Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi
Summary
This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results.
Abstract
The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; agar-block). It is well-accepted that soil-block microcosms yield higher decay rates, fewer moisture issues, lower variability among studies, and higher thresholds of preservative toxicity. Soil-block testing is thus the more utilized technique and has been standardized by American Society for Testing and Materials (ASTM) (method D 1413-07). The soil-block design has drawbacks, however, using locally-variable soil sources and in limiting the control of nutrients external (exogenous) to the decaying tissues. These drawbacks have emerged as a problem in applying this method to other, increasingly popular research aims. These modern aims include degrading lignocellulosics for bioenergy research, testing bioremediation of co-metabolized toxics, evaluating oxidative mechanisms, and tracking translocated elements along hyphal networks. Soil-blocks do not lend enough control in these applications. A refined agar-block approach is necessary.
Here, we use the brown rot wood-degrading fungus Serpula lacrymans to degrade wood in agar-block microcosms, using deep Petri dishes with low-calcium agar. We test the role of exogenous gypsum on decay in a time-series, to demonstrate the utility and expected variability. Blocks from a single board rip (longitudinal cut) are conditioned, weighed, autoclaved, and introduced aseptically atop plastic mesh. Fungal inoculations are at each block face, with exogenous gypsum added at interfaces. Harvests are aseptic until the final destructive harvest. These microcosms are designed to avoid block contact with agar or Petri dish walls. Condensation is minimized during plate pours and during incubation. Finally, inoculum/gypsum/wood spacing is minimized but without allowing contact. These less technical aspects of agar-block design are also the most common causes of failure and the key source of variability among studies. Video publication is therefore useful in this case, and we demonstrate low-variability, high-quality results.
Protocol
Discussion
Using our agar-block set-up (Figure 1) Serpula lacrymans grew in direct contact with the gypsum surfaces and into wood blocks (Figure 2), leading to more than 60% weight loss in the control brown-rotted pine blocks (Figure 3). This easily satisfies the ASTM standard goal of >50% decay, and the average coefficient of variation (CV) in decay at was 0.055 at week 16. This data is published in Schilling7. Again, other fungi will require longer incubation in agar-block than in soil-block. Fo…
Divulgations
The authors have nothing to disclose.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Petri dishes | Nunc | 4014 | 25 x 150 mm | |
| Agar, Type A | Sigma | A4550 | ||
| Ammonium nitrate, NH4NO3 | Millinckrodt | 3436-12 | ||
| Potassium phosphate, KH2PO4 | J.T. Baker | 3246-01 | ||
| Magnesium sulfate 7-hydrate, MgSO4•7H2O | Sigma | 230391 | ||
| D-(+)-Glucose | Sigma | G8270 | Dextrose | |
| Boric acid, H3BO4 | Mallinckrodt | 2549-04 | ||
| Zinc sulfate 7-hydrate, ZnSO4•7H2O | Mallinckrodt | 8880-12 | ||
| Manganous chloride 4-hydrate, MnCl2•4H2O | J.T. Baker | 2540-04 | ||
| Copper(II) sulfate 5-hydrate, CuSO4•5H2O | Sigma | 209198 | ||
| Ammonium heptamolybdate 4-hydrate, (NH4)6Mo7O24•4H2O | Sigma-Aldrich | 431346 | ||
| Calcium chloride dihydrate, CaCl2•2H2O | Mallinckrodt | 4160-12 | ||
| Sodium chloride, NaCl | Mallinckrodt | 7581-12 | ||
| Ferrous sulfate 7-hydrate, FeSO4•7H2O | Mallinckrodt | 5056-12 | ||
| Pipet-aid | Drummond | 4-000-110 | Cordless EtOH the surface |
|
| 10 ml sterile polystyrene pipette | BD Biosciences | 357551 | ||
| Gutter Guard | Thermwell Products Co. | VX620 | Pre-scrubbed with soap Hardware store |
|
| Calcium sulfate hemihydrate, CaSO4•0.5H2O | Acros Organics | 385355000 | ||
| #4 cork borer | Boekel | 1601 | ||
| Parafilm “M” | Pechiney | PM-996 |
References
- ASTM D1413-07. Standard test method for testing wood preservatives by laboratory soil-block cultures. . Annual Book of ASTM Standards. , 185-192 (2007).
- Bravery, A. F. . A miniaturized wood block for the rapid evaluation of wood preservative fungicides. , (1978).
- Low, G. A., Young, M. E., Martin, P., Palfreyman, J. W. Assessing the relationship between the dry rot fungus Serpula lacrymans and selected forms of masonry. Int. Biodeterior. Biodegrad. 46, 141-150 (2000).
- Nicolas, D. Volume I (One/1) – Degradation and Protection of Wood (Syracuse Wood Science Series #5). Wood Deterioration and Its Prevention by Preservative Treatments. , (1973).
- Schilling, J. S. Effects of calcium-based materials and iron impurities on wood degradation by the brown rot fungus Serpula lacrymans. Holzforschung. 64, 93-99 (2010).
