Aflatoxin B1 contamination reduces the saponins content and anti-osteoporosis efficacy of the traditional medicine Radix Dipsaci.

ETHNOPHARMACOLOGICAL RELEVANCE: The Radix Dipsaci, a traditional Chinese medicine with a history spanning over 2000 years in China, is widely recognized for its hepatorenal tonic properties, musculoskeletal fortifying effects, fracture healing capabilities, and its frequent application in the treatment of osteoporosis. Like many traditional Chinese herbal medicines, preparations from Radix Dipsaci are at risk of contamination by harmful mycotoxins such as aflatoxin B1. AIMS OF THE STUDY: This study aims to evaluate the impact of aflatoxin B1 contamination on Radix Dipsaci in terms of changes in quality, efficacy of anti-osteoporosis and hepatorenal toxicity. MATERIALS AND METHODS: The contamination rates and levels of major mycotoxins were determined in 45 batches of Radix Dipsaci samples using UPLC-MS/MS analysis. The total saponin content and the levels of akebia saponin D in Radix Dipsaci and its decoctions were evaluated through high-performance liquid chromatography (HPLC) analysis. Differences in secondary metabolites between samples without any mycotoxin contamination (N-RD) and those contaminated solely by aflatoxin B1 (AFB1-RD) were compared using metabolomics sequencing and analysis. The anti-osteoporotic efficacy of Radix Dipsaci contaminated with aflatoxin B1 was assessed in a murine model of retinoic acid-induced osteoporosis by quantifying bone mineral content and bone mineral density using dual-energy X-ray absorptiometry. Additionally, the hepatorenal toxicity of Radix Dipsaci contaminated with aflatoxin B1 was evaluated using hematoxylin-eosin staining and enzyme-linked immunosorbent assay (ELISA). RESULTS: The results indicated that aflatoxin B1 (AFB1) was the most frequently detected mycotoxin, found in 37.7% of the Radix Dipsaci samples. AFB1 contamination significantly altered the secondary metabolites of Radix Dipsaci. Specifically, there was a notable decrease in the levels of total saponins and akebia saponin D in the AFB1-contaminated samples, which exhibited a negative correlation with the levels of AFB1 contamination. However, the administration of a water decoction from AFB1-contaminated Radix Dipsaci did not result in significant improvements in bone mineral density, bone mineral salt content, the trabecular number, trabecular area, proportion of trabecular bone volume/tissue volume and trabecular separation in an osteoporosis mouse model. Additionally, we observed that approximately 16.04% of AFB1 could migrate from the raw herbs into the decoction, leading to hepatocyte and kidney cell damage, as well as increased levels of the oxidative stress molecule malondialdehyde and pro-inflammatory cytokines in the liver and kidney tissues of the osteoporosis model mice. CONCLUSION: In summary, Radix Dipsaci is highly susceptible to mycotoxin contamination, particularly aflatoxin B1. The contamination of Radix Dipsaci with AFB1 not only impacts their saponin content and anti-osteoporosis effect but also induces hepatotoxicity and nephrotoxicity.

Simultaneous detection of multiple mycotoxins in Radix Dipsaci and estimation of exposure risk for consumers.

Like many traditional Chinese herbal medicines, preparations from Radix Dipsaci are at risk of contamination by harmful mycotoxins; however, there have been no reports of actual contamination. In this study, we developed an analytical method to simultaneously detect eight mycotoxins in Radix Dipsaci and estimate the exposure risk for consumers. We have developed an analytical method utilizing ultra-high performance liquid chromatography and tandem mass spectrometry to accurately determine the levels of AFB1, AFB2, AFG1, AFG2, OTA, ZEN, T-2 and ST mycotoxins in 45 batches of Radix Dipsaci sourced from major medicinal herb markets across five regions in China. We also analyzed migration of mycotoxins from the raw herbs into water decoction. Based on these results and data on human consumption of the herbal medicine, we estimated risk of exposure and acceptable exposure limits to mycotoxins in the Radix Dipsaci using the "margin of exposure (MOE)" method. Of the 45 batches of Radix Dipsaci, 48.89% contained at least one of the eight mycotoxins, 24.44% contained one, 17.78% contained two and 6.67% contained three. The most frequent mycotoxins were aflatoxin B1, present in 35.56% of batches (at 0.25-34.84 µg/kg); aflatoxin G1, 15.56% (1.99-44.05 µg/kg); and ochratoxin A, 22.22% (16.11-143.38 µg/kg). These three mycotoxins transferred from the raw herb into water decoction at respective rates of 20.20%, 29.14%, and 24.80%. The 95th percentile values of the MOE risk factors for health effects of AFB1 were below 10,000 at high doses but above 10,000 at low doses of Radix Dipsaci long-term treatment. With the reduction in duration of exposure years, the MOE values of AFB1 and AFG1 gradually reverted to within the acceptable range. The mean, 50th, and 95th percentile values of the MOE risk factors for health effects of OTA exceeded 10,000 regardless of whether consumers received a low or high dose of Radix Dipsaci treatment for durations ranging from 1 to lifetime. Based on this exposure and a typical human diet, we have estimated the respective 20-year exposure limits for Radix Dipsaci to be 5.821 µg/kg, 4.035 µg/kg, and 56.073 µg/kg for the three mycotoxins under consideration. Contamination with multiple mycotoxins is frequently observed in Radix Dipsaci, and the three most prevalent contaminants have been found to leach into water decoctions, thereby posing a potential health hazard for individuals consuming this herbal preparation. This work highlights the need to monitor herbal medicines for mycotoxin contamination in order to protect consumers.

Identification and characterization of Morganella morganii strain YC12-C3 and Enterococcus faecalis strain YC12-C10 and elucidation of its deoxynivalenol-degrading potential.

Deoxynivalenol ( DON) is one of the most harmful mycotoxins in food or feed or Traditional Chinese Medicine. An efficient and applicable method for the detoxification of DON is urgently developed. 1152 strains were isolated from the intestinal contents of crucian. Morganella morganii YC12-C3 and Enterococcus faecalis YC12-C10 were screened with the highest degradation rate of DON via HPLC methods. The optimal degradation condition of YC12-C3 and YC12-C10 is co-cultured 24 h and 36 h at 28 ℃ in LB medium with pH 7 and 1.0% inoculation dosage, respectively. LC-MS/MS and 1H NMR results show that YC12-C10 and YC12-C3 can transform DON to 3-deoxy-6-demethanol-DON, a new metabolite biotransformed from DON, by deoxidization at C3 hydroxy and de-methanal reaction at methanol moiety of C6. In addition, the DON-degradation in agricultural material assay showed that YC12-C10 and YC12-C3 can degrade 150 µg·kg-1 DON in Coix lacryma-jobi, with a degradation rate of 68.89% and 59.94%, respectively. This result shows that YC12-C10 and YC12-C3 have a sound efficiency in removing DON ability in Coix lacryma-jobi, providing a new strain resource and application technique for biological detoxification of DON in food or feed or TCM industry.

Evaluation of mycotoxins, mycobiota and toxigenic fungi in the traditional medicine Radix Dipsaci.

Medicinal herbs have been increasingly used for therapeutic purposes against a diverse range of human diseases worldwide. However, inevitable contaminants, including mycotoxins, in medicinal herbs can cause serious problems for humans despite their health benefits. The increasing consumption of medicinal plants has made their use a public health problem due to the lack of effective surveillance of the use, efficacy, toxicity, and quality of these natural products. Radix Dipsaci is commonly utilized in traditional Chinese medicine and is susceptible to contamination with mycotoxins. Here, we evaluated the mycotoxins, mycobiota and toxigenic fungi in the traditional medicine Radix Dipsaci. A total of 28 out of 63 Radix Dipsaci sample batches (44.4%) were found to contain mycotoxins. Among the positive samples, the contamination levels of AFB1, AFG1, AFG2, and OTA in the positive samples ranged from 0.52 to 32.13 µg/kg, 5.14 to 20.05 µg/kg, 1.52 to 2.33 µg/kg, and 1.81 to 19.43 µg/kg respectively, while the concentrations of ZEN and T-2 were found to range from 2.85 to 6.33 µg/kg and from 2.03 to 2.53 µg/kg, respectively. More than 60% of the contaminated samples were combined with multiple mycotoxins. Fungal diversity and community were altered in the Radix Dipsaci contaminated with various mycotoxins. The abundance of Aspergillus and Fusarium increased in the Radix Dipsaci contaminated with aflatoxins (AFs) and ZEN. A total of 95 strains of potentially toxigenic fungi were isolated from the Radix Dipsaci samples contaminated with mycotoxins, predominantly comprising Aspergillus (73.7%), Fusarium (20.0%), and Penicillium (6.3%). Through morphological identification, molecular identification, mycotoxin synthase gene identification and toxin production verification, we confirmed that AFB1 and AFG1 primarily derive from Aspergillus flavus, OTA primarily derives from Aspergillus westerdijkiae, ZEN primarily derives from Fusarium oxysporum, and T-2 primarily derives from Fusarium graminearum in Radix Dipsaci. These data can facilitate our comprehension of prevalent toxigenic fungal species and contamination levels in Chinese herbal medicine, thereby aiding the establishment of effective strategies for prevention, control, and degradation to mitigate the presence of fungi and mycotoxins in Chinese herbal medicine.

Variation in SSRs at different genomic regions and implications for the evolution and identification of Armillaria gallica.

Armillaria spp. are devastating forest pathogens. Due to its low pathogenicity and abundant genetic variation, Armillaria gallica exhibited a unique and beneficial symbiosis with Gastrodia elata, which was used as a traditional Chinese medicine. However, the variation and population structure of A. gallica populations have rarely been investigated. Hence, we analyzed the evolution and variation in simple sequence repeats (SSRs) in three Armillaria genomes: A. gallica, A. cepistipes, and A. ostoyae to assess the genetic diversity and population structure of 14 A. gallica strains. Genome analysis revealed that SSRs were more abundant in the intergenic region than the intron and exon region, as was the SSR density. Compared with other two genomes, SSR density was the lowest in exon region and largest in the intron region of A. gallica, with significant variation in genic region. There were 17 polymorphic markers in A. gallica genome was identified, with 26.7% in genic region, which is higher than that of 18.8% in the intergenic region. Moreover, a total of 50 alleles and 42 polymorphic loci were detected among these A. gallica strains. The averaged polymorphism information content (PIC) was 0.4487, ranged from 0.2577 to 0.6786. Both principal coordinate analysis (PCoA) and population structure analyses based on the genotype data of SSRs divided the strains into two clusters. The cluster I included all the strains from high-altitude G. elata producing areas and some low-altitude areas, while the strains in Cluster II originated from low-altitude G. elata producing areas. These results indicated that substantial genome-specific variation in SSRs within the genic region of A. gallica and provide new insights for further studies on the evolution and breeding of A. gallica.

Contamination of the traditional medicine Radix Dipsaci with aflatoxin B1 impairs hippocampal neurogenesis and cognitive function in a mouse model of osteoporosis.

BACKGROUND: Aflatoxin B1, which can penetrate the blood-brain barrier and kill neural cells, can contaminate traditional herbal medicines, posing a significant risk to human health. The present study examined cellular, cognitive and behavioral consequences of aflatoxin B1 contamination of the anti-osteoporotic medicine Radix Dipsaci. METHODS: A mouse model of osteoporosis was created by treating the animals with all-trans-retinoic acid. Then the animals were treated intragastically with water decoctions of Radix Dipsaci that contained detectable aflatoxin B1 or not. The animals were compared in terms of mineral density and mineral salt content of bone, production of pro-inflammatory factors, neurogenesis and microglial activation in hippocampus, as well as behavior and cognitive function. RESULTS: Contamination of Radix Dipsaci with aflatoxin B1 significantly reduced the medicine's content of bioactive saponins. It destroyed the ability of the herbal decoction to improve mineral density and mineral salt content in the bones of diseased mice, and it induced the production of the oxidative stress marker malondialdehyde as well as the pro-inflammatory cytokines interleukin-1ß and tumor necrosis factor-α. Aflatoxin B1 contamination inhibited formation of new neurons and increased the proportion of activated microglia in the hippocampus. These neurological changes were associated with anhedonia, behavioral despair, and deficits in short-term memory and social memory. CONCLUSION: Contamination of Radix Dipsaci with aflatoxin B1 not only eliminates the herbal decoction's anti-osteoporotic effects, but it also induces neurotoxicity that can lead to cognitive decline and behavioral abnormalities. Such contamination should be avoided through tightly regulated production and quality control of medicinal herbs.

Pathogen-driven Pseudomonas reshaped the phyllosphere microbiome in combination with Pseudostellaria heterophylla foliar disease resistance via the release of volatile organic compounds.

BACKGROUND: Continuous monocropping obstacles are common in plants, especially medicinal plants, resulting in disease outbreaks and productivity reductions. Foliar disease, mainly caused by Fusarium oxysporum, results in a severe decrease in the yield of Pseudostellaria heterophylla annually. Determining an effective biomethod to alleviate this disease is urgently needed to improve its productivity and quality. RESULTS: This study screened thirty-two keystone bacterial genera induced by pathogens in P. heterophylla rhizosphere soil under continuous monocropping conditions. Pseudomonas, Chryseobacterium, and Flavobacterium, referred to as the beneficial microbiota, were significantly attracted by pathogen infection. The P. palleroniana strain B-BH16-1 can directly inhibit the growth and spore formation of seven primary pathogens of P. heterophylla foliar disease by disrupting fusaric acid production via the emission of volatile organic compounds (VOCs). In addition, strain B-BH16-1 enhances the disease resistance of P. heterophylla by obliterating the pathogen and assembling beneficial microbiota. CONCLUSION: Pathogen-induced Pseudomonas reshaped phyllosphere microbial communities via direct antagonism of pathogens and indirect disruption of the pathogen virulence factor biosynthesis to enhance disease suppression and improve yields. These results show that inhibiting pathogen virulence biosynthesis to reshape the plant microbial community using disease-induing probiotics will be an innovative strategy for managing plant disease, especially under continuous monoculture conditions.

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.

The sweating process promotes toxigenic fungi expansion and increases the risk of combined contamination of mycotoxins in Radix Dipsaci.

Sweating is one of the most important processing methods of Chinese medicinal herbs. However, the high temperature and humidity environment required for sweating Chinese medicinal herbs makes it very easy for fungi to breed, especially toxigenic fungi. The mycotoxins produced by these fungi will then contaminate the Chinese medicinal herbs. In this study, we explored the changes in mycobiota, toxigenic fungi, and mycotoxins with and without sweating in Radix Dipsaci (RD), a typical representative of traditional Chinese medicine that requires processing through sweating. We also isolated and identified the toxigenic fungi from RD, whether they were subjected to sweating treatment or not, and examined their toxigenic genes and ability. The results showed that the detection rate of mycotoxins (aflatoxins, ochratoxins, zearalenone, and T-2 toxin) in RD with sweating was 36%, which was 2.25-fold higher than that in RD without sweating. We also detected T-2 toxin in the RD with sweating, whereas it was not found in the RD without sweating. The sweating process altered the fungal composition and increased the abundance of Fusarium and Aspergillus in RD. Aspergillus and Fusarium were the most frequently contaminating fungi in the RD. Morphological and molecular identification confirmed the presence of key toxigenic fungal strains in RD samples, including A. flavus, A. westerdijkiae, F. oxysporum and F. graminearum. These four fungi, respectively, carried AflR, PKS, Tri7, and PKS14, which were key genes for the biosynthesis of aflatoxins, ochratoxins, zearalenone, and T-2 toxin. The toxigenic ability of these four fungal strains was verified in different matrices. We also found that A. flavus, A. westerdijkiae, and F. oxysporum were isolated in RD both with sweating and without sweating, but their isolation frequency was significantly higher in the RD with sweating than in the RD without sweating. F. graminearum was not isolated from RD without sweating, but it was isolated from RD with sweating. These findings suggest that the sweating process promotes the expansion of toxigenic fungi and increases the risk of combined mycotoxin contamination in RD.

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.

Gut microbiota regulate stress resistance by influencing microglia-neuron interactions in the hippocampus.

Communication among the brain, gut and microbiota in the gut is known to affect the susceptibility to stress, but the mechanisms involved are unclear. Here we demonstrated that stress resistance in mice was associated with more abundant Lactobacillus and Akkermansia in the gut, but less abundant Bacteroides, Alloprevotella, Helicobacter, Lachnoclostridium, Blautia, Roseburia, Colidextibacter and Lachnospiraceae NK4A136. Stress-sensitive animals showed higher permeability and stronger immune responses in their colon, as well as higher levels of pro-inflammatory cytokines in serum. Their hippocampus also showed more extensive microglial activation, abnormal interactions between microglia and neurons, and lower synaptic plasticity. Transplanting fecal microbiota from stress-sensitive mice into naïve ones perturbed microglia-neuron interactions and impaired synaptic plasticity in the hippocampus, translating to more depression-like behavior after stress exposure. Conversely, transplanting fecal microbiota from stress-resistant mice into naïve ones protected microglia from activation and preserved synaptic plasticity in the hippocampus, leading to less depression-like behavior after stress exposure. These results suggested that gut microbiota may influence resilience to chronic psychological stress by regulating microglia-neuron interactions in the hippocampus.

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.

[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.

Reversible Adhesive Bio-Toe with Hierarchical Structure Inspired by Gecko.

The agile locomotion of adhesive animals is mainly attributed to their sophisticated hierarchical feet and reversible adhesion motility. Their structure-function relationship is an urgent issue to be solved to understand biologic adhesive systems and the design of bionic applications. In this study, the reversible adhesion/release behavior and structural properties of gecko toes were investigated, and a hierarchical adhesive bionic toe (bio-toe) consisting of an upper elastic actuator as the supporting/driving layer and lower bionic lamellae (bio-lamellae) as the adhesive layer was designed, which can adhere to and release from targets reversibly when driven by bi-directional pressure. A mathematical model of the nonlinear deformation and a finite element model of the adhesive contact of the bio-toe were developed. Meanwhile, combined with experimental tests, the effects of the structure and actuation on the adhesive behavior and mechanical properties of the bio-toe were investigated. The research found that (1) the bending curvature of the bio-toe, which is approximately linear with pressure, enables the bio-toe to adapt to a wide range of objects controllably; (2) the tabular bio-lamella could achieve a contact rate of 60% with a low squeeze contact of less than 0.5 N despite a ±10° tilt in contact posture; (3) the upward bending of the bio-toe under negative pressure provided sufficient rebounding force for a 100% success rate of release; (4) the ratio of shear adhesion force to preload of the bio-toe with tabular bio-lamellae reaches approximately 12, which is higher than that of most existing adhesion units and frictional gripping units. The bio-toe shows good adaptability, load capacity, and reversibility of adhesion when applied as the basic adhesive unit in a robot gripper and wall-climbing robot. Finally, the proposed reversible adhesive bio-toe with a hierarchical structure has great potential for application in space, defense, industry, and daily life.

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.