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Isolation and Characterization of Beauveria and Metarhizium Spp

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Isolation and Characterization of Beauveria and Metarhizium Spp Gürlek et al. Egyptian Journal of Biological Pest Control (2018) 28:50 Egyptian Journal of https://doi.org/10.1186/s41938-018-0055-y Biological Pest Control RESEARCH Open Access Isolation and characterization of Beauveria and Metarhizium spp. from walnut fields and their pathogenicity against the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae) Songül Gürlek1, Ali Sevim1, Fikriye Milletli Sezgin2 and Elif Sevim1* Abstract Entomopathogenic fungi (EPFs) play an important role for regulating insect pest populations, as they exist in many different ecosystems. Within these fungi, Beauveria and Metarhizium spp. genera include species that are the most commercially important. The aim of this study was to determine the diversity and distribution of Beauveria and Metarhizium spp. in walnut fields of Kırşehir, Turkey, and to evaluate their pathogenicity against Cydia pomonella (L.) (Lepidoptera: Tortricidae). To perform this, 90 soil samples were collected from walnut fields where Beauveria and Metarhizium spp. were isolated from these soils, using selective media. The isolated 40 fungi were characterized based on their morphological and molecular characteristics including Bloc and β-tubulin gene sequences. Also, eight selected fungi were tested against C. pomonella larvae under laboratory conditions. The fungal isolates were identified as Beauveria pseudobassiana (15), B. bassiana (12), Metarhizium robertsii (11), and M. brunneum (13). M. brunneum ELA-38 caused 83% mortality within 2 weeks after application of 1 × 108 conidia/ml. Consequently, Beauveria and Metarhizium spp. are the common component of the soils collected from walnut fields and some of fungi obtained from this work might be beneficial in the future biological control programs of C. pomonella. Keywords: Entomopathogenic fungi, Pathogenicity, Walnut, Codling moth, Cydia pomonella Background process begins with the attachment of conidial spores on Entomopathogenic fungi (EPFs) cause lethal infections in the external body surface of insects (cuticle). their host, and they are the natural regulator of many in- Soil is an important reservoir for EPFs, and these soil sect pests including those living in soil by epizootics fungi have a great importance in terms of biological con- (Goettel et al. 2005). These microorganisms have attracted trol. Many EPFs such as B. bassiana, M. anisopliae, and remarkable attention for their usage in biological control Paecilomyces spp. spend a significant period of their life programs of insect pests in both agriculture and forestry cycle in soil (Jackson et al. 2000). The local isolates have as environmentally safe agents (Lacey et al. 2015; Strasser many advantages in comparison to exotic isolates be- et al. 2010). Among these fungi, Beauveria bassiana cause they may have ecological compatibility with the (Balsamo) Vuillemin and Metarhizium anisopliae pest species. This reduces the effect of the used agents (Metschn.) Sorokin are the most studied fungi in terms of against non-target organisms (Gulsar Banu et al. 2004). commercial production (Goettel et al. 2005; Meyling and The codling moth, Cydia pomonella L. (Lepidoptera: Eilenberg 2007). In the majority of the EPFs, the infection Tortricidae), is an important pest of many agricultural fruits such as apple, quince, pear, peach, and walnut * Correspondence: [email protected] worldwide. It may cause 100% crop losses when it is not 1 Ahi Evran University, Faculty of Engineering and Architecture, Genetic and controlled (Mamay and Yanık 2013). In Turkey, the con- Bioengineering, 40100 Kırşehir, Turkey Full list of author information is available at the end of the article trol of this pest depends upon chemical insecticides © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Gürlek et al. Egyptian Journal of Biological Pest Control (2018) 28:50 Page 2 of 6 (Republic of Turkey, Ministry of Agriculture and colonies from a single colony were transferred on an- Welfare 2010). The use of these chemical insecticides other PDAY for further investigation. All purified fungal has harmful effects on environment and humans. isolates were stored in 15% glycerol at − 20 °C. Moreover, pest species in agriculture and forestry gain re- sistance to such chemical insecticides used (Mota-Sanchez et al. 2008). Besides, some biological control agents such Identification of fungal isolates as the parasitoids (Liotryphon caudatus, Bassus ruwpes, Firstly, fungal isolates were morphologically character- and Mastrus ridibundus) and the entomopathogens ized based on some fungal structures obtained from (C. pomonella granulosis virus, Bacillus thuringiensis, monosporic pure cultures. Colony morphologies, co- Beauveria bassiana, Nosema carpocapsae,andnidia, and conidiogenous cell shapes were used for initial Steinerma sp.) have showed some potential (Mills characterization. All isolates were identified based on 2005; Zimmermann et al. 2013). the identification key of Humber (1997). Morphological EPFs can be adapted to their environmental conditions identification of the isolated fungi was confirmed by such as certain climatic conditions and habitat types molecular characterization, using gene sequences of the (Sevim et al. 2012). Therefore, it is important to isolate nuclear intergenic region (bloc) and beta-tubulin (Bt). and identify local fungal strains for controlling insect Genomic DNAs were extracted from monosporic fungal pests in related areas. cultures, using the microbial DNA isolation kit (MO-BIO, The present study aimed to isolate and characterize in- Carlsbad, CA, USA) according to the manufacturer’s rec- digenous soil-borne EPFs from soil samples collected ommendations. For Beauveria isolates, approximately from walnut fields near Kırşehir, Turkey. In addition, the 1500-bp segments of bloc gene region was amplified by pathogenicity of the isolated fungi against C. pomonella the primer pairs of B5.1F (5′-CGACCCGGCCAACT larvae under laboratory conditions was evaluated. ACTTTGA-3′) and B3.1R (5′-GTCTTCCAGTACCA CTACGCC-3′) as described in the paper of Rehner et al. Methods (2006). PCR conditions were adapted according to Rehner Sampling et al. (2006). For Metarhizium isolates, an approximately Ninety soil samples were collected from a depth of about 1300-bp segments of Bt gene region were amplified by 20 cm. Before collection, the surface layer of soil was re- PCR, using the primer pairs of T1 (5′-AACATGCGT moved. A total of five soil samples was taken from each GAGATTGTAAGT-3′) and T22 (5′-TCTGGATGT collection site and placed in a plastic bag and trans- TGTTGGGAATCC-3′) based on O’Donnell and Cigelnik ported to the laboratory. The five collected soil samples (1997). PCR amplifications for Bt gene region were per- of each site were placed together in a plastic bag and formed in a total volume of 50 μl, which included 5 μl Taq mixed completely. Small stones and some rough parts DNA polymerase reaction buffer, 3 μl MgCl2, 1.5 μl were removed (Sevim et al. 2010a). Finally, 1-g soil sam- 10 pmol of each primers, 1.5 μl10mMdNTPmix,1μl ple was used to isolate EPFs. 5U/μlofTaq DNA polymerase, 1 μl genomic DNA, and 35.5 μldH2O. Reactions were first incubated for 5 min at Isolation of Beauveria and Metarhizium spp. 95 °C, and then, 35 cycles were performed as follows: One gram of a given soil sample and 10 ml of the steril- 1 min at 94 °C, 1 min at the annealing temperature of 55 ° ized distilled water were mixed in 15-ml test tubes, C, and 2 min at 72 °C. Reactions were then incubated at which were vortexed for 10 min to obtain homogenous 72 °C for another 5 min. − − solution. Then, a serial dilution from 10 1 to 10 7 for After performing all amplifications, PCR products each soil sample was prepared to isolate a single colony were separated on 1.0% agarose gel and stained with eth- of fungi. Two hundred fifty microliters of the obtained idium bromide. The amplified PCR products were soil extracts from each tube was spread on selective viewed under UV light to see correct amplification. Fi- medium (SDA (Sabouraud Dextrose Agar) containing nally, PCR products were sent to the MACROGEN for 0.2 μg/ml dodine (N-dodecylguanidine monoacetate), sequencing. The sequencing process was performed by 100 μg/ml chloramphenicol, and 50 μg/ml streptomycin amplification primers. The obtained sequences were sulphate) and incubated at 28 °C for 2 weeks (Goettel used to carry out phylogenetic analysis. and Inglis 1997). At the end of the incubation period, For Beauveria isolates, the isolated fungi were com- growing single colonies were transferred on other SDA pared to the known Beauveria species, using bloc se- plates to get pure cultures. One hundred microliters of quences according to Rehner et al. (2011)toconfirm conidial suspension (1 × 105 conidia/ml) for each fungal correct identification. For Metarhizium isolates, the isolate was plated on PDAY (1% yeast extract and potato isolated fungi were compared to the fungal isolates and dextrose agar) and incubated at 28 °C for 1 week to se- species used in the study of Bischoff et al. (2009)using lect colonies derived from a single colony. The selected Bt sequences. Gürlek et al. Egyptian Journal
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