Bacterial soft rot has caused more crop losses worldwide than any other bacterial disease. Current methods of inhibiting bacterial soft rot, such as using chemicals, proved to be inefficient and not environmentally-friendly. In order to develop a more environmentally-friendly and cost effective product to curb bacterial soft rot, tannic acid and green tea extract were tested for their effectiveness against the plant pathogen and causative agent for soft rot, Pectobacterium carotovorum. This is a poster we used for the Search for SEAMEO Young Scientist competition, held in Penang, Malaysia. Thank you!

1. Inhibition of Bacterial Soft Rot
Chay Hui Xiang, Ng De Rong Tony, Lai Tian Lang Hwa Chong Institution, Singapore
ABSTRACT
According to Bhat et al. (2010), bacterial soft rot has caused more crop losses worldwide than any other bacterial disease. Current methods of inhibiting bacterial soft rot using
chemicals and antibiotics are not cost-effective and not environmentally-friendly (Kuehny et al., 1998; Rahman et al., 2012). In order to develop a more environmentally-friendly and
cost-effective product to curb bacterial soft rot, tannic acid and green tea extract were tested for their effectiveness against the plant pathogen and causative agent for soft rot,
Pectobacterium carotovorum. Experiments included a preliminary antibacterial test using the well diffusion assay, and the confirmatory colony count test. Results of both tests
confirmed that both tannic acid and green tea were effective in inhibiting bacteria in the logarithmic stage of growth. As an application, tannic acid and green tea extract were swabbed
on lettuce and chye sim infected with P. carotovorum in the stationary phase of growth. The incidence of soft rot in pieces of vegetables was lower in the presence of tannic acid and
green tea extract, when compared with the control without extracts. These findings suggest a viable solution to the problem of bacterial soft rot using natural substances such as tannic
acid and green tea, thus reducing economic losses due to plant diseases and ensuring sustainability in the production of food crops.
HYPOTHESES
OBJECTIVES
1. To investigate the effectiveness of tannic acid solution and green tea extract in
inhibiting the growth of P. carotovorum.
2. To test the ability of these substances to reduce soft rot formation in lettuce and
chye sim infected with P. carotovorum.
We hypothesise that tannic acid and green tea are inhibitory towards P. carotovorum
and thus are able to reduce soft rot formation in vegetables.
INTRODUCTION AND BACKGROUND
Soft rot is caused by the Gram-
negative bacterium, Pectobacterium
carotovorum, that enters the plant,
often through wounds or natural
openings (stomata or lenticels)
Within the plant, the
bacterium multiplies and
produces increasing amounts
of pectolytic enzymes.
The enzyme breaks down
pectin found in the middle
lamella of the plant cell,
eventually causing
breakdown and maceration
of tissues.
Bacterial soft rot has caused more crop losses worldwide
than any other bacterial disease.
The bacterium has a large host range, including genera from
nearly all plant families.
$100 million crop losses per year!
PROBLEM STATEMENT
• Chemicals and antibiotics have been used to inhibit bacterial soft rot and prevent
post-harvest losses (Olivier et al., 1999).
• Their use is heavily limited by their prices and their adverse effects on human
health and environment (Margosan et al., 1997).
• Sodium hypochlorite and antibiotics such as streptomycin and chloramphenicol
(Kuehny et al., 1998) are less cost-effective.
• Using chemicals pose hazards to terrestrial and aquatic lives due to their residual
toxicity (Rahman et al., 2012).
• More environmentally-friendly and cost-effective methods in curbing bacterial soft
rot are needed.
RESEARCH METHODS
PRELIMINARY WELL DIFFUSION TESTPREPARATION OF GREEN TEA AND TANNIC ACID
• Green tea leaves were ground in
saline solution.
• The mixture was centrifuged.
• The supernatant was filter-sterilised.
GREEN TEA TANNIC ACID
• Tannic acid was dissolved
in saline solution.
• The solution was filter-
sterilised.
CONFIRMATORY COLONY COUNT TEST INHIBITION OF SOFT ROT IN LETTUCE AND CHYE SIM
P. carotovorum was
inoculated into LB
broth and incubated
overnight.
The bacterial culture
was swabbed on the
surface of a Mueller-
Hinton agar plate.
Wells were created in the agar
in triplicates and filled with
• green tea or tannic acid (test)
• saline (negative control)
• bleach (positive control)
Plates were incubated.
Equal volumes of P. carotovorum culture
were added to LB broth and
• green tea or tannic acid (test)
• saline (negative control)
• bleach (positive control)
Tubes were incubated overnight to allow
for growth of P. carotovorum.
Serial ten-fold dilution was performed with
saline solution and plated on LB agar.
Plates were incubated.
/bleach
TEST CONTROL
7.9 ml LB
0.1 ml bacteria0.1 ml bacteria
2 ml extract 2 ml saline /
bleach
7.9 ml LB
P. carotovorum was mixed with
• tannic acid or green tea (test)
• saline (control)
Lettuce (Lactuca sativa) and chye
sim (Brassica chinensis var.
parachinensis) were cut into pieces
and swabbed with the test or
control mixtures.
They were checked at two-day
intervals for soft rot development.
The number of pieces with soft rot
and percentage area covered with
soft rot were determined.

2. RESULTS AND FINDINGS
PRELIMINARY WELL DIFFUSION TEST
Figure 1: Well diffusion test.
Wells were filled with:
(1) green tea
(2) tannic acid
(3) bleach (positive control)
(4) saline solution (negative control)
Green tea and tannic acid resulted in zones of inhibition
of growth of P. carotovorum. No zone of inhibition was
observed for saline solution (negative control).
Figure 2: Well
diffusion test showed
that tannic acid and
green tea resulted in
zones of inhibition.
CONFIRMATORY COLONY COUNT TEST
Figure 3: The colony count test
showed that tannic acid and green
tea showed inhibition towards the
growth of P. carotovorum.
Difference in the mean colony
counts among all 3 set-ups was
significant (Kruskal-Wallis p value
of 0.003).
INHIBITION OF SOFT ROT IN LETTUCE AND CHYE SIM
Figure 5: A lower percentage area of lettuce occupied by soft
rot was observed in the presence of tannic acid and green tea,
compared to the control. The difference in percentage area
was insignificant (ANOVA test p value of 0.208).
Figure 6: A lower percentage of pieces of chye sim
showing soft rot was observed in the presence of
tannic acid and green tea, compared to the control.
Figure 8: (a) Chye sim swabbed with P. carotovorum and green tea extract showed no signs of
bacterial soft rot. (b) Chye sim swabbed with only P. carotovorum displayed soft rot.
Figure 7: A lower percentage area of chye sim occupied by
soft rot was observed in the presence of tannic acid and green
tea, compared to the control. The difference in percentage
area was significant (ANOVA test p value of 0.012).
Figure 4: The colony count test for (a) saline (b) green tea (c) tannic acid, and
(d) bleach showed a reduction in the mean colony count in the presence of
green tea and tannic acid, compared to saline
Inhibition of Bacterial Soft Rot
(a) (b) (c) (d)
(a) (b)
CONCLUSIONS AND RECOMMENDATIONS
• Tannic acid and green tea were effective in inhibiting the growth of P. carotovorum.
• Both substances showed greater inhibition towards actively dividing cells in the
logarithmic phase of growth, compared to cells in the stationary phase of growth that
infected a host plant.
• A limitation was the varying thickness of lettuce pieces that could affect the time for the
formation of soft rot. This was overcome by selecting pieces that had similar thickness.
• For further work, spent green tea can be used to determine the effectiveness in the
inhibition of soft rot bacterium. Fruit peels that contain tannic acid may be used, further
reducing the cost. Inhibition of soft rot in vegetables can be tested in vivo.
REFERENCES AND ACKNOWLEDGEMENTS
We are grateful to Hwa Chong Institution for providing the facilities to carry out this work; our mentor, Mrs Goh-Yip Cheng Wai, for her guidance throughout this project;
Mdm Lim Cheng Fui and Mr Xie Shun Quan of the SRC Biology lab for their help. We also thank the Ministry of Education, Singapore for the constructive feedback given to us.
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SUSTAINABILITY
ENVIRONMENTAL
Environmentally-friendly substances were
used, reducing the use of toxic,
non-biodegradable pesticides
Chay Hui Xiang, Ng De Rong Tony, Lai Tian Lang Hwa Chong Institution, Singapore
SOCIAL
The price of food crops may be lowered,
making them more affordable, thus reducing
hunger and starvation
ECONOMICAL
Potential of increasing crop yield and boosting
the agricultural economy as farmers benefit
from higher agricultural productivity