Materials and Methods

Screening of onion genotypes.

Isolate Rh070927 of R. solani AG 8 was used for this study. The isolate was obtained originally from roots of a symptomatic (stunted) onion plant in the Columbia Basin of Oregon, and demonstrated to be pathogenic on onion (Patzek et al., 2013). Inoculum of R. solani AG 8 was increased, prepared, and quantified as described by Paulitz and Schroeder (2005). In summary, 500 cm³ of whole oat (Avena sativa) kernels were mixed with 500 ml distilled water in 2-L glass flasks, and sealed with aluminum foil. Each flask was autoclaved twice for 90 min, about 24 h apart. The sterilized oats in each flask were then inoculated with 5-mm-diameter colonized agar plugs taken from a 1-week-old potato dextrose agar culture of R. solani AG 8. Flasks were incubated in the dark at 25 °C for 6 weeks. The contents in each flask were shaken once a week to facilitate complete colonization of the oat kernels. The inoculum was air-dried on Kraft paper and then stored in plastic zip-lock bags at 4 °C in the dark until quantification and use. Within a few days of an experiment, oat inoculum was ground in a coffee grinder and sieved to obtain particle sizes of 250 to 1000 μm diameter. Inoculum density was quantified by dilution plating onto 2% water agar.

A sandy loam soil collected from an uncultivated portion of a grower-cooperator’s field in the Columbia Basin was steam-pasteurized for 1 h at 60 °C, and steamed again for the same duration 24 h later. The soil was then air-dried on Kraft paper, sieved to a particle size of ≤2 mm, and stored in buckets sealed with airtight lids. Four seeds of each of the onion genotypes or cultivars were planted in a plastic cone-tainer (cone-shaped, 4-cm diameter, and 21-cm long; Steuwe and Sons, Inc., Tangent, OR) filled with 150 g of pasteurized soil. Ground R. solani AG 8-colonized oat seeds (1% w/w for a total soil weight) (Paulitz and Schroeder, 2005) were mixed with the soil and then saturated with 50 ml of water just before planting.

Thirty-five onion genotypes or cultivars (9 from Bejo Seeds, Inc., Oceano, CA; 17 from Bayer CropScience Vegetable Seeds, Parma, ID; and 9 from Seminis Vegetable Seeds, Inc., Oxnard, CA) were tested for relative resistance to R. solani AG 8 in a growth chamber set at 15 ± 1 °C (Table 1). The four seeds planted in each of five replicate cone-tainers were covered with a thin layer (8 g) of pasteurized soil. Five replicate cone-tainers without R. solani AG 8 served as a control treatment for each onion genotype, to provide a 35 × 2 factorial (inoculation × onion genotype, respectively) treatment design. Cone-tainers were arranged in a randomized complete block design in plastic trays. Each plastic tray was covered with Kraft paper for seven days to conserve soil moisture and promote onion emergence. Plants were irrigated with 25 ml of water and 25 ml of 1/3-strength Hoagland’s solution (macroelements only) every 3 to 4 d until harvest. Onion seedlings in each cone-tainer were thinned to two seedlings 3 weeks after planting. Plant height (cm), root length (cm), and total dry biomass (g) were measured for the two seedlings in each inoculated and noninoculated cone-tainer for each genotype 45 d after planting. The experiment was repeated.

Table 1.

Percent reduction in plant height, root length, and total dry biomass of plants of each of 35 onion genotypes inoculated with the onion stunting pathogen, Rhizoctonia solani anastomosis group 8, compared with noninoculated control plants of each genotype in each of two trials in a growth chamber set at 15 ± 1 °C.

View Table

Data analysis.

Pearson’s correlation coefficients for plant height, root length, and total plant biomass were calculated using JMP Version 11 Pro (SAS Institute Inc., Cary, NC). The percentage reduction in plant height, root length, and total biomass of inoculated plants of each onion genotype were calculated as a measurement of disease severity based on plant height, root length, and total biomass of the noninoculated plants of each genotype. Analysis of variance (ANOVA) was used to determine the genotype effect on reduction in plant height, root length, and total biomass using JMP. Data were rank transformed (Conover and Iman, 1981) since the assumptions for parametric analyses were not met even after transforming the data using the Box–Cox transformation approach in JMP. Treatment means were compared by using Fisher’s protected least significant difference (lsd). Mean plant height, root length, and total biomass of inoculated vs. control plants of each genotype were compared using Student’s t test.

Results and Discussion

The correlation coefficients for overall reduction in onion plant height, root length, and total biomass of plants inoculated with R. solani AG 8 compared with noninoculated plants for all 35 onion genotypes revealed a highly significant correlation of plant height with total plant biomass (r = 0.79 and 0.78 at P < 0.001 in Expt. 1 and 2, respectively) and root length (r = 0.75 and 0.70 at P < 0.001 in Expt. 1 and 2, respectively), as well as between root length and total biomass (r = 0.69 and 0.61 at P < 0.001 in Expt. 1 and 2, respectively). Disease severity caused by R. solani AG 8 was expressed as the degree of stunting (Anstis et al., 2012; Patzek et al., 2013), i.e., the reduction in height of onion plants inoculated with R. solani AG 8 vs. that of noninoculated plants of the same genotype.

The average reduction in plant height across the 35 onion genotypes was 40% ± 21% (mean ± sd) in Expt. 1, and 38% ± 15% in Expt. 2. Although the degree of reduction in plant height ranged from 24% ± 30% (for the onion cv. Lasalle) to 62% ± 17% (R14882) in Expt. 1, and 22% ± 40% (PX07713218) to 53% ± 15% (Montblanc) in Expt. 2, the reduction in plant height caused by R. solani AG 8 did not differ significantly among genotypes in either experiment (Table 1). Comparing inoculated vs. noninoculated plants within genotypes using Student’s t test revealed that the height of plants for 14 onion genotypes inoculated with R. solani AG 8 in Expt. 1 (Lasalle, PX07713218, R14881, R14883, R14885, R14886, R14887, R14888, SN296, SN307, SN368, SN435, SN483, and SN559), and 7 genotypes in Expt. 2 (PX07713218, R14882, R14888, R14889, SN232, SN272, and SN307) were not reduced by the pathogen, i.e., these genotypes demonstrated potential partial resistance to the pathogen. Of these entries, the height of three (R14888, PX07713218, and SN307) were unaffected by R. solani AG 8 in both experiments (Table 1). Nine genotypes in Expt. 1 (Lasalle, Montblanc, R14885, SN325, SN368, SN483, SN435, SN559, and SN709), and 11 genotypes in Expt. 2 (Barbaro, Divino, Elbrus, PX07713218, R14882, R14886, R14888, R14889, SN131, SN232, and SN483) were stunted ≤33% compared with noninoculated plants, which equated to a stunting severity rating of 1 in the onion yield loss study of Sharma-Poudyal et al. (2015). Twenty-six genotypes in Expt. 1 and 24 genotypes in Expt. 2 displayed foliar stunting of 34% to 62% and 34% to 53%, respectively, which equated to a stunting severity rating of 2 as described by Sharma-Poudyal et al. (2015).

The lack of significant differences between inoculated vs. control plants for some genotypes in only one of the two experiments may reflect the limited number of plants evaluated, the large variance in dependent variables measured, and/or inherent variability within some of the genotypes evaluated with R. solani AG 8. For example, the reduction in plant height for inoculated plants of R14882 was 62% ± 17% in Expt. 1, which was highly significant, but was only 26% ± 22% in Expt. 2, which was not significant. However, what is not accounted for in this study is that differences in plant height between inoculated and noninoculated plants of the same genotype likely would have increased if the onions were grown for a longer duration, since severity of stunting in onion bulb crops in the Columbia Basin tends to be expressed most distinctly 2 to 2.5 months after planting, at about the five-true leaf stage of growth in spring-sown onions (Sharma-Poudyal et al., 2015). A stunting severity of 26% equated to a severity rating of 1 in the yield loss study by Sharma-Poudyal et al. (2015), which resulted in a significant reduction in marketable bulb weight of 10% to 47% in onion bulb crops in the Columbia Basin, depending on the cultivar and field conditions.

Growing onion seedlings in soil inoculated with R. solani AG 8 resulted in a significant reduction in root length for some genotypes. The average reduction in root length across genotypes was 40% ± 39% in Expt. 1, ranging from 9% ± 49% for R14889 to 76% ± 10% for SN232 and 76% ± 8% for Sterling. In Expt. 2, the average reduction in root length was 52% ± 34%, with a range of 14% ± 34% for SN325 to 74% ± 35% for Sterling. The extent of reduction in onion root length caused by R. solani AG 8 did not differ significantly among genotypes in Expt. 1, but was significant in Expt. 2 (Table 1). Mean root length of inoculated vs. noninoculated control plants was not affected significantly by the pathogen for 26 of the onion genotypes in Expt. 1. SN325 had the least reduction in root length, 14% ± 34% (which differed significantly from that of 17 other genotypes), and SN232 had the greatest reduction in root length, 57% ± 19%. In Expt. 2, root length of 18 onion genotypes was not reduced significantly by R. solani AG 8. The root length of 16 of the 35 genotypes (Barbaro, Divino, Elbrus, PX07713218, R14882, R14885, R14886, R14889, SN131, SN272, SN296, SN307, SN325, SN368, SN483, and SN709) was not reduced by R. solani AG 8 in either experiment (Table 1), indicating potential partial resistance of these genotypes to root pruning or spear-tipping by this pathogen.

The reduction in total onion plant biomass caused by R. solani AG 8 in Expt. 1 ranged from 18% ± 49% for Elbrus to 69% ± 15% for Sterling, with an average of 45% ± 24% among the 35 genotypes (Table 1). However, the reduction in onion biomass caused by R. solani AG 8 did not differ significantly among genotypes in Expt. 1. In Expt. 2, reduction in total plant biomass ranged from 29% ± 26% for SN232 to 79% ± 8% for Sterling, and averaged 53% ± 19% across all 35 genotypes, with significant differences among genotypes. In Expt. 1, total plant biomass of 19 onion genotypes was not affected by the pathogen, i.e., did not differ between inoculated and noninoculated plants, whereas in Expt. 2 this was true for only 7 genotypes (Table 1). In Expt. 2, the minimum biomass reduction was 31% ± 8% for R14886, which did not differ significantly from that of 19 other onion genotypes. Plant biomass of each of PX07713218, R14885, and R14888 was not reduced by inoculation with R. solani AG 8 in either experiment (Table 1).

Some onion genotypes (Lasalle, PX07713218, R14883, R14885, R14886, R14887, SN307, SN368, SN435, SN483, and SN559 in Expt. 1; and PX07713218, R14882, R14888, R14889, and SN272 in Expt. 2) showed no significant reduction in plant height, root length, and total biomass when inoculated with R. solani AG 8. The lack of consistency in reactions of some genotypes across the two experiments demonstrated the difficulty of screening onion plants for resistance to this pathogen with a limited number of plants. Screening onion cultivars using a much greater number of plants, and perhaps a range in inoculum levels, could facilitate more robust detection of differences in resistance among genotypes. However, resistance to Rhizoctonia spp. in other plant species generally is rare, and diseases caused by Rhizoctonia spp. usually are managed most effectively with disease management strategies other than planting resistant cultivars, e.g., appropriate crop rotations, fungicide seed treatments (Panella and Ruppel, 1996), fungicide soil treatments (Sharma-Poudyal et al., 2013c), cultural practices (Sharma-Poudyal et al., 2013a), etc.

Of the 35 genotypes evaluated, 3, 16, and 3 demonstrated partial resistance to R. solani AG 8 in both trials for plant height, root length, and total biomass, respectively. Only four genotypes displayed partial resistance to R. solani AG 8 for at least two of the three growth parameters: plant height, root length, and biomass of PX07713218 were not affected by the pathogen in either trial; and R14885, R14888 and SN307 displayed partial resistance in both trials for two of the three growth parameters, and in one trial for the third parameter. These four genotypes could be used in onion breeding programs to develop cultivars partially resistant to stunting caused by R. solani AG 8. The fact that a majority of the 35 onion genotypes screened in this study were susceptible to R. solani AG 8 corroborates empirical observations of onion growers in the Columbia Basin that all storage type cultivars grown under conditions conducive to onion stunting in this region can develop patches of stunted plants, as well as the yield loss study by Sharma-Poudyal et al. (2015) which documented significant yield reductions caused by onion stunting for each of six cultivars commonly grown in the Columbia Basin. It may be necessary to screen more diverse Allium germplasm, e.g., wild Allium relatives, to identify resistance to onion stunting that can be incorporated into cultivars suitable for the Columbia Basin and other regions of the world where onion stunting occurs, such as Australia (Anstis et al., 2012; Wicks et al., 2011).