[Inhibitory effect of three strains of biocontrol microbes on pathogens causing rhizome rot of Polygonatum cyrtonema].

Rhizome rot is one of the main disease in the cultivation of Polygonatum cyrtonema, and it is also a global disease which seriously occurs on the perennial medicinal plants such as Panax notoginseng and P. ginseng. There is no effective control method at present. To identify the effects of three biocontrol microbes(Penicillium oxalicum QZ8, Trichoderma asperellum QZ2, and Brevibacillus amyloliquefaciens WK1) on the pathogens causing rhizome rot of P. cyrtonema, this study verified six suspected pathogens for their pathogenicity on P. cyrtonema. The result showed that Fusarium sp. HJ4, Colletotrichum sp. HJ4-1, and Phomopsis sp. HJ15 were the pathogens of rhizome rot of P. cyrtonema, and it was found for the first time that Phomopsis sp. could cause rhizome rot P. cyrtonema. Furthermore, the inhibitory effects of biocontrol microbes and their secondary metabolites on three pathogens were determined by confrontation culture. The results showed that the three tested biocontrol microbes significantly inhibited the growth of three pathogens. Moreover, the secondary metabolites of T. asperellum QZ2 and B. amyloliquefaciens WK1 showed significant inhibition against the three pathogens(P<0.05), and the effect of B. amyloliquefaciens WK1 sterile filtrate was significantly higher than that of high tempe-rature sterilized filtrate(P<0.05). B. amyloliquefaciens WK1 produced antibacterial metabolites to inhibit the growth of pathogens, and the growth inhibition rate of its sterile filtrate against three pathogens ranged from 87.84% to 93.14%. T. asperellum QZ2 inhibited the growth of pathogens through competition and antagonism, and P. oxalicum QZ8 exerted the inhibitory effect through competition. The research provides new ideas for the prevention and treatment of rhizome rot of P. cyrtonema and provides a basis for the di-sease control in other crops.

[Effects of moso bamboo (Phyllostachys edulis) expansion on soil microbial community in evergreen broad-leaved forest]. / æ¯›ç«¹æ‰©å¼ å¯¹å¸¸ç»¿é˜”å¶æž—åœŸå£¤å¾®ç”Ÿç‰©ç¾¤è½çš„å½±å“.

The special eco-physiological characteristics of moso bamboo (Phyllostachys edulis) facilitate their fast invasion in nature ecosystems. The widespread expansion of moso bamboo causes degradation of adjacent forest ecosystem and change of landscape, as well as soil properties and microbial community composition. However, how moso bamboo expansion affects soil microbial composition is far from fully understood. Herein, we selected four moso bamboo expansion transects with three forest types at the Anji Lingfeng temple forest farm, Zhejiang Province, including evergreen broadleaved forest (BLF), mixed P. edulis and broadleaved forest (MEF) and P. edulis forest (PEF). We examined the effects of moso bamboo expansion on soil properties and soil microbial phospholipid fatty acids (PLFAs). Our results showed that soil pH was higher in moso bamboo forest than in MEF and BLF by 0.37 and 0.32 unit. In contrast, soil organic carbon, ammonium, and nitrate contents significantly decreased. Biomass of soil microbial groups displayed a decreasing trend except arbuscular mycorrhizal fungi, and the microbial richness index (SR) and diversity index (H) decreased significantly. In summary, moso bamboo expansion affected soil nutrient and carbon inputs, which was an important factor affecting soil microbial community structure. Results of redundancy analysis showed that changes of soil organic carbon and ammonium content were the main factors driving soil microbial community.

Cytochrome P450 Monooxygenases CYP6AY3 and CYP6CW1 Regulate Rice Black-Streaked Dwarf Virus Replication in Laodelphax striatellus (Fallén).

The small brown planthopper, Laodelphax striatellus (Fallén), is an important agricultural pest that causes significant losses by sucking and transmitting multiple plant viruses, such as rice black-streaked dwarf virus (RBSDV). Insecticides are commonly used to control planthoppers and cause the induction or overexpression of cytochrome P450 monooxygenases (P450s) from the CYP3 and CYP4 clades after insecticide application. However, little is known about the roles of insecticides and P450s in the regulation of viral replication in insects. In this study, RBSDV-infected L. striatellus were injected with imidacloprid, deltamethrin, pymetrozine, and buprofezin, respectively. The insecticide treatments caused a significant decrease in RBSDV abundance in L. striatellus. Treatment of piperonyl butoxide (PBO), an effective inhibitor of P450s, significantly increased the RBSDV abundance in L. striatellus. Fourteen P450 candidate genes in the CYP3 clade and 21 in the CYP4 clade were systematically identified in L. striatellus, and their expression patterns were analyzed under RBSDV infection, in different tissues, and at different developmental stages. Among the thirty-five P450 genes, the expression level of CYP6CW1 was the highest, while CYP6AY3 was the lowest after RBSDV infection. Knockdown of CYP6CW1 and CYP6AY3 significantly increased the virus abundance and promoted virus replication in L. striatellus. Overall, our data reveal that CYP6CW1 and CYP6AY3 play a critical role in the regulation of virus replication in L.striatellus.

Comprehensive Identification and Characterization of Long Non-coding RNAs Associated With Rice Black-Streaked Dwarf Virus Infection in Laodelphax striatellus (Fallén) Midgut.

Long non-coding RNAs (lncRNAs) are involved in a variety of biological functions through transcriptional and post-transcriptional regulation. However, little is known about their functions in the process of insect mediated virus transmission. In the present study, we analyzed using RNA-Seq, the lncRNAs that were differentially expressed in response to Rice black-streaked dwarf virus (RBSDV) infection in Laodelphax striatellus (Fallén) midgut. A total of 13,927 lncRNAs were identified and over 69% were assigned to intergenic regions. Among them, 176 lncRNAs were differentially expressed and predicted to target 168 trans-regulatory genes. Ten differentially expressed lncRNAs were selected and their expression changes were validated by RT-qPCR. KEGG analysis showed that these target genes were enriched in the essential biological process, such as Purine metabolism, Valine, leucine and isoleucine degradation, and Fatty acid elongation. The expression levels of the differentially expressed lncRNAs and the predicted target genes that were significantly enriched in the Human papillomavirus infection pathway were analyzed by RT-qPCR. The results showed that several lncRNAs were co-expressed with their target genes. One of the lncRNAs called MSTRG15394 and its target gene, encoding a secreted protease inhibitor (PI), were up-regulated at the transcriptional level after RBSDV infection. Knockdown of MSTRG15394 could down-regulate the PI expression at mRNA level. Inhibition of either MSTRG15394 or PI expression by RNA interference promoted RBSDV accumulation in L. striatellus midgut. Our finding provides new insights into the function of lncRNAs in regulating virus infection in an important insect vector.

[Effects of Phyllostachys edulis cultivation on soil bacterial and fungal community structure and diversity]. / æ¯›ç«¹ç§æ¤å¯¹åœŸå£¤ç»†èŒå’ŒçœŸèŒç¾¤è½ç»“æž„åŠå¤šæ ·æ€§çš„å½±å“.

This study examined how soil bacterial and fungal communities responded to the cultivation history of Moso bamboo in Anji and Changxing counties, Huzhou, Zhejiang, China. Soil samples (0-20 and 20-40 cm) were taken from bamboo plantations subjected to different cultivation histories and analyzed the community structures of soil bacterial and fungal by PCR-DGGE methods. It was found that soil bacterial and fungal communities varied greatly with the development of bamboo plantations which converted from Masson pine forest or formed via invading adjacent broadleaf shrub forest. Soil bacterial community structures exhibited a greater response to bamboo cultivation time than fungal community, but bacteria structure of surface soil displayed an ability of resiliency to disturbance and the tendency to recover to the original state. The cultivation time, sampling site and soil layer significantly affected the biodiversity of soil bacteria and fungi, especially the latter two factors. Redundancy analysis (RDA) of soil properties and bacteria or fungi communities showed that there were no accordant factors to drive the alteration of microbial structure, and the first two axes explained less than 65.0% of variance for most of the sampling sites and soil layers, indicating there existed soil parameters besides the five examined that contributed to microbial community alteration.

[Effects of different fertilization regimes on soil fungal communities under Phyllostachys violascens stand]. / 不同施肥模式对雷竹林土壤真菌群落特征的影响.

In order to investigate the effect of fertilization on soil fungal community of Phyllostachys violascens stand, we characterized the abundance and community structure of soil fungi using the methods of terminal restriction fragment length polymorphism (T-RFLP) and real-time quantitative PCR. The experimental treatments included blank control (CK), chemical fertilizer (CF), organic manure (M), mixture of chemical fertilizer and organic manure (CFM) and mixture of chemical fertilizer and organic manure plus mulching (CFMM). The results showed that fungal community structure and diversity were markedly affected by different fertilization regimes. In 0-20 cm soil layer, CF and CFMM treatments resulted in a significant reduction of Shannon and evenness indices. Soil fungi under CK, M, CFMM treatments in 0-20 cm soil layer and CK, CF, CFMM treatments in 20-40 cm soil layer formed independent community structures. Soil fungal abundance showed significantly positive correlation with the concentrations of soil total nitrogen, ammonium nitrogen and nitrate nitrogen. Organic matter, ammonium nitrogen, available phosphorus and available nitrogen concentrations were the main factors that influenced the variation of fungal community composition. The above findings indicated that 0-20 cm and 20-40 cm soil layers had different characteristics in response to different fertilization regimes under P. violascens stand. The soil fungal community in the 0-20 cm layer was mainly affected by soil organic matter, while that in the 20-40 cm layer was sensitive to the input of chemical fertilizer. The effect of fertilization on the diversity of fungal community mainly occurred in the 0-20 cm soil layer.

[Effects of Phyllostachys edulis invasion of native broadleaf forest on soil fungal community].

To investigate variation of soil fungal community in response to invasion of Phyllostachys edulis into native broadleaf forest, we characterized the community structure and the abundance of fungi in soil under bamboo (BB), mixture forest of bamboo and broadleaf (MF) and broadleaf forest (BL) using terminal restriction fragment length polymorphism (T-RFLP) and real-time quantitative PCR. The results showed that the most obvious difference in the soil fungal community structure was observed between the BB and BF stands, followed by that between the MF and BL. Shannon index and evenness index of soil fungi were higher in the MF than in the BB and BL. pH and NH4+-N content were the most important environmental gradients on the distribution of fungal community under BB, while NO3(-)-N content significantly affected the distribution of the fungal community under BL. The abundance of fungi in BL was significantly higher than that in BB and MF, and the fungi abundance showed a negative correlation with soil pH but a positive correlation with NO3(-)-N content. These results implied that heterotrophic nitrification driven by fungi could occur in soil of BL, and this process might be changed by the bamboo invasion.

[The impact of S.gordonii on P. gingivalis on the form of biofilm].

PURPOSE: To investigate the impact of S.gordonii on the ultrastructure of P. gingivalis biofilm and on the amount of P. gingivalis in biofilm. METHODS: P. gingivalis and/or S.gordonii grew on the culture slides to form single P. gingivalis biofilm and heterotypic biofilm of P.gingivalis-S.gordonii. Then the ultrastructure of the 2 kinds of film were examined by scanning electron microscope, and the amount of P. gingivalis in the biofilm was detected by qPCR. Statistical analysis was performed using pair t test with SPSS 13.0 statistical package. RESULTS: At 72 h, the amount of heterotypic biofilm was much more than that of the single P. gingivalis biofilm. Moreover, the structure of the heterotypic biofilm was more regular and with more pore space compared to the single P. gingivalis biofilm. Compared to single P. gingivalis biofilm, the amount of P. gingivalis in heterotypic biofilm was 5.4, 3.8 and 4.4 fold at 24, 48 and 72 h, respectively. CONCLUSIONS: The growth of P. gingivalis was promoted by S. gordonii in the form of heterotypic biofilm compared to single P. gingivalis biofilm.

[Effects of intensive management on abundance and composition of soil N2-fixing bacteria in Phyllostachys heterocycla stands].

Denaturing gradient-gel electrophoresis and real-time quantitative PCR (qPCR) were employed to determine the effects of intensive management on soil N2-fixing bacteria in a moso bamboo (Phyllostachys heterocycla) plantation. Soil samples were collected from the moso bamboo stands receiving 0 (CK), 10, 15, 20, and 25 years of intensive management. It was found that intensive management caused a strong decrease in soil pH but a general increase in soil available nutrients. The structure of the N2-fixing bacterial communities in the soils having received 10 and 25 years of intensive management were quite similar to that from the CK; however, those from 15 and 20 years of intensification differed from the CK. With increasing time of intensive management, the abundance and diversity of the nifH gene at first decreased and then increased, with the minimum values being observed after 15 years of intensive management, indicating the eventual resiliency of N2-fixing bacteria to disturbance induced by intensive management. Redundancy analysis indicated that soil available potassium, available nitrogen, nitrite nitrogen, and ammonium nitrogen were more closely related to the changes of N2-fixing bacterial community structure compared with the other soil indices measured. In conclusion, the soil N2-fixing bacterial community was negatively affected by intensive management in the short term, but could recover in the long term.

[Effects of bamboo charcoal on the growth of Trifolium repens and soil bacterial community structure].

The effects of addition rates (0, 3% and 9%) and particle sizes (0.05, 0.05-1.0 and 1.0-2.0 mm) of bamboo charcoal on the growth of Trifolium repens and soil microbial community structure were investigated. The results showed that bamboo charcoal addition greatly promoted the early growth of T. repens, with the 9% charcoal addition rate being slightly better than the 3% charcoal addition rate. The effects of different particle sizes of bamboo charcoal on the growth of T. repens were not different significantly. Growth promotion declined with time during 120 days after sowing, and disappeared completely after 5 months. DGGE analysis of the bacterial 16S rDNA V3 fragment indicated that bamboo charcoal altered the soil bacterial community structure. The amount and Shannon diversity index of bacteria in the bamboo charcoal addition treatments increased compared with CK. The quantitative analysis showed that the amount of bacteria in the treatment with bamboo charcoal of fine particle (D < 0.05 mm) at the 9% addition rate was significantly higher than in the other treatments. The fine bamboo charcoal had a great effect on soil bacteria amount compared with the charcoal of other sizes at the same addition rate.

[Characteristics of soil ammonia-oxidation microbial communities in different subtropical forests, China].

To investigate the effects of different forest stands in subtropical China on the communities of soil ammonia-oxidizing microorganisms, we characterized the abundance of ammonia-oxidizing archaea (AOA) and bacteria (AOB), and the community structure of AOA in soils under stands of broad-leaved (BF) , Chinese fir (CF) , Pinus massoniana (PF) and moso bamboo (MB) forests using real-time quantitative PCR and denaturing gradient gel electrophoresis (DGGE). The results showed that the AOA gene copy numbers (1.62 x 10(6)-1.88 x 10(7) per gram of dry soil) were significantly higher than those of AOB genes (2.41 x 10(5)-4.36 x 10(5) per gram of dry soil). Significantly higher soil AOA abundance was detected in the MB than that in the CF (P < 0.05), and the latter was significantly higher than that in the BF and PF soils (P < 0.05). There were no significant differences in the soil AOB abundance among the four forest stands. As indicated by DGGE pattern, soil AOA species varied among the four forest stands. There was a difference in the soil AOA communities between the CF and MB stands. The AOA demonstrated a competitive advantage over the AOB in the soils under these major subtropical forests. Soil pH, concentrations of soil available potassium and organic carbon as well as the forest type were the main factors that influence the variation of AOA community structure and diversity.

Identification of genes required for Cf-dependent hypersensitive cell death by combined proteomic and RNA interfering analyses.

Identification of hypersensitive cell death (HCD) regulators is essential to dissect the molecular mechanisms underlying plant disease resistance. In this study, combined proteomic and RNA interfering (RNAi) analyses were employed to identify genes required for the HCD conferred by the tomato resistance gene Cf-4 and the Cladosporium fulvum avirulence gene Avr4. Forty-nine proteins differentially expressed in the tomato seedlings mounting and those not mounting Cf-4/Avr4-dependent HCD were identified through proteomic analysis. Among them were a variety of defence-related proteins including a cysteine protease, Pip1, an operative target of another C. fulvum effector, Avr2. Additionally, glutathione-mediated antioxidation is a major response to Cf-4/Avr4-dependent HCD. Functional analysis through tobacco rattle virus-induced gene silencing and transient RNAi assays of the chosen 16 differentially expressed proteins revealed that seven genes, which encode Pip1 homologue NbPip1, a SIPK type MAP kinase Nbf4, an asparagine synthetase NbAsn, a trypsin inhibitor LeMir-like protein NbMir, a small GTP-binding protein, a late embryogenesis-like protein, and an ASR4-like protein, were required for Cf-4/Avr4-dependent HCD. Furthermore, the former four genes were essential for Cf-9/Avr9-dependent HCD; NbPip1, NbAsn, and NbMir, but not Nbf4, affected a nonadaptive bacterial pathogen Xanthomonas oryzae pv. oryzae-induced HCD in Nicotiana benthamiana. These data demonstrate that Pip1 and LeMir may play a general role in HCD and plant immunity and that the application of combined proteomic and RNA interfering analyses is an efficient strategy to identify genes required for HCD, disease resistance, and probably other biological processes in plants.

[Rationality for dynamic detection of shikonin of Arnerbia euchroma in industrial process].

OBJECTIVE: To discover reasons for great loss of shikonin during the concentrating process of percolate of Arnerbia euchroma (Royle) Johnst and develop a reasonable method for determination of shikonin. METHODS: Shikonin was selected as index, optimized chromatographic condition was used for analyzing the affection of alcohol content and crude drug content of sample solution on determination of shikonin, furthermore, reasonable sample preparation and determination methodology was examined and defined. RESULTS: The optimized chromatographic condition was as follows: shikonin was analyzed with a Zorbax Extend C-18 column (4.6 mm x 250 mm, 5 microm), methanol: water (82: 18) as the mobile phase, the column was maintained at 35 degrees C, the flow rate was 1.0 mL min(-1), detection wavelength was set at 516 nm and the time for analysis reduced from 40 min to 24 min. Alcohol content of sample solution influenced determination results significantly, peak area of equal content shikonin in low alcohol content (<40%) was only about 20% - 30% of that of high alcohol content (>70%), the reasonable content of sample solution were 0.0167 - 0.083 g mL(-1) with alcohol content above 40%. The method showed good linearity, precision, reproducibility and accuracy. CONCLUSION: The alcohol content of sample solution correlated with peak area closely for the first time, which indicate another important reason for "great loss" of shikonin during concentration process is that too much low ethanol content in test solution leads too much low results. The new method can detect shikonin more effectively and more reasonably and can monitor production process with high efficiency and low cost.

[Evolvement of soil bacterial community in intensively managed Phyllostachys praecox stand and related affecting factors].

By using PCR-DGGE and redundancy analysis (RDA), this paper studied the variations of soil bacterial community structure during the intensive management of Phyllostachys praecox stand, and analyzed the soil factors affecting the diversity of the bacterial community. The results showed that after the transformation of paddy field into P. praecox stand, the Shannon and richness indices of soil bacterial community increased significantly. Long-term intensive management of P. praecox stand altered the community structure of soil bacteria, reflecting in the changes of main bacterial populations and the great decrease after an initial increase of the diversity indices of soil bacterial community. Soil pH, soil total nitrogen, and soil available nitrogen and potassium could explain 76.1% of the total variation of soil bacterial community, and soil pH showed the greatest effect though it was not significant, indicating that the evolvement of soil bacterial community under the long-term intensive management of P. praecox stand was the result of the synergistic effects of several soil factors.

Development of a virus-induced gene-silencing system for functional analysis of the RPS2-dependent resistance signalling pathways in Arabidopsis.

Virus-induced gene silencing (VIGS) offers a rapid and high throughput technique platform for the analysis of gene function in plants. Although routinely used in some Solanaceous species, VIGS system has not been well established in Arabidopsis thaliana (L.) Heynh. We have recently reported some factors that potentially influence tobacco rattle virus (TRV)-mediated VIGS of phytoene desaturase (PDS) and actin gene expression in Arabidopsis. In this study, we have further established that the Agrobacterium strain used for agro-inoculation significantly affects the VIGS efficiency. Strain GV3101 was highly effective; C58C1 and LBA4404 were invalid, while EHA105 was plant growth stage-dependent for TRV-induced gene silencing. Furthermore, the VIGS procedure optimised for the PDS gene was applied for the functional analysis of the disease resistance gene RPS2-mediated resistance pathway. Silencing of RPS2 led to loss of resistance to the otherwise avirulence strain of Pseudomonas syringae pv. tomato DC3000 carrying the avirulence gene AvrRpt2. Silencing of RIN4, a RPS2 repressor gene, gave rise to conversion of compatible interaction to incompatible. Silencing of NDR1, RAR1 and HSP90, known to be required for the RPS2-mediated resistance, resulted in loss of the resistance, while silencing of EDS1 and SGT1b, which are not required for the RPS2-mediated resistance, caused no change of the resistance. These results indicate that the optimised procedure for the TRV-based VIGS is a potentially powerful tool for dissecting the signal transduction pathways of disease resistance in Arabidopsis.

TCD study of hemodynamic changes in PCA response to photic stimulation.

OBJECTIVES: During visual stimulation, the elevated metabolism rate will couple with increase of blood flow velocity(BFV) in posterior cerebral artery(PCA). This study with TCD was aimed to investigate whether the coupling might change according to the different vasoneuronal conditions. METHODS: Ninety-nine volunteers including 24 hypertension(HT) patients and 2 patients suffering from both HT and diabetes mellitus (DM) were enrolled in this trial. BFV and pulse indexes(PI) in P2 segments of PCA on both sides were monitored during visual stimulation. RESULTS: In all subjects, Mean BFV increased and PI went down in response to visual stimulation. The percentages of changes (deltaV and deltaP) of both mean BFV and PI were larger in young group( < 55 years old) than in old one(> or = 55 years old). There was significant positive correlation between deltaV and deltaP. Multivariated regression analysis did not show HT and DM, but age related to deltaV(deltaP). We did not find significant difference of deltaV(deltaP) between left and right sides. CONCLUSIONS: Blood flow velocity in PCA P2 segment increased due to decreased cerebrovascular resistance during visual stimulation and the response weakened with aging of the patient.