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First Report of Alternaria alternata Causing Leaf Blight in Pistacia terebinthus in Spain

López-Moral, A., Agustí-Brisach, C., Raya-Ortega, M. C., Roca, L. F., Lovera, M., Arquero, O., Trapero, A.
Plant disease 2018 v.102 no.11 pp. 2374
Alternaria alternata, DNA-directed RNA polymerase, Juglans regia, Pistacia lentiscus, Pistacia terebinthus, Pistacia vera, Prunus dulcis, color, conidia, containers, culture media, drought tolerance, fungi, hosts, indigenous species, internal transcribed spacers, lactic acid, leaf blight, leaves, mycelium, nut crops, pathogenicity, pathogens, photoperiod, protocols, relative humidity, rootstocks, runoff, sodium hypochlorite, spraying, summer, tap water, tissues, Spain
Pistacia terebinthus is a native plant from the western Mediterranean and abundant in Spain. It has been used as a rootstock of P. vera in the main growing regions of this crop in the world because it confers a degree of drought resistance (Gijón et al. 2009). In summer 2017, leaf blight symptoms were observed on P. terebinthus plants in a commercial nursery located in Almeria province (Andalusia region, Southern Spain). Large necrotic lesions occupying the entire leaf surface were consistently observed in ≈90% of P. terebinthus plants. Affected leaves were washed with tap water, surface sterilized for 1 min in 10% sodium hypochlorite, and washed twice with sterile distilled water (SDW). Small pieces from the margin of necrotic tissues were plated onto potato dextrose agar (PDA), acidified with 0.06% lactic acid, and incubated at 24°C with a 12-h photoperiod for 5 days. An Alternaria sp. was consistently isolated from necrotic leaf tissues. Fungal colonies were transferred to PDA and incubated as described above. One representative isolate (ColPat-420) was first identified based on its morphology as A. alternata (Woudenberg et al. 2013). This isolate produced on PDA abundant aerial mycelia, light to dark gray in color. Conidia were brown, septate, ovoid or elliptical, simple or grouped in chains, (16.3 to) 21.8 (to 27.5) × (7.5 to) 9.3 (to 11.3) μm, with two to four transverse septa and zero, one, or two longitudinal septa. The isolate ColPat-420 was identified molecularly by sequencing the internal transcribed spacer region (ITS) and RNA polymerase second largest subunit (RPB2) with primers ITS4/ITS5 (White et al. 1990) and RPB2-5F2/fRPB2-7cR (Woudenberg et al. 2013), respectively. ITS (accession no. MG859248) and RPB2 sequences (accession no. MG873562) were deposited at GenBank. These sequences showed high similarity to the ITS (100%) and RPB2 (98.8%) sequences of A. alternata isolate CBS 916.96 (accession nos. KF465761 and KC584375, for ITS and RPB2, respectively). For pathogenicity tests, healthy leaves of P. terebinthus, wounded and unwounded, were inoculated by spraying them to runoff with a 10⁵ conidia/ml suspension of the isolate ColPat-420 and incubating in humid chambers (56 × 18 × 41-cm plastic containers) at 24°C and 100% relative humidity for 15 days in darkness. Similarly, noninoculated leaves sprayed with SDW served as a control. There was one humid chamber per treatment (wounded and unwounded) with 15 replicated leaves. The experiment was conducted twice. All inoculated leaves showed necrotic irregular round lesions (from 0.2 to 2.5 cm in diameter) with abundant mycelium over the surface 15 days after inoculation, and A. alternata was reisolated, completing Koch’s postulates. No symptoms were observed on control leaves. In addition, pathogenicity tests using the same isolate were conducted with leaves of other hosts of A. alternata such as Juglans regia, P. lentiscus, P. vera, and Prunus dulcis, following the same protocol. Similar symptoms were observed in all of them, and the fungus was also reisolated. In all cases, A. alternata was not reisolated from noninoculated leaves. Although A. alternata is well-known as a pathogen of P. vera as well as of other nut crops (Michailides et al. 2016), to our knowledge, this pathogen has been described only recently affecting P. terebinthus in Turkey (Ozkilinc et al. 2017). Therefore, this study represents the first report of A. alternata causing leaf blight symptoms in P. terebinthus in Spain.