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ARS Home » Southeast Area » Fort Pierce, Florida » U.S. Horticultural Research Laboratory » Subtropical Insects and Horticulture Research » Research » Research Project #420741


Location: Subtropical Insects and Horticulture Research

2015 Annual Report

1a. Objectives (from AD-416):
1. Chemical Ecology: Develop semiochemical-based control methods for citrus pests, particularly Asian citrus psyllid (ACP). 1a. Develop behavioral assays and antennogram methods for key citrus pests to identify conspecific odors that mediate mate location, recognition or aggregation and use these compounds for control and monitoring. 1b. Insect-Plant Interactions: Identification of host and nonhost compounds that affect host selection, location or repellency. 2. Biological Control: Develop new and improve existing biological control methods for ACP and sharpshooter species. 2a. Establish natural enemies of ACP in Florida. 2b. Identify new viral pathogens of ACP and sharpshooter species, and evaluate the potential of these as management strategies. 3. Host Plant Resistance: Develop host plant resistance in citrus to key pests. 3a. Identify sources of plant resistance to ACP in citrus germplasm. 3b. Develop novel control methods based on disrupting key processes in pest biology.

1b. Approach (from AD-416):
Behavioral assays and antennogram (EAG/EAD) methods will be developed for key citrus pests to identify conspecific odors that mediate mate location, recognition or aggregation. Methods for monitoring and control using these compounds will be developed. Host and nonhost compounds will be identified that affect host selection, location or repellency through the use of gas chromatography (GC), GC-EAD, and GC-mass spectroscopy (GC-MS). To increase natural biological control of the Asian citrus psyllid, haplotypes of the parasitoid Tamarixia radiata will be characterized and released as deemed appropriate. Viral pathogens of Asian citrus psyllid and sharpshooter species will be identified by genomic methods and evaluated as management strategies. Existing citrus germplasm will be screened for host plant resistance to Asian citrus psyllids under greenhouse and field conditions. Genes and proteins involved in key biological processes such as salivary sheath formation will be studied to develop novel control strategies that block these processes.

3. Progress Report:
Mating disruption of the citrus leafminer was developed based on extensive field work resulting in a new product that has been refined and applied to approximately 3,000 acres of commercial citrus for control of citrus leafminer and citrus canker disease in 2015. Based on 2014 results, the pheromone loading rate was increased for 2015. Results and cost/benefit analyses are pending. The Product Development Committee of the Citrus Research and Development Foundation subsidized the launch through collaboration between ARS, University collaborators and a private company along with 3 commercial citrus groves. Adoption by industry was limited by production capability of the manufacturer. Development of attractants for the Asian citrus psyllid (ACP) has been difficult due to the paucity of chemoreceptors on the antenna and the fact that multiple sensory modalities are involved in ACP orientation. Initial attempts to demonstrate antennal response to a panel of volatile compounds known to be released by leaves of citrus and related plants were negative. However, ACP antennae responded strongly to breakdown products of common citrus leaf volatiles, ß-ocimene and citral. We discovered that these compounds degrade in air to smaller molecules including formic and acetic acids. Mixture and a mixture-amount experiment designs identified an optimal 2-component blend of the two acids. Probing by ACP was greatest on a wax matrix containing a 2:1 blend of formic:acetic acid. Addition of a third compound increased attractiveness. Synthetic ligands of olfactory binding proteins isolated from the antennae of Asian citrus psyllid (ACP) were shown to be highly attractive to ACP. These synthetic compounds enhanced the attractiveness of naturally-occurring host plant volatiles. Novel behavioral assays were developed to quantify the probing level of ACP exposed to the test materials. These synthetic compounds show potential as attractants to ACP by themselves and as enhancers of naturally occurring host plant volatiles. Progress was also made towards developing dispensers of attractant volatiles for ACP management. More ACP were captured in yellow sticky traps equipped with scent dispensers than in scentless controls. A prototype device that can be used to dispense the spores of pathogenic microorganisms to Asian citrus psyllid (ACP) was developed. Tests conducted in the greenhouse with free-flying psyllids showed that ACP will visit the dispenser and infect other psyllids dwelling on potted citrus trees. The dispenser can be hung from the branches of citrus and other host plants and will be useful for suppressing ACP populations in areas where insecticidal control is problematic, such as yards, parks, business zones and abandoned commercial orchards. A prototype laser-based system that can identify and kill target insect species was developed. The system recognizes the target insect species based on its wingbeat frequency. After the target insect is detected, it is killed with a low energy laser beam. The system could detect and identify flying Asian citrus psyllid (ACP) from a distance of 5 m; the laser pulse was sufficient to kill ACP. The system can be used to prevent insect vectors of pathogenic organisms from entering buildings such as greenhouses, human dwellings, and livestock sheds. The system can also be used (without the laser pulse) to census both pest and beneficial insects moving from one area to another. Genomes from two important hemipteran pests were completed as part of the national i5K Insect Genome Initiative: the glassy-winged sharpshooter and the Asian citrus psyllid. Data are publically available through the National Center for Biotechnology Information (NCBI). These are the first genomes for any leafhopper or psyllid. Working groups were established to improve annotations. The transcriptomes from these insect pests are being used by the larger research community and by ARS for development of Ribonucleic acid interference (RNAi) products for pest suppression in citrus and grapevines. Two significant products that suppress Asian citrus psyllid (ACP) and Diaprepes weevils were approved for patents. Development of an efficient delivery system for double-stranded Ribonucleic acid (dsRNA) into citrus trees and grapevines was completed. Efficacy of delivery of dsRNAs to citrus trees was evaluated over 8 months after application as a foliar spray, injection, or root drench. Also, citrus seedlings in pots received a soil drench and were transplanted to the field. The dsRNA products were detectable at 30 days post-treatment in leaf samples. In one set of trees studied, trees treated with dsRNA had better health characteristics compared with untreated trees. Colonization and oviposition by Asian citrus psyllid (ACP) were reduced on Poncirus trifoliata compared with susceptible citrus. Accessions of Poncirus trifoliata consistently resistant to colonization were identified. Research to determine factors responsible for resistance is underway. An investigation was concluded on Asian citrus psyllid (ACP) bionomics on orange jasmine. Populations were present year-round and pose risk to commercial citrus as psyllid reservoirs. Releases of three new haplotypes of the parasitoid Tamarixia radiata did not reduce ACP populations. Repetitive pruning of orange jasmine hedges may reduce biological control by the ACP parasitoid Tamarixia radiata. A new, effective behavioral assay was developed for investigating Asian citrus psyllid (ACP) attraction to plant volatiles. Mediocre response rates by ACP to volatiles have often been observed using traditional assay methods such as glass Y-tube olfactometers. Large percentages of ACP (e.g., 80 percent or more) consistently responded to attractants using the new assay. The assay is of value to chemical ecologists investigating attractants and repellents for the psyllid. Guava oil extracts, young leaves and mature leaves from five guava cultivars all exhibited repellency to Asian citrus psyllid (ACP). Oil extracts from young and to a greater extent, mature guava leaves were repellent. Sulfur compounds associated with guava may be repellent but guava oil extracts containing no sulfur compounds were repellent to ACP. Nine of 34 accessions of Bacillus thuringiensis showed toxicity to Asian citrus psyllid (ACP) adults. Accessions from USDA-ARS Beltsville were processed for testing by Iowa State University and sent to U.S. Horticulture Research Laboratory (USHRL) to be screened for toxicity. The bacteria were mixed into a sugar water diet, placed into a membrane sachet and fed to adult psyllids. Depending on dose, ACP feeding on toxic accessions slowly died with 100 percent mortality sometimes occurring within 4 to 5 days. Gut-binding proteins are being explored as an adjuvant for increasing speed of mortality. An inoculation program was developed to evaluate resistance to citrus greening disease in citrus transformed to express antimicrobial peptides and defensive proteins. Individual transformed plants were infested with huanglongbing (HLB)+ psyllids for two-weeks in a no-choice arrangement. Plants were then held for 6 months in a greenhouse with an open infestation of greening-infected infected psyllids. Over 7,000 transformed scion or rootstock accessions have been screened. A novel insecticide, cyantraniliprole, inhibited Asian citrus psyllid (ACP) feeding on citrus and reduced transmission of citrus greening disease. Psyllids exposed to leaves treated with this insecticide died more slowly than those exposed to a conventional insecticide. Antifeedants might have value in reducing spread of citrus greening disease. A system for delivery of bactericide to citrus was developed. Greenhouse experiments demonstrated penetration of topically applied compounds to the phloem. Successful treatment of citrus trees in commercial groves was demonstrated in ten Florida locations using grapefruit, Valencia orange or Hamlin orange. Treatments were applied in late summer through fall and evaluation of bacterial titer and plant health showed that some treatments were effective in reducing bacterial titer and improving tree health. Monitoring and second year treatments are continuing to determine impact on fruit quality and fruit yield and to support regulatory approval through interaction with private industry through an established Cooperative Research and Development Ageement (CRADA). We characterized peptides that bind to Asian citrus psyllid epithelial gut membrane. These are being screened for ability to block acquisition of the greening disease pathogen through the gut.

4. Accomplishments
1. Successful therapy for citrus greening disease delivers bactericide. There is an urgent need for methods to maintain the productivity of citrus trees infected with citrus greening disease. ARS researchers at Fort Pierce, Florida developed bactericide formulations in topical sprays capable of penetrating citrus tree bark and reaching the phloem where the greening bacteria reside. Field trials reduced the titer of the causal organism Candidatus Liberibacter asiaticus and improved tree vigor with undetectable nontarget effects on beneficial insects and honeybees. A Cooperative Research and Development Agreement was established; the ARS-industry team was awarded a major grant for field trials over 2 years.

2. A simple mating disruption device applied to 3,000 acres of citrus to control citrus leafminer and citrus canker disease. Mating disruption is a highly specific and environmentally benign method of pest control that has been pursued by ARS entomologists at Fort Pierce, Florida for citrus leafminer and the associated citrus canker disease. A simple elastomeric dispenser containing one component of the female sex pheromone was deployed in 2014 and 2015 to approximately 3,000 acres of commercial citrus groves in Florida. Results show prolonged trap catch disruption and reduction in leafminer infestations. The Citrus Research and Development Foundation is funding a two-year study of efficacy of this approach.

3. Novel approach blocks salivary sheath formation of an insect vector. Citrus greening disease continues to devastate citrus production in Florida and worldwide. ARS entomologists at Fort Pierce, Florida identified a strategy to block feeding by the vector, the Asian citrus psyllid, by inhibiting formation of the insect’s salivary sheath. Pure salivary sheaths were isolated and analyzed leading to identification of inhibitors of sheath synthesis that prevented psyllid feeding when applied to the leaf surface. Sheath disruptors are now being evaluated in laboratory and greenhouse trials.

4. Double-stranded Ribonucleic acid (dsRNA) molecules delivered to citrus trees control Asian citrus psyllid and a weevil. The use of dsRNA to induce Ribonucleic acid interference (also known as gene silencing) is a potentially powerful and highly specific tool for environmentally appropriate control of major pests. ARS entomologists at Fort Pierce, Florida in collaboration with the Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA, Brazil), showed that dsRNA products caused significant mortality, cessation of feeding, and reduced egg production when insects fed on treated citrus plants. Tests will now be expanded to validate this approach and identify a pathway to a commercial product.

5. Breakdown products of plant odors attract the Asian citrus psyllid. Attractants and/or repellents have long been sought for detection, monitoring and control of the Asian citrus psyllid (ACP), the vector of citrus greening disease. ARS entomologists at Fort Pierce, Florida discovered that common citrus odors spontaneously break down in air to smaller molecules, two of which are highly stimulatory to ACP antenna. Electroantennography revealed that ß-ocimene and citral degrade to compounds such as formic and acetic acids that elicit consistently high antennal responses. A bioassay was developed to allow ACP adults to probe beads of wax substrate containing varying proportions and amounts of formic acid, acetic acid, ß-ocimene and citral. A blend of 2:1 formic:acetic acid elicited the most probing and may form the basis of an attract-and-kill approach for ACP.

6. Environmental fate of foliar application of double-stranded Ribonucleic acid (dsRNA) products in citrus and soil. Nontarget effects of topically applied genetic constructs is a concern for researchers and consumers. ARS researchers at Fort Pierce, Florida in collaboration with the Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA, Brazil), demonstrated that double-stranded ribonucleic acid (dsRNA) was not detectable from foliage at three months post-treatment or from soil at 3 days after treatment. Rapid degradation of dsRNA in the environment and the high degree of specificity of dsRNA for pathogen or insect genes, suggest that nontarget effects of exogenously applied dsRNA is not a major concern.

7. New assay developed for investigating attractants and repellents of Asian citrus psyllid (ACP). ARS entomologists at Fort Pierce, Florida developed a behavioral assay for investigating ACP attraction to plant volatiles. Plant material of interest was contained in a large vial with a small entranceway. A positive response requires ACP to find the entrance, enter the vial, and settle on the plant material. High, consistent response rates (80 to 85 percent) occurred when vials were baited with young citrus leaves. But when a repellent was applied around a vial’s entrance, a response rate of only 8 percent occurred. The assay will be used to study attractants and repellents for Asian citrus psyllid.

8. Synthetic ligands of olfactory binding proteins attract Asian citrus psyllid (ACP). Attractants and/or repellents have long been sought for detection, monitoring and control of the Asian citrus psyllid (ACP), the vector of citrus greening disease. ARS entomologists at Fort Pierce, Florida demonstrated that synthetic compounds that bind with olfactory binding proteins isolated from ACP antennae are attractive to the psyllid and enhanced the attractiveness of naturally-occurring host plant volatiles. These findings will be used to develop scent baits to enhance detection and monitoring of ACP.

9. Pathogen spore dispenser to suppress Asian citrus psyllid (ACP) populations. Asian citrus psyllid (ACP) residing in residential areas and abandoned orchards can spread huanglongbing (HLB) to commercial orchards. Insecticidal treatment of these areas is problematic. ARS entomologists at Fort Pierce, Florida developed a prototype device to dispense spores of pathogenic micro-organisms to ACP. Greenhouse tests with free-flying ACP showed that ACP will visit the dispenser and infect other psyllids dwelling on potted citrus trees. The dispenser can be hung from the branches of citrus and other host plants and will be useful for suppressing ACP populations in yards, parks, business zones and abandoned commercial orchards.

Review Publications
Ruan, C., Hall, D.G., Liu, B., Duan, Y., Li, T., Hu, H., Fan, G. 2015. Host-choice behavior of Diaphorina citri Kuwayama (Hemiptera: Psyllidae) under laboratory conditions. Journal of Insect Behavior. doi: 10.1007/s10905-015-9488-2.
Ammar, E., Richardson, M.L., Abdo, Z., Hall, D.G., Shatters, R.G. 2014. Differences in stylet sheath occurrence and the fibrous ring (sclerenchyma) between xCitroncirus plants relatively resistant or susceptible to adults of the Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae). PLoS One. doi: 10.1371/PO.2014-0110919.
Ammar, E., Hall, D.G., Alvarez, J.M. 2015. Effect of Cyantraniliprole, a novel insecticide, on the inoculation of Candidatus Liberibacter asiaticus associated with citrus huanglongbing by the Asian citrus psyllid (Hemiptera: Liviidae). Journal of Economic Entomology. doi: 10.1093/jee/tov016.
Patt, J.M., Stockton, D., Meikle, W.G., Setamou, M., Agenor, M., Adamczyk, J.J. 2014. Innate and conditioned responses to chemosensory and visual cues in Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), vector of Huanglongbing pathogens. Insects. 5(4):921-941.
George, J., Morse, W.C., Lapointe, S.L. 2015. Morphology and sexual dimorphism of the weevil Myllocerus undecimpustulatus undatus (Coleoptera: Curculionidae). Annals of the Entomological Society of America. 108(3):325-332.
Lapointe, S.L., Keathley, C.P., Stelinski, L.L., Urrutia, W.H., Mafra-Neto, A. 2015. Disruption of the leafminer Phyllocnistis citrella (Lepidoptera: Gracillariidae) in citrus: effect of blend and placement height, longevity of disruption and emission profile of a new dispenser. Florida Entomologist. 98(2):742-748.
Willett, D..S., Stelinski, L.L., Lapointe, S.L. 2015. Using response surface methods to explore and optimize mating disruption of the leafminer Phyllocnistis citrella (Lepidoptera: Gracillariidae). Frontiers in Ecology and Evolution. 3:30.
Ammar, E., Hentz, M.G., Hall, D.G., Shatters, R.G. 2015. Ultrastructure of wax-producing structures on the integument of the melaleuca psyllid Boreioglycaspis melaleucae (Hemiptera: Psyllidae) and honeydew excretion behavior in males and females. PLoS One. doi: 10.1371/PO.2015-0121354.
Ammar, E., Hall, D.G., Shatters, R.G. 2015. Ultrastructure and development of the new stylets inside pre-molting first instar nymphs of the Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae). Florida Entomologist. 98:373-376.
Hall, D.G., George, J., Lapointe, S.L. 2015. Further investigations on colonization of Poncirus trifoliata by the Asian citrus psyllid. Crop Protection. 72:112-118.
Lapointe, S.L. 2015. A tribute to Dr. Anthony C. Bellotti and his contributions to cassava entomology. Florida Entomologist. 98(2):810-814.