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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.

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

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 C- orH-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.

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.

Chrysanthemum white rust disease, caused by the basidiomycete Puccinia horiana, is one of the most damaging diseases in the commercial production of Chrysanthemum × morifolium, an important ornamental cut flower crop worldwide. This pathogen, as well as the soybean rust fungus Phakopsora pachyrhizi, is exceptional among rust diseases in terms of the number of studies reporting fungicide

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.

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.