Sunflower (Helianthus annuus L.) plants with symptoms of interveinal chlorosis were observed in the summer of 2013 in one field in Cadiz (Spain) where the performance of 30 hybrids was assessed. Symptoms affected 80% of the hybrids with incidence as high as 90%. Chlorosis and yellowing near the leaf margin were visible at floral initiation, and they progressed from the lower to upper leaves. Mottled leaves were observed near the top of the plants. On severely affected leaves, chlorotic patches enlarged, coalesced, and large areas of the leaves became necrotic and dried. Cross sections of the lower stem showed a brown discoloration of the vascular system. The fungus that was consistently isolated from stem and petiole tissues of sunflower plants was morphologically identified as Verticillium dahliae Kleb. (Vd) (5) and molecularly confirmed by PCR amplification of the 526-bp band (4). The race of the isolates was determined in a greenhouse experiment at 18 to 28°C from February to April 2014. Isolates 1-13 and 2-13 of Vd, obtained from two of the hybrids in 2013, one of them being Transol, were independently inoculated to 1-month-old plants of each of three sunflower genotypes: the susceptible hybrid Transol and the inbred lines HA89 (carrying the V1 gene for resistance to Vd) (2) and HAR5 (resistant to other diseases but with unknown reaction to Vd). Plants were inoculated by immersing roots in a suspension of 106 conidia per ml for 30 min. Inoculated plants were individually transplanted to 1-liter pots filled with sand/silt. Roots of the control treatments were immersed in water. Six replications (pots) were established for each isolate × genotype combination, according to a complete randomized 3 × 3 factorial design. Five weeks after inoculation, symptoms developed in 100% of the plants in the three sunflower genotypes. Severity of symptoms (SS) in each plant was assessed as percentage of foliar tissue affected. Significantly higher SS occurred on inoculated plants as compared to non-inoculated plants, which did not develop symptoms. Mean disease severity on inoculated plants was 80% (averaged across isolates and genotypes). A significant effect of genotypes was obtained. Mean SS averaged across isolates were 98, 73, and 69% for HAR5, HA89, and Transol, respectively. When stem tissues from the three sunflower genotypes were sampled and incubated on potato dextrose agar at 25°C, the mycelial growth of Vd was confirmed for the inoculated plants but not for the control plants. Isolates of Vd infecting the resistant inbred line HA89 have only been identified in Argentina (1) and the United States (3). To our knowledge, this is the first report of a race overcoming the V1 gene in HA89 in Europe. This poses a risk to commercial sunflower breeding programs in European countries. References: (1) A. B. Bertero de Romano and A. Vázquez. Page 177 in: Proc. 10th Int. Sunf. Conf., Surfers Paradise, Australia, 1982. (2) G. N. Fick and D. E. Zimmer. Crop Sci. 14:895, 1974. (3) T. Gulya. Helia 30:115, 2007. (4) J. Mercado-Blanco et al. Plant Dis. 87:1487, 2003. (5) W. E. Sackston. Plant Dis. Rep. 41:885, 1957.
Rapid PCR-based Detection of Phytoplasmas from Infected Plants