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.

[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.

[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.

[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.

[Screening of zearalenone-degrading bacteria and analysis of degradation conditions].

Zearalenone(ZEN) is a mycotoxin produced by Fusarium, possessing estrogen-like effects, carcinogenicity, and multiple toxicities. To seek more efficient and practical agents for biological detoxification and broaden their application, this study isolated 194 bacterial strains from the moldy tuberous root of Pseudostellaria heterophylla, which were co-cultured with ZEN. An efficient ZEN-degrading strain H4-3-C1 was screened out by HPLC and identified as Acinetobacter calcoaceticus by morphological observation and molecular identification. The effects of culture medium, inoculation dose, culture time, pH, and temperature on the degradation of ZEN by H4-3-C1 strain were investigated. The mechanism of ZEN degradation and the degrading effect in Coicis Semen were discussed. The degradation rate of 5 µg·mL~(-1) ZEN by H4-3-C1 strain was 85.77% in the LB medium(pH 6) at 28 ℃/180 r·min~(-1) for 24 h with the inoculation dose of 1%. The degradation rate of ZEN in the supernatant of strain culture was higher than that in the intracellular fluid and thalli. The strain was inferred to secret extracellular enzymes to degrade ZEN. In addition, the H4-3-C1 strain could also degrade ZEN in Coicis Semen. If the initial content of ZEN in Coicis Semen was reduced from 90 µg·g~(-1) to 40.68 µg·g~(-1), the degradation rate could reach 54.80%. This study is expected to provide a new strain and application technology for the biological detoxification of ZEN in food processing products and Chinese medicinal materials.

The Biosynthesis of Heterophyllin B in Pseudostellaria heterophylla From prePhHB-Encoded Precursor.

Plant cyclic peptides (CPs) are a large group of small molecule metabolites found in a wide variety of plants, including traditional Chinese medicinal plants. However, the majority of plant CPs have not been studied for their biosynthetic mechanisms, including heterophyllin B (HB), which is one of the characteristic chemical components of Pseudostellaria heterophylla. Here, we screened the precursor gene (prePhHB) of HB in P. heterophylla and functionally identified its correctness in vivo and in vitro. First, we developed a new method to screen the precursors of HB from 16 candidate linear peptides. According to transcriptome sequencing data, we cloned the genes that encoded the HB precursor peptides and confirmed that the prePhHB-encoded precursor peptide could enzymatically synthesize HB. Next, we generated the transgenic tobacco that expressed prePhHB, and the results showed that HB was detected in transgenic tobacco. Moreover, we revealed that prePhHB gene expression is positively correlated with HB accumulation in P. heterophylla. Mutations in the prePhHB gene may influence the accumulation of HB in P. heterophylla. These results suggest that HB is ribosomally synthesized and posttranslationally modified peptide (RiPP) derived from the precursor gene prePhHB-encoded precursor peptide, and the core peptide sequence of HB is IFGGLPPP in P. heterophylla. This study developed a new idea for the rapid identification of Caryophyllaceae-type CP precursor peptides via RNA-sequencing data mining.

[Study on commodity specification and grade standard and quality evaluation of Galla Chinensis].

This paper is aimed to study the commodity specification and grade standard of Galla Chinensis,for standardizing market order and guide the market circulation,and provide a basis for standardization of Galla Chinensis. With 33 samples of Galla Chinensis from market as the object of the research,the herbal textual research and market research were carried out. Then the grading indicator were selected by the descriptive statistics,principal component analysis and cluster analysis,combining with production practice,the commodity specification and grade standard of Galla Chinensis were set out. The data of moisture,ash,gallic acid as the internal index were used to analyze the relationship between the quality difference between grades and the appearance characters and the intrinsic composition. Herbal textual research and market research found that the high quality of Galla Chinensis characterized with large,complete,wall thick,grayish brown characteristics,and Galla Chinensis could be divided into gallnuts and horned gall. Through principal component analysis and cluster analysis,combining actual production and herbal record,screening,the length,diameter,single weight,the number of 500 g were determined as 4 grading indicators,the commodity specification was divided into two: gallnuts and horned gall,the grades was divided into two: selected goods and mixed goods. The result of correlation analysis showed there was no significant correlation between the internal index and the appearance characters of Galla Chinensis. The result of multiple comparison showed that the ash content of the selected goods was smaller than that of the mixed goods,but it did not reach significant. The content of gallic acid of the selected goods and the mixed goods of gallnuts were higher than selected goods of horned gall,and higher than mixed goods of horned galls. Using the length,diameter,single weight,the number of 500 g as the appearance characters index. Combining with the herbal textual research and market research,we have divided two specifications and two grades for the commodity specification and grade standard of Galla Chinensis,which can provide a basis for industry standards and national standards.

[Variation and regularity of volatile oil constituents in fruits of national medicine Cinnamomum migao].

OBJECTIVE: To compare the constituents of the volatile oil in Cinnamomum migao from different regions of southwest in China in order to evaluate the quality of C. migao. METHOD: GC-MS was employed to analyze the constituents of the volatile oil in C. migao. RESULT: The volatile oil compositions of C. migao collected from 27 of cultivation regions were obviously different. Based on the chemical differences of the volatile oil compositions, C. migao was divided into four chemotype, they were eucalyptol, eucalyptol -cyclohexene, eucalyptol -alpha-terpineol, and eucalyptol -sabinene. The eucalyptol-type was cultivated in Luodian, guizhou province and Funing regions, Yunan province. The eucalyptol-cyclohexene-type was cultivated in Zhengfeng and Wangmo regions, Guizhou province. The type of eucalyptol, eucalyptol -sabinene and eucalyptol -alpha-terpineol were cultivated in Ceheng and Libo regions, Guizhou province. CONCLUSION: Combined with the geographical distribution, It is indicated that the volatile oil compositions in fruit of C. migao may have some relations to the specie itself characteristics and different elevations environment.