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Methods of Studying Soil Micro-organisms
Methods to study soil micro-organisms can be classified in two major categories. These categories are biochemical and molecular techniques (Kirk et al., 2004). The first group includes culturing methods, sole carbon source utilisation patterns and fatty acid analysis.
The culturing method will be dealt below. The second group is based on the nucleic acids of micro-organisms. Several techniques of this group exist. One of these techniques, the denaturing gradient gel electrophoresis (DGGE), is described here after the culturing method.
Culturing Method
Methods of culturing micro-organisms are based on their artificial growth on various nutrient rich selective or non selective sources. Different techniques are employed to isolate, purify, preserve and study the organisms. Culturing methods include dilution plating for bacteria and fungi, soil plating for mainly fungi, immersion methods for in situ active hyphal growth and direct hyphal plating (Parkinson and Coleman, 1991). These cultural techniques are generally inexpensive and provide information on viable culturally or active populations (Kirk et al., 2004). Population richness of the cultivated organisms is also determined by total microbial counts. Cultivation can also be targeted to specific groups of micro-organisms by using selective media.
Selective Media
Selective media are artificial media that allow only specific types of micro-organisms to grow. These media contain favourable substances and factors to select specific organisms and inhibiting substances and factors to prevent growth of undesirable organisms. In the preparation of these selective media different carbon and nitrogen sources are added. Examples of these energy sources to the micro-organisms are dextrose, peptone, yeast extract, casamino acids, glucose, sucrose and asparagine (Cuppels and Kelman, 1974; Nesmith and Jenkins, 1978; Gould et al., 1985; Hagedorn et al., 1987). Antimicrobial substances added include penicillin, tyrothricin, chloromycetin, 2, 3, 5 triphenyl tetrazolium chloride, polymyxin, vancomycin, bacitracin, benomyl, chloroneb, cycloheximide, pentachloronitrobenzene, pimaricin, dichloran, chloramphenicol, chlorothalonil, anisomycin, crystal violet, trimethoprim, nystatin, orthophenylphenol, streptomycin, fradiomycin and kanamycin (Cuppels and Kelman, 1974; Nesmith and Jenkins, 1978; Chen and Echandi, 1981; Edelstein, 1982; Gould et al., 1985; Hagedorn et al., 1987; Dietrich and Lamar, 1990; Otoguro et al., 2001). Growing conditions such as temperature, pH and incubation periods are also manipulated to increase the selectivity of media (Hagedorn et al., 1987; Dietrich and Lamar, 1990; Stevenson et al., 2004).
However, culturing methods are limited to laboratory growth conditions and by the selective nature of artificial nutrients used for culturable, fast growing and dominant organisms (Ranjard et al., 2000; Kirk et al., 2004; Bing-Ru et al., 2006). Therefore, for more reliable results, culturing methods should be used in combination with other techniques.
Molecular Techniques
Molecular techniques are based on nucleic acid sequence or genetic makeup of microorganisms. Genetic material from these micro-organisms can be extracted directly from the sample materials of different origins, i.e. aquatic, food, clinical and soil samples (Picard et al., 1992). Several techniques have been developed to detect and characterise micro-organisms using extracted genetic material (DNA). Probes that detect a unique sequence of a target organism can be designed and have been used successfully (Pickup, 1991). Restriction fragment length polymorphism (RFLP) involves enzymes that digest restricted fragments of DNA followed by the attachment of probes (Karp et al., 1996).
However, one of the limitations for RFLP is that a large amount of quality DNA is required. To overcome this limitation in the case of low amounts of DNA, methods that amplify DNA were developed. Such methods are the randomly amplified polymorphic DNA (RAPD), arbitrary primed PCR (AP-PCR), DNA amplification fingerprinting (DAF) and amplified fragment length polymorphism (AFLP) (Karp et al., 1996). Results from RFLP, AFLP, RAPD, AP-PCR and DAF are used in detection and diversity studies (Karp et al., 1996; Powell et al., 1996; Suzuki et al., 1997).
One of the most commonly used nucleic acid techniques in analysis of microbial diversity is the determination of sequences of 16S ribosomal RNA (rRNA) genes encoded by rDNA (Hill et al., 2000). The 16S molecule is suitable for such studies because it is universal, conserved, easily amplified and rapidly sequenced (Hill et al., 2000). Methods that use16S-rRNA gene sequence analysis for microbial diversity studies include cloning and sequencing, denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), dot blots, single strand conformational polymorphism (SSCP), and terminal restriction fragment length polymorphism (T-RFLP) (Rondon et al., 1999). The DGGE method is described below.
Chapter 1 General Introduction
References
Chapter 2 Literature Review
1. Introduction
2. Description of White Button Mushrooms [Agaricus bisporus (Lange) Imbach]
3. White Button Mushroom Production
4. Cultivation of White Button Mushrooms
5. Microbiological Properties of Casing Soil
6. The Common Casing Material – Peat
7. Research on Alternative Materials to Peat
8. Pathogens of White Button Mushrooms
9. Biological Control (Biocontrol) Potential of Bacteria in the Casing
10. Methods of Studying Soil Micro-organisms
12. References
Chapter 3 Microbial Dynamics of Different Casing Materials in the Production of White Button Mushrooms [Agaricus bisporus (Lange) Imbach]
-Abstract
-Introduction
-Materials and Methods
-Mushroom Growing Unit
-Growing Mushrooms
-Casing Materials
-Sampling
-Plating and Enumeration of Bacteria
-Isolation of Bacteria and DNA Extraction
-PCR Amplification and Sequencing
-Phenol Extraction and Quantification
-Experimental Design and Statistical Analysis
-Results
-Identification of Bacteria
-Enumeration of Bacteria in Unmixed Materials
-Quantification of Total Soluble Phenolic Compounds
-Enumeration of Bacteria in Casing Media (Peat and Mixtures)
-Discussion
-References
Chapter 4 Bacterial Profiling of Casing Materials for the White Button Mushroom [Agaricus bisporus (Lange) Imbach] Using Denaturing Gradient Gel Electrophoresis
-Abstract
-Introduction
-Materials and Methods
-Casing Materials
-DNA Extraction
-PCR Amplification
-DGGE
-Band Excision, Purification and Sequencing
-Gel Analysis and Phylogenetic Tree
-Results
-Bacterial Populations
-Effect of Pasteurisation
-Population Similarities
-Sequencing and Phylogeny
-Discussion
-References
Chapter 5 Effect of Casing Bacteria on Growth Stimulation and Mushroom Yield and Disease Suppression by Bacteria and Yeasts from Casing, Compost and Mushrooms
-Abstract
-Introduction
-Materials and Methods
-In Vitro Growth Stimulation
-Pot trial: Bacteria and Yield
-Compost and Casing Preparation
-Inoculum Preparation and Application
-Semi-Commercial Growth Stimulation, Pinning and Yield Trial
-Compost and Casing
-Inoculum Preparation and Inoculation
-In Vitro Biocontrol Analysis
-Results
-Growth Stimulation
-Casing Bacteria and Mushroom Yield
-Semi-Commercial Trial of Growth Stimulation, Pinning and Yield
-Discussion
-References
Chapter 6 Abundance, Diversity and Phylogenetic Profiles of Bacteria, Fungi and Yeasts in Compost, Casing and on Mushrooms
-Abstract
-Introduction
-Materials and Methods
-Compost, Casing and Mushroom Samples
-Microbial Enumeration
-DNA Extraction
-PCR Amplification
-DGGE, Excision of Bands and Reamplification of DNA
-Gel Analysis and Phylogenetic Tree
-Statistical Analysis
-Results
-Total Counts of Bacteria, Fungi and Yeasts
-Pure Culture Identification Using Sequencing
-Microbial Profile of Compost, Casing and Mushroom Samples on DGGE
-Population Similarities Between Compost, Casing and Mushroom Ecology
-Discussion
-References
Chapter 7 General Discussion
References
