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Handheld Raman spectroscopy for the early

detection of plant diseases: Abutilon mosaic
virus infecting Abutilon sp.

Article in Analytical methods · March 2016

DOI: 10.1039/C6AY00381H

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 Volume 8 Number 17 7 May 2016 Pages 3399–3638

Analytical
Methods
www.rsc.org/methods

ISSN 1759-9660

PAPER
Valerian Ciobotă et al.
Handheld Raman spectroscopy for the early detection of plant diseases:
Abutilon mosaic virus infecting Abutilon sp.
Analytical
Methods
PAPER

Handheld Raman spectroscopy for the early

detection of plant diseases: Abutilon mosaic virus
Cite this: Anal. Methods, 2016, 8, 3450
infecting Abutilon sp.
Sivaprasad Yeturu,†a Paul Vargas Jentzsch,†ab Valerian Ciobotă,*c Ricardo Guerrero,a
Patricia Garridoa and Luis A. Ramosa

Received 6th February 2016
Accepted 23rd March 2016
DOI: 10.1039/c6ay00381h
www.rsc.org/methods
Plant diseases have a direct impact on the productivity of crops, and therefore the early detection of
diseases is crucial. Abutilon mosaic virus (AbMV) (family Geminiviridae; genus Begomovirus) is an
important virus infecting ornamental crops throughout the world. Abutilon hybridum showing bright
yellow mosaic symptoms were observed in gardens in Tumbaco, Ecuador. The infection was confirmed
by Polymerase Chain Reaction (PCR) using degenerate begomovirus primers. The amplicon (!500 bp)
was sequenced, submitted to NCBI (KP877621) and showed 68.7–100% and 72.7–100% sequence
identity with other begomoviruses at nucleotide and amino acid levels, respectively. In order to evaluate
Raman spectroscopy as a diagnostic tool for AbMV infection, spectra of leaves from healthy and infected
plants were recorded. Raman signals of carotenoids are the most important features and there is
a significant decrease in the intensity of these bands when the plant is infected. The difference in the
intensity of the bands, particularly the one at 1526 cm"1, is proposed as a basis for the early detection of
viral infection in plants.

spread throughout the world.3–5 It was rst puried and

Introduction
It is a well-known fact that plant diseases have a direct impact
on the productivity of crops. Disease-causing agents can be
classied into noninfectious and infectious disease-causing
agents.1 Most of the concern arises due to infectious disease-
causing agents (e.g., viruses, viroids, mycoplasma, bacteria,
fungi, nematodes or other parasites), because the disease can
spread to neighboring crops and large areas can be affected.
Due to their characteristics, virus infections in plants are feared
worldwide. They can spread very fast in crops and cause
important losses in both crop yield and quality.
Abutilon hybridum is an ornamental shrub belonging to the
family Malvaceae, and it is widely cultivated in tropical and sub-
tropical areas of the world. It is one of the largest shrubs among
approximately 150 species of broadleaf evergreens in the
mallow family. Abutilon mosaic virus (AbMV), a bipartite bego-
movirus, is one of the rst viruses described in scientic
literature.2 AbMV is propagated in ornamental plants and has
a
Ecuadorian Agency for Quality Assurance in Agriculture, AGROCALIDAD, Av.
Interoceánica km 14 1/2, 170184 Tumbaco, Ecuador
b
Departamento de Ciencias Nucleares, Facultad de Ingenierı́a Quı́mica y
Agroindustria, Escuela Politécnica Nacional, Ladrón de Guevara E11-253, 170525
Quito, Ecuador
c
Rigaku Analytical Devices, Pasedagplatz 3-4, 13088 Berlin, Germany. E-mail:
Valerian.Ciobota@rigaku.com
† These authors contributed equally to this work.
characterized by Abouzid and Jeske.6 AbMV has a circular,
single stranded DNA (ssDNA) bipartite genome belonging to the
family Geminiviridae and genus Begomovirus, and the virus is
transmitted by whitey (Bemisia tabaci).7 AC2 is a major
pathogenicity determinant for geminiviruses and able to acti-
vate transcription in one hybrid yeast assay (Aberle & Jeske,
unpublished results), which is necessary for systemic infection,8
but not essential for any of the three modes of viral replication
(complementary strand replication (CSR), rolling circle repli-
cation (RCR) and recombination-dependent replication
(RDR)).9 Begomovirus is the largest genus with more than 180
species and several unassigned isolates.10 Many begomoviruses
have a bipartite genome that consists of DNA-A and DNA-B,
each approximately 2.7 Kb in size. DNA-A typically contains six
open reading frames (ORF): AV1 (known as AR1; coat protein,
CP) and AV2 (known as AR2; A V2 protein or movement protein,
MP) on the virion-sense strand; AC1 (known as AL1; replication
protein, Rep), AC2 (known as AL2; transcriptional activator,
TrAP), AC3 (known as AL3; replication enhancer, REn) and AC4
(known as AL4; AC4 protein) on the complementary-sense
strand. The coat protein (CP) of geminiviruses is a multifunc-
tional protein required for a range of functions associated with
encapsidation, accumulation of ssDNA, insect transmission
and both intra- and inter-cellular movement,11 while DNA-B is
involved in the systemic viral movement, symptom expression
and host range determination.12–14 Abutilon is an ornamental
garden plant which is associated with different begomoviruses

3450 | Anal. Methods, 2016, 8, 3450–3457 This journal is © The Royal Society of Chemistry 2016
Paper Analytical Methods

such as Abutilon mosaic virus-Hawaii (AbMV-HW), Abutilon it is also important that no sample preparation is involved, thus
mosaic Bolivia virus (AbMBoV), Abutilon mosaic Brazil virus the analysis can be made in a very short time and without
(AbMBV), Sida micrantha mosaic virus (SimMV), Sida leaf curl special materials and equipment. The detection of virus infec-
virus (SiLCV) and Abutilon mosaic India virus (AbMIV).15 Some tions in plants is a challenging task and Raman measurements
other begomoviruses are also found in different parts of the could provide essential information. The virus infection could
world, i.e., Rhynchosia rugose golden mosaic virus (RhRMV) on be detected not only by direct measurement, but also by
Rhynchosia minima, Sida golden mosaic virus (SiGMV) on Pha- measuring the symptoms the plant is showing, even if they are
seolus vulgaris, Sida yellow mottle virus (SiYMoV) on Malvastrum not visible to the naked eye.
coromandelianum, Okra yellow mosaic Mexico virus (OYMMV) on In this work, we report the natural occurrence of AbMV in
Corchorus siliquosus, Corchorus yellow spot virus (CoYSV) on ornamental Abutilon. The virus was isolated and compared to
Corchorus siliquosus, and Jatropha mosaic virus (JaMV) on other closely related viruses in other countries. In addition, the
Jatropha. The geminiviruses of DNA encode 5–7 proteins are Raman spectra of the leaves of healthy and infected plants were
host machineries and process to establish a productive infec- recorded using a portable Raman spectrometer, and the most
tion. The above interactions are reprogramme of plant cell cycle important bands were assigned and discussed. The early
and transcriptional controls, inhibit cell death pathways, detection of virus infection in Abutilon by means of Raman
interfere with cell signalling and protein turnover, and suppress spectroscopy is proposed for in situ screening analysis.
defense pathways.16
The great variety of virus affecting plants worldwide oen
results in dramatic reduction in productivity. As happens with Experimental
other plant diseases, the fast detection of a virus infection is Identication and characterization of AbMV
crucial in order to control the spread of the disease. Although
Sample collection. During November 2014, bright yellow
conventional molecular methods are very useful in the detec-
mosaic symptoms were observed on leaves of Abutilon (Fig. 1) in
tion and identication of viruses, they require special facilities,
a garden (n ¼ 10) in Tumbaco, Pichincha province of Ecuador.
equipment as well as expensive supplies. Moreover, considering
Leaf samples were brought to the laboratory under cold condi-
that agricultural elds are usually located in remote areas,
tions and stored at "80 $ C until processed for DNA extraction.
screening analytical methods for the detection of virus infec-
Total DNA extraction. Total genomic DNA was extracted from
tions in plants should be investigated. Therefore Raman spec-
healthy and symptomatic leaf samples of Abutilon using
troscopy arises as an interesting option to be applied as
a DNeasy Plant Mini Kit (Qiagen, USA), according to the man-
a screening technique.
ufacturer's instructions. The DNA quality and concentration
Raman spectroscopy is used to analyze the inelastic scat-
were measured using a NanoDrop ND-1000 spectrophotometer
tering of monochromatic light (usually from a laser). Light
(NanoDrop Technologies, Wilmington, DE).
interacts with the sample and a very small proportion of the
Polymerase chain reaction (PCR). Total DNA was extracted
photons suffer inelastic scattering, resulting in shis (up or
from two asymptomatic and eight symptomatic leaves and was
down) in their wavelengths. These shis provide information
subjected to PCR using CP gene specic primers (AV494 –
on the vibrational modes of the substances present in the
forward – GCC(C/T)AT(G/A)TA(T/C)AG(A/G)AAGCC(A/C)AG and
sample, thus allowing the characterization of the sample. As
AC1048 – reverse – GG(A/G)TT(A/G/T)GA(G/A)GCATG(T/A/C)
explained by Schmitt and Popp,17 the great development of
GTACATG).34 Amplicons of the expected size (500 bp) were
Raman spectroscopy in the last few decades was triggered by
detected in the case of diseased plant samples. The PCR
important advances in the efficient ltration of the elastic
program was as follows: 94 $ C for 5 min, 35 cycles at 94 $ C for 30
scattered light, among other technological advances. Currently,
Raman spectroscopy has applications in almost all disciplines
of natural sciences.17 Since it is a non-destructive technique, it
was used in the characterization of materials in several areas
including microbiology, arts, archaeology, geology, atmo-
spheric sciences and planetary exploration, among many
others.18–26 Application of Raman spectroscopy was included in
the quality control of certain products. Application of Raman
spectroscopy in forensic research was also proposed in the
past.27–31 Tip enhanced Raman scattering (TERS), one of the
variations of Raman spectroscopy, was successfully used to
investigate a single tobacco mosaic virus.32 Surface Enhanced
Raman Scattering (SERS) combined with chemometrics was
also suggested for the detection of certain virus infections.33
These two studies conrmed the great potential of the varia-
tions of Raman spectroscopy. Fig. 1 Abutilon mosaic virus infected Abutilon hybridum plant
Modern portable Raman spectrometers offer particular showing bright yellow mosaic symptoms (left: sick plant and right:
advantages for in situ measurements. For in situ measurements, healthy plant).

This journal is © The Royal Society of Chemistry 2016 Anal. Methods, 2016, 8, 3450–3457 | 3451
Analytical Methods
s, 52 $ C for 1 min, 72 $ C for 1 min, and nal extension of 72 $ C
for 10 min in a thermal cycler (Bio-Rad, USA). Amplied
products were resolved following electrophoresis through a 1%
agarose gel containing SYBR® Safe (Invitrogen, USA).
Sequencing and cluster analysis. The amplied PCR
product was puried by using a QIAquick PCR Purication Kit
(Qiagen, USA) and sequenced on both strands at Macrogen,
Seoul, Korea. Multiple sequence alignments were generated
using BioEdit (version 5.09).35 Both nucleotide and amino
acid sequences of coat protein genes of different begomovi-
rus species were compared and the corresponding
cluster tree was generated. Phylogenetic relationships were
determined by the neighbor-joining method with 1000
bootstrap replicates in MEGA version 4.02.36 The coat protein
genes of other known begomoviruses were collected from
GenBank.37
Raman spectroscopic measurements of leaves and data
processing
Fully developed leaves of healthy, early-stage infected and full
infected plants were measured. The term “early-stage infected”
is used to refer to leaves in which the symptoms of the disease
are invisible to the naked eye. These leaves, with a healthy
appearance, were collected from plants showing the symptoms
of the virus infection in other leaves and are considered to
represent leaves from plants at an early stage of infection. The
term “full infected” denotes leaves of a fully infected plant with
the leaves clearly showing the symptoms of the disease. These
terms were arbitrarily adopted in order to achieve an appro-
priate qualitative differentiation on the degree of infection of
the plant. All leaves for Raman measurements were collected
from plants in the same garden eld. The leaves were removed
from the plants in order to ensure that leaves of similar size
were measured. A total of 20, 19 and 15 leaves from healthy,
early-stage infected and full infected plants, respectively, were
measured in situ. One measurement was performed for each
leaf. For the measurements, the leaves were put in contact with
the spectrometer nose.
The Raman measurements were performed using a hand-
held Raman spectrometer, Progeny™ (Rigaku Raman Tech-
nologies, Wilmington, MA, USA), equipped with a 1064 nm
Nd:YAG laser. The size of the laser spot was 25 mm. The Raman
scattered light was detected by using a Peltier cooled InGaAs
photodiode array. The total exposure time for a Raman spec-
trum was 8 s, and the laser power was 200 mW. The acquired
spectra were automatically baseline corrected by the handheld
spectrometer soware. The accuracy and precision of the
Raman bands of the acquired spectra were below 3 cm"1 and
1.5 cm"1, respectively.
In order to evaluate the variability in the intensity of the
Raman features of the spectra of the leaves (healthy, early-stage
infected and full infected), a boxplot based on the intensity of
the strongest Raman band was obtained using “R” soware.38
Statistical analysis was carried out using Statistica 8 Soware.
Data were subjected to analysis of variance (ANOVA) followed by
the Tukey HSD test.
3452 | Anal. Methods, 2016, 8, 3450–3457
Paper
Results and discussion
Identication and characterization of AbMV
The eight AbMV-infected plants showing bright yellow mosaic
symptoms on Abutilon leaves (Fig. 1) were collected from
a garden in Tumbaco, Pichincha province in Ecuador. The total
DNA extracted from the eight infected and two healthy abutilon
leaves were subjected to PCR using the coat protein gene of
begomoviruses, resulting in an amplicon with the expected size
of !500 bp (Fig. 2). The PCR products were puried and
sequenced in both directions with gene specic primers and
BLAST analysis. The sequenced product was deposited in Gen-
Bank (Accession no. KP877621). The CP gene of AbMV was 500
nucleotides long and coded for a protein of 163 amino acids.
The coat protein gene sequence of our AbMV isolate was
compared with those of other reported begomoviruses (Gen-
Bank Accession no. HQ588901, X15983, U51137, AF058013,
HM236370, GQ357649, JN411687, DQ318929, KJ174331,
DQ875868, KF723260, HM585445, FN434438, AJ557450 and
DQ641707) at nucleotide and amino acid levels, respectively.
The coat protein gene of AbMV shared 68.7–100% identity at
the nucleotide level and 72.7–100% at amino acid levels with
reported begomovirus isolates (Table 1). The cluster tree at the
nucleotide level was constructed using MEGA version 4.1 and it
further showed clustering of KP877621 (AbMV-Ecuador) with
X15983 (AbMV-Germany), HQ588901 (AbMV-India), AF058013
(AbMV-USA) and U51137 (AbMV-HW-USA), forming a separate
clade (clade I). HM236370 (RhRGMV-Cuba), GQ357649 (SiGMV-
Florida), JN411687 (SiYMoV-Cuba), DQ318929 (OYMMV-Mex-
ico) and DQ875868 (CoYSV-Mexico) segregated into a separate
clade (clade II). KJ174331 (JaMV-Dominican Republic) with
KF723260 (JaMV-Jamaica) formed into another separate clade
(clade III). Furthermore, a separate clade (clade IV) was formed
by FN434438 (AbMBV-Brazil) and AJ557450 (SimMV-Brazil),
whereas HM585445 (AbMBoV-Bolivia) and DQ641707 (SiLCV-
Viet Nam) formed individual clades (see Fig. 3).
The begomoviruses considered in this work, which belong to
the family Geminiviridae, are one of the largest groups of plant
viruses. They are bipartite genome viruses and show a systemic
Fig. 2 Agarose gel electrophoretic analysis of amplicons of PCR for
the detection of AbMV in Abutilon. Lane M-1 Kb DNA ladder; lane 1, 2,
3, 4-AbMV infected abutilon plant samples; lane 5-healthy abutilon
plant sample.
This journal is © The Royal Society of Chemistry 2016
Paper Analytical Methods

spread in different parts of the infected plants; in some plants

KP877621 HQ588901 X15983 U51137 AF058013 HM236370 GQ357649 JN411687 KJ174331 DQ318929 DQ875868 KF723260 HM585445 FN434438 AJ557450 DQ641707
they are restricted to the phloem and in others they invade
many tissue types. The disease incidence depends on the

73.9
73.3
75.1
73.3
72.7
75.1
75.1
76.3
76.9
75.1
75.1
76.9
78.1
74.5
74.5
100
Sequence identities of the studied isolates at amino acid (above the diagonal) and nucleotide (below the diagonal) levels, respectively, with other reported begomoviruses

interaction between the plant host cells and viral gene products.
The role of DNA-A and DNA-B genome components in bego-
moviruses in phloem and non-phloem transmission was

71.4
94.6
95.2
94.6
92.8
92.8
95.8
95.2
95.8
95.2
95.2
95.2
95.2
90.5
97.6
100
studied in the past.39,40 The AbMV found in Ecuador and re-
ported in this work, belongs to the subgroup-III and is trans-
mitted by Bemisia tabaci.41 For the detection of AbMV-Ecuador

70.2
94.6
95.2
94.6
92.8
92.8
95.8
95.2
95.8
95.2
95.2
95.2
95.2
90.5

88.7
100
degenerate universal primers were used, since in earlier reports
these primers were successfully used to identify begomovi-
ruses.42–44 The degenerate primers consisted of mixtures of
similar but not identical primers with base changes in one or

71.4
88.7
89.3
89.9
86.9
86.9
89.9
89.3
91.1
89.3
89.9
89.9
89.3

82.4
82.8
100
more positions. They serve as universal primers that amplify
a DNA fragment from all begomoviruses. Narayana et al. re-
ported that the begomoviruses also infected different hosts,
71.4
95.8
95.2
95.8

92.8

96.4

96.4
96.4

82.2
87.7
88.9
100

100

such as Phaseolus vulgaris, Jatropha curcas, Euphorbia pulcherima

94

97

97

and Codiaeum varigatum.45

In the Tumbaco gardens, where the bright yellow mosaic
symptoms were observed in Abutilon, the disease incidence was
71.4
96.4

96.4

98.2
97.6
98.2
96.4

89.1
82.6
86.4
88.1
100
100
97

97

94

more than 90%. During the sample collection, B. tabaci vectors

were observed on leaves, as previously reported.41 Later, the
infected plants were properly removed to eradicate the infection
71.4
96.4

96.4

98.2
97.6
98.2
96.4

89.1
82.6
86.4
88.1
100
100

and avoid further spread. To the best of our knowledge, this is

97

97

94

the rst report of the natural occurrence of AbMV on Abutilon in

Tumbaco, Ecuador.
72.2
95.8
95.2
95.8

92.8

96.4

90.5
90.5

83.2
87.9
90.1
100
94

97

97

97

Interpretation of the Raman spectroscopic information

71.6
97.6

97.6
95.8
94.6
98.8
98.2

91.9
93.3
93.3
91.7
81.8
87.7
87.9

The average Raman spectra of healthy leaves as well as early

100
97

infection stage leaves (called “early-stage infected” leaves in this

work) and leaves of a fully infected plant (called “full infected”
leaves in this work), without normalization and normalized on
70.6
96.4

95.2

98.2

94.6
91.7
92.9
92.9
91.3
82.4
88.3
100
97

97

94

87

the band at 1526 cm"1, are shown in Fig. 4a and b, respectively.

The assignment of the most important bands occurring in the
average Raman spectra of healthy, early-stage infected and full
infected leaves is discussed in the following.
97.6

97.6
95.8
94.6

93.5
94.6
90.3
91.7
91.7
91.1
82.4
87.9
87.5
100

72
97

Taking into account that carotenoids are strong Raman

scatterers, it is expected that the Raman spectra of the leaves are
dominated by bands of carotenoids. As anticipated, strong
68.7
95.8
95.2
95.8
93.4

92.7
91.9
90.9
90.3
90.5
90.5
89.7
82.6
87.7
88.1
100

Raman signals of carotenoids are observed in the recorded

spectra. Based on previously published Raman spectroscopic
data,46,47 the bands occurring at 1526, 1155 and 1003 cm"1 can
94.6

94.6

95.8

92.9
92.1
92.7

91.7
92.1
92.1
91.1

88.1
88.3
100

70
94

82

unambiguously be assigned to the C]C stretching, C–C

stretching and in-plane CH3 rocking modes of carotenoids. The
69.4
98.8
98.2

92.1

91.1
91.1
96.4
96.8

92.7
93.1

91.3

90.7
82.4
87.9
87.7
100

bands at 1388, 1328, 1266, 1210, 1187 and 958 cm"1 can also be
attributed to carotenoids, since the Raman spectra of some
carotenoids (e.g., b-carotene and lutein) show similar Raman
features.46,48,49 The band at 1388 cm"1 can be assigned to a CH3
93.3
92.3

91.3
91.3

69.2
99.4

98.2
96.6

93.3

91.7

91.1

88.7
88.5
100

97

83

bending mode. It is known that a CH3 bending mode appears in

the vicinity of 1380 cm"1 as a weak Raman band, unless the CH3
group is directly attached to C]C, C^C, C]O or to an
69.4
96.2
96.6

92.7
91.7

90.9
90.9
98.4
98.2

93.1

91.1

90.7
82.6
88.1
87.7
100

aromatic ring. In these cases the band appears as medium to

strong.50 In general, the structure of carotenoids shows various
HM236370

HM585445
HQ588901

DQ318929
DQ875868

DQ641707
GQ357649

FN434438
KP877621

KF723260
AF058013

JN411687
KJ174331

AJ557450

CH3 groups directly attached to C]C, but the band for this
Isolates

U51137
X15983
Table 1

bending is weak compared to the strongest signals in the

spectra. Spectroscopic data published in the past50,51 suggest the

This journal is © The Royal Society of Chemistry 2016 Anal. Methods, 2016, 8, 3450–3457 | 3453
Analytical Methods
Fig. 3 Cluster tree constructed using the neighbor joining method of MEGA version 4.1. The cluster tree based on the coat protein gene
nucleotide sequences of AbMV isolates with those of other reported begomoviruses.
assignment of bands at 1328 and 1266 cm"1 to ]CH rocking
modes. In addition, the band at 1187 cm"1 is also assigned to
a C–C stretching mode, while the band at 1210 cm"1 seems to
be related to a CH deformation mode and/or a C–C stretching
mode. The weak band at 958 cm"1 is assigned to a ]CH
wagging mode of carotenoids.
On the basis of earlier reports,52 the weak bands at 1353,
1287 and 913 cm"1 as well as the shoulders at 1553 and 986
cm"1 were attributed to chlorophyll. However, the inuence of
other weak bands that occur for certain carotenoids at !1350
and !1280 cm"1 (ref. 46) cannot be ruled out. The intensity of
the bands of chlorophylls is remarkably weaker than the
intensity of characteristic carotenoids bands. Therefore, the
chlorophyll Raman features can only be observed in the average
Raman spectra of leaves from healthy plants and plants with an
earlier-stage infection. In the case of fully infected plants, the
Raman features of chlorophyll are too weak to appear.
Other constituent substances that are usually present in
vegetable materials which could contribute Raman signals to
the spectra of leaves are cellulose, hemicellulose and lignin. It is
known that hemicellulose and lignin are amorphous, while
cellulose is semicrystalline with crystallinity degrees reaching
levels as high as 65% in certain woods.53 Hemicelluloses are
expected to be intimately associated with cellulose. According to
Agarwal and Ralph,54 hemicellulose does not contribute any
specic Raman feature compared to those associated with
cellulose. The strongest and most representative bands for
cellulose occur at 1094 and 1120 cm"1,55 and none of these
bands are visible in the average spectra of leaves. On the other
hand, characteristic bands of lignin are visible.
Lignin is a complex and heterogeneous biopolymer
composed primarily of p-hydroxyphenyl (H), guaiacyl (G) and
3454 | Anal. Methods, 2016, 8, 3450–3457
Paper
syringyl (S) units.56 However, some researchers suggested that
the biosynthesis of lignin may be much more complex than
previously thought, including other units, especially when
mutant and transgenic plants are considered.57 Since the
S : G : H ratio (if only these three units are considered) can
change, the Raman bands of lignin can appear in specic
ranges, as reported by Sun et al.56 Taking into account these
considerations and the published data, the bands at 1665, 1629
and 1605 cm"1 were attributed to lignin. The band at 1665 cm"1
can be assigned to either C]C or C]O stretching modes. The
band at 1629 cm"1 is assigned to a C]C stretching mode, while
the band at 1605 cm"1 is assigned to a ring quadrant stretching
mode. The presence of certain essential oils may affect the
Raman spectra, especially in the region 1700–1600 cm"1, since
many terpene compounds show strong Raman bands in this
spectral region.58 Contributions of this kind to the observed
bands in the Raman spectra of leaves cannot be ruled out.
Bands in the vicinity of !1450 cm"1 (1460 and 1440 cm"1)
are assigned to bending modes of CH3 and CH2 groups. Several
compounds contain CH2 and CH3 groups in their structure, and
therefore it is not possible to attribute these bands to a specic
compound or chemical species.
Comparing the average spectra of healthy, early-stage infec-
ted and full infected leaves, it is possible to observe that there is
a general reduction in band intensity when the plant is infected,
as shown in Fig. 4a. The statistical evaluation of spectroscopic
data was performed using the band intensity of carotenoids at
1526 cm"1 (the strongest one). The mean % standard deviation
(minimum–maximum) values for the three groups of leaves are
presented:
Healthy: 6588.98 % 2035.94 (3264.68–10 633.57)
Early-stage infected: 4221.66 % 1961.88 (1847.44–8701.57)
This journal is © The Royal Society of Chemistry 2016
Paper
Fig. 4 Average Raman spectra of leaves from healthy plants (blue),
early-stage infected plants (green), and full infected plants (red): (a)
without normalization; and (b) normalized on the band at 1526 cm"1.
The term “early-stage infected” is used to refer to an early-stage
infection of the plant in which the symptoms of the disease are not
easily visible. The term “full infected” is used to denote a stage in which
the plant is fully infected and the leaves clearly show the symptoms of
the disease.
Full infected: 1939.39 % 1196.76 (906.79–5559.69)
The difference in intensity can be easily observed in the
boxplot of Fig. 5, in which the median, the interquartile range
and the maximum/minimum are clearly depicted. A comple-
mentary evaluation using one-way ANOVA and Tukey's pairwise
was performed and the results are depicted in Table 2. The
results of one-way ANOVA show that effects ‘between groups’
are signicantly different (P < 0.0001). Tukey's pairwise
comparisons among the means of each variable demonstrated
highly signicant differences. As shown in Table 2, the proba-
bility was lower than 0.01 for all cases and this indicates
differences at levels higher than 99%. According to these
results, the intensity of the band at 1526 cm"1 could be used in
a screening analytical method for the early detection of diseases
in plants, in this case, a virus infection.
In general, a screening analytical method for fast evaluation
of the state of health of a plant by leaf measurements should
This journal is © The Royal Society of Chemistry 2016
Analytical Methods
Fig. 5 Boxplot of the band intensity at 1526 cm"1 (the strongest
Raman band of all the spectra of leaves).
consider indicator compounds related to the health of the
plant. Intuitively, chlorophyll seems to be the rst option as an
indicator compound, due to its abundance in leaves. However,
carotenoids are also present in leaves and, despite their color
being masked by chlorophyll in photosynthetic tissues, they
show strong Raman signals, even stronger than chlorophyll
signals. The chemical structure of carotenoids is particularly
interesting from the point of view of Raman spectroscopy;
carotenoids have an allowed p–p* transition in the visible
region. Therefore, their resonance Raman spectra are obtained
when the appropriate wavelength of the laser excitation (in the
visible spectral region) is used, resulting in strong enhance-
ments of Raman bands.59 The enhancement of Raman bands of
carotenoids was also observed when the Raman spectra were
recorded using a near-infrared excitation at 1064 nm, but the
proposed mechanism is different (a p-electron/phonon
coupling).60 The enhancement of Raman bands explains why
the strongest signals in the Raman spectra of leaves are due to
carotenoids instead of chlorophyll. A logical question emerges:
can the content of carotenoids in leaves, i.e., the intensity of
a representative Raman band of carotenoids, be a health indi-
cator of the plant, concretely, indicate whether there is a virus
infection or not? The antioxidant properties of carotenoids are
well known61 and the protective effect of these compounds in
a specic bacterium (Deinococcus radiodurans) was also reported
in the past.62 Moreover, the role of carotenoids in plants as
accessory pigments for photosynthesis, in the protection
against photooxidation and as structural determinants in
plastid pigment–protein complexes, is well known, too.63 These
aspects suggest that decreasing contents of carotenoids could
be related to a health detriment in plants. Therefore, it would be
justied to exploit the intensity of carotenoid bands to evaluate
the health of plants. However, the contents of carotenoids in
leaves (of a specic plant species) can be inuenced by other
factors such as soil, nutrients, seasons, among other environ-
mental variables. How could a normal (healthy) content of
carotenoids for a specic plant species be established? An
appropriate data basis, which considers all the relevant factors,
would be necessary. With this data basis, a range for the content
Anal. Methods, 2016, 8, 3450–3457 | 3455
Analytical Methods
Table 2 Results of one-way ANOVA and Tukey's pairwise analysis
One-way ANOVA
Sum of sqrs df
Between groups 1.87031 & 108 2 9.35157 & 107
Within groups 1.68089 & 108 51
Tukey's pairwise
Variables Mean difference
Healthy–early-stage 2367.324753
infected
Healthy–full 4649.589527
infected
Early-stage infected– 2282.264774
full infected
a
Probability lower than 0.01 indicates differences at levels higher than 99%.
of carotenoids, expressed as the intensity of a selected Raman
band, could be established for different regions and seasons.
It is important to consider that abnormal contents of
carotenoids can also be a consequence of other diseases. This is
the reason why low contents of carotenoids in leaves should not
be considered as an exclusive symptom of the infection by
AbMV or other viruses of the genus Begomovirus. Therefore, for
the early detection of a specic disease, the intensity of the
representative Raman band of carotenoids should not be
considered alone and additional information must be taken
into account. Since Raman spectroscopy provides rapid, inex-
pensive and in situ measurements, it could be incorporated into
plant disease early-warning systems. If Raman spectra show
abnormally low contents of carotenoids in a routine control, the
conrmation of a disease can be obtained by other conventional
methods in the laboratory (e.g., for viruses, PCR analysis). A
system of this kind could be very useful for national sanitary
control institutions and help them to respond rapidly in case of
emergencies.
Conclusions
The natural occurrence of Abutilon mosaic virus (AbMV) in
ornamental Abutilon was identied in Ecuador. The present
study (AbMV-Abutilon-Ecuador) isolate is closely related to the
isolates such as AbMV-Germany, AbMV-India, AbMV-USA and
AbMV-HW-USA and also related to nearby countries like
AbMBV-Brazil whereas AbMBoV-Bolivia is a totally different
group. The virus is easily transmitted through B. tabaci vector
and the possibility of genetic recombination with the indige-
nous begomoviruses needs to be characterized in the future.
The results suggest that Raman spectroscopy can be used for
the fast and early detection of plant diseases. There is no
question about positive implications and the great benets of
Raman spectroscopy for agriculture, since in situ measurements
can be performed and a fast diagnosis of the plant can be ob-
tained. This study provides preliminary results on the
3456 | Anal. Methods, 2016, 8, 3450–3457
Paper
Mean square F Probability
28.37 5.208 & 10"9
3.29586 & 106
Tukey's statistical Probabilitya
5.489 0.0009683
10.78 0.0001232
5.292 0.001429
application of portable Raman spectroscopy for the detection of
plant (Abutilon) infection by a specic virus (AbMV). Future
studies should evaluate other plants and other viruses, and even
study the possibility to distinguish different virus infections
from each other. In addition, environmental factors (climate,
altitude, etc.) can be a source of variation in the composition of
leaves and other plant tissues. These factors should also be
a subject of study.
Acknowledgements
S. Y., P. V. J. and R. G. acknowledge the Prometeo Project of the
Secretariat for Higher Education, Science, Technology and
Innovation (SENESCYT) of the Republic of Ecuador for the
support of the investigation in Ecuador. P. G. and L. A. R.
acknowledge the Ecuadorian Agency for Quality Assurance in
Agriculture, AGROCALIDAD.
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