Fungal Crop Research Articles

Cover Feature: Synthesis and Antifungal Activities of Glucosamine Aromatic Derivatives Against Four Phytopathogenic Fungi of Crops (Chem. Biodiversity 11/2024)

Cover Feature: Synthesis and Antifungal Activities of Glucosamine Aromatic Derivatives Against Four Phytopathogenic Fungi of Crops (Chem. Biodiversity 11/2024)

Field-scale gene flow of fungicide resistance in Pyrenophora teres f. teres and the effect of selection pressure on the population structure.

The effectiveness of fungicides to control foliar fungal crop diseases is being diminished by the increasing spread of resistances to fungicides. One approach that may help to maintain efficacy is remediation of resistant populations by sensitive ones. However, the success of such approaches can be compromised by re-incursion of resistance through aerial spore dispersal; although, knowledge of localized gene flow is lacking. Here, we report on a replicated mark-release-recapture field experiment with several treatments set up to study spore-dispersal-mediated gene flow of a mutated allele that confers demethylase inhibitor resistance in Pyrenophora teres f. teres (Ptt). Artificial inoculation of the host, barley (Hordeum vulgare), was successful across the 12-ha trial, where the introduced sensitive- and resistant-populations were, respectively, 6- and 13-fold the DNA concentration of the native Ptt population. Subsequent disease pressure remained low which hampered spread of the epidemic to such extent that gene flow was not detected at, or beyond 2.5 m from source points. In the absence of gene flow, plots were assessed for treatment effects; fungicide applied to populations that contained 14.3% of allele mutation increased in frequency to 24.5%, whereas sensitive populations had no change in structure. Untreated controls of native Ptt population remained genetically stable, yet untreated controls that were inoculated with sensitive Ptt had half the resistance frequency of the native population structure. The trial demonstrates the potential for management to remediate fungicide resistant pathogen populations, where localized gene flow is minimal; to safeguard chemical crop protection into the future.

Synthesis and Antifungal Activities of Glucosamine Aromatic Derivatives Against Four Phytopathogenic Fungi of Crops.

A series of diversified glucosamine derivatives (3a-3y) was synthesized and their antifungal activity was examined against four kinds of phytopathogens, Fusarium graminearum (F. graminearum), Fusarium moniliforme (F. moniliforme), Curvularia. lunata (C. lunata), and Rhizoctonia solani (R. solani) which cause seriously economic losses worldwide by affecting crops. The compound 3o showed remarkable antifungal activity against F. graminearum with EC50 value of 3.96 μg/mL, compared to the standard drug triadimefon (10.1 μg/mL). 3D-QSAR model with the statistically recommended values (r2=0.915, q2=0.872) showed that positive charge group or bulky group in the benzyl ring was favorable for the antifungal activity. Enzyme activity assays confirmed that 3o had a moderate inhibition of trehalase with inhibition rate of 51.4 % at 5 μg/mL, which is comparable to those of commercial inhibitor validamycin A with inhibition rate of 83.3 %. Molecular docking analysis revealed that 3o also had a hydrogen bond interaction with key amino acid residue compared to validoxylamine.

Genomic Signatures of Domestication in a Fungus Obligately Farmed by Leafcutter Ants.

The naturally selected fungal crop (Leucoagaricus gongylophorus) farmed by leafcutter ants shows striking parallels with artificially selected plant crops domesticated by humans (e.g. polyploidy, engorged nutritional rewards, and dependence on cultivation). To date, poorly resolved L. gongylophorus genome assemblies based on short-read sequencing have constrained hypotheses about how millions of years under cultivation by ants shaped the fungal crop genome and potentially drove domestication. We use PacBio HiFi sequencing of L. gongylophorus from the leafcutter ant Atta colombica to identify 18 putatively novel biosynthetic gene clusters that likely cemented life as a cultivar (e.g. plant fragment degradation, ant-farmer communication, and antimicrobial defense). Comparative analyses with cultivated and free-living fungi showed genomic signatures of stepwise domestication transitions: (i) free-living to ant-cultivated: loss of genes conferring stress response and detoxification; (ii) hyphal food to engorged nutritional rewards: expansions of genes governing cellular homeostasis, carbohydrate metabolism, and siderophore biosynthesis; and (iii) detrital provisioning to freshly cut plant fragments: gene expansions promoting cell wall biosynthesis, fatty acid metabolism, and DNA repair. Comparisons across L. gongylophorus fungi farmed by 3 leafcutter ant species highlight genomic signatures of exclusively vertical clonal propagation and widespread transposable element activity. These results show how natural selection can shape domesticated cultivar genomes toward long-term ecological resilience of farming systems that have thrived across millennia.

Plant Disease: A Growing Threat to Global Food Security

The escalating global population has led to an increased demand for both quantity and quality in food production. Throughout history, plant diseases have posed significant threats to agricultural output by causing substantial food losses annually while also compromising product quality. Accurate identification of pathogens, clarifying the pathogenic mechanism of pathogens, and understanding the interaction between pathogens and hosts are important for the control of plant diseases. This Special Issue, “Research Progress on Pathogenicity of Fungi in Crops”, belongs to the section “Pest and Disease Management” of Agronomy. It contains research papers on the identification and phylogeny of fungal pathogens, the molecular genetics of plant fungal pathogens, the molecular mechanisms of fungal pathogenicity, and the molecular basis of the interaction between fungi and crops. These studies encapsulate efforts to understand disease systems within current genomics, transcriptomics, proteomics, and metabolomics studies, highlighting research findings that could be future targets for crop disease and pest control. The studies presented in this Special Issue promote the progress of fungal pathogenicity research in crops and provide a scientific basis for future disease control, which is of great significance for sustainable agricultural development and global food security.

The alleviation of difenoconazole-induced kidney injury in common carp (Cyprinus carpio) by silybin: Involvement of inflammation, oxidative stress, and apoptosis

Triazole fungicides, such as difenoconazole (DFZ), are frequently used to control fungus in crops that pollute water. The common carp (Cyprinus carpio) (hereafter referred to as “carp”) is an excellent bio-indicator of water quality. The seeds of the silymarin plant contain a flavonolignan called silybin (SYB), which is used to treat liver disease. To explore SYB's involvement in DFZ-triggered kidney damage in carps, an H&E assay was conducted, and ROS level was also examined. The results demonstrated that SYB alleviated DFZ-induced destruction of kidney tissue structure in carps, as well as alleviating the elevation of kidney ROS level in carps. RT-qPCR and Western blot were used to detect inflammation-, oxidative stress- and apoptosis-related factors at mRNA level and protein level. The experimental findings indicated that relative to the DFZ group, SYB + DFZ co-treatment reduced inflammation-related mRNA level of il-6, il-1β and tnf-α, elevated mRNA level of il-10. It also reduced protein expression levels of NF-κB and iNOS. In addition, SYB + DFZ co-treatment reduced DFZ-induced increase in the oxidative stress-related mRNA indicators sod and cat, and decreased the protein expression levels of Nrf2 and NQO1. SYB reduced the DFZ-induced increase in pro-apoptotic gene Bax mRNA and protein expression levels and the DFZ-induced decrease in anti-apoptotic gene Bcl-2 mRNA and protein expression levels. In summary, SYB potentially mitigates DFZ-induced kidney damage in carp by addressing inflammation, oxidative stress, and apoptosis. Our results establish a theoretical foundation for the clinical advancement of freshwater carp feeds.

Recent advances in virulence of a broad host range plant pathogen Sclerotinia sclerotiorum: a mini-review.

Sclerotinia sclerotiorum is a typical necrotrophic plant pathogenic fungus, which has a wide host range and can cause a variety of diseases, leading to serious loss of agricultural production around the world. It is difficult to control and completely eliminate the characteristics, chemical control methods is not ideal. Therefore, it is very important to know the pathogenic mechanism of S. sclerotiorum for improving host living environment, relieving agricultural pressure and promoting economic development. In this paper, the life cycle of S. sclerotiorum is introduced to understand the whole process of S. sclerotiorum infection. Through the analysis of the pathogenic mechanism, this paper summarized the reported content, mainly focused on the oxalic acid, cell wall degrading enzyme and effector protein in the process of infection and its mechanism. Besides, recent studies reported virulence-related genes in S. sclerotiorum have been summarized in the paper. According to analysis, those genes were related to the growth and development of the hypha and appressorium, the signaling and regulatory factors of S. sclerotiorum and so on, to further influence the ability to infect the host critically. The application of host-induced gene silencing (HIGS)is considered as a potential effective tool to control various fungi in crops, which provides an important reference for the study of pathogenesis and green control of S. sclerotiorum.

Novel Isoxazolylpyrimidine Derivatives: Design, Synthesis, Antifungal Activity and In‐Silico Studies

AbstractThe advancement of environmentally friendly chemical synthesis in both academic and corporate research has recently taken on enormous relevance. Pesticides are substances, either chemical or biological, that are used to get rid of or lessen the impact of pests. To control the fungi in crops a new set of isoxazolylpyrimidine derivatives were planned, synthesized, and characterized. The compounds indicated were synthesized in high yields, and their structures were determined using elemental analysis, 1H NMR, 13C NMR, and LCMS. HPLC was used to assess the purity of the synthesized molecules, and the purity of some compounds were found to be >98 % a/a while a couple were found to be >95 % a/a. The synthesized compounds were assessed for their antifungal activity. Among these, compounds 5d, 5h, and 5l exhibit substantial antifungal action and considerably suppress the growth of Rhizoctonia solani, Sclerotium rolfsii, and Macrophominaphaseolina. Macrophominaphaseolina inhibition percentages were 77.60, 70.49, 69.03, Sclerotium rolfsii inhibition percentages were 78.73, 74.03, 68.44, and Rhizoctonia solani inhibition percentages were 81.77, 76.29, and 71.01 for compounds 5 d, 5 h and 5 l respectively. The plausible mechanism of fungal inhibitory activities of rationally designed isoxazolyl pyrimidine derivatives is attributed to the blocking of the oxygen activation of HEME iron in membrane‐bound 14α‐demethylase. The presence of pyrimidine unit in designed materials is a vital factor for inducing antifungal activities, which was further corroborated through the density functional theory (DFT),molecular docking studies and molecular dynamics simulation studies.

Exploring sustainable management by using green nano-silver to combat three post-harvest pathogenic fungi in crops

Global crop protection and food security have become critical issues to achieve the ‘Zero Hunger’ goal in recent years, as significant crop damage is primarily caused by biotic factors. Applying nanoparticles in agriculture could enhance crop yield. Nano-silver, or AgNPs, have colossal importance in many fields like biomedical, agriculture, and the environment due to their antimicrobial potential. In this context, nano-silver was fabricated by Citrus medica L. (Cm) fruit juice, detected visually and by UV–Vis spectrophotometric analysis. Further, AgNPs were characterized by advanced techniques. UV–Vis spectroscopic analysis revealed absorbance spectra at around 487 nm. The zeta potential measurement value was noted as -23.7 mV. Spectral analysis by FT-IR proved the capping of the acidic groups. In contrast, the XRD analysis showed the Miller indices like the face-centered cubic (fcc) crystalline structure. NTA revealed a mean size of 35 nm for nano-silver with a 2.4 × 108 particles mL−1 concentration. TEM analysis demonstrated spherical Cm-AgNPs with 20–30 nm sizes. The focus of this research was to evaluate the antifungal activity of biogenic AgNPs against post-harvest pathogenic fungi, including Aspergillus niger, A. flavus, and Alternaria alternata. The Cm-AgNPs showed significant antifungal activity in the order of A. niger > A. flavus > A. alternata. The biogenic Cm-AgNPs can be used for the inhibition of toxigenic fungi.

Eicosane: An antifungal compound derived from Streptomyces sp. KF15 exhibits inhibitory potential against major phytopathogenic fungi of crops

In the present scenario, food security is of major concern due to exponentially increasing population and depleted crop production. The fungal diseases have contributed majorly to the scarcity of staple food products and economic loss worldwide. This problem could be tackled by preventing the crop loss during both pre and post-harvest seasons. During the current investigation, the bioactive compound eicosane was extracted from Streptomyces sp. KF15, subjected to purification and identified based on mass spectrometry and NMR analysis. The evaluation of in-vitro antifungal activity was done by poisoned food method, SEM analysis and growth pattern analysis. The bioactive compound eicosane with molecular weight of 282.5475 g/mol was purified by column chromatography and the straight chain hydrocarbon structure of CH3CH2(18)CH3 was elucidated by NMR analysis. In poisoned food assay, eicosane effectively inhibited the radial growth of all tested fungal pathogens; F. oxysporum was found to be the most sensitive with 24.2%, 33.3%, 42.4%, and 63.6% inhibition at 25–100 μg/ml concentrations. The SEM micrograph established clear differences in the morphology of eicosane treated fungi with damaged hyphae, flaccid mycelium and collapsed spores as compared to the tubular, turgid and entire fungi in control sample. Finally, the growth curve assay depicted the right side shift in the pattern of eicosane treated fungi indicating the delay in adapting to the conditions of growth and multiplication. The findings of this study encourage further research and development towards the novel antifungal drugs that can act against major phytopathogens.

Impact of ontogenic changes on the dynamics of a fungal crop disease model motivated by coffee leaf rust.

Ontogenic resistance has been described for many plant-pathogen systems. Conversely, coffee leaf rust, a major fungal disease that drastically reduces coffee production, exhibits a form of ontogenic susceptibility, with a higher infection risk for mature leaves. To take into account stage-dependent crop response to phytopathogenic fungi, we developed an SEIR-U epidemiological model, where U stands for spores, which differentiates between young and mature leaves. Based on this model, we also explored the impact of ontogenic resistance on the sporulation rate. We computed the basic reproduction number [Formula: see text], which classically determines the stability of the disease-free equilibrium. We identified forward and backward bifurcation cases. The backward bifurcation is generated by the high sporulation of young leaves compared to mature ones. In this case, when the basic reproduction number is less than one, the disease can persist. These results provide useful insights on the disease dynamics and its control. In particular, ontogenic resistance may require higher control efforts to eradicate the disease.

Identification of Acetomycin as an Antifungal Agent Produced by Termite Gut-Associated Streptomycetes against Pyrrhoderma noxium.

Plant fungal pathogen Pyrrhoderma noxium is responsible for the destructive and invasive disease of brown root rot currently affecting the city of Brisbane, Australia. In order to address this issue, environmentally friendly and safe alternatives to chemical control are preferred due to the city's public setting. Antifungal natural products are ideal candidates as biological control alternatives and can be detected through investigating the metabolomes of microbial symbionts. Within this study, an NMR-based metabolomics approach was applied to fermentation extracts obtained from 15 termite gut-associated streptomycetes. By analysing the NMR spectra, six of the extracts which displayed similar chemical profiles exhibited antifungal activity against the P. noxium pathogen. The major compound within these extracts was identified as acetomycin using NMR and X-ray crystallography analyses. This is the first reporting of acetomycin as a potential natural product fungicide, particularly as an antifungal agent against P. noxium. Inhibitory activity was also found against other important fungal crop pathogens, including Aspergillus niger, Botrytis cinerea, and Alteranaria alternata. Further experimentation using a woodblock test found inhibitory activity on the growth of the P. noxium pathogen for up to 3 weeks and a significant difference in the integrity of the woodblocks when conducting compression strength tests after 6 weeks. Therefore, acetomycin may be used as a biological control agent and natural product fungicide against P. noxium.

Fungal photoinactivation doses for UV radiation and visible light-a data collection.

Nearly two million people die each year from fungal infections. Additionally, fungal crop infections jeopardize the global food supply. The use of 254 nm UVC radiation from mercury vapor lamps is a disinfection technique known to be effective against all microorganisms, and there are surveys of published UVC sensitivities. However, these mainly focus on bacteria and viruses. Therefore, a corresponding overview for fungi will be provided here, including far-UVC, UVB, UVA, and visible light, in addition to the conventional 254 nm UVC inactivation. The available literature was searched for photoinactivation data for fungi in the above-mentioned spectral ranges. To standardize the presentation, the mean log-reduction doses were retrieved and sorted by fungal species, spectral range, wavelength, and medium, among others. Additionally, the median log-reduction dose was determined for fungi in transparent liquid media. Approximately 400 evaluable individual data sets from publications over the last 100 years were compiled. Most studies were performed with 254 nm radiation from mercury vapor lamps on Aspergillus niger, Candida albicans, and Saccharomyces cerevisiae. However, the data found were highly scattered, which could be due to the experimental conditions. Even though the number of individual data sets seems large, many important fungi have not been extensively studied so far. For example, UV irradiation data does not yet exist for half of the fungal species classified as "high priority" or "medium priority" by the World Health Organization (WHO). In addition, researchers should measure the transmission of their fungal suspensions at the irradiation wavelength to avoid the undesirable effects of either absorption or scattering on irradiation results.

Experimental investigation of the effect of tebuconazole on three biomarkers of innate immunity in the house sparrow (Passer domesticus).

Triazoles are among the most widely used fungicides in the world due to their efficacy against fungal crop diseases and their broad spectrum of action. Intensive use of triazoles has resulted in residual contamination in different compartments of agroecosystems and exposes non-target species to potential sublethal effects. Triazoles are known to be immunomodulators in medicine and therapeutic treatments, but very little data is available on their potential effect on immune parameters of non-target vertebrate species living in agroecosystems. In this study, we experimentally examined the impact of tebuconazole on three immune biomarkers (haemagglutination titre (HA), haemolysis titre (HL), and haptoglobin concentration (Hp)), as well as on the body condition of house sparrows (Passer domesticus). Our results suggest that tebuconazole had very little, if any, effect on the studied immune parameters. However, further studies are needed to better assess the effect of tebuconazole on bird immunity because (1) experimental individuals were kept under optimal conditions and the impact of tebuconazole on immunity may occur under suboptimal conditions, (2) only one concentration of tebuconazole was tested and its effect could be dose-dependent and (3) other complementary immunological biomarkers should be studied, given the complexity of the vertebrate immune system. Current knowledge on the potential effects of triazoles on the immunity of wild farmland vertebrates is still largely insufficient. Further physiological and immune studies should be conducted to better understand the effect of triazole fungicides on farmland birds.

Green Synthesis of Zinc Oxide Nanoparticles using Eucalyptus Lanceolata Leaf Litter in the Control of Bacterial and Fungal Crop Diseases

Nanotechnology is a significant area of scientific inquiry that has garnered considerable attention from scholars in the contemporary period. Various plant materials are being explored as potential resources in green chemistry-based techniques for producing metal Nanoparticles (NPs). This is in response to the adverse impacts of synthetic chemicals, which contribute to significant abiotic climate change concerns in modern agriculture. The primary categories of crop diseases are those resulting from bacterial and fungal infestations. Once a plant becomes infected, it can pose a significant danger to the growth of crops, resulting in a decrease in yield and quality. Furthermore, such infections can adversely affect food safety, creating a health concern for humans. This work aimed to investigate the process of Green Synthesis and Characterisation of Zinc Oxide Nanoparticles (GSC-ZnO-NP) utilizing an extract derived from Eucalyptus lanceolatus (specifically, leaf litter). The paper intended to control bacterial and fungal crop diseases using the GSC-ZnO-NP in conjunction with Eucalyptus lanceolatus extracts. The sample's crystalline structure and surface characteristics have been evaluated using X-ray Diffraction (XRD) and Scanning Electron Microscopy (SEM). The antimicrobial activity of the proposed GSC-ZnO-NP was evaluated against bacterial and fungal isolates from infected crops. The progressive augmentation in magnitude ranging from 10° to 30° in XRD signifies the emergence of ZnO-NPs characterized by distinct and well-defined crystalline configurations. Also, the antimicrobial efficacy of the Zn-NPs extract was comparable to or surpassed that of Fluconazole, suggesting the prospective use of this environmentally-friendly manufactured nanomaterial as a substitute or adjunct therapy for bacterial and fungal infections caused by these microorganisms in crops.

Molecular Targets of Herbicides and Fungicides─Are There Useful Overlaps for Fungicide Discovery?

New fungicide modes of action are needed for fungicide resistance management strategies. Several commercial herbicide targets found in fungi that are not utilized by commercial fungicides are discussed as possible fungicide molecular targets. These are acetyl CoA carboxylase, acetolactate synthase, 5-enolpyruvylshikimate-3-phosphate synthase, glutamine synthase, phytoene desaturase, protoporphyrinogen oxidase, long-chain fatty acid synthase, dihydropteroate synthase, hydroxyphenyl pyruvate dioxygenase, and Ser/Thr protein phosphatase. Some of the inhibitors of these herbicide targets appear to be either good fungicides or good leads for new fungicides. For example, some acetolactate synthase and dihydropteroate inhibitors are excellent fungicides. There is evidence that some herbicides have indirect benefits to certain crops due to their effects on fungal crop pathogens. Using a pesticide with both herbicide and fungicide activities based on the same molecular target could reduce the total amount of pesticide used. The limitations of such a product are discussed.

Recombination triggers fungal crop disease.

Recombination triggers fungal crop disease.

Evidence that the domesticated fungus Leucoagaricus gongylophorus recycles its cytoplasmic contents as nutritional rewards to feed its leafcutter ant farmers

Leafcutter ants farm a fungal cultivar (Leucoagaricus gongylophorus) that converts inedible vegetation into food that sustains colonies with up to millions of workers. Analogous to edible fruits of crops domesticated by humans, L. gongylophorus has evolved specialized nutritional rewards—swollen hyphal cells called gongylidia that package metabolites and are consumed by ant farmers. Yet, little is known about how gongylidia form, and thus how fungal physiology and ant provisioning collectively govern farming performance. We explored the process of gongylidium formation using advanced microscopy to image the cultivar at scales of nanometers, and both in vitro experiments and in silico analyses to examine the mechanisms of gongylidia formation when isolated from ant farmers. We first used transmission electron, fluorescence, and confocal microscopy imaging to see inside hyphal cells. This imaging showed that the cultivar uses a process called autophagy to recycle its own cellular material (e.g. cytosol, mitochondria) and then shuttles the resulting metabolites into a vacuole whose continual expansion displaces other organelles and causes the gongylidium cell’s bulging bulb-like appearance. We next used scanning electron microscopy and light microscopy to link this intracellular rearrangement to the external branching patterns of gongylidium cells as they clump together into edible bundles called staphyla. We next confirmed that autophagy plays a critical role in gongylidium formation both: (1) in vitro as gongylidium suppression occurred when isolated fungal cultures were grown on media with autophagy inhibitors, and (2) in silico as differential transcript expression (RNA-seq) analyses showed upregulation of multiple autophagy gene isoforms in gongylidia relative to undifferentiated hyphae. While autophagy is a ubiquitous and often highly derived process across the tree of life, our study reveals a new role for autophagy as a mechanism of functional integration between ant farmers and their fungal crop, and potentially as a signifier of higher-level homeostasis between uniquely life-time committed ectosymbionts.

Make the environment protect you from disease: elevated CO2 inhibits antagonists of the fungus-farming termite symbiosis

Fungus-farming termite colonies maintain monoculture fungus combs in underground chambers without apparent problems with diseases. Multiple lines of defense contribute to the suppression or removal of antagonists of the symbiosis, but the role of the termite-manipulated environment within mounds has yet to be tested. Specifically, termite mounds have extremely high levels of CO2 compared to atmospheric levels. We tested the effect of 5% CO2 on the growth of fungal crops from Macrotermes bellicosus colonies, generalist fungi that could challenge the symbiosis, as well as a specialist stowaway fungus, Pseudoxylaria. For sporulating fungi, we also quantified the effects on conidia production. We found that elevated CO2 significantly reduces mycelial growth and conidia production of the generalist fungi Aspergillus sp., Beauveria bassiana, and Metarhizium brunneum, whereas it overall had a net positive effect on the growth of the fungal crop Termitomyces and Pseudoxylaria; albeit, with variation between fungal strains within genera. Our findings point to elevated CO2 being of adaptive significance to the fungus-farming termite symbiosis as an additional layer of defense that helps keep termite fungus gardens free from fungal infections. The mound-building activities that make termites ecosystem engineers may thus also generate environmental conditions that impact the fate of fungi inhabiting the extended phenotypes that massive termite mounds represent.

Evaluation of single and combined effects of mancozeb and metalaxyl on the transcriptional and biochemical response of zebrafish (Danio rerio)

Mancozeb and metalaxyl are fungicidal agents frequently used in combination to control fungi in crops that may affect non-target organisms when entering ecosystems. This study aims to evaluate the environmental effects of Mancozeb (MAN) and Metalaxyl (MET), alone and in combination, on zebrafish (Danio rerio) as an experimental model. The oxidative stress biomarkers and the transcription of genes involved in detoxification in zebrafish (Danio rerio) were assessed after co-exposure to MAN (0, 5.5, and 11 μg L−1) and MET (0, 6.5, and 13 mg L−1) for 21 days. Exposure to MAN and MET induced a significant increase in the expression of genes related to detoxification mechanisms (Ces2, Cyp1a, and Mt2). Although Mt1 gene expression increased in fish exposed to 11 μg L−1 of MAN combined with 13 mg L−1 of MET, Mt1 expression was down-regulated significantly in other experimental groups (p < 0.05). The combined exposure to both fungicides showed synergistic effects in the expression levels that are manifested mainly at the highest concentration. Although a significant (p < 0.05) increase in alkaline phosphatase (ALP) and transaminases (AST and ALT), catalase activities, the total antioxidant capacity, and malondialdehyde (MDA) contents in the hepatocytes of fish exposed to MAN and MET alone and in combination was detected, lactate dehydrogenase (LDH), gamma-glutamyl transferase (GGT) activities, and hepatic glycogen content decreased significantly (p < 0.05). Overall, these results emphasize that combined exposure to MET and MAN can synergistically affect the transcription of genes involved in detoxification (except Mt1 and Mt2) and biochemical indicators in zebrafish.