Endophytic fungi from Lycium chinense Mill and characterization of two new Korean records of Colletotrichum.

Chinese boxthorn or matrimony vine (Lycium chinense Mill) is found primarily in southeastern Europe and Asia, including Korea. The dried ripe fruits are commonly used as oriental medicinal purposes. Endophytic fungi were isolated from surface sterilized tissues and fruits of the medicinal plant in 2013 to identify the new or unreported species in Korea. Among 14 isolates, 10 morphospecies were selected for molecular identification with the internal transcribed spacer (ITS) gene. Phylogenetic analysis revealed that all isolates belonged to Ascomycota including the genera Acremonium, Colletotrichum, Cochliobolus, Fusarium, Hypocrea and Nemania. Two Colletotrichum species were identified at the species level, using three genes including internal transcribed spacer (ITS), glycerol-3-phosphate dehydrogenase (GAPDH) and Actin (ACT) for PCR and molecular data analysis along with morphological observations. The fungal isolates, CNU122031 and CNU122032 were identified as Colletotrichum fructicola and C. brevisporum, respectively. Morphological observations also well supported the molecular identification. C. brevisporum is represented unrecorded species in Korea and C. fructicola is the first record from the host plant.

Thermally tunable-focus lenticular lens using liquid crystal.

A thermally tunable focusing lenticular liquid crystal (LC) lens array was fabricated using a polymer LC component, including a polarizer that produces linearly polarized light. The focal length in the proposed structure could be tuned by temperature-adjusted applied voltage to a transparent heater in a lenticular LC lens cell because it alters the birefringence of the LC and varies the difference in refractive index between the LC and the polymer. The results showed that the focal length of the E7 LC used varied continuously with temperature from 5.6 to 8.7 mm from 25°C to 54°C, respectively. The proposed lenticular LC lens has potential use in photonic devices such as biological imaging, phone cameras, and optical sensors.

Description of Streptomyces neopeptinius sp. nov., an actinobacterium with broad spectrum antifungal activities.

A streptomycete strain producing broad-spectrum antifungal substances was taxonomically characterized. The strain, designated KNF 2047(T) (= SH-09(T) = KCTC 10586BP(T)), was found to form extensively branching aerial and substrate mycelia, and produce spiny-ornamented spores with loose spiral chains. The whole cell hydrolyzates contained major amount of LL-diaminopimelic acid. The major fatty acids of the phospholipids were saturated and branched fatty acids containing 14~17 carbons, and the major isoprenoid quinones were hexa-and octa-hydrogenated menaquinones with 9 isoprene units. The phylogenetic analysis using the 16S rRNA gene indicated that the strain belongs to the genus Streptomyces but forms an independent phyletic line. These results clearly demonstrate that strain KNF2047(T) forms a new center of taxonomic variation within Streptomyces, for which the name Streptomyces neopeptinius sp. nov. is proposed.

Alternaria brassicifolii sp. nov. Isolated from Brassica rapa subsp. pekinensis in Korea.

A new species belonging to the genus Alternaria was isolated from the necrotic leaf spots of Brassica rapa subsp. pekinensis in Yuseong district, Daejeon, Korea. It is an occasional isolate, not an etiological agent, which is morphologically similar to A. broccoli-italicae, but differs in conidial size and conidiophore shape. Phylogenetic analysis using the sequence datasets of the internal transcribed spacer (ITS) region of the rDNA, glyceraldehyde-3-phosphate dehydrogenase (gpd), and plasma membrane ATPase genes showed that it is distantly related to A. broccoli-italicae and closely related to Alternaria species in the section Pseudoalternaria, which belonged to a clade basal to the section Infectoriae. Morphologically, the species is unique because it produces solitary conidia or conidial chains (two units), unlike the four members in the section Pseudoalternaria that produce conidia as short branched chains. It exhibits weak pathogenicity in the host plant. This report includes the description and illustration of A. brassicifolii as a new species.

Xylarinic acids A and B, new antifungal polypropionates from the fruiting body of Xylaria polymorpha.

Two new polypropionates designated as xylarinic acids A and B were isolated from the fruiting body of Xylaria polymorpha. Their structures were established as 4,6,8-trimethyl-2,4-decadienoic acid and 2,4,6-trimethyl-2-octenoic acid, respectively, on the basis of extensive spectroscopic analysis. Both compounds displayed significant antifungal activity against plant pathogenic fungi Pythium ultinum, Magnaporthe grisea, Aspergillus niger, Alternaria panax, and Fusarium oxysporium, whereas they did not show antibacterial and cytotoxic effect.

Antifungal cyclopeptolide from fungal saprophytic antagonist Ulocladium atrum.

The saprophytic fungus Ulocladium atrum Preuss is a promising biological control agent for Botrytis cinerea in greenhouse- and field-grown crops. However, despite its known potent antifungal activity, no antifungal substance has yet been reported. In an effort to characterize the antifungal substance from U atrum, we isolated an antibiotic peptide. Based on extensive spectroscopic analyses, its structure was established as a cyclopeptolide with a high portion of N-methylated amino acids, and its 1H and 13C chemical shifts were completely assigned based on extensive 1D and 2D NMR experiments. Compound 1 exhibited potent antifungal activity against the plant pathogenic fungus Botrytis cinerea and moderate activity against Alternaria alternate and Magnaporthe grisea.

CaWRKY2, a chili pepper transcription factor, is rapidly induced by incompatible plant pathogens.

WRKY family proteins are a class of plant-specific transcription factors involved in stress response signaling pathways. In this study a gene encoding a putative WRKY protein was isolated from a pepper EST database (http://genepool.kribb.re.kr). The cDNA, named Capsicum annuum WRKY2 (CaWRKY2), encodes a putative polypeptide of 548 amino acids, containing two WRKY domains with zinc finger motifs and two potential nuclear localization signals. Northern blot analyses showed that CaWRKY2 mRNA was preferentially induced during incompatible interactions of pepper plants with PMMoV, Pseudomonas syringae pv. syringae 61, and Xanthomonas axonopodis pv. vesicatoria race 3. Furthermore, CaWRKY2 transcripts were strongly induced by wounding and ethephon treatment, whereas only moderate expression was detected following treatment with salicylic acid and jasmonic acid. CaWRKY2 was translocated to the nucleus when a CaWRKY2-smGFP fusion construct was expressed in onion epidermal cells. CaWRKY2 also had transcriptional activation activity in yeast. Taken together our data suggest that CaWRKY2 is a pathogen-inducible transcription factor that may have a role in early defense responses to biotic and abiotic stresses.

Prevalence, Characterization, and Mycotoxin Production Ability of Fusarium Species on Korean Adlay (Coix lacrymal-jobi L.) Seeds.

Adlay seed samples were collected from three adlay growing regions (Yeoncheon, Hwasun, and Eumseong region) in Korea during 2012. Among all the samples collected, 400 seeds were tested for fungal occurrence by standard blotter and test tube agar methods and different taxonomic groups of fungal genera were detected. The most predominant fungal genera encountered were Fusarium, Phoma, Alternaria, Cladosporium, Curvularia, Cochliobolus and Leptosphaerulina. Fusarium species accounted for 45.6% of all species found; and, with phylogenetic analysis based on the combined sequences of two protein coding genes (EF-1α and ß-tubulin), 10 Fusarium species were characterized namely, F. incarnatum (11.67%), F. kyushuense (10.33%), F. fujikuroi (8.67%), F. concentricum (6.00%), F. asiaticum (5.67%), F. graminearum (1.67%), F. miscanthi (0.67%), F. polyphialidicum (0.33%), F. armeniacum (0.33%), and F. thapsinum (0.33%). The Fusarium species were then examined for their morphological characteristics to confirm their identity. Morphological observations of the species correlated well with and confirmed their molecular identification. The ability of these isolates to produce the mycotoxins fumonisin (FUM) and zearalenone (ZEN) was tested by the ELISA quantitative analysis method. The result revealed that FUM was produced only by F. fujikuroi and that ZEN was produced by F. asiaticum and F. graminearum.

First Report of Leaf Spot on Japanese Plum Caused by an Alternaria sp. in Korea.

Japanese plum (Prunus salicina Lindley) is a deciduous tree in the family Rosaceae. In Korea, this plant is widely distributed in orchards as an important stone fruit as well as in gardens as an ornamental tree because of their abundant white blossoms. Every September to November since 2001, leaf spots were observed on Japanese plum in a garden in Cheongyang, Chungnam District, Korea. Early symptoms consisted of small, brown spots that were 2 to 5 mm in diameter. Later, the leaf lesions became circular or irregular, dark brown, expanded to 15 mm in diameter, and resulted in discoloration with necrosis on twisted leaves that was followed by defoliation. In November, older lesions sometimes appeared blackish brown as sporulation occurred on the lesions. The causal fungus was isolated from diseased leaves and cultured on potato dextrose agar. A culture has been placed in the CABI Herbarium (IMI Accession No. 387139). Conidial dimension averaged 34 × 12 µm. On the basis of morphological characteristics of conidia and conidiophores, the causal fungus was identified as a small-spored species of Alternaria as described by E. G. Simmons (1). Pathogenicity tests were conducted by inoculating slightly wounded and nonwounded leaves with a conidial suspension adjusted to 1 × 106 conidia/ml. Four leaves per each experiment were either wounded or not and inoculated with a spore suspension. The eight leaves were placed in a moist chamber at 25°C. After 6 to 10 days, small brown spots appeared on 87% of the wounded and nonwounded leaves. Control leaves sprayed with distilled water did not develop any symptoms. The causal fungus was consistently reisolated from the leaf spots. Results from pathogenicity tests were similar in a repeated test. It is possible that small-spored Alternaria spp. isolates are host specific (2). Eight Alternaria spp., including A. alternata, A. tenuis, A. tenuissima, and A. citri, have been found to cause black spot on fifteen Prunus spp. in China, Japan, Hong Kong, Libya, Mexico, Australia, and the United States (2). Further studies on the host-specific toxin production, geographical distribution, and host ranges for the species of Alternaria isolated from Japanese plum are in progress. To our knowledge, this is the first report of leaf spot on Japanese plum (P. salicina) caused by a small-spored Alternaria sp. in Korea. References: (1) E. G. Simmons. Mycotaxon 55:79, 1995. (2) K. Inoue and H. Nasu. J. Gen. Plant Pathol. 66:18, 2002.

Characterization and Pathogenicity of Alternaria burnsii from Seeds of Cucurbita maxima (Cucurbitaceae) in Bangladesh.

In the course of survey of endophytic fungi from Bangladesh pumpkin seeds in 2011~2012, two strains (CNU111042 and CNU111043) with similar colony characteristics were isolated and characterized by their morphology and by molecular phylogenetic analysis of the internal transcribed spacer, glyceraldehydes-3-phosphate dehydrogenase (gpd), and Alternaria allergen a1 (Alt a1) sequences. Phylogenetic analysis of all three sequences and their combined dataset revealed that the fungus formed a subclade within the A. alternata clade, matching A. burnsi and showing differences with its other closely related Alternaria species, such as A. longipes, A. tomato, and A. tomaticola. Long ellipsoid, obclavate or ovoid beakless conidia, shorter and thinner conidial size (16~60 [90] × 6.5~14 [~16] µm) distinguish this fungus from other related species. These isolates showed more transverse septation (2~11) and less longitudinal septation (0~3) than did other related species. Moreover, the isolate did not produce any diffusible pigment on media. Therefore, our results reveal that the newly recorded fungus from a new host, Cucurbita maxima, is Alternaria burnsii Uppal, Patel & Kamat.

Occurrence of Leaf Blight on Cosmos Caused by Alternaria cosmosa in Korea.

In 2011, a leaf blight disease was observed on cosmos (Cosmos bipinnatus) leaves in Nonsan, Korea. The causal pathogen was isolated and identified based on morphological and molecular approaches. Morphological characteristics of the pathogen matched well with the Alternaria cosmosa and also easily distinguishable from Alternaria zinniae reported from cosmos seeds by producing branched beak. Phylogenetically, the pathogen could not be distinguished from A. passiflorae based on the sequence analysis of a combined data set of Alt a1 and gpd genes. However, A. passiflorae was distinguished from the present species by having conidiophores with 4 to 5 conidiogenous loci. The results indicate that the present Alternaria species is A. cosmosa. Pathogenicity tests revealed that the isolate was pathogenic to the leaves of Cosmos bipinnatus. This is the first report of Alternaria blight disease caused by A. cosmosa on cosmos in Korea.

Involvement of hydrogen peroxide in repression of catalase in TMV-infected resistant tobacco.

Catalases are ubiquitous, and play a role in plant defense against pathogens. We have reported that catalase mRNA and enzyme activity are repressed in the vicinity of hypersensitive tobacco lesions following TMV infection. We wished to identify the signals involved in this repression. Inoculation with TMV reduced catalase levels 26 to 28 h following infection, coincident with the known timing of endogenous salicylic acid (SA) accumulation. Application of SA caused a transient reduction in Ngcat1 mRNA level and catalase activity 4 to 6 h after treatment. However, repression was also observed in transgenic plants harboring the salicylate hydroxylase gene (NahG) and in TMV susceptible plants, which do not accumulate SA following TMV infection. In the same blots there was no induction of PR-1 or enhanced expression of H2O2-inducible glutathione-S-transferase and found that exposure to H2O2 also repressed Ngcat1 mRNA. Our findings suggest that repression of catalase transcription may be caused by the accumulation of H2O2 rather than of SA.

A New Record of Penicillium raphiae Isolated from Agricultural Soil of Ulleung Island, Korea.

A fungal isolate EML-NCP01 was recovered from agricultural soil in Ulleung Island, Korea. Phylogenetic analysis of internal transcribed spacer and ß-tubulin genes identified the isolate as the Penicillium species P. raphiae. Morphologically, the EML-NCP01 isolate was identical to the previous description of P. raphiae. The species presented here has not been reported in Korea.

First Record of Alternaria simsimi Causing Leaf Spot on Sesame (Sesamum indicum L.) in Korea.

Leaf spot disease was observed in sesame (Sesamum indicum L.) during 2009 and 2010 in Korea. The pathogen was identified as Alternaria simsimi based on morphological and cultural characteristics. The morphological identification was well supported by phylogenetic analysis of the ribosomal DNA-internal transcribed spacer region. A. simsimi isolates caused spot symptoms on leaves and stems of sesame plants 2 wk after artificial inoculation, which were similar to those observed in the field. This is the first record of leaf spot disease in Korea caused by A. simsimi.

Characterization and Pathogenicity of Alternaria vanuatuensis, a New Record from Allium Plants in Korea and China.

Alternaria from different Allium plants was characterized by multilocus sequence analysis. Based on sequences of the ß-tubulin (BT2b), the Alternaria allergen a1 (Alt a1), and the RNA polymerase II second largest subunit (RPB2) genes and phylogenetic data analysis, isolates were divided into two groups. The two groups were identical to representative isolates of A. porri (EGS48-147) and A. vanuatuensis (EGS45-018). The conidial characteristics and pathogenicity of A. vanuatuensis also well supported the molecular characteristics. This is the first record of A. vanuatuensis E. G. Simmons & C. F. Hill from Korea and China.

A Novel Alternaria Species Isolated from Peucedanum japonicum in Korea.

We isolated and examined a new Alternaria sp., which causes leaf spots on Peucedanum japonicum in Korea, by using molecular and morphological methods. Phylogenetic analysis based on a combined internal transcribed spacer region analysis and two protein-coding genes (gpd and Alt a1) demonstrated that the causal fungus was most closely related to A. cinerariae and A. sonchi, and relevant to A. brassicae. However, conidial morphology indicated that it is a novel species within the genus Alternaria, and therefore we have assigned the fungus a new name in this study.

New Records of Endophytic Paecilomyces inflatus and Bionectria ochroleuca from Chili Pepper Plants in Korea.

Two new species of endophytic fungi were encountered during a diversity study of healthy tissues of chili pepper plants in Korea. The species were identified as Paecilomyces inflatus and Bionectria ochroleuca based on molecular and morphological analyses. Morphological descriptions of these endophytic isolates matched well with their molecular analysis. In the present study, detailed descriptions of internal transcribed spacer regions and morphological observations of these two fungi are presented.

A New Record of Penicillium cainii from Soil in Korea.

Twenty Penicillium isolates were recovered during the investigation of fungal community in the soil samples collected from Wando (Jeonnam Province, Korea). Among them, one species was identified and described as P. cainii based on phylogentic analysis of internal transcribed spacer and ß-tubulin (BT2) genes and morphological characteristics. This is a first report of P. cainii in Korea.

Taxonomic Re-evaluation of Colletotrichum gloeosporioides Isolated from Strawberry in Korea.

For the past two decades, the causal agent of anthracnose occurring on strawberry in Korea was considered Colletotrichum gloeosporioides. However, the recent molecular analysis has shown that the genus Colletotrichum has undergone many taxonomic changes with introduction of several new species. As a result, it revealed that C. gloeosporioides indeed consisted of more than 20 distinct species. Therefore, the Korean pathogen isolated from strawberry should be reclassified. The shape and size of the conidia of the pathogen were not distinctly different from those of C. gloeosporioides and C. fructicola, but it differed in shape of the appressoria. A combined sequence analysis of partial actin, glycer-aldehydes-3-phosphate dehydrogenase genes, and the internal transcribed spacer regions showed that the strawberry isolates formed a monophyletic group with authentic strains of C. fructicola. On the basis of these results, the anthracnose fungi of the domestic strawberry in Korea were identified as C. fructicola and distinguished from C. gloeosporioides.

Incidence, Molecular Characteristics and Pathogenicity of Gibberella fujikuroi Species Complex Associated with Rice Seeds from Asian Countries.

Gibberella fujikuroi species complex (GFSC) was isolated from rice (Oryza sativa L.) seed samples from ten Asian countries and investigated for incidence of GFSC, molecular characteristics, and pathogenicity. Regardless of geographic origin, GFSC was detected with incidences ranging from 3% to 80%. Four species, Fusarium fujikuroi, F. concentricum, F. proliferatum, and F. verticillioides, were found to show an association with rice seeds, with F. fujikuroi being the predominant species. In phylogenetic analyses of DNA sequences, no relationship was found between species, isolates, and geographic sources of samples. Unidentified fragments of the ß-tubulin gene were observed in ten isolates of F. fujikuroi and F. verticillioides. With the exception of three isolates of F. fujikuroi, F. fujikuroi, F. proliferatum, and F. verticillioides were found to have FUM1 (the fumonisin biosynthetic gene); however, FUM1 was not found in isolates of F. concentricum. Results of pathogenicity testing showed that all isolates caused reduced germination of rice seed. In addition, F. fujikuroi and F. concentricum caused typical symptoms of bakanae, leaf elongation and chlorosis, whereas F. proliferatum and F. verticillioides only caused stunting of seedlings. These findings provide insight into the characteristics of GFSC associated with rice seeds and might be helpful in development of strategies for management of bakanae.