Through curiosity, Grandpa went on to study the behaviour of fusarium in an artificial liquid medium. A simple medium, in which he varied sugar (glucose) and nitrogen (ammonium salt) concentrations in opposite ways, as occurred in the plant. This gave the following results:
During the first few days, the fungus developed normally in all the media, with the cultures displaying a white, fluffy appearance. On the morning of the fourth day there was a surprise: the four flasks containing medium n° 1 (the richest in sugar and poorest in nitrogen) had turned bright red overnight, the others retaining their white appearance. On the morning of the fifth day, a second surprise was found: the four flasks containing medium n° 2 had also turned bright red. And night after night, medium to medium, the same thing happened. The last medium, very poor in sugar and rich in N, was the only exception to the rule. It retained its white appearance until the end of the experiment.
Microscopic study of the various media showed that in media very rich in sugar, the fusarium developed in an unusual way. Although it normally takes the form of long overlapping filaments (the mycelium), in this case it was in the form of small round, isolated cells, giving it the appearance of a yeast, the little fungus which transforms sugars into alcohol and is so useful to our winegrowers. And that is how, without really intending to, Grandpa may have found the answer to the previous two questions.
Question n° 1: why is the plant inoculated with fusarium initially rejuvenated?
Fusarium is a so-called "imperfect" fungus. It is imperfect because the only method of reproduction it is known to have is vegetative reproduction. This makes it unclassifiable, since fungi are classified according to their method of reproduction.
Microscopic observation then led to the next question: is this imperfect fungus related to yeasts? In other words, does it have a habit of transforming sugars into alcohol in the same way as yeasts do?
- which would explain the low level of sugars in diseased plants and the involuntary rejuvenation imposed by the parasite.
- which would also explain why plants treated with GA and rich in sugar, seem to adapt to its presence better than plants treated with IAA.
Question n° 2: why did the plant age suddenly later on?
Following the advice of the laboratory director, Grandpa also extracted and purified the pigment responsible for this sequence of surprises. This pigment was doubtless already known because it gives old cultures of fusarium a red-violet colour which is so routine that it goes unnoticed. This pigment, when purified, crystallises into long sharp needles. It has obvious colloidal properties: soluble in acid (it then takes on this red colour we mentioned), it flocculates in an alkaline medium, taking on a much less attractive purple colouring. In the end, a powerful oxidation-reduction agent synthesised by the ageing fungus, may be the source of "vascular symptoms of fusariosis", the brown colouring taken on by the sap carrying vessels in plants suffering from fusariosis.
By contributing to the breakdown of conducting vessels in roots, this pigment may prevent these organs from functioning normally, thereby depriving the plant of N. By contributing to the breakdown of these same vessels in the stems, does it promote the increase in sugar concentrations observed in the leaves of the most affected plants, the sugars synthesised in these organs being unable to migrate normally to other tissues? Since the plant or fungus cannot be asked, Grandpa had to be content to leave a question mark hanging over these possibilities, which was perfectly logical.
Remark: One thing is certain: the media used above were much less rich in sugar at the end than at the beginning of the experiment, growth of the fungus alone being inadequate to explain the intensity of the reaction. Grandpa tried to perform alcohol assays, but did not manage to assay anything at all.