Research Project: Identifying Vulnerabilities in Vector-host-pathogen Interactions of Grapevine and Citrus Pathosystems to Advance Sustainable Management Strategies

Location: Crop Diseases, Pests and Genetics Research

2024 Annual Report

Objectives
Objective 1: Develop genomic resources and application of multi-omic approaches for understanding microbial systematics and pathogenesis [NP303 C1, C2, PS1A, PS1B, PS2A, PS2B]. Sub-objective 1A: Expand whole genome sequence databases of Xylella fastidiosa (Xf) and “Candidatus Liberibacter asiaticus” (CLas) strains. Sub-objective 1B: Characterize metagenomes of Xylella spp. and “Ca. Liberibacter spp.” infected samples using machine learning (ML) focusing on improvement of taxonomic identification. Sub-objective 1C: Identify genetic determinants of Xf host range using a transposon mutagenesis and high-throughput sequencing approach. Sub-objective 1D: Identify genetic determinants of Xf persistence and survival under different climatic conditions. Objective 2: Develop phenomic approaches to identify environmental and plant determinants of pathogen infection [NP303, C1, C2, C3, PS1A, PS1B, PS2B, PS3A]. Sub-objective 2A: Characterize host response of citrus to S. citri infection that can influence co-infection of CLas by the ACP. Sub-objective 2B: Develop simplified metabolomic profiles for different grapevine cultivars and associate them with observed resistance to fungal pathogens, Xf, and associated diseases. Sub-objective 2C: Characterize response of different grapevine cultivars to Xf infection using RNAseq. Objective 3: Characterize microbiomes of pathogen-infected grapevine and citrus as well as associated insect vectors [NP303, C1, C2, PS1A, PS1B, PS2A, PS2C]. Sub-objective 3A: Describe the phytobiome of healthy, Xf-infected, and fungal canker pathogen-infected grapevines, and relate to host physiological status. Sub-objective 3B: Describe the phytobiome of healthy and CLas-infected citrus plants. Sub-objective 3C: Describe the microbiomes of sharpshooter vectors of Xf. Sub-objective 3D: Describe the microbiome in the Asian citrus psyllid vectors of CLas. Objective 4: Elucidate vector-pathogen-crop interactions to disrupt pathogen transmission [NP303, C2, C3, PS2B, PS2D, PS3A, PS3B]. Sub-objective 4A: Elucidate a time course of Xf bacterial colonization and exopolysaccharide attachment formation in functional foregut of sharpshooters. Sub-objective 4B: Characterize ultrastructure of the precibarial valve in the functional foregut of sharpshooters, and its possible role in Xf transmission over time.

Approach
Objective 1. The genomic underpinnings of pathogenesis can be determined for diseases caused by Xf and CLas by use of multi-omic approaches. Next Generation Sequencing (NGS)technologies will be used to generate giga bp level DNA sequence data sets which will be subjected to datamining through machine learning (ML) approaches to develop new and unique biological information. Genomic determinants of host susceptibility will be examined by mutagenesis and bioassays of tolerant versus susceptible host cultivars. Persistence of Xf will be studied under ambient and low temperature conditions using transcriptome sequencing and mutational validation of gene functions. Objective 2. Through measurements of growth, performance, and composition of grapevines and citrus under different pathogen challenge conditions, environmental and host susceptibilities to pathogen infection can be identified. Because citrus stubborn disease and huanglongbing are caused by phloem-restricted insect-vectored bacteria (Spiroplasma citri and CLas, respectively), pre-infection of S. citri will be examined to test if pathogen competition can reduce plant infectivity and/or susceptibility to CLas. Metabolomics of grapevines inoculated with Xf and fungal pathogens will be studied for specific chemical profiles and molecular attributes that could be help identify host susceptibility or resistance traits. Similarly, transcriptome analysis will be conducted on susceptible, tolerant, and resistant cultivars of grapevines challenged by Xf to better understand host plant resistance and improve disease mitigation of Xf diseases. Objective 3. An exploration of the microbiomes of grapevines and citrus infected by Xf and CLas, respectively, along with their insect vectors, will identify microorganisms and insect endosymbionts that may be used or developed to mitigate or reduce spread of Xf and CLas. Phytobiomes of grapevines inoculated by Xf and fungi will be examined by NGS to determine microbial community shifts correlated to host physiology. Phytobiomes of citrus infected by CLas will be examined by NGS to identify prophage(s) that can be used to differentiate and identify CLas populations and other microbes. Microbiomes of insect vectors of Xf and CLas will be examined by NGS technologies to identify insect endosymbionts. This information will be used in studies to reduce vector fitness and/or propensity of transmission. Objective 4. Xf attachment in the foregut of the blue green sharpshooter (BGSS) depends on exo-polysaccharide adhesives secreted by Xf and the ultrastructure of the functional foregut, especially the precibarial valve, in the vector. Functional foregut of BGSS exposed to grapevines infected by a mild versus a virulent strain of Xf will be examined by scanning electron microscopy (SEM) to determine if extent of bacterial colonization is correlated with disease virulence. Time course acquisition access periods and light and transmission microscopy will be used to ascertain if foregut morphology (grooves and invaginations) and bacterial adhesion to the cuticular lining of the functional foregut influence Xf transmission.

Progress Report
This report documents progress for project 2034-22000-015-000D, titled, “Identifying Vulnerabilities in Vector-host-pathogen Interactions of Grapevine and Citrus Pathosystems to Advance Sustainable Management Strategies”, which started in April 2022. In support of Sub-objective 1A, ARS scientists in Parlier, California, expanded genomic science expertise through application of next generation sequencing and PCR to analyze the plant pathogen Xylella fastidiosa (Xf) in samples from grapevines, pecans, plums, and blueberries obtained during a field trip to Georgia. Pure cultures of grapevine Xf strains were obtained from grapevine samples that exhibited Pierce’s disease symptoms. For Sub-objective 1B, ARS scientists used USDA SciNet High-Performance Computers (HPCs) for bacterial genome assembly and metagenomic analyses using DNA samples collected from Xf-infected tissues from almond, grape, pecan, plum, and strawberry. Bacterial genome assembly was compared between Linux-based and Windows-based programs for efficiency and accuracy. Different metagenomic classifiers (e.g. kaiju vs. kragen) were compared and evaluated for further microbiome research. In support of Sub-objective 1C, ARS scientists evaluated random Xf mutant populations after inoculation into grapevines to identify genes important for Xf infection in different host plants. For Sub-objective 1D, ARS scientists identified genes with different expression levels after Xf strains were exposed to cold temperatures to better understand the ability of this pathogen to survive different environmental conditions. In support of Sub-objective 2A, ARS scientists in Parlier, California, revealed details of citrus host responses to different pathogens by evaluating symptoms of Spiroplasma citri (Sc)-infected citrus trees in Ducor, McFarland, and San Emidio, California, using purified DNA to confirm Sc infections and to confirm symptoms in citrus trees were not caused by ‘Candidatus’ Liberibacter asiaticus (CLas, the pathogen associated with huanglongbing or HLB) or C. L. africanus (CLaf, the pathogen associated with African greening). A new isolate of Citrus yellow vein clearing virus (CYVCV), called HH from Hacienda Heights, California, was discovered through these efforts. Apparently two introduction events of CYVCV occurred in California because the HH strain had a genotype similar to other CYVCV isolates from Tulare, California, although sequencing analyses revealed its genome was in a different genotype group. Ongoing research on CYVCV symptomology in different citrus cultivars and other plant hosts showed infection associated with decreased volatile terpenes/terpenoids (e.g. a-phellandrene, a-terpinolene, limonene, p-cymene, linalool, and citral, which are known to repel whiteflies) levels resulting in lower repellency rather than attraction for citrus whiteflies that performed better on CYVCV-infected lemon plants. for Sub-objective 2B, ARS scientists continued to characterize differences in cultivar responses to pathogen infections by sampling field-grown grapevines infected with different fungal canker pathogens (including Diplodia seriata, Eutypa lata, and Neofusicoccum parvum), pulverizing the tissue, and performing chemical and DNA extractions. Biochemical evaluations by chromatography are proceeding, and upon completion, will ascertain if specific grapevine cultivar biochemical shifts due to infection occur between pathogens. In support of Sub-objective 2C, ARS scientists completed RNA sequencing from grapevines to characterize responses of different cultivars to Xf infections. RNA sequencing data analysis is ongoing. In support of Sub-objective 2D, ARS scientists in Parlier, California, continued development of tissue culture/transformation/genome editing platform using Thompson Seedless as the model table grape/raisin grape cultivar. Embryonic calli were successfully induced, propagated, and maintained using unopened/juvenile leaves as starting materials, and protoplasts were tested for transfection. As the genome editing target, grapevine gene CYP734A15 was successfully cloned from Thompson Seedless genomic DNA; this gene was used to transform Arabidopsis to verify its brassinosteroid inactivation function. In support of Sub-objective 3A, ARS scientists in Parlier, California, assessed shifts in phytobiome community composition of grapevines that were uninfected or infected with Xylella fastidiosa (Xf), pathogenic fungi (Diplodia seriata, Eutypa lata, Neofusicoccum parvum, or Phaeomoniella chlamydospora), or biological control fungi (Trichoderma species). In addition to assessing physiological changes associated with infection status in root, stem, and foliar tissues, total genomic DNA from those samples was sequenced to determine shifts in phytobiome composition. Preliminary results indicate that phytobiomes were altered according to the pathogen, especially around inoculation sites as well as in foliar tissues of plants infected by E. lata or Xf. Shifts in community composition were associated with changes in grapevine physiology. For Sub-objective 3B, ARS scientists described microbiome communities associated with HLB-infected trees, and in citrus tree DNA samples from Mexico and Texas, USA. Giga-size DNA sequence data for each sample were generated through next generation sequencing. Metagenomic analyses revealed variations of bacteriomes among different geographical locations. In support of Sub-objective 3C, ARS scientists in Parlier, California, determined the microbiome of Xf vectoring insects, Graphocephala atropunctata (blue-green sharpshooter) and Kolla paulula, using SciNet HPC resources. Different assembly techniques (CLC genome workbench vs. Linux-based Bowtie2-spades) were compared and evaluated for microbiome data quality. For Sub-objective 3D, ARS scientists described microbiome communities associated with CLas in the Asian citrus psyllid (ACP), a major vectoring insect of CLas, by testing DNA from samples collected in West Hills, California. In support of Sub-objective 3E, ARS scientists examined soil and rhizosphere microbiomes of grapevines following sequencing of DNA from rhizosphere, bulk soil, and endosphere samples of four grapevine rootstock cultivars (039-16, 101-14MG, Freedom, and Salt Creek), analysis of the 16S barcode region remains to be completed. In support of Sub-objective 4A, ARS scientists in Parlier, California, investigated colonization of Xylella fastidiosa (Xf) in sharpshooters by examining BGSS heads for Xf colonization. Differences by acquisition access period (AAP) day and strain were observed. These differences were likely due to removal behaviors performed by the insects and not Xf strain acquisition differences. For Sub-objective 4B, ARS scientists characterized the role of the precibarial valve in Xylella fastidiosa (Xf) transmission examining sharpshooter heads imaged by light microscopy. Although examinations by transmission electron microscopy continue, preliminary results confirmed hypotheses on the anatomy and operation of the precibarial valve relative to Xf bacteria being “frozen” below, but not above, the valve. An invagination was found to be present and determined to be involved in both valve operation and likely Xf transmission.

Accomplishments
1. Regional origin of Citrus yellow vein clearing virus detected in Tulare, California. Citrus yellow vein clearing virus (CYVCV) is an exotic citrus pathogen first detected in the western hemisphere in 2022 in dooryard citrus trees in Tulare, California, which triggered quarantine to prevent its spread to citrus orchards by insect vectors. ARS researchers in Parlier, California, sequenced the entire genome of four isolates and differentiated them into two strains, one originating from South Asia/Middle East and the other from China. This suggested two pathways of entry by propagation. Regulatory agencies were notified to remain vigilant and continue efforts in informing the public to only buy and plant citrus trees that have a certified label showing they are pathogen free.

2. Genomic resources of ‘Candidatus Liberibacter asiaticus’ strain from a historical endemic region. Citrus HLB is threating citrus production worldwide. A comprehensive understanding of ‘Candidatus Liberibacter asiaticus’ (CLas) diversity around the world is important for huanglongbing disease (HLB) management. ARS scientists in Parlier, California, collaborated with scientists in Guangdong, China, to sequence the genome of CLas strain GDCZ from a historical HLB endemic region in China. This genome sequence filled gaps in the HLB research database and provides a valuable resource for disease management including pathogen detection and quarantine applications.

3. Genome assembled for potential biological control agent, Trichoderma harzianum strain PAR3. Biopesticides are preferred over chemical treatments for sustainable pest management. Mycopesticides are biopesticides with fungi as the active ingredient that include strains of the fungus Trichoderma harzianum. ARS scientists in Parlier, California, discovered the local strain of T. harzianum called PAR3 and sequenced and assembled its genome, which was made publicly available. Genomic resources like this one are required to advance detailed systematic and taxonomical characterization of fungi including strains of Trichoderma like PAR3, which could be developed as mycopesticides.

4. Diversity of ‘Candidatus Liberibacter asiaticus’ (CLas) in Texas revealed through phage sequence analyses. Texas is the third largest citrus production state in the United States. Citrus huanglongbing disease (HLB) caused by ‘Candidatus Liberibacter asiaticus’ (CLas) was detected in Texas in 2012, but CLas diversity information in Texas is limited. To fill this knowledge gap, ARS scientists in Parlier, California, collaborated with scientists at Texas A&M University to collect and analyze field samples in southern Texas. Phage sequence analyses indicated that there are two different genotypes/strains in Texas. Information from this study helped understand the epidemiological relevance of CLas diversity in Texas and could be used in HLB management.

5. Plant pathogen priming elicits physiological changes resulting in reduced fungal disease symptoms. Grapevines become infected with multiple pathogens simultaneously, but little is known about how one infection may impact the progression of another. ARS researchers in Parlier, California, observed host physiological changes due to a bacterial, fungal, or nematode infection and observed how these changes limited the growth of a lesion of the fungal trunk pathogen Neofusicoccum parvum. The findings showed that grapevine pathogen priming elicited physiological changes resulting in reduced fungal trunk disease symptoms. This should be considered in management plans targeting fungal trunk pathogens.

6. Identification of Pseudomonas species causing bacterial blast in California almond orchards. Bacterial blast and bacterial canker diseases caused by Pseudomonas pathogens are extremely damaging to stone fruit and nut crops, especially during cold and wet winters. In the years 2019, 2020, 2021, and 2023, weather conditions in California led to severe blast disease, and emergency approval for Pseudomonas control products was necessary. However, limited information was available on Pseudomonas species causing blast disease in these crops and their susceptibility to available control products. Collaboration between scientists with ARS in Parlier, California, and at the University of California, Davis, identified several Pseudomonas species causing disease in almond and measured susceptibility to copper and kasugamycin, which are approved for bacterial blast control. This research provided essential information for effective control of bacterial blast in almonds.

7. Mitogenome of the cottony ash psyllid, a vector of the pathogen causing emerging ash decline disease. Cottony ash psyllid (CAP) was identified as a major pest of ash trees in the plains of North America as it may vector a devasting ‘Candidatus Liberibacter’ pathogen, which causes tree decline and death. However, little is known about the genetics of this insect pest including the genome of its mitochondrion, or mitogenome. ARS scientists in Parlier, California, sequenced the CAP genome and characterized its mitogenome. Mitogenome data are valuable for systematic and taxonomy studies of CAP and other psyllid species.

Review Publications
Bao, Y., Zhang, Q., Zhu, H., Pei, Y., Zhao, Y., Li, Y., Ji, P., Du, D., Peng, H., Xu, G., Wang, X., Yin, Z., Ai, G., Liang, X., Dou, D. 2024. Metformin blocks BIK1-mediated CPK28 phosphorylation and enhances plant immunity. Journal of Advanced Research. https://doi.org/10.1016/j.jare.2024.02.025.
Hao, J., Zhang, X., Qiu, S., Song, F., Lyu, X., Ma, Y., Peng, H. 2024. Species diversity, nitrogen fixation, and nutrient solubilization activities of endophytic bacteria in pea embryos. Applied Sciences. 14(2). Article 788. https://doi.org/10.3390/app14020788.
Cheng, Y., Meng, R., Niu, S., Peng, H., Jing, M. 2024. Protocol for quantitative evaluation of misfolded protein degradation using Agrobacterium-mediated expression system in Nicotiana benthamiana. STAR (Structured Transparent Accessible Reproducible) Protocols. 5(2). Article 103034. https://doi.org/10.1016/j.xpro.2024.103034.
De Leon, V.S., Chen, J., McCollum, G., Park, J., Louzada, E.S., Setamou, M., Kunta, M. 2024. Diversity of 'Candidatus Liberibacter asiaticus' strains in Texas revealed by prophage sequence analyses. Plant Disease. 108(6):1455-1460. https://doi.org/10.1094/PDIS-09-23-1994-SR.
Zheng, Y., Li, C., Xu, P., Liu, C., Chen, J., Deng, X., Zheng, Z. 2023. Genome sequence resource for “Candidatus Liberibacter asiaticus” strain GDCZ from a historical HLB endemic region in China. BMC Genomic Data. 24. Article 63. https://doi.org/10.1186/s12863-023-01160-3.
Sun, Y., Yokomi, R.K. 2023. Whole genome sequence of Citrus yellow vein clearing virus CA1 isolate. BMC Research Notes. 16. Article 166. https://doi.org/10.1186/s13104-023-06443-7.
Tayengwa, R., Westenskow, S.R., Peng, H., Hulbert, A.K., Neff, M.M. 2024. Genetic interactions between BEN1- and cytochrome P450-mediated brassinosteroid inactivation. Physiologia Plantarum. 176(1). Article e14141. https://doi.org/10.1111/ppl.14141.
Zhang, Y., Wang, J., Pi, L., Wang, N., Peng, H., Yin, Z., Dou, D. 2024. PUB40 attenuates Phytophthora capsica resistance by destabilizing the MEK2-SIPK/WIPK cascade in Nicotiana benthamiana. Phytopathology Research. 6. Article 31. https://doi.org/10.1186/s42483-024-00249-6.
Wallis, C.M. 2024. Characterization of data observing Meloidogyne incognita, Neofusicoccum parvum, and Xylella fastidiosa infection effects on development of grapevine phenolic compound levels and resistance to subsequent Neofusicoccum parvum. Data in Brief. 54. Article 110301. https://doi.org/10.1016/j.dib.2024.110301.
Sun, Y., Yokomi, R.K. 2024. Genotype sequencing and phylogenetic analysis revealed the origins of Citrus yellow vein clearing virus California isolates. Viruses. 16(2). Article 188. https://doi.org/10.3390/v16020188.
Gorman, Z.J., Chen, J., Perez De Leon, A.A., Wallis, C.M. 2023. Comparison of assembly platforms for the assembly of the nuclear genome of Trichoderma harzianum strain PAR3. BMC Genomics. 24. Article 454. https://doi.org/10.1186/s12864-023-09544-6.