Diagnostiek van Erwinia amylovora (BO-33.03-001-002)

To reduce the inhibition of growth of E. amylovora cells on semi-selective media (SNA-L, CCT, King’sB and TSA) a semi-automated plating technique was used to compare the standard plating assay from Naktuinbouw (plating using a Drigalsky spatel): spiral plating. This apparatus can plate bacterial suspensions from the center of the agar plate to the rim, thus diluting the suspension by a standard method and enlarging the distance between colonies. This might lead to less inhibition of the bacterial colonies.

After selection of the best media (SNA-L and CCT), samples were obtained from Naktuinbouw and spiked with E. amylovora. Results were compared with TaqMan analyses of the samples, which showed a good correlation. After standardization, 20 samples of Naktuinbouw, from which several were suspected from the presence of E. amylovora, were analysed using the spiral plater. The spiral plater proved to be slightly superior due to more separately located colonies, the use of (far) less plates to analyze samples and a better standardization. A drawback is, that only diluted samples can be used.

In collaboration with ACW Switzerland (Duffy, Porthier, and others) the genomic sequence of E. amylovora was searched for unique carbon source enzyme complexes which would be able to digest these C-sources but not (in general) by saprophytic bacteria, present in samples of fruit trees, suspected from fire blight (Kawanishi et al. 2011). It appeared after searching of the genomic sequence of E. amylovora, that there were no indication of enzyme sequences, involved in the metabolism of rare carbon sources. This was supported by the finding, that a saprophytic bacterial isolate was able to digest approximately the same C-sources as E. amylovora (Stockwell et al. 2010). Therefore we decided not to search for alternative C-sources to complement the current media for culturing E. amylovora.