Isolation and identification of Arbutus unedo L. fungi endophytes and biological control of Phytophthora cinnamomi in vitro.

Strawberry tree (Arbutus unedo, Ericaceae) is an evergreen tree with a circum-Mediterranean distribution. It has a great ecological and economic importance as a source of bioactive compounds with industrial applications and for fruit production. This study aims to characterize the fungi microbiome of this forestry species in order to develop biological control strategies in the increasing orchard production area. For this purpose, fungi endophytes were isolated from wild strawberry tree plants, and a molecular identification was carried out. In vitro assays were carried out to evaluate and characterize the antagonism of some endophytes. Among the several fungi endophytes isolated from strawberry tree (a total of 53 from 20 genera), a Trichoderma atroviride strain proved to have antagonism effect against several phytopathogens, including Alternaria alternata, Botrytis cinerea, Glomerella cingulata, and Mycosphaerella aurantia. This antagonism was particularly effective against Phytophthora cinnamomi, causing a reduction in growth of about 80% on this invasive oomycete. An enzymatic assay revealed the production of several enzymes by T. atroviride, such as cellulases, chitinases, glucosidases, alkaline phosphatases, and proteases, which is one of the several mechanisms known to be involved on Trichoderma biological control ability. The enzymatic activity, in particular that of cell wall-degrading enzymes, was accentuated when in a dual culture with P. cinnamomi. The production of serine proteases, aspartyl proteases, metalloproteases, and cysteine proteases was also detected in an experiment carried out in liquid medium, suggesting the involvement of these proteases on Trichoderma mycoparasitism mechanisms. Finally, in a three-way interaction with in vitro strawberry tree plants, the T. atroviride strain identified on this study (Au50) was able to protect the plants against P. cinnamomi, thus proving its potential as a biological control agent.