6.120
BIO-PCR FOR DETECTION OF AGROBACTERIUM TUMEFACIENS BIOVAR 1 AND 2 IN ORNAMENTAL PLANTS
BIO-PCR FOR DETECTION OF AGROBACTERIUM TUMEFACIENS BIOVAR 1 AND 2 IN ORNAMENTAL PLANTS A VAN ZAAYENl , R HOOFTMANl , JRCM VAN BECKHOVEN2, PM DE VRIES2AND JM VAN DER WOLF2 l inspection Service for Floriculture and Arboriculture (NAKB), P.O.Box 135, 2370 AC Roelofarendsveen, The Netherlands. 2, DLO Research institute for Plant Protection (IPO-DLO), P.O. Box 9060, 6700 GW Wageningen, The Netherlands. Background and objectives Infection of nurseries of rose, aster and chrysanthemum with Agrobacterium tumefaciens, (Atum), the causal organism of crown gall, can have important financial consequences. To avoid introduction and dissemination of this pathogen in plant propagation material, reliable detection methods are required. A PCR-amplification, using primers against sequences of the ipt-gene [2], present on the Ti-plasmid encoding one of the virulence factors, turned out to be specific for detection of Atum. Detection of Atum, directly in plant extract lacks sensitivity, due to the precence of PCRinhibitors, while DNA-purification is often laborious. This paper describes two BIO-PCR procedures for simple and specific detection of biovar 1 and 2, based on the enrichment of the target bacteria and PCR-amplification without the need of DNA-purification [1]. To evaluate the BIO-PCR procedures and to develop a sampling strategy, the procedures were used to study the internal spread of Atum in ornamental plants and the distribution of Atum in green houses with naturally infected plantlets. Materials and methods For detection of biovar 1, plant extracts were mixed with liquid S-medium, based on the selective medium, as described by Schroth et al.(Phytopathology 55: 645-647). After four days of incubation, enriched extracts were plated on the non-selective TYA medium (Brisbane and Kerr, 1983; J. Appi. Bacteriol. 54: 425-431). After one day of incubation total number of cells from the TYA plates was suspended in 2 mi of water and processed for PCR-amplification. For detection of biovar 2, plant extracts were plated on the agar medium BKIIe (Brisbane and Kerr, 1983) and incubated for three days. The total number of cells from the BKIIe plates was suspended in 2 ml of water and processed for PCR-amplification. Suspensions were 20-fold diluted in 5 mM NAOH and boiled. The touch down PCR-amplification was based on the ipt-primers directed against conserved parts of the T-DNA [2] and resulted in a 427 bp PCR product. Amplification products were separated and visualised on an acrylamide gel treated with silver stain. An internal control (170 bp) was added to the reaction mixture, resulting in an amplification product of 170 bp, to recognize false negative reactions due to components inhibiting the PCR-reaction. Results and conclusions Described BIO-PCR procedures were useful for testing of plant propagation material. They enabled detection of low concentrations of culturable cells of Atum in latently infected plant material within one week without the need of extracting DNA. For biovar 1, a detection level of c.10 cfu per ml of plant extract and for biovar 2, a detection level of 100-1000 cfu per ml of plant extract was found. The procedures were shown to be highly specific for Atum. From 26 biovar 1 strains tested, 25 grew in liquid S-medium and all tested 23 biovar 2 strains grew on BK]ie medium although some strains grew slowly. Use of the internal standard did not significantly affect the sensitivity of PCR. Atum was detected in 30% of stems of symptomiess Solidago plants and in 40% of stems of symptomiess rose plants, grown in contaminated greenhouses. The BIO-PCR procedures have also been used to demonstrate the internal movement of Atum in chrysanthemum, aster and rose, after stem and root inoculation. References 1. Van der Wolf JM, van Beckhoven JRCM, De Vries PM. In: Proc. Of the 9th int. Conf. on Plant Path. Bacteria, Madras, India, august 26-29, 1996 (in press.) 2. Haas JH, Moore LW, Ream W, Manulis S, 1995. Applied and Environmental Microbiology 61(8): 2879-2884.