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.
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.
This protocol applies to woody and non-woody substrates, as outlined, as well as to oven- or air-dried material. Read through the protocol first, however, before set-up. There are several points raised that may apply to your study, and these points (underlined) require planning. Also, note that there are two published agar-block methods that are occasionally used, one the British Standard 838 and another following an International Research Group on Wood Protection (IRG-WP) paper submitted by Bravery (1978). Our resembles method 838, with modifications primarily in the microcosm design and the control of the agar medium, but again, both approaches are often avoided due to historic moisture control issues in wood blocks, causing anoxia and variability. A good review of these test methods that includes discussion of agar-block designs, including the 838 standard, can be found in Nicholas (1973).
1) Preparing Microcosms
Microcosms for these trials are 1 cm taller (deeper) than typical petri dishes, increasing head space above wood blocks. They are filled with a modest and exact amount of agar in order to control absolute nutrient amounts, in addition to their concentration, and to keep wood blocks well away (>3 mm) from the lid. The agar used in this case of gypsum testing is a low-calcium Type A agar; however, we show representative results using Blakeslee’s medium, the ATCC recommended medium for maintaining the test isolate of Serpula lacrymans (Wulfen: Fries) Schroeter strain EMPA 65 (ATCC 32750).
This design keeps plant tissues away from agar contact and away from the dish lid and walls. Variable wetting of lignocellulosic substrates is the key source of variability in agar-block tests. Wetting to increase moisture content creates anoxia and suppresses or even halts aerobic biodegradation. It also creates a problem for anyone studying oxidative mechanisms of brown and white rot fungi responsible for wood decomposition. Condensation on plate lids is an issue if free water droplets form and wet the substrate. Likewise, wood and other tissues will ‘wick’ water rapidly from agar when in contact, leading to moisture contents in excess of 80% (dry wt. basis) and halting aerobic degradation. Tissues must be distanced from these water sources, allowing the filamentous fungus to find, connect, and control moisture within the substrate.
2) Preparing ‘Block’ Substrates
These protocols have been developed for solid wood, but are adaptable for other plant tissues. Mass loss is the standard measure for decay progress in wood degraded by filamentous fungi. Thus, our approach uses oven-dry weights pre- and post-decay to determine mass loss. However, for any bioenergy research, where the focus is on the plant tissue chemistry, many find that air drying tissues is preferable. We show here protocols for preparing your agar-block cultures to use oven-dried starting material, but give the alternative information to air dry and also to process powder instead of solid substrates.
3) Inoculating & Labeling
Inoculating agar-block microcosms is more time-consuming than inoculating soil-block jars. For us, we count on each inoculation taking 3 min. To petri dishes containing agar, this is the point of addition for mesh, wood, any exogenous nutrient sources (here, gypsum pellets), and the fungus. There is increased chance for contamination because of the amount of time the lid is open and the number of visits inside. There are also several key mistakes that are commonly made at this stage, and these are best covered by coupling video with text. Watch the video.
4) Incubating & Harvesting
Plates can be incubated to suit your fungus, but should be kept in a biological incubator if possible, to avoid condensation due to temperature fluctuations. Time series harvests are done aseptically, except for the last harvest. If these intermediate harvests are done after growth on the substrates is significant, handling the plates is easier because hyphae cross link substrates.
5) Interpreting Results
Lignocellulosic tissues will usually decay slower in agar-block designs, but at this point you should have relatively low variability even at moderate decay levels. You should also have moderate (20-50%), not high moisture in control tissues.
Figure 1. Agar-block microcosm, as set up in this demonstration, before incubation.
Figure 2. Serpula lacrymans colonizing pine wood blocks resting on plastic mesh to elevate blocks above agar-contact. This mycelium represents an important connection between wood and exogenous nutrient/element sources, and controlling these exogenous material sources in the agar or as solid materials is a key advantage of agar-block design, versus soil-block design.
Figure 3. Mean % weight loss, as a measure of extent of wood decay by Serpula lacrymans after 5 and 15 weeks incubation with pine in agar-block microcosms. Treatment were none (Ca-free), 5 mM CaCl2 added to agar (CaCl2), >99% pure gypsum (CaSO4), or 1% iron-amended gypsum. Protected ANOVA means comparisons were using Tukey’s tests, with α=0.05. For each harvest, bars under the same letter are not significantly different. Error bars = standard deviation. Published in Schilling (2010).
Figure 4. Overhead picture (A) of all five replicates at week 15 of decay by the same brown rot test fungus, Serpula lacrymans, as well as the blocks (B) removed and oven-dried. Note the lack of melanization in Ca-free treatment, the yellowing in pure calcium treatments, and the rust appearance in iron-amended treatment. Treatments are labeled as in Figure 3, with the control in (B) being non-inoculated blocks for comparison.
Figure 5. Overhead picture from a different trial using a higher iron concentration medium, Blakeslee’s malt agar. Note the loss of observed melanization, compared with Figure 4. Blocks removed and weighed showed no treatment effect on weight loss. This is presented as a demonstration of the influence of exogenous components of these block trials. These effects would not be testable in soil-block jar designs, where these exogenous inputs are too difficult to control.
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. For reference, we have successfully run similar designs in several past experiments with a variety of other fungal species, often using scanning electron microscopy with electron dispersive spectroscopy (SEM-EDS) and inductively coupled plasma spectroscopy (ICP-OES) to verify connectivity with exogenous substrates and translocation of calcium and other elements into wood8,9.
In addition to low variability, there are two other results that our agar-block design reveals. First, there was a treatment effect (Figure 3), where our calcium additions, whether to the agar as CaCl2 or as pure gypsum, inhibited decay by this fungus. This is a useful result because others have theorized calcium enhances decay based on studies with real-world materials like mortar and plaster. Instead, we see decay rates rebound with the addition of iron to the gypsum, suggesting iron, not calcium, is key. The second useful result, however, is not quantified but is instead the color of the mycelia (Figure 4). There are significant and obvious melanization differences in external hyphae among treatments, and researchers have previously noted ‘rust’ melanization when encountering this fungus on building materials (eg. Lowet al. 2000). In our more recent studies, we found that these effects are lost using a high-iron medium (Figure 5). Collectively, it suggests that this fungus utilizes iron, not calcium, in these materials and that earlier studies, with ‘rusty’ mycelia reported, were observing iron effect, not calcium effect.
Overall, these results and a lack of treatment effects using high-iron agar are a very strong demonstration of the control that the agar-block design can provide the researcher, especially in light of the alternative soil-block approach.
The authors have nothing to disclose.
Material Name | Tipo | 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 |