Re-Identification of Aspergillus Subgenus Nidulantes Strains and Description of Three Unrecorded Species From Korea.

Aspergillus subgenus Nidulantes with nine section forms the second largest subgenus of the fungi that comes under the genus Aspergillus. Species in this group of fungi are important as they are reported to play several important roles in the environment including influencing air quality in confined spaces, food spoilage, production of mycotoxins as well as in human pathogenicity. In the present study, 53 strains of Aspergillus subgenus Nidulantes (section: Nidulantes & Usti) isolated from Korea and preserved at the Korean Agricultural Culture Collection (KACC) were subjected to re-identification by using a combined dataset of partial ß-tubulin (BenA), Calmodulin (CaM) gene sequences as well as their morphological data. We confirmed 14 species from 53 isolates in Korea. Of them, eleven species were reported in Korea previously (A. amoenus, A. baeticus, A. calidoustus, A. creber, A. insuetus, A. jensenii, A. nidulans, A. protuberus, A. sydowii, A. tabacinus and A. unguis), and three species (A. griseoaurantiacus, A. puulaauensis and A. sublatus) were previously unreported from Korea. We detailed the characteristic features of these three species, that remain unexplored in Korea.

Re-identification of Colletotrichum gloeosporioides Species Complex Isolates in Korea and Their Host Plants.

The Colletotrichum gloeosporioides species complex includes many phytopathogenic species, causing anthracnose disease on a wide range of host plants and appearing to be globally distributed. Seventy-one Colletotrichum isolates in the complex from different plants and geographic regions in Korea were preserved in the Korean Agricultural Culture Collection (KACC). Most of them had been identified based on hosts and morphological features, this could lead to inaccurate species names. Therefore, the KACC isolates were re-identified using DNA sequence analyses of six loci, comprising internal transcribed spacer, gapdh, chs-1, his3, act, and tub2 in this study. Based on the combined phylogenetic analysis, KACC strains were assigned to 12 known species and three new species candidates. The detected species are C. siamense (n = 20), C. fructicola (n = 19), C. gloeosporioides (n = 9), C. aenigma (n = 5), C. camelliae (n = 3), C. temperatum (n = 3), C. musae (n = 2), C. theobromicola (n = 2), C. viniferum (n = 2), C. alatae (n = 1), C. jiangxiense (n = 1), and C. yulongense (n = 1). Of these, C. jiangxiense, C. temperatum, C. theobromicola and C. yulongense are unrecorded species in Korea. Host plant comparisons showed that 27 fungus-host associations are newly reported in the country. However, plant-fungus interactions need to be investigated by pathogenicity tests.

Re-Identification of Aspergillus Subgenus Circumdati Strains in Korea Led to the Discovery of Three Unrecorded Species.

Aspergillus is one of the largest and diverse genera of fungi with huge economical, biotechnological, and social significance. Taxonomically, Aspergillus is divided into six subgenera comprising 27 sections. In this study, 235 strains of Aspergillus subgenus Circumdati (section: Candidi, Circumdati, Flavi, Flavipedes, Nigri, and Terrei) preserved at the Korean Agricultural Culture Collection (KACC) were analyzed and re-identified using a combined dataset of partial ß-tubulin (BenA), Calmodulin (CaM) gene sequences and morphological data. We confirmed nineteen species to be priorly reported in Korea (A. neotritici, A. terreus, A. floccosus, A. allahabadii, A. steynii, A. westerdijkiae, A. ochraceus, A. ostianus, A. sclerotiorum, A. luchuensis, A. tubingensis, A. niger, A. welwitschiae, A. japonicus, A. nomius, A. tamarii, A. parasiticus, A. flavi, and A. oryzae). Among the studied strains, three species (A. subalbidus, A. iizukae, and A. uvarum), previously unreported or not officially documented, were discovered in Korea, to the best of our knowledge. We have given a detailed description of the characteristic features of the three species, which remain uncharted in Korea.

Re-identification of Colletotrichum acutatum Species Complex in Korea and Their Host Plants.

Colletotrichum acutatum species complex is one of the most important groups in the genus Colletotrichum with a high species diversity and a wide range of host plants. C. acutatum and related species have been collected from different plants and locations in Korea and deposited into the Korean Agricultural Culture Collection (KACC), National Institute of Agricultural Sciences since the 1990s. These fungal isolates were previously identified based mainly on morphological characteristics, and a limitation of molecular data was provided. To confirm the identification of species, 64 C. acutatum species complex isolates in KACC were used in this study for DNA sequence analyses of six loci: nuclear ribosomal internal transcribed spacers (ITS), betatubulin 2 (TUB2), histone-3 (HIS3), glyceraldehyde3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), and actin (ACT). The molecular analysis revealed that they were identified in six different species of C. fioriniae (24 isolates), C. nymphaeae (21 isolates), C. scovillei (12 isolates), C. chrysanthemi (three isolates), C. lupini (two isolates), and C. godetiae (one isolate), and a novel species candidate. We compared the hosts of KACC isolates with "The List of Plant Diseases in Korea", previous reports in Korea and global reports and found that 23 combinations between hosts and pathogens could be newly reported in Korea after pathogenicity tests, and 12 of these have not been recorded in the world.

Lipid ROS- and Iron-Dependent Ferroptotic Cell Death in Unicellular Algae Chlamydomonas reinhardtii.

The phenomenon of heat stress leading to ferroptosis-like cell death has recently been observed in bacteria as well as plant cells. Despite recent findings, the evidence of ferroptosis, an iron-dependent cell death remains unknown in microalgae. The present study aimed to investigate if heat shock could induce reactive oxygen species (ROS) and iron-dependent ferroptotic cell death in Chlamydomonas reinhardtii in comparison with RSL3-induced ferroptosis. After RSL3 and heat shock (50 °C) treatments with or without inhibitors, Chlamydomonas cells were evaluated for cell viability and the induction of ferroptotic biomarkers. Both the heat shock and RSL3 treatment were found to trigger ferroptotic cell death, with hallmarks of glutathione-ascorbic acid depletion, GPX5 downregulation, mitochondrial dysfunction, an increase in cytosolic calcium, ROS production, lipid peroxidation, and intracellular iron accumulation via heme oxygenase-1 activation (HO-1). Interestingly, the cells preincubated with ferroptosis inhibitors (ferrostatin-1 and ciclopirox) significantly reduced RSL3- and heat-induced cell death by preventing the accumulation of Fe2+ and lipid ROS. These findings reveal that ferroptotic cell death affects the iron homeostasis and lipid peroxidation metabolism of Chlamydomonas, indicating that cell death pathways are evolutionarily conserved among eukaryotes.

Recombinant engineered phage-derived enzybiotic in Pichia pastoris X-33 as whole cell biocatalyst for effective biocontrol of Vibrio parahaemolyticus in aquaculture.

Vibrio parahaemolyticus is a major seafood-borne pathogen causing significant economic losses in aquaculture systems. Therefore, application of phage encoded enzymes, particularly endolysin, as a new strategy for effective biocontrol and therapeutic agent against bacterial diseases. In the present study, we synthesized endolysin gene (Vplys60) of bacteriophage qdv001 and biochemically characterized by expressing in Pichia pastoris X-33. In addition to, we also investigated the anti-biofilm and anti-vibriosis activity of Pichia-expressing Vplys60 against vibrio challenged in vivo aquaculture model, Artemia franciscana. The result indicated that the predicted molecular size of Pichia expressed Vplys60 was approximately 28 kDa as verified by SDS-PAGE and zymogram. Vplys60 manifested stable activity over broad range of pH (6-10), temperatures (37-75 °C) and salinity (100-600 mM NaCl). Biochemical and in silico analysis revealed that addition of calcium ion (Ca2+) enhanced the lytic activity of Vplys60 whereas other metal ions inhibited the activity. Additionally, calcium-dependent Vplys60 has showed a strong amidase activity by cleaving the peptidoglycan of V. parahaemolyticus. Our data also showed that Vplys60 (75 µg/ml) significantly inhibits biofilm formation (91.6%) and significantly reduced the bacterial population. The in vivo challenge study showed enhanced survival rate in combination with reduced vibrio load in Artemia after administration of Pichia-expressing Vplys60.

Complete mitochondrial genome sequence of a xerophilic fungus, Aspergillus pseudoglaucus.

Aspergillus pseudoglaucus is a xerophilic filamentous fungus which can produce various secondary metabolites. Here, we reported the complete mitochondrial genome sequence of A. pseudoglaucus isolated from Meju, a soybean brick in Korea. Its mitochondrial genome was successfully assembled from raw reads sequenced using MiSeq by Velvet and SOAPGapCloser. Total length of the mitochondrial genome is 53,882 bp, which is third longest among known Aspergillus mitochondrial genomes and encoded 58 genes (30 protein-coding genes including hypothetical ORFs, two rRNAs, and 26 tRNAs). Nucleotide sequence of coding regions takes over 66.6% and overall GC content is 27.8%. Phylogenetic trees present that A. pseudoglaucus is located outside of section Nidulantes. Additional researches will be required for clarifying phylogenetic position of section Aspergillus.

Genome Sequence of Bacillus vallismortis TD3, a Salt-Tolerant Strain Isolated from the Sediments of a Solar Saltern in Tamil Nadu, India.

Various Bacillus spp. capable of producing enzymes with industrially desirable properties have been isolated from adverse environments. Here, we announce the 3.91-Mbp draft genome sequence of a moderately salt-resistant Bacillus vallismortis strain, TD3, capable of producing several industrially relevant enzymes.

Genome Sequence of a Moderately Halophilic Bacillus cereus Strain, TS2, Isolated from Saltern Sediments.

We report the 5.3-Mbp genome sequence of Bacillus cereus strain TS2, which was isolated from the sediments of a solar saltern in southern India. Genome analysis of B. cereus TS2, a salt-resistant strain, will improve our understanding of how B. cereus, a food pathogen, responds to hyperosmotic stress.

Bicarbonate supplementation enhances growth and biochemical composition of Dunaliella salina V-101 by reducing oxidative stress induced during macronutrient deficit conditions.

The unicellular marine alga Dunaliella salina is a most interesting green cell factory for the production of carotenes and lipids under extreme environment conditions. However, the culture conditions and their productivity are the major challenges faced by researchers which still need to be addressed. In this study, we investigated the effect of bicarbonate amendment on biomass, photosynthetic activity, biochemical constituents, nutrient uptake and antioxidant response of D. salina during macronutrient deficit conditions (N-, P- and S-). Under nutrient deficit conditions, addition of sodium bicarbonate (100 mM) significantly increased the biomass, carotenoids including ß-carotene and lutein, lipid, and fatty acid content with concurrent enhancement of the activities of nutrient assimilatory and carbonic anhydrase enzymes. Maximum accumulation of carotenoid especially ß-carotene (192.8 ± 2.11 µg/100 mg) and lipids (53.9%) was observed on addition of bicarbonate during nitrate deficiency compared to phosphate and sulphate deficiency. Supplementation of bicarbonate reduced the oxidative stress caused by ROS, lowered lipid peroxidation damage and improved the activities of antioxidant enzymes (SOD, CAT and APX) in D. salina cultures under nutrient stress.

Oral administration of lyophilized Dunaliella salina, a carotenoid-rich marine alga, reduces tumor progression in mammary cancer induced rats.

Dunaliella salina is a photosynthetic cell factory used for the commercial production of food additives, cattle stock feed and cosmetics as well as active ingredients for pharmaceutical industries. The investigation of the in vivo antitumor activity of D. salina lyophilized powder (DSLP) against 7,12-dimethylbenz(a)anthracene (DMBA) induced mammary carcinogenesis in female Wistar rats indicated a dose-dependent effect of DSLP. We studied the effect of DSLP at two different dosages of 500 and 1000 mg per kg bw on DMBA induced mammary cancer in rats by measuring the status of antioxidant enzymes, phase I and phase II detoxification enzymes, lipid peroxidation, and glycoconjugated proteins and by investigating the expression pattern of cell proliferation (Ki-67), hormonal receptor (ER, PR and HER2) status by immunohistochemical analysis, and apoptotic (caspase-3 and -9) and pro-inflammatory (COX-2) markers by colorimetric analysis. After 16 weeks of the study, we observed 100% tumor formation (including high tumor incidence and tumor volume) and a significant increase in the level of hormonal receptors, cell proliferation, and pro-inflammatory and apoptosis markers in tumor-bearing animals compared to the control. The oral administration of DSLP (1000 mg per kg bw) to the DMBA treated animals showed up to 83.4% reduction of tumors and effectively restored the levels of biochemical markers in the mammary tissues in addition to the downregulation of the expression of molecular markers. In conclusion, DSLP was found to show a chemopreventive effect against breast cancer induced in rats through the suppression of cell proliferation and the induction of apoptosis.

Systematic functional analysis and application of a cold-active serine protease from a novel Chryseobacterium sp.

Psychrotolerant bacteria isolated from natural and artificially cold environments were screened for synthesis of cold-active protease. The strain IMDY showing the highest protease production at 5°C was selected and phylogenetic analysis revealed that IMDY as novel bacterium with Chryseobacterium soli(T) as its nearest neighbor. Classical optimization enhanced the protease production from 18U/mg to 26U/mg and the enzyme was found to be active at low temperature, activity enhanced by CaCl2, inhibited by PMSF, stable against NaCl, and its activity retained in the presence of surfactants, organic solvents and detergents. On testing, the meat tenderization, myofibril fragmentation, pH, and TBA values were favorable in IMDY-protease treated meat compared to control. SDS profiling and SEM analysis also showed tenderization in meat samples. Hence, this study proposes to consider the cold-active protease from Chryseobacterium sp. IMDY as a pertinent candidate to develop potential applications in food processing industry.

Cold Stress Tolerance in Psychrotolerant Soil Bacteria and Their Conferred Chilling Resistance in Tomato (Solanum lycopersicum Mill.) under Low Temperatures.

The present work aimed to study the culturable diversity of psychrotolerant bacteria persistent in soil under overwintering conditions, evaluate their ability to sustain plant growth and alleviate chilling stress in tomato. Psychrotolerant bacteria were isolated from agricultural field soil samples colleced during winter and then used to study chilling stress alleviation in tomato plants (Solanum lycopersicum cv Mill). Selective isolation after enrichment at 5°C yielded 40 bacterial isolates. Phylogenetic studies indicated their distribution in genera Arthrobacter, Flavimonas, Flavobacterium, Massilia, Pedobacter and Pseudomonas. Strains OS211, OB146, OB155 and OS261 consistently improved germination and plant growth when a chilling stress of 15°C was imposed and therefore were selected for pot experiments. Tomato plants treated with the selected four isolates exhibited significant tolerance to chilling as observed through reduction in membrane damage and activation of antioxidant enzymes along with proline synthesis in the leaves when exposed to chilling temperature conditions (15°C). Psychrotolerant physiology of the isolated bacteria combined with their ability to improve germination, plant growth and induce antioxidant capacity in tomato plants can be employed to protect plants against chilling stress.

Analysis of the MAT1-1 and MAT1-2 Gene Ratio in Black Koji Molds Isolated from Meju.

Aspergillus luchuensis is known as an industrially important fungal species used for making fermented foods such as awamori and shochu in Japan, makgeolli and Meju in Korea, and Pu-erh tea in China. Nonetheless, this species has not yet been widely studied regarding mating-type genes. In this study, we examined the MAT1-1 and MAT1-2 gene ratio in black koji molds (A. luchuensis, Aspergillus niger, and Aspergillus tubingensis) and in Aspergillus welwitschiae isolated from Meju, a fermented soybean starting material for traditional soy sauce and soybean paste in Korea. The number of strains with the MAT1-1 locus was 2 of 23 (A. luchuensis), 6 of 13 (A. tubingensis), 21 of 28 (A. niger), and 5 of 10 (A. welwitschiae). Fungal species A. tubingensis and A. welwitschiae showed a 1 : 1 ratio of MAT1-1 and MAT1-2 mating-type loci. In contrast, A. luchuensis revealed predominance of MAT1-2 (91.3%) and A. niger of MAT1-1 (75%). We isolated and identified 2 A. luchuensis MAT1-1 strains from Meju, although all strains for making shochu in Japan are of the MAT1-2 type. These strains may be a good resource for breeding of A. luchuensis to be used in the Asian fermented-food industry.

Astaxanthin from psychrotrophic Sphingomonas faeni exhibits antagonism against food-spoilage bacteria at low temperatures.

Food production and processing industry holds a perpetual relationship with microorganisms and their by-products. In the present study, we aimed to identify beneficial cold-adapted bacteria devoid of any food spoilage properties and study their antagonism against common food-borne pathogens at low temperature conditions. Ten isolates were obtained on selective isolation at 5 °C, which were spread across genera Pseudomonas, Sphingomonas, Psychrobacter, Leuconostoc, Rhodococcus, and Arthrobacter. Methanol extracts of strains were found to contain several bioactive metabolites. Among the studied isolates, methanol extracts of S. faeni ISY and Rhodococcus fascians CS4 were found to show antagonism against growth of Escherichia coli, Proteus mirabilis, Enterobacter aerogenes, Listeria monocytogenes and Vibrio fischeri at refrigeration temperatures. Characterization of the abundant yellow pigment in methanol extracts of S. faeni ISY through UV-Vis spectrophotometry, high performance liquid chromatography (HPLC) and mass spectrometry (LC-MS) revealed the presence of astaxanthin, which, owing to its presence in very large amounts and evidenced to be responsible for antagonistic activity of the solvent extract.

Psychrotolerant Endophytic Pseudomonas sp. Strains OB155 and OS261 Induced Chilling Resistance in Tomato Plants (Solanum lycopersicum Mill.) by Activation of Their Antioxidant Capacity.

Studies on chilling stress damage and its mitigation through microorganisms in members of family Solanaceae is limited, despite their economic importance. We studied chilling stress alleviation in tomato plants colonized by psychrotolerant bacterial strains Pseudomonas vancouverensis OB155-gfp and P. frederiksbergensis OS261-gfp. Log phase cultures of bacterial strains were coated on surface-sterilized seeds (bacterization) before sowing and nonbacterized (control) seeds were coated with sterile bacterial growth medium. All plants were grown at temperatures of 30 and 25°C and at the end of 4 weeks, chilling treatment (12 and 10°C) was imposed for 1 week on half of the bacterized and control plants. Under normal conditions (30 and 25°C), no significant difference was observed in antioxidant activity, proline accumulation, and expression of cold acclimation genes in tomato leaf tissues of both control and bacterized plants. However, plants exposed to temperatures of 12 and 10°C were found to decrease in robustness and nutrient uptake, accompanied by increased membrane damage. Chilling resistance in bacterized plants was evident from reduced membrane damage and reactive oxygen species levels, improved antioxidant activity in leaf tissues, and high expression of cold acclimation genes LeCBF1 and LeCBF3 compared with control plants. Confocal microscopy confirmed effective colonization and intercellular localization of cold-adapted bacterial strains OB155-gfp and OS261-gfp.

Synthesis and larvicidal activity of low-temperature stable silver nanoparticles from psychrotolerant Pseudomonas mandelii.

Applications based on silver nanoparticles (AgNPs) are limited by low temperatures, which cause aggregation of the nanoparticle fraction, leading to reduced efficacy of their products. We aimed at studying AgNP synthesis by psychrotolerant bacteria, its stability under long-term storage, and larvicidal activity under low-temperature conditions. Electron and atomic force microscopy studies revealed that 6 among 22 psychrotolerant isolates synthesized AgNPs with an average diameter of 1.9-14.1 nm. Pseudomonas mandelii SR1 synthesized the least-sized AgNPs with an average diameter of 1.9-10 nm, at temperatures as low as 12 °C without aggregate formation, and the synthesized nanoparticles were stable for up to 19 months of storage period. On studying their larvicidal activity, LC90 (lethal concentration) values against Anopheles subpictus and Culex tritaeniorhynchus larvae were at 31.7 and 35.6 mg/L, respectively. Stable non-aggregate AgNPs at low-temperature conditions from P. mandelii SR1, coupled with their larvicidal property, can be applied to control larval populations in water bodies located in seasonal or permanently cold environments.