Our Entomopathogen Fungi lab team constantly gathers soil samples from different fields to identify the type of fungi and/or bacteria that may be there, treating the crops in a biological and organic manner, preventing them from being affected by plagues and diseases that would otherwise have to be treated with chemical solutions.
The productive dynamic is conducted under an Intraplot System, which means going to the orchards, taking soil samples, isolating, multiplying the microorganisms of interest, and then returning them to the same soil.
The identification process for soil fungi and/or bacteria, as well as the multiplication of microorganisms (entomopathogenic fungi and Trichoderma spp), is conducted as follows:
Larvae and/or insects are inserted in the different soils and a “tracking” process is activated for 5 days. The insect commonly used is the wax moth larva (Galleria mellonella), since it does not have the new immunologic defenses, therefore any external agent or contaminant will make it sick, and it is precisely the cause of infection what will tell us if the soil is suitable for crops or not.
The first day starts with the so called “turning” period, where larvae are forced to move across the soil, end to end. On the second day, the direction is changed. The more times larvae move across the soil, the better the result of the study. This is performed for 5 days, at environmental temperature. On the 6th day, larvae are removed from the soil and locked up in a humid chamber for 3 or 4 more days, at 20 to 25°C. In this process larvae die, and we are able to identify the cause of death because of the colors shown on their intersegment joints. For instance, if they died because of fungi or parasites such as Beauveria, we will identify white mycelium formation. If they died because of Metarhizium, they will show a very particular olive green color. If they died because of nematodes, they will show a shiny color.
Once the microorganism is identified, it is isolated, controlled, and compared to the target plagues or diseases, according to the time lapse and percentage of infection. The higher the infection percentage in the lowest time possible, the better the field efficiency is qualified.
As a standard, under laboratory condition, strains with percentages equal or higher than 80% within 8–12 days are released. On the field, conditions are variable due to several factors such as the time of application, the broth pH, calibration, level of expertise of the user, among others.
Once the induction stage of the microorganism is finished, the inoculation phase starts, which consists in extracting the parent strain — fungus in pure state — which is isolated, they are sterilized and left in a culture between 15°C and 24°C for 10 to 20 days.
Once the culture period ends, fungi are inserted into the substrate which will be applied to the soil. Then, they are dissolved in a mixture of rice and sterilized water at 121.1°C for 60 minutes in autoclave.
After identifying the kind of fungus or bacteria that caused the death of larvae, it is extracted. Once this stage is finished, we have to introduce the fungi into the substrate which will be applied to the soil. Then, they are dissolved in a mixture of rice and water, which is sterilized at 121°C in order to guarantee no other kind of contaminants are present. Once cooled and sterilized, a bag is filled with this mixture, which is the culture broth of the fungus/bacteria strain.
Once cooled and sterilized, the inoculation or sowing with the chosen strain takes place, under a laminar flow cabinet (air purification up to 99.9%).
It is important to necessarily comply with the sterilization and air contaminant control phase. Otherwise, unwanted contaminants — such as spp fungi, spp bacteria, spp yeasts — could be obtained.
Next, the inoculated substrate will stay in an incubation room with artificial light (no sun light) for 10 to 15 days, at a controlled temperature of 20–25°C, putting special emphasis on stirring the mixture in bags once a day.
In this stage, unwanted contaminants or low growth could be also observed, due to failures in the sterilization or inoculation process.
Each contaminant must be immediately removed through the sterilization process in autoclave. This way, there is no exposure of the environment or people to unwanted sanitary risks.
Once the 10-to-15-day incubation period ends, the product is still highly humid in the substrate, which is between 80% and 95% humidity.
The pre-drying process aims to reduce the amount of humidity to a range between 40%–50%, which is achieved after 10 to 12 days under environmental conditions.
After reaching the aforementioned range, the product is evaluated in terms of purity (>95%), viability (>95%) and conidium content (>1×109).
The final product in the bag must be homogeneous in color, without alterations or fermentations. It must show a fungi odor and according to the characteristics described for each microorganism.
Once the quality control process is approved, the substrate is packed in 800-gram bags with double vacuum sealing, which is finally sealed at a pressure of -1 bar.
This process, of which the purpose is reducing humidity and sealing the product, is performed to stabilize the doses and be able to store them for 30 days unrefrigerated or for 6 months under 4°C–6°C conditions.
Depending on the kind of plague or disease, the application is performed during irrigation when the target is the root of the plant, or through spraying if the risk of disease is in the air.
Once applied, through sprayers or through irrigation, fungi start reproducing through conidium formation, which spread by the wind or by the means of any other mechanical action, whether through people or any other agent such as insects, animals, rain, etc., resulting in the natural spreading of the fungi.
This way, soil, organic matter, and substrate start to interrelate introducing living organisms, such as weed and blueberry plants, interacting with the roots of the plants and the fungi, as well. The latter recognize plants as hosts, generating mycotoxins that plants recognize and integrate into their system.
Because of this natural, 100% organic process, plants will not suffer from plagues or diseases, because the microorganisms will be present across the whole plant’s system, which is finally translated into the highest quality organic and conventional fruit, to deliver the best berries to our customers around the world, every day of the year.
