Int. J. Indust. Entomol. Vol. 25, No. 2, 2012, pp. 195~203
International Journal of
Industrial Entomology
Purification and Identification of Paenibacillus sp., Isolated from Diseased
Larvae of Allomyrina dichotoma (Linnaeus, 1771) (Coleoptera: Scarabaeidae) in Insect Farms
Tae Hwa Kang1,2, Sang Hoon Han1,2, Hang Yeon Weon1, Young Bo Lee1, Namjung Kim1, Sung Hee Nam1,
and Hae Chul Park1,*
1
National Academy of Agricultural Science, Suwon, Gyeonggi, Korea
The first two authors contributed equally
2
(Received 20 November 2012; Accepted 2 January 2013)
In reared populations of Allomyrina dichotoma, commercial insects, the skin of last instar larvae was
changed softer with opaque white, and infested grubs
eventually died. To clarify the cause of the symptom,
we collected the larvae of A. dichotoma from five farms
and examined their intestinal bacterial florae using
pyrosequencing technique. From those results, a member of Paenibacillus was found only in the larvae showing the symptom of disease. Through PCR analysis
using a Paenibacillus specific primer set, we obtained
the partial 16S rRNA gene sequence and confirmed
the microbe as Paenibacillus sp. For clear identification, a whole guts was extracted from each larva showing the sign of the disease and incubated at 70oC for
15 min to isolate spore forming bacteria. After then,
each content of guts was cultured on MYPGPNAL agar
medium(12.5 µg/ml of nalidixic acid) at 30oC. The 16S
rRNA gene sequence analysis for the isolated bacteria
showed that they were closely related to P. rigui(97.9%
similarity), to P. chinjuensis(96.1% similarity), and to
P. soli(95.3% similarity). Additional tests including
API test and cellular fatty acid composition analysis
were performed, but the strain couldn't be identified
at species level, suggesting it may represent novel species of the genus Paenibacillus.
Introduction
Today, there are various kind of hobby as the quality of
life as well as economic development is improved. Keeping insects such as dynastid beetles, stag beetles, crickets,
and ants is becoming more popular. In Japan, of pet
insects, the size of stag beetles market approached to over
two or three hundred billion KRW, and the insect pet market size is estimated as forty billion KRW in Korea(Choi
et al., 2011). Allomyrina dichotoma (Linnaeus, 1771)(Fig.
1) has a large portion in Korean insect pet market, and it
is being sold at various store such as insect specialty
Key words: Allomyrina dichotoma, Milky disease,
Pyrosequencing, 16S rRNA, Paenibacillus sp.
*To whom the correspondence addressed
Department of Agricultural Biology, National Academy of
Agricultural Science, Suwon, Gyeonggi, Korea.
Tel: +82-31-290-8535; E-mail: culent@korea.kr
http://dx.doi.org/10.7852/ijie.2012.25.2.195
Fig. 1. Male Allomyrina dichotoma. The species has a large
portion in Korean insect pet market.
196
Tae Hwa Kang et al.
reached at between 2 × 109 and 5 × 109(Zhang et
al.,1997).
The object of this study is to purify and identify the
caused bacteria of the disease appearing in Korean A.
dichotoma larvae. For this, firstly, the intestinal bacterial
florae of the healthy and the diseased larvae were surveyed. On the basis of the bacterial florae, we tried to
search different points between the healthy and the diseased larva and, lastly, to purify and to identify the
microbe appearing only in the diseased larvae.
Materials and Methods
Fig. 2. Symptoms of the bacterial disease in Allomyrina
dichotoma. (1) healthy larva; (2) rough epidermis; (3) the body
color changes to milky white, and the body becomes soften; (4)
finally, the larva dies.
stores and hypermarkets.
In 2010, however, a pandemic disease spreads at several
insect farms, and in 2012, a case of mass mortality of Japanese rhinoceros beetles showed through the whole country of Korea. The symptoms of the disease mainly emerge
at the third instar-end larvae of the beetle, and the larva
dies before or after making pupa room. The symptoms of
the disease were as follows(Fig. 2): (1) rough epidermis;
(2) the body color changes from shiny white to milky
white, and the body becomes soften; (3) finally, the larva
dies. In general, when the larva of the beetle was bred at
an insect farm, about 60 individuals with fermented sawdust were put into 120L plastic box. In this case, if a diseased larva is found in the rearing box, almost all
individuals die of a disease which is similar to a bacterial
infection.
Several bacterial diseases were reported in insects,
such as American Foulbrood caused by Paenibacillus
larvae in European honeybee(Apis mellifera Linnaeus,
1758), Sotto disease by Bacillus thuringiensis in silkworm(Bombix mori (Linnaeus, 1758)), milky disease by
Paenibacillus popilliae and P. lentimorbus in Scarabaeid
beetles, and amber disease by Serratia entomophila and
S. proteamaculans in Scarabaed beetles(Dutky, 1940;
Grkovic et al., 1995; Han et al., 2008; Ibrahim et al.,
2010; Zhang et al., 1997). Most of the diseases were
caused by Bacillus and Paenibacillus, and the only one
case, amber disease by Serratia spp. Specially, milky
disease appearing in Scarabaeid beetles is very similar to
that of Korean A. dichotoma in the symptoms, which the
body color of A. dichotoma larvae was changed to milky
white and died when the number of spore of P. popilliae
Genomic DNA extraction from the gut of A. dichotoma
larvae
For DNA extraction, A. dichotoma larva was washed as
follows: the larva was soaked in 70% ethanol for 1 min
and then in 0.8% HOCl solution for 1 min. And solution
residues were removed by sinking in DW for 1 min. The
larva was dissected in a sterilized petri dish. The gut
extracted from the larva was mashed by a mortar.
Genomic DNA was extracted from 200 mg of the
mashed gut material with QiaAmp DNA mini
kit(Qiagen, Germany) according to the manufacturer’s
instruction.
Pyrosequencing on the gut microbiota of A. dichotoma
larvae
The intestinal bacterial florae was surveyed in three
healthy(two individuals from insect farm in Yuseong of
Daejeon, and one from Hwingseong of Gangwondo) and
three diseased larvae(each individual from Hwingseong of
Gangwondo, Namyangju of Gyeonggido, and Yeongam
of Jeollanamdo) by using a pyrosequencing technique.
For pyrosequencing, bacterial 16S rRNA genes were
amplified
using
the
primers
V1-9F(5-X-ACGAGTTTGATCMTGGCTCAG-3) and V3-541R(5-XAC-WTTACCGCGGCTGCT GG-3)(Chun et al., 2010)
where X denotes a 7–11 nucleotide long barcode followed
by a common linker AC. PCR amplification was performed using the following PCR protocol: initial denaturation(5 min at 94°C), 30 cycles at 94°C for 30 s, 55°C
for 30 s, and 72°C for 60 s and the final extension at 72°C
for 5 m. The PCR amplification curves were constructed
by sampling the PCR product at 5 cycle intervals and
quantifying it using a Qubit 2.0 fluorometer and Qubit
dsDNA BR assay kit(Invitrogen, USA). The PCR products obtained at 20 cycles and 30 cycles were selected.
The PCR products were gel-purified with the QIAquick
Gel extraction kit(Qiagen, Germany) and subjected to
pyrosequencing, which was performed by National Instru-
Purification and identification of Paenibacillus sp. from A. dichotoma
mentation Center for Environmental Management(Seoul,
Korea) using a 454 GS FLX Titanium Sequencing System(Roche, Germany), according to the manufacturer’s
instructions. Pyrosequencing data were analyzed using the
Mothur software package(version 1.23.1)(Schloss et al.,
2009). Pre-filtered flowgrams of the pyrosequencing
reads were clustered using the PyroNoise algorithm(Quince et al., 2011), and chimeric sequences were
removed using UCHIME(Edgar et al., 2011). UCHIME
was performed in De novo mode.
Polymerase chain reaction using Paenibacillus specific
primer
Paenibacillus sp. which may be one of causative pathogens of A. dichotoma larva was detected from the gut of
the larvae from Yuseong of Daejeon. Thus, with Paenibacillus specific primer set, PAEN515F(Shida et al.,
1997) and RNA1484R(Petterson et al., 1999), PCR detection was conducted for the larvae collected from insect
farms of six regions, Hwingseong of Gangwondo, Namyangju of Gyeonggido, Yeongdong of Chungcheongnamdo, Yuseong of Daejeon, Yeongam of Jeollanamdo,
and Hwasun of Jeollanamdo. Amplification conditions for
both reactions were denaturation at 94oC for 5 min followed by 25 cycles of denaturation at 94oC for 1 min,
annealing at 58oC for 1.5 min and extension at 72oC for
1.5 min followed by a final extension at 72oC for 5 min.
Purification of the target bacteria
The gut was extracted from the larva which Paenibacillus
sp. was detected by Paenibacillus specific primer. The gut
was grinded with 10 ml of Phosphate Buffered Saline
(PBS, pH 7.4) and then 500 µl of the specimen was inoculated in 30 ml MYPGP media(10 g of Mueller Hinton,
15 g of Yeast extract, 3 g of K2HPO4, 2 g of Glucose,and
1 g of Sodium pyruvate for 1 Liter DW). After over-night
incubation(30ºC, 180 rpm), the bacteria culture was incubated in water bath at 70oC for 15 min. And then 100 µl of
bacterial culture was inoculated into 30 ml of fresh
MYPGP media containing Nalidixic acid(12.5 ug/ml).
After over-night incubation(30ºC, 180 rpm), the bacterial
culture was diluted tenfold up to 10-9 and then each of
50 µl from 10-7, 10-8, and 10-9 dilutants was spread on
MYPGPNAL agar media. After over-night incubation(30ºC), several single colonies were picked and spread
on fresh MYPGPNAL agar media.
Identification of the target bacterium
Identification of Paenibacillus sp. was carried out by
using four methods following as 16S rRNA gene
sequence analysis, gram staining, API test, and cellular
fatty acid composition. 16S rRNA gene sequence was
197
amplified by using universal primer set, 27F and
1492R(amplicon size about 1,400 bp). For more clear
results, we designed the internal primer set PAEN793R(5'AGGCGGAGTG CTTACTGTGT-3') and PAEN540F(5'CGCACTGAAAACTGGATGAC-3') using Primer3 software(Rozen and Skaletsky, 2000). 16S rRNA gene was
amplified by two parts, such as the former region amplified by 27F-PAEN793R primer set and the latter region by
PAEN540F-1492R primer set. Amplification conditions
for both reactions were denaturation at 94oC for 5 min followed by 35 cycles of denaturation at 94oC for 30 min,
annealing at 58oC for 30 min and extension at 72oC for 30
min followed by a final extension at 72oC for 5 min. DNA
sequencing of the amplicons were performed by Macrogen(Seoul, Korea). Determined sequences were aligned
and analyzed under molecular evolutionary genetic analysis software, MEGA 5(Tamura et al., 2011). API test and
cellular fatty acid composition analyses were performed
by Korean Culture Center of Microorganisms(Seoul,
Korea). API 50CHB kit was used for API test. Resulting
data on each analysis were compared with apiweb(http://
apiweb.biomerieux.com/ servlet/identify/) for API test
and Microbial Identification System(MIDI, 1999) for cellular fatty acid composition.
Results and Discussion
Detection of Paenibacillus species from the infected A.
dichotoma larva
Pyrosequencing is commonly used for the analyses on the
bacterial florae of the various environment such as soil,
swamp, and freshwater as well as the intestinal tube and
the feces of organism, or for the comparison of the bacterial florae from two or three different environmental
region(Boissière et al., 2012; Kim et al., 2010; Matcher et
al., 2011; Nacke et al., 2011; Shi et al., 2010; Wong et al.,
2011). In insects, several works on the gut bacterial florae
were performed(Boissière et al., 2012; Shi et al., 2010;
Wong et al., 2011). Considering above cases, we tried to
compare the gut bacterial florae of three diseased larvae of
A. dichotoma from four regions including Yuseong of
Daejeon which the larval disease has broken out with
those of three healthy larvae. In the results, the larval gut
bacteria were classified by total 28 taxa, of which three
phylum, Firmicutes, Proteobacteria and Cyanobacteria,
showed high species diversity(Fig. 3).
On the basis of the results, the bacterial florae between
the healthy and the diseased larvae were compared with
each other using principal coordinates analysis and
seven clustering methods such as barycurtis, morisitahorn, thetayc, jclass, jest, sorclass, sorest, and the results
198
Tae Hwa Kang et al.
Fig. 3. Phylum and genus composition of the gut from Allomyrina dichotoma larvae(Star mark: diseased larva). Larval gut bacteria florae were composed as total 28 taxa, of which Firmicutes,
Proteobacteria and Cyanobacteria showed high species diversity.
Fig. 4. Clustering analysis based on seven algorithm on bacterial florae of larval gut collected from insect farms of four
region(Star mark: diseased larva).
Fig. 5. Principal coordinate analysis on bacterial florae of larval gut collected from insect farms of four region(Star mark:
diseased larva).
on each clustering method were integrated by jacknife
sample clustering method. In principal coordinate analysis and clustering method, however, the gut bacterial
florae between the healthy and the diseased larvae did
not showed clear different points(Figs. 4 and 5). In the
larvae only from Yuseong of Daejeon, Paenibacillus sp.
was detected as very small density of 0.1%. Several species of Paenibacillus were known as the famous disease
causative pathogen in insects(Gardener, 2004). P. larvae
is a causative bacteria of American Foulbrood in European honeybee(Genersch, 2008; Han et al., 2008), and in
Scarabaeid beetles, milky disease was caused by P. popilliae and P. lentimorbus, which are used for biological
control agents against on Japanese beetle(Popillia japonica Newman, 1841) in USA(Dutky, 1940; Pettersson et
al., 1999). In the point of view, Paenibacillus sp.
detected from the A. dichotomus larvae might be a causative pathogen of the larval disease. Thus, we conducted
PCR detection on A. dichotoma larvae from insect farm
of six region using Paenibacillus specific primer set,
PAEN515F(Shida et al, 1997) and RNA1484R(Petterson
et al, 1999). In the result, Paenibacillus sp. was detected
only in the larvae from Yuseong of Daejeon(Fig. 6). The
PCR result suggested strong possibility that Paenibacillus sp. might be a causative baciteria. Therefore, we tried
to purify Paenibacillus sp. from the gut of A. dichotoma
larvae for more detailed works, such as the identification
of the bacteria, and the infection test on A. dichotoma
larvae.
Purification and identification of Paenibacillus sp. from A. dichotoma
199
Fig. 7. Gram staining on purified Paenibacillus sp. The bacteria is rod type and appeare in violet color.
Table 1. NCBI accession number of 16S rRNA gene
sequences of 18 Paenibacillus species
No
Fig. 6. Detection of Paenibacillus sp. using Paenibacillus specific primer set on Allomyrina dichotoma larvae from insect
farms of six regions (Yuseong: A-1, A-2, B-10, B-11, B-12, B13, B-14; Yeongdong: A-3, A-4, A-5, A-6, A-7; Yeongam: A8, A-9, A-10, A-11, A-12; Hwasun: A-13, A-14, A-15, A-16;
Namyangju: B-1, B-2, B-3, B-4, B-5; Hwingseong: B-6, B-7,
B-8, B-9). Paenibacillus sp. was detected only in the larvae
from Yuseong of Daejeon.
Purification and identification of Paenibacillus species
from the infected A. dichotoma larva
For purifying Paenibacillus sp., the gut materials of A.
dichotoma larvae were cultured in MYPGPNAL media
containing 0.1% nalidixic acid(12mg/1ml), which inhibit
a subunit of DNA gyrase and induce formation of relaxation complex analogue(Emmerson and Jones, 2003).
And then, the conical tube was heated as 70oC in water
bath. It is known that Paenibacillus bacteria formed
endospores in poor environment, such as nutritionally
unfavorable local conditions via dormancy (Nicholson et
al., 2000; Nicholson, 2002). Therefore, except only
endospore forming bacteria, most of all bacteria were sterilized by heat. But, it was known that Paenibacillus bacteria have resistance on the antibiotics(Han et al., 2008).
The cultured bacteria were purified by culturing MYPGPNAL agar media. Purified bacteria were examined under a
light microscopy after Gram staining. Paenibacillus are
rod type and Gram positive bacteria, which showed violet
at Gram stain(Barenfanger et al., 2001). In the result of
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Scientific Name
Paenibacillus
azoreducens
P. chinjuensis
P. chitinolyticus
P. daejeonensis
P. dendritiformis
P. ehimensis
P. elgii
P. gansuensis
P. ginsengisoli
P. koreensis
P. larvae
P. lentimorbus
P. naphthalenovorans
P. riguii
P. soli
P. thiaminolyticus
P. validus
P. popilliae
Bacillus subtilis
Strain No.
CM1
WN9
HSCC596T
AP-20
CIP105967T
KCTC3748
SD17
B518
Gsoil1638
YC300
DSM7030
ATCC14707
PR-N1
WPCB173
DCY03
IFO15656
JCM9077
ATCC14706
DSM10
NCBI
Accession Remarks
No.
AJ272249
AF164345
AB045100
AF290916
AY359885
AY116665
AY090110
AY839866
AB245382
In-group
AF130254
AY530294
AB073199
AF353681
EU939688
DQ309072
AB073197
AB073203
AB073198
AJ276351 Out-group
Gram staining, the purified bacteria were rod type and
Gram positive(Fig. 7).
The identification is based on 16S rRNA gene sequence
as well as API test and cellular fatty acid composition. In
16S rRNA gene sequence, we analyzed adding 16 rDNA
200
Tae Hwa Kang et al.
Fig. 8. Neighbour joining tree of 16 s rRNA gene of Paenibacillus spp. Paenibacillus sp. is the closest to P. rigui showing as
2.0% branch length.
sequences of 18 Paenibacillus species from NCBI in our
data matrix(Table 1). In neighbor joining tree, Paenibacillus sp. purified from the gut of A. dichotoma larvae
showed as about 2.0% branch length from P. rigui
detected from Woopo Swamp(Changnyeong, Gyeongsangnamdo, Korea; Baik et al., 2011) and about 6.0%
from P. popilliae, the causative bacteria of milky disease
in Scarabaeid beetles(Fig. 8). In sequence divergence, the
bacteria showed as 2.1% divergence from P. rigui, 3.9%
from P. chinjuensis, and 4.7% from P. soli(Table 2).
Therefore, the bacterium seems to be the closest to P.
rigui. In the identification using the homology of 16S
rRNA gene sequence, the identification criterion by
Stackebrandt and Goebel(1994) was generally accepted,
who referred that 97% similarity is the cut-off for species
identification. After that, however, Drancourt et al.(2000)
suggested that 99% cut-off criterion is used for species
identification, and 97% similarity might be used only for
genus level identification. In recent, Janda and
Abbott(2007) suggested that it could be identified as same
species when 16S rDNA sequence showed as at least 99%
similarity, and the ideal score for same species is 99.5%
similarity. Therefore, it might be difficult that Paenibacillus sp. purified from A. dichotoma larvae is treated as a
same species using only 97.9% 16S rDNA sequence similarity.
Physiological trait test for Paenibacillus sp. purified
from A. dichotoma larvae was carried out using API
50CHB kit(bioMérieux). In the result, the bacteria was
positive for 25 of 50 conditions(L-Arabinose, D-Xylose, β
Methyl-xyloside, Galactose, D-Glucose, D-Fructose,
Rhamnose, Mannitol, α Methyl-D-mannoside, α MethylD-glucoside, N Acetyl glucosamine, Amygdaline, Arbutine, Esculine, Salicine, Cellobiose, Maltose, Lactose,
Melibiose, Saccharose, Trehalose, Melezitose, D-Raffinose, β Gentiobiose, D-Turanose; Table 3). The similarity
test on this result was performed under apiweb. In the
result, we could not find any data to be consistent with our
data, and the closest matched bacterium was Paenibacillus mucerans which showed as 62.1% similarity. Therefore, the result by API test was unsuitable for the
identification of Paenibacillus sp.
In cellular fatty acid composition, it was confirmed that
total of 15 fatty acids were distributed in cell wall. Among
them, 15:0 ANTEISO fatty acid showed the highest ratio
as 64.65%, and the next was 16:0 ISO as 8.58%, 15:0 ISO
as 7.50%, 14:0 ISO as 4.91%, and 16:0 as 4.36%(Table
4). In the result comparing our data with MIDI data, the
data showed similarity of >90% were absent.
Considering the results on above three analyses, 16S
rDNA sequence, API test, and cellular fatty acid composition, only 16S rDNA sequence data provided taxonomic
position of Paenibacillus sp. which is closely related to P.
rigui showing 97.9% sequence similarity. But the similarity was consistent with the species identification criterion suggested by Stackebrandt and Goebel(1994),
although the score is insufficient to recent criterion(Drancourt et al., 2000; Janda and Abbott, 2007). For more
clear identification, we are conducting DNA-DNA
hybridization for comparison with closely related species,
P. rigui and P. soli. If these closely related species showed
less than 70% similarity in the result of DNA-DNA
hybridization(Wayne et al., 1987), we could identify
Paenibacillus sp. as a new species.
In this study, we focused on the purification and identification of Paenibacillus sp. from the gut of the diseased
A. dichotoma larvae. Through further study, we will try to
clearly identify the bacteria and to test pathogenicity of
the bacteria on A. dichotoma larvae. Although this bacterium was purified from the diseased larvae, it is difficult
to judge it as the causative pathogen. Generally, milky disease caused by P. popilliae was expressed by increasing
number of endospore(Zhang et al., 1997). In survey on the
related references, however, we found that milky disease
was caused by several members of Paenibacillus and
Bacillus, such as P. popilliae, P. lentimorbus, P. lentimorbus var. maryland, P. lentimorbus var. australis, Bacillus
euloomarahae, and B. fribourgensis, which are known as
endospore forming bacteria, or including endospore sim-
P. riguii_WPCB173
P. chinjuensis_WN9
P. soli_DCY03
P. validus_JCM9077
P. ehimensis_KCTC3748
2
3
4
5
6
P. popilliae_ATCC14706
P. koreensis_YC300
9
10
0.056 0.047 0.041 0.042 0.038
5
6
8
9
0.063 0.054 0.048 0.050 0.045 0.008 0.050 0.018 0.072
0.062 0.053 0.062 0.063 0.065 0.064 0.074 0.066
0.060 0.047 0.042 0.044 0.041 0.010 0.044
7
10
P. gansuensis_B518
13
12
0.065 0.052 0.058 0.065 0.066 0.061 0.069 0.058 0.066 0.066 0.066 0.067
0.064 0.058 0.062 0.066 0.065 0.068 0.072 0.067 0.009 0.074 0.010
11
13
P. larvae_DSM7030
P. chitinolyticus_HSCC596T
P. ginsengisoli_Gsoil1638
P. daejeonensis_AP-20
P. azoreducens_CM1
Bacillus subtilis_DSM10
15
16
17
18
19
20
14
15
16
17
18
19
0.144 0.131 0.135 0.130 0.134 0.130 0.139 0.127 0.125 0.135 0.122 0.127 0.137 0.119 0.133 0.129 0.127 0.130 0.125
0.080 0.070 0.066 0.069 0.070 0.067 0.076 0.063 0.073 0.075 0.072 0.072 0.067 0.070 0.059 0.055 0.053 0.063
0.089 0.073 0.077 0.075 0.082 0.066 0.084 0.065 0.067 0.072 0.066 0.069 0.059 0.066 0.081 0.059 0.073
0.069 0.065 0.068 0.077 0.077 0.062 0.083 0.063 0.062 0.071 0.063 0.063 0.066 0.063 0.070 0.063
0.067 0.054 0.049 0.051 0.047 0.054 0.056 0.053 0.066 0.062 0.064 0.067 0.041 0.063 0.056
0.068 0.063 0.060 0.064 0.055 0.063 0.056 0.066 0.068 0.070 0.068 0.068 0.066 0.070
P. dendritiformis_CIP105967T 0.065 0.058 0.062 0.066 0.067 0.065 0.075 0.067 0.008 0.073 0.006 0.013 0.067
P. lentimorbus_ATCC14707
12
14
4
0.051 0.044 0.038 0.041
0.047 0.034 0.044
3
P. thiaminolyticus_IFO15656 0.063 0.054 0.062 0.062 0.065 0.064 0.074 0.066 0.004 0.072
P. elgii_SD17
8
11
2
0.039 0.037
0.021
1
P. naphthalenovorans_PR-N1 0.056 0.051 0.047 0.044 0.029 0.043
Paenibacillus sp._Nal8
1
7
Scientific Name_Strain Number
No
Table 2. 16s rRNA gene sequence divergence of Paenibacillus spp
20
Purification and identification of Paenibacillus sp. from A. dichotoma
201
202
Tae Hwa Kang et al.
Table 3. The result of API 50CHB kit (+: positive reaction; −: negative reaction)
No
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
API
CHB
control
Glycerol
Ertythritol
D-Arabinose
L-Arabinose
+
Ribose
D-Xylose
+
L-Xylose
Adonitol
β Methyl-xyloside
+
Galactose
+
D-Glucose
+
D-Fructose
+
D-Mannose
L-sorbose
Rhamnose
+
Dulcitol
Inositol
Mannitol
+
Sorbitol
α Methyl-D-mannoside +
α Methyl-D-glucoside +
NAcetylglucosamine +
Amygdaline
+
Arbutine
+
API Kit
No
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
API
CHB
Esculine
+
Salicine
+
Cellobiose
+
Maltose
+
Lactose
+
Melibiose
+
Saccharose
+
Trehalose
+
Inuline
Melezitose
+
D-Raffinose
+
Amidon
Glycogene
Xylitol
β Gentiobiose +
D-Turanose
+
D-Lyxose
D-Tagatose
D-Fucose
L-Fucose
D-Arabitol
L-Arabitol
Gluconate
2 ceto-gluconate 5 ceto-gluconate API Kit
Table 4. The result of cellular fatty acid composition
No
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Name
10 : 0
12 : 0
13 : 0 ISO
13 : 0 ANTEISO
14 : 0 ISO
14 : 0
15 : 0 ISO
15 : 0 ANTEISO
15 : 0
16 : 0 ISO
16 : 0
17 : 0 ISO
17 : 0 ANTEISO
17 : 1 w6c
17 : 0
%
0.13
0.15
0.13
0.21
4.91
1.77
7.50
64.65
2.73
8.58
4.36
1.12
3.45
0.18
0.14
ilar materials(Steinkraus and Tachiro, 1967). In this point
of view, it was considered that Paenibacillus sp. forming
endospore from A. dichotoma larvae might be a disease
causative bacteria.
Acknowledgement
This work was supported by the Rural Development of
Administration, Republic of Korea (Grant No. PJ907197)
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