Effects of ammonium sulphate on the community structure and biomass of ectomycorrhizal fungi in a Norway spruce stand in southwestern Sweden

1997 ◽  
Vol 75 (10) ◽  
pp. 1628-1642 ◽  
Author(s):  
Ola Kårén ◽  
Jan-Erik Nylund
Keyword(s):  
Polymerase Chain Reaction ◽  
Community Structure ◽  
Norway Spruce ◽  
Ammonium Sulphate ◽  
Ectomycorrhizal Fungi ◽  
Organic Layer ◽  
Chain Reaction ◽  
Sulphur Deposition ◽  
Restriction Profiles ◽  
Polymerase Chain

Effects of nitrogen and sulphur deposition on the community structure and biomass of ectomycorrhizal fungi in a Norway spruce (Picea abies (L.) Karst.) forest were examined following fertilization with ammonium sulphate. The treated plots had been fertilized three times a year from 1988 with (NH4)2SO3 (100 kg N and 114 kg S ∙ ha−1 ∙ year−1). Sampling was carried out in the organic layer of the fertilized and control plots in late autumn in 1992 and 1993. The fungal biomass was estimated in 1992 using ergosterol analysis. The nitrogen treatment reduced the fine-root biomass (to 49% of the control) but did not decrease the mycorrhizal frequency (close to 100%) or concentration of ergosterol in fine roots. To identify mycorrhizas fungal ribosomal DNA was amplified using the polymerase chain reaction and digested with endonucleases. Of the 58 samples analysed, 21 different restriction profiles could be distinguished. Only four of the restriction profiles matched the restriction patterns of the dominant sporocarps on the site. It is suggested that nitrogen deposition will primarily change the community structure of ectomycorrhizal fungi, whereas the number of species may be less affected than has been previously inferred from sporocarp inventories. Key words: air pollution, biomass, diversity, ectomycorrhiza, identification, polymerase chain reaction.

scholarly journals Deconstructing the Polymerase Chain Reaction II: an improved workflow and effects on artifact formation and primer degeneracy

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7121
Author(s):  
Ankur Naqib ◽  
Silvana Poggi ◽  
Stefan J. Green
Keyword(s):  
Polymerase Chain Reaction ◽  
Community Structure ◽  
Microbial Community ◽  
Annealing Temperature ◽  
Degenerate Primer ◽  
Chain Reaction ◽  
Degenerate Primers ◽  
Polymerase Chain ◽  
Dna Template ◽  
Microbial Dna

Polymerase chain reaction (PCR) amplification of complex microbial genomic DNA templates with degenerate primers can lead to distortion of the underlying community structure due to inefficient primer-template interactions leading to bias. We previously described a method of deconstructed PCR (“PEX PCR”) to separate linear copying and exponential amplification stages of PCR to reduce PCR bias. In this manuscript, we describe an improved deconstructed PCR (“DePCR”) protocol separating linear and exponential stages of PCR and allowing higher throughput of sample processing. We demonstrate that the new protocol shares the same benefits of the original and show that the protocol dramatically and significantly decreases the formation of chimeric sequences during PCR. By employing PCR with annealing temperature gradients, we further show that there is a strong negative correlation between annealing temperature and the evenness of primer utilization in a complex pool of degenerate primers. Shifting primer utilization patterns mirrored shifts in observed microbial community structure in a complex microbial DNA template. We further employed the DePCR method to amplify the same microbial DNA template independently with each primer variant from a degenerate primer pool. The non-degenerate primers generated a broad range of observed microbial communities, but some were highly similar to communities observed with degenerate primer pools. The same experiment conducted with standard PCR led to consistently divergent observed microbial community structure. The DePCR method is simple to perform, is limited to PCR mixes and cleanup steps, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible.

scholarly journals Etiology and Real-Time Polymerase Chain Reaction-Based Detection of Gremmeniella- and Phomopsis-Associated Disease in Norway Spruce Seedlings

2006 ◽  
Vol 96 (12) ◽  
pp. 1305-1314 ◽  
Author(s):  
Isabella Børja ◽  
Halvor Solheim ◽  
Ari M. Hietala ◽  
Carl Gunnar Fossdal
Keyword(s):  
Polymerase Chain Reaction ◽  
Sequence Analysis ◽  
Real Time ◽  
Norway Spruce ◽  
Internal Transcribed Spacer ◽  
Chain Reaction ◽  
Large Tree ◽  
Nursery Stock ◽  
European Race ◽  
Polymerase Chain

In spring 2002, an unusual disease outburst was recorded on Norway spruce seedlings in southeast Norway. Extensive damage was recorded on 1- and 2-year-old Norway spruce seedlings that either had wintered in nursery cold storage or had been planted out in autumn 2001. The damage was characterized by leader shoot dieback and stem necroses on the upper or lower part of the shoot from 2001. Gremmeniella abietina and a Phomopsis sp. frequently were isolated from the diseased seedlings. Internal transcribed spacer (ITS) ribosomal (r)DNA sequence analysis and random amplified microsatellites profiling indicated that the G. abietina strains associated with diseased nursery seedlings belonged to the large-tree type (LTT) ecotype of the European race of G. abietina var. abietina, and inoculation tests confirmed their pathogenicity on Norway spruce. Based on ITS rDNA sequence analysis, the Phomopsis strains associated with diseased seedlings did not represent any characterized Phomopsis spp. associated with conifers. The Phomopsis sp. was not pathogenic in inoculation tests, indicating that it may be a secondary colonizer. ITS-based real-time polymerase chain reaction assays were developed in order to detect and quantify G. abietina and Phomopsis in the nursery stock. We describe here the G. abietina-associated shoot dieback symptoms on Norway spruce seedlings and conclude that the unusual disease outburst likely was related to the G. abietina var. abietina epidemic caused by the LTT on large Scots pines in 2001.

scholarly journals Use of Restriction Fragment Length Polymorphism, Immunoblotting, and Polymerase Chain Reaction in the Differentiation of Avian Poxviruses

2003 ◽  
Vol 15 (2) ◽  
pp. 141-150 ◽  
Author(s):  
Theodros Tadese ◽  
Willie M. Reed
Keyword(s):  
Polymerase Chain Reaction ◽  
Profile Analysis ◽  
Protein Profile ◽  
Species Differentiation ◽  
Immunogenic Protein ◽  
Chain Reaction ◽  
Restriction Patterns ◽  
Field Isolates ◽  
Restriction Profiles ◽  
Polymerase Chain

Restriction deoxyribonucleic acid (DNA) fragment profile analysis coupled with immunogenic protein profile analysis has provided useful information in determining the differences between vaccine strains and field isolates of fowlpox virus (FPV). The DNA of strains examined in this study clearly fell into 3 minor groups of restriction patterns similar but distinct from one another: restriction patterns exhibited by the vaccine strains except 1 vaccine strain, Vac-82; restriction profiles indicated by Vac-82 and field isolates FI-38 and FI-42; and restriction patterns indicated by field isolates FI-43, FI-51, FI-54, and FI-56. Furthermore, when the strains were analyzed and compared by immunoblotting analysis, they showed group differences similar to the differences in restriction profiles. Both techniques provided high sensitivity in verifying differences between vaccine strains and field isolates of FPV. The disparity found in restriction fragments or immunogenic protein profile between vaccine strains and field isolates does not exclude the appreciable high degree of DNA sequence conservation and homology. However, the minor disparity observed in these strains suggests a molecular basis for why vaccinated commercial flocks could have continually been infected by variant strains of FPV. A rapid and sensitive polymerase chain reaction method, which amplified a product from the 4b core protein gene of the FPV genome, was developed for identification and differentiation of members of the genus Avipoxvirus. Whereas total DNA from either vaccine strains or field isolates was used as template for amplifying a predicted product of 578 or 1409 bp, only cleavage of the amplified product (1409 bp) represented an additional detection technique for species differentiation. An attempt to distinguish between strains on the basis of amplification product was partially successful.

scholarly journals Deconstructing the Polymerase Chain Reaction II: An improved workflow and effects on artifact formation and primer degeneracy

2019 ◽  
Author(s):  
Ankur Naqib ◽  
Silvana Poggi ◽  
Stefan J Green
Keyword(s):  
Polymerase Chain Reaction ◽  
Community Structure ◽  
Microbial Community ◽  
Annealing Temperature ◽  
Degenerate Primer ◽  
Chain Reaction ◽  
Degenerate Primers ◽  
Polymerase Chain ◽  
Dna Template ◽  
Microbial Dna

Polymerase chain reaction (PCR) amplification of complex microbial genomic DNA templates with degenerate primers can lead to distortion of the underlying community structure due to inefficient primer-template interactions leading to bias. We previously described a method of deconstructed PCR (“PEX PCR”) to separate linear copying and exponential amplification stages of PCR to reduce PCR bias (Green et al. 2015). In this manuscript, we describe an improved deconstructed PCR (“DePCR”) protocol separating linear and exponential stages of PCR and allowing higher throughput of sample processing. We demonstrate that the new protocol shares the same benefits of the original and show that the protocol dramatically and significantly decreases the formation of chimeric sequences during PCR. By employing PCR with annealing temperature gradients, we further show that there is a strong negative correlation between annealing temperature and the evenness of primer utilization in a complex pool of degenerate primers. Shifting primer utilization patterns mirrored shifts in observed microbial community structure in a complex microbial DNA template. We further employed the DePCR method to amplify the same microbial DNA template independently with each primer variant from a degenerate primer pool. The non-degenerate primers generated a broad range of observed microbial communities, but some were highly similar to communities observed with degenerate primer pools. The same experiment conducted with standard PCR led to consistently divergent observed microbial community structure. The DePCR method is simple to perform, is limited to PCR mixes and cleanup steps, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible.

scholarly journals Simera: Modelling the PCR Process to Simulate Realistic Chimera Formation

2016 ◽  
Author(s):  
Ben Nichols ◽  
Christopher Quince
Keyword(s):  
Experimental Data ◽  
Polymerase Chain Reaction ◽  
Community Structure ◽  
Chain Reaction ◽  
Future Studies ◽  
Target Dna ◽  
Detection Software ◽  
Polymerase Chain ◽  
Diversity Studies ◽  
Principal Method

AbstractPolymerase Chain Reaction (PCR) is the principal method of amplifying target DNA regions and, as such, is of great importance when performing microbial diversity studies. An unfortunate side effect of PCR is the formation of unwanted byproducts such as chimeras. The main goal of the work covered in this article is the development of an algorithm that simulates realistic chimeras for use in the evaluation of chimera detection software and for investigations into the accuracy of community structure analyses. Experimental data has helped to identify factors which may cause the formation of chimeras and has provided evidence of how influential these factors can be. This article makes use of some of this evidence in order to build a model with which to simulate the PCR process. This model helps to better explain the formation of chimeras and is therefore able to provide aid to future studies that intend to use PCR.

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