**1. Introduction**

102 Zoology

Xu, C. F., Xia, Y. H., Li, K.-B. & Ke, C. (1988). Further study of the transmission of citrus

Yang, Y., Huang, M., Beattie, G. A.C., Xia, Y., Ouyang, G. & Xiong, J. (2006). Distribution,

Spain, November 1986

pp. 343-352, ISSN 0967-0874

huanglongbing by a psyllid, *Diaphorina citri* Kuwayama, *Proceedings of 10th Conference of the International Organization of Citrus Virologists (IOCV)*, pp. 243-248,

biology, ecology and control of the psyllid *Diaphorina citri* Kuwayama, a major pest of citrus: A status report for China. *International Journal of Pest Management*, Vol. 52,

> Synthetic chemical pesticides remained the mainstay of pest eradication for more than 50 years. However, insecticide resistance, pest resurgence, safety risks for humans and domestic animals, contamination of ground water, decrease in biodiversity, and other environmental concerns have encouraged researchers for the development of environmentally benign strategies for pest control including the use of biological control agents. Naturally occurring biological control agents are important regulatory factors in insect populations. Many species are employed as biological control agents of insect pests in glass-house and row crops, orchards, ornamentals, range, turf and lawn, stored products, and forestry and for the abatement of pest and vector insects of veterinary and medical importance (Burges, 1981; Lacey & Kaya, 2000; Tanada & Kaya, 1993).

> The application of microorganisms for control of insect pests was proposed by notable early pioneers in invertebrate pathology such as Agostino Bassi, Louis Pasteur, and Elie Metchnikoff (Steinhaus, 1956, 1975). These biological control agents such as viruses, bacteria, protozoa, nematodes and most fungi exert considerable control of target populations.

> Among micro-organisms, entomopathogenic fungi constitute the largest single group of insect pathogens. Generally, two groups of fungi are found to cause diseases in insects. Entomopathogenic fungi belong to the orders Entomophthorales and Hypocreales (formerly called Hyphomycetes). Several other entomopathogenic fungi from other taxonomic groups are also known. Until now, over 700 species of fungi are known to infest insects (Wraight et

<sup>\*</sup>Corresponding Author

Current Status of Entomopathogenic Fungi

bound Pr1 (Shah et al, 2007).

disease within the insects (Khetan, 2001).

as Mycoinecticides and Their Inexpensive Development in Liquid Cultures 105

structural studies of *M. anisopliae* penetration sites on *Manduca sexta* larvae have shown high levels of Pr1 coincident with hydrolysis of cuticular proteins (Goettel et al, 1989; St. Leger et al, 1989). Pr1 inhibition studies also showed delayed mortality in *M. sexta* larvae, resulting from delayed penetration of the cuticle (St. Leger et al, 1988). Furthermore, construction of a *M. anisopliae* strain with multiple copies of the gene encoding Pr1 and over-expressing the protease resulted in 25% reduction of time to death among *M. sexta* compared to those infected by the wild-type strain (St. Leger et al, 1996). Furthermore, it has also been reported that successive *in vivo* passage enhanced the capacity of the fungus to cause infection (Daoust et al, 1982; Hussain et al, 2010b), which ultimately increased the activity of spore

After penetration through the cuticle, the conidia invade into the hemocoel to form a dense mycelial growth (Zimmerman, 1993). Along with penetration, fungi also produce secondary metabolites, derivatives from various intermediates, some of which have insecticidal activities (Vey et al, 2001). It has been experimentally proved that the entomopathogens producing these toxins, infection has been shown to result in more rapid host death (McCauley et al, 1968), compared to strains that do not produce these metabolites (Kershaw et al, 1999; Samuels et al, 1988). The insecticidal properties of destruxins, cyclic depsipeptide toxins from *Metarhizium* spp*,* described by Kodaira, (1961) are shown to be produced in wax moth and silkworm larvae by Roberts, (1966) and Suzuki et al, (1971), Furthermore, these toxins have been tested against various insects (Roberts, 1981). Currently, over 28 different destruxins have been described, mostly from *Metarhizium* spp*,* with varying levels of activities against different insects (Vey et al, 2001). The level of destruxin has been correlated with virulence (Al-Aïdroos & Roberts, 1978) and host specificity (Amiri-Besheli et al, 2000). Studies on the activities of destruxins have also shown modulation of the host cellular immune system, including prevention of nodule formation (Huxham et al, 1989; Vey et al, 2001) and inhibition of phagocytosis (Vilcinskas et al, 1977) among infected insects. Destruxins are produced as the mycelium grows inside the insect. Other representative toxins produced by entomopathogenic fungi include oosporein, beauvericin, and bassianolide from *Beauveria* spp. (Eyal et al, 1994; Gupta et al, 1994; Suzuki et al, 1977), efrapeptins (Dtolypin) from *Tolypocladium* spp. (Weiser & Matha, 1988), and hirsutellin from *Hirsutella thompsonii* (Mazet & Vey, 1995)**.** Inside the insect haemocoel, the fungus switches from filamentous hyphal growth to yeast-like hyphal bodies that circulate in the hemolymph. The proliferation of these hyphal bodies occurs through budding (Boucias & Pendland, 1982). Later the fungus switches back to a filamentous phase and invades internal tissues and organs (Mohamed et al, 1978; Prasertphon & Tanada, 1968). The fungus later erupts through the cuticle and an external mycelium covers all parts of the host and formed infective spores under appropriate environmental conditions (Boucias & Pendland, 1982; McCauley et al, 1968;). Under suboptimal conditions, some fungi form resting structures inside the cadaver as in the case of *Nomuraea rileyi* under conditions of low relative humidity and temperature (Pendland, 1982). The life cycle of the fungus is completed when the hyphal bodies sporulate on the cadaver of the host. The external hyphae produce conidia that ripen and are released into the environment. This allows horizontal transmission of the

Among 85 genera of entomopathogenic fungi only six species are commercially available for field application (Table 1). However, comparatively few have been investigated as potential mycoinsecticides. Fungal pathogens particularly *B. bassiana, I. fumosorosea* and *M. anisopliae* 

al, 2007). Such insect killing fungi present major advantages. Firstly, they are important natural enemies of arthropods (Chandler et al, 2000), capable of infecting them directly through the integument. Secondly, cultivation of those fungi and production of infective conidia are easy and fairly cheap (Roberts & Hajek, 1992). Finally, entomogenous fungi can be found under different ecological conditions (Ferron, 1978).

Unique among entomopathogens, fungi do not have to be ingested and can invade their hosts directly through the exoskeleton or cuticle. Therefore, entomopathogenic fungi can infect non-feeding stages such as eggs and pupae. The insect cuticle is the first barrier against biological insecticides. Insect cuticle mainly formed from three layers such as, epicuticle, procuticle and epidermis. Each layer has different chemical structure and properties (Juárez & Fernández, 2007). The epicuticle is very thin (0.1–3 μm) and multilayered. The outermost surface layer of the epicuticle is the lipid layer, it is mostly resistant to enzyme degradation and exhibits characteristic such as water barrier properties (Hadley, 1981); unless physically disrupted, it can help to prevent passage of cuticle degrading fungal enzymes. The site of invasion among insects is often between the mouthparts, intersegmental folds or through spiracles. At these sites, locally high humidity promotes conidial germination and the cuticle is non-sclerotised and more easily penetrated (Clarkson & Charnley, 1996; Hajek & St. Leger 1994).

Conidia upon landing on a potential host, initiates a series of steps that could lead to a compatible (infection) or a non-compatible (resistance) reaction. In a compatible reaction, fungal recognition and attachment proceed to germination on the host cuticle. Once the epicuticle is breached, progress by the penetration peg through the cuticle may be more or less direct via penetrant hyphae, penetrant structures may also extend laterally (Hajek & St. Leger, 1994). Fargues (1984) proposed adhesion to occur at three successive stages: (1) adsorption of the fungal propagules to the cuticular surface; (2) adhesion or consolidation of the interface between pre-germinant propagules and the epicuticle; (3) fungal germination and development at the insect cuticular surface, until appresorium is developed to start the penetration stage. Zacharuk (1970b) proposed an active adhesion process for *M. anisopliae* after detecting epicuticle dissolution and mucoid material penetrating the pore canals. Infection will proceed after a successful penetration has been achieved.

In terrestrial environment, fungal conidial germination proceeds with the formation of germ tube (Boucias & Pendland, 1991) or appressorium (Madelin et al, 1967; Zacharuk, 1970a), which forms a thin penetration peg that breaches the insect cuticle via mechanical (turgor pressure) or enzymatic means (proteases) (Zacharuk, 1970b). Exocellular mucilage, proposed to enhance binding to the host cuticle, is also secreted by several entomogenous fungi during the formation of infective structures (Boucias and Pendland, 1991). In *M. anisopliae*, appressorium formation, hydrophobins, and the expression of cuticle-degrading proteases are triggered by low nutrient levels (St. Leger et al, 1992), demonstrating that the fungus senses environmental conditions or host cues at the initiation of infection. The production of cuticle-degrading enzymes, chitinases, lipases and proteases, has long been recognized as important determinant of the infection process in various fungi, facilitating penetration as well as providing nourishment for further development (Charnley, 1984; Dean & Domnas, 1983; Hussain et al, 2010b; Samsináková et al, 1971;). Among the proteases found in entomopathogenic fungi, the spore bound Pr1 has been well characterized and its role in cuticle invasion has been established (Hussain et al, 2010b; St. Leger, 1994). Ultra-

al, 2007). Such insect killing fungi present major advantages. Firstly, they are important natural enemies of arthropods (Chandler et al, 2000), capable of infecting them directly through the integument. Secondly, cultivation of those fungi and production of infective conidia are easy and fairly cheap (Roberts & Hajek, 1992). Finally, entomogenous fungi can

Unique among entomopathogens, fungi do not have to be ingested and can invade their hosts directly through the exoskeleton or cuticle. Therefore, entomopathogenic fungi can infect non-feeding stages such as eggs and pupae. The insect cuticle is the first barrier against biological insecticides. Insect cuticle mainly formed from three layers such as, epicuticle, procuticle and epidermis. Each layer has different chemical structure and properties (Juárez & Fernández, 2007). The epicuticle is very thin (0.1–3 μm) and multilayered. The outermost surface layer of the epicuticle is the lipid layer, it is mostly resistant to enzyme degradation and exhibits characteristic such as water barrier properties (Hadley, 1981); unless physically disrupted, it can help to prevent passage of cuticle degrading fungal enzymes. The site of invasion among insects is often between the mouthparts, intersegmental folds or through spiracles. At these sites, locally high humidity promotes conidial germination and the cuticle is non-sclerotised and more easily penetrated (Clarkson &

Conidia upon landing on a potential host, initiates a series of steps that could lead to a compatible (infection) or a non-compatible (resistance) reaction. In a compatible reaction, fungal recognition and attachment proceed to germination on the host cuticle. Once the epicuticle is breached, progress by the penetration peg through the cuticle may be more or less direct via penetrant hyphae, penetrant structures may also extend laterally (Hajek & St. Leger, 1994). Fargues (1984) proposed adhesion to occur at three successive stages: (1) adsorption of the fungal propagules to the cuticular surface; (2) adhesion or consolidation of the interface between pre-germinant propagules and the epicuticle; (3) fungal germination and development at the insect cuticular surface, until appresorium is developed to start the penetration stage. Zacharuk (1970b) proposed an active adhesion process for *M. anisopliae* after detecting epicuticle dissolution and mucoid material penetrating the pore canals.

In terrestrial environment, fungal conidial germination proceeds with the formation of germ tube (Boucias & Pendland, 1991) or appressorium (Madelin et al, 1967; Zacharuk, 1970a), which forms a thin penetration peg that breaches the insect cuticle via mechanical (turgor pressure) or enzymatic means (proteases) (Zacharuk, 1970b). Exocellular mucilage, proposed to enhance binding to the host cuticle, is also secreted by several entomogenous fungi during the formation of infective structures (Boucias and Pendland, 1991). In *M. anisopliae*, appressorium formation, hydrophobins, and the expression of cuticle-degrading proteases are triggered by low nutrient levels (St. Leger et al, 1992), demonstrating that the fungus senses environmental conditions or host cues at the initiation of infection. The production of cuticle-degrading enzymes, chitinases, lipases and proteases, has long been recognized as important determinant of the infection process in various fungi, facilitating penetration as well as providing nourishment for further development (Charnley, 1984; Dean & Domnas, 1983; Hussain et al, 2010b; Samsináková et al, 1971;). Among the proteases found in entomopathogenic fungi, the spore bound Pr1 has been well characterized and its role in cuticle invasion has been established (Hussain et al, 2010b; St. Leger, 1994). Ultra-

Infection will proceed after a successful penetration has been achieved.

be found under different ecological conditions (Ferron, 1978).

Charnley, 1996; Hajek & St. Leger 1994).

structural studies of *M. anisopliae* penetration sites on *Manduca sexta* larvae have shown high levels of Pr1 coincident with hydrolysis of cuticular proteins (Goettel et al, 1989; St. Leger et al, 1989). Pr1 inhibition studies also showed delayed mortality in *M. sexta* larvae, resulting from delayed penetration of the cuticle (St. Leger et al, 1988). Furthermore, construction of a *M. anisopliae* strain with multiple copies of the gene encoding Pr1 and over-expressing the protease resulted in 25% reduction of time to death among *M. sexta* compared to those infected by the wild-type strain (St. Leger et al, 1996). Furthermore, it has also been reported that successive *in vivo* passage enhanced the capacity of the fungus to cause infection (Daoust et al, 1982; Hussain et al, 2010b), which ultimately increased the activity of spore bound Pr1 (Shah et al, 2007).

After penetration through the cuticle, the conidia invade into the hemocoel to form a dense mycelial growth (Zimmerman, 1993). Along with penetration, fungi also produce secondary metabolites, derivatives from various intermediates, some of which have insecticidal activities (Vey et al, 2001). It has been experimentally proved that the entomopathogens producing these toxins, infection has been shown to result in more rapid host death (McCauley et al, 1968), compared to strains that do not produce these metabolites (Kershaw et al, 1999; Samuels et al, 1988). The insecticidal properties of destruxins, cyclic depsipeptide toxins from *Metarhizium* spp*,* described by Kodaira, (1961) are shown to be produced in wax moth and silkworm larvae by Roberts, (1966) and Suzuki et al, (1971), Furthermore, these toxins have been tested against various insects (Roberts, 1981). Currently, over 28 different destruxins have been described, mostly from *Metarhizium* spp*,* with varying levels of activities against different insects (Vey et al, 2001). The level of destruxin has been correlated with virulence (Al-Aïdroos & Roberts, 1978) and host specificity (Amiri-Besheli et al, 2000). Studies on the activities of destruxins have also shown modulation of the host cellular immune system, including prevention of nodule formation (Huxham et al, 1989; Vey et al, 2001) and inhibition of phagocytosis (Vilcinskas et al, 1977) among infected insects. Destruxins are produced as the mycelium grows inside the insect. Other representative toxins produced by entomopathogenic fungi include oosporein, beauvericin, and bassianolide from *Beauveria* spp. (Eyal et al, 1994; Gupta et al, 1994; Suzuki et al, 1977), efrapeptins (Dtolypin) from *Tolypocladium* spp. (Weiser & Matha, 1988), and hirsutellin from *Hirsutella thompsonii* (Mazet & Vey, 1995)**.** Inside the insect haemocoel, the fungus switches from filamentous hyphal growth to yeast-like hyphal bodies that circulate in the hemolymph. The proliferation of these hyphal bodies occurs through budding (Boucias & Pendland, 1982). Later the fungus switches back to a filamentous phase and invades internal tissues and organs (Mohamed et al, 1978; Prasertphon & Tanada, 1968). The fungus later erupts through the cuticle and an external mycelium covers all parts of the host and formed infective spores under appropriate environmental conditions (Boucias & Pendland, 1982; McCauley et al, 1968;). Under suboptimal conditions, some fungi form resting structures inside the cadaver as in the case of *Nomuraea rileyi* under conditions of low relative humidity and temperature (Pendland, 1982). The life cycle of the fungus is completed when the hyphal bodies sporulate on the cadaver of the host. The external hyphae produce conidia that ripen and are released into the environment. This allows horizontal transmission of the disease within the insects (Khetan, 2001).

Among 85 genera of entomopathogenic fungi only six species are commercially available for field application (Table 1). However, comparatively few have been investigated as potential mycoinsecticides. Fungal pathogens particularly *B. bassiana, I. fumosorosea* and *M. anisopliae* 

Current Status of Entomopathogenic Fungi

(Ypsilos & Magan, 2005).

**2. Materials and methods** 

HCl (0.1%) and NaOH (10%).

**fungi** 

**2.1 Culturing of entomopathogenic fungi**

as Mycoinecticides and Their Inexpensive Development in Liquid Cultures 107

propagules of entomopathogenic fungi. Corn steep solid and cotton seed flour with yeast extract and KCl, NaCl etc., optimized blastospore production under water stress conditions

Entomopathogenic fungi infect insects in an aggressive manner by secreting cuticle degrading enzymes such as esterases, lipases, N-acetylglucosaminidases and chitinases (St. Leger et al., 1986). However, the production of extracellular protease Pr1, a major virulence determinant, plays an important role in the success of entomopathogenic fungi in insect penetration, which leads to the subsequent pathogenicity in the target host (Hussain et al., 2010b; Shah and Butt 2005). Previously, agro-industrial by-products such as corn-steep liquor and molasses have been used as alternative growth substrates to produce exopolysaccharides (Fusconi et al., 2008; Sutherland, 1996). Sugarcane molasses (SM), an industrial by-product rich in fermentable sugars, was proposed as a nutritious medium to produce bacterial cellulose by *Zoogloea* sp. (Paterson-Beedle et al., 2000). While, the dried powder of corn steep liquor was used as an inexpensive substitute for beef extract in the medium, which enhanced the lipase production of the strain of *Serratia marcescens* (Zhao et al., 2010). In the past, there is no report on the activity of extracellular protease Pr1 enzyme from the spores of entomopathogenic fungi cultivated from rice, previously grown on media with different composition. The current study is initiated in order to evaluate the effects of three different sources of nitrogen and two sugar sources in different combinations and concentrations in order to determine i) production in liquid media ii) activity of extracellular

The entomopathogenic fungi *M. anisopliae* (EBCL 02049), *B. bassiana* (EBCL 03005) and *I. fumosorosea* (EBCL 03011) were originally isolated from *C. formosanus* in China. The strains were deposited at European Biological Control Laboratory, France. The strains were successively sub-cultured on Potato Dextrose Agar (PDA, Difco Laboratories, Detroit, MI, US) at 26 ± 0.5 ºC, in complete darkness. Fungal strains maintenance was identical to our previous study, where it was extensively described (Hussain et al., 2009). In brief, 24-day-old spores of

**2.2 Influence of liquid media composition on the** *in vitro* **growth of entomopathogenic** 

The three sources of nitrogen: peptone (Sigma), yeast extract (Sigma) and corn steep liquor (Shanghai Xiwang Starch Sugar Co., Ltd.), and two sources of sugar: glucose (Sigma) and sugar molasses, were used in different combinations as shown in Table 2. In all the treatments, the following salts were used at the concentration, CaCl2 . 2H2O (0.06%), KCl (0.28%), MgCl2 . 6H20 (0.16%), MgSO4 . 7H2O (0.2%), NaHCO3 (0.03%) and NaH2PO4 . H2O (0.1%). To avoid reactions among the salts, they were prepared in compatible pairs: CaCl2. 2H2O with KCl; MgCl2. 6H20 with NaH2PO4. H2O; MgSO4. 7H2O with NaHCO3. The other precautionary measures to avoid precipitation were adopted as described by Leite et al. (2005). Media preparations were finalized by adjusting the pH to 6.2, with filter-sterilized

studied strains cultivated on PDA were used as inoculum in all the growth media.

protease Pr1 of the locally isolated strains of entomopathogenic fungi.

are being evaluated against numerous agricultural and urban insect pests. Several species belonging to order IIsoptera (Hussain et al., 2010a ; Hussain et al., 2011), Lepidoptera (Hussain et al., 2009), Coleoptera (Ansari et al., 2006), Hemiptera (Leite et al., 2005) and Diptera (St. Leger et al., 1987) are susceptible to various fungal infections. This has led to a number of attempts to use entomopathogenic fungi for pest control with varying degrees of success.


(Bhattacharyya et al., 2004)

Table 1. Commercial formulations of entomopathogenic fungal pesticides

The majority of fungal production systems consist of two stages system in which fungal inoculum of hyphal bodies is produced in liquid culture and then transferred to a solid substrate for production of aerial spores (Devi, 1994). For practical use of entomopathogenic fungi as bio-insecticides at each stage, it is necessary to develop culture medium and method that produce high concentrations of viable and virulent propagules at low cost (Jackson, 1997). These goals can be achieved by using the most favorable inexpensive components for fungal growth at the lowest concentration that afford high yield. Most common compounds for fungal entomopathogens include agro-industrial by-products such as corn steep liquor (Zhao et al., 2010) and sugarcane molasses (Hussain et al., 2011). Our previous investigations showed that both the by-products stimulate the growth of the propagules of entomopathogenic fungi. Corn steep solid and cotton seed flour with yeast extract and KCl, NaCl etc., optimized blastospore production under water stress conditions (Ypsilos & Magan, 2005).

Entomopathogenic fungi infect insects in an aggressive manner by secreting cuticle degrading enzymes such as esterases, lipases, N-acetylglucosaminidases and chitinases (St. Leger et al., 1986). However, the production of extracellular protease Pr1, a major virulence determinant, plays an important role in the success of entomopathogenic fungi in insect penetration, which leads to the subsequent pathogenicity in the target host (Hussain et al., 2010b; Shah and Butt 2005). Previously, agro-industrial by-products such as corn-steep liquor and molasses have been used as alternative growth substrates to produce exopolysaccharides (Fusconi et al., 2008; Sutherland, 1996). Sugarcane molasses (SM), an industrial by-product rich in fermentable sugars, was proposed as a nutritious medium to produce bacterial cellulose by *Zoogloea* sp. (Paterson-Beedle et al., 2000). While, the dried powder of corn steep liquor was used as an inexpensive substitute for beef extract in the medium, which enhanced the lipase production of the strain of *Serratia marcescens* (Zhao et al., 2010). In the past, there is no report on the activity of extracellular protease Pr1 enzyme from the spores of entomopathogenic fungi cultivated from rice, previously grown on media with different composition. The current study is initiated in order to evaluate the effects of three different sources of nitrogen and two sugar sources in different combinations and concentrations in order to determine i) production in liquid media ii) activity of extracellular protease Pr1 of the locally isolated strains of entomopathogenic fungi.
