Online International Interdisciplinary Research Journal, {Bi-Monthly}, ISSN2249-9598, Volume-III, Issue-V, Sept-Oct 2013

Production of Hydrolytic Enzymes by Garlic (Allium Sativum L.) Bulb

Fungal Pathogens
N.M.Ghangaonkar
Dept. of Botany, C.T.Bora College, Shirur-412210, Pune (MS), India

Abstract
Present study aims to study the production of hydrolytic enzymes like pectinases and
cellulases by the fungi associated with garlic bulbs from storages. These fungi play an
important role in deterioration of the bulbs in storage. The role of pectolytic and
cellulolytic enzymes in degradation of bulbs in storages is an established fact. Hence, for
studying the production of hydrolytic enzymes like cellulases and pectinases, eight fungal
species associated with garlic bulbs from storage were tested on substrate and non-
substrate medium. Among these Aspergillus niger, Alternaria porri, Fusarium
oxysporum, Stemphylium botryosum and Botrytis alli were found to be more vigorous in
producing these enzymes on substrate containing media. This ability of fungi proves their
pathogenecity to garlic bulbs.
KEYWORDS: cellulases, fungi, garlic, hydrolytic enzymes, pectinases

Introduction
Garlic (Allium sativum L.) is most important bulb crop grown in India. In particular bulbs
are associated and infected by number of fungal pathogens in fields as well as in storages
(Chantarasnit and Phanichyakarn, 1986). Due to these associations of fungi bulbs get
significantly damaged and destroyed resulting in bulb rots and bulb necrosis (Georgieva
and Kotev ,1977). Role of extracellular, hydrolytic enzymes produced by these fungi
during the process of bulb deterioration has been considered to be an important ability to
confirm the pathogenecity (Sanz, 1978). The present work deals with the production of
these extracellular hydrolytic enzymes by garlic bulb fungi. In particular production of
pectinase and cellulase enzymes were studied.s

Materials and methods

In order to study the production of pectinases and cellulases fungi were grown on
substrate and non substrate media. Non substrate medium is considered as a control
which is glucose nitrate. In case of pectinase production fungi were grown on pectin
nitrate broth media and for cellulase production they were grown on carboxymethyl
cellulose (CMC) broth medium as substrates media. The culture filtrates of the fungi
were tested for enzyme production by using viscometric assay method. In this method
6ml of 1% substrate medium in 0.2molar. acetate buffer with pH 5.2 and 4ml of enzyme
source(culture filtrates) were taken in Ostwald viscometer and were thoroughly mixed
and incubated at 350c.temperature. The effluent time of mixture at various intervals was
recorded and percent loss of viscosity was calculated. The enzyme activity was expressed
in relative viscosity units which is time required for 50% loss of viscosity of the reaction
mixture. The method was adopted as described by Mahadevan (1974).

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 Online International Interdisciplinary Research Journal, {Bi-Monthly}, ISSN2249-9598, Volume-III, Issue-V, Sept-Oct 2013

Results
Table1: Effect of substrate and non-substrate media on pectolytic enzyme
production in garlic bulb fungi

Fungi Culture filtrates from non- Culture filtrates from substrate

substrate medium. medium.
% viscosity loss after min. % viscosity loss after min.
20 min. 40 min. 60 min. 20 min. 40 min. 60 min.

Alternaria 10.04 17.08 26.03 26.06 51.60 84.03

porri
Aspergillus 19.02 29.06 30.03 50.00 66.06 93.03
niger
Fusarium 20.00 25.02 32.00 60.00 81.02 97.05
oxysporum
Botrytis 30.00 40.02 45.03 76.06 78.07 98.09
alli
Penicillium 19.25 21.01 32.09 39.02 52.08 63.08
sp.
Macrophomina 25.00 35.00 37.08 58.04 64.05 73.06
phaseolina
Cladosporium 18.00 28.01 34.07 43.03 72.02 77.17
alli
Stemphylium 20.00 26.03 32.05 28.20 43.12 66.16
botryosum

Fig: Effect of substrate and non-substrate media on pectolytic enzyme production in

garlic bulb fungi

Effect of Non-substrate medium on pectinase production

50
45
% viscosity loss after min.

40
35
30
25
20
15
10
5
0 20 min
40 min
60 min

Fungi

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 Online International Interdisciplinary Research Journal, {Bi-Monthly}, ISSN2249-9598, Volume-III, Issue-V, Sept-Oct 2013

Effect of substrate medium on Pectinase production

120
% viscosity loss

100
80
60
40
20
0 20
40
60

fungi

Table 2: Effect of substrate and non-substrate media on cellulolytic enzyme

production in garlic bulb fungi
Fungi Culture filtrates from non- Culture filtrates from substrate
substrate medium. medium.
% viscosity loss after min. % viscosity loss after min.
20 min. 40 min. 60 min. 20 min. 40 min. 60 min.

Alternaria 36.08 59.09 64.04 42.04 69.00 70.09

porri
Aspergillus 26.09 37.06 57.00 35.02 43.00 63.02
niger
Fusarium 42.08 48.03 64.04 55.02 57.37 78.62
oxysporum
Botrytis alli 48.06 60.08 65.08 56.01 69.01 74.01
Penicillium sp. 30.03 38.03 48.07 32.00 42.06 54.04
Macrophomina 38.03 57.04 59.02 41.00 61.09 65.02
phaseolina
Cladosporium 31.05 33.07 39.02 42.15 47.13 50.22
alli
Stemphylium 38.22 50.01 55.12 60.18 70.11 75.00
botryosum

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 Online International Interdisciplinary Research Journal, {Bi-Monthly}, ISSN2249-9598, Volume-III, Issue-V, Sept-Oct 2013

Fig: Effect of substrate and non-substrate media on cellulolytic enzyme Production

in garlic bulb fungi.

Effect of Non-substrate medium on cellulase production

70
% viscosity loss after min.

60
50
40
30
20
10
0 20
40
60

fungi

Effect of substrate medium on cellulase production

90
80
% viscosity loss after min.

70
60
50
40
30
20
10
0 20
40
60

fungi

From observations it has been clear that all fungi tested for their ability to produce
enzymes can actively synthesize the cellulases and pectinases on subrate containing
medium than non-substrate medium. In particular cellulases were regrously produced on
by Fusarium oxysporium which was followed by Stemphylium botriosum, Botrytis alli,
and Alternaria porri subsequently. Similarly, pectinase production was maximum done
by Botrytis alli which was followed by Fusarium oxysporium, Aspergillus niger, and then
Alternaria porri. These results confirm their paathogenicity towards garlic bulbs.

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 Online International Interdisciplinary Research Journal, {Bi-Monthly}, ISSN2249-9598, Volume-III, Issue-V, Sept-Oct 2013

Discussion
From results it is clear that cellulase production was induced on carboxymethyl cellulose
(CMC) broth which was previously adopted by Rees and Mandels (1957), Singh and
Husain (1962) and Rees and Levinson (1952). They found maximum production of
enzymes on it as compared to other media. Similarly, pectin nitrate medium was used for
pectinase production as a substrate medium which was previously used by Punde in 1972
for Alternaria sp. Glucose nitrate was used as non-substrate medium and it showd that it
does not induces the enzyme production as was proved by Gupta and Rautela in1966.
They found that presence of glucose in any concentration was inhibitory to enzyme
secretion by Alternaria sp. they also concluded that fungus requires optimum
concentration of pectin from 0.75% to 1.0% for enzyme production which is used in
present studies.
Cellulase production was maximum by Fusarium oxysporium in presence of CMC
medium which was already studied by Sarabhai and Husain in 1904. Stemphylium
botryosum , Botrytis alli, and Alternaria porri also enhanced production of cellulases in
presence of CMC medium. Cladosporium alli and Aspergillus niger could produce
enzyme moderately which was reported by Husain and Rich in1958 and Rees and
Mandels in1957 respectively. They also confirmed that Aspergillus niger was more active
on CMC than on glucose nitrate medium. High Pectinase production was seen in Botrytis
alli which was followed by Fusarium oxysporium in presence of pectin nitrate medium
which was studied by Subramanian in 1960. Both enzymes were actively produced on
pectin containing media as substrate which show resemblance with the study of Grber et-
al done in 1965.

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