Journal of Plant Pathology (2014), 96 (1), 29-34 Edizioni ETS Pisa, 2014 Abassi et al.

29

MANGANESE DEFICIENCY IS ASSOCIATED WITH HISTOLOGICAL CHANGES IN DATE

PALM FRONDS SHOWING BRITTLE LEAF DISEASE SYMPTOMS

R. Abassi1, A. Namsi2, M. Bennasri1, H. Ben Abdalah2, S. Ben Mâachia2, Z. Ouerghi1 and N. Duran-Vila3
1 Faculté des Sciences Mathématiques, Physiques et Naturelles de Tunis, Département de Biologie FST Campus Universitaire,
2092 El Manar Tunis, Tunisia
2 Centre Régional de Recherche en Agriculture Oasienne de Degache, BP 62, 2260 Degache, Tunisia
3 Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias, Apartado oficial,

46113 Moncada (Valencia) Spain

Running title: MFC symptoms associated with Mn deficiency

SUMMARY important element of oasis agriculture is the date palm

Several diseases, pests and physiological disorders
threat date palm life. Among them, Brittle leaf disease or,
in French, Maladie des feuilles cassantes (MFC) is a lethal
disease that has acquired alarming proportions since the
1960s, when it was first observed in southern Tunisia. Up
to now no causal agent has been found. Previous studies
have reported that brittle fronds are deficient in manga-
nese but the mechanisms involved in symptom expression
have not yet been identified. In this study, an anatomical
investigation was carried out in order to identify the dele-
terious effect of MFC on the development and structure of
palm fronds. Results showed that in old healthy fronds the
major tissue is schlerenchyma, which is characterized by
very thick lignified cell walls and is often associated with
conducting tissue. Indeed, large vessels are surrounded
by schlerenchyma fibers that are strongly lignified when
compared with those of young fronds. Diseased young
fronds have almost the same shape as the healthy young
fronds. Nevertheless, in old diseased fronds MFC induces
important structural changes. Xylem and phloem vessels
are larger, taking more space through the fibers that are
smaller in number and size, and tighter. The larger diame-
ter of the lumen and the lower thickness of the fibers pres-
ent in the fronds in advanced disease stages, decreases the
lignin content of cells walls, leading to an increased frond
friability, which gives “the brittle leaf disease” its name.
Key words: fibers, lignification, maladie des feuilles cas-
santes, MFC.
INTRODUCTION
Oasis agro-ecosystems of southern Tunisia cover about
40,000 ha (Namsi et al., 2007) playing an important role
in diversification of agricultural crops (fruit trees, vegeta-
bles and fodder) that grow under date palms. The most
Corresponding author: N. Duran-Vila
Fax: +34.963424001
E-mail: nduran@ivia.es
(Phœnix dactylifera L.), considered as one of the oldest
species in agricultural systems. The Tunisian heritage of
date palms is around 4.310.000 trees ranking 4th in the
Maghreb (Sedra, 2003) and allowing date exports to 56
countries (Triki et al., 2003). Date palms have a great socio-
economic potential, thus the problems (soil, water, disease
control, etc.) limiting their performance need to be kept
under scrutiny. Biotic and abiotic disorders threaten date
palm life.
Brittle leaf disease (MFC, from the French “Maladie
des Feuilles Cassantes”), described in the 1960s in south-
ern Tunisia, is of concern for the performance and survival
of affected oases. MFC can cause the decay of date palms,
whose number increased from around 300 to more than
36,000 in 2002 (Namsi et al., 2007). Early symptoms ap-
pear as softening and yellowing of the fronds that subse-
quently become brittle, brown and necrotic. MFC-affected
palms show an abnormal shiny yellowing, often accompa-
nied by loss of flexibility (Fig. 1A). The fronds, yellowish
at first, develop longitudinal streaks and bending of the leaf
blade, dry out and become wavy and brown with a burned
appearance (Fig. 1B). At the last stage, fronds dry out, nec-
rotize and fall off when is windy (Fig. 1C). At a more ad-
vanced stage, the color of the spines changes from yellow
to brown and they also become very brittle.
A multidisciplinary approach is necessary to understand
and control the MFC syndrome. The brittleness remains
a specific symptom, but no information is available on the
mechanisms and the type of physiological disorder causing
it. A proteomic study showed differences in the quantity of
several proteins including the manganese-binding PSBO
and PSBP proteins, components of the oxygen-evolving
complex of photosystem II, that decrease in affected tis-
sue, whereas other proteins increase, suggesting a stress
response (Marqués et al., 2011). It has also been found that
manganese (Mn) deficiency generates an oxidative stress
with and increase of total superoxide dismutase (SOD)
activity associated with a decrease of MnSOD RNA and
an increase of FeSOD and Cu/Zn-SOD RNAs (Saidi et
al., 2012). In addition, a study focusing on the phenolic
compounds present in the cell wall under Mn deficiency
30 MFC symptoms associated with Mn deficiency
Fig. 1. Progressive symptoms characteristic of the Brittle leaf disease (MFC, from the French “Maladie des feuilles cassantes”). A.
View of a symptomatic palm. B. Early first stage of the disease. C. Advanced disease stage.
showed that leaves presenting moderate symptoms, had an
enhanced extracellular accumulation of p-hydroxybenzoic
acid, p-hydroxycinnamic acid and p-hydroxybenzaldehyde
and a significant decrease in acetophenones, particularly
20-hydroxy-40, 50-dimethoxyacetophenone and aceto-
syringone (Latreche and Rahmania, 2011).
To further understand the MFC phenomenon, mineral
analysis of healthy and affected date palm leafs was per-
formed, followed by an anatomical observation to discern
the MFC effect on the cell wall structure and to clarify the
mechanism underlying frond fragility.
MATERIALS AND METHODS
Study area. The study was performed at the Chemsa oa-
sis, located in the Tozeur region. This was an experimental
plot that had been designed to define MFC symptoms on
palms and fronds and to elaborate a rating scale. Further-
more, it provided a source of samples for further analyses
in three selected sites.
Sampling technique. At each site containing MFC-af-
fected palms, three date palms were selected and identi-
fied according to the severity of MFC symptoms: (i) three
apparently healthy palms (AH) (no visible symptoms); (ii)
three palms showing early MFC symptoms (I1); and (iii)
three palms at the final MFC stage (I2). Three symptom-
less/healthy samples (H) were collected from another site
where MFC had never been observed. All samples were
collected from 20-year-old trees of cv. Deglet Nour.
Mineral analysis. Mn, iron (Fe) and zinc (Zn) con-
tents were determined according to Pauwels et al. (1992).
Healthy and symptomatic fronds were carefully washed,
oven-dried at 60°C for three days and ground into fine
powder with mortar and pestle. Then, 1 g of each sam-
ple was transferred to a beaker containing 20 ml HNO3
Journal of Plant Pathology (2014), 96 (1), 29-34
and kept in a fume cupboard for three days. The suspen-
sion was heated on a hot plate for 24 h, and concentrated
HNO3 was added intermittently until it turned colorless.
Then, samples were cooled and transferred into 100-ml
volumetric flask and made up to the mark by adding dis-
tilled water. Finally, suspensions were filtered and trans-
ferred into an analytic bottle for atomic absorption spec-
trophotometer and flame photometric analysis (NovAA
400, Analytic Jena AG, Germany). The data were subject-
ed to ANOVA and Newman and Keuls statistical analysis
(STATISTICA Software 5.1).
Tissue sections. Fronds from symptomless and symp-
tomatic date palms were collected, sectioned using a freez-
ing microtome, stained with acetocarmine and observed
under a light Reichert LKB microscope connected to a
camera.
RESULTS AND DISCUSSION
Mn deficiency and its effects. The correlation between
Mn content and symptom severity was highly significant
(Fig. 3A), with clear differences between healthy (H)
palms and those of the three MFC stages (AH, I1, I2) with
low Mn contents. The differences in Mn content from one
stage to another suggest the existence of a relationship be-
tween symptom severity and a progressive Mn deficiency.
The average Mn content in the AH stage was close to 12
ppm and below the deficiency level (estimated to be 20
ppm), reinforcing the hypothesis that the MFC syndrome
might be the result of a nutritional imbalance occurring
before the onset of symptoms (Namsi et al., 2007).
In disease stages I1 and I2, the average Mn content was
below 7 and 5 ppm, respectively. It seems that in these
stages certain physiological functions are probably dis-
turbed and the palm is unable to regulate the cellular ex-
change of nutrients, causing constraints at the level of cell
Journal of Plant Pathology (2014), 96 (1), 29-34 Abassi et al. 31

Fig. 2. Frond blades from a healthy palm (A) and from palms
(B,C,D) affected by MFC. A. Healthy frond (H); B. Appar-
ently healthy frond (AH). C. Frond from a palm at the early
disease stage (I1). D. Frond from a palm at an disease ad-
vanced stage (I2).

metabolism across the entire tree. Zinc (Zn) and iron (Fe)
contents in fronds from healthy and diseased date palms
support this assumption.
According to the literature, Mn interacts with Zn and
Fe in various plant physiological aspects (Pearson and
Rengel, 1997; Kim and Guerinot, 2007). In the materi-
als tested, the average Zn content in healthy tissues was
around 10 ppm but it increased considerably in MFC-
affected tissues, reaching about 27 ppm in the final MFC
stage (I2) (Fig. 3B). Paradoxically, Zn excess seems to be a
consequence of Mn deficiency, because symptoms of zinc
excess were not observed in MFC-affected palms. Thus,
competition between Mn and Zn appears to induce pro-
tein degradation and inhibits the accumulation of other
mineral elements because the excess of the second ele-
ment has negative effects on membrane permeability (De
Magalhaes et al., 2004).
Iron (Fe) content in diseased tissues ranged from 160
ppm in AH to 200 ppm in stage I2, whereas it was much
higher in healthy tissues (300 ppm) (Fig. 3C). Thus, there
is no relationship between the disease and Fe content.
However, a correlation between the three disease stages
and Fe content was observed.
Mn, Zn and Fe are known to be major cofactors of en-
zymes such as deoxygenases, peroxidases and superoxide
dismutase that are involved in the degradation, detoxifi-
cation of harmful substances and remediation of reactive
oxygen species (Rawyler et al., 2002). All these effects may
create mineral imbalances that may cause a disruption of
cation exchange, Fe in particular, which tends to increase
after a sharp decrease. Thus, competition among cations
seems to play a crucial role in this imbalance. In addition,
Fig. 3. Manganese (Mn), Zinc (Zn) and iron (Fe) content in
competition between Mn and Fe could explain this phe- fronds from healthy and diseased palms. Healthy (H); appar-
nomenon. When the Mn content decreases in tissues of ently healthy (AH); early disease stage (I1); advanced disease
MFC-diseased palm stages, the Fe content increases (Keh- stage (I2). Standard deviation was calculated with the data of
res et al., 2002). As a consequence of all these imbalances three repetitions.
32 MFC symptoms associated with Mn deficiency Journal of Plant Pathology (2014), 96 (1), 29-34

ep
f
bv

xy m
sv
ph

fs

A x245
x213 B

C x267 D

E x222 F

G x18 H I x528 J
Fig. 4. Cross-sections from healthy (A-B) and MFC-affected (C-D-E-F) fronds. A. Healthy young frond. B. Healthy aged frond.
C. Diseased young frond. D. Diseased aged frond. E. Frond in an advanced disease stage. F. Frond in the final disease stage. G.
Overall frond showing the area where the histological sections were made. H. Fiber of healthy frond. I. Fiber of frond at the first
disease stage. J. Fiber of frond at an advanced disease stage. Symbols: bv, big vessel; ep, epidermis; f, fiber; m, mesophyll cells; ph,
phloem; sv, small vessel; xy, xylem; fs, fibrous sheath.
Journal of Plant Pathology (2014), 96 (1), 29-34
and oxidative stresses, the general architecture of palm
tissue is altered (Shainberg et al., 2000).
Histological effects. The structure of cross-sectioned
fronds comprises mesophyll and parenchyma tissues, large
phloem and xylem vessels surrounded by a fibrous sheath,
and small vessels and fibers (Fig. 4A). Young healthy
fronds in an early stage of growth and differentiation, sec-
tioned through the middle (Fig. 4G), were composed of
two layers of upper epidermal cells and one layer of lower
epidermal cells which varied in shape and size. Epidermal
cells of the lower surface were slightly smaller than those
of the upper surface. At this early stage of growth, the
cuticle covering the upper epidermal cells was thin. Fibers
were not uniform and vessels showed differences in size,
shape and a thick cell wall.
Old healthy fronds (Fig. 4B) were more advanced in
terms of differentiation and development than the younger
ones. Differences in the thickness of epidermal cell walls
of the upper and lower surfaces were evident, those of the
upper layers being thicker than the lower ones. Schleren-
chyma cells had very thick and lignified cell walls often
associated with vessel tissues. Xylem was well defined and
could be clearly distinguished from the phloem. Lignin
was more apparent in big vessels of old fronds that are
surrounded by schlerenchyma fibers (see arrows in Fig.
4B). Small vessels were very clear, transparent and well
structured. Thick-walled fibers were well lignified and
widely distributed in the lower mesophyll tissue. These
fibers are rich in lignin, providing rigidity to the overall
leaflet structure (Reddy and Yang, 2005), but its synthesis/
accumulation changes with the development and growth
of the tissue (Kerem and Hadar, 1993). The diameter of
fiber lumen was another important factor to be consid-
ered with old fibers having a smaller lumen and higher
cell wall thickness (Fig. 4H) than young fibers. All this
indicates the existence of a high level of lignification pro-
viding rigidity to the frond. In fact, the walls of fibers of
many monocotyledons develop through successive ligni-
fication steps, with lignin contributing to the strength of
the fronds and woody stems and to the waterproofing of
xylem elements (Latreche and Rahmania, 2011).
Young fronds of MFC-affected palms (Fig. 4C) have
the same anatomical structure as young fronds of healthy
palms (Fig. 4A). Indeed, mesophyll, epidermal thickness,
vessels and fibers of MFC-affected fronds had similar
structure and architecture than those of from healthy
counterparts. In early growth stages, no major anatomi-
cal differences were observed. Nevertheless, in old MFC-
affected leaflets (Fig. 4D) important structural changes
were observed: (i) the upper surface is composed of one
layer of epidermal cells; (ii) the mesophyll thickness ob-
served in transverse sections of MFC-affected fronds is
slightly lower than that of young fronds; (iii) vessels have
an oval shape and have lost their normal arrangement; (iv)
xylem and phloem vessels are larger and have gained more
Abassi et al. 33
space through the fibers that became smaller and tighter.
Thus, the number and size of fiber cells was smaller and
fiber thickness had decreased from the early to old stage
in MFC-affected fronds.
Fibers of palm fronds in advanced MFC stage had a
larger lumen diameter and smaller thickness (Fig. 4E, 4I)
presumably due to a reduction of lignin content of the cell
wall, which was likely to be responsible for the increased
frond fragility (Abdul Khalil et al., 2006), that gives the
brittle leaf disease its name. In the final MFC stage a fur-
ther decrease in the lignin of the fibers was observed (Fig.
4F) with dark and poorly structured fiber cells (Fig. 4J).
This cellular disintegration was accompanied by a decrease
in elasticity and plasticity of the cell walls, due to an altera-
tion in the structure of cellulose and lignin that support
these properties (Mauseth, 1988). Nutritional imbalance or
reduced availability of minerals may be the ultimate cause
of cell wall degradation (Osono and Takeda, 2001).
Mn deficiency can derive from its unavailability in the
soil or limited uptake. The present results show that MFC
has a serious effect on the frond structure. First, a min-
eral imbalance occurs, disturbing the general metabolism
of the plant that affects cell wall integrity and alters the
structural rigidity of schlerenchyma tissues. The observed
modification of lignin in the fibers may be a consequence
of mineral disorders. The abnormalities in the frond struc-
ture and vascular tissues are probably the result of modi-
fied metabolic reactions catalyzed by Mn and, perhaps,
other trace elements. Further studies need to be carried
out for a better assessment and significance of the modifi-
cation shown in cell wall structure of MFC-affected palms.
ACKNOWLEDGEMENTS
The work was supported by Centre Régional de Re-
cherches en Agriculture Oasienne à Degache (Tunisia) and
grant AGL2012-32429 from the Ministerio de Economia y
Competividad (Spain).
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