Evidence for a calcium-binding site on the eggshell of Globodera rostochiensis with a role in hatching

Two inhibitors of hatching in Globodera rostochiensis, ruthenium red and lanthanum, have been shown to bind to the eggshell using the techniques of microdensitometry for ruthenium red and X-ray microanalysis for lanthanum. Neither inhibitor penetrated or adhered to unhatched or hatched viable juveniles. Scatchard analysis for binding of lanthanum and ruthenium red to eggshells gave dissociation constants (K) of K_La 32.5 ± 14.0 μM and K_Rured 33.5 ± 5.0 μM respectively. Both values are within the 95% fiducial limits of those shown to cause 50% inhibition of hatch in previous work. Pretreatment with sodium hypochlorite separated an outer part of the eggshell from an inner region which exclusively bound ruthenium red. It is the inner lipoprotein layer that is believed to include the membranes controlling the permeability of the tylenchid eggshell. The rate of binding of ruthenium red was similar for intact and isolated eggshells with 50% binding occurring after 6.11 ± 0.91 min and 4.95 ± 2.38 min but the latter gave a significantly higher maximum binding suggesting that rupture of the eggshells made available additional binding sites on their inner surface. The binding of ruthenium red to the eggshells was pH dependant over most of the range pH 2.8–8.5 with 50% binding, given with its standard deviation, occurring at pH 5.75 ± 0.85. Competitive binding of lanthanum influenced the binding of ruthenium red to the eggshells from which Scatchard analysis gave K_la of 176 ± 79 μM. Similarly, calcium influenced the binding but this caused a biphasic plot with high and low affinity binding sites of K“_ca of 0.423 ± 1.16 μm and K‘_ca of 1078 ± 462 μM. The existence of a high affinity site for calcium that also binds ruthenium red, suggests that the eggshell membrane includes a calcium binding glycoprotein as found in some other receptor mechanisms.     

The uptake of calcium prior to the hatching of the second-stage juvenile of Globodera rostochiensis

Energy dispersive X-ray microanalysis of 83 eggs of G. rostochiensis for calcium content showed that juveniles from eggs exposed to active hatching factor in potato root diffusate for 48 h contained significantly more calcium than those exposed for this time to the same diffusate inactivated by autoclaving, or to water. This occurred despite a slightly greater calcium concentration in the autoclaved than in the untreated diffusate; both contained more calcium than the water. Eggshells removed from stimulated eggs also contained more calcium than those from unstimulated eggs. The calcium content of juveniles and eggshells exposed to inactivated diffusate was similar to their corresponding values for eggs soaked in water. A similar analysis was made of freeze-dried samples of fluid taken from the matrix surrounding the eggs in cysts exposed to water or to active root diffusate. This showed a significantly greater calcium concentration in the stimulated cysts after 48 h exposure. The concentration subsequently decreased over the succeeding 72 h however, suggesting that the rate of calcium uptake by the stimulated eggs exceeded that of diffusion into the cyst. This uptake of calcium by eggs of G. rostochiensis exposed to a hatching stimulus seems pertinent to recent evidence that the active factor in potato root diffusate may initiate hatching through a calcium-mediated process.     

Resolution of natural hatching factors for golden potato cyst nematode, Globodera rostochiensis

The hatching responses of Globodera rostochiensis (golden potato cyst nematode) to purified and partially-purified preparations of natural (including the potato glycoalkaloids solanine and α-chaconine) and artificial hatching factors (HFs) were bimodal. At least 10 HFs, mostly anionic, were resolved from potato root leachate by a combination of gel permeation and ion-exchange chromatography. Whereas potato roots were the principal source of HFs, haulm leachate also contained such chemicals. Root leachate from aseptically-grown potato plants lacked several HFs which were present in conventionally-produced leachate.     

Changes in the adenine nucleotide content of cysts of Globodera rostochiensis associated with the hatching of juveniles

The content of AMP, ADP and ATP within single cysts of Globodera rostochiensis (60–80 fig dry weight) was determined as ATP to an accuracy of ± 10^-11 mol by a bioluminescent technique, after microenzymic methods had been used to phosphorylate AMP and ADP to ATP. Results for a total of 120 cysts showed that a change occurs in the adenylate energy charge of their contents after they have been exposed to potato root diffusate. Cysts in water had a mean adenylate energy charge of 0–63 (s.E. ± 0–04), but a randomly selected group of cysts after 24 h treatment with potato root diffusate had a significantly lower mean of 0–49±0–04. In a second, similar experiment, cysts in diffusate for only 8 h had an energy charge of 0*55 ± 0–03, but this value was not significantly less than the corresponding mean of o-6i ±0–03 of cysts that remained in water. The results indicate an effect on the metabolism of the unhatched juveniles that occurs too soon after the addition of diffusate to be directly due to any increase in locomotor activity. Apparently, the primary action of the hatching factor had affected many juveniles within 24 h of the addition of potato root diffusate to the cyst.     

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.     

Probability of Globodera rostochiensis Spread on Equipment and Potato Tubers

The probability of spreading cysts of Globodera rostochiensis on farming equipment and potato tubers was investigated in naturally infested field plots. The number of cysts recovered from soil that adhered to equipment differed significantly between different pieces of equipment. These differences were related to initial nematode density and, in most cases, to the volume of soil that adhered to the equipment. At an initial density of 0.04 egg/cm³ of soil, significantly more cysts were recovered from a potato digger than from a potato hiller, cultivator, or plow. At an initial density of 0.90 egg/cm³ of soil, significantly more cysts were recovered from the plow than from the other equipment. Although the population density was 22 times greater, only 10 times more cysts adhered 3 to equipment used in soil with a density of 0.90 egg/cm³ of soil than when used in soil infested at 0.04 egg/cm³. The number of potato tuber samples (4.5 kg) that contained cysts with viable eggs was positively correlated with the initial densities of G. rostochiensis in soil in which they were produced. The percentage of tuber samples with cysts containing viable eggs was 10-12% for tubers harvested from soil with densities less than 1 egg/cm³ and 30-76% for tubers harvested from soil with densities greater than 4 eggs/cm³ of soil.     

A dispenser for the rapid delivery of standard inocula of the cyst nematode Globodera rostochiensis

A simple machined dispenser is described, capable of providing rapid repetitive delivery of standard volumes of potato cyst nematode cysts. Standard volumes of cysts of mixed sizes, drawn from unsieved populations of G. rostochiensis, contain titres of infective eggs and larvae which are largely independent of cyst numbers; the method reduces inoculum variability compared with other cyst inoculation techniques. Standard volumes of cysts of mixed sizes may be used directly as the inoculum for plant resistance testing programmes without the operator error associated with hand-held scoops or the need for the subjective and time consuming selection of groups of individual cysts.     

Water relations during desiccation of cysts of the potato-cyst nematode Globodera rostochiensis

The loss during desiccation of osmotically active water (OAW), which freezes during cooling to -45 degrees C, and osmotically inactive water (OIW), which remains unfrozen, from the cysts of the potato cyst nematode, Globodera rostochiensis, was determined using differential scanning calorimetry. Exotherms and endotherms associated with non-egg compartments were not detected after 5 min desiccation at 50% relative humidity and 20 degrees C. The pattern of water loss from the cysts indicates that water is lost from compartments outside the eggs first, that nearly all the non-egg water is OAW and that the OIW content of the cyst is contained within the eggs. Water is lost from the eggs only after the OAW content outside the eggs falls below that within the eggs. Both OAW and OIW are lost from the eggs during desiccation but the eggs retain a small amount of OIW. Other animals which survive some desiccation but which are not anhydrobiotic will tolerate the loss of OAW but not the loss of their OIW. Anhydrobiotic animals can survive the loss of both their OAW and a substantial proportion of their OIW.     

Effects of the nematicide oxamyl on life cycle stages of Globodera rostochiensis

Hatched second stage juveniles of Globodera rostochiensis were found to be more sensitive than either unhatched juveniles or adult males to low concentrations of oxamyl (EC₅₀ value for activity 0.5 μg oxamyl ml^-1). Development of juveniles in roots was significantly inhibited by 2.0 μg oxamyl ml^-1 probably due to impairment of normal feeding activity. The behavioural events necessary for orientation of juveniles towards roots for invasion and orientation of males towards females for fertilisation were found to be impaired by 0.5 and 1.0 μg oxamyl ml^-1 respectively. A comparison with previous work on Meloidogyne incognita showed G. rostochiensis juveniles to be more sensitive to oxamyl.     

Possible involvement of calcium ions in the hatching of Schistosoma mansoni eggs in water

The possibility of involvement of calcium ions in the hatching of Schistosoma mansoni eggs in water is described. The hatching of S. mansoni eggs under low osmotic pressure was partially inhibited by EGTA (5 mM), lanthanum chloride (1-5 mM), and ruthenium red (0.1-1 mM). The reagents used in these experiments were not toxic to the eggs however, because miracidia hatched normally when the reagents were removed.     

Evidence for symplastic involvement in the radial movement of calcium in onion roots

The pathway of Ca(2+) movement from the soil solution into the stele of the root is not known with certainty despite a considerable body of literature on the subject. Does this ion cross an intact, mature exodermis and endodermis? If so, is its movement through these layers primarily apoplastic or symplastic? These questions were addressed using onion (Allium cepa) adventitious roots lacking laterals. Radioactive Ca(2+) applied to the root tip was not transported to the remainder of the plant, indicating that this ion cannot be supplied to the shoot through this region where the exodermis and endodermis are immature. A more mature zone, in which the endodermal Casparian band was present, delivered 2.67 nmol of Ca(2+) mm(-1) treated root length d(-1) to the transpiration stream, demonstrating that the ion had moved through an intact endodermis. Farther from the root tip, a third zone in which Casparian bands were present in the exodermis as well as the endodermis delivered 0.87 nmol Ca(2+) mm(-1) root length d(-1) to the transpiration stream, proving that the ion had moved through an unbroken exodermis. Compartmental elution analyses indicated that Ca(2+) had not diffused through the Casparian bands of the exodermis, and inhibitor studies using La(3+) and vanadate (VO(4)(3-)) pointed to a major involvement of the symplast in the radial transport of Ca(2+) through the endodermis. It was concluded that in onion roots, the radial movement of Ca(2+) through the exodermis and endodermis is primarily symplastic.     

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.     

A calcium-binding sialoglycoprotein associated with an apparent eggshell membrane of Globodera rostochiensis

Energy dispersive X-ray microanalysis has been used to study the binding of two inhibitors of hatching in Globodera rostochiensis, ruthenium red and lanthanum, to a calcium binding site on the eggshell. Eggs were treated with either buffered ruthenium red or buffer only, and the levels of ruthenium and calcium that could be detected from the surface of eggshells were measured before and after repeated etching in a plasma oven. Counts for ruthenium increased during etching to a maximum after 8–12 min, just before the eggshell was eroded from the juvenile. The calcium levels on eggshells binding ruthenium red were suppressed relative to those from buffer. Similar experiments with lanthanum showed a significant increase in this inhibitor after 10 min etching and an accompanying suppression of calcium levels. The nature of the binding site was probed by measuring ruthenium red binding after pretreatment with selected enzymes. A non-specific protease, pronase, reduced the subsequent binding of this inhibitor but the site was resistant to trypsin in the presence of calcium. Neuraminidase also had an effect suggesting that the protein is a sialoglycoprotein. Phospholipase A2 but not Phospholipase C influenced the subsequent binding of ruthenium red. Phospholipase A₂ caused a loss of hatching ability and the degradation of a phospholipid from the eggshell. Ruthenium red binding was also reduced by treatment of eggshells with detergents or sonication. Apparently a calcium-binding sialoglycoprotein forms part of the integral proteins of an eggshell membrane located on the inner surface of the eggshell.     

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.     

Functional analysis of pathogenicity proteins of the potato cyst nematode Globodera rostochiensis using RNAi

RNA interference (RNAi) has been used widely as a tool for examining gene function and a method that allows its use with plant-parasitic nematodes recently has been described. Here, we use a modified method to analyze the function of secreted beta-1,4, endoglucanases of the potato cyst nematode Globodera rostochiensis, the first in vivo functional analysis of a pathogenicity protein of a plant-parasitic nematode. Knockout of the beta-1,4, endoglucanases reduced the ability of the nematodes to invade roots. We also use RNAi to show that gr-ams-1, a secreted protein of the main sense organs (the amphids), is essential for host location.     

A Lignan-type Stress Compound in Potato Infected with Nematode (Globodera rostochiensis)

Rustmicin, a new antibiotic active against the wheat stem rust fungus, was isolated from a cultured broth of Micromonospora chalcea 980-MC₁. Rustmicin showed strong inhibitory activity against the wheat stem rust fungus both in vitro and in pot tests in a greenhouse with MIC being 1 and 0.8/ig/ml, respectively. Its structure was determined by NMR spectroscopy to be a new 14-membered macrolide antibiotic lacking sugar substituents.     

Reproduction of Globodera rostochiensis on Transformed Roots of Solanum tuberosum cv. Desiree

Transformed roots of the susceptible potato Solanum tuberosum L. cv. Desiree were inoculated with second-stage juveniles (J2) of Globodera rostochiensis pathotype Rol. Adult males emerged after 3-4 weeks and matings with females occurred. After 8 weeks gentle pressure on the eggs of maturing females released the J2 which were viable. Because this technique enables the production of vigorously growing roots with numerous laterals, it may be suitable for obtaining a high yield of sterile potato cyst nematodes.