The role of pectinase enzyme in the development of soft rot caused by Pseudomonas fluorescens in the purple variety of onions (Allium cepa)

African Journal of Microbiology Research Vol. 3 (4) pp. 163-167 April, 2009
Available online http://www.academicjournals.org/ajmr
ISSN 1996-0808 ©2009 Academic Journals



Ful Length Research Paper

The role of pectinase enzyme in the development of
soft rot caused by Pseudomonas fluorescens in the
purple variety of onions (Allium cepa)

Simbo Aboaba

Dept of Botany and Microbiology, University of Lagos, Lagos, Nigeria. E-mail: simboaboaba@yahoo.com.

Accepted 9 March, 2009

Pectic enzyme production was induced in vitro by culturing the pathogen (P flourescens) in minimal
salt medium containing citrus pectin as the only carbon source and in vivo by inoculating the pathogen
into healthy onion bulbs. The spent broth and onion rot extract were obtained and enzyme detection
was done using viscometric method and cup plate assay. The crude enzyme was partially purified by
precipitating with ammonium sulphate and dialyzed against water at 4oC for 24 h before use. The
activity of the purified enzyme was determined based on the macerating ability on Onion and Potato
discs and later by estimation of reducing sugars (galacturonic acid) level. The type of cleavage reaction
of the enzyme was determined using the thiobarbituric acid reaction and the presence of antimicrobial
substance in the rot tissue was also investigated. The pathogen produced a hydrolytic polygalactu-
ronase in vitro and in vivo. The purified enzyme showed appreciable activity with all the assay methods
used. Activity was highest at pH 5 in the culture filtrate sample and at pH 4 in the rot extract. Maceration
was observed in potato and onion discs. The detection of increasing concentration of polyphenols in
the rot tissue during rot development demonstrated the plant’s response to the pathogen.

Key words: Onion, maceration, pectinase, polyphenol and rot.


INTRODUCTION

A characteristic feature of many phytopathogenic organi-
cosidic bond is split, i.e. hydrolytic or trans-eliminative
sms is their ability to produce an array of enzymes capa-
cleavage (Bateman, 1966). The activity of the type of
ble of degrading the complex polysaccharides of the plant
induced enzyme may be influenced by environmental
cel wal and membrane constituents. These enzymes are
factors.
usual y produced inductively and are extracel ular, highly
It has been found that Botrytis cinerea was capable of
stable and present in infected host tissue (Arinze, 1978).
producing different 0 – 1.4 pectic-degrading enzymes in
The most widely studied of these enzymes are those that
vitro depending on the pH of the culture medium (Damle,
can degrade pectic substances and cel ulose (Arinze,
1952).
1978). The cementing substances in vegetables induce
The production of pectic enzyme in vivo is usual y pro-
the formation of pectinase which hydrolyze pectin to pro-
ven by the removal of infected tissue from the sick plant,
duce a mushy consistency (Jay, 2005).
purification of the crude enzyme and determination of its
The involvement of pectic enzymes in the degradation
ability to reproduce disease symptoms in a healthy plant.
of pectic constituents of cel wal s and of the middle lam-
Evidence of pectinase activity can sometimes be obtai-
el a of plant tissues has been reported for diverse types
ned microscopical y as the invaded cel s wil be seen to
of diseases such as soft rot, dry rot, wilts, blights and leaf
separate along the line of the middle lamel a and subse-
spots which are caused by pathogenic agents such as
quent loss of their staining ability (Woods, 1972). Physi-
fungi, bacteria and nematodes (Bateman and Mil ar,
cal y, this manifests as tissue maceration as injury and
1966). Pectic enzyme activities of bacteria have been
death of unplasmolysed cel s would have occurred.
reported during rot development in onions (Obi and Ume-
Bashan (1974) found that polygalacturonase lyase from
zurike, 1981). In most cases, pectic substances are the
Erwinia chyrsanthemi, purified to homogeneity, caused
main inducers in culture media and they can be differen-
cel death and maceration in disks of potato tissue.
tiated based on the mechanism by which the 0 - 1.4 gly-
Plants however, resist invasion of pathogenic agents

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164 Afr. J. Microbiol. Res.







through different mechanisms. One of these is the prod-
also performed, as described by Oguntuyo (1981). Agar medium
uction of antimicrobial substances, usual y phenolic com-
containing 2% pectin in 0.1 M citrate buffer was prepared. Wel s
pounds. There is evidence that phytoalexins are produ-
were made in the agar medium into which the enzyme sample was
ced during soft rot development of sweet potato caused
pipetted. The development and the diameter of a white precipitate
surrounding the wel within 5 min was used as a measure of enzy-
by Botryodiplodu theobromae and other fungi (Arinze,
me activity. Autoclaved enzyme samples were used as control.
1978). In this study, the ability of Pseudomonas fluore-

scens, a plant pathogen implicated in the soft rot of onion

bulbs to produce pectic enzymes in vitro and in vivo was
Effect of pH on enzyme activity
investigated.


The effect of pH on enzyme activity was determined by performing

the enzyme assay with reaction mixtures of which the pH ranged
MATERIALS AND METHODS
from 4 - 9. The enzyme activity was expressed as viscometric units.


Preparation of crude enzyme sample in vitro


Thiobabituric acid reaction
The method of Arinze (1978) was used. Briefly, P. flourescens (109

cfu/ml) was inoculated into conical flasks that contained minimal
The reaction mixture was prepared as described above and incu-
salt medium of the fol owing composition: (NH4)2SO4, 2.0 g; MgSO4,
bated at 25°C for 1 h. Fol owing incubation, 2.5 ml of a mixture
0.2 g; NaCl, 5.0 g; Ca(NO3)2.2H2O, 0.2 g; FeSO4, 0.01 g; K2PO4,
comprising 2.5 ml of 1 NHCl and 5 ml of 0.04 M thiobarbituric acid
5.5 g; KH2PO4, 0.5 g; 2% citrus pectin and 1000 ml distil ed water.
was added and the reaction mixture was mixed thoroughly. The
The flasks were incubated in a rotary shaker (60 rpm) at 25°C for 7
capped test tube was then placed into a water bath for 30 min,
days. The cultures were then filtered through a mil ipore filter (pore
cooled and the absorbance was read at 515 ?m for pectin hydro-
size of 45 µm) and the filtrate was dialysed against distil ed water at
lase activity and at 550 ?m for transeliminase activity. An autocla-
4°C for 24 h using a visking cel ulose tube, before being stored at -
ved enzyme sample was used for blank reading.
20°C.




Preparation of crude enzyme sample in vivo
Macerating enzyme activity


Healthy onion bulbs were surface sterilized according to the method
The ability of the enzyme samples to separate cel s in the tissue
of Oguntuyo (1981). Discs (0.5 cm in diameter) were cut on the first
was determined in a histological study using potato and onion tis-
leave base of the bulb using a sterile cork borer. P. fluorescens (109
sues, as described by Oguntuyo (1981).
cfu/ml) was introduced into the cavities using a sterile disposable

syringe. The bulbs were placed in sterile plastic jars and incubated

at room temperature for 7 days. The rot extract was obtained by
Release of reducing groups
removing the rot tissue and mixing it with 0.1 M phosphate buffer

(pH 7), containing 0.2 M NaCl and 0.001 M dithiothreitol in a ratio of
The method of Nelson and Somogyi (1944) was used to determine
1:10 (tissue: buffer). NaCl was used to extract the protein in the
the split of the 0 - 1.4 glycosidic linkages in a pectic substance by a
tissue and dithiothreitol to prevent oxidation by oxidative enzymes
hydrolytic reaction and the release of reducing groups were
present in the tissue (Arinze, 1978). The tissue was homogenized
determined both quantitatively and qualitatively. The results were
and filtered under vacuum. The sample was then dialyzed and
obtained from a standard curve of galacturonic acid (5 – 50 µg/ml).
stored as described above.
The affinity of the substrate for the enzyme both in vitro and in vivo,

and the ability to release free galacturonic acids were determined.

Enzyme purification



To purify the enzyme, ammonium sulphate was added to the pre-
Determination of total polyphenol
viously prepared in vitro and in vivo enzyme samples to obtain satu-

ration levels of 40, 60, 80, 90 and 100% (Green and Hughes,
Total polyphenol was obtained from healthy and diseased onion
1955). Addition of salt was carried out at 25°C in a water bath. The
tissues by homogenizing the tissues with methanol. A filtrate was
precipitate obtained at each saturation level was removed after
obtained under vacuum to which the same volume of petroleum
al owing it to stand for 30 min fol owed by centrifugation at 10 000 g
ether was added. This process was repeated four times and the
for 30 min at 4°C. The supernatant was used for the next higher
ether was evaporated to 10 ml in a rotary evaporator (Model Bibby,
saturation level. The precipitate at each level was dissolved in a
RE 200). The extract was final y shaken with the same volume of
smal volume of water and dialysed against water at 4°C for 24 h
petroleum ether and stored at 4°C prior to use. For polyphenol
before being stored at -20°C.
estimation, the method of Oguntuyo (1982) was used. The metha-

nol extract (1 ml) was diluted in 7.5 ml of distil ed water in a test

tube and 1 ml of saturated Na2CO3 and 0.5 ml of Folin-Denis reag-
Enzyme activity assays
ent was added. The mixture was left to stand for 1 h after which it

was centrifuged at 10 000 × g for 5 min and the precipitate was
A viscometric method was used as described by Oguntuyo (1981).
removed. The extinction value was read at 700 ?m (Zeiss PM6
Viscometers were calibrated against water, the flow of which repre-
digital spectrophotometer) against a reagent blank prepared with
sented 100% viscosity reduction. The autoclaved enzyme served
methanol. The result was expressed as µg tannic acid equivalent/g
as the control. Enzyme activity was expressed in viscometric units
fresh weight. The effect of incubation period (in days) and P.
defined as 100/t where t is time in minutes taken for 50% loss in
fluorescens inoculum concentration on the polyphenol content were
viscosity of the reaction mixture. In addition, cup-plate assays were
likewise assessed.

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Aboaba 165






(Figure 1). The development of a white precipitate in the
cup-plate assay also confirmed the presence of the
enzyme produced by P. fluorescens (result not shown).


Classification of the enzyme

The thiobarbituric acid reaction showed that the enzyme
is a hydrolytic polygalacturonase, as there was maximum
absorbance at 515?m. The cup-plate assay and the
increasing concentration of reducing sugars (galacturonic
acid) in the culture filtrate confirmed this. The hydrolase
was active at pH 5 for the culture filtrate and at pH 4 for
rot extract, as determined by viscometry (Figure 2).


Macerating activity


The maceration of onion and potato discs within 1 h

showed the activity of the enzyme (Plate 1).
Figure 1. Pectic enzyme activity over a 10 day incubation period.




Release of reducing groups


Enzyme activity of the in vitro enzyme sample exhibited
maximum activity at a substrate concentration of 0.2%
(w/v) pectin (14.1 µm km value) and 0.5% (w/v) pectin
concentration (22.5 µm km value) for the in vivo enzyme
sample (Figure 3)


Polyphenol detection

Polyphenols were detected in healthy and infected onion
tissues. The rot tissue from artificial y inoculated bulbs
showed an appreciable increase from 59.28 µg tannic
acid equiv/g at time of inoculation to 112.20 µg tannic
acid equiv/g tissue after 7 days of incubation (Figure 4). It
was also observed that the polyphenol content increased
as the P. fluorescens inoculum size increased (43 µg tan-
nic acid equiv/g with 102 cfu/ml to 110 µg tannic acid

equiv/g with 1010 cfu/ml) (results not shown).


Figure 2. Effect of pH on enzyme activity.


DISCUSSION



The ability of P. fluorescens, implicated in the soft rot of
RESULTS
onion bulbs to produce pectic enzymes in vitro and in

vivo was investigated. The bacterium produced a protein-
Detection of pectic enzyme activity
eous substance in the culture medium containing pectin

as the only carbon source. Reducing sugars were also
In vitro studies showed that a pectic enzyme was induc-
detected in the medium after incubation. This gave an
tively produced by P. fluorescens in culture medium con-
indication of the presence of a pectic enzyme that sho-
taining citrus pectin as shown by viscometry. Optimal
wed optimal activity at 2% (w/v) pectin concentration and
activity was achieved with pectin as the sole carbon sour-
in rot tissue extract during in vivo studies. The viscome-
ce. The enzyme was also detected in the rot tissue extra-
tric assay confirmed this and suggests a role for the pec-
ct as it could reduce viscosity of pectin after 30 min. The
tic enzyme in soft rot development in plants.
activity of the enzyme produced in vitro and in vivo every
Pectic substances are the primary constituents of the
24 h during a 10 day incubation period showed that there
middle lamel a and are structural elements in the primary
was an initial increase, fol owed by a slight decrease and
cel wal and microorganisms therefore require specific
then an increase towards the end of the incubation period
enzymes for the degradation of cel wal to penetrate it.

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166 Afr. J. Microbiol. Res.









Figure 3. Rate of release of free galacturonic acid at different
substrate concentrations.





Figure 4. Polyphenol content in infected tissue.




In this study, the purified P. fluorescens enzyme was

Plate 1. Photomicrograph showing intact cel s (top slide),
found to be a hydrolytic polygalacturonase as there was
col apsed cel s (middle slide) and area of advancing lesion
clearing around wel s in the cup-plate assay and it
(infected tissue and intact cel s) (bottom slide).
showed macerating activity on potato and onion discs.

This hydrolase was active at pH 5 in the culture filtrate


and at pH 4 in the onion rot extract. Ulrich (1975) found
Plant pathogens have evolved a means to recognize the
an endo-polygalacturonase produced by B. cepacia to
chemical structure in the plant cel wal and elaborate
have an optimum pH range of 4 – 4.6. The increasing
enzymes on the various cel wal components (Bateman
amount of reducing sugars in the medium during growth
and Basham, 1976). Many pectolytic bacteria have been
confirmed the presence and activity of the enzyme, a
implicated in soft rot diseases of plants. P. fluorescens
manifestation of its involvement in soft rot development.
was found to produce a pectic enzyme causing storage
This was not surprising as enzyme activity was found to
soft rot of potato tubes. Obi and Umezurike (1981) also
increase during growth in the shake cultures and in the
reported the elaboration of a pectic enzyme by Burkhol-
onion bulbs as the storage period increased. Inoculation
deria (Pseudomonas) cepacia in diseased onions. In
size was also found to be proportional to extent of rot
addition, such enzymes are produced by a variety of phy-
development. The histological study of rot tissue at areas
topathogens (Doyle et al., 2001).
of advancing lesions showed complete disintegration of

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Aboaba 167






the tissue (Plate 1). Pectic enzymes that have the ability
Bel TA, Etchel s JL, Wil iam CF, Porter WL (1962). Inhibitors of
to split the 1, 4 bonds between the galacturonic acid
pectinase and cel ulase by plants. Botan Gaz 123: 220-223.
moieties in the pectic fraction of the cel wal remain the
Damle VP (1952). Enzymatic study of certain parasitic fungi. J. Indian
Botan Soc. 31: 13-35.
only enzyme confirmed to cause plant tissue maceration.
Doyle MP, Beuchat LR, Montvil e TJ (2001). Food Microbiology,
The polygalacturonate lyase from Erwinia chrysanthemi
Fundamentals and Frontiers. 2nd Edition. ASM Press. pp. 593-627.
caused maceration and cel death in discs of potato
Green AA, Hughes WL (1955). Protein fractionation on the basis of
tissue (Basham, 1974).
solubility in aqueous solution of salts and organic solvents. In.
Methods of Enzymology. Academic Press, New York pp. 67-146.
The role of pectic enzymes in many plant diseases can-
Huang MC, Agris MC (1979). Effect of scar skin and dapple apple
not be over-emphasized (Oguntuyo, 1981; Obi and Ume-
diseases on certain phenolic compounds in apple. Phytopathol. 69:
zurike, 1981). The control of such conditions therefore
35-40.
can be solved by appropriate enzyme inhibition strate-
Jay ME (2005). Microbial Spoilage of Fruits and Vegetables. In: Modern
Food Microbiology. 4th Edition.CBS Publishers, New Delhi. pp. 187-
gies. It is known that plants produce antagonistic subs-
198.
tances as defense mechanisms against plant pathogens.
Nelson N, Somogyi M (1944). A photometric adaptation of the Somogyi
The ability to overpower the pathogen depends on many
method for the determination of glucose. J. Biol. Chem. 153: 375-
factors. Extracts of plant tissues have been found to
380.
Obi SK, Umezurike GM (1981). Pectic enzyme activities of bacteria
contain substances known to inhibit pectic enzymes in
associated with rotted onions (Al ium cepa). Appl. Environ.
vitro (Bel et al., 1962). These substances have been
Microbiol., 42 (4): 585-589.
found to be mostly phenols and their oxidation products.
Oguntuyo OO (1982). Studies on microbial spoilage of onions (Al ium
Their free radicals react readily with biochemical consti-
cepa L). Ph.D thesis, University of Lagos.
Omidiji O (1985). The toxicity of phenolic extracts from purple variety
tuents such as –NH2 and OH groups, thereby preventing
onions to pathogenic and non pathogenic bacteria. Biologia Africana.
or limiting some infections (Mahadevan et al., 1965).
2: 28-35.
Extracts of plant tissues have been found to contain
Stratil VL, Klejdus B, Kuban V (2006). Determination of total content of
substances known to inhibit pectic enzymes in vitro. Omi-
phenolic compound and their antioxidant activity in vegetables-
evaluation of spectrometric methods. J. Agric. Food Chem., 54: 607-
diji (1985) reported that phenolic extract from the inner
616.
scales of purple onions can inhibit growth of pathogenic
Ulrich JM (1975). Pectic enzymes of Pseudomonas cepacia and
P. fluorescens in broth and on agar plates.
penetration of polygalacturonase into cel s. Physiol. Plant. Pathol.
In this study, polyphenols were detected in healthy and
5(1): 37-44.
Anurag PRS, Sanwal GG (2009). Microbial pectate lyases: charac-
diseased tissues, but were at a higher concentration in
terization and enzymological properties. World J. Microbiol.
infected tissue. Huang and Agrios (1979) had found hig-
Biotechnol. 25(1): 1-14.
her amounts of phenols in infected than healthy tissue in
skin disease of apples. It is also believed that the change
in colour that accompanies injury or infection may be due
to the presence of polyphenols. Onion juice and vapors
are one of the best characterized antimicrobial systems in
plants. They contain the phenolic compounds protocate-
chnic acid and catechol, which are believed to contribute
to their antimicrobial activity (Doyle et al., 2001).
In conclusion, it is clear from this study that although P.
fluorescens had the ability to produce pectic enzyme
used for invasion of healthy tissue, it could not be cont-
rol ed by the antimicrobial polyphenols of the plant tissue.


REFERENCES

Adam MR, Moss MO (2008). Microbiology of Primary Food
Commodities. In. Food Microbiology. 3rd Edition. RSC Publishing,
UK. pp. 119-157.
Arinze AE (1978). Studies on the storage rot of sweet potato (Ipomea
batatas L and Lam) by Botryodiplodia theobromae Pat and other
fungi. PhD thesis, Imperial Col ege, London
Basham HG (1974). The role of pectolytic enzymes in the death of plant
cel s. Ph. D thesis, Cornel University, Ithaca, New York.
Bateman DF (1966). Hydrolytic and trans-eliminative degradation of
pectic substances by Fusarium solani f phaseoli. Phytopathol. 56:
238-244.
Bateman DF, Mil ar RL (1966). Pectic enzymes in tissue degradation.
Ann. Rev. Phytopathol. 4: 119-146.
Bateman DF, Basham HG (1976). Degradation of plant cel wal s and
membranes by microbial enzymes. Physiol. Plant. Pathol. 4: 316-35.

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