*2.4. Bioassay*

Conidia of the two-week-old PDA cultures of the identified isolates were removed by a scalpel then suspended in sterile distilled water containing Tween-80 (0.02%). The concentrations of 10<sup>2</sup> to 10<sup>8</sup> conidia/mL from each isolate were separately prepared based on the preliminary tests. Early fourth instar larvae of *C. suppressalis* were randomly selected and separately dipped in the serial concentrations of each isolate while the control larvae were dipped in an aqueous solution of 0.02% Tween-80 alone. The bioassays were done in three replicates with ten larvae per replication, and the larvae were maintained at the rearing condition for the whole bioassay period. Mortality was recorded within 7 days, and LC50 values were determined using POLO-Plus software. For calculation of LT50, mortality was recorded until the death of all larvae at 10<sup>8</sup> conidia/mL concentration.

#### *2.5. Hydrophobin Protein Extraction and Estimation*

Hydrophobin content was determined according to the method described by Ying and Feng [43]. Conidia from the two-week-old cultures were added to 1 mL of 2% SDS aqueous solution containing β-mercaptoethanol (5%, Merck) and incubated in a boiling water bath for 10 min before being centrifuged at 22,000× *g* and 4 ◦C for 10 min. The supernatant was removed, and conidia were rinsed twice in distilled water to eliminate SDS (Merck) soluble proteins. Samples were incubated in 1 mL formic acid at zero temperature for 2 h before being centrifuged at 22,000× *g* and 4 ◦C for 10 min. The supernatant was transferred into fresh tubes, and 0.5 mL of distilled water was added to the samples. Afterward, 0.75 mL of 45% NaOH solution added to the mixture and maintained at 4 ◦C overnight. The proteins were separated from the supernatant by centrifugation at 22,000× *g* and 4 ◦C for 10 min. The extractable proteins of formic acid were rinsed twice with ethanol solution (3:1, *v*/*v*) and then dissolved in 2% SDS to quantitatively determine the amount of protein as mg/mL of conidia using the procedure of Lowry et al. [44].

#### *2.6. Liquid Culture for Enzyme Production of the Isolates*

The liquid media used for biochemical production of the extracellular enzymes contained; 0.02% of KH2PO4; 0.01% of CaCl2; 0.01% of MgSO4; 0.02% of Na2HPO4; 0.01% of ZnCl2 and 0.01% of yeas<sup>t</sup> extract (Merck). The media were inoculated with 1 mL of 10<sup>8</sup> conidia/mL concentration of each isolate separately and 5% (weight) of larval cuticle was

added to each flask containing liquid media. Then the flasks were kept on a rotatory shaker (70 rev/min) for 8 days at 25 ± 1 ◦C [3].

#### *2.7. Sample Preparations for Enzymatic Assays*

After 8 days, the culture flasks were harvested by centrifugation at 10,000× *g* for 30 min and washed in ice-cold Tris-HCl (25 mM, pH 8). Weighed mycelia were ground to a fine powder, suspended in DW, homogenized and centrifuged at 22,000× *g* and 4 ◦C for 30 min to obtain the supernatant of enzyme assay [12].

#### 2.7.1. Assay of Proteases

Activities of subtilisin-like (Pr1) and trypsin-like (Pr2) as the two key fungal proteases were determined by the specific substrates of succinyl-(alanine) 2-prolinephenylalanine-*p*nitroanilide and benzoylphenylalanine-valine-arginine-*p*-nitroanilide (Sigma-Aldrich, Co., Vienna, Austria), respectively. The reaction mixture contained 100 μL of Tris-HCl buffer (20 mM, pH 8), 30 μL of each substrate separately and 20 μL of enzyme solution. The mixture was incubated at 25 ◦C for 10 min, then 100 μL of trichloroacetic acid (TCA, 30%) was added, and the absorbance was recorded at 405 nm [3].

## 2.7.2. Lipase Assay

Lipase assay was done using the method of Tsujita et al. [45]. Fifty microliters of *p*-nitrophenyl butyrate (27 mM, Sigma-Aldrich, Co., Vienna, Austria) as substrate, 20 μL enzyme solution and 100 μL of Tris-HCl buffer (20 mM, pH 7) were incorporated and incubated at 37 ◦C for 5 min. Then, 100 μL of NaOH (1 N) was added to each tube, and the absorbance was recorded at 405 nm.

## 2.7.3. Endochitinase Assay

Twenty microliters of the enzyme solution were added to 50 μL of 0.5% colloidal chitin (Sigma-Aldrich, Co., Vienna, Austria) as substrate and 100 μL of Tris-HCl buffer (20 mM, pH 7). Then, the samples were incubated in a water bath of 30 ◦C for 60 min. Then, 100 μL of dinitrosalisylic acid (DNS, Sigma-Aldrich, Vienna, Austria) was added, the incubation was prolonged for 10 min at boiling water, and the absorbance was recorded at 545 nm [46].

## 2.7.4. Exochitinase Assay

The activity of exochitinase was assayed by 200 μL of *p*-nitrophenyl-N-acetyl-β-Dglucosaminide (pNPg; Sigma-Aldrich, Co., Vienna, Austria) solution (1 mg pNPg per mL of distilled water) as substrate, 25 μL of enzyme solution and 500 μL of Tris-HCl (25 mM, pH 7) which was incubated at 40 ◦C for 20 h. Then, the mixture was centrifuged at 13,000 rpm at 4 ◦C, and the supernatant was added to 200 μL of sodium tetraborate-NaOH buffer (125 mM, pH 10) before to read the absorbance at 400 nm. The extinction coefficient of 18.5 Mm−<sup>1</sup> −cm<sup>−</sup><sup>1</sup> was considered for activity calculation based on the following formula:

Volume activity (U/mL) = [ ΔOD (OD test–OD blank) × Vt × df]/(18.5 × t × 1.0 × Vs) where, Vt = total volume; Vs = sample volume; 18.5 = millimolar extinction coefficient of *p*-nitrophenol under the assay condition; 1.0 = lightpath length (cm); t = reaction time; and df = dilution factor [46].

## *2.8. Protein Assay*

The method of Lowry et al. [44] was used to determine the content of protein in the provided samples. Twenty microliters of the enzyme solution were added into 100 μL of reagen<sup>t</sup> (Ziest Chem. Co., Tehran, Iran) and incubated for 30 min before reading the absorbance at 545 nm.

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

To measure thermotolerance for conidial germination, 100 μL of conidial suspensions (5 × 10<sup>6</sup> conidia/mL) from each isolate was transferred to 1.5 mL tubes and kept in a thermal cycler adjusted to 45 ◦C. After 1 h and 2 h, 20 μL of conidial suspensions were removed and plated (without spreading) on PDA. Finally, plates were maintained at 25 ◦C, and conidial germination was counted after 24 h by microscopic observation. Moreover, 20 μL of a conidial suspension (5 × 106 conidia/mL) was plated (without spreading) on PDA and kept at 5 ◦C to determine the germination after 7 and 14 days in cold condition. In both experiments, the conidial control suspensions were inoculated on PDA at 25 ◦C. The relative percent germination was estimated by comparing conidial germination to untreated isolates, and at least 100 conidia were counted for each treatment in every test [27].

## *2.10. Statistical Analysis*

Probit analysis was done to determine LC50 and LT50 values at the corresponding 95% confidence interval (CI) values by using POLO-Plus software. Biochemical data and germination of conidia were compared by one-way analysis of variance (ANOVA) followed by Tukey's test. Differences among control and treatments were statistically considered at a probability of less than 5% and marked by different letters.

#### **3. Results and Discussion**

#### *3.1. Screening and Identification of Fungi*

A total of 38 fungal specimens were collected from *C. suppressalis* larvae, which were naturally infected by fungi in the rice fields of northern Iran. The specimens were morphologically identified as *Akanthomyces lecanii* (×5 isolates), *Akanthomyces muscarius* (×4 isolates), *Aspergillus* sp. (×1 isolate), *Beauveria bassiana* (×21 isolates), *Hirutella subulata* (×2 isolates), *Metarhizium anisopliae* complex (×3 isolates) and *Trichoderma* sp. (×2 isolates) (Table 1, Figure 1). Among all specimens, it was the first report of the natural occurrence of *H. subulata* in Iran. In *A. lecanii*, conidiogenous cells were phialidic, phialides approximately small, length size of 11–16 μm and width size of 1.4–2 μm, aculeate and strongly tapering, solitary or in whorls 3–6, conidial shape ellipsoidal-cylindrical, length size 4.1–5.2 μm and width size of 1.3–2.1 μm (Table 1, Figure 1a). In *A. muscarious*, conidiogenous cells were phialidic; phialides burned straight on prostrate hyphae or on secondary branches, phialides generally tall and slender and longer than *A. lecanii*, length size of 28–35 μm and width size of 1.6–2 μm, conidial shape cylindrical and longer than *A. lecanii*, length size 6.5–9.5 μm and width size of 1.5–1.9 μm (Table 1, Figure 1b). The major difference between the species of *Akanthomyces* is the shape and size of phialides that our specimens matched accurately match the description given by Zare and Gams [33]. In *Aspergillus* sp. conidial shape was globose, length size 1.8–2.3 μm and width size of 1.8–2.3 μm (Table 1, Figure 1c). In addition, the Conidial shape of *B. bassiana* isolates was globose with a length size of 2.1–3.2 μm and width size of 2–3 μm; conidiogenous cells were phialidic; the phialides were flask-shaped, swollen at the base or near the base and tapering at the apex. In addition, the conidiogenous cells were usually solitary or in a cluster of up to five (Table 1, Figure 1d), which appeared typical of those described by other researchers [28,34,35]. The major difference between the species of *Beauveria* is the shape and size of conidia [28]. In *H. subulata*, conidiogenous cells were phialidic, phialides scattered, and the lower phialides were narrow ellipsoid; the conidial shape was ovoid and in a chain, length size 5.5–6.9 μm and width size of 3.9–5.1 μm (Table 1, Figure 1e). Our isolates were compared with Yoon et al. [37], and significant differences were not observed among them. The conidial shape of *M. anisopliae* isolates was oblong oval with a length size of 6.8–7.8 μm and a width size of 2.6–3.7 μm (Table 1, Figure 1f). Conidia were the only morphological particular that reliably distinguishes several *Metarhizium* species [39]. In *Trichoderma* sp. conidial shape was globose, length size 2.2–3.1 μm and width size of 2.1–2.9 μm (Table 1, Figure 1g).




Note: final tested isolates were renamed after the submission of the ITS sequence to the GenBank database.

**Table 1.** *Cont.*

**Figure 1.** Morphological characteristics (conidium and phialid) of the fungus associated with the larvae of *Chilo suppressalis*. (**a**) *Akanthomyces lecanii*, (**b**) *Akanthomyces muscarius*, (**c**) *Aspergillus* sp., (**d**) *Beauveria bassiana*, (**e**) *Hirutella subulate*, (**f**) *Metarhizium anisopliae* complex, (**g**) *Trichoderma* sp. Bars are 20 micrometers.

The ITS5-5.8S-ITS4 region was used for molecular analysis, confirming identifications of the fungal isolates. The amplified ITS region from all specimens showed approximately 600 bp-sized fragments, and the samples were sequenced and compared in the GenBank database. Results of the ITS sequence data were consistent with those obtained using morphological studies. After submission to the GenBank database, the fungal isolates were renamed based on the given code (Table 1). In the current study, *B. bassiana* was the most frequent detected fungus in rice collected from fields similar to others studies that have shown *B. bassiana* as the most widespread entomopathogenic fungi in the endemic Moroccan forests of *Argania spinosa*, Switzerland, Spain, China and Southern Italy [22,47–50]. Moreover, *B. bassiana* has been reported as the major pathogen of insects in more than 200 species that have been identified from the soil and dead insects in nature [24,25,51,52].
