Jun Myoung Yu

Fire blight and black shoot blight are two major bacterial diseases that affect apple and pear production in Korea and are caused by Erwinia amylovora and E. pyrifoliae, respectively. These diseases have recently reached epidemic levels, heightening the risk of coinfection within orchards and even individual trees. Traditional detection methods often fail to distinguish between these pathogens due to their similar characteristics and disease symptoms creating an urgent need for improved detection tools. In this study, we developed a novel droplet digital polymerase chain reaction (ddPCR) technique that specifically quantifies E. amylovora, with a detection range of 103 to 107 cfu/ml for cell culture templates and copies/ml for genomic DNA templates. The ddPCR platform is equipped with two fluorescence channels (FAM and HEX/VIC) and was further applied to develop a duplex ddPCR method for the simultaneous detection and absolute quantification of both pathogens. The method was tested on mixed DNA and cell cultures of E. amylovora and E. pyrifoliae and successfully quantified both pathogens within a single reaction. Moreover, duplex ddPCR effectively identified both pathogens in fruits artificially inoculated with various bacterial mixtures. This study provides valuable insights for addressing the cooccurrence of Erwinia diseases in orchards and offers a promising approach for precise diagnosis in plant disease management.

Erwinia amylovora, the causal agent of fire blight, poses a serious threat to several rosaceous plants, especially apples and pears. In this study, a spontaneous streptomycin-resistant E. amylovora strain (EaSmR) was isolated under laboratory conditions. Compared with the parental strain TS3128, the EaSmR strain exhibited high resistance to streptomycin (>100,000 µg/mL) and showed a significant reduction in both swimming and swarming motility. To investigate the mechanisms underlying streptomycin resistance, the genome of EaSmR was sequenced, and four single nucleotide variants (SNVs) were identified in comparison with the EaSmR genome with TS3128. Two genes in EaSmR were found to contain SNVs relative to TS3128, including a point mutation at codon 43 in the rpsL gene, the primary target of streptomycin, which was identified as the cause of the resistance. Additionally, three other point mutations were detected within the gene encoding type I methionyl aminopeptidase (MetAP1), resulting in an amino acid substitution from serine to valine (S76V). Furthermore, we analyzed the nonsynonymous single nucleotide variants (nsSNVs) between the EaSmR isolate and the reference type strain, CFBP1430. A total of 111 nsSNVs were found in EaSmR, including three stop-gain mutations, across 102 genes, which likely account for potential differences between the Korean strain TS3128 (EaSmR) and the reference strain CFBP1430. Whole-genome sequencing of EaSmR reveals significant genetic changes and provides valuable insights into the role of single nucleotide variants in antibiotic resistance and altered physiological traits. As the first report of a laboratory-induced, streptomycin-resistant E. amylovora strain from South Korea, this study provides essential insights into resistance mechanisms and highlights key genomic differences that may contribute to the unique characteristics of the Korean strain, establishing a valuable foundation for future disease management strategies.

Erwinia pyrifoliae is a gram-negative bacterial pathogen that commonly causes black shoot blight in pear and apple tree. Although the pathogenicity of this bacterial species is very similar to E. amylovora, there is no specific explanation of its pathogenic genes and mechanisms. In this study, our investigation into E. pyrifoliae pathogenicity involved generating seven YKB12327 mutant strains using Tn5 transposon mutagenesis. Observations revealed weakened growth rate and loss of pathogenicity in these mutants. Whole-genome sequencing and alignment analysis identified transposon insertions within the coding sequences of five strains and in the intergenic region of two strains. Annotation analysis elucidated genes directly or indirectly associated with pathogenicity. Notably, mutant strain MT16 displayed a transposon insertion mutation in the cyclic-di-GMP phosphodiesterase (pdeF) gene, a key player in bacterial signaling, governing microbial behavior and adaptation to environmental changes. Our findings provide insights into the genetic regulation of E. pyrifoliae pathogenicity, suggesting potential avenues for further research aimed at understanding and controlling this bacterial pathogen by targeting pdeF to mitigate apple black shoot blight disease.

Dragon fruit (Selenicereus undatus) is a valuable fruit crop in tropical and subtropical regions. It is renowned for its nutritional benefits, such as high sodium, potassium, and vitamin levels, and as a source of prebiotics and antioxidants (Balendres et al. 2019). In July 2023, anthracnose symptoms on stems were detected on dragon fruit plants in Jeju, South Korea. The typical anthracnose symptoms, such as sunken necrotic lesions (5-20 mm in diameter), were seen on the mature stems. The disease incidence ranged from 10% to 12% among the three surveyed greenhouses. To isolate the causative organism, infected stem samples were surface sterilized, cut into small pieces, and placed on potato dextrose agar (PDA). After two days of incubation at 24ºC, white hyphae appeared on the PDA around the plant tissues. Isolates CNU H23009 and CNU H23010 were purified from a single hypha under a stereoscope (e-Xtra Figure 1). Conidial morphology was examined from two-day-old fungal cultures grown on V8 juice agar. The conidia were transparent, aseptate, cylindrical to clavate, with a rounded apex and base, and measured 11.9 - 16.85 × 5.17 - 6.91 μm (mean = 15.28 × 5.93 μm, n = 30). No appressoria was observed. Morphological characteristics indicated the isolates were Colletotrichum sp. matching the description of the C. gloeosporioides species complex (Weir et al. 2012). To further identify the isolates, genomic DNA was extracted and the ribosomal internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and actin (ACT) were amplified using ITS1/ITS4, GDF/GDR, and ACT-512F/ACT-783R, respectively (Weir et al. 2012). Based on phylogenetic analysis, the isolates clustered with C. aenigma (strains ICMP18608, ICMP18686, CSH2, and QSG1), with 71% bootstrap support, as determined using the maximum parsimony method in PAUP 4.0 (e-Xtra Figure 2). Based on morphological and molecular characteristics, isolates were identified as C. aenigma. Sequences of CNU H23009 and CNU H23010 were deposited in GenBank with accession numbers OR535144 and OR535145 for ITS, OR540725 and OR540726 for GAPDH, and OR540723 and OR540724 for ACT. The pathogenicity was tested on healthy dragon fruit stems using wound inoculation with mycelial plugs of the CNU H23009 isolate. Controls were inoculated with PDA plugs. The plants were covered with plastic bags to maintain humidity and incubated in a greenhouse at 25ºC. After two days, necrotic spots had developed on the inoculated tissues; after four days, black, irregular, and sunken necrotic lesions similar to those seen in the field were observed. No symptoms occurred in the controls. C. aenigma was re-isolated from the artificially inoculated plants and re-identified based on conidial morphology. The pathogenicity test was repeated three times with three replications for each treatment. Previous studies have reported that C. aenigma, C. gloeosoporioides, C. siamense, C. truncatum, and C. karsti cause anthracnose in dragon fruit. However, C. aenigma has been reported only in Thailand (Balendres et al. 2019; Meetum et al. 2015). To our knowledge, this is the first report of C. aenigma causing anthracnose in dragon fruit in Korea.

Soil salinization is one of the leading threats to global ecosystems, food security, and crop production. Plant growth-promoting rhizobacteria (PGPRs) are potential bioinoculants that offer an alternative eco-friendly agricultural approach to enhance crop productivity from salt-deteriorating lands. The current work presents bacterial strain CNUC13 from maize rhizosphere soil that exerted several PGPR traits and abiotic stress tolerance. The strain tolerated up to 1000 mM NaCl and 30% polyethylene glycol (PEG) 6000 and showed plant growth-promoting (PGP) traits, including the production of indole-3-acetic acid (IAA) and siderophore as well as phosphate solubilization. Phylogenetic analysis revealed that strain CNUC13 was Microbacterium azadirachtae. Maize plants exposed to high salinity exhibited osmotic and oxidative stresses, inhibition of seed germination, plant growth, and reduction in photosynthetic pigments. However, maize seedlings inoculated with strain CNUC13 resulted in significantly improved germination rates and seedling growth under the salt-stressed condition. Specifically, compared with the untreated control group, CNUC13-treated seedlings exhibited increased biomass, including fresh weight and root system proliferation. CNUC13 treatment also enhanced photosynthetic pigments (chlorophyll and carotenoids), reduced the accumulation of osmotic (proline) and oxidative (hydrogen peroxide and malondialdehyde) stress indicators, and positively influenced the activities of antioxidant enzymes (catalase, superoxide dismutase, and peroxidase). As a result, CNUC13 treatment alleviated oxidative stress and promoted salt tolerance in maize. Overall, this study demonstrates that M. azadirachtae CNUC13 significantly enhances the growth of salt-stressed maize seedlings by improving photosynthetic efficiency, osmotic regulators, oxidative stress resilience, and antioxidant enzyme activity. These findings emphasize the potential of utilizing M. azadirachtae CNUC13 as a bioinoculant to enhance salt stress tolerance in maize, providing an environmentally friendly approach to mitigate the negative effects of salinity and promote sustainable agriculture.

Black shoot blight disease caused by Erwinia pyrifoliae has serious impacts on quality and yield in pear production in Korea; therefore, rapid and accurate methods for its detection are needed. However, traditional detection methods require a great deal of time and fail to achieve absolute quantification. In the present study, we developed a droplet digital polymerase chain reaction (ddPCR) method for the detection and absolute quantification of E. pyrifoliae using a pair of species-specific primers. The detection range was 103 - 107 copies/ml (DNA templates) and cfu/ml (cell culture templates). This new method exhibited good linearity and repeatability and was validated by absolute quantification of E. pyrifoliae DNA copies from samples of artificially inoculated immature pear fruits. Here, we present the first study of ddPCR assay for the detection and quantification of E. pyrifoliae. This method has potential applications in epidemiology and for the early prediction of black shoot blight outbreaks.

The unwanted detachment of organs such as flowers, leaves, and fruits from the main body of a plant (abscission) has significant effects on agricultural practice. Both timely and precise regulation of organ abscission from a plant is crucial as it influences the agricultural yield. The tomato (Solanum lycopersicum) has become a model system for research on organ abscission. Here, we characterized four tomato natural abscission variants named jointless (j), functionally impaired jointless (fij), functionally impaired jointless like (fij like), and normal joint (NJ), based on their cellular features within the flower abscission zones (AZ). Using eight INFLORESCENCE DEFICIENT IN ABSCISSION (SlIDA) genes and eight HAESA genes (SlHAE) identified in the genome sequence of tomato, we analyzed the pattern of gene expression during flower abscission. The AZ-specific expression for three tomato abscission polygalacturonases (SlTAPGs) in the development of flower AZ, and the progression of abscission validated our natural abscission system. Compared to that of j, fij, and fij like variants, the AZ-specific expression for SlIDA, SlIDL2, SlIDL3, SlIDL4, and SlIDL5 in the NJ largely corelated and increased with the process of abscission. Of eight SlHAE genes examined, the expression for SlHSL6 and SlHSL7 were found to be AZ-specific and increased as abscission progressed in the NJ variant. Unlike the result of gene expression obtained from natural abscission system, an in silico analysis of transcriptional binding sites uncovered that SlIDA genes (SlIDA, SlIDL6, and SlIDL7) are predominantly under the control of environmental stress, while most of the SlHSL genes are affiliated with the broader context in developmental processes and stress responses. Our result presents the potential bimodal transcriptional regulation of the tomato IDA-HAE module associated with flower abscission in tomatoes.

Salinity is among the most significant abiotic stresses that negatively affects plant growth and agricultural productivity worldwide. One ecofriendly tool for broadly improving plant tolerance to salt stress is the use of bio-inoculum with plant growth-promoting rhizobacteria (PGPR). In this study, a bacterium strain CNUC9, which was isolated from maize rhizosphere, showed several plant growth-promoting characteristics including the production of 1-aminocyclopropane-1-carboxylate deaminase, indole acetic acid, siderophore, and phosphate solubilization. Based on 16S rRNA and recA gene sequence analysis, we identified strain CNUC9 as Burkholderia pyrrocinia. Out of bacterial determinants to elicit plant physiological changes, we investigated the effects of volatile organic compounds (VOCs) produced by B. pyrrocinia CNUC9 on growth promotion and salinity tolerance in Arabidopsis thaliana. Higher germination and survival rates were observed after CNUC9 VOCs exposure under 100 mM NaCl stress. CNUC9 VOCs altered the root system architecture and total leaf area of A. thaliana compared to the control. A. thaliana exposed to VOCs induced salt tolerance by increasing its total soluble sugar and chlorophyll content. In addition, lower levels of reactive oxygen species, proline, and malondialdehyde were detected in CNUC9 VOCs-treated A. thaliana seedlings under stress conditions, indicating that VOCs emitted by CNUC9 protected the plant from oxidative damage induced by salt stress. VOC profiles were obtained through solid-phase microextraction and analyzed by gas chromatography coupled with mass spectrometry. Dimethyl disulfide (DMDS), methyl thioacetate, and 2-undecanone were identified as products of CNUC9. Our results indicate that optimal concentrations of DMDS and 2-undecanone promoted growth in A. thaliana seedlings. Our findings provide greater insight into the salt stress alleviation of VOCs produced by B. pyrrocinia CNUC9, as well as potential sustainable agriculture applications.