Partial Characterization of Bacteriophages from Indonesia and Its Potency as Biocontrol of Xanthomonas oryzae pv. oryzae

Partial Characterization of Bacteriophages from Indonesia and Its Potency as Biocontrol of Xanthomonas oryzae pv. oryzae

Novelty statement: 

Bacterial leaf blight on rice is caused by phytobacteria Xanthomonas oryzae pv. oryzae (Xoo). Bacteriophages isolated from the soil can infect the Xoo. The phage XooX1IDN and XooX2IDN are the first myovirus isolated in Indonesia possess a head and non-contractile tail particle with double-stranded DNA. We found that both phages are potentially able to control.

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 decrease by 50 to 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 80 ºC, 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 a family of Myoviridae. Biocontrol assay showed that the BLB pathogen’s growth decreased significantly, suggesting that both phages may be used as biological control agents for BLB disease.

Keywords: Bacterial leaf blight; Phage therapy; Myoviridae; Xanthomonas oryzae

Introduction

Xanthomonas oryzae pathovar (pv.) oryzae (Xoo) is a Gram-negative bacteria found in rice fields in rice-producing countries, including Indonesia. This bacterium is a causative agent of bacterial leaf blight (BLB), a destructive bacterial disease that 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  (called “Kresek” disease), and It decreases 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 from 6 to 90 percent in some rice varieties (Singh et al., 1980; Bhutto et al., 2018).

Numerous studies have reported the management strategies to control bacterial leaf blight, such as chemical control, genetic resistance, and biological control (Kim et al., 2016). Several studies have identified plant genes that confer resistance against X. oryzae through the plant breeding using series of resistance genes (R genes), which was designated from the Xa genes of rice cultivars (Degrasi et al., 2010). Unfortunately, this strategy is ineffective due to the ability of the pathogen to form new and more virulent pathotypes. In addition, the diversity and gene mutation mechanism in X. oryzae trigger the breakdown of the resistance genes of rice (Ponciano et al., 2003; Keller et al., 2000; Shanti et al., 2010).  Biological control thus seems to be an alternative way to manage this disease cost-effectively, sustainable and eco-friendly (Gnanamanickam, 2009). Among the biological control agents, the use of bacteriophage could be a promising control technique known as phage therapy (Addy et al., 2012).

Bacteriophage is a virus that infects and multiplies within bacterial host cells, lysis along with the development of bacteriophage particles in specific host cells, and obligately attacks narrow bacterial strain (Beaudoin et al., 2012). Recently, the use of phage as an approach to control bacterial pathogens has been highly attractive since some reports proved the potency of phage to control bacterial hosts (Svircev et al., 2018). Ralstonia phage RsoM1USA has been found to have the ability to inhibit the growth of R. solanacearum, a bacterial wilt pathogen on several crops (Addy et al., 2019). Moreover, Ahmad et al. (2014) isolated CP1 and CP2 bacteriophages that able to control X. axonopodis pv citri on citrus. Mostly, bacteriophage can be easily isolated from the soil, irrigation water around infected crops (Kalpage and Costa, 2014; Bielke et al., 2012), and from the symptomatic plant parts (Ritchie and Klos, 1977).

Although, bacteriophage is easily to explore; however, the selection step becomes a crucial point in exploitation for phage therapy (Addy et al., 2012a; Svircev et al., 2018). It is because the bacterial host exhibits alteration of virulence after phage infection, such as the production of plant toxins and an 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 the exploration of bacteriophage as biological control agent of X. oryzae pv. oryzae. About ten bacteriophages have been isolated from Vietnam and Thailand (Kovács et al., 2019)), from China (Dong et al., 2018), Japan (Kuo et al., 1967), or from India (Ranjani et al., 2018). None of the studies have reported bacteriophage of X. oryzae isolated from Indonesia. Therefore, this study is aimed to explore the bacteriophage as an initial step before using it as biological control agents for the first time from Indonesia.

Materials and Methods

Bacterial strain

Xanthomonas oryzae XooJ2 was isolated from the infected leaves in the rice field, showing “Kresek” symptom and was routinely cultured on yeast extract dextrose agar (YDA) at 28°C for 72 hours (Wilson et al., 1993). The bacterium was determined through several biochemical tests such as the KOH solubility assay (Suslow et al., 1982), catalase test (Shankara et al., 2017), starch hydrolysis assay (Cowan, 1974), and pathogenicity test using cultivar Logawa (Guo et al., 2012). Besides, confirmation was done by detecting the presence of a 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 minutes, followed  by denaturation at 96°C for 1 min, 55°C for 3 mins, 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 of 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. 2018). Briefly, soil samples were suspended with 2 ml of sterile water and shaken for 24 hours. One milliliter suspension was taken and filtered through a 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-hour-old bacterial culture was used as a host for phages propagation. Pure bacteriophage particles were stored at 4°C until used in further testing (Addy et al., 2019). In addition to these, 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 were mixed with 9.5 µl sterile distilled water, 2 µl enzyme buffer, and 0.5 µl restriction enzyme (EcoRV). 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 hours 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) were added, and the samples were boiled for 5 min. The 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) were spotted on top of the double-layered YDA plate. The formation of a clear zone on the spotting area indicated that the bacteria were susceptible to the phage. Potentially susceptible strains were tested further by serial dilution plaque assay to determine if they were truly susceptible to the phage (Ahmad et al., 2017).

Bacteriophages Stability Assays

Xanthomonas phages were tested for their stability on environmental factors such as temperature and pH (Iriate et al., 2007). Briefly, 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 between 30°C to 80°C. While determination the effect of pH, bacteriophage particles in SM buffer were 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 were then added to the bacterial suspension at m.o.i of 0.01, 0.1, 1.0, and 10, respectively. The plate was incubated inside the Microplate reader SH-1000 (Corona Electric, Japan) with slow agitation. 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 hours. This experiment was repeated three times with three replication for each m.o.i treatment (Addy et al., 2019).

Results

The Bacterial Leaf Blight Pathogen

The isolate XooJ2 was isolated from 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 result shows that the bacterium XooJ2 exhibited yellow, round in shape, convex, smooth surface, and flat edge colonies while 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 was also producing a leaf blight symptom 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). In addition, the transmission electron microscopy 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). 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 band around 7.0 kbp (Fg. 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 after incubating the particles at  80°C  (Fig.  5A).  Moreover, the phage XooX1IDN and XooX2IDN still could to give a plaque although the particles were treated with all pH ranges (Fig. 5B). In addition, both phages, XooX1IDN and XooX2IDN were able to infect and form plaques on the XooJ2 lawn but not on R. solanacearum DT3 (Table 1).

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 on 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 infected with phage XooX11IDN and XooX2IDN (at all m.o.i), the growth of the XooJ2 was inhibited until 24 to 27 hours post-incubation compared to control that the initial increase was detected after 6 hours post-incubation. 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 a 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 its stability on temperatures, pH, plaque and particle morphology, host specificity, genome size, and structural protein profile. According to the transmission electron microscope examination, both phages possessed similar phage morphology to a phage that consists of a head and non-contractile tail (presented by a short neck; Fig. 3). In addition, both phages, XooX1IDN and XooX2IDN are also possessed typical nucleic acid of myovirus that is double strands 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-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; Ogunyemi et al., 2019; Dong et al., 2018), 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 exhibit 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 loss their infectivity after treatment with 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 (Jonezyk, 2011). In the study, it also revealed that all bacteriophages still stable after treatment at various pH conditions, both in acidic and basic conditions, because bacteriophage infectivity was still maintained even though it was treated at various pH levels (Fig. 5B). However, bacteriophages tend to be stable in a pH range of 6 to 8. This phenomenon was also reported to the phage XOF4 that stable to grow in a 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). In the other hand, phage XooX1IDN and XooX2IDN are the specific phages that infect only X. oryzae. This is the typical phenomenon of bacteriophage and becomes the advantage of using phage as biological control agents since the phage only infect very narrow and specific bacterial strain (Ranjani et al., 2018; Elhalag et al., 2018; Dong et al., 2018).

The potency of phage XooX1IDN and XooX2ID to control XooJ2 was also tested to see how potent these two bacteriophages in suppressing the growth of the host XooJ2, qualitatively and quantitatively. The result demonstrated that XooX1IDN and XooX2IDN are able to control and inhibit the growth of X. oryzae. Although, some cells are shown steady growth phenomena, however, the growth of the cells remains significantly lower than control (Fig. 6), which indicates the equilibrium between lysis and cell growth was established or that phage-resistant cells growth rate might be decrease resulting the host population at a relatively low level. A similar result was previously reported when phage ΦRSL1 infected R. solanacearum (Fujiwara et al., 2011), when phage Xoo-sp2 infected X. oryzae (Dong et al., 2018), or when phage RsoM1USA infected R. solanacearum (Addy et al., 2019).

Utilization and use phage for biological control strategy has been widely reported as phage therapy against pathogenic bacteria (Fujiwara et al., 2011; Addy et al., 2012; Elhalag et al., 2018). This phage therapy should contribute to enhance the advantages in controlling bacterial leaf blight and reducing the use of conventional pesticides, which are beneficial 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 use the phage for biocontrol to increase the 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 are stable to growth at a maximum temperature of 60°C indicating that these bacteriophages are suitable to use as a biological control agent of bacterial leaf blight on rice.

Acknowledgment

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.

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