PCR-based detection of the causal agent of watermark disease in willows (Salix spp.).

The watermark disease, caused by Brenneria salicis (formerly Erwinia salicis), is of significant concern wherever tree-forming willows are grown or occur naturally. The movement of infected, asymptomatic cuttings is a major cause of pathogen dispersal. A reliable and sensitive diagnostic procedure is necessary for the safe movement of willow planting material. We derived primers from the nucleotide sequence of the 16S rRNA gene of B. salicis for the development of a PCR to detect this pathogen. One set of primers, Es1a-Es4b, directed the amplification of a 553-bp fragment from B. salicis genomic DNA as well as B. salicis cells. PCR products were not observed when genomic DNA was tested for 27 strains of other, related plant-associated bacteria. Genomic fingerprinting by amplification fragment length polymorphism of B. salicis strains, originating from four different countries, and related Brenneria, Pectobacterium, and Erwinia strains revealed a very high similarity among the B. salicis genomes, indicating that the spread of the pathogen is mainly due to the transportation of infected cuttings. The PCR had to be preceded by a DNA extraction in order to detect the pathogen in the vascular fluid of willows. The minimum number of cells that could be detected from vascular fluid was 20 CFU/ml. The PCR assays proved to be very sensitive and reliable in detecting B. salicis in willow plant material

Biodegradation of poly(3-hydroxyalkanoates) in anaerobic sludge and characterization of a poly(3-hydroxyalkanoates) degrading anaerobic bacterium

The degradation of samples of poly(3-hydroxybutyrate) [P(3HB)], copolymers of 3-hydroxybutyrate and 10 and 20% 3-hydroxyvalerate [P(3HB-co-3HV)], and poly(3-hydroxyoctanoate-co-3-hydroxydecanoate) [P(3HO-co-3HD)] in anaerobic sludge was investigated by gravimetry, changes in molecular weight and mechanical properties. After 123 days, the mass of P(3HB) decreased by 15%. P(3HB-co-3HV) showed 7 and 11% mass loss, while P(3HO-co-3HD) showed no change in mass. The average molecular weight of all samples decreased with incubation rime bur this decrease was not correlated to the loss of mass, and is to be attributed to abiotic hydrolysis. The tensile strength of the P(3HB) and P(3HB-co-3HV) samples decreased with incubation time, but elongation remained practically unchanged. By enumeration, it was shown that anaerobic P(3HB) degrading bacteria were enriched during the incubation period. An anaerobic bacterium LMG 16094, able to degrade P(3HB) and P(3HB-co-19% 3HV) in pure culture, but not P(3HB-co-97% 3HV), P(3HO-co-3HD) or poly(epsilon-caprolactone), was isolated and characterized by fatty acid analysis. Its cells contained mainly straight chain, saturated and unsaturated fatty acids, with a high portion of fatty acids with 16 carbons, and did not contain hydroxylated or branched fatty acids. Its 16 rDNA gene sequence was found to be related to those of members of Clostridium group I (Collins er al., 1994). In a simple in vitro test using strain LMG 16094 in pure culture, an extruded P(3HB-co-12% 3HV) film lost 10% of its initial mass within 21 days of anaerobic incubation