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

5 Conclusions and perspectives

Quantification of the active micro-organisms in the 13 MPPP on the market in Denmark in January 2004 generally revealed the abundance of active micro-organisms to be within a comparable range of the abundance specified in all the products based on the bacteria Bacillus thuringiensis and Streptomyces griseoviridis and in 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 general evaluation and especially for the evaluation of efficacy of the products.

The number of active micro-organisms was determined by CFU counting and 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 real and due to low viability or lower abundance of spores in the products.

Molecular analysis and production of crystalline inclusions containing d-endotoxin verified the identity of Bacillus thuringiensis as the active micro-organisms in Dipel ES, Bactimos L and Vectobac 12AS. Of the 63 isolates tested, all were determined to belong to the B. cereus group and 62 produced the crystals. The active micro-organism in Mycostop was identified as Streptomyces umbrinus and not S. griseoviridis as stated by the producer. The active micro-organisms in Mycotal and Vertalec were identified as Lecanicillium muscarium and L. longisporum, respectively, and not Verticillium lecanii as stated by the producer. The active micro-organisms in the remaining MPPP were identified to the species specified by the producers. These changes in species identity accentuate the necessity to use the same strain for risk evaluation.

Contaminating micro-organisms were not detected in any of the MPPP based on bacteria and in the products: Binab TF WP, Mycotal, Vertalec, and BotaniGard ES and 22WP. In Rotstop and in one batch of Tri002 low numbers, just above the detection limit, of contaminating bacteria were found. However, in five batches of three products based on Trichoderma spp.: Supresivit, Tri002 and Tri003, significant numbers of contaminating bacteria were found. For Supresivit it was well above the number expected from the information specified by the producer, while for the Tri002 and Tri003, the producer specified a higher number of contaminating bacteria in the product and carrying material than was actually found in this study. 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. Not all containers of the collected MPPP were labelled with the information required for authorization for marketing. The missing information included batch number and date of production and expiry.

The results have shown that the identity of the micro-organisms are affected by the changing taxonomy of micro-organisms, including details of taxonomic keys used and the 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 and the occurrence of contaminating micro-organisms deviated from the producers' information, which merits further attention. The method of quantification of active micro-organisms may be further optimized, especially regarding separation of spores prior to plating.