Biocontrol Science and Technology

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A review on the genus Metarhizium as an

entomopathogenic microbial biocontrol agent
with emphasis on its use and utility in Mexico

Carolina Brunner-Mendoza, María del Rocío Reyes-Montes, Soumya

Moonjely, Michael J Bidochka & Conchita Toriello

To cite this article: Carolina Brunner-Mendoza, María del Rocío Reyes-Montes, Soumya
Moonjely, Michael J Bidochka & Conchita Toriello (2019) A review on the genus Metarhizium as
an entomopathogenic microbial biocontrol agent with emphasis on its use and utility in Mexico,
Biocontrol Science and Technology, 29:1, 83-102, DOI: 10.1080/09583157.2018.1531111

To link to this article: https://doi.org/10.1080/09583157.2018.1531111

Published online: 05 Oct 2018.

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BIOCONTROL SCIENCE AND TECHNOLOGY
2019, VOL. 29, NO. 1, 83–102
https://doi.org/10.1080/09583157.2018.1531111

A review on the genus Metarhizium as an entomopathogenic

microbial biocontrol agent with emphasis on its use and utility
in Mexico
Carolina Brunner-Mendozaa, María del Rocío Reyes-Montesa, Soumya Moonjelyb,
Michael J Bidochkab and Conchita Torielloa
a
Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de
México, Ciudad de México, México; bDepartment of Biological Sciences, Brock University, St. Catharines,
Canada

ABSTRACT ARTICLE HISTORY

Metarhizium is a genus of entomopathogenic fungi that was initially Received 11 June 2018
classified into three species and varieties. More recently, DNA Returned 24 September 2018
sequencing has improved the phylogenetic resolution of Accepted 28 September 2018
Metarhizium which now includes 30 species. The insect host
KEYWORDS
ranges vary within the genus and some species such as Fungi; Clavicipitaceae;
M. robertsii have broad host ranges, while others such as entomopathogenic;
M. acridum show a narrow host range and are restricted to the biological control
order Orthoptera. Metarhizium spp. are ubiquitous naturally
occurring soil inhabiting fungi, and some are rhizosphere
colonisers and their diversity has been attributed to various
selective factors (habitat type, climatic conditions, specific
associations with plants and insect hosts). Metarhizium have been
used for the biological control of insect pests that affect
economically important agricultural crops and have been tested
under laboratory and field conditions for the control of insect
vectors of human disease, showing the effectiveness of the
fungus against the target pest. In Mexico, Metarhizium species
have been used for the control of insect pests such as the
spittlebug (Hemiptera: Cercopidae), and locusts (Orthoptera) that
affect crops such as corn, bean and sugarcane. Biosafety studies,
such as dermal and intragastric tests in mammalian models have
also been carried out to ensure safety to humans and other
animals. Metarhizium shows great promise as an alternative to
chemical insecticides that has relatively low impact on human
health and the environment. Key features of Metarhizium for
biocontrol of insects are outlined with special reference to their
utility in Mexico.

Introduction
Metarhizium Sorokin (Hypocreales: Clavicipitaceae) is a genus of ascomycetous fungi, dis-
tributed worldwide and recognised as a biological control agent of insects (Goettel, Eilen-
berg, & Glare, 2005). The species within this genus inhabit the soil as saprobes, as

CONTACT Conchita Toriello toriello@unam.mx Departamento de Microbiología y Parasitología, Facultad de

Medicina, Universidad Nacional Autónoma de México, Ciudad de México 04510, México
© 2018 Informa UK Limited, trading as Taylor & Francis Group
84 C. BRUNNER-MENDOZA ET AL.

rhizosphere inhabitants (Hu & St. Leger, 2002), as endophytes (Behie, Zelisko, &
Bidochka, 2012; Wyrebek, Huber, Sasan, & Bidochka, 2011) and also show complex sym-
bioses as pathogens of insects (Vega et al., 2009), and antagonism of fungal plant patho-
gens (Sasan & Bidochka, 2012). Moreover, phylogenetic analysis has shown that the genus
is more closely related to fungal grass endosymbionts Claviceps and Epichloë than to
animal pathogens (Gao et al., 2011; Spatafora, Sung, Sung, Hywel-Jones, & White,
2007). These characteristics suggest that Metarhizium may have evolved from a plant sym-
biont lineage. Many of the species have not left the role as plant symbionts, subsequently
acquiring the ability to infect and kill insects as an adaptation that allowed access to this
specialised source of nitrogen (Barelli, Moonjely, Behie, & Bidochka, 2016). However,
other species have specialised as insect pathogens and are poor plant colonisers. It is
apparent that several Metarhizium species have multifunctional lifestyles that include
their role as insect pathogens and as plant symbionts. However, this review will focus
on the utility of these fungi as insect pathogens, a role for which they are most notable,
particularly in the context of their use in Mexico.

Nomenclature and taxonomy of Metarhizium spp.

The species was initially described by Metschinkoff as Entomophthora anisopliae, based on
the name of its insect host, the scarab beetle, Anisopliae austriaca. In 1880, it was renamed
Isaria destructor. Three years later, Sorokin proposed the generic name Metarhizium. The
species M. album, M. brunneum and M. chrysorrheae, were described before the classifi-
cation by Tulloch (1976) and Rombach, Humber, and Evans (1987). Initial classifications
(Figure 1) were based on morphological features including the size and shape of the
conidia. At first, three species were distinguished as M. album, M. anisopliae, and M. flavo-
viride, each one with corresponding varieties. The inclusion of molecular data (isozyme,
RAPD, RFLP analysis) and physiological characteristics allowed the elucidation of
cryptic species or different varieties within the genus (Bidochka, McDonald, Leger, &
Roberts, 1994; Cobb & Clarkson, 1993; Curran, Driver, Ballard, & Milner, 1994;
Fungaro, Vieira, Pizzirani-Kleiner, & Azevedo, 1996). It was not until a taxonomic revi-
sion of the genus Metarhizium, based on conidial size, RAPD patterns, and a phylogenetic
analysis using the Internal Transcribed Spacer (ITS) regions of the ribosomal DNA
(rDNA) – region 28S (D3) – revealed ten monophyletic clades (Driver, Milner, &
Trueman, 2000). However, most relationships in M. anisopliae and M. flavoviride were
not fully resolved, due to the lack of informative sites in the ITS regions. Bischoff,
Rehner, and Humber (2006) described M. frigidum, and three years later, utilised multi-
locus phylogenetic analysis (EF-1α, RPB1, RPB2 and β-tubulin) to clarify the taxonomic
status within the M. anisopliae complex (Bischoff, Rehner, & Humber, 2009). They
found that 5′ EF-1α DNA sequence variation allowed for a higher degree of phylogenetic
resolution of the terminal clades within the M. anisopliae sensu lato complex. Also note
that the species M. anisopliae sensu stricto was retained as part of the complex. Generally,
publications pre 2007 with M. anisopliae are sensu lato while those after that time are
usually sensu stricto. The results of these studies and the conidial sizes of certain strains
supported the monophyly of nine terminal taxa within the M. anisopliae species
complex. At present, multilocus analysis of the genus Metarhizium has distinguished 30
species (both asexual and sexual states), including the majority of species recognised in
 BIOCONTROL SCIENCE AND TECHNOLOGY 85

Figure 1. Four historical Metarhizium classifications since Tulloch (1976), Rombach et al. (1987) and up
to the most recent molecular classification by Kepler et al. (2014). 1 = Sorokin (1883). 2 = Metschnikoff
(1879); Petch (1931); Tulloch (1976), Rombach et al. (1987) and Samson (1974). 3 = Driver et al. (2000).
4 = Bischoff et al. (2006) y (2009). 5 = Kepler et al. (2014); 5.1 = M. anisopliae complex; 5.2 = M. flavovir-
ide complex; 5.3 Core Metarhizium; 5.4 = Metarhizium. *Teleomorph. ^Host (Squamata:
Chamaeleonidae).

Metacordyceps as well as Nomuraea, and those in the more recently described genus Cha-
maeleomyces (Kepler, Humber, Bischoff, & Rehner, 2014). Novel Metarhizium species are
still being described such as M. dendrolimatilis (Chen, Han, Liang, Liang, & Jin, 2017)
(Figure 1) and new lineages such as Mani1, Mani2 and Mani 3 within the M. anisopliae
s.l. have been found in Mexican and Brazilian strains (Brunner-Mendoza, Moonjely,
Reyes-Montes, Toriello, & Bidochka, 2017; Rezende, Zanardo, da Silva Lopes, Delalibera,
& Rehner, 2015).

Fungal features
The genus Metarhizium includes anamorphic states that do not produce synnemata and
the recently added Metarcordyceps-like teleomorphic states (Sung et al., 2007). Conidio-
phores are branched but are occasionally simple in some species, with apices of branches
bearing one to several phialides that may be truncate or elongate. Conidia may be hyaline,
lilac, brown or green, and form chains (Kepler et al., 2014). These structures vary in shape
(cylindrical, globose, ellipsoidal) and size (from 4.0−14.5 × 2.0−5.0 µm). M. majus is the
species with the largest conidia (8.5−14.5 × 2.5−2.0 µm) and M. acridum is the species
with the smallest conidia (4.0−5.5 × 2.0−3.0 µm) (Bischoff et al., 2009; Driver et al.,
2000). Conidia are usually the only distinguishing morphological features within this
genus, however conidia morphology is indistinguishable between closely related species,
such as M. anisopliae (Figure 2), M. brunneum, M. pingshaense and M. robertsii. Morpho-
logical features among Metarhizium species can be imprecise, as there can often be an
86 C. BRUNNER-MENDOZA ET AL.

Figure 2. Metarhizium anisopliae (EH-473/4). A. Conidia; B. Mature colony (9 days) in potato dextrose
agar at 28°C.

overlap of traits among different species (Kepler & Rehner, 2013). On agar media, Metar-
hizium mature colonies (8–9 days) are described as predominantly dark green, light green,
white or brownish, or even as bicolour in the centre (Figure 3). The morphology of the
colonies can differ depending on the artificial media type (Kamp & Bidochka, 2002),
senescence (Wang, Butt, & St, & Leger, 2005), and other factors. The edges may be
white with variable thickness while the reverse image (on a Petri dish) may be brownish,
orange, and yellow or white (Fernandes et al., 2010).

Habitat associations and host range

Metarhizium is an extremely diverse genus and diversity may be associated with habitat,
climate conditions, plants and/or insect host. Metarhizium are mesophilic fungi that grow
at temperatures between 10 and 40°C, with optimal temperature for germination and
growth between 25 and 30°C (Roberts & Campbell, 1977), and a thermal death near
50°C (Walstad, Anderson, & Stambaugh, 1970), although some strains show cold-active
growth (15°C) while others are heat tolerant (up to 35°C) (Bidochka, Kamp, Lavendar,
Dekoning, & De Croos, 2001). Although there was no evidence of a link between latitude
and cold or heat tolerance in those studies, cold–active isolates were indeed found in more
northern sites, whereas no cold-active isolates could be found below 43.5° latitude, while
some isolates from tropical regions were able to grow at temperatures above 35°C
(Bidochka, Kasperski, & Wild, 1998). Recently, Metarhizium isolates have been identified
from China with the ability to grow in gradients from 27 to 5°C, this may be related to the
environment from which they were isolated; they also observed a decrease in the richness
of species with an increasing elevation (Masoudi, Koprowski, Bhattarai, & Wang, 2018).
 BIOCONTROL SCIENCE AND TECHNOLOGY 87

Figure 3. Morphological differences in Metarhizium strains in PDA at 27°C, 14 old cultures. A. M. robertsii
(ARSEF 2575); B. M. acridum (7486); C. M. brunneum (2974); D. M. flavoviride (380189); E. M. guizhouense
(B77-ai); F. M. pingshaense).

Furthermore, numerous studies have demonstrated that species vary across different
regions, M. robertsii and M. brunneum being the dominant species in Ontario and
Western Siberia (Kryukov et al., 2017; Wyrebek et al., 2011), M. brunneum in Oregon
and M. brunneum and M. flavoviride in Denmark (Fisher, Rehner, & Bruck, 2011;
Keyser, De Fine Licht, Steinwender, & Meyling, 2015; Steinwender et al., 2014); M. aniso-
pliae in Mexico (Brunner-Mendoza, Moonjely, et al., 2017) and Brazil (Rezende et al.,
2015), and M. pingshaense in Japan (Nishi, Hasegawa, Iiyama, Yasunaga-Aoki, & Shi-
mitzu, 2011). On the other hand, the Metarhizium community composition has also
been linked to forested and agricultural habitats (Bidochka et al., 2001; Inglis, Duke,
Goettel, & Kabaluk, 2008; Kepler, Ugine, Maul, Cavigelli, & Rehner, 2015; Rocha,
Inglis, Humber, Kipnis, & Luz, 2013; Steinwender et al., 2014). Distribution and abun-
dance of Metarhizium species are both related to the presence of certain types of plants
(Kepler et al., 2015; Wyrebek et al., 2011).
Studies of diversity and genetic population structure in agricultural soils in Mexico
(Guanajuato), using morphological and molecular techniques (EF-1α, ITS and β-
tubulin) have revealed a greater diversity of species of Metarhizium (M. anisopliae,
M. robertsii and M. pingshaense), infecting white grubs (Coleoptera: Melolonthidae), as
compared to species of another insect pathogenic fungus, Beauveria (only B. pseudobassi-
ana) (Carrillo-Benítez, Guzmán-Franco, Alatorre-Rosas, & Enríquez-Vara, 2013). In
another study, Pérez-González et al. (2014) assessed the relative abundance and diversity
of entomopathogenic fungi across 11 different geographical locations in Mexican agricul-
tural soils, and found that Metarhizium was less frequent, compared to the abundance of
88 C. BRUNNER-MENDOZA ET AL.

Beauveria isolates. In contrast, in a study in the state of Oaxaca, M. anisopliae s.s. was the
most abundant species in soil and leaf samples taken from sugarcane plantations (Hernán-
dez-Domínguez et al., 2016). Metarhizium isolates from infected Aeneolamia postica
(Hemiptera: Cercopidae) from sugarcane plantations in the state of Tabasco (in Southern
Mexico), showed intra-specific variability in RAPD markers and ITS-rDNA sequences
(Bautista-Gálvez & González-Cortés, 2005). Furthermore, in a recent study of Metarhi-
zium strains from the ‘Centro Nacional de Referencia de Control Biológico (CNRCB)’
of the ‘Secretaria de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación
(SAGARPA)’ from different states (Veracruz, Oaxaca, San Luis Potosi) and hosts (Coleop-
tera, Hemiptera, Lepidoptera) were phylogenetically placed, using 5′ TEF, as M. anisopliae,
M. robertsii, M. guizhouense and M. pinghaense (Brunner-Mendoza, Moonjely, et al.,
2017). All of these data show the ample biodiversity from the genus Metarhizium in
Mexico.
The anamorphic stages of some Metarhizium species have a broad insect host range.
For example M. robertsii, can infect over 200 insect species representing the orders
Orthoptera, Dermaptera, Hemiptera, Diptera, Hymenoptera, Lepidoptera and Coleoptera
(Zimmermann, 2007). Other Metarhizium species or genotypes are restricted to certain
arthropods. For example M. acridum and M. album show a narrow host range restricted
to the Orthoptera and Hemiptera, respectively (Wang, Kang, Lu, Bai, & Wang, 2012).
Genomic analysis suggests Metarhizium divergence first as specialists (M. acridum,
M.album) then more recently a transition to generalists (M. anisopliae, M. brunneum
and M. robertsii) (Hu et al., 2014). Recently, there are some accounts of M. viride and
M. granulomatis as aggressive pathogens of chameleons (Reptilia) (Samson, 1974;
Sigler, Gibas, Kokotovic, & Bertelsen, 2010).

Mechanism of infection
The infection mechanism of Metarhizium begins when susceptible insects come in contact
with conidia from anthropogenically dispersed conidial suspensions or conidia found in
the soil (anthropogenic or natural) (Hesketh, Roy, Eilenberg, Pell, & Hails, 2010).
Conidia attach to the host insect cuticle via hydrophobic interactions (Ortiz-Urquiza &
Keyhani, 2013; Thomas & Read, 2007). The main proteins involved in this are conidial
surface hydrophobins one of which is coded by ssgA (St. Leger, Staples, & Roberts,
1992). Specific adhesins such as Mad1 (Metarhizium adhesion 1) also contribute to attach-
ment of conidia on host surface (Wang & St. Leger, 2007a). Conidial germination on the
insect cuticle is dependent on a variety of biotic factors such as insect cuticular hydrocar-
bons as well as abiotic factors (e.g. temperature, solar radiation, and humidity) (Boucias &
Pendland, 1991; Ortiz-Urquiza & Keyhani, 2013). Most entomopathogenic fungi require
high humidity (>90%) for germination. Some Metarhizium isolates show germination,
growth and conidiation up to 37°C and may tolerate up to 4 h of exposure to UV-B radi-
ation (Braga, Flint, Miller, Anderson, & Roberts, 2001; Okuno, Tsuji, Sato, & Fujisaki,
2012). After the germination stage, the fungus develops a hold-fast structure called an
appressorium where cAMP and Ca+2 ion signals are involved (Clarkson, Screen, Bailey,
Cobb, & Charnley, 1998). Beneath the appressoria a penetration peg is formed that pene-
trates the host cuticle (Boucias & Pendland, 1991). The penetration process is aided by the
production of several cuticular hydrolytic enzymes including proteases, chitinases and
 BIOCONTROL SCIENCE AND TECHNOLOGY 89

lipases. Contrary to some misconceptions, insect cuticle is predominantly composed of

protein while chitin is found in lesser amounts. Respectively, Metarhizium produces
several protein degrading enzymes the most significant of which, in insect pathogenesis,
is a subtilisin-like protease (Pr1). However a plethora of other protein degrading
enzymes are also produced and include a thermolysin-like metalloproteinase, a trypsin-
like serine protease (Pr2), and exo-acting peptidases (St. Leger, Joshi, & Roberts, 1998)
that degrade cuticular protein and also play a significant role in acquiring nutrients, avoid-
ing host defense by degrading antifungal proteins, and regulating the micro-environ-
mental pH (St. Leger, Nelson, & Screen, 1999).
Following penetration of the cuticle, the fungal hyphae enter the insect hemocoel which
triggers host defense mechanisms, such as the production of phenoloxidases, and also the
activation of hemocytes that release bioactives and accomplish phagocytosis, encapsula-
tion or nodulation to combat mycosis. Insect Pathogen Recognition Receptors (PRR’s)
such as peptidoglycans and β-glucan-binding proteins interact with fungal Pathogen
Associated Molecular Patterns (PAMP’s) such as mannans and fungal β-glucans and
initiate defense reactions (Butt, Coates, Dubovskiy, & Ratcliffe, 2016). Once inside the
host, fungal morphology changes from hyphae to yeast-like blastospores. Blastospores
multiply in the haemocel and invade other tissues, while the fungus continues the
uptake of nutrients. Here Metarhizium secretes acid trehalase directed at trehalose
hydrolysis, the main sugar found in insect haemolymph (Schrank & Vainstein, 2010).
Some Metarhizium strains produce secondary metabolites (destruxins) that facilitate
pathogenesis (Samuels, Charnley, & Reynolds, 1988) and induce flaccid paralysis,
causing cellular alterations and malfunction of the middle intestine, malpighian tubules
and muscle tissues (Dumas, Robert, Pais, Vey, & Quiot, 1994), blocking H+ ATPase
activity (Muroi, Shiragami, & Takatsuki, 1994) and interacting with Ca2+ channels
(Samuels et al., 1988). The destruxins of the Metarhizium genus and other entomopatho-
genic fungi are categorised into six major groups: A through F (Wang et al., 2012). Some of
these compounds are linked to virulence and host specificity in this genus (Amiri-Besheli,
Khambay, Cameron, Deadman, & Butt, 2000). The secretion of destruxin A could cause an
adversely effect in the insect immune response, but not enough to kill the host Ríos-
Moreno et al., 2016). Metarhizium isolates that produce larger amounts of destruxins
are more virulent (Sowjanya, Padmaja, & Murthy, 2008). Furthermore, some destruxins
act as immune modulators suppressing the insect-host immune response (Pal, St. Leger,
& Wu, 2007; Wang et al., 2012).
Fat body is one of the first tissues colonised by the pathogen and muscle tissue is the last
(Schneider, Widmer, Jacot, Kölliker, & Enkerli, 2012). Here the fungus accumulates cellu-
lar mass and growth continues until the insect is ramified with mycelia. When the internal
contents have been consumed, the fungus then develops structures that re-emerge from
the insect cadaver, once again producing conidia which disseminate around the
mummified insect (Boomsma, Jensen, Meyling, & Eilenberg, 2014; Butt et al., 2016).
Metarhizium has evolved expanded gene families of proteases, chitinases, cytochrome
P450s, polyketide synthases, and nonribosomal peptide synthetases for cuticle-degra-
dation, detoxification and toxin biosynthesis that may facilitate their ability to adapt to
heterogenous environments (Gao et al., 2011). Generalist species of Metarhizium are
characterised by expansion of protein-families, compared to species with restricted host
range (Hu et al., 2014).
90 C. BRUNNER-MENDOZA ET AL.

Some arthropods have developed adaptative behavioural response to prevent contact

with fungal inoculum. For example, social insects, such as the ant, Lasius japonicus
(Hymenoptera: Formicidae), prevent the spread of Metarhizium conidia in their colonies
through grooming behaviour (Okuno et al., 2012). Inhibition of insect grooming behav-
iour, as well as cellular and humoral immunity by synthetical or natural insecticides, sig-
nificantly promotes fungal killing (Fisher, Castrillo, Donzelli, & Hajek, 2017; Quintela &
Mccoy, 1997). Likewise, the locust Schistocerca gregaria (Orthoptera: Acrididae) infected
with M. acridum can raise its body temperature by basking in the sun in a process called
behavioural fever (Blanford & Thomas, 1999) in order to eliminate the fungus.

Development as a biological microbial control agent

A substantial number of mycoinsecticides and mycoacaricides have been developed
worldwide since 1960. Beauveria bassiana, Metarhizium, Isaria fumosorosea and B. brong-
niartii are the most common active ingredients in 171 products, out of which 47 are
Metarhizium-based products, commercially-available world-wide (Faria & Wraight,
2007). Mycoinsecticides are products formulated with living propagules of entomopatho-
genic fungi with the addition of an inert ingredient, a substance or an adjuvant that facili-
tates its handling, application and effectiveness (García de León & Mier, 2010). There are
several kinds of formulations, such as soluble powders, humectant powders, dispersible
and water-soluble granulate and aqueous suspensions. The method of application, formu-
lation and biotic and abiotic factors in the environment all play an important role in
efficacy, persistence and spatial distribution of fungal propagules.
In 1991 the first collection of entomopathogenic fungi in Mexico was conducted with
the expressed purpose of promoting and maintaining a reference collection of native
strains for the generation of scientific and technological research in order to develop bio-
logical control programmes. It was not until 1993 that an entomopathogenic fungus
(Metarhizium spp.) was used to control grasshopppers and spittlebugs in three sugar
cane crops (Jalisco, San Luis Potosí and Veracruz) (SAGARPA, 1999). Particularly, M. ani-
sopliae strains were obtained from spittlebugs (Hemiptera: Cercopideae) and for the con-
idial massive production and field application a biphasic system (growth in liquid culture
and subsequently inoculated into a solid substrate such as corn, wheat or rice) was carried
out. Nowadays, the collection of entomopathogenic fungi from the CNRCB (CHE-
CNRCB) has more than 170 native Metarhizium spp. strains, and due to the enormous
diversity of Metarhizium species present in Mexico, the interest of native strains for
insect control campaigns has increased (SAGARPA, 2015). There are several commercial
formulations whose active ingredient is Metarhizium (such as Meta-Sin, Bio-Blast, and
Fitosan-M) (García de León & Mier, 2010; Tamez-Guerra et al., 2001), and there are
recently approximately 31 laboratories in different states in Mexico that massively
produce Metarhizium strains for their commercialisation (SAGARPA, pers. comm.).
Metarhizium spp. have been applied mainly in insect control campaigns in which the
SAGARPA, the SENASICA (Servicio Nacional de Sanidad, Inocuidad y Calidad Agroali-
mentaria) – through the ‘Dirección General de Sanidad Vegetal’ (DGSV) –, and every state
government collaborate. M. anisopliae is mainly used in Mexican agricultural areas in the
Gulf of Mexico and the Pacific coastal plains to control spittlebugs (Hemiptera: Cercopi-
dae), a major pest that reduces the production and the quality of sugarcane fields
 BIOCONTROL SCIENCE AND TECHNOLOGY 91

(Bautista-Gálvez & González-Cortés, 2005). According to SENASICA (2017) M. anisopliae

has been applied to control the spittlebug in sugarcane fields in different states of Mexico.
However, M. anisopliae is being applied to control sugarcane spittlebugs in many private
sugarmills in states such as Veracruz, Tamaulipas, Oaxaca, etc. but accurate data are not
available. M. acridum has been applied to control a complex of grasshopper species (Bra-
chystola magna, B. mexicana, Melanoplus differentialis and S. purpurascens) that seriously
affect maize, beans, sorghum, soybean and pumpkin crops (Barrientos-Lozano & Alma-
guer-Sierra, 2009; SENASICA, 2012). SENASICA (2017) reports that approximately
41,698 ha have been treated with M. acridum to control different locust and grasshopper
species (Schistocerca piceifrons piceifrons, Melanoplus sp., Sphenarium sp., Brachystola sp)
in different states of Mexico. Unfortunately, the information concerning the results of
fungal mycoinsecticide applications in Mexican plantations are seldom published or
found in provisional reports very difficult to access.
In addition, several laboratory and field studies have demonstrated the effectiveness and
in some cases the potential of M. anisopliae in the control of arthropods responsible for
diseases of medical importance such as malaria (Bukhari, Takken, & Koenraadt, 2011;
Scholte, Knols, & Takken, 2006), dengue fever (Garza-Hernández et al., 2013; Lobo, Rodri-
gues, & Luz, 2016; Reyes-Villanueva et al., 2011) and Chagas disease (Flores-Villegas et al.,
2016; Vázquez-Martínez, Cirerol-Cruz, Torres-Estrada, & López, 2014), Lyme disease
(Benjamin, Zhioua, & Ostfeld, 2002), as well as in the control of tick populations in
cattle (Alonso-Díaz et al., 2007; Fernández-Salas et al., 2017; Kaaya, Samish, Hedimbi,
Gindin, & Glazer, 2011). Additionally, Metarhizium species have been evaluated alone
and in combination with sublethal doses of commercial chemical formulation against
cockroaches (Pachamuthu & Kamble, 2000; Sharififard, Mossadegh, Vazirianzadeh, &
Latifi, 2016), termites (Hussain, Ahmed, & Shahid, 2011; Ravindran, Qiu, & Sivaramak-
rishnan, 2015; Wright, Raina, & Lax, 2005) and house flies (Ong, Ahmad, Ab Majid, &
Jaal, 2017; Sharififard, Mossadegh, Vazirianzadeh, & Zarei-Mahmoudabadi, 2011). In
Mexico, recent research focuses on M. anisopliae as a mycoinsecticide for Meccus pallidi-
pennis and Triatoma dimidiata, insect vectors of Chagas disease (Flores-Villegas et al.,
2016; Vázquez-Martínez et al., 2014); and for control of Boophilus microplus and Rhipice-
phalus microplus, ticks on infested cattle (Alonso-Díaz et al., 2007; Fernández-Salas et al.,
2017).

Behaviour in the environment

An important consideration in the use of entomopathogenic fungi for biological control is
their ability to persist in the environment via horizontal transmission between infected
and healthy hosts (Roberts & Hajek, 1992). While low persistence of the fungus in the
environment is desirable to reduce potentially negative impacts, increased survivability
of the conidia over time could ensure greater commercial stability and efficiency for appli-
cation in the field (Zimmermann, 2007). Under natural conditions, the persistence and
mobility of conidia of Metarhizium is subject to a great range of biotic and abiotic
factors (temperature, humidity, solar radiation, pH, soil microorganisms, plants, and
invertebrates) (Lacey et al., 2015). The prevalence and persistence of fungi is also
affected by crop plant species and tillage practices (Jaronski & Jackson, 2008).
92 C. BRUNNER-MENDOZA ET AL.

Studies of persistence and viability of Metarhizium in agricultural areas are scarce. In

Puebla, Mexico, M. acridum was applied in an amaranth (Amaranthus hypochondriacus)
cultivation for the biological control of the grasshopper Sphenarium purpurascens
(Orthoptera: Pyrgomorphidae). Results showed that the fungus was viable in soil 66
weeks post- application, and that the airborne conidial concentrations and vegetative
cover decreased until complete disappearance after 8 months (Guerrero-Guerra et al.,
2013). In Australia, M. anisopliae var. anisopliae (now M. anisopliae) and M. anisopliae
var. lepidiotum (now M. lepidiotae) were applied to control Dermolepida albohirtum
(Coleoptera: Scarabaeidae) and Lepidiota spp. (Coleoptera: Scarabaeidae) in sugarcane
fields. A small proportion of conidia survived for 3.5 years in all sites and formulations.
Rainfall and soil type had negligible effects on persistence (Milner, Samson, & Morton,
2003). However, other factors present in the rhizospheric microenvironment have an
impact on fungal persistence in soil. Recent studies demonstrated the ability of Metarhi-
zium strains to form stable associations with plants, both as rhizosphere colonisers and
endophytes. Field studies conducted using M. anisopliae (ARSEF 1080) revealed that
the fungal concentration in rhizospheric soil of cabbage was greater (105 propagules/g)
after several months when compared to non-rhizospheric soil (103 propagules/g) (Hu &
St. Leger, 2002). Similar observations were reported for M. anisopliae (F52) in Picea
abies, in which the fungus persisted significantly better in the rhizosphere than bulk soil
(Bruck, 2005). Further fungal persistence studies are needed for a better understanding
of its environmental impact and a more efficient use of microbial agents for biocontrol.
The issue of persistence is an important one and somewhat perplexing. Metarhizium
can be found globally in soils at relatively high levels. However, when tracking genetically
tagged isolates the persistence in soils modulates and in many cases the isolates disappear.
Mexico has extremely diverse habitats, from deserts, scrubland, grasslands to forests
and tropical rainforests. The survivability of fungal conidia in the varied habitats would
be of considerable importance considering the targeted insect pests. Unfortunately, the
survivability of Metarhizium strains under various environmental conditions is greatly
understudied.

Strain improvement
Several biotic and abiotic factors can have a significant impact on the efficiency of Metar-
hizium strains as biocontrol agents for field applications. It is possible to genetically engin-
eer desirable traits into Metarhizium that affects persistence and improves virulence.
Integration of additional copies of a constitutively over-expressed insect cuticle degrading
protease (Pr1A) into M. anisopliae resulted in increased virulence. The resultant trans-
genic strain when applied to target insect Manduca sexta (tobacco hornworm), showed
25% reduction in survival time (LT50) compared to parental strain (St. Leger, Joshi,
Bidochka, & Roberts, 1996). Hemolymph induced expression of the insect specific neuro-
toxin (AaIT) from the scorpion Androctonus australis in M. anisopliae (ARSEF 549)
increased fungal pathogenicity 22-fold and 9-fold against M. sexta caterpillars and adult
Aedes aegypti (yellow fever mosquito) respectively (Wang & St. Leger, 2007b). Transfer
of the esterase gene (MestI) from the generalist insect pathogen M. robertsii to the
specific locust pathogen M. acridum expanded the host range of M. acridum (Wang,
Fang, Wang, & St. Leger, 2011). Recombinant strains of Metarhizium have been developed
 BIOCONTROL SCIENCE AND TECHNOLOGY 93

that target malarial parasites in mosquitos (Fang et al., 2011) which have a great potential
to control livestock and human malaria. Recombinant technologies have also been used to
improve stress resistance in fungi which is a critical factor that affects persistence of field
applications. Expression of melanin in M. anisopliae resulted in enhanced resistance to
UV and high temperatures (Tseng, Chung, & Tzean, 2011). Integration of highly
efficient archaeal photolyase gene from into M. robertsii genome improved the photo-
repair ability of the fungus, consequently improving resistance to sunlight (Fang &
St. Leger, 2012). Alternatively, the virulence of Metarhizium spp. could be improved by
changing the culturing or growth conditions. M. robertsii displayed increased virulence
to Tenebrio molitor when subjected to transient anoxia or grown under nutrient limited
conditions (Oliveira & Rangel, 2018).

Biosafety of Metarhizium spp.

A biosafety review of the entomopathogenic fungus M. anisopliae was performed by Zim-
mermann (2007) where on the basis of all data presented, concluded that this fungus was
considered safe with minimal risk to vertebrates, human and the environment.
Due to the negative effects on the environmental and human health of chemical insec-
ticides, it is essential to expedite testing of biopesticides before release into the environ-
ment (Toriello, 2003). Several countries and international organisations (such as the
International Organisation for Biological Control and the United States Environmental
Protection Agency) have promoted and developed procedures for the registration of
microbial agents used as pesticides. Based on the proposal made in 1981 by the World
Health Organization for the registration of a bioinsecticide, the following data and tests
were considered: product analysis, residue analysis, toxicology, the effects on non-target
organisms, environmental fate, efficiency, and functionality (Siegel, 1997). In Mexico,
the ‘Comisión Federal para la Protección contra Riesgos Sanitarios’ (COFEPRIS) regulates
the requirements, specifications and procedures for the importation and distribution of
organisms used for biological control through SAGARPA, SENASICA, and the DGSV,
‘Secretaría de Economía’ (SE), and the ‘Secretaria de Salud’ (SS) (Toriello & Mier,
2007). There is currently a regulation on the registration of biopesticides in which data
such as the identity and composition of the microbial pesticide (scientific name, inert
ingredients), physicochemical properties (colour, pH), biological properties of the agent
(degree of specificity), toxicological information (oral and dermal toxicity), ecotoxicologi-
cal information (effects on terrestrial and aquatic flora and fauna), and stability studies
(product life) are required (COFEPRIS, 2005). Several biosafety tests have been performed
on these fungi and other biological control microbial agents, concerning their toxic
characteristics, as well as their environmental safety, for example, on the ecosystem stab-
ility, due to their persistence in the environment (Zimmermann, 1993), and there are
questions regarding the introduction of exotic strains that must be considered (Lockwood,
1993).
Some Mexican fungal strains, used in biological control, have been evaluated for poten-
tial health hazards such as their safety to mammals, using acute oral intragastric tests in
mice, and through short-term exposure via the dermal route (Brunner-Mendoza,
Navarro-Barranco, León-Mancilla, Pérez-Torres, & Toriello, 2017; Mier et al., 2005).
Other studies have assessed the pathogenicity and toxicity of M. anisopliae in mice
94 C. BRUNNER-MENDOZA ET AL.

(Toriello, Navarro-Barranco, Martínez, & Mier, 1999, 2006), or the evaluation of acute
gastric exposure of M. acridum (Toriello et al., 2009). Generally, these studies show low
risk of Metarhizium strains in mammalian models. There are reports that Metarhizium
is allergenic but without the presence of any major adverse effects on the manufacturing
staff or applicators (Zimmermann, 2007). Unfortunately, few studies have assessed health
hazards specifically in Mexican strains of Metarhizium spp.
A few reports have shown Metarhizium as the causative fungus in scarce human infec-
tions. For example, an ocular keratitis caused by Metarhizium spp., was observed in an 18-
year-old otherwise healthy male from Colombia (De García, Arboleda, Barraquer, &
Grose, 1997), a keratomycosis in a 36-year-old female librarian who wore extended-
wear soft contact lenses (Jani, Rinaldi, & Reinhart, 2001), a keratitis in a 12-year-old
girl who wore contact lens (Motley, Melson, & Mortensen, 2011) and, more recently, a
52-year-old female that required a corneal transplant (Showail, Kus, Tsui, & Chew,
2017); a 47-year-old woman and a 50-year-old man both from US (Goodman et al.,
2018). A Sklerokeratitis was observed in 52-year-old woman from rural Australia
(Amiel, Chohan, Snibson, & Vajpayee, 2008), and also a 76-year-old male from Japan
with a prolonged systemic steroid treatment for chronic rheumatoid arthritis (Eguchi
et al., 2015). Likewise, a possible disseminated infection in a 9-year-old boy with a 5-
year history of pre-B-cell acute lymphoblastic leukemia that despite antifungal treatment
(liposomal amphotericin and 5-flucytosine), the patient eventually died (Burgner et al.,
1998), and two cases of sinusitis in a 36-year-old male and a 79-year-old female, appar-
ently immunocompetent (Revankar et al., 1999), were also reported. In over a century,
only these few cases in humans (mostly immunocompromised) have been reported, sup-
porting the low risk of infection by Metarhizium spp. when used for the biological control
of insect pests.
However, research must continue on this area of biosafety as we are dealing with living
organisms as biological control agents The effects on non-target organisms, vertebrates,
mammals and human health must continue while using these fungi in biological control.

Conclusions
This review focuses on the role of Metarhizium as an insect pathogen. Several studies have
shown that Metarhizium is a viable alternative to chemical pesticides, due to its low
environmental impact and low risk to mammals. The use of Metarhizium as a biological
control agent in Mexico is incipient, hence the need to access the diversity of Metarhizium
strains for their potential application in Mexican agro-ecosystems.

Acknowledgments
Brunner-Mendoza C acknowledges the scholarship and financial support provided by the Consejo
Nacional de Ciencia y Tecnología (CONACyT-346729), as well for the support and training from
the Posgrado en Ciencias Biológicas, of the Universidad Nacional Autónoma de México (UNAM).
The authors gratefully acknowledge to Dr. Ayala-Zermeño for his help on SAGARPA data. Toriello
C acknowledge the financial support by Programa de Apoyo a Proyectos de Investigación e Inno-
vación Tecnológica (PAPIIT)-Dirección General de Asuntos de Personal Académico (DGAPA),
UNAM, project IT202012, and CONACyT project PDCPN 2015 PN 1247.
 BIOCONTROL SCIENCE AND TECHNOLOGY 95

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
Brunner-Mendoza C acknowledges the scholarship and financial support provided by the Consejo
Nacional de Ciencia y Tecnología (CONACyT-346729), as well for the support and training from
the Posgrado en Ciencias Biológicas, of the Universidad Nacional Autónoma de México (UNAM);
PAPIIT (Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica)-DGAPA
(Dirección General de Asuntos del Personal Académico).

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