*3.4. Extracellular Enzymes*

The conidia of entomopathogenic fungi attach to the cuticle of host insects, germinate and penetrate to the hemocoel with the assistance of extracellular enzymes, such as chitinases, proteases and lipases [58]. Trypsin (Pr1) and subtilisin-like (Pr2) proteases are the primitive synthesized enzymes to simplify penetration of the hyphae into the host body. Then, synthesis of the chitinases increases the penetration efficiency [59], and finally, lipases involved in hydrolyzing lipid derivatives within the cuticle and facilitating the infection of host cells [60]. Our results revealed differences in the activities of extracellular enzymes between the fungal isolates. Isolates BBAL1, BBRR1, BBLN2, BBLN1 and BBLD2 demonstrated the highest activity of Pr1 while the least activity was observed in ASAI, TSRT and TSAH isolates (Figure 2). In the case of Pr2, BBRR1, BBLN2, BBLD2 and BBLN1, isolates showed the highest activity (Figure 2). The highest activity of lipase was recorded in BBLD4, BBSI, BBLN2 and BBRR1 isolates (Figure 3). BBRR1, BBLN2,

BBAL1 and BBLN1 isolates showed the highest activity of exochitinase (Figure 4). In the case of endochitinase, the highest activity was obtained in BBRR1, BBLN2, BBBL1, BBAL1 and BBLN1 isolates (Figure 4). The higher Pr1 activity in the given isolates indicates the capability of protein digestion by these isolates in the initial stages of infection, so the efficiency of this enzyme may ensure the success of other enzymes to feasible penetration through insect cuticle. Charnley and St. Leger [61] believe in facilitating the cuticle infiltration by the proteases produced during invasion prior to chitinases during later steps. They concluded the major role of proteases in cuticle penetration compared to chitinases. Ramzi and Zibaee [12] demonstrated the different levels of proteinases, chitinase and lipase produced by *B. bassiana*, *M. anisopliae*, *L. lecanii* and *I. fumosoroseus* in the larvae *C. suppressalis* in which the isolates with the highest enzymatic activity led to the higher mortality Lu et al. [1] showed, the higher levels of protease and chitinase produced by ZJLSP09 isolate of *Lecanicilium* sp. in comparison with ZJLA07 and ZJLP08 isolates which were related to mortality in *Diaphorina citri* Kuwayana (Hemiptera: Psyllidae). Maqsoudi et al. [62] reported that the isolate of *B. bassiana* with the higher activity of proteases and chitinases led to the lower LC50 and LT50 values against *Pseudococcus viburni* Signoret (Hemiptera: Pseudococcidae). In our study, no clear correlation was obtained between lipase production and virulence of isolates, similar to earlier studies [12,23,63,64]. This conclusion on lipase may be more obvious in the case of BBLN2, which is the only isolate with higher virulence and lipase activity. Other isolates with higher virulence showed lower lipase activity. It seems that lipases are more important in the utilization of integument lipids for fungal development, not necessarily penetration. In contrast, the isolates with the higher virulence demonstrated the higher activity of proteases and chitinases, mainly BBLN1, BBLN2 and BBRR1. These findings apparently disclosure the correlation between efficiency of extracellular enzymes and higher virulence of the entomopathogenic fungi. Such isolates properly or rapidly penetrate through host cuticle with efficient cleavage of polypeptide and carbohydrate bonds then achieve hemocoel to continue the latter steps of infection. It should be mentioned that this process is accompanied by better production of blastospores and secondary metabolites within the host hemocoel to impose virulence on infected individuals.

**Figure 2.** *Cont*. 22

**Figure 2.** Activities of the proteases (U/mg protein, Mean ± SE) in the liquid culture media of the entomopathogenic fungi in the presence of *C. suppressalis* cuticle. Statistical differences are shown by different letters (Tukey's test, *p* ≤ 0.05).

**Figure 3.** Activity of the lipase (U/mg protein, Mean ± SE) in the liquid culture media of the entomopathogenic fungi in the presence of *C. suppressalis* cuticle. Statistical differences are shown by different letters (Tukey's test, *p* ≤ 0.05).

**Figure 4.** Activities of the chitinases (U/mg protein, Mean ± SE) in the liquid culture media of the entomopathogenic fungi in the presence of *C. suppressalis* cuticle. Statistical differences are shown by different letters (Tukey's test, *p* ≤ 0.05).

#### *3.5. Effects of Thermotolerance and Cold Activity on Conidial Germination*

The inactivation and delay of conidia germination caused by heat and cold as the most important environmental factors reduce the efficiency of the entomopathogenic fungi as the biocontrol agents from both the virulence against host and persistence in ecosystems. Selection of the entomopathogenic fungi that tolerate thermal fluctuations te is necessary before field application [21,27,65]. The effect of thermotolerance on the germination rate of the conidia of the collected isolates in the current study have shown in Table 5. Fifteen isolates demonstrated a germination rate of more than 50% after 1 h exposure to 45 ◦C, while only one isolate exhibited a high tolerance after 2 h (Table 5). After 2 h of exposure, the isolate thermotolerance could be divided into three classes: low (below 30%), moderate (between 30% and 60%) and high tolerance (above 60%). Among the isolates, *Aspergillus* sp. (ASAI) showed high tolerance. Moderate tolerances were observed in four isolates of *B. bassiana* (BBLN1, BBAL1, BBLN2 and BBLD2), three isolates of *M. anisopliae* (MASA, MAAI and MAAL) and two isolates of *Trichoderma* sp. (TSAH and TSRT). The other isolates were low tolerance to the heat of 45 ◦C. Similar results have been reported by Lee et al. [27] as the rate of the conidial germination in *B. bassiana*, *M. anisopliae* and *Lecanicillium attenuatum* significantly reduced after 2 h at 45 ◦C. Rivas et al. [66] demonstrated the significant lower conidial germination of *Lecanicillium* isolates after incubation at 32 ◦C. The susceptibility of *Metarhizium* isolates to high temperatures (45 ◦C) was demonstrated by Rangel et al. [65]. Exposure to 35 ◦C for 10 min harmed the conidial germination of *B. bassiana,* but the *M. anisopliae* isolate germinated readily at this temperature [67]. Generally, the optimal temperature for conidia germination and growth of entomopathogenic fungi is between 23 and 28 ◦C. The growth was reduced above 30 ◦C, and it was totally inhibited above 34 ◦C [20,27,65–67]. Our results imply only *Aspergillus* sp. isolates as the highly thermotolerant isolate has although it had no virulence against the larvae of *C. suppressalis*. Finally, the cold activity of fungal isolates was examined through the treatment of the conidia at 5 ◦C for one and two weeks. All the isolates showed high activity (above 80%) at 5 ◦C for both time intervals except for *Aspergillus* sp. (ASAI) (Table 5). Lee et al. [27] reported the high cold activity at 7–14 days for almost all collected isolates. Based on earlier reports, most entomopathogenic fungi have high cold activity, although germination and sporulation may be delayed or stopped at a cold temperature [21,27,68,69]. Such a property may be important in the survival of entomopathogenic fungi in cold periods of the year.


**Table 5.** Thermotolerance and cold activity of the entomopathogenic fungi collected from the larvae of *Chilo suppressalis*.


**Table 5.** *Cont.*

Note: Statistical differences are shown by different letters.
