Assessment of the arbuscular mycorrhizal fungal community in roots and rhizosphere soils of Bt corn and their non-Bt isolines

Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) in combination with sequencing of amplified partial 18S ribosomal DNA was used to assess the effects of transgenic Bt corn 5422Bt1 (event Bt 11) and 5422CBCL (event MON810) on the community structure of a non-target microorganism, namely the arbuscular mycorrhizal fungi (AMF) Glomus in corn roots and rhizosphere soils, relative to their non-Bt isolines 5422 (conventional parent) and 5422wx (conventional hybrid). AMF colonization in roots of different corn genotypes was also assessed using microscopic visualization. No adverse effect was detected on the indigenous AMF colonization of the roots of Bt hybrids 5422Bt1 and 5422CBCL. Two-way indicator species analysis (TWINSPAN^®) and detrended correspondence analysis (DCA) of the DGGE data from corn roots presented differences between Bt and non-Bt corn isolines (5422Bt1 vs. 5422wx, and 5422CBCL vs. 5422wx). However, differences were also recognized between the two non-Bt corn cultivars (5422 vs. 5422wx), and between the two Bt corn lines (5422Bt1 vs. 5422CBCL) in roots. Our results suggest that corn genotypes may have a greater influence on the AMF community structure of plant roots and rhizosphere soils than other factors, such as the age of the growing plants.     

Bacterial community in plant residues in a Japanese paddy field estimated by RFLP and DGGE analyses

Plant residues (PRs) are “hot spots” of microbial activities in soil. PRs with the size more than 0.5 mm were collected from a Japanese paddy field during rice cultivation period (from May to September) and fractionated into four categories by size (>4, 2-4, 1-2, and 0.5-1 mm) using sieves. Restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE) patterns were compared among the fractions after DNA extraction from the PRs and PCR amplification. The total amount of PRs with the size over 0.5 mm decreased in the field with the first-order kinetics (r²=0.810, p<0.01) with time from rice transplanting to harvest. RFLP analysis showed that the bacterial community structure in PRs with the 0.5-2 mm fraction was different from that in PRs with the >2 mm fraction and the latter community structure changed after the midseason drainage. In contrast, the DGGE patterns of the bacterial community in the PRs indicated the succession from June to September during rice cultivation forming three major groups irrespective of the fraction size. Sequence analysis of DGGE bands showed that Firmicutes (clostridia), α-, γ-, δ-Proteobacteria (myxobacteria), Nitrospira, Acidobacteria, Bacteroidetes, Verrucomicrobia and Spirochaetes were predominant members in the PRs irrespective of fraction size.     

Storage method affects disease suppression of flax wilt induced by composts

Compost can have a disease suppressive effect, but compost research is constrained by the fact that repetition of experiments with a similar batch of compost is impossible, since storage affects the organic material including the microbial communities. The objective of this study was to test the hypothesis that differential changes in microbial community structure and associated microbial activities after various storage methods (drying, freezing and cooling) lead to differential changes in the disease suppressive ability of compost material with respect to Fusarium oxysporum f. sp. lini induced by mixes of composts with peat substrate (20/80%, vol./vol.). A significant (P<0.0001) storage method × compost interaction was found with respect to suppression of Fusarium wilt of flax, indicating that the effect of storage type on disease suppression is compost-dependent. For seven composts storage had no (13 cases) or a significantly positive effect (eight cases) on disease suppression and for 1 compost there was a significant negative effect of storage on disease suppression. Significant changes in microbial activity and 16S-rDNA DGGE banding patterns of the composts were observed as a result of all tested ways of storage and these changes could be related to changes in disease suppression: relatively strong changes in microbial activity and bacterial composition due to storage had a relatively strong effect on disease suppression. The cool storage treatment (4 °C) resulted in the least deviation in disease suppression from the fresh compost, although the freezing treatment gave the most reliable results with the lowest standard deviation.     

Phosphate retention on soils in the central valley of Chile

The retention of phosphate and the content of available phosphate were estimated by a new method with monobasic sodium phosphate (pH 4.5) and the Olsen's method, respectively, on the Principal soils in the central valley in Chile. Trumaos (Andosols) are, in general, distinctly in the value of P-retention than other soils with the exception of trumaos of alluvial origins. This characteristic of trumaos can be employed for the identification of trumaos among r soils and alluvial trumaos among ordinary ones. The P-retention value of volcanic ash soils of decreases with the crystallization of the amorphous inorganic materials. According to the data of P-retention, certain rojos arcillosos (red clays) are presumed to have surface horizons of volcanic ash origins. Granitic rojos arcillosos are notably low in P-retention compared with other arcillosos. The organic matter content and P-retention value of soils are closely related to each other, suggesting that both organic matter and phosphate are retained by soils on the same a erials though by different mechanisms.

The content of available phosphate in surface horizons varied from soil to soil and no difference was noticeable among the soil groups of trumaos, rojos arcillosos, and paddy soils. Also no relation was found between the P-retention value and available phosphate content. However, in lower horizons where plant roots are scarce or absent, no available phosphate was found in trumaos in contrast to the presence of an appreciable amount of phosphate in rojos arcillosos. Phosphate of native trumaos appear to be practically insoluble. The surface soil rich in available phosphate was limited to cultivated fields, suggesting that the available phosphate was largely due to the remnant of applied fertilizers and partly to the remains of organic phosphorus from plants and soil organisms. The content of available phosphate tends to decrease with time after application of phosphates, and a significant negative correlation coefficient was obtained between the amount of available phosphate and the P-retention value of soils at least by three months after application of phosphate. The higher the P-retention value of soils, the less the content of available phosphate, and the faster the fixation of phosphate.     

Application of DGGE analysis to the study of bacterial community structure in plant roots and in nonrhizosphere soil

To analyze the structure of bacterial communities in spinach roots and in the nonrhizosphere soil, we used PeR-amplified 16S rRNA gene fragments separated by denaturing gradient gel electrophoresis (DGGE). DGGE revealed a large number of band patterns, which were ascribed to various bacterial species composing each of the bacterial communities. The pattern from the roots was less complex than that from the soil. It is considered that DGGE analysis is suitable for studies of bacterial community structure in soil-plant ecosystems.     

长期施肥对黑土农田土壤微生物群落的影响

基于中国科学院海伦农业生态试验站长期定位试验区,应用实时荧光定量PCR(Real-time PCR)和变性梯度凝胶电泳(DGGE)技术研究了无施肥(NF)、单施N、P化肥(NP)以及化肥配施有机猪粪肥(NPM)等3种长期施肥措施对黑土区玉米田土壤微生物群落密度和结构的影响.Real-time PCR方法定量NF、NP及NPM措施土壤细菌群落基因组DNA质量分别为381、1 351和1 773 ng g-1干土,真菌群落基因组DNA质量分别113.3、127.3和20.6 ng g-1干土,真菌与细菌的比率分别为0.31、0.09和0.01,NPM措施显著低于另两种施肥方式(p＜0.05).DGGE方法研究表明,NP和NPM措施不能改善土壤细菌和真菌群落的多样性、均匀性及优势菌优势程度;但主成分分析结果显示NP和NPM措施均可改变土壤细菌和真菌群落的构成,且真菌群落的变化更为显著;聚类分析结果显示NP和NPM措施下细菌群落结构较相近,其相似系数为0.89,真菌群落中NP措施与NF措施相近,相似系数为0.63,高于NP与NPM措施的相似系数0.51.上述结果表明有机猪粪肥的长期施用可以显著降低黑土农田土壤真菌与细菌的比率,且明显地改变土壤细菌和真菌群落的结构.     

Structural and functional diversity of bacterial community in soil treated with the herbicide napropamide estimated by the DGGE,CLPP and r/K-strategy approaches

Napropamide is one of the most commonly used herbicide in agricultural practice and can exhibit toxic effect to soil microorganisms. Therefore, the main objective of this study was to examine the genetic and functional diversity of microbial communities in soil treated with napropamide at field rate (FR, 2.25 mg kg⁻¹ of soil) and 10 times the FR (10 × FR, 22.5 mg kg⁻¹ of soil) by the denaturing gradient gel electrophoresis (DGGE) and the community level physiological profile (CLPP) methods. In addition, the r/K-strategy approach was used to evaluate the effect of this herbicide on the community structure of the culturable soil bacteria. DGGE patterns revealed that napropamide affected the structure of microbial community; however, the richness (S) and genetic diversity (H) values indicated that the FR dosage of napropamide experienced non-significant changes. In turn, the 10 × FR dosage of herbicide caused significant changes in the S and H values of dominant soil bacteria. DGGE profiles suggest an evolution of bacteria capable of degrading napropamide among indigenous microflora. Analysis of the CLPPs indicated that the catabolic activity of microbial community expressed as AWCD (average well-color development) was temporary positively affected after napropamide application and resulted in an increase of the substrate richness (S_R) as well as functional biodiversity (H) values. Analysis of the bacterial growth strategy revealed that napropamide affected the r- or K-type bacterial classes (ecotypes). In treated-soil samples K-strategists dominated the population, as indicated by the decreased ecophysiological (EP) index. Napropamide significantly affected the physiological state of culturable bacteria and caused a reduction in the rate of colony formation as well as a prolonged time of growth rate. Obtained results indicate that application of napropamide may poses a potential risk for soil functioning.     

Growth of hydrogenotrophic and acetoclastic methanogens on substrate from rice plant callus cells in anaerobic soil: an estimation to the role of slough-off root cap cells to their growth

Flooded paddy fields are the major anthropogenic sources of methane (CH₄) emission, and organic materials of rice plant origin were estimated to be important as its source. This study used rice (Oryza sativa L. cv, Yukihikari) callus cells as a model material for slough-off root cap cells, and carbon-13 (¹³C)-labelled callus cells were subjected to decomposition in aerobic and anaerobic soil microcosms for 56 days. DNA was extracted from a soil incubated with carbon-12 (¹²C)- and ¹³C-callus cells and subjected to buoyant density gradient centrifugation to identify methanogenic archaeal species that assimilated carbon from the callus cells. ¹³C-labelled 16S rRNA gene (16S rDNA) fragments from methanogenic archaea were not polymerase chain reaction (PCR)-amplified in heavy fractions under aerobic soil conditions, while they were successfully done from day 3 onwards under anaerobic soil conditions. Eighty-four denaturing gradient gel electrophoresis (DGGE) bands in heavy fractions were sequenced, revealing that they were members of Methanosarcina spp. (20 clones), Methanosaeta spp. (18 clones), Methanocella spp. (25 clones), Methanomicrobiales (10 clones), Methanobacterium spp. (7 clones) and Cluster ZC-I (2 clones). They included hydrogenotrophic and acetoclastic methanogens and were phylogenetically different from those residing in rice roots and, presumably, from those assimilating root exudate and mucilage-derived carbon. This study indicates that carbon of slough-off root cap cells propagates specific methanogenic species in rice rhizosphere under anaerobic soil conditions and thus augments the diversity of the total rhizospheric methanogenic community.     

鸡粪好氧堆肥过程中氨氧化古菌群落结构的动态变化

应用聚合酶链式反应-变性梯度凝胶电泳（PCR DGGE）技术,研究了好氧堆肥过程氨氧化古菌（ammonia oxidizing archaea, AOA）的群落结构和多样性变化。结果表明,不同堆肥时期鸡粪好氧堆肥AOA菌群的群落结构发生了明显的变化。与AOA HH 2(GU2258721)亲缘关系较近的b条带(相似性96%)和未培养泉古菌属［uncultured crenarchaeote NM 152(HQ8752251)］的m条带(相似性99%)是堆肥过程一直存在的AOA菌属。条带c、 b、 f、 i和条带m、 k、 l、 n代表的种属分属两个大类。AOA群落的Shannon Weiner指数（H）、 均匀度指数（EH）均表现为第30 d＞第5 d＞第25 d第45 d＞第3 d第12 d＞第1 d第15 d。对AOA种群数据与不同时期堆体温度、 pH、 全氮、 铵态氮、 亚硝态氮和硝态氮等环境因子的冗余分析表明,堆体温度、 全氮、 亚硝态氮和硝态氮对氨氧化古菌群落演替有着显著的影响（P0.05）,AOA的群落结构在堆肥第1 d、 第5 d、 第25 d、 第30 d、 第45 d变化较大。说明这些参数能有效控制鸡粪好氧堆肥过程中AOA的群落结构。