The Occurrence of Two Species of Entomophthorales (Entomophthoromycota), Pathogens of Sitobion Avenae and Myzus Persicae (Hemiptera: Aphididae), in Tunisia
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Hindawi Publishing Corporation BioMed Research International Volume 2013, Article ID 838145, 7 pages http://dx.doi.org/10.1155/2013/838145 Research Article The Occurrence of Two Species of Entomophthorales (Entomophthoromycota), Pathogens of Sitobion avenae and Myzus persicae (Hemiptera: Aphididae), in Tunisia Ibtissem Ben Fekih,1,2 Sonia Boukhris-Bouhachem,1 Jørgen Eilenberg,3 Mohamed Bechir Allagui,1 and Annette Bruun Jensen3 1 Plant Protection Laboratory of National Institute of Agricultural Research of Tunisia, Rue Hedi´ Karray, 2049 Ariana, Tunisia 2 National Institute of Agronomy of Tunisia, University of Carthage, 43, Avenue Charles Nicolle, 1082 CiteMahraj´ ene,` Tunisia 3 Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark Correspondence should be addressed to Ibtissem Ben Fekih; [email protected] Received 1 March 2013; Revised 22 April 2013; Accepted 28 May 2013 Academic Editor: Ameur Cherif Copyright © 2013 Ibtissem Ben Fekih et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The natural occurrence of entomophthoralean fungi pathogenic towards aphids on cereal and potato crops was investigated in the years 2009, 2010, and 2011. Infected aphids were sampled in three bioclimatic zones in Tunisia (Beja, Cap bon, and Kairouan) and fungal species were determined based on morphological characters such as shape, size, and number of nuclei in the primary conidia. Polymerase Chain Reaction (PCR) on the internal transcribed spacer 1 region (ITS1) was used to verify morphological determination. Both methods gave consistent results and we documented for the first time the natural occurrence of two fungal species from the order Entomophthorales (phylum Entomophthoromycota), Pandora neoaphidis and Entomophthora planchoniana. Both fungi were recorded on the aphid species Sitobion avenae and Myzus persicae onbarleyearsandpotatoleaves,respectively. Moreover, natural mixed infections by both species (P. neo aphi di s and E. planchoniana) were documented on the target aphids. This investigation provides basic information of entomopathogenic fungi infecting economically important aphids in Tunisia. 1. Introduction sustainable control strategy to manage pest insects like aphids [6]. Field observations have shown that aphid populations Aphids (Hemiptera: Aphididae) are one of the most impor- are commonly regulated by a range of natural enemies, such tant groups of insect pests in agriculture. They weaken their as predators, parasitoids, and also fungal pathogens [7]. In host plants in diverse ways by causing direct damage as temperate regions, fungal species from the phylum Ento- phloem feeders and also by indirect damage as plant virus mophthoromycota are important pathogens of aphids [8– vectors [1]. A total of approximately 4000 aphid species have 10]. Their ability to cause epizootics among their host insects been described, of which 157 species have been reported within a short time makes them potentially valuable for pest in Tunisia [2]. Most of the identified species in Tunisia are control and by that an element in future IPM systems [11–15]. considered to be pests and cause significant yield losses to A new taxonomical revision assigned entomophthoralean important crops such as cereals [3]. Furthermore, dissemi- fungi under the phylum Entomophthoromycota with three nation of the Potato Virus Y (PVY) by aphids is considered new orders, Entomophthorales, Neozygitales, and Basidiobo- as big problem in Tunisian potato fields [4]. The develop- lales [9]. The most common species worldwide infecting ment of insecticide resistance among aphids has stimulated aphids belong to the Entomophthorales, particularly to the an interest in developing alternative methods of control families Entomophthoraceae and Ancylistaceae, and the [5]. Integrated pest management (IPM) can be seen as a Neozygitales represented by the family Neozygitaceae [9]. 2 BioMed Research International The aphid pathogenic species have been documented almost 2.2.3. Scanning Electron Microscopy. Infected aphids were worldwide with most records from temperate climatic zones examined with an environmental scanning electron micro- [8, 16–18]. Little is known, however, about the natural occur- scope (ESEM) aiming to obtain information about detailed rence of these fungal pathogens in North Africa. A previous structures of the fungi. Samples were carbon coated using a study in Egypt recorded twelve entomopathogenic fungal conductive carbon disk and observation was done through speciesofwhichsevenbelongedtoEntomophthoromycota the ESEM model QUANTA 200I-D7827 with tungsten (W) [19]. The approach using entomopathogenic fungi in biologi- filament electron source. cal control is a new field in Tunisia. So far, only few studies on two Fusarium (Ascomycota) species on the artichoke 2.3. Molecular Characterization: DNA Extraction and PCR aphid species Capitophorus elaeagni have been performed in Amplification. Genomic DNA from 14 infected aphids Tunisia [20, 21]. Thus, basic knowledge about occurrence, (Myzus persicae and Sitobion avenae)andonehealthySitobion distribution, and prevalence over time of entomophthoralean avenae taken from rearing chamber and considered as a fungi in Tunisia is completely lacking. In this study we wanted negative control was extracted using a Chelex extraction to explore the natural occurrence of entomophthoralean protocol [25]. DNA extraction was done by adding 20 L fungi in relation to Sitobion avenae [22], among important phosphate buffered saline (PBS PH7.2) and 5 Lproteinase aphid species infesting barley ears and Myzus persicae [22], a K (10 mg/mL) to each 1.5 mL Eppendorf tube and the aphids common pest aphids infesting potato fields in Tunisia. were homogenized with a DNA-free pestle. After a quick spin at 10,500 ×g for 30 s, 100–200 L (depending on aphid 2. Materials and Methods size) of a 10% Chelex solution was added and the samples ∘ were incubated overnight at 56 C. Next day the samples were ∘ 2.1. Sampling. The investigations of mycoses in aphid pop- incubated at 94 Cfor15minandafteraspinat10,500×g ulation (apterae and alate specimens) were done in three for 30 s, the supernatants were transferred to new Eppendorf regions of Tunisia: in the north west, Beja: site of the Regional − ∘ ∘ ∘ tubes and stored at 20 C. Field Crop Research Center (36 44 00 N, 9 11 00 E), a sub- PCR was performed on the internal transcribed humidarea;inthenortheast,Capbon:SiteofSoliman ∘ ∘ spacer 1 (ITS 1) using two genus specific forward primers (36 40 40 N, 10 28 20 E), situated in the semiarid area of for Entomophthora and Pandora,respectively:Ml2: theregion;inthecenter,Kairouan:SiteofSidiMahmoud 5 -GCAACGGATCATCATGTAA-3 and PnCNf: 5 - ∘ ∘ (35 37 07 N, 9 55 34 E), a continental zone with arid cold TTTGGGTTTAAATAGAAGGTTGA-3 and reverse prim- winter. Barley and potato fields infested with aphids were ers Nu-5.8S-3 :5-ACTACGTTCTTCATCGATGA-3 [10] used for random sampling of fungal infected aphids between and PnCNr: 5 -AGGCAAAGCCTAGAGCACTT-3 (unpu- March and June of the years 2009, 2010, and 2011. Aphid bished). The primers were chosen to detect and confirm the cadavers with symptoms of fungal infection were placed into identity of the fungi infecting the field collected aphids. ventilated plastic boxes and carried to the laboratory. Positive DNA controls provided from ARSEF Collection of Entomopathogenic Fungal Cultures: ARSEF 2583: Pandora 2.2. Morphological Characterization. Fungus identification neoaphidis,isolatedfromtheaphidspeciesAcyrthosiphon wasbasedontheshape,size,andnucleinumbersinthe pisum (USA, 1988) and ARSEF 6918: Entomophthora muscae, primary conidia [23, 24]. The number of aphids subject to isolated from the Dipteria Coenosia tigrina (Denmark, 1999) morphological identification of fungal infection was 730 for and negative water controls were included in each set of PCR M. persicae and 980 for S. avenae. reactions. PCR amplifications were performed in 50 Lreaction volumes containing 2 L of chelex-extracted DNA 1 : 1 or 2.2.1. Sample Preparation. The aphid cadavers with fresh diluted 1 : 10, 10 LPhusionHFBuffer(5× 7.5 mM MgCl2), conidiophores were inverted over a glass slide in moist boxes ∘ 10 mM dNTPs, 0.5 M of each primer, 0.5 U Phusion High- at 20 C to allow conidia ejection for first 5 hours and then Fidelity DNA Polymerase (Finnzymes, Espoo, FI). For both 8 hours. Subsequently, some of the cadavers were stored ∘ primers, the PCR conditions were denaturation at 98 Cfor individually in 96% ethanol to be used in later molecular ∘ 30 s, followed by 38 cycles of denaturation at 98 Cfor10s, examination. Light and electron microscopic studies were ∘ ∘ annealing at 55 CforML2or60C for PnCNf for 20 s, and used for the morphological determination of the fungal ∘ ∘ extension at 72 C for 1 min, with a final extension at 72 Cfor species. 10 min. The size of the PCR amplifications was estimated by electrophoresis on a 1.5% agarose gel in 0.5 × TBE, and the 2.2.2. Light Microscopy. Conidia projected on the slide were products visualized with EZ-Vision (AMRESCO LLC, USA). mountedineitherlacticacidoraceto-orceinformeasuring Sequencing was used to verify the species identity based size or counting nuclei numbers, respectively. The shape and on the length of the PCR product, in particular for the