First Report of Campylocarpon fasciculare Causing Black Foot Disease of Grapevine in Turkey

Abstract

Soil-borne fungal diseases have become an important problem in grapevine nurseries of the Aegean region (western Turkey) in recent years. Reduced vigor, black vascular streaking in basal ends, blackish-sunken necrotic root lesions, and young vine death were observed in 15 grapevine nurseries of Manisa city in May 2011 and 2012. To determine the causal agents, symptomatic young grapevine (Vitis vinifera cv. Sultana 7) plants (grafted on 1103 Paulsen) were collected from nurseries (8 to 10 plants from each). Symptomatic basal end tissues were surface disinfested with 95% ethanol and flame sterilized. The internal tissues were plated onto potato dextrose agar amended with tetracycline (0.01%). Campylocarpon-like fungi were isolated (with 37.9% isolation frequency) from only one nursery (corresponding to 6.7% of all surveyed nurseries). Fungal colonies were incubated for 21 days in the dark to induce sporulation. Fungal colonies produced cottony aerial mycelium and turned chocolate-brown to dark brown on PDA. Abundant macroconidia were observed at branched conidiophores on long and cylindrical phialides. Microconidia were not observed. Macroconidia were generally 2 to 4 septate, cylindrical and slightly curved, with the following dimensions: 2 septate: 33.5 to 40.7 × 6.1 to 7.6 μm (mean: 35.9 × 6.8 μm), 3 septate: 36.2 to 43.4 × 6.6 to 8.3 μm (mean: 37.3 × 7.6 μm), and 4 septate: 48.9 to 53.5 × 7.6 to 8.3 μm (mean: 50.7 × 8.0 μm). Fifty macroconidia were measured. Morphologically, the isolates resembled the published description of Campylocarpon fasciculare Schroers, Halleen & Crous (2,4). For molecular identification, fungal DNA was extracted from mycelium and ribosomal DNA fragments (ITS1, 5.8S ITS2 rDNA), β-tubulin, and histone H3 genes, amplified with ITS 4-5, Bt 2a-2b, and H3 1a-1b primers (3,5), and sequenced. Sequences were compared with those deposited in GenBank. The isolate (MBAi45CL) showed 99% similarity with Campylocarpon fasciculare isolates AY677303 (ITS), AY377225 (β-tubulin), and JF735502 (histone H3). The DNA sequences were deposited into GenBank under accessions KJ573392, KJ573393, and KJ573394 for ITS, β-tubulin, and Histone H3 genes, respectively. To fulfill Koch's postulates, pathogenicity tests were conducted under greenhouse conditions on own-rooted grapevines (Vitis vinifera) cv. Sultana 7. Plants were removed from the rooting bench and the roots were slightly trimmed and submerged in a 107 ml–1 conidial suspension of the isolate for 60 min (5). After inoculation, the rooted cuttings were planted in 1-liter bags containing a mixture of soil, peat, and sand (2:1:1, v/v/v), and maintained in the greenhouse (24°C. 16/8-h day/night, 75% RH). Ten plants were inoculated with the isolate and five plants were submerged in sterile distilled water (control). After 4 months, young vines were examined for vascular discoloration, reduced root biomass, blackish lesions, and recovery of fungal isolates. The experiment was repeated twice. Blackish-brown discoloration of xylem vessels and necrosis in the basal ends was visible in the inoculated plants but not in the control plants. The pathogen was successfully re-isolated from 69.1% of the inoculated plants. This report is important for the new studies aiming at black foot disease control in Turkey viticulture. References: (1) A. Cabral et al. Phytopathol. Mediterr. 51:340, 2012. (2) P. Chaverri et al. Stud. Mycol. 68:67, 2011. (3) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (4) F. Halleen et al. Stud. Mycol. 50:431, 2004. (5) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.