Rosa Jaime Frias

Pistachio (Pistacia vera) is one of the most widely cultivated nut crops in California with approximately 115,000 ha of bearing trees. In recent years, Phytophthora root and crown rot has emerged as a severe and widespread disease of pistachio, particularly in orchards established on the industry-standard University of California Berkeley (UCBI) rootstock. To promote sustainable management strategies against Phytophthora root and crown rot, this study investigated the relative tolerance of three commercial rootstocks (UCBI [Pistacia atlantica × P. integerrima], Pioneer Gold I [PGI; P. integerrima × P. integerrima], and Platinum [P. integerrima × P. atlantica]) to Phytophthora niederhauserii, Phytophthora mediterranea, and Phytophthora taxon walnut. Stem and soil inoculation experiments were conducted, and lesion lengths on rootstocks stems, root loss, and crown rot development were assessed to determine differences in rootstock susceptibility. Platinum consistently exhibited significantly smaller lesion lengths, a lower percentage of root loss, and little to no crown rot development relative to UCBI and PGI rootstocks. In contrast, UCBI and PGI were confirmed to be highly susceptible to Phytophthora root and crown rot across all experiments. These findings demonstrate clear differences in rootstock tolerance and support the use of Platinum rootstocks in areas prone to Phytophthora root and crown rot infections caused by P. niederhauserii, P. mediterranea, or Phytophthora taxon walnut.

Several fungal species in the Botryosphaeriaceae family are associated with trunk diseases of major agricultural crops, such as almond and grapevine. These diseases can lead to considerable yield losses in vineyards and orchards. Botryosphaeriaceae can also infect plant material and seedlings during nursery operations. Latent infections in contaminated propagation materials play an important role in the spread of these fungal pathogens. Accurate identification and early detection of these pathogens are crucial for improving disease management. Molecular tools, such as real-time PCR, are widely used as rapid and sensitive approaches to improve disease diagnostics, allowing early detection and quantification of target pathogens. In this study, we developed two SYBR Green qPCR assays for the specific detection and quantification of Botryosphaeria dothidea and species within the Neofusicoccum parvum-ribis complex. Assays were validated using artificially inoculated and naturally infected grapevine and almond plants. We assessed the specificity of the assays on 79 isolates, including various Botryosphaeriaceae and off-target species, such as other pathogens associated with grapevine trunk diseases. Target pathogens were successfully detected in all artificially inoculated plants, as well as in most naturally infected field samples tested. These results demonstrate that both SYBR Green qPCR assays are specific, selective, and sensitive, validating their use for detecting B. dothidea and N. parvum-ribis species in wood samples. This cost-effective tool can be applied for accurate detection of these pathogens from both symptomatic and asymptomatic plants, enabling large-scale analyses and early screening to promote the production of healthy planting material.

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.