Evaluation of Solanaceous Species as Nonhost Trap Crops for Globodera pallida

Globodera pallida was originally obtained from infested fields in Shelley, Idaho. The identity was confirmed by morphological and molecular methods (Skantar et al., 2007). The nematode was reared on the susceptible potato ‘Desiree’ or ‘Russet Burbank’ under greenhouse conditions at 18°C and 16-hour light:8-hour dark photoperiod for 12 weeks. Dried cysts were recovered from soil by elutriator extraction, dried, and stored at 4°C until experimental use (USDA-APHIS, 2009).

Seeds of the solanaceous species under investigation were acquired from the U.S. National Plant Germplasm System (NPGS) via GRIN-GLOBAL. The following seeds were received from NPGS in Fall 2019: Solanum aethiopicum (accession Grif 14165), Solanum macrocarpon (accession PI 441914), Solanum quitoense (accession PI 489701), Solanum retroflexum (accession PI 634755), and Solanum douglasii (accession W6 56881). Seeds were planted into potting mix and grown for four weeks following emergence. Susceptible potato positive controls included ‘Desiree’ and ‘Russet Burbank’, which were grown for four weeks as tissue cultured plantlets in standard media (Murashige and Skoog, 1962).

Experiments were initiated when four-week-old tissue culture potato plantlets or seedlings from potting soil were transplanted into terracotta clay pots containing soil (Dandurand and Knudsen, 2016). Soil consisted of a 2:1 ratio of Lane Mountain 20/30 industrial silica sand to Prosser-series silt loam soil (WSU-IAREC, Prosser, WA) that had been dried and sieved through a 5 mm mesh (Dandurand and Knudsen, 2016). Soil was autoclaved twice for 90 min at 121°C prior to use. Greenhouse conditions were maintained between 16°C and 20°C with 60% relative humidity and a 16-hour light:8-hour dark photoperiod. Pots were watered daily to maintain soil moisture. Osmocote slow-release fertilizer (The Scotts Company, Marysville, OH) was applied at planting. Jack's Classic All Purpose 20-20-20 water soluble fertilizer (JR Peters Inc., Allentown, PA) was applied weekly. Bioworks SuffOil-X horticultural oil (Bioworks, Victor, NY) was applied weekly to prevent thrips infestation.

The host assay was performed in 10-cm-diameter terra cotta pots containing approximately 500 g soil. Pots were inoculated with the Idaho population of Globodera pallida that had been reared on susceptible potato under greenhouse conditions, and had completed diapause. Cysts were sealed within sterile 6 cm² nylon mesh (McMaster Carr, Elmhurst, IL) bags with 250 μm opening, with edges sealed by a hand sealer (ULINE Tabletop Impulse Sealer). Prior to inoculation, cysts were surface sterilized in 0.3% hypochlorous bleach for 5 min followed by five thorough rinses with sterile deionized water (Nour et al., 2003). The initial infestation rate was approximately 5 eggs/g soil, or 12 cysts per bag with an average of 200 eggs/cyst. Cyst bags were placed in the root zone of transplanted seedlings. Susceptible potatoes ‘Russet Burbank’ and ‘Desiree’ were included as positive controls. Bare soil unplanted pots were included as negative controls. Six replicate pots were grown in randomized complete block design for 12 weeks. The experiment was then terminated. Plants were cut off at the soil surface. Soil and root samples were dried for two weeks and then collected for cyst extraction. The host assay trial was repeated.

Cysts were extracted using the elutriator system (USDA-APHIS, 2009). Following extraction, cysts were floated in acetone to further remove debris (Brodie et al., 1976). Samples were then examined with a dissecting scope (Leica M80, Leica Microsystems, Wetzlar, Germany) where any progeny cysts were counted. A species that had zero progeny cysts in all replicates was determined to be a nonhost of G. pallida. Cyst bags were recovered from the host assay and three cysts per replicate were crushed in 100 μl sterile deionized water in a well of a 96-well plate (VWR® tissue culture plates, Radnor, PA) to determine average remaining encysted eggs. Eggs were counted using an inverted microscope (Leica DMi1, Leica Microsystems, Wetzlar, Germany). Remaining encysted egg viability was determined by the acridine orange staining method (Pillai and Dandurand, 2019). Non-viable eggs were counted with an inverted fluorescent microscope (Leica DMi8, Leica Microsystems, Wetzlar, Germany). Remaining viable eggs were calculated as total eggs – stained nonviable eggs. Percentage viability was calculated as (total eggs – stained nonviable eggs) / total eggs × 100.

Hatching assays were conducted using root exudates. Plants for root exudate collection were grown in 15-cm-diameter terra cotta pots containing 1,200 g soil. Susceptible potatoes ‘Desiree’ and ‘Russet Burbank’ were included as positive controls. An unplanted bare soil pot was included as a negative control. Four replicate pots were grown in randomized complete block design. Root exudates were collected at four weeks and at six weeks, following transplant into soil. Pots were not watered 24 hours prior to root exudate collection. Root exudates were collected by soil leaching, modified from Widdowson and Wiltshire (1958). Soil was first saturated with deionized water one hour prior to diffusate collection. Pots were then balanced on plastic cups placed beneath their drainage holes. Deionized water was slowly added to each pot until approximately 30 ml of diffusate was collected in the cup. The diffusate samples were then vacuum filtered, first through a 0.45-μm-pore filter (Corning disposable vacuum filter, Corning, NY) to remove soil particles and debris and then through a 0.22-μm-pore filter to remove microbes (Corning disposable vacuum filter, Corning, NY). The resulting root exudates were then frozen at −20°C until use.

In vitro hatching assays were performed within two months of collecting the exudates. The assays utilized cysts of the Idaho G. pallida population as described above. Cysts were surface sterilized in 0.3% hypochlorous bleach for five min before being thoroughly rinsed five times in sterile deionized water (Nour et al., 2003). Cysts were then hydrated for 48 hours in a 1:1 solution of sterile deionized water and 0.5% gentamicin. Cysts were then crushed to release the eggs. Approximately 100 eggs in 100 μl of 0.5% gentamicin solution were pipetted into wells of a 96-well plate. Initial eggs containing juveniles and initial released juveniles were counted using an inverted microscope (Leica DMi1, Leica Microsystems, Wetzlar, Germany). Root exudate, 100 μl, was applied to each well. There were three wells (technical replicates) per replicate of diffusate. Plates were incubated at 18°C for two weeks. Juveniles were then counted. The percentage hatch was calculated using the following equation: (Final J2s-Initial J2s) / Initial Eggs × 100. The root exudate collection and hatching assay experiment was repeated.

Statistical analysis was performed using the SAS statistical package (SAS Institute Inc., Cary, NC). Hatch, viability, and egg densities were analyzed with a generalized mixed linear model (PROC GLIMMIX) and means separation by least squares means. To meet the assumptions of a normal distribution, the host assay progeny cyst data was transformed as log₁₀ (x +1) in order to be analyzed using PROC GLM and means separation by Tukey's HSD.

Reproduction of G. pallida was high in the susceptible potato varieties ‘Desiree’ and ‘Russet Burbank’ but no reproduction was observed in any of the solanaceous species tested (P < 0.0001) (Table 1). Trial two susceptible potato varieties also had significant cyst reproduction (Table 2). Like the bare soil negative control, all solanaceous species investigated, other than potato, had zero cysts in all replicates in both trials and are thus considered nonhosts for G. pallida.

Remaining encysted eggs recovered from cysts used in inoculation in the host assay revealed that certain solanaceous species caused significantly more hatch than others (P < 0.0001) (Table 1). The bare soil control (158.4 ± 9.9 remaining eggs per cyst) was statistically the same as S. aethiopicum (160.4 ± 9.6 remaining eggs per cyst), S. douglasii (135.8 ± 8.1 remaining eggs per cyst), and S. macrocarpon (147.8 ± 8.2 remaining eggs per cyst). The susceptible potato varieties ‘Desiree’ (77.9 ± 4.7 remaining eggs per cyst) and ‘Russet Burbank’ (70.9 ± 12.7 remaining eggs per cyst) had about 50% less remaining encysted eggs than the bare soil, and were statistically the same as S. quitoense (96.9 ± 7.9 remaining eggs per cyst) and S. retroflexum (91.2 ± 9.3 remaining eggs per cyst). This indicates that S. quitoense and S. retroflexum induced hatch and thus may be useful as trap crops while S. aethiopicum, S. douglasii, and S. macrocarpon did not induce hatch and are not suitable trap crops. Data from trials one and two could not be combined due to interactions between trial and treatment. The trial two host assay had the same trends except that in this trial, the numbers of remaining encysted eggs with S. quitoense were significantly greater than the susceptible potato controls by 26% (Table 2).

In trial one, the percentage viability of the remaining eggs per cyst significantly differed in some treatments (P = 0.0014) (Table 1). Percentage viability of remaining eggs for the bare soil treatment (73.7% ± 1.7) was not statistically different for S. aethiopicum (65.2% ± 2.2), S. douglasii (62.4% ± 5.7), S. retroflexum (61.5% ± 2.9), and susceptible potato controls ‘Desiree’ (66.1% ± 5.0) and ‘Russet Burbank’ (56.3% ± 5.7). The percentage viability of remaining eggs for S. quitoense (48.0% ± 2.3) was significantly less than that of the bare soil control and susceptible potato ‘Desiree’ by 22%. The percentage viability of remaining eggs for S. macrocarpon (52.6% ± 5.3) was significantly less than that of the bare soil control. Compared to the controls, S. macrocarpon and S. quitoense are the only species which appear to affect egg viability. Data from trials one and two could not be combined due to interactions between trial and treatment. In trial two, viability of remaining encysted eggs was not significantly different among treatments (Table 2).

The mean remaining viable eggs per cyst was also significantly different in some treatments (P < 0.0001) (Table 1). The greatest remaining viable eggs were in the bare soil control (89.1 ± 5.0 viable eggs per cyst) and S. aethiopicum (81.9 ± 6.0 viable eggs per cyst). The greatest reduction in viable eggs was in the susceptible potato controls ‘Russet Burbank’ (33.8 ± 3.7 viable eggs per cyst) and ‘Desiree’ (43.9 ± 5.3 viable eggs per cyst), S. macrocarpon (46.7 ±10.1 viable eggs per cyst) and S. quitoense (32.8 ± 4.2 viable eggs per cyst). Compared to bare soil, S. macrocarpon and S. quitoense reduced viable eggs by 48% and 63%, respectively. Solanum retroflexum and S. douglasii were statistically the same as that of S. macrocarpon and ‘Desiree’. The host assay in trial two was similar to trial one except that viable eggs were less in the bare soil control than with S. aethiopicum (Table 2). Also, S. quitoense did not reduce viable eggs as much as the potato (Table 2).

The in vitro hatching assay using root exudates collected at four weeks revealed significant differences in hatch among the treatments (P = 0.0021) (Fig. 1A). Solanum quitoense caused 20% egg hatch and S. douglasii caused 18% egg hatch and were statistically the same as the susceptible potato controls ‘Desiree’ with 26% hatch and ‘Russet Burbank’ with 23% hatch. The bare soil control diffusate had approximately 1% egg hatch. Solanum aethiopicum with 4% hatch, S. macrocarpon with 4% hatch, S. retroflexum with 11% hatch was not significantly different than the bare soil. The S. retroflexum hatch was also not significantly different than the S. quitoense and S. douglasii hatches.

Figure 1:

The hatching assay using root exudates collected at six weeks also showed significant differences among treatments (P = 0.0013) (Fig. 1B). The susceptible potato control ‘Desiree’ caused 33% hatch while ‘Russet Burbank’ caused 18% hatch. Solanum quitoense with 17% hatch, S. retroflexum with 16% hatch, and S. douglasii with 11% hatch caused hatch statistically the same as that of ‘Russet Burbank’. The bare soil control hatch rate was 1%. Solanum aethiopicum with 4% hatch, S. macrocarpon with 2% hatch, and S. douglasii with 11% hatch were not significantly different than the bare soil control. Solanum douglasii hatch was also not significantly different from ‘Russet Burbank’.

Trials one and two of the hatching assays could not be combined due to interactions between treatment and trial. The trial two hatching assay showed similar trends to trial one for the root exudates collected at 4 weeks, in that S. douglasii (29% hatch), S. quitoense (53% hatch), and S. retroflexum (36% hatch) caused hatch like that of the susceptible potato controls ‘Desiree’ (57% hatch) and ‘Russet Burbank’ (39% hatch) (Fig. 2). However, in trial 2 for the root exudates collected at 6 weeks, S. douglasii (8% hatch) was not significantly different from the bare soil (2% hatch) (Fig. 2).

Figure 2: