1. IFAS recommendations for ACP management

2. Today’s presentation
Overview of recent research on ACP management with soil-applied neonics
Followup confirmatory surveys for insecticide resistance
Current ACP management recommendations
Ongoing work by IFAS entomologists
Everywhere Huanglongbing exists in the world, insecticide use has been one of the major components of psyllid/Huanglongbing management programs. In developing psyllid management programs, questions of importance have included :
We’ve used EPG as a tool to answer these questions with the goal of providing better guidance on the use of insecticides as part of an ACP/HLB management program.

3. Season-long ACP control
(foliar applications to prevent pesticide resistance to neonics shown in orange)
Tree size
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
Reset
(<3’) P A B
1-2 yr
(3-5’)
3-5 yr
(5-9’)
bearing B   A
A= Admire (imidacloprid); B=Belay (clothianidin); P=Platinum (thiamethoxam)

4. Refining Use Rates of Soil-applied Neonicotinoids
Dr. Kevin Langdon (Graduated Dec 2017)
Refine use rates based on tree size?
Dilution of product in larger tree canopies
How much product is needed in a leaf to control ACP?

5. Amount of imidacloprid needed in leaf to control ACP?
Setamou et al correlated psyllid control with 200PPB imidacloprid in leaf tissue
Since been used as the standard target dose in plants for psyllid control studies

6. Correlating application rate with imidacloprid concentration in leaf
Based on tree size, how much soil-applied imidacloprid is needed to reach 200 ppb in leaf tissues?
Trees treated in grove
Leaves analyzed for imidacloprid concentration using LC-MS-MS
Correlate psyllid populations with known concentrations in plant tissues to validate ppb target threshold

7. Correlating application rate with imidacloprid concentration in leaf
At CREC groves, psyllids found reproducing on trees with >10 PPM (10,000 ppb) in leaf tissues
Resistance assay performed using vial assay method
RR = 2.4 (negligible)

8. How much imidacloprid needed in leaf tissue to control psyllids?
Tim Ebert (Post-doc)
Used EPG to correlate neonic residues in plants with psyllid mortality/feeding
~50PPM imidacloprid in leaf reduced phloem-related feeding behaviors by 85% (July 2015)
Uneven distribution in leaf?
Questions like uneven distribution in the leaf had us second guessing our results, so we turned to additional laboratory studies to confirm these results.

9. How much imidacloprid needed in leaf tissue to control psyllids?
Kevin Langdon (PhD student)
Artificial diet bioassay to develop LC50 for psyllid exposure to imidacloprid via feeding
Compared feeding LC50 to contact (vial assay) LC50

10. Langdon & Rogers 2017, JEE 110(5): 2229-2234.
Response of laboratory susceptible D. citri strain to imidacloprid by ingestion and contact.
a. Number of adult D. citri tested.
b. Parts per million (ppm) active ingredient.
c. Ratio of ingestion LC50 divided by contact LC50.
Average concentration in leaves from commercial groves = 7 PPM; range <1PPM – 30PPM.
LC90 for feeding mortality = 62.0 PPM (62,000 PPB), much higher than the 200 PPB threshold suggested
Suggests feeding deterrence, not mortality, is the mechanism for soil-applied neonic protection of young citrus trees.

11. Response of laboratory and field collected D
Response of laboratory and field collected D. citri to imidacloprid by ingestion and contact in 2016.
The ingestion to contact ratio for the Vero Beach population is much greater than the Lake Placid population, despite the fact the resistance ratio is much greater for both the ingestion and contact assay for psyllids from the Lake Placid site. What this shows is that there is shift in resistance occurring, due to sublethal effects on feeding deterrence that the contact vial assay is not able to discern. But more importantly is the shift in LC90 values for feeding mortality seen in the field. The levels of imidacloprid needed to cause mortality are much higher than can be achieved within a plant under typical grove conditions. So there is a tremendous amount of selection pressure that exists for product failures in the field right now and indeed we think we are seeing just that.
a. Number of adult D. citri tested.
b. Test of differences in mortality at the mean dose level where means differ significantly at α ≤ (Contact: 19.5 ppm; Ingestion: 97.7 ppm).
c. Parts per million (ppm) active ingredient.
d. Ratio of ingestion LC50 divided by contact LC50 by location.
e. Percent mortality in negative control containing no diet at 72h.
Langdon & Rogers 2017, JEE 110(5):

12. Response of laboratory and field collected D
Response of laboratory and field collected D. citri to imidacloprid by ingestion and contact in 2016.
The ingestion to contact ratio for the Vero Beach population is much greater than the Lake Placid population, despite the fact the resistance ratio is much greater for both the ingestion and contact assay for psyllids from the Lake Placid site. What this shows is that there is shift in resistance occurring, due to sublethal effects on feeding deterrence that the contact vial assay is not able to discern. But more importantly is the shift in LC90 values for feeding mortality seen in the field. The levels of imidacloprid needed to cause mortality are much higher than can be achieved within a plant under typical grove conditions. So there is a tremendous amount of selection pressure that exists for product failures in the field right now and indeed we think we are seeing just that.
a. Number of adult D. citri tested.
b. Test of differences in mortality at the mean dose level where means differ significantly at α ≤ (Contact: 19.5 ppm; Ingestion: 97.7 ppm).
c. Parts per million (ppm) active ingredient.
d. Ratio of ingestion LC50 divided by contact LC50 by location.
e. Percent mortality in negative control containing no diet at 72h.
Langdon & Rogers 2017, JEE 110(5):

13. Response of laboratory and field collected D
Response of laboratory and field collected D. citri to imidacloprid by ingestion and contact in 2016.
The ingestion to contact ratio for the Vero Beach population is much greater than the Lake Placid population, despite the fact the resistance ratio is much greater for both the ingestion and contact assay for psyllids from the Lake Placid site. What this shows is that there is shift in resistance occurring, due to sublethal effects on feeding deterrence that the contact vial assay is not able to discern. But more importantly is the shift in LC90 values for feeding mortality seen in the field. The levels of imidacloprid needed to cause mortality are much higher than can be achieved within a plant under typical grove conditions. So there is a tremendous amount of selection pressure that exists for product failures in the field right now and indeed we think we are seeing just that.
a. Number of adult D. citri tested.
b. Test of differences in mortality at the mean dose level where means differ significantly at α ≤ (Contact: 19.5 ppm; Ingestion: 97.7 ppm).
c. Parts per million (ppm) active ingredient.
d. Ratio of ingestion LC50 divided by contact LC50 by location.
e. Percent mortality in negative control containing no diet at 72h.
Langdon & Rogers 2017, JEE 110(5):

14. Findings from Langdon’s Dissertation Research
Greater sensitivity of ACP to neonics through contact vs ingestion exposure
Foliar sprays are more effective in killing
Should be considered for future stewardship of neonic products
Failure to achieve lethal concentrations of neonics in leaf tissue following soil applications
Activity primarily through feeding deterrence

15. Findings from Langdon’s Dissertation Research
Persistence of neonic concentrations in leaf tissues <1 PPM through 12 weeks post-application
Sublethal exposures that may promote resistance development
Data from this study suggest resistance development to neonics at all study sites
Need to look at more grove locations!!!

16. Annual Survey to Monitor For Insecticide Resistance
Lukasz Stelinski Lab
Four purposes:
Monitor for insecticide resistance in D. citri field populations
Study the changes that occur over time in the insecticidal response of D. citri
Determine the natural variation in D. citri insecticidal response
Refine spray schedules
The monitoring program is part of the Statewide Citrus Health Management Area program for coordinated insecticidal spray regimes

17. Average LD50 Resistant Ratios
RR50 = Field Population LD50 / Laboratory Strain LD50
2009: ratios were on the rise for some major classes of insecticides
2013 and 2014 fell back to susceptible levels
Hypothesis: Improved spray regimes through CHMA were working
RR50 ratios from 2009 suggested reduced susceptibility to a few of the main classes of insecticides used to control D. citri.
Greatest RR50 was 34-fold for imidacloprid for the La Belle collection site
Years , populations were evaluated with a diagnostic dose
2013, we re-established LD50 estimates
New person was taking over the survey
Switched from mouth aspiration to vacuuming
Studies were conducted and found no differences in LD50 estimates between mouth aspirated and D-Vac
Resistance ratios RR = R-LD50 ÷ S-LD50
Statistical test = overlap of 95% fiducial limits AND
Other statistical tests with P<0.05 are described by Robertson et al. (2007) in the book “Bioassays with arthropods”
*Average of RR50 from all sites surveyed

18. 2017 Resistance monitoring in 6 locations in Florida (LD95s)
(Lukasz Stelinski)
LC95RR
MOA 1A,1B
MOA 3
MOA
Class
Site of action 1
OPs & carbamate
AChE inhibitors
Nerve action 3
pyrethroid
Sodium channel 4A
Neonicoti-noid
Nicotinic AChR modulator 4D
butenolide 28
diamide
Ryanodine receptor modulator nerves/muscles
LC95RR
MOA 4A
MOA 4D
MOA 28

19. Dr. Lukasz Stelinski lab, 2017
Group 4A
(Admire Pro)

20. Dr. Lukasz Stelinski lab, 2017
Group 4A
(Platinum)

21. Dr. Lukasz Stelinski lab, 2017
Group 1B
(Dimethoate)

22. Dr. Lukasz Stelinski lab, 2017
Group 1B
(Lorsban) ND ND

23. Dr. Lukasz Stelinski lab, 2017
Group 3
(Danitol) ND ND

24. Dr. Lukasz Stelinski lab, 2017
Group 4D
(Sivanto) ND

25. Dr. Lukasz Stelinski lab, 2017
Group 28
(Exirel) ND ND

26. Dr. Lukasz Stelinski lab, 2017
Group 5
(Delegate)

27. Psyllid Populations (Florida)

28. HLB infection rates in new plantings
Groves ~2 years of age (2014)
Psyllid pressure
HLB infection range
Average infection rate
# of groves
1 - low
0 – 4.05%
1.02% 20
2 – medium
1.73 – 65.6%
17.7%
3 - high
0 – 36.9%
20.96% 21
2017: Growers reporting HLB infection rates of 20-30% in plantings 1-2 years old

29. New MAC planting (CREC 2017)

30. Current status of insecticide resistance in Florida populations of Asian citrus psyllid and emerging management strategies
Lukasz L. Stelinski1, Philip Stansly2, Jawwad Qureshi3, and Michael Rogers1
University of Florida, Entomology and Nematology Department, CREC1, SWREC2, IREC3

31. How should neonicotinoids be used for young tree protection in the face of resistance?
Soil applications do not deliver sufficient neonicotinoid toxicant into the plant to kill resistant or semi-resistant ACP—application failures are occurring now
Sub-lethal concentrations of neonicotinoids persist in non-bearing citrus trees during the period when ACP are not effectively controlled, further exacerbating resistance selection

32. How should neonicotinoids be used for young tree protection in the face of resistance?
Moving forward, growers choosing to continue use of soil-applied neonics should closely monitor their groves to ensure the soil applications are still providing effective “control”
Keeping plants “psyllid free”

33. How should neonicotinoids be used for young tree protection in the face of resistance?
If control failures are observed after applications of soil-applied neonicotinoids, we recommend using neonicotinoids only as foliar sprays in new plantings along with a rigorous rotation with other foliar-applied chemistries (recommendation from 1 registrant)
e.g., whitefly example from Europe

34. Current consequences of resistance
Preliminary investigations indicate that at least 6 months of completely ceasing selection pressure will be required for ACP populations to fall back to susceptible levels
After populations reverse to susceptibility, they may remain forever ‘primed’ to becoming resistant faster than was required initially, making rigorous resistance management a top priority into the future
A rotation of at least 5 modes of action in sequence has been shown to effectively prevent development of resistance
First bullet point, just wanted to leave the door open that we cant guarantee resistance is everywhere, but it is in all places we looked.

35. Future Monitoring and Managing Pesticide Resistance in ACP Populations
Funding has been provided by APHIS to greatly expand resistance monitoring effort throughout the state
Repeated monitoring will be conducted in selected CHMAs
Product rotations will be recommended by CHMA based on results of resistance monitoring

38. ACP Management Summary
Pay attention to level of control when spraying
Pull back on use of a particular MOA if control failures observed
May need to switch from soil to foliar applied neonics if control is not sufficient
ROTATION is key
5 MOA’s in sequence preferred
Kaolin clay (dry season)
Reflective mulch

39. Questions?

40. Response of laboratory and field collected D
Response of laboratory and field collected D. citri to imidacloprid by ingestion and contact in 2016.
The ingestion to contact ratio for the Vero Beach population is much greater than the Lake Placid population, despite the fact the resistance ratio is much greater for both the ingestion and contact assay for psyllids from the Lake Placid site. What this shows is that there is shift in resistance occurring, due to sublethal effects on feeding deterrence that the contact vial assay is not able to discern. But more importantly is the shift in LC90 values for feeding mortality seen in the field. The levels of imidacloprid needed to cause mortality are much higher than can be achieved within a plant under typical grove conditions. So there is a tremendous amount of selection pressure that exists for product failures in the field right now and indeed we think we are seeing just that.
a. Number of adult D. citri tested.
b. Test of differences in mortality at the mean dose level where means differ significantly at α ≤ (Contact: 19.5 ppm; Ingestion: 97.7 ppm).
c. Parts per million (ppm) active ingredient.
d. Ratio of ingestion LC50 divided by contact LC50 by location.
e. Percent mortality in negative control containing no diet at 72h.
Langdon & Rogers 2017, JEE 110(5):