Combined Elevation of Temperature and CO2 Levels Impacts the Production and Sugar Composition of Aphid Honeydew

Honeydew is the keystone of many interactions between aphids and their predators, parasitoids, or mutualistic partners. Despite the crucial importance of honeydew in the aphid-ant mutualism, very few studies have investigated the potential impact of climate change on its production and composition. Here, we quantified changes in the sugar compounds and in the amount of honeydew droplets released by Aphis fabae reared on Vicia faba plants, under elevated levels of temperature and/or CO2. A combined elevation of these two abiotic factors increased honeydew production as well as the total amount in sugars, in particular the concentration of fructose and melezitose. Increased amount of sugars in phloem sap under elevated CO2 conditions, along with a raise of aphid metabolism and sap ingestion to compensate for water loss under elevated temperatures might explain these observed changes increase in honeydew production and sugar content. A higher amount of excreted honeydew coupled with a higher concentration in melezitose and fructose are expected to enhance both the feeding behavior and the laying of a recruitment trail by ant foragers, thereby reinforcing the ant-homopteran mutualism under a scenario of elevated temperature and CO2 levels. We discuss about the enhancing and counteracting effects of climate change on other biological agents (gut microorganisms, predators, parasitoids) that interact with aphids in a complex multitrophic system.

CsAGA1 and CsAGA2 Mediate RFO Hydrolysis in Partially Distinct Manner in Cucumber Fruits

A Raffinose family oligosaccharides (RFOs) is one of the major translocated sugars in the vascular bundle of cucumber, but little RFOs can be detected in fruits. Alpha-galactosidases (α-Gals) catalyze the first catabolism step of RFOs. Six α-Gal genes exist in a cucumber genome, but their spatial functions in fruits remain unclear. Here, we found that RFOs were highly accumulated in vascular tissues. In phloem sap, the stachyose and raffinose content was gradually decreased, whereas the content of sucrose, glucose and fructose was increased from pedicel to fruit top. Three alkaline forms instead of acid forms of α-Gals were preferentially expressed in fruit vascular tissues and alkaline forms have stronger RFO-hydrolysing activity than acid forms. By inducible gene silencing of three alkaline forms of α-Gals, stachyose was highly accumulated in RNAi-CsAGA2 plants, while raffinose and stachyose were highly accumulated in RNAi-CsAGA1 plants. The content of sucrose, glucose and fructose was decreased in both RNAi-CsAGA1 and RNAi-CsAGA2 plants after β-estradiol treatment. In addition, the fresh- and dry-weight of fruits were significantly decreased in RNAi-CsAGA1 and RNAi-CsAGA2 plants. In cucurbitaceous plants, the non-sweet motif within the promoter of ClAGA2 is widely distributed in the promoter of its homologous genes. Taken together, we found RFOs hydrolysis occurred in the vascular tissues of fruits. CsAGA1 and CsAGA2 played key but partly distinct roles in the hydrolysis of RFOs.

Generous hosts: Growth of ‘Candidatus Liberibacter asiaticus’ 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 GC-MS, 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 to uninfected phloem sap, we found that the organic acids implicated in the 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 the translocation of TCA cycle intermediates within the phloem. The net result, localized availability of organic acids likely benefits the 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’.

Detection of Oxytetracycline in Citrus Phloem and Xylem Saps Using Europium-Based Method

Oxytetracycline (OTC) has been used for the control of several plant diseases and was recently approved for the control of Huanglongbing, the citrus greening disease. Huanglongbing is caused by the phloem limited ‘Candidatus Liberibacter asiaticus’. Determination of OTC in the xylem and phloem of citrus plants is of great interest as they are the main routes of translocation in citrus. In addition, the determination of the level of OTC in the phloem sap is necessary for the control of the ‘Ca. L. asiaticus’ pathogen, which resides in the phloem. Herein, we demonstrated that the level of OTC in the citrus phloem and xylem saps obtained using the centrifugation method can be successfully measured using the europium (Eu) method directly or with cleanup by solid-phase extraction (SPE). Recovery of OTC from spiked sap samples purified by solid-phase extraction (SPE) was higher than 90%, while recovery from saps without SPE cleanup were nearly 100%. The ‘Ca. L. asiaticus’-infected leaf and phloem sap samples showed higher inhibition of the fluorescence intensity of the OTC standard compared to non-infected control leaf and phloem samples. In agreement with this finding, the levels of phenols and flavonoids in ‘Ca. L. asiaticus’-infected leaves were higher than those controls and were shown to interfere with the Eu method. Therefore, the SPE cleanup step only improved OTC recovery from leaf samples containing the interfering compounds. The Eu method was then used to determine OTC levels in the phloem and xylem sap of OTC-treated plants, and the results were similar whether measured directly or after SPE. Visualization under ultraviolet light (400 nm) showed the presence of OTC in citrus xylem and phloem saps with and without the use of SPE.

Effect of Soy Leaf Flavonoids on Pea Aphid Probing Behavior

Flavonoids detected in soybean Glycine max (L.) Merr. (Fabaceae) cause various alterations in the metabolism, behavior, and development of insect herbivores. The pea aphid Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae) poses potential threat to soybeans, but the effect of individual flavonoids on its feeding-associated behavior is relatively unknown. We monitored probing behavior (stylet penetration activities) of A. pisum on its preferred host plant, Pisum sativum L. untreated (control) and treated with 0.1% ethanolic solutions of flavonoids apigenin, daidzein, genistein, and kaempferol. We applied the electrical penetration graph (electropenetrography, EPG) technique, which visualizes the movements of aphid stylets within plant tissues. None of the applied flavonoids affected the propensity to probe the plants by A. pisum. However, apigenin enhanced the duration of probes in non-phloem tissues, which caused an increase in the frequency and duration of stylet mechanics derailment and xylem sap ingestion but limited the ingestion of phloem sap. Daidzein caused a delay in reaching phloem vessels and limited sap ingestion. Kaempferol caused a reduction in the frequency and duration of the phloem phase. Genistein did not affect aphid probing behavior. Our findings provide information for selective breeding programs of resistant plant cultivars to A. pisum.

Loss of The Phloem-Expressed Sugar Transporter VST1 Reduces Aphid Performance In Watermelon

Aphids can damage plants through sugar withdrawal by hijacking sugar metabolism and transport genes to increase their sugar sucking ability. Blocking plant diseases and pest access to nutrients has emerged as an exciting strategy for improving disease and insect resistance in plants. Our previous work identified a shift in the localization of the vacuolar sugar transporter (VST1) that contributes to sucrose (Suc) and glucose (Glc) unloading in the phloem of sweet watermelons. In this study, the potential role of VST1 in the response to aphid infestation was investigated. Loss of VST1 function adversely impacted aphid settling and honeydew production. The vst1 mutant displayed less aphid-induced hydrogen peroxide accumulation and cell death than wild-type (WT) plants. Additionally, the expression of the VST1 gene was induced by aphids. The mutation of VST1 reduced Suc and Glc accumulation in the phloem, indicating that Suc and Glc are important carbohydrate substrates in phloem sap that are transported by VST1 to support aphid propagation and infestation. Taken together, our results demonstrated that the mutation of VST1 by genome editing can decrease aphid performance in watermelon by blocking the sugar supply obtained from phloem sap.

Metagenomic Investigation Uncovers Presence of Probiotic-Type Microbiome in Kalparasa® (Fresh Unfermented Coconut Inflorescence Sap)

The phloem sap tapped from unopened inflorescence (spadix) of coconut palm using a novel collecting device, “coco-sap chiller,” has been branded Kalparasa® (henceforth as Kalparasa in the text) to distinguish its properties not found in sap harvested by traditional methods. To know its hitherto unidentified microbiome profile, we employed high-throughput sequencing to uncover the bacteriome and mycobiome in fresh and 12-h fermented samples. Fresh Kalparasa had a pH of 7.2, which dropped to 4.5 after 12 h, signifying fermentation of the sap. Diversity analysis indicated fresh Kalparasa having higher bacterial species than the fermented one. Contrary to this, fresh sap had lower fungal/yeast diversity than the fermented sample. Fresh Kalparasa had relatively higher abundance of probiotic-type Leuconostoc genus followed by equal proportions of Gluconobacter, Acetobacter, and Fructobacillus. The 12-h fermented Kalparasa showed a significant increase in Gluconobacter with a sharp decrease in Leuconostoc. Mycobiome data revealed fresh Kalparasa to be preponderant in Saccharomyces and Hanseniaspora genera of yeasts while the fermented sap had higher representation of Hanseniaspora and Cortinarius and lesser Saccharomyces. This suggested that the fermentation of Kalparasa was probably driven by symbiotic culture of bacteria and yeasts (SCOBY), particularly acetic acid bacteria and non-Saccharomyces yeasts. The bacteriome-function predictions highlighted the enrichment of glycerophospholipid, ABC transporters, purine, and pyrimidine metabolisms. Based on our findings, Kalparasa containing large population of Leuconostoc mesenteroides, Fructobacillus fructosus, Saccharomyces cerevisiae, and Hanseniaspora guilliermondii can be promoted as a healthy “unfermented” plant edible food containing live probiotic-type microbiome during its consumption.

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.

The Effectiveness of Physical and Chemical Defense Responses of Wild Emmer Wheat Against Aphids Depends on Leaf Position and Genotype

The bird cherry-oat aphid (Rhopalosiphum padi) is one of the most destructive insect pests in wheat production. To reduce aphid damage, wheat plants have evolved various chemical and physical defense mechanisms. Although these mechanisms have been frequently reported, much less is known about their effectiveness. The tetraploid wild emmer wheat (WEW; Triticum turgidum ssp. dicoccoides), one of the progenitors of domesticated wheat, possesses untapped resources from its numerous desirable traits, including insect resistance. The goal of this research was to determine the effectiveness of trichomes (physical defense) and benzoxazinoids (BXDs; chemical defense) in aphid resistance by exploiting the natural diversity of WEW. We integrated a large dataset composed of trichome density and BXD abundance across wheat genotypes, different leaf positions, conditions (constitutive and aphid-induced), and tissues (whole leaf and phloem sap). First, we evaluated aphid reproduction on 203 wheat accessions and found large variation in this trait. Then, we chose eight WEW genotypes and one domesticated durum wheat cultivar for detailed quantification of the defense mechanisms across three leaves. We discovered that these defense mechanisms are influenced by both leaf position and genotype, where aphid reproduction was the highest on leaf-1 (the oldest), and trichome density was the lowest. We compared the changes in trichome density and BXD levels upon aphid infestation and found only minor changes relative to untreated plants. This suggests that the defense mechanisms in the whole leaf are primarily anticipatory and unlikely to contribute to aphid-induced defense. Next, we quantified BXD levels in the phloem sap and detected a significant induction of two compounds upon aphid infestation. Moreover, evaluating aphid feeding patterns showed that aphids prefer to feed on the oldest leaf. These findings revealed the dynamic response at the whole leaf and phloem levels that altered aphid feeding and reproduction. Overall, they suggested that trichomes and the BXD 2,4-dihydroxy-7- methoxy-1,4-benzoxazin-3-one (DIMBOA) levels are the main factors determining aphid resistance, while trichomes are more effective than BXDs. Accessions from the WEW germplasm, rich with trichomes and BXDs, can be used as new genetic sources to improve the resistance of elite wheat cultivars.