Keywords

6.1 Introduction

Entomopathogenic fungi (EF) are responsible for epizootics that often regulate insect pest populations. The genera Metarhizium (Hypocreales : Clavicipitaceae ), Beauveria (Hypocreales: Cordycipitaceae ), and Isaria (Hypocreales: Cordycipitaceae) are the fungi most frequently used in biological control programs. The asexual spore , named conidium, is an anamorphic and primary form essential in the life cycle of many filamentous fungi (Osherov and May 2001). Mostly, entomopathogenic fungal infection starts with the attachment of conidia to the insect cuticle , and then progresses to conidial germination, penetration, vegetative growth (as hyphal bodies), conidiogenesis and, finally, dissemination (Lacey et al. 2001; Pedrini 2018).

Temperature is an abiotic factor that influences all fungal development stages, from primary processes such as biochemical and cellular reactions in conidia to overall infection and fungus-host interaction (Zimmermann 1982; Cabanillas and Jones 2009) (Fig. 6.1). Generally, three thermal conditions are considered for fungal development, i.e., low (below 11 °C), intermediate (11–28 °C) and high temperatures (above 28 °C) (Vidal et al. 1997). Under this classification, EF are considered mesophilic because they develop well between 10 and 40 °C, with optimal growth between 25 and 35 °C (Crisan 1973; Fargues et al. 1997; Vidal et al. 1997; Dimbi et al. 2004). Metarhizium rileyi (= Nomurea rileyi ) presented optimum growth at 25 °C (Ignoffo et al. 1976). Similar studies with Metarhizium spp. strains other than M. rileyi showed optimum growth between 11 and 35 °C (Ouedraogo et al. 1997), whereas B. bassiana strains grew over a wider temperature range, from 8 to 35 °C, exhibiting optimal growth at temperatures as low as 20 °C and as high as 30 °C (Fargues et al. 1997).

Fig. 6.1
figure 1

Effect of non-optimal temperatures for fungal growth in various processes of conidia life cycle

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Thermotolerance is defined as the ability to withstand relatively hot (or cold) conditions. Viability in thermotolerance above 40 °C was observed in several strains of Metarhizium spp. (Hedgecock et al. 1995; Li and Feng 2009). However, the growth of some strains of Beauveria spp. was inhibited after 2 h at 45 °C. On the other hand, cold activity was reported for both Beauveria spp. and Metarhizium spp., B. bassiana being able to grow at temperatures as low as 5 °C and some M. anisopliae strains at 8 °C, due to their cold adaptation (McCammon and Rath 1994; Croos and Bidochka 2001; Fernandes et al. 2008; Santos et al. 2011).

This physical factor is very important to regulate all development processes, since the beginning of germination up to conidial sporulation (Edelstein et al. 2005; Keyser et al. 2014) (Fig. 6.1). Thus, the screening of fungal strains thermotolerant to high and low temperatures, together with the molecular and genetic characterization, and the investigation on formulations to increase fungal performance are key factors to distinguish strains with high potential to be used in biological control programs of arthropods (Dillon and Charnley 1990; Fernandes et al. 2010; Oliveira et al. 2018).

6.2 Thermal Characteristics and Geographical Origin

Interactions between different environmental abiotic factors, spore germination and other physiological traits in fungi were early reported by Gottlieb (1950). EF are ubiquitous in soils worldwide, from the Arctic to the tropics (Zimmermann 2007), thus different thermal behavior in fungi isolated from different geoclimatic origins and/or from diverse hosts might be expected. In fact, Isaria fumosorosea (= Paecilomyces fumosoroseus ) strains from tropical or subtropical origin (Cuba, USA, India, Nepal, and Pakistan) demonstrated high tolerance to upper limits (optimal development at 32–35 °C) than European strains (optimal growth at 25–28 °C). Conversely, European strains isolated from temperate areas are able to grow at 8 °C and show to be more tolerant to low temperature than those fungal strains originated from tropical or subtropical regions (Vidal et al. 1997; Fargues and Bon 2004).

Some studies reported no thermal variability between B. bassiana strains isolated from temperate or tropical areas from Europe, Africa, Asia and America (Fargues et al. 1997, Devi et al. 2005, Rangel et al. 2005, Borisade and Magan 2014). Although M. anisopliae strains from the tropical region (Africa) were susceptible to germinate at 15 °C (Dimbi et al. 2004), M. anisopliae isolates from Ontario (Canada) showed large variation in both growth rate and conidial production at temperatures between 8 and 22 °C. This latter study shows that M. anisopliae isolated from forested areas were cold-active, while the isolates from agricultural areas showed an ability for growth at high temperatures and resilience to UV exposure (Bidochka et al. 2001).

Regarding cold activity, fungal strains isolated far from the equator presented higher relative germination under cold conditions than strains originated from near the equator. Beauveria bassiana isolated from higher latitudes were cold-active, however, there was not a similar correlation for heat (Fernandes et al. 2008). Thermal characteristics and geographical origins coincide with conditions during natural epizootics between EF and hosts. Persistence in the environment indicates certain adaptation as it was reported for Isaria spp., which showed to be well adapted to semiarid region (Cabanillas and Jones 2009). Finally, the specific pathogen M. acridum , virulent against desert locusts, is able to tolerate temperatures up to 42 °C, and thus to avoid the behavioral fever developed by host in an attempt to stop the development of the fungus, i.e., a successful strategy to avoid infection by thermosensitive EF such as B. bassiana (Elliot et al. 2002).

6.3 Culture Conditions and Conidial Thermotolerance

The production of conidia by EF is influenced mostly by culture conditions (viz., temperature, pH, water activity, aeration) and media composition (viz., carbon and nitrogen sources, metal ions) (Hallsworth and Magan 1996; Ibrahim and Jenkinson 2002; Ying and Feng 2006; Rangel et al. 2008). Elevated temperatures reduce spore viability, growth, germination and virulence (Anderson and Smith 1972; McCammon and Rath 1994; Inglis et al. 1996; Rangel et al. 2010; Tumuhaise et al. 2018). It is possible, however, to optimize both conidial production and thermotolerance by efficient culture conditions and/or supply of culture media (Ouedraogo et al. 1997; Cabanillas and Jones 2009; Kim et al. 2010c). Accordingly, Isaria spp. showed greater tolerance (from 20 to 30 °C) after growing on Sabouraud Dextrose Agar Yeast extract (SDAY) than fungi grown on Sabouraud Maltose Agar (SMA) (Cabanillas and Jones 2009). Submerged cultures of I. fumosorosea grown in Sabouraud Dextrose Broth (SDB) were able to develop well from 20 to 34 °C, showing optimal growth at 28 °C. In solid fermentations, however, these strains grew optimally at 25 °C (Esther et al. 2013). Although the excess of heat on conidia causes unviability in the derived fungal propagules, it is possible to improve thermotolerance from 26 to 30 °C by increasing the sucrose content in the culture media (McClatchie et al. 1994). Conidia of M. robertsii produced on potato, dextrose, agar and yeast extract (PDAY) medium, containing low concentrations of salicylic acid, demonstrated increased tolerance to heat (Rangel et al. 2012). Millet grain was used as a substrate to produce conidia by B. bassiana and M. anisopliae , potentially enhancing conidial thermotolerance of fungi grown on a massive production system (Kim et al. 2011).

Metarhizium acridum grows on agar medium and produces both aerial and microcycle conidia. Tolerance of both propagules was compared at 40–45 °C, showing that microcycle conidia were more heat resistant than normal aerial conidia (Zhang et al. 2010). Microcycle conidiation is defined as a process in which the germination of spores directly produces the formation of conidia, without the intervention of an intermediate mycelial growth. This microcycle conidiation can be induced by manipulation of environmental conditions, especially culture conditions that are stressful to fungi (Hanlin, 1994; Bosch and Yantorno 1999; Zhang et al. 2010; Jung et al. 2014).

A relationship between thermotolerance and hydrophobicity can be traced. Employing a siloxane-mediated conidial collection method based on hydrophobicity, it is possible to classify conidia from B. bassiana and M. anisopliae into two groups with different thermotolerance (Kim et al. 2010b). Similar results were observed for species with hydrophobic conidia such as B. bassiana, M. brunneum , M. robertsii , M. anisopliae and I. fumosorosea , which were more thermotolerant than species with hydrophilic conidia such as Tolypocladium cylindrosporum , T. inflatum , Simplicillium lanosoniveum , Lecanicillium aphanocladii , Aschersonia placenta and A. aleyrodis (Souza et al. 2014). The sugar content (types and concentrations) used as carbon source in culture media for conidial production may affect both conidial thermotolerance and hydrophobin-like or formic-acid-extractable (FAE) protein content (Ying and Feng 2004), (see Sect. 6.5).

6.4 Effect of Abiotic Factors on Conidial Germination

The interaction of abiotic factors (temperature, humidity, light, pH) are important to germination, dispersion, and development of fungal conidia (Glare et al. 1986; Jaronski 2010; Osherov and May 2001; Oliveira et al. 2018). Water is fundamental to start conidial germination. However, the interaction between water and temperature can reduce the viability and/or limit conidial viability of some EF, e.g., B. bassiana, M. anisopliae , I. farinosa and I. fumosorosea require humidity and optimum temperature conditions to their development (Hallsworth and Magan 1999; Devi et al. 2005; Borisade and Magan 2014).

Humidity and temperature are key factors in activation of metabolic pathways allowing the nutrients mobilization required for conidial germination, but are also important during storage periods, longevity and persistence of quiescent conidia (Daoust and Roberts 1983; Dillon and Charnley 1990, Bouamama et al. 2010). These factors also affect physiological interactions between the host and pathogen (Walstad et al. 1970; Luz and Fargues 1997; James et al. 1998). The ultraviolet (UV) radiation (UV-A and UV-B) also affects conidial germination of M. acridum and M. robertsii , limiting entomopathogenic fungal development, however increasing thermotolerance by accumulating trehalose and mannitol (Braga et al. 2001; Pereira-Junior et al. 2018; Rangel and Roberts 2018).

6.5 Thermal Effects and Metabolic Processes

EF have mechanisms to overcome and circumvent thermal effects (Rangel et al. 2010; Tseng et al. 2011; Keyser et al. 2014), triggering signal transduction and metabolic pathways that synthesize the molecules that will ultimately protect the fungal cells from damage caused by heat exposure (Farrell and Rose 1967; Ying and Feng 2004; Zhang et al. 2011; Liao et al. 2014; Wang et al. 2017, 2018). Among them, fungal proteins of hydrophobic nature associated to cell walls are often linked with thermal protecting functions. Hydrophobins are small proteins important for fungal growth and development (Wösten and Vocht 2000). Hydrophobin-like or formic-acid-extractable (FAE) proteins were studied in aerial conidia of B. bassiana and I. fumosorosea based on thermotolerance. FAE proteins provide hydrophobicity to conidia, exhibiting different composition between B. bassiana and I. fumosorosea. For both fungi, conidial viabilities decreased after exposure to heat stress (48 °C for up to 150 min), perhaps as a result of different conidial structure related to FAE proteins (Ying and Feng 2004). Zhang et al. (2011) characterized structurally the cell wall carbohydrates in B. bassiana, and demonstrated that targeted gene knockouts lacking β-1,3-glucanosyltransferase destabilize the cell wall and decreased germination after 1 to 4 h of heat shock at temperatures >40 °C.

Trehalose is a disaccharide that accumulates in fungi during stress situations, such as adverse growth conditions, heat, and hyperoxidative shock. Thus, along with other polyols these molecules are known as stress metabolites (Van Laere 1989; Fillinger et al. 2001; Liu et al. 2009). Polyols accumulation is associated with thermotolerance by helping in the stabilization of structure (and function) of proteins and enzymes at high temperatures (Kim and Lee 1993). Accumulation of glycerol , erythritol , arabitol , mannitol , and trehalose in conidia of M. anisopliae , B. bassiana, and I. farinosa under different culture age (up to 120 days), temperature (5–35 °C) and pH (2.9–11.1) were reported by Hallsworth and Magan (1996). Also, high accumulation of both trehalose and mannitol were observed in abiotic stressed conidia of M. acridum , suggesting they are part of a mechanism that the fungus uses to attain its high tolerance to UV-B radiation and heat (Rangel and Roberts 2018).

The heat stress also triggers the production of toxic reactive oxygen species (ROS) , favoring oxidative stress in fungal propagules (Zhang and Feng 2018). Catalase is an antioxidant enzyme characterized in B. bassiana (Pedrini et al. 2006) that showed to be an important regulator of conidial thermotolerance (Wang et al. 2013). The relationship between oxidative stress and elevated culture temperature also was reported for Aspergillus niger (Bai et al. 2003). The heat shock proteins (HSPs) are also associated with tolerance to heat: overexpressing the gene encoding for HSP25 in M. robertsii increased fungal growth under heat stress either in nutrient-rich medium or on insect wings, and also enhanced the tolerance of heat shock-treated conidia to osmotic stress (Liao et al. 2014).

6.6 Conidial Formulation and Thermotolerance

Formulations preserve the viability of conidia exposed to environmental stresses, improving the efficiency of fungal propagules in microbial control (Faria and Wraight 2007). Conidial formulations based on oil or oil-in-water emulsions are investigated because the combination of conidia with oils improved their performance against heat stress (Malsam et al. 2002; Mendonça et al. 2007; Barreto et al. 2016; Paixao et al. 2017; Oliveira et al. 2018).

Oil-based formulations of M. anisopliae s.s. were used to improve both germination and appressorium production in conidia used for tick control (Barreto et al. 2016; Alves et al. 2017), and also to protect conidia against the effect of solar radiation (Alves et al. 1998) and high temperatures (McClatchie et al. 1994). Conidia of M. anisopliae s.l. and M. robertsii formulated on either vegetable or mineral oils were more tolerant to heat stress than those either unformulated or formulated on carboxymethylcellulose gel (Paixao et al. 2017). M. anisopliae and B. bassiana viability also increases when fungi are formulated in emulsifiable oil (Oliveira et al. 2018), and vegetable oil improved both performance and thermotolerance of B. bassiana (Kim et al. 2010a). Thus, formulation is considering a very important tool to manage heat stress on conidia.

6.7 Conclusion

Temperature is a key factor that limits survival of entomopathogenic fungal conidia used in biological control programs. As detailed in this chapter, most of the investigations in this area have concentrated in: (i) fungal screening for thermotolerance, based on geographical origin of the strains (McCammon and Rath 1994; Morley-Davies et al. 1996; Fargues et al. 1997; De Croos and Bidochka 1999; Devi et al. 2005), (ii) test of tolerance to low or high temperature (Fernandes et al. 2008; Paixão et al. 2017), (iii) appropriate culture medium for conidial production (Hallsworth and Magan 1999; Cabanillas and Jones 2009; Esther et al. 2013), (iv) formulations to increase conidia thermotolerance and protection (Hedgecock et al. 1995; Barreto et al. 2016; Paixão et at. 2017), and (v) biological/molecular characteristics and mechanisms that mediate stress tolerance (Liu et al. 2009; Fernandes et al. 2010; Rangel et al. 2018). On the basis of the literature available, we can conclude that EF are promising tools against many arthropods (Zimmermann 2007; Faria and Wraight 2007). However, additional research is still needed mostly in both screening of thermotolerant strains and formulation types of fungal propagules, to circumvent the negative effects of abiotic factors that potentially limits their efficacy, thus improving the use of EF in biological control programs.