Target-site Resistance Mutations Research Articles

Residual toxicity of pesticide active substance commonly used in tomato crop on the predator Nesidiocoris tenuis (Heteroptera: Miridae)

AbstractNesidiocoris tenuis (Reuter) (Heteroptera Miridae) is a key biological control agent of the tomato leaf miner Tuta absoluta (Meyrick). However, in cases of prey scarcity, this predator switches into a phytophagous pest causing ring injuries in stems of tomato plants. The aim of this study was to assess the side effects of dry residues and explore the presence of target site resistance mutations of 10 insecticides, two bioinsecticides, an acaricide and a fungicide widely applied in IPM, on adults of N. tenuis collected from two different regions of Crete; the Western and the Eastern side of the island during two successive years. Lethal toxicity trials were performed on sprayed tomato stems and toxicity of adults of N. tenuis was recorded 2 h, 7 and 14 days after exposure. At both populations, chlorpyrifos, acetamiprid and thiamethoxam were moderately harmful at the application day and slightly harmful 7 days after the application. Only chlorpyrifos maintained its toxicity for more than 7 days. All the rest tested pesticides (emamectin benzoate, abamectin, chlorantraniliprole+abamectin, chlorantraniliprole, deltamethrin, tebufenpyrad, spinosad, Bacillus thuringiensis var kurstaki, metaflumizone and myclobutanil) were classified as harmless. Additionally, analysis of insecticide target site sequences indicated the absence of resistance mutations in both populations. Overall, our findings illustrate the impact of 15 pesticides and their residual toxicity on the mortality rates of N. tenuis adults.

Syntenic analysis of ACCase loci and target-site-resistance mutations in cyhalofop-butyl resistant Echinochloa crus-galli var. crus-galli in Japan.

Recently, suspected cyhalofop-butyl-resistant populations of allohexaploid weed Echinochloa crus-galli var. crus-galli were discovered in rice fields in Aichi Prefecture, Japan. Analyzing the target-site ACCase genes of cyhalofop-butyl helps understand the resistance mechanism. However, in E. crus-galli, the presence of multiple ACCase genes and the lack of detailed gene investigations have complicated the analysis of target-site genes. Therefore, in this study, we characterized the herbicide response of E. crus-galli lines and thoroughly characterized the ACCase genes, including the evaluation of gene mutations in the ACCase genes of each line. Four suspected resistant lines collected from Aichi Prefecture showed varying degrees of resistance to cyhalofop-butyl and other FOP-class ACCase inhibitors but were sensitive to herbicides with other modes of action. Through genomic analysis, six ACCase loci were identified in the E. crus-galli genome. We renamed each gene based on its syntenic relationship with other ACCase genes in the Poaceae species. RNA-sequencing analysis revealed that all ACCase genes, except the pseudogenized copy ACCase2A, were transcribed at a similar level in the shoots of E. crus-galli. Mutations known to confer resistance to FOP-class herbicides, that is W1999C, W2027C/S and I2041N, were found in all resistant lines in either ACCase1A, ACCase1B or ACCase2C. In this study, we found that the E. crus-galli lines were resistant exclusively to ACCase-inhibiting herbicides, with a target-site resistance mutation in the ACCase gene. Characterization of ACCase loci in E. crus-galli provides a basis for further research on ACCase herbicide resistance in Echinochloa spp. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The host plant strongly modulates acaricide resistance levels to mitochondrial complex II inhibitors in a multi-resistant field population of Tetranychus urticae

The two-spotted spider mite Tetranychus urticae is a polyphagous pest with an extraordinary ability to develop acaricide resistance. Here, we characterize the resistance mechanisms in a T. urticae population (VR-BE) collected from a Belgian tomato greenhouse, where the grower was unsuccessful in chemically controlling the mite population resulting in crop loss. Upon arrival in the laboratory, the VR-BE population was established both on bean and tomato plants as hosts. Toxicity bioassays on both populations confirmed that the population was highly multi-resistant, recording resistance to 12 out of 13 compounds tested from various mode of action groups. DNA sequencing revealed the presence of multiple target-site resistance mutations, but these could not explain resistance to all compounds. In addition, striking differences in toxicity for six acaricides were observed between the populations on bean and tomato. The highest difference was recorded for the complex II inhibitors cyenopyrafen and cyflumetofen, which were 4.4 and 3.3-fold less toxic for VR-BE mites on tomato versus bean. PBO synergism bioassays suggested increased P450 based detoxification contribute to the host-dependent toxicity. Given the involvement of increased detoxification, we subsequently determined genome-wide gene expression levels of VR-BE on both hosts, in comparison to a reference susceptible population, revealing overexpression of a large set of detoxification genes in VR-BE on both hosts compared to the reference. In addition, a number of mainly detoxification genes with higher expression in VR-BE on tomato compared to bean was identified, including several cytochrome P450s. Together, our work suggests that multi-resistant field populations can accumulate a striking number of target-site resistance mutations. We also show that the host plant can have a profound effect on the P450-associated resistance levels to cyenopyrafen and cyflumetofen.

Characterization of imazamox-resistant shattercane (Sorghum bicolor L.) populations from Kansas

AbstractShattercane is a problematic summer annual grass weed species in regions that produce grain sorghum. Three shattercane populations (DC8, GH4, and PL8) collected from sorghum fields from northwestern Kansas survived the field-use rate (52 g ha−1) of postemergence-applied imazamox. The main objectives of this research were to 1) confirm and characterize the level of resistance to imazamox in putative imazamox-resistant (IMI-R) shattercane populations, 2) investigate the underlying mechanism of resistance, and 3) determine the effectiveness of postemergence herbicides for controlling IMI-R populations. A previously known imazamox susceptible (SUS) shattercane population from Rooks County, KS, was used. All three putative populations exhibited a 4.1-fold to 6.0-fold resistance to imazamox compared with the SUS population. The ALS gene sequences from all IMI-R populations did not reveal any known target-site resistance mutations. A pretreatment with malathion, which inhibits cytochrome P450, followed by imazamox at various doses, reversed the resistance phenotype of the PL8 population. In a separate greenhouse study, postemergence treatments with nicosulfuron, quizalofop, clethodim, and glyphosate resulted in ≥96% injury to all IMI-R populations. The lack of known ALS target-site mutations and the reversal of resistance phenotype by malathion suggest the possibility of metabolism-based resistance to imazamox in PL8 shattercane population.

Tissue-specific transcriptome analyses in Drosophila provide novel insights into the mode of action of the insecticide spinosad and the function of its target, nAChRα6.

The insecticides spinosad and imidacloprid are neurotoxins with distinct modes of action. Both target nicotinic acetylcholine receptors (nAChRs), albeit different subunits. Spinosad is an allosteric modulator, that upon binding initiates endocytosis of its target, nAChRα6. Imidacloprid binding triggers excessive neuronal ion influx. Despite these differences, low-dose effects converge downstream in the precipitation of oxidative stress and neurodegeneration. Using RNA-sequencing, we compared the transcriptional signatures of spinosad and imidacloprid, at low-dose exposures. Both insecticides cause up-regulation of glutathione S-transferase and cytochrome P450 genes in the brain and down-regulation in the fat body, whereas reduced expression of immune-related genes is observed in both tissues. Spinosad shows unique impacts on genes involved in lysosomal function, protein folding, and reproduction. Co-expression analyses revealed little to no correlation between genes affected by spinosad and nAChRα6 expressing neurons, but a positive correlation with glial cell markers. We also detected and experimentally confirmed nAChRα6 expression in fat body cells and male germline cells. This led us to uncover lysosomal dysfunction in the fat body following spinosad exposure, and a fitness cost in spinosad-resistant (nAChRα6 null) males - oxidative stress in testes, and reduced fertility. Spinosad and imidacloprid share transcriptional perturbations in immunity-, energy homeostasis-, and oxidative stress-related genes. Low doses of other neurotoxic insecticides should be investigated for similar impacts. While target-site spinosad resistance mutation has evolved in the field, this may have a fitness cost. Our findings demonstrate the power of tissue-specific transcriptomics approach and the use of single-cell transcriptome data. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Incomplete reproductive barriers and genomic differentiation impact the spread of resistance mutations between green- and red-colour morphs of a cosmopolitan mite pest.

Pesticide resistance represents a clear and trackable case of adaptive evolution with a strong societal impact. Understanding the factors associated with the evolution and spread of resistance is imperative to develop sustainable crop management strategies. The two-spotted spider mite Tetranychus urticae, a major crop pest with worldwide distribution and a polyphagous lifestyle, has evolved resistance to most classes of pesticides. Tetranychus urticae exists as either a green- or a red-coloured morph. However, the extent of genetic divergence and reproductive compatibility vary across populations of these colour morphs, complicating their taxonomic resolution at the species level. Here, we studied patterns of genetic differentiation and barriers to gene flow within and between morphs of T. urticae in order to understand the factors that influence the spread of resistance mutations across its populations. We derived multiple iso-female lines from Tetranychus populations collected from agricultural crops. We generated genomic and morphological data, characterized their bacterial communities and performed controlled crosses. Despite morphological similarities, we found large genomic differentiation between the morphs. This pattern was reflected in the incomplete, but strong postzygotic incompatibility in crosses between colour morphs, while crosses within morphs from different geographical locations were largely compatible. In addition, our results suggest recent/on-going gene flow between green-coloured T. urticae and T. turkestani. By screening the sequences of 10 resistance genes, we found evidence for multiple independent origins and for single evolutionary origins of target-site resistance mutations. Our results indicate that target-site mutations mostly evolve independently in populations on different geographical locations, and that these mutations can spread due to incomplete barriers to gene flow within and between populations.

Multiple Resistance to Three Modes of Action of Herbicides in a Single Italian Ryegrass (Lolium multiflorum L.) Population in China

Italian ryegrass (Lolium multiflorum L.), a cross-pollinated grass, is gradually becoming a predominant weed in wheat fields in China and is evolving resistance to many groups of herbicides. The aim of this study is to determine the resistance levels of a single L. multiflorum population from a wheat field in Henan Province China, to three modes of action (MoAs) of herbicides and to further characterize the potential resistance mechanisms. This L. multiflorum population evolved multiple herbicide resistances to pyroxsulam [acetolactate synthase (ALS)], pinoxaden [acetyl-CoA carboxylase (ACCase)] and isoproturon [photosystem II (PSII)]. Target-site resistance (TSR) mutations (Pro-197-Gln, Pro-197-Thr, and Trp-574-Leu) and non-target-site resistance (NTSR) mediated by cytochrome P450 monooxygenase (CYP450) genes were associated with pyroxsulam resistance. Pinoxaden resistance was conferred by two TSR mutations, which referred to a rare Ile-2041-Val mutation and a common Ile-1781-Leu mutation but with two different nucleotide substitutions (CTA/TTA). CYP450- and glutathione-S-transferase (GST)-mediated resistances were the main resistance mechanisms for this multiple herbicide-resistant (MHR) population to the PSII inhibitor isoproturon. This is the first case of a single L. multiflorum population evolving multiple resistance to three herbicide MoAs (ALS, ACCase and PSII) in China. Diverse resistance mechanisms including TSR and NTSR mean L. multiflorum exhibits a high degree of resistance plasticity.

Physiological Fitness Associated to ACCase Target-Site Resistance Enhances Growth and Reproduction in Phalaris brachystachys

Short-spike canarygrass (Phalaris brachystachys (Link.)) from Iranian wheat fields has developed resistance to acetyl-coenzyme A carboxylase (ACCase) inhibitors due to a target-site mutation (Ile-1781-Thr). Target-site resistance mutations may confer pleiotropic effects in weeds. In this paper, the possible effect of the Ile-1781-Thr mutation on the ecological fitness during life cycles in P. brachystachys plants was investigated. ACCase genes of P. brachystachys populations resistant (R) and susceptible (S) to ACCase inhibitors were sequenced and the vegetative growth and reproductive characteristics of the plants were assessed. In the final growth stage (217 days after planting—DAP), R sub-population plants were 30 cm taller than the S plants. Additionally, the R sub-population produced up to 12 leaves and 2.8 tillers more per plant, and accumulated double the dry weight (2850 g m−2) compared to the S sub-population. The leaf area index (LAI) of the R sub-population was 1.1 times higher than that of the S sub-population. In addition, the net assimilation rate (NAR) and plant growth rate (PGR) between 114 and 182 DAP of the R sub-population were 0.11 and 13 g m−2 d−1 higher than the S sub-population, but the relative growth rate (RGR) was similar between R and S sub-populations. The number of spikes (6 vs. 3.8), the spike length (8.4 vs. 5.5), and number of seeds per plant (1276 vs. 751 seed plant−1) of the R sub-population were higher than the S ones, but the weight (3.25 g) and size (11.6 mm2) of 1000 seeds were similar between populations. The R sub-population of P. brachystachys exhibited higher plant growth and reproductive parameters than the S one, which may increase the frequency of resistance in the population in the absence of adequate weed-control methods.

Community structure and insecticide resistance of malaria vectors in northern-central Myanmar

BackgroundMyanmar is one of the six countries in the Greater Mekong Subregion (GMS) of Southeast Asia. Malaria vectors comprise many Anopheles species, which vary in abundance and importance in malaria transmission among different geographical locations in the GMS. Information about the species composition, abundance, and insecticide resistance status of vectorial systems in Myanmar is scarce, hindering our efforts to effectively control malaria vectors in this region.MethodsDuring October and November 2019, larvae and adult females of Anopheles mosquitoes were collected in three sentinel villages of Banmauk township in northern Myanmar. Adult female mosquitoes collected by cow-baited tent collection (CBTC) and adults reared from field-collected larvae (RFCL) were used to determine mortality rates and knockdown resistance (kdr) against deltamethrin using the standard WHO susceptibility test. Molecular species identification was performed by multiplex PCR and ITS2 PCR, followed by DNA sequencing. The kdr mutation at position 1014 of the voltage-gated sodium channel gene was genotyped by DNA sequencing for all Anopheles species tested.ResultsA total of 1596 Anopheles mosquitoes from seven morphologically identified species groups were bioassayed. Confirmed resistance to deltamethrin was detected in the populations of An. barbirostris (s.l.), An. hyrcanus (s.l.), and An. vagus, while possible resistance was detected in An. annularis (s.l.), An. minimus, and An. tessellatus. Anopheles kochi was found susceptible to deltamethrin. Compared to adults collected by CBTC, female adults from RFCL had significantly lower mortality rates in the four species complexes. A total of 1638 individuals from 22 Anopheles species were molecularly identified, with the four most common species being An. dissidens (20.5%) of the Barbirostris group, An. peditaeniatus (19.4%) of the Hyrcanus group, An. aconitus (13.4%) of the Funestus group, and An. nivipes (11.5%) of the Annularis group. The kdr mutation L1014F was only detected in the homozygous state in two An. subpictus (s.l.) specimens and in a heterozygous state in one An. culicifacies (s.l.) specimen.ConclusionsThis study provides updated information about malaria vector species composition and insecticide resistance status in northern Myanmar. The confirmed deltamethrin resistance in multiple species groups constitutes a significant threat to malaria vector control. The lack or low frequency of target-site resistance mutations suggests that other mechanisms are involved in resistance. Continual monitoring of the insecticide resistance of malaria vectors is required for effective vector control and insecticide resistance management.Graphical

Genomic insights into the evolution of Echinochloa species as weed and orphan crop

As one of the great survivors of the plant kingdom, barnyard grasses (Echinochloa spp.) are the most noxious and common weeds in paddy ecosystems. Meanwhile, at least two Echinochloa species have been domesticated and cultivated as millets. In order to better understand the genomic forces driving the evolution of Echinochloa species toward weed and crop characteristics, we assemble genomes of three Echinochloa species (allohexaploid E. crus-galli and E. colona, and allotetraploid E. oryzicola) and re-sequence 737 accessions of barnyard grasses and millets from 16 rice-producing countries. Phylogenomic and comparative genomic analyses reveal the complex and reticulate evolution in the speciation of Echinochloa polyploids and provide evidence of constrained disease-related gene copy numbers in Echinochloa. A population-level investigation uncovers deep population differentiation for local adaptation, multiple target-site herbicide resistance mutations of barnyard grasses, and limited domestication of barnyard millets. Our results provide genomic insights into the dual roles of Echinochloa species as weeds and crops as well as essential resources for studying plant polyploidization, adaptation, precision weed control and millet improvements.

Repeated origins, widespread gene flow, and allelic interactions of target-site herbicide resistance mutations.

Causal mutations and their frequency in agricultural fields are well-characterized for herbicide resistance. However, we still lack understanding of their evolutionary history: the extent of parallelism in the origins of target-site resistance (TSR), how long these mutations persist, how quickly they spread, and allelic interactions that mediate their selective advantage. We addressed these questions with genomic data from 19 agricultural populations of common waterhemp (Amaranthus tuberculatus), which we show to have undergone a massive expansion over the past century, with a contemporary effective population size estimate of 8 x 107. We found variation at seven characterized TSR loci, two of which had multiple amino acid substitutions, and three of which were common. These three common resistance variants show extreme parallelism in their mutational origins, with gene flow having shaped their distribution across the landscape. Allele age estimates supported a strong role of adaptation from de novo mutations, with a median age of 30 suggesting that most resistance alleles arose soon after the onset of herbicide use. However, resistant lineages varied in both their age and evidence for selection over two different timescales, implying considerable heterogeneity in the forces that govern their persistence. Two such forces are intra- and inter-locus allelic interactions; we report a signal of extended haplotype competition between two common TSR alleles, and extreme linkage with genome-wide alleles with known functions in resistance adaptation. Together, this work reveals a remarkable example of spatial parallel evolution in a metapopulation, with important implications for the management of herbicide resistance.

Selectivity and molecular stress responses to classical and botanical acaricides in the predatory mite Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseiidae).

Acaricide application remains an integral component of integrated pest management (IPM) for the two-spotted spider mite Tetranychus urticae. Species and strains of phytoseiid predatory mites vary significantly in their response to acaricides. For the success of IPM, it is imperative to identify the determinants of selectivity and molecular stress responses of acaricides in predatory mites. The three classical acaricides bifenazate, cyflumetofen, and fenbutatin oxide did not affect the survival and fecundity of Phytoseiulus persimilis regardless of the route of exposure. Selectivity of the orange oil and terpenoid blend-based botanical acaricides was low via a combination of direct exposure, acaricide-laced diet, and residual exposure but improved when limiting exposure only to diet. To gain insights into the molecular stress responses, the transcriptome of P. persimilis was assembled. Subsequent gene expression analysis of predatory mites orally exposed to fenbutatin oxide and orange oil yielded only a limited xenobiotic stress response. In contrast, P. persimilis exhibited target-site resistance mutations, including I260M in SdhB, I1017M in CHS1, and kdr and super-kdr in VGSC. Extending the screen using available Phytoseiidae sequences uncovered I136T, S141F in cytb, G119S in AChE, and A2083V in ACC, well-known target-sites of acaricides. Selectivity of the tested botanical acaricides to P. persimilis was low but could be enhanced by restricting exposure to a single route. Differential gene expression analysis did not show a robust induced stress response after sublethal exposure. In contrast, this study uncovered target-site mutations that may help to explain the physiological selectivity of several classical acaricides to phytoseiid predators.

Impact of a Novel W2027L Mutation and Non-Target Site Resistance on Acetyl-CoA Carboxylase-Inhibiting Herbicides in a French Lolium multiflorum Population.

Herbicides that inhibit acetyl-CoA carboxylase (ACCase) are among the few remaining options for the post-emergence control of Lolium species in small grain cereal crops. Here, we determined the mechanism of resistance to ACCase herbicides in a Lolium multiflorum population (HGR) from France. A combined biological and molecular approach detected a novel W2027L ACCase mutation that affects aryloxyphenoxypropionate (FOP) but not cyclohexanedione (DIM) or phenylpyraxoline (DEN) subclasses of ACCase herbicides. Both the wild-type tryptophan and mutant leucine 2027-ACCase alleles could be positively detected in a single DNA-based-derived polymorphic amplified cleaved sequence (dPACS) assay that contained the targeted PCR product and a cocktail of two discriminating restriction enzymes. Additionally, we identified three well-characterised I1781L, I2041T, and D2078G ACCase target site resistance mutations as well as non-target site resistance in HGR. The non-target site component endowed high levels of resistance to FOP herbicides whilst partially impacting on the efficacy of pinoxaden and cycloxydim. This study adequately assessed the contribution of the W2027L mutation and non-target site mechanism in conferring resistance to ACCase herbicides in HGR. It also highlights the versatility and robustness of the dPACS method to simultaneously identify different resistance-causing alleles at a single ACCase codon.

Insecticide resistance status and mechanisms in Aedes aegypti populations from Senegal.

Aedes aegypti is the main epidemic vector of arboviruses in Africa. In Senegal, control activities are mainly limited to mitigation of epidemics, with limited information available for Ae. aegypti populations. A better understanding of the current Ae. aegypti susceptibility status to various insecticides and relevant resistance mechanisms involved is needed for the implementation of effective vector control strategies. The present study focuses on the detection of insecticide resistance and reveals the related mechanisms in Ae. aegypti populations from Senegal.Bioassays were performed on Ae. aegypti adults from nine Senegalese localities (Matam, Louga, Barkedji, Ziguinchor, Mbour, Fatick, Dakar, Kédougou and Touba). Mosquitoes were exposed to four classes of insecticides using the standard WHO protocols. Resistance mechanisms were investigated by genotyping for pyrethroid target site resistance mutations (V1016G, V1016I, F1534C and S989P) and measuring gene expression levels of key detoxification genes (CYP6BB2, CYP9J26, CYP9J28, CYP9J32, CYP9M6, CCEae3a and GSTD4).All collected populations were resistant to DDT and carbamates except for the ones in Matam (Northern region). Resistance to permethrin was uniformly detected in mosquitoes from all areas. Except for Barkédji and Touba, all populations were characterized by a susceptibility to 0.75% Permethrin. Susceptibility to type II pyrethroids was detected only in the Southern regions (Kédougou and Ziguinchor). All mosquito populations were susceptible to 5% Malathion, but only Kédougou and Matam mosquitoes were susceptible to 0.8% Malathion. All populations were resistant to 0.05% Pirimiphos-methyl, whereas those from Louga, Mbour and Barkédji, also exhibited resistance to 1% Fenitrothion. None of the known target site pyrethroid resistance mutations was present in the mosquito samples included in the genotyping analysis (performed in > 1500 samples). In contrast, a remarkably high (20-70-fold) overexpression of major detoxification genes was observed, suggesting that insecticide resistance is mostly mediated through metabolic mechanisms. These data provide important evidence to support dengue vector control in Senegal.

Effects of a novel combination of two mutated acetolactate synthase (ALS) isozymes on resistance to ALS-inhibiting herbicides in flixweed (Descurainia sophia)

AbstractFlixweed [Descurainia sophia(L.) Webb ex Prantl] is a notorious broadleaf weed that is widely distributed in winter wheat–growing areas of China and has evolved resistance to tribenuron-methyl mainly due to target-site resistance (TSR) mutations in acetolactate synthase (ALS). In the current research, twoALSgenes were identified in tribenuron-methyl–susceptible (TS) or tribenuron-methyl–resistant (TR)D. sophia. Resistance mutations of Asp-376-Glu and Pro-197-Ala were identified on ALS1 and ALS2 isozymes in TRD. sophia, respectively. The TRD. sophiaevolved 10,836.3-fold resistance to tribenuron-methyl and displayed cross-resistance to multiple ALS-inhibiting herbicides with different chemical structures. Dose response experiments and ALS activity assay indicated that two mutated ALS isozymes contributed differentially in resistance to tribenuron-methyl, flucetosulfuron, and pyribenzoxim. In addition, the relative expression level of theALS1gene was 2.2- and 1.6-fold higher thanALS2genes in TRD. sophiaat 1 and 7 d after tribenuron-methyl treatment, respectively. In contrast, the relative expression level ofALS1andALS2in TSD. sophiais similar. This is the first research that explored different roles of ALS isozymes in resistance to ALS-inhibiting herbicides, which might provide a new perspective for the weed resistance management.

Contrasting levels of genetic predictability in the evolution of resistance to major classes of fungicides.

The evolution of resistance has been seen across all major classes of xenobiotics, including antimicrobial drugs and agricultural pesticides. This repeated emergence of resistance is a case of phenotypic parallel evolution, but often the parallelism extends to the molecular level too, with multiple species gaining the same mutation in response to the same chemical treatment. We review the degree of repeatability in target-site resistance mutations affecting different classes of site-specific agricultural fungicides used in crop protection, comparing the extent to which resistance in different pathogen species has evolved via the same or different mutations. For all major fungicide target sites, substantial levels of molecular parallel evolution can be seen, with at least one mutation recurring in over 50% of species. Target-site mutations appear to be most repeatable in cytochrome b, target site of quinone-outside inhibitor fungicides, and least predictable for CYP51, target site of the azoles. Intermediate levels of repeatability are seen for the MBC target site β-tubulin, and the SDHI target site succinate dehydrogenase. Repeatability may be lower where there are selective trade-offs between resistance and pleiotropic fitness penalties, or differing levels of cross-resistance across members of a fungicide class; or where single mutations confer only partial resistance, and epistatic interactions between multiple mutations result in a rugged fitness landscape. This affects the predictive power of in vitro mutation studies, and has practical implications for resistance monitoring strategies and diagnostic methods.

Monitoring and mechanisms of insecticide resistance in Spodoptera exigua (Lepidoptera: Noctuidae), with special reference to diamides

The beet armyworm, Spodoptera exigua, is a major lepidopteran pest of global importance in cultivation of numerous crops including cotton, maize, soybean, onion, cabbage, and ornamentals. It has evolved resistance to different insecticides. However, the current status of insecticide resistance in S. exigua has not been well examined in China. In this study, concentration-mortality responses of S. exigua to seven insecticides, including chlorantraniliprole, tetraniliprole, methoxyfenozide, indoxacarb, chlorfenapyr, emamectin benzoate and beta-cypermethrin were evaluated. The results showed that most of the tested populations had developed moderate to high resistance to chlorantraniliprole, with resistance ratios ranging from 6.3 to 2477.3-fold. Our results also showed that chlorantraniliprole have cross-resistance with tetraniliprole in S. exigua. The AY19 population collected from Anyang in Henan Province in 2019 exhibited a high resistance level to beta-cypermethrin (RR = 277.5). Methoxyfenozide and chlorfenapyr were highly effective against all of the tested populations with resistance ratios (RR) ranging from 0.1 to 2.2-fold. One of the tested populations showed moderate resistance to indoxacarb and emamectin benzoate. We detected the known ryanodine receptor target site resistance mutation, I4743M, in the field populations of S. exigua with different levels of diamide resistance.

Cytochrome P450 Herbicide Metabolism as the Main Mechanism of Cross-Resistance to ACCase- and ALS-Inhibitors in Lolium spp. Populations From Argentina: A Molecular Approach in Characterization and Detection

Knowledge about the mechanisms of herbicide resistance provide valuable insights into evolving weed populations in response to selection pressure and should be used as a basis for designing management strategies for herbicide-resistant weeds. The selection pressure associated with reactive management against glyphosate-resistant Lolium spp. populations would have favored the herbicide resistance to ACCase- and ALS-inhibitors. This work was aimed to determine the sensitivity of 80 Argentinean Lolium spp. populations to ALS- and ACCase-inhibitor herbicides for use in wheat or barley and to study the mechanisms of resistance involved. Sensitivity to pinoxaden and iodosulfuron-mesosulfuron were positively correlated (r = 0.84), even though both affect different target sites. Inhibitors of cytochrome P450 monooxygenases (P450s) increased the sensitivity to pinoxaden and iodosulfuron-mesosulfuron in 94% of herbicide-resistant populations and target-site ACCase resistance mutations were detected only in two cases. Polymorphic variants were obtained with a pair primer designed on P450 sequences, cluster analysis discriminated around 80% of susceptible and P450-metabolic resistant plants sampled from a single population or different populations. Five markers corresponding to herbicide sensitivity were identified to be significantly associated with phenotypic variance in plants. Resistance to ALS- and ACCase-inhibitor herbicides were closely related, challenging the rotation of herbicides of both sites of action as a practice against resistance. In that sense, the use of pinoxaden and iodosulfuron-mesosulfuron would have provoked a selection on P450 genes that conduced a convergence of P450-metabolism based resistant Lolium spp. populations, which was detected by markers in a contribution to elucidate the molecular basis of this type of resistance.

Identification and characterization of striking multiple-insecticide resistance in a Tetranychus urticae field population from Greece.

Tetranychus urticae is a notorious crop pest with a worldwide distribution that has developed resistance to a wide range of acaricides. Here, we investigated the resistance levels of a T. urticae population collected from an ornamental greenhouse in Peloponnese, Greece, and analyzed its resistance mechanisms at the molecular level. Toxicological assays showed resistance against compounds with different modes of action, with resistance ratios of: 89-fold for abamectin; > 1000-fold for clofentezine; > 5000-fold for etoxazole; 27-fold for fenpyroximate and pyridaben; 20- and 36-fold for spirodiclofen and spirotetramat, respectively; and 116- and > 500-fold for cyenopyrafen and cyflumetofen, respectively. Bioassays with synergists indicated the involvement of detoxification enzymes in resistance to abamectin, but not to cyflumetofen and spirodiclofen. RNA sequencing (RNA-seq) analysis showed significant over-expression of several genes encoding detoxification enzymes such as cytochrome P450 monooxygenases and UDP-glycosyltransferases, which have been previously associated with acaricide resistance. Known target-site resistance mutations were identified in acetyl-choline esterase, chitin synthase 1 and NDUFS7/psst, but putative novel resistance mutations were also discovered in targets such as glutamate-gated chloride channel subunit 3. Interestingly, target-site resistance mutations against pyrethroids or bifenazate were not identified, possibly indicating a recent reduced selection pressure in Greece, as well as a possible opportunity to rotate these chemistries. We identified and characterized a striking case of multiple acaricide resistance in a field population of T. urticae. Exceptionally strong resistance phenotypes, with accumulation of multiple resistance mutations and over-expression of P450s and other detoxification genes in the same field population are reported.

QTL mapping using microsatellite linkage reveals target-site mutations associated with high levels of resistance against three mitochondrial complex II inhibitors in Tetranychus urticae

The acaricides cyflumetofen, cyenopyrafen, and pyflubumide act as inhibitors of the mitochondrial electron transport system at complex II (succinate dehydrogenase; SDH), a new mode of action in arthropods. The development and mechanisms of low-level resistance against cyenopyrafen and cyflumetofen have been previously reported in Tetranychus urticae. In the present study, we investigated high levels of resistance against three SDH inhibitors in T. urticae field populations and clarify the genetic basis of resistance using quantitative trait locus (QTL) analysis. First, we constructed a microsatellite linkage map comprising 64 markers assembled into three linkage groups (LGs) with total length of 683.8 cM and average marker spacing of 11.03 cM. We then used the linkage map to perform QTL mapping, and identified significant QTLs contributing to resistance to cyflumetofen (one QTL on LG1), cyenopyrafen (one QTL on LG3), and pyflubumide (two QTLs on LG1 and LG3). The QTL peaks on LG1 for cyflumetofen and pyflubumide overlapped and included the SdhB locus. For cyenopyrafen resistance, the QTLs on LG3 included the SdhC locus. For cyflumetofen resistance, we found an I260T mutation in SdhB. For pyflubumide and cyenopyrafen resistance, we detected I260V and S56L substitutions in SdhB and SdhC, respectively, by direct sequencing. Both I260 in SdhB and S56 in SdhC were present in highly conserved regions of the ubiquinone binding site formed at the interface among SdhB, SdhC, and SdhD. Mutations at these positions have been implicated in resistance against fungicides that act as Sdh inhibitors in various pathogens. Therefore, we consider these mutations to be target-site resistance mutations for these acaricidal SDH inhibitors.