American Journal of Plant Sciences, 2011, 2, 276-281
doi:10.4236/ajps.2011.22030 Published Online June 2011 (http://www.SciRP.org/journal/ajps)
Copyright © 2011 SciRes. AJPS
Mango Malformation: I. Toxin Production
Associated with Fusarium Pathogens
Wafaa Haggag M.1, Hazza M.2, Sehab A.1, Abd El-Wahab M.1
1Department of Plant Pathology National Research Center, Dokki, Cairo, Egypt; 2 Science Faculty, Botany Department, Banha Uni-
versity, Banha, Egypt.
Email: wafaa_haggag@yahoo.com
Received April 2nd, 2011; revised May 4th, 2011; accepted May 18th, 2011.
ABSTRACT
Eight Fusarium species i.e. F. subglutinans, F. solani, F. oxyspoum, F. sterilihyphosum, F. proliferatum, F. monili-
forme, F. avena and F. chlamydspore isolated from mango malformed disease were tested for their ability to cause
mango malformation disease and their production of moniliformin and total fumonisins (FB1 + FB2) using HPLC. A
evaluated for moniliformin production, seven isolates were toxin producers, the production levels ranging from 0.51 to
8.90 µg/ml. The higher levels were produced by Fusarium subglutinans (8.51 µg/ml). Moderate concentrations of
moniliformin was produced by F.moniliforme (6.90 µg/ml), F. oxysporum (6.30 µg/ml), F. proliferatum (4.10 μg/ml)
and F. sterilihyphosum (1.10 μg/ml). Separation and identification of Fumonisin that was isolated from the pathogen-
causing disease are made by (HPLC). A evaluated for total fumonisin production (FB1 + FB2), seven isolates were
toxin producers, the production levels ranging from 0.10 to 8.30 µg/ml. The higher levels were produced by F. monili-
forme (8.30 µg/ml. Moderate concentrations of fumonisin was produced by F .proliferatum (0.64 µg/ml) and F. subglu-
tinans (0.50 µg/ml). Strong positive correlations between moniliformin and total fumonisins (FB1 + FB2) activities and
malformation disease incidence by F. subglutinans, F. solani, F. oxyspoum, F. sterilihyphosum, F. proliferatum was
observed.
Keywords: Fusarium, Mango Malformation, Moniliformin and Fumonisins
1. Introduction
Mango (Mangifera indica L.) is the most important fruit
grown in tropical and subtropical region of the world.
Mango (Mangifera indica L.) is the most important fruit
crop in Egypt. Mango Malformation is one of the most
destructive mango diseases [1,2]. Losses due to malfor-
mation have not been accurately assessed because yield
loss is not a linear function of disease severity [3]. Dur-
ing a survey of mango plantations in Sindh for investi-
gating the association of fungi with mango malformation
disease (MMD), six fungal species viz., Fusarium nivale
(Fr.) Ces, F. oxysporium, F. moniliforme, F. semitectum,
Alternari alternata and Aspergillus niger were isolated
and identified on the basis of their colony characteristics
and conidial morphology [4]. F. sterilihyphosum and F.
proliferatum are first report association with mango
malformation in [5]. As noted by [6] mango malforma-
tion probably involves two principles;1) the malforma-
tion inducing principle (MIP) which works through im-
balance in growth substance and in conditioning of
cells; 2)The toxic principle (TP) which causes growth
retardation and toxicity symptoms. Fuarium species,
particularly F. moniliforme var subglutinans, are likely
source of MIP and TP, and the causal agent of disease.
Singh and Dhillon [7] proposed that ethylene might play
role in mango malformation by suppression of apical
dominance, causing more isodiametric growth of rachi-
des and shortening and thickening of secondary branches
of malformed panicles. Ram [8] stated that the most
probable role of malformin in the causation of mango
malformation might be mediated through alteration of
membrane permeability leading to efflux of IAA or its
metabolite. He indicates the possibility that malformin
antagonizes IAA action through efflux action of auxins
of the malformed cells causing loss of apical dominance
at a very early stage of panicle development. Malformed
mango panicles have been shown to contain reduced lev-
els of auxin [7,9,10]. Strains of Fusarium proliferatum, F.
subglutinans, F. anthophilum, F. annulatum, F. succisae,
F. beomiforme, F. dlamini, F. napiforme, and F. nygamai
from a variety of substrates and geographic areas were
Mango Malformation: I. Toxin Production Associated with Fusarium Pathogens277
tested for the production of fumonisin B1 in culture.
None of the cultures of F. subglutinans, F. annulatum, F.
succisae, or F. beomiforme produced fumonisin B1 in
culture. Strains of F. proliferatum produced fumonisin
B1 in amounts ranging from 155 to 2936 ppm, of the
species tested, F. proliferatum is the most important
producer of fumonisin B1 because of its association with
corn and animal mycotoxicose such as porcine pulmo-
nary edema. F. napiforme and F. nygamai also may be
important because of their association with the food
grains millet and sorghum [11].
Thus, objective of the present study is to study the pro-
duction of toxins by pathogens involved in the causation
of malformation.
2. Materials and Methods
Fusarium species isolated from mango malformed dis-
ease were tested for their ability to cause malformation.
Mango seedlings cv. Sedekia (two years old) was inocu-
lated with culture filtrate of Fusarium spp. by injection
of the apical buds. Sterilized water was used as a control.
Transplanted seedlings were monitored for development
of malformation. At the end of the experiment (120 days),
all surviving seedlings were examined for apical disease
symptoms. Data were recorded on symptoms manifesta-
tion as diseases incidence and severity (from 1 - 4 scale).
2.1. Determination of Moniliformin and
Fumonisin
Cultures were initially grown on agar slants for 7 - 10
days. A slant was macerated in 27 ml of sterile water.
Aliquots of (2.5 ml) of the resulting suspension were
added to 250 ml Erlenmeyer flasks containing 50 ml of
inoculated medium made up of ultrapure water (1 L),
NH4Cl (3 g), FeSO4·7H2O (0.2 g). MgSO4·7H2O (2 g).
KH2PO4 (2 g) peptone (2 g), yeast extract (2 g) malt ex-
tract (2 g ) and glucose (20 g) after 48 h of incubation in
the dark at 28˚C on rotary shaker (220 rpm, 3.81 cm
throw), the suspension was macerated and employed as
moniliformin [12]. For production of moniliformin 2.5
ml aliquots were added to 250 ml Erlenmeyer flasks con-
taining 50ml of production medium consisting of ul-
trapure water, (NH4)2HPO4 (1 g). KH2PO4 (3 g), MgSO4-
7H2O (0.2 g) NaCL (5 g), Sucrose (40 g) and Glycerol
(10 g), final pH (6.2). The flasks were covered in alumi-
num foil to protect the toxin from the light and incubated
for 10 days [13].
2.1.1. Monilf ormin Analysis
A 500 µl aliquots of culture filtrate was loaded onto a
waters sep-pak®RP-18column pre-conditioned with wa-
ter. The column was eluted with 2 ml of ion-pair solution
(990:10 mix of 85:15 water/acetonitrile and 100:48:1.1
MKH2PO4/BU4N+OH [14]. A 100 µl aliquot was chro-
matographed on a Lichrosorb®RP-18 250*4.6 MM HPLC
with the ion—pair solution described above. The monili-
formin was detected with the same detector as above and
concentration evaluated by integration at 230 nm. Con-
centrations were determined by reference to calibration
curves established with standard provided by P. Scott of
Health Canada [15].
2.1.2. Fumoni si n Anal y s i s
The cultured were filtered as above and analysis was
performed on each replicate flask respectively as follows:
A1 ml aliquot of filtrate was applied to abondElut Certify
П® (200 mg, varian) column preconditioned by aspiring
methanol (6 ml) and water (6 ml) under vacuum. The
minicolumns were then washed with water (6 ml) and
methanol (6 ml). Fumonisins were eluted with 0.1%
trifluoroacetic acid (TFA) in methanol (3 ml). Fumonisin
was quantified by HPLC as follows [15]. The TFA/
methanol/methanol fraction from the clean-up column
was concentrated to dryness and taken up in 1ml of
methanol. A20 µL aliquot was transferred to 2 ml vial
and dried under stream of nitrogen. The residue was re-
dissolved in 100 µL of 0.05 M sodium borate buffer, pH
8.3 (adjusted with 1NHCl). A freshly prepared solution
of 4-fluoro-7-nitrobenzenofurazan (NBD-F) [100µL of
22 Mm NBD-F (Molecular probes Inc.) in 95% ethanol]
was added. After heating for 70 Secs at 70˚C, the solu-
tion was quenched in an ice bath and made up to 500 µL
with a 1:1 mixture of HPLC mobile phases A(0.05 M
NaH2PO4/Methanol adjusted to pH 6.3 with 2N NaoH,
1:1) and B (acetonitrile/ H2O, 8.2). A 20 µL aliquot was
injected in duplicate onto aLichrosorb®5 µm RP-18250
MM × 4.6 MM column on a varian vista®5500 HPLC
with a varian vista® CDS-401 data system. An 11 min
linear geadient of о to 100% B followed by a 2 min pla-
teau was run at a flow rate of 1 mL/min. the derivatized
fumonisins were detected by their fluorescence at 490
nm after excitation at 450 nm (detector: water® model
420-E) are agent blank produced two main peaks at 6.3
min and 12.3 min. Concentrations were determined by
refernce to calibration curves of fumonisin isolated in our
laboratory (Miller et al., 1994).
2.2. HPLC Analysis and Method Development
The HPLC system consisted of a ternary solvent pump
(Gynkotek model 480), auto sampler (Gynkotek Gina
50), decade electrochemical detector with a glassy carbon
electrode (Antec) and a diode array detector (Gynko-
tek340S). Gynko soft software V5.60 was used to control
the HPLC system and for data acquisition and analysis.
The equipment was supplied by Dionex Softron (Idstein,
Germany). Three columns, i.e. Multosphere C18 (3 μm;
Copyright © 2011 SciRes. AJPS
Mango Malformation: I. Toxin Production Associated with Fusarium Pathogens
278
125, 4 mm ID), Phenomenex Synergy MAX-RP C12
80A with TMS end-capping (4 μm; 150, 4.6 mm ID) and
Phenomenex Synergi Polar RP (ether linkedphenyl phase
with polar end-capping) were tested for the chroma-
tographic separation of the above-mentioned substances.
The Multosphere column was purchased from CS, Langer
wehe, Germany and Phenomenex, Aschaffenburg, Ger-
many supplied the Phenomenex columns. Peak identity
was determined by means of retention time and UV
spectra that were recorded for all samples in the range
200 - 400 nm.
3. Result and Discussion
Biochemical tests were carried out for studying the pro-
duction of toxins by Fusarium pathogens involved in the
causation of malformation. Eight fungi viz. F. subgluti-
nans, F. solani, F. oxyspoum, F. sterilihyphosum, F. pro-
liferatum, F. moniliforme, F. avena and F. chlamydspore
were tested using susceptible Sadekia cultivar as inocu-
lated as apical injection with culture filtrate (Table 1).
Data pertaining to artificial inoculations revealed that
effort to produce disease by apical injection with culture
filtrate. For Fusarium subglutinans proved to be the
dominant fungus with 100% sample’s infection. Fungi F.
oxysporum, F. sterilihyphosum and F. proliferatum
showed moderate infection in induced typical malforma-
tion symptoms in inoculated mango seedlings.
Toxin production by Fusarium spp: determination
of moniliformin and fumonisin
The ability of different Fusarium isolates to produce
moniliformin was determined by grown the strains on
liquid culture media (Table 2 and Figure 1). Monili-
formin was the secondary metabolite mostly produced by
the in vitro cultures of the Fusarium isolates analyzed
Table 1. Comparative virulence of selected Fusarium iso-
lates on inoculated mango cv. Sedekia seedlings.
Injection buds with culture filtrate
Treatment Disease incidence % Disease severity
F. subglutinans 100 4.0
F. solani 0.0 0.0
F. oxyspoum 25.0 0.3
F. sterilihyphosum 25.0 2.0
F. proliferatum 25.0 1.3
F.moniliforme 0.0 0.0
F.avena 0.0 0.0
F.chlamydspore 0.0 0.0
LSD 12.0 0.5
Table 2. Moniliform and fumonisin production by Fusa-
rium isolates in liquid media.
Concentration of
fumonisin µg/ml
Concentration of
moniliformin µg/ml
Isolates
ND 6.30
F. oxysporum
0.64 4.10
F. proliferatum
.
0.16 3.88
F. avenacum
ND 0.60
F. chlamydospore
0.50 8.51
F. subglutinans
ND 1.10
F. sterilihyphosum
0.10 ND
F. solani
8.30 6.90
F. moniliforme
ND = not detected.
using HPLC. A evaluated for moniliformin production,
seven isolates were toxin producers, the production lev-
els ranging from 0.51 to 8.90 µg/ml. There were found
differences in moniliformin production among the Fusa-
rium dependent on the species.
The higher levels were produced by F. subglutinans
(8.51 µg/ml). Moderate concentrations of moniliformin
was produced by F.moniliforme (6.90 µg/ml), F. ox-
ysporum (6.30 µg/ml), F. proliferatum (4.10 μg/ml) and
F. sterilihyphosum (1.10 μg/ml). The lowest concentra-
tion was obtained by F. chlamydospore (0.60 µg/ml).
Fumonisin was the secondary metabolite mostly pro-
duced by the in vitro cultures of the Fusarium isolates
analyzed using HPLC (Table 2 and Figure 2). A evalu-
ated for total fumonisin production (FB1 + FB2), seven
isolates were toxin producers, the production levels
ranging from 0.10 to 8.30 µg/ml. There were found dif-
ferences in fumonisin production among the Fusarium
dependent on the species. The higher levels were pro-
duced by F. moniliforme (8.30 µg/ml). Moderate con-
centrations of moniliformin was produced by F. prolif-
eratum (0.64 µg/ml) and F. subglutinans (0.50 µg/ml),
The lowest concentration was obtained by F. solani (0.10
µg/ml).
F. subglutinans and F. moniliforme appear to have
high level and different toxin profiles. This suggests that
thèse fungi can cause malformation, necrosis via the
production of phytotoxic metabolites. Some isolates of
the species i.e. F. subglutinans, F. sterilihyphosum, F.
oxysporum and F. proliferatum that were highly toxic to
mango seedlings and produce moniliformin, suggesting
that this toxins can be involved. Fusarium mycotoxins
continue to occur in agricultural commodities as a result
of fungal contamination hence presenting serious animal
and human health problems. Various Fusarium species
Copyright © 2011 SciRes. AJPS
Mango Malformation: I. Toxin Production Associated with Fusarium Pathogens
Copyright © 2011 SciRes. AJPS
279
have recently been found to produce several mycotoxins
as moniliformin [9,11]. Moniliformin is formed in many
cereals by a number of Fusarium species that include,
besides F. moniliforme, F. avenaceum. F. subglutinans,
F. proliferatum and others [4]. Malformin-like sub-
stances are somehow involved in the causation of mal-
formation, with malformin-stimulated ethylene produc-
tion [10] causing a hormonal imbalance and conse-
quently disturbed metabolism inducing malformation.
Related, recently described species that have been
Figure 1. HPLC of moniliformin production by Fusarium isolates in culture media.
Mango Malformation: I. Toxin Production Associated with Fusarium Pathogens
280
Figure 2. HPLC of fumonisin production by Fusarium isolates in culture media.
shown to produce fumonisins are F. dlamini, F. napi-
forme and F. nygamai [11]. FB1 concentrations formed
by F. moniliforme and F. proliferatum usually exceed
those of FB2. The co-occurrence of these toxins may
have synergistic harmful effects on the overall toxicity of
the isolates, and may be a greater problem than initially
lyte anticipated. Different Fusarium species dominated at
different stages of development and a good correlation
Copyright © 2011 SciRes. AJPS
Mango Malformation: I. Toxin Production Associated with Fusarium Pathogens281
was found between fumonisins and the presence of F.
moniliforme and F. proliferatum. The occurrence of very
high levels of fumonisin B# in some samples was corre-
lated with the presence of strains producing abundant
fumonisin B# in the laboratory [16].
4. Acknowledgements
This manuscript funded from the project “New applied
approaches to promote productivity and Quality of some
fruit crops (Mango)” National Research Centre, 2007 to
2010.
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