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Applied and Environmental Microbiology, June 2004, p. 3512-3520, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3512-3520.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Detection and Quantification of Airborne Conidia of Fusarium circinatum, the Causal Agent of Pine Pitch Canker, from Two California Sites by Using a Real-Time PCR Approach Combined with a Simple Spore Trapping Method

Wolfgang Schweigkofler,¹ Kerry O'Donnell,² and Matteo Garbelotto^1*

Department of Environmental Science, Policy, and Management, University of California, Berkeley, Berkeley, California 94720,¹ Microbial Genomics Research Unit, National Center for Agricultural Utilization Research, Peoria, Illinois 61604²

Received 3 December 2003/ Accepted 18 February 2004

Pinus radiata (Monterey pine), a tree native to coastal California and Mexico, is widely planted worldwide for timber production. A major threat to Monterey pine plantations is the fungal disease pine pitch canker, caused by Fusarium circinatum (Hypocreales). We present a novel trapping approach using filter paper in combination with a rapid molecular method to detect the presence of inoculum in the air. The assay is also useful for diagnosing the presence of the pathogen on plants. The test is based on the F. circinatum specific primer pair CIRC1A-CIRC4A, which amplifies a 360-bp DNA fragment in the intergenic spacer region of the nuclear ribosomal operon. Real-time PCR was used to calculate the number of fungal spores present in each reaction mixture by comparing the threshold cycle (Ct) of unknown spore samples to the Ct values of standards with known amounts of F. circinatum spores. The filter paper method allows prolonged and more sensitive spore sampling in the field compared to traditional traps using petri dishes filled with selective medium. A field test at two sites in coastal California infested with pine pitch canker was carried out during the summer and fall of 2002. Spore counts were in the range of ca. 1 x 10³ to ca. 7 x 10⁵/m², with the highest spore counts in the fall, suggesting a seasonal fluctuation.

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* Corresponding author. Mailing address: ESPM (Department of Environmental Science, Policy, and Management), 151 Hilgard Hall, University of California, Berkeley, Berkeley CA 94720. Phone: (510) 643-6412. Fax: (510) 643-5436. E-mail: matteo{at}nature.berkeley.edu.

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Applied and Environmental Microbiology, June 2004, p. 3512-3520, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3512-3520.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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