toxicity on fungal cells and, in particular, the capacity to alter the mushrooms pseudo-tissues. Although, in the above assays WLIP showed a lower activity when compared to tolaasin I the results suggested the possible role of the two LDPs in the symptom caused by the mushrooms bacterial pathogens. The use of biological and model membranes permitted to evaluate the mechanism of the substances toxicity. Both LDPs were able to lyse red blood cells via transmembrane pore formation with a C50 of 4.3 and 34.6 g/ml for WLIP and tolaasin, respectively. Pore radius, estimated with the Renkin theoretical approach, was 2.1 and 1.5 nm for tolaasin and WLIP, respectively. Furthermore, both LDPs were also able to form channels in lipid vesicles and the activity was dependent on liposomes composition. In particular, the toxicity increased with the sphingomyelin content in the membrane. Studies of dynamic light scattering suggested a detergent-like activity for WLIP at high concentration (>30 g/ml). This effect was not detected for tolaasin up to 56 g/ml. TIR-ATR experiments showed an increase in the helical content of tolaasin after absorption into the membrane. The orientation of the tolaasin -helical fraction showed an angle of 24° with respect to the lipid acyl chains, suggesting that tolaasin is not laying on the membrane surface but it is inserted into the lipid core. The financial supports of projects “Biological characterisation of the tolaasin and WLIP, lipodepsipeptides of Pseudomonas tolaasii and P. “reactans”, PAT Fondo Progetti (Project AgriBi) and MURST ex 60% are acknowledged.
USE OF PCR FOR THE DETECTION OF Clavibacter michiganensis subs. insidiosus in lucerne seeds
Bryxiová M., Kůdela V.
Research Institute of Crop Production, Drnovská 507, 161 06 Prague 6 – Ruzyně, Czech Republic
Bacterial wilt, caused by Clavibacter michiganensis subs. insidiosus (Cmi), is a serious disease of lucerne. The pathogen is of North American origin and has spread to many other countries. Cmi is an EPPO A2 quarantine pest, i.e., it is the quarantine pest present in some areas within the region. Seed transmission is virtually the only way to introduce the pathogen to previously free areas. A reliable and rapid method is needed in practice to check seed lots for infection. The objective of this study was to determine whether a polymerase chain reaction (PCR) based assay could be used to detect Cmi in lucerne seeds. Samples of 0.2 g from a seed lot were prepared, ground in a mortar and suspended with 5 ml culture medium. One millilitre of sterile water (as a negative control) and 1 ml of Cmi suspension at concentrations of 108, 107, 106, 105, 104, 103 and 102 cfu were added to each 5 ml seed macerate. The macerates were shaken at room temperature for 24 h. Total genomic DNA from macerates was isolated using a NucleoSpin kit (Macherey-Nagel). DNA primers were designed and used to detect Cmi in isolated DNA. Cmi was reliably detected in seed macerates which contained from 108 to 102 cfu. The detection of Cmi in lucerne seeds was enhanced using the PCR method.
The Presence of Phytoplasma in Black Currant Infected with the Blackcurrant Reversion Disease.
J. Spak, J. PRibylova, D. Kubelkova and V. Spakova
Department of Plant Virology, Institute of Plant Molecular Biology, Academy of Sciences of the Czech Republic, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic E-mail: firstname.lastname@example.org
A phytoplasma infection of the black currant cultivar Karlštejnský dlouhohrozen showing symptoms of the severe Russian (R) form of the blackcurrant reversion disease (BCRD) was identified in the Czech Republic in 2003. The size of phytoplasma bodies found by electron microscopy in phloem of leaf midribs was 530-750 nm. The phytoplasma infection was confirmed by the polymerase chain reaction (PCR) with the universal primer pair R16F1/R16R0, followed by PCR with the primer pair fU5/rU3. An amplification product of about 880 bp was obtained and sequenced. A comparison of its sequence with sequences available in the GenBank confirmed the classification of the phytoplasma in the 16SrI (Aster yellows group). The plant was also tested by RT-PCR for the presence of the Blackcurrant reversion virus (BRV), the causal agent of BCRD. A virus-specific 481 nt cDNA fragment of BRV was obtained and sequenced. The comparison of this sequence with sequences available in the GenBank proved BRV infection in this plant. Moreover, rhabdovirus-like particles were found in the plant by electron microscopy. This is the first evidence of the natural occurrence of phytoplasma infection in the black currant. This research was supported by the grant OC 853.001 Ministry of Education, Youth and Sports of the Czech Republic.
Thermodynamic sequence screening and genome probing revealed Chenopodium-systemic (CS) European variants of potato virus S unrelated to Andean PVS strain.
JAROSLAV MATOUŠEK,1, PETRA KOZLOVÁ,1,4 , JIŘÍ PTÁČEK,2 JORG SCHUBERT,3 and PETR DĚDIČ,2
1Institute of Plant Molecular Biology, AS CR, České Budějovice, Czech Republic, 2Institute for Potato Research, Havlíčkův Brod, CR, 3Federal Centre for Breeding Research, Aschersleben, Germany; 4South Bohemian University, Biological Faculty, České Budějovice, C R.
In our previous work, we reported a broad natural sequence variability among closely related Central European PVS isolates by analysis of cDNAs derived from 3'-terminal portions of PVS genomes using RT PCR and