Morphological and Molecular Characterization of Alternaria sect. Ulocladioides/Ulocladium Isolated from Citrus Fruits in Upper Egypt

Abstract

Citrus is the most important crop in Upper Egypt. 150 infected samples were collected from citrus samples (Navel orange, Tangerine and Lemon) in Upper Egypt, 50 samples from each fruit. Twenty-two isolates representing three species of Alternaria belong to A. sect. Ulocladioides and A. sect. Ulocladium were isolated on dichloran chloramphenicol- peptone agar (DCPA) medium at 27°C. Tangerine samples were more contaminated with Alternaria followed by Navel orange and no Alternaria species appeared from Lemon samples. Based on the Alt a1 the phylogenetic analysis identified the isolates as Alternaria atra, Alternaria botrytis and Alternaria oudemansii. The pathogenicity of the isolates was tested by inoculation of healthy navel orange, the resulted data showed that all tested isolates were pathogenic to healthy navel orange with different degrees ranged from 31.5±1 - 20±1 mm and A. oudemansii had a low virulent effect. The mycotoxins ability of tested isolates indicated that about 83% of the isolates were TeA toxin producers with amount ranged from 1.54 - 18.47 ug/ml.

Full text

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*Corresponding author: E-mail: m.hussein@sci.svu.edu.eg;
Asian Journal of Biochemistry, Genetics and Molecular
Biology
5(4): 30-41, 2020; Article no.AJBGMB.60766
ISSN: 2582-3698
Morphological and Molecular Characterization of
Alternaria sect. Ulocladioides/Ulocladium Isolated
from Citrus Fruits in Upper Egypt
Youssuf A. Gherbawy
1
, Thanaa A. Maghraby
1
, Karima E. Abdel Fattah
1
and Mohamed A. Hussein
1*
1
Botany and Microbiology Department, Faculty of Science, South Valley University, Qena 83523,
Egypt.
Authors’ contributions
This work was carried out in collaboration among all authors. Author YAG designed the study,
performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript.
Authors TAM and KEA managed the analyses of the study. Author MAH managed the literature
searches. All authors read and approved the final manuscript.
Article Information
DOI: 10.9734/AJBGMB/2020/v5i430138
Editor(s):
(1)
Dr. Arulselvan Palanisamy, Muthayammal College of Arts and Science, India.
Reviewers:
(1) S. Kannadhasan, Cheran College of Engineering, India.
(2)
J. Judith Vijaya, Loyola College, India.
Complete Peer review History:
http://www.sdiarticle4.com/review-history/60766
Received 20 July 2020
Accepted 26 September 2020
Published 20 October 2020
ABSTRACT
Citrus is the most important crop in Upper Egypt. 150 infected samples were collected from citrus
samples (Navel orange, Tangerine and Lemon) in Upper Egypt, 50 samples from each fruit. Twenty-
two isolates representing three species of Alternaria belong to A. sect. Ulocladioides and A. sect.
Ulocladium were isolated on dichloran chloramphenicol- peptone agar (DCPA) medium at 27°C.
Tangerine samples were more contaminated with Alternaria followed by Navel orange and no
Alternaria species appeared from Lemon samples. Based on the Alt a1 the phylogenetic analysis
identified the isolates as Alternaria atra, Alternaria botrytis and Alternaria oudemansii. The
pathogenicity of the isolates was tested by inoculation of healthy navel orange, the resulted data
showed that all tested isolates were pathogenic to healthy navel orange with different degrees
ranged from 31.5±1 - 20±1 mm and A. oudemansii had a low virulent effect. The mycotoxins ability
of tested isolates indicated that about 83% of the isolates were TeA toxin producers with amount
ranged from 1.54 - 18.47 ug/ml.
Original Research Article

Gherbawy et al.; AJBGMB, 5(4): 30-41, 2020; Article no.AJBGMB.60766
31
Keywords: Citrus; Alternaria; pathogenicity; TeA toxin.
1. INTRODUCTION
Citrus fruits widely used as edible fruits
belonging to citrus and related genera of the
family Rutaceae (orange family) [1]. It serves as
the main source of vitamins, minerals elements
and sugar; hence, it controls the building process
of human bodies [2]. The citrus fruit is attacked
by a number of pathogens from bloom to
harvesting stage and subsequently by post-
harvest pathogens that affect fruit yield and
considerably deteriorate the fruit quality [3]. The
common postharvest fungi of fruits include
Alternaria sp., Aspergillus sp., Fusarium sp., and
Penicillium sp. [4].
Alternaria species cause four different diseases
of citrus: Alternaria brown spot of tangerines
(ABS), Alternaria leaf spot of rough lemon,
Alternaria black rot of many citrus fruits, and
Mancha foliar on Mexican lime. Alternaria brown
spot affects many tangerines and their hybrids
and produces lesions on and abscission of
immature fruit and leaves, and corky lesions on
mature fruit [5]. Some species of Ulocladium
(synonymy: Alternaria) were plant pathogens,
Leaf blight caused by U. atrum is an important
disease on potato, causing considerable damage
worldwide [6,7,8]. In addition, conidia of U. atrum
were able to survive on the surface of grapevine
bark, leaves, inflorescences and berries [9].
Coviello et al. [10] reported that the major fruit
disease affecting fig and smut in dried fruits
caused by Alternaria rot [A. alternata, A. atra
(synonymy: Ulocladium atrum) and other
Alternaria spp.
Ulocladium species closely resemble some of the
saprotrophic Alternaria species based on
extensive morphological and phylogenetic
studies [11]. Phylogenetic studies place
Ulocladium convincingly within Alternaria,
suggesting that the latter is the correct
classification for these species [11]. Nineteen
former Ulocladium species have been distributed
among three main sections within the enlarged
genus Alternaria based on phylogenetic data: A.
sect. Pseudoulocladium, A. sect. Ulocladioides,
and A. sect. Ulocladium [12]. For Alternaria
species, the Alt a 1 sequence supported the
separation of groups of Alternaria spp. and
related taxa into several species-groups [13].
Although of Alt a1 in fungal biology remains
unclear, some evidence has suggested that the
role of Alt a 1 can be related to virulence and
fungal infection pathogenicity [14]. Lately,
sequencing of alternative regions has been
explored, such as a segment of an
endopolygalacturonase (endoPG) gene the
Alternaria major allergen 1 (Alt a1) gene and two
anonymous noncoding regions, OPA10-2 and
OPA1-3 [15,16].
Several Alternaria species were exhibited
virulence activities against different crops [17,18].
Abass [19] showed that Ulocladium (synonyomy
Alternaria) succeeded to invade wounded and
unwounded date palm fruits. Species of
Alternaria are well known for the production of a
variety of about 70 toxic secondary metabolites
[20], some of them recognized as mycotoxins,
such as alternariol (AOH), alternariol
monomethyl ether (AME), tenuazonic acid (TeA),
altenuene (ALT), and altertoxins I, II, III (ATX-I, -
II, -III) [21,22]. Alternaria mycotoxins have been
frequently isolated and reported in fruits and
vegetables, such as tomatoes, citrus fruits,
Japanese pears, carrots, barley, oats, olives,
mandarins, melons, peppers, apples,
raspberries, cranberries, grape, wheat, and
other grains [23]. Tenuazonic acid (TA) is one of
the major mycotoxins produced by some
Alternaria species [24]. TeA has been reported to
be acutely toxic for several animals such as
mice, chickens, and dogs, and it has been
associated with human haematological
disorders like Onyalai and in various
Alternaria-contaminated crops, fruits, and
vegetables [25].
This work focused on characterization of
Alternaria species from infected citrus samples
with attention to pathogenicity and Tenuazonic
acid activities.
2. MATERIALS AND METHODS
2.1 Collection of Samples
A total of one hundred and fifty samples of
infected citrus (Navel orange and tangerine)
were collected from five Governorates in Upper
Egypt (Aswan, Luxor, Qena, Sohag and Assuit),
Thirty samples from each. Each sample was put
in a sterile polyethylene bag sealed and
transferred to the mycological laboratory.
Samples were kept in a cool place during storage
5°C till fungal analysis.

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32
2.2 Isolation and Morphological
Identification of Alternaria spp.
The dilution plate method was used for isolation
the fungal strains from navel orange, tangerine
and lemon as described by Christensen [26]. A
defined weight of each infected citrus samples
placed in a sterile conical flask containing 100 ml
of sterile distilled water. Flasks were shaken by
hand in a rotating motion for 10 minutes. One m1
of desired dilution will be transferred aseptically
into each of sterile Petri-dishes and followed by
addition of about 15 to 20 m1 of liquefied
Dichloran chloramphenicol- peptone agar
(DCPA) medium. The dishes were rotated by
hand in abroad swirling motion to ensure uniform
distribution of homogenates. Three replicates
were prepared for each sample and cultures
were incubated at 27°C for 5 - 7 days.
Morphological identification based on length of
primary, secondary conidiophores, shapes, sizes
and colours of conidia was done for each isolate
grown on Synthetic Nutrient Poor Agar plates at
22°C for 7 days according to Woudenberg et al.
[11]. Further criteria also including colony texture,
colour and diameter of fungal colony were
estimated on Potato Dextrose Agar (PDA) plates
at 25°C for 7 days.
2.3 Molecular Identification of Alternaria
Isolates
2.3.1 DNA extraction
For DNA extraction, fungal isolates were cultured
in 250 ml flasks containing 50 ml Potato
Dextrose Broth (PDB) for 2-3 days using a rotary
shaker for 25°C at 120-150 rpm. The mycelium
was harvested by filtration, frozen at -80°C.
Mycelium was ground in liquid nitrogen using
sterile mortar to obtain homogeneous fine
powder. Afterwards, DNA extracted from 50 mg
of mycelium powder using acetyl
trimethylammonium bromide (CTAB) according
to Mohammadi et al. [27]. The resulted DNA
pellet dissolved in 1 ml of TE buffer (10 mM Tris,
1 mM EDTA, pH 7.5) as described by Moeller et
al. [28]. The DNA quantity and quality checked
by electrophoresis on a 0.8% agarose gel
revealed with ethidium bromide and visualized by
UV trans-illumination.
2.3.2 PCR amplification and sequencing
Amplification of Alt a 1 gene was
conducted using primer pairs; Alt-for (5-
ATGCAGTTCACCACCATCGC-3) and Alt-rev (5
ATGCAGTTCACCACCATCGC-3 [13]. Each
PCR reaction was performed in a 25 μL mixture
that contained 5 μL of the master mix (buffer,
dNTP, Taq DNA polymerase, 2 mM MgCl2), 1μL
of the template DNA, 0.5μL of both forward and
reverse primers and the volume was completed
to 25 μL with PCR water. Amplification was
performed in a thermal cycler (Flexigene,
Techne, Cambridge, UK). PCR) cycles for Alt a1
gene were using the following cycling protocol:
initial denaturation at 94°C for 1 min, 35
amplification cycles of 94 °C for 30 s, 57°C for 30
s and 72°C for 1 min, and a final extension at
72°C for 10 min. PCR product was observed in a
1.4% agarose gel, stained with ethidium bromide
and visualized with UV transilluminator. Amplified
products were purified, quantified and
sequencing in Macrogen (South Korea).
2.3.3 Phylogenetic analysis
Sequencing data were edited using Chromas
Lite, aligned and clustered by Mega 6.0 [29]. The
Stemphyllium genus is closely related to
Alternaria. Therefore, phylogenetic tree was
rooted with Stempphyllium callistephi
(AY563276) as out groups. The phylogenetic
reconstruction was done using the neighbor
joining (NJ) algorithm, with bootstrap values
calculated from 1,000 replicate runs, using the
software routines included in the MEGA
software.
2.4 Pathogenicity Test of the Selected
Strains
Pathogenicity test was carried out as described
by Baiyewu et al. [30] and Chukwuka et al. [31].
The selected healthy fruits of mature
"Washington" Navel orange was surface
sterilized by 1% Sodium hypochlorite solution for
5 min, then washed by rinsing these fruits in
sterilized distilled water for three times and dried
with sterilized filter paper. Inoculation procedures
were performed under sterile conditions by
inserting 5 mm agar disc of the tested fungus into
holes (5mm diameter and 5mm depth) made in
surface of fruit using a sterilized cork borer. Agar
discs of each tested fungi (about 5 mm diameter)
were taken from the margin of 10 days old PDA-
cultures of the tested fungi and placed into the
wound. After inoculation, the holes were plugged
with the removed pieces of the peel. Three
replicates, each of three fruits, were used for
each tested fungi and control treatment was
carried out by inoculated a plug of sterile PDA.
The inoculated fruits were placed in black

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33
polyethylene bags, and then incubated in the
dark at 25°C for 3 weeks. Following incubation,
fruit were cut in half and the fungal growth was
determined by measuring the diameter of lesion
produced in mm.
2.5 Mycotoxin Production
The strains were cultured on autoclaved rice at
40% moisture. The rice was inoculated with a
5mm disk of 1 week old (PDA) and incubated at
25°C for 3 weeks in the dark [32]. The cultural
material was homogenized with 30 ml of
methanol and filtered through a Whatman filter
paper (no.1). The filtrate was clarified with 60 ml
of 20% ammonium sulphate. Twenty ml was
adjusted to pH 2 with 6 N HCl and extracted
twice for TA with 15 ml of chloroform. TA was
then partitioned into 10 ml of 5% sodium
bicarbonate, acidified to pH 2 again, and
extracted twice with 10 ml of chloroform. The
chloroform extracts were combined, washed with
7.5 ml of water, and evaporated to dryness. The
residue was made up to 1 ml with methanol and
analyzed for TeA by HPLC combined with UV-
detection at 280 nm [32].
2.6 Statistical Analysis
Results data were subjected to analysis of
variance (ANOVA) using the Statistical Analysis
System. Means were separated by Duncan’s
multiple range test at P < 0.05 level.
3. RESULTS
3.1 Morphological and Molecular
Characteristics of Alternaria spp.
Twenty-two isolates representing three Alternaria
spp. belong to A. sect. Ulocladioides and A. sect.
Ulocladium were isolated from 150 samples of
citrus fruits on plates of DCPA media at 27
o
C.
Alternaria botrytis recovered from 10 and 8% of
the samples comprising 75 and 28.57% of
Alternaria from Navel orange and Tangerine
samples, respectively. A. oudemansii was
isolated from Tangerine samples only
constituting 64.24% of total Alternaria. In the last
place come A. atra which isolated from 2% of
Navel orange and Tangerine samples comprising
25 and 7.14% of Alternaria, respectively (Table 1
and Fig. 1).
Table 1. Average total counts (ATC), number of cases of isolation (NCI, out of 50 samples) and
occurrence remarks (OR) of Alternaria species recovered from 150 samples of navel orange
and tangerine and citrus fruits on Dichloran chloramphenicol- Peptone Agar (DCPA) medium
on at 27°C
Genera and species
Navel orange
Tangerine
Lemon
ATC
%C
%F
NCI
OR
ATC
%C
%F
NCI
OR
-
Alternaria atra
300 25 2 1 R 150 7.14 2 1 R -
Alternaria botrytis
900 75 10 5 L 600 28.57 8 4 L -
Alternaria oudemansii - - - - - 1350 64.28 12 6 L
-
Average total count 1200 100% 2100 100%
-
Occurrence remark: or (out of 50 samples): H = high occurrence from 25–50 cases, M = moderate occurrence
from 12–24 cases, L = low occurrence from 3–6 cases and R = rare occurrence from 1–2 cases
Fig. 1. Conidial shapes of different Alternaria species, A. atra (A), A. botrytis (B) and A.
oudemansii (C)

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34
3.2 Molecular Characterization of
Alternaria Species
Alt a1 gene was successfully amplified from
Alternaria strain recovered from citrus fruits
samples. All the sequences for Alt a1 gene were
deposited in the GenBank and their accession
numbers were indicated in Table 2. The highly
resolution Alt a1 gene dataset separated
Alternaria species into three groups. First group
contain A. botrytis (4 strain), second group
contain A. oudemansii (4 strain) and third group
contain A. atra (2 strain) (Fig. 2).
Phylogenetic analysis has been performed on
the present 10 Alternaria (synonymy Ulocladium
spp.) nucleotide sequences with the other
Ulocladium spp. published in the GenBank and
the results of this analysis are show in Fig (2).
Phylogenetic tree was generated from 27
sequences including 5 U. botrytis, 5 U.
oudemansii, 3 U. atrum and 13 Ulocladium spp.
sequence in GenBank in addition to the out-
group sequence Stemphyllium vesicarium.
First group comprised 4 strain of A. botrytis
(SVUAbo160, SVUAbo161, SVUAob162 and
SVUAbo163) clustered together clustered
with the U. botrytis AY563317 (synonymy:
A. botrytis) obtained from GenBank with
bootstrap (86%). Second group comprised 2
strain of A. atra (SVUAat158 and
SVUAat159) clustered together clustered with
the Ulocladium atrum AY563318 (synonymy: A.
atra) obtained from GenBank with a
bootstrap support (41%). Third group comprised
4 isolates (SVUAou164, SVUAou165,
SVUAou166 and SVUAou167) of A. oudemansii
clustered together clustered with U. oudemansii
FJ266514 (synonymy: A. oudemansii) obtained
from GenBank. The tree showed a well-
supported relationship (76% bootstrap) between
U. oudemansii (FJ266514) from GenBank and
four isolates (SVUAou164, SVUAou165,
SVUAou166 and SVUAou167) that based on
morphological features were identified as A.
oudemansii.
Fig. 2. Phylogenetic tree of Alternaria strains isolated from citrus fruits based on Alt a1 gene
sequence data. The numbers above branches indicate bootstrap values

3.3
Pathogenicity of the
Species
Ten of Alternaria
isolates were evaluated for their
pathogenicity on healthy Navel orange citrus
fruits. The obtained results of the pathogenicity
test revealed that all isolates caused black rot
symptoms of the oranges with average lesion
size ranged from 20 to 31.5 mm. A
tested isolates, the highest average lesion size
was 31.5 mm which achieved by
(SVUAob161).
The other isolates including
atra
(SVUAat158 and SVUAat159),
(SVUAbo160, SVUAbo162 and SVUAbo163)
exhibited virulent capacity wi
th lesion size ranged
30 to 31 mm. Whereas the lowest lesion size
was produced by
A. oudemansii
(SVUAou164, SVUAou165, SVUAou166 and
SVUAou167) with lesion size ranged from 20
25.5 mm (Table 2 and Fig. 3).
3.4 Mycotoxin Production
Six strains we
re chosen for tenuazonic acid toxin
production. The HPLC analysis of the standard
metabolites was done for characterization and
Fig. 3. Dendrogram
showing relationships among 10 isolates of
Gherbawy et al.; AJBGMB, 5(4): 30-41, 2020
; Article no.
35
Pathogenicity of the
Alternaria
isolates were evaluated for their
pathogenicity on healthy Navel orange citrus
fruits. The obtained results of the pathogenicity
test revealed that all isolates caused black rot
symptoms of the oranges with average lesion
size ranged from 20 to 31.5 mm. A
mong the
tested isolates, the highest average lesion size
was 31.5 mm which achieved by
A. botrytis
The other isolates including
A.
(SVUAat158 and SVUAat159),
A. botrytis
(SVUAbo160, SVUAbo162 and SVUAbo163)
th lesion size ranged
30 to 31 mm. Whereas the lowest lesion size
A. oudemansii
isolates
(SVUAou164, SVUAou165, SVUAou166 and
SVUAou167) with lesion size ranged from 20
-
re chosen for tenuazonic acid toxin
production. The HPLC analysis of the standard
metabolites was done for characterization and
quantitative determination of mycotoxins
recovered from culture filtrates of different
strains. About 83% of the isolates showed
ability to produce TeA toxin and only one strains
of Alternaria oudemansii
(SVUAou164) failed to
give any detectable amount
of toxin. The tested
isolates exhibited significant TeA activities and
the detected amounts of TeA
between 1.45 to 18 ug/ml. The maximum
concentration of TA toxin (18 ug/ml) was
obtained from A. botrytis
(SVUAbo161) followed
by A. botrytis
(SVUAbo160) with amount 15.01
ug/ml. The detected TeA amount from
(SVUAat158) and (SVUAat159)
8.75 and 15.01 ug/ml, respectively. The minimum
TeA amount was showed by
A. oudemansii
(SVUAou165) isolate (1.45 ug/ml) (Table 3 and
Fig. 4).
4. DISCUSSION
This study is the comprehensive research for
identification an
d genetic diversity of
Ulocadioides and A. sect.
Ulocladium
affecting the citrus fruits in Upper Egypt. In this
study, three Alternaria
species identified as
atra, A. botrytis and A. oudemansii
showing relationships among 10 isolates of
Alternaria
spp. based on
pathogenicity test
; Article no.
AJBGMB.60766
quantitative determination of mycotoxins
recovered from culture filtrates of different
strains. About 83% of the isolates showed
the
ability to produce TeA toxin and only one strains
(SVUAou164) failed to
of toxin. The tested
isolates exhibited significant TeA activities and
toxin ranged
between 1.45 to 18 ug/ml. The maximum
concentration of TA toxin (18 ug/ml) was
(SVUAbo161) followed
(SVUAbo160) with amount 15.01
ug/ml. The detected TeA amount from
A. atra
isolates were
8.75 and 15.01 ug/ml, respectively. The minimum
A. oudemansii
(SVUAou165) isolate (1.45 ug/ml) (Table 3 and
This study is the comprehensive research for
d genetic diversity of
A. sect.
Ulocladium
species,
affecting the citrus fruits in Upper Egypt. In this
species identified as
A.
atra, A. botrytis and A. oudemansii
were
spp. based on

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36
Table 2. Origin of sample collection, Code, accession numbers, name of the pathogen identified and measurement of lesion expansion and rank of
the Navel oranges condition 21 days after incubation from each sample
No. of
isolate
Code of isolate
Molecular identification
Substrate
Origin
Measurement (mm)
Pathogenicity/
virulence
Accession
number
0 - Control Navel orange
Assuit 7.5 A -
1 SVUAat158
A. atra
Tangerine
Aswan 30±1
B MT711112
2 SVUAat159
A. atra Tangerine
Sohag 31±1
B MT711113
3 SVUAbo160
A botrytis Tangerine
Qena 31±1
B MT711114
4 SVUAbo161
A. botrytis Navel orange
Qena 31.5±1
B MT711115
5 SVUAbo162
A. botrytis
Navel orange
Assuit 30±1
B MT711116
6 SVUAbo163
A. botrytis
Tangerine
Aswan 30±1
B MT711117
7 SVUAou164
A. oudemansii Tangerine
Luxor 20±1
C MT711118
8 SVUAou165
A. oudemansii Tangerine
Aswan 25.5±1
C MT711119
9 SVUAou166
A. oudemansii
Tangerine
Aswan 21±1
C MT711120
10 SVUAou167
A. oudemansii
Navel orange
Assuit 20±1
C MT711121
Means with different letters are significantly different from control (P < 0.05)
A: healthy, no visible symptoms (nonvirulent), B (high virulent) and C (moderate virulent)

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Table 3. Teuanzonic acid toxin production by Alternaria strains
Tenuazonic acid concentration (ug/ml)
Fungal species
Code of isolate
8.75 Alternaria atra SVUAat158
10.87 Alternaria atra SVUAat159
15.01 Alternaria botrytis SVUAbo160
18.47 Alternaria botrytis SVUAbo161
0.00 Alternaria oudemansii SVUAou164
1.54 Alternaria oudemansii SVUAou165
Fig. 4. TeA level from A. botrytis
associated with citrus fruits collected from the
markets in Upper Egypt. Our results were
identical with several literatures [33,34].
Alternaria sp. causes black rot and massive
deterioration of citrus fruits [35,36]. Uzuegbu
and Emifoniye [37] in their work of postharvest
fungal spoilage of some Nigerian fruits and
vegetables isolated Alternaria in 40% of the total
samples. Zora [38], who was recorded four
species Ulocladium species (synonymy
Alternaria) including U. atrum, U. botrytis, U.
charatum and U. chlamydosporum) from soils
and palm fields in Iraq. Our results indicated that
Alternaria species belonging to A. sect.
Ulocadioides and A. sect. Ulocladium groups
was weakly appeared and this in agreement with
[13] who reported that Ulocladium atrum
(synonymy A. atra) and Ulocladium botrytis
(synonymy A. botrytis) were poorly supported.
A phylogenetic analysis of small-spored, citrus-
associated Alternaria isolates was recently
completed and included the 10 morphospecies
[39]. Using morphological characters in Alternaria
identification is not enough to discriminate
among common small-spored species [16,40].
Therefore, the DNA analysis is needed for
accurate identification and characterization of the
species targeting the Alt a 1 gene has been
developed for the rapid detection of DNA by PCR
amplification for phylogenetic analysis of
Alternaria and related genera [41,42]. Alt a 1 is
expressed by both Alternaria and other members
of the Pleosporaceae family, including the
allergenic species (Stemphylium, Ulocladium,
Nimbya and Embellisia) [13,43,44]. The
phylogenetic analysis of our isolates with other
strain from Genebank illustrated that our isolates
grouped in three species namely A. atra
(synonymy U. atrum), A. botrytis (U. botrytis) and
A. oudemansii (U. oudemansii). The obtained
result are in agreement with the past study by
Hong et al. [13] indicated that bootstrap support
for Ulocladium group was low <50% and
Ulocladium group was divided into two
monophyletic groups, one of which was
composed of A. cheiranthi, E. indefessa and U.
chartarum and the other was composed of U.
cucurbitae, U. botrytis, and U. atrum. The
analysis of 13 species of Ulocaldium (synonymy
Alternaria) using Alt a1 and Gpd sequences,
Wang et al. [45] showed that Ulocladium species
is divided into two distinct monophyletic clades
with high bootstrap values. Clade 1 included nine
Ulocladium species, while clade 2 includes four
species of Ulocladium. Also, Runa et al. [46]
described the phylogenetic relationship of 13
species of Ulocladium with other related species
of Alternaria, Embellisia, and Stemphylium based
on sequences of Alt a1 and Gpd and they
revealed that ten species of Ulocladium clustered
into a core Ulocladium group but Ulocladium
alternariae and U. oudemansii clustered together
in a second clade sister to and immediately basal
to the primary Ulocladium clade.
The obtained results of the pathogenicity test
showed that all of the Alternaria isolates caused

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38
significant black rot on the healthy Navel orange,
some isolates are highly pathogenic on Navel
orange, for example, A. botrytis (SVUAbo161)
had a mean lesion size of 31.5 mm. The current
results are in a good agreement with many other
published researches showed the virulence of
Alternaria species and their association with fruit
rot on different plant hosts such as date palm,
pistachio and citrus fruits [47,48,49]. Our results
are in agreement with Abass [19] indicated that
the results of pathogenicity test on date palm
fruits of Barheee cultivar revealed the as lesion
sizes of Ulocladium (synonymy Alternaria) were
1.97 and 1.75 mm in wounded and unwounded
treatments, respectively. Peever et al. [50]
reported that 92% of Alternaria isolates
recovered from citrus were pathogenic to
detached Minneola leaves. Our findings also
revealed high variability in virulence among the
Alternaria isolates. Several workers have already
reported pathogenic variability among isolates of
Alternaria spp. [51,52]. Bukar et al. [35] who
reported that different spoilage types were
observed when the healthy oranges were re-
inoculated with the pure isolates of the
pathogens. Mojerlou and Safaie [53] claimed that
all Alternaria isolates caused black rot to Navel
and Valencia oranges cultivars and significant
differences were observed between cultivars and
among isolates. Nemsa et al. [54] studies the
pathogenicity of nine isolates of Alternaria
against Fortune mandarin. He demonstrated that
6 isolates were considered as pathogens on
unwounded fruits with different degrees of
aggressiveness as low (33.3% of isolates),
medium (33.3%) and high (33.3%).
In this study 83% of tested Alternaria species
were tenuazonic acid producers with different
concentrations. The genus Alternaria is well
known for its ability to secrete a wide range of
toxins, which can be either specific for the host or
common for several hosts. TeA is considered to
be having highest toxicity amongst the
mycotoxins produced by Alternaria [55,56].
Several studies demonstrated that various
isolates of Alternaria species isolated from
different sources were TeA producers [25,57,58,
59,60]. Wei et al. [61] found TeA as the most
recurrent toxin in all dried fruits with
concentration in the range of 6.9–5665.3 μg/kg.
Davis et al. [62] and Kinoshita et al. [63]
screened 185 strains of Alternaria species and
found the wide-spread occurrence of tenuazonic
acid (TA). Sixty-five percent of feed samples
were contaminated with TeA up to 1983 µg/kg
[64,65,66]. Previous studies suggest that strains
may differ in their mycotoxin secretion [67,68,
69].
5. CONCLUSION
Alternaria atra, A. botrytis and A. oudemansii
were recovered from citrus samples in Upper
Egypt and Tangerine samples were more
contaminated with Alternaria spp. All tested
isolates were pathogenic to healthy navel orange
and A. oudemansii was less virulent, about 83%
of Alternaria isolates were TeA toxin producers.
COMPETING INTERESTS
Authors have declared that no competing
interests exist.
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