Although the influences of temperature and water activity on the production of Alternaria toxins were studied extensively, the influence of other abiotic factors was neglected so far. In this study the effects of the two important factors, pH-value and C:N ratio, were elucidated for the first time and it was shown that mycotoxin production was highly affected by these factors.
In general, the regulation of most of fungal toxins is very complex and one special regulator does not exist. As shown by many studies before, pH value exerts a great influence on the production of different mycotoxins. Although the optimal pH value has to be determined individually for each mycotoxin, the production of most of the mycotoxins is increased at acidic pH values. ([Keller et al. 1997
]) reported highest sterigmatocystin production in Aspergillus nidulans
and highest aflatoxin production in A. parasiticus
at pH 4.0. By buffering ammonium growth media in a range from pH 4 to 8, they observed an approximately 5-fold decrease in mycotoxin production in both Aspergillus
spp. with increasing pH. According to the detected mycotoxin concentrations they observed that the transcript levels of the aflatoxin/sterigmatocystin pathway genes ver-1
first appeared at acidic pH values and only later or not at all in neutral or alkali conditions. Similarly, much higher levels of ochratoxin are produced by A. ochraceus
in the lower pH range, with a 4-fold reduction in levels being observed when the pH of the growth medium was increased from pH 4 to pH 10 ([O'Callaghan et al. 2006
]). Likewise, a low pH regulates deoxynivalenol production in Fusarium graminearum
([Gardiner et al. 2009
]). ([Gardiner et al. 2009
]) observed that a decrease of the initial pH from pH 4.0 to 3.5 led to a 2-fold increase in deoxynivalenol concentration. They also showed that an initial pH between 2.4 and 3.1 was required to induce deoxynivalenol production when F. graminearum
was previously grown in non-producing medium.
In agreement with our shaking flask results they reported a rapid acidification of the growth medium due to ammonium consumption ([Morton and Macmillan 1954
]; [Jernejc and Legiša 2001
]) followed by a increase to pH 8. They suggested that the drop in pH prior to the start of deoxynivalenol biosynthesis may be required to release a possible inhibition by the global pH regulatory system mainly mediated by the transcription factor PacC. PacC homologues play a role in the regulation of many mycotoxins, e.g. fumonisin production of F. verticillioides
([Flaherty et al. 2003
]) and sterigmacystin biosynthesis in A. nidulans
([Delgado-Virgen and Guzman-de-Peña 2009
]). Consequently, it is possible that alternariol production is regulated by a PacC homologue as well. Applying a blast search of PacC of A. nidulans
(GenBank: CAA67063.1) against the sequenced and annotated genome of the close relative Alternaria brassicicola
) revealed that A. brassicicola
harbors a PacC homologue as well (jgi|Altbr1|4090|AB04090.1, E-Value: 3.25E-97). To clarify whether alternariol biosynthesis is regulated by a PacC homologue the identification of the biosynthesis cluster is necessary. Nevertheless, our results from the bioreactor studies showed that in contrast to ([Gardiner et al. 2009
]) a drop in pH is not necessary to induce alternariol production because the pH was kept constant in the bioreactor. Furthermore, alternariol production in shaking flasks started after the end of growth phase when the pH of the medium was already increased. So, in A. alternata
mycotoxin production is induced by low pH values, but a change in pH is not necessary.
With respect to the C:N ratio experiments it was already shown before that this ratio is important for the production of secondary metabolites. ([Casas Lopéz et al. 2003
]) studied the influence of different C:N ratios on lovastatin production. They found that the presence of excess carbon under nitrogen limitation greatly enhanced the rate of production of lovastatin and determined an optimal C:N ratio of ~40. They stated that lovastatin production is associated with the nitrogen limited stationary growth phase when excess carbon can be channeled into secondary metabolism. The same can be applied to alternariol production. [Brzonkalik et al. (2011a
]) showed clearly the secondary metabolite character of alternariol which is produced in the stationary growth phase after nitrogen depletion. An excess of carbon has a positive impact on alternariol production but does not affect growth rate or yield. In contrast to alternariol tenuazonic acid is formed growth associated and contains nitrogen. Therefore, an increase in carbon concentration does not alter TA production. These results clearly emphasize the different regulatory mechanisms for both mycotoxins and were supported by the results of the bioreactor cultivation.