1a. Objectives (from AD-416):
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.
1b. Approach (from AD-416):
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.
3. Progress Report:
Progress was achieved toward all objectives during this first year of a 5-year project. Progress (Obj.#1) included commercialization of a mating disruption product for control of citrus leafminer (an important contributor to the incidence and severity of citrus canker disease), discovery of phagostimulants for the Asian citrus psyllid (vector of citrus greening disease), and progress towards identifying host plant volatile compounds that elicit behavioral responses from weevil pests. Sources of host plant resistance were identified in Poncirus trifoliata, a near-Citrus relatives and hypotheses were generated regarding the mechanism of resistance in that taxon. Regarding, the interaction between a parasitoid wasp and a fungal pathogen of Asian citrus psyllid was investigated. Field epizootics were observed and pathogens collected. Bioassays were developed to screen bactericides for control of the causal agent of Citrus greening disease. Bactericide activity of compounds including a novel peptide was demonstrated. Applications in commercial citrus groves were conducted to optimize bactericide penetration of plant tissues. Improvement in tree health and reduction of disease titer were confirmed. Novel peptides were discovered including one with bactericidal activity and others that bind to psyllid gut epithelium and block disease acquisition by the psyllid vector. Double-stranded-Ribonucleic acid (dsRNA) molecules previously shown to induce psyllid mortality through oral delivery were shown to reduce nymph development and reduce disease acquisition. These advances are detailed below: Plants hormones mediate plant responses to stress. Citrus trees treated with salicylic acid emitted greater amounts of odor compounds and produced very high levels of methyl salicylate. Asian citrus psyllid (ACP) aggregations (greater than 10 psyllids) occurred more frequently on leaves treated with methyl jasmonate compared with untreated sprigs. Treatment with plant hormones may induce the release of biologically active compounds that can be used to enhance detection and monitoring of Asian citrus psyllid (ACP). Multiple sensory modalities influence Asian citrus psyllid (ACP) orientation. These include a strong visual orientation to yellow, possible response to olfactory stimuli and, we now show, response to feeding stimulants (phagostimulants) perceived only after alighting. Initial attempts to demonstrate antennal response to volatile compounds 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 demonstrated that these compounds degrade spontaneously in air to smaller molecules including formic and acetic acids that elicit strong antennal responses. These compounds are not involved in orientation by olfaction but do stimulate probing and feeding responses in psyllids. Mixture designs and response surface models identified an optimal 3-component blend of the two acids and para-cymene that elicits probing and production of psyllid salivary sheaths. We are pursuing an attract-and-kill formulation that incorporates visual, olfactory and gustatory stimuli. We are also considering the potential for combining this approach with a repellent. Nine of 17 repellents evaluated were effective against ACP in the lab. Guava oil extracts and leaves from five guava cultivars repelled ACP. Sulfur compounds associated with guava had previously been shown to repel psyllids but other guava constituents were also repellent. A pheromone-based product for mating disruption of citrus leafminer became commercially available to citrus growers in Florida, California and elsewhere. The product is the result of collaboration between Agriculture Research Service (ARS) researchers and partners in industry, academia and citrus production. Through application of advanced statistical methods, we identified optimal pheromone formulations, developed and tested delivery systems and guided product development and release. The product provides citrus growers worldwide with a new, environmentally benign tool for control of citrus leafminer and citrus canker disease. Behavioral and electrophysiological methods identified a set of candidate volatile compounds present in peach and other host plants susceptible to feeding by the Sri Lankan weevil. These will be investigated for use in traps and lures. Asian citrus psyllid (ACP) infestation was reduced on Poncirus trifoliata compared with susceptible Citrus genotypes. Six Poncirus trifoliata cultivars reduced ACP infestations compared with sweet orange. Research was initiated on plant chemicals responsible for resistance including volatiles and secondary plant compounds such as flavonoids. Trichomes of young leaves of Poncirus trifoliata were described that may deter oviposition. Nine genotypes susceptible to colonization by ACP were compared for suitability as host plants for mass-rearing the psyllid. Based on plant architecture and growth characteristics in conjunction with ACP performance, the following were the best candidates: alemow, curry leaf, lime, orange jasmine, and sour orange. An inoculation program was developed to evaluate plant resistance to the bacterium responsible for citrus greening disease. Individual plants were infested with infected psyllids for two-weeks and held for 6 months in a greenhouse with an open infestation of infected psyllids. Over 8,600 scion or rootstock accessions have been subjected to inoculations involving more than 170,000 psyllids. On-going research indicates that no-choice inoculation alone is effective 79% of the time when large percentages of psyllids are infected. Transmission by Asian citrus psyllid (ACP) of the bacterium responsible for Citrus greening disease increased when young leaves were present. Citrus should be protected from psyllid infestations whenever trees are producing new leaves. Feeding behavior of Asian citrus psyllid (ACP) on a range of citrus and near-citrus relatives was characterized by electronic feeding monitor. Waveforms produced by healthy adult psyllids and psyllids infected with the greening pathogen were recorded on healthy citrus and citrus infected with the pathogen. Results are being analyzed. A computer program was developed to automatically classify insect feeding patterns involved in pathogen transmission. This analysis contributed to discovery of previously unrecognized feeding states and can be used to characterize plant resistance mechanisms, reduce time and effort required to analyze insect feeding, and facilitate development of novel intervention strategies to disrupt pathogen transmission. Two large epizootics of the entomopathogenic fungus Hirsutella citriformis attacking Asian citrus psyllid (ACP) were observed. Samples were submitted to Agriculture Research Service in Ithaca, New York, and shared with ARS in Peoria, Illinois, for in-vitro cultures. Serial propagations of the culture were unsuccessful and the culture was lost. Research will reconvene this fall when the fungus is most prevalent. Wasp foraging behavior was observed to select candidate insectary plants to support Tamarixia radiata, a tiny wasp that parasitizes Asian citrus psyllid. The wasp obtained nectar only from flowers with exposed nectaries and from plants with extra-floral nectar glands. The wasp readily ingested sugars in nectar and insect honeydew, and recognized scents associated with nectar. Introduction of insectary plants could enhance biological control of ACP. A laboratory study examined interactions between Tamarixia radiata and spores of Isaria fumosorosea, a fungus that attacks Asian citrus psyllid (ACP). Isaria fumosorosea decreased oviposition, abundance, and reproductive success of Tamarixia radiata. The wasp is most active in late spring and summer. The fungus is most active in the fall and early spring. Nineteen of 28 accessions of Bacillus thuringiensis had some toxicity to adult ACP. Depending on dose, psyllids feeding on toxic accessions slowly died with 100% mortality sometimes occurring within 4 to 5 days. Gut-binding proteins are being explored as an adjuvant for increasing speed of mortality. In collaboration with ARS in Ithaca, New York, Asian citrus psyllid colonies were established from individual females collected at locations across Florida. Iso-female lines are being evaluated and compared for their genetic ability to acquire and transmit the bacterium responsible for citrus greening disease. Pathogen acquisition varied between lines from 5 to >45%. Double-stranded Ribonucleic acid molecules induced Asian citrus psyllid (ACP) mortality when taken up by the psyllid in an artificial feeding system by selectively silencing an essential gene function. ACP fed on plants that produced these dsRNA molecules showed reduced nymphal development and reduced acquisition of the pathogen. The transformed plants were produced by infecting citrus trees with a non-pathogenic virus engineered to produce the specific RNA molecules. Bioassays were developed and validated for screening molecules for bactericide activity against the causal agent of citrus greening disease. The bioassays identified a novel peptide that reduced detectable citrus greening disease bacteria by 90% within citrus leaves after 6 days (invention disclosure forthcoming), and three peptides that bind to the psyllid gut epithelium and inhibited pathogen acquisition by the psyllid. Commercial grove evaluation of bactericide formulations were conducted to optimize penetration into plant tissues. Significant improvement in tree health and reduction in pathogen titer were documented; data were used to obtain Florida state crisis approval for use in commercial citrus production.
1. A new tool delivered for fight against citrus greening disease. Bactericide application technology became available to Florida citrus growers in 2016 to fight citrus greening disease. Agriculture Research Service (ARS) researchers at Fort Pierce, Florida, demonstrated that bactericide formulations designed to penetrate citrus trees and reach the phloem reduced bacterial titer and improved tree health. Data provided to the State of Florida and federal Environmental Protection Agency (EPA) were the basis for approval of these bactericides for use in Florida. It is estimated they are now being used on the majority of Florida’s commercial citrus orchards.
2. Promising peptide with unique mode of action effective against citrus greening disease. Agriculture Research Service (ARS) researchers at Fort Pierce, Florida, identified a novel peptide that kills the bacterium associated with the greening disease. The peptide entered citrus phloem and reduced pathogen titer by approximately 90 percent in intact citrus leaf assays. Further analysis showed the peptide to be active against other bacteria including Escherichia coli. Based on sequence and mode of action, this peptide represents a new class of antibacterial peptides that could be used to fight a number of bacterial pathogens in agriculture and/or human health.
3. Protect young citrus leaves from Asian citrus psyllid to limit citrus greening disease. Research on the Asian citrus psyllid and citrus greening disease showed that transmission greatly increased when young leaves were present. Agriculture Research Service (ARS) researchers at Fort Pierce, Florida, found that citrus was more likely to contract the disease if young leaves were present. Based on this finding, citrus should be protected from psyllid infestations whenever trees are producing new leaves to reduce spread and severity of citrus greening. The research was highlighted on the Entomological Society of America website and featured in AgResearch Magazine.
4. A new class of behaviorally active compounds stimulate feeding in Asian citrus psyllid. An effective trap has long been sought for the Asian citrus psyllid. The psyllid is strongly attracted to the color yellow but is only weakly attracted by odors. Agriculture Research Service (ARS) researchers at Fort Pierce, Florida, discovered a set of compounds that induce the psyllid to probe and feed after alighting upon a potential host. An optimal blend of 3 compounds was identified that induced psyllids to attempt to feed on an inert wax substrate. A retrievable eradication device is envisioned that capitalizes on 3 sensory modalities: visual attraction to yellow, olfactory attraction to citrus odors and gustatory response to phagostimulants.
5. Mating disruption device marketed for control of citrus leafminer and citrus canker disease. The citrus leafminer, a small moth, causes direct damage to citrus and facilitates spread of citrus canker disease. Agriculture Research Service (ARS) researchers at Fort Pierce, Florida, worked with industry, academic and commercial citrus partners to develop and commercialize a mating disruption product based on the sex pheromone of female leafminers. Advanced statistical methods identified optimal pheromone formulations. Experiments conducted in commercial citrus groves tested delivery systems and guided product development and release. The product provides citrus growers worldwide with a new, environmentally benign, nontoxic tool for control of the citrus leafminer and citrus canker disease.
5. Significant Activities that Support Special Target Populations:
Hall, D.G., Albrecht, U., Bowman, K.D. 2016. Transmission rates of ‘Ca. Liberibacter asiaticus’ by Asian citrus psyllid are enhanced by the presence and developmental stage of citrus flush. Journal of Economic Entomology. 109:558-563.
Stover, E., Inch, S., Richardson, M., Hall, D.G. 2016. Conventional citrus of some scion/rootstock combinations show field tolerance under severe huanglongbing disease pressure. HortScience. 51:127-132.
Edwin, E., Vasantha-Srinivasan, P., Senthil-Nathan, S., Thanigaivel, A., Ponsankar, A., Selin-Rani, S., Kalaivani, K., Hunter, W.B., Duraipandiyan, V., Al-Dhabi, N. 2016. Effect of andrographolide on phosphatases activity and cytotoxicity against Spodoptera litura. Invertebrate Survival Journal. pp. 13153-13163.
Yang, C., Powell, C., Duan, Y., Shatters, R.G., Lin, Y., Zhang, M. 2016. Mitigating citrus huanglongbing via effective application of antimicrobial compounds and thermotherapy. Crop Protection Journal. 84:150-158.