RDA-Genebank and Digital Phenotyping for Next-Generation Research on Plant Genetic Resources.

The National Agrobiodiversity Center under the Rural Development Administration (RDA) in Jeonju, Republic of Korea stands as the foremost national genebank in the country. Over the years, the National Agrobiodiversity Center has remained committed to enriching its collection with foreign genetic resources, elevating its status to a world-class plant genetic resources (PGR)- holding genebank. Currently, several steps are being undertaken to improve the accessibility of the collection to national as well as international researchers, improve the data available on the resources, and amend the passport information for the accessions. With the implementation of the Nagoya Protocol, the origin of genetic resources is being highlighted as an important input in the passport information. The RDA-Genebank actively responds to the Nagoya Protocol by supplementing passport data for resources lacking information on their origin. In addition, a large number of conserved resources are continuously multiplied, and agronomic traits are investigated concurrently. With the traditional methods of characterization of the germplasm requiring a significant amount of time and effort, we have initiated high-throughput phenotyping using digital techniques to improve our germplasm data. Primarily, we have started adding seed phenotype information followed by measuring root phenotypes which are stored under agronomic traits. This may be the initial step toward using largescale high-throughput techniques for a germplasm. In this study, we aim to provide an introduction to the RDA-Genebank, to adopted international standards, and to the establishment of high-throughput phenotyping techniques for the improvement of passport information.

Brassica biodiversity conservation: prevailing constraints and future avenues for sustainable distribution of plant genetic resources.

The past decade has seen an observable loss of plant biodiversity which can be attributed to changing climate conditions, destroying ecosystems to create farmlands and continuous selective breeding for limited traits. This loss of biodiversity poses a significant bottleneck to plant biologists across the globe working on sustainable solutions to address the current barriers of agricultural productivity. Plant genetic resources centers or genebanks that conserve plant germplasm can majorly contribute towards addressing this problem. Second only to soybean, Brassica remains the largest oil-seed crop and is cultivated across 124 countries, and FAO estimates for a combined gross production values of broccoli, cabbages, cauliflower, mustard and rape seeds stands at a staggering 67.5 billion US dollars during the year 2020. With such a global status, wide variety of uses and more recently, growing importance in the health food sector, the conservation of diverse genetic resources of Brassica appeals for higher priority. Here we review the current status of Brassica conservation across plant genebanks. At present, at least 81,752 accessions of Brassica are recorded to be conserved in 148 holding institutes spread across only 81 countries. Several aspects that need to be addressed to improve proper conservation of the Brassica diversity was well as dissemination of germplasm are discussed. Primarily, the number of accessions conserved across countries and the diversity of Brassica taxa most countries has been highly limited which may lead to biodiversity loss in the longer run. Moreover, several practical challenges in Brassica germplasm conservation especially with respect to taxonomic authorities have been discussed. The current review identifies and highlights areas for progress in Brassica conservation, which include but are not limited to, distribution of conserved Brassica biodiversity, challenges faced by conservation biologists, conservation methods, technical hurdles and future avenues for research in diverse Brassica species.

Glucosinolate Diversity Analysis in Choy Sum (Brassica rapa subsp. chinensis var. parachinensis) Germplasms for Functional Food Breeding.

The aim of this study was to analyze glucosinolates (GSLs) in germplasm that are currently conserved at the RDA-Genebank. The analysis focused on the glucosinolate diversity among the analyzed germplasms, with the goal of identifying those that would be most useful for future breeding efforts to produce nutritionally rich Choy sum plants. In total, 23 accessions of Choy sums that possessed ample background passport information were selected. On analyzing the glucosinolate content for 17 different glucosinolates, we observed aliphatic GSLs to be the most common (89.45%) and aromatic GSLs to be the least common (6.94%) of the total glucosinolates detected. Among the highly represented aliphatic GSLs, gluconapin and glucobrassicanapin were found to contribute the most (>20%), and sinalbin, glucoraphanin, glucoraphasatin, and glucoiberin were detected the least (less than 0.05%). We identified one of the accessions, IT228140, to synthesize high quantities of glucobrassicanapin and progoitrin, which have been reported to contain several therapeutic applications. These conserved germplasms are potential bioresources for breeders, and the availability of information, including therapeutically important glucosinolate content, can help produce plant varieties that can naturally impact public health.

Quantification and Diversity Analyses of Major Glucosinolates in Conserved Chinese Cabbage (Brassica rapa L. ssp. pekinensis) Germplasms.

The genebank at the National Agrobiodiversity Center (RDA-Genebank, Jeonju, Republic of Korea), conserves approximately 8000 germplasms of Brassica spp., of which Chinese cabbage (Brassica rapa L. ssp. pekinensis) is one of the major crops actively used as food in Northeast Asia, including Korea, as the main ingredient for kimchi. Glucosinolates are a major class of compounds in Chinese cabbage that are responsible for their distinctive flavor, and RDA-Genebank is constantly building a related database (DB) to select suitable germplasms required by consumers and provide resources for breeding programs. In this study, ten glucosinolates were analyzed in sixty Chinese cabbage germplasms. Six aliphatic glucosinolates were the major components, accounting for 85.00% to 91.98% of total glucosinolates in each germplasm. Among them, gluconapin (333.26 to 23,501.58 µmol∙kg-1 DW) was highly represented, followed by glucobrassicanapin (545.60 to 10,344.70 µmol∙kg-1 DW) and progoitrin (155.28 to 8536.51 µmol∙kg-1 DW). In addition, we selected germplasms with a high content of each studied glucosinolate. To analyze the diversity and distribution of glucosinolates among the studied germplasms, Pearson's correlation was performed, and the related results were interpreted through their biosynthetic pathways. The k-means clustering indicated four optimal clusters, which were confirmed through principal component analysis. Orthogonal projection to latent structure discriminant analysis (OPLS-DA) was also performed on the status (landrace and cultivar) and origin (Korea, China, Taiwan, and Japan) passport data of the germplasms, followed by the calculation of variable importance in the projection (VIP) values. These results are part of a continuous series of studies to analyze the glucosinolates of Brassica germplasms that are being conserved at RDA-Genebank. We aim to provide related results through a public platform accessible to everyone and thereby improve the distribution of Brassica germplasms.

Pepper Mild Mottle Virus: An Infectious Pathogen in Pepper Production and a Potential Indicator of Domestic Water Quality.

Pepper (Capsicum spp.; Family: Solanaceae; 2n = 24) is an important crop cultivated worldwide for the consumption of its fresh and dried processed fruits. Pepper fruits are used as raw materials in a wide variety of industrial processes. As a multipurpose vegetable crop, there is a need to increase the yield. However, yield productivity of pepper is severely constrained by infectious plant pathogens, including viruses, bacteria, fungi, and oomycetes. The pepper mild mottle virus (PMMoV) is currently one of the most damaging pathogens associated with yield losses in pepper production worldwide. In addition to impacts on pepper productivity, PMMoV has been detected in domestic and aquatic water resources, as well as in the excreta of animals, including humans. Therefore, PMMoV has been suggested as a potential indicator of domestic water quality. These findings present additional concerns and trigger the need to control the infectious pathogen in crop production. This review provides an overview of the distribution, economic impacts, management, and genome sequence variation of some isolates of PMMoV. We also describe genetic resources available for crop breeding against PMMoV.

Genome-Wide Association Studies of Seven Root Traits in Soybean (Glycine max L.) Landraces.

Soybean [Glycine max (L.) Merr.], an important oilseed crop, is a low-cost source of protein and oil. In Southeast Asia and Africa, soybeans are widely cultivated for use as traditional food and feed and industrial purposes. Given the ongoing changes in global climate, developing crops that are resistant to climatic extremes and produce viable yields under predicted climatic conditions will be essential in the coming decades. To develop such crops, it will be necessary to gain a thorough understanding of the genetic basis of agronomic and plant root traits. As plant roots generally lie beneath the soil surface, detailed observations and phenotyping throughout plant development present several challenges, and thus the associated traits have tended to be ignored in genomics studies. In this study, we phenotyped 357 soybean landraces at the early vegetative (V2) growth stages and used a 180 K single-nucleotide polymorphism (SNP) soybean array in a genome-wide association study (GWAS) conducted to determine the phenotypic relationships among root traits, elucidate the genetic bases, and identify significant SNPs associated with root trait-controlling genomic regions/loci. A total of 112 significant SNP loci/regions were detected for seven root traits, and we identified 55 putative candidate genes considered to be the most promising. Our findings in this study indicate that a combined approach based on SNP array and GWAS analyses can be applied to unravel the genetic basis of complex root traits in soybean, and may provide an alternative high-resolution marker strategy to traditional bi-parental mapping. In addition, the identified SNPs, candidate genes, and diverse variations in the root traits of soybean landraces will serve as a valuable basis for further application in genetic studies and the breeding of climate-resilient soybeans characterized by improved root traits.

Leaf Mustard (Brassica juncea) Germplasm Resources Showed Diverse Characteristics in Agro-Morphological Traits and Glucosinolate Levels.

Leaf mustard, characterized by its purple/red/green leaves with a green/white midrib, is known for its thick, tender, and spicy leaves with a unique taste and flavor. There were only a few studies reported on leaf mustard for its morphological and biochemical traits from Korea. A total of 355 leaf mustard accessions stored at the GenBank of the National Agrobiodiversity Center were evaluated for 25 agro-morphological traits and seven intact glucosinolates (GSLs). The accessions showed a wide variation in terms of most of the traits. The quantitative agro-morphological traits varied from 16.0 (leaf length) to 48.7% (petiole width) of the coefficient of variation (CV). The highest variation was observed in glucoiberin (299.5%, CV), while the total GSL showed a CV of 66.1%. Sinigrin, followed by gluconapin and gluconasturtiin, was the most abundant GSL, accounting for as high as 75% of the total GSLs, while glucobrassicanapin and glucoiberin were the least abundant, contributing 0.7% and 0.1% on average, respectively. Sinigrin had a positive significant correlation with all GSLs but gluconasturtiin, while glucobarbarin and gluconasturtiin were highly positively correlated to each other, but least correlated with other GSLs. The leaf length was negatively correlated with sinigrin and glucoiberin. The width of the petiole showed a positive correlation with gluconapin, glucobrassicanapin, and glucobrassicin, while the length of the petiole had a negative correlation with sinigrin, glucobrassicanapin, glucoiberin, glucobrassicin, and the total GSLs. A higher width of the midrib was associated with higher contents of gluconapin, glucobrassicanapin, and glucobrassicin. A PCA analysis based on the agro-morphological traits showed that the first and second principal components accounted for 65.2% of the overall variability. Accessions that form a head tend to exhibit a longer leaf length, a larger plant weight, a thicker midrib, and higher widths of the midrib, petiole, and leaf. The GSLs showed inconsistent inter-and intra-leaf variation. Accessions that identified for various traits in their performance, such as, for example, Yeosu66 and IT259487 (highest total glucosinolates) and IT228984 (highest plant weight), would be promising lines for developing new varieties.

High-Throughput Phenotypic Characterization and Diversity Analysis of Soybean Roots (Glycine max L.).

Soybean (Glycine max L.) is a crop native to Northeast Asia, including China, Korea, and Japan, but currently cultivated all over the world. The National Agrobiodiversity Center in Korea at the Rural Development Administration (RDA) conserves approximately 26,000 accessions and conducts characterizations of its accessions, to accumulate new information. Roots are essential organs of a plant, providing mechanical support, as well as aiding water and nutrient acquisition. Currently, not much information is available in international gene banks regarding root characterization. We studied the root phenotype of 374 soybean accessions, using a high-throughput method. Eight root morphological traits (RMT) were studied and we observed that the surface area (SA), number of forks (NF), and number of tips (NT) had a positive correlation with total length (LENGTH), and that link average length (LAL) and other traits all had a negative correlation. Additionally, the correlation between seed traits (height, width, and 100-seed weight) and root traits was confirmed for the first time in this experiment. The germplasms were divided into three clusters by k-means clustering, and orthogonal projections to latent structures discriminant analysis (OPLS-DA) was used to compare clusters. The most distinctive characteristics between clusters were total lateral average length (LAD) and total lateral average length (DIAM). Cluster 3 had the highest LENGTH, SA, NF, and NF, whereas cluster 1 had the smallest LENGTH, SA, and NF. We selected the top 10 accessions for each RMT, and IT208321, IT216313, and IT216137 were nominated as the best germplasms. These accessions can be recommended to breeders as materials for breeding programs. This is a preliminary report on the characterization of the root phenotype at an international gene bank and will open up the possibility of improving the available information on accessions in gene banks worldwide.

Genome-Wide Association Study of Resistance to Phytophthora capsici in the Pepper (Capsicum spp.) Collection.

One of the most serious pepper diseases is Phytophthora blight, which is caused by Phytophthora capsici. It is crucial to assess the resistance of pepper genetic resources to Phytophthora blight, understand the genetic resistances, and develop markers for selecting resistant pepper materials in breeding programs. In this study, the resistance of 342 pepper accessions to P. capsici was evaluated. The disease severity score method was used to evaluate the phenotypic responses of pepper accessions inoculated with the KCP7 isolate. A genome-wide association study (GWAS) was performed to identify single nucleotide polymorphisms (SNPs) linked to P. capsici (isolate KCP7) resistance. The pepper population was genotyped using the genotype-by-sequencing (GBS) method, and 45,481 SNPs were obtained. A GWAS analysis was performed using resistance evaluation data and SNP markers. Significantly associated SNPs for P. capsici resistance at 4 weeks after inoculation of the GWAS pepper population were selected. These SNPs for Phytophthora blight resistance were found on all chromosomes except Chr.05, Chr.09, and Chr.11. One of the SNPs found on Chr.02 was converted into a high-resolution melting (HRM) marker, and another marker (QTL5-1) from the previous study was applied to pepper accessions and breeding lines for validation and comparison. This SNP marker was selected because the resistance phenotype and the HRM marker genotype matched well. The selected SNP was named Chr02-1126 and was located at 112 Mb on Chr.02. The Chr02-1126 marker predicted P. capsici resistance with 78.5% accuracy, while the QTL5-1 marker predicted resistance with 80.2% accuracy. Along with the marker for major quantitative traits loci (QTLs) on Chr.05, this Chr02-1126 marker could be used to accurately predict Phytophthora blight resistance in pepper genetic resources. Therefore, this study will assist in the selection of resistant pepper plants in order to breed new phytophthora blight-resistant varieties.

Metabolic Analysis of the Development of the Plant-Parasitic Cyst Nematodes Heterodera schachtii and Heterodera trifolii by Capillary Electrophoresis Time-of-Flight Mass Spectrometry.

The cyst nematodes Heterodera schachtii and Heterodera trifolii, whose major hosts are sugar beet and clover, respectively, damage a broad range of plants, resulting in significant economic losses. Nematodes synthesize metabolites for organismal development and social communication. We performed metabolic profiling of H. schachtii and H. trifolii in the egg, juvenile 2 (J2), and female stages. In all, 392 peaks were analyzed by capillary electrophoresis time-of-flight mass spectrometry, which revealed a lot of similarities among metabolomes. Aromatic amino acid metabolism, carbohydrate metabolism, choline metabolism, methionine salvage pathway, glutamate metabolism, urea cycle, glycolysis, gluconeogenesis, coenzyme metabolism, purine metabolism, pyrimidine metabolism, and tricarboxylic acid (TCA) cycle for energy conversion (ß-oxidation and branched-chain amino acid metabolism) energy storage were involved in all stages studied. The egg and female stages synthesized higher levels of metabolites compared to the J2 stage. The key metabolites detected were glycerol, guanosine, hydroxyproline, citric acid, phosphorylcholine, and the essential amino acids Phe, Leu, Ser, and Val. Metabolites, such as hydroxyproline, acetylcholine, serotonin, glutathione, and glutathione disulfide, which are associated with growth and reproduction, mobility, and neurotransmission, predominated in the J2 stage. Other metabolites, such as SAM, 3PSer, 3-ureidopropionic acid, CTP, UDP, UTP, 3-hydroxy-3-methylglutaric acid, 2-amino-2-(hydroxymethyl-1,3-propanediol, 2-hydroxy-4-methylvaleric acid, Gly Asp, glucuronic acid-3 + galacturonic acid-3 Ser-Glu, citrulline, and γ-Glu-Asn, were highly detected in the egg stage. Meanwhile, nicotinamide, 3-PG, F6P, Cys, ADP-Ribose, Ru5P, S7P, IMP, DAP, diethanolamine, p-Hydroxybenzoic acid, and γ-Glu-Arg_divalent were unique to the J2 stage. Formiminoglutamic acid, nicotinaminde riboside + XC0089, putrescine, thiamine 2,3-dihydroxybenzoic acid, 3-methyladenine, caffeic acid, ferulic acid, m-hydrobenzoic acid, o- and p-coumaric acid, and shikimic acid were specific to the female stage. Overall, highly similar identities and quantities of metabolites between the corresponding stages of the two species of nematode were observed. Our results will be a valuable resource for further studies of physiological changes related to the development of nematodes and nematode-plant interactions.

Nutritional Metabolites of Red Pigmented Lettuce (Lactuca sativa) Germplasm and Correlations with Selected Phenotypic Characters.

Lettuce is an important dietary source of bioactive phytochemicals. Screening and identification of the health beneficial metabolites and evaluating the relationships with phenotypic characters can help consumers adjust their preferences for lettuce plant types. Thus, we explored the major health-beneficial individual metabolites and antioxidant potential of 113 red pigmented lettuce leaf samples. A UV-Vis spectrophotometer and UPLC-DAD-QTOF/MS (TQ/MS) instruments were used for the identification and quantification of metabolites and antioxidant activity accordingly. The metabolites were quantified against their corresponding external standards. The contents of metabolites varied significantly among lettuce samples. Cyanidin 3-O-(6″-O-malonyl)glucoside (4.7~5013.6 µg/g DW), 2,3-di-O-caffeoyltartaric acid (337.1~19,957.2 µg/g DW), and quercetin 3-O-(6″-O-malonyl)glucoside (45.4~31,121.0 µg/g DW) were the most dominant in red pigmented lettuce samples among anthocyanins, hydroxycinnamoyl derivatives, and flavonols, respectively. Lettuces with dark and very dark red pigmented leaves, circular leaf shape, a strong degree of leaf undulation, and highly dense leaf incisions were found to have high levels of flavonoids and hydroxycinnamoyl derivatives. Principal component analysis was used to investigate similarities and/or differences between samples, and the partial least square discriminant analysis classified them into known groups. The key variables that contributed highly were determined. Our report provides critical data on the bioactive constituents of red pigmented lettuce to breeders developing varieties with enhanced bioactive compounds and to nutraceutical companies developing nutrient dense foods and pharmaceutical formulations.

Transcriptome Analysis and miRNA Target Profiling at Various Stages of Root-Knot Nematode Meloidogyne incognita Development for Identification of Potential Regulatory Networks.

Root-knot nematodes (RKNs) are a group of plant-parasitic nematodes that cause damage to various plant species and extensive economical losses. In this study, we performed integrated analysis of miRNA and mRNA expression data to explore the regulation of miRNA and mRNA in RKNs. In particular, we aimed to elucidate the mRNA targets of Meloidogyne incognita miRNAs and variations of the RKN transcriptome during five stages of its life cycle. Stage-wise RNA sequencing of M. incognita resulted in clean read numbers of 56,902,902, 50,762,456, 40,968,532, 47,309,223, and 51,730,234 for the egg, J2, J3, J4, and female stages, respectively. Overall, stage-dependent mRNA sequencing revealed that 17,423 genes were expressed in the transcriptome of M. incognita. The egg stage showed the maximum number of transcripts, and 12,803 gene transcripts were expressed in all stages. Functional Gene Ontology (GO) analysis resulted in three main GO classes: biological process, cellular components, and molecular function; the detected sequences were longer than sequences in the reference genome. Stage-wise selected fragments per kilobase of transcript per million mapped reads (FPKM) values of the top 10 stage-specific and common mRNAs were used to construct expression profiles, and 20 mRNAs were validated through quantitative real-time PCR (qRT-PCR). Next, we used three target prediction programs (miRanda, RNAhybrid, and PITA) to obtain 2431 potential target miRNA genes in RKNs, which regulate 8331 mRNAs. The predicted potential targets of miRNA were generally involved in cellular and metabolic processes, binding of molecules in the cell, membranes, and organelles. Stage-wise miRNA target analysis revealed that the egg stage contains heat shock proteins, transcriptional factors, and DNA repair proteins, whereas J2 includes DNA replication, heat shock, and ubiquitin-conjugating pathway-related proteins; the J3 and J4 stages are represented by the major sperm protein domain and translation-related proteins, respectively. In the female stage, we found proteins related to the maintenance of molybdopterin-binding domain-containing proteins and ubiquitin-mediated protein degradation. In total, 29 highly expressed stage-specific mRNA-targeting miRNAs were analyzed using qRT-PCR to validate the sequence analysis data. Overall, our findings provide new insights into the molecular mechanisms occurring at various developmental stages of the RKN life cycle, thus aiding in the identification of potential control strategies.

Assessment of Metabolic Profiles in Florets of Carthamus Species Using Ultra-Performance Liquid Chromatography-Mass Spectrometry.

The genus Carthamus is a diverse group of plants belonging to the family Compositae. Florets of Carthamus species exhibit various colors, including white, yellow, orange, and red, which are related to their metabolite compositions. We aimed to investigate the metabolites accumulated in florets of three wild (C. lanatus, C. palaestinus, and C. turkestanicus) and one cultivated (C. tinctorius) species of safflower at three developmental stages. Metabolites were extracted from freeze-dried florets using 70% methanol; qualification and quantification were carried out using liquid chromatography quadrupole time-of-flight mass spectrometry in positive and negative ion modes followed by extraction of the peaks. Fifty-six metabolites, including phenylpropanoids, chalcones, isoflavonoids, flavanones, flavonols, flavones, and other primary metabolites, were identified for the first time in safflower wild species. The orange florets contained high abundances of safflomin A, anhydrosafflor yellow B, and baimaside, whereas white/cream and light-yellow pigmented florets had high abundances of 1,5-dicaffeoylquinic acid, luteolin 7-O-glucuronide, and apigenin 7-O-ß-D-glucuronide. The principal component analysis clearly distinguished the samples based on their pigment types, indicating that color is a dominant factor dictating the identity and amount of the metabolites. Pearson correlation data based on levels of metabolites showed that orange and yellow florets were significantly correlated to each other. White and cream pigmented species were also highly correlated. Comparison between three developmental stages of safflower wild species based on their metabolite profile showed inconsistent. The findings of this study broaden the current knowledge of safflower metabolism. The wide diversity of metabolites in safflower materials also helps in efforts to improve crop quality and agronomic traits.

First Report of Fusarium Wilt Caused by Fusarium equiseti on Cabbage (Brassica oleracea var. capitate L.) in Korea.

Cabbage (Brassica oleracea var. capitate L.) is an important vegetable crop that is widely cultivated throughout the world. In August 2019, wilting symptoms on cabbage (stunted growth, withered leaves, and wilted plants) were observed in a cabbage field of Pyeongchang, Gangwon Province, with an incidence of 5 to 10%. To identify the cause, symptomatic root tissue was excised, surface-sterilized with 70% ethanol, and rinsed thrice with sterile distilled water. The samples were dried on blotter paper, placed onto potato dextrose agar (PDA), and incubated at 25°C for 1 week. Five morphologically similar fungal isolates were sub-cultured and purified using the single spore isolation method (Choi et al. 1999). The fungus produced colonies with abundant, loosely floccose, whitish-brown aerial mycelia and pale-orange pigmentation on PDA. Macroconidia had four 4 to six 6 septa, a foot-shaped basal cell, an elongated apical cell, and a size of 20.2 to 31.8 × 2.2 to 4.1 µm (n = 30). No microconidia were observed. Chlamydospores were produced from hyphae and were most often intercalary, in pairs or solitary, globose, and frequently formed chains (6.2? to 11.7 µm, n = 10). Based on these morphological characteristics, the fungus was identified as Fusarium equiseti (Leslie and Summerell 2006). A representative isolate was deposited in the Korean Agricultural Culture Collection (KACC48935). For molecular characterization, portions of the translation elongation factor 1-alpha (TEF-1α) and second largest subunit of RNA polymerase II (RPB2) genes were amplified from the representative isolate using the primers pair of TEF-1α (O'Donnell et al. 2000) and GQ505815 (Fusarium MLST database), and sequenced. Searched BLASTn of the RPB2 sequence (MT576587) to the Fusarium MLST database showed 99.94% similarity to the F. incarnatum-equiseti species complex (GQ505850) and 98.85 % identity to both F. equiseti (GQ505599) and F. equiseti (GQ505772). Further, the TEF-1α sequence (MT084815) showed 100% identity to F. equiseti (KT224215) and 99.85% identity to F. equiseti (GQ505599), respectively. Therefore, the fungus was identified as F. equiseti based on morphological and molecular identification. For pathogenicity testing, a conidial suspension (1 × 106 conidia/ml) was prepared by harvesting macroconidia from 2-week-old cultures on PDA. Fifteen 4-week-old cabbage seedlings (cv. 12-Aadrika) were inoculated by dipping roots into the conidial suspension for 30 min. The inoculated plants were transplanted into a 50-hole plastic tray containing sterilized soil and maintained in a growth chamber at 25°C, with a relative humidity of >80%, and a 12-h/12-h light/dark cycle. After 4 days, the first wilt symptoms were observed on inoculated seedlings, and the infected plants eventually died within 1 to 2 weeks after inoculation. No symptoms were observed in plants inoculated with sterilized distilled water. The fungus was re-isolated from symptomatic tissues of inoculated plants and its colony and spore morphology were identical to those of the original isolate, thus confirming Koch's postulates. Fusarium wilt caused by F. equiseti has been reported in various crops, such as cauliflower in China, cumin in India, and Vitis vinifera in Spain (Farr and Rossman 2020). To our knowledge, this is the first report of F. equiseti causing Fusarium wilt on cabbage in Korea. It This disease poses a threat to cabbage production in Korea, and effective disease management strategies need to be developed.

Chitin Biosynthesis Inhibition of Meloidogyne incognita by RNAi-Mediated Gene Silencing Increases Resistance to Transgenic Tobacco Plants.

Meloidogyne incognita is a devastating plant parasitic nematode that causes root knot disease in a wide range of plants. In the present study, we investigated host-induced RNA interference (RNAi) gene silencing of chitin biosynthesis pathway genes (chitin synthase, glucose-6-phosphate isomerase, and trehalase) in transgenic tobacco plants. To develop an RNAi vector, ubiquitin (UBQ1) promoter was directly cloned, and to generate an RNAi construct, expression of three genes was suppressed using the GATEWAY system. Further, transgenic Nicotiana benthamiana lines expressing dsRNA for chitin synthase (CS), glucose-6-phosphate isomerase (GPI), and trehalase 1 (TH1) were generated. Quantitative PCR analysis confirmed endogenous mRNA expression of root knot nematode (RKN) and revealed that all three genes were more highly expressed in the female stage than in eggs and in the parasitic stage. In vivo, transformed roots were challenged with M. incognita. The number of eggs and root knots were significantly decreased by 60-90% in RNAi transgenic lines. As evident, root galls obtained from transgenic RNAi lines exhibited 0.01- to 0.70-fold downregulation of transcript levels of targeted genes compared with galls isolated from control plants. Furthermore, phenotypic characteristics such as female size and width were also marginally altered, while effect of egg mass per egg number in RNAi transgenic lines was reduced. These results indicate the relevance and significance of targeting chitin biosynthesis genes during the nematode lifespan. Overall, our results suggest that further developments in RNAi efficiency in commercially valued crops can be applied to employ RNAi against other plant parasitic nematodes.

A Metabolic Perspective and Opportunities in Pharmacologically Important Safflower.

Safflower (Carthamus tinctorius L.) has long been grown as a crop due to its commercial utility as oil, animal feed, and pharmacologically significant secondary metabolites. The integration of omics approaches, including genomics, transcriptomics, metabolomics, and proteomics datasets, has provided more comprehensive knowledge of the chemical composition of crop plants for multiple applications. Knowledge of a metabolome of plant is crucial to optimize the evolution of crop traits, improve crop yields and quality, and ensure nutritional and health factors that provide the opportunity to produce functional food or feedstuffs. Safflower contains numerous chemical components that possess many pharmacological activities including central nervous, cardiac, vascular, anticoagulant, reproductive, gastrointestinal, antioxidant, hypolipidemic, and metabolic activities, providing many other human health benefits. In addition to classical metabolite studies, this review focuses on several metabolite-based working techniques and updates to provide a summary of the current medical applications of safflower.

Characterization of a novel antibacterial N-acyl amino acid synthase from soil metagenome.

In an effort to isolate novel natural antibiotics, we searched for antibacterial long-chain N-acyl amino acid synthase (NAS) genes from 70,000 soil metagenome clones by Bacillus subtilis-overlaying screening. In an antibacterial cosmid clone, YS92B, a single gene nasYPL was responsible for the production of the Nas. nasYPL was 903 bp long, and the deduced amino acid sequence showed the highest 71% identity with a hypothetical protein from Massilia niastensis. Phylogenetic analysis demonstrated that NasYPL belongs to Group 1 Nas. Heterologous expression of the same nasYPL gene in Escherichia coli and two Pseudomonas strains (P. putida and P. koreensis) conferred antibacterial activities against Listeria monocytogenes, Staphylococcus epidermidis, and Bacillus subtilis. Mass spectral analysis of the antibacterial fractions identified 7 peaks corresponding to long-chain N-acyl tyrosine, 5 peaks to N-acyl phenylalanine, and 3 peaks to N-acyl leucine (or isoleucine) derivatives linked with 7 fatty acids, indicating enzymatic products derived by NasYPL. Therefore, NasYPL expression by host-specific manner may provide applicable antibacterial characteristics to biotechnologically important Pseudomonas strains.

Correction to: Release of overexpressed CypB activates ERK signaling through CD147 binding for hepatoma cell resistance to oxidative stress.

The original version of this article contained a mistake. The bands for HA Tag and t-ERK in Figs. 2d, 2h, 3d are incorrect. The author informs that these errors had no influence in the scientific content of the paper. The corrected figures (Figs. 2 and 3) are given below.

Characterization of an inhibitor-resistant endo-1,4-ß-mannanase from the gut microflora metagenome of Hermetia illucens.

OBJECTIVE: Hermetia illucens is a voracious insect scavenger that efficiently decomposes food waste. To exploit novel hydrolytic enzymes from this insect, we constructed a fosmid metagenome library using unculturable H. illucens intestinal microorganisms. RESULTS: Functional screening of the library on carboxymethyl cellulose plates identified a fosmid clone with a product displaying hydrolytic activity. Fosmid sequence analysis revealed a novel mannan-degrading gene (ManEM17) composed of 1371 base pairs, encoding 456 amino acids with a deduced 54 amino acid N-terminal signal peptide sequence. Conceptual translation and domain analysis revealed that sequence homology was highest (46%) with endo-1,4-ß-mannosidase of Anaerophaga thermohalophila. Phylogenetic and domain analysis indicated that ManEM17 belongs to a novel ß-mannanase containing a glycoside hydrolase family 26 domain. The recombinant protein (rManEM17) was expressed in Escherichia coli, exhibiting the highest activity at 55 °C and pH 6.5. The protein hydrolyzed substrates with ß-1,4-glycosidic mannoses; maximum specific activity (5467 U mg-1) occurred toward locust bean gum galactomannan. However, rManEM17 did not hydrolyze p-Nitrophenyl-ß-pyranosides, demonstrating endo-form mannanase activity. Furthermore, rManEM17 was highly stable under stringent conditions, including polar organic solvents as well as chemical reducing and denaturing reagents. CONCLUSIONS: ManEM17 is an attractive candidate for mannan degradation under the high-organic-solvent and protein-denaturing processes in food and feed industries.

Isolation and characterization of a halotolerant and protease-resistant α-galactosidase from the gut metagenome of Hermetia illucens.

Hermetia illucens is a voracious insect scavenger, decomposing food waste efficiently. To survey novel hydrolytic enzymes, we constructed a fosmid metagenome library using unculturable intestinal microorganisms from H. illucens in our previous study (Lee et al., 2014). Functional screening of the library on carboxymethyl cellulose plates identified a fosmid clone the product of which displayed hydrolytic activity. Sequence analysis of the fosmid revealed a novel α-galactosidase gene, Agas2. The Agas2 gene is composed of 2,007 base pairs encoding 668 amino acids with a deduced 25 amino acid N-terminal signal peptide sequence. The conceptual translation and domain analysis of Agas2 showed the highest sequence identity (84%) with the putative α-galactosidase of Dysgonomonas sp. HGC4, exhibiting well-conserved domain homology with glycosyl hydrolase family 97. Phylogenetic analysis indicated that Agas2 may be a currently uncharacterized α-galactosidase. The recombinant protein, rAgas2, was successfully expressed in E. coli. rAgas2 showed the highest activity at 40 °C and pH 7.0. It displayed great pH stability within a pH range of 5-11 for 15 h at 4 °C. rAgas2 was highly stable under stringent conditions, including polar organic solvents, non-ionic detergents, salt, and proteases. rAgas2 hydrolyzed α-d-galactose substrates, showing the maximum enzymatic activity toward p-nitrophenyl α-d-galactopyranoside (specific activity 128.37 U/mg). However, rAgas2 did not hydrolyze substrates linked with ß-glucose moieties. Overall, Agas2 may be an attractive candidate for the degradation of α-galactose family oligosaccharides in high-salt, protease-rich and high-organic-solvent processes.