Development of a Bacteriophage Cocktail against Pectobacterium carotovorum Subsp. carotovorum and Its Effects on Pectobacterium Virulence.

Pectobacterium carotovorum subsp. carotovorum is a necrotrophic plant pathogen that secretes plant cell wall-degrading enzymes (PCWDEs) that cause soft rot disease in various crops. Bacteriophages have been under consideration as harmless antibacterial agents to replace antibiotics and copper-based pesticides. However, the emergence of bacteriophage resistance is one of the main concerns that should be resolved for practical phage applications. In this study, we developed a phage cocktail with three lytic phages that recognize colanic acid (phage POP12) or flagella (phages POP15 and POP17) as phage receptors to minimize phage resistance. The phage cocktail effectively suppressed the emergence of phage-resistant P. carotovorum subsp. carotovorum compared with single phages in in vitro challenge assays. The application of the phage cocktail to napa cabbage (Brassica rapa subsp. pekinensis) resulted in significant growth retardation of P. carotovorum subsp. carotovorum (P < 0.05) and prevented the symptoms of soft rot disease. Furthermore, phage cocktail treatments of young napa cabbage leaves in a greenhouse environment indicated effective prevention of soft rot disease compared to that in the nonphage negative control. We isolated 15 phage-resistant mutants after a phage cocktail treatment to assess the virulence-associated phenotypes compared to those of wild-type (WT) strain Pcc27. All mutants showed reduced production of four different PCWDEs, leading to lower levels of tissue softening. Ten of the 15 phage-resistant mutants additionally exhibited decreased swimming motility. Taken together, these results show that the phage cocktail developed here, which targets two different types of phage receptors, provides an effective strategy for controlling P. carotovorum subsp. carotovorum in agricultural products, with a potential ability to attenuate P. carotovorum subsp. carotovorum virulence. IMPORTANCE Pectobacterium carotovorum subsp. carotovorum is a phytopathogen that causes soft rot disease in various crops by producing plant cell wall-degrading enzymes (PCWDEs). Although antibiotics and copper-based pesticides have been extensively applied to inhibit P. carotovorum subsp. carotovorum, the emergence of antibiotic-resistant bacteria and demand for harmless antimicrobial products have emphasized the necessity of finding alternative therapeutic strategies. To address this problem, we developed a phage cocktail consisting of three P. carotovorum subsp. carotovorum-specific phages that recognize colanic acids and flagella of P. carotovorum subsp. carotovorum. The phage cocktail treatments significantly decreased P. carotovorum subsp. carotovorum populations, as well as soft rot symptoms in napa cabbage. Simultaneously, they resulted in virulence attenuation in phage-resistant P. carotovorum subsp. carotovorum, which was represented by decreased PCWDE production and decreased flagellum-mediated swimming motility. These results suggested that preparations of phage cocktails targeting multiple receptors would be an effective approach to biocontrol of P. carotovorum subsp. carotovorum in crops.

Comparative Genomic Analysis of Pathogenic Factors of Pectobacterium Species Isolated in South Korea Using Whole-Genome Sequencing.

In this study, we conducted whole-genome sequencing with six species of Pectobacterium composed of seven strains, JR1.1, BP201601.1, JK2.1, HNP201719, MYP201603, PZ1, and HC, for the analysis of pathogenic factors associated with the genome of Pectobacterium. The genome sizes ranged from 4,724,337 bp to 5,208,618 bp, with the GC content ranging from 50.4% to 52.3%. The average nucleotide identity was 98% among the two Pectobacterium species and ranged from 88% to 96% among the remaining six species. A similar distribution was observed in the carbohydrate-active enzymes (CAZymes) class and extracellular plant cell wall degrading enzymes (PCWDEs). HC showed the highest number of enzymes in CAZymes and the lowest number in the extracellular PCWDEs. Six strains showed four subsets, and HC demonstrated three subsets, except hasDEF, in type I secretion system, while the type II secretion system of the seven strains was conserved. Components of human pathogens, such as Salmonella pathogenicity island 1 type type III secretion system (T3SS) and effectors, were identified in PZ1; T3SSa was not identified in HC. Two putative effectors, including hrpK, were identified in seven strains along with dspEF. We also identified 13 structural genes, six regulator genes, and five accessory genes in the type VI secretion system (T6SS) gene cluster of six Pectobacterium species, along with the loss of T6SS in PZ1. HC had two subsets, and JK2.1 had three subsets of T6SS. With the GxSxG motif, the phospholipase A gene did locate among tssID and duf4123 genes in the T6SSa cluster of all strains. Important domains were identified in the vgrG/paar islands, including duf4123, duf2235, vrr-nuc, and duf3396.

Characterization of Xanthomonas citri pv. glycines Population Genetics and Virulence in a National Survey of Bacterial Pustule Disease in Korea.

Xanthomonas citri pv. glycines (Xcg) is a major pathogen of soybean (Glycine max) in South Korea, despite the availability of soybean varieties with some resistance. We conducted a nationwide survey of the incidence and severity of bacterial pustule caused by Xcg. The percentage of infected fields was 7% to 17% between 2015 and 2017. We characterized the diversity of a nationwide collection of 106 Xcg isolates based on avrBs3 banding patterns. The isolates fell into 11 groups, each represented by a type strain; only two of these were similar to isolates collected from 1999 to 2002. The diversity of Xcg strains increased and the dominant strains changed between 1999 and 2017, with three new type strains comprising 44% of the isolates examined in 2012 to 2017. Pathogenicity tests did not show evidence for a shift in the races or aggressiveness of Xcg strains. Korean soybean cultivars, including the widely-grown Daewon cultivar, were susceptible to the 11 new type strains. The cultivar CNS, which carries the rxp resistance gene, was susceptible to most type strains, including two representing 83% of the Korean Xcg strains. In contrast, Williams 82, which also carries rxp, showed resistance to at least five type strains. Collectively, these results suggest that Williams 82 has resistance loci in addition to rxp. The widespread distribution of Xcg, the high virulence of the current endemic strains, and the low resistance of most Korean soybean cultivars collectively favor widespread disease in Korea in years that are favorable to pustule development.

Pan-Genome Analysis of Effectors in Korean Strains of the Soybean Pathogen Xanthomonas citri pv. glycines.

Xanthomonas citri pv. glycines is a major pathogen of soybean in Korea. Here, we analyzed pathogenicity genes based on a comparative genome analysis of five Korean strains and one strain from the United States, 8ra. Whereas all six strains had nearly identical profiles of carbohydrate-active enzymes, they varied in diversity and number of candidate type III secretion system effector (T3SE) genes. The five Korean strains were similar in their effectors, but differed from the 8ra strain. Across the six strains, transcription activator-like effectors (TALEs) showed diverse repeat sizes and at least six forms of the repeat variable di-residue (RVD) sequences, with differences not correlated with the origin of the strains. However, a phylogenetic tree based on the alignment of RVD sequences showed two distinct clusters with 17.5 repeats, suggesting that two distinct 17.5 RVD clusters have evolved, potentially to adapt Xcg to growth on distinct soybean cultivars. The predicted effector binding elements of the TALEs fell into six groups and were strongly overlapping in sequence, suggesting evolving target specificity of the binding domains in soybean cultivars. Our findings reveal the variability and adaptability of T3SEs in the Xcg strains and enhance our understanding of Xcg pathogenicity in soybean.

Comparative Pathogenicity and Host Ranges of Magnaporthe oryzae and Related Species.

Host shifting and host expansion of fungal plant pathogens increases the rate of emergence of new pathogens and the incidence of disease in various crops, which threaten global food security. Magnaporthe species cause serious disease in rice, namely rice blast disease, as well as in many alternative hosts, including wheat, barley, and millet. A severe outbreak of wheat blast due to Magnaporthe oryzae occurred recently in Bangladesh, after the fungus was introduced from South America, causing great loss of yield. This outbreak of wheat blast is of growing concern, because it might spread to adjacent wheat-producing areas. Therefore, it is important to understand the host range and population structure of M. oryzae and related species for determining the evolutionary relationships among Magnaporthe species and for managing blast disease in the field. Here, we collected isolates of M. oryzae and related species from various Poaceae species, including crops and weeds surrounding rice fields, in Korea and determined their phylogenetic relationships and host species specificity. Internal transcribed spacer-mediated phylogenetic analysis revealed that M. oryzae and related species are classified into four groups primarily including isolates from rice, crabgrass, millet and tall fescue. Based on pathogenicity assays, M. oryzae and related species can infect different Poaceae hosts and move among hosts, suggesting the potential for host shifting and host expansion in nature. These results provide important information on the diversification of M. oryzae and related species with a broad range of Poaceae as hosts in crop fields.

Distribution of Pectobacterium Species Isolated in South Korea and Comparison of Temperature Effects on Pathogenicity.

Pectobacterium, which causes soft rot disease, is divided into 18 species based on the current classification. A total of 225 Pectobacterium strains were isolated from 10 main cultivation regions of potato (Solanum tuberosum), napa cabbage (Brassica rapa subsp. pekinensis), and radish (Raphanus sativus) in South Korea; 202 isolates (90%) were from potato, 18 from napa cabbage, and five from radish. Strains were identified using the Biolog test and phylogenetic analysis. The pathogenicity and swimming motility were tested at four different temperatures. Pectolytic activity and plant cell-wall degrading enzyme (PCWDE) activity were evaluated for six species (P. carotovorum subsp. carotovorum, Pcc; P. odoriferum, Pod; P. brasiliense, Pbr; P. versatile, Pve; P. polaris, Ppo; P. parmentieri, Ppa). Pod, Pcc, Pbr, and Pve were the most prevalent species. Although P. atrosepticum is a widespread pathogen in other countries, it was not found here. This is the first report of Ppo, Ppa, and Pve in South Korea. Pectobacterium species showed stronger activity at 28°C and 32°C than at 24°C, and showed weak activity at 37°C. Pectolytic activity decreased with increasing temperature. Activity of pectate lyase was not significantly affected by temperature. Activity of protease, cellulase, and polygalacturonase decreased with increasing temperature. The inability of isolated Pectobacterium to soften host tissues at 37°C may be a consequence of decreased motility and PCWDE activity. These data suggest that future increases in temperature as a result of climate change may affect the population dynamics of Pectobacterium.

Plasmid composition and the chpG gene determine the virulence level of Clavibacter capsici natural isolates in pepper.

The gram-positive bacterial species Clavibacter capsici causes necrosis and canker in pepper plants. Genomic and functional analyses of C. capsici type strain PF008 have shown that multiple virulence genes exist in its two plasmids. We aimed to identify the key determinants that control the virulence of C. capsici. Pepper leaves inoculated with 54 natural isolates exhibited significant variation in the necrosis. Six isolates showed very low virulence, but their population titres in plants were not significantly different from those of the highly virulent isolates. All six isolates lacked the pCM1Cc plasmid that carries chpG, which has been shown to be required for virulence and encodes a putative serine protease, but two of them, isolates 1,106 and 1,207, had the intact chpG elsewhere in the genome. Genomic analysis of these two isolates revealed that chpG was located in the pCM2Cc plasmid, and two highly homologous regions were present next to the chpG locus. The chpG expression in isolate 1,106 was not induced in plants. Introduction of chpG of the PF008 strain into the six low-virulence isolates restored their virulence to that of PF008. Our findings indicate that there are at least three different variant groups of C. capsici and that the plasmid composition and the chpG gene are critical for determining the virulence level. Moreover, our findings also indicate that the virulence level of C. capsici does not directly correlate with bacterial titres in plants.

Identification of New Isolates of Phytophthora sojae and the Reactions of Korean Soybean Cultivars Following Hypocotyl Inoculation.

Phytophthora root and stem rot (PRSR) caused by Phytophthora sojae is one of the most destructive diseases of soybean. PRSR recently became an issue as soybean cultivation in paddy fields increased in South Korea. The management of PRSR mainly involves R-gene-mediated resistance, however, little is known about the resistance in Korean cultivars. Major Korean soybean cultivars were investigated for the presence or absence of R-gene-mediated resistance to four P. sojae isolates, two of which were new isolates. Isolate-specific reactions were observed following P. sojae inoculation. Of 21 cultivars, 15-20 cultivars (71.4-95.2%) showed susceptible reaction for each isolate. Ten cultivars were susceptible to all the isolates, and six cultivars were identified to have R-gene-mediated resistance to one or two isolates. The results of this study would provide a framework for the discovery of resistant cultivars, development of new cultivars resistant to P. sojae, and investigation of pathogenic diversity of P. sojae population in South Korea.

Prediction of Host-Specific Genes by Pan-Genome Analyses of the Korean Ralstonia solanacearum Species Complex.

The soil-borne pathogenic Ralstonia solanacearum species complex (RSSC) is a group of plant pathogens that is economically destructive worldwide and has a broad host range, including various solanaceae plants, banana, ginger, sesame, and clove. Previously, Korean RSSC strains isolated from samples of potato bacterial wilt were grouped into four pathotypes based on virulence tests against potato, tomato, eggplant, and pepper. In this study, we sequenced the genomes of 25 Korean RSSC strains selected based on these pathotypes. The newly sequenced genomes were analyzed to determine the phylogenetic relationships between the strains with average nucleotide identity values, and structurally compared via multiple genome alignment using Mauve software. To identify candidate genes responsible for the host specificity of the pathotypes, functional genome comparisons were conducted by analyzing pan-genome orthologous group (POG) and type III secretion system effectors (T3es). POG analyses revealed that a total of 128 genes were shared only in tomato-non-pathogenic strains, 8 genes in tomato-pathogenic strains, 5 genes in eggplant-non-pathogenic strains, 7 genes in eggplant-pathogenic strains, 1 gene in pepper-non-pathogenic strains, and 34 genes in pepper-pathogenic strains. When we analyzed T3es, three host-specific effectors were predicted: RipS3 (SKWP3) and RipH3 (HLK3) were found only in tomato-pathogenic strains, and RipAC (PopC) were found only in eggplant-pathogenic strains. Overall, we identified host-specific genes and effectors that may be responsible for virulence functions in RSSC in silico. The expected characters of those genes suggest that the host range of RSSC is determined by the comprehensive actions of various virulence factors, including effectors, secretion systems, and metabolic enzymes.

Colanic Acid Is a Novel Phage Receptor of Pectobacterium carotovorum subsp. carotovorum Phage POP72.

The emergence and widespread nature of pathogen resistance to antibiotics and chemicals has led to the re-consideration of bacteriophages as an alternative biocontrol agent in several fields, including agriculture. In this study, we isolated and characterized a novel bacteriophage, POP72, that specifically infects Pectobacterium carotovorum subsp. carotovorum (Pcc), which frequently macerates agricultural crops. POP72 contains a 44,760 bp double-stranded DNA genome and belongs to the family Podoviridae. To determine the phage receptor for POP72, a random mutant library of Pcc was constructed using a Tn5 transposon and screened for resistance against POP72 infection. Most of the resistant clones had a Tn5 insertion in various genes associated with colanic acid (CA) biosynthesis. The phage adsorption rate and CA production decreased dramatically in the resistant clones. Complementation of the clones with the pUHE21-2 lacI q vector harboring genes associated with CA biosynthesis restored their sensitivity to POP72, as well as their ability to produce CA. These results suggest that CA functions as a novel phage receptor for POP72. The application of POP72 protected Chinese cabbage from Pcc infection, suggesting that phage POP72 would be an effective alternative antimicrobial agent to protect agricultural products from Pcc.

Timing of Fusarium Head Blight Infection in Rice by Heading Stage.

Fusarium graminearum causes the devastating plant disease Fusarium head blight and produces mycotoxins on small cultivated grains. To investigate the timeframe of F. graminearum infection during rice cultivation, a spore suspension of F. graminearum was applied to the rice cultivars Dongjin 1 and Nampyeongbyeo before and after the heading stage. The disease incidence rate was the highest (50%) directly after heading, when the greatest number of flowers were present, while only 10% of the rice infected 30 days after heading showed symptoms. To understand the mechanism of infection, an F. graminearum strain expressing green fluorescent protein (GFP) was inoculated, and the resulting infections were visually examined. Spores were found in all areas between the glume and inner seed, with the largest amount of GFP detected in the aleurone layer. When the inner part of the rice seed was infected, the pathogen was mainly observed in the embryo. These results suggest that F. graminearum migrates from the anthers to the ovaries and into the seeds during the flowering stage of rice. This study will contribute to uncovering the infection process of this pathogen in rice.

Purification and Characterization of Pasteuricin Produced by Staphylococcus pasteuri RSP-1 and Active against Multidrug-Resistant Staphylococcus aureus.

Staphylococcus aureus is an important pathogen implicated in various diseases, including staphylococcal food poisoning. Bacteriocins are considered safe and effective antimicrobial substances for the prevention of the growth of pathogenic bacteria. In this article, we describe the purification and characterization of pasteuricin, a novel bacteriocin produced by Staphylococcus pasteuri RSP-1. A cell-free supernatant of S. pasteuri RSP-1 exerted strong antimicrobial activity against staphylococci, including methicillin-resistant S. aureus (MRSA), and gram-positive bacteria. The loss of antimicrobial activity upon treatment with proteolytic enzymes confirmed the proteinaceous nature of pasteuricin. A rapid and pronounced bactericidal effect of pasteuricin was confirmed by a live-dead bacterial viability assay. To our knowledge, pasteuricin is the first reported S. pasteuri bacteriocin that inhibits S. aureus. Because pasteuricin is characterized by strong antimicrobial activity and high stability, it has potential as an alternative antimicrobial agent to antibiotics.

Simple Detection of Cochliobolus Fungal Pathogens in Maize.

Northern corn leaf spot and southern corn leaf blight caused by Cochliobolus carbonum (anamorph, Bipolaris zeicola) and Cochliobolus heterostrophus (anamorph, Bipolaris maydis), respectively, are common maize diseases in Korea. Accurate detection of plant pathogens is necessary for effective disease management. Based on the polyketide synthase gene (PKS) of Cochliobolus carbonum and the nonribosomal peptide synthetase gene (NRPS) of Cochliobolus heterostrophus, primer pairs were designed for PCR to simultaneously detect the two fungal pathogens and were specific and sensitive enough to be used for duplex PCR analysis. This duplex PCR-based method was found to be effective for diagnosing simultaneous infections from the two Cochliobolus species that display similar morphological and mycological characteristics. With this method, it is possible to prevent infections in maize by detecting infected seeds or maize and discarding them. Besides saving time and effort, early diagnosis can help to prevent infections, establish comprehensive management systems, and secure healthy seeds.

Improved multiplex PCR primers for rapid identification of coagulase-negative staphylococci.

Coagulase-negative staphylococci (CNS) are opportunistic pathogens that are currently emerging as causative agents of human disease. Though CNS are widespread in the clinic and food, their precise identification at species level is important. Here, using 16S rRNA sequencing, 55 staphylococcal isolates were identified as S. capitis, S. caprae, S. epidermidis, S. haemolyticus, S. pasteuri, S. saprophyticus, S. warneri, and S. xylosus. Although 16S rRNA sequencing is universally accepted as a standard for bacterial identification, the method did not effectively discriminate closely related species, and additional DNA sequencing was required. The divergence of the sodA gene sequence is higher than that of 16S rRNA. To devise a rapid and accurate identification method, sodA-specific primers were designed to demonstrate that species-specific multiplex polymerase chain reaction (PCR) can be used for the identification of CNS species. The accuracy of this method was higher than that of phenotypic identification; the method is simple and less time-consuming than 16S rRNA sequencing. Of the 55 CNS isolates, 92.72% were resistant to at least one antibiotic, and 60% were resistant to three or more antibiotics. CNS isolates produced diverse virulence-associated enzymes, including hemolysin (produced by 69.09% of the isolates), protease (65.45%), lipase (54.54%), lecithinase (36.36%), and DNase (29.09%); all isolates could form a biofilm. Because of the increasing pathogenic significance of CNS, the efficient multiplex PCR detection method developed in this study may contribute to studies for human health.

Lateral flow assay-based bacterial detection using engineered cell wall binding domains of a phage endolysin.

The development of a cost-effective and efficient bacterial detection assay is essential for diagnostic fields, particularly in resource-poor settings. Although antibodies have been widely used for bacterial capture, the production of soluble antibodies is still expensive and time-consuming. Here, we developed a nitrocellulose-based lateral flow assay using cell wall binding domains (CBDs) from phage as a recognition element and colloidal gold nanoparticles as a colorimetric signal for the detection of a model pathogenic bacterium, Bacillus cereus (B. cereus). To improve conjugation efficiency and detection sensitivity, cysteine-glutathione-S-transferase-tagged CBDs and maltose-binding protein-tagged CBDs were produced in Escherichia coli (E. coli) and incorporated in our assays. The sensitivity of the strip to detect B. cereus was 1×104 CFU/mL and the overall assay time was 20min. The assay showed superior results compared to the antibody-based approach, and did not show any significant cross-reactivity. This proof of concept study indicates that the lateral flow assay using engineered CBDs hold considerable promise as simple, rapid, and cost-effective biosensors for whole cell detection.

Genomic characterization of bacteriophage vB_PcaP_PP2 infecting Pectobacterium carotovorum subsp. carotovorum, a new member of a proposed genus in the subfamily Autographivirinae.

Bacteriophage vB_PcaP_PP2 (PP2) is a novel virulent phage that infects the plant-pathogenic bacterium Pectobacterium carotovorum subsp. carotovorum. PP2 phage has a 41,841-bp double-stranded DNA encoding 47 proteins, and it was identified as a member of the family Podoviridae by transmission electron microscopy. Nineteen of its open reading frames (ORFs) show homology to functional proteins, and 28 ORFs have been characterized as hypothetical proteins. PP2 phage is homologous to Cronobacter phage vB_CskP_GAP227 and Dev-CD-23823. Based on phylogenetic analysis, PP2 and its homologous bacteriophages form a new group within the subfamily Autographivirinae in the family Podoviridae, suggesting the need to establish a new genus. No lysogenic-cycle-related genes or bacterial toxins were identified.

OmpF of Pectobacterium carotovorum subsp. carotovorum Pcc3 is required for carocin D sensitivity.

Carocin D is a bacteriocin produced by Pectobacterium carotovorum subsp. carotovorum Pcc21. Carocin D inhibits the growth of P carotovorum subsp. carotovorum and closely related strains. Pectobacterium carotovorum subsp. carotovorum is a causative bacterium for soft rot disease and leads to severe economic losses. Bacteriocins recognize and interact with a specific membrane protein of target bacteria as a receptor. To identify the receptor responsible for carocin D recognition, mutants that underwent a phenotypic change from carocin D sensitivity to carocin D insensitivity were screened. Based on Tn5 insertions, carocin D sensitivity was dependent on expression of the outer membrane protein OmpF. The insensitivity of the mutant (Pcc3MR) to carocin D was complemented with ompF from carocin D-sensitive strains, not from carocin D-resistant strains. The selectivity between sensitive and resistant strains could be attributed to variation in OmpFs in the cell-surface-exposed regions. Based on sequence analysis and complementation assays, it appears that carocin D uses OmpF as a receptor and is translocated by the TonB system. According to previously reported translocation mechanisms of colicins, OmpF works along with the TolA system rather than the TonB system. Therefore, the current findings suggest that carocin D is imported by a unique colicin-like bacteriocin translocation system.

Clavibacter michiganensis subsp. capsici subsp. nov., causing bacterial canker disease in pepper.

Clavibacter michiganensis is a Gram-stain-positive bacterium with eight subspecies. One of these subspecies is C. michiganensis subsp. michiganensis, which causes bacterial canker disease in tomato. Bacterial strains showing very similar canker disease symptoms to those of a strain originally classified as C. michiganensis have been isolated from pepper. In this paper, we reclassified strains isolated from pepper. On the basis of phylogenetic analysis with 16S rRNA gene sequences, the strains isolated from pepper were grouped in a separate clade from other subspecies of C. michiganensis. Biochemical, physiological and genetic characteristics of strain PF008T, which is the representative strain of the isolates from pepper, were examined in this study. Based on multi-locus sequence typing and other biochemical and physiological features including colony color, utilization of carbon sources and enzyme activities, strain PF008T was categorically differentiated from eight subspecies of C. michiganensis. Moreover, genome analysis showed that the DNA G+C content of strain PF008T is 73.2 %. These results indicate that PF008T is distinct from other known subspecies of C. michiganensis. Therefore, we propose a novel subspecies, C. michiganensis subsp. capsici, causing bacterial canker disease in pepper, with a type strain of PF008T (=KACC 18448T=LMG 29047T).

A Novel Bacteriophage Targeting Cronobacter sakazakii Is a Potential Biocontrol Agent in Foods.

Cronobacter sakazakii is an important pathogen that causes high mortality in infants. Due to its occasional antibiotic resistance, a bacteriophage approach might be an alternative effective method for the control of this pathogen. To develop a novel biocontrol agent using bacteriophages, the C. sakazakii-infecting phage CR5 was newly isolated and characterized. Interestingly, this phage exhibited efficient and relatively durable host lysis activity. In addition, a specific gene knockout study and subsequent complementation experiment revealed that this phage infected the host strain using the bacterial flagella. The complete genome sequence analysis of phage CR5 showed that its genome contains 223,989 bp of DNA, including 231 predicted open reading frames (ORFs), and it has a G+C content of 50.06%. The annotated ORFs were classified into six functional groups (structure, packaging, host lysis, DNA manipulation, transcription, and additional functions); no gene was found to be related to virulence or toxin or lysogen formation, but >80% of the predicted ORFs are unknown. In addition, a phage proteomic analysis using SDS-PAGE and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) revealed that seven phage structural proteins are indeed present, supporting the ORF predictions. To verify the potential of this phage as a biocontrol agent against C. sakazakii, it was added to infant formula milk contaminated with a C. sakazakii clinical isolate or food isolate, revealing complete growth inhibition of the isolates by the addition of phage CR5 when the multiplicity of infection (MOI) was 10(5).

Isolation and Characterization of Bacteriophages Against Pseudomonas syringae pv. actinidiae Causing Bacterial Canker Disease in Kiwifruit.

Pseudomonas syringae pv. actinidiae causes bacterial canker disease in kiwifruit. Owing to the prohibition of agricultural antibiotic use in major kiwifruit-cultivating countries, alternative methods need to be developed to manage this disease. Bacteriophages are viruses that specifically infect target bacteria and have recently been reconsidered as potential biological control agents for bacterial pathogens owing to their specificity in terms of host range. In this study, we isolated bacteriophages against P. syringae pv. actinidiae from soils collected from kiwifruit orchards in Korea and selected seven bacteriophages for further characterization based on restriction enzyme digestion patterns of genomic DNA. Among the studied bacteriophages, two belong to the Myoviridae family and three belong to the Podoviridae family, based on morphology observed by transmission electron microscopy. The host range of the selected bacteriophages was confirmed using 18 strains of P. syringae pv. actinidiae, including the Psa2 and Psa3 groups, and some were also effective against other P. syringae pathovars. Lytic activity of the selected bacteriophages was sustained in vitro until 80 h, and their activity remained stable up to 50°C, at pH 11, and under UV-B light. These results indicate that the isolated bacteriophages are specific to P. syringae species and are resistant to various environmental factors, implying their potential use in control of bacterial canker disease in kiwifruits.