Composition and diversity of soil microbial communities change by introducing Phallus impudicus into a Gastrodia elata Bl.-based soil.

BACKGROUND: The Gastrodia elata Bl. is an orchid, and its growth demands the presence of Armillaria species. The strong competitiveness of Armillaria species has always been a concern of major threat to other soil organisms, thus disrupting the equilibrium of soil biodiversity. Introducing other species to where G. elata was cultivated, could possibly alleviate the problems associated with the disequilibrium of soil microenvironment; however, their impacts on the soil microbial communities and the underlying mechanisms remain unclear. To reveal the changes of microbial groups associated with soil chemical properties responding to different cultivation species, the chemical property measurements coupled with the next-generation pyrosequencing analyses were applied with soil samples collected from fallow land, cultivation of G. elata and Phallus impudicus, respectively. RESULTS: The cultivation of G. elata induced significant increases (p < 0.05) in soil pH and NO3-N content compared with fallow land, whereas subsequent cultivation of P. impudicus reversed these G. elata-induced increases and was also found to significantly increase (p < 0.05) the content of soil NH4+-N and AP. The alpha diversities of soil microbial communities were significantly increased (p < 0.01) by cultivation of G. elata and P. impudicus as indicated with Chao1 estimator and Shannon index. The structure and composition of soil microbial communities differed responding to different cultivation species. In particular, the relative abundances of Bacillus, norank_o_Gaiellales, Mortierella and unclassified_k_Fungi were significantly increased (p < 0.05), while the abundances of potentially beneficial genera such as Acidibacter, Acidothermus, Cryptococcus, and Penicillium etc., were significantly decreased (p < 0.05) by cultivation of G. elata. It's interesting to find that cultivation of P. impudicus increased the abundances of these genera that G. elata decreased before, which contributed to the difference of composition and structure. The results of CCA and heatmap indicated that the changes of soil microbial communities had strong correlations with soil nutrients. Specifically, among 28 genera presented, 50% and 42.9% demonstrated significant correlations with soil pH and NO3-N in response to cultivation of G. elata and P. impudicus. CONCLUSIONS: Our findings suggested that the cultivation of P. impudicus might have potential benefits as result of affecting soil microorganisms coupled with changes in soil nutrient profile.

Chemistry, Biological Activities, and Pharmacological Properties of Gastrodin: Mechanism Insights.

Gastrodin, a bioactive compound derived from the rhizome of the orchid Gastrodia elata, exhibits a diverse range of biological activities. With documented neuroprotective, anti-inflammatory, antioxidant, anti-apoptotic, and anti-tumor effects, gastrodin stands out as a multifaceted therapeutic agent. Notably, it has demonstrated efficacy in protecting against neuronal damage and enhancing cognitive function in animal models of Alzheimer's disease, Parkinson's disease, and cerebral ischemia. Additionally, gastrodin showcases immunomodulatory effects by mitigating inflammation and suppressing the expression of inflammatory cytokines. Its cytotoxic activity involves the inhibition of angiogenesis, suppression of tumor growth, and induction of apoptosis. This comprehensive review seeks to elucidate the myriad potential effects of Gastrodin, delving into the intricate molecular mechanisms underpinning its pharmacological properties. The findings underscore the therapeutic potential of gastrodin in addressing various conditions linked to neuroinflammation and cancer.

Analyses of Multilocus Sequences and Morphological Features Reveal Ilyonectria Species Associated with Black Rot Disease of Gastrodia elata.

Black rot is a common disease of Gastrodia elata, causing serious threats to G. elata production. In this study, a total of 17 Cylindrocarpon-like strains were isolated from G. elata black rot tissues. Multilocus sequence analyses based on ITS, HIS, TEF, and TUB combined with morphological characterizations were performed to identify six Ilyonectria species, including four new species, Ilyonectria longispora, I. sinensis, I. xiaocaobaensis, and I. yunnanensis, and two known species, I. changbaiensis and I. robusta. The pathogenicity of 11 isolates comprising type strains of the four new species and representative isolates from each of the six species was tested on healthy tissues of G. elata. All isolates were pathogenic to G. elata tissues, and symptoms were identical to black rot disease, confirming that our isolates were the causal agents of black rot disease of G. elata.

First report of the complete mitochondrial genome of 3 beetles (Coleoptera: Scarabaeidae) harming Gastrodia elata (Asparagales: Orchidaceae).

Gastrodia elata Blume, a valuable traditional Chinese medicine with significant clinical and nutritional importance, is a fungal heterotrophic orchid. We present the first report of the mitochondrial genome structure and characteristics of 3 Scarabaeidae pests affecting G. elata: Sophrops peronosporus Gu & Zhang, Anomala rufiventris Kollar & Redtenbacher, and Callistethus plagiicollis Fairmaire. Each mitogenome contained 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs), and a control region, with no gene rearrangements observed. All 21 tRNAs, except trnS1 that lacks a dihydrouridine, had a stable cloverleaf secondary structure. Maximum likelihood and Bayesian inference analyses based on the 13 PCGs produced 2 topologically similar phylogenetic trees, both of with high nodal support. Larvae of these Scarabaeidae pests cause substantial damage by gnawing on the tubers and roots of G. elata, leading to reduced yield and compromised quality. These findings contribute to phylogenetic studies of Scarabaeidae, expand knowledge of G. elata pests, and offer valuable reference materials for their identification and control.

The Effect of Fermentation on the Chemical Constituents of Gastrodia Tuber Hallimasch Powder (GTHP) Estimated by UHPLC-Q-Orbitrap HRMS and HPLC.

OBJECTIVE: To compare the effect of fermentation on the chemical constituents of Gastrodia Tuder Halimasch Powder (GTHP), to establish its fingerprinting and multicomponent content determination, and to provide a basis for the processing, handling, and clinical application of this herb. METHODS: Ultra-high-performance liquid chromatography-quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was used to conduct a preliminary analysis of the chemical constituents in GTHP before and after fermentation. High-performance liquid chromatography (HPLC) was used to determine some major differential components of GTHP and establish fingerprints. Cluster analysis (CA), and principal component analysis (PCA) were employed for comprehensive evaluation. RESULTS: Seventy-nine compounds were identified, including flavonoids, organic acids, nucleosides, terpenoids, and others. The CA and PCA results showed that ten samples were divided into three groups. Through standard control and HPLC analysis, 10 compounds were identified from 22 peaks, namely uracil, guanosine, adenosine, 5-hydroxymethylfurfural (5-HMF), daidzin, genistin, glycitein, daidzein, genistein, and ergosterol. After fermentation, GTHP exhibited significantly higher contents of uracil, guanosine, adenosine, 5-hydroxymethylfurfural, and ergosterol and significantly lower genistein and daidzein contents. CONCLUSIONS: The UHPLC-Q-Orbitrap HRMS and HPLC methods can effectively identify a variety of chemical components before and after the fermentation of GTHP. This study provides a valuable reference for further research on the rational clinical application and quality control improvement of GTHP.

GelFAP v2.0: an improved platform for Gene functional analysis in Gastrodia elata.

BACKGROUND: Gastrodia elata (tianma), a well-known medicinal orchid, is widely used to treat various kinds of diseases with its dried tuber. In recent years, new chromosome-level genomes of G.elata have been released in succession, which offer an enormous resource pool for understanding gene function. Previously we have constructed GelFAP for gene functional analysis of G.elata. As genomes are updated and transcriptome data is accumulated, collection data in GelFAP cannot meet the need of researchers. RESULTS: Based on new chromosome-level genome and transcriptome data, we constructed co-expression network of G. elata, and then we annotated genes by aligning with sequences from NR, TAIR, Uniprot and Swissprot database. GO (Gene Ontology) and KEGG (Kyoto Encylopaedia of Genes and Genomes) annotations were predicted by InterProScan and GhostKOALA software. Gene families were further predicted by iTAK (Plant Transcription factor and Protein kinase Identifier and Classifier), HMMER (hidden Markov models), InParanoid. Finally, we developed an improved platform for gene functional analysis in G. elata (GelFAP v2.0) by integrating new genome, transcriptome data and processed functional annotation. Several tools were also introduced to platform including BLAST (Basic Local Alignment Search Tool), GSEA (Gene Set Enrichment Analysis), Heatmap, JBrowse, Motif analysis and Sequence extraction. Based on this platform, we found that the flavonoid biosynthesis might be regulated by transcription factors (TFs) such as MYB, HB and NAC. We also took C4H and GAFP4 as examples to show the usage of our platform. CONCLUSION: An improved platform for gene functional analysis in G. elata (GelFAP v2.0, www.gzybioinformatics.cn/Gelv2 ) was constructed, which provides better genome data, more transcriptome resources and more analysis tools. The updated platform might be preferably benefit researchers to carry out gene functional research for their project.

Whole genome sequencing and analysis of Armillaria gallica Jzi34 symbiotic with Gastrodia elata.

BACKGROUND: Armillaria species are plant pathogens, but a few Armillaria species can establish a symbiotic relationship with Gastrodia elata, a rootless and leafless orchid, that is used as a Chinese herbal medicine. Armillaria is a nutrient source for the growth of G. elata. However, there are few reports on the molecular mechanism of symbiosis between Armillaria species and G. elata. The genome sequencing and analysis of Armillaria symbiotic with G. elata would provide genomic information for further studying the molecular mechanism of symbiosis. RESULTS: The de novo genome assembly was performed with the PacBio Sequel platform and Illumina NovaSeq PE150 for the A. gallica Jzi34 strain, which was symbiotic with G. elata. Its genome assembly contained ~ 79.9 Mbp and consisted of 60 contigs with an N50 of 2,535,910 bp. There were only 4.1% repetitive sequences in the genome assembly. Functional annotation analysis revealed a total of 16,280 protein coding genes. Compared with the other five genomes of Armillaria, the carbohydrate enzyme gene family of the genome was significantly contracted, while it had the largest set of glycosyl transferase (GT) genes. It also had an expansion of auxiliary activity enzymes AA3-2 gene subfamily and cytochrome P450 genes. The synteny analysis result of P450 genes reveals that the evolutionary relationship of P450 proteins between A. gallica Jzi34 and other four Armillaria was complex. CONCLUSIONS: These characteristics may be beneficial for establishing a symbiotic relationship with G. elata. These results explore the characteristics of A. gallica Jzi34 from a genomic perspective and provide an important genomic resource for further detailed study of Armillaria. This will help to further study the symbiotic mechanism between A. gallica and G. elata.

Transcriptomic heterochrony and completely cleistogamous flower development in the mycoheterotrophic orchid Gastrodia.

Cleistogamy, in which plants can reproduce via self-fertilization within permanently closed flowers, has evolved in > 30 angiosperm lineages; however, consistent with Darwin's doubts about its existence, complete cleistogamy - the production of only cleistogamous flowers - has rarely been recognized. Thus far, the achlorophyllous orchid genus, Gastrodia, is the only known genus with several plausible completely cleistogamous species. Here, we analyzed the floral developmental transcriptomes of two recently evolved, completely cleistogamous Gastrodia species and their chasmogamous sister species to elucidate the possible changes involved in producing common cleistogamous traits. The ABBA-BABA test did not support introgression and protein sequence convergence as evolutionary mechanisms leading to cleistogamy, leaving convergence in gene expression as a plausible mechanism. Regarding transcriptomic differentiation, the two cleistogamous species had common modifications in the expression of developmental regulators, exhibiting a gene family-wide signature of convergent expression changes in MADS-box genes. Our transcriptomic pseudotime analysis revealed a prolonged juvenile state and eventual maturation, a heterochronic pattern consistent with partial neoteny, in cleistogamous flower development. These findings indicate that transcriptomic partial neoteny, arising from changes in the expression of conserved developmental regulators, might have contributed to the rapid and repeated evolution of cleistogamous flowers in Gastrodia.

Chemical composition and anticonvulsant activities of herb pair of Gastrodia elata Blume-Acorus tatarinowii Schott decoction on experimentally induced seizures in mice.

Epilepsy is a serious public health problem in the world. At present, over 30% of affected patients remain refractory to currently available treatment. Medicinal plants as pharmaceuticals and healthcare treatments have been frequently used in the management of epilepsy in China for many centuries. Gastrodia elata-Acous tatarinowii (GEAT), as a classic and most commonly used herb pair in traditional Chinese medicine (TCM), has been employed to control seizures for thousands of years. However, the animal experiment data on its anticonvulsant effect is limited in the literature. Thus, this study aimed to reveal the therapeutic actions of GEAT decoction against seizures in mice. UHPLC-MS/MS was performed to analyze the chemical components of GEAT decoction. The mice were given GEAT decoction for 7 days, and MES, PTZ, and 3-MP injection was given 30 min after the last administration. Video monitoring was performed for comparisons. In addition, the PTZ-induced kindling models were conducted to investigate the seizure severity, anxiety and cognitive profile, inflammation, and oxidative stress parameters in mice. The results showed that GEAT decoction dose-dependently protected mice against MES, 3-MP, and PTZ-induced acute seizures. Furthermore, GEAT decoction significantly ameliorated seizure severity, decreased the accumulation of inflammatory mediators TNF-α, IL-1ß, and IL-6, mitigated oxidative stress, as well as alleviated anxious-like behavior and cognitive deficits in PTZ-kindled mice. These results suggest that GEAT decoction possesses certain anticonvulsant properties, which might be clinically useful as phytotherapy alone or as an adjunct therapy for the prevention and treatment of seizures and epilepsy.

Two Decarestrictine Analogs from the Soil-Derived Fungus Penicillium sp. YUD18003 Associated with Gastrodia elata.

Two new decarestrictine analogs decarestrictine P and penicitone, together with eight known homologous compounds were isolated from the soil fungus from the rhizosphere of Penicillium sp. YUD18003 related to Gastrodia elata. Their different structures include a decanolides decartestridine P and a long-chain polyhydroxyketone penicitone. The structures of new compounds were determined by nuclear magnetic resonance (NMR) spectroscopic analysis and high resolution electrospray ionization mass spectrometry (HR-ESI-MS), while their absolute configurations were determined by spectroscopic methods, DP4+ probability analysis, modified Snatzke's method and electron circular dichroism (ECD) calculations. All compounds were evaluated for antimicrobial activities.

Melatonin Treatment Maintains the Quality of Fresh-Cut Gastrodia elata under Low-Temperature Conditions by Regulating Reactive Oxygen Species Metabolism and Phenylpropanoid Pathway.

The application of melatonin (MT) has been shown to improve the quality during the storage of fruits and vegetables. The primary objective of this study is to investigate the effects of MT on the quality of fresh-cut Gastrodia elata during low-temperature (4 °C) storage. The results indicated that MT treatment not only suppressed the respiratory rate and malondialdehyde content but also slowed down the decline in total acidity and total soluble solids, effectively inhibiting microbial growth and enhancing the product safety of fresh-cut G. elata. The treatment with MT reduced the superoxide anions and hydrogen peroxide production, as well as inhibiting the activity and expression of peroxidase and polyphenol oxidase. Additionally, it led to increased activity and the expression of antioxidant-related enzymes, including superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, and dehydroascorbate reductase, while also resulting in elevated levels of ascorbic acid and glutathione. Furthermore, the treatment with MT induced an increase in the total phenolic and flavonoid content of fresh-cut G. elata and enhanced the activity and expression of key enzymes involved in the phenylpropanoid pathway (phenylalanine ammonia-lyase, cinnamate-4-hydroxylase, 4-coumarate: CoA ligase). In summary, MT enhances the antioxidant capacity by activating both the ROS metabolism and phenylpropanoid pathway, thus maintaining the quality of fresh-cut G. elata.

Bioactivity Profiling and Quantification of Gastrodin in Gastrodia elata Cultivated in the Field versus Facility via Hyphenated High-Performance Thin-Layer Chromatography.

Gastrodia elata (Orchidaceae) is native to mountainous areas of Asia and is a plant species used in traditional medicine for more than two thousand years. The species was reported to have many biological activities, such as neuroprotective, antioxidant, and anti-inflammatory activity. After many years of extensive exploitation from the wild, the plant was added to lists of endangered species. Since its desired cultivation is considered difficult, innovative cultivation methods that can reduce the costs of using new soil in each cycle and at the same time avoid contamination with pathogens and chemicals are urgently needed on large scale. In this work, five G. elata samples cultivated in a facility utilizing electron beam-treated soil were compared to two samples grown in the field concerning their chemical composition and bioactivity. Using hyphenated high-performance thin-layer chromatography (HPTLC) and multi-imaging (UV/Vis/FLD, also after derivatization), the chemical marker compound gastrodin was quantified in the seven G. elata rhizome/tuber samples, which showed differences in their contents between facility and field samples and between samples collected during different seasons. Parishin E was also found to be present. Combining HPTLC with on-surface (bio)assays, the antioxidant activity and inhibition of acetylcholinesterase as well as the absence of cytotoxicity against human cells were demonstrated and compared between samples.

Draft Genome and Biological Characteristics of Fusarium solani and Fusarium oxysporum Causing Black Rot in Gastrodia elata.

Gastrodia elata is a valuable traditional Chinese medicinal plant. However, G. elata crops are affected by major diseases, such as brown rot. Previous studies have shown that brown rot is caused by Fusarium oxysporum and F. solani. To further understand the disease, we studied the biological and genome characteristics of these pathogenic fungi. Here, we found that the optimum growth temperature and pH of F. oxysporum (strain QK8) and F. solani (strain SX13) were 28 °C and pH 7, and 30 °C and pH 9, respectively. An indoor virulence test showed that oxime tebuconazole, tebuconazole, and tetramycin had significant bacteriostatic effects on the two Fusarium species. The genomes of QK8 and SX13 were assembled, and it was found that there was a certain gap in the size of the two fungi. The size of strain QK8 was 51,204,719 bp and that of strain SX13 was 55,171,989 bp. Afterwards, through phylogenetic analysis, it was found that strain QK8 was closely related to F. oxysporum, while strain SX13 was closely related to F. solani. Compared with the published whole-genome data for these two Fusarium strains, the genome information obtained here is more complete; the assembly and splicing reach the chromosome level. The biological characteristics and genomic information we provide here lay the foundation for further research on G. elata brown rot.

Ancient Horizontal Gene Transfers from Plastome to Mitogenome of a Nonphotosynthetic Orchid, Gastrodia pubilabiata (Epidendroideae, Orchidaceae).

Gastrodia pubilabiata is a nonphotosynthetic and mycoheterotrophic orchid belonging to subfamily Epidendroideae. Compared to other typical angiosperm species, the plastome of G. pubilabiata is dramatically reduced in size to only 30,698 base pairs (bp). This reduction has led to the loss of most photosynthesis-related genes and some housekeeping genes in the plastome, which now only contains 19 protein coding genes, three tRNAs, and three rRNAs. In contrast, the typical orchid species contains 79 protein coding genes, 30 tRNAs, and four rRNAs. This study decoded the entire mitogenome of G. pubilabiata, which consisted of 44 contigs with a total length of 867,349 bp. Its mitogenome contained 38 protein coding genes, nine tRNAs, and three rRNAs. The gene content of G. pubilabiata mitogenome is similar to the typical plant mitogenomes even though the mitogenome size is twice as large as the typical ones. To determine possible gene transfer events between the plastome and the mitogenome individual BLASTN searches were conducted, using all available orchid plastome sequences and flowering plant mitogenome sequences. Plastid rRNA fragments were found at a high frequency in the mitogenome. Seven plastid protein coding gene fragments (ndhC, ndhJ, ndhK, psaA, psbF, rpoB, and rps4) were also identified in the mitogenome of G. pubilabiata. Phylogenetic trees using these seven plastid protein coding gene fragments suggested that horizontal gene transfer (HGT) from plastome to mitogenome occurred before losses of photosynthesis related genes, leading to the lineage of G. pubilabiata. Compared to species phylogeny of the lineage of orchid, it was estimated that HGT might have occurred approximately 30 million years ago.

Neuroprotection and Mechanism of Gas-miR36-5p from Gastrodia elata in an Alzheimer's Disease Model by Regulating Glycogen Synthase Kinase-3ß.

Alzheimer's disease (AD) is currently the most common neurodegenerative disease. Glycogen synthase kinase 3ß (GSK-3ß) is a pivotal factor in AD pathogenesis. Recent research has demonstrated that plant miRNAs exert cross-kingdom regulation on the target genes in animals. Gastrodia elata (G. elata) is a valuable traditional Chinese medicine that has significant pharmacological activity against diseases of the central nervous system (CNS). Our previous studies have indicated that G. elata-specific miRNA plays a cross-kingdom regulatory role for the NF-κB signaling pathway in mice. In this study, further bioinformatics analysis suggested that Gas-miR36-5p targets GSK-3ß. Through western blot, RT-qPCR, and assessments of T-AOC, SOD, and MDA levels, Gas-miR36-5p demonstrated its neuroprotective effects in an AD cell model. Furthermore, Gas-miR36-5p was detected in the murine brain tissues. The results of the Morris water maze test and western blot analysis provided positive evidence for reversing the learning deficits and hyperphosphorylation of Tau in AD mice, elucidating significant neuroprotective effects in an AD model following G. elata RNA administration. Our research emphasizes Gas-miR36-5p as a novel G. elata-specific miRNA with neuroprotective properties in Alzheimer's disease by targeting GSK-3ß. Consequently, our findings provide valuable insights into the cross-kingdom regulatory mechanisms underlying G. elata-specific miRNA, presenting a novel perspective for the treatment of Alzheimer's disease.

Assessment of the Effects of Structural Modification of Gastrodia elata Polysaccharide on Anti-Breast Cancer Activity Using Asymmetrical Flow Field-Flow Fractionation.

Gastrodia elata ("Tian Ma" in Chinese) is used as a food and medical ingredient in traditional Chinese medicine. In this study, to enhance the anti-breast cancer activity of Gastrodia elata polysaccharide (GEP), GEPs were modified via sulfidation (SGEP) and acetylation (AcGEP). The physicochemical properties (such as solubility and substitution degree) and structural information (such as molecular weight Mw and radius of gyration Rg) of GEP derivatives were determined by Fourier transformed infrared (FTIR) spectroscopy and asymmetrical flow field-flow fractionation (AF4) coupled online with multiangle light scattering (MALS) and differential refractive index (dRI) detectors (AF4-MALS-dRI). The effects of the structural modification of GEP on the proliferation, apoptosis, and cell cycle of MCF-7 cell were studied systematically. The ability of MCF-7 cell for the uptake of GEP was studied by laser scanning confocal microscopy (LSCM). The results suggested that the solubility and anti-breast cancer activity of GEP were enhanced and the average Rg and Mw of GEP decreased after chemical modification. The AF4-MALS-dRI results showed that the chemical modification process simultaneously caused the degradation and aggregation of GEPs. The LSCM results revealed that more SGEP can enter the MCF-7 cell interior compared with AcGEP. The results indicated that the structure of AcGEP could play a dominating role in antitumor activity. The data obtained in this work can be used as a starting point for investigating the structure-bioactivity of GEPs.

Effects of polysaccharides from Gastrodia elata on the immunomodulatory activity and gut microbiota regulation in cyclophosphamide-treated mice.

BACKGROUND: Cyclophosphamide (CTX) is a widely used chemotherapeutic agent for the treatment of malignant tumors and autoimmune diseases. However, it can cause immunosuppression and damage the intestinal mucosa. The development of new agents to counteract these side effects is becoming increasingly important. Previous studies have shown that the polysaccharides from Gastrodia elata (GEPs) have strong immune-enhancing effects; however, their functions regarding the intestines and the underlying mechanism are still unclear. In this study, the effects of GEPs on immunomodulatory activity, intestinal barrier function, and gut microbiota regulation were investigated in a mouse model of CTX-induced immunosuppression. RESULTS: Gastrodia elata polysaccharides attenuated the CTX-induced decrease in organ indices of the thymus and spleen, and promoted the secretion of immune-related cytokines and immunoglobulins in the serum. They also improved the intestinal pathology and restored the intestinal barrier function by elevating the expression of intestinal tight junction proteins, occludin and ZO-1. Moreover, GEPs restored the composition and abundance of the gut microbiota and increased the short-chain fatty acid (SCFA) content in the colon. The abundance of SCFA-producing bacteria (Muribaculaceae, Prevotellaceae, and Bacteroidaceae) also increased. CONCLUSIONS: Gastrodia elata polysaccharides can effectively alleviate immunosuppression and regulate the intestinal barrier integrity and the structure of gut microbiota in CTX-treated mice. They may be used as ingredients to develop functional foods for intestinal health. © 2023 Society of Chemical Industry.

[Screening of an Armillaria gallica strain for Gastrodia elata cultivation].

This study aims to screen a strain from Armillaria for the cultivation of Gastrodia elata. Specifically, Armillaria strains were isolated from different producing areas of G. elata and identified. Based on the growth characteristics of the strains and the experiment on the cultivation of G. elata, an optimal A. gallica strain was screened out. The specific process is as follows. The fungus-gro-wing materials of G. elata were collected from four producing areas and the Armillaria strains were isolated(G,Y,S,H). The strains were then identified based on morphological observation and phylogeny analysis and the commonly used strains were determined. The sucrase genotypes of the strains were identified according to our previous research findings, and the growth characteristics of the strains, such as growth rate, diameter, dry weight, and polysaccharide content of the rhizomorphs, were measured. According to the biological characteristics and sucrase genotypes, two strains were selected for the cultivation of G. elata. The tuber yield and the content of gastrodin and p-hydroxybenzyl alcohol in the tuber of G. elata were measured to select the optimal strain. The results showed that the four strains were all A. gallica. The rhizomorphs of strains G and H of the same sucrase genotype had larger/higher length, growth rate, diameter, branch number, dry weight, and polysaccharide content than those of strains S and Y of the same sucrase genotype. The tuber yield and the total content of gastrodin and p-hydroxybenzyl alcohol in tuber of G. elata cultivated with strain H were 6.528 kg·m~(-2) and 0.566%, respectively, which were 4.58 and 1.30 folds those of G. elata cultivated with strain S. Strains H and S were screened out from four strains of A. gallica based on the growth characteristics and sucrase genotype. According to the tuber yield and content of total gastrodin and p-hydroxybenzyl alcohol in the tuber of G. elata, strain H was identified as the optimal one. The findings in this study are expected to lay a basis for cultivating G. elata with high yield and quality of tubers.

[Application of strigolactone analogs in storage of Gastrodia elata].

This study explored the preservation effect of strigolactone analogs on Gastrodia elata tubers and screened out the suitable preservation measures of G. elata to provide a safer and more effective method for its storage and preservation. Fresh G. elata tubers were treated with 7FGR24, 2,4-D isooctyl ester, and maleic hydrazide, respectively. The growth of flower buds, the activities of CAT, and MDA, and the content of gastrodin and p-hydroxybenzyl alcohol were measured to compare the effects of different compounds on the storage and preservation of G. elata. The effects of different storage temperatures on the preservation of 7FGR24 were compared and analyzed. The gibberellin signal transduction receptor gene GeGID1 was cloned, and the effect of 7FGR24 on the expression level of GeGID1 was analyzed by quantitative polymerase chain reaction(qPCR). The toxicity of the G. elata preservative 7FGR24 was analyzed by intragastric administration in mice to evaluate its safety. The results showed that compared with 2,4-D isooctyl ester and maleic hydrazide, 7FGR24 treatment had a significant inhibitory effect on the growth of G. elata flower buds, and the CAT enzyme activity of G. elata was the highest, indicating that its preservation effect was stronger. Different storage temperatures had different effects on the preservation of G. elata, and the preservation effect was the strongest at 5 ℃. The open reading frame(ORF) of GeGID1 gene was 936 bp in length, and its expression level was significantly down-regulated after 7FGR24 treatment, indicating that 7FGR24 may inhibit the growth of flower buds by inhibiting the gibberellin signal of G. elata, thereby exerting a fresh-keeping effect. Feeding preservative 7FGR24 had no significant effect on the behavior and physiology of mice, indicating that it had no obvious toxicity. This study explored the application of the strigolactone analog 7FGR24 in the storage and preservation of G. elata and preliminarily established a method for the storage and preservation of G. elata, laying a foundation for the molecular mechanism of 7FGR24 in the storage and preservation of G. elata.

[Cloning and gene function of dicarboxylate-tricarboxylate carrier protein in Gastrodia elata].

The gene GeDTC encoding the dicarboxylate-tricarboxylate carrier protein in Gastrodia elata was cloned by specific primers which were designed based on the transcriptome data of G. elata. Bioinformatics analysis on GeDTC gene was carried out by using ExPASY, ClustalW, MEGA, etc. Positive transgenic plants and potato minituber were obtained by virtue of the potato genetic transformation system. Agronomic characters, such as size, weight, organic acid content, and starch content, of potato minituber were tested and analyzed and GeDTC gene function was preliminarily investigated. The results showed that the open reading frame of GeDTC gene was 981 bp in length and 326 amino acid residues were encoded, with a relative molecular weight of 35.01 kDa. It was predicted that the theoretical isoelectric point of GeDTC protein was 9.83, the instability coefficient was 27.88, and the average index of hydrophilicity was 0.104, which was indicative of a stable hydrophilic protein. GeDTC protein had a transmembrane structure and no signal peptide and was located in the inner membrane of mitochondria. The phylogenetic tree showed that GeDTC was highly homologous with DTC proteins of other plant species, among which GeDTC had the highest homology with DcDTC(XP＿020675804.1) in Dendrobium candidum, reaching 85.89%. GeDTC overexpression vector pCambia1300-35Spro-GeDTC was constructed by double digests, and transgenic potato plants were obtained by Agrobacterium-mediated gene transformation. Compared with the wild-type plants, transgenic potato minituber harvested by transplanting had smaller size, lighter weight, lower organic acid content, and no significant difference in starch content. It is preliminarily induced that GeDTC is the efflux channel of tricarboxylate and related to the tuber development, which lays a foundation for further elucidating the molecular mechanism of G. elata tuber development.