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Potential of Azadirachtin for Managing Black Cutworms and Japanese
Beetle Grubs in Turf
Justine George and Daniel A. Potter
Dept. of Entomology
S-225 Agricultural Science Building N.
University of Kentucky
Lexington, KY 40546-0091
USA
Keywords: Popillia japonica, Agrotis ipsilon, botanical insecticide, neem
Abstract
Botanical insecticides have potential for managing insect pests with low
hazard to humans and the environment. Azadirachtin, a limonoid extracted from
the seeds of the neem tree, Azadirachta indica (Meliaceae), acts as an insect growth
regulator and antifeedant against many crop pests. We evaluated two commercial
azadirachtin formulations, Azatrol EC and Azatin XL (1.2 and 3.0%, respectively)
against two important turfgrass pests: black cutworms [BCW], Agrotis ipsilon, and
Japanese beetle [JB], Popillia japonica. Feeding on azadirachtin-sprayed creeping
bentgrass caused molting disorders and death of early-instar BCW, and slowed
feeding and stunted the growth of late instars. Azadirachtin also showed systemic
activity against early instar BCW fed bentgrass treated via a root soak, and some
deterrence of late instars in the field. Application to Kentucky bluegrass, followed by
irrigation, killed 2nd-instar JB at 5 times label rate, but label rates did not provide
control in the greenhouse or field. Azadirachtin residues in turf did not deter egg-
laying by JB. This work indicates that azadirachtin will suppress BCW if applied
when early instars are present, but probably will not effectively control late instars
or root-feeding grubs.
INTRODUCTION
Azadirachtin, a limonoid extracted from the seeds of the neem tree, Azadirachta
indica (Fam: Meliaceae), has antifeedant and growth regulatory effects against many
insect pests (Isman, 1990; Mordue (Luntz) et al., 2005). Feeding inhibition by
azadirachtin results from inhibition of sugar receptors coupled with stimulation of
deterrent receptor cells (Mordue (Luntz) et al., 2005). Azadirachtin also disrupts insect
hormonal systems causing molting and reproductive aberrations (Schmutterer, 1990).
Azadirachtin-based insecticides are labeled for outdoor landscapes, but at present they are
rarely used on turf (Potter, 2007).
Several characteristics of azadirachtin suggest it could be used to suppress insect
pests on sites such as sport fields where use of conventional insecticides may be
restricted. It has low mammalian toxicity and low impact on beneficial species (Mordue
(Luntz) et al., 2005). Root-systemic activity has been shown in some plants (Arpaia and
van Loon, 1993; Sundaram et al., 1995) and acropetal and basipetal systemic movement
reportedly also occurs in turfgrass (G. Custis, PBI/Gordon, pers. commun.). Azadirachtin
topically applied to Japanese beetle [JB], Popillia japonica, grubs (Coleoptera:
Scarabaeidae) disrupted development to the adult stage (Ladd et al., 1984). It also exhibits
good efficacy against many leaf-eating caterpillar pests of crops (Immaraju, 1998).
Azadirachtin insecticides deter feeding by adult JB (Baumler and Potter, 2007), so it is
plausible that their residues might also deter egg-laying in turfgrass.
Direct or systemic activity of azadirachtin against foliage-feeding caterpillars or
root-feeding white grubs in turf has not previously been documented. We evaluated direct
and systemic activity of commercial azadirachtin products against black cutworms
[BCW], Agrotis ipsilon (Lepidoptera: Noctuidae), and conducted greenhouse and field
trials to assess activity on early and late-instar JB grubs. We also tested if azadirachtin
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Proc. IIn
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IC on Turfgrass
Eds.: J.C. Stier et al.
Acta Hort. 783, ISHS 2008
residues in turf deter oviposition by adult JB. Our goal was to determine the potential of
azadirachtin as an "organic" alternative for suppressing turf insect pests.
MATERIALS AND METHODS
General Methods
Two azadirachtin formulations, Azatrol (PBI/Gordon, Kansas City, MO)
containing 1.2% azadirachtin, and Azatin (OHP, Mainland, PA) containing 3%
azadirachtin, were evaluated. Azatrol was used in all trials because its label lists turfgrass
as a target site. The Azatin label lists BCW and adult JB as target pests, but not
specifically in turfgrass, so that product was included in some, but not all trials. Each
product was applied at its highest label rate (4.165 L product/ha for Azatrol; 1.169 L
product/ha for Azatin) unless indicated otherwise. Creeping bentgrass (Agrostis
stolonifera ‘Penncross’) was used for trials with BCW; trials with JB used Kentucky
bluegrass (Poa pratensis ‘Midnight’). Turf cores (15 cm diameter, unless stated
otherwise) used for greenhouse trials were pulled from stands of each grass at the
University of Kentucky Spindletop Research Farm, near Lexington, KY. Field trials were
done in those stands, which were maintained without insecticides under typical mowing,
fertility, and irrigation regimes (Prater et al., 2006; George et al., 2007). BCW were
obtained from a commercial insectary and held on artificial diet (Prater et al., 2007) until
used. JB adults and grubs were field-collected as needed. Choice trials were analyzed by
paired t-tests; other trials were analyzed by one-way ANOVA followed by Dunnett’s test
for comparing treatments versus control (α = 0.05).
Black Cutworm [BCW] Trials
1. Choice Test Using Azadirachtin-Sprayed Clippings. Azatrol or Azatin were sprayed
on foliage of potted creeping bentgrass cores in the greenhouse and allowed to dry for 6 h.
Two tillers each from treated and untreated cores were stuck to the base of a 14.5 cm
diameter Petri dish with tape. Moistened filter paper was stuck to the lid of each dish and
a moist dental wick was added to maintain humidity. One 5th instar was introduced in the
middle of each dish. Dishes were held in darkness at 25ºC and assays were terminated in
1−3 h, after the BCW had consumed about 2/3 of its choice of grass. Each treatment was
replicated 12 times. Percentage of each choice consumed was arcsine square-root
transformed before analysis.
2. Effects on Development and Survival of Early and Late Instars. Azatrol was
sprayed on bentgrass cores at labeled rate and left to dry for 4 h on leaf blades. Clippings
were cut and fed ad libitum to BCW in 9 cm Petri plates at 27ºC, as above. There were
two sets of trials, starting with either 10 neonates (<1 d old) per dish, or five 4th instars per
replicate in individual dishes. Surviving larvae were transferred to new plates with fresh
clippings from the same sprayed cores every 2 d. Larvae were individually weighed and
numbers of frass pellets were counted after 2, 4, and 7 d. The cores were watered (about 1
cm) 1 d after the first clippings were cut and every 2 d thereafter. Number of individuals
from the 4th instar trial that had pupated was recorded after 7 d. Pupae were weighed,
sexed, and held until adults had emerged. Frass counts, larval survival and weight, and
days to pupation were analyzed as described above.
3. Curative Control of Late Instars. Eight 4th instars per replicate were introduced into
potted creeping bentgrass cores in the greenhouse and allowed 5 h to settle in the thatch.
The cores were then treated using hand sprayer (20 ml spray per cylinder) using Azatrol
at labeled rate. Rims of pots were smeared with petroleum jelly and covered with mesh to
prevent escapes. After 72 h, the turf cores were drenched with 750 ml of soap disclosing
solution (Prater et al., 2007) and BCW that surfaced (normal response to soap drench)
were collected and counted.
4. Systemic Treatments. Creeping bentgrass cores were maintained in the greenhouse
and trimmed (2.54-cm height) every 5 d. Cores to be treated were removed from the pots,
placed in plastic trays in a solution of Azatrol (33.4 ml per 3.785 L for six cores), the
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label tank mix rate for spraying turf at 1.69 L/acre. The solution was not allowed to touch
the grass or thatch, so could only be absorbed through the roots. The cores were left to
soak for 48 h and then repotted to soil. Control cores were soaked in water. Clippings
were harvested from each core and challenged with 10 neonate 1st instars (per dish), and
five 4th instars (in individual dishes) per replicate as before. Larvae were provided with
fresh clippings from the same cores every 2 d to prevent larvae from being food-limited.
Data were taken after 2, 4, and 7 d. On each date the numbers of live, moribund, or dead
larvae were recorded. All survivors were weighed on final day to test for any sublethal
effect on weight gain.
5. Choice Trials with 4th Instars in the Field. Galvanized metal enclosures (91 × 46 ×
15 cm) were driven 2 cm into the aforementioned creeping bentgrass stand. The frames
divided into two equal sections with a 5 cm untreated buffer zone in between. One section
treated with Azatrol or Azatin at labeled rate and the other section sprayed with water.
When residues had dried, 25 active 4th instars were placed on the buffer zone, and
enclosures were covered with wire mesh to exclude bird predation. The plots were
drenched using soap solution after 5 d and numbers of BCW that surfaced from treated,
buffer, and control sections were recorded.
Japanese Beetle [JB] Trials
1. Choice Tests for Oviposition in Treated versus Untreated Turf. The hypothesis that
Azatrol or Azatin residues deter JB females from ovipositing in treated turf was tested in
choice trials (George et al., 2007). Test arenas were circular clear plastic bins (19 cm
high, 20 cm diameter) with a raised poly-foam floor into which four holes were cut. Cores
of Kentucky bluegrass (5.1-cm diameter, 7-cm deep, 4-cm grass height) were inserted
into waxed paper cups that were set in the holes such that beetles could walk between
them and burrow into the turf. The cores were spaced every 90° around the bin.
Six replicates were run for Azatrol versus untreated, Azatin versus untreated, and
untreated versus untreated. Dosages were mixed with water at label rate and 20 ml
solution was applied to each of the two opposite turf cores in each bin. The other two
cores received water only. Eight JB females were added to each bin 1 d after the turf was
treated. An apple piece was provided as food after 2 d. Cores were broken apart after 4 d
and eggs and females were counted. To test if females’ exposure to residues impaired
their viability, JB found in the two cores of each treatment were tested in a “fly-off” assay
(George et al., 2007). Numbers of eggs and viable beetles were compared between
treatments within trials.
2. Efficacy of Azadirachtin against JB Grubs. Greenhouse and field trials tested effects
of azadirachtin products on grub survival and viability in turf. For the greenhouse trial,
Kentucky bluegrass cores (10 cm diameter, 12 cm tall) were each inoculated with 10
healthy field-collected 2nd instars. The next day, the turf was sprayed with either 1× or 5×
the labeled rate (six replicates) for both the products, followed by 2 cm of irrigation. The
turf cores were broken apart after 10 d and numbers of live grubs were recorded. Grub
viability was assessed by placing them on moist, 4-cm-deep peat moss in plastic trays and
recording the percentage that burrowed beneath the surface in 10 min, the normal
response (George et al., 2007).
Separate field trials were done with 2nd or 3rd instars. For both trials, PVC
enclosures (15.2 cm diameter, 17.5 cm height) were driven 15 cm into the aforementioned
Kentucky bluegrass stand, with six replicates per treatment. Ten, healthy field-collected
2nd or 3rd instars were added to each enclosure on 8 September 2006. The cores were
sprayed with labeled rates of either Azatin or Azatrol the next day, followed by 2 cm
irrigation. Plots were sampled after 14 d. All live grubs from each enclosure were brought
to the laboratory and tested for viability (ability to burrow down) as above.
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RESULTS
Black Cutworm [BCW] Trials
1. Choice Test Using Azadirachtin-Sprayed Clippings. Mean (± SE) percentage of
foliage consumed after 1 h was 86.7 ± 9.0 versus 16.7 ± 9.4 for untreated versus Azatin-
sprayed tillers, respectively (t = 5.4, df = 8, P < 0.001), and 96.0 ± 3.0 versus 21.5 ± 10.8
for untreated versus Azatrol, respectively (t = 5.7, df = 9, P < 0.001. After 3 h,
percentages consumed were 100 and 15.8 ± 9.6 for untreated versus Azatin, and 97.3 ±
1.9 and 25.5 ± 12.2 for untreated versus Azatrol; both comparisons were significant by
paired t- test (P < 0.001).
2. Effects on Development and Survival of Early and Late Instars. First instars
provided Azatrol-sprayed clippings stopped feeding, none of them molted, and 100% died
within 5 d. First instars given untreated clippings fed normally and all had molted to the
2nd instar by 5 d (data not shown). Fourth instars provided treated clippings showed
reduced frass production (Table 1), indicative of reduced feeding. All control larvae
appeared healthy after 7 d, whereas 23% (7/30) of those fed on sprayed grass were dead
or moribund by 7 d (Table 1).
3. Curative Control of Late Instars. Curative treatment of 4th instars in turfgrass cores
was not effective. Similar numbers of larvae were recovered via soap drench from
Azatrol-treated and untreated cores (6.0 ± 0.4 versus 7.8 ± 0.2 respectively; F = 14.8, df
=1, 11, P =0.01).
4. Systemic Treatments. Neonates provided with clippings from creeping bentgrass
cores treated via root soak showed very little feeding. Mean (± SE) numbers surviving
after 5 d were 1.0 ± 0.4 and 9.7 ± 0.2 for treated versus untreated cores, respectively (t =
4.0, df = 11, P < 0.001), suggesting that azadirachtin was systemically translocated
through the roots. Fourth instars provided clippings from systemically-treated cores
showed little mortality by 4 d, but by 7 d, their mortality averaged 2.0 ± 0.3 (out of five
per replicate) versus 0% for untreated cores (F = 60, df = 1, 11, P < 0.001). There was a
trend for their 7-d weights to be lower on clippings from treated cores than from controls
(239 ± 11 versus 309 ± 18 mg, respectively (F = 4.5, df = 1, 11, P = 0.08).
5. Choice Tests with 4th Instars in the Field. More BCW were recovered from the sides
of the field enclosures with untreated turf than from sides treated with Azatin (mean
values were 6.9 ± 1.5 versus 3.4 ± 0.5, respectively; t = 2.14, df = 7, P < 0.05). Feeding
damage also appeared greater in the untreated plots (authors’ observation). For
comparisons with Azatrol, however, there was no difference in mean number of BCW
recovered from untreated versus treated turf (4.5 ± 0.8 versus 4.3 ± 0.8, respectively; t =
0.2, df = 7; P = 0.4).
Japanese Beetle [JB] Trials
1. Choice Tests for Oviposition in Treated versus Untreated Turf. Similar numbers of
eggs were laid in creeping bentgrass cores with fresh azadirachtin residues as in untreated
turf (Table 2). Total number of eggs deposited per chamber also did not differ
significantly, nor did exposure to azadirachtin reduce viability of females in the fly-off
assays (Table 2).
2. Efficacy of Azadirachtin against JB Grubs. Both rates of Azatin caused significant
mortality of 2nd instars in the greenhouse trial within 10 d (Fig. 1). Mean number of viable
grubs were 42.3 and 51.3% lower, respectively, at the 1× and 5× rates, compared to the
control (F = 13.2; df = 2, 17; P < 0.01). Dead grubs in the treated turf appeared brownish
or pale yellowish. Azatrol was not as effective. Mean numbers of live grubs recovered
from those cores were 7.8 ± 0.5, 5.7 ± 0.6, and 7.7 ± 0.6 for untreated, Azatrol 1×, and
Azatrol 5× cores, respectively (F = 4.1, df = 2, 17; P < 0.05).
Neither product applied at label rate significantly controlled 2nd instar JB grubs in
the field. Numbers of live grubs recovered did not differ between treated and control plots
(F = 1.46, df = 2, 17; P = 0.27; Fig. 2). Numbers viable enough to burrow down into
moist peat moss also were similar between treatments (F = 2.0; df = 2, 17; P = 0.18; Fig.
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2). Treatments targeting 3rd instars also were ineffective. Mean numbers of live grubs
recovered 14 d after treatment were 12.3 ± 1.0, 11.7 ± 1.1, and 9.5 ± 1.0 for Azatrol,
Azatin, and untreated plots, respectively (F = 1.97; df = 2, 17; P = 0.19), with no
difference in their viability assessed by burrowing capability (data not shown). More than
10 grubs were recovered from some cores reflecting those that were introduced, plus the
natural field population.
DISCUSSION
Feeding on Azatrol-treated creeping bentgrass clippings resulted in 100%
mortality of 1st instars, suggesting that azadirachtin insecticides could suppress BCW in
sport fields or other turf sites if applications coincided with presence of young larvae.
This might require multiple applications because azadirachtin residues have limited
persistence in the field (Mordue et al., 2005). Such treatments could be timed by
monitoring moth flight with traps and commercial pheromone lure (Hong and
Williamson, 2004). Our data suggest that at labeled rates, commercial azadirachtin
products are too slow-acting to effectively control large BCW once damage appears.
Although fresh residues of both products deterred feeding by BCW in laboratory choice
tests, they did not cause BCW to abandon treated turf in our field enclosures.
This is the first published report that azadirachtin has root-systemic activity in
turfgrass. Such treatment effectively controlled 1st-instar BCW and reduced weight gain
and survival of 4th instars. However, the tank mix rate used for the root soak in that trial
would normally be applied at 3.79−7.57 L per 92.9 m2, so our methodology likely
resulted in greater uptake of azadirachtin than would occur at field application rates.
Some systemic activity may also have occurred in trial testing effects on development and
survival of late instars, but since the grass was first sprayed and watered 1 d later it is not
possible to separate direct from systemic activity in that assay.
Although azadirachtin residues have antifeedent activity against JB adults
(Baumler and Potter, 2007) in our choice tests they did not deter oviposition, nor did they
affect flight capability of females exposed to treated turf. Ladd et al. (1984) showed that
topically-applied azadirachtin killed early-instar JB grubs and disrupted normal
development of later instars. High rates killed some 2nd instars in our greenhouse trials,
but label rates did not control late instars in the field. This suggests that, despite irrigation,
the azadirachtin did not penetrate the thatch and soil at rates high enough to provide
control. Azadiachtin may nevertheless have potential for grub control if ways can be
found to deliver a lethal dose to the root zone.
ACKNOWLEDGEMENTS
We thank C.T. Redmond for technical support and B. Rajeev (Univ. of Kentucky)
and G. Custis (PBI/Gordon) for critically reading the manuscript. Partial support provided
by a grant from the U.S. Golf Association. This is paper no. 07-08-022 of the Kentucky
Agricultural Experiment Station.
Literature Cited
Arpaia, S. and van Loon, J.J.A. 1993. Effects of azadirachtin after systemic uptake into
Brassica oleracea on on larvae of Pieris brassicae. Entomol Exp. Appl. 66: 39-45.
Baumler, R.E. and Potter, D.A. 2007. Knockdown, residual, and antifeedant activity of
pyrethroids and home landscape bioinsecticides against Japanese beetles (Coleoptera:
Scarabaeidae) on linden foliage. J. Econ. Entomol. 100: 451-458.
George J., Redmond, C.T., Royalty, R.N. and Potter, D.A. 2007. Residual effects of
imidacloprid on Japanese beetle (Coleoptera: Scarabaeidae) oviposition, egg hatch,
and larval viability in turfgrass. J. Econ. Entomol. 100: 431-439.
Hong, S.C. and Williamson, R.C. 2004. Comparison of sticky wing and cone pheromone
traps for monitoring seasonal abundance of black cutworm larvae and adults on golf
courses. J. Econ. Entomol. 97: 1666-1670.
Immaraju, A.J. 1998. The commercial use of azadirachtin and its integration into viable
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pest control programmes. Pestic. Sci. 54: 285-289.
Isman, M.B., Koul, O., Luczynski, A. And Kaminski, J. 1990. Insecticidal and antifeedant
bioactivities of neem oils and their relationship to azadirachtin content. J. Agric. Food
Chem. 38: 1406-1411.
Ladd, T.L, Warthen, J.D. and Klein, M.G. 1984. Japanese beetle (Coleoptera:
Scarabaeidae): The effects of azadirachtin on the growth and development of the
immature forms. J. Econ. Entomol. 77: 903-905
Mordue (Luntz), A.J., Morgan, E.D. and Nisbet, A.J. 2005. Azadirachtin, a natural
product in insect control. Comp. Mol. Insect Sci. 6: 117-135
Potter, D.A. 2007. Managing insect pests of sport fields: What does the future hold? Acta
Hort. In Press.
Prater, C.A., Redmond, C.T., Barney, W., Bonning, B. and Potter, D.A. 2006. Microbial
control of black cutworm (Lepidoptera: Noctuidae) in turfgrass using Agrotis ipsilon
multiple nucleopolyhedrovirus. J. Econ. Entomol. 1129-1137.
Schmutterer, H. 1990. Properties and potential of natural pesticides from the neem tree,
Azadirachta indica. Annu. Rev. Entomol. 35: 271-297.
Sundaram, K.M.S. and Campbell, R. 1995. Uptake, translocation, persistence and fate of
azadirachtin in aspen plants and its effect on pestiferous two-spotted spider mite. Crop
Prot. 14: 415-421.
Tables
Table 1. Mortality of BCW fed azadirachtin-treated or untreated creeping bentgrass
clippings from the onset of the 4th instar and frass production indicative of reduced
feeding on treated grass.
No. dead or moribund (out of
five) after1No. frass pellets produced
during days
Treatment 2 d 4 d 7 d 1−2 3−4
Control 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 273 ± 25 210 ± 16
Azatrol 0.3 ± 0.3 0.5 ± 0.3 1.2 ± 0.6 184 ± 38 134 ± 22
t (P)2Ns ns ns 1.94 (0.04) 2.67 (0.01)
1Data are means (± SE)
2 two-sample t-tests (one-sided, 10 df) comparing treated versus control
Table 2. Numbers of eggs deposited by P. japonica in treated versus untreated turf cores
during 4-d choice tests, total eggs per chamber, and viable females after exposure to
each treatment combination.
Choice test results
Mean (± SE) eggs per
two turf cores Mean (± SE) per
chamber
Comparison (A
versus B)
A
B
t
P Total
eggs1Viable
females2
Azatrol
versus untreated 3.7 ± 3.1 5.8 ± 2.4 0.52 0.31 9.5 ± 3.7 6.2 ± 0.3
Azatin
versus untreated 3.8 ± 1.1 5.0 ± 2.5 0.51 0.32 8.8 ± 3.0 5.8 ± 0.7
Untreated versus
Untreated 6.0 ± 2.2 9.0 ± 1.8 0.92 0.20 15.0 ± 2.4 5.8 ± 0.7
1ANOVA for total eggs: F = 1.0; df = 2, 10; P = 0.4
2ANOVA for viable females: F = 0.09; df = 2, 10; P = 0.90
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Figures
Control Azatrol X Azatrol 5X Azatin X Azatin 5X
0
2
4
6
8
10
No of grubs (Mean ± SE)
Viable grubs Dead grubs
Fig. 1. Mean (± SE) numbers of viable and dead P. japonica grubs recovered from
Kentucky bluegrass cores 10 d after spraying with azadirachtin products at 1× or
5× label rate, with post-treatment irrigation, in the greenhouse. Ten 2nd instars
were introduced into each core 1 d before treatment.
Control Azatrol Azatin
0
2
4
6
8
10
No of grubs (Mean ± SE)
Total grubs Viable grubs
Fig. 2. Mean (± SE) numbers of total and viable P. japonica grubs recovered from
Kentucky bluegrass field plots 14 d after applying azadirachtin products at
labeled rates, with post-treatment irrigation. Ten 2nd instars were introduced into
each plot 1 d before treatment.
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