J Pest Sci (2017) 90:839–854 DOI 10.1007/s10340-017-0849-9

REVIEW

Use of entomopathogenic fungi for the control of stored-product insects: can fungi protect durable commodities? Christos I. Rumbos1 • Christos G. Athanassiou1

Received: 28 October 2016 / Revised: 2 March 2017 / Accepted: 13 March 2017 / Published online: 25 March 2017  Springer-Verlag Berlin Heidelberg 2017

Abstract Entomopathogenic fungi are considered promising microbial control agents for the control of postharvest insects, and their evaluation for this purpose has lately attracted a significant amount of research. They are naturally occurring, environmentally safe organisms that infect insects by contact. Insect fungal pathogens have a broad spectrum of hosts, can be mass-produced easily, rapidly and economically and can be applied with the same technical means as conventional contact insecticides. In this context, the most studied fungal species for the control of stored-product insect species are Beauveria bassiana and Metarhizium anisopliae. Both fungal species have a wide host range and have been tested against most of the major stored-product insect pests. The effect of biotic and abiotic factors on the virulence and success of entomopathogenic fungi in storage insect control, as well as the combined application of these agents with other pest control technologies, is reviewed here. Temperature and relative humidity influence the efficacy of entomopathogenic fungi, often with variable results, whereas the combined use of entomopathogenic fungi with diatomaceous earth in many cases has a synergistic or additive effect. Alternative methods of entomopathogenic fungi application, based on the lure-and-kill approach, are proposed, and future challenges for the use of entomopathogenic fungi against stored-product insects are highlighted. Communicated by N. Desneux. & Christos G. Athanassiou [email protected] 1

Laboratory of Entomology and Agricultural Zoology, Department of Agriculture, Crop Production and Rural Environment, University of Thessaly, Phytokou str., 38446 Volos, Magnesia, Greece

Keywords Entomopathogenic fungi
Stored-product insects
Biological control
Microbial control

Key message •

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The virulence of entomopathogenic fungi against storage insects varies considerably, depending on the target species and life stage, as well as the fungal isolate. Several fungal strains are effective at relatively dry conditions that commonly prevail in stored-product environments. New formulations (e.g. electrostatically charged powders), as well as the integration of entomopathogenic fungi in innovative control systems (e.g. autodissemination lure-and-kill technique), can improve their performance and merit further research.

Background Stored-product insects are responsible for considerable qualitative and quantitative losses during the storage of durable dry commodities throughout the world (White 1995; Mason and McDonough 2012). Post-harvest insect pest management has been mainly focused on chemical and, to a lesser extent, on physical and biological control methods (Subramanyam and Hagstrum 1995; Zettler and Arthur 2000). However, the increased demands of consumers and regulatory authorities for reduced use of pesticides, as well as the development of insect resistance to many of the commonly used insecticides, force the food industry to decrease reliance on chemical control (Hagstrum and Flinn 1995). Moreover, physical control, based

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on the manipulation of the physical attributes of the storage environment, e.g. heat treatment and freezing, is often limited by the required increased energy inputs (Adler 2010; Fields et al. 2012). Biological control uses natural enemies of insect pests, such as predators, parasitoids and pathogens, to reduce insect populations below the economic threshold level. The history of biological control in post-harvest and storedproduct insects is long and goes back to the beginning of the twentieth century. Between 1910 and 1930, parasitoids such as Venturia canescens Gravenhorst (Hymenoptera: Ichneumonidae), Lariophagus distinguendus Fo¨rster (Hymenoptera: Pteromalidae) and Habrobracon hebetor Say (Hymenoptera: Braconidae) were first identified as natural enemies of stored-product insects, and their ability to successfully suppress populations of these pests was evaluated (Buchwald and Berliner 1910; Froggatt 1912; Ryabov 1926; Flanders 1930). Historically, the evaluation of the use of insect pathogens against stored-product insects also begins at the same period, with the isolation and identification of the bacterial pathogen Bacillus thuringiensis Berliner (Bacillales: Bacillaceae) from the Mediterranean flour moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) (Berliner 1915). Since then, a considerable amount of research has been dedicated to the investigation of potential biological control agents against post-harvest insect pests (Brower et al. 1996; Scho¨ller et al. 1997; Moore et al. 2000). The application of macro-biological control agents (i.e. predators and parasitoids) for the control of post-harvest insects, despite its proven effectiveness (Brower et al. 1996), is likely to have important limitations. In this regard, the suppression of insect populations from predators and parasitoids is slow and requires long periods to be effective (Scho¨ller et al. 1997). Moreover, their application requires careful timing, so that the host is present in adequate numbers at the time of release, which means that a certain infestation level is required. If, however, beneficials are released too late and the number of target insect pests is high, extremely high numbers of beneficials must be released to successfully control the pests (Flinn and Scholler 2012). Furthermore, the mass rearing of parasitoids and parasites employs mass-rearing facilities and specialized technical staff, which boost their production cost (Brower et al. 1996). The potential contamination of the treated food commodities by insect fragments from the released insects is another limitation for their general use, which should be considered carefully, in conjunction with consumer’s acceptance, while in the case of coexistence of several target species, multiple predators and/or parasitoids should be reared and released, as many of them are species specific (Brower et al. 1996). It is generally considered that microbial control agents are more compatible with dry durable commodities and

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grain storage systems, where several stored-product insect species usually coexist. Insect pathogens have a broad spectrum of hosts, in contrast to beneficial insects, many of which are host specific, although others can be effective for a wide variety of target species (Kavallieratos et al. 2006, 2014). Moreover, most microbial agents can be mass-produced in vitro on industrial scale more easily, rapidly and economically than most macro-biological control agents and can be applied with the same technical means as chemical protectants (Wraight and Hajek 2009). Microbial control agents usually affect less nontarget organisms, such as mammals and humans (Brower et al. 1996). Yet, insect pathogens have long storage time and can provide long-term protection (Athanassiou et al. 2008), whereas their hosts are not known to develop easily resistance (Moore et al. 2000). Due to the significant amount of research on the field of microbial control agents, there are already numerous examples of effective control of stored-product insects with microbials. The bacterium B. thuringiensis, the most commercially successful pathogen used for insect control, is currently registered for application on stored grains in many parts of the world (Lord et al. 2007), while its efficacy has been demonstrated against several stored-product insects (Oppert et al. 2011; Yilmaz et al. 2012; Nouri-Ganbalani et al. 2016). Also, several viruses have been isolated from stored-product insects, mainly from Lepidopteran species (Brower et al. 1996), and have been evaluated against post-harvest insect species (Hunter 1970; Vail et al. 1991). Entomopathogenic nematodes have been also evaluated against most major storedproduct insects with variable results (Mbata and ShapiroIlan 2005; Rumbos and Athanassiou 2012, 2016; Negrisoli et al. 2013), whereas limited research has been conducted with entomopathogenic protozoa (Lord 2003). Entomopathogenic fungi are promising microbial control agents for insect pests in storage environment. They include fungal species, which induce disease symptoms and cause lethal infections to insects and control, in this way, insect populations in nature (Burges 1981; Carruthers and Soper 1987; Moore et al. 2000; Batta 2016a). Entomopathogenic fungi are naturally occurring organisms, distributed in a wide range of habitats, including agricultural, pasture and urban habitats (Lacey et al. 1996; Chandler et al. 1997; Sa´nchez-Pena et al. 2011). Since they exist in nature, entomopathogenic fungi have low environmental impact and are generally considered environmentally safe agents with low mammalian toxicity (Saik et al. 1990; Siegel and Shadduck 1990; Cox and Wilkin 1996; Moore et al. 2000; Batta 2016a). They infect insects by contact, i.e. fungal conidia attach on the host cuticle, germinate and infection hyphae invade their hosts by direct penetration of the host exoskeleton or cuticle (Clarkson and Charnley 1996; Srivastava et al. 2009; Stephou et al. 2012; Ortiz-Urquiza and

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Keyhani 2013). Fungal penetration is enhanced by the combined action of enzymes that are secreted by the fungi and mechanical force (Charnley and St Leger 1991; Srivastava et al. 2009). Death of the insect comes as a result of the combined action of fungal toxins, starvation and physiological disruption (Clarkson and Charnley 1996). When the insect dies, the fungus develops in the cadaver and sporulation occurs on the external part of the body of the dead host, reintroducing in this way more inoculum in the fungus–insect system (Goettel and Inglis 1997). Entomopathogenic fungi are usually isolated from soil or insects (Meyling and Eilenberg 2007; Quesada-Moraga et al. 2007). In order to study the natural occurrence of entomopathogenic fungi, larvae of highly susceptible insect species, such as the greater wax moth, Galleria mellonella L. (Lepidoptera: Pyralidae), and the yellow mealworm beetle, Tenebrio molitor L. (Coleoptera: Tenebrionidae), are added as bait to soil samples to recover fungal isolates (Zimmermann 1986; Pilz et al. 2008). However, highly virulent fungal entomopathogenic strains are more frequently isolated using as bait larvae of the insect to be controlled rather than G. mellonella (Prior 1991; Klingen and Haukeland 2006). Alternatively, insects naturally infected by fungi can be collected in the field, and the fungal species can be cultured and identified in the laboratory. Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) has been isolated from more than 700 species of insect hosts (Inglis et al. 2001). In one of the few studies available on the occurrence of entomopathogenic fungi on post-harvest insects, B. bassiana was the only widespread entomopathogenic fungus in Kenya, isolated from the maize weevil, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae), Tribolium and Carpophilus species (Oduor et al. 2000). Recently, Barra et al. (2013) isolated entomopathogenic fungi from rhizospheric soil using adults of Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae) as bait insects and reported that Paecilomyces and Metarhizium were the most abundant genera isolated. During the last two decades, entomopathogenic fungi have attracted a lot of attention for the control of stored-product insects, and recent studies have demonstrated their effectiveness against major stored-product insect pests, in numerous studies that examined a wide variety of application scenarios. In total, seven species of entomopathogenic fungi have been tested against a wide range of stored-product insect species, as presented in Table 1.

Major species of entomopathogenic fungi against stored-product insects Most studies with post-harvest insects have been conducted with isolates of B. bassiana and, to a lesser extent, Metarhizium anisopliae (Metschnikoff) Sorokin (Ascomycota:

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Hypocreales) (Table 1). Both fungal pathogens have a wide host range and have been tested against most of the major stored-product insect species, under various types of conditions and commodities. Beauveria bassiana This species is by far the most widely tested entomopathogenic fungus for the control of stored-product insects. Already from 1977, Ferron (1977) first reported the successful infection of adults of the bean weevil, Acanthoscelides obtectus Say (Coleoptera: Bruchidae), from B. bassiana, whereas a few years later, Searle and Doberski (1984) reported the complete control of adults of the sawtoothed grain beetle, Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae), after 26 days of exposure to a soil isolate of B. bassiana at concentrations higher than 104 conidia ml-1. Since then, different isolates of B. bassiana have been tested with variable results against major storage insect pests (Hluchy´ and Samsˇinˇa´kova´ 1989; Adane et al. 1996; Hidalgo et al. 1998; Rice and Cogburn 1999; Smith et al. 1998, 1999; Ekesi et al. 2000; Kassa et al. 2002; Padı´n et al. 2002; Cherry et al. 2005; Rodrı´guez-Go´mez et al. 2009; Batta et al. 2010; Kavallieratos et al. 2014). Adane et al. (1996) evaluated the virulence of ten B. bassiana isolates formulated as powder against S. zeamais in stored maize and reported significant differences among the isolates with respect to virulence, with mortality ranging between 37 and 100% 11 days after exposure to 108 conidia ml-1. Similarly, significant differences were recorded between the median lethal time (LT50) values of eight isolates of B. bassiana against adults of the cowpea weevil, Callosobruchus maculatus (F.) (Coleoptera: Bruchidae), in stored cowpea in single-dose time–mortality bioassays, with LT50 values varying between 3.11 and 6.13 days following immersion of the insects in suspensions with 108 conidia ml-1 (Cherry et al. 2005). Rice and Cogburn (1999) investigated the insecticidal activity of a conidial powder of B. bassiana against adults of the rice weevil, Sitophilus oryzae (L.) (Coleoptera: Curculionidae), the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae), and the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), on three types of food substrates and recorded mortality levels higher than 80% at the high doses for all species and substrates after 21 days of exposure. The pathogenicity and virulence of B. bassiana isolates vary remarkably among the host species and the life stage of the target pest. For instance, T. castaneum is considered resistant to B. bassiana infection, due to the benzoquinonecontaining defensive cuticular secretions that it produces, which can inhibit fungal growth (Pedrini et al. 2015; OrtizUrquiza and Keyhani 2016). Kassa et al. (2002) tested 11

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Table 1 Entomopathogenic fungal species that have been evaluated for the control of stored-product insect pests Order

Target insect pest

Infected stage

Fungus and order

References

Coleoptera

Acanthoscelides obtectus

Adult

Beauveria bassiana

Ferron (1977)

Oryzaephilus surinamensis

B. bassiana B. bassiana B. bassiana, Metarhizium anisopliae

Crespo et al. (2002) Padı´n et al. (2002) Rodrı´guez and Pratissoli (1990)

Adult

B. bassiana

Dal Bello et al. (2006)

Adult

B. bassiana

Searle and Doberski (1984)

Adult

B. bassiana

Throne and Lord (2004)

Egg

B. bassiana

Lord (2009a)

Adult

B. bassiana

Wakefield et al. (2010)

Adult

B. bassiana, M. anisopliae

Shafighi et al. (2014)

Larva

B. bassiana

Lord (2007b)

Callosobruchus maculatus

Adult

B. bassiana, M. anisopliae

Cherry et al. (2005)

Caryedon serratus

Adult

M. anisopliae

Ekesi et al. (2001)

Cryptolestes ferrugineus

Adult

B. bassiana

Lord (2007b)

Egg

B. bassiana

Lord (2009a)

Adult

B. bassiana

Wakil and Schmitt (2015)

Hypothenemus hampei

Adult

B. bassiana

Varela and Morales (1996)

Lasioderma serricorne

Larva

B. bassiana

Lord (2007b)

Prostephanus truncatus

Rhyzopertha dominica

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Adult Adult Adult

Adult

B. bassiana

Smith et al. (1999, 2006)

Adult

B. bassiana, B. brongniartii, M. anisopliae, Paecilomyces sp.

Kassa et al. (2002)

Adult

B. bassiana

Meikle et al. (2001)

Adult

B. bassiana, M. anisopliae

Bourassa et al. (2001)

Adult

M. anisopliae

Athanassiou et al. (2008)

Adult

B. bassiana, M. anisopliae

Batta (2008)

Adult

M. anisopliae

Kavallieratos et al. (2006)

Adult

B. bassiana, M. anisopliae

Shafighi et al. (2014)

Adult

B. bassiana

Vassilakos et al. (2006)

Adult

B. bassiana

Moino et al.(1998)

Adult

B. bassiana

Rice and Cogburn (1999)

Adult

B. bassiana, M. anisopliae

Mahdneshin et al. (2009)

Adult Egg

M. anisopliae B. bassiana

Batta (2005) Lord (2009a)

Adult

B. bassiana

Wakil et al. (2011, 2012)

Adult

Purpureocillium lilacinum

Barra et al. (2013)

Adult

B. bassiana

Wakil and Schmitt (2015)

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Table 1 continued Order

Target insect pest Sitophilus oryzae

Sitophilus granarius

Sitophilus zeamais

Sitotroga cerealella Tenebrio molitor

Tribolium castaneum

Infected stage

Fungus and order

References

Adult

B. bassiana

Sheeba et al. (2001)

Adult

M. anisopliae

Batta (2004)

Adult

B. bassiana, M. anisopliae, Verticillium lecanii, Isaria farinosa (=Paecilomyces farinosus)

Dal Bello et al. (2001)

Adult

B.bassiana, M.anisopliae, Isaria fumosorosea (=Paecilomyces fumosoroseus)

Kavallieratos et al. (2014)

Adult

B. bassiana

Padı´n et al. (2002)

Adult

B. bassiana, I. fumosorosea

Ramaswamy et al. (2009)

Adult

M. anisopliae

Athanassiou et al. (2008)

Adult

B. bassiana, M. anisopliae

Batta (2008)

Adult

M. anisopliae

Kavallieratos et al. (2006)

Adult

B. bassiana

Vassilakos et al. (2006)

Adult Adult

B. bassiana B. bassiana

Moino et al. (1998) Rice and Cogburn (1999)

Adult

B. bassiana

Dal Bello et al. (2006)

Adult

B.bassiana

Stephou et al. (2012)

Adult

B. bassiana

Lord (2007b)

Adult

B. bassiana

Hluchy´ and Samsˇinˇa´kova´ (1989)

Adult

B. bassiana

Hansen and Steenberg (2007)

Adult

B. bassiana

Athanassiou and Steenberg (2007)

Adult

B. bassiana

Adane et al. (1996)

Adult

B. bassiana

Hidalgo et al. (1998)

Adult

B. bassiana, M. anisopliae, Paecilomyces sp.

Kassa et al. (2002)

Adult

B. bassiana

Meikle et al. (2001)

Adult

B. bassiana

Adult

B. bassiana

Moino et al. (1998) Rodrı´gues and Pratissoli (1990)

Adult

P. lilacinum

Barra et al. (2013)

Adult

B. bassiana

Lord (2007b)

Adult

B. bassiana, M. anisopliae, Nomuraea rileyi

Ekesi et al. (2000)

Larva

B. bassiana

Larva, adult

B. bassiana

Safavi et al. (2007) Rodrı´guez-Go´mez et al. (2009)

Larva

B. bassiana

Batta et al. (2010)

Adult

B. bassiana

Adult

B. bassiana

Padı´n et al. (1997) Padı´n et al. (2002)

Adult Adult

B. bassiana, I. fumosorosea B. bassiana, M. anisopliae

Ramaswamy et al. (2009) Batta (2008)

Adult

B. bassiana, M. anisopliae

Shafighi et al. (2014)

Adult

B. bassiana

Rice and Cogburn (1999)

Larva

B. bassiana

Akbar et al. (2004, 2005)

Egg

B. bassiana

Lord (2009a)

Adult

B. bassiana

Wakil and Schmitt (2015)

Larva

B. bassiana

Lord (2007a)

Larva, adult

B. bassiana

Lord (2009b)

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Table 1 continued Order

Target insect pest Tribolium confusum

Lepidoptera

Psocoptera

Infected stage

Fungus and order

Larva

M. anisopliae

Michalaki et al. (2006)

Larva, adult

I. fumosorosea

Michalaki et al. (2007)

Adult

M. anisopliae

Kavallieratos et al. (2006)

Adult Adult

P. lilacinum B. bassiana

Barra et al. (2013) Stephou et al. (2012)

Plodia interpunctella

Larva

B. bassiana

Lord (2007b)

Ephestia kuehniella

Larva

I. fumosorosea

Michalaki et al. (2007)

Liposcelis paeta

Adult

B. bassiana

Wakil and Schmitt (2015)

isolates of B. bassiana from Ethiopia against adults of S. zeamais and the larger grain borer, Prostephanus truncatus Horn (Coleoptera: Bostrychidae), and reported that P. truncatus was more susceptible to the fungal pathogen than S. zeamais. Similarly, Smith et al. (1998) found that P. truncatus was more susceptible to B. bassiana than S. zeamais. Moreover, R. dominica was more susceptible to B. bassiana than S. oryzae (Rice and Cogburn 1999). In a recent genomic analysis, in which the whole genome of a B. bassiana strain was sequenced, Xiao et al. (2012) found and mapped many species-specific virulence genes that may explain part of the observed variance in the efficacy of B. bassiana against various storage insects. Regarding the life stage, Rodrı´guez-Go´mez et al. (2009) reported [90% mortality for adults of T. molitor, whereas larval mortality ranged between 15 and 80% after 6 and 11 days of exposure, respectively, to B. bassiana conidia cultured on various media. The increased susceptibility to B. bassiana of T. molitor adults compared to larvae was attributed by the authors to the enhanced conidia attachment on the adult cuticle surface, due to its nature and the presence of particular substances, such as mucilage (Hajek and St Leger 1994), as well as to the improved mycelia penetration, through the abundant spiracles, pore canals and intersegmental spaces of the adult’s cuticle (Rodrı´guez-Go´mez et al. 2009). Ecdysis and the formation of new cuticle after each larvae molting (Vestergaard et al. 1999), as well as the reduced immune function in aged individuals, have also been linked to the higher susceptibility to infection in adults (Rodrı´guez-Go´mez et al. 2009). Recently, Kavallieratos et al. (2014) evaluated the effectiveness of a B. bassiana isolate against S. oryzae, with different application methods, i.e. application of the conidial suspension on food and spraying of adults with or without food, and concluded that the application method exerts a significant role on the effectiveness of

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References

entomopathogenic fungi as grain protectants. Moreover, Hidalgo et al. (1998) found that among different formulations of B. bassiana, an oil-based fungal preparation was found more effective than others. Batta (2016a) proposed that a mixture of aqueous and oil formulations should be more appropriate, in order to provide the required water for conidial germination. Apparently, the selection of highly virulent isolates that can be applied against a mixture of stored-product insects is a precondition for the successful control of insects in grain storage systems. Still, most of the efficacy of aqueous formulations has not been investigated in detail for the fungal species available, as most of the studies are focused on either dry or oil formulations. Beauveria brongniartii Few studies with B. brongniartii (Saccardo) Petch (Ascomycota: Hypocreales) against stored-grain insects are available. A B. brongniartii isolate, originally isolated from a spider, caused 85% mortality to P. truncatus adults 4 days after exposure to 107 conidia ml-1 of the fungal pathogen and was equally effective with 10 isolates of B. bassiana (Kassa et al. 2002). Rodrigues and Pratissoli (1990) reported 6-month protection of maize and bean grains from damage by S. zeamais and A. obtectus, respectively, following treatment with B. brongniartii and M. anisopliae at a dose of 108 conidia ml-1. Metarhizium anisopliae The virulence of M. anispopliae on stored-product insect species has been found to vary remarkably among different fungal strains. Ekesi et al. (2001) tested five M. anisopliae isolates in laboratory bioassays and reported complete control of the groundnut bruchid, Caryedon serratus Olivier (Coleoptera: Bruchidae), by one isolate, whereas the

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other isolates tested were not effective. Several studies have been conducted using M. anisopliae isolates against S. oryzae with variable results. Dal Bello et al. (2001) reported low virulence of three M. anisopliae isolates against adults of S. oryzae, with mortality levels ranging between 20 and 50% 1 month after exposure to 7 9 106 conidia ml-1. In contrast, Kavallieratos et al. (2014) reported complete control of S. oryzae adults 14 days after application of M. anisopliae on adults, with or without food, at dose rates of 1.77 9 107 and 1.77 9 108 conidia ml-1. In another study, adult mortality of S. oryzae was high and ranged from 73.3 to 86.7% with M. anisopliae conidia formulated in charcoal and oven ash and applied either before or after pest infestations, respectively, indicating that different isolates of the same fungus can have variable infectivity against the same insect species (Batta 2004). Differently formulated M. anisopliae conidia caused different levels of mortality of adults of R. dominica (Batta 2005). Conidia suspended in distilled water or formulated in invert emulsion or wheat flour caused 56.7, 93.3 and 86.7% mortality, respectively, 7 days after treatment (Batta 2005). The virulence of M. anisopliae against larvae of T. confusum was moderate, with mortality levels in all cases lower than 55% after 7 days of exposure even at 8 9 1010 conidia kg-1 wheat or flour (Michalaki et al. 2006). Using the same M. anisopliae isolate and two fungal preparations, a conidial suspension and a conidial powder, Kavallieratos et al. (2006) reported varying mortality levels against adults of R. dominica, S. oryzae and T. confusum, depending on the insect species, the dose and the fungal preparation. In that study, two conidial preparations were tested, a conidial powder and a suspension, the powder being more effective than the suspension in the case of S. oryzae and T. confusum, whereas the opposite was observed for R. dominica. For a M. anisopliae isolate, at various conditions, it was shown that R. dominica was more tolerant than S. oryzae (Kavallieratos et al. 2006; Athanassiou et al. 2008). Kavallieratos et al. (2008) investigated the factors that affected the attachment of M. anisopliae conidia and reported different degrees of attachment to different body parts of T. confusum adults. Conidia were found on all body parts examined, i.e. elytra, femur, tibia, tarsus and sternites VI and VII of T. confusum adults; however, the highest conidia number was recorded on the sternites (Kavallieratos et al. 2008). Isaria spp. The first reports of Isaria spp. (formerly Paecilomyces spp.) against post-harvest insects evaluated Isaria farinosa (Holmskjold) Fries (Ascomycota: Hypocreales) (formerly Paecilomyces farinosus (Holmskjold) Brown and Smith)

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and an Isaria sp. isolate against S. oryzae (Dal Bello et al. 2001) and S. zeamais and P. truncatus (Kassa et al. 2002), respectively. Isaria fumosorosea (Ifr) Wize (formerly Paecilomyces fumosoroseus (Wize) Brown and Smith) (Ascomycota: Hypocreales) was effective against T. confusum and E. kuehniella, but its effectiveness was highly dependent on the target species and life stage, exposure interval and temperature (Michalaki et al. 2007). Survival of S. oryzae adults after application of I. fumosorosea varied depending on the application method but was not significantly affected by dose (Kavallieratos et al. 2014). For example, of the application methods examined in that study, direct application of the fungus on S. oryzae adults was more effective than contact of the insects with the substrate, which was previously treated with the fungus. Other entomopathogenic fungi Ekesi et al. (2000) found that Nomuraea rileyi (Farlow) Sampson (Ascomycota: Hypocreales) caused 33–95% adult mortality of the Angoumois grain moth, Sitotroga cerealella (Olivier) (Lepidoptera: Gelechiidae), in sorghum after 7 days of exposure. In the highest dose rate tested (5.2 9 107 conidia g-1), the mortality caused by N. rileyi was similar to the mortality caused by pirimiphos-methyl at a rate of 10 ppm (Ekesi et al. 2000). Lecanicillium lecanii Zare and Gams (Ascomycota: Eurotiales) (formerly Verticillium lecanii Zimmermann) caused low mortality (20%) of S. oryzae adults 30 days after spraying adults with a suspension containing 7 9 106 spores ml-1 of the fungus (Dal Bello et al. 2001). Recently, Barra et al. (2013) evaluated the virulence of 20 isolates of Purpureocillium lilacinum (Thom) Luangsa-ard, Houbraken, Hywel-Jones and Samson (formerly Paecilomyces lilacinus (Thom) Samson) against adults of T. confusum, S. zeamais and R. dominica and reported highly variable results, depending on the fungal isolate and the target species. Briefly, the mortality percentages 20 days after exposure fluctuated between 35–90, 10–50 and 15–65 for T. confusum, S. zeamais and R. dominica, respectively, suggesting that virulence may be expressed differently among various isolates (Barra et al. 2013). Combination of entomopathogenic fungal species The use of several antagonists instead of a single one is likely to provide better biocontrol in terms of efficacy (Baker and Cook 1982). The combined application of two biocontrol agents can be advantageous, as it may allow the control of multiple target species or provide consistent control and improve efficacy across a broad range of environmental conditions. In this framework, Dal Bello

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et al. (2001) studied the efficacy of a mix of B. bassiana and M. anisopliae isolates against S. oryzae and found that this combined application did not enhance the mortality compared to the single applications of each isolate alone. However, a mixture of dry conidia of B. brongniartii and M. anisopliae on maize gave high mortality of S. zeamais and effective control for a period of 6 months (Rodrigues and Pratissoli 1990).

Effect of biotic and abiotic factors on the virulence of entomopathogenic fungi Researchers early understood that the virulence and success of entomopathogenic fungi in controlling post-harvest insects vary depending on the environmental conditions, with temperature and humidity being the key elements for their efficacy. As far as relative humidity is concerned, the results available are often contradictory. It is generally believed that entomopathogenic fungi are favored by high humidity/moisture conditions. For example, Searle and Doberski (1984) identified humidity as the major factor determining whether B. bassiana spores will germinate and infect O. surinamensis adults and reported reduced mortality at humidity levels lower than 90%. This effect has also been noted in some field insect pests, apart from stored-product insects. Relative humidity significantly affected infection of larvae of the beet armyworm, Spodoptera exigua Hu¨bner (Lepidoptera: Noctuidae), by M. anisopliae, as mortality increased with the increase in relative humidity (Han et al. 2014). However, there are cases where the efficacy of entomopathogenic fungi against post-harvest insects was not affected by low humidity conditions. Ferron (1977) reported that the infection of A. obtectus adults by B. bassiana is independent from the humidity levels, but the development of fungal hyphae on the cadavers is possible only at humidity levels near saturation. Similarly, Akbar et al. (2004) did not find significant differences between two levels of relative humidity, i.e. 55 and 75%, in the efficacy of B. bassiana against T. castaneum larvae. Other scientists have also reported humidity-independent infection of post-harvest insects by entomopathogenic fungi (Rodrigues and Pratissoli 1990; Adane et al. 1996). Recent reports correlate the improved efficacy of entomopathogenic fungi with relatively reduced humidity levels (Lord 2005, 2007a, b; Michalaki et al. 2006; Athanassiou and Steenberg 2007). For example, Lord (2005) reported that the mortality of R. dominica treated with B. bassiana was increased with the decrease in relative humidity. Similarly, the mortality of the granary weevil, Sitophilus granarius L. (Coleoptera: Curculonoidae), in wheat treated with B. bassiana was higher at

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55% than at 75% relative humidity at 20 and 25 C (Athanassiou and Steenberg 2007). At the same time, M. anisopliae was generally more effective at 55% than at 75% relative humidity against T. confusum larvae (Michalaki et al. 2006). These findings are of practical interest, as stored-product environments are often dry; thereafter, these conditions could prove to be an advantage for the insecticidal activity of entomopathogenic fungi. At the same time, increased humidity/moisture levels in durable stored products are not desired, due to rapid microbial infection and rapid deterioration of the commodity. Hence, in contrast to what was believed in the past, some fungal strains can be more effective at relatively dry conditions, as in the case of durable stored products. Nevertheless, in this regard, the fungal strain plays an important role, as there are strains that are highly virulent at low relative humidity levels (Lord 2005, 2007a, b; Athanassiou and Steenberg 2007). The mechanisms on which some strains are more effective at dry conditions are poorly understood, and probably high efficacy is related to increased water stress of the target insects. Moreover, high relative humidity values may reduce the stability and persistence of fungal conidia, as it has been shown for Metarhizium flavoviride Gams and Rozsypal (Deuteromycotina: Hyphomycetes) (Hedgecock et al. 1995; Moore et al. 1996; Hong et al. 1998). Temperature plays a significant role for the effectiveness of entomopathogenic fungi. It is widely accepted that high temperatures affect negatively conidial viability and germination (Daoust and Roberts 1983; Moore et al. 1996; Horaczek and Viernstein 2004). There are few available data, though, on the effect of temperature on the virulence of entomopathogenic fungi. Generally, different fungal species have different temperature requirements. For instance, regarding several strains of B. bassiana, the optimum temperature for conidial germination and vegetative growth is around 25 C (Walstad et al. 1970; Fargues et al. 1997; Ekesi et al. 1999). Beauveria bassiana has been found to be more effective at 26 C than at 30 C against R. dominica, S. oryzae (Vassilakos et al. 2006) and S. granarius (Athanassiou and Steenberg 2007) in stored wheat. Similar optimum temperatures were reported for B. bassiana against R. dominica by Lord (2005). Isaria fumosorosea was more effective at 20 C than at 25 C (Michalaki et al. 2007), while the effectiveness of M. anisopliae against T. confusum larvae was increased with the increase in temperature (Michalaki et al. 2006). Biotic variables are also important factors that determine the insecticidal effect of entomopathogenic fungi for the control of stored-product insects. Although limited information is available on the impact of commodity on the efficacy of entomopathogenic fungi, it appears that the type of stored product is determinative for their effectiveness.

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Metarhizium anisopliae is more effective against T. confusum larvae on wheat than on flour, probably due to the reduced attachment of fungal spores on the insect’s cuticle in the presence of flour particles, but also due to feeding patterns of the exposed insects (Michalaki et al. 2006). Similar results on the effect of commodity on the efficacy of B. bassiana were shown by Rice and Cogburn (1999). Commodity can affect also the number of attached conidia on the grain kernels, i.e. significantly higher numbers of conidia were observed on wheat treated with M. anisopliae dry conidia compared to barley and rice (Kavallieratos et al. 2008). The conidia retention ability of the treated grains may have an effect on insect mortality, as it has been shown already for diatomaceous earth particles (Kavallieratos et al. 2005).

Combination of entomopathogenic fungi with other technologies The simultaneous application of different pest management strategies may, in many cases, provide improved pest control compared to single applications of each technology alone (Throne 1989). Moreover, the high dose rates of entomopathogenic fungi required in several cases to achieve successful biocontrol render their application in commercial scale in grain storage facilities practically and economically unattractive. Therefore, the combination of entomopathogenic fungi with other pest control technologies could be a realistic alternative. Varietal resistance, diatomaceous earths, insecticides, botanicals or macrobiocontrol agents have been tested in combination with entomopathogenic fungi with various results. Combination of entomopathogenic fungi with diatomaceous earth The combined use of entomopathogenic fungi with diatomaceous earth (DE) has been thoroughly investigated against a broad spectrum of stored-product insect species. Lord (2001) was the first to report a clear synergism between B. bassiana and diatomaceous earth against adults of R. dominica and O. surinamensis using a commercial DE formulation. Since then, various studies have confirmed the synergistic or additive effect between B. bassiana and DE against many major stored-product insect species, such as T. castaneum (Akbar et al. 2004), S. granarius (Athanassiou and Steenberg 2007), S. oryzae (Dal Bello et al. 2006; Vassilakos et al. 2006), A. obtectus (Dal Bello et al. 2006) and R. dominica (Lord 2005; Vassilakos et al. 2006; Riasat et al. 2011; Wakil et al. 2011, 2012). Increased fungal efficacy after simultaneous application with DE was shown also for M. anisopliae against S.

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oryzae and R. dominica on wheat and maize; however, the observed additive effect was not so clear as in the case of B. bassiana and DEs (Athanassiou et al. 2008). Lord (2001) attributed synergism to the changes in the chemistry of the insect’s cuticle when DE is present, which may influence the ability of conidia to attach, germinate and penetrate the host. Moreover, in some cases there has been an increase in the number of fungal conidia attached to the insect’s cuticle in the presence of DE (Lord 2001; Akbar et al. 2004); however, the increased conidial attachment in the presence of DE is not considered to play a significant role in the fungus performance (Lord 2001). In an earlier study, Stephou et al. (2012) used Agrobacterium-mediated transformation to create a strain of B. bassiana with green fluorescent protein (GFP) and found that fluorescence of the fungus on the external part of the cuticle of S. oryzae and T. confusum was increased when the fungus was applied with DE, as compared to the application of the fungus alone. Hence, conidial attachment is increased under the simultaneous presence of DEs. Apparently, conidial attachment, germination and adherence to the insects’ cuticle are affected by the characteristics of the external cuticular part, i.e. the texture, but also the lipid composition of that area (Lord 2001; Akbar et al. 2004; Stephou et al. 2012). DE seems to play an important role exactly in this area, by blocking the lipids that have a fungistatic effect (Smith and Grula 1981; Sosa-Gomez et al. 1997; Lord 2001). Also, when DE was present, B. bassiana persisted for a longer period (Stephou et al. 2012). However, this is not always the case; the efficacy of M. anisopliae against larvae of T. confusum on wheat and flour was not always increased, but sometimes decreased by the presence of a DE formulation (Michalaki et al. 2006). Varying results were also obtained after the combined application of M. anisopliae and DE against R. dominica, S. oryzae and T. confusum adults (Kavallieratos et al. 2006) and after application of I. fumosorosea and DE against larvae and adults of T. confusum and larvae of E. kuehniella (Michalaki et al. 2007). In the case of M. anisopliae, the presence of DE in grains treated with fungus increased in many cases the fungal persistence (Athanassiou et al. 2008). In that study, R. dominica mortality was significantly higher in grains treated with M. anisopliae and DE, rather than the fungus alone 6 months after application. Especially for the case of M. anisopliae, Moore and Higgins (1997) found that silica caused a detrimental effect to fungal conidia, in terms of reducing germination, probably through desiccation. Thus, it is likely that DE, which is mainly composed of amorphous silica, affects the conidia of M. anisopliae, resulting in reduction in the fungal efficacy against the target species. Vassilakos et al. (2006) observed that at low fungal rates of B. bassiana, the addition of DE did not increase its efficacy

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against R. dominica, whereas with the increase in fungal rates an additive effect was noted. Based on these results, the authors suggested that for B. bassiana there is a ‘‘critical conidial concentration,’’ which differentiates the effect of DE on the fungal efficacy, and above which an additive effect is more likely to occur. These findings make evident that additional experimentation is warranted to elucidate the effect of DE on the efficacy of entomopathogenic fungi and reveal the factors that affect their interaction. Overall, the reasons for the increase in efficacy of some fungi in the presence of DE against insects, at least in the case of B. bassiana, are poorly understood. We assume that this increase in efficacy was mainly due to the fact that the DE particles in the cuticle enhance fungal attachment or the DE increases fungal viability and conidial germination. Different DE formulations have been tested so far in combination with entomopathogenic fungi, such as InsectoTM (Athanassiou and Steenberg 2007), SilicoSec (Kavallieratos et al. 2006; Michalaki et al. 2006, 2007; Vassilakos et al. 2006; Athanassiou and Steenberg 2007; Wakil et al. 2012; Shafighi et al. 2014), DEBBM (Wakil et al. 2011), PyriSec (Athanassiou and Steenberg 2007), and Protect-It (Lord 2001, 2005; Athanassiou et al. 2008). However, in most cases, the DE effect on the entomopathogenic fungus was not affected by the type of DE (Athanassiou et al. 2006; Athanassiou and Steenberg 2007), which clearly indicates that all DEs are likely to have the same effect. In contrast, temperature and relative humidity levels are critical for the insecticidal effect of both entomopathogenic fungi and DEs. High temperatures usually have a negative impact on the conidial germination and subsequently the fungal efficacy (Moore and Higgins 1997; Lord 2005); however, for B. bassiana, this effect can be moderated when the fungus is applied simultaneously with DEs (Athanassiou and Steenberg 2007). As mentioned above, entomopathogenic fungi usually require high humidity levels to germinate and sporulate (Roberts and Campbell 1977), and this effect is not, in most cases, affected by the presence of DE (Athanassiou and Steenberg 2007). Combination of entomopathogenic fungi species with insecticides In order to improve biological control of stored-product insect pests, several researchers have evaluated the combined use of efficient entomopathogenic fungi isolates with insecticides in the context of an integrated pest management approach. Dal Bello et al. (2001) reported that the combined application of a mix of a B. bassiana and a M. anisopliae isolate with fenitrothion against S. oryzae caused the highest mortality compared to the fungal

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mixture and the organophosphate insecticide alone. Wakil et al. (2012) demonstrated the potential of the combined application of B. bassiana and the neonicotinoid thiamethoxam for long-term grain protection against R. dominica. Similarly, the application of B. bassiana was efficiently combined with DE and imidacloprid against R. dominica, T. castaneum, the rusty grain beetle, Cryptolestes ferrugineus Stephens (Coleoptera: Laemophloeidae), and the Psocoptera species Liposcelis paeta Pearman (Psocoptera: Liposcelididae), but their effectiveness was closely related to the insect species (Wakil and Schmitt 2015). Based on the above studies, it becomes evident that the combined effect of entomopathogenic fungi with low mammalian toxicity insecticides may have some potential for further development in the future and merits additional investigation. Combination of entomopathogenic fungi species with various technologies In a laboratory trial, Hansen and Steenberg (2007) described the effect of the combined use of the parasitoids L. distinguendus and Anisoptermalus calandrae (Howard) (Hymenoptera: Pteromalidae) with B. bassiana against S. granarius and reported that the two larval parasitoids were susceptible to infection by the fungus, when the fungus was applied as a surface treatment. Therefore, a lure-and-kill approach was proposed by the authors, where the fungus is applied in a trap to prevent parasitoids from being exposed to the fungus. Moreover, the fungus was found virulent for the parasitoid species, which should be seriously taken into account when different biocontrol methods are to be applied at the same time. Throne and Lord (2004) evaluated the combined use of B. bassiana with resistant oat cultivars that prolong the immature developmental period of O. surinamensis against this species. According to their results, the application of the fungus did not affect the oat varietal resistance to O. surinamensis, i.e. the presence of the fungus did not affect in any case the duration of larval development; however, it did affect the number of progenies produced (Throne and Lord 2004). Based on these results, the authors suggest that B. bassiana could be used to control this species in not-cleaned stored oats. Beauveria bassiana has been integrated also with botanicals, namely rosemary oil and azadirachtin; however, its efficacy against T. castaneum larvae has either been stable (not enhanced) or even reduced (Akbar et al. 2005). The authors attributed the reduction in fungal efficacy to the repellent effect that azadirachtin exerts to T. castaneum, due to which larval movement and conidial attachment were reduced (Akbar et al. 2005). Finally, Lord (2009b) evaluated the efficacy of B. bassiana against larvae and

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adults of T. castaneum with reduced oxygen (5 ± 1%) and increased carbon dioxide (40 ± 2%). Based on their results, the gas modifications affected both the fungus and the insect, as conidial germination and growth rate of the fungus, as well as the beetle development rate were all reduced (Lord 2009b). Overall, the modified atmosphere tested improved the insect control by the fungus, indicating the potential of these two technologies to be integrated (Lord 2009b).

Formulation and commercialization of entomopathogenic fungi The efficacy of entomopathogenic fungi against storedproduct insects depends highly on the formulation used, as it affects conidial viability, uptake from the host insect and subsequently insect mortality. A successful mycopesticide formulation must be easy to apply and has improved storage characteristics and enhanced biocontrol efficacy (Wraight et al. 2001). Different carriers have been tested and proposed for the formulation of entomopathogenic fungi used against storage insects (Hidalgo et al. 1998; Smith et al. 1999; Batta 2004, 2016a, b; Batta et al. 2010). In general, liquid formulations of entomopathogenic fungi are less studied than dry formulations (usually dusts) (Batta 2016a). Several studies investigated the use of dry conidia of entomopathogenic fungi for the control of stored-product insects (Rodrigues and Pratissoli 1990; Adane et al. 1996; Hidalgo et al. 1998). However, airborne conidia are related to potential human health hazards; therefore, alternative formulations have been proposed. Hidalgo et al. (1998) investigated different formulations of B. bassiana against S. zeamais, i.e. a talc-based dustable powder, an oil suspension and a fat pellet formulation, whereas Smith et al. (1999) evaluated the use of hydrogenated rapeseed oil as a carrier for conidia of B. bassiana against P. truncatus. Batta et al. (2010) studied the efficacy of two isolates of B. bassiana when applied in an invert emulsion formulation against T. molitor and reported higher mortality of T. molitor larvae when the invert emulsion was used compared with aqueous conidial suspensions. Recently, electrostatically charged powders have been proposed as potential carriers for the delivery of entomopathogenic fungi against post-harvest insects. These substances are chemically inert, non-hygroscopic electrostatic micropowders that show potential for use as a carrier in bait stations and autodissemination systems against stored-product insects (Nansen et al. 2007; Baxter et al. 2008; Wakefield et al. 2010). Nansen et al. (2007) examined the uptake and behavioral responses of O. surinamensis to an electrostatically charged powder harvested

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from the Brazilian Carnauba palm, Copernica cerifera Martius (Palmae), and suggested that considerable amounts of this dust were taken up and retained over several days from the beetles. Similar results were shown with the same substance for P. interpunctella (Baxter et al. 2008). Based on these results, Baxter (2008) integrated B. bassiana spores in an autodissemination system for the control of P. interpunctella and reported that the conidia co-formulation with the electrostatic powder increased conidia uptake by the moths. In this autodissemination system, the spores of the fungal pathogen were horizontally transferred using the carnauba wax between conspecifics during normal social behavior such as mating or aggregation (Baxter 2008). The carnauba wax, formulated as EntostatTM, is already in use in stored-product protection as a means for autodissemination of pheromones for mating disruption (Trematerra et al. 2013). Also, this material has been proved highly synergistic for the insecticidal efficacy of the organophosphorus compound pirimiphos-methyl, for the control of S. oryzae and O. surinamensis (Athanassiou et al. 2016). Recently, Storm et al. (2016) reported that kaolin can be used as a synergistic co-formulant of B. bassiana for the control of O. surinamensis and S. granarius. Nevertheless, this combination showed no synergistic effect in the case of T. confusum. It is generally considered that this synergism can be attributed to the same factors that have been reported above for the combination of fungi with DEs, though not fully clarified yet. Moreover, the combined effect of Entostat, kaolin and B. bassiana was found to be very effective for the control of P. truncatus (Nboyine et al. 2015; Acheampong et al. 2016). The potentials of ‘‘charged’’ dusts that contain conidia should be evaluated in more detail, as this technology will end up in ‘‘active’’ bioinsecticides that are attached to the insects’ cuticle, or can be transferred among individuals of the target species (Wakefield 2006; Wakefield et al. 2010; Athanassiou et al. 2016; Storm et al. 2016). Several commercial products based on entomopathogenic fungi are available on the market worldwide against a broad spectrum of target pests, with the majority of them being based on B. bassiana and M. anisopliae (De Faria and Wraight 2007). Among more than 170 commercial products, De Faria and Wraight (2007) reported only one (Boverosil, Czech Republic) that claims activity against stored-product insects, which is currently out of production. In EU, entomopathogenic fungi as plant protection products fall within the scope of Directive 91/414/ EEC (European Commission 1991). Two B. bassiana (ATCC-74040, GHA1) and one I. fumosorosea (Apopka 97) strains are currently registered in the EU and listed in Annex I; however, none of these strains is registered for application against stored-product insects.

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The future of entomopathogenic fungi in storedproduct protection Based on the available literature reported in this review, it becomes evident that the use of entomopathogenic fungi has strong potential against stored-product insects. Nevertheless, numerous research challenges remain. One of the major drawbacks in using entomopathogenic fungi is the contradicting results and efficacy levels obtained with different fungal strains. For instance, the efficacy of M. anisopliae strains against S. oryzae varies from low virulence (Dal Bello et al. 2001) to complete control (Kavallieratos et al. 2014). Different fungal strains, usually isolated locally in the region of experimentation, give variable results. Better documentation and characterization of the strains used in the various bioassays would be a valuable tool for the future exploitation of entomopathogenic fungi. Molecular tools, such as DNA fingerprinting or sequencing, could enable the differentiation of the fungal strains used, could clarify the taxonomic relationships among strains or even discriminate between aggressive and non-aggressive entomopathogenic fungal strains. Another crucial disadvantage of the use of entomopathogenic fungi is the enormous amount of formulated conidia needed for application. In this context, autodissemination, or related co-formulants may provide a viable solution to this problem. Moreover, the direct application of fungal propagules on durable, edible stored products, such as grains and related amylaceous commodities, may not be fully acceptable by the consumer. Therefore, alternative, innovative methods of application of entomopathogenic fungi should be evaluated against post-harvest insects. The lure-and-kill technique, based on the attraction of the target pest using semiochemicals (pheromones or kairomones) in a bait station, where the pest would pick up a lethal dose of conidia, could be an appropriate alternative use of entomopathogenic fungi in storage facilities against post-harvest insects (Soper 1978; Smith et al. 1999; Baxter 2008; Baxter et al. 2008; Wakefield et al. 2010). Such a pest management approach would reduce the need for broad-scale applications of fungal conidia and the human health and consumer concerns related to entomopathogenic fungi-treated products. The use of an adhesive carrier, such as electrostatically charged dusts, for the fungal conidia in a bait station could increase the success of the method through the autodissemination of the entomopathogen, i.e. conidia would stick more easily and in sufficient quantities to the attracted individuals, which would transmit the fungus throughout the rest of the population. Therefore, future research should focus on the development of biopesticide products based on entomopathogenic fungi for the specific control of stored-product insects and the integration of entomopathogenic fungi in innovative dissemination and control systems.

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Author contributions Christos I. Rumbos and Christos G. Athanassiou conducted the review of the existing literature and wrote the manuscript. Both authors read and approved the final manuscript. Acknowledgements We would like to thank Frank H. Arthur and James E. Throne for reading the manuscript prior to submission. Compliance with ethical standards Conflict of interest Christos Rumbos and Christos Athanassiou declare that there have no conflicts of interest. Christos Rumbos and Christos Athanassiou conceived the work and wrote the manuscript. Research involving animal and human rights The research did not involve human participants and/or animals. Ethical approval This article does not contain any studies with human participants performed by any of the authors.

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