**1. Introduction**

The rice striped stem borer, *Chilo suppressalis* Walker (Lepidoptera: Crambidae), is an economic pest of rice, annually causing significant losses in Asia, southern America and northern Africa [1]. The larvae feed intensively on rice stems and cause "whitehead" and "dead-heart" of the seedlings, which directly decrease the overall yield of rice [2]. The main control measure to suppress the *C. suppressalis* population is the wide-spraying of synthetic insecticides, including diazinon, Padan® and Reagent®. Nevertheless, *C. suppressalis* has developed resistance to these insecticides on one hand and resulted in environmental pollution, food residuals and toxicity on non-target organisms on the other hand [3,4]. These concerns should change the managemen<sup>t</sup> strategies of chemical insecticides toward biocontrol agents like entomopathogens. Among the entomopathogens used to manage the population of insect pests, entomopathogenic fungi cause epizootics among insect pests and appear as the prevalent natural pathogens to regulate population fluctuations of pests and subsequent losses [5]. Their presence in almost all terrestrial and aquatic ecosystems, as well as way of infection by producing different extracellular enzymes and by releasing toxic secondary metabolites, has led to the success of entomopathogenic fungi to affect noxious arthropods in agriculture, forestry and livestock [5,6].

There are many reports on the efficacy of different entomopathogenic fungi, including *Akanthomyces lecanii*, *Akanthomyces muscarious*, *Aspergillus* spp., *Beauveria bassiana*, *Isaria fumosorosea*, *Isaria sinclairii*, *Metarhizium anisopliae*, *Metarhizium rileyi*, *Nomuraea rileyi*, *Pecilomyces lilacinus* and *Purpureocillium lilacinum* against lepidopteran pests such as *Chilo suppressalis*, *Spodoptera litura*, *Spodoptera frugiperda*, *Spodoptera exigua*, *Ostrinia nubilalis*,

**Citation:** Shahriari, M.; Zibaee, A.; Khodaparast, S.A.; Fazeli-Dinan, M. Screening and Virulence of the Entomopathogenic Fungi Associated with *Chilo suppressalis* Walker. *J. Fungi* **2021**, *7*, 34. https://dx.doi.org/ 10.3390/jof7010034

Received: 21 October 2020 Accepted: 16 November 2020 Published: 7 January 2021

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*Helicoverpa armigera*, *Helicoverpa zea*, *Plutella xylostella*, *Duponchelia fovealis*, *Agrotis ipsilon*, *Pieris rapae*, *Trichoplusia ni*, *Ocinara varians*, *Galleria mellonella*, *Plodia interpunctella*, *Ephestia kuehniella* [7–19]. These agents have generally shown to be safe for humans with the least effects on non-targets while they are relatively sensitive to environmental conditions, mainly heat, cold and UV radiation, so it is imperative to find isolates adaptable to these constraints for formulation and field application [20–23].

Exogenous isolates of the entomopathogenic fungi that were commercialized as pest biocontrol agents in different countries may be ineffective on some pests due to environmental suitability and strain differences related to the host [24]. Therefore, the application of local isolates may be a promising choice mainly in case of ecological suitability with pest species and lower hazards on non-target organisms compared to exotic strains [22,25–27]. These points were verified by several studies that demonstrate the virulence of isolates belonging to the same fungal species could be different because of genetic variations occurring in a specific geographical distribution [28–30]. The provinces of Guilan and Mazandaran are located in the north of Iran with high humidity, moderate annual temperatures and heavy rainfall, in which these conditions are appropriate for entomopathogenic fungi [5]. The rice fields of northern Iran, known as a reservoir of *C. suppressalis* [31], can represent ideal sites to study the existence of entomopathogenic fungi with natural enzootics to *C. suppressalis*, so the aims of our study were to; (a) isolate and identify different entomopathogenic fungi from fungus-infected *C. suppressalis* larvae, (b) evaluate the virulence of these fungi against the larvae of *C. suppressalis*, (c) examine the infection process of these isolates by the production of extracellular secretions and (d) compare the conidial germination of the fungal isolates after exposure to heat and cold.

#### **2. Materials and Methods**

#### *2.1. Collection and Morphological Identification*

The collection sites were all the municipal regions of Guilan and Mazandaran provinces in the north of Iran (Mazandaran and Guilan, Iran) with the highly cultivated area of rice. In each site, the remained stems of rice within the paddy fields were opened, and the infected larvae of *C. suppressalis* were collected and kept in sterile centrifuge tubes. The infected larvae were recognized according to the mycelial growth outside the larval body. Once the samples were transferred to the laboratory, the larvae were surface disinfected with sodium hypochlorite (2%) for 3 min and rinsed three times in sterile distilled water [27]. The larvae were then transferred on potato dextrose agar (PDA, Merck) plates and incubated at 25 ◦C for 2–4 days for fungal development. Afterward, the fungal mycelia were picked up and transferred to fresh PDA plates for purification. Finally, single-spore cultures were gathered according to the method described by Fang [32] and cultured on PDA slants. All collected specimens were inoculated on PDA plates and incubated at 25 ◦C in the dark for 14 days. For microscopic examination, mycelia and conidia from fungal specimens were mounted on a sliding glass and observed at 100× magnification on a phase-contrast microscope (Canon INC DS126311, Taiwan). Morphological identification of the specimens was made based on conidial morphology, shape, color and size based on the following literature: *Akanthomyces* spp. isolates [28,33,34], *Beauveria* spp. Isolates [28,34,35], *Hirsutella* spp. isolates [34,36–38] and *Metarhizium* spp. isolates [34,39].

#### *2.2. Genomic DNA Extraction and PCR*

DNA extraction was done using the protocol of Montero-Pau et al. [40]. Briefly, the mass mycelia of the specimen grown in PDA media were transferred to the 1.5 mL tubes containing 100 μL of alkaline lysis buffer (0.2 mM disodium ethylene diamide tetraacetic acid, 25 mM NaOH, pH 8.0, Merck) and centrifuged for 30 min at 2000× *g*. Then, the tubes were incubated at 95 ◦C for 30 min and cooled on ice for five min. Finally, 100 μL of Tris-HCl solution (Sigma-Aldrich, Vienna, Austria; 40 mM, pH 5.0) was added to the tubes, vortexed and maintained at −20 ◦C. The extracted solution was used as a template for PCR.

To amplify the internal transcribed spacers (ITS5-5.8S-ITS4), ITS5 (5GGAAGTAAAAG TCGTAACAAGG3) and ITS4 (5-TCCTCCGCTTATTGATATGC-3) primers were synthesized as previously described [41]. The PCR reaction mixture consisted of 12.5 μL of master mix (Including 10× PCR buffer. MgCl2, dNTPS TaqPolymerase, CinnaClone, Tehran, Iran), 7.5 μL of double-distilled H2O, 1 μL of each primer and 3 μL of DNA solution. PCR was carried out using a thermal cycler (Eppendorf Personal, Darmstadt, Germany) with the following reaction parameters: an initial denaturation for 2 min at 94 ◦C, 30 cycles of 94 ◦C for 30 s, 53 ◦C for 30 s and 72 ◦C for 1 min and a final extension for 5 min at 72 ◦C. Amplified PCR products were visualized by electrophoresis on 1% agarose gels. The PCR products were sent to a sequencing service company (Royan Zistagene Co., Tehran, Iran) for purification and sequencing. Finally, sequences were compared with other fungi using the BLAST search tool in NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
