Fly infestations threaten farming and livestock operations, contributing to animal irritation and the spread of serious diseases. In this study, the application of Bacillus velezensis PHP1601 (Bacillales: Bacillaceae) treatments (105 and 1010 endospore g−1) to pig manure slurries was evaluated as a means of controlling fly reproductive cycles. Two cycles of fly emergence were evaluated over a 33 days period for each replicated trial. For both treatments, the first emergence event resulted in a significant reduction in the percentage of flies emerging compared to an un-treated control. Extended monitoring of the biocontrol containers revealed that fly emergence was completely eliminated by the time that a second round of fly emergence was observed for the control. A B. velezensis-specific real-time PCR method was developed and used to assess the population dynamics of the applied biocontrol agent over the course of the experiment. Strain PHP1601 remained viable in the manure and cell numbers increased by several orders of magnitude. REP-PCR fingerprinting was used to confirm the clonality of endospores recovered from the manure. Fly species recovered from the trials were identified by cytochrome oxidase gene barcode sequencing. Several species of veterinary and medically significant flies were identified. They were all deemed to be susceptible to treatments with PHP1601 and constituted part of the strain's host range. The study demonstrated the effectiveness of B. velezensis PHP1601 as a promising biocontrol agent for controlling fly infestations under conditions similar to its intended use.
Livestock and poultry farming generate considerable quantities of manure, which promotes the development of fly infestations (Tangtrakulwanich et al. 2015). Fly infestations negatively impact farming operations through pathogen transmission and animal irritation, leading to significant economic losses (Meerburg et al. 2007; Taylor et al. 2012). The control of fly populations within animal production facilities is an important goal for promoting animal health and ensuring food safety. Increasingly, eco-friendly alternatives to traditional chemical-based control methods that pose risks to human health and the ecosystem have been advocated (Brown 2006; Manrakhan and Addison 2014; Hinckle and Hogsette 2021). Biological control strategies provide opportunities for establishing sustainable control measures that safeguard against and reduce losses related to fly infestations in a non-toxic manner that does not compromise the safety of livestock, farm workers or the environment.
Fly species commonly associated with fly infestation include house flies, Musca domestica (Linnaeus) (Diptera: Muscidae), stable flies, Stomoxys calcitrans (Linnaeus) (Diptera: Muscidae), and blowflies (Diptera: Calliphoridae). House flies, for instance, are notorious carriers of numerous pathogens, including bacteria, viruses, and parasites, which they can transmit to animals and humans (Hinckle and Hogsette 2021). Stable flies can inflict painful bites, which can cause stress and discomfort to livestock, leading to reduced feed intake, weight loss, and decreased milk production (Taylor et al. 2012). Blowflies can cause flystrike or myiasis, whereby they lay their eggs on wounds, leading to maggot infestations and complications in livestock (Khater and Geden 2018). This can result in economic losses attributed to poor hide or wool quality, reduced weight gain, and decreased livestock fertility (Soyelu and Masika 2009). Since nuisance flies in animal production facilities can negatively impact animal welfare and disrupt operations, establishing effective control measures is paramount.
Advertisement
Bacterial species of the Bacillus subtilissensu lato group (Bacillales: Bacillaceae) are gaining attention as candidate fly biocontrol agents (Torres et al. 2022; Ramesar and Hunter 2023b). Many members of the B. subtilis species complex have achieved "generally regarded as safe" status and are non-pathogenic to humans or animals (Abdallah et al. 2019; Lin et al. 2020). In a recent study, B. velezensis PHP1601 (Bacillales: Bacillaceae) was shown to be effective in controlling Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae) fly larvae through the activity of its lipopeptide biosurfactants (Ramesar and Hunter 2023a). Lipopeptide fractions containing surfactin homologues demonstrated the highest levels of larvicidal activity (Ramesar and Hunter 2023b). Larvicide bioassays (240 h) using purified surfactin resulted in an LC50 of 9.87 µg g−1. Bacillus velezensis PHP1601 was, therefore, considered a promising candidate for controlling problematic flies associated with farming operations and was selected for further evaluation.
Piggery operations provide an ideal environment for fly proliferation (Meerburg et al. 2007); hence, a study was initiated with the aim of assessing the biocontrol potential of B. velezensis PHP1601 treatments in controlling fly infestation associated with pig manure. Trials were established at an operational piggery at the University of KwaZulul-Natal's Ukulinga Research Farm, South Africa, where endospore treatments were applied to pig manure slurry samples and evaluated for their efficacy in disrupting fly reproductive cycles. Additional objectives included determining the potential host range of flies susceptible to B. velezensis PHP1601, and assessing the population dynamics of the applied biocontrol agent over the course of the experiments.
Materials and methods
Bacterial strains
Bacillus velezensis PHP1601 was obtained from Andermatt PHP (Pty) Ltd (Strathdean Farm, Nottingham Road, 3280, KwaZulu-Natal, South Africa). Reference Bacillus strains used in the study were sourced from an in-house culture collection within the Discipline of Microbiology, School of Life Sciences, University of KwaZulu-Natal (Pietermaritzburg, 3209, KwaZulu-Natal, South Africa) and included B. velezensis FZB42T, B. subtilis DSM10T and B. cereus DFbc. Strains were routinely cultured on tryptone soy agar (TSA) (30 °C for 18 h) or in tryptone soy broth (TSB) (Biolab, Merck, Germany) at 28 °C for 18 h at 200 rpm. Master cultures of each strain preserved in 20% (w v−1) glycerol solution were stored at − 80 °C. Sporulation was promoted by culturing strains in 100 m1 of glucose omitted Schaeffer's broth as described by Leighton and Doi (1971).
Trial design
Manure trials were conducted at a piggery facility located at Ukulinga Research Farm, University of KwaZulu-Natal, South Africa (29°39′45''S, 30°24′10''E) during the summer months of December 2021 to March 2022 when fly activity was at its height. The trial design was adapted from Nurita and Hassan (2013) and utilised standardised 1 l black plastic bucket containers (12 cm tall × 10.5 cm wide) (Figure S1). Each container lid had a 3 cm hole at it centre, to which a clear plastic bottle (14 cm tall × 4.5 cm wide) was glued. The base of each bottle was removed and covered with fine gauze net material, which allowed air to diffuse into the container while preventing flies and larvae from escaping. This allowed the emergence of flies to be observed and recorded over time.
Advertisement
Manure trial
Approximately 12–14 kg of pig manure was collected from the facility and mixed in a 15 l plastic bucket for 10 min to create a homogenous mixture. This mixture was kept in a closed container for three days at ambient temperature (~ 26 °C) to assess for the emergence of fly larvae, which could interfere with the investigation. The dry mass of the manure was determined by drying 5 × 20 g samples at 75 °C (Labotec EcoTherm Industrial Oven, Labotec, RSA) for two days, to facilitate accurate endospore dosages to be calculated and applied (Wong et al. 1999).
Prior to setting up each treatment, the manure sample was homogenised as described previously. Four hundred grams of unamended manure was added to each container and five replicates established for each treatment. Two treatments of B. velezensis PHP1601 were evaluated, i.e., 105 and 1010 endospores g−1. These treatments were prepared by amending the manure with 10 ml of an endospore suspension, in deionised water. Initial endospore titres of PHP1601 cultured in Schaeffer's broth were determined using a bacterial counting chamber (Neubauer, Marienfeld, Germany) before being adjusted to the desired concentration via centrifugation and dilution. Controls used included an autoclaved 1010 endospore g−1 treatment, and an endospore-free control amended with 10 ml of deionised water. They were included to substantiate any differences in fly emergence observed between treatments. Strips of shredded black plastic refuse bag (~ 5.0 g) were introduced into each container to provide a surface for fly visitation and oviposition. An additional set of controls and treatments were prepared to assess changes in moisture content at various intervals (0, 16 and 33 days) to supplement qPCR and endospore quantification assays without disturbing the trial itself.
Prior to starting the trial, buckets were grouped in five replicate sets containing a representative of each treatment and control. These were placed at several sites within the piggery environs and opened to attract flies and promote oviposition. Over a five days period, bucket sets were switched between locations daily to increase the likelihood of an even distribution of fly visitation. Subsequently, buckets were covered and relocated to an unused pen within the piggery for the duration of the trial (33 days). Buckets were monitored for fly emergence every three days, as evidenced by the appearance of flies within the clear plastic bottle attached to the container lid. The bottles could be unscrewed to take samples for counting or identification purposes.
Fly identification
Emerging flies were collected and sorted into morphologically similar groups. Representative specimen samples (~ 100 mg) were homogenised by liquid nitrogen maceration. Thereafter, DNA was extracted following the Thermo Scientific GeneJet DNA extraction kit (Thermo Fisher Scientific, USA) protocol for rodent tails. Flies were identified by PCR amplification and sequence analysis of the cytochrome oxidase gene according to the method of Radzevičiūtė et al. (2017). All PCR reactions were prepared using DreamTaq Green PCR Master Mix reagents (Thermo Fisher Scientific, USA).
qPCR detection and quantification of B. velezensis in pig manure slurries
A qPCR method based on B. velezensis specific primers designed by Dunlap (2019) was used to detect and monitor B. velezensis populations in pig manure, as a proxy for monitoring changes in B. velezensis PHP1601 concentration. At 0, 16 and 33 days intervals, 2 g of manure slurry was sampled from three replicates of each treatment for DNA extraction and qPCR analysis. Additionally, DNA from duplicate sets of pig manure spiked with 100–109 cells g−1 of B. velezensis PHP1601 and 109 cells g−1 of B. velezensis FZB42T were extracted to determine DNA extraction efficiency.
Prior to DNA extraction, manure samples were treated to remove exogenous DNA and water soluble contaminants. Briefly, 100 mg of manure suspended in 1.5 ml saline (NaCl) solution (0.8% w v−1) was vortexed for 1 min to dissolve exogenous DNA. Thereafter, particulate matter was collected by centrifugation (6000 × g for 5 min). This washing step was repeated three times. Microbial DNA was extracted from pelleted cells and particulate matter using a DNeasy PowerSoil kit (Qiagen, Germany) as per the manufacturer’s instructions and recommendation for hard to lyse cells. An amendment was made to the sample homogenisation step, which included bead-beating samples at 7 m s−1 for 1 min (Bead Blaster 24, Benchmark Scientific, USA). Eluted DNA was quantitatively and qualitatively assessed using UV spectral analysis (Nanodrop, Thermo Fisher Scientific, USA), and agarose (1.5% w v−1) gel electrophoresis at 80 V for 100 min in TAE buffer (40 mM Tris, 20 mM Acetic acid, 1 mM EDTA, pH 8.3).
A qPCR hydrolysis probe was designed using Primer3 (https://bioinfo.ut.ee/primer3-0.4.0/primer3/) to align within the B. velezensis specific 2-keto-3-deoxygluconate kinase (kdgK) gene region. Potential probe sequences were queried in silico against a sample of 75 B. velezensis genomes (NCBI GenBank, https://www.ncbi.nlm.nih.gov/) using the Blastn algorithm (https://blast.ncbi.nlm.nih.gov/) to confirm specificity. The resulting probe, Bvel-P, was synthesised incorporating a 5' FAM fluorophore and a 3' Blackhole quencher 1 (BHQ1) to give a final sequence of (5’-3') FAM-TCCCCAGTCTGCTGACCCACT-BHQ1 (Inqaba BioTech Laboratories, Pretoria, South Africa).
All qPCR reactions contained per 20 µl: 1 × HOT FIREPol Probe qPCR Mix (ROX) Plus (Solis BioDyne, Estonia), 0.4 mM Bvel-F (5'CCTTTGCGTTTTGTTACCC-3') and Bvel-R (5’-CACATCAATTCCTTCTCC-3') primers, 0.2 mM of Bvel-P hydrolysis probe, and 1 ng µl−1 of template DNA. Reactions were performed using a QuantStudio 5 Real-time PCR system, which included an initial denaturation step at 95 °C for 12 min followed by 40 cycles of 95 °C for 10 s and 60 °C for 1 min. QuantStudio Scientific Design and Analysis software (v. 1.5.1) (Thermo Fisher Scientific, USA) was used to normalise amplification curves against the ROX dye and to determine Ct values, qPCR efficiencies and SE values. Controls included a no template control to assess PCR contamination, and non-specific amplification controls using genomic DNA from B. subtilis DSM10T and B. cereus DFbc to confirm primer specificity. Bacillus velezensis qPCR standards (1–109 cells per reaction) were included in triplicate, in each set of qPCR reactions for quantification purposes. Standards were prepared from an overnight culture of B. velezensis PHP1601 (TSB, 30 °C, 150 rpm) that was quantified using a bacterial counting chamber (Neubauer, Marienfeld, Germany) prior to DNA extraction according to a CTAB method described by Minarovičová et al. (2018).
Since endospores of B. velezensis were not lysed efficiently using the DNeasy PowerSoil kit, a plate count method was used to quantify viable endospores present in manure samples collected in triplicate. Briefly, a serial dilution (10−1–10−8) of 1 g of manure in saline solution was prepared in McCartney bottles and heat treated at 80 °C for 20 min in a water bath to eliminate vegetative cells and select for endospores. Aliquots (100 µl) from each dilution were spread-plated onto TSA and incubated for 18 h at 30 °C. The resulting colonies, within a range of 30–300, were picked off from the highest dilutions plated and suspended in 100 µl of nuclease-free water and heated at 94 °C for 10 min to lyse the cells and release their DNA. Cellular debris was removed by centrifugation (15,000 × g for 10 min) and 1 µl of the supernatant was used to identify colonies of B. velezensis as per the method of Dunlap (2019). Isolates that tested positive as B. velezensis were fingerprinted according to the REP-PCR method described by Versalovic et al. (1994), and compared to B. velezensis PHP1601. REP-PCR fingerprints were resolved by agarose gel (1.5% w v−1) electrophoresis at 80 V for 100 min using TBE buffer (89 mM tris, 89 mM boric acid, 2 mM EDTA, pH 8.3).
Statistical analysis
The manure trials were performed comprising of five replicates of each treatment and control. Fly antagonism was assessed by comparing the averaged total number of flies emerged in each treatment to fly emergence events recorded for the controls. The trial was repeated two weeks after the first trial to substantiate the results acquired. Statistical analysis was performed using R (v 4.2.1) (R Core Team 2022) through the RStudio integrated development environment (v 2022.07.1, build 554). Fly emergence data was initially assessed for normality using the Shapiro-Wilks test. Thereafter, one-way ANOVA followed by Tukey's HSD test was performed to determine statistical differences (P < 0.01) between treatments and the controls tested. The data presented for cell and endospore concentrations enumerated from manure treatments were determined from three replicates sampled at each sampling interval and reported as the mean ± SE per gram (dry weight) of manure. The Kruskal–Wallis test followed by the Conover-Iman test for pairwise comparisons were performed to determine statistical differences between cell and endospore treatments using the “conover.test” package (v 1.1.5) in R (Dinno 2017).
Results
Influence of PHP1601 treatments on fly oviposition
Prior to conducting the manure trial, the influence of the different treatments and controls on fly oviposition levels was assessed. The average number of eggs laid between the controls and treatments after 24 h of the buckets being left open ranged between 46.80 ± 12.94 and 53.40 ± 4.88. However, no statistical difference in the number of eggs laid were detected (F3, 16 = 0.05, P > 0.01). Subsequently, it was concluded that neither the controls nor treatments introduced a bias in fly attraction or oviposition that could interfere with the results acquired from the trials.
Manure biocontrol trial 1
The effect of the endospore treatments on fly emergence is shown in Fig. 1a. The first indication of fly antagonism was a delay to the start of fly emergence observed in the endospore treatments compared to the controls. Fly emergence was evident from 6 to 21 days for the controls, whereas fly emergence in the 105 and 1010 endospore g−1 treatments was observed from 15–21 to 12–21 days, respectively. Significant differences (F3, 16 = 52.84, P < 0.001) in the total number of emergent flies was evident between the controls and treatments over this period (Fig. 1b). On average, the highest number of flies that emerged were from the endospore free (43.00 ± 4.60 flies) and non-viable endospore controls (39.80 ± 2.73 flies). Both controls displayed significantly higher levels of fly emergence (all P < 0.001) than each of the endospore treatments. This substantiated that neither the presence of the endospore biomass nor the use of deionised water to prepare the endospore solutions negatively impacted the ability of the eggs or larvae or pupae to develop into flies. Overall, the total number of flies that emerged in endospore treatments were 5.01–12.44 times lower than in the controls. The levels of fly emergence for the two endospore treatments, over the duration of the trial, were not significantly different (P = 0.808). However, it was observed that the 105 endospore g−1 treatment required a longer time (three days) for fly emergence to be detected.
Fig. 1
Effect of B. velezensis PHP1601 endospore treatments on fly emergence trends over a 24 days period (a), and averaged total number of flies counted during the primary fly emergence event (b). Error bars represent SE. Means denoted by a different letter indicate significant differences between treatments (P < 0.01). Based on fly emergence patterns observed for the controls, the primary fly emergence event was judged to occur from 6 to 21 days
×
The trial ended abruptly after 24 days due to unforeseen animal interference of the trial area, resulting in the destruction of many of the trial containers. At this stage in the investigation, evidence substantiated that the treatments of B. velezensis PHP1601 significantly inhibited fly emergence.
Manure biocontrol trial 2
To verify the findings of the first fly emergence trial, the trial was repeated. Two fly emergence events were observed in the controls, whereas fly emergence associated with the endospore treatments were only evident during the first emergence event (Fig. 2a). Over the course of the trial, significant differences in fly emergence was observed (F5, 25 = 33.20, P < 0.001) (Fig. 2b). During the primary fly emergence interval, significant differences in fly emergence were observed whereby the 105 endospore g−1 treatment had the lowest number of flies when compared to the controls (all P < 0.01). In contrast, fly numbers associated with the 1010 endospore g−1 treatment were not statistically different to either of the controls or the 105 endospore g−1 treatment (P > 0.01) during the primary emergence period. This indicated that the 105 endospore g−1 treatment was the best treatment tested and that the biocontrol effectiveness could be negatively impacted when a threshold dosage was exceeded.
Fig. 2
Effect of B. velezensis PHP1601 endospore treatments of on fly emergence trends over a 33 days period (a), and averaged total number of flies counted during each fly emergence event (b). Error bars represent SE. Means denoted by a different letter indicate significant differences in fly emergence between treatments (P < 0.01) across the primary and secondary fly emergence events. Asterisks indicate treatments that had no fly emergence. Based on fly emergence patterns observed for the controls and treatments, primary fly emergence was judged to occur from 6 to 24 days and a secondary emergence event from 27 to 33 days
×
Fly emergence during the second emergence event was limited to the controls and showed a significant increase in the numbers of flies emerged (all P < 0.001) compared to their respective primary emergence events (Fig. 2b). This indicated that the trial design was suitable for a population of flies to complete their life cycle. However, all larvae in the 105 and 1010 endospores g−1 treatments of B. velezensis PHP1601 were confirmed to be dead upon inspection at the 33 days interval. This confirmed that the B. velezensis PHP1601 treatments are effective in controlling various fly populations.
Identification of the host range of flies antagonised by B. velezensis PHP1601
Seven distinct fly morphologies were distinguished when fly emergence was recorded over the course of the second field trial. These flies were identified based on analysis of their CO1 gene sequence as L. cuprina – the Australian blowfly, Dacus bivittatus (Bigot) (Diptera: Tephritidae) – the common fruit fly, Sarcophaga ruficornis (Fabricius) (Diptera: Sarcophagidae) – the flesh fly, Megaselia scalaris (Loew) (Diptera: Phoridae) – the scuttle fly, Muscina stabulans (Fallén) (Diptera: Muscidae) – the false stable fly, Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae) – oriental latrine fly, and Synthesiomyia nudiseta (Wulp) (Diptera: Muscidae). Sequences were deposited into GenBank with the following accession numbers: OP355338.1, OP356210.1, OP356702.1, OP356703.1, OP356704.1, OP356705.1 and OP356706.1 respectively.
All fly species were found in the control groups. However, only L. cuprina, D. bivittatus, S. ruficornis, C. megacephala, S. nudiseta and M. stabulans were observed in the treatments during the first fly emergence event (Figure S2). Subsequently, all larvae in the treatments were assessed to have been deceased by the 33 days interval. Hence, indicating their susceptibility to the B. velezensis PHP1601 treatments.
Growth of B. velezensis PHP1601 in pig manure slurries
The B. velezensis cell standard prepared using the CTAB method produced DNA of high quality (A260:280: 1.82 ± 0.02, A260:230: 1.35 ± 0.16) suitable for qPCR analysis. The qPCR standard curve (10 – 109 cells per reaction) yielded a straight line curve ( y = − 3.388x + 35.961, R2 = 0.999) that had good qPCR efficiency (97.307%) (Figure S3). The limit of detection was ten cells per reaction and concentrations of B. velezensis within the 10–109 cells per reaction range could be quantified.
DNA extracted from B. velezensis spiked manure (1 – 109 cells g−1) had detectable levels of co-extracted organic compounds (A260:280: 1.68 ± 0.03, A260:230: 0.95 ± 0.21), which was considered to be a consequence of the complex nature of the manure constituents. Attempts to further purify the DNA as per the kit’s recommendations were not successful.
qPCR successfully detected B. velezensis in the 104 to 109 cells g−1 spiked manure standards and reaffirmed their respective concentrations (Figure S4). No endogenous B. velezensis were detected in unamended manure samples by qPCR assays or culturing, and were considered to not impact qPCR analysis. This indicated that the limits of B. velezensis detection and quantification are within the 104 to 109 cells g−1 range. Additionally, the qPCR derived concentration of B. velezensis FZB42T in the spiked manure control (1.27 ± 0.15 × 109 cells g−1) was similar to that acquired from the corresponding B. velezensis PHP1601 spiked manure standard (1.15 ± 0.31 × 109 cells g−1), which indicated that variance in DNA extraction or qPCR sensitivity between different B. velezensis strains were unlikely. Subsequently, the B. velezensis-specific qPCR protocol was considered suitable to assess the population dynamics of the best-performing treatment (i.e., 105 endospore g−1) in pig manure.
Throughout the study, no evidence of B. velezensis cells, nor endospores, was detected in the dead endospore control, whereas, in the treatment, the population increased significantly (\(\chi_{4}^{2}\) = 13.52, p < 0.01) by several orders of magnitude over a 16 days period (Table 1). This indicated that the endospores germinated and were in a bioactive state. By the 16 days interval, ~ 0.62% of the B. velezensis population had sporulated, which is interesting as it suggests that nutrient exhaustion had not yet been reached. By the end of the trial (33 days) there was a substantial decrease in the concentration of vegetative cells (99.48%) (P < 0.001), accompanied by increased sporulation levels to 2.48 ± 0.77 × 108 endospores g−1 (sporulated population: 98.94%) (P < 0.001). Furthermore, REP-PCR fingerprinting indicated that the genomic profiles of the B. velezensis colonies recovered were identical to that of the B. velezensis PHP1601, which confirmed that the population change determined for B. velezensis was attributable to the PHP1601 strain (Fig. 3). The amplification of the ~ 169 bp B. velezensis specific KdgK region detected in all qPCR reactions was verified by agarose gel electrophoresis (Figure S5).
Table 1
Population dynamics of B. velezensis over the course of a 33 days manure trial
Time (d)
105 endospore g−1 treatment
Dead endospore control
Cells (cells g−1)
Endospores (endospore g−1)
Cells (cells g−1)
Endospores (endospore g−1)
0
ND
1.14 ± 0.19 × 105 a
ND
ND
16
5.04 ± 0.17 × 108 b
3.17 ± 0.31 × 107 c
ND
ND
33
2.66 ± 0.16 × 106 d
2.48 ± 0.77 × 108 e
ND
ND
Cell concentration was determined using qPCR, whereby all Ct values were determined once the respective amplification curves passed the threshold of 0.04 within 40 PCR cycles. The concentration of endospore of B. velezensis were determined using a plate count method and verified using B. velezensis-specific qPCR. When no amplification occurred or no endospores of B. velezensis were isolated, it was interpreted that B. velezensis was not detected (ND). The data represents the mean ± SE of concentrations of cells or endospore per gram (dry weight) of manure determined from three replicates. Letters indicate significant differences (P < 0.01) between the cell and endospore treatments achieved over the duration of the trial (33 days)
Fig. 3
REP-PCR genomic fingerprints of representative B. velezensis colonies isolated from a 105 endospore g−1 manure treatment at 0, 16 and 33 days. Bacterial colonies were confirmed to be strains of B. velezensis using species-specific qPCR targeting the kdgK gene, prior to selection for REP-PCR. Bacillus velezensis PHP1601 was included as a reference control (PR), along with a no template control (No) and 10 kb molecular weight ladders (L). Representative fingerprints of isolates recovered at 0, 16 and 33 days are shown in lanes 1–3, 4–6 and 7–9, respectively
×
Discussion
Manure trials were conducted to assess the effectiveness of B. velezensis PHP1601 as a viable biocontrol agent against fly species commonly associated with a piggery setting. Many biocontrol candidates have been reported to exhibit poor insect antagonism and/or inconsistent results when assessed under farm, semi-field or field trial conditions due to factors such as changes in temperature, RH, desiccation, and exposure to UV radiation (Besset-Manzoni et al. 2019). Therefore, the manure trials undertaken were considered an important milestone in assessing the biocontrol candidacy of B. velezensis PHP1601 under field conditions that were representative of their intended use.
The trials were conducted using closed containers to house the various treatments and contain the flies that emerged, whilst still being subjected to the prevailing temperatures found within the piggery. This approach was adopted to ensure that a reasonable level of consistency was retained within, and between, replicated treatments whilst limiting the influence of independent variables (e.g., moisture content, repeated fly visitation and oviposition) that could have interfered with the findings of the trial. This allowed the trial to be operated at a smaller scale and yet incorporate essential elements of a field trial.
Pig manure slurry was considered a suitable medium for the trials because it is known to attract flies and contributes to the development of fly infestations (Tangtrakulwanich et al. 2015). Chicken and cow manure slurries are also important nutritional sources for flies, which contribute to the development of fly infestations if left unattended (Cruz-Vázquez et al. 2007; Tangtrakulwanich et al. 2015). Therefore, these types of manure could be used as suitable media for future field trial assessments.
The first trial showed a significant reduction of fly emergence by both endospore treatments tested (Fig. 1a). This was considered to be a positive indication that PHP1601 could elicit a fly antagonistic effect under the prevailing environmental conditions. Similar results were obtained when the trial was repeated (Fig. 2a). When the second trial was extended beyond 24 days, it was found that none of the survivors of the first fly emergence event associated with the endospore treatments were able to produce a second generation of flies and all remaining larvae were deceased by the end of the trial. Hence, the larvicidal effect of B. velezensis PHP1601 was described as a chronic antagonistic effect rather than an acute one. As far as the authors are aware, no similar studies have been reported in the literature. Therefore, this constitutes the first report of a B. velezensis exhibiting real-world potential as a fly biocontrol agent.
In both sets of trials, the endospore treatments displayed statistically similar levels of fly inhibition when compared to each other. The 105 endospores g−1 treatment consistently displayed the most prolonged period for fly emergence, and the lowest numbers for fly emergence. As such, it was considered the more effective of the two treatments tested. Generally, higher doses of biocontrol agents are associated with higher levels of pest antagonism (Fite et al. 2019). However, in previous studies with B. velezensis PHP1601 it was noted that the feeding behaviour of Lucilia cuprina larvae was negatively impacted at elevated cell concentrations resulting in larvae progressively moving away from the bioassay medium (Ramesar and Hunter 2023b). This was attributed to an accumulation of lipopeptide compounds, which may have reduced the palatability of the medium. Therefore, we hypothesise that when B. velezensis PHP1601 was applied to the pig manure slurry at the lower endospore concentration (105 endospores g−1), the substrate medium was more palatable, allowing the larvae to feed for more extended periods resulting in a more effective larvicidal response. It was likely that after a specific threshold concentration of B. velezensis PHP1601 or lipopeptides was reached in the pig manure slurries, no further biocontrol benefit could be achieved from higher dosages. This finding is promising as it suggests that lower application dosages of B. velezensis PHP1601 are preferable for effective fly antagonism.
qPCR assays indicated that B. velezensis PHP1601 endospores germinated, grew and multiplied in the pig manure slurry, reaching > 108 cells g−1 by the 16 days interval (Table 1). This is promising because it showed that the strain could grow in a medium that accommodates its target for biocontrol, i.e., fly species (Tangtrakulwanich et al. 2015). It also indicates that strain PHP1601 was metabolically active and likely produced lipopeptide biosurfactants, which brought about the fly antagonism, as reported previously in controlled in vitro laboratory studies (Ramesar and Hunter 2023b). To confirm this, analytical methods for the detection and quantification of lipopeptide biosurfactants in environmental samples still need to be developed and evaluated in future studies.
REP-PCR profiling of colonies derived from endospores recovered from the pig manure confirmed that they were clonally identical to PHP1601 (Fig. 3). This supported the notion that the fly antagonism could be attributed to the application of the B. velezensis PHP1601. Furthermore, the ability to recover endospores of B. velezensis PHP1601 allows it to be isolated and quantified, which is attractive from a population dynamics and environmental persistence point of view.
qPCR has been used as an efficient means of detecting and quantifying microbes in environmental sources, provided that the microbes are amenable to DNA extraction methods. Using species-specific PCR primers targeting the KdgK gene, a qPCR method was developed that integrated a hydrolysis probe to detect and quantify B. velezensis in manure samples. The limits of detection and quantification was similar to those reported in the literature for B. velezensis qPCR studies (Zhang et al. 2018). Bacillus velezensis is a species commonly studied for its biocontrol potential against many phytopathogens. Subsequently, the qPCR protocol was considered a valuable tool that could be used in other studies related to the quantification of B. velezensis. Future research would involve exploring means to efficiently extract DNA from B. velezensis endospores in environmental samples to allow for qPCR quantification.
Fly species that fell into the potential host range of B. velezensis PHP1601 included: L. cuprina, D. bivittatus, S. ruficornis, C. megacephala, S. nudiseta and M. stabulans. Strains of S. nudiseta, C. megacephala, L. cuprina, and Sarcophaga sp. are commonly attracted to the stench of faecal material and their presence in the trial was not unexpected (Nurita and Hassan 2013). These flies are considered nuisance pests and can pose severe threats to farming practices. Lucilia cuprina and S. ruficornis are both causative agents of myiasis or flystrike, which are significant threats to the livestock industry (Azevedo et al. 2015). These flies contribute to economic losses from reduced milk production from dairy farms, decreased animal and livestock fertility and reduced hide quality (Mukandiwa et al. 2012). Dacus spp. are recognised as common pests found in fruit farms in South Africa (Grové and de Beer 2014). Further economic loss is attributed to the cost of purchase and application of chemical pesticides, which are losing effectiveness in controlling these species due to insect resistance (Mukandiwa et al. 2012). Therefore, the ability of B. velezensis PHP1601 to control these fly species is promising and warrants that the strain undergoes further field trial evaluation. A comprehensive evaluation of its fly host range is also recommended.
As a result of the significant degree of fly antagonism observed in both trials, B. velezensis PHP1601 is considered a promising candidate for the biocontrol of fly species. qPCR assays indicated that the strain was viable and active in the manure slurries, increasing in cell concentration by several orders of magnitude over the course of the experiment. These findings indicate the strain could be detected and monitored if applied under field conditions. The potential host range of the B. velezensis PHP1601 was found to include fly species of economic and veterinary importance. This highlights the potential for this strain to be used in a variety of fly biocontrol applications and, therefore, it warrants further investigation in this regard.
Acknowledgements
This research was funded by Agricultural Sector Education Training Authority of South Africa and Andermatt Plant Health Products.
Declarations
Conflicts of interest
The authors declare that there are no conflicts of interest.
Ethical approval
Ethical approval was not required for any aspect relevant to the content of this article.
Informed consent
The authors consent to the publication of this article.
Consent to participate
Not applicable. Research involving humans or vertebrate animal testing were not performed.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
