Enzyme production by endophytes of Brucea javanica

Journal of Agricultural Technology
Enzyme production by endophytes of Brucea javanica



Y.W. Choi, I.J. Hodgkiss and K.D. Hyde*

Centre for Research in Fungal Diversity, Department of Ecology & Diversity, The University
of Hong Kong, Pokfulam Road, Hong Kong SAR, PR China

Choi, Y.W., Hodgkiss, I.J and Hyde, K.D. (2005). Enzyme production by endophytes of
Brucea javanica. Journal of Agricultural Technology 1: xx-xx.

Twenty-one endophytic isolates from Brucea javanica were tested for their ability to produce
extracellular cellulase and extracellular and intracellular amylase, ligninase, pectinase and
xylanase. The same fungi were tested for their ability to cause weight loss in wood blocks. All
fungi produced amylase and cellulase, while only one sterile mycelium produced ligninase and
no isolates produced pectinase. The enzyme tests indicate that most endophytes are degraders
of the simpler sugars and cellulose available in recently dead leaves and possibly wood. Only
one slow growing species of sterile mycelium however, appeared to be capable of degrading
lignin that would be available in dead wood. No fungi appeared to be latent pathogens. A
discussion of enzyme production in relation to possible roles of endophytes is provided.

Key words: endophytes, enzyme production, latent pathogens, ligninases.

Introduction

Brucea javanica (Simarubeaceae) is a tropical, 3 m high, woody shrub,
clothed in yellow fluff. It is found from southern China to northern Australia.
The seeds are used in Traditional Chinese Medicine for the treatment of
dysentery, malaria and cancer (Lin et al., 1990), while the leaves are used in
folk medicine for poultices on boils, ringworm, scurf, centipede bites and
enlarged spleens (Perry, 1980). We are interested in establishing the
composition of endophytic communities within B. javanica plants and their
functional roles, either before or after plant death, and whether the endophytes
possess any of the medicinal properties of the plant.

This report is part of a study where we isolated endophytes from B.
javanica. We selected 21 unique taxa of fungal endophytes and examined their
ability to produce enzymes in vitro. Qualitative assays were used to screen the
endophytic fungi for their ability to produce lignocellulose degrading and other
enzymes, including amylase, cellulase, ligninase, pectinase and xylanase. The
enzyme tests may help us to understand the functional roles of endophytes and

*Corresponding author: K.D.Hyde; e-mail: kdhyde@hkucc.hku.hk

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test whether fungi can change their mode of life from an endophyte, to a
saprobe or pathogen. Weight loss tests using wood blocks were also used to
compare the results of the enzyme tests and investigate the effectiveness of the
wood degrading enzymes produced by the endophytes (Pointing et al., 2003;
Bucher et al., 2004).

Methods and materials

Twenty-one taxa of endophytic fungi, comprising 7 species identified to
genus, 8 identified to a major taxonomic group, and 7 unidentified sterile
mycelia were selected to perform the enzyme assays and are listed with their
collection details in Table 1. The endophytic fungi were isolated from stems,
branches or leaves of B. javanica from different sites and during different
seasons. The endophytes selected included potential pathogens, such as
Colletotrichum spp., Fusarium spp., Phomopsis spp. and Phoma sp., as well as
unidentified species of coelomycetes, hyphomycetes, xylariaceous taxa and
sterile mycelia.

Table 1. Brucea endophytes used in this study.

Name Code
HKUCC
Location Collection
Date
No.
Colletotrichum sp.
B10810
6055
North Queensland, Australia June, 1999
Colletotrichum sp.
L27103
6061
Hong Kong
September, 1999
Fusarium sp.
S29905
6063
Hong Kong
September, 1999
Phomopsis sp.
S12803
6056
North Queensland, Australia June, 1999
Phomopsis sp.
S29904
6062
Hong Kong
September, 1999
Phoma sp. B25603
6058
Hong
Kong
September,
1999
Coelomycete sp. 4
B0605
5881
Hong Kong
March, 1999
Coelomycete sp. 6
S2502
5883
Hong Kong
March, 1999
Coelomycete sp. 8
S3601
5885
Hong Kong
March, 1999
Hyphomycete sp. 4
S0903a
7118
Hong Kong
March, 1999
Hyphomycete sp. 3
L0302
5866
Hong Kong
March, 1999
Hyphomycete sp. 2
L0901
5658
Hong Kong
March, 1999
Xylariaceae sp.
B25605
6059
Hong Kong
September, 1999
Xylariaceae sp.
S26807
6060
Hong Kong
September, 1999
Sterile Mycelia
B23904
6942
Hong Kong
September, 1999
Sterile Mycelia
S10702
6054
North Queensland, Australia June, 1999
Sterile Mycelia
S11303
7119
North Queensland, Australia June, 1999
Sterile Mycelia
S11309
7120
North Queensland, Australia June, 1999
Sterile Mycelia
S13602
6057
North Queensland, Australia June, 1999
Sterile Mycelia
S31105
6064
Hong Kong
September, 1999
Sterile Mycelia
S31106
6965
Hong Kong
September, 1999

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Journal of Agricultural Technology
Growth rates

The growth rates of the fungi were measured in triplicate on PDA.

Enzyme assays

Five different enzyme assays, plus weight loss tests were conducted to
investigate the abilities of the endophytes to produce wood degrading and other
enzymes. Enzyme production can be separated into intracellular and
extracellular production. The cellulase test only measured extracellular
cellulase production, while the other tests measured both intracellular and
extracellular enzyme production.
Extracellular enzyme production ratio = the ratio of clear zone diameter
to that of colony diameter.
The extracellular enzymatic reactions of the following tests were
classified into 4 types:

Strong reaction: the extracellular enzyme ratio was higher than or equal to 2.
Medium reaction: the extracellular enzyme ratio was less than 2 but more than 1.
Weak reaction: the extracellular enzyme ratio was equal to or less than 1.
No reaction: there is no reaction at all.

For intracellular enzymes (those endophytes with extracellular enzyme
ratio equal to or less than 1), the diameter of the clear zone was used as a
measurement of the amount of enzyme production.

Amylase

Starch agar was prepared and autoclaved. The test fungi were inoculated
onto the agar plates and incubated for ten days. The plates were flooded with a
dilute iodine solution (Lugol’s iodine). After flooding with iodine, the starch
stains blue-black and the zone of degradation around the colonies is either
stained brown or remains colourless (Peterson and Bridge, 1994).

Starch solution was prepared by dissolving 10 g soluble starch in 50 ml
distilled water. This was stirred to give an even paste and then heated at 70-
80ºC for 1-2 minutes before adding to medium.

Cellulase

Cellulose azure agar was used to test for the presence of cellulase. Water
agar (1.6% agar in d.H2O) was prepared and 10 ml aliquots were transferred to

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glass tubes, autoclaved, and then allowed to solidify. Cellulose azure (1%),
yeast extract (0.1%) and agar (1.6%) were prepared and autoclaved. This was
allowed to cool until viscous, mixed gently, and 1 ml aliquots were transferred
aseptically onto the surface of the solidified water agar as an overlay.

The test fungi were inoculated and an uninoculated bottle was retained as
a control. The bottle caps were loosely fitted to allow adequate gaseous
exchange. They were incubated at 25ºC in darkness and examined daily for 10
days. When cellulase degrades cellulose azure agar, the blue colour at the top
layer migrates to the lower layer. This cellulolytic reaction can be divided into
4 classes (Pointing, 1999):

Strong reaction (3+): the blue colour migrates to the bottom of the universal bottle in 10
days and the blue colour is decolourised afterwards.
Medium reaction (2+): the blue colour migrates to the bottom of the universal bottle in
10 days and the blue colour is not decolourised afterwards.
Weak reaction (+): the blue colour migrates to half the depth of the universal bottle in
10 days.
No reaction (~): there is no blue colour migration at all.

Ligninase

Poly R agar was made with 02% Poly R, 0.2% glucose, 01% yeast extract and
1.6% agar. The test fungi were inoculated onto the agar plates. Clearance can
be considered as an indication of ligninase production (Pointing, 1999).

Pectinase

Pectin agar was prepared and autoclaved. The test fungi were inoculated
onto the agar plates and incubated for 7 to 10 days. The colony diameters were
recorded. The agar plates were flooded with 0.1 M aqueous malic acid and left
to stand at room temperature for 1 hour. The malic acid was drained and the
plates were flooded with 0.1% aqueous ruthenium red and left for 2 days at
4ºC. The ruthenium red was drained off and the plates were washed for 1 hour
in distilled water. They were washed with 0.1% aqueous ammonium
persulphate to increase contrast. The agar plates are pink after staining and
dark pink zones around colonies indicate pectinase activity (Peterson and
Bridge, 1994).

Xylanase

Xylan agar was prepared with 1% xylan, 0.1% yeast extract and 1.6%
agar, and autoclaved. The test fungi were inoculated onto the agar plates.
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Journal of Agricultural Technology
Clearance can be considered as an indication of xylan utilization (Pointing,
1999). Dilute iodine solution was used to stain the agar plates, and a yellow-
opaque area around colonies indicated xylan degradation as compared to a
reddish purple colour for undegraded xylan.

Weight loss test

Debarked branches of Brucea javanica were cut into 3 cm test blocks.
They were soaked in water for 24 hours and dried at 60ºC for 48 hours. Test
blocks were picked randomly, weighed and marked with a number (from 1 to
142) to obtain the initial dry weight. They were then wrapped with aluminium
foil and autoclaved twice (121ºC, 15 minutes).

Corn meal agar (CMA) plates were prepared and the test fungi were
inoculated separately onto the agar plates. Each test fungus had 5 replicates and
they were incubated at 25ºC for 10 days. The test blocks were placed onto agar
plates with growing mycelia. The plates were incubated at 25ºC for 6 weeks.
The test blocks were removed and dried at 60ºC for 48 hours. The final dry
weight of the test blocks were obtained and the percentage weight loss was
then calculated.
Percentage weight loss = (Initial dry weight – final dry weight) / Initial
dry weight × 100%

Results

Enzyme assay

Amylase

All strains produced amylase, one sterile mycelium (S11309) had a
strong reaction (an extracellular enzyme ratio of 3.9), five strains had medium
reactions and 15 had weak reactions (Table 2). Thirteen strains produced
intracellular amylase. Hyphomycete sp. 2 and 3 produced the highest amounts
of intracellular amylase.
No purple reaction with Lugol’s solution occurred under the growing
mycelium, while in some species clear halos extended 3-4 mm beyond the
growing hyphae. The amylase production was therefore internal and external.
A clear zone beyond the mycelia (extracellular enzyme production ratio greater
than 1) indicated that external amylase was produced to degrade starch, while
those that only produced a clear zone below the colonies (extracellular enzyme
production ratio less than 1) were indicative of internal amylase production.

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Table 2. Summary of 5 enzymatic tests, weight loss assay and average growth
rates.


s
e

s
e

a
se
a
th
x
a
n
w
t
e

Ta
loss
ra
Amyl
Cellula
Weight
Ligninase
Pectinase
Xyla
Gro

1 2 3 4 5 6
7
Coelomycete sp. 4
0.9
-*
-
-
0.9 (4) 15.3
5.6
Coelomycete sp. 6
0.8
2+ (6) -
-
1.1 (4) 23.9
8.6
Coelomycete sp. 8
0.9
2+ (6) -
-
1 (6)
20.3
8.8
Colletotrichum sp.
0.9
2+ (9) -
-
1.1 (4) 23.3
9.1
Colletotrichum sp.
0.9
2+ (6) -
-
1.1 (4) 22.4
10.2
Fusarium
sp. 1.1
- - - - 13.4
6.4
Hyphomycete sp. 2
1
+ (13) -
-
1 (6)
19.9
10
Hyphomycete sp. 3
1
2+ (8) -
-
1 (4)
12.6
9.8
Hyphomycete
sp.
4
0.9
+
(9)
- - - 17.6
9.2
Phoma sp.
0.6
2+ (6) -
-
0.9 (4) 13.9
12.1
Phomopsis
sp. 0.8
+
(13)
- - - 17.5
10
Phomopsis
sp. 1.3
- - - 1.2
(8)
14.8
6.4
Sterile
mycelium
1.3
2+
(6)
- - 1.4
(8)
23.2
3.2
Sterile
mycelium
0.8
- - - - 19.5
6.4
Sterile
mycelium
1 +
(8)
- - - 18.8
6.4
Sterile
mycelium
1.5
+
(10)
- - 1.8
(10) 18 3.2
Sterile
mycelium
0.8
+
(10)
- - 1
(4)
14.4
10.8
Sterile
mycelium
0.5
- - - - 13.7
10
Sterile mycelium
3.9
-
1.2 (8) -
6.4 (5) 4.9
1.1
Xylariaceae sp.
1.1
+ (13) -
-
1 (8)
23.4
8.1
Xylariaceae
sp. 0.6
- - - 1
(4)
17.6
12.1
Negative
control
- - - - 0 0
Trametes versicolor 0.9
+ (6)
0.68 (4) 1 (7)
1.2(6) 59.7
11.6
1. Extracellular enzyme production rate (clear zone in cm, colony diameter in cm) over
10 days.
2. Strength of reaction: 2+ = medium; + = weak; - no reaction in (x) days.
3. As 1. but in (x) days.
4. As 3.
5. As 3.
6. Actual weight loss %.
7. Average growth rate mm/day.
*10 days if not stated



Cellulase

Fourteen endophytes produced cellulase (Table 2). None had a strong
reaction, seven had medium reactions (2+) and seven had weak reaction (+).
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Journal of Agricultural Technology
Ligninase

Only one endophyte, sterile mycelium strain (S11309), produced
extracellular ligninase (Table 3). Over 8 days it extracellular enzyme
production was 1.2 and it produced a clear zone at a rate of 0.3 cm per day.
Trametes versicolor produced intracellular ligninase with a clear zone
production of 1.1 cm per day and over 4 days its ratio was 0.68. When
comparing the amount of ligninase produced, Trametes versicolor, the positive
control, was a better producer of ligninase than strain S11309.

Pectinase

None of the endophytes were found to produce pectinase. The positive
control Trametes versicolor produced extracellular pectinase, with a clear zone
of 1.2 cm diameter per day, and an extracellular ratio of 1.1.

Xylanase

Fifteen of the endophytes tested produced extracellular xylanase, but
only one had a strong reaction (6.4-Strain S11309), six had medium reactions
(1.1-1.8) and eight had weak reactions (0.9-1) (Table 2). Eight strains were
intracellular producers of xylanase. Colletotrichum species B10810 and
L27103, Phoma species (B25603) and the xylariaceous taxa (B25605)
produced higher amounts of extracellular or intracellular xylanase than the
other strains.

Wood block weight loss test

The results of the wood block weight loss tests are shown in Table 3. The
“actual weight loss %” was calculated by deducing the average weight loss due
to leaching in the negative control (11.8%) from the average weight loss due to
each isolate. Six of the endophytes caused more than 20% weight loss, and the
other 15 caused less than 20% weight loss. One strain, the sterile mycelium
strain S11309 caused only 4.9% weight loss. The average growth rates of the
endophytes on corn meal agar plates are also shown for comparison in Table 3.


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Comparison of results of enzymatic tests and the wood block weight
loss test

From the combined results of the five enzymatic tests and wood block
weight loss tests shown in Table 4 it can be seen that those endophytes that
caused more than 20% weight loss produced all three types of enzyme, i.e.
amylase, cellulase and xylanase. There were no clear patterns of enzyme
production in those taxa that produced less than 20% weight loss. Two of the
three hyphomycetes tested (L0901 and L0302), which caused less than 20%
weight loss, produced cellulase, xylanase and amylase. Trametes versicolor,
which is a white rot fungus, produced all types of enzymes tested in this study,
and caused a 59.7% weight loss.

Discussion

Methodology

Priest (1984) showed that there were several possible regulatory
mechanisms in enzyme production, including enzyme induction. In the enzyme
production tests used in this study, cellulose, pectin, Poly-R, starch and xylan
were used as substrates to induce enzyme production. They were mixed with
low nutrient content agar that had just enough nutrient for mycelial growth to
spread across the agar plates.
Decay
caused
by
Gendarme colossus has previously been found to be
dependent on time of incubation and temperature (Adaskaveg et al., 1995).
Weight loss increased as the incubation time increased and the optimal
temperature for the highest weight loss was 30 to 40ºC. In this study, the
containers were 90 mm Petri dishes, which are not air-tight and the incubation
time was 6 weeks. As a result, the incubation temperature was never greater
than 25ºC. As the wood branches used in this study were low in weight (~ 0.1
g), and since most of the endophytes tested were fast-growing fungi, a shorter
incubation time, 6 weeks, was chosen.

Enzyme production

In order to establish the functional role of endophytes it would be useful
to establish their patterns of substrate utilization and which enzymes they
produce (Carroll and Petrini, 1983). If they are weak parasites or latent
pathogens they may produce proteinase and pectinase (Brett, 1990b; Reddy et
al., 1997), while if they are mutualistic, eventually being saprobes they are
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Journal of Agricultural Technology
likely to produce cellulase, mannanase and xylanase (Pointing, 1999). We have
therefore tested the ability of endophytes from within seedlings to produce
amylase, cellulase, mannanase, proteinase and xylanase in order to allude
possible roles.

Amylase

All endophytes tested in this study were found to degrade soluble starch.
The results are similar to the findings of Adaskaveg et al. (1991), who found
that the white and brown rot fungi utilized starch in palm wood. These fungi
also utilized starch when grown on starch agar media. Endophytes are likely to
be the first colonizers of dead plant tissue as they are already living within the
plant when it is still alive (Guo et al., 1998; Fröhlich et al., 2000). Plant tissues
store starch as a food source and this starch is one of the most easily digestive
food sources within plant tissues. When the plant dies, the starch becomes
available and the endophytes can consume the starch before other new
colonizers appear.

Cellulase and xylanase

Endophytes that degraded xylan, are also likely to have the ability to
degrade cellulose and are discussed together here. Xylan (hemicellulose) and
cellulose occupy between 25 to 40% and 40 to 50% of the wood mass
respectively. After all simple food sources, such as glucose and starch, are used
up, wood degrading fungi will start to degrade the cell wall components. As a
result, endophytes that produce both cellulases and hemicellulases, such as
xylanases should have the ability to compete with other types of fungi
surviving on dead wood and leaves (Carroll and Petrini, 1983).

Pectinase

No endophytes tested had the ability to degrade pectin. Only the positive
control, Trametes versicolor, degraded pectin. Pectic substances are
predominantly located in the middle lamella and primary wall and they are
highly susceptible to degradation under relatively mild conditions (Brett,
1990b), but in small amounts (Tsoumis, 1991).





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Ligninase

Only one of the endophytes tested (S11309) and the positive control,
Trametes versicolor produced ligninase. This indicates that most of the
endophytes in the plant tissues may not have the ability to degrade lignin. It is
thought that most endophytes start to grow when the plant is weakened or dies.
This and other studies (e.g. Carroll and Petrini, 1983) indicate that endophytes
appear to be able to degrade more simple substrates, such as starch, cellulose
and hemicelluloses, rather than very complex substrates like lignin.

Strain S11309 is a slow growing endophyte (1.1 mm/day) that had very
high extracellular enzyme production ratios in xylanase (6.4) and amylase (3.9)
tests and a higher ratio (1.2) in the ligninase test than the positive control
Trametes versicolor (0.68). All of the enzymes tested were produced
externally. The fungus digested most of the food sources offered to it. This
implies that this slow growing fungus is able to produce a wide range of
enzymes externally.

Pathogenesis

The production of fungal enzymes by fungi in nature has a role in their
pathogenicity or degradative capacity (Archer and Wood, 1995). Sieber et al.
(1991), Carroll and Petrini (1983) and Savorie and Gourbiere (1989) found that
the endophyte Leptostroma strongly produced extracellular cellulases,
indicative of its ability to digest cell wall components. Sieber et al. (1991) also
stated that the fervent production of extracellular cellulases together with that
of pectinases in the Leptostroma endophyte could imply that the fungus is well
equipped for both penetration of living cells and decomposition of dead tissues.
Brett (1990a) stated that the major enzymes involved in microorganisms
attacking living higher plant tissues are pectic enzymes. Pectic enzymes are
induced in the presence of pectic substances by both pathogenic fungi and
pathogenic bacteria.

No endophyte tested in this study had the ability to degrade pectin. Pectic
substances are predominantly located in the primary wall, the middle lamella in
small proportions (or are absent in older wood), and cambial tissue, where they
form the membrane that separates the young daughter cells produced by the
cambium (Tsoumis, 1991). If an endophyte can degrade pectic substances, this
implies that the fungus is likely to be a latent pathogen. As the result of this
study, where only Trametes versicolor, a wood degrading fungus, degraded
pectin, the endophytes tested are unlikely to be latent pathogens, even though
some belong to pathogenic genera (e.g. Colletotrichum spp.).
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