Novel Detection and Quantification Approach of Erwinia amylovora In Vitro and In Planta Using SYBR Green-Based Real-Time PCR Assay.

Fire blight, caused by the bacterial pathogen Erwinia amylovora, is a highly destructive disease of apple and pear. Because the apple tree gets systemically infected with E. amylovora and eventually dies, E. amylovora is a considerably important pathogen in the orchard that requires long-term management. In addition, it is crucial to prevent the spread of the pathogen by expeditious diagnosis. In this study, via comparative approaches to the genome sequences of the strains of various Erwinia spp., we designed specific primers targeting a hypothetical gene that is single copy and located in the chromosomal DNA of E. amylovora. This primer set specifically amplified the DNA of E. amylovora but no other bacteria, including E. pyrifoliae, Pectobacterium spp., Pantoea spp., and Dickeya chrysanthemi. Furthermore, the SYBR Green-based real-time PCR using the primer set allowed accurate estimation of the population of E. amylovora. Developing a rapid and accurate diagnostic method using the novel primer set enables effective defense against pathogen spread through continuous monitoring and quick response.

A New Approach Using the SYBR Green-Based Real-Time PCR Method for Detection of Soft Rot Pectobacterium odoriferum Associated with Kimchi Cabbage.

Pectobacterium odoriferum is the primary causative agent in Kimchi cabbage soft-rot diseases. The pathogenic bacteria Pectobacterium genera are responsible for significant yield losses in crops. However, P. odoriferum shares a vast range of hosts with P. carotovorum, P. versatile, and P. brasiliense, and has similar biochemical, phenotypic, and genetic characteristics to these species. Therefore, it is essential to develop a P. odoriferum- specific diagnostic method for soft-rot disease because of the complicated diagnostic process and management as described above. Therefore, in this study, to select P. odoriferum-specific genes, species-specific genes were selected using the data of the P. odoriferum JK2.1 whole genome and similar bacterial species registered with NCBI. Thereafter, the specificity of the selected gene was tested through blast analysis. We identified novel species-specific genes to detect and quantify targeted P. odoriferum and designed specific primer sets targeting HAD family hydrolases. It was confirmed that the selected primer set formed a specific amplicon of 360 bp only in the DNA of P. odoriferum using 29 Pectobacterium species and related species. Furthermore, the population density of P. odoriferum can be estimated without genomic DNA extraction through SYBR Green-based real-time quantitative PCR using a primer set in plants. As a result, the newly developed diagnostic method enables rapid and accurate diagnosis and continuous monitoring of soft-rot disease in Kimchi cabbage without additional procedures from the plant tissue.

Changes in the Composition and Microbial Community of the Pepper Rhizosphere in Field with Bacterial Wilt Disease.

Bacterial wilt caused by Ralstonia solanacearum is considered one of the most harmful diseases of pepper plants. Recently, research on plant disease control through the rhizosphere microbiome has been actively conducted. In this study, the relationship with disease occurrence between the neighboring plant confirmed by analyzing the physicochemical properties of the rhizosphere soil and changes in the microbial community. The results confirmed that the microbial community changes significantly depending on the organic matters, P2O5, and clay in the soil. Despite significant differences in microbial communities according to soil composition, Actinobacteriota at the phylum level was higher in healthy plant rhizosphere (mean of relative abundance, D: 8.05 ± 1.13; H: 10.06 ± 1.59). These results suggest that Actinobacteriota may be associated with bacterial wilt disease. In this study, we present basic information for constructing of healthy soil in the future by presenting the major microbial groups that can suppress bacterial wilt.

Discrimination and Detection of Erwinia amylovora and Erwinia pyrifoliae with a Single Primer Set.

Erwinia amylovora and Erwinia pyrifoliae cause fire blight and black-shoot blight, respectively, in apples and pears. E. pyrifoliae is less pathogenic and has a narrower host range than that of E. amylovora. Fire blight and black-shoot blight exhibit similar symptoms, making it difficult to distinguish one bacterial disease from the other. Molecular tools that differentiate fire blight from black-shoot blight could guide in the implementation of appropriate management strategies to control both diseases. In this study, a primer set was developed to detect and distinguish E. amylovora from E. pyrifoliae by conventional polymerase chain reaction (PCR). The primers produced amplicons of different sizes that were specific to each bacterial species. PCR products from E. amylovora and E. pyrifoliae cells at concentrations of 104 cfu/ml and 107 cfu/ml, respectively, were amplified, which demonstrated sufficient primer detection sensitivity. This primer set provides a simple molecular tool to distinguish between two types of bacterial diseases with similar symptoms.

Understanding the ontogeny and succession of Bacillus velezensis and B. subtilis subsp. subtilis by focusing on kimchi fermentation.

Bacillus subtilis and B. velezensis are frequently isolated from various niches, including fermented foods, water, and soil. Within the Bacillus subtilis group, B. velezensis and B. subtilis subsp. subtilis have received significant attention as biological resources for biotechnology-associated industries. Nevertheless, radical solutions are urgently needed to identify microbes during their ecological succession to accurately confirm their action at the species or subspecies level in diverse environments, such as fermented materials. Thus, in this study, previously published genome data of the B. subtilis group were compared to exploit species- or subspecies-specific genes for use as improved qPCR targets to detect B. velezensis and B. subtilis subsp. subtilis in kimchi samples. In silico analyses of the selected genes and designed primer sequences, in conjunction with SYBR Green real-time PCR, confirmed the robustness of this newly developed assay. Consequently, this study will allow for new insights into the ontogeny and succession of B. velezensis and B. subtilis subsp. subtilis in various niches. Interestingly, in white kimchi without red pepper powder, neither B. subtilis subsp. subtilis nor B. velezensis was detected.

New insight and metrics to understand the ontogeny and succession of Lactobacillus plantarum subsp. plantarum and Lactobacillus plantarum subsp. argentoratensis.

Lactobacillus plantarum is one of the most extensively studied Lactobacillus species because of its presence in a variety of environmental niches, versatility, and metabolic capabilities, resulting in the use of this organism in many industrial applications. However, although extensive effort has been invested in screening this species from a variety of habitats, a reliable and accurate method for studying the succession and ontogeny of this organism in complex ecosystems is still required to confirm the activity of L. plantarum at the subspecies level. Therefore, in this study, novel subspecies-specific genes for the quantitative detection of two L. plantarum subspecies were identified by comparative genomic analysis. The specificity of primer sets for selected genes specific to each targeted microbe was confirmed in kimchi samples. Interestingly, in all the kimchi samples at 4 °C, the presence of L. plantarum subsp. argentoratensis was not observed. Hence, we found that low temperatures markedly affected the ontogeny of L. plantarum subsp. argentoratensis during kimchi fermentation. Subsequently, this touchstone method will offer new insight and metrics to understand the ontogeny and succession of L. plantarum subsp. plantarum and L. plantarum subsp. argentoratensis in various niches.

Mushrooms collected from deogyu mountain, muju, Korea and their antioxidant activity.

Mushrooms collected from Deogyu mountain, Korea, in 2011, were identified as four classes, four orders, 13 families, 22 genera, and 33 species. In particular, agaricales was most abundant and comprised more than 70%. Their antioxidant activities were estimated using three different bioassay methods, the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radical scavenging assay, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, and reducing power assay. As a result, the methanol extracts of Stereum ostrea, Laetiporus sulphureus var. miniatus, and Tyromyces sambuceus exhibited potent antioxidant activity in all bioassays tested.

Species belonging to genus protodaedalea in Korea.

One rare and interesting species collected from Seorak-san, Inje-gun; Yeonyeop-san, Hongcheon-gun; Daeam-san, Yanggu-gun, Gangwon-do; Pocheon-gun, Gyeonggi-do; Songni-san, Boeun-gun; Joryeong-san, Goesan-gun, Chungcheongbuk-do and Sobaeksan, Yeongju-si, Gyeongsangbuk-do is described and illustrated in detail. The species "Protodaedalea hispida Imazeki" and genus "Protodaedalea Iamzeki" has not been previously recorded in Korean fungal flora. The specimens have been deposited in the Herbarium Conservation Center of the National Academy of Agricultural Sciences.

Tectella patellaris from Korea.

One rare and interesting species collected from Gyeryong-san, Chungnam Province is described and illustrated in detail. The species "Tectella patellaris (Fr.) Murr." and genus "Tectella Earle" is a first record for Korean fungal flora. Specimens cited here have been deposited in the Herbarium Conservation Center of National Academy of Agricultural Sciences.