Characterisation of Pseudomonadaceae populations in Tuber melanosporum wild truffieres

Vilanova, X1*.; Sanchez, M1.; Viñas, M2. Morcillo, M. 1

1*Micología Forestal & Aplicada S. L. Address* Rambla Arnau de Vilanova , 6 local D 08800 Vilanova i la Geltrú (Barcelona-Spain). E-mail* xaviervilanova@micofora.com URL* www.micofora.com 2Giro Joint Research Unit IRTA-UPC, IRTA, Torre Marimon, E-08140-Caldes de Montbui (Barcelona-Spain).

Abstract In the last decade several surveys have revealed an important role of Pseudomonas genus as a Mycorrhizal Helper Bacteria (MHB). Moreover, the bioaugmentation of environmental isolated Pseudomonas strains to the substrate at inoculation time in different plant species throughout mycorrhization assays have been related on the occurrence of a greater number of mycorrhiza of Tuber genus. In this work, a selective culture medium was designed to isolate bacteria belonging to Pseudomonaceae family from soil samples taken from two different wild truffières. The amount of culturable Pseudomonadaceae populations was monitored using the selective medium throughout one year in different wild truffières. A total of eight samples per monitored truffières were taken every 40 days for the two monitored truffières. In the same area, non-truffle producing evergreen oaks were used as negative controls being sampled simultaneously to the monitored truffières. The results revealed both a low diversity of colonial morphology and a similar abundance of culturable Pseudomonadaceae strains on the selective-designed agar medium throughout the one-year survey in both monitored truffières (6.44 x 104 ± 3.54 x 104 CFU · g

-1and 5.71 x 104 ± 3.25 x 104 CFU · g

-1 respectively (± standard deviation of n=8). Non-producing trees

showed similar colonial morphologies to those found from the truffières, but less abundant (2.54 x 104 ± 2.54 x 104 CFU · g-1of soil (± standard deviation of n=8)). Finally, thirty samples from both truffières and controls were characterized by means of 16SrRNA gene-based PCR-DGGE (Polymerase Chain Reaction (PCR) with a subsequent Denaturing Gradient Gel Electrophoresis (DGGE). We obtained a low diversity (1-4 DGGE bands) in all the studied samples (both truffières and controls). It is noteworthy that most of detected bands migrated mainly in two defined positions from the DGGE gel, corresponding to a two different ribotypes belonging to Pseudomonas chlororaphis and Stenotrophomonas spp. respectively. KEYWORDS

Pseudomonas, MHB, Tuber melanosporum, wild truffieres.