A CAPS test allowing a rapid distinction of Penicillium expansum among fungal species collected on grape berries, inferred from the sequence and secondary structure of the mitochondrial SSU-rRNA

作者：Garcia Carole; La Guerche Stephane; Mouhamadou Bello; Ferandon Cyril; Labarere Jacques; Blancard Dominique; Darriet Philippe; Barroso Gerard^*

来源：International Journal of Food Microbiology, 2006, 111(3): 183-190.

DOI：10.1016/j.ijfoodmicro.2005.10.004

• Penicillium expansion
• Penicillium thomii
• Emericella nidulans
• CAPS test
• SSU-rRNA
• mitochondria
• grape berry
• geosmin
• secondary structure

Penicillium expansum is a fungal species highly damageable for the postharvest conservation of numerous fruits. In vineyards, this fungus is sometimes isolated from grape berries where its presence may lead to the production of geosmin, a powerful earthy odorant, which can impair grapes and wines aromas. However, the discrimination of R expansum from related fungi is difficult because it is based on ambiguous phenotypic characters and/or expensive and time-consuming molecular tests. In this context, the complete sequences and secondary structures of Penicillium expansum and Penicillium thomii mitochondrial SSU-rRNAs were achieved and compared with those of two other phylogenctically related Ascomycota: Penicillium chrysogenum and Emericella nidulans. The comparison has shown a high conservation in size and sequence of the core and of the variable domains (more than 80% of nt identity) of the four SSU-rRNAs, arguing for a close phylogenctic relationship between these four species of the Trichocomaceae family. Large (from 10 to 18 nt) inserted/deleted (indel) sequences were evidenced in the V1, V5 and V6 variable domains. The size variations (10 to 18 nt) of the VI indel sequence allowed the distinction of the four species; the V5 indel (15 nt) was specifically recovered in E. nidulans; the V6 indel (16 nt), shared by the three Penicillium species, was lacking in E. nidulans. A couple of conserved primers (UI/R2) were defined to generate a PCR product containing the VI to V5 variable domains. This product contained the two regions of the four SSU-rRNAs showing the highest rates of nt substitutions, namely the V2 variable domain and, surprisingly, a helix (HI 7) of the core. The H 17 sequence was shown to specifically possess in P expanstan a recognition site for the Cla I restriction endonuclease. Hence, this enzyme generates a digestion pattern of the PCR product with two bands (350 bp+500 bp), specific to P. expansum and easily separable by agarose gel electrophoresis. This leads to a CAPS test usable for P. expansion discrimination among grape berries isolated filamentous fungi. The CAPS test was validated by a comparative analysis involving 29 strains belonging to 17 species currently isolated from grape berries in the Bordeaux vineyards.

• 出版日期2006-10-1