Reproduction of Meloidogyne arenaria race 2 on Flue-cured tobacco possessing resistance genes Rk1 and/or Rk2

A population of M. arenaria race 2 collected from a flue-cured tobacco field in Halifax County, VA was maintained on susceptible tomato (Solanum lycopersicum L.) variety ‘Rutgers’ in greenhouses at the main Virginia Tech campus in Blacksburg, VA and at the Southern Piedmont Agricultural Research and Extension Center (SPAREC) near Blackstone, VA. The species identity had been previously confirmed by differential host testing (Taylor and Sasser, 1978) and was verified by examination of more than 40 perineal patterns from the population (Eisenback, 1985). Three greenhouse experiments were conducted in 2018 at SPAREC with an additional trial in 2019 at the main Virginia Tech Campus in Blacksburg, VA in order to evaluate the impact of resistance genes Rk1 and/or Rk2 on parasitism of flue-cured tobacco by M. arenaria. Experiments were arranged in randomized complete block designs with seven replications, except for the test in Blackstone in September, 2018, which had six replications. These experiments assessed resistance to M. arenaria in a panel of five flue-cured tobacco entries: Hicks (susceptible to the four major tropical root-knot nematode species, M. incognita, M. javanica, M. arenaria, and M. hapla); K326 (homozygous for Rk1); T-15-1-1 (homozygous for Rk2); CC 13 (homozygous for Rk1 and heterozygous for Rk2); and STNCB-2-28 (homozygous for both Rk1 and Rk2). Seed of K326 and CC 13 were donated by Cross Creek Seed, Raeford, NC, while seed of the remaining four entries were produced in the SPAREC flue-cured tobacco nursery in 2016 and 2017. Seed were germinated in organic vermiculite (The Epsoma Company, Millville, NJ) and four to five week old seedlings were transplanted to individual 7.6 cm clay pots containing a 2:1 mixture of steam sterilized sandy loam field soil with Profile Greens-Grade Porous Ceramic soil amendment (Profile Products, Buffalo Grove, Il). Seedlings with four to six true leaves were transplanted into 15 cm clay pots. Egg inoculum for greenhouse trials was collected from infested roots using the method of Hussey and Barker (1973). Eggs were collected in 1 L of tap water, counted with a compound microscope at X 20 to 40, and an egg suspension calibrated to contain 125 eggs/mL of suspension immediately prior to inoculation. Plants were inoculated with a 40 mL aliquot containing 5,000 nematode eggs applied directly to the root mass during transplanting. Plants were maintained in greenhouses at ambient air temperatures of 20 to 33°C, with natural lighting.

Plant roots were washed free of soil substrate 28 days following inoculation and blotted dry; the aerial portion of the plants were discarded. Fresh root weights were recorded and the whole root system was evaluated for galling according to the gall count index developed by Taylor and Sasser (1978), in which gall counts are ranked as follows: 0 galls present = 0; 1–2 galls present = 1; 3–10 galls present = 2; 11–30 galls present = 3; 31–100 galls present = 4; and more than 100 galls present = 5. Roots were cut into 4 to 6 cm long sections and thoroughly mixed. Root-knot presence in roots was assessed in three 1 g subsamples from each plant. Feeder roots were cleared in 1% sodium hypochlorite and stained with 0.005% acid fuchsin (Byrd et al., 1983). Roots were examined and nematodes were counted with a stereomicroscope at X10 to 40. For egg mass counts, two 1 g subsamples were stained in 0.15 g/L Phloxine-B (Daykin and Hussey, 1985) for approximately 5 min and were counted with a stereomicroscope at X10 to 40. Eggs were extracted from the remaining root system in the manner described above and counted at X40 using an inverted compound microscope. The reproductive index for each plant was calculated by dividing the final egg count (P_f) by the initial egg inoculum number (P_i).

Field trials were conducted in 2018 and 2019 in a flue-cured tobacco field in Palmer Springs, VA infested with a population of M. arenaria race 2 in order to compare the flue-cured tobacco entries described above. Trials were arranged in randomized complete block designs with 11 replications in 2018 and 10 replications in 2019. Treatments were randomized independently each year. Plots consisted of single 16.1 m long rows spaced 1.2 m apart. Plots were mechanically transplanted, fertilized, and maintained in accordance with the recommendations of Virginia Cooperative Extension (Reed et al., 2018).

Initial soil nematode population densities were estimated each year based on soil samples collected between bed formations and transplanting. Final population densities were determined after final samples have been collected 18 weeks after transplant. Twenty-four 2 cm by 16 cm soil samples were collected from each plot and bulked. Nematode counts were performed at the Virginia Tech Nematode Diagnostic and Assay Lab on the Virginia Tech Blacksburg Campus. Bulked samples were initially hand mixed to reduce aggregates, sifted, and a 250 cubic centimeter subsample from each plot was subjected to nematode extraction using a mechanical elutriator, sugar flotation, and decantation sieving (Barker, 1985). Two plants were destructively sampled from each plot beginning three weeks after transplanting, and every three weeks until 18 weeks after transplanting. Soil was washed free of the root systems of the sampled plants and galling was assessed as described above. Fresh weights of the entire root systems were recorded and fibrous feeder roots were separated from lignified structural roots. The number of nematodes present in roots, egg masses on roots, and eggs per gram of root were determined for the fibrous feeder root portion of the root system of each plant. Egg mass production was evaluated by counting Phloxine-B stained egg masses (Daykin and Hussey, 1985) on three 1 g subsamples at X10 to 40. Numbers of nematodes present in roots were determined by clearing two 1 g subsamples of feeder roots with sodium hypochlorite and staining the roots with acid fuchsin (Byrd et al., 1983) that were examined with a stereomicroscope at X10 to 40. Eggs were extracted from the remaining feeder roots by agitation in 1% sodium hypochlorite (Hussey and Barker, 1973). Eggs were suspended in 1 L of tap water and two 10 mL aliquots were counted at X40 with a compound microscope. These counts were used to calculate the approximate number of nematodes extracted from the known mass of feeder roots, which was then used to calculate the number of eggs per gram of feeder root for the entire root system.

Data were transformed (log₁₀ (x + 1)) prior to statistical analysis. Data from all trials were subjected to analysis of variance (ANOVA) using PROC GLM in SAS (version 9.4; SAS Institute, Cary, NC). Differences among treatment means were identified using Fisher’s protected Least Significant Difference test (P ≤ 0.05).

Results varied across the four greenhouse trials conducted in 2018 and 2019, so each trial was analyzed independently. Significant differences in root galling were found in all four trials. Galling was lowest on entries CC 13 and STNCB 2-28 in every experiment, and highest on the susceptible entry Hicks in all but the April–May 2018 trial (Table 1). Galling of CC 13 and STNCB 2-28 was significantly less than that of other entries in all tests except the April–May 2018 trial, and in the trial conducted in 2019, in which galling of CC 13 was similar to that of K326. Galling was significantly lower on CC 13 and STNCB 2-28 compared to entries K326 and T-15-1-1, which respectively possess Rk1 and Rk2 alone, in all studies but the April–May 2018 trial; in this trial, galling of K326 was similar to that of CC 13 and STNCB 2-28. Galling was significantly lower on entry K326 than T-15-1-1 in two of the four trials, and galling of both of these entries was significantly lower than that of Hicks in two trials. However, in the trial conducted in Blackstone from April to May of 2018, galling of Hicks was significantly less than that of T-15-1-1.

Nematodes per gram of root varied considerably among trials, but were typically lowest on CC 13 and/or STNCB 2-28 (Table 2). Roots of CC 13 and STNCB 2-28 always contained significantly fewer nematodes than susceptible entry Hicks, except in the April–May trial conducted in 2018, in which CC 13 was the only entry with significantly fewer nematodes in roots relative to the other four entries. Significantly fewer nematodes were present in the roots of both K326 and T-15-1-1 than Hicks in the trial conducted in Blackstone in September of 2018, and K326 had fewer nematodes in roots than Hicks in the trial conducted in 2019. Significantly fewer nematodes were present in the roots of entries CC 13 and STNCB 2-28 than entries K326 and T-15-1-1 in the trials conducted in both locations in September of 2018.

Egg mass counts differed significantly in all trials. CC 13 and STNCB 2-28 always had significantly fewer egg masses compared to Hicks, and less than K326 in three trials (Table 3). Egg mass production was lower on both CC 13 and STNCB 2-28 than T-15-1-1 in one trial, although in another trial, CC 13 had significantly fewer egg masses than T-15-1-1, while egg mass production on STNCB 2-28 was intermediate. Significantly fewer egg masses were present on T-15-1-1 relative to Hicks in two trials, and in only one trial did K326 exhibit significantly lower egg mass production than Hicks.

Reproduction varied dramatically across trials. Egg production was significantly lower on CC 13 and STNCB 2-28 than on all other entries in the trial conducted in September of 2018 in Blacksburg, and along with K326, was less than that of Hicks in the trial conducted from April to May in 2018 in Blackstone (Table 3). Egg counts were low (not exceeding 22 eggs per gram of root) across all entries in the trial conducted in September of 2018 in Blackstone, but both Hicks and T-15-1-1 had significantly fewer eggs per gram of root than CC 13 in this trial. There was no consistent trend in relative egg production among the susceptible entry and those possessing either Rk1 or Rk2 alone in the three trials where significant differences were present. No differences in egg production were found among entries in the trial conducted from April to May of 2019 in Blackstone, in which egg counts were also relatively low.

Reproductive indices for all four entries possessing Rk1 and/or Rk2 were significantly lower than that of susceptible Hicks in the trial conducted April to May of 2018 in Blackstone (Table 3). In the trial conducted in Blacksburg in September of 2018, the reproductive indices of T-15-1-1, CC 13, and STNCB 2-28 were significantly lower than those of Hicks and K326, while CC 13 and STNCB 2-28 were also significantly lower than T-15-1-1. In the remaining trials, reproduction was low and significant differences in reproductive indices were not detected (Table 3).

No root galling was observed on the roots of plants sampled at 3 and 6 weeks after transplanting in 2018 (Table 4). Limited galling was observed on the root systems (less than 10 galls per root system) of all entries at 9 and 12 weeks, but no significant differences were found. Galling of STNCB 2-28 was significantly less than that of Hicks, K326, and T-15-1-1 at 15 weeks, and was significantly less than that of K326 at 18 weeks.

In 2019, galling was observed on the roots of plants sampled at 3 and 6 weeks after transplanting, but no significant differences were found among entries; no more than one or two galls were observed on root systems (Table 4). CC 13 and STNCB 2-28 both had significantly less galling than T-15-1-1 at 9 weeks, and at this timepoint galling of CC 13 was also significantly less than that of entry K326, while galling of susceptible Hicks was intermediate. Galling was significantly less on both CC 13 and STNCB 2-28 relative to K326 and Hicks at 12 weeks after transplanting, while at 18 weeks, STNCB 2-28 had significantly less root-galling than Hicks, K326 and T-15-1-1; no significant differences were observed at 15 weeks.

Significantly fewer nematodes were present in the roots of T-15-1-1 than CC 13 3 weeks after transplanting in 2018, whereas in 2019, fewer nematodes were present in the roots of CC 13 than Hicks, K326, and T-15-1-1 (Table 5). No differences in nematodes abundance were found at 6 weeks in either year, and in 2018, no significant differences were present at 9 weeks. In 2019, CC 13 had significantly fewer nematodes present in roots than K326. In both years, starting at 12 weeks, STNCB 2-28 had the fewest nematodes in roots; with the exception of 15 weeks in 2018, STNCB 2-28 always had significantly fewer nematodes present in roots than Hicks, and often relative to K326 as well, while other entries were typically intermediate in nematode abundance. The number of nematodes present in the roots of entry CC 13, which is homozygous for Rk1 and heterozygous for Rk2, was never significantly different from that of Hicks.

Egg masses and/or eggs were not observed on any entry at 3 or 6 weeks after transplanting in either year, and in both years no significant differences were observed at 9 weeks after transplanting, when reproduction was first observed (Table 6). In both years, STNCB 2-28 typically had the fewest egg masses present, although there were notable exceptions to this trend, particularly at 12 weeks in 2018, when K326 had significantly fewer egg masses present than Hicks, while all other entries experienced intermediate egg mass production. No single entry consistently exhibited the highest egg mass numbers in either year. In 2019, egg production on STNCB 2-28 was significantly less than on K326, while no significant differences were found at this timepoint in 2018. At 15 weeks in 2018, egg production was significantly lower on CC 13, STNCB 2-28, and susceptible Hicks relative to T-15-1-1, whereas at the same timepoint in 2019, egg counts were significantly lower for CC 13 and STNCB 2-28 relative to Hicks. No significant differences in egg production were found at 18 weeks in either year.

Reproductive indices were not calculated for field trials because the amount of egg inoculum present in the soil at transplanting can only be speculated upon based on pre-plant root-knot second-stage juvenile abundance. Mean initial and final soil M. arenaria counts are presented in Table 7. In our 2018 study, M. arenaria juveniles were not present at detectable levels in pre-plant soil samples from 71% of the plots, while following termination of the study, M. arenaria juveniles were not present at detectable levels in soil nematode extracts from 10% of the studied plots. In 2019, M. arenaria juveniles were not present at detectable levels in pre-plant soil nematode extracts from 53% of plots, while following termination of the 2019 study, M. arenaria juveniles were not present at detectable levels in soil nematode extracts from 56% of the studied plots.