Changes in phenol oxidase and peroxidase levels
in cocoyam tubers of different postharvest ages infected
by Sclerotium rolfsii sacc
Summary
Cmde aqueous extracts from the peripheral rot zone of cocoyam tubers
infected by Sclerofium rolfsii sacc were shown to be inhibitory to dialysed
in vivo polygalacturonase (PG) of the pathogen. The PG inhibitory action,
Introduction
In Nigeria, Sclerotium rolfsii sacc has been recorded as
one of the major pathogens of cocoyams [8, 91. It has been
observed that certain varieties of cocoyam tubers were least
susceptible to biodegradation than others particularly at cer-
tain postharvest ages. Many infected plant tissues particu-
larly locally infected and resistant tissue show a common
shift in the metabolic pattern that includes accumulation of
an array of secondary substances and activation of peroxi-
dase and other phenol oxidizing enzymes [3]. Studies of
the phenolic compounds and phenolase enzymes in apple
leaves that are resistant to apple scab (Venturia inaequulis)
have shown that resistance is related to the levels of oxi-
dized phenols. It has been reported that certain cell wall
degrading enzymes are inactivated by oxidized phenolics
[2, 111. These observations suggest that it is in fact the
quinones produced by the action of the phenolase that are
responsible for disease resistance. Increased concentration
of oxidative enzymes like phenol oxidase and/or peroxi-
dase near the infected zones has been reported for bean
leaves [12], potato [15] and has further been implicated in
disease resistance 1141.
In this paper, we report on the role of oxidative enzymes
(phenol oxidase and peroxidase) in the decay of cocoyam
tubers by Sclerotium rolfsii by estimating the levels of phe-
nol oxidase, peroxidase and PG inhibitory activities in co-
coyam tubers of three major postharvest ages infected by
the pathogen. The significance of the distributions is dis-
cussed in relation to susceptibility or resistance to rotting in
cocoyam tubers of different postharvest ages.
Materials and methods
Sources of Fungus and plant materials
The culture of Sclerotium rolfsii used in this investigation was
isolated from cocoyam tubers. The cocoyam tubers, (Colocusiu es-
~ Xunrhosomu sugirrifolium L. S C H O ~ were
culenta L. S C H Oand ) tracts served as the control.
obtained from a local farm at Assah Ubirielem in ORSU Local
Government Area of Imo State, Nigeria.
Department of Botany, University of Port Harcourt, Nigeria
Nahrung 40 (1996) Nr. 1, S.25-27 Q VCH Verlagsgesellschaft mbH. D-69451 Weinheim 1996
N. C. Ohazurike and A. E. Arinze
phenol oxidase and peroxidase activities were higher in cocoyam tubers of
the Xunthosomu sugittifolium varieties than in those of the Coolocusiu
esculentu varieties. The levels of phenol oxidase, pemxidase and PG inhi-
bitory activities also decreased as the postharvest age of the tubers in-
creased.
Preparation of cocoyam rot extract PG of the pathogen
Rot extract PG or in vivo PG of the fungus was prepared and
assayed as previously described [9]. The method of purification
was ammonium sulphate precipitation. The enzyme solution was
fractionally precipitated with solid ammonium sulphate at 25 "C.
The precipitates were dissolved in water and dialysed against dis-
tilled water for at least 16 h at 5 "C.
PG inhibition by crude extracts
One gram tissue from the advancing edge of the rot zone of
each variety of cocoyam tubers was extracted with 10 ml 0.1 M
phosphate buffer pH 7 and 0.2 M NaCI, that is, in the proportion
of I g tissue to 10 ml buffer. The tissue was homogenized in a
blender and filtered through several layers of muslin cloth. The
extract was concentrated to 0.5 ml by means of a rotary evaporator
and made up to 4 m l with water. The effect of the extract on the
activity of the dialysed in vivo PG was determined viscometrically
as described [I]. The reaction mixture consisted of 4 ml 1% so-
dium polypectate (Napp), l ml dialysed rot extract or in vivo PG,
and 1 ml of the aqueous extract. 1 ml water was substituted for the
aqueous extract 1 ml of the aqueous extract. 1 ml water was substi-
tuted for the aqueous extract for the control.
Estimation of phenol oxidase activities
The colorimetric method of estimating the activity of the en-
zyme based on the formation of a dark coloured compound from
catechol was used. Tissue extracts were prepared using a modifica-
tion of the method described [7].The oxidase in the extract was
first activated by adding ammonium sulphate 14,51 in the follow-
ing mixture: 0.2 ml of dialysed rot extract, 0.6 ml of water, 0.2 ml
of M (NH&S04 and 0.1 ml of 0.1 M sodium citrate buffer
pH 7. The mixture stood for 20 min at 20 "C after which 1 ml
0.1 M citrate buffer pH 7 and 1 ml 10 M catechol were succes-
sively added and the volume m d e up to 6 ml with water. The
mixture was transferred quickly to a cuvette and the increase in
absorbance at 495 nm over a 60 s period was determined in a spec-
trophotometer. Enzyme activity was expressed as change in absor-
bance (A) per ml extract per min at 495 nm at 25 "C. Boiled ex-
Estimation of peroxidase activities
Peroxidase assay was based on the same principle as phenol
oxidase [6].After activation. 1 ml of the enzyme mixture was di-
luted to 1000 ml with water and to this was added successively the
0027-769X/96/010.7-0025$10.00+.25/0 25
following: 1 ml sodium citratc-phosphate buffer pH 4. I , 0.4 ml of
0.1% P-phenylenediamine diluted to 6 ml with distilled water. The
iiiixture was transferred to a spectrophotometer and peroxidase ac-
tivity measured as change in absorbance per 0.001 ml extract per
niin at 485 nm.
from cocoyam tubers of the Xunthosorna scrgittifoliuni vari-
eties were more inhibitory on the PG than extracts from
cocoyam tubers of the Colocusia rsculenta varieties.
Phenol oxidase activities

Results Results (Table 2) showed that phenol oxidase activity

Rot extract PG of S. roifsii
l h e rot extract PG was obtained and purified by ammo-
nium sulphate precipitation as described under methods.
Precipitates occurred at 80% and 90% fractions. The dia-
lysed 90% fraction was then used in the phenol oxidase
peroxidase and PG inhibition assay.
was higher in cocoyam tubers of one day postharvest age
in all the varieties than in cocoyam tubers of the three
months and six months postharvest ages. The phenol oxi-
dase activity was also higher in Xutithosomti sagittifoliurn
varieties than in the Colocasia esculentu varieties.
Peroxidase activities

Results (Table 3) showed that peroxidase activity was

PG inhibition by extracts of cocoyam tubers
The results (Table 1 ) indicated that extracts from co-
coyam tubers of one day postharvest age have more inhibi-
tory action on dialysed in vivo PG of the pathogen than
extracts from cocoyam tubers of three months and six
months postharvest ages. From the results also, extracts
highest in cocoyam tubers of one day postharvest age and
least in cocoyam tubers of six months postharvest age. The
difference in peroxidase activity in the two postharvest ages
was more pronounced than in the case of phenol oxidase
activity was highest in the tubers of Xanthosomcr sagirtifo-
lium varieties and least in tubers of the C. rsculmto vari-
eties.

Table 1. Determination of the levels (units] of inhibition of dialysed in vivo PG enzyme of S. rolfsii by aqueous extract from peripheral
zone of rot in varieties of cocoyam tubers of different postharvest ages

C. e.~culentavarieties X. sngittifolium varieties Mean

“Coeoindia” “Akonoke” “Edeu hie” “Edeocha”

~ ~~

One day postharvest 87.5 91.9 107.3 97.3 96

Three months postharvest 79.7 83.8 102.0 89.3 88.7
Six months postharvest 70.8 77.0 97.9 82.5 82.0.5

Mean 79.3 84.2 102.4 89.7

LSD = 72.13 = 0.0.5

Table 2. Determination of levels [units] of phenol oxidase in varicties of cocoyam tubers of different postharvest ages

C. esculenra varieties X . sogirrifoliicm varieties Mean

“Cocoindia” “A konoke” “Edeuhie” “Edeocha”

~

One day postharvest 0.983 1 .0.5 1.85 I .45 1.33

Three months postharvest 0.043 0.98 I .79 1.37 I .27
Six months postharvest 0.732 0.752 0.913 0.813 0.80

Mean 0.89 0.93 0.90 1.21

LSD (0.05) - 5.78

Table 3. Detemiination of the levels (units] of peroxidase in varieties of cocoyam tubers of difterent postharvest ages

C rJculentu varieties X . scigirrifoliurn varieties Mean

“Cocoindia” “Akonoke” “Edeu hie” “Edeoch a”

One day postharvest 41 1.2 422.9 492.8 456.8 440.9

Three rnoiitlis postharvest 390.8 393.7 443.6 428.7 414.2
Six months postharvest 323.4 369.1 420.2 406.7 380.0

Mean 375.1 395.4 452.2 430.7

LSD (0.05) = 61.90

26 Nahmng 40 (1996) Nr. I . S. 25-27

Discussion References

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[2] DEVERALL, B. J., and R. K. S. WOOD, Ann. Appl. Biol. 49
ities in the cocoyam tubers of the Xunrhosomo sugittifolium (1961) 473-487.
varieties than in the Colocasiu esculenru varieties. Changes [3] GOODMAN, R. N., K. KIRALand K. R. WOOD,The biochemis-
in the level of oxidative enzymes particularly phenol oxi- try and physiology of plant diseases. pp. 433. University of
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of oxidation and the ability of the fungus to inactivate the BERGMEYER, pp. 895-897. Academic Press, New York 1963.
oxidases could account for this. The differential distribution [7] MAXWELL, D. P.. and D. F. BATEMAN. Phytopathology 56
(1966) 132-136.
of the activities of these enzymes in varieties of cocoyam
[K] NWUFO,M. I., and A. 0. FAJOLA,J. Root Crops 7 (1981)
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bolic responses that have been associated with the defence [9] OHAZURIKI:., N. C., and A. E. ARINLR,Fitopatol. bras. 17
mechanisms of the hosts. (1992) 393-398.
The studies further revealed decreasing PG inhibition, [IO] OHAZURIKE, N. C., Ph.D Dissertation. University of Port Har-
phenol oxidase and peroxidase activities as the postharvest court, Nigeria, 1994.
age of the cocoyam tubers increased. Previous research [ I I ] PATIL.S. S., and A. E. DIMOND,Phytopathology 57 (1967)
(lo] showed increasing extent of decay of cocoyam tubers 676-682.
as the postharvest age increased and more rot in 1121 RUDOLPH,K., and M. A. STAHMAN, Plant Physiology 41
C. esculentu varieties than in X. sugitrifnliurn varieties. It (1966) 386-394.
[ 13) SACHER, L. A,, G. H. N. TOWERSand D. D. DAVIFS, Pyto-
has also been reported [ 161 that biochemical defences may chemistry 1 1 (1972) 2383.
be present all the time in a healthy plant or they may be 1141 STAHMANN, M. A,, B. C. CLAREand W. WOODRIJRRY, Plant
produced at a particular age and stage of development - Physiology 41 (1966) 1505-1512.
thus accounting for variation in susceptibility with age. In- [ 151 TOMIYAMA, K.,and M. A. SATHAMANN, Plant Physiology 39
creased susceptibility or decrease in oxidative enzyme ac- ( 1 964) 483-490.
tivity with postharvest age involves physiological changes [ 161 WHIVEY,P. J., Microbial Plant Pathology, p. 160. Pica Press,
in the host tissues which make them more susceptible sub- New York 1977.
strates for rapid development of the pathogen. Hydrolysis
of starch to sugar with age is a good example. Increase in Dr. N. C. OHAXRIKE,Department of Biology, A. 1. C. E. Owcrri,
the activities of enzyme associated with carbohydrate Imo Stale. Nigeria
breakdown have been reported in potato tuber slices when
aged artificially I131. Changes in susceptibility are there-
fore more likely to be the result of changes in the metabo-
lism of the host cells as they age and these differ in various Received 5 July 1995
hosts. These possibilities are further being investigated. Revised manuscript received 25 October 1995

Nahrung 40 (1996) Nr. I . S. 25-27 27