Sarah Choi & Neelah Hassanzadeh
Age 17 | Langley, British Columbia
Canada-Wide Science Fair Excellence Award: Senior Silver Medal | Dalhousie University $2,500 Entrance Scholarship | UBC Science $2,000 Entrance Award | University of New Brunswick Canada-Wide Science Fair $2,500 Scholarship | University of Ottawa $2,000 Entrance Scholarship | Western University $2,000 Entrance Scholarship
INTRODUCTION
The common synthetic fertilizer’s ability to increase nitrate levels is mitigated by the leaching of nitrate into aquatic ecosystems and waterways; this contamination eventually causes eutrophication and pollutes groundwater (Dubey & Townsend, 2004). Synthetic fertilizer application has also been a contributor to increased nitrous oxide emissions (Sanders, 2012). A novel solution is using black soldier fly larvae (BSFL) frass as organic fertilizer.
Black soldier flies are native to North America, and their frass —the solid excrement of insect larvae—contains the only plant-digestible form of chitin. This chitin naturally produces antimicrobial peptides when under environmental stress (Sistrunk, 2016), acting as a protective barrier. The chitin does this by making mineral nutrients inaccessible to pathogens as well as by blocking the release of pathogenic mycotoxins (Wan, Zhang, & Stacey, 2008); this capability has been proven to be highly beneficial for insects, although it is not yet fully understood in its effects for plants (Behie & Bidochka, 2013). Frass also contains nitrifying bacteria and nitrogen-fixing bacteria, which partake in the nitrogen cycle and assist in the plants’ uptake of nitrogen (Behie & Bidochka, 2013). It has been shown that there are more greenhouse gases, like nitrous oxide, in areas where more synthetic fertilizer is applied to the soil (Hawkinson, 2005), whereas BSFL frass can store carbon and nitrogen in the soil (Lovett et al., 2002). BSFL frass also prevents atmospheric loss of nitrogen and groundwater contamination through the nitrogen fixation by the bacteria (Lovett et al., 2002). The atmospheric nitrogen cannot be directly assimilated by plants, which is why there must be a nitrogen fixation process by bacteria to make the nitrogen available for plant uptake. In frass, bacteria like Bacillus and Pseudomonas help fix atmospheric bacteria while other nitrifying bacteria make nitrogen within the soil accessible to plants for photosynthesis (Zahn, 2017). Nitrifying bacteria convert nitrogen in its ammoniacal form its nitrate form, which allows for more efficient root uptake by the plant (Alattar, Alattar, and Popa, 2016). The nitrifying and nitrogen-fixing bacteria are critical to horticultural production because fixed nitrogen is a limited nutrient in most environments (Zahn, 2017). Furthermore, this assimilation of nitrate to plant roots (Figure 1) makes soils more resilient to floods, droughts, and land degradation processes.
Figure 1. Impact of nitrifying and nitrogen-fixing bacteria in frass on the nitrogen cycle.
Frass also consists of safer chemicals for human exposure, whereas synthetic fertilizers often are composed of harmful substances such as cadmium, uranium, and arsenic, all of which are carcinogens as well as triggers for developmental problems in children (Sharma & Singhvi, 2017). In addition to all these beneficial components, if BSFL frass possesses defensive properties for plants, it could be a viable alternative to chemical fertilizers for plant growth. This study aims to determine if BSFL frass defends against plant pathogens, thereby increasing soil fertility. Although chitin has been shown to provide positive effects for insects (Zahn, 2017), this study aims to solidify this role of chitin and bacteria in the protection of plants specifically, and frass’s benefits as a fertilizer. The first objective of this study is to investigate whether common fungi, Rhizoctonia solani and Fusarium oxysporum, will grow in frass. Secondly, this study examines the effects of frass as a fertilizer and as a protective barrier against the common pathogen, Pythium myriotylum. This study also examines a combination of frass with other organic fertilizers, such as Trichoderma, which is a non-pathogenic fungus that stimulates plant growth and nutrient uptake, and humic acid, which raises the nutrient-holding capacity of soil (Gonsalves & Ferreira, 1993). The addition of other organic fertilizers will allow for a comparison between frass and these more common fertilizers, in addition to an assessment of their combined effects.
HYPOTHESIS
It is our hypothesis that the treatments with frass will show no bioaccumulation of pathogens in the petri dishes and will demonstrate better plant health and growth, whereas control groups will have little to no positive effect.
METHODS
Part I: Transmission of Disease to BSFL Frass
Culturing the Plant Pathogens: A 7 mm plug of Fusarium oxysporum was obtained from Kwantlen Polytechnic University. The plug was transferred to a petri dish with potato dextrose agar (PDA) medium in a microbiological safety cabinet to culture fungi; this process was repeated for Rhizoctonia solani. Five petri dishes were set up for each fungus and stored in an incubator at 27°C for one week.
Retrieving Frass: Sixteen plastic cups were filled with 20 BSFL: eight for both F. oxysporum and R. solani, with four cups receiving the regular fruit and vegetable diet without fungus (control), and four cups with the diet containing fungus. The amount of diet varied per day based off the Enterra diet chart for BSFL larvae. The BSFL received food every two days. After a week, frass was collected from the cups and placed into 16 petri dishes with PDA. The petri dishes were stored in an incubator at 27°C for one week.
Part I was repeated for a second trial to ensure precision in the results.
Part II: Growth of Valentino Green Bush Bean Plants with Frass
Part IIa: Arranging Plants for Fertilizer Trial (no Pythium)
Valentino green bush bean plants were used for Part II and were obtained as seeds. Nine different treatment groups of fertilizers (Table 1) were tested, with five replicates per treatment. All individuals were planted in pots (4 in tall) with sterile soil. Treatments were mixed with 300 mL of water and given to the plant. Each individual was given approximately 20 mL of water every three days. For 56 days, all plants were given 8 hours of light and then 16 hours of darkness each day, which is a standard light/dark interval for plant studies (Shin, Song, & Thompson, 2011). They were held in an incubation room set to 25°C.
Table 1. An outline of the composition of each treatment groups for Parts IIa and IIb.
Part IIb: Arranging Plants for Disease Trial (with Pythium)
Five different treatment groups of fertilizers with Pythium myriotylum (Table 1) were tested, with five replicates per treatment. The cultured fungus was scraped using a lab spatula and mixed evenly throughout the soil for the disease groups before the plants were potted. All individuals were planted in pots (4 in tall) with sterile soil and were given 300 mL of water. Each individual was given approximately 20 mL of water every three days. Each disease group had one whole plate of P. myriotylum (90 mm diameter). For 56 days, all plants were given 8 hours of light and then 16 hours of darkness.
All water used was processed through a reverse osmosis (RO) filtration system. For both the disease and fertilizer trials, heights of the plants were recorded every fourth day. Wet and dry biomasses, number of leaves, and width of leaves were tested after 56 days. The pH and electrical conductivity of the soil were tested using a multiparameter probe, and the nitrate concentration was tested using a nitrate meter.
RESULTS
Part I: Transmission of Disease to BSFL Frass
Both trials demonstrated that the F. oxysporum and the R. solani fungi did not grow in frass. In the first trial, after about a week, there was only frass in the plate with no growth of anything observed. In the second trial, a grey fluffy substance had grown. After further analysis using PCR, it was determined that the substance was not linked to Fusarium nor Rhizoctonia, and it was also not a plant pathogen.
Part II: Growth of Valentino Green Bush Bean Plants with Frass
Biomass and Heights
After 56 days in both the fertilizer and disease trials, the treatments with frass showed higher heights and biomasses compared to both the control and the treatments with the chemical fertilizer (Figures 2 and 3). In the fertilizer trial, the treatment of frass and humic acid showed the highest mean height of approximately 61.02 cm (Table 2). In the disease trial, the treatment of the combination of frass, humic acid, and Trichoderma showed the highest mean height of 52.34 cm. The treatment with the greatest dry biomass was the treatment of frass, humic acid, and Trichoderma, with a mean dry biomass of 13.18 g. Treatments containing frass improved plant biomass with disease by five-fold in comparison to the positive control. A Tukey-Kramer test showed that both biomass and height data for fertilizer and disease trials demonstrated significant results (p < 0.05).
Figure 2. Plants’ growth in height from Part IIa.
Figure 3. Plants’ growth in height from Part IIb.
Nitrate Concentration, pH, and Electrical Conductivity
In the fertilizer trial, the nitrate concentration was highest in the treatment of frass and humic acid, with 224 ppm (Table 2). In the disease trial, the treatment with frass, humic acid, and Trichoderma had the highest mean nitrate concentration of 228 ppm, and the following treatment was only frass with a mean nitrate concentration of 212 ppm. A Tukey-Kramer test showed that both of those treatments were found to be in the same statistical group, indicating that the two treatments both increased nitrate in similar amounts and are not significantly different. Treatments with frass had higher mean nitrate amounts compared to soil with no treatment. The optimum pH of 6.5 to 7 for bacterial populations (Perry, 2003) was achieved for all treatments with frass, while all other groups without frass, such as the control group, had pH values less than 6.5. The optimum EC is 0.6 to 0.7 mS/cm according to Behie & Bidochka (2013). In comparison to the soil with disease, which had the electrical conductivity (EC) of 0.362 mS/cm, the soil with disease and treatments with frass had mean EC values ranging from 0.658 mS/cm to 0.662 mS/cm.
Table 2. Collection of data from all trials and statistical analysis of nitrate concentration for Part IIb. F = frass, H = humic acid, T = Trichoderma, C = chemical fertilizer.
DISCUSSION
This study demonstrates that BSFL frass does not transmit disease and defends against it, thereby a consistent addition of frass to soil can contribute to the prevention of fungal disease from pathogens like Rhizoctonia, Fusarium, and Pythium. When testing for the transmission of disease in Part I, there was no growth of R. solani or F. oxysporum observed in either of the two trials, suggesting that the disease from the fungi (in the diet) was metabolized by the larvae and converted into safer chemicals for the environment. Thus, it is our interpretation that BSFL frass can be safely used as organic fertilizer without it being contaminated.
Part II of the study further demonstrated the beneficial impacts of frass on plant health against Pythium. Pythium blight causes highly destructive turfgrass disease, which is one of the most common reasons for crop failure. While this condition was not observed in any pots with treatments including frass, some pots from the positive control showed the appearance of the disease; this confirmed our hypothesis regarding frass’s ability to prevent bioaccumulation of the pathogen. Figure 3 shows that the heights were greater for treatment groups with frass, suggesting that immune receptors within the chitin successfully blocked pathogens from harming the plant (Zahn, 2017). The addition of different organic fertilizers with frass was effective as frass allows carbon dioxide and nitrates to stay in the soil (Lovett et al., 2002) (which allows the plant to access nutrients) while the humic acid enables the soil to have a greater capacity of holding water (Gonsalves & Ferreira, 1993). This improved capacity for accessing nutrients and moisture may explain the increased biomasses and heights for treatments including frass. These results comply with the theory that there is a positive relationship between biomass and the production of heavier and healthier fruits (Ceresini, 1999). The greater amount of nitrogen in the treatments containing both frass and humic acid compared to the treatment with frass demonstrates the ability of humic acid to improve the nitrogen uptake by plants. Treatments with frass also had more optimal pH ranges, which is important for beneficial bacterial populations in frass to develop and promote plant growth (Perry, 2003). The pH results correspond with the high amounts of nitrate in the treatment groups with frass, because a greater amount of nitrifying and nitrogen-fixing bacteria allows more nitrogen to be absorbed by the plant. Since all treatments with frass had higher nitrate amounts compared to soil with no treatment, it was shown that frass was able to increase nitrogen content in soil.
CONCLUSION
Our study can conclude that frass can improve plant growth without the transmission of plant-pathogenic disease from compost waste diet. Not only does frass improve soil fertility and defend against pathogens, but it does so without harming the environment. Furthermore, frass is available on the market for consumers at a lower, more competitive price than competing conventional synthetic fertilizers (Zahn, 2017). BSFL frass is inexpensive to mass produce and consume because of how easily available it is throughout the year, allowing for mass quantities of larvae to be bred in a controlled environment. Frass also contains safer chemicals for human exposure. In addition, the production of frass occurs in a cycle where waste crops such as fruit can be used to feed the BSFL that generate the frass, then that frass can then be used in the production of new crops. Therefore, the production method is more self-sustaining and economically advantageous than that of synthetic fertilizers which use many non-renewable resources, such as fossil fuels (Woods, Williams, Hughes, Black, & Murphy, 2010). Thus, BSFL frass is safer for the consumer, less expensive, more effective, environmentally sustainable, and can be applied less frequently for a long-lasting effect so that consumers do not have to contribute much time to its usage. By using this organic fertilizer that can defend against plant pathogens, the frass will not only help with providing essential nutrients for the plant in a sustainable way, but also act as a substitute for synthetic fertilizers that pollute the environment.
ACKNOWLEDGEMENTS
We would like to thank Kwantlen Polytechnic University for providing support and facility for this project.
REFERENCES
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SARAH CHOI
I am a senior student at R.E. Mountain Secondary School interested in research and biological studies. This year was my first time competing in the Canada-Wide Science Fair with my project about how a North American species (Black Soldier Fly) of insect frass can be used as a safe and effective biofertilizer for many crops, providing benefits to both the plant and the environment - all with a production process that is economically advantageous and self-sustainable through the recycling of food compost wastes. I was able to conduct my trial as a research intern in the Institute of Sustainable Horticulture at Kwantlen Polytechnic University in Langley, BC. I have worked in this lab since September of 2017, and my experiences there and at the national science fair sparked my interest in biology and research, which I hope to further pursue in the future. Along with my partner, I was honoured to receive a silver medal at the Canada-Wide Science fair, the NSERC Young Innovator award, and the Association of Professional Biology Provincial (BC) Award for the highest BC standing with a biology project.
NEELAH HASSANZADEH
My name is Neelah Hassanzadeh and I am a senior highschool student. I started volunteering at the Institute of Horticulture (KPU) in June 2018 and continued to work at ISH throughout the year. Sarah and I decided to start our own project about Black Soldier Fly Larvae frass as an organic fertilizer that is able to defend against harmful pathogens in October 2018. We worked on this project under the supervision of Dr. Sepideh Tahriri Adabi, a research scientist at ISH. Now, I continue to work as a research assistant at ISH and aspire to become a nurse.