Longitudinal Transcriptomic, Proteomic, and Metabolomic Response of Citrus sinensis to Diaphorina citri Inoculation of Candidatus Liberibacter asiaticus.

Huanglongbing (HLB) is a fatal citrus disease that is currently threatening citrus varieties worldwide. One putative causative agent, Candidatus Liberibacter asiaticus (CLas), is vectored by Diaphorina citri, known as the Asian citrus psyllid (ACP). Understanding the details of CLas infection in HLB disease has been hindered by its Candidatus nature and the inability to confidently detect it in diseased trees during the asymptomatic stage. To identify early changes in citrus metabolism in response to inoculation of CLas using its natural psyllid vector, leaves from Madam Vinous sweet orange (Citrus sinensis (L.) Osbeck) trees were exposed to CLas-positive ACP or CLas-negative ACP and longitudinally analyzed using transcriptomics (RNA sequencing), proteomics (liquid chromatography-tandem mass spectrometry; data available in Dryad: 10.25338/B83H1Z), and metabolomics (proton nuclear magnetic resonance). At 4 weeks postexposure (wpe) to psyllids, the initial HLB plant response was primarily to the ACP and, to a lesser extent, the presence or absence of CLas. Additionally, analysis of 4, 8, 12, and 16 wpe identified 17 genes and one protein as consistently differentially expressed between leaves exposed to CLas-positive ACP versus CLas-negative ACP. This study informs identification of early detection molecular targets and contributes to a broader understanding of vector-transmitted plant pathogen interactions.

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.

Genetic diversity and population structure of the Asian citrus psyllid in China.

The Asian citrus psyllid (ACP) is the main vector of Citrus Huanglongbing, the most damaging citrus disease, causing significant financial losses in the citrus industry. Global warming has expanded the habitat of this pest, allowing it to continue its northward migration to China. Population genetic information of ACP is fundamentally essential for species management. This study investigated the genetic diversity and population structure of Chinese ACP using the mitochondrial cytochrome oxidase subunit I gene by dataset comprised 721 sequences from 27 geographic sites in China. Low haplotype diversity (0.323 ±â€…0.022) and low nucleotide diversity (0.00071 ±â€…0.00007) were observed in the entire population, which may indicate recent founder events. Twenty-three haplotypes were identified and clustered into 2 haplogroups: haplogroup I and haplogroup II. Haplogroup II included only 2 unique haplotypes, which occurred exclusively in the Southwest China ACP population. Genetic differentiation analyses were also indicative of Southwest China population was significantly differentiated from the remaining populations. Demographic history analysis showed that ACP population in China has experienced demographic expansion. Our results provided a better understanding of the genetic distribution patterns and structures of ACP populations in China.

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).

Abnormal cell wall structure caused by boron nutrient imbalance in orchards could affect psyllid feeding behaviour, resulting in epidemic variation of Asian citrus psyllid.

The control of Huanglongbing (HLB), one of the most destructive pests of citrus, relies heavily on the reduction of Asian citrus psyllid (ACP), Diaphorina citri Kuwayama. An in-depth understanding of ACP feeding behaviours among citrus plants is urgent for comprehensive management of orchards. An investigation was conducted in 37 citrus orchards in HLB epidemic areas, sampling shoots in the area with aggregation feeding of ACP (ACPf) and shoots in a neighbouring area without ACP feeding (CK), to study the interaction between leaf chemical composition and ACP psyllid feeding behaviours. Results of FTIR showed a strong absorption peak intensity, mainly representing functional groups originating from cell wall components in the leaf with ACP feeding. As compared with the control, cell wall components, such as alkali-soluble pectin, water-soluble pectin, total soluble pectin, cellulose, and hemicellulose, of the cell wall of ACPf increased by 134.0%, 14.0%, 18.0%, 12.5%, and 20.35%, respectively. These results suggest that cell wall mechanical properties significantly decreased in the term of decreases in pectin performance and cellulose mechanical properties. In addition, there was a remarkably lower boron (B) content in leaves and cell wall components with ACP feeding. Further analysis indicated that leaf B content significantly affected leaf cell wall components. Taken together, we provide evidence to demonstrate that the regional distribution of nutrient imbalance in orchards could affect psyllid feeding behaviour by weakening the cell wall structure, resulting in epidemic variation in ACP. This could help us to understand the management of psyllid infections in orchards with unbalanced nutrition.

Detection of Candidatus Liberibacter asiaticus and five viruses in individual Asian citrus psyllid in China.

Introduction: Asian citrus psyllid (ACP, Diaphorina citri) is an important transmission vector of "Candidatus Liberibacter asiaticus" (CLas), the causal agent of Huanglongbing (HLB), the most destructive citrus disease in the world. As there are currently no HLB-resistant rootstocks or varieties, the control of ACP is an important way to prevent HLB. Some viruses of insect vectors can be used as genetically engineered materials to control insect vectors. Methods: To gain knowledge on viruses in ACP in China, the prevalence of five RNA and DNA viruses was successfully determined by optimizing reverse transcription polymerase chain reaction (RT-PCR) in individual adult ACPs. The five ACP-associated viruses were identified as follows: diaphorina citri bunyavirus 2, which was newly identified by high-throughput sequencing in our lab, diaphorina citri reovirus (DcRV), diaphorina citri picorna-like virus (DcPLV), diaphorina citri bunyavirus (DcBV), and diaphorina citri densovirus-like virus (DcDV). Results: DcPLV was the most prevalent and widespread ACP-associated virus, followed by DcBV, and it was detected in more than 50% of all samples tested. DcPLV was also demonstrated to propagate vertically and found more in salivary glands among different tissues. Approximately 60% of all adult insect samples were co-infected with more than one insect pathogen, including the five ACP-associated viruses and CLas. Discussion: This is the first time these viruses, including the newly identified ACP-associated virus, have been detected in individual adult ACPs from natural populations in China's five major citrus-producing provinces. These results provide valuable information about the prevalence of ACP-associated viruses in China, some of which have the potential to be used as biocontrol agents. In addition, analysis of the change in prevalence of pathogens in a single insect vector is the basis for understanding the interactions between CLas, ACP, and insect viruses.

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.

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.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Survival analyses of Diaphorina citri immatures on young citrus orchard in São Paulo, Brazil / Análises de sobrevivência de imaturos de Diaphorina citri em pomar jovem de citros em São Paulo, Brasil

The survival of eggs and nymphs of Diaphorina citri Kuwayama (Hemiptera: Liviidae) was estimated on young plants (< 1 year) of Citrus sinensis (cultivar Valencia) grafted on Citrus limonia in 3 seasons (Winter and Spring 2013; and Autumn 2014) in Ribeirão Preto, São Paulo State, Brazil. Young shoots with eggs were selected from natural or artificial infestations. Eggs, small nymphs (1st to 3rd instar), and large nymphs (4th and 5th instar) were counted every 3 days. Shoots with large nymphs were caged until the emergence of the adults. The lowest eggs and nymph survival were observed during Winter (0.0%), and Autumn (0.4%); the highest survival rates were recorded during Spring (12.2%). The biotic factor of mortality identified was the parasitism of large nymphs by Tamarixia radiata Waterston (Hymenoptera: Eulophidae), observed in all seasons. Air temperatures (minimum and maximum), and air relative humidity (minimum) could not explain the immature survival differences between Spring (12.2%) and Autumn (0.4%). Rainfall during Spring (165 mm) was higher than during Autumn (48 mm) and may have favored the survival of D. citri in the Spring. Adverse abiotic factors seem to be more important on immature D. citri survival than the biotic factor (parasitism).(AU)

A sobrevivência de ovos e ninfas de Diaphorina citri Kuwayama (Hemiptera: Liviidae) foi estimada em plantas jovens (< 1 ano) de Citrus sinensis (cultivar Valencia) enxertadas em Citrus limonia em 3 estações (inverno e primavera de 2013, e outono de 2014) em Ribeirão Preto, estado de São Paulo, Brasil. Ramos novos com ovos foram selecionados a partir de infestações naturais ou artificiais. Ovos, ninfas pequenas (ínstar 1 ao 3) e ninfas grandes (ínstar 4 e 5) foram contados a cada 3 dias. Ramos com ninfas grandes foram colocados em gaiolas até a emergência dos adultos. As menores sobrevivências de ovos e ninfas foram observadas no inverno (0,0%) e no outono (0,4%), e a maior sobrevivência ocorreu na primavera (12,2%). O principal fator biótico de mortalidade identificado foi o parasitismo de ninfas grandes por Tamarixia radiata Waterston (Hymenoptera: Eulophidae), observado em todas as estações. As temperaturas (mínima e máxima) e umidade relativa (mínima) do ar não explicaram as diferenças nas sobrevivências de imaturos entre a primavera (12,2%) e o outono (0,4%). As chuvas na primavera (165 mm) foram maiores que no outono (48 mm) e podem ter favorecido a sobrevivência de D. citri na primavera. Fatores abióticos adversos parecem ser mais importantes na sobrevivência de imaturos de D. citri que o fator biótico (parasitismo).(AU)