First Report of Cylindrocarpon liriodendri on Kiwifruit in Turkey

During a routine survey of diseases of kiwifruit (Actinidia chinensis Planch.) cv. Hayward conducted in autumn of 2009 in Ardeşen, Rize Province (eastern Black Sea Region, Turkey), symptoms of a new disease were observed in five locations. Affected trees showed leaf wilting that frequently led to the death of the trees. Symptoms at ground level included necrotic lesions on woody tissues of both the rootstock and roots. Small pieces from necrotic wood and root tissues were surface disinfested and plated onto potato dextrose agar (PDA) medium amended with 0.5 g liter^-1 of streptomycin sulfate and incubated for 7 days at 25°C in the dark. Isolates were transferred to PDA and presumptively identified as a Cylindrocarpon sp. by morphology and conidial characteristics. The isolates were transferred to PDA and Spezieller Nährstoffarmer Agar (SNA) and then incubated at 25°C for 10 days with a 12-h photoperiod. On PDA, the isolates developed floccose to felted mycelium, which varied in color from brown-yellow to sepia. On SNA, all isolates produced microconidia measuring 6.25 to 15 (9.6) × 2.5 to 5 (3.02) μm and macroconidia of one-septate measuring 7.5 to 20 (13.3) × 2.5 to 5 (3.8) μm, two-septate measuring 12.5 to 25 (20.7) × 3.25 to 5 (4.58) μm, and three-septate measuring 16.3 to 30 (11.04) × 3.75 to 5 (4.82) μm. Chlamydospores 7.5 to 11.3 (9.78) μm were intercalary or terminal in the mycelium, single or occasionally in chains. Identity of these isolates was determined by a multiplex PCR system using a set of three pairs of specific primers (Mac1/MaPa2, Lir1/Lir2, and Pau1/MaPa2) (1), which generated a product size of 253 bp, which is characteristic of Cylindrocarpon liriodendri J.D. MacDonald and E.E. Butler, in agreement with morphological features (2). Additionally, the internal transcribed spacers regions (ITS1 and ITS4) of rDNA were obtained for isolates 10K-TR1 and 10K-TR2 and deposited in GenBank (Accession Nos. HQ113122 and HQ113123). These sequences showed high similarity (98%) with the sequence of C. liriodendri (GenBank Accession No. DQ718166). A pathogenicity test was conducted using isolate 10K-TR1 and repeated twice. Six 8-month-old callused and rooted cuttings of kiwifruit cv. Hayward were surface disinfested for 1 min in a 1.5% sodium hypochlorite solution, washed twice with sterile distilled water (SDW), and inoculated by dipping their roots for 30 min in a spore suspension of the fungus (1 × 10⁶ conidia ml^-1) obtained from 30-day-old colonies grown on PDA. Six control cuttings were dipped in SDW. Two weeks later, cuttings were drench inoculated with 50 ml of the designated spore suspension to guarantee root infection and controls were drenched again with SDW. Plants were maintained in a greenhouse with a temperature range of 25 to 30°C. Four months after inoculation, the inoculated plants developed wilting and root symptoms similar to those observed in natural infections and C. liriodendri was reisolated, completing successfully Koch's postulates. No symptoms were observed on the control plants. To our knowledge, this is the first report of C. liriodendri on kiwifruit trees in Turkey. References: (1) S. Alaniz et al. Plant Dis. 93:821, 2009. (2) F. Halleen et al. Stud. Mycol. 55:227, 2006.