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Research Project: IPM Methods for Insect Pests of Orchard Crops

Location: Subtropical Insects and Horticulture Research

2018 Annual Report

Objectives
Objective 1: Discover, develop and implement semiochemical-based control and monitoring methods for key pests of orchard crops including, but not limited to, Asian citrus psyllid, citrus leafminer, citrus canker disease, and the Diaprepes root weevil. Sub-objective 1a: Identify physiologically active odorants and attractant blends for detection and monitoring of ACP. Sub-objective 1b: Complete large scale tests and promote adoption of CLM mating disruption. Sub-objective 1c: Identify attractants for DRW and Sri Lankan weevil. Objective 2: Identify sources of resistance and characterize traits and mechanisms conferring plant resistance to the Asian citrus psyllid in Citrus and near-Citrus relatives. Sub-objective 2a: Identify and determine the underlying mechanism of resistance in Poncirus trifoliata to oviposition by Asian citrus psyllid (ACP). Sub-objective 2b: Describe feeding behavior of ACP on susceptible and resistant citrus and near-citrus germplasm. Objective 3: Develop and implement new and improved biological control strategies for key pests of citrus, including Asian citrus psyllid, using existing and new natural enemies. Sub-objective 3a: Biological control of Asian citrus psyllid by Hirsutella citriformis. Sub-objective 3b: Development of an autodisseminator of entomopathogens to suppress ACP populations. Objective 4: Develop and implement control of key pests and vectors including, but not limited to, Asian citrus psyllid by 1) identifying interdiction points in key biological processes through genomics, proteomics and metabolomics, 2) identifying inhibitors (dsRNA, peptides, chemicals), and 3) developing delivery methods, e.g., transgenic plants and topical applications of exogenous compounds. Sub-objective 4a: Combining molecular/cellular biology (including targeted and omics level research) with bioassays to identify interdiction molecules including but not limited to dsRNAs (as RNAi inducers), peptides, peptidomimetics and RNA aptamers that block key molecular events in targeted processes such as, but not limited to, salivary sheath formation, specific digestive processes, and/or disease transmission. Sub-objective 4b: Develop delivery strategies for interdiction molecules.

Approach
Insect-plant interactions are varied and complex. The processes of host location, selection, feeding, and oviposition are only broadly understood, and for relatively few species. In the case of recent arrivals of invasive pests of orchard crops, these aspects of pest biology are not understood in the detail required to design appropriate, novel, and environmentally sound management strategies, such as the following examples. Information-transmitting odors (semiochemicals) can often be inexpensively synthesized and used to interfere with insect pest behavior. Also, understanding the physical or biochemical basis for plant resistance to insects allows engineering or selection of crop varieties with endogenous resistance. In the case of invasive vectors of plant pathogens, lack of understanding of the mechanisms of pathogen transmission (i.e., acquisition, retention and inoculation) further impedes progress in pest management. These mechanisms are also complicated, and are layered onto the complex biological processes described above. The objectives of this project focus on both vector and non- vector pests in orchards. They address discovery, study and utilization of: 1) semiochemicals and other physical or chemical bases of host plant resistance, 2) mechanisms involved in host plant resistance in compatible near-Citrus germplasm, 3) new biological control agents and novel utilization of known ones, and 4) key biological processes that represent opportunities for interdiction of insect-host interactions. Together, these projects aim to design all-new biological control and non-pesticidal management strategies. An advantage of these approaches is their compatibility with existing, especially pesticidal, methods in citriculture. Several of the approaches are broadly applicable to a range of subtropical orchard crops.

Progress Report
Progress was achieved toward all objectives during this third year of a 5-year project. Development of a commercially marketed product for mating disruption of citrus leafminer was essentially completed. Substantial progress was made in combining components that exploit Asian citrus psyllid (ACP) sensory modalities in an attract-and-kill device. The device incorporates a bright yellow exterior with attractant odorant to bring ACP to a wax matrix that includes a phagostimulant blend to induce feeding and an insecticide that provides quick knockdown. The device has been tested in environmental chambers and field cages. ACP mortality was consistently high. The device does not become fouled with dead psyllids and nontarget insects, lizards, toads, etc. as do traditional yellow sticky card traps. Field longevity of the device was estimated to be 3 months or longer. Electroantennogram and olfactometer studies indicated that Sri Lankan weevils respond to hexanol, a common plant odor. Field tests are underway to determine attraction under field conditions and estimate optimal concentration for a trap. Scent lures for Asian citrus psyllid (ACP) were formulated. Young sweet orange shoots were sprayed with methyl jasmonate, a plant signaling compound that elicits plant defense responses. Odors emitted by shoots following treatment were analyzed. Scent lures were formulated based on odor compounds responsible for most of the variation between treated and untreated shoots and tested in 3-D traps from the Florida Department of Plant Industry. More ACP were attracted to traps with sweet orange scent than to lures based on other citrus types. Poncirus trifoliata (trifoliate orange) is resistant to oviposition by Asian citrus psyllid (ACP). Laboratory assays showed that Poncirus leaves emit volatiles that discourage oviposition, and candidate chemicals responsible were identified. Field surveys of 24 citranges (hybrids between Poncirus and sweet oranges) revealed none displayed reduced ACP infestations. The research is being extended beyond the citranges. Research was abandoned on formulations of Hirsutella citriformis for biocontrol of Asian citrus psyllid (ACP) due to retirement of a primary ARS collaborator at Peoria, Illinois. Contingency research was pursued. In vivo and in vitro cultures were established in collaboration with ARS at Ithaca, New York to elucidate the biology of the fungus and to phenotype five isolates. An epizootic of the fungus was discovered that facilitated field evaluations of seasonal phenology of the pathogen. A new potential pathogen of ACP in Florida was discovered and tentatively identified as a species of Sporothrix. A collaborative study with University of California and Florida Department of Agriculture showed that the bacterium responsible for citrus greening disease remained detectable for up to six weeks in Asian citrus psyllid (ACP) captured in traps containing propylene glycol. Growers and regulators wanting to monitor ACP for the pathogen will benefit if traps with propylene glycol are used. Although infected ACP can be detected using yellow sticky cards, traps can only be left outdoors for a minimal time period because heat and sunlight degrade bacterial DNA. A study with greenhouse seedlings was completed on transmission rates of citrus greening disease by Asian citrus psyllid (ACP). Seedlings exposed for 2 weeks to 20 adult ACP from infected colonies developed the disease 77% of the time. Transmission rates were positively correlated with the percentage of ACP that tested positive for the pathogen but not correlated with titers of the pathogen. Variability in transmission rates may have been influenced by differences in sex ratios or ages of adults. Although seedlings were treated with an insecticide after inoculation and remained ACP-free thereafter, the presence and abundance of nymphs from eggs laid during the inoculation period may have influenced transmission rates. Changes in sunlight are known to affect the biology and behavior of the Asian citrus psyllid. Research conducted in a glasshouse insectary with psyllids caged on plants under different light regimes. However, percentages of eggs that hatched and/or of nymphs that developed to adult were positively affected by increases in light. Significant positive correlations were found between numbers of adults produced on plants and solar radiation, light intensity and daily hours of illumination. A team of ARS researchers collected male and female pairs of the Asian citrus psyllid (ACP) throughout Florida, maintained these as separate genetic lines in the lab, and tested them for competency as vectors of the citrus greening pathogen. ACP populations varied in their ability to acquire and transmit the pathogen. The ability or inability to transmit the pathogen was passed from parents to offspring for several generations, proving that ACP genes regulate the spread of the pathogen. These populations varying in transmission competency may lead scientists to new ways of halting the spread of citrus greening disease. An evaluation was completed of mosquito repellents and essential plant oils as deterrents of the Asian citrus psyllid. Sixteen of 22 candidate insect repellents were significantly repellent in laboratory assays, but none were 100% repellent. Dose-repellency experiments were conducted with three repellents – for each, a concentration of 13% generally reflected a threshold below which repellency was insignificant and above which no significant increase in repellency occurred. In greenhouse studies with eight repellents applied directly to plants as 5 or 25% solutions, reductions in numbers of psyllids infesting plants occurred but this was attributed mainly to plant damage caused by the repellents and not actual repellency. Field evaluation was conducted on citrus trees infected with a Citrus Tristeza Virus (CTV) vector engineered to deliver double-stranded ribonucleic acid (dsRNA) molecules identified by ARS at Ft. Pierce. Further evaluation of these dsRNAs was conducted using a single leaf bioassay and direct delivery of small stable RNA analogs that carry a 2'-deoxy-2'-fluoro-beta-D-arabinose sugar modification. The analogs bind messenger ribonucleic acid (mRNAs) resulting in ribonuclease H-mediated destruction of matching messenger RNA. In this assay, greater mortality was observed in ACP feeding on leaves treated with the mRNA-binding analogs compared with controls. Methods of delivery to citrus using field-deployable strategies are now being evaluated. We used 3-D printing to construct devices to deliver bioactive molecules to whole citrus trees. Devices were attached to trees to systemically deliver currently available bactericides and novel bactericidal proteins to kill the huanglongbing-causing bacteria within citrus vascular tissues. An agrichemical company is interested in this technology and is supporting a CRADA that provides funds for laboratory/greenhouse/field evaluation of this method. ARS researchers at Ft. Pierce, Florida, developed and led a team effort that resulted in the awarding of a $1 million grant from the Defense Advanced Research Projects Agency to develop a synthetic bacterial species related to the bacterium that causes citrus greening. This new strain will be designed to be nonpathogenic, i.e., not induce citrus greening symptoms, and to outcompete the pathogenic bacteria that cause huanglongbing. This project has already developed and cloned a synthetic genome into yeast and demonstrated its integrity. Experiments now underway will transplant the artificial genome into a bacterium to displace the bacterium’s natural genome. We also demonstrated methods for direct delivery of HLB-causing bacteria into citrus and periwinkle. This is the first successful mechanical delivery of the pathogen to citrus and paves the way for delivering the synthetic cross-protecting strain once it is established. ARS researchers at Ft. Pierce, Florida, led an initiative to assemble ARS scientists from multiple ARS locations to develop an ARS Grand Challenge proposal. This Grand Challenge was awarded and has resulted in a collective multidisciplinary ARS team working together to integrate research efforts. The effort has resulted in development of novel and integrated strategies for control of citrus greening, e.g., collective development of a proposal for evaluation of a new spray technology awarded through the USDA Multi-Agency program. An improved genome and official gene set of the Asian citrus psyllid were completed. The Open Source Datasets are hosted at and the NCBI database. Key biological pathways were built, and multiple genes annotated for immunity, digestion, salivary gland enzyme, and insecticide resistance. Improved accuracy of gene sequences aided selection of effective biopesticides for psyllid control. Patents were submitted. Double-stranded Ribonucleic acid was used to produce genetically modifified (GMO) citrus to evaluate potential to suppress psyllids, reduce bacteria transmission and improve citrus tree yield. Genomic data were used to produce antisense oligonucleotides that suppress bacteria. The regenerating trees have been rooted in tissue culture and shown to be transgene positive. They have not yet been tested for expression of the inserted construct. A patent has been submitted. Trials were initiated with two commercial companies on a new insect detection and control technology called the ‘Photonic Fence’, to be aimed at controlling Asian citrus psyllid. This technology uses a number of light modalities, precision optics, and sophisticated software to detect, identify, track, and kill flying pest insect species. The system can be programmed to kill only certain pest species and is highly specific.

Accomplishments
1. A novel attract-and-kill device to provide long-lived control of the Asian citrus psyllid, vector of the deadly citrus greening disease. ARS researchers at Ft. Pierce, Florida, combined visual, olfactory and gustatory cues in a single device that attracts the psyllids and then induces them to attempt to feed on a toxic wax substrate. The device provides consistent psyllid mortality over a period of at least 3 months and does not become fouled with insects and nontarget organisms as do traditional yellow sticky card traps. The device is expected to provide an alternative to insecticide sprays applied directly to the citrus crop thereby reducing environmental and health concerns.

2. Proper application of commercially available bactericides provide yield improvements in huanlongbing (HLB)-infected citrus trees in Florida. The efficacy of two bactericides (oxytetracycline and streptomycin) registered for use against the HLB-causing bacterium is limited due to the difficulty of delivering these to the tree vascular tissue (phloem) where the bacteriua are located. ARS researchers at Ft. Pierce, Florida, demonstrated significant improvement in crop yield after one year of antibiotic applications when combined with certain adjuvants that facilitate penetration. A Cooperative Research and Development Agreement (CRADA) has been established with an agricultural chemical company to optimize the bactericide/adjuvant blend and to promote rapid commercialization.

U.S. DEPARTMENT OF AGRICULTURE

Subtropical Insects and Horticulture Research: Fort Pierce, FL