Time point- and plant part-specific changes in phloem exudate metabolites of leaves and ears of wheat in response to drought and effects on aphids.

Alterations in the frequency and intensity of drought events are expected due to climate change and might have consequences for plant metabolism and the development of plant antagonists. In this study, the responses of spring wheat (Triticum aestivum) and one of its major pests, the aphid Sitobion avenae, to different drought regimes were investigated, considering different time points and plant parts. Plants were kept well-watered or subjected to either continuous or pulsed drought. Phloem exudates were collected twice from leaves and once from ears during the growth period and concentrations of amino acids, organic acids and sugars were determined. Population growth and survival of the aphid S. avenae were monitored on these plant parts. Relative concentrations of metabolites in the phloem exudates varied with the time point, the plant part as well as the irrigation regime. Pronounced increases in relative concentrations were found for proline, especially in pulsed drought-stressed plants. Moreover, relative concentrations of sucrose were lower in phloem exudates of ears than in those of leaves. The population growth and survival of aphids were decreased on plants subjected to drought and populations grew twice as large on ears compared to leaves. Our study revealed that changes in irrigation frequency and intensity modulate plant-aphid interactions. These effects may at least partly be mediated by changes in the metabolic composition of the phloem sap.

Sieve element occlusion: Interactions with phloem sap-feeding insects. A review.

Phloem sieve element (SE) occlusion has been hypothesized for decades to be a mechanism of resistance against phloem sap-feeding insects. Few studies have tested this hypothesis although it is likely a widespread phenomenon. This review focuses on SE occlusion by callose and P-proteins. Both are reversible, which would allow the plant to defend itself against phloem sap-feeders when SEs are penetrated and resume normal function when the insects give up and withdraw their stylets. Callose (ß-1,3 glucans with some ß-1,6 branches) serves many roles in plant physiology in many different tissues, each being under the control of different callose synthase genes; only callose deposited in SE sieve pores is relevant to SE occlusion. The amount of callose in sieve pores (and consequently how much it impedes sap flow) is determined by the balance in activity between callose synthase and ß-1,3 glucanase. Sieve pore callose deposition has been shown to provide resistance to some phloem sap-feeders in a few studies, and in one, the difference in resistance between a susceptible and resistant rice variety was due to the ability or inability of the insect to upregulate the plants' ß-1,3 glucanase that degrades the callose deposition. P-proteins occur only in dicotyledons and include a variety of proteins, not all of which are involved in SE occlusion. In some plants, P-proteins form distinct bodies in mature functional SEs. In papilionid legumes, these discrete bodies, called forisomes can expand and contract. In their expanded state, they effectively plug SEs and stop the flow of sap while in their contracted state, they provide negligible resistance to sap flow. Expansion of forisomes is triggered by an influx of Ca2+ into the SE. Penetration of a legume (Vicia faba) SE by a generalist aphid not adapted to legumes triggers forisome expansion which occludes the SE and prevents the aphid from ingesting sap. In contrast, a legume specialist aphid, Acyrthosiphon pisum, does not trigger forisome expansion and readily ingests sap from V. faba. P-protein bodies in SEs of non-legumes do not appear to be involved in SE occlusion. In most dicotyledons, P-proteins do not form discrete bodies, but rather occur as filamentous aggregations adhering to the parietal margins of the SE and in response to damage, are released into the lumen where they are carried by the flow of sap to the downstream sieve plate where they back up and clog the sieve pores. Their effectiveness at actually stopping the flow of sap is controversial. In one study, they seemed to provide little resistance to the flow of sap while in other studies, they provided considerable resistance. In response to injury in melon, they completely stop the flow of sap, and in an aphid-resistant melon, penetration of SEs by the melon aphid, Aphis gossypii, triggers P-protein occlusion which prevents the aphids from ingesting sap. The first P-protein described, PP1, occurs only in the genus Cucurbita, and although it has been often cited to function as a SE occlusion protein, experimental evidence suggests it does not play a significant role in SE occlusion. The most common strategy for phloem sap-feeders to mitigate P-protein occlusion seems to be avoid triggering it. A widely cited in vitro study suggested that aphid saliva can reverse P-protein occlusion, but a subsequent study demonstrated that saliva was ineffective at reversing P-protein occlusion in vivo. Lastly, SE callose deposition in wheat triggered by Russian wheat aphid has been hypothesized to create an artificial sink that benefits the aphid, but additional studies are needed to test that hypothesis.

EPG analysis of stylet penetration preference of woolly apple aphid on different parts of apple trees.

Woolly apple aphid (WAA), Eriosoma lanigerum (Hausmann), is an important global pest that feeds on Malus species. We studied the feeding preference of WAA on apple trees in the field for two consecutive years and in the laboratory we used electronic penetration graphs (EPG) to record the stylet penetration behavior of WAA on different parts of apple trees. We found that in the field WAA fed primarily on twigs and branches, not on leaves and fruits. Six EPG waveforms were produced during WAA probing on shoots, trunks and leaves of apple trees, including the non-penetration wave (np), the stylet pathway phase wave (C), the intracellular feeding wave (pd), the xylem feeding wave (G), waves indicative of release of saliva into the phloem (E1), and a wave indicative of ingestion from phloem (E2). In the laboratory, aphids only successfully fed on shoots, trunks and leaves, not on fruits. The EPG parameters on the phloem of shoots were significantly higher than those on trunks, indicating WAA prefer to feed on shoots. These laboratory findings explain the relative field feeding preference of WAA on different parts of apple trees, which occurs primarily on branches, barks, and young twigs in orchards, especially on young twigs.

Vectorizing Pro-Insecticide: Influence of Linker Length on Insecticidal Activity and Phloem Mobility of New Tralopyril Derivatives.

To improve the proinsecticidal activity and phloem mobility of amino acid-tralopyril conjugates further, nine conjugates were designed and synthesized by introducing glutamic acid to tralopyril, and the length of the linker between glutamic acid and tralopyril ranged from 2 atoms to 10 atoms. The results of insecticidal activity against the third-instar larvae of P. xylostella showed that conjugates 42, 43, 44,and 45 (straight-chain containing 2-5 atoms) exhibited good insecticidal activity, and their LC50 values were 0.2397 ± 0.0366, 0.4413 ± 0.0647, 0.4400 ± 0.0624, and 0.4602 ± 0.0655 mM, respectively. The concentrations of conjugates 43-45 were higher than that of conjugate 42 in the phloem sap at 2 h, and conjugate 43 showed the highest concentration. The introduction of glutamic acid can improve phloem mobility. The in vivo metabolism of conjugates 42 and 43 was investigated in P. xylostella, and the parent compound tralopyril was detected at concentrations of 0.5950 and 0.3172 nmol/kg, respectively. According to the above results, conjugates 42 and 43 were potential phloem mobile pro-insecticide candidates.

Transcriptomics identifies key defense mechanisms in rice resistant to both leaf-feeding and phloem feeding herbivores.

BACKGROUND: Outbreaks of insect pests in paddy fields cause heavy losses in global rice yield annually, a threat projected to be aggravated by ongoing climate warming. Although significant progress has been made in the screening and cloning of insect resistance genes in rice germplasm and their introgression into modern cultivars, improved rice resistance is only effective against either chewing or phloem-feeding insects. RESULTS: In this study, the results from standard and modified seedbox screening, settlement preference and honeydew excretion tests consistently showed that Qingliu, a previously known leaffolder-resistant rice variety, is also moderately resistant to brown planthopper (BPH). High-throughput RNA sequencing showed a higher number of differentially expressed genes (DEGs) at the infestation site, with 2720 DEGs in leaves vs 181 DEGs in sheaths for leaffolder herbivory and 450 DEGs in sheaths vs 212 DEGs in leaves for BPH infestation. The leaf-specific transcriptome revealed that Qingliu responds to leaffolder feeding by activating jasmonic acid biosynthesis genes and genes regulating the shikimate and phenylpropanoid pathways that are essential for the biosynthesis of salicylic acid, melatonin, flavonoids and lignin defensive compounds. The sheath-specific transcriptome revealed that Qingliu responds to BPH infestation by inducing salicylic acid-responsive genes and those controlling cellular signaling cascades. Taken together these genes could play a role in triggering defense mechanisms such as cell wall modifications and cuticular wax formation. CONCLUSIONS: This study highlighted the key defensive responses of a rarely observed rice variety Qingliu that has resistance to attacks by two different feeding guilds of herbivores. The leaffolders are leaf-feeder while the BPHs are phloem feeders, consequently Qingliu is considered to have dual resistance. Although the defense responses of Qingliu to both insect pest types appear largely dissimilar, the phenylpropanoid pathway (or more specifically phenylalanine ammonia-lyase genes) could be a convergent upstream pathway. However, this possibility requires further studies. This information is valuable for breeding programs aiming to generate broad spectrum insect resistance in rice cultivars.

Plasmodesmata play pivotal role in sucrose supply to Meloidogyne graminicola-caused giant cells in rice.

On infection, plant-parasitic nematodes establish feeding sites in roots from which they take up carbohydrates among other nutrients. Knowledge on how carbohydrates are supplied to the nematodes' feeding sites is limited. Here, gene expression analyses showed that RNA levels of OsSWEET11 to OsSWEET15 were extremely low in both Meloidogyne graminicola (Mg)-caused galls and noninoculated roots. All the rice sucrose transporter genes, OsSUT1 to OsSUT5, were either down-regulated in Mg-caused galls compared with noninoculated rice roots or had very low transcript abundance. OsSUT1 was the only gene up-regulated in galls, at 14 days postinoculation (dpi), after being highly down-regulated at 3 and 7 dpi. OsSUT4 was down-regulated at 3 dpi. No noticeable OsSUTs promoter activities were detected in Mg-caused galls of pOsSUT1 to -5::GUS rice lines. Loading experiments with carboxyfluorescein diacetate (CFDA) demonstrated that symplastic connections exist between phloem and Mg-caused giant cells (GCs). According to data from OsGNS5- and OsGSL2-overexpressing rice plants that had decreased and increased callose deposition, respectively, callose negatively affected Mg parasitism and sucrose supply to Mg-caused GCs. Our results suggest that plasmodesmata-mediated sucrose transport plays a pivotal role in sucrose supply from rice root phloem to Mg-caused GCs, and OsSWEET11 to -15 and OsSUTs are not major players in it, although further functional analysis is needed for OsSUT1 and OsSUT4.

Distribution pattern of entry holes of the tree-killing bark beetle Polygraphus proximus.

Bark beetles attack their hosts at uniform intervals to avoid intraspecific competition in the phloem. Bark texture and phloem thickness also affect bark beetle attacks, and the bark characteristics are not spatially homogeneous; therefore, the distribution patterns of entry holes can demonstrate an aggregated distribution. Polygraphus proximus Blandford (Coleoptera: Scolytinae) is a non-aggressive phloephagous bark beetle that feeds on Far Eastern firs. They have caused mass mortality in Russia and Japan. However, the distribution pattern of entry holes of P. proximus and spatial relationships with bark characteristics have not been studied. Thus, we investigated the distribution pattern of entry holes of P. proximus. The distribution of entry holes was significantly uniform in most cases. As the attack density increased, an aggregated distribution pattern within a short distance (< 4.0 cm) was observed. The rough bark had a significantly higher number of entry holes than the remaining bark. The distribution pattern of entry holes demonstrated a significantly aggregated spatial association with rough bark. Finally, rough bark around knots had significantly thicker phloem than the remaining barks. These suggest that P. proximus may preferentially attack rough bark to reproduce in the thicker phloem under a rough bark surface.

An Aphid-Secreted Salivary Protease Activates Plant Defense in Phloem.

Recent studies have reported that aphids facilitate their colonization of host plants by secreting salivary proteins into host tissues during their initial probing and feeding. Some of these salivary proteins elicit plant defenses, but the molecular and biochemical mechanisms that underlie the activation of phloem-localized resistance remain poorly understood. The aphid Myzus persicae, which is a generalized phloem-sucking pest, encompasses a number of lineages that are associated with and adapted to specific host plant species. The current study found that a cysteine protease Cathepsin B3 (CathB3), and the associated gene CathB3, was upregulated in the salivary glands and saliva of aphids from a non-tobacco-adapted (NTA) aphid lineage, when compared to those of a tobacco-adapted lineage. Furthermore, the knockdown of CathB3 improved the performance of NTA lineages on tobacco, and the propeptide domain of CathB3 was found to bind to tobacco cytoplasmic kinase ENHANCED DISEASE RESISTANCE 1-like (EDR1-like), which triggers the accumulation of reactive oxygen species in tobacco phloem, thereby suppressing both phloem feeding and colonization by NTA lineages. These findings reveal a novel function for a cathepsin-type protease in aphid saliva that elicits effective host plant defenses and warranted the theory of host specialization for generalist aphids.

Intraspecific competition counters the effects of elevated and optimal temperatures on phloem-feeding insects in tropical and temperate rice.

The direct effects of rising global temperatures on insect herbivores could increase damage to cereal crops. However, the indirect effects of interactions between herbivores and their biotic environment at the same temperatures will potentially counter such direct effects. This study examines the potential for intraspecific competition to dampen the effects of optimal temperatures on fitness (survival × reproduction) of the brown planthopper, Nilaparvata lugens [BPH] and whitebacked planthopper, Sogatella furcifera [WBPH], two phloem-feeders that attack rice in Asia. We conducted a series of experiments with increasing densities of ovipositing females and developing nymphs on tropical and temperate rice varieties at 25, 30 and 35°C. Damage from planthoppers to the tropical variety was greater at 30°C compared to 25°C, despite faster plant growth rates at 30°C. Damage to the temperate variety from WBPH nymphs was greatest at 25°C. BPH nymphs gained greater biomass at 25°C than at 30°C despite faster development at the higher temperature (temperature-size rule); however, the effect was apparent only at high nymph densities. WBPH survival, development rates and nymph weights all declined at ≥ 30°C. At about the optimal temperature for WBPH (25°C), intraspecific crowding reduced nymph weights. Temperature has little effect on oviposition responses to density, and intraspecific competition between females only weakly counters the effects of optimal temperatures on oviposition in both BPH and WBPH. Meanwhile, the deleterious effects of nymph crowding will counter the direct effects of optimal temperatures on voltinism in BPH and on body size in both BPH and WBPH. The negative effects of crowding on BPH nymphs may be decoupled from resource use at higher temperatures.

Glucosylation prevents plant defense activation in phloem-feeding insects.

The metabolic adaptations by which phloem-feeding insects counteract plant defense compounds are poorly known. Two-component plant defenses, such as glucosinolates, consist of a glucosylated protoxin that is activated by a glycoside hydrolase upon plant damage. Phloem-feeding herbivores are not generally believed to be negatively impacted by two-component defenses due to their slender piercing-sucking mouthparts, which minimize plant damage. However, here we document that glucosinolates are indeed activated during feeding by the whitefly Bemisia tabaci. This phloem feeder was also found to detoxify the majority of the glucosinolates it ingests by the stereoselective addition of glucose moieties, which prevents hydrolytic activation of these defense compounds. Glucosylation of glucosinolates in B. tabaci was accomplished via a transglucosidation mechanism, and two glycoside hydrolase family 13 (GH13) enzymes were shown to catalyze these reactions. This detoxification reaction was also found in a range of other phloem-feeding herbivores.

Changes in reflectance of rice seedlings during planthopper feeding as detected by digital camera: Potential applications for high-throughput phenotyping.

Damage to grasses and cereals by phloem-feeding herbivores is manifest as nutrient and chlorophyll loss, desiccation, and a gradual decline in host vigour. Chlorophyll loss in particular leads to a succession of colour changes before eventual host death. Depending on the attacking herbivore species, colour changes can be difficult to detect with the human eye. This study used digital images to examine colour changes of rice seedlings during feeding by the brown planthopper, Nilaparvata lugens (Stål) and whitebacked planthopper, Sogatella furcifera (Horváth). Values for red (580 nm), green (540 nm) and blue (550 nm) reflectance for 39 rice varieties during seedling seed-box tests were derived from images captured with a digital camera. Red and blue reflectance gradually increased as herbivore damage progressed until final plant death. Red reflectance was greater from plants attacked by the brown planthopper than plants attacked by the whitebacked planthopper, which had proportionately more green and blue reflectance, indicating distinct impacts by the two planthoppers on their hosts. Analysis of digital images was used to discriminate variety responses to the two planthoppers. Ordination methods based on red-green-blue reflectance and vegetation indices such as the Green Leaf Index (GLI) that included blue reflectance were more successful than two-colour indices or indices based on hue, saturation and brightness in discriminating between damage responses among varieties. We make recommendations to advance seed-box screening methods for cereal resistance to phloem feeders and demonstrate how images from digital cameras can be used to improve the quality of data captured during high-throughput phenotyping.

Combining Transient Expression and Cryo-EM to Obtain High-Resolution Structures of Luteovirid Particles.

The Luteoviridae are pathogenic plant viruses responsible for significant crop losses worldwide. They infect a wide range of food crops, including cereals, legumes, cucurbits, sugar beet, sugarcane, and potato and, as such, are a major threat to global food security. Viral replication is strictly limited to the plant vasculature, and this phloem limitation, coupled with the need for aphid transmission of virus particles, has made it difficult to generate virus in the quantities needed for high-resolution structural studies. Here, we exploit recent advances in heterologous expression in plants to produce sufficient quantities of virus-like particles for structural studies. We have determined their structures to high resolution by cryoelectron microscopy, providing the molecular-level insight required to rationally interrogate luteovirid capsid formation and aphid transmission, thereby providing a platform for the development of preventive agrochemicals for this important family of plant viruses.

Neonicotinoids in excretion product of phloem-feeding insects kill beneficial insects.

Pest control in agriculture is mainly based on the application of insecticides, which may impact nontarget beneficial organisms leading to undesirable ecological effects. Neonicotinoids are among the most widely used insecticides. However, they have important negative side effects, especially for pollinators and other beneficial insects feeding on nectar. Here, we identify a more accessible exposure route: Neonicotinoids reach and kill beneficial insects that feed on the most abundant carbohydrate source for insects in agroecosystems, honeydew. Honeydew is the excretion product of phloem-feeding hemipteran insects such as aphids, mealybugs, whiteflies, and psyllids. We allowed parasitic wasps and pollinating hoverflies to feed on honeydew from hemipterans feeding on trees treated with thiamethoxam or imidacloprid, the most commonly used neonicotinoids. LC-MS/MS analyses demonstrated that both neonicotinoids were present in honeydew. Honeydew with thiamethoxam was highly toxic to both species of beneficial insects, and honeydew with imidacloprid was moderately toxic to hoverflies. Collectively, our data provide strong evidence for honeydew as a route of insecticide exposure that may cause acute or chronic deleterious effects on nontarget organisms. This route should be considered in future environmental risk assessments of neonicotinoid applications.

Defence gene expression and phloem quality contribute to mesophyll and phloem resistance to aphids in wild barley.

Aphids, including the bird cherry-oat aphid (Rhopalosiphum padi), are significant agricultural pests. The wild relative of barley, Hordeum spontaneum 5 (Hsp5), has been described to be partially resistant to R. padi, with this resistance proposed to involve higher thionin and lipoxygenase gene expression. However, the specificity of this resistance to aphids and its underlying mechanistic processes are unknown. In this study, we assessed the specificity of Hsp5 resistance to aphids and analysed differences in aphid probing and feeding behaviour on Hsp5 and a susceptible barley cultivar (Concerto). We found that partial resistance in Hsp5 to R. padi extends to two other aphid pests of grasses. Using the electrical penetration graph technique, we show that partial resistance is mediated by phloem- and mesophyll-based resistance factors that limit aphid phloem ingestion. To gain insight into plant traits responsible for partial resistance, we compared non-glandular trichome density, defence gene expression, and phloem composition of Hsp5 with those of the susceptible barley cultivar Concerto. We show that Hsp5 partial resistance involves elevated basal expression of thionin and phytohormone signalling genes, and a reduction in phloem quality. This study highlights plant traits that may contribute to broad-spectrum partial resistance to aphids in barley.

Using Aphids to Measure Turgor Pressure Inside Sieve Elements.

The phloem is the long-distance transport system for photoassimilates within the plant. The vulnerability of the phloem tissue to blockage and damage makes it technically difficult to study, which has made it one of the least understood tissues. Transport of solution through the phloem appears to be by osmotically driven bulk flow, making an understanding of phloem hydrostatic pressure important in our comprehension of phloem flow mechanics. Here we describe a method of making in vivo direct transient measurements of phloem hydrostatic pressure using excised aphid stylets to directly access the phloem.

Reduced phloem uptake of Myzus persicae on an aphid resistant pepper accession.

BACKGROUND: The green peach aphid (GPA), Myzus persicae, is economically one of the most threatening pests in pepper cultivation, which not only causes direct damage but also transmits many viruses. Breeding aphid resistant pepper varieties is a promising and environmentally friendly method to control aphid populations in the field and in the greenhouse. Until now, no strong sources of resistance against the GPA have been identified. Therefore the main aims of this study were to identify pepper materials with a good level of resistance to GPA and to elucidate possible resistance mechanisms. RESULTS: We screened 74 pepper accessions from different geographical areas for resistance to M. persicae. After four rounds of evaluation we identified one Capsicum baccatum accession (PB2013071) as highly resistant to M. persicae, while the accessions PB2013062 and PB2012022 showed intermediate resistance. The resistance of PB2013071 resulted in a severely reduced uptake of phloem compared to the susceptible accession, as determined by Electrical Penetration Graph (EPG) studies. Feeding of M. persicae induced the expression of callose synthase genes and resulted in callose deposition in the sieve elements in resistant, but not in susceptible plants. CONCLUSIONS: Three aphid resistant pepper accessions were identified, which will be important for breeding aphid resistant pepper varieties in the future. The most resistant accession PB2013071 showed phloem-based resistance against aphid infestation.

Parallel adaptations and common host cell responses enabling feeding of obligate and facultative plant parasitic nematodes.

Parallel adaptations enabling the use of plant cells as the primary food source have occurred multiple times in distinct nematode clades. The hallmark of all extant obligate and facultative plant-feeding nematodes is the presence of an oral stylet, which is required for penetration of plant cell walls, delivery of pharyngeal gland secretions into host cells and selective uptake of plant assimilates. Plant parasites from different clades, and even within a single clade, display a large diversity in feeding behaviours ranging from short feeding cycles on single cells to prolonged feeding on highly sophisticated host cell complexes. Despite these differences, feeding of nematodes frequently (but certainly not always) induces common responses in host cells (e.g. endopolyploidization and cellular hypertrophy). It is thought that these host cell responses are brought about by the interplay of effectors and other biological active compounds in stylet secretions of feeding nematodes, but this has only been studied for the most advanced sedentary plant parasites. In fact, these responses are thought to be fundamental for prolonged feeding of sedentary plant parasites on host cells. However, as we discuss in this review, some of these common plant responses to independent lineages of plant parasitic nematodes might also be generic reactions to cell stress and as such their onset may not require specific inputs from plant parasitic nematodes. Sedentary plant parasitic nematodes may utilize effectors and their ability to synthesize other biologically active compounds to tailor these common responses for prolonged feeding on host cells.

Arabidopsis ACTIN-DEPOLYMERIZING FACTOR3 Is Required for Controlling Aphid Feeding from the Phloem.

The actin cytoskeleton network has an important role in plant cell growth, division, and stress response. Actin-depolymerizing factors (ADFs) are a group of actin-binding proteins that contribute to reorganization of the actin network. Here, we show that the Arabidopsis (Arabidopsis thaliana) ADF3 is required in the phloem for controlling infestation by Myzus persicae Sülzer, commonly known as the green peach aphid (GPA), which is an important phloem sap-consuming pest of more than fifty plant families. In agreement with a role for the actin-depolymerizing function of ADF3 in defense against the GPA, we show that resistance in adf3 was restored by overexpression of the related ADF4 and the actin cytoskeleton destabilizers, cytochalasin D and latrunculin B. Electrical monitoring of the GPA feeding behavior indicates that the GPA stylets found sieve elements faster when feeding on the adf3 mutant compared to the wild-type plant. In addition, once they found the sieve elements, the GPA fed for a more prolonged period from sieve elements of adf3 compared to the wild-type plant. The longer feeding period correlated with an increase in fecundity and population size of the GPA and a parallel reduction in callose deposition in the adf3 mutant. The adf3-conferred susceptibility to GPA was overcome by expression of the ADF3 coding sequence from the phloem-specific SUC2 promoter, thus confirming the importance of ADF3 function in the phloem. We further demonstrate that the ADF3-dependent defense mechanism is linked to the transcriptional up-regulation of PHYTOALEXIN-DEFICIENT4, which is an important regulator of defenses against the GPA.

Cucurbit extrafascicular phloem has strong negative impacts on aphids and is not a preferred feeding site.

Cucurbits have long been known to possess two types of phloem: fascicular (FP) within vascular bundles and extrafascicular phloem (EFP) surrounding vascular bundles and scattered through the cortex. Recently, their divergent composition was revealed, with FP having high sugar content consistent with conventional phloem, but EFP having much lower sugar levels and a very different proteome. However, the evolutionary advantages of possessing both FP and EFP have remained unclear. Here, we present four lines of quantitative evidence that together support the hypothesis that FP represents a typical phloem and is an attractive diet for aphids, whereas aphids avoid feeding on EFP. First, aphid stylet track endings were more abundant near the abaxial FP element of minor veins, suggesting a feeding preference for FP over EFP. Second, sugar profiles from stylet exudates were wholly consistent with FP origins, further supporting preference for FP and avoidance of EFP. Third, supplementation of EFP exudate into artificial diets confirmed an aversion to EFP in choice experiments. Finally, EFP exudate had negative effects on aphid performance. On the basis of aphids' inability to thrive on EFP, we conclude that EFP is atypical and perhaps should not be classed as a phloem system.