Characterization of the mechanism of action of the fungicide fenpicoxamid and its metabolite UK-2A.

BACKGROUND: Fenpicoxamid is a new fungicide for control of Zymoseptoria tritici, and is a derivative of the natural product UK-2A. Its mode of action and target site interactions have been investigated. RESULTS: UK-2A strongly inhibited cytochrome c reductase, whereas fenpicoxamid was much less active, consistent with UK-2A being the fungicidally active species generated from fenpicoxamid by metabolism. Both compounds caused rapid loss of mitochondrial membrane potential in Z. tritici spores. In Saccharomyces cerevisiae, amino acid substitutions N31K, G37C and L198F at the Qi quinone binding site of cytochrome b reduced sensitivity to fenpicoxamid, UK-2A and antimycin A. Activity of fenpicoxamid was not reduced by the G143A exchange responsible for strobilurin resistance. A docking pose for UK-2A at the Qi site overlaid that of antimycin A. Activity towards Botrytis cinerea was potentiated by salicylhydroxamic acid, showing an ability of alternative respiration to mitigate activity. Fungitoxicity assays against Z. tritici field isolates showed no cross-resistance to strobilurin, azole or benzimidazole fungicides. CONCLUSION: Fenpicoxamid is a Qi inhibitor fungicide that provides a new mode of action for Z. tritici control. Mutational and modeling studies suggest that the active species UK-2A binds at the Qi site in a similar, but not identical, fashion to antimycin A. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Alveolarides: Antifungal Peptides from Microascus alveolaris Active against Phytopathogenic Fungi.

Three novel cyclodepsipeptides, alveolarides A (1), B (2), and C (3), each possessing the rare 2,3-dihydroxy-4-methyltetradecanoic acid unit and a ß-phenylalanine amino acid residue, along with the known peptide scopularide were isolated and identified from the culture broth of Microascus alveolaris strain PF1466. The pure compounds were evaluated for biological activity, and alveolaride A (1) provided strong in vitro activity against the plant pathogens Pyricularia oryzae, Zymoseptoria tritici, and Ustilago maydis. Moderate activity of alveolaride A was observed under in planta conditions against Z. tritici, Puccinia triticina, and Phakopsora pachyrhizi. Structures of 1, 2, and 3 were determined by detailed analysis of NMR (1D and 2D) and mass spectrometry data. The partial absolute configuration of alveolaride A (1) was established.

MALDI-MS Imaging Analysis of Fungicide Residue Distributions on Wheat Leaf Surfaces.

Improved retention and distribution of agrochemicals on plant surfaces is an important attribute in the biological activity of pesticide. Although retention of agrochemicals on plants after spray application can be quantified using traditional analytical techniques including LC or GC, the spatial distribution of agrochemicals on the plants surfaces has received little attention. Matrix assisted laser desorption/ionization (MALDI) imaging technology has been widely used to determine the distribution of proteins, peptides and metabolites in different tissue sections, but its application to environmental research has been limited. Herein, we probed the potential utility of MALDI imaging in characterizing the distribution of three commercial fungicides on wheat leaf surfaces. Using this MALDI imaging method, we were able to detect 500 ng of epoxiconazole, azoxystrobin, and pyraclostrobin applied in 1 µL drop on the leaf surfaces using MALDI-MS. Subsequent dilutions of pyraclostrobin revealed that the compound can be chemically imaged on the leaf surfaces at levels as low as 60 ng of total applied in the area of 1 µL droplet. After application of epoxiconazole, azoxystrobin, and pyraclostrobin at a field rate of 100 gai/ha in 200 L water using a track sprayer system, residues of these fungicides on the leaf surfaces were sufficiently visualized. These results suggest that MALDI imaging can be used to monitor spatial distribution of agrochemicals on leaf samples after pesticide application.

Transmission of the G143A QoI-resistance point mutation through anastomosis in Magnaporthe grisea.

BACKGROUND: Soon after the introduction of Qo inhibitor fungicides in 1996, the point mutation leading to the amino acid exchange glycine to alanine at the 143 position of the mitochondrial cytochrome b gene was identified as the main cause of resistance. The present study describes the role of anastomosis in the transmission of the G143A mutation in Magnaporthe grisea. RESULTS: Two M. grisea mutants were co-cultivated on oatmeal agar and also co-inoculated on barley leaves. The mutants differed by the presence of the G143A mutation in one isolate and a disrupted AOX gene by insertion of a hygromycin gene in the other (M-145). Specific resistant (r) or sensitive (s) phenotypes of 409 monosporic cultures were determined on media amended with either hygromycin (H) or azoxystrobin (S) plus SHAM. The phenotypes identified reflected not only the phenotypes of mutants M-145 and G143A but also the wild-type parent phenotype HsSs and a new HrSr isolate. CONCLUSION: Identification of the M. grisea phenotypes HrSr and HsSs suggests that anastomosis occurred during co-cultivation and co-inoculation of the mutants M-145 and G143A, allowing the transfer of the G143A point mutation from the QoI-resistant isolate to the susceptible isolate.

Phoslactomycins from Streptomyces sp. MLA1839 and their biological activities.

Phoslactomycins H (1) and I (2), two new members of the phoslactomycin class of chemistry, were isolated from Streptomyces sp. MLA1839 on the basis of their antifungal activities. Their structures were elucidated using extensive NMR spectroscopy and mass spectrometry. Phoslactomycin H (1) featured a rare and unprecedented N,N-dimethylamine substitution at C-4 and existed as a hydroxy acid rather than the more common lactone. Herein, we report the structure of these compounds and their biological activities.

Mitochondrial genome sequences and molecular evolution of the Irish potato famine pathogen, Phytophthora infestans.

The mitochondrial genomes of haplotypes of the Irish potato famine pathogen, Phytophthora infestans, were sequenced. The genome sizes were 37,922, 39,870 and 39,840 bp for the type Ia, IIa and IIb mitochondrial DNA (mtDNA) haplotypes, respectively. The mitochondrial genome size for the type Ib haplotype, previously sequenced by others, was 37,957 bp. More than 90% of the genome contained coding regions. The GC content was 22.3%. A total of 18 genes involved in electron transport, 2 RNA-encoding genes, 16 ribosomal protein genes and 25 transfer RNA genes were coded on both strands with a conserved arrangement among the haplotypes. The type I haplotypes contained six unique open reading frames (ORFs) of unknown function while the type II haplotypes contained 13 ORFs of unknown function. Polymorphisms were observed in both coding and non-coding regions although the highest variation was in non-coding regions. The type I haplotypes (Ia and Ib) differed by only 14 polymorphic sites, whereas the type II haplotypes (IIa and IIb) differed by 50 polymorphic sites. The largest number (152) of polymorphic sites was found between the type IIb and Ia haplotypes. A large spacer flanked by the genes coding for tRNA-Tyr (trnY) and the small subunit RNA (rns) contained the largest number of polymorphic sites and corresponds to the region where a large indel that differentiates type II from type I haplotypes is located. The size of this region was 785, 2,666 and 2,670 bp in type Ia, IIa and IIb haplotypes, respectively. Among the four haplotypes, 81 mutations were identified. Phylogenetic and coalescent analysis revealed that although the type I and II haplotypes shared a common ancestor, they clearly formed two independent lineages that evolved independently. The type II haplotypes diverged earlier than the type I haplotypes. Thus our data do not support the previous hypothesis that the type II lineages evolved from the type I lineages. The type I haplotypes diverged more recently and the mutations associated with the evolution of the Ia and Ib types were identified.

A Two-Phase Resistance Response of Venturia inaequalis Populations to the QoI Fungicides Kresoxim-Methyl and Trifloxystrobin.

The class of fungicides acting as respiration inhibitors by binding to the Qo center of cyto-chrome b (QoIs) are in wide use for the management of apple scab caused by Venturia inaequalis. In order to assess responses of V. inaequalis populations to treatments with QoIs, sensitivities of isolates were determined for germinating conidia or for mycelial colonies developing from germinating conidia. Under both test conditions, inhibitory potencies of kresoxim-methyl and trifloxystrobin were largely equivalent. V. inaequalis populations treated with QoIs in a commercial and an experimental orchard both responded with significant shifts toward declining QoI sensitivities. However, the population responses were quantitative in nature, and highly resistant isolates indicative of a cytochrome b target site mutation were not detected. V. inaequalis populations from both orchards investigated also were fully resistant to sterol de-methylation-inhibiting fungicides (DMIs) such as fenarimol and myclobutanil, but isolate sensitivities to QoIs and DMIs were largely unrelated. Performance tests with kresoxim-methyl and trifloxystrobin at the experimental orchard diagnosed as DMI-resistant revealed that the quantitative shift toward declining QoI sensitivities did not constitute the status of practical QoI resistance. In contrast to these quantitative responses, emergence of qualitative QoI resistance was documented for V. inaequalis in an orchard in North Germany, which had been treated intensively with a total of 25 QoI applications over four consecutive seasons. Isolates retrieved from the orchard were highly resistant to both kresoxim-methyl and trifloxystrobin and were characterized as G143A cytochrome b mutants. The results indicated that the paths of QoI resistance can be both quantitative and qualitative in nature. A similar phenomenon has not been described before. Circumstantial evidence suggests that the quantitative phase of V. inaequalis population responses to QoIs might be succeeded by a quantitative selection of highly resistant G143A target-site mutants.

Impact of alternative respiration and target-site mutations on responses of germinating conidia of Magnaporthe grisea to Qo-inhibiting fungicides.

Qo-inhibiting fungicides act as respiration inhibitors by binding to the Qo center of cytochrome b. Sensitivities of fungi to Qo inhibitors can be influenced by the induction of alternative respiration or by mutational changes of the cytochrome b target site. Previous studies on both mechanisms in Magnaporthe grisea (Hebert) Barr were focused on the mycelial stage of the pathogen. The present study describes the expression and impact of both resistance mechanisms during the stage of conidia germination. In the absence of a host, alternative respiration provided a >500-fold rescue from azoxystrobin during the germination of conidia derived from four wild-type isolates of M. grisea. This rescue potential during conidia gemination was substantially more pronounced than for mycelial growth. However, the pronounced effectiveness of alternative respiration during conidia germination was not apparent when barley leaves were protected with azoxystrobin prior to inoculation with conidia. In a comparison of a wild-type strain and an alternative respiration-deficient mutant, azoxystrobin efficacies in suppressing symptom development differed by a factor of two, with full disease control achieved for both genotypes at 1 microg ml(-1) azoxystrobin. In contrast, conidia derived from two QoI-resistant target site mutants were highly resistant to azoxystrobin and trifloxystrobin and fully capable of infecting leaf surfaces protected with 10 microg ml(-1) of azoxystrobin. Both target-site mutants had emerged spontaneously in the presence of high azoxystrobin doses when residual mycelial growth was supported by alternative respiration. The effective silencing of alternative respiration in protective applications of Qo-inhibiting fungicides might constitute a strategy of slowing the emergence of highly resistant target site mutants.

Characterization of spontaneous mutants of Magnaporthe grisea expressing stable resistance to the Qo-inhibiting fungicide azoxystrobin.

The class of Qo-inhibiting fungicides (QoIs) act as respiration inhibitors by binding to the Qo center of cytochrome b. The longevity of these fungicides has been challenged by the selection of fungal sub-populations resisting high doses of QoI fungicides, with a G143A amino acid exchange in the cytochrome b target site identified as the most common cause of resistance. In contrast, the mechanism of alternative respiration, as another mechanism of fungal QoI resistance, has thus far not been affiliated with practical resistance. In the present study, azoxystrobin-resistant mutants of Magnaporthe grisea were generated and characterized. Emergence of these spontaneous mutants was facilitated when resting melanized mycelia were allowed to escape full inhibition by azoxystrobin. This escape was related to the intactness of alternative respiration, indicating that residual expression of this rescue mechanism was involved in the spontaneous emergence of target-site mutants. The two mutants characterized resisted high doses of the QoI, azoxystrobin, with resistance factors exceeding 1,000. Two different mutations of the cytochrome b gene were identified as exchanges of guanine, leading to a G143A or a G143S amino acid exchange. Resistance of both target-site mutants remained stable during four consecutive disease cycles in the absence of azoxystrobin. Several parameters tested to measure fitness penalties inherent to the mutational changes revealed that the G143A mutant was not compromised. In contrast, the conidia production of the G143S mutant was significantly lower under both saprophytic and pathogenic conditions of reproduction.

Characterization of Colletotrichum graminicola Isolates Resistant to Strobilurin-Related QoI Fungicides.

Isolates of Colletotrichum graminicola were collected from annual bluegrass or bent grass turf in Japan and the United States, and their sensitivities to QoI fungicides (QoIs) as well as their cytochrome b sequences were characterized. Five isolates sampled from turf treated repeatedly with azoxystrobin were highly QoI resistant under both in vivo and in vitro test conditions. The nucleotide sequences of a large cytochrome b gene segment involving the binding site of QoIs were fully homologous for all resistant isolates and contained the G143A target site mutation known to confer QoI resistance in other pathogens. QoI-sensitive isolates collected prior to treatments with QoIs were more diverse with regard to their cytochrome b gene sequences and their phenotype responses to QoIs. All wild-type isolates retained a glycine in position 143 of cytochrome b. Three of the four QoI-sensitive isolates were, in addition, distinguished by leucines in positions 95, 130, and 141, which were exchanged to threonine in all resistant but also in one of the sensitive isolates. In addition to a more pronounced divergence of cytochrome b sequences, the sensitive wild-type isolates also were diverse with regard to the induction of alternative respiration in response to QoI action, as indicated by comparisons of QoI sensitivities displayed in the absence or presence of the alternative oxidase inhibitor salicylhydroxamic acid. These different phenotype responses expressed under in vitro test conditions had no or only a slight impact on anthracnose control in protective applications of azoxystrobin. Isolate responses in vitro were very similar for trifloxystrobin, indicating cross-resistance among the class of QoIs. Our results imply that C. graminicola falls into the class of pathogens with a potential for rapid selection of highly QoI-resistant phenotypes. Frequent monitoring of population sensitivities will be required to determine the status of population responses toward practical QoI resistance.

Disruption of the alternative oxidase gene in Magnaporthe grisea and its impact on host infection.

Plants and numerous fungi including Magnaporthe grisea protect mitochondria from interference by respiration inhibitors by expressing alternative oxidase, the enzymatic core of alternative respiration. The alternative oxidase gene AOXMg of M. grisea was disrupted. Several lines of evidence suggested that the disruption of AOXMg was sufficient to completely curb the expression of alternative respiration. In the infection of barley leaves, several AOXMg-minus and, thus, alternative respiration-deficient mutants of M. grisea retained their pathogenicity without significant impairment of virulence. However, differences between the wild-type strain and an AOXMg-minus mutant were apparent under oxidative stress conditions generated by the treatment of infected barley leaves with the commercial respiration inhibitor azoxystrobin. Symptom development was effectively suppressed on leaves infected with the alternative respiration-deficient mutant, while lesions on leaves infected with the wild-type strain continued to develop at much higher inhibitor doses. However, respective lesions rarely developed to the stage of full maturity. The results did not conform to a previous model implying that expression of alternative respiration is silenced during pathogenesis by the presence of constitutive plant antioxidants. Rather, alternative respiration provided protection from azoxystrobin during both saprophytic and infectious stages of the pathogen. The nature of similar oxidative stress conditions in the ecology of M. grisea remains an open question.