Physiological, biochemical and molecular characterization of the brown film of Lentinula edodes (Berk.) Pegler and its interaction with Trichoderma sp.

Abstract

The cultivation cycle of shiitake has several stages and the formation of brown film is the most important, since it is a strategic step in the production of mushrooms. Many studies have already shown the relation of the activity of some enzymes in the brown film formation, mainly the ligninases. Another ally in the understanding of this process is the study of gene expression of L. edodes strains that behave differently in the brown film formation. During the production of mushrooms, a very important factor is the contamination, being the fungus of the genus Trichoderma the most studied. The control of this pathogen is accomplished through the adoption of aseptic techniques, however, it is known that the brown cover can also aid in the control, acting as a physical barrier. In this sense, scanning electron microscopy (SEM) is a powerful technique in the study of host-pathogen interaction. Given the relevance of the brown film in shiitake, this study investigated the dynamics of this process at the physiological, biochemical and molecular levels. Our results showed that all the enzymes showed activity in different culture conditions, and the lignin peroxidase presents the highest activity when compared to other enzymes, for both strains. In addition, the high activity of tyrosinase in the UFLA-LE6 strain at the beginning of the cultivation may explain the fact that this strain forms the brown film faster than UFLA-LE5 strain and presents high levels of productivity. However, we observed that this same strain is more susceptible to environmental changes, such as temperature and pH; different from the UFLA-LE5 strain, which presented a greater range of growth in environments considered atypical for a good development. On the issue of antagonism with Trichoderma sp., the UFLA-LE6 strain produced a brown line in the zone of interaction with the pathogen, which has already been reported as a resistance strategy and is probably related to melanin synthesis. With SEM we detected an increase in infection over time, direct penetration of the pathogen in the mushroom and evidence of mycoparasitism. In the transcriptomic analysis, we have seen that the UFLA-LE5 strain has more upregulated genes compared to the UFLA-LE6 strain. This fact may seem contradictory; however, it may suggest a failure of this strain to control the level of genetic expression. Some of the genes studied with RT-qPCR exhibited a decrease in expression on the outside sample of UFLA-LE6 strain at 90 days. This profile can be explained by the speed and stability with which the brown film is formed in this strain, so, after that process such genes may no longer be required and therefore not expressed.