First Report of Charcoal Rot Caused by Macrophomina phaseolina in Sunflower in Turkey.

Charcoal rot symptoms were observed on 2-month-old oilseed sunflower plants (Helianthus annuus L.) in the Eskişehir Province of Turkey in June 2009. The disease was observed in 70% of the fields surveyed and incidence ranged from 10 to 50%. Symptoms were first observed in plants approaching physiological maturity and consisted of silver-gray lesions girdling the stem at the soil line, reduced head diameter compared with noninfected plants, and premature plant death. Pith in the lower stem was completely absent or compressed into horizontal layers. Black, spherical microsclerotia were observed in the pith area of the lower stem, underneath the epidermis, and on the exterior of the taproot. The internal stem had a shredded appearance. Later, the vascular bundles became covered with small, black flecks or microsclerotia of the fungus. Forty plant samples were collected from 10 fields. After surface sterilization with 2% NaOCl, outer tissues sampled from diseased tissues (2 to 3 mm long) of root and stems were removed and transferred to potato dextrose agar containing 250 mg liter of chloramphenicol. Petri plates were incubated for 7 days at 26 ± 2°C in the dark. Ninety-eight percent of the fungal colonies were identified as Macrophomina phaseolina (Tassi) Goidanich based on gray colony color, colony morphology, and the size of the microsclerotia, which ranged from 80 to 90 μm in diameter, from both infected sunflowers and compared with pure cultures (3). All resulting cultures produced abundant microsclerotia. The only other sunflower pathogen known to form microsclerotia is Verticillium dahliae Kleb., whose microsclerotia are irregular in shape and 15 to 50 μm in diameter. Sequence-related amplified polymorphisms technique was used for diversity of M. phaseolina since it has proven to be more informative than amplified fragment length polymorphism, random amplified polymorphic DNA, and simple sequence repeat (2). Results showed a high level of genetic diversity (60%) among the 26 isolates of M. phaseolina. Sequencing of the internal transcribed spacer region (1) showed high homology (>96%) to M. phaseolina (GenBank Accession No. HQ380051). Pathogenicity tests for 20 isolates of M. phaseolina were carried out on three commercially used cultivars, SANAY, TUNCA, and TR-3080. Groups of 10 seedlings were grown separately in an autoclaved peat/soil mixture in 30-cm-diameter plastic pots in a greenhouse at 30 ± 2°C. Soil infestation was performed 1 day before sowing. Two-week-old cultures on barley medium (4) were blended in distilled sterile water and adjusted to 10 sclerotia ml. Each pot received 250 ml of inoculant. Each treatment had three replications. Three pots for each cultivar were left uninoculated. Within 3 weeks, five to seven inoculated plants in each pot died. Identical disease symptoms were observed 30 days after inoculation; on the control plants no symptoms were observed. Microsclerotia were produced after 7 weeks at the stem base on 85% of the surviving plants. To our knowledge, this is the first report of M. phaseolina in sunflower in Turkey. References: (1) B. D. Babu et al. J. Plant Dis. Prot. 96:797, 2007. (2) H. Budak et al. Theor. Appl. Genet. 109:280, 2004. (3) P. Holliday and E. Punithalingam. No. 275 in: Description of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1970. (4) M. R. Omar et al. J. Plant Dis. Prot. 114:196, 2007.