Diaphorina Citri Kuwayama Research Articles

Evaluation of sampling methods of Diaphorina citri Kuwayama (Hemiptera: Liviidae) in two citrus growing areas

Efficient, easy to implement and low-cost monitoring methodologies are necessary to obtain information on arthropod pest populations and to implement the most convenient and timely phytosanitary control practices. To optimize the sampling and monitoring of the Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), an insect associated with the transmission of Candidatus Liberibacter asiaticus and Ca. L. americanus, which cause Huanglongbing (HLB), a disease with a great impact on citrus orchards, three methods of adult sampling (yellow sticky traps, sweep net, and stem tap) were evaluated. For immature sampling, vegetative shoots were checked. The results demonstrate that the population density at the time of sampling affects the effectiveness and sensitivity of the sampling methods. Yellow sticky traps capture more adults and are the only effective method at low psyllid densities. Stem tap and sweep net are less expensive methods; however, they do not detect adults nor correlate with the number of nymphs and eggs in vegetative shoots when adult density is low. For adults, an optimal sample size was determined for each method. For yellow sticky traps, 3 to 5 traps for a 2-hectare plot with weekly frequency are recommended. For immatures, it is recommended to estimate the percentage of infestation by inspecting 45 to 55 vegetative shoots well-distributed within a 2-hectare plot, as a practical measure for farmers and extensionists to monitor D. citri.

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

Climate covariate selection influences MaxEnt model predictions and predictive accuracy under current and future climates

The performance and transferability of species distribution models (SDMs) depends on a number of ecological, biological, and methodological factors. There is a growing body of literature that explores how the choice of climate covariate combinations and model parameters can affect predictive performance, but relatively few that delve into covariate reduction methods and the optimisation of model parameters, and the resulting spatial and temporal transferability of those models. The present work used the citrus pest, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), to illustrate how MaxEnt models trained on the insect’s native range in Asia varied in their predictions of climatic suitability across the introduced range when eight different covariate reduction methods were applied during model building. Additionally, it showed how model sensitivity varied across these different covariate combinations using three sets of independently validated occurrence points in the invaded range of the psyllid. Climatically suitable areas for D. citri differed by as much as two-fold between the best and worst-performing models in selected areas. Great care should be taken in the selection of the highest-performing predictor combinations and model parameter settings for SDMs, particularly in the case of invasive species where the assumption of environmental equilibrium is likely violated in the introduced range. Understanding how the predictive ability of SDMs can be influenced by the methodological choices made during the model building phase is vital to ensuring that ecological and invasion management programmes do not over- or underestimate climatic suitability and subsequent invasion risk.

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

Repellency, Toxicity, and Chemical Composition of Plant Essential Oils from Myrtaceae against Asian Citrus Psyllid, Diaphorina citri Kuwayama (Hemiptera Liviidae).

Diaphorina citri Kuwayama (D. citri) is one of the major pests in the citrus industry, which spreads Citrus Huanglongbing disease. It has developed resistance to chemical insecticides. Therefore, searching for greener solutions for pest management is critically important. The main aim of this study was to evaluate the repellent and insecticidal efficacy of essential oils (EOs) from four species of Myrtaceae plants: Psidium guajava (PG), Eucalyptus robusta (ER), Eucalyptus tereticornis (ET), and Baeckea frutescens (BF) against D. citri and to analyze their chemical compositions. GC-MS analysis was performed, and the results indicated that the EOs of PG, ER, ET, and BF were rich in terpenoids, ketones, esters, and alcohol compounds. The repellent rate of all four EOs showed that it decreased with exposure time but increased with the concentration of EOs from 80.50% to 100.00% after treating D. citri for 6 h with four EOs at 100% concentration and decreased to 67.71% to 85.49% after 24 h of exposure. Among the compounds from the EOs tested, eucalyptol had the strongest repellent activity, with a 24 h repellency rate of 100%. The contact toxicity bioassay results showed that all EOs have insecticidal toxicity to D. citri; the LC50 for nymphs was 36.47-93.15 mL/L, and for adults, it was 60.72-111.00 mL/L. These results show that when PG is used as the reference material, the ER, ET, and BF EOs have strong biological activity against D. citri, which provides a scientific basis for the further development of plant-derived agrochemicals.

Insecticide Efficacy of Green Synthesis Silver Nanoparticles on Diaphorina citri Kuwayama (Hemiptera: Liviidae).

Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a vector of Liberibacter asiaticus Jagoueix et al. and Liberibacter americanus Teixeira et al., causal agents of the critical yellow dragon disease or Huanglongbing (HLB), which affects citrus production worldwide. Recently, green synthetic nanoparticles have emerged as a potential alternative to control of agricultural insect pests. The insecticide effect of silver nanoparticles (AgNPs) on 2nd instar nymphs of D. citri under laboratory and greenhouse conditions was evaluated. Mortality was recorded 24, 48, and 72 h after application on D. citri nymphs under both laboratory and greenhouse conditions. The laboratory results showed that AgNPs caused 97.84 and 100% mortality at 32 and 64 ppm, respectively, 72 h after treatment. In the greenhouse, AgNPs caused 78.69 and 80.14% mortality using 64 and 128 ppm 72 h after application. This research is the first to evaluate the green synthesis AgNPs on D. citri and are a promising strategy to control the pest.

Fungi associated with the Asian citrus psyllid, Diaphorina citri Kuwayama, in northeastern Mexico

ABSTRACT Diaphorina citri Kuwayama (Asian citrus psyllid, ACP) is a devastating pest due to transmission of Liberibacter spp. to citrus plants. We identified presumed entomopathogenic fungi (EF) naturally associated with field-collected adult ACP, both killed (mycosed) and alive, at 14 field sites in the Gulf Coastal Plain of NE Mexico. On dead ACP, five species of EF (Hypocreales) were observed: Hirsutella citriformis Speare (Ophiocordycipitaceae), Cordyceps javanica (Frieder. & Bally) Kepler, Shrestha & Spatafora, Beauveria cf. bassiana (Bals.) Vuill., and Akanthomyces cf. lecanii (Cordycipitaceae); also, Niveomyces J.P.M. Araújo & C. de Bekker (Cordycipitaceae), a hyperparasite of Ophiocordycipitaceae, in this case H. citriformis. The most prevalent fungi on field ACP were H. citriformis and C. javanica, on dead and on live, incubated insects, respectively. In laboratory bioassays, C. javanica and H. citriformis conidia from cultures killed >60% of ACP adults. H. citriformis, C. javanica, and A. lecanii killed >60% nymphs. Niveomyces was observed only growing on H. citriformis fructifications on field-killed ACP; it was absent from live incubated insects, indicating Niveomyces is a mycoparasite on H. citriformis. Niveomyces cultures (ARSEF 10230) on agar grew as a Pleurodesmospora synanamorph; its LSU rRNA region D1–D2 sequences (Genbank OM780101) were 99.81% identical to Niveomyces sequences. Conidia from Niveomyces cultures were entomopathogenic on ACP nymphs and adults. These are the first reports of Niveomyces associated with H. citriformis; of the in vitro dimorphic growth (synanamorphs) Niveomyces/Pleurodesmospora; and of experimental verification of entomopathogenicity of Niveomyces. Understanding the interactions of these fungi will support sustainable ACP management.

Development of a sterile insect technique as a control strategy for the Asian citrus psyllid: establishing the effect of sterilizing X-rays on fecundity, fertility, and survival.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is a major pest of citrus due to its role as the vector of the bacterium that causes huanglongbing. In commercial citrus, ACP control currently relies on the application of insecticides, which may not be sustainable long-term, nor practical in urban areas. The sterile insect technique (SIT) is an alternative strategy in which large numbers of pests are reared, sterilized using radiation, and then released into the field to compete with wild individuals for matings, suppressing population growth. As a fundamental step toward the development of SIT for ACP, this study sought to identify the optimum radiation dose required to sterilize ACP without affecting their survival and mating capacity. Virgin adult ACP of both sexes were subjected to doses of X-ray irradiation ranging from 40 to 480 Gy, then paired with a nonirradiated mate and allowed to produce offspring. Fecundity was estimated as the number of eggs laid, and fertility as the proportion of those eggs that hatched. Females were more radio-sensitive than males, exhibiting a major drop in fecundity at even the lowest dose and 100% sterility at 80 Gy. In contrast, a fivefold higher dose (400 Gy) did not achieve complete sterility in males, with around 5% offspring survival. However, F1 progeny of males exposed to 320 Gy or higher were subsequently found to be 100% sterile. This confirmation of inherited sterility suggests that balancing the sterilizing effects of radiation against its mortality-inducing effects may warrant further evaluation.

Enterobacteriaceae as a Key Indicator of Huanglongbing Infection in Diaphorina citri.

Extensive microbial interactions occur within insect hosts. However, the interactions between the Huanglongbing (HLB) pathogen and endosymbiotic bacteria within the Asian citrus psyllid (ACP, Diaphorina citri Kuwayama) in wild populations remain elusive. Thus, this study aimed to detect the infection rates of HLB in the ACP across five localities in China, with a widespread prevalence in Ruijin (RJ, 58%), Huidong (HD, 28%), and Lingui (LG, 15%) populations. Next, microbial communities of RJ and LG populations collected from citrus were analyzed via 16S rRNA amplicon sequencing. The results revealed a markedly higher microbial diversity in the RJ population compared to the LG population. Moreover, the PCoA analysis identified significant differences in microbial communities between the two populations. Considering that the inter-population differences of Bray-Curtis dissimilarity in the RJ population exceeded those between populations, separate analyses were performed. Our findings indicated an increased abundance of Enterobacteriaceae in individuals infected with HLB in both populations. Random forest analysis also identified Enterobacteriaceae as a crucial indicator of HLB infection. Furthermore, the phylogenetic analysis suggested a potential regulatory role of ASV4017 in Enterobacteriaceae for ACP, suggesting its possible attractant activity. This research contributes to expanding the understanding of microbial communities associated with HLB infection, holding significant implications for HLB prevention and treatment.

Reduction of Wolbachia in Diaphorina citri (Hemiptera: Liviidae) increases phytopathogen acquisition and decreases fitness.

Wolbachia pipientis is a maternally inherited intracellular bacterium that infects a wide range of arthropods. Wolbachia can have a significant impact on host biology and development, often due to its effects on reproduction. We investigated Wolbachia-mediated effects in the Asian citrus psyllid, Diaphorina citri Kuwayama, which transmits Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus greening disease. Diaphorina citri are naturally infected with Wolbachia; therefore, investigating Wolbachia-mediated effects on D. citri fitness and CLas transmission required artificial reduction of this endosymbiont with the application of doxycycline. Doxycycline treatment of psyllids reduced Wolbachia infection by approximately 60% in both male and female D. citri. Psyllids treated with doxycycline exhibited higher CLas acquisition in both adults and nymphs as compared with negative controls. In addition, doxycycline-treated psyllids exhibited decreased fitness as measured by reduced egg and nymph production as well as adult emergence as compared with control lines without the doxycycline treatment. Our results indicate that Wolbachia benefits D. citri by improving fitness and potentially competes with CLas by interfering with phytopathogen acquisition. Targeted manipulation of endosymbionts in this phytopathogen vector may yield disease management tools.

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.

Identification of 'Candidatus Liberibacter asiaticus', the Huanglongbing Bacterium, in Citrus from Colombia.

'Candidatus Liberibacter' spp. are the most prevalent microorganisms in the citrus plant, associated with citrus huanglongbing, which are transmitted by psyllid vectors. In Colombia, the vector Diaphorina citri Kuwayama has been reported in different regions, but 'Ca. Liberibacter asiaticus' (CLas) has only been detected in insect vectors, not in citrus host plants. To identify the presence and quantify the pathogen in citrus tissues, we employed a combined strategy that involved three techniques based on polymerase chain reaction (PCR). First, we used endpoint PCR with specific primers for CLas (OI1/OI2c) to confirm the infection. Second, we used qPCR with specific primers CIT295a/CIT298 designed on 16S rDNA gene regions to quantify the pathogen load. Finally, we used droplet digital PCR (ddPCR) to determine the copy number of the pathogen in citrus tissues using the β-subunit of the ribonucleotide reductase gene (nrdB) that is specific to CLas. We identified the presence of CLas in citrus plants for the first time in Colombia and quantified its titer in the plant tissue. We employed ddPCR and qPCR to provide crucial information for the country's disease management, control strategies, and general crop health.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Electrophysiological and behavioral responses of Tamarixia radiata (Hymenoptera: Eulophidae) to volatiles of nymphal Diaphorina citri (Hemiptera: Liviidae)

Abstract Huanglongbing (HLB), a devastating citrus disease caused by Candidatus Liberibacter asiaticus, is efficiently vectored by the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). Tamarixia radiata (Waterston) plays a crucial role as an ectoparasitoid, preying on D. citri nymphs. By collecting and identifying headspace volatiles from fifth instar nymphs of D. citri using a gas chromatograph–mass spectrometer (GC–MS), we obtained a collection of 9 volatile compounds. These compounds were subsequently chosen to investigate the electrophysiological and behavioral responses of female T. radiata. At a concentration of 10 μg/μl, 9 compounds were compared with cis-3-hexen-1-ol (control), resulting in trans-2-nonenal inducing the highest relative electroantennogram (EAG) value, followed by hexanal, heptanal, n-heptadecane, tetradecanal, n-tetradecane, n-pentadecane, 1-tetradecanol, and 1-dodecanol. The top 5 EAG responses of female T. radiata to these compounds were further investigated through EAG dose–response experiments. The results showed positive dose–responses as concentrations increased from 0.01 to 10 μg/μl. In Y-tube olfactometer bioassays, female T. radiata exhibited a preference for specific compounds. They were significantly attracted to tetradecanal at a concentration of 10 µg/µl and trans-2-nonenal at 0.01 µg/µl, while no significant attraction was observed toward hexanal, heptanal, or n-heptadecane. Our report is the first to demonstrate that volatiles produced by D. citri nymphs attract T. radiata, which suggests that this parasitoid may utilize nymph volatiles to locate its host.

Pan-Genome Analysis of Wolbachia, Endosymbiont of Diaphorina citri, Reveals Independent Origin in Asia and North America.

Wolbachia, a group of Gram-negative symbiotic bacteria, infects nematodes and a wide range of arthropods. Diaphorina citri Kuwayama, the vector of Candidatus Liberibacter asiaticus (CLas) that causes citrus greening disease, is naturally infected with Wolbachia (wDi). However, the interaction between wDi and D. citri remains poorly understood. In this study, we performed a pan-genome analysis using 65 wDi genomes to gain a comprehensive understanding of wDi. Based on average nucleotide identity (ANI) analysis, we classified the wDi strains into Asia and North America strains. The ANI analysis, principal coordinates analysis (PCoA), and phylogenetic tree analysis supported that the D. citri in Florida did not originate from China. Furthermore, we found that a significant number of core genes were associated with metabolic pathways. Pathways such as thiamine metabolism, type I secretion system, biotin transport, and phospholipid transport were highly conserved across all analyzed wDi genomes. The variation analysis between Asia and North America wDi showed that there were 39,625 single-nucleotide polymorphisms (SNPs), 2153 indels, 10 inversions, 29 translocations, 65 duplications, 10 SV-based insertions, and 4 SV-based deletions. The SV-based insertions and deletions involved genes encoding transposase, phage tail tube protein, ankyrin repeat (ANK) protein, and group II intron-encoded protein. Pan-genome analysis of wDi contributes to our understanding of the geographical population of wDi, the origin of hosts of D. citri, and the interaction between wDi and its host, thus facilitating the development of strategies to control the insects and huanglongbing (HLB).

Effector enrichment by Candidatus Liberibacter promotes Diaphorina citri feeding via Jasmonic acid pathway suppression.

Citrus huanglongbing (HLB) is a devastating disease caused by Candidatus Liberibacter asiaticus (CLas) that affects the citrus industry. In nature, CLas relies primarily on Diaphorina citri Kuwayama as its vector for dissemination. After D. citri ingests CLas-infected citrus, the pathogen infiltrates the insect's body, where it thrives, reproduces, and exerts regulatory control over the growth and metabolism of D. citri. Previous studies have shown that CLas alters the composition of proteins in the saliva of D. citri, but the functions of these proteins remain largely unknown. In this study, we detected two proteins (DcitSGP1 and DcitSGP3) with high expression levels in CLas-infected D. citri. Quantitative PCR and Western blotting analysis showed that the two proteins were highly expressed in the salivary glands and delivered into the host plant during feeding. Silencing the two genes significantly decreased the survival rate for D. citri, reduced phloem nutrition sucking and promoted jasmonic acid (JA) defenses in citrus. By contrast, after overexpressing the two genes in citrus, the expression levels of JA pathway-associated genes decreased. Our results suggest that CLas can indirectly suppress the defenses of citrus and support feeding by D. citri via increasing the levels of effectors in the insect's saliva. This discovery facilitates further research into the interaction between insect vectors and pathogens. © 2024 Society of Chemical Industry.

Identification and Distribution of the 'Candidatus Liberibacter asiaticus'-Asian Citrus Psyllid Pathosystem in Saudi Arabia.

Citrus greening disease was first reported in Saudi Arabia during the 1970s when characteristic foliar and fruit symptoms were observed in commercial citrus groves. However, 'Candidatus Liberibacter asiaticus' (CLas) was not detected in symptomatic trees until 1981 to 1984 when CLas-like cells were observed by transmission electron microscopy in leaves collected from symptomatic citrus groves in southwestern Saudi Arabia. Despite the anticipated establishment of the CLas-Asian citrus psyllid (ACP) (Diaphorina citri Kuwayama) pathosystem, CLas presence has not been verified in suspect trees nor have ACP infestations been documented. Given the recent expansion of citrus production in Saudi Arabia, a systematic country-wide survey was carried out to determine the potential CLas distribution in the 13 citrus-growing regions of the country. Citrus trees were surveyed for the presence of CLas-psyllid vector(s) and characteristic disease symptoms in commercial and urban citrus trees. Adult psyllids collected from infested citrus trees were identified as ACP based on morphological characteristics. Real-time quantitative PCR amplification (qPCR) of the CLas β-subunit of the ribonucleotide reductase (RNR) gene from citrus leaf and fruit samples and/or ACP adults revealed that trees were positive for CLas detection in 10 of the 13 survey regions; however, CLas was undetectable in ACP adults. Phylogenetic and single nucleotide polymorphism (SNP) analyses of a PCR-amplified, cloned fragment of the CLas 16S rRNA gene (∼1.1 kbp) indicated Saudi Arabian isolates were most closely related to Florida, U.S.A., isolates. Analysis of climate variables indicated that the distribution of the ACP-CLas pathosystem observed in Saudi Arabia was consistent with published predictions of terrains most likely to support establishment.

Identification and characterization of two P450 enzymes from Citrus sinensis involved in TMTT and DMNT biosyntheses and Asian citrus psyllid defense.

The homoterpenes (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT) are the major herbivore-induced plant volatiles that help in defense directly by acting as repellants and indirectly by recruiting insects' natural enemies. In this study, DMNT and TMTT were confirmed to be emitted from citrus (Citrus sinensis) leaves infested with Asian citrus psyllid (Diaphorina citri Kuwayama; ACP), and two cytochrome P450 (CYP) genes (CsCYP82L1 and CsCYP82L2) were newly identified and characterized. Understanding the functions of these genes in citrus defense will help plan strategies to manage huanglongbing caused by Candidatus Liberibacter asiaticus (CLas) and spread by ACP. Quantitative real-time PCR (qPCR) analysis showed that CsCYP82L1 and CsCYP82L2 were significantly upregulated in citrus leaves after ACP infestation. Yeast recombinant expression and enzyme assays indicated that CsCYP82L1 and CsCYP82L2 convert (E)-nerolidol to DMNT and (E,E)-geranyllinalool to TMTT. However, citrus calluses stably overexpressing CsCYP82L1 generated only DMNT, whereas those overexpressing CsCYP82L2 produced DMNT and TMTT. Furthermore, ACPs preferred wild-type lemon (Citrus limon) over the CsCYP82L1-overexpressing line in dual-choice feeding assays and mineral oil over TMTT or DMNT in behavioral bioassays. Finally, yeast one-hybrid, electrophoretic mobility shift, and dual luciferase assays demonstrated that CsERF017, an AP2/ERF transcription factor, directly bound to the CCGAC motif and activated CsCYP82L1. Moreover, the transient overexpression of CsERF017 in lemon leaves upregulated CsCYP82L1 in the absence and presence of ACP infestation. These results provide novel insights into homoterpene biosynthesis in C. sinensis and demonstrate the effect of homoterpenes on ACP behavior, laying a foundation to genetically manipulate homoterpene biosynthesis for application in huanglongbing and ACP control.

Guardians of Citrus Groves: An In-Depth Analysis of Neem Leaf Extract's Effects on Asian Citrus Psyllid Attractiveness, Diaphorina citri kuwayama, And its Potential for Integrated Pest Management

Background: The pervasive impact of fungal diseases on wheat crops necessitates the exploration of sustainable and safe pest control alternatives. Neem (Azadirachta indica) is renowned for its medicinal properties and is being investigated for its potential as a biopesticide in agriculture. Objective: To evaluate the antifungal efficacy of Azadirachta indica leaf extracts on fungal pathogens in wheat crops, aiming to offer an environmentally benign alternative to chemical fungicides. Methods: Neem leaves were collected, sterilized, dried, and ground to powder. This powder was subjected to solvent extraction using methanol, diethyl ether, chloroform, petroleum ether, and distilled water. The resulting extracts were tested for antifungal activity against Diaphorina citri kuwayama using the agar well diffusion method. Wheat plants treated with the extracts were monitored for fungal disease incidence and yield changes. Results: Methanolic extracts of Azadirachta indica showed a significant zone of inhibition of 34.67 mm against Diaphorina citri kuwayama. Additionally, methanolic extracts displayed a zone of inhibition of 31.6±2.04 mm, while the control showed 31.33±0.58 mm. The MIC values ranged from 35 µg/ml for methanol to 24 µg/ml for distilled water, with the control at 20 µg/ml. Treatment of wheat crops with these extracts resulted in reduced fungal contamination and enhanced yield. Conclusion: The study confirms that Azadirachta indica leaf extracts have substantial antifungal properties, demonstrating potential as a natural and environmentally friendly pest management strategy in wheat production. This approach aligns with sustainable agricultural practices and could have a beneficial impact on human health by reducing harmful chemical residues in food.