i. entomopathogenic fungi as biocontrol...

Review of literature

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I. Entomopathogenic fungi as biocontrol agents

In the era before microscopes, fungi visible to the naked eye helped to

gave birth to invertebrate pathology as a modern field of study. Early

observations of diseases in useful insects, the honey bee and the silkworm,

included documentation of mycoses. Biological control using entomopathogenic

fungi is especially promising as these microorganisms present unique

mechanisms of action during the infection and colonization in the host (Charnley,

1997). Both general and particular historical aspects of fungal entomopathogens

and their use as microbial control agents have been thoroughly reviewed by

Boucias and Pendland (1998); Roberts and St. Leger (2004). A comprehensive

international program facilitated a wide array of studies that serve to illustrate

well the marriage of basic and applied research needed to develop a fungal

pathogen for use as a microbial control agent (Castrillo et al., 2005).

Development of an effective mycoinsecticide depends on selection of an isolate

that is highly virulent for the target host and which is genetically and biologically

stable (Milner et al., 2002). Comparison of entomopathogens with conventional

chemical pesticides depends on their efficiency and cost. In addition to

efficiency, there are advantages in using microbial control agents, such as human

safety and other non-target organisms; pesticide residues are minimized in food

and biodiversity would be increased in managed ecosystems (Shahid et al.,

2012).

More than 700 known fungal species from 100 genera have adopted an

entomopathogenic lifestyle and occupy a unique, highly specialized nutritional


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niche in the kingdom Fungi, encompassing more than 80,000 species.

Entomopathogenicity arose and was lost in many phyla during fungal evolution.

With the principal exception of the higher basidiomycetes, entomopathogenic

species are found within every major fungal lineage and in almost all ecosystems.

The largest numbers of fungal species that are pathogenic to insects belong to the

order Hypocreales (Dikarya, Ascomycota, Pezizomycotina, Sordariomycetes,

Hypocreomycetidae) (Molnar et al., 2010). Most of the taxonomic groups that

can parasitize insects contain entomopathogenic genera, such as Metarhizium,

Beauveria, Verticillium, Nomuraea, Entomophthora, and Neozygites, to name a

few (Deshphande, 1999). They are being developed as BCAs (biocontrol agents)

for the biological control of pests (Burges, 1998; Butt et al., 1999) and were

comprehensively listed (Butt et al., 2001).

Entomopathogenic fungi were reported to infect a wide range of insects

including lepidopterous larvae, aphids and thrips, which are of great concern in

agriculture worldwide (Roberts and Humber, 1981). Aspergillus flavus, an

entomopathogenic fungus, was reported to be pathogenic against mosquito

species Aedes fluviatilis and Culex quinquefasciatus by Moraes et al. (2001) and

Aspergillus clavatus, was reported to be pathogenic to Aedes aegypti L.,

Anopheles gambiae and Culex quinquefasciatus (Diptera) by Seye et al. (2009).

Metarhizium anisopliae (Metsch.) Sorokin and other entomopathogenic fungi are

being examined as potential biological insect control agents (Lacey et al.,

2001).Metarhizium anisopliae and Beauveria bassiana have been studied and

applied in controlling of rice Brown planthoppers, rice bug, coconut beetle,

grasshoppers, termite (Chinh et al. 2001; Thuy et al. 2001; Loc, 1997a,b; Loc et

al., 1999, 2001, 2002, 2004, 2005). The susceptibility of Agrilus planipennis


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Fairmaire (Coleoptera) to entomopathogenic fungi Beauveria bassiana and

Metarhizium anisopliae was reported by Liu and Bauer (2006). El-Sinary and

Rizk (2007) reported pathogenecity of Beauveria bassiana on Galleria

melonella. M. anisopliae was found to be more virulent towards Redpalm weevil

(Rhynchophorus ferrugineus) (Coleoptera) compared to B. bassiana in a study

conducted by Gindin et al. (2006). Both the fungi were reported to be pathogenic

to tomato spider mite (Tetranychus evansi) (Acari: Tetranychidae) (Bugeme et

al., 2008). Prasad et al. (2010) reported pathogenecity of Beauveria bassiana

against larvae of Helicoverpa armigera (Lepidoptera). Metarhizium anisopliae

spp. were reported to be pathogenic to insects belonging to different orders which

include Grasshopper (Orthoptera) (Thomas et al. 1997), Termites (Isoptera)

(Andrew 2000), locusts (Schistocerca gregaria) (Bateman and Luke, 2000)

Spittlebug, Mahanarva posticata (Homoptera) (Miller et al. 2004), Broad mite

(Polyphagotarsonemus latus Bank) (Nugroho and Ibrahim.2004), Epilachna

beetle (Hemisepilachna vigntioctopunctata) (Padmaja and Gurvinder Kaur,

1998), Scab mite (Psoroptes ovis) (Sarcoptiformes) (Lekimme et al. 2008),

Ocinara varians (lepidoptera) (Hussain et al. 2009), Spider mite (Tetranychus

urticae) (Bugeme et al. 2009), black citrus aphid (Toxoptera citricidus) and citrus

pyrilla (Diaphorina citri) (Nguyen et al. 2010), housefly, Musca domestica L.

(diptera) (Sharififard et al. 2011). Bioefficacy of Metarhizium anisopliae against

Dysdercus cingulatus (Fab.) (Hemiptera: Pyrrhocoridae), Oxycarenus

hyalinipennis (Costa) (Hemiptera: Lygaeidae), Aphis craccivora (Koch)

(Homoptera: Aphididae), Mylabris pustulata (Thunb.) (Coleoptera: Meloidae),

Pericallia ricini Fab. (Lepidoptera: Arctiidae), Spodoptera litura (Fab.)


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and Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) was reported by

Sahayaraj and Francis, (2010) and he suggested the efficient use of the fungus for

the control of Helicoverpa armigera. M. anisopliae displays a remarkably broad

host range, spanning from insects to ticks and other members of the class

Arachnida (Chandler et al., 2000; Roberts and St. Leger, 2004). The

pathogenicity of M. anisopliae isolate obtained from dead cadavers of mole

formula cricket (Gryllotalpa orientalis) (Orthoptera) was reported to be

pathogenic to cockroach (Periplaneta americana) by Wakil et al. (2012).

M. anisopliae has been reported as pathogen for stored product pests by

Khashaveh et al. (2008). Therefore, this fungus holds great potential for use as

biological control agent (Butt et al., 2001). Virulence (speed of kill) of a fungal

entomopathogen against a particular host insect depends on biological properties

of the specific isolate-host combination, together with factors such as fungal dose

and further, intrinsic and extrinsic factors affect the actual pattern and extent of

fungal growth in vivo was poorly understood, according to him. Variation in

virulence between isolates, species and doses is determined more by quantitative

rather than qualitative differences in fungal growth kinetics (Anderson et al.,

2011).

II. Biocontrol of cockroach

Cockroaches, described as second only to termites in economic

importance, have been the object of major control efforts by the pest control

industry. Cockroaches live in a wide range of environments around the world.

Pest species of cockroaches adapt readily to a variety of environments, but prefer

warm conditions found within buildings. It is proven or suspected carrier of the

organisms causing viral diseases such as poliomyelitis (Prado et al., 2002) and


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also diarrhea, dysentery, cholera, leprosy, plague, typhoid fever (Czajka et al.,

2003). Cockroaches have been shown to be linked with allergic reactions in

humans and leave chemical traces in their fecal matter as well as emitting

airborne pheromones for swarming and mating and these chemical trails transmit

bacteria on surfaces (Prado et al., 2002). P. americana (American Cockroach) is

the well-known pest species ubiquitous throughout the world and is an important

reason for the need to eliminate this vermin is that sensitization to cockroaches is

associated with asthma (Rabito, 2011). There are numerous parasites and

predators of cockroaches, but few of them have proven to be highly effective for

biological control of pest species (Kaakeh et al., 1996, 1997). Cockroaches are

primarily controlled through the use of synthetic organic insecticides (Organo

phosphates, pyrethroids, carbamates) but many factors including insecticide

resistance, concerns about human and environmental safety and increased

developmental cost of new insecticides have intensified the search for new

control methods. Pathogenicity of Beauveria bassiana against P. americana was

studied and the need for potential alternative for the use of biologically based

insecticides, such as those containing entomopathogenic fungi was expressed by

Murali Mohan et al. (1999). German cockroaches have developed resistance to a

wide range of insecticides including organochlorine, organophosphate and

pyrethroid insecticides (Scott et al., 1990). The situation warrants development of

alternate methods of management of this house hold pest and to minimize the use

of chemical pesticides in an attempt to safe guard human health and environment.


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There are few reports which address the ability of M. anisopliae to infect

cockroaches but no information on mechanism of insect kill was reported.

Defense reaction in the form of nodule formation was reported in P. americana

up on injection of M. anisopliae conidia (Gunnarsson and Lackie, 1985). A

commercial formulation of M. anisopliae has been developed for cockroach

control in US by the Eco science Corporation (Kaakeh et al., 1996). Most of the

work was done keeping the focus on mortality of the target pest caused by the

fungus in view of the urgent need for more active and virulent fungal strains and

identifying target specific isolates of the pathogen causing pest mortality at a

higher rate.

Horizontal transmission of pathogens within the same target species

called as autodissemination, and it has been shown to be useful for the biocontrol

of insect pest from several different insect orders. Mortality due to initial fungal

application maybe followed by horizontal transmission within the target

population a phenomenon that may be attributable to the gregarious behavior of

cockroaches (Kaakeh et al., 1996). Hernandez et al. (2008) reported that

Cockroach (P. americana) adults were more susceptible to infection by M.

anisopliae under high relative humidity compared with laboratory conditions.

Tove Steenberg and Karl Martin (1999) reported field collected German

cockroaches infected with pathogenic fungi. Horizontal transmission of fungal

infection among healthy and infected cockroaches was studied in German

cockroach Blattella germanica by Kaakeh et al. (1996) and was reported as a

successful mode of autodissemination among German cockroaches (Quesada-

Moraga et al., 2004). Successful mortality due to horizontal transmission of M.

anisopliae infection among termites in a colony was reported by Andrew (2000).


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Autodissemination of fungal conidia of some entomophthoralean species while

the host insect is still alive was reported in flies infected with Strongwellsea

castrans and thrips infected with Entomophthora thripidum (Pell et al., 2001).

Infection by M. anisopliae causing sub-lethal reproductive effects on the female

German cockroaches and its potential to decrease the pest status (Quesada-

Moraga et al., 2004) in which treated females showed an effect on oothecal

production, oothecal hatch and nymphal production. This would bring a downfall

in the populations of the insect in the locality. Horizonital transmission of M.

anisopliae among the ectoparasitic mites of the genus Psoroptes was reported

when the uninfected live mites were brought in contact with infected cadaver

(Brooks and Wall, 2005). The capacity of M. anisopliae to get horizontally

transmitted among the adults of Ceratitis capitata was evaluated in the laboratory

tests (Quesada-Moraga et al., 2008). Efficient treatments were reported to be

important for horizontal transmission of the pathogen from diseased to healthy

insects, causing epizootics in field populations and a longer-term control effect.

Behavioral characteristics of cockroaches, such as aggregation (Kaakeh et al.,

1996) and hiding preference for spots with high humidity, may favor the

horizontal transmission of the fungus (Lopes and Alves, 2010).

III. Insecticidal activity of destruxin

Metarhizium anisopliae was reported to produce some cyclic peptide

toxins, destruxins, which exhibit a variety of insecticidal actions by Sharif et al.

(2010) and thirty-eight destruxin analogues have been reported to date (Schrank

and Vainstein, 2010). M. anisopliae infects insects by penetrating the cuticle and

produce destruxin, and represents a pathogen for many insect species (Sewify

and Hashem 2001; Ihara et al., 2003). The insecticidal properties of destruxins,


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cyclic depsipeptide toxins from Metarhizium spp., was described by Kodaira

(1961) and shown to be produced in wax moth and silkworm larvae by Roberts

(1966) and Suzuki et al. (1971). Destruxins constitute a large class of peptide

related compounds comprising a hydroxyl acid and five amino acids joined by

amide and ester linkages (Suzuki et al., 1970). Some strains of M. anisopliae

produce the cyclodepsipeptidic toxin, dtx in combination with other cyclic

depsipeptides and hydrophobins (Gillespe and Claydo, 1989). The toxic

secondary metabolites that are extracellularly secreted play an important role in

pathogenesis (Kershaw et al., 1999). All the naturally occurring dtxs and its

analogues are active against wide range of insect pests (Pedras et al., 2002). For

entomopathogens producing these toxins, infection has been shown to result in

more rapid host death (McCauley et al., 1968) compared to strains that do not

produce these metabolites (Samuels et al., 1988; Kershaw et al., 1999). Inter- and

intra-specific variation in destruxin production was detected in Metarhizium and

may be important in determining virulence and/or specificity against insects.

Some weakly to moderately pathogenic strains were highly pathogenic when

injected into Galleria mellonella larvae, demonstrating the importance of the

cuticle as a barrier to fungal infection (Amiri-Besheli et al., 1999). The pure

forms of dtxs are more active than crude dtx as reported in Agrostis segetum

(Thomsen and Eilenberg, 2000). On the other hand, Skrobek and Butt (2005) in

their studies on the effect of crude extract of dtx from M. anisopliae individually

on human and insect cell lines expressed the opinion that testing of crude

destruxin offers an alternative approach and is recommended when assessing the

risks of metabolites for registration purposes. Some strains of M. anisopliae,

however, grow profusely in their hosts without inducing symptoms of toxicosis,


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and death of the insect occurs slowly (Samuels et al., 1988). These observations

suggest intra-specific variation in toxin production. Destruxins A and E are more

toxic than the others (Dumas et al., 1994), so the relative amounts of these

destruxins could influence the speed of kill (i.e. virulence), and possibly

specificity. These reports turned the focus towards the cyclo depsipeptidic

mycotoxin, destruxin produced by this fungus and revealed its bright perspective

for insect pest control. Destruxins are produced by various fungi, and a direct

relationship has been established between destruxin production and the virulence

of the entomopathogen Metarhizium anisopliae.

Different application strategies are adopted for testing the effect of dtx

on various insect species. The most widely used one is injection or forced feeding

as demonstrated in Manduca sexta (Samuels et al., 1988; Vey and Quiot, 1989;

Dumas et al., 1994). Insecticidal activity up on topical application is dependent

on the target insect species. Fargues et al. (1986) noticed that dtx has no

unsecticidal properties on the larvae of Galleria mellonella when applied

topically while others reported contact toxicity in pests like Empoasca vitis,

Phaedon cochleariae (Poprawski et al., 1994; Amiri et al., 1999).

Insecticidal activity of dtxs was examined in insects belonging to all

orders of insect (Pedras et al., 2002). Toxins were administered by topical

application, forced ingestion, immersion or injection to larvae or adult insects.

Dtxs cause initial titanic paralysis, which at lethal doses leads to death of the

insect (Samuels et al., 1988).

Studies for establishing the role of dtx in the pathogenicity of M.

anisopliae suggested a correlation between in vitro production of dtx and fungal

virulence (Fargues et al., 1986; Kershaw et al., 1999) with dtxs acting as


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virulence factors by facilitating penetration of pathogen into the host (Samuels et

al., 1988). According to the studies of Wang et al. (2003) the mutant strain

devoid of the dtx production ability has been observed to possess the ability to

kill G. mellonella as fast as the wild type strain. It has been reported that non-

toxin producing strains of M. anisopliae still exhibited high pathogenecity

against different insect hosts (Kershaw et al., 1999; Amiri et al., 2000). In this

regard, two virulence strategies are proposed i.e. the ‘toxin strategy’ and ‘growth

strategy’ (Valadares-Inglis and Peberdy, 1998). This indicated that the toxin

producing strain showed limited growth in the insect haemolymph but produced

dtx in sufficient quantities to cause host death. On the other hand, non-toxin

producing strain implemented copious growth in the haemolymph to cause

disruption of homoestasis and starvation leading to host death (Valadares-Inglis

and Peberdy, 1998). Larvae of Phaedon cochleariae were found to be more

susceptible to infection by M. anisopliae if it was used in conjunction with a

crude dtx mixture (Amiri et al., 2000). Therefore, exploring the interaction

between the lower doses of dtx and the fungus, M. anisopliae becomes important

for improving mycoinsecticides and decreasing the risks of the same in the

environment. Fungi producing secondary metabolites, derivatives from various

intermediates in primary metabolism, some of which have insecticidal activities

were also reported (Vey et al., 2001). Huxam et al., (1989) reported the inhibition

of haemocytic activation of freely circulating cells associated with immune

response in Periplaneta americana by destruxin when the later was injected in to

the haemocoel.


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IV. Compatibility of entomopathogenic fungi with pesticides, fungicides

and botanicals

Combined utilization of selective insecticides in association with fungal

pathogens can increase the efficiency of control by reduction of the amount of

applied insecticides, minimizing environmental contamination hazards and pest

resistance (Moino and Alves, 1998; Quintela and McCoy 1998). Conidial

survival may be affected either by environmental factors (Furlong and Pell, 1997)

or by bio-pesticides and/or chemical products used to protect crop plants

(Anderson and Roberts 1983; Loria et al., 1983; Alves and Lecuona, 1998).

Many experiments have been carried out aiming to detect side effects of

pesticides on entomopathogenic fungi (Olmert and Kenneth, 1974; Gardner and

Storey, 1985; Neves et al., 2001). The use of incompatible pesticides with

enthomopatogenic fungal propagules and products may inhibit the development

and reproduction of biocontrol agents, and this negatively affect the efficacy of

IPM programme (Duarte et al., 1992; Malo, 1993). Most of the studies on

compatibility evaluated the effect only on vegetative growth and sporulation,

disregarding conidial germination in compatibility studies. Germination is also an

important criterion to evaluate compatibility of pesticides with entomopathogenic

fungi in vitro (Anderson and Roberts, 1983). Neves et al. (2001) pointed out the

importance of condial germination in compatibility studies and emphasized that

the inhibition of this initial step affect the plain development of the fungus in the

field because this fungal structure is responsible for initiating the disease on

insect pests. Success of a pest control programme using entomopathogenic fungi,

however, depends on conidial survival in the field environment. Todorova et al.

(1998) reinforced the importance of pesticides influencing conidial germination,


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since they are responsible for the occurrence of first disease foci in the field.

Duarte et al. (1992) pointed out the importance in considering the antagonistic

effect of the pesticides on all developmental phases of entomopathogenic fungi

since these products may affect the bio-insecticide potential as well as the

occurrence of epizootics. Several studies have contributed information for the

choice of pesticides with more selective action on the entomopathogens, and

most of them were conducted under laboratory conditions (Castinerias et al.

1991; Silva et al., 1993). One of the options for improving the efficacy of the

entomopathogenic fungi is combined application with sublethal doses of

insecticides (Hiromori and Nishigaki, 1998). The knowledge of the compatibility

between the entomopathogenic fungi and pesticides may facilitate the choice of

these products in Integrated Pest Management (IPM) programs. Lecanicillium

muscarium, an insect pathogen that is used commercially to control greenhouse

pests and is a candidate species for the control of B. tabaci, was suggested to be

applied sequentially with imidacloprid, IPM Strategy (Cuthbertson et al., 2005).

The German cockroach, Blattella germanica (L.), is an important

structural pest controlled primarily with synthetic organic insecticides

(organophosphates, pyrethroids, and carbamates) (Rust et al., 1993; Benson and

Zungoli, 1997). Compatibility between M. anisopliae and insecticides can lead to

a reduced use of insecticides (Quintela and McCoy1997) for cockroach control

there by reducing human exposure (Sanyang and Van-Emden, 1996) in the urban

structures. The potential impact in the use of M. anisopliae and insecticides

combination has been evaluated only once in a German cockroach control

program (Kaakeh et al., 1997). Widespread resistance to these insecticides in

populations of this cockroach (Rust et al., 1993; Valles and Yu, 1996; Holbrook


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et al., 1999), and concern about human safety and the environment have

motivated investigations of alternative methods of cockroach control. Pesticides

can also act in a positive manner in combination with entomopathogens where, at

sublethal doses they interact with the latter causing or activating infectious

diseases by stress, or turning the insects more susceptible to the action of

microbial toxins (Batista Filho et al., 2001). To harness the benefits of

entomopathogenic fungus their compatibility with insecticides becomes decisive

for combined use, while the potential inhibitory effects of insecticides on the

entomopathogenic fungus cannot be ignored (Amutha et al., 2010).

a. Pesticides

There are numerous examples where the application of chemical

pesticides has enhanced the efficacy of entomopathogens against insect pests

(Kruger and McCoy (1997); Kaakeh et al. (1997); Gardner and Kinard (1998).

James and Elzen (2001) and Alizadeh et al. (2007) reported that imidacloprid had

no negative effect on B. bassiana. The increased virulence in the combination of

entomopathogenic fungus M. anisopliae with imidacloprid against the dengue

vector Aedes aegypti (Paula et al., 2011). Nasirian et al., (2006) reported that the

fipronil and imidacloprid gel baits completely killed the German cockroaches

under laboratory conditions in ingested bait method. It was also found to exert

synergistic effect on insects at sublethal or lethal dose either as spray mixture or

as bait with conidia of the fungus and a number of laboratory studies have

reported efficacy of fipronil and imidacloprid gel baits in control of cockroach

infestation but only a few field studies have been done so far according to

Gardner and Kinard (1998).


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Monocrotophos was reported to be compatible to B. bassiana (Umadevi

et al., 2003). Mochi et al. (2005) reported that the toxic action of pesticides on

the fungus in soil is small, suggesting that this bioagent can be used in

combination with pesticides without compromising its activity. The fungal

(Fusarium semitectum) formulation sprayed in combination with pesticide,

monocrotophos was reported to be safe against broad mite,

Polyphagotarsonemus latus (Mikunthan and Manjunatha, 2009). Enhanced lethal

effect of M. anisopliae on Periplaneta americana was observed when applied in

combination with chlorpyrifos (Wakil et al., 2012). Amutha et al. (2010)

suggested Quinalphos and chlorpyrifos to be used in combination with B.

bassiana which were proved to be less toxic to the fungal pathogen. Shafa Khan

et al. (2012) concluded that B. bassiana and M. anisopliae were most sensitive to

chlorpyrifos but, imidachloprid, monocrotophos, quinalphos were recommended

to be highly safe and most compatible to the same fungi.

Chlorpyrifos had been reported to strongly inhibit the growth and

sporulation of B. bassiana in a dose-dependent manner even at concentrations

lower than recommended rates of field use (Rao, 1989). Earlier reports by

Ambethgar (2003) indicate that chlorpyrifos and monocrotophos were slightly

harmful to B. bassiana at normal field dose. Oliveira et al. (2003) reported 100%

inhibition of the germination of B. bassiana using chlorpyrifos. Masarat (2009)

reported that strong inhibition of the growth of B. bassiana by chlorpyrifos and

endosulfan.

Investigations by Muhammed Ramzan Asi et al. (2010) revealed that

chlorpyriphos was most detrimental to M. anisopliae. Strategies have been

employed to increase efficiency and to accelerate insect mortality by combining


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entomopathogenic fungi with sub lethal doses of chemical insecticides and

botanicals. Chlorpyrifos was also reported to be toxic to adults of German

cockroach by Abd-Elghafar et al. (1992). Pachamuthu et al. (2000) found a

significant interaction between the entomopathogenic fungi and commercial

pesticide where significant differences in the LT50 values was observed when M.

anisopliae was used singly than in combination with chlorpyrifos comparatively .

The susceptibility level and insecticide resistance mechanisms of German

cockroach to organochlorated, organophosphate, carbamate and pyrethroid

insecticide groups have been studied by Nasirian (2010). The insecticide

resistance status in three hospital-collected strains of the German cockroach

using four commonly used insecticides from different classes by Limoee et al.

(2011) revealed the resistance of German cockroach to chlorpyrifos.

Based on fungal respiratory activity, the toxic action of a range of

pesticides (acaricides, fungicides, insecticides and herbicides) on M. anisopliae

in the soil is small, suggesting little negative impact on the fungal activity

resulting from their use (Mochi et al., 2005). Fungal biological control agents and

selective insecticide may act synergistically increasing the efficiency of the

control, allowing the lower doses of insecticides, preservation of natural enemies,

minimizing environmental pollution and decreasing the likelihood of

development of resistance to either agent (Boman, 1980; Moino and Alves, 1998;

Ambethgar, 2009). Efforts have been made to enhance the biological activity of

M. anisopliae by integrating it with sub lethal doses of chemical insecticides

(Zurek et al., 2002).


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b. Fungicides

An important factor to be considered in favour of entomopathogenic

fungi is that to date there have been no reports of development of resistance

(Santos et al., 2007). Klingen and Haukeland (2006) provide a comprehensive

review of the effects of agrochemicals on entomopathogenic fungi and concluded

that insecticides and herbicides were not generally harmful to fungal growth,

while fungicides were sometimes harmful. Studies on mite control in viticulture

have found copper-based fungicides to be more IPM-compatible than alternatives

such as carbamates in sparing predatory phytoseiid mites (Morando et al., 1996;

Rumbos et al., 2000). Ropek and Para (2002) showed that copper fungicides

inhibit the growth and infectivity of Verticillium lecanii, that can be important for

aphid control in citrus. Mani et al. (1995) found that exposure to copper

oxychloride reduced the longevity and fecundity of the citrus mealybug

parasitoid Leptomastix dactylopii (Hymenoptera: Encyrtidae). A glasshouse pot

trial by McLean et al. (2001) confirmed that Trichoderma harzianum, an

effective biocontrol agent of the onion white rot pathogen Sclerotium cepivorum,

was sensitive to mancozeb. Rebollar et al. (1996) reported that benomyl and

mancozeb showed a high degree of growth inhibition of Verticillium lecanii

fungus. Durán et al. (2004) mention that benomyl, dimethomorph-mancozeb,

chlorothalonil, propineb, mancozeb, and mancozeb-cymoxanil mixture

fungicides significantly affect germination and growth of B. bassiana while

fosetyl-Al, propamocarb, and copper oxychloride do not. It is interesting that one

fungicide (fosetyl-aluminium) appeared to stimulate mycelial growth of

Lecanicillium longisporum. Synergism has been identified between

entomopathogenic fungus and insecticides (Kaakeh et al., 1997; Shah et al.,


27

2007); therefore, it is possible that such trends may be present between some

fungicides and fungal biocontrol agents (BCAs). For example, Kouassi et al.

(2003) reported that application of B. bassiana followed by fungicide application

(metalaxyl, mancozeb and copper oxide) could synergize insecticidal activity.

Fungicides most effectively influenced the respiratory activity of M. anisopliae,

with copper oxychloride and mancozeb being the active ingredients that most

affected CO2 production by the fungus (Mochi et al., 2005). The background

information about the different degrees of entomopathogenic fungi showing

fungicide tolerance was also reported (Maribel et al., 2010). Mancozeb and

copper oxychloride were reported to be incompatible to B. bassiana and M.

anisopliae and caused complete inhibition of vegetative growth and spore

germination (Shafa Khan et al., 2012).

c. Botanicals

In recent years there has been an attempt to replace the synthetic

insecticides with less expensive, locally available, ecologically safe and socio-

friendly options including botanicals (Ban-wo and Adamu, 2003; Ogendo et al.,

2006; Talukder, 2006; Is-man, 2007). Jayaraj (1988) hinted the possibility of

combining botanicals with microbial for enhanced efficacy against insect pests.

The commercial plant based pesticides were well tolerated by B. bassiana where

neemgold and biospark were relatively very safe followed by exodos (Sahayaraj

et al., 2011). Vyas et al. (1992) reported that, neemark, a biopesticide of neem

was well tolerated by M. anisopliae. Neem oil, a biofertilizer was reported to be

detrimental to the germination of the Beauveria bassiana conidia and moderately

toxic to M. anisoplaie the combination of neem oil and B. bassiana conidia was

suggested not to be used in IPM programs by Hirose et al. (2001). Islam et al.


28

(2010) reported combined application of an entomopathogenic fungus and a

botanical insecticide may benefit from both, and it has proven effective for the

control of B. tabaci on eggplant. Neem is one of the general-purpose botanical

pesticides used in organic agriculture. It is widely used around the world today

either as a stand-alone treatment (Nadia et al., 1996; Kumar et al., 2005; Kumar

and Poehling, 2006) or in conjunction with synthetic pesticides or

entomopathogens (Depieri et al., 2005; Filotas et al., 2005; Mohan et al., 2007).

Azadirachtin enhances the vegetative growth and spore germination of B.

bassiana and M. anisopliae (Shafa Khan et al., 2012). The synergism between

the botanical insecticide, azadicarchtin and destruxin, a mycotoxin that was

extracted from M. anisopliae in a joint action against cotton aphid, Aphis gossypii

was reported by Fei Yi et al. (2012).

d. Pesticides used against Cockroaches

The German cockroach, B. germanica (L.), is controlled primarily with synthetic

organic insecticides (organophosphates, pyrethroids, and carbamates) (Schal and

Hamilton, 1990; Rust et al., 1993; Benson and Zungoli, 1997). Among the

pyrethroid compounds, deltamethrin and cypermethrin are often used in the form

of miraculous Chinese chalk stick, (locally named as Lakshman rekha), powder

and liquid to ward off the kitchen insects (Das and Sudip, 2006). Boric acid

(H3BO3) has been used as an insecticide for many years, especially against

cockroaches (Mallis, 1969). Recently, its use has been limited because it is a

slow-acting poison (Cornwell, 1976). Several weeks may be required to produce

a significant population reduction in the German cockroach, Blattella germanica

(L.) and a major resistance problem against many of the latter insecticides,

thereby greatly diminishing their usefulness and historical data have shown that


29

B. germanica has the capability to develop resistance to most insecticides in

wide-scale use (Cochran, 1989, 1995a). As a result, interest has again centered on

lesser-used compounds, including boric acid (Cochran, 1995b). It was reported

that boric acid when used along with B. bassiana increased the mortality rates in

American cockroach compared to when boric acid alone was used (Hernandez et

al., 2008). Boric acid is especially effective when used as part of an ongoing

integrated pest management (IPM) program according to Quarles (2001).

Widespread resistance to the insecticides used for the control the populations of

American cockroach (Scott et al., 1990; Rust et al., 1993; Holbrook et al., 1999;

Valles and Yu, 1996), and concern about human safety and the environment have

motivated investigations of alternative methods of cockroach control.

V. Insect host – pathogen interaction and mechanism of insect kill

The interaction between the entomopathogenic fungus M. anisopliae

and the host insect is a multifactorial process that culminates with the death of

the insect and include adhesion of conidia to the surface of the insect,

germination, penetration, invasive growth, and conidiation which are the

different stages of the pathogenic process and vary considerably in their mode of

action and virulence (Hernandez et al., 2010; Shahid et al., 2012). Death of the

infected host usually occurs during colonization of the hemocoel, where in the

host suffers depletion of nutrients, or starvation, as was shown in Culex pipiens

quinquefasciatus larvae infected with the oomycete Lagenidium giganteum

(Domnas et al., 1974). Inside the insect hemocoel the fungus switches from

filamentous hyphal growth to yeast-like hyphal bodies or protoplasts that

circulate in the hemolymph and proliferate via budding (Boucias and Pendland,

1982). The fungus later erupts through the cuticle and an external mycelium


30

covers all or parts of the host and formation of infective spores ensues under

appropriate environmental conditions (McCauley et al., 1968; Boucias and

Pendland, 1982). Both Beauveria bassiana and Metarhizium anisopliae have

been shown to produce metabolites within insect hosts with effects ranging from

paralysis to immunosuppression (Hajek and St. Leger, 1994; Hung and Boucias,

1992; Kershaw et al., 1999). Kershaw et al. (1999) hypothesized that differences

in isolate virulence can be attributed to the position of the phenotype of a

particular isolate occupies on a continuum between two main strategies; an

isolate may produce a large amount of toxins or may focus their energy into

vegetative growth. In addition to the toxic effects of metabolites, fungi could kill

insects via vegetative growth, with death occurring when fungal hyphae penetrate

vital organs, block the flow of hemolymph, or sap the nutritive resources from

the host (Clarkson and Charnley, 1996). Aspergillus flavus when injected in to

haemocoel of the German cockroach invaded various internal organs which was

observed in tissue sections that include alimentary canal, fatbody etc. (Pathak

and Kulshrestha, 1998).

The insect host is not a passive player in the infection process and neither is

death imminent once infection has been initiated where insects employ both

cellular and humoral defenses to combat microbial infection in which the

infected fungus encounters activated host defense mechanisms from the time it

attaches and attempts to penetrate the cuticle and even after successful

penetration it encounters hemocytes of host hemocoel engaged in encapsulation,

nodule formation, and phagocytic activities (Vey and Gotz, 1986; Gillespie et al.,

1997). Activation of the host’s innate defense system follows upon its detection

of the invading fungus via changes in the properties of the cuticle basement


31

membrane (Gunnarson, 1988) and substances associated with the fungal cell wall

(Unestam and Soderhall, 1977; Butt et al., 1996;). In most cases entomogenous

fungi are able to overcome host defenses by continuing to grow even after having

been phagocytized and by suppressing the spreading ability of granulocytes,

which prevents nodule formation (Hung et al., 1993). Efficient use of nutrients in

the haemolymph while combating the insect’s blood-borne defences will be

critical to successful parasitism (Xia et al., 2000). Quesada-Moraga et al. (2006)

reported that some proteins extracted from two M. anisopliae and B. bassiana

isolates gave significant mortalities against Spodoptera litura. These researches

revealed a bright perspective for pest control. However, the expensive costs and

latent danger to humans limited the toxins to use extensively and thereby,

exploring the interaction between mycotoxins and entomopathogenic fungi

become important for improving mycoinsecticides and decreasing the risks of

mycotoxins.

Dubovskii et al. (2008) reported a significant increase of the generation of

reactive oxygen species (ROS) and activities of enzymatic antioxidants in lymph

of the honeycomb moth Galleria mellonella L. at development of the process of

encapsulation of nylon implants. This has been established at a very early stage

when a nylon implant was pierced in to the cuticle and a capsule is formed on its

surface. Decrease of the enzymatic antioxidant activities in the insect hemocytes

were revealed after the implant incorporation. Oxidative stress during the viral

pathogenesis of insect cell lines was described previously by Wang et al. (2001).

Changes of superoxide dismutase (SOD) and glutathione-S-transferase (GST)

activities as well as of the content of SH-containing compounds were studied in

hemolymph of the Vairimorpha ephestiae microsporidian-infected greater wax


32

moth Galleria mellonella larvae by Lozinskaya et al., (2004) and it has been

reported that activity of the antioxidant system and generation of free radicals in

hemolymph of G. mellonella larvae change depending on the stage of

microsporidian development in the insect organism. The key role of ROS in

encapsulation and elimination of Plasmodium in the mosquito Anopheles was

reported by Mendoza et al. 2002 and Kumar et al. 2003. Reduced glutathione

(GSH) constitutes a second line in insect immunity as it plays a role in the

detoxification of toxins in insect body from concomitant oxidative stress (Nappi

and Vass, 2001; Kumar et al., 2003). The studies were carried out which indicate

participation of hemocytes in the ROS production in insects (Whitten and

Ratcliffe, 1999; Glupov et al., 2001; Nappi and Christensen, 2005) and it was

suggested that the ROS formed during encapsulation can participate in

elimination of parasite at the expense of the high reactional capability (Carton et

al., 2008). Reactive oxygen species (ROS) are used by insect as cytotoxic

materials against invading pathogens and parasites (Fang, 1999; Peterson and

Lukhart, 2006). Lyakhovich et al. (2006) viewed that various antioxidants that

were present in the insect may decrease the level of lipid peroxidation. The key

role in maintenance of the oxidation–reduction balance in hemolymph at

development of the encapsulation process is played by non-oxidative

antioxidants (Dubovskii et al., 2008). Dubovskiy et al. (2008) reported the

increased activities of SOD, GST, malondialdehyde and RSSR/RSH ratio and

decrease in catalase activity on the first and following days after bacterial

infection by Bacillus thuringiensis indicating the increased levels of oxidative

stress in the midgut of Galleria mellonella larvae. Elevated levels of lipid

peroxidation and protein oxidation was observed in Aedes caspius mosquitoes


33

when infected with Bacillus thuringiensis indicate the induction of oxidative

stress (Ahmed, 2012).

Oxidative stress arises as the result of ischemia- or anoxia-reperfusion in

various animal systems (Fuller et al., 1988; Halliwell and Gutteridge, 1989;

Ruuge et al., 1991). It is generally accepted that damage occurs early during the

oxygen reperfusion phase, due to the formation of reactive oxygen species (ROS)

from various sources, and that antioxidant systems are key to the removal of

these reactive species to prevent subsequent damage from their activity.

Surprisingly, little attention has been given to insects in this regard. Many studies

addressed oxidative stress and antioxidant defenses in response to plant pro-

oxidants or insecticides (Ahmad, 1992; Aucoin et al., 1995), where antioxidants

were essential for their detoxification, or the prevention of damage from their

activity. Insects are prone to the normal burden of oxidative stress associated

with an aerobic life style. The value of an antioxidant enzyme system depends

upon its location relative to where the oxygen radicals are generated (Natraj and

Dalibor, 2006). A variety of animals, including a large number of insect species,

undergo stresses comparable to ischemia-reperfusion as a natural part of their life

cycles. Detoxification mechanism must involve a right balance between the

formation and detoxification of reactive oxygen species (ROS). Insects have

evolved a complex antioxidant mechanism to overcome the toxic effects of ROS.

The antioxidant defense is primarily constituted by the enzymatic actions of

glutathione peroxidase (GPX), catalase (CAT), superoxide dismutase (SOD), and

ascorbate peroxidase. The cellular antioxidant status determines the susceptibility

to oxidative damage and is usually altered in response to oxidative stress

(Halliwell and Gutteridge, 1999). Alterations in the antioxidant enzyme activities


34

and increased oxidative damage have been observed in various insects that were

infected with various pathogens. The detection, characterization and analysis of

the role of ROS were well established in both normal and pathological processes

of cellular metabolism. All cellular components are susceptible to attack by ROS,

but the major multifold effects include membrane peroxidation, loss of ions,

protein cleavage, DNA inactivation, damage and strand breakage (Wolff et al.,

1986). One method by which ROS can cause cell death is by initiating and

propagating lipid peroxidation, which results in the loss of cell membrane

structure leading to increased permeability to ions and fluids (Jamieson, 1989).

To minimize the potential threats of ROS, the cells are equipped with numerous

antioxidant defense systems. Their function is to maintain low steady state levels

of ROS and other radicals in the cell, a process involving precise regulation of

their location and amount. The antioxidant enzymes, such as superoxide

dismutase (SOD, E.C. 1.15.1.1), catalase (CAT, E.C. 1.11.1.6) and peroxidase

(POX, E.C. 1.11.1.7) form a part of the defence system (Joanisse and Storey,

1996 a,b). Insects appear to rely on ascorbate POX (APOX, E.C. 1.11.1.11)

activity, which catalyses the oxidation of ascorbic acid with the concurrent

reduction of hydrogen peroxide (Mathews et al., 1997). SOD dismutates

superoxide anions directly (McCord and Fridovich 1969), but in this process,

potentially toxic hydrogen peroxide (H2O2) is generated.

Catalases and peroxidases are the most important enzymes that degrade

peroxide into water and oxygen. These enzymes and superoxide dismutase are

the first lines of cell defence against ROS. Antioxidant defence was measured by

activities of SOD, CAT and APOX. CAT and POX, more appropriately the

specific APOX, act to remove these peroxides. The key step in oxidative stress is


35

the production of reactive oxygen species (ROS) which initiate a variety of auto-

oxidative chain reactions on membrane unsaturated fatty acids and proteins,

producing lipid peroxides and protein carbonyls respectively resulting in a

cascade of reactions ultimately leading to destruction of organelles and

macromolecules (Jamieson, 1989). Hydroxyl radical (OH.), hydrogen peroxide

(H2O2) and superoxide radical (O2.-), the ubiquitous products of single electron

reductions of dioxygen, are amongst the most reactive compounds known to be

produced during oxidative stress (Dietz et al., 1999). The detection,

characterization and analysis of the role of reactive oxygen species (ROS) was

well established in both normal and pathological processes of cellular

metabolism. ROS were considered as probable cytotoxic agents responsible for

destruction of pathogenic organisms in insect haemolymph (Komarov et al.,

2009). The toxic effect of a cyclodepsipeptide, destruxin secreted by M.

anisopliae and its mode of action on the lepidopteran pest Spodoptera litura was

shown to be countered by the antioxidant enzymes to an extent governed by the

concentration and time of treatment (Sowjanya and Padmaja, 2008a, 2008b).

Oxidative stress is caused by free radicals such as reactive oxygen

species (ROS), which includes superoxide (O2.-), peroxyl, alkoxyl, hydroxyl and

nitric oxide. ROS are characterized by presence of an unpaired electron in their

outer orbit. In addition to these ROS radicals in living organisms, there are other

ROS non-radicals such as the singlet oxygen (1O2), hydrogen peroxide and

hypochlorous acid (Pietta, 2000). Small quantities of ROS are formed

spontaneously under normal conditions as byproducts of redox processes such as

oxidative phosphorylation in the mitochondria and β- oxidation of fatty acids.

However, the production of ROS is increased when the organism is subjected to


36

irradiation, chemicals or infection (Knopowski et al., 2002). Overproduction of

ROS damages cellular lipids, nucleic acids, proteins and leads to lipid

peroxidation, genome instability or gene mutation; protein carbonyl formation

and enzymatic inactivity resulting in degenerative processes leading to aging

(Martin et al., 1996; Berlett and Standtman, 1997; Finkel and Halbroook, 2000).

To defend against the ROS formed, animal cells use three enzymes, superoxide

dismutase, catalase and glutathione peroxidase. Superoxide dismutase converts

superoxide anion to oxygen and hydrogen peroxide. Catalase reduces hydrogen

peroxide to water and oxygen (Fridovich, 1978). Glutathione peroxidase

neutralizes hydrogen peroxide by taking hydrogens from two glutathione

molecules resulting in two H2O and one molecule of an oxidized form of

glutathione (Gaikwad et al., 2010). Lipid peroxidation is one of the major

outcomes of free radical-mediated injury to tissue. Studies have revealed the

susceptibility of the cellular components from the attack of ROS, but the major

multifold effects are manifested in the form of loss of ions and protein cleavage.

The generation of ROS like OH– and O2– radicals disintegrates biomembranes by

lipid peroxidation which is a general mechanism of stress induced responses in

living systems (Panda et al., 2003). Ultrastructural effects of crude destruxin on

the salivary gland were reported by Sowjanya et al. (2008), where characteristic

changes including detachment of microvilli, epithelial cell vacuolization was

observed through transmission electron microscopy of Spodoptera litura. ROS

include oxygen ions, free radicals and peroxides, both inorganic and organic and

are generally very small and highly reactive, because of the presence of unpaired

electrons (Aslanturk et al., 2011). Studies reported that organophosphate

pesticides caused lipid peroxidation and the alterations in the antioxidant defense


37

enzymes of insect (Gupta et al., 2010; Wu et al., 2011). Many researchers who

are interested in studying changes in the redox status rely on GSH levels as a

reliable indicator of an organism’s redox status (Eun Kyung Go et al., 2007).

Numerous antioxidants are known to forestall oxidative damage or to limit its

propagation. GSH, is a most abundant and an important intracellular thiol redox

regulator that plays a major role in both the maintenance of redox status and in

the protection of cells from electrophilic and oxidative attacks (Dickinson et al.,

2002). These antioxidants can act to detoxify the ROS upto a certain level

beyond which the ROS induced damage leads to larval mortality.

i. entomopathogenic fungi as biocontrol...

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