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Evaluation of an F1 (NIL3 x Viroflay) and F2 (Viroflay x Califlay) population progenies for resistance to Pfs race 5
Plant materials, planting, inoculation and rating
In the first experiment of the study, we evaluated two spinach hybrids (NIL1 x Viroflay and NIL3 x Viroflay) for incomplete resistance response to Pfs race 5. The hybrids were each developed from a cross between Viroflay (identified to be universally susceptible to all reported Pfs races), and individual open-pollinated near-isogenic lines, NIL1 and NIL3. NIL1 is resistant to Pfs race 5 and reported to have resistance allele at the RPF1 locus. Similarly, NIL3 is resistant to Pfs race 5 and reported to possess resistance allele at the RPF3 locus. The hybrids (NIL1 x Viroflay and NIL3 x Viroflay), together with checks, Califly and Lion, were considered in this evaluation (Table 1). Califlay has resistance allele at the RPF3 locus while Lion has resistance allele at both RPF1 and RPF3 loci (Feng et al. 2018a). Plants from the genotypes were established by direct seeding in 3-inch pottymix-filled pots in three replicates (pots) per cultivar. Each replicate had five plants, making a total of 15 plants per genotype in a trial. The evaluation was conducted in six independent trials. The plants were grown in the greenhouse (of the Rosen Center at the University of Arkansas, Fayetteville, Arkansas) following standard procedure for two weeks and inoculated following the routine inoculation method (Feng et al. 2014) (Fig. 1).
Table 1
Disease response of a set of spinach genotypes to downy mildew Pfs race 5 under greenhouse conditions in six independent tests
| | | Cotyledon infectiona | | |
Genotype | RPF1 Alleles | RPF3 Alleles | Expected | Observed | Disease incidence (%) | Disease severity (%)b |
Viroflay | rr | rr | + | + | 100.0 | 87.2 |
Califlay | rr | RR | - | - | 0.0 | 0.0 |
F1 (NIL3 x Viroflay)c | rr | Rr | ? | - | 0.0 | 0.0 |
F1 (NIL1 x Viroflay)c | Rr | rr | - | - | 0.0 | 0.0 |
NIL3 | rr | RR | - | - | 0.0 | 0.0 |
NIL1 | RR | rr | - | - | 0.0 | 0.0 |
Lion | Rr | Rr | - | - | 0.0 | 0.0 |
aEach test consisted of 3 replicates with 15 plants per genotype. Cotyledons: ‘+’ indicates > 85% of the seedlings showed evidence of infection and sporulation after 7 days, ‘-’ indicates < 15% of the seedlings showed evidence of infection and sporulation after 7 days. ? = means reaction undetermined |
bDisease severity was calculated using the mean disease response (severity) of all replicates in all tests for each genotype based on true leaf infection. True leaves were rated on a scale of 0 to 4, 0 (no symptom and 0% disease severity), 1 (1–25% chlorosis and necrosis), 2 (26–50% chlorosis and necrosis), 3 (51–75% chlorosis and necrosis), and 4 (76–100% chlorosis and necrosis) respectively. |
cThe F1 is used for investing for incomplete resistance. |
RR = homozygous resistance; Rr = heterozygous resistance; rr = homozygous susceptible |
Briefly, Pfs sporangia were retrieved from sporulating spinach leaves and filtered through four layers of cheesecloth. Each pot of plants was inoculated with 20 ml (105 sporangia/ml) of inoculum using a Badger basic spray gun (model 250). The inoculated plants were immediately transferred to the dew chamber (100% relative humidity) for incubation at 18°C for 24 h. The plants were then moved to the growth chamber at 19°C with a 12 h light/12 h dark regime for 5 days and transferred back to the dew chamber for 24 h to induce sporulation. Qualitative evaluation was based on the presence or absence of sporulation on cotyledons and true leaves; as resistant (-) or as susceptible (+), respectively. Quantitative evaluation of true leaves was conducted as previously described (Irish et al. 2003). Briefly true leaves were evaluated on a standard scale of 0 to 4, with 0 = no sporulation; 1 = up to 25% leaf area covered with sporulation; 2 = 26 to 50% leaf area covered with sporulation; 3 = 51 to 75% leaf area covered with sporulation; and 4 = 76 to 100% leaf area covered with sporulation. The disease incidence and severity on each cultivar was calculated using the standard method as thus:
Downy mildew disease incidence (DI) on the genotypes was calculated based on the number of cotyledons infected: + indicates > 85% infected, - indicates < 15% infected, and ± indicates an intermediate reaction. Disease severity (DS) on the cultivars was calculated using the mid-point of the range of each disease category (0–4) with the formula: DS = [(A*0) + (B*12.5) +(C*37.5) + (D*62.5) + (E*87.5)] / (A + B + C + D + E), where A, B, C, D, and E represents (0–4). The downy mildew response data (severity) was used for assessing the degree of resistance and susceptibility to estimate or capture resistance level in the hybrid.
Evaluation of F2 population progenies
For the second experiment of the study, two F2 population progenies from a cross of Viroflay x Califlay consisting of 142 and 63 individuals, and parents (Viroflay and Califlay) were evaluated in the greenhouse of the Rosen Center at the University of Arkansas, Fayetteville. Arkansas. Califlay is identified to be resistant to Pfs race 5 and reported to possess resistance allele at the RPF3 locus. The F2 breeding populations were developed in USDA, California by Dr. Beiquan Mou. In this study, plants were grown following standard procedure by direct seeding in rows inside 25 cm x 50 cm plastic trays filled with the sunshine potting soil (Sun Gro Horticulture, Canada). Seeds were planted in 10 rows per tray and 15–20 seeds per row. Two trays were used for each population with each tray receiving at least 120 seeds of the F2 populations. The parents were grown by direct seeding as well in a tray. The inoculation protocol for downy mildew followed the standard procedure and plants were scored qualitatively based on the cotyledon infection assay (Feng et al. 2014; Irish et al. 2003).
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Characterization of resistance to Pfs race 19 in commercial cultivars
Plant materials, planting, inoculation and rating
In this experiment, a selected set of 39 contemporary commercial cultivars with sources of resistance to the previously described races was evaluated under greenhouse/growth chamber conditions at the Rosen Center of the University of Arkansas, Fayetteville. The commercial cultivars were part of the panel previously evaluated in the field to the prevalent Pfs races in California and Arizona (Clark et al. 2020; Dhillon et al. 2019; 2020b.). Based on the standard pathogenicity assay, it is hypothesized that the RPF3 locus confers resistance to Pfs race 5 (Feng et al. 2018b). Thus, at least 3 different cultivar/genotypes (Califlay, NIL3, and Whale) known to contain resistance allele(s) at the RPF3 locus were used as negative control, and one cultivar (Viroflay) known to be universally susceptible to all known Pfs races, was considered as positive control.
To characterize downy mildew resistance in the commercial cultivars, the experiment was carried out in two independent trials. Plants were grown in rows inside trays for each test with each tray containing positive (Viroflay) and negative (Califlay) controls. At least, two-week-old seedlings were used for inoculations in this study and plants were inoculated following standard procedure (Feng et al. 2014; Irish et al 2003). Disease severity (DS) on the cultivars was calculated using the mid-point of the range of each disease category (0–4) with the formula: DS = [(A*0) + (B*12.5) +(C*37.5) + (D*62.5) + (E*87.5)] / (A + B + C + D + E), where A, B, C, D, and E represents (0–4) as described previously.
Data analysis
The resulting downy mildew response data were subjected to descriptive statistical analysis and analysis of variance. The analysis was done in EXCEL (Microsoft) and JMP Pro 15 (SAS Institute, Cary NC). The Tukey’s test (Honestly Significant Difference) was used for the mean separation at P = 0.05