Asian Citrus Psyllid Diaphorina Citri Research Articles

The Asian Citrus Psyllid (<i>Diaphorina citri</i>) in Africa: using MaxEnt to predict current and future climatic suitability, with a focus on potential invasion routes

The Asian Citrus Psyllid (ACP) (Diaphorina citri Kuwayama, 1908) (Hemiptera: Psyllidae) is a major citrus pest. The species has been introduced to West and East Africa, but has not yet spread to southern Africa, where it could have a devastating impact on citrus farming and livelihoods. A proactive response is key to mitigating the species’ impacts, particularly the ongoing monitoring of potential invasion routes and entry points into South Africa. Species distribution models (SDMs) were developed under current and future climates for ACP in Africa, and these models were used to (1) determine where the species likely poses a threat, (2) identify potential invasion routes into South Africa, and (3) assess how these factors will be affected under climate change. The SDMs indicated that there is an almost contiguous band of suitable climate along the east coast of Africa that joins the species’ current range in East Africa to South Africa, and under aggressive climate change a potential route of invasion through Namibia and Botswana. Much of South Africa is climatically suitable for the species, but under climate change, climatically suitable areas are likely to shift further inland. The spread of ACP into South Africa is unlikely to be prevented, but the outputs of the present models will inform monitoring activities and assist with preparations to respond to this predicted biological invasion.

The Asian Citrus Psyllid (<i>Diaphorina citri</i>) in Africa: using MaxEnt to predict current and future climatic suitability, with a focus on potential invasion routes

The Asian Citrus Psyllid (ACP) (Diaphorina citri Kuwayama, 1908) (Hemiptera: Psyllidae) is a major citrus pest. The species has been introduced to West and East Africa, but has not yet spread to southern Africa, where it could have a devastating impact on citrus farming and livelihoods. A proactive response is key to mitigating the species’ impacts, particularly the ongoing monitoring of potential invasion routes and entry points into South Africa. Species distribution models (SDMs) were developed under current and future climates for ACP in Africa, and these models were used to (1) determine where the species likely poses a threat, (2) identify potential invasion routes into South Africa, and (3) assess how these factors will be affected under climate change. The SDMs indicated that there is an almost contiguous band of suitable climate along the east coast of Africa that joins the species’ current range in East Africa to South Africa, and under aggressive climate change a potential route of invasion through Namibia and Botswana. Much of South Africa is climatically suitable for the species, but under climate change, climatically suitable areas are likely to shift further inland. The spread of ACP into South Africa is unlikely to be prevented, but the outputs of the present models will inform monitoring activities and assist with preparations to respond to this predicted biological invasion.

Potent and selective ‘genetic zipper’ method for DNA-programmable plant protection: innovative oligonucleotide insecticides against Trioza alacris Flor

Chemical insecticides increased the chemical burden on natural ecosystems posing environmental health risk factor. The urgent need for a more sustainable and ecological approach has produced many innovative ideas, including eco-friendly ‘genetic zipper’ method (or CUAD platform) based on contact oligonucleotide insecticides. Oligonucleotide insecticides have enjoyed success recently on many sternorrhynchans showing highly adaptable structure for distinct insect pest species and selective mode of action. In this article, we describe the efficiency of the oligonucleotide insecticides (briefly, olinscides or DNA insecticides) Alacris-11 and Laura-11, as well as their combined use in mixture (1:1), designed for control of bay sucker (Trioza alacris Flor), an important psyllid pest of noble laurel (Laurus nobilis L.). These olinscides are based on short unmodified antisense DNA oligonucleotides that target ITS2 between 5.8S rRNA and 28S rRNA in pre-rRNA (Laura-11) and 28S rRNA region in mature 28S rRNA and pre-rRNA (Alacris-11). The maximum pest mortality, observed on the 14th day of the experiment, comprised 95.01 ± 4.42% for Alacris-11, 97.16 ± 2.48% for Laura-11, and 98.72 ± 1.14% for their mixture (1:1). The control oligonucleotide CTGA-11 did not cause any significant mortality (9.38 ± 0.57%), emphasizing selectivity in the action of oligonucleotide insecticides. The results show potent and specific nature of oligonucleotide insecticides for pest control and open up new frontiers in control of economically important psyllids in agriculture and forestry, including Asian citrus psyllid (Diaphorina citri Kuwayama) and many others. Scientists can easily adopt ‘genetic zipper’ method for plethora of insect pests because DNA is a programmable molecule and provides game-changing characteristics for plant protection.Graphical

Agroecological Alternatives for Pest and Disease Management in Mexican Lime [Citrus aurantifolia (Christm.) Swingle] Cultivation

agroecological alternatives for the integrated management of recurrent pests in Mexican lime (Citrus aurantifolia); as well as to establish possible synergies with the commercial bioinsecticide. Design/methodology/approach: This experiment was conducted in two phases: 1) visual monitoring and pre-identification of pests and fungal diseases in the Mexican lime crop, and 2) application and evaluation of the effect of application of commercial bioinsecticide (Biocanela) and mineral broths (bordeaux broth and sulfocalcium broth) alone and mixtures. Results: The pests with highest incidence were thrips (Pezothrips kellynus), Asian citrus psyllid (Diaphorina citri), whitefly (Bemisia tabaci), fruit fly (Drosophila melanogaster), and red spider mite (Tetranychus urticae), while the main fungal diseases were sooty mold (Capnodium citri), red algae (Cephaleuros virescens), citrus greasy spot (Mycosphaerella citri), and anthracnose (Colletotrichum acutatum). The mixture of mineral broths with the bioinsecticide Biocanela showed the highest fungicide and insecticide activity, and repellency on various pests and diseases of the Mexican lemon crop. Limitations on study/implications: Generate scientific knowledge regarding to the best agroecological alternatives for the integrated management of citrus pests and diseases that are economically and environmentally profitable. Findings/conclusions: Application of the mixture of sulfocalcium broth and Biocanela showed repellency effectiveness against pests such as thrips (Pezothrips kellyanus), Asian citrus psyllid (Diaphorina citri), whitefly (Bemisia tabaci), and fruit fly (Drosophila melanogaster). Likewise, it was effective against sooty mold (Capnodium citri), red algae (Cephaleuros virescens), and citrus greasy spot (Mycosphaerella citri), while the effect was lighter against anthracnose (Colletotrichum acutatum). Additionally, it induces new, healthy shoots in Mexican lemon trees.

Occurrence and unlimited spread of Diaphorina citri Kuwayama (Hemiptera: Liviidae) in Tanzania: A potential economic threat to the citrus industry

Citrus production is a cornerstone of Tanzania's agricultural sector, supporting economic development and food security. However, the industry faces a critical threat from the Asian citrus psyllid (Diaphorina citri Kuwayama), the primary vector of the destructive Huanglongbing (HLB) disease. This study investigates the distribution, abundance, and environmental drivers of D. citri and the African citrus triozid (Trioza erytreae) in major citrus-growing regions of Tanzania. Surveys conducted between March and May 2023 revealed the presence of D. citri in the Tanga and Morogoro regions, including the first documented occurrence in the Tanga region. Conversely, T. erytrae was confined to the Arusha region. Altitude emerged as a significant factor influencing psyllid abundance. D. citri populations decreased with increasing altitude, while T. erytrae exhibited the opposite trend. Temperature also showed correlations with psyllid populations, though additional research is needed to fully elucidate these relationships. Citrus variety significantly influenced D. citri abundance, with Late Valencia (Kitenesi) being the most susceptible. Importantly, several varieties showed no presence of D. citri, offering potential sources of resistance. The intensity of new flush growth was strongly correlated with psyllid abundance. These findings highlight the urgent need for region-specific and altitude-tailored citrus pest management strategies. The identification of potentially HLB-resistant citrus varieties provides a promising avenue for breeding programs. This research offers critical insights to protect Tanzania's citrus industry and ensure the sustainable production of this vital economic crop. Keywords: Citrus, Citrus greening, Diaphorina citri Kuwayama, Huanglongbing, Trioza erytreae, Distribution, Altitude, Tanzania

Differential expression of “Candidatus Liberibacter solanacearum” genes and prophage loci in different life stages of potato psyllid

Psyllid species, including the potato psyllid (PoP) Bactericera cockerelli (Sulc) (Triozidae) serve as host and vector of “Candidatus Liberibacter spp.” (“Ca. Liberibacter”), which also infects diverse plant hosts, including citrus and tomato. Psyllid transmission of “Ca. Liberibacter” is circulative and propagative. The time of “Ca. Liberibacter” acquisition and therefore vector life stage most competent for bacterial transmission varies by pathosystems. Here, the potato psyllid-“Ca. Liberibacter solanacearum” (CLso) pathosystem was investigated to dissect CLso-prophage interactions in the tomato plant and PoP-psyllid host by real-time quantitative reverse transcriptase amplification of CLso genes/loci with predicted involvement in host infection and psyllid-CLso transmission. Genes/loci analyzed were associated with (1) CLso-adhesion, -invasion, -pathogenicity, and -motility, (2) prophage-adhesion and pathogenicity, and (3) CLso-lysogenic cycle. Relative gene expression was quantified by qRT-PCR amplification from total RNA isolated from CLso-infected 1st–2nd and 4th–5th nymphs and teneral adults and CLso-infected tomato plants in which CLso infection is thought to occur without SC1-SC2 replication. Gene/loci expression was host-dependent and varied with the psyllid developmental stage. Loci previously associated with repressor-anti-repressor regulation in the “Ca Liberibacter asiaticus”-prophage pathosystem, which maintains the lysogenic cycle in Asian citrus psyllid Diaphorina citri, were expressed in CLso-infected psyllids but not in CLso-infected tomato plants.

A limited concentration range of diaphorin, a polyketide produced by a bacterial symbiont of the Asian citrus psyllid, promotes the in vitro gene expression with bacterial ribosomes.

Diaphorin is a polyketide produced by "Candidatus Profftella armatura" (Gammaproteobacteria: Burkholderiales), an obligate symbiont of a devastating agricultural pest, the Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae). Physiological concentrations of diaphorin, which D. citri contains at levels as high as 2-20 mM, are inhibitory to various eukaryotes and Bacillus subtilis (Firmicutes: Bacilli) but promote the growth and metabolic activity of Escherichia coli (Gammaproteobacteria: Enterobacterales). Our previous study demonstrated that 5-mM diaphorin, which exhibits significant inhibitory and promoting effects on cultured B. subtilis and E. coli, respectively, inhibits in vitro gene expression utilizing purified B. subtilis and E. coli ribosomes. This suggested that the adverse effects of diaphorin on B. subtilis are partly due to its influence on gene expression. However, the result appeared inconsistent with the positive impact on E. coli. Moreover, the diaphorin concentration in bacterial cells, where genes are expressed in vivo, may be lower than in culture media. Therefore, the present study analyzed the effects of 50 and 500 µM of diaphorin on bacterial gene expression using the same analytical method. The result revealed that this concentration range of diaphorin, in contrast to 5-mM diaphorin, promotes the in vitro translation with the B. subtilis and E. coli ribosomes, suggesting that the positive effects of diaphorin on E. coli are due to its direct effects on translation. This study demonstrated for the first time that a pederin-type compound promotes gene expression, establishing a basis for utilizing its potential in pest management and industrial applications.IMPORTANCEThis study revealed that a limited concentration range of diaphorin, a secondary metabolite produced by a bacterial symbiont of an agricultural pest, promotes cell-free gene expression utilizing substrates and proteins purified from bacteria. The unique property of diaphorin, which is inhibitory to various eukaryotes and Bacillus subtilis but promotes the growth and metabolic activity of Escherichia coli, may affect the microbial flora of the pest insect, potentially influencing the transmission of devastating plant pathogens. Moreover, the activity may be exploited to improve the efficacy of industrial production by E. coli, which is often used to produce various important materials, including pharmaceuticals, enzymes, amino acids, and biofuels. This study elucidated a part of the mechanism by which the unique activity of diaphorin is expressed, constructing a foundation for applying the distinct property to pest management and industrial use.

The beta pore-forming bacterial pesticidal protein Tpp78Aa1 is toxic to the Asian citrus psyllid vector of the citrus greening bacterium

The Asian citrus psyllid (ACP) Diaphorina citri transmits the causative agent of huanglongbing, or citrus greening disease, that has decimated global citrus production. Pesticidal proteins derived from bacteria such as Bacillus thuringiensis (Bt) can provide effective and environmentally friendly alternatives for management of D. citri, but few with sufficient toxicity to D. citri have been identified. Here, we report on the toxicity of 14 Bt-derived pesticidal proteins from five different structural groups against D. citri. These proteins were selected based on previously reported toxicity to other hemipteran species and on pesticidal protein availability. Most of the proteins were expressed in Escherichia coli and purified from inclusion bodies or His-tag affinity purification, while App6Aa2 was expressed in Bt and purified from spore/crystal mixtures. Pesticidal proteins were initially screened by feeding psyllids on a single dose, and lethal concentration (LC50) then determined for proteins with significantly greater mortality than the buffer control. The impact of CLas infection of D. citri on toxicity was assessed for selected proteins via topical feeding. The Bt protein Tpp78Aa1 was toxic to D. citri adults with an LC50 of approximately 204 µg/mL. Nymphs were more susceptible to Tpp78Aa1 than adults but no significant difference in susceptibility was observed between healthy and CLas-infected nymphs or adults. Tpp78Aa1 and other reported D. citri-active proteins may provide valuable tools for suppression of D. citri populations.

Risk of spread of the Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae) in Ghana.

The impact of invasive species on biodiversity, food security and economy is increasingly noticeable in various regions of the globe as a consequence of climate change. Yet, there is limited research on how climate change affects the distribution of the invasive Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera:Liviidae) in Ghana. Using maxnet package to fit the Maxent model in R software, we answered the following questions; (i) what are the main drivers for D. citri distribution, (ii) what are the D. citri-specific habitat requirements and (iii) how well do the risk maps fit with what we know to be correctly based on the available evidence?. We found that temperature seasonality (Bio04), mean temperature of warmest quarter (Bio10), precipitation of driest quarter (Bio17), moderate resolution imaging spectroradiometer land cover and precipitation seasonality (Bio15), were the most important drivers of D. citri distribution. The results follow the known distribution records of the pest with potential expansion of habitat suitability in the future. Because many invasive species, including D. citri, can adapt to the changing climates, our findings can serve as a guide for surveillance, tracking and prevention of D. citri spread in Ghana.

Comparison of Insecticide Sprays in Alternate Rows and in All Rows on Asian Citrus Psyllid Control and Huanglongbing Progress in Sweet Orange Orchards.

One strategy to reduce huanglongbing (HLB) is controlling its insect vector, the Asian citrus psyllid (ACP) Diaphorina citri, by preventive insecticide sprays. The recommendation is to spray insecticide in all rows (conventional spray [CONV]), but some growers empirically spray in alternate rows (ALT) to increase the spray frequency without increasing the operating cost. Therefore, this work compared the effect of ALT with CONV on the ACP population and HLB incidence. The spray deposition (amount of metallic copper per leaf area), coverage (percentage of water-sensitive paper area covered by spray), and efficacy (ACP mortality) of each treatment were also evaluated on both sides of the trees. Two field trials were performed: Trial #1 compared ALT every 7 days (ALT7) with CONV every 14 days (CONV14), and trial #2 compared different spray frequencies of ALT with CONV every 7 days (CONV7). In trial #1, no differences were observed in the ACP population or HLB progress between ALT7 and CONV14 after 5 years. In trial #2, ALT7 presented the highest percentage of ACP and cumulative HLB incidence than CONV7 and ALT every 3 to 4 days, after 2 years. Hence, when the frequency of ALT was half the frequency of CONV, similar results were observed. Spray deposition, coverage, and efficacy were similar between tree sides in CONV, but they were uneven in ALT, resulting in higher values on the tree side that directly received the spray. Insecticide spray should be performed with the frequency enough to keep new shoots protected during their growth.

The Amino Acid Transporter PtCAT7 and Ammonium Nutrition Enhance the Uptake of Thiamethoxam in Citrus Rootstock Seedlings.

Thiamethoxam (THX), when applied to the soil, can be taken up by citrus roots and subsequently transported to the leaves, providing effective protection of plants against the Asian citrus psyllid (Diaphorina citri Kuwayama). In this study, the field experiments showed that the coapplication of THX and nitrogen fertilizer (AN) did not affect THX uptake in six-year-old citrus plants. However, their coapplication promoted THX uptake in three-year-old Potassium trifoliate rootstocks and relieved the inhibition of AN at a higher level on plant growth characteristics, including biomass and growth of root and stem. RNA-seq analysis found that THX induced upregulation of a cationic amino acid transporter (PtCAT7) in citrus leaves. PtCAT7 facilitated THX uptake in the yeast strain to inhibit its growth, and the PtCAT7 protein was localized on the plasma membrane. Our results demonstrate that THX and N fertilizer can be coapplied and PtCAT7 may be involved in THX uptake in citrus.

Spider venom neurotoxin based bioinsecticides: A novel bioactive for the control of the Asian citrus psyllid Diaphorina citri (Hemiptera)

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is a key vector of the phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas) associated with huanglongbing (HLB), the most serious and currently incurable disease of citrus worldwide. Here we report the first investigation into the potential use of a spider venom-derived recombinant neurotoxin, ω/κ-HxTx-Hv1h (hereafter HxTx-Hv1h) when delivered alone or when fused to snowdrop lectin (Galanthus nivalis agglutinin; GNA) to control D. citri. Proteins, including GNA alone, were purified from fermented transformed yeast Pichia pastoris cultures. Recombinant HxTx-Hv1h, HxTx-Hv1h/GNA and GNA were all orally toxic to D. citri, with Day 5 median lethal concentrations (LC50) derived from dose-response artificial diet assays of 27, 20 and 52 μM, respectively. Western analysis of whole insect protein extracts confirmed that psyllid mortality was attributable to protein ingestion and that the fusion protein was stable to cleavage by D. citri proteases. When applied topically (either via droplet or spray) HxTx-Hv1h/GNA was the most effective of the proteins causing >70 % mortality 5 days post treatment, some 2 to 3-fold higher levels of mortality as compared to the toxin alone. By contrast, no significant mortality or phenotypic effects were observed for bumble bees (Bombus terrestris L.) fed on the recombinant proteins in acute toxicity assays. This suggests that HxTx-Hv1h/GNA has potential as a novel bioinsecticide for the management of D. citri offering both enhanced target specificity as compared to chemical pesticides and compatibility with integrated pest management (IPM) strategies.

Association of Non-host Crop Plants with Mandarin in Host Location and Survival of Diaphorina citri Kuwayama (Hemiptera: Psyllidae).

Research efforts have been made to develop novel tactics, such as those targeting behavioral control, for management of the Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae), vector of the causal agent of citrus Huanglongbing. Here, we investigated whether association of "Ponkan" mandarin (Citrus reticulata) with volatiles from non-host crops: avocado, passion fruit or coffee, alters host location by the Asian citrus psyllid; and whether they can be temporary hosts for the Asian citrus psyllid. In wind tunnel assays, we found that the association of mandarin seedling with avocado plant volatiles reduced in 30% the number of psyllids sitting on host plants compared to the mandarin alone. In contrast, passion fruit plant volatiles facilitated host location by psyllids, which found mandarin seedlings faster than when exposed to mandarin alone. The association with coffee volatiles did not alter the attractiveness of mandarin to the Asian citrus psyllid. Survival and half-lethal time (LT50) of D. citri fed on non-host plants were longer than those insects with water only, but shorter than those fed on mandarin. Among the non-host plants, D. citri performed better in coffee, followed by avocado and passion fruit plants. Our results indicate that the association of mandarin with avocado plant can be beneficial for Asian citrus psyllid management.

Effect of Individual Protective Covers on Young ‘Valencia’ Orange (Citrus sinensis) Tree Physiology

Huanglongbing (HLB), an important citrus disease, causes many physiological and anatomical changes such as phloem dysfunction, imbalance in carbohydrate partitioning, decrease in leaf chlorophyll, and nutritional imbalances in the affected trees, ultimately resulting in tree decline. In Florida, HLB is associated with phloem-limited bacteria Candidatus Liberibacter asiaticus (CLas), and it is vectored by the Asian citrus psyllid (Diaphorina citri). No cure for HLB has been found, and most of the HLB management efforts have been focused on vector control or exclusion, improved nutrient management, and the use of HLB-tolerant rootstocks. Individual protective covers (IPCs) are a type of psyllid exclusion tool that is increasingly used by growers for HLB management of newly planted citrus trees. However, no studies have evaluated their influence on citrus tree physiology. This study investigated the effect of IPCs and different rates of insecticides on CLas infection and different physiological attributes, including soluble (glucose, fructose, and sucrose) and nonsoluble (starch) carbohydrates, leaf chlorophyll, and leaf macronutrients and micronutrients over 2.5 years of field growth. The treatments (tree cover and insecticides rate) were applied in newly planted ‘Valencia’ sweet orange (Citrus sinensis) trees grafted on ‘Cleopatra’ (C. reticulata) rootstock. The IPCs prevented CLas transmission and accumulation of foliar starch, sucrose, and glucose commonly associated with HLB. IPC-covered trees had more leaf chlorophyll-a and chlorophyll-b than noncovered trees and more leaf nitrogen (N) and zinc (Zn). Our findings suggest that IPCs effectively prevent CLas infection and maintain the physiological health of young citrus trees under heavy HLB pressure. Therefore, IPCs are recommended as an important component of integrated pest management for this devastating disease.

Fixed Spraying Systems Application in Citrus Orchards: Nozzle Type and Nozzle Position Effects on Droplet Deposition and Pest Control

Pesticide application is an essential means of controlling plant diseases and pests in citrus orchards. In recent years, fixed spraying systems have gradually been used as alternatives to traditional sprayers and manual sprayers in some hilly citrus orchards. In this paper, influences of fixed system spraying parameters, such as droplet size and spraying height, on spraying quality were elucidated and analyzed. The performances of two nozzle types, pressure-swirl nozzles and fixed spray plate sprinklers, were assessed and compared by effective droplet coverage ratio (DCR), droplet distribution uniformity coefficient of variation (CV), and droplet penetration ratio (DPR). The results showed that appropriately increasing droplet size and spraying height could improve the DCR and distribution uniformity of pressure-swirl nozzles. The DCR and distribution uniformity of fixed spray plate sprinklers had a positive correlation with droplet size, while spraying height had no significant effect on these variables. Additionally, with the increase in droplet size, DPR initially increased and then gradually decreased. The optimized results showed that the optimal parameters for pressure-swirl nozzles were a droplet size of 240 μm and spraying height of 100 cm, while for fixed spray plate sprinklers, the results were a droplet size of 240 μm and spraying height of 50 cm. Comparison results showed that the spraying quality of fixed spray plate sprinklers was better overall, with values of DCR, CV, and DPR being 37.15%, 24.20%, and 71.67%, respectively, while the corresponding values for pressure-swirl nozzles were 39.65%, 35.41%, and 56.02%. Based on the above results and the occurrence rule of citrus pests and disease, the optimal spraying parameters of fixed spraying systems were selected to control the Asian citrus psyllid Diaphorina citri. Furthermore, the effect of fixed spraying systems on controlling Diaphorina citri reached the maximum at 3 days after spraying, which was 97.83%, and the effect declined at 14 days after spraying, which was 85.47%. This study provides valuable scientific references for guiding the application of fixed spraying systems in hilly citrus orchards.

Diaphorin, a polyketide produced by a bacterial endosymbiont of the Asian citrus psyllid, adversely affects the in vitro gene expression with ribosomes from Escherichia coli and Bacillus subtilis.

Diaphorin is a polyketide produced by "Candidatus Profftella armatura" (Gammaproteobacteria), an obligate mutualist of an important agricultural pest, the Asian citrus psyllid Diaphorina citri (Hemiptera). Our previous study demonstrated that diaphorin, at physiological concentrations in D. citri, inhibits the growth and cell division of Bacillus subtilis (Firmicutes) but promotes the growth and metabolic activity of Escherichia coli (Gammaproteobacteria). This unique property of diaphorin may aid microbial mutualism in D. citri, potentially affecting the transmission of "Candidatus Liberibacter spp." (Alphaproteobacteria), the pathogens of the most destructive citrus disease Huanglongbing. Moreover, this property may be exploited to promote microbes' efficiency in producing industrial materials. However, the mechanism underlying this activity is unknown. Diaphorin belongs to the family of pederin-type compounds, which inhibit protein synthesis in eukaryotes by binding to eukaryotic ribosomes. Therefore, as a first step to assess diaphorin's direct influence on bacterial gene expression, this study examined the effect of diaphorin on the in vitro translation using ribosomes of B. subtilis and E. coli, quantifying the production of the green fluorescent protein. The results showed that the gene expression involving B. subtilis and E. coli ribosomes along with five millimolar diaphorin was 29.6% and 13.1%, respectively, less active than the control. This suggests that the diaphorin's adverse effects on B. subtilis are attributed to, at least partly, its inhibitory effects on gene expression. Moreover, as ingredients of the translation system were common other than ribosomes, the greater inhibitory effects observed with the B. subtilis ribosome imply that the ribosome is among the potential targets of diaphorin. On the other hand, the results also imply that diaphorin's positive effects on E. coli are due to targets other than the core machinery of transcription and translation. This study demonstrated for the first time that a pederin congener affects bacterial gene expression.

Effects of Candidatus Liberibacter asiaticus infection on metagenome of Diaphorina citri gut endosymbiont

Asian citrus psyllid (Diaphorina citri, D. citri) is the important vector of “Candidatus Liberibacter asiaticus” (CLas), associated with Huanglongbing, the most devastating citrus disease worldwide. CLas can affect endosymbiont abundance of D. citri. Here, we generated the high-quality gut endosymbiont metagenomes of Diaphorina citri on the condition of CLas infected and uninfected. The dataset comprised 6616.74 M and 6586.04 M raw reads, on overage, from CLas uninfected and infected psyllid strains, respectively. Taxonomic analysis revealed that a total of 1046 species were annotated with 10 Archaea, 733 Bacteria, 234 Eukaryota, and 69 Viruses. 80 unique genera in CLas infected D. citri were identified. DIAMOND software was used for complement function research against various functional databases, including Nr, KEGG, eggNOG, and CAZy, which annotated 84543 protein-coding genes. These datasets provided an avenue for further study of the interaction mechanism between CLas and D. citri.

Bacterial Pesticidal Protein Mpp51Aa1 Delivered via Transgenic Citrus Severely Impacts the Fecundity of Asian Citrus Psyllid, Diaphorina citri.

The Asian citrus psyllid (ACP) Diaphorina citri vectors the causative agent of citrus greening disease that has the capacity to decimate citrus production. As an alternative and more sustainable approach to manage D. citri than repeated application of chemical insecticides, we investigated the potential use of the bacteria-derived pesticidal protein, Mpp51Aa1, when delivered by transgenic Citrus sinensis cv. Valencia sweet orange or Citrus paradisi cv. Duncan grapefruit. Following confirmation of transcription and translation of mpp51aa1 by transgenic plants, no impact of Mpp51Aa1 expression was seen on D. citri host plant choice between transgenic and control Duncan grapefruit plants. A slight but significant drop in survival of adult psyllids fed on these transgenic plants was noted relative to those fed on control plants. In line with this result, damage to the gut epithelium consistent with that caused by pore-forming proteins was only observed in a minority of adult D. citri fed on the transgenic Duncan grapefruit. However, greater impacts were observed on nymphs than on adults, with a 40% drop in the survival of nymphs fed on transgenic Duncan grapefruit relative to those fed on control plants. For Valencia sweet orange, a 70% decrease in the number of eggs laid by adult D. citri on transgenic plants was noted relative to those on control plants, with a 90% drop in emergence of progeny. These impacts that contrast with those associated with other bacterial pesticidal proteins and the potential for use of Mpp51Aa1-expressing transgenic plants for suppression of D. citri populations are discussed. IMPORTANCE Pesticidal proteins derived from bacteria such as Bacillus thuringiensis are valuable tools for management of agricultural insect pests and provide a sustainable alternative to the application of chemical insecticides. However, relatively few bacterial pesticidal proteins have been used for suppression of hemipteran or sap-sucking insects such as the Asian citrus psyllid, Diaphorina citri. This insect is particularly important as the vector of the causative agent of citrus greening, or huanglongbing disease, which severely impacts global citrus production. In this study, we investigated the potential of transgenic citrus plants that produce the pesticidal protein Mpp51Aa1. While adult psyllid mortality on transgenic plants was modest, the reduced number of eggs laid by exposed adults and the decreased survival of progeny was such that psyllid populations dropped by more than 90%. These results provide valuable insight for potential deployment of Mpp51Aa1 in combination with other control agents for the management of D. citri.

Evaluation of Artemisia absinthium L. Essential Oil as a Potential Novel Prophylactic against the Asian Citrus Psyllid Diaphorina citri Kuwayama

Interest in developing novel crop protectants has increased in the recent decade due to the harmful effects of synthetic pesticides on humans and the environment. Diaphorina citri threatens the citrus industry worldwide and is the primary vector of phloem-limited bacterium (HLB). However, there is no available cure for HLB. Diaphorina citri management mainly depends on the use of synthetic insecticides, but their massive application leads to resistance in pest populations. Therefore, alternative pest management strategies are needed. Our results indicated that fewer D. citri adults settled on plants treated with AAEO than on control 48 h after release. The psyllids fed on citrus leaves treated with AAEO significantly reduced the honeydew production compared to the control. The AAEO showed potent ovicidal activity against the D. citri eggs with LC50 5.88 mg/mL. Furthermore, we also explored the fitness of D. citri on AAEO-treated and untreated Citrus sinensis by using two-sex life table tools. Our results revealed that the intrinsic rate of increase (r) was higher on untreated seedlings (0.10 d−1) than those treated with an LC20 concentration of AAEO (0.07 d−1). Similarly, the net reproductive rate (R0) was higher for untreated seedlings (14.21 offspring) than those treated (6.405 offspring). Furthermore, the AAEO were safer against Aphis mellifera, with LC50 35.05 mg/mL, which is relatively higher than the LC50 24.40 mg/mL values against D. citri. The results indicate that AAEO exhibits toxic and behavioral effects on D. citri, which can be a potential candidate for managing this pest.

Evaluation of Trunk Injection Techniques for Systemic Delivery of Huanglongbing Therapies in Citrus

The bacterial pathogen associated with citrus huanglongbing (HLB) resides in the phloem of affected trees. The widespread abundance of the vector in Florida, the Asian citrus psyllid (Diaphorina citri), and the location of the pathogen in the tree vascular tissue limits the efficacy of foliar-applied therapies. Trunk injection is a crop protection strategy that applies therapeutic compounds directly into the tree vascular system, enabling their systemic distribution within the tree. However, limited information is available on the most effective methodology for implementing trunk injection at the commercial scale and the extent of damage inflicted by the injection. In this study, 5-year-old HLB-affected ‘Midsweet’ sweet orange (Citrus sinensis) trees were injected with the insecticide imidacloprid, the antibacterial oxytetracycline, or water. Injections occurred in Jun and Oct 2020 using three trunk injection techniques. Trees were monitored for external wounding and internal damage associated with injection, as well as tree health, bacterial titers, and yield for two production seasons. Low-pressure injection caused the least damage; however, it was less effective at delivering the tested compounds than medium- or high-pressure injection. Despite causing the greatest extent of external and internal damage at the injection site, injection of oxytetracycline significantly improved tree health, reduced bacterial titers, and increased yield in the two seasons of this study. Imidacloprid injection caused less wound damage but did not result in any lasting benefits to the trees. These results suggest that trunk injection of oxytetracycline could be an effective strategy for managing HLB and that the damage inflicted by this crop protection strategy can be reduced by selecting a suitable injection technique.