1. NERI National Environmental Research Institute University of Aarhus Denmark www.dmu.dk The fungus Trichoderma spp. on vegetables Anne Winding, senior scientist, Ph.D. Department of Environmental Chemistry and Microbiology, NERI Frederiksborgvej 399, 4000 Roskilde. e-mail: aw@dmu.dk Results Specific PCR-primers for the fungus Trichoderma spp. was found selective by testing against 12 Trichoderma strains and 16 closely related fungal strains (Table 1). No Trichoderma spp. was found on the surface of any of the 155 vegetables tested (tomatoes, cucumber, broccoli, celery) with the used techniques based on cultivation followed by PCR amplification of DNA (Table 2). The preliminary results indicate that the use of T. harzianum in MPCA cause limited exposure to the consumer. Further and other studies Vegetables Determine presence of Trichoderma spp. by PCR directly on the vegetables with optimized method Develop specific molecular detection of used MPCA T. harzianum strain and determine presence on vegetables Human exposure through inhalation Another part of the project determines presence of Trichoderma spp. in inhalation air. Trichoderma spp. was present when MPCA was used by tomato producers. Identification of these Trichoderma to the applied T. harzianum strain will be attempted. Introduction Trichoderma spp. is a naturally occurring fungi in soil and T. harzianum is an active ingredient in Microbial Pest Control Agents (MPCA) active against root pathogenic fungi. The MPCA is administered to the plants by watering. The fungus establishes in the root zone and exerts its beneficial effect by general increase of resistance against pathogenic fungi. The natural occurrence of Trichoderma spp. on vegetables and any possible survival of the introduced T. harzianum from the applied MPCA to the vegetables for consumption are poorly described despite the importance for risk assessment. Acknowledgement Danish EPA through ‘Pesticidforskningsprogrammet’ financed the project. V. M. Hansen and A. M. Madsen, NFA, established contact to the farmers. B. M. Hansen and N. B. Hendriksen, AU, sampled the vegetables. J. Eilenberg, N. V. Meyling, B. Jensen and M. Lubeck, KU-Life are acknowledged for kind donation of fungal strains. A. G. Holm-Jensen provided technical assistance. References Hagn, A., S. Wallisch, V. Radl, J. C. Munch, M. Schloter. 2007. A new cultivation independent approach to detect and monitor common Trichoderma species in soil. J. Microbiol. Meth. 69:86-92. Williams, J., Clarkson, JM, Mills, PR, Cooper, RM. 2003. A selective medium for quantitative reisolation of Trichoderma harzianum from Agaricus bisporus compost. Appl. Env. Microbiol. 69:4190-4191. Gardening /fieldCrop Trichoderma MPCA applied Detection limit (cfu/g) cCucumber - 1.2 eCucumber - 1.5 gTomato - 3.5 gTomato - 4 h + 2 h + 3 fBroccoli - 15 fBroccoli - 15 bBroccoli - 15 fCelery - 13 fCelery - 15 Objective Assess the occurrence of Trichoderma spp. and T. harzianum from Microbial Pest Control Agents on vegetables for consumption for input to risk assessment of MPCA Specific objectives Testing and implementation of specific PCR-primers targeting Trichoderma spp. Quantify Trichoderma spp. on vegetables grown in greenhouses and in open fields. Table 1: Fungal strains tested for amplification with the Trichoderma specific PCR-primers. The primers were uTf/uTr (Hagn et al. 2007) and aligned only to Trichoderma spp. when tested by BLAST. Table 2: Vegetables investigated for occurrence of Trichoderma spp. Cucumber and tomatoes were grown in greenhouse, broccoli and celery in the fields. The MPCA Supresivit was applied to greenhouse ‘h’ 8 and 14 weeks prior to sampling. Methods Extraction of microorganisms from vegetables Peel of tomatoes and cucumbers; 25 g subsamples of broccoli, and an outer stalk of each celery were weighed and beaten in a Stomacher Lab Blender for 2 x 1 min. together with 100 ml MilliQ H 2 O. The liquid in the bags were plated on agar plates and DNA was extracted from colonies by freeze-thaw-boiling followed by centrifugation. Cultivation Trichoderma harzianum Specific Media (THSM) (Williams et al. 2003) was used. PCR conditions Each PCR tube contained a total volume of 25 µl, with 13.25 µl DNase-free water, 2.5 µl of MgCl 2 (25 mM), 2.5 µl 10×PCR buffer (+NH 4 SO 4 -MgCl 2 ), 2.5 µl deoxynucleoside triphosphate (0.25 mM), 1µl each of primer (5 pmol µl-1), 0.5 µl Taq DNA polymerase (1U µl-1; Fermentas) and 1 µl sample as template. The PCR- conditions were optimized to the following: 3 min at 95°C; 30 cycles of 30 sec. at 94°C, 30 sec. at 58°C, 30 sec. at 72°C; 10 min at 72°C; and final cooling at 4°C. FungiStrain PCR (uTf/uTr) T. longibrachiatumDSMZ 768+ T. virensDSMZ 1963+ T. hamatumDSMZ 63055+ T. harzianumDSMZ 63059+ T. koningiiDSMZ 63060+ T. polysporumDSMZ 63062+ T. virideDSMZ 63065+ T. harzianumSupresivit+ T. harzianumTRI002+ T. harzianumTRI 003 batch 0031 and 0043- T. harzianum & polysporumBinap (ATCC20475 & 20476)+ Phlebiopsis giganteaRotstop- Verticillium lecaniiMycotal and Vertalec- Beauveria bassiana Botany Gard 22WP, GHA-5, and KVL 07-172- Paecilomyces fumascroseusKVL 04-35- Metarhizium anisopliaeKVL 07-83- Clonostachys roseaIBT 7519 GR1, IBT 7839 GR3- Fusarium langsethiaeIBT 9955 F26, IBT 8051 F27- Epichloe typhinaE8- Neonectria galligena418, 420- Vegetables PCR primer specificity