The overall goal of this procedure is to inoculate an aerobic wood degrading fungus in a Petri dish microcosm where wood moisture and exogenous substrates are tightly controlled. This is accomplished by first preparing microcosms with exact volumes of known edia. Then the wood to be degraded, and any exogenous solid substrates are assembled on top of a plastic grid on the agri surface without allowing contact among substrates.
The microcosm is then inoculated and incubated with the test fungus. Following incubation, woodblock are harvested to determine mass loss and to prepare tissues for analyses. Ultimately, results can be obtained that show the degree of wood biodegradation and the role of exogenous element sources through mass loss measurements, observations of mycelial morphology, and targeted elemental analyses like inductively coupled plasma spectroscopy.
Hi, my name is Brooke Jacobson from the laboratory of Dr.Jonathan Schilling in the Department of Bioproducts and Biosystems Engineering at the University of Minnesota. Today we're going to show your a procedure for using Petri dishes as microenvironments for the plant tissue decomposition by aerobic fungi. We use this procedure in our lab to study the mechanisms of fungal lignocellulose decomposition.
Lignocellulose is the plant tissue comprised of lignin, cellulose and hemi cellulose. The tissue we study most often is wood mistakes with the less technical aspects of auger block design are the most common causes of failure and the key sources of variability among studies making video publication of our auger block set up useful. We know from experience that this procedure yields high quality, low variability results.
So let's get started. For these experiments, use Petri dishes that are one centimeter deeper than typical Petri dishes to increase the headspace above the woodblock. In the microcosm.
Begin microcosm preparation by making enough a edia for 20 milliliters per Petri dish for calcium and iron free. Type a agar at 15 grams of agar to a 500 milliliter volumetric flask containing approximately 400 milliliters of deionized water amended with supplements. Use previously prepared stock solutions to add micronutrients to the mixture.
Once nutrients are added, fill the volumetric flask to the 500 milliliter line. Next, transfer the media to a 1000 milliliter flask. The autoclave at 121 degrees Celsius and 16 PSI for 20 minutes.
Take care not to exceed a volume of 500 milliliters per flask to avoid having agri solidify before test plates are administered. Following autoclaving, allow the media to cool to the touch to minimize condensation. Minimizing moisture is critical in these experiments as moisture content or mc.
Over 80%will cause anoxia in tissues. Limit decay by aerobic fungi and increase variability once.Cool. Use a portable pipette aid and 10 milliliter sterile polystyrene pipettes to aseptically.
Transfer the agar to a high stack of plates in a biosafety cabinet. Next generously cut plastic mesh grids to fit inside the plate bottoms. After cutting and thoroughly washing grids with soap and water, prepare mesh grids for drying and autoclaving for this demonstration.
Southern yellow pine or SYP is used as it represents an important source of lumber. In residential housing in the us, use non-treated lumber and cut 19 by 19 millimeter knot free blocks from a single longitudinal cut along the grain. Cut this length into 19 millimeter cubed blocks.
Split the 19 millimeter cubed blocks in half along the grain using a chisel and hammer instead of a saw. This makes one rough block edge to face down on the plastic mesh in the agri block microcosm and a smooth top edge to label. Label the smooth edge of the blocks with a pencil, cut enough blocks to satisfy the treatments, as well as the non inoculated controls that will serve both as contamination monitors and as baseline data samples conditioned in parallel for oven dried material, place samples in aluminum whey pans and into a convection oven at 100 degrees Celsius for 48 hours.
Transfer the samples from the oven to a desiccate to cool them once.Cool. Pre weigh the samples to determine the initial oven dry weight. Finally, prepare the samples including plastic grids and wood for autoclave by tightly wrapping with foil to minimize wetting.
This does not require that each sample be wrapped individually, but that steam exposure is low and consistent. Autoclave the labeled samples at 121 degrees Celsius and 16 PSI for one hour. In a sterile biosafety cabinet, assemble empty AEs, A source plate for inoculum mesh plant substrate exogenous materials, a sharpie paraform strips, forceps, and transfer tools.
The next step of the inoculation is to add the items to the agri, contain Petri dishes to minimize contamination risk. Flame transfer tools and forceps with 95%ethanol as they are used. First, add the plastic mesh to the petri dish.
Then add the wood substrate here. Two wood blocks are added along the length of the mesh so that data can be paired from the early and late harvests. These blocks are added side by side with grain and facing the point of the source of fungal inoculum.
Next, add the exogenous materials.Here. The role of gypsum is being tested on decay by a filamentous fungus. Therefore, the fungus should encounter these gypsum discs before reaching the wood.
One gypsum disc is added between each block and inoculum point on top of the mesh. Take care to avoid contact between the discs in the wood while keeping them close together. For the fungal inoculum, use a number four cork Boer with seven millimeter diameter to make plugs from two week cultures grown in 20 milliliters of agar.
This helps to control the inoculum volume. For non inoculated controls, it is best to add a plug from a sterile plate. Do not allow contact between inoculum and either the exogenous substrates or the wood, but place them close together.
Use one inoculum plug per block. Seal the dishes with param that has been cut with a razor hold plates horizontal and wrap param in a smooth continuous motion. Finally, label the dish lids using an alcohol resistant marker labeling block numbers directly over respective substrates.
Also draw circles over the gypsum discs. Transfer the plates carefully to a biological incubator to avoid jostling the contents and to minimize condensation due to temperature fluctuations. The conditions used here are at 20 degrees Celsius and in the dark perform time series harvests aseptically except for the last harvest.
At the week five harvest point, remove the entire treatment lot. Spray each bottom and top with 70%ethanol and use flamed forceps to remove material inside the biosafety cabinet. Take pictures before destroying the agri plates, focusing on any morph F or melon.
Then remove blocks to be treated. Roll away any excess pha, but be careful not to lose any decayed material oven. Dry blocks in aluminum whe pans and determine mass loss using the fresh and dry weights of the controls to monitor for excessive wicking.
Mass loss data helps gauge the decay progress and is important data. If elemental concentrations are to be calculated on a gram weight basis, percentage data must be normalized. For statistics, label and track any dark brown blocks, which are clearly waterlogged.
Finally destroy the cultures in autoclavable bags, saving plastic support mesh and any other components that can be recycled for use in future experiments. Using the agri block microcosm setup, pine wood blocks rest on plastic mesh to elevate blocks above agri contact. Circular Crains grew in direct contact with the gypsum surfaces and into the wood blocks.
This mycelium represents an important connection between wood and exogenous nutrient element sources. In controlling these exogenous material sources in the agar or a solid materials is a key advantage of a agri block design versus soil block design mean percent weight loss as a measure of extent of wood decay by ser crain's after five and 15 weeks. Incubation with pine in agro block microcosms is shown here.
Treatments were none. Five millimolar calcium chloride added to agar greater than 99%pure gypsum or 1%iron amended gypsum for each harvest bars under the same letter are not significantly different. With the error bars representing the standard deviation, more than 60%weight loss was observed in the control brown rotted pine blocks, which satisfies the American Society for Testing and Materials standard goal of greater than 50%decay.
The average coefficient of variation in decay was 0.055 at week 16. In addition to low variability, a treatment effect was observed. Fungal decay was inhibited with calcium additions, whether to the agar as calcium chloride or as pure gypsum.
Interestingly, decay rates rebound with the addition of iron to the gypsum. Suggesting iron not calcium is critical in observing the five replicates at week 15 of decay. By the same brown rot, test fungus, circular lamins, the color of the mycelia reveals mein.
Differences in external hyphae among treatments, blocks were removed and up and dried. Revealing the lack of mein in calcium free treatment. The yellowing in pure calcium treatments and the rust appearance in iron amended treatment.
The control represents non inoculated blocks for comparison. Interestingly, the mein effects are lost using a high iron medium. Collectively, it's suggests that this fungus utilizes iron, not calcium.
In these materials, We've just shown you how to use an auger block setup to successfully test the biodegradation of plant tissues by aerobic filamentous fungi. When doing this procedure, it's important to remember to keep the substrates from coming in contact with one another, especially the plant tissue and the auger minimize moisture content from the auger and the platelets, and double check to make sure that the contents of each plate have not shifted before incubation. Keeping these variables in mind, you can control the decay environment much better than you can when using a soil block setup.
You can generate data with a statistical power needed to answer your research questions. So that's it. Thanks for watching and good luck with your experiments.
