Baseline susceptibility of Anopheles gambiae to clothianidin in northern Ghana.

BACKGROUND: Clothianidin, an insecticide with a novel mode of action, has been deployed in the annual indoor residual spraying programme in northern Ghana since March 2021. To inform pragmatic management strategies and guide future studies, baseline data on local Anopheles gambiae sensu lato (s.l.) susceptibility to the clothianidin insecticide were collected in Kpalsogu, a village in the Northern region, Ghana. METHODS: Phenotypic susceptibility of An. gambiae mosquitoes to clothianidin was assessed using the World Health Organization (WHO) insecticide resistance monitoring bioassay. The WHO cone bioassays were conducted on mud and cement walls sprayed with Sumishield 50 wettable granules (WG) (with clothianidin active ingredient). Daily mortalities were recorded for up to 7 days to observe for delayed mortalities. Polymerase chain reaction (PCR) technique was used to differentiate the sibling species of the An. gambiae complex and also for the detection of knock down resistance genes (kdr) and the insensitive acetylcholinesterase mutation (ace-1). RESULTS: The WHO susceptibility bioassay revealed a delayed killing effect of clothianidin. Mosquitoes exposed to the cone bioassays for 5 min died 120 h after exposure. Slightly higher mortalities were observed in mosquitoes exposed to clothianidin-treated cement wall surfaces than mosquitoes exposed to mud wall surfaces. The kdr target-site mutation L1014F occurred at very high frequencies (0.89-0.94) across all vector species identified whereas the ace-1 mutation occurred at moderate levels (0.32-0.44). Anopheles gambiae sensu stricto was the most abundant species observed at 63%, whereas Anopheles arabiensis was the least observed at 9%. CONCLUSIONS: Anopheles gambiae s.l. mosquitoes in northern Ghana were susceptible to clothianidin. They harboured kdr mutations at high frequencies. The ace-1 mutation occurred in moderation. The results of this study confirm that clothianidin is an effective active ingredient and should be utilized in malaria vector control interventions.

Accumulation of mutations in the AP2 homoeologs causes suppression of anther extrusion with altered spike and culm development in hexaploid wheat.

Cleistogamy or closed flowering is a widely used trait in barley (Hordeum vulgare) breeding because it reduces the risk of fungal infection in florets at anthesis. Cleistogamy in barley is caused by a point mutation within the microRNA172 (miR172) target site of the Cly1 gene, which encodes the Apetala2 (AP2) transcription factor. Because cleistogamy is not apparent in cultivars of hexaploid wheat (Triticum aestivum), a strategy to develop cleistogamous wheat was proposed by inducing point mutations in all three AP2 homoeologs, which are the wheat orthologs of barley Cly1. In this study, we investigated the effects of miR172 target site mutations on wheat cleistogamy using double mutants by combining three previously obtained mutant alleles (AP2-A1, D1 and D2) in a near-isogenic background. The AP2-D2 allele had the greatest effect on reducing the anther extrusion rate and lodicule size compared with the other two mutant alleles. The double mutant containing the AP2-A1 and AP2-D2 alleles had a much greater suppression of anther extrusion by reducing the lodicule size than the single AP2-D2 mutant, suggesting cumulative effects of the two mutant alleles. In addition, both single and double mutants exhibited compact spikes and shorter plant heights due to reduced rachis and culm internodes in the upper parts. The presence or absence of the wild-type AP2-B homoeolog had no significant effect on phenotype. This study provides insights into the cumulative effects of mutant AP2 alleles in suppressing open flowering and provides a basis for further research on the development of complete cleistogamy in hexaploid wheat. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01458-9.

Pro197Ser and the new Trp574Leu mutations together with enhanced metabolism contribute to cross-resistance to ALS inhibiting herbicides in Sinapis alba.

White mustard, (Sinapis alba), a problematic broadleaf weed in many Mediterranean countries in arable fields has been detected as resistant to tribenuron-methyl in Tunisia. Greenhouse and laboratory studies were conducted to characterize Target-Site Resistance (TSR) and the Non-Target Site Resistance (NTSR) mechanisms in two suspected white mustard biotypes. Herbicide dose-response experiments confirmed that the two S. alba biotypes were resistant to four dissimilar acetolactate synthase (ALS)-pinhibiting herbicide chemistries indicating the presence of cross-resistance mechanisms. The highest resistance factor (>144) was attributed to tribenuron-methyl herbicide and both R populations survived up to 64-fold the recommended field dose (18.7 g ai ha-1). In this study, the metabolism experiments with malathion (a cytochrome P450 inhibitor) showed that malathion reduced resistance to tribenuron-methyl and imazamox in both populations, indicating that P450 may be involved in the resistance. Sequence analysis of the ALS gene detected target site mutations in the two R biotypes, with amino acid substitutions Trp574Leu, the first report for the species, and Pro197Ser. Molecular docking analysis showed that ALSPro197Ser enzyme cannot properly bind to tribenuron-methyl's aromatic ring due to a reduction in the number of hydrogen bonds, while imazamox can still bind. However, Trp574Leu can weaken the binding affinity between the mutated ALS enzyme and both herbicides with the loss of crucial interactions. This investigation provides substantial evidence for the risk of evolving multiple resistance in S. alba to auxin herbicides while deciphering the TSR and NTSR mechanisms conferring cross resistance to ALS inhibitors.

Increased metabolism in combination with the novel cytochrome b target-site mutation L258F confers cross-resistance between the Qo inhibitors acequinocyl and bifenazate in Tetranychus urticae.

Acequinocyl and bifenazate are potent acaricides acting at the Qo site of complex III of the electron transport chain, but frequent applications of these acaricides have led to the development of resistance in spider mites. Target-site resistance caused by mutations in the conserved cd1- and ef-helices of the Qo pocket of cytochrome b has been elucidated as the main resistance mechanism. We therefore monitored Qo pocket mutations in European field populations of Tetranychus urticae and uncovered a new mutation, L258F. The role of this mutation was validated by revealing patterns of maternal inheritance and by the independently replicated introgression in an unrelated susceptible genetic background. However, the parental strain exhibited higher resistance levels than conferred by the mutation alone in isogenic lines, especially for acequinocyl, implying the involvement of strong additional resistance mechanisms. This was confirmed by revealing a polygenic inheritance pattern with classical genetic crosses and via synergism experiments. Therefore, a genome-wide expression analysis was conducted that identified a number of highly overexpressed detoxification genes, including many P450s. Functional expression revealed that the P450 CYP392A11 can metabolize bifenazate by hydroxylation of the ring structure. In conclusion, the novel cytochrome b target-site mutation L258F was uncovered in a recently collected field strain and its role in acequinocyl and bifenazate resistance was validated. However, the high level of resistance in this strain is most likely caused by a combination of target-site resistance and P450-based increased detoxification, potentially acting in synergism.

Target gene mutations endowed cross-resistance to acetolactate synthase-inhibiting herbicides in wild Brassica juncea.

Wild Brassica juncea is a troublesome weed that infests wheat fields in China. Two suspected wild B. juncea populations (19-5 and 19-6) resistant to acetolactate synthase (ALS) inhibitors were collected from wheat fields in China. To clarify their resistance profiles and resistance mechanism, the resistance levels of populations 19-5 and 19-6 to ALS-inhibiting herbicides and their underlying target-site resistance mechanism were investigated. The results showed that the 19-5 population exhibited resistance to tribenuron-methyl, pyrithiobac­sodium and florasulam, while the 19-6 population was resistant to tribenuron-methyl, pyrithiobac­sodium, imazethapyr and florasulam. Using the homologous cloning method, two ALS genes were identified in wild B. juncea, with one gene (ALS1) encoding 652 amino acids and the other (ALS2) encoding 655 amino acids. Pro-197-Arg mutation on ALS2 and Trp-574-Leu mutation on ALS1, together with the combination of these two mutations in a single plant, were observed in both 19-5 and 19-6 populations. ALS2 enzymes carrying the Pro-197-Arg mutation were cross-resistant to tribenuron-methyl, pyrithiobac­sodium, imazerthapyr and florasulam, with resistance index (RI) values of 6.23, 32.81, 7.97 and 1162.50, respectively. Similarly, ALS1 enzymes with Trp-574-leu substitutions also displayed high resistance to these four herbicides (RI values ranging from 132.61 to 3375.00). In addition, the combination of Pro-197-Arg (ALS2) and Trp-574-Leu (ALS1) mutations increased the resistance level of the ALS enzyme to ALS inhibitors, with its RI values 3.83-214.19, 6.88-37.34, 1.91-31.82 and 2.03-5.90-fold higher than a single mutation for tribenuron-methyl, pyrithiobac­sodium, imazerthapyr and florasulam, respectively. Collectively, Pro-197-Arg mutation on ALS2, Trp-574-Leu mutation on ALS1 and the combination of Pro-197-Arg (ALS2) and Trp-574-Leu (ALS1) mutations in wild B. juncea could endow broad-spectrum resistance to ALS inhibitors, which might provide guides for establishing effective strategies to prevent or delay such resistance evolution in this weed.

A naturally evolved mutation (Ser59Gly) in glutamine synthetase confers glufosinate resistance in plants.

Glufosinate is an important and widely used non-selective herbicide active on a wide range of plant species. Evolution of resistance to glufosinate in weedy plant species (including the global weed Eleusine indica) is underway. Here, we established the molecular basis of target site glufosinate resistance in Eleusine indica. Full-length E. indica glutamine synthetase (GS) iso-genes (EiGS1-1, 1-2, 1-3, and EiGS2) were cloned, and expression of EiGS1-1 and EiGS1-2 was higher than that of EiGS2. A novel point mutation resulting in a Ser59Gly substitution in EiGS1-1 was identified in glufosinate-resistant plants. Rice calli and seedlings transformed with the mutant EiGS1-1 gene were resistant to glufosinate. Purified mutant EiGS1-1 expressed in yeast was more tolerant to glufosinate than the wild-type variant. These transgenic results correlate with a more glufosinate-resistant GS in the crude tissue extract of resistant versus susceptible E. indica plants. Structural modelling of the mutant EiGS1-1 revealed that Ser59 is not directly involved in glufosinate binding but is in contact with some important binding residues (e.g. Glu297) and especially with Asp56 that forms an intratoroidal contact interface. Importantly, the same Ser59Gly mutation was also found in geographically isolated glufosinate-resistant populations from Malaysia and China, suggesting parallel evolution of this resistance mutation.

Long-term survey and characterization of cyflumetofen resistance in Tetranychus urticae populations from Turkey.

The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae) is the most economically important mite pest in agricultural areas and chemical acaricides are widely used to control T. urticae populations. Cyflumetofen is a recently introduced acaricide that inhibits the mitochondrial electron transport chain at complex II (succinate dehydrogenase, SDH), which represents the most recently developed mode of action for mite control worldwide. In the present study, started upon the launch of cyflumetofen in Turkey, a five-year survey was performed to monitor cyflumetofen susceptibility in 28 T. urticae populations collected from agricultural fields across the country. The first resistance case that might cause control failure in practical field conditions was uncovered in 2019, three years after the registration of cyflumetofen. In addition, an extremely resistant population (1722-fold resistance) was also detected towards the end of 2019. Cyflumetofen resistance did not decrease in the laboratory after relaxation of selection pressure for over one year in field-collected populations, suggesting the absence of a fitness cost associated with resistance in these populations. Next to phenotypic resistance, metabolic and physiological mechanisms underlying the decreased susceptibility were also investigated. Synergism assays showed the involvement of P450 monooxygenases in cyflumetofen resistance. Downregulation of carboxylesterases as resistance mechanism, is underpinned by the fact that pre-treatment with esterase inhibitor DEF decreased cyflumetofen toxicity in field-collected strains. Furthermore, a novel H258L substitution in the subunit B of complex II was uncovered in a field population. In silico modeling of the new mutation suggested that the mutation might indeed influence toxicity to complex II inhibitors cyenopyrafen and pyflubumide, but most likely not cyflumetofen. However, further studies are needed to uncover the exact role of this mutation in resistance to this new class of complex II inhibitors.

Tribenuron-methyl metabolism and the rare Pro197Phe double mutation together with 2,4-D metabolism and reduced absorption can evolve in Papaver rhoeas with multiple and cross herbicide resistance to ALS inhibitors and auxin mimics.

Multiple resistance mechanisms to ALS inhibitors and auxin mimics in two Papaver rhoeas populations were investigated in wheat fields from Portugal. Dose-response trials, also with malathion (a cytochrome P450 inhibitor), cross-resistance patterns for ALS inhibitors and auxin mimics, alternative herbicides tests, 2,4-D and tribenuron-methyl absorption, translocation and metabolism experiments, together with ALS activity, gene sequencing and enzyme modelling and ligand docking were carried out. Results revealed two different resistant profiles: one population (R1) multiple resistant to tribenuron-methyl and 2,4-D, the second (R2) only resistant to 2,4-D. In R1, several target-site mutations in Pro197 and enhanced metabolism (cytochrome P450-mediated) were responsible of tribenuron-methyl resistance. For 2,4-D, reduced transport was observed in both populations, while cytochrome P450-mediated metabolism was also present in R1 population. Moreover, this is the first P. rhoeas population with enhanced tribenuron-methyl metabolism. This study reports the first case for P. rhoeas of the amino acid substitution Pro197Phe due to a double nucleotide change. This double mutation could cause reduced enzyme sensitivity to most ALS inhibitors according to protein modelling and ligand docking. In addition, this study reports a P. rhoeas population resistant to 2,4-D, apparently, with reduced transport as the sole resistance mechanism.

Investigating molecular mechanisms of insecticide resistance in the Eastern Democratic Republic of the Congo.

BACKGROUND: Malaria vector control in the Democratic Republic of the Congo is plagued by several major challenges, including inadequate infrastructure, lack of access to health care systems and preventative measures, and more recently the widespread emergence of insecticide resistance among Anopheles mosquitoes. Across 26 provinces, insecticide resistance has been reported from multiple sentinel sites. However, to date, investigation of molecular resistance mechanisms among Anopheles vector populations in DRC has been more limited. METHODS: Adult Anopheles gambiae sensu lato (s.l.) and Anopheles funestus s.l. were collected from two sites in Sud-Kivu province and one site in Haut-Uélé province and PCR-screened for the presence of 11 resistance mutations, to provide additional information on frequency of resistance mechanisms in the eastern DRC, and to critically evaluate the utility of these markers for prospective country-wide resistance monitoring. RESULTS: L1014F-kdr and L1014S-kdr were present in 75.9% and 56.7% of An. gambiae s.l. screened, respectively, with some individuals harbouring both resistant alleles. Across the three study sites, L43F-CYP4J5 allele frequency ranged from 0.42 to 0.52, with evidence for ongoing selection. G119S-ace1 was also identified in all sites but at lower levels. A triple mutant haplotype (comprising the point mutation CYP6P4-I236M, the insertion of a partial Zanzibar-like transposable element and duplication of CYP6AA1) was present at high frequencies. In An. funestus s.l. cis-regulatory polymorphisms in CYP6P9a and CYP6P9b were detected, with allele frequencies ranging from 0.82 to 0.98 and 0.65 to 0.83, respectively. CONCLUSIONS: This study screened the most up-to-date panel of DNA-based resistance markers in An. gambiae s.l. and An. funestus s.l. from the eastern DRC, where resistance data is lacking. Several new candidate markers (CYP4J5, G119S-ace1, the triple mutant, CYP6P9a and CYP6P9b) were identified, which are diagnostic of resistance to major insecticide classes, and warrant future, larger-scale monitoring in the DRC to inform vector control decisions by the National Malaria Control Programme.

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.

Variation in mutations providing resistance to acetohydroxyacid synthase inhibitors in Cyperus difformis in China.

Cyperus difformis has evolved resistance to pyrazosulfuron-ethyl and other acetohydroxyacid synthase (AHAS) inhibitors in paddy fields in China. To understand the distribution of resistance and the mutations involved, 38 populations collected were from 7 provinces and compared. Application of pyrazosulfuron-ethyl at 30 g a.i. ha-1 identified 16 populations that survived, demonstrating resistance to this herbicide. Two exons of 498 and 1428 bp in length and a 1228-1233-bp intron of AHAS were cloned by genome walking, and three pairs of primers were designed to amplify eight conserved regions in this gene. In the 16 resistant (R) populations, five different types of mutations in the conserved region of the AHAS gene were identified: Pro-197-Ser, Pro-197-Arg, Pro-197-Leu, Asp-376-Glu, and Trp-574-Leu. Three R populations, YX15-22, YX12-10 and YX15-38, were chosen for in vitro AHAS activity assays, and the results showed that AHAS from YX15-22 carrying the Pro-197 mutation was insensitive to pyrazosulfuron-ethyl (resistance index (RI) = 310.0) and penoxsulam (RI = 10.0), whereas the enzyme from YX12--10 and YX15-38 was insensitive to pyrazosulfuron-ethyl, penoxsulam, imazapic and bispyribac­sodium (RI values ranging from 4.3 to 4462.0). AHAS inhibitor cross-resistance bioassays showed that YX12-10 and YX15-38 had cross-resistance to all of the tested herbicides (RI values ranging from 5.8 to 3321.9), while the YX15-22 population only had resistance to pyrazosulfuron-ethyl (RI = 827.4) and penoxsulam (RI = 6.6). This study clarified the distribution of resistant C. difformis in China and the different cross-resistance patterns given by various mutation types of AHAS.

Susceptibility of fall armyworm, Spodoptera frugiperda (J.E.Smmith), to eight insecticides in China, with special reference to lambda-cyhalothrin.

Fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), is the main destructive insect pest of grain crops that occurs in all maize growing regions of the Americas. It has rapidly invaded the Southern China since January 2019. However, the current status of insecticide resistance in S. frugiperda has not been reported in China. In this study, we determined the susceptibility of eight populations of FAW to eight insecticides by an artificial diet incorporation method. The results showed that among eight insecticides, emamectin benzoate, spinetoram, chlorantraniliprole, chlorfenapyr, and lufenuron showed higher toxicity to this pest, while lambda-cyhalothrin and azadirachtin exhibited lower toxicity. Susceptibility of S. frugiperda to indoxacarb was significantly different (10.0-fold for LC50) across the various geographic populations. To investigate the biochemical mechanism of FAW to lambda-cyhalothrin, we performed the synergism tests and the results showed that piperonyl butoxide (PBO) and triphenyl phosphate (TPP) produced a high synergism of lambda-cyhalothrin effects in the two field populations. Sequencing of the gene encoding the acetylcholinesterase (AChE) gene in the two field populations identified two amino acid mutations, all of which have been shown previously to confer resistance to organophosphates (OPs) in several arthropod species. The results of this study provided valuable information for choosing alternative insecticides and for insecticide resistance management of S. frugiperda.

Identification and functional characterization of a novel acetyl-CoA carboxylase mutation associated with ketoenol resistance in Bemisia tabaci.

Insecticides of the tetronic/tetramic acid family (cyclic ketoenols) are widely used to control sucking pests such as whiteflies, aphids and mites. They act as inhibitors of acetyl-CoA carboxylase (ACC), a key enzyme for lipid biosynthesis across taxa. While it is well documented that plant ACCs targeted by herbicides have developed resistance associated with mutations at the carboxyltransferase (CT) domain, resistance to ketoenols in invertebrate pests has been previously associated either with metabolic resistance or with non-validated candidate mutations in different ACC domains. A recent study revealed high levels of spiromesifen and spirotetramat resistance in Spanish field populations of the whitefly Bemisia tabaci that was not thought to be associated with metabolic resistance. We confirm the presence of high resistance levels (up to >640-fold) against ketoenol insecticides in both Spanish and Australian B. tabaci strains of the MED and MEAM1 species, respectively. RNAseq analysis revealed the presence of an ACC variant bearing a mutation that results in an amino acid substitution, A2083V, in a highly conserved region of the CT domain. F1 progeny resulting from reciprocal crosses between susceptible and resistant lines are almost fully resistant, suggesting an autosomal dominant mode of inheritance. In order to functionally investigate the contribution of this mutation and other candidate mutations previously reported in resistance phenotypes, we used CRISPR/Cas9 to generate genome modified Drosophila lines. Toxicity bioassays using multiple transgenic fly lines confirmed that A2083V causes high levels of resistance to commercial ketoenols. We therefore developed a pyrosequencing-based diagnostic assay to map the spread of the resistance alleles in field-collected samples from Spain. Our screening confirmed the presence of target-site resistance in numerous field-populations collected in Sevilla, Murcia and Almeria. This emphasizes the importance of implementing appropriate resistance management strategies to prevent or slow the spread of resistance through global whitefly populations.

Bed bugs, Cimex lectularius L., exhibiting metabolic and target site deltamethrin resistance are susceptible to plant essential oils.

Pyrethroid resistance has been a major hurdle limiting the effective control of bed bugs (Cimex lectularius L.). Alternative approaches that include the use of plant essential oils (EOs) have been proposed for effective management of bed bugs. However, EO resistance level comparisons between pyrethroid susceptible and resistant bed bug populations have not been previously conducted. The goal of this study was twofold: (i) determine deltamethrin resistance levels and associated resistance mechanisms in the field-collected Knoxville strain and (ii) quantify resistance levels of the Knoxville strain to five EOs (thyme, oregano, clove, geranium and coriander), their major insecticidal constituents (thymol, carvacrol, eugenol, geraniol and linalool) and an EO-based product (EcoRaider®). First, we found that the Knoxville strain was 72,893 and 291,626 fold more resistant to topically applied deltamethrin in comparison to the susceptible Harlan strain at the LD25 and LD50 lethal dose levels, respectively. Synergist bioassays and detoxification enzyme assays revealed significantly higher activity of cytochrome P450 and esterase enzymes in the resistant Knoxville strain. Further, Sanger sequencing revealed the presence of the L925I mutation in the voltage-sensitive sodium channel α subunit gene. The Knoxville strain that possesses both enzymatic and target site deltamethrin resistance, however, did not show any resistance to EOs, their major insecticidal constituents and EcoRaider® in topical bioassays (resistance ratio of ~1). In conclusion, this study demonstrated that a deltamethrin-resistant strain of bed bugs is susceptible to EOs and their insecticidal constituents.

Fenoxaprop-P-ethyl and mesosulfuron-methyl resistance status of shortawn foxtail (Alopecurus aequalis Sobol.) in eastern China.

Resistance to the acetyl-coenzyme A carboxylase (ACCase)- and acetolactate synthase (ALS)- inhibiting herbicides in shortawn foxtail (Alopecurus aequalis) has been reported in wheat fields of eastern China. To better understand the distribution of the resistant populations and the occurrence of the target-site mutations, 74 populations collected from Anhui, Jiangsu and Shandong province were surveyed, and the ACCase and ALS gene fragments, encompassing all the documented mutant codon positions, were amplified and sequenced. Plants from 37 and 34 populations survived fenoxaprop-P-ethyl and mesosulfuron-methyl treatment at 62.1 g a.i. ha-1 and 9 g a.i. ha-1 respectively, with different survival rates. Twenty-seven populations exhibited multiple resistance to fenoxaprop-P-ethyl and mesosulfuron-methyl. Whole-plant dose-response experiments showed that the resistance index ranged from 6.2 to 167.8 for fenoxaprop-P-ethyl, and from 7.8 to 139.5 for mesosulfuron-methyl. Four ACCase (I1781L, I2041N, I2041T and D2078G) and four ALS (P197R, P197S, P197T and W574 L) resistance mutations were detected respectively. Individuals containing two amino acid substitutions were also found. D2078G and W574 L were predominant ACCase and ALS gene mutations respectively. This study has shown that fenoxaprop-P-ethyl and mesosulfuron-methyl resistance was prevalent in A. aequalis in eastern China, and target site mutations in the ACCase and ALS gene were one of the most common mechanisms.

A point mutation in the glutamate-gated chloride channel of Plutella xylostella is associated with resistance to abamectin.

The diamondback moth, Plutella xylostella, is a global pest of cruciferous vegetables. Abamectin resistance in a field population of P. xylostella was introgressed into the susceptible Roth strain. The resulting introgression strain Roth-Abm showed 11 000-fold resistance to abamectin compared with Roth. An A309V substitution at the N-terminus of the third transmembrane helix (M3) of the glutamate-gated chloride channel of P. xylostella (PxGluCl) was identified in Roth-Abm. The frequency of the V309 allele of PxGluCl was 94.7% in Roth-Abm, whereas no such allele was detected in Roth. A subpopulation of Roth-Abm was kept without abamectin selection for 20 generations to produce a revertant strain, Roth-Abm-D. Abamectin resistance in Roth-Abm-D declined to 1150-fold compared with Roth, with the V309 allele frequency decreased to 9.6%. After treatment of the Roth-Abm-D strain with 80 mg/l abamectin the V309 allele frequency in the survivors increased to 55%. This demonstrates that the A309V mutation in PxGluCl is strongly associated with a 10-fold increase in abamectin resistance in Roth-Abm relative to Roth-Abm-D. Homology modelling and automated ligand docking results suggest that the A309V substitution allosterically modifies the abamectin-binding site, as opposed to directly eliminating a key binding contact. Other resistance mechanisms to abamectin in Roth-Abm are discussed besides the A309V mutation of PxGluCl.

Resistance of American sloughgrass (Bechmannia syzigachne) populations to ACCase-inhibiting herbicides involves three different target site mutations from China.

American sloughgrass [Beckmannia syzigachne (Steud.) Fernald] is a problematic annual grass weed in winter wheat fields of China, which causes great loss of wheat yield. Repeated use of acetyl-CoA carboxylase (ACCase)-inhibiting herbicides during the last two decades to control this weed has been selected for resistance in American sloughgrass in Jiangsu province. In this study, whole-plant dose-response assays were conducted to investigate the level of resistance in four resistant American sloughgrass populations (LY, JH, BYJ and BYP) to four ACCase-inhibiting herbicides belonging to aryloxyphenoxypropionates, cyclohexanediones, and phenylpyrazolines groups under greenhouse conditions. Based on resistance factor (RF), three populations, LY, BYJ and BYP, were highly resistant to fenoxaprop-P-ethyl, clodinafop propargyl, sethoxydim and pinoxaden. JH plants exhibited resistance to fenoxaprop-P-ethyl and clodinafop propargyl, but showed much lower RF values for sethoxydim and pinoxaden. Molecular analysis of resistance revealed that resistance in all the four populations was target site-based. Results confirmed that substitutions of Ile-1781-Leu, Ile-2041-Asn and Asp-2078-Gly, respectively, in LY, JH and BYJ/BYP, are responsible for diverse sensitivity to different ACCase-inhibiting herbicides in these populations. The substitution at position 1781 had been reported, while it is the first report of Ile-2041-Asn and Asp-2078-Gly mutations that corresponded to resistance in American sloughgrass.

Distribution, frequency and molecular basis of clethodim and quizalofop resistance in brome grass (Bromus diandrus).

BACKGROUND: Brome grass (Bromus diandrus Roth) is prevalent in the southern and western cropping regions of Australia, where it causes significant economic damage. A targeted herbicide resistance survey was conducted in 2020 by collecting brome grass populations from 40 farms in Western Australia and subjecting these samples to comprehensive herbicide screening. One sample (population 172-20), from a field that had received 12 applications of clethodim over 20 years of continuous cropping, was found to be highly resistant to the acetyl-CoA carboxylase (ACCase)-inhibiting herbicides clethodim and quizalofop, and so the molecular basis of resistance was investigated. RESULTS: All 31 individuals examined from population 172-20 carried the same resistance-endowing point mutation causing an aspartate-to-glycine substitution at position 2078 in the translated ACCase protein sequence. A wild-type susceptible population and the resistant population had similar expression levels of plastidic ACCase genes. The level of resistance to quizalofop, either standalone or in mixture with clethodim, in population 172-20 was lower under cooler growing conditions. CONCLUSION: Target-site resistance to ACCase-inhibiting herbicides, conferred by one ACCase mutation, was selected in all tested brome plants infesting a field with a history of repeated clethodim use. This mutation appears to have been fixed in the infesting population. Notably, clethodim resistance in this population was not detected by the farmer, and a high future incidence of quizalofop resistance is anticipated. Herbicide resistance testing is essential for the detection of evolving weed resistance issues and to inform effective management strategies. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Mapping and Characterization of Target-Site Resistance to Cyclic Ketoenol Insecticides in Cabbage Whiteflies, Aleyrodes proletella (Hemiptera: Aleyrodidae).

Cabbage whitefly, Aleyrodes proletella L., is an invasive hemipteran pest of cruciferous plants, particularly field brassica crops. Its importance has been increased over the last decade, particularly in European countries. The control of cabbage whiteflies largely relies on the application of synthetic insecticides, including tetronic and tetramic acid derivatives such as spiromesifen and spirotetramat (cyclic ketoenol insecticides), acting as insect growth regulators targeting acetyl-CoA carboxylase (ACC). In 2019, reduced efficacy against cabbage whiteflies of ketoenol insecticides at recommended label rates has been reported. Subsequently we collected field samples of A. proletella in different European countries and confirmed the presence of ketoenol resistance in laboratory bioassays. Reciprocal crossing experiments revealed an autosomal dominant trait, i.e., heterozygotes express a fully resistant phenotype. Transcriptome sequencing and assembly of ACC variants from resistant strains revealed the presence of an ACC target-site mutation, A2083V, as previously described and functionally validated in Bemisia tabaci (A2084V in A. proletella). Next, we employed a molecular genotyping assay to investigate the geographic spread of resistance and analyzed 49 populations collected in eight European countries. Resistance allele frequency was highest in the Netherlands, followed by Germany. Finally, we provide a proposal for the implementation of appropriate resistance management strategies.

A novel mutation in mitochondrial cytochrome b conferring resistance to bifenazate in two-spotted spider mite Tetranychus urticae Koch (Acarina: Tetranychidae).

BACKGROUND: The two-spotted spider mite Tetranychus urticae causes significant damage to ornamental, cotton, sugarcane and horticultural crops in Australia. It has a long history of developing resistance to many acaricides including bifenazate. A mutation in the conserved cd1- and ef-helices of the Qo pocket of cytochrome b is recognized as the primary mechanism of bifenazate resistance. To investigate the resistance mechanisms against bifenazate in Australian two-spotted spider mite, we sequenced the complete mitochondrion genome of five mite strains including a susceptible and bifenazate-resistant strain. RESULTS: We identified a novel mutation D252N in the G126S background at cytochrome b being the cause of bifenazate resistance in a bifenazate-resistant strain, Bram. We validated the role of this mutation combination by reciprocal crosses between a bifenazate resistant and susceptible strain. By doing these crosses we confirmed the pattern of inheritance was maternal. Additionally, mitochondrial heteroplasmy was not observed by single mite genotyping of the mutations in cytb in a known bifenazate-resistant strain Bram. The phylogenetic analysis with the complete mitochondrion genome sequences revealed that Australian two-spotted spider mite strains are closely related to the green form of T. urticae found in China. CONCLUSIONS: The novel mutation D252N found in the cytochrome b in the G126S background was revealed to be the main cause of bifenazate resistance in the Australian T. urticae strain Bram. © 2024 Society of Chemical Industry.