First Report of Alternaria Leaf Blight Caused by Alternaria alternata on Phedimus aizoon in China.

Phedimus aizoon is native to east Asian countries that including China, Siberia, Korea, Mongolia, and Japan. In China, the plant is highly valued for use in folk medicine, for detoxification and analgesia, blood pressure, hemostasis, and used as an ornamental. In August 2021, a leaf spot and blight disease were observed on P. aizoon in a 120-ha field in Pizhou, Jiangsu Province, China where disease incidence reached 90%, and almost every leaf was withered. Early symptoms appeared as dark brown lesions on leaf margins that enlarged and coalesced to form large necrotic areas. In efforts to determine the cause of the disease, ten symptomatic leaves were randomly collected from ten different plants at the site. Diseased leaf pieces that measured 5 mm2 were disinfected in 75% ethyl alcohol for 30 s and 7% NaOCl for 60 s, rinsed three times in sterile distilled water, and placed on potato dextrose agar (PDA). Ten fungal isolates obtained by single-spore isolations were selected for further study. These isolates produced colonies that measured 70 to 82 mm in diameter after 7 days growth on PDA. Colonies were black to brown in color with gray-white aerial hyphae on their surfaces. The isolates produced conidia that were ovate to pear-shaped, brown to black in color, with 1 to 4 transverse septa and 0 to 1 oblique septa, smooth surfaced, parietal cells extending into the beak, and measured 10 to 35.5 × 5.0 to 12.5 µm. Conidiophores were brown, erect or curved, branched, with pronounced spore marks, and measured 7.5 to 37.5 × 2.5 to 5.0 µm. All ten fungal isolates were morphologically similar to Alternaria alternata (Simmons 2007). Two representative isolates FC01 and FC02 were used for molecular identification. The internal transcribed spacer (ITS) region, RNA polymerase second largest subunit (RPB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1), and Alternaria major allergen (Alt a 1) were amplified with the primers ITS4/ITS5, RPB2-5F2/RPB2-7CR (Khodaei and Arzanlou 2013), gpd1/gpd2, EF1-728F/EF1-986R (Nishikawa and Nakashima 2020) and Alt-for/Alt-rev (Woudenberg et al. 2015). The resulting sequences were deposited in GenBank (ITS, ON584560, ON564492; RPB2, ON729984, ON703241; GAPDH, ON652866, ON652867; TEF1, ON652868, ON652869; Alta1, ON652870, ON652871). Phylogenetic analyses showed 100% identity between FC01 and FC02 and the type strain CBS 916.96. Thus, the fungus was identified as A. alternata based on morphology and molecular analysis. Pathogenicity tests were done by spraying conidial suspensions containing 106 conidia per ml of A. alternata isolates FC01 and FC02 on leaves of five healthy P. aizoon plants, separately. Five control plants were sprayed with distilled water and both sets of plants covered with plastic bags and placed in a greenhouse maintained at 25° C. Plastic bags were removed from plants after 48 h. Dark brown lesions developed on inoculated plants after 16 days and control plants remained symptomless. The pathogenicity tests were conducted three times. A. alternata was reisolated and identified based on morphological and molecular traits, thus fulfilling Koch's postulates. To our knowledge, this is the first report of A. alternata causing leaf blight on P. aizoon in China and worldwide. Based on the plant's medicinal value, further studies should be directed toward control of this disease.

First report of Alternaria alternata causing leaf blight on Actaea dahurica in China.

In July 2019, leaf blight on Actaea dahurica, a plant with high value in Chinese traditional medicine, was discovered in a 2 ha planting area in Heilongjiang Province (129.6°E, 44.6°N), China. Disease incidence was 90% in the field. Symptoms consisted of irregular black spots with gray margins on both sides of the leaf, often at the leaf margin, mostly on the older leaves. To isolate the pathogen, ten diseased leaves were randomly collected, surface disinfested, and 5 x 5 mm segments were removed from the margin of the lesions. Leaf segments were placed onto potato dextrose agar (PDA) and incubated at 25 ℃ for 7 days. Ten pure cultures with the same morphological characteristics were obtained from three leaves showing typical symptoms. Cultures on PDA initially had a cottony mycelium, white-gray to gray. After two to three weeks of growth, mycelium color changed from gray to black. Conidiophores were clustered, dark at the base, tapering to the apex, born from simple sublates, unbranched, with 1 to 5 septa, and 70.4-530.3 × 5-7.5 µm in size. Conidia were 12.5-82.5 × 5.2-20.3 µm, usually in chains, had 2 to 8 transverse septa, 0 to 4 longitudinal or oblique septa, and a smooth brown surface. Simple, pale, vimineous or verrucous beaks developed from the apical cells with 0 to 4 septa. The morphological characteristics were consistent with Alternaria species (Simmons, 2007). To fulfill Koch's postulates, pathogenicity tests were carried out on three-month-old A. dahurica plants. A spore suspension was prepared from PDA cultures of isolates SM0101 and SM0102 and adjusted to 105 spores/mL using a hemocytometer. Each leaf was sprayed with 2 mL of the spore suspension, then incubated at 25 ℃ for 7 days. The same number of healthy A. dahurica plants were sprayed with sterile water as a control. After 7 days, small brown necrotic spots appeared on inoculated plants, but the control group showed no symptoms. A fungus with the same characteristics as that used for inoculation was re-isolated from the lesions. This experiment was replicated three times, and the results of each experiment were consistent. Genomic DNA was extracted from isolates SM0101 and SM0102 and used for PCR amplification of the rDNA internal transcribed spacer regions (ITS), RNA polymerase II gene (RPB2) and Alternaria allergen a 1 (Alt a 1) gene sequences using the primer pairs ITS1/ITS4 (White et al. 1990), RPB2-5F2/RPB2-7CR (Khodaei and Arzanlou, 2013) and Alt-for/Alt-rev (Hong et al. 2005), respectively. The ITS (OL703042, OL616086), RPB2 (OL703043, OL898416), and Alt a 1 sequences (OL616087, OL898415) were deposited in GenBank. The sequences obtained in this study had the highest match to corresponding sequences of Alternaria alternata CBS 916.96 (AF347031, KC584375, AY563301). For isolate SM0101 the matches were ITS (461/461 bp), RPB2 (897/985 bp), and Alt a 1 (488/488 bp). For isolate SM0202 the matches were ITS (457/457 bp), RPB2 (893/985 bp), and Alt a 1 (484/484 bp). A phylogenetic analysis was performed using MEGA7 software. The alignment included sequences from 16 ex-type Alternaria species and the two isolates causing leaf blight on A. dahurica. Branch supports were calculated with 1,000 bootstrap replicates, and phylogenetic inference was performed using the maximum likelihood estimation. The fungus isolated from A. dahurica clustered with A. alternata. This is the first report of A. alternata on A. dahurica in the world. This report will help to identify the disease symptoms in the field and provides a basis for research into the occurrence, distribution, and control of leaf blight on A. dahurica.

Comparison of Two Different Astragali Radix by a ¹H NMR-Based Metabolomic Approach.

Astragali Radix (AR) is a commonly used herbal drug in traditional chinese medicine and is widely used for the treatment of diabetes, cardiovascular diseases, nephropathy, and neuropathy. The main source of AR in China is the dried root of Astragalus membranaceus var. mongholicus (Bge.) Hsiao, and both cultivated and wild ARs are used clinically. A systematic comparison of cultivated AR (GS-AR) and wild AR (SX-AR) should be performed to ensure the clinical efficacy and safety. In this study, the chemical composition of the two different ARs, which were collected in the Shanxi (wild) and Gansu (cultivated) provinces, were compared by NMR-based metabolic fingerprint coupled with multivariate analysis. The SX-AR- and GS-AR-induced metabolic changes in the endogenous metabolites in mice were also compared. The results showed that SX-AR and GS-AR differed significantly not only in the primary metabolites but also in the secondary metabolites. However, alterations among the endogenous metabolites in the serum, lung, liver, and spleen were relatively small. This study provided a novel and valuable method for the evaluation of the consistency and diversity of herbal drugs, and further studies should be conducted on the difference in polysaccharides as well as the biological effects between the two kinds of AR.

Transcriptome sequencing of Codonopsis pilosula and identification of candidate genes involved in polysaccharide biosynthesis.

BACKGROUND: Codonopsis pilosula (Franch.) Nannf. is one of the most widely used medicinal plants. Although chemical and pharmacological studies have shown that codonopsis polysaccharides (CPPs) are bioactive compounds and that their composition is variable, their biosynthetic pathways remain largely unknown. Next-generation sequencing is an efficient and high-throughput technique that allows the identification of candidate genes involved in secondary metabolism. PRINCIPAL FINDINGS: To identify the components involved in CPP biosynthesis, a transcriptome library, prepared using root and other tissues, was assembled with the help of Illumina sequencing. A total of 9.2 Gb of clean nucleotides was obtained comprising 91,175,044 clean reads, 102,125 contigs, and 45,511 unigenes. After aligning the sequences to the public protein databases, 76.1% of the unigenes were annotated. Among these annotated unigenes, 26,189 were assigned to Gene Ontology categories, 11,415 to Clusters of Orthologous Groups, and 18,848 to Kyoto Encyclopedia of Genes and Genomes pathways. Analysis of abundance of transcripts in the library showed that genes, including those encoding metallothionein, aquaporin, and cysteine protease that are related to stress responses, were in the top list. Among genes involved in the biosynthesis of CPP, those responsible for the synthesis of UDP-L-arabinose and UDP-xylose were highly expressed. SIGNIFICANCE: To our knowledge, this is the first study to provide a public transcriptome dataset prepared from C. pilosula and an outline of the biosynthetic pathway of polysaccharides in a medicinal plant. Identified candidate genes involved in CPP biosynthesis provide understanding of the biosynthesis and regulation of CPP at the molecular level.

[Effect of adjuvant therapy with Dingguier umbilical paste on intestinal flora of diarrhea infants and its action characteristics].

The applicator therapy is a unique method to treat infant diarrhea in traditional Chinese medicines and widely applied in clinical practice. Currently, many researchers have proved the rationality of the therapy based on the traditional Chinese medicine mechanism and on the data from clinical practice, but its action mechanism is uncertain at present. In this study, with the assistance of pediatric practitioners, the automated ribosomal intergenic-spacer analysis (ARISA) was adopted to study the effect of the adjuvant therapy with Dingguier umbilical paste on intestinal flora of diarrhea infants, in which Dingguier umbilical paste served as the adjuvant therapy in oral traditional Chinese medicines and fecal samples of infants with different diarrhea symptoms were collected and used as the study materials. The results showed that the adjuvant therapy had a significant effect on the shift of intestinal flora, which was associated with the decrease in the similarity difference to the normal control group and the increase in the number of operational taxonomic units (OTUs) shared with the normal control group. Additionally, adjuvant therapy with Dingguier umbilical paste also showed long action duration and increased OTUs number. These results indicated that Dingguier umbilical paste has the effect in restoring the micro-ecosystem of unbalanced intestinal bacteria. Intestinal flora may be one of major targets for the applicator therapy for the infant diarrhea, but not for the single oral traditional Chinese medicine for infant diarrhea.

(1)H NMR based metabolomic study of the antifatigue effect of Astragali Radix.

Astragali Radix (Huangqi), a well-known traditional Chinese medicinal plant, has been used for centuries as a reinforcing vital energy herb in China. In this study, the antifatigue effect of Astragali Radix was investigated by an (1)H NMR based metabolomic approach coupled with multivariate statistical analysis. The results indicated that oral administration of Astragali Radix at a dose of 3 g kg(-1) body weight could significantly prolong the exhaustive swimming time of rats, and alter the serum and urine metabolome. The rats treated by Astragali Radix extracts showed higher levels of glucose, creatine, glycine, citrate, guanidinoacetate, allantoin, dimethylglycine, dimethylamine (DMA), creatinine, betaine and malate and lower levels of lactate, choline species, O-acetylated glycoproteins (OAG), glycerol, ß-OH-butyrate, α-ketoglutarate, trimethylamine (TMA) and hippurate. And these metabolic changes indicated that Astragali Radix facilitated recovery from fatigue by regulating the glycometabolism, lipid metabolism and energy metabolism. Chemical analysis showed that various components were present in the Astragali Radix extracts, and the bioactive compounds responsible for the antifatigue activity should be further investigated.

[Status and analysis of Astragali Radix resource in China].

To explore the status of the resources of Astragali Radix, a survey on its germplasm resources was carried out. Some conclusions can be drawn for Astragali Radix: the major source is the cultivated Astragalus mongolicus. The new major cultivation areas for A. mongolicus and A. membranaceus are Shandong and Gansu province. The semi-wildly planting model in Shanxi province maintains the genuine trait of Astragali Radix, but its yield is limited, and now a combination model has been developed. The major problems for Astragali Radix are the selection of planting sites, the rot root and difficulty in collecting and processing. Several developmental proposals for Astragali Radix were put forward including rational distribution of planting areas, establishment of standard system, development and standardization of producing technologies.

Metabolomic profiling of the antitussive and expectorant plant Tussilago farfara L. by nuclear magnetic resonance spectroscopy and multivariate data analysis.

This study aims to find metabolites responsible for antitussive and expectorant activities of Tussilago farfara L. by metabolomic approach. Different parts (roots, flower buds, and leaves) of the title plant were analyzed systematically. The in vivo study revealed that the leaves and flower buds had strong antitussive and expectorant effects. Then ¹H NMR spectrometry together with principal component analysis (PCA) and partial least squares discriminant (PLS-DA) analysis were used to investigate the compounds responsible for the bioactivities. PCA was used to find the differential metabolites, while PLS-DA confirmed a strong correlation between the observed effects and the metabolic profiles of the plant. The result revealed that chlorogenic acid, 3,5-dicaffeoylquinic acid, and rutin may be closely related with the antitussive and expectorant activities. The overall results of this study confirm the benefits of using metabolic profiling for screening active principles in medicinal plants.

[Metabolomic study of flower buds of Tussilago farfara in different development stages by GC-MS].

OBJECTIVE: Plant metabolomics combined with GC-MS was used to investigate metabolic fingerprinting of Tussilago farfara at different growth stages. METHOD: Dried Samples were extracted by two-phase solvent system to obtain polar and nonpolar parts, which were subjected to GC-MS analysis. Metabolites were identified by NIST data base search and comparison with the authentic standards. The data were introduced into SIMCA-P 11.0 software package for multivariate analysis after pretreatment. RESULT: Fifty-four metabolites were identified, including 35 polar metabolites and 19 nonpolar compounds. The score plot for PCA showed clear separation of the different development stages of flower buds of T. farfara, showing a trend of gradual change. Samples of October, November, December were in close proximity on the plot, indicating that the metabolome of these three periods was similar, samples from September (early development) and March (after flowering) were far away, showing big chemical differences. Content comparison results of some representative metabolites reveals that, the content of proline, lysine and linoleic acid increased gradually to the highest in the medium term, but sharply decreased to the lowest after flowering; the content of malic acid and citric acid were the lowest in the medium term; sucrose content decreased gradually, and then reached the lowest level after blooming. CONCLUSION: It is obvious that metabolites of the early development and flowering stage were quite different with those of the traditional harvest time, suggesting that they can not be used as traditional medicine. This study will provide a research basis for harvest time determination and bioactive compounds of T. farfara.

Anti-depressant effects of Xiaoyaosan on rat model of chronic unpredictable mild stress: a plasma metabonomics study based on NMR spectroscopy.

OBJECTIVES: To investigate the antidepressant effects of Xiaoyaosan (XYS) in a chronic unpredictable mild stress (CUMS) depression model. METHODS: The changes in behaviour and plasma metabolic profiles were investigated after four-week CUMS exposure and treatment. Drugs were administered during the four-week period of CUMS, with the healthy group serving as negative controls, and the fluoxetine and venlafaxine groups serving as positive controls. Plasma samples were collected at 28th day, and the plasma metabolic profiling was measured using NMR, followed by multivariate analysis. KEY FINDINGS: Exposure to CUMS for four weeks caused depression-like behaviour in rats, as indicated by significant decreases in weight gain, sucrose consumption and locomotor activity. Eleven potential biomarkers, including seven in the Carr-Purcell-Meiboom-Gill spectra, five in the diffusion-edited spectra, and one in both were identified. It was found that trimethylamine-N-oxide, alanine, ß-hydroxybutyrate, valine, leucine/isoleucine, low-density lipoprotein/very low-density lipoprotein and lipids were lower and phosphatidylcholine, high-density lipoprotein, choline and N-acetyl glycoproteins were higher in CUMS-treated rats, as compared with controls. XYS significantly suppressed behavioural changes and attenuated plasma metabolite changes. CONCLUSIONS: XYS produced an obvious antidepressant effect, and the metabonomic approach benefits estimation of the pharmacodynamic action of traditional Chinese medicine prescriptions.

Metabolic fingerprinting of Tussilago farfara L. using ¹H-NMR spectroscopy and multivariate data analysis.

INTRODUCTION: The flower bud of Tussilago farfara L. is widely used for the treatment of coughs, bronchitis and asthmatic disorders in traditional Chinese medicine. In Europe, the plant has been used as herbal remedies for virtually the same applications, but the leaves are preferred over flowers. OBJECTIVE: To systematically evaluate the chemical profiles of Tusssilago farfara leaves and flowers along with the identification of the polar and non-polar metabolites. METHODOLOGY: Metabolic profiling carried out by means of ¹H-NMR spectroscopy and multivariate data analysis was applied to crude extracts from flowers and leaves. Metabolites were identified directly from the crude extracts through one-dimensional and two-dimensional NMR spectra. RESULTS: A broad range of metabolites were detected without any chromatographic separation. Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) of ¹H-NMR data provided a clear separation between the samples. The corresponding loadings plot indicated that higher levels of phenylpropanoids, amino acids, organic acids and fatty acids, as well as lower levels of sugars, terpenoids and sterols were present in the leaves, as compared with flowers. For the flowers, more phenylpropanoids were present in fully open flowers, while more sugars and fatty acids were present in flower buds. CONCLUSION: NMR spectra (one- and two-dimensional) are useful for identifying metabolites, especially for the overlapped signals. The NMR-based metabolomics approach has great potential for chemical comparison study of the metabolome of herbal drugs.

Urinary metabonomic study using a CUMS rat model of depression.

Chronic unpredictable mild stress (CUMS) is a well-validated model of depression. In this study, a urinary metabonomics method based on the NMR spectrometry was used to study the metabolic perturbation in CUMS-induced rat depression model. With pattern recognition analysis, a clear separation of CUMS rats and healthy controls was achieved, and nine endogenous metabolites contributing to the separation were identified. CUMS-treated rats were characterized by the increase of glycine, pyruvate, glutamine, and asparagines, as well as the decrease of 2-oxoglutarate, dimethylglycine, citrate, succinate, and acetate. The urinary biochemical changes related to the metabolic disturbance in CUMS induced depression, and the possible correlations with live qi stagnation in traditional Chinese medicine are discussed. The work shows that CUMS is a reliable model for studying depression, and the noninvasive urinary metabolomic method is a valuable tool to investigate the biochemical pertubations in depression as an early diagnostic means.

Metabolomic profiling of the flower bud and rachis of Tussilago farfara with antitussive and expectorant effects on mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Flower bud of Tussilago farfara L. is widely used for the treatment of cough, bronchitis and asthmatic disorders in the Traditional Chinese Medicine. However, due to the increasing demands, adulteration with rachis is frequently encountered in the marketplace. No report demonstrated the chemical and pharmacological differences between flower bud and rachis before. MATERIALS AND METHODS: The water extracts were orally administrated to mice. Ammonia induced mice coughing model was used to evaluate the antitussive activity. The expectorant activity was evaluated by volume of phenol red in mice's tracheas. Metabolites were identified directly from the crude extracts through 1D- and 2D-NMR spectra. A metabolic profiling carried out by (1)H NMR spectroscopy and multivariate data analysis was applied to crude extracts from flower bud and rachis. RESULTS: Flower bud significantly lengthened the latent period of cough, decreased cough frequency caused by ammonia and enhanced tracheal phenol red output in expectorant evaluation. Principal component analysis (PCA) yielded good separation between flower bud and rachis, and corresponding loading plot showed that the phenolic compounds, organic acid, sugar, amino acid, terpene and sterol contributed to the discrimination. CONCLUSIONS: These findings provide pharmacological and chemical evidence that only flower bud can be used as the antitussive and expectorant herbal drug. The high concentration of chlorogenic acid, 3,5-dicaffeoylquinic acid, rutin in flower buds may be related with the antitussive and expectorant effects of Flos Farfara. To guarantee the clinical effect, rachis should be picked out before use.

[Comparative study of the clinical effects of salvia miltiorrhiza injection and shengmai injection on chronic hepatitis B].

OBJECTIVE: To observe the effect of Salvia miltiorrhiza Injection (SMI) and Shengmai Injection (SI) on liver function and fibrosis related indexes in patients with chronic hepatitis B. METHODS: Seventy-nine chronic hepatitis B patients were randomly divided into the SMI group (n=47) and the SI group (n=32), they were treated with SMI and SI respectively on the basis of conventional treatment. The therapeutic course was 35 days for both groups. The changes of main symptoms and physical signs were observed, and indexes of liver function and fibrosis including serum hyaluronidase, laminin, III type precollagen (PC-III) and IV type collagen (IV-C) were investigated before and after treatment. RESULTS: Symptoms, physical signs and liver functions were improved obviously in both SMI and SI groups, SI showed better effect than SMI (P < 0.05). The four liver fibrosis indexes declined significantly in the SMI group after treatment (P < 0.05), but no obvious change of those was found in the SI group (P > 0.05), showing significant difference between the two groups (P < 0.05). Conclusion SMI is effective in improving liver function and inhibiting liver fibrosis, and SI has even better effect in improving liver function than SMI, though it shows no anti-liver fibrosis effect.

[Influence of gentian leaf blight on the output and quality of Chinese gentian].

OBJECTIVE: To study the influecnce of gentian leaf blight on the output and quality of rough gentian. METHOD: The same grade seedlings were transplanted, disease of every plant was investigated in autumn and the output of gentian was determined. HPLC was applied to determine the content of gentiopicroside and swertiamarin. RESULT: The output decreased with the aggravation of the disease, and the decrease was obvious when the index of disease was above 60. The content of gentiopicroside and swertiamarin began to drop when the index of disease was above 70. CONCLUSION: The loss of output and the drop of quality are relatively heavy when the disease is serious. The loss of income is not obvious when the index of disease is under 60.