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LC50 of the Peptide Produced by the Entomopathogenic Fungus Nomuraea rileyi (Farlow) Samson Active Against Third Instar Larvae of Anticarsia gemmatalis (Lep.: Noctuidae)

Abstracts

The entomopathogenic fungus Nomuraea rileyi (Farlow) Samson produced a peptide active against Anticarsia gemmatalis 3rd instar larvae. To produce this peptide, N. rileyi was cultivated aerobically in Saboraud, maltose, yeast-extract broth at 26 ± 1ºC for 12 days, after which the medium was filtered and separated in a liquid/liquid extractor, concentrated and the peptide purified chromatographically. The crystals obtained were kept refrigerated until needed for LC50 analysis. The LC50 of this peptide against A. gemmatalis 3rd instar larvae was determined in triplicate experiments using solutions containing 1.0, 0.2, 0.1, 0.01, 0.001 and 0.0001 mg/ml of N. rileyi peptide. The results of these experiments were used to calculate a linear equation in which Y = 6,81176 + 1,01382 * LOGx , giving a LC50 value of 0.0163 mg/ml.

Anticarsia gemmatalis; Nomuraea rileyi; integrated control; secondary metabolite; toxins


Este trabalho objetivou determinar a CL50 de um peptídeo produzido pelo fungo N. rileyi, para larvas de 3º ínstar de A. gemmatalis. O peptídeo foi produzido através de fermentação aerada, em meio SMY, sob condições controladas por 12 dias. O metabólito foi purificado, utilizando-se de sistemas de cromatografia. Os cristais obtidos foram então armazenados a baixa temperatura para posterior análise da atividade biológica. Os experimentos foram conduzidos em 3 repetições, com tratamentos que consistiram de soluções com 0,0001; 0,001; 0,01; 0,1; 0,2 e 1,0 mg/mL. Através de análise de regressão chegou-se a equação da reta Y = 6,81176 + 1,01382 * LOGx e um valor para CL50 de 0,0163 mg/mL.


LC50 of the Peptide Produced by the Entomopathogenic Fungus Nomuraea rileyi (Farlow) Samson Active Against Third Instar Larvae of Anticarsia gemmatalis (Lep.: Noctuidae)

Sideney Becker Onofre1* * Author for correspondence ; Raul Riveros Gonzalez2; Cláudio Luiz Messias3 João Lúcio Azevedo4 and Neiva Monteiro de Barros4

1 Paraná Federal Center of Technological Education - CEFET-PR; Microbiology Laboratory; becker@qualinet.com.br; Pato Branco - PR - Brazil. 2 University of Caxias do Sul; Physical-Chemistry Laboratory; Caxias do Sul - RS - Brazil. 3 University Campinas State; Genetic and Evolution Laboratory; cmessias@unicamp.com.br; Campinas - SP - Brazil. 4 University of Caxias do Sul; Departament of Biological Sciencs and Biotechnology Institute; n.barros@terra.com.br; Caxias do Sul - RS - Brazil

ABSTRACT

The entomopathogenic fungus Nomuraea rileyi (Farlow) Samson produced a peptide active against Anticarsia gemmatalis 3rd instar larvae. To produce this peptide, N. rileyi was cultivated aerobically in Saboraud, maltose, yeast-extract broth at 26 ± 1oC for 12 days, after which the medium was filtered and separated in a liquid/liquid extractor, concentrated and the peptide purified chromatographically. The crystals obtained were kept refrigerated until needed for LC50 analysis. The LC50 of this peptide against A. gemmatalis 3rd instar larvae was determined in triplicate experiments using solutions containing 1.0, 0.2, 0.1, 0.01, 0.001 and 0.0001 mg/ml of N. rileyi peptide. The results of these experiments were used to calculate a linear equation in which Y = 6,81176 + 1,01382 * LOGx , giving a LC50 value of 0.0163 mg/ml.

Key words: Anticarsia gemmatalis; Nomuraea rileyi, integrated control, secondary metabolite, toxins

INTRODUCTION

Many fungi produce secondary metabolites, which act on other organisms, sometimes causing inhibition of growth, disease and even death. Examples of such metabolites include the aflatoxins produced by some Aspergillus flavus strains (Diener and Davis, 1969), ochratoxin produced by A. ochraceus (Myokey et al., 1969; Kodaira, 1969) and the toxins and antibiotics produced by members of the genus Penicillium. Some entomopathogenic fungi produce metabolites, which can affect other microorganisms and insects (Onofre, et al., 1999), e.g. the fungus Metarhizium anisopliae produces an insecticidal cyclodepsipeptide called destruxin, which inhibits the growth of various bacterial strains (Kodaira, 1962; Kaijiang and Roberts, 1986; Dumas et al., 1995; Jegorov et al., 1995). Fungi such as Beauveria bassiana, Paecilomyces fumosoroseus and Fusarium moniliforme also produce cyclodepsipeptides, including beauvericin and the enniatin complex (Kucera and Sansinakova 1968; West and Buggs, 1968; Hamil et al., 1969; Richard et al., 1995; Logrieco et al., 1996). Studies have reported that the fungus Nomuraea rileyi produces metabolites active against insects (Ignoffo et al., 1976; Wasti and Hartmann, 1978; Kucera and Sansinakova 1968; Mohamed and Nelson, 1984; Ye et al., 1993), including some metabolites showing toxic activity against the larvae of Heliothis zea, H. virescens (Mohamed and Nelson, 1984) and Bombyx mori (Ye et al., 1993).

Defoliating caterpillars are important pests of Brazilian soybeans and beans, and among these Anticarsia gemmatalis (the soybean caterpillar) being the most important (Costa, 1958; Redaelli, 1960; Bertels and Ferreira, 1973; Corseuil et al., 1974). This pest can be controlled using chemical insecticides and biological agents such as virus, bacteria and fungi (Ignoffo et al., 1976) and it has been shown that N. rileyi can be used for the biological control of A. gemmatalis when applied during the first stages of larval development (Ignoffo et al., 1976). The aim of the present work was to isolate and purify a peptide produced by N. riley and to study the effect of this peptide on insect mortality.

MATERIALS AND METHODS

Fungal Strain: Nomuraea rileyi(Farlow) Samson strain SA-86101 (Biological Control Division, Biotechnology Institute, University of Caxias do Sul, Grande do Sul, Brazil) was isolated from Anticarsia gemmatalis (Lep.: Noctuidae).

Media: Saboraud, maltose, yeast-extract (SMY) broth (4% maltose, 1% peptone and 0.5% yeast extract, pH 6.0) was autoclaved, cooled and inoculated as shown in Fig. 1.


Peptide production and isolation:N. rileyi strain SA-86101 was grown in SMY medium as described by Ignoffo et al. (1976) and used to produce a conidial suspension containing about 2.4x109 conidia/ml, which was inoculated into vessels containing 12 liters of SMY broth. Incubation was at 26 ± 1oC for 12 days with constant aeration, after which the culture medium was filtered to remove mycelia and passed through several successive Whatman Nº 1 filter papers. NR-tox1 was separated in a liquid/liquid extractor using filtered culture medium : dichlorometane (10:1) and concentrated in a rotary evaporator at 35oC, the final fractions containing about 135mg/l of fungal metabolites and other residual material. Metabolites were isolated using G60 silica Gel (0.063 to 0.20 mm) column chromatography with a solvent (chloroform :methanol : ethyl acetate, 18:1:1) ratio of 100:1 (w/w), 5ml fractions being collected and analyzed using thin layer chromatography (TLC). Similar fractions were mixed and analyzed by infrared spectroscopy (IR). After successive purification, crystals were obtained, which were maintained at 20oC until needed for chemical and biological analysis.

Bioassays: The peptide described above was diluted in distilled water to produce solutions containing 1.0, 0.2, 0.1, 0.01, 0.001 and 0.0001 mg/ml and sprayed onto soybean leaves at application rates of 100, 20, 10, 1 and 0.1 mg/cm2. The leaves were dried in trays for 30 min in a laminar flow chamber, each tray containing a different peptide concentration and application rate. For the bioassay 50, A. gemmatalis 3rd instar larva were placed in each tray and incubated at ~25oC under a 12h photoperiod. The mortality rates were observed each day. Control experiments were conducted as above except that leaves were sprayed with distilled water instead of protein solution. Three replicates were made for each treatment and the larvae were kept until they either died or pupated. Data were analyzed using Probit analysis (Sokal, 1958; Finney, 1971) and LC50 values calculated.

RESULTS AND DISCUSSION

Chemical analysis showed that the N. rileyi metabolite (NR-tox1) active against A. gemmatalis had a positive ninhidrin reaction, a melting point of 244.4oC and was soluble in water, ethanol and methanol. Infrared and ultraviolet spectral data (Figures 2 and 3) coincided with those expected for an oligopeptide.



Table 1 and Fig. 4 show the insecticidal activity of the isolated peptide on A. gemmatalis 3rd instar larvae. The highest mortality rates varied between 82.66% and 80.00 for the 1.0, 0.2, and 0.1 mg/ml peptide concentrations, with no statistically significant differences between them (Table 1), although they were all significantly different to the other concentrations (0.01, 0.001 and 0.0001 mg/ml) which gave significantly lower mortality rates. The 40% mortality rate given by the 0.01 mg/ml concentration was significantly different to the 0.001 and 0.0001 mg/ml concentrations, but significantly lower than the mortality rate given by the higher concentrations.


Mortality data submitted to linear regression resulted in the equation: Y = 6,81176 + 1,01382 * LOGx which gave the estimated LC50 value as 0.0163 mg/ml with a 95% confidence interval of 0.0100 to 0.0266 (p=5%) These results for the N. riley peptide agree with those reported by YE, et al., (1993) who demonstrated that purified peptides extracted from fungal culture media showed insecticidal activity against B. mori, Prodenra litura and Pieris rapae larvae. Mohamed and Nelson (1984) reported that N. riley crude extracts caused 42, 48 and 72 h mortality rates of 23.3, 44.5 and 68.9% for H. virensens larvae and 28.7; 53.8 and 78.3% for H. zea larvae.

The insecticidal activity of the N. rileyi peptide described in this paper demonstrated similar toxicity to that of other metabolites produced by entomopathogenic fungi such as B. bassiana, which produced beauvericin and the enniatin complex, both of which were effective against Calliphora erythrocephala and Aedes aeggypti larvae (Grove and Pople, 1980). In a study on the effect of beauvericin on Spodoptera exigua larvae, Boucias et al. (1994) showed that 86% of treated larvae exhibited tetanic paralysis within 6 hours of treatment and mortality rates of about 26% after 24h, 45% after 36h and 73% after 72h. The entomopathogenic fungus M. anisopliae also produced toxins, known as destruxins, which have been shown to be toxic to the larvae of A. aegypti, Galleria mellonella, Delia antiqua, Cetonia aurata, Oryctes rhinoceros, Choristoneura fumiferana, Schistocerca gregaria, Periplaneta americana and Aedes albopictus (Crisan, 1971; Poprawski et al., 1995; Fargues, et al., 1985; Fargues et al., 1986; Huxman et al., 1989; Kopecky et al., 1995; James et al., 1995; Brousseau et al., 1996).

Although the antimicrobial and insecticidal activity of N. riley metabolites is well documented, the chemical structure and mechanism of action of these metabolites are as yet undefined. Such metabolites are important potential instruments to encourage the use of entomopathogenic fungi in pest control, promoting an integrated approach to pest control. These metabolites could be used as insecticides or antibiotics, with some having the potential for large-scale production for insect control in the field. However, perhaps the most important use of insecticidal metabolites of entomopatho-genic fungi is in the investigation of insect mortality genes and the use of this knowledge to produce more virulent fungal strains. Insertion into plants of genes for insecticidal metabolites, generating plants less susceptible to insect attack, is an important area for future research.

ACKNOWLEDGMENTS

This work formed part of an M.Sc. Thesis presented at the Biotechnology Institute, University of Caxias do Sul (UCS), Brazil. We would like to thank the UCS, the Paraná Federal Center of Technological Education and CAPES for financial support.

RESUMO

Este trabalho objetivou determinar a CL50 de um peptídeo produzido pelo fungo N. rileyi, para larvas de 3º ínstar de A. gemmatalis. O peptídeo foi produzido através de fermentação aerada, em meio SMY, sob condições controladas por 12 dias. O metabólito foi purificado, utilizando-se de sistemas de cromatografia. Os cristais obtidos foram então armazenados a baixa temperatura para posterior análise da atividade biológica. Os experimentos foram conduzidos em 3 repetições, com tratamentos que consistiram de soluções com 0,0001; 0,001; 0,01; 0,1; 0,2 e 1,0 mg/mL. Através de análise de regressão chegou-se a equação da reta Y = 6,81176 + 1,01382 * LOGx e um valor para CL50 de 0,0163 mg/mL.

Received: September 04, 2000;

Revised: April 16, 2001;

Accepted: October 17, 2001.

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  • *
    Author for correspondence
  • Publication Dates

    • Publication in this collection
      23 Oct 2002
    • Date of issue
      Sept 2002

    History

    • Reviewed
      16 Apr 2001
    • Received
      04 Sept 2000
    • Accepted
      17 Oct 2001
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