Bioinformatic Analysis of the Beauvericin Gene from Beauveria bassiana and Insecticidal Effect on Spodoptera littoralis (Boisd)

Citation :Egypt. Acad. J. Biolog. Sci. ( C. physiology and Molecular biology ) Vol.7(2)pp89-99 (2015) Egyptian Academic Journal of Biological Sciences is the official English language journal of the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of Sciences Ain Shams University. Physiology & molecular biology journal is one of the series issued twice by the Egyptian Academic Journal of Biological Sciences, and is devoted to publication of original papers that elucidateimportant biological, chemical, or physical mechanisms of broad physiological significance. www.eajbs.eg.net Provided for non-commercial research and education use.

Death occurs due to toxin production by the fungus and/or multiplication to inhabit the entire insect body (Goettel et al., 2010).Entomopathogenic fungi are prolific producers of bioactive secondary metabolites (Isaka et al., 2003;Molnar et al., 2010), which are predicted to play key roles as virulence factors for fungi, infecting arthropods (Rohlfs and Churchill, 2011).Metabolites produced by entomopathogenic fungi would serve one or more of the following functions: (1) toxic to the host and help to cause death; (2) aid the fungus overcome host defense; (3) suppress competition from other pathogens and saprophytes on the insect cadaver; (4) provide a defense outside the host against other organisms (Charnley, 2003).The beauvericin synthetase purified from B. bassiana (Peeters et al., 1988).Beauvericin belongs to the cyclic non ribosomal hexadepsi peptide family of natural products, originally isolated from entomopathogenic fungi, including B. bassiana (Hamill, et al. 1969).B. bassiana produces several toxic compounds (Strasser et al., 2000;Vey et al., 2001).A majority of these insecticidal molecules are low molecular weight biologically active secondary metabolites (Zimmermann, 2007).Beauvericin, bassianin, bassianolide, beauverolides, beauveriolides, tenellin, oosporein (Strasser et al., 2000;Vey et al., 2001), oxalic acid (Roberts, 1981) bassiacridin (Quesada-Moraga and Vey, 2004) are some of these important metabolites.These compounds also contributed in B. bassiana pathogenicity as they act as immune suppressors and host specific toxins (Von Dohren, 2004).

Cultivation of Beauveria bassiana isolate:
The entomopathogenic fungi; B. bassiana (AUMC 9896) Egyptian isolate was isolatedin Bio-insectcide Production Unit, Plant Protection Research Institute and was identified in Mycological Center, Faculty of Science, Assiut University.Sahar and Moharram (2014).The isolate was cultured on Czapek Dox Brothmedium for 5 days at 25 o C and aseptically filtered through sterile filter paper.

PCR Amplification: 1-Primer design:
A simple way for the primer design was used based on the alignment of

2-Rearing of insect:
The Cotton Leaf worm, S. littoralis larval were obtained from the department of cotton leaf worm, Plant Protection Research Institute.

Bioassay
The crude of toxin, was extracted from isolate of B. bassiana and four concentrations prepared (100%, 75%, 50% and 25%).500 µl.From each dilution were added to 5 gm of diet were put into plastic container (15 cm diameter) were contains 20 larvae and were covered with muslin cloth for aeration.The larvae were left to feed on treated diet for 48 h., then mortality percentages were recorded, the survival larvae were transferred to feed on untreated diet.Mortality percentages were corrected (Ortiz-Urquiza et al., 2010), the lethal concentration of 50 and 90 % from treated was calculated by Probit analysis (Finney, 1971).

RESULTS
Total DNA was extracted from B. bassiana EG-isolate.The integrity of total DNA was estimated by ethidium bromide agarose gel electrophorasis assay, and the purity of the total DNA obtained which was 1.8 measured by spectrophotometer A260/280 absorbance ratio for indicating high yield and purity of the extracted DNA.Following DNA extraction the beauvericin gene in the isolate genome was amplified using PCR technique.1µl of DNA was mixed with PCR reaction mixture, taq DNA polymerase and (Forward and Reverse) primers directly which designed to amplify the beauvericin gene.

Extraction and amplification of DNAbeauvericin gene:
Total DNA was extracted from B. bassiana Egyptian isolate.The integrity of total DNA was estimated by ethidium bromide re, taq DNA polymerase and (Forward and Reverse) primers directly which designed to amplify the beauvericin gene.

Electrophorasis analysis of PCR product:-
The size of the PCR product amplified from B. bassiana was estimated after running in 1% agarose gel electrophorasis by comparing its electrophorasis mobility with those of standard DNA marker as shown in (Fig. 1).

Nucleotide Sequence analysis:-
The nucleotide sequence of the PCRamplified fragment for the beauvericin gene of B. bassiana EG-isolate was done to determine the relationship with other B. bassiana isolates registered in GenBank (Table 1).The sequencing assembly was done by analyzed the sequence results generated by the forward and reverse sequencing primers with the software program sequencing analysis.Nucleotides were found to be 636bp from beauvericin genome sequence (Fig. 2).

Insecticidal effect of toxins crude extraction
Toxins crude extraction due to isolate of B. bassiana investigated against 3 rd inster larvae of S. littoralisas shown in Table (2).After 1 day, the crude of B. bassiana gave 5.00%, 10.50%, 17.00% and 30.00% mortality in the concentrations 25, 50, 75 and 100%, respectively.The second day the percentage of mortality were 15.50%, 30%,55.50%and 60.50%in the concentrations 25, 50, 75 and 100%, respectively.After 4 days, the percentage of mortality were 51.00%, 57.50%, 79.00% and 96.50% in the concentrations 25, 50, 75 and 100%, respectively.Goettel et al., 2010 reported that entomopathogenic fungus, B. bassiana is a broad host range entomopathogen that plays an importantrole in the control of insect populations in nature.This fungus is the most widely used fungal species available commercially.It is generally found on infected insects both in temperate and tropical areas throughout the world (Zimmermann, 2007).Duringits pathogenic phase, the developing hyphae directly penetrate the insect integument by producing extracellular enzymes (Fan et al., 2007) and B. bassiana produces several toxic compounds (Strasser et al., 2000;Vey et al., 2001).A majority of these insecticidal molecules are low molecular weight biologically active secondary metabolites (Zimmermann, 2007).Beauvericin, bassianin, bassianolide, beauverolides, beauveriolides, tenellin, oosporein and oxalic acid.These compounds also contributed in B. bassiana pathogen city as they act as immune suppressors and host specific toxins (Von Dohren, 2004).Among them, Beauvericin is the most important compound which was isolated first from B. bassiana.Not all isolates of B. bassiana produce beauvericin (Frappier et al., 1975;Zimmermann, 2007), but other species produce this compound like Fusarium Spp.(Hamill, et al 1969 andLogrieco, et al. 1998).Beauvericin carries insecticidal properties.

DISCUSSION
Molecular methods based on PCR and DNA sequences of PCR products can greatly reduce the amount of time needed for identifying and characterization of any isolate.Polymerase Chain Reaction (PCR) technique is reported to be ingenious technique in molecular biology that allow rapid and specific amplification of DNA present in very small amounts in complex mixtures of nucleic acids, so it is a powerful technique developed for detection any gene.Viaud et al., 1996 determined to talgenome size of B. bassiana by PCR amplification which considered highly sensitive process give desirable results.Concentration of DNA and particularly annealing temperature of primers has high importance in successful PCR amplification.For this, temperature gradient of annealing temperature from 56°C to 65°C was used in thermal cycler and 58°C was showed to be the optimum temperature, Wang et al., (2003) used this temperature as optimum temperature for amplifying the pr1 gene, our results are agree with them and disagree with Viaud et al. 1996 who used the 65°C as annealing temperature.
Beauvericin was confirmed as the active compound from B. bassiana against Artimiasalina, which was considered a model organism to study insecticidal activity.Subsequently, the insecticidal effect of beauvericin on a microgram level was investigated on Calliphora erythrocephala, Aedesaegypti, Lygus spp., Spodoptera frugiperda and Schizaphis graminum (Grove and Pople, 1980;Jestoi, 2008. Leland et al., 2005).Wang and Xu(2012) found that beauvericin was a strong insecticidal activity against a broad spectrum of insect pests.the insecticidal mechanism of beauvericin is still worth investigated.There are few reports about the insecticidal mechanism of beauvericin.Despite similarities between the chemical structures of beauvericin and other mycotoxins, beauvericin is more effective (Grove and Pople, 1980) and may have a unique mechanism of action.The discovery of the active mechanism of beauvericin against insects will be helpful to find new commercial insecticidal agents, reduce the threat of insecticidal agents to human cells.

Fig. 4 :
Fig. 4: Phylogenetic tree of beauvericin gene; the dendrogram displaying the percentage of sequence homology between beauvericin gene from B. bassiana Eg. isolate and the other three B. bassiana published in GenBank.Translation of nucleotide sequence of beauvericin gene from B. bassiana Eg.Isolate :-The predict numbers of amino acids were produced from translation of beauvericin gene nucleotide sequence were 211 amino acids (Figs. 5&6).

Fig. 7 :
Fig. 7: Multiple amino acid sequence alignment of beauvericin gene from B. bassiana Eg.Isolate with the corresponding amino acid sequence of beauvericin gene from three B. bassiana isolates available in Gen Bank.

Table 2 :
Corrected a cumulative mortality percentages of 3 rd instars larvae of S. littoralis after feeding on synthetic diet treated with metabolic toxins crude