BIOTROPIA Vol. 28 No. 3,2021: 231 - 238 DOI: 10.11598/btb.2021.28.3.1344 

GROWTH INHIBITION OF FUNGAL PLANT PATHOGENS 
BY ANTAGONIST BACTERIA USING DUAL CULTURE ASSAYS 

YAN RAMONA* 

ZTchool $Biology, Facalg of Mathematics and Sciences, Uniuersitas Udqana 
" Integrated Laborat09 for Biosciences and Biotechnology, Uniuersitas Udqana 

Received 6 April 2020 /Accepted 26 July 2020 

ABSTRACT 

There has been excessive use of chemical-based pesticides globally resulting in significant environmental 
consequences with adverse effects o n  human health. Therefore, more sustainable environment friendly 
alternative solutions are needed to counter such environmental and health challenges. Development of 
biocontrol method is a sustainable solution to these challenges. The main objective of this research was to 
investigate the efficacy of potential antagonist bacteria to inhibit important fungal plant pathogens in uitro by 
applying dual culture assays o n  potato dextrose agar F D A )  or trypticase soya agar (TSA) media, with a view to 
isolate and screen potential antagonists for development of future biocontrol agents. The target pathogens were 
Fasariam o y p o r m ,  Ceratoystis sp., Apergilasj7au.w and Apergillz~s niger, commonly found to infect horticultural 
plants in Bedugul village in north Bali island. The antagonist bacteria were isolated from various sources such as 
soil of rhizosphere zone and roots of lettuce plants and mature compost. The potential of antagonist candidates 
was screened o n  the basis of inhibitory activity against targeted fungal pathogens. Bacterial antagonists with 
highest zone of inhibition were identified up to genus level using biochemical tests, and the results were matched 
with those specified in the Bergey's Manual of determinative bacteriology. Fifteen bacterial isolates were 
successfully isolated from various sources, and 60% of these isolates showed antagonistic activity in vitm against 
fungal pathogens with various degree of inhibition. This indicated the initial potential to develop as biocontrol 
agents. Based on preliminary identification, genera of BaciIIas and Psezldomonas were found to be the predominant 
isolates and in addition genus Acinetobacter was also identified in this study. 

Keywords: Antagonistic, A.pergiIlas $au.us, AspergiIIas niger, Ceratoystis sp., Dual Culture Assay, Fmrizlm 
0 ~ ~ p 0 m / m  

INTRODUCTION 

Chemical-based pesticides have been used to 
control plant pathogens in farming practices for 
decades, and its application in agricultural sector 
into the future wdl continue in the short to the 
medium term (Bueno e t  al. 2017), despite the 
pollution challenges. These chemical-based 
solutions such as pesticides are used to protect 
agricultural crops from attack by fungi, insects, 
weeds, and rodents as well as lengthen the 
lifespan of farming products or prevent spoilage 
(I<umar e t  al. 2012). Application of chemical 
pesticides is also common in non-agricultural 

*Corresponding author, email: yan-ramona@unud.ac.id 

sectors, such as sport facilities, shampoos for 
animals, and boats (Nicolopoulou-Stamati e t  al. 
2016). Long term application of chemical based 
pesticides in agricultural and non-agricultural 
sectors has resulted environment and health 
impact in everyday life. The residue of such 
harmful compounds is now found in soil, 
waterways, agricultural crops, milk and meat 
causing human health challenges (Kim e t  aL. 
2016). The presence of these chemical 
contaminations in soil also frequently kills 
beneficial microbiota of soil, such as mycorrhiza 
and plant growth promoting rhzobacteria 
beyond the target pathogens. Furthermore, 
increase in resistance of plant pathogens may 
occur as a result of excessive application of such 



BIOTROPIA Vol. 28 No. 3,2021 

chemical compounds in farming practices 
(Lucas e t  al. 2015). This may also result in 
increased dose or innovations in new pesticides 
that are needed to control the same pathogens 
(Fernandes et al. 2010). Other studies have also 
reported negative impacts of pesticide exposure 
to the consumer and community (Andersson 
2014). In terms of health impacts pesticides 
studies indicate neurological effects, diabetes, 
respiratory diseases, fetal diseases, genetic 
ksorders and cancer in humans (Andersson 
2014; IOm et al. 2017). Methyl bromide is an 
example of a widely used broad spectrum 
pesticide which was phased out in developed 
countries without an alternative (UNEP 2014). 
In addition it is claimed that these chemicals 
contribute to greenhouse gas emission (Heimpel 
et aI. 2013). 

Although above serious challenges exist, 
pesticides use in farming practices will continue 
and totally phasing out of such compounds will 
take time until better sustainable non-toxic 
alternatives are found (Bueno et al. 2017). 
Biological based sustainable technologies are 
being intensively researched towards the 
development of alternative methods to control 
plant pathogens worldwide. Integrated Pest 
Management (IPM), including the use of 
biological control agents to control pests and 
pathogens is one of the most favorable and 
sustainable solution paths globally. 

In Bali, particularly in Bedugul Village, crop 
rotation in -farming practices has been-applied 
for years. Horticultural crops planted in this 
village include tomato, lettuce, cabbage, potato, 
and broccoli. At the time of sample collection, 
t h s  farm was planted with lettuce plants, and 
therefore, the bacterial antagonists were isolated 
from soil in the rhizosphere zone and roots of 
lettuce plants from this area. In addition to these 
sources, we also isolated antagonists from vermi 
compost collected from a composting site in 
Denpasar city where earth worms were used as 
starter in the process of composting. Previous 
literatures for example in Gehan e t  a/. (2018) 
have reported that mature composts (including 
vermi compost) are rich in antagonists of fungal 
pathogens. 

Based on the above rationale, the goal of t h s  
study focused on isolation, screening, and 
identification of potential of bacterial biocontrol 
agents to inhibit plant pathogens, with a view 

toward the development of biocontrol agents, so 
that chemical-based fungicide use in the future 
can be reduced. The primary objective of t h s  
study was to investigate the effectiveness of 
bacterial antagonist isolates to inhibit the in vitro 
growth of several fungal plant pathogens 
(Fasam'am oqporam, Cerato ystis sp., AspepiIIas 

flavas, and Aspegjills niger), commonly found to 
infect horticultural plants (including lettuce 
plants) in Bali, particularly in Bedugul area 
(Suryanti et aL. 2013) and neighboring villages of 
Bedugul, including I<embang Merta village 
(Wulansari et al. 201 5). 

MATERIALS AND METHODS 

Fungal Pathogens 

Fungal pathogens (Fasam'am oypomm, 
Cerato ysth sp., A.pe@I/as yavzls, and Ape@IIas 
niger) were obtained from stock culture 
collections of the Mycology Laboratory, School 
of Biology, Universitas Udayana, Bali. These 
fungal isolates were identified in our previous 
study from samples collected in Bedugul Village 
(Suryanti et aI. 2013). For regular use, these 
pathogens were subcultured on fresh PDA. For 
long-term storage, they were grown for 48 hours 
at ambient temperature on potato dextrose agar, 
cut into 1 x 1 cm2, placed in sterile distilled 
water, and stored at 4 "C. 

Sample Collection and Isolation of Potential 
Bacterial Antagonists 

Antagonists of plant pathogens were isolated 
from several sources, namely mature vermi- 
compost (a composting site in Denpasar City), 
soils sampled from rhizhosphere in lettuce farm 
in Bedugul, Bali, and rhizoplane (roots) of 
lettuce plants from Bedugul, Bali. Isolation of 
bacterial antagonists from these sources was 
conducted by applying serial dilution and plating 
method on potato dextrose agar (PDA) or 
trypticase soya agar (TSA). Samples weighing 10 
g were dissolved in saline solution (0.85% w/v 
NaC1) and volume was adjusted to 100 mL to 
obtain dilution rate of lo-', and stomached for 
20 minutes to release attached microbes on 
particles of soil or other samples. These samples 
were then further diluted in saline solution up to 

and 0.1 mL of sample from each dilution 



Growth inhibition of fungal plant pathogens by bacterial antagonists - Yan Ramona 

was then spread onto either potato dextrose agar 
(PDA) or trypticase soya agar (TSA), followed 
by incubation at ambient temperature for 24 -72 
hours. Colonies that could be distinguished 
appeared on media (at dilution rates of 10" - 

following 48 hours incubation at ambient 
temperature and were streaked for single colony 
isolation to obtain pure culture isolates. These 
pure cultures were then suspended in trypticase 
soya broth (TSB) or potato dextrose broth 
(PDB) with 30% glycerol in the medium, and 
stored at -80°C (as stock cultures) until needed 
for further experiments. 

In vitro Dual Culture Assay for Screening 
Potential Antagonists 

Isolates of bacteria obtained previously were 
spot inoculated in triplicates onto the periphery 
of PDA or TSA plates (corresponding to the 
medlum on which they were isolated). Plugs 
(1 cm2) of fungal pathogens were then placed in 
the center of the plates followed by incubation 
at ambient temperature or approximately at 
25 "C for 1 to 7 days, after which time the 
inhibition zone between the antagonists and 
pathogens were measured from three different 
angles using a vernier calipers, and then 
averaged. Three replications were made in 
the experiment to obtain representative data. 
Isolates showing inhibition zone were 
subcultured on fresh PDA or TSA and stored at 
4 OC for further study. For long term storage, 
these isolates were stored in TSB with 30% 
glycerol and stored at -80 "C. 

Preliminary Identification of Biocontrol 
Agent Candidates 

Bacterial isolates showing in vitro antagonistic 
activity against tested plant fungal pathogens 
were preliminarily identified up to genus level on 
the basis of their morphological characteristics 
and specific selected tests (for soil bacterial 
identification) and the results were matched with 
those indicated in Bergey's Manual of Systematic 
Bacteriology (Krieg & Holt 1984; Holt e t  al. 
1994). The tested characteristics of bacterial 
antagonist candidates were: Gram stain and cell 
morphology, oxidative/fermentative properties, 
spore stain, motility, flagella stain, oxidase, 
urease, and catalase production, ability to 
hydrolyze casein, UV fluorescence, levan 

production, gelatin hydrolysis, casein hydrolysis, 
and starch hydrolysis. The procedures 
(preliminary identification of soil bacteria up to 
genus level) of all tests followed the steps 
specified in Ramona (2003), Suryanti e t  al. (2013) 
and Wulansari e t  al. (201 5). 

RESULTS AND DISCUSSION 

A total of fifteen (15) distinguishable 
bacterial isolates were successfully isolated from 
this study. Of these, 60% of the isolates showed 
antagonistic activity in vitro against one or more 
tested fungal pathogens with various degree of 
inhibition (Fig. 1 and Table 1). Rhizosphere 
zone of lettuce plants appeared to be the best 
source of potential antagonists. Most bacterial 
antagonists isolated in this study were obtained 
from such zone and they showed various degree 
of in vitro inhibition of plant pathogens 
(Table 1). Surprisingly, only 2 isolates from 
lettuce roots inhibited the plant pathogen in vho. 
Bacterial isolates obtained from vermi-compost 
inhibited the in vitro growth of all fungal 
pathogens used in the study with various degree 
of inhibition (Table 1). It can be seen in Table 1 
that isolates A4, A5, T I ,  and T2 which were 
identified as BaciIIzts or P~eztdomonas (Table 2 
and 3) appeared to have the widest spectrum of 
in vitro inhibition. These isolates inhibited the 
growth of all tested fungal pathogens with 
various degree of inhibition, depending on the 
species of pathogens. These results provided us 
with initial indication of antagonists with 
potential to be developed as biocontrol agents. 
T h s  can now be targeted for further greenhouse 
or field trial scale studies for validation and 
effective application. The inhibition zone 
produced by these bacterial antagonists against 
the targeted fungal pathogens varied between 
0.86 f 0.11 cm and 3.20 f 0.00 cm (Table 1). 
Isolate A1 whch was identified to belong to 
genus of Pseudomonas only inhbited the 
growth of Fzt~aiztm o.yqbormm, with inhibition 
zone of 1.83 f 0.15 cm (Table 1 and 2). The 
o r i p  of this isolate was the rhzosphere zone of 
lettuce plants. 

The limited number of isolates obtained in 
t h s  study (Table 1, 2, and 3) was partly due to 
the primary focus on budding overall knowledge 
and basic tools related to in uitro cultivation of 



BIOTROPIA Vol. 28 No. 3,2021 

beneficial antagonistic microbes against fungal 
pathogens of Horticultural crops and from this 
foundation then subsequently build a wider 
screening system for extensive isolation of 
biocontrol agents. Previous reports indicated 
that an optimal optimized screening system is 
essential for in vitro cultivation from wider 
substrates (Weller 1988) and this study advanced 
this objective. Our results from this initial study 
were in line with Malleswari (2014) and Suthar e t  
al. (2016) who reported similar numbers of 
antagonist isolates from soil samples. In 
synthetic media, such as TSA or PDA 
(considered as general media for bacteria and 
fungi, respectively) genera of Bacillus, 
Pmdomonas, and some genera belonging to 
enterobacteriaceae commonly grow well on such 
media and dominate during culturing. Bacteria 
or fungi with specific or unidentified need will 
not show growth response on such media and 
has been reviewed previously by Pham and Kim 
(2012) and as suggested only 1% of soil bacteria 
may be culturable on synthetic media. 
Therefore, there is a need for further 
improvement of methods as well as content and 
quality of synthetic media so that wider range of 
soil bacteria become culturable and therefore 
increase the probability to obtain potential 
isolates for biocontrol development. Pham and 

IGm (2016) proposed a novel method for 
bacterial cultivation by applying polycarbonate 
permeable membrane of trans well plates in 
liquid cultures with common synthetic media to 
mimic the actual environmental conditions. By 
applying such method they increased numbers 
of culturable isolates by more than 20%. Such 
methods will be built on the foundations of this 
study in future strategies. 

The dual culture assays applied in our study 
(following isolation of antagonist candidates) 
appeared to be very convenient to initially 
indicate antagonism of bacterial antagonists 
against plant fungal pathogens. These results will 
have to be evaluated and correlated for their 
effectiveness in further greenhouse or field 
scales experiments. Overall, this is a classical 
method and has been used effectively to isolate 
and initially screen bacterial antagonists of 
fungal plant pathogens in vitro. Other studies 
recently applied this method to obtain bacterial 
antagonist candidates and subsequently 
identified the same to target fungal pathogens 
(Suthar e t  al. 2016; Wang e t  a l  2019); and 
Manandhar e t  al. 2019). This indicates that dual 
culture method is still a reliable approach to 
initially screen and identify antagonistic activity 
of a bacterial isolate against fungal or bacterial 
pathogens. 

digwe 1 Some examples of ia .uitw antaganism between bacterial isolates obtained in this study and the tested 
pathogens. (a) P s 8 ~ h m o w  sp. vs C ~ ~ k o p i f  sp., @) Ps81domona sp. vs AspergdIhs niggt", (c)~ B~ILzfs sp. vs 
RFpqdII~@aas and (d). B(dn'&s sp. vs Ffian'itrn o x ~ p m r n  
Note: Arrowheads show the inhibition zones between the bacterial antagonists and tested fungal pathogens. 



Growth inhibition of fungal plant pathogens by bacterial antagonists - Yan Ramona 

Table 1 Average diameter of in vitro inhibition zones (on PDA or TSA media) of bacterial antagonist candidates on 
several plant pathogens ( F m v i u m  o q ~ o r z l m ,  Ceratoystis sp., A.pergilusj7auas, and A.pegiIIas niger) 

Diameter of inhibition zones o n  fungal pathogens (cm)* 
Isolate codes Source of antagonists 

F. oq.porzlm Cerato ystis sp. A. j7auu.s A. niger 
A1 (PDA) Lettuce rhizosphere 1.83 f 0.15 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 
A4 (PDA) Lettuce rhizosphere 1.06 f 0,06 2.20 f 0.10 1.50 f 0.10 1.60 f 0.11 
A5 (PDA) Lettuce rhizosphere 3.20 f 0.00 2.46 f 0.1 1 1.33 f 0.11 1.30 f 0.10 
A6 (PDA) Lettuce rhizosphere 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 
T I  (PDA) Vermi-compost 0.96 f 0.06 1.80 f 0.06 1.13 f 0.11 1.13 f 0.11 
T2 (PDA) Vermi-compost 0.86 f 0.11 1.83 f 0.06 1.26 f 0.06 0.90 + 0.10 
S1 (TSA) Lettuce rhizosphere 1.63 f 0.06 0.00 f 0.00 3.36 f 0.30 0.00 f 0.00 
S2 (TSA) Lettuce rhizosphere 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 
S3 (TSA) Lettuce rhizosphere 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 1.13 f 0.15 
F1 (TSA) Roots of lettuce 1.50 f 010 0.00 f 0.00 2.70 f 0.10 0.00 f 0.00 
F2 (TSA) Roots of lettuce 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 
F3 (TSA) Roots of lettuce 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 
R1 (TSA) Roots of lettuce 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 
R2 (TSA) Roots of lettuce 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 
R3 (TSA) Roots of lettuce 0.00 f 0.00 0.00 f 0.00 1.60 f 0.17 0.00 f 0.00 

Notes: * Values in Table 1 f standard deviation are averages of triplicates; PDA (Potato Dextrose Agar) and TSA 
(Trypticase Soy Agar) were media used in the dual culture assays. 

The relative average inhibition zones shown 
in Table 1 varied from 0.0 to 3.36 cm, and these 
apparently were affected by types and the origin 
of the isolates. The zone of inhibition was also 
determined by species of fungal pathogens 
tested. Generally, bacterial isolates obtained 
from rhizosphere of lettuce plants was found to 
be antagonistic against one or more fungal 
pathogens tested in this study (Table 1). Similar 
results were also reported by Ramona (2003) 
and Wang et al. (2019) who found abundant 
antagonist isolates in the rhizosphere zones of 
various plants. This indicates that rhizosphere 
area can still be considered as a reliable source 
of bacterial antagonists of plant pathogens for 
development of biocontrol agents. In addition 
to rhizosphere zone, antagonists can also be 
isolated from many sources, such as mature 
compost or surface of plant roots (rhizoplane) 
(Table 1). Some researchers have also often 
isolated these antagonists from suppressive soil 
(Schlatter e t  al. 2017), suppressive compost 
(Gehan eta/. 2018), or from contaminated media 
in the laboratory (Ramona 2003). 

The origin of the antagonist isolates is not 
the most important aspect in the development 
of biocontrol agents. The effectiveness of these 
antagonists in protecting horticultural plants 
from attack by certain pathogens either in 

greenhouse or field applications is apparently 
more important consideration than their origins. 
To increase the effectiveness of antagonist 
isolations, ideally these antagonists should be 
isolated from places where these agents are to 
be applied, as they are already well-adapted in 
such areas of plant systems (TVtupps 2001; 
Ganeshan & I<umar 2005; Dukare e t  al. 201 8). 

Spectrum of fungal pathogen inhibition in 
vitro of the bacterial isolates obtained from this 
study varied among species (Table 1). Some 
isolates were able to control only one tested 
fungal pathogen, while the other controlled two 
or more pathogens (Table 1). Generally, 
bacterial antagonists isolated from verrni- 
compost and rhizosphere zone of lettuce plants 
appeared to have wider spectrum of fungal 
pathogen inhibition in vitro when compared to 
those isolated from other sources. Surprisingly, 
isolates obtained from Rhizoplane (hair-root 
surfaces) of lettuce plants were found to be less 
effective in inhibiting the four fungal pathogens 
in vitro than isolates obtained from other sources 
(Table 1). This phenomenon was contradictory 
to that reported by Wei et a/. (1996) and Walia et 
al. (2013), but in line with that reported by Oka 
(2004). Although some isolates in Table 1 
showed a wide spectrum of fungal pathogen 
inhibition, these isolates will have to be further 



B I O T R O P I A  Vol. 28 No. 3 , 2 0 2 1  

tested to study their effectiveness when applied 
in glasshouse or field trials. Ramona (2003) 
reported that a positive correlation between the 
results of in vitro bioassay and those of 
glasshouse or field trials may not be necessary. 
The reasons for these were extensively reviewed 
by Whipps (2001) and O'Brien (2016) and wider 
testing is essential following the screening for 
practical application. 

Although observable inhibition zones were 
clearly indicated in the in vitro bioassay Fable 1 
and Fig. I), the mechanisms by which bacterial 
antagonists may inhibit the fungal pathogens 
(for examples: antibiosis, siderophore 
production, or other mechanisms) was still 
inconclusive. Besides that, the effect of this 
antagonism (whether it was lethal or fungi static) 
has not yet been answered and w d  be 
considered in future studies. 

Bacterial isolates with abllity to inhibit the 
growth of at least one tested fungal pathogen 
were initially identified up to genus level, based 
on the method of soil bacterial identification 
specified in Ramona (2003) and Suryanti e t  al. 
(2013). The results are shown in Table 2 and 
Table 3. Based on these results, the genus name 
of the isolates was determined. One of the 

Pseadomonas isolates showed fluorescence 
properties under UV light, in addition to the 
results of other tests generally used in the initial 
preliminary identification of soil bacteria, and 
therefore, it was identified as P. flzlorescence (Table 
2). Among the 9 bacterial antagonists, 5 isolates 
belong to BaciIIas sp. as all of them produce 
endospore and showed characteristics of genus 
BaciIIas as shown in Table 3. 

P r e h n a r y  identification of the isolates 
obtained from this study showed that genera of 
BaciIIas and Pseadomonas were predominant 
bacterial isolates contained in all samples (Table 
2). This is in line with that reported by ICumari 
and IUlanna (2016) and Soare e t  al. (2019). The 
presence of these two bacterial genera in the soil 
or other samples is relatively more abundant 
than others and is partly due to simpler 
requirements for growth of these groups when 
compared to others (Raaijmakers e t  al. 2010). 
The ability of BaciIIus sp. to produce endospore 
also contributes to the survival of this genus in 
adverse environmental conditions (ICumari & 
IUlanna 201 6). Besides Ban'IIas and Pseadomonas, 
genus of Acinetobacter was also isolated in this 
study (Table 2). 

Table 2 Preliminary identification of Gram-negative bacterial antagonists 

Isolate Gram Flagella UV Starch Levan Gelatin Preliminary 
O / F  Motility Oxidase Pigment 

codes stain Position fluorescence hydrolysis production hydrolysis Identity 

A1 negative 0 + Polar + + - Pseudomonas sp. 
A4 negative 0 + Polar + + + - P.~uorescence 
TI negative 0 + Polar + + - Pseudomonas sp. 
S1 negative 0 ND + Creamy Acinetobacter sp. 

Note: O/F: Oxidative/Fermentative. 

Table 3 Preliminary identification o f  Gram-positive bacterial antagonists 

Isolate Gram Flagella Starch Casein Preliminary 
O / F  Motility Catalase Pigment Spore Urease colony 

Codes stain position Hydrolysis hydrolysis Identity 

A5 positive 0 + Polar + + + + + dry BaciIIus sp. 
T2 positive 0 + Polar + + + + + dry BaciIIus sp. 
S3 positive 0 + Polar + + + + dry Ban'IIus sp. 
F1 positive 0 + + + + dry BaciIIus sp. 
R3 positive 0 + polar + + + + + dry BaciIIus sp. 

Note: O/F: Oxidative/Fermentative. 



Growth inhibition of fungal plant pathogens by bacterial antagonists - Yan Ramona 

CONCLUSION 

Nine out of 15 (60 Yo) bacterial isolates 
showed antagonistic activities against several 
tested fungal pathogens in the in vitro dual 
culture assay, although the mechanisms by 
which these biocontrol candidates inhibit the 
fungal pathogen is still inconclusive, and 
therefore further study is needed to specifically 
elucidate the mechanisms (such as antibiosis, 
production of siderophore, etc.) of biocontrol. 
In the initial and preliminary identification of 
these antagonists, it was found that genera of 
BaciIIus and P~eudomonas were predominant 
isolates found in all samples. Besides these, 
genus of Acinetobacter was also isolated and 
identified in this study. 

ACKNOWLEDGEMENTS 

The author wishes to acknowledge laboratory 
of Mycology, School of Biology, Universitas 
Udayana for the provision of cultures of fungal 
pathogens. The Laboratory of Illicrobiology, 
School of Biology, Universitas Udayana, and the 
Integrated Laboratory for Biosciences and 
Biotechnology, Universitas Udayana should also 
be acknowledged for the provision of facilities 
and equipment during this project. Special 
gratitudes also go to Prof I<alidas Shetty 
(NDSU-USA) for his proofreading and criticism 
on this manuscript. 

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