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First Report of Phoma exigua var. heteromorpha Causing Oleander Dieback in Spain

During the autumn of 2003, a new disease was detected in oleander (Nerium oleander L.) nurseries in Valencia and Murcia in eastern Spain. Affected leaves showed ovoid or ellipsoid necrotic spots. Necrotic lesions were also observed on stems and lateral shoots that resulted in severe blight and defoliation. In some cases, severe infections caused the death of plants. Isolations from symptomatic leaves and stems onto potato dextrose agar (PDA) supplemented with 0.5 g liter^-1 of streptomycin sulfate (PDAS) consistently yielded dark olivaceous fungal colonies. For sporulation, these isolates were transferred to potato carrot agar (PCA) and incubated at 25°C for 15 days with a 12-h photoperiod. Abundant pycnidia (200 μm in diameter) developed superficially or immersed in the culture medium. Conidia were hyaline, ellipsoid or cylindrical, guttulate, and occasionally, one septate. Conidial dimensions were 6.1 to 9.6 × 2.2 to 3.2 μm (average 6.2 × 2.8 μm). The addition of a drop of concentrated NaOH to the cultures gave a blue-green pigmentation to the agar changing to brown-red. On the basis of cultural characteristics and fungal morphology, the isolates were identified as Phoma exigua Desmaz. This identification was confirmed by sequencing the complete internal transcribed spacer regions 1 and 2, including the 5.8S ribosomal DNA of isolate Pho 6 (GenBank Accession No. AY899262). This sequence was identical to sequences in GenBank from well-characterized strains of P. exigua (1). Pathogenicity tests were conducted on 9-month-old oleander plants (cv. Splendens Gigantium) using three isolates of P. exigua from different locations. Two methods of inoculation were used. Leaves were spray inoculated with an aqueous suspension (1.5 × 10⁵ conidia per ml) of conidia harvested from 15-day-old cultures grown on PCA, or a 5-mm-diameter agar disc, cut from the margin of an 8-day-old culture growing on PCA, was inserted mycelium side down in a stem wound made with a sterile scalpel and sealed with Parafilm. Controls were inoculated with sterile distilled water or sterile PCA discs. There were five replicates for each isolate and inoculation method with an equal number of uninoculated plants. After inoculation, all plants were covered separately with plastic bags for 48 h to maintain high humidity. Plants were maintained in a greenhouse at 20 to 30°C. Within 5 to 15 days after inoculation, symptoms developed that were similar to those observed in the nurseries. The fungus was reisolated from the stems and leaves of all inoculated plants, completing Koch's postulates. On the basis of ITS sequence data and the host from which they were isolated, our isolates were identified as P. exigua var. heteromorpha (Sch. et Sacc.) Noordeloos et Boerema (2,3). This disease has been previously reported to cause severe damage to oleander in France, California, Italy, and the Netherlands. To our knowledge, this is the first report of P. exigua var. heteromorpha on oleander in Spain.

References: (1) E. C. A. Abeln et al. Mycol. Res. 106:419, 2002. (2) M. E. Noordeloos and G. H. Boerema. Versl. Meded. Plziektenk. Dienst. Wageningen 166:108, 1988. (3) H. A. van der AA et al. Persoonia 17:435, 2000.