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. 2023 Feb;39(1):88-107.
doi: 10.5423/PPJ.OA.08.2022.0121. Epub 2023 Feb 1.

Identification and Characterization of Pseudomonas syringae pv. syringae, a Causative Bacterium of Apple Canker in Korea

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Identification and Characterization of Pseudomonas syringae pv. syringae, a Causative Bacterium of Apple Canker in Korea

Seunghee Lee et al. Plant Pathol J. 2023 Feb.

Abstract

In the present investigation, bacterial isolates from infected apple trees causing apple canker during winter were studied in the northern Gyeongbuk Province, Korea. The pathogen was identified as Pseudomonas syringae pv. syringae (Pss) through various physiological and biochemical characterization assays such as BIOLOG, gas chromatography of fatty acid methyl esters, and 16S rRNA. Bioassays for the production of phytotoxins were positive for syringopeptin and syringomycin against Bacillus megaterium and Geotrichum candidum, respectively. The polymerase chain reaction (PCR) method enabled the detection of toxin-producing genes, syrB1, and sypB in Pss. The differentiation of strains was performed using LOPAT and GATTa tests. Pss further exhibited ice nucleation activity (INA) at a temperature of -0.7°C, indicating an INA+ bacterium. The ice-nucleating temperature was -4.7°C for a non-treated control (sterilized distilled water), whereas it was -9.6°C for an INA- bacterium Escherichia coli TOP10. These methods detected pathogenic strains from apple orchards. Pss might exist in an apple tree during ice injury, and it secretes a toxin that makes leaves yellow and cause canker symptoms. Until now, Korea has not developed antibiotics targeting Pss. Therefore, it is necessary to develop effective disease control to combat Pss in apple orchards. Pathogenicity test on apple leaves and stems showed canker symptoms. The pathogenic bacterium was re-isolated from symptomatic plant tissue and confirmed as original isolates by 16S rRNA. Repetitive element sequence-based PCR and enterobacterial repetitive intergenic consensus PCR primers revealed different genetic profiles within P. syringae pathovars. High antibiotic susceptibility results showed the misreading of mRNA caused by streptomycin and oxytetracycline.

Keywords: GATTa; LOPAT; apple canker; ice nucleation activity; syringomycin; syringopeptin.

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Conflict of interest statement

Conflicts of Interest

No potential conflict of interest relevant to this article was reported.

Figures

Fig. 1
Fig. 1
The life cycle of Pseudomonas syringae pv. syringae (Pss) on the apple tree. Bacteria grow on the surface of stems and leaves (epiphytically) during the early spring season. The primary inoculum originates from bacteria located in dormant buds. The Pss population is mainly present on the buds and leaf surfaces. Canker formation occurs on the stem following pathogen colonization. In summer, Pss causes lesions on leaves leading to yellowing and dryness symptoms, and sap oozes out from the stem canker. When the temperature falls in autumn, the bark bursts or gets peeled off as the water level of the apple tree decreases, and bacteria with ice nucleation activity, which is believed to cause winter damage, survive in a dormant stage in the apple tree.
Fig. 2
Fig. 2
Disease survey of Pseudomonas syringae pv. syringae (Pss) from various orchards in Korea. (A) The disease survey has been conducted from various places (Mungyeong, Yecheon, Yeongju, Bonghwa, Andong, and Yeongyang) in the northern Gyeongbuk Province, Korea. Infected samples were collected from the above places (marked in red color) to isolate the pathogens. (B) Percentage (%) of disease incidence of apple canker caused by Pss at various apple orchards in Gyeongbuk Province, Korea. Observations were made from 20 apple trees. Bars with the same letters indicate no statistical difference between the two orchards, according to the least significant difference test (P < 0.05).
Fig. 3
Fig. 3
Typical symptoms of apple canker disease on apple trees at different stages. (A) Bacterial canker symptoms on the stem are associated with gummosis spreading. (B) Formation of cracks on the bark of the stem where the infection moves from the epidermis and begins to induce a canker on the stem. (C) Oozing out the sap from the cracked portion on the stem. (D) Bacteria have moved internally into the wood tissues, causing browning of the tissues after the bark has peeled off. (E) Oozing out the sap from the trunk of the tree in severe cases (indicated by arrows). Cross-section of the infected root (F), graft region (G), and stem (H) of applewood showing visual symptoms (browning) of apple canker.
Fig. 4
Fig. 4
Morphological characteristics of Pseudomonas syringae pv. syringae isolates observed using transmission electron microscopy. (A) Rod-shaped bacterial cells. (B) A single bacterium with two flagella. (C) An enlarged image of the bacterium with two flagella. (D) Dividing bacterium. The experiment was carried out at least twice, with three replicates per treatment. Scale bars = 2.0 μm (A), 0.5 μm (B, D), 0.2 μm (C).
Fig. 5
Fig. 5
Phylogenetic analysis of Pseudomonas syringae pv. syringae (Pss) based on 16S rRNA sequences. Phylogenetic relationships of Pss were determined based on similarity using the MEGA-X program. WSPS007 is the observed isolate; Re_1 and Re_2 WSPS007 were re-isolated from the pathogenicity test.
Fig. 6
Fig. 6
Pathogenicity test of Pseudomonas syringae pv. syringae (Pss) WSPS007 cell suspensions by foliar spray and soil drench methods on detached leafy branches of an apple tree. (A) Development of disease symptoms on leaves of detached leaves 7 days after inoculation (DAI) by foliar spray method. Inoculated leaves showed yellow halos around the black spots, whereas no symptoms appeared on non-inoculated (control) leaves and stems. (B) Symptoms on bottom leaves (shown in red square) appear as yellowing, 14 days after inoculation by using the soil drench method. In enlarged images, the bottom (indicated by an arrow) and upper leaves of the infected plant appeared healthy. No symptoms were observed in non-treated plants (control). (C) This disease symptoms (indicated by arrows) appeared on the stem two months after inoculation. The experiment was performed two times with six replicates per treatment.
Fig. 7
Fig. 7
Expression of toxin-producing genes and repetitive element sequence-based polymerase chain reaction (PCR) and enterobacterial repetitive intergenic consensus PCR in Pseudomonas syringae pv. syringae (Pss) isolates from apple canker infected tissues from various apple orchards in the Gyeongbuk Province, Korea. (A) Expression of toxin-producing genes of Pss induced by plant signal molecules, syrB1 (syringomycin biosynthesis enzyme), recA (principal sigma factor), hrpZ (harpin), and sypB (syringopeptin synthesis). (B) Three types of banding patterns were formed as a result of the random PCR, and these patterns were grouped using the NTSYS program. The index of the pattern was determined according to the presence or absence of a band, and the pattern was grouped using unweighted pair group method with arithmetic mean. ERIC, enterobacterial repetitive intergenic consensus; REP, repetitive element sequence.

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