Embed Size (px)
Citation preview
Biochemical Characterization of Gram-Positive andGram-Negative Plant-Associated Bacteria with Micro-RamanSpectroscopy
MATHEWS L. PARET, SHIV K. SHARMA,* LISA M. GREEN, and ANNE M. ALVAREZDepartment of Plant and Environmental Protection Sciences, College of Tropical Agriculture and Human Resources, University of Hawaii atManoa, 3190 Maile Way, St. John Plant Sciences Building 315, Honolulu, Hawaii 96822 (M.L.P., A.M.A.); and Hawaii Institute of Geophysics
and Planetology, School of Ocean and Earth Science and Technology, University of Hawaii at Manoa, 2525 Correa Road, Honolulu, Hawaii
96822 (S.K.S., L.M.G.)
Raman spectra of Gram-positive and Gram-negative plant bacteria have
been measured with micro-Raman spectrometers equipped with 785 and
514.5 nm lasers. The Gram-positive bacteria Microbacterium testaceum,
Paenibacillus validus, and Clavibacter michiganensis subsp. michiganensis
have strong carotenoid bands in the regions 1155–1157 cm�1 and 1516–
1522 cm�1 that differentiate them from other tested Gram-negative
bacteria. In the Raman spectrum of Gram-positive bacteria Bacillus
megaterium excited with 785 nm laser, the Raman bands at 1157 and 1521
cm�1 are weak in intensity compared to other Gram-positive bacteria,
and these bands did not show significant resonance Raman enhancement
in the spectrum recorded with 514.5 nm laser excitation. The Gram-
positive bacteria could be separated from each other based on the bands
associated with the in-phase C¼C (v1) vibrations of the polyene chain of
carotenoids. None of the Gram-negative bacteria tested had carotenoid
bands. The bacteria in the genus Xanthomonas have a carotenoid-like
pigment, xanthomonadin, identified in Xanthomonas axonopodis pv.
dieffenbachiae, and it is a unique Raman marker for the bacteria. The
representative bands for xanthomonadin were the C–C stretching (v2)
vibrations of the polyene chain at 1135–1136 cm�1 and the in-phase C¼C
(v1) vibrations of the polyene chain at 1529–1531 cm�1, which were
distinct from the carotenoid bands of other tested bacteria. The tyrosine
peak in the region 1170–1175 cm�1 was the only other marker present in
Gram-negative bacteria that was absent in all tested Gram-positives. A
strong-intensity exopolysaccharide-associated marker at 1551 cm�1 is a
distinguishable feature of Enterobacter cloacae. The Gram-negative
Agrobacterium rhizogenes and Ralstonia solanacearum were differentiated
from each other and other tested bacteria on the basis of presence or
absence and relative intensities of peaks. The principal components
analysis (PCA) of the spectra excited with 785 nm laser differentiated the
various strains of bacteria based on the unique pigments these bacteria do
or do not possess. Raman spectroscopy of diverse plant bacteria that are
pathogenic and non-pathogenic to plants, and isolated from plants and
soil, indicates the possibilities of using the method in understanding plant–
bacterial interactions at the cellular level.
Index Headings: Micro-Raman spectroscopy; Plant bacteria; Principal
component analysis; PCA; Microbacterium testaceum; Clavibacter mich-
iganensis subsp. michiganensis; Ralstonia solanacearum; Xanthomonas
axonopodis pv. dieffenbachiae.
INTRODUCTION
Bio-chemical characterization and differentiation of bacteriato genera and species level by Raman spectroscopy, includingnormal Raman, resonance Raman, and surface-enhancedRaman scattering, have been a highly active area of researchfor many years.1–7 Laser Raman spectroscopy is a method foridentifying compounds within biological cells and can provide
supplementary information on cell composition. Bacteria canbe differentiated by these components. Numerous Ramanreference database papers on biological molecules are currentlyavailable and these could aid in effective characterization of thebio-molecules associated with the diverse types of bacteria.8,9
However, studies on characterization of diverse genera ofplant-pathogenic bacteria by Raman spectroscopy are few innumber. Bacteria that are plant pathogenic or associated withplants are diverse in their habitats and their effects on planthealth have been well documented.10,11 The total annualworldwide crop loss due to plant diseases caused by bacteria,fungi, viruses, and nematodes is about $220 billion.10 Thus,there is a major need for identifying and developing new toolsfor characterization of plant bacteria, which are one of themajor disease-causing organisms.
Bacteria are classified into two major groups based on cellwall structure, e.g., Gram positive (Gþ) and Gram-negative(G�). The Gþ bacterial cell walls have a murein-containingthick peptidoglycan layer that is a polymer of disaccharidescross-linked by short peptide fragments. The G� bacterial cellwalls have a thin peptidoglycan layer between the plasmamembrane and a phospholipid lipopolysaccharide outermembrane.12 In this study, we studied various plant-pathogenicand plant-associated Gþ and G� bacteria, respectively,belonging to diverse genera including Microbacterium testa-ceum, Paenibacillus validus, Bacillus megaterium, and Clav-ibacter michiganensis subsp. michiganensis (Gþ); and Erwiniachrysanthemi, Enterobacter cloacae, Agrobacterium rhizo-genes, Ralstonia solanacearum, and Xanthomonas axonopodispv. dieffenbachiae (G�). This study is an evaluation of Ramanspectroscopy as a new method for rapid and nondestructivescreening of a few representative plant-pathogenic and plant-associated bacteria.
MATERIALS AND METHODS
Bacterial Strains Used and Source of Isolation. The Gþbacteria used in the current study, M. testaceum strain Gut4, P.validus strain CC87, B. megaterium strain CC65 and C.michiganensis subsp. michiganensis strain A518-2 were,respectively, isolated from anthurium (Anthurium andraea-num), soil, soil, and tomato (Lycopersicon esculentum). TheG� bacteria used in the current study, E. chrysanthemi strainA5608, E. cloacae strain A5149, A. rhizogenes strain A3040,R. solanacearum strain A4515, and Xanthomonas axonopodispv. dieffenbachiae strain D150, were, respectively, isolatedfrom pineapple (Ananas comosus), ginger (Zingiber officinale),
Received 29 July 2009; accepted 21 January 2010.* Author to whom correspondence should be sent. E-mail: [email protected].
Volume 64, Number 4, 2010 APPLIED SPECTROSCOPY 4330003-7028/10/6404-0433$2.00/0
� 2010 Society for Applied Spectroscopy
carrot (Daucus carota), ginger (Z. officinale), and anthurium(A. andraeanum).
Preparation of the Bacterial Samples. Bacterial strainsfrom our collection were streaked on Nutrient Agar (NA;Bactot Nutrient Agar, Difco Laboratories, MI) with a plasticloop and incubated at 28 8C for 48 to 72 h. Two or threecolonies of each strain were picked from NA with a plastic loopand transferred to 2 mL of sterile distilled water (dH2O) in atest tube, vortexed for 10 s, and adjusted spectrophotometri-cally (Spectronic 20; Bausch and Lomb Inc.; Rochester, NY) toOD600nm ¼ 0.1. One milliliter (1 mL) of this sample was thentransferred to a 1.5 mL microfuge tube and centrifuged at14000 rpm for three minutes. The supernatant was discardedand the pellet resuspended in 1 mL 0.85% NaCl followed byspinning the cells down as mentioned above. The cells weresubsequently washed twice in 1 mL dH2O to remove anyculture media, if present, and finally suspended in 100 lLsterile dH2O for analysis.
Raman Instrumentation. The Raman measurements pre-sented here were excited by an Invictus 785 nm (near infrared)laser and measured with a fiber-coupled micro-Raman RXNsystem (Kaiser Optical Systems Inc., Ann Arbor, MI). Polishedmirror aluminum sheet Anolux III (Anomet, Inc. Ontario,Canada), 25 3 25 mm2 and 0.5 mm in thickness, was used asthe substrate to minimize interference of the Raman andfluorescence from normally used glass slides.13,14 Thealuminum sheet substrate was cleaned with methanol and 5lL of the bacterial samples were spotted for micro-Ramananalysis. The samples were air-dried for 30 minutes beforetaking the spectra. The laser power at the sample was kept at 20mW and the exposure time was 60 s. A 503 objective was usedfor the analysis. All the bacterial samples were further analyzedusing a Renishaw inVia Raman microscope (Renishaw Inc.,Hoffman Estates, IL) equipped with a 514.5 nm (green) argonion laser. For this analysis, the laser power at the sample waskept at 3.6 mW; the exposure time was 20 s and a 503objective was used.
Spectral Analysis. The analysis of the spectra collected
with the 785 nm laser excitation was carried out with GRAMS/AI software (Thermo Scientific, Waltham, MA). The spectrawith the 514.5 nm laser excitation were measured using WiRE2.0 Raman Software (Renishaw Inc., Hoffman Estates, IL) andthe spectra were analyzed with GRAMS/AI software. For eachbacterial strain tested, ten Raman spectra were taken with the785 nm laser source and five Raman spectra were taken withthe 514.5 nm laser source, followed by substrate subtraction,averaging, and baseline correction. For principal componentanalysis (PCA), the baseline-corrected 785 nm Raman spectraof bacteria were cropped in the 400 to 1800 cm�1 spectralrange and unity-normalized. PCA was implemented with theMatlab princomp function running under Matlab R2007b withthe Statistics Toolbox (The Mathworks, Inc., Natick, MA).Dendrograms were generated with Matlab pdist (usingMahalanobis distance), linkage (using shortest distance), anddendrogram (full tree) functions, operating on PCA scoresgenerated by princomp.
RESULTS AND DISCUSSION
The Raman spectra of Gþ and G� bacteria excited with the785 nm laser are illustrated in Figs. 1 and 2, respectively.Figures 3 and 4 show the Raman spectra of Gþ and G� bacteriameasured with 514.5 nm laser excitation. The positions ofRaman lines and tentative band assignments of all the Gþ andG� bacteria spectra excited with the 785 nm and 514.5 nmlasers are given in Tables I and II, respectively. ResonanceRaman spectra of the Gþ bacteria and X. axonopodis pv.dieffenbachiae excited with the 514.5 nm laser in the 1800 to3200 cm�1 region are shown in Fig. 5, and the tentativeassignments of the combination and overtone bands aresummarized in Table III.
Figure 1a shows Raman spectrum of M. testaceum withdistinct bands at 758 cm�1 of tryptophan, 961 cm�1 of C–N
FIG. 1. Raman spectra of Gram-positive bacteria excited with the 785 nmlaser: (a) Microbacterium testaceum (Mte), (b) Paenibacillus validus (Pva), (c)Bacillus megaterium (Bme), and (d) Clavibacter michiganensis subsp.michiganensis (Cmm).
FIG. 2. Raman spectra of Gram-negative bacteria excited with the 785 nmlaser: (a) Erwinia chrysanthemi (Ech), (b) Enterobacter cloacae (Ecl), (c)Agrobacterium rhizogenes (Arh), (d) Ralstonia solanacearum (Rso), and (e)Xanthomonas axonopodis pv. dieffenbachiae (Xad).
434 Volume 64, Number 4, 2010
stretch, 1156 and 1517 cm�1 of carotenoids, 1274 cm�1 ofamide III, 1392 cm�1 of CH3 bending, and 1605 cm�1 forphenylalanine when excited with the 785 nm laser.5,8,9,15,16
The presence or absence of these peaks and the intensities ofthese peaks in relation to other Gþ and G� bacteria can be usedfor differentiation of M. testaceum as indicated in Figs. 1 and 2.However, numerous unique bands in the spectrum of M.testaceum (Fig. 3a) when excited with the 514.5 nm laserclassifies M. testaceum into a very different group. Mediumintensity bands at 958 cm�1 of C–N stretch and 1213 cm�1 ofC–C6H5 stretch of phenylalanine and tryptophan in protein aredistinguishable features for M. testaceum.17 Carotenoidbands15 at 1155 and 1516 cm�1 are confirmed when excitedwith the 514.5 nm laser (Fig. 3a). M. testaceum is currentlybeing studied as a promising biological control agent for abacterial blight disease in anthuriums caused by X. axonopodispv. dieffenbachiae.18 M. testaceum was isolated from theguttation fluids of the leaf surface of anthurium and lives inclose association with X. axonopodis pv. dieffenbachiae.Distinguishable Raman spectra of these two bacteria could beof great use in further studies of the epidemiology and theinteractions between the pathogen and its potential biologicalcontrol agent.
When excited with the 785 nm laser, the Raman spectrum ofP. validus (Fig. 1b) shows distinguishable features, e.g., astrong intensity doublet consisting of bands at 832 and 857cm�1 representing ‘‘exposed’’ tyrosine and ‘‘buried’’ tyrosine,respectively.16 In addition, the C¼O ester of lipids8 at 1737cm�1 is unique among all other tested Gþ and G� bacteria.Other bands of significance in the 785 nm excited spectrum ofP. validus are of guanine at 669 cm�1, COC stretch at 901cm�1, C–N stretch at 957 cm�1, carbohydrates, C–C, C–O, –C–OH deformations at 1057 cm�1, carotenoids at 1157 and 1522cm�1, amide III at 1260 cm�1, C–H bend in proteins at 1349cm�1, and CH3 bend of proteins at 1389 cm�1.5,8,15,16 Thepresence of carotenoid bands15 is further confirmed at 1156 and1519 cm�1 by the 514.5 nm laser-excited spectrum as indicatedin Fig. 3b.
The Raman spectrum in Fig. 1c shows the features of B.megaterium that distinguish it from the other Gþ bacteriaspectra excited with the 785 nm laser. Relative intensities ofbands at 830 and 855 cm�1 represent ‘‘exposed’’ and ‘‘buried’’tyrosine; 902 cm�1 for COC stretch; 1210 cm�1 of C–C6H5
stretch; phenylalanine and tryptophan in protein; 1320 cm�1 ofamide III in protein; and 1521 cm�1 representing carotenoidsare key features.15,17,19,20 The CH3 bending8 at 1385 cm�1 isunique to B. megaterium and P. validus and is absent from allthe tested G� bacteria spectra. The Raman data of the 514.5 nmlaser-excited Raman spectra in Fig. 3c show a mediumintensity 1126 cm�1 band of ¼C–C¼ unsaturated fatty acidsin lipids,20 which is a distinguishable feature among all Gþ andG� bacteria. A medium intensity CH2 deformation band17 at1315 cm�1 is another important feature among Gþ bacteria.The carotenoid bands15 are present at 1156 and 1518 cm�1 asseen in Fig. 3c. However, the intensity of carotenoid bands isweaker in B. megaterium than in all other Gþ bacteria. Thebacterium is one of the largest among eubacteria (approxi-mately 5 lm); it is spore forming, and colonies form in chainswith a sticky polysaccharide on its cell wall, making it highlyresistant to environmental stress.21 The lack of strongresonance enhancement of the carotenoid bands when excitedwith the 514.5 nm laser may be due to this unique characteristicof the cell. B. megaterium has a unique pattern in thecarotenoid region and is distinguishable from all other Gþand G� based on these spectral features. Raman spectroscopicstudies of many carotenoids have revealed strong carotenoidbands in the regions 1500–1550 and 1150–1170 cm�1 due toin-phase C¼C (v1) and C–C stretching (v2) vibrations of thepolyene chain.22 In addition, in-plane rocking modes of CH3
groups attached to the polyene chain and coupled with C–Cbonds are seen as a peak in the 1000 to 1020 cm�1 range. Thewave number location of these bands and, in particular, the (v1)band is strongly dependent on the length of the carotenoidchain and can be used as a marker for identifying uniquecharacters of this soil-borne bacterium.22,23
The bands at 1336 and 1555 cm�1 representing adenine and
FIG. 3. Raman spectra of Gram-positive bacteria excited with the 514.5 nmlaser: (a) Microbacterium testaceum (Mte), (b) Paenibacillus validus (Pva), (c)Bacillus megaterium (Bme), and (d) Clavibacter michiganensis subsp.michiganensis (Cmm).
FIG. 4. Raman spectra of Gram-negative bacteria excited with the 514.5 nmlaser: (a) Erwinia chrysanthemi (Ech), (b) Enterobacter cloacae (Ecl), (c)Agrobacterium rhizogenes (Arh), (d) Ralstonia solanacearum (Rso), and (e)Xanthomonas axonopodis pv. dieffenbachiae (Xad).
APPLIED SPECTROSCOPY 435
TA
BL
EI.
Ten
tati
ve
Ra
ma
nb
an
da
ssig
nm
ents
(cm�
1)
of
the
Gra
m-p
osi
tiv
ea
nd
Gra
m-n
egat
ive
pla
nt
ba
cter
iaex
cite
dw
ith
the
78
5n
mla
ser.
Gra
m-p
osi
tiv
eb
act
eria
:M
icro
bac
teri
um
test
ace
um
(Mte
)st
rain
Gu
t4,
Pa
enib
acil
lus
vali
du
s(P
va
)st
rain
CC
87
,B
aci
llu
sm
ega
teri
um
(Bm
e)st
rain
CC
65
,a
nd
Cla
vib
act
erm
ich
igan
ensi
ssu
bsp
.m
ich
igan
ensi
s(C
mm
)st
rain
A5
18
-2.
Gra
m-n
egat
ive
ba
cter
ia:
Erw
inia
chry
san
them
i(E
ch)
stra
inA
56
08,
En
tero
bact
ercl
oac
ae
(Ecl
)st
rain
A5
14
9,A
gro
bac
teri
um
rhiz
og
enes
(Arh
)st
rain
A3
04
0,
Ral
sto
nia
sola
na
cea
rum
(Rso
)st
rain
A4
51
5,a
nd
Xa
nth
omo
nas
ax
on
opo
dis
pv.
die
ffen
bac
hia
e(X
ad)
stra
inD
15
0.a
Gra
m-p
osi
tive
bac
teri
aG
ram
-neg
ativ
ebac
teri
a
Ban
das
sig
nm
ents
Mte
Gu
t4P
va
CC
87
Bm
eC
C6
5C
mm
A5
18
-2E
chA
56
08
Ecl
A5
14
9A
rhA
30
40
Rso
A4
51
5X
adD
15
0
––
55
2w
,bd
––
––
55
2w
,bd
–C
–S
–S
–C
bo
nds6
57
9w
,bd
–5
79
w,b
d–
57
5w
,bd
––
––
Car
boh
yd
rate
s6
596
w,b
d–
––
––
–594
vw
–P
hosp
hat
idyli
nosi
tol8
62
1w
62
0w
62
0m
61
9w
62
0w
62
0w
62
0w
62
0w
62
0w
Ph
eny
lala
nin
e9
64
2w
64
2m
64
2m
64
3v
w6
43
w6
43
w6
43
w,b
d6
42
w6
40
wT
yro
sin
e9
––
66
2w
––
––
––
C–
Sst
retc
hin
gm
od
eo
fcy
stin
e8
–6
69
w,b
d–
66
9v
w6
69
w6
68
w,b
d–
––
Gu
anin
e16
––
681
w–
––
–680
vw
–R
ing
bre
athin
gm
odes
inth
eD
NA
bas
es8
72
6m
72
5m
72
4m
72
5w
72
5w
,bd
72
5m
72
0m
72
5m
–A
den
ine,
acet
yl
coen
zym
e-A
2,9
,16
75
8w
–7
48
w–
75
8w
,bd
75
8w
,bd
–7
58
vw
75
7w
Try
pto
ph
an9
78
3m
78
2m
78
2m
78
1m
78
2m
78
2s
78
0s
78
3m
78
0w
Gu
anin
e,cy
tosi
ne,
ura
cil,
citr
icac
id9,1
6
81
2w
––
80
7v
w–
81
0m
––
–T
yro
sin
e1
–8
32
s8
30
m,b
d–
82
9m
82
5m
82
5m
83
0m
,bd
82
5w
,bd
‘‘E
xp
ose
d’’
tyro
sin
e16
86
0w
85
7s,
sh8
55
m,b
d8
53
vw
85
3m
85
4m
85
4m
85
3m
,bd
–‘‘B
uri
ed’’
tyro
sin
e16
––
87
9w
,sh
87
4v
w–
––
––
Try
pto
ph
and-
ring
23
90
0w
90
1m
90
2m
,bd
–8
99
w,b
d9
02
m,b
d9
00
w9
01
m,b
d–
CO
Cst
r.16
––
93
2w
–9
40
m,b
d–
93
1w
–9
35
w,b
dC
–C
BK
str.
,a
-hel
ix(p
rote
in)1
7,
C–O
–C
gly
cos
bo
nd
(car
bo
hy
dra
tes)
17
96
1w
95
7w
95
1w
––
––
––
C–
Nst
r.5,
(v4)
C–
C–H
ou
t-o
f-p
lan
eb
end
of
the
po
lyen
ech
ain
of
caro
ten
oid
s/x
anth
om
on
adin
38
–9
81
w9
80
m9
84
m–
–9
80
m9
81
vw
,bd
–C
–C
str.
b-sh
eet
(pro
tein
s)8,¼
CH
ben
din
g(l
ipid
s)8
10
03
s1
00
3s
10
03
s1
00
3m
10
03
s1
00
3s
10
03
s1
00
3s
10
03
sP
hen
yla
lan
ine9
10
32
w–
–1
03
1w
,sh
10
31
m1
03
1w
10
32
w1
03
2w
10
31
mC
–H
in-p
lan
e,p
hen
yla
lan
ine9
–1
05
7s
10
57
w1
05
1w
,bd
––
––
–C
arb
oh
yd
rate
s,C
–C
,C
–O
,–
C–
OH
def
.5
––
1064
w1071
w–
––
1067
w,s
h–
C–N
str.
(pro
tein
)17,
chai
nC
–C
str.
(lip
ids)
17
––
––
10
89
w,b
d–
––
–P
O2�
str.
(DN
A/R
NA
)17,
chai
nC
–C
str.
(lip
ids)
17,
C–O
,C
–C
str.
(car
bohydra
tes)
17
11
01
m,s
h1
10
3s
11
03
s,b
d1
09
7v
w,
11
02
w,b
d1
10
2m
,sh
10
97
s1
10
1m
11
01
m,b
d–
Car
boh
yd
rate
sC
–C
,C
–O
,–
C–
OH
def
.5
11
31
w,s
h1
13
1w
11
25
m,s
h1
13
1w
11
25
m,s
h1
12
7w
11
27
m,s
h1
12
6m
,sh
–¼
C–
C¼
(unsa
tura
ted
fatt
yac
ids
inli
pid
s)20
––
––
11
56
w–
––
–1
15
6v
s1
15
7v
s1
15
7m
11
57
s–
––
–1
13
5s
C–
Cst
r.(v
2)
vib
rati
ons
of
the
poly
ene
chai
nof
caro
tenoid
s/xan
thom
onad
in15,2
2,3
8
––
––
11
72
w1
17
5w
,sh
11
70
w,b
d1
17
2m
,bd
11
72
wT
yro
sin
e3
11
92
w,s
h–
–1
19
3w
,bd
––
––
–1
21
1w
12
09
w1
21
0w
,sh
12
10
w,b
d1
20
9w
,sh
12
10
w,s
h1
21
3w
–1
20
6w
C–
C6H
5st
retc
h,
phen
yla
lanin
e,tr
ypto
phan
(pro
tein
)17
12
74
m,s
h1
26
0m
,sh
12
54
w,b
d1
27
0w
12
53
m,s
h1
25
1m
,sh
12
34
m1
24
5w
,sh
,1
25
6m
,sh
12
71
w,s
hA
mid
eII
I16,1
9
––
––
––
–1
30
8w
,sh
13
13
w,s
hC
H2
twis
t(l
ipid
s)17
13
20
w–
13
20
m,s
h–
13
22
vs
––
13
21
m,s
h1
32
1w
,sh
Am
ide
III
(Pro
tein
)20
––
13
32
w,s
h–
––
–1
33
2w
,sh
–C
–H
ben
d(p
rote
in)5
13
37
m,b
d–
13
41
m1
33
6s,
bd
13
36
s,b
d1
33
7v
s,b
d1
33
7v
s,b
d1
33
9s,
bd
13
39
s,b
dA
den
ine2
4
13
52
w,s
h1
34
9m
,bd
––
––
––
13
60
w,s
h,
13
72
w,s
hC
–H
ben
d(p
rote
in)5
,6
436 Volume 64, Number 4, 2010
tryptophan, respectively, indicated in Fig. 1d, are key featuresin the spectrum of C. michiganensis subsp. michiganensis andthe relative intensities of these bands are higher than in otherGþ bacteria.24 The carotenoid bands15 are at 1157 and 1522cm�1 (Fig. 1, trace d) and are confirmed when excited with the514.5 nm laser (Fig. 3d). Relative intensities of amide III peakat 1267 cm�1 and amide I peak at 1662 cm�1 can also be usedto distinguish C. michiganensis subsp. michiganensis fromother Gþ bacteria. The carotenoid bands, in particular the (v1)band of 1522 cm�1, distinguish C. michiganensis subsp.michiganensis from all other bacteria and can be assigned as aunique marker. Numerous carotenoids have been described inC. michiganensis subsp. michiganensis previously, includinglycopene and cryptoxanthene (yellow pigmented colonies),spirilloxanthin and lycopene (pink pigmented colonies), b-carotene, cryptoxanthene, and canthaxanthine (orange pig-mented colonies), and a mutant has been known to onlypossess lycopene (red pigmented colonies).25,26 The C.michiganensis subsp. michiganensis is a seed-borne pathogenand causes a bacterial canker disease in tomatoes. Identificationof the pathogen from culture plates has always been difficultbecause of the growth of numerous saprophytes when tomatoseed samples are plated on culture media.27 An evaluation ofthe Raman spectra of these saprophytes in comparison with theunique bands identified in C. michiganensis subsp. michiga-nensis and identification of distinguishable markers will be keycontributions to the tomato industry.
E. chrysanthemi excited with the 785 nm laser shows aRaman spectrum with unique bands among all the testedG� bacteria (Fig. 2a). These are the 575 cm�1 band ofcarbohydrates and the 1089 cm�1 band associated with PO2
�
stretch of DNA/RNA, the chain C–C stretch of lipids, andC–O, C–C stretch of carbohydrates.6,17 Figure 2a shows amedium intensity band of E. chrysanthemi at 1156 cm�1 that isunique among all the tested G� bacteria. Even though this bandwas in the region for carotenoid bands15 in other bacteria,associated bands in the region 1516–1522 cm�1 were notpresent. This indicates that they are not carotenoid bands andthis is further confirmed by the absence of these bands whenthe bacterial sample was excited with the 514.5 nm laser asseen in Fig. 4a. A key feature of the E. chrysanthemi Ramanspectrum exhibited in Fig. 2a is the tyrosine band16 at 853cm�1, which is of significantly higher intensity than thecorresponding bands in other G� bacterial spectra. In addition,the phenylalanine band9 at 1003 cm�1 and the C–H in-planerocking and phenylalanine band9 at 1031 cm�1 are of higherintensities than other G� bacteria. The ¼C–C¼ of unsaturatedfatty acids in lipids band20 at 1125 cm�1 also shows higherrelative intensity than in other G� bacteria spectra. E.chrysanthemi did not have any major spectral features whenexcited with the 514.5 nm laser, as shown in Fig. 4a. It gave alot of fluorescence, and this was similar to results for A.rhizogenes and E. cloacae. The E. chrysanthemi is the causalagent of heart rot disease of pineapple. Major outbreaks of thedisease have been a major concern for the quarantine divisionin Hawaii, and preventing entry of the pathogen frominternational supplies of the planting material has been a keyarea in management of the disease. A major focus of currentresearch is differentiating E. chrysanthemi from closely relatedE. carotovora subsp. carotovora, which is associated with softrot in many ornamental and crop plants worldwide.28 Theidentified Raman markers are promising for testing of plantT
AB
LE
I.C
on
tin
ued
.
Gra
m-p
osi
tive
bac
teri
aG
ram
-neg
ativ
ebac
teri
a
Ban
das
sign
men
tsM
teG
ut4
Pv
aC
C8
7B
me
CC
65
Cm
mA
51
8-2
Ech
A5
60
8E
clA
51
49
Arh
A3
04
0R
soA
45
15
Xad
D1
50
13
92
m,s
h1
38
9w
13
85
m–
––
13
95
m,b
d–
13
94
w,s
hC
H3
ben
din
g8
14
19
w–
14
14
w,
14
18
w–
––
–1
41
7w
,sh
–C
OO�
sym
met
ric
str.
5
14
50
s1
45
3v
s1
45
1s
14
50
m,b
d1
45
0v
s,b
d1
45
0v
s,b
d1
45
0v
s,b
d1
45
0v
s,b
d1
45
0m
,bd,
14
58
mC
H2
ben
din
g(l
ipid
s)1
15
17
vs
15
22
vs
15
21
m1
52
2v
s–
––
–1
53
1v
sIn
-ph
ase
C¼
C(v
1)
vib
rati
ons
of
the
po
lyen
ech
ain
of
caro
tenoid
s/xan
thom
onad
in15,2
2,3
8
15
53
w–
–1
55
5w
15
55
w1
55
1s
15
54
w–
–T
ryp
top
han
24
15
77
m1
57
4w
,bd
15
67
w,
15
75
m1
56
7w
,1
57
3w
15
75
w,
15
85
w,s
h1
57
6m
,sh
15
75
w,
15
87
w1
57
5m
15
80
w,s
h,
15
87
w,s
hE
PS
-ass
oci
ated
ban
ds1
5
16
05
w,s
h1
60
9w
,sh
16
09
m,s
h–
16
06
w,s
h1
60
6w
,sh
16
07
w,s
h1
60
7w
,sh
16
11
w,s
hP
hen
yla
lan
ine1
6
16
24
w,s
h1
62
3w
,sh
16
18
m,s
h1
61
4w
16
19
w1
61
9w
–1
62
2m
,sh
16
18
w,s
h,
16
25
w,s
hT
yro
sin
e16
16
60
s,b
d1
66
0s,
bd
16
58–
16
69
s,b
d1
66
2m
,bd
16
59
vs
16
56–
16
69
vs,
bd
16
56–
16
62
vs,
bd
16
58
–1
67
0v
s,b
d1
66
5–
167
9m
,bd
Am
ide
1,
C¼
Cst
r.16
––
17
27
w–
17
23
w,s
h1
72
7w
,sh
–1
72
7m
,sh
17
29
w,s
hE
ster
gro
up
8
–1
73
7s
––
––
––
–C¼
Oes
ter
(lip
ids)
8
29
39
vs
29
40
vs
29
38
vs
29
37
vs
29
37
vs
29
36
vs
29
38
vs
29
36
vs
29
35
vs
CH
3an
dC
H2
str.
19
aA
bb
rev
iati
on
s:v
w:
ver
yw
eak
,w
:w
eak
,m
:m
ediu
m,
s:st
rong
,v
s:v
ery
stro
ng
,b
d:
bro
ad,
sh:
sho
uld
er,
EP
S:
exo
po
lysa
cch
arid
e.
APPLIED SPECTROSCOPY 437
TA
BL
EII
.T
enta
tiv
eR
am
an
ba
nd
ass
ign
men
ts(c
m�
1)
of
the
Gra
m-p
osi
tiv
ea
nd
Gra
m-n
egat
ive
pla
nt
ba
cter
iaex
cite
dw
ith
the
51
4.5
nm
lase
r.G
ram
-po
siti
ve
ba
cter
ia:
Mic
rob
acte
riu
mte
sta
ceu
m(M
te)
stra
inG
ut4
,P
aen
ibac
illu
sva
lid
us
(Pv
a)
stra
inC
C8
7,
Ba
cill
us
meg
ate
riu
m(B
me)
stra
inC
C6
5,
an
dC
lavi
ba
cter
mic
hig
anen
sis
sub
sp.
mic
hig
anen
sis
(Cm
m)
stra
inA
51
8-2
.G
ram
-neg
ativ
eb
act
eria
:E
rwin
iach
rysa
nth
emi
(Ech
)st
rain
A5
60
8,E
nte
rob
act
ercl
oac
ae
(Ecl
)st
rain
A5
14
9,
Agr
ob
acte
riu
mrh
izo
gen
es(A
rh)
stra
inA
30
40
,R
alst
on
iaso
lan
acea
rum
(Rso
)st
rain
A4
51
5,
an
dX
an
thom
on
asa
xo
no
pod
isp
v.die
ffen
bach
iae
(Xad
)st
rain
D1
50
.a
Gra
m-p
osi
tive
bac
teri
aG
ram
-neg
ativ
ebac
teri
a
Ban
das
sign
men
tsM
teG
ut4
Pv
aC
C8
7B
me
CC
65
Cm
mA
51
8-2
Ech
A5
60
8E
clA
51
49
Arh
A3
04
0R
soA
45
15
Xad
D1
50
––
––
––
53
3v
w5
39
vw
53
2m
S–
Sst
r.,
CO
Cg
lyco
sid
icri
ng
def
.16
–597
vw
594
vw
––
––
––
Phosp
hat
idyli
nosi
tol8
–6
19
vw
62
5v
w–
––
––
62
2w
Ph
eny
lala
nin
e9
64
2v
w6
44
vw
–6
42
w6
39
vw
64
8v
w–
64
3v
w6
43
wT
yro
sin
e9
66
4v
w6
69
vw
66
9v
w6
70
vw
––
–6
72
vw
–G
uan
ine1
6
––
––
––
––
677
wR
ing
bre
athin
gm
odes
inth
eD
NA
bas
es8
713
vw
726
vw
–730
vw
730
vw
––
726
vw
–A
den
ine,
acet
yl
coen
zym
e-A
2,9
,16
–7
58
vw
––
––
74
8v
w7
57
vw
75
9w
Try
pto
ph
an9
783
w783
m784
m784
m–
781
w–
782
m783
mG
uan
ine,
cyto
sine,
ura
cil,
citr
icac
id2,1
6
–8
31
m,b
d8
28
w8
26
w–
––
83
0w
83
3w
‘‘E
xp
ose
d’’
tyro
sin
e16
86
3w
86
4w
,sh
85
8w
––
–8
51
vw
85
3w
85
3w
‘‘B
uri
ed’’
tyro
sin
e16
–8
97
w,
90
8w
–8
98
w–
89
0v
w9
03
vw
88
9w
–C
OC
str.
16
95
8m
96
0m
95
1w
96
2w
––
––
96
0w
,sh
C–
Nst
r.5
(v4)
C–C
–H
out-
of-
pla
ne
ben
dof
the
po
lyen
ech
ain
of
caro
ten
oid
s/x
anth
om
on
adin
38
10
03
s1
00
4s
10
04
m1
00
4s
10
03
w1
00
3w
10
03
m1
00
4s
10
04
sIn
-pla
ne
rock
ing
mo
des
of
CH
3g
roup
sat
tach
edto
the
po
lyen
ech
ain
of
caro
ten
oid
s/x
anth
om
on
adin
coup
led
wit
hC
–C
bo
nd
s(v
3)
15,2
2,3
8
–1033
vw
1033
w1033
vw
––
–1032
m1033
mC
–H
in-p
lane,
phen
yla
lanin
e17
–1
05
6w
,1
10
3m
–1
10
0v
w–
––
10
95
vw
–C
arb
oh
ydra
tes,
C–C
,C
–O
,–
C–
OH
def
.5
–1
12
9v
w1
12
6m
11
32
vw
––
11
24
vw
11
27
w–
¼C
–C¼
(unsa
tura
ted
fatt
yac
ids
inli
pid
s)20
11
55
vs
11
56
vs
11
56
m1
15
7v
s–
––
–1
13
6v
sC
–C
str.
(v2)
vib
rati
on
so
fth
ep
oly
ene
chai
no
fca
rote
no
ids/
xan
tho
mo
nad
in15,2
2,3
8
––
11
70
w,s
h–
––
11
80
vw
11
73
w1
17
2w
Ty
rosi
ne3
11
91
m,s
h–
11
94
vw
11
91
w,s
h–
––
––
12
13
w,s
h1
20
6v
w,s
h1
21
0v
w–
––
––
12
09
w,s
hC
–C
6H
5st
r.,
phen
yla
lanin
e,tr
ypto
phan
(pro
tein
)17
12
80
m,
12
90
w,s
h1
26
3v
w,
12
83
m1
23
1w
,1
26
7w
12
67
w1
24
4(w
)1
24
2(w
)1
26
8v
w1
23
4m
,bd
12
42
w,b
dA
mid
eII
I19,2
9
13
13
(w)
13
12
w1
31
5m
13
15
w1
30
4v
w–
13
14
vw
13
13
m1
31
3m
CH
2tw
ist
(lip
ids)
17
–1
33
8w
–1
33
9w
––
–1
33
9w
13
39
wA
den
ine2
4
13
60
w1
36
1w
13
61
m,
13
77
vw
1356
w–
––
1361
m1362
mC
–H
ben
din
g(p
rote
in)5
,6
–1
39
3v
w1
39
8w
––
–1
40
3w
14
02
wC
OO�
sym
met
ric
str.
5,8
14
50
m,b
d1
45
0m
,bd
,1
47
7w
,sh
14
53
m,b
d1
44
9m
,bd
14
49
w,b
d1
44
8w
14
48
m,b
d1
45
2m
,bd
14
53
m,b
dC
H2
ben
din
g(l
ipid
s)1
––
1494
vw
––
–1491
w,s
h–
1496
vw
Aden
ine,
guan
ine2
4
––
––
––
15
05
vw
––
Cy
tosi
ne8
15
16
vs
15
19
vs
15
18
w1
52
2v
s–
––
–1
52
9v
sIn
-ph
ase
C¼
C(v
1)
vib
rati
on
so
fth
ep
oly
ene
chai
no
fca
rote
no
ids/
xan
tho
mo
nad
in15,2
2,3
8
15
85
w1
58
0w
,bd
15
88
m1
58
1w
––
15
82
vw
15
88
m,b
d1
58
5w
EP
S-a
sso
ciat
edb
and
s15
16
03
(w)
–1
60
8v
w–
––
–1
60
4w
,sh
16
04
vw
Ph
eny
lala
nin
e16
––
16
16
vw
––
––
16
22
w,s
h1
62
5v
wT
yro
sin
e16
––
16
43
vw
––
––
16
41
w,s
h–
Th
ym
ine1
9
16
64
m1
66
7m
,bd
16
57
w,b
d1
66
2m
,bd
16
55
w,b
d1
65
8w
16
72
m,b
d1
66
6s,
bd
16
67
w,b
dA
mid
eI,
C¼
Cst
r.16
–1
73
1v
w–
––
––
––
Est
erg
roup
8
–1
74
0w
––
––
––
–C¼
Oes
ter
(lip
ids)
8
29
37
vs
29
37
vs
29
38
vs
29
37
vs
29
37
vs
29
39
vs
29
40
vs
29
37
vs
29
36
vs
CH
3an
dC
H2
str.
19
aA
bb
rev
iati
on
s:v
w:
ver
yw
eak
,w
:w
eak
,m
:m
ediu
m,
s:st
ron
g,
vs:
ver
yst
ron
g,
bd
:b
road
,sh
:sh
ould
er,
EP
S:
exop
oly
sacc
har
ide.
438 Volume 64, Number 4, 2010
samples for the presence of E. chrysanthemi and furtherdifferentiation from other closely related organisms.
A strong-intensity exopolysaccharide (EPS) associatedband15 at 1551 cm�1 is the key feature of E. cloacae spectrumin comparison with all other tested bacteria shown in Figs. 1and 2. E. cloacae shares the guanine band16 at 668 cm�1 withE. chrysanthemi and P. validus; the same band is either absentor of weaker intensity in other tested bacteria. The tyrosineband1 at 810 cm�1 is a distinctive feature of E. cloacae amongG� bacteria. The E. cloacae does not have any major spectralfeatures when excited with the 514.5 nm laser, as shown in Fig.4b, although it gave a lot of fluorescence. The E. cloacae is anopportunistic pathogen and is known to be associated withapples, elm trees, coconut, corn, edible ginger, and onionbulbs.29 It is also a human pathogen and causes burn, wound,and urinary tract infections.12 The EPS are carbohydratepolymers that are secreted by a wide variety of bacteria and canremain associated with the cell wall to form a bound capsulelayer or can be released as extracellular slime.30,31 The EPSalso play an important role in pathogenesis in manyenterobacterial species. A strong EPS-associated peak could
serve as a major identifiable marker in E. cloacae and couldhelp us to learn more about the unique features of thispathogen, which infects both humans and plants.
The genus Agrobacterium comprises numerous soil-inhab-iting plant pathogens known for their natural ability to transferDNA to plants. This function has been utilized in biotechnol-ogy as one of the most important tools for genetic modificationof plants since its first description as a natural vector.32 Themajor pathogens in this group are A. rhizogenes, which induceshairy root of apple; A. tumefaciens, which causes crown gall ofgrapes and woody plants; and A. rubi, which causes cane gallof raspberries and blackberries. The Raman spectrum of A.rhizogenes is compared with the spectra of other bacteria (Figs.1 and 2). The distinctive feature of A. rhizogenes in the 785 nmexcited spectrum is the presence of a band at 1234 cm�1
representing amide III19 as seen in Fig. 2c. The bands at 980cm�1 representing C–C stretching b-sheet of proteins and¼CHbending of lipids, and at 1395 cm�1 representing CH3 bending,are unique among the G� bacteria tested.8 Major distinguish-able features of the A. rhizogenes spectrum in comparison withall other bacteria are the absence of many bands (e.g., 575, 668,758, 810, 957, 1057, 1089, 1135, 1156, 1192, 1251, 1274,1322, 1349, 1385, 1392, 1521, 1531, 1619, and 1727 cm�1) asshown in Figs. 1 and 2, which are of medium or strongintensities in other bacteria tested. In addition, relativeintensities of numerous bands (620, 643, 669, 720, 780, 825,954, 900, 931, 1003, 1032, 1101, 1127, 1337, 1395, 1450, and1656 cm�1) were variable for many bacteria when compared tothose of the A. rhizogenes spectrum. The Raman spectrum of A.rhizogenes did not show any carotenoid bands in either the 785or 514.5 nm laser-excited spectra, as indicated by the absenceof Raman fingerprints of carotenoid bands15 in the regions1155–1157 cm�1 and 1516–1522 cm�1 in Figs. 2 and 4.However, the carotenoid bands were present in the Ramanspectra of all tested Gþ bacteria. The phenylalanine band9 of A.rhizogenes at 1003 cm�1 is weaker than in the spectra of E.chrysanthemi, E. cloacae, R. solanacearum, M. testaceum, P.validus, and B. megaterium, as shown in Figs. 1 and 2. A.rhizogenes did not have any major spectral features whenexcited with the 514.5 nm laser, as shown in Fig. 4c, althoughit gave a lot of fluorescence.
The R. solanacearum strain tested does not have any uniqueRaman fingerprint to distinguish it from other bacteria whenexcited with the 785 nm laser (Fig. 2d). However, as indicatedin Fig. 4d, distinguishable bands at 782 and 1173 cm�1 areobserved when excited with the 514.5 nm laser; the 782 cm�1
TABLE III. Tentative vibrational Raman band assignments in the region 1800–3200 cm�1 of plant bacteria excited with the 514.5 nm laser. Gram-positive bacteria: Microbacterium testaceum (Mte) strain Gut 4, Paenibacillus validus (Pva) strain CC87, Clavibacter michiganensis subsp. michiganensis(Cmm) strain A518-2, and Gram-negative bacteria: Xanthomonas axonopodis pv. dieffenbachiae (Xad) strain D150.a
Gram-positive bacteriaGram-negative bacteria
Xad D150 Band assignments38Mte Gut4 Pva CC87 Cmm A518-2
2003 w – – – 2v3
2118 w – – – v2 þ v4
2157 s 2155 m 2161 w – v2 þ v3
2307 s 2310 m 2315 m 2267 s 2v2
2476 w – – – v1 þ v4
2515 s 2529 m 2525 m – v1 þ v3
2665 s 2673 s 2673 s 2656 s v1 þ v2
3029 m – – – 2v1
a Abbreviations: w: weak, m: medium, s: strong.
FIG. 5. Resonance Raman spectra in the 1800–2800 cm�1 region of bacteriacontaining carotenoids and xanthomonadin excited with the 514.5 nm laser (a)Microbacterium testaceum (Mte), (b) Paenibacillus validus (Pva), (c) Bacillusmegaterium (Bme), (d) Clavibacter michiganensis subsp. michiganensis(Cmm), and (e) Xanthomonas axonopodis pv. dieffenbachiae (Xad).
APPLIED SPECTROSCOPY 439
band represents guanine, cytosine, uracil, and citric acid, andthe 1173 cm�1 band represents tyrosine.2,3,16 R. solanacearumis one of the most devastating pathogens in the tropics, sub-tropics, and temperate regions of the world; it infests numerouscrops including tomato, eggplant, peanut, banana, potato,ginger, and mulberry. R. solanacearum race 3 biovar 2 strainshold great significance for the USA as it is classified as a selectagent under the Agricultural Bioterrorism Act of 2002. Thestrain A4515 is representative of strains infecting edible gingerin Hawaii. Identifying the mechanisms of the close associationof this soil bacterium with the plant root exudates is the key tounderstanding the successful colonization of R. solanacearumand invasion into the plants. The Raman metabolic profiling ofR. solanacearum under diverse nutritional conditions will helpus to understand more of this complex system. This should leadto the formulation of strategies for management of the diseaseby identifying non-supportive nutritional systems for thispathogen.
X. axonopodis pv. dieffenbachiae showed totally uniqueRaman fingerprints among all bacteria tested in our study. Thestrong bands at 1135 and 1531 cm�1 shown in Fig. 2e areunique and do not represent the usual carotenoid bands33,34 ofbacteria assigned in numerous publications in Raman spec-troscopy. The same bands were confirmed when excited withthe 514.5 nm laser and appeared at 1136 and 1529 cm�1, asshown in Fig. 4e. The X. axonopodis pv. dieffenbachiaebelongs to the bacterial family Xanthomonads and all speciesin this family possess the pigment xanthomonadin, which isstructurally similar to carotenoids.35,36 These pigments arebrominated, aryl-polyene, yellow, water-insoluble pigmentsthat are associated exclusively with the outer membrane of thecell wall of the Xanthomonads. The biological role of thexanthomonadin is to protect the organism from photo-biological damage and it may also be of significance inepiphytic survival of the pathogen in numerous host plants.36,37
Here we describe the first report of the Raman fingerprints ofthe pigment xanthomonadin in X. axonopodis pv. dieffenba-chiae by Raman spectroscopy. Further work is in progress toevaluate numerous pathovars of the bacterium to confirmwhether it could be assigned as a Raman marker for the genusXanthomonas.
Resonance Raman spectra of carotenoids and xanthomona-
din-containing bacterial samples excited with the 514.5 nmlaser were also analyzed in the 1800–3200 cm�1 region (Fig.5), and the tentative band assignments38 of 2v3, v1þv2, v1þv3,v2þv3, v2þv4, 2v1, and 2v2 are given in Table III. M. testaceumshowed strong vibrational bands associated with carotenoids at2157, 2307, 2515, 2665, and 3029 cm�1 corresponding tov2þv3, 2v2, v1þv3, v1þv2, and 2v1 modes. P. validus and C.michiganensis subsp. michiganensis showed comparativelyweaker bands than M. testaceum. B. megaterium did not showany bands in this region. X. axonopodis pv. dieffenbachiaeshowed a strong-intensity band at 2267 cm�1 corresponding to2v2 overtone, which is distinct from the bands associated withother bacteria. These assignments further distinguish Gþbacteria from the G� X. axonopodis pv. dieffenbachiae.
Principal component analysis on the Raman spectral data ofnine plant bacteria measured with the 785 nm laser excitationin the 400 to 1800 cm�1 range showed that the first three PCsexplained 88% of the variance and four PCs explained 92.7%of the variance. Figure 6 shows a PCA plot of the first threePCs of the plant bacteria tested in this study. The three-dimensional perspective presented in Fig. 6 shows cleardistinction between the bacteria. The principal components ofall the G� bacteria that do not contain any pigment lined upconsistently and along with the Gþ bacteria B. megaterium(Bme), as shown in Fig. 6. The Raman spectrum of B.megaterium has weak carotenoid bands compared with otherGþ bacteria as seen in Figs. 1 and 3, and this may be the reasonthat it clusters with G� bacteria, albeit with marginaldifferences. The Gþ bacteria C. michiganensis subsp. michi-ganensis (Cmm), M. testaceum (Mte), and P. validus (Pva) andthe G� bacteria Xanthomonas campestris pv. dieffenbachiae(Xad) clearly separate out in the PCA analysis.
Because it is difficult to fully present PCA data in a singleperspective of three-dimensional PC space, a dendrogram39
was constructed based on Mahalanobis distances40 (dM)between the groups of scores for the first four PCs (Fig. 7),which better illustrates the discrimination between the bacterialspecies. As observed in the PCA plot in Fig. 6, the G� bacteriawith no pigments cluster closer together below ;0.5 dM (Fig.7). Some intermixing between these G� bacteria is noted; oneof the dM of R. solanacearum (Rso) is close to the E.
FIG. 7. Dendrogram showing classification between different bacterial speciesbased on Mahalanobis distances between scores of first four principalcomponents.
FIG. 6. Three-dimensional PCA plot showing discrimination between plantbacteria (�) Arh: Agrobacterium rhizogenes; (*) Bme: Bacillus megaterium;(n) Cmm: Clavibacter michiganensis pv. michiganensis; (¤) Ech: Erwiniachrysanthemi; (¤) Ecl: Enterobacter cloacae; (§) Mte: Microbacteriumtestaceaum; (&) Pva: Paenibacillus validus; (&) Rso: Ralstonia solanacea-rum; and (,) Xad: Xanthomonas axonopodis pv. dieffenbachiae).
440 Volume 64, Number 4, 2010
chrysanthemi (Ech) cluster, and one of the dM of the A.rhizogenes (Arh) is close to the Rso cluster. However, there isno mixing with Gþ bacteria containing carotene or with G�bacteria X. campestris pv. dieffenbachiae (Xad) containing thexanthomonadin pigment. Bacterial strains with these uniquepigments (C. michiganensis subsp. michiganensis, M. testa-ceum, P. validus, and X. campestris pv. dieffenbachiae)separate out easily in the dendrogram from the strains that donot have these pigments (A. rhizogenes, B. megaterium, E.cloacae, E. chrysanthemi, and R. solanacearum). Based on thisstudy it is confirmed that Raman spectroscopy cannot be usedto definitively separate out Gþ from the G� bacteria; however,there is a good prospect of clear distinction between strainswith these unique pigments, which further validates theuniqueness of the Raman bands identified in these bacteriaspecies.
CONCLUSION
Plant-pathogenic and plant-associated bacteria are bio-chemically characterized by Raman spectroscopy and can beseparated out on the basis of the presence or absence of variousRaman peaks that serve as identifiable markers for the testedbacteria. In addition, the intensities of various peaks give usadditional information on a variety of aspects related tobacteria–plant interactions in diverse environments. Theidentification of xanthomonadin in Xanthomonas axonopodispv. dieffenbachiae by a micro-Raman spectroscope equippedwith both 785 and 514.5 nm lasers is a significant finding inour study. The in-phase C¼C (v1) and C–C stretching (v2)vibrations of the polyene chain of carotenoids separated Gram-positive and Gram-negative plant-associated bacteria. The PCAanalysis of the 785 nm excited spectra differentiate variousstrains based on the unique pigments these bacteria do or donot possess, but clear separation into Gram-positive and Gram-negative classification is not possible. Raman spectroscopyoffers the science of plant bacteriology a tool to furtherunderstand the intricate mechanisms involved in host–pathogeninteractions.
ACKNOWLEDGMENTS
Our sincere thanks to Anupam K. Mishra and Tayro Acosta for theirvaluable help in setting up Raman experiments and help in analyzing the data,and Asoka S. deSilva for culturing all the tested bacteria used in our study. Weare grateful to Dr. Barry Lienert and Dr. David Bates for their assistance withPCA analysis of the Raman data. The project was partially funded by USDASpecial Grants program for Tropical and Subtropical Agricultural Research(Award No. 2004-34135-15191) and USDA-ARS/Minor Crops Research(Award No. 59-5320-1-525). Raman instruments used in the present work werefunded by the Army Research Laboratory under a cooperative agreement(WA11MF-07-2-0002). The authors would like to thank the associate editorand the reviewer for their constructive suggestions. The authors would also liketo thank Nancy Hulbirt and May Izumi for their valuable help with figures andediting, respectively. This is SOEST contribution No. 7888 and HIGPcontribution No. 1835.
1. C. Xie and Y. Li, J. Appl. Phys. 93, 2982 (2003).2. J. D. Gelder, P. Scheldeman, K. Leus, M. Heyndrickx, P. Vandenabeele, L.
Moens, and P. D. Vos, Anal. Bioanal. Chem. 389, 2143 (2007).3. E. C. Lopez-Diez and R. Goodacre, Anal. Chem. 76, 585 (2004).4. A. Sengupta, M. Mujacic, and E. J. Davis, Anal. Bioanal. Chem. 386, 1379
(2006).
5. M. L. Laucks, A. Sengupta, K. Junge, E. J. Davis, and B. D. Swanson,Appl. Spectrosc. 59, 1222 (2005).
6. M. Culha, A. Adiguzel, M. M. Yazici, M. Kahraman, F. Sahin, and M.Gulluce, Appl. Spectrosc. 62, 1226 (2008).
7. A. A. Kamnev, P. A. Tarantilis, L. P. Antonyuk, L. A. Bespalova, M. G.Polissiou, M. Colina, P. H. E. Gardiner, and V. V. Ignatov, J. Mol. Struct.536–564, 199 (2001).
8. Z. Movasaghi, S. Rehman, and I. U. Rehman, Appl. Spectrosc. Rev. 42,493 (2007).
9. J. D. Gelder, K. D. Gussem, P. Vandenabeele, and L. Moens, J. RamanSpectrosc. 38, 1133 (2007).
10. G. N. Agrios, Plant Pathology (Elsevier Academic Press, Burlington, MA,2005), 5th ed.
11. G. L. Schuman and C. J. D’Acry, Essential Plant Pathology (AmericanPhytopathological Society Press, St. Paul, MN, 2006).
12. J. G. Holt, N. R. Kreig, P. H. A. Sneath, J. T. Staley, and S. T. Williams,Bergey’s Manual of Determinative Bacteriology (Lippincott Williams andWilkins, Baltimore, MD, 2000), 9th ed.
13. P. V. Zinin, A. Misra, L. Kamemoto, Q. Yu, and S. K. Sharma, J. Opt. Soc.Am. B 24, 2779 (2007).
14. A. K. Misra, S. K. Sharma, L. Kamemoto, P. V. Zinin, Q. Yu, N. Hu, andL. Melnick, Appl. Spectrosc. 63, 373 (2009).
15. C. Sandt, T. Smith-Palmer, J. Pink, and D. Pink, Appl. Spectrosc. 62, 975(2008).
16. J. D. Gelder, K. D. Gussem, P. Vandenabeele, M. Vancanneyt, P. D. Vos,and L. Moens, Anal. Chim. Acta 603, 167 (2007).
17. L. J. Goeller and M. R. Riley, Appl. Spectrosc. 61, 679 (2007).
18. A. M. Alvarez, P. J. Toves, and T. S. Vowell, APSnet Feature (Feb. 2006),http://www.apsnet.org/online/feature/anthurium.
19. U. Neugebauer, U. Schmid, K. Baumann, W. Ziebuhr, S. Kozitskaya, V.Deckert, M. Schmitt, and J. Popp, ChemPhysChem 8, 124 (2007).
20. K. C. Schuster, E. Urlaub, and J. R. Gapes, J. Microbiolog. Meth. 42, 29(2000).
21. C. Mitchell, S. Iyer, J. F. Skomurski, and J. C. Vary, Appl. Environ.Microbiol. 52, 64 (1986).
22. H. Schulz, M. Baranska, and R. Baranski, Biopolymers 77, 212 (2005).
23. H. Schulz and M. Baranska, Vib. Spectrosc. 43, 13 (2007).
24. Q. Wu, T. Hamilton, W. H. Nelson, S. Elliott, J. F. Sperry, and M. Wu,Anal. Chem. 73, 3432 (2001).
25. S. Saperstein and M. P. Starr, Biochem. J. 57, 273 (1954).
26. S. Saperstein, M. P. Starr, and J. A. Filfus, J. Gen. Microbiol. 10, 85(1954).
27. W. S. Kaneshiro, C. Y. Mizumoto, and A. M. Alvarez, Eur. J. Plant Pathol.116, 45 (2006).
28. W. S. Kaneshiro, M. Burger, B. G. Vine, A. S. deSilva, and A. M. Alvarez,Plant Disease 92, 1444 (2008).
29. K. A. Nishijima, A. M. Alvarez, P. R. Hepperly, M. H. Shintaku, L. M.Keith, D. M. Sato, B. C. Bushe, J. W. Armstrong, and F. T. Zee, PlantDisease 88, 1318 (2004).
30. J. A. Leigh and D. L. Coplin, Ann. Rev. Microbiol. 46, 307 (1992).
31. I. K. Toth, L. Pritchard, and P. R. J. Birch, Ann. Rev. Phytopathol. 44, 305(2006).
32. J. Schell and M. Van Montagu, Basic Life Sci. 9, 159 (1977).
33. W. F. Howard, Jr., W. H. Nelson, and J. F. Sperry, Appl. Spectrosc. 34, 72(1980).
34. J. R. Goodwin, L. M. Hafner, and P. M. Fredericks, J. Raman Spectrosc.37, 932 (2006).
35. M. P. Starr, C. L. Jenkins, L. B. Bussey, and A. G. Andrewes, Arch.Microbiol. 113, 1 (1977).
36. W. W. C. Chun, The Plant Health Instructor (2002) DOI: 10.1094/PHI-A-2000-0824-01, http://www.apsnet.org/education/advancedplantpath/topics/xanthomonadins/default.htm.
37. W. Chun, J. Cui, and A. Poplawsky, Physiol. Mol. Plant Pathol. 51, 1(1997).
38. R. J. H. Clark, N. R. D’Urso, and P. F. Zagalsky, J. Am. Chem. Soc. 102,6693 (1980).
39. K. V. Mardia, J. T. Kent, and J. M. Bibby, Multivariate Analysis(Academic Press, San Diego, CA, 1979).
40. P. C. Mahalanobis, Proc. Natl. Inst. Sci. India 2, 49 (1936).
APPLIED SPECTROSCOPY 441
