evaluate the usefulness of the frequently used csp1 marker for resistance detection and epidemiologic stratification among A. fumigatus isolates. The phylogenetic distribution of csp1 types mostly correlates with A. fumigatus population structure and is also meaningful for narrowly defined cases of azole resistance phenotypes. Nevertheless, outcrossing of csp1 into other clades is also observed. The csp1 type alone, therefore, does not sufficiently discriminate genetic clades and should not be used as the sole marker in epidemiologic studies.

Climate Change influence in fungi

Abstract The International Panel on Climate Change (IPCC, 2013) and almost the entire scientific community agreed to consider the Mediterranean Area as one of the most vulnerable regions in the world to the impacts of global warming. In this scenario, heat waves, defined by the World Meteorological Organization as five or more consecutive days of heat reaching a daily maximum temperature at least 5 °C higher than the average maximum temperature, have been recently foreseen to be particularly frequent and intense in Portugal. As a consequence, agriculture constitutes one of the most sensitive sectors that could be affected by the climate change. Among the xenobiotics contaminating agricultural crops, mycotoxins (MY) are the most challenging since their presence represents an economic burden due to crops loss and a serious health effects related for animal and human with severe repercussions. Many fungal plant pathogens species from Aspergillus and Fusarium genera produce MY harmful to animal and human health. The contamination of cereals grains is commonly reported, and the climate change scenario can also influence significantly the contamination during postharvest phase having implications in food security and safety aspects. Additionally, it is foreseen that specific species increase due to climate change leading to higher use of azole-based fungicides. Azole-based fungicides are the most used antifungals to control fungi and MY production in crops. However, excessive and long-term use of azole fungicides in agriculture has led to the emergence of acquired azole resistance in some plant pathogenic fungi and also trigger in potential pathogenic fungi for humans and the toxigenic potential. Overall, climate changes can trigger crops contamination by toxigenic fungal species, increase human exposure to mycotoxins, promote the acquired azole resistance and, consequently, the occurence of life-threatening infections.

Azole resistance in Aspergillus species: promising therapeutic options

ABSTRACT Introduction Azoles are the first-line antifungal agents used for the treatment of Aspergillus infection. There is an increasing concern for azole resistance all over the world mainly from agricultural fungicide use. Choosing safe and effective antifungal regimens has become a challenge. Areas covered Here, the authors review the epidemiology, mechanisms, and detection of azole resistance along with management options for azole-resistant Aspergillus infection, including new antifungal agents under development. Expert opinion Routine global epidemiological surveillance is required to understand azole resistance prevalence. Azole-resistant Aspergillus infections are associated with high mortality. No good therapeutic options are currently available. High index of suspicion of resistance is required if a patient is not responding to 4–7 days of azole therapy, particularly in the areas of resistance. Susceptibility testing for Aspergillus is not routinely available in many parts of the world, which makes it difficult to diagnose azole resistance in Aspergillus infection. There are several new antifungal classes with novel mechanisms of action; clinical trials are ongoing.

Potential absorption of mercury-contaminated substrate by Trichoderma sp isolated from Brazil Nuts and Amazon Soil

Mercury is an inorganic contaminant with serious harmful consequences to the environment. There has been a continuous rise in its level due to industrialization and other anthropogenic activities, such as the burning of coal and petroleum products, use of mercurial fungicides in agriculture and mercury catalyst in industries, and production of waste by paper industries. Five strains of Trichoderma sp., a filamentous fungi, were used in this study to evaluate their resistance to high concentrations of mercury for the purpose of using them for bioremediation. The solid culture medium used was prepared with malt agar 2% with pH 7.0 in which the strains of Trichoderma sp. were inoculated. The minimum inhibitory concentration (MIC) of the selected Trichoderma sp. isolates was calculated considering the time for growth and concentration of the mercury salt (Hg(NO3)2). At a mercury concentration of 50 mg/mL, maximum growth was first observed in TCH 1 (89.42 ± 0.63 mm) followed by TCH 2 isolate (87.33 ± 0.58 mm). At this concentration, all isolates reached the maximum mycelia growth. When the concentration of 200 mg/L Hg(NO3)2 was used, complete growth inhibition of the isolates was observed. Scanning electron microscopy suggested that differences in sporulation between the control and mercury treatment groups. In conclusion, it can be stated that Trichoderma isolates have great potential for bioremediation of sources contaminated with mercury.

Mutations introduced in susceptibility genes through CRISPR/Cas9 genome editing confer increased late blight resistance in potatoes

The use of pathogen-resistant cultivars is expected to increase yield and decrease fungicide use in agriculture. However, in potato breeding, increased resistance obtained via resistance genes (R-genes) is hampered because R-gene(s) are often specific for a pathogen race and can be quickly overcome by the evolution of the pathogen. In parallel, susceptibility genes (S-genes) are important for pathogenesis, and loss of S-gene function confers increased resistance in several plants, such as rice, wheat, citrus and tomatoes. In this article, we present the mutation and screening of seven putative S-genes in potatoes, including two DMR6 potato homologues. Using a CRISPR/Cas9 system, which conferred co-expression of two guide RNAs, tetra-allelic deletion mutants were generated and resistance against late blight was assayed in the plants. Functional knockouts of StDND1, StCHL1, and DMG400000582 (StDMR6-1) generated potatoes with increased resistance against late blight. Plants mutated in StDND1 showed pleiotropic effects, whereas StDMR6-1 and StCHL1 mutated plants did not exhibit any growth phenotype, making them good candidates for further agricultural studies. Additionally, we showed that DMG401026923 (here denoted StDMR6-2) knockout mutants did not demonstrate any increased late blight resistance, but exhibited a growth phenotype, indicating that StDMR6-1 and StDMR6-2 have different functions. To the best of our knowledge, this is the first report on the mutation and screening of putative S-genes in potatoes, including two DMR6 potato homologues.

Extensive Genetic Diversity and Widespread Azole Resistance in Greenhouse Populations of Aspergillus fumigatus in Yunnan, China.

ABSTRACTAspergillus fumigatus is the main cause of invasive aspergillosis (IA) with a high annual global incidence and mortality rate. Recent studies have indicated an increasing prevalence of azole-resistant A. fumigatus (ARAF) strains, with agricultural use of azole fungicides as a potential contributor. China has an extensive agricultural production system and uses a wide array of fungicides for crop production, including in modern growth facilities such as greenhouses. Soils in greenhouses are among the most intensively cultivated. However, little is known about the occurrence and distribution of ARAF in greenhouse soils. Here, we investigated genetic variation and triazole drug susceptibility in A. fumigatus from greenhouses around metropolitan Kunming in Yunnan, southwest China. Abundant allelic and genotypic variations were found among 233 A. fumigatus strains isolated from nine greenhouses in this region. Significantly, ∼80% of the strains were resistant to at least one medical triazole drug, with >30% showing cross-resistance to both itraconazole and voriconazole. Several previously reported mutations associated with triazole resistance in the triazole target gene cyp51A were also found in our strains, with a strong positive correlation between the frequency of mutations at the cyp51A promoter and that of voriconazole resistance. Phylogenetic analyses of cyp51A gene sequences showed evidence for multiple independent origins of azole-resistant genotypes of A. fumigatus in these greenhouses. Evidence for multiple origins of azole resistance and the widespread distributions of genetically very diverse triazole-resistant strains of A. fumigatus in greenhouses calls for significant attention from public health agencies.IMPORTANCE The origin and prevalence of azole-resistant Aspergillus fumigatus have been attracting increasing attention from biologists, clinicians, and public health agencies. Current evidence suggests agricultural fungicide use as a major cause. In southwest China, greenhouses are used to produce large amounts of fruits, flowers, and vegetables for consumers throughout China as well as those in other countries, primarily in southeast Asia. Here, we found a very high frequency (∼80%) of triazole-resistant A. fumigatus in our sample, the highest reported so far, with a significant proportion of these strains resistant to both tested agricultural fungicides and medical triazole drugs. In addition, we found novel allelic and genotypic diversities and evidence for multiple independent origins of azole-resistant genotypes of A. fumigatus in greenhouse populations in this region. Our study calls for a systematic evaluation of the effects of azole fungicide usage in greenhouses on human health.

Prevalence of Azole-Resistant Aspergillus fumigatus is Highly Associated with Azole Fungicide Residues in the Fields.

Triazole resistance in Aspergillus fumigatus is a growing public health concern. In addition to its emergence in the therapy of invasive aspergillosis by triazole medicines, it has been frequently detected in agricultural fields all over the world. Here, we explore the potential link between residues of azole fungicides with similar chemical structure to triazole medicines in soil and the emergence of resistant A. fumigatus (RAF) through 855 500 km2 monitoring survey in Eastern China covering 6 provinces. In total, 67.3%, 15.2%, 12.3%, 2.9%, 1.5%, 0.4%, and 0.3% of the soil samples contained these five fungicides (tebuconazole, difenoconazole, propiconazole, hexaconazole, and prochloraz) of 0-100, 100-200, 200-400, 400-600, 600-800, 800-1000, and >1000 ng/g, respectively. The fractions of samples containing RAF isolates were 2.4%, 5.2%, 6.4%, 7.7%, 7.4%, 14.3%, and 20.0% of the samples with total azole fungicide residues of 0-100, 100-200, 200-400, 400-600, 600-800, 800-1000, and >1000 ng/g, respectively. We find that the prevalence of RAFs is positively (P < 0.0001) correlated with residual levels of azole fungicides in soils. Our results suggest that the use of azole fungicides in agriculture should be minimized and the intervals between treatments expanded to reduce the selective pressure toward the development of resistance in A. fumigatus in agricultural fields.

Effects of Agricultural Fungicide Use on Aspergillus fumigatus Abundance, Antifungal Susceptibility, and Population Structure.

Antibiotic resistance is an increasing threat to human health. In the case of Aspergillus fumigatus, which is both an environmental saprobe and an opportunistic human fungal pathogen, resistance is suggested to arise from fungicide use in agriculture, as the azoles used for plant protection share the same molecular target as the frontline antifungals used clinically. However, limiting azole fungicide use on crop fields to preserve their activity for clinical use could threaten the global food supply via a reduction in yield. In this study, we clarify the link between azole fungicide use on crop fields and resistance in a prototypical human pathogen through systematic soil sampling on farms in Germany and surveying fields before and after fungicide application. We observed a reduction in the abundance of A. fumigatus on fields following fungicide treatment in 2017, a finding that was not observed on an organic control field with only natural plant protection agents applied. However, this finding was less pronounced during our 2018 sampling, indicating that the impact of fungicides on A. fumigatus population size is variable and influenced by additional factors. The overall resistance frequency among agricultural isolates is low, with only 1 to 3% of isolates from 2016 to 2018 displaying resistance to medical azoles. Isolates collected after the growing season and azole exposure show a subtle but consistent decrease in susceptibility to medical and agricultural azoles. Whole-genome sequencing indicates that, despite the alterations in antifungal susceptibility, fungicide application does not significantly affect the population structure and genetic diversity of A. fumigatus in fields. Given the low observed resistance rate among agricultural isolates as well the lack of genomic impact following azole application, we do not find evidence that azole use on crops is significantly driving resistance in A. fumigatus in this context.IMPORTANCE Antibiotic resistance is an increasing threat to human health. In the case of Aspergillus fumigatus, which is an environmental fungus that also causes life-threatening infections in humans, antimicrobial resistance is suggested to arise from fungicide use in agriculture, as the chemicals used for plant protection are almost identical to the antifungals used clinically. However, removing azole fungicides from crop fields threatens the global food supply via a reduction in yield. In this study, we survey crop fields before and after fungicide application. We find a low overall azole resistance rate among agricultural isolates, as well as a lack of genomic and population impact following fungicide application, leading us to conclude azole use on crops does not significantly contribute to resistance in A. fumigatus.