PÓT TESTS OF NEMATICIDES AGAINST POTATO-ROOT EELWORM

Details are given of nematicidal tests, directed against the potato-root eelworm, in 81. glazed pots of infested soil. The soil was sampled a few weeks after injection to secure data on the kill achieved, by counting the eelworm larvae hatching in root diffusate. A potato tuber was then planted in each pot and yielded information on phytotoxic or soil-amendment effects. A second soil sample at the end of the season showed the degree to which the eelworm population had recovered after treatment. With only duplicate pots, and single batches of 100 cysts from each pot, clear-cut results were achieved with D–D mixture and ethylene dibromide. In the present pilot test, the data were handled by ordinary analysis of variance methods, by which the lowest significant percentage kill was 73%, but the methods could be adapted for purposes of probit analysis.     

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.     

Observations on the biology of the potato-root eelworm, Heterodera schachtii Schmidt

Results are presented of experiments on the effect of low temperature and of soil type upon the potato-root eelworm, Heterodera schachtii.
In some experiments exposure of cysts, to low temperatures had a definite lethal effect, but in others the cysts were unharmed. The reason for this variation was not apparent. Exposure of the larvae of die nematode to even a relatively slight degree of frost resulted in the death of the larvae, provided mat die surrounding medium was completely frozen. In this respect the larvae appear to differ markedly from those of the beet strain of H. schachtii. That the larvae had actually been killed was shown by the fact that they produced no cysts on the roots of potato plants and also by their almost immediate absorption of iodine.
It was confirmed that H. schachtii cysts produced on the roots of potato plants grown in a heavy medium are smaller in size and fewer in number than those formed on the roots of plants grown in a light medium. The heavy medium used was pure clay, the light one pure sand, while equal numbers of eelworm larvae served in each case as inoculum. The difference in cyst size was significant, and fewer larvae emerged from the cysts formed in clay than from those produced in sand.     

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 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.     

Control of the potato-root eelworm, Heterodera rostochiensis Wollenweber, by allyl isothiocyanate, the mustard oil of Brassica nigra L

Previous work showed that, in the presence of root excretioci of certain crucifen, larvae of the potato-root eelworm do not emerge from the cysts when stimulated by potato-root excretions; a similar lack of response is also shown in the presence of dilute solutions of allyl isothiocyanate, the mustard oil of black mustard. The present paper describes a small-scale field trial in which this mustard oil is applied with peat to the drills at the time of planting. An increase in yield of about 100% resulted from a dressing of the oil equivalent to o.1 cwt/acre.     

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 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.     

The resistance of potato varieties to Heterodera schachtii Schmidt, the potato-root eelworm

The potato varieties Epicure and Doon Star permitted the production of fewer and smaller cysts of Heterodera schachtii on their roots than the varieties Golden Wonder and Majestic, when all were grown under standard conditions. Under the stimulus of potato-root excretion the number of larvae which emerged from Epicure and Doon Star cysts was less than that from Majestic or Golden Wonder cysts of the same volume. Accepting Lapage's (1938) criteria of resistance to nematode infestation in animals and applying them to plants, it is concluded that this deleterious effect of the variety on the eelworm is probably of the nature of a degree of resistance to eelworm attack specific to each variety and is physiological and not anatomical in action. Of the varieties tested, Epicure is judged the most resistant, closely followed by Doon Star. Majestic is fairly susceptible but not so much so as Golden Wonder on which the eelworm forms large cysts that are extremely productive of larvae.     

Influence of earthworms on larval emergence in the potato-root eelworm, Heterodera rostochiensis Wollenweber

Hatching experiments were carried out with potato-root eelworm cysts ( a ) from plant pots containing different numbers of earthworms, and ( b ) from worm casts. Cysts from these sources differed from controls in the following ways: (1) more larvae emerged, per cyst; (2) the larvae emerged sooner; (3) more cysts produced larvae; (4) the number of emerging larvae, per cyst, increased with the number of earthworms.
Examination of the contents of cysts isolated from worm casts showed that, compared with controls, 145% more eggs hatched; it also indicated some of the limitations of the 'per-cyst' basis of expressing results.     

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.     

MOVEMENT OF EELWORMS

The optimum crumb sizes for movement of potato-root eelworm larvae in a sandy loam, a heavy clay and a peat soil were 150–250 and 250–400 μ. Mobility was very similar in clay and sandy loam, in both of which there was an optimum suction for movement. In the peat, however, mobility increased with suction and no optimum suction was established. Larvae may be able to move in peat at high suctions because friction between the larvae and the peat crumbs is less than between clay or sand crumbs. Larvae moved to the wet end of a moisture gradient in sand, the number increasing with the steepness of the gradient. The rate of spread of larvae in sand 150–250 μ diameter varied between 2 and 3 cm. a day, depending on suction. As pore size increases, any upward movement in a moisture gradient is opposed by falling under gravity. Larvae do not respond to a moisture gradient or fall under gravity in sand where the width of the pore approximates to the diameter of the larva. The presence of host roots also counteracted the response to a moisture gradient; the degree of orientation to the roots increased with the time the roots were in the sand. Direct observation on larvae, newly emerged from cysts, in the presence of host plant roots, suggests that larvae orientate themselves at a distance from the root and do not reach the root by random movement. Many of the movements of eelworms are explicable by considering the relationship between pore size, eelworm diameter and water distribution, and a diagram relates movement and various soil factors.     

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 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.     

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.     

Evidence for the involvement of calcium in the hatching of Globodera rostochiensis

Low concentrations of ruthenium red and lanthanum chloride inhibited hatching of juveniles from eggs in cysts of Globodera rostochiensis in potato root diffusate. Pro-bit analysis for 31 cysts at seven concentrations of ruthenium red showed that 50% inhibition with 95% fiducial limits occured at 47 ± 23 μm; a similar value of 59 ± 14 μm was obtained using eggs removed from cysts. Results for 10 to 20 cysts at six concentrations of lanthanum chloride suggested a somewhat higher value for 50% inhibition of 110 ± 83 μM. In contrast hatching of eggs in cysts of Heterodera schachtii in water was unaffected by 5 ITIM lanthanum chloride and 625 μM ruthenium red, concentrations which cause over 90% inhibition of hatch in G. rostochiensis.
Two calcium ionophores synergised hatching of a 1971 population of G. rostochiensis in dilute diffusate. Optimal concentrations of 2 μM for A23187 and 10 μM for BrX537A increased the hatch from 17 ± 3–6 juveniles/cyst to 114 ± 44 juveniles/cyst and 138 ± 40 juveniles/cyst respectively. Ionophores in the absence of diffusate hatched very few eggs of this population but caused a greater hatch in a second (1975) population which gave a high hatch in water of 43 ± 10 juveniles/cyst. This was increased by A23187 to 181 ± 41 juveniles/cyst. The results with both the inhibitors and the ionophores suggest that hatching in G. rostochiensis might be a calcium-mediated process.     

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.     

In-Vitro and In-Vivo Effects of Aldicarb on Survival and Development of Heterodera schachtii

Aqueous solutions of 5-500 μg/ml aldicarb inhibited hatching of Heterodera schachtii. Addition of hatching agents, zinc chloride, or sugarbeet root diffusate, to the aldicarb solutions did not decrease the inhibition of hatching. When cysts were removed from the aldicarb solufions and then treated for 4 wk in sugarbeet root diffusate, larvae hatched and emerged. Treatments of newly hatched larvae of H. schachtii with 5-100 μg/ml aldicarb depressed later development of larvae on sugarbeet (Beta vulgaris). Similar treatments with aldicarb sulfoxide had less effect on larval development, and aldicarb sulfone had no effect. Numbers of treated larvae that survived and developed were inversely proportional to concentration (0.1-5.0 μg/ml) and duration (0-14 days) of aldicarb treatments. Development of H. schachtii on sugarbeet grown in aldicarb-treated soil was inversely proportional to the concentration of aldicarb in the tested range of 0.75 - 3.0 μg aldicarb/g of soil. Transfer of nematode-infected plants to soil with aldicarb retarded nematode development, whereas transfer of plants first grownin treated soil to nematode-infested soil only slightly suppressed nematode development. Development of H. schachtii was inhibited in slices of storage roots of table beet (B. vulgaris), sugarbeet and turnip, (Brassica rapa), that had grown in soil treated with aldicarb.     

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.     

The hatching factor of the potato-root eelworm

1. The hatching factor of the potato-root eelworm was concentrated from potato-root leachings by adsorption on charcoal. The crude material, extracted from charcoal with acetone, was purified by partition between ethyl acetate and m-potassium dihydrogen phosphate, and ethyl acetate and a solution of potassium metabisulphite (disulphite). Neutral material was removed by extraction from phosphate buffer at pH7.8. The yield of purified material, after chromatography on silica, DEAE-cellulose and Perlon columns, was 18mug./l. of potato-root leachings, and 0.04% by weight of the acetone-soluble material extracted from charcoal; 20% of the activity of the original potato-root leachings was recovered. 2. The purified material was a colourless gum. On paper chromatograms it gave a single weakly fluorescing spot, which reacted with 2,4-dinitrophenylhydrazine and aniline-xylose reagents. It was confirmed that the hatching factor is an acid, and it is inactivated at values above pH9. Other properties were a ready oxidation by permanganate and reaction with certain carbonyl reagents. Colour tests for alpha- and beta-unsaturated gamma-lactones were negative. The hatching factor showed no light-absorption maxima at wavelengths above 210mmu; absorption bands in the infrared region (670-4000cm.(-1)) are given. Our purified material, although a gum, gave a similar hatch to a ;crystalline' product at 640 times its concentration.