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Biocontrol Science and Technology
ISSN: 0958-3157 (Print) 1360-0478 (Online) Journal homepage: http://www.tandfonline.com/loi/cbst20
Infection and mortality of Microtheca
ochroloma (Coleoptera: Chrysomelidae) by Isaria
fumosorosea (Hypocreales: Cordycipitaceae)
under laboratory conditions
Cecil O. Montemayor, Pasco B. Avery & Ronald D. Cave
To cite this article: Cecil O. Montemayor, Pasco B. Avery & Ronald D. Cave (2016) Infection
and mortality of Microtheca ochroloma (Coleoptera: Chrysomelidae) by Isaria fumosorosea
(Hypocreales: Cordycipitaceae) under laboratory conditions, Biocontrol Science and
Technology, 26:5, 605-616, DOI: 10.1080/09583157.2015.1126222
To link to this article: http://dx.doi.org/10.1080/09583157.2015.1126222
Accepted author version posted online: 08
Mar 2016.
Published online: 09 Mar 2016.
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RESEARCH ARTICLE
Infection and mortality of Microtheca ochroloma (Coleoptera:
Chrysomelidae) by Isaria fumosorosea (Hypocreales:
Cordycipitaceae) under laboratory conditions
Cecil O. Montemayor, Pasco B. Avery and Ronald D. Cave
Indian River Research & Education Center, University of Florida, Institute of Food and Agricultural Sciences,
Fort Pierce, FL, USA
ABSTRACT
Microtheca ochroloma Stål, the yellowmargined leaf beetle, is a pest
in crucifer crops during the late fall and winter months in Florida. On
organic farms, it is difficult to control due to the restricted use of
insecticides, in addition to the lack of specific natural enemies.
The objective of this study was to evaluate a blastospore-
formulated product of Isaria fumosorosea (PFR-97
TM
20% WDG)
against this beetle. In the first experiment, four of the beetle’s life
stages were treated with a suspension of 3 × 10
7
blastospores/ml.
Mean corrected mortality of treated insects was significantly
higher in 1st and 3rd instars than in the egg, pupal, and adult
stages. Larvae infected by I. fumosorosea exhibited reduced
growth and unsuccessful molting. The second experiment
quantified mortality of first instars of M. ochroloma by four
concentrations of PFR-97
TM
. Mean corrected larval infection/
treatment was significantly (2.6 times) higher with a concentration
of 4 g of product per 100 ml of water compared to concentrations
of 1–3 g per 100 ml of water. Different factors that might have
affected the pathogenicity of I. fumosorosea against M. ochroloma
are discussed.
ARTICLE HISTORY
Received 10 March 2015
Returned 8 April 2015
Accepted 26 November 2015
KEYWORDS
Yellowmargined leaf beetle;
biological control;
entomopathogenic fungus;
survival time; lethal
concentration
1. Introduction
Microtheca ochroloma Stål, the yellowmargined leaf beetle, is a serious pest in crucifer
crops during the late fall and winter months in Florida (Ameen & Story, 1997). Since
1947, this adventive species had become established throughout most of the southern
United States. Its main damage is defoliation; however, tubers can also be damaged
when infestations are high. On organic farms, it is difficult to control M. ochroloma par-
tially due to the lack of specific natural enemies of M. ochroloma in the United States
(Fasulo, 2005). Currently, there is no pest management program available for organic
growers to control this pest in the United States.
Biological control by entomopathogenic fungi may potentially be used to manage
M. ochroloma on organic farms (Dos Anjos et al., 2007). Isaria fumosorosea Wize (=Pae-
cilomyces fumosoroseus), an entomopathogenic fungi with worldwide distribution, is well
© 2016 Taylor & Francis
CONTACT Pasco B. Avery pbavery@ufl.edu Indian River Research & Education Center, University of Florida,
Institute of Food and Agricultural Sciences, 2199 South Rock Road, Fort Pierce, FL 34945, USA
BIOCONTROL SCIENCE AND TECHNOLOGY, 2016
VOL. 26, NO. 5, 605–616
http://dx.doi.org/10.1080/09583157.2015.1126222
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documented for its effectiveness against many pest insects (Lacey, Kirk, Millar, Mercadier,
& Vidal, 1999; Osborne & Landa, 1992; Vidal, Lacey, & Fargues, 1997), including chry-
somelid beetles such as the Colorado potato beetle, Leptinotarsa decemlineata (Say)
(Bajan, 1973), Pyrrhalta luteola (Mueller), Spaethiella sp. (Humber, Hansen, & Wheeler,
2009), Diabrotica undecimpunctata Mannerheim, and Acalymma vittata (Fabricius)
(Rogers, 2012). Balusu and Fadamiro (2013) evaluated the bioinsecticide NOFLY®
(I. fumosorosea strain FE 9901) on adults and larvae of M. ochroloma. Results showed
higher larval mortality compared to the control 5 d post-exposure; however, the
larval and adult mortality were only 50% and 14%, respectively, over the 9-d exposure
period.
In 1986, a strain of I. fumosorosea named Apopka 97 was isolated in Apopka (Orange
County), FL from Phenacoccus sp. (Hemiptera: Pseudococcidae) (Vidal, Osborne, Lacey,
and Fargues, 1998). The strain is registered under the commercial name PFR-97
TM
20%
WDG
®
[chemical family: microbial insecticide, chemical name: I. fumosorosea Apopka
Strain 97 (ATCC 20874)] by the manufacturer Certis USA, in Columbia, MD. It is rec-
ommended for biological control of insect and mite pests of vegetables, fruits, and
other food crops. In this study, I. fumosorosea Apopka 97 strain was evaluated as a poten-
tial biological control agent of M. ochroloma.
2. Materials and methods
2.1 Stock colony
Adults and larvae of M. ochroloma were collected from White Rabbit Acres certified
organic farm in Vero Beach, FL and transported to the laboratory at the Hayslip Biological
Control Research and Containment Laboratory at the Indian River Research and Edu-
cation Center in Ft. Pierce. The colony was maintained on turnip leaves in plastic boxes
(27 × 15 × 8 cm, Ziploc®) with screen mesh openings in the walls for ventilation held at
25°C, with 50% relative humidity (RH) under a 10 h light (L): 14 h dark (D) photoperiod.
2.2 Fungus
PFR-97
TM
20% WDG (a.i. I. fumosorosea Apopka strain 97 20%, inert ingredients 80%)
was provided by Certis USA in a 0.45 kg bag (Lot: 0833004401) in the form of desicca-
tion-tolerant formulated granules of I. fumosorosea blastospores. The bag contained 1 ×
10
9
colony-forming units/g.
2.3 Plant material
Turnip Seven Top (Greens) (Brassica rapa L. var. rapifera) seeds were seeded in 72-hole
trays containing a sterilised soil mixture (Fafard
®
germination mix, Agawam, MA) inside a
greenhouse. Seedlings were transplanted 2 weeks later into 3.8 L plastic pots containing
soil mix Fafard
®
3B (Agawam, MA). Plants were fertilised weekly with 400 ml per pot
of liquid fertiliser (Miracle Grow
®
24N-8P-16 K).
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2.4 Experiment 1. Susceptibility of beetles to infection by PFR-97
TM
Five stages of M. ochroloma, egg, 1st instar, 3rd instar, pupa, and adult, were removed
from the laboratory colony for exposure to a concentration of 1 g of PFR-97
TM
in 100
ml of sterile distilled water (∼3×10
7
blastospores/ml). The fungal suspension was pre-
pared in a beaker for 30 min and allowed to settle for 20 min until the supernatant con-
taining blastospores and the inert material of the product separated. The initial blastospore
concentration was determined by counting the number of blastospores per ml using a dis-
posable plastic Neubauer hemocytometer, C-Chip DHC-N01 (NanoEnTek Inc., Seoul,
Korea).
Each suspension was poured separately into 180 ml Nalgene™(Nalge Nunc Intl.,
Rochester, NY) spray bottles for application to test insects. The supernatant was
applied to groups of 10 insects per stage housed in separate Petri dishes (60 × 75 mm, Fish-
erbrand®) with moistened filter paper (55 mm Ø diameter, Whatman®) on the bottom
dish. A 2.5 cm
2
Ø piece of turnip leaf was placed on top of the filter paper and each
group of insects per life stage with their respective piece of leaf received 3 sec of application
(∼2.5 ml) on each side of the leaf. The sprayed leaf was not removed from the Petri dish,
and starting 3 d after treatment, new non-sprayed leaves were added daily to each Petri
dish but not removed. Dishes were sealed with Parafilm® and placed into a growth
chamber held at 25°C, 60% RH under a 14 h L: 10 h D photoperiod.
The blastospore deposition density was determined by placing a plastic cover slip
among the test insects during the application of the fungal suspension, after which the
number of blastospores per mm
2
was determined. Viability of the blastospores was deter-
mined by taking 100 µl from a 10
−3
serial dilution of the product, spreading it on potato
dextrose agar (PDA) in Petri dishes, and maintaining the dishes under the same environ-
mental conditions as the tested insects. After the plates were incubated for 12–16 h at 25°C
and 100% RH, percentage viability was determined by viewing 200 spores. Spores were
considered to have germinated if a germ tube formed. This procedure was repeated for
each experimental repetition, and the percentage viability for all repetitions ranged
from 87% to 89%.
Fungal treatments were applied to 5–10 replicate dishes with 10 beetles of the same life
stage per replicate. Control treatments consisted of 3–5 replicates (Trial 1) and 5 replicates
(Trial 2) in which the test insect stages were sprayed with sterile distilled water only. Mor-
tality was checked daily for 7 d following the fungal application. Infection rate was deter-
mined by using the control mortality as a correction factor (Abbott, 1925). Morphological
traits unique to I. fumosorosea in dead insects (see below for method) were used to confirm
infection. The experiment was conducted twice (Trials 1 & 2) on separate occasions.
2.5 Experiment 2. Infectivity of the most susceptible beetle stage by four
concentrations of PFR-97
TM
The goal of this experiment was to compare the infectivity of four concentrations of PFR-
97
TM
in the most susceptible stage of M. ochroloma, which was determined in Experiment
1. The fungal treatment concentrations were 1, 2, 3, and 4 g of PFR-97
TM
per 100 ml of
sterile distilled water, and distilled water only was sprayed in the control. Each treatment
was applied (see method below) to groups of ten 1st instars in separate Petri dishes with
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moistened filter paper on the bottom. A 2.5 cm
2
piece of turnip leaf was placed on top of
the filter paper. There were 10 replicate dishes (100 insects total) for each fungal treatment
concentration and 5 replicates (50 insect’s total) for the control. The Petri dishes were then
sealed and placed in an environmentally controlled chamber set to 25°C, 60% RH, and 10
h L: 14 h D photoperiod. Assessment of mortality and confirmation of infection was the
same as described above. The experiment was conducted once.
To confirm infection in Experiment 1, dead insects were removed daily and transferred
directly to Petri dish plates containing a mixture of PDA, dodine, streptomycin, and chlor-
ophenacol (Meyling, 2007); dishes were then sealed with Parafilm® and placed in the same
chamber as above. The fungal phenotype unique to I. fumosorosea growing on the insect
stage was recorded as infection confirmation. Confirmation of the I. fumosorosea pheno-
type was often difficult due to contamination by saprophytic fungi. Therefore, to minimise
fungal contaminants in Experiment 2, dead insects were surface sterilised in 70% ethanol
for a few seconds before being placed on the PDA-mixture plates. Unconfirmed infection
was assigned to dead insects in which I. fumosorosea could not be identified because of its
absence or contamination by other fungi.
2.6 Statistical analysis
Percentages were subjected to angular transformation prior to analysis. Corrected mor-
tality and infection rates were not significantly different (P= 0.172) between the two
trials of Experiment 1. Therefore, the data were combined and re-analysed using an
ANOVA, and means were separated by a Student–Newman–Keuls (SNK) test (α=
0.05) to detect significant differences among life stages. In Experiment 2, the corrected
mortality was used to assess the effect of each fungal concentration on the most susceptible
stage of M. ochroloma, with treatment means separated by the SNK test (α= 0.05). All tests
were performed with PROC GLM in SAS v. 9.2 (SAS Institute Inc. 2002, Cary, NC). Sur-
vival times (ST
10
and ST
25
) were determined using Kaplan–Meier survival analysis, and
significant differences among treatments (life stage) were identified using a Wilcoxon
signed rank test (α= 0.05) performed with JMP® Pro 11 Discovering JMP (SAS Institute
Inc. 2013, Cary, NC).
3. Results
3.1 Experiment 1
For 1 g of PFR-97
TM
in 100 ml of water, the concentration of blastospores was 3.0 ± 0.1 ×
10
7
blastospores/ml. Mean blastospore deposition density was 1, 043 ± 181.5 blastospores/
mm
2
and viability was > 80% for all suspensions of I. fumosorosea.
The most susceptible stage of M. ochroloma to the Apopka 97 strain of I. fumosorosea
was the larva (Figure 1). Mean corrected mortality rates of eggs, pupae, and adults were
not significantly different. In contrast, mean corrected mortality rates of the 1st and 3rd
instars were significantly higher at 30.7 and 38.6% (F= 17.77; df = 4, 56; P< 0.0001).
Mean infection rates for 1st and 3rd instars were significantly higher (F= 12.19; df = 4,
35; P< 0.0001) than those for the egg, pupal, and adult stages; however, only 17 and
20% of the infections of the 1st and 3rd instar were confirmed as I. fumosorosea
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(Figure 2). Mortality of the 1st instar was observed beginning 3 d after treatment, at which
time the larvae were molting to the 2nd instar. Mortality in the 3rd instar was observed
beginning 1 d after treatment.
The ST
10
(4 d) and ST
25
(5 d) for the 1st instar were significantly higher (χ
2
= 4.13; df =
1; P= 0.0121) than that (2 d and 3 d, respectively) for the 3rd instar (Figure 3). The sur-
vival rates of 1st and 3rd instars after 7 d were 61 and 51%, respectively. The ST
50
value
could not be determined due to the low mortality rate of larvae exposed to the 1 g treat-
ment concentration of PFR-97
TM
. The ST models were not significant for eggs, pupae, or
adults (P> 0.05).
Figure 1. Mean corrected mortality of M. ochroloma by PFR-97
TM
20% WDG at 3.0 × 10
7
blastospores/
ml 7 d after application. Bars (± SEM) with different letters within each stage are significantly different
(SNK test, P< 0.05).
Figure 2. Mean percent corrected infection of M. ochroloma by PFR-97
TM
20% WDG at 3.0 × 10
7
blas-
tospores/ml 7 d after application. Bars (± SEM) with the same letter are not significantly different (SNK
test, P> 0.05).
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3.2 Experiment 2
The concentration and deposition of blastospores for the four experimental concen-
trations of PFR-97
TM
are reported in Table 1. The 1st instar of M. ochroloma was selected
for this experiment based on the results of Experiment 1. There was 0–4% mortality in the
control treatments per concentration; therefore, the corrected mortality in all the fungal
treatments was considered to be caused by infection with I. fumosorosea. Mean infection
rate was significantly higher by 2.6 times in the 4 g concentration treatment than in the 1,
2, and 3 g concentration treatments (F= 3.76, df = 3, 36; P= 0.0191) (Figure 4). Confirmed
infection comprised 90–100% of the mortality at the two highest concentrations.
4. Discussion
This is the first investigation evaluating the efficacy of the Apopka strain of I. fumosorosea
blastospores against all life stages of M. ochroloma under laboratory conditions. The
fungus had a low, insignificant ovicidal effect with an egg mortality rate of 3%
(Figure 1). Although the ovicidal effect was low, the fungal residues on the eggs and on
the leaf surface may have a significant impact on the emerging neonates. There is a
Figure 3. (Colour online) Survival plot for first and third instars of M. ochroloma treated with PFR-97
TM
for 7 d under laboratory conditions.
Table 1. Concentration and deposition of blastospores in four suspensions of I. fumosorosea (PFR-97™)
in Experiment 2.
PFR-97
TM
Concentration ± SE
a
Deposition ± SE
a
(g/100 ml of water) (blastospores/ml) (blastospores/mm
2
)
1 2.2 ± 0.1 × 10
7
779 ± 150.5
2 3.8 ± 0.2 × 10
7
1088 ± 174.1
3 8.4 ± 0.7 × 10
7
4157 ± 962.6
4 1.1 ± 0.0 × 10
8
6658 ± 881.6
a
Standard error.
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great deal of variability and discussion concerning the ovicidal effect of I. fumosorosea on
various insect pests. Rodrigues-Rueda and Fargues (1980) showed that I. fumosorosea had
high ovicidal activity on eggs of the moths Mamestra brassicae (Linneaus) and Spodoptera
littoralis (Boisduval), but no significant effect was reported on eggs of Plutella xylostella
(Linneaus) (Maketon, Orosz-Coghlan, & Jaengarun, 2008). Tigano-Milani, Carneiro, de
Faria, Frazăo, and McCoy (1995) reported that isolates of I. fumosorosea infected < 40%
of the eggs of the leaf beetle Diabrotica speciosa (Germar). In contrast, Lacey et al.
(1999) reported a low but significant mortality (10–20%) of eggs of the whiteflyBemisia
tabaci (Gennadius) treated with PFR-97
TM
.
Larvae of M. ochroloma in the 1st and 3rd instars experienced the highest infection
rates among all the insect life stages tested (Figure 1). The unconfirmed infections may
be attributed to the procedure of transferring dead insects to Petri dishes without first
surface sterilising the insects. This might have resulted in the rapid growth of sapropha-
gous fungi, thus slowing the growth of any I. fumosorosea present and not allowing its
expression of diagnostic morphological features.
Larvae infected by I. fumosorosea exhibited reduced growth (Figure 5a–d) and unsuccessful
molting in which the exuvia remained attached to the new integument and played a role in
overall mortality. Hussain, Tian, He, and Ahmed (2009)showedareductionintheconsump-
tion and growth of all instars of the silk moth Ocinara varians Walker when I. fumosorosea
strain 03011-C3.19A was applied. A reduction in feeding was also reported by Fargues,
Delmas, and Lebrun (1994) in the Colorado potato beetle infected by Beauveria bassiana
(Balsamo) Vuillemin. Mortality and growth rate reduction may be attributed to the production
of toxins by the fungus, mechanical disruption of the structural integrity of membranes by the
growth of hyphae, and dehydration of cells from the loss of fluids (Asaff, Cerda-Garcia-Rojas,
& de la Torre, 2005;Ferron1981; Tefera & Pringle, 2003).
Figure 4. Mean percent corrected infection of first-instar M. ochroloma at four concentrations of PFR-
97
TM
7 d after application. Bars (± SEM) followed by the same letter are not significantly different (P>
0.05).
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Microtheca ochroloma larvae pupate within a net-like case which only has direct contact
with the cuticle of the pupa at the apex of the body. This net-like case may serve as a phys-
ical barrier to the deposition of blastospores on the cuticle of the pupa, which is necessary
to initiate infection. For this reason, low mortality and infection rates of pupae were
observed.
Adults of M. ochroloma were not affected by I. fumosorosea, possibly because the hard
cuticle is composed primarily of a higher degree of cross-linked proteins and chitin than
that of the immature stages, which provides greater strength and hardness to the exoske-
leton and functions as a formidable barrier to blastospore germination (Klowden, 2007).
Only 4% mortality of adult beetles was recorded in the fungal treatment, compared to
none in the control treatment (Figures 1–2), but the mortality in the treatment cannot
be confidently attributed to the fungus since there was no confirmed infection.
However, Michalaki, Athanassiou, Steenberg, and Buchelos (2007) reported low mortality
of adults of the confused flour beetle, Tribolium confusum Jacquelin du Val, after exposure
to I. fumosorosea.
In Experiment 2, there was a well-defined positive correlation between fungus concen-
tration and mortality rates for the 1st instars of M. ochroloma. The highest confirmed
infection rates were achieved with the 4 g concentration treatment, which corresponded
to the highest concentration of blastospores/ml and deposition of blastospores/mm
2
(Table 1). However, this concentration treatment achieved only 29% infection in the lab-
oratory and infection rates in the field may be expected to be lower. According to the
results of this study, higher concentrations of PFR-97
TM
should be tested in the laboratory,
since it seems that infection rates in the 1st instar increase as the concentration of blastos-
pores/ml increases (Figure 4). Since this bioassay was conducted once, the accuracy of the
Figure 5. (Colour online) (a) Reduction in the growth of larvae of M. ochroloma infected by I. fumosoro-
sea;(b–d) Unsuccessful molting by a larva of M. ochroloma infected with I. fumosorosea: (b) head par-
tially out; (c) exuvia attached to the dorsal part of the body; (d) larva starting to pull out from the tip of
the abdomen.
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data observed in this study should be considered preliminary, and additional tests are war-
ranted to account for natural variation in cohorts of PFR-97
TM
to confirm its ‘true’mor-
tality effect on the target organism at different concentrations. Robertson, Preisler, Ng,
Hickle, and Gelertner (1995), after conducting several screening bioassays using cohorts
of the same fungal population against a target insect, concluded that the conventional
practice of using ratios of one lethal concentration to another in screening bioassay
studies may lead to erroneous conclusions if natural variations and subsequent gener-
ations of the same genetic strain are unknown.
Entomopathogenic fungi are being explored as an alternative to chemical insecticides
for use against pest insects feeding on cole crops (Dos Anjos et al., 2007; Klingen,
Hajek, Meadow, & Renwick, 2002; Oliveira et al., 2011; Sudirman, Prayogo, Yanimar, &
Ginting, 2008). Glucosinolates, found mainly in the family Brassicaceae, are plant com-
pounds that can affect plant–insect-pathogen interactions (Hopkins, van Dam, & van
Loon, 2009; Rask et al., 2000). Inhibition of fungal pathogens due to the presence of gluco-
sinolates has been observed in the laboratory (Inyang, Butt, Beckett, & Archer, 1999;
Sudirman et al., 2008; Vega, Dowd, McGuire, Jackson, & Nelsen, 1997). In addition, iso-
thiocyanates, produced by cole plants when damaged mechanically or attacked by arthro-
pods (Bones & Rossiter, 1996; Rask et al., 2000), have been shown to inhibit germination
and growth of Metarhizium brunneum Petch, B. bassiana, and I. fumosorosea on agar
plate’sin vitro (Inyang et al., 1999; Sudirman et al., 2008; Vega et al., 1997). In our
study, it is possible that the cut and consumed turnip leaves contained the fungitoxic iso-
thiocyanates that might have negatively affected the pathogenicity of the fungus. This
possibility may play a significant role for the need of a higher concentration of
I. fumosorosea blastospores to obtain greater insect mortality. However, after comparing
both in vitro and in vivo tests, Klingen et al. (2002) noted that although isothiocyanates
can inhibit M. brunneum in vitro, no fungal inhibition was found when using a more rea-
listic fungus/plant/soil microcosm. Therefore, based on our results, more research is war-
ranted to determine if the high concentration of I. fumosorosea blastospores (4 times the
label rate) predicted in our laboratory study can actually be reduced when applied under
field conditions.
The unconfirmed infection rate in Experiment 2 was lower compared to that in Exper-
iment 1 because the insects that died in Experiment 2 were surface sterilised with alcohol
for a few seconds before placing them on the PDA. The unconfirmed infection rate in
Experiment 2 may be reduced even more by using the polymerase chain reaction tech-
nique to identify the presence of PFR-97
TM
strain in the dead insects, as has been con-
ducted in other studies (Hoy, Singh, & Rogers, 2010; Meyer, 2007; Meyer, Hoy, &
Boucias, 2008).
Once blastospores were deposited on the integument of the insect, death of the host and
the appearance of fungal infection in the 1st instar of M. ochroloma began 3 d following
application. Similar results were reported by Tounou et al. (2003) in nymphs of the green
leafhopper, Empoasca decipiens Paoli, which began dying 3 d after treatment with
I. fumosorosea strain Pfr12. However, there will be a higher probability of deposition
and subsequent germination of blastospores when higher concentrations of the fungal sus-
pensions are applied, thereby killing a greater number of insects compared to lower
concentrations.
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In summary, the larval stage of M. ochroloma is the most susceptible stage to
I. fumosorosea. Larvae suffered reduced growth and unsuccessful molting in which the
exuvium remained attached to the new integument. Due to the potential inhibitory inter-
action of glucosinolates in the tissues of the plant consumed by the beetle larvae and
adults, it does not seem to be an economical and cost-effective strategy for
I. fumosorosea to be applied alone in the field on crucifers for managing this pest.
Higher concentrations than those tested may need to be applied in the field to have a
higher efficacy than that obtained under laboratory conditions; however, the screening
bioassay was only conducted once and cannot account for variation in the fungal biopes-
ticide. In this study, I. fumosorosea was shown to have low efficacy against M. ochroloma
adults when applied directly on the beetle, but no information has been reported about its
effect on leaf consumption by larvae and adults. Another aspect of this fungal interaction
with the beetle pest where further research is warranted is to determine what effect the
infection may have on the fecundity of the second generation of beetles after being
exposed to I. fumosorosea, which could potentially reduce the overall population and
plant damage. Also, B. bassiana, which was reported infecting M. ochroloma in Brazil
(Dos Anjos et al., 2007; Oliveira et al., 2011), should be tested because it may be more
pathogenic under field conditions against this beetle. Therefore, further laboratory and
field evaluations should be conducted to determine any effect I. fumosorosea or other ento-
mopathogenic fungi may have on herbivory rate or fecundity. If results show promise,
then the best fungal candidate could potentially be incorporated into an IPM strategy
for increasing field efficacy against this insect and decreasing plant damage.
Acknowledgements
We are grateful to Valerie Quant, owner of White Rabbit Acres in Vero Beach, FL, for allowing the
collection of beetles on her farm. We thank Rodrigo Diaz for assistance with data analysis and Susan
Webb and Edward Skvarch for their critiques and suggestions that made substantial improvements
to this study. In addition, we acknowledge the Ministry of Economy and Finances of Panama and
the Florida Department of Agriculture and Consumer Services for being the major sponsors of this
research.
Disclosure statement
No potential conflict of interest was reported by the authors.
References
Abbott, W. S. (1925). A method of computing the effectiveness of an insecticide. Journal of
Economic Entomology,18, 265–267.
Ameen, A. O., & Story, R.N. (1997). Feeding preferences of larval and adult Microtheca ochroloma
(Coleoptera: Chrysomelidae) for crucifer foliage. Journal of Agricultural Entomology,14, 363–
368.
Asaff, A., Cerda-Garcia-Rojas, C., & de la Torre, M. (2005). Isolation of dipicolinic acid as an insec-
ticidal toxin from Paecilomyces fumosoroseus.Applied Microbiology and Biotechnology,68, 542–
547.
Bajan, C. (1973). Paecilomyces fumoso-roseus (Wize) pathogenic agent of the Colorado beetle
(Leptinotarsa decemlineata Say). Ekologia Polska,21, 705–713.
614 C.O. MONTEMAYOR ET AL.
Downloaded by [University of Florida] at 07:33 22 March 2016
Balusu, R., & Fadamiro, H. Y. (2013). Susceptibility of Microtheca ochroloma (Coleoptera:
Chrysomelidae) to botanical and microbial insecticide formulations. Florida Entomologist,96,
914–921.
Bones, A. M., & Rossiter, J. T. (1996). The myrosinase-glucosinolate system, its organization and
biochemistry. Physiologia Plantarum,97, 194–208.
Dos Anjos, J., Rosalino, P., Sausen, C. D., Do Prado, L., Egewarth, R., Soares, V., and Bastos, S. T.
(2007), ‘Fungos entomopatogênicos em Diabrotica speciosa eMicrotheca ochroloma (Col.,
Chrysomelidae) em hortalizas [Entomopathogenic fungi in Diabrotica speciosa and
Microtheca ochroloma (Col.Chrysomelidae) in leafy greens]’,Techinal Report, Universidade
Federal de Santa Maria, Santa Maria, Brazil.
Fargues, J., Delmas, J. C., and Lebrun, R. A. (1994). Leaf consumption by larvae of the Colorado
potato beetle (Coleoptera: Chrysomelidae) infected with the entomopathogen, Beauveria bassi-
ana.Journal of Economic Entomology, 87, 67–71.
Fasulo, T. R. (2005). Yellowmargined leaf beetle, Microtheca ochroloma Stål (Insecta: Coleoptera:
Chrysomelidae), University of Florida, IFAS Extension. Retrieved from http://edis.ifas.ufl.edu/
pdffiles/IN/IN62500.pdf
Ferron, P. (1981). Pest control by the fungi Beauveria bassiana and Metarhizium. In H. D. Burges
(Ed.), Microbial control pests and plant diseases (pp. 1970–1980). New York: Academic Press.
Hopkins, R. J., van Dam, N. M., & van Loon, J. A. (2009). Role of glucosinulates in insect–plant
relationships and multitrophic interactions. Annual Review of Entomology,54,57–83.
Hoy, M. A., Singh, R., & Rogers, M. E. (2010). Evaluations of a novel isolate of Isaria fumosorosea
for control of the Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae). Florida
Entomologist,93,24–32.
Humber, R. A., Hansen, K. S., and Wheeler, M. M. (2009). USDA-ARS Collection of
Entomopathogenic Fungal Cultures (ARSEF), ARSEF-Catalog of Species. Retrieved from
http://arsef.fpsnl.cornell.edu/mycology/catalogs/Catalog.pdf.
Hussain, A., Tian, M., He, Y., & Ahmed, S. (2009). Entomopathogenic fungi disturbed the larval
growth and feeding performance of Ocinara varians (Lepidoptera: Bombycidae) larvae. Insect
Science,16, 511–517.
Inyang, E. N., Butt, T. M., Beckett, A., & Archer, S. (1999). The effect of crucifer epicuticular waxes
and leaf extracts on the germination and infectivity of Metarhizium anisopliae conidia.
Mycological Research,103, 419–426.
Klingen, I., Hajek, A., Meadow, R., & Renwick, J. A. A. (2002). Effect of brassicaceous plants on the
survival and infectivity of insect pathogenic fungi. BioControl,47, 411–425.
Klowden, M. J. (2007). Physiological systems in insects. Integumentary systems. Moscow, ID:
Academic Press Elsevier.
Lacey, L. A., Kirk, A. A., Millar, L., Mercadier, G., & Vidal, C. (1999). Ovicidal and larvicidal activity
of conidia and blastospores of Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes)
against Bemisia argentifolii (Homoptera: Aleyrodidae) with a description of a bioassay system
allowing prolonged survival of control insects. Biocontrol Science and Technology,9,9–18.
Maketon, M., Orosz-Coghlan, P., & Jaengarun, J. (2008). Field evaluation of Isaria fumosorosea on
controlling the diamondback moth (Plutella xylostella) in Chinese kale. Phytoparasitica,36, 260–
263.
Meyer, J. M. (2007). Microbial associates of the Asian citrus psyllid and its two parasitoids: symbionts
and pathogens. Ph.D. dissertation, University of Florida, Gainesville.
Meyer, J. M., Hoy, M. A., & Boucias, D. G. (2008). Isolation and characterization of an Isaria fumo-
sorosea isolate infecting the Asian citrus psyllid in Florida. Journal of Invertebrate Pathology,99,
96–102.
Meyling, N. V. (2007). Methods for isolation of entomopathogenic fungi from the soil environment
–laboratory manual. [Metoder til isolering af insektpatogene svampe fra jorden –en laborator-
iemaunal], Department of Ecology, Faculty of Life Sciences, University of Copenhagen. Retrieved
from http://orgprints.org/11200/.
Michalaki, M. P., Athanassiou, C. G., Steenberg, T., & Buchelos, C. T. (2007). Effect of Paecilomyces
fumosoroseus (Wize) Brown and Smith (Ascomycota: Hypocreales) alone or in combination with
BIOCONTROL SCIENCE AND TECHNOLOGY 615
Downloaded by [University of Florida] at 07:33 22 March 2016
diatomaceous earth against Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae)
and Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). Biological Control,40, 280–286.
Oliveira, F. R., Dequech, S. T., Sturza, V. S., Walker, M. P., Guths, C., Bolzan, A., …Cocco, D.
(2011). Ocorrência de Beauveria bassiana em Microtheca ochroloma Stål no cultivo da couvechi-
nesa (Brassica chinensis L.). 12° SICONBIOL, Simpósio de Controle Biológico –18 a 21 de julho
de 2011.
Osborne, L. S., & Landa, Z. (1992). Biological control of whiteflies with entomopathogenic fungi.
Florida Entomologist,75, 456–471.
Rask, L., Andréasson, E., Ekbom, B., Eriksson, S., Pontoppidan, B., & Meijer, J. (2000). Myrosinase:
gene family evolution and herbivore defense in Brassicaceae. Plant Molecular Biology,43,93–
113.
Robertson, J. L., Preisler, H. K., Ng, S. S., Hickle, L. A., & Gelertner, W. D. (1995). Natural variation:
A complicating factor in bioassays with chemical and microbial pesticides. Journal of Economic
Entomology,88,1–10.
Rodrigues-Rueda, D., & Fargues, J. (1980). Pathogenicity of entomopathogenic hyphomycetes,
Paecilomyces fumosoroseus and Nomuraea rileyi, to eggs of noctuids, Mamestra brassicae and
Spodoptera littoralis.Journal of Invertebrate Pathology,36, 399–408.
Rogers, M. A. (2012). Efficacy of biopesticides for organic management of cucumber beetles, Ph.D.
dissertation. Knoxville: University of Tennessee.
Sudirman, L. I., Prayogo, Y., Yanimar, Y., & Ginting, S. (2008). Effect of leaf litters and soils on via-
bility of entomopathogenic fungi Beauveria bassiana (Bals.) Vuill. HAYATI Journal of
Biosciences,15,93–98.
Tefera, T., & Pringle, K. L. (2003). Food consumption by Chilo partellus (Lepidoptera: Pyralidae)
larvae infected with Beauveria bassiana and Metarhizium anisopliae and effects of feeding
natural versus artificial diets on mortality and mycosis. Journal of Invertebrate Pathology,84,
220–225.
Tigano-Milani, M. S., Carneiro, R. G., de Faria, M. R., Frazăo, H. S., & McCoy, C. W. (1995).
Isozyme characterization and pathogenicity of Paecilomyces fumosoroseus and P. lilacinus to
Diabrotica speciosa (Coleoptera: Chrysomelidae) and Meloidogyne javanica (Nematoda:
Tylenchidae). Biological Control,5, 378–382.
Tounou, A. K., Agboka, K., Poehling, H. M., Raupach, J. K., Langewald, J., Zimmermann, G., &
Borgemeister, C. (2003). Evaluation of the entomopathogenic fungi Metarhizium anisopliae
and Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) for control of the green leaf-
hopper Empoasca decipiens (Homoptera: Cicadellidae) and potential side effects on the egg para-
sitoid Anagrus atomus (Hymenoptera: Mymaridae). Biocontrol Science and Technology,13, 715–
728.
Vega, F. E., Dowd, P. F., McGuire, M. R., Jackson, M. A., & Nelsen, T. C. (1997). In vitro effects of
secondary plant compounds on germination of blastospores of the entomopathogenic fungus
Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes). Journal of Invertebrate
Pathology,70, 209–213.
Vidal, C., Lacey, L. A., & Fargues, J. (1997). Pathogenicity of Paecilomyces fumosoroseus
(Deuteromycotina: Hyphomycetes) against Bemisia argentifolii (Homoptera: Aleyrodidae)
with a description of a bioassay method. Journal of Economic Entomology,90, 765–772.
Vidal, C., Osborne, L. S., Lacey, L. A., & Fargues, J. (1998). Effect of host plant on the potential of
Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) for controlling the silverleaf
whitefly, Bemisia argentifolii (Homoptera: Aleyrodidae) in greenhouses. Biological Control,12,
191–199.
616 C.O. MONTEMAYOR ET AL.
Downloaded by [University of Florida] at 07:33 22 March 2016
