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.

Rhizopus oryzae causes the leaf rot disease of Epimedium sagittatum in Guizhou, China.

Epimedium sagittatum is a collective term for herbaceous plants belonging to the family Berberidaceae. Their dried leaves and stems have significant therapeutic effects on tumor inhibition, hypertension control, and coronary heart disease (Ke et al. 2023; Zhao et al. 2019). In 2021 and 2022, plants with similar leaf rot symptoms ranging from 30% to 55% was observed on E. sagittatum in Congjiang County, Guizhou province. The initial symptoms of the disease manifest locally on the leaf, with yellowing on the surface edge of the affected tissue, browning in the middle part, and brown-white discoloration in the innermost part (Supplementary Figure S1B). As the disease progresses, the entire infected leaf gradually softens, while the veins remain intact (Supplementary Figure S1C). Ultimately, the leaf withers and dehisces. The nine samples with typical symptoms were collected from Congjiang County, Guizhou province (26.598°N, 106.707°E). Twenty-seven fungi were isolated, including ten isolates of Rhizopus and seventeen isolates of seven other genera. On isolate YYH-CJ-17 many sporangia were formed and turned to a brown-gray to black color on potato dextrose agar medium (PDA) after culturing 5 days under dark at 25 ℃ (Supplementary Figure S2A and S2B). The branches of mycelium were finger-shaped or root-shaped. The sporangium was spherical or nearly spherical, 60-250 µm in diameter, and sporangiospores were elliptical or spherical and 4-8 µm in diameter. The obtained 547 bp ITS fragment (accession OR225970) and 1231 bp EF-1α region (accession OR242258) from isolate YYH-CJ-17 were compared with NR database using the BLAST tool provided by NCBI, which revealed more than 99.5% identity (query cover more than 98%) with the sequences of ITS (accessions MF522822.1) and EF-1α (accession AB281541.1) of Rhizopus oryzae Went & H.C. Prinsen Geerlings (Gao et al. 2022; Zhang et al. 2022). The phylogenetic tree constructed with the ITS and EF-1α gene sequences demonstrates that strain YYH-CJ-17 clusters with R. oryzae in the same branch and the bootstrap value was greater than 99% (Supplementary Figure S3). Based on the morphological characteristics and ITS and EF-1a sequences, the isolate YYH-CJ-17 is identified as R. oryzae. Pathogenicity tests were performed on detached healthy leaves and living plants of E. sagittatum. Healthy leaves of E. sagittatum were subjected to inoculation with isolate YYH-CJ-17 with 5 × 105 CFU mL-1 concentration in sterile culture dishes. The progression of the disease was marked by the gradual softening of the infected leaves and the expansion of the lesions, which ultimately produced black-brown sporangium (Supplementary Figure S4A). Furthermore, the E. sagittatum living plants were sprayed with 5 × 105 CFU mL-1 conidial suspension of isolate YYH-CJ-17, with ddH2O as a negative control, and then were cultivated at 25℃ and 90% humidity for 21 days in the greenhouse. This assay found that the E. sagittatum leaves treated with isolate YYH-CJ-17 exhibited the same symptoms observed on plants in fields (Supplementary Figure S4B). The fungus re-isolated from the inoculated leaves were identified as R. oryzae by ITS sequencing and were blasted with NR database, which highest matched with the sequence of ITS (accessions MF522822.1) mentioned above, thus fulfilling Koch's postulates. R. oryzae has been identified as a causative agent of a diverse array of host diseases, including leaf mildew of tobacco, fruit rot of yellow oleander and pears, and soft rot of bananas (Farooq et al. 2017; Khokhar et al. 2019; Kwon et al. 2012; Pan et al. 2021). To the best of our knowledge, this is the first report of leaf rot on E. sagittatum caused by R. oryzae in China, which will provide clear prevention and management target for the leaf rot disease of E. sagittatum.

A Probiotic Bacillus amyloliquefaciens D-1 Strain Is Responsible for Zearalenone Detoxifying in Coix Semen.

Zearalenone (ZEN) is a mycotoxin produced by Fusarium spp., which commonly and severely contaminate food/feed. ZEN severely affects food/feed safety and reduces economic losses owing to its carcinogenicity, genotoxicity, reproductive toxicity, endocrine effects, and immunotoxicity. To explore efficient methods to detoxify ZEN, we identified and characterized an efficient ZEN-detoxifying microbiota from the culturable microbiome of Pseudostellaria heterophylla rhizosphere soil, designated Bacillus amyloliquefaciens D-1. Its highest ZEN degradation rate reached 96.13% under the optimal condition. And, D-1 can almost completely remove ZEN (90 µg·g-1) from coix semen in 24 h. Then, the D-1 strain can detoxify ZEN to ZEM, which is a new structural metabolite, through hydrolyzation and decarboxylation at the ester group in the lactone ring and amino acid esterification at C2 and C4 hydroxy. Notably, ZEM has reduced the impact on viability, and the damage of cell membrane and nucleus DNA and can significantly decrease the cell apoptosis in the HepG2 cell and TM4 cell. In addition, it was found that the D-1 strain has no adverse effect on the HepG2 and TM4 cells. Our findings can provide an efficient microbial resource and a reliable reference strategy for the biological detoxification of ZEN.

Effect of symbiotic fungi-Armillaria gallica on the yield of Gastrodia elata Bl. and insight into the response of soil microbial community.

Armillaria members play important roles in the nutrient supply and growth modulation of Gastrodia elata Bl., and they will undergo severe competition with native soil organisms before colonization and become symbiotic with G. elata. Unraveling the response of soil microbial organisms to symbiotic fungi will open up new avenues to illustrate the biological mechanisms driving G. elata's benefit from Armillaria. For this purpose, Armillaria strains from four main G. elata production areas in China were collected, identified, and co-planted with G. elata in Guizhou Province. The result of the phylogenetic tree indicated that the four Armillaria strains shared the shortest clade with Armillaria gallica. The yields of G. elata were compared to uncover the potential role of these A. gallica strains. Soil microbial DNA was extracted and sequenced using Illumina sequencing of 16S and ITS rRNA gene amplicons to decipher the changes of soil bacterial and fungal communities arising from A. gallica strains. The yield of G. elata symbiosis with the YN strain (A. gallica collected from Yunnan) was four times higher than that of the GZ strain (A. gallica collected from Guizhou) and nearly two times higher than that of the AH and SX strains (A. gallica collected from Shanxi and Anhui). We found that the GZ strain induced changes in the bacterial community, while the YN strain mainly caused changes in the fungal community. Similar patterns were identified in non-metric multidimensional scaling analysis, in which the GZ strain greatly separated from others in bacterial structure, while the YN strain caused significant separation from other strains in fungal structure. This current study revealed the assembly and response of the soil microbial community to A. gallica strains and suggested that exotic strains of A. gallica might be helpful in improving the yield of G. elata by inducing changes in the soil fungal community.

Comparative Analysis of the Expression of Resistance-Related Genes Respond to the Diversity Foliar Pathogens of Pseudostellaria heterophylla.

The foliar disease, which is the primary complex disease of Pseudostellaria heterophylla, can be caused by multiple co-infecting pathogens, resulting in a significant reduction in yield. However, there is a lack of research on the relationship between co-infection of various pathogens and the response of resistance-related genes in P. heterophylla. Through the use of 18S rDNA sequencing and pathogenicity testing, it has been determined that Fusarium oxysporum, Alternaria alternata, Arcopilus aureus, Botrytis cinerea, Nemania diffusa, Whalleya microplaca, and Cladosporium cladosporioides are co-infecting pathogens responsible for foliar diseases in P. heterophylla. Furthermore, the qRT-PCR analysis revealed that F. oxysporum, A. alternata, B. cinerea, A. aureus, N. diffusa, Schizophyllum commune, C. cladosporioides, and Coprinellus xanthothrix upregulated ten, two, three, four, seven, thirteen, five, one, and six resistance-related genes, respectively. These findings suggest that a total of 22 resistance-related genes were implicated in the response to diverse fungi, and the magnitude and frequency of induction of resistance-related genes varied considerably among the different fungi. The aforementioned gene associated with resistance was found to be implicated in the response to multiple fungi, including PhPRP1, PhBDRN15, PhBDRN11, and PhBDRN3, which were found to be involved in the resistance response to nine, five, four, and four fungi, respectively. The findings indicate that the PhPRP1, PhBDRN15, PhBDRN11, and PhBDRN3 genes exhibit a broad-spectrum resistance to various fungi. Furthermore, the avirulence fungi C. xanthothrix, which is known to affect P. heterophylla, was found to prime a wide range of resistance responses in P. heterophylla, thereby enhancing its disease resistance. This study provided insight into the management strategies for foliar diseases of P. heterophylla and new genetic materials for disease-resistant breeding.

[Common diseases and drug use characteristics of Chinese herbal medicines and suggestions].

Based on the data of 56 kinds of diseases and drug use in 100 kinds of cultivated Chinese herbal medicines, this paper used frequency analysis method to count the types of diseases and their drug use characteristics, and systematically analyzed the status of drug registration and monitoring standards for disease prevention and control of Chinese herbal medicines. The results showed that 14 diseases such as root rot, powdery mildew, and drooping disease were common in the production of Chinese herbal medicines. Among the 99 pesticides reported, 67.68% were chemically synthesized, 23.23% were biological pesticides, and 9.09% were mineral pesticides. Among the reported pesticides, 92.93% of them were low toxic, with relative safety. However, 70% of the production drugs were not registered in Chinese herbal medicines, and the phenomenon of overdose was serious. The current pesticide residue monitoring standards does not match well with production drugs in China. Although the matching degree between Maximum Residue Limit of Pesticide in Food Safety National Standard(GB 2763-2021) and production drugs is more than 50%, there are few varieties of Chinese herbal medicines covered. The matching degree between Chinese Pharmacopoeia(2020 edition), Green Industry Standard of Medicinal Plants and Preparations(WM/T2-2004), and production drugs is only 1.28%. It is suggested to speed up the research and registration of Chinese herbal medicine production and further improve the pesticide residue limit standard combined with the actual production, so as to promote the high-quality development of Chinese herbal medicine industry.

[Cloning and functional verification of PhAEP gene, a key enzyme for biosynthesis of heterophyllin A in Pseudostellaria heterophylla].

This paper aimed to study the role of asparagine endopeptidase(AEP) gene in the biosynthesis mechanism of cyclic peptide compounds in Pseudostellaria heterophylla. The transcriptome database of P. heterophylla was systematically mined and screened, and an AEP gene, tentatively named PhAEP, was successfully cloned. The heterologous function verification by Nicotiana benthamiana showed that the expression of the gene played a role in the biosynthesis of heterophyllin A in P. heterophylla. Bioinformatics analysis showed that the cDNA of PhAEP was 1 488 bp in length, encoding 495 amino acids with a molecular weight of 54.72 kDa. The phylogenetic tree showed that the amino acid sequence encoded by PhAEP was highly similar to that of Butelase-1 in Clitoria ternatea, reaching 80%. The sequence homology and cyclase active site analysis revealed that the PhAEP enzyme may specifically hydrolyse the C-terminal Asn/Asp(Asx) site of the core peptide in the HA linear precursor peptide of P. heterophylla, thereby participating in the ring formation of the linear precursor peptide. The results of real-time quantitative polymerase chain reaction(RT-qPCR) showed that the expression level of PhAEP was the highest in fruits, followed by in roots, and the lowest in leaves. The heterophyllin A of P. heterophylla was detected in N. benthamiana that co-expressed PrePhHA and PhAEP genes instantaneously. In this study, the PhAEP gene, a key enzyme in the biosynthesis of heterophyllin A in P. heterophylla, has been successfully cloned, which lays a foundation for further analysis of the molecular mechanism of PhAEP enzyme in the biosynthesis of heterophyllin A in P. heterophylla and has important significance for the study of synthetic biology of cyclic peptide compounds in P. heterophylla.

Chronic exposure to aflatoxin B1 increases hippocampal microglial pyroptosis and vulnerability to stress in mice.

BACKGROUND: Chronic aflatoxin B1 (AFB1) exposure may increase the risk of multiple neuropsychiatric disorders. Stress is considered one of the main contributors to major depressive disorder. Whether and how chronic AFB1 exposure affects vulnerability to stress is unclear. METHODS: Mice were exposed for three weeks to AFB1 (100 µg/kg/d) and/or chronic mild stress (CMS). The vulnerability behaviors in response to stress were assessed in the forced swimming test (FST), sucrose preference test (SPT), and tail suspension test (TST). Microglial pyroptosis was investigated using immunofluorescence, enzyme-linked immunosorbent assays, and western blot assay in the hippocampus of mice. Hippocampal neurogenesis and the effects of AFB1-treated microglia on proliferation and differentiation of neural stem/precursor cells (NSPCs) were assessed via immunofluorescence in the hippocampus of mice. RESULTS: Mice exposed to CMS in the presence of AFB1 exhibited markedly greater vulnerability to stress than mice treated with CMS or AFB1 alone, as indicated by reduced sucrose preference and longer immobility time in the forced swimming test. Chronic aflatoxin B1 exposure resulted in changes in the microglial morphology and increase in TUNEL+ microglia and GSDMD+ microglia in the hippocampal dentate gyrus. When mice were exposed to both CMS and AFB1, pyroptosis-related molecules (such as NLRP3, caspase-1, GSDMD-N, and interleukin-1ß) were significantly upregulated in the hippocampus. These molecules were also significantly enhanced by AFB1 in primary microglial cultures. AFB1-treated mice showed decrease in the numbers of BrdU+, BrdU-DCX+, and BrdU-NeuN+ cells in the hippocampal dentate gyrus, as well as the percentages of BrdU+ cells that were NeuN+ in the presence or absence of CMS when compared with vehicle-treated mice. The combination of AFB1 and CMS exacerbated these effects to an even greater extent. The number of DCX+ cells correlated negatively with the percentage of ameboid microglia, TUNEL+ microglia and GSDMD+ microglia in the hippocampal dentate gyrus. AFB1-treated microglia suppressed the proliferation and neuronal differentiation of NSPCs in vitro. CONCLUSION: Chronic AFB1 exposure induces microglial pyroptosis, promoting an adverse neurogenic microenvironment that impairs hippocampal neurogenesis, which may render mice more vulnerable to stress.

[Contamination status and exposure risk of mycotoxins in Coicis Semen].

By investigating the contamination status and predicting the exposure risk of mycotoxin in Coicis Semen, we aim to provide guidance for the safety supervision of Chinese medicinal materials and the formulation(revision) of mycotoxin limit standards. The content of 14 mycotoxins in the 100 Coicis Semen samples collected from five major markets of Chinese medicinal materials in China was determined by UPLC-MS/MS. The probability evaluation model based on Monte Carlo simulation method was established after Chi-square test and One-way ANOVA of the sample contamination data. Health risk assessment was performed on the basis of margin of exposure(MOE) and margin of safety(MOS). The results showed that zearalenone(ZEN), aflatoxin B_1(AFB_1), deoxynivalenol(DON), sterigmatocystin(ST), and aflatoxin B_2(AFB_2) in the Coicis Semen samples had the detection rates of 84%, 75%, 36%, 19%, and 18%, and the mean contamination levels of 117.42, 4.78, 61.16, 6.61, and 2.13 µg·kg~(-1), respectively. According to the limit standards in the Chinese Pharmacopoeia(2020 edition), AFB_1, AFs and ZEN exceeded the standards to certain extents, with the over-standard rates of 12.0%, 9.0%, and 6.0%, respectively. The exposure risks of Coicis Semen to AFB_1, AFB2, ST, DON, and ZEN were low, while 86% of the samples were contaminated with two or more toxins, which needs more attention. It is suggested that the research on the combined toxicity of different mycotoxins should be strengthened to accelerate the cumulative exposure assessment of mixed contaminations and the formulation(revision) of toxin limit standards.

Volatiles from Pseudomonas palleroniana Strain B-BH16-1 Suppress Aflatoxin Production and Growth of Aspergillus flavus on Coix lacryma-jobi during Storage.

Semen coicis is not only a traditional Chinese medicine (TCM), but also a typical food in China, with significant medical and healthcare value. Because semen coicis is rich in starch and oil, it can be easily contaminated with Aspergillus flavus and its aflatoxins (AFs). Preventing and controlling the contamination of semen coicis with Aspergillus flavus and its aflatoxins is vital to ensuring its safety as a drug and as a food. In this study, the endosphere bacteria Pseudomonas palleroniana strain B-BH16-1 produced volatiles that strongly inhibited the mycelial growth and spore formation activity of A. flavus. Gas chromatography-mass spectrometry profiling revealed three volatiles emitted from B-BH16-1, of which 1-undecene was the most abundant. We obtained authentic reference standards for these three volatiles; these significantly reduced mycelial growth and sporulation in Aspergillus, with dimethyl disulfide showing the most robust inhibitory activity. Strain B-BH16-1 was able to completely inhibit the biosynthesis of aflatoxins in semen coicis samples during storage by emitting volatile bioactive components. The microscope revealed severely damaged mycelia and a complete lack of sporulation. This newly identified plant endophyte bacterium was able to strongly inhibit the sporulation and growth of Aspergillus and the synthesis of associated mycotoxins, thus not only providing valuable information regarding an efficient potential strategy for the prevention of A. flavus contamination in TCM and food, but potentially also serving as a reference in the control of toxic fungi.

Genome Resource for Acinetobacter schindleri H4-3-C1: An Endophyte of Pseudostellaria heterophylla with Degradation Activity to Toxins Produced by Fungal Pathogens.

Acinetobacter schindleri is an endophyte of Pseudostellaria heterophylla, a traditional Chinese herbal plant. It has high degradation activity to toxins produced by fungal pathogen Fusarium graminearum. Here, we deployed PacBio single-molecule real-time long-read sequencing technology to generate a complete genome assembly for the Acinetobacter schindleri H4-3-C1 strain and obtained 1.59 Gb of clean reads. These reads were assembled to a single circular DNA chromosome with a length of 3,265,024 bp, and no plasmid was found in the genome. Totals of 3,193 coding sequences, 91 transfer RNA, 21 ribosomal RNA, and 75 small RNAs were identified in the genome. This high-quality genome assembly and gene annotation resource will facilitate the excavation of the zearalenone degradation gene and provide valuable resources for preventing and controlling toxigenic fungal diseases of P. heterophylla. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Akebia saponin D protects hippocampal neurogenesis from microglia-mediated inflammation and ameliorates depressive-like behaviors and cognitive impairment in mice through the PI3K-Akt pathway.

Given the ability of akebia saponin D (ASD) to protect various types of stem cells, in the present study, we hypothesized that ASD could promote the proliferation, differentiation, and survival of neural stem/precursor cells (NSPCs), even in a microglia-mediated inflammatory environment, thereby mitigating inflammation-related neuropsychopathology. We established a mouse model of chronic neuroinflammation by exposing animals to low-dose lipopolysaccharide (LPS, 0.25 mg/kg/d) for 14 days. The results showed that chronic exposure to LPS strikingly reduced hippocampal levels of PI3K and pAkt and neurogenesis in mice. In the presen of a microglia-mediated inflammatory niche, the PI3K-Akt signaling in cultured NSPCs was inhibited, promoting their apoptosis and differentiation into astrocytes, while decreasing neurogenesis. Conversely, ASD strongly increased the levels of PI3K and pAkt and stimulated NSPC proliferation, survival and neuronal differentiation in the microglia-mediated inflammatory niche in vitro and in vivo. ASD also restored the synaptic function of hippocampal neurons and ameliorated depressive- and anxiety-like behaviors and cognitive impairment in mice chronically exposed to LPS. The results from network pharmacology analysis showed that the PI3K-AKT pathway is one of the targets of ASD to against major depressive disorder (MDD), anxiety and Alzheimer's disease (AD). And the results from molecular docking based on computer modeling showed that ASD is bound to the interaction interface of the PI3K and AKT. The PI3K-Akt inhibitor LY294002 blocked the therapeutic effects of ASD in vitro and in vivo. These results suggested that ASD protects NSPCs from the microglia-mediated inflammatory niche, promoting their proliferation, survival and neuronal differentiation, as well as ameliorating depressive- and anxiety-like behaviors and cognitive impairment by activating the PI3K-AKT pathway. Our work suggests the potential of ASD for treating Alzheimer's disease, depression and other cognitive disorders involving impaired neurogenesis by microglia-mediated inflammation.

Soil Microbial Communities Affect the Growth and Secondary Metabolite Accumulation in Bletilla striata (Thunb.) Rchb. f.

Bletilla striata (Thunb.) Rchb.f. is a perennial herb belonging to the Orchidaceae family. Its tubers are used in traditional Chinese medicine to treat gastric ulcers, inflammation, silicosis tuberculosis, and pneumogastric hemorrhage. It has been reported that different soil types can affect the growth of B. striata and the accumulation of secondary metabolites in its tubers, but the biological mechanisms underlying these effects remain unclear. In this study, we compared agronomic traits and the accumulation of secondary metabolites (extractum, polysaccharide, total phenol, militarine) in B. striata grown in sandy loam or sandy clay soil. In addition, we compared physicochemical properties and microbial communities between the two soil types. In pot experiments, we tested how irradiating soil or transplanting microbiota from clay or loam into soil affected B. striata growth and accumulation of secondary metabolites. The results showed that sandy loam and sandy clay soils differed significantly in their physicochemical properties as well as in the structure and composition of their microbial communities. Sandy loam soil had higher pH, SOM, SOC, T-Ca, T-N, T-Mg, T-Mn, T-Zn, A-Ca, A-Mn, and A-Cu than sandy clay soil, but significantly lower T-P, T-K, T-Fe, and A-P content. Sandy loam soil showed 7.32% less bacterial diversity based on the Shannon index, 19.59% less based on the Ace index, and 24.55% less based on the Chao index. The first two components of the PCoA explained 74.43% of the variation in the bacterial community (PC1 = 64.92%, PC2 = 9.51%). Similarly, the first two components of the PCoA explained 58.48% of the variation in the fungal community (PC1 = 43.67%, PC2 = 14.81%). The microbiome associated with sandy clay soil can promote the accumulation of militarine in B. striata tubers, but it inhibits the growth of B. striata. The accumulation of secondary metabolites such as militarine in B. striata was significantly higher in sandy clay than in sandy loam soil. Conversely, B. striata grew better in sandy loam soil. The microbiome associated with sandy loam soil can promote the growth of B. striata, but it reduces the accumulation of militarine in B. striata tubers. Pot experiment results further confirmed that the accumulation of secondary metabolites such as militarine was higher in soil transplanted with loam microbiota than in soil transplanted with clay microbiota. These results may help guide efforts to improve B. striata yield and its accumulation of specific secondary metabolites.

Gasdermin D-mediated microglial pyroptosis exacerbates neurotoxicity of aflatoxins B1 and M1 in mouse primary microglia and neuronal cultures.

Aflatoxin B1 (AFB1) disrupts the blood-brain barrier by poisoning the vascular endothelial cells and astrocytes that maintain it. It is important to examine whether aflatoxin B1 or its metabolite, aflatoxin M1 (AFM1), affect microglia, which as the "immune cells" in the brain may amplify their damaging effects. Here we evaluated the toxicity of AFB1 and AFM1 against primary microglia and found that both aflatoxins decreased the viability of primary microglia and increased the leakage of lactate dehydrogenase, gamma-H2AX expression, nuclear lysis, necrosis and apoptosis in a dose-dependent manner. The potential contribution of microglia to the toxic effects of aflatoxins was assessed in transwell co-culture experiments involving microglia, neurons, astrocytes, oligodendrocytes or neural stem/precursor cells. And we found that the toxic effects of both aflatoxins on various types of nervous system cells were greater in the presence of microglia than in their absence. We also found that both aflatoxins induced gasdermin D-mediated microglial pyroptosis and inflammatory cytokine expression by activating the NLRP3 inflammasome. Blockade of gasdermin D activity in AFB1- or AFM1-treated primary microglia using dimethyl fumarate (DMF) reduced the release of IL-1ß, IL-18 and nitric oxide, as well as the neurotoxicity of microglia-conditioned medium to neurons, astrocytes, oligodendrocytes and neural stem/precursor cells. These data suggested that the toxicity of AFB1 and AFM1 on various cells of the central nervous system is due, remarkably, the gasdermin D-mediated microglial pyroptosis exacerbates their neurotoxicity.

[Thoughts and suggestions on ecological cultivation of Gastrodia elata].

Due to the special biological characteristics, Gastrodia elata suffers from high resource consumption and low utilization rate in modern agricultural production, which significantly block the green and healthy development of this industry. Based on the theory and technology in ecological cultivation of Chinese medicinal materials, this study analyzed the challenges in ecological cultivation of G. elata, such as waste of fungus material, a few cultivation modes available, continuous cropping obstacles, frequent occurrence of diseases, and poor stability of ecological structure. According to the production practice, the following suggestions were proposed for ecological cultivation of G. elata: following the principle of environmental protection and no pollution, selecting suitable habitats to yield high-quality medicinal materials, committing to green control of diseases and pests, upgrading industrial structure to maximize the benefits, establishing a sound mechanism for protecting the genetic diversity of wild G. elata, carrying out simulative habitat cultivation to improve medicinal material quality, adopting science-based planning of fungus resources to relieve forestry pressure, enhancing the recycling and utilization of fungus materials, and applying diversified cultivation modes to improve the stability of ecological structure. The result is expected to provide a reference for the quality development of G. elata industry.

[Isolation, identification, and pathogenicity research of brown rot pathogens from Gastrodia elata].

Brown rot is a common disease in the cultivation and production of Gastrodia elata, but its pathogens have not been fully revealed. In this study, the pathogenic fungi were isolated and purified from tubers of 77 G. elata samples with brown rot. Pathogens were identified by the pathogenicity test and morphological and molecular identification. The pathogenicity of each pathogen and its inhibitory effects on Armillaria gallica were compared. The results showed that 119 strains of fungi were isolated from tubers of G. elata infected with brown rot. Among them, the frequency of separation of Ilyonectria fungi was as high as 42.01%. The pathogenicity test showed that the pathogenicity characteristics of six strains of fungi were consistent with the natural symptoms of brown rot in G. elata. The morphological and molecular identification results showed that the six strains belonged to I. cyclaminicola and I. robusta in the Nectriaceae family of Sordariomycetes class, respectively. Both types of fungi could produce pigments, conidia, and chlamycospore, and the growth rate of I. cyclaminicola was significantly higher than that of I. robusta. The comparison of pathogenicity showed that the spots formed by I. cyclaminicola inoculation were significantly larger than those of I. robusta inoculation, suggesting I. cyclaminicola was superior to I. robusta in pathogenicity. The results of confrontation culture showed that I. cyclaminicola and I. robusta could signi-ficantly inhibit the germination and cordage growth of A. gallica. A. gallica also inhibited the growth of pathogens, and I. cyclaminicola was less inhibited as compared with I. robusta. The results of this study revealed for the first time that I. cyclaminicola and I. robusta were the pathogens responsible for G. elata brown rot.

[Utilization of used fungus-growing materials of Gastrodia elata].

This study aims to explore the resource utilization of used fungus-growing materials produced in the cultivation of Gastrodia elata. To be specific, based on the production practice, this study investigated the recycling mechanism of used fungus-growing materials of G. elata by Phallus inpudicus. To screen edible fungi with wide adaptability, this study examined the allelopathic effects of Armillaria mellea secretions on P. impudicus and 6 kinds of large edible fungi and the activities of enzymes related to degradation of the used fungus-growing materials of G. elata. The results showed that P. impudicus can effectively degrade cellulose, hemicellulose, and lignin in used fungus-growing materials of G. elata. The cellulase activity of A. mellea was significantly higher than that of P. impudicus, and the activities of lignin peroxidase, polyphenol oxidase, and xylanase of P. impudicus were significantly higher than those of A. mellea, which was the important reason why A. mellea and P. impudicus used different parts and components of the used fungus-growing materials to absorb carbon sources and develop ecological niche differences. The growth of P. impudicus was significantly inhibited on the used fungus-growing materials of G. elata. The secretions of A. mellea had allelopathic effects on P. impudicus and other edible fungi, and the allelopathic effects were related to the concentration of allelopathy substances. The screening result showed that the growth and development of L. edodes and A. auricular were not significantly affected by 30% of A. mellea liquid, indicating that they had high resistance to the allelopathy of A. mellea. The results showed that the activities of extracellular lignin peroxidase, polyphenol oxidase, and xylanase of the two edible fungi were similar to those of P. impudicus, and the cellulase activity was higher than that of P. impudicus. This experiment can be further verified by small-scale production tests.

Pathogen-Mediated Assembly of Plant-Beneficial Bacteria to Alleviate Fusarium Wilt in Pseudostellaria heterophylla.

Fusarium wilt (FW) is a primary replant disease that affects Pseudostellaria heterophylla (Taizishen) and is caused by Fusarium oxysporum, which occurs widely in China under the continuous monocropping regime. However, the ternary interactions among the soil microbiota, P. heterophylla, and F. oxysporum remain unknown. We investigated the potential interaction relationship by which the pathogen-mediated P. heterophylla regulates the soil and the tuberous root microbiota via high-throughput sequencing. Plant-pathogen interaction assays were conducted to measure the arrival of F. oxysporum and Pseudomonas poae at the tuberous root via qPCR and subsequent seedling disease incidence. A growth assay was used to determine the effect of the tuberous root crude exudate inoculated with the pathogen on P. poae. We observed that pathogen-mediated P. heterophylla altered the diversity and the composition of the microbial communities in its rhizosphere soil and tuberous root. Beneficial microbe P. poae and pathogen F. oxysporum were significantly enriched in rhizosphere soil and within the tuberous root in the FW group with high severity. Correlation analysis showed that, accompanied with FW incidence, P. poae co-occurred with F. oxysporum. The aqueous extract of P. heterophylla tuberous root infected by F. oxysporum substantially promoted the growth of P. poae isolates (H1-3-A7, H2-3-B7, H4-3-C1, and N3-3-C4). These results indicated that the extracts from the tuberous root of P. heterophylla inoculated with F. oxysporum might attract P. poae and promote its growth. Furthermore, the colonization assay found that the gene copies of sucD in the P. poae and F. oxysporum treatment (up to 6.57 × 1010) group was significantly higher than those in the P. poae treatment group (3.29 × 1010), and a pathogen-induced attraction assay found that the relative copies of sucD of P. poae in the F. oxysporum treatment were significantly higher than in the H2O treatment. These results showed that F. oxysporum promoted the colonization of P. poae on the tuberous root via F. oxysporum mediation. In addition, the colonization assay found that the disease severity index in the P. poae and F. oxysporum treatment group was significantly lower than that in the F. oxysporum treatment group, and a pathogen-induced attraction assay found that the disease severity index in the F. oxysporum treatment group was significantly higher than that in the H2O treatment group. Together, these results suggest that pathogen-mediated P. heterophylla promoted and assembled plant-beneficial microbes against plant disease. Therefore, deciphering the beneficial associations between pathogen-mediated P. heterophylla and microbes can provide novel insights into the implementation and design of disease management strategies.

[Pesticide residues in Chinese medicinal materials and suggestions for safety].

The present study counted the frequency of detection technologies and monitoring frequency of pesticide species by frequency analysis based on the 28 258 pieces of data on pesticide content of Chinese medicinal materials in CNKI, calculated the detection rate and exceeding rate of different types of pesticides, and systematically analyzed the pesticide residue pollution of Chinese medicinal materials. The results showed that there were 40 types of pesticides with detection rates higher than 10%, where new pesticides such as organochlorines and nicotine accounted for 55%, and organic phosphorus, pyrethroids, and carbamates accounted for 17.5%, 15.0%, and 12.5%, respectively. Seventeen types of pesticides exceeded the standard to varying degrees, including 12 types(70.59%) with exceeding rates not higher than 5%, four types(23.53%) with exceeding rates in the range of 5%-10%, and one type(5.88%) with an exceeding rate higher than 10%. As revealed by the analysis results of the past five years, the pesticide residue pollution of Chinese medicinal materials showed a downward trend. Compared with the conditions at worst, organochlorines decreased by about 2/3 in detection rate and 47.23% in exceeding rate, carbamates by about 1/2 in detection rate and 10.78% in exceeding rate, organic phosphorus by 3/4 in detection rate and 7.22% in exceeding rate, pyrethroids by 1/2 in detection rate and 11.05% in exceeding rate, and other types by about 1/2 in detection rate but not exceeded the standard. In general, pesticide residues in Chinese medicinal materials and safety have been significantly improved. However, pesticide residues are still important factors affecting the quality and safety of Chinese medicinal materials. It is suggested to further improve the control standards of pesticide residues in Chinese medicinal materials, strengthen the monitoring of pesticides used in practical production, and promote the ecological planting mode to facilitate the high-quality development of the Chinese medicinal material industry.

[Methods for synchronous detection of 14 mycotoxins in Pseudostellariae Radix and investigation on its contamination].

This study aimed to establish a method for synchronous detection of 14 mycotoxins in Pseudostellariae Radix and investigate its contamination with mycotoxins, so as to provide technical guidance for monitoring the quality of Chinese medicinal materials and medication safety. The sample was extracted with 80% acetonitrile in an oscillator for 1 h, purified using the modified QuEChERS purifying agent(0.1 g PSA + 0.3 g C_(18) + 0.3 g MgSO_4), and separated on a Waters HSS T3 chromatographic column(2.1 mm×100 mm, 1.8 µm). The gradient elution was carried out with 0.1% formic acid in water and acetonitrile, followed by the scanning in the multi-reaction monitoring(MRM) mode and the analysis of mycotoxin contamination in 26 Pseudostellariae Radix samples. The recovery rates of the established method were within the range of 82.17%-113.6%, with the RSD values less than 7% and the limits of quantification(LOQ) being 0.019-0.976 µg·kg~(-1). The detection rate of 14 mycotoxins in 26 batches of medicinal materials was 53.85%. The detection rate of sterigmatocystin(ST) was the highest, followed by those of zearalenone(ZEN), aflatoxin G_2(AFG_2), fumonisin B_1(FB_1), HT-2 toxin, and nivalenol(NIV). Their respective detection rates were 38.46%, 26.92%, 23.08%, 11.54%, 11.54%, and 7.69%, with the pollution ranges being 1.48-69.65, 0.11-31.05, 0.11-0.66, 0.28-0.83, 20.86-42.56, and 0.46-1.84 µg·kg~(-1), respectively. The established method for the detection of 14 mycotoxins is accurate, fast and reliable. The research results have very important practical significance for guiding the monitoring and prevention and control of exogenous fungal contamination of Chinese medicinal materials.