Occurrence in Elx, SE Spain of Inflorescence Rot Disease of Date

J. Phytopathology 153, 417–422 (2005)
2005 Blackwell Verlag, Berlin
Laboratory of Plant Pathology, Department of Marine Sciences and Applied Biology, University of Alicante, Alicante, Spain
Occurrence in Elx, SE Spain of Inflorescence Rot Disease of Date Palms Caused
by Mauginiella scaettae
S. K. Abdullah1, L. Asensio2, E. Monfort2, S. Gomez-Vidal2, J. Palma-Guerrero2, J. Salinas2, L. V. LopezLlorca2, H.-B. Jansson2 and J. Guarro3
AuthorsÕ addresses: 1Biology Department, College of Science, University of Basrah, Basrah, Iraq; 2Laboratory of Plant
Pathology, Department of Marine Sciences and Applied Biology, University of Alicante, Apto. 99, 03080 Alicante, Spain;
3
Microbiology Unit, Medicine School, Rovira i Virgili University, C/Sant Llorenç, 21, 43201 Reus, Spain (correspondence to
H.-B. Jansson. E-mail: [email protected])
Received March 23, 2005; accepted May 13, 2005
Keywords: date palm, inflorescence rot, Mauginiella scaettae, internal transcribed spacer sequencing, Spain
Abstract
Fungal inflorescence rot of date palms, caused by
Mauginiella scaettae, was detected at all four sites surveyed in Elx, SE Spain. There was a higher incidence
of the disease in male than in female inflorescences.
The pathogen responsible for the disease was isolated
and identified as M. scaettae. Sequencing of the internal transcribed spacer (ITS) region of this fungus
demonstrated that it is close to Phaeosphaeria.
crop occurred in 1983 in the Katif province, Saudi Arabia (Zaid et al., 2002).
Our aims were to identify the causal agent of inflorescence rot disease occurring on date palms at the Elx
grove in south-east (SE) Spain, to determine the incidence of the disease in different palm plantations, and to
find a possible anamorph–teleomorph connection by analysis of the internal transcribed spacer (ITS) sequences.
Materials and Methods
Area of study
Introduction
The occurrence of date palm inflorescence rot, also
called ÔKhamedjÕ in North Africa, has been reported in
most date palm-growing areas of the world. This disease
and its causal fungus were reported for the first time by
Cavara (1925a,b) in Libya. The disease was reported
subsequently in other North African countries (Chabrolin, 1928; Munier, 1955; Calcat, 1959), and has also
been reported in Iraq (Allison, 1952; Hussain, 1958; AlAni et al., 1971), Egypt (Michael and Sabet, 1970) and
from the Arabian Peninsula (Abu Yaman and Abu
Blam, 1971; Djerbi, 1982). The major cause of inflorescence rot is considered to be the fungus Mauginiella
scaettae Cavara (Hussain, 1958; Al-Ani et al., 1971).
However, other fungi such as F. oxysporum Schldl.
emend. Snyder & Hansen, F. moniliforme Sheld. aggregate and F. solani (Mart.) Appel & Wollenw. aggregate
Snyder & Hansen, Thielaviopsis paradoxa (de Seynes)
Höhn. and Trichothecium roseum (Pers.:Fr.) Link, have
also been commonly found associated with date palm
rotted inflorescences (Brown and Butler, 1938;
El-Behadili et al., 1977; Rattan and Al-Dboon, 1980).
Inflorescence rot of palms is considered to be of major
economic importance in Iraq and Saudi Arabia. Severe
outbreaks occurred in Basrah, Iraq, in 1948–1949 and
1977–1987, causing 80% loss of the annual harvest (AlHassan and Waleed, 1977). Losses of up to 70% of the
This study was conducted during May–June 2004 in
Elx, province of Alicante, SE Spain, which is very
close to the Mediterranean coast (047¢30¢¢W, 389¢–
382¢N). The mean number of rainy days during the
period January–March is 4 days/month with mean
annual rainfalls ranging between 14 and 25 mm, and
for the months April–May, the mean number of rainy
days is 5 and 4 days, respectively, and the mean
annual rainfall during the same period ranges between
30 and 24 mm, respectively (Elx Meteorological
Station, Alicante, Spain). Date palm (Phoenix dactylifera L.), a native of North Africa, was introduced into
southern Spain and is now widely distributed throughout the country in extensive palm groves. The only
two palm groves with commercial value in Europe are
located in Elx and Orihuela, SE Spain (Galiana and
Agulló, 1983). This plant provides dates for food and
leaves, especially white (etiolated) young leaves, for
religious purposes (Masalles et al., 1988). The ÔpalmeralÕ (palm forest or palm grove) at Elx, known as the
ÔPalmeral of EuropeÕ, was declared as a World Heritage Site by UNESCO in 2000. It consists of several
scattered date palm plantations with genetically different trees. According to the last survey (1998), the total
number of adult palms in this palmeral was estimated
to be approximately 200 000 (Orts and López-Jiménez,
2003).
www.blackwell-synergy.com
Abdullah et al.
418
Table 1
Incidence of inflorescence rot in date palms at four sites in Elx (SE Spain)
Male inflorescences
Site
Female inflorescences
Number with Number without Percentage with Number with
Number
Percentage
Total inflorescences
symptoms
symptoms
symptoms
symptoms
without symptoms with symptoms (% with symptoms)
Algorós
Camino
Universidad
Conservatorio
61
39
35
71
40
9
46
10
60.4
81.3
43.2
87.7
Survey for the incidence of inflorescence rot
A survey on the incidence of the disease was conducted in four date palm plantation sites at Elx (Table 1).
Four sectors of land (50 · 50 m) each containing 100
date palms were selected at each site. Date palms with
or without inflorescence rot symptoms were scored in
each sector and the percentage of inflorescences of
male and female palms showing inflorescence rot
symptoms, as well as the total percentage of disease
symptoms, was determined.
Isolation of the pathogen
Twenty small pieces (5 cm long) from each infected
inflorescence examined were surface-sterilized with 5%
sodium hypochlorite for 10 min and then washed three
times with sterile distilled water. The surface-sterilized
pieces were blotted dry on sterilized filter paper, and
10 pieces were placed in plates containing 1.3% malt
extract agar (Scharlau, Barcelona, Spain) supplemented with chloramphenicol (50 lg/ml), and 10 pieces
were placed on top of moist sterilized filter paper in
Petri dishes. Both sets were incubated at 25C in the
dark.
Pathogenicity test
Four monosporic cultures from arthroconidia of individual mass cultures described above were obtained
and grown on potato carrot agar (PCA; 20 g potato,
20 g carrot, 20 g agar, 1 l distilled water) and one of
these was used for pathogenicity tests. Conidial suspensions were prepared in sterile water, and conidial
concentrations were quantified microscopically using a
Neubauer chamber and adjusted to a concentration of
106 conidia/ml with sterile distilled water containing
0.02% Tween 20. Three detached and opened healthy
spathes were surface-sterilized with 95% ethanol, and
the upper external surface of the spathe was removed
so that the flowers and strands were exposed to the
atmosphere. The detached spathes were inoculated by
spraying the inoculation sites (flowers and strands)
with either 20 ml of the conidial suspensions or water
(controls) using an atomizer. Inoculated spathes were
kept in plastic boxes and incubated at 25C in the
dark. Spathes were examined for lesions after 4 days
of incubation.
Re-isolation of the pathogen
To satisfy Koch’s postulates, pieces of inoculated flowers and strands were removed from the sites showing
16
20
22
22
68
65
134
62
19.0
23.5
14.1
26.2
41.6
44.4
24.1
56.4
disease symptoms, surface-sterilized for 10 min with
5% sodium hypochlorite, washed three times with sterile distilled water and plated on PCA. Plates were
incubated at 25C and the subsequent growth of the
pathogen was recorded.
Molecular biology study
Isolates Four monosporic isolates obtained as axenic
cultures from infected inflorescences of date palms were
used for genomic DNA analysis. Isolates 1, 2 and 4 were
obtained from infected male inflorescences, whereas isolate 3 was obtained from an infected female inflorescence.
All strains were obtained from the Algorós site in Elx.
Genomic DNA extraction The fungal isolates were
inoculated with three plugs (5 · 5 mm) taken from the
edge of a growing colony and grown in 250 ml flasks
containing 100 ml medium consisting of 0.3% yeast
extract, 0.3% malt extract, 0.5% peptone, 2% dextrose
and incubated for 3–4 days at 25C on a rotary shaker
(119 rpm). The mycelium was collected by filtration
(Millipore, Madrid, Spain; 0.22 lm), frozen in liquid
nitrogen in Eppendorf tubes and lyophilized. DNA
was extracted following the protocol of Zolan and
Pukkila (1986), and was stored at )20C until used.
Polymerase chain reaction The ITS1-F (5¢-CTTGGTCATTTAGAGGAAGTAA-3¢) and ITS4 (5¢-TCCTCCGCTTATTGATATGC-3¢) primers (Invitrogen,
Barcelona, Spain) were used for amplification of the
ITS region (White et al., 1990; Gardes and Bruns,
1993). Amplification was carried out in a volume of
40 ll, containing 4 ll of 10X Termo buffer, 4 ll dNTP
(0.2 mm final concentration), 3.2 ll MgCl2 (2 mm),
0.1 ll of Taq DNA polymerase (0.5 U/ml) and 2 ll of
each of the primers (0.5 lm) in sterile double-distilled
water to 20 ll, and 20 ll DNA template to each tube.
The polymerase chain reaction (PCR) was performed in
a GenAmp 9700 thermal cycler (Perkin-Elmer, MJ
Research, Waltham, NJ, USA) using the following conditions: initial denaturation for 8 min at 95C, 35 cycles
of 30 s of denaturation at 95C, 20 s of annealing at
53C and 60 s of extension at 72C; this was followed
by final extension for 5 min at 72C. The PCR products
were separated on a 1.2% agarose gel, stained with ethidium bromide and viewed with ultraviolet light. PCRamplified fragments were purified using QIAquickPCR
(Qiagen, Amersham, Piscataway, NJ, USA) columns
following the manufacturer’s instructions.
Inflorescence Rot Disease of Date Palms
DNA sequencing PCR purified products were quantified
using Hoechst fluorochrome (Sigma, Madrid, Spain)
and calf thymus DNA according to Ausubel et al.
(2002) and then amplified using Big Dye Terminator
mix (Applied Biosystems, Madrid, Spain) according to
the specifications of the manufacturer, and samples
were sequenced in an ABI-PRISM 3100 sequencer
(Applied Biosystems). After alignment of the ITS
sequences, these were submitted to the NCBI GenBank
database (http://www.ncbi.nlm.nih.gov/blast) and compared with existing ITS sequences.
Results
Field symptoms
Date palm spathes first showed inflorescence rot symptoms when they began to emerge in early spring. These
symptoms were observed on the external surface of
unopened spathes as brownish or rusty-coloured
lesions (Fig. 1). The internal side of the spathe facing
Figs. 1–4 Symptoms of inflorescence rot of date palm, Phoenix
dactylifera. (1) External surface of
an unopened spathe showing
brownish lesions. (2) Split spathe
with disease symptoms at the top.
(3) Split spathe at a later stage of
infection showing complete
destruction of flowers and strands.
(4) Severely infected spathes which
remain unopened and become
dry. Scale bars ¼ 10 cm
419
the infected flowers and strands showed similar but
milder symptoms. When the infected spathe split,
symptoms appeared mostly near the top of the spathe
(Fig. 2), and thereafter a complete destruction of the
flowers and strands occurred (Fig. 3). Severely affected
spathes at an early stage remained unopened and
became dry (Fig. 4). The incidence of the disease is
presented in Table 1. Infection in male palms in the
four sites surveyed ranged between 43 and 87%,
whereas in female palms it ranged between 14 and
26%. In general, male inflorescences were affected
more than female inflorescences. The incidence of
infection differed at the four sites, the highest incidence
was at the Conservatorio site (56%) and the lowest at
Universidad (24%).
The pathogen
Attempts were made to isolated the causal agent from
20 spathes (14 males and six females) showing inflores-
Abdullah et al.
420
cence rot, of which 16 yielded pure cultures of
M. scaettae. The four remaining spathes displayed a
mixed infection with M. scaettae and other fungi, such
as F. oxysporum, F. solani, T. roseum and Botrytis spp.
Mauginiella scaettae Colonies white, up to 4 cm (diameter) on PCA after 10 days of growth at 25C. Mycelium immersed and superficially composed of branched
hyaline septate hyphae. Reverse, at first creamy to pale
brown becoming black in old cultures. Sporulation
abundant, homogeneous, with powdery appearance.
Immersed hyphae 1.5–2.5 lm wide, aerial hyphae
3–4 lm wide. Arthroconidia produced by segmentation
of the aerial hyphae, unicellular or multicellular,
hyaline glistening white in mass, non-septate conidia
6–8 · 2.5–4 lm; 1, septate conidia 6–14 · 3–4 lm; 2,
septate conidia 16–22 · 3.5–4 lm; 3, septate conidia
12–26 · 3.5–5 lm; 4, septate conidia 24–26 ·
3.5–4 lm; 5, septate conidia 24–28 · 3.5–4 lm and 6,
septate conidia up to 35 lm long (Cavara, 1925a;
Figs 5 and 6).
Pathogenicity test
Detached inflorescences that were inoculated with
spore suspensions of M. scaettae developed clear
Fig. 7 Date palm with healthy spathe 4 days after inoculation with
Mauginiella scaettae (arrows) showing inflorescence rot symptoms.
H2O arrow shows mock inoculated controls. Scale bar ¼ 5 cm
inflorescence rot symptoms (Fig. 7) after 4 days. Pure
cultures with abundant arthroconidia, a characteristic
feature of M. scaettae, were re-isolated from all artificially inoculated spathes. Mock inoculated controls
displayed neither symptoms nor arthroconidia.
ITS region sequences
The ITS sequences, varying from 564 to 606 bp, of the
four monosporic isolates of M. scaettae tested, showed
93–94% homology with those of several Phaeosphaeria
spp. The highest similarities were found for P. triglochinicola, P. juncina and P. nodorum, followed by other
species of Phaeosphaeria for all isolates sequenced. Of
the 559 common base pairs of the four M. scaettae isolates sequenced, there was a discrepancy in only one
base pair. Only the sequence of a M. scaettae isolate
201 (CBS 117551) from a diseased female inflorescence
was published (GenBank accession number AY965895).
Discussion
Figs. 5–6 Mauginiella scaettae isolated from symptomatic date palm
inflorescence. (5) Growth on potato carrot agar (PCA), potato dextrose agar (PDA) and malt extract agar (MEA). (6) Hyphae and
conidia of the fungus. Scale bars ¼ 25 lm
Our results showed that the inflorescence rot disease
was present in date palms of the four sites studied.
The survey also revealed that male inflorescences, in
general, were more affected than female inflorescences,
Inflorescence Rot Disease of Date Palms
which agrees with previous reports (Al-Ani et al.,
1971; Al-Roubaie et al., 1987). The high incidence of
inflorescence rot disease among male palms may be
attributed to the fact that male palms receive little
attention and because the old infected inflorescences
are not removed regularly. Al-Ani et al. (1971) demonstrated that the disease spread easily to adjacent palms
when the conditions for development of the disease
were favourable. These authors also demonstrated that
the pathogen is mainly preserved as mycelium in infected inflorescences remaining on palms from the previous season or remain within infected leaf bases.
Al-Roubaie et al. (1987) suggested that the primary
infection by M. scaettae probably occurred during the
early stage of floral bud formation and prior to the
envelope development of the spathes and to their hardening. It is worth mentioning that date palms planted
for gardening purposes in Elx and Alicante are usually
free from symptoms of the disease. The reason for this
may be attributed to good management strategies (i.e.
leaf pruning and removal of old inflorescences immediately after harvest).
At Elx palm groves, the availability of rain prior to
the stage of flower bud formation (January–March)
and during the early stage of bud formation (March–
April) is probably responsible for creating favourable
conditions for fungal growth, when hyphae between
the leaf bases can grow and infect newly developed
inflorescences. Several outbreaks of the disease have
been recorded in areas subjected to prolonged periods
of rain and cold weather, 2–3 months before the emergence of spathes (Al-Ani et al., 1971; Al-Hassan and
Waleed, 1977; Djerbi, 1983; Zaid et al., 2002).
In the course of this study we observed that four
of 20 inflorescences infected by M. scaettae were
colonized by another fungus identified as either F. oxysporum, F. solani, T. roseum or Botrytis sp. However,
none of the above species was isolated alone from
infected inflorescences. Therefore, further studies are
needed to investigate their possible role in inflorescence
rot disease of date palms. Several fungi other than
M. scaettae, such as Fusarium spp., Ceratocystis paradoxa and T. roseum, have been reported in several
instances of inflorescence rot disease of date palm and
considered of minor importance (Brown and Butler,
1938; El-Behadili et al., 1977; Rattan and Al-Dboon,
1980; Al-Roubaie et al., 1987).
The ultrastructure of the cell wall and the hyphal
septa, together with the diazonium blue B test have
shown that M. scaettae represents an anamorph of an
unknown ascomycete (van der Walt and Hopsu-Havu,
1976; von Arx et al., 1982). The results of analysis of
the ITS sequencing data indicate that M. scaettae is
closely related to species of Phaeosphaeria I. Miyake
clade B, and in particular to P. triglochinicola, which
belongs to subclade B4 according to Camara et al.
(2002). The majority of species of Phaeosphaeria form
pseudoparenchymatous ascomata with bitunicate asci,
which mainly occur on monocotyledonous plants
(Holm, 1957; Barr, 1987; Shoemaker and Babcock,
421
1989). Molecular studies on the phylogeny of Leptosphaeria sensu stricto and Phaeosphaeria by Camara
et al. (2002) indicated that the structure of the peridial
wall, the anamorph and, to a lesser extent, their hosts
are taxonomically predictive at the genus level. Anamorphs of Phaeosphaeria are distributed in different
form-genera including Hendersonia Berk., Phaeoseptoria Speg., Septoria Sacc., Septoriella Oudem. and
Stagonospora (Sacc.) Sacc. (Leuchtmann, 1984). Moreover, several Phaeosphaeria, including members of
subclade B4, from diverse, non-grass monocotyledonous families are characterized by unknown anamorphs (Camara et al., 2002). Becasue M. scaettae is a
pathogen of date palm (P. dactylifera), which is a nongrass monocotyledonous plant, this mitosporic fungus
may represent an anamorph of a Phaeosphaeria species. The addition of a new anamorph and host to
Phaeosphaeria would support the suggestion by
Camara et al. (2002) that a convergent evolution has
taken place in the evolutionary history of this group of
fungi.
Acknowledgement
This work was supported by a grant (AGL 2000-0342-P4-02) from
the Spanish Ministry of Science and Technology.
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