Progress report for LS22-367
Project Information
The rapidly growing global population and expansion in the agriculture sector has resulted in the generation of large amount of agricultural wastes which are rich in lignocelluloses.. Biological recycling strategy to increase resource and efficiency while maintaining ecological sustainability is a potential solution. In this application, we propose interdisciplinary systems research approaches through utilization of mushroom to convert the lignocellulosic biomass to fruiting bodies with high nutritional quality and medicinal value against many ailments including gastrointestinal nematode (GIN) parasites. Furthermore, spent mushroom substrate (SMS) remaining after mushroom harvest has high levels of organic matter and minerals and possess a great potential to be utilized in agricultural and horticultural sectors and further contribute to reduce the use of non-renewable resources. White-rot fungi, one type of edible mushrooms, is proposed to serve as a pretreatment tool for delignification by using agricultural residues as the substrate biomass resources. The mushroom enzymatically converts the substrate or compost into mushroom that has multiple uses as food and medicine in humans and against gastro intestinal nematode parasites in livestock. The SMS recovered can be used as feed, organic fertilizer and soil remediation to supply nutrients and increases the water-holding capacity. The proposed work provides an opportunity for research and extension to develop and champion adoption of sustainable production practices tailored to the needs of small and limited resource farmers to recycle nutrients from abundantly available resources back into value added products. The findings expect to lay the ground for more research to establish sustainable bio recycling and valorization of agricultural waste in the southern United States and the nation at large
1. Evaluate physiochemical and nutritional quality of mushroom, substrate, and spent mushroom substrate.
2. Evaluate application of spent mushroom substrate as soil fertilizer for cultivation of horticultural crop.
3. Determine the ability of mushroom and spent mushroom substrate as a nematicide against the gastrointestinal nematode parasite Barber Pole worm in small ruminants.
Cooperators
Research
Cultivation of Oyster mushroom on crop residues for use as a nematicide against Haemonchus contortus in small ruminants
Obtention of crop residue
Sufficient quantities of crop residues and grass hays used in the cultivation of Oyster mushroom (Pleurotus ostreatus) were collected from the Randolph Research Farm of Virginia State University (VSU) located in the Tri-Cities area of Central Virginia (37.1°N; 77.3°W) at an elevation of 45 m above sea level. The agricultural residues were stored in big plastic bags and stored in a cold dark room (temperature 10 ± 5 °C, relative humidity 20–30%) until used.
Substrate preparation
Collected sun-dried residues and grass hays were ground with a cyclone hammer mill (~2 cm). These substrates were then soaked separately in water overnight to ensure they absorbed sufficient moisture and were allowed to dry to obtain an average moisture content of 65%. Substrates filled in autoclavable bags were pasteurized at 121 °C and 15 psi for 20 min, prior to inoculation with spawn.
Spawn Cultivation: The culture of oyster mushroom (Pleurotus ostreatus) was received from an organic commercial farm and further maintained on wheat grain medium - 17 °C. Cultivation of spawn was done in a temperature and humidity controlled growth chamber.
Mushroom cultivation and harvesting
After pasteurization, substrate-filled bags with micro holes in them for respiration were inoculated with grain spawn with a weight percentage of about 1% of the wet weight of substrate. These bags were placed on shelves in a room for cultivating mushroom where temperature is maintained at 25 ± 2 °C with a thermostat. Oyster mushroom was cultivated on corn stover, edamame straw, Switch and Timothy grass hays. When the bags became white due to colonization by fungal mycelium, visible beneath the transparent polythene sheets, the bags are poked with holes to induce the fruiting body formation. The temperature and humidity (80–90%) of the growing room was maintained by a humidifier and occasional spraying of water in the morning and afternoon. Fruiting bodies developed were harvested in two flushes from each bag. The mushroom cultivation process which will be completed soon.
Chemical analyses of mushroom, substrates and spent mushroom substrate
The fruiting body samples harvested at 2nd flush will be freeze dried. The dried materials will be ground and subsampled for analysis. Total proteins (%), total carbohydrates (%), fats (%), crude fiber (%), and ash (%) will be estimated following standard procedure of AOAC. The difference 100-(% Moisture +% Crude protein +% Crude fat +% Crude fiber +%Ash) will be used for the estimation of total carbohydrate.
Preparation of oyster mushroom extract: The freeze dried oyster mushroom samples will be ground to a powder using a laboratory grinder. The ground sample (170 g) will be extracted with both ethanol and water and the extracts evaluated for their anthelminthic activity
Haemonchus contortus egg recovery: Donor goats selected from VSU research goat herd will be harnessed and feces collected. Fecal egg reduction will be confirmed by fecal egg count using the McMaster egg counting technique. Samples of fecal eggs recovered will be processed using wash, centrifugation and filtration.
Egg hatch assay: Recovered eggs will be washed twice in saline and the concentration of eggs determined by McMaster assay. Hundred eggs/well will be distributed in 48 wells culture plates. Four different concentrations of Oyster mushroom ethanol or water extracts will be added to triplicate wells. Pure Albendazole (99.8%) (Sigma, USA) dissolved in dimethyl sulfoxide (DMSO) will be used as a positive control while water and ethanol will be used as negative controls. The plates will be incubated at 22-23 °C in a humidified incubator (85-90%) with some modification. Incubation will be done for 72 hours. The number of hatched larvae will be counted in each well and the percentage hatch for each treatment and control wells will be calculated.
Larval development and viability assay: Recovered eggs will be hatched to first stage larval without addition of the mushroom extract. Larval killing assay will be evaluated by washing hatched larvae in normal saline and transferring them to triplicate wells containing concentrated (10X) Earl’s salt solution (Sigma Aldrich) supplemented with yeast extract. Appropriate control wells containing Earl’s medium, purified water, ethanol, Albendazole and Ivermectin will also be included in the assay. Plates will be incubated for seven days in conditions described above and monitored daily for larval viability or degradation by microscopic analysis using a compound microscope. Viability will be evaluated using observed movement/motility as seen under a compound microscope with and without shaking. At the end of seven days, live, dead and degraded larvae will be counted and percentage mortality calculated for each well.