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.     

Electrical penetration graphic waveforms in relation to the actual positions of the stylet tips of Nilaparvata lugens in rice tissue

The stylet penetration behavior of Nilaparvata lugens (Hemiptera: Delphacidae) in rice plants (Oryza sativa) was evaluated through the use of an electrical penetration graph (EPG). To accomplish this, we classified the EPG signals into seven different waveforms, np, N1, N2, N3, N4-a, N4-b, and N5, according to their shapes, amplitudes, and frequencies. The N4-b waveform was always preceded by N3 and N4-a, in that order. Continuous honeydew excretion only occurred during the N4-b period, and the honeydew deposited on a filter paper containing ninhydrin reagent during the N4-b period was stained violet. The tips of the stylets that were severed in the N3, N4-a, and N4-b periods were in the phloem region of rice. Moreover, the flow of plant sap after stylectomy only occurred during the N4-b period. Finally, sucrose was the only carbohydrate component identified when HPLC analysis of the plant sap was conducted. On the other hand, honeydew excretion hardly occurred during the N5 period and the tips of the stylets that were severed during the N5 period were located in the xylem region of rice. Based on the location of the stylets and honeydew excretion, the EPG waveforms for the stylet penetration behaviors of N. lugens were assigned to the following groups; np: non-penetration of stylets, N1: penetration initiation, N2: salivation and stylet movement, N3: an extracellular activity near the phloem region, N4-a: an intracellular activity in phloem region, N4-b: phloem sap ingestion, and N5: activity in the xylem region.     

Plant penetration by pea aphids (Acyrthosiphon pisum) of different plant range

Plant penetration by the stylets of six clones of the pea aphid, Acyrthosiphon pisum, on Vicia faba (acceptable to all clones) and Pisum sativum (acceptable to 3/6 clones) was investigated by the DC electrical penetration graph technique. In a 10 h recording period, 93% of 144 aphids exhibited sustained feeding on phloem sap. Significant interclonal differences were observed for the incidence of potential drops (indicative of brief punctures of plant cells) and the duration of waveform E1 (insect salivation into a sieve element). In addition, the total duration of the sieve element phase and the duration of completed bouts of sustained feeding differed between the two test plants, in a fashion varying between clones. However, these differences could not be related to the acceptability of plants to the different aphid clones. The duration of the stylet pathway phase preceding the first sustained feeding on phloem sap did not vary significantly with either aphid clone or plant. It is concluded that the resistance of P. sativum to certain A. pisum clones does not arise from factors impeding either stylet penetration through the plant tissues or the maintenance of feeding from the sieve elements. It is proposed that host plant affiliation of A. pisum may be mediated primarily by specific olfactory or gustatory cues, before the aphid initiates stylet penetration of the plant.     

Comparative metabolomics of the interaction between rice and the brown planthopper

Introduction

The interactions between plants and insect herbivores are complex and multifaceted. Rice and its specialist insect pest the brown planthopper (BPH), Nilaparvata lugens Stål (Hemiptera: Delphacidae) constitute an ideal system for studying plant–insect interactions.

Objectives

Combined metabolomics analyses of rice plant and BPH were conducted to understand the mechanism of host rice plant defense and BPH insect response.

Methods

Metabolite dynamics in rice leaf sheath and BPH honeydew was investigated using the gas chromatography–mass spectrometry (GC–MS) method. The GC–MS data were analyzed by principal component analysis and partial least squares-discriminant analysis.

Results

Twenty-six metabolites were detected in the leaf sheath extracts. Rice leaf sheath metabolomics analysis results show that BPH feeding induces distinct changes in the metabolite profiles of YHY15 and TN1 plants. These results suggest that BPH infestation enhance fatty acid oxidation, the glyoxylate cycle, gluconeogenesis and the GABA shunt in TN1 plants, and glycolysis and the shikimate pathway in YHY15. We propose that the BPH15 gene mediates a resistance reaction that increases the synthesis of secondary metabolites through the shikimate pathway. Thirty-three metabolites were identified in BPH honeydew. Honeydew metabolomics analysis results show that when BPH insects were fed on resistant YHY15 plants, most of the amino acids in honeydew were significantly decreased compared to those of BPH fed on TN1 plants. Based on metabolomics results, we propose that BPH feeding on resistant YHY15 plants would enhance amino acid absorption. At the same time, urea was significantly increased in BPH fed on YHY15.

Conclusion

Metabolomics study is valuable in understanding the complex and multifaceted interaction between plants and insect herbivores and provide essential clue for development of novel control BPH strategies.

     

Electrical penetration graphs indicate that tricin is a key secondary metabolite of rice,inhibiting phloem feeding of brown planthopper,Nilaparvata lugens

Tricin (5,7,4′‐trihydroxy‐3′,5′‐dimethoxyflavone) is a valuable secondary metabolite which is widely present in gramineous plants, including cultivated rice (Oryza sativa L.) (Poaceae). It can defend the rice plant against damage by the brown planthopper (BPH), Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), one of the most important pests of rice. This study was conducted to elucidate the mechanisms of action of tricin on BPH feeding behavior. BPH feeding behavior in resistant (Rathu Heenati, RHT) and susceptible (Taichuang native 1, TN1) rice varieties and artificial diets was monitored using the electrical penetration graph (EPG) technique. Tricin concentrations in leaves of varieties RHT and TN1 were quantitatively analyzed by liquid chromatography, coupled to tandem mass spectrometric techniques. Six (NP and N1‐5) and four (NP, N1, N2, and N4) types of waveforms occurred during feeding on rice plants and artificial diets, respectively. The tricin concentration of rice varieties was correlated with total and average durations of N4. Moreover, EPG data indicated that tricin significantly increased the duration of non‐probing and pathway periods and strongly inhibited phloem ingestion (N4). The inhibition was strongly dose dependent, resulting in complete suppression of activity in the phloem region when the tricin concentration was increased to 1 g l^?1. This study revealed that tricin disturbed the feeding behavior of BPH mainly by increasing the non‐probe period and inhibiting phloem ingestion. We confirmed the hypothesis that tricin is a ‘stylet probing stimulant’ of rice planthoppers as proposed in previous studies. The information on the ecological effect of tricin from this study may be useful to clarify the resistance mechanism against BPH of RHT and other tricin‐containing rice varieties.     

Salivation into sieve elements in relation to plant chemistry: the case of the aphid Sitobion fragariae and the wheat, Triticum aestivum

Extended sieve element salivation (E1 waveform in the electrical penetration graph) is a characteristic activity during early sieve element punctures, particularly in resistant plants. In order to explore a chemically-mediated mechanism of resistance associated with sieve element salivation, we compared the pattern of feeding behaviour of the aphid, Sitobion fragariae (Walker), on two cultivars of the wheat Triticum aestivum L., with different concentrations of hydroxamic acids (Hx). During 24 h of electronic monitoring, aphids dedicated over 50% of the total time to phloem ingestion from the sieve elements. Total time allocated to E1 in the experiment, time to first E1 within the experiment, time allocated to E1 before a sustained phloem ingestion (E2) and the contribution of sieve element salivation to the phloem phase (E1/[E1+E2]) were significantly higher in the high-Hx cultivar. The increased salivation in plants with higher contents of Hx suggests the existence, at least in this system, of a chemically-mediated sieve element constraint.     

Stylet penetration by larvae of the greenhouse whitefly on cucumber

Probing behaviour of Trialeurodes vaporariorum (Westwood) larvae was monitored using the DC electrical penetration graph (EPG) technique on the host plant cucumber. EPGs were recorded for 16 h, simultaneously with honeydew excretion using a honeydew clock. Three waveforms were distinguished: a pathway waveform (C), and two phloem waveforms, one with a high (H), and one with a low frequency (L) signal. The C waveform mainly occurred in the crawler stage of the 1st instar larvae. EPGs recorded from larvae during and after moulting indicated that the process involves stylet withdrawal; hence the stylets of each new instar need to penetrate again from the leaf surface to the phloem.All sessile stages, from L1 to pre-pupa, spent almost their entire time in waveforms H and L. These waveforms alternated more frequently in the early instars than during the later ones, in which the H waveform became predominant. The H waveform was highly correlated with honeydew excretion and thus phloem sap ingestion. The L waveform was not related to honeydew excretion but EPGs indicated that the stylet tips remain in a sieve element during both waveforms. Periods of honeydew production demonstrated a delay of 30–40 min in relation to the onset and end of H and L waveforms. This delay is presumably related to the time needed for food passing through, or emptying of, the insect's gut. From the 1st instar to the pre-pupa, the frequency of excreted honeydew droplets decreased but their size increased, causing a net increase of the excretion rate.     

Effects of experimental stress factors on probing behaviour by aphids

Probing behaviour of Aphis fabae Scopoli and Rhopalosiphum padi (L.) was tested in different stress situations normally occurring in aphid-plant studies such as interruption of feeding or starvation, transfer to a new plant, and attachment to the electrode wire. The DC electrical penetration graph (EPG) technique and a honeydew clock were used to collect data on behavioural effects of these stress conditions. As a general effect, an interruption of feeding behaviour acted as a reset, i.e., the same sequence and time course of probing events were shown, irrespective the interruptions duration, from 1–100 min. Nevertheless, some minor differences were found, especially in A. fabae. Increased interruption times (deprivation from the host plant) stimulated the aphids to insert their stylets earlier. When A. fabae was put back on its host plant after a one min interruption phloem feeding started earlier than with longer interruption times, but only when it was put back to the same plant and feeding site on which it fed before. It is concluded that this effect is at least partly due to memory of previous probing/feeding experience on the plant as it vanishes with longer interruption times. This explanation also holds for phloem salivation (E1) before starting sustained sap ingestion, which was reduced on the previous feeding site, but only after the one min interruption in A. fabae. The aphid-plant specificity appeared high in these effects. Both aphids were somewhat affected by wiring, resulting in earlier probing, longer total pathway phase, and less and later phloem feeding (as reflected by honeydew excretion). Thus confirming, that the evaluation of EPG results can be improved with supplementary data from free aphids.     

不同水稻品种上白背飞虱取食行为的EPG分析

【目的】为探明不同水稻品种（丰源优272、R9810-T、华恢1号、明恢63、麻糯谷和Rathu Heenati）对白背飞虱Sogatella furcifera的抗性差异机理,利用刺吸电位技术(electronic penetration graph, EPG)记录了该虫在这6个品种三叶期稻苗上的取食行为。【方法】结合特定的EPG波形,考察了9个非韧皮部指标和22个韧皮部指标。【结果】在总记录时间8 h内,白背飞虱在品种Rathu Heenati上非刺探波np的总时间最长,其次是麻糯谷上非刺探波np的总时间,这两者间差异显著且都显著地长于其他4个品种的np总时间(P<0.05);该虫在Rathu Heenati上路径波Nc的总时间为8 523.41 s,是感虫品种明恢63 Nc总时间的2.24倍;该虫在Rathu Heenati上吸食韧皮部汁液N4-b的总时间显著地比其他品种短(P<0.05)。华恢1号和R9810-T上所有的EPG指标和感虫品种明恢63的EPG指标都没有显著的差异(P≥0.05)。白背飞虱在丰源优272上单次分泌水溶性唾液N4-a的平均时间更长,并伴随长时间的韧皮部取食。【结论】由此推测,品种Rathu Heenati可能存在不利于白背飞虱取食的忌避成分,并且在韧皮部外组织和韧皮部组织都存在抗性因子;在麻糯谷上仅存在忌避成分;然而,品种华恢1号和R9810-T可能对白背飞虱不具有明显的抗性;丰源优272可能是比明恢63更为感虫的品种。结合介体昆虫取食行为与传播持久性病毒的关系,这些结果也为利用抗白背飞虱品种控制南方水稻黑条矮缩病提供了参考。     

Penetration of faba bean sieve elements by pea aphid does not trigger forisome dispersal

Immediately after their stylets penetrate a phloem sieve element, aphids inject saliva into the sieve element for approximately 30–60 s before they begin to ingest phloem sap. This salivation period is recorded as waveform E1 in electrical penetration graph (EPG) monitoring of aphid feeding behavior. It has been hypothesized that the function of this initial period of phloem salivation is to reverse or prevent plugging of the sieve element by one of the plant's phloem defenses: formation of P‐protein plugs or callose synthesis in the sieve pores that connect adjacent sieve elements. This hypothesis was tested using the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae), and faba bean, Vicia faba L. (Fabaceae), as a model system, and the results do not support the hypothesis. In legumes, such as faba bean, P‐protein plugs in sieve elements are formed by dispersal of proteinaceous bodies called forisomes. Contrary to the hypothesis, the great majority of sieve element penetrations by pea aphid stylets do not trigger forisome dispersal. Thirteen sieve elements were cryofixed early in phloem phase before the aphids could complete their salivation period and the forisomes were not dispersed in any of the 13 samples. However, in these samples, the aphids completed on average a little over half of their normal E1 salivation period before they were cryofixed. Thus, it is possible that sieve element penetration triggered forisome dispersal in these samples but the abbreviated period of salivation was still sufficient to reverse dispersal. To rule out this possibility, 17 sieve elements were cryofixed during R‐pds, which are an EPG waveform associated with sieve element penetration but without the characteristic E1 salivation that occurs during phloem phase. In 16 of the 17 samples, the forisomes were not dispersed. Thus, faba bean sieve elements usually do not form P‐protein plugs in response to penetration by pea aphid stylets. Consequently, the characteristic E1 salivation that occurs at the start of each phloem phase does not seem to be necessary to prevent a plugging response because penetration of sieve elements during R‐pds does not trigger forisome dispersal despite the absence of E1 salivation. Furthermore, as P‐protein plugs do not normally form in response to sieve element penetration, E1 salivation that occurs at the start of each phloem phase is not a response to development of a P‐protein plug. Thus, the E1 salivation period at the beginning of the phloem phase appears to have function(s) unrelated to phloem sealing.     

Lipid profiles reveal different responses to brown planthopper infestation for pest susceptible and resistant rice plants

Introduction

Brown planthopper (BPH) is the most destructive insect pest for rice, causing major reductions in rice yield and large economic losses. More than 31 BPH-resistance genes have been located, and several of them have been isolated. Nevertheless, the metabolic mechanism related to BPH-resistance genes remain uncharacterized.

Objectives

To elucidate the resistance mechanism of the BPH-resistance gene Bph6 at the metabolic level, a Bph6-transgenic line R6 (BPH-resistant) and the wild-type Nipponbare (BPH-susceptible) were used to investigate their lipid profiles under control and BPH treatments.

Methods

In conjunction with multivariate statistical analysis and quantitative real-time PCR, BPH-induced lipid changes in leaf blade and leaf sheath were investigated by GC–MS-based lipidomics.

Results

Forty-five lipids were identified in leaf sheath extracts. Leaf sheath lipidomics analysis results show that BPH infestation induces significant differences in the lipid profiles of Nipponbare and R6. The levels of hexadecanoic acid, methyl ester, linoleic acid, methyl ester, linolenic acid, methyl ester, glycidyl palmitate, eicosanoic acid, methyl ester, docosanoic acid, methyl ester, beta-monolinolein, campesterol, beta-sitosterol, cycloartenol, phytol and phytyl acetate had undergone enormous changes after BPH feeding. These results illustrate that BPH feeding enhances sterol biosynthetic pathway in Nipponbare plants, and strengthens wax biosynthesis and phytol metabolism in R6 plants. The results of quantitative real-time PCR of 5 relevant genes were consistent with the changes in metabolic level. Forty-five lipids were identified in the leaf blade extracts. BPH infestation induces distinct changes in the lipid profiles of the leaf blade samples of Nipponbare and R6. Although the lipid changes in Nipponbare are more drastic, the changes within the two varieties are similar. Lipid profiles in leaf sheath brought out significant differences than in leaf blade within Nipponbare and R6. We propose that Bph6 mainly affects the levels of lipids in leaf sheath, and mediates resistance by deploying metabolic re-programming during BPH feeding.

Conclusion

The results indicate that wax biosynthesis, sterol biosynthetic pathway and phytol metabolism play vital roles in rice response to BPH infestation. This finding demonstrated that the combination of lipidomics and quantitative real-time PCR is an effective approach to elucidating the interactions between brown planthopper and rice mediated by resistance genes.

     

Effect of methoxyphenols on grain aphid feeding behaviour

Methoxyphenols might be important in the resistance of cereals to aphids. Electrical penetration graph (EPG) recordings were used to determine the effect of caffeic, ferulic and sinapic acids, and scopoletin on the feeding behaviour of the grain aphidSitobion avenae (F.). Aphids on wheat seedlings treated systemically with these phenols showed reduced ingestion of phloem sap and salivation into sieve elements in most cases. The earlier pathway phases of probing were prolonged. Moreover increase in number of probes as well as reduction of total time of probing was observed. In addition, no O-demethylase activity was found in homogenates of aphids fed on moderately-resistant (phenolic rich) or susceptible (phenolic poor) wheat varieties. The significance of these results for understanding the resistance of cereals to aphids is discussed.     

Distinct Expression Profiles and Different Functions of Odorant Binding Proteins in Nilaparvata lugens St?l

Background

Odorant binding proteins (OBPs) play important roles in insect olfaction. The brown planthopper (BPH), Nilaparvata lugens Stål (Delphacidae, Auchenorrhyncha, Hemiptera) is one of the most important rice pests. Its monophagy (only feeding on rice), wing form (long and short wing) variation, and annual long distance migration (seeking for rice plants of high nutrition) imply that the olfaction would play a central role in BPH behavior. However, the olfaction related proteins have not been characterized in this insect.

Methodology/Principal Findings

Full length cDNA of three OBPs were obtained and distinct expression profiles were revealed regarding to tissue, developmental stage, wing form and gender for the first time for the species. The results provide important clues in functional differentiation of these genes. Binding assays with 41 compounds demonstrated that NlugOBP3 had markedly higher binding ability and wider binding spectrum than the other two OBPs. Terpenes and Ketones displayed higher binding while Alkanes showed no binding to the three OBPs. Focused on NlugOBP3, RNA interference experiments showed that NlugOBP3 not only involved in nymph olfaction on rice seedlings, but also had non-olfactory functions, as it was closely related to nymph survival.

Conclusions

NlugOBP3 plays important roles in both olfaction and survival of BPH. It may serve as a potential target for developing behavioral disruptant and/or lethal agent in N. lugens.     

Salivary secretions by aphids interacting with proteins of phloem wound responses

Successful phloem feeding requires overcoming a number of phloem-related plant properties and reactions. The most important hurdle is formed by the phloem wound responses, such as coagulating proteins in the phloem sieve elements of the plant and in the capillary food canal in the insect's mouth parts, i.e. the stylets. It seems that in order to prevent protein clogging inside a sieve element, ejection of watery saliva plays an important role. This ejection is detected in the electrical penetration graph (EPG) as E1 salivation and always precedes phloem sap ingestion. During this feeding from sieve elements, another regular and concurrent salivation also occurs, the watery E2 salivation. This E2 saliva is added to the ingested sap and, it probably prevents phloem proteins from clogging inside the capillary food canal. Whatever the biochemical mode of action of the inhibition of protein coagulation might be, in some plants aphids do not seem to be able to prevent clogging, which may explain the resistance to aphids in these plants. The relevance of this hypothesis is demonstrated by new experimental results and is related to new EPG results from plants with phloem-located resistance.     

Effects of previous plant infestation on sieve element acceptance by two aphids

Probing behaviour as affected by a previous infestation was studied in two aphid species on their respective host plants. Probing behaviour by Aphis fabae (reported to have beneficial effects when living in colonies) and Rhopalosiphum padi (without known beneficial effects) was studied using the electrical penetration graph (EPG) technique. In A. fabae the main effects were longer and more continuous sap ingestion, and less salivation into sieve elements before sap ingestion. These suggest phloem factors. Nevertheless, mesophyll and non-vascular tissues are likely to be involved to a lesser extent, as reflected by fewer non-probing periods before the first phloem phase on previously colonised leaves as compared to clean leaves. Total honeydew production increased on a previously colonised leaf due to the prolonged sap ingestion periods but the excretion rate was not affected, indicating that the ingestion rate remained unaltered. R. padi did not show responses to previous colonisation. It is hypothesized that the changes in probing behaviour are due to changed plant properties, chemical contents of sieve element sap and/or physiological changes induced by the saliva from the colony.     

Development of 25 near-isogenic lines (NILs) with ten BPH resistance genes in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.): production,resistance spectrum,and molecular analysis

Key message

A first set of 25 NILs carrying ten BPH resistance genes and their pyramids was developed in the background of indica variety IR24 for insect resistance breeding in rice.

Abstract

Brown planthopper (Nilaparvata lugens Stal.) is one of the most destructive insect pests in rice. Development of near-isogenic lines (NILs) is an important strategy for genetic analysis of brown planthopper (BPH) resistance (R) genes and their deployment against diverse BPH populations. A set of 25 NILs with 9 single R genes and 16 multiple R gene combinations consisting of 11 two-gene pyramids and 5 three-gene pyramids in the genetic background of the susceptible indica rice cultivar IR24 was developed through marker-assisted selection. The linked DNA markers for each of the R genes were used for foreground selection and confirming the introgressed regions of the BPH R genes. Modified seed box screening and feeding rate of BPH were used to evaluate the spectrum of resistance. BPH reaction of each of the NILs carrying different single genes was variable at the antibiosis level with the four BPH populations of the Philippines. The NILs with two- to three-pyramided genes showed a stronger level of antibiosis (49.3–99.0%) against BPH populations compared with NILs with a single R gene NILs (42.0–83.5%) and IR24 (10.0%). Background genotyping by high-density SNPs markers revealed that most of the chromosome regions of the NILs (BC₃F₅) had IR24 genome recovery of 82.0–94.2%. Six major agronomic data of the NILs showed a phenotypically comparable agronomic performance with IR24. These newly developed NILs will be useful as new genetic resources for BPH resistance breeding and are valuable sources of genes in monitoring against the emerging BPH biotypes in different rice-growing countries.

     

Acceptability of different species of Brassicaceae as hosts for the cabbage aphid

The probing and feeding behaviour of the cabbage aphid, Brevicoryne brassicae (L.), (Homoptera, Aphididae) was studied on several plant species that represented various levels of acceptability: Sinapis alba L. (a permanent host plant), Capsella bursa-pastoris (L.) Med., Thlaspi arvense L., Lunaria annua L., Erysimum cheiranthoides L. (accidental host plants), Vicia faba L. (a non-host plant), using the electrical penetration graph technique (EPG). B. brassicae on V. faba did not show any patterns related to penetration of phloem vessels. Stylet penetration was deterred on L. annua and E. cheiranthoides where non-penetration prevailed, the periods of sap ingestion were short or did not occur, the percentage of time spent in the phloem was consistently low (5–6%) and E1 salivation predominated. The pathway activities were not suppressed on C. bursa-pastoris and T. arvense and the aphids spent an average of 3 h in the phloem during the 8-h experiment. However, a considerable delay between finding and accepting the phloem and a substantial proportion of E1 salivation (20–30% of all phloem activities) indicated a deterrent factor in the sieve elements of these plants. Aphid probing and sap ingestion were rarely interrupted on S. alba. The results of this study suggest that the deterrent agents vary in activity and may hinder stylet penetration at different levels (epidermis, parenchymatous tissues and/or phloem elements), depending on the plant species.