Discovery and biological profile of pyridachlometyl.

Pyridachlometyl is a novel tubulin dynamics modulator fungicide developed by Sumitomo as a new agent designed to tackle fungicide resistance. Pyridachlometyl is being developed as a first-in-class molecule with an anti-tubulin mode of action, the chemical structure of which is characterized by a unique tetrasubstituted pyridazine ring. The first commercial product 'Fuseki flowable' received initial registration in 2023 in Japan. The concepts of the discovery project, optimization of chemical structures, and biological profiles are reviewed herein. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

In vitro and in planta investigation of succinate dehydrogenase inhibitor resistance conferred by target-site amino acid substitutions in Puccinia horiana, chrysanthemum white rust.

BACKGROUND: Resistance to succinate dehydrogenase inhibitor (SDHI) fungicides has been reported in some rust fungi within Pucciniales. However, measuring the resistance factors conferred by a specific substitution at the target site is difficult for most species because of the difficulty in performing in vitro experiments and the complexity of the binuclear state in these obligate parasites. We focused on Puccinia horiana because it easily forms homozygous basidiospores that are sensitive to SDHIs during in vitro germination, whereas the uredospores of other rust fungi are less sensitive. RESULTS: We identified two substitutions, SdhC-I88F and SdhD-C125Y, that drive SDHI resistance in Pu. horiana. Using basidiospore germination inhibition tests, we measured the resistance factors for six SDHI fungicides in Pu. horiana isolates harboring SdhC-I88F substitutions, wherein orthologous substitutions were most frequently observed in SDHI-resistant Pucciniales, such as soybean rust (Phakopsora pachyrhizi). The resistance factors were high for penthiopyrad and benzovindiflupyr (>150), moderate for oxycarboxin and inpyrfluxam (10-30), and low for mepronil and fluxapyroxad (3-10). The most potent SDHI against SdhC-I88F-harboring isolates was inpyrfluxam, with a half-maximal effective concentration (EC50) of 0.0082 mg L-1 owing to its high intrinsic activity. SdhD-C125Y played a minor, but significant role in increasing the resistance factors (one- to tenfold increases), depending on the individual SDHIs. CONCLUSION: This study is the first to use basidiospore germination inhibitory tests to quantify the resistance factors for SDHI-resistant Pucciniales. Owing to its homozygous binucleate nature and the high availability of basidiospores, Pu. horiana is useful for investigating SDHI resistance in Pucciniales. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Using Mass Spectrometry Imaging to Visualize Pesticide Accumulation and Time-Dependent Distribution in Fungicide-Coated Seeds.

Pesticide seed treatment provides efficient crop protection in the early season and enables a reduction in the quantity of fungicides used later. Hence, it has been a practical application for crop protection in major crop sectors such as corn, soybean, wheat, and cotton. The chemicals on pesticide-treated seeds may show different distributions depending on the structure of the seeds and the physical properties of the chemicals, but they have not been well studied because of a lack of versatile analytical tools. Here, we used mass spectrometry imaging to visualize the distribution of a fungicide (ethaboxam) in corn and soybean seeds coated with it. Contrasting distribution patterns were noted, which are likely dependent on the seed structure. We also obtained information on fungicide distribution after the seedings, which will contribute to a better understanding of the fungicide delivery pathway within plants. Using this new analytical method, we were able to obtain hitherto unavailable time-dependent, dynamic information on the ethaboxam. We expect that this method will be a useful tool with widespread applications in pesticide development and use. Copyright © 2023 Shuichi Shimma, Hiromi Saito, Takuya Inoue, and Fukumatsu Iwahashi. This is an open-access article distributed under the terms of Creative Commons Attribution Non-Commercial 4.0 International License, which permits use, distribution, and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

New Abscisic Acid Derivatives Revealed Adequate Regulation of Stomatal, Transcriptional, and Developmental Responses to Conquer Drought.

The phytohormone abscisic acid (ABA) plays an important role in plant stress response, mainly against desiccation. Hence, ABA receptor agonists may function as agents to enhance drought tolerance in crops. ABA exhibits diverse functions that impact plant development and are regulated by various ABA receptor subfamilies. Indeed, we previously reported that 3'-alkyl ABAs exhibit diverse receptor specificities and that 3'-butyl ABA induced a drought stress response without eliciting growth inhibitory effects in Arabidopsis seedlings. Thus, to further investigate plant responses induced by 3'-butyl ABA, as well as the receptors that control the opposing stress and growth responses, we designed new 3'-alkyl ABA derivatives. In addition to the 3'-alkyl chain, a cyclopropyl group was attached to position 3 of ABA to occupy the C6 cleft in the ABA-binding pocket of the receptors, which served to increase the binding affinity and specificity to a certain receptor set. Additionally, the inhibitory activity of pyrabactin resistance 1 (PYR1) and PYR1-like (PYL1) proteins against type 2C protein phosphatase increased following incorporation of the 3-cyclopropyl group in all tested 3'-alkyl ABAs. Interestingly, 3'-butyl ABA induced the highest tolerance against drought stress, compared with 3-cyclopropyl derivatives. To investigate the molecular mechanism underlying the effects elicited by different chemical treatments, those of ABA derivatives on stomatal closure, growth, and gene expression were studied. Evaluation of the receptors activated by ABA derivatives and the plant responses revealed the induction of PYR1, PYL1, PYL2, and PYL5, mediated stomatal closure, and regulated transcription, consequently leading to drought tolerance in plants.

Metabolomic analysis of Schoenoplectus juncoides reveals common markers of acetolactate synthase inhibition among paddy weeds.

Despite the increase in pressure for reducing the use of chemical pesticides in agriculture, herbicides remain one of the efficient tools for augmenting food production. Various herbicide-resistant weeds against most herbicidal modes of action (MoA) are emerging worldwide, and therefore, the necessity of developing herbicides with novel MoA is increasing. Toward this, rigid methods of determining MOA that can be applied for various weeds species are required. Despite the existence of weed species with resistance to acetolactate synthase (ALS) inhibitors, inhibition of ALS remains one of the most widely used herbicidal MoAs containing more than 50 commercial active ingredients. Here, we aimed to identify marker metabolites that are indicative of ALS inhibition. We performed non-targeted and targeted metabolomics using ALS inhibitor-treated Schoenoplectus juncoides. We identified internal metabolite markers for ALS inhibition, with excellent selectivity for ALS inhibitors and herbicides with different MOAs in various weed species. These markers will enable us to evaluate ALS inhibitory activity of chemicals in vivo in a wide variety of weed species.

Microtiter plate test using liquid medium is an alternative method for monitoring metyltetraprole sensitivity in Cercospora beticola.

BACKGROUND: Metyltetraprole is a new quinone outside inhibitor (QoI) fungicide showing potent activity against QoI-resistant fungi that possess the G143A cytochrome b mutation, which confers resistance to existing QoIs such as trifloxystrobin. For its sustainable use, monitoring of metyltetraprole sensitivity is necessary and the establishment of appropriate methodology is important in each pathogen species. RESULTS: In Cercospora beticola, the causal agent of sugar beet leaf spot, some isolates were less sensitive to metyltetraprole (EC50 > 1 mg L-1 , higher than the saturated concentration) using the common agar plate method, even with 100 mg L-1 salicylhydroxamic acid, an alternative oxidase inhibitor. However, microtiter tests (EC50 < 0.01 mg L-1 ), conidial germination tests (EC50 < 0.01 mg L-1 ) and in planta tests (>80% control at 75 mg L-1 run-off spraying) confirmed that all tested isolates were highly sensitive to metyltetraprole. For trifloxystrobin, G143A mutants were clearly resistant upon microtiter plate tests (median EC50 > 2 mg L-1 ) and distinct from wild-type isolates (median EC50 < 0.01 mg L-1 ). Notably, mycelium fragments were usable for the microtiter plate tests and the test was applicable for isolates that do not form sufficient conidia. Our monitoring study by microtiter plate tests did not indicate the presence of metyltetraprole-resistant C. beticola isolates in populations in Hokkaido, Japan. CONCLUSION: The microtiter tests were revealed to be useful for monitoring the sensitivity of C. beticola to metyltetraprole and trifloxystrobin. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Sensitivity of Botrytis cinerea to fenpyrazamine in Japan and its disease control efficacy against the low-sensitive isolate.

The baseline sensitivity of Botrytis cinerea to fenpyrazamine was evaluated using 323 isolates collected in Japan prior to its launch. In this study, the isolates were classified as "sensitive" and "low-sensitive" according to their mycelial growth on 10 mg/L fenpyrazamine. However, their EC50 values for the germ-tube elongations from conidia were not significantly different between these two classes. In both a pot test and a field trial, diseases caused by the sensitive and low-sensitive isolates were effectively controlled by fenpyrazamine.

Physiological effects of mandestrobin.

Mandestrobin is a novel and potent fungicide with a methoxyacetamide structure, and inhibits complex III on the mitochondrial respiratory chain of fungi. It is widely accepted that some fungicides, including QOIs and SDHIs, have additional physiological effects on treated plants. In this study, we evaluated the physiological effects of mandestrobin both in the field and the laboratory. Mandestrobin treatment increased the yield of Brassica napus by an average of 6.3% in the field under disease-free conditions. Mandestrobin treatment delayed chlorophyll degradation and the senescence of B. napus leaf discs, and excised Arabidopsis thaliana leaves in darkness. Analyses of transcriptome and gene ontology enrichment of mandestrobin-upregulated genes showed that chlorophyll degradation genes and jasmonate-related genes were downregulated while salicylate-related genes were upregulated by mandestrobin treatment. A possible mechanism by which mandestrobin triggered the physiological effects observed in the field and the laboratory was discussed.

Pyridachlometyl has a novel anti-tubulin mode of action which could be useful in anti-resistance management.

BACKGROUND: Fungicide resistance is a growing problem affecting many crop pathogens owing to the low success rate in finding novel chemical classes of fungicides. Pyridachlometyl is a new fungicide that seems to belong to a new chemical class of tubulin polymerization promoters. RESULTS: Pyridachlometyl exhibited potent antifungal activity against a broad range of fungal species belonging to the phyla Ascomycota and Basidiomycota. No cross-resistance was observed with other fungicide classes, such as ergosterol biosynthesis inhibitors, respiratory inhibitors, or tubulin polymerization inhibitors in Zymoseptoria tritici. Pyridachlometyl-resistant strains were obtainable by UV mutagenesis in Z. tritici and Penicillium digitatum. Mutations in tubulin-coding genes were found in all laboratory mutants but the patterns of mutation were distinct from that of tubulin polymerization inhibitors, such as benzimidazole fungicides. CONCLUSION: Pyridachlometyl is a promising new tool for disease control. However, strict resistance management strategies should be recommended for the practical use of pyridachlometyl. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Antifungal activity of metyltetraprole against the existing QoI-resistant isolates of various plant pathogenic fungi: Metyltetraprole against QoI-R isolates.

BACKGROUND: Metyltetraprole is a novel quinol oxidation site of Complex III inhibitor (QoI) fungicide that inhibits mitochondrial electron transport at the Qo site of the cytochrome bc1 complex. Previous reports have demonstrated that it is also active against the QoI-resistant (QoI-R) isolates of Zymoseptoria tritici and Pyrenophora teres with the mutations G143A and F129L in their cytochrome b gene, respectively. Further studies on cross-resistance between metyltetraprole and existing QoIs were performed using an increased number of isolates of Z. tritici, P. teres, Ramularia collo-cygni, Pyrenophora tritici-repentis, and several other plant pathogenic fungi. RESULTS: Differences in the EC50 values between the wild-type and QoI-R isolates with the mutations G143A or F129L were always smaller for metyltetraprole compared to those for the existing QoIs, and they were never greater than five in terms of resistance factor. The 2-year field experiments showed that the metyltetraprole treatment did not increase the percentage of QoI-R isolates likely to harbor the G143A mutation in a Z. tritici population. CONCLUSION: The unique behavior of metyltetraprole against the existing QoI-R isolates was confirmed for all tested pathogen species. Our results provide important information to establish a fungicide resistance management strategy using metyltetraprole in combination or alternation with other fungicides. © 2019 Society of Chemical Industry.

Discovery of metyltetraprole: Identification of tetrazolinone pharmacophore to overcome QoI resistance.

Quinone outside inhibitors (QoIs) are one of the major agricultural fungicide groups used worldwide. However, the development of resistance by different pathogenic species associated with specific mutation at the target gene site is becoming a critical issue for the sustainable use of QoIs. The authors aimed to design a novel QoI molecule to overcome the aforementioned issue. A rational approach to avoid steric hindrance between the QoI molecule and the mutated target site was successfully employed. The resulting compound, metyltetraprole, is characterized by 3-substituted central ring with a tetrazolinone moiety, the key structure to retain potent activity against QoI-resistant mutants. Metyltetraprole is a promising new fungicide under commercial development, and its development in this study has paved the way to overcoming resistance to QoI fungicides.

Abscisic Acid Derivatives with Different Alkyl Chain Lengths Activate Distinct Abscisic Acid Receptor Subfamilies.

The plant hormone abscisic acid (ABA) regulates the development of various plant organs including seeds, roots, and fruits, and significantly contributes to abiotic stress responses, especially to drought. Since recent climate changes are adversely affecting crop cultivation, enhancement of plant stress tolerance by regulation of ABA signaling would be an important strategy. In the plant genome, ABA receptors are encoded by multiple genes constituting three subfamilies; however, functional differences among them remain unclear. To enhance desired effects of ABA, the biological functions of the receptor family warrant clarification. This study aimed to determine the functional differences among ABA receptors in plants. We screened small-molecule ligands binding to specific receptors, using a chemical array. In vitro evaluation of hit compounds using 11 Arabidopsis ABA receptors revealed that (+)-3'-alkyl ABAs served as agonists for different receptors depending on the length of their 3'-alkyl chains. Combinatorial in vitro and physiological effects of these compounds on the stomata, seeds, and seedlings indicated that, along with subfamily III, receptors of subfamily II are important to induce strong drought responses. Among (+)-3'-alkyl ABAs assessed herein, (+)-3'-butyl ABA induced a transcriptional response and stomatal closure but only slightly inhibited seed germination and growth, suggesting that it enhances drought tolerance. In silico docking simulation and site-directed mutagenesis revealed the amino acid residues contributing to the selective agonist activity of the (+)-3'-alkyl ABAs. These results provide novel insights into the structure and biological effects of 3'-derivatives of ABA and a basis for agrochemical development.

Metyltetraprole, a novel putative complex III inhibitor, targets known QoI-resistant strains of Zymoseptoria tritici and Pyrenophora teres.

BACKGROUND: Metyltetraprole is a new fungicide with a unique tetrazolinone-moiety and a similar side chain to a known quinone outside inhibitor (QoI), pyraclostrobin. In this study we describe a unique bioactivity of metyltetraprole on QoI-resistant strains of Zymoseptoria tritici and Pyrenophora teres. RESULTS: Metyltetraprole exhibited potent antifungal activity against Ascomycetes; it was especially effective against Z. tritici and P. teres in seedling pot tests. Metyltetraprole was also effective in field tests with QoI-resistant mutants. Antifungal activity tests using field strains of Z. tritici and P. teres showed that the performance of metyltetraprole was unaltered by QoI, succinate dehydrogenase inhibitor (SDHI), and sterol 14α-demethylation inhibitor (DMI) resistance. However, the mitochondrial activity test indicated that the compound inhibits the respiratory chain via complex III. CONCLUSION: Metyltetraprole is a novel fungicide that is highly effective against a wide range of fungal diseases, including important cereal diseases. Although metyltetraprole most likely inhibits the respiratory chain via complex III, it remains effective against QoI resistant strains. Therefore, metyltetraprole is considered as a novel fungicidal agent for controlling diseases affecting cereal crops and overcoming pathogen resistance to existing fungicides. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Features of interactions responsible for antifungal activity against resistant type cytochrome bc1: A data-driven analysis based on the binding free energy at the atomic level.

Quinone outside inhibitors (QoIs), which inhibit the mitochondrial respiratory system by binding to the Qo site of Complex III in fungi, are widely used as pesticides with broad spectrum antifungal activity. However, excessive use of QoIs leads to pesticide resistance through mutation of amino acid residues in the Qo site. Recently, metyltetraprole, a novel QoI that is effective against wild-type and resistant mutant fungi, was developed. Interestingly, metyltetraprole has a very similar structure to other QoIs, azoxystrobin and pyraclostrobin, which do not act on resistant mutants. However, it is unknown how slight structural differences in these inhibitors alter their effectiveness towards fungi with amino acid mutations in the Qo site of Complex III. Therefore, we studied the features of interactions of inhibitors effective towards resistant mutants by quantitatively comparing the interaction profiles of three QoIs at the atomic level. First, we reproduced the binding affinity by the thermodynamic integration (TI) method, which treated explicitly environmental molecules and considered the pseudo-binding pathway. As such, a good correlation (R2 = 0.74) was observed between the binding free energy calculated using the TI method and experimentally observed pIC50 value in 12 inhibitor-target pairs, including wild-type and mutant Complex III in two fungal species, Zymoseptoria tritici and Pyrenophora teres. Trajectory analysis of this TI calculation revealed that the effectiveness against resistant mutant fungi strongly depended on the interaction of constituent parts of the inhibitor disposed near the active center of the target protein. Specifically, the key in the effectiveness against resistant mutant fungi is that the corresponding component part, tetrazolinone moiety of metyltetraprole, traded off Coulomb and van der Waals interactions in response to subtle changes in the binding pose.

Highly Accurate Detection and Identification Methodology of Xenobiotic Metabolites Using Stable Isotope Labeling, Data Mining Techniques, and Time-Dependent Profiling Based on LC/HRMS/MS.

A generally applicable method to discover xenobiotic metabolites is important to safely and effectively develop xenobiotics. We propose an advanced method to detect and identify comprehensive xenobiotic metabolites using stable isotope labeling, liquid chromatography coupled with benchtop quadrupole Orbitrap high-resolution tandem mass spectrometry (LC/HRMS/MS), data mining techniques (alignment, peak picking, and paired-peaks filtering), in silico metabolism prediction, and time-dependent profiling. The LC/HRMS analysis was carried out using Arabidopsis T87 cultured cells treated with unlabeled or with 13C- or 2H-labeled 2,4-dichlorophenoxyacetic acid (2,4-D). Paired-peak filtering enabled the accurate detection of 83 candidates for 2,4-D metabolites without any false positive peaks derived from solvents or the biological matrix. We confirmed 10 previously reported 2,4-D metabolites and identified 16 novel 2,4-D metabolites. Our method provides accurate detection and identification of comprehensive xenobiotic metabolites and represents a potentially useful tool for elucidating xenobiotic metabolism.

Melandrium yellow fleck bromovirus infects Arabidopsis thaliana and has genomic RNA sequence characteristics that are unique among bromoviruses.

Melandrium yellow fleck bromovirus (MYFV) systemically infected Arabidopsis thaliana, although the susceptibility of several A. thaliana accessions to MYFV differed from their susceptibility to the other two bromoviruses infecting A. thaliana. We constructed full-length cDNA clones of MYFV genomic RNAs 1, 2, and 3 and determined their complete nucleotide sequences. Similar to Broad bean mottle bromovirus, (1) the 5'-terminal nucleotide of the MYFV genomic RNAs was adenine, and (2) the "D-arm" was absent from the tRNA-like structure in the 3' untranslated regions (UTRs) of MYFV RNAs. As unique characteristics, MYFV RNA3 lacked the poly(A) tract in the intercistronic region and contained a directly repeated sequence of about 200 nucleotides and polypyrimidine tracts of heterogeneous lengths in the 5' UTR. Co-infection experiments using RNA3 clones with or without the duplicated sequence demonstrated that the duplication contributed to the competitive fitness of the virus in Nicotiana benthamiana.

Genetic analysis of a host determination mechanism of bromoviruses in Arabidopsis thaliana.

Brome mosaic virus (BMV) and Spring beauty latent virus (SBLV) are closely related, tripartite RNA plant viruses. In Arabidopsis thaliana, BMV shows limited multiplication whereas SBLV efficiently multiplies. Such distinct multiplication abilities have been observed commonly in all Arabidopsis accessions tested. We used this model system to analyze the molecular mechanism of viral resistance in plants at the species level. Unlike SBLV, BMV multiplication was limited even in protoplasts and a reassortment assay indicated that at least viral RNA1 and/or RNA2 determine such distinct infectivities. By screening Arabidopsis mutants with altered defense responses, we found that BMV multiplies efficiently in cpr5-2 mutant plants. This mutation specifically enhanced BMV multiplication in protoplasts, which depended on the functions of RNA1 and RNA2. In the experiment using DNA vectors to express BMV replication proteins encoded by RNA1 and RNA2, BMV RNA3 accumulation in cpr5-2 protoplasts was similar to that in wild-type Col-0 protoplasts, despite significant reduction of accumulation levels of replication proteins, suggesting that cpr5-2 mutation could enhance BMV multiplication independently of increased accumulation, therefore enhanced translation and stabilization, of the replication proteins.