Plant Physiology and Biochemistry 70 (2013) 246e251

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Plant Physiology and Biochemistry

journal homepage: www.elsevier.com/locate/plaphy

Research article

Biochemical and physiological responses of oil palm to bud rot caused

by Phytophthora palmivora
Andrs Leonardo Moreno-Chacn a, Jhonatan Eduardo Camperos-Reyes a,
Rodrigo Andrs vila Diazgranados a, Hernn Mauricio Romero b, *
a

Oil Palm Biology and Breeding Research Program, Colombian Oil Palm Research Center, Cenipalma, Calle 20A # 43A-50, piso 4, Bogota, Colombia
Department of Biology, Universidad Nacional de Colombia and Oil Palm Biology and Breeding Research Program, Colombian Oil Palm Research Center,
Cenipalma, Calle 20A # 43A-50, piso 4, Bogota, Colombia
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 22 January 2013
Accepted 14 May 2013
Available online 5 June 2013

In recent years, global consumption of palm oil has increased signicantly, reaching almost 43 million
tons in 2010. The sustainability of oil palm (Elaeis guineensis) cultivation has been compromised because
of the bud rot disease whose initial symptoms are caused by Phytophthora palmivora. There was a signicant incidence of the disease, from an initial stage 1 of the disease to the highest stage 5, that affected
photosynthetic parameters, content of pigments, sugars, polyamines, enzymatic antioxidant activities,
phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and b-(1,3) glucanase (b-Gluc, EC 3.2.1.39). In healthy
palms photosynthesis was 13.29 mmol CO2 m
2 s
1 in average, while in stage 5 the average photosynthesis was around 3.66 mmol CO2 m
2 s
1. Additionally, total chlorophyll was reduced by half at the last
stage of the disease. On the contrary, the contents of putrescine, spermine and spermidine increased
three, nine and twelve times with respect to stage 5, respectively. Antioxidant enzyme activities, as well
as the phenylalanine ammonia-lyase and b-(1,3) glucanase showed an increase as the severity of the
disease increased, with the latter increasing from 0.71 EAU in healthy palms to 2.60 EAU in plants at stage
5 of the disease. The peroxidase (POD, EC 1.11.1.7) enzymatic activity and the content of spermidine were
the most sensitive indicators of disease.
2013 Elsevier Masson SAS. All rights reserved.

Keywords:
Elaeis guineensis
Photosynthesis
Polyamines
Enzymatic antioxidant system
b-(1,3) Glucanase

1. Introduction
Oil palm cultivation is expanding. In 2011, the total global oil
palm production area in the world was estimated at nearly 13.6
million hectares, of which 267,000 ha are in Colombia, distributed
in four regions: the Western region around the Municipality of
Tumaco, Nario; the Northern region, located near the Atlantic
Coast; the Central region in the valley of the Magdalena River; and
the Eastern region in the foothills of the Eastern Mountain Range
[1]. Colombia is the largest palm and kernel oil producer in Latin
America and the fth largest producer in the world, accounting for
2.0% of total world production [1].

Abbreviations: EAU, enzyme activity units; TFF, fresh leaf tissue; AOS, active
oxygen species; PA, polyamine; CAT, catalase; POD, peroxidase; SOD, superoxide
dismutase; GR, glutathione reductase; PAL, phenylalanine ammonia-lyase; b-Gluc,
b-(1,3) glucanase.
* Corresponding author. Tel.: 57 1 2086300x410; fax: 57 1 3681152.
E-mail addresses: amoreno@cenipalma.org (A.L. Moreno-Chacn), jcamperos@
cenipalma.org
(J.E.
Camperos-Reyes),
ravila@cenipalma.org
(R.A.
vila
Diazgranados), hmromeroa@unal.edu.co, hromero@cenipalma.org (H.M. Romero).
0981-9428/$ e see front matter 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.plaphy.2013.05.026

In Latin America, oil palm cultivation has been compromised

because of the bud rot disease (BR), which has destroyed large
plantations such as La Arenosa in Urab, Colombia, in the 1960s and
more recently has destroyed nearly 35,000 ha around Tumaco, Nario, Colombia. In the affected plantations, over 90% of productive oil
palms have been affected by the disease often resulting in plant
death, The bud rot disease has become the main cause of deterioration and loss of plantations in different regions of Colombia La Arenosa in Turbo (Colombia), Dernpasa (Brazil) and many plantations in
Ecuador [2]. These losses result in a decline in rural investments in
the affected regions and give rise to massive job losses in the plantations as well as in the local support and services industries.
It has been established that BR is caused by the oomycete,
Phytophthora palmivora [2]. The infection occurs in the soft tissue of
the spear leaves and in the elongation and maturation zones in the
upper third of the bud, and spreads to neighboring spear leaves.
These tissues provide ideal conditions for P. palmivora to colonize
white leaf tissue, sporulating and causing repeated infections that
result in the colonization of the bud by P. palmivora. Once colonized
the bud tissues become susceptible to invasion by opportunistic
microorganisms and the resulting decaying tissue attracts insects

A.L. Moreno-Chacn et al. / Plant Physiology and Biochemistry 70 (2013) 246e251

such as Rhynchophorus palmarum, that stimulate the decomposition process leading to the end of the productive life of the palm [3].
The problems associated with BR are the result of a physiological
disorder compounded with one or more types of stress [4] that
might trigger susceptibility to microorganisms that normally are
not pathogenic. Phytophthora blight caused by P. palmivora is a
devastating disease and a major constraint in the production of
cucurbits, peppers, tomatoes, and several other vegetable crops
throughout the world. The pathogen causes plant wilt, root rot,
crown rot, seedling damping-off, leaf and stem blight, and fruit rot
and results in signicant yield and quality loss [5]. The efcacy of
current strategies for management of this disease is limited. No
single fungicide has consistently and effectively suppressed losses
related to P. palmivora epidemics.
Survival strategies of seedlings under Phytophthora attack have
been extensively studied in many woody plants. The mechanisms of
resistance or defense against Phytophthora vary; therefore, the strategies for managing the disease also vary in terms of duration and
effectiveness. Some studies suggest that P. palmivora and other
Phytophthora species produce a structurally-related extracellular
family of proteins called elicitins that induce a hypersensitive
response, excitation of enzymatic control mechanisms of reactive
oxygen species and other biochemical changes in various species [6].
The production of various active oxygen species (AOS) is a
common event in all organisms living under aerobic conditions.
AOS are toxic intermediates that result from successive oneelectron steps in the reduction of molecular O2 and are routinely
generated at low levels by plant cells in chloroplasts, mitochondria,
and in other cell compartments involved in reductioneoxidation
processes.
When plants are attacked by pathogens they respond by activating a variety of defense mechanisms, including the rapid production and accumulation of AOS, primarily the superoxide anion
(O2
) and hydrogen peroxide (H2O2). Multiple roles for active oxygen in plant defense have been suggested, including acting as
second messengers, as substrates in synthesis of structural defense
components, and as the toxic agents that cause plant cell and
pathogen death. These studies have documented a strong correlation between production of AOS and the occurrence of the hypersensitive reaction (HR), the most common and dramatic defense
reaction in plants, as well as of systemic acquired resistance (SAR), a
non-specic induced resistance response [5,7].
Plants are able to defend themselves against pathogen attacks
by triggering defense reactions that include physical, chemical and
enzymatic responses. This induced resistance might occur when
plant cell membranes recognize general or specic signal molecules from pathogens during the pathogenesis interaction. In
addition, the pathogens enzymatic arsenal can degrade the plant
cell wall and liberate oligosaccharide fragments that, in turn, could
amplify the resistance response in the plant. General pathogen
derived signal molecules, which are known as pathogenassociation molecular patterns (PAMP), include fungal cell wall
fragments (oligosaccharins), lipopolysaccharides, peptidoglycans,
and glycoproteins. When PAMPs are perceived by a host membrane
receptor, they induce a non-host resistance in the plant that can
reduce or eliminate plant infection by the pathogen [5].

247

Stress situations may result in cellular changes that involve a

large number of responses. It has been documented that Polyamine
(PA) biosynthesis is affected by diverse stress conditions. Under
biotic stress, defense mechanisms, that induce the PA in response to
a pathogen attack, tend to curb the spread of the pathogen [7].
These stimuli promote the synthesis of defense compounds, resistance structures, physical and chemical barriers, etc. [8]. It is known
that there is a positive response of PA synthesis to fungi [9], viral
pathogens and mycorrhizae [10]. In this regard, it is known that, for
example, spermine is not a good indicator of defense against
pathogens [11], but it is very important for resistance to viruses
[12].
Another important observation regarding the implications of
the signaling pathways with PA involved in the response to biotic
stress, suggests that the accumulation of hydrogen peroxide as a
result of PA catabolism and nitric oxide induced by spermine/
spermidine, plays an important role as a sign of plant-pathogen
interaction [13]. Also, it has been observed that in oil palm PAs, at
meristematic level, increase in response to bud rot disease [14].
It has been established that the exogenous application of PA, as
well as its biosynthesis inhibitors, have an effect on the progression
of diseases [15]. Thus, the exogenous application of PAs increases
endogenous PAs and this practice has become a management
alternative against diverse types of stress [16]. Gathered evidence
has shown that the exogenous application of PAs has a positive
effect on cell integrity, reverses the growth inhibition caused by the
stress, generates the expression of genes that lead to osmotic
regulation, reduces the root content of hydrogen peroxide and superoxide and increases antioxidant enzyme activity. Therefore, it
becomes evident that the application or accumulation of PA acts as
elicitor of genes that are involved in resistance to biotic stress [16].
This paper aims to describe the changes in physiological parameters (gas exchange, water use efciency), metabolites (sugars,
chlorophylls, carotenoids, PA) and phenylalanine ammonia-lyase,
b-(1,3) glucanase and antioxidant enzyme activity, related to the
response of oil palm to bud rot disease. These results can be used to
start the characterization of the plant-pathogen relationship in the
oil palm -P. palmivora system for future new long-lasting and more
efcient strategies for the agronomic management of the disease.
Furthermore, these results could be the foundation for a breeding
program leading to the development of disease resistant varieties.

2. Results
2.1. Gas exchange
Gas exchange variables (Table 1) show a dramatic drop of the
photosynthetic potential from 13.29 mmol CO2 m
2 s
1 in healthy
palms to 3.66 mmol CO2 m
2 s
1 in palms at stage 5 of the disease.
Stomatal conductance and transpiration in healthy palms is four
times that of diseased palms at stage 5. The water use efciency of
diseased palms at stage 5 is half that of healthy palms. That is, there
is a highly signicant decrease in both photosynthetic capacity and
water use efciency as the severity level of the disease increases,
which is accentuated at stage 5 and is less at stages 1 and 3.

Table 1
Gas exchange in oil palm at different bud rot disease stages. Mean  standard deviation.
Healthy
Max. photosynthesis (mmol CO2 m
2 s
1)
Stomatal conductance (mmol H2O m
2 s
1)
Transpiration (mmol H2O$m
2 s
1)
WUE (mmol CO2/mmol H2O)

13.29
281.83
3.92
3.6






Stage 1
1.08
50.26
1.04
0.8

12.49
243.04
3.45
3.7

Stage 3





1.41*
59.32**
0.73*
0.6n.s

9.85
196.58
3.12
3.2

Stage 5





1.79**
60.96**
0.72**
0.6*

3.66
70.49
1.51
1.5






3.58**
60.63**
1.12**
0.24*

t-student test with no statistically signicant differences (n.s), signicant (*), highly signicant (**) differences between healthy palms and disease stages 1, 3 and 5 with P  0.05.

248

A.L. Moreno-Chacn et al. / Plant Physiology and Biochemistry 70 (2013) 246e251

2.2. Biochemical determinations

There is a signicant decrease in the content of chlorophyll A
and B (Table 2) from the stage 1, with a content reduction of about
40% with respect to stage 0, and remains stable at stages 3 and 5.
The total chlorophyll content (Table 2) also decreased as the disease
severity increased. At stage 1, this content decreases by about 13%
and over 45% at stages 3 and 5, compared to stage 0. Carotenoid
content (Table 2) showed a tendency to increase as the severity of
the disease progressed. Carotenoid content increased by about 60%
at stage 1, 100% at stage 3 and 140% at stage 5, compared to stage
0 of the disease. This suggests an energy targeting strategy via the
xanthophyll cycle.
The contents of total and reducing sugars (Table 3) increased as
the severity level increased. Total sugars at stages 1, 3 and 5
increased about twofold compared with stage 0. Reducing sugars at
stage 1 increased fourfold, and vefold at stages 3 and 5, compared
with stage 0. Total sugars at stages 3 and 5 are lower than the
reducing sugars. That is, there is an accumulation of photoassimilates and the translocation of sugars from the aerial part of
the palm is inhibited. Total protein (Table 3) was not affected by
increased severity levels of the disease.
Among the analyzed polyamines (Table 3), putrescine only
increased at stage 5; spermidine and spermine increased from the
stage 1, and their concentration increased twofold at stage 5.
The palm showed an increase in antioxidant enzyme activity
(Table 3) of catalase, peroxidase, ascorbate peroxidase, glutathione
reductase, superoxide dismutase and the enzyme activity of PAL
and b-Gluc as the level of severity of BR increased. Comparing stage
0 with stage 1, there was a slight increment in the recorded enzyme
activities, which suggests low inoculum pressure, high enzyme
basal state or a low production of reactive oxygen species (ROS);
PAL activity at this stage is similar to that of stage 0, suggesting that
the phenylpropanoid pathway had not yet been activated. Similar
results were obtained with b-1,3-glucanase, whose activity was not
affected at this initial stage. On the contrary, the GR activity did
show a large increase at this stage, which is indicative of the
importance of the glutathione redox system in the early stages of
the disease. At stage 5 there is a signicant growth point, suggesting that at this point the pathogen had activated the consortium of enzymatic responses in the plant. Finally, stage 5 shows
a response very similar to that of stage 3, only slightly more variable
as a result of the progress of the disease in the tissue, as at this point
the disease seems to be not so controlled.
3. Discussion
The overall results support the conclusion that the stage 1 of the
bud rot disease has no signicant effect on the physiology and
metabolism of oil palm. Thus, gas exchange parameters, putrescine,
enzyme activity, CAT, POD, SOD, PAL and b-Gluc did not change
signicantly (Table 3). However BR affected the content of chlorophylls, carotenoids, total and reducing sugars and GR activity,
which suggests that the pathogen must have a slow penetration

strategy based on the glutathione redox cycle in the apoplast [17],

and does not caused an imbalance in the cytosolic energy cycles.
Stages 3 and 5 showed an increase in the overall enzyme activity
CAT, POD, SOD, GR, PAL, and b-Gluc, and in the content of carotenoids, spermidine, spermine, putrescine and sugars, but a substantial decline in photosynthetic parameters and chlorophyll
levels. At these stages of the disease the effect of the pathogen
reached the cytosolic redox balances and triggered the plant
response mechanisms [18].
The translocation of photoassimilates to other parts of the plant
was interrupted because of the low photosynthetic capacity and
inefcient use of water, as well as an accumulation of sugars in the
leaves (Tables 1e3). Therefore, photosynthesis levels (Table 1)
decreased as the severity of the disease increased which could have
been caused by the accumulation of photoassimilates (sugars)
(Table 3) in the chloroplasts that inhibited carboxylase activity of
rubisco. This reduction of rubisco activity, together with the
reduction of chlorophyll A levels and increased chlorophyll B
(Table 2), made the photosynthetic process less efcient [19,20].
The decline in photosynthetic capacity caused by the increased
severity of the disease could also be indirectly seen in the increased
carotenoid levels (Table 2), which mitigated the large energy ows
reaching the palm that could not be assimilated by chlorophyll or
by the electron transport chain of photosynthesis, and might have
been received by xanthophyll cycle carotenoids [21]. However, it
seems that the plant tried to protect itself by reducing its transpiration and stomatal conductance (Table 1) to prevent excess loss of
water during the photosynthetic process.
Oil palms responded to BR by inducing polyamine accumulation
as a mechanism to reduce the spread of the pathogen [10]. The
stimuli generated promoted the synthesis of defense compounds,
resistance structures, chemical and physical barriers, etc. [9]. For
example, spermine accumulation has been associated with stimulation of the activity of two important proteins in the activation of
mitogen-activated protein kinases (MAPKs), involved in plant defense mechanisms, wound-induced protein kinase (WIPK) and
salicylic acid-induced protein kinases (SA-SIPK) [11]. Also, it has
been observed that the exogenous application of spermine actives
MAPKs, increases hypersensitive reaction mechanisms, induces
systemic response and cell death [21]. However, there are still many
gaps in our understanding of the PA role in the Elaeis guineensise
Phytophthora palmivora interaction. However, the role of PA in the
response of plants to pathogens has been studied in, for example,
the interaction of Hordeum vulgare L. with Puccinia hordei [23].
Here, PA accumulated, particularly spermidine, in infected leaves.
H. vulgare also showed accumulation of PA in the leaves after the
infection with Blumeria graminis f. sp. Hordei [10,24].
Another important observation regarding the implications of
signaling pathways with PA involved in the response to biotic stress
suggests that the accumulation of hydrogen peroxide as a result of
PA catabolism and spermine/spermidine-induced nitric oxide play
an important role as a signal of the plant-pathogen interaction [13].
The progression of the disease also allowed us to see a concerted
response of the antioxidant enzyme and defense systems in the

Table 2
Chlorophyll and carotenoid contents in oil palm at different stages of the bud rot disease. Mean  standard deviation.
Healthy
Chlorophyll A (ng Chlorophyll A/mgTFF)
Chlorophyll B (ng Chlorophyll B/mgTFF)
Total chlorophyll (ng Chlorophyll T/mgTFF)
Carotenoid (ng carotenoid/mgTFF)
Chlorophyll A/Chlorophyll B
Total carotenoids/Chlorophyll

4.4
8.8
13.2
1.5
0.50
0.16








0.2
0.3
0.4
0.3
0.01
0.06

Stage 1
2.4
7.3
9.6
2.8
0.32
0.30








0.3**
0.3**
0.5**
0.7**
0.02**
0.12**

Stage 3
2.6
5.3
7.8
2.7
0.51
0.34








0.2**
0.8**
0.8**
0.7**
0.10n.s
0.14**

Stage 5
3.0
4.8
7.8
3.7
0.62
0.37








0.3
0.3**
0.4**
0.7**
0.08**
0.08**

t-student test with no statistically signicant differences (n.s), signicant (*), highly signicant (**) differences between healthy palms and disease stages 1, 3 and 5 with P  0.05.

A.L. Moreno-Chacn et al. / Plant Physiology and Biochemistry 70 (2013) 246e251

249

Table 3
Contents of sugars, polyamines, antioxidant enzyme activity, phenylalanine ammonia-lyase and b-1,3-glucanase in oil palm at different stages of the bud rot disease.
Mean  standard deviation.
Healthy
Total sugars (mg sugar/g TFF)
Reducing sugars (mg sugar/g TFF)
Putrescine (nmoles/g TFF)
Spermine (nmoles/g TFF)
Spermidine (nmoles/g TFF)
AscorbatePeroxidase (nmolesascorbate oxide/(min mg protein))
Catalase (mmoles H2O2/(min mg protein))
Peroxidase (D Abs. 436 nm/(D min mg protein))
Glutathione reductase (nmoles NADPH/(min mg protein)
Superoxide dismutase (50% Inhibition cytochrome C/(mgprotein min))
Phenylalanine ammonia-lyase (nmolescinnamic acid/(min mg protein))
b-1,3-Glucanase (mg glucose/(min mg protein))

3.3
1.8
25.72
28.98
48.09
169.1
3.3
295.3
31.0
2206.7
3314.4
0.7

Stage 1














0.2
0.2
6.19
10.37
12.72
35.5
0.4
89.92
6.0
470.3
144.1
0.2

7.1
8.6
33.18
163.16
222.30
413.0
6.2
552.1
76.9
3324.3
3516.1
0.8

Stage 3













0.3**
0.6**
12.76n.s
81.85*
100.17*
515.5n.s
0.6**
62.8**
8.3**
660.9**
316.4n.s
0.3n.s

7.3
10.2
39.34
139.88
405.70
405.6
11.3
714.4
133.4
5440.8
4199.9
1.2

Stage 5













0.2**
1.2**
13.16*
28.58**
148.59**
166.0*
0.6**
114.2**
35.6**
1199.2**
309.8**
0.6*

9.4
10.2
83.46
270.21
583.06
639.4
16.7
24181
142.1
7621.5
5180.4
2.6














1.4**
1.2**
37.40**
74.70**
256.72*
279.3**
1.5**
21523**
54.0**
1942.2**
610.7**
0.9**

t-student test with no statistically signicant differences (n.s), signicant (*), highly signicant (**) differences between healthy palms and disease stages 1, 3 and 5 with P  0.05.

most advanced stages of the disease. The altered photosynthesis

cycles generally result in excessive energy transfer to oxygen,
which favors the formation of reactive oxygen species, which are
controlled by a set of enzymatic reactions and some metabolites.
Catalase is not a chloroplastic enzyme, which regulates the levels of
hydrogen peroxide. Its importance lies in its high specicity and
ability to sense minor or nano-molar changes in the substrate. The
response found for this enzyme, which increases its activity by
four-fold at stage 5 of the disease with respect to stage 0, suggests
an increase of hydrogen peroxide outside the chloroplast as a result
of the pathogen penetration that goes along with the progression of
the disease, i.e. there is a mechanism that drives the production of
oxygen reactive species to control the spread of the microorganism
at stages 3 and 5 of the disease. Yu et al. [25], report the increase of
CAT activity as a response to the pathogens entering the chloroplasts. Gayoso et al. [26], in Capsicum annuum, and Chen et al. [27],
in Glycine max found a direct relationship between the progression
of Phytophthora and catalase activity, which is evidence that this
enzyme is key in protecting plants against pathogen attacks.
The study of the enzymes of the ascorbate/glutathione cycle that
takes place in the chloroplasts, involving superoxide dismutase,
ascorbate peroxidase and glutathione reductase shows the effectiveness of the transfer of energy to oxygen and the level of preparation to respond to abrupt changes in energy transfer efciency.
The activity of these three enzymes increases as the severity of the
disease increases, thus there is an increment in the production of
the superoxide radical, which is an intermediate state of oxygen
during its energy assimilation process and one of the cycle regulation points. This suggests an alteration in the energy cycles of
photosynthesis, which is typical of plants with systemic response to
disease progression. Additionally, peroxidases, enzymes located in
various cell organelles, are indicative of the evolution of their
substrates, i.e. donors (peroxides) and receptors (phenols). The
activity of this enzyme in bud rot-infected palms increase as the
disease severity increases, suggesting that the synthesis of the two
substrates had been promoted. This had already been observed in
other enzymes for hydrogen peroxide. Peroxidase activity has a
great inuence on the condensation of phenols that ultimately
form lignin, cellulose and suberin structures. Therefore, the activation of the enzyme is an essential step in controlling the spread of
the pathogen. Similar results were obtained by Dutsadee & Nunta
[28] in Hevea brasiliensis inoculated with P. Palmivora and Gayoso
et al. [26], in C. annuum L. var.annuum inoculated with Phytophthora
capsici.
Our result suggest that the oil palm defense mechanism involves
the activation of PAL, a key enzyme of the phenylpropanoid
pathway, from which phenolic metabolites are synthesized and so
the spread of the pathogen is reduced. PAL activation is associated

with systemic acquired resistance (SAR) as well as the b-1,3glucanase which is responsible for glucose release from homopolysaccharide cellulose, producing a series of
1,3/1, 6-glucan
oligosaccharides that induce a wide range of responses in plants.
Thus, the activation of both enzymes in response to BR might be a
key component of the response of the palm to P. palmivora attack
and must be further research in light of the potential for selection of
genotypes more resistant to BR. These results were conrmed by
Bailey et al. [29], in Theobroma cacao infected with Phytophthora
megakarya and by other studies [30].
The results show that oil palm responds to BR by activating
several enzymes of the antioxidant system and also with the increase in the activity of some defense related enzymes. The activation of both types of enzymes is an indicator of the recognition that
the palm is making of a pathogen, however, despite such recognition; the plant fails to overcome the attack and is greatly affected in
its primary and secondary metabolism. Further research is needed
in order to understand the mechanisms by which the pathogen is
overcoming the defense mechanisms of the plant. It could be a
timing issue, in which by the time the plant nally recognizes the
pathogen; the pathogen is so generally spread that the palm cannot
survive the disease. On the other hand, the idea of a complex in
which P. palmivora causes the initial damage and is then followed by
opportunistic pathogens that are the nal responsible for the plant
destruction, could answer the question of why despite the general
defense response of the plant, the BR disease takes over the plant.
It is plausible that the defense mechanism we measured were
triggered by the opportunistic pathogens, but the initial culprit
could not be recognized by the susceptible palms.
4. Materials and methods
The study was conducted in the plantation Oleaginosas las
Brisas S.A, located in the municipality of Puerto Wilches
(Santander e Colombia), at an altitude of 125 m.a.s.l with an
average temperature of 34  C, relative humidity of 70.5%, 3.852 mm
annual rainfall, and tropical rainforest agro-ecological conditions.
In collaboration with the plantation, a plot with a high incidence
of bud rot disease (incidence >64%) was selected, with 4-year old
planting material Deli x Ekona (E. guineensis). A general census was
conducted in the selected plot using CENIPALMA scale for bud rot
[3,31]. Based on the census, nine random palm samples were taken
at stages 0 (healthy), 1, 3 and 5, and measurements were taken for
gas exchange and biochemical indicators in the frond number
three, according to the palm phyllotaxis. The symptoms of the BR
have been described [3,31]. There is a fairly constant overall
symptom in the early stages of disease development, but it may
develop differentially depending on the situations in which the

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A.L. Moreno-Chacn et al. / Plant Physiology and Biochemistry 70 (2013) 246e251

palms are affected. In the healthy state, the emerging leaf is clean
and without any lesion. In state 1, lesions account for between
0.1%e20% of the emerging leaf; at state 3 the lesions cover between
40.1%e60% of the emerging leaf; and in state 5 lesions are spread
between the 80.1%e100% of the emerging leaf [3,31]. Currently,
there is a strategic plan in place for managing the disease [31];
however, the palms intervened have had long periods of recovery
and reincorporation into the productive cycle. Also, they may
become a major pathogen source for future plantings.
4.1. Photosynthetic capacity and gas exchange
Measurements of gas exchange (maximum photosynthesis,
transpiration, stomatal conductance) and water use efciency were
made using the LICOR 6400 infrared gas analyzer (IRGA). The
samples were taken between 8 a.m. and 11 a.m. in three leaets of
frond 3, with 1000 mmol m
2 s
1 of PAR saturation.
4.2. Biochemical determinations
Leaets of the middle third of frond number three were cut into
small pieces and quickly placed in liquid nitrogen and stored
at
80  C until the time of processing. For analysis, tissues were
macerated in liquid nitrogen and biochemical measurements were
taken from the macerated substance using the Synergy Mx (BioTek)
equipment.
4.2.1. Chlorophyll A, B, and total carotenoids
Total chlorophyll and carotenoids were extracted from 15.0 mg
fresh leaf tissue with 80% acetone (v/v) at
20  C; the determination was carried out according to [32]. The content was expressed
as mg pigment/g fresh tissue.
4.2.2. Sugars
The determination of total sugars was made with 15.0 mg of
fresh leaf tissue [33] at 4  C. The content was expressed as mg
sugar/g fresh tissue. The determination of reducing sugars was
made with 15.0 mg of fresh leaf tissue [34,35] at 4  C. The content
was expressed as mg sugar/g fresh tissue.
4.2.3. Polyamines
The determination of putrescine, spermidine and spermine was
made with 1.5 g of fresh leaf tissue [14]. The content was expressed
as nmoles polyamine/g fresh tissue.
4.2.4. Antioxidant enzyme activity
The extraction of catalase (CAT, EC.1.11.1.6) and peroxidase (POD,
EC 1.11.7.1) was carried out on 250.0 mg of fresh tissue with 50 mM
sodium phosphate buffer, 3% (p/v) PVP-40, pH 6.80 at 4  C. Catalase
enzyme activities (CAT, EC 1.11.1.6) were determined using the
permanganometric method [36], the specic enzyme activity was
dened as mmoles H2O2/(min mg protein) at 37  C and the peroxidase enzyme activity (POD-EC.1.11.1.7) was determined using the
O-dianisidine method [37] and the enzyme activity unit was
dened as D Abs. 436 nm/(D min mg protein) at 37  C.
The extraction of ascorbate peroxidase (APX, EC 1.11.1.11) and
glutathione reductase (GR, EC 1.6.4.2) was carried out on 500.0 mg
of fresh tissue with TriseHCl 50 mM buffer, 10 mM EDTA-Na2 pH
7.60 at 4  C and the enzyme activity of ascorbate peroxidase was
recorded using spectrophotometric monitoring [38]; the enzyme
activity unit of ascorbate peroxidase was dened as nmoles
ascorbate oxidase/(min mg protein) at 25  C. The glutathione
reductase activity was measured using the method of Yannarelli
et al. [39], and the enzyme activity unit was dened as nmoles
NADPH/(min mg protein) at 25  C.

The extraction of superoxide dismutase (SOD, EC1.15.1.1) was

carried out on 500.0 mg of fresh tissue with TriseHCl 100 mM
buffer pH 7.50 at 4  C and the activity was determined using inhibition of cytochrome c oxidation [40], the enzyme activity was
dened as the amount of enzyme capable of inhibiting 50% of cytochrome c oxidation/(mg protein min) at 560 nm at 25  C.
4.2.5. Enzyme activity of phenylmethylammonia-lyase
The extraction of phenylmethylammonia-lyase (PAL, EC 4.3.1.5)
was carried out using 500.0 mg of fresh tissue with sodium borate
buffer 100 mM pH 6.5 at 4  C [41]. This specic enzyme activity was
reported as: nmoles cinnamic acid/(min mg protein) at 37  C.
4.2.6. Enzyme activity of b-1,3-glucanase
The extraction of b-1,3-glucanase (b-Gluc, EC 3.2.1.6) was carried
out with 500.0 mg of tissue with sodium phosphate buffer 100 mM,
3% (p/v) PVP-40, pH 6.50 at 4  C. The activity was determined using
the colorimetric quantication of glucose enzymatically released
from lamirarin [42]. The concentration of glucose released was
determined using the NelsoneSomogyi method [34,3]. The enzyme
activity unit b-1,3-glucanase was dened as UbGC mg glucose/
min. This specic enzyme activity is the unit of activity per milligram of protein UAEbGluc mg glucose/(min mg protein) at 37  C.
4.2.7. Protein content
The total soluble protein content was determined by the method
of Bradford [43].
Data generated were analyzed using one-way ANOVA student t
test in SAS statistical software version 9.1. Statistically signicant
differences between healthy palms and each severity stage of the
disease, with P  0.05.
Acknowledgments
The authors wish to thank Oleaginosas las Brisas S.A, the
members of the Research Team of the Biology and Breeding Program of CENIPALMA and the staff of the Palmar de la Vizcana
Research Center for their contribution to this research. The research
was funded by the Ministry of Agriculture and Rural Development
and by the Oil Palm Promotion Fund (FFP) managed by Fedepalma.
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