Fungal Biology 122 (2018) 1069e1076

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Fungal Biology
journal homepage: www.elsevier.com/locate/funbio

Metarhizium rileyi biopesticide to control Spodoptera frugiperda:

Stability and insecticidal activity under glasshouse conditions
Erika Paola Grijalba a, Carlos Espinel a, Paola Emilia Cuartas a, *, Martha Liliana Chaparro a,
Laura Fernanda Villamizar a, b
a
n Colombiana de Investigacio
Corporacio
n Agropecuaria - Corpoica, Centro de Investigacio
, kilo

n Tibaitata
, Cundinamarca,

metro 14 vía Mosquera-Bogota
Colombia
b
AgResearch, Forage Science, Lincoln Research Centre, Private Bag 4749, Christchurch 8140, New Zealand

a r t i c l e i n f o a b s t r a c t

Article history: The insect-pathogenic fungus Metarhizium rileyi is highly sensitive to nutritional and environmental con-
Received 7 March 2018 ditions which makes it difficult to produce as a stable biopesticide. In this study, a Colombian isolate of this
Received in revised form fungus was produced in bulk, and conidia were formulated as an emulsifiable concentrate (EC). The stability
27 July 2018
of formulated conidia was studied. Conidial viability was maintained at >85 % viability for 12 m under
Accepted 29 August 2018
Available online 19 September 2018
refrigeration and for >three months at 18
C. The pH values were stable, while contaminant content was
significantly reduced. The efficacy of the EC to control Spodoptera frugiperda (Smith) was correlated with the
Corresponding Editor: Christian Voigt storage time using different mathematical models, and conservative values of six and 12 months at 8
C and
18
C respectively, were established. Finally, the EC was evaluated in maize plants under glasshouse con-
Keywords: ditions. The LC50 and LC90 were estimated to be 1.17  104 and 4.03  106 conidia/mL respectively and a 57 %
Bioproduct reduction in recent damage of plants was achieved. This study demonstrated the potential of M. rileyi
Fall armyworm formulated as EC to control S. frugiperda in maize. Therefore, it is necessary to continue developing this
Formulation biopesticide, in order to deliver a new tool to be integrated in pest management programs.
Nomuraea rileyi
© 2018 British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Shelf life

1. Introduction There is a great variety of chemical insecticides used to control

Fall armyworm, Spodoptera frugiperda (Smith, 1797) (Lepidop-
tera: Noctuidae), has a wide geographical distribution, being found
throughout North and South America (Andrews, 1980; Murúa et al.
2006). In January 2016, the pest was first reported in Nigeria from
where it has since spread to other regions of Africa and the au-
thorities have declared a regional emergency (Goergen et al. 2016).
S. frugiperda is a polyphagous insect, with approximately 186 host
plants including corn, rice, cotton, peanut, alfalfa, potato, tomato,
soybean and sorghum (Casmuz et al. 2010). The main management
strategy is chemical control, which generates a high dependence
with an approximate cost of US$500 to US$600 million per year,
directly affecting a grower's production costs (Valicente and Souza,
2009).
* Corresponding author.
E-mail addresses: egrijalba@corpoica.org.co (E.P. Grijalba), cespinel@corpoica.
org.co (C. Espinel), pcuartas@corpoica.org.co (P.E. Cuartas), mlchaparro@corpoica.
org.co (M.L. Chaparro), laura.villamizar@agresearch.co.nz (L.F. Villamizar).
this insect. The most commonly used classes of pesticide are car-
bamates, organophosphates, and pyrethroids. To humans, con-
sumption of maize and other crops that have been sprayed with
these insecticides presents potential carcinogenic and neurotoxicity
risks due to bio-accumulated pesticide residues (Flores, 2010). In
addition, continued use of these chemicals causes disturbance in the
dynamics of natural enemies, problems of pest resistance to these
compounds, health risks for workers who apply the products in the
field and environmental contamination (Valicente and Souza, 2009).
Biological control of S. frugiperda includes the use of natural
enemies and pathogens, such as Bacillus thuringiensis (Bt), which
are sprayed on crops as biopesticides. Transgenic plants expressing
the Cry protein of Bt produce a type of host plant resistance and are
possibly the most used tool to reduce insect populations (Siebert
et al. 2008). However, the use of pesticides over a long period,
along with geographic and climatic factors, has led to the devel-
opment of insecticide resistance in different populations (Jakka
et al. 2014; Sousa et al. 2016).
The use of entomopathogenic fungi within the genera Beauveria,
Metarhizium, and Isaria provides another biological alternative to

https://doi.org/10.1016/j.funbio.2018.08.010
1878-6146/© 2018 British Mycological Society. Published by Elsevier Ltd. All rights reserved.
1070 E.P. Grijalba et al. / Fungal Biology 122 (2018) 1069e1076
control S. frugiperda, but one which has been less commercialized
(Santos, 2014). Metarhizium rileyi (Farlow) Kepler S.A. Rehner &
Humber (Ascomycetes: Hypocreales), previously known as
Nomuraea riley, is an entomopathogenic fungus with great poten-
tial to control S. frugiperda and other noctuid pests (Ignoffo, 1981;
Ignoffo and Boucias, 1992; Sanchez-Pen ~ a, 2000; Martins et al.
2005). However, M. rileyi is highly sensitive to nutritional and
environmental conditions compared with other entomopathogenic
fungi (Edelstein et al. 2004), which may affect the microbiological
characteristics and virulence of the conidia (Caro et al. 2005) as well
as their stability in storage.
The Colombian isolate of M. rileyi codified as Nm06 was selected
for development because it caused 95 % mortality among third
instar S. frugiperda (Bosa et al. 2004). Subsequently a solid
fermentation system for mass production of conidia was developed
(Arriero, 2002; Caro et al. 2005) and more recently, conidia were
formulated as an emulsifiable concentrate (EC) (Za
rate et al. 2010).
To continue developing this biopesticide, stability under storage
conditions and insecticidal activity of formulated conidia should be
determined.
Despite the increasing sales and use of biopesticides, there are
still limitations that restrict more widespread uptake, such as
delayed mortality, difficulties of production, product cost, lack of
appropriate formulations, and reputation based on previous poor
performance of biopesticides (Glare et al. 2016). The principal re-
quirements for commercial registration of bioproducts are the
microorganism identity, product concentration, shelf life, recom-
mended dose, and field efficacy. It is particularly important to
establish an extended shelf life (period of survival of the microbe in
the product) as this underpins the technical and economic feasi-
bility of each product. However, stability under storage conditions
is influenced by many factors, including genotype of the microor-
ganism used as the active ingredient, formulation and abiotic fac-
tors such as temperature, relative humidity and packaging (Santos
et al. 2012).
The objectives of this work were to characterize the M. rileyi
biopesticide formulated as an emulsifiable concentrate and deter-
mine the microbiological, physico-chemical and biological param-
eters affecting stability. These parameters were evaluated during a
12 m storage period. Finally, the insecticidal activity and lethal
concentrations of the biopesticide were also determined under
glasshouse conditions, as a first step to establish future experi-
ments for determination of field rates and recommendations for
field application.
2. Material and methods
2.1. Microorganism and culture medium
M. rileyi isolate codified as Nm06 (CORPOI-
CA_BGMICB_Nm06_2001) is an accession of the Germoplasm Bank
of Microorganisms with Biological Control Interest of Corpoica,

” Research Center (Mosquera, Colombia). This
located at “Tibaitata
fungus was isolated from a natural infected larva of S. frugiperda
collected in the field in a maize crop at the department of Meta
(Colombia). The fungus was recovered from the collection, where it
is preserved frozen (70
C) in a cryopreserving solution (glycerol
15 %), by plating onto malt and yeast extract agar (YM) plates and
grown by incubation at 24
C.
2.2. Production and formulation of M. rileyi Nm06
Conidia production was carried out by following the method-
ology described by Caro et al. (2005). Metallic trays containing
150 g of rice and 150 mL of protein hydrolysate (8 %) were sterilized
and inoculated with 20 mL of conidia suspension adjusted to a
1  106 conidia/mL. Inoculated trays were incubated at 25
C for
10 d while the fungus grew and sporulated reaching a yield of
3.9  109 conidia/g of dry substrate. Dry conidia were harvested
directly by brushing the sporulated substrate and the spores
powder obtained was mixed with a liquid carrier containing
vegetable oil and emulsifying agents to form an emulsifiable
concentrate EC with a final concentration of 1.5  109 conidia/mL.
2.3. Insect rearing
S. frugiperda larvae used for bioassays were obtained from a
colony established at the Corpoica's Research Center “Tibaitata
”
Bogota (Colombia). This colony was established with larvae
collected in a maize crop located at the municipality of Cerete


rdoba, Colombia). Larvae were fed with artificial diet based on
(Co
wheat germ (Greene et al. 1976) and were maintained in controlled
conditions at 28
C and 60 % relative humidity (RH), with a natural
photoperiod of 12 h.
2.4. Storage stability study for the EC formulation based on M. rileyi
Nm06 conidia
Experimental design was completely randomized with repeated
measures in time and three replicates per treatment. The experi-
mental units consisted of 15 mL capacity glass vials containing
10 mL of the formulation EC. Vials were tightly closed with a rubber
stopper. One group of 15 vials was stored at 8
C ± 2
C and another
group at 18
C ± 2
C for 12 m. Before storage (time zero) and every
three months, three vials of each treatment were removed from the
incubators and microbiological variables (germination and content
of contaminants) were evaluated as well as the pH and the insec-
ticidal activity.
2.4.1. Microbiological properties
2.4.1.1. Germination. Samples of 1 mL were removed from each
vial, diluted in 9.0 mL of 0.1 % Tween 80 solution (101) and mixed
vigorously to form a homogeneous emulsion. Serial dilutions were
then prepared (102 and 103). The final dilutions were inoculated
on three Petri dishes with Malt extract and Yeast Extract Agar (YM)
supplemented with Benomyl (0.00008 %). Inoculated plates were
incubated at 25
C and, after 48 h, a square of agar of approximately
1 cm2 was removed and placed on a glass slide. A drop of Lacto-
phenol blue was added to the agar surface, allowed to distribute for
30 s and covered with a glass cover slide. Percentage germination
was then determined by assessing growth of hyphae from 100
spores for each plate at 40 magnification. Each plate served as a
replicate with three replications per treatment (Ekesi et al. 1999).
2.4.1.2. Contaminant content. Content of contaminants was esti-
mated using the methodology described by Quiroga et al. (2011),
with some modifications. All previously prepared dilutions were
plated onto Petri dishes with Nutritive Agar (NA) for estimation of
bacteria and onto Potato Dextrose Agar (PDA) supplemented with
antibiotic (0.1 %) for estimation of molds. Plates with NA were
incubated at 28
C for 24 h and plates with PDA were incubated at
25
C for seven days. Dilutions 102 and 103 were also inoculated
on plates of YM agar with antibiotic (0.1 %) incubated at 28
C for
48 h to determine the content of contaminant yeasts. Colony
forming units (CFU) were counted and results were expressed as
CFU/mL. Contaminant content (CC) was calculated applying the
following formula:
 E.P. Grijalba et al. / Fungal Biology 122 (2018) 1069e1076
CCðCFU=mLÞ ¼ B þ M þ Y
where B ¼ bacteria content (CFU/mL), M ¼ mold content (CFU/mL),
and Y ¼ yeast content (CFU/mL).
2.4.2. Physical and chemical properties
Before storage and after 12 m, three 1 mL samples per treatment
were mixed with 9 mL of distilled and deionized water and shaken
vigorously to emulsify. Subsequently, the pH was measured with a
potentiometer (Consort®).
2.4.3. Biological properties
2.4.3.1. Insecticidal activity. To evaluate the insecticidal activity of
the EC formulation, a bioassay under laboratory conditions was
carried out following the methodology described by Caro et al.
(2005). The experimental design included three treatments: the
control where larvae were inoculated with sterile water (T1) and
treatments T2 and T3 where larvae were inoculated with recon-
stituted samples of M. rileyi formulation EC stored at 8
C and 18
C,
respectively. Treatments T2 and T3 were diluted in distilled water
and adjusted to 1  107 conidia/mL. Leaves of castor-oil plants
(Ricinus communis) previously washed and disinfected with sodium
hypochlorite (0.5 %) were sprayed on both sides with 2 mL of the
corresponding treatment and allowed to dry before being cut into
square pieces of approximately 2 cm2. Three experimental units
(replicates) were established per treatment. Each replicate con-
sisted of 10 plastic containers (473.2 mL capacity) each containing
one fragment of inoculated leaf and one second instar larva of
S. frugiperda. The containers were incubated for 14 d at 25
C, with
an un-inoculated fresh piece of leaf added every four days. After
14 d from inoculation, larvae were examined and classified as dead
or alive and symptoms of M. rileyi infection in all dead individuals
(rigid body, mummified with white mycelia or light green and
powdery sporulation) were noted.
Mortality results in the fungal treatments were corrected for the
mortality in the control treatment using the Schneider e Orelli
formula to estimate efficacy (Zar, 1999):
Efficacyð%Þ ¼ ð%T  %CÞ=ð100  %CÞ  100
where %T corresponds with the mortality in the fungal treatment
and %C corresponds with the mortality in the control treatment.
2.5. Mathematical estimation of shelf life for the EC formulation
based on M. rileyi Nm06
Conidia insecticidal activity expressed as efficacy was correlated
with the storage time using different mathematical models to
determine the formulation shelf life. Shelf life has been defined as
the period over which the product efficacy is higher than or equal to
80 % (Go
mez et al., 2013).
Evaluated mathematical models were (Prandini et al. 2009):
Zero order: y ¼ kt
First order: Ln (y) ¼ kt
Second order: 1/y ¼ kt
Higuchi: y¼k√t
Kosmeyer-Peppas: y ¼ ktn
Polynomial 25: y ¼ k þ kt þ kt2 þ kt3þ...ktn
where y corresponds to variables germination or insecticidal ac-
tivity, t corresponds with storage time (months) and k corresponds
with degradation constant.
1071
2.6. Biopesticide evaluation under glasshouse conditions
2.6.1. Lethal concentrations (LC50 and LC90)
The experiment was organized as a randomized complete block
design of six treatments with three replicates per treatment. Each
experimental unit consisted of a furrow of 3 m length containing 20
maize (Zea mays) plants. Treatments T1 to T5 were the fungal
formulation adjusted to five different conidial concentrations: 104,
105, 106, 107 and 108 conidia/mL, respectively, and T6 was the con-
trol. Twenty days after plants emerged, treatments were applied
using a manual sprayer to atomize and apply the fungal suspension
at a rate of 2 mL per plant. After one hour, when the plants were dry,
two second instar larvae of S. frugiperda were added to each plant.
Two days after the infestation, all larvae were collected after
destructive harvesting of the plants. Larvae were placed individu-
ally in plastic 473 mL containers containing one castor-oil-plant leaf
fragment as a food source. Containers were maintained at the lab-
oratory (25±2
C, 60 % HR) and mortality was assessed daily. The
results were analyzed using Probit analysis with the software POLO-
PLUS 2.0, (LeOra software) in order to establish the 50 % and 90 %
lethal concentrations (LC50 and LC90).
2.7. Recent damage reduction in maize plants
This experiment was established following the methodology
previously described to determine the lethal concentrations.
Experimental design was randomized complete block with two
treatments and three replicates per treatment. Treatment T1 con-
sisted of two applications of the fungal biopesticide, 20 and 27 d
after plant emergence, at the rate of 1.3  1012 conidia/ha (corre-
sponding to the LC93 determined in the laboratory). Treatment T2
was the control without application. The plants were assessed and
damage was evaluated 2, 4, 6, 8, 11, 13 and 15 d after the first
application (daa). Recent damage was defined as the presence of
areas with scraping, holes and fresh frass on the newest leaf (Lasa

mez et al. 2013).
et al. 2007; Go
2.8. Statistical analysis
Results were analyzed for normality and variance equality using
Shapiro Wilk and Bartlett tests with 95 % confidence. Parametric
data from the variables germination, contaminant content and pH
were analyzed by one-way ANOVA and the mean values were
compared and differentiated using Tukey test (95 %) or Least Sig-
nificant Difference - LSD (95 %). Mean values of insecticidal activity
were differentiated by the non-parametric KruskaleWallis test
(95 %). All analyzes were carried out with the software Statistix
version 8.1.
3. Results and discussion
3.1. Storage stability of the EC formulation based on M. rileyi
conidia
Changes in the microbiological, physico-chemical and biological
properties of the biopesticide after storage in different conditions
will be described in this section.
3.1.1. Microbiological properties
3.1.1.1. Germination. Conidia formulated as EC and stored at 8
C
was very stable, maintaining germination higher than 85 %, with no
significant reduction in viability during the 12 m of storage
(F ¼ 2.14, df ¼ 6, 14, P ¼ 0.1129) (Fig. 1). These germination values
are considered adequate for biopesticides based on entomopatho-
genic fungi, in accordance with international standards (Jenkins
1072 E.P. Grijalba et al. / Fungal Biology 122 (2018) 1069e1076
and Grzywacz, 2000). Conidia in the EC stored at 18
C maintained a
high germination rate until the sixth month of storage (78 %) and
then showed a significant drop in viability (F ¼ 16.1, df ¼ 6, 14,
P ¼ 0.0000).
These results show the effect of storage temperature on M. rileyi
germination, with a greater drop in conidia germination when
temperature is increased. This behavior is related to greater active
metabolism at high temperatures which produces toxic metabo-
lites that affect conidial viability and cause cell death (Marin ~ o et al.
2004). Cellular metabolism decreases under cold storage condi-
tions, which prevents the production of toxic metabolites and
depletion of nutrients, thus extending the shelf life of the micro-
organisms (Sabaratnam and Traquair, 2002).
The effect of storage temperature observed in this study is
similar to that reported by different authors who have evaluated
the stability of different formulations based on entomopathogenic
fungi which have been stored at different temperatures. In most
cases, viability losses are directly proportional to the temperature,
leading to the recommendation for storage under refrigeration
(Hedgecock et al. 1995; Alves et al. 2002; Marin ~ o et al. 2004; Z
arate
et al. 2010; Kim et al. 2011).
However, oil based formulations have shown a positive effect by
improving the survival of some microorganisms in storage condi-
tions. For example, Alves et al. (2002) evaluated the stability of
Metarhizium anisopliae conidia in seven oil based formulations,
which were stored for 10 months at 10
C. All formulations main-
tained viability of the conidia at levels higher than 90 % for the
period of the experiment. Similarly, Bhanu Prakash et al. (2015)
assessed the stability of seven oil based M. anisopliae conidial for-
mulations which were stored at 4
C for 6 m. These authors found
that formulations incorporating sesame oil and sunflower oil were
the most stable, but germination in all formulations was signifi-
cantly reduced to <60 %. In the present study, formulated conidia in
the EC remained viable after storage at 8
C for 12 m, time
considered adequate for marketing of this type of bioproducts
(Ignoffo, 1981).
When storage temperature was increased to 18
C, the EC
maintained conidia germination at an acceptable level (>85 %) only
for 2.5 m, less time than that obtained under cold conditions.
Germination percentage was reduced by 17 % and 61 % after 6 and
9 m of storage respectively, a similar reduction to that obtained by
other formulations at this temperature. For example, Batta (2003)
developed an inverted emulsion to formulate M. anisopliae con-
idia and evaluated its stability at 20
C, finding that germination
Fig. 1. Germination of M. rileyi conidia formulated as emulsifiable concentrate and
stored at 8
C and 18
C. Values with the same letter did not present significant dif-
ferences according to the Tukey Test (95 %). The statistical analysis was performed
separately for each temperature.
was significantly reduced from 88 % to 44 % after 4.6 m of storage.
Additionally, Za
rate et al. (2010) evaluated the stability of three
emulsifiable oil based formulations of Nomuraea rileyi (now
M. rileyi) conidia, stored for two months at 10
C, 20
C and 30
C
and detected a reduction in germination directly related to time of
storage and temperature. Germination percentage was reduced to
zero after two months of storage at 30
C for the three formulations.
At 20
C, reduction in germination ranged between 20 and 50 % but
there was no reduction in germination when the formulations were
stored at 10
C.
3.1.1.2. Contaminants. There was a significant reduction of con-
taminatory microorganisms in the EC formulation after storage at
8
C and 18
C for 12 m. The density of microorganisms other than
M. rileyi (bacteria, molds and yeasts) was significantly reduced from
1.83  104 to 7.8  103 CFU/mL (F ¼ 71.7, df ¼ 1, 4, P ¼ 0.0011) at
8
C, and from 1.83  104 to 4.66  103 CFU/mL (F ¼ 90.8, df ¼ 1, 4,
P ¼ 0.0007) at 18
C. These results suggest that this oil based
formulation limits the growth of and may cause mortality of con-
taminatory microorganisms that could have a negative effect on the
active ingredient (M. rileyi conidia) during storage, interfere with its
insecticidal activity, or be a risk for the health of workers who apply
the product or even a risk to the public who consume products
treated with the fungus (Ravensberg, 2011). Reduction of con-
taminatory microbes during storage is a property of EC formula-
tions, due to their low moisture content and limited oxygen
availability. The low or near zero water content in the formulation
inhibits the growth of contaminatory microorganisms which
require water to assimilate the nutrients and maintain an active
metabolism (Tortora et al. 2007). Most bacterial contaminants
multiply with values of water activity around 0.99 and die when
this value declines (Varo
and Segura, 2009). In addition, the amount
of dissolved oxygen in an oil based formulation will be very low
creating stress for aerobic microbial contaminants that require
oxygen to carry out their metabolic processes and growth (Barreiro
and Sandoval, 2006).
Generally, the acceptance limit for contaminant microbial con-
tent or product purity in biopesticides is established by the pro-
ducer or based on the specific requirements for registration in each
country. However, a standard maximum level 0.1 % of the active
ingredient concentration is recognized (Ravensberg, 2011). In the
present work the EC formulation stored at either temperature,
maintained a contaminatory microbe concentration below this
level in the M. rileyi formulation which maintained microbiological
quality for 12 m of storage.
3.1.2. Physical and chemical properties
3.1.2.1. pH. The pH of the EC formulation stored for 12 months at
8
C remained unchanged after diluting the product with water.
However, when the formulation was stored at 18
C, the pH
significantly increased (Table 1) (F ¼ 31.4, df ¼ 3, 8, P ¼ 0.0001),
suggesting that this parameter was affected by the storage tem-
perature. However, all the pH values obtained during the storage
stability study ranged between 7.0 and 7.4 and no changes were
Table 1
pH of M. rileyi conidia formulated as emulsifiable concentrate and stored at 8
C and
18
C. Values with the same letter did not present significant differences according
to the LDS Test (95 %).
Time (months) Storage temperature
8
C 18
C
0 7.06 ± 0.021 (b) 7.06 ± 0.021 (b)
12 7.00 ± 0.058 (b) 7.37 ± 0.08 (a)
 E.P. Grijalba et al. / Fungal Biology 122 (2018) 1069e1076
observed in the oil based carrier that could suggest chemical
degradation capable of changing the pH of the emulsion (Formiga
et al. 2007). It is important to note that studied fungal isolate is
tolerant to a wide pH range from 4 to 8 (Aguirre, 2006), which
suggests that the viability of the conidia would not be affected at
the time of using the product if it is mixed with good quality water.
3.1.3. Biological properties
3.1.3.1. Insecticidal activity. Larvae with signs of fungal infection
(rigid larvae, cottony white mycelia, pale green sporulation)
(Ce
spedes et al. 2008) were found in all treatments following
application of the EC biopesticide. The EC stored at 8
C maintained
insecticidal activity against second instar larvae for 12 m, with a
final efficacy of 96.43 % (F ¼ 0.03, df ¼ 4, 10, P ¼ 0.9973). Insecticidal
activity of the biopesticide was significantly reduced to 80 %
(F ¼ 9.11, df ¼ 4, 10, P ¼ 0.0023) after 9 m of storage at 18
C and
then maintained stable until 12 m (79.05 %). The biological stability
of the formulation is possibly related to the liquid medium used to
suspend the conidia. Vega-Aquino et al. (2010) found insecticidal
activity of M. rileyi against S. frugiperda varied between 90 % and
100 %, depending on the oil used (vegetal or mineral). Bateman
et al. (1993) considered that oil based formulations improved
persistence and insecticidal activity of entomopathogenic fungi
(McClatchie et al. 1994; Vimala Devi and Prasad, 1996; Ummidi and
Vadlamani, 2014; Barreto et al. 2016) concluded that, for fungi such
as Beauveria spp. and Metarhizium spp., the lipophilic character of
this delivery system favors conidia adhesion to the insect cuticle
and protects the microorganism from desiccation, temperature and
ultraviolet radiation.
3.2. Mathematical estimation of shelf life for the EC formulation
based on M. rileyi conidia
In this work, efficacy of insecticidal activity was correlated with
storage time using different mathematical models and the best fit
was achieved with a polynomial model (Table 2). The shelf life was
defined as the time for efficacy of this bioinsecticide to reach a value
of 80 %, the threshold established based on the value reported by
Jenkins and Grzywacz (2000) as the minimum desirable biological
activity for a bioinsecticide evaluated under controlled conditions.
Shelf-lives were estimated using the generated equation and are
presented in Table 3. In general, shelf life was inversely propor-
tional to temperature. The mathematical model predicted that this
biopesticide would maintain its insecticidal activity at a level equal
or higher than 80 % for 26.2 m when stored 8
C. This estimated
shelf-life is higher than the 18 m considered necessary for the
marketing of fungal biopesticide products (Couch and Ignoffo,
1981). The shelf-life obtained, approximately 2 y in cool condi-
tions, significantly exceeds values reported for other oil-based
formulations with fungi. For example, Bhanu Prakash et al. (2015)
assessed the efficacy of three formulations based on M. anisopliae
against Spodoptera litura (Fabricius, 1775) (Lepidoptera: Noctuidae)
and determined a maximum shelf-life of six months at 4
C, as after
this time efficacy of the formulation was reduced to unacceptable
values between 70 and 73 %.
Table 2
Mathematical models used to correlate biopesticide efficacy vs. storage time and corresponding squared correlation coefficients (R2).
Storage temperature (ºC) Mathematical models
Zero order First order
8±2 0.7575 0.7577
18 ± 2 0.7515 0.7517
1073
The shelf-life estimated for the EC stored at 18
C was 11 m. This
result was confirmed in the bioassay where the biopesticide stored
for 12 m was tested and resulted in 79.05 % efficacy. The shelf-life
obtained was higher than a number of other entomopathogenic
fungal biopesticides which had been stored from 18
C to 20
C
(Table 4), although it is not known whether these reported values
were estimated based on the viability and/or efficacy. One of the
few works estimating the shelf life based on the biopesticide effi-
cacy in the reported by Batta (2004), who evaluated the relation-
ship between shelf-life and efficacy for M. anisopliae formulations
and found that they were stable for four months at 20
C, main-
taining levels of efficacy between 70 and 80 % against larvae of the
rice weevil, Sitophilus oryzae.
Even bioassays showed that insecticidal activity of the EC
formulation stored at 18
C remained, equal or greater than the
minimum limit of 80 % for nine months, the germination dropped
below 85 % since the third month of storage (78 %), being signif-
icantly reduced after ninth month (68 %). If the minimum value of
85 % germination recommended by Jenkins and Grzywacz (2000)
is used to establish the shelf-life, the product would expire after
3 m of storage at room temperature. However, the bioassays
showed that although the product reduced the conidia germina-
tion (evaluated after 48 h of incubation), the efficacy against
S. frugiperda larvae was stable and equal or higher than the
acceptance limit for around 11 m. This result suggests that stored
formulated product contained viable conidia able to develop the
insecticidal activity against the insect pest, but the storage con-
ditions possibly induced a latency state slowing the germination
process.
The shelf life estimated in the present work for the EC formu-
lation based on M. rileyi and stored at 18
C needs to be validated
with a real-time study. However, results explained above have
demonstrated the potential of this formulation which seems to
overcome the high sensitivity of M. rileyi conidia to both biotic and
abiotic conditions in comparison with other species of entomopa-
thogenic fungi (Edelstein et al. 2004). The sensitivity of this fungus
to storage conditions, added to the difficulties of production
(Arriero, 2002; Caro et al. 2005), could be related to the absence of
registered bioproducts based on this fungal species in Colombia
(ICA, 2017) and United States (EPA, 2017).
3.3. Determination of lethal concentrations (LC50 and LC90) under
glasshouse conditions
All maize plants had typical signs of S. frugiperda larval damage
following infestation. The youngest leaves close to the bud or
growing point had the characteristic signs of damage caused by first
instars larvae (leaf scraping and translucent spots produced by
larvae damaging the epidermis of the leaves) (García et al. 2002).
Results of larvae mortality were used to calculate the lethal
concentrations of the evaluated formulation (EC) by Probit analysis.
The P values were higher than 0.05 (Table 5), accepting the hy-
pothesis of a linear correlation between dose and larvae mortality;
in addition, the low values of Chi-square (c2) suggested homoge-
neity among the data, which also indicated that the experimental
distribution was adjusted to the theoretical (Zar, 1999).
Second order Higuchi Korsemeyer Peppas Polynomial
0.7578 0.4083 0.7475 0.8228
0.7499 0.5536 0.7419 0.8122
1074 E.P. Grijalba et al. / Fungal Biology 122 (2018) 1069e1076

Table 3
Polinomyal model equation and shelf-life estimation, defined as the moment which efficacy reaches 80 %.

Storage temperature (
C) Model equation Shelf life (months)

8±2 Y ¼ 0.0292  2e0.0029x þ 100.19 26.20

18 ± 2 Y ¼ 0.1673  2e0.083x þ 101.18 11.00

Table 4
Shelf-lives for some commercial biopesticides base on entomopathogenic fungi.

Company Country Formulation Name Microorganism Concentration Shelf life/ References

(conidia/g or Conditions
mL)

Natural Control Colombia Wettable Vercani WP Lecanicillium 1  108 6 m/Room https://naturalcontrol.com.co/wp-content/uploads/2015/10/ficha-

Ltda powder lecanii temperature tecnica-vercani.pdf
(Colombia)
Soluciones Colombia Wettable Bovetropico
Beauveria 1  10⁹ 12 m/Room http://smdeltropico.com/fichas-tecnicas/FICHA_TECNICA_
Microbianas powder WP bassiana temperature BOVETROPICO.pdf
del Tropico Wettable Paecilotropico
Paecilomyces 1  10⁹ 12 m/Room http://smdeltropico.com/fichas-tecnicas/FICHA_TECNICA_
ltda powder WP lilacinus temperature PAECILOTROPICO.pdf
BioSafe Canada Wettable BioCeres WP
Beauveria 1  1010 12 m/4
C http://anatisbioprotection.com/en/news/bioceres-bio-insecticide.
Systems powder bassiana, strain 6 m/21
C html
ANT-03
Belchim Crop UK Emulsifiable Naturalis L Beauveria 2  107 12 m/Room http://belchim.co.uk/pdf/Label/NATURALIS-L_Label.pdf
Protection concentrate bassiana ATCC temperature
Limited 74040
BioWorks Inc. USA Wettable Mycotrol WPO Beauveria 4  1012 13 m/Room https://www.bioworksinc.com/products/shared/product-shelf-
powder bassiana strain temperature life.pdf
GHA
Wettable Botanigard B. bassiana 4  1010 14 m/Room https://www.bioworksinc.com/products/shared/product-shelf-
powder 22WP GHA temperature life.pdf
Emulsifiable BotaniGard ES B. bassiana 5  1010 18 m/Room https://www.bioworksinc.com/products/shared/product-shelf-
concentrate GHA temperature life.pdf
Emulsifiable Mycotrol ESO Beauveria 2  1010 18 m/Room https://www.bioworksinc.com/products/shared/product-shelf-
concentrate bassiana temperature life.pdf
Certis USA Dispersible PFR-97 Isaria 1  109 12 m/Cool http://www.kellysolutions.com/erenewals/documentsubmit/
granules fumosorosea conditions KellyData%5CND%5Cpesticide%5CMSDS%5C70051%5C70051-19%
Apopka Strain 3 m/Room 5C70051-19_PFR___97_20__WDG_12_20_2011_3_44_00_PM.pdf
97 temperature
BAST Africa Emulsifiable Green Muscle Metarhizium 2  1010 36 m/4
C http://www.fao.org/ag/locusts/oldsite/PDFs/meetings/MycopestE.
concentrate EC anisopliae 24 m/10 pdf
e20
C:
12 m/30
C:
1 m/40
C:
Koppert Brasil Wettable Metarril WP Metarhizium 5  108 1 m/24 http://koppert.com.br/produtos/metarril/
powder anisopliae e28
C
12 m/4
C
Brasil Wettable Boveril WP® Beauveria 2  109 1 m/24 http://koppert.com.br/assets/fichas/boveril.pdf
powder bassiana e26
C
6 m/0e4
C
12 m/4
C

Larvae mortality was related to conidial concentration in the
sprayed suspension, which produced a median (50 %) lethal
concentration (LC50) of 1.17  104 conidia/mL and a 90 % lethal
concentration (LC90) of 4.03  106 conidia/mL (Table 5). These
concentrations were similar to those reported previously by
Santos (2014), who used the same isolation as unformulated
conidia suspensions and obtained a LC50 of 1.3  104 conidia/mL
and a LC90 of 1.1  106 conidia/mL. The similarity of these results
suggests that formulation of the fungus as an emulsifiable
concentrate did not affect the insecticidal activity. The values
obtained were also similar to those described for other
Colombian isolates of the same fungal species evaluated on
S. frugiperda larvae, with LC50s ranging from 9.8  103 to
1.2  104 conidia/mL (Bosa et al. 2004).
The isolate M. rileyi Nm06, active ingredient in the EC formu-
lation evaluated in the present work, appeared to be more patho-
genic than other isolates of this fungal species. For example, using
M. rileyi Vimala-Devi (1994) produced an LC50 of 9.3  106 conidia/
mL against S. litura second instar larvae, whereas Rajan and
Muthukrishnan (2009) using M. rileyi, produced an LC50 of
2.8  107 conidia/mL against larvae of Cnaphalocrocis medinalis
(Guene
e, 1854) (Lepidoptera: Crambidae).

Table 5
Lethal concentrations of M. rileyi Nm06 formulated as an emulsifiable concentrate and evaluated over second instar larvae of S. frugiperda under glasshouse conditions.

Biopesticide LC50 (Con/mL) Confidence limits LC50 (Con/mL) LC90 (Con/mL) P Heterogeneity

Lower Upper c2 df
4 2 4 6
EC M. rileyi Nm06 1.17  10 3.1  10 5.58  10 4.03  10 0.764 1.169 3
 E.P. Grijalba et al. / Fungal Biology 122 (2018) 1069e1076
The pathogenicity of formulated M. rileyi Nm06, expressed as
LC50, was higher than that of other entomopathogenic fungi
against S. frugiperda larvae. Beauveria bassiana isolate Bb18,
M. anisopliae isolate Ma91, and a commercial product based on
B. bassiana produced LC50 values between 8.18  106 and
2.53  109 conidia/mL (García et al. 2011; Gonza
lez-Maldonado
et al. 2015). These results suggest that the evaluated isolate
Nm06 is highly virulent and has potential to be used under field
conditions. A lower LC50 allows use of fewer conidia or a smaller
quantity of formulated product to obtain efficient control of the
pest, which minimizes production and application costs. One of
the key issues to ensure the successful adoption of biopesticides
is its competitiveness in terms of costs when compared to
chemical products (Marrone, 2014), and this parameter is related
to the recommended application dose and determined by the
virulence of the selected active ingredient (Gupta and Dikshit,
2010; Go
mez et al. 2013).
3.4. Reduction of recent larval damage by the application of the
formulated (EC) M. rileyi Nm06 under glasshouse conditions
The percentage of recent damage caused by the larvae on maize
plants was estimated to determine the efficacy of applying the EC
formulation based on M. rileyi Nm06 for control of S. frugiperda
larvae under glasshouse conditions. For the first week of evaluation
(days 2, 4, 6 and 8), the damage level oscillated between 93.3 % and
100 %, with no significant difference between treatments and the
control (for day 2: F ¼ 1.00, df ¼ 1, 4, P ¼ 0.3739 and for days 4, 6 and
8: F ¼ 4.00, df ¼ 1, 4, P ¼ 0.1161) (Fig. 2).
At day 11, a significant decrease (F ¼ 1339, df ¼ 1, 4, P < 0.0001)
was observed in the percentage of damaged plants treated with the
EC in comparison with the control plants, with values of 50 % and
100 % (Fig. 2), respectively. By days 13 and 15 from treatment,
damage continued to drop to levels of 23.3 % for plants treated with
the EC, while the control treatment had more than 80 % of plants
damaged. The efficacy of formulated M. rileyi Nm06 conidia was
confirmed by the recovery of sporulated cadavers. These results
were similar to those obtained by Go
mez et al. (2013), who main-
tained damage caused by S. frugiperda close to 15 % by the appli-
cation of a biopesticide based on a Colombian isolate of S. frugiperda
nucleopolyhedrovirus (SfMNPV).
The percentage of recent damage obtained in the treated plants
after two biopesticide applications and after the day 11 of evalua-
tion was less than the threshold established for this pest's economic
impact (35 %) (Ferna
ndez, 2002), which confirms the high potential
of this formulation to be used in an integrated pest management
program to reduce the losses caused by the S. frugiperda.
Fig. 2. Reduction of larval recent damage by the application of the formulated (EC)
M. rileyi Nm06 under glasshouse conditions. The statistical analysis was performed
separately for each evaluation time. Treatments with the same letter did not present
significant differences (95 %).
1075
4. Conclusions
An oil-based EC formulation stabilized M. rileyi Nm06 conidia
for survival in storage and a combined analysis of all quality pa-
rameters, allowed to stablish shelf-lives of six and 12 months at
18
C and 8
C, respectively. The formulation maintains physical-
chemical properties, a low contaminant content, viability and
insecticidal activity, at adequate levels to ensure a satisfactory
performance against pests. The resultant biopesticide also
demonstrated potential to control fall armyworm in maize. This
research provides the background needed to continue with the
next steps of development by standardizing and scaling up the
production and formulation processes and determining effective
application rates and frequencies of application under field
conditions.
Conflicts of interest
The authors declare that they have no conflict of interest.
Acknowledgments
The authors express their gratitude to the Colombian Corpora-
tion for Agricultural Research (Corpoica), the Ministry of Agricul-
ture and Rural Development, the Administrative Department of
Science and Technology (Colciencias) and the National Federation
of Cereal growers (Fenalce), by funding the project “Scaling up and
evaluation of shelf life and the efficacy of two insecticides for
Spodoptera frugiperda management in the productive system
of maize", within this work was developed. We are also very
grateful to Dr. Trevor Jackson for his helpful suggestions and
recommendations.
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