Soil Sampling for mycology

 

In the first round of prototypes I ideated on storage systems for foraged items during a mushroom foray. These are more obvious to the traditions of mushroom hunting where you are collecting large and small fungi samples as you move throughout the forest. These are alternatives to the traditional use of a basket or bag and were created with functionality and wearability.  Building on these alternative collection systems, I am working on other ways to gain an understanding of the fungi in the ecosystem beyond the  harvesting of mushrooms.

In talking to mycologists and reading literature on fungi, there is actually a large amount of fungi in the form of mycelium, spores and hyphae, rather than the fruiting body, the mushroom. Soil sampling is suggested as a way to gain a picture of the fungi that is the in the environment, regardless of the mushroom, with estimates of 10^6 spores per gram of soil, with several species present.

The methods of isolating and identifying these species include soil dilution and plating, in which the sample is placed on agar to germinate spores and hyphal extraction in which larger bits of hyphae are extracted from the sample and placed in agar. The plating method was created by mycologist, John Henry Warcup, who was able to demonstrate that fungi could be isolated by placing a soil sample directly on an agar plate, allowing hyphal tips to grow from the soil.   Fungi that is endophytic (growing in) or epiphytic (growing on) on a substrate can also be extracted, isolated and identified after collection. For example, to extract epiphytes, a leaf that contains potential fungi can be washed, with the water that is rinsed off the leaf raised in agar to check for spore germination. To access the endophytic fungi, the exterior of the leaf is first sterilized to remove any bacteria, and then ground using mortar and pestle or cut up to expose the interior. This can then be mixed with water to be plated onto agar.  These processes are done later on in a sterile lab environment to prevent cross contaminations. Rather than thinking about this process of extraction and identification, I am more interested in rethinking about this soil sample collection process which is done while you are out in the environment.

Based on a soil sampling report put forth by the EPA to assess VOC (volatile organic compounds) in soils, four main factors play into an effective soil samples which include effervescence, sample size, holding times and percent solids. Effervescence is the formation of bubbles when the sample comes into contact with an acidic preservative. If the bubbling occurs, the sample needs to remain unpreserved since it could become deleterious to the soil. Sample size ranges, but is suggested to be at 40 mL. Holding time is the length of time to hold an unpreserved sample before it is tested, which the document advises a 48 hour period where the sample can sit on ice before entering the lab for testing. Percent solids is the total amount of solid material within a sample, this is to make sure that the sample has enough material to be analyzed back in the lab. Although these procedures and factors may be more relevant to analyzing VOC content, this could provide guidelines for soil sampling for fungi.

In guides for soil sampling specific to mycology, the process is changed slightly in order to preserve the fungi content. One of these guides is the Handbook for Mycological Methods put out by the Food and Agricultural Organization of the United Nations for study of the impact on mycology on coffee production. The handbook provides specifications for collecting fungi from a tree, air, and soil.

Collecting endophytic fungi from a tree:

“Cut three shoots from different parts of each tree sampled on the proximal side (side closest to the main stem) of the second node from the apical meristem (the growing point) of each shoot. At the lab, re-cut the stem to a length of about 2 to 3 cm starting from the proximal end of the second internode (the smooth stem between nodes). Surface sterilize the segments in a strong solution of hypochlorite (5% available chlorine) for 5 min. Shake several times. Decant the sterilent after the appointed time and replace twice with sterile water. After decanting the rinse water, blot on sterile filter paper in a sterile petri dish. Using 70% alcohol and a flame to sterilize your instruments, re-cut about half of a cm from each end of the segments with a scalpel in another sterile petri dish containing sterile filter paper while holding it with some strong forceps to yield segments 1 to 2cm in length. Gently push up to five segments in a radiate pattern into a plate of D/2G18 and incubate. “

Air sampling, as the FAO handbook suggests, is taken with exposed agar plates in order to determine settling of the spore load while the coffee bean is drying or sitting in a facility.  This technique they have adopted is chosen based on the type of fungi they are studying – those that affect the coffee production process. As a result, this technique will most likely capture comparisons of fungi in different environments to yield a quantitative comparison rather than a qualitative one.

With soil sampling, the handbook notes what information can be attained through  this process.

“Although commonly employed, taking a single ‘grab sample’ of soil is not a very useful activity. Any two samples whether separated by a meter or a kilometer will be different because they cannot be the same. What is of interest is not the fact of the difference but ascribing the difference to some feature of ecological significance.

As such the soil sample is as follows:

Use a strong spatula to remove about 20g of soil into a sterile bottle of an appropriate size after brushing away loose material and the uppermost layer of soil (usually 1 to 3 cm) until the small fibrous roots are exposed. Sample the soil around these roots. Away from the coffee stem sample at the same depth as the ‘0’ sample. To sample in the orchard, the first sample, labeled ‘0’, is taken within 10 cm of the main stem of a coffee plant, the second, labeled ‘1’, is taken below the edge of the foliage of the same plant and the third, labeled ‘2’, is taken along the line defined by the first two but at a point such that it is as far as possible away from any coffee plant but still within the plantation system. A fourth sample (label ‘3’) may be taken near to but outside of the plantation for reference.

In this process, four samples are taken for analysis at different points from the coffee plant, one near the root, one near the foliage, one within the plantation, and one outside of the plantation. This analysis would thus provide a perspective on the impact of fungi on the coffee plant itself in relation to distance away from the tree.

In the soil sampling section, it is also recommended that the samples be air dried if not immediately analyzed.  This would allow for proper storage before the dilution process to isolate and identify fungi within the sample.

Other things that are used for sampling include surfaces such as cement and brick in which a swabbing technique is utilized to collect a sample.

Other techniques that are included in this guide are for surfaces such as cement and brick in which a swabbing technique is utilized and for insects. The guide recommends that insects be captured live and placed in a petri dish containing a enumeration media (like agar). Any fungi in the fecal matter and footprints that the insect leave behind could then germinate in the dish. The insect can then be crushed and diluted to test for mycological content 🙁

In Progress of Mycology, most soil fungi are between the top 10 -30 cm of soil, after litter and humus is cleared from the top. In this book, the sample is supposed to be representative of a site, thus “composite samples are obtained by mixing equal amounts of material taken from soil samples collected over a wide area”.   One technique also uses Scotch tape to take successive samples from a soil surface.

The Handbook of Industrial Mycology provides another source for sampling fungi for mycology, called baiting, in which substrata can be collected from the space to provide a habitat for the fungi or placed in a container that has specific environmental conditions to encourage growth (moist chambers). Fallen and dead wood can also be collected for further analysis, especially if decay is present. These small stem samples are them split and placed in a media before incubating to germinate fungi.

Although there are a lot of variations of how to collect and process the soil samples, it is still an important technique to be used to study fungi presence out in the field, especially for those that might exist without mushrooms. In these soil sampling methods, the collector is usually required to dig and scoop a sample of the soil into a collection chamber. This routine is repeated several times and in several locations depending on the nature of the study. Since my project is focused on citizen science, the goal is to just provide an overall species list of fungi that is present in an area during a foray in order to count frequency.  This could mean that a soil sample is collected at designated points at intervals (for example, collect a sample every 100 meters) or whenever the participant finds a mushroom, they can take a sample. Although the latter is less methodological and might be less precise, when working with citizen science, there is a balance to maintain between lowering barriers to entry and creating a rigorous procedure. This was stated during an interview with one of the club mycologists regarding their process with DNA sequencing and changing the process to accommodate various fidelities during testing. *find dat quote* There might also be a preference to collection methods such that instead of keeping separate containers to prevent cross contamination of samples (FAO coffee production and fungi), it may be easier and beneficial to create one larger sample using multiple smaller samples collected throughout a designated area.

This has lead to work on some initial designs for a soil sampling shoe based on other work during the ideation process. This concept was initially inspired by soil and dirt becoming trapped in the treads of the shoes during a hike – can shoes be designed with intentional dirt trapping to collect soil samples? This passive collection would utilize a gesture (stepping) with wearable technology worn on the foot.

Below are some animated sketches that speculate possible (not necessarily feasible) ways to actuate this concept. soilshoe_tube

Shoe tube: soil sample is collected through a tube

soilshoe_flap

Flap: a flap on the ball of the foot opens up to scoop up soil soilshoe_auger

Auger: an auger located on the heel collects soilsoilshoe_heely

Wheel: wheel with scoops rotates to scoop up soilsoilshoe_multipletubes

Multiple tubes: multiple samples can be collected through stepping

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