Research Project: Development of Applied Management Systems for Diseases of Perennial Crops with Emphasis on Vector-Borne Pathogens of Grapevine and Citrus

Location: Crop Diseases, Pests and Genetics Research

2023 Annual Report

Objectives
Grapes and citrus are major crops in California with an estimated value of $8.7 billion per year. Inputs for protection against pathogens and insect pests can be significant, and often environmentally friendly control strategies are lacking. To address the plant disease management challenges confronted by grape and citrus producers in California, research will focus on the following four objectives and associated sub-objectives. Objective 1: Create novel grapevine and citrus pathogen identification and disease diagnosis methods. Subobjective 1A: Develop novel species identification techniques for Xylella fastidiosa (Xf) and fungal pathogens of grapevines including utilizing FAME profiling and phenotype microarrays. Subobjective 1B: Improve diagnostics of citrus pathogens through field deployable technologies such as isothermal amplification, robotics, and artificial intelligence. Subobjective 1C: Develop sensitive and accurate detection systems for grape and citrus pathogens using genomic information. Objective 2: Elucidate vector feeding and movement behaviors associated with transmission and spread of Xylella fastidiosa. Subobjective 2A: Compare vector probing behaviors of blue-green sharpshooter (BGSS), with or without Xf, on Pierce’s disease (PD)-resistant or -susceptible grapevines. Subobjective 2B: Develop an electropenetrography (EPG)-based Resistance Index (EPG-RI) to rapidly detect grapevine resistance to Xf inoculation behaviors of vectors. Subobjective 2C: Determine the role of glassy-winged sharpshooter (GWSS) nymphs in spreading Xf within vineyards. Subobjective 2D: Develop a flexible individual-based model to evaluate implications of results from studies conducted during completion of this project on pathogen spread. Objective 3: Develop vibrational control methods for grapevine pests for integration into vineyard management practices. Subobjective 3A: Evaluate female BGSS re-mating receptivity and communication. Subobjective 3B: Determine efficacy of natural tremulatory signals in disrupting mating of the BGSS. Subobjective 3C: Assess BGSS male attractiveness to playback of female vibrational signals. Subobjective 3D: Develop methods for transmission of GWSS and BGSS disruptive signals to crops and ground vegetation. Objective 4: Develop sustainable management tactics for pests and diseases of grapevine and citrus. Subobjective 4A: Develop formulations to improve the use of region-specific biological control strains of Trichoderma spp. as a disease management tool for combating bacterial and fungal diseases of grapevines. Subobjective 4B: Determine susceptibility of vine mealybug (VMB) to soil-applied imidacloprid. Subobjective 4C: Target bacterial endosymbionts for control of VMB. Subobjective 4D: Evaluate sub-lethal effects of soil-applied imidacloprid on GWSS fecundity, survival, and movement behavior. Subobjective 4E: Determine distribution and genetic diversity of citrus tristeza virus (CTV) in citrus in California and assess the environmental/economic impact for future deployment of genetically-engineered (GE)-CTV.

Approach
The approach is to synergistically exploit weak links between main components of grapevine and citrus pathosystem (pathogen, vector, plant) and insect pests to induce an unstable or neutral interaction that can lead to disruption of destructive processes affecting grape and citrus production. First, disease management requires accurate, sensitive, and cost-effective diagnostic tests to identify causal agents. The most recent genomic information will be used to improve pathogen detection methods that are based on DNA sequences, whereas studies of pathogen phenotypes will lead to development of novel complimentary diagnostic methods. Second, developing plant disease management strategies requires a fundamental understanding of pathogen spread. Mathematical models will be used to evaluate the role of environmental factors on pathogen spread and to simulate management approaches. Experiments will compare insect vector feeding behaviors on resistant and susceptible plants and evaluate the role of juvenile stages in pathogen spread. Third, management of insect-transmitted pathogens requires novel sustainable methods for suppressing vector population growth. Methods to disrupt mating by interfering with insect vector behaviors will be developed. A final objective will assess current control methods to minimize risk of insecticide resistance, develop novel molecular technology targeting vector endosymbionts, identify biological control agents of fungal cankers, and evaluate safety and efficacy of genetically-engineered viruses to manage citrus diseases. The research will benefit grape and citrus growers by addressing current needs and developing novel technology to meet the demand for sustainable farming practices.

Progress Report
Progress was made on Objective 1 towards creating novel pathogen identification and disease diagnosis methods. In support of Sub-objective 1A, researchers in Parlier, California, obtained fatty acid methyl ester (FAME) profiles for 4-16 isolates each of seven different fungal pathogen species, and for seven strains of Xylella fastidiosa (Xf). FAME profiles for Xf were different in strains that belong to different subspecies. Comparative analysis can be used to test the robustness of the FAME technique to identify pathogen species and within-species groups of pathogen strains when paired with genomic sequence data. For Sub-objective 1B, researchers in Parlier, California, successfully detected ‘Candidatus Liberibacter asiaticus’ from crude extracts of infected citrus leaf tissue, and from crude extracts of Asian citrus psyllid using recombinant polymerase amplification and lateral flow test strip techniques. These testing methods are rapid, sensitive, and do not require extensive sample processing. Under Sub-objective 1C, researchers used whole genome sequence information to develop and enhance polymerase chain reaction protocols for detection of Xylella taiwanensis, the pathogen causing pear leaf scorch disease. Two primer sets were designed based on a 7-copy gene found in the complete genome sequence of Xt strain PLS229, and are being tested for sensitive detection of this pathogen. In support of Objective 2 and Agreement 60-2034-0002, researchers in Parlier, California, used a patented hand-held spectrophotometer to examine the internal leaf structure of young lemon plants inoculated with citrus yellow vein clearing virus as an early detection tool for this pathogen. Further progress was made on Objective 2 to understand vector feeding and movement behaviors associated with transmission and spread of Xf. Under Sub-objective 2C, researchers in Parlier, California, quantified vertical movement of glassy-winged sharpshooter nymphs from plants in the understory up into the canopy of grapevines. Most nymphs moved up into the vine canopy shortly after egg hatch. Under Sub-objective 2D, researchers wrote an individual based spatially-explicit model on the spread of insect-transmitted plant pathogens that mirrors the behavior of standard Susceptible-Infected-Removed models. This model has been uploaded to SCINet for preliminary simulations. Progress was made on Objective 3 towards development of vibrational control methods for grapevine pests. In support of Sub-objective 3A, researchers in Parlier, California, tested the efficacy of candidate tremulatory signals in disrupting mating of the blue-green sharpshooter (BGSS). Playback of female signals in intervals of 40, 20, and 10 seconds between signals did not disrupt mating of BGSS. Current experiments are evaluating whether a reduced silent gap between signals (5 sec) disrupts mating. Researchers also tested whether BGSS males are attracted to playback of female vibrational signals. Results showed that males perceive and reply to artificial playback of female signals, and attempt to locate the source of the signals (electromagnetic shaker attached to plants). Although none of the tested males successfully located the source of the artificial signal during trials, analysis of BGSS communication during the mate searching phase indicate that timing between a male call and female reply is important. Future experiments will compare the efficacy of random playbacks of female signals with timely replies (manual reply) in attracting males to the source. If effective, automated playback systems will be developed for trap-and-kill of BGSS in vineyards. Progress was made on Objective 4 towards developing sustainable management tactics for pests and diseases of grapevine and citrus. For Sub-objective 4A, researchers in Parlier, California, collected 40 fungal isolates from grapevine tissues from vineyards throughout California. Of these, 20 were tentatively identified as Trichoderma spp. based on morphology or genetic sequences. These Trichoderma isolates had varied capacity to limit fungal pathogen growth in co-plating assays. Initial greenhouse work found that 12 Trichoderma spp. isolates from California could limit growth of fungal pathogens and potentially Xf. Additionally, eight isolates are under testing in the field for efficacy to protect pruning wounds from fungal infection. For Sub-objective 4B, researchers monitored uptake of imidacloprid in grapevines for quantification of insecticide concentrations in plant tissues. Interactions of glassy-winged sharpshooter with imidacloprid treated plants were also described. For Sub-objective 4C, draft genomes were assembled and annotated for the primary vine mealybug symbiotic bacteria that will be targeted to reduce insect fitness. Under Sub-objective 4E, researchers surveyed citrus for Citrus tristeza virus (CTV) in twelve major citrus-growing counties of California, estimated the CTV incidence per county, and identified the CTV strain present.

Accomplishments
1. Surveys of Citrus tristeza virus (CTV) in California identified strain T30 as a potential viral vector for Huanglongbing (HLB) disease control. Citrus tristeza virus (CTV) has been one of the most economically important pathogens of citrus worldwide. However, in California, there are several CTV strains that are prevalent in citrus without causing any disease. These mild strains are being explored as a vehicle to deliver disease control agents such as antimicrobial peptides and RNA Interference (RNAi) in citrus against the serious invasive pathogen of Huanglongbing (HLB) disease and its vector, Asian citrus psyllid (ACP). ARS researchers at Parlier, California, surveyed citrus in the major citrus-growing counties of California and determined that CTV incidence was low (less than 1 percent) in the Central Valley, highest (11 to 35 percent) in the interior regions of southern California and was not detected in desert regions (Coachella and Imperial) of the state. More than 95 percent of the CTV was the T30 strain or a mixture of T30 and other strains. This suggests that the best CTV strain to develop as a disease control delivery system for deployment in California is T30, as this strain is the most prevalent and does not cause disease. This information will inform researchers developing disease control for HLB and ACP, and federal and state regulators assessing risks associated with release of engineered CTV to control HLB and ACP.

2. Protocols for rearing and handling vine mealybug facilitate pest management research. The vine mealybug is one of the most challenging pests of grapevine to control in California, with table grape growers spending up to $500 an acre annually for control. ARS researchers in Parlier, California, developed protocols for rearing and handling vine mealybug in the laboratory. Methods for rearing vine mealybug are essential to support studies on genome sequencing, identification of biological control agents, and for evaluating susceptibility to insecticides.

3. Novel Trichoderma strains from California vineyards provide biological control of grapevine diseases. Some biological control organisms such as Trichoderma species are commercially available to protect grapevine pruning wounds, but these strains are not necessarily adapted to the environmental conditions of vineyards in California. ARS researchers in Parlier, California, isolated and screened 40 unique fungi from Californian vineyards and conducted testing for biological control activities. Twelve Trichoderma spp. isolates were found that could limit growth of fungal pathogens and potentially Xylella fastidiosa. Researchers will be able to develop these strains with industry partners to provide commercial biocontrol products for disease management in California vineyards.

4. Female black widow spiders communicate through web vibrations for territorial rivalry. Female black widow spiders (Latrodectus hesperus) can be a phytosanitary pest in crops destined for domestic and international markets. Black widows are solitary predators of arthropods with no tolerance for intruders on the webs, suggesting that separation of black widow webs could be driven by natural territorial rivalry between females. However, female-female communication behaviors in this species remains unknown. ARS scientists at Parlier, California, demonstrated that female black widow spiders exhibit territorial rivalry, and that female-female rivalry is mediated by vibrational signals through the web. Understanding the communication behaviors used for competition in black widow spiders will facilitate development of novel methods to prevent spiders from colonizing crops. This will benefit growers and fruit packers who are concerned about black widow spiders in produce.

5. Development of new detection methods for Xylella taiwanensis improves pathogen surveillance capabilities. Xylella taiwanensis (Xt) is a bacterial pathogen that causes pear leaf scorch disease (PLSD) in Taiwan. The pathogen is a quarantine pathogen and has not been detected in the United States. Accurate and sensitive detection of Xt is critical for disease management and for international quarantine and pathogen surveillance efforts. ARS scientists in Parlier, California, and collaborators from Taiwan developed five Xt-specific molecular detection assays. These detection assays will facilitate the current disease management efforts in Taiwan and contribute to pathogen surveillance around the world where Xt introduction is a concern, including the United States.

6. Glassy-winged sharpshooter feeding behavior provides an additional measurement of plant resistance. Xylella fastidiosa (Xf) is an important pathogen in grapevine that is spread by insect feeding and causes major losses in California vineyard production. Xf is inoculated directly into plant xylem vessels during feeding of insect vectors such as the glassy-winged sharpshooter. ARS scientists in Parlier, California, used electropenetrography (EPG) methods to compare glassy-winged sharpshooter feeding behavior on disease-resistant wild Vitis champinii versus disease-susceptible Vitis vinifera grapevines (cultivar Chardonnay). Results showed that sharpshooters performed more inoculation-associated behaviors in more xylem cells on Chardonnay than on V. champinii. This demonstrated that EPG can detect grapevine resistance to vector inoculation of Xf, which adds to the understanding of disease resistance that currently includes bacterial infection and symptom development processes. Future research could use EPG to screen grapevines for this novel type of resistance in breeding programs and germplasm collections.

7. High temperatures during the growing season impact Pierce’s disease risk. Pierce’s disease of grapevine caused by Xylella fastidiosa has continued to significantly cause economic impact to the grape industry in California for decades. Colder winters in some growing regions limit pathogen survival and recurrence in perennial crops, but little is known about how climate factors affect Pierce’s disease in hotter climates such as the California Central Valley. ARS scientists in Parlier, California, created a conceptual model of temperature-dependent disease severity based on summer and winter temperatures representative of California grape growing regions, and experimentally tested disease recurrence rates when infected plants experienced high summer temperatures. Results show that disease recurrence is very high under these conditions, which highlights the need for region-specific management practices that do not rely on vine recovery over the winter in areas where summer temperatures are high. This information is important for vineyard managers making decisions about when to remove infected vines and when to implement insecticide sprays for vector control.

8. Blue-green sharpshooters have altered feeding behavior after feeding on Xylella fastidiosa-infected grapevines. Blue-green sharpshooters (BGSS) are efficient vectors of Xylella fastidiosa (Xf), which causes Pierce’s disease of grapevine, a serious problem in vineyard production with significant economic impact. To understand pathogen spread in the environment it is important to understand the influence of different feeding sources on insect vector behavior. ARS scientists in Parlier, California, used electropenetrography (EPG) to compare BGSS feeding behavior on healthy grapevine after previously feeding on either basil, infected grapevine, or healthy grapevines. Insects performed the largest amount of Xf-inoculation behavior when previously fed on healthy grapevines, least for basil, and intermediate for infected grapevine. This difference may be due to acquisition of non-pathogenic microbes from healthy grapevine, causing greater clogging of the insect mouthparts and altering behavior. This EPG study expanded our understanding of the interactions between vectors and the microbes they carry, which can result in technologies to limit pathogen spread.

9. Glassy-winged sharpshooters use superpropulsion of excreted waste to survive on low-energy food sources. Sharpshooter vectors of the plant pathogenic bacterium Xylella fastidiosa (Xf) feed exclusively on plant xylem sap, an extremely low-density nutrient source (95 percent water) that generates large volumes of droplet excreta. How excretion dynamics impact the ability of these insects to obtain adequate energy from high-volume feeding on low nutrient density food sources is largely unexplored. ARS scientists at Parlier, California, collaborated with scientists at Georgia Institute of Technology, Georgia, using a combination of mathematical models, computational fluid dynamics, and biophysical experiments, to demonstrate for the first time in a living organism that these insects use a process called superpropulsion to expel excreta droplets in an energy-efficient manner. This process is energetically cheaper than other excretion mechanisms, and likely contributes to the ability of these insects to survive on low energy food sources. From an engineering perspective, the biological insights from this study can inform designs of energy-efficient self-cleaning structures and microfluidic ejectors for powering small, soft robots for industrial and agricultural automation.

10. Fatty acid methyl ester profiling provides a non-genetic approach to pathogen species identification. Fatty acid methyl ester profiling (FAME) is an easily-automated assay for identifying pathogens that can serve as an alternative culture-independent method to DNA based technologies. Fast and accurate pathogen identification is necessary to ensure correct diagnosis before potentially costly control methods are implemented. ARS researchers in Parlier, California, developed methods using FAME profiling to identify grapevine fungal pathogens to the species level. This technology will provide additional tools for plant diagnostic laboratories to verify pathogen species and strains, which will enhance management recommendations.

Review Publications
Roddee, J., Backus, E.A., Cervantes, F.A., Hanboonsong, Y. 2023. Xylella fastidiosa inoculation behaviors (EPG X wave) are performed differently by blue-green sharpshooters based on infection status of prior probing host. Journal of Economic Entomology. 116(3):697-712. https://doi.org/10.1093/jee/toad043.
Sisterson, M.S., Uchima, S.Y., Wallis, C.M., Krugner, R. 2022. Glassy-winged sharpshooters cease feeding and avoid plants treated with sub-lethal doses of the neonicotinoid insecticide imidacloprid. Journal of Economic Entomology. 116(1):240-248. https://doi.org/10.1093/jee/toac201.
Krugner, R., Espindola, C., Justus, N., Hatton, R.L. 2023. Web vibrations in intraspecific contests of female black widow spiders, Latrodectus hesperus. Environmental Entomology. 52(2):169-174. https://doi.org/10.1093/ee/nvad003.
Sisterson, M.S., Dwyer, D.P., Uchima, S.Y. 2022. Evaluation of alfalfa fields and pastures as sources of Spissistilus festinus (Hemiptera: Membracidae): Quantification of reproductive and nutritional parameters. Environmental Entomology. 52(1):119-128. https://doi.org/10.1093/ee/nvac104.
Backus, E.A., Patterson, W.C. 2023. Investigation of an expanded, lumped-element model of electropenetrography (EPG) and the accuracy of the traditional R and emf model. Computers and Electronics in Agriculture. 208. Article 107771. https://doi.org/10.1016/j.compag.2023.107771.
Challita, E.J., Sehgal, P., Krugner, R., Bhamla, M.S. 2023. Droplet superpropulsion in an energetically constrained insect. Nature Communications. 14. Article 860. https://doi.org/10.1038/s41467-023-36376-5.
Burbank, L.P., Ochoa, J.C. 2022. Evidence for elicitation of an oxidative burst in Vitis vinifera by Peptides derived from a Xylella fastidiosa cold shock protein peptide csp20. PhytoFrontiers. 2(4):339-341. https://doi.org/10.1094/PHYTOFR-05-22-0048-SC.
Wei, W., Burbank, L.P. 2022. Toxin-Antitoxin system HigBA in Xylella fastidiosa is expressed at low temperature but does not impact virulence or grapevine recovery in greenhouse trials. PhytoFrontiers. https://doi.org/10.1094/PHYTOFR-02-22-0009-R.
Su, C., Fung, J., Chang, R., Chang, C., Jan, F., Shih, H., Chen, J. 2023. TaqMan quantitative-PCR detection of Xylella taiwanensis in Taiwan. Plant Disease. https://doi.org/10.1094/PDIS-01-23-0064-RE.
Huang, J., Dai, Z., Zheng, Z., da Silva, P., Kumagai, L., Xiang, Q., Chen, J., Deng, X. 2021. Bacteriomic analyses of Asian citrus psyllid and citrus samples infected with “Candidatus Liberibacter asiaticus” in southern California and Huanglongbing management implications. Frontiers in Microbiology. 12. Article 683481. https://doi.org/10.3389/fmicb.2021.683481.
Wallis, C.M. 2022. Exploring potential induction of grapevine (Vitis spp.) root phenolic compounds by ring nematodes, Mesocriconema xenoplax. BMC Research Notes. 15:375. https://doi.org/10.1186/s13104-022-06262-2.