Amanita ponderosa, Malençon & R.Heim

Arraiano-Castilho, Ricardo, Silva, Ana Cristina, Vila-Viçosa, Carlos, Castro, Mário Rui, Morgado, Luís Neves & Oliveira, Paulo, 2022, The Amidella clade in Europe (Basidiomycota: Amanitaceae): clarification of the contentious Amanita valens (E. - J. Gilbert) Bertault and the importance of taxon-specific PCR primers for identification, Cryptogamie, Mycologie 20 (6), pp. 139-157 : 146

MOLECULAR PROBES FOR DISCRIMINATION FROM AMANITA PONDEROSA

The use of ITS and LSU primers specific for Amanita pseudovalens comb. nov., stat. nov. on the Ode01-Ode16 samples indicated that all DNA extracts from the Luzianes A and B collections (Ode1-8 and Ode10-12) and from the Portalegre collection (Ode16) were A. pseudovalens comb. nov., stat. nov. ( Fig. 6 ). All extracts that did not amplify with these primers amplified with the positive control primer sets (not shown). Similar results were obtained with some of the herbarium material ( Appendix 2 ).

Figures

FIG. 6. — Results of PCR amplifications using discriminant probes for Amanita pseudovalens comb. nov., stat. nov.: A, ITS region: ITS3-ApsIr3 (all samples positive with concurring control with primers ITS3-ITS4, not shown); B, LSU region: NLC2R-ApsLr2 (all positive with control NLC2R-LR5); Ode15 and Ode16 were negative and positive, respectively, in another amplification (not shown); M, molecular marker; neg. designates the negative control (water); pos. designates the positive control (the P01 extract).

APPENDIX 2. — Example of a matched comparison of Amanita ponderosa Malençon & R.Heim (ApoIf2-ITS4) and A. pseudovalens comb. nov., stat. nov. (ITS3- ApsIr3) for the same set of samples representing these two species, along with others of A. curtipes E.-J.Gilbert and A. lepiotoides Barla, and an unknown sample (SM-DB 61). The whole contents of each 20 µL PCR reactions were loaded, to help detect faint signals. Negative controls (neg.) with water instead of DNA. Amanita ponderosa is represented by the valens type (G73) and three samples from Spring 2010 (B07, P08, B10). Amanita pseudovalens is represented by the pseudovalens type (G74), the two MPU samples, Ode05 and Ode07. The Amanita curtipes samples (M03, M04 and M05) may produce a weak co-migrating band with the ITS3-ApsIR3 primer pair, and the A. lepiotoides samples (G08 and Eusk) were negative in both cases. Positive control reactions were not attempted because herbaria materials may be contaminated with other fungi.