Electrical Penetration Graph Analysis Research Articles

Tomato Aphid (Aphis gossypii) Secreted Saliva Can Enhance Aphid Resistance by Upregulating Signaling Molecules in Tomato (Solanum lycopersicum).

This study investigated the impact of Aphis gossypii watery saliva on the induction of tomato (Solanum lycopersicum) plant resistance. To examine the role of A. gossypii saliva, we collected watery saliva from A. gossypii after a 48 h feeding period on an artificial diet. SDS-PAGE resolving gel 12% was used to separate the salivary proteins. Relative expression of gene analysis revealed that the intrusion of A. gossypii saliva dripping onto S. lycopersicum leaves triggered robust defense responses mediated by a signaling molecule, i.e., salicylic acid, while the signaling molecule's jasmonic acid-dependent defense responses were moderately activated. Aphid saliva infiltrated S. lycopersicum leaves slowed the intrinsic rate of population growth of A. gossypii and significantly reduced the number of nymphs produced daily, compared to untreated leaves. During a choice test with untreated S. lycopersicum, aphids showed a repellent response towards saliva-infiltrated S. lycopersicum. Moreover, the (EPG) electrical penetration graph analysis demonstrated that the eating pattern of A. gossypii compared to untreated S. lycopersicum, that had been exposed to saliva was negatively impacted. These results provide compelling evidence for the involvement of salivary components of A. gossypii in inducing resistance against aphids in S. lycopersicum plants. Furthermore, the study underscores the crucial role of watery saliva in the intricate interactions between aphids and plants. The activation of pathways was also part of the defensive response (jasmonic acid (JA), salicylic acid (SA) signaling molecules). The findings of this research deliver valuable insights into the potential of watery aphid saliva as a natural defense mechanism against aphid infestations in S. lycopersicum crops.

Transcriptome profiling in rice reveals a positive role for OsNCED3 in defense against the brown planthopper, Nilaparvata lugens

9-cis-epoxycarotenoid dioxygenase (NCED) is the rate-limiting enzyme for abscisic acid (ABA) biosynthesis in higher plants. In rice, OsNCED3 was shown to promote ABA synthesis, and improve abiotic stress tolerance, but the function of OsNCED3 in regulating rice defense against the brown planthopper (Nilaparvata lugens; BPH) has been unclear. In this study, several parameters were used to assess rice resistance to BPH, including the average injury level, the functional plant loss index, and electrical penetration graph analysis. Rice lines overexpressing OsNCED3 (OE) were more resistant to BPH than the wild-type cv. Zhonghua11 (WT). Transcriptome analysis was performed on WT, OE, and a RNAi transgenic line silenced for OsNCED3; these three lines were either infested or non-infested with BPH. Seventeen RNA libraries were compared, and most of the differentially expressed genes (DEGs) were upregulated. The number of DEGs in the RNAi line infested with BPH was significantly higher than the OE, and WT lines, and many DEGs were related to the stress response, and biosynthesis of jasmonic acid. This study shows that overexpression of OsNCED3 in rice improves resistance to BPH, and has potential merit in rice breeding programs.

Cowpea aphid resistance in cowpea line CB77 functions primarily through antibiosis and eliminates phytotoxic symptoms of aphid feeding

Cowpea (Vigna unguiculata) is one of the most important crops in semiarid areas of the world, where it thrives in hot, dry conditions. While cowpea is able to withstand abiotic stresses, it suffers serious losses from biotic antagonists, including infestation by the cowpea aphid (Aphis craccivora). Cowpea aphid infestations are highly destructive, especially on young plants. However, it is unclear whether cowpea aphid damage is the result of aphids having phytotoxic effects on their hosts, or simple density effects. To better understand cowpea aphid damage and the potential for resistance traits to mitigate aphid impacts, we evaluated phenotypic changes in cowpea in response to variable aphid densities and systemic versus local infestations. Low aphid densities induced leaf distortions and pseudogalling, suggesting that cowpea aphids are phytotoxic to cowpea. Resistance to the cowpea aphid has been previously identified in an African cowpea germplasm, and near isogenic lines (NILs) containing resistance quantitative trait loci (QTL) were generated in the California blackeye cultivar background. Using a series of performance assays, we determined that resistance conferred by the two QTL counteracts aphid phytotoxicity and severely limits aphid growth and fecundity. Using choice assays, a preference by cowpea aphids for the susceptible NIL was observed. Electrical penetration graph analysis revealed that the resistance phenotype includes weak surface level deterrence and strong phloem-based resistance that manifests during the sap ingestion phase. Our study provides evidence of phytotoxic traits in A. craccivora while identifying a viable means of counteracting aphid damage and reproductive potential through resistance.

Feeding Behavior of Riptortus pedestris (Fabricius) on Soybean: Electrical Penetration Graph Analysis and Histological Investigations.

Simple SummaryRiptortus pedestris (Fabricius) (Hemiptera: Alydidae) is a major soybean pest with the peak population occurrence during the seed maturity stage from pod filling to harvest. Soybean pods and/or seeds are required for R. pedestris development. However, the feeding strategies employed by this stink bug to feed on soybean are still not clear. In the present study, we recorded the feeding behaviors of R. pedestris on soybean using electropenetrography (EPG). The biological meaning of each waveform was confirmed by histological examination of plant tissues containing stylets or salivary sheath. In total, five phases of waveforms were identified: non-probing, pathway (Rp1), xylem sap ingestion (Rp2), salivation and ingestion (Rp3), and interruption (Rp4). Xylem ingestion (Rp2) was observed during R. pedestris feeding on soybean leaflets, stems, and pods, demonstrating that the stink bug was ingesting xylem sap from vascular tissue. Cell rupture (salivation/ingestion, Rp3) was only recorded during R. pedestris feeding on cotyledon and pods. Histological images showed that stylet tips and the salivary sheath were positioned in the tissues of cotyledon and pods. Taken together, our results demonstrate that R. pedestris uses a cell-rupture strategy to acquire nutrients from soybean pods and/or seeds while utilizing salivary sheath tactics to obtain water from xylem sap. These findings provide insightful information to understand the interactions between R. pedestris and the soybean plant. Riptortus pedestris (Fabricius) is a major agricultural pest feeding on soybean pods and seeds. The large populations occur during seed maturity stages from pod filling to harvest. Its infestation results in shriveled and dimpled seeds while vegetative structures (leaflet and stem) remain green, known as “Stay Green” syndrome. Additional evidence also demonstrates that soybean pods and seeds are required for Riptortus pedestris development. However, the feeding behavior strategies employed by this stink bug to feed on soybean plants are still not clear. In the present study, the feeding behaviors of R. pedestris on soybean plants were recorded by electropenetrography (EPG), and a waveform library was created for this species. A total of five phases of waveforms—nonprobing, pathway (Rp1), xylem sap ingestion (Rp2), salivation and ingestion (Rp3), and interruption (Rp4)—were identified. Non-probing waveforms Z and NP and pathway (Rp1) were found in all tested plant structures (leaflet, stem, cotyledon, and pods). Waveform Rp2 (xylem sap ingestion, xylem ingestion) was primarily recorded during R. pedestris feeding on leaflets and stems, while Rp3 (salivation/ingestion) was only observed during feeding on cotyledon and pods. Histological examinations confirmed that correlation between Rp2 and stylet tip positioning in the xylem vessel in leaflets and stems. Stylet tips end in the tissues of cotyledon and pods when Rp3 is recorded. Taken together, our results demonstrate that R. pedestris ingests xylem sap from vegetative tissues of soybean (leaflet and stem) via a salivary sheath strategy to obtain water. It mainly acquires nutrients from soybean pods and/or seeds using cell-rupture tactics. This study provided insightful information to understand the field occurrence patterns of “Stay Green” syndrome, which may have important implications for pest control.

Local and systemic effect of azadirachtin on host choice and feeding activity of Macrosiphum rosae on rose plants

Many studies in the last decades addressed the antifeedant and repellent effects of neem (Azadirachta indica)-based pesticides on arthropod herbivores with azadirachtin as the leading active ingredient. However, results for aphids regarding repellence and feeding were often contradictory and mainly based on laboratory studies with self-made neem extracts in high concentrations. Moreover, no data are available on the presence of azadirachtin in the phloem of treated plants—the tissue that most aphids feed on. We investigated how the application of the commercial neem product NeemAzal-T/S onto only one plant half (upper or lower) affects nymphs of the aphid Macrosiphum rosae in clip cages on the untreated plant half. Results for aphid mortality indicate that active ingredients of NeemAzal-T/S are translocated both upwards and downwards in rose plants and that active ingredients are transported in the phloem. Furthermore, we investigated whether NeemAzal-T/S has a repellent or antifeedant effect on M. rosae. A choice test in the greenhouse as well as the quantification of honeydew excretion and electrical penetration graph analysis do not show differences in settling or feeding on untreated and NeemAzal-T/S-treated rose plants. Collectively, our data show that the effect of approved concentrations of NeemAzal-T/S on M. rosae is based on the toxicity after feeding on treated plants, not on starvation.

Rice virus release from the planthopper salivary gland is independent of plant tissue recognition by the stylet.

The transmission of plant viruses by arthropod vectors is closely related to feeding behavior. For persistently transmitted viruses, virus release means that virus moves through the salivary gland microvillus barriers of insects into plant via the stylet. However, whether virus release is dependent on plant tissue and component recognition by the stylet is unclear. In this study, the small brown planthopper (SBPH) and two rice viruses transmitted by it were used as a model to explore this question. After the viruliferous insects penetrated a stretched membrane without plant tissue structure and ingested liquid food (rice sap, nutrient solution or water), both viruses were detected in the liquid food after only a 6 min inoculation access period, suggesting that the viruses were released from SBPH salivary gland independent of plant tissue and component recognition by the stylet. In subsequent electrical penetration graph (EPG) analysis, N4a-like and N4b-like waveforms, similar to N4a (phloem salivation before ingestion) and N4b (sieve element ingestion), were observed during SBPH penetrating the membrane, exhibiting normal feeding activity of planthopper on membrane, which further demonstrated that virus release from salivary gland was along with feeding activity, without the stylet sensing plant tissue. EPG analysis and identification of salivary proteins indicated more active feeding behavior and efficient salivation in viruliferous planthoppers. These results suggest that the rice virus is released from insect salivary gland independent of plant tissue and component recognition by the stylet, and the simple virus release mode facilitates virus transmission by vectors. © 2020 Society of Chemical Industry.

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.

Reduction of Plant Suitability for Corn Leaf Aphid (Hemiptera: Aphididae) Under Elevated Carbon Dioxide Condition.

In the current context of global climate change, atmospheric carbon dioxide (CO2) concentrations are continuously rising with potential influence on plant-herbivore interactions. The effect of elevated CO2 (eCO2) on feeding behavior of corn leaf aphid, Rhopalosiphum maidis (Fitch) on barley seedlings Hordeum vulgare L. was tracked using electrical penetration graph (EPG). The nutrient content of host plant and the developmental indexes of aphids under eCO2 and ambient CO2 (aCO2) conditions were also investigated. Barley seedlings under eCO2 concentration had lower contents of crude protein and amino acids. EPG analysis showed the plants cultivated under eCO2 influenced the aphid feeding behavior, by prolonging the total pre-probation time of the aphids (wandering and locating the feeding site) and the ingestion of passive phloem sap. Moreover, fresh body weight, fecundity and intrinsic population growth rate of R. maidis was significantly decreased in eCO2 in contrast to aCO2 condition. Our findings suggested that changes in plant nutrition caused by eCO2, mediated via the herbivore host could affect insect feeding behavior and population dynamics.

Plant-mediated indirect effects of two viruses with different transmission modes on Bemisia tabaci feeding behavior and fitness

Viral infections can induce phenotypic modifications on plant cues that mediate host plant colonization by vectors. It has been postulated that virus transmission mode influences the way in which virus infection alters the vector´s probing behavior and fitness on plants in order to optimize transmission. We evaluated the indirect effects of tomato plant infection by a semipersistently transmitted crinivirus, Tomato chlorosis virus (ToCV), and a persistently transmitted begomovirus, Tomato severe rugose virus (ToSRV), on the biological parameters and probing behavior of Bemisia tabaci MEAM1, using the electrical penetration graph (EPG) technique. There was a deleterious effect on the viability of nymphs developed on ToCV- or ToSRV-infected tomato plants, but no effects on reproductive parameters of emerged adults, in comparison to mock-inoculated plants. The EPG analysis showed a delay in starting to probe and fewer probing attempts on ToCV-infected plants, suggesting that ToCV infection reduces plant palatability. No virus-indirect effect was observed on the insect´s probing behavior in the phloem tissue. Our combined results show that tomato infection by viruses with two different transmission modes affected the development of the whitefly vector similarly, with neutral effects on probing behavior parameters associated with virus acquisition in the phloem, and thus no apparent benefits for virus spread.

Whitefly feeding behavior and retention of a foregut-borne crinivirus exposed to artificial diets with different pH values.

Transmission of plant viruses by phytophagous hemipteran insects encompasses complex interactions underlying a continuum of processes involved in virus acquisition, retention and inoculation combined with vector feeding behavior. Here, we investigated the effects of dietary pH on whitefly (Bemisia tabaci) feeding behavior and release of Lettuce infectious yellows virus (LIYV) virions retained in the vector's foregut. Electrical penetration graph analysis revealed that variables associated with whitefly probing and ingestion did not differ significantly in pH (4, 7.4, and 9) adjusted artificial diets. To investigate virus retention and release, whiteflies allowed to acquire LIYV virions in a pH 7.4 artificial diet were fed pH 4, 7.4, or 9 virion-free artificial (clearing) diets. Immunofluorescent localization analyses indicated that virions remained bound to the foreguts of approximately 20%-24% of vectors after they fed on each of the 3 pH-adjusted clearing diets. When RNA preparations from the clearing diets were analyzed by reverse transcription (RT) nested-PCR and, in some cases, real-time qPCR, successful amplification of LIYV-specific sequence was infrequent but consistently repeatable for the pH 7.4 diet but never observed for the pH 4 and 9 diets, suggesting a weak pH-dependent effect for virion release. Viruliferous vectors that fed on each of the 3 pH-adjusted clearing diets transmitted LIYV to virus-free plants. These results suggest that changes in pH values alone in artificial diet do not result in observable changes in whitefly feeding behaviors, an observation that marks a first in the feeding of artificial diet by whitefly vectors; and that there is a potential causal and contingent relationship between the pH in artificial diet and the release/inoculation of foregut bound virions.

Characterization and genetic dissection of resistance to spotted alfalfa aphid (Therioaphis trifolii) in Medicago truncatula.

Aphids cause significant yield losses in agricultural crops worldwide. Medicago truncatula, a model legume, cultivated pasture species in Australia and close relative of alfalfa (Medicago sativa), was used to study the defence response against Therioaphis trifolii f. maculate [spotted alfalfa aphid (SAA)]. Aphid performance and plant damage were compared among three accessions. A20 is highly susceptible, A17 has moderate resistance, and Jester is strongly resistant. Subsequent analyses using A17 and A20, reciprocal F1s and an A17×A20 recombinant inbred line (RIL) population revealed that this moderate resistance is phloem mediated and involves antibiosis and tolerance but not antixenosis. Electrical penetration graph analysis also identified a novel waveform termed extended potential drop, which occurred following SAA infestation of M. truncatula. Genetic dissection using the RIL population revealed three quantitative trait loci on chromosomes 3, 6, and 7 involved in distinct modes of aphid defence including antibiosis and tolerance. An antibiosis locus resides on linkage group 3 (LG3) and is derived from A17, whereas a plant tolerance and antibiosis locus resides on LG6 and is derived from A20, which exhibits strong temporary tolerance. The loci identified reside in regions harbouring classical resistance genes, and introgression of these loci in current medic cultivars may help provide durable resistance to SAA, while elucidation of their molecular mechanisms may provide valuable insight into other aphid–plant interactions.

Overexpression of IRM1 Enhances Resistance to Aphids in Arabidopsis thaliana

Aphids are insects that cause direct damage to crops by the removal of phloem sap, but more importantly they spread devastating viruses. Aphids use their sophisticated mouthpart (i.e. stylet) to feed from the phloem sieve elements of the host plant. To identify genes that affect host plant resistance to aphids, we previously screened an Arabidopsis thaliana activation tag mutant collection. In such mutants, tagged genes are overexpressed by a strong 35S enhancer adjacent to the natural promoter, resulting in a dominant gain-of-function phenotype. We previously identified several of these mutants on which the aphid Myzus persicae showed a reduced population development compared with wild type. In the present study we show that the gene responsible for the phenotype of one of the mutants is At5g65040 and named this gene Increased Resistance to Myzus persicae 1 (IRM1). Overexpression of the cloned IRM1 gene conferred a phenotype identical to that of the original mutant. Conversely, an IRM1 knockout mutant promoted aphid population development compared to the wild type. We performed Electrical Penetration Graph analysis to investigate how probing and feeding behaviour of aphids was affected on plants that either overexpressed IRM1 or contained a knockout mutation in this gene. The EPG results indicated that the aphids encounter resistance factors while reaching for the phloem on the overexpressing line. This resistance mechanism also affected other aphid species and is suggested to be of mechanical nature. Interestingly, genetic variation for IRM1 expression in response to aphid attack was observed. Upon aphid attack the expression of IRM1 was initially (after 6 hours) induced in ecotype Wassilewskija followed by suppression. In Columbia-0, IRM1 expression was already suppressed six hours after the start of the infestation. The resistance conferred by the overexpression of IRM1 in A. thaliana trades off with plant growth.

Electrical Penetration Graph Analysis of the Feeding Behavior of Soybean Aphids on Soybean Cultivars With Antibiosis

The soybean aphid, Aphis glycine Matsumura (Hemiptera: Aphididae), is a major pest of soybean. In the current study, we used the Electrical Penetration Graph technique to study feeding behavior of soybean aphids on antibiotic-resistant soybean lines KS1621, KS1613, and KS1642, and a susceptible soybean line, KS4202. We observed that soybean aphids spent significantly shorter periods of time in the sieve element phase but slightly more times in nonprobing phases in all three resistant lines than in the susceptible control. Our study suggests that resistance factors exist in the phloem of the resistant soybean lines, and that these lines may contain antixenosis in addition to antibiosis.

Survival rate and stylet penetration behavior of current Korean populations of the brown planthopper, Nilaparvata lugens, on resistant rice varieties

To understand the development of host plant resistance-breaking ability of the current BPH populations in Korea, we conducted nymphal survivorship tests and electrical penetration graph (EPG) studies on susceptible and resistant rice varieties with four different BPH populations, which were collected in the early 1980s (S-BPH) and in 2005, 2006, and 2007. The S-BPH had low survival rates on resistant rice varieties carrying either Bph1 or bph2. However, the current BPH populations have high resistance-breaking ability on the varieties with their elevated survival rates, whereas their survival rates were still low on the other resistant varieties, Gayabyeo ( Bph1 the other unknown gene) and Rathu Heenati ( Bph3). The EPG analysis also revealed that the ratio of BPH that could reach the phloem sap ingestion waveform (N4-b) within 15 h on the resistant rice varieties containing Bph1 or bph2 was higher in the current BPH populations (16.7–50%) than in the S-BPH population (0-4.2%). However, the pre-reaching time from the penetration start to the first N4-b waveform in the current BPH populations was significantly longer on resistant varieties ( Bph1 or bph2) than on susceptible varieties. Furthermore, the total duration of N4-b waveform was significantly shorter on the resistant varieties. From these results, we suggest that, although the current BPHs collected in Korea have a high resistance-breaking ability through the increase of survival rate on resistant rice varieties carrying either Bph1 or bph2, they still have some difficulties feeding on the phloem sap of the resistant rice varieties.

The role of sinigrin in host plant recognition by aphids during initial plant penetration

AbstractPlant penetration behaviour (probing) of the cabbage aphid, Brevicoryne brassicae, and the pea aphid, Acyrthosiphon pisum, was studied on excised leaves of broad beans, Vicia faba, kept in water or in a 1% aqueous solution of sinigrin. Using the DC EPG (Electrical Penetration Graph) technique it was shown that the cabbage aphid on sinigrin‐untreated bean leaves showed numerous short probes into epidermis and mesophyll. None of these aphids showed either phloem salivation or ingestion waveforms on untreated leaves. In contrast, on sinigrin‐treated bean leaves, 35% of the probing time was spent on phloem sap ingestion (E2) and almost all aphids reached phloem vessels and started feeding. The duration of phloem salivation before phloem ingestion and the mean duration of phloem ingestion periods were similar on a host and a sinigrin‐treated non‐host plant. However, the total probing time by B. brassicae was 10% longer, the total phloem sap ingestion time was twice as long, and the time to the first phloem phase within a probe was three times shorter on the host plant compared to sinigrin‐treated broad beans. Acyrthosiphon pisum also responded to the addition of sinigrin to broad beans, but in this case sinigrin acted as a deterrent. On sinigrin‐treated leaves, A. pisum terminated probes before ingestion from phloem vessels, and none of these aphids showed phloem salivation and ingestion on treated leaves. Glucosinolates were detected in the mesophyll cells of the brassicaceous plant, Sinapis alba. Based on this finding and in addition to the foregoing EPG analysis of aphid probing on these plants and broad beans, our hypothesis is that aphids may recognise their host plants as soon as they probe the mesophyll tissue and before they start ingestion from phloem vessels.

Electrical Penetration Graph analysis reveals population differentiation of host‐plant probing behaviors within the aphid species Uroleucon ambrosiae

Abstract Uroleucon ambrosiae collected from the eastern and the southwestern United States were studied in relation to host‐plant probing. In the field, eastern aphids are highly specific on Ambrosia trifida, while southwestern aphids feed on Ambrosia trifida plus many other species in the family Asteraceae. Electrical penetration graphs of insects on the principal host, A. trifida, and an additional host of southwestern populations, Heterotheca subaxillaris, revealed regional differentiation in host‐associated aphid behaviors, specifically in the first phase of subcuticular probing, and in phloem finding activities. Eastern aphids used A. trifida more efficiently than southwestern aphids, but were demonstrably less vigorous in their probing activities on H. subaxillaris.