Phytoparasitica (2015) 43:437447

DOI 10.1007/s12600-014-0446-x

Non-specific lipid transfer proteins (ns-LTPs) from maize

induce resistance in pearl millet against downy mildew disease
S. Manjula & M. Murali & G. R. Shivamurthy &
K. N. Amruthesh

Received: 7 May 2014 / Accepted: 1 December 2014 / Published online: 20 December 2014
# Springer Science+Business Media Dordrecht 2014

Abstract Non-specific Lipid Transfer Proteins were

isolated and purified from maize seeds using combinations of ammonium sulphate precipitation and cat-ion
exchange chromatography followed by HPLC. Two
peptide segments were obtained by MALDI-TOF
MS analysis, which were designated as ns-LTP M1
and ns-LTP M2. Both the ns-LTPs were treated to pearl
millet seeds at different concentrations for 6 h. Among
the concentrations tested, ns-LTP M1 (50 g/ ml)
showed a maximum seed germination of 94% and
1320 seedling vigor followed by ns-LTP M2. The
maximum downy mildew disease protection of 62%
was offered by ns-LTP M1 (50 g/ ml) followed by
ns-LTP M2 which offered 58% protection.
Furthermore, peroxidase (POX) and lipoxigenase
(LOX) enzymes related to plant resistance metabolism
were also increased considerably after ns-LTP M1
treatment. POX activity was up to two folds when
compared to susceptible control seedlings. Thus the
present investigation suggests that the maize LTPs could
enhance downy mildew disease resistance in pearl millet
through the induction of defense mediated enzymes.

S. Manjula : M. Murali : G. R. Shivamurthy :

K. N. Amruthesh (*)
Applied Plant Pathology Laboratory, Department of Studies
in Botany, University of Mysore, Manasagangotri,
Mysore 570 006 Karnataka, India
e-mail: dr.knamruthesh@botany.uni-mysore.ac.in
K. N. Amruthesh
e-mail: dr.knamruthesh@gmail.com

Key words ns-LTPs . maize . pearl millet . downy

mildew . induction of resistance . defence related
enzymes

Introduction
With the increasing concern regarding environmental
protection and human health, alternative disease management strategies have been employed. One of the
important options for managing disease is to induce
resistance in the host to exploit its innate immunity by
specific biotic or abiotic elicitors and has been considered as a potential strategy for plant disease management in recent years. A number of chemical compounds
and microorganisms are reported to induce resistance
against plant diseases (Walters et al. 2005). Lipid transfer proteins (LTPs) are small, basic and abundant proteins in higher plants, which are known as nonspecific
transporters of lipid molecules with low molecular mass
(Carvalho and Gomes 2007; Yeats and Rose 2008). The
expression of LTPs can be induced by abiotic and/or
biotic stresses such as cold, water deficit, NaCl, heavy
metal treatment, or pathogen invasion (Thoma et al.
1994; Molina et al. 1993; Pearce et al. 1998).
LTPs have been isolated or cloned from a number of
monocotyledons and dicotyledonous plants including
Arabidopsis (Finkina et al. 2007), bean (Clark and
Bohnert 1999), maize (Tchang et al. 1988), rice
(Vignols et al. 1994) and wheat (Flemming et al.
1992). LTPs in plants are thought to play key roles in
many biological processes, such as cutin biosynthesis,

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defense response (Lee et al. 2009), plant signalling

(Blein et al. 2002; Maldonado et al. 2002) and seed
maturation (Thoma et al. 1994). Studies from several
research groups showed that LTPs take a certain effect in
plant defense mechanisms (Carvalho and Gomes 2007).
Over expression of GtLTP1 in tobacco plant improved
tolerance against Botrytis cinerea demonstrating
GtLTP1 is a useful molecular tool for genetic engineering of disease resistance plants (Kiba et al. 2012).
Similarly, there are reports that Harpin, a protein
elicitor from the bacteria Erwinia amylovora, was first
reported in 1992 (Wei et al. 1992) and it activated both
hypersensitive response (HR) defenses and growth systems in many important crops (Qiu et al. 2002). A
protein elicitor isolated from S. graminicola induced
activation of phenyl alanine ammonia lyase (PAL) and
peroxidase (POX) activity (Sharathchandra et al. 2006)
and homologous proteins like INF1, NPP1 (Baillieul
et al. 2003), cryptogein, PcF (Orsomando et al. 2003),
PB90 (Wang et al. 2003), megaspermin and quercinin
(Koehl et al. 2003) with the similar biological activity
have been found in other Phytophthora spp., an
oomycete plant pathogen. The protein elicitor PebC1
from Botrytis cinerea strain BC-4-2-2-1 promoted seedling growth, drought tolerance in wheat and also induce
disease resistance against gray mold fungus in tomato
(Zhang et al. 2010).
Pearl millet [Pennisetum glaucum (L.) R. Br.] is one
of the major staple crops of arid and semi arid tropics of
the world, which provides food, fodder and nutritional
security to millions of people. It is also known as poor
mans crop and provides basic sustainable living in the
semi-arid regions of the world. Pearl millet is grown
annually on more than 29 million ha in arid and semiarid tropical regions of Asia, Africa and Latin America.
India is the largest producer of pearl millet in Asia, both
in terms of area (about 9 million ha) and production (8.3
million tons) with an average productivity of 930 Kg/ ha
during the past three years (Khairwal 2008; Anonymous
2013). Downy mildew disease caused by Sclerospora
graminicola (Sacc.) Schroeter is a major biotic constraint of pearl millet and it causes yield loss up to 4060% as the grain is replaced by leaf like structures
(Thakur et al., 1999; Anonymous 2013). The downy
mildew disease of pearl millet is being managed by
various methods by using both biotic and abiotic source
(Murali et al. 2012, 2013; Mythrashree et al. 2013)
However, there are instances wherein the pathogen develops resistance to fungicides as well as residue

Phytoparasitica (2015) 43:437447

problems and breakdown of host resistance (Thakur

et al. 1999). Thus, an effective downy mildew disease
management is important to prevent yield loss.
However, so far there has been no report on induction
of resistance by ns-LTPs from maize against downy
mildew disease of pearl millet. In this study, an attempt
was made to analyse effect of seed treatment with maize
ns-LTPs on the activity of defense related enzymes like
peroxidase and lipoxigenase during host-pathogen interaction in pearl millet.

Materials and methods

Seed materials
Maize seed samples (Maize-NAH-1137) were obtained
from Zonal Agriculture Research Station, V.C. Farm,
Mandya and the same were used for purification of nsLTPs for biological studies.
Host plant
Seeds of pearl millet that are highly susceptible (7042S)
and highly resistant (IP18296) to downy mildew disease
were obtained from the International Crops Research
Institute for the Semi-Arid Tropics (ICRISAT),
Patencheru, India. The seeds were obtained under a
Material Transfer Agreement and were used throughout
the study.
Source of pathogen and inoculum preparation
S. graminicola was isolated from pearl millet cv. 7042S
grown in a heavily infested field. The pathogen was
maintained on its susceptible host until use. Leaves
of pearl millet showing profuse sporulation of
S. graminicola on the abaxial side were collected
in the evening from the plants maintained in field
conditions were thoroughly washed under running
tap water to remove sporangia. The leaves were then
blot-dried, cut into small pieces and maintained in a
moist chamber for sporulation. The following morning,
fresh sporangia were washed into distilled water. For use
as inoculum, the zoospores concentration was adjusted
to 4x104/ ml using Haemocytometer (Safeeulla 1976)
and used as source of inoculum in green house studies.

Phytoparasitica (2015) 43:437447

Isolation and purification of ns-LTPs from Maize seeds

The ns-LTPs from maize seed samples were isolated
using the modified procedure of (Gorjanovic et al.
2005). Hundred grams of seeds were weighed and ground
into fine powder using a mixer. The resulting flour was
extracted for 16 h in 200 ml of 0.1M sodium acetate
buffer of pH 5.5 at 4 C. The slurry was filtered through
a cheese cloth and the filtrate was centrifuged at 12,000 g
for 30 min. The collected supernatant was filtered using
Whatmann No.1 filter paper and the filtrate was subjected
to 30% ammonium sulphate precipitation followed by
centrifugation. The resulting pellet was discarded, subsequently (NH4)2SO4 was added to the supernatant to get
70% saturation. Following precipitation and centrifugation, the pellet was dissolved in minimal volume of
starting buffer and centrifuged at 12,000 g for 30 min.
The crude extract of protein was desalted using desalting
column to remove the ammonium sulphate, and the
desalted protein sample was loaded onto a CM-cellulose
column (2.6 cm internal diameter and 10 cm long) which
was pre-equilibrated with 0.1M sodium acetate buffer of
pH 5.5. The unbound proteins were collected in a fraction
tube with a flow rate of 3 ml/15 min using starting buffer,
while the bound basic proteins were eluted using 1M
NaCl in the same buffer. Protein elutions were monitored
using UV-visible spectrophotometer at 280 nm.
The peak fractions with more absorbance from
cation-exchange chromatography were pooled, desalted
and concentrated by lyophilisation. The lyophilized protein was dissolved in 0.1 M Sodium acetate buffer pH
5.5 and subjected to RP-HPLC (Shimadzu LC 20AP,
M20A PDA detector, Japan) Phenomenex (Luna, USA)
column ID (30X 250 mm 10 , 100 A0) for further
purification using modified HPLC procedure of
Finkina et al. (2007). The Column was run with mobile
phase A-Acetonitrile and Mobile phase B-0.1% trifluro
acetic acid. Proteins were eluted as follows, Isocratic
elution from 0-3 min 95% B and 5%A, linear gradient
elution from 3-35 min 95-54% B, from 35-40 min 5495% B, followed by Isocratic elution from 40-45 min of
A 5% and B 95% at flow rate of 22 ml/ min. The
proteins were monitored at 280 nm and peak fractions
were further subjected to MALDI/ MS analysis.

439

(Ultraflextreme, BrukerDaltonics Germany). All the

samples were analyzed in the positive mode using an
acceleration voltage of 20 kV and external calibration,
providing a mass accuracy of 0.1% or better. The matrix
used was -cyano 4-hydroxy cinnamic acid (Sigma, St.
Louis, MO) at a concentration of 15 mg/ mL in 50%
CH3CN, 0.1% TFA. 1 l of protein and 1 l of the
matrix solution were mixed on the sample plate and
allowed to dry at room temperature before being loaded
into the instrument. Data from 1000 laser pulses were
averaged for each mass spectrum to improve the signal
to noise ratio.
Seed treatment with purified ns-LTPs
Highly susceptible (7042S cv.) pearl millet seeds were
treated separately with ns-LTP M1 (9 kDa) and ns-LTP
M2 (7 kDa) from maize at two different concentrations
viz. 10, 25 and 50 g/ ml (w/v) by soaking method and
were kept at 252 C in a rotary shaker for 6 h at 100
rpm/ min to facilitate the penetration of the protein into
the seeds. Seeds soaked in sterile distilled water (SDW)
for same time served as control.
Evaluation of maize ns-LTP extracts on pearl millet seed
germination and seedling vigour
Pearl millet seeds (cv. 7042S) treated with purified nsLTPs and untreated seeds were plated equidistantly on
two layers of moistened blotting paper discs in the Petri
dishes to evaluate percentage of germination (Singh and
Gopinath, 1985). Another set of treated and untreated
seeds were tested through between paper methods to
record seedling vigour (Abdul Baki and Anderson,
1973). The experiment consisted of four replications of
100 seeds and was repeated thrice. The length of the root
and shoot of individual seedling was measured to determine the vigour index. The vigour index was calculated
using the formula:
Vigour index Mean root length Mean shoot length
 Germination percentage

Mass spectrometry

Evaluation of maize ns-LTPs on pearl millet downy

mildew disease incidence under greenhouse conditions

The molecular mass of the proteins were determined by MALDI-TOF MS on a mass spectrometer

The ns-LTPs which offered enhanced seed quality parameters were selected to evaluate its efficacy against

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Phytoparasitica (2015) 43:437447

induction of downy mildew disease protection under

greenhouse conditions. The inducer treated and untreated seeds were sown in earthen pots (9x9 inch diameter)
containing 2:1:1 red soil, sand (red sandy soil) and
farmyard manure (FYM) which was previously
autoclaved. Two-day-old seedlings were challenge inoculated by whorl inoculation method with zoospore
suspension of S. graminicola at a concentration of
40,000 zoospores/ ml prepared as described earlier.
Challenge inoculated plants were maintained under
greenhouse conditions (90- 95% RH and 20-25 C)
and observed for disease development. There were
three replicates (20 pots/ replicate) per treatment with
20 seeds per pot and repeated thrice. The pearl
millet plants were rated diseased when they showed
any one of the typical downy mildew symptoms
such as sporulation on the abaxial leaf surface, chlorosis, stunted growth and green ear formation.
Downy mildew disease incidence was recorded at
30 days after sowing (DAS) and percent protection
was calculated using formula,
Downy mildew disease protection % :

C  T  100
C

where C represents per cent downy mildew disease

incidence in control and T represents per cent downy
mildew disease incidence in treated plants.
Biochemical studies
Sampling of seedlings
The maize ns-LTP M1 treated pearl millet seeds along
with controls were placed on Petri plates lined with
moistened blotter discs and incubated at 252 C.
Two-day-old seedlings were carefully removed without
damaging the roots and dipped in S. graminicola spore
suspension at 4x104 zoospores/ ml. The seedlings (susceptible, susceptible maize LTP M1 treated and resistant) were harvested at 0, 3, 6, 9, 12, 24, 48 and 72 hours
after post inoculation (hpi) and used for biochemical
studies.

Temporal pattern of Peroxidase (POX) activity

One gram fresh weight of seedlings harvested at above
mentioned time intervals were macerated with 0.2 M

sodium phosphate buffer (pH 6.5) in a pre-chilled

mortar and pestle. The homogenate was centrifuged
at 10,000 rpm for 15 min at 4 C to get the
supernatant. POX activity was determined following
the method of Hammerschmidt et al. (1982). The
reaction mixture of 3 ml consisted of 0.25% (v/v)
guaiacol in 10 mM potassium phosphate buffer (pH 6.9)
containing 10 mM hydrogen peroxide. Five microlitres
of crude extract was added to initiate the reaction,
which was followed calorimetrically at 470 nm.
POX activity was expressed as the increase in absorbance at 470 nm/ mg protein/ min. The experiment was repeated three times taking three replicates
each time.

Temporal pattern of Lipoxygenase (LOX) activity

One gram fresh weights of the seedlings were homogenized in 0.2 M sodium phosphate buffer (pH 6.5) containing 1% polyvinylpyrrolidone (PVP), 0.1% TritonX100 and 0.04% sodium meta-bisulfite. The homogenate
was centrifuged at 9000 g for 20 min at 4 C and the
supernatant was used as the enzyme source. Enzyme
activity was measured by monitoring the appearance of
the conjugated dienehydroperoxide at 234 nm. Linoleic
acid was used as substrate, which was prepared according to the standard method (Axelrod et al. 1981).
Activity was recorded for 3 min using a spectrophotometer. The enzyme activity was expresses in terms of
mmol quinone formed/ min/ mg protein. The experiment was repeated three times taking three replicates
each time.

Protein estimation
Protein content in extracts was estimated by the dye
binding method (Bradford, 1976) using bovine serum
albumin (Sigma) as a standard.

Statistical analysis
Each experimental data was subjected to analysis of
variance (ANOVA) using SPSS Inc16.0. Significant
effects of treatments were determined by the magnitude
of the F value (P 0.05). Treatment means were separated by Tukeys HSD test.

Phytoparasitica (2015) 43:437447

Results
Isolation and purification of ns-LTPs from Maize seeds
The protein extracted from maize seeds (NAH-1137)
was subjected to ammonium sulphate fractionation; the
pellet that precipitated at 30-70% saturation was
redissolved in starting buffer and subjected to cationexchange chromatography. Bound basic proteins were
eluted with the addition of 1 M NaCl in the starting
buffer. The two major peak fractions were observed
when read at 280 nm in UV spectrophotometer
(Fig. 1A). The purity and molecular weight of the proteins in the two fractions were analyzed by SDSPAGE
(16% acrylamide gel) under reducing conditions and F2
fraction showed approximately 7-9 kDa protein bands
with respect to the standard marker. When this F2- fraction
was subjected to RP HPLC for further purification, numbers of peaks with LTP activity were observed between
20-30 minutes. The Two major peaks (P1 and P2) of LTP
activity were further analyzed by Mass spectrometry

441

(Fig. 1B). Mass spectrometric analysis of HPLC purified

protein of P1 revealed the presence of major molecular ion
masses at m/z of 7292.8 and P2 revealed the presence of
minor molecular ion masses at m/z of 9034.5. Thus the
HPLC purified maize protein contains both ns-LTP M1
and ns-LTP M2 family. The P1 exhibit a major component
ns-LTP M2 corresponds to 7 kDa proteins with molecular
mass of 7292.8 Da and P2 exhibit minor component of
ns-LTP M1 corresponds to 9 kDa protein of molecular
mass 9034.5 Da (Fig. 1C and 1D).
Effect of maize ns-LTPs on pearl millet seed
germination and seedling vigor
Purified maize ns-LTPs were tested for their effect on
seed quality parameters in different concentrations. The
purified maize ns-LTPs treated to pearl millet susceptible seeds significantly enhanced germination and seedling vigor to varying degrees (Table 1). The percent seed
germination and seedling vigor varied slightly with
different ns-LTPs at various concentrations. Among

Fig. 1 Chromatographic methods of Maize protein, (A) Isolation of basic ns-LTP from CM cellulose cation-exchange chromatography. (B)
Purification of F2 fraction from preparative RP-HPLC. (C&D) MALDI chromatogram of P1 & P2 fraction of RP-HPLC

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Phytoparasitica (2015) 43:437447

Table 1 Effect of maize ns-LTPs on seed germination and seedling vigor of pearl millet
Source of ns-LTP

% germination

Vigor index

10 g/ ml

25 g/ ml

50 g/ ml

10 g/ ml

ns-LTP M1

900.62a

930.28a

940.40a

12116.72a

12786.21a

13207.86a

ns-LTP M2

12382.21b

9683.86c

Control

880.64

ab

890.40

910.70

890.40

920.28

890.40

25 g/ ml

10815.19

9683.86

50 g/ ml

11543.74

9683.86

Values are means of four independent replicates. indicate standard errors. Means followed by the same letter(s) within the same column are
not significantly (p 0.05) different according to Tukeys HSD.

the two maize ns-LTPs tested for seed quality parameters, maize ns-LTP M1 at 50 g/ ml concentration
showed highest seed germination of 94% and 1320
seedling vigor, followed by maize ns-LTP M2 with
92% seed germination, 1238 seedling vigor at same
concentration. The germination and vigor of pearl millet
seeds in response to purified maize ns-LTP treatments
were higher in all the tested concentrations over the
control seeds which recorded 89% seed germination
and 968 seedling vigor. Among the three different nsLTPs concentrations tested for improved seed quality
parameters, 50 g/ ml concentration was significant
than the other two concentration.
Effect of maize ns-LTPs on pearl millet downy mildew
disease protection under greenhouse conditions
The ability of purified maize ns-LTPs in managing the
pearl millet downy mildew disease was assessed under
green house conditions by treating the susceptible pearl
millet seeds for 6 h with purified maize ns-LTPs at 50
g/ ml concentration. Among the two ns-LTPs tested
maize ns-LTP M1 offered maximum protection of 62%
against downy mildew disease followed by maize nsLTP M2 which offered 58% protection (Fig. 2). In
general, significant downy mildew disease protection
under green house conditions were observed in both
the maize ns-LTPs tested over the susceptible control
which offered 97.75% disease incidence.
Biochemical studies
Temporal pattern of Peroxidase (POX) activity
A significant difference of POX activity was observed in
inducer treated and control seedlings at different time
intervals. POX enzyme activities were higher in

pathogen inoculated seedlings when compared with

uninoculated ones. In all the samples tested there was
a steady increase in enzyme activity up to 48 hpi and
decreased thereafter (Fig. 3). The highest POX activity
of 84.2 U was observed at 48 hpi in resistant inoculated
seedlings followed by maize ns-LTP M1 induced susceptible inoculated seedlings after treatment, which
showed 75.2 U of POX activities. In time-course measurements of POX activity (from 0 to 72 hpi), purified
maize ns-LTP M1 treated seedlings showed more POX
enzyme activity in both inoculated and un-inoculated
susceptible seedlings over the susceptible inoculated
and uninoculated controls. There was up to two fold
increase in POX activity in maize ns-LTP M1 treated
inoculated and uninoculated sample over the control,
but the activity was lesser when compared to resistant
samples.
Temporal pattern of Lipoxygenase (LOX) activity
A significant difference in LOX activity was observed in
resistant, maize ns-LTP M1 treated and control seedlings at the different time intervals tested (Fig. 4). The
maximum enzyme activity of 64.5 U was observed in
resistant inoculated seedlings at 24 hpi. An increase in
LOX enzyme activity was observed in purified maize
ns-LTP M1 treated seedlings when compared with untreated susceptible controls. Purified maize ns-LTP M1
treatment induced the LOX activity in both inoculated
and uninoculated susceptible seedlings as early as 3 hpi
and reached maximum at 24 hpi which was maintained
thereafter. The rate of increase was more pronounced in
the maize ns-LTP M1 treated seedlings after inoculation
with the pathogen which reached maximum at 24 hpi
with 59.8 U when compared with maize ns-LTP M1
treated uninoculated seedlings which showed 54.2 U of
enzyme activity at same time point. While, susceptible

Phytoparasitica (2015) 43:437447

443

Fig. 2 Effects of purified maize ns-LTPs on downy mildew disease

protection in peal millet under green house conditions.Values are
means of four independent replicates. Vertical bars indicate SE.

Means followed by the same letter(s) within the same column are
not significantly (p 0.05) different according to Tukeys HSD

inoculated seedlings showed a maximum of 26.4 U of

LOX activities at 24 hpi which was lesser than the maize
ns-LTP M1 treated and resistant inoculated seedlings.

The present study was aimed at purification and molecular determination of a protein elicitor from maize and
evaluation of its efficacy in pearl millet seedlings against
downy mildew disease. The purified maize ns-LTPs

promoted seed germination and seedling vigor in pearl

millet and also induced disease resistance against downy
mildew. LTPs have been isolated or cloned from a
number of monocotyledons and dicotyledonous plants
including Arabidopsis, bean, maize, rice and wheat
(Tchang et al. 1988; Vignols et al. 1994; Clark and
Bohnert, 1999; Flemming et al. 1992). In the present
investigation we isolated, purified and determined the
molecular masses of the ns-LTPs from Zea mays (maizeNAH-1137). For purification and molecular mass determination of LTPs we employed extraction, ammonium

Fig. 3 Temporal pattern of accumulation of POX enzyme in pearl

millet seedlings upon ns- LTP M1 treatment. RU-Resistant uninoculated; RI- Resistant inoculated; STU- Susceptible treated uninoculated; STI- Susceptible treated and inoculated; SU-Susceptible

uninoculated; SI- Susceptible inoculated. Values are means of three

independent replicates. Vertical bars indicate SE. Means followed
by the same letter(s) within the same column are not significantly
(p 0.05) different according to Tukeys HSD

Discussion

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Phytoparasitica (2015) 43:437447

Fig. 4 Temporal pattern of accumulation of LOX enzyme in pearl

millet seedlings upon ns- LTP M1 treatment. RU-Resistant uninoculated; RI- Resistant inoculated; STU- Susceptible treated uninoculated; STI- Susceptible treated and inoculated; SU-Susceptible

uninoculated; SI- Susceptible inoculated. Values are means of three

independent replicates. Vertical bars indicate SE. Means followed
by the same letter(s) within the same column are not significantly (p
0.05) different according to Tukeys HSD

sulphate fractionation, cation-exchange chromatography, followed by RP-HPLC and mass spectrometric

procedures. The two main families of plant ns-LTPs
are identified by their molecular masses of about 9 and
7 kDa (Kader 1996) named as ns-LTPs type 1 and 2,
respectively (Neumann et al. 1995). Non-specific LTP 1
and ns-LTP 2 are present in the seeds of the most
economically important cereals like barley (Douliez
et al. 2000), wheat (Jones and Marinac, 2000), rice
(Monnet et al. 2001) and maize (Liu et al. 2002). In
our investigation also maize seeds contain both the nsLTP 1 and ns-LTP 2 which corresponds to 9 and 7 kDa
proteins respectively.
Plant ns-LTPs are also thought to be defense proteins
towards pathogens, because some ns-LTPs are able to
inhibit fungal and microbial pathogens in vitro (GarciaOlmedo et al. 1995). In addition, ns-LTP 1 shares some
structural and non-specific lipid binding properties with
elicitins from Phytophthora species, which induces a
hypersensitive response. Both ns-LTP 1 and elicitin are
commonly bound to elicitin receptors on the plasma
membrane, suggesting that ns-LTP 1 can modulate
intracellular signal transduction (Buhot et al. 2001;
Blein et al. 2002). The seed germination and seedling
vigor were enhanced in maize ns-LTPs treated seedlings when compared to control. The maize ns-LTP
M1 treated pearl millet seeds showed a maximum of
94% and seedling vigor of 1320 followed by maize
ns-LTP M2 at 50 g/ mL concentration. Likewise,

seed treatment with biotic elicitors enhanced seed

germination and seedling vigor in various crop plants
(Murali et al. 2013; Mythrashree et al. 2013). LTPs
can also play a role in long-distance systemic signalling in plants, which indicate that plant ns-LTPs can
function both in direct plant defense as antimicrobial
agents and in regulation of plant immune responses
(Sarowar et al. 2009). The results corroborates with
the present findings, where purified maize ns-LTPs
induced downy mildew disease resistance under
greenhouse conditions. The results are also in accordance with the reports of Bu et al. (2014), where a
fungal protein elicitor PevD1 induces Verticillium wilt
resistance in cotton. In the same way Gentian lipid
transfer protein homolog with antimicrobial properties
confers resistance to Botrytis cinerea in transgenic
tobacco plants (Kiba et al. 2012).
Disease resistance in plants is associated with the
activation of wide array of defence responses that serve
to prevent pathogen infection. These mechanisms include pre-existing physical and chemical barriers as well
as inducible defence responses in the form of induction
of defence-related enzymes that are activated upon pathogen infection. The interaction between the pathogen
and host plant induces some changes in cell metabolism,
primarily in the enzyme activities, including that of
phenylalanine ammonia lyase (PAL), peroxidase
(POX), polyphenol oxidase (PPO) and lipoxygenase
(LOX) (Thipyapong and Steffens 1997; Mythrashree

Phytoparasitica (2015) 43:437447

et al. 2013). Similarly in the present study, pearl millet

seeds treated with maize ns-LTP M1 induces the activity
of defense related enzymes like POX and LOX upon
challenge inoculation with the pathogen. Peroxidases
are recorded as one of the first enzymes responding
and providing fast defense against plant pathogens.
Our results are in accordance with the report of Bu
et al. (2014) where afungal protein elicitor PevD1 induces defense enzymes, including PAL, POX, and PPO
in cotton plants. In the present investigation, POX activity increased in maize ns-LTP M1 treated seedlings
upon pathogen infection when compared with untreated
controls. The figures specify that the level of POX
activity remained high at 48 hpi after which the enzyme
activity gradually dropped in both inoculated and uninoculated samples. In the same way increase in POX
activity was reported in tomato plants treated with Peb
C1 protein elicitor from B. Cinerea (Zhang et al. 2010).
In our study, there was increase in POX activity in the
maize ns-LTP M1 treated seedlings upon challenge
inoculation with the pathogen compared to uninoculated susceptible seedlings. In a similar way chickpea seedlings exposed to cell wall protein of Fusarium
oxysporum f. sp. ciceri, showed enhanced synthesis of
phenols, pathogenesis-related proteins and activities of
PAL and POX relative to water treated controls (Saikia
et al. 2006). Our results also revealed that the level of
LOX enzyme was considerably higher in maize ns-LTP
M1 treated seedlings compared to susceptible untreated
control seedlings. High activity of LOX was recorded
in maize ns-LTP M1 treated and resistant challenge
inoculated seedlings. A number of previous studies
have shown that enhanced enzyme content of POX
and LOX is associated with induced resistance against
a broad range of pathogens (Zhang et al. 2010; Bu
et al. 2014). An increase in POX and LOX activities
may be facilitating pearl millet seedlings to prevent
the invasion by pathogen. Thus maize ns-LTP M1
promoted POX and LOX activities and induced
downy mildew disease resistance against S. graminicola
in pearl millet.
Acknowledgements The authors are grateful to the University
Grants Commission (UGC) for awarding Major Research Project
to corresponding author for financial assistance. Authors are also
thankful to Indian Council of Agricultural Research (ICAR), All
India Co-ordinated Pearl Millet Improvement Project (AICPMIP),
Government of India, Mysore Center, Institution of Excellence
(IOE) Project Authorities, University of Mysore, Mysore, India
and Department of Botany DST-FIST for research facilities.

445

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