Corky ringspot disease (CRS), also commonly referred to as “spraing” in Europe, is an important potato necrotic tuber syndrome caused by
The spread of CRS is a widespread concern within the global potato industry. Both TRV and SRN co-occur throughout much of Europe (Hooker, 1981) and have been observed in more than ten states within the USA since first being observed in Florida in 1946 (Allen, 1963; David et al., 2010; Eddins et al., 1946; Gudmestad et al., 2008; Kirk et al., 2008; Thomas, 1976; Weingartner and Shumaker, 1990). Even small numbers of SRN (3 nematodes/250 cm3 of soil) can cause substantial CRS damage to a potato crop if left untreated (Mojtahedi et al., 2001). Mitigation of CRS in production settings is currently achieved by treating potato fields with expensive, non-selective pesticides that reduce the abundance of the SRN vector and planting virus insensitive cultivars (Yellareddygari et al., 2018) derived from seed growers’ fields that have been certified as being SRN and TRV free (Hafez and Sundararaj, 2009). Heavy usage of systemic pesticides is undesirable due to grower expense and negative impact on the environment; thus, development of potato cultivars that are resistant to TRV infection is an ideal solution. Breeding programs focused on this goal have developed and released several potato cultivars with TRV resistance (Brown et al., 2009, 2000; Dale, 1989; Dale and Solomon, 1988; Robinson and Dale, 1994; Shumaker et al., 1984).
Unfortunately, once a field has been affected by CRS, remediation is extremely difficult. Although bulk application of fumigants (1,3-dicholopropene, aka Telone II) and systemic nematicides (oxamyl, aka Vydate C-LV) can effectively reduce the population of the SRN vector in the soil, complete eradication is nearly impossible. SRN are mobile organisms that can survive deep in the soil profile re-emerging after pesticide application has subsided (Weingartner, 1983). Further complicating matters is the fact that TRV has one of the broadest host ranges of any virus (Boydston et al., 2004; Cooper and Harrison, 1973; Mojtahedi et al., 2003) and can lay dormant in weeds for years only to reappear when potato is cropped again.
Strategies to remediate land affected by CRS generally involve moving infected fields out of potato production and cropping varieties of alfalfa var. “Vernema” or Scotch spearmint var. “770” (Banttari et al., 1993; Mojtahedi et al., 2002) as rotation crops. These plant species are an adequate food source for SRN but cannot serve as hosts for TRV (Mojtahedi et al., 2002). Similar to other parasitic nematodes, SRN molts several times throughout its lifecycle, effectively shedding the virus with each molt (Robinson and Harrison, 1989). Greenhouse and field experiments suggest that if immature nematodes are not exposed to a virus-infected food source, the nematode population will no longer harbor the virus after several generations (Mojtahedi et al., 2002) and CRS symptoms will diminish (Banttari et al., 1993). Unfortunately, due to the specialized equipment needs and market factors, crop rotations that regularly include alfalfa and spearmint are not adequately profitable for potato growers.
Using greenhouse pot experiments, we have examined the potential of the TRV resistant potato cultivar, Castle Russet, to cleanse soils containing viruliferous SRNs of TRV infection relative to a known host (tobacco), a non-host (alfalfa), and an unrelated popular potato variety (Russet Burbank). The objectives of this study were to assess how host plant genotype, cultivation time, and nematode inoculation pressure influenced nematode fecundity and the presence of TRV through multiple generations of SRN.
Two independent experiments were carried out in a greenhouse at the USDA-ARS Temperate Tree Fruit and Vegetable Research Unit in Prosser, WA. The average temperature during the trials was 25°C with a 12-hour photoperiod. The plants were grown in 10 L plastic pots filled with a reconstituted sandy silt loam soil composed of 84% sand, 10% silt and 6% clay (Brown et al., 2006). The soil was steam pasteurized before use.
For both experiments, tobacco var. “Samsun NN” seed was bulk sown into a 10 cm2 plastic pot filled with Sunshine #1 potting mix (Sun-Gro Horticulture Distributor Inc., Bellevue, WA) and plants were allowed to develop in a growth chamber (25°C; 12 h photoperiod). Alfalfa var. “Vernema” was direct seeded into Sunshine potting mix #1 in a 72-cell tray. In Experiment 1, plants of Russet Burbank and Castle Russet (aka POR06V12-3) were propagated first in tissue culture. The tissue culture media was prepared by dissolving in 1 L of double distilled water 4.41 g of MS Modified BC Potato Basal Medium, 8 g of Plant TC agar (Product Numbers M516 & A111, PhytoTechnology Laboratories Shawnee Mission, KS), and 25 g of sucrose. The mixture was autoclaved at 120°C for 20 min. Tissue culture plantlets were kept for 1.5 months in the tissue culture media to develop a massive root system, and were then transferred to a 72-cell tray in Sunshine potting mix # 1. The foliage was pruned to induce vigorous growth. In Experiment 2, disease-free nuclear tubers were produced from tissue cultured plants and stored at 5.5°C until the dormancy of the tubers was broken.
A viruliferous population of
An overview of the experimental procedure can be observed in Figure 1. Experiment 1 was performed in April 2016. For this experiment, 10 tobacco, 30 alfalfa, 18 Russet Burbank and 28 Castle Russet were removed from the potting mix, roots washed, and then transplanted into pots containing the sandy silt loam soil. Two weeks after transplanting, 60 viruliferous
One month post inoculation a number of replicates were destructively harvested and processed for evaluation of TRV infection. The number of replicates harvested was as follows: 10 tobacco, 10 alfalfa, 6 Russet Burbank and 6 Castle Russet for Experiment 1; 5 tobacco, 5 alfalfa, 5 Russet Burbank and 5 Castle Russet for Experiment 2. After plants were removed from the pots, a 250 cm³ sample of soil was collected and nematodes were isolated from the soil by sieving and sugar-centrifugal flotation (Jenkins, 1964). Nematodes were counted and returned to their original pots. Oostenbrink’s nematode reproduction factor (Rf=Final nematode count/Initial nematode population count) was calculated from these count numbers (Oostenbrink, 1966). Next, a bait plant (tobacco) was transplanted into the soil that remained in each of the harvested pots to ascertain if the nematodes remained viruliferous.
The harvested plants were washed and split into foliar, root and stolon/tuber (if present) sub-samples. For each sub-sample, approximately 0.5 to 1 g of tissue was grounded in grinding bags (Agdia, Inc., Elkhart IN). A total nucleic acid extraction was performed using a modified Dellaporta procedure (Crosslin and Hamlin, 2011). Nucleic acid pellets were resuspended in 400 μl of UltraPure distilled water (Invitrogen, Carlsbad, CA). The standardized RT-PCR reactions were conducted in 25 μl reactions that contained 12.5 μl of PCR mix (2× Reaction Mix from SuperScript® III One-Step RT-PCR System with Platinum®
Two months post inoculation, the above procedure was repeated for the following number of replicates: 10 alfalfa, 6 Russet Burbank and 6 Castle Russet for Experiment 1; 5 alfalfa, 5 Russet Burbank and 5 Castle Russet for Experiment 2. Here, plants were destructively harvested, the nematode Rf was calculated from soil samples, and new tobacco bait plants were added. In addition, the aforementioned bait plants were destructively harvested and evaluated for TRV infection by RT-PCR. Three months post inoculation, the above procedure was performed on the remaining replicates (10 alfalfa, 6 Russet Burbank and 16 Castle Russet) for Experiment 1; and (5 alfalfa, 5 Russet Burbank, and 5 Castle Russet) for Experiment 2. Again, plants were destructively harvested, the Rf was calculated from soil samples, and new tobacco bait plants were added. Additionally, the bait plants from the two-month evaluation were also tested. Four months post inoculation the bait plants from the three-month evaluation were destructively harvested and tested for TRV infection.
All statistical analyses were performed within the R statistical computing environment (R Core Team, 2018). Generalized linear models were constructed to evaluate the influence of plant host (plant), exposure period (months), and inoculation pressure (experiment) on nematode count, Rf and TRV detection using RT-PCR. Nematode count was modeled as a negative binomial variable using the glm.nb function in the MASS library (Venables et al., 2002). Rf value was log transformed and modeled using the lm function in the base R package (R Core Team, 2018). To eliminate the confounding effects of zero values in log transformation an offset (half the minimum value found in the dataset) was added to all values. RT-PCR detection of TRV in bait plants was modeled as a binomial variable using the MASS library (Venables et al., 2002). The relationship between these variables was modeled using the following equation:
In which
For each characteristic (nematode count, Rf value, proportion of foliar and root TRV infection) groupings of genotype at the final sampling time point within each experiment were determined using Tukey’s honest significant distance (
Raw data and analysis scripts used to generate tables and figures can be downloaded at the following Zenodo repository:
The capacity of tobacco var. Samsun NN, alfalfa var. “Vernema”, Russet Burbank potato and Castle Russet potato to sustain populations of viruliferous SRN was investigated in two independent greenhouse trials that differed by SRN inoculation pressure (Fig. 1). Overall, plant host (
Statistics from both likelihood ratio
Average nematode count, reproduction factor (Rf) values and proportion of foliage and root tissue samples with TRV infection at the final sampling time point.
Plant | Experiment | Nematode count | Rf value | Foliage TRV infection (%) | Root TRV infection (%) |
---|---|---|---|---|---|
Alfalfa | 1 | 126.40b | 84.26b | 0a,b | 0b |
Burbank | 1 | 20.00d | 13.33b | 33a,b | 50a,b |
Castle | 1 | 119.87c | 79.91b | 0b | 0b |
Tobacco | 1 | 720.80a | 480.53a | 80a | 100a |
Alfalfa | 2 | 6.00d | 0.22c | 0b | 20a |
Burbank | 2 | 64b | 2.41b | 100a | 100a |
Castle | 2 | 19.6c | 0.73b,c | 0b | 0a |
Tobacco | 2 | 768a | 28.98a | 100a | 80a |
Notes: Experiment 1 was inoculated 60 nematodes/pot, whereas Experiment 2 was inoculated using 1,060 nematodes/pot. The final measurement of Nematode count and Rf value was performed after three months. The final measurement of TRV infection in foliage and root tissues was performed after 4 months. a,b,c,dSuperscripted letter assignments (a-d) reflect groupings determined using Tukey’s honest significant difference within the experiment (
The virulence of SRNs was evaluated over a three-month period by using RT-PCR to detect TRV from total nucleic acids extracted from foliage and root tissue of tobacco bait plants. The presence of TRV was significantly influenced by plant host (