International Journal of Systematic and Evolutionary Microbiology (2015), 65, 1860 – 1865
DOI 10.1099/ijs.0.000186
Chryseobacterium shandongense sp. nov.,
isolated from soil
Fan Yang,† Hong-ming Liu,† Rong Zhang, Ding-bin Chen, Xiang Wang,
Shun-peng Li and Qing Hong
Correspondence
Qing Hong
[email protected]
Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life
Sciences, Nanjing Agricultural University, Nanjing, Jiangsu 210095, PR China
YF-3T is a Gram-stain-negative, non-motile, non-spore-forming, yellow – orange, rod-shaped
bacterium. Optimal growth conditions were at 30 8C, pH 7.0 and 1 % (w/v) NaCl. Phylogenetic
analysis, on the basis of the 16S rRNA gene sequence, showed that strain YF-3T was closely
related to the strains Chryseobacterium hispalense AG13T and Chryseobacterium taiwanense
Soil-3-27T with 98.71 % and 96.93 % sequence similarity, respectively. Strain YF-3T contained
MK-6 as the main menaquinone and had a polyamine pattern with sym-homospermidine as the
major component. Its major polar lipid was phosphatidylethanolamine. The dominant fatty acids
of strain YF-3T were iso-C15 : 0, iso-C17 : 0 3-OH, summed feature 9 (comprising iso-C17 : 1v9c
and/or C16 : 0 10-methyl) and summed feature 3 (comprising C16 : 1v7c and/or C16 : 1v6c). The
DNA G þ C content of strain YF-3T was 37 mol%. The DNA –DNA relatedness levels between
strain YF-3T and the most closely related strains, C. hispalense AG13T and C. taiwanense
Soil-3-27T, were 31.7 ^ 2.1 % and 28.4 ^ 5.4 %, respectively. Based on these results, a novel
species named Chryseobacterium shandongense sp. nov. is proposed. The type strain is YF-3T
( ¼ CCTCC AB 2014060T ¼ JCM 30154T).
The genus Chryseobacterium represents a genera with one
of the fastest growing number of species (Herzog et al.,
2008), which are found in a wide variety of environments.
The genus Chryseobacterium was first described by Vandamme et al. (1994) and at the time of writing it contains
88 species with validly published names (http://www.
bacterio.net/). In 1994 – 2000, the genus only comprised
six species: Chryseobacterium balustinum, Chryseobacterium
gleum, Chryseobacterium indologenes, Chryseobacterium
indoltheticum, Chryseobacterium meningosepticum and
Chryseobacterium scophthalmum. However, the number of
species increased by 45 and 33 between 2001 and 2010,
and 2011 and 2014, respectively. These newly discovered
members of the genus Chryseobacterium are distributed
in a variety of environments, such as roots (Park et al.,
2006), a lake (Joung & Joh, 2011), clinical samples (Vaneechoutte et al., 2007), soil (Li & Zhu, 2012), sludge (Pires
et al., 2010), raw milk (Hantsis-Zacharov & Halpern,
2007), the midgut of insects (Kampfer et al., 2010a), food
products (including raw cow’s milk, fish, poultry and
lactic acid beverages) (Hantsis-Zacharov et al., 2008) and
†These authors contributed equally to this work.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA
gene sequence of strain YF-3T is KJ644318.
Four supplementary figures and one supplementary table are available
with the online Supplementary Material.
1860
human clinical sources (Yassin et al., 2010). In this study,
strain YF-3T was isolated from soil from Qingdao, Shandong province, China (358 359 –378 099 N 1198 309–
1218 009 E).
During the isolation of strains, 10 g of soil was added to a
flask containing 100 ml trypticase soy broth (TSB; 17.0 g
pancreatic digest of casein, 3.0 g soy peptone, 2.5 g dextrose, 5.0 g NaCl, 2.5 g K2HPO4; pH 7.0 ^ 0.2) and incubated at 30 8C at 150 r.p.m. for 2 days. Trypticase soy agar
(TSA; per litre distilled water: 17.0 g pancreatic digest of
casein, 3.0 g soy peptone, 2.5 g dextrose, 5.0 g NaCl, 2.5 g
K2HPO4, 15.0 g agar; pH 7.0 ^ 0.2) plates were spread
with 0.1 ml diluted soil suspension and incubated at
30 8C for 2 days. A yellow 2 orange colony was selected,
purified and then the isolate was cultivated at 30 8C on
the same medium and preserved in 20 % (v/v) glycerol at
2 80 8C.
Strain YF-3T was cultivated on TSA plates at 30 8C for 2 days.
The presence of flexirubin-type pigments was investigated by
noting whether a colour shift occurred when the colony was
flooded with 20 %(w/v) KOH (Fautz & Reichenbach, 1980).
Gram staining was performed by the modified method of
Gerhardt et al. (1994). Cell motility was determined according the procedure described by Smibert & Krieg (1994).
A colony of strain YF-3T was picked from the last quadrant
streak after it was grown on TSA at 30 8C for 24 h. Then
the cell morphology and dimensions were determined by
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Chryseobacterium shandongense sp. nov.
transmission electron microscopy (H-7650; Hitachi) (Fig.
S1, available in the online Supplementary Material).
Growth at different temperatures (4, 20, 25, 30, 37 and
42 8C) and pH 4 –10 (at 1 pH unit intervals) was assessed
in TSB, the pH range was buffered with citrate/phosphate
buffer or Tris/HCl buffer (Breznak & Costilow, 1994). Salt
tolerance was investigated on TSB supplemented with 0 –
9 % (w/v) NaCl (at 1 % intervals). The OD600 value was
determined after 3 days of incubation in order to evaluate
the growth of strain YF-3T. In addition, growth on Luria –
Bertani (LB; 10.0 g peptone, 5.0 g yeast extract, 10.0 g NaCl,
15.0 g agar; pH 7.0 ^ 0.2) agar, R2A (0.5 g proteose peptone,
0.5 g yeast extrct, 0.5 g casamino acids, 0.5 g glucose, 0.5 g
soluble starch, 0.3 g sodium pyruvate, 0.3 g K2HPO4, 0.05 g
MgSO4.7H2O, 15.0 g agar; pH 7.0 ^ 0.2) agar, nutrient
agar (NA; 5.0 g peptone, 3.0 g beef extract, 5.0 g NaCl,
15.0 g agar; pH 6.8 ^ 0.2), cetrimide agar (CA; 20.0 g pancreatic digest of gelatin, 1.4 g MgCl, 10.0 g K2SO4, 0.3 g cetrimide, 15.0 g agar; pH 7.2 ^ 0.2), Simmons’ citrate (SC; 5.0 g
NaCl, 2.0 g sodium citrate, 1.0 g NH4H2PO4, 1.0 g K2HPO4,
0.2 g MgSO4, 0.08 g bromothymol blue, 15.0 g agar; pH
6.9 ^ 0.2) agar and MacConkey agar (17.0 g peptone, 3.0 g
proteose tryptone, 10.0 g lactose, 1.5 g bile salts, 5.0 g NaCl,
0.001 g crystal violet, 0.03 g neutral red, 13.5 g agar; pH
7.1 ^ 0.2) was also evaluated.
Catalase activity was determined by bubble production
with 3 % (v/v) H2O2. Oxidase activity was assayed using
filter-paper discs (grade 388; Sartorius) impregnated with
a 1 % (w/v) solution of N,N,N9,N9-tetramethyl-p-phenylenediamine (Sigma-Aldrich). A positive result was indicated
by the development of a blue – purple colour after applying
biomass to the filter paper. Antibiotic susceptibility tests
were performed with the disc (Hangzhou Tianhe Microbial
Reagent) diffusion method on TSA plates incubated at
30 8C for 2 days. Strains were considered sensitive
when the diameter of the inhibition zone was $ 10 mm
(Jorgensen & Ferraro, 2009). The hydrolysis of DNA, cellulose, starch and casein were investigated as described by
Smibert & Krieg (1994). Basic biochemical, enzyme activity
and carbon source tests were performed using the API
20NE, API 20E and API ZYM systems (bioMerieux), and
GN2 MicroPlates (Biolog), according to the manufacturers’
instructions.
For the analysis of whole-cell fatty acids, strain YF-3T and
the reference strains, Chryseobacterium hispalense AG13T
and Chryseobacterium taiwanense Soil-3-27T, were grown
on TSA at 30 8C for 24 h. The biomass, which was always
harvested from the same sector (the last quadrant streak)
was freeze-dried, then the fatty acid methyl esters were
extracted according to the standard procedure of the
Microbial Identification System (MIDI) (Sasser, 1990).
Extracts were analysed using a Hewlett Packard model
6890 gas chromatograph equipped with a flame-ionization
detector (Kampfer & Kroppenstedt, 1996) and a 5 %
phenyl-methyl-silicone capillary column. The extraction
of the respiratory quinones was carried out from freezedried cell material according to the method of
http://ijs.sgmjournals.org
Collins et al. (1977) and determined by HPLC (Tamaoka
et al., 1983). Polar lipids were extracted from 100 mg of
freeze-dried cell material using a chloroform/methanol/
0.3 % (w/v) aqueous NaCl mixture 1 : 2 : 0.8 (by vol.) (Tindall et al., 2007). Then polar lipids were recovered into the
chloroform phase by adjusting the chloroform/methanol/
0.3 % aqueous NaCl (w/v) mixture to a ratio of 1 : 1 : 0.9
(by vol.). Polar lipids were separated by two-dimensional
silica gel TLC. Total lipid material was detected using
molybdophosphoric acid and specific functional groups
were detected using spray reagents specific for defined
functional groups (Tindall et al., 2007) (DSMZ service)
(Fig. S2). Cells of strain YF-3T used for polyamine analysis
were grown on TSB, harvested at the late exponential
growth phase and lyophilized. Extraction of polyamines
was performed as described by Busse & Auling (1988)
and analysis was conducted using the HPLC equipment
described by Stolz et al. (2007).
The genomic DNA of strain YF-3T was extracted and purified according to the method described by Sambrook &
Russell, (2001) and the DNA G þ C content was determined by reversed-phase HPLC (Tamaoka & Komagata,
1984) using Escherichia coli K-12 as a standard. DNA –
DNA hybridizations were performed at 50 8C, with photobiotin-labelled probes in microplate wells, as described by
Ezaki et al. (1989). A bioassay plate reader (HTS 7000;
Perkin Elmer) was used to measure the fluorescence, and
reciprocal experiments were performed on each pair of
strains investigated. The 16S rRNA gene of strain YF-3T
was amplified with the bacterial universal primers 27F
and 1492R (Lane, 1991). The PCR products were purified
using an AxyPrep PCR Purification kit (AxyGen) and were
cloned into a pMD 19-T Vector. The purified plasmid
DNA was sequenced with an automated sequencer
(model 3730; Applied Biosystems). Pairwise sequence
similarity was calculated with the known sequences using
the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/;
Kim et al., 2012). Phylogenetic analysis was performed
by using MEGA version 5.0 (Tamura et al., 2011) after
multiple alignments of data using CLUSTAL_X (Thompson
et al., 1997). Distances were determined through distance
options based on Kimura’s two-parameter system
(Kimura, 1980). Unrooted trees were reconstructed
via neighbour-joining (Saitou & Nei, 1987) (Fig. 1),
maximum-parsimony (Fitch, 1971) (Fig. S3) and maximum-likelihood (Felsenstein, 1981) (Fig. S4) methods.
Confidence values for the branches of phylogenetic trees
were calculated according to bootstrap analyses (based
on 1000 resamplings) (Felsenstein, 1985).
YF-3T was Gram-stain-negative, rod-shaped (0.5 –0.7 mm
in width, 2.1 – 2.3 mm in length), non-spore-forming and
non-motile. Colonies of strain YF-3T on TSA plates were
yellow –orange, circular, convex, smooth, translucent and
shiny, and the colonies were not visible as single entities
after prolonged incubations. The optimal growth conditions for YF-3T were 30 8C, 1 % (w/v) NaCl and pH 7.0
in TSB. Good growth also occurred on LB agar and R2A
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1861
F. Yang and others
C. gwangjuense THG-A18T (JN196134)
85*
C. geocarposphaerae 91A-561T (HG738132)
*
0.01
C. defluvii B2T (AJ309324)
99*
*
‘C. yeoncheonense’ DCY67 (JX141782)
C. aahli T68T (JX287893)
*
‘C. massiliae’ 90B (AF531766)
*
C. gambrini 5-1St1aT (AM232810)
C. daecheongense CPW 406T (AJ457206)
*
C. wanjuense R2A10-2T (DQ256729)
*
*
C. taiwanense BCRC 17412T (DQ318789)
C. gregarium P 461/12T (AM773820)
*
C. hagamense RHA2-9T (DQ673672)
C. camelliae THG C4-1T (JX843771)
71*
Chryseobacterium shandongense YF-3T (KJ644318)
98*
C. hispalense AG13T (EU336941)
C. taeanense PHA3-4T (AY883416)
96*
*
C. taichungense CC-TWGS1-8T (AJ843132)
C. vietnamense GIMN1.005T (HM212415)
100
C. gleum ATCC 35910T (ACKQ01000057)
C. arthrosphaerae CC-VM-7T (FN398101)
86*
C. indologenes LMG 8337T (AM232813)
C. nakagawai NCTC 13529T (JX100822)
98*
C. lactis NCTC 11390T (JX100821)
C. viscerum 687B-08T (FR871426)
Ornithobacterium rhinotracheale LMG 9086T (L19156)
Fig. 1. Neighbour-joining phylogenetic tree according to 16S rRNA gene sequences indicating the relationship of strain YF3T to closely related species of the genus Chryseobacterium. Asterisks indicate branches that were also recovered using the
maximum-parsimony and maximum-likelihood algorithms. Bootstrap values of more than 70 % (based on 1000 replications)
are shown at branching points. The 16S rRNA gene sequence of Ornithobacterium rhinotracheale LMG 9086T was used as
an outgroup. Bar, 0.01 substitutions per nucleotide position.
agar; weak growth occurred on NA; no growth occurred
on CA, SC agar or MacConkey agar. YF-3T was resistant
to tenebrimycin, streptomycin, amikacin, oxacillin,
kanamycin, aztreonam and gentamicin, but sensitive to
chloromycetin, cefotaxime, spectinomycin, minocycline,
levofloxacin, cefuroxime, cefoperazone, cefoxitin, norfloxacin, furadantin, ciprofloxacin, midecamycin, polymyxin B,
vancomycin, ofloxacin, erythromycin, clindamycin, tetracycline, benzylpenicillin, cefazolin, cefepime, ampicillin,
ceftriaxone, trimethoprim-sulfamethoxazole, ceftazidime,
cefalotin and piperacillin. The morphological, cultural,
physiological and biochemical characteristics of strain
YF-3T are listed in the species description. The differences
between strain YF-3T and the reference strains
(C. hispalense AG13T and C. taiwanense Soil-3-27T) are
presented in Table 1.
The predominant fatty acids of strain YF-3T ($ 5 %) were
iso-C15 : 0, iso-C17 : 0 3-OH, summed feature 9 (comprising
iso-C17 : 1v9c and/or C16 : 0 10-methyl) and summed feature
3 (comprising C16 : 1v7c and/or C16 : 1v6c), which was
1862
consistent with those of its closest phylogenetic neighbours
grown under the same conditions. Smaller amounts of
anteiso-C15 : 0 and iso-C15 : 0 3-OH were also present. The
detailed fatty acid composition of strain YF-3T is shown
in Table 2 where it is compared with that of the reference
strains C. hispalense AG13T and C. taiwanense Soil-3-27T.
The polar lipid profile of strain YF-3T consisted of the predominant compounds phosphatidylethanolamine, five
unknown lipids and two unknown aminolipids. The
main respiratory quinone was menaquinone MK-6. Polyamine analysis indicated that sym-homospermidine
[43.2 mmol (g dry weight)21] was the major component
and that minor amounts of spermidine [3.1 mmol (g dry
weight)21] and spermine [2.8 mmol (g dry weight)21]
and traces of 1,3-diaminopropane, cadaverine and
putrescine [, 0.1 mmol (g dry weight)21] were also present,
which is consistent with the characteristics of other members of the genus Chryseobacterium (Hamana & Matsuzaki,
1990; Kampfer et al., 2003). The DNA G þ C content of
strain YF-3T was 37 mol%.
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Chryseobacterium shandongense sp. nov.
Table 1. Differential biochemical characteristics of strain
YF-3T and type strains of closely related species of the genus
Chryseobacterium
T
Strains: 1, Chryseobacterium shandongense sp. nov. YF-3 ; 2,
C. hispalense AG13T; 3, C. taiwanense BCRC 17412T. All data are
from this study. þ , Positive; 2, negative; W , weakly positive.
Characteristic
API 20 NE strips
Reduction of nitrate
L -Malic acid
b-Glucosidase
Utilization of (GN 2 plate):
L -Serine
Histidine
Dextrin
D -Fructose
L -Fucose
D -Galactose
Gentiobiose
a-D -Glucose
Maltose
D -Mannose
L -Rhamnose
D -Sorbitol
D -Glucuronic Acid
Inosine
L -Proline
Glycyl-L -aspartic acid
Turanose
Enzymic activities (API ZYM strip)
Esterase (C4)
Esterase lipase (C8)
Lipase (C14)
Trypsin
a-Chymotrypsin
N-Acetyl-b-glucosaminidase
Cystine aminopeptidase
1
2
Strains: 1, Chryseobacterium shandongense sp. nov. YF-3T; 2,
C. hispalense AG13T; 3, C. taiwanense BCRC 17412T. All data are
from this study. Values are percentages of total fatty acids. Fatty
acids amounting to less than 1 % of the total fatty acids are not
shown. TR , Trace (less than 1 %); 2 , not detected/reported.
3
Fatty acid
2
2
þ
þ
þ
2
2
2
2
þ
þ
þ
þ
þ
2
þ
þ
þ
2
2
þ
2
þ
þ
þ
þ
2
2
þ
þ
þ
þ
þ
þ
þ
þ
þ
2
þ
2
2
þ
þ
þ
2
2
2
2
2
2
2
2
2
2
2
2
þ
2
2
2
þ
þ
þ
2
2
2
2
2
þ
2
2
þ
2
2
2
2
2
W
W
Þ
W
A nearly full-length 16S rRNA gene sequence (1478 bp) of
strain YF-3T was determined. Similarity analysis of 16S
rRNA gene sequences showed that strain YF-3T was most
closely related to C. hispalense AG13T (98.71 %) and
C. taiwanense Soil-3-27T (96.93 %). Phylogenetic analysis
based on sequence similarities of the 16S rRNA gene indicated there was a relationship between strain YF-3T and
members of the genus Chryseobacterium (Fig. 1). DNA –
DNA hybridization with its closest relatives was carried out
to determine further the taxonomic status of YF-3T. In the
present study, the levels of DNA –DNA relatedness between
strain YF-3T and C. hispalense AG13T and C. taiwanense Soil3-27T were found to be 31.7 ^ 2.1 % and 28.4 ^ 5.4 %,
respectively (Table S1), far below the value of 70 % that is
commonly accepted to define a novel species (Wayne et al.,
1987).
http://ijs.sgmjournals.org
Table 2. Fatty acid compositions of strain YF-3T and closely
related species of the genus Chryseobacterium
C16 : 0
iso-C15 : 0
iso-C17 : 0
anteiso-C15 : 0
iso-C15 : 0 3-OH
iso-C17 : 0 3-OH
C16 : 0 3-OH
C18 : 1v9c
C20 : 1v9c
Summed features:
3*
9†
1
2
3
1.44
47.06
1.31
42.48
1.96
44.17
1.17
TR
TR
2.85
2.74
15.06
1.33
1.04
1.71
1.93
2.68
13.59
1.02
2
2
2
5.57
13.76
10.54
19.25
9.47
16.16
TR
TR
4.31
17.92
TR
*Summed feature 3 contains C16 : 1v7c and/or C16 : 1v6c.
†Summed feature 9 contains iso-C17 : 1v9c and/or C16 : 0 10-methyl.
Therefore, on the basis of these phylogenetic, phenotypic
and chemotaxonomic data, strain YF-3T represents a
novel species of the genus Chryseobacterium, for which
the name Chryseobacterium shandongense sp. nov. is
proposed.
Description of Chryseobacerium shandongense
sp. nov.
Chryseobacterium shandongense (shan.dong.en9se. N.L.
neut. adj. shandongense pertaining to Shandong province,
the location of the soil sample from which the type strain
was isolated).
Cells are Gram-stain-negative, non-motile, non-sporeforming, rod-shaped, and approximately 0.5 – 0.7 mm in
width and 2.1 – 2.3 mm in length. Best growth occurs on
TSA; good growth occurs on LB agar and R2A agar;
weak growth occurs on NA; no growth occurs on CA, SC
agar or MacConkey agar. Colonies grown for 24 h on
TSA are about 3 mm in diameter, circular with a shiny surface and entire edges, yellow – orange (flexirubin-type, nondiffusible), translucent and mucoid. Growth conditions are
25 – 37 8C (optimum 30 8C), at pH 5.0 –8.0 (optimum 7.0),
with 0 –5 % (w/v) NaCl (optimum 1 %). Positive for the
hydrolysis of DNA, starch and casein and for oxidase and
catalase activities. Negative for the reduction of nitrate
and cellulose hydrolysis. In API 20E and 20NE kits,
positive for citrate utilization, acetoin production, indole
production, aesculin hydrolysis and gelatin hydrolysis,
but negative for arginine dihydrolase, urease, lysine
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F. Yang and others
decarboxylase, ornithine decarboxylase, H2S production
and tryptophan deaminase. Acid is produced from D -mannose, but not from mannitol, inositol, sorbitol, melibiose,
amygdalin, arabinose, N-acetylglucosamine or potassium
gluconate. In GN2 Microplates, utilization of dextrin,
glycogen, Tween 40, Tween 80, adonitol, i-erythritol,
D -fructose, L -fucose, gentiobiose, a-D -glucose, a-lactose,
lactulose, maltose, D -psicose, raffinose, L -rhamnose,
sucrose, turanose, xylitol, monomethyl succinate, acetic
acid, cis-aconitic acid, D -galactonic acid, lactone, D -galacturonic acid, D -glucosaminic acid, a-hydroxybutyric acid,
b-hydroxybutyric acid, p-hydroxyphenylacetic acid, aketobutyric acid, DL -lactic acid, malonic acid, propionic
acid, quinic acid, succinic acid, L -alaninamide, L -alanine,
L -asparagine, L -aspartic acid, L -glutamic acid, glycyl-L aspartic acid, glycyl-L -glutamic acid, L -proline, L -pyroglutamic acid, inosine, thymidine, 2,3-butanediol, DL -a-glycerol,
glucose 1-phosphate, glucose 6-phosphate and N-acetyl-D glucosamine is positive, but not utilization of a-cyclodextrin, N-acetyl-D -galactosamine, L -arabinose, D -arabitol,
cellobiose, D -galactose, myo-inositol, methyl b-D -glucoside,
trehalose, methyl pyruvate, citric acid, formic acid, D gluconic acid, D -glucuronic acid, c-hydroxybutyric acid,
itaconic acid, a-ketoglutaric acid, a-ketovaleric acid, D saccharic acid, sebacic acid, succinamic acid, glucuronamide, D -alanine, L -alanyl-glycine, L -phenylalanine, D serine, L -threonine, DL -carnitine, c-aminobutyric acid,
phenyethylamine, putrescine or 2-aminoethanol. In API
ZYM tests, alkaline phosphatase, esterase (C4), esterase
lipase (C8), lipase (C14), leucine aminopeptidase, valine
aminopeptidase, acid phosphatase, naphthol-AS-BI-phosphoamidase, a-glucosidase, N-acetyl-b-glucosaminidase,
cystine aminopeptidase (weak), trypsin (weak) and a-chymotrypsin (weak) activities are present, but a-galactosidase, b-galactosidase, b-glucuronidase, a-mannosidase
and a-fucosidase activities are absent. Menaquinone-6 is
the main respiratory quinone. The predominant fatty
acids ($ 5 %) are iso-C15 : 0, iso-C17 : 0 3-OH, summed feature 9 (comprising iso-C17 : 1v9c and/or C16 : 0 10-methyl)
and summed feature 3 (comprising C16 : 1v7c and/or
C16 : 1v6c). Polar lipids consist of phosphatidylethanolamine, five unidentified lipids and two unidentified aminolipids. sym-Homospermidine is the predominant
polyamine, but minor amounts of spermidine and spermine are also present.
The type strain is YF-3 ( ¼ CCTCC AB 2014060 ¼ JCM
30154T), which was isolated from farmland soil collected
from Qingdao city, Shandong province, PR China. The
DNA G þ C content of the type strain is 37 mol%.
T
T
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