These microorganisms were isolated and identified as fungal endophytes and tested for their performance to compete against R. solani using in vitro dual culture assays. We tested the ability of antagonistic fungal isolates to excrete volatile substances and evaluated the effect of filtrates of liquid cultures of all fungal isolates on the mycelial growth of R. solani. Finally, we evaluated the antagonism under greenhouse conditions. Rhizoctonia solani R14 and Phomopsis sp. R24 strains were isolated from infected potato plants from a field in August 2007 in Montreal region (Canada). Fungal endophytes (E1, E2 E8, E13, and E18) were

isolated from the leaves Ponatinib manufacturer of Norway maples in October 2007 in Montreal based on the methods described by Berg et al. (2005). These endophytes were evaluated for antagonism against R. solani. Fungal strains were identified by PCR and sequencing of internal transcribed spacer (ITS) regions of rDNA. Mycelia, grown in liquid potato dextrose broth at 25 °C, were harvested by filtration and used to extract DNA using the plant DNA extraction kit (Qiagen, Canada). PCR was performed using primers ITS1 and ITS4 to amplify ITS regions of seven isolates (R14, R44, E1, E2, E8, E13, and E18)

(Tables 1 and 2). Amplification reactions were carried out in a volume of 50 μL using the Dream Taq kit (Fermentas, Olaparib cost Canada) according to the manufacture’s recommendations. PCR was performed using a Mastercycler (Eppendorf, Canada) following the programme: 5 min at 94 °C, followed by 29 cycles of 30 s at 94 °C, 30 s at 59 °C ID-8 and 1 min at 72 °C, and 7 min at 72 °C. PCR amplicons were sequenced at the Genome Quebec Innovation Center (Montreal, Canada). Sequences were blasted using the nucleotide blast search at NCBI. Sequences were deposited in EMBL under

accession numbers FN646616–FN646622. Morphological observations such as colony growth, colour, type of mycelia, size, and form arrangement of conidia were used to confirm molecular data (Alexopoulos et al., 1996). Fungal isolates were screened for their ability to suppress the mycelial growth of R. solani strain R14 by in vitro dual culture assays on potato dextrose agar (PDA) (Lahlali et al., 2007). Each combination of pathogen/antagonist was replicated 10 times and plates were randomly placed in the dark and incubated at 25 °C until the PDA medium was completely covered with pathogen mycelia. As negative controls, 10 Petri dishes were inoculated only with an R. solani agar disc and a water agar disc. The radial mycelial growth of R. solani towards the antagonistic fungus (Ri) and that on a control plate (Rc) were measured and the mycelial growth inhibition was calculated according to the formula: (Rc−Ri)/Rc × 100. Statistical analyses were performed with anova using the sas statistical package (SAS Institute, Cary, NC). When the effect was found to be significant, the LSD was performed for mean separation at P≤0.05.