THE HATCHING RESPONSES OF SOME ROOT EELWORMS OF THE GENUS HETERODERA

An account is given of investigations on the hatching responses, under laboratory conditions, of nine species or varieties of Heterodera , namely, the beet, cabbage, clover, Galeopsis , carrot, hop, potato, oat and pea eelworms. In the first seven of these, marked differences occurred in hatching responses from cysts incubated in root leachings from various plants, these differences being virtually diagnostic for the eelworm species concerned. In general, good responses to leachings from host plants occurred, whereas there was little or no response in non-host leachings; but cases of response to non-hosts and of failure to respond to hosts were encountered. Promising results were obtained in the analysis, by bio-assay, of mixtures of some of these eelworm species. No appreciable response occurred from cysts of oat and pea eelworms incubated in host leachings, but there was some evidence that such a response did occur from pea eelworm cysts under field conditions. Curves for rate of hatching of seven species and for hatching in diluted leachings in five species were found to be of the form described by Fenwick for potato eelworm. Results are given of some experiments on the effect of age of plant on potency of the root leachings, and on the loss of potency during storage.     

The effects of desiccation and root diffusates from potato and mustard on eggs of the white potato cyst nematode Globodera pallida

Following exposures to potato root diftusate of between 6 and 12 h, desiccation killed a proportion of juveniles in eggs of G. pallida and affected the hatching behaviour of survivors. In hatching tests of 9 wk duration, more juveniles hatched in the final wk from cysts, which were soahed and dried alternately for 9 wk than from cysts soaked in tap water for the final 2 wk only. Mustard root diffusate prevented eggs previously stimulated by potato root diffusate from hatching, but it did not alleviate the effects of desiccation.     

A Comparison of the Hatching of Juveniles from Cysts of Heterodera schachtii and H. trifolii

The effects of root diffusates of selected plants within the families Chenopodiaceae and Cruciferae and the hatching agent zinc chloride were tested for their effects on hatching and emergence of juveniles from cysts of Heterodera schachtii and a race of H. trifolii parasitic on Chenopodaceae and Cruciferae in The Netherlands. Although all diffusates strongly stimulated hatching of juveniles of H. schachtii, their effects on H. trifolii were less evident.     

THE BIO-ASSAY OF POTATO-ROOT DIFFUSATE

The effect of diluting potato-root diffusate is investigated. It is found that for any given sample the hatching ability is inversely proportional to the logarithm of its dilution and there is, moreover, for each sample a 'threshold' value of dilution beyond which it is inactive. The slope of dilution curves for different diffusates are all parallel. Use is made of these relationships in estimating the strength of any given sample.     

THE EMERGENCE OF LARVAE FROM FREE EGGS OF THE POTATO-ROOT EELWORM HETERODERA ROSTOCHIENSIS (WOLL.)

A technique for conducting hatching experiments on eggs freed from cysts is described. The form of the hatching response was found to be similar to that of eggs contained within cysts, but the response of the free eggs to the hatching stimulus was slightly more rapid. Investigations into factors affecting free egg hatching showed that it was necessary to presoak cysts before extracting the eggs from them for hatching. Eggs taken from dry cysts or from cysts that had been opened or cracked before presoaking did not respond to diffusate. When free eggs and whole cysts were exposed to the same graded series of dilutions of diffusate, the L.A. values (i.e. log concentrations of hatching factor) derived from plotting the hatching curves were in very close agreement.     

THE STIMULATORY PROPERTIES OF SOME ORGANIC SUBSTANCES ON CYSTS OF THE BEET EELWORM, HETERODERA SCHACHTII SCHMIDT

Techniques for determining the influence of various substances on the rate of larval emergence from cysts of the beet eelworm are described. There is no significant difference between the rates of larval emergence in glutamic acid, galactinol, inositol and water; the rate of larval emergence in beet diffusate, on the other hand, was significantly higher. Studies of larval emergence in carbohydrates suggest that the cyst population used in the experiments was heterogeneous, consisting of two types of cyst which have different reactions to stimulation at different concentrations. The significance of root exudates in relation to beet eelworm is discussed.     

Effect of Liquid Nutrient Culture,Vacuum Distillation and Dialysis on Hatching Activity of Sugar Beet Root Diffusate for Heterodera schachtii

Roots of sugar beets grown in liquid culture excrete substances that stimulate egg hatch and emergence of larvae from cysts of Heterodera schachtii. Their hatching effect is comparable to that of sugar beet root diffusate leached from soil-grown sugar beet plants. Consequently, liquid culture provides a way of obtaining H. schachtii hatch-stimulant free of contaminants from soil. Root diffusate, concentrated 50-fold or dried by vacuum distillation, retained hatching activity. The active principle of diffusate is dialyzable with a diffusion rate between those of inorganic salts and compounds with molecular weights greater than 15,000.     

THE EMERGENCE OF LARVAE FROM CYSTS OF THE BEET EELWORM, HETERODERA SCHACHTII SCHMIDT, IN AQUEOUS SOLUTIONS OF ORGANIC AND INORGANIC SUBSTANCES

The rate of larval emergence from cysts of the beet eelworm in a variety of aqueous solutions containing organic and inorganic substances is significantly higher than the emergence rate in water. It is suggested that differences between larval emergence rates in monoamino-monocarboxylic amino-acids may be related to the lipid solubility of these substances and their ability to penetrate the egg membranes. The larval emergence rate in fructose, glucose, sucrose and maltose was significantly higher than that in water, but in raffinose, arabinose and xylose the rate of emergence was no higher than in water. A high rate of larval emergence occurred in sodium chloride, potassium chloride and mercuric chloride, but not in magnesium chloride or calcium chloride. Experiments with several other organic solutions are described. There is an optimum concentration for larval emergence in beet diffusate. The osmotic pressure of the diffusate when maximum emergence occurred was 0·48 atm. Measurements of shrinkage of unhatched larvae in various concentrations of urea, sodium chloride and sucrose showed that decreasing rates of emergence at higher concentrations may be due to changes in the unhatched larvae brought about: by an osmotic effect. High concentrations of beet diffusate may have a similar effect.     

The Effects of Hot Water Treatments on Survival of Heterodera schachtii

Cysts of Heterodera schachtii were treated in a water bath at constant temperatures ranging from 45 - 62.5 C for 1 sec to 28 hr. Treated and untreated cysts were incubated 8 weeks in sugarbeet root diffusate at 24 C to measure emergence of surviving larvae. Within the temperature range of 49 - 54 C, the minimum lethal temperature was proportional to the log time of treatment. No larvae emerged from cysts exposed 10 sec at 60 C. Although treatment of cysts for 8 hr at 45 C significantly reduced emergence, increasing the treatment period to 28 hr did not completely suppress emergence.     

Plant-Induced Hatching of Eggs of the Soybean Cyst Nematode Heterodera glycines

Root diffusate from soybean plants caused greater hatching of Heterodera glycines eggs during vegetative growth of the host, but the activity declined with plant senescence. Chelation of the root diffusate with ethylenediamine tetraacetic acid (EDTA) significantly increased hatching activity for H. glycines eggs. Diffusate from leafless plants caused little hatching, whereas treatment of intact plants with the growth regulators gibberellin and kinetin had no effect on the hatching activity of root diffusate. Treating H. glycines eggs with zinc chloride and root diffusate reduced egg hatching from zinc chloride alone. Levels of zinc in the root diffusate were insufficient to induce egg hatch, based on analysis by atomic absorption spectrophotometry. The enzymatic activity of leucine aminopeptidase in H. glycines eggs was not altered by treatment with chelated or nonchelated root diffusate.     

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.     

Partial purification of hatching activity for Globodera rostochiensis from potato root diffusate

The potential of several chromatographic methods for isolating hatching factors for potato cyst nematodes from potato root diffusate was investigated using a bioassay based on emergence of juveniles from cysts. Gel filtration provided an overall estimate of molecular weight of 437 Da for the hatching activity and ion exchange chromatography indicated that at least 60% of the recovered activity was anionic in nature. Material less polar than the hatching activity could be removed by passing potato root diffusate through a reversed-phase Sep-Pak C₁₈ cartridge and the elutant showed 83.3 ± 4.4% (mean of 32 cysts) of the initial hatching activity. High performance liquid chromatography (HPLC) with a reversed phase, C₁₈ column and gradient elution (0–80% CH₃OH in water) confirmed that much of the hatching activity was polar and that it was not retained by this method of separation. A weak anion exchange resin achieved slight retention of much of the hatching activity and an ion pairing reagent lowered the polarity sufficiently to allow some retention in subsequent reversed phase HPLC on a C_IS column. Both ion exchange and ion pairing HPLC suggested that hatching activity was not chromatographed as a single compound and indicated that fractions able to influence the nucleolus of the nucleus within the dorsal pharyngeal gland cell did not always show hatching activity.     

Host Status of Different Potato (Solanum tuberosum) Varieties and Hatching in Root Diffusates of Globodera ellingtonae

Globodera ellingtonae was detected in Oregon in 2008. In order to make decisions regarding the regulation of this nematode, knowledge of its biology is required. We determined the host status of a diversity of potato (Solanum tuberosum) varieties in soil-based experiments and identified hatching stimulants in in vitro hatching assays. ‘Russet Burbank,’ ‘Desiree,’ ‘Modac,’ ‘Norland,’ ‘Umatilla,’ and ‘Yukon Gold’ were good hosts (RF > 14) for G. ellingtonae. Potato varieties ‘Maris Piper,’ ‘Atlantic,’ and ‘Satina,’ all which contain the Ro1 gene that confers resistance to G. rostochiensis, were not hosts for G. ellingtonae. In in vitro hatching assays, G. ellingtonae hatched readily in the presence of diffusates from potato (PRD) and tomato (Solanum lycopersicum; TRD). Egg hatch occurred in an average of between 87% and 90% of exposed cysts, with an average of between 144 and 164 juveniles emerging per cyst, from PRD- and TRD-treated cysts, respectively. This nematode hatched rapidly in the presence of PRD and TRD, with at least 66% of total hatch occurring by day 3 of exposure. There was no dose-response of egg hatch to concentrations of PRD or TRD ranging from 1:5 to 1:100 diffusate to water. When G. ellingtonae was exposed to root diffusates from 21 different plants, hatch occurred in 0% to 70% of exposed cysts, with an average of between 0 to 27 juveniles emerging per cyst. When root diffusate-exposed cysts were subsequently transferred to PRD to test viability, root diffusates from arugula (Eruca sativa), sudangrass (Sorghum bicolor subsp. drummondii), and common vetch (Vicia sativa) continued to inhibit egg hatch compared with the other root diffusates or water in which hatch occurred readily (60 to 182 juveniles emerging per cyst). Previously known hatching stimulants of G. rostochiensis and G. pallida, sodium metavanadate, sodium orthovanadate, and sodium thiocyanate, stimulated some egg hatch. Although, Globodera ellingtonae hatched readily in PRD and TRD and reproduced on potato, the pathogenicity of this nematode on potato remains to be determined.     

The influence of crucifers and mustard oil on the emergence of larvae of the potato-root eelworm, Heterodera rostochtensis Wollenweber

Hatching experiments were carried out with cysts of the potato-root eelworm. The effect of solutions of potato-root excretion mixed with root excretions of six types of crucifer seedlings was studied. With cress, white mustard, and black mustard, the emergence of larvae in the mixed excretions was very much less than that from control cysts in potato-root excretion alone. On subsequent return to potato-root excretion alone, larval emergence was found to be unimpaired in the case of white and black mustards; in the case of cress, however, the total emergence differed significantly from control. Similar effects, of both types, were obtained with certain dilutions of ally isothiocyanate, the mustard oil of black mustard seed, in potato-root excretion.     

Changes in the second stage juveniles of Globodeva rostochiensis prior to hatching in response to potato root diffusate

cAMP levels in eggs of G. rostochiensis and the diameter of the nucleolus of the nucleus within the dorsal pharyngeal gland cell of the second stage juvenile have been measured as indicators of the response of the nematode to the hatching stimulus in potato root diffusate. The nucleolus increased from 2.72 ± 0.103 μm for unhatched individuals to 3.28 ± 0.14 μm and 3.88 ± 0.15 μm after soaking eggs in potato root diffusate for 3 and 4 days respectively. Juveniles expressed from unstimulated eggs in water to potato root diffusate for 4–5 days showed a similar increase in size of the nucleolus to 3.94 ±0.15 μm but those released into water for this time had smaller nucleoli of 3.20 ± 0.98 μm. The change in diameter of the nucleolus is probably related to the accumulation of secretions in this gland cell before hatching. Preliminary results with dibutyryl analogues of CAMP and cGMP showed some inhibition of hatch in 10% potato root diffusate. Theophylline had a similar effect but NaF was dissimilar in that the effect of this inhibitor was not reversible. A standard radioimmunoassay showed that significant changes in cAMP levels occurred in the unhatched juveniles within cysts after treatment with potato root diffusate for 2.5 or 8 h compared with values for cysts kept in water. This change occurs before other known responses of the juveniles to potato root diffusate and it defines the period of interest for future work on the initial action of hatching factor.     

Effects of Selected Nematicides on Hatching of Heterodera schachtii

Aldicarb, carbofuran, fensulfothion, and phenamiphos were tested in concentrations of 1-100 μg/ml for their effects on hatching of Heterodera schachtii. Exposure of cysts to 1 μg aldicarb or carbofuran/ml stimulated hatch whereas phenamiphos and, to a lesser degree, fensulfothion inhibited hatch. Addition of aldicarb to sugarbeet root diffusate or 4 mM zinc chloride suppressed activities of these hatching agents. Transfer of cysts previously treated with aldicarb or carbofuran to zinc chloride or water rapidly initiated hatch which finally exceeded the hatch from cysts not treated with the nematicides.     

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.     

THE POTATO-EELWORM HATCHING FACTOR

The hatching factor from potato root diffusate has many of the properties of eclepic acid from tomato root and Solanum nigrum (Russell, Todd & Waring, 1949), as well as the same biological activity. No comparison of the specific activity of the two products is possible because of differences in the assay techniques, but the best preparations of both showed maximal hatching activity at about 0.1 mg./1. It is found that the potato preparations, which are freely soluble in water, are simultaneously inactivated and rendered insoluble by brief exposure to caustic alkali. The product of alkali treatment has been crystallized.     

Changes in the oxygen consumption of cysts of Globodera rostochiensis associated with the hatching of juveniles

The oxygen consumption of single cysts (90–110 /tg dry wt) was measured with an oxygen electrode microrespirometer. The mean oxygen consumption of nine cysts after 7 days in tap-water, was 0–48 + 0–05 mm³ 0₂ mg dry wt^-1 h^-1. After transfer to potato root diffusate for 1 day the mean oxygen consumption of the same cysts showed a significant increase to 159±7% of the rate recorded before they were removed from water. After 3 and 7 days in diffusate the corresponding means were 131±9% and 127±12% respectively. Cysts that remained in water throughout the experiments did not show any significant change in their oxygen consumption from the rate recorded after 7 days. The initial increase in oxygen uptake after the addition of diffusate was shown not to be due to the presence of microorganisms. Comparison of hatching data with the changes in oxygen consumption of similar cysts after 24 h in diffusate suggests that the increased oxygen uptake cannot be attributed solely to locomotor activity of the juveniles during the hatching process. The increased rate of respiration may precede other known changes that follow after the juveniles within a cyst are stimulated to hatch.