Project description:Septoria tritici blotch (STB) is caused by the ascomycete Zymoseptoria tritici and one of the predominating diseases in wheat (Triticum aestivum) in Europe. The control of STB is highly reliant on frequent fungicide applications. The primary objective of this study was to assess sensitivity levels of Z. tritici to different fungicide groups. The fungicides included in this study were epoxiconazole, prothioconazole-desthio, tebuconazole, and fluxapyroxad. A panel of 63 isolates from Estonia, Latvia, and Lithuania, and 10 isolates from Finland were tested. Fungicide sensitivity testing was carried out as a bioassay analyzing single pycnidium isolates on different fungicide concentrations. The average EC50 value in Baltic countries and Finland to epoxiconazole was high ranging from 1.04 to 2.19 ppm. For prothioconazole-desthio and tebuconazole, EC50 varied from 0.01 to 0.24 ppm, and 1.25 to 18.23 ppm, respectively. The average EC50 value for fluxapyroxad varied from 0.07 to 0.33 ppm. To explain the range of sensitivity, the samples were analyzed for CYP51 and Sdh mutations, as well as cytb G143A, CYP51 overexpression, and multidrug resistance (MDR). Frequencies of ZtCYP51 mutations D134G, V136A/C, A379G, I381V, and S524T in the Finnish-Baltic region were lower than in other European countries, but have increased compared to previous years. The frequency of cytb G143A conferring strobilurin resistance also augmented to 50-70% in the Z. tritici populations from Estonia, Finland, Latvia, and Lithuania. No Sdh mutations were found in this study, and neither strains of MDR phenotypes. However, we found a strain harboring a previously unknown transposon insertion in the promoter of the MFS1 gene, involved in drug efflux and multi-drug resistance. This new insert, however, does not confer an MDR phenotype to the strain.
Project description:The increasing emergence of fungicide-resistant pathogens requires urgent solutions for crop disease management. Here, we describe a structural investigation of new fungicides obtained by combining strobilurin and succinate dehydrogenase inhibitor pharmacophores. We identified compounds endowed with very good activity against wild-type <i>Pyricularia oryzae</i>, combined in some cases with promising activity against strobilurin-resistant strains. The first three-dimensional model of <i>P. oryzae</i> cytochrome <i>bc1</i> complex containing azoxystrobin as a ligand was developed. The model was validated with a set of commercially available strobilurins, and it well explains both the resistance mechanism to strobilurins mediated by the mutation G143A and the activity of metyltetraprole against strobilurin-resistant strains. The obtained results shed light on the key recognition determinants of strobilurin-like derivatives in the cytochrome <i>bc1</i> active site and will guide the further rational design of new fungicides able to overcome resistance caused by G143A mutation in the rice blast pathogen.
Project description:As the incidence of fungicide resistance in plant pathogens continues to increase, control of diseases and the management of resistance would be greatly aided by rapid diagnostic methods. Quantitative allele-specific PCR (ASqPCR) is an ideal technique for the in-field analysis of fungicide resistance as it can quantify the frequency of mutations in fungicide targets. We have applied this technique to the fungal pathogen Blumeria graminis f. sp. tritici (Bgt), the causal agent of wheat powdery mildew. In Australia, strobilurin-resistant Bgt was first discovered in 2016. Molecular analysis revealed a nucleotide transversion in the cytochrome b (cytb) gene in the cytochrome bc1 enzyme complex, resulting in a substitution of alanine for glycine at position 143 (G143A). We have developed an in-field ASqPCR assay that can quantify both the resistant (A143) and sensitive (G143) cytb alleles down to 1.67% in host and Bgt DNA mixtures, within 90 min of sample collection. The in situ analysis of samples collected during a survey in Tasmania revealed A143 frequencies ranging between 9-100%. Validation of the analysis with a newly developed laboratory based digital PCR assay found no significant differences between the two methods. We have successfully developed an in-field quantification method, for a strobilurin-resistant allele, by pairing the ASqPCR assay on a lightweight qPCR instrument with a quick DNA extraction method. The deployment of these type of methodologies in the field can contribute to the effective in-season management of fungicide resistance.
Project description:Ramularia leaf spot caused by the fungus <i>Ramularia collo-cygni</i>, has recently become widespread in Estonian barley fields. Currently, disease control in barley fields relies on SDHI and DMI fungicides, which might be threatened by <i>R. collo-cygni</i> isolates that are well-adapted to fungicide pressure. In a two-year study, 353 <i>R. collo-cygni</i> isolates were collected from spring barley fields in Estonia. A total of 153 <i>R. collo-cygni</i> isolates were examined for sensitivity to azoles (DMIs; prothioconazole-desthio, epoxiconazole, mefentrifluconazole) and succinate dehydrogenase inhibitors (SDHIs; boscalid, fluxapyroxad). Epoxiconazole was the least effective and a new fungicide mefentrifluconazole was the most effective DMI. Among SDHIs, fluxapyroxad was more effective than boscalid. Also, single <i>R. collo-cygni</i> isolates with high resistance to tested fungicides occurred, which could affect fungicide control of the pathogen. The entire collection of <i>R. collo-cygni</i> was analysed for mutations in fungicide target proteins. Six mutations were identified in <i>CYP51</i> gene, the most dominant being I381T, I384T, and S459C. Also, numerous point mutations in the <i>SdhC</i> gene were present. The mutation G143A in strobilurin target protein CytB dominates in over 80% of the <i>R. collo-cygni</i> population, confirming the low efficacy of strobilurin fungicides in barley disease control.
Project description:Strobilurin fungicides are widely used in agricultural production due to their broad-spectrum and fungal mitochondrial inhibitory activities. However, their massive application has restrained the growth of eukaryotic algae and increased collateral damage in freshwater systems, notably harmful cyanobacterial blooms (HCBs). In this study, a strobilurin fungicide-degrading strain, <i>Hyphomicrobium</i> sp. strain DY-1, was isolated and characterized successfully. Moreover, a novel esterase gene, <i>strH</i>, responsible for the de-esterification of strobilurin fungicides, was cloned, and the enzymatic properties of StrH were studied. For trifloxystrobin, StrH displayed maximum activity at 50°C and pH 7.0. The catalytic efficiencies (<i>k</i> <sub>cat</sub>/<i>K<sub>m</sub></i> ) of StrH for different strobilurin fungicides were 196.32 ± 2.30 μM<sup>-1</sup> · s<sup>-1</sup> (trifloxystrobin), 4.64 ± 0.05 μM<sup>-1</sup> · s<sup>-1</sup> (picoxystrobin), 2.94 ± 0.02 μM<sup>-1</sup> · s<sup>-1</sup> (pyraclostrobin), and (2.41 ± 0.19)×10<sup>-2 </sup>μM<sup>-1</sup> · s<sup>-1</sup> (azoxystrobin). StrH catalyzed the de-esterification of a variety of strobilurin fungicides, generating the corresponding parent acid to achieve the detoxification of strobilurin fungicides and relieve strobilurin fungicide growth inhibition of <i>Chlorella</i> This research will provide insight into the microbial remediation of strobilurin fungicide-contaminated environments.<b>IMPORTANCE</b> Strobilurin fungicides have been widely acknowledged as an essential group of pesticides worldwide. So far, their residues and toxic effects on aquatic organisms have been reported in different parts of the world. Microbial degradation can eliminate xenobiotics from the environment. Therefore, the degradation of strobilurin fungicides by microorganisms has also been reported. However, little is known about the involvement of enzymes or genes in strobilurin fungicide degradation. In this study, a novel esterase gene responsible for the detoxification of strobilurin fungicides, <i>strH</i>, was cloned in the newly isolated strain <i>Hyphomicrobium</i> sp. DY-1. This degradation process detoxifies the strobilurin fungicides and relieves their growth inhibition of <i>Chlorella</i>.
Project description:The ascomycete Zymoseptoria tritici is the causal agent of Septoria leaf blotch on wheat. Disease control relies mainly on resistant wheat cultivars and on fungicide applications. The fungus displays a high potential to circumvent both methods. Resistance against all unisite fungicides has been observed over decades. A different type of resistance has emerged among wild populations with multidrug-resistant (MDR) strains. Active fungicide efflux through overexpression of the major facilitator gene MFS1 explains this emerging resistance mechanism. Applying a bulk-progeny sequencing approach, we identified in this study a 519-bp long terminal repeat (LTR) insert in the MFS1 promoter, a relic of a retrotransposon cosegregating with the MDR phenotype. Through gene replacement, we show the insert as a mutation responsible for MFS1 overexpression and the MDR phenotype. Besides this type I insert, we found two different types of promoter inserts in more recent MDR strains. Type I and type II inserts harbor potential transcription factor binding sites, but not the type III insert. Interestingly, all three inserts correspond to repeated elements present at different genomic locations in either IPO323 or other Z. tritici strains. These results underline the plasticity of repeated elements leading to fungicide resistance in Z. tritici and which contribute to its adaptive potential. IMPORTANCE Disease control through fungicides remains an important means to protect crops from fungal diseases and to secure the harvest. Plant-pathogenic fungi, especially Zymoseptoria tritici, have developed resistance against most currently used active ingredients, reducing or abolishing their efficacy. While target site modification is the most common resistance mechanism against single modes of action, active efflux of multiple drugs is an emerging phenomenon in fungal populations reducing additionally fungicides' efficacy in multidrug-resistant strains. We have investigated the mutations responsible for increased drug efflux in Z. tritici field strains. Our study reveals that three different insertions of repeated elements in the same promoter lead to multidrug resistance in Z. tritici. The target gene encodes the membrane transporter MFS1 responsible for drug efflux, with the promoter inserts inducing its overexpression. These results underline the plasticity of repeated elements leading to fungicide resistance in Z. tritici.
Project description:<i>Zymoseptoria tritici</i> is the causative agent of <i>Septoria tritici</i> blotch (STB), which costs billions of dollars annually to major wheat-producing countries in terms of both fungicide use and crop loss. Agricultural pathogenic fungi have acquired resistance to most commercially available fungicide classes, and the rate of discovery and development of new fungicides has stalled, demanding new approaches and insights. Here we investigate a potential mechanism of targeting an important wheat pathogen <i>Z. tritici via</i> inhibition of <i>N</i>-myristoyltransferase (NMT). We characterize <i>Z. tritici</i> NMT biochemically for the first time, profile the <i>in vivo Z. tritici</i> myristoylated proteome and identify and validate the first <i>Z. tritici</i> NMT inhibitors. Proteomic investigation of the downstream effects of NMT inhibition identified an unusual and novel mechanism of defense against chemical toxicity in <i>Z. tritici</i> through the application of comparative bioinformatics to deconvolute function from the previously largely unannotated <i>Z. tritici</i> proteome. Research into novel fungicidal modes-of-action is essential to satisfy an urgent unmet need for novel fungicide targets, and we anticipate that this study will serve as a useful proteomics and bioinformatics resource for researchers studying <i>Z. tritici</i>.
Project description:Effects of fungicide treatments on non-target fungi in the phyllosphere are not well known. We studied community composition and dynamics of target (Puccinia striiformis) and non-target fungi in wheat that was heavily infected with yellow rust. Mycobiotas in bulk leaf samples and individual leaves were studied by metabarcoding targeting the internal transcribed spacer-1 (ITS1) region of the ribosomal DNA. The amount of yellow rust in individual samples was quantified by qPCR (quantitative PCR). In addition, septoria tritici blotch (Zymoseptoria tritici), powdery mildew (Blumeria graminis), tan spot (Pyrenophora tritici-repentis), and yellow rust (P. striiformis) were visually evaluated. We showed how fungal communities were affected by three different broad-spectrum fungicides that had been applied at different timings and doses to control Puccinia striiformis. We showed that fungal content was relatively constant even after fungicide treatments. Principal component analysis demonstrated that communities from fungicide-treated plots could be separated from the communities in non-treated plots. We observed effects of fungicide treatments on fungal communities using different dose, timing and products. Some fungi, including the target organism P. striiformis were effectively controlled by most of the fungicide applications whereas some yeasts and also P. tritici-repentis increased after treatments. We demonstrated the feasibility of using metabarcoding as a supplement to visual assessments of fungicide effects on target as well as non-target fungi.