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.

A high-throughput multilocus-amplicon sequencing panel to monitor insecticide resistance in fall armyworm (FAW) Spodoptera frugiperda (Lepidoptera: Noctuidae).

BACKGROUND: Fall armyworm (FAW), Spodoptera frugiperda, is a highly polyphagous crop pest that has spread over the world rapidly and invaded Australia in 2020. Globally, FAW has been reported to be resistant to several insecticides permitted in Australia. Timely resistance diagnosis is critical for integrated pest management-based control of FAW in Australia. RESULTS: We developed a multi-amplicon panel by next-generation sequencing (multiamplicon-seq) to identify known insecticide resistance mutations in Australian FAW with high throughput and low cost. The panel included nine known mutations causing insecticide resistance in FAW and four gene mutations causing insecticide resistance in several insect species, not yet reported in FAW. We sequenced 36 plates (96-well) in one MiSeq flow cell with easy sequencing library preparation. We found that Australian FAW carried a very high proportion of the F290V mutation in the acetylcholinesterase (AChE) gene that causes resistance to organophosphate and carbamate insecticides. Furthermore, FAW has a GABA-activated chloride channel mutation, A301Q in the RDL gene. The sequencing-based platform provided evidence of a duplication in the AChE gene. Here several single nucleotide polymorphisms (SNPs) within the 476-bp amplicon of the AChE gene demonstrated 100% heterozygosity across samples and some individuals carried two haplotypes with the F290V mutation. CONCLUSION: Molecular surveillance by multiamplicon-seq will increase capacity for early detection and future resistance monitoring in highly dispersed Australian FAW. It can provide timely resistance information and has the potential to play an important role in the resistance management of FAW. © 2023 Society of Chemical Industry.

Mutation (G275E) of nAChR subunit Foα6 associated with spinetoram resistance in Australian western flower thrips, Frankliniella occidentalis (Pergande).

Western flower thrips, Frankliniella occidentalis is an economically important agricultural pest. It causes damage by feeding and oviposition or indirectly by plant virus transmission. Australian F. occidentalis are resistant to many insecticides including spinosad and the related chemical spinetoram. Spinetoram resistance to F. occidentalis has been recently reported in three different Australian States, however, mechanisms conferring that resistance have not been investigated. To identify the mechanisms underlying resistance to spinetoram in F. occidentalis, we sequenced the genomic region of nicotinic acetylcholine receptor Foα6 in number of spinosad and spinetoram resistant field-populations. We found that a single nucleotide substitution (G to A) in exon 9 of the α6 subunit was present in resistant strains (G275E) and absent from susceptible. By examining field populations we consider the G275E mutation is the major cause of resistance to spinetoram in Australian F. occidentalis. We developed a real-time PCR diagnostic assay to quickly identify resistant alleles in field-populations. The method was used to test spinetoram resistant F. occidentalis collected from Australian cotton during the 2018-2019. Results show thrips tested carried the G275E mutation and the resistance allele was unusually widely distributed. The wide distribution of G275E mutation was not expected because spinetoram is not extensively used in Australian cotton. We speculate resistance may relate to extensive chemical use in crops nearby such as horticulture where thrips are often targeted for control. Our molecular diagnostic assay can provide timely and precise resistance frequency information that can support sustainable chemical use including spinetoram based IPM.

Development of abamectin resistance in Tetranychus urticae in Australian cotton and the establishment of discriminating doses for T. lambi.

Since the 1980s Tetranychus urticae Koch has dominated Australian cotton due to its ability to develop resistance. Here we give screening data for a range of chemicals tested against T. urticae including abamectin, bifenthrin, diafenthiuron, etoxazole and propargite and speculate why abamectin resistance emerged without warning. Abamectin resistance was not detected in T. urticae in Australian cotton before season 2007-2008 when a few resistant individuals were detected in a single strain. Resistance was detected again in season 2010-2011 and continued to be detected in every subsequent season comprising 80% of strains tested in 2018-2019. We speculate the reason may relate to prophylactic abamectin use to prevent mite flare with Creontiades dilutes Stål mirid sprays. With the introduction of transgenic Bt-cotton, spraying significantly reduced and anecdotally Tetranychus lambi became more abundant. Although T. lambi may now be more common than T. urticae its response to chemical controls is completely unknown. Tetranychus lambi conspecific dose responses were established to support resistance monitoring against abamectin, diafenthiuron and propargite that generated discriminating dose (DD) estimates of 0.0007 g/L abamectin, 0.03 g/L diafenthiuron and 0.7 g/L propargite. These DD were used in season 2018-2019 but resistance was not detected against any product including abamectin. The reason why T. lambi may now dominate despite T. urticae being still resistant is speculated and thought related to the progressive reduction in insecticide use in Australian cotton and/or the changing weed complex in the transgenic cotton era.

The Management of Insect Pests in Australian Cotton: An Evolving Story.

The Australian cotton industry progressively embraced integrated pest management (IPM) to alleviate escalating insecticide resistance issues. A systems IPM approach was used with core principles that were built around pest ecology/biology and insecticide resistance management; together, these were integrated into a flexible, year-round approach that facilitated easy incorporation of new science, strategies, and pests. The approach emphasized both strategic and tactical elements to reduce pest abundance and rationalize decisions about pest control, with insecticides as a last resort. Industry involvement in developing the approach was vital to embedding IPM within the farming system. Adoption of IPM was facilitated by the introduction of Bt cotton, availability of selective insecticides, economic validation, and an industry-wide extension campaign. Surveys indicate IPM is now embedded in industry, confirming the effectiveness of an industry-led, backed-by-science approach. The amount of insecticide active ingredient applied per hectare against pests has also declined dramatically. Though challenges remain, pest management has transitioned from reactively attempting to eradicate pests from fields to proactively managing them year-round, considering the farm within the wider landscape.

Spinosad resistance, esterase isoenzymes and temporal synergism in Frankliniella occidentalis (Pergande) in Australia.

Spinosad has been widely used in Australia to control western flower thrips Frankliniella occidentalis (Pergande) but spinosad usefulness is now compromised by resistance. Here we studied a highly spinosad resistant strain of F. occidentalis to explore if esterases had a role in spinosad resistance. Enhanced esterase activity in pressured spinosad-resistant F. occidentalis was confirmed via PAGE electrophoresis and estimated to be approximately three times higher than that in a susceptible strain. Spinosad-esterase inhibition data in the resistant strain, showed a concentration effect with significant esterase-spinosad binding occurring at spinosad concentrations from 6.2× 10(-7) to 1.5× 10(-5) M. Similarly, a spinosad-piperonyl butoxide (PBO) inhibition curve showed a concentration effect, with significant esterase-PBO binding occurring in the resistant strain at PBO concentrations between 3.3× 10(-5) M and 8.4× 10(-4) M. No binding of esterase to spinosad or PBO occurred in the susceptible strain. Results of bioassays in which spinosad resistant F. occidentalis were sprayed with a 4h delayed release formulation of cyclodextrin-complexed spinosad with immediately available PBO demonstrated that spinosad resistance was significantly reduced from 577 to 72-fold. With further development the PBO synergism of spinosad using a delayed release formulation, similar to that used here, may provide effective control for spinosad resistant F. occidentalis. Temporal synergism of spinosad may prove to be effective tactic for the control of spinosad resistant F. occidentalis where the main resistance mechanism involved has been confirmed to be esterase based.

Quantification of the pirimicarb resistance allele frequency in pooled cotton aphid (Aphis gossypii Glover) samples by TaqMan SNP genotyping assay.

BACKGROUND: Pesticide resistance monitoring is a crucial part to achieving sustainable integrated pest management (IPM) in agricultural production systems. Monitoring of resistance in arthropod populations is initially performed by bioassay, a method that detects a phenotypic response to pesticides. Molecular diagnostic assays, offering speed and cost improvements, can be developed when the causative mutation for resistance has been identified. However, improvements to throughput are limited as genotyping methods cannot be accurately applied to pooled DNA. Quantifying an allele frequency from pooled DNA would allow faster and cheaper monitoring of pesticide resistance. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate a new method to quantify a resistance allele frequency (RAF) from pooled insects via TaqMan assay by using raw fluorescence data to calculate the transformed fluorescence ratio k' at the inflexion point based on a four parameter sigmoid curve. Our results show that k' is reproducible and highly correlated with RAF (r >0.99). We also demonstrate that k' has a non-linear relationship with RAF and that five standard points are sufficient to build a prediction model. Additionally, we identified a non-linear relationship between runs for k', allowing the combination of samples across multiple runs in a single analysis. CONCLUSIONS/SIGNIFICANCE: The transformed fluorescence ratio (k') method can be used to monitor pesticide resistance in IPM and to accurately quantify allele frequency from pooled samples. We have determined that five standards (0.0, 0.2, 0.5, 0.8, and 1.0) are sufficient for accurate prediction and are statistically-equivalent to the 13 standard points used experimentally.

Evidence of superclones in Australian cotton aphid Aphis gossypii Glover (Aphididae: Hemiptera).

BACKGROUND: Aphis gossypii is an important pest of cotton that has developed resistance to many chemicals used for its control. Any lack of understanding of its genetic structure, resistance status and host plant specialisation hampers effective management. RSULTS: Eight microsatellite markers were genotyped for a collection of Australian A. gossypii field isolates from 55 plant species from major Australian cotton-producing regions. The aphid's pirimicarb resistance status linked to the ACE1 (acetylcholinesterase) S431F mutation was determined by PCR-RFLP. Overall, the genetic diversity was low and there were only 13 multilocus genotype (MLG) groups found in a total of 936 aphids, suggesting asexual reproduction. Three MLGs (Aust-01, Aust-02 and Aust-04) represented 78% of all aphids tested. MLGs Aust-01 (41%) and Aust-02 (18%) were linked to the ACE1 S431F mutation and found on cotton and a range of hosts. Aust-04 (19%) hosted mainly on cotton (but also Asteraceae and Malvaceae) was predominantly susceptible to pirimicarb. Given their abundance and widespread occurrence, these three clones were considered to be superclones. CONCLUSION: The study demonstrated that any strategy to control A. gossypii and manage pirimicarb resistance should target A. gossypii strains of all MLG types residing on any plant species and not just cotton

The influence of sublethal deposits of agricultural mineral oil on the functional and numerical responses of Phytoseiulus persimilis (Acari: Phytoseiidae) to its prey, Tetranychus urticae (Acari: Tetranychidae).

Occasional pesticide application in integrated pest management to at least part of a crop requires that any biological control agents must re-invade previously sprayed areas in order that resurgent pests can be constrained. The ability of the phytoseiid predatory mite Phytoseiulus persimilis to feed on adult two-spotted spider mite (TSSM) Tetranychus urticae on excised leaf discs in both control conditions and in a treatment with a sub lethal residue of agricultural mineral oil (AMO) was assessed. The predator exhibited a Type II functional response with the asymptote significantly higher in the AMO conditions due to the fact that the prey grew slower and reached a smaller size in this treatment. In terms of prey volume eaten, the satiation level of the predator was unchanged by the AMO deposits. The numbers of eggs produced by adult P. persimilis females at densities of 4, 8 and 16 TSSM adult females/disc in the control were significantly higher than those in the AMO treatment, but were similar for the higher density levels, 32 and 64 prey per disc. Thus the functional response in terms of volume of prey eaten explained the numerical response in terms of predator eggs produced. The presence of AMO deposits when the prey were at high density had no effect on predator efficiency (volume eaten) but resulted in a lower intake than that in control conditions when there was a greater distance between prey.