A Single Latent Plant Growth-Promoting Endophyte BH46 Enhances Houttuynia cordata Thunb. Yield and Quality.

Plant growth-promoting endophytes (PGPE) can effectively regulate plant growth and metabolism. The regulation is modulated by metabolic signals, and the resulting metabolites can have considerable effects on the plant yield and quality. Here, tissue culture Houttuynia cordata Thunb., was inoculated with Rhizobium sp. (BH46) to determine the effect of BH46 on H. cordata growth and metabolism, and elucidate associated regulatory mechanisms. The results revealed that BH46 metabolized indole-3-acetic acid and induced 1-aminocyclopropane-1-carboxylate deaminase to decrease ethylene metabolism. Host peroxidase synthesis MPK3/MPK6 genes were significantly downregulated, whereas eight genes associated with auxins, cytokinins, abscisic acid, jasmonic acid, and antioxidant enzymes were significantly upregulated. Eight genes associated with flavonoid biosynthesis were significantly upregulated, with the CPY75B1 gene regulating the production of rutin and quercitrin and the HCT gene directly regulating the production of chlorogenic acid. Therefore, BH46 influences metabolic signals in H. cordata to modulate its growth and metabolism, in turn, enhancing yield and quality of H. cordata.

Endophytic Pseudomonas fluorescens promotes changes in the phenotype and secondary metabolite profile of Houttuynia cordata Thunb.

The interactions between microbes and plants are governed by complex chemical signals, which can forcefully affect plant growth and development. Here, to understand how microbes influence Houttuynia cordata Thunb. plant growth and its secondary metabolite through chemical signals, we established the interaction between single bacteria and a plant. We inoculated H. cordata seedlings with bacteria isolated from their roots. The results showed that the total fresh weight, the total dry weight, and the number of lateral roots per seedling in the P. fluorescens-inoculated seedlings were 174%, 172% and 227% higher than in the control seedlings. Pseudomonas fluorescens had a significant promotional effect of the volatile contents compared to control, with ß-myrcene increasing by 192%, 2-undecanone by 203%, decanol by 304%, ß-caryophyllene by 197%, α-pinene by 281%, bornyl acetate by 157%, γ-terpinene by 239% and 3-tetradecane by 328% in P. fluorescens-inoculated H. cordata seedlings. the contents of chlorogenic acid, rutin, quercitin, and afzelin were 284%, 154%, 137%, and 213% higher than in control seedlings, respectively. Our study provided basic data to assess the linkages between endophytic bacteria, plant phenotype and metabolites of H. cordata to provide an insight into P. fluorescens use as biological fertilizer, promoting the synthesis of medicinal plant compounds.

An assembled bacterial community associated with Artemisia annua L. causes plant protection against a pathogenic fungus.

The microorganisms associated with a plant influence its growth and fitness. These microorganisms accumulate on the aerial and root surfaces of plants, as well as within the plants, as endophytes, although how the interaction between microorganisms protects the plant from pathogens is still little understood. In the current study, the impact of assembled the bacterial communities against the pathogenic fungus to promote Artemisia annua L. growths was investigated. We established a model of bacterium-fungus-plant system. Eight bacterial strains and a fungal pathogen Globisporangium ultimum (Glo) were isolated from wild A. annua roots and leaves, respectively. We assembled the six-bacteria community (C6: Rhizobium pusense, Paracoccus sp., Flavobacterium sp., Brevundimonas sp., Stenotrophomonas sp., and Bacillus sp.) with inhibition, and eight-bacteria community (C8) composing of C6 plus another two bacteria (Brevibacillus nitrificans and Cupriavidus sp.) without inhibition against Glo in individually dual culture assays. Inoculation of seedlings with C8 significantly reduced impact of Glo. The growth and disease suppression of A. annua seedlings inoculated with C8 + Glo were significantly better than those of seedlings inoculated with only Glo. C8 had more inhibitory effects on Glo, and also enhanced the contents of four metabolites in seedling roots compared to Glo treatment only. Additionally, the inhibitory effects of root extracts from A. annua seedlings showed that Glo was most sensitive, the degree of eight bacteria sensitivity were various with different concentrations. Our findings suggested that the non-inhibitory bacteria played a vital role in the bacterial community composition and that some bacterial taxa were associated with disease suppression. The construction of a defined assembled bacterial community could be used as a biological fungicide, promoting biological disease control of plants.

Latent Pathogenic Fungi in the Medicinal Plant Houttuynia cordata Thunb. Are Modulated by Secondary Metabolites and Colonizing Microbiota Originating from Soil.

Latent pathogenic fungi (LPFs) affect plant growth, but some of them may stably colonize plants. LPFs were isolated from healthy Houttuynia cordata rhizomes to reveal this mechanism and identified as Ilyonectria liriodendri, an unidentified fungal sp., and Penicillium citrinum. Sterile H. cordata seedlings were cultivated in sterile or non-sterile soils and inoculated with the LPFs, followed by the plants' analysis. The in vitro antifungal activity of H. cordata rhizome crude extracts on LPF were determined. The effect of inoculation of sterile seedlings by LPFs on the concentrations of rhizome phenolics was evaluated. The rates of in vitro growth inhibition amongst LPFs were determined. The LPFs had a strong negative effect on H. cordata in sterile soil; microbiota in non-sterile soil eliminated such influence. There was an interactive inhibition among LPFs; the secondary metabolites also regulated their colonization in H. cordata rhizomes. LPFs changed the accumulation of phenolics in H. cordata. The results provide that colonization of LPFs in rhizomes was regulated by the colonizing microbiota of H. cordata, the secondary metabolites in the H. cordata rhizomes, and the mutual inhibition and competition between the different latent pathogens.

Endophytic fungi stimulate the concentration of medicinal secondary metabolites in houttuynia cordata thunb.

Endophytic fungi usually establish a symbiotic relationship with the host plant and affect its growth. In order to evaluate the impact of endophytic fungi on the Chinese herbal medicinal plant Houttuynia cordata Thunb., three endophytes isolated from the rhizomes of H. cordata, namely Ilyonectria liriodendra (IL), unidentified fungal sp. (UF), and Penicillium citrinum (PC), were co-cultured individually with H. cordata in sterile soil for 60 days. Analysis of the results showed that the endophytes stimulated the host plant in different ways: IL increased the growth of rhizomes and the accumulation of most of the phenolics and volatiles, UF promoted the accumulation of the medicinal compounds afzelin, decanal, 2-undecanone, and borneol without influencing host plant growth, and PC increased the fresh weight, total leaf area and height of the plants, as well as the growth of the rhizomes, but had only a small effect on the concentration of major secondary metabolites. Our results proved that the endophytic fungi had potential practical value in terms of the production of Chinese herbal medicines, having the ability to improve the yield and accumulation of medicinal metabolites.

GC-MS analyses of the volatiles of Houttuynia cordata Thunb.

GC-MS is the basis of analysis of plant volatiles. Several protocols employed for the assay have resulted in inconsistent results in the literature. We developed a GC-MS method, which were applied to analyze 25 volatiles (α-pinene, camphene, ß-pinene, 2-methyl-2-pentenal, myrcene, (+)-limonene, eucalyptol, trans-2-hexenal, γ-terpinene, cis-3-hexeneyl-acetate, 1-hexanol, α-pinene oxide, cis-3-hexen-1-ol, trans-2-hexen-1-ol, decanal, linalool, acetyl-borneol, ß-caryophyllene, 2-undecanone, 4-terpineol, borneol, decanol, eugenol, isophytol and phytol) of Houttuynia cordata Thunb. Linear behaviors for all analytes were observed with a linear regression relationship (r2>0.9991) at the concentrations tested. Recoveries of the 25 analytes were 98.56-103.77% with RSDs <3.0%. Solution extraction (SE), which involved addition of an internal standard, could avoid errors for factors in sample preparation by steam distillation (SD) and solidphase micro extraction (SPME). Less sample material (≍0.05g fresh leaves of H. cordata) could be used to determine the contents of 25 analytes by our proposed method and, after collection, did not affect the normal physiological activity or growth of H. cordata. This method can be used to monitor the metabolic accumulation of H. cordata volatiles.

[Correlation analysis of nutrients and microorganisms in soils with polyphenols and total flavonoids of Houttuynia cordata].

The relationship of nutrients and microorganisms in soils with polyphenols and total flavonoids of Houttuynia cordata were investigated by measuring nutrients, enzyme activity, pH, concentrations of microbe phospholipid fatty acids (PLFAs) in soils, and determining concentrations of polyphenols and total flavonoids of H. cordata. The research is aimed to understand characteristics of the planting soils and improve the quality of cultivated H. cordata. The soils at different sample sites varied greatly in nutrients, enzyme activity, pH, microbic PLFAs and polyphenols and all flavonoids. The content of total PLFAs in sample sites was following: bacteria > fungi > actinomyces > nematode. The content of bacteria PLFAs was 37.5%-65.0% at different sample sites. Activities of polyphenol oxidease, concentrations of available P and content of PLFAs of bacteria, actinomyces and total microorganisms in soils were significantly and positively related to the concentrations of polyphenols and total flavonoids of H. cordata, respectively (P < 0.05) . The Content of fungi PLFAs in soils was significantly and negatively related to concentrations of polyphenols and total flavonoids of H. cordata, respectively (P < 0.05). This study provides evidence that effectiveness of the soil nutrient, which may be improved due to transformation of soil microorganisms and enzymes to N and P in the soils, was beneficial to adaptation of H. cordata adapted to different soil conditions, and significantly affects metabolic accumulation of polyphenols and flavonoids of H. cordata.

Quality evaluation of Houttuynia cordata Thunb. by high performance liquid chromatography with photodiode-array detection (HPLC-DAD).

A new, validated method, developed for the simultaneous determination of 16 phenolics (chlorogenic acid, scopoletin, vitexin, rutin, afzelin, isoquercitrin, narirutin, kaempferitrin, quercitrin, quercetin, kaempferol, chrysosplenol D, vitexicarpin, 5-hydroxy-3,3',4',7-tetramethoxy flavonoids, 5-hydroxy-3,4',6,7-tetramethoxy flavonoids and kaempferol-3,7,4'-trimethyl ether) in Houttuynia cordata Thunb. was successfully applied to 35 batches of samples collected from different regions or at different times and their total antioxidant activities (TAAs) were investigated. The aim was to develop a quality control method to simultaneously determine the major active components in H. cordata. The HPLC-DAD method was performed using a reverse-phase C18 column with a gradient elution system (acetonitrile-methanol-water) and simultaneous detection at 345 nm. Linear behaviors of method for all the analytes were observed with linear regression relationship (r(2)>0.999) at the concentration ranges investigated. The recoveries of the 16 phenolics ranged from 98.93% to 101.26%. The samples analyzed were differentiated and classified based on the contents of the 16 characteristic compounds and the TAA using hierarchical clustering analysis (HCA) and principal component analysis (PCA). The results analyzed showed that similar chemical profiles and TAAs were divided into the same group. There was some evidence that active compounds, although they varied significantly, may possess uniform anti-oxidant activities and have potentially synergistic effects.

[Chemical constituents of Psammosilene tunicoides and bacteriostatic activity].

OBJECTIVE: To study the chemical constituents and bacteriostatic activity of Psammosilene tunicoides. METHOD: Such methods as silica gel column chromatography and gel column chromatography were adotped separate and purify the compounds, and their structures were indentified on the basis of their spectral data and physicochemical properties. RESULT: Ten compounds were separated from ethanol extracts and identified as methyl-4-hydroxybenzoate (1), N-methylsaccharin (2), 3-hydroxy-4-methoxybenzoic acid (3), germanicol (4), tricosanoic acid (5), octacosane (6), amber acid (7), succinic acid (8), stellarine A (9), and oleanane-12-ene-3alpha, 16alpha-two hydroxy-23,28-acid (10). CONCLUSION: All compounds except compound 10 were separated from P. tunicoides for the first time. Compounds 8 and 9 showed the bacteriostatic activity to a certain extent.

[Researches on relationship between genetic differentiation and chemical variation of Cinnamomum migao].

OBJECTIVE: To study the relationship between the genetic diversity and chemical variation of Cinnamomum migao. METHOD: ISSR marker technique was used to research the genetic structure of 9 population, GC-MS was used to analyze the main ingredients of the volatile oil in C. migao. RESULT: The analysis on the main ingredients of the volatile oil showed that there were significant or extremely significant differences in 9 populations. The minimum variation index of population was Yunnan Funing and the maximum variation index of population was Guangxi Yueye. ISSR marker analysis showed that the average of polymorphic loci percentage (P) was 42.41%, expected heterozygosity (H) was 0.181 0, Shannon's information index (I) was 0.293 8, the Nei's genetic diversity (H(s)) in the group was 0.188 9, genetic differentiation index (G(st)) was 2.269 1. The relationship between the genetic diversity and chemical variation showed that there was no significant correlation between the main ingredients of the volatile oil and 4 indexes of genetic structure of C. migao. CONCLUSION: The genetic diversity of C. migao was relatively high at the population levels, while it is low within the population levels, the relationship between chemical variation and genetic diversity was not obvious, that may indicate that other factors causes the chemical variation of C. migao.

Chemical Compositionand Anti-acetyl cholinesterase Activity of Flower Essential Oils of Artemisiaannuaat Different Flowering Stage.

The chemical composition of the essential oils of flower at the pre-flowering, full-flowering and post-flowering stage of A. annua was analyzed by GC and GC/MS and sixty-two components were identified. The main compounds in the pre-flowering oil were ß-myrcene (37.71%), 1, 8-cineole (16.11%) and camphor (14.97%). The full-flowering oil contained predominantly caryophyllene (19.4%), germacrene D (18.1%), camphor (15.84%), 1, 8-cineole (10.6%) and (Z)-ß-farnesene (9.43%). The major constituents identified in the post-flowering oil were camphor (16.62%), caryophyllene (16.27%), ß-caryophyllene oxide (15.84%), ß-farnesene (9.05%) and (-)-spathulenol (7.21%). The variety of anti-AChE activity of flower oil of A. annua at three flowering stage might be a result of the variety of the content and interaction of those terpenoids with anti-AChE activity. The greatest acetylcholinesterase inhibitory activity (IC50 = 0.13 ± 0.02 mg mL(-1)) was exhibited by the essential oil of flower of A. annua at post-flowering stage.

[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.