Beruflich Dokumente
Kultur Dokumente
DOI 10.1007/s12600-014-0446-x
Received: 7 May 2014 / Accepted: 1 December 2014 / Published online: 20 December 2014
# Springer Science+Business Media Dordrecht 2014
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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Mass spectrometry
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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C T 100
C
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.
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
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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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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
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Means followed by the same letter(s) within the same column are
not significantly (p 0.05) different according to Tukeys HSD
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
Discussion
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