First Report of Polystigma amygdalinum Causing Red Leaf Blotch of Almond in California

California produces approximately 80% of the world's almonds [Prunus dulcis (Miller) D. A. Webb]. In May of 2024, yellow spots and orange to dark red-brown blotches, were detected in an almond orchard (Nonpareil, Monterey, and Fritz cvs.) in Merced County. Similar symptoms were observed during June of 2024 from 12 additional orchards in Fresno, Madera, Merced, San Joaquin, and Stanislaus counties. Disease incidence in orchards varied from a few infected leaves to nearly 100%. Symptoms were characteristic of red leaf blotch caused by the fungal pathogen Polystigma amygdalinum. Leaves from all orchards were collected and taken to the laboratory in sealed resealable bags to conduct morphological and DNA analyses to identify the pathogen involved. For molecular identification, we used a quantitative real-time PCR detection method, and a primer pair (PamyI2F4/PamyI2R2) previously developed for P. amygdalinum (Zúñiga et al. 2018). Following direct DNA extraction from necrotic leaf tissues, the assay confirmed the detection of P. amygdalinum in symptomatic leaves. In addition, four lesions on separate leaves from different orchards were selected for genomic DNA extraction and sequencing of the internal transcribed spacer region (ITS) using ITS1F/ITS4 primers (Gardes and Bruns 1993) and a segment of the 28S region of the large subunit (LSU) ribosomal RNA gene using LR5 and LR0R primers (Guterres et al. 2018). Resulting sequences (ITS: GenBank Accession PV491257-PV491260; LSU: GenBank Accession PV487836-PV487836) were compared to reference sequences (Habibi et al. 2015; Guterres et al. 2018) using BLAST analysis. The ITS sequences of the Californian isolates were 100% identical to at least one of P. amygdalinum reference strains MH205935 or KC756362; LSU sequences were at least 99.46% similar to that of P. amygdalinum reference strain (KM111540.1). Morphological analyses were done by mounting sections of necrotic leaves on microscopic slides for observation under a light microscope (Leica DM2000 LED Microscope, Wetzlar, Germany). Conidiomata consisted of globose pycnidia, 80-120 μm diam. Conidia were hyaline, aseptate, filiform, 22.1 to 36.9 × 1.6 to 2.5 μm (n = 60). Perithecia formed in fallen leaves during January and were flask-shaped and contained clavate asci with eight ascospores. Asci were 95.4 to 129.5 × 18.3 to 25.1 μm. Ascospores were aseptate, ovoid to cylindrical with one end round and one end slightly acute, sometimes slightly constricted in the middle, hyaline, and measured 15.2 to 20.7 × 6.1 to 8.4 μm. Pathogenicity tests were conducted on potted, 1-year-old almond trees (cv. Monterey and Nonpareil) under greenhouse conditions. An ascospore suspension of 1 × 104 ascospores·ml-1 was obtained by crushing fallen almond leaves that contained mature perithecia of P. amygdalinum. The emerging leaflets from 10 plants of each cultivar were spray-inoculated with the ascospore suspension and then covered with plastic bags for 48hrs. After 40 days, typical symptoms of red leaf blotch appeared on leaves of all inoculated plants for each cultivar and no symptoms occurred in the control plants. Koch's postulates were satisfied after confirming P. amygdalinum from symptomatic leaves through molecular identification as described above. This is the first detection of P. amygdalinum from almond in California, the United States, and North America. These findings improve our knowledge of the etiology and biology of red leaf blotch of almond in California.