Toxicology Reports 3 (2016) 473–480
Contents lists available at ScienceDirect
Toxicology  Reports
j ourna l ho me  page: www.elsev ier .com/ locate / toxrep
Evalua s  a
alfalfa  
Gadi  V.P. kash
a Montana Stat ox 65
b Kagoshima U
a  r  t  i  c  l
Article history:
Received 14 A
Received in re
Accepted 4 Ma
Available onlin
Keywords:
Low risk insecticides
Insect pathogenic fungi
Efficacy
Lethal concentration
Mortality rate
eopte
auses
in  Mo
ica,  th
my  in
re  kn
examined  the  six  commercially  available  biorational  insecticides  against  H.  postica  under  laboratory  con-
dition:  Mycotrol® ESO  (Beauveria  bassiana  GHA),  Aza-Direct® (Azadirachtin),  Met52® EC (Metarhizium
brunneum  F52),  Xpectro  OD® (B. bassiana  GHA  +  pyrethrins),  Xpulse  OD® (B. bassiana  GHA + Azadirachtin)
and  Entrust  WP® (spinosad  80%).  Concentrations  of 0.1,  0.5,  1.0,  and  2.0  times  the  lowest  labelled  rates
were  tested  for all products.  However,  in the  case  of Entrust  WP,  additional  concentrations  of  0.001
1. Introdu
Alfalfa w
culionidae)
sativa L. (Fa
[1]. H. posti
ting, but ca
adults dam
lowering cr
the most da
substantial 
Heavily infe
skeletonize
larvae survi
retarding re
a density o
∗ Correspon
E-mail add
http://dx.doi.o
2214-7500/© 
nc-nd/4.0/)and  0.01  times  the  lowest  label  rate were  also  assessed.  Mortality  rates  were  determined  at  1–9 days
post  treatment.  Based  on  lethal  concentrations  and  relative  potencies,  this  study  clearly  showed  that
Entrust  was  the  most  effective,  causing  100%  mortality  within  3 days  after  treatment  among  all  the
tested  materials.  With  regard  to  other biorational,  Xpectro  was the second  most  effective  insecticide  fol-
lowed by Xpulse,  Aza-Direct,  Met52,  and  Mycotrol.  Our  results  strongly  suggested  that  these  biorational
insecticides  could  potentially  be applied  for  H. postica  control.
© 2016  The  Author(s).  Published  by Elsevier  Ireland  Ltd.  This  is an open  access  article  under  the CC
BY-NC-ND  license.  (http://creativecommons.org/licenses/by-nc-nd/4.0/)
ction
eevil Hypera postica (Gyllenhall) (Coleoptera: Cur-
, is the most destructive insect pest of alfalfa Medicago
baceae) in the intermountain west of the United States
ca not only decreases yield and quality of the first cut-
n also harm subsequent cuttings [2]. Both larvae and
age terminals, foliage and new crown shoots, thereby
op yield and quality [3]. However, the larvae caused
mage [4]. During severe infestations, larvae can cause
defoliation, resulting in severe first cutting losses [5].
sted fields may  appear silver or white, with most leaves
d or consumed entirely [1]. If large numbers of adults or
ve until harvest, they damaged stems and crown buds,
growth [6]. A decrease in stem elongation occurred at
f 30–100% of the smallest larval density [7]. Residual
ding author.
ress: reddy@montana.edu (G.V.P. Reddy).
effects from severe damage decrease plant vigor, resulting in lower
stand density and poor yields in subsequent harvests [2].
Although H. postica is native to Europe it was inadvertently
introduced into the western United States in the early 1900s [8],
and into the eastern United States in the late 1940s [9]. In Mon-
tana, alfalfa is the second most important crop after small grains
[10]. Alfalfa growers in Montana first began to notice H. postica
during spring 2013 when the weevil caused considerable damage
and yield losses [10]. In addition, alfalfa weevils caused economic
damage in irrigated fields in the Yellowstone and Missouri river val-
leys in Montana [10]. Insecticidal treatment are economical when
a larval population average between 1.5–2.0 larvae/stem, or 20 lar-
vae/sweep [11]. In 2014 and 2015, H. postica outbreak occurred
in Valier, Montana. Even though H. postica does the most damage
before the first cutting [12], considerable damage was also noticed
even after the first harvest.
To date, other than classical biological control, insecticide
applications and early harvesting are the most common man-
agement strategies for alfalfa weevil [13]. However, most of the
chemical insecticides used to manage this pest are extremely
rg/10.1016/j.toxrep.2016.05.003
2016 The Author(s). Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license. (http://creativecommons.org/licenses/by-tion  of  toxicity  of  biorational  insecticide
weevil
 Reddya,∗, Frank  B.  Antwia, Govinda  Shresthaa,  Ta
e University, Western Triangle Agricultural Research Center, 9546 Old Shelby Rd., P.O. B
niversity, Faculty of Education, Korimoto 1-20-6, Kagoshima, 890-0065, Japan
 e  i  n  f  o
pril 2016
vised form 4 May  2016
y 2016
e 5 May  2016
a  b  s  t  r  a  c  t
The  alfalfa  weevil,  Hypera  postica  (Col
(Fabaceae).  While  H. postica  usually  c
damaging  levels  of the pest  persisted  
tional  insecticides  can  control H. post
including  pollinators  and  natural  ene
the best  alternative  options,  as  they against  larvae  of  the
i  Kuriwadab
6, Conrad, MT  59425, USA
ra:  Curculionidae),  is  a major  pest  of  alfalfa  Medicago  sativa  L.
 the  most  damage  before  the  first  cutting,  in  summer  of  2015
ntana  well  after  the first  harvest  of  alfalfa.  Although  conven-
ese  chemicals  have  adverse  effects  on non-target  organisms
sects.  In this  context,  use  of  biorational  insecticides  would  be
own  to pose  less  risk  to non-target  organisms.  We  therefore
474 G.V.P. Reddy et al. / Toxicology Reports 3 (2016) 473–480
Table 1
Materials and application rates used for the laboratory bioassays against Hypera postica larvae.
Treatment Chemical name Trade name Concentrations (ml/l) Source
T1 Untreated control – – –
T2  spinosad (Saccharopolyspora spinosa) Entrust® WP 0.000091, 0.00091, 0.0091,
0.0455, 0.091, and 0.182
Dow Agro Science LLC, Indianapolis, IN
T3  Metarhizium brunneum F52 Met52® EC 0.072, 0.36, 0.72, and 1.44 Novozymes Biologicals, Salem, VA
T4  Beauveria bassiana GHA Mycotrol ESO® 0.072, 0.36, 0.72, and 1.44 LAM International, Butte, MT
T5  Azadirachtin (extracts from Azadirachta indica)  Aza-Direct® 0.144, 0.72, 1.44, and 2.88 Gowan Company, Yuma, AZ
T6  B. bassiana GHA + pyrethrins Xpectro® OD 0.25, 1.25, 2.5, and 5.0 LAM International, Butte, MT
T7  B. bassiana GHA + cold pressed Neem extract Xpulse® OD 0.072, 0.36, 0.72 and 1.44 LAM International, Butte, MT
hazardous to bees [14,15], and other beneficial insects like
the parasitoids Bathyplectes curculionis (Thomson) (Hymenoptera:
Ichneumonidae) and Oomyzus incertus Ratzburg (Hymenoptera:
Eulophidae) [16]. Increasing concerns for environmental safety and
insecticide resistance arising from a frequent use of synthetic insec-
ticides affect the long-term feasibility of the current strategy of
alfalfa weevil management [17]. Consequently, many alfalfa grow-
ers in north central and central Montana are looking for more
environmental friendly control methods for managing this destruc-
tive pest.
In this context, as a green alternative to synthetic insecticides,
use of biorational insecticides would be the best alternative options
because these insecticides are usually considered low-risk agents
having the features of low mammalian toxicity as well as less
impact on non-target organisms [18]. The biorational insecticides
include the use of naturally derived compounds from plants or
microbes such as spinosyns and azadirachtin, living organisms
(insect pathogenic fungi) such as Beauveria bassiana (Bals.) Vuill
(Ascomycota: Hypocreales) and Metarhizium brunneum (anisopliae)
(Metsch.) Sorokin (Ascomycota: Hypocreales) or the combined for-
mulation of these insecticides [18]. In recent years, a number of
biorational insecticides are commercially available and have been
used or tested against variety of pest species such as aphids [19],
thrips [20], and coleopteran pests [21,22]. No attempts have been
made so far to study the effects of these insecticides on H. postica
control except the studies by Hedlund and Pass [23] and Sakurai
et al. [24], who showed the infection of H. postica with B. bassiana,
and M. brunneum.  This study therefore aimed to evaluate the toxic-
ity of biorational insecticides against H. postica under the laboratory
conditions.
2. Materials and methods
2.1. Rearing of insects
H. postica larvae were collected from alfalfa fields in Valier, Mon-
tana, USA, using sweep nets in July 2015 and taken to the laboratory.
Larvae were placed in collapsible cages (12 × 10 × 10 cm), fed alfalfa
foliage, and held at 22 ± 2 ◦C, 70–80% RH and an approximately
14:10 h L:D photoperiod. Field-collected larvae were separated by
instar as described by Harcourt [25]. The instars ranged from first
to fourth instars. The first instar is light yellow or tan in color
with a darker head and about 1 mm long while the second instar
is yellowish-brown with their head deepening to black, third and
Fig. 1. Percen
bassiana GHA)
GHA  + Azadiratage mortality of 2nd instar larvae of Hypera postica treated with different concent
, Aza-Direct® (Azadirachtin), Met52® EC (Metarhizium brunneum F52), Xpectro OD® (Be
chtin) and Entrust WP® (spinosad 80%) at days 1–9.rations (log) of biorational insecticides: Mycotrol® ESO (Beauveria
auveria bassiana GHA + pyrethrins), Xpulse OD® (Beauveria bassiana
G.V.P. Reddy et al. / Toxicology Reports 3 (2016) 473–480 475
Table  2
Lethal concentrations and relative potencies of Hypera postica larvae to biorational insecticides.
Treatments Day LC50 (g a.i./L) C. I. (95%) P > 2 Relative Potency (LC50;S/LC50;T)a
Entrust 2 0.0000123 1.42538 × 10−5–0.0000432 0.9054 1
Mycotrol ESOb 4 0.163602 0.159619–0.167686 1.0000 7.52 × 10−5
Met52 ECc 4 0.23434 0.14327–1.17575 0.7271 5.25 × 10−5
Aza-Direct 4 0.08146 0.04730–0.12666 0.0221 0.000151
Xpulse  ODd 4 0.01417 0.00573–0.02360 0.0229 0.000868
Xpectro OD 4 0.00109 0.0001385–0.00229 0.4189 0.011284
Entrust  WP 4 NDe ND ND ND
Mycotrol ESO 5 0.10845 0.08129–0.16632 0.2299 0.000113
Met52  EC 5 0.03441 0.02316–0.04705 0.0421 0.000357
Aza-Direct 5 0.01758 0.00407–0.03324 0.0317 0.0007
Xpulse  OD 5 0.00371 0.0005850–0.00663 0.5906 0.003315
Xpectro OD 5 0.00172 0.00157–0.00188 1.0000 0.007151
Entrust  WP 5 ND ND ND ND
a Ratios of the lethal concentrations of standard insecticide (Entrust WP)  to the treatments at 50% mortality.
b 2 × 1013 viable spores per quart with weight estimate of 4.78 × 1012 grams per spore.
c 5 × 1010 viable conidia per gram of active ingredient and contains 5.5 × 109 colony forming units (CFU)/gram of product.
d Beauveria bassiana Strain GHA (0.06%) contains ≥1 × 1011 viable spores per quart.
e ND, no data due to single response values (100% mortality), and therefore could not be determined by statistical analysis as well as lethal ratios.
fourth instar size is up to 9 mm long, are bright green with shiny
black head capsule, and have a white stripe down the halfway point
of their rears. Second instars were used for all tests.
2.2. Biorational insecticides
Biorational insecticides tested were of commercial formulations
(Table 1) and were stored dried at 4–5 ◦C until diluted to the desired
concentrations for use. The concentrations used in the study were
0.1, 0.5, 1.0 and 2.0 times the lowest label rate. However, in case
of Entrust product, we prepared additional concentrations of 0.001
and 0.01 times the lowest label rate since this product has been
known for high toxicity.
2.3. Toxicity tests
Toxicity tests were performed in the laboratory from 15 July
through August 2015 when larvae from field populations were
available. Materials were applied via contact at the desired concen-
trations (see Table 1 for rates). For each replicate, five larvae were
transferred onto a disk of Whatman No. 1 filter paper (9 cm diam-
eter, Whatman quantitative filter paper, ashless, Sigma-Aldrich, St.
Louis, Missouri, USA) in a 9 cm disposable Petri dish where they
were topically treated with the test material.
Each Petri dish also contained three alfalfa stems about 5 cm
long, each with 6–8 leaves as larval food. Six replicate Petri dishes,
containing a total of 30 larvae were treated using a Sprayer
Fig. 2. Probit analysis on median lethal time (LT50) of Mycotrol® ESO (Beauveria bassiana GHA) treated 2nd instar larvae of Hypera postica at different concentrations.
476 G.V.P. Reddy et al. / Toxicology Reports 3 (2016) 473–480
Fig. 3. Pro
Fig
(Sprayco, L
were treate
dishes were
rearing of in
2.4. Statisti
SAS 9.4 
was used tobit analysis on median lethal time (LT50) of Met52® EC (Metarhizium brunneum F52) treat
. 4. Probit analysis on median lethal time (LT50) Aza-Direct® (Azadirachtin) treated 2nd 
ivonia, MI)  with 1 ml  of a test material [26]. Controls
d with 1.0 ml  of tap water. Following application, Petri
 held under the same laboratory conditions as used for
sect. Larval mortality was assessed daily for nine days.
cal analyses
was used in analyzing the data [27]. Abbott’s formula
 adjust for control mortality [28], Sigma Plot 13.0 (SPSS
Inc., Chicag
log concent
lethal value
used to det
Among 
mortality o
caused at 0
dition, we e
or 4 and 5 ded 2nd instar larvae of Hypera postica at different concentrations.
instar larvae of Hypera postica at different concentrations.
o, IL) for plotting the graphs of mortality (%) versus
ration, and PROC PROBIT procedure for estimating the
s (LC50s). Comparison of the 95% confidence limits was
ermine differences in lethal values [29–31].
the different products, Entrust product caused 100%
f H. postica larvae within 3 days, while other products
–100% mortality at 4–9 days (Fig. 1). Based on this con-
stimated LC50 of Entrust and other products at 2 days
ays post treatment respectively (Table 2).
G.V.P. Reddy et al. / Toxicology Reports 3 (2016) 473–480 477
Fig. 5. Probit analysis on median lethal time (LT50) of Xpectro® (B. bassiana GHA + pyrethrins) treated 2nd instar larvae of Hypera postica at different concentrations.
Extra binomial variations due to genetic and environmental
influences that caused poor fit were accounted for by multiply-
ing the variances by the heterogeneity factor (2/k-2), where k is
the number of concentrations [27,31,32]. Relative potencies for the
treatments were compared using the lethal concentrations [30].
Because mortality rates of the all tested materials increased over
time (Figs. 2–7), treatments were also analyzed for LT50 using the
program Probit-MSChart [33]. Mortality response (in probits) was
regressed against log10 day.
Fig. 6. Probit analysis on median lethal time (LT50) of Xpulse® (B. bassiana GHA + azadirachtin) treated 2nd instar larvae of Hypera postica at different concentrations.
478 G.V.P. Reddy et al. / Toxicology Reports 3 (2016) 473–480
F nd ins
3. Results
3.1. Mortal
Among 
mortality to
tality at day
products, su
plants) and
pathogenic
took 6–7 da
trations (Fi
for example
postica larv
3.2. Lethal 
In overa
materials a
ear regressi
(log10 day) 
increased w
Generall
tions for al
were foun
ucts, highe
obtained fo
time for En
other prod
Met52 (103
Direct (71.9
2.56–3.94 d
(Fig. 5).
3.3. Lethal 
The leth
in Table 2. 
tions. Entru
compared t
LC50 value 
centrations
effective bi
Met52, and
Furtherm
Entrust and
and Xpectro
tality (Tabl
tenc
 2).
cussi
ostic
 Uni
 due
 out
 the
yed 
r) (Zy
es ha
 thre
parts
emie
cides
es fro
his s
 with
trus
t (sp
 to b
ad, th
ically
y to m
men
osad
% of 
lts. W
of En
ful, 
 high
tors 
 [40,ig. 7. Probit analysis on median lethal time (LT50) of Entrust® (spinosad) treated 2
ity percentage
all tested biorational insecticides, Entrust caused high
 H. postica larvae, acting rapidly and reaching 100% mor-
 3 across all concentrations (Fig. 1). However, for other
ch as, Aza-Direct, (naturally derived compounds from
, Xpulse and Xpectro (combined formulation of insect
 fungi and naturally derived compounds from plants)
ys to kill 100% of H. postica larvae across all concen-
g. 1). Furthermore, insect pathogenic fungus products
 Met52 and Mycotrol took 5–9 days to kill 100% of H.
ae across all concentrations (Fig. 1).
time (LT50)
ll, the toxicity results of contact bioassay with all tested
gainst second instars of H. postica showed good lin-
on relationship between mortality (in probit) and time
after treatment (Figs. 2–7). The mortality rate (in probit)
ith log10 day for all examined products.
y, lethal time decreased with increasing concentra-
l treatments. However, the differences in lethal time
d between the products. Among the tested prod-
st lethal time (122.7–164.7 h; 5.11–6.86 days) was
r Mycotrol (Fig. 2) in contrast to the lowest lethal
trust (18.1–27.8 h; 0.75–1.16 days) (Fig. 7). For the
ucts, the second highest lethal time was  found for
.6–148.8 h; 4.32–6.2 days) (Fig. 3) followed by Aza-
–111.3 h; 2.996–4.64 days) (Fig. 4), Xpulse (61.4–94.6 h;
ays) (Fig. 6) and Xpectro (43.6–73.9 h; 1.82–3.08 days)
concentration (LC )
had po
(Table
4. Dis
H. p
in the
largely
carried
pest in
emplo
(Arthu
enemi
nomic
many 
ural en
insecti
enemi
In t
postica
cies, En
Entrus
known
Spinos
intrins
toxicit
that hy
to spin
and 86
ful resu
effect 
be help
causes
regula
postica50
al concentrations for each tested material are depicted
Generally, there was a good fit to the model assump-
st was found the most effective biorational insecticide
o all other tested materials, since Entrust had a lowest
(Table 2). Among other products, based on lethal con-
 estimated at day 4 and day 5, Xpectro was  second most
orational insecticide followed by Xpulse, Aza-Direct,
 Mycotrol (Table 2).
ore, we computed relative potencies at day 2 for
 at days 4 and 5 for Mycotrol, Met52, Aza-Direct, Xpulse,
, using Entrust as the standard insecticide at 50% mor-
e 2). The result showed that none of the treatments
needed as t
In this st
bassiana + a
ing mortali
Mycotrol (B
effect, they
ment. Over
[22]. Patho
tial candida
Fungal path
insecticides
subsequent
[43,44]. Hatar larvae of Hypera postica at different concentrations.
ies, which were at par when compared with Entrust
on
a rapidly became the most devastating pest of alfalfa
ted States following its invasion in the 1940s, [34]
 to an absence of specialized natural enemies. The USDA
 a large-scale biological control program against this
 late 1950–1970s [35]. Seven parasitoid species were
[36] in addition to the fungus Zoophthora phytonomi
gomycetes: Entomophthorales) [37]. Although natural
ve brought the H. postica population below the eco-
shold level in other places, it is still a serious pest in
 of Montana. This pattern may  be due to a lack of nat-
s in these areas. Exploring the potential of biorational
 to manage H. postica may  protect these same natural
m the adverse effects of conventional insecticides.
tudy, Entrust (spinosad) caused 100% mortality of H.
in 3 days after treatment. Based on the relative poten-
t was the most effective among the treatments. While
inosad) was  effective against H. postica, this chemical is
e harmful to natural enemies, particularly parasitoids.
e active ingredient in Entrust, has been observed to be
 toxic to pollinators especially bees, though it has low
any beneficial insects [38]. Williams et al. [39] reported
opteran parasitoids are significantly more susceptible
 than predatory insects, with 78% of laboratory studies,
field studies reporting a moderately harmful, or harm-
hile further laboratory and field studies examining the
trust (spinosad) on the parasitoids of H. postica would
the need for these parasitoids may be low, since Entrust
 mortality to H. postica.  Although many insect growth
have been tested and found to be effective against H.
41], further cost-benefit analyses of these products are
hey seem expensive to use given the level of crop loss.
udy, Xpectro® (B. bassiana + pyrethrins) and Xpulse® (B.
zadirachtin) mixture products were effective in caus-
ty in H. postica.  Although the tested fungal pathogens
. bassiana), and Met52 (M. brunneum)  have delayed
 both caused 100% mortality within 9 days of treat-
 a thousand pathogens have been isolated from insects
gens associated with major insect pests are poten-
tes for development into microbial insecticides [42].
ogens have a different mode of action than synthetic
, killing their hosts through infection that leads to the
 production of insecticidal toxins, such as destruxins
rcourt et al. [45] reported that H. postica larvae were
G.V.P. Reddy et al. / Toxicology Reports 3 (2016) 473–480 479
found to be infected by a fungal entomopathogen (Entomoph-
thora phytonomi Arthur) which significantly reduced the weevil
population in Canada. However, Millstein et al. [46] reported the
importance of conidial discharge and relative humidity in Erythrina
sp. infecting
that conidia
M. anisoplia
Clonostachy
postica adu
and L. lecan
Aza-Dire
mortality 7–
with Oroum
of neem see
(Meliaceae)
high morta
stages of H. 
Yardim et a
effects on th
(Coleoptera
number of p
In gener
various ent
tica. Howev
be obtained
insecticides
able in the U
research is n
non-target 
Conflict of 
The auth
Acknowled
This wo
gram of the
Education p
award # 14
culture, Mu
Control of P
231844]. An
expressed i
necessarily
Agriculture
(USDA). We
of statistica
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Morad
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 Tharp
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sis) M H. postica in Kentucky, USA. Mustafa et al. [47] reported
l suspensions of entomopathogenic fungi B. bassiana,
e,  Lecanicillium lecanii (Zimm.) Zare and W.  Gams and
s rosea (Link: Fr.) isolates had significant effects on H.
lt mortality, with isolates of B. bassiana, M. anisopliae,
ii being more effective on adults than C. rosea.
ct (extracts from Azadirachta indica)  also caused 100%
9 days after treatment in this study. These results agree
chi and Lorra [48] who reported that aqueous extracts
d kernels and leaves, and chinaberry Melia azedarach L.
 leaves applied to alfalfa leaves in the laboratory caused
lity and strong growth-disturbing effects in the larval
postica, with most larvae dying before or during molting.
l. [49] reported that neem (azadirachtin) had significant
e larvae of another alfalfa weevil, Hypera variabilis Hbst.
: Curculionidae) but insignificant effects on the total
redators in alfalfa fields in Turkey.
al, our study showed that the tested materials including
omopathogenic fungi can be used to manage H. pos-
er, it remains to be seen if similar levels of control can
 under field conditions. Most of the naturally derived
 used in this study are currently commercially avail-
nited States, and could be adopted by growers. Further
eeded to determine the impact of these insecticides on
insects and natural enemies (e.g. bees and parasitoids).
interest
ors declare that there are no conflicts of interest.
gements
rk was supported by Professional Development Pro-
 USDA-Western Sustainable Agriculture Research and
roject #2014-38640-22175/Utah State University sub
0867038; and USDA National Institute of Food and Agri-
ltistate Project W3185, The Working Group Biological
est Management Systems of Plants [Accession number#
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