C H A P T E R

29
Metarhizium
Tarun Kumar Patel
Sant Guru Ghasidas Government P.G. College, Kurud, Dhamtari, India

1. Introduction

The use of insect pathogens as one of the major components in the biological control of
crop pests for integrated pest management is gaining general acceptance as a realistic goal
and has stimulated research on their effective utilization. Various entomopathogens and their
products are currently in use to control insect pests; these offer certain advantages over
conventional chemical insecticides.

1.1 Entomopathogenic organisms

The organisms that can act as a parasite of insects and kills or seriously disables them are
known as “entomopathogen.” The term entomopathogenic has a Greek origin in which
“entomon” refers to insect, and “pathogenic” means disease causing. Since they are
considered natural mortality agents and environmentally safe, there is worldwide interest
in the use of entomopathogenic organisms for biological control of insects and other
arthropod pests. A large number of entomopathogenic organisms have been known and
characterized. The entomopathogenic organism includes bacteria, viruses, fungi, nema-
todes, and protozoan.
The entomopathogenic fungi (EPF) Metarhizium anisopliae (Metschnikoff) Sorokin has
emerged as excellent biological control agents for soil-inhabiting insects (Klein and Lacey,
1999; Mazodze and Zvoutete, 1999; Choo et al., 2002; Milner et al., 2003; Ansari et al., 2004).
Metarhizium, the second-most common EPF after Beauveria, is a soil-borne fungus, showing
worldwide distribution. It infects predominantly soil-inhabiting insects. The genus
Metarhizium consists of over 200 species with a wide host range, including arachnids and
five orders of insects (Boucias and Pendland, 1998).

Beneficial Microbes in Agro-Ecology

https://doi.org/10.1016/B978-0-12-823414-3.00029-0 593 © 2020 Elsevier Inc. All rights reserved.
594 29. Metarhizium

2. Classification and taxonomic status of entomopathogenic fungi

More than 750 species of fungi from about 90 genera are known as insect pathogens (ento-
mopathogen), with many subspecies, pathotypes, strains, and isolates within some of these
isolates (Humber, 1997, Bielikova et al. 2012). Many genera of EPFs previously grouped
under the anamorphic class Hyphomycetes have been reclassified in the order Hypocreales
under phylum Ascomycota. The anamorphic states of these fungi are filamentous and fast
reproduce asexually by conidia generally formed aerially on conidiophores arising from
the substrate. Hypocreales are among the most commonly encountered insect pathogens
because cadavers often remain intact for long periods and the external mycelium is conspic-
uous. An overview of entomopathogenic fungal taxa from kingdom to genus level is given in
Table 2.3 (Scholte et al., 2004).
The phylum Zygomycota has two classes, Trichomycetes and Zygomycetes. The most
important entomopathogens in the Zygomycota belong to the order Entomophthorales.
The order consists of six genera comprising of about 200 species of EPFs (Scholte et al., 2004).
Deuteromycetes, one of the important classes of fungi, has a well-defined morphological
group known as Hyphomycetes consisting of many genera of EPF. All the members of this
group are filamentous fungi that reproduce by conidia, generally formed on conidiophores
(Mar et al., 2012). They have a wide host ranges and generally invade the host through the
external integument; however, infection through the digestive tract may also be possible
(Scholte et al., 2004).

3. Strategies for the isolation of Metarhizium

The classical microbiological methods have been used traditionally to isolate and identify
Metarhizium. Members of Metarhizium are facultative pathogens and are relatively easily
grown as pure culture on various-defined or semidefined culture mediums. However,
many nonpathogenic microorganisms quickly colonize insect cadavers, especially if they
are not already colonized by any pathogen. Consequently, it is often a challenge to determine
whether a fungus isolated from an insect cadaver was responsible for the insect’s death. The
isolation of Metarhizium can be achieved by various methods, based on source, type, condi-
tion, and suitability of the inoculum. However, the isolation methods may be grouped into
two major types: (a) Culture plate method and (b) Insect bait method (Table 29.1).

3.1 Culture plate method

Generally, Sabouraud’s dextrose agar with yeast extract (SDAY) supplemented with strep-
tomycin sulfate and penicillin is routinely used for cultivation by direct plating through serial
dilution. EPF may be isolated directly from insect cadavers on which the fungus has already
sporulated. If external sporulation has not occurred, cadavers may be placed in a suitable
environment with high relative humidity for proper conidiation. It is desirable to surface ster-
ilize the cadavers with sodium hypochlorite (0.5%e5%, v/v) and ethanol (70%, v/v) to
remove potential superficial contaminants. However, the results might be unsatisfactory if
the integrity of the cadaver integument is poor. For insects that have died of mycosis, vege-
tative growth and/or sporulation on the surface of the integument usually occurs within a
few days at 20e25
C under high conditions of humidity. Once adequate growth is observed
II. Fungi
 3. Strategies for the isolation of Metarhizium 595
TABLE 29.1 An overview of entomopathogenic
fungal taxa (from kingdom to genus
level).
Fungi

Chytridiomycota
Chytridiomycetes
Blastocladiales
Coelomycetaceae

Coelomomyces
Zygomycota
Zygomycetes
Entomophthorales
Ancylistaceae
Conidiobolus

Entomophthoraceae
Entomophthora
Erynia
Trichomycetes
Harpellales
Legeriomycetaceae

Smittum
Deuteromycetes (Hyphomycetes)
Culicinomyces
Beauveria
Metarhizium
Tolypocladium

Adopted from Scholte, E.-J., Knols, B.G.J., Samson, R.A., Takken, W.

2004. “Entomopathogenic fungi for mosquito control: a review.”
J. Insect Sci. 4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC528879/.

on cadavers, the fungus can be transferred to an appropriate medium. If conidiogenesis is

observed over the cadaver, conidia can be streaked on to an agar medium directly or conidia
can be suspended in buffer or water before being streaked. Another method requires the
homogenization of cadavers followed by serial dilution plating (Fig. 29.1) of the homogenate
on appropriate growth medium.
Maintenance of sterile environment and suppression of crosscontamination of sample is
more difficult using the aforementioned routine culture medium. In addition, inhibition of
contaminating fast-growing saprophytic fungi is more problematic than bacteria (Chase

II. Fungi
596 29. Metarhizium

FIGURE 29.1 The serial dilution plating technique for the isolation and enumeration of Metarhizium.

et al., 1986). Metarhizium are considered to be poor competitors relative to other soil borne
fungi. Rapid growing fungi from the genera like Trichoderma, Mucor, and Rhizopus can cover
Metarhizium colonies making isolation rather more difficult or impossible. Thus, the direct
isolation of entomopathogenic fungi from soil usually depends on the use of a selective
medium. The most accepted isolation method is the soil dilution plate method. Undesirable
fungi of soil may be inhibited by fungicides and other chemicals such as rose bengal,
benomyl, dodine, and cyclohexamide in amended selective media (Liu et al., 1993).
Media based on the differential activity of the fungicide, dodine (N-dodecylguanidine
monoacetate), have been successfully used to isolate Metarhizium spp. from soil or insect
cadavers. An oatmeal agar medium amended with dodine suppresses growth of contami-
nating fungi and support growth of Metarhizium spp. (Beilharz et al., 1982).
Addition of dodine in oatmeal agar medium results in improved isolation of EPF. In addi-
tion, media amendment with crystal violet enhances the contrast of fungal colonies. Howev-
er, 460 mg/L dodine and 380 mg/L of the fungicide benomyl was found suitable for effective
isolation of M. anisopliae (Chase et al., 1986). Whereas, some isolates of M. anisopliae were
inhibited at 300 mg/L of dodine, a modified formulation with a reduced concentration of
dodine (10 mg/L) and 500 mg/L of cyclohexamide led to improved recovery of Metarhizium
from soil (Liu et al., 1993). Rangel et al. (2010) developed a low-cost medium using lesser
amount of dodine. Some other dodine-free medium were also developed recently which
favored isolation of different species of EPF (Fernandes et al., 2010; Posadas et al., 2012).

3.2 Insect bait method

Insect baits may be used to indirectly isolate fungi from soil. In general, Metarhizium are
considered to be weak saprophytes, but since they possess the ability to infect living insects,
they can gain access to a living insect relatively free of competitors. Although larvae of the
greater wax moth (Galleria mellonella) are most commonly used, larvae of other insects such
as the large flour beetle (Tribolium destructor) and the pine bark beetle (Acanthocinus aedilis)

II. Fungi
 4. Identification and characterization of entomopathogenic fungi 597
may also be utilized for the selection of potent fungal isolates. After the recommendation of
the insect bait method for the selective isolation of EPF by Zimmermann (1986), a number of
studies have been carried out on isolation EPF using insect baits. Further, a set of recommen-
dations was published by the International Organization for Biological Control (IOBC) for
isolation of EPF using insect baits. The use of insect as bait might vary with the target fungi
to be isolated (Klingen et al., 2002).

4. Identification and characterization of entomopathogenic fungi

Several methods have been used to describe and differentiate the variations encountered
within the species of Metarhizium. These include macro- and microscopic characteristics
including colony/growth characteristics, morphological characteristics of spores and
sporangia, growth or nutrient requirements (Samson, 1981). Furthermore, other approaches,
including immunotaxonomic and chemotaxonomic methods, have also been used, neverthe-
less with limited success (Bidochka et al., 1994). It is obvious that taxonomic procedures are
becoming more and more complex with the identification of new species. However, it is
general perception that some advanced and reliable molecular techniques may be needed,
in addition to the traditional morphological characteristics, for the characterization and
identification of fungi at species level. In addition to morphological and biochemical param-
eters, the molecular techniques based on polymorphism of DNA using RAPD, metabolite
profile, extracellular protein profile, etc. could also be employed.

4.1 Bioassay against insect pests

Bioassay of Metarhizium can be performed using conidial suspension of fungal isolates.
Different growth stages of insect viz. egg, larva, pupae, or adult insect could be undertaken
for bioassay. The insects were first surface sterilized with any appropriate surface sterilizing
agent (e.g., sodium hypochlorite) followed by completely removal of surface sterilizing agent
using sterile distilled water. The larvae were subsequently dried by blotting with
UV-irradiated tissue paper and subsequently treated by dipping them in conidial suspen-
sions with gentle shaking for a minute to evenly distribute the conidia over the entire surface
of larvae. The larvae were fed on sterilized suitable food material.
Dong et al. (2009) bioassayed the conidial suspension of M. anisopliae on termite Odonto-
termes formosanus and found that hyphae invaded the integument and body cavity of the
termite, muscles and fat tissue were decomposed and absorbed by hyphae. The hyphae
seriously destroyed hemolymph, tissues, pipelines, formed net like structure and produced
large number of conidia in the body of the pest (Dong et al., 2009). Ansari et al. (2011) found
that the dry conidia of EPF have potential for the biocontrol of adult Culicoides nubeculosus
(biting midge).

4.2 Spodoptera litura as model insect pest for bioassay

Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), the common cutworm, is an
economically serious and polyphagous pest reported to attack on a wide range of food crops
(about 112 crops belonging to 44 families worldwide and 60 crops in India) (Moussa et al.,

II. Fungi
598 29. Metarhizium

1960). Indiscriminate use of chemicals to increase field produce has led to the development of
resistance to a wide range of insecticides in S. litura. Since other control measures are inade-
quate when applied alone, an integrated approach for the control of insect pest might prove
to be a feasible solution (Ramakrishnan et al., 1984; Armes et al., 1997; Seth and Sharma, 2001;
Shiff, 2002; Alphey et al., 2010, Guedes et al., 2016).

4.2.1 Host range of Spodoptera litura

Different species of Spodoptera, a polyphagous pest, show wide hosts range (Brown and
Dewhurst, 1975) covering over 40 families of plants having economic importance, of which
S. litura alone affects at least 87 species (Salama et al. 1971). Among the main crop species
attacked by S. litura in the tropics are Colocasia esculenta, cotton, flax, groundnuts, jute,
lucerne, maize, rice, soyabeans, tea, tobacco, and a number of vegetable crops viz. aubergines,
brassica, capsicum, cucurbit vegetables, phaseolus, potatoes, sweet potatoes, vigna, etc.

4.2.2 Life cycle of S. litura

Generally, the females insect lay 1000e2000 eggs in masses of 100e300 on the lower sur-
face of leaf of host plant (Miyahara et al., 1971) and cover them with hair-like scales of the
abdomen. A high temperature and low humidity adversely affect the fecundity of number
of eggs. Newly laid eggs of S. litura were reported to survive cold exposure to as lower as
1
C for eight days. Partially developed eggs survived longer than newly laid ones under
equivalent conditions. The eggs hatch in about 4 days in warm conditions, or up to
11e12 days in winter. The larvae pass through six instars in 15e23 days at 25e26
C. The
early larval stage (first to third instar) feed in groups, leaving the opposite epidermis of
the leaf intact. Later stage of the larvae (fourth to sixth instar) spend daytime hiding in the
soil near the host plant and feed at night and early in the morning. The pupal period is spent
in earthen cells in the soil and lasts about 11e13 days at 25
C. Longevity of adults is about
4e10 days, being reduced by high temperature and low humidity. The life cycle, therefore,
may be completed in about five weeks.

5. Secondary metabolites

An array of insecticidal and other bioactive secondary metabolites, e.g., destruxins, swain-
sinone, cytochalasin C has been identified in Metarhizium.

5.1 Destruxins
Destruxins (Dxs) are a class of insecticidal, antiviral, and phytotoxic cyclic depsipeptides
that are also studied for their toxicity to cancerous cell lines (Liu and Tzeng, 2012). They are
the most prevalent secondary metabolites produced by Metarhizium considered as important
virulence factors accelerating the deaths of infected insects. There are about 38 Dxs or Dx an-
alogs divided chemically into five groups from Dx A to E (Hu et al., 2006). Some Dxs, especially
destruxin A, B, and E (DA, DB, and DE), show insecticidal activities (Cavelier et al., 1998,
Thomsen and Eilenberg, 2000). Structure of destruxin A, destruxin B and destruxin E is
depicted in Fig. 29.2. Destruxin B had suppressive effects on hepatitis B virus surface antigen
gene expression in human hepatoma cells (Chen et al., 1997; Schrank and Vainstein, 2010).

II. Fungi
 5. Secondary metabolites 599

FIGURE 29.2 Structure of destruxins: destruxin A (A), destruxin B (B) and destruxin-C (C). Source: Data from
PubChem, URL: https://pubchem.ncbi.nlm.nih.gov.

II. Fungi
600 29. Metarhizium

TABLE 29.2 List of various destruxin and related information.

Name CID Molecular formula MW (g/mol)

Destruxin A2 157382 C28H45N5O7 563.68620

Destruxin A 122810 C29H47N5O7 577.71278

Destruxin A 71308749 C29H47N5O7 577.71278
Destruxin B26 157514 C29H49N5O7 579.72866
Destruxin B 3084477 C29H49N5O7 579.72866
Destruxin A 635789 C29H49N5O7 579.72866
Destruxin E 107863 C29H47N5O8 593.71218

Destruxin B 428373 C30H51N5O7 593.75524

Destruxin B 9873299 C30H51N5O7 593.75524
Destruxin B 73444 C30H51N5O7 593.75524
Destruxin B 24837370 C30H51N5O7 593.75524
Destruxin B (9CI) 23306078 C30H51N5O7 593.75524
AG-H-18542 157513 C29H49N5O8 595.72806

Destruxin E1 5 157510 C30H49N5O8 607.73876

Destruxin B1 127623 C31H53N5O7 607.78182
Homodestruxin B 163819 C31H53N5O7 607.78182
Homodestruxin B (9CI) 13890767 C33H53N5O7 607.78182
Homodestruxin B 23451520 C31H53N5O7 607.78182
Homodestruxin B 21607148 C31H53N5O7 607.78182

AG-H-18540 157511 C29H47N5O9 609.71158

AG-H-18541 157512 C31H51N5O9 637.76474
Pseudodestruxin B 9986956 C36H55N5O7 669.85120
Pseudodestruxin A 10078314 C37H57N5O7 683.87778
Destruxin E 107863 C29H47N5O8 593.71218

Source: Data from PubChem, URL: https://pubchem.ncbi.nlm.nih.gov.

These metabolites play an important role to weaken the host immune defense, damage the
muscular system and the malpighian tubules, affecting excretion and leading to feeding and
mobility difficulties (Kershaw et al. 1999; Pal et al., 2007). Infected insects usually seek places
with higher temperature in order to increase body temperature and so inhibit the develop-
ment of the infecting microorganism (Elliot et al., 2002). Therefore, the action of the destruxins
reducing host mobility would also impair the defense mechanism. Indeed Metarhizium
isolates that produce higher quantities of destruxins are more virulent (Sree et al., 2008;
Schrank and Vainstein, 2010). A list of destruxins and their related informations (chemical
IDs (CID), molecular formulae, and molecular weights (MW)) are given in Table 29.2.

II. Fungi
 7. Mechanism of pathogenicity in insect 601

6. Evolution of entomopathogenesis
The full genome of all the major EPFs is not available yet. Nevertheless, some of the EPFs
have been thoroughly investigated. Gene-level studies are limited only for the virulence,
conidiation, and stress related genes of selected EPFs. Metarhizium proteinases genes (Pr1
and Pr2) and adhesins (Mad1 and Mad2) were found associated with the pathogenesis. It
was also reported that protease inhibitors reduces the virulence in Metarhizium (Butt et al.,
2013). In M. anisopliae var. anisopliae strain ARSEF 2575 (recently renamed Metarhizium
robertsii; Bischoff et al., 2009), disruptions of key genes for the biosynthesis of serinocyclins
(Krasnoff et al., 2007) and the mutagenic NG-391 compound (Krasnoff et al., 2006) demon-
strated that none of these is required for virulence of the fungus against Spodoptera exigua
(Moon et al., 2008; Donzelli et al., 2010).
In a recent study Xiao et al. (2012) reported 45 nonribosomal peptide synthetase (NRPS),
polyketide synthase (PKS) and terpenoid synthase/cyclase genes in the B. bassiana genome,
which is more than Cordyceps militaris but less than those in Metarhizium spp. Three of the
putative biosynthesis clusters are highly conserved in the entomopathogens, but interestingly
absent in other fungi. This finding suggests that the evolution of entomopathogenicity in
fungi may be associated with the production of secondary metabolites.

7. Mechanism of pathogenicity in insect

EPF infects susceptible hosts via direct penetration through the cuticle. The infection
process can be divided as follows: conidia adherence to the host cuticle through hydrophobic
interactions and mucilaginous substances, germination of conidia, germ tube differentiation
into apressoria, development of a penetration peg and cuticle penetration, differentiation of
hyphae into mycelia in the hemolymph, host colonization, extrusion to the host cadaver
surface and conidiophores formation, and conidia production (Schrank and Vainstein, 2010).
The infection initiates with the deposition of conidia on the host cuticle followed by
adhesion and, under proper conditions of humidity and temperature, spore germination.
Adhesion is pivotal to the initiation of the infection and involves hydrophobic interactions
between the spore surface proteins (as hydrophobins) and the lipid layer that covers
arthropod cuticle (Fang et al., 2007). Lipid degradation has recently been reported to be
related to prepenetration growth of M. anisopliae on the host cuticle (Jarrold et al., 2007). These
molecules are present in the epicuticle, the first barrier against arthropod pathogenic micro-
organism, suggesting the importance of lipolytic enzymes in initial infection stages. Lipases
produced by M. anisopliae are suggested to take part in this process (Silva et al., 2005).
Moreover, lipases could be identified in the conidia surface. In fact, the degradation of the
lipid layer of the host could be important for the recognition of a susceptible host as well
as for the production of the initial molecules which support the initiation of the germination
of the conidia (Beys da Silva et al., 2010). Adhesin protein MAD1 was shown to be important
for the attachment of the conidia coming in contact to the host surface. It has been suggested
that MAD1, a mucilaginous hydrophilic protein, binds with the hydrophobins present in the
cuticle of insects and degrades it to facilitate the conidial germination (Wang and St. Leger,
2007; Schrank and Vainstein, 2010).The second stage of pathogenesis requires penetration of
germinating hyphae through the host cuticle. To achieve this, the EPFs secrete proteolytic,

II. Fungi
602 29. Metarhizium

chitinolytic and lipolytic enzymes and degrade major constituents of the cuticle (proteins,
chitin, and lipids) (Lubeck et al., 2008; Boldo et al., 2009).
After the fungus has breached the first line of defense (cuticle), successful infection may be
established only if the fungus overcomes the insects’ innate immune response. Most arthro-
pods respond in both a cellular and humoral manner to fungal infection, with immune acti-
vation occurring as early as the start of cuticle degradation at the penetration point (Schrank
and Vainstein, 2010). Fungi have two main strategies for overcoming host defense responses,
the development of cryptic growth forms that are effectively masked from the arthropod
defense responses, and the production of immune modulating substances that suppress the
host defense system. The production of a collagen-like protein (MCL1) by Metarhizium has
been suggested to be a part of an evasion mechanism in response to insect immune system
(Schrank and Vainstein, 2010). As the colonization inside host proceeds the nutrients become
depleted, the fungus emerges out by producing aerial hyphae followed by conidia on the
surface of the cadaver of dead insects (Small and Bidochka, 2005).

8. Enzymes associated with pathogenesis or virulence

It is inferred that the production of enzymes that disrupt the integrity of the hosts will have a
strong selective advantage for pathogens. The enzymes that can fully degrade chitin into
N-acetylglucosamine monomers are divided into N-acetylglucosaminidases and chitinases
(Schrank and Vainstein, 2010). N-acetylglucosaminidases catalyze the release of
N-acetylglucosamine (GlcNAc) monomers from GlcNAc dimers or from the nonreducing termi-
nus of GlcNAc multimers. Chitinases can be classified into two major classes: endochitinases that
cleave the chitin polymer at any point inside the fiber and exochitinases that cleave from the
nonreducing end of the polymer and release GlcNAc dimers. Six different chitinases (30, 33,
43.5, 45, 60 and 110 kDa) have already been characterized in M. anisopliae (St. Leger et al., 1991;
Pinto et al., 1997; Kang et al., 1999), however, only one M. anisopliae chitinase has been experimen-
tally shown to participate in the infection process (Da Silva et al., 2005). A 43.5 kDa chitinase
secreted from M. anisopliae strain E6 induced by the Boophilus microplus cuticle was also detected
by immunoproteomics. IgG antispore surface proteins could be used for searching for proteins
possibly involved in early stages of fungus versus tick infection followed by LC-MS/MS Santi
et al. (2009). Considering the number of chitinases secreted by Metarhizium, it is expected that
these enzymes act directly and synergistically along with other hydrolases throughout the infec-
tion process in order to solubilize the host cuticle, providing nutrition and allowing fungal pene-
tration to ramify and colonize the entire host (Schrank and Vainstein, 2010).

9. Characterization of cyclodepsipeptides

Cyclic peptides comprise a large number of natural products and synthetic compounds
showing potential application in biotechnology and medicine. Thin layer chromatography
(TLC) is classically used to detect the presence of destruxins in extracts of fungal cultures
by fluorescence indicators or oxidizing reagents (Gupta et al., 1989, Venkatasubbaiah et al.,
1994). High performance liquid chromatography (HPLC) with UV detection is readily used
for analysis of destruxins due to the absence of strong chromophores in their structure (Ped-
ras et al., 2002). These metabolites have been extracted using organic solvents from culture
filtrates of potential fungal strains following fractionation using silica gel chromatography

II. Fungi
 10. Applications of bioformulations of Metarhizium and their metabolic products 603
and/or reversed phase HPLC (Buchwaldt and Jensen, 1991). Chen et al. (1999) quantified
various destruxins by HPLC using calibration curves of purified destruxins.
Mass spectrometry (MS) has as much structural elucidation potential as NMR; however, it
is not widely used due to the lack of development of mass spectrometry structure elucidation
based workflows (Bouslimani et al., 2014). The advanced MS has several orders of magnitude
more sensitive than NMR and can be used to more efficiently analyze complex biological
mixtures. The major reasons for MS not being frequently applied to structure elucidation
are the availability of a limited number of established rules that can be applied. Nevertheless,
bioinformatics approaches and various data bases are helpful to overcome these limitations
(Bouslimani et al., 2014).
Loutelier et al. (1995) found fast atom bombardment mass spectrometry (FAB-MS) as a
very useful method for analysis of destruxins in crude fungal extracts. The modern method
for quantitative analysis of these compounds is typically carried out using liquid chromatog-
raphy separation combined with MS fitted with an ionization source, like electrospray ioni-
zation (ESI) or atmospheric pressure chemical ionization (APCI) (Ngoka and Gross, 1999;
Manicke et al., 2009). Generally, a separation device is directly coupled online with the MS
for the separation of various compounds present in the sample. However, prior separation
is not necessary in some cases, and it may be replaced by a simpler extraction step. Current
approaches including direct injection of the sample into the MS from sample plate and/or the
spotting of samples using a matrix for analysis by Matrix Assisted Laser Desorption/Ioniza-
tion (MALDI) are much popular (Ngoka and Gross, 1999).
The chemical structures of destruxins A and B were established by paper chromatography,
and the structure was confirmed by total synthesis (Suzuki et al. 1966; Tamura et al., 1964).
The structures of hydroxydestruxin B, hydroxyl-homo-destruxin B, and b-D-glucopyranosyl
hydroxyl-destruxin B were elucidated using MS and NMR data (Pedras et al., 1999, 2001),
whereas structural characterization of other naturally occurring destruxins have been
performed through MS and NMR techniques. The fragmentation patterns obtained by disso-
ciation of depsipeptide ester bond by applied electric field were used to assign the structures
of destruxins and cyclodepsipeptides (Suzuki et al. 1966; Suzuki and Tamura 1972; Ayer and
Penea-Rodriguez, 1987).
There are very few reports of rapid and high-throughput analysis for large number of sam-
ples (Fesik et al., 1987). A number of factors, such as sample carryover, data acquisition, and
processing require sincere considerations for rapid analysis in efficient and reproducible
manner (Ngoka and Gross, 1999). However, a survey of literature reveals that many of the
known cyclodepsipeptides from entomopathogenic fungi are yet to be sequence through MS.

10. Applications of bioformulations of Metarhizium and their

metabolic products

10.1 Biopesticide
Metarhizium species may be introduced in the field as conidia, mycelia fragments or
blastospores in aqueous solutions. Sunlight, temperature, humidity, substratum, and chemi-
cal pesticides, however, influence the stability, sensitivity, and persistence of the conidia.
These factors independently or collectively affect naturally occurring and introduced fungal
inoculum either directly or indirectly through the insect host. For example, high temperatures
can inhibit fungal growth and even cause lysis within the insect haemocoel (Butt et al. 1994).
604 29. Metarhizium

Metarhizium species can be introduced into the field as conidia, mycelial fragments or
blastospores in aqueous solutions. Various biotic and abiotic factors viz. sunlight, tempera-
ture, humidity, substrate, and chemical pesticides, however, influence the stability, sensi-
tivity, and persistence of conidia. These factors independently or inclusively influence the
natural fungal biome and introduced directly or indirectly through the host insect.
The extensive acceptance and use of fungi-based biopesticides depends on improvements
in the development of formulations that could increase virulence, extend the viability of the
pathogen and improve the efficiency of the application, with a prolonged persistence in the
field. Unfortunately, little progress has been made in improving existing formulations.
However, oil-based formulations are promising for the control of some pests. It is assumed
that the oil reduces the dependence of the saturated conditions normally required for success-
ful infections (Bateman et al., 1993). However, the mode of action of the oil to facilitate
pathogenicity in the field is not clear. It is possible that the oil releases nutrients or fungistatic
compounds diluted in the epicuticle that could otherwise stimulate or inhibit germination
and infection (Ibrahim et al., 1999).
The commercial formulation of Metarhizium is already used to control insect pests of
various crops, viz. in Brazil to control the parasites of sugar cane (spittlebug), the area treated
only with M. anisopliae is estimated at about 1 million hectares (Faria and Wraight, 2007).
They are used to control crop pests and vectors of various diseases such as mosquitoes
and flies. The Metarhizium can also be grown endophytically in plant tissues, stimulating
research into new plant protection measures. The use of personalized formulations of fungal
entomopathogens with enzymes can improve endophytic behavior, and this may increase the
effectiveness of plant protection strategies against herbivorous parasites (Krell et al., 2018).
Metarhizium are powerful biological control agents and do not need to be ingested to infect
host organisms because they can invade the cuticle through secretarial hydrolytic enzymes.
Furthermore, they can control the insect’s immune system through the diverse array of
secondary metabolites. Therefore, these can be used to control a wide variety of insects using
various inoculation/treatment mechanisms for insects with different feeding mechanisms
(e.g., suction, biting, or borers).

10.2 Plant growth promoter activity

Metarhizium species have recently been found to be plant rhizosphere associates as well as
insect pathogens. Plant roots treated with Metarhizium grew comparatively faster, and the den-
sity of plant root hairs also increases. They mycelia of Metarhizium grows within root cortical
cells as well as between the intercellular spaces with no apparent damage to the plant and pro-
motes the proliferation of root hairs. So, they can also colonize plant roots endophytically, thus
showing potential as a plant endosymbiont (Sasan and Bidochka, 2012). Fungal entomopath-
ogens, Metarhizium brunneum, promote the growth of wheat following their endophytic estab-
lishment within plants after treatment of seeds. It subsequently accords numerous benefits,
which primarily including enhancement of plant growth. M. brunneum was able to promote
various plant-growth parameters viz. height of the plant, root length, and root or shoot weight,
etc. When Metarhizium species are inoculated or applied in agricultural fields they show pref-
erentially associates with the plant rhizosphere (Jaber, 2018; Vega, 2018).
Because of their abundance, rhizospheric Metarhizium could have enormous environ-
mental impact, with coevolutionary implications (Liao et al., 2014). Metarhizium are also

II. Fungi
 12. Conclusion 605
able to transfer nutrients like nitrogen to their plant hosts. Recent studies have identified
genes and their encoded transporters involved in the movement of essential nutrients like
nitrogen, phosphorous, and nonlimiting soil nutrients between fungi and the host plant
(Behie and Bidochka, 2014). Metarhizium are also beneficial to mitigate high salt induced
oxidative stress condition and improve the quantity and quality of crops (Khan et al., 2012).

10.3 Suppression of disease-causing pathogens

It is reported that Metarhizium act as disease antagonist can suppress pathogens following
plant colonization as well. Fusarium culmorum is one of the main causal agents for crown and
root rot in wheat. Use of endophytic Metarhizium suppresses the probability of infection and
hence reduces the occurrence of crown and root rot disease. Furthermore, endophytic coloni-
zation in wheat crop with fungal entomopathogen resulted in a significant reduction in the
incidence, development and severity of the disease (Jaber, 2018).

11. Future prospects

Microbial control has evolved with a better understanding of the pathogens, their hosts,
and their environments. The major obstacle in exploiting fungi for arthropod control is
that they kill their hosts rather too slow. Even highly virulent isolates take two to five
days to kill an insect, and infected hosts can survive much longer, depending on dose and
environmental conditions (Alonso-Dıaz et al., 2007). Wang and St. Leger (2007) have devel-
oped a method to accelerate the killing speed modifying M. anisopliae to express a neurotoxin
from the scorpion Androctonus australis. The toxin dramatically increased pathogenicity and
virulence, and the modified fungus achieved the same mortality rates compared to the
wild type in tobacco hornworm at 22-fold lower doses, and, at certain concentrations,
reduced the survival times of infected mosquitoes by >40%. Boldo et al. (2009) have also
accelerated the killing rate by super expressing the chitinase of M. anisopliae. They reported
more than 20% reduction in the time required to kill the host, suggesting that over expression
of chitinase increases the efficiency of pest killing (Schrank and Vainstein, 2010). The charac-
terization of hydrolytic enzymes of Metarhizium at the gene level is important in ascertaining
the function of these in pathogenicity.
Fungal entomopathogens have been proposed as ecologically more suitable alternatives for
biocontrol of insect pests. Unfortunately, its effectiveness is still limited by its susceptibility to
ultraviolet (UV) light and low humidity. The development of various application mechanisms
and selections of improved endophytic strains of Metarhizium could overcome the traditional
obstacles that prevent the widespread adoption of fungal entomopathogens and also provide
ecological friendly alternative for the management of pests and plant pathogens.

12. Conclusion
Metarhizium plays the multiple ecological roles that could further be played by entomopa-
thogenic fungi. The chapter presents several areas that should receive focused attention to in-
crease the probability of success for making use of Metarhizium species, an effective
alternative to chemical control. Bearing such additional roles in mind while developing these

II. Fungi
606 29. Metarhizium

fungi as microbial agents could improve the value of many commercially available mycoin-
secticides. Additional research is needed to leverage these basic findings toward improve-
ments in microbial control.
These newly understood attributes provide possibilities to use fungi in multiple roles. In
addition to arthropod pest control, some fungal species could simultaneously suppress plant
pathogens and plant parasitic nematodes as well as promote plant growth. A greater under-
standing of fungal ecology is needed to define their roles in nature and evaluate their limita-
tions in biological control. More efficient mass production, formulation, and delivery systems
must be devised to supply an ever-increasing market. More testing under field conditions is
required to identify effects of biotic and abiotic factors on efficacy and persistence. Lastly,
greater attention must be paid to their use within integrated pest management programs;
in particular, strategies that incorporate fungi in combination with arthropod predators
and parasitoids need to be defined to ensure compatibility and maximize efficacy.
The Metarhizium carries out the multiple ecological functions. There are several areas that
should be focused for further investigation to promote the Metarhizium species as an effective
alternative to chemical pesticides. Considering the various additional roles into account in the
development of these fungi, microbial agents could improve the value of many commercially
available mycoinsetticides. Further research is needed to take advantage of these basic
outcomes toward improvements in the control of microbes.
These recently included attributes offer the possibility of using Metarhizium fungi in
multiple roles other than biopesticides. Greater understanding of fungal ecology is needed
to define their roles in nature and to assess their limits in biological control. More efficient
methods for the production, formulation and delivery systems must be designed to provide
a growing market. Further trials in field conditions are needed to identify the effects of biotic
and abiotic factors on efficacy, viability and endurance. Finally, more attention should be
paid to use of Metarhizium in integrated pest management programs to ensure compatibility
and maximize effectiveness.

References
Alonso-Díaz, M.A., García, L., Galindo-Velasco, E., Lezama-Gutierrez, R., Angel-Sahagún, C.A., Rodríguez-
Vivas, R.I., Fragoso-Sánchez, H., July 20, 2007. Evaluation of metarhizium anisopliae (Hyphomycetes) for the
control of Boophilus microplus (Acari: ixodidae) on naturally infested cattle in the Mexican tropics. Vet. Parasitol.
147 (3e4), 336e340. https://doi.org/10.1016/j.vetpar.2007.03.030.
Alphey, L., Benedict, M., Bellini, R., Clark, G.G., Dame, D.A., Service, M.W., Dobson, S.L., April 2010. Sterile-Insect
methods for control of mosquito-borne diseases: an analysis. Vector Borne Zoonotic Dis. 10 (3), 295e311. https://
doi.org/10.1089/vbz.2009.0014.
Ansari, M.A., Vestergaard, S., Tirry, L., Moens, M., February 2004. Selection of a highly virulent fungal isolate,
metarhizium anisopliae CLO 53, for controlling hoplia philanthus. J. Invertebr. Pathol. 85 (2), 89e96. https://
doi.org/10.1016/j.jip.2004.01.003.
Ansari, M.A., Pope, E.C., Carpenter, S., Ernst-Jan Scholte, Butt, T.M., January 10, 2011. Entomopathogenic fungus as a
biological control for an important vector of livestock disease: the Culicoides biting midge. PloS One 6 (1), e16108.
https://doi.org/10.1371/journal.pone.0016108.
Armes, N.J., Wightman, J.A., Jadhav, D.R., Ranga Rao, G.V., 1997. Status of insecticide resistance in Spodoptera litura
in Andhra Pradesh, India. Pestic. Sci. 50 (3), 240e248. https://doi.org/10.1002/(SICI)1096-9063(199707)
50:3<240::AID-PS579>3.0.CO;2-9.
Ayer, W.A., Pena-Rodriguez, L.M., May 1, 1987. Metabolites produced by alternaria brassicae, the black spot pathogen
of canola. Part 1, the phytotoxic components. J. Nat. Prod. 50 (3), 400e407. https://doi.org/10.1021/np50051a010.

II. Fungi
 References 607
Bateman, R.P., Carey, M., Moore, D., Prior, C., 1993. The enhanced infectivity of metarhizium flavoviride in oil formulations
to desert locusts at low humidities. Ann. Appl. Biol. 122, 145e152. https://doi.org/10.1111/j.1744-7348.1993.tb04022.x.
Behie, S.W., Bidochka, M.J., November 2014. Nutrient transfer in plant-fungal symbioses. Trends Plant Sci. 19 (11),
734e740. https://doi.org/10.1016/j.tplants.2014.06.007.
Beilharz, V.C., Parbery, D.G., Swart, H.J., January 1, 1982. Dodine: a selective agent for certain soil fungi. Trans. Br.
Mycol. Soc. 79 (3), 507e511. https://doi.org/10.1016/S0007-1536(82)80043-0.
Beys da Silva, Walter, O., Santi, L., Schrank, A., Vainstein, M.H., January 1, 2010. Metarhizium anisopliae lipolytic
activity plays a pivotal role in Rhipicephalus (Boophilus) microplus infection. Fungal Biology 114 (1), 10e15.
https://doi.org/10.1016/j.mycres.2009.08.003.
Bidochka, M.J., McDonald, M.A., St Leger, R.J., Roberts, D.W., February 1994. Differentiation of species and strains of
entomopathogenic fungi by random amplification of polymorphic DNA (RAPD). Curr. Genet. 25 (2), 107e113.

Bieliková, L., Landa, Z., Osborne, L.S., Curn, V., January 29, 2012. Characterization and identification of entomopa-
thogenic and mycoparasitic fungi using RAPD-PCR technique. Plant Protect. Sci. 38 (1), 1e12. https://doi.org/
10.17221/4813-PPS.
Bischoff, J.F., Rehner, S.A., Humber, R.A., July 2009. A multilocus phylogeny of the metarhizium anisopliae lineage.
Mycologia 101 (4), 512e530. https://doi.org/10.3852/07-202.
Boldo, J.T., Junges, A., Amaral, K.B.do, Staats, C.C., Vainstein, M.H., Schrank, A., October 2009. Endochitinase CHI2
of the biocontrol fungus metarhizium anisopliae affects its virulence toward the cotton stainer bug dysdercus
peruvianus. Curr. Genet. 55 (5), 551e560. https://doi.org/10.1007/s00294-009-0267-5.
Boucias, D.G., Pendland, J.C., 1998. Entomopathogenic fungi: fungi imperfecti. In: Boucias, D.G., Pendland, J.C. (Eds.),
Principles of Insect Pathology. Springer US, Boston, MA, pp. 321e364. https://doi.org/10.1007/978-1-4615-4915-4_10.
Bouslimani, A., Sanchez, L.M., Garg, N., Dorrestein, P.C., May 15, 2014. Mass spectrometry of natural products: current,
emerging and future technologies. Nat. Prod. Rep. 31 (6), 718e729. https://doi.org/10.1039/C4NP00044G.
Brown, E.S., Dewhurst, C.F., June 1975. The genus Spodoptera (Lepidoptera, Noctuidae) in africa and the near east.
Bull. Entomol. Res. 65 (2), 221e262. https://doi.org/10.1017/S0007485300005939.
Buchwaldt, L., Jensen, J.S., January 1, 1991. HPLC Purification of Destruxins Produced by Alternaria Brassicae in Culture
and Leaves of Brassica Napus. Phytochemistry 30 (7), 2311e2316. https://doi.org/10.1016/0031-9422(91)83638-2.
Butt, T.M., Greenfield, B.P.J., Greig, C., Maffeis, T.G.G., Taylor, J.W.D., Piasecka, J., Dudley, E., et al., December 13,
2013. Metarhizium anisopliae pathogenesis of mosquito larvae: a verdict of accidental death. PloS One 8 (12),
e81686. https://doi.org/10.1371/journal.pone.0081686.
Butt, T.M., Hajek, A.E., Humber, R.A., 1994. The effect of temperature on growth and survival of protoplasts of the
gypsy moth pathogen entomophaga maimaiga. J. Invertebr. Pathol. 64, 74e75.
Cavelier, F., Jean, V., André, F., Haraux, F., Sigalat, C., Traris, M., Vey, A., 1998. Natural cyclopeptides as leads for
novel pesticides: tentoxin and destruxin. Pestic. Sci. 52 (1), 81e89. https://doi.org/10.1002/(SICI)1096-
9063(199801)52:1<81::AID-PS666>3.0.CO;2-H.
Chase, A.R., Osborne, L.S., Ferguson, V.M., June 1, 1986. Selective isolation of the entomopathogenic fungi Beauveria
bassiana and metarhizium anisopliae from an artificial potting medim. Fla. Entomol. 69 (2), 285e292.
Chen, H.C., Chou, C.K., Sun, C.M., Yeh, S.F., May 1997. Suppressive effects of destruxin B on hepatitis B virus surface
antigen gene expression in human hepatoma cells. Antivir. Res. 34 (3), 137e144.
Chen, J.-W., Liu, B.-L., Tzeng, Y.-M., January 8, 1999. Purification and quantification of destruxins A and B from
metarhizium anisopliae. J. Chromatogr. A 830 (1), 115e125. https://doi.org/10.1016/S0021-9673(98)00849-8.
Choo, H.Y., Kaya, H.K., Jin, H., Lee, D.W., Kim, H.H., Lee, S.M., Choo, Y.M., April 1, 2002. Entomopathogenic
nematodes (Steinernema spp. and Heterorhabditis bacteriophora) and a fungus Beauveria brongniartii for biolog-
ical control of the white grubs, ectinohoplia Rufipes and exomala orientalis, in Korean golf courses. BioControl 47
(2), 177e192. https://doi.org/10.1023/A:1014559729607.
Dong, C., Zhang, J., Huang, H., Chen, W., Hu, Y., 2009. Pathogenicity of a new China variety of metarhizium aniso-
pliae (M. Anisopliae var. Dcjhyium) to subterranean termite Odontotermes formosanus. Microbiol. Res. 164 (1),
27e35. https://doi.org/10.1016/j.micres.2006.11.009.
Donzelli, B.G.G., Krasnoff, S.B., Churchill, A.C.L., Vandenberg, J.D., Gibson, D.M., April 2010. Identification of a
hybrid PKS-NRPS required for the biosynthesis of NG-391 in metarhizium robertsii. Curr. Genet. 56 (2), 151e162.
Elliot, S.L., Blanford, S., Thomas, M.B., August 7, 2002. Host-pathogen interactions in a varying environment: temperature,
behavioural fever and fitness. Proc. Biol. Sci. 269 (1500), 1599e1607. https://doi.org/10.1098/rspb.2002.2067.
Fang, W., Pei, Y., Bidochka, Michael J., April 2007. A regulator of a G protein signalling (RGS) gene, Cag8, from the
insect-pathogenic fungus metarhizium anisopliae is involved in conidiation, virulence and hydrophobin synthe-
sis. Microbiology 153 (Pt 4), 1017e1025. https://doi.org/10.1099/mic.0.2006/002105-0.

II. Fungi
608 29. Metarhizium

Faria, M.R. de, Wraight, S.P., December 1, 2007. Mycoinsecticides and mycoacaricides: a comprehensive list with
worldwide coverage and international classification of formulation types. Biol. Contr. 43 (3), 237e256. https://
doi.org/10.1016/j.biocontrol.2007.08.001.
Ferguson, C.M., Barton, D.M., Harper, L.A., Swaminathan, J., van Koten, C., Hurst, M.R.H., 2012. Survival of Yersinia
entomophaga MH96 in a pasture ecosystem and effects on pest and non-target invertebrate populations. N. Z.
Plant Protect 65, 166e173.
Fernandes, É.K.K., Keyser, C.A., Rangel, D.E.N., Foster, R.N., Roberts, D.W., September 1, 2010. CTC medium: a
novel dodine-free selective medium for isolating entomopathogenic fungi, especially metarhizium acridum,
from soil. Biol. Contr. 54 (3), 197e205. https://doi.org/10.1016/j.biocontrol.2010.05.009.
Fesik, S.W., Bolis, G., Sham, H.L., Olejniczak, E.T., April 7, 1987. Structure refinement of a cyclic peptide from
two-dimensional NMR data and molecular modeling. Biochemistry 26 (7), 1851e1859. https://doi.org/
10.1021/bi00381a010.
Guedes, R.N.C., Smagghe, G., Stark, J.D., Desneux, N., 2016. Pesticide-induced stress in arthropod pests for
optimized integrated pest management programs. Annu. Rev. Entomol. 61, 43e62. https://doi.org/10.1146/
annurev-ento-010715-023646.
Gupta, S., Roberts, D.W., Renwick, J.A.A., March 1, 1989. Preparative isolation of destruxins from metarhizium
anisopliae by high performance liquid chromatography. J. Liq. Chromatogr. 12 (3), 383e395. https://doi.org/
10.1080/01483918908051742.
Hu, Q.-B., Ren, S.-X., Wu, J.-H., Chang, J.-M., Musa, P.D., October 2006. Investigation of destruxin A and B from 80
metarhizium strains in China, and the optimization of cultural conditions for the strain MaQ10. Toxicon 48 (5),
491e498. https://doi.org/10.1016/j.toxicon.2006.06.018.
Humber, R.A., 1997. Chapter V-1 - fungi: identification. In: Lacey, L.A. (Ed.), Manual of Techniques in Insect Pathol-
ogy. Academic Press, London, pp. 153e185. https://doi.org/10.1016/B978-012432555-5/50011-7.
Ibrahim, L., Butt, T.M., Beckett, A., Clark, S.J., July 1, 1999. The germination of oil-formulated conidia of the insect
pathogen, metarhizium anisopliae. Mycol. Res. 103 (7), 901e907. https://doi.org/10.1017/S0953756298007849.
Jaber, L.R., December 2018. Seed inoculation with endophytic fungal entomopathogens promotes plant growth and
reduces crown and root rot (CRR) caused by Fusarium culmorum in wheat. Planta 248 (6), 1525e1535. https://
doi.org/10.1007/s00425-018-2991-x.
Jackson, T.A., Pearson, J.F., O’Callaghan, M., Mahanty, H.K., Willocks, M.J., 1992. Pathogen to product-development
of Serratia entomophila enterobacteriaceae) as a commercial biological control agent for the New Zealand grass
grub (costelytra zealandica). In: Jackson, T.A. (Ed.), Use of Pathogens in Scarab Pest Management. Travis R. Glare.
http://agris.fao.org/agris-search/search.do?recordID¼US201301763694.
Jarrold, S.L., Moore, D., Potter, U., Keith Charnley, A., February 1, 2007. The contribution of surface waxes to
pre-penetration growth of an entomopathogenic fungus on host cuticle. Mycol. Res. 111 (2), 240e249. https://
doi.org/10.1016/j.mycres.2006.10.007.
Kang, S.C., Park, S., Lee, D.G., May 1, 1999. Purification and characterization of a novel chitinase from the entomo-
pathogenic Fungus, Metarhizium anisopliae. J. Invertebr. Pathol. 73 (3), 276e281. https://doi.org/10.1006/
jipa.1999.4843.
Khan, A.L., Hamayun, M., Khan, S.A., Kang, S.-M., Khan Shinwari, Z., Kamran, M., Ur Rehman, S., Kim, J.-G., Lee, I.-J.,
April 2012. “Pure culture of metarhizium anisopliae LHL07 reprograms soybean to higher growth and mitigates salt
stress. World J. Microbiol. Biotechnol. 28 (4), 1483e1494. https://doi.org/10.1007/s11274-011-0950-9.
Klein, M.G., Lacey, lawrence a., June 1, 1999. An attractant trap for autodissemination of entomopathogenic fungi
into populations of the Japanese beetle popillia japonica (Coleoptera: scarabaeidae). Biocontrol Sci. Technol. 9
(2), 151e158. https://doi.org/10.1080/09583159929730.
Klingen, I., Eilenberg, J., Meadow, R., September 1, 2002. Effects of farming system, field margins and bait insect on
the occurrence of insect pathogenic fungi in soils. Agric. Ecosyst. Environ. 91 (1), 191e198. https://doi.org/
10.1016/S0167-8809(01)00227-4.
Krasnoff, S.B., Sommers, C.H., Moon, Y.-S., Donzelli, B.G.G., Vandenberg, J.D., Churchill, A.C.L., Gibson, D.M.,
August 25, 2006. Production of Mutagenic Metabolites by Metarhizium Anisopliae. Research-article. https://
doi.org/10.1021/jf061405r.
Krasnoff, S.B., Keresztes, I., Gillilan, R.E., Szebenyi, D.M.E., Donzelli, B.G.G., Churchill, A.C.L., Gibson, D.M.,
November 29, 2007. “Serinocyclins A and B, Cyclic Heptapeptides from Metarhizium Anisopliae.”. Review-
article https://doi.org/10.1021/np070407i.
Krell, V., Jakobs-Schoenwandt, D., Vidal, S., Patel, A.V., 2018. Cellulase enhances endophytism of encapsulated meta-
rhizium brunneum in potato plants. Fungal Biol. 122 (5), 373e378. https://doi.org/10.1016/j.funbio.2018.03.002.

II. Fungi
 References 609
Lacey, L.A., Grzywacz, D., Shapiro-Ilan, D.I., Frutos, R., Brownbridge, M., Goettel, M.S., November 2015. Insect path-
ogens as biological control agents: back to the future. J. Invertebr. Pathol. 132, 1e41. https://doi.org/10.1016/
j.jip.2015.07.009.
Liao, X., O’Brien, T.R., Fang, W., Leger, R.J.S., August 2014. The plant beneficial effects of metarhizium species corre-
late with their association with roots. Appl. Microbiol. Biotechnol. 98 (16), 7089e7096. https://doi.org/10.1007/
s00253-014-5788-2.
Liu, B.-L., Tzeng, Y.-M., December 2012. Development and applications of destruxins: a review. Biotechnol. Adv. 30
(6), 1242e1254. https://doi.org/10.1016/j.biotechadv.2011.10.006.
Liu, Z.Y., Milner, R.J., McRae, C.F., Lutton, G.G., November 1, 1993. The use of dodine in selective media for the isola-
tion of metarhizium spp. from soil. J. Invertebr. Pathol. 62 (3), 248e251. https://doi.org/10.1006/jipa.1993.1107.
Loutelier, C., Hubert, M., Marcual, A., Lange, C., Mercadier, J.-L., Cavelier, F., Verducci, J., Jacquier, R., 1995. Gas-phase reac-
tivity of protonated cyclodepsipeptidic toxins: the study of a systematic fragmentation pathway under fast-atom
bombardment desorption. Rapid Commun. Mass Spectrom. 9 (15), 1512e1515. https://doi.org/10.1002/
rcm.1290091510.
Lubeck, I., Arruda, W., Souza, B.K., Stanisçuaski, F., Carlini, C.R., Schrank, A., Vainstein, M.H., May 1, 2008.
Evaluation of metarhizium anisopliae strains as potential biocontrol agents of the tick Rhipicephalus (Boophilus)
microplus and the cotton stainer dysdercus peruvianus. Fungal Ecol. 1 (2), 78e88. https://doi.org/10.1016/
j.funeco.2008.09.002.
Manicke, N.E., Kistler, T., Ifa, D.R., Cooks, R.G., Zheng, O., February 1, 2009. High-throughput quantitative analysis
by desorption electrospray ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 20 (2), 321e325. https://
doi.org/10.1016/j.jasms.2008.10.011.
Mar, T.T., Suwannarach, N., Lumyong, S., December 2012. Isolation of entomopathogenic fungi from northern
Thailand and their production in cereal grains. World J. Microbiol. Biotechnol. 28 (12), 3281e3291. https://
doi.org/10.1007/s11274-012-1139-6.
Mazodze, R., Zvoutete, P., November 1, 1999. Efficacy of metarhizium anisopliae against heteronychus licas (scara-
baedae: dynastinae) in sugarcane in Zimbabwe. Crop Protect. 18 (9), 571e575. https://doi.org/10.1016/S0261-
2194(99)00061-7.
Milner, R.J., Samson, P., Morton, R., August 1, 2003. Persistence of conidia of metarhizium anisopliae in sugarcane
fields: effect of isolate and formulation on persistence over 3.5 years. Biocontrol Sci. Technol. 13 (5), 507e516.
https://doi.org/10.1080/0958315031000140965.
Miyahara, Y., Tanaka, A., Wakikado, T., 1971. Seasonal changes in the number and size of the egg masses of Prodenia
litura. Jpn. J. Appl. Entomol. Zool. http://agris.fao.org/agris-search/search.do?recordID¼US201302254734.
Moon, Y.-S., Donzelli, B.G.G., Krasnoff, S.B., McLane, H., Griggs, M.H., Cooke, P., Vandenberg, J.D., Gibson, D.M.,
Churchill, A.C.L., July 2008. Agrobacterium-mediated disruption of a nonribosomal peptide synthetase gene in
the invertebrate pathogen metarhizium anisopliae reveals a peptide spore factor. Appl. Environ. Microbiol. 74
(14), 4366e4380. https://doi.org/10.1128/AEM.00285-08.
Moussa, M.A., Zaher, M.A., Kotby, F., 1960. Abundance of the cotton leaf worm, Prodenia litura (F.), in relation to
host plants. I. Host plants and their effect on biology (Lepidoptera : agrotidae-Zenobiinae). Bull. Soc. Entomol.
Egypte 44. https://www.cabdirect.org/cabdirect/abstract/19630500077.
Ngoka, L.C., Gross, M.L., August 1999. Multistep tandem mass spectrometry for sequencing cyclic peptides in an ion-trap
mass spectrometer. J. Am. Soc. Mass Spectrom. 10 (8), 732e746. https://doi.org/10.1016/S1044-0305(99)00049-5.
Pal, S., Raymond, J., Leger, S., Wu, L.P., March 23, 2007. Fungal peptide destruxin A plays a specific role in suppress-
ing the innate immune response in Drosophila melanogaster. J. Biol. Chem. 282 (12), 8969e8977. https://doi.org/
10.1074/jbc.M605927200.
Pedras, M.S.C., Zaharia, I.L., Gai, Y., Smith, K.C., Ward, D.E., October 20, 1999. Metabolism of the Host-Selective
Toxins Destruxin B and Homodestruxin B: Probing a Plant Disease Resistance Trait. Rapid-communication.
https://doi.org/10.1021/ol991042z.
Pedras, M.S.C., Zaharia, I.L., Gai, Y., Zhou, Y., Ward, D.E., January 16, 2001. In planta sequential hydroxylation and
glycosylation of a fungal phytotoxin: avoiding cell death and overcoming the fungal invader. Proc. Natl. Acad.
Sci. U.S.A. 98 (2), 747e752. https://doi.org/10.1073/pnas.98.2.747.
Pedras, M.S.C., Irina Zaharia, L., Ward, D.E., March 2002. The destruxins: synthesis, biosynthesis, biotransformation,
and biological activity. Phytochemistry 59 (6), 579e596.
Pinto, A.de S., Barreto, C.C., Vainstein, M.H., Schrank, A., Ulhoa, C.J., April 1, 1997. Purification and characterization
of an extracellular chitinase from the entomopathogen metarhizium anisopliae. Can. J. Microbiol. 43 (4), 322e327.
https://doi.org/10.1139/m97-045.

II. Fungi
610 29. Metarhizium

Posadas, J.B., Comerio, R.M., Jorge, I., August 2012. Mini, ana L. Nussenbaum, and Roberto E. Lecuona. “A novel
dodine-free selective medium based on the use of cetyl trimethyl ammonium bromide (CTAB) to isolate Beauveria
bassiana, metarhizium anisopliae sensu lato and paecilomyces lilacinus from soil. Mycologia 104 (4), 974e980.
https://doi.org/10.3852/11-234.
PubChem, URL: https://pubchem.ncbi.nlm.nih.gov.
Ramakrishnan, N., Saxena, V.S., Dhingra, S., 1984. Insecticide-resistance in the population of Spodoptera litura (F.) in
Andhra Pradesh. Pesticides 18 (9), 23e27.
Rangel, D.E.N., Dettenmaier, S.J., Fernandes, É.K.K., Roberts, D.W., January 1, 2010. Susceptibility of metarhizium
spp. and other entomopathogenic fungi to dodine-based selective media. Biocontrol Sci. Technol. 20 (4),
375e389. https://doi.org/10.1080/09583150903518370.
Salama, H.S., Dimetry, N.Z., Salem, S.A., 1971. On the host preference and biology of the cotton leaf worm Spodop-
tera littoralis bois. Z. Angew. Entomol. 67 (1e4), 261e266. https://doi.org/10.1111/j.1439-0418.1971.tb02122.x.
Samson, R.A., 1981. Identification: entomopathogenic deuteromycetes. Microb. Cont. Pests Plant Dis. 1970-1980.
http://agris.fao.org/agris-search/search.do?recordID¼US201302855628.
Santi, L., Silva, W.O.B.da, Pinto, A.F.M., Schrank, A., Vainstein, M.H., December 2009. “Differential immunoproteo-
mics enables identification of metarhizium anisopliae proteins related to Rhipicephalus microplus infection. Res.
Microbiol. 160 (10), 824e828. https://doi.org/10.1016/j.resmic.2009.09.012.
Sasan, R.K., Bidochka, M.J., January 2012. The insect-pathogenic fungus metarhizium robertsii (clavicipitaceae) is also
an endophyte that stimulates plant root development. Am. J. Bot. 99 (1), 101e107. https://doi.org/10.3732/
ajb.1100136.
Scholte, E.-J.,B.G.J.K., Samson, R.A., Takken, W., June 23, 2004. Entomopathogenic fungi for mosquito control: a
review. J. Insect Sci. 4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC528879/.
Schrank, A., Vainstein, M.H., December 15, 2010. Metarhizium anisopliae enzymes and toxins. Toxicon 56 (7),
1267e1274. https://doi.org/10.1016/j.toxicon.2010.03.008 highlights in toxinology: Biodiversity in Toxins - Tools
for Biological Research and Drug Development.
Seth, R.K., Sharma, V.P., June 1, 2001. Inherited sterility BY SUBSTERILIZING radiation IN spodoptera litura
(lepidoptera: noctuidae): bioefficacy and potential for pest suppression. Fla. Entomol. 84, 183.
Shiff, C., April 1, 2002. Integrated approach to malaria control. Clin. Microbiol. Rev. 15 (2), 278e293. https://doi.org/
10.1128/CMR.15.2.278-293.2002.
Silva, W.O.B., Mitidieri, S., Schrank, A., Vainstein, M.H., January 1, 2005. Production and extraction of an extracel-
lular lipase from the entomopathogenic fungus metarhizium anisopliae. Process Biochem. 40 (1), 321e326.
https://doi.org/10.1016/j.procbio.2004.01.005.
Silva, M.V.da, Santi, L., Staats, C.C., da Costa, A.M., Colodel, E.M., David Driemeier, Vainstein, M.H., Schrank, A.,
April 2005. Cuticle-induced endo/exoacting chitinase CHIT30 from metarhizium anisopliae is encoded by an
ortholog of the Chi3 gene. Res. Microbiol. 156 (3), 382e392. https://doi.org/10.1016/j.resmic.2004.10.013.
Small, Cherrie-Lee, N., Bidochka, M.J., March 2005. Up-regulation of prl, a subtilisin-like protease, during conidiation
in the insect pathogen metarhizium anisopliae. Mycol. Res. 109 (Pt 3), 307e313.
Sree, K.S., Padmaja, V., Murthy, Y.L.N., 2008. Insecticidal activity of destruxin, a mycotoxin from metarhizium
anisopliae (Hypocreales), against Spodoptera litura (Lepidoptera: Noctuidae) larval stages. Pest Manag. Sci. 64
(2), 119e125. https://doi.org/10.1002/ps.1480.
St Leger, R.J., Cooper, R.M., Charnley, A.K., November 1, 1991. Characterization of chitinase and chitobiase produced
by the entomopathogenic fungus metarhizium anisopliae. J. Invertebr. Pathol. 58 (3), 415e426. https://doi.org/
10.1016/0022-2011(91)90188-V.

II. Fungi