Diagnosis of ink disease of chestnut by molecular identification of associated Phytophthora species.
For diagnostic proposes of ink disease, chestnut orchards with symptoms of decline or sudden death of trees were sampled by soil baiting techniques and selective agar media (P10VPH). A total of 36 Phytophthora isolates were obtained. One isolate per tree and 3 or 2 isolates from the soil of the same plant were collected for molecular identification. Genomic DNA was extracted from all the isolates and from the reference strain P. cinnamomi CECT 2965. The ribosomal regions ITS1, 5.8S and ITS2 were amplified with the universal primer pairs ITS6 and ITS4 by PCR. The amplified fragment (900 bp) was digested with the restriction enzymes MspI, AluI and TaqI. Two different patterns of fingerprinting were obtained with the enzymes TaqI and AluI (type I and II) and 3 different patterns with MspI (type I, Ia, II). The fingerprinting of each isolate was compared in the CABI database by public web access. Type I and Ia (14 isolates) were assigned to P. cinnamomi and type II (4 isolates) was assigned to P. cambivora. Molecular methods provide a rapid means of Phytophthora species identification associated with ink disease of chestnut and will provide a useful tool for etiological and epidemiological studies of this important disease of chestnut.