[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.

First report of root rot caused by Fusarium armeniacum on American ginseng in China.

American ginseng (Panax quinquefolius) is an important medicinal plant cultivated in China since the 1980s. Its dried roots are used for food, health care products, and medicine in China (Yuan et al. 2010). Root rot caused by Fusarium spp. was a major disease, with 33 to 41% incidence surveyed in main production areas of Wendeng County (121.80 °E, 37.09 °N) in Shandong Province, China in 2016 to 2019. Symptoms included soft, water-soaked, dark brown to black lesions on the roots. Lesions progressed and the inner parts gradually disintegrated. One-year-old diseased roots were collected in September 2016. Symptomatic tissues were surface-sterilized in 75% ethanol for 30 s and 0.8% NaOCl for 3 min, rinsed in sterile water, plated on potato dextrose agar (PDA), and incubated at 25°C in darkness. Single colonies were then obtained and transferred to carnation leaf agar (CLA) (Burgess et al. 1993) for growth at 25°C with a 12-h photoperiod. Colonies cultured on PDA for 7 days were white to light pink, turning to apricot pigmentation in color. After 30 days on CLA, the colonies produced elongate, falcate macroconidia having 3 to 5 septa, with a long, tapering and curved apical cell, and having the size ranging from 31.1 to 45.6 µm long x 4 to 4.6 µm wide. Microconidia were zero to 1septate, ellipsoid to ovoid and varied in size from 9.5 to 16.8 µm long x 3 to 3.2 µm wide. Chlamydospores formed abundantly, in chains or clusters. This fungus was identified as F. armeniacum (Burgess et al. 1993). Identification was confirmed by sequencing three DNA regions including the internal spacer ribosomal DNA (ITS), elongation factor 1α and ß-tubulin genes (Lu et al. 2019). The three DNA regions (MN417271, MG457199, and MN427653) had 100% homology to the sequences of F. armeniacum (KJ737378, HM744664 and HQ141640) (Wang et al. 2015, Yli-Mattila et al. 2011). Pathogenicity tests were conducted on 1- to 2-year-old bare roots and 2-year-old whole plants. For root inoculation, 14 healthy roots were inoculated with two mycelial PDA plugs/root. After 3 to 10 days at 25°C, all the inoculated roots showed water-soaked and root rot symptoms while no lesions were observed in the control roots. For plant inoculation, eight seedlings planted in pots filled with sterilized soil were inoculated by pouring a conidial suspension of 1×105 conidia/ml at 30 ml/pot. Eight seedlings inoculated with sterilized water served as the controls. After 90 days, only 37.5% of the roots survived with typical root rot symptoms whereas the control plants remained symptomless. F. armeniacum was re-isolated from symptomatic roots but not from the control roots. Besides F. armeniacum, F. solani and F. oxysporum that have been reported to be associated with American ginseng root rot in China and Canada (Reeleder et al. 2002; Punja et al. 2008) were also obtained from the diseased root samples in this study. However, the development of root rot caused by F. armeniacum was much more rapid and its symptoms were more severe. Moreover, F. armeniacum could directly infect American ginseng with no wound requirement. F. armeniacum was previously reported on Glycine max (Leguminosae) (Ellis et al. 2012), Platycodon grandiflorus (Campanulaceae) (Wang et al. 2015) and natural grasses (Poaceae) (Nichea et al. 2015). This is the first report of F. armeniacum causing root rot on American ginseng in China. As this species is more virulent to American ginseng, more research is needed to work on this disease.

[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.

[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.

[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.

The relationship between shifts in the rhizosphere microbial community and root rot disease in a continuous cropping American ginseng system.

The root rot disease causes a great economic loss, and the disease severity usually increases as ginseng ages. However, it is still unclear whether the disease severity is related to changes in microorganisms during the entire growing stage of American ginseng. The present study examined the microbial community in the rhizosphere and the chemical properties of the soil in 1-4-year-old ginseng plants grown in different seasons at two different sites. Additionally, the study investigated ginseng plants' root rot disease index (DI). The results showed that the DI of ginseng increased 2.2 times in one sampling site and 4.7 times in another during the 4 years. With respect to the microbial community, the bacterial diversity increased with the seasons in the first, third, and fourth years but remained steady in the second year. The seasonal changing of relative abundances of bacteria and fungi showed the same trend in the first, third, and fourth years but not in the second year. Linear models revealed that the relative abundances of Blastococcus, Symbiobacterium, Goffeauzyma, Entoloma, Staphylotrichum, Gymnomyces, Hirsutella, Penicillium and Suillus spp. were negatively correlated with DI, while the relative abundance of Pandoraea, Rhizomicrobium, Hebeloma, Elaphomyces, Pseudeurotium, Fusarium, Geomyces, Polyscytalum, Remersonia, Rhizopus, Acremonium, Paraphaeosphaeria, Mortierella, and Metarhizium spp. were positively correlated with DI (P < 0.05). The Mantel test showed that soil chemical properties, including available nitrogen, phosphorus, potassium, calcium, magnesium, organic matter, and pH, were significantly correlated to microbial composition. The contents of available potassium and nitrogen were positively correlated with DI, while pH and organic matter were negatively correlated with DI. In summary, we can deduce that the second year is the key period for the shift of the American ginseng rhizosphere microbial community. Disease aggravation after the third year is related to the deterioration of the rhizosphere microecosystem.

[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.

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.

[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.