Circadian oscillations in Trichoderma atroviride and the role of core clock components in secondary metabolism, development, and mycoparasitism against the phytopathogen Botrytis cinerea.

Circadian clocks are important for an individual's fitness, and recent studies have underlined their role in the outcome of biological interactions. However, the relevance of circadian clocks in fungal-fungal interactions remains largely unexplored. We sought to characterize a functional clock in the biocontrol agent Trichoderma atroviride to assess its importance in the mycoparasitic interaction against the phytopathogen Botrytis cinerea. Thus, we confirmed the existence of circadian rhythms in T. atroviride, which are temperature-compensated and modulated by environmental cues such as light and temperature. Nevertheless, the presence of such molecular rhythms appears to be highly dependent on the nutritional composition of the media. Complementation of a clock null (Δfrq) Neurospora crassa strain with the T. atroviride-negative clock component (tafrq) restored core clock function, with the same period observed in the latter fungus, confirming the role of tafrq as a bona fide core clock component. Confrontation assays between wild-type and clock mutant strains of T. atroviride and B. cinerea, in constant light or darkness, revealed an inhibitory effect of light on T. atroviride's mycoparasitic capabilities. Interestingly, when confrontation assays were performed under light/dark cycles, T. atroviride's overgrowth capacity was enhanced when inoculations were at dawn compared to dusk. Deleting the core clock-negative element FRQ in B. cinerea, but not in T. atroviride, was vital for the daily differential phenotype, suggesting that the B. cinerea clock has a more significant influence on the result of this interaction. Additionally, we observed that T. atroviride clock components largely modulate development and secondary metabolism in this fungus, including the rhythmic production of distinct volatile organic compounds (VOCs). Thus, this study provides evidence on how clock components impact diverse aspects of T. atroviride lifestyle and how daily changes modulate fungal interactions and dynamics.

Spontaneous circadian rhythms in a cold-adapted natural isolate of Aureobasidium pullulans.

Circadian systems enable organisms to synchronize their physiology to daily and seasonal environmental changes relying on endogenous pacemakers that oscillate with a period close to 24 h even in the absence of external timing cues. The oscillations are achieved by intracellular transcriptional/translational feedback loops thoroughly characterized for many organisms, but still little is known about the presence and characteristics of circadian clocks in fungi other than Neurospora crassa. We sought to characterize the circadian system of a natural isolate of Aureobasidium pullulans, a cold-adapted yeast bearing great biotechnological potential. A. pullulans formed daily concentric rings that were synchronized by light/dark cycles and were also formed in constant darkness with a period of 24.5 h. Moreover, these rhythms were temperature compensated, as evidenced by experiments conducted at temperatures as low as 10 °C. Finally, the expression of clock-essential genes, frequency, white collar-1, white collar-2 and vivid was confirmed. In summary, our results indicate the existence of a functional circadian clock in A. pullulans, capable of sustaining rhythms at very low temperatures and, based on the presence of conserved clock-gene homologues, suggest a molecular and functional relationship to well-described circadian systems.