Plant Phloem Research Articles

Niche construction and niche choice by aphids infesting wheat ears

The niche of aphids is largely defined by their consumption of plant phloem sap and its composition, including nutrients and specialized metabolites. Niche construction is the change of the environment by organisms, which may influence the fitness of these organisms and their offspring. To better understand interactions between plants and aphids, it is necessary to investigate whether aphids modify the chemical composition of the phloem sap of their host plants and whether conspecifics are affected by previous infestation. In the current study, ears of wheat (Triticum aestivum) plants were infested with clonal lineages of the English grain aphid (Sitobion avenae) or were left uninfested. The metabolic composition of ear phloem sap exudates was analyzed through amino acid profiling and metabolic fingerprinting. Aphids of the clonal lineages were either put on previously aphid-infested or on uninfested ears and their colony sizes followed over time. Furthermore, it was investigated whether aphids choose one treatment group over another. Sitobion avenae infestation affected the relative concentrations of some metabolites in the phloem exudates of the ears. Compared to uninfested plants, the relative concentration of asparagine was higher after aphid infestation. Colonies grew significantly larger on previously aphid-infested ears, which the aphids also clearly chose in the choice experiment. The pronounced positive effect of previous infestation on aphid colonies indicates niche construction, while the choice of these constructed niches reveals niche choice by S. avenae on wheat. The interplay between these different niche realization processes highlights the complexity of interactions between aphids and their hosts.

Leptodelphax maculigera (Hemiptera: Delphacidae) Harbors the Corn Stunt Complex Pathogens.

The African planthopper Leptodelphax maculigera (Hemiptera: Delphacidae) has been recently reported in many places in Brazil in association with maize. Its occurrence in maize production fields in Brazil has brought concerns to the corn production chain regarding the possibility of this planthopper to be a vector for maize bushy stunt phytoplasma (MBSP), corn stunt spiroplasma (Spiroplasma kunkelii), maize rayado fino virus (MRFV), and maize striate mosaic virus (MSMV). The phytoplasma and spiroplasma, which are bacteria belonging to the class Mollicutes, and the two viruses are associated with the corn stunt disease complex. Given the presence of the African planthopper species and the corn stunt complex in Brazil, we further investigated the abundance of this planthopper species in the State of Santa Catarina, Brazil, and whether the planthopper can carry the four pathogens. We inspected 12 maize production fields in different municipalities in the state for 20 weeks, using two yellow sticky traps for each maize field. The sticky traps were replaced weekly. A total of 130 specimens of L. maculigera were captured, with a great discrepancy in quantity among locations and weeks. We detected the mollicute MBSP and the viruses MRFV and MSMV in L. maculigera, whereas S. kunkelii was absent in the assessed African planthopper samples. The molecular detection of the phytoplasma and the viruses in field-collected African planthoppers is strong evidence that this insect species has the ability to acquire those pathogens through feeding from the phloem of diseased maize plants. Nonetheless, transmission capacity needs to be experimentally proven to assert L. maculigera as a vector for the corn-stunting pathogens.

(Invited) Carbon Nanostructures for Targeted Delivery and Health Monitoring in Plants

To meet the demand for food and bioenergy of a rapidly growing global population requires novel and convergent approaches to enhance photosynthetic organism function. Nanotechnology is enabling the development of targeted and species independent tools for advancing plant bioengineering and precision agriculture.My research group has developed carbon nanotube-based sensors capable of real-time sensing of plant signaling molecules. These nanosensors enabled the creation of plant sentinels that communicate the health status of plants to electronic devices. Near-infrared (nIR) fluorescent single-walled carbon nanotubes (SWCNTs) were designed and interfaced with plant leaves to report hydrogen peroxide (H2O2), a key signaling molecule associated with the onset of plant stress. The sensor nIR fluorescence response (>900 nm) is quenched by H2O2 with selectivity against other stress-associated signaling molecules and within the plant physiological range (10–100 H2O2 μM). In vivo remote nIR imaging of H2O2 sensors enabled optical monitoring of plant health in response to stresses including UV-B light (−11%), high light (−6%), and a pathogen-related peptide (flg22) (−10%), but not mechanical leaf wounding (<3%). The sensor’s high biocompatibility was reflected on similar leaf cell death (<5%) and photosynthetic rates to controls without SWCNT. These optical nanosensors report early signs of stress and will improve our understanding of plant stress communication, provide novel tools for precision agriculture, and optimize the use of agrochemicals in the environment.We also developed targeted carbon-based nanomaterials as tools for precise chemical delivery (carbon dots, CDs) and gene delivery platforms (SWCNTs) to plant organelles (chloroplasts) and vasculature (phloem). A biorecognition approach of coating the nanomaterials with a rationally designed chloroplast targeting peptide improved the delivery of CDs with molecular baskets (TP-β-CD) for delivery of agrochemicals and of plasmid DNA coated SWCNT (TP-pATV1-SWCNT) from 47% to 70% and from 39% to 57% of chloroplasts in leaves, respectively. Plants treated with TP-β-CD (20 mg/L) and TP-pATV1-SWCNT (2 mg/L) had a low percentage of dead cells, 6% and 8%, respectively, similar to controls without nanoparticles, and no permanent cell and chloroplast membrane damage after 5 days of exposure. Orthogonally, the surfaces of carbon dot nanocarriers were functionalized with sucrose, enabling rapid and efficient foliar delivery into the plant phloem, a vascular tissue that transports sugars, signaling molecules, and agrochemicals through the whole plant. The chemical affinity of sucrose molecules to sugar membrane transporters on the phloem cells enhances the uptake of biocompatible carbon dots with β-cyclodextrin molecular baskets (suc-β-CD) that can carry a wide range of agrochemicals. The nanoparticle fluorescence emission properties allowed detection and monitoring of rapid translocation (<40 min) in the vasculature of wheat leaves by confocal and epifluorescence microscopy. The suc-β-CDs more than doubled the delivery of chemical cargoes into the leaf vascular tissue. The sucrose coating of nanoparticles approach enables unprecedented targeted delivery to roots with ≈70% of phloem-loaded nanoparticles delivered to roots.The use of plant biorecognition molecules mediated delivery provides an approach for guiding nanocarriers with their cargoes to plant organelles, cells and tissues and whole plants and engineering safer and efficient agrochemical and biomolecule delivery tools. Communication and actuation of plants mediated by nanosensors and targeted nanostructures can turn plants into technology and lead to a more precise and sustainable agriculture with reduced environmental impact.

Serological and Molecular Detection of Citrus Tristeza Virus: A Review.

Citrus tristeza virus (CTV) is a globally pervasive and economically significant virus that negatively impacts citrus trees, leading to substantial reductions in fruit yield. CTV occurs within the phloem of infected plants, causing a range of disease phenotypes, such as stem pitting (SP), quick decline (QD), and other detrimental diseases. Research on CTV is challenging due to the large size of its RNA genome and the diversity of CTV populations. Comparative genomic analyses have uncovered genetic diversity in multiple regions of CTV isolates' genomes, facilitating the classification of the virus into distinct genotypes. Despite these challenges, notable advancements have been made in identifying and controlling CTV strains through serological and molecular methods. The following review concentrates on the techniques of nucleic acid identification and serological analysis for various CTV isolates, assisting in the comparison and evaluation of various detection methods, which are crucial for the effective management of CTV diseases, and so contributes to the innovation and development of CTV detection methods.

Feeding specialization shapes the bottom‐up effect of arbuscular mycorrhizal fungi across a plant–aphid–parasitoid system

Societal Impact StatementArbuscular mycorrhizal fungi (AMF) impact the relationships between plants, aphids (insects that feed on plant phloem), and their natural enemies (insects that prey on or parasitize aphids). The presence of AMF influences the growth and population of different aphid species and affects the development of aphid‐killing wasps and their attraction to plants. This research has been conducted also considering the insects' feeding strategy and their feeding specialization. This study provides novel perspectives on how these fungi shape interactions in the natural world, offering potential insights for the development of sustainable pest management strategies in agriculture.Summary Arbuscular mycorrhizal fungi (AMF) are major root symbionts regulating plant physiology. Their presence affects the performance of aboveground insect herbivores in relation to their feeding strategy and their feeding specialization. For example, the effect of the arbuscular mycorrhizal (AM) symbiosis on chewing insects, positive for specialists and negative for generalists, has been previously demonstrated. Conversely, the impact of AMF on phloem‐suckers with relatively different levels of specialization remains unexplored. We tested the influence of the AM Funneliformis mosseae on the fitness of the specialist aphid Acyrthosiphon pisum and the generalist aphid Myzus persicae on Vicia faba plants. Further, we investigated the effects of AMF on the higher trophic level, the aphid parasitoids Aphidius ervi (specialist) and Aphidius colemani (generalist), by evaluating plant attractiveness and parasitoid fitness. To support the results of behavioral and biological bioassays we characterized the photosynthetic parameters, the volatilome and the transcriptome of tested plants. Mycorrhizal plants proved unsuitable for the generalist M. persicae but enhanced the fitness of the specialist A. pisum. The AM symbiosis had no effects on the behavioral response of A. colemani and enhanced the attraction and fitness of A. ervi. Volatilome and transcriptome profiling corroborated the results of bioassay highlighting a bottom‐up effect of the AMF across a plant–aphid–parasitoid system.

Not just candy: A herbivore‐induced defence‐related plant protein in honeydew enhances natural enemy fitness

Abstract Herbivore feeding often increases secondary metabolite production in plants. These herbivore‐induced plant proteins might end up in honeydew excreted by phloem‐feeding insects. This is important because honeydew is one of the most abundant and accessible carbohydrate sources for natural enemies in many agroecosystems and these proteins can thus mediate many tri‐trophic interactions. Here, we hypothesized that defensive metabolites induced in the phloem by herbivory accumulate in the honeydew excreted by phloem‐feeding insects and, consequently, affect the fitness of the herbivores' natural enemies that feed on it. We used a tri‐trophic system consisting of citrus plants, the mealybug Planococcus citri and its primary parasitoid Anagyrus vladimiri. First, we assessed A. vladimiri fitness when fed on P. citri honeydew. We then collected honeydew of seven phloem‐feeding insects, including P. citri, and analysed their protein content. Finally, we analysed the effect of superoxide dismutase (SOD), an antioxidant enzyme associated with plant defences that was commonly found in the analysed honeydews, on A. vladimiri fitness. The fitness of A. vladimiri increased when fed on honeydew compared to a sucrose‐based diet, demonstrating that honeydew can contain compounds that benefit natural enemies. Proteomic analyses showed that defence‐related plant proteins were present in honeydew of seven phloem‐feeding insects analysed. Among these, the enzyme SOD was present in honeydew of all of them. Moreover, the levels of SOD were 10‐fold higher in the phloem of plants infested by P. citri than in that of uninfested plants. SOD was also actively excreted in P. citri honeydew, and we proved that it increases the fecundity of the parasitoid A. vladimiri. We conclude that enzymatic proteins induced by herbivory in the phloem and involved in plant defence, accumulate in the honeydew excreted by phloem‐feeding insects and, contrary to the current paradigm, at least some of these can have positive effects on the third trophic level. Read the free Plain Language Summary for this article on the Journal blog.

Citrus-mediated gene silencing of cytochrome P450 suppresses insecticide resistance and increases mortality in Diaphorina citri.

Asian citrus psyllid, Diaphorina citri, is a hemipteran that vectors the causal pathogen of citrus greening disease, or huanglongbing (HLB). HLB is a tree killing disease that has severely limited citrus production globally. Unfortunately, there is no cure for this disease, and mitigation depends on multiple insecticide applications to reduce vector populations. Silencing of cytochrome P450 expression associated with detoxification enzymes by RNA interference is known to increase susceptibility of D. citri to insecticides. However, dsRNA was previously introduced into psyllids by topical applications. The possible application of this technology for pest management will require effective field delivery of the dsRNA. Therefore, we evaluated a virus vector (Citrus tristeza virus; 'mild strain' T36) to deliver gene silencing directly to this sap-sucking insect via plant phloem. Citrus macrophylla plants inoculated with CTV expressing a truncated consensus sequence of CYP450 (CTV-tCYP450) constantly produced small interfering RNA in the plant phloem that targeted five cytochrome p540 (CYP450) genes in D. citri. Insecticide susceptible D. citri reared on citrus infected with CTV-tCYP450 were subsequently more susceptible to imidacloprid, fenpropathrin, carbaryl, and chlorpyrifos than those reared on citrus infected with wildtype CTV or non-infected negative controls. Additionally, nymph survival and adult lifespan were significantly reduced when psyllids were reared on CTV-tCYP450 citrus plants compared with controls. Interestingly, similar results were obtained after one and two generations of rearing. Finally, field-collected psyllids from areas with known broad-spectrum insecticide resistance were rendered more susceptible to imidacloprid and fenpropathrin after feeding on CTV-tCYP450 citrus trees as compared with those reared on controls. The integration of citrus-mediated RNA inference targeting psyllid detoxification enzymes could function as a resistance management tool and reduce insecticide input in an integrated pest management program for HLB. © 2024 Society of Chemical Industry.

Development And Acceptability of Banana (Esp. Damilig-Musa Acuminata) Fiber as Alternative Source of Natural Fabric

Banana fiber yarn is a natural best fiber derived from the inner bark (or phloem) of banana plants. It is a very eco-friendly material, which also has the advantage of being completely biodegradable. This yarn is also known as banana silk, due to the shiny and silky aspect of the most precious fibers, or Musa yarn, from the Musa genre to which the banana plant belongs. The study will focus on the Development and Acceptability of Banana (Esp. Damilig-Musa Acuminata) Fiber as Alternative Source of Natural Fabric. The panel of experts will evaluate the following namely: DOST personnel, Garments instructor, fashion designer/ textile experts and Faculty and garments fashion and design students of Isabela State University – Ilagan Campus. The purpose of this research is to develop and test the effectiveness of banana (Damilig-musa acuminata), specifically its pseudo-stem. The researcher aims to unlock the following queries: 1. What are the processes used in the production of yarn? 2. What will be the outcome of the develop banana (Damilig-musa acuminata) after the process of fiber extraction in terms of texture, appearance and durability. 3. What will be the assessment and acceptability of the banana yarn (Damilig-musa acuminata) after weaving process is completed in terms of colorfastness, crease resistant and fabric shrinkage The descriptive type of research will applied in the study with the set of survey questionnaire as the main instrument in gathering data. The weighted mean will also utilizes to determine the general acceptability of the study. The study will use the five-point Likert rating scale. The process will conduct in accordance to Development and Acceptability of Banana (Esp. Damilig-Musa Acuminata) Fiber as Alternative Source of Natural Fabric. The procedures are harvesting, peeling, retting, and sun drying, grouping, spinning and weaving. Evaluation results will show that the project obtained an overall mean of 4.38 to the development of fiber and 3.45 to the level of acceptability of fabric which means that the Development and Acceptability of Banana (Esp. Damilig-Musa Acuminata) Fiber as Alternative Source of Natural Fabric is Moderately Acceptable to the panel of experts based on the criteria checklist. Based on the results of the experiments, it can be concluded that banana fibers can be used as an alternative source of natural fibers because they have several inherent advantages over cotton fiber. Because of the abundance of raw material in nature each year, banana fiber-based yarns and fabrics are less expensive and more sustainable. Because of the low fiber weight and strength loss, softening banana fibers by boiling in distilled water was found to be a better treatment.

Leafhopper salivary carboxylesterase suppresses JA-Ile synthesis to facilitate initial arbovirus transmission in rice phloem

Plant jasmonoyl-L-isoleucine (JA-Ile) is a major defense signal against insect feeding, but whether or how insect salivary effectors suppress JA-Ile synthesis and thus facilitate viral transmission in the plant phloem remains elusive. Insect carboxylesterases (CarEs) are the third major family of detoxification enzymes. Here, we identify a new leafhopper CarE, CarE10, that is specifically expressed in salivary glands and is secreted into the rice phloem as a saliva component. Leafhopper CarE10 directly binds to rice jasmonate resistant 1 (JAR1) and promotes its degradation by the proteasome system. Moreover, the direct association of CarE10 with JAR1 clearly impairs JAR1 enzyme activity for conversion of JA to JA-Ile in an in vitro JA-Ile synthesis system. A devastating rice reovirus activates and promotes the co-secretion of virions and CarE10 via virus-induced vesicles into the saliva-storing salivary cavities of the leafhopper vector and ultimately into the rice phloem to establish initial infection. Furthermore, a virus-mediated increase in CarE10 secretion or overexpression of CarE10 in transgenic rice plants causes reduced levels of JAR1 and thus suppresses JA-Ile synthesis, promoting host attractiveness to insect vectors and facilitating initial viral transmission. Our findings provide insight into how the insect salivary protein CarE10 suppresses host JA-Ile synthesis to promote initial virus transmission in the rice phloem.

Physiological and developmental disturbances caused by Botryosphaeria dieback in the annual stems of grapevine.

Botryosphaeria dieback is a grapevine trunk disease caused by fungi of the Botryosphaeriaceae family, which attacks more specifically the woody tissues. The infection leads to different symptoms including a severe form with a leaf drop as well as premature plant death. Botryosphaeria dieback causes major economic losses, since no effective treatment is yet available. A better understanding is necessary to find solutions to fight this disease. In this study, our objective was to characterize the "leaf drop" form by (1) looking for the presence of pathogens in the basal internodes of stems, (2) quantifying blocked vessels by tylosis and/or gummosis, and (3) describing the impact of the disease on vine physiology (gene expression and metabolome) and development (establishment and functioning of the cambium and phellogen) at the level of xylem and phloem of basal stem internodes. Our study has shown that Botryosphaeriaceae were present in both phloem and xylem of the basal internodes of the annual stem, with xylem vessels obturated. We have also clearly demonstrated that gene expression and metabolite profiles were strongly modified in both xylem and phloem of diseased plants. Differences in stems between healthy (control, C) and diseased (D) plants were low at flowering (vines not yet symptomatic), higher at the onset of symptom expression and still present, although less marked, at full disease expression. qRT-PCR analysis showed in both phloem and xylem an overexpression of genes involved in plant defense, and a repression of genes related to meristematic activity (i.e. vascular cambium and phellogen). Metabolomic analysis showed specific fingerprints in stems of healthy and diseased plants from the onset of symptom expression, with an increase of the level of phytoalexins and mannitol, and a decrease of 1-kestose one. At the structural level, many alterations were observed in internodes, even before the onset of symptoms: a classical obstruction of xylem vessels and, for the first time, a disorganization of the secondary phloem with an obstruction of the sieve plates by callose. The disease modifies the development of both secondary phloem (liber) and phellogen. Altogether, this study combining different approaches allowed to highlight deep vine dysfunction in the internodes at the base of stems, that may explain vine decline due to Botryosphaeria dieback.

Towards sustainable and ecofriendly polymer composite materials from bast fibers: a systematic review

Abstract This systematic review focuses on the exploration and advancement of sustainable and eco-friendly polymer composite materials derived from bast fibers. Bast fibers, obtained from the phloem of certain plants like flax, hemp, jute, and kenaf, represent a renewable and environmentally benign resource. Their integration into polymer based composites has gained significant attention due to the growing environmental concerns and the need for sustainable material development. The importance of this study lies in its comprehensive examination of bast fibers as viable alternatives to the synthetic fibers in polymer composite materials. By harnessing the natural strength, light weight, and biodegradability of bast fibers, this review contributes to the creation of materials that are not only environmentally sustainable but also possess enhanced mechanical properties suitable for various industrial and domestic applications.

Leafhopper salivary vitellogenin mediates virus transmission to plant phloem

Salivary effectors of piercing-sucking insects can suppress plant defense to promote insect feeding, but it remains largely elusive how they facilitate plant virus transmission. Leafhopper Nephotettix cincticeps transmits important rice reovirus via virus-packaging exosomes released from salivary glands and then entering the rice phloem. Here, we report that intact salivary vitellogenin of N. cincticeps (NcVg) is associated with the GTPase Rab5 of N. cincticeps (NcRab5) for release from salivary glands. In virus-infected salivary glands, NcVg is upregulated and packaged into exosomes mediated by virus-induced NcRab5, subsequently entering the rice phloem. The released NcVg inherently suppresses H2O2 burst of rice plants by interacting with rice glutathione S-transferase F12, an enzyme catalyzing glutathione-dependent oxidation, thus facilitating leafhoppers feeding. When leafhoppers transmit virus, virus-upregulated NcVg thus promotes leafhoppers feeding and enhances viral transmission. Taken together, the findings provide evidence that viruses exploit insect exosomes to deliver virus-hijacked effectors for efficient transmission.

Protective and curative effect of an experimental biocomplex against Flavescence dorée phytoplasma infection and transmission by Scaphoideus titanus under laboratory conditions

Flavescence dorée (FD) is one of the most devastating diseases threatening grapevine cultivation in Europe. The causal agents are phytoplasmas, which reside in phloem vessels of infected plants and in the body of leafhopper vectors, the most important being Scaphoideus titanus. Current control mainly relies on vector management, which is often insufficient to prevent from disease outbreaks. Suitable methods to interfere with phytoplasmas in planta are constantly sought to support insecticide sprays. We tested a biocomplex containing trace elements and soluble sugars, with potential systemic spread and antimicrobial activity, with the aim to verify its capability to penetrate plant phloem and to interfere with phytoplasma survival in vector-inoculated plants, using two FD isolates (FD-C and FD-D). The penetration into the plant phloem, as well as its acquisition by phloem-feeding vectors, were tested using an iridium-marked biocomplex. Phytoplasma infection was detected by real Time PCR and compared in plants exposed to biocomplex treatment before inoculation (short-time and mid-time preventive application), and to treatment after inoculation (curative application). We also assessed if the biocomplex interferes with the vector competence of S. titanus, by conducting phytoplasma transmission trials with sequential inoculation steps, where untreated and biocomplex-treated grapevine plantlets were sequentially offered to infectious leafhoppers. Foliar treatment with the biocomplex supported the penetration of phloem tissues, and the product was successfully acquired by the experimental phytoplasma vector Euscelidius variegatus after feeding on the phloem of treated plants. When the biocomplex was applied on grapevines, it induced a significant reduction of phytoplasma infection regardless of FD isolate, both as a preventive and curative treatment, indicating an antibacterial activity in the plant. Moreover, sequential inoculation experiments showed a decrease of S. titanus inoculation rates after vector exposure to a biocomplex-treated plant, suggesting a reduction of transmission efficiency that was higher using the FD-C rather than FD-D isolate. Our results indicate the tested biocomplex as a promising tool for the creation of anti-phytoplasma formulations to support vector management in FD containment.

Targeted Delivery of Sucrose-Coated Nanocarriers with Chemical Cargoes to the Plant Vasculature Enhances Long-Distance Translocation.

Current practices for delivering agrochemicals are inefficient, with only a fraction reaching the intended targets in plants. The surfaces of nanocarriers are functionalized with sucrose, enabling rapid and efficient foliar delivery into the plant phloem, a vascular tissue that transports sugars, signaling molecules, and agrochemicals through the whole plant. The chemical affinity of sucrose molecules to sugar membrane transporters on the phloem cells enhances the uptake of sucrose-coated quantum dots (sucQD) and biocompatible carbon dots with β-cyclodextrin molecular baskets (suc-β-CD) that can carry a wide range of agrochemicals. The QD and CD fluorescence emission properties allowed detection and monitoring of rapid translocation (<40min) in the vasculature of wheat leaves by confocal and epifluorescence microscopy. The suc-β-CDs more than doubled the delivery of chemical cargoes into the leaf vascular tissue. Inductively coupled plasma mass spectrometry (ICP-MS) analysis showed that the fraction of sucQDs loaded into the phloem and transported to roots is over 6.8 times higher than unmodified QDs. The sucrose coating of nanoparticles approach enables unprecedented targeted delivery to roots with ≈70% of phloem-loaded nanoparticles delivered to roots. The use of plant biorecognition molecules mediated delivery provides an efficient approach for guiding nanocarriers containing agrochemicals to the plant vasculature and whole plants.

Gene silencing of cathepsins B and L using CTV-based, plant-mediated RNAi interferes with ovarial development in Asian citrus psyllid (ACP), Diaphorina citri.

Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a vector of the bacteria Candidatus Liberibacter americanus (CLam) and Candidatus Liberibacter asiaticus (CLas), which are phloem-restricted and associated with the most important and destructive worldwide citrus disease, Huanglongbing (HLB). Currently, no cure for HLB has been described. Therefore, measures have focused on reducing D. citri populations. In these insects, cathepsin B (DCcathB) and L (DCcathL) enzymes play an important role in digestion, and are involved in embryogenesis, immune defense, and ecdysis. In this study, we used a CTV-based vector to deliver dsRNA (CTV-dsRNA) into Citrus macrophylla plants targeting DCcathB and DCcathL genes in D. citri that fed on the phloem of these CTV-RNAi infected plants. Subsequently, we evaluated expression of DCcathB and DCcathL genes as well as the Vitellogenin (Vg) gene by RT-qPCR in D. citri fed on CTV-dsRNA occurring in plant phloem. It was found that a defective phenotype in D. citri females as a result of knockdown of DCcathB and DCcathL genes mediated by CTV dsRNA. These results showed that Psyllids fed on plants treated with the CTV-dsRNA exhibited downregulation of the Vg gene, one of the most important genes associated with embryogenic and female development, which was associated with dsRNA-mediated silencing of the two cathepsin genes. Based on our findings, a CTV-based strategy for delivering RNAi via plants that targets DCcathB and DCcathL genes may represent a suitable avenue for development of dsRNA-based tools to manage D. citri that limits the spread of HLB.

The aphid Myzus persicae (Hemiptera: Aphididae) acquires chloroplast DNA during feeding on host plants.

Aphids (Hemiptera: Aphididae) extract nutrients from host plant phloem via stylets that facilitate salivation and sap uptake. When navigating to the phloem, aphids periodically puncture nonvascular cells and sample cell contents, but rarely cause significant cell damage. As a result, aphids are considered "stealthy" feeders. In contrast, insects that do cause damage, such as chewing herbivores, will take up host cell contents-including DNA-into their guts. Researchers can use molecular barcoding methods to identify recent host use patterns of chewing herbivores. This information is valuable for both pest management and basic ecological studies. Because of their stealthy feeding style, it was assumed that host plant DNA could not be recovered from aphids and other Sternorrhyncha. However, several recent studies document host plant DNA uptake by psyllids, which feed in a similar manner to aphids. Therefore, we hypothesized that aphids may also acquire DNA from host plants. Since aphids puncture and sample cytosol contents from cells, we predicted that aphids would be most likely to acquire DNA from chloroplasts. To test this, we performed host feeding and host transfer experiments with Myzus persicae (Sulzer), then used PCR to recover and sequence a region between the trnT and trnF genes from acquired chloroplast DNA. We found that M. persicae readily acquires chloroplast DNA, even prior to phloem contact, and that fragment sizes sufficient for host plant identification can be recovered. Our work suggests that molecular gut content analysis is a viable tool for studying aphid-host interactions.

Identification and evaluation of potential inhibitor molecules against TcyA from Candidatus Liberibacter asiaticus

Of the two putative amino acid binding periplasmic receptors of ABC transporter family in Candidatus Liberibacter asiaticus (CLas), cystine binding receptor (CLasTcyA) has been shown to mainly express in phloem of citrus plant and is a target for inhibitor development. The crystal structure of CLasTcyA in complex with substrates has been reported earlier. The present work reports the identification and evaluation of potential candidates for their inhibitory potential against CLasTcyA. Among many compounds, selected through virtual screening, and MD simulation, pimozide, clidinium, sulfasalazine and folic acid showed significantly higher affinities and stability in complex with CLasTcyA. The SPR studies with CLasTcyA revealed significantly higher binding affinities for pimozide and clidinium (Kd, 2.73 nM and 70 nM, respectively) as compared to cystine (Kd, 1.26 µM). The higher binding affinities could be attributed to significantly increased number of interactions in the binding pocket as evident from the crystal structures of CLasTcyA in complex with pimozide and clidinium as compared to cystine. The CLasTcyA possess relatively large binding pocket where bulkier inhibitors fit quite well. In planta studies, carried out to assess the effect of inhibitors on HLB infected Mosambi plants, showed significant reduction in CLas titre in plants treated with inhibitors as compared to control plants. The results showed that pimozide exhibited higher efficiency as compared to clidinium in reducing CLas titre in treated plants. Our results showed that the inhibitor development against critical proteins like CLasTcyA can be an important strategy in management of HLB.

Arsenophonus Interacts with Buchnera to Improve Growth Performance of Aphids under Amino Acid Stress.

Amino acids play a crucial role in the growth and development of insects. Aphids cannot ingest enough amino acids in plant phloem to meet their requirements, and therefore, they are mainly dependent on the obligate symbiont Buchnera aphidicola to synthesize essential amino acids. Besides Buchnera, aphids may harbor another facultative symbiont, Arsenophonus, which alters the requirement of the cotton-melon aphid Aphis gossypii for amino acid. However, it is unclear how Arsenophonus regulates the requirement. Here, we found that Arsenophonus ameliorated growth performance of A. gossypii on an amino acid-deficient diet. A deficiency in lysine (Lys) or methionine (Met) led to changes in the abundance of Arsenophonus. Arsenophonus suppressed the abundance of Buchnera when aphids were fed a normal amino acid diet, but this suppression was eliminated or reversed when aphids were on a Lys- or Met-deficient diet. The relative abundance of Arsenophonus was positively correlated with that of Buchnera, but neither of them was correlated with the body weight of aphids. The relative expression levels of Lys and Met synthase genes of Buchnera were affected by the interaction between Arsenophonus infections and Buchnera abundance, especially in aphids reared on a Lys- or Met-deficient diet. Arsenophonus coexisted with Buchnera in bacteriocytes, which strengthens the interaction. IMPORTANCE The obligate symbiont Buchnera can synthesize amino acids for aphids. In this study, we found that a facultative symbiont, Arsenophonus, can help improve aphids' growth performance under amino acid deficiency stress by changing the relative abundance of Buchnera and the expression levels of amino acid synthase genes. This study highlights the interaction between Arsenophonus and Buchnera to ameliorate aphid growth under amino acid stress.

The genome of Candidatus phytoplasma ziziphi provides insights into their biological characteristics

Phytoplasmas are obligate cell wall-less prokaryotic bacteria that primarily multiply in plant phloem tissue. Jujube witches’ broom (JWB) associated with phytoplasma is a destructive disease of jujube (Ziziphus jujuba Mill.). Here we report the complete ‘Candidatus Phytoplasma ziziphi’ chromosome of strain Hebei-2018, which is a circular genome of 764,108-base pairs with 735 predicted CDS. Notably, extra 19,825 bp (from 621,995 to 641,819 bp) compared to the previously reported one complements the genes involved in glycolysis, such as pdhA, pdhB, pdhC, pdhD, ackA, pduL and LDH. The synonymous codon usage bias (CUB) patterns by using comparative genomics analysis among the 9 phytoplasmas were similar for most codons. The ENc-GC3s analysis among the 9 phytoplasmas showed a greater effect under the selection on the CUBs of phytoplasmas genes than mutation and other factors. The genome exhibited a strongly reduced ability in metabolic synthesis, while the genes encoding transporter systems were well developed. The genes involved in sec-dependent protein translocation system were also identified.The expressions of nine FtsHs encoding membrane associated ATP-dependent Zn proteases and Mn-SodA with redox capacity in the Ca. P. ziziphi was positively correlated with the phytoplasma concentration. Taken together, the genome will not only expand the number of phytoplasma species and provide some new information about Ca. P. ziziphi, but also contribute to exploring its pathogenic mechanism.

Transcriptomic alterations in the sweet orange vasculature correlate with growth repression induced by a variant of citrus tristeza virus.

Citrus tristeza virus (CTV, family Closteroviridae) is an economically important pathogen of citrus. CTV resides in the phloem of the infected plants and induces a range of disease phenotypes, including stem pitting and quick decline as well as a number of other deleterious syndromes. To uncover the biological processes underlying the poorly understood damaging symptoms of CTV, we profiled the transcriptome of sweet orange (Citrus sinensis) phloem-rich bark tissues of non-infected, mock-inoculated trees and trees singly infected with two distinct variants of CTV, T36 or T68-1. The T36 and T68-1 variants accumulated in the infected plants at similar titers. With that, young trees infected with T68-1 were markedly repressed in growth, while the growth rate of the trees infected with T36 was comparable to the mock-inoculated trees. Only a small number of differentially expressed genes (DEGs) were identified in the nearly asymptomatic T36-infected trees, whereas almost fourfold the number of DEGs were identified with the growth-restricting T68-1 infection. DEGs were validated using quantitative reverse transcription-PCR. While T36 did not induce many noteworthy changes, T68-1 altered the expression of numerous host mRNAs encoding proteins within significant biological pathways, including immunity and stress response proteins, papain-like cysteine proteases (PLCPs), cell-wall modifying enzymes, vascular development proteins and others. The transcriptomic alterations in the T68-1-infected trees, in particular, the strong and persistent increase in the expression levels of PLCPs, appear to contribute to the observed stem growth repression. On the other hand, analysis of the viral small interfering RNAs revealed that the host RNA silencing-based response to the infection by T36 and that by T68-1 was comparable, and thus, the induction of this antiviral mechanism may not contribute to the difference in the observed symptoms. The DEGs identified in this study promote our understanding of the underlying mechanisms of the yet unexplained growth repression induced by severe CTV isolates in sweet orange trees.