1Department of Agricultural Biology, Chungbuk National University, Cheongju, 363-763, Korea (sdwoo@cbnu.ac.kr)

Pine wilt is the most important disease of pine trees in Korea, Japan and China. The pathogen causing this disease, the pinewood nematode (Bursaphelenchus xylophylus), is transmitted vectored by adults of some cerambycid beetle species and the Japanese pine sawyer, Monochamus alternatus, is the major vector species in Korea. Although chemical insecticides have been used to kill vector insect and thus prevent transmission of the pathogen, the efficacy is not good. In Japan, to control this insect, an entomopathogenic fungus was studied and developed as an insecticide. This is thought to be the convenient and effective method to control M. alternatus. Recently, there are several reports about the pinewood nematode is vectored by also the pine sawyer, M. saltuarius, in Korea. The objective of this study, therefore, was to isolate and identify entomopathogenic fungi from to control it. We collected M. saltuarius cadaver the cadaver of M. saltuarius and then screened several fungi colonies. The pathogenicity of each fungus was tested using oak longicorn beetle, Moechotypa diphysis, as substitutive insect. M. diphysis is also serious pest to various trees in forest. As the result, only one of them showed high pathogenicity against M. diphysis. Selected fungus was identified by microscopic examination and DNA analysis. Pathogenicity was also evaluated to M. saltuarius

1. lsolation of fungi Fungi were isolated directly from cadavers supporting fungal sporulation, using a semi-selective medium consisting of potato dextrose agar (PDA; DifcoTM, USA) containing PENICILLIN-STREPTOMYCIN SOLUTION (Sigma, USA). 2. Culture condition Fungi were grown on potato dextrose agar (PDA; DifcoTM, USA) in the dark at 25 and store at 4 until ℃ ℃use. 3. Insects Adults M. saltuarius and Moechotypa diphysis were field-collected and used to bioassay.

Fig. 1. Selection of fungus from cadaver of M. saltuarius. Growth of the selected fungus on PDA medium at 25 for 3days (A) and 14days (B).℃

Several fungal colonies were isolated from the cadaver and their pathogenicities were assayed against to Moechotypa diphysis for the selection of entomopathogenic fungus. As a result, a kind of fungus was obtained and named as MsW1. Colonies of MsW1 was usually slow growing, downy, at first white but later often becoming yellow to pinkish.

Fig. 2. Phase contrast (A) and scanning electron (B) micrographs of MsW1. Conidia and mycelia were mounted from plates and examined under X400 on a Nikon microscope. For the SEM, samples were mounted on stubs, coated with gold and observed in a Carl Zeiss LEO-1530 SEM.

MsW1 was characterized by the sympodial development of single-celled conidia (ameroconidia) on a geniculate or zig-zag rachis.

108 107 106 105 104

Days o

n 1

00%

mort

ality

conidia/ml

Fig. 3. Pathogenicity of MsW1 against to Moechotypa diphysis adults.The pathogenicity was evaluated as till times that mortality reaches to 100%. Conidia of MsW1 obtained from 14-day-old PDA plate were suspended in 0.02% Tween-80 solution. Spores were prepared from 1 × 108 conidia/ml to 1 × 104 conidia/ml by diirect counting in a haemocytometer. M. diphysis were inoculated by dipping for 10~15 sec into the spore suspension and were maintained in a plant culture chamber at 25 .℃

Mortality of M. diphysis showed 100% at all concentarions and the pathogenicity increased with increasing conidial concentrations.

Fig. 3. Moechotypa diphysis adults infected with MsW1 at 5 days (A) and 10 days (B) after death.The pathogenicity of MsW1 was also confirmed against M. saltuarius adults, but it needs more in detail and repeatedly.

P15’-AAGCTTCGACATGGTCTG–3’

P35‘-GGAGGTGGTGAGGTTCTGTT-3’

0 524

P55‘-AGGAGAGAGCTCGACGGTCA-3’

333

Fig. 5. Location and sequence of PCR primers for differentiation of the Beauveria spp.

Identification of enotomopathogenic fungi was conducted by previous reported primers, P1 (forward), P3 (backward), and P5 (backward) (Dwayne et al. 1996). PCR with the P1-P3 primer set could discriminate isolates of Beauveria spp from other entomopathogenic fungi with a PCR product of about 500bps. The P1-P5 primer set could identify B. bassiana strain specifically from Beauveria spp. with a approximately 330bp within P1-P3 PCR product.

M 1 2

600bp

500bp

400bp

300bp

200bp

P1-P3

P1-P5

Fig. 6. Agarose gel electrophoresis of P1-P3 and P1-P5 PCR products from MsW1. Genomic DNA was extracted using an I-genomic BYF DNA extraction mini Kit (Intron Co., Korea). The reaction parameters for P1-P3 primer set were as follows; initial denaturation for 3 min at 94 ℃, followed by 35 cycles of 94℃ for 30sec, 53 ℃ for 30sec, and 72 ℃ for 45sec, and a final 10-min extension at 72 ℃. The reaction parameters for P1-P5 primer set were same with those of P1-P3 except the annealing temperature of 50 ℃. Lane M, 100 bp size marker; lane 1, P1-P3 PCR ; lane 2, P1-P5 PCR.

The result of PCR with P1-P3 primer set indicated that MsW1 is belonged to Beauveria spp. To discriminate the species of MsW1 more precisely, PCR was performed with the P1-P5 primer set. As the result, a specific PCR product of about 330bp was amplified. This suggests that MsW1 is B. bassiana.

Entomopathogenic fungus, MsW1, was isolated from M. saltuarius and was identified with B. bassiana.

The pathogenicity of MsW1 against Moechotypa diphysis, as substitutive insect, showed 100% mortality at the tested all concentrations of conidia.

We are investigating the potential of MsW1 as a biocontrol agent for M. saltuarius.

Dwayne et. al. 1996. J. Invertebr. Pathol. 67:289-299.

Mitsuaki Shimazu. 2004. J. Appl. Entomol. 39(3):485-490

The cabbage armyworm, Mamestra brassicae, is an important

insect pest of vegetables and ornamental plants in Asia and

Europe. Several nucleopolyhedroviruses (NPVs) have been isolated

from M. brassicae and considered useful biological-control agents

for M. brassicae. However, Korean strain of M. brassicae NPV

(MbNPV) is poorly characterized. For the practical use of NPV as

a control agent of noctuid pests, it is necessary to identify and

characterize the virus strain in advance. The objective of our study

was the pathogenic, morphologic and genetic characterization of a

MbNPV isolate (MbNPV-K1) derived from a diseased larva of M.

brassicae

found in Korea.

Fig. 4. Specific PCR amplification of the

polyhedrin gene region.

Lane M, 100bp size marker; lane 1,

MbNPV-K1; lane 2, Mamestrin.

1Department of agricultural Biology, Chungbuk National University, Cheongju, Korea, sdwoo@cbnu.ac.kr, 2School of Agricultural Biotechnology, Seoul National

University, Seoul, Korea, and 3 College of Natural Resources and Life Science, Dong-A University, Busan, Korea

Approximately 0.96Kb DNA fragment of the polyhedrin gene

including its flanking regions was successfully amplified. The

amplified PCR product was cloned into a pGemT PCR cloning

vector and sequenced. The sequencing results showed that the

PCR product was a fragment of corresponding to the previous

reported polyhedrin gene.

Proteins from the polyhedra were subjected to a 12% SDS-PAGE

and stained with Coomassie brilliant blue. Lane 1, polyhedrin of

MbNPV-K1; lane 2, polyhedrin of Mamestrin; lane 3, polyhedrin

of Autographa californica NPV; lane 4, polyhedrin of Spodoptera

exigua NPV; lane 5, polyhedrin of Bombyx mori NPV; lane M,

molecular mass markers. Molecular weight of polyhedrin of

MbNPV-K1 was about 31kDa and similar that of Mamestrin.

The 958 nt long DNA sequences encompassing the entire coding

region and 5’, 3’ non-coding flanking sequences were determined.

Nucleotide sequence analysis indicated the presence of an open

reading frame of 741 nucleotides which could encode 247 amino

acids with a predicted molecular mass of 31 kDa. The nucleotide

and amino acid sequences within the coding region of MbNPV-K1

polyhedrin shared 99.0% similarity with the polyhedrin gene from

previous reported MbNPVs (table 1).

Table 1. Comparison of nucleotide and amino acid sequence

identities (%) of the polyhedrin gene of MbNPV-K1 with those of

other MbNPVs

The morphology of polyhedra was observed using SEM and TEM.

The lethal dose and time were determined using M. brassicae larvae

by droplet-feeding bioassay. The polyhedrin gene was amplified by

PCR and sequenced. All results were compared with those of the

previous commercialized MbNPV (Mamestrin) as a control.

(A) (B)

Fig. 1. Scanning electron micrographs of the polyhedra of MbNPV-

K1 (A) and Mamestrin (B).

(A) (B)

Fig. 2. Transmission electron micrographs of the polyhedra of

MbNPV-K1 (A) and Mamestrin (B).

The morphology of polyhedra and the structure of polyhedrin gene

did not show any significant difference between two viruses. But

the size of polyhedra of MbNPV-K1 was slightly bigger than the

control. The value of lethal concentration (LC50) of MbNPV-K1

against 3rd instar larvae of M. brassicae was 15 times higher than

the control. The lethal time was also shorter than the control

The higher pathogenicity of MbNPV-K1 provides the possibility of

the development of effective viral insecticide using this for the

control of M. brassicae..

M 1 2 3 4 5

- 30kDa

45kDa -34kDa -

26.5kDa -19.5kDa -

- 31kDa

1kbp -

M1

958bp

Fig. 3. SDS-PAGE analysis of the polyhedral proteins.

MbNPV-K1GenBank Accession No.

AB198073 M20927 E08532

Nucleotides 741bp 99% 99% 99%

Amino acids 247aa 99% 99% 99%

Virus LC50 (PIBs/larva)

MbNPV-K1 3.915 × 103

Mamestrin 6.002 × 104

Table 2. Values of lethal concentration(LC50) of MbNPV-K1 and

Mamestrin against 3rd instar larvae of M. brassicae.

VirusConcentrations

(PIBs/larva)LT50(95%CL) LT95(95%CL)

MbNPV-K1

1.0 × 107 4.43 (4.16-4.18) 6.98 (6.46-7.75)

1.0 × 106 5.46 (5.18-5.74) 8.54 (7.90-9.49)

1.0 × 105 6.44 (5.95-6.94) 9.24 (8.36-11.30)

1.0 × 104 6.17 (5.59-6.61) 9.31 (8.23-11.28)

Mamestrin

1.0 × 107 4.16 (3.92-4.40) 6.18 (5.71-6.92)

1.0 × 106 5.14 (4.88-5.39) 7.83 (7.30-8.59)

1.0 × 105 6.44 (5.95-6.94) 9.24 (8.30-11.30)

1.0 × 104 8.67 (7.46-10.08) 15.57 (11.15-21.84)

5’-ATG CGC TGG TAT AAT TTA GAA GAT -3’

5’-CATG TCA ATA AAA TAC TTA CGT ATC A-3’

MbPol-F primer

MbPol-R primer

Polyhedrin Gene

Table 3. Values of lethal times of MbNPV-K1 and Mamestrin

against 3rd instar larvae of M. brassicae.

Doyle et. al. 1990. App. and Enviro. Microbio. 59:2704-2710

Hughes et. al. 1997. J. Gen. Virol. 78:1801-1805

Kwon et. al. 2005. Korean J. Appl. Entomol. 44:225-230

Mukawa and Goto. 2006. J. Gen. Virol. 87:1491-1500

Rovesti et. al. 2000. J. Invertebr. Pathol. 75:2-8

The polyhedra of MbNPV-K1 were larger (1.5 ~ 2.3um across) than

those of Mamestrin (1.3~1.9um across). This suggest that MbNPV-

K1 would possess more virions than Mamestrin. Bar markers

represent 1 ㎛ .

Each virion contains multiple nucleocapsids. The size and number of

contained virions between two viruses did not show any significant

difference. Bar markers represent 0.2 ㎛ .

107

106

105

104

p. i. days

No

. of

dea

d

larv

ae

107

106

105

104

No

. of

dea

d

larv

ae

Fig. 5. Pathogenicity of

MbNPV-K1(A) and

Mamestrin(B) against 3rd

instar larvae of M.

brassicae.

Larvae of M. brassicae were inoculated by a modified droplet-

feeding method. Experiments were replicated three times with 25

larvae per treatment. Larvae were observed daily for

mortality until 20 days after inoculation.