10 J. Trop. Plant Pests Dis. Vol. 24, No. 1 2024: 10–16 J. Trop. Plant Pests Dis.
Vol. 24, No. 1, March 2024
Pages: 10–16
ISSN: 1411-7525
E-ISSN: 2461-0399
DOI : 10.23960/j.hptt.12410-16
SHORT COMMUNICATION
Detection of the presence of bacteria causing grain rot disease (Burkholderia glumae)
in some rice seed producers in South Sulawesi, Indonesia
Abdul Rahman
3
, Rahmat Jahuddin
1
, Andi Khusnul Fatima Bahar
2
, Ahmad Yani
2
, & Baharuddin
Patandjengi
2
Manuscript received: 2 January 2023. Revision accepted: 19 July 2023. Available online: 11 December 2023.

ABSTRACT
One of the constraints in rice production is grain rot disease caused by Burkholderia glumae, which can be carried by seeds
grain. An observation to determine the presence of B. glumae in different grain yield classes of seeds was conducted by taking
samples derived from several seed producers in South Sulawesi. This research was carried out by first taking seed samples
at the South Sulawesi Agricultural Technology Study Center (BPTP), Tungro Research Workshop, South Sulawesi Main
Seed Center (Balai Benih Induk), PT. Sang Hyang Seri, PT. Pertani and PT. Harmoni were then tested at the Agricultural
Quarantine Laboratory of Makassar. Based on the observations, it was concluded that all samples in the sowing seed class
tested positive for B. glumae, supported by an average percentage of disease incidence of 25.13%, namely foundation seeds
from the Balai Benih Induk, as well as foundation seeds and stock seeds from AIAT.
Key words: B. glumae, extension seed, foundation seed, stock seed

Corresponding author:
Baharuddin Patandjengi ([email protected])
1
Department of Agrotechnology, Faculty of Agriculture,
Makassar Islamic University. Jl. Perintis Kemerdekaan No.
9. Kota Makassar, Sulawesi Selatan, Indonesia 90245.
2
Department of Plant Protection, Faculty of Agriculture,
Hasanuddin University. Jl. Perintis Kemerdekaan, Kota
Makassar, Sulawesi Selatan, Indonesia 90245.
3
Indonesian Quarantine Agensy, Jl. Harsono RM No.3. Jakarta
Selatan, Indonesia 12560.
INTRODUCTION
The high demand for rice must be supported
by increased rice production. According to data from
the Central Statistics Agency of Indonesia (CSA), the
productivity of the national rice yield in 2023 reached
52,59 quintals per hectare. One of the factors affecting
rice productivity is the quality of seeds and the use of
disease-free seeds. Increasing rice productivity does not
always run smoothly and tends to encounter problems.
One of these problems is plant diseases. Several new
diseases have been discovered in Indonesia, including
bacterial grain rot caused by Burkholderia glumae
(Wahidah et al., 2019; Sahlan et al., 2023). The presence
of B. glumae can also result in various diseases,
including sheath rot and seedling rot, commonly referred
to as panicle blight (Nandakumar et al., 2009; Ham et
al., 2011).
Grain rot disease in rice was first reported in Japan
in 1950 and has since become one of the important
diseases affecting rice plants worldwide (Zhou-qi et
al., 2016). B. glumae is one of the most damaging seed-
borne pathogens in many rice-producing regions. The
most severe infections have been linked to yield losses
ranging from 15% to 80% (Fang et al., 2009). According
to Regulation No. 51 of 2015 from the Minister of
Agriculture of the Republic of Indonesia, B. glumae has
been detected in rice plantations located on the Java,
Sumatra, and Kalimantan islands. This disease is more
prevalent in rice grains, earning it the name “grain rot”
in several Asian countries (Wamishe et al., 2014).
The spread of B. glumae must be monitored
because this pathogen affects both the quality and
quantity of rice. B. glumae infestation in rice plantations
in Indonesia has not yet reached the severity of bacterial
leaf blight caused by Xanthomonas oryzae and blast
disease caused by Pyricularia oryzae (Handiyanti et
al., 2018). In terms of ecological conditions, Indonesia
is an ideal location for the spread of bacterial grain rot,
given its hot and dry climate and high precipitation
levels. The presence of high nighttime temperatures
and frequent precipitation are environmental factors
that tend to exacerbate this disease (Karki et al., 2012).
The presence of bacterial grain rot disease has
existed since 1987, but since then, there have been no
reports indicating that this disease has caused severe
damage (Indonesian Ministry of Agriculture, 2015). The

Rahman et al. Detection of the presence of bacteria causing grain rot disease 11
presence of B. glumae has reportedly spread to several
areas in Indonesia such as Sulawesi, Java, North Sumatra
(Aflaha et al., 2020; Widarti et al., 2020; Hasibuan et
al., 2018). Since 2015, however, the existence of this
disease has begun to be reported again in several regions
of Indonesia. Due to global climate change, B. glumae
has been identified as an emerging pathogen in several
countries. Importing seeds from countries where rice
grain rot has recently been prevalent can serve as a
source of new inoculum (Handiyanti et al., 2018).
Molecular detection was performed to identify
the presence of B. glumae in multiple seed samples.
Molecular biology-based identification is considered
more accurate than morphological identification. Using
molecular identification techniques, the identities of
various plant diseases, including those whose pathogens
could not be isolated on artificial media, were revealed.
The Polymerase Chain Reaction (PCR) technique
is one of the molecular detection methods. PCR, a
highly sensitive technique, allows the detection of
low-abundant, slow-growing, or non-culturable cells
(Schaad et al., 2003). This method has a high level of
sensitivity and can be completed in a shorter amount
of time than other methods commonly used to detect
pathogens (Venbrux et al., 2023).
In this article, we provide an overview of grain
rot diseases and the incidence of the disease in the
fields of six South Sulawesi-based rice seed producers.
Additionally, we determined that grain rot disease
caused by B. glumae was detected using the PCR
technique with specific primers in some rice seed
producers in South Sulawesi.
MATERIALS AND METHODS
Research Site. The research was conducted at the
Agricultural Quarantine Laboratory of Makassar,
Indonesia.
Seed Sampling. The rice seed samples weighed 100
g per seed class in six seed sources, including three
seed-producing government agencies (BP= The South
Sulawesi Agricultural Technology Study Center (BPTP);
LT= Lolit Tungro (Tungro Research Workshop); BB=
Main Seed Center (Balai Benih Induk)) and three private
producing companies (SS= PT. Sang Hyang Seri; PT=
PT. Pertani; HR= PT. Harmoni). Seed classes were
further categorized as 1= Foundation seed, 2= Stock
seed, and 3= Extension Seeds. The combination of the
following symbols:
SS1=Foundation seed samples from PT. Sang
Hyang Seri;
SS2=Stock seed samples from PT. Sang Hyang
Seri;
SS3=Extension seed samples from PT. Sang
Hyang Seri;
PT3=Extension seed samples from PT. Pertani;
HR3=Extension seed samples from PT. Harmoni;
LT1=Foundation seed samples from Lolit
Tungro;
LT3=Extension seed samples from Lolit Tungro;
BP1=Foundation seed samples from BPTP;
BP2=Stock seed samples from BPTP;
BP3=Extension seed samples from BPTP;
BB1=Foundation seed samples from Main Seed
Center;
BB2=Stock seed samples from Main Seed
Center;
BB3=Extension seed samples from Main Seed
Center.
Disease Incidence of Symptomatic Rice Grains. The
observation of seed morphology involved selecting
seeds exhibiting symptoms of stripes or brown spots on
the surface of rice grains from each seed class. From a 1
kg sample for each seed class from every seed producer,
400 seeds were randomly selected to calculate the
percentage of symptom occurrence by distinguishing
between symptomatic and asymptomatic seeds. The
percentage of symptomatic seeds was calculated using
the formula:
DI
b
a
100%#=
DI=Disease Incidence;
a=The number of symptomatic seeds;
b=The total number of seeds observed.
Morphology and Molecular Identification of B.
glumae. The observation of seed morphology involved
selecting seeds that exhibited symptoms of stripes or
brown spots on the surface of rice grains from each
type of seed source.
Extraction of each sample was performed to
obtain total DNA, utilizing the DNeasy Plant Mini Kit
(QIAGEN) protocol. DNA amplification was carried out
using PCR reagents, including pure Taq bead ready to go
(GE Health), a pair of specific primers (1418S-1418A),
and nuclease-free water. The specific primers used to
amplify the DNA of each bacterial isolate were 1418S:
5’-GCG ATA TGG CAA GAC GCA AA-3 and Primer
1418A: 5’-AGT CAT ACC CTT TGT CAG CGT-3’

12 J. Trop. Plant Pests Dis. Vol. 24, No. 1 2024: 10–16
Table 1 showed that the intensity of attack
symptoms on the extension seeds was higher than on the
stock and foundation seeds. Extension seeds from PT.
Sang Hyang Seri had the highest percentage, reaching
32.25%, followed by 22% for stock seeds, and 16.25%
for foundation seeds from Lolit Tungro.
B. glumae DNA Amplification by PCR. The presence
of B. glumae was successfully detected by PCR. The
presence of ±575 bp DNA bands confirmed positive
amplification reactions in six seed samples (Figure 2).
Figure 2 showed the samples from Balai Benih
Induk tested positive for the stock seed and extension
seed samples. Samples from AIAT and PT. Sang Hyang
Seri tested positive only for the extension seeds, while
samples from Lolit Tungro (foundation and stock
seeds), PT. Harmoni (extension seeds), and PT. Pertani
(extension seeds) also tested positive. However, the
foundation seed samples from Balai Benih Induk, as
well as the foundation seeds and stock seeds from
AIAT, tested negative using the PCR detection test for
B. glumae bacteria that causes seed rot disease.
The disease that causes grain rot by B. glumae
is one of the bacteria that causes significant crop loss
worldwide. A study conducted by Mulaw et al. (2018)
reported that 45 out of 175 samples of bacterial panicle
(Aflaha et al., 2020).
The DNA amplification process was conducted
using a PCR Thermal Cycler machine (Applied Bio
System). The amplification reaction consisted of
denaturation at 94 ºC for 30 s, annealing at 58 ºC for 20 s,
and extension at 72 ºC for 30 s. This cycle was repeated
29 times, with the final extension at 72 ºC for 5 min.
The amplified DNA bands were visualized
through electrophoresis using 1.5% agarose at 90 volts
for 45 min. DNA band visualization was carried out
with Sybr green staining. The DNA marker employed
was a 100 bp ladder (Thermo), mixed with 5 μL, 2 μL
of loading buffer 5×, and 2 μL Sybr green. Detection
was performed using UV light on the gel documentation
system (UVP UPLAND CA). The presence of B. glumae
was confirmed by observing DNA bands with a size of
571 bp (Aflaha et al., 2020).
RESULTS AND DISCUSSION
Infestation and Morphology of Rice Seeds Infected
with B. glumae. Observation of seed morphology was
conducted by selecting seeds exhibiting symptoms of
stripes or brown spots on the surface of the rice grains
from each type of seed source (Figure 1).
Figure 1. Differences in samples of rice seeds. A. Symptoms of grain rot; B. Healthy seeds.
A B
Seed sources
The percentage of symptoms (%)
Foundation seed Stock seed Extension seed
PT Sang Hyang Seri (SS) 11.75 22.00 32.25
PT Pertani (PT) - - 25.75
PT Harmoni (HR) - - 26.50
Lolit Tungro (LT) 16.25 - 27.50
BPTP (BP) 6.00 17.50 18.25
Balai Benih Induk (BB) 8.25 14.25 20.50
Table 1. Percentage of symptomatic seeds

Rahman et al. Detection of the presence of bacteria causing grain rot disease 13
blight in Arkansas were caused by B. glumae, and
no disease was found to be caused by B. gladioli. B.
glumae infects rice grains, invades the plumule through
stomata and wounds, and spreads in the spaces between
parenchyma cells during the germination process.
Bacterial proliferation in the plumule supports bacteria
to produce toxic compounds such as toxoflavin, which
can cause the grain to rot (Zhou-qi et al., 2016). One of
the infected grains is sterile or causes the grains to turn
brown at the base (Goto et al., 1988).
The results of molecular detection showed
that not all seed classes were positively infected by
B. glumae. Samples from the seed class all tested
positive for B. glumae bacteria. Therefore, it becomes
a concern for seed producers in producing extension
seeds. This information will assist seed producers in
selecting the seeds to be produced. B. glumae, which
was previously only a minor pathogen, has increased
its status to become a major pathogen in rice. This is
related to favorable weather changes, namely warm
night conditions and high humidity and rainfall during
the growing season (Zhou-qi et al., 2016; Suharti et al.,
2017). Geographically, countries with semi-tropical and
tropical climates are relatively vulnerable to B. glumae
attacks because the optimum temperature for the growth
of this bacterium is relatively high, ranging from 30 to
35 ºC (Ham et al., 2011).
According to Pedraza et al. (2018), the primary
means of transmission for B. glumae is via seeds
that have been contaminated. The presence of the
pathogen in the soil or on infected plants poses a risk
of seed contamination during different stages, including
harvesting, processing, and storage (Kouzai & Akimoto-
Tomiyama, 2022). The aforementioned scenario may
lead to the extensive dissemination of the pathogen
among seed-producing enterprises. Furthermore, it
has been observed that B. glumae exhibits the ability
to endure and maintain its population during both
the vegetative and reproductive stages of rice plants
(Pedraza et al., 2018). Consequently, in situations where
the initial contamination stems from the seeds, the
pathogen possesses the ability to endure, reproduce, and
spread throughout the complete life cycle of the plant,
encompassing the pivotal stage of seed production.
The introduction and sustained presence of
the pathogen within seed-producing companies are
influenced by its existence in the environment, including
the soil and other plant species. B. glumae exhibits a broad
spectrum of plant invasion capabilities, encompassing
both monocotyledonous and dicotyledonous plant
species (Compant et al., 2008). This implies that the
bacteria exhibit the ability to persist in the soil through
the process of infecting and establishing colonies within
various host plants. Therefore, rice is widely regarded
as the plant species most vulnerable to invasion by B.
glumae.
Apart from environmental factors, the
pathogenicity of B. glumae is significantly influenced
by the presence of a type 3 secretion system (T3SS)
(Wallner et al., 2021). The pathogenicity of B. glumae is
contingent upon the utilization of quorum sensing (QS)
mechanisms to facilitate the production and secretion
of toxoflavin, a compound that is primarily responsible
for the extensive harm inflicted upon rice crops (Ham
et al., 2011). This phenomenon entails the ability of
bacteria to engage in intercellular communication and
synchronize their assault on the host plant, thereby
potentially facilitating its dissemination.
Rice bacterial diseases are relatively difficult
to control because the pathogen and its genetic
MK+12 76543 101198 1213
!575 bp
500 bp
250 bp
Figure 2. Results of B. glumae DNA amplification from rice seeds. M, DNA Marker 1 Kb (Thermo Scientific
USA); Rice seed samples: 1. PT3; 2. HR3; 3. LT1; 4. LT3; 5. BP1; 6. BP2; 7. BP3; 8. SS1; 9. SS2; 10.
SS3. 11. BP3; 12. BP2; 13. BP1.

14 J. Trop. Plant Pests Dis. Vol. 24, No. 1 2024: 10–16
characteristics are easily mutated, especially its virulence
against rice varieties. In the United States, it was
reported that there were more than 400 strains isolated
from rice plantations in various states (Nandakumar et
al., 2009). There are variations in virulence from highly
virulent to low virulence. B. glumae is known to produce
a toxin that is suspected as a virulence factor (Jeong et
al., 2003). Currently, there is no standard technique for
forecasting bacterial diseases, especially for the tropics,
so the exact timing of control cannot be determined.
The main control of rice diseases caused by
bacteria is by using resistant varieties. The resistance of
rice plants to B. glumae is highly dependent on the type
of variety used (Amirullah et al., 2020). Furthermore,
the effective implementation of environmental
management strategies is also of paramount importance
in mitigating the spread of B. glumae. These measures
include agricultural techniques such as crop rotation,
weed control, and soil management, which collectively
strive to reduce the prevalence of the pathogen in the
surrounding ecosystem (Akimoto-Tomiyama, 2021).
The use of resistant varieties is the main option
for controlling rice grain rot, but until now, there has
been no report on resistant varieties. The utilisation
of pathogen-free seeds is the primary preference due
to the inherent characteristics of the pathogen carried
by the seeds. However, it should be remembered that
even seeds that do not show disease symptoms are not
necessarily free from B. glumae. Therefore, the health
factor of the seeds must also be one of the parameters
of seed testing, in addition to agronomic factors.
The implementation of comprehensive seed
testing protocols is a crucial measure in protecting the
agriculture sector against inadvertent transmission of B.
glumae-infected seeds. Seed-producing companies can
enhance their biosecurity measures and guarantee the
provision of uncontaminated seeds to farmers by adopting
sophisticated detection technologies, conducting routine
inspections, engaging in collaboration with relevant
stakeholders, and making investments in employee
training.
CONCLUSION
The symptom of stripes or brown spots on the
grain surface of rice was found in all six different seed-
producing companies and all classes. According to the
results of molecular detection with specific primers,
some of the samples were infected by B. glumae. The
highest proportion of symptomatic seeds in every seed
resource was shown by the extension seed class. To
figure out the taxonomy of the bacteria at the level of
individual strains, sequencing reads need to be matched
to the bacteria.
ACKNOWLEDGMENTS
We would like to thank the members of the
Agricultural Quarantine Laboratory of Makassar and
the six rice producers in South Sulawesi BPTP, Tungro
Research Workshop, South Sulawesi Main Seed
Center (Balai Benih Induk), PT. Sang Hyang Seri, PT.
Pertani, and PT. Harmoni) for their assistance during
the experiment.
FUNDING
This study was supported by Ministry of
Research, Technology and Higher Education Indonesia
through Matching Fund 2022 program.
AUTHORS’ CONTRIBUTIONS
BP and RJ considered and planned the experiment.
AR and AY carried out monitoring rice fields in six
different locations and taking seeds samples. AR
performed molecular work and analysis. AY collecting
data on the plant damage area caused by B. glumae.
AKFB perform analysis and interpreting the plant
damage and prepared the manuscript. The authors
provided responses and comments on the research flow,
data analysis and interpretation as well as shape of the
manuscript. All the authors have read and approved the
final manuscript.
COMPETING INTEREST
We declare there are no relevant financial or
nonfinancial competing interests to report.
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