Potential use of newly isolated bacteriophage as a biocontrol against Acidovorax citrulli.

Acidovorax citrulli, the gram-negative bacteria that causes bacterial fruit blotch (BFB), has been responsible for huge worldwide economic losses in watermelon and melon production since 1980. No commercial cultivar resistant to BFB has been reported. Of the two reported genotypes of A. citrulli, genotype I is the main causal agent of BFB in melon and genotype II causes disease in watermelon. After the isolation of the first bacteriophage against A. citrulli (ACP17), efforts have been made to isolate bacteriophages with wider host ranges by collecting samples from watermelon, pumpkin, and cucumber. The newly isolated phage ACPWH, belonging to the Siphoviridae family, has a head size of 60 ± 5 nm and tail size of 180 ± 5 nm, and can infect 39 out of 42 A. citrulli strains. ACPWH has genome size of 42,499 and GC content of 64.44%. Coating watermelon seeds with bacteriophage ACPWH before soil inoculation with A. citrulli resulted in 96% germination and survival, compared to 13% germination of uncoated control seeds. These results suggest that phage ACPWH may be an effective and low-cost biocontrol agent against BFB.

Potential Solutions Using Bacteriophages against Antimicrobial Resistant Bacteria.

Bacteriophages are viruses that specifically infect a bacterial host. They play a great role in the modern biotechnology and antibiotic-resistant microbe era. Since the discovery of phages, their application as a control agent has faced challenges that made antibiotics a better fit for combating pathogenic bacteria. Recently, with the novel sequencing technologies providing new insight into the nature of bacteriophages, their application has a second chance to be used. However, novel challenges need to be addressed to provide proper strategies for their practical application. This review focuses on addressing these challenges by initially introducing the nature of bacteriophages and describing the phage-host-dependent strategies for phage application. We also describe the effect of the long-term application of phages in natural environments and other bacterial communities. Overall, this review gathered crucial information for the future application of phages. We predict the use of phages will not be the only control strategy against pathogenic bacteria. Therefore, more studies must be done for low-risk control methods against antimicrobial-resistant bacteria.

Identification of a Cupin Protein Gene Responsible for Pathogenicity, Phage Susceptibility and LPS Synthesis of Acidovorax citrulli.

Bacteriophages infecting Acidovorax citrulli, the causal agent of bacterial fruit blotch, have been proven to be effective for the prevention and control of this disease. However, the occurrence of bacteriophage-resistant bacteria is one of hurdles in phage biocontrol and the understanding of phage resistance in this bacterium is an essential step. In this study, we aim to investigate possible phage resistance of A. citrulli and relationship between phage resistance and pathogenicity, and to isolate and characterize the genes involved in these phenomena. A phage-resistant and less-virulent mutant named as AC-17-G1 was isolated among 3,264 A. citrulli Tn5 mutants through serial spot assays and plaque assays followed by pathogenicity test using seed coating method. The mutant has the integrated Tn5 in the middle of a cupin protein gene. This mutant recovered its pathogenicity and phage sensitivity by complementation with corresponding wild-type gene. Site-directed mutation of this gene from wild-type by CRISPR/Cas9 system resulted in the loss of pathogenicity and acquisition of phage resistance. The growth of AC-17-G1 in King's B medium was much less than the wild-type, but the growth turned into normal in the medium supplemented with D-mannose 6-phosphate or D-fructose 6-phosphate indicating the cupin protein functions as a phosphomannos isomerase. Sodium dodecyl sulfa analysis of lipopolysaccharide (LPS) extracted from the mutant was smaller than that from wild-type. All these data suggest that the cupin protein is a phosphomannos isomerase involved in LPS synthesis, and LPS is an important determinant of pathogenicity and phage susceptibility of A. citrulli.

Biocontrol of Soft Rot Caused by Pectobacterium odoriferum with Bacteriophage phiPccP-1 in Kimchi Cabbage.

Pectobacterium odoriferum has recently emerged as a widely infective and destructive pathogen causing soft-rot disease in various vegetables. Bacteriophage phiPccP-1 isolated from Pyeongchang, South Korea, showed lytic activity against P. odoriferum Pco14 and two other Pectobacterium species. The transmission electron microscopy and genome phylograms revealed that phiPccP-1 belongs to the Unyawovirus genus, Studiervirinae subfamily of the Autographivirinae family. Genome comparison showed that its 40,487 bp double-stranded DNA genome shares significant similarity with Pectobacterium phage DU_PP_II with the identity reaching 98% of the genome. The phiPccP-1 application significantly inhibited the development of soft-rot disease in the mature leaves of the harvested Kimchi cabbage up to 48 h after Pco14 inoculation compared to the untreated leaves, suggesting that phiPccP-1 can protect Kimchi cabbage from soft-rot disease after harvest. Remarkably, bioassays with phiPccP-1 in Kimchi cabbage seedlings grown in the growth chamber successfully demonstrated its prophylactic and therapeutic potential in the control of bacterial soft-rot disease in Kimchi cabbage. These results indicate that bacteriophage phiPccP-1 can be used as a potential biological agent for controlling soft rot disease in Kimchi cabbage.

Transport of Phage in Melon Plants and Inhibition of Progression of Bacterial Fruit Blotch.

Bacterial fruit blotch (BFB) is an economically important disease in melons and watermelons for which no effective control method is available. Application of phytobacterium-infecting phage has been evaluated as an alternative means of preventing bacterial diseases in plants. Coating of seeds with bacteriophages infecting Acidovorax citrulli, the causal agent of BFB, is effective for controlling the disease, as shown in our previous study. We evaluated the transport of bacteriophage ACPWH from soil to the leaves of melon plants, and we also evaluated its effect on BFB. Leaves of melon plants were spray-inoculated with A. citrulli, and bacteriophage ACPWH was added to soil after symptoms had developed. ACPWH was detected by PCR in foliar tissue 8 h after addition to soil. DAPI-stained ACPWH accumulated at the leaf tip after 24 h. Melon treated with ACPWH showed 27% disease severity, compared to 80% for the non-treated control, indicating that ACPWH can be used to control BFB.

Isolation, Characterization, and Application of a Bacteriophage Infecting the Fish Pathogen Aeromonas hydrophila.

Bacteriophages are increasingly being used as biological control agents against pathogenic bacteria. In the present study, we isolate and characterize bacteriophage Akh-2 from Geoje Island, South Korea, to evaluate its utility in controlling motile Aeromonas septicemia. Akh-2 lysed four of the seven Aeromonas hydrophila strains tested. Transmission electron microscopy analysis showed that Akh-2 belongs to the Siphoviridae family, with head and tail sizes of 50 ± 5 and 170 ± 5 nm, respectively. One-step growth curve analysis revealed that the phage has a latent period of 50 ± 5 min and a burst size of 139 ± 5 plaque-forming units per infected cell. The phage appeared stable in a pH range of 6-8 and a temperature range of -80 to 46 °C. Based on next-generation sequencing analysis, its genome is 114,901 bp in size, with a 44.22% G + C content and 254 open reading frames. During an artificial induction of the disease, loach (Misgurnus anguillicaudatus) treated with Akh-2 showed an increased survival rate and time compared with the non-treated control. Our results suggest that Akh-2 is a potential biological agent for the treatment of Aeromonas infections in fish.

First Isolation and Molecular Characterization of Bacteriophages Infecting Acidovorax citrulli, the Causal Agent of Bacterial Fruit Blotch.

Bacteriophages of Acidovorax citrulli, the causal agent of bacterial fruit blotch, were isolated from 39 watermelon, pumpkin, and cucumber leaf samples collected from various regions of Korea and tested against 18 A. citrulli strains. Among the six phages isolated, ACP17 forms the largest plaque, and exhibits the morphology of phages in the Myoviridae family with a head diameter of 100 ± 5 nm and tail length of 150 ± 5 nm. ACP17 has eclipse and latent periods of 25 ± 5 min and 50 ± 5 min, respectively, and a burst size of 120. The genome of ACP17 is 156,281 base pairs with a G + C content of 58.7%, 263 open reading frames, and 4 transfer RNA genes. Blast search and phylogenetic analysis of the major capsid protein showed that ACP17 has limited homology to two Stentrophomonas phages, suggesting that ACP17 is a new type of Myoviridae isolated from A. citrulli.

Isolation and Characterization of a Bacteriophage Preying an Antifungal Bacterium.

Several Bacillus species were isolated from rice field soils, and 16S rRNA gene sequence analysis showed that Bacillus cereus was the most abundant. A strain named BC1 showed antifungal activity against Rhizoctonia solani. Bacteriophages infecting strain BC1 were isolated from the same soil sample. The isolated phage PK16 had an icosahedral head of 100 ± 5 nm and tail of 200 ± 5 nm, indicating that it belonged to the family Myoviridae. Analysis of the complete linear dsDNA genome revealed a 158,127-bp genome with G + C content of 39.9% comprising 235 open reading frames as well as 19 tRNA genes (including 1 pseudogene). Blastp analysis showed that the proteins encoded by the PK16 genome had the closest hits to proteins of seven different bacteriophages. A neighbor-joining phylogenetic tree based on the major capsid protein showed a robust clustering of phage PK16 with phage JBP901 and BCP8-2 isolated from Korean fermented food.