First report of Cercospora leaf spot caused by Cercospora chenopodii on Spinacia oleracea in the U.S.A.

In November 2017, leaf spots were observed in a fresh market spinach crop of cultivar SV2157VB in New Jersey at ∼20% incidence but not in the adjacent crop of Unipack-151. Pale tan lesions (3 to 8 mm in diameter) had gray sporulation at the centers. Symptomatic leaf pieces were surface-sterilized in 0.6% NaOCl for 60 s, triple-rinsed in sterilized water, dried, and plated onto PDA and water agar (each with 100 µg of chloramphenicol/ml). Seven isolates all produced hyaline, straight to slightly curved conidia, mostly two- to three-septate, 20.0 to 82.0 µm (45.2 ± 11.3 µm, mean ± standard deviation) × 6.0 to 11.0 µm (8.1 ± 1.0 µm) (n = 312), with no significant differences among isolates. Spore characteristics matched those of Cercospora spp. (Świderska-Burek 2015). The consensus sequence of the calmodulin gene (cmdA) amplified from all seven isolates using primers CALDF1 and CALDR (Lawrence et al. 2013) (GenBank accession no. MN422444) was 99.78% identical to that of Cercospora cf. chenopodii (JX142839.1), which is not differentiated from C. chenopodii by this gene (Groenewald et al. 2013). Actin (actA) primers ACT-512F and ACT-783R generated an identical sequence for the isolates (MN422442) that was 99.55% identical to that of C. chenopodii (JX143081.1) (Carbone and Kohn 1999). The translation elongation factor 1-α (TEF1-α) sequence of the isolates (MN422445) had 99.45% identity to the sequence of a voucher specimen of C. chenopodii (JX143327.1). C. beticola-specific primers CbCALF and CbCALR did not amplify DNA of these isolates, only DNA of control isolates of C. beticola (Knight et al. 2019). Based on a phylogenetic tree inferred from concatenated actA, cmdA, ITS rDNA, and TEF1-α sequences, the isolates clustered with the voucher strain C. chenopodii CBS 132620. Six isolates were each tested for pathogenicity on four 33-day-old plants of each of spinach cultivars Mandolin, SV2157VB, Unipack-151, and Viroflay; sugar beet cultivar KDH4-9 (USDA National Plant Germplasm System PI 683513, homozygous susceptible to C. beticola), red beet cultivars Red Ace and Ruby Queen, and Swiss chard cultivar Silverado. Plants were enclosed in plastic bags for 24 h, atomized with a spore suspension (1 × 10⁴ conidia/ml) prepared from clarified V8 agar plates of each isolate, and returned to the bags for 24 h. Four non-inoculated control plants of each cultivar were atomized with water as control treatments. Beet and chard plants were placed in a greenhouse and spinach plants in a growth chamber with 10 h lighting/day to avoid bolting under 16-h summer days in Washington. Symptoms had not developed after 7 days, at which time spinach plants were moved to the greenhouse. By 9 days, leaf spots resembling those on the original leaves were observed on Mandolin and SV2157VB. By 14 days, there were leaf spots on all four spinach cultivars. Symptoms were most severe on SV2157VB (18.3 ± 2.0% mean ± standard error of leaf area), followed by Mandolin and Viroflay (8.8 ± 1.6%), and then Unipack-151 (2 ± 0.5%). Symptoms did not develop on non-inoculated spinach or inoculated and non-inoculated sugar beet, table beet, and Swiss chard. Cercospora could only be reisolated from symptomatic spinach plants, with the isolates confirmed as C. chenopodii based on actA sequences. Although C. beticola has been reported to cause Cercospora leaf spot of spinach (Knight et al. 2018), this is the first report of C. chenopodii causing a leaf spot of spinach in the United States. Research is needed to evaluate impacts of this species on additional spinach cultivars and management options.