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

2024 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
This report documents progress for project 2034-22000-014-000D, titled, “Development of Applied Management Systems for Diseases of Perennial Crops with Emphasis on Vector-Borne Pathogens of Grapevine and Citrus”, which started in February 2022. In support of Objective 1, ARS researchers in Parlier, California continued to develop new methods for disease diagnosis in grapevine and citrus. Under Sub-objective 1A, ARS researchers tested methods to diagnose unidentified fungi by comparing chemical signatures based on fatty acid methyl esterase profiling (FAME), and growth characteristics on different nutrient sources with phenotype microarrays (PM). Using FAME and PM profiles, common fungal pathogens of grapevine could be identified and differentiated. Under Sub-objective 1B, ARS researchers collaborated with the Alliance of Pest Control Districts to improve sampling protocols for reliable detection of the pathogen ‘Candidatus Liberibacter asiaticus’ (CLas) in citrus. Additionally, under Sub-objective 1B and subordinate agreement 60-2034-0002, ARS researchers tested the Croptix hand-held, light-based device for early detection of plant physiological changes caused by citrus yellow vein clearing virus (CYVCV). They also tested CYVCV components for potential use as detection targets in rapid screening tests (ELISA and immunostrips). Suppoting Sub-objective 1C, ARS researchers developed new PCR tests for Xylella fastidiosa strains that cause almond leaf scorch and pecan leaf scorch. These tests were designed to be more sensitive than previous PCR tests and are currently being tested on X. fastidiosa strains from California and Georgia. Under Sub-objective 1D, progress was made on developing a new molecular test for detection of Grapevine leaf roll virus 3. This test is under laboratory validation with viral RNA and infected grapevine tissue. For Objective 2, ARS researchers made progress on understanding insect vector feeding and movement behaviors related to Xylella fastidiosa transmission. Under Sub-objective 2A, ARS researchers recorded blue-green sharpshooter feeding on X. fastidiosa-infected grapevines of susceptible or resistant varieties using electropenetrography (EPG). After feeding, insects were tested for presence or absence of X. fastidiosa to compare feeding behaviors with pathogen acquisition. Under Sub-objective 2C, ARS researchers measured horizontal movement of glassy-winged sharpshooter nymphs between plants. In support of Sub-objective 2D, they created detailed computer simulation to track spread of insect-transmitted plant pathogens through an agricultural region to better understand pest movement on a large scale. Simulation models can be used to predict the impact of pathogen or vector invasion events or introduction of specific management practices. Under Objective 3, progress was made towards the development of vibrational control methods for grapevine pests. Under Sub-objective 3B, ARS researchers tested playback of vibrational signals from female blue-green sharpshooters to evaluate effectiveness of these signals for disruption of blue-green sharpshooter mating. Results indicate successful mating disruption by broadcasting a female signal immediately following a male’s signal. Under Sub-objective 3C, researchers made progress on testing feasibility of sending vibrational signals throughout plants to attract and physically trap a male at the signal location. Currently, efforts are ongoing to map the ground transmission of vibrational signals through a test vineyard using wires placed below the plant roots. In support of Objective 4, progress was made on developing sustainable management tactics for pests and diseases of grapevine and citrus. Under Sub-objective 4A, ARS researchers continued testing new fungal biological control strains to improve management of grapevine trunk diseases and important insect pests such as mealybugs and sharpshooters. When applied to pruning wounds, six Trichoderma fungal strains eliminated existing fungal infections and remained long-term within the plant tissues. They also continued screening of newly identified insect-pathogenic fungi for potential to reduce mealybug populations via delayed feeding and insect development. Under Sub-objective 4B, researchers evaluated effects of sub-lethal doses of the insecticide imidacloprid on vine mealybug survival and reproduction. Under Sub-objective 4E, ARS researchers identified and sequenced a strain of Citrus tristeza virus type T30. This strain causes quick decline of citrus grown on sour orange rootstock, which was not previously seen for virus type T30. In related work, they initiated monitoring of three-cornered alfalfa hopper (vector for grapevine red blotch virus) movement between alfalfa fields and vineyards. Additionally, researchers initiated a study to count insects on yellow panel sticky traps using machine learning.

Accomplishments
1. Improved detection of Huanglongbing disease agent through novel sampling methods. Huanglongbing (HLB) disease is caused by the bacterial pathogen ‘Candidatus Liberibacter asiaticus’ (Clas). Early detection and prompt response to infections are key factors for eradication of Clas where this high-consequence plant pathogen threatens the U.S. citrus industry. Sampling citrus trees for detection of Clas is challenging because the pathogen is not evenly distributed in an infected tree. ARS researchers in Parlier, California, in collaboration with the Alliance of Pest Control Districts compared different sampling methods for optimal pathogen detection. Sampling fruit-connected stem tissue from all four sides of each tree was found to give the most consistent detection of Clas compared with other sampling methods currently in use by regulatory agencies. This new development in sampling methods is expected to significantly improve early detection efforts and facilitate Clas eradication in California.

2. Discovery of Trichoderma strains for effective biological control of grapevine trunk diseases. Grapevine trunk diseases caused by fungal pathogens are a significant problem for grape production. Chemical control options for fungal pathogens are limited and unsustainable in many regions due to human health and environmental concerns. Biological control agents provide an important alternative to chemical pesticides due to their ability to provide long-term, sustainable disease control. ARS researchers in Parlier, California, discovered novel Trichoderma species of fungi from California vineyards and tested their biological control activity to manage grapevine trunk diseases. Six of these new biological control strains provided disease control equivalent to a commercial fungicide and were able to survive in grapevines throughout the entire growing season. These new Trichoderma strains provide an effective biopesticide option for grape growers in California to control grapevine trunk diseases.

3. Xylella fastidiosa strains from different growing regions cause similar disease severity. Xylella fastidiosa is a bacterial pathogen that causes significant disease problems for grape cultivation in California. Hundreds of X. fastidiosa strains were collected from vineyards in California in the last few years, but there was no information about disease severity caused by most of these strains. ARS researchers in Parlier, California, compared disease severity in plants infected with 71 different X. fastidiosa strains from all California grape-growing regions. No significant difference in disease severity between different strains was found. This research provides important information for managing X. fastidiosa in vineyards because it shows that all pathogen strains cause the same amount of disease and can be managed in a similar way.

4. Vine mealybug life stage determines survival following insecticide treatment. Vine mealybug is the number one pest of grapevine in California. Control of this pest relies on applications of systemic insecticides, and overreliance on a few active ingredients resulted in reduced efficacy of these products. Insects can reduce chemical exposure by not feeding on treated plants, and it is unclear whether observed insecticide failure is due to higher tolerance to the chemical, or reduced feeding by the insects under certain conditions. To better understand failures of chemical control products, ARS researchers in Parlier, California, tested vine mealybugs in the laboratory for susceptibility to the systemic neonicotinoid insecticide imidacloprid. High concentrations of imidacloprid applied to grapevines did not kill mature female vine mealybugs or prevent them from producing viable eggs, whereas low concentrations of imidacloprid did kill newly hatched insects. This information is important for understanding insecticide efficacy for controlling vine mealybugs and is expected to improve insecticide application strategies in vineyard management.

5. Citrus yellow vein clearing virus is likely spread via plant propagation in California. Several important citrus pathogens are transmitted through grafting and plant propagation. The best control method for these pathogens is to use pathogen-free plant materials acquired from certified citrus nurseries, but citrus propagation in backyards and urban settings is less strictly controlled. ARS researchers in Parlier, California, in collaboration with the California Department of Food and Agriculture determined that approximately 25% of dooryard citrus in the area sampled was infected by citrus viroids that are transmitted solely by grafting and propagation of plant material. Thirteen percent of samples collected were infected with the emerging pathogen citrus yellow vein clearing virus (CYVCV), and approximately one third of the CYVCV-infected trees were also infected with graft-transmitted citrus viroids. Presence of CYVCV in the same trees as graft-transmitted pathogens suggests that plant propagation is likely causing a significant amount of CYVCV spread in California, in addition to insect transmission. These results could be used by regulatory agencies for strengthening quarantine efforts around CYVCV-infected sites.

6. Cost-effective identification of Xylella fastidiosa using biochemical methods. The bacterial pathogen Xylella fastidiosa causes several plant diseases that have significant impacts on agricultural production. DNA-based testing is currently the fastest method to identify X. fastidiosa strains but can be cost-prohibitive on a large scale. ARS researchers in Parlier, California, developed biochemical methods known as fatty acid methyl ester (FAME) analyses for X. fastidiosa identification. These methods allow rapid (less than 1 hour), cost-effective (less than $2) identification, and differentiation of specific X. fastidiosa strains. This method could be applied to other pathosystems to facilitate pathogen identification and disease management in cases where DNA-based identification is cost-prohibitive or otherwise not possible.

Review Publications
Maguvu, T.E., Frias, R.J., Hernandez-Rosas, A.I., Holtz, B.A., Niederholzer, F.J., Duncan, R.A., Yaghmour, M.A., Culumber, C.M., Gordon, P.E., Vieira, F.C., Rolshausen, P.E., Adaskaveg, J.E., Burbank, L.P., Lindow, S.E., Trouillas, F.P. 2024. Phylogenomic analyses and comparative genomics of Pseudomonas syringae associated with almond (Prunus dulcis) in California. PLOS ONE. 19(4). Article e0297867. https://doi.org/10.1371/journal.pone.0297867.
Burbank, L.P., Gomez, L., Shantharaj, D., Abdelsamad, N.A., Vasquez, K., Burhans, A.N., Ortega, B.C., Helm-Rodriguez, S.D., Strickland, J.A., Krugner, R., De La Fuente, L., Naegele, R.P. 2024. Virulence comparison of a comprehensive panel of Xylella fastidiosa Pierce’s disease isolates from California. Plant Disease. 108(6):1555-1564. https://doi.org/10.1094/PDIS-09-23-1923-RE.
Roddee, J., Wangkeeree, J., Backus, E.A., Hanboonsong, Y. 2023. Probing behavior of the leafhopper analyzed through DC electropenetrography and microscopy. Journal of Insect Physiology. 151. Article 104584. https://doi.org/10.1016/j.jinsphys.2023.104584.
Cooper, A.M., Jameson, S.B., Pickens, V., Osborne, C., Backus, E.A., Silver, K., Mitzel, D.N. 2023. An electropenetrography waveform library for the probing and ingestion behavior of Culex tarsalis on human hands. Insect Science. 31(4):1165-1186. https://doi.org/10.1111/1744-7917.13292.
Burbank, L.P., Sisterson, M.S., Wei, W., Ortega, B.C., Luna, N.R., Naegele, R.P. 2023. High growing season temperatures limit winter recovery of grapevines from Xylella fastidiosa infection – implications for epidemiology in hot climates. Plant Disease. 107(12):3858-3867. https://doi.org/10.1094/PDIS-03-23-0492-RE.
Sisterson, M.S., Uchima, S.Y. 2023. Planococcus ficus (Hemiptera: Pseudococcidae) movement and demography: Methods for generating cohorts for laboratory studies. Journal of Economic Entomology. 117(1):118-126. https://doi.org/10.1093/jee/toad210.
Ahmed, M., Hu, J.S., Strickland, J.A., Krueger, R., Shannon, C., Zhang, A. 2024. Reproductive behavior and development of the global insect pest cotton seed bug. Insects. Insects 2024, 15(1), 65;. https://doi.org/10.3390/insects15010065.
Zhai, Y., Gnanasekaran, P., Iftikhar, R., Turner, K., Van Tassel, D., Cassetta, E., Lubin, T., Pappu, H.R. 2024. Identification and molecular characterization of Dahlia common mosaic virus from Silphium spp., a new natural host of the virus. PhytoFrontiers. https://doi.org/10.1094/PHYTOFR-12-23-0156-SC.
Zhai, Y., Gnanasekaran, P., Pappu, H.R. 2024. Development of a CRISPR/SHERLOCK-based method for rapid and sensitive detection of selected pospiviroids. Viruses. 16(7). Article 1079. https://doi.org/10.3390/v16071079.
Wallis, C.M., Chen, J. 2024. Fatty acid methyl ester profiling of Californian Xylella fastidiosa strains. PhytoFrontiers. 4(3):277-281. https://doi.org/10.1094/PHYTOFR-09-23-0120-SC.