Interpretable classification for multivariate gait analysis of cerebral palsy.

BACKGROUND: The Gross Motor Function Classification System (GMFCS) is a widely used tool for assessing the mobility of people with Cerebral Palsy (CP). It classifies patients into different levels based on their gross motor function and its level is typically determined through visual evaluation by a trained expert. Although gait analysis is commonly used in CP research, the functional aspects of gait patterns has yet to be fully exploited. By utilizing the gait patterns to predict GMFCS, we can gain a more comprehensive understanding of how CP affects mobility and develop more effective interventions for CP patients. RESULT: In this study, we propose a multivariate functional classification method to examine the relationship between kinematic gait measures and GMFCS levels in both normal individuals and CP patients with varying GMFCS levels. A sparse linear functional discrimination framework is utilized to achieve an interpretable prediction model. The method is generalized to handle multivariate functional data and multi-class classification. Our method offers competitive or improved prediction accuracy compared to state-of-the-art functional classification approaches and provides interpretable discriminant functions that can characterize the kinesiological progression of gait corresponding to higher GMFCS levels. CONCLUSION: We generalize the sparse functional linear discrimination framework to achieve interpretable classification of GMFCS levels using kinematic gait measures. The findings of this research will aid clinicians in diagnosing CP and assigning appropriate GMFCS levels in a more consistent, systematic, and scientifically supported manner.

Identification and characterization of Brevibacillus halotolerans B-4359: a potential antagonistic bacterium against red pepper anthracnose in Korea.

Our study aimed to identify potential biocontrol agents (BCAs) against major phytopathogens under in vitro conditions by screening the Freshwater Bioresources Culture Collection (FBCC), Korea. Of the identified 856 strains, only 65 exhibited antagonistic activity, among which only one representative isolation, Brevibacillus halotolerans B-4359 was selected based on its in vitro antagonistic activity and enzyme production. Cell-free culture filtrate (CF) and volatile organic compounds (VOCs) of B-4359 were shown to be effective against the mycelial growth of Colletotrichum acutatum. Notably, B-4359 was found to promote spore germination in C. acutatum instead of exhibiting a suppressive effect when the bacterial suspension was mixed with the spore suspension of C. acutatum. However, B-4359 showed an excellent biological control effect on the anthracnose of red pepper fruits. Compared to other treatments and untreated control, B-4359 played a more effective role in controlling anthracnose disease under field conditions. The strain was identified as B. halotolerans using BIOLOG and 16S rDNA sequencing analyses. The genetic mechanism underlying the biocontrol traits of B-4359 was characterized using the whole-genome sequence of B-4359, which was closely compared with related strains. The whole-genome sequence of B-4359 consisted of 5,761,776 bp with a GC content of 41.0%, including 5,118 coding sequences, 117 tRNA, and 36 rRNA genes. The genomic analysis identified 23 putative secondary metabolite biosynthetic gene clusters. Our results provide a deep understanding of B-4359 as an effective biocontrol agent against red pepper anthracnose for sustainable agriculture.

Evaluation of Trichoderma atroviride and Trichoderma longibrachiatum as biocontrol agents in controlling red pepper anthracnose in Korea.

Anthracnose disease is a serious threat to red pepper crops in Korea and many other countries, resulting in considerable yield losses. There are now no effective control techniques available except for fungicide sprays, which may directly impact consumers. This study aims to investigate the biological activity of Trichoderma isolates in controlling red pepper anthracnose caused by Colletotrichum acutatum in vitro and in the field. Out of 11 Trichoderma isolates screened for biocontrol agents against three fungal pathogens, including C. acutatum; two effective Trichoderma isolates, T. atroviride ATR697 (ATR697) and T. longibrachiatum LON701 (LON701) were selected for further investigation. Using the overlapping plates experiment, it was discovered that the volatile organic compounds (VOCs) produced by ATR697 strongly inhibited C. acutatum mycelial growth to a larger extent than the isolate LON701. A cellophane membrane experiment has shown that mycelial growth of C. acutatum was inhibited by 36% and 27% when treated with ATR697 and LON701, respectively. Culture filtrates (CFs) of two Trichoderma isolates inhibited the mycelial growth of C. acutatum in vitro. When red peppers were treated with spore suspensions of LON701 and ATR697, the disease severity (%) was 44.1% and 55.8%, respectively, in a curative method; while the disease severity (%) was 5% and 11.6%, in LON701- and ATR697-treated red peppers, respectively, in a preventive method. These results showed the suppression of disease severity (%) was relatively higher in the preventive method than in the curative method. Furthermore, Trichoderma isolates ATR697 and LON701 were resistant to commercial chemical fungicides in vitro, indicating these strains may also be used synergistically with a chemical fungicide (pyraclostrobin) against the growth of C. acutatum. There was no difference in the inhibition rate (%) of the pathogen between the treatment with LON701 alone and LON701+pyraclostrobin. Based on in vitro findings, ATR697 and LON701 played a role in effectively controlling red pepper anthracnose in field conditions, with LON701 treatment resulting in a disease rate of 14% when compared to ATR697, chemical, and non-treated controls. Overall, our study showed the ability of Trichoderma isolates to control red pepper anthracnose and their potential to develop as novel biocontrol agents to replace chemical fungicides for eco-friendly, sustainable agriculture.

Pre-sliding of femoral neck system improves fixation stability in pauwels type III femoral neck fracture: a finite element analysis.

BACKGROUND: Femoral neck fractures are a common injury in older adults and their management presents a significant challenge for orthopedic surgeons. The Femoral Neck System (FNS) was recently introduced for the fixation of femur neck fractures. Although neck shortening was reduced with the FNS, the complication rates were not reduced. Thus, improvements to enhance fixation stability should be made for the FNS. We hypothesized that (1) the pre-sliding technique and (2) the use of longer anti-rotation screw would increase fracture stability. This study aimed to determine the change in fracture stability using the pre-sliding technique and long anti-rotation screw in the FNS for fixation of Pauwels type III femoral neck fractures. METHODS: Finite element models of Pauwels type III femoral neck fracture fixed with pre-sliding FNS and 5-mm longer anti-rotation screw were established. The models were subjected to normal walking load. The material properties of the elements belonging to the bone were mapped by assigning the formulation with the computed tomography Hounsfield unit. RESULTS: Pauwels type III femoral neck fractures fixed with pre-slided FNS showed better fracture stability, decreasing fracture gap and sliding by 14% and 12%, respectively, under normal walking load. No element of cortical bone in any of the models had an absolute value of principal strain that exceeded 1%. The peak von Mises stress (VMS) of the implants ranged from 260 to 289 MPa, and the highest peak VMS value was 50% lower than the yield strength of the titanium alloy (800 MPa). The longer anti-rotation screw did not affect fracture stability. CONCLUSIONS: The pre-sliding technique using the FNS showed higher fracture stability than the standard fixation technique for a Pauwels type III femoral neck fracture. The longer anti-rotation screw did not contribute significantly to fixation stability. As this finite element analysis considered the inhomogeneous mechanical property of the bone, it offered equivalent mechanical conditions to investigate the components of interest.

Trajectory of bolt and length of plate in femoral neck system determine the stability of femur neck fracture and risk of subsequent subtrochanteric fracture : a finite element analysis.

BACKGROUND: This study aimed to analyze the differences in the stability of fractures, stress distribution around the distal-most screw according to the length of the plate and the trajectory of the bolt in Pauwels type III femoral neck fracture using the femoral neck system (FNS). METHODS: Finite element models of Pauwels type III femoral neck fractures were established with surgical variations in the trajectory of the bolt (central, inferior, valgus, and varus) and length of the lateral plate (1- and 2-hole plate). The models were subsequently subjected to normal walking and stair-climbing loads. RESULTS: The screw-holding cortical bone in subtrochanter in the model with a 2-hole plate and the bolt in the inferior trajectory and the models with 1-hole or 2-hole plate and the bolt in valgus trajectory had shown greater maximum principal strain than the models with central or varus trajectories. The gap and sliding distance on the fracture surface were larger with inferior or varus trajectories of the bolt and smaller with the valgus trajectory of the bolt under both loads, compared to those of the central trajectory. CONCLUSION: For the fixation of Pauwels type III femoral neck fracture, the trajectory of the FNS bolt and the length of the plate affect the mechanical stability of the fracture and the strain of cortical bone around the distal-most screw. The surgical target should stay on the central trajectory of the bolt and the 2-hole plate's mechanical benefits did not exceed the risk.

Identification and Characterization of Diplodia parva and Diplodia crataegicola Causing Black Rot of Chinese Quince.

Fungal isolates from infected Chinese quince trees were found to cause black rot in Yeongcheon, Gyeongsangbuk Province, Korea. The quince leaves withered and turned reddish-brown and fruits underwent black mummification. To elucidate the cause of these symptoms, the pathogen was isolated from infected leaf and fruit tissues on potato dextrose agar and Levan media. Several fungal colonies forming a fluffy white or dark gray mycelium and two types of fungi forming an aerial white mycelium, growing widely at the edges, were isolated. Microscopic observations, investigation of fungal growth characteristics on various media, and molecular identification using an internal transcribed spacer, ß-tubulin, and translation elongation factor 1-α genes were performed. The fungal pathogens were identified as Diplodia parva and Diplodia crataegicola. Pathogenicity tests revealed that the pathogen-inoculated fruits exhibited a layered pattern, turning brown rotting; leaves showed circular brown necrotic lesions. The developed symptoms were similar to those observed in the field. Fungal pathogens were reisolated to fulfill Koch's postulates. Apples were inoculated with fungal pathogens to investigate the host range. Strong pathogenicity was evident in the fruits, with browning and rotting symptoms 3 days after inoculation. To determine pathogen control, a fungicidal sensitivity test was conducted using four registered fungicides. Thiophanate-methyl, propineb, and tebuconazole inhibited the mycelial growth of pathogens. To the best of our knowledge, this is the first report on the isolation and identification of the fungal pathogens D. parva and D. crataegicola from infected fruits and leaves of Chinese quince, causing black rot disease in Korea.

First Report of Cladosporium cladosporioides Causing Leaf Spot of Aralia cordata var. continentalis in Korea.

Aralia cordata var. continentalis (Kitag), commonly known as Japanese spikenard, is an upright herbaceous perennial medicinal plant effective in relieving pain. It is also consumed as a leafy vegetable. Leaf spots and blight symptoms on A. cordata resulting in defoliation were observed in July 2021 from a research field with a disease incidence of nearly 40-50% from 80 plants in Yeongju, Korea. Brown spots with chlorotic halos first appear on the upper leaf surface (Fig. 1A). In the later stage, spots enlarge and coalesce; resulting in the leaves to dry-off (Fig. 1B). To isolate the causal agent, small pieces of diseased leaves displaying the lesion were surface-sterilized by 70% ethanol for 30 s and rinsed twice with sterile distilled water (SDW). Later, the tissues were crushed in a sterile 2.0-ml Eppendorf tube with a rubber pestle in SDW. The suspension was serially diluted and spread on potato dextrose agar (PDA) medium, incubated at 25°C for 3 days. A total of 3 isolates were obtained from the infected leaves. Pure cultures were obtained by the monosporic culture technique (Choi et al. 1999). After 2 to 3 days of incubation with a 12-h photoperiod, the fungus initially produced gray mold colonies in olive color, and the edges of the mold appeared white with a velvety texture after 20 days (Fig. 1C). Microscopic observations revealed small, single-celled, rounded, and pointed conidia that measured 6.67 ± 0.23 µm × 4.18 ± 0.12 µm (length × width) (n=40 spores) (Fig. 1D). On the basis of its morphology, the causal organism was identified as Cladosporium cladosporioides (Torres et al. 2017). For molecular identification, pure colonies of three single-spore isolates were used for DNA extraction. A fragment of the ITS, ACT, and TEF1-α were amplified using the primers ITS1/ITS4 (Zarrin et al. 2016), ACT-512F/ACT-783R, and EF1-728F/EF1-986R, respectively, by PCR (Carbone et al. 1999). The DNA sequences from all three isolates (GYUN-10727, GYUN-10776, and GYUN-10777) were identical. The resulting ITS (ON005144), ACT (ON014518), and TEF1-α (OQ286396) sequences from the representative isolate GYUN-10727 were 99 to 100% identical to the C. cladosporioides (ITS: KX664404, MF077224; ACT: HM148509; TEF1-α: HM148268, HM148266). The phylogenetic dendrogram was constructed from the comparative analysis of ITS, ACT, and TEF1-α gene sequences, showing the relationship between Cladosporium cladosporioides and related Cladosporium species (Fig. 2). The isolate GYUN-10727 has been deposited in Korean Agricultural Culture Collection (KACC 410009), and used as a representative strain in this study. For the pathogenicity test, healthy fresh leaves (3 leaves per plant) of 3-months-old A. cordata plants in pots were spray inoculated with conidial suspensions (1 × 104 conidia/mL) of GYUN-10727, which was obtained from a 7-day-old PDA culture. Leaves sprayed with SDW were considered as control. After 15 days of incubation at 25°C ± 5°C under greenhouse conditions, necrotic lesions were observed on the inoculated A. cordata leaves, while control leaves did not develop any disease symptoms. The experiment was performed twice with three replicates (pots) per treatment. The pathogen was re-isolated from the symptomatic A. cordata leaves, but not from control plants, to fulfill Koch's postulates. The re-isolated pathogen was identified by PCR. Cladosporium cladosporioides has been reported to cause diseases in sweet pepper (Krasnow et al. 2022) and garden peas (Gubler et al. 1999). To our knowledge, this is the first report of C. cladosporioides causing leaf spots of A. cordata in Korea. The identification of this pathogen will help develop strategies to efficiently control the disease in A. cordata.

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.

Characterization and evaluation of Bacillus subtilis GYUN-2311 as a biocontrol agent against Colletotrichum spp. on apple and hot pepper in Korea.

Crop plants are vulnerable to a variety of diseases, including anthracnose, caused by various species of Colletotrichum fungi that damages major crops, including apples and hot peppers. The use of chemical fungicides for pathogen control may lead to environmental pollution and disease resistance. Therefore, we conducted this research to develop a Bacillus subtilis-based biological control agent (BCA). B. subtilis GYUN-2311 (GYUN-2311), isolated from the rhizosphere soil of an apple orchard, exhibited antagonistic activity against a total of 12 fungal pathogens, including eight Colletotrichum species. The volatile organic compounds (VOCs) and culture filtrate (CF) from GYUN-2311 displayed antifungal activity against all 12 pathogens, with 81% control efficiency against Fusarium oxysporum for VOCs and 81.4% control efficacy against Botryosphaeria dothidea for CF. CF also inhibited germination and appressorium formation in Colletotrichum siamense and C. acutatum. The CF from GYUN-2311 showed antifungal activity against all 12 pathogens in different media, particularly in LB medium. It also exhibited plant growth-promoting (PGP) activity, lytic enzyme activity, siderophore production, and the ability to solubilize insoluble phosphate. In trials on apples and hot peppers, GYUN-2311 effectively controlled disease, with 75 and 70% control efficacies against C. siamense in wounded and unwounded apples, respectively. Similarly, the control efficacy of hot pepper against C. acutatum in wounded inoculation was 72%. Combined application of GYUN-2311 and chemical suppressed hot pepper anthracnose to a larger extent than other treatments, such as chemical control, pyraclostrobin, TK®, GYUN-2311 and cross-spraying of chemical and GYUN-2311 under field conditions. The genome analysis of GYUN-2311 identified a circular chromosome comprising 4,043 predicted protein-coding sequences (CDSs) and 4,096,969 bp. B. subtilis SRCM104005 was the strain with the highest average nucleotide identity (ANI) to GYUN-2311. AntiSMASH analysis identified secondary metabolite biosynthetic genes, such as subtilomycin, bacillaene, fengycin, bacillibactin, pulcherriminic acid, subtilosin A, and bacilysin, whereas BAGEL analysis confirmed the presence of competence (ComX). Six secondary metabolite biosynthetic genes were induced during dual culture in the presence of C. siamense. These findings demonstrate the biological control potential of GYUN-2311 against apple and hot pepper anthracnose.

Ultrasonic fatigue analysis of 3D-printed carbon fiber reinforced plastic.

The development of 3D printing technologies using composite materials has revolutionized additive manufacturing. Using these technologies, various products can be fabricated with strengths beyond the limits of the strength of the polymer used. However, although parts manufactured using carbon fiber reinforced plastic (CFRP) 3D printing have excellent characteristics, research on their durability is lacking, making their application difficult in the real industry. In this study, an ultrasonic fatigue test was conducted on a CFRP material manufactured by 3D printing to evaluate fatigue performance. Because of the characteristics of CFRP, the strength varies depending on the orientation angle of the carbon fiber, and the durability also varies. Therefore, an experiment on three types of specimens mixed in the bi-direction and uni-direction of 0° and 90° was conducted. For the ultrasonic fatigue test, a specimen design with a special shape is required according to the resonance frequency and dynamic modulus of the material. To this end, a specimen was designed based on measurements of the physical properties of the material according to the angle of the fiber, which were verified by Finite element method (FEM) modal analysis, and the fatigue life was estimated through an actual experiment. The fatigue failure life was simulated by FEM fatigue analysis considering the measured fatigue test results and the derived anisotropic properties simultaneously. Additionally, based on the advantages of CFRP 3D printing, which adjusts the fiber pattern, we fabricated a specimen with a concentric pattern to derive the fatigue life and calculate the actual life improvement. Based on the results of this study, the specific rigidity of the CFRP parts can be optimized by adjusting the fiber pattern. Additionally, the results of this study can aid in the analysis of the fatigue characteristics of 3D-printed CFRP materials.

Occurrence of phenotypic variation in Paenibacillus polymyxa E681 associated with sporulation and carbohydrate metabolism.

We report the phenotypic variation in Paenibacillus polymyxa E681 (E681), a plant growth-promoting rhizobacterium (PGPR) isolated from a winter barley root in Korea. Phenotypic variation (F-type) occurred when E681 (B-type) was grown in the media, and F-type was generated from B-type. B- and F-types were characterized by their morphological, Biolog, and GC-MIDI analyses. F-type cells altered the original biological capacity of B-type cells on endospore and flagella formation, changes in pH in culture, and carbon utilization. In growth curve analysis, B-type variants recovered bacterial growth as the variation occurred after the decline phase, but F-type variants did not. To determine this cause, we conducted comparative proteome analysis between B- and F-types using two-dimensional gel electrophoresis (2-DE). Of the identified proteins, 47% were involved in glycolysis and other metabolic pathways associated with carbohydrate metabolism. Therefore, our findings provide new knowledge on the mechanism of phenotypic variation and insights into agricultural biotechnology.

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.

Mechanical effects of surgical variations in the femoral neck system on Pauwels type III femoral neck fracture : a finite element analysis.

AIMS: In this study, we aimed to explore surgical variations in the Femoral Neck System (FNS) used for stable fixation of Pauwels type III femoral neck fractures. METHODS: Finite element models were established with surgical variations in the distance between the implant tip and subchondral bone, the gap between the plate and lateral femoral cortex, and inferior implant positioning. The models were subjected to physiological load. RESULTS: Under a load of single-leg stance, Pauwels type III femoral neck fractures fixed with 10 mm shorter bolts revealed a 7% increase of the interfragmentary gap. The interfragmentary sliding, compressive, and shear stress remained similar to models with bolt tips positioned close to the subchondral bone. Inferior positioning of FNS provided a similar interfragmentary distance, but with 6% increase of the interfragmentary sliding distance compared to central positioning of bolts. Inferior positioning resulted in a one-third increase in interfragmentary compressive and shear stress. A 5 mm gap placed between the diaphysis and plate provided stability comparable to standard fixation, with a 7% decrease of interfragmentary gap and sliding distance, but similar compressive and shear stress. CONCLUSION: Finite element analysis with FNS on Pauwels type III femoral neck fractures revealed that placement of the bolt tip close to subchondral bone provides increased stability. Inferior positioning of FNS bolt increased interfragmentary sliding distance, compressive, and shear stress. The comparable stability of the fixation model with the standard model suggests that a 5 mm gap placed between the plate and diaphysis could viably adjust the depth of the bolt. Cite this article: Bone Joint Res 2022;11(2):102-111.

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.

Enhancement of seed germination and microbial disinfection on ginseng by cold plasma treatment.

BACKGROUND: This study aimed to investigate the effect of cold plasma treatment on the improvement of seed germination and surface sterilization of ginseng seeds. METHODS: Dehisced ginseng (Panax ginseng) seeds were exposed to dielectric barrier discharge (DBD) plasma operated in argon (Ar) or an argon/oxygen mixture (Ar/O2), and the resulting germination and surface sterilization were compared with those of an untreated control group. Bacterial and fungal detection assays were performed for plasma-treated ginseng seeds after serial dilution of surface-washed suspensions. The microbial colonies (fungi and bacteria) were classified according to their phenotypical morphologies and identified by molecular analysis. Furthermore, the effect of cold plasma treatment on the in vitro antifungal activity and suppression of Cylindrocarpon destructans in 4-year-old ginseng root discs was investigated. RESULTS: Seeds treated with plasma in Ar or Ar/O2 exhibited a higher germination rate (%) compared with the untreated controls. Furthermore, the plasma treatment exhibited bactericidal and fungicidal effects on the seed surface, and the latter effect was stronger than the former. In addition, plasma treatment exhibited in vitro antifungal activity against C. destructans and reduced the disease severity (%) of root rot in 4-year-old ginseng root discs. The results demonstrate the stimulatory effect of plasma treatment on seed germination, surface sterilization, and root rot disease suppression in ginseng. CONCLUSION: The results of this study indicate that the cold plasma treatment can suppress the microbial community on the seed surface root rot in ginseng.

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.

Molecular changes associated with spontaneous phenotypic variation of Paenibacillus polymyxa, a commonly used biocontrol agent, and temperature-dependent control of variation.

There has been a growing interest in deploying plant growth-promoting rhizobacteria (PGPR) as a biological control agent (BCA) to reduce the use of agrochemicals. Spontaneous phenotypic variation of PGPR, which causes the loss of traits crucial for biocontrol, presents a large obstacle in producing commercial biocontrol products. Here, we report molecular changes associated with phenotypic variation in Paenibacillus polymyxa, a PGPR widely used for biocontrol worldwide, and a simple cultural change that can prevent the variation. Compared to B-type (non-variant) cells of P. polymyxa strain E681, its phenotypic variant, termed as F-type, fails to form spores, does not confer plant growth-promoting effect, and displays altered colony and cell morphology, motility, antagonism against other microbes, and biofilm formation. This variation was observed in all tested strains of P. polymyxa, but the frequency varied among them. RNA-seq analysis revealed differential regulation of many genes involved in sporulation, flagella synthesis, carbohydrate metabolism, and antimicrobial production in F-type cells, consistent with their pleiotropic phenotypic changes. F-type cells's sporulation was arrested at stage 0, and the key sporulation gene spo0A was upregulated only in B-type cells. The phenotypic variation could be prevented by altering the temperature for growth. When E681 was cultured at 20 °C or lower, it exhibited no variation for 7 days and still reached ~ 108 cfu/mL, the level sufficient for commercial-scale production of biocontrol products.