Use of 16S rRNA Gene Terminal Restriction Fragment Analysis To Assess the Impact of Solids Retention Time on the Bacterial Diversity of Activated Sludge

Terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes was used to investigate the reproducibility and stability in the bacterial community structure of laboratory-scale sequencing batch bioreactors (SBR) and to assess the impact of solids retention time (SRT) on bacterial diversity. Two experiments were performed. In each experiment two sets of replicate SBRs were operated for a periods of three times the SRT. One set was operated at an SRT of 2 days and another set was operated at an SRT of 8 days. Samples for T-RFLP analysis were collected from the two sets of replicate reactors. HhaI, MspI, and RsaI T-RFLP profiles were analyzed using cluster analysis and diversity statistics. Cluster analysis with Ward's method using Jaccard distance and Hellinger distance showed that the bacterial community structure in both sets of reactors from both experimental runs was dynamic and that replicate reactors were clustered together and evolved similarly from startup. Richness (S), evenness (E), the Shannon-Weaver index (H), and the reciprocal of Simpson's index (1/D) were calculated, and the values were compared between the two sets of reactors. Evenness values were higher for reactors operated at an SRT of 2 days. Statistically significant differences in diversity (H and D) between the two sets of reactors were tested using a randomization procedure, and the results showed that reactors from both experimental runs that were operated at an SRT of 2 days had higher diversity (H and D) at the 5% level. T-RFLP analysis with diversity indices proved to be a powerful tool to analyze changes in the bacterial community diversity in response to changes in the operational parameters of activated-sludge systems.     

Effects of short solids retention time on microbial community in a membrane bioreactor

Effects of operating lab-scale nitrifying membrane bioreactors (MBR) at short solids retention times (SRT = 3, 5 and 10d) were presented with focus on reactor performance and microbial community composition. The process was capable of achieving over 87% removal of ammonia and 95% removal of chemical oxygen demand (COD), almost regardless of SRT. The denaturing gradient gel electrophoresis (DGGE) analysis shown that bacterial communities evolved in time in a similar way at different SRT. The results of clone library analysis indicated that Betaproteobacteria was the dominant bacterial group in all the reactors but there were significant difference of species for different SRT with higher species diversity at longer SRT. Ammonia and COD removal efficiencies were not correlated with the number of bacterial species or their diversity.     

Interpreting 16S rDNA T-RFLP Data: Application of Self-Organizing Maps and Principal Component Analysis to Describe Community Dynamics and Convergence

Interpreting the large amount of data generated by rapid profiling techniques, such as T-RFLP, DGGE, and DNA arrays, is a difficult problem facing microbial ecologists. This study compares the ability of two very different ordination methods, principal component analysis (PCA) and self-organizing map neural networks (SOMs), to analyze 16S-DNA terminal restriction-fragment length polymorphism (T-RFLP) profiles from microbial communities in glucose-fed methanogenic bioreactors during startup and changes in operational parameters. Our goal was not only to identify which samples were similar, but also to decipher community dynamics and describe specific phylotypes, i.e., phylogenetically similar organisms, that behaved similarly in different reactors. Fifteen samples were taken over 56 volume changes from each of two bioreactors inoculated from river sediment (S2) and anaerobic digester sludge (M3) and from a well-established control reactor (R1). PCA of bacterial T-RFLP profiles indicated that both the S2 and M3 communities changed rapidly during the first nine volume changes, and then became relatively stable. PCA also showed that an HRT of 8 or 6 days had no effect on either reactor communtity, while an HRT of 2 days changed community structure significantly in both reactors. The SOM clustered the terminal restriction fragments according to when each fragment was most abundant in a reactor community, resulting in four clearly discernible groups. Thirteen fragments behaved similarly in both reactors, eight of which composed a significant proportion of the microbial community as judged by the relative abundance of the fragment in the T-RFLP profiles. Six Bacteria terminal restriction fragments shared between the two communities matched cloned 16S rDNA sequences from the reactors related to Spirochaeta, Aminobacterium, Thermotoga, and Clostridium species. Convergence also occurred within the acetoclastic methanogen community, resulting in a predominance of Methanosarcina siciliae-related organisms. The results demonstrate that both PCA and SOM analysis are useful in the analysis of T-RFLP data; however, the SOM was better at resolving patterns in more complex and variable data than PCA ordination.     

Comparison of Two Fingerprinting Techniques, Terminal Restriction Fragment Length Polymorphism and Automated Ribosomal Intergenic Spacer Analysis, for Determination of Bacterial Diversity in Aquatic Environments

We investigated bacterial diversity in different aquatic environments (including marine and lagoon sediments, coastal seawater, and groundwater), and we compared two fingerprinting techniques (terminal restriction fragment length polymorphism [T-RFLP] and automated ribosomal intergenic spacer analysis [ARISA]) which are currently utilized for estimating richness and community composition. Bacterial diversity ranged from 27 to 99 phylotypes (on average, 56) using the T-RFLP approach and from 62 to 101 genotypes (on average, 81) when the same samples were analyzed using ARISA. The total diversity encountered in all matrices analyzed was 144 phylotypes for T-RFLP and 200 genotypes for ARISA. Although the two techniques provided similar results in the analysis of community structure, bacterial richness and diversity estimates were significantly higher using ARISA. These findings suggest that ARISA is more effective than T-RFLP in detecting the presence of bacterial taxa accounting for <5% of total amplified product. ARISA enabled also distinction among aquatic bacterial isolates of Pseudomonas spp. which were indistinguishable using T-RFLP analysis. Overall, the results of this study show that ARISA is more accurate than T-RFLP analysis on the 16S rRNA gene for estimating the biodiversity of aquatic bacterial assemblages.     

Comparison of two fingerprinting techniques, terminal restriction fragment length polymorphism and automated ribosomal intergenic spacer analysis, for determination of bacterial diversity in aquatic environments

We investigated bacterial diversity in different aquatic environments (including marine and lagoon sediments, coastal seawater, and groundwater), and we compared two fingerprinting techniques (terminal restriction fragment length polymorphism [T-RFLP] and automated ribosomal intergenic spacer analysis [ARISA]) which are currently utilized for estimating richness and community composition. Bacterial diversity ranged from 27 to 99 phylotypes (on average, 56) using the T-RFLP approach and from 62 to 101 genotypes (on average, 81) when the same samples were analyzed using ARISA. The total diversity encountered in all matrices analyzed was 144 phylotypes for T-RFLP and 200 genotypes for ARISA. Although the two techniques provided similar results in the analysis of community structure, bacterial richness and diversity estimates were significantly higher using ARISA. These findings suggest that ARISA is more effective than T-RFLP in detecting the presence of bacterial taxa accounting for <5% of total amplified product. ARISA enabled also distinction among aquatic bacterial isolates of Pseudomonas spp. which were indistinguishable using T-RFLP analysis. Overall, the results of this study show that ARISA is more accurate than T-RFLP analysis on the 16S rRNA gene for estimating the biodiversity of aquatic bacterial assemblages.     

Phylogenetic diversity, host-specificity and community profiling of sponge-associated bacteria in the northern Gulf of Mexico

Background

Marine sponges can associate with abundant and diverse consortia of microbial symbionts. However, associated bacteria remain unexamined for the majority of host sponges and few studies use phylogenetic metrics to quantify symbiont community diversity. DNA fingerprinting techniques, such as terminal restriction fragment length polymorphisms (T-RFLP), might provide rapid profiling of these communities, but have not been explicitly compared to traditional methods.

Methodology/Principal Findings

We investigated the bacterial communities associated with the marine sponges Hymeniacidon heliophila and Haliclona tubifera, a sympatric tunicate, Didemnum sp., and ambient seawater from the northern Gulf of Mexico by combining replicated clone libraries with T-RFLP analyses of 16S rRNA gene sequences. Clone libraries revealed that bacterial communities associated with the two sponges exhibited lower species richness and lower species diversity than seawater and tunicate assemblages, with differences in species composition among all four source groups. T-RFLP profiles clustered microbial communities by source; individual T-RFs were matched to the majority (80.6%) of clone library sequences, indicating that T-RFLP analysis can be used to rapidly profile these communities. Phylogenetic metrics of community diversity indicated that the two sponge-associated bacterial communities include dominant and host-specific bacterial lineages that are distinct from bacteria recovered from seawater, tunicates, and unrelated sponge hosts. In addition, a large proportion of the symbionts associated with H. heliophila were shared with distant, conspecific host populations in the southwestern Atlantic (Brazil).

Conclusions/Significance

The low diversity and species-specific nature of bacterial communities associated with H. heliophila and H. tubifera represent a distinctly different pattern from other, reportedly universal, sponge-associated bacterial communities. Our replicated sampling strategy, which included samples that reflect the ambient environment, allowed us to differentiate resident symbionts from potentially transient or prey bacteria. Pairing replicated clone library construction with rapid community profiling via T-RFLP analyses will greatly facilitate future studies of sponge-microbe symbioses.     

Diversities of coral-associated bacteria differ with location, but not species, for three acroporid corals on the Great Barrier Reef

Patterns in the diversity of bacterial communities associated with three species of Acropora ( Acropora millepora, Acropora tenuis and Acropora valida ) were compared at two locations (Magnetic Island and Orpheus Island) on the Great Barrier Reef to better understand the nature and specificity of coral–microbial symbioses. Three culture-independent techniques demonstrated consistent bacterial communities among replicate samples of each coral species, confirming that corals associate with specific microbiota. Profiles were also conserved among all three species of Acropora within each location, suggesting that closely related corals of the same genus harbor similar bacterial types. Bacterial community profiles of A. millepora at Orpheus Island were consistent in samples collected throughout the year, indicating a stable community despite temporal changes. However, DGGE and T-RFLP profiles differed on corals from different reefs. Nonmetric multidimensional scaling of T-RFLP profiles showed that samples grouped according to location rather than coral species. Although similar sequences were retrieved from clone libraries of corals at both Magnetic and Orpheus Island, differences in the relative dominant bacterial ribotypes within the libraries drive bacterial community structure at different geographical locations. These results indicate certain bacterial groups associated specifically with corals, but the dominant bacterial genera differ between geographically-spaced corals.     

A study of the relationship among sludge retention time,bacterial communities,and hydrolytic enzyme activities in inclined plate membrane bioreactors for the treatment of municipal wastewater

Inclined plate membrane bioreactors (ip-MBRs) have been proposed as a highly effective method in wastewater treatment. With the help of settling enhancer inclined plates, dense excess sludge can be kept in the mainstream of the process, and consequently, suitable sludge mass can be maintained in the membrane tank. In this study, the relationship among sludge retention time (SRT), bacterial communities, and hydrolytic enzyme activities was investigated. Two identical bench-scale ip-MBRs were operated 1 year in real municipal wastewater treatment. Multidimensional scaling (MDS) plots of terminal restriction fragment length polymorphism (T-RFLP) fingerprints showed similar changes in the bacterial communities in terms of bacterial members and abundance over time in both the reactors, which was primarily caused by the changes of wastewater composition. However, the impact of SRT revealed significant differences in the dominant bacterial communities when both the reactors were operated with a largely different SRT (infinite SRT and SRT of 20 days). The sequences of bacterial 16S rRNA gene were classified into six libraries of A–F. The largest group of sequences belonged to the phylum Proteobacteria. The phylum Bacteroidetes was dominant in the seed sludge retrieved from the conventional activated sludge (CAS) as Flavobacterium-like bacterium was dominantly observed. Under the MBR operation (libraries B–F), bacterial communities belonging to the phyla Proteobacteria and Chloroflexi were dominant. Most of them may be responsible for protein degradation because aminopeptidase activity increased in proportion with the abundance of these bacteria.     

Community structure analyses are more sensitive to differences in soil bacterial communities than anonymous diversity indices

Changes in the diversity and structure of soil microbial communities may offer a key to understanding the impact of environmental factors on soil quality in agriculturally managed systems. Twenty-five years of biodynamic, bio-organic, or conventional management in the DOK long-term experiment in Switzerland significantly altered soil bacterial community structures, as assessed by terminal restriction fragment length polymorphism (T-RFLP) analysis. To evaluate these results, the relation between bacterial diversity and bacterial community structures and their discrimination potential were investigated by sequence and T-RFLP analyses of 1,904 bacterial 16S rRNA gene clones derived from the DOK soils. Standard anonymous diversity indices such as Shannon, Chao1, and ACE or rarefaction analysis did not allow detection of management-dependent influences on the soil bacterial community. Bacterial community structures determined by sequence and T-RFLP analyses of the three gene libraries substantiated changes previously observed by soil bacterial community level T-RFLP profiling. This supported the value of high-throughput monitoring tools such as T-RFLP analysis for assessment of differences in soil microbial communities. The gene library approach also allowed identification of potential management-specific indicator taxa, which were derived from nine different bacterial phyla. These results clearly demonstrate the advantages of community structure analyses over those based on anonymous diversity indices when analyzing complex soil microbial communities.     

土壤细菌DNA提取及多样性分析的T-RFLP方法

获得高质量的土壤总DNA是土壤细菌生态学的关键步骤之一.实验通过综合应用两个试剂盒(Soilmaster kit和DNA IQTM系统)的优点进行土壤样品总DNA的提取,结果证明该方法是一种快速、有效、灵敏、稳定的土壤DNA提取方法.另外尝试将16S rDNA序列和T-RFLP(Terminal restriction fragment 1ength polymorphism)技术引入土壤细菌DNA群落多样性的研究中,证明T-RFLP是一种有力的土壤细菌多样性分析工具.     

Community Structure Analyses Are More Sensitive to Differences in Soil Bacterial Communities than Anonymous Diversity Indices

Changes in the diversity and structure of soil microbial communities may offer a key to understanding the impact of environmental factors on soil quality in agriculturally managed systems. Twenty-five years of biodynamic, bio-organic, or conventional management in the DOK long-term experiment in Switzerland significantly altered soil bacterial community structures, as assessed by terminal restriction fragment length polymorphism (T-RFLP) analysis. To evaluate these results, the relation between bacterial diversity and bacterial community structures and their discrimination potential were investigated by sequence and T-RFLP analyses of 1,904 bacterial 16S rRNA gene clones derived from the DOK soils. Standard anonymous diversity indices such as Shannon, Chao1, and ACE or rarefaction analysis did not allow detection of management-dependent influences on the soil bacterial community. Bacterial community structures determined by sequence and T-RFLP analyses of the three gene libraries substantiated changes previously observed by soil bacterial community level T-RFLP profiling. This supported the value of high-throughput monitoring tools such as T-RFLP analysis for assessment of differences in soil microbial communities. The gene library approach also allowed identification of potential management-specific indicator taxa, which were derived from nine different bacterial phyla. These results clearly demonstrate the advantages of community structure analyses over those based on anonymous diversity indices when analyzing complex soil microbial communities.     

An evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure and dynamics

A systematic evaluation of the value and potential of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure has been undertaken. The reproducibility and robustness of the method has been assessed using environmental DNA samples isolated directly from PCB-polluted or pristine soil, and subsequent polymerase chain reaction (PCR) amplification of total community 16S rDNA. An initial investigation to assess the variability both within and between different polyacrylamide gel electrophoresis (PAGE) runs showed that almost identical community profiles were consistently produced from the same sample. Similarly, very little variability was observed as a result of variation between replicate restriction digestions, PCR amplifications or between replicate DNA isolations. Decreasing concentrations of template DNA produced a decline in both the complexity and the intensity of fragments present in the community profile, with no additional fragments detected in the higher dilutions that were not already present when more original template DNA was used. Reducing the number of cycles of PCR produced similar results. The greatest variation between profiles generated from the same DNA sample was produced using different Taq DNA polymerases, while lower levels of variability were found between PCR products that had been produced using different annealing temperatures. Incomplete digestion by the restriction enzyme may, as a result of the generation of partially digested fragments, lead to an overestimation of the overall diversity within a community. The results obtained indicate that, once standardized, T-RFLP analysis is a highly reproducible and robust technique that yields high-quality fingerprints consisting of fragments of precise sizes, which, in principle, could be phylogenetically assigned, once an appropriate database is constructed.     

16S rRNA gene analyses of bacterial community structures in the soils of evergreen broad-leaved forests in south-west China

Bacterial community structure was studied in humus and mineral soils of evergreen broad-leaved forests in Ailaoshan and Xishuangbanna, representing subtropical and tropical ecosystems, respectively, in south-west China using sequence analysis and terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes. Clone sequences affiliated to Acidobacteria were retrieved as the predominant bacterial phylum in both forest soils, followed by those affiliated to members of the Proteobacteria, Planctomycete and Verrucomicrobia. Despite higher floristic richness at the Xishuangbanna forest than at the Ailaoshan forest, soil at Xishuangbanna harbored a distinctly high relative abundance of Acidobacteria-affiliated sequences (80% of the total clones), which led to a lower overall bacterial diversity than at Ailaoshan. Bacterial communities in humus and mineral soils of the two forests appeared to be well differentiated, based on 16S rRNA gene phylogeny, and correlations were found between the bacterial T-RFLP community patterns and the organic carbon and nutrient contents of the soil samples. The data reveal that Acidobacteria dominate soil bacterial communities in the evergreen broad-leaved forests studied here and suggest that bacterial diversity may be influenced by soil carbon and nutrient levels, but is not related to floristic richness along the climatic gradient from subtropical to tropical forests in south-west China.     

Seasonal Fluctuations of Bacterial Community Diversity in Agricultural Soil and Experimental Validation by Laboratory Disturbance Experiments

Natural fluctuations in soil microbial communities are poorly documented because of the inherent difficulty to perform a simultaneous analysis of the relative abundances of multiple populations over a long time period. Yet, it is important to understand the magnitudes of community composition variability as a function of natural influences (e.g., temperature, plant growth, or rainfall) because this forms the reference or baseline against which external disturbances (e.g., anthropogenic emissions) can be judged. Second, definition of baseline fluctuations in complex microbial communities may help to understand at which point the systems become unbalanced and cannot return to their original composition. In this paper, we examined the seasonal fluctuations in the bacterial community of an agricultural soil used for regular plant crop production by using terminal restriction fragment length polymorphism profiling (T-RFLP) of the amplified 16S ribosomal ribonucleic acid (rRNA) gene diversity. Cluster and statistical analysis of T-RFLP data showed that soil bacterial communities fluctuated very little during the seasons (similarity indices between 0.835 and 0.997) with insignificant variations in 16S rRNA gene richness and diversity indices. Despite overall insignificant fluctuations, between 8 and 30% of all terminal restriction fragments changed their relative intensity in a significant manner among consecutive time samples. To determine the magnitude of community variations induced by external factors, soil samples were subjected to either inoculation with a pure bacterial culture, addition of the herbicide mecoprop, or addition of nutrients. All treatments resulted in statistically measurable changes of T-RFLP profiles of the communities. Addition of nutrients or bacteria plus mecoprop resulted in bacteria composition, which did not return to the original profile within 14 days. We propose that at less than 70% similarity in T-RFLP, the bacterial communities risk to drift apart to inherently different states.     

Terminal Restriction Fragment Length Polymorphism Data Analysis for Quantitative Comparison of Microbial Communities

Terminal restriction fragment length polymorphism (T-RFLP) is a culture-independent method of obtaining a genetic fingerprint of the composition of a microbial community. Comparisons of the utility of different methods of (i) including peaks, (ii) computing the difference (or distance) between profiles, and (iii) performing statistical analysis were made by using replicated profiles of eubacterial communities. These samples included soil collected from three regions of the United States, soil fractions derived from three agronomic field treatments, soil samples taken from within one meter of each other in an alfalfa field, and replicate laboratory bioreactors. Cluster analysis by Ward's method and by the unweighted-pair group method using arithmetic averages (UPGMA) were compared. Ward's method was more effective at differentiating major groups within sets of profiles; UPGMA had a slightly reduced error rate in clustering of replicate profiles and was more sensitive to outliers. Most replicate profiles were clustered together when relative peak height or Hellinger-transformed peak height was used, in contrast to raw peak height. Redundancy analysis was more effective than cluster analysis at detecting differences between similar samples. Redundancy analysis using Hellinger distance was more sensitive than that using Euclidean distance between relative peak height profiles. Analysis of Jaccard distance between profiles, which considers only the presence or absence of a terminal restriction fragment, was the most sensitive in redundancy analysis, and was equally sensitive in cluster analysis, if all profiles had cumulative peak heights greater than 10,000 fluorescence units. It is concluded that T-RFLP is a sensitive method of differentiating between microbial communities when the optimal statistical method is used for the situation at hand. It is recommended that hypothesis testing be performed by redundancy analysis of Hellinger-transformed data and that exploratory data analysis be performed by cluster analysis using Ward's method to find natural groups or by UPGMA to identify potential outliers. Analyses can also be based on Jaccard distance if all profiles have cumulative peak heights greater than 10,000 fluorescence units.     

Chilling and cultivar type affect the diversity of bacterial endophytes colonizing sweet pepper (Capsicum anuum L.)

A climate chamber experiment was conducted to assay the effect of low temperatures (chilling) on the diversity of bacteria colonizing the endospheres of two thermophilic sweet pepper (Capsicum anuum L.) cultivars, Milder Spiral and Ziegenhorn Bello. Structural diversity was analyzed by 16S rRNA-based terminal restriction fragment length polymorphism (T-RFLP) analysis and by the generation of 16S rRNA gene libraries to determine dominant community members in T-RFLP profiles. Cultivable community members colonizing lines Milder Spiral and Ziegenhorn Bello were identified by 16S rRNA gene analysis. T-RFLP profiles and 16S rRNA gene libraries revealed a high heterogeneity of community composition due to chilling and suggested further the existence of cultivar-specific communities. The majority of isolates obtained from the cultivar Milder Spiral were assigned as high-G+C Gram-positive bacteria (Microbacterium sp., Micrococcus sp., Rhodococcus sp.) and Firmicutes (Staphylococcus sp.). Of the isolated endophytes obtained from cultivar Zeigenhorn Bello, 93% were affiliated with Staphylococcus aureus and Bacillus sp. (Firmicutes). The experimental set-up was suited to demonstrate that chilling and cultivar type can influence the diversity of bacterial endophytes colonizing sweet pepper. We propose additional chilling experiments to investigate the effect of chilling on functional, plant-beneficial abilities of bacterial endophytes associated with low-temperature-sensitive crops, such as sweet pepper.     

Comparison of Bacterial Communities in New England Sphagnum Bogs Using Terminal Restriction Fragment Length Polymorphism (T-RFLP)

Wetlands are major sources of carbon dioxide, methane, and other greenhouse gases released during microbial degradation. Despite the fact that decomposition is mainly driven by bacteria and fungi, little is known about the taxonomic diversity of bacterial communities in wetlands, particularly Sphagnum bogs. To explore bacterial community composition, 24 bogs in Vermont and Massachusetts were censused for bacterial diversity at the surface (oxic) and 1 m (anoxic) regions. Bacterial diversity was characterized by a terminal restriction fragment length (T-RFLP) fingerprinting technique and a cloning strategy that targeted the 16S rRNA gene. T-RFLP analysis revealed a high level of diversity, and a canonical correspondence analysis demonstrated marked similarity among bogs, but consistent differences between surface and subsurface assemblages. 16S rDNA sequences derived from one of the sites showed high numbers of clones belonging to the Deltaproteobacteria group. Several other phyla were represented, as well as two Candidate Division-level taxonomic groups. These data suggest that bog microbial communities are complex, possibly stratified, and similar among multiple sites.     

Terminal restriction fragment length polymorphism data analysis for quantitative comparison of microbial communities

Terminal restriction fragment length polymorphism (T-RFLP) is a culture-independent method of obtaining a genetic fingerprint of the composition of a microbial community. Comparisons of the utility of different methods of (i) including peaks, (ii) computing the difference (or distance) between profiles, and (iii) performing statistical analysis were made by using replicated profiles of eubacterial communities. These samples included soil collected from three regions of the United States, soil fractions derived from three agronomic field treatments, soil samples taken from within one meter of each other in an alfalfa field, and replicate laboratory bioreactors. Cluster analysis by Ward's method and by the unweighted-pair group method using arithmetic averages (UPGMA) were compared. Ward's method was more effective at differentiating major groups within sets of profiles; UPGMA had a slightly reduced error rate in clustering of replicate profiles and was more sensitive to outliers. Most replicate profiles were clustered together when relative peak height or Hellinger-transformed peak height was used, in contrast to raw peak height. Redundancy analysis was more effective than cluster analysis at detecting differences between similar samples. Redundancy analysis using Hellinger distance was more sensitive than that using Euclidean distance between relative peak height profiles. Analysis of Jaccard distance between profiles, which considers only the presence or absence of a terminal restriction fragment, was the most sensitive in redundancy analysis, and was equally sensitive in cluster analysis, if all profiles had cumulative peak heights greater than 10,000 fluorescence units. It is concluded that T-RFLP is a sensitive method of differentiating between microbial communities when the optimal statistical method is used for the situation at hand. It is recommended that hypothesis testing be performed by redundancy analysis of Hellinger-transformed data and that exploratory data analysis be performed by cluster analysis using Ward's method to find natural groups or by UPGMA to identify potential outliers. Analyses can also be based on Jaccard distance if all profiles have cumulative peak heights greater than 10,000 fluorescence units.     

生物肥与甲壳素和恶霉灵配施对香蕉枯萎病的防治效果

通过盆栽试验研究了生物肥与甲壳素和恶霉灵配施防治香蕉枯萎病效果,试验结果表明,生物肥与恶霉灵配施(H+F)处理香蕉枯萎病病情指数最高,生物肥与甲壳素配施(C+F)处理病情指数最低。单独生物肥处理防病效果为32.8%,生物肥与甲壳素配施处理为42.5%,而生物肥与恶霉灵配施加重了香蕉枯萎病病情。Biolog Eco微平板研究发现,AWCD(平均每孔颜色变化率)和Shannon等4个多样性指数变化趋势与防病效果相反:防病效果好的处理,土壤细菌功能多样性指数反而低,经检测发现病原真菌(Fusarium oxysporum f. sp. cubense)可利用Biolog Eco微平板上碳源底物并发生颜色变化,干扰测定结果。T-RFLP分析土壤细菌DNA多样性,对照(灭菌生物肥)土壤中TRFs末端限制性片段最少,生物肥与甲壳素配施处理最多。与网上数据库比较,生物肥与甲壳素配施增加了土壤中芽胞杆菌种类,与恶霉灵配施降低了芽胞杆菌种类。分析发现,T-RFLP和Biolog的主成份分析载荷图具有较高一致性。因此,生物肥与生物农药甲壳素配施,从生态角度控制土传病害,优势互补,提高了土壤细菌多样性,改善了土壤细菌群落结构,有利于提高防病效果。