Filamentous fungi cause a wide range of superficial and invasive infections, and antifungal susceptibility assay is essential for guiding effective therapy. Standard broth microdilution methods rely on microconidia-based inocula; however, many clinically important moulds produce insufficient microconidia due to intrinsic characteristics or culture conditions, limiting the applicability of conventional testing and often necessitating empirical treatment. To overcome this limitation, we developed an alternative method using precisely quantified mycelial cell suspensions for MIC determination. The method was evaluated using 115 fungal strains and compared with traditional microconidia-based testing. High concordance was observed between the two approaches, with agreement rates of 90% or higher across three common dermatophytes causing tinea infections and three representative fungi responsible for invasive infections. This mycelial-based approach provides a reliable and practical alternative for antifungal susceptibility testing of poorly sporulating moulds and may improve the efficiency and accessibility of MIC testing in clinical laboratories.

The increasing resistance to conventional antifungal agents, such as Amphotericin B (AmB), has led to a growing demand for alternative therapeutic approaches for Candida albicans, an opportunistic fungal pathogen responsible for infections in immunocompromised patients. This study aimed to evaluate the effectiveness of photodynamic inactivation (PDI) in combination with AmB for controlling C. albicans growth, particularly in its yeast and hyphal forms, and to assess the impact of multiple PDI doses. C. albicans (ATCC 90028) was cultured in yeast and hyphal suspensions that were adjusted to 108 CFU/mL and treated with AmB at varying concentrations (0.065–1.04 μg/mL), with and without PDI. PDI was performed using the photosensitizer curcumin (2.5 μM), activated by a 450 nm LED light source at a fluence of 15 J/cm2. The effect of single and repeated PDI doses was evaluated in the fungal biomolecules, which were assessed using Fourier transform infrared (FTIR) spectroscopy. Optical density (OD) measurements quantified fungal growth reduction at 540 nm. The combination of AmB and PDI significantly reduced C. albicans growth, achieving a 75% reduction in the yeast form and an 87.5% reduction in the hyphal form. Two doses of PDI further enhanced antifungal efficacy, particularly against hyphae, which exhibited higher sensitivity to treatment. These findings suggest that PDI enhances the antifungal action of AmB, particularly in more resistant C. albicans forms such as hyphae and biofilms. The observed synergistic effect supports the potential use of PDI as an effective strategy to combat antifungal resistance in clinical applications.

Backgroundin vitro susceptibility testing for the non-sporulating fungus Madurella mycetomatis is performed with a hyphal suspension as starting inoculum and a viability dye for endpoint reading. Here we compared the performance of four different viability dyes for their use in in vitro susceptibility testing of M. mycetomatis.MethodsTo compare the reproducibility and the agreement between the viability dyes 2,3-bis-(2-methoxy-4-nitro-5-sulfphenyl)-2H-tetrazolium-5-carboxanilide salt (XTT), resazurin, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) and luciferin, the in vitro susceptibilities of 14 genetically diverse M. mycetomatis isolates were determined for itraconazole and amphotericin B. The reproducibility, agreement, price and ease of use were compared.ResultsEach of the four dyes gave highly reproducible results with >85.7% reproducibility. Percentage agreement ranged between 78.9% and 92.9%. Resazurin was the most economical to use (0.0009 €/minimal inhibitory concentration [MIC]) and could be followed in real time. Luciferin omitted the need to transfer the supernatant to a new 96-well plate, but cost 6.07 €/MIC.ConclusionAll four viability dyes were suitable to determine the in vitro susceptibility of M. mycetomatis against itraconazole and amphotericin B. Based on the high reproducibility, high percentage agreement, price and possibility to monitor in real time, resazurin was the most suited for routine in vitro susceptibility testing in the diagnostic laboratory in mycetoma-endemic countries. Because luminescence could be measured directly without the need to transfer the supernatant to a new 96-well plate, luciferin is suitable for drug-screening campaigns.Lay summaryTo determine the in vitro susceptibility testing in the non-sporulating fungus Madurella mycetomatis, a viability dye is needed for endpoint reading. In this study we tested the viability dyes XTT, resazurin, MTS and luciferin for their use in in vitro susceptibility testing. It appeared that they all could be used but there were differences in time to result and costs associated with them.

Soybean (Glycine max [L.] Merr.) samples from commercial fields in Decatur and Spencer counties, Indiana were submitted to the Purdue Plant and Pest Diagnostic Lab in August to October 2022. Plants exhibited whole-leaf to interveinal chlorosis of the foliage, red to dark brown external lesions on the crown spreading from the soil-line upward, and severe root rot. In the fields, patches of diseased plants were observed, with greater than 50% of the plants affected and yield loss up to 50%. Orange to red perithecia were present on the exterior of symptomatic stem tissue and ranged in size from 329 to 433 × 232 to 306 µm (n = 10). Stems were surface sterilized in 10% Clorox (0.825% NaOCl) for 1 min, then rinsed with sterile distilled water and dried. In a laminar flow hood, sections of symptomatic stem tissue were plated on using quarter-strength potato dextrose agar (QPDA) and incubated under fluorescent lights on a 12-hr light/dark cycle at 20°C. After 6 days, fungal colonies with fluffy aerial hyphae, which were white near the colony margins and orange to burnt-red near their center, grew uniformly from the stem tissue plated. Elongate, cylindrical hyaline conidia with zero to three septations measuring 45.5 to 73.8 × 4.4 to 6.7 µm (n = 22) grew in clusters from symptomatic stem tissue within the plate. Perithecia developed after 14 days. Falcate, hyaline ascospores with one to two septa measuring 29.4 to 54.7 × 4.6 to 6.8 (n = 23) µm developed within the perithecia. Calonectria ilicicola Boedijn & Reitsma was confirmed based on morphological characteristics (Padgett et al. 2015). Isolate PPDL 22-01457B was used for DNA extraction using the ZR Fungal/Bacterial DNA Miniprep kit (Zymo Research, Irvine, CA). The internal transcriber region (ITS), actin (ACT) and β-tubulin (TUB2) genes were amplified (Carbone and Kohn 1999; Glass and Donaldson 1995; O'Donnell and Cigelnik 1997; White et al. 1990). Amplicons were sent for Sanger sequencing (Genewiz, Inc., South Plainfield, NJ), submitted to Genbank, and assigned accession numbers ITS: OQ932995, Actin: OR484986, and β-tubulin: OR546281. Sequences were analyzed using the NCBI BLASTn tool with results showing 99.5 to 100% identical to C. ilicicola (GenBank accessions LC500063, OQ303403, CP085825, respectively). To perform Koch's Postulates, 90 soybean seeds (CP3620E) were planted in potting media (Berger, Saint-Modeste, Quebec, Canada) in a seed flat with 45 of the plants used as controls and grown under grow lights for 16hr light/8hr dark at 20℃. Individual seedling crowns were inoculated 3 days post-emergence with a 5 to 10 ml spore and hyphal suspension that was scraped from the surface of a 14-day old QPDA culture after adding 300 mL deionized (DI) to each plate grown at 20 to 22°C. The control plants received sterile-DI water. Plants were covered in a plastic bag for 72 h. Plant stems were sprayed with sterile-DI water once a day for seven days. Symptoms were observed after four days, but significant crown rot and lesions developed after two weeks before wilting and dying. Calonectria iliciola was isolated uniformly from symptomatic plants and identified morphologically. Control plants showed no symptoms. Inoculations were repeated 3 times with similar results. As of fall 2023, red crown rot has been confirmed in Adams and Rush counties in Indiana. Red crown rot has been confirmed in several Midwest states (Kleczewski et al. 2019, Neves et al. 2023), but the extent of its distribution and disease management strategies are still limited.

The orphan but highly virulent pathogen Pythium insidiosum causes pythiosis in humans and animals. Surgery is a primary treatment aiming to cure but trading off losing affected organs. Antimicrobial drugs show limited efficacy in treating pythiosis. Alternative drugs effective against the pathogen are needed. In-house drug susceptibility tests (i.e., broth dilution, disc diffusion, and radial growth assays) have been established, some of which adapted the standard protocols (i.e., CLSI M38-A2 and CLSI M51) designed for fungi. Hyphal plug, hyphal suspension, and zoospore are inocula commonly used in the drug susceptibility assessment for P. insidiosum. A side-by-side comparison demonstrated that each method had advantages and limitations. Minimum inhibitory and cidal concentrations of a drug varied depending on the selected method. Material availability, user experience, and organism and drug quantities determined which susceptibility assay should be used. We employed the hyphal plug and a combination of broth dilution and radial growth methods to screen and validate the anti-P. insidiosum activities of several previously reported chemicals, including potassium iodide, triamcinolone acetonide, dimethyl sulfoxide, and ethanol, in which data on their anti-P. insidiosum efficacy are limited. We tested each chemical against 29 genetically diverse isolates of P. insidiosum. These chemicals possessed direct antimicrobial effects on the growth of the pathogen in a dose- and time-dependent manner, suggesting their potential application in pythiosis treatment. Future attempts should focus on standardizing these drug susceptibility methods, such as determining susceptibility/resistant breakpoints, so healthcare workers can confidently interpret a result and select an effective drug against P. insidiosum.

The objectives of this study were to determine the qualitative production of extracellular enzymes produced by ten endophytic fungi and to investigate their antifungal potential against some phytopathogenic fungi namely, Alternaria alternata, Botrytis cinerea, Fusarium oxysporum and Rhizoctonia solani. In addition, the endophytic fungal isolates were screened for enzyme production by plate assay method. All endophytic fungi were able to produce proteases and cellulases with different levels except Alternaria alternata. Meanwhile, Fusarium, Alternaria, Nigrospora and Phoma species produced amylase. None of the tested endophytic fungi showed laccase production. Endophytic fungi filtrates revealed variable antifungal activities against the tested phytopathogenic fungi with Curvularia lunata filtrate being the most effective. This filtrate induced 48% and 80% growth inhibition of B. cinerea and R. solani, respectively. The phytochemical analysis of the endophytic fungi crude extract disclosed the presence of alkaloids and terpenoids. Morphological observations by optical microscope showed uncommon hyphal deformation and enlargement of cytoplasmic vacuoles of R. solani when treated with C. lunata filtrate. Furthermore, C. lunata (hyphal suspension and filtrate) was examined to control root rot caused by R. solani on faba bean plants in vivo. Both antagonistic treatments significantly reduced root rot severity.

HomePlant DiseaseVol. 104, No. 11First Report of Damping-Off on Cucumis melo Caused by Pythium irregulare in South Korea PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Damping-Off on Cucumis melo Caused by Pythium irregulare in South KoreaMi-Jeong Park, Chang-Gi Back, and Jong-Han ParkMi-Jeong Park†Corresponding author: M. J. Park; E-mail Address: mijpark@korea.krhttp://orcid.org/0000-0001-7452-3478Horticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Wanju 55365, Republic of Korea, Chang-Gi BackHorticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Wanju 55365, Republic of Korea, and Jong-Han ParkHorticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Wanju 55365, Republic of Korea AffiliationsAuthors and Affiliations Mi-Jeong Park † Chang-Gi Back Jong-Han Park Horticultural and Herbal Crop Environment Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Wanju 55365, Republic of Korea Published Online:17 Sep 2020https://doi.org/10.1094/PDIS-03-20-0507-PDNAboutSectionsView articlePDFPDF PlusSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat View articleMuskmelons (Cucumis melo L.) are among the most popular cucurbits worldwide and are cultivated in Korea for their sweet and juicy taste. In April 2018, wilting and damping-off symptoms were observed in melon seedlings planted in a greenhouse located in Jeonju, South Korea (35°52′37.7″N, 127°05′10.6″E). Seven days after planting, approximately 10% of the 10,000 seedlings at the two-true-leaf stage were wilted, and brown, water-soaked lesions were observed on stems and roots at or below soil level in wet soil. Plants in advanced stages of this disease wilted completely, damped off, and died. Sections (10 mm2) from 10 diseased stems were surface disinfected with 1% sodium hypochlorite for 30 s, followed by 70% ethanol for 30 s, and rinsed in sterilized distilled water for 1 min. Each section was placed on water agar medium. A small block of agar containing a single hyphal tip of the fungus was taken from the margin of the colony on water agar under the microscope and transferred to new medium. Pure fungal cultures with same growth pattern were obtained from all the sections. A representative culture sample was deposited in the Korean Agricultural Culture Collection (accession no. KACC 48924). On potato dextrose agar (PDA), colonies were white and cottony with a rosette pattern. The isolate grew between 5 and 35°C, with an optimum range of 25 to 30°C, and fully colonized a PDA plate in 3 days. The main hyphae were 2 to 5 μm in width. Hyphal swellings were globose to subglobose, or irregular, intercalary, or terminal, and 10 to 20 μm in diameter. Oogonia were globose, terminal, or intercalary, and 15 to 20 μm in diameter. Antheridia were one to two per oogonium and were mostly monoclinous, sometimes diclinous. Oospores were globose, mostly aplerotic, sometimes plerotic, and 15 to 19 μm in diameter. On the basis of this morphological data, the isolate was tentatively identified as Pythium irregulare Buisman (Van der Plaats-Niterink 1981). For molecular identification of the isolate, genomic DNA was extracted from the mycelial mats, and the ITS rDNA and cox2 gene regions were amplified using primers ITS1/ITS4 (White et al. 1990) and Cox2-F/Cox2-RC4 (Choi et al. 2015), respectively. Sequences from this isolate have been submitted to GenBank with accession numbers MN955406 and MN953048. NCBI BLAST searches showed 99% identity to P. irregulare CBS 250.28 (ex-neotype culture) in both ITS rDNA (AY598702, 944/946 base pairs) and cox2 gene (GU071760, 552/556 base pairs) sequences. A pathogenicity test of the isolate was performed on 4-week-old muskmelon seedlings (Earl's Talent cultivar). The underground portions of each plant were inoculated with hyphal suspensions of the isolate, which were poured into each pot. Plants treated with sterile water served as controls. After inoculation, plants were maintained in a glass greenhouse at 25°C. The soil in each pot was continuously kept moist. Five days after inoculation, inoculated seedlings showed characteristic symptoms of damping-off disease. Infected seedlings wilted, and water-soaked lesions appeared on the lower stems, near the soil line. The symptoms observed in this pathogenicity test were similar to those observed in the greenhouse. Control plants displayed no symptoms. The pathogenicity test was repeated twice. The same organism was reisolated from inoculated plants, confirming Koch's postulates. P. irregulare is known to have a broad host range, including cucurbits (Farr and Rossman 2020). Occurrences of disease caused by P. irregulare on melons have been previously recorded in regions of North America, including the United States and Canada. To our knowledge, this is the first report of damping-off caused by P. irregulare on melons in Korea. The pathogen presents a potential threat to South Korean melon seedling production.The author(s) declare no conflict of interest.

In Korea, okra (Abelmoschus esculentus [L.] Moench) is cultivated on small family farms and experimental plots (0.5 ha). In August 2014 and 2015, okra plants of cultivar ‘Green Hope’, grown in the farms of Iksan, Korea, were observed for leaf spot symptoms. Leaf spots were circular to irregular-shaped, initially brown, and subsequently turned reddish-brown with a yellowish margin. On leaves, stromata were brown, small, and composed of a few swollen hyphal cells. The conidiophores were fasciculate, olivaceous to brown, paler toward the apex, straight to minimally curved, geniculate, 30 to 250 µm long, 3.5 to 5.0 µm wide, one- to five-septate, and with conspicuous conidial scars. Conidia (n = 50) were hyaline, acicular, subacute to obtuse at the apex, truncate to obconically truncate at the base, 2- to 20-septate, 34 to 280 × 3.4 to 6.5 µm, and with a thickened, darkened hilum at the base. The morphological characteristics were consistent with Cercospora malayensis F. Stevens & Solheim (Chupp 1953). To obtain a pure conidial suspension, conidia were collected from 50 infected leaves using sterile forceps, placed in an Eppendorf tube containing sterile water, streaked onto the surface of 2% water agar plates supplemented with 100 mg/liter of streptomycin sulfate, and transferred onto potato dextrose agar (PDA). After 7 days of incubation, fungal colonies (10 isolates) appeared off-white and flat with aerial mycelia. Voucher specimens were housed in the Korea University herbarium (KUS-F28701), and one isolate was deposited in the Korean Agricultural Culture Collection (KACC47769). Additional fungal confirmation was conducted using the internal transcribed spacer (ITS), translation elongation factor 1-α (EF), and actin (ACT) gene sequences (Groenewald et al. 2013). A BLASTn search using ITS and ACT sequences showed >99% identity with C. malayensis ITS and ACT sequences (KM489070 and KY082664), whereas EF sequences showed 97.8% identity with C. malayensis EF gene (KY082663). The ITS, EF, and ACT sequences of the isolate were deposited in GenBank (MH129519, MH129517, and MH129515). Five 4-month-old okra plants (Green Hope) were spray-inoculated with C. malayensis (KACC47769) hyphal suspensions obtained from 2-week-old colonies grown on PDA at 25°C and transferred to a greenhouse (25°C) with a 12-h photoperiod. Five control plants were inoculated with sterile water. Symptoms observed on the C. malayensis inoculated plants were 100% identical with infected field conditions. C. malayensis was reisolated from symptomatic leaf tissues, and the identity was confirmed by microscopic studies. The pathogenicity test was repeated twice. Okra leaf spots associated with C. malayensis have previously been reported from several countries but not Korea (Farr and Rossman 2019). To our knowledge, this is the first report of C. malayensis on okra in Korea. Because okra hectares may increase, the disease poses a threat to okra cultivation in Korea.

Potato tubers (cultivar ‘Austrian Crescent’) displaying a dry rot decay symptom and internal sclerotia were received for diagnosis. Isolations were attempted from symptomatic material, and Sclerotinia sclerotiorum was frequently recovered. Pathogenicity tests for S. sclerotiorum in potato tubers were completed using agar plugs with S. sclerotiorum placed over tuber wounds and through injecting stolon end with a S. sclerotiorum hyphal suspension. Both inoculation methods induced decay symptoms, although these were observed less frequently with the agar plug inoculation (37.5%) compared with hyphal injection (100%). S. sclerotiorum was consistently reisolated from inoculated symptomatic tubers. This symptom is rare, but potato growers should be aware that tuber infection is possible when white mold is present.

BackgroundAlthough fungal infections play a central role in severely ill and immunosuppressed patients, in contrast to antibiotics immunomodulatory effects of antifungals have not been sufficiently investigated. The present study sets out to compare the effect of different antimycotics on immunologic reaction towards mold in vitro.Materials/methodsAspergillus fumigatus ATCC204305 was used to develop a model of invasive aspergillosis in vitro in whole blood. Since autoclaved hyphal suspension demonstrated the most potent cytokine release, they were used for the further study including blood of 20 male volunteers. Impact on IL-6, IL-8 and TNF-α time concentration profiles by 5 mg/mL conventional and liposomal amphotericin B, 20 mg/mL fluconazole, 5 mg/mL voriconazole, 2 mg/mL posaconazole or saline solution was investigated over 4 h of incubation at 37 °C.ResultsCompared to baseline, cytokine levels increased by addition of hyphal suspension over 4 h approximately: 54-fold for IL-6, 1000-fold for IL-8 and 270-fold for TNF-α. While conventional amphotericin B further increased IL-6 and to a smaller extent IL-8 levels, this was not the case for its liposomal formulation. Congruently amphotericin B increased cytokines in blood without fungus substantially. Fluconazole reduced cytokine increase for all three cytokines compared to stimulation with hyphae without antifungal agent.ConclusionsOur data indicate significant differences in the immunomodulatory potency of different antimycotics. While fluconazole had the highest anti-inflammatory potential, conventional amphotericin even increased cytokine release. This preliminary information might have clinical implication, since cytokine dysregulation plays a major role in the pathogenesis and outcome of fungal infections. Clinical studies are warranted to confirm our findings.

Pak choy ( Brassica campestris ssp. chinensis L.) is the major vegetable crop cultivated in China. During December 2017, leaf rot was frequently observed on leaves of pak choy in greenhouses of Shanghai, China. Diseased leaf samples were plated onto acidified potato dextrose agar and fungal cultures were isolated and identified as Rhizopus oryzae , based on morphological features and molecular identification. Definitive identification as R. oryzae was based on the comparative molecular analysis of rRNA gene sequences. Blast analysis revealed 99% similarity with R. oryzae . Pathogenicity was determined by inoculating healthy pak choy leaves and plants with hyphal suspensions of R . oryzae. The fungus was re-isolated from developing similar lesions on the inoculated plants and identified as similar to the inoculated fungus, thus fulfilling Koch’s postulates. This is the first report of fungal leaf rot caused by R. oryzae on pak choy in China.

Hot pepper is an exceedingly popular vegetable crop in China and is cultivated on 737 hectares in Beijing. A severe leaf disease with typical symptoms of spots with grey centres and dark brown borders was observed on hot pepper plants in Beijing in the winter of 2016. More than 50% of all plants in the region were infected. Fungal cultures were isolated from naturally infected leaf tissue, and identified as Stemphylium lycopersici based on morphological features, cultural characteristics and molecular identification by sequencing the ITS, gpd and cmdA genes. Pathogenicity was determined by inoculating healthy pepper plants with hyphal suspensions, and the fungus was re-isolated from developing lesions on the inoculated plants, thus fulfilling Koch’s postulates. This is the first report of natural infection by S. lycopersici causing leaf spot on hot pepper in China.

Lonicera vidalii Franch. & Sav. (family Caprifoliaceae) is a native tree in Korea and Japan. It is a rare deciduous tree of the forest community, mainly occurring in the southern part of Korea. In September 2017, leaf spots on trees, typical symptoms caused by cercosporoid fungi, were observed in Hongneung Arboretum, Seoul, Korea (37°35′37″N, 127°02′38″E). Initial symptoms presented as orbicular to irregular, pale- to dark-brown leaf spots typically 0.2 to 0.9 cm in diameter, which in some cases merged to form larger blotches across the surface of mature leaves. At infection sites, septate, branched, hyaline to pale brown, smooth, and 2 to 2.5 μm wide hyphae of the fungus were seen emerging through the stomata at the adaxial leaf side. Stromata were large and well-developed, globular, olivaceous brown, 30 to 50 µm in diameter, and emerging through stomatal openings. Conidiophores were aggregated in loose to dense fascicles through stomatal openings, subcylindrical, proliferating sympodially, straight to slightly geniculate, one- to four-septate, 15 to 53 × 3.5 to 5.5 μm, and smooth. Conidiogenous cells were terminal, cylindrical, proliferating sympodially, and 12.5 to 27 × 3.5 to 5.5 μm. Conidia were solitary, guttulate cylindrical to narrowly obclavate, straight to mildly curved, obtuse to rounded at the apex, one- to four-septate, variable in length, and 19 to 97 × 2 to 4 μm. Three monoconidial isolates were deposited in the culture collection (CDH) of the National Institute of Forest Science, Korea (CDH2017-15 to CDH2017-17). To ensure the identity of the fungus, genomic DNA was extracted, and sequence comparisons were made based on the partial nucleotide sequences of internal transcribed spacer (ITS) rDNA, translation elongation factor-1 alpha (EF-1α), actin, and RNA polymerase II second largest subunit (rpb2), using the respective primer pairs ITS1/ITS4, EF1-728F/EF1-986R, ACT-512F/ACT-783R, and RPB2-5f2/fRPB2-7cR (Nakashima et al. 2016). The sequences obtained were deposited in GenBank (MG712852 to MG712854, MG712861 to MG712863, MG712858 to MG712860, and MG930772 to MG930774, respectively). For a phylogenetic analysis, sequences from different gene regions (ITS, EF-1α, actin, and rpb2) obtained from GenBank were aligned, concatenated, and analyzed as a single dataset based on a maximum likelihood analysis. This Korean cercosporoid fungus was positioned in a clearly distinct lineage, provisionally representing an undetermined species of Pseudocercospora, which is closest to Pseudocercospora stephanandrae (Tak. Kobay. & H. Horie) C. Nakash. & Tak. Kobay., MUCC 914 (NR 111469 in ITS, GU384526 in EF-1α, GU320516 in actin, and KX462658 in rpb2). Sequence comparisons revealed that this Korean pathogen differed from P. stephanandrae at 4 of 477 characters (∼1.0%) in the ITS, 3 of 294 (∼1.0%) in the EF-1α, 4 of 190 (∼2.1%) in the actin, and 43 of 686 (∼6.3%) in the rpb2 sequences. In addition, this Korean pathogen is morphologically distinct from P. stephanandrae by having larger stromata as well as longer conidiophores and conidial size (Nakashima and Kobayashi 2000). Based on morphology and molecular analyses, the fungus was tentatively identified as Pseudocercospora sp. (Guo and Hsieh 1995; Nakashima et al. 2016). Pathogenicity was demonstrated by hyphal suspensions from 15-day-old cultures onto leaflets of L. vidalii and then maintaining them in a growth chamber (22°C and 80% relative humidity). Controls were treated with sterile water. Distinct leaf spot symptoms on the inoculated leaflets were developed, and the controls remained symptomless. The pathogen, recovered from symptomatic leaflets, was morphologically identical to those observed in the field. Lonicera-Pseudocercospora associations were recorded in China, Japan, Taiwan, Korea, New Zealand, and Brazil. However, the leaf spot infection associated with Pseudocercospora sp. on L. vidalii has not been recorded worldwide (Farr and Rossman 2017). This is the first report of leaf spot caused by Pseudocercospora sp. on L. vidalii globally as well as in Korea. The disease poses a threat to ornamental plantings and naturally growing plants of Lonicera spp. in Korea.

Background: Dermatophytes are responsible for the majority of the fungal infections involving skin, hair and nails. There has been a remarkable increase in the number of fungal infections especially in those people whose immune system is compromised by aging, HIV infection, organ transplantation or cancer therapy. Objective: The aim of this study was to compare both broth microdilution method & disk diffusion method for in-vitro activity of some antifungal drugs (Terbinafine, Fluconazole, Itraconazole) against different species of dermatophytes. Patients and Method: This study was performed on 50 dermatophyte isolates recovered from various clinical specimens (skin, hair and nail) collected from dermatology outpatient clinic of Ain Shams University Hospital. All samples were cultured on sabarouds. Isolates recovered from SDA were subcultured on Potato Dextrose Agar (PDA) & incubated at 28°C for 7 days to enhance sporulation. The growth was harvested in sterile saline & the conidial and hyphal suspension was adjusted to 0.5 macfarland. Then antifungal susceptibility was done using: Disk diffusion (DD) method and Broth micro dilution (BMD) method. Results: There was a highly significant agreement between the antifungal susceptibility testing of fluconazole, itraconazole and terbinafine by disk diffusion method and Broth micro-dilution method. In our studyagreement between both methods for itraconazole was 1.00 (kappa), for terbinafine was 0.947, and for fluconazole was 0.878. The factors that may affect the results of BMD or DD are type and size of inoculum, composition of the media, temperature and duration of incubation and disc strength. Conclusion: There was a highly significant agreement between the antifungal susceptibility testing of fluconazole,

New Guinea impatiens, Impatiens hawkeri W. Bull, is widely cultivated as a potted plant and garden plant. In July 2013, hundreds of young plants (cv. Fanfare) showing symptoms of leaf spot with approximately 50% incidence were found in polyethylene tunnels in Yongin City, Korea. Leaf spots were circular to oblong, reaching 6 mm or more in diameter. The spots were initially uniformly brown to reddish brown, turning gray with reddish brown margin. Diseased plants defoliated prematurely and were abandoned without marketing due to signs of discoloration and yellowing on leaves. A cercosporoid fungus was consistently observed in association with disease symptoms. Stromata were brown, small, and composed of a few swollen hyphal cells. Conidiophores were emerging through the cuticle, fasciculate (n = 2 to 20), olivaceous to brown, paler toward the apex, straight to mildly curved, geniculate, 30 to 260 μm long, 3.5 to 5 μm wide, 1- to 6-septate, and with conspicuous conidial scars. Conidia were hyaline and acicular. Smaller conidia were straight and longer conidia were mildly curved. Conidia were subacute to obtuse at the apex, truncate to obconically truncate at the base, 2- to 18-septate, 30 to 320 × 3.5 to 5.5 μm, and with thickened, darkened hila at the base. Morphological characteristics of the fungus were consistent with the previous reports of Cercospora fukushiana (Matsuura) W. Yamam. (1). Voucher specimens were housed in the Korea University herbarium (KUS). An isolate from KUS-F27438 was deposited in the Korean Agricultural Culture Collection (Accession No. KACC47640). Fungal DNA was extracted with DNeasy Plant Mini Kits (Qiagen Inc., Valencia, CA). The complete internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 (4) and sequenced. The resulting sequence of 497 bp was deposited in GenBank (Accession No. KJ620981). This showed >99% similarity with sequence of C. fukushiana (EF600954) on I. balsamina from Korea. Isolate of KACC47640 was used in the pathogenicity tests. Hyphal suspensions were prepared by grinding 3-week-old colonies grown on PDA with distilled water using a mortar and pestle. Five plants were inoculated with hyphal suspensions and five plants were sprayed with sterile distilled water. The plants were covered with plastic bags to maintain a relative humidity of 100% for 24 h and then transferred to a 25 ± 2°C greenhouse with a 12-h photoperiod. Typical symptoms of necrotic spots appeared on the inoculated leaves 10 days after inoculation, and were identical to the symptoms observed in the field. C. fukushiana was re-isolated from symptomatic leaf tissues, confirming Koch's postulates. No symptoms were observed on water-inoculated control plants. Previously, leaf spots of Impatiens spp. associated with C. apii, C. balsaminae, and C. fukushiana have been reported (1,2,3). To our knowledge, this is the first report of C. fukushiana on I. hawkeri in Korea. Our observations in the nurseries of I. hawkeri suggest that low humidity with good ventilation as well as plant hygiene in greenhouses might be main strategies for preventing this disease.

Melia azedarach L., called chinaberry, is native to Southeast Asia and Australia. The trees are commonly planted as ornamentals in the southern part of Korea. In October 2010, a leaf spot disease was observed on trees for the first time in Wando, Korea. Further surveys conducted from 2010 to 2012 showed that the disease occurs on trees in Jeju, Seogwipo, and Tongyeong cities as well as Wando county with nearly 100% incidence. Leaf spots were circular to semicircular, later becoming angular, small, pale brown in the center with a dark brown margin, and later becoming milky white. Leaf spots sometimes coalesced to blight the entire leaf and were capable of rapidly defoliating whole trees in late September. Fruiting was amphigenous, but mostly hypogenous. Stromata were substomatal, globular, dark brown, and 25 to 70 μm in diameter. Conidiophores were densely fasciculate, pale olivaceous to pale brown, substraight to mildly curved, not geniculate, 10 to 30 μm long, 2.5 to 4.5 μm wide, and aseptate or uniseptate. Conidia were pale olivaceous, generally darker than conidiophores, cylindric to obclavate, substraight in shorter ones, curved to mildly sinuous in longer ones, obconically truncate at the base, obtuse at the apex, 2- to 14-septate, 16 to 120 × 3 to 5 μm, guttulate, and had inconspicuous hila. Morphological characteristics of the fungus were consistent with the previous descriptions of Pseudocercospora subsessilis (Syd. & P. Syd.) Deighton (2). Voucher specimens (n = 6) were deposited in the Korea University Herbarium (KUS). An isolate from KUS-F25395 was deposited in the Korean Agricultural Culture Collection (KACC45688). The complete internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 (3) and sequenced. The resulting sequence of 517 bp was deposited in GenBank (Accession No. JX993904). A BLAST search in GenBank revealed that the sequence shows >99% similarity (1 bp substitution) with a sequence of P. subsessilis ex M. azedarach from Cuba (GU269815). For pathogenicity tests, hyphal suspensions were prepared by grinding 3-week-old colonies grown on potato dextrose agar with distilled water using a mortar and pestle. Five 3-year-old chinaberry trees were inoculated with hyphal suspensions using a fine haired paint brush. Three healthy trees of the same age, serving as controls, were sprayed with sterile water. The plants were covered with plastic bags to maintain 100% relative humidity for 24 h and then transferred to a greenhouse. Typical symptoms of necrotic spots that appeared on the inoculated leaves 10 days after inoculation were identical to the ones observed in the field. P. subsessilis was reisolated from symptomatic leaf tissues, confirming Koch's postulates. No symptoms were observed on control plants. The disease has been reported in several Asian countries as well as in Cuba and the United States (1). To our knowledge, this is the first report of leaf spot on chinaberry caused by P. subsessilis in Korea. The observed high incidence and severity suggest that this disease can be a limiting factor in utilizing this tree species as ornamentals in public areas.

Sweet basil, Ocimum basilicum L., is a fragrant herb belonging to the family Lamiaceae. Originated in India 5,000 years ago, sweet basil plays a significant role in diverse cuisines across the world, especially in Asian and Italian cooking. In October 2008, hundreds of plants showing symptoms of leaf spot with nearly 100% incidence were found in polyethylene tunnels at an organic farm in Icheon, Korea. Leaf spots were circular to subcircular, water-soaked, dark brown with grayish center, and reached 10 mm or more in diameter. Diseased leaves defoliated prematurely. The damage purportedly due to this disease has reappeared every year with confirmation of the causal agent made again in 2011. A cercosporoid fungus was consistently associated with disease symptoms. Stromata were brown, consisting of brown cells, and 10 to 40 μm in width. Conidiophores were fasciculate (n = 2 to 10), olivaceous brown, paler upwards, straight to mildly curved, not geniculate in shorter ones or one to two times geniculate in longer ones, 40 to 200 μm long, occasionally reaching up to 350 μm long, 3.5 to 6 μm wide, and two- to six-septate. Conidia were hyaline, acicular to cylindric, straight in shorter ones, flexuous to curved in longer ones, truncate to obconically truncate at the base, three- to 16-septate, and 50 to 300 × 3.5 to 4.5 μm. Morphological characteristics of the fungus were consistent with the previous reports of Cercospora guatemalensis A.S. Mull. & Chupp (1,3). Voucher specimens were housed at Korea University herbarium (KUS). An isolate from KUS-F23757 was deposited in the Korean Agricultural Culture Collection (Accession No. KACC43980). Fungal DNA was extracted with DNeasy Plant Mini DNA Extraction Kits (Qiagen Inc., Valencia, CA). The complete internal transcribed spacer (ITS) region of rDNA was amplified with the primers ITS1/ITS4 and sequenced. The resulting sequence of 548 bp was deposited in GenBank (Accession No. JQ995781). This showed >99% similarity with sequences of many Cercospora species, indicating their close phylogenetic relationship. Isolate of KACC43980 was used in the pathogenicity tests. Hyphal suspensions were prepared by grinding 3-week-old colonies grown on PDA with distilled water using a mortar and pestle. Five plants were inoculated with hyphal suspensions and five plants were sprayed with sterile distilled water. The plants were covered with plastic bags to maintain a relative humidity of 100% for 24 h and then transferred to a 25 ± 2°C greenhouse with a 12-h photoperiod. Typical symptoms of necrotic spots appeared on the inoculated leaves 6 days after inoculation, and were identical to the ones observed in the field. C. guatemalensis was reisolated from symptomatic leaf tissues, confirming Koch's postulates. No symptoms were observed on control plants. Previously, the disease was reported in Malawi, India, China, and Japan (2,3), but not in Korea. To our knowledge, this is the first report of C. guatemalensis on sweet basil in Korea. Since farming of sweet basil has recently started on a commercial scale in Korea, the disease poses a serious threat to safe production of this herb, especially in organic farming.

The genus Acanthus (Acanthaceae) includes ~30 herbaceous, perennial species grown for their attractive foliage and flower spikes. Between June and December 2009 the CDFA Plant Pest Diagnostics Lab in Sacramento, CA received multiple leaf spot disease samples on Acanthus spinosus and A. mollis, commonly known as bear's breeches. Samples were collected four times from two nurseries in Santa Barbara County. Disease was observed in nearly 100% of the plants inspected. Leaf spots were brown, roundish to elliptical, and 1 to 4 mm in diameter. Older spots often developed grayish centers and often coalesced, leading to large necrotic areas. Conidiophores were fasciculate, amphigenous, light brown to olivaceous, multiseptate, geniculate, and had distinctive spore scars. Conidia were hyaline, straight to slightly curved with tapered tips and truncate bases. Conidia were solitary, multiseptate (1 to 10) and 48 to 160 × 2.5 to 5 μm (average 100 × 3.9 μm). Colonies obtained from single conidial isolates were established on acidified potato dextrose agar (APDA). Morphologically, the causal agent was identified as Cercospora diantherae Ellis and Kellerm (1), a species synonymous with C. apii sensu lato (2). The C. apii sensu lato complex includes three morphologically similar taxa, C. apii, C. beticola, and C. apiicola (3). Sequence analysis of the internal transcribed spacer region from the Acanthus isolate confirmed it belongs to the C. apii complex (GenBank HQ328503). Multiplex PCR to distinguish species within the complex was also performed on the isolate (3). A 176-bp fragment was only observed in the PCR reaction containing the C. beticola primers. To confirm pathogenicity, hyphal suspensions were used to inoculate healthy leaves of A. mollis plants potted in 3.7-liter containers. Hyphal suspensions were obtained by grinding 3-week-old colonies grown on APDA with distilled water using a mortar and pestle. Both sides of healthy leaves and petioles were sprayed with ~40 ml of the suspension. Five plants were inoculated with C. beticola and five plants were sprayed with sterile water. Plants were incubated in a dew chamber for 48 h and then transferred to a 25°C growth chamber with a 12-h photoperiod. The experiment was repeated. Five days after inoculation, small necrotic leaf spots developed on the leaves. After 14 days, the spots had enlarged and the leaves began to turn yellow. Over time, the spots coalesced leading to large necrotic areas, especially along the leaf margins. Petiole spots, not seen on field samples, were seen on laboratory inoculated plants. Sporulation of C. beticola occurred within most of the spots and the pathogen was successfully reisolated from all inoculated leaves. No foliar symptoms developed on any of the control plants. Worldwide, C. beticola is a destructive pathogen of sugar beet (4), and has also been reported on a number of other plant hosts (3). To our knowledge, this is the first report of C. beticola causing a leaf spot disease on a host in the Acanthaceae family. This strain has been deposited into the culture collection at Centraalbureau voor Schimmelcultures.

Pseudallescheria boydii (Scedosporium apiospermum) is a saprophytic fungus widespread in the environment, and has recently emerged as an agent of localized as well as disseminated infections, particularly mycetoma, in immunocompromised and immunocompetent hosts. We have previously shown that highly purified α-glucan from P. boydii activates macrophages through Toll-like receptor TLR2, however, the mechanism of P. boydii recognition by macrophage is largely unknown. In this work, we investigated the role of innate immune receptors in the recognition of P. boydii. Macrophages responded to P. boydii conidia and hyphae with secretion of proinflammatory cytokines. The activation of macrophages by P. boydii conidia required functional MyD88, TLR4, and CD14, whereas stimulation by hyphae was independent of TLR4 and TLR2 signaling. Removal of peptidorhamnomannans from P. boydii conidia abolished induction of cytokines by macrophages. A fraction highly enriched in rhamnomannans was obtained and characterized by NMR, high performance TLC, and GC-MS. Preparation of rhamnomannans derived from P. boydii triggered cytokine release by macrophages, as well as MAPKs phosphorylation and IκBα degradation. Cytokine release induced by P. boydii-derived rhamnomannans was dependent on TLR4 recognition and required the presence of non-reducing end units of rhamnose of the rhamnomannan, but not O-linked oligosaccharides from the peptidorhamnomannan. These results imply that TLR4 recognizes P. boydii conidia and this recognition is at least in part due to rhamnomannans expressed on the surface of P. boydii.

Injection inoculation protocols for fruit body formation of Cordyceps militaris (C. militaris) were investigated to improve the incidence of infection in the silkworm species Bombyx mori (B. mori). Injection, with suspensions of C. militaris hyphal bodies into living silkworm pupae, was used to test for fruit body production. Use of Daeseungjam rather than Baegokjam or Keumokjam varieties of B. mori is thought to be suitable for infection by C. militaris. From mounting, nine-day-old to 11-day-old pupae showed the best incidence of infection with a 100 μL injection volume. Silkworm pupae injected with a hyphal suspension concentration of more than 2 × 105 colony-forming unit (cfu) recorded a greater than 96% incidence of infection. Also, fruit bodies of C. militaris were induced and produced at a light intensity between 500 and 1,000 lx.