Characterization of Lethal Toxin-Producing Genes in Amanita brunneitoxicaria and PCR-based Detection of Deadly Poisonous Amanitas in the Section Phalloideae

A recent discovery of Amanita brunneitoxicaria revealed that this mushroom and A. exitialis are the main causative agents of fatal mushroom poisoning in Thailand. Poisonous amatoxins present are bicyclic octapeptides encoded by the MSDIN family and macrocyclized by prolyl oligopeptidase B enzyme. Diversity of the MSDIN toxin-associated genes in A. brunneitoxicaria however, remains unexplored. Therefore, this study aimed to characterize the MSDIN family members in A. brunneitoxicaria in comparison with A. exitialis based on whole genome sequencing, followed by gene annotation using bioinformatic tools. Identification of conserved core peptides of the MSDIN family revealed the presence of α-amanitin and β-amanitin as endogenous toxins in A. brunneitoxicaria. In addition to the major amatoxins, amanexitide and phallacidin were also found in A. exitialis. Phylogenetic analyses of the MSDIN family members showed that an unknown peptide present in A. brunneitoxicaria is clustered with phalloidin. On the basis of PCR detection, a modified forward primer was incorporated into the PCR reactions to obtain clear and distinctive DNA fragments of approximately 300 bp from three deadly poisonous mushrooms of A. brunneitoxicaria, A. exitialis and A. fuliginea in the section Phalloideae. Hence, they could be distinguished from other edible and less toxic species in the same genus. Such rapid and accurate identification of the target region of the MSDIN family from the lethal mushrooms is relevant as means to reduce mortality. HIGHLIGHTS Amanita brunneitoxicaria and A. exitialis are responsible for most cases of fatal mushroom poisoning in Thailand During wild mushroom foraging, A. brunneitoxicaria is often mistaken for the edible species of A. vaginata A. brunneitoxicaria and other lethal aminitas contain amatoxin-producing genes encoded by the MSDIN family PCR-based detection of the target region of the MSDIN family can be used to distinguish the lethal aminitas in the section Phalloideae This rapid detection is useful for timely and appropriate medical treatment of mushroom intoxication GRAPHICAL ABSTRACT