Mango Malformation: I. Toxin Production Associated with Fusarium Pathogens

doi:10.4236/ajps.2011.22030

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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)

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: [email protected]

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;

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

Mango Malformation: I. Toxin Production Associated with Fusarium Pathogens

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

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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