Resistance In Field Populations Research Articles

Current situation and forecasting of resistance evolution to lambda-cyhalothrin in Spanish medfly populations.

BACKGROUNDThe control of the Mediterranean fruit fly Ceratitis capitata (Wiedemann) in Spanish field populations mainly relies on the insecticides lambda‐cyhalothrin and spinosad as bait sprays. However, their sustainable used is compromised by the development of lambda‐cyhalothrin resistance and the detection of spinosad resistant alleles. In addition, the use of lure‐and‐kill traps covered with deltamethrin has increased in the last years. It is thus urgent to predict the impact that the combination of both pyrethroids will have in the evolution of lambda‐cyhalothrin resistance and how they could be combined with spinosad so as to establish proper resistance management programs.RESULTSToxicity bioassays were performed to analyze the current levels of lambda‐cyhalothrin resistance in field populations, proving that it has remained stable in the last decade. An evolutionary model was established to explore the weight of selected parameters in the evolution of lambda‐cyhalothrin resistance in C. capitata and to forecast resistance development under different resistance management scenarios. Our results highlight the importance of fitness cost and inheritance to fit the experimental results. The analyses predicted that the rotation of lambda‐cyhalothrin and spinosad, when deltamethrin traps are also deployed in the field, will slow down the evolution of resistance, especially when cross‐resistance between both pyrethroids is considered.CONCLUSIONLambda‐cyhalothrin resistance has not increased in the last decade, probably due to the alternation of this insecticide with spinosad. Our modelling results indicate that the best option to avoid an increase in lambda‐cyhalothrin resistant alleles, considering that deltamethrin use is growing, would be to continue combining their use with spinosad. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Overexpression of ATP-binding cassette transporter Mdr49-like confers resistance to imidacloprid in the field populations of brown planthopper, Nilaparvata lugens.

The brown planthopper (BPH), Nilaparvata lugens (Stål), is the most severe pest attacking rice crops using sucking mouthparts. It causes significant damages to rice growth and food production worldwide. With the long-term and wide use of insecticides, field populations of BPH have developed resistance to many insecticides. Here, we showed that upregulation of an ATP-binding cassette transporter gene NlMdr49-like contributes to imidacloprid resistance in field populations of BPH. A comparative transcriptome analysis was performed to evaluate the gene expression in two field populations (JXSG18 and YNTC18). Compared with a susceptible strain (Sus), 202 upregulated genes and 170 downregulated genes were identified in both field populations. Functional enrichment analysis revealed that the differentially expressed genes (DEGs) are mainly linked to metabolic process and transmembrane transport. Among the candidate DEGs, NlMdr49-like was significantly upregulated in both field populations. Based on the genome and transcriptome of BPH, the full-length complementary DNA of NlMdr49-like was sequenced and its molecular characteristics were analyzed. Expression pattern analysis of various tissues showed that NlMdr49-like was predominantly expressed in midgut and Malpighian tubules which are important excretion organs. Knocking down NlMdr49-like reduced BPH resistance to imidacloprid, but did not affect its resistance to the other nine insecticides (chlorpyrifos, thiamethoxam, nitenpyram, dinotefuran, sulfoxaflor, triflumezopyrim, ethiprole, buprofezin and pymetrozine). Furthermore, a transgenic strain of Drosophila melanogaster overexpressing NlMdr49-like was less susceptible to imidacloprid. Our findings indicate that upregulation of NlMdr49-like is another mechanism contributing to imidacloprid resistance in N. lugens. This result is helpful to further understand the resistance mechanism of N. lugens to imidacloprid. © 2021 Society of Chemical Industry.

Cypermethrin and ivermectin resistance in field populations of Rhipicephalus sanguineus sensu lato (Latrielle, 1806) collected from dogs in south India.

The abuse of acaricides for control of Rhipicephalus sanguineus sensu lato ticks led to a strong selection pressure for acaricide resistance. Data on acaricide resistance in R. sanguineus s.l. populations from India is meager though veterinarians frequently report treatment failures. The present study was aimed to characterize the level of resistance in R. sanguineus s.l. against most commonly used drugs, cypermethrin and ivermectin in Andhra Pradesh, south India. Fourteen adult female tick populations were collected from naturally infested dogs at veterinary clinics, residence and stray dog in nine state municipal corporations/municipalities. Information on the history of dog treatment with acaricides was obtained by interviewing dog owners. The larval packet test (LPT) and larval immersion test (LIT) were used on the larvae of ticks to test for resistance to cypermethrin and ivermectin, respectively. Mortality data of each tick population was analyzed by probit analysis. Corresponding to the most susceptible population, thirteen (92.6%) and six (42.9%) tick populations were regarded as resistant to cypermethrin and ivermectin, respectively. The phenotypic level of cypermethrin (resistance ratios at LC50% varied from 1.55 to 13.29) and ivermectin (resistance ratios at LC50% ranged from 1.16 to 4.79) resistance varied distinctly between the populations. Additionally, all the populations were tested using PCR to demonstrate the frequency of the point mutation in sodium channel gene that corresponds with resistance of R. sanguineus s.l. to cypermethrin. A nucleotide substitution (T2134C) on domain III segment VI of the sodium channel gene was noticed in phenotypically resistant tick populations on DNA sequencing. Ivermectin resistance in the brown dog ticks is reported for the first time from India.

Status of Cry1Ac and Cry2Ab2 resistance in field populations of Helicoverpa zea in Texas, USA.

Helicoverpa zea is a major target pest of Bt corn and Bt cotton. Field-evolved resistance of H. zea to Cry1 and Cry2 proteins has been widely reported in the United States. Understanding the frequency of resistance alleles in a target insect is critical for Bt resistance management. Despite multiple cases of practical resistance to Cry proteins having been documented in H. zea, there are no data on the current status of alleles conferring resistance to Cry1Ac and Cry2Ab2 in field populations of this pest. During 2018-2019, a total of 106 F2 families for Cry1Ac and 120 F2 families for Cry2Ab2 were established using mass mating and light trap strategy. We screened 13,568 and 15,360 neonates using a discriminatory dose of Cry1Ac and Cry2Ab2, respectively. The results showed that 93.4% and 35.0% of the F2 families could survive on the discriminatory dose of Cry1Ac and Cry2Ab2, respectively. The estimated resistance allele frequency for Cry1Ac in H. zea ranged from 0.4150 to 0.4975 and for Cry2Ab2 ranged from 0.1097 and 0.1228. These data indicate that the frequency of alleles conferring resistance to Cry1 and Cry2 proteins in H. zea in Texas are high. In addition, our data suggest the resistance to Cry1Ac and Cry2Ab2 in the screened families of H. zea varies from recessive to dominant. The information in this study provides precise estimates of Cry resistance allele frequencies in H. zea and increases our understanding of the risks to the sustainability of Bt crops.

Spinosad resistance in field populations of melon fly, Zeugodacus cucurbitae (Coquillett), in Hawaii.

BACKGROUNDControl of Zeugodacus cucurbitae, a serious agricultural pest worldwide, often includes or is dependent on the use of spinosad‐based insecticides. This is especially the case in Hawaii, where GF‐120, a protein bait containing spinosad as the active ingredient, has been in use as a key integrated pest management (IPM) tool against this Tephritid for the last two decades. Here, we report on resistance to spinosad [resistance ratios (RRs) and median lethal concentration (LC50)] in Hawaii's populations of Z. cucurbitae.RESULTSHigh resistance was found in populations from three farms on Oahu (RR = 102–303; LC50 = 191–567 mg L–1) and in a population from Maui (RR = 8.50; LC50 = 15.9 mg L–1). These will be problematic for control given that the most concentrated dilution ratio on the GF‐120 label is 96 mg L–1 of spinosad (1 part GF‐120 to 1.5 parts water). Background resistance in a naïve wild population from the Island of Hawaii (RR = 2.73; LC50 = 5.1 mg L–1) was relatively low compared with a spinosad‐susceptible laboratory colony (LC50 = 1.87 mg L–1). Resistance in the three Oahu and one Maui populations declined over generations in the absence of spinosad but remained elevated in some cases. Moreover, melon flies collected from one of the Oahu farms 1 year after the cessation of spinosad use revealed high persistence of resistance.CONCLUSIONCompared with a 2008 survey of spinosad resistance, our findings indicate a 34‐fold increase in resistance on one of the Oahu farms over 9 years. The evolution and persistence of high levels of resistance to spinosad in Z. cucurbitae in Hawaii highlights the need for alternative control tactics, particularly rotation of active ingredients. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Field Evolved Resistance in Earias vittella (Lepidoptera: Noctuidae) From Punjab, Pakistan Against Commercial Formulations of Bacillus thuringiensis kurstaki.

Transgenic commercial cotton expressing Bacillus thuringiensis (Bt) Cry endotoxins or vegetative Vip toxins provide protection to cotton against bollworm attack. Continuous exposure of these targeted pests to cry toxins and to Bt commercial spray formulations has resulted in the development of resistance through natural selection. Spotted bollworm Earias vittella (Noctuidae: Lepidoptera) is considered to be one of the most destructive pests of cotton and okra crops in South Asia including Pakistan and has developed resistance to various synthetic insecticides. In the present study, the level of resistance in field populations of the spotted bollworm E. vittella against Bt Cry toxins has been evaluated for the first time. We collected twelve populations of E. vittella from three districts of Punjab, Pakistan for testing against four commercial Bt formulations containing different strains of B. thuringiensis subspecies kurstaki (Btk) with a range of Cry toxins. Low to high levels of resistance were found in the field populations compared with a laboratory-reared susceptible population of E. vittella (resistance ratios 6 to 111-fold). These results suggest that E. vittella has developed resistance against different Cry toxins after continuous exposure to Bt cotton in field. In order to prevent field control failures, regular insecticide resistance monitoring programs are required together with the use of integrated management approaches, including the use of Bt cotton varieties expressing two or more toxins to delay the development of resistance against Bt toxins in E. vittella.

Efficacy of entomopathogenic nematodes against Tuta absoluta

BackgroundThe South American tomato pinworm, Tuta absoluta (Meyrick) is one of the most serious insect pests of tomato plants. Current control strategies for T. absoluta primarily involve the use of insecticides, but increasing resistance in field populations has prompted research on alternative control measures. Biological control with entomopathogenic nematodes (EPNs) can be an alternative or one component of an Integrated Pest Management programme. In foliar application, EPNs encounter many factors that affect their survival and efficacy. This paper reports investigations into some of these factors for several EPN species; relative humidity (RH), temperature, time required by EPNs to enter a leaf and number of applications in whole leaf bioassays on tomato leaves. ResultsRH was the most important factor; EPNs efficacy and survival decreased as RH declined. Steinernema feltiae was the most effective species followed by Steinernema carpocapsae then Heterorhabditis bacteriophora. Steinernema carpocapsae survived better at low RH than S. feltiae. The two Steinernema species induced similar mortality at 25 °C, but S. feltiae was more virulent at lower temperatures (15 and 20 °C) while S. carpocapsae was more virulent at higher temperatures (30 and 35 °C). First and second instar larvae of T. absoluta required more applications compared to third and fourth instar larvae. ConclusionThe species of EPN used to control T. absoluta requires careful selection in relation to the temperature and relative humidity environment of the crop to maximise efficacy.

Biochemical characterization of insecticide resistance in field population of major JE vectors from northern districts of West Bengal, India

Biochemical characterization of insecticide resistance in field population of major JE vectors from northern districts of West Bengal, India

First Report on the Mechanisms of Insecticide Resistance in Field Populations of the Small Hive Beetle in Florida

The small hive beetle (Aethina tumida Murray) is a serious threat to beekeeping and crops that rely on honeybees for pollination. The small hive beetle not only causes significant damage to honeybees by feeding on pollen and honey, attacking bee brood and causing stored honey to ferment, but also might serve as a vector of diseases. In addition, the small hive beetle has developed resistance to the pyrethroid and organophosphate insecticides registered for control of honeybee pests in the United States. The development of resistance in small hive beetle populations is a great concern to the beekeeping industry; thus, there is an urgent need for strategies to manage that resistance. Therefore, we used synergist probes to determine the mechanisms of resistance in a small hive beetle population to these insecticides. Our studies on the toxicity of insecticides alone or with the synergists piperonyl butoxide (PBO) and S,S,S,-tributyl phosphorotrithionate (DEF) suggested that mixed-function oxidases and esterases were the major resistance factors to these insecticides in a studied population of the small hive beetle. In contrast, there was no synergism with diethyl maleate (DEM), triphenyl phosphate (TPP) and formamidine. Therefore, glutathione-S-transferase, carboxylesterase and target site were not involved in insecticide resistance in the small hive beetle. Rotation of classes of insecticides (with different modes of action) and metabolic synergists were suggested for the development of successful resistance management programs. To the best of our knowledge, this is the first study of the mechanisms of resistance in small hive beetle populations in Florida and suggests an urgent need for alternative control strategies for these serious pests of honeybee colonies.

Development of a PacBio Long-Read Sequencing Assay for High Throughput Detection of Fungicide Resistance in Zymoseptoria tritici.

Fungicide resistance has become a challenging problem in management of Septoria tritici blotch (STB), caused by Zymoseptoria tritici, the most destructive disease of winter wheat throughout western and northern Europe. To ensure the continued effectiveness of those fungicides currently used, it is essential to monitor the development and spread of such resistance in field populations of the pathogen. Since resistance to the key families of fungicides used for STB control (demethyalation inhibitors or azoles, succinate dehydrogenase inhibitors or SDHIs and Quinone outside Inhibitors or QoIs) is conferred through target-site mutations, the potential exists to monitor resistance through the molecular detection of alterations in the target site genes. As more efficient fungicides were developed and applied, the pathogen has continuously adapted through accumulating multiple target-site alterations. In order to accurately monitor these changes in field populations, it is therefore becoming increasingly important to completely sequence the targeted genes. Here we report the development of a PacBio assay that facilitates the multiplex amplification and long-read sequencing of the target gene(s) for the azole (CYP51), SDHI (Sdh B, C, and D), and QoI (cytochrome b) fungicides. The assay was developed and optimised using three Irish Z. tritici collections established in spring 2017, which capture the range of fungicide resistance present in modern European populations of Z. tritici. The sequences obtained through the PacBio assay were validated using traditional Sanger sequencing and in vitro sensitivity screenings. To further exploit the long-read and high throughput potential of PacBio sequencing, an additional nine housekeeping genes (act, BTUB, cal, cyp, EF1, GAPDH, hsp80-1, PKC, TFC1) were sequenced and used to provide comprehensive Z. tritici strain genotyping.

Identifying potential novel resistance to the foliar disease \u2018Scald\u2019 (Rhynchosporium commune) in a population of Scottish Bere barley landrace (Hordeum vulgare L.)

Barley ‘Scald’ is an economically damaging fungal disease that is a global problem, causing significant yield and economical losses in the UK barley feed and malting industries. Presently, a limited number of Rhynchosporium resistance genes exist, but selective pressures on the fungi cause the demand for new sources of resistance. Landraces, such as the Scottish Bere barley, hold potential sources of resistance that can be utilised, with farmers providing anecdotal evidence of resistance in field populations of Bere. This study analysed 131 heritage cultivars and landrace lines, including 37 Bere lines, to screen for resistance using both detached leaf assays (DLAs) and field experiments. Results showed that Bere lines produced smaller, but more necrotic, lesions for the majority of isolates in the DLAs, as well as smaller scores when visually assessed in field conditions. Whilst the infection patterns of the lines differed between isolates and experimental conditions, three Bere lines were identified as consistently showing reduced levels of infection (45 A 23, 58 A 36 Eday, and 8-125). Using genome-wide association analysis, we were able to identify a number of genomic regions associated with reduced infection symptoms, four in regions associated with known resistance genes, but another four associated with new regions that contain promising candidate genes. Further analysis of these new regions and candidate genes should be undertaken to identify targets for future disease-resistance breeding.

Basis and monitoring of methoxyfenozide resistance in the South American tomato pinworm Tuta absoluta

The South American tomato pinworm, Tuta absoluta (Meyrick, 1917) (Lepidoptera: Gelechiidae), is heavily targeted by insecticide applications. Methoxyfenozide is highly effective against T. absoluta with reduced side effects to natural enemies. This work aimed to (1) genetically and biochemically characterize resistance of the T. absoluta GBN population to methoxyfenozide, (2) establish cross resistance profiles with other insecticide groups and (3) monitor resistance in populations with the goal of improving T. absoluta insecticide resistance management (IRM). Methoxyfenozide resistance was completely recessive, polyfactorial and autosomal. Effective dominance revealed that 10 mg methoxyfenozide/L would be enough to eliminate susceptible homozygotes and heterozygotes, thus used to diagnose resistance in field populations. The synergism of methoxyfenozide toxicity in the resistant population for PBO (SR = 95×), DEF (SR = 51×) and DEM (SR = 45×), suggested monooxygenases, esterases and glutathione S-transferases as resistance mechanisms. However, only monooxygenase activity appeared to be involved in methoxyfenozide resistance. Resistance ratio for methoxyfenozide (2352-fold) after selection and cross-resistance ratios of a lab-selected GBN strain (“GBN-Sel”) were significant relative to a susceptible strain “JDR1-Sus” for tebufenozide (656-fold), cartap hydrochloride (10.68-fold), deltamethrin (4.70-fold), abamectin (2.65-fold), lufenuron (2.22-fold) and indoxacarb (1.92-fold), with negative cross-resistance to spinetoram (0.32-fold). Evidence of control failures was observed in 10 field populations of T. absoluta (mortalities between 13 and 76%), and all populations showed frequencies of resistant phenotypes (percentage survivorship ranging between 4 and 96%). A rational basis for managing resistance to bisacylhydrazines is discussed, along with details of recommended T. absoluta resistance management tactics.

Soybean aphid infestation and crop yield in relation to cultivar, foliar insecticide, and insecticidal seed treatment in South Dakota

The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), remains an important pest of soybean, Glycine max (L.) Merr., in areas of the Midwestern United States and southern Canada. Options for managing soybean aphid are currently limited to insecticides and aphid-resistant varieties. Farmers commonly employ a prophylactic treatment of soybean seed with a neonicotinoid insecticide such as thiamethoxam and often a remedial, late-season application of foliar insecticides such as the pyrethroid, λ-cyhalothrin. Environmental concerns about detrimental side effects of neonicotinoids and the substantiation of pyrethroid resistance in field populations of soybean aphid have strengthened the case for planting aphid-resistant soybean cultivars. We conducted a three-year field study in eastern South Dakota, United States, that evaluated commercially-available active substances and enhanced plant materials for soybean aphid management that included a thiamethoxam seed treatment, spray application of λ-cyhalothrin, and an aphid-resistant soybean cultivar containing a pyramid of the Rag1 and Rag2 aphid-resistance genes. Soybean aphid infestations had sustained, economically injurious levels in 2015, but not the other two years. The three management tactics independently reduced soybean aphid population densities. Thiamethoxam and the resistant cultivar suppressed cumulative aphid-days (CAD) in all three years, whereas the λ-cyhalothrin spray decreased CAD in 2015 and 2017. By far, the resistant cultivar had the greatest impact on soybean aphid populations, reducing CAD by 28- to 150-fold year−1, with mean numbers of aphids much less than 100 soybean aphids plant−1. Thiamethoxam seed treatment reduced CAD by 1.7- to 3.5-fold year−1, and λ-cyhalothrin reduced it from 2.0- to 5.6-fold year−1. Soybean yield was not affected by treatments in 2015. Yield was greater for the susceptible than for the resistant cultivar in 2016, and yield was greater for plots without foliar insecticide in 2017. Implications for the use of these management tactics against soybean aphid are discussed.

Phenotypic and molecular analyses in rice weevil, Sitophilus oryzae (Linneaus) (Coleoptera: Curculionidae): identification of a super kdr mutation, T929I, conferring resistance to deltamethrin.

The rice weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae) is a cosmopolitan pest of stored cereal grains and other commodities globally. Infestations caused by S. oryzae makes grains unsuitable for consumption, processing, and export. Deltamethrin, a synthetic pyrethroid insecticide, is widely used in major grain storages in India as a prophylactic treatment to control this pest. However, recurrent use of this insecticide had led to genetic resistance in S. oryzae, questioning its ongoing use at the current recommended concentration. Dose response analysis of resistant (Delta-R) and susceptible (Lab-S) strains of S. oryzae collected from grain storages across southern India, revealed that Delta-R was 134-fold more resistant than the Lab-S at median lethal concentration (LC50 ). A concentration of 180 ppm over 48 h effectively discriminated 16 resistant field populations from Lab-S with per cent resistance ranging from 8.72% to 75.86%. Exposing all the resistant populations to 1000 ppm over 48 h identified 12 populations with strongly resistant individuals and confirmed the existence of two distinct resistance phenotypes, 'weak' and 'strong' in S. oryzae. Furthermore, sequence analysis of the voltage-gated sodium channel (vgsc) gene in Delta-R identified a single target site mutation, T929I conferring resistance in S. oryzae. CAPS (Cleaved Amplified Polymorphic Sequence) marker analysis of this allele confirmed that frequency of resistance is high (up to 0.96) supporting the results of phenotypic analysis. Both phenotype and molecular marker analyses clearly demonstrated that deltamethrin at 180 and 1000 ppm can be used to discriminate weakly and strongly resistant populations in S. oryzae, respectively. Resistance diagnostics based on the mutation, T929I, supports our phenotypic data and indicates that resistance to deltamethrin in S. oryzae is prevalent in southern parts of India, stressing the need to identify a synergist or suitable alternatives. © 2021 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.

Detection of Phosphine Resistance in Field Populations of Four Key Stored-Grain Insect Pests in Pakistan.

Simple SummaryThe resistance of coleopteran stored-product pests to phosphine fumigation is becoming a global phenomenon. However, there is limited literature available and a lack of knowledge on this issue in Pakistan. Thus, in the current study, we estimated the status of phosphine resistance among ten populations of Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae), Sitophilus granarius (L.) (Coleoptera: Curculionidae), Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) and Trogoderma granarium Everts (Coleoptera: Dermestidae), originated from different regions of Pakistan. Laboratory-susceptible populations of all insect species were also considered in the tests. Concentration–response bioassays were conducted for each insect species. All of the tested populations of each species were found to be resistant to phosphine. However, they varied with regard to their levels of resistance. Overall, R. dominica exhibited the highest resistance level, followed by T. castaneum, T. granarium and S. granarius. Although phosphine is effective against several stored-product pests, the development of resistance may lead to failures in its application in Pakistan.In Pakistan, the control of stored-product insect pests mainly relies on the use of phosphine gas along with other control tactics. The aim of this study was to determine the level of phosphine resistance among ten differently located populations of the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae), the granary weevil, Sitophilus granarius (L.) (Coleoptera: Curculionidae), the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) and the khapra beetle, Trogoderma granarium Everts (Coleoptera: Dermestidae). Laboratory-susceptible populations of all insect species were also considered in the experiments. Concentration–response bioassays were conducted for each species. All of the tested populations (10 out of 10) of each species were found to be resistant to phosphine, but varied in their level of resistance. Probit analysis estimated LC50 at 2.85, 1.90, 2.54 and 2.01 ppm for laboratory-susceptible populations of R. dominica, S. granarius, T. castaneum and T. granarium, respectively. Against R. dominica, the highest and lowest resistance levels were observed in the Rahim Yar Khan (LC50 at 360.90 ppm) and Rawalpindi (LC50 at 210.98 ppm) populations, respectively. These resistant populations were 126.67- and 74.02-fold more resistant than the laboratory population. The Multan and Lahore populations of S. granarius exhibited the maximum (LC50 at 122.81 ppm) and minimum (LC50 at 45.96 ppm) resistance levels, respectively, i.e., they were 64.63- and 24.18-fold more resistant than the laboratory population. The Layyah population of T. castaneum showed the maximum resistance level (LC50 at 305.89 ppm) while the lowest was observed in the Lahore population (LC50 at 186.52 ppm), corresponding to 120.42- and 73.43-fold more resistant than the laboratory population, respectively. Regarding T. granarium, the Layyah population showed the maximum resistance level (LC50 at 169.99 ppm) while the Lahore population showed the minimum resistance (LC50 at 74.50 ppm), i.e., they were 84.57- and 37.06-fold more resistant than the laboratory population, respectively. Overall, R. dominica presented the highest resistance level, followed by T. castaneum, T. granarium and S. granarius. The current study suggests that the application of phosphine may not be an adequate control strategy for the management of the above tested insect pests in Pakistan.

Multifungicide resistance profiles and biocontrol in Lasiodiplodia theobromae from mango fields

Stem-end rot (SER) caused by Lasiodiplodia theobromae is a major postharvest disease of mango. To better understand the occurrence of multifungicides resistance in field populations, the sensitivity to five fungicides was investigated in 224 L. theobromae isolates collected from the mango growing region of Hainan Province in 2014–2018. The biocontrol activity of Bacillus amyloliquefaciens HBR-2 against resistant phenotypes of L. theobromae was evaluated using in vitro and in vivo tests. Our studies have established the baseline sensitivity of L. theobromae to difenoconazole, prochloraz and iprodione. Most isolates tested were sensitive to difenoconazole, prochloraz and iprodione. The overall resistance frequencies for difenoconazole, prochloraz and iprodione were 24.11, 15.18 and 1.34%, respectively. In addition, the overall resistance frequencies of L. theobromae for carbendazim and pyraclostrobin were 71.43% and 57.14%, respectively. In Hainan, 16 fungicide-resistant phenotypes were detected: 29.02, 60.27, 7.14, and 0.45% of the isolates were resistant to one, two, three, and four fungicides, respectively. The novel isolate B. amyloliquefaciens HBR-2 was found to have strong antagonistic activity against different resistant phenotypes of L. theobromae. The cell culture broth was the most effective treatment for controlling mango fruit rot in vivo. Multifungicide-resistant isolates of L. theobromae were widespread in Hainan. Biocontrol could be a valuable additional tool to manage the SER of mango fruit in the future. These findings would be useful in designing and implementing fungicide resistance management spray programs for the control of SER in mango.

The detoxification enzymes activity profile in susceptible Aedes and Culex mosquitoes

The status of susceptible mosquito is important for monitoring and managing insecticide resistance in field population. The purpose of this study is to profile the differential expression level of enzymatic activities of metabolic enzymes exhibited in the mosquito species of Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus from the susceptible strain. The fourth instar larvae of each strain were subjected to biochemical assay. The total protein content and enzymatic activities of Glutathione S-transferase (GST), α-esterase (α-est), β-esterase (β-est) Cytochrome P450 (Cyt P450) and Acetylcholinesterase (AChE) from each mosquito strains were elucidated. Significant difference (P<0.05) was detected between the total protein content between all species of susceptible strain mosquitoes. One-way ANOVA showed that the specific enzymatic activities of GST, α-est and Cyt P450 of all test mosquitoes were significantly different upon comparison with each species (P<0.05). The mean of enzymatic activities of insensitive AChE showed no significant difference upon comparison with each other (P>0.05). The enzymatic activities of β-est shows no significant difference between Ae. aegypti and Cx. quinquefasciatus mosquitoes (P>0.05) but the enzymatic activity of β-est in Ae. albopictus manifested significant difference upon comparison with the enzymatic activities of the other two test species (P<0.05). The results obtained may provide more information about the enzymatic activities of metabolic enzymes in Ae. aegypti, Ae. albopictus and Cx. quinquefasciatus mosquitoes which might be beneficial for public sector for the application of proper vector control measures.

Novel diamide resistance-linked mutation in Korean Spodoptera exigua and a LAMP assay based on a mutation-associated intronic InDel

Recently, resistance to diamide insecticides has been reported in various lepidopteran pests, including Spodoptera exigua. Six field populations and a local population were tested, and results revealed high levels of diamide resistance. We selected a diamide-resistant strain, showing LC50 values 28,950- and 135,286-fold higher than those of a susceptible strain against chlorantraniliprole and flubendiamide, respectively. However, G4946E mutation in the ryanodine receptor, one of the well-known diamide resistance mechanisms, was not found in the tested resistant field populations. Instead, through genome sequencing, we found an I4790M mutation and some InDels, particularly a 29-bp insertion in the neighboring intron that was associated with this resistant allele. By using this distinct region, resistant allele diagnostic primers were designed and applied in a lamp loop-mediated isothermal amplification (LAMP) assay and general PCR. The applicable temperature of the LAMP assay was from 63 to 65 °C for 2 h with four primers. Addition of a loop primer further increased the amplification efficiency. LAMP was feasible with a broad range of DNA concentrations, with a minimum detectable concentration of 100 pg. A DNA releasing method comprising 5 min of incubation at 95 °C allowed DNA detection from some larva and adult samples. The combination of this DNA releasing technique and LAMP resulted in a rapid (up to 100 min) and accurate diagnostic strategy that could be applied to monitor and manage diamide resistance in S. exigua.

Functional PPO2 mutations: co-occurrence in one plant or the same ppo2 allele of herbicide-resistant Amaranthus palmeri in the US mid-south.

Protoporphyrinogen IX oxidase 2 (PPO2) inhibitors are important for the management of glyphosate- and acetolactate synthase-resistant Palmer amaranth [Amaranthus palmeri (S.) Wats.]. The evolving resistance to PPO inhibitors is of great concern. We surveyed the evolution of resistance to fomesafen in the US Mid-south and determined its correlation with the known functional PPO2 target-site mutations (TSM). The 167 accessions analyzed were grouped into five categories, four resistant (147) and one susceptible (20). Arkansas accessions constituted 100% of the susceptible group while the Missouri accessions comprised 60% of the most resistant category. The majority of Mississippi accessions (88%) clustered in the high-survival-high-injury category, manifesting an early-stage resistance evolution. One hundred and fifteen accessions were genotyped for four known TSMs; 74% of accessions carried at least one TSM. The most common single TSM was ΔG210 (18% of accessions) and the predominant double mutation was ΔG210 + G399A (17%). Other mutations are likely less favorable, hence are rare. All TSMs were detected in three accessions. Further examination revealed that 9 and two individuals carried G399A + G210 and G399A + R128G TSM in the same allele, respectively. The existence of these combinations is supported by molecular modeling. Resistance to PPO inhibitors is widespread across the Mid-southern USA. Highly resistant field populations have plants with multiple mutations. G399A is the most prone to co-occur with other ppo2 mutations in the same allele. Mutation at R128 in the configuration of the PPO2 catalytic domain restrains the co-occurrence of R128G with ΔG210, making ΔG210 + G399A the most plausible, tolerable functional mutation combination to co-occur in the same ppo2 allele.