J. Crop Prot. 2014, 3 (2): 159-171______________________________________________________ Research Article Pathogenicity of Lecanicillium longisporum (Ascomycota: Hypocreomycetidae) on the aphid Cinara pini (Hemiptera: Lachnidae) in laboratory conditions Amir Hossein Nazemi1, Gholamhossein Moravvej*1, Javad Karimi1 and Reza Talaei-Hassanlouei2 1. Department of Plant Protection, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran. 2. Department of Plant Protection, Faculty of Agricultural Sciences and Engineering, University of Tehran, Karaj, Iran. Abstract: The aphid species, Cinara pini (Linnaeus, 1758) reported in our previous work as a new aphid on pinus trees for Iran, was described using the classic method and through analysis of COI gene sequence. In the next step, we addressed the efficiency of the entomopathogenic fungus, Lecanicillium longisporum (Zimm.) Zare and Gams strain LRC 190, on the aphid. The fungus was administered to the second instar nymphs and adults using topical application procedure. The results indicated that the entomopathogen caused 90% mortality in adults over seven days at a concentration of 108 spores/ml, while the same control 7 level was achieved for nymphs by 8 × 10 spores/ml. The LC50 values were obtained as 1.2 × 106 and 6.9 × 105 spores/ml for adults and nymphs, respectively. The present study suggests that the entomopathogenic fungus, L. longisporum could be considered as a potential candidate in biocontrol programs of C. pini. This is the first report on the pathogenicity of L. longisporum on C. pini. Keywords: Biocontrol, Insect pathology, DNA Barcode, Pathogenecity Introduction12 life histories, including potentially long term parthenogenesis. Most-parsimonious Molecular methods have provided additional phylogenetic trees were inferred from partial Downloaded from jcp.modares.ac.ir at 10:16 IRST on Tuesday September 28th 2021 valuable characters for the resolution of (905-coding-bp) sequences of elongation factor taxonomic problems and the discovery of new 1α (EF-1α) and complete (675-bp) sequences of species within the Aphididae (Foottit, 1997). cytochrome oxidase 2 (COII) The Cinara curtis DNA barcoding has been proposed as a genus was studied by Normark (2000), who used standardized approach to species delimitation in DNA sequences of nuclear elongation factor 1α numerous groups of living organisms and COII to reconstruct evolutionary relationship (Hajibabaei et al., 2007) including insects (Floyd among species of Lachnidae. Most authors have et al., 2009). The aphid family Lachnidae (c. 320 included the genera Lachnus, Moculoachnus, species), sister group to the economically Pterochloroides, Tuberolachnus, and devastating family Aphididae (c. 3300 species), Nippolachnus within a tribe Lachnini (Blackman encompasses a diverse array of associations with and Eastop, 1994; Czylok, 1990; Ilharco and host plants and attendant hymenopterans and of Harten, 1987; Lampel and Burgener, 1987). There is no evidence to support monophyly of this group of aphids. Universal primers have Handling Editor: Dr. Mohammad Mehrabadi ________________________________ been developed for conserved mtDNA * Corresponding author, e-mail: [email protected] sequences flanking regions useful for Received: 18 November 2012, Accepted: 2 December 2013 phylogenetic and population studies (Folmer et Published online: 4 December 2013 159 Pathogenicity of Lecanicillium longisporum __________________________________________ J. Crop Prot. al., 1994; Lunt et al., 1996; Simon et al., 1994). production of blastospores within the Taxonomic and phylogenetic studies have used haemocoel; ramification of the mycelia and mitochondrial or nuclear genes as cytochrome invasion of tissues, which finally causes the oxidase I and II (CO-I and CO-II), nuclear death of the host. The last step is the production elongation factor 1α, and leucine tRNA of conidia on the surface of the cadaver (Askary (Normark, 2000). Favert and Voegtlin (2004) et al., 1999). In addition to the nutrient provided an important molecular phylogenetic utilisation and tissue digestion during infection analysis of Cinara species, associated with of insect hosts, many entomopathogenic fungi pinyon pines in the southwestern United States, produce toxic substances which may play a using CO-I and nuclear elongation factor 1α crucial role in the death process (Gindin et al., DNA sequences. In recent years, DNA 1994). Many isolates of Lecanicillium have barcoding has attracted attention of the shown high pathogenicity to several species of researchers for studying different groups of aphids such as Aphis gossypii (Glover), insects. DNA barcoding is able to identify the Macrosiphum euphorbiae (Thomas) and Myzus sample in all stages of life by using short DNA persicae (Sulzer) (Alavo et al., 2002; Askary et sequences (e. g. eggs, larvae, nymphs or pupae) al., 1998; Kim et al., 2001). In addition, recent and it is an approach for identifying many studies have demonstrated that the genus invertebrate taxa which their morphological Lecanicillium, including the isolate of L. identification is problematic due to lack of longisporum (as the active ingredient of availability of taxonomic keys for immature life Vertalec®), have suppressive activity against stages. The standard sequence used for this powdery mildews (Askary et al., 1998; Kim et purpose is a fragment of 5´ end of mitochondrial al., 2007; Kiss, 2003; Miller et al., 2004; COI gene that is amplified by the "universal Romero et al., 2003). This suggests that these primers"(Folmer et al., 1994). Several studies fungi may play a dual role, controlling both showed that it is a reliable tool for the molecular aphids and powdery mildews (Askary et al., identification of Lepidoptera, Hymenoptera, 1998; Kim et al., 2007; Kim et al., 2008). Coleoptera and Diptera species (Hajibabaei et The rising costs of traditional chemical al., 2006). pesticides, increasing resistance of insects to As a new aphid species in the region, these products and their undesirable effects on attempt was made to focus on the the environment (Corey et al., 1993; entomopathogenic fungus, Lecanicillium Mukanganyama et al., 2003; Scott et al., 1998) Downloaded from jcp.modares.ac.ir at 10:16 IRST on Tuesday September 28th 2021 longisporum as a biocontrol agent for aphid have led to renew efforts to search for novel population management.Until recently, the approaches to insect pest management (El- form-genus Verticillium contained a wide Salam and El-Hawary, 2011). In the last variety of species with diverse host ranges, decades, biological control, including the use of including arthropods, nematodes, plants and entomopathogenic fungi, has emerged as a fungi. However, the genus has been redefined strategy to control aphids, especially in high- using rDNA sequencing, placing all insect value crops. These have been viewed as a pathogens into the new genus Lecanicillium substitute or complement to traditional (Zare and Gams, 2001). Lecanicillium spp. use chemical measures. Aphids seem to be both mechanical forces and hydrolytic enzymes susceptible hosts for entomopathogenic fungi to directly penetrate the insect integument and because of their morphological, biological and the cell wall of the fungal plant pathogen ecological characteristics (Steinkraus, 2006). (Goettel et al., 2008). Lecanicillium spp. Due to a global trend toward application of usually use a general pathway of pathogenesis COI gene as DNA barcode, we attempted to for entomopathogenic mitosporic fungi provide information about this gene sequence including adherence to the host cuticle by of C. pini, as a new aphid species for Iranian conidia; germination; penetration of the cuticle; fauna collected in a previous work (Nazemi et 160 Nazemi etal. ______________________________________________________ J. Crop Prot. (2014) Vol. 3 (2) al., 2013). The main purpose of the present ethidium bromide and its image was saved. The research is to study the pathogenicity of L. PCR product was purified and sent to Macrogen longisporum against this new urban pest. Inc. for sequencing. Materials and Methods Sequencing and analysis Chromatograms were checked using the BioEdit Aphid collection software (Hall, 1999). The resulting sense and The aphid Cinara pini (Linnaeus) was collected antisense sequences were edited and used in from Pinus mugo Turra in Mashhad region in alignment. Representative sequences for all the northeast of Iran during spring 2009. The known Cinara groups were retrieved from classic method using the available keys GenBank and included in the alignment file. (Blackman and Eastop, 2006) diagnosed it as Sequences were aligned using the default settings the genus Cinara. Final confirmation of the of Clustal W (Thompson et al., 1994). species was made by Olivera Petrovic- Unweighted parsimony analysis of the alignments Obradovic from Belgrade University, Serbia. was conducted with PAUP*4.0b2 (Swofford, 1999). For the analyses, gaps were treated as Molecular study procedure missing characters, and the reliability of trees was DNA extraction tested with a bootstrap test (Felsenstein, 1985). Genomic DNA from the aphid was extracted Parsimony bootstrap analysis included 100 re- using a Bioneer kit (Bioneer Co. Daejeon, samplings using the branch and bound algorithm. South Korea). Individual aphids were kept at - The most appropriate model of sequence 20 °C for at least 12 h, crushed using a evolution was determined using hierarchical micropestle in 180 µl lysis buffer and