1. Please state project objectives and what work was done this quarter to address them:
I. Near term field management
(a) Develop methods to time management actions
Working with 6 commercial citrus growers throughout central Florida, we have been developing a robust dataset to describe the seasonal phenology (life cycle) of lebbeck mealybug populations. Describing the seasonal life cycle of this pest helps determine key timepoints for management of its population in relation to tree development and/or time of year. In addition to intense sampling to describe the annual seasonal biology of lebbeck mealybug, we are also testing rudimentary pheromone traps to see if we can attract male mealybugs into traps. Reducing the number of males in a mating population can reduce offspring production by females (data shown in a previous report). We are hoping to determine if traps based on pheromones can help detect populations and their relative phenology in the future, which could enable growers to fine tune management actions. Starting in January 2021, we observed zero to very low mealybug population until late March 2021 from all citrus groves sampled in Florida. However, since onset of spring season (April 2021), we have been recording greater mealybug population densities in the citrus groves with mealybug populations peaking in late spring 2021 (June 2021), before a steady decline in population up till July 2021 (Figure 1). Immatures appear to reach a population peak in early Spring (May 2021), while crawler populations appears to reach a peak in late Spring 2021 (June 2021) (Figure 1). We also observed that mealybugs were more likely to be located around the calyx and peduncle of fruit set and developing fruits. They feed on the developing citrus fruit causing premature fruit abortion and/or fruit necrosis in 2 weeks which will result in yield loss (Figure 2).
Figure 2. Abundance of different life stages of lebbeck mealybugs inn central Florida
Figure 2: infested developing fruit over 2 week period
(b) Expand laboratory insecticide and adjuvant screening.
EPF data presented in previous report.
Adjuvant screening: Preliminary trials have been conducted with adjuvants alone to determine their lethality to lebbeck mealybug. A total of 10 adjuvants, including and 4 from Helena Agri Enterprises were mixed with DI water at label rates and sprayed until dripping on Volk lemon leaves with 2nd and 3rd instar mealybugs attached. 8 of the adjuvants resulted in significantly greater mealybug mortality over a 7-day period compared to a DI water control, and were comparable to the morality achieved by the insecticide Delegate (Spinetoram) applied at maximum label rate.
All adjuvants and Delegate were also tested against adults and larvae of Cryptolaemus montrouzieri to determine their potential impact on known predators of lebbeck mealybug. 3 of the adjuvants resulted in significantly higher C. montrouzieri larvae mortality compared to a DI water control, while none of the adjuvants resulted in higher C. montrouzieri adult mortality. The insecticide Delegate resulted in 35% mortality of C. montrouzieri larvae, and 100% mortality of C. montrouzieri adults, both significantly greater than a DI water control.
Next steps involve testing adjuvants against mealybug ovisacs and combining adjuvants with Delegate to determine if the combination of adjuvant and insecticide results in increased mealybug instar and ovisac mortality.
c. Evaluate promising materials in open grove setting
The insecticides Malathion and Assail (Acetamiprid) were tested on an ad hoc basis on heavily infested young citrus trees. Trees were part of a separate experiment and became naturally infested with lebbeck mealybug. Both Malathion and Assail were applied at maximum label rate, and the number of living mealybugs of all life stages at 3, 6, and 15 days post application were compared to the number of living mealybugs pre-application.
Both Malathion and Assail resulted in close to 100% mortality of adults and instars. However, in 35% of ovisacs, at least some eggs survived treatment and crawlers emerged in the following days. These results suggest that Malathion and Assail can be useful to knock down infestations of lebbeck mealybug, but that populations may rebound without additional treatments.
We have additional trials planned pending appropriate infestation levels at 3 locations.
(1d) Ant Management
We are testing a variety of methods to remove red imported fire ants (Solenopsis invicta) from citrus groves. Four experimental treatments were tested and compared to an untreated control: Chlorpyrifos drench followed by a Bifenthrin drench after 3 months, Clinch ant bait (Abamectin) applied every 3 months, Extinguish ant bait (S-methoprene) applied every 3 months, and spot treating ant colonies with hot water.
Fire ant abundance was assessed by counting the number of surviving colonies, and by determining presence/absence of foragers with cookie baits. Natural enemy abundance was assessed by dissecting mealybug clusters for predator larvae. Sampling for each of these methods took place every 2 weeks.
Preliminary data show that both insecticidal baits and hot water treatments significantly reduced fire ant colony abundance initially, while the chlorpyrifos drench had no effect on colony abundance. However, fire ant colony abundance rebounded in the hot water treatments after 3 months and has returned to close to the same levels as control plots. Colony abundance in both bait treatments has remained consistently low. Finally, although Chlorpyrifos had no effect on colony abundance, once bifenthrin was applied, colony abundance dropped and has continued to trend downwards, although this treatment still remains statistically similar to control plots.
Predator populations within mealybug clusters were unaffected by treatment, and numerous predators have been found in mealybug clusters across all treatment types.
Future work includes retreating hot water plots and continuing to monitor colony abundance for a total of 1 year. Ant forager identification and quantification is currently underway.
II. Long term management
a. Assessment of predator- what is currently in the system, can they be enhanced, how to implement use of predators alongside insecticide use for ACP and mealybugs
Predators currently present in the system:
Using molecular gut content analysis, we have identified a broader suite of predatory arthropods, including several species of spider, that consume lebbeck mealybug than anticipated. We are still extracting DNA from the remaining field-collected predators and anticipate having more to share with the regards to this sub area in our next report.
Predators available for purchase or present locally for potential augmentative control:
Predators who directly consume prey:
The commercially available predators Cryptolaemus montrouzieri, Orius insidiosus, Adalia bipuctata, Hippodamia convergens, and Chrysoperla carnea have been screened using no-choice assays to determine if they will feed on 2nd and 3rd instar lebbeck mealybugs, and on mealybug ovisacs. Additionally, several wild-caught and lab reared predators have also been screened, including the coccinellids Diomus austrinus, Coccinella septempunctata, and Olla v-nigrum; larva of the genus Ceraeochrysa (colloquially called trash bugs); and the earwig Euborellia annulipes.
For commercially available predators, only Cryptolaemus montrouzieri consistenty consumed both lebbeck mealybug instars and ovisacs. Chrysoperla carnea consumed larvae at comparable rates to C. montrouzieri, but only infrequently consumed ovisacs. Adalia bipuctata and Hippodamia convergens ate only a few instars, and no ovisacs. Finally, Orius insidiosus consumed virtually no instars, and no ovisacs.
For wild-caught and lab-reared predators, both Ceraeochrysa larvae and Euborellia annulipes consumed instars and ovisacs at high rates, and although not quite as consistently as C. montrouzieri. Diomus austrinus and Coccinella septempunctata consumed a few larvae, and only infrequently consumed ovisacs. Olla v-nigrum ate very few instars and no ovisacs.
From our results, only Cryptolaemus montrouzieri shows promise as a commercially available biological control agent for lebbeck mealybug, although Ceraeochrysa larvae and Euborellia annulipes may act as natural biological control in the grove. Chrysoperla carnea may be useful for controlling mealybug larvae, but not reproductive adult females.
Future work will consist of conducting choice assays with predators that did feed on lebbeck mealybug, as well as testing greater numbers of all predators to increase sample size.
Cryptolaemus montrouzieri were tested in the field against a lebbeck mealybug infestation on an ad hoc basis. A grower experienced a lebbeck mealybug infestation inside a Citrus Under Protective Screening (CUPS) grove and ordered Cryptolaemus montrouzieri to release to control the outbreak. Trees infested with mealybugs were marked and the infestation recorded as low or high. 10,000 C. mountrouzieri were released to cover an area of ~3,150 mature trees, ~100 of which were infested. Marked trees were reassessed 2 weeks after release.
After 2 weeks, most of the infested trees had visibly reduced mealybug abundance, and in many cases, all mealybugs had been consumed. In approximately half of infested trees, Cryptolaemus montrouzieri larvae could be seen feeding on mealybug clusters. The majority of mealybug clusters that remained undamaged either appeared to have died on their own accord or were composed of very few individuals. While preliminary, these results indicate that Cryptolaemus montrouzieri can effectively be used control infestations of lebbeck mealybug in enclosed settings.
Limited numbers of Anagyrus pseudococci, a commonly used parasitoid for commonly occurring mealybugs in greenhouses, were tested to determine if they would parasitize lebbeck mealybug adults and ovisacs. A. pseudococci adults were placed in arenas with adult lebbeck mealybugs, or ovisacs and left for 40-48 hrs. Surviving parasitoids were then transferred to adult Citrus mealybugs and given an opportunity to parasitize them, to act as a positive control. Mealybugs were checked for parasitism after 16 days. Most parasitoids died in arenas with lebbeck mealybugs, and none parasitized adults or ovisacs. However, half of those that survived went on to parasitize Citrus mealybug adults. These preliminary results suggest that A. pseudococci will not readily parasitize lebbeck mealybug, however further trials with more individuals and life stages of mealybugs are needed.
b. Determine how to implement mealybug management concurrent with other pest management programs
Ongoing population monitoring shows that, to date, there is no correlation between flush development and population development of lebbeck mealybug. We had anticipated that this pest, because it feeds in a similar manner to ACP, may have populations that were in sync with flush development, however flush phenology does not appear to be directly tied to mealybug population growth. What we do see, however, is a large increase in population concurrent with fruit set and development. Large populations have been documented developing at the calyx junction, often leading to fruit death and abortion of fruit well before physiological fruit drop. This is still an ongoing area of work; however, we have been able to confirm that fruit development is a key period to target management actions, and several growers have been successful in keeping populations of mealybugs down while protecting their fruit from heavy impacts of this pest by targeting appropriate, systemic materials during this time point. We still have several months remaining in this study, but the data generated so far have been crucial to understanding when to time management actions.
c. Determine what insecticide chemistries inhibit feeding
We are continuing to tease apart the feeding biology of lebbeck mealybug, which is the integral step to enable this sub area to be completed.
d. Develop tools to minimize spread
Killing crawlers with isopropyl alcohol
Different concentrations of isopropyl alcohol were tested to determine how lethal they are to 1st instar lebbeck mealybugs. 50%, 70%, and 90% solutions of isopropanol were sprayed onto mealybug crawlers placed on cloth swatches and compared to a DI water control. Both 1 spray at each concentration, and 2 sprays at each concentration were tested. Mortality was assessed after 5, 10, and 15 minutes. The test was repeated, this time assessing mortality at 30 min, 1hr, and 2hrs. For all concentrations of isopropanol, 1 spray resulted in significantly greater mealybug mortality compared to the control. However, several mealybug crawlers remained alive and active after a single spray for all concentrations. 2 sprays of each concentration resulted in almost 100% mortality or incapacitation at all time points.
Killing ovisacs with isopropyl alcohol
50%, and 70% solutions of isopropanol were also tested against lebbeck mealybug ovisacs to assess lethality. Single ovisacs were sprayed both 1 and 2 times with each concentration and compared to a DI water control. Mortality of the adult females was assessed at 0, 3, and 5 days, and ovisacs were held for up to 3 weeks to see if crawlers emerged.
Adult mortality was high by the end of 5 days for both concentrations and spray regimes and was significantly greater than the DI water control. However, most ovisacs remained viable, and crawlers emerged within several days for all treatments. Our results suggest that isopropanol is effective against adults but does not reliably penetrate the ovisac to kill eggs.
Next steps involve testing 90% isopropanol against ovisacs, and more thoroughly soaking ovisacs with each different concentration to determine if any amount of isopropanol will result in reliable egg mortality.
Using steam to kill adult mealybugs and ovisacs
Adult mealybugs and ovisacs may be accidentally transferred from infested groves on tools and equipment. This is a particular concern for Individual Protective Covers (IPCs), where ovisacs can easily be transferred to new young trees if IPCs are reused. Steam treatments may be an effective method of sanitizing equipment and killing both adults and ovisacs.
Using a steam cabinet on-station, we steam treated adults and ovisacs at 100, 120, and 130 degrees Fahrenheit for 5, 10, and 15 minutes to determine mortality. Previous preliminary experiments showed steam treatments at 130 degrees Fahrenheit for 15 and 30 minutes resulted in 100% mortality. We also tested ovisacs inside of a folded IPC to determine if ovisacs within bags would be more insulated from heat and moisture during steam treatments. For ovisacs in IPCs, 120 degrees for 10 and 15 minutes, 130 degrees for 5 and 10 minutes, 135 degrees for 5 minutes, and 140 degrees for 5 minutes were all tested.
Mortality was assessed at 0, 3, and 5 days after treatment, and ovisacs were held for an additional 2 weeks to see if crawlers emerged. For ovisacs and adults without an IPC, adult mealybug and ovisac mortality was not significantly different than the control at 100 degrees F for all time points. Crawlers emerged from all ovisacs at this temperature. At 120 degrees F for 5 minutes, mortality was also functionally 0%, and crawlers emerged. However, mortality rose to 100% at 120 degrees F for 10 and 15 minutes, and no crawlers survived. At 130 degrees F, mortality was 100% for all time points with no crawlers. Inside of an IPC, there were no crawlers and mortality was 100% at 130 degrees for 10 minutes, and at 140 degrees for 5 minutes. All other time points had functionally 0% mortality, and crawlers emerged.
It appears that IPCs insulate ovisacs and adults from steam treatments and require additional time or higher temperatures to sterilize adults and eggs. Currently, exposed ovisacs will be sterilized at 120 degrees for 10 minutes, and at 130 degrees for 5 minutes. Inside of an IPC, ovisacs will be sterilized at 130 degrees for 10 minutes, and 140 degrees for 5 minutes.
A final test will be conducted with ovisacs inside IPCs at 120 degrees to determine how much time is required to achieve 100% mortality at this temperature.
Future work will include sanitation procedures for large equipment and vehicles to develop recommendations for growers and scouts.
2. Please state what work is anticipated for next quarter:
Many of the updates provided in the previous sections are updates to ongoing research. These projects will continue to make progress over the next quarter.
3. Please state budget status (underspend or overspend, and why):
We are currently on track with our spending for this project.