Pest Management Science

BACKGROUNDThe soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a pest of soybean in North America that can cause significant yield loss when outbreaks are not managed. Current management tactics primarily rely on inexpensive pyrethroids, but the sustainability of this option is threatened by insecticide-resistance in A. glycines populations across the Upper-Midwest United States. Field-evolved resistance is associated with mutations in the voltage-gated sodium channel subunit h1 (vgsc-h1) gene. RESULTSFour double-stranded RNA (dsRNA) molecules, each matching the sequence of a vgsc-h1 transcript variant ("Specific dsRNAs"), were topically applied to aphids with a genotype carrying the corresponding allele. The mortality of pyrethroid resistant aphids exposed to a Specific dsRNA increased in a dose-dependent manner when applied alone or with a constant concentration of lambda-cyhalothrin, plateauing at 1000 ng ul-1. Synergism was detected between two of four combinations of the Specific dsRNAs and lambda-cyhalothrin. These results were mirrored by the topical application of a single dsRNA with the consensus sequence of all vgsc-h1 variants ("Combined dsRNA"). Mortality was consistently higher in aphids treated with either Specific dsRNA or the Combined dsRNA, alone or with lambda-cyhalothrin, compared to insecticide alone. The number of nymphs produced per female treated with the Specific or Combined dsRNA alone decreased significantly compared to untreated controls. CONCLUSIONThis study demonstrates that the topical application of dsRNAs targeting vgsc-h1 increases the susceptibility and reduces the reproductive capacity of pyrethroid resistant soybean aphids, potentially providing a novel tool for the management of insecticide-resistant aphid populations.

Testing of insecticides and insecticide-based vector control tools relies on the use of bioassays whereby insects are exposed and mortality, or another relevant entomological endpoint, is measured. To be able to compare results between products and across time and space standard laboratory strains are used. Insecticide-susceptible strains are often used as comparators in studies involving insecticide-resistant populations. Kisumu is a long-established, widely-used insecticide-susceptible strain of Anopheles gambiae. In this study, 16 facilities across different countries exposed their Kisumu colonies aged 3-5 days to four insecticides (permethrin, alpha-cypermethrin, DDT and pirimiphos-methyl) at a range of concentrations in WHO tube assays to assess susceptibility. Insecticide-treated filter papers were made at the Liverpool School of Tropical Medicine and sent to testing centres, along with a standard protocol and testing conditions requirements, to standardise testing as much as possible. A first observation was that DDT results were affected by the use of Risella oil, the WHO-recommended carrier oil but immiscible in acetone, leading to uneven coating of papers and increased variability in bioassay results (40x variance between colonies). Bioassay data generated by the testing facilities suggest a wide range of susceptibility to different insecticides between colonies of Kisumu: LC50 values between colonies differed, though not significantly, by up to 4x for permethrin, 6x for pirimiphos-methyl, and 15x for alpha-cypermethrin. Some colonies are classified as insecticide resistant based on the WHO definition of <98% mortality on exposure to a discriminating concentration. The assumption that Kisumu is a standard susceptible strain, consistent between facilities is challenged, which has implications when comparing results between studies. We propose genotypic analysis of the colonies to identify signs of resistance markers indicative of contamination from resistant strains or wild mosquitoes, and generation of guidance to support the maintenance and monitoring of susceptible mosquito strains.

Background: Resistance to protoporphyrinogen oxidase (PPO) inhibiting herbicides is mainly driven by diverse target-site mutations, reducing the effectiveness of this site of action in row crop systems. Fendioxypyracil is a newly developed PPO inhibitor with high intrinsic grass and broadleaf activity, but its performance against resistant populations and target-site enzyme variants remains insufficiently characterized. Results: Enzyme assays using PPO2 from Amaranthus palmeri and Setaria viridis demonstrated that fendioxypyracil maintained low IC50 values across a broad range of resistance associated mutations, including dG210 deletion and G210, R128, and G399 substitutions, whereas oxadiazon, tiafenacil, and saflufenacil showed substantial loss of potency. Greenhouse dose response experiments confirmed strong fendioxypyracil efficacy, with susceptible and G399A populations controlled at <3 g ai/ha, while dG210 and R128G populations showed only moderate shifts in sensitivity but remained effectively controlled at the recommended rate. Transgenic Arabidopsis thaliana expressing resistant PPX2 alleles exhibited faster and more severe injury with fendioxypyracil compared to saflufenacil. Field trials conducted in a PPO resistant Amaranthus palmeri population demonstrated that fendioxypyracil provided consistent weed control and density reduction, matching the performance of trifludimoxazin and saflufenacil while exceeding that of fomesafen. Conclusion: Fendioxypyracil provides robust and broad-spectrum activity against PPO resistant Amaranthus populations and target mutant enzymes, maintaining efficacy across diverse mutation backgrounds. These results demonstrate its potential as an effective tool for managing PPO inhibitor resistance and sustaining weed control in row-crop production systems.

Many economically important insect pests reproduce through asexual or partially asexual life cycles, yet how reproductive mode influences insecticide resistance management remains unclear. The choice of resistance management strategy has been suggested to differ for sexual and asexual pests. For instance, current IRAC guidance suggests that pesticide mixtures are less effective in non-mating pests than in sexually reproducing populations. Here, stochastic evolutionary simulations are used to compare resistance evolution under sequences and mixtures across four reproductive modes observed in pests of economic importance: sexual reproduction, obligate parthenogenesis, cyclical parthenogenesis and haplodiploidy. Contrary to current expectations, mixtures are not disadvantaged in asexual populations and, in some cases, lead to delayed resistance evolution compared to sexually reproducing populations. These differences arise as the result of reduced genetic recombination which constrained the assembly and spread of multi-resistant genotypes. Overall, these findings suggest that mixtures remain a viable resistance management strategy for pests with asexual reproduction.

BACKGROUNDIn 2023, soybean growers in Eaton County, Michigan, reported repeated failures to control common ragweed (Ambrosia artemisiifolia L.) with (PPO)-inhibiting herbicides, sulfentrazone and fomesafen, in non-genetically modified soybeans. This study aimed to investigate resistance levels and mechanisms of resistance in two suspected resistant populations (R1 and R2). RESULTSDose-response assays revealed resistance to sulfentrazone in both populations, with LD50 values 24 and 36-fold higher than the susceptible population and reduced sensitivity to fomesafen. Nanopore sequencing identified two PPO2 target-site substitutions at codon 98: Arg- 98-Leu (R98L) and a novel Arg-98-Gln (R98Q) mutation. Both substitutions were associated with reduced herbicide binding affinity in computational modelling simulations. R98Q substitution conferred strong and selective resistance in the PPO2 enzyme inhibition assays, supporting its role as a key resistance mechanism. CONCLUSIONThis study reports the first field occurrence of the R98Q substitution in PPO2 of A. artemisiifolia populations and demonstrates its association with to PPO inhibitor resistance. These findings highlight the rapid evolution of target-site resistance and the need for continued monitoring and rapid diagnostic tools to detect emerging mutations. Further research is needed to clarify the contribution of additional resistance mechanisms.

IntroductionProgress against malaria has stalled since 2015, with insecticide resistance a key driver. Spatial emanators release volatile insecticides into the air, exposing mosquitoes through a route distinct from the tarsal contact used by treated nets, indoor residual spraying, and standard bioassays. Transfluthrin is currently monitored using the WHO bottle bioassay, which combines contact and vapour exposure and cannot isolate airborne effects. A scalable, vapour-only method is needed to characterise susceptibility to volatile pyrethroids. MethodsWe adapted the WHO tube bioassay to deliver non-contact transfluthrin vapour exposure without contacting treated papers. Non-blood-fed female Anopheles were exposed to acetone control or transfluthrin papers at 0.005, 0.1 and 2 mg/paper. Knockdown was recorded over 60 minutes and mortality at 24 hours. Testing spanned three laboratories (LSTM, AIRID and KEMRI) using susceptible reference and resistant lab colonies. Dose response models estimated EC{square}{square} for knockdown (EC{square}{square}KD) and mortality (EC{square}{square}Mort), with resistance ratios (RR) against the local susceptible reference. ResultsThe assay generated clear concentration- and time-dependent responses. At LSTM, Kisumu gave an EC{square}{square}KD of 0.052 and EC{square}{square}Mort of 0.054 mg/paper; Siaya was comparable (RR{square}{square}KD 1.17, RR{square}{square}Mort 0.89), whereas Tiassale 13 and KDR showed reduced susceptibility (RR{square}{square}KD 2.42 and 2.85; RR{square}{square}Mort 5.56 and 6.65). At AIRID, the resistant Cove strain did not reach 50% knockdown or mortality. At KEMRI, Siaya showed reduced susceptibility (RR{square}{square}KD 3.36/RR{square}{square}Mort 5.65). Kisumu at AIRID and KEMRI was comparable to LSTM (EC{square}{square}KD 0.007/EC{square}{square}Mort 0.030; EC{square}{square}KD 0.044/EC{square}{square}Mort 0.023). Despite inter-laboratory variation, susceptible strains remained distinguishable from resistant ones. Lower RRs than for contact pyrethroids suggest contact-based phenotypes may not predict vapour susceptibility. ConclusionThe assay provides a practical benchtop method using standard WHO hardware, distinguishing susceptible from resistant strains across three laboratories. Twenty-four-hour mortality is the recommended endpoint, 60-minute knockdown secondary. Not yet validated, it provides a foundation for further validation and adaptation to other actives.

1- Biological control of mosquitoes using aquatic predators offers a sustainable alternative to chemical insecticides, yet the specific predator and prey functional traits that govern consumption efficacy remain poorly quantified at a global scale. 2- We conducted a global meta-analysis of 755 effect sizes across 59 studies to evaluate how predator identity (fish vs. odonate naiads), body size, dietary guilds, and prey characteristics influence consumption rates. Using multilevel models and robust publication-bias corrections, we quantified predation efficiency, expressed throughout as the consumption rate (CR, larvae predator -1 h -1 ), while accounting for methodological variations across experimental designs. The primary literature itself proved geographically skewed towards Asia (chiefly India), with Africa, the Americas, and Europe markedly under-represented. 3- Grouping predators solely by broad taxonomic identity concealed the central pattern in our data. Although naiads outperformed fish when compared directly within the same studies, this taxon-level difference was driven almost entirely by non-mosquitofish species, the least efficient predator group overall. Mosquitofish (\textit{Gambusia} spp.) and dragonfly naiads were statistically indistinguishable from one another, indicating that dietary specialisation, not taxonomic identity, is the stronger predictor of predation efficacy. Predator body size strongly and positively predicted consumption rates in naiads---driven primarily by dragonflies---but showed no significant or negative relationship in fish. 4- Methodological traits heavily structured the extreme heterogeneity observed across studies; notably, exposure time acted as a severe rate-suppressor, where prolonged assays drastically underestimated per-hour consumption rates due to satiety or handling constraints. Nevertheless, a combined model incorporating all significant ecological moderators simultaneously explained a substantial share of the between-study variance, confirming that predator-prey dynamics in these systems are highly predictable from functional traits. 5- Effective biological control cannot rely on broad taxonomic assumptions but requires evidence-based trait-matching. Management programs should prioritise body size when deploying insect predators, selecting the largest individuals within species known to consume mosquito larvae and favour insectivorous fish species over generalists. Crucially, because short-term laboratory assays artificially inflate efficacy, multi-duration assessments are essential to accurately scale up biocontrol predictions from experimental arenas to complex, real-world ecosystems.

A parasitic mite of honey bee brood (Tropilaelaps mercedesae), is spreading through populations of Apis mellifera honey bees in new regions and poses a major threat to honey bee health. Despite its clear threat, the biology of this mite is poorly understood, with gaps on such fundamental issues as how fast its populations can grow. This leaves the beekeeping world underprepared to plan for its arrival and management. In this study, we documented the growth of T. mercedesae populations in untreated A. mellifera colonies in Thailand and South Korea, and did the same for another parasitic mite (Varroa destructor) when possible. We found that the population growth of T. mercedesae was variable but could reach high levels (daily r of 0.010, 0.036, and 0.057), while the population growth of V. destructor (r = 0.021) matched previous estimates. Our results indicate that T. mercedesae populations can grow rapidly but they do not always attain this potential. Based on our results, humidity should be studied as a potential driver of population growth. If future work can reveal key drivers of T. mercedesae population growth, this would help predict infestations and help design management strategies that exploit the pest's biological vulnerabilities.

Herbicide resistance threatens effective weed control in modern agriculture, particularly in grass weeds such as Alopecurus myosuroides and Lolium multiflorum. Cinmethylin is a pre-emergence herbicide with a novel mode of action that inhibits plastidial fatty acid thioesterases (FATs), enzymes essential for fatty acid biosynthesis. Although no cases of field resistance to cinmethylin have been reported, its resistance risk has not been fully assessed. In this study, we biochemically characterized defined amino acid substitutions in FAT A and FAT B to evaluate their effects on cinmethylin inhibition profile. Some substitutions in FAT A reduced inhibition in vitro, with mutations at residue R171 causing the largest shifts in sensitivity. However, these highly resistant variants required multiple specific nucleotide polymorphisms and are therefore predicted to be unlikely to arise in weed populations. In FAT B, sensitivity shifts were generally moderate. Importantly, most substitutions that reduced cinmethylin sensitivity also impaired enzymatic activity, suggesting limited viability in planta. Overall, these results indicate that while theoretical target-site resistance mechanisms exist, the practical risk of rapid resistance evolution to cinmethylin is low, supporting its value for integrated grass weed management

Neurotoxic chemicals, including pyrethroids, neonicotinoids, and chlorinated hydrocarbons, have been effective for mosquito control; however, their continued use has resulted in widespread insecticide resistance. This resistance has necessitated increased insecticide application, a phenomenon known as the pesticide treadmill, which poses significant risks to both environmental and human health. Consequently, there is a growing need for sustainable alternatives with minimal adverse impacts. Although plant-based repellents have shown promise, many are formulated as topical products that may be costly, inconvenient, or culturally unacceptable in some communities. Despite the documented mosquito-repellent properties of Hyptis suaveolens, its traditional use relies largely on fresh plant material, which is limited by seasonality and may not provide year-round protection. In addition, evidence on practical, sustained-release delivery systems for plant-derived mosquito repellents remains limited. Candle wax represents a readily available and culturally familiar medium that may facilitate the gradual release of volatile repellent compounds during combustion without requiring direct skin application. This study therefore evaluated the mosquito-repellent efficacy of Hyptis suaveolens extracts infused into candle wax as a simple plant-based delivery system. Leaves of H. suaveolens were collected, air-dried, ground, and extracted using ethanol and water. The crude extracts were incorporated into melted candle wax at a 5:10 concentration, then solidified and burned in a mosquito-infested room for 30 minutes. Results indicated that rooms with infused candles had significantly fewer mosquitoes than the control room. These findings support the mosquito-repellent properties of Hyptis suaveolens and demonstrate that its incorporation into candle wax is a promising plant-based delivery system for mosquito control, warranting further evaluation under field conditions.

Personal insect repellents can offer an effective means of preventing mosquito bites. Despite a wide variety of products that claim to repel mosquitoes, relatively few studies have systematically compared their performance through standardized tests. In Australia, chemical-based repellents must be registered with the Australian Pesticides and Veterinary Medicines Authority (APVMA); however, many unregistered products with unverified effectiveness remain commercially available. Here, we tested 55 insect repellents and 5 non-repellent perfumes and deodorants sold in Australia for their protection against Aedes aegypti mosquito landings (as a proxy for bites) using arm-in-cage assays across eight human subjects. We tested 43 topical repellents (containing 18 different primary active ingredients), four wristbands, three clothing stickers, three ultrasonic devices, one garment, one repellent applied to fabric, two perfumes, two deodorants and one antiperspirant. Thirteen repellents, including all wristbands, stickers and ultrasonic devices did not provide complete protection against mosquito landings for any human subject, while a further sixteen provided complete protection for 1 hr or less on average. Repellents containing DEET, oil of lemon eucalyptus, picaridin and IR3535 as the primary active ingredient were the only products that provided more than 2 hours of complete protection, but with significant variation among products with the same active ingredient. Only 7/30 essential oil-based topical repellents and perfumes provided complete protection for greater than 1 hr. Nearly half (25/52) of the repellents subject to registration with the APVMA were unregistered despite being advertised or labelled as mosquito repellents, and these tended to be less effective than registered products. Only 5/28 repellents met or exceeded their claimed protection time. This study highlights the prevalence of unregistered and ineffective mosquito repellents on the Australian market while providing evidence for products and active ingredients that do protect against mosquito bites under laboratory conditions.

Fungal and oomycete plant pathogens are major drivers of yield losses worldwide and are spurring the search for sustainable alternatives to synthetic pesticides. Algae, in general, and microalgae, in particular, represent a promising source of bioactive compounds for crop protection. Despite this, their efficacy across different host-pathogen systems remains poorly characterised. This study evaluated the biocontrol potential of the aqueous extract of Chlorella sorokoniana against three economically important phytopathogens using complementary in vitro, ex vivo, and in planta assays. The strongest activity was observed against Magnaporthe oryzae, with the extract reducing fungal growth by approximately 70% in vitro, inhibiting appressorium formation by 55%, suppressing lesion development on detached rice leaves by more than 75%, and reducing rice blast severity by 64.5% as a preventive foliar treatment. In contrast, the extract showed little or no direct in vitro antifungal activity against Pythium ultimum and Rhizoctonia solani, yet it significantly reduced disease severity in planta by 13-37% and 48-55%, respectively. The contrasting responses among pathosystems suggest that C. sorokoniana aqueous extracts act through different mechanisms depending on the pathogen and the crop, combining direct antifungal activity against M. oryzae with plant-associated protective effects against soil-borne pathogens. These findings highlight the importance of evaluating candidate biocontrol products using complementary in vitro and in planta approaches, as laboratory antimicrobial assays alone may substantially underestimate their agricultural potential. The broad-spectrum protection achieved with an unrefined aqueous extract further supports C. sorokoniana as a promising source of sustainable crop protection products and provides a strong foundation for future mechanistic studies, formulation development, and field validation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/742703v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@394716org.highwire.dtl.DTLVardef@6a208corg.highwire.dtl.DTLVardef@17f0724org.highwire.dtl.DTLVardef@adda96_HPS_FORMAT_FIGEXP M_FIG C_FIG

IntroductionChanges in vector control strategies alter the selection pressures exerted on natural populations of Anopheles gambiae s.l. and may influence the dynamics of insecticide resistance mechanisms. However, the consequences of discontinuing indoor residual spraying (IRS) campaigns on the evolution of Ace-1-mediated resistance remain poorly documented under natural conditions. This study aimed to assess the spatiotemporal evolution of resistance to pirimiphos-methyl following the cessation of IRS and to investigate evidence consistent with the existence of a biological cost associated with Ace-1-mediated resistance. MethodsNatural populations of An. gambiae s.l. were collected between 2017 and 2023 in three districts in Burkina Faso that had undergone IRS campaigns (Kampti, Solenzo, and Kongoussi). Susceptibility tests with pirimiphos-methyl (0.25%) were conducted out following WHO protocols, and a subsample of exposed mosquitoes was genotyped to detect the Ace-1 G119S mutation. Spatiotemporal trends in mortality, allele frequencies and genotypes were analyzed according to the pre-IRS, IRS, and post-IRS periods. The association between the Ace-1 genotype and survival following exposure to pirimiphos-methyl was assessed using logistic regression, while the concordance between phenotypic and molecular indicators of resistance was examined using Spearmans correlation. ResultsThe susceptibility of An. gambiae s.l. populations to pirimiphos-methyl was gradually restored after the discontinuation of IRS at all sites. At the same time, the frequencies of the Ace-1 119S resistance allele declined sharply, particularly in Kampti and Solenzo, while they remained low in Kongoussi throughout the study period. Mosquitoes carrying resistant genotypes had a significantly higher probability of survival after exposure to pirimiphos-methyl than susceptible homozygotes, with resistant homozygotes (RR) exhibiting the greatest survival advantage (OR = 27.62; 95% CI: 6.91-110.45; p < 0.001). A significant negative correlation was observed between the frequency of the Ace-1 119S allele and phenotypic mortality ({rho} = -0.48; p = 0.033), indicating a concordance between the two indicators of resistance. The progressive decline in allele frequencies, the decreasing prevalence of resistant genotypes, and the concomitant restoration of susceptibility are field observations consistent with the existence of biological costs associated with Ace-1-mediated resistance. ConclusionThis study provides field evidence consistent with the existence of biological costs associated with Ace-1-mediated resistance in natural populations of An. gambiae s.l. These results underscore the value of an adaptive resistance management strategy based on alternating selection pressures and could guide future vector control strategies, particularly if indoor residual spraying campaigns or other interventions relying on organophosphates were reintroduced.

Bacterial leaf blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), causes yield losses exceeding 50% in affected areas, including the Bagre rice plain in Burkina Faso. Genome-edited (GEd) rice lines have been successful in tackling BB. Modifications in the Xoo virulence protein target site upstream of three SWEET susceptibility genes in two elite rice varieties, IR64 and Ciherang-Sub1, have been demonstrated to confer broad-spectrum resistance to Asian and East African Xoo strains. Here, we evaluate the potential of the GEd lines as a solution for BB management in Burkina Faso. We challenged the GEd lines against five locally collected Burkinabe Xoo strains under controlled green-house conditions and assessed their agro-morphological performance under field conditions representative of local agroecological conditions. Greenhouse pathogen assays demonstrated that GEd IR64 and Ciherang-Sub1 lines were resistant to all tested local Xoo strains across three successive generations. We identified TalC as the primary disease-causing effector in the local Xoo populations. Irrigated field trials conducted over two seasons in the Kou Valley, Burkina Faso, revealed absence of agro-morphological penalties in GEd lines compared to their parental wild-type lines. Observed trait variation was attributable to environmental fluctuations rather than genomic modifications. Collectively, our findings demonstrate that genome editing of the rice lines does not impose growth penalties, and support the suitability of GEd IR64 and Ciherang-Sub1 for large-scale adoption in Burkina Faso, pending multi-location validation and introgression into locally adapted varieties.

Plant-parasitic nematodes (PPNs) are among the most destructive agricultural pests worldwide, causing significant economic losses across diverse cropping systems. Soil fumigation, the most common management strategy, is costly, detrimental to soil health, and increasingly restricted due to environmental and regulatory concerns. As a result, there is a critical need for alternative, sustainable approaches for PPN control. Solanum sisymbriifolium is resistant to several Meloidogyne species and represents a promising source of natural nematicidal compounds. In this study, we evaluated the effects of S. sisymbriifolium extracts on Meloidogyne chitwoodi and M. hapla, two economically important nematodes. Compounds were extracted using solvents of increasing polarity and then reconstituted in water. The water-solubilized extracts were then used in bioassays to assess their effects on nematode egg hatching, egg viability, and second-stage juvenile (J2) survival. Egg hatching and J2 viability of both Meloidogyne species were consistently affected by compounds in the 1-butanol fraction. Further characterization of these compounds may enable the development of novel, environmentally sustainable alternatives to conventional nematode management strategies.

Pesticides remain indispensable for global food security, yet their use must be reconciled with the preservation of biodiversity. Despite advances in developing safer pesticides, their sublethal effects and synergistic potential with co-formulants in tank-mixtures, including inert spray adjuvants, often remain poorly understood in beneficial insects like bees. In this study, we assessed the effects of acute oral exposure to field-realistic doses of SilwetTM L-77 (an organosilicone adjuvant; 0.1-2.5%), acetamiprid (a cyano-substituted neonicotinoid; 6-150 ng/bee), and sulfoxaflor (a sulfoximine; 3-27 ng/bee) on gustatory responsiveness, associative olfactory learning, and short-term memory retention in honeybees (Apis mellifera L.). While we found no significant evidence of interaction effects at the exposure concentrations tested, the highest doses of SilwetTM L-77 and sulfoxaflor alone significantly reduced gustatory responsiveness. Furthermore, all three agrochemicals alone weakly affected associative learning without impacting memory retention. Although adjuvant-insecticide mixtures did not synergistically impair bee cognition, these adjuvants can impact sensory perception. Moving beyond mortality-based assessments and including sensory-cognitive thresholds is essential for a more holistic understanding of pollinator health.

The tomato leafminer, Tuta absoluta, poses a severe global agricultural threat due to its rapid leaf-mining behavior and swift development of resistance to conventional chemical pesticides. While microbial chitinases are potent biopesticides, their field efficacy is limited by environmental degradation and the short exposure window before larvae penetrate leaf tissues. This study evaluates a stimuli-responsive, controlled-release nanobiopesticide system utilizing a novel chitinase from newly characterized Serratia marcescens GBS19. A 61.1 kDa chitinase (GBS19_ChiA) was heterologously expressed in Escherichia coli and purified to a specific activity of 215.01 U/mg. The enzyme was immobilized onto starch-coated silica nanoparticles designed for target-triggered release via host alpha-amylase. Genomic profiling and R-based kinetic modeling were integrated to evaluate the efficacy of purified and immobilized forms against T. absoluta. Immobilization enhanced thermal and pH stability, with the nanocarrier maintaining 85% activity over 10 weeks. In larval bioassays, immobilization increased mortality from 21.9% to 59.4% (5000 U/mL) by day 3, reaching 62.5% by day 6. Genomic analysis identified an expansive secretome and a Type VI Secretion System (T6SS), characterizing GBS19 as a multi-pronged pathogen. Kinetic modeling established that while immobilized enzymes are effective, the 2.5-hour exposure time on T. absoluta requires the synergistic action of chitinases (ChiA/B/C) to reach the lethal desiccation threshold before larvae establish protective mines. Starch-coated silica nanoparticles significantly improve chitinase stability and delivery. However, overcoming the rapid penetration of T. absoluta necessitates a whole-cell or multi-enzyme synergistic approach to outpace larval behavioural defences.

BACKGROUNDReduced control of giant ragweed (Ambrosia trifida L.) with protoporphyrinogen oxidase (PPO)-inhibiting herbicides was recently reported in two southern Illinois populations, VRC and TMS. The objectives of this study were to assess resistance to postemergence-applied PPO inhibitors in VRC and TMS, evaluate VRC response to acetolactate synthase (ALS)- and enolpyruvyl shikimate phosphate synthase (EPSPS)-inhibiting herbicides, and identify target-site mechanisms associated with PPO-inhibitor resistance. RESULTSBased on LD estimates, VRC resistance ratios ranged from 1.1- to 3.7-fold for lactofen and 2.2- to 6.9-fold for fomesafen relative to PPO-sensitive populations SIU and DSO. In TMS, LD estimates were 257.4 g ai ha {superscript 1} for lactofen and 318.4 g ai ha {superscript 1} for fomesafen. Glyphosate LD estimates exceeded three times the labeled field rate in VRC, SIU, and DSO, whereas VRC and SIU had higher cloransulam-methyl LD estimates than DSO. Whole-transcriptome sequencing identified polymorphisms in PPX1 and PPX2; however, only PPO2 R98Q altered a catalytic-domain binding-pocket residue and was considered likely to contribute to resistance in VRC. R98Q was absent in TMS, and PPX1 and PPX2 expression did not differ among populations. CONCLUSIONVRC and TMS have evolved resistance to lactofen and fomesafen, and VRC also exhibited reduced sensitivity to cloransulam-methyl and glyphosate. PPO2 R98Q is novel in A. trifida and, to our knowledge, represents the first report of this mutation associated with PPO-inhibitor resistance in plants. The absence of target-site alterations in TMS suggests a potential non-target-site basis for resistance. These findings highlight the need for integrated, diversified management to further reduce herbicide selection pressure.

Xylosandrus crassiusculus (Motschulsky), the granulate ambrosia beetle, was one of the first highly-destructive ambrosia beetles introduced into the southern U.S in the 1970s where it was found in South Carolina (Kovach 1986). The Redbay ambrosia beetle, Xyleborus glabratus Eichhoff, was first detected in the U.S. in South Georgia in 2002. This beetle and its associated fungi, the laurel wilt fungus Raffaelea laurelensis and others have caused substantial destruction to native redbay (Persea borbonia) in GA, SC, FL and elsewhere. This beetle-pathogen complex also poses a threat to commercial avocado production in the U.S., Central and South America as well as to valuable other Persea spp. and related plants (Laureacea) that are known hosts. As an addition here, 10 years of the spring appearances (Fig.1) of X. crassiusculus in North Florida is offered for future comparisons. A second unusual appearance is the finding and working with UV mulch and ethanol, as a surprising attraction of X. crassiusculus and other ambrosia beetles including X. glabratus. It was also found that the ambrosia beetles do not respond to yellow and green as expected by most. Also, adding burlap was found to be attractive (increases dead and dying appearing trees) as is silver metallic like UV mulch, while camouflage (camo) was found to work like yellow and green. These occurrences led to the invention and development of UV mulch with new traps to better monitor ambrosia beetles. New traps led to new uses for yellow, green and camo to monitor and decrease damage and losses from ambrosia beetles. The data are presented as evaluated and appear in the figures, discussion and a supplemental section. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/733798v1_fig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1006101org.highwire.dtl.DTLVardef@1e0a3d6org.highwire.dtl.DTLVardef@1244d1borg.highwire.dtl.DTLVardef@423cb7_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1:C_FLOATNO Relative timing of annual emergence of Xylosandrus crassiusculus in north Florida. Collected over 10 years using 5 Baker traps with a 10% ethanol/water solution. Data are from years as marked. Note: data from year 2003 was not collected. C_FIG

Accurately determining the mating status of the agricultural fruit fly pest Ceratitis capitata, commonly known as Medfly, is essential for timely and effective eradication efforts. To overcome the limitations of subjective DAPI-based staining assessments of females captured in Jackson dry traps and Multilure liquid traps, we developed a multi-tier molecular diagnostic method that unequivocally detects mating status using DNA probes targeting the male-specific Y114 locus on the Y-chromosome of the species. Our protocol integrates morphological evaluation with increasingly sensitive molecular assays through the following steps: 1) A preliminary quality assessment of the specimens physical condition, DNA preservation, and mating status using conventional PCR followed by agarose electrophoresis (cPCR); 2) Quantification and real-time detection of sperm presence via quantitative PCR (qPCR); and 3) Detection of trace sperm amounts through droplet digital PCR (ddPCR). This PCR-based framework is designed for samples collected in the field, enabling accurate analysis of specimens exposed to adverse environmental conditions and varying levels of preservation after 2- and 3-weeks weathering times in traps. It allows quantitative determination of mating status even when sperm concentrations are extremely low, such as during transient copulation, and achieves detection limits down to approximately 14 spermatozoa in a mated female. By accounting for variable specimen quality and the performance characteristics of each molecular platform, this tiered approach ensures highly sensitive and unequivocal detection of mated females. The methodology can be used to assist eradication efforts across the C. capitata geographic range through the timely detection of mated females, halting their expansion and establishment into novel regions reducing control and eradication costs.