Yucel et al. Egyptian Journal of Biological Pest Control (2018) 28:39 Egyptian Journal of https://doi.org/10.1186/s41938-018-0043-2 Biological Pest Control RESEARCH Open Access Determination of fungal pathogens of Hypera postica (Gyllenhall) (Coleoptera: Curculionidae): isolation, characterization, and susceptibility Birgul Yucel1, Celalettin Gozuacik2, Donus Gencer1, Ismail Demir1* and Zihni Demirbag1 Abstract Background: Fungal pathogens of Hypera postica (Gyllenhall) (Coleoptera: Curculionidae) were collected from the vicinities of Adana and Igdir in Turkey. The pathogenicity of the fungal isolates against the pest were investigated. According to morphologic (colony morphology, spore shape) and molecular (sequences of ITS1-5.8S ITS2 region and EF1-α, Bloc, and RPB1 genes) characterizations, the isolates were identified as Beauveria bassiana (HpI-2, HpI-6, HpI-7, HpI-10, HpA-3, HpA-4, HpA-5) and Beauveria pseudobassiana (HpI-4). All these strains were isolated from H. postica for the first time. In order to determine pathogenesis of all isolates on the target pest, bioassays were conducted against larvae and adults, as screening of (1 × 107 conidia/ml) and dose-response (1 × 105,1×106, 1×107,1×108, conidia/ml), under laboratory conditions. The fungal isolates, closely related to each other, yielded significantly varied mortalities on larvae and adults. H. postica larvae were found more susceptible than adults to the fungal isolates in all tests. The highest mortality rates (100 and 98%) for larvae and adults, respectively, were obtained by B. bassiana strainHpA-5within14daysat1×108 conidia/ml concentration. The median lethal 4 concentration (LD50) of HpA-5 required to kill the larvae and adults of H. postica at concentrations of 2.37 × 10 and 1.4 × 105 conidia/ml, respectively. These results are promising; therefore, the B. bassiana strain HpA-5 can potentially be used against H. postica. Keywords: Hypera postica, Microbial control, Beauveria bassiana, Isolation, Susceptibility Background productive nitrogen fixer in legumes, making it useful Alfalfa, Medicago sativa L., also called Lucerne, Purple in long-term rotations as a soil builder to provide Medic, and Trefoil, is a perennial flowering plant of the nitrogen. pea family (Fabaceae) and cultivated as significant forage Alfalfa weevil, Hypera postica (Gyllenhall) (Coleoptera: crop lasting longer period than any other crop. Alfalfa Curculionidae), is the most devastating insect pest of al- has a very great product potential, and it is also one of falfa, and a few closely related legumes which feeding the most palatable and nutritious forage crops. Due to terminals and new crown shoots thereby lowering crop the high protein and vitamin content, alfalfa is the yield and quality (Reddy et al. 2016). Anay and Kornosor main component of the diet of dairy cattle. Alfalfa is (2000) recorded H. postica on alfalfa at Adana in Turkey. among the most valuable and cultivated plants world- This pest occurs at several times of the growing season wide (Mustafa et al. 2014). Also, it is one of the more and reduces forage production in many ways. Heavily infested areas may appear silver or white; most of the leaves are turned into skeletons or completely consumed (Radcliffe and Flanders 1998). If large numbers of larvae * Correspondence: [email protected] or adults survive until harvest, they damage crown buds 1Department of Biology, Faculty of Sciences, Karadeniz Technical University, and stems, retarding regrowth (Fick 1976). Residual ef- 61080 Trabzon, Turkey fects from severe damage decrease plant vigor, resulting Full list of author information is available at the end of the article © 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. Yucel et al. Egyptian Journal of Biological Pest Control (2018) 28:39 Page 2 of 8 in poor yields in subsequent harvests and lower stand conidial suspension of 1 × 106 conidia/ml was prepared, density (Fick and Liu 1976). Several types of control have plated on PDAY, and incubated at 28 °C for 1 week under been used to decrease the economic damage incurred by 12-h L and 12-h D photoperiod. After 6–7 days, a single this pest such as cultural, chemical, and biological control colony was cut out, transferred to a fresh PDAY medium, methods. Increasing concerns for insecticide resistance and incubated at 28 °C for 2–3 weeks until the plates and environmental safety arising from a frequent use of were fully overgrown. Single colony spore suspensions synthetic insecticides affect the long-term control feasi- were stored in entomopathogen culture collection at bility of alfalfa weevil management (Regev et al. 1983). Microbiology Laboratory, Karadeniz Technical University, Consequently, many alfalfa producers are looking for Trabzon, Turkey. more environmentally friendly control methods all over the world to take over this destructive pest. Morphological identification Biological control by entomopathogenic fungi (EPF) is The appearances of fungal infection on larvae and an attractive alternative to the use of traditional pesti- adults, colony morphology, spore size, and spore shape cides, mainly because these fungi are safer to animals, of fungal isolates were used in the first identification plants, and the environment. EPF play an important role process. Spore sizes were measured, using a phase-contrast in the regulation of insect populations in Turkey as well microscope. Initial identification was made according to as all over the world (Sevim et al. 2010). Besides, reports the key of Humber (1997). The fungal cultures were depos- related to fungi are quite limited, so there is a need to ited in the Microbiology Laboratory at Karadeniz Technical evaluate the potential of fungi as a group of biological University (Trabzon, Turkey). control agents against this pest. Limited attempts have been carried out to study the effects of EPF on the con- Molecular characterization of isolates trol of H. postica, including the studies of Hedlund and Sequencing of the ITS1-5.8S-ITS2 region between 18 S Pass 1968 and Sakurai et al. 1998 who recorded the in- and 28 S rRNA subunits and the partial sequencing of fection of H. postica with Beauveria bassiana and EF1-α, Bloc,andRPB1 genes was conducted to confirm Metarrhizium brunneum. Most of the studies, conducted the identity of the isolates. Hyphae and spores, obtained for the control of H. postica, showed that a negative in- from pure cultures, were inoculated into flasks containing fluence of the pest on alfalfa is still continuing. 100 ml of potato dextrose broth (PDB medium, Difco, NJ, In the present study, the pathogenicity of eight EPFs, USA), and the cultures were incubated at 25 °C in a rotary isolated from H. postica, was tested against both the lar- shaker (GFL 3031) at 230 rpm for 1 week under 12/12 vae and adults of the pest under laboratory conditions. photoperiod. After the incubation, cultured media were filtered, mycelial mass was harvested on sterile filter paper, Methods and each sample was frozen in liquid nitrogen and then Collection of the insect crushed. Isolation of genomic DNA was extracted, using H. postica larvae and adults were collected from alfalfa the ZR Fungal/Bacterial DNA MiniPrep (50, ZYMO RE- fields in Adana and Igdir, Turkey, between 2014 and 2015 SEARCH) from 50 mg of crushed mycelium. The ITS1-5. and transferred to the laboratory in plastic boxes. The in- 8S-ITS2 region of each fungal isolate was amplified, using sects were checked once a week, and those showed fungal the primer pair of ITS5, as a forward primer and ITS4 as a diseases or death symptoms were transferred to a moist reverse primer (White et al. 1990) in a 50-μlreaction chamber for 7 days to stimulate fungal sporulation. The volume containing 10 μl Phusion HF DNA polymerase re- naturally infected collected larvae were also transferred action buffer, 200 μMofeachdNTPs,0.05nmolofeach from the field to the laboratory in eppendorf tubes. Fun- opposing amplification primer, 1 unit Phusion DNA poly- gus isolation was made from mycosed larvae and adults. merase (Thermo Scientific), and 50 ng genomic DNA. Healthy individuals were selected and used for bioassays. PCR conditions were as follows: initial denaturation at 98 °C for 30 s; 35 cycles of 98 °C for 30 s, annealing at Isolation of fungi 55 °C for 30 s, and extension at 72 °C for 30 s; and final Fungi were isolated from infected larvae and adults extension at 72 °C for 10 min. by cutting out portions of mycosed cadavers and cul- Approximately 1200 bp segments of EF1-α,1500bpseg- tured on potato dextrose agar medium with 1% yeast ment of Bloc (the nuclear intergenic region), and 700 bp extract (PDAY medium, Merck, Darmstadt, Germany) segments of RBP1 (RNA polymerase II largest subunit) including 50 μg/ml ampicillin and 50 μg/ml tetracycline were amplified and sequenced for further characterization (AppliChem) to prevent bacterial growth. The cultures of Beauveria isolates, according to the study of Rehner et were incubated for 1–2 weeks at 25–28 °C to facilitate al. (2011). PCR conditions were adapted, essentially as de- growth and sporulation. Then, all isolates were subcultured scribed earlier (Rehner and Buckley 2005 and Rehner from a single colony to acquire a pure colony. Therefore, et al. 2011). The primers used in this study are given Yucel et al. Egyptian Journal of Biological Pest Control (2018) 28:39 Page 3 of 8 in Table 1. PCR products were loaded on 1.0% agarose surface sterilized by dipping into 1% sodium hypochlorite gels stained with ethidium bromide and scanned under UV for 3 min followed by 70% ethanol for 3 min and washed light for visualization.