Improved draft reference genome for the Glassy-winged Sharpshooter (Homalodisca vitripennis), a vector for Pierce's disease

Homalodisca vitripennis (Hemiptera: Cicadellidae), known as the glassy-winged sharpshooter, is a xylem feeding leafhopper and an important agricultural pest as a vector of Xylella fastidiosa, which causes Pierce’s disease in grapes and a variety of other scorch diseases. The current H. vitripennis reference genome from the Baylor College of Medicine's i5k pilot project is a 1.4-Gb assembly with 110,000 scaffolds, which still has significant gaps making identification of genes difficult. To improve on this effort, we used a combination of Oxford Nanopore long-read sequencing technology combined with Illumina sequencing reads to generate a better assembly and first-pass annotation of the whole genome sequence of a wild-caught Californian (Tulare County) individual of H. vitripennis. The improved reference genome assembly for H. vitripennis is 1.93-Gb in length (21,254 scaffolds, N50 = 650 Mb, BUSCO completeness = 94.3%), with 33.06% of the genome masked as repetitive. In total, 108,762 gene models were predicted including 98,296 protein-coding genes and 10,466 tRNA genes. As an additional community resource, we identified 27 orthologous candidate genes of interest for future experimental work including phenotypic marker genes like white. Further, as part of the assembly process, we generated four endosymbiont metagenome-assembled genomes (MAGs), including a high-quality near complete 1.7-Mb Wolbachia sp. genome (1 scaffold, CheckM completeness = 99.4%). The improved genome assembly and annotation for H. vitripennis, curated set of candidate genes, and endosymbiont MAGs will be invaluable resources for future research of H. vitripennis.

Improved draft reference genome for the Glassy-winged Sharpshooter (Homalodisca vitripennis), a vector for Pierce's disease

Homalodisca vitripennis (Hemiptera: Cicadellidae), known as the glassy-winged sharpshooter, is a xylem feeding leafhopper and an important agricultural pest as a vector of Xylella fastidiosa, which causes Pierce's disease in grapes and a variety of other scorch diseases. The current H. vitripennis reference genome from the Baylor College of Medicine's i5k pilot project is a 1.4-Gb assembly with 110,000 scaffolds, which still has significant gaps making identification of genes difficult. To improve on this effort, we used a combination of Oxford Nanopore long-read sequencing technology combined with Illumina sequencing reads to generate a better assembly and first-pass annotation of the whole genome sequence of a wild-caught Californian (Tulare County) individual of H. vitripennis. The improved reference genome assembly for H. vitripennis is 1.93 Gb in length (21,254 scaffolds, N50 = 650 Mb, BUSCO completeness = 94.3%), with 33.06% of the genome masked as repetitive. In total, 108,762 gene models were predicted including 98,296 protein-coding genes and 10,466 tRNA genes. As an additional community resource, we identified 27 orthologous candidate genes of interest for future experimental work including phenotypic marker genes like white. Further, as part of the assembly process, we generated four endosymbiont metagenome-assembled genomes (MAGs), including a high-quality near complete 1.7-Mb Wolbachia sp. genome (1 scaffold, CheckM completeness = 99.4%). The improved genome assembly and annotation for H. vitripennis, curated set of candidate genes, and endosymbiont MAGs will be invaluable resources for future research of H. vitripennis.

Isolation protocol trials for grapevine bacteriosis (Pierce's disease) agent Xylella fastidiosa Wells et al. during research monitoring of the Republic of Crimea

Xylella fastidiosa Wells et al. causes grapevine bacteriosis and is among most hazardous malicious bacterial phytopathogens affecting a wide variety of important crops and ornamental plants. The agent colonises plant xylem and transmits with insects feeding on xylem sap. The insect vectors of X. fastidiosa belong to the order Hemiptera, suborder Auchenorrhyncha, families Cicadellidae, Cercopidae, Aphrophoridae and Cicadidae. A phytosanitary control survey conducted by the All-Russian Research Institute for Plant Quarantine in 2014 identified a high risk of X. fastidiosa introduction and adaptation in the Russian Federation. The Crimean Peninsula is a potential introduction area of the Pierce’s disease agent due to suitable climatic conditions, the ample availability of major host plants, as well as insect vectors. During a research monitoring in 2018-2020, the Institute assessed the phytosanitary status of the Crimean territory. The survey sampled vegetative parts of grapevines, stone fruits (peach, cherry, plum, merry, almond), selected essential-oil and ornamental shrubs and trees. Diagnostic procedures were performed in accordance with the international standards. A three-year phytosanitary survey of the Crimean plantations revealed no presence of X. fastidiosa. Two protocols of sample preparation and DNA extraction from various substrates have been tested. A real-time PCR-based protocol was proved highly specific for zero false positive and nonspecific rates.

Landscape Epidemiology of Xylella fastidiosa in the Balearic Islands

Xylella fastidiosa (Xf) is a vascular plant pathogen native to the Americas. In 2013, it was first reported in Europe, implicated in a massive die-off of olive trees in Apulia, Italy. This finding prompted mandatory surveys across Europe, successively revealing that the bacterium was already established in some distant areas of the western Mediterranean. To date, the Balearic Islands (Spain) hold the major known genetic diversity of Xf in Europe. Since October 2016, four sequence types (ST) belonging to the subspecies fastidiosa (ST1), multiplex (ST7, ST81), and pauca (ST80) have been identified infecting 28 host species, including grapevines, almond, olive, and fig trees. ST1 causes Pierce’s disease (PD) and together with ST81 are responsible for almond leaf scorch disease (ALSD) in California, from where they were introduced into Mallorca in around 1993, very likely via infected almond scions brought for grafting. To date, almond leaf scorch disease affects over 81% of almond trees and Pierce’s disease is widespread in vineyards across Mallorca, although producing on average little economic impact. In this perspective, we present and analyze a large Xf-hosts database accumulated over four years of field surveys, laboratory sample analyses, and research to understand the underlying causes of Xf emergence and spread among crops and wild plants in the Balearic Islands. The impact of Xf on the landscape is discussed.

Allopatric plant pathogen population divergence following disease emergence

Within the landscape of globally distributed pathogens, populations differentiate via both adaptive and non-adaptive forces. Individual populations are likely to show unique trends of genetic diversity, host-pathogen interaction, and ecological adaptation. In plant pathogens, allopatric divergence may occur particularly rapidly within simplified agricultural monoculture landscapes. As such, the study of plant pathogen populations in monocultures can highlight the distinct evolutionary mechanisms that lead to local genetic differentiation. Xylella fastidiosa is a plant pathogen known to infect and damage multiple monocultures worldwide. One subspecies, Xylella fastidiosa subsp. fastidiosa was first introduced to the USA ∼150 years ago, where it was found to infect and cause disease in grapevines (Pierce’s disease of grapevines, PD). Here, we studied PD-causing subsp. fastidiosa populations, with an emphasis on those found in the USA. Our study shows that following its establishment in the USA, PD-causing strains likely split into populations in the East and West Coast. This diversification has occurred via both changes in gene content (gene gain/loss events) and variations in nucleotide sequence (mutation and recombination). In addition, we reinforce the notion that PD-causing populations within the USA acted as the source for subsequent subsp. fastidiosa outbreaks in Europe and Asia. IMPORTANCE Compared to natural environments, the reduced diversity of monoculture agricultural landscapes can lead bacterial plant pathogens to quickly adapt to local biological and ecological conditions. Because of this, accidental introductions of microbial pathogens into naïve regions represents a significant economic and environmental threat. Xylella fastidiosa is a plant pathogen with an expanding host and geographic range due to multiple intra- and inter-continental introductions. X. fastidiosa subsp. fastidiosa, infects and causes disease in grapevines (Pierce’s disease of grapevines; PD). This study focused on PD-causing X. fastidiosa populations, particularly those found in the USA but also invasions into Taiwan and Spain. The analysis shows that PD-causing X. fastidiosa has diversified via multiple co-occurring evolutionary forces acting at an intra- and inter-population level. This analysis enables a better understating of the mechanisms leading to the local adaptation of X. fastidiosa, and how a plant pathogen diverges allopatrically after multiple and sequential introduction events.

Control of Pierce’s Disease Through Areawide Management of Glassy-Winged Sharpshooter (Hemiptera: Cicadellidae) and Roguing of Infected Grapevines

The General Beale Pilot Project serves as a case study for the use of areawide monitoring and treatment programs for glassy-winged sharpshooter (GWSS), Homalodisca vitripennis (Germar), and monitoring and roguing programs for grapevines infected with Xylella fastidiosa, to achieve regional management of Pierce’s disease. The Project is located in southeast Kern County, CA, and contains ~2,800 ha of citrus and grapevines grown within approximately 50 km2. For nearly 20 yr, an average of 470 traps have been used to monitor GWSS populations regionally by the California Department of Food and Agriculture, and to inform coordinated, areawide treatments by the USDA-APHIS Areawide Treatment Program to overwintering GWSS in citrus. Grape growers were responsible for treating their own vineyards, and for the roguing of infected grapevines based on surveys provided by the University of California. Herein, we provide a history of the General Beale Pilot Project, broken down into six eras based on levels of Project success, which incorporate data on GWSS captures, pesticide use, and disease incidence. We describe patterns of success related to the regional coordination of effective treatment and roguing programs that can be used by grape and neighboring citrus growers for areawide management of Pierce’s disease. We conclude by describing current and future challenges for Pierce’s disease management, including pesticide availability and resistance, GWSS refuges, the inability to detect and rogue infected vines in the year they become infected, and the sustainability of voluntary programs that rely on public funding.

Genetic analysis reveals an east-west divide within North American Vitis species that mirrors their resistance to Pierce’s disease

Pierce’s disease (PD) caused by the bacterium Xylella fastidiosa is a deadly disease of grapevines. This study used 20 SSR markers to genotype 326 accessions of grape species collected from the southeastern and southwestern United States, Mexico and Costa Rica. Two hundred sixty-six of these accessions, and an additional 12 PD resistant hybrid cultivars developed from southeastern US grape species, were evaluated for PD resistance. Disease resistance was evaluated by quantifying the level of bacteria in stems and measuring PD symptoms on the canes and leaves. Both Bayesian clustering and principal coordinate analyses identified two groups with an east-west divide: group 1 consisted of grape species from the southeastern US and Mexico, and group 2 consisted of accessions collected from the southwestern US and Mexico. The Sierra Madre Oriental mountain range appeared to be a phylogeographic barrier. The state of Texas was identified as a potential hybridization zone. The hierarchal STRUCTURE analysis on each group showed clustering of unique grape species. An east-west divide was also observed for PD resistance. With the exception of Vitis candicans and V. cinerea accessions collected from Mexico, all other grape species as well as the resistant southeastern hybrid cultivars were susceptible to the disease. Southwestern US grape accessions from drier desert regions showed stronger resistance to the disease. Strong PD resistance was observed within three distinct genetic clusters of V. arizonica which is adapted to drier environments and hybridizes freely with other species across its wide range.

Xylella fastidiosa and Glassy-Winged Sharpshooter Population Dynamics in the Southern San Joaquin Valley of California

Xylella fastidiosa is a vector-transmitted bacterial plant pathogen that affects a wide array of perennial crops, including grapevines (Pierce’s disease). In the southern San Joaquin Valley of California, epidemics of Pierce’s disease of grapevine were associated with the glassy-winged sharpshooter, Homalodisca vitripennis. During the growing season, rates of X. fastidiosa spread in vineyards are affected by changes in pathogen distribution within chronically infected grapevines and by vector population dynamics. Grapevines chronically infected with X. fastidiosa rarely tested positive for the pathogen prior to July, suggesting vector acquisition of X. fastidiosa from grapevines increases as the season progresses. This hypothesis was supported by an increase in number of X. fastidiosa-positive glassy-winged sharpshooters collected from vineyards during July through September. Analysis of insecticide records indicated that vineyards in the study area were typically treated with a systemic neonicotinoid in spring of each year. As a result, abundance of glassy-winged sharpshooters was typically low in late spring and early summer, with abundance of glassy-winged sharpshooter adults increasing in late June and early July of each year. Collectively, the results suggest that late summer is a crucial time for X. fastidiosa secondary spread in vineyards in the southern San Joaquin Valley, because glassy-winged sharpshooter abundance, number of glassy-winged sharpshooters testing positive for X. fastidiosa, and grapevines with detectable pathogen populations were all greatest during this period.

Deep Learning Neural Network Prediction Method Improves Proteome Profiling of Vascular Sap of Grapevines during Pierce’s Disease Development

Plant secretome studies highlight the importance of vascular plant defense proteins against pathogens. Studies on Pierce’s disease of grapevines caused by the xylem-limited bacterium Xylella fastidiosa (Xf) have detected proteins and pathways associated with its pathobiology. Despite the biological importance of the secreted proteins in the extracellular space to plant survival and development, proteome studies are scarce due to methodological challenges. Prosit, a deep learning neural network prediction method is a powerful tool for improving proteome profiling by data-independent acquisition (DIA). We explored the potential of Prosit’s in silico spectral library predictions to improve DIA proteomic analysis of vascular leaf sap from grapevines with Pierce’s disease. The combination of DIA and Prosit-predicted libraries increased the total number of identified grapevine proteins from 145 to 360 and Xf proteins from 18 to 90 compared to gas-phase fractionation (GPF) libraries. The new proteins increased the range of molecular weights, assisted in the identification of more exclusive peptides per protein, and increased identification of low-abundance proteins. These improvements allowed identification of new functional pathways associated with cellular responses to oxidative stress, to be investigated further.

Deep learning neural network prediction method improves proteome profiling of vascular sap of grapevines during Pierce’s disease development

Plant secretome studies have shown the importance of plant defense proteins in the vascular system against pathogens. Studies on Pierce’s disease of grapevines caused by the xylem-limited bacteria Xylella fastidiosa (Xf) have detected proteins and pathways associated to its pathobiology. Despite the biological importance of the secreted proteins in the extracellular space to plant survival and development, proteome studies are scarce due to technical and technological challenges. Deep learning neural network prediction methods can provide powerful tools for improving proteome profiling by data-independent acquisition (DIA). We aimed to explore the potential of this strategy by combining it with in silico spectral library prediction tool, Prosit, to analyze the proteome of vascular leaf sap of grapevines with Pierce’s disease. The results demonstrate that the combination of DIA and Prosit increased the total number of identified proteins from 145 to 360 for grapevines and 18 to 90 for Xf. The new proteins increased the range of molecular weight, assisted on the identification of more exclusive peptides per protein, and increased the identification of low abundance proteins. These increases allowed the identification of new functional pathways associated with cellular responses to oxidative stress to be further investigated.