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).     

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.     

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.     

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.     

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.     

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.     

Effect of Initial Nematode Density on Managing Globodera rostochiensis with Resistant Cultivars and Nonhosts

Cropping systems in which resistant potato cultivars were grown at different frequencies in rotation with susceptible cultivars and a nonhost (oats) were evaluated at four initial nematode population densities (Pi) for their ability to maintain Globodera rostochiensis at a target level of <0.2 egg/cm³ of soil. At a Pi of 0.1 to 1 egg/cm³ of soil, cropping systems with 2 successive years of a resistant cultivar every 3 years of potato production reduced and maintained G. rostochiensis at <0.2 egg/cm³ of soil. At a Pi of 1 to 4 eggs/cm³ of soil, 2 successive years of a resistant cultivar followed by 1 year of oats for every 4 years of production were necessary to reduce and maintain G. rostochiensis populations at <0.2 egg/cm³ of soil. At a Pi greater than 4 eggs/cm³ of soil, 2 successive years of a resistant cultivar plus 1 year of oats reduced G. rostochiensis densities to <0.2 egg/cm³ of soil, but the population increased above that density after cropping 1 year to a susceptible cultivar. The numbers of cysts and eggs per cyst in the final population (Pf) of G. rostochiensis were influenced by initial density and the frequency of growing a susceptible cultivar in a cropping system. The lowest number of cysts and eggs per cyst in the final G. rostochiensis population occurred with a cropping system consisting of 2 successive years of a resistant cultivar followed by oats with a susceptible cultivar grown the fourth year of production.     

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.     

Effects of Host Resistance on Second-stage Juveniles and Adult Males of Globodera rostochiensis

Potato cultivars Katahdin (susceptible) and Rosa (resistant) were exposed to infective second-stage juveniles (J2) of Globodera rostochiensis for varying periods of time, after which root systems were washed and plants were placed in Hoagland''s solution to assess J2 egression and male emergence. After transfer to liquid culture, many J2 egressed from both cultivars, but significantly more egressed from the resistant Rosa than from Katahdin. Juveniles that egressed from Rosa invaded a second host, resistant or susceptible, in significantly fewer numbers than did juveniles that egressed from Katahdin. Also, significantly fewer males developed in and emerged from resistant host roots, relative to susceptible ones. These effects of resistance may be an important component of the tolerance to invasion by G. rostochiensis exhibited by Rosa.     

Lipid Composition of Cyst Stages of Globodera rostochiensis

Lipid compositional analysis was conducted on the white, yellow, and brown cyst stages of Globodera rostochiensis (golden cyst nematode). Triacylglycerols were the largest lipid fraction in all stages examined, ranging from 55-75% of total lipid. Ethanolamine phosphoglycerides and choline phosphoglycerides were present in high amounts in all cyst fractions, with a total phospholipid content of 20%, 14.7%, and 12.8% in the white, yellow, and brown cyst stages, respectively. Sterols, steryl esters, sphingomyelin, and cardiolipin were found in minor amounts in all three cyst stages and showed greater changes than other classes of lipids relative to cyst stage. The fatty acid compositions of the three cyst stages were similar. Eicosenoic acid (20:1) and arachidonic acid (20:4) were found in higher concentrations than other fatty acids in all cyst preparations; vaccenic acid (18:1) occurred at the third highest concentration. More than 78% of total fatty acids were unsaturated at all cyst stages, and more than 60% were of C20 or longer chain length. The lipid profile of all three cyst stages is consistent with invertebrate adaptation to low-temperature environments.     

Management of Globodera rostochiensis as Influenced by Nematode Population Densities and Soil Type

The effects of aldicarb, oxamyl, 1,3-D, and plastic mulch (solarization) on soil population densities of the golden nematode (GN) Globodera rostochiensis was assessed in field and microplot experiments with different soil types. Oxamyl was evaluated in both soil and foliar treatments, whereas aldicarb, 1,3-D, and solarization were applied only to soil. Soil applications of aldicarb and oxamyl resulted in reduced nematode populations after GN-susceptible potatoes in plots with initial population densities (Pi) of > 20 and 7.5 eggs/cm³ soil, respectively, but nematode populations increased in treated soil when Pi were less than 20 and 7.5 eggs/cm³soil. In clay loam field plots with Pi of 19-76 eggs/cm³ soil, nematode densities increased even with repeated foliar applications of oxamyl, whereas nematode populations at Pi greater than 76 eggs/cm³ soil were reduced by foliar oxamyl. Treatment with 1,3-D or solarization, singly or in combination, reduced GN soil population densities regardless of soil type or Pi. Temperatures lethal to GN were achieved 5 cm deep under clear plastic but not 10 or 15 cm deep.     

Major Sperm Protein Genes from Globodera rostochiensis

Three genes in the major sperm protein (MSP) gene family from the potato cyst nematode Globodera rostochiensis were cloned and sequenced. In contrast to the absence of introns in Caenorhabditis elegans MSP genes, these genes in G. rostochiensis contained a 57 nucleotide intron, with normal exon-intron boundaries, in the same relative location as the intron in Onchocerca volvulus. The MSP genes of G. rostochiensis had putative CAAT, TATA, and polyadenylation signals. The predicted G. rostochiensis MSP gene product is 126 amino acids long, one residue shorter than the products in the other species. The comparison of MSP amino acid sequences from four diverse nematode species suggests that O. volvulus, Ascaris suum, and C. elegans may be more closely related to each other than they are to G. rostochiensis.     

The Influence of Potato Cultivar on Lipid Content and Fecundity of Bolivian and British Populations of Globodera rostochiensis

The influence of host cultivar on the lipid levels provided by a female to her progeny was investigated with Oil Red O stain and a quantitative image analyzer. A population of Globodera rostochiensis was multiplied at Toralapa Field Station in Bolivia on 25 different potato cultivars grown in that country. The mean neutral lipid content of newly formed second-stage juveniles varied significantly with cultivar over a 200% range. The corresponding range was only 18% and 28% for the same Bolivian and a UK population of G. rostochiensis, respectively, when both completed reproduction concurrently on 10 pot-grown European cultivars in the United Kingdom. Egg numbers per female varied with host for Bolivian cultivars that lack known partial resistance to Globodera spp. There was a 15-fold range between the most and least fecund nematode-host combinations (Kosi and Gendarme). The Bolivian G. rostochiensis population showed only a 2-fold range in mean eggs per cyst when grown on European cultivars in the UK. The fatty acid profiles of lipids from Bolivian G. rostochiensis cysts reared on Bolivian potato cultivars were dominated by C₂₀ (37-64%) and C₁₈ (28-46%) fatty acids and ranged from C₁₄ to C₂₂. The three major fatty acids detected were C_20:4:, C_20:1, and C_18:1. Few differences between cultivars were observed. For a UK population of G. rostochiensis reared on ssp. tuberosum, higher relative percentages of C₁₈ and monounsaturated fatty acids and lower relative percentages of C₂₀ and polyunsaturated fatty acids were found.     

Globodera rostochiensis Population Density Decline in Relation to Spatial Distribution around Resistant Potato Plants

Golden nematode, Globodera rostochiensis (GN) population decline under resistant potatoes was related to cyst distance from plants 23 cm apart in rows 92 cm apart. GN decline, determined by sampling an infested field planted to the resistant cultivar ''Yankee Chipper'', was 81.8% in cores 11.5 cm from plants within rows. Decline was 27.4% at 23 cm from plants between rows and 36.6% at 46 cm. Population decline of juveniles in cysts added to soil in bags was 90.3% for cysts 11.5 cm from plants within rows planted to the resistant cultivar ''Rosa''. Decline between rows was 83.5, 76.9, and 60.4% at 11.5, 23.0, and 46.0 cm from plants, respectively. Maximum decline within for rows 30.5, 46.0, 61.0, and 92.0 cm apart, respectively. Decline under fallow was 43.5%, signif- which peaked 7 weeks AE. There was no effect of soil depth on population decline at any sampling position. Decreasing row spacing resulted in 79.9, 74.2, 73.4, and 66.1% GN population decline for rows 30.5, 46.0, 61.0, and 92.0 cm apart, respectively. Decline under fallow was 43.5%, significantly less than under potatoes. Potato root weight between rows was negatively correlated with row spacing and positively correlated with GN population decline.     

Control of Globodera rostochiensis in Relation to Method of Applying Nematodes

Soaking potato tuber pieces for 15 min in 8,000 μg/ml of oxamyl just before planting reduced the number of Globodera rostochiensis cysts that developed on potato roots, but this treatment was phytotoxic. Five foliar applications of 1.12 kg a.i./ha of oxamyl or carbofuran at 10-day intervals beginning when 90% of the plants had emerged suppressed increase in G. rostochiensis densities. Similar foliar applications of phenamiphos were ineffective in controlling G. rostochiensis. Soil applications (in the row at planting) of aldicarb, carbofuran, phenamiphos, ethoprop, and oxamyl at 5.6 kg a.i./ha reduced the numbers of white females that developed on potato roots, but only those treatments involving aldicarb and oxamyl suppressed G. rostochiensis population increase. Combined soil and foliar treatments did not provide any advantage over soil treatment alone, as soil applications of 5.6 kg a.i./ha alone were equal to, or better than, combined soil (3.4 kg a.i./ha) and foliar (2.2 kg a.i./ha) applications in controlling G. rostochiensis.     

Life Cycle of the Golden Cyst Nematode,Globodera rostochiensis,in Quebec,Canada

In 2006, the golden cyst nematode, Globodera rostochiensis, was discovered in the province of Quebec, Canada. We report here the life cycle of G. rostochiensis under the climatic conditions of southwestern Quebec. Only one full generation was completed per year under these latitudes. On susceptible potato cv. Snowden, G. rostochiensis needed a minimum of 579 growing degree units (GDU) (base 5.9°C) to complete its life cycle and the first mature cysts were observed 42 to 63 days after planting (DAP). In soil, second-stage juveniles (J2) were first observed 14 to 21 DAP, whereas both white females on roots and males in soil appeared synchronously after 35 to 42 days. The duration of the life cycle was affected by temperature but not by soil type. A second wave of hatching systematically occurred later in the season and a second generation of males was observed during the 2011 growth season. No complete second cycle was observed before plant senescence. Climate change and later maturing cultivars/crops could allow the development of a full second generation in the future.     

Molecular Characterization of Aldolase from Heterodera glycines and Globodera rostochiensis

Fructose-bisphosphate aldolase (EC 4.1.2.13) is a key enzyme in glycolysis. We have characterized full-length coding sequences for aldolase genes from the cyst nematodes Heterodera glycines and Globodera rostochiensis, the first for any plant-parasitic nematode. Nucleotide homology is high (83% identity), and the respective sequences encode 40 kDa proteins with 89% amino acid identity. Genomic sequences contain six introns located at identical positions in both genes. Intron 4 in the H. glycines gene is >500 bp. Partial genomic sequences determined for seven other cyst nematode species reveal that the large fourth intron is characteristic of Heterodera but not Globodera aldolase genes. Total aldolase-like specific activity in homogenates from H. glycines was 2-fold lower than in either Caenorhabditis elegans or Panagrellus redivivus (P = 0.001). Activity in H. glycines samples was higher in juvenile stages than in adults (P = 0.003). Heterodera glycines aldolase has Km = 41 µM and is inhibited by treatment with carboxypeptidase A or sodium borohydride.     

The novel GrCEP12 peptide from the plant-parasitic nematode Globodera rostochiensis suppresses flg22-mediated PTI

The potato cyst nematode Globodera rostochiensis is a biotrophic pathogen that secretes effector proteins into host root cells to promote successful plant parasitism. In addition to the role in generating within root tissue the feeding cells essential for nematode development,¹ nematode secreted effectors are becoming recognized as suppressors of plant immunity.^2-4 Recently we reported that the effector ubiquitin carboxyl extension protein (GrUBCEP12) from G. rostochiensis is processed into free ubiquitin and a 12-amino acid GrCEP12 peptide in planta. Transgenic potato lines overexpressing the derived GrCEP12 peptide showed increased susceptibility to G. rostochiensis and to an unrelated bacterial pathogen Streptomyces scabies, suggesting that GrCEP12 has a role in suppressing host basal defense or possibly pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) during the parasitic interaction.³ To determine if GrCEP12 functions as a PTI suppressor we evaluated whether GrCEP12 suppresses flg22-induced PTI responses in Nicotiana benthamiana. Interestingly, we found that transient expression of GrCEP12 in N. benthamiana leaves suppressed reactive oxygen species (ROS) production and the induction of two PTI marker genes triggered by the bacterial PAMP flg22, providing direct evidence that GrCEP12 indeed has an activity in PTI suppression.     

Fungal Parasites of the Potato Cyst Nematode Globodera rostochiensis: Isolation and Reinfection

Fungal parasitism of eggs of the potato cyst nematode Globodera rostochiensis was < 1, 3, and 17% at three sites in Sweden. The fungi isolated most frequently from infected eggs were a Septocylindrium-like fungus ( 19 %), Exophiala spp. (17 %), and Cylindrocarpon spp. (13 %). Verticillium suchtasporium was isolated from infected eggs at a low frequency (4%). In laboratory experiments V. suchlasporium infected 93% of the eggs within cysts after 10 days on dilute corn meal agar. This species showed chitinase and protease activity. Infection of eggs by the Septocylindrium-like fungus was moderate, whereas Cylindrocarpon destructans and Cladosporium cladosporoides did not infect eggs. No chitinase activity was found in these fungi, but protease activity was recorded in all. Growth of the fungi in cysts did not influence the number of physiologically disordered eggs.     

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.