Aphid Resistance Research Articles

Diverse patterns of constitutive and inducible overexpression of detoxifying enzyme genes among resistant Aphis glycines populations

Understanding the mechanisms of pyrethroid resistance is essential to the effective management of pesticide resistance in Aphis glycines Matsumura (Hemiptera: Aphididae). We mined putative detoxifying enzyme genes in the draft genome sequence of A. glycines for cytochrome oxidase P450 (CYP), glutathione-S-transferase (GST) and esterases (E4 and carboxylesterases-CES). Aphids from clonal populations resistant to pyrethroids from three sites in Minnesota, USA, were screened against a diagnostic LC99 concentration of either λ-cyhalothrin or bifenthrin and detoxifying enzyme genes expression in survivors was analyzed by qPCR. Their expression profiles were compared relative to a susceptible clonal population. We found 61 CYP (40 full-length), seven GST (all full-length), seven E4 (five full-length) and three CES (two full-length) genes, including 24 possible pseudogenes. The detoxifying enzymes had different expression profiles across resistant aphid populations, possibly reflecting differences in the genetic background and pyrethroid selection pressures as the number of constitutively overexpressed detoxifying enzyme genes was correlated with the level of resistance. Our findings will strengthen the understanding of the pyrethroid resistance mechanisms in A. glycines.

Screening of cowpea accessions for cowpea aphid resistance

ABSTRACT Cowpea (Vigna unguiculata) genotypes may preserve important characteristics of potential use for the genetic breeding of this species. This study aimed to characterize sources of genetic resistance to the cowpea aphid (Aphis craccivora), among accessions maintained at the cowpea germplasm bank of the Universidade Federal do Ceará (Brazil), in order to suggest crossing options for obtaining superior cultivars. Twenty-one local accessions were assessed together with two genotypes as resistant checks and two others as susceptible checks. A randomized complete block design, with four replications, was used. Counts of live adults and nymphs per plant were used to evaluate the genotypic resistance and, based on the ranking of these data, the effective resistance was estimated. The applied approach showed to be effective to discriminate the genetic variability for cowpea aphid resistance, identifying antibiosis as the probable resistance mechanism. Four genotypic groups were identified: resistant, intermediate, susceptible and highly susceptible. Thus, for a short-time cultivar release strategy, backcrosses are suggested between the BRS Guariba cultivar (recurrent parental, with good agronomic traits) and aphid-resistant accessions, such as Enrica Pobre (CE-36), Das Almas (CE-07), CE-51 (selected within CE-13) and Ritinha (CE-08). However, for a long-term breeding strategy, crossings between the most dissimilar accessions are suggested, using resistant genitors (e.g. BRS Guariba, Enrica Pobre, Das Almas, CE-51 and Ritinha) and others with good commercial and agronomic attributes.

Evaluation of cowpea (<em>Vigna unguiculata</em> L. Walp) genotypes for yield and associated traits

Twenty cowpea (Vigna unguiculata) genotypes were evaluated for thrip and aphid resistance and yield and associated traits. The associated parameters studied were Thrip score, aphid score, plant height, number of branches, number of clusters, number of pods, seeds per pod and yield and the result obtained showed significant differences . From the result obtain it can be concluded that GCP-400, GCP-387 and GCP-287 had the best results in most of the parameters evaluated.

The sorghum epicuticular wax locus Bloomless2 reduces plant damage in P898012 caused by the sugarcane aphid

AbstractThe sugarcane aphid [Melanaphis sacchari] has been rapidly spreading in the southern United States with devastating impacts on sorghum [Sorghum bicolor (L.) Moench]. Previous studies have shown the sorghum epicuticular wax mutants, in particular a bloomless2 (bm2) mutant and a sparse bloom mutant, were resistant to greenbugs [Schizaphis graminum], a type of aphid. In this study we sought to determine if five different genotypes, which all have mutations in the Bm2 locus, differ for sugarcane aphid resistance as compared to their respective wild‐type plants (P954035, P898012, and Tx7078). Two greenhouse studies with artificial infestation and one field study under natural infestation were conducted. Aphid numbers on the second leaf from the top and the lowest green leaf, plant damage, and plant growth stage were assessed at weekly intervals. The Greenhouse Test 2 and the Field test were the most effective at determining plant resistance and susceptibility to the sugarcane aphid. Bm2 mutants in the P898012 background had greater aphid damage than their wild type plants. These findings suggest that the Bm2 locus has a role in reducing aphid damage in the P898012 background yet has a limited role in the Tx7078 and P954035 backgrounds.

The Arabidopsis PAD4 Lipase-Like Domain Is Sufficient for Resistance to Green Peach Aphid.

Plants have evolved mechanisms to protect themselves against pathogenic microbes and insect pests. In Arabidopsis, the immune regulator PAD4 functions with its cognate partner EDS1 to limit pathogen growth. PAD4, independently of EDS1, reduces infestation by green peach aphid (GPA). How PAD4 regulates these defense outputs is unclear. By expressing the N-terminal PAD4 lipase-like domain (PAD4LLD) without its C-terminal EDS1-PAD4 (EP) domain, we interrogated PAD4 functions in plant defense. Here, we show that transgenic expression of PAD4LLD in Arabidopsis is sufficient for limiting GPA infestation but not for conferring basal and effector-triggered pathogen immunity. This suggests that the C-terminal PAD4 EP domain is necessary for EDS1-dependent immune functions but is dispensable for aphid resistance. Moreover, PAD4LLD is not sufficient to interact with EDS1, indicating the PAD4-EP domain is required for stable heterodimerization. These data provide molecular evidence that PAD4 has domain-specific functions.

Genetic Transformation and siRNA-Mediated Gene Silencing for Aphid Resistance in Tomato

We explored the ability of RNA interference (RNAi) to silence the Acetylcholinesterase 1 (Ace 1) gene in aphid Myzus persicae and developed transgenic tomato plants resistant to aphid infestation. Three plasmid constructs, T-449: a single Ace 1 fragment (forward orientation), T-452: two Ace 1 fragments (reverse and forward orientations), and T455: a single inverted Ace 1 fragment, were developed and transformed into two tomato cultivars, Jamila and Tomaland. PCR, northern blotting, and small interfering RNAs (siRNA) analysis were performed to validate the success of Agrobacterium-mediated transformation. The efficiency of transformation was highest for the T-452 construct. In vivo effects of the transformed constructs were confirmed in feeding experiments, and there was significant downregulation of the Ace 1 gene. In addition, an aphid challenge assay was conducted to investigate the siRNA-mediated silencing of the target gene (Ace 1) in the inhibition of fecundity in M. persicae. We found that the plants that were transformed with the T-452 vector had 37.5% and 26.4% lower fecundity at 27 °C in the Jamila and Tomaland, respectively. Our results strongly indicated that the plant-mediated silencing of aphid-RNA might be a robust and effective approach for developing pest and disease resistant in plants.

Cucumber mosaic virus 2b proteins inhibit virus-induced aphid resistance in tobacco.

SummaryCucumber mosaic virus (CMV), which is vectored by aphids, has a tripartite RNA genome encoding five proteins. In tobacco (Nicotiana tabacum), a subgroup IA CMV strain, Fny‐CMV, increases plant susceptibility to aphid infestation but a viral mutant unable to express the 2b protein (Fny‐CMV∆2b) induces aphid resistance. We hypothesized that in tobacco, one or more of the four other Fny‐CMV gene products (the 1a or 2a replication proteins, the movement protein, or the coat protein) are potential aphid resistance elicitors, whilst the 2b protein counteracts induction of aphid resistance. Mutation of the Fny‐CMV 2b protein indicated that inhibition of virus‐induced resistance to aphids (Myzus persicae) depends on amino acid sequences known to control nucleus‐to‐cytoplasm shuttling. LS‐CMV (subgroup II) also increased susceptibility to aphid infestation but the LS‐CMV∆2b mutant did not induce aphid resistance. Using reassortant viruses comprising different combinations of LS and Fny genomic RNAs, we showed that Fny‐CMV RNA 1 but not LS‐CMV RNA 1 conditions aphid resistance in tobacco, suggesting that the Fny‐CMV 1a protein triggers resistance. However, the 2b proteins of both strains suppress aphid resistance, suggesting that the ability of 2b proteins to inhibit aphid resistance is conserved among divergent CMV strains.

Accumulation of Carboxylate and Aromatic Fluorophores by a Pest-Resistant Sweet Sorghum [Sorghum bicolor (L.) Moench] Genotype.

The sugary juice from sweet sorghum [Sorghum bicolor (L.) Moench] stalks can be used to produce edible syrup, biofuels, or bio-based chemical feedstock. The current cultivars are highly susceptible to damage from sugarcane aphids [Melanaphis sacchari (Zehntner)], but development of new cultivars is hindered by a lack of rapid analytical methods to screen for juice quality traits. The mechanism of aphid resistance/tolerance is also largely unknown, though the importance of defense phytochemicals has been suggested. The purpose of this study was to develop low-cost methods sensitive to fluorescent fingerprints in sweet sorghum juice, which is a complex mixture of saccharides, carboxylates, polyphenols, and metal ions. Of primary juice components, tryptophan and trans-aconitic acid were the highest intensity contributors to the overall fluorescence and UV/visible absorbance, respectively, while tyrosine and polyphenols contributed to a less extent. In a test of 24 sweet sorghum cultivars, tryptophan and tyrosine contents were the highest in the aphid-susceptible hybrid N109A x Chinese, while sucrose, trans-aconitic acid, and polyphenols were the highest in the resistant line No. 5 Gambela. This suggests that the accumulation of carboxylate (trans-aconitic acid) and polyphenolic secondary products in No. 5 Gambela may contribute to its aphid resistance, thus allowing it to maintain sucrose production. Rapid detection of these chemical signatures could be used to prescreen the breeding material for potential resistance and juice quality traits, without analytical separation required for metabolomics.

The FATTY ACID DESATURASE2 Family in Tomato Contributes to Primary Metabolism and Stress Responses.

The conversion of oleic acid (C18:1) to linoleic acid (C18:2) in the endoplasmic reticulum is critical to the accumulation of polyunsaturated fatty acids in seeds and other tissues, and this reaction is catalyzed by a Δ12-desaturase, FATTY ACID DESATURASE2 (FAD2). Here, we report that the tomato (Solanum lycopersicum) genome harbors two genes, SlFAD2-1 and SlFAD2-2, which encode proteins with in vitro Δ12-desaturase activity. In addition, tomato has seven divergent FAD2 members that lack Δ12-desaturase activity and differ from canonical FAD2 enzymes at multiple amino acid positions important to enzyme function. Whereas SlFAD2-1 and SlFAD2-2 are downregulated by biotic stress, the majority of divergent FAD2 genes in tomato are upregulated by one or more stresses. In particular, SlFAD2-7 is induced by the potato aphid (Macrosiphum euphorbiae) and has elevated constitutive expression levels in suppressor of prosystemin-mediated responses2 (spr2), a tomato mutant with enhanced aphid resistance and altered fatty acid profiles. Virus-induced gene silencing of SlFAD2-7 in spr2 results in significant increases in aphid population growth, indicating that a divergent FAD2 gene contributes to aphid resistance in this genotype. Thus, the FAD2 gene family in tomato is important both to primary fatty acid metabolism and to responses to biotic stress.

Morphological and chemical markers associated with Acyrthosiphon pisum tolerance in hybrid pea lines

Aim of study: Determining morphological and chemical markers in hybrid lines of winter forage pea (Pisum sativum subsp. arvense L.) associated with Acyrthosiphon pisum tolerance for breeding programs.Area of study: The experimental field of the Institute of Forage Crops, Pleven, Bulgaria, during the 2016-2018 period.Material and methods: Six hybrid lines of winter forage pea were studied for tolerance to A. pisum. The field trial was conducted using a long-plot design and a natural background of soil (leached chernozem) supplied with major nutrients. An entomological net for sweeping was used once a week for aphid number recording. Stem height and leaf number were recorded and chemical composition was determined at the flowering stage. The coefficient of variation concerning aphid density was calculated and the stability and adaptability of lines was evaluated.Main results: Hybrid lines 6 and 12A were stable, widely adapted to the changing environmental conditions and the aphid density was statistically the lowest (31.1 and 36.8 individuals/m2, respectively). A significant positive interaction was found between aphid density and plant height, leaf area, protein, and phosphorus content. Lines 6 and 12A had lower stems (74.7 and 82.5 cm), smaller leaf areas (571.13 and 657.39 cm2/plant), lower protein and P contents, and these markers defined them as aphid tolerant.Research highlights: Incorporation of plant markers of pea lines, less preferred by aphids, is an efficient tool for improving breeding programs for aphid resistance.

Quantitative trait locus for resistance to the aphid Rhopalosiphum padi L. in barley (Hordeum vulgare L.) is not linked with a genomic region for gramine concentration

Gramine is an indole alkaloid found in certain grass species. Results of previous studies have diverged as to whether this compound might cause resistance to aphids or not. In a breeding program aiming to introduce resistance to the cereal pest Rhopalosiphum padi L. in barley, a cultivar has been crossed with the progenitor of cultivated barley, Hordeum vulgare ssp. spontaneum, and an F1-derived population of doubled haploid (DH) lines was screened both for seedling gramine concentration and resistance to R. padi. The resistance was measured as individual aphid growth in the laboratory. The present study aims to determine if there is a genetic relationship between aphid growth retardation and gramine concentration. To do so, the lines were genotyped with a 384 SNP oligonucleotide pool assay and QTL analyses were performed for both traits. A previously identified aphid resistance locus on the distal part of chromosome 2HS was thereby confirmed, with resistance inherited from H. v. ssp. spontaneum, whereas one or more QTL for gramine concentration were potentially indicated on chromosome 3H, thus corroborating that the two traits are not linked genetically.

The Polycomb protein LHP1 regulates Arabidopsis thaliana stress responses through the repression of the MYC2-dependent branch of immunity.

Polycomb repressive complexes (PRCs) have been traditionally associated with the regulation of developmental processes in various organisms, including higher plants. However, similar to other epigenetic regulators, there is accumulating evidence for their role in the regulation of stress and immune-related pathways. In the current study we show that the PRC1 protein LHP1 is required for the repression of the MYC2 branch of jasmonic acid (JA)/ethylene (ET) pathway of immunity. Loss of LHP1 induces the reduction in H3K27me3 levels in the gene bodies of ANAC019 and ANAC055, as well as some of their targets, leading to their transcriptional upregulation. Consistently, increased expression of these two transcription factors leads to the misregulation of several of their genomic targets. The lhp1 mutant mimics the MYC2, ANAC019, and ANAC055 overexpressers in several of their phenotypes, including increased aphid resistance, abscisic acid (ABA) sensitivity and drought tolerance. In addition, like the MYC2 and ANAC overexpressers, lhp1 displays reduced salicylic acid (SA) content caused by a deregulation of ICS1 and BSMT1, as well as increased susceptibility to the hemibiotrophic pathogen Pseudomonas syringae pv. tomato DC3000. Together, our results indicate that LHP1 regulates the expression of stress-responsive genes as well as the homeostasis and responses to the stress hormones SA and ABA. This protein emerges as a key chromatin player fine tuning the complex balance between developmental and stress-responsive processes.

Aphid resistance in Capsicum maps to a locus containing LRR-RLK gene analogues

Key messageA QTL for aphid resistance on pepper chromosome 2 was identified and validated. This QTL affects aphid survival and reproduction, and was fine mapped to a locus containing LRR-RLK analogues.Myzus persicae is one of the most threatening insect pests that adversely affects pepper (Capsicum) cultivation. Resistance to aphids was previously identified in Capsicum baccatum. This study aimed at elucidating the genetics of aphid resistance in C. baccatum. A QTL analysis was carried out for M. persicae resistance in an F2 population derived from an intraspecific cross between a highly resistant plant and a susceptible plant. Survival and reproduction were used as resistance parameters. Interval mapping detected two QTLs affecting aphid survival (Rmpas-1) and reproduction (Rmprp-1), respectively, both localized in the same area and sharing the same top marker on chromosome 2. Use of this marker as co-factor in multiple-QTL mapping analysis revealed a second, minor QTL (Rmprp-2) only affecting aphid reproduction, on chromosome 4. Fine mapping confirmed the effects of Rmpas-1 and Rmprp-1 and narrowed the major QTL Rmprp-1 down to a genomic region of 96 kb which is predicted to encode four analogues of resistance genes of the receptor-like kinase family containing a leucine-rich repeat domain (LRR-RLKs). This work provides not only initial information for breeding aphid-resistant pepper varieties, but also forms the basis for future molecular analysis of gene(s) involved in aphid resistance.

Gene pyramids and the balancing act of keeping pests at bay.

This article comments on: Kamphuis LG, Klingler JP, Jacques S, Gao L-l, Edwards OR, Singh KB. 2019. Additive and epistatic interactions between AKR and AIN loci conferring bluegreen aphid resistance and hypersensitivity in Medicago truncatula. Journal of Experimental Botany 70, 4887-4902.

Additive and epistatic interactions between AKR and AIN loci conferring bluegreen aphid resistance and hypersensitivity in Medicago truncatula.

Aphids, including the bluegreen aphid (BGA; Acyrthosiphon kondoi), are important pests in agriculture. Two BGA resistance genes have been identified in the model legume Medicago truncatula, namely AKR (Acyrthosiphon kondoi resistance) and AIN (Acyrthosiphon induced necrosis). In this study, progeny derived from a cross between a resistant accession named Jester and a highly susceptible accession named A20 were used to study the interaction between the AKR and AIN loci with respect to BGA performance and plant response to BGA infestation. These studies demonstrated that AKR and AIN have additive effects on the BGA resistance phenotype. However, AKR exerts dominant suppression epistasis on AIN-controlled macroscopic necrotic lesions. Nevertheless, both AKR and AIN condition production of H2O2 at the BGA feeding site. Electrical penetration graph analysis demonstrated that AKR prevents phloem sap ingestion, irrespective of the presence of AIN. Similarly, the jasmonic acid defense signaling pathway is recruited by AKR, irrespective of AIN. This research identifies an enhancement of aphid resistance through gene stacking, and insights into the interaction of distinct resistance genes against insect pests.

Inheritance Study of Aphid ( Aphis craccivora KOCH) and Rosette Resistance in Groundnut ( Arachis hypogaea L .)

This study was conducted to investigate the gene effects, heritability, genetic advance and number of effective factors controlling the inheritance of aphid and rosette resistance and other quantitative characters. Two aphid resistance, one rosette resistance, one aphid susceptible and one rosette susceptible lines were used as parents to develop F1s, F2s, BC1P1 and BC1P2. The seventeen generations obtained were evaluated along with three checks in three replications using randomized complete block design. The three parameter model was adequate to explain variations observed in the inheritance of days to fifty percent flowering, plant height, number of seeds per plant and shelling percentage. Non allelic interaction was significant with high narrow-sense heritability as obtained for rosette disease incidence, rosette severity index, number of matured pods per plant and net pod yield. It is possible to expect advance for these characters in further segregating generations. Wide ranges of narrow and broad sense heritability accompanied with moderate to high genetic advance except for aphid infestation index were obtained for the characters studied. The number of effective factors revealed that the understudied characters were governed by mono, oligo, and polygenes.

Overexpression of CmMYB15 provides chrysanthemum resistance to aphids by regulating the biosynthesis of lignin

MYB transcription factors are widely involved in the development of and physiological processes in plants. Here, we isolated the chrysanthemum R2R3-MYB family transcription factor CmMYB15, a homologous gene of AtMYB15. It was demonstrated that CmMYB15 expression was induced by aphids and that CmMYB15 could bind to AC elements, which usually exist in the promoter of lignin biosynthesis genes. Overexpression of CmMYB15 in chrysanthemum enhanced the resistance of aphids. Additionally, the content of lignin and the expression of several lignin biosynthesis genes increased. In summary, the results indicate that CmMYB15 regulates lignin biosynthesis genes that enhance the resistance of chrysanthemum to aphids.

Bioactivity-guided isolation of rosmarinic acid as the principle bioactive compound from the butanol extract of Isodon rugosus against the pea aphid, Acyrthosiphon pisum.

Aphids are agricultural pest insects that transmit viruses and cause feeding damage on a global scale. Current pest control practices involving the excessive use of synthetic insecticides over many years have resulted in aphid resistance to a number of pesticides. In nature, plants produce secondary metabolites during their interaction with insects and these metabolites can act as toxicants, antifeedants, anti-oviposition agents and deterrents towards the insects. In a previous study, we demonstrated that the butanol fraction from a crude methanolic extract of an important plant species, Isodon rugosus showed strong insecticidal activity against the pea aphid, Acyrthosiphon pisum. To further explore this finding, the current study aimed to exploit a bioactivity-guided strategy to isolate and identify the active compound in the butanol fraction of I. rugosus. As such, reversed-phase flash chromatography, acidic extraction and different spectroscopic techniques were used to isolate and identify the new compound, rosmarinic acid, as the bioactive compound in I. rugosus. Insecticidal potential of rosmarinic acid against A. pisum was evaluated using standard protocols and the data obtained was analyzed using qualitative and quantitative statistical approaches. Considering that a very low concentration of this compound (LC90 = 5.4 ppm) causes significant mortality in A. pisum within 24 h, rosmarinic acid could be exploited as a potent insecticide against this important pest insect. Furthermore, I. rugosus is already used for medicinal purposes and rosmarinic acid is known to reduce genotoxic effects induced by chemicals, hence it is expected to be safer compared to the current conventional pesticides. While this study highlights the potential of I. rugosus as a possible biopesticide source against A. pisum, it also provides the basis for further exploration and development of formulations for effective field application.

UDP-glucosyltransferases potentially contribute to imidacloprid resistance in Aphis gossypii glover based on transcriptomic and proteomic analyses

The cotton aphid, Aphis gossypii Glover, is a destructive global crop pest. Control of A. gossypii has relied heavily on the application of chemical insecticides. The cotton aphid has developed resistance to numerous insecticides, including imidacloprid, which has been widely used to control cotton pests in China since the 1990s. Our objective was to investigate the potential role of UDP-glycosyltransferases (UGTs) in imidacloprid resistance based on transcriptomic and proteomic analyses of field-originated imidacloprid-resistant (IMI_R) and -susceptible (IMI_S) A. gossypii clones. The transcriptomic and proteomic analyses revealed that 12 out of 512 differentially expressed genes and three out of 510 differentially expressed proteins were predicted as UDP-glycosyltransferase (UGT). Based on quantitative real-time PCR analysis, nine UGT genes, UGT343A4, UGT344A15, UGT344A16, UGT344B4, UGT344C7, UGT344C9, UGT344N4, UGT 24541, and UGT7630, were up-regulated in the IMI_R clone compared to the IMI_S clone. Meanwhile, UGT344A16, UGT344B4, UGT344C7, and UGT344N4 were overexpressed at the protein level based on western blot analysis. Furthermore, knockdown of UGT344B4 or UGT344C7 using RNA interference (RNAi) significantly increased sensitivity to imidacloprid in the IMI_R clone. In conclusion, UGTs potentially contributed to imidacloprid resistance in A. gossypii originating from cotton-growing regions of China. These results provide insights into the way we study insecticide resistance in cotton aphids.

Accessing a Russian Wheat Aphid Resistance Gene in Bread Wheat by Long-Read Technologies.

Russian wheat aphid (RWA) ( Kurdjumov) is a serious invasive pest of small-grain cereals and many grass species. An efficient strategy to defy aphid attacks is to identify sources of natural resistance and transfer resistance genes into susceptible crop cultivars. Revealing the genes helps understand plant defense mechanisms and engineer plants with durable resistance to the pest. To date, more than 15 RWA resistance genes have been identified in wheat ( L.) but none of them has been cloned. Previously, we genetically mapped the RWA resistance gene into an interval of 0.83 cM on the short arm of chromosome 7D and spanned it with five bacterial artificial chromosome (BAC) clones. Here, we used a targeted strategy combining traditional approaches toward gene cloning (genetic mapping and sequencing of BAC clones) with novel technologies, including optical mapping and long-read nanopore sequencing. The latter, with reads spanning the entire length of a BAC insert, enabled us to assemble the whole region, a task that was not achievable with short reads. Long-read optical mapping validated the DNA sequence in the interval and revealed a difference in the locus organization between resistant and susceptible genotypes. The complete and accurate sequence of the region facilitated the identification of new markers and precise annotation of the interval, revealing six high-confidence genes. Identification of as the most likely candidate opens an avenue for its validation through functional genomics approaches.