First Report of Phomopsis longicolla on Cocklebur (Xanthium strumarium) in Croatia

Cocklebur (Xanthium strumarium L.; family Asteraceae) is a widespread weed species in eastern Croatia found especially in arable crops including soybean. Symptoms of disease appear after the plants reach physiological maturity. Stems and branches are completely blighted, and on the surface, are covered with minute, black pycnidia embedded in the epidermal tissue of the host and are especially numerous around nodes. More than 100 plants with symptoms were examined. From each plant with symptoms, three pieces of symptomatic tissues (5 to 10 mm) were disinfected and placed on potato dextrose agar (PDA), pH 4.5 and 25°C with a 12/12 h of light/dark regimen. The cultural and morphological characteristics of the fungi isolated from X. strumarium corresponded with those described (1) for Phomopsis longicolla Hobbs isolated from soybean. P. longicolla frequency was 3%, while other isolates belonged to other Phomopsis species. To confirm the morphological identification of isolates, molecular identification was performed. DNA of four isolates was extracted from 7-day-old monoconidial cultures grown on PDA. The internal transcribed spacer (ITS) regions of ribosomal DNA were amplified with universal primer ITS4 and ITS5 and sequenced (M-Medical Genenco, Rome). The sequences were aligned with the multiple sequence alignment program ClustalW, showing 100% similarity among them, and a sequence (GenBank Accession No. EF026104) was compared with the ITS sequences available on the database, revealing that it is identical to many P. longicolla isolates. To confirm Koch's postulate, cocklebur plants were infected in the field by applying mycelial plugs (5 mm in diameter) from the margin of 6-day-old cultures to the plant stem. The inoculation point was internodal at the mid-stem. After inoculation, plugs were covered with a piece of cotton wool and aluminum foil. Stem lesions were measured 10 days after inoculation. Mean stem lesions were 15 to 21 mm. A pathogenicity test was also done on soybean cv. Tisa (21-day-old) seedlings by applying mycelium plugs (5 mm) with a sterile scalpel on previously wounded hypocotyls. The inoculate point was covered with wet cotton wool and aluminum foil. After 10 days, mean stem lesions were 18 to 30 mm. The pathogen was always reisolated from the stem lesions. Control plants inoculated by PDA plugs did not exhibit any symptoms. There is a report of P. longicolla on cocklebur in the United States (2) and on other plants from the Asteraceae family. Other weeds such as Abutilon theophrasti Med., were shown to be a host of fungal pathogens belonging to the Phomopsis/Diaporthe complex of soybean (3). Our results also confirm that cocklebur could be a natural inoculum source for Phomopsis seed decay of soybean caused primarily by P. longicolla. However, to our knowledge, this is the first report of P. longicolla being isolated from naturally infected cocklebur in Croatia. References: (1) T. W. Hobbs et al. Mycologia 77:535, 1985. (2) K. W. Roy et al. Can. J. Plant Pathol. 19:193, 1997. (3) K. Vrandecic et al. Plant Pathol. 53:251, 2004.