1. health and fitness
2. disease

European Journal of Academic Essays 1(9): 20-23, 2014
ISSN (online): 2183-1904
ISSN (print): 2183-3818
www.euroessays.org
Alternaria alternata Isolated From Lemons (Citrus
lemon) in Libya
El-Gali, Zahra Ibrahim
Department of Plant Protection, Faculty of Agriculture, Omer Al-Mukhtar University, El-Beida Libya.
[email protected]
Abstract: Lemons (Citrus lemon Burmann) is one of the citrus fruits of Libya. Blight symptoms was observed on plants were
grown in homes gardens. I isolated and examined a fungus which causes leaf lesions and blight and fruits drop on lemons by
using morphological method. Baseded on classification keys, the pathogen was compared on basis of morphological and
characteristics, the recovered isolate from the plant tissues was identified as Alternaria alternata (Fr.) Keissler, and identified
was confirmed by pathogenicity test.
Keywords: Lemons, Blight, Alternaria alternata, Morphology, Pathogenicity, Libya.
_______________________________________________________________________________________________________________
1. Introduction
Citrus occupies a prominent position in fruit industry of the
world. Libya is among the leading citrus growing countries of
the world and earns substantial amount of foreign exchange
annually. In Libya, citrus is the largest group of fruits
produced over an area of 10 000 hectares with a total
production of 84 Mt. annually, which is very low as
compared to other citrus growing countries. Diseases are one
of the major factors which impede the fruit yield and quality.
Among diseases, Alternaria leaf spot caused by Alternaria
spp. one of the most devastating and occurs throughout citrus
growing countries of the world [1,2] including Libya.
Alternaria is a ubiquitous fungal genus associated with a
wide variety of substrates including seeds, plants, agricultural
products, animals, soil, and the atmosphere. Most species are
well-known and significant plant pathogens causing a range
of diseases and post-harvest rots of numerous agronomic and
ornamental plants [3]. Some species are commonly correlated
with deterioration of painted wall surfaces [4], found as
saprophytes in soil, and decaying plant tissues, or as
endophytes in various plants [5,6]. In addition, several taxa
have emerged as animal or human pathogens [7]. Alternaria
species have been generally identified and classified based on
cultural and conidial morphology [8.9.10]. Alternaria spp.
cause several diseases of citrus, including Alternaria brown
spot of tangerine, leaf and fruit spot of rough lemon and
Rangpur lime, affecting Mexican lime, and black rot of fruit
of several Citrus spp. [11,12]. Species of Alternaria also
commonly occur as saprophytes on all aboveground tissues
of citrus trees and also may colonize citrus leaves as
endophytes (T. L. Peever and L. W. Timmer, unpublished
data). In addition to the diseases mycotoxins produced by
Alternaria and specifically by Alternaria alternata are
numerous. Among them the most widely studied are:
tenuazonic acid (TA), alternariol (AOH), alternariol
monomethyl ether (AME), altenuene (ALT), alter toxin I and
ten toxin [13,14].
Lemons was the most planting in homes gardens in El-Beida,
Libya and the blight symptoms was observed on leaves and
fruits. Therefore the objective of this study included
isolation, purification and identification of pathogenic fungus
causing leaf and fruit blight disease of lemons.
2. Materials and Methods
2.1. Field observations and symptoms.
Lemons plants showing infection on leaves and fruits were
collected from the experimental area of the homes gardens,
in El-Beida city at 25 km from the coast, with an elevation
ranging from 612 ± 8.4 meters above sea level between lat
32º 45ʼ and 30°16’ N, and long 21°42’ and 21°37.9’ E.
Maximum and minimum mean temperatures are 25°C and
10°C, respectively. Annual rainfall averages 650 mm and is
most abundant during December to February. Soil of the site
is a typical clay soil, composed of 11.8% sand, 35.2% silt
and 50.1% clay. Digital photographs of leaf and fruit
symptoms were recorded using a Sony DSC-S700 model
digital camera.
2.2. Collection and isolation of the fungus.
Naturally infected fruit and leaves were collected and
processed for isolation of causal disease in the laboratory of
Department of Plant Pathology, Faculty of Agriculture. A
special medium known as Potato Dextrose Agar (PDA) at
24°C was used for isolation of pathogen and observed
periodically for fungal growth.. The isolated fungal was
Corresponding Author: El-Gali, Zahra Ibrahim, Dept. of Plant Protection, Fac. of Agricultrue, Email: [email protected]
European Journal of Academic Essays 1(9): 20-23, 2014
purified and multiplied which was later on identified as fungi
on the basis of morphology characteristics.
(Olympus CX21), Germany). A slide culture technique was
also used to observe the morphology of the isolated fungi.
The isolates were identified initially by
2.3. Microscopic Analysis
The fungi were grown on PDA at 25oC for 7-9 days, and the
conidia of the isolates were examined under a microscope
comparing morphological and cultural characteristics (i.e.,
size of conidia, number of cells/conidia, color of conidia, and
type of mycelia to those described in [10, 15,16].
Severe fruit infections, especially shortly after petal fall,
result in the drop of young fruitlets (Fig. 3-a). Remaining
fruit were appeared surrounded by brown lesions at the fruit
base. (Fig. 3-b). On occasion, A. alternata is able to
penetrate the citrus rind and cause localized necrosis, but this
is relatively rare. Fruit are susceptible to Alternaria blight for
4 months after petal fall. Even when the fruit are no longer
susceptible, some fruit may fall as the result of earlier
infections, especially if the lesions are near the fruit stem.
Severely affected fruit abscise reducing yield, and blemishes
on the remaining fruit greatly diminishing marketability.
2.4. Pathogenicity test:
Lemon leaves were washed in running water and surface
disinfected for 1 min in 1% Sodium hypochlorite. Then
washed with sterile distilled water and dried with sterile filter
paper. Twenty leaves were inoculated with a mycelial plug (5
mm in diameter) of A. alternata culture placed over the intact
skin. Twenty other non-inoculated leaves served as control.
The leaves were placed in humid chamber at room
temperature.
3. Results and Discussion
3.1. Description of Symptoms
Alternaria attacks young fruit, leaves and twigs, producing
brown-to-black lesions surrounded by a yellow halo. On
young leaves, the disease produces minute brown to black
spots. On the other hand Symptoms was appear in as a die
back after infection. Lesions usually continue to expand and
large areas of the leaf was killed (Fig. 1) even without tissue
colonization. These changes caused by toxins that plays a
role in disease development [17,18].
Figure 3. Symptoms on twigs (a) and young fruits (b).
3.2. Description of Species
3.2.1. Cultural characteristics.
Typical cultural and morphological characteristics are shown
in Figure 4. On PDA plates colonies were velvety. The
isolates first developed grayish with a very thin white margin,
and cottony texture (Fig. 4-a). Colonies later developed
color, the colors became dark olive as fungal age increased
(Fig. 4-b).
Figure 1. Spot and blight symptoms or die back on leaf
Leaf lesions are generally circular but will often have a tail,
following the leaf vein which gives the lesions an eye-spot
appearance (Fig. 2). The necrosis extends along the veins as
the toxin spreads in vascular tissues. Lesions enlarge as
leaves mature and can vary in size from 1-10 mm (0.04-0.4
inches) and will be larger if the infection occurred earlier in
the season. If Alternaria blight is severe, the leaves may drop.
Figure 4. a. Alternaria alternata isolated from leaf and
young fruits (at narrow) and b. colony morphology on PDA
medium.
3.2.2. Morphology of fungus
The isolated fungus corresponded in morphology to
Alternaria alternata described by Mycelium is septate (Fig.
5-a) and the conidia are variable in size and shape, but most
often short and ellipsoid to oval, tapering in the lower half.
Figure 2. Circular lesions on leaf
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European Journal of Academic Essays 1(9): 20-23, 2014
Alternaria is easily identified by the shape of its conidia,
which are large, ovoid to obclavate, dark-colored
(melanized), multicellular with longitudinal and transverse
septations (phaeodictyospores). Conidia are produced in
single or branched chains on short conidiophores (Fig 5-b)
and showed 3-8 transverse and longitudinal septa (Fig. 5-c).
Conidia of the isolates were catenated in long and sometimes
branched chains of 8 to 12 spores. The spores were
obclavate, dark olive, with 4 to 8 trans-verse and 0 to 2
longitudinal or oblique septa (Figure 5b and c). The conidia
were on average measured (22 to 38) × (5.8 to 11.2) μm.
Conidia are short ellipsoid to oval, tapering in the lower half
into a narrow tail extension. The upper part which was
materialized by a very short beak well rounded ending
abruptly appears allowing the formation of new spores, thus
furnishing evidence of catenulation. Primary conidiophores
arise directly from hyphae at the PDA surface; they can be
simple or branched. The asexual spores of the fungus are
thick-walled, multicellular, and pigmented and thus tolerate
adverse conditions like dry weather. These results confirmed
the isolated pathogen as Alternaria alternata.
role in pathogenesis and is responsible for leaf necrosis
[17,18]..The fungi were reisolated from the lesions of the
diseased leaves and were identical to the original isolates.
Figure 6. Symptoms of lesion and blight disease on lemon
leaf after inoculation. None inoculated leaf (left); inoculated
leaf (right).
References
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Pathogenicity test:
To confirm pathogenicity, mycelial plugs containing conidia were
used as inoculum. Lemon leaves were inoculated in the
laboratory with A. alternata and maintained in a moist
chamber for 2 to 5 days. Brown, corky lesions resembling
symptoms that occurred in the field were observed
surrounding the inoculation sites beginning around day 1
accompanied by blight symptoms or die back (Fig. 6).
Symptoms were not observed on control leaves not
inoculated with fungus. Symptoms was appear in as little as
24 h after infection. Lesions usually continue to expand and
large areas of the leaf may be killed by the host-selective
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a toxin which exhibits high host specificity, plays a major
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