Abstract
Larval frass from insects which consists of larval excrement, exoskeleton, and undigested diet, is a rich source of organic material and microorganisms. Despite its potential value, research on frass valorisation in agriculture is limited. In this study, single-layer agar (SLA) and double-layer agar (DLA) in vitro bioassays were conducted to evaluate the effect of water-based black soldier fly larvae (BSFL) frass extracts from two different diets on the growth of six plant pathogens: Alternaria solani, Botrytis cinerea, Fusarium oxysporum, Phytophthora capsici, Rhizoctonia solani and Sclerotinia sclerotiorum. The results showed that frass extract from Gainesville house fly diet strongly or completely inhibited the growth of all tested plant pathogens in both SLA and DLA bioassays, while frass extract from fruit/vegetable/bakery/brewery diet strongly inhibited the mycelial growth of A. solani, B. cinerea, and S. sclerotiorum, and moderately inhibited the mycelial growth of P. capsici in both bioassays. For both diets and bioassays, 0.22 μm microfiltered frass extracts which are free of microorganisms showed generally no effect on the growth of the pathogens indicating that growth inhibition is caused by frass-inhabiting microorganisms. Both SLA and DLA bioassays revealed strong antagonistic effect of microorganisms inhabiting BSFL frass against the plant pathogens B. cinerea, A. solani, R. solani, P. capsici, F. oxysporum and S. sclerotiorum. Moreover, the study showed the impact of the BSFL diet on the antagonistic effect of frass extract. In future work, the antagonistic effect of frass extracts against the above-mentioned pathogens will be tested in vivo. BSFL frass could eventually find applications for the control of plant diseases.
1 Introduction
Significant losses of horticultural products are caused by a multitude of pathogens and represent a major challenge in both open-field and greenhouse growing systems (Savary et al., 2012). Horticultural crops are susceptible to a wide range of fungal pathogens against which synthetic fungicides are widely used. Synthetic fungicides have multiple drawbacks such as lack of long-term efficacy and potential adverse effects on human health and the environment (Aktar et al., 2009; Mahmood et al., 2016). In this context, environmentally friendly approaches for efficient pre- and post-harvest disease management are required (Niu et al., 2020). Among the different environmentally friendly approaches, biocontrol of plant pathogens via microorganisms with antagonistic activities is an attractive option.
Hermetia illucens commonly known as the black soldier fly has great potential to efficiently convert organic matter into a high-value source of protein and fat that provides sustainable solutions for both organic waste management and food security (Kim et al., 2021; Singh and Kumari, 2019). Frass is the remaining biomass from growing black soldier fly larvae (BSFL); frass consists of undigested food waste, larval excrement, and chitin from larval molting. Larval frass is a rich source of organic material that makes it a valuable amendment to improve soil fertility (Barragán-Fonseca et al., 2022; Lopes et al., 2022). Studies reported the potential of BSFL frass to replace commercial fertilizers in horticultural crops as sweet potato (Ipomoea batatas) (Romano et al., 2022) and maize (Zea mays) (Beesigamukama et al., 2020). Chitin-based products were reported to improve crop yields by promoting plant growth (Sharp, 2013). Among the mechanisms involved, the capacity of chitin to induce plant defense responses against biotic stress was pointed out (Sharp, 2013). Insect frass was reported to stimulate systemic resistance in plants due to the presence of beneficial microbes or eliciting molecules as chitin (Barragán-Fonseca et al., 2022; Poveda, 2021; Schmitt and de Vries, 2020). Besides improving soil fertility and plant growth/health, larval frass is a rich source of microorganisms (Bruno et al., 2019; Gold et al., 2020; Jiang et al., 2019; Wynants et al., 2019) that could have antagonistic activities against plant pathogens. Currently, few studies have investigated the antagonistic effect of frass on plant pathogens.
In this study, single-layer agar (SLA) and double-layer agar (DLA) in vitro bioassays were conducted to evaluate the effect of water-based frass extracts from two different diets on the growth of six tomato (Solanum lycopersicum) plant pathogens [Alternaria solani (the causal agent of early blight), Botrytis cinerea (the causal agent of grey mold), Fusarium oxysporum (the causal agent of Fusarium wilt), Phytophthora capsici (one of the causal agents of buckeye rot), Rhizoctonia solani (the causal agent of Rhizoctonia root rot) and Sclerotinia sclerotiorum (the causal agent of Sclerotinia rot); MAPAQ, 2023]. Control of these plant pathogens is mainly based on the use of synthetic pesticides registered in Canada, good cultural practices, and tolerant cultivars (Health Canada, 2023; MAPAQ, 2023).
2 Materials and methods
Diets
Two diets were employed in this study: Gainesville house fly diet (GV) at 70% humidity (used as a reference diet; Hogsette, 1992) and a complex fruit/vegetable/bakery/brewery diet (FVBB) at 70% humidity. GV composed in fresh weight of 50% wheat bran, 30% alfalfa meal and 20% cornmeal. FVBB composed of 39% fruits (5% tomato, 7% orange, 3% apple, 7% bell pepper, 5% pineapple, 2% strawberry, 2% cantaloupe, 2% pear, 2% banana, 2% grape and 2% lemon), 36% vegetables (10% lettuce, 5% potato, 3% cabbage, 2% onion, 3% celery, 3% leek, 2% cauliflower, 3% broccoli, 3% carrot and 2% corn), 15% bread and 10% spent brewer’s grains. They were all fresh fruits, vegetables and bread purchased in March 2021 from a local supplier (Tout Prêt Inc., Sainte-Foy, Québec, QC, Canada) and then shredded (<2 mm) using an industrial food processor (Rietz disintegrator, model: RA2-8-K322; Bepex Company, TX, USA) and stored at −30 °C until required. Both diets have been previously shown to support high larval growth and high bioconversion rates of organic feedstock in larval biomass (Arabzadeh et al., 2022).
Frass
The fly colony was maintained at the Laboratoire de recherche en sciences aquatiques (LARSA) of Université Laval (Québec, QC, Canada). For the production of larvae, adult black soldier fly that emerged from a common cohort of pupae were kept in a knitted nylon mesh insect cage (650 μm opening; BugDorm Insect Rearing Cage Model 4M3030, MegaView Science Co., Ltd., Taichung, Taiwan) under lighting to induce mating. Fluted cardboard strips (2 cm × 3 cm) were placed over fermenting chicken feed as an attractant. Cardboard strips containing eggs clutches were removed daily from the cages and suspended above 160 g (wet weight) of each diet in containers (17.5 cm × 19 cm) covered with a double horticultural netting (Novagryl P-30 horticultural mesh, Dubois Agrinovation, Saint-Rémi, QC, Canada) for 24 h. The cardboards were then removed, and the containers were placed in an incubator [27 °C, 70% relative humidity, and fluorescent lighting with a photoperiod of 12:12 (L:D)] for 5 days. The substrate of the larvae was uniformly moistened during the first four days using a fine mist spray bottle. Following hatching, the samples were transferred onto a sieve (mesh opening of 2 mm) and placed over an empty container. A bright light was positioned above the container to encourage larvae to move away from the surface of the substrate and pass through the sieve. Thereafter, 800 five-day-old larvae were manually counted and transferred to each of the three round polypropylene containers (17.5 cm diameter, 19 cm height) with 800 g of each diet previously hydrated to 70% humidity. The rearing of BSFL lasted 10 days (40% of the larvae had transitioned into the prepupal stage by this time); frass was sampled by sieving (4 mm; Fisher Scientific co., MA, USA) and immediately used for SLA and DLA bioassays. Frass extracts were prepared by mixing fresh frass (10 g) in 100 mL of sterile physiological saline solution (0.5% NaCl) under agitation (150 rev/min, 27 °C) for 60 min followed by filtering through 8 layers of sterile cheesecloth (Arabzadeh et al., 2022).
Plant pathogens
Plant pathogens (A. solani, B. cinerea, F. oxysporum, R. solani, S. sclerotiorum and P. capsici) tested in this study were graciously provided by the Laboratoire d’expertise et de diagnostic en phytoprotection (MAPAQ, Québec, QC, Canada). They were cultured at room temperature (22.5 °C) on potato dextrose agar (PDA; Becton, Dickinson and Company, Sparks, MD, USA; 39 g/L of distilled water).
Schema of single-layer and double-layer agar bioassays.
Citation: Journal of Insects as Food and Feed 10, 10 (2024) ; 10.1163/23524588-20230022
Single-layer agar bioassay
The schema describing the SLA bioassay is shown in Figure 1. Frass extract from GV diet or FVBB diet [0.22 μm microfiltered (to remove microorganisms) or not] or sterile physiological saline solution (control) was incorporated to warm (48 °C) PDA at the final concentration of 1% (w:v, frass:PDA). PDA (20 mL) containing either frass extract, microfiltered frass extract, or sterile physiological saline solution was poured into Petri dishes (100 mm × 15 mm; VWR International, Mississauga, ON, Canada). PDA plug (10 mm diameter) covered with actively growing mycelium of each pathogen tested was placed in the center of the agar plate. After an incubation period of 7 days in darkness at room temperature, the mycelial radial growth of each pathogen was measured in mm with a ruler as the average of four diameters of the thallus. Mycelial disk diameter (10 mm) was not considered in the diameter of the radial growth. The experiment was conducted as a completely randomized design with three replicates; a Petri dish being the experimental unit.
Double-layer agar bioassay
The schema describing the DLA bioassay, also known as dual-culture overlay assay, is shown in Figure 1. Frass extract from GV diet or FVBB diet [0.22 μm microfiltered (to remove microorganisms) or not] or sterile physiological saline solution (control) was incorporated to warm (48 °C) PDA 10% (3.9 g/L of distilled water) supplemented with 13.5 g/L of agar (CRITERION™ Agar, Hardy Diagnostics, Santa Maria, CA, USA) at the final concentration of 1% (w:v, frass:PDA). PDA 10% (20 mL) containing either frass extract, microfiltered frass extract or sterile physiological saline solution was then poured into Petri dishes (100 mm × 15 mm). This first layer was incubated (obscurity) at room temperature for 48 h. A second layer (10 mL) of PDA 10% supplemented with 13.5 g/L of agar was poured onto the first layer. The Petri dishes were then inverted and incubated (obscurity) at 4 °C for 24 h. After incubation, a PDA plug (10 mm diameter) covered with actively growing mycelium of each pathogen was placed in the center of the agar (on the second layer) plate and Petri dishes were incubated for 7 days in darkness at room temperature. Mycelial radial growth of each pathogen was measured in mm as previously described. The experiment was conducted as a completely randomized design with three replicates; a Petri dish being the experimental unit.
Statistical analysis
Analyses of variance (ANOVAs) were performed on the data using R (R Core Team, 2021). Treatment means were compared using Tukey’s test (
3 Results
Unfiltered frass extracts from GV diet and FVBB diet strongly (inhibition rate > 60%) inhibited the mycelial growth of A. solani as compared to the control in both SLA and DLA bioassays (Figure 2). In both bioassays, unfiltered frass extract from GV diet inhibited more strongly the mycelial growth of A. solani as compared to frass extract from FVBB diet (Figure 2). Filtered frass extracts showed no effect on the growth of A. solani (Figure 2).
Effect of 0.22 μm filtered and unfiltered frass extracts from Gainesville diet (GV) and fruit/vegetable/bakery/brewery waste-based diet (FVBB) on mycelial growth of Alternaria solani according to single-layer agar (a) and double-layer agar (b) in vitro bioassay. Each value represents the mean of three replicates ± standard deviation. Means with the same letter are not significantly different according to Tukey’s test (
Citation: Journal of Insects as Food and Feed 10, 10 (2024) ; 10.1163/23524588-20230022
The effect of frass extracts on the mycelial growth of B. cinerea is presented in Figure 3. As for A. solani, unfiltered frass extracts from GV diet and FVBB diet strongly inhibited the mycelial growth of B. cinerea as compared to the control in both SLA and DLA bioassays. Filtered frass extract from GV diet was shown in the DLA bioassay to significantly inhibit the growth of the fungus (Figure 3b).
Effect of 0.22 μm filtered and unfiltered frass extracts from Gainesville diet (GV) and fruit/vegetable/bakery/brewery waste-based diet (FVBB) on mycelial growth of Botrytis cinerea according to single-layer agar (a) and double-layer agar (b) in vitro bioassay. Each value represents the mean of three replicates ± standard deviation. Means with the same letter are not significantly different according to Tukey’s test (
Citation: Journal of Insects as Food and Feed 10, 10 (2024) ; 10.1163/23524588-20230022
Mycelial growth of S. sclerotiorum was strongly (SLA bioassay) or completely (DLA bioassay) inhibited by unfiltered frass extracts from GV diet and FVBB diet (Figure 4). Unfiltered frass extract from GV diet was shown in the SLA bioassay to inhibit more strongly the mycelial growth as compared to that from FVBB diet. Filtered frass extracts, except frass extract from GV diet in DLA bioassay (weak increase of mycelial growth), showed no effect on the growth of S. sclerotiorum (Figure 4).
Effect of 0.22 μm filtered and unfiltered frass extracts from Gainesville diet (GV) and fruit/vegetable/bakery/brewery waste-based diet (FVBB) on mycelial growth of Sclerotinia sclerotiorum according to single-layer agar (a) and double-layer agar (b) in vitro bioassay. Each value represents the mean of three replicates ± standard deviation. Means with the same letter are not significantly different according to Tukey’s test (
Citation: Journal of Insects as Food and Feed 10, 10 (2024) ; 10.1163/23524588-20230022
Unfiltered frass extracts from both diets significantly reduced P. capsici mycelial growth in SLA and DLA bioassays (Figure 5). Unfiltered frass extract from GV diet strongly or completely inhibited mycelial growth while unfiltered frass extract from FVBB diet caused a moderate (30% < inhibition rate < 50%) inhibition of mycelial growth as compared to the control. Filtered frass extracts showed no effect on the growth of P. capsici in both SLA and DLA bioassays (Figure 5).
Effect of 0.22 μm filtered and unfiltered frass extracts from Gainesville diet (GV) and fruit/vegetable/bakery/brewery waste-based diet (FVBB) on mycelial growth of Phytophthora capsici according to single-layer agar (a) and double-layer agar (b) in vitro bioassay. Each value represents the mean of three replicates ± standard deviation. Means with the same letter are not significantly different according to Tukey’s test (
Citation: Journal of Insects as Food and Feed 10, 10 (2024) ; 10.1163/23524588-20230022
Mycelial growth of F. oxysporum was strongly inhibited by unfiltered frass extracts from both GV diet and FVBB diet in SLA bioassay (Figure 6). In DLA bioassay, unfiltered frass extract from GV diet caused a complete inhibition of mycelial growth while unfiltered frass from FVBB caused a moderate inhibition. Filtered frass extracts showed no effect on the growth of F. oxysporum (Figure 6).
Effect of 0.22 μm filtered and unfiltered frass extracts from Gainesville diet (GV) and fruit/vegetable/bakery/brewery waste-based diet (FVBB) on mycelial growth of Fusarium oxysporum according to single-layer agar (a) and double-layer agar (b) in vitro bioassay. Each value represents the mean of three replicates ± standard deviation. Means with the same letter are not significantly different according to Tukey’s test (
Citation: Journal of Insects as Food and Feed 10, 10 (2024) ; 10.1163/23524588-20230022
The effect of frass extracts on R. solani mycelial growth is presented in Figure 7. Unfiltered frass extracts from both diets strongly inhibited the mycelial growth in SLA. Frass extract from FVBB diet caused a weak inhibition of mycelial growth in DLA while frass extract from GV diet caused a complete inhibition. Filtered frass extracts showed no effect on the growth of R. solani (Figure 7).
Effect of 0.22 μm filtered and unfiltered frass extracts from Gainesville diet (GV) and fruit/vegetable/bakery/brewery waste-based diet (FVBB) on mycelial growth of Rhizoctonia solani according to single-layer agar (a) and double-layer agar (b) in vitro bioassay. Each value represents the mean of three replicates ± standard deviation. Means with the same letter are not significantly different according to Tukey’s test (
Citation: Journal of Insects as Food and Feed 10, 10 (2024) ; 10.1163/23524588-20230022
4 Discussion
Both SLA and DLA are fast, simple and easy to perform bioassays that are extensively used to evaluate the antimicrobial/antagonistic activity of microorganisms or organic matters such as compost (Bouchard-Rochette et al., 2022; Koné et al., 2010; Kouki et al., 2012; Ros et al., 2005; Zouari et al., 2020). These bioassays were used herein to evaluate the antagonistic activity of BSFL frass extracts against different plant pathogens.
Unfiltered frass extract from GV diet strongly or completely inhibited the growth of all the tested pathogens in both SLA and DLA diet bioassays. Unfiltered frass extract from FVBB diet strongly or completely inhibited the mycelial growth of A. solani, B. cinerea and S. sclerotiorum and moderately inhibited the mycelial growth of P. capsici in both bioassays. The growth inhibition observed results mainly from the presence of antagonistic microorganisms in BSFL frass since 0.22 μm microfiltered frass extracts which are free of microorganisms showed generally no antagonistic activity indicating that in situ compounds with antifungal/anti-oomycete activity are absent or not enough concentrated to affect the growth of the pathogens.
Microorganisms can exert in vitro antagonistic activity through (1) the secretion of toxic compounds (antibiosis) such as antifungal/anti-oomycete compounds, (2) parasitism, or (3) competition for food or space (Boro et al., 2022; Vos et al., 2014). SLA bioassay allows evaluating the antagonistic activity of microorganisms through antibiosis, parasitism, or competition (Bruno et al., 2019; Gold et al., 2020; Jiang et al., 2019; Wynants et al., 2019) while DLA bioassay is a well-established method to test microorganisms such as bacteria for their ability to produce antimicrobial compounds/antibiotics (Bouchard-Rochette et al., 2022). Unfiltered frass extract from GV diet showed strong/complete inhibition of mycelial growth of A. solani, B. cinerea, S. sclerotiorum, F. oxysporum, P. capsici and R. solani in both SLA and DLA bioassays. This indicates that microorganisms secreting toxic compounds against all the tested pathogens are present in frass extract from GV diet. However, the presence of antagonistic microorganisms acting through parasitism or competition for food/space cannot be excluded. Frass extract from FVBB diet showed a strong and a weak (inhibition rate of 4.3%) inhibition of R. solani in SLA and DLA, respectively. This suggests that the inhibiting effect of FVBB diet frass extract against R. solani observed in SLA most likely results from the presence of antagonistic microorganisms acting through parasitism or competition for food/space in FVBB diet frass.
Based on EU regulations ((EU) No. 2021/1925), heat treatment (70 °C, 60 min) is proposed to reduce harmful organisms in insect frass. This practice was shown to decrease the relative abundance of pathogenic bacteria as those from the Enterobacteriaceae family (Van Looveren et al., 2022; Watson et al., 2021) but could also decrease populations of microorganisms promoting plant growth/health. Among the microorganisms recognized for their antagonistic activity against plant pathogens, species of the genus Bacillus were reported in insect frass (Arabzadeh et al., 2023; Gold et al., 2020; Green, 2023; Van Looveren et al., 2022). Bacillus is the most largest endospore-forming bacteria genus (Fritze, 2004). Endospores are not only resistant to extrem conditions including high temperatures, but the germinaton of endospores could be stimulated by heat treatments (Leuschner and Lillford, 1999). In this context, it would be interesting in future work to study the effect of heat treatment on the antagonistic activity of frass extracts considering that, depending on the microorganisms involved, heat treatment could have negative impact on the antagonistic activity of frass.
In this study, SLA and DLA bioassays revealed strong antagonistic effect of microorganisms inhabiting frass against the plant pathogens B. cinerea, A. solani, R. solani, P. capsici, F. oxysporum and S. sclerotiorum. Moreover, the study showed the impact of the BSFL diet on the antagonistic effect of frass extracts suggesting that antagonistic microorganisms are present in the diet used to rear BSFL. In future work, the antagonistic effect of frass extracts against the above-mentioned pathogens will be tested in vivo. Frass could eventually find applications for the control of plant diseases.
Corresponding author; e-mail: russell.tweddell@fsaa.ulaval.ca
Acknowledgements
We thank Organisation for Economic Co-operation and Development (OECD), Natural Sciences and Engineering Research Council of Canada and Premier Tech Technologies Ltd for financial support. We also acknowledge Julien Vivancos from the Ministère de l’Agriculture, des Pêcheries et de l’Alimentation du Québec (MAPAQ) for providing the plant pathogens.
Conflict of interest
The authors declare no conflict of interest.
OECD disclaimer
This paper was given at the workshop Development of standard research methodologies for the mass rearing of insects fed waste organic residues for the production of novel animal feeds, which took place in Quebec, Canada, on 12-16 June 2022, and which was sponsored by the OECD Co-operative Research Programme: Sustainable Agricultural and Food Systems whose financial support made it possible for the author to participate in the workshop.
The opinions expressed and arguments employed in this paper are the sole responsibility of the authors and do not necessarily reflect those of the OECD or of the governments of its Member countries.
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