Comparison of ethanol-baited trap designs for ambrosia beetles in orchards in the eastern United States.

Ambrosia beetles (Coleoptera: Curculionidae: Scolytinae) are among the most devastating pests of orchards, nurseries, and forests. Improving trap design and ethanol lures for capturing ambrosia beetles is necessary to develop effective monitoring and management strategies. In this 2-year study, we assessed 4 trap designs and 3 commercially formulated ethanol lures to refine trapping methods tailored for orchard environments in the eastern United States. Our investigation included orchards in 2 regions, Georgia (pecan orchards) and New York (apple orchards), targeting major ambrosia beetle (Coleoptera: Curculionidae) pest species such as Xylosandrus crassiusculus (Motschulsky), X. compactus (Eichhoff), X. germanus (Blandford), and Anisandrus maiche (Stark). Among the trap designs evaluated, clear sticky cards were most effective for capturing ambrosia beetles across orchard locations. Notably, in Georgia, sticky cards paired with specific low-release ethanol lures demonstrated enhanced capture of X. crassiusculus and X. compactus, 2 key ambrosia beetle pests found infesting young pecan trees. Similarly, in New York, sticky cards baited with low-release ethanol lures captured the highest rates of X. germanus and A. maiche, thus indicating its suitability for diverse ambrosia beetle populations. Overall, our study provides practical implications for tailoring trapping protocols to optimize ambrosia beetle management strategies in orchard settings.

Influence of daily temperature maximums on the development and short-distance movement of the Asian citrus psyllid.

Temperature is a key factor in insect biology and ecology. Climate change is driving insect exposure to temperature extremes and understanding the effect of extreme temperatures on the biology of invasive agricultural pests will be key to predicting the effect of temperature increases. Here, we simulated diurnal cycles with different lengths of exposure times to maximum temperatures experienced in summer in different locations of California on the survivorship and development of the Asian citrus psyllid (ACP) (Diaphorina citri Kuwayama). ACP is the invasive vector of Huanglongbing disease (HLB), a lethal bacterial pathogen of citrus which is currently spreading in the Los Angeles, California basin. We also tested the effect of high or low humidity at high temperatures on ACP survival and development and the effect of high temperatures on short-distance dispersal. ACP were able to complete their life cycle in all temperature treatments (28-43 °C) except in daily cycles when 43 °C was maintained for 6 h. Temperature and exposure time significantly decreased adult emergence above 40 °C. High temperatures significantly increased development time with longer development as exposure times to high temperatures increased. The interaction between low humidity and high temperature increased the number of emerging adults and decreased developmental times. ACP short-distance dispersal increased over time but was not affected by temperature. These results indicate that ACP are capable to develop in temperatures higher than previously reported, suggesting that increasing temperatures may reduce the invasive capacity of ACP in regions where maximum daily temperatures are increasing along with the duration of such temperatures throughout the day.

Salicylic acid mediated immune response of Citrus sinensis to varying frequencies of herbivory and pathogen inoculation.

BACKGROUND: Plant immunity against pathogens and pests is comprised of complex mechanisms orchestrated by signaling pathways regulated by plant hormones [Salicylic acid (SA) and Jasmonic acid (JA)]. Investigations of plant immune response to phytopathogens and phloem-feeders have revealed that SA plays a critical role in reprogramming of the activity and/or localization of transcriptional regulators via post-translational modifications. We explored the contributing effects of herbivory by a phytopathogen vector [Asian citrus psyllid, Diaphorina citri] and pathogen [Candidatus Liberibacter asiaticus (CaLas)] infection on response of sweet orange [Citrus sinensis (L.) Osbeck] using manipulative treatments designed to mimic the types of infestations/infections that citrus growers experience when cultivating citrus in the face of Huanglongbing (HLB) disease. RESULTS: A one-time (7 days) inoculation access period with CaLas-infected vectors caused SA-associated upregulation of PR-1, stimulating defense response after a long period of infection without herbivory (270 and 360 days). In contrast, while repeated (monthly) 'pulses' of 7 day feeding injury by psyllids stimulated immunity in CaLas-infected citrus by increasing SA in leaves initially (up to 120 days), long-term (270 and 360 days) repeated herbivory caused SA to decrease coincident with upregulation of genes associated with SA metabolism (BMST and DMR6). Similarly, transcriptional responses and metabolite (SA and its analytes) accumulation in citrus leaves exposed to a continuously reproducing population of D. citri exhibited a transitory upregulation of genes associated with SA signaling at 120 days and a posterior downregulation after long-term psyllid (adults and nymphs) feeding (270 and 360 days). CONCLUSIONS: Herbivory played an important role in regulation of SA accumulation in mature leaves of C. sinensis, whether or not those trees were coincidentally infected with CaLas. Our results indicate that prevention of feeding injury inflicted by D. citri from the tritrophic interaction may allow citrus plants to better cope with the consequences of CaLas infection, highlighting the importance of vector suppression as a component of managing this cosmopolitan disease.

Cold acclimation increases Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae) survival during exposure to freezing temperatures.

The Asian citrus psyllid, Diaphorina citri, is the vector of the pathogenic bacteria that causes Huanglongbing, the most devasting disease affecting citrus worldwide. As cultivation of citrus tends to expand northward, the tolerance of psyllids to freezing temperatures needs to be investigated. While mortality of D. citri to cold temperature has been previously studied, much less is known regarding the acclimation potential of psyllids to cold temperatures. We first evaluated cold resistance of D. citri depending on color morph and sex. Subsequently, we compared mortality of D. citri at -4 °C for 10 hours between unacclimated psyllids, psyllids that were gradually acclimated to cold over 1 or 2 weeks, and psyllids intermittently acclimated also for 1 or 2 weeks. We did not find major differences in cold resistance between color morphs and sex. We found that after 1 week of gradual acclimation, the survival of psyllids at -4 °C increased to 86%, compared to 20% in the control group. Survival did not increase after the second week of gradual acclimation. Although intermittent acclimation improved survival compared to the control group, it was less effective than gradual acclimation with a survival at 30% and 70% after 1 and 2 weeks of acclimation, respectively, although this difference with gradual acclimation was not significant at week 2. These data show that gradual cold acclimation allows D. citri to survive brief periods below freezing. It may serve as a mechanism responsible for increasing northern establishment of this pest.

Wind Speed and Direction Drive Assisted Dispersal of Asian Citrus Psyllid.

Wind directly influences the spread of vector-borne plant pathogens by driving the passive dispersal of vectors to potentially new areas. Here, we evaluated the effect of wind speed and direction on the dispersal of the Asian citrus psyllid (ACP), Diaphorina citri (Kuwayama) (Hemiptera: Psyllidae), the vector of the bacteria causing huanglongbing (HLB), a lethal disease of citrus. The effect of different wind speeds on short or long-distance dispersal of ACP was investigated using a high-speed wind tunnel under laboratory conditions. The effect of wind direction on ACP dispersal under field conditions was evaluated using custom-made wind vane-style traps. In wind tunnel assays, ACP remained on plants until wind treatments reached ≥48 km/h when psyllids were mostly dislodged from plants and moved by the wind. For a short-distance, wind-driven movement (movement by the wind from one plant to another), the effect of wind speed was not significant at any of the wind speed treatments tested. Wind vane traps placed in a Florida citrus grove captured significantly more ACP on the windward side, suggesting that ACP were moved with the wind. The number of ACP found on the windward side of traps was significantly higher from May to August. These results indicate that ACP is likely to disperse with prevailing wind direction and that settled ACP may become dislodged and moved at random by high wind speeds occurring in areas of significant citrus production (southern California, Florida, or Texas).

High Temperatures Decrease the Flight Capacity of Diaphorina citri Kuwayama (Hemiptera: Liviidae).

Diaphorina citri Kuwayama (Hemiptera: Liviidae), commonly known as Asian citrus psyllid (ACP), is an invasive insect pest and the vector of the bacterium causing Huanglongbing (HLB), a lethal disease of citrus. In the United States, ACP has been established in all citrus-producing zones, all of which have different environmental conditions. The spread of ACP and, more importantly, HLB, has progressed differently depending on the state, with more rapid spread in Florida and Texas than in California. Climatic variations between the regions are likely a strong factor in the difference in the rate of spread. Despite this, it is unknown how the flight capacity of D. citri is influenced by high temperatures (>30 °C) and subsequently, low humidity experienced in California but not in Texas or Florida. In this study, by using a custom-made, temperature-controlled flight mill arena, we assessed the effect of high temperatures on the flight capacity and flight propensity of D. citri under low (20-40%) and high (76-90%) relative humidity conditions. We found that temperature and humidity influence the propensity to engage in short or long-distance flight events. Psyllids exposed to temperatures above 43 °C only performed short flights (˂60 s), and a high relative humidity significantly decrease the proportion of long flights (≥60 s) at 26 and 40 °C. The flight capacity for insects who engaged in short and long flights was significantly affected by temperature but not by humidity. For long flyers, temperature (in the 26-43 °C range) was negatively correlated with distance flown and flight duration. The most favorable temperature for long dispersion was 26 °C, with suboptimal temperatures in the range of 32-37 °C and the least favorable temperatures at 40 and 43 °C. In conclusion, D. citri is able to fly in a broad range of temperatures and efficiently fly in high and low humidity. However, temperatures above 40 °C, similar to those experienced in semi-arid environments like Southern California or Arizona, are detrimental for its flight capacity.

A Multimodal Attract-and-Kill Device for the Asian Citrus Psyllid Diaphorina citri (Hemiptera: Liviidae).

Phytophagous insects, including Asian citrus psyllids (Diaphorina citri Kuwayama), use multiple sensory modalities (vision, olfaction, and gustation,) to locate and accept host plants. We explored incorporation of several sensory cues into a multi-modal attract-and-kill device (AK device) using a three-dimensional shape to increase visibility, as well as elements of color, attractant, phagostimulant, UV reflectant, and toxicant. Attraction of adult D. citri to the device was mediated by a combination of a highly reflective yellow cylinder, a UV reflectant compound (magnesium oxide), and an odorant blend as a short-range attractant. The device surface was coated with a slow-release wax matrix (SPLAT™) augmented with a phagostimulant consisting of a 3-component blend (formic acid, acetic acid, and para-cymene) and an insecticide (ß-cyfluthrin). Psyllids landing on the device attempted to feed from the wax matrix, became intoxicated, died, and fell from the device. The device remained fully active over a period of 12 weeks partly because dead psyllids or nontargets did not adhere to the surface as occurs on adhesive yellow sticky cards, the industry standard. Laboratory and field assays showed that the device attracted and killed significantly more adult D. citri than ordinary yellow sticky cards. This device or a future iteration based on the design elements of this device is expected to contribute to sustainable and environmentally appropriate management of D. citri by exploiting the psyllid's innate behavioral responses to visual, olfactory, and gustatory stimuli.

Evaluation of semiochemical based push-pull strategy for population suppression of ambrosia beetle vectors of laurel wilt disease in avocado.

Ambrosia beetles (Coleoptera: Curculionidae: Scolytinae and Platypodinae) bore into tree xylem to complete their life cycle, feeding on symbiotic fungi. Ambrosia beetles are a threat to avocado where they have been found to vector a symbiotic fungus, Raffaelea lauricola, the causal agent of the laurel wilt disease. We assessed the repellency of methyl salicylate and verbenone to two putative laurel wilt vectors in avocado, Xyleborus volvulus (Fabricius) and Xyleborus bispinatus (Eichhoff), under laboratory conditions. Then, we tested the same two chemicals released from SPLAT flowable matrix with and without low-dose ethanol dispensers for manipulation of ambrosia beetle populations occurring in commercial avocado. The potential active space of repellents was assessed by quantifying beetle catch on traps placed 'close' (~5-10 cm) and 'far' (~1-1.5 m) away from repellent dispensers. Ambrosia beetles collected on traps associated with all in-field treatments were identified to species to assess beetle diversity and community variation. Xyleborus volvulus was not repelled by methyl salicylate (MeSA) or verbenone in laboratory assays, while X. bispinatus was repelled by MeSA but not verbenone. Ambrosia beetle trap catches were reduced in the field more when plots were treated with verbenone dispensers (SPLAT) co-deployed with low-dose ethanol dispensers than when treated with verbenone alone. Beetle diversity was highest on traps deployed with low-dose ethanol lures. The repellent treatments and ethanol lures significantly altered the species composition of beetles captured in experiment plots. Our results indicate that verbenone co-deployed with ethanol lures holds potential for manipulating ambrosia beetle vectors via push-pull management in avocado. This tactic could discourage immigration and/or population establishment of ambrosia beetles in commercial avocado and function as an additional tool for management programs of laurel wilt.

Effects of Wind, Temperature, and Barometric Pressure on Asian Citrus Psyllid (Hemiptera: Liviidae) flight behavior.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is the vector of the bacterium responsible for huanglongbing, a deadly plant disease affecting citrus worldwide. We investigated the effects of wind direction and speed on flight duration and direction of D. citri, as well as the effects of temperature and barometric pressure on sustained flight duration of D. citri. Experiments were performed with laboratory flight mills and wind tunnels. Flight activity of D. citri increased with increasing temperature. Of the few insects that flew at 18°C, most performed short duration flights (<60 s). When exposed to temperatures between 21 and 28°C, D. citri performed long duration flights (>60 s). In addition, the distance covered increased with temperature. Interestingly, males were more sensitive to cold temperature and flew significantly shorter distances than females at 21 and 25°C. Barometric pressure recorded before and during the flight mill experiment suggested that decreasing pressure reduced distance flown by D. citri. Flight direction was strongly influenced by wind. In wind tunnel experiments where psyllids were challenged to reach citrus leaf flush positioned either downwind or upwind, most D. citri moved downwind when exposed to continuous airflow. In a subsequent experiment, we challenged psyllids to pulsed wind blowing at higher speeds. In this case, most psyllids progressed upwind, suggesting upwind movement by psyllids during pauses within pulsed airflow. Collectively, the results indicate that D. citri are able to modify their flight behavior in response to abiotic factors.

Bacterial phytopathogen infection disrupts belowground plant indirect defense mediated by tritrophic cascade.

Plants can defend themselves against herbivores through activation of defensive pathways and attraction of third-trophic-level predators and parasites. Trophic cascades that mediate interactions in the phytobiome are part of a larger dynamic including the pathogens of the plant itself, which are known to greatly influence plant defenses. As such, we investigated the impact of a phloem-limited bacterial pathogen, Candidatus Liberibacter asiaticus (CLas), in cultivated citrus rootstock on a well-studied belowground tritrophic interaction involving the attraction of an entomopathogenic nematode (EPN), Steinernema diaprepesi, to their root-feeding insect hosts, Diaprepes abbreviatus larvae. Using belowground olfactometers, we show how CLas infection interferes with this belowground interaction by similarly inducing the release of a C12 terpene, pregeijerene, and disconnecting the association of the terpene with insect presence. D. abbreviatus larvae that were not feeding but in the presence of a CLas-infected plant were more likely to be infected by EPN than those near uninfected plants. Furthermore, nonfeeding larvae associated with CLas-infected plants were just as likely to be infected by EPN as those near noninfected plants with D. abbreviatus larval damage. Larvae of two weevil species, D. abbreviatus and Pachnaeus litus, were also more attracted to plants with infection than to uninfected plants. D. abbreviatus larvae were most active when exposed to pregeijerene at a concentration of 0.1 µg/µl. We attribute this attraction to CLas-infected plants to the same signal previously thought to be a herbivore-induced plant volatile specifically induced by root-feeding insects, pregeijerene, by assessing volatiles collected from the roots of infected plants and uninfected plants with and without feeding D. abbreviatus. Synthesis. Phytopathogens can influence the structuring of soil communities extending to the third trophic level. Field populations of EPN may be less effective at host-finding using pregeijerene as a cue in citrus grove agroecosystems with high presence of CLas infection.

Flight Capacities and Diurnal Flight Patterns of the Ambrosia Beetles, Xyleborus glabratus and Monarthrum mali (Coleoptera: Curculionidae).

We compared the flight activity of Xyleborus glabratus Eichhoff, vector and symbiont of the causal agent of laurel wilt disease (Raffaelea lauricola), with a native species Monarthrum mali (Fitch) using flight mills. Flight mills were operated either for 24 h or for three 3-h time intervals. During the 3-h interval experiment, the shortest time to flight initiation for X. glabratus occurred at 1600-1900 hours. The average flight time and total flying distance during 1600-2100 hours were also higher than those quantified during the other two recording times investigated. However, total flight duration and proportion of fliers was highest at 1000-1300 hours. We compared several flight parameters. About 64.0% of tested X. glabratus flew <20 m. During 24-h recording periods, M. mali flew longer distances than X. glabratus. Over 50.0% of M. mali flew over 100 m on the flight mill. Xyleborus glabratus flight activity was greatest between 1200 and 1800 hours, while M. mali flew most frequently between 1500 and 2100 hours. Monarthrum mali flew more than five times more frequently than X. glabratus, and their longest single flight distance (37.5 ± 12.5 m) and total flight distance (213.7 ± 85.5 m) were greater than those of X. glabratus. These data will be useful for development of species-specific control and monitoring protocols for these ambrosia beetles based on greater understanding of their flight capacities and associated invasion distance.

Differential Response of a Local Population of Entomopathogenic Nematodes to Non-Native Herbivore Induced Plant Volatiles (HIPV) in the Laboratory and Field.

Recent work has shown the potential for enhanced efficacy of entomopathogenic nematodes (EPN) through their attraction to herbivore induced plant volatiles. However, there has been little investigation into the utilization of these attractants in systems other than in those in which they were identified. We compared (E)-ß-caryophyllene and pregeijerene in the highbush blueberry (Vaccinium corymbosum) agroecosystem in their ability to enhance the attraction of EPN to and efficacy against the system's herbivore, oriental beetle (Anomala orientalis). The relative attractiveness of (E)-ß-caryophyllene and pregeijerene to a local isolate of the EPN species Steinernema glaseri was tested in a six-arm olfactometer in the laboratory to gather baseline values of attraction to the chemicals alone in sand substrate before field tests. A similar arrangement was used in a V. corymbosum field by placing six cages with assigned treatments and insect larvae with and without compound into the soil around the base of 10 plants. The cages were removed after 72 h, and insect baits were retrieved and assessed for EPN infection. The lab results indicate that in sand alone (E)-ß-caryophyllene is significantly more attractive than pregeijerene to the local S. glaseri isolate Conversely, there was no difference in attractiveness in the field study, but rather, native S. glaseri were more attracted to cages with G. mellonella larvae, no larvae, and cages with the blank control and G. mellonella larvae.