Project : USDA ARS

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Research Project: IPM Methods for Insect Pests of Orchard Crops

Location: Subtropical Insects and Horticulture Research

2019 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
An attract-and-kill device was developed that exploits Asian citrus psyllid (ACP) response to a combination of visual, olfactory, and taste stimuli. The device incorporates a bright yellow exterior with attractant odorant to bring ACP to a wax matrix that includes a stimulatory blend to induce feeding and an insecticide that provides quick knockdown. The device was tested in field cages and in small scale field trials. ACP mortality was consistently high. The device does not become fouled with dead psyllids and nontarget insects as do traditional yellow sticky card traps. Field longevity of the device was estimated to be 3 months or longer. Development of a commercially marketed product for mating disruption of citrus leafminer was essentially completed. The scientist in charge of the project retired and the project has been terminated. Electroantennogram and olfactometer studies indicated that Sri Lankan weevils responded to hexanol, a common plant odor. The scientist in charge of the project retired and the project has been terminated. 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 scientists in charge of the project retired and the project has been terminated. In vivo and in vitro cultures of Hirsutella citriformis were established in collaboration with another ARS location to elucidate the biology of the fungus and to phenotype five isolates. The scientist in charge of the project retired and the project has been terminated. Field trials in residential areas were conducted in south Texas with collaborators from Texas University. The field trials showed that placing autodispensers of fungal spores that kill Asian citrus psyllid in backyard trees was as effective as whole tree spraying with a commercial formulation of the fungal spores. The fungal spores do not remain viable for longer than two weeks, so ways must be found to improve spore survivability over longer periods of time. The most effective control molecules we could identify were two antimicrobial peptides, not gut binding peptides, so we redirected to advance the delivery of the antimicrobial peptides to citrus. We designed and printed tree delivery devices and used them to deliver one antimicrobial peptide. This resulted in a greater than 90% reduction in the Candidatus Liberibacter asiaticus (CLas) bacterium in 1 meter potted citrus trees one week after application that persisted for at least four weeks. ARS researchers are now working with a partner to develop commercially viable delivery devices and will initiate field evaluations during the next year. An ARS scientist in Ft. Pierce, Florida, developed new Ribonucleic acid interference (RNAi) and Morpholino type-antisense oligonucleotide products to reduce Asian citrus psyllid (ACP) and Candidatus Liberibacter asiaticus (CLas). They increased psyllid mortality by 70-100% and reduced huanglongbing (HLB)- and Zebra chip-causing bacteria loads by 40-70% in plants. These were patented and a commercial company partner for antisense oligonecleotide products was found. A commercial partner to produce RNAi to suppress ACP is being sought. Two sets of transgenic citrus have been developed, each expressing a different proven antimicrobial peptide. One set was shown to induce Asian citrus psyllid mortality in adults that fed on the plants for one week. Further evaluations are underway for analysis of acquisition and transmission of Candidatus Liberibacter asiaticus (CLas). Slow release clay to extend crop protection times was shown to deliver product over a 12-month period in glasshouse trials. A field trial is scheduled. Clay product is commercially available, and information is dispersed through presentations to industry and grower groups. Field trials at a local public display garden are identifying which plant species can be grown to attract and sustain the natural enemies of the Asian citrus psyllid in target landscapes. This ‘conservation biological control’ strategy will be useful for suppressing psyllid populations in situations where chemical spray controls are not practical or permitted, such as residential landscapes and border areas of commercial citrus groves. The approaches developed in these trials will be used to attract and conserve natural enemies of Asian citrus psyllid in residential and commercial landscapes and along the edges of commercial citrus groves. Trials were conducted with a commercial company on a new insect detection and control technology called the ‘Photonic Fence’, to be used for controlling insect vectors of pathogenic organisms, including Asian citrus psyllid and mosquitoes. 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. The trials demonstrated that the system could efficiently identify, track, and kill the yellow fever mosquito, Aedes aegypti. The system was not as competent in tracking Asian citrus psyllid and refinements in the optical detection system are underway.

Accomplishments
1. Oligonucleotides suppress citrus greening & Asian citrus psyllid. Bacterial pathogens in plants are difficult to target due to biofilms of gram negative microbes. A set of novel Morpholino type - antisense oligonucleotides which could move through the biofilm and kill bacteria were developed and patented by ARS scientists in Ft. Pierce, Florida. These were shown to reduce bacteria titers in bacteria infected citrus and potato. These products provide potential treatments for suppressing Huanglongbing disease in citrus, and may provide new management products to manage pathogens of citrus trees.

2. Oligonucleotides disrupt psyllid endosymbionts. Insect vectors of plant pathogens, like psyllids and bacterial plant pathogens, are the number one problem in citrus production. A set of antisense oligonucleotides were developed and patented by ARS scientists at Ft. Pierce, Florida, under a cooperative research agreement with a commercial company. The oligonucleotides are highly specific, using complimentary gene sequences to suppress Asian citrus psyllids, or the psyllid’s endosymbionts, thereby disrupting psyllid biology. These oligonucleotides can also bind and kill pathogenic bacteria in plants, like citrus trees. ARS researchers are collaborating with an agricultural chemical company to move these towards commercialization and may provide new management products to reduce Huanglongbing, and reduce the Asian citrus psyllid and other psyllid vectors of plant pathogens.

U.S. DEPARTMENT OF AGRICULTURE

Subtropical Insects and Horticulture Research: Fort Pierce, FL