Protocol

Author

Alain Pierret

Additional Authors:

C.E. Pankhurst, B.G. Hawke & J.M. Kirby

Overview

Protocol to determine the metabolic potential and functional diversity of the bacterial communities associated with plant roots located within macropores

Background

Up to 80% of the plant root system may be preferentially located within or closely associated with the macropores (within 1-10 mm), a zone defined as the macropore sheath (Stewart et al., 1999). It has been demonstrated (Pierret et al., 1999; Pankhurst et al., 2002) that the macropore sheath is both chemically and microbiologically different from the bulk soil. More research is required to understand the relationship between the size and activity of the microbial communities within the macropore sheath and root distribution within the soil profile, with increasing soil depth. This protocol explains how to sample and characterize these microbial communities.

Materials/Equipment

• Scalpel
• Magnifying lamp (optional)
• Dissecting microscope
• Phosphate buffered saline (PBS) solution made up of:
  • 8.5 g NaCl,
  • 6.8 g KH₂PO₄ and
  • 11.4 g K₂HPO₄ per litre.
• Selective agar media (described by Pankhurst et al. (1995))
• 25 C incubator
• Plating medium containing:
  • 1.0 g peptone,
  • 1.0 g yeast extract,
  • 0.5 g sodium glycerophosphate,
  • 15.0 g agar and
  • 5 ml glycerol per litre.
• Oxoid Pseudomonas agar base
• Antibiotic supplements:
  • 10 mg cetrimide,
  • 10 mg fucidin and
  • 50 mg cephaloridine per litre.
• Sterile petri dishes
• Magnetic stirrer
• Biolog plates
• Biolog MicroStation plate reader

Units, terms, definitions

Macropore sheath (MPS) soil

Macropore root (MR) soil

Colony Forming Units (CFU)

The MPS refers to the soil around macropores in which roots are predominantly located (Stewart et al., 1999)

Procedure

I – Collection and dissection of soil specimens

• Collect undisturbed soil samples in the field
• Carefully break apart soil samples to expose the macropores
• Using a scalpel, with the aid of a magnifying lamp if necessary, dissect soil from the 1 to 3 mm region surrounding the exposed macropores – This soil is referred to as macropore sheath soil (MPS, Fig. 1) (Stewart et al., 1999).
• For a comparison with bulk soil, dissect soil at a distance > 10 mm away from the macropores (Fig. 1).
• Within larger macropores (4-6 mm diameter), collect small soil aggregates (< 0.5 mm diameter) that are often attached to plant roots. These aggregates are referred to as macropore root (MR) soil (Fig. 2).

Figure 1 Roots (R) within a macropore. Macropore sheath (MPS) soil includes the inside of the macropore wall and soil extending 1-3 mm into the soil matrix. Bulk soil (BS) is collected >10 mm from the macropore.
Figure 2 Clumping of roots (white arrow) within a macropore. The black arrows show small soil aggregates that are associated with the roots within the macropore. These aggregates are referred to as macropore root (MR) soil. II – Enumeration of microbial functional groups Preparation of culturable populations

• Prepare a phosphate buffered saline (PBS) solution that contains 8.5 g NaCl, 6.8 g KH₂PO₄ and 11.4 g K₂HPO₄ per litre.
• Prepare three replicate one gram samples of MPS, MR and bulk soil and add them to 9 ml PBS.
• Shake the sample + PBS mixture at 250 rpm for 30 min prior to serial dilution.

Preparation of selective media, Inoculation and incubation

• To estimate aerobic bacteria, fungi and actinomycetes, use the selective agar media described by Pankhurst et al. (1995). Incubate at 25 C for 4-7 days.
• To estimate aerobic spore-forming bacteria (mostly Bacillus spp.), heat a designated set of diluted suspensions at 80 C for 15 min prior to plating them onto a medium containing 1.0 g peptone, 1.0 g yeast extract, 0.5 g sodium glycerophosphate, 15.0 g agar and 5 ml glycerol per litre. Incubate at 25 C for 4-7 days.
• To estimate aerobic Pseudomonas spp. use Oxoid Pseudomonas agar base plus antibiotic supplements (cetrimide 10 mg, fucidin 10 mg and cephaloridine 50 mg per litre). Incubate at 25 C for 4-7 days.
• To estimate cellulolytic bacteria and fungi use the media and most-probable-number (MPN) method described by Gupta and Roper (1994). Incubate at 25 C for 4-7 days.
• To estimate ammonia and nitrite oxidizing bacteria use the media and MPN method described by Schmidt and Belser (1994). Incubate at 25 C for 4 weeks.
• To estimate Pythium spp. use the VP3 medium and procedure described by Ali-Shtayeh et al. (1986). Incubate at 25 C for 4-7 days.

III – Substrate utilization patterns using Biolog GN plates (Biolog, Hayward, CA)

• Bring an aliquot of the PBS soil suspension used for enumeration of microbial functional groups in the MPS, MR and bulk soil to a final dilution of 10 ³ with PBS.
• Add 50 milliliters of this suspension to a sterile Petri dish and stir it with the aid of a magnetic stirrer and flea and use to inoculate the triplicate Biolog plates.
• Inoculate each of the 96 wells in an individual Biolog plate with 150 l of soil suspension.
• Incubate plates at 25 C for 2, 48 and 72 hours.
• Read light absorbance (590 nm) after 24, 48 and 72 hours incubation using a Biolog MicroStation plate reader.

  Interpretation of results:

  The formation of purple color occurs when the microbes can utilize the carbon source of a given well and begin to respire. The respiration of the cells in the community reduces a dye that is included with the carbon source. The reaction patterns are therefore proportional to the optical density values of individual wells at 590 nm (OD590) from which is derived the numbers of CFUs in each well. The most popular method of analysis of the data is via Principle Components Analysis (PCA) of average well color development (AWCD) data. Community-level physiological profiles are assessed for key characteristics such as, (i) pattern development (similarity), (ii) rate of color change in each well and (iii) richness of well response (diversity).

Notes and troubleshooting tips

• Ensure there will be adequate room in the incubator throughout the extended period during which samples need to be kept.

Links to resources and suppliers

Oxoid Pseudomonas agar base

Biolog GN plates – Biolog, Hayward, CA

Literature references

Ali-Shtayeh M S, Len L-H C and Dick M W 1986 An improved method andmedium for quantitative estimates of Pythium species from soil. Trans. Brit. Mycol. Soc. 86, 39-47.

Gupta V V S R and Roper M M 1994 A most-probable-number method to enumerate the populations of cellulolytic bacteria and fungi in soils. In Soil Biota: Management in Sustainable Framing Systems, Poster Papers. Ed.

Pankhurst C E. pp 115-117. CSIRO, East Melbourne.

Pankhurst C E, Hawke B G, McDonald H J, Kirkby C A, Buckerfield J C, Michelsen P, O’Brien K A, Gupta V V S R and Doube B M 1995 Evaluation of soil biological properties as potential bioindicators of soil health. Aust. J. Expt. Agric. 35, 1015-1028.

Schmidt E L and Belser L W 1994 Autotrophic nitrifying bacteria. In Methods of Soil Analysis. Part 2, Microbiological and Biochemical Properties. Editorial committee R W Weaver chair. Pp 159-178.

Soil Science Society of America Inc, Madison WI.

Stewart J B, Moran C J and Wood J T 1999 Macropore sheath: quantification of plant root and soil macropore association. Plant Soil 211, 59-67.

Health, safety & hazardous waste disposal considerations

• Consult an MSDS prior to handling any of the chemicals used in this protocol.