[Changes in physicochemical property and microbial community of Pseudostellaria heterophylla soil at diffe-rent fallow ages]. / 太子参不同休耕年限土壤理化特征和微生物群落变化.

Pseudostellaria heterophylla is authentic traditional Chinese herbal medicine in Fujian Province. P. hete-rophylla suffers from serious consecutive monoculture problems. Fallow can alleviate such problems, but the mecha-nism is still unclear. In this study, high-throughput sequencing was performed to analyze the changes in soil microbial community structure and diversity in the P. heterophylla soil at different fallow ages as well as their relationships with soil physicochemical properties and phenolic acids. The results showed that fungal community diversity decreased but bacterial community diversity increased in fallow soils compared with the control soil of P. heterophy-lla. For bacterial communities, the relative abundance of Acidobacteria increased, while that of Proteobacteria and Actinobacteria decreased in fallow soils. For fungal communities, the relative abundance of dominant phyla had no significant difference between fallow and control soils. Soil acidity and organic matter content showed a trend of weakening and decreasing, respectively, with the increases of fallow years. In addition, with the increases of fallow years, the content of phenolic acids in soil, including benzoic acid and salicylic acid, showed significant decrease, while some other phenolic acids such as p-coumaric acid were accumulated obviously. Taken together, fallow could efficiently ameliorate the structure of soil microbial community and soil properties of P. heterophylla, and thus alleviate the effects of continuous cropping.

[Effects of Rehmannia glutinosa consecutive monoculture on the community structure and diversity of phyllosphere bacteria]. / åœ°é»„è¿žä½œå¯¹å¶é™ ç»†èŒç¾¤è½ç»“æž„åŠå¤šæ ·æ€§çš„å½±å“.

Rehmannia glutinosa, a perennial herbaceous species, belongs to the family Scrophularia-ceae. As a staple medicinal material, its tuberous roots are highly valued in traditional Chinese medicine. However, R. glutinosa suffers from serious consecutive monoculture problems in production, which leads to a decline in both productivity and quality. Phyllosphere bacteria, the most abundant component of phyllosphere microorganisms, play crucial roles in plant growth and health. Characterization of phyllosphere bacteria could provide new insights into the mechanisms of consecutive monoculture problems and their control measures. Meanwhile, the varied taxa could be served as an important indicator of consecutive monoculture problems. The barcoded pyrosequencing of 16S rDNA genes combined with a culture-dependent approach was applied to characterize the shifts of bacterial community structure and diversity in the phyllosphere under consecutive monoculture of R. glutinosa. The results showed that consecutive monoculture clearly affected bacterial community structure in the phyllosphere. The phyllosphere bacterial communities of the two-year monocultured (TY) and the diseased plants (DP) were more similar, and different from the one-year monocultured (OY). The evenness, Shannon and Simpson diversity indices were significantly lower in TY and DP than in OY. Species annotation showed that bacterial community in R. glutinosa phyllosphere mainly consisted of Proteobacteria (91.2%), Firmicutes (5.1%) and Actinobacteria (3.7%). There was no significant difference in the number of detected bacterial taxa. However, Proteobacteria was significantly increased while Firmicutes and Actinobacteria were significantly decreased under consecutive monoculture. At the genus level, the relative abundances of genera Exiguobacterium, Bacillus and Arthrobacter, potentially beneficial microorganisms, were significantly higher in OY than that in TY and DP, but it was opposite for the genus Pseudomonas. The results from the culture-dependent approach and pathogenicity test showed that Pseudomonas plecoglossicida D9, widely isolated from the diseased leaves, was highly pathogenic to leaves. In conclusion, R. glutinosa monoculture resulted in distinct phyllosphere bacterial community variation with the accumulation of pathogen loads at the expense of beneficial microorganisms, which could contribute to the occurrence of leaf disease symptoms,and aggravate R. glutinosa replant disease in a monoculture regime.

[Effects of biochar and sheep manure on rhizospheric soil microbial community in continuous ratooning tea orchards]. / æ–½ç”¨ç”Ÿç‰©è´¨ç‚­å’Œç¾Šç²ªå¯¹å®¿æ ¹è¿žä½œèŒ¶å›­æ ¹é™ åœŸå£¤å¾®ç”Ÿç‰©çš„å½±å“.

Long-term continuous ratooning of tea could lead to serious soil acidification, nutritional imbalance, and the deterioration of the rhizosphere micro-ecological environment. Understanding the effects of biochar and sheep manure on the growth of tea plants and the rhizosphere microbial community structure and function would provide theoretical basis to improve the soil micro-ecological environment of continuous ratooning tea orchards. Biolog technology combined with phospholipid fatty acid (PLFA) approaches were employed to quantify the effects of biochar (40 t·hm-2) and sheep manure on the growth of 20 years continuous ratooning tea plants, soil chemical properties, and the soil microbial community structure and function. The results showed that after one year treatment, biochar and sheep manure both improved soil pH and nutrition, and significantly enhanced tea production. Compared with the routine fertilizer application (CK), the biochar and sheep manure treatments significantly increased the carbon metabolic activity (AWCD) and microorganism diversity in the rhizosphere soils, and increased the relative utilization of the carbon sources such as amines, carbohydrates, and polymers. The total PLFA concentrations in the biochar and sheep manure treatments were significantly increased by 20.9% and 47.5% than that in the routine fertilizers application. In addition, sheep manure treatment significantly decreased the saturated/monosaturated fatty acids In conclusion, biochar and sheep manure could alleviate soil acidification, enhance soil nutrition and the growth of tea plants. Both management strategies could increase the soil microbial activity and biomass, enhance the diversity, and improve the microbial community structure, which could be taken as effective measures to regulate the rhizosphere micro-environment of tea plants.

Determination of arsenic species in Solanum Lyratum Thunb using capillary electrophoresis with inductively coupled plasma mass spectrometry.

A simple and highly efficient interface to couple capillary electrophoresis with inductively coupled plasma mass spectrometry by a microflow polyfluoroalkoxy nebulizer and a quadruple ion deflector was developed in this study. By using this interface, six arsenic species, including arsenite, arsenate, monomethylarsonic acid, dimethylarsinic acid, arsenobetaine, and arsenocholine, were baseline-separated and determined in a single run within 11 min under the optimized separation conditions. The instrumental detection limit was in the range of 0.02-0.06 ng/mL for the six arsenic compounds. Repeatability expressed as the relative standard deviation (n = 5) of both migration time and peak area were better than 2.5 and 4.3% for six arsenic compounds. The proposed method, combined with a closed-vessel microwave-assisted extraction procedure, was successfully applied for the determination of arsenic species in the Solanum Lyratum Thunb samples from Anhui province in China with the relative standard deviations (n = 5) ≤4%, method detection limits of 0.2-0.6 ng As/g and a recovery of 98-104%. The experimental results showed that arsenobetaine was the main speciation of arsenic in the Solanum Lyratum Thunb samples from different provinces in China, with a concentration of 0.42-1.30 µg/g.

[Effects of different amendments on contents of phenolic acids and specific microbes in rhizosphere of Pseudostellaria heterophylla.] / ä¸åŒæ”¹è‰¯æŽªæ–½å¯¹å¤ªå­å‚æ ¹é™ åœŸå£¤é šé ¸å«é‡åŠç‰¹å¼‚èŒç¾¤çš„å½±å“.

Pseudostellaria heterophylla is a perennial herbaceous plant in the family Caryophyllaceae. The tuberous roots of P. heterophylla are highly valued in traditional Chinese medicine and have a high market demand. However, extended monoculture of P. heterophylla results in a significant decline in the biomass and quality, and escalates disease and pest problems. Therefore, it is important to understand the underlying mechanism and biocontrol methods for consecutive monoculture problems. With "Zheshen 2" as an experimental material, the changes in the contents of main nutrients in soil, phenolic acids and specific microbes under monoculture and different amendments were analyzed by using high performance liquid chromatography (HPLC) and qPCR. The results showed that consecutive monoculture of P. heterophylla led to a decrease in yield by 43.5% while the microbial fertilizer treatment and the paddy-upland rotation could relieve the consecutive monoculture problems. Available nitrogen, available phosphorus, available potassium and total potassium were significantly higher in the consecutively monocultured soils than in the newly planted soils. But consecutive monoculture resulted in soil acidification. HPLC analysis showed that conse-cutive monoculture of this plant did not lead to a consistent accumulation of soil phenolic acids. At middle stage of root expansion and at harvest stage, most of phenolic acids were even higher in the newly planted soils than in the consecutively monocultured soils. Furthermore, qPCR analysis showed that the amounts of three specific pathogens identified previously (i.e. Fusarium oxysporum, Talaromyces helicus, Kosakonia sacchari) were significantly higher in the consecutively monocultured soils than in the newly planted soils. However, the microbial fertilizer treatment and the paddy-upland rotation resulted in a significant decline in the population of these specific pathogens and improved the soil environment. In conclusion, the consecutive monoculture problems of P. heterophylla may be due to the rapid proliferation of host-specific pathogens, rather than the deficiency of soil nutrients and the autotoxicity of allelochemicals in root exudates. The results in this study could provide the theoretical basis to explore the underlying mechanism of replanting disease of P. heterophylla and its biocontrol strategies.

Transcriptome-wide identification of the genes responding to replanting disease in Rehmannia glutinosa L. roots.

The development of the medicinal plant Rehmannia glutinosa L. are severely declined when are replanted on the soil of the preceding crops being themselves. The biological basis of this so called "replanting disease" is unknown. Here, we have exploited the parallel sequencing capacity of both RNA-seq and DGE technology to ascertain what genes are responsive to the replanting disease in roots of R. glutinosa. RNA-seq analysis generated 99,708 non-redundant consensus sequences from the roots of the first year (R1) and the second year (R2) replanted R. glutinosa plants. From this set, a total of 48,616 transcripts containing a complete or partial encoding region was identified. Based on this resource, two DGE tag libraries were established to capture the transcriptome differences between the R1 and R2 libraries. Finally, a set of 2,817 (1,676 up- and 1,141 down-regulated) differentially transcribed genes was screened, and 114 most strongly differentially transcribed genes were identified by DGE analysis between first year and replanted plants. Furthermore, a more detailed examination of 16 selected candidates was carried out by qRT-PCR. The indication was that replanting could promote Ca(2+) signal transduction and ethylene synthesis, resulting in forming of the replanting disease. We analyzed the biomass indexes of replanted R. glutinosa roots by irrigating Ca(2+) signal blockers. The results suggested that the alleviation of the disease impairment could be the decrease of Ca(2+) signal transduction. This study provided a global survey of the root transcriptome in replanted R. glutinosa roots at the tuberous root expansion stage. As a result, a number of candidate genes underlying the replanting disease have been identified.

Transcriptome/degradome-wide identification of R. glutinosa miRNAs and their targets: the role of miRNA activity in the replanting disease.

Rehmannia glutinosa, a traditional Chinese medicine herb, is unable to grow normally in a soil where the same species has recently been cultivated. The biological basis of this so called "replanting disease" is unknown, but it may involve the action of microRNAs (miRNAs), which are known to be important regulators of plant growth and development. High throughput Solexa/Illumina sequencing was used to generate a transcript library of the R. glutinosa transcriptome and degradome in order to identify possible miRNAs and their targets implicated in the replanting disease. A total of 87,665 unigenes and 589 miRNA families (17 of which have not been identified in plants to date) was identified from the libraries made from a first year (FP) and a second year (SP) crop. A comparison between the FP and SP miRNAs showed that the abundance of eight of the novel and 295 of the known miRNA families differed between the FP and SP plants. Sequencing of the degradome sampled from FP and SP plants led to the identification of 165 transcript targets of 85 of the differentially abundant miRNA families. The interaction of some of these miRNAs with their target(s) is likely to form an important part of the molecular basis of the replanting disease of R. glutinosa.

[Molecular cloning and expression analysis of an Aux/IAA gene (RgIAA1) from Rehmannia glutinosa].

To clone and analyze a member of the Auxin/indole-3-acetic acid (Aux/IAA) gene family, RgIAA1, from Rehmannia glutinosa. The transcriptional EST database of R. glutinosa was used to clone the new Aux/IAA gene by cDNA probe of AtIAA14. Bioinformatics was applied to analyze the sequence characteristics of RgIAA1 protein and construct phylogenetiC trees. Quantitative RT-PCR has been applied to detect the transcription level of RgIAA1 in seven tissues as well as in leaves under three stresses. The results showed that, the cDNA sequence of RgIAA1 contains 903 bp was obtained. The open reading frame (ORF) of RgIAA1 was 681 bp encoding 226 amino acids, which has typical structural domains and characteristic sequence of Aux/IAA family proteins. RgIAA1 showed the highest expression level in unfolded leaf, followed by the stem. And the expression of RglAA1 was quickly decreased with leaf growing up. The transcription level increased under continuous cropping conditions while it reduced both in salinity and waterlogging stresses. RgIAA1, an Aux/IAA gene from R. glutinosa has been obtained for the first time, which can lay the foundation for further studies about its molecular function in development and responses to stress.

[Characteristics of soil microbial community under different vegetation types in Wuyishan National Nature Reserve, East China].

By using Biolog Ecoplate system, this paper studied the structure and functional diversity of soil microbial community under different vegetation types in Wuyishan National Nature Reserve, aimed to probe into the effects of vegetation type on the diversity of soil microbial community. The results showed that the soil chemical properties, soil enzyme activities, and average well color development (AWCD) were higher in natural forest than in planted forest, and were the lowest in abandoned field. The AWCD reflecting soil microbial activity and functional diversity was increased with increasing incubation time, but there existed significant differences among different vegetation types. The carbon sources mostly used by soil microbes were carbohydrates and carboxylic acids, followed by amino acids, phenolic acids and polymers, and amines had the lowest utilization rate. The Simpson index, Shannon index, Richness index and McIntosh index in natural forest were holistically higher than those in planted forest. Principal component analysis (PCA) identified 2 principal component factors in relation to carbon sources, explaining 56.3% and 30.2% of the variation, respectively. The carbon sources used by soil microbial community differed with vegetation types. Amino acids and amides were the two main carbon sources separating the 2 principal component factors. The results of this study could provide basis for further approaching the relationships between vegetation diversity and soil microbial community diversity.

Identification of autotoxic compounds in fibrous roots of Rehmannia (Rehmannia glutinosa Libosch.).

Rehmannia is a medicinal plant in China. Autotoxicity has been reported to be one of the major problems hindering the consecutive monoculture of Rehmannia. However, potential autotoxins produced by the fibrous roots are less known. In this study, the autotoxicity of these fibrous roots was investigated. Four groups of autotoxic compounds from the aqueous extracts of the fibrous roots were isolated and characterized. The ethyl acetate extracts of these water-soluble compounds were further analyzed and separated into five fractions. Among them, the most autotoxic fraction (Fr 3) was subjected to GC/MS analysis, resulting in 32 identified compounds. Based on literature, nine compounds were selected for testing their autotoxic effects on radicle growth. Seven out of the nine compounds were phenolic, which significantly reduced radicle growth in a concentration-dependent manner. The other two were aliphatic compounds that showed a moderate inhibition effect at three concentrations. Concentration of these compounds in soil samples was determined by HPLC. Furthermore, the autotoxic compounds were also found in the top soil of the commercially cultivated Rehmannia fields. It appears that a close link exists between the autotoxic effects on the seedlings and the compounds extracted from fibrous roots of Rehmannia.

[Differential protein analysis on the root response of rice with high phosphorous uptake efficiency to low phosphorous stress].

A comparative proteomics analysis was performed to identify the molecular response of a rice cultivar (Oryza sative cv. 'IRRI71331') with high phosphorous (P) uptake efficiency to low P stress. The hydroponically grown rice plants were provided with two levels of P (0.5 mg x L(-1) and 10 mg x L(-1)) supplied in quarter strength Kimura solution, and the root total proteins extracted on the 3rd and 6th day of treatments were separated by two-dimensional gel electrophoresis (2-DE). Comparing with the control (10 mg x L(-1) of P), a total of 29 protein spots under low P stress (0.5 mg x L(-1)) showed differences in their relative abundance, among which, 17 were higher, 11 were lower, and 1 was novel on the 3rd day, and 8 were induced, 19 were suppressed, 1 was disappeared, and 1 had no obvious change on the 6th day. Ten differentially expressed protein spots were identified by MALDI-TOF/MS, and searched in protein databases. According to the putative functions, the identified proteins were classified into four groups, i.e., signal transduction (glycine-rich RNA-binding protein, phosphate starvation response regulator-like), gene expression (putative pre-mRNA splicing factor, putative AAA-metalloprotease), metabolism (adenylosuccinate lyase, serpin, S-adenosylmethionine synthetase, MYB transcription factor-like protein), and ion transport (cation-transporting ATPase, sarcoplasmic reticulum protein). The identified proteins were involved in various physiological responses to enhance stress resistance, such as signal recognition and transduction, RNA cleavage, degradation of denatured protein, and ion transportation and cellular ion balance. The serine protease inhibitor and S-adenosylmethionine synthetase and the MYB transcription factor-like protein, which were the key proteins associated with P deficiency--tolerance of other species, were affected by the same stress for rice. The results indicated that the tolerance to low P stress was controlled by a complex signal transduction and metabolism regulation network in rice root system.

[Isolation of sperm cells from rice pollen tubes].

Isolation of sperm cells from higher plants is a basis for studying the mechanism of double fertilization. In this study, the isolation of rice sperm cells from pollen tube was conducted. When fresh pollen grains from nearly blooming flowers were put into a medium containing 20% sucrose, 10% polyethylene glycol 4500 (PEG 4500), 0.05% CaCl2, 0.01% boric acid, over 40% pollen grains germinated and formed a pollen tube. After pollen tubes were transformed into a broken solution containing 8% mannitol, the tubes broke and released tube cytoplasm including two sperm cells. However, both sperm cells were enrapt in the cytoplasm and could not be identified. When 0.5% cellulase and pectinase were added into the broken solution, two sperm cells were released from cytoplasm. Both sperm cells could be collected using micromanipulator. We also tried to isolate sperm cells using in vivo-in vitro method: styles were pollinated and pollen tubes were allowed to grow for 40 min in vivo. Then styles were cut near ovary and floated in the same medium above-mentioned for 1 h until tubes emerged from the cut end. The styles with pollen tube were transformed into the broken solution and released the content including two sperm cells.

[Effects of fertilizer application on contents of 2-undecanone and soluble carbonhydrate in Houttuynia cordata with different fertilizers].

OBJECTIVE: To investigate the effect of five kinds of fertilizers at three application levels on the content of 2-undecanone and carbohydrate in Houttuynia cordata. METHOD: A single factor randomized block design was used to investigate the content of 2-undecanone and carbohydrate in the plant. RESULT: The results showed that the content of 2-undecanone was the highest both in aerial and underground parts of H. cordata, which was fertilized with complex fertilizers served in conventional way, having the content 18.6 microg g(-1) and 26.0 microg g(-1) respectively. In addition, 2-undecanone contents in aerial parts of H. cordata (14.9 microg g(-1)) fertilized with manure of human were also higher than that with chemical fertilizer, pig and duck manures, but no significant difference were found among the other treatments in aerial or underground parts of the plants, respectively. The results also demonstrated that fertilized with organic fertilizer might be beneficial to enhance the quality of sugar in H. cordata, mainly including the contents of total sugar, solutable sugar, fructose and reduced sugar in the plants, especially with manure of human and pig. CONCLUSION: As the result of this study and the related previous research on yield of H. cordata were considered, the fertilizing ways for increasing quality of H. cordata should take the manure of human as a main fertilizer and mix with the other organic fertilizers, complex fertilizers and chemical ones may be needed to balance the plant nutrient. In the field practice, the amount of organic fertilizer including 108,000 kg hm(-2) human mature, together with some high-efficient complex fertilizer and a small amount of quick-acting chemical fertilizer is recommended.