Assessment of the genetic diversity of Argentinean isolates of Beauveria bassiana (Ascomycota: Hypocreales) using ISSR markers

2.1 Origin of biological material

Isolates of B. bassiana were obtained from infected insects of the orders Hemiptera, Coleoptera and Dermaptera and from soil samples of agricultural crops, collected in several localities of Buenos Aires, Corrientes and Tucumán provinces that correspond to three different agro-ecological regions (Pampas, Northeast and Northwest, respectively) (Toledo et al., 2008). Cultures were preserved in the mycological collections of Centro de Estudios Parasitológicos y de Vectores- CEPAVE (La Plata, Buenos Aires, Argentina) and the USDA-Agricultural Research Service Collection of Entomopathogenic Fungal Cultures-ARSEF (Ithaca, New York, USA), where they were admitted under their respective accession numbers (Table 1).

2.2 DNA extraction

DNA was extracted from fungal cultures developed in 2% Malt Extract Agar (MEA) medium after 7 d incubation at 25 °C in darkness (Zhang et al., 2010). The quality of genomic DNA was assessed by electrophoresis on 0.7% w/v agarose gels supplemented with ethidium bromide (100 ng ml⁻¹ final concentration). The amount of DNA was estimated by comparison with a molecular marker control of known concentration (Lambda Phage Genome digested with Hind III – Promega Biotech) using the image analyzer (SYNGENE GeneTools).

2.3 Diversity analysis

After evaluating a total of eleven ISSR markers, 3′ anchored and 5′ anchored, four were selected to perform the analysis of 36 isolates of B. bassiana (Table 2). Amplification reactions including DNA free control were carried out in a 15 µL volume of reaction, containing 10× reaction buffer (500 mM KCl; 100 mM Tris–HCl, pH 9.0 at 25 °C; 1% Triton X-100), 2 mM (primers KA5 and 826) or 2.5 mM (primers BA3 and CA5) of MgCl₂, 0.25 mM each dNTP, 60 ng of each primer, 1 U Taq DNA polymerase and 100 ng of template DNA (reagents Inbio Highway®, Tandil, Buenos Aires, Argentina). PCR reactions were performed on a MJ Research (PTC-150 MiniCycler) programmed as follows: initial denaturation at 94 °C for 3 min, followed by 30 cycles of denaturation at 94 °C for 45 s, annealing at 48 °C for 45 s and extension at 72 °C for 3 min, with an additional final extension step at 72 °C for 5 min.

PCR products were resolved by electrophoresis on 1.5% w/v agarose gels supplemented with ethidium bromide (100 ng ml⁻¹ final concentration) using as an internal standard the DNA marker 100–1000 bp (Inbio Highway®, Tandil, Buenos Aires, Argentina) and photographed. The ISSR banding patterns generated were analyzed with the SYNGENE GeneTools software and a dendrogram was built using the similarity matrix of DICE and the Unweighted Pair Group Method with Arithmetic Mean (UPGMA), with a 5% tolerance.

2.4 Molecular identification

The identity of the isolates of B. bassiana previously characterized on the basis of morphological characters (Toledo et al., 2007, 2008) was confirmed by the amplification of the ITS, which was performed on seven of the 36 isolate. They were selected on the basis of their phenotypic differences, geographical origin, host and location in the dendrogram generated through the analysis of diversity. Universal primers ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) were used for the isolates Bb147 and Bb189 and ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 for the remaining isolates (White et al., 1990). The PCR program consisted in an initial cycle of denaturation at 94 °C for 4 min; followed by 33 cycles of denaturation at 94 °C for 1 min, 45 s at 56 °C, 1 min at 72 °C and a final elongation step at 72 °C for 5 min. The reaction mixture contained 1× reaction buffer (500 mM KCl; 100 mM Tris–HCl, pH 9.0 at 25 °C; 1% Triton X-100 without magnesium); 1.5 mM MgCl₂, 0.2 mM each dNTP, 0.3 μM each primer; 1U of T-Plus DNA Polymerase (Highway Molecular Biology-INBIO-UNICEN) and 50 ng of template DNA per reaction, in a final volume of 15 μL. Amplification products were resolved by electrophoresis on a 1% w/v agarose gel supplemented with ethidium bromide (100 ng ml⁻¹ final concentration).

Amplified fragments were precipitated by adding a volume of isopropanol and 0.1 volumes of sodium acetate 3 M. The mixture was incubated at −18 °C for 12 h and then centrifuged at 15,000g for 15 min. DNA pellet was washed with ethanol 70%, subsequently to be dried and dissolved in sterile distilled water. Amplicons were sequenced at Macrogen Inc. (Seoul, Korea) according to the method described by Sanger et al. (1977). Sequences were edited using the program BioEdit version 7.0.9.0 (Hall, 1999) and they were used to perform a phylogenetic analysis that included the sequences of the isolates of Beauveria from this work with sequences of some species for the genus available at the GenBank database (www.ncbi.nlm.nih.gov). Two representatives of each species of B. bassiana (AY531984.1 and NR_111594.1, the last one designated as neotype by Rehner et al. (2011), B. brongniartii (HQ880770.1 and DQ376245.1), B. amorpha (HQ880804.1 and HQ880806.1), B. caledonica (HQ880818.1 and HQ880821.1), and B. malawiensis (DQ376247.1 and HQ880825.1) were used as in-group, while the sequence of a representative of Lecanicillium saksenae (AB360351.1) was used as out-group. Sequences were aligned with the ClustalW tool of the program Mega5 (Tamura et al., 2011), assuming a gap opening penalty of 15 and a gap extension penalty of 6.66. The alignment was edited automatically by the software Gblock Version 0.91b (Talavera and Castresana, 2007; Tamura et al., 2011), setting to 2 the minimum length of a block and leaving all the other options by default. The phylogenetic study was carried out using the Bayesian inference and analysis of maximum likelihood. To do this, the best substitution model was selected using the Akaike Information Criterion (AIC) (Akaike, 1973) in the jModelTest software version 2.1.7 (D̀’arriba et al., 2012), and the parameters of the chosen model were used in the subsequent analysis. The maximum likelihood analysis was carried out with the PhyML software v.3.0 (Guindon and Gascuel, 2003) using the BioNJ algorithm to obtain starting trees whose topologies were then optimized by both nearest neighbor interchange (NNI) and sub-tree pruning and regrafting (SPR) algorithms. The statistical support for the nodes was evaluated through 1000 bootstrap replicates. For the Bayesian analysis, the software MrBayes v.3.2 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003) was used to perform two independent Metropolis-coupled Markov chain Monte Carlo (MCMCMC) with four chains through 1,000,000 generations, with trees in each chain sampled every 100th generation. The first 25% of trees were removed as burn-in and posterior probabilities were determined from the remaining trees.

3.1 Diversity analysis

Primers 826, BA3, CA5 and KA5 (Table 2), selected on the basis of the number of bands amplified and their stability, generated a total of 101 fragments, 92 of which were polymorphic. Primers generated between 10 and 44 bands (namely, 826 = 10; BA3 = 20; CA5 = 27; KA5 = 44) whose sizes ranged from 300 to 1700 bp. The banding patterns generated by combining the fingerprints generated with each primer were analyzed by agreeing to take a 70% of similarity as court, and in this way, it was noted that each isolate had a different haplotype (Fig. 1).

Close

Fig. 1

(A) Dendrogram built using the similarity matrix of DICE and the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) which includes 36 isolates of Beauveria bassiana; (B) ISSR amplification patterns of nine isolates (Bb061, Bb083, Bb113, Bb175, Bb55, Bb001, Bb119, Bb146 and Bb189) generated with the primers BA3 (a), CA5 (b), 826 (c) and KA5 (d). M = DNA marker 100–1000 bp. * Isolates selected for the specific identification from the sequencing of the ITS.

Export to PPT

3.2 Molecular identification

The ITS amplicons of seven representative isolates of B. bassiana had a size between 487 bp and 568 bp and their sequences were deposited in the databases DDBJ/EMBL/GenBank under the accession numbers KU702657.1 (Bb069), KU702658.1 (Bb077), KU702659.1 (Bb112), KF308683.1 (Bb147), KU702660.1 (Bb151), KU702661.1 (Bb153) and KT952326.1 (Bb189). From the alignment of the nucleotide sequences, the software Gblock selected 499 reliable characters to be used in the phylogenetic study. The best substitution model according to Akaike Information Criterion (AIC), was the General Time-Reversible model with Invariant sites (GTR + I). By incorporating the chosen substitution model of molecular evolution into the phylogenetic analysis, under the maximum likelihood approach in PhyML v.3.0 and Bayesian inference in MrBayes v.3.2, similar tree topologies were obtained. Fig. 2 shows the Bayesian phylogram, in which it is noted that all the isolates included in this study were grouped together with two reference isolates of B. bassiana (AY531984.1 and NR_111594.1 = neotype) with high supporting values (posterior probabilities = 1 and bootstrap = 99). Furthermore, B. brongniartii, B. amorpha, B. caledonica and B. malawiensis, all morphologically-related species, formed independent clades.

Close

Fig. 2

Phylogenetic tree generated by the Bayesian analysis of the ITS sequences of seven Argentinean isolates of Beauveria bassiana and related species. Lecanicillium saksenae was used as out-group. The isolates corresponding to this work are highlighted in bold. Below each of the reference isolates detailed its number of access to GenBank. The probability values later ≥0.7 and bootstrap ≥70% are represented above and below of the internodes, respectively. The bar at the bottom indicates the number of substitutions per site.

Export to PPT