Final Internship Report
Author: Elias Andraos
Mitacs Accelerate Internship
For BIOPONIX Agriculture Corp.,
Montreal, QC
Background Information
BIOPONIX AG Corp. produces the Bioponix growing system, consisting of a planter, a soil conditioner, and a liquid fertilizer (Figure 1). The aim of the system is to deliver higher yields and higher quality produce than organic field agriculture by providing an optimal environment for symbiotic bacteria and fungi. Other purported benefits include increased growth rate, biomass and resistance to biotic and abiotic stresses (“Our System,” 2020).
Figure 1: Schematic representation of experimental setup. The BIOPONIX system consists of a 20 l reservoir filled every other day with water treated with the BIOPONIX brand soil conditioner. A cassette insert sits on top of the reservoir with a layer of rock wool in the bottom providing an air gap. Above this gap, the cassette is filled with 5 l of a compost, peat and perlite potting mix. Units were fertilized with a solid 4:4:4 fertilizer. For the field comparison, an equal volume of potting mix was placed in a hole in the ground (or in a mound for the cucumbers).
One group of symbionts that the Bioponix system aims to foster is the arbuscular mycorrhizal fungi (AMF). In exchange for photosynthates, AMF help plants withstand abiotic stresses such as nutrient scarcity and drought as well as biotic stresses such as disease (Baum et al., 2015).
An outdoor demonstration was conducted with six crops (strawberry, tomato, pepper, cucumber, basil and lettuce) grown in Bioponix planters compared to those grown in field plots.
Research Goals Achieved and Experimental Limitations
All major research goals were achieved by comparing the Bioponix system to field agriculture. Despite the late start date (July 14th) and the early first frost (October 3rd), all six crops were grown to maturity, though the tomato and pepper fruits did not fully ripen. Most crops were grown from seedlings purchased from a nursery. Lettuce was grown from seed. Plants were monitored during the season by measuring stem elongation and taking chlorophyll readings with a SPAD meter. We planned to check the stomatal conductance, an indicator of leaf gas and water exchange, but the instrument was unavailable due to repairs, so no data was collected.
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We planned to compare crop growth in the Bioponix system with crops grown in a conventional greenhouse, but no space was available in the McGill greenhouse due to renovations. Therefore, the analysis will focus on a side-by-side comparison of the Bioponix system and field agriculture in an outdoor environment.
Most of the planned analysis were completed. Due to time constraints, I chose to compare the AMF colonization of strawberry plants only, which had 4.5 times greater yield in the Bioponix than the adjacent field environment. However, I was not able to analyze the polyphenol and ascorbic acid content of harvested produce because the test reagents did not arrive on time.
Research Outcomes
Visual monitoring of plants throughout the growing season showed no significant difference in the dates of first appearance of reproductive structures in cucumber and pepper plants, which were in a vegetative growth stage when the seedlings were transplanted into the Bioponix units or the field. Plants in the field treatment were adequately watered from rainfall, and no plants in any treatment were wilted or showed other signs of water stress throughout the growing season.
Figure 2: SPAD readings and relative growth rates of all crops. Relative growth rate was calculated using stem elongation measurements. Error bars are standard error of three replicates (two in the case of cucumber). Individual figures are available in appendix.
Monitoring of plants throughout the growing season with a SPAD-meter showed that nutrient deficiencies became apparent in tomato, lettuce and cucumber plants in the Bioponix planters more quickly than in the field (Error! Reference source not found.). This is expected due to the smaller soil volume. However, after fertilizer application around calendar day 230 (35 days after transplanting) the SPAD values of the Bioponix plants recovered to the same level as the field-grown plants. Growth rates of all crops in both treatments decreased as they reached the reproductive growth stages and physiological maturity. This was expected due to the shorter days, lower temperatures and natural life cycle of these annual plants. There was no obvious difference in the growth rates between treatments.
Total yield was greater in the Bioponix units than the field for the strawberry, basil and cucumber crops (Figure 3). This difference was most pronounced in the strawberry crops, so this crop was chosen for the AMF analysis in its root system. Root image processing using WinRhizo showed that plants had greater root surface area per plant in Bioponix units than the field. Moreover, pepper, strawberry, lettuce and cucumber crops grown in Bioponix units had significantly more root surface area per plant than the field-grown crops (Figure). Roots of diameter < 0.5 mm were excluded in order to reduce the sampling bias, as fine roots are difficult to recover from field soil due to their small size. There is more variation in the root surface area of basil than other crops because it was difficult to measure the complex, fibrous, matted root system of the basil plant. The higher yield in strawberry and lettuce plants in the Bioponix units correlates with a higher root surface area.
Figure 3: Yield per plant, normalised to the total yield from the field. All tomatoes were graded as unmarketable as they did not ripen before the first frost. Significantly different (p<0.05) total yields are marked with an asterisk, error bars are 1 SE. Absolute yield values are available in appendix.
Figure 4: Root surface area per plant, as estimated by WinRhizo. Roots of diameter < 0.5 mm were excluded in order to reduce the sampling bias, as more fine roots were lost during sampling in the field. Significantly different (p<0.05) treatment pairs are marked with an asterisk, error bars are 1 SE.
Dry root biomass was significantly higher for pepper, basil and lettuce crops in the Bioponix units than the field, while dry shoot biomass was significantly higher in basil and lettuce from the Bioponix units than the field (Error! Reference source not found.a). This means that the higher root surface area in strawberry, pepper and cucumber plants is due to a lower average root diameter, not a larger total root mass.
Shoot to root ratios were significantly higher for tomato and pepper crops in the Bioponix units (Figure 5b). This means that the higher yields in strawberry, basil and cucumber are not due to increased investment into root biomass.
Figure 5: A) Ratio of shoot and root dry biomass from crops grown in Bioponix system divided by field biomass. A ratio under 1 indicates a higher biomass in the field group, while a ratio over 1 indicates a higher biomass in the Bioponix group. Ratios consisting of significantly different values are marked with an asterisk. B) Shoot to root dry biomass ratio of various crops grown in Bioponix system and in field soil. Significantly different (p<0.05) treatment pairs are marked with an asterisk, error bars are 1 SE.
Observed AMF colonization rates were 15 ± 3% in the field and 2 ± 2% in the Bioponix units. This means that the significantly higher yield observed in the Bioponix strawberries was correlated with a lower AMF colonization. Colonization rates of intentionally inoculated strawberry plants reported in the literature vary between 25 – 75 % (Chávez and Ferrera-Cerrato, 1990; Norman et al., 1996), so the AMF colonization rate observed in the field strawberries was lower than is typical. The lower colonization across both treatments may be due to the lack of nutrient or water stress, as plants are known to initiate AMF symbiosis when under biotic or abiotic stress. It may also be explained by the late inoculation, as it is unknown if any AMF were present in the growing medium that the strawberry seedlings were purchased in.
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An alternative explanation for the higher strawberry yield is the higher nutrient availability due to the higher fertilizer use efficiency of the Bioponix system, as well as the higher total applied fertilizer.
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I observed, but did not quantify, crop diseases during the study. Downy mildew was observed in all basil plots, necessitating an early harvest (Figure ). Also, tomato plants in both field and Bioponix plots suffered from Septoria leaf spot as well as from blossom end rot. While there was no qualitative difference between treatments in terms of disease appearance, a quantitative difference in disease impact may be a source of variation that made the treatments more similar with respect to yield and biomass.
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As transplanted seedlings already have a partially developed root system, this did not allow me to distinguish the full effect of the Bioponix planters and field environment on crop root development from seed to physiological maturity. It will be informative to repeat the trial and grow the crops from seed, vegetative material (e.g., strawberry runners of a uniform age/size) or young transplants (e.g., tomato seedlings with 2-4 compound leaves).
Figure 7: Various diseases observed in field and Bioponix plots. All diseases were seen in field and Bioponix plots. A) Downy mildew on basil in a field plot. The whole plot was lost to this infection, and other plots were harvested early when symptoms spread. B) Blossom end rot on tomato in Bioponix unit. C) Septoria leaf spot on Bioponix tomato.
Methods
Site Description and Materials
Both Bioponix above-ground units and in-ground field crops were located in field 109W at the Emile A. Lods Agronomy Research Centre (45°’25’ N, -73° 55’W) in Ste-Anne-de-Bellevue, Quebec. The soil types in this field are Chicot (Gleyed Melanic Brunisol) and Chateauguay (Gleyed Eluviated Melanic Brunisol).
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Plant varieties were chosen based on seedling availability: “Better Boy” tomatoes Solanum lycopersicum (indeterminate), “Just Sweet” peppers Capsicum annuum (yellow miniature bell peppers), “Buttercrunch” lettuce Lactuca sativa var. capitata (Bibb type), “Straight Eight” cucumber Cucumis sativus, unknown cultivars of everbearing strawberries Fragaria × ananassa and sweet basil Ocimum basilicum. All seedlings were purchased from Pépinière de l'Ouest Inc. and Pépinière Cramer Inc. in Notre-Dame-de-L’Ile Perrot.
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Experimental Design
Bioponix units were planted on the 14th of July. Units were filled with 5 L of a 1:1 mixture of Sun-Gro Professional Growing mix (85% sphagnum peat moss, 15% perlite) and Fafard Marine Compost (details in Appendix). 20 ml (about 5 g) of MYKE Vegetable and Herb Mycorrhizae and 20 ml (about 10 g) of Gaia Green Vermicompost were added as inoculants.
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Tomato, pepper and cucumber seedlings were planted one per cassette insert i.e. 2 per Bioponix unit. Basil was planted one cluster of (2 to 5) plants per cassette, as they could not be separated. Only one cassette was planted per unit for the strawberry trials, as only 6 individual plants could be sourced total. Lettuce was sown at the recommended density (5 mm spacing) in two rows per cassette. Tomato and strawberry plants already had reproductive structures present in the purchased seedlings, which were trimmed to promote vegetative growth after transplanting.
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1 L of soil conditioner in water (2 mL/L) was applied to the mixture after planting, then the reservoirs were filled with soil conditioner in water. Planters were topped up with Sun-Gro Professional Growing mix after a few days when the soil had settled.
For the field trial, crops were planted in 3 m by 3 m plots separated by 1 m. Field experiment plots 1-12 were planted on the 15th, plots 13-17 on the 18th of July. For all crops except cucumber, holes of ca. 30 cm diameter and 20 cm depth were hand-dug and filled with 5 L of the same mixture as the Bioponix units. Cucumber seedlings were planted one plant per mound on mounds of the same mixture ca. 20 cm high and ca. 30 cm in diameter. After planting, the soil around the plant was watered to saturation.
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Gaia Green 4:4:4 solid fertilizer was applied at two points during the growing season at a rate of 20 g per plant. Bioponix units received additional Bionova Veganics Grow 3-2-4 liquid fertilizer, at a season total of 12 L per plant.
Methods of Analysis
Chlorophyll content was done with a SPAD-502 meter (Konica-Minolta, Japan) on three fully developed non-senescent leaves. Each recorded value is the average of the three measurements. Blocks of each treatment were measured alternatingly in order to avoid bias due to changing weather conditions.
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Stem elongation was measured from the point where the stem emerged from the ground to the farthest shoot apical meristem. In the case of strawberry plants, the longest distance from the crown to the center of a trifoliate leaf was used.
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Samples were prepared for AMF colonization measurements by heating in 2.5% KOH for 2 min followed by rinsing and acidification in 1 M HCl for 5 min. Samples were then stained in 0.01% acid fuchsine in 1:1:1 lactic acid, glycerol and deionized water. After heating to set the colour, samples were rinsed and preserved in 1:1 glycerol and deionized water (Sobat and Whalen, 2022). Colonization was then quantified using the gridline intersection method (Giovannetti and Mosse, 1980) under a dissection microscope with 60× magnification (ZEISS SteREO Discovery V8, Germany).
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Calculations
Relative growth rate was calculated according to Equation 1 (Hoffmann and Poorter, 2002), adapted to use stem elongation instead of biomass.
Equation 1: Relative growth rate calculation
Statistical Analysis
All error bars shown are standard errors. Statistical significance between treatments was assessed using a one-tailed heteroscedastic t-test.
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Impact of Partner Organization on Research
The Bioponix units were set up and planted according to the instructions of members of the partner organization, who assisted with the planting. They also provided advice on the timing of the fertilizer treatments and suggested the additional application of liquid fertilizer to the Bioponix units to resolve the visual nutrient deficiency symptoms.
Benefits to Partner Organization
The yield advantage of the Bioponix system compared to field agriculture was demonstrated for some crops. This data will help the company market their growing system.
The insight that the higher yield in strawberry was not caused by greater AMF symbiosis may point to other growth-enhancing properties of the Bioponix system, which merit further investigation by BIOPONIX and its research partners.
Future Research Plans
Comparing Bioponix container agriculture to crop production in field soil is important because most of the organic vegetable production occurs in field environments. However, it remains important to compare the Bioponix system with other container systems, such as conventional container agriculture or hydroponic agriculture. Future research might focus on these comparisons to provide more compelling evidence to users of container-based technologies.
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References
Baum, C., El-Tohamy, W., Gruda, N., 2015. Increasing the productivity and product quality of vegetable crops using arbuscular mycorrhizal fungi: A review. Sci. Hortic. 187, 131–141. https://doi.org/10.1016/j.scienta.2015.03.002
Chávez, M.G., Ferrera-Cerrato, R., 1990. Effect of Vesicular-Arbuscular Mycorrhizae on Tissue Culture- derived Plantlets of Strawberry. HortScience 25, 903–905. https://doi.org/10.21273/HORTSCI.25.8.903
Giovannetti, M., Mosse, B., 1980. An Evaluation of Techniques for Measuring Vesicular Arbuscular Mycorrhizal Infection in Roots. New Phytol. 84, 489–500. https://doi.org/10.1111/j.1469- 8137.1980.tb04556.x
Hoffmann, W.A., Poorter, H., 2002. Avoiding Bias in Calculations of Relative Growth Rate. Ann. Bot. 90, 37–42. https://doi.org/10.1093/aob/mcf140
Norman, J.R., Atkinson, D., Hooker, J.E., 1996. Arbuscular mycorrhizal fungal-induced alteration to root architecture in strawberry and induced resistance to the root pathogenPhytophthora fragariae. Plant Soil 185, 191–198. https://doi.org/10.1007/BF02257524
Our System [WWW Document], 2020. . Bioponixag. URL https://www.bioponixag.com/our-system (accessed 1.13.23).
Sobat, E., Whalen, J.K., 2022. Mycorrhizal colonization associated with roots of field-grown maize does not decline with increasing plant-available phosphorus. Soil Use Manag. 38, 1370–1379. https://doi.org/10.1111/sum.12786
Appendix
Appendix Figure 1: Individual graphs for Figure 2. SPAD readings and relative growth rates of all crops. Relative growth rate was calculated using stem elongation measurements. Error bars are standard error of three replicates (two in the case of cucumber)
Fafard Sea Compost
INGREDIENTS
Compost, sphagnum peat moss, seaweed, shrimp flour
GUARANTEED MINIMUM ANALYSIS Total Nitrogen (N) - 1.2%
Available Phosphoric Acid (P2O5) - 0.7% Soluble Potash (K2O) - 0.6%
Calcium (Ca) - 1%
Magnesium (Mg) - 0.2% Sulphur (S) - 0.025%
Iron (Fe) - 0.1%
Zinc (Zn) - 0.01%
Boron (B) - 0.001%
Minimum Organic Matter - 33% Maximum Moisture - 55%
