A total of 24 isolates of Phytophthora infestans were tested and analyzed for their resistance to metalaxyl fungicides. Sensitivity to metalaxyl was determined by growing isolates on 20% V8 medium amended with 0, 5, and 100 microg/ml metalaxyl. Four isolates among the 24 tested were resistant to metalaxyl. Eleven isolates were intermediate and nine isolates were sensitive. Amplified fragment length polymorphism (AFLP) assay was used to identify the amplification products of resistant isolates. As a result, selected fragments were cloned, sequences and primer pairs were developed which linked to metalaxyl insensitivity in P. infestans using competitive PCR.
Clone Cells ; DNA* ; Genetic Markers* ; Phytophthora infestans* ; Phytophthora* ; Polymerase Chain Reaction

Efficacy of resistance induction by the bacterial isolates Pseudomonas putida (TRL2-3), Micrococcus luteus (TRK2-2) and Flexibacteraceae bacterium (MRL412), which were isolated from the rhizosphere of plants growing in Jeju Mountain, were tested in a greenhouse. The disease severity caused by Phytophthora infestans was effectively reduced in the potato plants pre-inoculated with bacterial isolates compared with those of the untreated control plants growing in a greenhouse. In order to estimate the level of protection by the bacterial isolates, Mancozeb WP (Diesen M(R), Kyong nong) and DL-3-amino butyric acid (BABA) were pre-treated, whereas Dimethomorph WP (Forum(R), Kyong nong) and phosphonic acid (H3PO3) were post-treated the challenge inoculation with the pathogen. Disease severities of chemical pre-treated as well as post-treated plants were reduced compare to those of the untreated. The disease reduction in the plants pre-treated with Mancozeb WP was the highest, whereas that of post-treated with Dimethomorph WP was the lowest. The yields of plants pre-inoculated with three bacterial isolates were greatly increased than those of control plants. These results suggest that biological control by bacterial isolates might be an alternative strategy against late blight disease in potato plants growing in greenhouse.
Butyric Acid ; Cytophagaceae ; Micrococcus luteus ; Phytophthora infestans* ; Phytophthora* ; Pseudomonas putida ; Rhizosphere ; Solanum tuberosum*

Many members of the Saprolegniales (Oomycete) cause mycoses and disorders of fishes, of which Achlya and Saprolegnia are most ubiquitous genera worldwide. During a survey of the diversity of freshwater oomycetes in Korea, we collected seven isolates of Achlya, for which morphological and molecular phylogenetic analyses enabled them to identify as Achlya americana and Achlya bisexualis. In Korea, only a species of Achlya, A. prolifera, has been previously found to cause seedling rot on rice (Oryza sativa), but none of the two species have been reported yet. Importantly, A. bisexualis was isolated from a live fish, namely rice fish (Oryzias sinensis), as well as freshwater, and this is the first report of Achlya-causing mycoses on freshwater fishes in Korea. The presence of A. americana and A. bisexualis on live fish in Korea should be closely monitored, as considering the well-known broad infectivity of these species it has the potential to cause an important emerging disease on aquaculture industry.
Achlya ; Aquaculture ; Fishes ; Fresh Water ; Korea ; Mycoses ; Oomycetes ; Saprolegnia ; Seedlings

Docosahexaenoic acid (DHA) has many unique physiological functions such as promoting the development of brain and retina in infants. Therefore, it is widely applied to food, pharmacy, breeding and other industries. To obtain engineered strains of Aurantiochytrium limacinum SR21 suitable for industrial application with increased lipid and DHA production, we designed a simple, fast, accurate and high-throughput screening method based on Nile red staining of oil droplets. First, ultraviolet C (UVC) mutagenesis was used to generate a random mutant library of A. limacinum. Second, screening conditions were optimized including staining conditions of Nile red and the sorting criterion. Thereby, three putative high-lipid mutants (D03432, D05106 and D01521) were selected from the mutant library containing 3 648 mutated clones. The three mutants grew faster and produced higher amounts of lipids and DHA compared to wild type (WT). In 100 mL cultures, the lipid content of D03432 and D05106 mutants reached 64.74% and 63.13% of dry cell weight respectively, whereas the wild strain exhibited only 43.19%. DHA yield in these two mutants were even 2.26-fold and 2.37-fold higher than that of the wild strain. Experiment with 5 L fermentor further confirmed that D03432 and D05106 mutants had better performance than the wild strain on DHA yield (45.51% and 66.46% more than that of the wild strain, respectively), and demonstrated their high potential for industrial application. This work not only generated several high-DHA content mutants with high potential for industrial use, but also provided vital guidance for high-throughput screening of lipid hyper-accumulating mutants in other oil-producing microorganisms.
Bioreactors ; Docosahexaenoic Acids ; Mutagenesis ; Staining and Labeling ; Stramenopiles

Filamentous microalga Tribonema sp. has the advantages of highly resistance to zooplankton-predation, easy harvesting, and high cellular lipid content, in particular large amounts of palmitoleic acid (PA) and eicosapentaenoic acid (EPA). Therefore, Tribonema sp. is considered as a promising biomass feedstock to produce biodiesel and high-value products. In this work, we studied the effect of different concentrations of nitrogen (NaNO₃: 255-3 060 mg/L), phosphorus (K₂HPO₄: 4-240 mg/L), iron ((NH₄)₃FeC₁₂H₁₀O₁₄: 0.6-12 mg/L) and magnesium (MgSO₄: 7.5-450 mg/L) on the biomass, lipid content, and fatty acid composition of Tribonema sp. FACHB-1786, aiming at enhancing cell lipid productivity. The growth of Tribonema sp. had a positive correlation with the concentration of magnesium, and the maximum biomass of Tribonema sp. (under the condition of 450 mg/L MgSO₄) was 8.09 g/L, much greater than those reported in previous studies using the same and other Tribonema species under autotrophic conditions. Different nitrogen concentrations exerted no significant effect on algal growth (P > 0.05), but a higher nitrogen concentration resulted in a greater amount of lipid in the cells. The maximum volumetric productivities of total lipids (319. 6 mg/(L·d)), palmitoleic acid (135.7 mg/(L·d)), and eicosapentaenoic acid (24.2 mg/(L·d)) of Tribonema sp. were obtained when the concentrations of NaNO₃, K₂HPO₄, (NH₄)₃FeC₁₂H₁₀O₁₄, and MgSO₄ were 765 mg/L, 80 mg/L, 6 mg/L, and 75 mg/L, respectively. This study will provide a reference for substrate optimization for Tribonema sp. growth and lipid production.
Biofuels ; Biomass ; Lipids ; Microalgae ; Nitrogen ; Stramenopiles

The effect of four sub-lethal concentrations (400, 800, 1,200 and 1,600 microg/ml) of three amino acids such as isoluecine, aspartic acid and phenylalanine on vegetative growth and sexual and asexual reproduction of Achlya racemosa, A. proliferoides and Saprolegnia furcata was investigated. The density of vegetative growth and diameters of vegetative colonies of species of the Oomycetes fungi decreased with rising the concentration of the applied amino acid. Vegetative hyphae of treated fungi almost appeared branched in case of S. furcata, thick in case of A. racemosa and distorted in case of A. proliferoides as compared with control. The different treatments with amino acids depressed both sporangial formation and discharge, which were dependent on the tested species of zoosporic fungi, the amino acid and its dosage. Phenylalanine was the most effective amino acid in inhibiting sporulation and S. furcata was the most sensitive fungal species. Aspartic acid and isoleucine stimulated germination of discharged spores through the formation of germlings. Gemmae formation by the three fungi was reduced at the low concentrations of amino acids and nearly missed at high concentrations. Sex organs (oogonia and antheridia) were affected partly; rudiment oogonia were observed at low concentrations (400 and 800 microg/ml) and disappeared at higher concentrations, whereas antheridial branch formation was stimulated as the fungi were treated with isoleucine and to some extent phenylalanine.
Achlya* ; Amino Acids* ; Aspartic Acid ; Fungi ; Germination ; Hyphae ; Isoleucine ; Oogonia ; Oomycetes ; Phenylalanine ; Reproduction, Asexual ; Saprolegnia* ; Spores

The effects of different concentrations of three amino acids as carbon and or nitrogen sources on mycelial dry weights, changes in pH values of synthetic medium, ammonia secretion and extracellular protease activity by three zoosporic fungi, pathogens of fish and shellfish, were studied. As compared with the control, the addition of isoleucine and aspartic acid as nitrogen sources were generally stimulative for mycelial dry weight production whereas phenylalanine was inhibitory irrespective to the tested fungal species. When amino acids served as carbon and nitrogen sources, the mycelial dry weights of the three fungi were increased (mostly non-significantly) relative to untreated control but weights were decreased as the concentrations of the three amino acids raised. The addition of individual amino acids as carbon and nitrogen sources to the medium significantly increased pH values of the medium comparable to the control. The addition of each of the three amino acids as carbon and nitrogen sources to the medium significantly induced ammonia secretion by the three species of zoosporic fungi. Ammonia secretion in synthetic medium amended with amino acids as nitrogen source raised by the three zoosporic fungi relative to untreated control except in case of Achlya racemosa treated with isoleucine. Extracellular protease activity was almost promoted in case of Achlya proliferoides and Saprolegnia furcata cultures treated with isoleucine and aspartic acid individually in presence of glucose and vice versa in case of phenylalanine. However, extracellular protease activity of A. racemosa decreased compared with the control at various concentrations of isoleucine and both phenylalanine and aspartic acid assumed inconsistent effects. Extracellular protease activity of the three zoosporic fungi in the medium devoid of glucose varied depending upon zoosporic fungal species, the tested amino acid and the applied concentrations. The values of protease activity were approximately less two folds than that obtained in presence of glucose.
Achlya ; Amino Acids* ; Ammonia* ; Aspartic Acid ; Carbon ; Fungi ; Glucose* ; Hydrogen-Ion Concentration ; Isoleucine ; Nitrogen ; Oomycetes* ; Phenylalanine ; Saprolegnia ; Shellfish ; Weights and Measures

A total of 187 endophytic fungi were isolated from 11 plant species, which were collected from 11 locations in Korea. Their antifungal activities were screened in vivo by antifungal bioassays after they were cultured in potato dextrose broth and rice solid media. Antifungal activity against plant pathogenic fungi such as Magnaporthe grisea (rice blast), Corticium sasaki (rice sheath blight), Botrytis cinerea (tomato gray mold), Phytophthora infestans (tomato late blight), Puccinia recondita (wheat leaf rust), and Blumeria graminis f. sp. hordei (barley powdery mildew) was determined in vivo by observing the inhibition of plant disease development. Twenty (11.7%) endophytic fungi fermentation broths were able to control, by more than 90%, at least one of the six plant diseases tested. Among 187 liquid broths, the F0010 strain isolated from Abies holophylla had the most potent disease control activity; it showed control values of more than 90% against five plant diseases, except for tomato late blight. On the other hand, fourteen (7.5%) solid culture extracts exhibited potent disease control values of more than 90% against one of six plant diseases. The screening results of this study strongly suggested that metabolites of plant endophytic fungi could be good potential sources for screening programs of bioactive natural products.
Abies ; Biological Assay ; Biological Products ; Botrytis ; Fermentation ; Fungi* ; Glucose ; Hand ; Korea ; Lycopersicon esculentum ; Magnaporthe ; Mass Screening* ; Phytophthora infestans ; Plant Diseases ; Plants* ; Solanum tuberosum

Nannochloropsis has been considered as a promising feedstock for biodiesel production in recent years. To improve its lipid productivity, heavy-ion irradiation mutagenesis, an effectively breeding method used in plants and microorganisms was applied in Nannochloropsis oceanica OZ-1. After large-scale screening using Imaging-PAM and microplate-reader, two mutants (HP-1 and HP-2) with higher growth rate were isolated from the wild type N. oceanica. Subsequently analysis showed that after 18 days of cultivation biomass accumulation of the HP-1 and HP-2 mutant was increased by 18% and 26% respectively compare to the wild type. Total lipid productivity of the HP-1 and HP-2 mutant was 295 mg/(L x d) and 275 mg/(L x d), respectively, whereas that of the wild type was 247 mg/(L x d). Both mutants showed significantly advantage over their wild type concerning biomass accumulation and lipid productivity.
Heavy Ions ; Lipids ; biosynthesis ; Microalgae ; growth & development ; metabolism ; radiation effects ; Mutagenesis ; genetics ; Mutation ; genetics ; Stramenopiles ; genetics ; metabolism ; radiation effects

Nitrogen source is one of the important factors that affect the microalgae growth and lipid accumulation. We studied the effects of various nitrogen sources (i.e. NaNO3, CO(NH2)2, NH4Cl and CH3COONH4) and amount on the growth density, lipid yield, and eicosapentaenoic acid (EPA) content of Nannochloropsis oculata by single factor experimental method. The results show that N. oculata preferred NH4+ as nitrogen source rather than NO3- and CO(NH2)2. NH4+ could promote the growth and lipid accumulation of N. oculata. With the increase of nitrogen concentration, the biomass and the content of polyunsaturated fatty acid (PUFA) increased, but the content of lipid decreased. CH3COONH4 was the most suitable for growth, accumulation of lipid and EPA of N. oculata among the four investigated nitrogen sources. The optimal concentration was 5.29 mmol/L.
Eicosapentaenoic Acid ; metabolism ; Lipids ; biosynthesis ; isolation & purification ; Microalgae ; growth & development ; metabolism ; Nitrogen ; analysis ; metabolism ; Stramenopiles ; growth & development ; metabolism