Progress report for FNE21-970
In this research time our main objective is to successfully cultivate Chicken of the Woods indoors utilizing methods already practiced or easily accessible to any size mushroom farm. By showing the viability of growing COTW indoors, and making the methods and outcome publicly available, we believe that many mushroom growers will start bringing this high-value mushroom to market.
The PCR (polymerase chain reaction) work detailed in this application may not significantly add to the scientific knowledge of Chicken of the Woods DNA. Instead our second primary objective is to confirm previous lab-based research. We will confirm that identified primer sequences are associated with physical trains present in fruiting bodies, or growing mushrooms. This research will also make this knowledge more available to small mushroom growers interested in adding PCR technology to their labs, which is a growing trend.
A tertiary objective is to disseminate information about Chicken of the Woods. We aim to inform both the general public about this delicious mushroom, and to inform farmers of the feasibility of cultivating it indoors. This is why we believe outreach is essential to the success of this project, and to the future use of the research outcomes.
This research seeks to provide much needed guidance to small mushroom farmers who want to diversify their consumer options with the healthy, visually appealing COTW mushroom. We will address the problem of the information gap – how do we take lab research on COTW and make it viable for small mushroom farms. Adopting a new variety of fungi can put a small mushroom farm at an advantage in their region. Though mushrooms were called ‘the poor man’s meat’ in Stalin’s Russia, today the high-end market value of COTW makes it an ideal variety for small farms looking for higher value sales. Chicken of the Woods is a sought after fungi which has gained popularity as foraging has grown as a hobby in the United States and fetches $25 per pound at markets. It is not cultivated indoors and not commercially available. As mushrooms become a more common staple in consumers fridges, customers are seeking out variety. Consider that shiitakes only started being produced in America in very limited amounts in the 1970’s, and today they are 25% of the mushroom market. You have probably enjoyed them in a meal this year, even if you are not a mushroom enthusiast. We hope to see COTW rise in familiarity and popularity in our lifetime.
Two reasons why this project is important are the quest for knowledge, and the need for more variety available for the small mushroom farmer. Additionally, we believe that the health benefits and culinary range of COTW will help its rise in popularity if it is viable to produce. This species gets its colorful name because of its chicken like consistency, making it a favorite meat-substitute for vegetarians and meat eaters alike. Additionally, those seeking health benefits from their food will find the studies on antioxidants in COTW compelling. The rise in individuals pursuing natural healthy food and vegetarian alternatives in America give COTW a large market potential. Most small mushroom farms would love to trial new species regularly, but do not have the time or space to dedicate to something that may not be fruitful. We hope that through this grant we an gather and publish enough data to educate small farmers on the viability of taking the next step in cultivating COTW.
Success in this project will not be judged by how many varieties we grow, by the biological efficiency (BE), or even by the quality of taste of the product. It is in the act of testing methodology and recording data on these factors that we will provide useful information to both the scientific mycological community and the small mushroom farmer. Increasing the chances that it can be grown indoors adds a year-round high market-value crop to their offerings. Through the results of our research small farmers will know what works and what does not when it comes to growing COTW. They will be given advice that will lead them to make an educated decision as to whether adding this variety of mushroom to there market offerings is feasible. Most mushroom farmers do not have this on their radar, so the concept will be new and exciting to many.
For the scientific community, our work will aid in confirming previous PCR research on the correlation between the presence of certain primer sequences and their associated physical attributes. Confirming this research will aid both the more lab-focused mycologist who wishes to study perhaps the medicinal or other characteristics of COTW, and the mushroom grower looking to perfect strains with PCR technology. To be clear, since PCR technology is relatively new and not well known, the primer sequences identified should tie to color and other traits in the fully grown mushroom. We will confirm that a certain sequence has this physical trait. In the future this knowledge will help others modify COTW DNA to make this trait more prominent.
- - Technical Advisor
Our first objective is to Test the commercial viability of known isolated samples of COTW. We will accomplish this by growing our samples on agar, transferring them to high-quality grain spawn for two generations, before finally attempting to fruit the mushrooms on hardwood sawdust, hardwood sawdust and straw, as well as mineral enriched hardwood sawdust. Additionally we will preform a series of PCR applications of our samples DNA and have it sequenced at a 3rd party lab.
After procuring two commercially available isolated strains of Chicken of the Woods in liquid culture (LC) form, each sample will grown on five petri dishes using a standard Potato Dextrose Agar (PDA) solution as the substrate. During this period the petri dishes healthy mycelium growth will be photo documented with a digital camera.
After two to four weeks the petri dishes should be fully colonized by the mycelium. We will then prepare five 1 quart mason jars for each strain of high-quality grain spawn. The mycelium on the petri dishes will be transferred into these new jars. This is a standard procedure for mushroom farms. Our grains spawn will be made up of 80% rye berries fully hydrated to their field capacity (50% grain, 50% water), 19% hydrated saw dust (40% sawdust, 60% water), and 1% gypsum (1g). To prevent contamination of endospores in our samples the rye berries will be soaked for 18 hours in clean water to encourage any existing endospores to germinate. Once the grain has been soaked it will be packed with the rest of the substrate materials (hydrated sawdust, gypsum) and loaded into 1 quart mason-style jars, equipped with synthetic filter disks (0.03 microns) to allow gas-exchange during the grow while keeping out contamination. After the jars have been loaded with the substrate they will be placed into pressure cookers and sterilized at 20 PSI for 1 hour. The pressure cookers will be allowed to cool to room temperature, which can take up to 24 hours. Once the pressure cooker has cooled it will be taken into the lab. Once in the lab we will follow our standard decontamination and cleaning procedure, which includes cleaning the room with anti-bacterial cleaners and allowing a horizontal HEPA flowhood (capable of filtering particles down to 0.03 microns 99.997% of the time) to run for 1 hour.
Once the room has been prepared we will transfer the mycelium from the petri dishes to the substrate contained inside the mason jars. The transfers will be done in front of the HEPA flow hood using sterile gloves, a surgical mask, and a sterile scalpel to ensure that no other bacteria of fungi are able to contaminate the samples as they are transferred into the mason jars. While the mason jars are growing, we will take daily photos with a digital camera to photo document the mycelium growth process. The resulting product from this process will be known as our first generation of grain spawn.
After two to four weeks the mycelium from the first generation should have completely colonized the grain spawn in the mason jars. We will select one jar from each strain to transfer to the next generation of grain. We will produce seven more 1 quart jars of grain spawn produced using the procedure described above. Once the grain has been prepared we will use the sterile transfer technique described above to transfer the one quart jar from the first generation to seven new second generation of quart jars.
After an additional two to four weeks, the second generation of mycelium will have completed growing. These second generation mason jars will be transferred to six 5lb mushroom grow bags equipped it 0.03 micron filter patches. Each grow bag will be filled with one of the three substrates previously mentioned (sawdust, mineral enriched sawdust, grain and mineral enriched sawdust). The end result will be two grow bags of each sawdust spawn per strain, resulting in 12 grow bags (60 lbs) of substrate to fruit. Sawdust will be hydrated in a large plastic container a mixture of 40% water and 60% sawdust. Once the sawdust is hydrated it will be allowed to continue absorbing water for one hour. Finally the additives (1% gypsum, 20% rye berries) will be added.
All sawdust substrate will be sterilized at 20 PSI for 2 hours and 30 minutes. Sterile transfers will be done from the mason jars to the grow bags using the technique described above. Bags will be allowed to colonize for two to four weeks before being taken into the fruiting green house.
In the greenhouse the bags will be injected with 300 ml of cold water at a temperature of 40C. The greenhouse will maintain a relative humidity of 99% to 95% electronically controlled by an ultrasonic humidification system. The temperature will only be partially controlled, exposing the samples to the natural temperature cycles of the eastern panhandle of West Virginia. A evaportave cooler that keeps the room from exceeding temperatures above 25C. Temperatures will be recorded and logged electronically via a DHT-22 temperature and relative humidity sensor.
In the fruiting greenhouse the grow bags will be be cut into with a sterilized blade. The cuts will be one cm in length, and be three per bag. Photos of the grow bags will taken daily with a digital camera to record the fruiting process. When the mushrooms have completely fruited they will be harvested by hand by twisting the base of the mushroom away from the bag. The mushroom fruiting bodies will be weighed fresh on a digital scale. The biological efficiency (BE) of the grow will be determined using Stamens (2000).
After computing the BE for each strain and substrate pairing, we will be deduce the commercial viability of each strain and substrate type.
For each strain we grow out on petri dishes we will additionally preform a PCR test using an OpenPCR (OpenPCR Inc) machine to test for the existence of previously identified PCR Primer Sequence in the DNA of our samples. Samples will be transferred and cleaned in .5ml sample tubes. DNA will be extracted using Borges et al. (1990) using a lysis buffer for suspension. For our purposes we will be using the following previously identified PCR primer chains ITS1, ITS2, ITS3, and ITS4. Our PCR . Once we have obtained confirmation of successful PCR amplification process the samples will be sent to a 3rd party lab to preform DNA sequencing via the fluorescent dye-terminator method.
The second objective is to collect wild strains for further research and study. We will place various ads in physical publications as well as social media to alert people of our project. People will be instructed to send us wild mushroom samples of COTW along with the location they found them,
Once we have isolated the wild sample on PDA petri dishes, we will then transfer them to agar slants in 50ml test tubes. These slants will contain the PDA solution used above as well as a hardwood piece of wood. The hardwood PDA slants will then be stored at ~40F for later trials of COTW.
To begin our project we first created 100 plates of potato dextros agar (PDA) growth medium were prepared using a ratio of 1:25 PDA to distilled water. The PDA medium was sterilized in a pressure cooker for 30 minutes at 15 PSI. When the grown medium was measured to have cooled to 121°F it was transferred petri dishes in front of a laminar flow hood. The petris were then sealed with parafilm and sealed in a plastic bag for later use.
Two commercially available liquid culture syringes were acquired and cloned to a sterile PDA medium prepared earlier. The cultures were cloned under a laminar flow hood using sterile technique. Approximately .1 ml of liquid culture was applied to each petri dish. The dishes were then closed with paraflim and set in the incubation room where they could be observed. The first sample contaminated with a green contamination, most likely trichoderma. The second petri achieved full colonization for later use.
To facilitate grain spawn production six 1-quart wide-mouth mason jars and fourteen 5lb bags were filled with fully hydrated (60% water) rye berries. The rye berries were prepared by soaking them in water for 24 hours to germinate endospores that may potentially be in the rye. Then the rye berries were boiled for 10 minutes and then allowed to air dry in a strainer. Once the rye berries have cooled to 75°F they were loaded into the bags and mason jars. The bags were sterilized in a pressure cooker for 2 hours at 15 PSI. The jars were sterilized for 1 hour at 15 PSI.
Using the prepared grain spawn the commercially available sample of colonized COTW was cloned to a sterile mason jar of rye berry grow medium. A sterile scalpel was used to take a tissue sample from the colonized petri dish and place it into the jar of rye berries. When the jar was fully colonized it was divided into six bags of rye berry grow medium under a laminar flow hood using sterile technique.
Twenty-two samples of COTW were collected from the public, these samples were cloned in our laboratory under a laminar flow hood following sterile technique. The samples were washed with 70% isopropyl alcohol solution before being taken under the laminar flow hood to be cloned. Once washed the sample was broken by hand into two parts, the first part is discarded while the second part is held under the airflow from the hood. Using a sterile scalpel a 1mm biopsy is made to the inside of the COTW sample that has been exposed to the flow hood after breaking the sample into two parts. The tissue biopsy is placed into the prepared petri dishes of PDA grow medium . The samples took one week to show signs of growth and four weeks to fully colonize the PDA medium. Eight of our samples contaminated with unidentified fungus or bacteria.
Using the same technique used for cloning the commercial samples 5 grain jars and 8 bags of grain spawn were produced using biopsies of the wild samples.
In summary at our current stage of our project we have successfully cloned and colonized 15 petri dishes of COTW on PDA, and 5 slants on PDA for longer term storage. We have brought 6 jars of COTW on rye berries to first generation colonization, and 14 bags of COTW on rye berries to second generation colonization. We have colonized COTW on sawdust substrate with 6 samples producing sclerocia (pre-fruiting body formation). We have seen contamination on 37% of our Volunteer Forager clones. Samples have been prepared for PCR testing taking place in year two.
Education & Outreach Activities and Participation Summary
Produced a one page flyer to inform the public about the project and solicit samples of COTW. These were displayed at our weekly farmers markets as well as at Peach Fest (Romney WV), Sunflower Fest (Winchester VA).
Farmers involved with the project were encouraged to discuss the project and its intended outcomes with the public at length when attending events.
A press release to inform the public about the project was shared via the Hampshire Review, The Mushroom Growers Newsletter, West Virginia Farm Bureau Newsletter, Cornell Small Farms Program email list, and to the WV Extension network via our grant advisor.
Instagram and Twitter were used to promote the study and the collection of samples as well. Three promoted ads ran on Instagram reaching 5,537 unique accounts.
Six farmers have reached out expressing interest in our results.
Our farm plans on commercially harvesting COTW pending success of this project.