Virulence Comparison of a Comprehensive Panel of Xylella fastidiosa Pierce's Disease Isolates from California.

Xylella fastidiosa, the causal agent of Pierce's disease of grapevine, has been found in all major grape-growing regions in California, U.S.A. Large collections of X. fastidiosa isolates are available from these areas, which enable comparative studies of pathogen genetic traits and virulence. Owing to the significant resource requirements for experiments with X. fastidiosa in grapevine, however, most studies use only a single isolate to evaluate disease, and it is not clear how much variability between isolates impacts disease development in experimental or natural settings. In this study, a comprehensive panel of X. fastidiosa isolates from all California grape-growing regions was tested for virulence in susceptible grapevine and in the model host plant, tobacco. Seventy-one isolates were tested, 29 in both grapevine and tobacco. The results of this study highlight the inherent variability of inoculation experiments with X. fastidiosa, including variation in disease severity in plants inoculated with a single isolate, and variability between experimental replicates. There were limited differences in virulence between isolates that were consistent across experimental replicates, or across different host plants. This suggests that choice of isolate within the X. fastidiosa subsp. fastidiosa Pierce's disease group may not make any practical difference when testing in susceptible grape varieties, and that pathogen evolution has not significantly changed virulence of Pierce's disease isolates within California. The location of isolation also did not dictate relative disease severity. This information will inform experimental design for future studies of X. fastidiosa in grapevine and provide important context for genomic research.

Droplet superpropulsion in an energetically constrained insect.

Food consumption and waste elimination are vital functions for living systems. Although how feeding impacts animal form and function has been studied for more than a century since Darwin, how its obligate partner, excretion, controls and constrains animal behavior, size, and energetics remains largely unexplored. Here we study millimeter-scale sharpshooter insects (Cicadellidae) that feed exclusively on a plant's xylem sap, a nutrient-deficit source (95% water). To eliminate their high-volume excreta, these insects exploit droplet superpropulsion, a phenomenon in which an elastic projectile can achieve higher velocity than the underlying actuator through temporal tuning. We combine coupled-oscillator models, computational fluid dynamics, and biophysical experiments to show that these insects temporally tune the frequency of their anal stylus to the Rayleigh frequency of their surface tension-dominated elastic drops as a single-shot resonance mechanism. Our model predicts that for these tiny insects, the superpropulsion of droplets is energetically cheaper than forming jets, enabling them to survive on an extreme energy-constrained xylem-sap diet. The principles and limits of superpropulsion outlined here can inform designs of energy-efficient self-cleaning structures and soft engines to generate ballistic motions.

Web Vibrations in Intraspecific Contests of Female Black Widow Spiders, Latrodectus hesperus.

Female black widow spiders, Latrodectus hesperus Chamberlin and Ivie (Araneae: Theridiidae), are solitary predators of arthropods with no tolerance for intruders on the webs. In California, L. hesperus are found in urban and agricultural settings and can be a phytosanitary pest in fresh produce. Spatial separation of L. hesperus webs could be determined by seasonal population densities, with territorial competition expected under high densities in the environment. However, little is known about female-female communication behaviors in this species. In 1-hr laboratory observations, displays of female-female rivalry included production of vibrational signals in a majority (20 of 30) of trials. The number of signals produced by both females was highest during the initial 10 min of trials, with signaling rate (time interval between signals) peaking during the 40-50 min observation period. The overall ratio of signals produced by the resident female and the introduced female was about 5:1, with the number of signals produced by the resident female higher than the number of signals produced by the introduced female. Analysis of rivalry signals showed a peak in magnitude (about 0.4 m/s) ranging from 6 to 23 Hz and smaller peaks at about 29, 38, and 47 Hz. Collectively, these results demonstrate that female L. hesperus exhibit territorial rivalry and that female-female rivalry is mediated by emission of vibrational signals through the web. Understanding the mechanisms of intraspecific competition in L. hesperus is required for elucidating interspecific interactions in the environment and may lead to development of novel methods to prevent spiders from colonizing crops.

Glassy-Winged Sharpshooters Cease Feeding and Avoid Plants Treated With Sub-Lethal Doses of the Neonicotinoid Insecticide Imidacloprid.

Insecticides are a primary means for suppressing populations of insects that transmit plant pathogens. Application of insecticides for limiting the spread of insect-transmitted plant pathogens is often most effective when applied on an area-wide scale. The glassy-winged sharpshooter is a vector of the bacterial pathogen Xylella fastidiosa, which causes numerous plant diseases including Pierce's disease of grapevine. The glassy-winged sharpshooter has been the subject of an area-wide suppression program in California for nearly two decades. Overreliance on a limited number of active ingredients including the neonicotinoid imidacloprid has resulted in increased levels of resistance to commonly applied products. In California, glassy-winged sharpshooters move between citrus, an important overwintering host, and vineyards. Accordingly, imidacloprid is routinely applied via the irrigation system in vineyards and citrus orchards. For soil applied applications, it may take days to weeks for concentrations in plants to increase to lethal doses. Further, as the dose of imidacloprid required to kill sharpshooters increases due to resistance, so too does the period that sharpshooters are exposed to sub-lethal doses. Response of glassy-winged sharpshooter to cowpea plants treated with sub-lethal doses of imidacloprid was evaluated by conducting no-choice and choice tests. In no-choice feeding assays, glassy-winged sharpshooters caged on plants treated with sub-lethal doses of imidacloprid ceased feeding and produced little excreta. Further, sub-lethal exposure to a range of doses over a 4-d period did not affect viability over a 9-wk post-exposure holding period on untreated plants. In choice-tests, glassy-winged sharpshooters avoided treated plants and were observed predominately on untreated plants. Results suggest that application of imidacloprid to vineyards and citrus orchards may push glassy-winged sharpshooters out of treated habitats rather than kill them.

Validation of a Novel Stereo Vibrometry Technique for Spiderweb Signal Analysis.

From courtship rituals, to prey identification, to displays of rivalry, a spider's web vibrates with a symphony of information. Examining the modality of information being transmitted and how spiders interact with this information could lead to new understanding how spiders perceive the world around them through their webs, and new biological and engineering techniques that leverage this understanding. Spiders interact with their webs through a variety of body motions, including abdominal tremors, bounces, and limb jerks along threads of the web. These signals often create a large enough visual signature that the web vibrations can be analyzed using video vibrometry on high-speed video of the communication exchange. Using video vibrometry to examine these signals has numerous benefits over the conventional method of laser vibrometry, such as the ability to analyze three-dimensional vibrations and the ability to take measurements from anywhere in the web, including directly from the body of the spider itself. In this study, we developed a method of three-dimensional vibration analysis that combines video vibrometry with stereo vision, and verified this method against laser vibrometry on a black widow spiderweb that was experiencing rivalry signals from two female spiders.

Insights Regarding Resistance of 'Nemaguard' Rootstock to the Bacterium Xylella fastidiosa.

'Nemaguard' is a commonly used rootstock for almond and stone fruits due to resistance to nematodes and enhanced scion vigor. Nemaguard also happens to be resistant to strains of Xylella fastidiosa that cause almond leaf scorch disease. Previous research showed that prior to June-budding, this rootstock can prevent infection of almond nursery stock by X. fastidiosa. Further, the rootstock also promotes recovery from infection in susceptible almond scions. Objectives of this study were to 1) compare movement and bacterial populations of X. fastidiosa in almond and Nemaguard, 2) determine whether the metabolic profile of infected versus noninfected plants of each species correspond with differences in pathogen distribution, and 3) evaluate the impact of feeding on Nemaguard on transmission efficiency and pathogen populations in insects. Results showed limited or no movement of X. fastidiosa beyond the point of mechanical inoculation in Nemaguard, whereas X. fastidiosa was detected in susceptible almond and isolated from plant samples distal to the point of inoculation. Large differences in the concentration of phenolic compounds between Nemaguard and almond were also found, although this was not impacted by infection status. After acquiring X. fastidiosa from infected plants, vector access periods of up to 14 days on Nemaguard neither reduced pathogen populations in vectors nor reduced transmission efficiency of X. fastidiosa to susceptible plants when compared with similar vector-access periods on susceptible grapevines. Results suggest Nemaguard, in spite of having high phenolic concentrations in its xylem, does not directly impact X. fastidiosa survival and that future research should focus on identification of potential physical traits that prevent bacterial attachment, multiplication, or movement within the plant.

Insect-to-insect horizontal transmission of a phytoreovirus in the absence of an infected plant host.

Vertical transmission of Homalodisca vitripennis reovirus (HoVRV) from glassy-winged sharpshooter (GWSS, Homalodisca vitripennis (Germar)) females to progeny occurred in laboratory assays at frequencies too low (2.6%-15.4%) to account for HoVRV incidence (90-100%) in field populations resident in citrus. Because citrus is immune to HoVRV and no plant host is known, horizontal transmission of HoVRV from insect-to-insect was evaluated. Exposure of colony-reared, virus-free test nymphs to HoVRV-infected source adults held in the same cage for 10 days on virus-immune cowpea resulted in HoVRV transmission (13.3%-30.7%) to test nymphs. HoVRV was not transmitted when exposure was indirect and required passive movement of virions through the xylem of immune citrus seedlings. Collectively, these results demonstrate direct insect-to-insect horizontal transmission of HoVRV, providing a plausible explanation for high incidence of HoVRV in GWSS field populations in the absence of efficient vertical transmission or a plant host.

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.

Complete Genome Sequence Data of Three Xylella fastidiosa subsp. multiplex Strains Isolated from Olive Trees in California, U.S.A.

Xylella fastidiosa is a xylem-limited bacterial plant pathogen that causes disease on numerous hosts. Additionally, X. fastidiosa asymptomatically colonizes a wide range of plant species. X. fastidiosa subsp. multiplex has been detected in olive (Olea europaea) trees grown in California, U.S.A., as well as in Europe. Strains of X. fastidiosa subsp. multiplex isolated from California olive trees are not known to cause disease on olive, although some can induce leaf-scorch symptoms on almond (Prunus dulcis). No genome assemblies currently exist for olive-associated X. fastidiosa subsp. multiplex strains; therefore, a hybrid assembly method was used to generate complete genome sequences for three X. fastidiosa subsp. multiplex strains (Fillmore, LM10, and RH1) isolated from olive trees grown in Ventura and Los Angeles counties of California.

Targeted Mutations in Xylella fastidiosa Affect Acquisition and Retention by the Glassy-Winged Sharpshooter, Homalodisca vitripennis (Hemiptera: Cicadellidae).

Xylella fastidiosa (Wells) is a xylem-limited bacterium that causes Pierce's disease of grapevines. The bacterium is transmitted by insect vectors such as the glassy-winged sharpshooter (GWSS), Homalodisca vitripennis (Germar). Experiments were conducted to compare the role of selected X. fastidiosa genes on 1) bacterial acquisition and retention in GWSS foreguts, and 2) transmission to grapevines by GWSS. Bacterial genotypes used were: mutants Xf-ΔpilG, Xf-ΔpilH, Xf-ΔgacA, and Xf-ΔpopP; plus wild type (WT) as control. Results showed that Xf-ΔpilG had enhanced colonization rate and larger numbers in GWSS compared with WT. Yet, Xf-ΔpilG exhibited the same transmission efficiency as WT. The Xf-ΔpilH exhibited poor acquisition and retention. Although initial adhesion, multiplication, and retention of Xf-ΔpilH in GWSS were almost eliminated compared with WT, the mutation did not reduce transmission success in grapevines. Overall, Xf-ΔgacA showed colonization rates and numbers in foreguts similar to WT. The Xf-ΔgacA mutation did not affect initial adhesion, multiplication, and long-term retention compared with WT, and was not significantly diminished in transmission efficiency. In contrast, numbers of Xf-ΔpopP were variable over time, displaying greatest fluctuation from highest to lowest levels. Thus, Xf-ΔpopP had a strong, negative effect on initial adhesion, but adhered and slowly multiplied in the foregut. Again, transmission was not diminished compared to WT. Despite reductions in acquisition and retention by GWSS, transmission efficiency of genotypes to grapevines was not affected. Therefore, in order to stop the spread of X. fastidiosa by GWSS using gene-level targets, complete disruption of bacterial colonization mechanisms is required.

Correction to: Transmission of the frequency components of the vibrational signal of the glassy-winged sharpshooter, Homalodisca vitripennis, within and between grapevines.

Unfortunately, Fig. 3 was incorrectly published in the original publication. The correct version of Fig. 3 is updated here.

Transmission of the frequency components of the vibrational signal of the glassy-winged sharpshooter, Homalodisca vitripennis, within and between grapevines.

The agricultural pest, Homalodisca vitripennis, relies on vibrational communication through plants for species identification, location, and courtship. Their vibrational signal exhibits a dominant frequency between 80 and 120 Hz, with higher frequency, lower intensity harmonics occurring approximately every 100 Hz. However, previous research revealed that not all harmonics are recorded in every signal. Therefore, how the female H. vitripennis vibrational signal changes as it travels through the plant was investigated. Results confirmed that transmission was a bending wave, with decreased signal intensity for increasing distance from the source; moreover, at distances of 50 cm, higher frequencies traveled faster than lower frequencies, suggesting that dispersion of H. vitripennis signal components may enable signaling partners to encode distance. Finally, H. vitripennis generates no detectable airborne signal (pressure wave), yet their low vibrational frequency components are detectable in neighboring plants as a result of leaf-to-air-to-leaf propagation. For instance, with isolated key female signal frequencies, 100 Hz was detected at a 10 cm gap between leaves, whereas 600 Hz was detectable only with a 0.1 cm gap. Together, these results highlight the complexity of vibration propagation in plants and suggest the possibility of the animals using the harmonic content to determine distance to the signaling H. vitripennis source.

Update of the Scientific Opinion on the risks to plant health posed by Xylella fastidiosa in the EU territory.

EFSA was asked to update the 2015 EFSA risk assessment on Xylella fastidiosa for the territory of the EU. In particular, EFSA was asked to focus on potential establishment, short- and long-range spread, the length of the asymptomatic period, the impact of X. fastidiosa and an update on risk reduction options. EFSA was asked to take into account the different subspecies and Sequence Types of X. fastidiosa. This was attempted throughout the scientific opinion but several issues with data availability meant that this could only be partially achieved. Models for risk of establishment showed most of the EU territory may be potentially suitable for X. fastidiosa although southern EU is most at risk. Differences in estimated areas of potential establishment were evident among X. fastidiosa subspecies, particularly X. fastidiosa subsp. multiplex which demonstrated areas of potential establishment further north in the EU. The model of establishment could be used to develop targeted surveys by Member States. The asymptomatic period of X. fastidiosa varied significantly for different host and pathogen subspecies combinations, for example from a median of approximately 1 month in ornamental plants and up to 10 months in olive, for pauca. This variable and long asymptomatic period is a considerable limitation to successful detection and control, particularly where surveillance is based on visual inspection. Modelling suggested that local eradication (e.g. within orchards) is possible, providing sampling intensity is sufficient for early detection and effective control measures are implemented swiftly (e.g. within 30 days). Modelling of long-range spread (e.g. regional scale) demonstrated the important role of long-range dispersal and the need to better understand this. Reducing buffer zone width in both containment and eradication scenarios increased the area infected. Intensive surveillance for early detection, and consequent plant removal, of new outbreaks is crucial for both successful eradication and containment at the regional scale, in addition to effective vector control. The assessment of impacts indicated that almond and Citrus spp. were at lower impact on yield compared to olive. Although the lowest impact was estimated for grapevine, and the highest for olive, this was based on several assumptions including that the assessment considered only Philaenus spumarius as a vector. If other xylem-feeding insects act as vectors the impact could be different. Since the Scientific Opinion published in 2015, there are still no risk reduction options that can remove the bacterium from the plant in open field conditions. Short- and long-range spread modelling showed that an early detection and rapid application of phytosanitary measures, consisting among others of plant removal and vector control, are essential to prevent further spread of the pathogen to new areas. Further data collection will allow a reduction in uncertainty and facilitate more tailored and effective control given the intraspecific diversity of X. fastidiosa and wide host range.

Plant Water Stress and Vector Feeding Preference Mediate Transmission Efficiency of a Plant Pathogen.

Pathogen spread by arthropod vectors is the outcome of pathogen-vector-plant interactions, as well as how these interactions are impacted by abiotic and biotic factors. While plant water stress impacts each component of the Pierce's disease pathosystem (Xylella fastidiosa Wells et al., insect vectors, and grapevines), the outcome of interactions in relation to pathogen spread is unknown. The objectives of this study were 1) to determine the role of plant water stress on vector acquisition and inoculation of X. fastidiosa under choice and no-choice conditions for source or recipient vines, and 2) to provide insights into the effects of vineyard irrigation regimes on spread of X. fastidiosa by using a host-vector epidemic model. Under no-choice conditions, pathogen acquisition increased as water stress increased in source plants, while inoculation was not affected by water status of recipient vines. Thus, under no-choice conditions, plant water stress increased transmission of X. fastidiosa. However, when vectors had a choice of an uninfected well-watered versus an infected water-stressed grapevine, transmission efficiency declined as water stress levels increased. While our experimental results produced wide uncertainty estimates, the epidemiological modeling suggested a non-linear relationship between water stress and pathogen spread: moderate water stress enhances pathogen spread but severe or no stress produce equivalent spread. In summary, both host plant condition and vector host preference interacted to determine transmission efficiency of X. fastidiosa.

Mating disruption of Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae) by playback of vibrational signals in vineyard trellis.

BACKGROUND: Glassy-winged sharpshooter (GWSS), Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae) is an important vector of the bacterium Xylella fastidiosa, the causal agent of Pierce's disease of grapevine. Area-wide insecticide applications have suppressed GWSS populations for ∼ 25 years, but reduced levels of insecticide susceptibility have been reported. Therefore, alternative methods of control are needed. Objectives of this study were to evaluate the efficacy of playback of vibrational mating communication signals for disrupting mating of GWSS in a natural vineyard setting and to evaluate spectral properties of signal transmission through vineyard trellis. RESULTS: Playback reduced mating of GWSS on grapevines. A total of 28 (of 134) male-female pairs mated in the control treatment (silence) and only one (of 134) pair mated when treated with the vibrational signal playback. Playback of vibrational signals through vineyard trellis was affected by distance from the signal source, with frequency composition being the highest at the source and lowest on vines positioned away from the source. Frequency composition in canes housing test insects decreased exponentially as distance from the source increased, whereas the relative amplitude of analyzed frequencies decreased linearly. CONCLUSION: Although further studies are needed prior to method implementation, data from this study continue to support integration of vibrational mating disruption with current methods to suppress GWSS populations. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

Effects of Energy Reserves and Diet on Glassy-Winged Sharpshooter Egg Maturation.

The glassy-winged sharpshooter is an invasive insect capable of transmitting the plant pathogen Xylella fastidiosa. As rates of pathogen spread are a function of vector abundance, identification of factors contributing to glassy-winged sharpshooter egg production will aid in predicting population growth. Here, effects of stored energy reserves and adult diet on glassy-winged sharpshooter egg maturation were evaluated. To estimate energy reserves available to adult females at the beginning of feeding assays, residuals from a regression of wet weight on size were used. Analysis of a subset of females sacrificed at the beginning of feeding assays, demonstrated that females with a positive residual wet weight had higher lipid content and carried more eggs than females with a negative residual wet weight. To evaluate effects of diet and energy reserves on egg maturation, energy reserves available to females entering feeding assays on cowpea and grapevine were estimated. For females held on cowpea, residual wet weight and quantity of excreta produced over a 6-d feeding period affected egg production. In contrast, for females held on grapevine, only residual wet weight affected egg production. Comparison of cowpea and grapevine xylem sap determined that eight amino acids were more concentrated in xylem sap from cowpea than from grapevine. Collectively, the results suggest that glassy-winged sharpshooter population growth within crop monocultures will not depend solely on the nutritional quality of the specific crop for producing mature eggs but also on the quantity of energy reserves accumulated by females prior to entering that crop habitat.

Design of a candidate vibrational signal for mating disruption against the glassy-winged sharpshooter, Homalodisca vitripennis.

BACKGROUND: The glassy-winged sharpshooter (GWSS), Homalodisca vitripennis, is an important pest of grapevines due to its ability to transmit Xylella fastidiosa, the causal agent of Pierce's disease. GWSS mating communication is based on vibrational signals; therefore, vibrational mating disruption could be an alternative to insecticides for suppression of the GWSS population. Our objectives were to identify spectral features of the female signal that elicit male signaling, design disruptive signals able to alter male perception and acceptance of a female, and determine the signal intensity required for future field applications. RESULTS: Male responses to playback of modified female signals were significantly reduced by 60-75% when part of the female signal spectral components above or below 400 Hz were deleted. Playback bioassays showed that transmission of an 80 Hz pure frequency tone to plants completely suppressed male signaling to female signal playback, even if the disruptive signal amplitude was 10 dB lower than the female signal playback. CONCLUSION: Although the mechanism underlying cessation of male signaling activity in the presence of disruption is not yet understood, results suggest that an 80 Hz vibrational signal should be tested in laboratory and field experiments to assess its efficacy in disrupting mating of GWSS. © 2017 Society of Chemical Industry.

Sequence polymorphism in an insect RNA virus field population: A snapshot from a single point in space and time reveals stochastic differences among and within individual hosts.

Population structure of Homalodisca coagulata Virus-1 (HoCV-1) among and within field-collected insects sampled from a single point in space and time was examined. Polymorphism in complete consensus sequences among single-insect isolates was dominated by synonymous substitutions. The mutant spectrum of the C2 helicase region within each single-insect isolate was unique and dominated by nonsynonymous singletons. Bootstrapping was used to correct the within-isolate nonsynonymous:synonymous arithmetic ratio (N:S) for RT-PCR error, yielding an N:S value ~one log-unit greater than that of consensus sequences. Probability of all possible single-base substitutions for the C2 region predicted N:S values within 95% confidence limits of the corrected within-isolate N:S when the only constraint imposed was viral polymerase error bias for transitions over transversions. These results indicate that bottlenecks coupled with strong negative/purifying selection drive consensus sequences toward neutral sequence space, and that most polymorphism within single-insect isolates is composed of newly-minted mutations sampled prior to selection.

Seasonal Fluctuations of Sap-Feeding Insect Species Infected by Xylella fastidiosa in Apulian Olive Groves of Southern Italy.

A study on seasonal abundance of Auchenorrhyncha species and their infectivity by Xylella fastidiosa in the Apulia region of Italy was conducted to identify ideal periods for monitoring and adoption of potential control measures against insect vectors. Adult populations of Auchenorrhyncha species were monitored monthly over a 2-yr period from five olive groves. A total of 15 species were captured, identified, and tested for presence of X. fastidiosa by polymerase chain reaction (PCR). For three species, Philaenus spumarius L., Neophilaenus campestris (Fallèn), and Euscelis lineolatus Brullé, positive reactions to X. fastidiosa were obtained, on average, in 16.3, 15.9 and 18.4% of adult insects, respectively. Philaneous spumarius was the dominant species (39.8% of total Auchenorrhyncha captured) with the highest adult abundance in summer months. Adult P. spumarius and N. campestris were first detected between March and May in both years, and all insects tested during these periods (year 1: n = 42, year 2: n = 132) gave negative reactions to X. fastidiosa by PCR. Similarly, first adults of E. lineolatus that appeared from October to November (year 1: n = 20, year 2: n = 15) tested negative for presence of X. fastidiosa Given the lack of transstadial and transovarial transmission of X. fastidiosa and considering that P. spumarius is univoltine, control measures against nymphal stages of P. spumarius should be investigated as means of population suppression to reduce spread of X. fastidiosa in olive groves.