An update of a green pesticide: Metarhizium anisopliae

Abstract

Metarhizium anisopliae (Hypocreales: Clavicipitaceae, M. anisopliae), as an invertebrate fungal pathogen, has played a significant role in the control of many agricultural pests and human disease vectors. M. anisopliae is typically used as a chemical in dry or liquid formulations of large numbers of aerial conidia. The conidia can directly infect arthropod pests by penetrating their cuticular layer. The availability of the complete genome of M. anisopliae and capable techniques for its transformation have brought several developments in its use as a pest controller. For prospects, more in-depth research to improve fermentation and formulation technologies is needed, while multistress-tolerant and engineering entomopathogenic strains are selected to promote the widespread acceptance and usefulness of M. anisopliae as a cost-effective fungal biopesticide. Here, we provide an overview of the pathogenesis and formulation strategies, as well as the host ranges and molecular approaches of increasing virulence and efficacy. Several studies of this fungus pathogen demonstrated that M. anisopliae can be an efficient biocontrol agent and has great potential for further research and development.

KEYWORDS:

• Metarhizium anisopliae
• pesticide
• pathogenesis
• toxin
• formulation
• virulence

1. Background

Agricultural pests, such as ticks, termites, grasshoppers, and locusts, have caused great economic losses in many countries and regions in the world (Nourrisson et al. 2017; Savary et al. 2019). Furthermore, human disease vectors, such as mosquitos, are responsible for about one million deaths annually (World Health Organization 2017). To date, chemical insecticides are the most frequently and widely used to control agricultural pests and human disease vectors (Gupta et al. 2019). However, the quantity and frequency of chemical pesticide spraying have increased annually, which has resulted in increased resistance among pests (Damalas and Koutroubas 2018). The large-scale use of chemical pesticides also pollutes the environment, including soil and water, and poses great risks to humans and animals (Deng et al. 2019a). An eco-friendly alternative to chemical pesticides is biopesticide, which has the characteristics of a wide range of sources, great potential for improvement, low drug resistance, and lower nontarget toxicity, including lack of mammalian toxicity (Walia et al. 2017). Entomopathogenic fungi are biological agents that do not produce harmful effects on the environment; thus, they could be called ‘green pesticides’ (Sandhu et al. 2017; Lovett et al. 2019a). Their high efficiency of killing hosts and great biodiversity represent many possibilities for biological control agents, which can help reduce the use of chemicals and improve sustainable development (Damalas and Koutroubas 2018).

Metarhizium anisopliae (M. anisopliae) is a common insect pathogen, belonging to Fungi, Ascomycota, Sordariomycetes, Hypocreales, Clavicipitaceae, Metarhizium (Lovett et al. 2019a). Recently, M. anisopliae has been assigned to M. anisopliae. It is widely distributed in the soil of plant rhizosphere or arthropod carcass as a saprophyte (Rehner and Kepler 2017). M. anisopliae can infect many important crop pests but also human disease vectors (Lovett et al. 2019a). Moreover, this fungus is considered asexual because no teleomorph has been observed, and conidia are its form of infection (Lovett et al. 2019a). The infection process of M. anisopliae includes adhesion, spore germination, appressorium formation, host penetration, in vivo development, and host death (Gao et al. 2011). However, compared to chemical pesticides, M. anisopliae has a slower killing speed, which takes approximately five days under ideal conditions (Fang et al. 2007). Furthermore, pest control efficiency is easily affected by environmental conditions, such as humidity, temperature, and ultraviolet radiation (Gašić and Tanović 2013). With a better understanding of the processes and molecular mechanisms of host infection, the virulence of M. anisopliae can be enhanced by genetic engineering or the development of a suitable formulation, which could greatly improve the application and efficacy of M. anisopliae (Lovett et al. 2019b). This review mainly introduces the host infection pathogenesis of M. anisopliae and the application and development of M. anisopliae in different regions of the world.

2. Historical perspective

The Metarhizium genus was initially composed of four varieties, including M. anisopliae, M. guizhouense, M. pingshaense, and M. taii (Bischoff et al. 2009). With the improvement of DNA sequencing technology, more than 30 species of the genus Metarhizium have been isolated and identified (Fernández-Bravo et al. 2021). In Russia, Elie Metchnikoff studied an insect disease of wheat cockchafers, and he called the disease green muscardine and determined that the infecting agent was M. anisopliae (Vega et al. 2009). Subsequent studies showed that the cereal cockchafer and sugar beet weevil could be killed via direct infection of M. anisopliae conidia (Zhang et al. 2018). Since then, hundreds of fungi have been identified and developed as biocontrol agents. M. anisopliae has been extensively studied because of its environmental friendliness, safety for humans and mammals, narrow host range, and ease of mass production (Zhang et al. 2018). It has been reported that M. anisopliae can infect a variety of arachnid and insect hosts, including agricultural pests and human disease vectors (Gopal et al. 2006). As we learn more about the pathogens, their hosts and their environment, microbial control has also evolved.

3. Pathogenesis of M. anisopliae

M. anisopliae infects susceptible hosts by penetrating the cuticle directly (Freimoser et al. 2005). For ease of description, the infection process can be divided into: (1) conidia adherence to the host cuticle; (2) conidia germination and development; (3) appressorium formation; (4) cuticle penetration; (5) colonization of hemolymph; (6) extrusion and sporulation (Figure 1) (Gao et al. 2011). Metarhizium spp. has species-specific genes in its infection pattern, such as the M. anisopliae adhesin-like protein 1 (Mad1) and high osmolarity glycerol (Hog) pathway Hog1 kinase genes during germination (San and Mun 2017). The major obstacle to insect infection is the complexity of the host's cuticle, which consists mainly of proteins and chitin (Charnley 2003). The secretion of protease is considered to be an important pathogenic factor for fungi attached to the cuticle layer (Butt et al. 2016).

Figure 1. Overview of the basic infection cycle depicted by M. anisopliae in invertebrates. The infection process can be divided into: (1) conidia adherence to the host cuticle; (2) conidia germination and development; (3) appressorium formation; (4) cuticle penetration; (5) colonization of hemolymph; (6) extrusion and sporulation.

3.1 Conidia adhesion

In the first stage of infection, conidia deposit on the host cuticle and then adhere (San and Mun 2017; Schrank and Vainstein 2010). There are hydrophobic proteins in the outer layer of conidia, which promote the adhesion of the hydrophobic epidermis and conidia (Ment et al. 2010a; Greenfield et al. 2014). The cell walls of conidia of M. anisopliae also contain exogenous lectins, which combine with epidermal glycoprotein of the host to form a specific bond and play a role in identifying the host (Samson et al. 1988). Adhesion refers to hydrophobic interactions between lipid layers covering the cutin of arthropods and spore surface proteins (such as hydrophobic proteins). Lipids in the cuticular layer are the first barrier of the arthropod against pathogenic microorganisms and strengthen the importance of lipid enzymes in the early stages of infection (Schrank and Vainstein 2010). Moreover, lipid degradation is related to the prepenetration growth of M. anisopliae on the host epidermis (Schrank and Vainstein 2010). The degradation of the host lipid layer is of great significance for the identification of susceptible hosts and the production of the first nutrient molecules that support the germination of conidia (Pedrini et al. 2007). Furthermore, lipase can be identified on the surface of conidia (Schrank and Vainstein 2010). The conidia of less-virulent strains have poor adhesion to the host epidermis (Beys-Da-Silva et al. 2013). Adhesin protein Mad1 is of great significance in the attachment of swelling conidia to the host surface (Wang and St Leger 2007b). It was speculated that Mad1 could replace degraded hydrophobins upon changes in the conidia cell wall (Wang and St Leger 2007b).

3.2 Conidia germination and development

When conidia adhere to the host cuticle, the germination of conidia is initiated, and different lengths of germ tubes are produced (Ment et al. 2010a). Trehalase can be observed in the early stage of germination and it utilizes trehalose, which is common in host hemolymph. The activity of trehalase may provide glucose for energy production (San and Mun 2017). Spores swell and produce germ tubes that differentiate into appressorium after germination (Lovett and St Leger 2015). The growth of the germ tube is strongly oriented to the epidermis, and the germ tube adheres to the upper epidermis surface through mucus secreted by the hypha apex. Then, the germ tube gradually elongates to produce infection pegs (Hui et al. 2004). Amino acids, proteins, sugars, lipids, and other general nutrients can germinate the conidia of M. anisopliae. Two adhesin-encoding genes, Mad1 and Mad2, are respectively related to the pathogenesis of insects or the colonization of plant roots (Butt et al. 2013). Mad1 protein is expressed in the early stage of insect infection, while Mad2 protein is expressed only after fungal hyphae emerge and conidiate on the insect cadaver. Mad1 protein is also in charge of cell division and proper cytoskeleton organization, and the deletion of the Mad1 gene causes a delay in the germination of conidia (Barelli et al. 2011).

3.3 Appressorium formation

The germ tube differentiates into the appressorium before penetrating the epidermis (Senthil-Nathan 2015). Appressorium contains a large number of mitochondria, Golgi bodies, endoplasmic reticulum, and ribosomes. Through exuberant attachment metabolism, M. anisopliae can concentrate mechanical and dissolve energy into a small part of the host epidermis, thus promoting fungal invasion. The appressorium is covered with a thick mucilage layer, and the morphology of the appressorium varies greatly, ranging from elliptic to irregular, presenting a mulberry shape due to mucous accumulation (Hui et al. 2004). The number of appressoria and its structure are related to the thallus itself, host, and nutritional conditions. It has been demonstrated that the ornithine decarboxylase (ODC1) gene and Metarhizium perilipin-like protein (MPL1) gene are responsible for the formation of appressoria. The ODC1 gene is upregulated during the germination of conidia and the differentiation of the germ tube to form the appressorium (Wang and St. Leger 2007c; Pulido et al. 2011). The MPL1 gene regulates lipid homeostasis and appressorium differentiation (San and Mun 2017). MPL1 is mainly expressed when the fungus is engaged in lipid accumulation (Wang and St Leger 2007b). Fang et al. proved that specific inhibitors of protein kinase A could delay the expression of cuticle degrading enzymes and the formation of appressorium (Fang et al. 2009). In addition, it is reported that protein kinase A is involved in ergosterol biosynthesis and maintains turgor pressure in the appressorium. This expansion pressure produces the pressure required to penetrate the host cuticle (San and Mun 2017).

3.4 Cuticle penetration

In the process of penetration, physical pressure and cuticle degradation are two key factors. Besides, the efficiency of physical pressure mainly depends on appressorium formation, and lipid droplet formation provides the major physical pressure. Protein, chitin, and wax are important compositions in insect cuticles. Protease plays an important role in the degradation of the host epidermis, and its activity is the main factor determining the infection ability of M. anisopliae (Fang et al. 2009). M. anisopliae produces subtilisin-like enzyme (PR1), trypsin-like enzyme (PR2), acid protease (PR3), chymotrypsin, and carboxypeptidase with different catalytic properties, which digest the arthropods’ protein-rich plasma membrane (Moraes et al. 2003). The types and amount of protein produced by M. anisopliae are unique to each host, which can explain its ability to infect many different hosts (San and Mun 2017). Trypsins are only produced in specific hosts, such as cockroaches and beetles (Santi et al. 2010a). Chitinase activity is regulated by chitin degradation products, that is, induction inhibition mechanism. Pretreatment of insect epidermis with PR1, which is related to the pathogenesis of M. anisopliae, can enhance the subsequent chitinase activity (Lin et al. 2011). Different chitinase subtypes are produced in different hosts (Senthil-Nathan 2015). P450 monoxygenase is a key factor for cuticle penetration. The importance of monoterminal oxidation to M. anisopliae is due to the alkyl branch at the β position causing steric inhibition of terminal oxidizing enzymes. The single cytochrome P450 monoxygenase family 52 (MrCYP52) is required by M. anisopliae for rapid hydrolysis of alkanes and that this provides nutrition for germination and formation of infection structures (St. Leger et al. 1986). In addition, besides destroying host antifungal proteins that block pathogen invasion, release amino acids, and generate amines to regulate pH, epidermal degradation enzymes can also use host proteins as nutrients (Hui et al. 2004). The optimal pH values of PR1 and PR2 are approximately 8, which are generated only under alkaline conditions (Stleger et al. 1987). Therefore, the alkalinity of the infected epidermis is a physiological signal of toxicity factors (Hui et al. 2004). In the case of nutritional-deficiency, PR1 penetrates the cuticle by hydrolyzing the cuticular proteins (Carneiro et al. 2015). Furthermore, the production of PR1 was inhibited by exogenous carbon and nitrogen sources (Santi et al. 2010a). PR3 is an acidic protease with low isoelectric point. At present, its composition and action mechanism are still unclear. Chitinase is also the main determinant of pathogenicity (Hui et al. 2004). Carbon competition between pathogens and hosts may determine the rate and extent of infection (Seyoum et al. 2002). Lipase exists on the surface of conidia, which can release free fatty acids by enhancing the hydrophobic interaction between conidia and host, and enhance the adhesion between conidia and host through its lipolytic activity, so as to promote the adhesion between conidia and host (San and Mun 2017). Protease, lipase, and chitinase degrade the cuticle together, enabling effective penetration and utilization of nutrients in the hemocoel of the host for successful infection (Santi et al. 2010b). St Leger et al. concluded that the epidermal degradation enzymes of M. anisopliae are only synthesized at a pH that can play an effective role, regardless of the presence of epidermal substances in the culture medium (Leger et al. 1998). In addition, the pH of the surrounding area changes during M. anisopliae infection, which enables the production of extracellular enzymes and their activities. Environmental pH also regulates the expression of toxic genes (Hui et al. 2004).

3.5 Colonization of hemolymph

Once the fungus breaks through the cuticular layer and the lower epidermis, it starts to invade and proliferate in the hemolymph, which may be restricted to the hemolymph until the death of the host (Seyoum et al. 2002). In vivo and in the culture of infected insects, M. anisopliae produces a family of cyclic peptide toxins called destruxins (DTXs) (Hsiao and Ko 2001). DTXs of M. anisopliae are the first cyclic peptide insect-borne fungal toxins to be systematically studied. Trehalases are produced to convert trehalose from host hemolymph into glucose for energy production, which may represent an adaptation to the host environment (Santi et al. 2010b). In addition, to protect conidia from ultraviolet radiation and reactive oxygen species formed at high temperatures, catalase and peroxidases are found on the surface of conidia (San and Mun 2017). Fungi can use sugar as nutrition and consume trehalose, thus reducing the utilization rate of sugar as a host nutrient (Santi et al. 2010b). Moreover, Mad1 protein initiates gene expression involved in the cell cycle, enabling hyphae to rapidly proliferate and differentiate in the hemolymph of the host (Wang and St. Leger 2007c). These proteins target the cytoskeleton and regulate cell division during the cell cycle (San and Mun 2017).

3.6 Extrusion and sporulation

With the development of host colonization, the nutrients are exhausted and the fungus produces hyphae, which will appear and produce conidia on the surface of the dead host (Gabarty et al. 2014). M. anisopliae then forms a denser network of green spores on the cadavers of infected hosts, ending the infection (San and Mun 2017).

4. Toxins

M. anisopliae produces a large number of fungal secondary metabolites, including DTXs, which induce membrane depolarization by opening Ca²⁺channels, resulting in paralysis and death of the host insects (Hsiao and Ko 2001). DTXs are a class of insecticidal, phytotoxic, and anti-viral cyclic peptide insect-borne fungal toxins and are the most deeply studied toxins of the entomopathogenic fungi (Schrank and Vainstein 2010). These compounds are composed of an α-hydroxy acid and five amino acids to form a cyclic hexadepsipeptide (Gabarty et al. 2014). To date, 38 DTXs have been reported and the majority are produced by M. anisopliae (Schrank and Vainstein 2010). DTXs can be divided chemically into five basic groups labeled A to E (Pedras et al. 2002). Destruxins A, E, and B show significant insecticidal activity (Sree et al. 2008). Both destruxins B (DB) and desmethyl-DB have phytotoxicity to the plants of Brassica (Hu et al. 2006). Moreover, it has been reported that DB can suppress the expression of the hepatitis B viral surface antigen in human hepatoma Hep3B cells, while the chromosomes of Hep3B cells carry integrated viral genes, which suggests that DB may have a promising future as a specific anti-HBV drug (Chen et al. 1997). Furthermore, these toxins assume a significant role in weakening host immune defense by damaging the Malpighian tubules and the muscular system, leading to feeding and affecting excretion as well as mobility difficulties (Schrank and Vainstein 2010). Infected insects often seek out warmer places to raise their body temperature, thereby inhibiting the growth of the infected microbes (Elliot and Thomas 2002). DTXs can reduce host mobility to undermine behavior defense mechanisms (Schrank and Vainstein 2010).

Swainsonine, a sugar analogue from M. anisopliae isolate F-3622, was first reported by Hino et al. (1985; Batta 2003; Roberts and Leger 2004). Swainsonine has great potential as a chemotherapeutic drug because its glycosylation changes are associated with the processes of some diseases (Sim and Perry 1997). In addition to its direct effects on oligosaccharide processing in cancer cells, it also has immunostimulating activity (Roberts and Leger 2004). Effects of swainsonine, such as enhancing lymphocyte proliferation and augmenting cytotoxicities of lymphokine-activated killer and natural killer cells, have been demonstrated (Sim and Perry 1997). In addition to its therapeutic potential, swainsonine is also widely used as a biochemical tool in studies of the nature and biological roles of N-linked oligosaccharides and alpha mannosidases (Sim and Perry 1997).

5. M. anisopliae as a Biological Control Agent

Mosquitoes transmit various pathogens, such as Plasmodium and dengue virus, causing illness and death in humans (Alkhaibari et al. 2016); World Health Organization 2020). Longevity, fecundity, and blood-feeding are the most important factors affecting the possibility of mosquitoes transmitting diseases (Scholte et al. 2006). It has been reported that M. anisopliae infection can reduce the survival time of mosquitoes, including Anopheles, Aedes, and Culex (Daoust and Roberts 1982; Riba et al. 1986a; Riba et al. 1986b; Scholte et al. 2003; Scholte et al. 2007; Lwetoijera et al. 2010; Paula et al. 2019; Choi et al. 2020; Shoukat et al. 2020; Vivekanandhan et al. 2020) (Figure 2). Scholte et al. contaminated Anopheles gambiae (Diptera: Culicidae, An. gambiae) with the oil-formulated conidia of M. anisopliae, and they found that M. anisopliae infection reduced fecundity and blood-feeding of the adult female malaria vector An. gambiae (Scholte et al. 2006). Similarly, Scholte et al. and Shoukat et al. reported that the fecundity of the female dengue virus vector Aedes aegypti (Diptera: Culicidae, Ae. aegypti) and Aedes albopictus (Diptera: Culicidae, Ae. albopictus) was reduced by M. anisopliae infection (Scholte et al. 2006; Shoukat et al. 2020). They also found that when Ae. aegypti females were coinfected with M. anisopliae and dengue virus type 2 (DENV-2), the infection rates of DENV-2 in the heads and midguts were significantly reduced (Scholte et al. 2007; Garza-Hernández et al. 2013). Moreover, a transgenic approach can be used to improve the virulence of M. anisopliae for mosquitoes or interfere with the development of pathogens in the mosquito (Wassermann et al. 2016). In addition, ticks are considered to be the main vectors of animal diseases, second only to mosquitoes as human disease vectors. These arthropods are distributed all over the world, and diseases spread by human ticks range from bacteria to protozoa, viruses, and even fungi (Parola and Raoult 2001). It has been demonstrated that M. anisopliae can kill the larvae, nymphs, and adults of many genera and species of ticks (Table 1) (Mwangi et al. 1995; Zhioua et al. 1997; Kirkland et al. 2004; Ment et al. 2010b; Ren et al. 2014; Beys-da-Silva et al. 2020; Molaei et al. 2020; Abuowarda et al. 2020). Therefore, M. anisopliae may have potential as a biocontrol agent for vector-borne disease control.

Figure 2. Mycotoxic effects of M. anisopliae. (A) M. anisopliae cultured on Potato Dextrose Agar Medium. At the initial stage, the colonies were white and hairy. In the sporulation stage, there were clumps of green conidia in the middle of the colony. (B) The cadavers of Aedes aegypti infected by M. anisopliae or not. (C) The cadavers of Anopheles stephensi infected by M. anisopliae or not.

Table 1. Agricultural pests and human disease vectors sensitive to M. anisopliae.

Family	Species	Selected references
Culicidae	Anopheles gambiae adults and larvae	Scholte et al. (2006); Opisa et al. (2019)
	Anopheles stephensi larvae	Riba et al. (1986b); Vivekanandhan et al. (2020)
	Anopheles arabiensis adults and larvae	Choi et al. (2020)
	Aedes aegypti adults and larvae	Riba et al. (1986b); Scholte et al. (2007); Vivekanandhan et al. (2020)
	Aedes albopictus adults and larvae	Scholte et al. (2007); Shoukat et al. (2020)
	Culex pipiens adults and larvae	Daoust and Roberts (1982); Choi et al. (2020); Bilal et al. (2012)
	Culex quinquefasciatus adults and larvae	Lwetoijera et al. (2010); Wassermann et al. (2016); Mohanty et al. (2008)
Ixodidae	Dermacentor variabilis adults	Ren et al. (2014)
	Rhipicephalus microplus larvae and adults	Ojeda-Chi et al. (2010); Beys-da-Silva et al. (2020)
	Rhipicephalus sanguineus nymphs and adults	Kirkland et al. (2004); Abuowarda et al. (2020)
	Rhipicephalus appendiculatus larvae, nymphs and adults	Mwangi et al. (1995)
	Ixodes scapularis larvae, nymphs and adults	Zhioua et al. (1997); Inyang et al. (2000)
	Haemaphysalis qinghaiensis adults, nymphs and adults	Ren et al. (2014); Albernaz et al. (2009)
	Hyalomma excavatum adults	Ment et al. (2010b)
Rhinotermitidae	Coptotermes formosanus	Yanagawa and Shimizu (2007); Keppanan et al. (2018)
	Coptotermes curvignathus	Pikkheng et al. (2009); Kin et al. (2017)
	Reticulitermes flavipes	Zober (1995)
	Reticulitermes speratus	Shimizu and Yamaji (2003); Mitaka et al. (2017)
	Heterotermes tenuis adults	Moino et al. (2002)
Termitidae	Odontotermes formosanus	Dong et al. (2009); Fu et al. (2020)
	Nasutitermes exitiosus	Hänel (1982)
Acrididae	Schistocerca gregaria adults, nymphs, eggs	Xia et al. (2000); Abdellaoui et al. (2020)
	Chortoicetes terminifera adults	Hunter et al. (2001)
	Locusta migratoria nymphs	Peng et al. (2008); Liu et al. (2019); Bilal et al. (2012)
	Oxya chinensis nymphs	Zober (1995); Abro et al. (2019)
Pyralidae	Ostrinia furnacalis larvae	Chiuo and Hou (1993)
	Ostrinia nubilalis larvae	Riba et al. (1986a)
	Diatraea saccharalis larvae	De Oliveira et al. (2018); Destéfano et al. (2004)
	Cnaphalocrocis medinalis larvae	Hong et al. (2017); Destéfano et al. (2004)
	Chilo partellus larvae	Tefera and Pringle (2003)
	Galleria mellonella larvae	Keppanan et al. (2017); Sbaraini et al. (2021)
Noctuidae	Spodoptera litura larvae	Sree et al. (2008)
	Spodoptera littoralis larvae	El Husseini (2019)
	Helicoverpa armigera larvae	Lawo et al. (2008); Santi et al. (2011)
	Hyblaea puera larvae	Remadevi et al. (2010)
Carposinidae	Carposina niponensis larvae	Yaginuma (1990)
Lymantriidae	Pantana phyllostachysae adults	Zhang et al. (2002)
Blattellidae	Blattella germanica adults	Lopes and Alves (2011); Chao et al. (2020)
Lasiocampidae	Dendrolimus punctatus larvae	Zhang et al. (2002)
Reduviidae	Triatoma infestans nymphs and adults	Luz et al. (1998)
	Triatoma sordida nymphs and adults	Rangel et al. (2020)
	Panstrongylus megistus nymphs and adults	Rangel et al. (2020)
Delphacidae	Nilaparvata lugens adults	Tang et al. (2019)
	Sogatella furcifera adults	Tang et al. (2019)
Tephritidae	Anastrepha ludens adults	Toledo-Hernández et al. (2018)
	Zeugodacus cucurbitae adults	Onsongo et al. (2019)
Bruchidae	Callosobruchus maculatus adults	Cherry et al. (2005); Ozdemir et al. (2020)
	Caryedon serratus adults	Ekesi et al. (2001)
House flies	Musca domestica larvae and adults	Oreste et al. (2016); Baker et al. (2018)
Tenebrionidae	Alphitobius diaperinus larvae and adults	Ment et al. (2010a); Cassiano et al. (2008); Inyang et al. (2000)
Pyrrhocoridae	Dysdercus peruvianus nymphs and adults	Santi et al. (2011)
Plutellidae	Plutella xylostella larvae	Zafar et al. (2020)
Aleyrodidae	Trialeurodes vaporariorum adults	Oreste et al. (2016)
Aphelinidae	Encarsia formosa larvae and adults	Oreste et al. (2016)
Thripidae	Frankliniella occidentalis adults	Li et al. (2021)
Curculionidae	Curculio nucum larvae	Sharififard et al. (2011)
Cerambycidae	Monochamus alternatus larvae and adults	Li et al. (2021)
Delphacidae	Peregrinus maidis adults	Toledo et al. (2010)
Bostrychidae	Rhyzopertha dominica adults	Ashraf et al. (2017); Saeed et al. (2020)
Tenebrionidae	Tribolium castaneum adults	Ashraf et al. (2017); Saeed et al. (2020)
Laemophloeidae	Cryptolestes ferrugineus adults	Ashraf et al. (2017); Saeed et al. (2020)
Liposcelididae	Liposcelis paeta adults	Ashraf et al. (2017); Saeed et al. (2020)
Phycitidae	Ephestia kuehniella larvae	Athanassiou et al. (2017)
Tortricidae	Thaumatotibia leucotreta adults	Mkiga et al. (2020); Acheampong et al. (2020)
Crambidae	Spoladea recurvalis adults	Opisa et al. (2019)
Gelechiidae	Tuta absoluta pupae	Contreras et al. (2014)
Tettigoniidae	Anabrus simplex nymphs	Rangel et al. (2021)

Termites are important pests that cause severe damage to crops, living trees and wood-based materials (Rath 2000). It has been reported that M. anisopliae can be used to control many termites (Table 1) (Hänel 1982; Zober 1995; Moino et al. 2002; Shimizu and Yamaji 2003; Yanagawa and Shimizu 2007; Dong et al. 2009; Pikkheng et al. 2009; Kin et al. 2017; Mitaka et al. 2017; Keppanan et al. 2018; Fu et al. 2020). The first report of termite fungal biological control agent is a single isolate C4-B, which is identified as M. anisopliae taxonomically (Wright et al. 2005). In addition, locusts represent probably the most striking of all insect pests and are abundant insects of deserts and dry grasslands (Scanlan et al. 2001). Traditionally, chemical pesticides have been used to control swarms and bands, but this is under strict scrutiny. M. anisopliae has been tested successfully against Schistocerca gregaria (Orthoptera: Acrididae), Chortoicetes terminifera (Orthoptera: Acrididae), Locusta migratoria (Orthoptera: Oedipodidae) and Oxya chinensis (Orthoptera: Acrididae) (Table 1) (Zober 1995; Xia et al. 2000; Hunter et al. 2001; Peng et al. 2008; Abro et al. 2019; Liu et al. 2019; Abdellaoui et al. 2020). In Australia, field trials have shown that efficacy depends on the application rate and vegetation cover, while the rate of development of both Metarhizium and the locust is affected by temperature (Hunter et al. 2001). However, one challenge of using M. anisopliae is to overcome the behavioral fever of locusts, which can inactivate the conidia (Hunter et al. 2001). It has been reported that heat-tolerant genes can be transferred into M. anisopliae by genetic engineering to overcome this challenge (San and Mun 2017; Keppanan et al. 2017). Thus, M. anisopliae has desirable attributes for the development of a mycoinsecticide against pests.

In addition, M. anisopliae has been applied to control Spodoptera litura (Lepidoptera: Noctuidae), Carposina niponensis (Lepidoptera: Carposinadae), Galleria mellonella (Lepidoptera: Pyralidae), Prodenia litura (Lepidoptera: Noctuidae), Pantana phyllostachysae (Lepidoptera: Lymantriidae), Blattella germanica (Blattodea: Ectobiidae), Dendrolimus punctatus (Lepidoptera: Lasiocampidae), Triatoma infestans (Hemiptera: Reduviidae), Helicoverpa armigera (Lepidoptera: Noctuidae), Hyblaea puera (Lepidoptera: Hyblaeidae), Callosobruchus maculatus (Coleoptera: Chrysomelidae), Musca domestica (Diptera: Muscidae), Dysdercus peruvianus (Hemiptera: Pyrrhocoridae), Anastrepha luden (Diptera: Tephritidae), Chilo partellus (Lepidoptera: Crambidae), Panstrongylus megistus (Hemiptera: Reduvidae), Monochamus alternatus (Coleoptera: Cerambycidae), Curculio nucum (Coleoptera: Curculionidae), Frankliniella occidentalis (Thysanoptera: Thripidae), Encarsia Formosa (Hymenoptera: Aphelinidae), Trialeurodes vaporariorum (Hemiptera: Aleyrodidae), Plutella xylostella (Lepidoptera: Plutellidae), Zeugodacus cucurbitae (Diptera: Tephritidae), Sogatella furcifera (Hemiptera: Delphacidae), Nilaparvata lugens (Hemiptera: Delphacidae), Ostrinia furnacalis (Lepidoptera: Crambidae), Ostrinia nubilalis (Lepidoptera: Crambidae), Diatraea saccharalis (Lepidoptera: Crambidae), Triatoma sordida (Hemiptera: Triatominae), Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), Spodoptera littoralis (Lepidoptera: Noctuidae), Alphitobius diaperinus (Coleoptera: Tenebrionidae), Pieris rapae (Lepidoptera: Paleidae), Plutella xylostella (Lepidoptera: Plutellidae), Dorysthenes granulosus (Coleoptera: Cerambycidae), Caryedon serratus (Coleoptera: Bruchidae), Peregrinus maidis (Hemiptera: Delphacidae), Rhyzopertha dominica (Coleoptera Bostrichidae), Tribolium castaneum (Coleoptera: Tenebrionidae), Cryptolestes ferrugineus (Coleoptera: Laemophloeidae), Liposcelis paeta (Psocoptera: Liposcelididae), Ephestia kuehniella (Lepidoptera: Pyralidae), Thaumatotibia leucotreta (Lepidoptera: Tortricidae), Spoladea recurvalis (Lepidoptera: Crambidae), Tuta absoluta (Lepidoptera: Gelechiidae), Anabrus simplex (Orthoptera: Tettigoniidae) (Table 1) (Sree et al. 2008; Ment et al. 2010b; Keppanan et al. 2017; Yaginuma 1990; Rustiguel et al. 2018; De Oliveira et al. 2018; Luz et al. 1998; Hong et al. 2017; Kirubakaran et al. 2014; Chiuo and Hou 1993; Destéfano et al. 2004; Tefera and Pringle 2003; Zhang et al. 2002; Guogui 2003; Cherry et al. 2005; Cassiano et al. 2008; Lawo et al. 2008; Remadevi et al. 2010; Lopes and Alves 2011; Santi et al. 2011; Sharififard et al. 2011; Cheng et al. 2016; Oreste et al. 2016; Baker et al. 2018; Toledo-Hernández et al. 2018; El Husseini 2019; Onsongo et al. 2019; Tang et al. 2019; Chao et al. 2020; Fite et al. 2020; Kim et al. 2020; Li et al. 2021; Ozdemir et al. 2020; Rangel et al. 2020; Rice et al. 2020; Zafar et al. 2020; Bilal et al. 2012; Mohanty et al. 2008; Ojeda-Chi et al. 2010; Sbaraini et al. 2021; Ekesi et al. 2001; Toledo et al. 2010; Ashraf et al. 2017; Saeed et al. 2020; Athanassiou et al. 2017; Mkiga et al. 2020; Acheampong et al. 2020; Opisa et al. 2019; Contreras et al. 2014; Rangel et al. 2021). In summary, due to its safety and lack of arthropod resistance, M. anisopliae has good potential in the control of agricultural pests and vectors of human diseases (Wang and St Leger 2007a).

6. Molecular approaches to improve the virulence and efficacy of M. anisopliae

Poor efficacy has hindered the application of Metarhizium; hence, we urgently need to find methods to maximize environmental persistence and virulence of Metarhizium (Wang and St Leger 2007a). One approach to improve virulence is genetic manipulation. M. anisopliae provides a large list of genes that can be combined in a single pathogen vector because they use different proteins to adhere to the host, penetrate host cuticles, and overcome host defenses (Lovett and St Leger 2018). Many natural and synthetic genes have been inserted into the genome of M. anisopliae to enhance its virulence and efficacy against pests (Table 2).

Table 2. The major approaches for modifying entomopathogens to enhance the virulence of M. anisopliae.

Modified strain	Principal targeted species	Selected references
Ma-AaIT	Hypothenemus hampei larvae	Pava-Ripoll et al. (2008)
	Manduca sexta caterpillars larvae	Wang and St Leger (2007a)
	Aedes aegypti adults	Wang and St Leger (2007a)
Ma-[SM1]8	Plasmodium falciparum and Anopheles gambiae adults	Fang et al. (2011)
Ma-scorpine	Plasmodium falciparum and Anopheles gambiae adults	Fang et al. (2011)
Ma-PfNPNA-1	Plasmodium falciparum and Anopheles gambiae adults	Fang et al. (2011)
Ma-[SM1]8:scorpine	Plasmodium falciparum and Anopheles gambiae adults	Fang et al. (2011)
Ma-Cat1	Plutella xylostella larvae	Morales Hernandez et al. (2010)
Ma-Vip3Aa1	Spodoptera litura larvae	Zhang et al. (2014)

The scorpion excitatory sodium channel blocker, Androctonus australis Hector insect toxin (AaIT), is an insect-specific neurotoxin (Lovett and St Leger 2018). The recombinant expression of AaIT in fungi can increase their virulence against insect pests and disease vectors (Deng et al. 2019b). M Pava-Ripoll et al. demonstrated that recombinant M. anisopliae AaIT-Ma549 was dramatically more virulent than wild-type against Hypothenemus hampei larvae (Pava-Ripoll et al. 2008). Wang et al. also showed that the high-level expression of AaIT toxin in M. anisopliae increased its fungal toxicity to larval Manduca sexta caterpillars by 22-fold and Ae. aegypti adults by 9-fold (Table 2) (Wang and St Leger 2007a).

Fang et al. engineered M. anisopliae to express the eight repeats of salivary gland and midgut peptide 1 ([SM1]₈); a single-chain human antibody that agglutinates sporozoites; an antimicrobial toxin scorpine; or a fusion protein of [SM1]₈ and scorpine. These recombinant fungi not only killed An. gambiae, but also reduced sporozoite counts of An. gambiae by 70% to 90%. M. anisopliaeexpressing scorpion and an [SM1]₈: scorpion fusion protein reduced the number of sporozoites by 98%, indicating that M. anisopliae-mediated inhibition of Plasmodium development may be a powerful weapon for malaria control (Fang et al. 2011).

M. anisopliae exhibits an increase in catalase-peroxidase activity during its germination and growth (Kawasaki and Aguirre 2001). Morales Hernandez et al. found that catalase overexpression can reduce the germination time and increase the pathogenicity of M. anisopliae (Morales Hernandez et al. 2010). Thus, they isolated the genomic copy of M. anisopliae catalase-1 (cat1) gene and overexpressed it in the fungus. They found that increased cat1 expression was accompanied by increased H₂O₂ tolerance and germination rate, all of which enhanced the virulence against Plutella xylostella larvae (Morales Hernandez et al. 2010).

In addition, wild M. anisopliae isolates showed no pathogenicity to Spodoptera litura larvae (Lee et al. 2006). Zhang et al. genetically modified M. anisopliae by inserting the insect midgut-specific toxin Vip3Aal gene of Bacillus thuringiensis. The expression of this toxin allows M. anisopliae to infect S. litura larvae through the ingestion of conidia rather than through the cuticle penetration (Zhang et al. 2014).

7. M. anisopliae formulations

In addition to strain selection and genetic modification, formulations can also have a significant impact on improving the efficacy of biological pesticides. An ideal formulation is helpful to the handling and application of biopesticides and reduces the chances of nontarget organisms coming into contact with fungal spores.

Bukhari et al. devised formulations of M. anisopliae spores in aqueous, dry, synthetic oil (ShellSol T and Ondina oil 917) for the control of Anopheles larvae (Table 3) (Bukhari et al. 2011). Among these formulations, synthetic oil is easily miscible and suitable for water surfaces and improves persistence (Abdul-Ghani et al. 2012). Their results have shown that the delivery system of fungal conidia suspended in ShellSol T solvent was significantly more virulent to An. gambiae and Anopheles stephensi (Diptera, Culicidae) larvae than that of unformulated conidia (Bukhari et al. 2011). However, dry and aqueous formulated M. anisopliae spores lost their pathogenicity quickly after being applied to the water surface (Bukhari et al. 2011).

Table 3. Selected examples of formulations using M. anisopliae in various habitats.

Formulation		Habitat	Principal targeted species	Selected references
Oil	ShellSol T	plastic containers	Anopheles gambiae larvae	Bukhari et al. (2011)
		plastic containers	Anopheles stephensi larvae	Bukhari et al. (2011)
		experimental plots	Phaedon cochleariae larvae	Inyang et al. (2000)
	Ondina oil 917	plastic containers	Anopheles gambiae larvae	Bukhari et al. (2011)
		plastic containers	Anopheles stephensi larvae	Bukhari et al. (2011)
	Mineral oil	culture medium	Rhipicephalus microplus larvae, nymphs and adults	Camargo et al. (2012)
	Mineral oil and diatomaceous earth	plastic cups	Aedes aegypti adults	Rodrigues et al. (2019)
	Cornstarch-oil	petri dishes	Diabrotica undecimpunctata adults	Pereira and Roberts (1991)
	Sunflower oil and Imidacloprid (IMI)	petri dishes	Conotrachelus psidii adults	Brito et al. (2008)
	Sunflower oil-in-water	paper cups	Aedes aegypti eggs	Albernaz et al. (2009)
	Oil-in-water	petri plates	Rhipicephalus Microplus females	Muniz et al. (2020)
	Soybean oil	agar plates	Dysdercus peruvianus	Rangel et al. (2020)
	Coconut/soybean oil	capped graduated tubes	Tetranychus cinnabarinus adults	Batta (2003)
		capped graduated tubes	Bemisia tabaci nymphs	Batta (2003)
	Peanut oil	potted grass	Rhipicephalus appendiculatus larvae, nymphs and adults	Kaaya and Hedimbi (2012)
		potted grass	Amblyomma variegatum larvae, nymphs, and adults	Kaaya and Hedimbi (2012)
	Canola oil	field paddock	Rhipicephalus Microplus	Leemon et al. (2008)
	Neem oil	natural field	Aedes aegypti larvae	Paula et al. (2019)
		natural field	Culex quinquefasciatus larvae and adults	Seye et al. (2012)
		experimental cage	Anopheles stephensi female	Dhar et al. (1996)
		experimental cage	Anopheles culicifacies female	Dhar et al. (1996)
		greenhouse conditions	Anopheles gambiae larvae	Okumu et al. (2007)
Dust	Diatomaceous earth	glass-capped dishes	Tribolium confusum larvae and adults	Michalaki et al. (2006)
		small cylindrical glass vial	Rhyzopertha dominica adults	Rice et al. (2019)
		small cylindrical glass vial	Sitophilus oryzae adults	Rice et al. (2019)
Other	Alginate	tightly sealed plastic tubes	Diabrotica undecimpunctata adults	Pereira and Roberts (1991)
	Aqueous	potted grass	Rhipicephalus appendiculatus adults and eggs	Kaaya and Hedimbi (2012)
		recipientes plásticos	Alphitobius diaperinus larvae and adults	Rohde et al. (2006)
	Granular formulations consisting of ground chicken feed pellets with conidial powder	petri dishes	Lesser mealworm larvae	Rice et al. (2019)
	Polymerized cellulose gel combined with surfactants	Polypropylene bags	Rhipicephalus microplus	Rice et al. (2019)

Inyang et al. took the third instar larvae of Phaedon cochleariae as the target host to study the effect of simulated rain on the persistence of oil formulations of M. anisopliae conidia when applied to oilseed rape foliage (Inyang et al. 2000). Their results suggested that sunflower oil formulations enhanced the infectivity of M. anisopliae for P. cochleariae in both laboratory and field experiments (Inyang et al. 2000). This viscous oil carrier could improve the adhesion of M. anisopliae conidia to plant surfaces and reduce displacement by rain (Inyang et al. 2000).

Oil carriers, such as sunflower, peanut, Ondina, soybean, canola and mineral oil, as well as cornstarch oil can transport the inoculum to parts of the insect's body, which is more conducive to germination than the application of conidia in Tween (Table 3) (Batta 2003; Paula et al. 2019; Sharififard et al. 2011; Pereira and Roberts 1991; Dhar et al. 1996; Okumu et al. 2007; Brito et al. 2008; Leemon et al. 2008; Albernaz et al. 2009; Kaaya and Hedimbi 2012; Seye et al. 2012; Camargo et al. 2012; Rodrigues et al. 2019; Muniz et al. 2020). Oil carriers also seem to improve spore germination at low relative humidities and prolong spore viability, even at high temperatures (Inyang et al. 2000).

Michalaki et al. utilized diatomaceous earth (DE) in combination with an M. anisopliae formulation to treat wheat/flour. Their research is the first work on the combination of M. anisopliae and DE (Michalaki et al. 2006). Kavallieratos et al. demonstrated that the combined use of DE and M. anisopliae could be of practical significance to pest control, and this combination could be used against adults of three stored-grain beetle species (Table 3). DEs can remove certain epidermis lipids from insects, allowing germinated M. anisopliae conidia to more easily infiltrate insect blood (Michalaki et al. 2006).

Alginates have been used to encapsulate fungal structures for the biocontrol of entomopathogens and weeds Pereira and Roberts (1991), (Walker and Connick 1983) found that the alginate formulations are very effective in preventing fungi from inactivation due to high temperatures and artificial solar radiation. Thus, they used an Alginates formula to control adult Diabrotica undecimpunctata (Table 3) (Pereira and Roberts 1991).

In addition to the above formulations, there are also other formulations that can improve the efficacy of M. anisopliae, such as aqueous, granular formulations consisting of ground chicken feed pellets with conidial powder and polymerized cellulose gel combined with surfactants (Kaaya and Hedimbi 2012; Walker and Connick 1983; Rohde et al. 2006; Reis et al. 2008; Rice et al. 2019).

8. Conclusions and future perspectives

M. anisopliae has been considered an important entomopathogenic fungus in the biocontrol of many pests and human disease vectors throughout the world since it was isolated and identified nearly 150 years ago. In this review, we mainly summarized the historical perspective, pathogenesis, toxins, M. anisopliae as a biological control, and molecular approaches to increase its virulence as well as the formulations of M. anisopliae. Generally, conidia, also known as asexual spores, are the main form of M. anisopliae that infects susceptible hosts. Once conidia touch the cuticular layer of a sensitive host, the infection begins with adhesion and germ tube formation. Then the expression of genes related to fungal infection structures, hydrolytic enzymes, and host defense mechanisms is triggered by both the insect pathogen and host. Normally, a sign of a successful fungal infection is the cleavage of the hosts’ epidermis. When the fungus arrives in the hemolymph through the growing hyphal, the host body will become colonized. M. anisopliae also produces numerous toxins, including DTXs and swainsonine. DTXs play an important role in weakening host immune defense, damaging the muscle system and Malpighian tubule, affecting host excretion, and resulting in feeding and mobility difficulties. The discoveries about the ecological roles displayed by M. anisopliae with multiple effects will help its eventual commercialization.

Although great progress has been made in the control of diseases and pests by using biopesticides, the development of fungal biological control agents still faces some problems. First, most biopesticide formulations, including spores, filaments, and other living preparations, cannot be easily combined with anti-corrosive ingredients, which affect the use and preservation of fungal pesticides. Second, the use of biopesticides is greatly affected by environmental conditions, thus leading to unstable efficacy. Third, compared with that of chemical pesticides, the price of biological pesticides is relatively high. Thus, researchers should make full use of the rich microbial resources in the world to screen out and select efficient strains and make continuous breakthroughs in technology to cultivate biocontrol strains with high virulence and productivity.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Declaration of interest statement

No potential conflict of interest was reported by the author(s).

Author contributions statement

Conception and design: SQD; analysis and interpretation of the data: ZYP, STH, JTC, NL, YW and AN; writing – original draft preparation: STH and SQD; writing – review and editing: ZYP, AN and SQD. All authors have agreed to be accountable for all aspects of the work and the published version of the manuscript.

Data availability statement

Data sharing is not applicable to this article as no new data were created or analysed in this study.

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

This research was supported by National Natural Science Foundation of China (82102432), Anhui Provincial Natural Science Foundation (2108085QH347), Research Fund Project of Anhui Medical University (2020xkj005) to DSQ.