Effects of a mixed-isolate mycorrhizal inoculum on the potato – potato cyst nematode interaction

Inoculation of microplants of potato cv. Golden Wonder with Vaminoc, a mycorrhizal inoculum of three arbuscular mycorrhizal fungi (Glomus spp.), resulted in an increase in in‐sand hatch of Globodera pallida, but not G. rostochiensis, within 2 weeks. By this time, mycorrhized plants also supported a larger number of feeding nematodes of both PCN species (50% higher for G. rostochiensis) than did non‐mycorrhized plants, with a higher proportion of the G. pallida population being fertilised females than for G. rostochiensis. After 12 weeks, the multiplication rate of G. rostochiensis on mycorrhized plants was significantly greater than on non‐mycorrhized plants, whereas no such difference was observed for G. pallida. The principal component of PCN multiplication affected by mycorrhization was increased cyst number per plant from 6 to 12 weeks. Over this period, there was no increase in cyst number per plant for either PCN species on non‐mycorrhized plants, whereas the value increased on mycorrhized plants for both G. rostochiensis (by almost 200%) and G. pallida (57%). Mycorrhization resulted in significant increases in the root and shoot dry weights of plants grown in the absence of PCN. Although mycorrhized plants carried a larger PCN burden than non‐mycorrhized plants when grown on PCN‐infested medium, as a result of the increased PCN multiplication rate, they produced larger root systems than did nonmycorrhized plants, suggesting increased tolerance to PCN of the mycorrhized plants, particularly to G. rostochiensis. Of morphological characters investigated in the absence of PCN, only stem height (increased) was significantly affected by mycorrhization. Colonisation by mycorrhizal fungi resulted in increased tuber yield both in the absence (significant increase) and presence (non significant) of PCN, as a result of increased tuber number per plant. These results are discussed in the light of the possible use of AMF as part of an integrated PCN management plan.     

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.     

Comparative Responses of Globodera rostochiensis and G. pallida to Hatching Chemicals

Globodera rostochiensis and G. pallida responded similarly to hatch stimulation by potato root leachate, but proportionally more second-stage juveniles (J2s) of G. rostochiensis hatched than of G. pallida in response to picrolonic acid, sodium thiocyanate, alpha-solanine, and alpha-chaconine. Fractionation of the potato root leachate identified hatching factors with species-selective (active toward both species but stimulating greater hatch of one species than the other), -specific (active toward only one species), and -neutral (equally active toward both species) activities. In a comparison of two populations of each of the two potato cyst nematode (PCN) species, however, greater similarity in response to the individual hatching factors was observed among populations of different species produced under the same conditions than among different populations of the same PCN species. Smaller numbers of species-specific and species-selective hatching factor stimulants and hatching inhibitors than of hatching factors were resolved. In a study to determine whether the different hatching responses of the two species to the same root leachate were associated with different ratios of species-selective and species-specific hatching factors, G. rostochiensis pathotype Ro1 exhibited greater hatch than did G. pallida pathotype Pa2/3 in response to leachate from older plants (more than 38 days old), while G. pallida exhibited greater hatch in response to leachate from younger plants (less than 38 days old); the response of G. pallida pathotype Pal with respect to plant age was intermediate between the other two populations. Combined molecular exclusion-ion exchange chromatography of the root leachates from plants of different ages revealed an increase in the proportion of G. rostochiensis-specific and -selective hatching factors as the plants aged.     

Dynamics and management of the white potato cyst nematode Globodera pallida in commercial potato crops

Eight trials were conducted in commercial potato fields infested with the white potato cyst nematode (wPCN, Globodera pallida) and one in a field infested with the yellow PCN (yPCN, Globodera rostochiensis). Our aims were to produce data to validate and refine a computer‐based program (The Model) for the long‐term management of PCN, to determine nematicide effectiveness and to assess rates of PCN population decline between potato crops. Prior to planting, each farmer applied an overall nematicide treatment to his field, except for ten untreated plots that were widely spaced to encompass a range of PCN population densities. Each untreated plot was paired with a similar plot in the adjacent treated area and all plots were intensively sampled for PCN population densities at planting (P_i) and again at harvest (P_f) when tuber yields were determined. Four trials were re‐sampled 2–4 years later to determine PCN population decline rates. Regressions that form the basis of ‘The Model’ and described the relationship between P_i and tuber yield and PCN population density at harvest were fitted to the results from both the untreated and nematicide treated plots. These regressions also enabled us to estimate the yield potential at each site in the absence of PCN and showed that nematicide treatment generally did not increase yield potential and that both tuber yield and PCN multiplication decreased with increasing P_i. However, there were major differences between sites and cultivars. When untreated, the yield of cv. Maris Piper was hardly affected in a highly organic soil with P_i > 200 eggs g^?1 whereas the yield of partially resistant cv. Santé was decreased from a potential of c. 60 t ha^?1 to c. 20 t ha^?1 in a light silt with P_i = 20 egg g^?1 soil. Similarly, untreated wPCN multiplication rates at a low P_i ranged from 46‐fold to >100‐fold. Nematicide effectiveness was estimated from the regressions and, at several sites, yield was decreased despite nematicide treatment. Control of wPCN multiplication was even poorer. In only two of seven trials planted with susceptible cultivars was more than 50% control achieved – maximum populations in treated plots usually exceeded 250 eggs g^?1. Partially resistant Santé decreased the multiplication rate of wPCN in the two trials where it was planted. An alternative analysis using Genstat indicated that The Model tended to underestimate the maximum multiplication rate and overestimate the maximum population density. When four sites were re‐sampled 2–4 years after harvest the populations of wPCN had declined by between 15% and 33.5% per annum with a mean of 26% per annum. Modelling indicated that rotations longer than 8 years were required to control wPCN unless other effective control measures, such as growing a partially resistant cultivar, were used.     

Image Analysis of the Growth of Globodera pallida and Meloidogyne incognita on Transgenic Tomato Roots Expressing Cystatins

An approach based on image analysis that enables rapid collection and analysis of nematode size and shape during growth is reported. This technique has been applied to assess Meloidogyne incognita and Globodera pallida during their development over 35 and 42 days, respectively, on transgenic tomato roots expressing the wild-type rice cystatin Oc-I or an engineered variant, Oc-IAD86. Morphometric values were established that subdivided enlarged saccate females from other life stages. Analysis of this data subset indicates that the size of females and the frequency with which they parasitize roots expressing a cystatin are reduced. Results also demonstrate that cystatins can influence the growth of G. pallida prior to the adult stage. Similar image analysis procedures should be generally applicable to the study of host status or erivironmental factors that influence growth rates of plant-parasitic nematodes.     

Relationship between Root Growth of Potato,Root Diffusate Production,and Hatching of Globodera rostochiensis

Hatching response of Globodera rostochiensis in potato root diffusate (PRD) collected by soaking individual potato, Solanum tuberosum, root systems in water for 2 hours was used to assess the relationship between root growth and PRD production. Resistant potato cultivars Hudson and Rosa were used as test plants. Maximum hatch occurred in PRD collected 3 weeks after plant emergence (AE) in the greenhouse, and declined after this time. Hatch was positively correlated with increased root weight only during the first 3 weeks AE. Hudson PRD was consistently more active than Rosa PRD in stimulating hatch, except when adjusted for root weight. Although the results indicated that cells at the root tip produced a more active PRD than cells located elsewhere, PRD appeared to be produced along the entire root. Differences in time length of the vegetative growth phase, extent of root growth, and volume of roots, rather than the production of a more active PRD per se, may explain why Hudson is more effective than Rosa in reducing G. rostochiensis population densities in soil.     

A Comparison of Three Molecular Markers for the Identification of Populations of Globodera pallida

Potato cyst nematodes cost the potato industry substantial financial losses annually. Through the use of molecular markers, the distribution and infestation routes of these nematodes can be better elucidated, permitting the development of more effective preventative methods. Here we assess the ability of three molecular markers to resolve multiple representatives of five Globodera pallida populations as monophyletic groups. Molecular markers included a region of the rbp-1 gene (an effector), a non-coding nuclear DNA region (the ITS region), and a novel marker for G. pallida, a ∼3.4 kb non-coding mitochondrial DNA (mtDNA) region. Multiple phylogenetic analysis methods were performed on the three DNA regions separately, and on a data set of these three regions combined. The analyses of the combined data set were similar to that of the sole mtDNA marker; resolving more populations as monophyletic groups, relative to that of the ITS region and rbp-1 gene region. This suggests that individual markers may be inadequate for distinguishing populations of G. pallida. The use of this new non-coding mtDNA marker may provide further insights into the historical distribution of G. pallida, as well as enable the development of more sensitive diagnostic methods.     

Effects of Inoculum Density and Egg Age on Establishment of Globodera rostochiensis Populations

The establishment of Globodera rostochiensis Rol populations was examined under greenhouse conditions. The probability of G. rostochiensis population establishment was calculated from the number of plants that produced new cysts with viable eggs following inoculation with various numbers of eggs of different ages. Probability of population establishment was positively correlated with inoculum density but was not affected by the age of eggs used in these experiments. The probability of G. rostochiensis establishment ranged from 5% at densities of 2 eggs/pot to 100% at densities of 25 eggs/pot or greater. At densities of 3 eggs/pot and beyond, there was no correlation between inoculum density and the number of viable eggs/new cyst. Also, the number of plants that produced new cysts was a function of inoculum density and not age of eggs. Juveniles from eggs 1 year old or older were equally as infective as were those from eggs in newly developed cysts (4 months old).     

The effect of the potato cyst nematode Globodera pallida on in vitro root growth of potato genotypes, differing in tolerance

Roots of eighteen potato genotypes, differing in tolerance of G. pallida, were grown from tuberpieces on agar in Petri dishes. Juveniles of G. pallida were inoculated directly onto root tips. Root length was measured at various times after inoculation. Inoculation reduced root growth within one day. At later stages, genotypes differed strongly in growth of inoculated roots. Between four and seven days after inoculation, growth of inoculated roots was not significantly correlated with growth of untreated roots, and was only poorly correlated with tolerance assessed in the greenhouse or in the field. However, multiple regression analysis revealed that the tolerance of the tested genotypes was associated with both the rate at which they induced hatching and the growth of roots after inoculation. The combination of these two variables accounted for high percentages explained variance.     

Effects of Host Resistance on the Fecundity of Globodera rostochiensis

The fecundity of Globodera rostochiensis (R₁A) females that developed on resistant Rosa and susceptible Katahdin potato cultivars were compared. Cysts collected from each cultivar were bulked, separated into four sizes (> 500 μm, 355-500 μm, 250-355 μm, and < 250 μm), and crushed to determine fecundity as measured by viable egg content (VEC). Fewer and generally smaller cysts developed on Rosa than on Katahdin. Although cyst size significantly (P = 0.01) influenced VEC, cyst age (8 or 13 weeks) had no effect. Regardless of size, cysts produced on Rosa contained significantly fewer viable eggs than did cysts produced on Katahdin. The fecundity of progeny from cysts produced on Rosa was significantly reduced compared with that of progeny from cysts produced on Katahdin. After two generations on Katahdin, the VEC of cysts from a population originating from Rosa was significantly less than that of cysts from a population originating from Katahdin, indicating that in the presence of a pure population of G. rostochiensis R₁A, the females that develop on the resistant cultivar Rosa represent a diminished rather than a superior selected population.     

Hatching Behavior of Potato Cyst Nematodes from the Canary Islands

The present work investigated early hatching differences in naturally occuring field populations and newly reared populations of potato cyst nematodes from the Canary Islands. Hatching behavior of the two species appears to be distinct, with more juveniles hatched from G. pallida that hatch earlier and over a shorter time than G. rostochiensis. The hatching rate of 3-year-old PCN populations was more than double (mean 44.5% ñ 1) that shown by newly reared populations (mean 19.1% ñ 12.5), and those that could be classified as pathotype Pa 1 (Pa 1 and P 13) were found to hatch particularly poorly. Significant differences were also observed in the juveniles released in tap water between newly reared populations of both species, with mean hatch significantly higher for G. rostochiensis. The results are discussed in relation to the implication that these findings may have for competition between the two species of PCN in the field.     

Decline of Globodera rostochiensis as Influenced by Potato Root Diffusate Movement and Persistence in Soil

Decline of Globodera rostochiensis populations occurring naturally in soil and those added to potato hills and furrow centers in nylon bags was correlated with root weight of Hudson, Rosa, and Katahdin potatoes at two locations in New York. Cysts in bags were added to soil at planting and at 1, 3, 5, 7, and 9 weeks after emergence (AE). Fallow decline required only 2-4 weeks in soil and did not increase with time. Decline due to growing potatoes was greater in hills than in furrow centers, and resistant Hudson potatoes stimulated greater nematode hatch for longer times in both hills and furrows than did resistant Rosa and susceptible Katahdin. Potato root diffusate (PRD) was produced in highest concentration early in the season; decreased egg hatch with time was probably the result of declining PRD production and inactivation of PRD in soil. Decreasing potato row spacing from 92 cm to 46 and 23 cm between rows increased G. rostochiensis decline in furrow centers, with the majority of decline occurring within 1-3 weeks AE. Replanting potatoes after 1 week of trap crop growth failed to favor population reduction over a single full season crop.     

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.     

Root Growth of Susceptible and Resistant Potato Cultivars and Population Dynamics of Globodera rostochiensis in the Field

Globodera rostochiensis population densities and potato root growth were measured in field plots of one susceptible and two resistant potato cultivars. Root growth and nematode densities were estimated from soil samples taken at three depths between plants within the rows, three depths 22.5 cm from the rows, and at two depths midway between rows (furrows). Four weeks after plant emergence (AE), nematode densities in the rows had declined 68% in plots of the susceptible cultivar and up to 75% in plots of both resistant cultivars. Significant decline in nematode densities in the furrows 4 weeks AE occurred only in plots of the susceptible cultivar. Total decline in nematode density in fallow soil was 50%, whereas in plots of the resistant cultivars, decline was more than 70% in the rows and more than 50% in the furrows. Nematode densities increased in the rows of the susceptible cultivar but declined in the furrows. We conclude that G. rostochiensis decline or increase is correlated with host resistance and the amount of roots present at any particular site.     

The Future of Nematology: Integration of New and Improved Management Strategies

The potential for managing plant-parasitic nenlatodes by combining two or more control strategies in an integrated program is examined. Advantages of this approach include the use of partially effective strategies and protection of highly effective ones vulnerable from nematode adaptation or environmental risk. Strategies can be combined sequentially from season to season or applied simultaneously. Programs that have several strategies available but that are limited in the true integration of control components are used as examples of current management procedures and the potential for their improvement. These include potato cyst nematodes in northern Europe, soybean cyst nematode in North Carolina, and root-knot nematodes on vegetable and field crops in California. A simplified model of the impact of component strategies on the nematode damage function indicates the potential for combining control measures with different efficacies to give acceptable nematode population reduction and crop protection. The likelihood for additive, synergistic, or antagonistic effects from combining strategies is considered with respect to the biological target and component compatibility.     

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.     

Localization by restriction fragment length polymorphism mapping in potato of a major dominant gene conferring resistance to the potato cyst nematode Globodera rostochiensis

Summary A major dominant locus conferring resistance against several pathotypes of the root cyst nematode Globodera rostochiensis was mapped on the linkage map of potato using restriction fragment length polymorphism (RFLP) markers. The assessment of resistance versus susceptibility of the plants in the experimental population considered was based on an in vivo (pot) and an in vitro (petri dish) test. By linkage to nine RFLP markers the resistance locus Gro1 was assigned to the potato linkage group IX which is homologous to the tomato linkage group 7. Deviations from the additivity of recombination frequencies between Gro1 and its neighbouring markers in the pot test led to the detection of a few phenotypic misclassifications of small plants with poor root systems that limited the observation of cysts on susceptible roots. Pooled data from both tests provided better estimates of recombination frequencies in the linkage interval defined by the markers flanking the resistance locus.