[Identification of Coptis chinensis root rot disease pathogenic Fusarium spp. fungi].

Root rot disease is vital disease of Coptis chinensis, it has bursted in most producing area in recent years, and has caused severe damage. To identify the pathogenic fungi, Fusarium spp. fungi were isolated from rot root, of which the pathogenic fungi were screened with inoculation on C. chinensis root and plant, and identified with molecular and morphological method. The 20 Fusarium spp. fungi were obtained, of which 5 displayed high pathogenicity. It was deduced that F. oxysporum, F. solani and F. tricinctum were the pathogen, possibly pioneer pathogen of C. chinensis root rot disease. Among which F. oxysporum was dominant and deserved to pay more attention. High temperature and high humidity can increase pathogenicity of Fusarium spp. So the global climate warming may lead to temperature rising of C. chinensis producing area and favor the pathogen fungi, which may be one of the main factors leading to bursting of C. chinensis root rot disease. To control the root rot, beside developing and using pesticide, producing base should be moved to a high altitude area.

[Diversity of culturable fungi isolated from Coptis chinensis rot root in Shizhu city of Chongqing].

Root rot disease restricts the Coptis chinensis industry in Shizhu of Sichuan province. To disclose fungi composition and pathogen in rot root,so as to prevent and treat the root rot disease,the C. chinensis rot root of 5 years from 4 areas in Shizhu were collected in 3 seasons respectively. The fungi were isolated and molecularly and morphological identified,followed with population statistics. 437 fungi were isolated,belonging to 5 subphylum,11 classes,16 orders,22 families and 28 genus respectively. There are great difference among the fungi compositions of different area,year and sampling season,while there was no obvious variation rule. Ilyonectria sp.,Pythium sp.,Phoma sp,Trichoderma sp.are dominant genus,while Pythium sp.,Ilyonectria sp.,Phoma sp.,Fusarium sp. may contain root rot pathogen. Antagonistic bacteria may be screened from the strains of Trichoderma sp. isolated.

Composition and diversity of rhizosphere fungal community in Coptis chinensis Franch. continuous cropping fields.

In this study, effects of continuous cropping on soil properties, enzyme activities, and relative abundance, community composition and diversity of fungal taxa were investigated. Rhizosphere soil from field continuously cropped for one-year, three-year and five-year by Coptis chinensis Franch. was collected and analyzed. Illumina high-throughput sequencing analysis showed that continuous cropping of C. chinensis resulted in a significant and continuous decline in the richness and diversity of soil fungal population. Ascomycota, Zygomycota, Basidiomycota, and Glomeromycota were the dominant phyla of fungi detected in rhizosphere soil. Fungal genera such as Phoma, Volutella, Pachycudonia, Heterodermia, Gibberella, Cladosporium, Trichocladium, and Sporothrix, were more dominant in continuously cropped samples for three-year and five-year compared to that for one-year. By contrast, genera, such as Zygosaccharomyces, Pseudotaeniolina, Hydnum, Umbelopsis, Humicola, Crustoderma, Psilocybe, Coralloidiomyces, Mortierella, Polyporus, Pyrenula, and Monographella showed higher relative abundance in one-year samples than that in three-year and five-year samples. Cluster analysis of the fungal communities from three samples of rhizosphere soil from C. chinensis field revealed that the fungal community composition, diversity, and structure were significantly affected by the continuous cropping. Continuous cropping of C. chinensis also led to significant declines in soil pH, urease, and catalase activities. Redundancy analysis showed that the soil pH had the most significant effect on soil fungal population under continuous cropping of C. chinensis.

[Research on bacteria microecology in root rot rhizosphere soil of Coptis chinensis produced in Shizhu city].

Illumina Hiseq 2500 high-throughput sequencing platform was used to study the bacteria richness and diversity, the soil enzyme activities, nutrients in unplanted soil, root-rot and healthy rhizophere soil of Coptis chinensis for deeply discussing the mechanism of the root-rot of C. chinensis. The high-throughput sequencing result showed that the artificial cultivation effected the bacteria community richness and diversity. The bacteria community richness in healthy and diseased rhizosphere soil showed significant lower than that of in unplanted soil (P<0.05) and declined bacteria diversity. The bacteria community richness in root-rot rhizosphere soil increased significantly than that of health and unplanted soil and the diversity was lower significant than that of unplanted soil (P<0.05). The results of soil nutrients and enzyme activities detected that the pH value, available phosphorus and urease activity decreased and the sucrase activity increased significantly (P<0.05). The content of organic carbon and alkaline hydrolysis nitrogen the catalase and urease activity in root rot soil samples was significantly lower than that of healthy soil samples (P<0.05). However, the contents of available phosphorus and available potassium were significantly in root-rot sample higher than that of healthy soil samples (P<0.05). Comprehensive analysis showed that the artificial cultivation declined the bacteria community richness and diversity. The bacteria community richness decreased significantly and the decreased diversity may be the cause of the root-rot. Meanwhile, the decrease of carbon and the catalase activity may be another cause of the root-rot in C. chinensis produced in Shizhu city, Chongqing province.

[Isolation and identification of berberine from endophytic fungi HL-Y-3].

The endophytic fungi HL-Y-3, which was isolated from the healthy leaves of Coptis chinensis, produced berberine when grown in the PDA culture medium. The presence of berberine was confirmed by the chromatographic and spectroscopic analyses. The yield of berberine was recorded as 9.313 µg•g⁻¹ by HPLC. The strain HL-Y-3 was identified as Alternaria sp.by morphological observation and 5.8S rDNA-ITS sequence analysis.The separation and purification of constituents were performed by PTLC. The mass spectrometry (MS) of the analyte was shown to be identical with authentic berberine.Further analysis with nuclear magnetic resonance (NMR) spectroscopy to showed that the chemical structure of the fungal berberine was identical with authentic berberine. The research provided new resources for the utilization of berberine.

[Bacteria community in different aged Coptis chinensis planting soil revealed by PCR-DGGE analysis].

In order to reveal the cause of disease occurred in the process of Coptis chinensis growth, this paper studied the bacterial species diversity index of different aged rhizospheric and non-rhizospheric soil planting normal or sick C. chinensis by using PCR-DGGE technique. The representative DGGE bands were chosen to be cloned, and sequenced, the phylogeny were constructed. The results showed that the bacterial communities were very different between the normal and diseased soil samples of C. chinensis, and the diversity index (H) of diseased soil samples were higher than that of normal soil samples. Sequencing analysis of representative cloned DGGE bands showed that the unculturable bacteria were the dominant groups, and bacteria belonged to genus Bacillus, Acidovorax, Acinetobacter, uncultured Kluyvera, and uncultured Comamonas were also existing, but the reported plant pathogenic bacteria were not found in the C. chinensis planting soil. The density and brightness of clone band d in diseased soil samples was higher than that in normal soil sample, and sequencing analysis showed that it belonged to genus Acidovorax. Obviously, during the process of C. chinensis growth, the rhizospheric bacteria population changed, and the quantity of bacteria belong Acidovorax increased, which probably resulted in the disease occurred during C. chinensis growth.

Evaluation of the in vitro and in vivo protective effects of unfermented and fermented Rhizoma coptidis formulations against lipopolysaccharide insult.

Lipopolysaccharide (LPS) can produce endotoxic shock by triggering the systemic inflammatory response. Here, we evaluated the in vitro and in vivo protective effects of unfermented and fermented Rhizoma coptidis (RC and FRC, respectively) against LPS-insult. In general, RC suppressed the LPS-induced expression of key inflammatory mediators in RAW264.7 cells, in a dose-dependent manner. Notably, FRC at a 20 µg/ml dose in combination with the probiotic used for fermentation showed more potent in vitro anti-inflammatory activities than that exhibited by the corresponding dose of RC. Moreover, oral treatment with FRC in association with the probiotic, but not oral administration of RC, significantly attenuated blood endotoxin and C-reactive protein (CRP) levels, and gut permeability, and significantly augmented the intestinal population of Bifodobacterium spp. and Lactobacillus spp. in LPS-treated animals. Our results demonstrate the beneficial impact of fermented RC in combination with the associated probiotic in combating LPS-insult both in vitro and in vivo.