First Report of Diaporthe phaseolorum on Sunflower (Helianthus annuus) in Croatia
Authors: Vrandecic K. , Cosic J. , Jurkovic D. , Duvnjak T. , Riccioni L. 
Published: September 2009

Sunflower (Helianthus annuus L.) is a crop that is grown worldwide for the production of edible oil. In Croatia, it has considerable economic significance. From 2004 to 2007, sunflower stems showed light-to-dark brown lesions of different sizes and shapes. The lesions were observed for the presence of pycnidia in affected areas. Isolations from infected tissue on potato dextrose agar (PDA) yielded in two fungal species. One, which was isolated in most cases, was the well known sunflower pathogen Diaporthe helianthi Munt. Cvet. Morphological characteristics, stromata pattern, formation of alpha and beta conidia, and ascostromata characteristic of the other isolated fungus matched the description of D. phaseolorum (Cooke & Ellis) Sacc. (2). D. phaseolorum frequency was 5%. On PDA, the fungus formed white, floccose, aerial mycelium that filled a petri dish (9 cm) in 6 days. D. phaseolorum produces conidiomata in black stromatic structures, which consist of pycnidia with alpha and beta conidia. The alpha conidia were unicellular, hyaline, ellipsoidal to fusiform, and 5.6 to 10.0 × 1.9 to 4.8 μm. The beta conidia were hyaline, elongated, filiform, straight, curved at one or both ends, and 11.7 to 27.6 × 0.7 to 2.0 μm. After 50 days, perithecia were formed. Asci were clavate and 27.64 to 40.1 × 5.70 to 8.2 μm. Eight ascospores formed within asci. Ascospores were two-celled, elliptic, hyaline, and slightly constricted at the septa, and 8.93 to 13.5 × 2.1 to 4.0 μm. Amplification and sequencing of the internal transcribed spacer (ITS) rDNA region were performed with ITS4 and ITS5 universal primers (3) on two isolates (Su9 and Su10) and data were deposited in GenBank (Accession Nos. GQ149763 and GQ149764). Comparison of sequences available in GenBank revealed that the ITS sequence was identical to D. phaseolorum found on Stokesia laevis Hill (Greene) (U11323/U11373) and identical to the strain CBS 116020 isolated from Aster exilis Elliot. (AY745018). On the basis of the obtained results of morphological characteristics and molecular approaches, the pathogen was identified as D. phaseolorum. Pathogenicity evaluation consisted of artificial infections on field-grown sunflower plants at the full button stage as described by Bertrand and Tourvielle (1). A leaf test was done by placing a mycelial plug of 5 × 5 mm from a cork borer of two isolates (Su9 and Su10) on the tip of the main vein. The inoculation site was covered with moistened, cotton wool and wrapped in aluminum foil to prevent the inoculum from drying out. Ten plants of each of the four replications were inoculated. Control plants were inoculated with pure PDA plugs. Lesions of 12 to 40 mm long were observed on the sunflower leaf 10 days after inoculation. Control plants did not develop symptoms. The pathogen was reisolated from the infected plants. To our knowledge, this is the first report of the finding of D. phaseolorum on sunflower in Croatia and we have no literature data about the occurrence of this fungus on sunflower in the world. References: (1) F. Bertrand and D. Tourvielle. Inf. Tech. CETIOM 98:12,1972. (2) E. Punithalingma and P. Holliday. No. 336 in: Descriptions of Pathogenic Fungi and Bacteria. CMI/CAB, Kew, Surrey, England, 1972. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, Inc., New York, 1990.