In the early spring season of the years 2013 and 2014, a survey has been conducted to investigate the symptoms of apple canker. These are similar to the symptoms of bacterial blight, in the apple orchards of various places (Yeongju, Yeongyang, Cheongsong, Mungyeong, Andong, and Yecheon) in the northern Gyeongbuk Province, Korea (Fig. 2A). For the disease survey analysis, observations have been made randomly by selecting 20 trees from each orchard. Disease symptoms have been observed during the early stage and late winter season.

Samples were collected from leaves and stems of 5-year-old disease-infected apple trees from various regions (Supplementary Table 1). The symptomatic leaf tissues were cut into squares using a scalpel and the surface disinfected by immersing them in 70% ethyl alcohol for 30 s, followed by treatment with 2% sodium hypochlorite (NaOCl) solution for 1 min and rinsing in sterile distilled water (SDW) two or three times. Thus, the surface-sterilized tissues were placed onto the King’s B agar (KB; 20 g protease peptone, 1.6 g K₂HPO₄, 1.5 g MgSO₄·7H₂O, 15 g agar, 15 ml glycerol, and water to make 1,000 ml volume) plates and incubated at 28°C for 2-3 days. To isolate pathogens from infected apple stems, the cross-sectioned stem at infected region and graft region were placed onto the KB agar plates for a few seconds (Supplementary Fig. 1). Then, the cultured plates were observed under UV light 48 h after incubating at 28°C to differentiate fluorescence colonies from non-fluorescence colonies. Thus the fluorescent colonies on the KB medium were selected and stored in glycerol (20%) at −50°C for long-term storage.

The representative bacterial isolate WSPS007 and reference strains (P. syringae pv syringae, P. syringae pv. morsprunorum, and P. syringae pv. persicae) were tested for LOPAT (levan production, oxidase reaction, pectolytic activity, arginine hydrolysis activity, and tobacco hypersensitivity), following the method described by Lelliott and Stead (1987). LOPAT tests are used to discriminate P. syringae from other species of fluorescent pseudomonads. To determine the levan production from sucrose, the bacterial colonies were inoculated onto Petri dishes containing nutrient agar (NA) supplemented with 5% sucrose and observed for levan production after incubating the Petri dishes at 28°C for 24-48 h. For the presence of oxidase (O), bacteria were cultured onto the KB medium for 48 h at 25°C. The colonies were picked up with a sterile loop and rubbed on a filter paper impregnated with 1% (w/v) solution of N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride. The appearance of a violet color within 10 s indicates a positive test whereas the absence of color or delayed color development indicates a negative test. Pectinase activity was determined on the crystal violet pectate medium. When the bacterial colonies show a jelly-like substance 7 days after incubation at 25°C, it indicates a positive test. The presence of arginine dihydrolase was detected by introducing a 48 h-old grown bacterial culture into two test tubes containing a semi-liquid medium. Once inoculated, one tube is overlaid with sterile mineral oil. Color variations, from orange-red to magenta-amarant after 2-5 days in anaerobic conditions, indicate a positive reaction. Tobacco hypersensitivity (HR) reaction was detected in tobacco leaves. Bacterial cell suspensions (10⁸ cfu/ml) were infiltrated into the mesophyll of a fully-grown tobacco leaf. Rapid glassy to white necrosis at the infiltrated area 24 h after inoculation indicates the ability to induce an HR.

For distinguishing pathovars within P. syringae, GATTa (gelatin and aesculine hydrolysis, tyrosinase activity, and tartrate acid metabolism) tests are commonly used (Lelliott and Stead, 1987). For gelatin hydrolysis, a 24 h-old bacterial culture was introduced into a tube containing a solidified medium consisting of 0.3% yeast extract, 0.5% peptone, and 12% gelatin. The characteristic liquefaction of gelatin after 7-14 days of incubation at 18°C is an indication of a positive result. For aesculin hydrolysis, bacteria grown for 24-48 h on NA were inoculated onto a semi-solid medium containing 1% peptone, 0.1% aesculine, 0.05% ferric citrate, and 2% agar. The brown color of the medium after 24-48 h incubation at 26-28°C indicates the presence of the aesculinase (Sneath, 1956). For tyrosinase activity, a 24-48-hour-old bacterial culture was inoculated onto a semi-solid medium containing 0.5% sucrose, 1% casamino acid, 0.1% L-tyrosine, 0.05% potassium phosphate, and 0.0125% magnesium sulfate. The pH of the medium was adjusted to 7.2 before adding 2% agar. Change in the color of the medium to red after 7-10 days of incubation at 26-28°C shows the presence of tyrosinase (Lelliott et al., 1966). For tartrate utilization, 24-48-hour-old-grown bacterial culture was inoculated in the broth (pH 7.0) containing 0.1% ammonium dihydrogen phosphate, 0.02% potassium chloride, 0.02% magnesium sulfate, 0.2% (w/v) sodium tartrate, and 1 ml of 4% alcohol bromothymol blue solution. After 21 days of incubation at 25°C, a color change in the medium from green to blue indicates a positive test result (Lelliott and Stead, 1987).

To determine the INA, bacterial colonies grown on NA plates for 48 h at 28°C were selected randomly. Escherichia coli TOP10 grown on the NA medium was used to measure INA, whereas SDW, deionized water (DI; ultra-pure grade water), and apple leaf juice were used as controls. Bacterial suspensions (10⁶ cfu/ml) were prepared with SDW, or DI, or a mixture of SDW + apple leaf juice in glass test tubes (15 ml). All test tubes were placed in a styrofoam box (30 × 20 × 15 cm), and the box was placed into a freezer at −70°C (Kim and Kim, 1997). The freezing point was measured using a Pt100 thermocouple (Tanaka Metal Jewelry). Strains were scored positive for INA if immediate ice formation occurred in test tubes. The freezing point in a test tube was represented as a peak on the graph. The freezing point for Pss was compared with controls, such as a tube with distilled water and a tube with Escherichia coli suspensions. The experiment was performed two times with six replicates per treatment.

For microscopic observation, the bacterial colony was picked up with a sterile loop, placed in SDW on a Formvar-coated copper grid, and stained with 2% uranyl acetate for negative staining (Kim et al., 2016). The preparation was examined under a HITACHI transmission electron microscope (TEM; JEOL, Tokyo, Japan). Morphological and biochemical properties were identified, evaluated, and compared as described in Bergey’s Manual of Systematic Bacteriology (Bergey et al., 1939) and the laboratory guide for the identification of plant pathogenic bacteria (Schaad et al., 2001).

The putative Pseudomonas isolate was tested for the utilization of 95 carbon sources using the BIOLOG program (Jeon et al., 2003). Briefly, the bacterial cells cultured on bacterial universal growth agar supplemented with 0.25% maltose and 0.9% thioglycolate at 28°C for 48 h were suspended in an inoculating fluid (0.4% NaCl, 0.03% Pluronic F-68, and 0.01% gellan gum), inoculated onto microplates (Biolog GP MicroPlate), and incubated at 28°C. After 24-48 h of incubation, the plates were read using a MicroLog 3-Automated MicroStation system (BIOLOG, Hayward, CA, USA). The bacterium was identified based on the MicroLog Gram-negative database (version 4.0, BiOLOG). Gas chromatography of fatty acid methyl esters (GC-FAME) was conducted to confirm bacterial identification. Bacteria were cultured on tryptic soy agar plates at 28°C for 48 h. The colonies were harvested and placed in screw-cap culture tubes, and 1 ml of saponification reagent (NaOH aqueous methanol) was added. A methylation reagent (hydrochloric acid in aqueous methanol) was added after heat treatment and fatty acids were extracted with extraction solvent (hexane/MTBE), mild base (10.8 g NaOH in 900 ml), and a saturated NaOH solution. The Sherlock system analyzed the fatty acid composition, followed by the generation of a similarity index for the isolates that corresponded to a microorganism in the database Sherlock version 3.1 (New York, DE, USA) (Kim et al., 2016).

The selected WSPS007 isolate was subjected to molecular identification based on the sequence homology of its 16S rRNA gene (Weisburg et al., 1991). The genomic DNA of WSPS007 isolate was isolated using a kit (Genomic DNA Extraction Kit for bacteria, iNtRON Biotechnology, Seongnam, Korea) as per the manufacturer’s instructions. The 16S rRNA gene was amplified using a polymerase chain reaction (PCR) with Taq DNA polymerase and with primer set 27F (5′-AGA GTT TGA TCM TGG CTC AG-3′) and 1492R (5′-GGY TAC CTT GTT ACG ACT T-3′) (Weisburg et al., 1991). The conditions for thermal cycling were as follows: denaturation at 94°C for 5 min followed by 30 cycles at 94°C for 1 min, annealing at 56°C for 1 min, and extension at 72°C for 1 min. At the end of the cycle, the reaction mixture was held at 72°C for 5 min and then cooled to 4°C. The PCR product obtained was sequenced by an automated sequencer (Genetic Analyzer 3130, Applied Biosystems, Foster City, CA, USA). The above primers were used for this purpose. The sequence was compared with the reference species of bacteria contained in a genomic database using an NCBI-BLAST tool. The re-isolated pathogens, such as Re_1 WSPS007 and Re_2 WSPS007 from the pathogenicity test were also subjected to molecular identification using the same method as above. Sequence alignment and phylogenetic tree construction were performed by maximum parsimony using a MEGA 4.0 program (Biodesign Institute, Tempe, AZ, USA).

To assess the pathogenicity of Pss on the apple plant, disease-free apple twigs (cv. Fuji) from 2-year-old apple trees were artificially inoculated with bacterial suspensions (10⁶ cfu/ml) by foliar spray and soil drench methods. For artificial inoculation, the pathogenic bacterial cell suspensions were prepared by spreading the suspensions on KB agar plates, incubating them for 48 h at 28°C, and adjusting to a final concentration (10⁶ cfu/ml) in SDW. For the foliar spray method, disease-free apple twigs with leaves were washed with water and dried. All the leaves on a twig were wounded with a sterile needle and inoculated with bacterial suspensions (10⁶ cfu/ml) of 50 ml by the foliar spray method, dried for some time, and incubated for 2 weeks at 28°C. Apple twigs sprayed with SDW served as a control. Disease symptoms were assessed 2 weeks after inoculation. For the soil drench method, a pathogenicity test was conducted on 2-year-old healthy apple plants grown in pots under greenhouse conditions at 24 ± 2°C. Apple plants were drenched with 500 ml bacterial suspensions (10⁶ cfu/ml) near the root region in pots. Pots drenched with SDW served as a non-treated control. Disease severity (%) was recorded 1 week after incubating the plants under humid conditions (70-80%) in the greenhouse. Pathogenicity was evaluated based on a disease rating index scale from 0 to 4 (where 0, no lesions; 1, leaves showing yellowing symptoms appear from 0 to 25%; 2, leaves showing yellowing from 26 to 50%; 3, leaves show yellow and then become brown from 51 to 75%; and 4, leaves showing blight and branch becomes wilt from 76 to 100%). Each experiment was performed at least two times with six replicates per treatment. To fulfill Koch’s postulates, the pathogen Pss was re-isolated from all symptomatic tissues to determine whether they are morphologically identical to the original isolates.

The Pss strains were screened for their ability to produce syringomycin and syringopeptin. To test syringomycin production, a procedure described by Toben et al. (1989) was followed. Pss strains were grown overnight in 5 ml of nutrient broth yeast extract. Bacterial cells were harvested by centrifugation and washed once with SDW. The bacterial suspensions were adjusted to a final concentration of 1 × 10⁶ cfu/ml and 10 μl aliquots of bacterial suspensions were inoculated onto syringomycin minimal agar plates and allowed to dry for 5 min. The plates were incubated for 5 days at 28°C and then lightly over-sprayed with the suspension of the arthrospores of the fungus Geotrichum candidum (KCTC6195), which is sensitive to syringomycin. As a result of syringomycin production, the fungal growth inhibition zone was measured 5 days after incubation at 28°C. The Pss strains were evaluated for syringopeptin production as described by Grgurina et al. (1996), but were modified to use peptone-glucose-NaCl agar (PGNA) instead of potato dextrose agar. Pss strains were cultured as described above, except the bacterial suspensions were transferred to PGNA plates and incubated for 4 days at 25°C. The PGNA plates were then over-sprayed with the cell suspensions of Bacillus megaterium Km (KACC13749), which is highly sensitive to syringopeptin. To avoid confusion in distinguishing between the syringomycin and syringopeptin inhibition zones, syringopeptin mutations were introduced into the strain BR334 (Zhang et al., 1995). The strain BR344 is a syrC mutant of B301D−R, which provides a clear bioassay for syringopeptin but does not produce syringomycin (Grgurina et al., 1996).

The toxin-producing genes, such as syrB1, recA, hrpZ, and sypB, were detected by PCR amplification method using a BIONEER AccuPower PCR premix kit (Bioneer Co., Daejeon, Korea). Primers used for PCR amplification were designed using the Laser Gene Expert Sequence Analysis Package (DNASTAR, Madison, WI, USA) and are listed in Table 1. PCR was completed within 35 cycles with annealing temperatures of 55°C, 52°C, 58°C, and 63°C for syrB1, sypB, recA, and hrpZ, respectively, with an initial denaturation step at 94°C for 5 min, extension at 94°C and 72°C for 30 and 1 min, respectively, and the final extension step at 72°C for 10 min. The amplified DNA samples were loaded on 1% agarose gel and electrophoresed using loading STAR for confirmation. The 100 bp DNA ladder (Dyne Bio, Seongnam, Korea) was used as a marker.

All the isolated strains were subjected to ERIC-PCR (enterobacterial repetitive intergenic consensus PCR) and REP-PCR (repetitive element sequence-based PCR) genomic fingerprinting using primer sets corresponding to REP (REP1R-I: 5′-IIIICGICGICATCIGGC-3′ and REP2-I: 5′-ICGICTTA TCIGGCCTAC-3′) and ERIC (ERIC1R: 5′-ATG TAA GCT CCT GGG GAT TCA C-3′ and ERIC2: 5′-AAG TAA GTG ACT GGG GTG AGC G3′) elements (Schaad et al., 2001). The reaction mixture (25 μl) consisted of a Taq reaction buffer containing 1.0 μl of template DNA, 100 pmol of each primer, 200 μM of each deoxynucleotide triphosphate, 2.0 mM MgCl₂, and 1.0 U of Taq DNA polymerase (Bioneer Co.). The PCR amplifications were performed in a MyGenie96/384 Thermal Block (Bioneer Co.) with the following parameters: initial denaturation at 95°C for 7 min, template denaturation at 94°C for 1 min, primer annealing at 52°C (ERIC) or 41°C (REP) for 1 min, and DNA extension for 8 min at 65°C. The PCR was repeated for 30 cycles, with a final extension step of 15 min at 65°C. Amplicons were detected by electrophoresis on a 1.5% agarose gel with 0.5× TAE buffer and visualized by a UV transilluminator after staining with ethidium bromide (1 μg/ml) solution.

To assess the antibiotic sensitivity, a total of nine antibiotics (oxolinic acid [WDG], validamycin-A [WSP], dithianon [WSP], validamycin-A [WDG], copper hydroxide [WP], streptomycin [WP], iminoctadine triacetate [SC], oxytetracycline [WSP], and kasugamycin [WDG]) were used against Pss. Approximately 100 μl bacterial suspension (10⁷ cfu/ml) cultured for 48 h was spread onto the freshly prepared NA medium. The sterile paper disks (6 mm in diameter) were impregnated with antibiotics in different ratios of concentrations (1:500, 1:1,000, and 1:2,000) and placed on agar plates. The discs loaded with SDW served as a negative control. A clear zone of inhibition was observed 48 h after incubating at 28°C and sensitivity to antibiotics was recorded.

In Korea, from 2013 onwards, apple trees at various locations showed disease symptoms causing apple cankers. The cause of this was identified and confirmed in this study. The symptoms that appeared were identical to the symptoms of bacterial infections, such as apple cankers caused by Pss (Araujo et al., 2020), which mainly affect leaves and stems. The percentage (%) of disease incidence of apple canker (from a total of 20 apple trees) varied from place to place in the Gyeongbuk Province, Korea (Fig. 2B). Disease incidence (%) in Cheongsong was recorded at a greater level than in other locations. The symptoms of apple canker at the initial stage appeared as yellowing of the leaves on branches of the plant (Supplementary Figs. 1B and 2A), especially during fruit formation (Supplementary Figs. 1D and 2C). Under severe conditions, the leaves completely wither and shed from the tree and start showing cracking symptoms (Supplementary Fig. 2E). The symptoms of apple canker were associated with gummosis spreading on the stem (Fig. 3A) which induced the formation of cracks on the bark of the stem (Fig. 3B) from which the sap oozes out (Fig. 3C), resulting in the spread of the bacteria in the tissue causing its browning (Fig. 3D). Under severe conditions, sap oozes out from the trunk of the tree with symptoms (Fig. 3E). Furthermore, apple canker symptoms extended to the infected roots, graft region, and stem, which could be visually observed in their cross-sections (Fig. 3F-H), followed by the collapse of the entire tree.

A total of 82 isolates were collected from different locations in the northern Gyeongbuk Province, Korea (Supplementary Table 1). Of these, only 50 isolates that exhibit fluorescence under UV light were considered pseudomonads (Supplementary Table 2); while the isolates which do not exhibit fluorescence are considered non-pseudomonads. Based on pathogenicity levels of various isolates from the orchards of Andong region of the Gyeongbuk Province, Korea (Supplementary Fig. 3), only one representative isolate (WSPS007) Pss was used for further studies. The isolate WSPS007 has been found to exhibit higher virulence causing in terms of disease severity (%) on detached apple leaves in comparison with other Pss isolates (Supplementary Table 3).

All the studied Pseudomonas syringae pathovars were gram-negative and the results of LOPAT and GATTa tests were reported in Table 2. In LOPAT tests, it was noticed that all the isolates were positive for levan production; the colonies were large white dome-shaped on the NSA medium. They showed negative results in oxidase, pectolytic capability, and arginine dihydrolase test. In the tobacco HR test, the tobacco leaves became hypersensitive 24 h after the introduction of the pathogen into the leaves (Table 2, Supplementary Fig. 4A). In GATTa tests, the gelatin showed liquefaction, and aesculin was hydrolyzed, which indicates that the reaction was positive, but no tyrosinase activity was observed (Table 2, Supplementary Fig. 4B). The strains were confirmed to be Pss.

We studied the impact of INA by adding Pss cell suspensions to SDW or deionized water, and a mixture of SDW and apple leaf juice. The result of the latter was compared with two controls. For all three samples, INA and the freezing point were found to be higher than that of E. coli and the control. When Pss cell suspensions were added to SDW, INA was found at −0.7°C; while the INA for E. coli and SDW was found at −9.6°C and −4.7°C, respectively (Supplementary Fig. 5A). When Pss cell suspensions were added to deionized water, INA was found at −2.8°C; while the INA for E. coli and sterilized deionized water was found at −5.9°C and −6.3°C, respectively (Supplementary Fig. 5B); and finally when Pss cell suspensions were added to a mixture of SDW and apple leaf juice, the ice nucleation temperature was found to be −0.3°C; while INA for E. coli and SDW + apple leaf juice was found at −2.3°C and −2.2°C, respectively (Supplementary Fig. 5C). The ice crystallization event is well known to be an uncertain phenomenon when carried out without nucleating agents. When added to aqueous solutions, the cells of Pss can exert accurate control of nucleation, which is a key step of the crystallization phenomenon. The freezing point of the three samples was 0°C, but the supercooling break was different for treated and control samples. This means that the degree of supercooling was reduced by 2.5°C upon the addition of Pss cell suspensions.

Bacterial colonies cultured on the KB solid medium appeared as translucent, closely white, circular, convex shape, and fluorescent under UV light. When viewed using a TEM, the bacterial population was found to be rod-shaped (Fig. 4A), approximately 2.0 μm (width) × 3.0 μm (length). An individual bacterium was observed with lophotrichous flagella at one end (Fig. 4B and C); whereas the dividing bacterium was observed with flagella (Fig. 4D). The BIOLOG system determined the classification of the strain as Pseudomonas syringae, because it closest matched from the reference results based on the utilization of various carbohydrates examined (Table 3). In particular, carbon sources, such as sorbitol, L-lactate, and erythritol, resulted in clear classification. When bacterial strains were subjected to FAMEs analysis, the strains were found to be close to P. syringae, and further, they showed a distinct difference from Erwinia sp. (Table 4). Comparing these traits to those mentioned in the BIOLOG database revealed that this strain had a match probability of 100% to P. syringae.

The WSPS007 isolate was further characterized by 16S rRNA gene sequencing. The 16S rRNA gene sequences suggested that the isolate WSPS007 (accession no. KF500097.1) belonged to the Pseudomonas genus and had the highest homology to Pss (99%). Thus, both morphological and molecular characteristics confirmed this species as Pss. In the phylogenetic tree, the isolate was clustered with other Pseudomonas sp., which are closely related to P. syringae (Fig. 5). Almost all isolates were identified as Pss with 99% homology (Supplementary Table 2).

Pathogenicity of the isolated pathovar Pss (WSPS007) was tested on detached stems of 2-year-old apple plants containing leaves (Fig. 6). When the leaves on the branch were inoculated with 50 ml of bacterial suspensions (10⁶ cfu/ml) by foliar spray, Pss started to induce apple canker symptoms on leaves of the branch, appearing as brown to black spots, which later turned to yellow and fell off 2 weeks after inoculation (Fig. 6A). The infected leaves showed the symptoms of diseased spots with yellow halos around them, whereas no symptoms were developed in non-inoculated (control) leaves. In the case of the inoculation by the soil drench method, the leaves at the top appeared healthy, whereas the bottom leaves showed disease symptoms (Fig. 6B). Initially, the leaves close to the soil became yellow, and the symptoms progressed from the bottom to the top. The yellowing symptoms gradually turned to blight symptoms. The symptoms appeared slowly in the upper part of the plant. The bark of the stem showed canker and longitudinal symptoms after two months (Fig. 6C). These symptoms mainly appeared near the grafting region. Then, the xylem of the bark canker showed browning symptoms. No disease symptoms were observed in the non-treated control. One week after inoculation, the pathogen was re-isolated from the symptomatic tissues and cultured onto the KB medium at 28°C for 3 days to fulfill Koch's postulates.

Pathogenic Pss strains were screened for the secretion of toxic substances, such as syringomycin and syringopeptin against the growth of G. candidum and B. megaterium, respectively, as indicator microorganisms (Supplementary Fig. 6). Out of 50 Pss strains screened for syringomycin production under in vitro conditions, 47 strains exhibited the formation of an inhibition zone against G. candidum, indicating that they secrete syringomycin, which might be due to the presence of syrP, whereas three strains (SHPS021, SHPS025, and SHPS057) did not show any inhibition zone (Supplementary Fig. 6A). Among all the Pss strains, the strain WSPS007 exhibited a greater inhibition zone (6.6 mm) than other strains. In the case of syringopeptin production, four strains (SHPS021, SHPS025, SHPS042, and SHPS057) did not show any inhibition zone, while other strains exhibited inhibition zones against the bacterium B. megaterium, 48 h after incubation on PGN agar plates, and 12 strains showed a minor level of inhibition zone (0.6-1.0 mm) (Supplementary Fig. 6B). On the other hand, the strain WSPS007 has been found to produce both syringopeptin and syringomycin at a greater level than other strains.

Expressions of syringomycin synthetase (syrB1) and syringopeptin synthetase (sypB) genes were observed in Pss (Fig. 7A). Similarly, the harpin-encoding hrpZ, which secretes “harpin” protein, enables Pss to elicit the HR in tobacco leaves. The principal sigma factor and housekeeping gene, recA, used for the determination of the ability of Pss to induce pathogenicity, was also expressed at an equal level to that of syrB1. The gene recA is required for DNA repair and homologous recombination.

Rep-PCR using REP and ERIC primers revealed differences in the genetic profiles of the tested Pseudomonas syringae pathovars (Fig. 7B). ERIC- and REP-PCR showed significant diversity in the banding patterns of all Pseudomonas strains from different infected apple orchards in the Gyeongbuk Province, Korea. The corresponding PCR products ranged approximately from 100 to 2,000 bp in size, and the banding patterns differed from strain to strain in both ERIC- and REP-PCR. Based on the secretion of syringopeptin, inhibition zones were formed, and the strains were grouped as follows: T1, the group that did not produce syringopeptin; T2, the group producing about 1-4 mm inhibition zone; and T3, the group producing >4 mm inhibition zone. The cumulative dendrogram of REP-PCR was constructed based on the REP and ERIC of P. syringae from different apple orchards.

In the antibiotic sensitivity test, the pathogenic Pss strain used in our study showed the highest sensitivity to oxytetracycline with an inhibition zone of 22.6 mm, the next highest sensitivity was to streptomycin with an inhibition zone of 15.8 at 1:2,000 dilutions, and the inhibition zone was 12.4 mm against oxolinic acid (Table 5). The pathogen Pss was strongly resistant to the remaining six antibiotics, for which no inhibition zone was observed.