J Phytopathol

ORIGINAL ARTICLE

The Association Between Leaf Malondialdehyde and Lignin

Content and Resistance to Spot Blotch in Wheat
Comfort S. Yusuf1,2, Ramesh Chand1, Vinod K. Mishra3 and Arun K. Joshi3,4
1
2
3
4

Department of Mycology & Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi 221005, India
Department of Biological Sciences, Faculty of Science, Adamawa State University, Mubi, Nigeria
Department of Genetics & Plant Breeding, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi 221005, India
International Maize and Wheat Improvement Center (CIMMYT), South Asia Regional Office, Agriculture Botany Division - 1st floor, Nepal Agriculture
Research Council (NARC), Khumaltar, Lalitpur, Nepal

Keywords
Bipolaris sorokiniana, lesion size, Triticum
aestivum
Correspondence
A. K. Joshi, International Maize and Wheat
Improvement Center (CIMMYT), South Asia
Regional Office, Agriculture Botany Division 1st floor, Nepal Agriculture Research Council
(NARC), Khumaltar, Lalitpur, Nepal.
E-mail: a.k.joshi@cgiar.org
Received: January 26, 2016; accepted: June
13, 2016.
doi: 10.1111/jph.12509

Abstract
Spot blotch (causative pathogen Bipolaris sorokiniana (Sacc.) Shoem) is a
common disease of wheat in the Eastern Gangetic Plains region of India.
The association of leaf malondialdehyde and lignin contents with the
severity of spot blotch disease was studied using a correlation analysis
based on a population of recombinant inbred lines bred from the cross cvs.
Yangmai 6 (resistant) 9 Sonalika (susceptible). The material was fieldtested over two consecutive years and inoculated artificially with a highly
virulent strain of the pathogen. Disease severity was assessed at three
growth stages around and after anthesis. Leaf lignin content tended to be
higher in the more resistant RILs, while the opposite was the case for leaf
malondialdehyde content. Lesion size showed a positive correlation with
disease severity and leaf malondialdehyde content, while disease severity
and leaf lignin content were negatively correlated with one another, as
were leaf malondialdehyde and leaf lignin content. Leaf malondialdehyde
and/or leaf lignin content could be informative as markers for selection for
higher levels of resistance against spot blotch in wheat.

Introduction
Wheat provides a significant proportion of the calorific intake by humans. Like all crops, its production
depends on the vagaries of climate and the occurrence
of disease. In wheat-producing regions where the
temperature and relative humidity are both high during the growing season (particularly the Indian subcontinent and parts of Latin America and Africa), one
of the major biotic constraints to wheat production is
the disease known as spot blotch (Chaurasia et al.
2000; Joshi et al. 2007a; Gurung et al. 2013; Singh
et al. 2015), caused by the fungus Bipolaris sorokiniana
(Sacc.) Shoem (Saari and Wilcoxson 1974; Joshi et al.
2007b). The disease affects >9 mha of the wheat crop
grown in the Indo-Gangetic plain (Joshi et al. 2007b;
Singh et al. 2015), reducing grain yield by up to 20%
(Saari 1998). The extent of yield loss is dependent on

2016 Blackwell Verlag GmbH

cultivar, sowing time, year, location and the environmental conditions pertaining around anthesis
(Sharma and Duveiller 2004). Complete genetically
determined resistance has yet to be identified, so even
those cultivars classified as resistant suffer some yield
reduction when the disease pressure is high (Joshi
and Chand 2002).
When exposed to stress, a plants survival often
depends on how rapidly it can respond (Maffei et al.
2007). One of the most effective of mechanisms
deployed to counter foliar pathogens is to isolate the
pathogen by the localized production of lignin, a
compound which strengthens the cell wall, rendering it more resistant to pathogen ingress (Moura
et al. 2010). The deposition of lignin has been implicated as a defence response of wheat to a number of
diseases (Moerschbacher et al. 1988; Bishop et al.
2002; Tronchet et al. 2010), while the inactivation

Malondialdehyde and lignin with resistance to spot blotch

of several gene-encoding enzymes involved in

monolignol synthesis has been shown to increase
the susceptibility of diploid einkorn wheat (Triticum
monococcum) to the fungal pathogen causing powdery mildew disease (Bhuiyan et al. 2009). An
enhancement in lignin synthesis, as well as certain
structural modifications to the molecule, can provide
a measure of protection against damage to cellular
membranes caused by a build-up of the reactive
oxygen species which are an inevitable result of cellular stress (Malen
cic et al. 2004). An increased lignin deposition in the spot blotch resistant lines of
wheat was recently shown by Eisa et al. (2013). The
compound malondialdehyde (MDA) is typically produced as a by-product of lipid peroxidation, and
thus is regarded as an indicator of cellular damage
(Dallagnol et al. 2011).
This study was conducted to compare the production of MDA in wheat lines of contrasting levels of
resistance to spot blotch. At the same time, an attempt
was made to quantify the synthesis of lignin (mg/g
drywt) during the development of disease symptoms.
The overall aim was to establish whether either MDA
production and/or the capacity to generate lignin
could be used as a resistance indicator in a wheat
breeding programme aiming to improve the level of
spot blotch resistance.
Material and Methods
Field trial and experimental material

A series of field trials were conducted during Rabi season of 2013/2014 and 2014/2015 at the Banaras
Hindu University Institute of Agricultural Sciences
(Varanasi, India). The local region is considered to be
a hot spot for spot blotch disease (Meena et al. 2014).
The wheat lines involved in the trial comprised a set
of 87 recombinant inbred lines (RILs) bred from the
cross cv. Yangmai 6 9 Sonalika, along with the two
parents. Kumar et al. (2009) have used the same set
of RILs as those described here (F8) to investigate the
genetic basis of spot blotch resistance in cv. Yangmai
6. The material was planted as a randomized block
with three replications keeping plot size of 3 m 3 rows
with row-to-row and plant-to-plant spacing of 20 and
5 cm, respectively. The plots were provided with the
locally recommended dose of NPK fertilizer (120 kg
N, 60 kg P2O5, 40 kg K2O per ha). While all P and K
was given at sowing time, nitrogen was in split dose;
1/2 at sowing, 1/4 at first irrigation (21 days after
sowing) and 1/4 at the time of second irrigation
(40 days after sowing).
2

C. S. Yusuf et al.

Five irrigations were given, first being at the 22nd

day of planting and the other four at an approximate
interval of around 20 days.
Artificial inoculation with B. sorokiniana

A highly virulent strain of B. sorokiniana (NABM MAT

1; NCBIJN128877) was used as inoculum. The pathogen was multiplied by culturing it on sorghum grain,
and an aqueous spore suspension of 104 spores per ml
was generated for spraying over the plants at ear emergence (GS 50, Zadoks et al. 1974). Spraying was carried
out in the evening to take advantage of lower ambient
temperatures, and the plots were subsequently irrigated
to produce the high humidity environment needed for
the rapid establishment of the disease.
Disease severity (%DS)

The severity of spot blotch disease was evaluated on

ten plants per line, using the scale described by Saari
and Prescott (1975). The lines were scored three
times: at GS 63 (early anthesis), GS 69 (late anthesis)
and GS 77 (late milk). The first digit (D1) of the score
indicates the extent of vertical disease progress on the
plant, while the second (D2) reflects the area of leaf
exhibiting disease symptoms. A %DS value was calculated from the expression (D1/9) (D2/9)100. The %
DS scores at the three growth stages were used to generate an area under the disease progress curve
(AUDPC), given by the summed values of {[(Yi+Y(i +
1))/2]9(t(i + 1)ti)}, where Yi represented the %DS at
time ti, and (t(i + 1)-ti) the number of days elapsed
between consecutive disease scores, following Shanner and Finney (1977).
Disease response and phenotyping of the RILs

The disease response of the RILs was categorized into

four reaction types: resistant (R, %DS < 30), moderately resistant (MR, 30 < %DS < 50), moderately susceptible (MS, 50 < %DS < 80) and susceptible (S, %
DS >80) (Joshi et al. 2004). The RILs were divided
into two phenotypic groups based on the parental
types to minimize the effect of plant type on the disease reaction. Thus, RILs forming club-shaped, short,
compact and golden coloured ears were classified as
cv. Yangmai 6 types (Fig. 1a), and those forming
pyramid-shaped, long, loose and reddish coloured
ears and darkly pigmented chaff as cv. Sonalika types
(Fig. 1b). The size of five spot blotch lesions on the
flag leaf of five individuals per RIL was estimated from
the product of their length and width (Bashyal et al.
2016 Blackwell Verlag GmbH

Malondialdehyde and lignin with resistance to spot blotch

C. S. Yusuf et al.

(a)

(b)

Fig. 1 Ears of (a) The susceptible RIL parent

cv. Sonalika, (b) the resistant parent cv. Yangmai 6.

2011), measured when flag leaf %DS in the cv. Sonalika plots exceeded 50%. Time to heading was taken
as the day on which 50% of ears within a RIL had
fully emerged from the boot of the leading tiller.
Thousand kernel weight was calculated as the mean
of a 1000 grain sample taken from each of the three
replicate plots per RIL (or parent).

1 ml 12M H2SO4, then 28 ml distiled water was added

and the solution was held at 30  0.5C for 1 h with
occasional stirring. After hydrolysis, (120  5C for
1 h), the solution was filtered while still hot, and the
quantity of acid-soluble lignin present in the filtrate
estimated spectrophotometrically at 205 nm (Fredrik
and Elisabeth 2011).

MDA and lignin content

Statistical analysis

To estimate leaf MDA content, leaf samples were collected both prior to and 72 h postinoculation, snap-frozen in liquid nitrogen and held at 80C until required.
A 100 mg sample of powdered leaf was homogenized
in 5 ml 0.1% trichloroacetic acid (TCA) and centrifuged
(10 000 9 g, 5 min, 4C). A 0.3 ml aliquot of the
supernatant was then mixed with 1.2 ml 0.5% thiobarbituric acid in 20% TCA and held at 95C for 30 min.
The reaction was stopped by chilling in an ice bath for
5 min, and the samples were re-centrifuged
(10 000 9 g, 10 min, 25C) and the absorbance of the
supernatant read at 532 nm. After subtracting the nonspecific absorbance at 600 nm, the MDA concentration
was estimated based on an extinction coefficient of
155 m/M/cm and expressed as nmol MDA per g leaf
fresh weight (Heath and Packer 1968).
To derive the lignin content (mg/g drywt) of the
leaf, its dry matter content was first determined by
drying at 50C for 24 h until a constant weight had
been reached. A 100 mg sample was then extracted in

All statistical analyses were performed using SAS v9.2

software (SAS Institute Inc., Cary, NC, USA). For overall comparison of the means of R, MR, MS and S type
of RILs, SPSS software was used. Data were first tested
for normality using the KolmogorovSmirnov test, and
the homogeneity of variance determined using the
Levene test (1960). A standard analysis of variance was
conducted on compliant data sets, and the means were
separated using Duncans multiple range test
(P 0.05). For data which did not satisfy both tests
even after log10 transformation, a KruskalWallis was
conducted, and treatment average ranks were separated
using the stepwise step-down multiple comparisons
method (Campbell and Skillings 1985) (P 0.05).

2016 Blackwell Verlag GmbH

Results
The conditions in the both growing seasons favoured
the development of spot blotch disease (Table 1). The
two RIL parents contrasted with respect to both %DS
3

Malondialdehyde and lignin with resistance to spot blotch

C. S. Yusuf et al.

Table 1 Mean performance of the parents and the cv. Yangmai 6/cv. Sonalika RILs with respect to components of disease resistance and other traits
over two independent trials
Biochemical components

Disease scores

Yield traits

Parents and RILs

Lignin mg/g drywt

MDA n molg-1freshwt

DS %

LS cm2

AUDPC

DH

TKW

Yangmai 6
Sonalika
7
9
10
11
27
38
46
51
2
4
8
33
34
66
19
21
22
62
71
76
112
113
25
36
40
57
58
111
115
120
LSD 95%

46.7e
27.2u
41.3m
45.0i
45.5h
43.4k
44.9i
43.6k
45.6h
52.6b
48.5c
44.2j
55.1a
48.9c
43.4k
46.3f
47.5d
21.9s
34.5b
33.0f
34.4b
33.4e
28.7m
31.0l
31.7j
27.9n
33.7e
31.2k
34.3c
22.4s
28.1n
37.5s
4.33

0.12u
0.25a
0.17o
0.08c
0.16p
0.06e
0.03o
0.10z
0.03o
0.03o
0.09c
0.10b
0.16r
0.02o
0.02o
0.05m
0.16p
0.23e
0.22f
0.20l
0.26a
0.15p
0.22h
0.25a
0.25a
0.20l
0.06d
0.18n
0.26a
0.22h
0.23d
0.22f
0.03

8.6v
88.9a
17.3t
25.9s
17.3t
17.3t
17.3t
17.3t
17.3t
17.3q
43.2o
34.5o
34.5o
34.5o
43.2p
43.2p
51.8h
51.8j
51.8j
60.5j
60.5j
60.5j
43.2i
43.2j
69.1d
60.5c
66.7d
60.4b
60.7a
60.5b
69.1b
66.7b
3.63

0.01c
2.1a
0.013c
0.01c
0.02c
0.02c
0.07c
0.013c
0.23c
0.01c
0.26c
0.2c
0.02c
0.013c
0.1c
0.02c
0.01c
0.03c
0.05c
0.016c
0.01c
0.17c
0.26c
0.36c
0.33c
0.02c
0.36c
0.25c
0.33 c
0.17c
0.33c
0.03c
0.40

181.5
885.6
142.6
172.8
164.2
164.2
164.2
142.6
164.2
164.2
298.1
263.6
337.0
285.2
354.3
259.3
392.2
453.7
393.2
544.4
423.5
362.9
483.9
453.7
606.9
604.9
596.3
574.7
596.3
574.7
756.2
596.3
53.1

77g
62.5p
67n
75e
70.5e
72c
73.5d
80.b
71h
70g
69l
75e
73f
73h
72.5h
76.5h
66o
68.5n
71g
69.5i
71h
71.5h
71.5g
72g
71g
77i
68k
68.5n
65m
74g
68n
73g
0.40

41.2a
34.6g
41.2a
48.2b
41.6a
29.4p
42.2a
28.8q
43.2a
41.6a
36g
36g
28.8q
38c
36.4g
32.8i
33.6h
23.6x
26.4t
34h
39.5b
38.4c
36.1m
36.1m
31.6l
33.2h
25.6u
28.4r
31.5m
32.2l
31.5l
37.5e
9.0

Disease
reaction

Parental type
in phenology

R
S
R
R
R
R
R
R
R
R
MR
MR
MR
MR
MR
MR
MS
MS
MS
MS
MS
MS
MS
MS
S
S
S
S
S
S
S
S

Y
S
YT
YT
ST
YT
ST
YT
ST
YT
ST
YT
YT
YT
ST
YT
ST
ST
YT
YT
YT
ST
ST
ST
ST
ST
YT
YT
ST
ST
ST
YT

MDA, Malondialdehyde; DS, Disease severity; LS, Lesion size; AUDPC, Area under disease progress curve; DH, Days to heading; TKW, Thousand kernel
weight; YT, Yangmai6 type; ST, Sonalika type; Means with the same superscript shows non- significant difference within the lines.

and AUDPC: the cross-season means for cv. Sonalika

were 88.9% (%DS) and 885.6 (AUDPC), while those
for cv. Yangmai 6 were, respectively, 8.6% and 181.5
(Table 1).
The RILs segregated as 17 S, 21 MS, 26 MR and 23
R (Fig. 2). Both %DS and AUDPC varied significantly
among the RILs (Fig. 3). With respect to lesion size,
cv. Sonalika performed markedly worse than cv.
Yangmai 6 (2.10 vs. 0.01 cm2) (Table 1). The traits
investigated, except days to heading, varied significantly among the RILs (Figs 3 and 4). The lowest
lesion size among the cv. Sonalika type RILs was
achieved by RIL-10 (0.02 cm2), and the worst-performing cv. Yangmai 6 type RIL was RIL-40 (lesion
4

size of 0.36 cm2). Overall, lesion size was smaller in

the cv. Yangmai 6 type resistant RILs than in the cv.
Sonalika type ones (Table 1). Thousand kernel weight
also varied among the RILs (Fig. 4, Table 1). The lignin content of the cv. Yangmai 6 leaf was significantly
greater than that of the cv. Sonalika leaf (47.7 vs.
27.2 mg per g dry wt., see Table 1). The MDA content
of cv. Yangmai and cv. Sonalika leaf was, respectively,
0.12 and 0.25 nmol per g. Both lignin content and
MDA varied significantly among the RILs (Fig. 3).
Correlations between the pairs of traits across the
RIL population are given in Table 2. The following
were statistically significant: %DS and LG
(r = 0.452), %DS and MDA (r = +0.252); AUDPC
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Malondialdehyde and lignin with resistance to spot blotch

C. S. Yusuf et al.

and lignin content (r = 0.243), lesion size and MDA

(r = +0.217), lesion size and %DS (r = +0.513) and
lesion size and AUDPC (r = +0.531).

30
26

Number of RILs

25

23
21

20

17

Discussion

15

Resistance to spot blotch in wheat is a quantitative

trait (Joshi et al. 2004), with the size of the pathogeninduced lesions on the host leaf proving to be the
most reliable way of quantifying the hosts response
(Bashyal et al. 2011). Here, cv. Sonalika, which is recognized as being highly susceptible, allowed the mean
spot blotch lesion size to grow some 20-fold larger
than those which developed on cv. Yangmai 6, and
the RILs displayed a range of susceptibility on the
basis of this parameter. A significant positive correlation existed between lesion size and both %DS and
AUDPC, which confirmed that lesion size is a robust
trait for selecting host resistance, as suggested by
Bashyal et al. (2011).
Apart from its spot blotch susceptibility, the early
flowering cv. Sonalika is well adapted to growing

10
5
0
Resistant

Moderately
Resistant

Moderately
Susceptible

Susceptible

Fig. 2 Frequency distribution of the four disease reaction categories in

the RIL population of the cross Yangmai 6 9 Sonalika.

and lignin content (r = 0.438), AUDPC and MDA

(r = 0.293), AUDPC and %DS (r = +0.937); lignin
content and MDA (r = 0.206); TKW and lesion size
(r = 0.345), TKW and %DS (r = 0.721), TKW and
AUDPC (r = 0.695), TKW and MDA (r = 0.266)
and TKW and lignin content (r = +0.310); lesion size
0.4

50

0.3

40

b
a

0.2

LG (unit)

MDA (nmol)

b
b

0.1

30
20
10
0

0.0
Susceptible
Lines

Moderately
Susceptible
Lines

Moderately
Resistant
Lines

Susceptible
Lines

Resistant
Lines

Moderately
Moderately Resistant Lines
Susceptible Resistant Lines
Lines

800

80
d

600
AUDPC

60
DS (%)

40

a
20

d
c

400

b
a

200
0

0
Susceptible
Lines

Moderately
Susceptible
Lines

Moderately
Resistant
Lines

Resistant
Lines

Susceptible
Lines

Moderately
Moderately Resistant Lines
Susceptible Resistant Lines
Lines

Fig. 3 The performance of RILs classified as R (resistant), MR (moderately resistant), MS (moderately susceptible) and S (susceptible) with respect to
%DS, AUDPC and MDA content across two independent trials for the cross Yangmai 6 9 Sonalika. Note: Similar letter above Mean  SE (harmonic
mean sample size=21.1; n > 17) represents insignificant difference in a, c (P 0.05; DMRT), b (P 0.05; KruskalWallis, stepwise step-down multiple
comparison), and d (Log10(X), P 0.05; DMRT.

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Malondialdehyde and lignin with resistance to spot blotch

75

C. S. Yusuf et al.

42

c
40

74

38

73
36

72

34
71

32
30

70
Susceptible

Moderately
Susceptible

Moderately
Resistant

Susceptible

Resistant

Moderately
Susceptible

Moderately
Resistant

Resistant

0.30

0.25
0.20
0.15

0.10

0.05
0.00
Susceptible

Moderately
Susceptible

Moderately
Resistant

Resistant

Fig. 4 The performance of RILs classified as R (resistant), MR (moderately resistant), MS (moderately susceptible) and S (susceptible) with respect to
heading date, thousand kernel weight and spot blotch lesion size across two independent trials for the cross Yangmai 6 9 Sonalika.

conditions in the Indian subcontinent; cv. Yangmai 6

reaches heading more than ten days later (Table 1).
The absence of significant correlation established here
between heading date and %DS was not in agreement
with the conclusions reached in other studies (van
Table 2 Correlation coefficient value r among LG, MDA, disease severity and yield traits
Traits
MDA
LG
DS
AUDPC
DH
TKW

LG
.206

DS
.252*
.452**

AUDPC
.293**
.438**
.937**

DH
.056
.015
.029
.017

TKW
.266*
.310**
.721**
.695**
.041

LS
.217*
.243*
.513**
.531**
.064
.345**

Probability 0.001 = ***, 0.01 = **, 0.05 = *. DH, Days to heading; TKW,
Thousand kernel weight; LS, Lesion size; DS, Disease severity; LG, Lignin;
MDA, Malondialdehyde; AUDPC, Area under disease progress curve.

Beueningen and Kohli 1990; Dubin et al. 1998) but

matched with that Joshi et al. (2002). Also, the mean
values of heading date were not significant between
resistant and susceptible lines. Thousand kernel
weight was negatively correlated with both %DS and
AUDPC, as also noted by both Meena et al. (2014)
and Khodarahmpour et al. (2011). As thousand kernel weight was significantly higher in the group of
resistant RILs compared to the susceptible ones, the
implication is that selection for grain size should lead
to genetic advance with respect to spot blotch resistance. This is important because the grains are only
bigger because susceptible genotypes are damaged by
the disease and so produce smaller grains
Plants constantly encounter threats from pathogens
and hence have developed multiple defence mechanisms, including oxidative burst, and deposition of lignin and callose into the cell wall. The enzymes

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C. S. Yusuf et al.

involved in lignin biosynthesis include phenylalanine

ammonia lyase (PAL), cimnnamyl alcohol dehydrogenase (CAD) and peroxidase (POX). An increase in the
content of these enzymes during pathogen infection is
an indication of their role in plant defence (Vance
et al. 1980; Nicholson and Hammerschmidt 1992;
Scott-Craig et al. 1995). Therefore, increased lignification is a common response of plants to attack by
pathogen (Vance et al. 1980). Lignification is one of a
number of defence mechanisms employed by plants
to inhibit the invasion of a pathogen. Certain sources
of spot blotch resistance have been shown to mount
an effective lignification response when challenged
with the spot blotch pathogen (Das et al. 2003; Eisa
et al. 2013). Here, the more resistant RILs developed
smaller disease lesions and exhibited a higher lignin
content than did the more susceptible ones (Fig. 3a),
supporting the view that cell wall thickening erects
a physical barrier to the spread of the pathogen
(Kimmins and Brown 1973). The peroxidation of
lipids is a generic response to stress; one of its products
is the compound MDA. As a result, MDA content has
been frequently taken as an indicator of lipid peroxidation (Malen
cic et al. 2004), and hence as a marker
of stress-induced cellular damage. Here, the more disease susceptible RILs tended to accumulate the highest quantities of MDA when the plants were
challenged by the spot blotch pathogen; furthermore,
the accumulation of MDA was positively correlated
with both %DS and lesion size.
In conclusion, it was observed that increased MDA
content, which is a product of LP resulting from spot
blotch stress, appeared to be responsible for increased
disease expressed as cell damage and eventual cell
death. And, therefore, low lignin content with high
MDA content can be used as biochemical marker in
selection for resistant lines against spot blotch disease
of wheat.
Acknowledgements
YC thanks Adamawa State University for financial support given under the auspices of the Tertiary Education
Trust Fund of Nigeria. Resources granted to AJ under
the CIMMYT CSISA project, jointly funded by the
BMGF and USAID, were employed to carry out this
study. The support given by Sabin Basi, CIMMYTNepal, in statistical analysis is gratefully acknowledged.
Conflict of interest
The authors declare that they have no conflict of
interest.
2016 Blackwell Verlag GmbH

Malondialdehyde and lignin with resistance to spot blotch

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