First report of Alternaria alternata complex causing leaf spot on almond in Spain.

In 2022, Spain was the third largest almond-producing country in the world (FAOSTAT, 2022). Starting the summer of 2021 a high incidence of brown leaf spots with grey centre on leaf margin accompanied by severe defoliation was recorded on almond trees (Prunus dulcis [Mill.] D. Webb). The affected area was about 200 ha in the region of Campos de Hellín (Castilla-La Mancha, Spain). The affected orchards were characterized by high planting density and irrigation. The disease affected the cultivars present, 'Laureanne' and 'Penta'. In some orchards, up to 90% defoliation was observed before harvest. Symptomatic leaves were collected and surface sterilized by inmersion in 1% NaOCl for 2 min, followed by rinsing in sterile distilled water, and air-drying. Small fragments of leaf tissue (approx. 5 mm) were cut from the margins of necrotic lesions and plated on potato dextrose agar with streptomycin (PDAS). The plates were incubated at 28°C for one week in the dark, then the fungal isolate was sub-cultured on new PDAS. Two individual strains (A10 and A12) were obtained from different leaves by single hyphal tip isolation. The isolates initially appeared greyish-white, later darkening to black or olive-green with a lighter border. Conidia were light brown and obclavate to elliptical, divided by transverse and vertical septa and measured 10-52 × 6-20 µm for both isolates (n = 100). Morphological characteristics resembled those of the small-spored group of the Alternaria genus (Simmons 2007). Gene sequences of ITS rDNA, GAPDH, RPB2, and TEF1 regions were amplified using primer pairs ITS1/ITS4 (White et al. 1990), GPD1/GPD2 (Berbee et al. 1999), RPB2-5F2/RPB2-7cR (Sung et al. 2007; Liu et al. 1999) and EF1-728F/EF1-986R (Carbone & John 1999) respectively. The PCR products from isolates A10 and A12 were sequenced and submitted to NCBI GenBank database with the accession numbers of PQ554678-79 and PQ559657-62. A maximum likelihood phylogenetic analysis based on the concatenated gene sequences was performed using IQ-TREE v1.6.12 (Minh et al., 2020). The analysis showed that the isolates were closely related to A. alternata complex (Woudenberg et al. 2015). The pathogenicity test were carried out with isolates A10 and A12 in two trials, one on leaves with hypodermic needle wounds and one on unwounded leaves. Inoculation was carried out by spraying a water suspension of 5·104 conidia/ml on leaves of healthy one-year-old pot-grown almond plants (cv. 'Laureanne', n = 5), control plants were sprayed with sterile water. Following inoculation, plants were covered with a plastic bag and keep in darkness for two days. Growth chamber conditions were 25 ± 2°C, 70 ± 25 % RH and 12-h photoperiod. Symptoms similar to those initially observed, appeared on the inoculated plants 8 days after inoculation. Both isolates showed similar aggressiveness. In addition, 29 days after inoculation, defoliation rates close to 40% were achieved with no difference between the trials with and without leaf wounds. The fungus isolated from the lesions showed the same morphology and sequences as the inoculated ones, confirming Koch's postulates. To our knowledge, this is the first report of A. alternata as a causal agent of leaf spot disease on almond trees in Spain. Considering the importance of almond tree cultivation and its expected increase in the area under intensive and irrigated cultivation in Spain, it is essential to pay more attention to this new pathogen.