Mango malformation: II. mangiferin changes associated with <i>fusarium</i> pathogens

doi:10.4236/as.2011.23038

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Vol.2, No.3, 291-296 (2011)

doi:10.4236/as.2011.23038

Agricultural Sciences

Mango malformation: II. mangiferin changes associated

with fusarium pathogens

Wafaa M. Haggag1*, Mahmoud Hazza2, Mohamed E. Abd El-Wahab1

1Department of Plant Pathology, National Research Center, Dokki, Cairo, Egypt;

2Science Faculty, Botany Department, Banha University, Egypt; Corresponding Author: [email protected]

Received 16 February 2011; revised 23 May 2011; accepted 25 July 2011.

ABSTRACT

Mangiferin (1,3,6,7-tetrahydroxy xanthone-C2-b-

D-glucoside) promoted vegetative growth and

exhibited inhibitory role on the occurrence of

malformation. Mangiferin changes associated

with mango malformation pathogens were fol-

lowed after inoculated mango seedlings (three

years) with malformation pathogens i.e. Fusa-

rium subglutinans, F. sterilihyphosum, F. ox-

ysporum and F. proliferatum. Mangiferin re-

mained at lower level in leaves of malformed

shoots as compared to healthy one. The floral

malformation was observed to be associated

with the reduction of mangiferin. S trong positive

correlations between mangiferin activity and

malformation incidence were observed. Mangiferin

level at panicle initiation may give a possible

estimate of malformation incidence in mango.

Keywords: Fusarium; Mangiferin; Mango

Malformation

1. INTRODUCTION

Mango (Mangifera indica L.) is the most important

fruit crop in Egypt. Mango ranks third in exports after

citrus and grapes. Egypt produces 232,000 tones of

mango annually and exports moderate amount (about

1500 tones) annually to 20 countries in near east and

Europe (FAO, 2007).

Mango Malformation is one of the most destructive

mango diseases [1]. Accumulation of mangiferin and

toxic metabolites of Fusarium moniliforme has been

suggested to be responsible for the malformation disease

of mango (Mangifera indica L.). Differences in the low-

and medium M, phenolic and steroidal compounds in

healthy and malformed florets of Mangifera indica, the

latter infested with Fusarium moniliforme var. subg luti-

nans are reported. Mangiferin, which are not normal

constituents of healthy florets were found in substantial

amounts in the diseased florets. Both mangiferin and 1,3,

6,7-tetrahydroxyxanthone were found to be potent anti-

fungal agents[2]. Accumulation of mangiferin, a natural

metabolite of Mangifera indica at the site of differenti-

ating buds, influences the changes from reproductive to

vegetative growth. Mangiferin in high concentration

suppressed the activity of peroxidase, catalase, α-amy-

lase and IAA-oxidase. Polyphenoloxidase and invertase

showed increased activity. Mangiferin accumulation

increased the rate of photosynthesis but lowered those of

transpiration and respiration. Mangiferin treatment in-

creased the contents of chlorophyll, carbohydrates, total

nitrogen, protein nitrogen, nucleic acids (RNA and

DNA) and indole-3yl-acaetic acid (IAA) [3]. Mangiferin

acts as a phytoalexin-like compound in M. indica. Non-

pathogenic strains of F. moniliforme isolated from maize

cob and banana fruits induced more mangiferin synthesis

than the pathogenic isolate from malformed mango

shoots in vivo. Activity of the mangiferin degrading en-

zymes, polyphenoloxidase of non-pathogenic strains of

F. moniliforme was higher than that of the pathogenic

strain. In vitro tests also confirmed rapid and massive

degradation of mangiferin by non-pathogenic strains.

Biochemical events associated with elicitation, degrada-

tion and accumulation of mangiferin determine the host

specificity of F. moniliforme in M. indica [4]. At the

stages of bud pre-emergence and initial differentiation,

mangiferin content in the leaves from the axils of which

healthy and malformed buds emerged did not show sig-

nificant differences although in the latter one mangiferin

content was higher. But in the fully developed panicles

at pre-blooming stage mangiferin content of the leaves

attached to the malformed panicle was in traces and sig-

nificantly lower than that of healthy one. Mangiferin

content in malformed panicles also did not differ sig-

nificantly at the initial stage. But the difference become

highly significant after influx of mangiferin from axil

leaves to the fully developed panicle at pre-blooming

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stage. Mangiferin due to its vegetative growth promoting

property tilted the hormonal balance of malformed pani-

cles in favour of vegetative growth resulting into trans-

formation of malformed florets into green leafy struc-

tures [5]. The mangiferin treated strains produced more

aerial hyphae but less pigment. Prolonged mangiferin

treatment affected the saprophytic ability of the strains

but improved its parasitism. The significance of mangiferin

induced changes in evolving the host-specific strain of

F. moniliforme of M. indicia lies in showing that an e-

cological disadvantage of survival in one niche (sapro-

phytic) may prove advantageous in another (parasite)

[8]. The fungal and mite populations were initially posi-

tively related to the mangiferin content and the disease

incidence. Further increase in mangiferin content re-

duced the fungal and mite populations [6]. The symp-

toms are the combined effects of aberrant host metabo-

lites produced in response to infection and phytotoxins

secreted by the pathogen. The pathogen has been identi-

fied as a physiological race of F. moniliforme (F.

moniliforme f. sp. mangifera) developed due to interac-

tion with the host metabolite, mangiferin for a prolonged

period. The disease cycle is greatly influenced with the

biochemical changes in the host tissues. Host metabo-

lites also effect the seasonal variation of population of

the pathogen Vis-a-Vis disease incidence. Proper bal-

ance of mangiferin and the Fusarium population is re-

quired for disease manifestation. Either by suppressing

or avoiding elicitation of hypersensitive reaction of the

host at the initial stage of infection, colonization by the

pathogen and subsequent symptom production could be

effected [3].Thus, objective of the present study is to

evaluate mangiferin change in mango plant due to infec-

tion with pathogenic fungi.

2. MATERIALS AND METHODS

Mango (Mangifera indica L.) seedling cv. Sedekia

(three years old) was inoculated with 105 colony form-

ing units of Fusarium spp. i.e. F. subglutinans , F. ster-

ilihyphosum, F. oxysporum and F. proliferatum, the

causal organisms of mango malformation as inoculated

soil. Sterilized water was used as a control. Transplanted

seedlings were monitored for development of malforma-

tion. At the end of the experiment (120 days), all surviv-

ing seedlings were examined for apical disease symp-

toms. Samples for mangiferin study were taken from

leaves one-two cm below the tip of young seedlings.

2.1. Isolation of Mangiferin

The leaves were macerated with acetone in a high

speed blender. After 4 h, the mixture was filtered and the

solvent was removed under pressure. The extractions

were poured into 100 ml distilled water and the suspen-

sion was successfully extracted with ether and ethyl

acetate. At the interface of the aqueous ethyl acetate,

brown solid mass was precipitated and this was collected

by filtration. The identity of the brown residue was de-

termined as mangiferin according to Ghosal et al. [2].

Subsequently, a small volume of the extract was filtered

through 13 mm membrane filter (0.45 µm; polypropyl-

ene) directly into HPLC sample vial for injection with-

out further dilution.

2.2. Mangiferin Analysis

Extract were done using HPLC system (prominence

Lc, Shimaduz, Kyoto, Japan) equipped with a Lichro-

spher 100RP-18 (5 µm) column (250 mm × 4 mm,

Merck, Darmstadt, Germany), a C18 guard column and a

photoiode-array detector (Shimadzu, SPD-M20A). THE

elution system (0.8 mL·min–1) involved 2 mM phospho-

ric acid in water (eluent A) and MeOH (eluent B). The

gradient was as follows: 0 min, 25% eluent B,0 - 40 min,

80% eluent B, linear the retention time and spectral

characteristics of each sample were compared to a ref-

erence sample of mangiferin (Extrasynthese, Lyon,

France) [7].

2.3. HPLC Analysis and Method

Development

The HPLC system consisted of a ternary solvent pump

(Gynkotek Model 480), autosampler (Gynkotek Gina

50), decade electrochemical detector with a glassy car-

bon electrode (Antec) and a diode array detector

(Gynkotek 340 S). 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; 125; 4 mm ID), Phenomenex Synergy

MAX-RP C12 80 A 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 chromatographic separation of the above-men-

tioned substances. The Multosphere column was purchased

from CS, Langer-wehe, Germany and Phenomenex, As-

chaffenburg, Germany supplied the Phenomenex col-

umns. Peak identify was determined by means of reten-

tion time and UV spectra that were recorded for all sam-

ples 250 nm. During method development, three solvent

gradients were tested: program I: 0 - 6 min (12% B), 7

min (18% B), 14 min (25% B), 19 min (40% B), 24 min

(50% B), 29 min (12% B) (solvent A = 2% acetic acid in

aqueous solution (v/v) and solvent B = acetonitrile);

program II: 0 min (5% B, 5% C); 4,5 min (6,5% B, 5%

C), 7 min (18% B), 14 min (25% B),19 min (40% B), 24

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min (50% B), 30 min (5% B, 5% C) [solvent A = 2%

acetic acid in aqueous solution (v/v), solvent B = ace-

tonitrileand solvent C = tetrahydrofurane (THF)]; pro-

gram III: identical to Program I except that solvent A

was purified water buffered to pH 4 with citrate buffer.

This program, in combination with the Synergy

MAX-RP C12 column, was used exclusively for electro-

chemical detection. In all cases a flow rate of 1 ml/min

was used. The injection volume was 20 μl for each

analysis and separations were carried out at room tem-

perature. Linear calibration lines for mangiferin were

compiled using standard series of six dilutions between

4.7 and 98.3 µg/ml. The repeatability of HPLC method

was determined by ten injections of the same sample of

extracted. The reproductively of the complete assay was

tested by means of ten sample preparations [8].

3. RESULT AND DISCUSSION

Changes in mangiferin (1,3,6,7-tetrahydroxy xanthone-

C2-b-D-glucoside) C10H10O11 of healthy and malformed

shoots of mango cultivar Sedekia (three years old) was

followed after inoculated with mango malformation

pathogens i.e. F. subglutinans, F. sterilihyphosum, F.

oxysporum and F. proliferatum. Data pertaining to artifi-

cial inoculations revealed that, Fusarium subglutinans

proved to be the dominant fungus with 100% sample’s

infection in inoculated soil (Table 1). Fungi F. ox-

ysporum, F. sterilihyphosum and F. proliferatum showed

moderate infection in induced typical malformation

symptoms in inoculated mango seedling and were re-

isolated. Changes in mangiferin activity in mango seed-

lings cv. Sedekia as response to infection with mango

malformation pathogens i.e. F. subglutinans, F. sterili-

hyphosum, F. oxysporum and F. proliferatum grown

under greenhouse was determined. Mangiferin activity

varied widely amongst different inoculated pathogens.

From the data given in Ta ble 1 and Figure 1, it is clear

that accumulation of mangiferin was maximum (0.96

µg/g–1 FW) in leaves of healthy shoots. Mangiferin re-

duced in mango shoot in response to infection with F.

sterilihyphosum by 0.52 µg/g–1 FW. The amount of

mangiferin is 0.15 µg/g–1 FW due to infection with F.

subglutinis. Mangiferin is detected as 0.12 µg/g–1 FW as

result of infection with F. proliferatum. It is clear that

the least amount of mangiferin is detected as result of

infection with F. oxysporum (0.0039 µg/g–1 FW). Strong

positive correlations between mangiferin activity and

malformation incidence were observed. Mangiferin pro-

moted vegetative growth and exhibited inhibitory role on

the occurrence of malformation. It was also observed

that the healthy had highest activity of mangiferin as

compared to infected ones. Results of the present study

Table 1. Status of mangiferin in mango shoot explants as

influenced by different inoculation with pathogenic

fungi.

Mangiferin (µg/g–1 FW)Malformation (%)

Treatment

0.96 ―

Control

0.12 100

F. subglutinans

0. 52 50

F. sterilihyphosum

0.0039 50

F. oxysporum

0.15 50

F. proliferatum

clearly indicate that level of mangiferin could be consid

ered as a potential biochemical indicator for the malfor-

mation disease of mango. Interaction among Fusarium

moniliforme and colonizing malformed mango panicles,

and mangiferin (1,3,6,7-tetrahydroxyx-anthone C2-ß-D

glucoside), a defensive metabolite of the host plant in

relation to floral malformation, was in- vestigated. The

fungal populations were initially positively related to the

mangiferin content and the disease incidence. Further

increase in mangiferin content reduced the fungal and

mite populations; however, the increase in infection rate

was not affected until the Fusarium population was too

low. The fungal conidia remained adhered to the body

surface of the mites inhabiting malformed panicles, and

its plating on potato dextrose agar showed a trail of fun-

gal colonies along the pathway of its movement.

Tyrolicus casei facilitated the ingress of the fungus into

the host cells while F. moniliforme served as the feed of

T.casei and increased its multiplication [6,9]. Mangiferin

acts as a phytoalexinlike compound in M. indica. Activ-

ity of the mangiferin degrading enzymes, poly-

phenoloxidase of non-pathogenic strains of F. monili-

forme was higher than that of the pathogenic strain. In

vitro tests also confirmed rapid and massive degradation

of mangiferin by non-pathogenic strains. The disease

incidence could be minimized by removing stress factor

(the pathogen) through removal of malformed plant parts

and supplying the malformed plants necessary micronu-

trients by spraying with mangiferin metal chelates

[10,11]. The biochemical significance of the changes in

these constituents, resulting from the hypersensitive re-

sponses in the host specie, is appraised in respect to the

Fusarium pathogens, the causal organisms of mango

alformation. m

W. M. Haggag et al. / Agricultural Science 2 (2011) 291-296

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Control

F. subglutinans

Openly accessible at

W. M. Haggag et al. / Agricultural Science 2 (2011) 291-296

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F. oxysporum

F. sterilihyphosum

W. M. Haggag et al. / Agricultural Science 2 (2011) 291-296

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F. proliferatum

Figure 1. Changes in Mangiferin (1,3,6,7-tetrahydroxy xanthone-C2-b-D-glucoside) C10H10O11 of healthy and malformed shoots of

mango cultivar Seddek when inoculated with Fusarium pathogens.

4. ACKNOWLEDGEMENTS

This manuscript funded from the project “New applied approaches

to promote productivity and Quality of some fruit crops (Mango)” PI:

Prof. Wafaa M. Haggag. National Research Centre, 2007 to 2010.

Openly accessible at

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