Plant-Entomopathogenic Fungi Interaction: Recent Progress and Future Prospects on Endophytism-Mediated Growth Promotion and Biocontrol.

Entomopathogenic fungi, often acknowledged primarily for their insecticidal properties, fulfill diverse roles within ecosystems. These roles encompass endophytism, antagonism against plant diseases, promotion of the growth of plants, and inhabitation of the rhizosphere, occurring both naturally and upon artificial inoculation, as substantiated by a growing body of contemporary research. Numerous studies have highlighted the beneficial aspects of endophytic colonization. This review aims to systematically organize information concerning the direct (nutrient acquisition and production of phytohormones) and indirect (resistance induction, antibiotic and secondary metabolite production, siderophore production, and mitigation of abiotic and biotic stresses) implications of endophytic colonization. Furthermore, a thorough discussion of these mechanisms is provided. Several challenges, including isolation complexities, classification of novel strains, and the impact of terrestrial location, vegetation type, and anthropogenic reluctance to use fungal entomopathogens, have been recognized as hurdles. However, recent advancements in biotechnology within microbial research hold promising solutions to many of these challenges. Ultimately, the current constraints delineate potential future avenues for leveraging endophytic fungal entomopathogens as dual microbial control agents.

Production of highly pure R,R-2,3-butanediol for biological plant growth promoting agent using carbon feeding control of Paenibacillus polymyxa MDBDO.

BACKGROUND: Chemical fertilizers have greatly contributed to the development of agriculture, but alternative fertilizers are needed for the sustainable development of agriculture. 2,3-butanediol (2,3-BDO) is a promising biological plant growth promoter. RESULTS: In this study, we attempted to develop an effective strategy for the biological production of highly pure R,R-2,3-butanediol (R,R-2,3-BDO) by Paenibacillus polymyxa fermentation. First, gamma-ray mutagenesis was performed to obtain P. polymyxa MDBDO, a strain that grew faster than the parent strain and had high production of R,R-2,3-BDO. The activities of R,R-2,3-butanediol dehydrogenase and diacetyl reductase of the mutant strain were increased by 33% and decreased by 60%, respectively. In addition, it was confirmed that the carbon source depletion of the fermentation broth affects the purity of R,R-2,3-BDO through batch fermentation. Fed-batch fermentation using controlled carbon feeding led to production of 77.3 g/L of R,R-2,3-BDO with high optical purity (> 99% of C4 products) at 48 h. Additionally, fed-batch culture using corn steep liquor as an alternative nitrogen source led to production of 70.3 g/L of R,R-2,3-BDO at 60 h. The fed-batch fermentation broth of P. polymyxa MDBDO, which contained highly pure R,R-2,3-BDO, significantly stimulated the growth of soybean and strawberry seedlings. CONCLUSIONS: This study suggests that P. polymyxa MDBDO has potential for use in biological plant growth promoting agent applications. In addition, our fermentation strategy demonstrated that high-purity R,R-2,3-BDO can be produced at high concentrations using P. polymyxa.

First Report of Binucleate Rhizoctonia AG-G Causing Root Rot of Japanese Bay Tree (Machilus thunbergii) in Korea.

Machilus thunbergii Sieb. & Zucc., commonly known as Japanese bay tree, is a large evergreen tree belonging to the Lauraceae family and is widely distributed in Asia, including Korea in subtropical and tropical forest areas (Wu et al., 2006). In April 2021, a root rot disease of 2-year-old Japanese bay trees was observed in a nursery on Wando Island in Korea. Tree roots exhibited brown to black discoloration, root rot, and deterioration, and leaves were severely wilted followed by plant death, with a disease incidence of approximately 30%. Symptomatic roots were surface sterilized with 1% NaOCl for 5 min and washed three times with distilled water. The root tissues were dried and plated on potato dextrose agar (PDA) and vegetable juice agar (V8) media. After 3-4 days of incubation at 25 ˚C, brown Rhizoctonia fungal-like colonies grew on both culture media. Hyphae of two representative isolates (CMML21-35 and CMML21-36) exhibited typical characteristics of Rhizoctonia, including a constriction of branching hyphae (Alvarez et al., 2013). In addition, two nuclei in each mycelial cell were observed after staining of mycelia with 0.1% Safranin O. The two isolates were identified as binucleate Rhizoctonia based on the microscopic observation. To confirm identification of the isolates, the internal transcribed spacer (ITS) and large subunit (LSU) regions were sequenced using two primer sets, ITS1/ITS4 and LROR/LR5 (White et al., 1990; Vilgalys and Hester 1990). BLASTn search analysis revealed that the ITS sequence of isolates had 99.66% (582 base pair matched of 584) sequence similarity with the sequences of binucleate Rhizoctonia (accession numbers JF519837 and AY927327, respectively) and the LSU sequence matched well with the sequence of Rhizoctonia sp. AG-G (accession number MN977413; similarity 99.56% and 910 base pair matched of 914). The sequences were deposited in GenBank under accession numbers OM049427 and OM049428 for ITS, OM679289 and OM679290 for LSU. Phylogenetic analysis of ITS and LSU regions revealed that the isolates grouped with binucleate Rhizoctonia anastomosis group AG-G (Teleomorph: Ceratobasidium sp.) with a high bootstrap value. Accordingly, the morphological and molecular characteristics confirmed the causal pathogen as binucleate Rhizoctonia AG-G (Jiang et al., 2016; Gonzalez et al. 2016). To test pathogenicity, a 2-year-old Japanese bay tree was inoculated by creating a hole in the soil near the root rhizosphere and placing 1.5g of ground mycelia obtained from a 5 day-old broth culture at two time points one week apart (Bartz et al., 2010). The control pot was inoculated with sterilized ddH2O. Inoculated and control plant pots were incubated in plastic boxes with 100% relative humidity at 25 ℃ for five days. After that, the pots were placed in the greenhouse at 23-25 ℃. One month post inoculation, initial disease symptoms were observed, and after two months, severe foliar wilting and eventual plant death occurred. The non-inoculated control remained healthy. The pathogen was re-isolated from infected roots, fulfilling Koch's postulates. The experiment was conducted three times with three replications. This is the first report of root rot of Japanese bay tree caused by binucleate Rhizoctonia AG-G in Korea and in the world. Previously, a pathogenic binucleate Rhizoctonia AG-G was isolated from colonized apple tree roots in orchards in Italy (Kelderer et al., 2012). The present study implies that this pathogen potentially causes a negative impact on the nursery and forest industries, thus further research on the screening for pathogenicity in other tropical and subtropical trees and also apple, which is an important crop in Korea, is needed.

First report of post-harvest tuber rot of Apios americana Medikus caused by Penicillium expansum in South Korea.

Apios americana Medikus, a perennial vine legume native to North America, is known as 'Indian potato' or 'Apios' for their underground tubers that are used for human consumption in Korea (Choi et al., 2017). These tubers are known to be rich in isoflavones as well as other secondary products to have known several medicinal properties (Chu et al., 2019). The harvested tubers in 2020 were observed to rot during storage of tubers for 4 months at 4°C in the genetic resource storage room at the Bioenergy Crop Research Institute, National Institute of Crop Science, Muan, Jeollanam-do, South Korea. The incidence of rot symptoms with blue mold was less than 1% per 20 kg box. Ten infected tubers were collected from several boxes and the lesions were cut into small pieces, and then surface sterilized in 1.5% NaClO for 2 minutes, followed by rinsing 3 times with sterilized water. To investigate their morphological characteristics, ten isolates were cultured in Malt Extract Agar (MEA) medium at 25° C for 5 days (Pitt and Hocking, 1988). The surface morphology of the mycelium had white or light green fluffy, and completely blue spores were formed after about 5 days. The conidia were one-stage branched with an elliptical shape, about 3.5 to 4.3 × 2.9 to 3.6 (mean 3.8 ± 0.3 × 3.2 ± 0.2) ㎛ in diameter (n=30). Genomic DNAs of the isolates were extracted using Solgent DNA Extraction Kit (Solgent, Daejeon, Korea), and then PCR products of the internal transcribed spacer (ITS1/ITS4) region and the beta-tubulin gene (Bt2a/Bt2b) were sequenced and analyzed (Glass and Donaldson, 1995). The BLASTn showed that the representative isolate had 99% homology with reference Penicillium expansum strain ICMP 2708 (ITS region and TUB2 gene) in NCBI GenBank. The sequences of the isolate were deposited in GenBank as accession numbers MZ636667 and MZ702813 for ITS and TUB2 genes, respectively. Based on the morphological characteristics and molecular analysis, the isolate was identified as P. expansum. Pathogenicity assays of the isolate were also performed using three tubers in three replicates inoculated with spore suspension (concentration, 1×106 conidia/mL) and compared with a control group inoculated with sterilized water. The inoculated Apios tubers were placed in a plastic box maintained in conditions of high humidity at 25°C. Five days after inoculation, the typical symptoms were observed on inoculated tubers, and no symptoms were observed in the control one. P. expansum was again isolated from artificially inoculated tubers to complete Koch's assumption. This is the first report of P. expansum causing tuber rot in A. americana in South Korea. As the cultivated area of Apios is increasing in Korea, it will be necessary to develop effective storage methods and management strategies for the control of storage diseases such as blue mold.

Cytogenetics and Consequences of Polyploidization on Different Biotic-Abiotic Stress Tolerance and the Potential Mechanisms Involved.

The application of polyploidy in sustainable agriculture has already brought much appreciation among researchers. Polyploidy may occur naturally or can be induced in the laboratory using chemical or gaseous agents and results in complete chromosome nondisjunction. This comprehensive review described the potential of polyploidization on plants, especially its role in crop improvement for enhanced production and host-plant resistance development against pests and diseases. An in-depth investigation on techniques used in the induction of polyploidy, cytogenetic evaluation methods of different ploidy levels, application, and current research trends is also presented. Ongoing research has mainly aimed to bring the recurrence in polyploidy, which is usually detected by flow cytometry, chromosome counting, and cytogenetic techniques such as fluorescent in situ hybridization (FISH) and genomic in situ hybridization (GISH). Polyploidy can bring about positive consequences in the growth and yield attributes of crops, making them more tolerant to abiotic and biotic stresses. However, the unexpected change in chromosome set and lack of knowledge on the mechanism of stress alleviation is hindering the application of polyploidy on a large scale. Moreover, a lack of cost-benefit analysis and knowledge gaps on the socio-economic implication are predominant. Further research on polyploidy coupling with modern genomic technologies will help to bring real-world market prospects in the era of changing climate. This review on polyploidy provides a solid foundation to do next-generation research on crop improvement.

Biocontrol of the causal brown patch pathogen Rhizoctonia solani by Bacillus velezensis GH1-13 and development of a bacterial strain specific detection method.

Brown patch caused by the basidiomycete fungus Rhizoctonia solani is an economically important disease of cool-season turfgrasses. In order to manage the disease, different types of fungicides have been applied, but the negative impact of fungicides on the environment continues to rise. In this study, the beneficial bacteria Bacillus velezensis GH1-13 was characterized as a potential biocontrol agent to manage brown patch disease. The strain GH1-13 strongly inhibited the mycelial growth of turf pathogens including different anastomosis groups of R. solani causing brown patch and large patch. R. solani AG2-2(IIIB) hyphae were morphologically changed, and fungal cell death resulted from exposure to the strain GH1-13. In addition, the compatibility of fungicides with the bacterial strain, and the combined application of fungicide azoxystrobin and the strain in brown patch control on creeping bentgrass indicated that the strain could serve as a biocontrol agent. To develop strain-specific detection method, two unique genes from chromosome and plasmid of GH1-13 were found using pan-genome analysis of 364 Bacillus strains. The unique gene from chromosome was successfully detected using both SYBR Green and TaqMan qPCR methods in bacterial DNA or soil DNA samples. This study suggests that application of GH1-13 offers an environmentally friendly approach via reducing fungicide application rates. Furthermore, the developed pipeline of strain-specific detection method could be a useful tool for detecting and studying the dynamics of specific biocontrol agents.

Fungi Associated with Postharvest Diseases of Sweet Potato Storage Roots and In Vitro Antagonistic Assay of Trichoderma harzianum against the Diseases.

Sweet potato is the 11th most important food crop in the world and an excellent source of nutrition. Postharvest diseases were monitored in sweet potato storage roots collected from the local markets in Korea during 2021. Several diseases including Fusarium surface and root rot, charcoal rot, dry rot, and soft rot were observed in the postharvest sweet potatoes. A total of 68 fungal isolates were obtained from the diseased samples, and the isolates were grouped into 8 different fungal colony types. Based on multilocus phylogeny and morphological analysis of 17 representative isolates, the isolates were identified as Fusarium oxysporum, F. ipomoeae, F. solani, Penicillium citrinum, P. rotoruae, Aspergillus wentii, Mucor variicolumellatus (Mu. circinelloides species complex), and Macrophomina phaseolina. F. oxysporum was the predominant pathogen as this is the most common pathogen of sweet potato storage roots causing the surface rot disease, and M. phaseolina caused the most severe disease among the pathogens. Dual culture antagonistic assays were evaluated using Trichoderma harzianum strains CMML20-26 and CMML20-27. The results revealed that the two strains showed strong antifungal activity in different ranges against all tested pathogens. This study provides an understanding of diverse postharvest diseases in sweet potatoes and suggests potential biocontrol agents to manage the diseases. In addition, this is the first report of sweet potato storage root rot diseases caused by A. wentii, and P. rotoruae worldwide.

Plant and Fungal Genome Editing to Enhance Plant Disease Resistance Using the CRISPR/Cas9 System.

Crop production has been substantially reduced by devastating fungal and oomycete pathogens, and these pathogens continue to threaten global food security. Although chemical and cultural controls have been used for crop protection, these involve continuous costs and time and fungicide resistance among plant pathogens has been increasingly reported. The most efficient way to protect crops from plant pathogens is cultivation of disease-resistant cultivars. However, traditional breeding approaches are laborious and time intensive. Recently, the CRISPR/Cas9 system has been utilized to enhance disease resistance among different crops such as rice, cacao, wheat, tomato, and grape. This system allows for precise genome editing of various organisms via RNA-guided DNA endonuclease activity. Beyond genome editing in crops, editing the genomes of fungal and oomycete pathogens can also provide new strategies for plant disease management. This review focuses on the recent studies of plant disease resistance against fungal and oomycete pathogens using the CRISPR/Cas9 system. For long-term plant disease management, the targeting of multiple plant disease resistance mechanisms with CRISPR/Cas9 and insights gained by probing fungal and oomycete genomes with this system will be powerful approaches.

Control of Plant Viral Diseases by CRISPR/Cas9: Resistance Mechanisms, Strategies and Challenges in Food Crops.

Protecting food crops from viral pathogens is a significant challenge for agriculture. An integral approach to genome-editing, known as CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR associated protein 9), is used to produce virus-resistant cultivars. The CRISPR/Cas9 tool is an essential part of modern plant breeding due to its attractive features. Advances in plant breeding programs due to the incorporation of Cas9 have enabled the development of cultivars with heritable resistance to plant viruses. The resistance to viral DNA and RNA is generally provided using the Cas9 endonuclease and sgRNAs (single-guide RNAs) complex, targeting particular virus and host plant genomes by interrupting the viral cleavage or altering the plant host genome, thus reducing the replication ability of the virus. In this review, the CRISPR/Cas9 system and its application to staple food crops resistance against several destructive plant viruses are briefly described. We outline the key findings of recent Cas9 applications, including enhanced virus resistance, genetic mechanisms, research strategies, and challenges in economically important and globally cultivated food crop species. The research outcome of this emerging molecular technology can extend the development of agriculture and food security. We also describe the information gaps and address the unanswered concerns relating to plant viral resistance mediated by CRISPR/Cas9.

First Report of Post-Harvest Tuber Rot of American Groundnut (Apios americana) Caused by Fusarium acuminatum.

Apios americana Medik, commonly known as American groundnut, is a leguminous perennial vine crop native to North America and is cultivated in Japan and Korea (Chu et al. 2019). Its tubers are edible and believed to be very nutritious, especially for women just after childbirth. The tubers also contain secondary metabolites, saponin and genistein, which is good for human health (Ichige et al. 2013). However, the storage of tubers at inappropriate temperatures and humidity levels can cause severe fungal infection, and adversely affect tuber quality. During March and April 2020, a white to pale-orange fungal mycelia were observed on stored American groundnut tubers, with 10 to 15% of seed tubers rotten. Infected tubers were collected, and fungal isolates were isolated on potato dextrose agar (PDA) using the single spore isolation method (Leslie and Summerell 2006). A pure culture (isolate JC20003) was obtained and stored at the Bioenergy Crop Research Institute, NICS, Muan, Republic of Korea. The fungus was cultured on PDA and V8 liquid media for 7 days at 25℃ to observe its morphological characteristics. The length and width of macroconidia ranged from 20.6 to 52.9 µm and 2.9 to 5.1 µm, respectively (n = 30). The microconidia were 8.5 to 14.9 µm and 2.3 to 4.2 µm in length and width, respectively (n = 30). Macroconidia were broadly falcate, strongly septate, 2 to 6 septations with dorsiventral curvature; chlamydospores were formed in chains; and microconidia were fusiform with 0 to 1 septation observed. Genomic DNA of the isolate was extracted using Solgent DNA extraction kit (Solgent, Daejeon, Korea), followed by PCR analysis using the internal transcribed spacer (ITS5/ITS4) and elongation factor (EF-1/EF2) genes (White et al. 1990; O'Donnel 2000). PCR products were sequenced and analyzed to confirm species identity (Yang et al. 2018). These sequences were deposited in GenBank (accession numbers MT703859/ITS and MT731939/EF). BLASTn search analysis showed 100% sequence similarity with Fusarium acuminatum (isolates N-51-1/ITS and WXWH24/EF). Based on morphological and molecular data analysis, the fungus was identified as F. acuminatum (Leslie and Summerell 2006; Marin et al. 2012). Pathogenicity tests were conducted on five tubers inoculated with 5 mm mycelial plugs with three replicates, while a non-mycelial plug served as the control. After 5 days of incubation in plastic containers at 25 °C with high humidity, typical symptoms developed. No symptoms were observed on the control tubers; F. acuminatum was re-isolated from artificially inoculated tubers to complete Koch's postulates. This is the first report on post-harvest tuber rot caused by F. acuminatum in Apios americana.

Occurrence of Sweetpotato (Ipomoea batatas) Wilt and Surface Rot Disease and Determining Resistance of Selected Varieties to the Pathogen in Korea.

Fusarium wilt and Fusarium surface rot caused by Fusarium oxysporum Schltdl are the major diseases of sweetpotato (Ipomoea batatas) and was surveyed in different locations (Cheongju, Heanam, Iksan, Icheon, Kimje, Nonsan, Yeoungam, and Yeoju) in Korea from 2015 to 2017 in the field, after harvesting and in storehouse. The wilt incidence in the early stage represented 17.9%, 5.9%, and 8.3% in 2015, 2016, and 2017, respectively. Samples were collected, and the causal organism was isolated on potato dextrose agar (PDA). Ten pure cultures were stored at the Sweetpotato Research Laboratory, Bioenergy Crop Research Institute, Muan, Korea. Morphological analysis, along with molecular phylogeny of the sequences of internal-transcribed spacer (ITS) and elongation factor 1-α (EF-1α) genes and their combined phylogenetic analysis, confirmed the isolates as the Fusarium oxysporum Schltdl. Pathogenicity tests were conducted on sweetpotato stems, and storage roots by artificially inoculation methods, and the most virulent isolate was selected as SPL18019. A rapid screening method on 21 selected varieties for resistant variety selection was applied on stems. The Pungwanmi was found resistant to Fusarium wilt, whereas Annobeni was the most susceptible. On the other hand, six varieties were used to test surface rot resistance, and Yulmi and Yesumi were resistant and susceptible, respectively, to Fusarium surface rot.

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.

Phylogenetic Placement and Morphological Characterization of Sclerotium rolfsii (Teleomorph: Athelia rolfsii) Associated with Blight Disease of Ipomoea batatas in Korea.

In this study, we aimed to characterize fungal samples from necrotic lesions on collar regions observed in different sweetpotato growing regions during 2015 and 2016 in Korea. Sclerotia appeared on the root zone soil surface, and white dense mycelia were observed. At the later stages of infection, mother roots quickly rotted, and large areas of the plants were destroyed. The disease occurrence was monitored at 45 and 84 farms, and 11.8% and 6.8% of the land areas were found to be infected in 2015 and 2016, respectively. Fungi were isolated from disease samples, and 36 strains were preserved. Based on the cultural and morphological characteristics of colonies, the isolates resembled the reference strain of Sclerotium rolfsii. Representative strains were identified as S. rolfsii (teleomorph: Athelia rolfsii) based on phylogenetic analysis of the internal transcribed spacer and large subunit genes along with morphological observations. To test the pathogenicity, sweetpotato storage roots were inoculated with different S. rolfsii strains. 'Yulmi' variety displayed the highest disease incidence, whereas 'Pungwonmi' resulted in the least. These findings suggested that morphological characteristics and molecular phylogenetic analysis were useful for identification of S. rolfsii.

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.

Characterization of Paecilomyces variotii and Talaromyces amestolkiae in Korea Based on the Morphological Characteristics and Multigene Phylogenetic Analyses.

During fungal diversity surveys of the order Eurotiales in Korea, two fungal strains, EML-DG33-1 and EML-NCP50, were isolated from samples of rat dung and fig tree leaf collected at a garden located in Gwangju in 2014. To complete the National Species List of Korea, it is a prerequisite to verify whether many questionable species, which were previously recorded but not confirmed, indeed present in Korea. Herein, the isolates were confirmed as undescribed species, Paecilomyces variotii and Talaromyces amestolkiae based on the combination of morphological and phylogenetic analyses of multigenes including the rDNA internal transcribed spacer, ß-tubulin, and RNA polymerase II subunit 2.

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.

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.

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.

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.