Fludioxonil Treatment Research Articles

Transcriptomic Analysis of Fludioxonil Resistance Mechanisms in Botrytis cinerea

Botrytis cinerea is a destructive plant pathogen that infects a wide range of economically important crops. Limiting pathogen infection during production and after harvest is largely dependent on fungicide applications; fungicide-resistant isolates of B. cinerea have been recovered from various hosts. Resistance of B. cinerea to fludioxonil has been associated with overexpression of transporter genes ( BcatrB and mfsM2) and mutations on histidine kinase proteins ( Bos1, Bchhk2, Bchhk17). To identify possible mechanisms associated with fludioxonil resistance, the genomic expression of three sensitive and three low-resistant isolates was studied. Overexpression of BcatrB was observed when comparing low-resistant and sensitive isolates but was not specific to the fludioxonil treatment. Seven amino acid substitutions and one deletion were identified in the transcription factor Bcmrr1 in low-resistant isolates, associated with overexpression of BcatrB. The L497 deletion, previously associated with highly resistant isolates (MDR1h), was observed in two low-resistant isolates. Other differentially expressed genes associated with transmembrane transport, oxidoreductase activity, and lipid metabolic processes could be key in understanding the fungicidal mechanism(s) of fludioxonil. Expression profiles were isolate-specific. Following fludioxonil exposure, two sensitive isolates of B. cinerea sensu stricto showed a change in gene expression levels associated with cell membrane and peroxidase activity. In one low-resistant isolate of B. cinerea group S, fludioxonil exposure resulted in the overexpression of stress response genes and MFS transporter Bcstl1; one sensitive and two low-resistant isolates showed no significant changes in gene expression profiles. This work provides insight into the effect of fludioxonil on B. cinerea and potential fungicide resistance mechanisms. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Risk assessment for resistance to fludioxonil in Corynespora cassiicola in Liaoning China

Cucumber corynespora leaf spot, caused by Corynespora cassiicola, is the primary disease of cucumber leaves in greenhouses in China. Fludioxonil is a phenylpyrrole fungicide that inhibits C. cassiicola growth. We studied the sensitivity of 170 isolates of C. cassiicola to fludioxonil and evaluated resistance risk. All of the isolates were sensitive to fludioxonil. The EC50 values ranged from 0.082 to 0.539 μg/mL with a mean of 0.207 ± 0.0053 μg/mL. Laboratory-created mutants with a high resistance factor to fludioxonil were genetically stable after 10 transfers and showed positive cross-resistance to iprodione and procymidone but not to azoxystrobin, carbendazim, pydiflumetofen, and prochloraz. There was no significant difference in mycelial growth and temperature adaptation between the mutant s and the sensitive isolates, except for pathogenicity and sporulation. The resistant isolates accumulated less glycerol than their parental isolates and were more sensitive to osmotic stress. The histidine kinase activity of the sensitive isolates was significantly inhibited compared to that of the resistant mutants. Sequence alignment of the histidine kinase gene CCos revealed that the mutants RTL4, RXM5, and RFS102 had point mutations at different sites that resulted in amino acid changes at G934E, S739F, and A825P in the CCos protein. The mutant RFS102 had an alanine deletion at site 824. After fludioxonil treatment, CCos expression by RFS20 was significantly lower than that of the parental isolate. Our findings demonstrate that C. cassiicola exhibits moderate resistance to fludioxonil.

Transcriptomic Analysis of Resistant and Wild-Type Botrytis cinerea Isolates Revealed Fludioxonil-Resistance Mechanisms.

Botrytis cinerea, the causal agent of gray mold, is one of the most destructive pathogens of cherry tomatoes, causing fruit decay and economic loss. Fludioxonil is an effective fungicide widely used for crop protection and is effective against tomato gray mold. The emergence of fungicide-resistant strains has made the control of B. cinerea more difficult. While the genome of B. cinerea is available, there are few reports regarding the large-scale functional annotation of the genome using expressed genes derived from transcriptomes, and the mechanism(s) underlying such fludioxonil resistance remain unclear. The present study prepared RNA-sequencing (RNA-seq) libraries for three B. cinerea strains (two highly resistant (LR and FR) versus one highly sensitive (S) to fludioxonil), with and without fludioxonil treatment, to identify fludioxonil responsive genes that associated to fungicide resistance. Functional enrichment analysis identified nine resistance related DEGs in the fludioxonil-induced LR and FR transcriptome that were simultaneously up-regulated, and seven resistance related DEGs down-regulated. These included adenosine triphosphate (ATP)-binding cassette (ABC) transporter-encoding genes, major facilitator superfamily (MFS) transporter-encoding genes, and the high-osmolarity glycerol (HOG) pathway homologues or related genes. The expression patterns of twelve out of the sixteen fludioxonil-responsive genes, obtained from the RNA-sequence data sets, were validated using quantitative real-time PCR (qRT-PCR). Based on RNA-sequence analysis, it was found that hybrid histidine kinase, fungal HHKs, such as BOS1, BcHHK2, and BcHHK17, probably involved in the fludioxonil resistance of B. cinerea, in addition, a number of ABC and MFS transporter genes that were not reported before, such as BcATRO, BMR1, BMR3, BcNMT1, BcAMF1, BcTOP1, BcVBA2, and BcYHK8, were differentially expressed in the fludioxonil-resistant strains, indicating that overexpression of these efflux transporters located in the plasma membranes may associate with the fludioxonil resistance mechanism of B. cinerea. All together, these lines of evidence allowed us to draw a general portrait of the anti-fludioxonil mechanisms for B. cinerea, and the assembled and annotated transcriptome data provide valuable genomic resources for further study of the molecular mechanisms of B. cinerea resistance to fludioxonil.

Toxic effects of fludioxonil on the growth, photosynthetic activity, oxidative stress, cell morphology, apoptosis, and metabolism of Chlorella vulgaris

Fludioxonil is widely used in the control of crop diseases because of its broad spectrum and high activity, but its presence is now common in waterways proximate to treated areas. This study examined the toxic effects and mechanisms of fludioxonil on the microalgal taxa Chlorella vulgaris. The results showed that fludioxonil limited the growth of C. vulgaris and the median inhibitory concentration at 96 h was 1.87 mg/L. Concentrations of 0.75 and 3 mg/L fludioxonil reduced the content of photosynthetic pigments in algal cells to different degrees. Fludioxonil induced oxidative damage by altering C. vulgaris antioxidant enzyme activities and increasing reactive oxygen species levels. Fludioxonil at 0.75 mg/L significantly increased the activity of antioxidant enzymes. The highest level of activity was 1.60 times that of the control group. Both fludioxonil treatment groups significantly increased ROS levels, with the highest increase being 1.90 times that of the control group. Transmission electron microscope showed that treatment with 3 mg/L fludioxonil for 96 h disrupted cell integrity and changed cell morphology, and flow cytometer analysis showed that fludioxonil induced apoptosis. Changes in endogenous substances indicated that fludioxonil negatively affects C. vulgaris via altered energy metabolism, biosynthesis of amino acids, and unsaturated fatty acids. This study elucidates the effects of fludioxonil on microalgae and the biological mechanisms of its toxicity, providing insights into the importance of the proper management of this fungicide.

Biological and molecular characterizations of field fludioxonil-resistant isolates of Fusarium graminearum

Fusarium head blight (FHB) predominately caused by F. graminearum, is an economical devastating disease for grain cereal crops especially on wheat. The phenylpyrrole fungicide fludioxonil exhibits excellent activity against F. graminearum and has been registered to control FHB in China. In this study, 6 fludioxonil-resistant (FludR) isolates of F. graminearum were identified from 2910 isolates collected from wheat cultivated field in Jiang Su, An Hui and Henan province of China in 2020. The sensitivity assay showed that resistance factor (RF) of FludR isolates ranges from 170.73 to >1000. In comparison with fludioxonil-sensitive (FludS) isolates, all of FludR isolates showed fitness defects in terms of mycelial growth, conidiation and virulence. Under fludioxonil treatment condition, the glycerol accumulation was obviously increased in FludS isolates, but was slightly increased in FludR isolates. Four FludR isolates exhibited increased sensitivity to osmotic stresses. Moreover, there is no positive cross-resistance between fludioxonil and other fungicides including phenamacril, carbendazim and tebuconazole. When treated with fludioxonil, the phosphorylation level of Hog1 was significantly decreased in the four FludR isolates, which was in contrast to the observation in the FludS and two FludR isolates where phosphorylation level of Hog1 was increased. Sequencing assay showed that the mutations were identified in different domains in FgOS1, FgOS2 or FgOS4 in FludR isolates. This was first reported that biological and molecular characterizations of field isolates of F. graminearum resistant to fludioxonil. The results can provide scientific directions for controlling FHB using fludioxonil.

Sensitivity of Colletotrichum nymphaeae to Six Fungicides and Characterization of Fludioxonil-Resistant Isolates in China.

Colletotrichum nymphaeae is the dominant species causing anthracnose disease of peach in China. In this study, 140 isolates of C. nymphaeae were assessed for their sensitivity to six fungicides. It was found that C. nymphaeae was highly resistant to carbendazim, procymidone, and boscalid but sensitive to pyraclostrobin and prochloraz. For fludioxonil, the fungus exhibited differential sensitivities (i.e., approximately 14% of isolates were resistant to fludioxonil and the resistance was stable). Fludioxonil-resistant isolates had a mean EC50 value of 2.2380 µg/ml, whereas the mean EC50 value was 0.0194 µg/ml in fludioxonil-sensitive isolates. The mean EC50 values of C. nymphaeae for pyraclostrobin and prochloraz were 0.0083 µg/ml and 0.002 µg/ml, respectively. No cross-resistance was observed between fungicides from different groups. Mycelial growth rate, control efficacy, and osmotic stress responses were significantly different (P < 0.05) between fludioxonil-sensitive (FluS) and -resistant (FluR) isolates, but no significant difference was observed (P > 0.05) in virulence and sporulation between FluS and FluR isolates. No mutation was detected in coding regions of the CnOs-1, Cal, Hk1, Hog1, TPI, and Mrr1 genes. Interestingly, with fludioxonil treatment, the expression of ABC transporter gene atrB was significantly overexpressed in some resistant isolates. However, overexpression of the atrB gene was not detected in one moderately and one highly resistant isolate, indicating that other unknown mechanisms may be involved. Current findings uncovered several effective chemicals and provided the foundation for designing management strategies to practically control peach anthracnose with the most effective demethylation inhibitor fungicides and quinone outside inhibitor fungicides.

Antifungal effects of low environmental risk compounds on development of pear postharvest diseases: Orchard and postharvest applications

In the main productive area of Argentina, decays caused by Botrytis cinerea, Alternaria alternata, and Alternaria spp. lead to important postharvest losses in pears during medium- and long-term storage. In view of the problems associated with the use of chemical synthetic fungicides, and attending to current demands for global production of safe food, in this work we evaluated the antimicrobial effect of different low-toxicity compounds, also known as ‘biorationals’ in the control of B. cinerea, A alternata, and Alternaria spp, postharvest diseases in pear (Pyrus communis L.). Commercial formulations of acibenzolar-s-methyl (ASM), algae extract (AE) from Ascophyllum nodosum, chitosan (CH), and potassium phosphite (KPhi) were evaluated after application on orchards (30, 60, and 90 days after full flower, and 10 days prior to harvest) and postharvest (to assess possible curative and preventive effects) in ‘Beurrè d'Anjou’ and ‘Packham's Triumph’ pear cultivars. Additionally, the antimicrobial behavior of the above biorationals was evaluated in vitro by determining their inhibitory activity (EC50) on B. cinerea and A alternata mycelial growth. Water was used as negative control, while the chemical fungicide fludioxonil was included in postharvest application experiments as positive control. Both postharvest decay control efficacy and effects on fruit quality parameters varied substantially among the biorationals assayed, depending on cultivar, time of application, and pathogen evaluated. In ‘Beurrè d'Anjou’, both in-orchard and curative postharvest applications of CH controlled the incidence of B. cinerea decays, while orchard applications of KPhi effectively controlled Alternaria spp decay incidence. In the ‘Packham's Triumph’ cultivar, both orchard and postharvest applications of ASM were effective in controlling decays caused by Alternaria spp and A. alternata, whereas orchard application of AE was effective in controlling B. cinerea incidence. Meanwhile, postharvest application of CH provided a curative effect for both B. cinerea and A. alternata infections and exhibited also preventive efficacy against the latter pathogen. ‘Beurrè d'Anjou’, but not ‘Packham's Triumph’, fruit firmness was increased (by 4–7 N) by all the compounds when applied postharvest. In most assay types and for the two cultivars, control fludioxonil treatment outperformed the efficacy of the compounds against both pathogens. For both B. cinerea and A. alternata spp, the EC50 values of the biorational compounds were considerably higher than those of fludioxonil, and in some cases they could not be determined. Thus, that although orchard control results were variable, the postharvest behavior of the biorationals evaluated was generally uniform, affording good control efficiency against A. alternata and exerting clear preventive effects in the two cultivars. The studied compounds might hence represent a useful alternative to traditional agrochemical postharvest practices, leading to a meaningful reduction in the use of chemical fungicides.

CsAtf1, a bZIP transcription factor, is involved in fludioxonil sensitivity and virulence in the rubber tree anthracnose fungus Colletotrichum siamense

In phytopathogenic fungi, the HOG MAPK pathway has roles in osmoregulation, fungicide sensitivity, and other processes. The ATF1/CREB-activating transcription factor Atf1 is a regulator that functions downstream of the HOG MAPK pathway. Here, we identified a gene, designated CsAtf1, that encodes a bZIP transcription factor in Colletotrichum siamense, which is the main pathogen that causes Colletotrichum leaf fall disease in rubber trees in China. CsAtf1 localizes to the nucleus. Its mRNA expression correlates positively with that of CsPbs2 and CsHog1 in the HOG MAPK pathway in response to activator (anisomycin), inhibitor (SB203580) and fludioxonil treatments. The CsAtf1 deletion mutant showed slightly retarded mycelial growth, small conidia, slow spore germination, and abnormal appressorium formation. This mutant showed the increased spore germination rate after fludioxonil treatment and more resistance to the fungicide fludioxonil than did the wild-type fungus. However, unlike deletion of Pbs2 or Hog1, which resulted in greater sensitivity to osmotic stress, the CsAtf1 deletion induced slightly increased resistance to osmotic stress and the cell wall stress response. The ΔCsAtf1 strain also exhibited significantly reduced virulence on rubber tree leaves. These data revealed that CsAtf1 plays a key role in the regulation of fludioxonil sensitivity and in pathogenicity regulation in C. siamense.

Downregulation of the ypdA Gene Encoding an Intermediate of His-Asp Phosphorelay Signaling in Aspergillus nidulans Induces the Same Cellular Effects as the Phenylpyrrole Fungicide Fludioxonil.

Many eukaryotic histidine-to-aspartate (His-Asp) phosphorelay systems consist of three types of signal transducers: a His-kinase (HK), a response regulator (RR), and a histidine-containing phosphotransfer intermediate (HPt). In general, the HPt acts as an intermediate between the HK and the RR and is indispensable for inducing appropriate responses to environmental stresses. In a previous study, we attempted but were unable to obtain deletion mutants of the ypdA gene in order to characterize its function in the filamentous fungus Aspergillus nidulans. In the present study, we constructed the CypdA strain in which ypdA expression is conditionally regulated by the A. nidulans alcA promoter. We constructed CypdA strains with RR gene disruptions (CypdA-sskAΔ, CypdA-srrAΔ, and CypdA-sskAΔsrrAΔ). Suppression of YpdA induced by ypdA downregulation activated the downstream HogA mitogen-activated protein kinase cascade. YpdA suppression caused severe growth defects and abnormal hyphae, with features such as enhanced septation, a decrease in number of nuclei, nuclear fragmentation, and hypertrophy of vacuoles, both regulated in an SskA-dependent manner. Fludioxonil treatment caused the same cellular responses as ypdA suppression. The growth-inhibitory effects of fludioxonil and the lethality caused by ypdA downregulation may be caused by the same or similar mechanisms and to be dependent on both the SskA and SrrA pathways.

Biotreatment and bacterial succession in an upflow immobilized cell bioreactor fed with fludioxonil wastewater.

The large quantities and the persistent nature of fungicide wastewaters have increased the efforts towards a sustainable technological solution. In this context, fludioxonil-contaminated wastewater was treated in an upflow immobilized cell bioreactor, resulting in chemical oxygen demand (COD) removal efficiency even higher than 80%, whereas the electrical conductivity (EC) of the effluent was gradually increased. Organic-F was mineralized by 94.0 ± 5.2%, which was in accordance with the high fludioxonil removal efficiency (95.4 ± 4.0%). In addition, effluent total Kjeldahl nitrogen (TKN) concentration reduced significantly during bioprocessing. A strong relationship among COD removal, TKN/total nitrogen removal, and effluent EC increase (p < 0.01) was identified. Despite the adequate aeration provided, effluent nitrite and nitrate concentrations were negligible. Illumina sequencing revealed a reduction in the relative abundances of Betaproteobacteria, Chloroflexi, Planctomycetes, and Firmicutes and an increase in the proportion of Alphaproteobacteria and Actinobacteria. A shift in bacterial communities occurred during fludioxonil treatment, resulting in the significant increase of the relative abundances of Empedobacter, Sphingopyxis, and Rhodopseudomonas (from 0.67 ± 0.13% at the start-up to 34.34 ± 1.60% at the end of biotreatment). In conclusion, the immobilized cell bioreactor permitted the proliferation of specialized activated sludge microbiota with an active role in the depuration of postharvest fungicides.

Phenylpyrrole fungicides act on triosephosphate isomerase to induce methylglyoxal stress and alter hybrid histidine kinase activity

Fludioxonil, a natural product of pyrrolnitrin, is a potent fungicide used on crops worldwide. Drug action requires the presence of a group III hybrid histidine kinase (HHK) and the high osmolarity glycerol (HOG) pathway. We have reported that the drug does not act directly on HHK, but triggers the conversion of the kinase to a phosphatase, which dephosphorylates Ypd1 to constitutively activate HOG signaling. Still, the direct drug target remains unknown and mode of action ill defined. Here, we heterologously expressed a group III HHK, dimorphism-regulating kinase 1 (Drk1) in Saccharomyces cerevisae to delineate fludioxonil’s target and action. We show that the drug interferes with triosephosphate isomerase (TPI) causing release of methylglyoxal (MG). MG activates the group III HHK and thus the HOG pathway. Drug action involved Drk1 cysteine 392, as a C392S substitution increased drug resistance in vivo. Drug sensitivity was reversed by dimedone treatment, indicating Drk1 responds in vivo to an aldehydic stress. Fludioxonil treatment triggered elevated cytosolic methylglyoxal. Likewise, methylglyoxal treatment of Drk1-expressing yeast phenocopied treatment with fludioxonil. Fludioxonil directly inhibited TPI and also caused it to release methylglyoxal in vitro. Thus, TPI is a drug target of the phenylpyrrole class of fungicides, inducing elevated MG which alters HHK activity, likely converting the kinase to a phosphatase that acts on Ypd1 to trigger HOG pathway activation and fungal cell death.

Effect of exogenous fludioxonil postharvest treatment on physiological response, physico-chemical, textural, phytochemical and sensory characteristics of pomegranate fruit

The study investigated the effects of fludioxonil (FLU) on the postharvest quality of pomegranate fruit (cv. Wonderful). Fruits were dipped in FLU concentrations (control, 150, 300 and 600 mg/L) and stored for 4 months at 5 °C and 90–95% relative humidity (RH) plus an additional 4 days at 20 °C and 65% RH. Effects of FLU were evaluated on physiological responses, quality and sensory attributes. Results showed that fruit weight loss and decay incidence were reduced by FLU treatment, with fruit treated with 600 mg/L concentration showing the best results. Fruit respiration rate was more influenced by storage duration than FLU concentration. The severity and occurrence of physiological disorders increased with storage duration but were more pronounced in fruits treated with FLU. Storage duration influenced fruit colour whereas aril colour was affected by FLU concentration. Untreated fruit showed better aril redness and chroma although treated fruit also had acceptable red colour. Chemical quality attributes of fruit juice were not significantly affected by FLU concentrations. Fruit treated with FLU had significantly (p < 0.05) higher total phenolic content during storage although lower ascorbic acid content was observed compared to untreated fruit. Treating fruit with FLU resulted in better sensory attributes with regards to crispness, juiciness and sweetness. Overall, this study showed that fruit treated with FLU at 600 mg/L had the best quality with respect to decay incidence, weight loss, total phenolics and sensory attributes.

Fludioxonil Induces Drk1, a Fungal Group III Hybrid Histidine Kinase, To Dephosphorylate Its Downstream Target, Ypd1.

Novel antifungal drugs and targets are urgently needed. Group III hybrid histidine kinases (HHKs) represent an appealing new therapeutic drug target because they are widely expressed in fungi but absent from humans. We investigated the mode of action of the widely utilized, effective fungicide fludioxonil. The drug acts in an HHK-dependent manner by constitutive activation of the HOG (high-osmolarity glycerol) pathway, but its mechanism of action is poorly understood. Here, we report a new mode of drug action that entails conversion of the HHK from a kinase into a phosphatase. We expressed Drk1 (dimorphism-regulating kinase), which is an intracellular group III HHK from the fungal pathogen Blastomyces dermatitidis, in Saccharomyces cerevisiae Drk1 engendered drug sensitivity in B. dermatitidis and conferred sensitivity upon S. cerevisiae In response to fludioxonil, Drk1 behaved as a phosphatase rather than as a kinase, leading to dephosphorylation of its downstream target, Ypd1, constitutive activation of the HOG pathway, and yeast cell death. Aspartic acid residue 1140 in the Drk1 receiver domain was required for in vivo phosphatase activity on Ypd1, and Hog1 was required for drug effect, indicating fidelity in HHK-dependent drug action. In in vitro assays with purified protein, intact Drk1 demonstrated intrinsic kinase activity, and the Drk1 receiver domain exhibited intrinsic phosphatase activity. However, fludioxonil failed to induce intact Drk1 to dephosphorylate Ypd1. We conclude that fludioxonil treatment in vivo likely acts on an upstream target that triggers HHK to become a phosphatase, which dephosphorylates its downstream target, Ypd1.

Rhizoctonia solani에 의한 인삼 모잘록병 방제용 종자처리 약제 선발

Damping-off is a critical disease on ginseng seedling, which caused by the fungal pathogen Rhizoctonia solani. The disease has been prevented by tolclofos-methyl for the last 20 years. However, the tolclofos-methyl usually detected on the harvested roots of 6-year-old ginseng. Herein, we tried to select an alternative pesticide which not only must be safe but also efficiently inhibits the fungal pathogen. Four fungicides (fludioxonil, flutolanil, pencycuron, and thifluzamide) were applied to their inhibition efficacy against the pathogen. In in vitro test, fludioxonil treatment showed 80% inhibition activity for 25 days. Thifluzamide and flutolanil showed the activity for 10 days. Pencycuron showed for 1 days. In addition, the fludioxonil was more effective to control the pathogen comparing to other three fungicides in field. The incidence of damping-off was reduced to 71% by fludioxonil treatment. The level of the fungicide residue in seedling was 0.44 mg/kg, which value will be a negligible level in final products after 5 years. Consequently, the fludioxonil is a conceivable alternative for tolclofos-methyl to cope with R. solani.

NikA/TcsC Histidine Kinase Is Involved in Conidiation, Hyphal Morphology, and Responses to Osmotic Stress and Antifungal Chemicals in Aspergillus fumigatus

The fungal high osmolarity glycerol (HOG) pathway is composed of a two-component system (TCS) and Hog1-type mitogen-activated protein kinase (MAPK) cascade. A group III (Nik1-type) histidine kinase plays a major role in the HOG pathway of several filamentous fungi. In this study, we characterized a group III histidine kinase, NikA/TcsC, in the life-threatening pathogenic fungus, Aspergillus fumigatus. A deletion mutant of nikA showed low conidia production, abnormal hyphae, marked sensitivity to high osmolarity stresses, and resistance to cell wall perturbing reagents such as congo red and calcofluor white, as well as to fungicides such as fludioxonil, iprodione, and pyrrolnitrin. None of these phenotypes were observed in mutants of the SskA response regulator and SakA MAPK, which were thought to be downstream components of NikA. In contrast, in response to fludioxonil treatment, NikA was implicated in the phosphorylation of SakA MAPK and the transcriptional upregulation of catA, dprA, and dprB, which are regulated under the control of SakA. We then tested the idea that not only NikA, but also the other 13 histidine kinases play certain roles in the regulation of the HOG pathway. Interestingly, the expression of fos1, phkA, phkB, fhk5, and fhk6 increased by osmotic shock or fludioxonil treatment in a SakA-dependent manner. However, deletion mutants of the histidine kinases showed no significant defects in growth under the tested conditions. Collectively, although the signal transduction network related to NikA seems complicated, NikA plays a crucial role in several aspects of A. fumigatus physiology and, to a certain extent, modulates the HOG pathway.

Effect of phenylpyrrole fungicide fludioxonil on morphological and physiological characteristics of Sclerotinia sclerotiorum

In this study, the effect of fludioxonil on morphological and physiological characteristics of Sclerotinia sclerotiorum has been investigated. The results indicated that fludioxonil had a strong inhibition on mycelia growth of S. sclerotiorum. After fludioxonil treatment, cell membrane permeability, glycerol content, POD and PAL activity increased markedly, but oxalate and EPS content significantly decreased. The protective and curative test of fludioxonil suggested that protective effect was better than curative either on leaves or on stems of oilseed rape. These results will increase our understanding of action mechanism of fludioxonil against S. sclerotiorum.

Orchard calcium and fungicide treatments mitigate effects of delayed postharvest fungicide applications for control of postharvest decay of pear fruit

Postharvest decay of pear fruit is commonly initiated at wounds that occur during harvest and handling, yet pears may be held in cold storage for considerable lengths of time before fungicides are applied to prevent postharvest decay. The efficacy of postharvest fungicide treatments declines as the time between harvest and fungicide treatment increases. Foliar sprays of calcium chloride applied to ‘Bosc’ pear trees beginning in mid-July and repeated every two weeks for a total of three applications slowed the increase in decay incidence observed when thiabendazole or fludioxonil treatments were delayed up to eight weeks after harvest. Increased decay incidence due to delayed postharvest fungicide application was also reduced by application of either pyraclostrobin + boscalid, thiophanate-methyl, or trifloxystrobin one week before harvest. Calcium chloride treatments followed by either of the pre-harvest fungicides provided the greatest decay suppression when postharvest fungicide treatments were delayed. Decay control by fludioxonil treatment was superior to that provided by thiabendazole when the fungicides were applied to fruit promptly after harvest, but differences between these two fungicides decreased as the delay in application approached eight weeks after harvest. A program of orchard calcium chloride followed by pre-harvest fungicide treatments can reduce overall decay incidence and mitigate the consequences of delays in postharvest fungicide treatment.

Biological control of Fusarium dry rot and other potato tuber diseases using Pseudomonas fluorescens and Enterobacter cloacae

Dry rot caused by various species of Fusarium is a disease of significant importance in potatoes. Field trials were conducted in 2005 and 2006 in New Brunswick, Canada to assess the efficacy of Pseudomonas fluorescens and Enterobacter cloacae applied as a seed treatment in suppressing Fusarium dry rot of potato ( Solanum tuberosum L.) ‘Russet Burbank’ under field conditions. In 2005, the trial consisted of five treatments namely (1) non-treated, non-inoculated control; (2) non-treated control inoculated with Fusarium sambucinum; (3) seed inoculated with F. sambucinum and treated with P. fluorescens; (4) seed inoculated with F. sambucinum and treated with E. cloacae; and (5) seed inoculated with F. sambucinum and treated with the fungicide fludioxonil. In 2006, a mustard meal treatment was added. After harvest, tubers were assessed for disease severity of dry rot in addition to other tuber diseases, including silver scurf ( Helminthosporium solani), and common scab ( Streptomyces scabiei). In addition, tubers were graded and assessed for total yield, tuber size, tuber number, and tuber weight. Significant reduction in dry rot severity was obtained with all treatments compared to the non-treated control inoculated with Fusarium sambucinum. The highest dry rot reduction averaged over the two years of the study was for the fludioxonil treatment (55.7%) followed by the treatment with mustard meal (47.5%; 2006 only), Pseudomonas fluorescens (35%) and Enterobacter cloacae (26.5%). All treatments significantly reduced the severity of common scab and silver scurf compared to the non-treated, non-inoculated control. In both years, on an average, seed treated with P. fluorescens and E. cloacae produced higher total number of tubers. Both total and marketable tuber yields were significantly higher for the E. cloacae treatment compared to the non-treated, inoculated control. The results of this study suggest that P. fluorescens, E. cloacae and mustard meal are viable options for controlling potato tuber diseases along with fludioxonil. This is the first study to investigate the effect of these bacteria on potato diseases under field settings.

Transcriptional profiling for Aspergillusnidulans HogA MAPK signaling pathway in response to fludioxonil and osmotic stress

In filamentous fungi, the His–Asp phosphorelay signaling system and HOG pathway are involved in the action of the fungicides, fludioxonil, and iprodione, as well as osmotic and oxidative stress responses. Aspergillus nidulans response regulators (RRs), SskA and SrrA, and histidine kinase (HK), NikA, are involved in the growth inhibitory effects of these fungicides. To gain further insights into the molecular basis for these signaling systems, we performed DNA microarray analyses of fludioxonil and osmotic stress responses in A. nidulans. A global expression analysis revealed that a large number of genes were modulated by fludioxonil treatment in an SskA-dependent manner, whereas SrrA hardly contributed to this modulation. The fludioxonil up-regulated or down-regulated genes (FUGs or FDGs, respectively) are also dependent on the HogA MAPK cascade. We found that the SskA–HogA pathway regulates expression of atfA gene encoding a transcription factor involved in conidia stress tolerance. From the results of microarray analyses, AtfA-dependent FUGs largely overlapped with HogA-dependent FUGs, suggesting that AtfA functions downstream of the HogA MAPK. A series of microarray analyses showed that the inferred SskA–HogA–AtfA pathway is implicated in the transcriptional response to osmotic stress as well as fludioxonil. The srrA atfA null double mutant turns off the SrrA and SskA–HogA–AtfA pathways and showed sensitivity to osmotic stress but no resistance to fludioxonil. Our data revealed that the growth inhibitory effect of fludioxonil depends on factors other than AtfA in spite of the fact that AtfA functions downstream of the HogA MAPK cascade. The complexity of the stress response in the His–Asp phosphorelay system followed by the HogA MAPK cascade is discussed.

Calcineurin, Mpk1 and Hog1 MAPK pathways independently control fludioxonil antifungal sensitivity in Cryptococcus neoformans

Fludioxonil is employed as an agricultural fungicide to control plant-pathogenic fungi such as Botrytis cinerea. Cryptococcus neoformans is a basidiomycetous human fungal pathogen that causes fatal disease in immunocompromised hosts. This paper demonstrates that three different signalling cascades regulate sensitivity of C. neoformans to fludioxonil. Fludioxonil inhibited growth of the serotype A sequence reference strain H99 but not that of the sequenced serotype D strain JEC21. In the drug-sensitive wild-type strain, fludioxonil exposure activated the Hog1 osmosensing pathway, and hog1Delta mutations conferred fludioxonil resistance. Fludioxonil treatment caused cell growth inhibition following cell swelling and cytokinesis defects in the sensitive wild-type but not in a hog1Delta mutant strain, suggesting that Hog1 activation results in morphological cellular defects. Fludioxonil exerted a fungistatic effect on the wild-type strain H99, but exhibited fungicidal activity against calcineurin mutant strains, indicating that the calcineurin pathway contributes to drug resistance in this fungus. Combination of fludioxonil and the calcineurin inhibitor FK506 synergistically inhibited C. neoformans growth. mpk1Delta MAPK mutant strains exhibited fludioxonil hypersensitivity, indicating that this pathway also contributes to drug resistance. These studies provide evidence that the broad-spectrum antifungal drug fludioxonil exerts its action via activation of the Hog1 MAPK pathway and provide insight into novel targets for synergistic antifungal drug combinations.