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Diversity and control of bacterial contamination of plants propagated in

temporary immersion bioreactor system

Article  in  Acta Horticulturae · March 2017

DOI: 10.17660/ActaHortic.2017.1155.65

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Diversity and control of bacterial contamination of

plants propagated in temporary immersion bioreactor
system
S. Klayraung1,a, P. Niamsup1, P. Poonnoy2 and N. Topoonyanont1
1Faculty of Science, Maejo University, Sansai, Chiangmai, Thailand;2Faculty of Engineering and Agro‐Industry,
Maejo University, Sansai, Chiangmai, Thailand.

Abstract
Microbial contamination can cause major loss to micropropagated plants. In this
research, we studied the genera of bacteria contaminated in sugarcane plantlets and
rice seedlings grown in different temporary immersion bioreactor (TIB) systems.
Samples were collected from the contaminated medium of a TIB system, used in the
plant tissue culture laboratory of the Faculty of Science, Maejo University (Chiangmai,
Thailand). All bacterial isolates were characterized by the 16S rRNA gene base
sequencing technique. A total of 24 bacterial isolates was obtained from sugarcane
plantlets of the Saccharum sp. ‘LK92-11’. These bacterial isolates were characterized
into 4 genera. It was found that the contamination in 20 L TIB container was caused
by Bacillus amyloliquifaciens, B. pumilus and B. subtilis, whereas in 700 mL TIB
container was by Methylobacterium sp., Paenibacillus sp. and Sphingomonas sp.; all
belonging to Firmicutes and Proteobacteria groups. These six bacteria species were
not endophytic bacteria of sugarcane, indicating that they were from the
environment. Meanwhile, in rice seeds germinated in TIB system, 48 bacterial
isolates were identified into 8 genera. These bacteria include Bacillus,
Curtobacterium, Microbacterium, Achromobacter, Acidovorax, Burkholderia,
Enterobacter, Klebsiella and Pantoea, belonging to Actinobacteria, Firmicutes and
Proteobacteria groups. Most of them have previously been reported as endophytic
bacteria in rice. These results suggest that the diversity of bacterial contamination in
TIB system depends on plant species and explant types, and it is also linked to
environmental conditions. Furthermore, procedures in preventing bacterial
contamination from various sources during sugarcane propagation in TIB system
were designed, based on the obtained results. Obviously, the level of bacterial
contamination decreased under controlled conditions and good practices in
laboratory.

Keywords: endophytes, laboratory, microorganisms, sanitary conditions, tissue culture

INTRODUCTION
Bacterial contamination can cause major losses of micropropagated plants, since the
contamination in plant tissue culture causes a pH change of culture media which has
important effects on the plant cell growth (Leifert and Cassells, 2001). The presence of
microbes in plant cultures usually results in increased culture mortality and the presence of
latent infections can result in variable growth, tissue necrosis, reduced shoot proliferation,
or even shoot culture death. Bacterial contamination in plant tissue culture can originate
from explants, laboratory environments, operators, or ineffective aseptic techniques (Leifert
and Cassells, 2001). In addition, different parts or stages of the plants, plant species and
sources of plants can also affect bacterial contamination in the plant tissue culture (Cornu
and Michel, 1987; Boxus and Terzi, 1988). Temporary immersion bioreactor (TIB) system is
a micropropagation procedure that employs the use of automation technique to control the

a
E-mail: srikanja@mju.ac.th

  Acta Hortic. 1155. ISHS 2017. DOI 10.17660/ActaHortic.2017.1155.65 439

Proc. VI Int. Symp. on Production and Establishment of Micropropagated Plants
  Eds.: M. Beruto and E.A. Ozudogru
rapid multiplication of plant cultures with potential applications to plant propagation at
industrial level (Poonnoy and Topoonyanont, 2012). Bacterial contamination in plant tissue
culture is a common problem which can slow down the development of all in vitro
techniques. The characterization of bacteria and the analyzing processes may provide
important information about the sources of contamination and how to prevent or reduce
bacterial contamination (Leifert et al., 1991). Therefore, the objectives of this study were to
investigate the sources of bacterial contamination and to find the best method for the
control of bacterial contamination in TIB cultivation system.

MATERIALS AND METHODS

Detection of bacteria

1. Bacterial population in laboratory.

The laboratory of plant tissue culture of the Faculty of Science at Maejo University was
investigated for detecting the bacterial density and distribution of bacterial strains in
various areas according to the planimetry reported in Figure 1. The ‘Settle Plate’ method
was used for detection. Nutrient agar (NA) containing nystatin was exposed to the air in the
various area of the laboratory for 15 min. The plates were then sealed and incubated at 37°C
for 48 h. The number of colonies was then counted. The number of microorganisms was
expressed as CFUs plate‐1. Thereafter, selected colonies were streaked onto NA plates for
purity check and purified bacteria were characterized. All bacterial isolates were stored in
20% glycerol stock.

Figure 1. Map of the investigated areas in the plant tissue culture laboratory of the Faculty
of Science, Maejo University.

2. Isolation of bacteria from contaminated TIB system.

Sugarcane (Saccharum sp. ‘LK92‐11’) received from Mitrphol Sugarcane and Sugar
Research and Development Company, Limited (Chaiyaphum, Thailand) were cultivated in
TIB system including 700 mL and 20 L containers. Liquid MS (Murashige and Skoog, 1962)

440
medium without plant growth regulators (HF medium) was used to cultivate sugarcane.
Rice seeds (Oryza sativa ST1) were also germinated in TIB bioreactor using liquid ½MS
medium without sucrose. Bacterial contaminants of TIB plants were isolated directly from
visibly contaminated plant cultures by spreading of bacterial growth on NA plates
containing nystatin. Plates were incubated at 37°C for 48 h. Bacterial purification was
obtained by re‐streaking on NA. All bacterial isolates were maintained in 20% glycerol
stock. The selected colonies were identified using a 16S rRNA base sequencing.

Identification of bacteria
Purified bacteria cultures were firstly characterized by their cell morphology using
Gram’s staining. The 16S rRNA gene was PCR amplified from the genomic DNA. The
bacterial DNA was isolated by a Genomic DNA Mini Kit (Geneaid, Taiwan). The DNA was
amplified with the universal primers 27F (5’‐AGAGTTTGATCMTGGCTCAG‐3’) and1522R (5’‐
AAGGAGGTGATCCRCCGCA‐3’). Sequencing of 16S rRNA gene was determined by FirstBASE
Laboratories, Malaysia. The sequences were aligned with those in the NCBI GenBank and the
similarities were determined by using the BLAST algorithm
(http://www.ncbi.nlm.nih.gov/BLAST).

Prevention of bacterial contamination in TIB system

The study of bacterial prevention using critical points analysis and commercial
chemicals was carried out. The plant tissue culture laboratory at the Faculty of Science, of
Maejo University (Chiangmai, Thailand), where the TIB systems were located, was managed
to have fumigation once a month and a daily cleaning of the floor by using an appropriate
disinfectant. The microbial population was determined by settle plate method. The data of
bacterial distribution were analyzed to indicate the critical steps of the use of the TIB
system. Furthermore, HF media with and without 2 mL L‐1 plant preservative mixture
(PPMTM) were provided to sugarcane shoots every 3 h for 15 min each. The temperature was
controlled at 25±2°C. The experiments were carried out during 2 weeks. The bacterial
contamination was determined by direct observation.

RESULTS
The average bacterial population in the investigated area is shown in Figure 2. The
results showed that the area of pathway (9), the preparation room (10), the autoclave room
(11) and the cloth changing room (3) contained high level of bacterial population.

Figure 2. Average of bacterial colonies per plates, found in the investigated areas of the
plant tissue culture laboratory (the numbers refer to the areas indicated in Figure
1).

441
 The bacterial isolates were purified and characterized by cell morphology. The 16S
rRNA genes of pure bacterial cultures were investigated. Figure 3 showed that the isolated
bacteria, found in the different area of laboratory, belonged to 17 genera, i.e., 8 genera of
Gram positive bacteria (Bacillus, Corynebacterium, Dermacoccus, Exiguobacterium, Kocuria,
Microbacterium, Micrococcus, and Staphylococcus) and 9 Gram negative bacteria
(Acinetobacter, Arthrobacter, Enhydrobacter, Enterobacter, Proteus, Pseudomonas,
Rhodococcus, Methylobacterium, and Roseomonas). It was also found that the distribution of
the bacterial species was the highest in the area of pathway and in the preparation room.

Figure 3. Distribution of bacterial species detected in the laboratory according to the
directions for entering in the TIB room.
Bacterial contaminants were isolated from sugarcane and rice seedling cultured in the
TIB system. Three characters of bacterial contaminants of sugarcane plant culture were
isolated from the 700 mL TIB container. The colors of the selected bacterial colonies were
pink, white and yellow. When their bacterial 16S rRNA base sequences were compared to
NCBI database, it was found that the bacteria belonged to Methylobacterium, Paenibacillus
sp. and Sphingomonas sp. In the 20 L TIB container, the medium was brown and opaque
media and three different colonies were obtained by using NA containing nystatin as
bacterial isolation medium. According to 16S rRNA genes, they were identified as Bacillus
amyloliquefaciens, B. pumilus and B. subtilis.
In case of rice TIB system, contaminated white turbid culture medium was observed.
A total of 48 isolates were obtained, characterized into 18 species according to the 16S rRNA
base sequences. The Gram positive bacterial contaminant belonged to the genera of Bacillus,
Curtobacterium and Microbacterium, whereas Gram negative bacteria were identified as
Achromobacter, Acidovorax, Burkhoderia, Enterobacter, Klebsiellaand Pantoeabelonging to
Actinobacteria, Firmicutes and Proteobacteria groups.

442
 The study on the prevention of bacterial contamination by a cleaning plan obviously
decreased the microbial population of the plant tissue culture laboratory (Figure 4). The
culture of sugarcane in the TIB system was further investigated after a cycle of cleaning
process. Sugarcanes cultivated in 20 L TIB using HF medium with and without PPMTM
showed no bacterial contamination in both of them.

Figure 4. Bacterial population counted before and after cleaning by fumigation and wipe up
with disinfectant in selected area where the area numbers were shown in Figure
1.

DISCUSSION
Bacterial contamination in the plant tissue culture laboratory was found in higher
content in the highly frequented areas, such as pathway, preparation room and cloth
changing room. Toivola et al. (2002) reported that the highest bacterial population was
detected in the heavily populated workplaces. A strong relationship between human activity
and microbial density in indoor air was reported in other studies (Qian et al., 2012; Jurado
et al., 2014). In addition, the high level of bacteria in cloth changing room was related to
human bodies as well as clothes which are a natural source for bacteria (Qian et al., 2012).
In our study, it was also found that the most bacterial contaminants in the 20 L TIB
system used to cultivate sugarcane were mainly Bacillus sp. The results indicated that the
environment in the laboratory was a major source of contamination of bacteria in the TIB
system. According to the results of bacterial distribution in the laboratory, the Bacillus
species were largely found in the investigated areas. Many researchers have indicated that
the sources of Bacillus in plant tissue culture are from the laboratory environment (Thomas,
2006, 2007; Odutayo et al., 2007). Thomas (2007) reported that Bacillus species, including
B. pumilus and B. subtilis were isolated from ethanol in plant tissue laboratories, whereas
Odutayo et al. (2007) found that B. cereus and B. subtilis were dominant in the laboratory
indoor air and in human skin. Therefore, every step of the plant tissue culture process
including explants handling, media preparation, subculturing, storage of sterile culture and
TIB sets, should be considered in order to prevent bacterial contamination. As for the use of
PPMTM as chemical biocide, the study suggests that it is not necessary to add this substance
into the culture media for bacterial contamination control because no difference was
observed with and without PPMTM in the medium. The most important aspect for working
with the TIB system is the good skill and practice of the operators in the plant tissue culture
laboratory. Furthermore, the sanitary state of the laboratory is another important factor for
the success of culture with TIB system.
It was also found that the major source of bacterial contamination in the 20 L TIB
system was coming from the environment. However, there were also endophytes in rice

443
cultures. The bacteria isolated from the contaminated rice TIB system were identified as
Bacillus, Curtobacterium, Microbacterium, Achromobacter, Acidovorax, Burkholderia,
Enterobacter, Klebsiella and Pantoea, and most of them have been reported to be endophytic
bacteria (Elbeltagy et al., 2000; Cottyn et al., 2001; Okunishi et al., 2005; Mano et al., 2006;
Mano and Morisaki, 2008; Jha and Kumar, 2009; Kaga et al., 2009).

CONCLUSIONS
In conclusion, the plant tissue cultured in 20 L TIB system has a high risk of bacterial
contamination, mainly caused by the laboratory environment. Guidelines for the prevention
of bacterial contamination should stress the importance of a good hygiene of the workers,
and a strict aseptic technique must always be kept in order to prevent cross contamination
from the environment into the plant tissue culture containers. Furthermore, the diversity of
bacterial contamination in TIB system depends on plant species, explant types and
environmental conditions.

ACKNOWLEDGEMENTS
The authors wish to thank the National Research Council of Thailand (NRCT) for the
research grants.

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