Overview
As Fusarium Wilt continues to wreak havoc on the banana industry, our goal with hardware is to create a practical product with which to implement our engineered biofungicidal rhizobacteria. We prioritized the simplicity in our design for farmers to effectively understand, attach, and use appropriately. We created a two-pronged approach by creating a pourable bottle of liquid inoculant and by designing an addon fertilizer injector for existing drip irrigation systems.
Addressing Issues
Application Methodology
Due to root turnover (the shifting of the root system of a banana tree searching for nutrients), our product must be applied regularly to maintain sufficient coverage and protection. Considering this, we will have to apply the liquid inoculant approximately 16 to 18 times per year for every banana tree, equating to roughly one application every 3 weeks. We discuss this after the section on Liquid Inoculant.
Horticulture Methods
Bananas are usually planted around 1.2 to 1.8 meters apart in large plantations; this equates to 3000 or more banana plants per acre (NHB). This density, along with plantation sizes reaching tens of acres in size, presents a large logistical issue. If our inoculant is to be applied manually, it would require multiple farmers long days of work to protect each banana plant. The distance and energy for the inoculant to be supplied to each farmer and poured directly onto the plant is simply impractical. Instead, it would be more applicable to automatically distribute the inoculate with a system or machine.
The Liquid Inoculant
Microbial inoculants, a mix of beneficial microorganisms and other beneficial ingredients, are widely used to improve soil conditions, crop productivity, and control pests and diseases (Xitebio, 2022). The inoculant helps create favorable conditions for both the plant and the microorganism, guiding to symbiosis between them. There are two types of inoculant, granular and liquid.
Specifically for liquid inoculants, both the concentration and acres treated per bottle are higher than granulated inoculants. Also with liquid inoculants, the microorganism can be applied onto the banana plant both prior to and after planting. Overall, using a liquid inoculant seems to be the most efficient method of delivering our probiotic into the banana plant rhizosphere.
Ingredients
Banana plants expend large amounts of resources when combating diseases like Fusarium Wilt. With this in mind, we designed the inoculant to improve probiotic root adhesion and plant growth by adding supplemental ingredients; these ingredients act as adjuvants that can potentially enhance the banana plant’s overall health and chance of survival against FOC.
Table 1: Liquid Inoculant Ingredients List.
| Ingredient | Amount (% in total volume) |
|---|---|
| Earthworm Castings | 5% |
| Plant-Derived Protein Hydrolysate | 5% |
| Kelp (Ascophyllum nodosum) extract | 10% |
| Manganese Sulfate (MnSO4) | 5% |
| Soluble Potash (K+) | 4% |
| Soluble Phosphate (H2PO4) | 5% |
| Nitrogen Sulfate | 4% |
| Malic Acid | 1% |
| Water | 61% |
| B. subtilis | 108~109 CFU/ml |
The hummus in the earthworm casting increases the soil's water retention, preventing the nutrients and probiotics inside the water from leaching away (Mara, 2022). Plant-derived protein hydrolysates increase the plant’s nutrient-use efficiency by alleviating the abiotic plant stress due to salinity, drought, and heavy metals, maintaining the plant’s growth pace under low nutrient availability, resulting in increased productivity and quality (Colla et al., 2017). In our case, the protein hydrolysates can help maintain the use efficiency of our probiotics, even in harsh conditions. Furthermore, kelp—Ascophyllum nodosum extracts enhance plant tolerance to biotic and abiotic stresses through both supplementation of stress-protective compounds taken up by the plant and through elicitation of systemic responses (Hines et al., 2021). Adding kelp to our liquid inoculant spreads the probiotics more thoroughly, coats the roots, and increases the use efficiency of our probiotics like the protein hydrolysates.
In addition, we also addressed manganese deficiency in bananas by adding manganese sulfate, an essential nutrient for plant growth (Brouder, S. et al.,2003). Manganese sulfate is highly soluble and suitable for soil and hydroponic applications (Brouder, S. et al.,2003). Besides manganese deficiency, bananas also suffered from potassium, phosphorus, and nitrogen deficiency (Wasylyk, 2022). Therefore, soluble potash, soluble phosphate, and nitrogen sulfate will all be added to the product.
We also considered the safety and potential side effects of our liquid inoculant. B. subtilis, a common, benign soil microbe and essentially considered to be nonpathogenic will not pose a threat to the banana (Olekar et at., 2012). Most of the ingredients are essentially fertilizers; however, studies showed that excessive amounts of fertilizers may still affect plant health (Weisenhorn & Hoidal, 2021). Hence, after referencing similar products, we decided to keep the concentration of all nutrients at 4%-5% (Roots inoculant, Dekker, 2016; Worm Tea, Microbe Brew).
Our Bacteria
To successfully implement the bacteria, it must also be able to survive long-term within the inoculant. We'll take advantage of the sporulation mechanism in our theorized chassis B. subtilis, a defense system against environmental stress (Tan & Ramamurthi, 2014). Sporulation turns the bacteria into an endospore and allows it to endure harsh environmental conditions, only to germinate when favorable conditions return. Utilizing B. subtilis’ spore-forming capabilities, our bacteria would be able to survive nearly indefinitely within the liquid inoculant, increasing its shelf-life significantly. To add these spores to our inoculant, the bacteria first must be grown up to 108~109 CFU/ml using a fed-batch culture with a nutrient-feeding bioreactor (Monteiro et al, 2005). Then, we can purify the endospore and combine it with the inoculant for packaging and delivery. When the bacteria is introduced to the soil around the banana plant, it reacts to sugars and amino acids in the soil, activating germination and resuming protein production and protection for the bananas (Paidhungat et al,. 2002).
Application Methodology
Due to the phenomenon of root turnover, the physical extent of the banana tree root system shifts and moves significantly; to account for this, our product must be applied with sufficient frequency to maintain the coverage of our bacteria over the roots. Taking Dr. Drenth’s advice to ensure the probiotics’ protectivity by covering the roots from an earlier stage, we decided to apply the liquid inoculant to the banana plant from the nursery stages and continue to do so throughout the plant’s whole lifespan to eliminate the possibility of unexpected infection caused by an insufficient amount of probiotics. This will allow better localization of our probiotics into the rhizosphere, much like how gut microbiota establish residency in the early stages of human life.
Frequency
Since the primary banana roots are the ones growing from the corm, the thicker secondary roots are produced from the primary (Weinert, 2022). Then, the feeder roots, or so-called root hairs, are produced on the secondary roots and take up nutrients from the soil (Weinert, 2022). In this case, they are the ones that will absorb some of our probiotics. Since the root hairs will only remain functional for three weeks before being replaced by new feeder roots, the application of our probiotics after the first four months will be at least once every three weeks (Weinert, 2022).
Contributing to Dr. Drenth’s suggestion, we researched the first stages of the banana lifespan. The first four months can also be seen as the plant establishment stage, which is the developmental period before the growing space is fully occupied (Naiman et al., 2005). To let our bacteria cover the whole root system and soil during this stage, the constant application of applying our liquid inoculant once in 10-14 days during the first four months when the root growth rate is the highest is essential (Greeneden, 2022).
According to our plan, we will have to apply the liquid inoculant approximately 18 times a year. Assuming 4 weeks in a month, and a once-every-two-week application regimen during the first four months after the banana plant is transferred into the field, the liquid inoculant will have to be applied 8 times in total [2 x 4 = 8]. Four months after the banana plant starts to grow until the harvest, the product will be applied 10 times. With eight months left in the banana cultivation cycle and a once-every-three-weeks regimen, we will have to apply the inoculant a further 4(8) / 3 ≈ 10 times. Therefore, the liquid inoculant must be applied 10+8 = 18 times per year. After the first year, our inoculant will be applied once every three weeks, so it will be applied (12 x 4)/3 = 16 times a year.
Dilution Ratio
Based on the application rate of other liquid inoculants available on the market, we estimate that 250ml of inoculant must be used per round of application. Before actual application into the soil, the inoculant is diluted at a 1:100 inoculant to water ratio to a final volume of 25L.
Application Summary
Though our theory states that an application of 18 times a year our plan requires actual experimentation within the field to prove its efficacy. If the plan does not work, we will improve our current method based on the experimental results to attain a practical plan which benefits the banana industry by preventing Foc infection.
Inoculant Integration
To integrate our inoculant into the field in an automated manner, we chose to combine our inoculant with existing drip irrigation systems. For contextualization of what drip irrigation systems are, click here to download the contextualizing document.
Diagram for the Fertilizer Injector design
Fertilizer injectors are used by growers to apply water-soluble fertilizers to plants. To decrease the amount of manual work and simplify the process for farmers, we integrated our liquid inoculant into the drip irrigation system, by connecting our fertilizer injector to the mainline of the system. We created a low-cost and efficient fertilizer injector as an addon for farmers using drip irrigation systems. In our design, a large tank filled with our inoculant will supply inoculant to the drip irrigation through the main irrigation pipe. With a low-cost setup using PVC pipes and custom hose fittings, the fertilizer injector can be attached to the main irrigation line. It should have a low assembly time and be cheap to attach.
There are three main components to our addon design: the inoculant tank, the manual gate valve, and the flow-through fertilizer injector. The inoculant tank, holding the liquid inoculant, has a capacity of 20L. With an estimation of 5-6L of liquid inoculant needed for one hectare per year, a full tank will be able to supply up to 4 hectares for a year. The hectares treated per tank can be increased by regularly refilling the tank through the screw cap with more inoculant. The screw cap can be accessed easily and creates a tight seal, decreasing the exposure of the inoculant when the tank is not being refilled. The top manual gate valve controls the flow of water going through the pipe. When turned off, water can simply flow through the main irrigation pipe without trouble. When turned on, however, the water is cut off and has to flow through the bottom pipe to continue flowing. As it passes through the alternate pipe, it flows through the flow-through fertilizer injector. Here, we utilize the Venturi Effect, where the faster the water, the lower the pressure. In the fertilizer injector, the water speeds up as it passes through the venturi constriction, lowering the water pressure and pulling the inoculant through the pipe and mixing it with water. After a specified amount of time has passed, the top manual gate valve should be turned off again and the bottom valve should also be closed to resume normal water flow and stop the inoculant from flowing.
Product Material Choice
High-density polyethylene (HDPE) will be used as the material for both the bottle and the fertilizer Injector design. The material is used as the standard for biopesticide storage for its recyclability and low cost (Mishra et al, 2020). We hope to use recycled HDPE to decrease the strain plastic has on the global climate. In addition, the piping is made of PVC (Polyvinyl Chloride), which is resistant to corrosion, chemicals, and failures. This, along with the ease of use and low price, makes it the best material for our purposes. Garden hose fittings are made out of either plastic or brass. Brass is an alloy of copper, zinc, and other materials, and it resists corrosion. These materials are also lost cost, and easily accessible. Garden hose fitting also consists of 2-way, 3-way, and 4-way tubes, allowing a single garden hose to be connected to one or more hoses with equal distribution of water, minimizing costs for extra hoses and fittings.
Future Possibilities
Through the meeting with team Thailand_RIS, they suggested that we apply the proteins that are produced by the bacteria, but not the bacteria itself. This alternative implementation method may help alleviate the pressure we faced from governmental regulation, which has a stricter standard for genetically modified organisms compared to proteins. Expanding on this method will also allow our product to be applied in fields much sooner than what would’ve been with genetically modified organisms.
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