Environmental Project, 982 – Quantification and Identification of Active Microorganisms in Microbial Plant Protection Products

Quantification and Identification of Active Microorganisms in Microbial Plant Protection Products

Summary

This project investigates all Microbial Plant Protection Products (MPPP) that were on the Danish market in January 2004. These products are regulated by Statutory order no. 533 of 18 June 2003, which is based on EU Directive 91/414/EEC.

The general objectives of the project were to check and evaluate the MPPP on the Danish market. Specific objectives were to verify the identity and quantity of the active micro-organisms, detect any microbial contamination and compare with information provided by the producers.

During collection of the MPPP it was noticed that not all the label information requested for authorization was provided on the labels. There was a lack of batch number and date of production and expiry.

The abundance of active micro-organisms in the MPPP was found to be within a comparable range of the abundance specified by the producer of all the products based on the bacteria Bacillus thuringiensis and Streptomyces griseoviridis. This was also the case for the products Binab TF WP and Rotstop with fungi as the active micro-organism. However, three products based on the fungi Trichoderma spp., two products based on Verticillium lecanii, and two products based on Beauveria bassiana contained significantly lower number of active micro-organisms than specified by the producers. The lower number might have implications for the risk assessment and especially for the efficacy evaluation of the products.

The number of active micro-organisms was determined by counting colony-forming units (CFU). This method holds some drawbacks like incomplete separation of clumping spores prior to plate spreading and low culturability at the incubation conditions. Especially, separation of spores can be optimized further. However, the lower abundance can be true and be due to low viability or lower abundance of spores in the products.

The identity of Bacillus thuringiensis as the active micro-organisms in Dipel ES, Bactimos L and Vectobac 12AS was verified by molecular analysis and production of crystalline inclusions containing d-endotoxin, which is the toxin responsible for the plant protection. Of the 63 isolates tested, all were determined to belong to the B. cereus subgroup of the genus Bacillus and 62 produced the crystals. The active micro-organism in Mycostop was identified as Streptomyces umbrinus and not S. griseoviridis as specified by the producer. The active fungus in Mycotal was identified as Lecanicillium muscarium and in Vertalec as L. longisporum. The active micro-organisms in these products have until now been known as Verticillium lecanii. However, a recent revision of this genus has led to segregation of the two strains into two new species. The active micro-organisms in the remaining MPPP based on fungi were identified to the species specified by the producers.

Contaminating micro-organisms were not detected in any of the MPPP that are based on bacteria and neither in the fungal products: Binab TF WP, Mycotal, Vertalec, and BotaniGard ES and 22WP. In Rotstop and in one batch of Tri002 a relatively low number of contaminating bacteria was found (just above the detection limit). However, in five batches of three products based on Trichoderma spp.: Supresivit, Tri002 and Tri003, significant numbers of contaminating bacteria were found. In Supresivit it was well above the number expected from the information specified by the producer. For Tri002 and Tri003 the producer had specified a higher number of contaminating bacteria in the product. Although contaminating bacteria were indeed found here, the level was not as high as specified by the producer. The preparation of these MPPP may be conducted in ways that have a higher chance of including bacteria, e.g. by the use of clay materials in the formulations. The presence of bacteria in a product with fungi as the active micro-organism may represent a problem if the bacteria affect the efficacy and proliferation of the active micro-organism or if the bacteria have health or environmentally related risks.

This project is the first conducted in Denmark with the aim of quantifying and identifying the active micro-organisms in all the MPPP on the Danish market. As far as we know it is also the first project of its kind being performed in the EU. The results have shown that the identities of the active micro-organisms in the products are affected by on-going changes in taxonomy of micro-organisms. Further, the identification depends on the details of taxonomic keys used and in particular the increased use of new molecular based techniques such as DNA sequence homology for species affiliation. For some of the MPPP the abundance of the active micro-organisms were lower than specified by the producers. The occurrence of contaminating micro-organisms in some products based on Trichoderma spp. was higher than specified by the producers, which merits further attention. The method of quantification of active micro-organisms may be further optimized especially regarding separation of spores prior to plating.