Environmental Project, 982 – Quantification and Identification of Active Microorganisms in Microbial Plant Protection Products – 2 Investigation of microbial plant protection products

Quantification and Identification of Active Microorganisms in Microbial Plant Protection Products

2 Investigation of microbial plant protection products

2.1 Collection of microbial plant protection products
2.2 Quantification
2.3 Contaminating bacteria

2.1 Collection of microbial plant protection products

The Chemical Inspection Service in the Danish EPA collected the MPPP on the Danish market through two rounds of visits to the distributors at January 13 and January 30, 2004, respectively. One to three independent batch numbers were sampled for each product.

2.2 Quantification

2.2.1 Colony-forming units

Bacillus thuringiensis

From each batch of the B. thuringiensis (Bt) products triplicates of one ml were aseptically transferred to Blue Cap bottles and diluted 10 times with 9 ml phosphate buffer (pH 7.0). Five mg proteinase (proteinase K recombinant PCR Grade cat. no.745 723, Roche Diagnostics GmbH, Mannheim, Germany), 125 μl 10% SDS (w/v) (lauryl sulfate, SL-4390, Sigma Chemical Co., Steinheim, Germany) and 82.5 mg Dowex (50 Wx8, 44505, Fluka Chemie AG, Buchs, Schwitzerland) were added to one-ml samples, which were stirred for 3.5 hours at room temperature. Initial experiments had shown this treatment to give the best separation of the spores (appendix a). Ten-fold dilutions in phosphate buffer, pH 7.0, were spread plated on triplicate agar plates with 100 μl on each plate. The media used was Tryptic Soy Agar (TSA) which consists of 30 g l^-1 Tryptic Soy Broth (02-200, Scharlau Chemie, Barcelona, Spain) solidified with agar (15 g l^-1) and autoclaved at 121°C for 20 min. TSA is the media recommended by the producers. The importance of media composition was compared by culturing Vectobac 12, batch number 140046, on TSA as well as T3 media which is known for its ability to support growth of bacteria from the Bacillus cereus subgroup of the genus Bacillus (Travers et al. 1987). The number of CFU was enumerated after incubation of the agar plates at room temperature for 2 days.

At the time of CFU counting, one colony was isolated from each counted plate, resulting in 9 individual colonies from each batch. In addition, 9 colonies were isolated from T3 media with Vectobac 12, batch number 140046. The colonies were further purified by re-streaking on TSA-plates and after re-growth cultured in 2 ml 1/10 Tryptic Soy Broth overnight. From 1.4 ml of this overnight culture, DNA was isolated by a freeze-thaw-boil method (Johnsen et al. 2002). The remaining 600 μl were stored at –80°C.

Fungi and Streptomyces griseoviridis

Fungal spores and hyphae and S. griseoviridis spores were extracted, separated and cultured on agar media as prescribed by the manufactures with modifications according to Table 2.1. The agar media used was 2% malt extract agar (MEA) (cat. no. 01-573 Scharlau Chemie, Barcelona, Spain) solidified with agar (15 g l^-1) and potato dextrose agar (PDA) (cat. no. 01-483 Scharlau Chemie, Barcelona, Spain). Both agars were autoclaved at 121°C for 20 min.

At the time of CFU counting, the morphology of the colonies was checked and representative colonies were isolated for identification.

Table 2.1: Extraction, separation and culturing of BCA products with fungi and actinomycetes.

BCA	Extraction and separation procedure	Agar media	Incubation conditions
Supresivit	•0.40 g product + 10 ml MQ H2O + 1 drop Tween80 •Shaking 15 min. in multi wrist shaker	2% MEA^a	room temperature
Binab TF WP	•0.40 g product + 10 ml MQ H2O + 1 drop Tween80 •Shaking 15 min. in multi wrist shaker	2% MEA^a	•room temperature
Tri002 & Tri003	•0.40 g product + 10 ml MQ H2O + 1 drop Tween80 •Shaking 15 min. in multi wrist shaker	2% MEA^a	•room temperature
Mycotal & Vertalec	•1.00 g product + 1 l MQ H2O •Stirring with stir bar for 30 min.	2% MEA^a	•room temperature
BotaniGard ES & BotaniGard 22WP	•5.00 g product + 95 ml 0,1% Tween80 •Shaking 15 min. in multi wrist shaker	PDA^b	•room temperature
Rotstop	•0.20 g product + 200 ml MQ H2O •Blending for 1 min. at max speed in Laboratory Blender	PDA^b	•28°C, 2-3 days
Mycostop	•0.15 g product + 150 ml MQ H2O •Stirring with stir bar for 30-60 min. •Blending for 1 min. at max speed in Laboratory Blender	PDA^b	28°C, min. 2 days

a:: Malt Extract Agar

b: Potato Dextrose Agar

2.2.2 Molecular identification of strains from the Bacillus cereus group

Bt is a member of the B. cereus group, which is a subgroup of the genus Bacillus. Other known members of this group are B. cereus, B. anthracis, B. mycoides, B. weihenstephanensis, and B. pseudomycoides. The genetic similarity of the isolated Bt strains was studied by PCR amplification of the internally transcribed sequence (ITS) between 16S and 23S rDNA, which is a highly variable sequence. The primers used were ITS-16S-1392-S-15 and ITS-23S-206-A-21 (Willumsen et al. under revision) and they target conserved sequences in 16S and 23S rDNA, respectively. The method is referred to as PCR-ITS and the resulting PCR-ITS products were analyzed by electrophoresis in 2% agarose gels.

2.2.3 Formation of crystalline inclusions containing delta-endotoxin in the Bacillus thuringiensis cultures

All Bt strains with insecticidal activity forms crystalline inclusions containing d-endotoxin in sporulated culture (Hansen et al. 1998). Hence, the isolated strains were tested for formation of crystals by the following procedure: The isolates were grown on T3 agar media at 30°C for 3 days to ensure sporulation. The sporulated cell cultures were then examined for the distinct shape of the crystalline inclusions containing d-endotoxin by wet mount phase contrast microscopy at 1000 times magnification.

2.2.4 Identification of fungi and actinomycetes

The colony morphology of the fungi Trichoderma harzianum, T. polysporum, Verticillium lecanii, Phlebiopsis gigantea, Beauveria bassiana and Streptomyces griseoviridis was visually inspected after growth on agar plates. For microscopy, the fungi were grown underneath cover slips placed on top of agar blocs of ca. 1 cm2. The fungal hyphae, mycelia and spores adhering to the coverslips were mounted on slides and studied by 400 times magnification. Incubating the plates at 30°C and 35°C and morphological characteristics facilitated distinguishing between T. harzianum and T. polysporum. Identification of the fungi was based on Domsch et al. (1980), and a taxonomic key by McCray (2004) further identified the Trichoderma species. In addition, available information in the data package submitted with the applications for authorization of the respective products was used.

One representative isolate of each fungal species and Streptomyces griseoviridis was identified by the identification service at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Braunschweig, Germany (Appendix b).

2.3 Contaminating bacteria

Whenever bacterial colonies were encountered on agar plates inoculated with fungi, the number of bacterial CFU was determined.