First Report of Rust Disease on Alcea rosea Caused by Puccinia modiolae in Korea.

Alcea rosea, in the family Malvaceae, is a biennial plant native to China and is grown typically for gardening in Korea (Lee 2003). Seven microcyclic Puccinia species have been reported on A. rosea: P. heterogenea, P. heterospora, P. lobata, P. malvacearum, P. platyspora, P. sherardiana, and P. modiolae (Demers et al. 2015; Aime and Abbasi 2018). In early May 2022, characteristic symptoms of rust were observed on four of ten seedlings of A. rosea purchased at a wholesale nursery (36°50'19.8″N, 128°55'28.7″E) in Bonghwa, Korea. Rust spots were present on almost 90% of the 1,000 seedlings of A. rosea in that nursery during our survey in late May. Through a distribution survey from June to July 2022, similar symptomatic leaves were additionally collected from the leaves of A. rosea grown in gardens at five sites in Gimcheon (two sites), Gumi (one), Seongju (one), and Busan (one). Spots were yellow-orange the center surrounded by chlorotic haloes on the adaxial leaf surface, and reddish-brown or dark brown pustules on the abaxial leaf surface. Over time, the spots enlarged and coalesced, causing the decay of large sections of the leaves, and heavily infected leaves fell early. Spermogonia, produced at the center of the chlorotic spot on the adaxial leaf surface, were subepidermal, obovoid, and 113.2-164.5 × 97.6-153.3 µm in size. Telia were reddish-brown to dark brown, round, mostly grouped, 0.28-0.61 mm in diameter, and mainly formed on the abaxial leaf surface but sometimes on the adaxial leaf surface also. Teliospores were two-celled, but rarely one- or three-celled, and were fusoid and 37. 6-110 × 12.4-21.5 µm in size; the wall was yellowish or almost colorless, smooth, 1.2-2.6 µm thick at the sides, and up to 7.4 µm thick at the apex. The morphological characteristics were similar to those of P. modiolae, although the teliospores in our study were longer than those observed by Aime and Abbasi (2018). For phylogenetic analysis, genomic DNA was extracted from the teliospores of each regional specimen. Partial 18S, internal transcribed spacer (ITS), and partial 28S sequences were amplified using primers NS1, ITS4, ITS5, and LR11. The PCR products were sequenced (Celemics, Seoul, Korea) and deposited in GenBank. The ITS-partial large subunit (LSU) sequence and 28S sequences had 100% homology with other P. modiolae sequences deposited in GenBank (accession numbers are shown in Fig. 2). In the phylogenetic trees of the ITS and LSU sequences, the isolates collected in this study were grouped with the reference sequences of P. modiolae, including the Korean isolate (ON631218) recently reported on Malva verticillata by Lee et al. (2022). For the pathogenicity test, the teliospores with germinating basidiospores were suspended in sterile distilled water and smeared on the upper surface of asymptomatic A. rosea leaves in August. The inoculated plants were sprayed with distilled water and kept in the dark with saturated moisture for 24 h in an isolated glass house of the Animal & Plant Quarantine Agent. After 2 weeks, typical rust spots and telia of P. modiolae were observed on the leaves of the inoculated plants, but not in the control plants, which were only sprayed with distilled, sterilized water and otherwise treated similarly to the inoculated plants. The results of this study show that the casual fungus is P. modiolae, which has been commonly found in A. rosea in Korea. To the best of our knowledge, this is the first report of P. modiolae in A. rosea in Korea.

Revisiting Rhytisma lonicericola: Morphological Characterization and Molecular Phylogenetic Analysis.

Rhytisma lonicericola was identified as a tar spot fungus on Lonicera sp. in 1902, and has since been recorded on several species of Lonicera in China, Japan, and Korea. Most of the previous records of R. lonicericola have been based on a list of disease occurrences in the absence of any formal morphological identification or molecular analyses. Using six newly obtained specimens collected in the past 2 years, we confirmed the tar spot fungus found on L. japonica in Korea as R. lonicericola based on morphological examinations and molecular phylogenetic analyses. This fungus was distinguished from R. xylostei, another tar spot fungus on Lonicera, by ascospore size and geographical distributions. We present detailed mycological information and, for the first time, DNA sequence data useful for the identification of R. lonicericola.

Kordyana commelinae Associated with White Smut-like Disease on Commelina communis and C. minor in Korea.

A fungus of the genus Kordyana, found on leaves of Commelina communis and C. minor exhibiting white smut-like symptoms, was identified as Kordyana commelinae based on morphological characteristics and two rDNA sequence analyses. We report the novel occurrence of the genus Kordyana in Korea and the association of K. commelinae with the host plant species. As well, we provide the necessary mycological information to resolve species delimitation and taxonomic problems of Kordyana.

Macruropyxis fraxini on Fraxinus rhynchophylla: Confirmation in the Korean Peninsula after 82 Years and the First Record in South Korea.

Macruropyxis fraxini has been recorded on several species of Fraxinus in China, Japan, Russia (Far East), and North Korea since its first recorded observation as a rust fungus on F. rhynchophylla in Jilin, China, in 1899. In the Korean Peninsula, the rust fungus was first recorded on F. rhynchophylla in 1935, based on four specimens collected at Mt. Kumgangsan, Gangwondo Province, in the North Korean territory. We confirmed this rust in the Korean Peninsula after 82 years. The rust fungus was identified based on morphological characteristics and a molecular phylogenetic analysis. This is the first record of M. fraxini in South Korea.

Transcriptome analyses of the ginseng root rot pathogens Cylindrocarpon destructans and Fusarium solani to identify radicicol resistance mechanisms.

BACKGROUND: The ascomycete fungi Cylindrocarpon destructans (Cd) and Fusarium solani (Fs) cause ginseng root rot and significantly reduce the quality and yield of ginseng. Cd produces the secondary metabolite radicicol, which targets the molecular chaperone Hsp90. Fs is resistant to radicicol, whereas other fungal genera associated with ginseng disease are sensitive to it. Radicicol resistance mechanisms have not yet been elucidated. METHODS: Transcriptome analyses of Fs and Cd mycelia treated with or without radicicol were conducted using RNA-seq. All of the differentially expressed genes (DEGs) were functionally annotated using the Fusarium graminearum transcript database. In addition, deletions of two transporter genes identified by RNA-seq were created to confirm their contributions to radicicol resistance. RESULTS: Treatment with radicicol resulted in upregulation of chitin synthase and cell wall integrity genes in Fs and upregulation of nicotinamide adenine dinucleotide dehydrogenase and sugar transporter genes in Cd. Genes encoding an ATP-binding cassette transporter, an aflatoxin efflux pump, ammonium permease 1 (mep1), and nitrilase were differentially expressed in both Fs and Cd. Among these four genes, only the ABC transporter was upregulated in both Fs and Cd. The aflatoxin efflux pump and mep1 were upregulated in Cd, but downregulated in Fs, whereas nitrilase was downregulated in both Fs and Cd. CONCLUSION: The transcriptome analyses suggested radicicol resistance pathways, and deletions of the transporter genes indicated that they contribute to radicicol resistance.

Survival Factor Gene FgSvf1 Is Required for Normal Growth and Stress Resistance in Fusarium graminearum.

Survival factor 1 (Svf1) is a protein involved in cell survival pathways. In Saccharomyces cerevisiae, Svf1 is required for the diauxic growth shift and survival under stress conditions. In this study, we characterized the role of FgSvf1, the Svf1 homolog in the homothallic ascomycete fungus Fusarium graminearum. In the FgSvf1 deletion mutant, conidial germination was delayed, vegetative growth was reduced, and pathogenicity was completely abolished. Although the FgSvf1 deletion mutant produced perithecia, the normal maturation of ascospore was dismissed in deletion mutant. The FgSvf1 deletion mutant also showed reduced resistance to osmotic, fungicide, and cold stress and reduced sensitivity to oxidative stress when compared to the wild-type strain. In addition, we showed that FgSvf1 affects glycolysis, which results in the abnormal vegetative growth in the FgSvf1 deletion mutant. Further, intracellular reactive oxygen species (ROS) accumulated in the FgSvf1 deletion mutant, and this accumulated ROS might be related to the reduced sensitivity to oxidative stress and the reduced resistance to cold stress and fungicide stress. Overall, understanding the role of FgSvf1 in F. graminearum provides a new target to control F. graminearum infections in fields.

Stress Tolerance and Virulence-Related Roles of Lipopolysaccharide in Burkholderia glumae.

The lipopolysaccharide (LPS) composed of lipid A, core, and O-antigen is the fundamental constituent of the outer membrane in gram-negative bacteria. This study was conducted to investigate the roles of LPS in Burkholderia glumae, the phytopathogen causing bacterial panicle blight and seedling rot in rice. To study the roles of the core oligosaccharide (OS) and the O-antigen region, mutant strains targeting the waaC and the wbiFGHI genes were generated. The LPS profile was greatly affected by disruption of the waaC gene and slight reductions were observed in the O-antigen region following wbiFGHI deletions. The results indicated that disruption in the core OS biosynthesis-related gene, waaC, was associated with increased sensitivity to environmental stress conditions including acidic, osmotic, saline, and detergent stress, and to polymyxin B. Moreover, significant impairment in the swimming and swarming motility and attenuation of bacterial virulence to rice were also observed in the waaC-defective mutant. The motility and virulence of O-antigen mutants defective in any gene of the wbiFGHI operon, were not significantly different from the wild-type except in slight decrease in swimming and swarming motility with wbiH deletion. Altogether, the results of present study indicated that the LPS, particularly the core OS region, is required for tolerance to environmental stress and full virulence in B. glumae. To our knowledge, this is the first functional study of LPS in a plant pathogenic Burkholderia sp. and presents a step forward toward full understanding of B. glumae pathogenesis.

Efficacy of Diphenyleneiodonium Chloride (DPIC) Against Diverse Plant Pathogens.

Many of the fungicides and antibiotics currently available against plant pathogens are of limited use due to the emergence of resistant strains. In this study, we examined the effects of diphenyleneiodonium chloride (DPIC), an inhibitor of the superoxide producing enzyme NADPH oxidase, against fungal and bacterial plant pathogens. We found that DPIC inhibits fungal spore germination and bacterial cell proliferation. In addition, we demonstrated the potent antibacterial activity of DPIC using rice heads infected with the bacterial pathogen Burkholderia glumae which causes bacterial panicle blight (BPB). We found that treatment with DPIC reduced BPB when applied during the initial flowering stage of the rice heads. These results suggest that DPIC could serve as a new and useful antimicrobial agent in agriculture.

Functional characterization of a putative DNA methyltransferase, EadM, in Xanthomonas axonopodis pv. glycines by proteomic and phenotypic analyses.

Xanthomonas axonopodis pv. glycines (Xag) is a phytopathogenic bacterium causing bacterial pustule disease in soybean. Functions of DNA methyltransferases have been characterized in animal pathogenic bacteria, but are poorly understood in plant pathogens. Here, we report that functions of a putative DNA methyltransferase, EadM, in Xag. An EadM-overexpressing strain, Xag(EadM), was less virulent than the wild-type carrying an empty vector, Xag(EV). Interestingly, the viable cell numbers of Xag(EadM) were much lower (10-fold) than those of Xag(EV) at the same optical density. Comparative proteomic analysis revealed that proteins involved in cell wall/membrane/envelope and iron-transport were more abundant. Based on proteomic analysis we carried out diverse phenotypic assays. Scanning electron microscopy revealed abnormal bacterial envelopes in Xag(EadM). Additionally, Xag(EadM) showed decreased stress tolerance against ciprofloxacin and sorbitol, but enhanced resistance to desiccation. Exopolysaccharide production in Xag(EadM) was also decreased. Production of siderophores, which are iron-chelators, was much higher in Xag(EadM). As in Xag, Escherichia coli expressing EadM showed significantly reduced (1000-fold) viable cell numbers at the same optical density. Thus, EadM is associated with virulence, envelope biogenesis, stress tolerance, exopolysaccharide production, and siderophore production. Our results provide valuable and fundamental information regarding DNA methyltransferase functions and their related cellular mechanisms in plant pathogenic bacteria.

Cooperative interactions between seed-borne bacterial and air-borne fungal pathogens on rice.

Bacterial-fungal interactions are widely found in distinct environments and contribute to ecosystem processes. Previous studies of these interactions have mostly been performed in soil, and only limited studies of aerial plant tissues have been conducted. Here we show that a seed-borne plant pathogenic bacterium, Burkholderia glumae (Bg), and an air-borne plant pathogenic fungus, Fusarium graminearum (Fg), interact to promote bacterial survival, bacterial and fungal dispersal, and disease progression on rice plants, despite the production of antifungal toxoflavin by Bg. We perform assays of toxoflavin sensitivity, RNA-seq analyses, lipid staining and measures of triacylglyceride content to show that triacylglycerides containing linolenic acid mediate resistance to reactive oxygen species that are generated in response to toxoflavin in Fg. As a result, Bg is able to physically attach to Fg to achieve rapid and expansive dispersal to enhance disease severity.

Identification of Genes Related to Fungicide Resistance in Fusarium fujikuroi.

We identified two genes related to fungicide resistance in Fusarium fujikuroi through random mutagenesis. Targeted gene deletions showed that survival factor 1 deletion resulted in higher sensitivity to fungicides, while deletion of the gene encoding F-box/WD-repeat protein increased resistance, suggesting that the genes affect fungicide resistance in different ways.

Transcriptome analyses to understand effects of the Fusarium deoxynivalenol and nivalenol mycotoxins on Escherichia coli.

Fusarium spp. cause many diseases in farming systems and can produce diverse mycotoxins that can easily impact humans and animals through the ingestion of food and feed. Among these mycotoxins, deoxynivalenol (DON) and nivalenol (NIV) are considered the most important hazards because they can rapidly diffuse into cells and block eukaryotic ribosomes, leading to inhibition of the translation system. Conversely, the effects of DON and NIV mycotoxins on bacteria remain unclear. We employed RNA-seq technology to obtain information regarding the biological responses of bacteria and putative bacterial mechanisms of resistance to DON and NIV mycotoxins. Most differentially expressed genes down-regulated in response to these mycotoxins were commonly involved in phenylalanine metabolism, glyoxylate cycle, and cytochrome o ubiquinol oxidase systems. In addition, we generated an overall network of 1028 up-regulated genes to identify core genes under DON and NIV conditions. The results of our study provide a snapshot view of the transcriptome of Escherichia coli K-12 under DON and NIV conditions. Furthermore, the information provided herein will be useful for development of methods to detect DON and NIV.

A Putative Transcription Factor pcs1 Positively Regulates Both Conidiation and Sexual Reproduction in the Cereal Pathogen Fusarium graminearum.

The plant pathogen Fusarium graminearum causes Fusarium head blight in cereal crops and produces mycotoxins that are harmful to animals and humans. For the initiation and spread of disease, asexual and sexual reproduction is required. Therefore, studies on fungal reproduction contribute to the development of new methods to control and maintain the fungal population. Screening a previously generated transcription factor mutant collection, we identified one putative C2H2 zinc-finger transcription factor, pcs1, which is required for both sexual and asexual reproduction. Deleting pcs1 in F. graminearum resulted in a dramatic reduction in conidial production and a complete loss of sexual reproduction. The pathways and gene ontology of pcs1-dependent genes from microarray experiments showed that several G-protein related pathways, oxidase activity, ribosome biogenesis, and RNA binding and processing were highly enriched, suggesting that pcs1 is involved in several different biological processes. Further, overexpression of pcs1 increased conidial production and resulted in earlier maturation of ascospores compared to the wild-type strain. Additionally, the vegetative growth of the overexpression mutants was decreased in nutrient-rich conditions but was not different from the wild-type strain in nutrient-poor conditions. Overall, we discovered that the pcs1 transcription factor positively regulates both conidiation and sexual reproduction and confers nutrient condition-dependent vegetative growth.

Microcyle conidiation in filamentous fungi.

The typical life cycle of filamentous fungi commonly involves asexual sporulation after vegetative growth in response to environmental factors. The production of asexual spores is critical in the life cycle of most filamentous fungi. Normally, conidia are produced from vegetative hyphae (termed mycelia). However, fungal species subjected to stress conditions exhibit an extremely simplified asexual life cycle, in which the conidia that germinate directly generate further conidia, without forming mycelia. This phenomenon has been termed as microcycle conidiation, and to date has been reported in more than 100 fungal species. In this review, first, we present the morphological properties of fungi during microcycle conidiation, and divide microcycle conidiation into four simple categories, even though fungal species exhibit a wide variety of morphological differences during microcycle conidiogenesis. Second, we describe the factors that influence microcycle conidiation in various fungal species, and present recent genetic studies that have identified the genes responsible for this process. Finally, we discuss the biological meaning and application of microcycle conidiation.

Biological Efficacy of Streptomyces sp. Strain BN1 against the Cereal Head Blight Pathogen Fusarium graminearum.

Fusarium head blight (FHB) caused by the filamentous fungus Fusarium graminearum is one of the most severe diseases threatening the production of small grains. Infected grains are often contaminated with mycotoxins such as zearalenone and trichothecences. During survey of contamination by FHB in rice grains, we found a bacterial isolate, designated as BN1, antagonistic to F. graminearum. The strain BN1 had branching vegetative hyphae and spores, and its aerial hyphae often had long, straight filaments bearing spores. The 16S rRNA gene of BN1 had 100% sequence identity with those found in several Streptomyces species. Phylogenetic analysis of ITS regions showed that BN1 grouped with S. sampsonii with 77% bootstrap value, suggesting that BN1 was not a known Streptomyces species. In addition, the efficacy of the BN1 strain against F. graminearum strains was tested both in vitro and in vivo. Wheat seedling length was significantly decreased by F. graminearum infection. However, this effect was mitigated when wheat seeds were treated with BN1 spore suspension prior to F. graminearum infection. BN1 also significantly decreased FHB severity when it was sprayed onto wheat heads, whereas BN1 was not effective when wheat heads were point inoculated. These results suggest that spraying of BN1 spores onto wheat heads during the wheat flowering season can be efficient for plant protection. Mechanistic studies on the antagonistic effect of BN1 against F. graminearum remain to be analyzed.

Development of a Selective Medium for the Fungal Pathogen Fusarium graminearum Using Toxoflavin Produced by the Bacterial Pathogen Burkholderia glumae.

The ascomycete fungus Fusarium graminearum is a major causal agent for Fusarium head blight in cereals and produces mycotoxins such as trichothecenes and zearalenone. Isolation of the fungal strains from air or cereals can be hampered by various other airborne fungal pathogens and saprophytic fungi. In this study, we developed a selective medium specific to F. graminearum using toxoflavin produced by the bacterial pathogen Burkholderia glumae. F. graminearum was resistant to toxoflavin, while other fungi were sensitive to this toxin. Supplementing toxoflavin into medium enhanced the isolation of F. graminearum from rice grains by suppressing the growth of saprophytic fungal species. In addition, a medium with or without toxoflavin exposed to wheat fields for 1 h had 84% or 25%, respectively, of colonies identified as F. graminearum. This selection medium provides an efficient tool for isolating F. graminearum, and can be adopted by research groups working on genetics and disease forecasting.