Composition Of Phloem Sap Research Articles

Phloem Sap Composition: What Have We Learnt from Metabolomics?

Phloem sap transport is essential for plant nutrition and development since it mediates redistribution of nutrients, metabolites and signaling molecules. However, its biochemical composition is not so well-known because phloem sap sampling is difficult and does not always allow extensive chemical analysis. In the past years, efforts have been devoted to metabolomics analyses of phloem sap using either liquid chromatography or gas chromatography coupled with mass spectrometry. Phloem sap metabolomics is of importance to understand how metabolites can be exchanged between plant organs and how metabolite allocation may impact plant growth and development. Here, we provide an overview of our current knowledge of phloem sap metabolome and physiological information obtained therefrom. Although metabolomics analyses of phloem sap are still not numerous, they show that metabolites present in sap are not just sugars and amino acids but that many more metabolic pathways are represented. They further suggest that metabolite exchange between source and sink organs is a general phenomenon, offering opportunities for metabolic cycles at the whole-plant scale. Such cycles reflect metabolic interdependence of plant organs and shoot-root coordination of plant growth and development.

'Candidatus Liberibacter' Pathosystems at the Forefront of Agricultural and Biological Research Challenges.

'Candidatus Liberibacter' Pathosystems at the Forefront of Agricultural and Biological Research Challenges.

Generous Hosts: 'Candidatus Liberibacter asiaticus' Growth in Madagascar Periwinkle (Catharanthus roseus) Highlights Its Nutritional Needs.

'Candidatus Liberibacter asiaticus', the putative causal agent of citrus greening, is not available in pure culture yet. In addition to trees of citrus and citrus relatives, 'Ca. L. asiaticus' can grow in Madagascar periwinkle (Catharanthus roseus). Using gas chromatography-mass spectrometry, we compared the phloem sap composition in sweet orange 'Valencia' (Citrus sinensis) and periwinkle plants after the infection with 'Ca. L. asiaticus'. Interestingly, in contrast to our previous studies of total leaf metabolites, we found that, compared with uninfected phloem sap, the organic acids implicated in the tricarboxylic acid cycle (TCA) cycle including citrate, isocitrate, succinate, fumarate, and malate were reduced significantly in the infected phloem saps of both species. As a result of the reduction of organic acids content, the pH of infected phloem saps was increased. We hypothesize that the bacterial growth induces the mitochondrial TCA cycle in parenchyma cells to produce more of these compounds to be used as a bacterial carbon source. Once these compounds reach a low level in the phloem sap, the bacterium may send a signal, yet to be identified, to initiate a feedback loop to further induce the TCA cycle. Phloem blockage might be another reason behind the reduced translocation of TCA cycle intermediates within the phloem. The net result, localized availability of organic acids, likely benefits bacterial growth and may explain the unequal distribution of 'Ca. L. asiaticus' within infected trees. These findings may help in designing media for the pure culturing of 'Ca. L. asiaticus'.

Effects of Arbuscular Mycorrhiza on Primary Metabolites in Phloem Exudates of Plantago major and Poa annua and on a Generalist Aphid.

Arbuscular mycorrhiza (AM), i.e., the interaction of plants with arbuscular mycorrhizal fungi (AMF), often influences plant growth, physiology, and metabolism. Effects of AM on the metabolic composition of plant phloem sap may affect aphids. We investigated the impacts of AM on primary metabolites in phloem exudates of the plant species Plantago major and Poa annua and on the aphid Myzus persicae. Plants were grown without or with a generalist AMF species, leaf phloem exudates were collected, and primary metabolites were measured. Additionally, the performance of M. persicae on control and mycorrhizal plants of both species was assessed. While the plant species differed largely in the relative proportions of primary metabolites in their phloem exudates, metabolic effects of AM were less pronounced. Slightly higher proportions of sucrose and shifts in proportions of some amino acids in mycorrhizal plants indicated changes in phloem upload and resource allocation patterns within the plants. Aphids showed a higher performance on P. annua than on P. major. AM negatively affected the survival of aphids on P. major, whereas positive effects of AM were found on P. annua in a subsequent generation. Next to other factors, the metabolic composition of the phloem exudates may partly explain these findings.

Why is phloem sap nitrate kept low?

Since the first description of phloem sap composition nearly 60 years ago, it is generally assumed that phloem sap does not contain nitrate and that there is little or no backflow of nitrate from shoots to roots. While it is true that nitrate can occasionally be absent from phloem sap, there is now substantial evidence that phloem can carry nitrate and furthermore, transporters involved in nitrate redistribution to shoot sink organs and roots have been found. This raises the question of why nitrate may or may not be present in phloem sap, why its concentration is generally kept low, and whether plant shoot-root nutrient cycling also involves nitrate. We propose here that phloem sap nitrate is not only an essential component of plant nutritional signaling but also contributes to physical properties of phloem sap and as such, its concentration must be controlled to ensure proper coordination of plant development and nutrient transport. This article is protected by copyright. All rights reserved.

Location, location, location: Feeding site affects aphid performance by altering access and quality of nutrients.

Aphid feeding behavior and performance on a given host plant are influenced by the plants’ physical and chemical traits, including structural characters such as trichomes and nutritional composition. In this study, we determined the feeding behavior and performance of soybean aphids (Aphis glycines) on the stem, the adaxial (upper), and the abaxial (lower) leaf surfaces during early vegetative growth of soybean plants. Using the electrical penetration graph technique, we found that aphids feeding on the stem took the longest time to begin probing. Once aphids began probing, the sieve elements were more conducive to feeding, as evidenced by less salivation on the stem than either leaf surface. In whole-plant assays, stems harbored higher aphid populations, and aphids had shorter development time on stems than the adaxial and the abaxial leaf surfaces. We compared trichome density and length on the stem, the adaxial, and the abaxial leaf surfaces to investigate whether plant trichomes affected aphid feeding and performance. There were higher density and longer trichomes on stems, which likely resulted in aphids taking a longer time to probe. Still a negative impact on aphid population growth was not observed. Analysis of phloem sap composition revealed that vascular sap-enriched exudates from stems had higher sugars and amino acids than exudates from leaves. In artificial diet feeding assays, the population of aphids reared on a diet supplemented with stem exudates was higher than on a diet supplemented with leaf petiole exudates which is in agreement with results of the whole-plant assays. In summary, our findings suggest that the performance of soybean aphids on a specific plant location is primarily driven by accessibility and the quality of phloem composition rather than structural traits.

Diurnal feeding as a potential mechanism of osmoregulation in aphids.

Diurnal variation in phloem sap composition has a strong influence on aphid performance. The sugar-rich phloem sap serves as the sole diet for aphids and a suite of physiological mechanisms and behaviors allow them to tolerate the high osmotic stress. Here, we tested the hypothesis that night-time feeding by aphids is a behavior that takes advantage of the low sugar diet in the night to compensate for osmotic stress incurred while feeding on high sugar diet during the day. Using the electrical penetration graph (EPG) technique, we examined the effects of diurnal rhythm on feeding behaviors of bird cherry-oat aphid (Rhopalosiphum padi L.) on wheat. A strong diurnal rhythm in aphids as indicated by the presence of a cyclical pattern of expression in a core clock gene did not impact aphid feeding and similar feeding behaviors were observed during day and night. The major difference observed between day and night feeding was that aphids spent significantly longer time in phloem salivation during the night compared to the day. In contrast, aphid hydration was reduced at the end of the day-time feeding compared to end of the night-time feeding. Gene expression analysis of R. padi osmoregulatory genes indicated that sugar breakdown and water transport into the aphid gut was reduced at night. These data suggest that while diurnal variation occurs in phloem sap composition, aphids use night-time feeding to overcome the high osmotic stress incurred while feeding on sugar-rich phloem sap during the day.

Impact of scion/rootstock reciprocal effects on metabolomics of fruit juice and phloem sap in grafted Citrus reticulata.

BackgroundRootstock has a significant impact on plant growth and development, including fruit maturation. However, the existence of mutual interaction between scion and rootstock is often neglected. To explore the origin of different fruit quality traits in citrus, we studied the effect of rootstock and the reciprocal interaction between scion and rootstock of nine combinations; three mandarin varieties grafted on three different rootstocks. We analyzed the metabolic profile of juice via gas and liquid chromatography-mass spectrometry (GC-MS and LC-MS, respectively). Additionally, we profiled phloem sap composition in the scion and the rootstock. Quality traits of fruit and their physio-chemical characteristics were also evaluated.ResultsFor all three cultivars, rootstock was found to affect fruit yield and biochemical fruit quality parameters (sugar and acidity) in interactions with the scions. In mandarin juice, eight of 48 compounds (two primary and six secondary) were related directly to the rootstock, and another seven (one primary and six secondary) were interactively affected by scion and rootstock. In scion and rootstock sap, six and 14 of 53 and 55 primary metabolites, respectively, were directly affected by the rootstock, while 42 and 33 were affected by rootstock interactively with scion, respectively.ConclusionIn this work, we show for the first time a reciprocal effect between rootstock and scion. Based on our results, the scion and rootstock interaction might be organ, distance or time dependent.

Collection of the phloem sap, pros and cons

ABSTRACTPhloem tissue is essential for the translocation of nutrients, water, energy, and signals in plants. In order to study the chemical composition of phloem sap, several methods have been used for its collection including the dipotassium ethylenediamine tetraacetic acid (K2-EDTA) exudation, incision, and the stylectomy by aphids. Each method has advantages and disadvantages. Unfortunately, there is no ideal method that can be used for all plants or to collect ultrapure phloem sap with no cellular contamination. However, K2-EDTA exudation is the most used method because it is easy, fast, and results in a high quantity of phloem sap. In woody plants, it is easy to separate the bark. Using the bark which contains the phloem tissue would avoid the contamination with xylem sap when phloem sap is collected. Lately, we developed a simple and a quick method for the collection of the citrus phloem sap depending on the centrifugation of the detached bark tissue. Here, I report the advantages of the centrifugation method over the K2-EDTA exudation in collecting phloem sap from citrus. To emphasize the purity of collected saps, phloem sap (from bark tissue) and xylem sap (from inner part of stem) were collected using both methods. Using gas chromatography mass spectrometry, the centrifugation method showed less artifact peaks than the K2-EDTA exudation indicating more pure saps were collected. For instant, less hexoses were detected in phloem sap and the absence of sucrose in xylem sap in centrifugation method than in K2-EDTA exudation. More importantly, centrifugation method allowed accurate estimation of the concentrations of metabolites. This method could be successfully used for the collection of saps of citrus and other trees until the invention of a more specific method to collect ultra-pure phloem sap.

Bacteriocyte Reprogramming to Cope With Nutritional Stress in a Phloem Sap Feeding Hemipteran, the Pea Aphid Acyrthosiphon pisum.

Nutritional symbioses play a central role in the ability of insects to thrive on unbalanced diets and in ensuring their evolutionary success. A genomic model for nutritional symbiosis comprises the hemipteran Acyrthosiphon pisum, and the gamma-3-proteobacterium, Buchnera aphidicola, with genomes encoding highly integrated metabolic pathways. A. pisum feeds exclusively on plant phloem sap, a nutritionally unbalanced diet highly variable in composition, thus raising the question of how this symbiotic system responds to nutritional stress. We addressed this by combining transcriptomic, phenotypic and life history trait analyses to determine the organismal impact of deprivation of tyrosine and phenylalanine. These two aromatic amino acids are essential for aphid development, are synthesized in a metabolic pathway for which the aphid host and the endosymbiont are interdependent, and their concentration can be highly variable in plant phloem sap. We found that this nutritional challenge does not have major phenotypic effects on the pea aphid, except for a limited weight reduction and a 2-day delay in onset of nymph laying. Transcriptomic analyses through aphid development showed a prominent response in bacteriocytes (the core symbiotic tissue which houses the symbionts), but not in gut, thus highlighting the role of bacteriocytes as major modulators of this homeostasis. This response does not involve a direct regulation of tyrosine and phenylalanine biosynthetic pathway and transporter genes. Instead, we observed an extensive transcriptional reprogramming of the bacteriocyte with a rapid down-regulation of genes encoding sugar transporters and genes required for sugar metabolism. Consistently, we observed continued overexpression of the A. pisum homolog of RRAD, a small GTPase implicated in repressing aerobic glycolysis. In addition, we found increased transcription of genes involved in proliferation, cell size control and signaling. We experimentally confirmed the significance of these gene expression changes detecting an increase in bacteriocyte number and cell size in vivo under tyrosine and phenylalanine depletion. Our results support a central role of bacteriocytes in the aphid response to amino acid deprivation: their transcriptional and cellular responses fine-tune host physiology providing the host insect with an effective way to cope with the challenges posed by the variability in composition of phloem sap.

Aphid infestation leads to plant part-specific changes in phloem sap chemistry, which may indicate niche construction.

Phloem sap quality can differ between and within plants, and affect the performance of aphids. In turn, aphid infestation may change the chemical composition and nutritional value of phloem sap. However, the effects of different aphid species on the overall phloem sap composition of distinct parts within plant individuals in relation to aphid performance remain unclear. To test the specificity of plant responses to aphids, we used two chemotypes of Tanacetum vulgare plants and placed the monophagous aphids Macrosiphoniella tanacetaria and Uroleucon tanaceti on different plant parts (stems close to the inflorescence, young and old leaves). Aphid population growth was determined and sugars, organic acids, amino acids and metabolic fingerprints of phloem exudates were analysed. Macrosiphoniella tanacetaria performed best on stems, whereas U.tanaceti performed best on old leaves, indicating differences in niche conformance. Aphid infestation led to distinct changes in the phloem exudate composition of distinct metabolite classes, differing particularly between plant parts but less between chemotypes. In summary, plant responses to aphids are highly specific for the chemotype, plant part, metabolite class and aphid species. These changes may indicate that aphids construct their own niche, optimizing the food quality on the plant parts they prefer.

Bemisia tabaci MED Population Density as Affected by Rootstock-Modified Leaf Anatomy and Amino Acid Profiles in Hydroponically Grown Tomato.

Bemisia tabaci is one of the most devastating pests in tomato greenhouse production. Insecticide resistance management for B. tabaci requires a novel approach that maximizes non-chemical methods for pest control. The aim of this study was to test the effects of rootstocks on B. tabaci populations in hydroponically grown tomato plants. In order to contribute to the better understanding of the mechanisms defining the attractiveness of plant to the aerial pest, the effects of rootstocks on leaf anatomy and the amino acid composition of phloem sap were assessed. A two-factorial experimental design was adopted using cultivars (rootstock cultivars and Clarabella) grown as either non-grafted or grafted with cultivar Clarabella as a scion. The rootstock cultivars included Arnold, Buffon, Emperador, and Maxifort. A reduction in B. tabaci density was observed using all rootstock cultivars. The number of adult individuals per leaf was 2.7–5.4 times lower on rootstock cultivars than on Clarabella. The number of large nymphs per square centimeter was at least 24% higher on non–grafted Clarabella compared with all other treatments. The leaf lamina thickness and mesophyll thickness were lower in self-grafted Clarabella than in non-grafted or in one grafted on rootstock cultivars; however, the extent of this reduction depended on the rootstock. The leaves with thinner laminae were generally less attractive to B. tabaci. Eighteen amino acids were detected in the exudates of phloem sap. In all treatments, the most abundant amino acid was γ-aminobutyric acid (GABA), followed by proline, serine, alanine, and histidine. The scion cultivar Clarabella was the most attractive to B. tabaci and had a higher content of leucine than did rootstock cultivars, and a higher content of lysine compared to Buffon and Maxifort. The features modified by rootstock such are changes in leaf anatomy can affect the attractiveness of plants to B. tabaci. Thus, the grafting of tomato could constitute a valuable tool in an integrated management strategy against this aerial pest.

Effects of intraspecific and intra-individual differences in plant quality on preference and performance of monophagous aphid species.

Plant chemistry is one of the main drivers of herbivore distribution. Monophagous herbivore species are highly specialized, but even within their only host species the chemistry varies. The herbivore's choice is initially mainly guided by volatile plant compounds. Once on the plant, particularly for aphids the phloem quality affects their performance. However, little is known about the intraspecific and intra-individual variation in phloem sap and their influences on monophagous aphids. To determine potential mechanisms involved in aphid colonization, we tested the effects of intraspecific chemical variation in Tanacetum vulgare, which produces different chemotypes, on the preference of two monophagous aphid species. Moreover, we measured the performance of the aphids on different plant parts (stem close to the inflorescence, young and old leaves) of these chemotypes and analyzed their phloem sap composition. Both species preferred the β-thujone (THU) over the trans-carvyl acetate (CAR) chemotype in dual-choice assays. Survival of Macrosiphoniella tanacetaria was neither affected by intraspecific nor intra-individual variation, whereas the reproduction was highest on stems. In contrast, Uroleucon tanaceti survived and reproduced best on old leaves of the preferred chemotype. The sugar, organic acid and amino acid composition pronouncedly differed between phloem exudates of different plant parts, but less between chemotypes. Unexpectedly, high concentrations of amino acids did not necessarily enhance aphid performance. These different performance optima may cause niche differentiation and, therefore, enable co-existence. In conclusion, the tremendous variation in plant chemistry even within one species can affect the distribution of highly specialized aphids at various scales aphid species-specifically.

Occurrence of free fatty acids in the phloem sap of different citrus varieties

ABSTRACTCandidatus Liberibacter asiaticus is a phloem restricted bacterium that causes citrus greening disease or huanglongbing (HLB), a major treat to commercial citrus production in Florida. It is transmitted by the Asian citrus psyllid, a phloem sap-feeding insect. Studies conducted on the composition of citrus phloem sap revealed the presence amino acids, organic acids and sugars and of low amounts of free fatty acids. In the present study, the phloem sap of 12 citrus varieties with different degrees of tolerance to HLB were extracted with ethyl acetate and analyzed by GC-MS after derivatization with boron trifluoride, a fatty acid-specific reagent. Nine free fatty acids were detected in all varieties. Of the 9 fatty acids detected, only capric acid was significantly different among varieties.

Metabolite signature of the phloem sap of fourteen citrus varieties with different degrees of tolerance to Candidatus Liberibacter asiaticus

Huanglongbing (HLB) disease in citrus is caused by the phloem-limited bacterium, Candidatus Liberibacter asiaticus (CLas) which is transmitted by the Asian citrus psyllids, a phloem sap feeder. Most of commercial citrus cultivars are susceptible to the HLB disease. However, some varieties were found to be relatively tolerant than other. Herein, we studied the phloem sap composition of fourteen varieties with different degrees of tolerance to CLas to identify metabolites that could be responsible for the tolerance or essential to grow CLas. The phloem sap was subjected to untargeted gas chromatography-mass spectrometry (GC-MS) metabolomics. After trimethylsilyl derivatization, more than seventy different metabolites were detected. The majority were sugars and they also were the most abundant metabolites. Most of amino acids were positively correlated with tolerance to CLas while several of the organic acids were negatively correlated with the tolerance. Fumaric acid was the only organic acid that is correlated positively. Although the concentrations of the main sugars (sucrose, fructose, and glucose) varied among varieties, they were not correlated with citrus tolerance or susceptibility to CLas. On the other hand, some of the low abundant sugars were positively correlated with citrus tolerance to CLas. Sugar acids and scyllo-inositol were negatively correlated with citrus tolerance to CLas. Development of commercial tolerant citrus varieties to HLB is highly recommended and further metabolomic studies are required to investigate other metabolites which cannot be detected by GC-MS and could be responsible for citrus tolerance to CLas.

Metabolomic comparative analysis of the phloem sap of curry leaf tree (Bergera koenegii), orange jasmine (Murraya paniculata), and Valencia sweet orange (Citrus sinensis) supports their differential responses to Huanglongbing

ABSTRACTOrange jasmine, Murraya paniculata and curry leaf tree, Bergera koenegii are alternative hosts for Diaphorina citri, the vector of Candidatus Liberibacter asiaticus (CLas), the pathogen of huanglongbing (HLB) in citrus. D. citri feeds on the phloem sap where CLas grows. It has been shown that orange jasmine was a better host than curry leaf tree to D. citri. In addition, CLas can infect orange jasmine but not curry leaf tree. Here, we compared the phloem sap composition of these 2 plants to the main host, Valencia sweet orange, Citrus sinensis. Phloem sap was analyzed by gas chromatography-mass spectrometry after trimethylsilyl derivatization. Orange jasmine was the highest in proteinogenic, non-proteinogenic amino acids, organic acids, as well as total metabolites. Valencia was the highest in mono- and disaccharides, and sugar alcohols. Curry leaf tree was the lowest in most of the metabolites as well as total metabolites. Interestingly, malic acid was high in Valencia and orange jasmine but was not detected in the curry leaf. On the other hand, tartaric acid which can prevent the formation of malic acid in Krebs cycle was high in curry leaf. The nutrient inadequacy of the phloem sap in curry leaf tree, especially the amino acids could be the reason behind the longer life cycle and the low survival of D. citri and the limitation of CLas growth on this host. Information obtained from this study may help in cultivation of CLas and development of artificial diet for rearing of D. citri.

Genetic variability of the phloem sap metabolite content of maize (Zea mays L.) during the kernel-filling period

Using a metabolomic approach, we have quantified the metabolite composition of the phloem sap exudate of seventeen European and American lines of maize that had been previously classified into five main groups on the basis of molecular marker polymorphisms. In addition to sucrose, glutamate and aspartate, which are abundant in the phloem sap of many plant species, large quantities of aconitate and alanine were also found in the phloem sap exudates of maize. Genetic variability of the phloem sap composition was observed in the different maize lines, although there was no obvious relationship between the phloem sap composition and the five previously classified groups. However, following hierarchical clustering analysis there was a clear relationship between two of the subclusters of lines defined on the basis of the composition of the phloem sap exudate and the earliness of silking date. A comparison between the metabolite contents of the ear leaves and the phloem sap exudates of each genotype, revealed that the relative content of most of the carbon- and nitrogen-containing metabolites was similar. Correlation studies performed between the metabolite content of the phloem sap exudates and yield-related traits also revealed that for some carbohydrates such as arabitol and sucrose there was a negative or positive correlation with kernel yield and kernel weight respectively. A posititive correlation was also found between kernel number and soluble histidine.

Nucleotides, micro- and macro-nutrients, limonoids, flavonoids, and hydroxycinnamates composition in the phloem sap of sweet orange

ABSTRACTCurrently, the global citrus production is declining due to the spread of Huanglongbing (HLB). HLB, otherwise known as citrus greening, is caused by Candidatus Liberibacter asiaticus (CLas) and is transmitted by the Asian citrus psyllids (ACP), Diaphorina citri Kuwayama. ACP transmits CLas bacterium while feeding on the citrus phloem sap. Multiplication of CLas in the phloem of citrus indicates that the sap contains all the essential nutrients needed for CLas. In this study, we investigated the micro- and macro-nutrients, nucleotides, and others secondary metabolites of phloem sap from pineapple sweet orange. The micro- and macro-nutrients were analyzed using inductively coupled plasma-mass spectroscopy (ICP-MS) and inductively coupled plasma-optical emission spectroscopy (ICP-OES). Nucleotides and other secondary metabolites analysis was accomplished by reversed phase HPLC coupled with UV, fluorescence detection, or negative mode electrospray ionization mass spectrometry (ESI-MS). Calcium (89 mM) was the highest element followed by potassium (38.8 mM) and phosphorous (24 mM). Magnesium and sulfur were also abundant and their concentrations were 15 and 9 mM, respectively. The rest of the elements were found in low amounts (< 2mM). The concentrations of ATP, ADP, and AMP were 16, 31, and 3 µ mole/Kg fwt, respectively. GTP, GMP. NAD, FMN, FAD, and riboflavin were found at concentrations below (3 µ mole/Kg fwt). The phloem was rich in nomilin 124 mM and limonin 176 µ mole/Kg fwt. Hesperidin, vicenin-2, sinensetin, and nobiletin were the most predominant flavonoids. In addition, several hydroxycinnamates were detected. The results of this study will increase our knowledge about the nature and the chemical composition of citrus phloem sap.

Amino acids implicated in plant defense are higher in Candidatus Liberibacter asiaticus-tolerant citrus varieties

ABSTRACTCitrus Huanglongbing (HLB), also known as citrus greening, has been threatening the citrus industry since the early 1900's and up to this date there are no effective cures for this disease. Field observations and greenhouse controlled studies demonstrated that some citrus genotypes are more tolerant to Candidatus Liberibacter asiaticus (CLas) pathogen than others. However, the mechanisms underpinning tolerance has not been determined yet. The phloem sap composition of CLas-tolerant and sensitive citrus varieties was studied to identify metabolites that could be responsible for their tolerance to CLas. The citrus phloem sap was collected by centrifugation and was analyzed with gas chromatography-mass spectrometry after methyl chloroformate derivatization. Thirty-three metabolites were detected in the phloem sap of the studied varieties: twenty 20 amino acids, eight 8 organic acids, and five 5 fatty acids. Interestingly, the levels of most amino acids, especially those implicated in plantdefense to pathogens such as phenylalanine, tyrosine, tryptophan, lysine, and asparagine were higher in tolerant varieties. Although the level of organic acids varied between cultivars, this variation was not correlated with citrus resistance to CLas and could be cultivar specific. The fatty acids were found in trace amounts and in most cases their levels were not significantly different among varieties. Better understanding of the mechanisms underpinning citrus tolerance to CLas will help in developing economically tolerant varieties.

Composition of citrus phloem sap and honeydew produced by the citrus phloem sap feeder, the Asian citrus psyllid, Diaphorina citri (Homopetera: Psyllidae)

The honeydew composition of Asian citrus psyllids (ACP), the vector of citrus Huanglongbing (HLB), was studied using gas chromatography-mass spectrometry (GC-MS). Honeydew samples were collected from healthy ACP that were reared on one-year Valencia trees inside an insectary at a University of Florida’s Citrus Research and Education Center (CREC) grove in Lake Alfred, FL, USA. Dried samples (1 mg) were mixed with 30 μL of methoxyamine hydrochloride solution in pyridine (2%) and allowed to react for 17 h at room temperature. After methoximation, silylation reactions were induced by adding 80 μL of N-methyl-N-trimethylsilyl) trifluoracetamide (MSTFA) for 2 h at room temperature and 0.5 μL of derivatized sample was injected into the GC-MS running in the full scan mode. To check for amino acids, a 10 mg of alkaline honeydew sample was reacted with methylchloroformate (MCF) in a mixture of pyridine and methanol. The MCF derivatives were extracted with chloroform and analyzed with GC-MS. The moisure content was determined by drying the honeydew samples to a constant weight at a 100 °C. The major honeydew composition was as follows: 74.5 ± 2.8 sucrose, 12.4 ± 0.5 D-fructose, 6.4 ± 3.0 mannose, 1.8 ± 0.6 trehalose, myo-inositol 2.8 ± 0.8, ribitol 0.5 ± 1, galactose 0.4 ± 0.2, quinic acid 0.4± 0.3, and malic acid 0.3 ± 0.1. The moisture content of honeydew was 22.6 ± 2.6 No amino acids were detected as TMS or as MCF derivatives.