During the late summer of 2003, a wilt disease of the weed Italian cockleburr (Xanthium italicum Mor.) was observed in the Basilicata Region of southern Italy. Diseased plants were growing near an apricot orchard in which some trees were severely affected by Verticillium wilt. The most characteristic symptoms of the wilt disease affecting Italian cockleburr were yellowing, stunting, and gradual wilting. Also, diagonal and cross sections of stems revealed brown discoloration of their vascular tissues. To elucidate the etiology of the disease, we attempted detection and identification of the causal agents using traditional and polymerase chain reaction (PCR)-based methods. Small pieces of petiole and stem tissues from diseased and asymptomatic plants were surface disinfested in NaOCl solution, rinsed in sterile distilled water, blotted dry, and plated onto water agar (WA) medium. Following incubation at 22°C, the emerging colonies were transferred to potato dextrose agar (PDA). Verticillium dahliae (one isolate) was consistently identified on the basis of its morphological features according to the description of Smith (2). Using PCR assays with the primer pair ITS5/ITS4 (3), which are directed to fungal nuclear ribosomal DNA (rDNA) repeat sequences, an amplification product of approximately 560 bp was obtained by using total DNA extracted from wilt-affected Italian cockleburr plant tissues (five plants examined) as well as fresh mycelium from the V. dahliae-infected Italian cockleburr pure culture-maintained isolate mentioned above. No visible PCR products were obtained with total DNA from asymptomatic Italian cockleburr plants. Sequence analysis of the ITS5/ITS4 amplicons revealed no differences in their nucleotide positions. The obtained sequence of the V. dahliae-infected Italian cockleburr isolate (GenBank Accession No. AJ865691) was then used as query sequences in a BLAST 2.0 search (1). Sequence of the southern Italian isolate proved to be identical to that of the Greek strain “76 Greece” of V. dahliae (GenBank Accession no. AF104926). To prove Koch's postulates, 10 healthy Italian cockleburr seedlings were experimentally inoculated by dipping trimmed roots in a single-conidial suspension (1.5 × 106 CFU/ml) obtained from 10-day-old colonies of the V. dahliae-infected Italian cockleburr pure culture-maintained isolate. After 4 weeks, all inoculated Italian cockleburr plants showed symptoms identical to those of naturally infected field-grown plants. V. dahliae was consistently reisolated from inoculated plants. Additional inoculation experiments revealed that pepper and eggplant were also susceptible to the V. dahliae-infected Italian cockleburr isolate showing typical Verticillium wilt symptoms. To our knowledge, this is the first report of the occurrence of Verticillium wilt of X. italicum. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) H. C. Smith. N.Z. J. Agric. Res. 8:450, 1965. (3) T. J. White et al. Pages 315–322 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.
Determination of the Response of Some Cotton Varieties to Cotton Wilt Disease Caused by Verticillium dahliae Kleb