Scanning electronic microscopy was conducted using cryo-SEM (Hitachi S-3000N microscope, Japan), operating between 10 and 15 kV on samples containing a thin layer of gold sputter coating. Strain R5-6-1 was cultivated on PDA medium for 5 days in the dark at 25 °C, during which time conidiophore and conidia formation started. The margin of the Selumetinib molecular weight culture was then sliced out. The operation was carried out carefully not

to deform the surface features of the culture. The fungal strain was cultured in PD broth for 4 days at 180 r.p.m. min−1 in an orbital shaker at 25 °C. Fungal DNA was extracted using the Multisource Genomic DNA Miniprep Kit (Axygen Bioscience, Inc.) following the manufacturer’s instructions. Primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) (White et al., 1990) were used for amplification of the fungal rDNA internal transcribed spacer (ITS) regions 1 and 2. The PCR reaction (50 μL total volume) contained 5 μL 10 × PCR buffer, 7 μL 25 mM Mg2+,

2 μL 2.5 mM dNTP, 2 μL of each primer (10 μM), 4 μL (0.5–10.0 ng) of total DNA, 1 μL Taq polymerase and 27 μL ddH2O. Thirty-five cycles were run, each consisting of a denaturation step at 94 °C (40 s), an annealing step at 54 °C (50 s) and an extension step at 72 °C (60 s). After the 35th cycle, a final 10-min extension step at 72 °C was performed. The reaction products were separated in a 1.0% w/v agarose gel and bands were stained with ethidium bromide. The PCR products were then purified using the DNA Gel Extraction Wnt antagonist Kit (Axygen Bioscience, Inc.) and sequenced in an ABI 3730 sequencer (Applied Biosystems) using the ITS1 and ITS4 primers. The sequences were subjected to a blast search and were

aligned using clustal x together with the next neighbors (i.e. sequences that had a negative probability e-value of 0.0 in a blast search against the GenBank database); the alignment was manually corrected in genedoc. The evolutionary distance was determined using the Jukes–Cantor model to construct a phylogenetic tree by the neighbor-joining method using phylogeny inference package (phylip, v 3.68). The resultant trees were analyzed using the program consense to calculate a majority rule consensus tree. The tree file was then displayed by treeview. Bootstrap (1000 replicates) analysis used SEOBOOT, DNADIST, NEIGHBOR PI3K inhibitor and CONSENSE in phylip. Sequence inspection of the ITS1, 5.8S rRNA gene and ITS2 regions showed 100% identity of H. oryzae isolates R5-6-1 and RC-3-1. The blast similarity search revealed that H. oryzae shared 96%, 95% and 95% identity with ITS 1 and 2 sequences of unidentified Harpophora spp. (AJ132541), Harpophora spp. (AJ132542) and Harpophora spp. (AJ010039), respectively. In order to relate H. oryzae to already known Harpophora sequences and species and other related genera in Magnaporthaceae, a phylogenetic analysis was performed. As shown in Fig. 1, the NJ tree grouped Harpophora spp.