Integration of volatile and non-volatile metabolite profile, and in vitro digestion reveals the differences between different preparation methods on physico-chemical and biological properties of Gastrodia elata.

Gastrodia elata Blume (G. elata) is a traditional medicinal and edible plant whose quality is significantly influenced by post-harvest processing. To obtain an optimal post-harvest processing method for G. elata, this study employed sensory evaluation, scanning electron microscopy (SEM), gas chromatography-ion mobility spectrometry (GC-IMS), and non-targeted metabolomics, in conjunction with an in vitro digestion model, to assess the impact of different processing and drying methods on the quality of G. elata. The findings showed that the steam treatment followed by heat pump drying resulted in the highest levels of total phenols, total flavonoids, and polysaccharides in G. elata, and caused more pronounced damage to its microstructure. This treatment also maintained the highest antioxidant activities and optimal acetylcholinesterase (AChE) inhibition capacity throughout in vitro digestion, meanwhile, effectively eliminating the unpleasant odor and achieving the highest sensory scores. Furthermore, non-targeted metabolomic analysis revealed noteworthy alterations in the metabolite profile of G. elata, mainly related to purine metabolism and the biosynthesis of amino acids pathways. This study provides valuable insights into the post-harvest processing of G. elata.

Characteristics and functions of volatile organic compounds in the tripartite symbiotic system of Gastrodia elata-Armillaria gallica-Rahnella aceris HPDA25.

Tripartite interactions among plants, fungi, and bacteria are critical for maintaining plant growth and fitness, and volatile organic compounds (VOCs) play a significant role in these interactions. However, the functions of VOCs within the niche of mycoheterotrophic plants, which represent unique types of interactions, remain poorly understood. Gastrodia elata, a mycoheterotrophic orchid species, forms a symbiotic relationship with specific Armillaria species, serving as a model system to investigate this intriguing issue. Rahnella aceris HPDA25 is a plant growth-promoting bacteria isolated from G. elata, which has been found to facilitate the establishment of G. elata-Armillaria symbiosis. In this study, using the tripartite symbiotic system of G. elata-Armillaria gallica-R. aceris HPDA25, we investigate the role of VOCs in the interaction among mycoheterotrophic plants, fungi, and bacteria. Our results showed that 33 VOCs of HPDA25-inducible symbiotic G. elata elevated compared to non-symbiotic G. elata, indicating that VOCs indeed play a role in the symbiotic process. Among these, 21 VOCs were accessible, and six active VOCs showed complete growth inhibition activities against A. gallica, while R. aceris HPDA25 had no significant effect. In addition, three key genes of G. elata have been identified that may contribute to the increased concentration of six active VOCs. These results revealed for the first time the VOCs profile of G. elata and demonstrated its regulatory role in the tripartite symbiotic system involving G. elata, Armillaria, and bacteria.

Diversity of Trichoderma species associated with the black rot disease of Gastrodia elata, including four new species.

Introduction: Trichoderma species establish symbiotic relationships with plants through both parasitic and mutualistic mechanisms. While some Trichoderma species act as plant pathogenic fungi, others utilize various strategies to protect and enhance plant growth. Methods: Phylogenetic positions of new species of Trichoderma were determined through multi-gene analysis relying on the internal transcribed spacer (ITS) regions of the ribosomal DNA, the translation elongation factor 1-α (tef1-α) gene, and the RNA polymerase II (rpb2) gene. Additionally, pathogenicity experiments were conducted, and the aggressiveness of each isolate was evaluated based on the area of the cross-section of the infected site. Results: In this study, 13 Trichoderma species, including 9 known species and 4 new species, namely, T. delicatum, T. robustum, T. perfasciculatum, and T. subulatum were isolated from the diseased tubers of Gastrodia elata in Yunnan, China. Among the known species, T. hamatum had the highest frequency. T. delicatum belonged to the Koningii clade. T. robustum and T. perfasciculatum were assigned to the Virens clade. T. subulatum emerged as a new member of the Spirale clade. Pathogenicity experiments were conducted on the new species T. robustum, T. delicatum, and T. perfasciculatum, as well as the known species T. hamatum, T. atroviride, and T. harzianum. The infective abilities of different Trichoderma species on G. elata varied, indicating that Trichoderma was a pathogenic fungus causing black rot disease in G. elata. Discussion: This study provided the morphological characteristics of new species and discussed the morphological differences with phylogenetically proximate species, laying the foundation for research aimed at preventing and managing diseases that affect G. elata.

Fermented Gastrodia elata Bl. Alleviates Cognitive Deficits by Regulating Neurotransmitters and Gut Microbiota in D-Gal/AlCl3-Induced Alzheimer's Disease-like Mice.

Alzheimer's disease (AD) is a common neurological disease with recognition ability loss symptoms and a major contributor to dementia cases worldwide. Gastrodia elata Bl. (GE), a food of medicine-food homology, has been reported to have a mitigating effect on memory and learning ability decline. However, the effect of GE fermented by Lactobacillus plantarum, Acetobacter pasteurianus, and Saccharomyces (FGE) on alleviating cognitive deficits in AD was not studied. Mice were randomly divided into six groups, control, model, donepezil, low, medium, and high doses of FGE, and D-Galactose/Aluminum chloride (D-Gal/AlCl3) was used to establish an AD-like mouse model. The results indicated that FGE could improve the production of neurotransmitters and relieve oxidative stress damage in AD-like mice, which was evidenced by the declined levels of amyloid-ß (Aß), Tau, P-Tau, acetylcholinesterase (AchE), and malondialdehyde (MDA), and increased acetylcholine (Ach), choline acetyltransferase (ChAT), and superoxide dismutase (SOD) levels in brain tissue. Notably, FGE could enhance the richness of the gut microbiota, especially for beneficial bacteria such as Lachnospira and Lactobacillus. Non-target metabolomics results indicated that FGE could affect neurotransmitter levels by regulating amino acid metabolic pathways to improve AD symptoms. The FGE possessed an ameliorative effect on AD by regulating neurotransmitters, oxidative stress levels, and gut microbiota and could be considered a good candidate for ameliorating AD.

Preparation and evaluation of gastrodin microsphere-loaded Gastrodia elata polysaccharides composite hydrogel on UVB-induced skin damage in vitro and in vivo.

Skin damage from sun exposure is a common issue among outdoor workers and is primarily caused by ultraviolet rays. Upon absorption of these rays, the skin will experience inflammation and cell apoptosis. This study explored the concept of 'Combination of medicine and adjuvant' by utilizing Gastrodia elata polysaccharide, a key component of Gastrodia elata Bl.|, to develop a new hydrogel material. Oxidized Gastrodia elata polysaccharide (OGEP) and carboxymethyl chitosan (CMCS) was use to prepare a biocompatible, biodegradable and self-healing hydrogel OGEP/CMCS (OC). And this hydrogel was further loaded with Gastrodin-containing microspheres (GAS/GEL) to create GAS/GEL/OGEP/CMCS (GGOC) hydrogel. Characterization studies revealed that OC and GGOC hydrogels exhibited favorable mechanical properties, antioxidant activity and biocompatibility. The experiments showed that OC and GGOC hydrogels could regulate mitochondrial membrane potential, prevent mitochondrial breakage, inhibit proinflammatory factors, prevent NF-κB protein activation and regulate apoptosis-related pathways. This study highlighted the application potential of Gastrodia elata polysaccharide as a 'Combination of medicine and adjuvant' and the anti-UVB damage effect of the prepared hydrogel.

Experimental study on the detection of Gastrodia elata by enzymatic recombinase amplification and immunochromatography.

OBJECTIVE: The objective of this research is to develop two methodologies, Enzymatic recombinase amplification (ERA) and Polymerase Chain Reaction (PCR) coupled with Lateral Flow Dipstick (LFD), for the swift authentication of Gastrodia elata. METHODOLOGY: Primers and nfo probes for the ERA of Gastrodia elata were developed based on the ITS2 genome sequences of Gastrodia elata and its counterfeits. Specific primers for the PCR analysis of Gastrodia elata were generated using the NCBI (National Center for Biotechnology Information) online platform. Through experimental validation, the optimal reaction system and conditions for both methodologies were established, and their efficacy was assessed. RESULTS: The methodologies developed herein are applicable for the targeted analysis of the medicinal species, Gastrodia elata. The sensitivity of the ERA-LFD detection method matched that of the conventional PCR-LFD approach, recorded at 1 ng µL-1. Consistency was observed in the results across three replicates of visualization test strips for both techniques. Upon evaluation, both the PCR-LFD and ERA-LFD methods demonstrated a total compliance rate of 100 %. CONCLUSION: The ERA-LFD and PCR-LFD methods facilitate reduced detection times and offer visual results. These techniques are particularly effective for on-site detection and quality control in the authentication of Gastrodia elata within traditional Chinese medicine markets and at the primary level of healthcare provision.

Research progress of Gastrodia elata Blume polysaccharides: a review of chemical structures and biological activities.

Gastrodia elata Blume (G. elata), listed as one of the 34 precious Chinese medicines, servers a dual purpose as both a medicinal herb and a food source. Polysaccharide is the main active ingredient in G. elata, which has pharmacological activities such as immune regulation, anti-oxidation, anti-cancer, anti-aging, neuroprotection and antibacterial activity and so on. The biological activities of G. elata polysaccharide (GPs) is closely related to its chemical structures. However, no a review has synthetically summarized the chemical structures and pharmacological activities of GPs. This study delves into the chemical structures, pharmacological action of GPs, offering insights for the future development an application of these compounds.

[Effects of soil relative water content on growth characteristics of Armillaria spp. and seedling production of Gastrodia elata f. glauca].

The seedling survival rate, yield, and individual weight of Gastrodia elata is closely related to the soil relative water content(RWC) and the growth characteristics of the associated fungi Armillaria spp. This study explored the effects of the soil RWC on the growth characteristics of Armillaria spp. and the seedling production of G. elata f. glauca, aiming to provide guidance for breeding G. elata f. glauca and selecting elite strains of Armillaria. According to the growth characteristics on the medium for activation, thirty strains of Armillaria were classified into 4 clusters. Two strains with good growth indicators were selected from each cluster and cultiva-ted with immature tuber(Mima) and the branches of the broad-leaved trees in a water-controlled box. The results showed that the Armillaria clusters with uniaxial branches of rhizoid cords, such as clusters Ⅲ and Ⅳ, were excellent clusters in symbiosis with G. elata f. glauca. The soil RWC had significant effects on the growth characteristics of Armillaria strains and the seedling survival rate, yield, and individual weight of G. elata f. glauca. The growth characteristics of Armillaria strains and the seedling survival rate, yield, and individual weight of G. elata f. glauca in the case of the soil RWC being 75% were significantly better than those in the case of other soil RWC. Cultivating Mima with elite strains of Armillaria, together with branches of broad-leaved trees, in the greenhouses with the artificial control of the soil RWC, can achieve efficient seedling production and Mima utilization of G. elata f. glauca.

Two new compounds isolated from the aerial parts of gastrodia elata blume.

Two new aromatic compounds, namely gastupdin A (1), and gastupdin B (2), together with three known compounds, arundin(3), phomosines B (4) and monocillin IV (5), were isolated from the aerial parts of Gastrodia elata Blume. The structures of the new compounds were confirmed through spectral analyses including NMR, HR-ESI-MS, ECD, UV, and IR. All isolated compounds were evaluated for their neuroprotective effects against 6-hydroxydopamine-induced cell death in Human Neuroblastoma Cells, with curcumin as the positive control, however, the activity of all compounds was weaker than the positive control, showing no significant activity.

Identification of geographical origins of Gastrodia elata Blume based on multisource data fusion.

INTRODUCTION: Identifying the geographical origin of Gastrodia elata Blume contributes to the scientific and rational utilization of medicinal materials. In this study, infrared spectroscopy was combined with machine learning algorithms to distinguish the origin of G. elata BI. OBJECTIVE: Realization of rapid and accurate identification of the origin of G. elata BI. MATERIALS AND METHODS: Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectra and Fourier transform near-infrared (FT-NIR) spectra were collected for 306 samples of G. elata BI. SAMPLES: Firstly, a support vector machine (SVM) model was established based on the single-spectrum and the full-spectrum fusion data. To investigate whether feature-level fusion strategy can enhance the model's performance, the sequential and orthogonalized partial least squares discriminant analysis (SO-PLS-DA) model was established to extract and combine two types of spectral features. Next, six algorithms were employed to extract feature variables, SVM model was established based on the feature-level fusion data. To avoid complicated preprocessing and feature extraction processes, a residual convolutional neural network (ResNet) model was established after converting the raw spectral data into spectral images. RESULTS: The accuracy of the feature-level fusion model is better as compared to the single-spectrum model and the fusion model with full-spectrum, and SO-PLS-DA is simpler than feature-level fusion based on the SVM model. The ResNet model performs well in classification but requires more data to enhance its generalization capability and training effectiveness. CONCLUSION: Sequential and orthogonalized data fusion approaches and ResNet models are powerful solutions for identifying the geographic origin of G. elata BI.

Diverse Mycena Fungi and Their Potential for Gastrodia elata Germination.

It remains to be determined whether there is a geographical distribution pattern and phylogenetic signals for the Mycena strains with seed germination of the orchid plant Gastrodia elata. This study analyzed the community composition and phylogenetics of 72 Mycena strains associated with G. elata varieties (G. elata. f. glauca and G. elata. f. viridis) using multiple gene fragments (ITS+nLSU+SSU). We found that (1) these diverse Mycena phylogenetically belong to the Basidiospore amyloid group. (2) There is a phylogenetic signal of Mycena for germination of G. elata. Those strains phylogenetically close to M. abramsii, M. polygramma, and an unclassified Mycena had significantly higher germination rates than those to M. citrinomarginata. (3) The Mycena distribution depends on geographic site and G. elata variety. Both unclassified Mycena group 1 and the M. abramsii group were dominant for the two varieties of G. elata; in contrast, the M. citrinomarginata group was dominant in G. elata f. glauca but absent in G. elata f. viridis. Our results indicate that the community composition of numerous Mycena resources in the Zhaotong area varies by geographical location and G. elata variety. Importantly, our results also indicate that Mycena's phylogenetic status is correlated with its germination rate.

Discovery of seed germinating fungi (Mycetinis scorodonius) from Gastrodia elata Bl. f. glauca S. chow in Changbai Mountain and examination of their germination ability.

Multi-generational asexual reproduction of Gastrodia elata Bl. will cause seedling species degeneration. Sexual reproduction of Gastrodia elata Bl. seed is an effective method to solve the problem of degeneration. The development of Gastrodia elata Bl. seeds cannot be separated from the germination fungus. However, there are few strains of germination fungus in production, and there is also the problem of species degradation in application for many years. It is very important for the sexual reproduction of Gastrodia elata Bl. to isolate more new strains of excellent germination fungus from the origin. This study used the Gastrodia elata Bl. f. glauca S. chow seeds germination vegetative propagation corms capture method to isolate its symbiotic germination fungus, and comprehensively identified the species of germination fungus by colony morphology, ITS, sporocarps regeneration and germination function, and compared the growth characteristics and germination ability with other germination fungus (Mycena purpureofusca, Mycena dendrobii and Mycena osmundicola). The germination fungus was isolated from the vegetative propagation corms of Gastrodia elata Bl. f. glauca S. chow seeds and named GYGL-1. After comprehensive identification, GYGL-1 was Mycetinis scorodonius. Compared with other germination fungus, GYGL-1 has fast germination speed, vigorous growth, and high germination ability for Gastrodia elata Bl. f. glauca S. chow seeds. Innovated the isolation method of Gastrodia elata Bl. seeds germination fungus, obtained the regenerated sporocarps of the germination fungus, and discovered that Mycetinis scorodonius has a new function of germinating Gastrodia elata Bl. f. glauca S. chow seeds, enriching the resource library of Gastrodia elata Bl. germination fungus.

Features of bacterial and fungal communities in the rhizosphere of Gastrodia elata cultivated in greenhouse for early harvest.

Symbiotic microbes are essential for developing and growing Gastrodia elata, an achlorophyllous orchid of high medicinal value. Recently, the cultivation of G. elata in greenhouses has been adopted in Korea to produce mature tubers in a short time. However, no studies have been conducted on the microbial community structure of G. elata cultivated in greenhouse environments. Therefore, we analyzed the temporal features of bacterial and fungal communities in the rhizosphere of G. elata at the juvenile [JT; 2 months after sowing (MAS)], young (YT; 6 MAS), and mature (MT; 11 MAS) tuber stages using culture-dependent and high-throughput sequencing technology. The richness and diversity of the bacterial and fungal communities decreased with tuber growth of G. elata. The symbiotic fungi Mycena sp. and Armillaria sp. as well as tuber extract inhibited the growth of various soil-inhabiting fungal and bacterial strains, indicating that G. elata and its symbiotic fungi play important roles in the selection of rhizosphere microbes. Mortierella rishikesha was the most abundant fungal species in the rhizosphere. We also identified the microorganisms potentially beneficial for G. elata development during greenhouse cultivation. Tubers and symbiotic fungi actively exert selective pressure on rhizosphere microbes, influencing the diversity and abundance of bacterial and fungal communities as G. elata grows. This study is a first report on the temporal microbial community structure of G. elata cultivated in greenhouse. The results on the associated microbiome of G. elata will help understand their beneficial interactions with G. elata and contribute to improvement in cultivation.

The preventive effect of Gastrodia elata Blume extract on vancomycin-induced acute kidney injury in rats.

BACKGROUND: Gastrodia elata Blume (GEB), a traditional medicinal herb, has been reported to have pharmacological effect including protection against liver, neuron and kidney toxicity. However, explanation of its underlying mechanisms remains a great challenge. This study investigated the protective effects of GEB extract on vancomycin (VAN)-induced nephrotoxicity in rats and underlying mechanisms with emphasis on the anti-oxidative stress, anti-inflammation and anti-apoptosis. The male Sprague-Dawley rats were randomly divided three groups: control (CON) group, VAN group and GEB group with duration of 14 days. RESULTS: The kidney weight and the serum levels of blood urea nitrogen and creatinine in the GEB group were lower than the VAN group. Histological analysis using hematoxylin & eosin and periodic acid Schiff staining revealed pathological changes of the VAN group. Immunohistochemical analysis revealed that the expression levels of N-acetyl-D-glucosaminidase, myeloperoxidase and tumor necrosis factor-alpha in the GEB group were decreased when compared with the VAN group. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells, phosphohistone and malondialdehyde levels were lower in the GEB group than VAN group. The levels of total glutathione in the GEB group were higher than the VAN group. CONCLUSIONS: The findings of this study suggested that GEB extract prevents VAN-induced renal tissue damage through anti-oxidation, anti-inflammation and anti-apoptosis.

Digestive characteristics of Gastrodia elata Blume polysaccharide and related impacts on human gut microbiota in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Gastrodia elata Blume is a traditional Chinese medicine with the effects of improving the deficiency of the body and maintaining health, and polysaccharide (GEP) is one of the effective ingredients to play these activities of G. elata. Traditionally, G. elata is orally administered, so the activities of GEP are associated with digestive and intestinal metabolism. However, the digestive behavior of GEP and its effects on the human gut microbiota are unclear and need to be fully studied. AIM OF THE STUDY: This study aimed to investigate the changes in structural characteristics of GEP during digestion and the related impacts of its digestive product on gut microbiota in human fecal fermentation, and to explain the beneficial mechanism of GEP on human health from the perspective of digestive characteristics and "gut" axis. MATERIALS AND METHODS: The changes of reducing sugars, free monosaccharides and physicochemical properties of GEP during digestion were investigated by GPC, HPLC, FT-IR, CD, NMR, SEM, and TGA. Moreover, polysaccharide consumption, pH value changes, SCFAs production, and changes in gut microbiota during fermentation were also discussed. RESULTS: During digestion of GEP, glucose was partially released causing a decrease in molecular weight, and a change in monosaccharide composition. In addition, the characteristics of GEP before and after digestion, including configuration, morphology, and stability, were different. The digestive product of GEP was polysaccharide (GEP-I), which actively participated in the fecal fermentation process. As the fermentation time increased, the utilization of GEP-I by the microbiota gradually increased. The abundance of probiotics such as Bifidobacterium, Collinsella, Prevotella, and Faecalibacterium was significantly increased, and the abundance of pathogenic Shigella, Dorea, Desulfovibrio, and Blautia was significantly inhibited, thereby suggesting that GEP has the potential to maintain human health through the "gut" axis. In addition, the beneficial health effects of GEP-I have also been observed in the influence of microbial metabolites. During the fermentation of GEP-I, the pH value gradually decreased, and the contents of beneficial metabolites such as acetic acid, propionic acid, and caproic acid significantly increased. CONCLUSION: The structure of GEP changed significantly during digestion, and its digestive product had the potential to maintain human health by regulating gut microbiota, which may be one of the active mechanisms of GEP.

Gastrodinol derivatives and prenylated flavones from the flower branch of Gastrodia elata.

Based on the research progress and traditional usage with whole herbal of the TCM "Tianma", chemical studies herein on the flower branch of Gastrodia elata were carried out in-depth and got 13 compounds including the gastrodinols (1-4), the flavonoid morins (5-8, 11-12), together with the specialist mulberrofurans (9, 13) and gastrodiamide (10) for the first time from the species. The antibacterial and cholinesterase inhibitory activities were then evaluated and the results showed that compounds 5, 11, 12, 13 have good activity against anti-methicillin-resistant Staphylococcus aureus, and compounds 9, 13 had good acetylcholinesterase inhibitory activity. All these results provide new chemical composition for better understanding the traditional application of "Tianma" and for exploring new pharmacological ingredients.

Biology of Callistethus plagiicollis (Coleoptera: Scarabaeoidea: Rutelinae) and comparative morphology of its larvae.

Larvae of the beetle subfamily Rutelinae are poorly described in the literature. Notably, the morphology of the larvae of Callistethus plagiicollis Fairmaire has not previously been analyzed. Here, we report for the first time that these larvae feed on the tubers and roots of Gastrodia elata Blume, an important traditional Chinese herbal medicine, which causes a reduction in the yield and economic value of G. elata. We employed scanning electron microscopy and light microscopy to investigate the morphology and occurrence regularity of egg, larvae, pupae, and adult specimens of C. plagiicollis collected from the G. elata planting base in Guizhou Province, China, with a focus on the ultrastructure of mature larvae. The results revealed one generation of C. plagiicollis per year in the study area and three instar stages of larvae. Mature larvae were identified by the following characteristics: raster without palidia with a large number of hamate setae, antennal apex containing seven sensilla basiconica, larval haptomerum containing eight sensilla styloconica and four enlarged heli, and seven longitudinally arranged stridulatory teeth on the stipes of the maxilla. The combination of scanning electron and light microscopy effectively revealed the difference between membranous and sclerotized structures, ensuring accurate identification of C. plagiicollis larvae. By determining the feeding characteristics and occurrence regularity of C. plagiicollis, this study has implications for improved pest management in G. elata crops. RESEARCH HIGHLIGHTS: We identified C. plagiicollis as a new pest of G. elata, a traditional Chinese medicine Scanning electron and light microscopy were combined to analyze the morphology of the mature larvae of C. plagiicollis for the first time We determined the feeding characteristics and occurrence regularity of C. plagiicollis, which can be used to develop effective pest management strategies.

Structural characterization, molecular dynamic simulation, and conformational visualization of a water-soluble glucan with high molecular weight from Gastrodia elata Blume.

Polysaccharides have been widely used in the development of natural drugs and health food. However, polysaccharide characterization lags due to inherently complicated features and the limitations of existing detection approaches. We aimed to provide new insight into the fine structure and conformational visualization of polysaccharides from Gastrodia elata Blume, a medicinal and edible plant. A water-soluble polysaccharide (GEP2-6) with the high molecular weight of 2.7 × 106 Da was first obtained, and its purity reached 99.2 %. Chemical and spectroscopic analyses jointly revealed that GEP2-6 was a glucan linked by α-(1 â†’ 4) and α-(1 â†’ 6) glycosidic bonds. After enzymolysis, the local structure of GEP2-6 included α-1,4-Glcp, α-1,6-Glcp, α-1,4,6-Glcp, and α-1-Glcp at a molar ratio of 31.27∶1.32∶1.08∶0.93. The glycosidic linkage pattern of repeating units was further simulated by a glycan database and spatial examination software. The good dissolution performance was interpreted by dynamics simulation and practical molecular characteristics. Spherical flexible chains and the porous stable conformation were corroborated using atomic force microscopy. In addition, GEP2-6 could effectively scavenge DPPH and hydroxyl radicals as a promising natural antioxidant. These efforts will contribute to the expansion of clinical applications of this G. elata polysaccharide and the structural elucidation for macromolecular polysaccharides combined with traditional and modern analysis techniques.

Decrease in beneficial bacteria and increase in harmful bacteria in Gastrodia seedlings and their surrounding soil are mainly responsible for degradation of Gastrodia asexual propagation.

Introduction: Asexual reproduction of Gastrodia elata Bl. f. glauca S. chow (GeB) produces degeneration with increasing number of GeB. Therefore, we analyzed the microorganisms of GeB seedlings and surrounding soil by Illumina Miseq high-throughput sequencing technology. Methods: In this study, Illumina Miseq high-throughput sequencing technology was applied to analyze the types and quantities of GeB seedlings and surrounding soil microorganisms in the first to third generations of asexual reproduction, isolated and identified the dominant strains of GeB in the first to third generations and screened the antagonistic bacteria of its pathogenic fungi, and evaluated the effects of beneficial bacteria on the production performance of seedlings planted with GeB. Results: With an increase in the number of asexual reproductive generations, the number of pathogenic fungi and bacteria in GeB seedlings and the surrounding soil increased, and the number of beneficial fungi and bacteria decreased. Pseudomonas sp., Agrobacterium rhizomes, and Herbaspirillum hiltneri were isolated and identified in the first generation, and Trichoderma harzianum, Penicillium viridiatum, Fusarium oxysporum, and Novosphingobium sp. Were isolated and identified in the third generation. Antagonistic strains of the three pathogenic bacterial strains were screened. In conclusion, beneficial bacteria significantly improved the production performance of asexual reproductive seedlings planted with GeB. Discussion: In conclusion, our findings suggested that the microorganisms of GeB seedlings and the surrounding soil change as the number of generations of GeB reproduction increases, disrupts the microecological balance of surrounding soil and endophytic microbiomes.This study provides a theoretical basis for the degradation of asexual reproduction in GeB.

First Report of Brown Rot Caused by Porogramme epimiltina on Gastrodia elata in China.

Gastrodia elata Blume is a valuable medicinal plant in China with great significance in medicine (Li et al. 2023). From 2022 to 2023, G. elata tuber rot occurred in about 50 households in the main cultivation areas of G. elata (27°39' N, 104°16' E) in Yiliang County, Zhaotong, Yunnan Province, southwest China. The planting area of G. elata was 776 ha, and the incidence rate was 10%. Symptoms present as light brown lesions on the surface of the tuber, sunken, soft and foul-smelling. Infected G. elata tubers were randomly collected from each household, packed into transparent plastic bags, and strains were isolated in the laboratory as follows. The tubers of 15 infected G. elata tubers were surface-sterilized with 0.5% NaOCl for 2 min, rinsed five times with sterile water, and dried. Symptomatic tissues from the margin between necrotic and healthy tissues were cut into 5 × 5 mm pieces, placed onto potato dextrose agar (PDA), and incubated at 28 ºC in the dark for 3 days. Hyphal tips of fungi growing from the samples were transferred onto new PDA plates and incubated until they produced conidia. Two fungal strains (Charliezhao 425 and 433) with the same morphological characteristics were obtained from the samples. Colonies were whitish and grew rapidly, irregularly turning pale orange at the edge or center of the mycelium pad on a two-week-old petri dish, and finally dark red，spore oval to spherical, 2.7 to 5.3 × 2.3 to 3.5 µm (n=50). The morphological characteristics of the isolates resembled Porogramme epimiltina (Mao et al. 2023; Kubayashi et al. 2001). Genomic DNA of two representative isolates (Charliezhao 425 and 433) was extracted using the DN14 cetyltrimethylammonium bromide rapid plant genome extraction kit (Aidlab Biotechnologies Co., Ltd, Beijing). The ITS and TEF1 genes were amplified by polymerase chain reaction using the primers ITS1/ITS4 (White et al. 1990) and EF1-983F/EF1-2218R (Rehner et al, 2005), respectively. All sequences were deposited in GenBank (accession no. OR905803, OR905804 for ITS, OR939812, OR939813 for TEF1). A BLASTN homology search with the ITS nucleotide sequences showed that they had 98.99 to 99.15% identity with P. epimiltina isolate OP997539 (588/594 bp) and isolate OP997539 (584/589 bp), respectively; and the TEF1 sequences had 95.41 to 95.59% % identity to isolates OP556566 (540/565 bp) and isolate OP556566 (542/567 bp), respectively. To complete Koch's hypothesis, the surfaces of 5 mature and healthy G. elata tubers were disinfected with 1% NaClO solution for 1 minute, rinsed with sterile water 5 times, and dried at 25 ℃ for 30 minutes. Conidial suspensions (106 spores/ml) were collected from two isolates (Charliezhao 425 and 433) and sprayed on G. elata tuber, and the control treated with distilled water. All G. elata tubers were incubated at 25℃ with 80% relative humidity. The experiment had three replicates. After 7 days of culture, there were obvious rotten and smelly on the inoculated tubers. No symptoms were observed in the control groups. The pathogen was re-isolated from all inoculated birch tubers and confirmed as P. epimiltina by morphological and molecular analysis, which fulfilled Koch's hypothesis. Gastrodia elata is a valuable and extensively used herbal Traditional Chinese Medicine with a wide range of clinical applications. As far as we know, this is the first report of P. epimiltina causing brown rot of G. elata in China.