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Entomopathogenic fungi as biocontrol agents

  • 29.09
  • Kunming Institute of Botany, CAS Kunming Yunnan China

Abstract and Figures

An attractive alternative method to chemical pesticides is the microbial biocontrol (MBCAs) agents. They are the natural enemies devastating the pest population with no hazard effects on human health and the environment. Entomopathogenic fungi has an important position among all the biocontrol agents because of its route of pathogenicity, broad host rang and its ability to control both sap sucking pests such as mosquitoes and aphids as well as pests with chewing mouthparts, yet they only cover a small percentage of the total insecticide market. Improvements are needed to fulfill the requirements for high market share. Entomopathogenic fungi differ from other microorganisms in their infection process: they directly breach the cuticle to enter the insect hemocoel, while other microorganisms enter by ingestion through mouth and then cause disease. Insect cuticle is mainly composed of chitin and protein surrounded by wax, lipid layer or fatty acids. Fungal pathogenesis mainly starts with the secretion of cuticle degrading enzymes. Some important and well known cuticle degrading enzymes are chitinase, protease and lipase which can degrade chitin, protein and lipid of the cuticle, respectively. In this review we collected literatures from different sources and we arranged them in a such a way to better to understand the process of insect pathogenicity of entomopathogenic fungi and to find ways to improve the virulence of wild strain fungi to shorten the killing time of the pest and to commercialize the entomopathogenic fungi. In this way, the market share of the fungal entomopathogenic fungi will increase and a decrease in the usage of synthetic chemical pesticides will automatically follow.
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Molecular Plant Breeding 2012, Vol.3, No.7, 63-79
Research Report Open Access
Entomopathogenic Fungi as Microbial Biocontrol Agent
Sehroon Khan , Lihua Guo , Yushanjiang Maimaiti , Mahmut Mijit , Dewen Qiu
Key Laboratory of Integrated Pest Management in Crops, Ministry of Agriculture, Institute of Plant Protection, Chinese Academy of Agricultural Sciences
(CAAS), Beijing, P.R. China
Corresponding authors email:; Authors
Molecular Plant Breeding, 2012, Vol.3, No.7 doi: 10.5376/mpb.2012.03.0007
Received: 25 Apr., 2012
Accepted: 11 May, 2012
Published: 20 May, 2012
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:
Khan et al., 2012, Entomopathogenic Fungi as Microbial Biocontrol Agent, Molecular Plant Breeding, Vol.3, No.7 63-79 (doi: 10.5376/mpb.2012.03.0007)
Abstract An attractive alternative method to chemical pesticides is the microbial biocontrol (MBCAs) agents. They are the natural
enemies devastating the pest population with no hazard effects on human health and the environment. Entomopathogenic fungi has
an important position among all the biocontrol agents because of its route of pathogenicity, broad host rang and its ability to control
both sap sucking pests such as mosquitoes and aphids as well as pests with chewing mouthparts, yet they only cover a small
percentage of the total insecticide market. Improvements are needed to fulfill the requirements for high market share.
Entomopathogenic fungi differ from other microorganisms in their infection process: they directly breach the cuticle to enter the
insect hemocoel, while other microorganisms enter by ingestion through mouth and then cause disease. Insect cuticle is mainly
composed of chitin and protein surrounded by wax, lipid layer or fatty acids. Fungal pathogenesis mainly starts with the secretion of
cuticle degrading enzymes. Some important and well known cuticle degrading enzymes are chitinase, protease and lipase which can
degrade chitin, protein and lipid of the cuticle, respectively. In this review we collected literatures from different sources and we
arranged them in a such a way to better to understand the process of insect pathogenicity of entomopathogenic fungi and to find ways
to improve the virulence of wild strain fungi to shorten the killing time of the pest and to commercialize the entomopathogenic fungi.
In this way, the market share of the fungal entomopathogenic fungi will increase and a decrease in the usage of synthetic chemical
pesticides will automatically follow.
Keywords Entomopathogenic fungi; Beauveria bassiana; Biocontrol agents; Enzymes; Pathogenesis
Integrated Pest Management (IPM) involves ins-
pection, identification and treatment of pests. The
treatment (when required) is carried out after inspection
and identification with an environmentally safe and
pest specific pesticide with limited persistence. Therefore
biological pest management is considered as an
important part of IPM. Biological control is an
important part of integrated pest management (IPM).
According to Oerke and Dehne (2004), insect pests
are responsible for an estimated 42% of all losses in
crop production. Extensive use of synthetic chemical
pesticides, insecticide resistance to chemical pesticides
(Ffrench-Constant et al., 2004), the resulting environ-
mental pollution, adverse effects on human health and
other organisms and the demand for reduced chemical
inputs in agriculture have provided an impetus to the
development of alternative forms of pest control
(Wilson and Tisdall, 2001). An attractive alternative
method to chemical pesticides is biocontrol (Nicholson,
2007) and the microbial biocontrol (MBCAs) agents
as the natural enemies of the pest population devastate
pests with no hazard effects on human health and
environment. As the microbial biocontrol agents have
complex mode of action, it’s very difficult for a pest to
develop resistance against MBCAs. The present
MBCAs are viruses, bacteria, nematodes, and fungi
and they are used throughout the world with great
advantage and success. But fungal biocontrol agents
are the most important among all the MBCAs due to
easy delivery, improving formulation, vast number of
pathogenic strains known, easy engineering techniques
and over-expression of endogenous proteins or exo-
genous toxins (St. Leger et al., 1996; Butt et al., 2001;
Wang and St. Leger, 2007; Federici et al., 2008; St.
Leger and Wang, 2010). Similarly, the entomopathogenic
Molecular Plant Breeding 2012, Vol.3, No.7, 63-79
fungi are important among all the biological control
agents due to its broad host range, route of
pathogenicity and its ability to control sap sucking
pests such as mosquitoes and aphids (Butt, 2002; Qazi
and Khachatourians, 2005; Thomas and Read, 2007;
Fan et al., 2007) as well as pests with chewing
mouthparts (Hajek and St. Leger, 1994; de Faria and
Wraight, 2007).
This review will help us understand entomopatho-
genic fungal virulence and present the most recent
improvements and achievements in the relevant field.
This will help us determine how to improve the
virulence of entomopathogenic fungi to shorten the
killing time of pest.
1 Entomopathogenic Fungi (EPF)
Entomopathogenic fungi (EPF) are widely distributed
with both restricted and wide host ranges which have
different biocontrol potentials against arthropods
insects and plant pathogenic fungi. Entomopathogenic
fungi were among the first organisms to be used for
the biological control of pests. More than 700 species
of fungi from around 90 genera are pathogenic to
insects (Khachatourians and Sohail, 2008). Most EPF
species are from the fungal divisions Ascomycota and
Zygomycota. The ascomycete fungi were previously
divided into two groups, the Ascomycota and the
Deuteromycota (Table 1). The Fungi Imperfecti of
Deuteromycota was known for having no sexual stage
was known called as. But later on, cultural and
molecular studies have demonstrated that some of
these ―imperfect fungi‖ (formally class Hyphomycetes
in the Deuteromycota) were in fact anamorphs (asexual
forms) of the Ascomycota within the order Hypocreales,
and Clavicipitaceae family (Fukatzu, 1997; Hodge,
2003; Krasnoff, 1995; Shimazu, 1998). Within the
Zygomycota, the most entomopathogenic species are
in the order Entomophthorales (Roy et al., 2006).
These fungi nutritionally may be saprotrophs that
colonize the rhizosphere and phyllosphere, endophytic
saprotrophs, hemibiotrophic, necrotrophic of plants,
entomopathogenic or mycoparasitic and some of them
have adopted more than one econutritional mode.
2 Life cycle of entomopathogenic fungi
The life cycle of EPF is composed of the spore which
geminates into mycelia and the mycelia in turnproduce
spores (sporemyceliaspore phases). The life cycle
of most entomopathogenic fungi consist of two phases:
a normal mycelia growth phase mostly outside the
host body and a yeast like budding phase mostly in the
hemocoel of host. The yeast-like, dimorphic mode of
growth in Beauveria bassiana was described by Alves
et al (2002); and the production of oblong blastospore-
like propagules in M. flavoviride was described by
Fargues et al (2002). The life cycle of M. anisopliae
under liquid culture conditions has also been
described (Uribe and Khachatourians, 2008).
Table 1 Classification of entomopathogenic fungi (Roy et al., 2006)
Molecular Plant Breeding 2012, Vol.3, No.7, 63-79
Beauveria bassiana in the absence of a specific insect
host grows through an asexual vegetative life cycle
consists of germination, filamentous growth and
formation of sympoduloconidia (Figure 1B). In the
presence of its host insect, Beauveria conidiospores
germinate on the surface of the cuticle of host and
penetrate the insect’s integument through the
germinated hyphal tubes where the fungus alters its
growth morphology to a yeast-like phase and produces
hyphal bodies by budding like growth, which circulate
in the haemolymph (Figure 1A) resulting in the host
death. The fungal growth then reverts back to the
typical hyphal form (the saprotrophic stage). The
ability to convert to the yeast-like phase may be a
prerequisite for pathogenicity.
Figure 1 Entomopathogenic dimorphic mode of growth
Note: A: Yeast-like parasitic phase during infection of suscep-
tible species; B: Saprobic phase shows filamentous hyphae
Entomopathogenic fungi, Verticillium lecanii, Beauveria
bassiana and Metarhizium anisopliae are intensively
studied as common natural enemies and important
epizootics of aphids and other agricultural pests
(Roberts and St. Leger, 2004; Thomas and Read, 2007;
Wang et al., 2004; Milner, 1997; Li and Sheng, 2007).
Beauveria bassiana (Balsamo) Vuilemin is one of the
major fungal entomopathogens infecting nearly 95%
of migratory alate aphids, especially M. persicae
(Chen et al., 2008). Beauveria bassiana and Verticillium
lecanii have dual biological control properties, i.e.
they are natural enemies of pests and also plant
pathogens (Bonnie et al., 2010; Goettel et al., 2008).
Koppert Biological Systems currently use Verticillium
lecanii (Zimm. Viegas) as an insect pathogen which
has been commercialized for controlling aphids (Faria
and Wraight, 2007) and it is effective in controlling
plant pathogenic fungi like powdery mildews (Askary
et al., 1998; Dik et al., 1998; Miller et al., 2004), rusts
(Spencer and Atkey, 1981), green molds (Benhamou
and Brodeur, 2000), Fusarium (Koike et al., 2007),
Verticillium dahliae (Kusunoki et al., 2006) and
Pythium ultimum (Benhamou and Brodeur, 2001).
Beauveria bassiana is reported to limit the growth of
plant pathogenic fungi in vitro, colonize endophytically
in numerous plants and induce systemic resistance
when pathogen infect the plant as well as reducing the
diseases caused by soil born plant pathogens like
Pythium, Rhizoctonia, and Fusarium (Ownley et al.,
2010). Mitosporic fungi are generally environmentally
friendly with negligible or low mammalian toxicity,
have no residual toxicity (Copping, 2004) and are
successful as mycoinsecticides against aphids (Faria
and Wraight, 2007; Milner, 1997; Shah and Pell, 2003).
To date, several mycopesticides have been developed
and used in several countries including the United
Kingdom and the United States (Table 2) (Goettel et
al., 2005; Kiss, 2003). These include Vertalec® based
on Lecanicillium longisporum (Petch) Zare & Gams
(formerly known as Verticillium lecanii (Zimm.) Viegas)
against aphids, Botanigard® based on Beauveria
bassiana (Bals.) Vuill. against aphids and whitefly;
however, they remain a small percentage of the total
insecticides. The major reason for the small market
share of these fungi as mycoinsecticides is its slow
killing rate and an increase in market share is directly
proportional to killing speed (St Leger and Wang, 2009).
Although these products have the advantage of a
restricted host range, this specificity is also one of the
limiting factors for their commercial use (Ownley et
al., 2004). Therefore, a mycopesticide with a wider
host range but with little to no influence on other
natural enemies of pests or beneficial organisms may
have a commercial advantage if it simultaneously
controls various pests and/or plant diseases (Wraight
and Carruthers, 1999).
Insect pathogenic fungi are different in pathogenicity
than bacteria and viruses in that they infect insects by
breaching the host cuticle. The cuticle is composed of
chitin fibrils embedded in a matrix of proteins, lipids,
pigments and N-acylcatecholamines (Richard et al.,
2010). They secrete extracellular enzymes proteases,
chitinases and lipases to degrade the major constituents
Molecular Plant Breeding 2012, Vol.3, No.7, 63-79
Table 2 Entomopathogenic fungi produced commercially and experimentally (Butt et al., 2001; Wraight et al., 2001; Copping, 2004;
Kabaluk and Gazdik, 2005; Zimmermann, 2007; Khachatourians, 1986)
Product/Trade name
Target pests
Mosquito larvae
Hirsutella thompsonii
Citrus rust mite
Spittle bug; Sugarcane frog
Nomuraea rileyi
Lepidopteran larvae
Verticillium lecanii
Aphids; Coffee green bug;
Greenhouse whitefly thrips
Beauveria bassiana
Mite; Coffee green bug
BotaniGard ES; Botani-
Gard 22WP
Laverlam International (formerly
Emerald BioAgriculture)
Mycotrol ES; Myco-
Laverlam International (formerly
Emerald BioAgriculture)
Aphids Spittle bug; Sugar-
Naturalis-L Andermatt
Troy Biosciences Inc
Arysta (formerly NPP, Calliope)
Racer BB
SOM Phytopharma
AMC Chemical/Trichodex
B. brongniartii (_B.
Beauveria Schweizer
Lbu (formerly Eric Schweizer
Greenhouse whitefly thrips
Mosquito larvae
Arysta (formerly NPP, Calliope)
Nitto Denko
Andermatt Biocontrol AG
of the cuticle (i.e. protein, chitin and lipids) and allow
hyphal penetration (Wang et al., 2005; Cho et al.,
2006). Extracellular lipases are also involved in
microbial virulence and play different roles in the
infection process (Stehr et al., 2003). The success-
fulness of infection was directly proportional to
secretion of exoenzymes (Khachatourians, 1996). It is
believed that both mechanical force and enzymatic
action are involved in the penetration of fungus to the
hemocoel of the insect. In this review, we take an
overview of the chitinase, protease and lipase with
their importance in the process of pathogenesis.
Besides exoenzymes, the entomopathogenic fungi are
reported to secrete toxin proteins and metabolites in
vitro and sometime in vivo as well. There are a
number of toxic compounds in the filtrate of entomo-
pathogenic fungi such as small secondary metabolites,
cyclic peptides and macromolecular proteins. Beauveria
bassiana is reported to produce low molecular
weight cyclic peptides and Cyclosporins A and C with
insecticidal properties such as beauvericin, enniatins,
bassianolide, (Roberts, 1981; Vey et al., 2001), Oos-
porein (a red-colored dibenzoquinone with antibiotic
activity against gram-positive bacteria), andcyclic
peptides with immunosuppressive activities. There are
also some insecticidal cyclic peptides like beauvericin
and bassianolide isolated from Beauveria bassiana.
Some of the strains of Beauveria bassiana are reported
to produce high molecular weight compounds with
Molecular Plant Breeding 2012, Vol.3, No.7, 63-79
toxic activity against pest, like hirsutellin A (Enrique
and Alain, 2004). However, the major obstacle
limiting the market share of these fungi as mycoin-
secticides is their slow killing speed and increase in
market share is directly proportional to killing speed
(St Leger and Wang, 2009).
A large number of studies were conducted to
potentiate and improve the virulence of entomopatho-
genic fungi to a greater extent than their individual
activities and thus increasing the market share. The
extensive transcriptomic and genetic study of entomo-
pathogenic fungal infection process revealed that a
number of different genes were involved in the
pathogenicity (Freimoser et al., 2003; Wang et al.,
2005; Cho et al., 2006a, 2006b, 2007) such as
chitinases (Bagga et al., 2004; Fang et al., 2005),
guanine nucleotide-binding proteins and its regulator
(Fang et al., 2007, 2008), adhesin which helps in
attachment of spore (Wang and St. Leger, 2007a), a
perilipin-like protein that regulates appressorium
turgor pressure and differentiation (Wang and St.
Leger, 2007b) and a cell protective coat protein
helping in escaping the pathogen from the host
immunity recognition (Wang and St. Leger, 2006).
Similarly, an increased virulence of the entomopatho-
genic fungi was observed with over-expression of
virulence genes such as subtilisin protease PR1A (St.
Leger et al., 1996), subtilisin protease PII gene
(Ahman et al., 2002), and hybrid chitinase containing
a chitin binding domain (Fan et al., 2007). Wang
and St. Leger (2007c) modified scorpion neurotoxin
peptide, AAIT using Metarhizium’s codon preferences
under the control of mc11 gene promoter for fungal
transformation. The LC50 of the transgenic strain
AaIT-Ma549 was reduced 22-fold against the Manduca
sexta and nine fold against the Aedes aegypti (Wang
and St. Leger, 2007c). AaIT-Ma549 was also tested
against the coleopteran coffee pest, Hypothenemus
hampei, reducing LC50 by 15.7-fold and the average
survival time by 20% (Pava-Ripoll et al., 2008).
3 Beauveria bassiana (Clavicipitaceae)
The entomopathogenic mitosporic ascomycete, Beau-
veria bassiana (Bals.) Vuill. is an important natural
pathogen of insects and it has been developed as a
microbial insecticide for use against many major
arthropod pests in agricultural, urban, forest, livestock
and aquatic environments (Charnley and Collins, 2007;
Faria and Wraight, 2007). It has been developed as a
microbial insecticide for use against many major
pests, including lepidopterans and orthopterans. About
33.9% of the mycoinsecticides is based on B. bassiana,
followed by Metarhizium anisopliae (33.9%), Isaria
fumosorosea (5.8%) and Beauveria brongniartii (4.1%)
(Faria and Wraight, 2007); however, to increase the
market share of B. bassiana, the killing speed which is
the major hindrance limiting their use as mycoinsec-
ticides should be accelerated, (St Leger and Wang,
2009). As natural strains of these fungi often lack
sufficient virulence or tolerance to adversity (Braga et
al., 2001a, 2001b; Ying and Feng, 2004; Rangel et al.,
2005), genetic manipulation is necessary to improve
their efficacy and ecological fitness (Roberts and St.
Leger, 2004; Fang et al., 2005). The importance of
the B. bassiana and B. brongniartii can be briefly
understood from table 3 below.
Research studies are carried out to optimize the
preparation and application of fungal inoculums
(Wraight et al., 2001) and (St Leger and Wang, 2009)
to improve the virulence of mycoinsecticides by
genetic modification. Based on this approach, most
studies on the virulence factors of entomopathogenic
fungi have been directed to elucidate the most relevant
cuticle degrading enzymes (Griesch and Vilcinskas,
1998; Khachatourians, 1996; St Leger et al., 1996),
because their over-expression in engineered strains
results in more fungi that are more deadly toward
insects (Fang et al., 2005; St Leger et al., 1996).
Similarly, over-expression of a chitinase gene (Bbchit1)
was found to enhance the virulence of Beauveria
bassiana against aphids (Myzus persicae) (Fang et
al., 2005). Importantly, different genes involved in
pathogenicity have been characterized from M.
anisopliae or B. bassiana and some of these genes
include cuticle degrading enzymes (Bagga et al., 2004;
Fang et al., 2005), G protein and its regulator (Fang et
al., 2007), adhesin that mediates spore attachment and
fungal differentiation (Wang and St. Leger, 2007a), a
perilipin-like protein that regulates appressorium
turgor pressure and differentiation (Wang and St.
Leger, 2007b), and a cell protective coat protein
Molecular Plant Breeding 2012, Vol.3, No.7, 63-79
Table 3 Examples of effects of B. bassiana and B. brongniartii (strains and formulations) on beneficial and non-target organisms (this
table was taken from Zimmermann, 2007)
Beneficial organism
Fungus (Strain/Formu-
Trials (L/F)
Amblyseius cucumeris
B. bassiana (Naturalis-
L, BotaniGard WP)
No detrimental effect when sprayed onto
excised cucumber leaves
Jacobson et al.,
Aphidius colemani; Orius
insidiosus; Phytoseiulus
Persimilis Encarsia formosa
B. bassiana (commercial
formulation, strain
Highly susceptible under laboratory condi-
tions, lower infection rates in greenhouse
Ludwig and
Oetting, 2001
Apis mellifera
B. bassiana
Conidia were applied in bee hives: low
mortality and no noticeable effect on
behavior, larvae and colony characteristics
Alves et al., 1996
Apis mellifera
B. bassiana (unformu-
lated Spore preparation)
B. bassiana reduced bee longevity at the
two highest concentrations tested and
caused mycosis at 106-108 spores per bee
Apis mellifera
B. bassiana (Naturalis-
L, Bio-Power)
30-day dietary and contact studies had no
significant effect; LC50 (23 days, ingestion)
9 285 µg/bee
Copping, 2004
Apis mellifera
B. brongniartii
No negative effects noticed
Wallner, 1988
Arthropod and nematode
B. bassiana (Naturalis-
Chlorpyrifos had a stronger negative
impact than the microbial treatment
Wang et al., 2001
Bembidion lampros; Agonum
B. bassiana
A negligible number was infected; low
susceptibility of both species
Riedel and Steen-
berg, 1998
Bombus terrestris
B. bassiana
Able to infect bumblebees; it appears that
there are no risks if the fungus is
incorporated into soil or sprayed onto
plants that are not attractive to bumblebees
Hokkanen et al.,
Carabidae; Calanthus
micropterus; Calanthus
piceus; Carabus violaceus;
Cychrus caraboides; Leistus
ruefescens; Nebria
brevicollis; Pterostichus
oblongopunctatus; P. niger
B. bassiana
No adverse effects noticed
Hicks et al., 2001
Carabidae; Staphylinidae
B. bassiana
Infection levels in adult ground beetles and
rove beetles were low (Carabidae max.
7.6% and Staphylinidae max. 7.0%); an
epizootic in the staphylinid Anotylus
rugosus (67%) and Gyrohypnus angustatus
(37%) was observed
Steenberg et al.,
Cephalonomia tarsalis
B. bassiana
3 h exposure to 100 and 500 mg/kg wheat
resulted in 52.5 and 68.6% mortality
Lord, 2001
Chrysoperla carnea
B. bassiana
Temperature, starvation and nutrition
stresses significantly affected the
susceptibility; nutrition stress caused the
most increase in adult and larval mortality
Donegan and
Lighthart, 1989
Coleomegilla maculate
B. bassiana (isolate
ARSEF 3113)
No mortality was observed
Pingel and
Lewis, 1996
Coleomegilla maculate
Eriopis connexa
B. bassiana (isolate
ARSEF 731)
Mortality after direct application of spores;
exposure via sprayed leaf surfaces resulted
in no infection
Magalhaes et al.,
Coleomegilla maculate
B. bassiana (10 isolates)
6 isolates were highly virulent, 3 isolates
caused low mortality
Todorova et al.,
Diadegma semiclausum
B. bassiana
Detrimental effects on cocoon production
and emergence depending on concentration
Furlong, 2004
Formica polyctena
B. brongniartii
No negative effects noticed
Dombrow, 1988
Molecular Plant Breeding 2012, Vol.3, No.7, 63-79
Continuing table 3
Beneficial organism
Fungus (Strain/Formu-
Trials (L/F)
Earthworms: Lumbricus
terrestris and others
B. brongniartii (com-
mercial product of
barley grains)
No effect in lab and in field noticed
Hozzank et al.,
Earthworms: Lumbricus
B. brongniartii
No effect on earth worms noticed
Zavadil, 1992
Earthworms: Aporrectodea
B. bassiana (Bb64)
No effect on hatching rate of cocoons
Nuutinen et al.,
Lysiphlebus testaceipe
Aphidius colmani
B. bassiana
No significant impacts on both parasitoids
Murphy et al.,
Megachile rotundata
B. bassiana (strain for
grasshopper control)
Spray-application of flowering alfalfa in
pots: female and male mortality averaged
9%; no difference in treatment and control;
however B. bassiana grew out from dead
Goettel and
Johnson, 1992
Nontarget arthropods
B. brongniartii
Only 1.1% of 10 165 collected insects and
spiders were infected
and Cerutti, 1986
Nontarget arthropods
B. brongniartii
1 671 nontarget specimens were collected:
3.4% of them were infected, mainly species
from Araneae, Thysanoptera, Homoptera,
Coleoptera and Lepidoptera
Back et al., 1988
Nontarget arthropods (major
predators, parasitoids and
pollinators on rangeland)
B. bassiana (strain
No statistical differences in the abundance
of aerial insects
Brinkman and
Fuller, 1999
Nontarget arthropods
B. bassiana (emulsi-
fiable concentrate)
From 3 615 invertebrates collected, only
2.8% became infected; B. bassiana could
be applied to forest soil without a
significant negative impact on forest-
dwelling invertebrate population
Parker et al.,
Non-target beetle
B. bassiana (strain SP
No detectable effects
Ivie et al., 2002
Perillus bioculatus
B. bassiana (six
5 isolates were highly pathogenic, isolate
IPP46 showed low pathogenicity
Todorova et al.,
Pimelia senegalensis,
Trachyderma hispida, Bracon
hebetor, Apoanagyrus lopezi
B. bassiana
No infection in P. senegalensis and T.
hispida ; 100% mortality in the parasitoids
B. hebetor and A. lopezi
Danfa et al.,
Poecilus versicolor
B. brongniartii
Melocont-WP, and
No significant negative effects on P.
versicolor could be observed
Traugott et al.,
Predatory mites: O. insidiosus,
A. colemani, Dacnusa sibiria,
Parasites: Encarsia Formosa,
Eretmocerus Eremicus,
Aphidoletes aphidimyza
B. bassiana (Botanigard
Can be used, not recommended during
application of B. bassiana used with
caution during application of B. bassiana
Shipp et al., 2003
Prorops nasuta
B. bassiana (3 isolates)
Strain 25 caused the lowest infection level
De La Rosa et
al., 2000
Serangium parcesetosum
B. bassiana
The predator had significantly lower
survivorship when sprayed with B.
bassiana than with P. fumosoroseus;
feeding on B. bassiana contaminated prey
caused 86% mortality
Poprawski et al.,
Molecular Plant Breeding 2012, Vol.3, No.7, 63-79
involved in evading host immune responses (Wang
and St. Leger, 2006).
4 Entomopathogenic fungal virulence enzymes
The initial interaction in the pathogenesis is mediated
by mechanical force, enzymatic processes and perhaps
certain metabolic acids. The enzymes responsible for
successful interaction with the host and environment
are listed in Table 4 (Khachatourians and Qazi, 2008).
The enzymes involved in pathogenesis of insects are
generally grouped in to proteases and peptidases,
chitinases and lipases.
4.1 Proteases and peptidases
Insect cuticle mainly is composed of chitin and
protein; hence proteases and peptidases of EPF are
important for the degradation of the insect cuticle,
saprophytic growth of the fungi, activation of the
prophenol oxidase in the hemolymph, and they act as
virulence factor. The fungi from which protein degra-
ding enzymes proteases, collagenases, and chymolea-
stases have been identified and characterized are A.
aleyrodis, B. bassiana, B. brongniartii, E. coronata,
Erynia spp., Lagenidium giganteum, Nomuraea rileyi,
M. anisopliae and V. lecanii (Charnley and St Leger
1991; Khachatourians, 1991, 1996; Sheng et al., 2006).
Joshi et al., (1995) cloned extracellular subtilisin-like
serine endoprotease (Pr1) from B. bassiana and
subtilisin-like protease (Pr1B) (Joshi et al., 1997) with
54% sequence homology to Pr1A. Screen and St Leger
(2000) found chymotrypsin (CHY1) of 374 amino acids
with pI of 5.07 and MW38279 from M. anisopliae.
Similarly, Freimoser et al (2005) identified some
overlapped gene responses with unique expression
patterns in response to cuticles from Lymantria dispar,
Blaberus giganteus and Popilla japonica and measured
gene expression responses to a number of insect
cuticles by using cDNA microarrays constructed from
an expressed sequence tags (EST) clone collection of
837 genes.
Small and Bidochka (2005) identified the sequence of
seven conidiation associated genes (cag) using
subtractive hybridization in M. anisopliae. Out of
which, cag7 was found to be essential for cuticle
degradation having encoded an extracellular
subtilisin-like proteinase (Pr1). Kim et al (1999)
described the gene structure and expression of a novel
B. bassiana protease (bassianin I) which is 1 137 bp
and 379 amino acids long. Bidochka and Melzer
(2000) reported genetic polymorphisms in three
Table 4 Entomopathogenic fungal protein encoding genes isolated and sequenced (Khachatourians and Qazi, 2008)
Metarhizium anisopliae
Superoxide dismutase
Shrank et al., 1993
Subtilisin like protease
Joshi et al., 1997
Pr1 (A-K)
Bagga et al., 2004
DNA binding protein
Screen et al., 1997
Nitrogen response regulator
Screen et al., 1998
Bogo et al., 1998
Kang et al., 1998
Chitin synthase
Nam et al., 1998
Baratto et al., 2003; 2006; Screen et al., 2001
Zinc carboxypeptidase
Joshi and St Leger, 1999
Bidochka et al., 2001
Zhao et al., 2006
Peptide synthetase
Bailey et al., 1996
Tryptophan synthetase
Staats et al., 2004
Beauveria bassiana
Joshi et al., 1995
Bassianin I
Kim et al., 1999
Serine endoprotease
Fang et al., 2002
Fang et al., 2005
Yokoyama et al., 2002
UV repair
Chelico et al., 2006
B. brongniartii
UV repair
Chelico et al., 2006
Molecular Plant Breeding 2012, Vol.3, No.7, 63-79
subtilisin-like protease isoforms (Pr1A, Pr1B, Pr1C)
from isolates of M. anisopliae and an extracellular
protease from B. bassiana (BBP) has also been
purified and characterized by Urtiz and Rice (2000).
The isoelectric point of BBP was 7.5 and it is 0.5 kDa
smaller than Pr1. Fang et al (2002) reported a cuticle-
degrading protease (CDEP-1) from B. bassiana pre-
dicting a protein of 377 amino acids (Mr 38 616, pI
8.302) with 1 134 bp. Southern analysis indicated that
CDEP-1 is a single-copy gene. St Leger et al (1996)
constructed an engineered mycoinsecticides based on
M. anisopliae by over-expressing the toxic protease
Pr1 from M. anisopliae genome to accelerate the
killing speed of M. anisopliae. The over-expression of
Pr1 in the hemolymph of M. sexta activates the
phenoloxidase system which causes 25% reduction
in the time of death and 40% reduction in food
4.2 Chitinases
The major component of insect cuticle is chitin,
therefore both endo and exo-chitinases play critical
roles in the cleavage of N-Acetylglucosamine (NAGA)
polymer of the insect cuticle into smaller units or
monomers. Khachatourians (1991) demonstrated that
the extracellular chitinases are virulence determinant
factors. Chitinolytic enzymes (N-acetyl-β-D-glucosa-
minidases and endochitinases were present in the
broth culture supplemented with insect cuticles from
M. anisopliae, M. flavoviride, and B. bassiana (St
Leger et al., 1996). The chitinase from M. anisopliae
consists of acidic (pI 4.8) proteins with molecular
masses 43.5 kDa and 45 kDa. The identified N-terminal
sequences of both bands were similar to an endochi-
tinase from Trichoderma harzianum. Valadares-Inglis
and Peberdy (1997) located chitinolytic enzymes in
enzymatically produced protoplasts and whole cells
(mycelia) of M. anisopliae. No significant induction
was observed from mycelia, yet protoplasts were
found to induce these enzymes significantly. The
majority of chitinolytic activity was cell-bound in both
whole cells and protoplast preparations, and the
activity was mainly located in the membrane fraction.
Kang et al (1998, 1999) reported a chitinase with
molecular mass of 60 kD from M. anisopliae grown in
a medium containing chitin as the sole carbon source
with an optimum pH of 5.0, which is different from
the chitinases values previously reported by St Leger
et al (1996) for endo-chitinases of 33.0, 43.5, and 45
kDa and exo-chitinases of 110 kDa. Screen et al (2001)
cloned the chitinase gene (Chit1) from M. anisopliae
sf. acridum ARSEF strain 324 and M. anisopliae sf.
anisopliae ARSEF strain 2575 (Chit1) using the pro-
moter of Aspergillus (gpd) for constitutive expression.
A 42 kD chitinase of M. anisopliae was expressed and
characterized in Escherichia coli by Baratto et al
(2003) using a bacteriophage T7- based promoter
expression vector. Baratto et al (2006) performed
transcriptional analysis of the chitinase chi2 gene of M.
anisopliae var. anisopliae and showed that it has 1
542 bp encoding for a deduced 419 amino acids.
Nahar et al (2004) reported that the extracellular
constitutive chitin deacetylase (CDA) secreted by M.
anisopliae converts chitin (a β-1, 4-linked N-acetyl-
glucosamine polymer) into its deacetylated form
chitosan (a glucosamine polymer). This CDA was not
inhibited by solubilized melanin.
Fang et al (2005) purified an endochitinase from
liquid cultures of B. bassiana supplemented with
chitin. Bbchit1 was 33 kD (pI 5.4) and the encoding
gene, Bbchit1, and its upstream regulatory sequences
were cloned based on N-terminal amino acid sequence.
Bbchit1 contains no introns and it is present as a
single copy in the B. bassiana genome. The amino
acid sequence of Bbchit1 is similar to that of the
endochitinase of Streptomyces avermitilis, S. coelicolor
and T. harzianum (Chit36Y), but not to EPFs that
reflect novel chitinases. Fang and co-worker (2005)
constructed a B. bassiana transformants (gpd-Bbchit1),
which overproduced Bbchit1 and had enhanced
4.3 Lipases
Although the major bulk components of the insect
cuticle are protein and chitin, the outermost epicuticular
surface layer are made up of a complex mixture of
non-polar lipids. Epicuticular lipids play a role in
chemical communication events (Blomquist and Vogt,
2003), and in keeping the cuticular surface dry which
affects insecticide and chemicals penetration (Hadley,
1981; Blomquist et al., 1987; Juárez, 1994). They
Molecular Plant Breeding 2012, Vol.3, No.7, 63-79
exhibit certain unique structural characteristics such as
relatively high molecular mass and chemical stability,
which is mainly due to specific physicochemical
properties such as length and branching of the carbon
chains (usually between 20 to more than 40 carbons),
as well as the position and the kind of functional
groups and double bonds. The most abundant
components are long chain HC, wax esters, fatty
alcohols and free or sterified fatty acids.
The insect epicuticle contains lipoproteins, fats, and
waxy layers which would be barriers to EPF without
the action of lipases and lipoxygenases, as some of
these structures have anti-fungal activities, and EFP
can’t use them as substrates (Khachatourians, 1996).
In addition, some chains of saturated fatty acids even
inhibit the growth of some EPF. Lord et al (2002)
showed a role for the lipoxygenase pathway through
eicosanoid-mediated cellular immune response to the
B. bassiana. James et al (2003) demonstrated that
conidial germination of B. bassiana and P. fumosoroseus
are affected by cuticular lipids and silverleaf whitefly
(B. argentifolii). The whitefly nymphs produce thick
coating of long-chain wax esters affecting spore
4.4 The function of phospholipases in cuticle
Since lipids represent major chemical constituents of
the insect cuticle, enzymes capable of hydrolyzing
these compounds, such as phospholipases, could be
expected to be involved in the cuticle disruption
processes that occur during host invasion. Phospho-
lipases are a heterogeneous group of enzymes that are
able to hydrolyze one or more ester linkages in
glycerophospholipids. The action of phospholipases
can result in the destabilization of membranes, cell
lysis and the release of lipid second messengers
(Ghannoum, 2000). These enzymes are categorized
according to the location of the ester link that is
cleaved (Figure 2). Although phospholipase B (PLB)
refers to an enzyme that can remove both sn-1 and
sn-2 fatty acids, this enzyme also has lysophospho-
lipase-transacylase activity.
Extracellular phospholipases have been implicated as
pathogenicity factors for bacteria, rickettsiae and
Figure 2 Sites of action of phospholipases
Note: A1, A2, B, C and D indicate cleavage sites of the corres-
ponding phospholipases (PLA1, PLA2, PLB, PLC and PLD)
protozoa. The type of phospholipase involved in
virulence varies with the organism. For example, C.
perfringens (Alape-Giron, 2000) secretes a phospholi-
pase C (PLC), whereas T. gondii secretes a phosphor-
lipase A (PLA). The importance of these enzymes,
especially PLB, for virulence has so far only been
verified in medically important fungi. PLB was
secreted by different clinically important fungal
species such as Candida albicans (Mukherjee, 2001),
Aspergillus fumigatus and Cryptococcus neoformans.
The role of PLB in the pathogenicity of entomopatho-
genic fungi remains to be determined, even in the
best-studied species M. anisopliae.
5 Advantages of using fungi as insecticides
The advantages of using fungi as insecticides are: (1)
They have high degree of specificity for controlling
pest without affecting beneficial insect predators and
non-harmful parasites. (2) They have no hazard effects
on environment or the health of mammals which is
normally affected by chemical insecticide applications.
(3) They have different ways of infection; hence insect
resistance cannot be developed and they can be used
as prolonged pest control. (4) They have genes for
secretion of insect toxins; hence they have high
potentials for further development by biotechnological
research. (5) Some of them have endophytic capability;
hence they can play important roles in the activation
Molecular Plant Breeding 2012, Vol.3, No.7, 63-79
of immune system. (6) High persistence in the envi-
ronment provides long-term suppression effects of
entomopathogenic fungi on pest.
6 Disadvantages of the fungi as biocontrol
(1) They have very slow killing rate: normally 2-3
weeks are required to kill the insects whereas
chemical insecticides may need only 2-3 hours. (2) As
the pathogenesis process of the fungi is bioprocess, it
requires specific conditions to be carried out, such as
specific temperature, humidity and period of light. (3)
They have high specificity in killing pests making
them a narrow host killer while a broad range killer
pesticide is required for commercialization, hence
additional control agents are needed for other pests. (4)
Their production is relatively expensive and the short
shelf life of spores necessitates cold storage. (5) The
persistence and efficacy of entomopathogenic fungi in
the host population vary in different insect species,
thus insect-specific application techniques need to be
optimized to retain long-term impacts. (6) They also
present potential risks to immunodepressive people.
Since centuries ago, fungi have always been used for
medicinal and other beneficial proposes and they are
just as important nowadays. In this review, we
summarized the advantages and applications of fungi
as a biopesticides, attempted and collected the know-
ledge about the entomopathogenic fungi as biocontrol
agents. We collected knowledge of the past and
present about entomopathogenic fungi to explore
ways to improve the abilities of entomopathogenic
fungi as a biocontrol agent. In this way, new ideas
and hypothesis will emerge which will farther help
developing the fungi’s capabilities as biocontrol
agents. New techniques will be developed which will
help manage the pest in a better way as the present
pathogenesis mechanism of fungi is slow and needs
improvement. Genetic and proteomic studies are
expected to be the main tools for the future develop-
ment of the entomopathogenic fungi as biocontrol
agents; however, in the near future, there will be
awider array of techniques become available to
biologists which will enable us to take full advantage
of entomopathogenic fungi.
This work was supported by International Cooperation Fund
(No. 2012DFR30810). We also acknowledge the China
Scholarship Council for the PhD scholarship.
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... Entomopathogenic fungi are microorganisms that are used first as a biological pest control agent. More than 700 species from 90 genera of fungi are pathogenic to insects (Khan, et al, 2012). Most species of entomopathogenic fungi derived from the division Ascomycota and Zygomycota. ...
... Its main constituent insect cuticle chitin and protein degradation by protease and a peptidase enzyme. Another role as a growth factor saprophytic fungi, propanol oxidase activation in the hemolymph and as a virulence factor of fungi (Khan, et al, 2012). The main component of insect cuticle is the chitin substance. ...
This research was conducted in two stages. The research method is the isolation and application of biopesticide. In the first stage aims to isolate entomopathogenic fungi Beauveria bassiana around Berastagi vegetable farms and grow on agar and corn mashed artificial medium. Testing the virulence of Beauveria bassiana in vitro in several types of vegetable pests before it is applied on agricultural land. In phase II aims to application B. bassiana in the vegetable patch. In the in vitro treatment, the ability to infect insects beauvria average reached 75%. , Controls on crop lands, in the third week began their Crocidolomia pests that attack the growing point so that the cabbage plants fail to bloom. Average cropland applied biopesticides B. bassiana has been no attack. At week 7 attacks on control plants reach 13 plants or 17.3%. In field applied while Beauveria bassiana attack reached six plants, or about 8%.
... Fungal-based biopesticides thus lend to both inoculative (applied at very low concentration and with an autonomous population increase) and augmentative (low concentration but the environment is modified to favour their development) applications (Cook 2000). (4) Entomopathogenic fungi have long persistence in the soil and therefore a long-term suppression activity (Khan et al. 2012). Mycoinsecticides are yet to make a dent in the biopesticide market considering the impetus given to biological control in IPM. ...
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Insect pathogenic fungi have a huge potential as microbial components of biopesticides which serve as benign components in plant protection. The infection cycle of these fungi is well known. Realising their potential and scope to improve their utility in phytomedicine, extensive work on the molecular biology of pathogenesis has been done in the past decade. Wet bench techniques like gene isolation, cloning and characterisation and gene knockout experiments to transcriptomics techniques like cDNA-AFLP, microarray, qPCR, cDNA, EST and SSH library construction, as well as whole genome sequencing and analysis of data with a suite of bioinformatic tools and pipelines integrated with several biological databases, were done to understand the process/processes involved at each stage of the infection cycle of the insect pathogenic fungi. These are in particular adherence of spores to the insect cuticle, factors that aid in coping with the physical stress conditions in the surrounding environment, formation of an infection peg, penetrance into the insect, factors that abet in overcoming insect defence systems and growth in the insect, production of toxic secondary metabolites that lead to insect death and surfacing out from the insect cadaver as well as sporulating to iterate the infection cycle on yet another insect. The picture that emerged is detailed in this chapter. The genes/proteins involved and the analyses that aided in their identification are described. Environmental genomics through multitag 454 pyrosequencing of rRNA sequence reads in deciphering the effect of the inundative application of an entomopathogenic fungus on the native soil fungal diversity is described. The chapter highlights the bioinformaticsbolstered investigation of the factors that influence the affectivity of insect pathogenic fungi as microbial biopesticides.
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Background: Bioassays evaluating entomopathogenic fungi (EPF) isolates for effective microbial control of whitefly are a fundamental part of the screening process for bioprotectants, but development of repeatable, robust bioassays is not straightforward. Currently, there is no readily available standardised method to test efficacy of EPF on whitefly. Here, we describe the calibration and use of a spray tower to deliver a standardised protocol to assess EPF activity; the method was validated using 18 EPF from four genera in tests against greenhouse whitefly, Trialeurodes vaporariorum (Westwood). Results: At 138 kPa, the sprayer delivered 0.062 mL mm-2 (620 L ha-1 ) and an even deposition of spray across the central 1590 mm2 of the spray area. Average conidial deposition for all EPF was 252 conidia mm-2 and equivalent to 2.5 x1012 conidia ha-1 at an application concentration of 1 x107 conidia mL-1 . Conidial deposition of a test Beauveria bassiana suspension increased with increasing application concentration. Egg laying by T. vaporariorum adults was restricted to 177mm2 using clip cages specifically designed to ensure that third instar T.vaporariorum received a uniform spray coverage. Nymphs occupied 373 ± 5 mm2 of the leaf after migrating during the first instar. Average T. vaporariorum mortality totaled 8-89% 14 days after application of 1x107 conidia mL-1 of each EPF isolate. Conclusion: Combining the calibrated sprayer and bioassay method provides a reliable, standardised approach to test the virulence of EPF against whitefly nymphs. This laboratory based assay is affordable, replicable and allows the user to alter the dose of conidia applied to the target. This article is protected by copyright. All rights reserved.
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Tujuan penelitian adalah untuk mengetahui keragaman fungi entomopatogen yang ada disekitarlahan tanaman sayuran Brastagi dan manfaatnya dalam pengendalian hama-hama yang menyerangsayuran, sehingga bisa dimanfaatkan guna menjaga kwalitas sayuran organik. Metode yangdigunakan adalah eksplorasi fungi dengan umpan larva serangga Tenebrio molitor, fungi yangmenginfeksi larva diisolasi dengan menggunakan media Potato dekstrosa agar (PDA), kemudiandilakukan identifikasi makroskopik terhadap fungi yang ditemukan serta mendeskripsikan manfaatfungi-fungi tersebut dalam perananya sebagai pengendalian hama terpadu secara hayati. Pada tahaplanjut fungi-fungi tersebut dapat dikembangkan untuk diaplikasikan di lapangan sehingga petanibisa mengendalikan hama sayuran tanpa penggunaan pestisida.
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Lipases are the industrially important biocatalysts, which are envisioned to have tremendous applications in the manufacture of a wide range of products. Their unique properties such as better stability, selectivity and substrate specificity position them as the most expansively used industrial enzymes. The research on production and applications of lipases is ever growing and there exists a need to have a latest review on the research findings of lipases. The present review aims at giving the latest and broadest overall picture of research and development on lipases by including the current studies and progressions not only in the diverse industrial application fields of lipases, but also with regard to its structure, classification and sources. Also, a special emphasis has been made on the aspects such as process optimization, modelling and design that are very critical for further scale-up and industrial implementation. The detailed tabulations provided in each section, which are prepared by the exhaustive review of current literature covering the various aspects of lipase including its production and applications along with example case studies, will serve as the comprehensive source of current advancements in lipase research. This review will be very useful for the researchers from both industry as well as academia in promoting lipolysis as the most promising approaches to intensified, greener and sustainable processes. This article is protected by copyright. All rights reserved.
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This study was carried out to investigate the insecticidal properties of Beauveria bassiana, Metarhizium anisopliae and Heterorhabditis bacteriophora for their virulence against different larval instars of Rhynchophorus ferrugineus (Olivier). Both fungi were either applied alone or in combination, with H. bacteriophora simultaneously or 1 and 2 weeks after fungal application; EPN were also applied alone. Moreover, assessment of host development, diet consumption, frass production and weight gain were observed at sub-lethal dose rates. In combined treatments, additive and synergistic interactions were observed. Synergism was observed more frequently in H. bacteriophora + B. bassiana combinations than in H. bacteriophora + M. anisopliae combinations, and was higher in early instars than old instars. In 2nd and 4th instars, synergy was noted in H. bacteriophora + B. bassiana combinations at 0, 7 and 14 d intervals and in 6th instar synergy was observed only in H. bacteriophora + B. bassiana combinations (at 0 and 7 d intervals). A decrease in pupation, adult emergence and egg hatching was enhanced in the combined treatments. Furthermore, reduced weights and variation in duration of insect developmental stages were observed among entomopathogens and enhanced in H. bacteriophora + B. bassiana combinations. Larvae treated with sub-lethal concentrations exhibited reductions in food consumption, growth and frass production and weight gain.
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The housefly Musca domestica is a worldwide insect pest that acts as a vector for many pathogenic diseases in both people and animals. The present study was conducted to evaluate the virulence of different local isolates of Beauveria bassiana, Metarhizium anisopliae and Isaria fumosorosea on M. domestica using two bioassay techniques: (1) adult immersion and (2) a bait method applied to both larvae and adults. The results showed evidence of a broad range of responses by both stages (larvae and adults) to the tested isolates of B. bassiana, M. anisopliae and I. fumosorosea. These responses were concentration-dependent, with mortality percentages ranging from 53.00% to 96.00%. Because it resulted in lower LC50 values and a shorter lethal time, B. bassiana (Bb-01) proved to be the most virulent isolate against both housefly larvae and adults. Sublethal doses of the tested isolates were also assessed to evaluate their effect on M. domestica fecundity and longevity. The fungal infections reduced housefly survival regardless of their sex and also decreased egg production in females.
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Bioefficacy of Beauveria bassiana (Balsamo) Vuillemin and Lecanicillium lecanii Zimmerman in comparison with their commercial formulations along with standard check insecticide, Fenvalerate 20 EC were evaluated against onion thrips, Thrips tabaci Lindeman under greenhouse as well as field conditions. The results revealed that the standard check fenvalerate 20 EC @ 0.0075 % showed significantly the highest cumulative corrected mortality of 97.84 % followed by commercial formulation of B. bassiana, Myco-Jaal @ 1 × 108 spores/mL which showed 80.90 % mortality. The laboratory cultured B. bassiana showed percent mortalities of 74.11, 71.69 and 78.48 % for the concentrations of 1.23 × 107, 1.23 × 106 and 1.23 × 108 spores/mL, respectively. However, these concentrations were statistically at par on all the days of observation. Thrips mortality gradually increased with the increase in concentrations of fungal preparations and days of observations. Similar trend was also observed in L. lecanii experiment. Under field conditions, Fenvalerate 20 EC @ 0.0075 % recorded highest mortality of T. tabaci (90.10 %) followed by commercial formulation of V. lecanii (Phule Bugicide @ 2 × 108 cfu/g) with 74.90 % mortality. All the concentrations of fungal concentrations gave low mortality ranging from 9.40 to 10.10 % and 7.10 to 7.40 % at 2 days after treatment (DAT) of B. bassiana and L. lecanii, respectively. The standard check of Fenvalerate 20 EC @ 0.0075 % was highly toxic and showed significantly maximum percent reduction (90.50 %) of T. tabaci population in both the experiments. The present study clearly shows that these entomopathogens may be integrated with existing integrated pest management (IPM) practices for management of T. tabaci.
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The use of microbial organisms as biological control agents has progressed significantly since Metschnikoff launched the first attempt at microbial insect control with Metarhizium anisopliae in 1879. Following the lead of Metschnikoff, entomopathogenic nematodes, fungi, bacteria and viruses have been extensively studied for commercialization and practical use as biopesticides in inundative releases against insect pests in various cropping systems. However, compared with chemical insecticides, these microbial products represent less than 2% of the total insecticide market share. Factors such as control efficacy, cost, formulation, shelf life, application techniques, and persistence have limited the commercial use of these microbial control agents in insect pest management. This review discusses research advances for entomopathogens, especially commercialization, formulation and application techniques, for microbial biocontrol of insect pests in the horticultural ecosystem.
Biological pesticides are increasingly finding therr place in IPM and increasing numbers of products are making therr way to the marketplace. Particularly in China, Latin America and Australia, implementation is proceeding on a large scale. However, in the USA and Europe, registration procedures for insect pathogens to be used for insect control have been established that requrre low levels of risk, resulting in costs of retarding the implementation of microbial agents. This book provides a review of the state of the art of studies on the envrronmental impact of microbial insecticides. It originates from a Society for Invertebrate Pathology Microbial Control Division Symposium .. Assessment of envrronmental safety of biological insecticides", organised in collaboration with the EU-ERBIC research project (FAIR5-CT97-3489). This symposium was initiated by Heikki Hokkanen and Chris Lomer, and was held at the SIP Annual Meeting in 2001 in The Netherlands. The emphasis in this book is on large scale use of microbial agents for insect control, demonstrating how this use has been proceeding with minimal envrron­ mental impact. This book is intended to be of use to regulatory authorities in determining whether further studies in eertain areas are necessary and how to conduct them if needed, or whether sufficient information has been collected already to permit fuH registration of many of these biological control agents.
Recent advances on mass culture, stabilization and formulation, and market potential of mycoinsecticides (Beauveria bassania [B. bassiana], B. brongniartii, Lagenidium giganteum, Metarhizium anisopliae, M. anisopliae var. acridum, Paecilomyces fumosoroseus and Verticillium lecanii), mycoherbicides (Colletotrichum gloeosporioides [Glomerella cingulata], Phytophthora palmivora, Puccinia canalichlata and Colletotrichum truncatum) and antagonistic yeasts (Candida oleophila and Cryptococcus albidus) for the control of many insect pests, weeds and postharvest diseases, are presented.
The bacterium Bacillus sphaericus and the fungus Entomophaga maimaiga did not affect Apis mellifera longevity and did not cause disease among treated bees. Both of these microorganisms are considered safe for honey bees. A technical powder and a formulated product of the bacterium Bacillus thuringiensis var. tenebrionis reduced bee longevity at the higher concentration tested (108 spores/ml sucrose syrup), but did not cause disease. The fungus Beauveria bassiana reduced bee longevity at the 2 highest concentrations tested and caused mycosis among treated bees at all 3 concentrations tested (106-108 spores per bee). -from Author
In this chapter we present as an example a specific case study from the ecological safety evaluation of the Hyphomycete fungi Metarhizium anisopliae (Metsch.) Sorokin and Beauveria bassiana (Bals.) Vuill., carried out in Finland during the ERBIC-research project. Only the part concerning the safety to bumblebees is presented here (for a full report see Hokkanen et al., 2003). Metarhizium anisopliae and Beauveria bassiana are two well-studied, commercialised, and commonly used entomopathogenic fungi (EPF), also occurring naturally in Finland. We decided to focus on bumblebees because they are the most important group of natural pollinators of crop plants and wild flowers in the temperate zone. While possible impacts of Metarhizium and Beauveria on the honeybee have been addressed by several authors in many publications, to our knowledge no earlier information exists on the possible impact of these fungal pathogens on bumblebees. These pollinators are also abundant in our model agroecosystem, turnip oilseed rape, which was chosen because both the key pest, the pollen beetle (Meligethes aeneus), and its natural enemy complex have been well studied under Scandinavian conditions. Background information was already available on the occurrence of deuteromycetous EPF in the model system (Vä nninen et al. 1989), on the persistence of augmented fungal propagules in cultivated soils of the study area in question (southern Finland) (Vänninen et al. 2000), and on the impact of entomopathogenic fungi on the pollen beetle (Hokkanen 1993). In addition, entomopathogenic fungi and nematodes are a possible future option for managing the soil-dwelling stages of the pollen beetle and other pests in this system via incorporation in the soil (Butt et al. 1994). Fungi can also be used against foliage-dwelling stages of the pest either by spraying, or when vectored by honeybees (Butt et al. 1998). Different application strategies for entomopathogens could therefore be considered, linked to differing non-target risk scenarios based on the impact of the application strategies on key components of the ecosystem.
Fungal disease of insects represents an important facet of the interaction between entomopathogenic fungi (EPF) and their hosts. Altogether some 90 genera and 700 species are involved with entomopathogenicity, but only a few members of the entomophthorales and hyphomycetes have been well studied (Khachatourians 1991). The long-range potential of the EPF in research is their application in insect pest management or biocontrol (Leathers et al. 1993). Commercialization of EPF for pest control requires understanding of physiological aspects of growth, metabolic activity, genetic basis of virulence and host specificity-challenges that were forecast to be met with the new biotechnology (Khachatourians 1986; Boucias 1988). With such knowledge, physiological manipulations, isolation of mutants with enhanced virulence, and construction of environmentally safe strains with limited persistence should be possible within the forseeable future.
Insects are members of the Arthropoda. Among the characteristics of this phyllum is the presence of an external skeleton or cuticle. Because of its location, the cuticle serves a variety of functions in addition to the skeletal roles of support and muscle anchorage. The defensive capability of the cuticle is clear since only one group of entomopathogens, the fungi, have acquired the ability to invade insects actively via this route. The other major groups of disease-causing microorganisms, the viruses and bacteria, are restricted primarily to the alimentary canal, where the midgut provides an exposed mucosal surface.