ABSTRACT
In 2021 and 2022, wilt symptoms were observed in lettuce (Lactuca sativa L.) fields in Yuma County, Arizona (AZ). Incidence was < 1% at all locations. Symptoms included stunting, yellowing and wilting of outer leaves. As disease progressed, outer leaves wilted and turned necrotic. In advanced stages, tap roots turned brown-gray, with few feeder roots. The crown remained intact until the plant collapsed. Symptomatic romaine and iceberg plants were collected from two sites. Necrotic roots were washed and then plated on amended corn meal agar (PARP) (Kannwischer et al. 1978). After 2-3 days, slow growing, coenocytic, submerged mycelia grew from these pieces. In culture, profuse oogonia formed with diameters of 30-39 (avg. = 33.7) µm and spiny protuberances (5-8 [avg. = 6.4] µm long) with thickened bases. Oospores were spherical and aplerotic, with diameters of 25-32 (avg. = 27.8) µm. Lettuce with identical symptoms from the Salinas Valley, California (CA) were also tested and similar isolates were recovered. Pathogenicity was tested using six AZ and one CA isolates. Inoculum was grown on barley seeds moistened with sterile distilled water, autoclaved three times (at 24 h intervals), then inoculated with colonized agar plugs and incubated at 20°C. Inoculum was used after two weeks. For each isolate, 12 3-week-old iceberg (cv. Speedway) and romaine (cv. Del Sol) plants were inoculated by placing 3-4 colonized barley seed next to the roots of the potted plants. Plants were maintained in a greenhouse at 24-26°C (daytime high) with ambient light. After eight days, all inoculated plants exhibited chlorotic lower leaves that later wilted. Both feeder roots and taproots showed brown-gray discoloration and were necrotic. Microscopy showed the presence of spiny oogonia in inoculated roots. Symptoms caused by the AZ and CA isolates were indistinguishable from each other. Isolations from necrotic tissue resulted in colonies morphologically identical to the original isolates. Twelve control plants inoculated with uncolonized barley seed developed no symptoms. DNA was extracted from all seven AZ and CA isolates pre-inoculation, and AZ isolate 2 recovered from both lettuce types post-inoculation for molecular characterization. The internal transcribed spacer (ITS) and cytochrome C oxidase subunit 2 (COX II) were amplified for the above isolates using primer sets ITS1/ITS4 (White et al. 1990) and FM66/FM58 (Villa et al. 2006), then sequenced. ITS of pre- and post-inoculated isolates for AZ (OQ054806 and OQ054807) and CA (OQ564388) matched 1078/1078 bases of Globisporangium uncinulatum (syn. Pythium uncinulatum; AY598712.2) with 99.8% identity. There were two SNPs in COX II for AZ isolate 1 (OR069239); all other isolates pre- and post- inoculation for AZ (OR069240 and OR069242) and CA (OR069241) uniformly matched 533/535 bases of G. uncinulatum (KJ595385.1) with 99.4% identity. Based on these molecular and morphological data, the isolates were identified as G. uncinulatum (Blok and Van Der Plaats-Niterink 1978; Van Der Plaats-Niterink 1981). To our knowledge, this is the first report of G. uncinulatum on lettuce in AZ. Designated as Pythium wilt, this disease is reported on lettuce in The Netherlands (Blok and Van Der Plaats-Niterink 1978), Japan (Matsuura, et al. 2010), and CA (Davis, et al. 1995). Arizona is an important lettuce growing region; if this disease becomes more prevalent, lettuce production in this region could be negatively impacted.
ABSTRACT
Commercial production of raspberry (Rubus ideaus) transplants is almost exclusively accomplished through clonal propagation. One system involves forcing young shoots to grow from roots. The shoots are cut and rooted in propagation trays and referred to as tray plants. Sanitation is important during tray plant production as this method carries some risk due to contamination by substrate pathogens. In May 2021, a new disease was observed on raspberry tray plant cuttings at one nursery location in California, and observed again in 2022 and 2023 but at a much lesser extent. Multiple cultivars were affected; however, up to 70% mortality was observed on cv. RH740.1. In less affected cultivars, mortality ranged from 5-20%. Symptoms included chlorotic leaves, lack of rooting, and blackening at the basal end of shoots, followed by death of the cutting. Affected propagation trays had inconsistent foliage and patchy growth. Chains of chlamydospores (two to eight spores in each chain) similar in morphology to Thielaviopsis species (Shew and Meyer 1992) were observed at the cut end of symptomatic tray plants using a microscope. Isolates were retrieved by incubating tissue on surface-disinfested (1% NaOCl) carrot discs in a humid chamber for 5 days until greyish black mycelium was observed (Yarwood 1946). Mycelium was transferred to acidified potato dextrose agar and formed a gray to black compact mycelial colony with both endoconidia and chlamydospores. Endoconidia were catenulate, single-celled with slightly rounded ends, colorless, and 10-20 µm x 3-5 µm in size; dark-colored chlamydospores were 10-15 µm x 5-8 µm in size. The ITS region of isolates 21-006 and 22-024 was amplified with ITS5 and ITS4 primers using a 48°C annealing temperature (White et al. 1990), Sanger sequenced (GenBank accession OQ359100) and yielded 100% match to Berkeleyomyces basicola accession MH855452. Pathogenicity was confirmed by dipping 80 grams of roots of cv. RH740.1 into a suspension of 106 conidia/mL of isolate 21-006 for 15 min. For the non-inoculated control, 80 grams of roots were dipped in water. Roots were then planted into trays of coir (Berger, Watsonville, CA). Six weeks after inoculation, twenty-four shoots were harvested from each treatment, stuck into propagation trays filled with coir and maintained in a humid chamber for 14-days to induce rooting. Tray plants were then harvested and assessed for root development, black basal shoot tips, and presence of chlamydospores. Forty-two percent of cuttings from the inoculated treatment had rotten basal tips and failed to root, in comparison to 8% of the cuttings from the non-inoculated control. Chlamydospores were visualized only on shoots that emerged from inoculated roots and B. basicola was isolated only from cuttings originating from inoculated roots. Post-inoculation isolates were confirmed as B. basicola using methods described above. To our knowledge, this is the first report of B. basicola infecting raspberry. Confirmation of this pathogen on tray plants is significant because of the potential impact this disease may have in commercial nursery production worldwide. In 2021, the value of the harvested raspberry crop in the U.S. totaled $531 M, of which California represented $421 M (USDA 2022). The value of the 2021 crop was highest in the U.S. ($531 M), followed by Russia ($512 M), Spain ($405 M) and Mexico ($332 M) (FAO 2021).
