Partial Characterization of Bacteriophages from Indonesia and its Potency
as Biocontrol of Xanthomonas oryzae pv. oryzae
Desi Rejeki1, Hardian Susilo Addy1,2,4* and Erlia Narulita1,3
1Post-Graduate Program, Study Program of Magister
Biotechnology, University of Jember, East Java 68121, Indonesia
2Division of Biology Molecule and Biotechnology,
Center for Development of Advanced Sciences and Technology, University of Jember, Jawa Timur, Indonesia
3Faculty of Teacher Training and Education, University of
Jember, Jember, East Java 68121, Indonesia
4Center of Excellence for Biotechnology of Plant Industry
(PU-BioTIn), University of Jember Indonesia
*For
correspondence: hsaddy.faperta@unej.ac.id
Received 07 December 2019;
Accepted 18 February 2020; Published 10 December 2020
Abstract
Bacterial leaf blight (BLB) is a disease
caused by Xanthomonas oryzae pv. oryzae
(Xoo) of rice in rice-producing countries including Indonesia and attack rice in all stages of growth. In the advanced, crop production will be decreased by up to 50–70%. Recently, the practical efforts to overcome the problem by using resistant
varieties, antibiotics, and sanitation; however, the ability
of the pathogen to
forms the new virulent pathotypes is
noteworthy. Alternatively, the pathogen could be environmental-friendly controlled by utilizing bacteriophages as
biological control agents because of their specific characteristics to their
bacterial hosts. This research aimed to obtain information
about the characteristic of the first isolated bacteriophages from Indonesia. The result showed that two bacteriophages had been isolated from soil in Arjasa Jember and soil in Gadingan
Situbondo, namely phage XooX1IDN and phage XooX2IDN, respectively. The two phages were inactivated at 80şC and stable
at pH within the range of 6 to 8. The phage XooX1IDN has a genome
size of approximately 39 kb, while phage XooX2IDN had a genome size 38 kb. Morphologically, both phages possessed the family of Myoviridae. Phage biocontrol in vitro assay showed that both phages significantly reduced the
growth of BLB pathogen, indicating that both phages potentially, as biological
control agents for BLB disease in rice. © 2021 Friends Science Publishers
Keywords: Bacterial leaf blight; Phage therapy; Myoviridae; Xanthomonas oryzae
Introduction
Xanthomonas oryzae pathovar (pv.) oryzae (Xoo)
is a Gram-negative bacterium found in field of
rice-producing countries including Indonesia. This bacterium is a causative
agent of bacterial leaf blight (BLB), a destructive bacterial disease that is prevalent among various rice
varieties in the rice growing countries including Indonesia
(Singh et al. 2015). Since the pathogen multiplies in
xylem and predominantly invades the vascular tissue, the most common symptom of
this disease is wilting, especially in young leaves namely “Kresek” disease and
decrease rice production (Nino-Liu et al. 2006). BLB remains a serious problem on rice production,
especially in Asia where the infection of pathogen results in enormous losses
of yield ranging 6 to 90 percents in
some rice varieties (Singh et al. 1980; Bhutto
et al. 2018).
Numerous
studies have reported the management strategies of bacterial leaf blight such as
chemical control, genetic resistance, and biological control (Kim et al. 2016). A number of studies have reported plant genes that confer resistance against X. oryzae through the plant
breeding using series of resistance gene (R genes), designated from the Xa
genes of rice cultivars (Degrasi
et al. 2010). Unfortunately, this strategy is ineffective due to the ability
of BLB pathogen to form a new and more
virulent pathotypes because of Xoo’s diversity and
gene mutation mechanism of X. oryzae
to breakdown the resistance genes of rice (Keller et al. 2000; Ponciano et al.
2003; Shanti et al. 2010).
Biological control thus seems to be an alternative way
to manage this disease being cost-effectively, sustainable
and eco-friendly (Gnanamanickam 2009). Among the alternative of biological control agent,
the use of bacteriophage could be a promising control technique, known as phage
therapy (Addy et al. 2012a).
Bacteriophage is a virus that infects and multiplies within bacterial host cells, causing lysis along with the development of bacteriophage
particles in specific host cells, and attacks a narrow bacterial strain (Beaudoin et al.
2007). Recently, the use of the phage as an approch to control bacterial
pathogens has been highly attractive since some reports proved the potency of phage to
control it bacterial host (Svircev et al. 2018).
Ralstonia phage RsoM1USA has been found to have ability to inhibit the growth
of Ralstonia solanacearum, a bacterial wilt pathogen on several crops (Addy et
al. 2019). Moreover, Ahmad et al.
(2014) isolated CP1 and CP2
bacteriophages that were able to control X. axonopodis pv citri on citrus. Mostly, bacteriophage
can be easily isolated from
the soil and irrigation water
around infected crops
(Bielke et al. 2012; Kalpage and Costa 2014) and from the symptomatic plant parts (Ritchie and
Klos 1977).
Although, bacteriophage is easy to explore; however, the selection of
bacteriophage isolates become crucial point in exploitation of
bacteriophage for phage therapy (Addy et
al. 2012a; Svircev et al. 2018). It is because bacterial host
cells exhibit the changes in virulence after infection by the phage such as
production of plant toxin and increase in virulence factors (Verheust et al. 2010). For example, infection of Ralstonia phage RSS1
increases the virulence of R.
solanacearum to be more destructive on tomato (Addy et al. 2012b). In contrast, phage XacF1 decreases the virulence of Xanthomonas axonopodis pv citri to infect citrus leaves (Ahmad et
al. 2014).
Several studies have been reported to explore bacteriophage as
biological control agent of X. oryzae pv.
oryze. About 10 bacteriophages have
been isolated from Vietnam and Thailand (Kovács et al. 2019), China (Dong et al. 2018), Japan () and India (Ranjani et al. 2018). None
of the study has been reported on the bacteriophage of X. oryzae isolated from Indonesia. Therefore, this study is aimed to explore the bacteriophage as an initial step prior to its use as biological
control agents for the first time from
Indonesia.
Materials and Methods
Bacterial strain
Xanthomonas
oryzae XooJ2 was isolated from the infected rice leaves (56-day-old plant
after transplanting) in the rice field showing “Kresek” symptoms and was
routinely cultured on yeast extract
dextrose agar (YDA) at 28°C for 72 h. The bacterium
was confirmed through several biochemical tests such as the KOH solubility assay, catalase test, starch hydrolysis
assay, and pathogenicity test using cultivar Logawa (Schaad et al. 2001). In addition, confirmation was done by detecting the presence of specific gene sequence in X. oryzae pv. oryzae was done through polymerase
chain reaction (PCR) using specific pair primer of JLXoo-230F (5'-CCT CTA TGA
GTC GGG AGC-3') and JLXoo-230R (5'-ACA CCG TGA TGC AAT GAA GA -3'). The GoTag PCR mixture (Promega, USA) was
subjected to a 35 cycles after pre-denaturation at 96°C for 5
min, followed by denaturation at 96°C for 1 min, 55°C for 3 min,
72°C for 1 min, and a final extension step at 72°C
for 7 min. The PCR
product was subjected to gel electrophoresis in a 1.5% (wt/vol) agarose gel in TAE, followed by staining with ethidium bromide (Lu et al. 2014).
Isolation and purification of
xanthomonas infecting bacteriophages
One gram
of soil samples,
collected from rice fields in District Arjasa, Regency, Jember and District Gadingan, Regency Situbondo, East
Java Province, Indonesia, were used for phage isolation using the basic enrichment
method (Addy et al. 2019). Briefly, soil sample was
suspended with 2 mL of sterile water and shaken for 24 h. One milliliter of
suspension was taken and filtered through 0.45-µm membrane filter (Steradisc, Krabo Co., Japan) and use
as phage lysate in plaque assay with XooJ2 as host. Bacteriophages were then purified as described by (Ahmad et al. 2017). Routinely, 24 h-old bacterial culture was used as host for phage’s
propagation. Pure bacteriophage particles
were stored at 4°C until used in further testing (Addy et al. 2019). The morphology of phages was assessed by transmission
electron microscopy.
Nucleic acid digestion and protein profile
To
determine the nucleic
acid type of bacteriophages, the genome of bacteriophages was digested with EcoRV restriction
enzyme according to the supplier’s instructions (Promega, USA). Eight
microliters of phage DNA suspension was mixed with 9.5 µL sterile distilled water, 2 µL enzyme buffer and 0.5 µL restriction enzyme (EcoRV or XbaI) the mixture was incubated at 37°C for 60 min. DNA fragments
were subjected
to gel electrophoresis in 1% agarose gel.
To determine
the protein profile, whole phage particles were subjected to Sodium
Dedoxyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis. Briefly, whole phage particles were
harvested using ultracentrifuge (Hitachi, Japan) at 4°C, 30.000 × g for 2 h and
equal volumes of sample buffer (0.5 M Tris-HCl (pH 7.2) buffer containing 4%
SDS, 10% β-mercaptoethanol,
20% glycerol, and 0.1% bromophenol blue) was added. The samples
were boiled for 5 min. Gel was then stained and
visualized using Coomassie brilliant blue dye.
Host specificity assay
To determine the host
specificity of phage XooX1IDN and XooX2IDN, the purified phage was subjected to spot testing using XooJ2 and R. solanacearum DT3 as the bacterial target. In this test, three
microliters of the phage suspension (103 PFU/mL) was spotted on top of the double-layered YDA plate. The formation of a clear
zone on the spotting area indicated that the bacterium were susceptible to the phage.
Potentially susceptible strains were tested further by serial dilution plaque
assay to determine whether they were truly susceptible to the phage (Ahmad et al. 2017).
Bacteriophages stability assays
Xanthomonas phages were tested
for their stability against environmental factors such as temperature and pH (Iriarte et
al. 2007). To determine the effect of temperature on the stability
and infectivity of bacteriophages, the purified
phage particles in SM buffer were incubated at different temperatures, 30°C to 80°C. While to determine the effect of pH, bacteriophage particles in SM buffer was adjusted to reach various pH
of 3 to 9 followed by incubation at room temperature. Phage number was
estimated by calculating plaque on the YDA plate using isolate XooJ2 as a host.
Biological control assay in vitro
To determine the effect of phages on XooJ2 (susceptible host), the growth
of XooJ2 in NB medium (in 24-well plates) at 28°C was monitored on the phage
XooX1IDN- and XooX2IDN-treated and untreated XooJ2. Briefly, the concentration of the overnight culture of XooJ2 was
adjusted with NB to initial
OD600 of 0.3, and 1.5 mL of the bacterial
suspension was added to each well of the 24-well plate. One hundred and
fifty microliters of phage suspension was then added at m.o.i of 0.01,
0.1, 1.0, and 10, respectively, and the plate was incubated inside Microplate
reader SH-1000 (Corona Electric, Japan) with slow shaking. SM buffer was
used as a phage control (m.o.i of 0). Bacterial growth was estimated by measuring
the absorbance at 600 nm every 180 mins for 36 h. This
experiment was repeated three times with three replications for each m.o.i treatment (Addy et al. 2019).
Results
The bacterial leaf blight pathogen
The isolate
XooJ2 was isolated from 56-day-old
rice from the
symptomatic leaf of bacterial leaf blight in Jember. The isolate XooJ2 was
purified and characterized by its biochemistry and molecular properties. The
bacterium XooJ2 exhibited yellow, round in shape, convex, smooth surface, and
flat edge colonies when grown on
Nutrient Agar (NA) media (Fig. 1A). Furthermore,
the genome of XooJ2 was subjected to PCR amplification using the Xanthomonas
oryzae pv. oryzae specific PCR primer and resulted in the predicted band with an approximate size of 230 bp (Fig. 1B). The
isolate XooJ2 also produced leaf blight
symptoms after re-inoculation to the rice leaf (Fig. 1C).
Morphology plaques
and phages, nucleic acid, and protein profile
Phage XooX1IDN and XooX2IDN, isolated from rice
fields in Jember and Situbondo, showed turbid
plaques (diameter 2 ± 1 mm) on tested medium (Fig. 2).
Transmission electron microscope revealed similar tailed forms of both
phages (Fig. 3). Analysis of
protein bands patterns through SDS-PAGE showed that all
bacteriophages had a
similar composition of more than 10 sub-units of
protein (Fig.
4A).
Fig. 1: Partial characteristic of bacterial host XooJ2, a pathogen of bacterial leaf blight on rice.
XooJ2 colonies on YDA medium exhibit yellow colonies (A), agarose gel electrophoresis of PCR product of 230 bp
using specific pair primer (B), and The leaves exhibit bacterial leaf blight symptoms in
the field and the result of the reinoculation assay (C)
Fig. 2:
Plaques morphology of
phage XooX1IDN and XooX2IDN on tested media
Fig. 3: Transmission electron microscopy of negatively
stained (A) XooX1IDN
and (B)
XooX2IDN
particles at 50-k fold magnification and at an acceleration voltage of 80 kV. Scale bar represents 50 nm
The genome of both bacteriophages of
non-digested endonuclease was more than 10,000 bp and was clearly digested with DNAse and
endonuclease restriction enzymes, but not RNAse (Fig. 4B). Moreover, EcoRV restriction enzyme provided
similar patterns except for the particular
Fig. 6: Effects of phage XooX1IDN and XooX2IDN on the
growth of XooJ2. (A)
The XooJ2 growth characteristic in NB medium inoculated with phage XooX1IDN and XooX2IDN at different
multiplicity of infection
(m.o.i).
Controls were medium with (Ck+) and without the host XooJ2 (Ck-). The culture
turbidity was observed at 24 h (bottom) and 48 h (upper) after phage
inoculation. (B) The growth curve of XooJ2 after inoculation with
bacterial host and phage at m.o.i of 0 (red), 0.01 (green), 0.1 (purple), 1 (blue), and 10 (orange), respectively. The XooJ2 cell density was monitored by measuring the absorbance at 600 nm every 3 h for 36 h. The
data are presented as the means from four replications for each m.o.i treatment. The
error bars indicate the standard deviation
band around 7.0
kbp (Fig. 4B, lane
4) and was predicted to have a genome size of
approximately about 39 kbp for phage XooX2IDN and about 38 kb for phage
XooX2IDN.
Effect of
temperature, pH and host specificity
Some
environmental factors such as temperature and pH contribute to the inactivation
of bacteriophage particles. The result showed that the number of phage XooX1IDN
and XooX2IDN particles began to decrease
after incubation of both phages at 60°C and no bacteriophage particles were
detected by incubating the particles at 80°C (Fig. 5A). Moreover, the phage XooX1IDN and XooX2IDN still formed plaques although the
particles have pre-incubated in suspensions of different pH levels (Fig. 5B). In addition, both phages, XooX1IDN and XooX2IDN only formed plaques on XooJ2 lawn but not on R. solanacearum DT3.
Inhibition of XooJ2 growth by
XooX1IDN and XooX2IDN in vitro
To evaluate the ability of phage XooX1IDN and XooX2IDN to
lyse XooJ2 in liquid culture, a growth
inhibition assay of host XooJ2 was performed as described under “Materials
and Methods”. The result showed that all XooJ2 cultures treated with
phages (at all m.o.i) were less turbid compare to the XooJ2 culture without
phages treatment (Fig. 6A). When XooJ2 cultures
were initially
Fig. 4: Analysis of phage XooX1IDN and XooX2IDN characteristics. (A) Structural protein profile of phage particles on SDS-PAGE, (B) Restriction profile of phages nucleic acid (1) after digestion with DNAseI (2), RNAseA
(3), and endonuclease EcoRV (4)
Fig. 5: Effect of (A) temperature and (B) pH
on the phage XooX1IDN and XooX2IDN particles stability
infected with phages (at all
m.o.i) of both XooX11IDN and XooX2IDN, growth of the XooJ2 was inhibited until
24 to 27 h post-inoculation compared to control that the growth that was
initially detected 6 h post-inoculation. Moreover, the growth of XooJ2
in liquid NB was significantly lower than in the cultures treated with the
phage XooX1IDN and XooX2IDN. However, the turbidity of XooJ2 cultures treated
with both phages at different m.o.i was not at significant level, compared to other m.o.is (Fig. 6B).
Discussion
Phages XooX1IDN and
XooX2IDN are the first Xoo-infecting phages
isolated from soil in rice field of
Jember and Situbondo, Indonesia. Both bacteriophages were studied further, such as stability on temperatures, pH, plaque and particle morphology, host specificity, genome size, and structural protein profile. According
to the transmission electron microscope examination, all phages have a phage
morphology similar to phage having head and non-contractile tail (presented by
short neck; Fig. 3). In addition, both phages, XooX1IDN and XooX2IDN are also possessed typical nucleic
acid of myovirus that is double-stranded DNA with an average genome size of about 38–39 kb (Fig. 4). According to
the morphology and nucleic acid type as mentioned on the guidelines of the
International Committee on Taxonomy of Viruses (ICTV) (Ackermann 2003), all
phages possess head and tailed
particles may belong to the families of Myoviridae,
Siphoviridae, or Podoviridae (Order Caudovirales).
Moreover, phages characterized by head and non-contractile tail (possesses
short or long neck) commonly belong to the family of Myoviridae. The similar
morphology and genome type were also reported to that myoviruses isolated from
paddy field in China (Chae et al. 2014; Dong et al. 2018; Ogunyemi et al. 2019), phages isolated from
tomato field in United State of America (Addy et al. 2019), or phage isolated from tomato in Japan (Fujiwara et al. 2008), which exhibited head and
non-contractile tail phage particles.
The
thermal stability of bacteriophages showed that the phage
infectivity drastically
decreased at the temperature of 60°C or more (Fig. 5A). Moreover, bacteriophages were completely lost their infectivity after
incubation at a temperature of 80°C. Probably this condition may
occur because the relationship of cross
sulfide in the capsid protein of denatured bacteriophages at higher
temperatures results in a loss of bacteriophage integrity (Jończyk et al.
2011). In the study, it was also revealed that all bacteriophages remained stable after
treatment at various pH conditions, both
in acidic and basic conditions, as the bacteriophage infectivity was
still maintained even though it was treated at various pH levels (Fig. 5B). However, bacteriophages tend to be more stable in a pH range of 6 to 8. This phenomenon was also reported for the phage XOF4
that remained stable after growth at pH range of 6 to 8 (Ranjani
et al. 2018). Temperature and pH contribute
to the inactivation of bacteriophage particles by damaging their structural
elements (Nobrega et al. 2016), phage aggregation, and
ability to penetrate host cells (Langlet et
al. 2007). On the other hand, phage XooX1IDN and XooX2IDN are the specific
phages that infect only X. oryzae. This is typical phenomenon of bacteriophage and become the advantage of
using phage as biological control agents since the phage only infect very
narrow and specific bacterial strain (Dong et
al. 2018; Elhalag et al. 2018; Ranjani et al. 2018).
The potency of phage
XooX1IDN and XooX2ID to control
XooJ2 was also tested to see how potent these two bacteriophages were, in suppressing the growth of the host XooJ2, qualitatively and quantitatively. The results demonstrated that XooX1IDN and
XooX2IDN were able to control and inhibit the growth of X. oryzae. Although some cells showed steady
growth phenomena,
however, the cells
growth remained significantly lower than control (Fig. 6), which indicates that equilibrium between lysis and cell growth was established or that
phage-resistant cell growth rate might be decreased resulting the host population at a
relatively low level. A similar result was
previously reported for phage ΦRSL1 infecting R. solanacearum (Fujiwara et al. 2011),
phage Xoo-sp2 infecting X.
oryzae (Dong et al. 2018), or phage RsoM1USA infecting R.
solanacearum (Addy et al. 2019).
Utilization and use of phage for
biological control strategy have been widely reported as phage therapy against
pathogenic bacteria (Fujiwara et al.
2011; Addy et al. 2012a; Elhalag et al. 2018). This phage therapy should
contribute to enhancing the advantages of
controlling bacterial leaf blight and reducing the use of conventional
pesticides, which are harmful to the environment, human and animal health. Therefore, several steps
must be examined during phage exploitation as biological control agent. All begins from the analysis of
phage-host interaction in vitro
followed by in vivo assay (Addy et al. 2012a). In this study, it is suggested that phage XooX1IDN and
XooX2IDN have the potency to be
used in controlling bacterial leaf disease. However, Dong et al. (2018) suggested that several studies must be done before
utilize the phage for biocontrol to increase safety and sufficient
implementation such as the host
range, safety aspect of phage application, and mass production condition of phages. Therefore, some studies still needed to ensure that phage XooX1IDN and
XooX2IDN
are the best phages for phage therapy against bacterial leaf blight disease on
rice, especially in Indonesia.
Conclusion
The XooX1IDN and XooX2IDN are the first Xanthomonas oryzae infecting
bacteriophages that belongs to the family of Myoviridae and have a double-stranded DNA as genome with
approximately about 39 kb and 38 kb in size. The bacteriophages remain stable by growth at
maximum temperature of 60°C, indicating that these bacteriophages are
suitable to use as biological control agent of bacterial leaf blight on rice.
Acknowledgments
This research was supported by Grant from The Directorate of Research and Community Service–Ministry of Research, Technology, and Higher
Education Republic of Indonesia with contract number 175/SP2H/LT/DRPM/2019.
Author Contributions
All authors conceived and designed
the research; DR performed the experiment; HSA and DR analysed the data and
wrote the paper.
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