Persistence of DNA of Gaeumannomyces graminis var. tritici in soil as measured by a DNA-based assay (original) (raw)
Related papers
Journal of Phytopathology, 2005
Linear mycelial growth rates of 70 isolates of Gaeumannomyces graminis var. tritici on agar medium amended or unamended with the fungicide silthiofam were not correlated. Mycelial growth rate was not influenced by the fungicide applied to the seed of the plants from, which the isolates originated. DNA polymorphism determined by randomly amplified polymorphic DNA (RAPD) polymerase chain reaction was used to assess genetic variation among isolates. Thirty RAPD markers generated with five arbitrary 10-mer primers revealed DNA polymorphism suitable for assessing variability in this fungal population. Cluster analysis of RAPD data identified two groups at the 54% similarity level. There was a significant relationship between the presence of 11 markers and sensitivity to silthiofam.
Mycological Research, 1996
A slot-blot hybridization assay for quantifying Gaeumannomyces graminis var. trifici, in diseased roots and in soil, using a specific and sensitive DNA probe (pGI58), has been developed. The pGI58 probe hybridized strongly to pathogenic isolates of G. graminis var. trifici, moderately to G. graminis var. avenae and did not hybridize to non-pathogenic G. graminis var. graminis and to a wide range of other soil fungi. The pG158 contains a dispersed highly repeated sequence which provides the high level of sensitivity needed for a quantitative diagnostic test. The amount of Gaeumannomyces graminis var. trifici in soil can be estimated directly from the test soil without growing the susceptible plants or culturing the fungus. pGI58 detects approximately 1'0 ng and 0'3 ng of G. graminis var. tri!ici DNA in I IJg total DNA extracted from diseased roots and from highly infested soil organic maHer, respectively. Both the high copy clone, pGI58 and a single copy clone, pG217, clearly distinguished the three varieties of G. graminis and can be used for accurate intra-specific classifications of G. graminis isolates. The pGI58 probe provides a tool for field researchers to determine levels of pathogenic isolates of G. graminis in soils and to study the ecology of this fungus.
Improved Method for Dna Isolation from Different Types of Soil Infested with Three Fungal Genera
In the current study, we describe a DNA isolation method that is based on an easy, quick polyvinylpolypyrrolidone-precipitation to release phytofungi from the soil, combined with lysozyme-RNase and -SDS lysis of the fungal population. DNA extracts were subjected to different techniques, including gel electrophoresis, restriction enzyme digestion, RAPD and ITS-PCR amplification. The proposed method yielded high-quality DNA, which was transparent, non-viscous and lacked visible contamination of RNA. Isolated DNA was efficiently digested with restriction enzymes. DNA extracted from soil was pure enough to be utilized at high concentrations for PCR amplifications. The extracted DNA was of high quality and allowed direct detection of specific genes by the polymerase chain reaction (PCR). The amplicon length of the fragment ITS4/ITS5, ranged in size from 550 to 680 bp. A polymerase chain reaction method used to detect soil-borne plant pathogens such as Fusarium spp., Rhizoctonia solani and Macrophomina phaseolina in the soil was developed and used with a range of soil textures. A direct method for the extraction of DNA from soil samples, which can be used for PCR-mediated diagnostics without a need for further DNA purification, was developed. The developed protocol seemed adequate to the range of soil textures that were artificially infested by a variety of soil-borne pathogens.
Direct quantification of fungal DNA from soil substrate using real-time PCR
Journal of Microbiological Methods, 2003
Detection and quantification of genomic DNA from two ecologically different fungi, the plant pathogen Fusarium solani f. sp. phaseoli and the arbuscular mycorrhizal fungus Glomus intraradices, was achieved from soil substrate. Specific primers targeting a 362-bp fragment from the SSU rRNA gene region of G. intraradices and a 562-bp fragment from the F. solani f. sp. phaseoli translation elongation factor 1 alpha gene were used in real-time polymerase chain reaction (PCR) assays conjugated with the fluorescent SYBRR Green I dye. Standard curves showed a linear relation (r 2 = 0.999) between log values of fungal genomic DNA of each species and real-time PCR threshold cycles and were quantitative over 4 -5 orders of magnitude. Realtime PCR assays were applied to in vitro-produced fungal structures and sterile and non-sterile soil substrate seeded with known propagule numbers of either fungi. Detection and genomic DNA quantification was obtained from the different treatments, while no amplicon was detected from non-seeded non-sterile soil samples, confirming the absence of cross-reactivity with the soil microflora DNA. A significant correlation ( P < 0.0001) was obtained between the amount of genomic DNA of F. solani f. sp. phaseoli or G. intraradices detected and the number of fungal propagules present in seeded soil substrate. The DNA extraction protocol and real-time PCR quantification assay can be performed in less than 2 h and is adaptable to detect and quantify genomic DNA from other soilborne fungi. D
Extraction of DNA from plant and fungus tissues in situ
BMC research notes, 2012
When samples are collected in the field and transported to the lab, degradation of the nucleic acids contained in the samples is frequently observed. Immediate extraction and precipitation of the nucleic acids reduces degradation to a minimum, thus preserving accurate sequence information. An extraction method to obtain high quality DNA in field studies is described. DNA extracted immediately after sampling was compared to DNA extracted after allowing the sampled tissues to air dry at 21°C for 48 or 72 hours. While DNA extracted from fresh tissues exhibited little degradation, DNA extracted from all tissues exposed to 21°C air for 48 or 72 hours exhibited varying degrees of degradation. Yield was higher for extractions from fresh tissues in most cases. Four microcentrifuges were compared for DNA yield: one standard electric laboratory microcentrifuge (max rcf = 16,000 × g), two battery-operated microcentrifuges (max rcf = 5,000 and 3,000 × g), and one manually-operated microcentrifu...
Rapid DNA Extraction for Screening Soil Filamentous Fungi Using PCR Amplification
Polish Journal of Environmental Studies
A simple and rapid procedure for efficiently isolating fungi DNA suitable for use as a template for PCR amplification and other molecular assays is described. The main advantages of the method are: (1) the mycelium is directly recovered from Petri-dish cultures; (2) the technique is rapid and relatively easy to perform , and (3) it allows for processing of around 50 samples during a single day; (4) it is inexpensive; (5) the quality and quantity of DNA obtained are suitable for molecular assays; (6) it can be applied to filamentous fungi from soil as well as from a fungi from other environmental sources; and (7) it does not require the use of expensive and specialized equipment or hazardous reagents.
DNA polymorphism and host range in the take-all fungus, Gaeumannomyces graminis
Mycological Research, 1999
Thirty-five isolates of Gaeumannomyces graminis were tested for ability to infect wheat, rye and oats, and for DNA polymorphisms using nuclear ribosomal DNA (rDNA) RFLP patterns and RAPD analysis. In general, the cereal-attacking isolates could be readily assigned to the rye-attacking (R) or non-rye-attacking (N) subgroups of var. tritici, or to var. avenae, on the basis of either of these molecular approaches. A small number of isolates gave anomalous rDNA RFLP patterns, but could nevertheless be assigned to one of the three groups by RAPD analysis. Two related G. graminis var. tritici isolates (T1-1 and T1-2) clearly grouped with the N rather than the R var. tritici subgroup on the basis of molecular analysis but were pathogenic to rye, indicating that the ability to infect this host may have arisen more than once. An earlier phylogenetic study of Gaeumannomyces involving DNA sequence analysis of the internal transcribed spacers of the rDNA indicated that several oat-infecting isolates originally classified as G. graminis var. tritici could be grouped with var. avenae isolates. From the rDNA RFLP and RAPD analysis described here, however, these isolates appear to be intermediate between var. avenae and var. tritici, although it is not clear whether they represent evidence of inter-varietal sexual hybridization.
Development of rapid DNA extraction and PCR amplification methods for fungi and parasitic plants
Zemdirbyste-Agriculture
The main culprits of catastrophic agricultural losses or crop mortality are phytopathogenic fungi and parasitic plants. To manage the plant pathogens, a simple and rapid disease diagnosis is needed. The aim of the experiment was to develop simple and rapid genomic DNA extraction and PCR amplification methods for fungi and parasitic plants. For the DNA extraction, mycelia from 16 fungi species and stems from two parasitic plants species were incubated in a lysis buffer and were homogenised using a sterilised wooden stick. The homogenates were incubated at 95°C temperature for 10 min. Crude extracts served as a template for the PCR amplification containing UKOD polymerase. The application of lysis buffer, mechanical and heat disruption resulted in a fast DNA extraction from fungi and parasitic plants. DNA amplification time is reduced when using Master Mix containing UKOD polymerase. The presented results confirm that these simple and rapid DNA extraction and PCR amplification methods are applicable to diverse fungi species and parasitic plants.
A rapid method for isolation of total DNA from pathogenic filamentous plant fungi
Genetics and Molecular Research, 2010
DNA isolation from some fungal organisms of agronomic importance is difficult because they have cell walls or capsules that are relatively unsusceptible to lysis. We have developed a fast DNA isolation protocol for Fusarium oxysporum, which causes fusarium wilt disease in more than 100 plant species, and for Pyrenochaeta terrestris, which causes pink root in onions. This protocol was based on the sodium dodecyl sulfate/ phe nol method, without β-mercaptoethanol and without maceration in liquid nitrogen; it uses phenol/chloroform extraction to remove proteins and co-precipitated polysaccharides. The A 260/280 absorbance ratios of isolated DNA were around 1.9, suggesting that the DNA fraction was pure and may be used for further analysis. Additionally, the A 260/230 values were higher than 1.8, suggesting negligible contamination by polysaccharides. The DNA isolated by this protocol is of sufficient quality for molecular applications; this tech nique could be applied to other organisms that have similar substances that hinder DNA extraction.