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

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.

Identification and Characterization of Bacillus tequilensis GYUN-300: An Antagonistic Bacterium Against Red Pepper Anthracnose Caused by Colletotrichum acutatum in Korea.

Anthracnose is a fungal disease caused by Colletotrichum species and has detrimental effects on many crops, including red pepper. This study used Bacillus tequilensis GYUN-300 (GYUN-300), which exhibit antagonistic activity against the fungal pathogen, Colletotrichum acutatum. This pathogen causes anthracnose that manifests primarily as a fruit rot in red pepper. There have been little efforts to identify antagonistic bacteria from mushrooms; this strain of bacteria was identified as B. tequilensis using BIOLOG and 16S rDNA sequencing analysis. The genetic mechanism underpinning the biocontrol traits of GYUN-300 was characterized using the complete genome sequence of GYUN-300, which was closely compared to related strains. GYUN-300 inhibited mycelial growth and spore germination of C. acutatum under in vitro conditions. Important antagonistic traits, such as siderophore production, solubilization of insoluble phosphate, and production of lytic enzymes (cellulase, protease, and amylase), were observed in GYUN-300, These trains promoted growth in terms of seed germination and vigorous seedling growth compared to the non-treated control. When red pepper fruits were treated with GYUN-300, the preventive and curative effects were 66.6 and 38.3% effective, respectively, in wounded red pepper fruits; there was no difference between the preventive and curative effects in non-wounded red pepper fruits. Furthermore, GYUN-300 was resistant to several commercial fungicides, indicating that GYUN-300 bacterial cells may also be used synergistically with chemical fungicides to increase biocontrol efficiency. Based on in vitro results, GYUN-300 played a role to control anthracnose disease effectively in field conditions when compared to other treatments and non-treated controls. The results from this study provide a better understanding of the GYUN-300 strain as an effective biocontrol agent against red pepper anthracnose; this form of biocontrol provides an environment-friendly alternative to chemical fungicides.

Distinct Transcriptional Programs Underlie Differences in Virulence of Isolates on Host Plants in a Fungal Pathogen, Colletotrichum gloeosporioides.

Susceptible host plants challenged by fungal pathogens can display different types of lesions, which can be attributed to environmental factors affecting the nature of interactions between the host and pathogen. During our survey of apple anthracnose in Korea, two distinct types of disease symptoms, designated as progressive (PS) and static symptoms (SS), were recognized. PS is a typical, rapidly enlarging symptom of apple anthracnose, while SS is a small, dark speck that does not expand further until the harvesting season. Isolation and genotyping of pathogens from disease lesions suggested that all of them belong to Colletotrichum gloeosporioides, a well-known causal agent of apple anthracnose. Two types of isolates were comparable in growth on media, spore germination and appressorium formation, virulence test on fruits at various temperature conditions. Furthermore, they were analyzed at the molecular level by a phylogenetic tree, RNA-seq, and expression of virulence gene. However, the SS isolates were defective in appressorium-mediated penetration into the underlying substratum. RNA-seq analysis of PS and SS isolates showed that distinct transcriptional programs underlie the development of different types of anthracnose symptoms in host plants. One downregulated gene in SS encoded isocitrate lyase is essential for disease development via its involvement in the glyoxylate cycle. It partly explains why SS is less virulent than PS on host plants. Overall, our work challenges the traditional view on the development of different lesion types and provides valuable insights into variations that exist in the pathogen population.

First Report of Leaf Blight of Adenophora triphylla var. japonica caused by Pseudomonas viridiflava in Korea.

Severe disease with leaf spots and necrotic symptoms were observed in Adenophora triphylla var. japonica (Regel) Hara (A. triphylla) during the survey in July 2020 on a field in Andong, Gyeongbuk province, Korea. It is a highly valued medicinal plant used to treat various diseases, including cough, cancer, and obesity. The infected plants initially showed spots with halo lesions, at later stages, enlarged and spread to the leaves, which the lesions becoming yellowing and chlorotic (Fig. 1). In some areas, disease incidence was up to 15% of the plants. The symptomatic samples were collected from A. triphylla and cut into 4 to 5 mm squares, surface-sterilized in 1% sodium hypochlorite for 1 min, rinsed three times, and macerated in sterile distilled water (SDW). They were spread onto nutrient agar (NA) plates and incubated at 28°C for 3 days. The representative bacterial strains selected for identification showed fluorescent colonies on King's medium B (KB). Fifteen isolates from independent samples were subjected to biochemical and pathogenicity tests. The isolates induced a hypersensitive reaction in tobacco leaves, gave a reaction in the anaerobe respiratory test, and were negative for levan, oxidase, arginine dihydrolase, gelatin hydrolysis, aesculin hydrolysis, and starch hydrolysis. The isolated strains presented the following LOPAT profile: - - + - +. The Biolog GN2 microplate and the Release 4.20 system putatively found the isolate to exhibit 93% similarity with the bacterium, Pseudomonas viridiflava. Likewise, analysis of FAME profiles using the Microbial identification system (Sherlock version 3.1) also characterized the representative bacterial strain as P. viridiflava with 87% similarity. The genomic DNA of the isolate was extracted, and the 16S rDNA sequence was amplified with a universal bacterial primer set (27F and 1492R). The sequence was submitted to GenBank under the accession number MT975233. BLASTn analysis yielded 99.79% identity with P. viridiflava strain RT228.1b (accession no. AY604846.1) and 99.72% similarity with P. viridiflava KNOX249.1b strain (accession no. AY604848.1). Phylogenetic dendrogram constructed from the comparative analysis of 16S rDNA gene sequences showing the relationship between P. viridiflava GYUN274 and related Pseudomonas species (Fig. 2). Pathogenicity tests were conducted three times on seedling of A. triphylla by spraying 50 ml of bacterial suspensions of a 24-h culture in KB medium (108 CFU/ml). The leaves inoculated with SDW alone did not develop symptoms; however, the plants treated with isolated bacterial suspensions developed halo and blight symptoms similar to those observed in the field 7 days post-inoculation. Finally, Koch's postulates were verified by re-isolating P. viridiflava from all symptomatic tissues and determined to be morphologically identical to the original isolates. To our knowledge, this is the first report of leaf blight disease of A. triphylla caused by P. viridiflava in Korea. Based on the observed symptoms, and identification by morphological characteristics, molecular data, and pathogenicity against the host plant, the proper control measures can be identified in future studies.

Identification and Characterization of Xanthomonas arboricola pv. juglandis Causing Bacterial Blight of Walnuts in Korea.

The present study describes the bacterial blight of walnut, caused by Xanthomonas arboricola pv. juglandis (Xaj) in the northern Gyeongbuk province, Korea. Disease symptoms that appear very similar to anthracnose symptoms were observed in walnut trees in June 2016. Pathogens were isolated from disease infected leaves, fruits, shoots, bud, flower bud of walnut, and cultured onto yeast dextrose carbonate agar plates. Isolated bacteria with bacterial blight symptoms were characterized for their nutrient utilization profiles using Biolog GN2 and Vitek 2. In addition, isolates were subjected to physiological, biochemical, and morphological characterizations. Furthermore, isolates were identified using 16S rDNA sequence analysis, and multi-locus sequence analysis using atpD, dnaK, efp, and rpoD. To confirm pathogenicity, leaves, fruits, and stems of 3-year-old walnut plants were inoculated with bacterial pathogen suspensions as a foliar spray. One week after inoculation, the gray spots on leaves and yellow halos around the spots were developed. Fruits and stems showed browning symptoms. The pathogen Xaj was re-isolated from all symptomatic tissues to fulfill Koch's postulates, while symptoms were not appeared on control plants. On the other hand, the symptoms were very similar to the symptoms of anthracnose caused by Colletotrichum gloeosporioides. When walnut plants were inoculated with combined pathogens of Xaj and C. gloeosporioides, disease symptoms were greater in comparison with when inoculated alone. Xaj population size was more in the month of April than March due to their dormancy in March, and sensitive to antibiotics such as oxytetracycline and streptomycin, while resistant to copper sulfate.

Characterization of Bacillus velezensis AK-0 as a biocontrol agent against apple bitter rot caused by Colletotrichum gloeosporioides.

Bacillus genus produces several secondary metabolites with biocontrol ability against various phytopathogens. Bacillus velezensis AK-0 (AK-0), an antagonistic strain isolated from Korean ginseng rhizospheric soil, was found to exhibit antagonistic activity against several phytopathogens. To further display the genetic mechanism of the biocontrol traits of AK-0, we report the complete genome sequence of AK-0 and compared it with complete genome sequences of closely related strains. We report the biocontrol activity of AK-0 against apple bitter rot caused by Colletotrichum gloeosporioides, which could lead to commercialization of this strain as a microbial biopesticide in Korea. To retain its biocontrol efficacy for a longer period, AK-0 has been formulated with ingredients for commercialization, named AK-0 product formulation (AK-0PF). AK-0PF played a role in the suppression of the mycelial growth of the fungicide-resistant pathogen C. gloeosporioides YCHH4 at a greater level than the non-treated control. Moreover, AK-0PF exhibited greater disease suppression of bitter rot in matured under field conditions. Here, we report the complete genome sequence of the AK-0 strain, which has a 3,969,429 bp circular chromosome with 3808 genes and a G+C content of 46.5%. The genome sequence of AK-0 provides a greater understanding of the Bacillus species, which displays biocontrol activity via secondary metabolites. The genome has eight potential secondary metabolite biosynthetic clusters, among which, ituD and bacD genes were expressed at a greater level than other genes. This work provides a better understanding of the strain AK-0, as an effective biocontrol agent (BCA) against phytopathogens, including bitter rot in apple.