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.

DNA degradation in Haplaxius crudus (Hemiptera: Cixiidae) and Diaphorina citri (Hemiptera: Liviidae) on yellow sticky traps in Florida.

Lethal bronzing (LB) and huanglongbing (HLB) are harmful plant diseases causing significant economic losses in Florida agriculture. Both diseases are caused by bacteria that are transmitted by Hemipteran insect vectors. Accurate detection of pathogens within insect vectors can help provide a better understanding of disease epidemiology. Monitoring of the vector of LB is done primarily using sticky traps within palm canopies. However, it is unknown how long pathogen and vector DNA remain intact under field conditions. If significant DNA degradation takes place over the course of days or weeks, there is a possibility of false negatives occurring when detecting pathogens from these surveys. This study determined how long Haplaxius crudus Van Duzee (Hemiptera: Cixiidae) and LB DNA could remain detectable on sticky traps under field conditions in Florida in winter and summer, using PCR and qPCR. Additionally, this study compared the DNA degradation of Diaphorina citri Kuwayama (Hemiptera: Liviidae) and Candidatus Liberibacter asiaticus (CLas), the causal agent of HLB. The results showed that DNA concentration and amplification rate declined as time on sticky traps increased. Degradation varied between different target genes. The amplification rate of insect genes from sticky trap samples suggests that sticky traps should be changed weekly in summer, and every 2 wk in winter for accurate H. crudus detection. Traps should be changed every 4 days for phytoplasma detection. Traps can be changed monthly for accurate D. citri and CLas detection. Based on these results, standard monitoring protocols can be implemented to more accurately detect vectors and pathogens.

Foliar Antibiotic Treatment Reduces Candidatus Liberibacter asiaticus Acquisition by the Asian Citrus Psyllid, Diaphorina citri (Hemiptera: Liviidae), but Does not Reduce Tree Infection Rate.

Huanglongbing (HLB), or citrus greening, is the most destructive disease of cultivated citrus worldwide. Candidatus Liberibacter asiaticus (CLas), the putative causal agent of HLB, is transmitted by the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). In Florida, D. citri was first reported in 1998, and CLas was confirmed in 2005. Management of HLB relies on the use of insecticides to reduce vector populations. In 2016, antibiotics were approved to manage CLas infection in citrus. Diaphorina citri is host to several bacterial endosymbionts and reducing endosymbiont abundance is known to cause a corresponding reduction in host fitness. We hypothesized that applications of oxytetracycline and streptomycin would reduce: CLas populations in young and mature citrus trees, CLas acquisition by D. citri, and D. citri abundance. Our results indicate that treatment of citrus with oxytetracycline and streptomycin reduced acquisition of CLas by D. citri adults and emerging F1 nymphs as compared with that observed in trees treated only with insecticides, but not with antibiotics. However, under field conditions, neither antibiotic treatment frequency tested affected CLas infection of young or mature trees as compared with insecticide treatment alone (negative control); whereas trees enveloped with mesh screening that excluded vectors did prevent bacterial infection (positive control). Populations of D. citri were not consistently affected by antibiotic treatment under field conditions, as compared with an insecticide only comparison. Collectively, our results suggest that while foliar application of oxytetracycline and streptomycin to citrus reduces acquisition of CLas bacteria by the vector, even high frequency applications of these formulations under field conditions do not prevent or reduce tree infection.

Complete de novo assembly of Wolbachia endosymbiont of Diaphorina citri Kuwayama (Hemiptera: Liviidae) using long-read genome sequencing.

Wolbachia, a gram-negative [Formula: see text]-proteobacterium, is an endosymbiont found in some arthropods and nematodes. Diaphorina citri Kuwayama, the vector of 'Candidatus Liberibacter asiaticus' (CLas), are naturally infected with a strain of Wolbachia (wDi), which has been shown to colocalize with the bacteria pathogens CLas, the pathogen associated with huanglongbing (HLB) disease of citrus. The relationship between wDi and CLas is poorly understood in part because the complete genome of wDi has not been available. Using high-quality long-read PacBio circular consensus sequences, we present the largest complete circular wDi genome among supergroup-B members. The assembled circular chromosome is 1.52 megabases with 95.7% genome completeness with contamination of 1.45%, as assessed by checkM. We identified Insertion Sequences (ISs) and prophage genes scattered throughout the genomes. The proteins were annotated using Pfam, eggNOG, and COG that assigned unique domains and functions. The wDi genome was compared with previously sequenced Wolbachia genomes using pangenome and phylogenetic analyses. The availability of a complete circular chromosome of wDi will facilitate understanding of its role within the insect vector, which may assist in developing tools for disease management. This information also provides a baseline for understanding phylogenetic relationships among Wolbachia of other insect vectors.

Annotation of putative circadian rhythm-associated genes in Diaphorina citri (Hemiptera: Liviidae).

The circadian rhythm involves multiple genes that generate an internal molecular clock, allowing organisms to anticipate environmental conditions produced by the Earth's rotation on its axis. Here, we present the results of the manual curation of 27 genes that are associated with circadian rhythm in the genome of Diaphorina citri, the Asian citrus psyllid. This insect is the vector for the bacterial pathogen Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus greening disease (Huanglongbing). This disease severely affects citrus industries and has drastically decreased crop yields worldwide. Based on cry1 and cry2 identified in the psyllid genome, D. citri likely possesses a circadian model similar to the lepidopteran butterfly, Danaus plexippus. Manual annotation will improve the quality of circadian rhythm gene models, allowing the future development of molecular therapeutics, such as RNA interference or antisense technologies, to target these genes to disrupt the psyllid biology.

Antibacterial FANA oligonucleotides as a novel approach for managing the Huanglongbing pathosystem.

Candidatus Liberibacter asiaticus (CLas), a bacterium transmitted by the Asian citrus psyllid, Diaphorina citri, is the causal agent of citrus greening disease, or Huanglongbng (HLB). Currently, vector population suppression with insecticides and tree removal are the most effective strategies for managing the HLB pathosystem. In this study, we assessed the bactericidal capabilities of 2'-deoxy-2'-fluoro-D-arabinonucleic acid antisense oligonucleotides (FANA ASO) both in vitro and in vivo by (1) confirming their capacity to penetrate insect cells, (2) silencing bacterial essential genes, and (3) quantifying reductions in bacterial titer and D. citri transmission. We confirmed that FANA ASO are able to penetrate insect cells without the use of a delivery agent. Expression of an essential gene in the D. citri endosymbiont, Wolbachia (wDi), significantly decreased by 30% following incubation with a wDi-specific FANA ASO. Viability of isolated wDi cells also decreased in response to the FANA ASO treatment. Delivery of a CLas-specific FANA ASO to infected adult D. citri in feeding assays resulted in significant silencing of a CLas essential gene. CLas relative density and transmission were significantly lower among D. citri fed FANA ASO in diet compared to untreated insects. Root infusions of a CLas-specific FANA ASO into infected Citrus trees significantly reduced CLas titer during a 30-day trial. Our results suggest that FANA ASO targeting insect-transmitted plant bacteria or insect endosymbionts may be useful tool for integrated management of agricultural pathogens.

Comparative transcriptome analysis of thiamethoxam susceptible and resistant Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), using RNA-sequencing.

Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), transmits the causal pathogen of huanglongbing and is a global pest of citrus. D. citri populations exhibit resistance to multiple insecticide modes of action in areas where these chemicals have been overused. We performed genome-wide transcriptional analysis for two field populations of D. citri (Wauchula and Lake Alfred, Florida, USA) that exhibit 1300-fold resistance to the neonicotinoid insecticide, thiamethoxam, and compared it to that of susceptible psyllids collected from the same area and without imposed selection. The Lake Alfred population responded to insecticide resistance by up-regulation of 240 genes and down-regulation of 148 others. The Wauchula population exhibited similar patterns to the Lake Alfred population with up-regulation of 253 genes and down-regulation of 115 others. Gene Ontology annotation associated with cellular processes, cell, and catalytic activity were assigned to differentially expressed genes (DEGs). The DEGs from Lake Alfred and Wauchula populations were mapped to Kyoto Encyclopedia of Gene and Genomes pathways and implicated enrichment of metabolic pathways, oxidative phosphorylation, extracellular matrix-receptor interaction, terpenoid backbone biosynthesis, and insect hormone biosynthesis in the resistant populations. Up-regulation of 60s ribosomal proteins, UDP-gluscoyltransferases, cytochrome c oxidases, and CYP and ABC transporters among thiamethoxam-resistant D. citri implicates a broad array of novel and conventionally understood resistance mechanisms.

Insecticide rotation scheme restores insecticide susceptibility in thiamethoxam-resistant field populations of Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), in Florida.

BACKGROUND: We investigated rotation using insecticides with multiple modes of action as a resistance management strategy for Asian citrus psyllid, Diaphorina citri, Kuwayama (Hemiptera: Liviidae), in Florida. The stability of thiamethoxam resistance was investigated in the laboratory by establishing populations of field-collected, resistant D. citri and rearing them under no insecticide exposure. Furthermore, recovery of susceptibility was investigated in the field by initiating rotation to insecticides in plots that previously were treated with consecutive thiamethoxam applications. RESULTS: The resistance ratio (RR) for thiamethoxam reached between 1266.29- and 1395.00-fold after three and four consecutive applications of thiamethoxam, respectively. However, the RR for thiamethoxam remained low (1.71-5.28-fold) under both rotations at both Lake Alfred and Wauchula. Thiamethoxam was cross-resistant with imidacloprid (RR site 1 = 1059.65-fold, RR site 2 = 1595.43-fold) and clothianidin (RR site 1 = 1798.78-fold, RR site 2 = 1270.57-fold) in the nonrotated treatment at both sites. There was very low cross-resistance to other insecticides with different modes of action. Both laboratory and field investigations indicated that susceptibility to thiamethoxam fully recovered after five D. citri generations. Expression of CYP4C67 was significantly increased in resistant populations. CONCLUSION: Our results revealed that D. citri populations develop a high level of resistance following only three or four consecutive neonicotinoid sprays; this was associated with subsequent product failure. Our data suggest that metabolic detoxification by cytochrome P450s contributes to thiamethoxam resistance in D. citri. Overall, the investigation demonstrated that resistance to thiamethoxam can be managed readily in populations of D. citri by rotating modes of action.

Near-Complete Genome Sequences of a Wolbachia Strain Isolated from Diaphorina citri Kuwayama (Hemiptera: Liviidae).

Wolbachia strains are one of three endosymbionts associated with the insect vector of "Candidatus Liberibacter asiaticus," Diaphorina citri Kuwayama (Hemiptera: Liviidae). We report three near-complete genome sequences of samples of Wolbachia from D. citri (wDi), with sizes of 1,518,595, 1,542,468, and 1,538,523 bp.

Peptide conjugated morpholinos for management of the huanglongbing pathosystem.

BACKGROUND: 'Candidatus Liberibacter asiaticus' (CLas) is the causal agent of the devastating citrus disease Huanglongbing (HLB) and is transmitted by the insect vector Diaphorina citri (Hemiptera: Liviidae). A potential approach for treating CLas infection is the use of synthetic nucleic acid-like oligomers to silence bacterial gene expression. Peptide conjugated morpholinos (PPMOs) targeting essential genes in CLas and the psyllid vector's endosymbiotic bacteria, Wolbachia (-Diaphorina, wDi), were evaluated using in vitro and in vivo assays. RESULTS: Expression of the wDi gyrA gene was significantly reduced following incubation of wDi cells with PPMOs. In addition, the viability of isolated wDi cells was greatly reduced when treated with PPMOs as compared to untreated cells. Feeding D. citri adults with a complementary PPMO (CLgyrA-14) showed significantly reduced (70% lower) expression of the CLas gyrA gene. CLas relative density was significantly lower in the psyllids fed with CLgyrA-14, when compared to untreated insects. Psyllids that were treated with CLgyrA-14 were less successful in transmitting the pathogen into uninfected plants, compared to untreated insects. CONCLUSION: The expression of essential genes in the D. citri symbiont, wDi and the HLB pathogen were suppressed in response to PPMO treatments. This study demonstrates the potential of PPMOs as a novel strategy for management of bacterial pathogens of fruit trees, such as HLB. © 2020 Society of Chemical Industry.

Fitness costs associated with thiamethoxam and imidacloprid resistance in three field populations of Diaphorina citri (Hemiptera: Liviidae) from Florida.

Insecticide resistance is an increasing problem in citrus production. The Asian citrus psyllid, Diaphornia citri Kuwayama, is recognized as one of the most important citrus pests worldwide and it has developed resistance in areas where insecticides have been overused. The development of insecticide resistance is often associated with fitness costs that only become apparent in the absence of selection pressure. Here, the fitness costs associated with resistance to thiamethoxam and imidacloprid were investigated in three agricultural populations of D. citri as compared with susceptible laboratory colonies. Results showed that all field populations had greater resistance than laboratory susceptible colonies. For both thiamethoxam and imidacloprid, a Candidatus Liberibacter asiaticus-positive (CLas+) colony was more susceptible than the CLas- colony. Resistance ratios ranged from 7.65-16.11 for imidacloprid and 26.79-49.09 for thiamethoxam in field populations as compared with a susceptible, CLas- laboratory strain. Among three resistant field populations, a significantly reduced net reproductive rate and finite rate of population increase were observed in a population from Lake Wales, FL as compared to both susceptible strains. The fecundity of field populations from Lake Wales, FL was statistically lower than both laboratory susceptible populations. Certain changes in morphological characteristics were observed among resistant, as compared, with susceptible strains. Our data suggest fitness disadvantages associated with insecticide resistance in D. citri are related to both development and reproduction. The lower fitness of D. citri populations that exhibit resistance to neonicotinoid insecticides should promote recovery of sensitivity when those populations are no longer exposed to thiamethoxam and/or imidacloprid in the field. The results are congruent with a strategy of insecticide rotation for resistance management.

Maternal Contribution of Candidatus Liberibacter asiaticus to Asian Citrus Psyllid (Hemiptera: Liviidae) Nymphs Through Oviposition Site Inoculation and Transovarial Transmission.

Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), transmits Candidatus Liberibacter asiaticus (Las), the bacterial pathogen putatively responsible for citrus huanglongbing. Multiple studies have shown psyllids acquire Las more frequently, and are more likely to inoculate susceptible plants, when they acquire Las as nymphs. Understanding the transmission of Las to nymphs is critical to the Las lifecycle. The objective of this study was to determine the transmission Las by female D. citri to their offspring. Two transmission pathways were quantified: horizontal transmission (acquisition of Las via feeding at the oviposition site) and vertical transmission (transovarial). Eggs of individual, infected females were transferred to an uninfected seedling to assess vertical transmission. In a second experiment, horizontal transmission was evaluated by replacing eggs laid by infected females with uninfected nymphs. Nymphs exposed to Las via horizontal transmission of the oviposition site were more likely to acquire Las than from vertical transmission. Las deposited in flush by an infected adult female feeding during oviposition was sufficient for infecting nymphs. Combined results of both experiments suggest that vertical transmission allows Las to spread in low amounts even when infected plant hosts are not available and that inoculation of the oviposition site provides a source of Las to developing nymphs via the plant phloem. These data support the hypothesis that transmission through infected plant material via maternal inoculation is a primary pathway of Las transmission between vector and host.

Insecticide toxicity associated with detoxification enzymes and genes related to transcription of cuticular melanization among color morphs of Asian citrus psyllid.

The Asian citrus psyllid (Diaphorina citri Kuwayama) is known to exhibit abdominal color polymorphisms. In the current study, susceptibility to four insecticides was compared among orange/yellow, blue/green and gray/brown color morphs of field collected D. citri. The LD50 values and 95% fiducial limits were quantified for each insecticide and color morph combination and ranged between 0.10 ng/µL (0.06-0.10) and 6.16 ng/µL (3.30-12.50). Second, we measured the detoxification enzyme activity levels of orange/yellow, blue/green and gray/brown color morphs for cytochrome P450, glutathione S-transferase, and general esterase. The mean P450 activity (equivalent units) was significantly lower in gray/brown (0.152 ± 0.006) and blue/green morphs (0.149 ± 0.005) than in the orange/yellow morphs (0.179 ± 0.008). GST activity (µmol/min/mg protein) was significantly lower in the orange/yellow morph (299.70 ±1.24) than gray/brown (350.86 ± 1.19) and blue/green (412.25 ± 1.37) morphs. The mean EST activity (µmol/min/mg protein) was significantly higher in blue/green (416.72 ± 5.12) and gray/brown morphs (362.19 ± 4.69) than in the orange/yellow morphs (282.56 ± 2.93). Additionally, we analyzed the relative expression of assortment genes involved in cuticular melanization and basal immunity. The transcripts of Dopa Decarboxylase and Tyrosine Hydroxylase were expressed higher in blue/green and gray/brown than orange/yellow morphs. The transcription results paralleled the susceptibility of D. citri to organophosphate, neonicotinoid and pyrethroid insecticides. GST and EST activity may also be correlated with low levels of insecticide susceptibility. Cuticular melanization could be a factor for the development of resistance to insecticides among different color morphs.

Genetic variation and potential coinfection of Wolbachia among widespread Asian citrus psyllid (Diaphorina citri Kuwayama) populations.

Wolbachia can profoundly influence the survival, reproduction, and defenses of insect hosts. These interactions could potentially be harnessed for managing pests or insect-transmitted diseases. Diaphorina citri Kuwayama is a phloem-feeding pest capable of transmitting the putative causal agent of citrus greening, Candidatus Liberibacter asiaticus (CLas). Like many insects, D. citri is also infected with Wolbachia (wDi). Recent studies indicate that the relative abundance of wDi could be associated with the abundance of CLas, and that wDi may contribute to regulating expression of phage lytic cycle genes in CLas, suggesting the need for better understanding of wDi biology in general. This study investigated the genetic diversity of wDi among D. citri in populations spanning eleven countries and two U.S. territories. Six Wolbachia genes, wsp, coxA, fbpA, ftsZ, gatB, and hcpA, were sequenced and compared across samples. Two prevalent wDi strains were identified across the samples, and screening of clone libraries revealed possible coinfection of wDi strains in specific populations. D. citri mitochondrial cytochrome oxidase subunit I gene (mtCOI) were more divergent between D. citri populations that were infected with different wDi strains or had different infection statuses (single infection vs. coinfection). While we could not eliminate the possibility that maternal transmission may contribute to such patterns, it is also possible that wDi may induce cytoplasmic incompatibility in their host. These findings should contribute to the understanding of wDi population ecology, which may facilitate manipulation of this endosymbiont for management of citrus greening disease worldwide.

Induced Systemic Resistance Against Citrus Canker Disease by Rhizobacteria.

Citrus canker, caused by Xanthomonas citri subsp. citri, is an important citrus disease that causes significant economic losses worldwide. All commercial citrus varieties are susceptible to citrus canker. Currently, chemical control with copper based products is the main approach to control X. citri subsp. citri dispersal and plant colonization. However, extensive use of copper compounds can result in copper-resistant strains and cause adverse effects on the environment. Alternatives to chemical control involve the activation of citrus immunity to control the disease. Here, we investigated the ability of multiple rhizobacteria to induce a systemic defense response in cultivar Duncan grapefruit. Burkholderia territorii strain A63, Burkholderia metallica strain A53, and Pseudomonas geniculata strain 95 were found to effectively activate plant defense and significantly reduce symptom development in leaves challenged with X. citri subsp. citri. In the priming phase, root application of P. geniculata induced the expression of salicylic acid (SA)-signaling pathway marker genes (PR1, PR2, PR5, and salicylic acid carboxyl methyltransferase [SAM-SACM]). Gene expression analyses after X. citri subsp. citri challenge showed that root inoculation with P. geniculata strain 95 increased the relative levels of phenylalanine ammonia lyase 1 and SAM-SACM, two genes involved in the phenylpropanoid pathway as well as the biosynthesis of SA and methyl salicylate (MeSA), respectively. However, hormone analyses by UPLC-MS/MS showed no significant difference between SA in P. geniculata-treated plants and control plants at 8 days post-beneficial bacteria root inoculation. Moreover, P. geniculata root-treated plants contained higher reactive oxygen species levels in aerial tissues than control plants 8 days post-treatment application. This study demonstrates that rhizobacteria can modulate citrus immunity resulting in a systemic defense response against X. citri subsp. citri under greenhouse conditions.

Horizontal Transmission of Intracellular Insect Symbionts via Plants.

Experimental evidence is accumulating that endosymbionts of phytophagous insects may transmit horizontally via plants. Intracellular symbionts known for manipulating insect reproduction and altering fitness (Rickettsia, Cardinium, Wolbachia, and bacterial parasite of the leafhopper Euscelidius variegatus) have been found to travel from infected insects into plants. Other insects, either of the same or different species can acquire the symbiont from the plant through feeding, and in some cases transfer it to their progeny. These reports prompt many questions regarding how intracellular insect symbionts are delivered to plants and how they affect them. Are symbionts passively transported along the insect-plant-insect path, or do they actively participate in the process? How widespread are these interactions? How does symbiont presence influence the plant? And what conditions are required for the new infection to establish in an insect? From an ecological, evolutionary, and applied perspective, this mode of horizontal transmission could have profound implications if occurring frequently enough or if new stable symbiont infections are established. Transmission of symbionts through plants likely represents an underappreciated means of infection, both in terms of symbiont epidemiology and the movement of symbionts to new host species.

Bacterial phytopathogen infection disrupts belowground plant indirect defense mediated by tritrophic cascade.

Plants can defend themselves against herbivores through activation of defensive pathways and attraction of third-trophic-level predators and parasites. Trophic cascades that mediate interactions in the phytobiome are part of a larger dynamic including the pathogens of the plant itself, which are known to greatly influence plant defenses. As such, we investigated the impact of a phloem-limited bacterial pathogen, Candidatus Liberibacter asiaticus (CLas), in cultivated citrus rootstock on a well-studied belowground tritrophic interaction involving the attraction of an entomopathogenic nematode (EPN), Steinernema diaprepesi, to their root-feeding insect hosts, Diaprepes abbreviatus larvae. Using belowground olfactometers, we show how CLas infection interferes with this belowground interaction by similarly inducing the release of a C12 terpene, pregeijerene, and disconnecting the association of the terpene with insect presence. D. abbreviatus larvae that were not feeding but in the presence of a CLas-infected plant were more likely to be infected by EPN than those near uninfected plants. Furthermore, nonfeeding larvae associated with CLas-infected plants were just as likely to be infected by EPN as those near noninfected plants with D. abbreviatus larval damage. Larvae of two weevil species, D. abbreviatus and Pachnaeus litus, were also more attracted to plants with infection than to uninfected plants. D. abbreviatus larvae were most active when exposed to pregeijerene at a concentration of 0.1 µg/µl. We attribute this attraction to CLas-infected plants to the same signal previously thought to be a herbivore-induced plant volatile specifically induced by root-feeding insects, pregeijerene, by assessing volatiles collected from the roots of infected plants and uninfected plants with and without feeding D. abbreviatus. Synthesis. Phytopathogens can influence the structuring of soil communities extending to the third trophic level. Field populations of EPN may be less effective at host-finding using pregeijerene as a cue in citrus grove agroecosystems with high presence of CLas infection.

Innate immune system capabilities of the Asian citrus psyllid, Diaphorina citri.

Citrus production worldwide is currently threatened by Huanglongbing, or citrus greening disease. The associated pathogen, Candidatus Liberibacter asiaticus (CLas), is transmitted by the Asian citrus psyllid, Diaphorina citri. Annotation of the D. citri genome revealed a reduced innate immune system lacking a number of antimicrobial peptides and the Imd pathway associated with defense against Gram-negative bacteria. We characterized this apparent immune reduction in survival assays in which D. citri were exposed to Gram-negative or Gram-positive bacteria. D. citri experienced significant mortality when exposed to Serratia marcescens (Gram-negative) through oral ingestion or by septic injury. Escherichia coli (Gram-negative) also caused significant D. citri mortality, but only when inoculated at high concentrations through oral ingestion or by septic injury. Neither Micrococcus luteus (Gram-positive) or Bacillus subtilis (Gram-positive) caused significant mortality as compared to controls in any experiment. E. coli titers increased rapidly following exposure, while M. luteus titer remained stable for 72 h. We demonstrate that D. citri is capable of defending against E. coli, a Gram-negative bacterium, despite lacking the Imd defense pathway. The tolerance of D. citri to M. luteus infection, yet inability to effectively clear infections, presents questions to efficacy of D. citri immune response to effectively clear Gram-positive infections.

Risk assessment of various insecticides used for management of Asian citrus psyllid, Diaphorina citri in Florida citrus, against honey bee, Apis mellifera.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is a major pest of citrus trees worldwide. A wide variety of insecticides are used to manage D. citri populations within citrus groves in Florida. However, in areas shared by citrus growers and beekeepers the use of insecticides may increase the risks of Apis mellifera  L. (Hymenoptera: Apidae) loss and contaminated honey. The objective of this research was to determine the environmental toxicity of insecticides, spanning five different modes of action used to control D. citri, to A. mellifera. The insecticides investigated were imidacloprid, fenpropathrin, dimethoate, spinetoram and diflubenzuron. In laboratory experiments, LD50 values were determined and ranged from 0.10 to 0.53 ng/µl for imidacloprid, fenpropathrin, dimethoate and spinetoram. LD50 values for diflubenzuron were >1000 ng/µl. Also, a hazard quotient was determined and ranged from 1130.43 to 10893.27 for imidacloprid, fenpropathrin, dimethoate, and spinetoram. This quotient was <0.447 for diflubenzuron. In field experiments, residual activity of fenpropathrin and dimethoate applied to citrus caused significant mortality of A. mellifera 3 and 7 days after application. Spinetoram and imidacloprid were moderately toxic to A. mellifera at the recommended rates for D. citri. Diflubenzuron was not toxic to A. mellifera in the field as compared with untreated control plots. Phenoloxidase (PO) activity of A. mellifera was higher than in untreated controls when A. mellifera were exposed to 14 days old residues. The results indicate that diflubenzuron may be safe to apply in citrus when A. mellifera are foraging, while most insecticides used for management of D. citri in citrus are likely hazardous under various exposure scenarios.

Tale of the Huanglongbing Disease Pyramid in the Context of the Citrus Microbiome.

The Huanglongbing (HLB) disease pyramid is composed of Liberibacters, psyllid vectors, citrus hosts, and the environment. The epidemiological outcomes for Liberibacter-associated plant diseases are collectively determined by the inherent relationships among plant-Liberibacters-psyllids, and how various environmental factors affect plant-Liberibacter-psyllid interactions. Citrus-Liberibacter-psyllid interactions occur in a complex microbiome system. In this review, we focus on the progress in understanding the HLB disease pyramid, and how the microbiome affects the HLB disease pyramid including the interaction between HLB and the citrus microbiome; the interaction between Liberibacters and psyllids; the interaction between Liberibacters and gut microbiota in psyllids; and the effect of HLB on selected above- and belowground citrus pathogens. Their implications for HLB management are also discussed.