Materials and Methods

Accessions of Rubus species were obtained by the Washington State University breeding program and used in crossability studies in 1996 and 1997 (Moore and Trople, 1999). The hybrids were planted in the field and evaluated for fertility, and only the most fertile hybrids were retained in the breeding program. Species and hybrids that were still available plus additional Rubus species accessions were evaluated for host status for P. penetrans in 2010–12 (Table 1). Generally, there was only a single genotype representing a species or hybrid. Rubus idaeus ‘Meeker’ was included as a control in each trial (Table 1). During each year of the study, tissue culture plants (clones) of Rubus species and hybrids were initiated from surface-sterilized shoots at Washington State University, Puyallup Research & Extension Center, Puyallup, WA. The tissue culture plants were placed on rooting media and then rooted in a greenhouse. Approximately 3 months after rooting, the tissue culture plants were transported to USDA-ARS Horticultural Crops Research Unit, Corvallis, OR, where they were transplanted into 3.7-L pots containing a steam-pasteurized 1:2 sand:loam mix. Plants were allowed to establish in pots for ≈1 month before inoculation with P. penetrans. During this time period plants were fertilized with 20N–8.8P–16.6K (J.R. Peters, Allentown, PA) twice a week and were grown in a greenhouse under long-day conditions (16-h photoperiod) with 26/18 °C day/night temperatures.

Table 1.

Sources and lineages of Rubus species and hybrids evaluated for host status to Pratylenchus penetrans.

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The P. penetrans population used in this experiment was obtained from a raspberry field in Lynden, WA, and maintained on raspberry and mint (Mentha piperita) in the greenhouse. Inoculum was obtained by extracting P. penetrans from roots under intermittent mist for 48 h (Ingham, 1994). At inoculation, ≈1800 (2010) or 2500 (2011 and 2012) mixed-stage individuals of P. penetrans were inoculated into the root zone in 1-mL aliquots placed in five 2.5-cm deep holes made around the base of the plant. The holes were filled after inoculation. Each Rubus species or hybrid was replicated eight times in each of three trials and plants were arranged in a randomized block design. The experimental duration after nematode inoculation was ≈4 months, during which time the plants were pruned and staked to manage growth and fertilized and grown in a similar environment as described previously.

At termination, the above-ground portion of the plant was removed and discarded. The contents of the pot were emptied onto a tray where the roots were shaken free of soil and a 50-g soil sample was collected and placed on a Baermann funnel and nematodes were extracted for 5 d (Ingham, 1994). Roots were washed free of soil and P. penetrans was extracted from the root system emanating from the original root ball by intermittent mist for 1 week (Ingham, 1994). The roots were then placed in a 70 °C oven for 1 week before determining dry weight. In 2010, the entire root system, including the original root ball, was weighed, whereas in 2011 and 2012, the original root ball was removed and only roots emanating from the original root ball were weighed. Pratylenchus penetrans recovered from soil and roots was enumerated using a dissecting microscope at 40× magnification and is expressed as number of P. penetrans per gram dry root and total number (soil plus root) of P. penetrans recovered per pot. In addition, reproduction factor (RF = final total P. penetrans population density/initial P. penetrans population inoculation density) values were calculated.

Root weight and nematode data were log₁₀(x + 1) transformed before analysis to control for heteroscedasticity; non-transformed data are shown. Trials were analyzed separately. Data were then analyzed for effects of block and plant type using a mixed linear model analysis of variance (ANOVA) with block as a random factor and plant type as a fixed factor. Means were separated using Tukey’s least significant differences (P < 0.05) (JMP 9.1; SAS Institute, Cary, NC).

Results and Discussion

We evaluated a diverse set of Rubus species in an attempt to identify a source of resistance to P. penetrans, which could potentially be introgressed into a commercially acceptable raspberry cultivar. There was a clone × trial interaction in the ANOVA, and therefore trials are presented separately (Table 2); however, similar trends were observed across trials. In all trials, R. niveus and R. leucodermis consistently supported the fewest total P. penetrans and P. penetrans/gram root. The RF values for these species indicate that both are poor hosts for P. penetrans (1 > RF > 0; Ferris et al., 1993). In 2010, R. sumatranus and R. odoratus supported similarly low P. penetrans population densities as R. niveus and R. leucodermis. All of these species supported lower population densities of P. penetrans than R. idaeus ‘Meeker’, the most commonly planted cultivar in Washington (Peerbolt Crop Management, 2013). When RF values were considered, only R. innominatus and R. parviflorus would be considered good hosts (10 > RF > 1 = good host) for P. penetrans in 2010 (Table 2). In 2011, R. sumatranus had similarly low P. penetrans/gram root as R. niveus and R. leucodermis; however, total P. penetrans was similar to those found in R. idaeus ‘Meeker’. For R. odoratus, completely opposite results were observed in 2011 compared with 2010 with R. odoratus supporting the second highest total P. penetrans population densities of any Rubus species and considered a good host for P. penetrans. In 2012, the same R. leucodermis selection evaluated in 2010 and 2011 continued to be resistant to P. penetrans. In addition, another selection of R. niveus, WSU 2263, was a similarly poor host for P. penetrans as R. niveus PI 606461.

Table 2.

Host status of Rubus species and hybrids to Pratylenchus penetrans (Pp).

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In 2010 and 2011, we evaluated the same selection of R. crataegifolius, ‘Jogkal’ (Hong et al., 1971) that Vrain and Daubeny (1986) evaluated for resistance to P. penetrans. Our finding regarding the host status of R. crataegifolius ‘Jogkal’ to P. penetrans was inconsistent across trials (Table 2). Vrain and Daubeny (1986) screened two R. crataegifolius selections, one from Japan and the other ‘Jokgal’ from Korea (Hong et al., 1971), both of which supported few P. penetrans with RF values less than 0.2. These combined results indicate that R. crataegifolius may be a useful source of resistance to P. penetrans.

There were few consistent trends in root weight, which would explain the ability of a Rubus species to serve as a good or poor host. In 2010, the average dry root weight across Rubus species was 9.33 (± 0.25) g. The Rubus species with the largest root systems were R. odoratus, R. leucodermis, R. sumatranus, and R. ideaus ‘Meeker’ with dry root weights ranging from 11.17 (± 0.43) g to 14.74 (± 0.9) g. Rubus niveus had the smallest root system, 7.03 (± 0.35) g, but was only different from the aforementioned species as well as R. innominatus. In 2011, the average dry root weight across Rubus species was 8.25 (± 0.44) g. Similar to 2010, R. odoratus had one of the largest root systems, 19.99 (± 2.12) g, which was not different from R. innominatus and R. coreanus. Rubus parvifolius had the smallest roots system, 3.56 (± 0.46) g, which was not different from the dry root weights of R. niveus, R. spectabilis, and R. pungens.

In addition to the Rubus species material, we evaluated a number of Rubus hybrids in 2011 and 2012 (Tables 1 and 2). The average dry root weights across hybrids were 6.18 (± 0.35) g and 4.08 (± 0.21) g in 2011 and 2012, respectively. The R. phoenicolasius (WSU 1475) and R. cockburnianus (WSU 1479) hybrids did not differ from R. idaeus ‘Meeker’ in population densities of P. penetrans in roots and total number in either trial. The R. lasiostylus (WSU 1474) and R. sumatranus (WSU 1414 and 1415) hybrids supported fewer P. penetrans than R. idaeus ‘Meeker’ in 2011 but high populations in 2012. WSU 1673, a selection from the cross of R. niveus × R. idaeus ‘Tulameen’, supported few P. penetrans in 2011 but very high densities in 2012. These combined results indicate that more research is needed to learn about the inheritance of the putative resistance.

The variability we observed across trials in final population densities of P. penetrans compared with initial population densities that have been reported by others. In our study, RF values for P. penetrans on ‘Meeker’ were 0.9 (± 0.2), 0.6 (± 0.1), and 2.1 (± 0.2) in 2010, 2011, and 2012, respectively. When Vrain and Daubeny (1986) considered the host status of R. idaeus ‘Willamette’ to P. penetrans in greenhouse trials across years, RF values were 2.3, 0.82, and 0.86. The host status of ‘Meeker’ to P. penetrans also varied across experimental venues with RF values of 1.7 and 0.20 in greenhouse and in microplots experiments (Vrain and Daubeny, 1986) and 1.3 in another greenhouse trial (Bristow et al., 1980). This observed variability across trials could be the result of a difference in environmental conditions and methodologies used in the experiments. Pratylenchus penetrans reproduces over a wide range of temperatures with a generation (egg to egg) requiring 46, 38, 28, 26, and 22 d at 17, 20, 30, 25, and 27 °C, respectively, under controlled conditions (Mizukubo and Adachi, 1997). The duration of three different experiments evaluating host status of Rubus species materials to P. penetrans was ≈100 d (Vrain and Daubeny, 1986), 110 d (Bristow et al., 1980), and 120 d (this study). Depending on the temperatures at which these experiments were conducted, anywhere from 2.6 to 5.5 P. penetrans generations could have occurred based on constant temperatures of 17 and 27 °C, respectively (Mizukubo and Adachi, 1997). Another difference could be initial inoculation densities used across experiments. In the aforementioned studies, initial inoculation densities ranged from 2.2 to 4.0 P. penetrans/g soil (Vrain and Daubeny, 1986), 5.0 P. penetrans/g soil (Bristow et al., 1980), and 0.7 to 1.0 P. penetrans/g soil in our study. However, inoculation density does not seem to be a plausible explanation for the variation because the lowest RF value reported for ‘Willamette’ by Vrain and Daubeny (1986), 0.82, occurred when plants were inoculated with the highest initial population density (4.0 P. penetrans/g soil). In fact, in our study, when the same initial inoculation density was used in 2011 and 2012 (1.0 P. penetrans/g soil), RF values were 0.6 and 2.1 on R. idaeus ‘Meeker’, respectively.

Pratylenchus penetrans is a significant production constraint in raspberry and having a commercially acceptable cultivar with resistance to P. penetrans would be a useful tool for raspberry growers to combat this pest. Our research demonstrates that some sources of R. niveus and R. leucodermis are poor hosts for P. penetrans and may provide a source of resistance genes that can be transferred to red raspberry. There was minimal sampling of variability within Rubus species and resistance may be present in specific accessions of other species. In addition, our results indicate that more research is needed to learn about the inheritance of the putative resistance. To truly understand the potential of R. niveus and R. leucodermis as sources of resistance, these species should evaluated in the field.