[Note]
Identification of an Entomopathogenic Fungus, Beauveria bassiana
SFB-205 toxic to the Green Peach Aphid, Myzus persicae
Jae Su Kim, Jong Yul Roh1, Jae Young Choi2,Sang Chul Shin3, Mun Jang Jeon4, and Yeon Ho Je1,*
AgroLife Research Institute (ARI), Dongbu HiTek Co. Ltd., Daejon 305-708, Republic of Korea
1Department of Agricultural Biotechnology, College of Agriculture & Life Science, Seoul National University, Seoul 151-742, Republic of Korea
2Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-742, Korea
3Division of Forest Insect Pests and Diseases, Korea Forest Research Institute, Seoul 130-712, Korea
4Department 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 sec- ond instars of Myzus persicae nymphs in the glass- house 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 M.
persicae at 4 days after application in the glasshouse.
The attachment of SFB-205 conidia on the surface of
M. persicae nymphs, and germination and penetration were observed using scanning electron microscopy.
SFB-205 was identified as Beauveria bassiana species through the comparison of 5.8 s rRNA genes. There were 24 polymorphisms between SFB-205 and the pre- viously reported isolate, B. bassiana ATCC74040 using six kinds of primer combinations in amplified frag- ment length polymorphism (AFLP) analysis. The B.
bassiana SFB-205 might be used as a practical biolog- ical control agent for the green peach aphid, M. per-
sicae in the field.
Key words: Beauveria bassiana, Entomopathogenic fungi, Aphid, Myzus persicae
Introduction
Entomopathogenic fungi are widely available as biolog-
ical control agents for controlling insect pests in agricul- ture 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., Paecil- omyces 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 fun- gal pathogens, consists of a thin outer epicuticle contain- ing lipids and proteins, and a thick procuticle consisting of chitin and proteins (Samson et al., 1988). Entomopatho- genic fungi utilize a combination of mechanical and enzy- matic mechanisms, and the secretion of insecticidal enzymes such as chitinases, proteases and lipases is known to play a major role in penetrating the insect cuti- cle (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 ento- mopathogenic fungus, Verticillium lecanii was examined as a microbial control agent (Ashouri et al., 2004).
In this study, to find an effective candidate for control- ling the green peach aphid, M. persicae, several ento-
Industrial Entomology
*To whom the correspondence addressed
Department of Agricultural Biotechnology, College of Agri- culture & Life Sciences, Seoul National University, Seoul 151- 742, Korea. Tel: +82-2-880-4706; Fax: +82-2-873-2319; E- mail: [email protected]
ined 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., SFV- 202 and SFV-206) were isolated from soil in Korea. The isolates were stored in 20% glycerol at −80oC (Forma- 86C, 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±1oC 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±1oC for 3 days.
Scanning electron microscopy
For scanning electron microscopy, conidia, blastospores and nymphs were collected and initially fixed in 2.5%
glutaraldehyde and 2% paraformaldehydein the phos- phate buffer (0.1 M; pH 7.2) for 2 h at 24oC. Subse- quently, they were fixed in a fresh fixative solution (for 24 h) at 4oC. Specimens were rinsed in three changes of buffer,10 ml each and then post-fixed in 1% OsO4 for 2 h.
The specimenswere washed twice in distilled water for 30 min, dehydrated ina graded ethanol series. Specimens were coated with gold-palladium (20:80) in a Polaron E5100 sputter coating instrument. Photographs were taken with a JEOLJSM-35 SEM at 2.0 KV.
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 com- plete medium, were incubated for 2 days under agitation at 27oC. The resulting mycelium was harvested by filtra- tion. To extract genomic DNA, 1 g of mycelium was
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 65oC 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 previ- ously 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 Anal- ysis 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 pro- gram as 1 for presence and 0 for absence to generate a
Primer Oligonucleotide sequence (5’−3’) Preselective primers
EcoRI+ 0 MseI+ 0 Selective primers EcoRI-AGC EcoRI-ACT MseI-CAC
MseI-CAG MseI-CAT MseI-CCA
MseI-CCG MseI-CCT
GAC TGC GTA CCA ATT C GAT GAG TCC TGA GTA A
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
Fig. 1.Insecticidal activities of several entomopathogenic fungi against second instars of M. persicae nymphs in the glasshouse. Conidia suspensions (1×106 conidia/ml) were sp- rayed 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).
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 main- tained in a chamber at 25oC, 40~50% RH and a 16:8 h L:D cycle. Red hot peppers (variety: Booja) were culti- vated in an automatically controlled glasshouse. Ten-foli- ate 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 treat- ment was compared using a one way analysis of variance (ANOVA) test at 0.05 of p-value.
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 high- est insecticidal activity about 32.7% efficacy in about 30% RH at 4 days after application. In laboratory con-
dition, hyphal growth of SFB-205 from the surface of the aphids was observed at 2 days after incubation, and yel- low color of conidia were formed at 5 days after incu- bation at 27±1oC (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).
Fig. 2.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. 3.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).
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-
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 struc- ture (Fig. 4).
Aphids such as M. persicae and A. gossypii are major harmful pests in Korea. Up to date, the previously com- mercialized chemical pesticides have been showing resis- tances 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 candidatesand have general morphological char- acteristics 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 expres- sion 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 SFB- 205 for commercialization was verified because it reduced the population density of aphids in initial times of appli- cation. 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 sup- ported by the Research and Development Project for Dongbu HiTek Co. Ltd., and the Brain Korea 21 project.
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