NERINational Environmental

Research InstituteUniversity of Aarhus

Denmarkwww.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.

AcknowledgementDanish 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.

ReferencesHagn, 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/field Crop

Trichoderma

MPCA applied

Detection limit

(cfu/g)

c Cucumber - 1.2

e Cucumber - 1.5

g Tomato - 3.5

g Tomato - 4

h Tomato + 2

h Tomato + 3

f Broccoli - 15

f Broccoli - 15

b Broccoli - 15

f Celery - 13

f Celery - 15

ObjectiveAssess 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.

MethodsExtraction of microorganisms from vegetablesPeel 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 H2O. The liquid in the bags were plated on agar plates and DNA was extracted from colonies by freeze-thaw-boiling followed by centrifugation.CultivationTrichoderma harzianum Specific Media (THSM) (Williams et al. 2003) was used. PCR conditionsEach PCR tube contained a total volume of 25 µl, with 13.25 µl DNase-free water, 2.5 µl of MgCl2 (25 mM), 2.5 µl 10×PCR buffer (+NH4SO4-MgCl2), 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.

Fungi StrainPCR

(uTf/uTr)T. longibrachiatum DSMZ 768 +

T. virens DSMZ 1963 +

T. hamatum DSMZ 63055 +

T. harzianum DSMZ 63059 +

T. koningii DSMZ 63060 +

T. polysporum DSMZ 63062 +

T. viride DSMZ 63065 +

T. harzianum Supresivit +

T. harzianum TRI002 +

T. harzianum TRI 003 batch 0031 and 0043 -

T. harzianum & polysporum Binap (ATCC20475 & 20476) +

Phlebiopsis gigantea Rotstop -

Verticillium lecanii Mycotal and Vertalec -

Beauveria bassianaBotany Gard 22WP, GHA-5, and KVL 07-172 -

Paecilomyces fumascroseus KVL 04-35 -

Metarhizium anisopliae KVL 07-83 -

Clonostachys rosea IBT 7519 GR1, IBT 7839 GR3 -

Fusarium langsethiae IBT 9955 F26, IBT 8051 F27 -

Epichloe typhina E8 -

Neonectria galligena 418, 420 -

Vegetables

PCR primer specificity