ISSN 2320-5407

International Journal of Advanced Research (2014), Volume 2, Issue 4 ,1216-1227

Journal homepage: http://www.journalijar.com

INTERNATIONAL JOURNAL
OF ADVANCED RESEARCH

RESEARCH ARTICLE

Screening and Characterization of Fungi and their associated Mycotoxins in some Fruit
Crops
M.I. Ammar and M. A. El-Naggar
Plant Pathology Research Institute, Agriculture Research Center, Giza,Egypt.
.

Manuscript Info
Manuscript History:
Received: 10 February 2014
Final Accepted: 22 March 2014
Published Online: April 2014

Key words:
Pomegranate. Fungi. Guava.
Mycotoxins.Fusarium .Alternaria

*Corresponding Author
M.I.Ammar

Abstract
The mycological analysis of five fruit crops samples revealed that,
the two genera Alternaria with nine species and Fusarium with eight species
were found to be the most dominant fungi on all selected fruits.
Pathogenicity test on pomegranate fruits revealed that A. alternata
reproduced the typical symptoms of black spots while A. alternata, A.
arbusti, P. funiculsoum and A. niger were pathogenic and reproduced the
typical symptoms of heart rot. On guava fruits, C. dematium, A. alternata, A.
raphani, Phoma sp., Fusarium sporotrichioides, F. proliferatum and F.
culmorum caused visible symptoms of fruit spot and fruit rot while, fruits
inoculated with B. theobromae and Phomopsis sp. exhibited stylar end rot
symptoms. Additionally, according to available literatures, this is the first
report of A. alternaria and P. funiculosum as causal pathogen of black spot
and heart rot of pomegranate fruits in Egypt respectively. Also, pathogenicity
trails reported for the first time stylar end rot disease of guava and C.
dematium as a causal pathogen of anthracnose in Egypt. Mycotoxin assay
revealed that eight Fusarium species were capable of producing detectable
levels of four major mycotoxins i.e. Deoxynivalenol (ranging from 7.8405.3g/L), Fumonisin (35.9-1121.0 g/L), T-2 toxin (0.0-55.2 g/L) and
Zearalenone (0.0-456.9 g/L). Nine Alternaria species was tested for
produce four kinds of mycotoxins i.e. Alternariol, Tenuazonic acid,
altenuene and Alternariol monomethyl ether. A. alternata associated with
fruit rot of guava was able to produce the four mycotoxins and other
remaining species was varied in mycotoxins production profile. All four
mycotoxins were apparently not produced by A. zinniae and A. infectoria.

Copy Right, IJAR, 2014,. All rights reserved

1. Introduction
In the past 20 years international trade in fresh fruits and vegetables has grown greatly and is presently a
multi-billion dollar business representing the major export for many developing countries (Barkai-Golan and Paster,
2008). One of the limiting factors that influence the fruits economic value, the relatively short shelf-life period
caused by attacked pathogens. Plant pathogens may infect fruits either prior to harvest under field conditions or after
harvest during transit and storage. The symptoms of infection may be observed at different period after harvest but
many pathogens may remain dormant for varying periods until favorable conditions become available for their
development, leading to visible symptoms. It is estimated that about 20-25% of the harvested fruits are decayed by
pathogens during post-harvest handling even in developed countries (Droby,2006 and Zhu, 2006). It should be noted
that for a total of 100,000 fungi, less than 10% are pathogenic for plants and around 100 species are responsible for
the majority of postharvest damage (Eckert and Ratnayake, 1983).Storage diseases lead to economic loss by
reducing quality and marketability of damaged fruit, or may result in complete loss of the stored fruit. Fruits contain

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high levels of sugars and nutrients element and their low pH values make them particularly desirable to fungal
decayed (Singh and Sharma, 2007). The common postharvest and storage fungi of fruits are Alternaria spp.,
Aspergillus spp., Fusarium spp., and Penicillium spp. (Bhale, 2011). Besides the losses in income to the fruit
marketers, in some cases host pathogen interactions provide a favorable environment and source for production of
many different compounds. Mycotoxins are a structurally diverse group of mostly small molecular weight
compounds, produced mainly by the secondary metabolism of some filamentous fungi, or molds under suitable
conditions (Zain,2011). The major mycotoxin-producing fungi are not aggressive pathogens in plants; however,
mycotoxins are produced by several genera in plants during the growing season when portals of entry are provided
and environmental conditions are appropriate and be continued or initiated in postharvest and stored products.
The majority of these toxins are produced by fungi of the genera, Aspergillus, Penicillium and Fusarium (BarkaiGolan and Paster, 2008). The toxigenic Fusarium and Alternaria species are often classified as field fungi, because
they require very high moisture content in the substrate for growth and mycotoxin synthesis. The storage fungi,
primarily species of AspergillusandPenicillium also grow well at lower moisture contents. Thus, Fusarium and
Alternaria usually represent a high mycotoxicological risk in preharvested or freshly harvested plant that are drying,
whereas toxigenic species of Aspergillus and Penicillium represent a higher risk for products in storage or being
used in food and feed processing (Harris and Mantle,2001; Harvey et al., 2001; Hussein and Brasel, 2001 and
Logrieco et al., 2002). In Egypt and under local markets in Najran, Saudi Arabia, there is relatively little information
related to the natural occurrence of fungi and mycotoxins in fruits. The present investigation was undertaken to find
out the association of field and storage fungal diseases with some important fruits and the mycotoxins mainly
synthesised by strains of toxigenic fungi.

2. Materials and Methods

2.1. Samples Collection
A survey of crop fungi were conducted on five economically important fruits i.e. Promegranate (Punica
granatum), Guava (Psidium guajava), apple (Pyrus malus), mango (Mangifera indica) and Citrus (Citrus sp.) during
2012, 2013 seasons. Naturally infected fruits of Guava, Pomegranate and Citrus were collected from orchards at
Biehera governorate, Egypt. Also, infected fruits were collected from local markets in Najran, Saudi Arabia.
Samples were brought to the laboratory in separate sterilized polythene bags, examined critically with respect to
symptomatology and sorted out for the isolation of the causal agents.
2.2. Isolation and identification of the associated fungi
The isolation and identification of the causal agents were performed for every single fruit. Fungal
pathogens responsible for fruit rotting were isolated from the flesh beneath the peal while fruits exhibit fruit spots
were isolated from the peel surface. Infected fruit tissues were surface sterilized in 1% sodium hypochlorite solution
for 1 min and rinsed twice in sterilized water. Using a sterile scalpel, tissue pieces composed of spots, halo, and
surrounding healthy tissue were placed onto potato dextrose agar (PDA) amended with tetracycline at 12g/ml, and
incubated at 25C.In some cases, rotting fruit samples were incubated in a moist chamber and mycelium of
individual fungal species transferred onto PDA plates and incubated for 3-7 days at a temperature of 25C.Fungal
colonies emerging from symptomatic tissue was picked up and transferred to new plates and left to grow for 5 to 7
days prior. Pure cultures were then obtained by single spore isolation and maintained on PDA slants for further
study. The fungi were observed under a microscope and identification of the pathogens was made with the help of
available literature (Barnett and Hunter, 1999 and Biligrami et al., 1991). The identification for all fungal isolates
was re-confirmed by the Microloge system (Biolog, Inc., Hayward, CA) at the National Research Central Lab.,
GSFMO, KSA for further identification(Grizzle, 2006 and Singh, 2009). Different frequencies of fungi on same
crop were noted.
2.3. Pathogenicity test
To fulfill Koch's postulates, fresh disease free samples were brought to the laboratory and surface sterilized
with 5% Ethanol. PDA-plugs, 5 mm in diameter, with seven-day-old actively grown mycelium were transferred to
skin wounds by pressing slightly pin-pricking the fruits to a 1 mm. depth with a sterile needle on superficially
disinfected fruits (three plug per fruit, 3fruit per fungus). In case of fungi associated with stylar end rot of guava,
mycelium growth plugs were transferred into the fruit calyx as well as on wounds made by a scalpel on previously
sterilized fruit surfaces. Following inoculation, the fruits were placed in plastic boxes supplemented with watered
tissue paper to maintain relative humidity and kept at 25 C. Fruits inoculated in the same way using PDA disks
were kept as control. Pathogenicity tests were conducted on fruits and were repeated three times (Tziros et al.,
2007).The artificially inoculated samples were examined daily and the extent of damage was recorded. Lesion
development on fruit was assessed by measuring the disease area in centimeters on each fruit. The pathogens were
reisolated and disease symptoms were clearly evident, the culture and symptoms signs were compared with original.

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2.4.Mycotoxins:
The different fungal isolates were propagated as pure culture in 100 ml SMKY broth (Sucrose 200 g, MgSO47H2O
0.5 g, KNO3 3 g, yeast extract 7 g) for 10 days. All fungal isolates had three replicates and incubated in dark
condition at 252C.
2.4.1.Fusarium mycotoxins assay
Microtitre plate enzyme-linked immunosorbent assay (ELISA) reader (automated Chem-well) and Fusarium
mycotoxins test kit (r-biopharm, Germany) were used to ELISA analyses. The samples were analyzed using the
Fumonisin, Zearalenone, T-2 and DON test procedure which was described by company(r-biopharm) producer
(Enzyme Immunoassay for the quantitative analysis of aflatoxins, 1999and Leszczynska et al., 2001). Ten ml of
blended fungal broth has been sub-sampled with 20ml of 70% methanol and vortex for 10 min by magnetic stirrer.
The extract was filtrated by Whatman no.1 filter paper and then diluted as 5ml filtered solution, 15ml distilled water
and 0.25ml Tween 20. The solution was mixed by magnetic stirrer for 2min. 50 l toxins (5, 10, 20, 45 ppb)
standard solutions and 50 l prepared test samples were added into separate wells of micro-titer plate, in duplicate.
Plates were incubated at room temperature. The liquid was then removed completely from the wells, the each well
was washed with 250 l washing PBS-Tween-Buffer (pH 7.2) and this was repeated two times. Subsequently,
enzyme substrate (50 l) and Chromogen (tetramethyl-benzidine, 50 l) were added to each well and incubated for
30min at room temperature in the dark. 100 l of the stop reagent (1M H2SO4) was added and the absorbance was
measured at 450nm in ELISA reader.
2.4.2. Penicillium mycotoxins assay
Penicillium isolate were examined to produce each of the Patulin, Citrinin, citreoviridin and penicillic acid. Culture
was blended for 2 min using a high speed homogenizer and filtered using glass filter paper. Patulin was extracted
from homogenized filtrate using acetonitrile: water (5:95 v:v) solution. The solvent was then evaporated at 35C
under vacuum. The dried residues were dissolved in 1 ml of acetonitrile: water (5:95 v:v) solution. HPLC was used
to quantify Patulin (Christian, 1990).
2.4.3. Aspegillus nigar mycotoxins assay
The tested isolate of A. nigar was examined to oxalic acid excretion. The tested isolate cultivated on SMKY broth
medium and oxalic acid concentration was determined by high performance Liquid chromatography (HPLC).
Separation of oxalic acid was carried out in a CLCC825 CM caption exchange column; mobile phase, 90% H2O and
10% CH3OH; flow rate, 1 ml/min and temperature 35C according to Ghorbani et al., 2007.
2.4.4. Aternaria mycotoxins assay
The different species of Alternaia was examined for mycotoxins production. Alternariol (AOH), Tenuazonic (TeA),
Altenuene (ALT), and Alternariol monomethyl ether(AME) was determined by Chromatography analysis according
to Nawaz et al., 1997.
2.5. Data analysis
Different data was recorded and expressed as means S.E. Significance of mean differences were statistically
compared using an analysis of variance (ANOVA) at the 5% probability level with individual pairwise comparisons
made using Tukeys HSD test through an SPSS v. 15.0 software package in Microsoft Windows 7 operating system.

3. Results and Discussion

3.1. Survey and isolated fungi
Survey study was conducted on five fruits i.e. Pomegranate, guava, mango, apple and citrus which
collected from mature fruits in the field of Egypt and from different markets of Najran region, Saudi Arabia. The
mycological analysis of five fruit crops samples revealed that, 31 fungal species belonging to nine genera were
associated with fruit symptoms. In general, the two genera Alternaria with 9 species and Fusarium with 8 species
were found to be the most dominant fungi on all fruits (Table 1). The post-harvest fungal diseases are responsible
for biodeterioration of tropical fruits pulp (Gadgile et al., 2009a,b). Guava and pomegranate fruits showed more
fungal association than other fruits although, the presence of fungal diseases on these fruits received little attention
and not well documented in Egypt so, our study received more attention to these fruits. Similarly some species were
thought to attack fruits long before harvest and exhibited host specificity i.e. stylar end rot of guava associated with
B. theobromae and Phomopsis psidii and black spots of pomegranate caused by A. alternata while others were
observed to be responsible for the postharvest, decay and deterioration as general pathogens(Table 1).
3.2. Symptoms on fruits
In a survey of pomegranate, we reported the appearance of novel symptoms of Alternaria spp. These symptoms
include black spots on fruit, ranging from a single lesion to lesions that cover more than 50% of the fruit surface
(Fig. 1). The damage is restricted to the peel surface while the edible tissue remains unaffected. In contrast, heart rot

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of pomegranate associated with Alternaria spp., and Penicillium funiculosum, in which the fruit rot, is restricted to
the internal area whereas the peel remains unaffected. Another fungus that is also isolated from pomegranate is
Aspergillus niger : symptoms on fruits appeared in two forms: spherical depressed spots occurred in scattered form
on the pericarp only (Fig.1a) and black rot restricted to internal fruit tissues. The heart rot decay caused by A. niger
is softer with exuded juice than that caused by Alternaria and symptoms reached the outer surface of fruit. In the
Mediterranean region, A. alternata, Penicillium spp. and A. niger are an important pathogens associated with black
spots and heart rot of pomegranate (Labuda et al., 2004; Tziros et al., 2007 and Pala et al.,2009).
Table(1)Relative frequency(%), Pathogenicity and symptoms of fungal species associated with four different
fruit crops during pre/postharvest.
Source
Host

Fungi

Symptoms
Field

% frequency* Pathogenicity*

Storages

Guava
(Psediumguajva)

Pomegranate
(Punicagranatum)

Egypt
Fruit canker
2.750.14e
A. arbusti
A. blumeae
Egypt
Fruit canker
2.10.06d
A .alternata
Egypt
Fruit canker
0.690.05a
A. bumsii
Egypt
Fruit spots
1.380.02c
A. alternata
Egypt
Black spots
6.130.02k
A. alternata
Egypt
Heart rot
1.380.02c
A. gaisen
Egypt
Fruit canker
0.690.01a
A .alternata
Yemen
Fruit blotch
2.770.04e
P. funiculosum
Egypt
Heart rot
2.770.04e
Aspergillus niger
unknown
Black rot; fruit spot
4.150.03j
C. trichellum
unknown
Fruit canker
2.770.04e
C. dematium
Egypt
Egypt
Anthracnose
1.380.05c
F. sporotrichioides
Egypt
Fruit spots; fruit rot
4.500.3h
F. oxysporum
Egypt
Fruit spots; fruit rot
0.690.03a
F. chlamidosporium
Egypt
Fruit spots; fruit rot
1.040.02b
F. proliferatum
Egypt
Fruit spots; fruit rot
1.040.02b
F. culmorum
Egypt
Fruit spots; fruit rot
1.040.02b
A. raphani
Egypt
Fruit spots; fruit rot
3.460.03f
A .alternaria
Egypt
Fruit spots; fruit rot
2.770.01e
Phoma sp.
Egypt
fruit rot
0.690.01a
A. zinniae
Egypt
Stylar end rot
2.770.02e
Phomopsis sp.
Egypt
Stylar end rot
5.190.01f
B. theobromae
Egypt
Stylar end rot
4.150.01e
Fusarium sp.
Egypt
Stylar end rot
0.690.0a
*Values are the mean S.E. of three replicates.
In the same column, means followed by the same letters are not significantly different (P 0.05).
**All F-values are significant at P >0.001.

0.540.02f
0.00.0a
0.00.0a
0.00.0a
0.860.01i
0.730.02j
0.00.0a
0.43 0.02c
5.540.02n
7.36 0.04q
0.00.0a
0.760.02j
1.400.02L
0.00.0a
0.950.03j
0.750.03j
1.250.03k
0.47 0.04c
0.750.02j
0.450.01c
0.00.0a
1.200.01c
1.450.01j
0.830.01c

Continued: Table (1) Relative frequency (%), Pathogenicity and symptoms of fungal species associated with
four different fruit crops during pre/postharvest.
Source

Mango
(Mangife
raindica)

Host

Fungi
Field

Storages

C. fragariae

C. gleosporioides
C. arachidis
A. infectoria

Pakistan
Pakistan
Pakistan
Egypt

Symptoms

% frequency*

Pathogenicity*

Anthracnose

2.080.05d

0.350.03b

Anthracnose
Anthracnose
Black Spot

4.150.02j
3.460.01f
7.270.04L

0.500.03d
0.00.0a
0.400.02b

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spp.)

Apple(Pyrusmal
us)

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C. musae
Botrytis cineria
F. equisti
F. langsethiae
A. alternata
Penicillium sp.
A.niger
A. citri

International Journal of Advanced Research (2014), Volume 2, Issue 4 ,1216-1227

France
France
France
France
France
France
France
Nourth
Africa
-

Fruit rot
Gray rot
Fruit rot
Fruit rot
Black spot
Fruit rot
Fruit rot
Black rot

1.040.02b
2.770.03e
1.040.02b
0.690.01a
1.380.02c
2.080.05d
0.690.05a
4.150.03j

P.digitatum
Egypt
Green rot
5.190.02j
A.niger
Egypt
Fruit rot
4.840.02i
ND: not determined
*Values are the mean S.E. of three replicates.
In the same column, means followed by the same letters are not significantly different (P 0.05).
**All F-values are significant at P >0.001.

0.00.0a
2.450.01m
0.670.01j
0.580.02f
0.380.02b
ND
ND
ND
ND
ND

Based on the findings of this study, the most important causal agent responsible for the fruits diseases of
guava are the fungi. Similarly some species were thought to attack fruits before harvest in the field as stylar end rot
symptoms which observed in Egypt on the nearly-matured fruits in the region lying just below and adjoining the
persistent calyx as brownish water-soaked area. Such area gradually increases in size and turn brown with
translucent margins and covered with concentric zones of dark colour pycnidia embedded in the disease tissues (Fig.
2.a) or covered with fungal growth (Fig. 2. , b,c). One of the pathogenic fungi that are associated with fruit rot of
guava is Fusarium spp. while, C. dematium was found to be associated with anthracnose symptoms of guava
fruits(Fig.2e).In general, the following fruit diseases have been reported: gray mold caused by Botrytis cinerea on
apple, Alternaria black spot (Alternaria alternata) of stored mango and apple fruits(Fig.2i), fruit rots on citrus and
apple(Penicillium spp. and Aspergillus niger respectively) and anthracnose on apple and mango(Fig.2.f)
(Colletotrichum spp.).

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Fig.1. Disease symptoms of collected pomegranate fruits. a, fruit spots (Aspergillus niger); b, c, Black spots
(Alternaria alternata) and d, e, f, fruit blotches (Alternaria alternata, Alternaria spp.).

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Fig. 2. Disease symptoms of different fruits collected from markets in Najran, Saudi Arabia and under field
condition in Egypt. a ,b and c, Stylar end rot of guava (Botryodiplodia theobromae , Phomopsis psidii) ; d, fruit rot
of guava (Alternaria spp.) ; e, Anthracnose of guava fruits (Colletotrichum dematium); f, Anthracnose of mango(C.
gleosporoides, Pakistan); j,h, Black rot of citrus (Alternaria citri; south Africa) and i, Black spots of apple Royal
Gala fruits (Alternaria alternata).
3.3. Proving pathogenicity
Different fungal species were established as the causative organisms of fruit diseases in the five crops tested fruits.
Disease symptoms were observed after seven day of incubation at 25C on pomegranates inoculated with Alternaria
alternata, A. arbusti, A. niger and P. funiculosum. No decay was observed on control fruit and fruit inoculated with
C. trichellum and other Alternaria species. The first four fungi were consistently reisolated from decayed fruits. On
fourth day of inoculation, Alternaria alternata reproduced the typical symptoms of black spot as brown to black
spots on the peel surface of fruits (without any internal decay symptoms).Initial symptoms in case of A. niger and P.
funiculosum revealed as necrosis on the fruit peel without sporulation. After 7days, more extensive necrosis
surrounded by water soaked area was observed in case of A. niger and spore mass was associated with the central
portion of necrosis area. Several Penicillium spp., Alternaria alternata and A. niger have been previously reported

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as a causal pathogens of heart rot of pomegranate fruits (Tziros et al., 2007; Bardas et al., 2009; Ezra et al., 2010;
Gat et al.,2012; Michailides et al., 2012 and Zhang et al., 2012). These species usually attack the fruit during
bloom/fruit set and as the fruit develops, the fungus spreads to the interior of the fruit causing black heart or heart
rot (Ezra et al., 2010; Gat et al., 2012 andYehia2013). According to available literatures, this is the first report of A.
alternaria as causal pathogen of black spot of pomegranate fruits and P. funiculosum, as fruit rot causal agent in
Egypt. Other Alternaria species from pomegranate fruits were non-pathogenic. A. alternata can be found as an
epiphytic saprophyte on all parts of the plant without causing any damage. Isolation of A. alternata from plants does
not indicate whether pathogenic isolates of this species are present (Farr et al., 1989). Based on literatures the
specificity of A. alternata pathotypes causing black spot is unknown while, Alternaria sp. isolated from any part of
the pomegranate plant and from other plant species can cause heart rot when it is introduced into the fruit (Ezra, et
al.2010). Of the many species belonging to Alternaria, there are also include opportunistic plant pathogens which
can affecting many cultivated plants in the fields and stored fruits and vegetables during post-harvest (Guo et al.,
2004).
In inoculation of guava fruits, C.dematium, A.alternata, A. raphani, Phoma sp.,Fusarium sporotrichioides,
F. proliferatum and F. culmorum caused visible symptoms as brown lesions on fruits appeared on the fourth day of
inoculation (ranging from 0.45-1.40mm in diameter). With disease developed, infection progressed to the fruit flesh
and fungal growth appears on the infected lesions (Sumia et al., 2006; Rao et al., 2012 and Zakaria et.al.,
2012).Inoculated fruits with B. theobromae and Phomopsis psidii isolated from guava fruits exhibited stylar end rot
symptoms showed disease symptoms similar to those found in field which showed on inoculated fruits 7 days after
inoculation (Rai, 1956 and Quintero and Urdaneta, 1997). The rest genera didnt induce any visible symptoms. In
the present study, F. oxysporum was found to be non-pathogenic to guava fruits however; F. oxysporum var. psidi
was reported to cause post-harvest rot (Sumia et al., 2006) and causing Fusarium wilt disease of guava plant.
Fusarium species causing fruit rot is considered opportunistic weak pathogen and often needed predisposing factors
(Nishijima, 1998). Pathogenicity trails reported for the first time stylar end rot disease of guava and C. dematium as
a causal pathogen of anthracnose in Egypt.
In general, pathogenicity test on other fruits revealed that, C. gleosporioides on mango, Botrytis cineria and A.
alternata on apple were pathogenic and showed anthracnose, gray mold and black spots symptoms respectively
(Singh and Sharma, 2007). Numerous cases have been reported in which single species of Colletotrichum infects
multiple hosts as C. domatium (Freeman et al.,1998; Dillard, 1992 and Sutton, 1992). In contrast, it is common to
find that several Colletotrichum species are associated with a single host as C. gleosporioides and C. fragariae
(Howard et al., 1992 and Freeman et al.,1998).
3.4. Mycotoxins production:
Mycotoxigenic fungi belong mainly to Fusarium, Alternaria, Aspergillus and Penicillium genera,other species are
known as uncommon toxigenic fungi. Fusarium and Alternaria usually represent a high mycotoxicological risk and
often classified as field fungi because they require very high moisture content for growth and their mycotoxinwere
formed in preharvested or freshly harvested plant. The toxigenic species of Aspergillus and Penicillium represent a
high horrible risk for products in storage or being used in food and feed processing and grow well at lower moisture
contents (Logrieco et al., 2003 and Barakai-Golan and Paster, 2008).
3.4.1. Fusarium mycotoxins:
We examined eight Fusarium species recovered from guava and apple fruits in producing four major mycotoxins.
Results of mycotoxin production by Fusarium species showed the quantification of four mycotoxins i.e.
Deoxynivalenol(DON) (ranging from 7.8-405.3g/L), Fumonisin (FUM)(35.9-1121.0 g/L), T-2 toxin(0.0-55.2
g/L) and Zearalenone (ZEA) (0.0-456.9 g/L).i.e. DON, FUM, T-2 and ZEA.
Out of 8 Fusarium species recovered, four species produced detectable levels of four mycotoxins in vitro i.e. F.
oxysporum, F. sorotrichioides, F. proliferatum and F. equiseti. All these species have been reported as producer of
these mycotoxinsand all of these were in matching with (Logrieco et al., 2002; Logrieco et al., 2003 and Rai et al.,
2012). In general Fusarium species synthesise different mycotoxins, some at very high concentration as FUM
produce by F. proliferatum ( up to 1121.0 g/L) and others species i.e. F. culmorum , chlamdosporium and F.
langsethiae were found unable to produce detectable amounts of T-2 toxin(Table 2).The economic importance of
Fusarium species is enhanced by their ability to synthesize harmful mycotoxins and efforts to control Fusarium
infections and prevent or eliminate the presence of its mycotoxins in foods have not met with a great deal of success
(Desjardins et al., 2000 and Barakai-Golan and Paster, 2008).

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Table (2).Profile of mycotoxins production by Fusarium strains from some fruit crops.
Mycotoxins concentration (g/L)*
(DON)
( FUM)
(T-2)
(ZEA)
F.culmorum
Guava
Egypt
11.10.06e
102514.4b
0.00.0f
253.3.73b
F.sporotrichioides
Guava
Egypt
23.50.31c
9852.89b
73.60.35a
121.60.35d
a
f
b
F.oxysporum
Guava
Egypt
405.30.17
45.60.35
55.20.12
90.80.46e
d
c
f
F.chlamdosporium
Guava
Egypt
17.40.23
6452.89
0.00.0
0.00.0j
b
a
c
F. proliferatum
Guava
Egypt
338.60.35
1121.00.58
17.00.58
456.90.52a
j
e
f
Fusarium sp
Guava
Egypt
1.250.03
325.10.058
0.00.0
195.30.17c
e
d
d
F. equiseti
Apple
France
11.40.23
546.832.9
14.50.29
30.60.35f
f
f
e
F. langsethiae
Apple
France
7.80.46
35.90.52
11.20.12
0.00.0j
F value**
390926.6
1137.01
10979.9
51808.16
-Deoxynivalenol(DON), Fumonisin (FUM), T-2 toxin, Zearalenone(ZEA) .
*Values are the mean S.E. of three replicates.
In the same column, means followed by the same letters are not significantly different (P 0.05).
**All F-values are significant at P >0.001.
Fusarium isolates

Host

Origin

3.4.2. Alternaria mycotoxins:

The results showed that all Alternaria species tested except of A. zinniae and A. infectoria in this study were capable
to produce one and/or several kinds of four mycotoxins i.e. Alternariol (AOH) Tenuazonic acid (TA), altenuene
(ALT) and Alternariol monomethyl ether (AME).
Only A. alternata from fruit rot of guava, produced in vitro considerable amounts of TA (3.2 g/L), AOH (5.2
g/L), AME (27.9 g/L) and ALT (4.9 g/L), while the black spot strain from pomegranate fruits produced TA
(12.5 g/L), AOH (7.5 g/L) and ALT (1.7 g/L) but unable to synthesize any detectable amount of AME (Table
3).In general, ALT (1.3 g/L), TA (5.81 g/L), AOH (4.8 g/L) and AME (32.5 g/L)were the only detectable
mycotoxins in A. arbusti, A. blumeae, A. A. bumstii and A. gaisen respectively. Alternaria alternata is a ubiquitous
necrotrophic fungus including at least seven pathogenic variants, each producing unique host-selective toxins which
play an important role in the pathogenesis of plants and causing disease on specific host plants (Kohmoto et al.,
1991; Hatta et al.,2002 and Ito et al., 2004). Five major Alternaria mycotoxins belong to three structural classes can
be found as natural contaminants in foodstuffs: the benzopyrene derivatives alternariol (AOH), alternariol mono
methyl ether (AME), altenuene (ALT), the tetramic acid tenuazonic acid (TA) and the perylene derivative alter toxin
I (ATX I) (Bottalico and Logrieco, 1998 and Barkai-Golan and Paster, 2008). Several authors revealed that
Alternariol and alternariol monomethyl ether have been isolated from many different cultures of the genus
Alternaria as A. alternata, A. arborescens, A. infectoria and A. tenuissima, among others (Andersen et al., 2002). In
general, only little toxicological data are available just for seven out of the 30 known Alternaria mycotoxins which
is insufficient for an assessment of the health risk for the consumer (Bundesinstitut fr Risikobewertung, BfR,
2003).
Table (3). Quantitative estimation of mycotoxins produces by Alternaria species in vitro.
Mycotoxins concentration (g/L)*
Alternaria
Host
Origin
Isolates
(AOH)
( TA)
(ALT)
(AME)
A. arbusti
Pomegranate
Egypt 0.00.0d
0.00.0d
1.30.30c
0.00.0d
d
b
d
A. blumeae
Pomegranate
Egypt 0.00.0
5.811.7
0.00.0
0.00.0d
c
d
c
A. bumsii
Pomegranate
Egypt 4.80.12
0.00.0
1.90.40
0.00.0d
d
d
d
A. gaisen
Pomegranate
Egypt 0.00.0
0.00.0
0.00.0
32.50.50a
b
d
b
A. raphani
Guava
Egypt 6.90.23
0.00.0
2.60.31
17.80.80c
c
c
a
A. alternata
Guava
Egypt 5.20.23
3.20.40
4.90.0
27.90.90b
d
d
d
A. zinniae
Guava
Egypt 0.00.0
0.00.0
0.00.0
0.00.0d
a
a
c
A. alternata
Pomegranate
Egypt 7.80.23
12.50.55
1.70.40
0.00.0d
d
d
d
A. infectoria
Mango
Egypt 0.00.0
0.00.0
0.00.0
0.00.0d
F value**
588.95
151.9
110.49
2922.4
- Alternariol (AOH) Tenuazonic acid (TA), altenuene (ALT) and Alternariol monomethyl ether (AME).
*Values are the mean S.E. of three replicates.
In the same column, means followed by the same letters are not significantly different (P 0.05).
**All F-values are significant at P >0.001.

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3.4.3. Aspergillus and Penicillium Mycotoxins

Investigation carried out on the presence of mycotoxins in one isolate of P. funiculosum , the causal
pathogen of heart rot of pomegranate revealed the occurrence of Patulin (PAT,25.6 g/L) and Penicillic acid (PAC,
11.9 g/L). On the other hand, P. funiculosum was found to be unable to produce any detectable amount of Citrinin
and citreoviridin (Table 4). A. niger associated with black rot of pomegranate found to be capable of producing
Oxalic acid (7.9 g/L). Aflatoxins AFB1, AFB2, AFG1 and AFG2 were not produced by A. niger used in our study
(Table 4).
Table (4). Profile of mycotoxin production by A.niger and P. funiculosum isolated from pomegranate fruits.
Fungal
Isolates

Host

Mycotoxins concentration (g/L)

Origin

A. niger

Pomegranate

Egypt

P. funiculosum

Pomegranate

Egypt

(oxalic acid)

( AFB1)

(AFB2)

(AFG1)

(AFG2)

7.9

0.0

0.0

0.0

0.0

PAT

CIT

CITV

PAC

--

25.6

0.0

0.0

11.9

--

- Aflatoxins: AFB1, AFB2, AFG1 and AFG2

-Patulin (PAT), Citrinin (CIT), Penicillic acid (PAC), citreoviridin (CITV).
The ability of several Penicillium and Aspergillus species involved in postharvest decay of fruits to produce such
mycotoxins has previously been demonstrated (Frisvad et al.,2006; Moslem et al., 2013 and Tancinov et al., 2013).
Samson and Pitt (1990) have been reported P. funiculosum as mycotoxin producer. Aspergillus niger can produce a
variety of mycotoxins, include oxalic acid crystals, kojic acid, and cyclic pentapeptides called malformins. A.niger
may be does not have the genetic structure to Aflatoxins secretion, despite the ability of some species in same genus
as A. flavus, A. parasiticus to Aflatoxins formation (Blumenthal, 2004). A. niger was found able to produce oxalic
acid in liquid media and to secrete this acid into the medium (Blumenthal,2004).The production of oxalic acid may
have a role in pathogenicity and ecology of fungi which reviewed by Dutton and Evans (1996). About the role of
oxalic acid in pathogenesis, it is believed to play a role in facilitating plant cell wall degradation also, substrate
mobilizing through plant cell wall i.e pectin(Tanaka and Nonaka, 1981; Ruijter et al.,1999 and Sharma et al.,
2011).This fact may explain the symptoms associated with black heart rot of pomegranate and water soaked area
associated with A. niger infection.

4. Conclusion
The investigation aimed to monitor and survey some fungal plant diseases on some fruit in the tropical region, which
has recorded for the first time as well as study their pathogenicity, symptoms and their possibility of mycotoxins
secretion that may be harmful to both humans and animals and adept the plant for infection.

5. Acknowledgement
Authors extended their thanks to Fruit and Woody Tree Diseases Dept., Egypt for providing the laboratory facilities.

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