Verticillium wilt, primarily caused by Verticillium dahliae Klebahn and V. albo-atrum Reinke & Berthold, affects a wide range of economically important crops. This disease is an increasing problem in areas where young mango trees are planted on land previously planted in vegetable crops. In 2008, symptoms of Verticillium wilt were observed in mango cvs. Kent and Osteen in the subtropical fruit-producing area of Málaga in southern Spain. In a new mango grove of cv. Kent, previously planted in potatoes and tomatoes, ~20% of 200 1-year-old trees had one-sided branch dieback. In many of these trees the symptoms expanded, leading to decline and eventual death. Cross sections of affected branches revealed brown vascular discoloration. Verticillium was isolated from surface-sterilized segments of symptomatic branches placed on acidic potato dextrose agar (PDA). Plates were incubated at 24°C. After 3 days, slow-growing colonies were transferred to PDA. Verticillium was similarly isolated from symptomatic potato plants grown in a nearby field. Identification of V. dahliae was initially based on morphology and further confirmed by molecular methods. All isolates tested produced microsclerotia, a defining feature that distinguishes V. dahliae from V. albo-atrum. For molecular characterization, V. dahliae specific primers 19 and 22 (1) and universal primers ITS1 and ITS4, which amplify the rRNA internal transcribed spacer (ITS) region (4), were used. Bands of expected size were amplified with both primer combinations. ITS fragments were sequenced and identical to the V. dahliae reference sequence (GenBank AY555948) (3). Pathogenicity assays were conducted with a selected isolate from mango using tomato plants from the susceptible line ‘Moneymaker’ and the near isogenic ‘Motabo’ line carrying the Ve gene conferring resistance to race 1 isolates. Five 1-month-old plants (four-leaf stage) were inoculated by root immersion in a suspension of 107 conidia/ml. Five control plants were mock inoculated with distilled water. As a positive control, five plants were inoculated with the previously described race 1 strain Dvd-T5 (2), which induces severe symptoms in susceptible tomato cultivars. Symptoms were scored visually at various time points up to 40 days by a 0 to 5 scale in which 0 = negligible chlorosis or wilting, 1 = chlorosis and wilting and/or curling in individual leaves, 2 = necrosis in leaves, 3 = at least one branch dead, 4 = wilt and/or chlorosis in upper leaves and/or two or more branches dead, and 5 = plant dead or all leaves and most of stem necrotic. The isolate from mango caused typical Verticillium wilt symptoms with a mean disease rating of 3.6 at 40 days postinoculation in both lines. The mean disease rating for Dvd-T5 in Moneymaker 40 days postinoculation was 4.0. V. dahliae was reisolated from symptomatic plants but not from noninoculated controls. To our knowledge, this is the first report of Verticillium wilt on mango in Spain. More problems with Verticillium wilt are expected because of the increasing planting of mango in fields previously dedicated to horticultural crops. References: (1) J. H. Carder et al. Modern Assays for Plant Pathogenic Fungi: Identification, Detection and Quantification. CAB International, Oxford, 1994. (2) K. F. Dobinson et al. Can. J. Plant. Pathol. 18:55, 1996. (3) M. P. Pantou et al. Mycol. Res. 109:889, 2005. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Amplification. Academic Press, San Diego, 1990.
First Report of Verticillium Wilt Caused by Verticillium nonalfalfae on Pelargonium grandiflorum in Italy