A soil fumigant increases American ginseng (Panax quinquefolius L.) survival and growth under continuous cropping by affecting soil microbiome assembly: a 4-year in situ field experiment.

IMPORTANCE: Numerous reports of soil fumigants and fungicides on annual crops exist; however, it is unclear whether the single application to perennial plants persistently improves plant growth and controls disease or whether it has a long-lasting impact on soil microbes. We found that soil fumigation enhances ginseng growth and suppresses root rot disease by reshaping the soil microbial community. Our findings benefit the agricultural development of ginseng and provide a theoretical basis for the prevention of ginseng diseases.

[Effect of light intensity on growth, accumulation of ginsenosides, and expression of related enzyme genes of Panax quinquefolius].

Appropriate light intensity is favorable for the photosynthesis, biomass accumulation, key enzyme activity, and secondary metabolite synthesis of medicinal plants. This study aims to explore the influence of light intensity on growth and quality of Panax quinquefolius. To be specific, sand culture experiment was carried out in a greenhouse under the light intensity of 40, 80, 120, and 160 µmol·m~(-2)·s~(-1), respectively. The growth indexes, photosynthetic characteristics, content of 6 ginsenosides of the 3-year-old P. quinquefolius were determined, and the expression of ginsenoside synthesis-related enzyme genes in leaves, main roots, and fibrous roots was determined. The results showed that the P. quinquefolius growing at 80 µmol·m~(-2)·s~(-1) light intensity had the most biomass and the highest net photosynthetic rate. The total biomass of P. quinquefolius treated with 120 µmol·m~(-2)·s~(-1) light intensity was slightly lower than that with 80 µmol·m~(-2)·s~(-1). The root-to-shoot ratio in the treatment with 120 µmol·m~(-2)·s~(-1) light intensity was up to 6.86, higher than those in other treatments(P<0.05),and the ginsenoside content in both aboveground and underground parts of P. quinquefolius in this treatment was the highest, which was possibly associated with the high expression of farnesylpyrophosphate synthase(FPS), squalene synthase(SQS), squalene epoxidase(SQE), oxidosqualene cyclase(OSC), dammarenediol-Ⅱ synthase(DS), and P450 genes in leaves and SQE and DS genes in main roots. In addition, light intensities of 120 and 160 µmol·m~(-2)·s~(-1) could promote PPD-type ginsenoside synthesis in leaves by triggering up-regulation of the expression of upstream ginsenoside synthesis genes. The decrease in underground biomass accumulation of the P. quinquefolius grown under weak light(40 µmol·m~(-2)·s~(-1)) and strong light(160 µmol·m~(-2)·s~(-1)) was possibly attributed to the low net photosynthetic rate, stomatal conductance, and transpiration rate in leaves. In the meantime, the low expression of SQS, SQE, OSC, and DS genes in the main roots might led to the decrease in ginsenoside content. However, there was no significant correlation between the ginsenoside content and the expression of synthesis-related genes in the fibrous roots of P. quinquefolius. Therefore, the light intensity of 80 and 120 µmol·m~(-2)·s~(-1) is beneficial to improving yield and quality of P. quinquefolius. The above findings contributed to a theoretical basis for reasonable shading in P. quinquefolius cultivation, which is of great significance for improving the yield and quality of P. quinquefolius through light regulation.

Application of vermicompost and biochar suppresses Fusarium root rot of replanted American ginseng.

Soil sterilization integrated with agronomic measures is an effective method to reduce soilborne replant diseases. However, the effect of vermicompost or biochar application after soil sterilization on soilborne diseases is poorly understood. A pot experiment was conducted in American ginseng to investigate the effects of vermicompost (VF), biochar (BF), and a combination of vermicompost and biochar (VBF) applied after soil sterilization on the incidence of Fusarium root rot using natural recovery (F) as control. After one growing season, the disease index of root rot, the phenolic acids, and the microbial communities of American ginseng rhizosphere soil were analyzed. The disease index of VF, BF, and VBF decreased by 33.32%, 19.03%, and 80.96%, respectively, compared with F. The highest bacterial richness and diversity were observed in the rhizosphere soil of VBF. Besides, VF and VBF significantly increased the relative abundance of beneficial bacteria (Pseudomonas, Lysobacter, and Chryseolinea) in the rhizosphere soil. Higher concentrations of vanillin, one of the phenolic acids in the roots exudates, were recorded in the rhizosphere soils of BF and VBF. The vanillin concentration showed a significant negative correlation with the disease index. To conclude, vermicompost improved the beneficial bacteria of the rhizosphere soil, while biochar regulated the allelopathic effect of the phenolic acids. The study proposes a combined application of biochar and vermicompost to the rhizosphere soil to control Fusarium root rot of replanted American ginseng effectively. KEY POINTS: Vermicompost improves the relative abundance of rhizosphere beneficial bacteria. Biochar inhibits the degradation of phenolic acids by adsorption. The combination of vermicompost and biochar enhances the disease control effect.

[Effects of nutrients deficiency on growth and saponin accumulation of American ginseng].

Mineral nutrient elements are the key factors to maintain the growth and quality of American ginseng. In order to understand the comprehensive effect of different nutrient elements deficiency on American ginseng, 2-year-old American ginsengs were cultivated by Hoagland solution(CK) or 10 different nutrients deficiency solution in sand culture. During the cultivation, the deficient symptom was observed. The plant height, leaf area, biomass, photosynthetic index, root activity, ginsenoside content were measured. The results showed that N, K or Fe deficiency could lead to leaves of American ginseng yellowing. Deficiency N, K, Ca, Mg and B were the main factors that decrease plant height and leaf area. The biomass of plant decreased significantly in all the nutrient deficient treatments(P<0.05)compared with control group, and N, K, Ca or Fe deficiency groups descended over 50%. In the absence of N, K and Fe elements, the P_n, G_s, C_i, T_r and chlorophyll of leaves were decreased mostly. The first three factors decreasing root activity were N, K and Ca deficiency. The effects of nutrient deficiency on saponins of American ginseng were different.Generally, N, P, B, Zn and Cu deficiency resulted the synthesis of saponins decreased significantly(P<0.05). This study contributed to clarify the demand characteristics of American ginseng for different nutrient elements,which is of great significance for the diagnose of nutrient deficiency, the rational fertilizer and the improvement of yield and quality of American ginseng.

[Investigation of aflatoxins,mycobiota,and toxigenic fungi during post-harvest handling of Ziziphi Spinosae Semen].

Ziziphi Spinosae Semen is one of the Chinese herbal medicine being susceptible to aflatoxins contamination. To investigate the sources of aflatoxins contamination and toxigenic fungi species on Ziziphi Spinosae Semen,32 samples were collected from multiple steps during the post-harvest processing in this study. Aflatoxins in these samples were determined by immunoaffinity column and HPLC coupled with post-column photochemical derivatization. The dilution-plate method was applied to the fungi isolation. The isolated fungi strains were identified by morphological characterization and molecular approaches. The results showed that aflatoxins were detected in 28 samples from every step during the processing of Ziziphi Spinosae Semen. Three samples were detected with aflatoxin B_1 and 2 samples with both aflatoxin B_1 and total aflatoxin exceeding the limit of Chinese Pharmacopoeia. Especially the samples from the washing step,with the highest detected amounts of AFB_1 and AFs were reached 94. 79,121. 43 µg·kg~(-1),respectively. All 32 samples were contaminated by fungi. The fungal counts on the newly harvested samples were 2. 20 × 10~2 CFU·g~(-1). Moreover,it increased as tphreocessing progresses,and achieved 1. 16×10~6 CFU·g~(-1) after washing. A total of 321 isolates were identified to 17 genera. Aspergillus flavus was the main source of aflatoxins during the processing and storage of Ziziphi Spinosae Semen. One isolate of A. flavus was confirmed producing AFB_1 and AFB_2. The fungal count was significantly increased by composting,and Aspergillus was the predominant genus after shell breaking. The contamination level of aflatoxins was increased by composting and washing.

Effects of maize rotation on the physicochemical properties and microbial communities of American ginseng cultivated soil.

The production of American ginseng (Panax quinquefolius L.) is severely limited by the replant disorders in China. Crop rotation with maize might reduce the replant problems, but little information is available on the effect of maize rotation on soil cultivated with ginseng. In this study, we analyzed nutrients, phenolic acids, and microbial communities in soils from the fields with continuous maize, mono-culture ginseng, and 1-, 3-, and 5-year maize rotation after ginseng. Pot experiments were also conducted to evaluate the performance of replanting ginseng in these soils. The results showed that Mn, Cu, and 5 phenolic acids in ginseng-cultivated soil were significantly decreased by maize rotation. A 5-year maize rotation significantly increased the relative abundance of beneficial soil bacteria, such as Arthrobacter, rather than decreasing the abundances of potential pathogenic genera. Clustering analysis revealed that the physicochemical properties and microbial communities of 3- and 5-year maize rotation soil were more similar to CM than to G soil. The biomass of replanted ginseng root was improved, and root disease was reduced over 3 years of maize rotation. Overall, the results showed that at least a 3-year maize rotation is needed to overcome the replant failure of American ginseng.

Occurrence of Toxigenic Fungi and Mycotoxins on Root Herbs from Chinese Markets.

Herbs derived from roots, leaves, flowers, or fruits of plants are unavoidably contaminated with fungi and mycotoxins during growth, harvest, and storage, thereby posing a health threat to humans. Especially, root herbs (RHs) are more easily contaminated with fungi and mycotoxins because the roots are in direct contact with the soil. Here, we investigated the occurrence of fungi, aflatoxins (AFs), and ochratoxin A (OTA) in eight RHs that are used as medicines, beverages, dietary supplements, and functional foods in China and other countries. Morphological observation and MultiGeneBlast (ß-tubulin and calmodulin) were used to identify the potentially toxigenic fungi. Of the 48 samples tested, all were contaminated by fungi, and 1,844 isolates belonging to 25 genera were detected. The genera Aspergillus and Penicillium, which contain potentially toxigenic fungal species, represented a frequency of 10 and 25%, respectively. Thirty-three isolates of Aspergillus flavus, Aspergillus parasiticus, Aspergillus niger, and Penicillium polonicum were arbitrarily selected for analysis of their toxigenic potential. Five of 13 isolates of A. flavus and 1 isolate of A. parasiticus produced AFs, whereas OTA production was not detected for any of the isolates of A. niger and P. polonicum. The occurrence of AFs and OTA in the 48 samples of eight RHs was tested by ultraperformance liquid chromatography-tandem mass spectrometry; 37.50% of samples from six RHs were contaminated with AFs and 16.67% of samples from four RHs were contaminated with OTA. Seven (14.58%) and four (8.33%) samples of ginseng, polygala, and liquorice exceeded the permissible limits of aflatoxin B1 and AFs, respectively. Because ginseng, polygala, and liquorice are widely used as herbs, dietary supplements, and functional foods, the high frequency of AF contamination of these herbs indicated by our current study warrant attention to raise public awareness.

Mycobiota and Mycotoxins in Traditional Medicinal Seeds from China.

The multi-mycotoxin occurrence for internal and superficial fungi contamination were comprehensively assessed in medicinal seeds used as food or beverage. Based on a polyphasic approach using morphological characters, ß-tubulin and ITS gene blast, a total of 27 species belonging to 12 genera were identified from surface-sterilized seeds. Chaetomium globosporum was most predominant (23%), followed by Microascus trigonosporus (12%) and Alternaria alternata (9%). With respect to superficial mycobiota, thirty-four species belonging to 17 genera were detected. Aspergillus niger and Penicillium polonicum were predominant (12% and 15%, respectively). Medicinal seed samples and potential toxigenic fungi were tested for ochratoxin A (OTA) and aflatoxins (AFB1, AFB2, AFG1, AFG2) using UPLC-MS/MS. Platycladi seeds were contaminated with AFB1 (52.0 µg/kg) and tangerine seed was contaminated with OTA (92.3 µg/kg). Subsequent analysis indicated that one A. flavus strain isolated from platycladi seed was able to synthesize AFB1 (102.0 µg/kg) and AFB2 (15.3 µg/kg). Two P. polonicum strains isolated from tangerine and lychee seeds were able to synthesize OTA (4.1 µg/kg and 14.8 µg/kg, respectively). These results identify potential sources of OTA and aflatoxins in medicinal seeds and allude to the need to establish permitted limits for these mycotoxins in these seeds that are commonly consumed by humans.

[Autotoxic effect of ginsenoside extrats on growth of American ginseng in different medium].

Ginsenosides are the abundant secondary metabolites in American ginseng (Panax quinquefolium), it could be released into soil through root exudation and decomposition during plant growth. This study determined ginsenoside contents in American ginseng cultivated soil by HPLC. Three ginsenosides, Rb1, Rb2 and Rd, were detected in the rhizosphere soil of 3-4 years old American ginseng cultivated in Huairou District, Beijing, and their contents were 0.80-3.19 mg x kg(-1). Correspondingly, the contents of the three ginsenosides in soil solution were 4-16 mg x L(-1) at field water-holding capacity of 20%. According to the field soil test data, we designed the concentration of ginsenosides for bioassays (0.2-125 mg x L(-1) in solution or 0.2-125 mg x kg(-1) in soil). The results showed that radicle lengths of American ginseng were reduced by 6%-23% in solution containing 0.2-125 mg x L(-1) ginsenoside extract, and a significant difference was observed at concentration of 125 mg x L(-1) (P < 0.05). The shoot lengths of American ginseng were not significantly inhibited by 0.2-125 mg x L(-1) ginsenosides extractions. After 20 days of growth in nutrient solution amended with 25 mg x L(-1) ginsenosides extraction, plant height of 3-year-old American ginseng seedling was decreased by 28% compared to the control, and the biomass of aerial parts was also reduced by 50% (P < 0.05). However, the growth of newly-grown fibrous root was not significantly inhibited. Comparatively, when American ginseng embryos were cultivated into sterile or non-sterile soil, neither radicle lengths nor shoot lengths were significantly affected by 0.2-125 mg x kg(-1) ginsenoside extracts. In conclusion, ginsenosides showed autotoxic effect on growth of American ginseng radicle and adult seedling, however, this effect was weakened in field soil.

Effects of Fusarium solani and F. oxysporum Infection on the Metabolism of Ginsenosides in American Ginseng Roots.

American ginseng (Panax quinquefolius L.) is a highly valuable herb widely used for medicinal treatments. Its pharmacologically important compounds are the ginsenosides, which are secondary metabolites in American ginseng root. The concentrations of ginsenoside in roots can be changed by fungal infection, but it is unclear what specific root tissues are impacted and whether the change is systemic. In this study, American ginseng roots were inoculated with two fungal pathogens (Fusarium solani or F. oxysporum) and the levels of six ginsenosides (Rb1, Rb2, Rc, Rd, Re, and Rg1) were then measured in the phloem and xylem around the discolored lesions and adjacent healthy areas of the root. Results indicated that the growth of Fusarium spp. was strictly limited to phloem, and correspondingly the ginsenoside concentration was only altered in this infected phloem. The concentration of Rg1, Rd, and Rc significantly changed in phloem tissues where F. solani was inoculated, while only Rg1 and Rd changed significantly after F. oxysporum inoculation. However, no changes of any ginsenoside occurred in either xylem or phloem tissue adjacent to the inoculation point. In addition, when two Fusarium spp. were grown on ginsenoside-amended Czapek medium, the majority of ginsenosides were depleted. Therefore, pathogenic Fusarium spp. may reduce ginsenoside levels by consuming them.

[Fungal composition in massa medicata fermentata based on culture dependent method and independent PCR-SSCP technique].

OBJECTIVE: To analyze the fungal composition in Massa Medicata Fermentata based on culture dependent method and independent PCR-SSCP technique. METHOD: Fungi were directly isolated from Massa Medicata Fermentata samples. The obtained strains were identified according to morphology and DNA sequence. Meanwhile the total fungal DNA was extracted from Massa Medicata Fermentata samples, the cultural independent PCR-SSCP technique based on ß-tubulin gene were used to identify the mycobiota. RESULT: According to cultural method, Aspergillus flavus and Rhizopus oryzae were present in Massa Medicata Fermentata samples, while A. flavus and A. niger were present in fried Massa Medicata Fermentata samples. In contrast, 5 species were obtained by PCR-SSCP technique, A. flavus was overlapped with fungal taxa derived from culture dependent method; A. ambiguu and A. s ivoriensis were dominant with relative abundance of 57% and 35% respectively, while the relative abundance of A. flavus was as low as 4%. None species was obtained from fried Massa Medicata Fermentata samples. CONCLUSION: PCR-SSCP based on ß-tubulin gene could distinguish fungi into species, culture dependent method combined with culture independent method could better understand the fungal composition associated with Massa Medicata Fermentata fermentation.

[Effect of continuous cropping and soil treatment on rhizosphere fungal community of Panax quinquefolium].

OBJECTIVE: To analyze the effects of continuous cropping and soil treatment on rhizosphere fungal community of Panax quinquefolium, and the correlation between rhizosphere fungal community and growth of P. quinquefolium. METHOD: Field plot trail of continuous cultivated P. quinquefolium was conducted, meanwhile continuous cropping soil was treated by organic carbon fertilizer plus methylene dithiocyanate and Kingbo respectively. The rate of seedling survival, root disease index and root weight were investigated. Dilution plating was used to analyze the rhizosphere fungal community, multiple comparisons including H, M(a) and J were performed between rhizosphere fungal community and growth of P. quinquefolium. RESULT: As to P. quinquefolium planted in continuous soil, the rate of seedling survival, root weight decreased, root disease index increased remarkably (P<0.05). Meanwhile the fungal counts decreased 50% -63% , the diversity index (H') decreased 39%-43%, while the ratio of Penicillium and Aspergillus increased. There was a significant negative correlation between rhizosphere fungal diversity and P. quinquefolium root disease index (r = -0.970, P=0.006). Organic carbon fertilizer plus methylene dithiocyanate treatment could improve the rate of seedling survival and root weight, and could partly increase the rhizosphere fungal diversity. CONCLUSION: In continuous cropping soil of P. quinquefolium, the rhizosphere fungal counts and diversity index decreased, meanwhile the fungal community composition changed. Organic carbon fertilizer plus methylene dithiocyanate treatment could improve the growth of continuous cultivated P. quinquefolium.

[Effect of nitrogen, phosphorus and potassium deficiency on content of phenolic compounds in exudation of American ginseng].

OBJECTIVE: Some of the phenolic compounds detected in the soil of commercially cultivated American ginseng could inhibit the seed germination and seedling growth of American ginseng. In this paper we studied the root exudation of American ginseng induced by deficiency of nitrogen, phosphorus and potassium on the content of phenolic compounds. METHOD: Two years old American ginsengs were cultured in hydroponic culture with different nutrient solution. The culture solution was collected after 14 days. The exudations of different polarities in the culture solution were enriched by the amberlite XAD4 and XAD7. The content of the total phenolic acids in the exudation was analyzed by Folin-Ciocalteu colorimetry; the contents of vanillic acid, p-coumaric acid and trans-cinnamic acid were detected and quantified by HPLC. RESULT: Both in the situation of nitrogen and potassium deficiency, the concentration of total phenolic compounds increased significantly in the exudation of American ginseng comparing with the complete nutrient solution (P < 0.05) , while decreased significantly under phosphorus deficient conditions (P < 0.05). The contents of the 3 autotoxic phenolic acids decreased significantly under nitrogen, phosphorus and potassium deficient conditions (P < 0.05). CONCLUSION: The contents of total phenolic compounds and the 3 autotoxic phenolics in the root exudation of American ginseng altered variously in the deficiency of nitrogen, phosphorus and potassium.

[Variation of ginsenosides in infected roots of American ginseng (Panax quinquefolium)].

UNLABELLED: To study the infected root of Panax quinquefolium on the contents of ginsenosides. METHOD: The contents of three major ginsenosides Rg1, Re and Rb1 were determined by HPLC compared quantitatively between the different degree infected roots and normal root in the phloem and xylem. RESULT: Rg1 in phloem and xylem of varying degrees infected root showed no significant difference, but Rb1 decreased 26.3% and 28.3% respectively in medium and serious infected roots comparing to normal root. Re in phloem of seriously infected roots decreased in xylem significantly. CONCLUSION: The results indicate that the variation of ginsenosides in different degrees infected roots exists and the proportion of Rg1, Re and Rb1 in the total ginsenosides changes.