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Product Requirements

The goal of our project was to create a solution that would really help cherry farmers. Therefore, it was very important for us to involve them in the development of PseuPomona and create a product that meets their needs. With the additional help of other stakeholders, we identified several key aspects that needed to be taken into account when developing a bacteria-based agrotech innovation:

The main requirement of farmers, which was emphasised many times during our meetings, was the ease of use and compatibility with the practices already used. Other aspects important from their point of view are storability and affordability. Additionally, Prof. Dr. Paolina Garbeva drew our attention to the appropriate choice of methods used for processing and formulation. It was pointed out to us that all the steps of the production process should be designed in a way to ensure the highest possible cell viability, guarantee the longest shelf life, and reduce any possibility of contamination. The number of viable cells is an especially important requirement. It should be high enough so that the newly introduced bacteria are not outcompeted by the native species present in the soil and are able to successfully colonise the rhizosphere. Moreover, incorporating mechanisms ensuring safety for humans and the environment was the main requirement of the risk assessors from the National Institute for Public Health and the Environment (RIVM).

PseuPomona

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Herman Capel, a cherry farmer, applying PseuPomona.

Features:

  • Easy to apply: Application is compatible with the standard practices used by farmers. No additional equipment is required.
  • Safe: The bacteria are equipped with kill switches that guarantee their containment at the site of application, preventing uncontrollable escape into the environment.
  • Controllable: Farmers can selectively kill the modified bacteria without harming the trees or other microorganisms.
  • Efficient: Only 10 g of product per tree is enough for protection.
  • Detectable: The presence of modified bacteria in the soil can be detected at any moment with a detection kit.
  • Long shelf life: Can be stored for up to 6 months at 4-10 °C.

Product Formulation

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Figure 1: Minimum viable product of PseuPomona.

The production of microbial inoculants that would guarantee the desired effect after application in the field is one of the main bottlenecks in the development of biostimulants. Such a product must be prepared in the form of a stable formulation that will ensure the highest possible cell viability and thus its effective performance [1, 2]. Drying is the most common technique used to preserve bacteria. This is mainly due to the fact that it significantly reduces the chance of contamination and elongates the product’s shelf life [1-3]. In the case of Gram-negative species, which do not form spores and thus are more susceptible to damage, additional measures improving the desiccation tolerance are necessary [1-3]. For P. fluorescens, freeze-drying in combination with the use of lactose as a protectant proved to significantly improve the viability of cells [4]. As such, it is possible to obtain a powder with the number of viable cells ranging from 108 to 1011 CFU/g [1, 4].

To get a complete picture of what bacteria-based agricultural products look like, in addition to diving into the data present in the literature, we also looked for products containing P. fluorescens that are already on the market (Table 1).

Table 1: Commercially available products based on Pseudomonas fluorescens.

Commercially available products based on Pseudomonas fluorescens

Company Product Viable cell # Formulation Application Shelf life and storage
Agri Life, India SheathGuard™ 1×108-9 CFU/g wettable or soluble powders Seed dressing, Soil application, Foilar application Up to 6 months from the date of manufacturing at 4-10°C
Seed2Plant Pseudomonas fluorescens Biofertilizer N/A N/A Seed dressing, Soil application, Foilar application Up to 6 months from the date of manufacturing at 4-10°C
Agri Life, India PSEUDO-PEP 108 CFU/g Talcum-based powdered formulation of Pseudomonas fluorescens Seed treatment, seedlings treatment, drip irrigation, mixed with manure/compost Storage in cool and dry conditions
Peptic Biosciences Ltd. Pseudomonas fluorescens Powder N/A water-soluble powder Drenching, spraying, drip irrigation N/A

Both literature and existing P.fluorescens-based products show that it is possible to obtain PseuPomona in a lyophilised formulation. This format ensures that bacteria can be stored over long periods of time while remaining active and practical for farmers. Based on these inputs, we ideated our solution and designed the minimum viable product (Figure 1).

Detection kit

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Contents of the PseuPomona detection kit.

To ensure that our solution is safe and easily detectable, the product includes a LAMP-based detection kit that can be used directly by the farmers to check the presence of the modified bacteria in their orchards. As with the development of the main product, it was very important for us to involve them in the design of the detection method. The requirements together with how we incorporated them into our solution are shown in Table 2. To read about the molecular characteristics of the assay, visit our Results page.

Table 2: Design requirements for the detection kit and their implementation in our product.

Commercially available products based on Pseudomonas fluorescens

Requirement Explanation Implementation in our product
Easy to perform The procedure should be easy to follow and perform. It should not involve the use of complex equipment. The user manual provides a clear step-by-step description of the process. Additionally, the procedure makes use of conventional equipment already present in an orchard, without the requirement of any specialized laboratory equipment.
Fast The testing procedure should not take too much time. The test takes around an hour of preparation and runs without the need of intervention for about 30 minutes.
Safe The method should not require the use of hazardous chemicals or other unsafe materials. All the components needed to perform the test are safe to use on-site.
Non-invasive Collecting the samples should not affect the surroundings. Samples used for the test can be collected using the standard equipment and procedure used by the farmers to check for nutrient content in the soil.
Clear The results should be easy to read with a clear outcome. The colorimetric results can be seen with the naked eye, without the need of complex measuring tools.
Sensitive The sensitivity of the test should be as high as possible, limiting the chances of falsely negative results. The sensitivity of our test is 96.6% of true positives/total positives [4] (while 97.6% with PCR)
Specific The specificity of the test should be as high as possible, limiting the chances of falsely positive results. The specificity of our test is 97.6% of true negatives/total negatives [4] (while 98.7% with PCR)

User manual

An on site instruction video showcasing the procedure of using PseuPomona on an orchard.

If the video is not loading, watch it here.


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Figure 2: Tools supplied with the detection kit.

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Figure 3: Tools needed for the detection not supplied with the kit.

Part 1 - adding PseuPomona to the soil:

  1. Mix the bacterial powder (±10 grams per tree) with water used for irrigation.
  2. Mix solution B (the inducer) with water (5 ml of solution B for every litre of water) and add the solution to your irrigation system to water the fruit trees as usual. Repeat this step daily.

Part 2 - testing of the soil samples:

  1. After 5 to 7 days, take soil samples from under the trees in the orchard to confirm the presence of PseuPomona. The trees should be chosen from different parts of the orchard. You can combine this step with soil sampling done for other purposes like measuring the concentration of nutrients.
  2. Transfer around 1 gram of soil to a 2 mL Eppendorf tube and mix it with 1.5 mL of DNAse free water for each ground sample. Make sure to mix vigorously to achieve a uniform mixture.
  3. Let it rest for 15 minutes.
  4. Place the test tubes in the hand centrifuge and centrifuge them for at least 5 minutes or until you can see sediment formed at the bottom of the tube.
  5. Transfer the liquid into a new 1.5 ml Eppendorf tube using a plastic pipette. Try not to collect the dirt sediment.
  6. Fill the thermos flask with water, plug in the induction heater, and bring the water to 90°C. Check the temperature with the thermometer.
  7. Place the test tubes in the water for 10 minutes, then disconnect the heater, wait 30 seconds, and take the test tubes out. Important: to prevent a burn, take the tube out with the tweezers.
  8. Place the test tubes in the hand centrifuge and centrifuge them for at least 5 minutes or until you can see sediment formed at the bottom of the tube.
  9. Remove most of the liquid while leaving a few microliters behind, close the tube and tap it for the sediment to mix with the remaining solution.
  10. Place two toothpicks in every test tube and move each toothpick to a separate test tube containing the LAMP reaction mix.
  11. Mix the toothpick thoroughly with the reaction mix, it out and close the tube.
  12. Thoroughly mix the contents of the LAMP reaction tube by quickly inverting it 6 times.
  13. Check the temperature of the water in the thermos flask with the thermometer and bring it to 65°C with the induction heater if necessary.
  14. Carefully place the LAMP reaction tubes in the water for 30 minutes, then disconnect the heater, and take the tubes out. Important: to prevent a burn, take the tube out with the tweezers.
  15. Compare the colour of the solution with the examples for positive and negative controls provided on the scheme below (positive means that live PseuPomona bacteria are present in the soil, and negative means that these bacteria are absent).

Schematic overview of the detection procedure:

detection scheme

Figure 4: A scheme of the detection procedure.

The next steps lead to the removal of the PseuPomona bacteria from the soil. Only proceed when you want to resume the normal flowering event of your trees. In the meantime, monitor the risk of upcoming frost events; make sure that all the sensors placed in the orchard are working.

Part 3 - the removal of PseuPomona from the soil:

  1. When you think the risk of frost events is low enough, mix powder C (the remover) with water and add the solution to your irrigation system to water the fruit trees as usual.
  2. After 5 to 7 days, take soil samples from under the trees in the orchard to confirm the absence of PseuPomona as described in part 2. The trees should be chosen from different parts of the orchard. You can combine this step with soil sampling done for other purposes like measuring the concentration of nutrients.

Note: The test’s sensitivity is 97.6% [4], meaning that there is a 2.4% risk to not detect our bacteria.


[1] Berninger, T., González López, Ó., Bejarano, A., Preininger, C., & Sessitsch, A. (2018). Maintenance and assessment of cell viability in formulation of non-sporulating bacterial inoculants. Microbial biotechnology, 11(2), 277-301. https://doi.org/10.1111/1751-7915.12880.

[2] O'Callaghan M. (2016). Microbial inoculation of seed for improved crop performance: issues and opportunities. Applied microbiology and biotechnology, 100(13), 5729-5746. https://doi.org/10.1007/s00253-016-7590-9.

[3] García A. H. (2011). Anhydrobiosis in bacteria: from physiology to applications. Journal of biosciences, 36(5), 939-950. https://doi.org/10.1007/s12038-011-9107-0J. Cabrefiga, J. Francés, E. Montesinos, A. Bonaterra, Improvement of a dry formulation of Pseudomonas fluorescens EPS62e for fire blight disease biocontrol by combination of culture osmoadaptation with a freeze-drying lyoprotectant, Journal of Applied Microbiology, Volume 117, Issue 4, 1 October 2014, Pages 1122-1131, https://doi.org/10.1111/jam.12582.

[4] Sadeghi, Y., Kananizadeh, P., Moghadam, S. O., Alizadeh, A., Pourmand, M. R., Mohammadi, N., Afshar, D., & Ranjbar, R. (2021). The Sensitivity and Specificity of Loop-Mediated Isothermal Amplification and PCR Methods in Detection of Foodborne Microorganisms: A Systematic Review and Meta-Analysis. Iranian journal of public health, 50(11), 2172-2182. https://doi.org/10.18502/ijph.v50i11.7571.

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