Int. J. Indust. Entomol. Vol. 17, No. 2, 2008, pp. 211~215
[Note]
International Journal of
Industrial Entomology
Identification of an Entomopathogenic Fungus, SFB-205 toxic to the Green Peach Aphid,
Beauveria bassiana
Myzus persicae
Jae Su Kim, Jong Yul Roh , Jae Young Choi , Sang Chul Shin , Mun Jang Jeon , and Yeon Ho Je 1
2
3
4
*
1,
AgroLife Research Institute (ARI), Dongbu HiTek Co. Ltd., Daejon 305-708, Republic of Korea 1 Department of Agricultural Biotechnology, College of Agriculture & Life Science, Seoul National University, Seoul 151-742, Republic of Korea 2 Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-742, Korea 3 Division of Forest Insect Pests and Diseases, Korea Forest Research Institute, Seoul 130-712, Korea 4 Department of Forest Resources, College of Life and Environmental Science, Daegu University, Kyeongsan 712-714, Korea (Received 20 October 2008; Accepted 05 December 2008)
To select entomopathogenic fungi controlling aphids effectively, several isolates were screened against second instars of nymphs in the glasshouse using conidia suspension at 1.0 ×105 conidia/ml. Among these isolates, SFB-205 conidia showed the highest insecticidal activity about 32.7% efficacy to at 4 days after application in the glasshouse. The attachment of SFB-205 conidia on the surface of nymphs, and germination and penetration were observed using scanning electron microscopy. SFB-205 was identified as species through the comparison of 5.8 s rRNA genes. There were 24 polymorphisms between SFB-205 and the previously reported isolate, ATCC74040 using six kinds of primer combinations in amplified fragment length polymorphism (AFLP) analysis. The SFB-205 might be used as a practical biological control agent for the green peach aphid, in the field. Myzus persicae
M.
persicae
M. persicae
Beauveria bassiana
B. bassiana
B.
bassiana
M. per-
sicae
Key words: Beauveria bassiana, Entomopathogenic fungi, Aphid, Myzus persicae
Introduction Entomopathogenic fungi are widely available as biolog*To whom the correspondence addressed Department of Agricultural Biotechnology, College of Agriculture & Life Sciences, Seoul National University, Seoul 151742, Korea. Tel: +82-2-880-4706; Fax: +82-2-873-2319; Email:
[email protected]
ical control agents for controlling insect pests in agriculture and forestry (Burges and Hall, 1982; Butt et al., 2001; Parker et al., 2002; Roberts and Hajek, 1992). Several species such as Beauveria sp., Metarhizium sp., Paecilomyces sp. and Verticillium sp. have been registered in EPA and commercialized (Charnley, 2003; Clarkson and Charnley, 1996; Shah and Pell, 2003). However, fungal insecticides constitute a very small percentage of the total insecticide market, although they are the only practical control agent against target insects that feed on crops by sucking (Hajek and St. Leger, 1994; St. Leger et al., 1996). Currently most entomopathogenic fungal products have been made by conidia (Butt et al., 2001; Kassa et al., 2008). The insect cuticle which is the first barrier to fungal pathogens, consists of a thin outer epicuticle containing lipids and proteins, and a thick procuticle consisting of chitin and proteins (Samson et al., 1988). Entomopathogenic fungi utilize a combination of mechanical and enzymatic mechanisms, and the secretion of insecticidal enzymes such as chitinases, proteases and lipases is known to play a major role in penetrating the insect cuticle (Charnley and St. Leger, 1991). The green peach aphid, Myzus persicae, is a notorious pest due to its ability to transmit plant viruses and to attack plants in the field. It readily infests vegetables and ornamental plants grown in greenhouses and feeds on hundreds of host plants in over 40 plant families like cucumber, potato, pepper, tomato, and so on (Jansson and Smilowitz, 1986). For controlling this pest, an entomopathogenic fungus, Verticillium lecanii was examined as a microbial control agent (Ashouri et al., 2004). In this study, to find an effective candidate for controlling the green peach aphid, M. persicae, several ento-
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mopathogenic fungi (including one Beauveria sp., one Paecilomyces sp., and two Verticillium sp.) were examined and the fungus which has the highest activity was identified through ribosomal RNA sequence analysis and AFLP (amplified fragment length polymorphism).
Materials and Methods Fungal strains and maintenance
Four fungal strains used in this study (Paecilomyces sp., SFP-198; Beauveria sp., SFB-205; Verticillium sp., SFV202 and SFV-206) were isolated from soil in Korea. The isolates were stored in 20% glycerol at − 80 C (Forma86C, Termo Electron Co.) as a conidium form. The conidia were propagated on Sabouraud dextrose agar medium supplemented with yeast extract at 0.5% (SDYA). They were incubated at 27 ± 1 C for 14 ~15 days. The conidia were harvested by scraping the plates with autoclaved brush and then dried to a moisture content of 5% using a vacuum freeze-drier (Jenkins et al., 1998). Liquid culture media were based on Sabouraud dextrose broth medium supplemented with yeast extract at 0.5% (SDYB) and incubated with shaking at 150 rpm at 27 ±1 C for 3 days. o
Primers for amplified fragment length polymorphism (AFLP) analysis Primer Oligonucleotide sequence (5’ − 3’) Preselective primers EcoRI + 0 GAC TGC GTA CCA ATT C MseI + 0 GAT GAG TCC TGA GTA A Table 1.
Selective primers EcoRI-AGC EcoRI-ACT MseI-CAC MseI-CAG MseI-CAT MseI-CCA MseI-CCG MseI-CCT
GAC TGC GTA CCA ATT CAGC GAC TGC GTA CCA ATT CACT GAT GAG TCC TGA GTA ACAC GAT GAG TCC TGA GTA ACAG GAT GAG TCC TGA GTA ACAT GAT GAG TCC TGA GTA ACCA GAT GAG TCC TGA GTA ACCG GAT GAG TCC TGA GTA ACCT
o
o
Scanning electron microscopy
For scanning electron microscopy, conidia, blastospores and nymphs were collected and initially fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in the phosphate buffer (0.1 M; pH 7.2) for 2 h at 24 C. Subsequently, they were fixed in a fresh fixative solution (for 24 h) at 4 C. Specimens were rinsed in three changes of buffer, 10 ml each and then post-fixed in 1% OsO for 2 h. The specimens were washed twice in distilled water for 30 min, dehydrated in a graded ethanol series. Specimens were coated with gold-palladium (20 : 80) in a Polaron E5100 sputter coating instrument. Photographs were taken with a JEOL JSM-35 SEM at 2.0 KV. o
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Identification of
Beauveria
sp.
The 5.8 s ribosomal RNA of SFB-205 was amplified, sequenced and analyzed through comparing with NCBI (National Center for Biotechnology Information) gene bank data.
AFLP analysis
Conidial suspensions of SFB-205 and the previously reported isolate, Beauveria bassiana ATCC74040 in complete medium, were incubated for 2 days under agitation at 27 C. The resulting mycelium was harvested by filtration. To extract genomic DNA, 1 g of mycelium was o
Insecticidal activities of several entomopathogenic fungi against second instars of M. persicae nymphs in the glasshouse. Conidia suspensions (1×106 conidia/ml) were sprayed on nymphs in red hot peppers using 0.1% Tween 80 as a spreader. Control indicates 0.1% Tween 20 treatment. No. of living nymphs was counted at 4 days after treatment. Different letters above error bars indicate significant difference (P < 0.05, One way ANOVA test). Fig. 1.
ground to a fine powder in liquid nitrogen and incubated in 800 ml DNA extraction buffer (100 mM Tris-HCl (pH 8.0), 25 mM EDTA, 1% SDS, 25 mM NaCl) at 65 C for 20 min. The genomic DNA was purified by phenol extraction. The genomic DNA was resuspended in TE buffer (10 mM Tris, 1 mM EDTA pH 8.0). AFLP fingerprinting was done according to a previously reported protocol using primers fluorescently labelled with Cy5 (De Muro et al., 2003; Devi et al., 2006). The used primers in this study were listed (Table 1). The final PCR products were loaded on a ReproGel Long Read (Amersham Biosciences GmbH, Freiburg, Germany) and analyzed on an ALFexpress II DNA Analysis System (Amersham Biosciences). As a size marker, ALFexpress Sizer 50.500 (Amersham Biosciences) was used in the gels (Fig. 4). Polymorphic fragments (bands) were scored by the program as 1 for presence and 0 for absence to generate a o
Beauveria bassiana toxic to M. persicae
213
Conidial infection of SFB-205 against second instars of M. persicae nymphs by observing with optical microscope (A) and scanning electron microscope (B). Panel A: Infected M. persicae nymphs were examined at 2 (b) and 5 (c) days after treatment comparing non-treated control (a). Panel B: Attachments of conidia on cuticles (a) and germination (b) were observed. Fig. 2.
binary matrix (Fig. 4). After that, tree was constructed using unweighted-pair group method using arithmetic average (UPGMA) clustering (Treecon, version 1.3 b) (Van de Peer and de Wachter, 1994).
Insecticidal activity against
Myzus persicae
Second instars of M. persicae nymphs were obtained from the insectary of Dongbu HiTeK Co. Ltd. They were maintained in a chamber at 25 C, 40 ~ 50% RH and a 16 : 8 h L : D cycle. Red hot peppers (variety: Booja) were cultivated in an automatically controlled glasshouse. Ten-foliate peppers were inoculated with second instars of M. persicae nymphs and the initial density per plant was examined before treatment of fungal culture products. They were sprayed one time using portable hand sprayer (Gardena 864, Germany). Bioassays were performed in triplicate. Insecticidal activity was evaluated among 3 to 10 days after application. The analysis of variance was conducted on normalized data. The mean of each treatment was compared using a one way analysis of variance (ANOVA) test at 0.05 of p-value.
dition, hyphal growth of SFB-205 from the surface of the aphids was observed at 2 days after incubation, and yellow color of conidia were formed at 5 days after incubation at 27 ± 1 C (Fig. 2A). The attachment of SFB-205 conidia on the surface of M. persicae and germination and penetration were observed using scanning electron microscopy (Fig. 2B). o
o
Results and Discussion To screen entomopathogenic fungi controlling aphids effectively, several isolates were assayed against second instars of M. persicae nymphs in the glasshouse (Fig. 1). Among these isolates, SFB-205 conidia showed the highest insecticidal activity about 32.7% efficacy in about 30% RH at 4 days after application. In laboratory con-
Morphology of SFB-205 conidia (A) and blastospores (B). SFB-205 was cultured in SDBY media at 27oC for 3 days. They were observed using optical microscope (a) and scanning electron microscope (b). Fig. 3.
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SFB-205 showed the white color hyphae and produced the yellow color conidia in SDYA medium. SFB-205 conidia were round shaped (Fig. 3A) and it is identical to the general morphology of Beauveria sp. conidia (Burges and Hall, 1982). The blastospores of SFB-205 were long and elliptical and two or three times bigger than the conidia in size (Fig. 3B). SFB-205 was identified as B. bassiana species through genetic analysis of 5.8 s rRNA genes by comparing sequences to the NCBI gene bank data (data not shown). To examine the genetic difference between SFB-205 and the previously commercialized strain, B. bassiana ACTC74040, analysis of amplified fragment length poly-
morphism (AFLP) was performed. There were 24 polymorphisms between two isolates using six kinds of primer combinations in AFLP analysis. A cluster analysis on the basis of the obtained AFLP markers demonstrated that SFB-205 and ATCC74040 were different in genetic structure (Fig. 4). Aphids such as M. persicae and A. gossypii are major harmful pests in Korea. Up to date, the previously commercialized chemical pesticides have been showing resistances on aphids and residual side effect on environment (Roberts and Hajek, 1992; Vandenberg, 1996). Therefore another control agents need to be developed on the view of mode of action. Low side effect and another mode of action are considered deeply. In this study, B. bassiana SFB-205 was picked out as an aphid-control agent. It showed superior insecticidal activity against M. persicae among candidates and have general morphological characteristics as B. bassiana. It was confirmed that SFB-205 was significantly different from the previously reported and commercialized strain, ATCC74040. Control of aphids should be achieved in early times of application because the life cycle of aphids is very short and growth rate is fast (Vandenberg, 1996). Slow expression of insecticidal activity is not practical strategy in case of aphids control. Hence, selection of active ingredients for mass production and formulation was focused on early times of efficacy against M. persicae in the glasshouse. In conclusion, practical control of aphids in the glasshouse could be achieved by using SFB-205. A potential of SFB205 for commercialization was verified because it reduced the population density of aphids in initial times of application. Next study will be focused on the selection of active ingredient for industrialization.
Acknowledgement This study was supported by General and Integrated Research for Pine Wilt Disease of Korea Forest Research Institute, Republic of Korea. This work was also supported by the Research and Development Project for Dongbu HiTek Co. Ltd., and the Brain Korea 21 project.
References
Analysis of amplified fragment length polymorphism (AFLP) between SFB-205 and B. bassiana ACTC74040. Fig. 4.
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