Development of Selected Tobacco Cyst Nematode Isolates on Resistant and Susceptible Cultivars of Flue-cured Tobacco

Tobacco cyst nematode (Globodera tabacum solanacearum) isolates were collected from 11 locations in Virginia, 3 in North Carolina, and 1 in Maryland. Isolates from each location were maintained and increased on flue-cured tobacco, Nicotiana tabacum cv K326. Plants of flue-cured tobacco cultivars K326 (susceptible) and NC567 (resistant) were each inoculated with 6,000 G. t. solanacearum eggs/plant. Tests were conducted over one (6 weeks) or two (14 weeks) generations of the nematode. Shoot and root weights and the number of nematodes present within a 1-g subsample of feeder roots were recorded at completion of the tests. Nematode counts were categorized by nematode life stage (vermiform, swollen, pyriform, and adult). Although significant differences in nematode development were detected among isolates, differences were not consistent across experiments. Results indicate similar virulence among G. t. solanacearum isolates on resistant and susceptible flue-cured tobacco cultivars. Therefore, plant breeders may effectively use a single G. t. solanacearum isolate when screening tobacco germplasm for resistance.     

Enhanced Hatching of Globodera tabacum solanacearum Juveniles by Root Exudates of Flue-cured Tobacco

Stimulation of hatching of a tobacco cyst nematode (Globodera tabacum solanacearum) by root exudates from resistant NC 567 and susceptible K 326 cultivars of flue-cured tobacco, Nicotiana tabacum, was investigated. Root exudates were collected by soaking seedlings in deionized water for 2 hours at 22 °C in the dark. Fifteen mature and uniformly sized cysts were exposed at 15, 20, or 25 °C to undiluted root exudate, root exudate diluted 1:1 or 1:3 with deionized water, or deionized water alone. Hatched juveniles were counted and removed at weekly intervals during 42 and 53 days of exposure in experiments conducted in 1994 and 1995, respectively. Root exudates from both susceptible cultivar K 326 and resistant cultivar NC 567 stimulated more hatching than deionized water at 25 °C in 1994, and at all three tested temperatures in 1995. In 1994, dilution of root exudates 1:3 reduced stimulation of hatching at 25 °C compared to undiluted exudate. Hatching at 25 °C was similarly stimulated by exposure to undiluted root exudate and exudate diluted 1:1. In 1995, both dilutions reduced stimulation of hatching by root exudates at all the temperatures.     

Shade Tobacco Yield Loss and Globodera tabacum tabacum Population Changes in Relation to Initial Nematode Density

Field microplot experiments were conducted from 1987 to 1992 to determine the relationship between fresh weight leaf yield of shade tobacco (Nicotiana tabacum) and initial density of Globodera tabacum tabacum (encysted J2 per cm³ soil). Initial nematode densities of 0.1 to 1,097 J2/cm³ soil were negatively correlated with leaf yield, total shoot weight, and normalized plant height 5 to 6 weeks after transplanting (r = -0.73, -0.73, and -0.52, respectively). Nonlinear damage functions were used to relate initial G. t. tabacum densities to the yield and shoot weight data. The model described leaf yield losses of < 5 % for initial nematode densities of less than 100 J2/cm³ soil. Densities above 100 J2 resulted in yields decreasing exponentially to a maximum yield loss of >40% at 500 to 1,000 J2/cm³ soil. A similar initial density tolerance threshold relationship was observed for total shoot weight. No threshold effect was evident for standardized plant height, which was a poor predictor of leaf yield. Globodera tabacum tabacum population increase over a growing season was described by a linear relation on a log/log plot (R² = 0.73).     

Genetics of Burley and Flue-Cured Tobacco Resistance to Globodera tabacum tabacum

Genotypes of burley (cultivars B-21 and B-49), flue-cured (line VA-81 and cultivar PD-4), and Connecticut broadleaf (cultivar C9) tobacco (Nicotiana tabacum) resistant (R) or susceptible (S) to the tobacco cyst nematode Globodera tabacum tabacum were crossed. F1 progeny of burley and susceptible broadleaf were selfed and backcrossed to produce additional progeny for evaluation of resistance in greenhouse experiments. Plants without adult female nematodes visible (×10 magnification) on the root surface 6 weeks after inoculation were classified as resistant, whereas those plants in which one or more females were evident were classified as susceptible. Segregation ratios for progeny of resistant and susceptible plants were not different from 3:1 and 1:1 for F2 (F1 × F1) and BC1 (F1 × S) lines, respectively, indicating that resistance in burley to G. t. tabacum is conferred by a single, dominant gene. Segregation ratios for resistance in crosses between nematode-resistant burley and flue-cured tobacco (F1 and F2 progeny) and between burley-flue-cured hybrids and broadleaf BC1 (F1 × S) and BC2 (BC1 × S) progeny were consistent with the assumption that resistance to G. t. tabacum in burley and flue-cured tobacco is conferred by the same or closely linked single, dominant gene(s).     

Early Crop Root Destruction for Management of Tobacco Cyst Nematodes

Prompt tillage after crop harvest was investigated as a cultural control for the tobacco cyst nematode, Globodera tabacum tabacum, on stalk-cut broadleaf cigar wrapper tobacco. Stalk stumps and roots remaining after harvest were destroyed by tilling immediately or from 2 to 6 wk after harvest in field experiments over 4 yr. Cyst nematode Pf/Pi ratios ranged from 0.65 to 1.62 when plants were tilled immediately after harvest and 1.13 to 5.88 when tillage was delayed. Nematode population development was monitored by inoculating plants in pots placed in fields with J2 in eggs and sampling over time (8 to 18 wk). Three generations per year were observed, and G. t. tabacum generation time was as short as 6 wk for each generation. Destroying stalks and root systems remaining after harvesting stalk-cut broadleaf cigar wrapper tobacco removes the host to preclude development of nematodes at the end of the second and entire third generation. Early tillage resulted in consistently lower tobacco cyst nematode populations than allowing viable roots to remain in fields for an additional 8 to 18 wk. This management tactic reduces the need for nematicide application to slow nematode population increases over time and can reduce losses due to infection by G. t. tabacum.     

Control of Globodera tabacum solanacearum by Alternating Host Resistance and Nematicide

The feasibility of alternating use of resistant vs. susceptible flue-cured tobacco cultivars to improve control of Globodera tabacum subsp, solanacearum (TCN) was investigated at two Virginia locations in 1984-86. Post-harvest TCN population densities were reduced in each year of the study when fenamiphos was used with a TCN-resistant cultivar (NC 567), relative to susceptible cultivars (K 326 or Mc 944). Using NC 567 with fenamipbos also reduced preplant TCN population densities in the next growing season. Egg population densities before planting in 1986 were significantly lower in plots planted with NC 567 in 1984, even when a susceptible cultivar had been planted in 1985. Use of fenamiphos with NC 567 in 1984 and 1985 further reduced preplant egg population densities in 1986. Economic returns were significantly greater in 1984 when NC 567 was used with fenamiphos, rather than a susceptible cultivar. Treatments involving fenamiphos and (or) NC 567 in 1984 and 1985 resulted in higher economic returns in 1986 than did treatments using a susceptible cultivar without fenamiphos in both previous years. Economic returns were highest in 1986 when fenamiphos and NC 567 were used in 1984 and 1985 and a susceptible cultivar was planted in 1986.     

Broadleaf Tobacco Yield Loss in Relation to Initial Globodera tabacum tabacum Population Density

Field microplot experiments were conducted from 1995 to 1998 to determine the relationship between fresh shoot weight of stalk-cut broadleaf and shade-grown cigar wrapper tobacco types (Nicotiana tabacum L.) and initial density of Globodera tabacum tabacum second stage juveniles (J2) per cm³ soil. Total shoot weight was negatively correlated with initial nematode densities of 12.3 to 747.3 J2/cm³ soil (r = -0.53 and -0.70 for broadleaf and shade-grown tobacco, respectively). Nonlinear damage functions were used to relate initial G. t. tabacum densities to shoot weight. The models described shoot weight losses of less than 14% or 39% for broadleaf and shade tobacco, respectively, at G. t. tabacum densities below 50 J2/cm³ soil. Total shoot weights were reduced by 40% and 60% of uninfested plots as preplant nematode densities approached maximum levels (>600 J2/cm³ soil) for broadleaf and shade tobacco, respectively. Globodera t. tabacum population increase over a growing season was described by a linear relation on a log/log plot (R² = 0.07 and 0.61 for broadleaf and shade, respectively). These experiments demonstrate that G. t. tabacum can directly reduce shoot weight of stalk-cut broadleaf tobacco. Broadleaf is more tolerant to nematode infection than shade tobacco, as shade tobacco shoot weight reductions were greater at the same initial nematode densities in the same years.     

Predisposition of Broadleaf Tobacco to Fusarium Wilt by Early Infection with Globodera tabacum tabacum or Meloidogyne hapla

In greenhouse experiments, broadleaf tobacco plants were inoculated with tobacco cyst (Globodera tabacum tabacum) or root-knot (Meloidogyne hapla) nematodes 3, 2, or 1 week before or at the same time as Fusarium oxysporum. Plants infected with nematodes prior to fungal inoculation had greater Fusarium wilt incidence and severity than those simultaneously inoculated. G. t. tabacum increased wilt incidence and severity more than did M. hapla. Mechanical root wounding within 1 week of F. oxysporum inoculation increased wilt severity. In field experiments, early-season G. t. tabacum control by preplant soil application of oxamyl indirectly limited the incidence and severity of wilt. Wilt incidence was 48%, 23%, and 8% in 1989 and 64%, 60%, and 19% in 1990 for 0.0, 2.2, and 6.7 kg oxamyl/ha, respectively. Early infection of tobacco by G. t. tabacum predisposed broadleaf tobacco to wilt by F. oxysporum.     

Effects of Continuous Cropping of Resistant and Susceptible Cultivars on Reproduction Potentials of Heterodera glycines and Globodera tabacum solanacearum

The reproductive potentials of Heterodera glycines (mixture of races 3 and 4 and unidentified races) and a tobacco cyst nematode Globodera tabacum solanacearum were studied in the field. The experiments involved four cultivars of soybean Glycine max and four cultivars of Nicotiana tabacum. The reproductive potential of the H. glycines population was high on Essex and Lee 74 soybean but low on Forrest and Bedford over the 3 years (1982-84) of continuous cropping. The reproductive potential of H. glycines was 12% on Forrest and 6% on Bedford in 1982 but increased to 37 and 35% in 1983 and to 71 and 41% in 1984, respectively, on these two cultivars. The reproductive potential of G. tabacum solanacearum was high on McNair 944 and Coker 319 tobacco cultivars and low on VA 81 and PD 4 over the 3 years of cropping. The reproductive potential of G. tabacum solanacearum on VA 81 and PD 4 was 18 and 17% in 1982, 7 and 16% in 1983, and 5 and 5% in 1984, respectively. The changes in reproductive potentials of H. glycines and G. tabacum solanacearum may be related to inherent genetic variability in the systems that control reproduction of the two cyst nematodes and nature of resistance incorporated in the soybean and tobacco cultivars.     

Interrelationships of Meloidogyne Species with Flue-cured Tobacco

Microplot and field experiments were conducted to determine relationships of population densities of Meloidogyne spp. to performance of flue-cured tobacco. A 3-yr microplot study of these interactions involved varying initial nematode numbers (P_i).and use of ethoprop to re-establish ranges of nematode densities. Field experiments included various nematicides at different locations. Regression analyses of microplot data from a loamy sand showed that cured-leaf yield losses on ''Coker 319'' for each 10-fold increase in P_i were as follows: M. javanica and M. arenaria—-13-19%; M. incognita—5-10%; M. hapla—3.4-5%; and 3% for M. incognita on resistant ''Speight G-28'' tobacco. A P_i of 750 eggs and larvae/500 cm³ of soil of all species except M. hapla caused a significant yield loss; only large numbers of M. hapla effected a loss. M. arenaria was the most tolerant species to ethoprop. Root-gall indices for microplot and most field-nematicide tests also were correlated negatively with yield. Relationships of P_i(s) and necrosis indices to yield were best characterized by linear regression models, whereas midseason numbers of eggs plus larvae (P_m) and sometimes gall indices vs. yield were better characterized by quadratic models. The relation of field P_m and yield was also adequately described by the Seinhorst model. Degrees of root galling, root necrosis, yield losses, and basic rates of reproduction on tobacco generally increased from M. hapla to M. incognita to M. arenaria to M. javanica.     

Trap Crops and Population Management of Globodera tabacum tabacum

Tobacco, eastern black nightshade, and tomato were grown for 3 to 13 weeks to assess differences in invasion, development, and soil density of Globodera tabacum tabacum (tobacco cyst nematode) in field plots and microplots over three seasons. Tobacco cyst nematodes invaded roots of resistant and susceptible tobacco, nightshade, and tomato. Nematode development was fastest in nightshade and slowest in tomato, and few adults developed in roots of nematode-resistant tobacco. Soil populations of tobacco cyst nematodes were reduced up to 80% by destroying nightshade or susceptible tobacco grown for 3 to 6 weeks. Nematode populations were reduced up to 96% by destroying tomato or resistant tobacco grown for 3 to 6 weeks. Timing of crop destruction was less critical with tomato and resistant tobacco, as nematode populations did not increase after 13 weeks of growth. These studies demonstrate that trap cropping, through crop destruction, can significantly reduce G. t. tabacum populations.     

Cereal Cyst Nematode (Heterodera avenae) on Oats. II. Early Root Development and Nematode Tolerance

The effect of Heterodera avenae infestation on early seminal and lateral root growth was examined in four oat genotypes differing in tolerance to H. avenae. Recently emerged seminal roots were inoculated with a range of H. avenae larval densities, then transferred a hydroponic system to remove the effect of later nematode penetration on root development. Intolerance to H. avenae was assessed in terms of impairment of seminal root extension resulting in fewer primary lateral roots emerging from the seminal root below the zone of juvenile penetration. Tolerant plants infested with H. avenae had longer lateral root systems than infested intolerant plants. The decline in lateral root growth below the penetration zone was partly offset by increased growth above. This did not contribute to tolerance, however, as there were no differences between cultivars for this feature. Nematodes induced earlier nodal root emergence in all cultivars. Nodal root development was most advanced on the most tolerant cultivar.     

Extraction of Cyst Nematodes from Organic Soils

The effects of extraction technique, sample size, soil moisture level, and overflow rate on recovery of Globodera rostochiensis and (or) Heterodera schachtii cysts from organic soils were investigated. A modified Fenwick can (MFC) and an underflow elutriator (UE) described in this paper were evaluated and compared for cyst recovery efficiency and amount of organic flotsam collected. The MFC and UE extracted similar numbers of cysts, but the UE collected 50% less flotsam than the MFC. Sample size was negatively correlated with cyst recovery and positively correlated with amount of flotsam. The amount of flotsam recovered with the MFC was correlated with overflow speed. Presoaking air dried samples for 30 minutes halved the amount of flotsam without affecting cyst recovery. Extracting cysts from wet soil without prior drying resulted in negligible recovery with both extraction techniques. There were no significant differences in cyst recovery of the two genera tested.     

Intraspecific Variability within Globodera tabacum solanacearum Using Random Amplified Polymorphic DNA

Random amplified polymorphic DNA (RAPDs) were used to investigate the intraspecific variability among 19 geographic isolates of Globodera tabacum solanacearum from eight counties in Virginia and one county in North Carolina. Globodera tabacum tabacum, G. t. virginiae, and the Mexican cyst nematode (MCN) were included as outgroups. Six primers were used and 119 amplification products were observed. Each primer yielded reproducible differences in fragment patterns that differentiated the isolates and species. Hierarchical cluster analysis was performed to illustrate the relatedness among isolates and species. The average Jaccard''s similarity index among isolates of G. t. solanacearum was 74%, possibly representing greater variation than that reported in the literature across different pathotypes of the potato cyst nematode, Globodera pallida, in studies where RAPD were also employed. The RAPD markers described here may be useful for the development of specific primers or probes that could improve the identification of TCN populations. Such improvements in the characterization of TCN genotypes would facilitate the effective deployment of existing and future resistant cultivars to control these economically important pests.     

Nicotine Content of Tobacco Roots and Toxicity to Meloidogyne incognita

The motility of Meloidogyne incognita second-stage juveniles (J2) and their ability to induce root galls in tomato were progressively decreased upon exposure to nicotine at concentrations of 1-100 μg/ml. EC₅₀ values ranged from 14.5 to 22.3 μg/ml, but J2 motility and root-gall induction were not eliminated at 100 μg/ml nicotine. Nicotine in both resistant NC 89 and susceptible NC 2326 tobacco roots was increased significantly 4 days after exposure to M. incognita. The increase was greater in resistant than in susceptible tobacco. Root nicotine concentrations were estimated to be 661.1-979.1 μg/g fresh weight. More M. incognita were detected in roots of susceptible than in roots of resistant tobacco. Numbers of nematodes within resistant roots decreased as duration of exposure to M. incognita was increased from 4 to 16 days. Concentrations of nicotine were apparently sufficient to affect M. incognita in both susceptible and resistant tobacco roots. Localization of nicotine at infection sites must be determined to ascertain its association with resistance.     

Stage-specific Monoclonal Antibodies to the Potato Cyst Nematode Globodera pallida Stone (Behrens)

Using standard hybridoma technology and hierarchical screening, monoclonal antibodies (MAbs) were obtained with specific reactivity against two developmental stages of Globodera pallida. The procedure was based on enzyme-linked immunosorbent assay (ELISA) with homogenates prepared from second-stage juveniles, young adult females, and potato roots. Hybridomas were formed by fusing myelomas with splenocytes derived from mice immunized with either infective juveniles or females of G. pallida. About 600 hybridoma lines were screened from the fusion involving the mouse immunized with juveniles. Two MAbs (LJMAbl &2) were identified with high reactivity toward second-stage juveniles but no reactivity with either potato roots or females of G. pallida. A total of 630 cell lines was screened from the corresponding fusion involving the spleen of a mouse receiving immunogens from adult female nematodes. One MAb (LFMAbl) was obtained with the required specificity against only adult female G. pallida. This work extends the application of monoclonal antibodies in nematology from valuable probes for research and species identification to recognition of developmental stages. These specific MAbs have potential value in plant breeding programs for screening for resistant lines unable to support nematode development.     

Hatch and Reproduction of Globodera tabacum tabacum in Response to Tobacco,Tomato, or Black Nightshade

The effects of broadleaf tobacco, tomato, and black nightshade on juvenile hatch and reproduction of Globodera tabacum tabacum were determined in laboratory and greenhouse experiments. Root exudates from nightshade stimulated greater egg hatch than those from either ''Rutgers'' tomato or ''86-4'' tobacco. Hatch was greater at higher proportions of root exudates for all three plant species. Root exudates from plants greater than 3 weeks old stimulated more hatch than younger plants. No regression relationships existed between plant age and nematode batch. In other experiments, hatch from eggs in cysts was higher for tomato and nightshade after 10 weeks in greenhouse pots compared to tobacco and bare soil. Numbers of second-stage juveniles in eggs in cysts produced from a previous generation on the same host were highest on nightshade and less on tomato and tobacco. Cysts of variable age recovered from field soil had increased hatch in both root exudates or water compared to recently produced cysts from plants in growth chambers. Globodera t. tabacum may be subject to both host and environmentally mediated diapause.     

Effects of Initial Nematode Density on Population Dynamics of Globodera rostochiensis on Resistant and Susceptible Potatoes

The influence of resistant and susceptible potato cultivars on Globodera rostochiensis population density changes was studied at different nematode inoculum levels (Pi) in the greenhouse and field. Soil in which one susceptible and two resistant cultivars were grown and fallow soil in pots was infested with cysts to result in densities of 0.04-75 eggs/cm³ soil. A resistant cultivar was grown in an infested field with Pi of 0.7-16.7 eggs/cm³ soil. Pi was positively correlated with decline of soil population densities due to hatch where resistant potatoes were grown in the greenhouse and in the field but not in fallow soil. However, Pi was not correlated with in vitro hatch of G. rostochiensis cysts in water or potato root diffusate. Under continuous culture o f a resistant cultivar, viable eggs per cyst declined 60-90% per plant growth cycle (4 weeks) and the number of cysts containing viable eggs had decreased by 77% after five cycles. The rate of G. rostochiensis reproduction on both resistant and susceptible cultivars was negatively correlated with Pi. These data were used to predict the effect of resistant and susceptible potato cultivars on G. rostochiensis soil population dynamics.     

Morphometrics of Globodera tabacum tabacum,G. t. virginiae,and G. t. solanacearum (Nemata: Heteroderinae)

A morphometric evaluation of second-stage juveniles (J2), males, females, cysts, and eggs of several isolates of the tobacco cyst nematode (TCN) complex, Globodera tabacum tabacum (GTT), G. t. virginiae (GTV), and G. t. solanacearum (GTS) is presented. Morphometrics of eggs, J2, and males are considerably less variable than of females and cysts. No measurements of eggs and J2 are useful for identification of the three subspecies. Distance from the median bulb and excretory pore to the head end in J2 and males is quite stable. Stylet knob width of males is useful for identifying GTV isolates and tail length in separating males of GTT isolates from GTV and GTS. Body length/width (L/W) ratio of females and cysts discriminates GTT from GTV and GTS; stylet knob width is an auxiliary character for identifying GTV. This subspecies complex has a continuum of values for the other characters. Data suggest a close relationship between GTV and GTS, which also occur in close proximity in Virginia.     

Effects and Dynamics of a Nematode Community on Maize

Relationships between nematode density and yield and between final and preplant population levels were examined in small maize plots on sandy soils in north-central Florida. Plant-parasitic nematodes present in the community included Belonolaimus longicaudatus, Criconemella sphaerocephala, Meloidogyne incognita, Paratrichodorus minor, Pratylenchus brachyurus, and a Xiphinema sp. Plant growth--including stand count, grain yield, stalk weight, and size of young plants--often was inversely correlated (P ≤ 0.05) with densities of B. longicaudatus and occasionally with P. brachyurus, but not with densities of other species or with a range of soil variables. More severe losses in grain yields from B. longicaudatus occurred in 1987 than in 1988, although mean preplant nematode densities in February were similar in both years (4.4 vs. 3.9/100 cm³ soil). Final population densities of most nematode species were linearly related (P ≤ 0.05) to densities measured at planting or earlier. These relationships were stronger (higher r²) with the ectoparasites B. longicaudatus and C. sphaerocephala than with the endoparasites M. incognita and P. brachyurus. No significant correlations were found between population densities of different nematode species.