First Report of Verticillium dahliae Causing Wilt in Pumpkin in Trinidad.

Abstract

Pumpkin (Cucurbita pepo L.) is an economically important crop in Trinidad. Production supplies local and export markets in Barbados, Antigua and Barbuda, Canada, the United States, and the United Kingdom. However, there has been a marked decline in pumpkin export over the last 3 years. Field infections and development of disease in transit have been identified as major factors in decreased export. Between November 2006 and December 2007 (day/night temperatures of 32 to 34°C and 25 to 27°C, respectively), symptoms of vascular wilt were observed in at least 17 pumpkin fields. Symptoms included discoloration (yellowing with subsequent browning) of the older leaves and stems followed by wilting, girdling, and dieback of vines prior to fruit maturity. Necrotic leaves remained attached to the stems. A light brown vascular discoloration in the absence of a sticky exudate was observed in the stems. Onset of symptoms was most apparent at the late flowering to early fruit development stage. All C. pepo lines appeared equally affected by the disease. Yield losses (reduction of fresh weight and proportion of immature fruits at harvest time) as a result of this disease were estimated at 30 to 80%. Accurate diagnosis and pathogen identification were based on symptomology, colony morphology on potato dextrose agar (PDA) and semiselective media (including presence of microsclerotia), pathogenicity tests, and molecular characterization of the rDNA region. Pathogenicity tests with five isolates (randomly selected but geographically distinct) were conducted on healthy pumpkin plants at the two-true-leaf stage. A spore suspension of 2.1 × 106 conidia/ml was used to inoculate eight seedlings grown in sterile potting mix (1). Plants inoculated with sterile distilled water served as negative controls. Plants inoculated with the spore suspension developed a vascular wilt and general decline 35 days postinoculation. The pathogen was reisolated from all inoculated pumpkin seedlings. Pathogenicity tests were repeated once. Colony morphology consistently conformed to established criteria for Verticillium dahliae (Kleb.) (2). PCR amplification with universal primers ITS4 and ITS5 (3) was carried out on fungal DNA extracted from pure cultures. Sequence comparison of amplicons of approximately 600 bp long (GenBank Accession No. EF377335) was carried out using MEGABLAST search ( www.ncbi.nlm.nih.gov/blast/ ) for highly similar sequences and Lasergene v7.2 (DNASTAR Inc. Madison, WI) software. Alignment data revealed the highest and most significant homology to Verticillium dahliae (GenBank Accession No. DQ282123) at 98.2%. V. dahliae has a wide host range and causes vascular wilt in a large number of economically important crops (2). Control strategies are complicated by the ability of the microsclerotia produced by V. dahliae to survive for more than a decade in soil despite the absence of a host (4). To my knowledge, this is the first report of Verticillium wilt affecting pumpkin in Trinidad.