Impact of phenolic compounds on progression of Xylella fastidiosa infections in susceptible and PdR1-locus containing resistant grapevines.

Pierce's disease is of major concern for grapevine (Vitis vinifera) production wherever the bacterial pathogen Xylella fastidiosa and its vectors are present. Long-term management includes the deployment of resistant grapevines such as those containing the PdR1 locus from the wild grapevine species Vitis arizonica, which do not develop Pierce's disease symptoms upon infection. However, little is understood about how the PdR1 locus functions to prevent disease symptom development. Therefore, we assessed the concentrations of plant defense-associated compounds called phenolics in healthy and X. fastidiosa-infected PdR1-resistant and susceptible grapevine siblings over time. Soluble foliar phenolic levels, especially flavonoids, in X. fastidiosa-infected PdR1-resistant grapevines were discovered to be significantly lower than those in infected susceptible grapevines. Therefore, it was hypothesized that PdR1-resistant grapevines, by possessing lowered flavonoid levels, affects biofilm formation and causes reduced X. fastidiosa intra-plant colonization, thus limiting the ability to increase pathogen populations and cause Pierce's disease. These results therefore reveal that differences in plant metabolite levels might be a component of the mechanisms that PdR1 utilizes to prevent Pierce's disease.

Inference of Bacterial Pathogen Instantaneous Population Growth Dynamics.

Although bacterial host colonization is a dynamic process that requires population growth, studies often focus on comparing bacterial populations at a given time point. However, this may not reflect the dynamics of the colonization process. Time-course assays provide important insights into the dynamics of host colonization but are laborious and may still lack resolution for immediate processes affecting populations. An alternative way to address this issue, using widely accessible tools (such as quantitative PCR [qPCR]), is to take advantage of the relationship between bacterial chromosomal replication and cell division to determine population growth status at the sampling time. Conceptually, the ratio between the number of copies at the origin of replication and that at the terminus of replication should be correlated with the measured bacterial growth rate. This peak-to-trough ratio (PTR) to estimate instantaneous population growth status was tested with the slow-growing plant-pathogenic bacterium Xylella fastidiosa. We found no correlation between PTR and the measured growth rate when using genome-level data but overall sequencing depth of coverage trends matched theoretical expectations. On the other hand, the population growth status of X. fastidiosa was predicted by PTR when using qPCR data, which was improved by the pretreatment of cells with a photoreactive DNA-binding dye. Our results suggest that PTR could be used to determine X. fastidiosa growth status both in planta and in insect vectors. We expect PTR will perform better with fast-growing bacterial pathogens, potentially becoming a powerful tool for easily and quickly assessing population growth status.

Discriminating between viable and membrane-damaged cells of the plant pathogen Xylella fastidiosa.

Xylella fastidiosa is a plant pathogenic bacterium with devastating consequences to several crops of economic importance across the world. While this pathogen has been studied for over a century in the United States, several aspects of its biology remain to be investigated. Determining the physiological state of bacteria is essential to understand the effects of its interactions with different biotic and abiotic factors on cell viability. Although X. fastidiosa is culturable, its slow growing nature makes this technique cumbersome to assess the physiological state of cells present in a given environment. PMA-qPCR, i.e. the use of quantitative PCR combined with the pre-treatment of cells with the dye propidium monoazide, has been successfully used in a number of studies on human pathogens to calculate the proportion of viable cells, but has less frequently been tested on plant pathogens. We found that the use of a version of PMA, PMAxx, facilitated distinguishing between viable and non-viable cells based on cell membrane integrity in vitro and in planta. Additional experiments comparing the number of culturable, viable, and total cells in planta would help further confirm our initial results. Enhancers, intended to improve the efficacy of PMAxx, were not effective and appeared to be slightly toxic to X. fastidiosa.

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.

Landscape Effects on Solenopsis invicta (Hymenoptera: Formicidae) and Geocoris spp. (Hemiptera: Geocoridae), Two Important Omnivorous Arthropod Taxa in Field Crops.

The economically important brown stink bug, Euschistus servus (Say) (Hemiptera: Pentatomidae), is a native pest of many crops in southeastern United States and insecticide applications are the prevailing method of population suppression. To elucidate biological control of E. servus populations, we investigated two egg predators' (red imported fire ants, Solenopsis invicta Buren (Hymenoptera: Formicidae), and Geocoris spp. (Hemiptera: Geocoridae)) responses to both local and landscape factors that may have influenced their combined ability to cause mortality in immature E. servus. We estimated the density of fire ants and Geocoris spp. on four major crop hosts-maize, peanut, cotton, and soybean-in 16 landscapes over 3 yr in the coastal plain of Georgia, USA. Both Geocoris spp. and fire ant populations were concentrated on specific crops in this study, maize and soybean for Geocoris spp. and peanut and cotton for fire ants, but the percentage area of specific crops and woodland and pasture in the landscape and year also influenced their density in focal fields. The crop specific density of both taxa, the influence of the percentage area of specific crops and woodland in the landscape, and the variability in density over years may have been related to variable alternative resources for these omnivores in the habitats. Despite the variability over years, differential habitat use of fire ants and Georcoris spp. may have contributed to their combined ability to cause E. servus immature mortality.

Xylella fastidiosa: Insights into an Emerging Plant Pathogen.

The bacterium Xylella fastidiosa re-emerged as a plant pathogen of global importance in 2013 when it was first associated with an olive tree disease epidemic in Italy. The current threat to Europe and the Mediterranean basin, as well as other world regions, has increased as multiple X. fastidiosa genotypes have now been detected in Italy, France, and Spain. Although X. fastidiosa has been studied in the Americas for more than a century, there are no therapeutic solutions to suppress disease development in infected plants. Furthermore, because X. fastidiosa is an obligatory plant and insect vector colonizer, the epidemiology and dynamics of each pathosystem are distinct. They depend on the ecological interplay of plant, pathogen, and vector and on how interactions are affected by biotic and abiotic factors, including anthropogenic activities and policy decisions. Our goal with this review is to stimulate discussion and novel research by contextualizing available knowledge on X. fastidiosa and how it may be applicable to emerging diseases.

Landscape Effects on Reproduction of Euschistus servus (Hemiptera: Pentatomidae), a Mobile, Polyphagous, Multivoltine Arthropod Herbivore.

Landscape factors can significantly influence arthropod populations. The economically important brown stink bug, Euschistus servus (Say) (Hemiptera: Pentatomidae), is a native mobile, polyphagous and multivoltine pest of many crops in southeastern United States and understanding the relative influence of local and landscape factors on their reproduction may facilitate population management. Finite rate of population increase (λ) was estimated in four major crop hosts-maize, peanut, cotton, and soybean-over 3 yr in 16 landscapes of southern Georgia. A geographic information system (GIS) was used to characterize the surrounding landscape structure. LASSO regression was used to identify the subset of local and landscape characteristics and predator densities that account for variation in λ. The percentage area of maize, peanut and woodland and pasture in the landscape and the connectivity of cropland had no influence on E. servus λ. The best model for explaining variation in λ included only four predictor variables: whether or not the sampled field was a soybean field, mean natural enemy density in the field, percentage area of cotton in the landscape and the percentage area of soybean in the landscape. Soybean was the single most important variable for determining E. servus λ, with much greater reproduction in soybean fields than in other crop species. Penalized regression and post-selection inference provide conservative estimates of the landscape-scale determinants of E. servus reproduction and indicate that a relatively simple set of in-field and landscape variables influences reproduction in this species.

Museum specimen data reveal emergence of a plant disease may be linked to increases in the insect vector population.

The emergence rate of new plant diseases is increasing due to novel introductions, climate change, and changes in vector populations, posing risks to agricultural sustainability. Assessing and managing future disease risks depends on understanding the causes of contemporary and historical emergence events. Since the mid-1990s, potato growers in the western United States, Mexico, and Central America have experienced severe yield loss from Zebra Chip disease and have responded by increasing insecticide use to suppress populations of the insect vector, the potato psyllid, Bactericera cockerelli (Hemiptera: Triozidae). Despite the severe nature of Zebra Chip outbreaks, the causes of emergence remain unknown. We tested the hypotheses that (1) B. cockerelli occupancy has increased over the last century in California and (2) such increases are related to climate change, specifically warmer winters. We compiled a data set of 87,000 museum specimen occurrence records across the order Hemiptera collected between 1900 and 2014. We then analyzed changes in B. cockerelli distribution using a hierarchical occupancy model using changes in background species lists to correct for collecting effort. We found evidence that B. cockerelli occupancy has increased over the last century. However, these changes appear to be unrelated to climate changes, at least at the scale of our analysis. To the extent that species occupancy is related to abundance, our analysis provides the first quantitative support for the hypothesis that B. cockerelli population abundance has increased, but further work is needed to link B. cockerelli population dynamics to Zebra Chip epidemics. Finally, we demonstrate how this historical macro-ecological approach provides a general framework for comparative risk assessment of future pest and insect vector outbreaks.

Spittlebugs as vectors of Xylella fastidiosa in olive orchards in Italy.

The recent introduction of Xylella fastidiosa in Europe and its involvement in the Olive Quick Decline Syndrome (OQDS) in Apulia (Salento, Lecce district, South Italy) led us to investigate the biology and transmission ability of the meadow spittlebug, Philaenus spumarius, which was recently demonstrated to transmit X. fastidiosa to periwinkle plants. Four xylem-sap-feeding insect species were found within and bordering olive orchards across Salento during a survey carried out from October 2013 to December 2014: P. spumarius was the most abundant species on non-olive vegetation in olive orchards as well as on olive foliage and was the only species that consistently tested positive for the presence of X. fastidiosa using real-time PCR. P. spumarius, whose nymphs develop within spittle on weeds during the spring, are likely to move from weeds beneath olive trees to olive canopy during the dry period (May to October 2014). The first X. fastidiosa-infective P. spumarius were collected in May from olive canopy: all the individuals previously collected on weeds tested negative for the bacterium. Experiments demonstrated that P. spumarius transmitted X. fastidiosa from infected to uninfected olive plants. Moreover, P. spumarius acquired X. fastidiosa from several host plant species in the field, with the highest acquisition rate from olive, polygala and acacia. Scanning electron microscopy (SEM) revealed bacterial cells resembling X. fastidiosa in the foreguts of adult P. spumarius. The data presented here are essential to plan an effective IPM strategy and limit further spread of the fastidious bacterium.

A chitinase is required for Xylella fastidiosa colonization of its insect and plant hosts.

Xylella fastidiosa colonizes the xylem network of host plant species as well as the foregut of its required insect vectors to ensure efficient propagation. Disease management strategies remain inefficient due to a limited comprehension of the mechanisms governing both insect and plant colonization. It was previously shown that X. fastidiosa has a functional chitinase (ChiA), and that chitin likely serves as a carbon source for this bacterium. We expand on that research, showing that a chiA mutant strain is unable to grow on chitin as the sole carbon source. Quantitative PCR assays allowed us to detect bacterial cells in the foregut of vectors after pathogen acquisition; populations of the wild-type and complemented mutant strain were both significantly larger than the chiA mutant strain 10 days, but not 3 days, post acquisition. These results indicate that adhesion of the chiA mutant strain to vectors may not be impaired, but that cell multiplication is limited. The mutant was also affected in its transmission by vectors to plants. In addition, the chiA mutant strain was unable to colonize host plants, suggesting that the enzyme has other substrates associated with plant colonization. Lastly, ChiA requires other X. fastidiosa protein(s) for its in vitro chitinolytic activity. The observation that the chiA mutant strain is not able to colonize plants warrants future attention to be paid to the substrates for this enzyme.

Competitive release and outbreaks of non-target pests associated with transgenic Bt cotton.

The adoption of transgenic Bt cotton has, in some cases, led to environmental and economic benefits through reduced insecticide use. However, the distribution of these benefits and associated risks among cotton growers and cotton-growing regions has been uneven due in part to outbreaks of non-target or secondary pests, thereby requiring the continued use of synthetic insecticides. In the southeastern USA, Bt cotton adoption has resulted in increased abundance of and damage from stink bug pests, Euschistus servus and Nezara viridula (Heteroptera: Pentatomidae). While the impact of increased stink bug abundance has been well-documented, the causes have remained unclear. We hypothesize that release from competition with Bt-susceptible target pests may drive stink bug outbreaks in Bt cotton. We first examined the evidence for competitive release of stink bugs through meta-analysis of previous studies. We then experimentally tested if herbivory by Bt-susceptible Helicoverpa zea increases stink bug leaving rates and deters oviposition on non-Bt cotton. Consistent with previous studies, we found differences in leaving rates only for E servus, but we found that both species strongly avoided ovipositing on H. zea-damaged plants. Considering all available evidence, competitive release of stink bug populations in Bt cotton likely contributes to outbreaks, though the relative importance of competitive release remains an open question. Ecological risk assessments of Bt crops and other transgenic insecticidal crops would benefit from greater understanding of the ecological mechanisms underlying non-target pest outbreaks and greater attention to indirect ecological effects more broadly.

Blocking the Transmission of a Noncirculative Vector-Borne Plant Pathogenic Bacterium.

The successful control of insect-borne plant pathogens is often difficult to achieve due to the ecologically complex interactions among pathogens, vectors, and host plants. Disease management often relies on pesticides and other approaches that have limited long-term sustainability. To add a new tool to control vector-borne diseases, we attempted to block the transmission of a bacterial insect-transmitted pathogen, the bacterium Xylella fastidiosa, by disrupting bacteria-insect vector interactions. X. fastidiosa is known to attach to and colonize the cuticular surface of the mouthparts of vectors; a set of recombinant peptides was generated and the chemical affinities of these peptides to chitin and related carbohydrates was assayed in vitro. Two candidates, the X. fastidiosa hypothetical protein PD1764 and an N-terminal region of the hemagglutinin-like protein B (HxfB) showed affinity for these substrates. These proteins were provided to vectors via an artificial diet system in which insects acquire X. fastidiosa, followed by an inoculation access period on plants under greenhouse conditions. Both PD1764 and HxfAD1-3 significantly blocked transmission. Furthermore, bacterial populations within insects over a 10-day period demonstrated that these peptides inhibited cell adhesion to vectors but not bacterial multiplication, indicating that the mode of action of these peptides is restricted to limiting cell adhesion to insects, likely via competition for adhesion sites. These results open a new venue in the search for sustainable disease-control strategies that are pathogen specific and may have limited nontarget effects.

A likelihood-based biostatistical model for analyzing consumer movement in simultaneous choice experiments.

Consumer feeding preference among resource choices has critical implications for basic ecological and evolutionary processes, and can be highly relevant to applied problems such as ecological risk assessment and invasion biology. Within consumer choice experiments, also known as feeding preference or cafeteria experiments, measures of relative consumption and measures of consumer movement can provide distinct and complementary insights into the strength, causes, and consequences of preference. Despite the distinct value of inferring preference from measures of consumer movement, rigorous and biologically relevant analytical methods are lacking. We describe a simple, likelihood-based, biostatistical model for analyzing the transient dynamics of consumer movement in a paired-choice experiment. With experimental data consisting of repeated discrete measures of consumer location, the model can be used to estimate constant consumer attraction and leaving rates for two food choices, and differences in choice-specific attraction and leaving rates can be tested using model selection. The model enables calculation of transient and equilibrial probabilities of consumer-resource association, which could be incorporated into larger scale movement models. We explore the effect of experimental design on parameter estimation through stochastic simulation and describe methods to check that data meet model assumptions. Using a dataset of modest sample size, we illustrate the use of the model to draw inferences on consumer preference as well as underlying behavioral mechanisms. Finally, we include a user's guide and computer code scripts in R to facilitate use of the model by other researchers.