The First Report of Septobasidium sp. Occurrence on Phyllostachys reticulata (Rupr.) K. Koch in South Korea and Molecular Phylogenetic Analysis
Article information
Abstract
In May 2024, a disease resembling felt disease was observed on the stems and branches of Phyllostachys reticulata, with brown coloration appearing on the infected areas, and samples were collected in Byeongbawi Rock and its surroundings, Gochang-gun, Jeonbuk-do. Phylogenetic analysis was performed based on internal transcribed spacer rDNA sequences obtained from the basidiomata. This fungus was classified as Septobasidium sp., showing 97.84% similarity to the Septobasidium sp. strain isolated from the Schisandra chinensis tree. The structural characteristics of the layers of mycelia mat were compared through detailed microscopic examination. Interestingly, while the mats layer structure for commonly known blight diseases are typically single-layered, the basidiomata observed on P. reticulata exhibited a 1 to 3 layered structure. This study represents the first record of felt disease on P. reticulata in Korea, providing a comprehensive report on its morphological and molecular characteristics.
Introduction
Felt disease is known to affect stems, branches, and leaves of various tree species, with scale insects parasitizing within the mycelial mat and establishing a symbiotic relationship (Henk, 2005; Lu and Guo, 2011). The order Septobasidiales, which causes such felt diseases, is taxonomically classified within the Pucciniomycetes, a subclass of plant pathogenic fungi that includes the well-known rust fungi (Bauer et al., 2006; Swann et al., 2001). Specifically, Septobasidium sp., which causes felt disease, proliferates by consuming the exudates of scale insects and forms a symbiotic relationship with them (Couch, 1931). Symbiosis is generally an advanced association or relationship between organisms of different species in which one or both of the interacting individuals benefit in some way (Elkhateeb et al., 2021). Scale insects feed on plant sap through their sucking mouthparts while the fungi obtain their nutrition from the scale insects. This represents a parasitic/host relationship that appears to be beneficial solely to the fungi, but the scale insects also gain an advantage like a protective shield (Henk, 2005; Henk and Vilgalys, 2007). While the occurrence of felt disease does not directly damage the trees, it can lead to secondary harm as the density of the symbiotic scale insects increases. These insects may then feed on the tree vascular fluid, potentially causing tree dieback and other related damage (Alexopoulos and Mims, 1979; Choi et al., 2016).
Currently, felt disease in Korea has been reported on 14 broadleaf tree species, including Septobasidium bogoriense Pat. affecting Prunus mume (Siebold) Siebold & Zucc tree [Chinese plum], and S. tanakae (Miyabe) Boedijn & B.A. Steinm. affecting trees of Juglans regia L. [Walnut], Morus alba L. [Mulberry], Carpinus laxiflora (Siebold & Zucc.) Blume [Loose-flower hornbeam], Quercus serrata Murray [Jolcham oak], Prunus salicina Lindl. [Jadu cherry], Paulownia coreana Uyeki [Paulownia], Actinidia arguta (Siebold & Zucc.) Planch. Ex Miq. [Kiwi], Euonymus japonicus Thunb [Evergreen spin-dletree], Prunus serrulata Lindl. f. spontanea (Maxim.) Chin S. Chang [Oriental flowering cherry], and Castanea crenata Siebold & Zucc [Castanea] (Korean Society of Plant Pathology, 2023). However, there is a complete lack of information regarding the molecular and genetic diversity. Recent studies have reported blight diseases caused by Septobasidium sp. on Schisandra chinensis (Turcz.) Baill. (Omiza), Diospyros kaki Thunb. (Persimmon), and Castanopsis sieboldii (Makino) Hatus. ex T. Yamaz. & Mashiba (Siebold's chinquapin), based on internal transcribed spacer (ITS) rDNA gene sequences (Choi et al., 2016; Ha et al., 2021; Lee et al., 2023).
In South Korea, the bamboo subfamily (Bambusoideae) predominantly consists of Phyllostachys reticulata (Rupr.) K. Koch, Sasa coreana Nakai, and Sasamorpha borealis (Hack.) Makino, which are among the most commonly occurring species in the region (Choi et al., 2017; Kim and Choi, 2024). Among them, the major plant diseases affecting P. reticulata include smut disease caused by Ustilago shiraiana Henn (Xiqiao, 1988), rust disease caused by Puccinia sp. (Yu et al., 2020), bamboo witches’ broom caused by Candidatus Phytoplasma asteris (Jung et al., 2006), and the bamboo flowering phenomenon resulting from physiological stress (Wu et al., 2023). In May 2024, light brown mycelial mats were observed on the stems and branches of P. reticulata, which is native to Byeongbawi Rock in Gochang-gun, Jeollabuk-do, causing aesthetic damage. This study aims to identify the organism responsible for this disease, representing the first report of its kind of the disease and its causal organism in South Korea.
Materials and Methods
Samples collection and external morphological characteristics.
Branch and stem samples with fungal mats were collected in May 2024. The collected samples were immediately placed in an icebox and transported to the laboratory within 2 hr. The morphological characteristics of the mycelial mats were observed by excising a portion of the bark with a sharp knife. Morphological characteristics were observed using an optical microscope (DM2500; Leica, Wetzlar, Germany) equipped with a scientific CMOS camera, and the size of hyphal mat layers from 13 randomly selected samples was measured using Mosaic 2.4 software (Tucsen, Fuzhou, China).
Genomic DNA extraction, polymerase chain reaction (PCR), and PCR-restriction fragment length polymorphism (RFLP).
Genomic DNA was extracted from newly growing mycelial mats treated under 80% humidity. After incubation in a controlled environment, newly formed basidiomata was aseptically excised. DNA was extracted according to the manufacturer's instructions using the ZR Fungal/Bacterial DNA MiniPrep Kit (Zymo Research Co., Irvine, CA, USA). To amplify the ITS region, including ITS1, 5.8S, and ITS2 genes, the primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) were used (White et al., 1990). DNA at a concentration of approximately 50 ng/μl was mixed with 10 pmole of each primer for the respective gene regions, and the final volume was adjusted to 25 μl with ster-ile distilled water, using EmeraldAmp GT PCR Master Mix (2× Pre-mix) (Takara Co., Tokyo, Japan). PCR for each gene region was performed with an initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 96°C for 30 sec, annealing at 57°C for 30 sec (for ITS1/ITS4), and extension at 72°C for 1 min. A final extension was carried out at 72°C for 10 min to complete the PCR process. Each PCR product (6 μl) was mixed with Dyne Loading STAR (1.2 μl; Dyne Bio Co., Seongnam, Korea) and subjected to electrophoresis on a 1.0% agarose gel at 100 V for 30 min. DNA bands were visualized under a UV transilluminator to confirm the presence of a single band. Additionally, the ITS rDNA gene amplification products were analyzed for molecular diversity using RFLP analysis with restriction enzymes Sau3A I, Alu I, and Rsa I (Ta-KaRa, Otsu, Japan). PCR-RFLP analysis were mixed with Dyne Loading STAR 1 μl and RFLP products 5 μl, loaded onto a 2.0% agarose gel, and electrophoresed at 100 V for 45 minutes. DNA band patterns were visualized using a UV transilluminator thereafter.
Sequencing and phylogenetic analysis.
The PCR products amplified from the ITS rDNA gene were purified using a PCR purification kit (Bioneer Co., Daejeon, Korea) and then subjected to direct sequencing by an external sequencing service provider (Macrogen Co., Seoul, Korea). The obtained sequences were analyzed and assembled using MEGA 11.0 (Tamura et al., 2021) to check for errors. Subsequently, sequences were compared with multiple gene sequences available in the National Center for Biotechnology Information (NCBI). A phylogenetic tree was constructed using the neighbor-joining method and bootstrap analysis with 1,000 replicates in the MEGA 11.0 program.
Results
Morphological characteristics.
The basidiomata observed in the forested area around Byeongbawi Rock in May appeared light brown or brown, and was noted to be slightly darker brown in July, 2 months later (Fig. 1). On the P. reticulata stems, the mycelial mats appeared as thin, rounded patches measuring 2.8–4.1×3.3–5.2 cm, adhering to the bark. In contrast, on the branches, the basidiomata exhibited polymorphic shapes and was formed on each branch, with dimensions ranging from 1.5–3.8×2.5–6.3 cm. Although other tree species were also native to the surrounding area, basidiomata was observed exclusively on P. bambusoides, indicating host specificity. The velvet-like mycelial mats initially had a smooth surface with new its hyphae growing at the edges, which gradually turned brown. In May, the surface of the mats was smooth and plump, but by July, the mats exhibited wrinkling and appeared somewhat desiccated. Microscopic examination revealed the presence of scale insects beneath the basidiomata. Measurement of the mycelial size from 13 randomly selected samples showed dimensions ranging from 1.2–4.3×1.8–6.4 μm. The basidiomata mats were categorized into three types: those consisting of either one, two or three layers (Fig. 2). The size of the mats, subiculum, and pillars, as well as the supporting structures, varied among the samples (Table 1). Additionally, the size of the mats ranged from a minimum of 360–530 μm to a maximum of 1,055–1,060 μm, regardless of the number of layers. Notably, the hyphae were observed exclusively on the outer surface of the bark and were not present on the inner surface. The basidiomata observed on the P. reticulata were morphologically similar to those of the genus Septobasidium (Couch, 1938; Gómez and Henk, 2004).
Occurrence of felt disease on Phyllostachys reticulata caused by Septobasidium sp. (A) Basidiomata formed on the stems. (B, C) Variation in basidiomata color by period (B: May, C: July). (D, E) Close-up observation of basidiomata on stems and branches. (F) Basidiomata newly grown on branches. (G) Presence of scale insects around basidiomata.
Basidiomata layer structure and morphological characteristics. (A) Hyphae of basidiomata. (B-D) Basidiomata layers exhibiting structural diversity, ranging from a one-layer structure to a three-layer structure.
Basidiomata layer structure of Septobasidium isolate causing Phyllostachys reticulata felt disease
Analysis of molecular and genetic diversity.
To identify the fungus occurring on P. reticulata stems and branches, the ITS rDNA gene region was amplified using PCR. As a result, all three isolates produced a single band of approximately 550–600 bp. RFLP analysis of the ITS rDNA amplification products using restriction enzymes Sau3A I, Alu I, and Rsa I revealed identical band patterns across all three isolates, indicating that the samples belong to the same strain. Sequence analysis of the ITS rDNA gene revealed no variation among the JB-sep-PB01, -PB02, and -PB03 isolates. The sequences were corrected and submitted to the NCBI GenBank (accession numbers: PQ270468− PQ270470). Additionally, a comparison of the ITS rDNA gene sequences with those of Septobasidium species available in the NCBI database showed a 97.84% similarity to the Septobasidium sp. strain isolated from S. chinensis tree in South Korea (HQ267956), indicating the closest phylogenetic relationship (Fig. 3). The sequences also showed a 97.13% similarity to the uncultured Septobasidiaceae strain (MH005909). Additionally, the similarity was 93.30% to the S. pallidum strain (MG231816) and 93.07% to the Septobasidium sp. strain (PP532761). The sequences showed a 90.20% similarity to the Septobasidium sp. strain (OR898810) associated with the felt disease of C. sieboldii tree in South Korea (Lee et al., 2023), an 89.11% similarity to the Septobasidium sp. strain (HQ267959) causing S. chinensis tree felt disease (Choi et al., 2016), and an 88.24% similarity to the Septobasidium sp. strain (MW843565) associated with D. kaki felt disease. Consequently, the basidiomata isolated from P. reticulata is tentatively classified as Septobasidium sp.
Phylogenetic relationship of Septobasidium based on an alignment of internal transcribed spacer (ITS) nucleotide sequences. Analysis was performed using MEGA 11.0 software with the neighbor-joining method and 1,000 bootstrap replicates for validation. The scale bar refers to a phylogenetic distance of 0.02 nucleotide substitutions per site.
Discussion
The felt disease occurring on P. reticulata in the Byeongbawi Rock area is tentatively classified as Septobasidium sp. based on morphological characteristics observed through microscopic examination, PCR-RFLP, and ITS rDNA gene sequence analysis. While most Septobasidiaceae species exhibit mats consisting of a single layer of basidiomata (Choi et al., 2016; Henk and Vilgalys, 2007; Lu and Guo, 2011; Ma et al., 2019), the Septobasidium observed on P. reticulata is characterized by having one, two or three layers of basidiomata, representing a significant morphological difference. Further research is needed to explore the implications of the number of basidiomata layers. Felt disease is closely associated with a symbiotic relationship with scale insects (Couch, 1938; Henk and Vilgalys, 2007). While the damage to the plant is mentioned as felt disease caused by Septobasidium species, the actual damage is considered more due to sap-sucking damage from insects rather than fungal parasitism (Alexopoulos and Mims, 1979). The Septobasidium basidiomata occurring on P. reticulata was observed to form exclusively on the outer surface of the its bark, without penetrating into the inner bark. This suggests that the damage to the plant is attributed to the feeding activities of scale insects rather than direct damage by the fungi. Similarly, the basidiomata observed in May appeared somewhat swollen, while by July it had dried out slightly. In 2023, Septobasidium sp. causing felt disease on C. sieboldii tree in Jeju Island was reported to exhibit vertical cracking of the basidiomata mats as temperatures rise (Lee et al., 2023). This observation is consistent with the characteristics of felt disease and the life cycle of scale insects, indicating a need for further research into this relationship. Although over 200 species of felt disease have been classified to date, most have not undergone ITS gene region sequence analysis (Couch, 1938; Gómez and Kisimova-Horovitz, 2001; Henk, 2005; Li and Guo, 2013). Additionally, the number of nucleotide sequences registered in the NCBI database for Septobasidium is significantly lower compared to other fungi. Furthermore, the lack of PCR-RFLP analysis for Septobasidium has made it challenging to distinguish molecular diversity among species. In South Korea, strains causing felt disease on S. chinensis tree, D. kaki tree, and C. sieboldii tree have been tentatively classified as Septobasidium sp. based on ITS rDNA gene sequences. Among these, the Septobasidium sp. strain causing felt disease on S. chinensis tree reported in 2016 shows 89.11% similarity to the strain isolated from P. reticulata (HQ267959) in this study. However, the Septobasidium sp. strain (HQ267956), which is registered in the NCBI database but not reported in the literature, shows 97.84% similarity. This suggests the presence of different two Septobasidium sp. strains on the same host, indicating the need for further research. The felt disease has been reported on 14 species of broadleaf host plants in South Korea, including S. bogoriense Pat., S. tanakae (Miyabe) Boedijn & B.A. Steinm., and Septobasidium sp. However, the felt disease identified on P. reticulata in this study, based on ITS rDNA gene analysis, PCR-RFLP analysis, and hyphal structure examination, has been determined to be a new type of Septobasidium sp. causing felt disease in Korea. This represents the first report of felt disease on P. reticulata.
Notes
Conflicts of Interest
No potential conflict of interest relevant to this article was reported.
Acknowledgments
We are grateful to Hyeong-Kee Park, Director of Jeonnam Tree Hospital, for his significant assistance in the preparation of this manuscript.