Growth and Secondary Metabolites

Beside aggressiveness on oilseed rape A- and B-type isolates of Phoma lingam can be distinguished by cultural traits. Production of secondary metabolites is a very useful criterion. However, A- and B-type also differ significantly with regard to radial growth on different media (refer to the upper left figures). A more rapid growth can be attributed to B-type-isolates (blue lines) compared to A-type isolates (red lines), which is even more pronounced on more nutrient poor media (comparison of malt ectract -upper chart- and vegetable-8 juice -lower chart). Data shown indicated the growth of A- and B-type reference isolates over a time period of 23 days. Radial growth is plotted on the y-axis. Looking at the figures showing A-type (left petri dish) and B-type (right petri dish) a pronounced branched growth of A-type isolates becomes obvious. However, due to variation of radial growth this trait is not very reliable for differentiation.

Of much better value is the trait pigment production in liquid medium, even there is also variation with at least respect to quantity of pigments. After 3-5 weeks of cultivation a brownish discolorisation of the media becomes obvious varying from light brown to dark brown. As seen in the bottom left figure all B-type isolates shown here (6 in number) display discolorization. The two A-type isolates (2nd and forth from the right) only show original color of the Czapek Dox culture filtrate. All isolates so far tested in our laboratory (several hundreds) possess the negative correlation between pigment production and aggressiveness on oilseed rape.

The correlation is positive considering the production of the host unspecific phytotoxins sirodesmins, which belongs to the chemical family of dioxo-piperazines. In other words A-type isolates produce sirodesmins but do not produce pigments and B-types vice versa. Sirodesmin production can be seen from the TLC-analysis shown here. After ethyl acetate extraction of the culture filtrates sirodesmins can be stained by AgNO3. Staining shows six sirodesmin derivates visiable as brownish bands. Analysed were two A-type isolates starting at the left of the TLC plate, followed by two B-type and an A-type isolate, clearly demonstrating this difference.
The property of sirodesmin production was used by a French group headed by Marie-Hélène Balesdent und Thierry Rouxel, who raised the terms TOX+ (A-types) and TOX- (B-types) (Balesdent et al. 1992). Even in this case we now know that these terms are also oversimplifing, due to other phytotoxins produced by B-type isolates. The terms SIRO+ and SIRO-0 should be mor appropriate instead. Work published by the Canadian biochemist Soledade Pedras and her polish colleague Piotr Kachlichki showed that phytotoxic compounds are produced by B-type isolates. Connected to this we should state from work of our own group that sirodesmins should not be considered as pathogenecity factors. Instead they should be considered as virulence factors (or better aggressiveness factors) because Sock & Hoppe (1999) showed that UV knock down mutants (factor 1,000 -10,000) were still pathogenic but showed significantly reduced lesions sizes on stems and cotyledons.