The innovation activity seeks to replace synthetic plant protection products with a biological preparation that is just as effective in controlling fungal diseases and insects in cruciferous crops.
It is important to reduce pollution in the field that derives from synthetic pesticides besides the leaching of nutrients in the conditions of integrated agriculture. Also, pesticide residue in the soil damages useful organisms who would control both the pests and the pathogens. During spraying, the used pesticide may spread from the field away to the surroundings through air by dust formed from soil particles and by water. Moreover, excessive use of pesticides brings along the resistance capability of pests against plant protection products, which causes new preparations to be put to use. These problems have in turn caused the need to design new innovative and alternative pest control methods.
The environmental trend in plant protection is moving towards the use of microbiological preparations, where the active ingredient is a tiny organism – a fungus or bacterium. Many bacteria secrete toxic compounds during their life cycle. When ingested by an insect, these compounds destroy the pest. There are also fungal organisms that specifically cause disease or death in insects. When an entomopathogenic fungus sporulates on the surface of the cuticle covering the insect’s body, it invades the insect’s body – therefore providing a good choice alternative for targeted pest control.
Using bees as carriers for biological plant protection
Honeybees, which carry biological powder preparations on flowers, are used as a novelty in the application of plant protection products to plants. The formulations contain various microorganisms, the effectiveness of which in controlling pests depends on environmental conditions such as temperature, humidity and sunshine.
The use of microbial organisms in pest control is more environmentally friendly than the use of synthetic preparations, but unfortunately the use of living organisms is more sensitive to environmental effects. The micro-organism may be effective against the pest, but if the environmental conditions are not suitable, the preparation may not work. For example, exposure to sunlight may render the formulation ineffective. In addition, it should be considered that if the preparation works against the pest, it is possible that beneficial insects may also be affected.
Spraying crops with large machinery is energy, water and time intensive. The option to use bees, which fly to the flowering plants anyway, carry the preparation to the flowering crops. This method has been tested both in greenhouses and in the open field. It has also been successfully tested in Estonia for the control of powdery mildew (grey mould) on garden strawberries.
As part of the experiments, we have applied the following two biological preparations:
🌼 BotaniGard 22WP Beauveria bassiana;
🌼 Met52 Metarrhizium anisopliae
For the preparation to work effectively on the target organism, it is important to find the right factors in the pollinator-crop-plant protection system:
Firstly, it is important that the formulation worn by the bee is effective against the pest but safe for the carrier and other insects that visit the flowers.
Secondly, the method can only be applied against the pest in flowering plants (i.e. plants the bloom). In the case of oilseed rape, it is the larvae of the beetle, not the adults, that is present in the flowers. This larvae causes real damage much earlier, at the bud development stage.
The third important element is that the bee applying the preparation should always choose to fly to the plant where the preparation is to be carried and/or applied. The preference of the bees, i.e. their food choices has a direct influence on the results of the transfer of the preparation.
The preparations we are testing contain spores of microorganisms common to our soils. Applying them to plants increases the number of microorganisms on the leaves, buds or flowers. This creates an unfavourable environment for those pathogens and infects them in the development phase with entomopathogens.
In 2017, patch test was carried out at the test field of the Estonian University of Life Sciences, where a biological preparation was used to repel beetles. The number of flowers of was evaluated on the test patches, which showed that it was somewhat higher on the test patches sprayed with the preparation. Beetles were also picked from the experimental field, then kept in Petri dishes with filter paper sprayed with the preparation to simulate contact. The beetles were fed with flowers from rapeseed. Mortality due to the preparation was observed 4-6 days after spraying.
The infectivity of the preparation was also tested in a laboratory experiment on honey bees, which were not significantly affected. It is assumed that the hairs covering bees do not allow the fungal spores to reach the cuticle.
In 2018, additional testing was carried out at the experimental field of the Estonian University of Life Sciences. 20 test plots were built in a 2 ha summer swede rape field. The test plots were sprayed with water, and with standard: 1x, 2x, 5x and 10x, accordingly. The test patches with different treatment norms were arranged in random order to minimize the effects of microclimatic conditions or soil variability. Beetles were also handpicked from the test plots to assess their mortality. Among beetles harvested after spraying, mortality was observed in the laboratory from the sixth day after spraying.
Beauveria bassiana infection observed in beetles. Photo credits: Eveli Eliaser
Test rigs covered with nets. Photo credits: Anne Must
In 2019, experiments were performed in insect-resistant pavilions (right photo) established in the field to assess the infestation of beetle larvae as bees carry the preparation to plants. In order to ensure correct results, the effect of the formula was evaluated both in test plot experiments as well as in lab conditions.
The results of three years work indicate that the beetles are infected with the entomopathogenic fungus contained in the biological preparation. At the same time, it does not cause high enough mortality to prevent crop loss. The best effects of this preparation are seen in the long run, especially in organic farming, to help reduce the population of disease-causing beetles in the area.
In 2020 and 2021, trials were set up in winter oilseed rape production fields where honey bees were used as carriers of BotaniGard 22WP on oilseed rape flowers. In the first year, the apiary was located at a distance of about 700 m from the oilseed rape field; in the second year, the apiaries were located in five different oilseed rape fields at a distance of 10-200 m from the field. We were hoping to find Beauveria bassiana spores on rape flowers. To do this, we collected the flowers at different distances from the bee side of the rapeseed field. We performed molecular analysis to identify spores on the flowers. Although the methodology worked with a control field, where we deliberately introduced a large amount of the preparation, we did not find any reliable evidence of the preparation having been transferred to the flowers by bees (the DNA method also detects DNA from dead spores). Probably many more hives should be used and the preparation should be added repeatedly during the day to increase the amount of material transferred to the flowers. Although we did not check whether the bees in the experimental hives visited the oilseed rape flowers, we observed active flight from the hives towards the field.
Graph below: A decrease in the number of larvae was observed in the tunnel test plots where bumble bees introduced either BotaniGard 22 WP, Met52EC or a mixture of both (kontroll – control, segu – mixture).
Adding a preparation to a dispenser installed in a honey bee hive. Photo credits: Kristel Karise.
Above – map from Estonian Land Registry (Maa-amet), with the location of the apiary marked with a yellow arrow and the flower sampling sites marked with red circles.
On the right, PCR results of flower samples collected from different distances in a field of canola using a primer suitable for the detection of B. bassiana (photo below). M, marker; POS, positive control (indicated by arrow); NEG, negative control. If the preparation was detected on the flowers, stripes similar to those of the positive control (at similar height) should have appeared under the number indicating the distance from the harvest date.
PCR results. Credits: Anne Must.
- The mortality rate of the formula is too low or too slow to achieve effective pest control, but in the long run it may help to reduce the number of pests in the next generation.
- Both preparations infect beetles, but the effect is too slow.
- Both preparations infect the larvae inhabited in the flowers of the crop.
- The number of larvae in the soil was reduced.
- Neither preparation is suitable for controlling beetles in the bud damage phase.
- These preparations could be used as a supportive method to reduce the oversized beetle population.
Honeybees living in miniature beehives with sprayed filter paper. Photo credits: Eveli Eliaser.
Effect on pollinators
No significant increase in mortality was observed in honeybees when exposed to flowers or filter paper containing the active substance BotaniGard, Beauveria bassiana. This is also confirmed by several scientific articles. When honeybees are used as inoculants, a method must be used to prevent the bees from passing through the powder container when entering the hive. The internal temperature of the hive is higher than necessary for the development of B. bassiana. When exposed to the formula, bees are not harmed significantly, as the hair covering the pollinator’s body prevents fungal spores from entering the bee’s cuticle and the bees’ habit of cleaning the body of pollen removes most of the fungal spores.
It is confirmed that the carry-over effect of the formula from the dispenser to the beehive is unlikely as the bees lose most of the portable powder during the flight. All the ingredients of the powder are of natural origin (clay, B. bassiana) and B. bassiana is also a fungus found in natural habitants. Therefore, honey taken from hives is marketable.
The evaluation of the efficacy of methods of precision plant protection on cruciferous crops was carried out in the framework of measure 16 “Cooperation” of the Estonian Rural Development Programme 2014-2020. The budget of the innovation activity was 100 000 € and the period was 06.03.2017 – 06.01.2022.
Estonian University of Life Sciences – Reet Karise, Marika Mänd, Anne Must, Angela Ploomi, Egle Liiskmann