Methods to Study Soil Biota ( microbiota, meso-macrofauna )

1. Methods to Study Soil Biota(microbiota, meso-macrofauna)

2. Physical Chemical Biological Soil Health

3. Decompose organic matter Cycle nutrients Release nutrients N2 fixation and mycorrhizae Suppress disease Improve soil structure Bioremediate contaminants Consume greenhouse gases Immobilize nutrients Cause disease Produce greenhouse gases Benefits and detriments of microbes in the rhizosphere Ecosystem Services of the Soil Biota

4. Microbiota

5. In a gram of soil….there are…. Billions of bacteria Millions of fungi Thousands of nematodes and protozoa In a gram of soil….there may be…. As many as 10,000 kinds of bacteria 5,000 kinds of fungi hundreds of kinds of nematodes and protozoa

6. Earthworms

7. METHODS FOR STUDYING SOIL MICROBES

8. Objective of Studying Soil Microbes • Isolating microbes • Analyzing microbial biomass and activity

9. ISOLATION OF SOIL MICROBES

10. Medium and Culture Lab. medium Natural habitat • Medium: Nutrients used to grow organisms outside of their natural habitats • Culture: The propagation of microorganisms using various media

12. Components of Media • Macronutrients (C, N, P, K) • Micronutrients (Fe, Mg, Ca, Na) • Vitamin

13. Types of Microbiological Media • Chemically defined media • The exact chemical composition is known • Complex (undefined) media • The exact chemical composition is not known • Often consist of plant or animal extracts, such as soybean meal, milk protein, etc.

14. Types of Microbiological Media • Solid Medium • A medium made from solid material • Liquid Medium • Components are dissolved in water and sterilized • Semisolid Medium • A medium to which has been added a gelling agent • Agar (most commonly used) • Gelatin • Silica gel (used when a non-organic gelling agent is required)

15. Purposes of Media • GENERAL • Nutrient • SELECTIVE & DIFFERENTIAL • N-free medium • ENRICHMENT • Medium plus specific compound • ASSAY • Starch agar, nitrate broth

16. Media Types • General purpose • Enriched • Selective • Differential

17. Selective & Differential Media • Selective media • Contain agents that inhibit the growth of certain bacteria while permitting the growth of others • Frequently used to isolate specific organisms from a large population of contaminants • Differential media • Contain indicators that react differently with different organisms (for example, producing colonies with different colors) • Used in identifying specific organisms

18. Enrichment Media • Encourages growth of desired microbe • Assume a soil sample contains a few phenol-degrading bacteria and thousands of other bacteria • Inoculate phenol-containing culture medium with the soil and incubate • Transfer 1 ml to another flask of the phenol medium and incubate • Transfer 1 ml to another flask of the phenol medium and incubate • Only phenol-metabolizing bacteria will be growing

19. ENRICHMENT OF Azotobacter

20. Inoculating An Agar Plate to Obtain Single Colonies

21. Mixed culture Pure culture

22. Streak Plate

23. Identifying Bacteria Cultures

24. Colony Morphology • Colony morphology • Color • Shape • Margin • Elevation

25. Colony Morphology

26. Colony morphology

27. Fig. 3.12.a

28. Fig. 3.12.b

29. Fig. 3.12.c

30. Fig. 20.3

31. IDENTIFICATION OF ISOLATED MICROBES

32. Conventional vs. Molecular Taxonomy • Phenotypic characteristics • includes physical, biochemical characteristics • two unrelated organisms could have the same phenotype • Phylogeny • 70% DNA hybridization? • 97% identity over 16S rRNA? • phenotypically distinct organisms may have identical 16S!

33. Phenotypic Characteristics Used In Conventional Taxonomy Morphology, Gram reaction, nutritional classification, cell wall, lipid, cell inclusions and storage products, pigments, carbon source utilization, nitrogen source utilization, sulfur source utilization, fermentation products, gaseous needs, temperature range, pH range, pathogenicity, symbiotic relationships, habitat.

35. G+C mol% • “ DNA base composition is one of the required characteristics for the minimum description of genera and species (Lévy-Frébault & Portaels, 1992; Goodfellow& O’Donnell,1993).” • Often, GC ratio of DNA is also used. • if two organisms that are thought to be closely related based on phenotypic criteria do not have similar GC values, then they are not in fact closely related.

36. % GC Content • The duplex structure of DNA : G-C and A-T • The content of G+C (mol% GC) varies within microorganisms • Thermal denaturation of DNA increased  increase in OD260. • Conclusions: • 1. G+C<5%  members of a species • 2. Similarity in %GC  does not imply relatedness • 3. Different in %GC  distantly related • 4. DNA base composition  useful measure of genetic heterogenity

37. ANALYZING MICROBIAL BIOMASS AND ACTIVITY

38. Methods for Determining Microbial Biomass and Activity (1) Field Samples Profile or Depth Basis Plant Association Replicate samples Composite samples Transportation, Mixing, Grinding, Subsampling, Dilution

39. Soil Sampling

40. Developing a Sampling Plan What sampling strategy (ex: random vs zone vs grid) should I use? What depths should I sample? How many samples should I collect?

41. Sampling Goal Determine an accurate average microbes content of a field (or a zone) by collecting sub-samples and compositing them.

42. Sampling Strategies Random Zone Grid Figure 1. (A) Aerial photograph (w/ random sampling), (B) Management zones, and (C) Field grids (Rains and Thomas, 2001). X X X X X X X X X X X X Advantages or disadvantages of each?

43. Sampling Strategies How to determine zones? Group by: Yield Remotely sensed indices Electrical Conductivity Nutrient levels Soil series Soil texture Topography • Which of these are fairly easy/cheap to obtain?

44. Accuracy Tight grid sampling (<300 ft grid) is more accurate and produces higher yield than soil series sampling. Overall, grid and zone sampling are more accurate than random sampling. Topographic sampling is less accurate than tight grids, but more accurate than 5 acre grids. Recommendation: First determine field variability with grid sampling. If field variability is low (< ~2-3 fold range), use grid sampling. If field variability is high, break into zones.

45. How Deep Should I Sample? 0-6 in. and 6-24 in. for nitrate, chloride, and sulfate related microbes’ activity 0-6 in. for phosphorus, metals, organic matter related microbes’ activity Figure 1. Soil sampling hand probes and augers.

46. Subsampling, what is it depend on? 1. Desired accuracy and 2. Desired precision

47. Does size of the field affect numbers of subsamples to collect? Figure 2. Relationship between field size and the number of subsamples required for a soil sample with an accuracy level of 15% and a confidence level of 80% (Swenson et al., 1984). Field size does not matter much!

48. How to Collect Soil • Sample depth • plowed fields • 6 inches to plow depth • no-till or pastures • 4 inches deep • Composite cores, mix well. • Fill a 1-pint soil test bag, clearly labeled with the field identification.

49. Too much or too little… Ideal volume

50. Information of Field Should be Given Lagoon sample Litter sample