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.

1. INHIBITION OF BACTERIAL SOFT ROT
Team members:
Chay Hui Xiang
Ng De Rong Tony
Lai Tian Lang
School:
Hwa Chong Institution
Singapore
Mentor:
Mrs Goh-Yip Cheng Wai

2. 2
ABSTRACT
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.
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 early stages 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.

3. 3
TABLE OF CONTENTS
Introduction and Background of Study Page 4
Research Methods Page 7
Results and Findings Page 9
Conclusions and Recommendations Page 13
References Page 14
ACKNOWLEDGEMENTS
We are grateful to Hwa Chong Institution for providing the facilities to carry out this
work, Mdm Lim Cheng Fui and Mr Xie Shun Quan of the Biology laboratory at the
Science Research Centre, Hwa Chong Institution, for helping us while conducting
our experiments, and most importantly our mentor, Mrs Goh-Yip Cheng Wai, who
provided guidance to us throughout this project.

4. 4
INTRODUCTION AND BACKGROUND OF STUDY
From 2014 to 2016, there will be 795 million chronically hungry people in the world.
Furthermore, the global population is expected to surge well over nine billion by the
end of 2050. As a result, the agricultural sector will have to produce 60 percent more
food globally within the same period. At the same time, roughly one-third of food
produced – 1.3 billion tonnes per year – is lost globally throughout the supply chain
(Food and Agriculture Organisation of the United Nations, 2015).
For decades, bacterial soft rot has caused more crop loss worldwide than any
other bacterial disease. The disease can be found on crops in the field, in transit and
in storage or during marketing. In total, this amounts to approximately 100 million
dollars lost every single year (Bhat, Masood, Bhat, Bhat, Razvi, Mir, Akhtar, Wani and
Habib, 2010). Bacterial soft rot is most commonly caused by the Gram-negative
bacteria, Pectobacterium carotovorum subsp. carotovorum (formerly known as
Erwinia carotovora subsp. carotovora) and Pseudomonas fluorescens (Agrios, 1997).
The bacteria chiefly attack succulent, tender tissues of storage organs such as fleshy
tubers, fruits, roots, bulbs, corms, and rhizomes, as well as bud, stem, petiole and leaf
stalk tissues (Babadoost, 1990). The host range is large, and includes various
commercially important plants including potatoes, carrots and cabbages. P.
carotovorum is a member of the family Enterobacteriaceae, along with other plant
pathogens such as Erwinia amylovora and human pathogens such as Salmonella
spp. and Yersinia spp. (Toth, Bell, Holeva and Birch, 2002). It enters plant tissues
primarily through wounds, often created by insect feeding or bruising at harvest.
Once in the plant tissue, the bacterium produces enzymes that break down the
pectic substances of the middle lamella, eventually causing breakdown and
maceration of the tissues (Gupta and Thind, 2006). This results in damaged crops and
lowered crop yields.
Fungal soft rot is also a serious disease that is affecting postharvest crops. Fungi
causing soft rot diseases include Penicillium expansum and Sclerotinia fructigena on
apples and Rhizopus on sweet potatoes (Moore-Landecker, 1996). The
sporangiospores of Rhizopus secrete pectinolytic and cellulolytic enzymes which
break down pectin in the middle lamella and cellulose of the cell walls, respectively,
eventually causing soft rot (Agrios, 1997). A common fungal disease in cabbages is
the gray mould rot caused by the fungus Botrytis cinerea (Acedo, 2010).
Umunna and Anselem (2014) reported that bacterial soft rot is one of the most
common diseases in potatoes. Other agents of diseases in potatoes are virus, fungi
and pests, but bacterial soft rot alone accounts for about 30% to 50% of this
loss. Furthermore, according to Acedo (2010), bacterial soft rot is the most important
postharvest disease in cabbages and another study conducted by Johnson (1999)
has proved bacterial soft rot to be one of the most severe postharvest diseases of
potatoes worldwide.

5. 5
In order to be sustainable, agriculture must meet the needs of the present and
future generations. Sustainable development has been defined from Our Common
Future, also known as the Brundtland Report, as development that meets current
needs, without compromising the ability of future generations to meet their needs
(WCED, 1987). Sustainability often consists of three dimensions: environmental, social
and economic sustainability. By reducing the spread of bacterial soft rot,
sustainability can be ensured environmentally, socially, and economically.
Firstly, environmental sustainability is discussed. The current use of chemicals to
prevent plant diseases is not a sustainable method, as these chemicals can
contaminate soil, water, turf, and other vegetation (Aktar, Sengupta, and
Chowdhury, 2009). Furthermore, toxins from these chemicals may adversely affect
terrestrial and aquatic lives via bioaccumulation and biomagnification. Human
health is also threatened. World-wide deaths and chronic diseases due to pesticide
poisoning are increasing. Hence, the use of substances naturally found in food, such
as tannic acid and green tea can be effective yet environmentally-friendly.
Secondly, social sustainability is considered. By reducing the spread of bacterial
soft rot, the overall crop yield will be increased. An increase in crop yield can thus
help to support the increasing global population, especially those who suffer from
poverty and malnourishment. Increasing global food supply will decrease food
prices, and enable people to better afford food to sustain their hunger.
Thirdly, economical sustainability is ensured. Today, the total amount of post-
harvest losses account to about $100 million (Bhat et al., 2010). Reducing the spread
of bacterial soft rot will boost the agriculture economy by leaps and bounds. With
higher crop yield, farmers will also benefit from higher agricultural productivity. On a
macroscopic level, countries which rely heavily on agriculture for export can
increase exports and profits, sustaining economic growth.
Problem Statement
Although universal methods of using chemicals and antibiotics to inhibit bacterial
soft rot and prevent post-harvest losses have already been developed, and some
chemicals are recommended (Olivier, MacNeil and Loria, 1999), their use is heavily
limited by their prices and their adverse effects on human health and environment
(Margosan, Smilanick, Simmons and Henson, 1997). For instance, the incidence of
soft rot could be reduced with the use of sodium hypochlorite, and antibiotics such
as streptomycin and chloramphenicol (Kuehny, Holcomb, Chang and Branch, 1998).
However, such chemicals have proven to be less cost-effective as they usually have
exorbitant price tags. In addition to these financial factors, using chemicals pose
hazards to terrestrial and aquatic lives due to their residual toxicity (Rahman, Khan,
Ali, Mian, Akanda, and Hamid, 2012). Chemicals are washed down of the plants by
rain flow to other parts on land, possibly ending up in river channels or lakes, causing
serious health hazards to nearby organisms. Therefore, increasing needs for a more

6. 6
environmentally friendly and cost-effective method in curbing bacterial soft rot,
have definitely surfaced.
Furthermore, although there are well-established procedures for the production
of transgenic plants with resistance towards these bacterial pathogens, and
considerable progress has been made using a range of new methodologies, there
are no current commercially available transgenic plant species with increased
resistance towards these bacteria (Wally and Punja, 2010).
Hence, there is an increasing need for a more environmentally-friendly and
effective method of reducing the spread of bacteria soft rot. Green tea (Camellia
sinensis) contains polyphenolic compounds with activity against a wide spectrum of
microbes. Many of the biological properties of green tea have been ascribed to the
catechin fraction, which constitutes up to 30% of the dry leaf weight (Taylor,
Hamilton-Miller and Stapleton, 2005). Green tea has been shown to have
antimicrobial effects against a variety of Gram positive and Gram negative bacteria,
for example, Escherichia coli, Salmonella spp., Staphylococcus aureus and
Enterococcus spp., some fungi (Candida albicans) and a variety of viruses (HIV,
herpes simplex, influenza) (Jigisha, Nishant, Navin and Pankaj, 2012; Steinmann, Buer,
Pietschmann and Steinmann, 2013). On the other hand, tannic acid has also been
shown to be very effective against various types of bacteria, for example,
Escherichia coli, Bacillus cereus, Pseudomonas aeruginosa, moulds such as
Aspergillus niger, Aspergillus flavus, Penicillium granulatum, and yeasts such as
Geotricum candidum, Yarrowia lypolitica, Rhodotorula rubra (Colak, Yapici and
Yapici, 2010).
Objectives and Hypotheses
The aim of this project is to investigate the effectiveness in using tannic acid
solution and green tea extract in inhibiting the growth of P. carotovorum. The ability
of these substances to reduce soft rot formation in lettuce and chye sim infected
with P. carotovorum is also tested.
We hypothesise that tannic acid and green tea are inhibitory towards P.
carotovorum and thus are able to reduce soft rot formation in vegetables.

7. 7
RESEARCH METHODS
Outline of methods
The outline of project was as follows:
1. Preparation of extracts
2. Well diffusion test
3. Colony count test
4. Inhibition of soft rot in lettuce and chye sim
Variables
Controlled variables were the temperature and duration of growth of the
bacterium and the dimensions of lettuce and chye sim. The independent variables
were the type of inhibitory agent (tannic acid or green tea), type of vegetable
(lettuce or Lactuca sativa, chye sim or Brassica chinensis var. parachinensis). The
dependent variables were the diameter of zone of inhibition, number of colony
forming units, percentage of area of lettuce and chye sim covered by soft rot, and
the percentage of pieces of chye sim with soft rot.
Preparation of tannic acid solution and green tea extract
5 g of green tea leaves was ground in 15 ml of saline using a mortar and pestle
and centrifuged at 7000 rpm for 10 min. The supernatant was collected and filter-
sterilised using a 0.45 μm microfilter. Tannic acid (Sigma-Aldrich) was dissolved in
deionised water to give a concentration of 1 mg/ml. The solutions were filter-sterilised
using a 0.45 μm microfilter.
Well diffusion test
P. carotovorum was first grown in 10 ml LB broth overnight at 30°C with shaking.
Using a sterile cotton swab, P. carotovorum broth culture was swabbed evenly on
the surface of a Mueller-Hinton agar plate. Wells were created in the agar and filled
with 80 μl of tannic acid solution or green tea extract. The positive control was
bleach and the negative control was saline solution. Triplicates were performed. The
plates were incubated at 30°C overnight and checked for zones of inhibition. The
diameter of zones of inhibition would give a preliminary indication if the tannic acid
and green tea were antibacterial towards P. carotovorum.
Colony count test
P. carotovorum was grown overnight in 10 ml LB broth at 30°C with shaking. 0.1 ml
bacterial culture was added to 2 ml of either tannic acid or green tea extract and
7.9 ml of LB broth. For the negative control set-up, saline was used, while 1% bleach
was used for the positive control set-up. Five replicates were prepared, and the

8. 8
tubes were incubated overnight at 30°C with shaking. The absorbance of the
bacterial culture in each tube was determined using a UV-vis spectrophotometer at
600 nm. The test and control were serial diluted based on the absorbance value. This
was done by transferring 0.1 ml of the test or control to 0.9 ml of saline. This was
repeated until 10-5 dilution. 0.1 ml of the diluted culture was spread on an LB agar
plate. The plates were incubated at 30°C overnight. The number of colonies on
each plate was recorded the next day. In this confirmatory colony count test, the
number of colony forming units in the test and control set-ups was compared, and
statistical significance was determined using the Kruskal-Wallis test.
Inhibition of soft rot in lettuce and chye sim
In the application, the ability of tannic acid and green tea extract to reduce
development of soft rot on lettuce and chye sim infected with P. carotovorum was
determined. P. carotovorum culture in the stationary phase of growth was mixed in a
ratio of 1:1 with the extracts or saline solution in the test and control, respectively. The
control using bleach was omitted as it was not feasible for bleach to be added to
vegetables for human consumption. The mixtures were left at room temperature for
30 min. Lettuce and chye sim pieces of dimensions of 4 cm x 4 cm were prepared
and swabbed with 20 μl of the test or control mixtures. Each piece was placed in a
small sterile Petri dish. 20 pieces were used for each test or control. The plates were
placed in an incubator at 30°C. They were checked at two-day intervals up to 14
days for development of soft rot. The percentage area covered with soft rot was
determined at the end of 14 days.

9. 9
RESULTS AND FINDINGS
Well diffusion test
Zones of inhibition were observed for tannic acid and green tea, thus showing
that these substances inhibited the growth of P. carotovorum. The results are shown
in Figures 1 and 2.
Figure 1: Well diffusion test for wells filled with (1) green tea, (2) tannic acid, (3)
bleach as positive control, and (4) saline as negative control. Green tea and tannic
acid resulted in zones of inhibition of growth of P. carotovorum.
Figure 2: Well diffusion test showed that both tannic acid and green tea resulted in
zones of inhibition around the wells.

10. 10
Colony count test
In the confirmatory test, the highest number of colony forming units was observed
in the saline control without extract. No colonies formed in the presence of bleach in
the positive control. There was an observable reduction in the number of colony
forming units in the presence of green tea and 1 mg/ml tannic acid. The Kruskal-
Wallis test showed that the difference in the mean number of colonies in the four
different set-ups was statistically significant, with a p value of 0.003. It was also
observed that tannic acid was slightly more effective than green tea in inhibiting the
growth of P. carotovorum, as that there was a slightly greater reduction in number of
colonies in the presence of tannic acid as compared to tea. The results are shown in
Figures 3 and 4.
Figure 3: The colony count test showed that both tannic acid and green tea
inhibited the growth of P. carotovorum.
(a) (b) (c) (d)
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 tea and
tannic acid, compared to saline.

11. 11
Inhibition of soft rot in lettuce (Lactuca sativa)
Figure 5 shows that the application of 1 mg/ml tannic acid and green tea
reduced the percentage surface area of lettuce covered with soft rot. However,
both these agents were not very effective in inhibiting P. carotovorum in the
stationary phase of growth. Despite an observable difference in the mean
percentage area in the tests and the control set-ups, the ANOVA test showed that
there was no significant difference among the three set-ups, with a p value of 0.208.
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.

12. 12
Inhibition of soft rot in chye sim (Brassica chinensis var. parachinensis)
The same trend was observed for chye sim. A higher percentage of pieces of
chye sim displayed soft rot in the control set-up with only P. carotovorum, compared
to those with the addition of tannic acid and green tea. This is shown in Figure 6. The
ANOVA test confirmed the significant difference among the set-ups, with a p value
of 0.012. The area occupied by soft rot in all the set-ups is shown in Figure 7.
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 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.

13. 13
The surface of vegetables with bacterial soft rot turned from green to brown in
colour. The chye sim piece became thinner and more translucent, to the extent that
veins could be clearly seen. This was caused by enzymes secreted by P.
carotovorum that broke down the middle lamella of the plant tissue. This is illustrated
in Figure 8.
(a) (b)
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 P. carotovorum only turned
brown and displayed soft rot.
These results strongly suggested that tannic acid and green tea extract could be
used to reduce formation of soft rot in vegetables, thus reducing their spoilage.
CONCLUSIONS AND RECOMMENDATIONS
From our study, it can be seen that both tannic acid and green tea were
effective against P. carotovorum. Both substances showed greater inhibition towards
actively dividing cells in the logarithmic phase of growth, as could be seen from the
colony count results, as compared to cells in the stationary phase of growth that
infected a host plant. In other words, tannic acid and green tea were likely to be
bacteriostatic in nature, inhibiting the reproduction of bacteria, rather than showing
a bactericidal effect. Nevertheless, there is still a good potential of using both tannic
acid and green tea in overcoming the problem of soft rot in plants.
Past research and experiments conducted have discovered several ways to
control bacterial soft rot. In 2006, scientists discovered a bacteriocin-like substance
which inhibited bacterial soft rot caused by Erwinia carotovorum in potato tubers via
the lysis of the bacteria (Cladera-Olivera, Caron, Motta, Souto and Brandelli, 2006).
The use of chemicals such as chlorine, bleach and citric acid also showed more
than 90% reduction in potential for bacterial soft rot (Bartz and Kelman, 1986).
However, the inhibitors used were either hard to mass-produce to meet the needs of
the agriculture industry, or were chemicals that could pose harm to consumers and
the environment. Our study used two easily obtainable and environmentally-friendly
substances, which could be useful in increasing crop yield.

14. 14
One of the limitations was the varying thickness of lettuce pieces that could
possibly affect the time for the formation of soft rot. This was overcome by selecting
pieces that had approximately similar thickness. Furthermore, the method of
application of tannic acid and green tea to plants cannot be utilised in large-scale
applications. The development of aerosols of these agents and direct spraying on
plants would be more feasible in large-scale applications.
Our findings will benefit farmers and those generally working in the agricultural
industry. Not only will they be able to understand the causes and effects of bacterial
soft rot, but also use cost effective and environmentally friendly extracts such as
green tea and tannic acid to tackle this imminent problem. Furthermore, people
living in poverty and facing malnourishment may also be able to benefit. As the
global food supply increases, prices for food will decrease, and these people can
better afford food to sustain their hunger. On a wider scale, countries which heavily
rely on agriculture for economic growth will benefit in the long run, as the output of
crop plantations and the revenue they earn will increase.
The use of natural substances which are inexpensive in reducing incidence of soft
rot in plants has important implications. It increases crop yield and ensures
sustainability in the production of food crops. Environmental protection is achieved
when the use of non-biodegradable, chemical substances which may pollute the
environment is reduced. This also leads to sustainable development.
For further work, spent green tea can be used to determine the effectiveness in
the inhibition of soft rot bacterium. Moreover, fruit peels that contain tannic acid
may be used instead of pure tannic acid, further reducing the cost. Finally, other
methods of applications such as spraying can be explored.
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