The adenosine triphosphate content of cysts of Globodera pallida and G. rostochiensis as a possible quantitative estimate of field populations

A rapid method is described for estimating the number of juveniles of Globodera pallida and G. rostochiensis in cysts collected from field soils. The technique uses a photometer to measure the adenosine triphosphate (ATP) content of the cysts by bioluminescence. Results for ten to twelve soil samples of 100 g taken from each of four fields showed that the number of juveniles that emerged when the samples were placed in potato root diffusate was significantly related to ATP content of a second series of cyst samples from these fields. Cysts from one field gave a higher ATP content per hatched juvenile than was recorded for the remaining three fields. There are several, untested, explanations of this discrepancy, but some evidence suggests that the hatching test and not the ATP method may have given an unreliable estimate of the viable egg content of cysts from this field. Further work may result in an enhanced precision and increased rapidity in estimating field populations of cyst nematodes by this technique.     

A technique to assess the efficacy of non-volatile nematicides against the potato cyst nematode Globodera rostochiensis

The assessment of larval emergence from cysts, second stage larval behaviour in soil, invasion of roots, development within roots and the final population provides a technique which can be used for evaluating the efficacy of any non-volatile organophosphate or carbamate nematicide.
Oxamyl delayed emergence from cysts, disorientated second stage larvae in soil, delayed and reduced invasion of roots, reduced the number of eggs in the final population and markedly reduced the final population of cysts.     

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.     

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.     

The ATP method for rapid assessment of the efficacy of a single application of a fumigant against Globodera spp. in field soils

Population estimates were carried out for Globodera pallida by both visual egg counts and measurement of the adenosine triphosphate (ATP) content of cysts from 26 fields in Northern England. The two methods agreed for seven non-fumigated fields but covariance analysis suggested that the visual assessment underestimated mortality for all 19 fields shortly after a single treatment with a fumigant nematicide. Both methods indicated that three fields were safe for cropping for potatoes but the ATP method suggested that cropping was possible on a further eight fields considered unsuitable by visual assessments. The results indicated that a high peat content contributed to the low efficacy of the fumigants in three fields. Comparison with previous results for the ATP method using cysts from 40 Dutch fields suggested that fumigation in early autumn was required for a satisfactory single treatment. The results show that the ATP method can assess the efficacy of a single application of a fumigant for economic control in time to allow retreatment when necessary or a change in the decision to plant potatoes the following season.     

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.     

Control of potato cyst nematode (Globodera rostochiensis) by nematicides and a resistant potato cultivar at four sites in Scotland

The control of potato cyst nematode (Globodera rostochiensis) by the oxime-carbamates aldicarb and oxamyl was tested in four fields in Scotland. Dazomet was tested in three of these fields and carbofuran in one. In untreated plots in the three most heavily infested fields Maris Piper (resistant) yielded better than Pentland Crown (non-resistant). All nematicides increased the yields of both potato cultivars but had a greater effect on the yield of Pentland Crown. Dazomet increased yields of tubers most. Heavy nematode infestation reduced yield of tubers more in a sandy soil than in two sandy loams. In a field with few potato cyst nematodes nematicides did not significantly affect tuber yields. Although the nematicides greatly increased yields, they were not completely effective in controlling potato cyst nematodes. In treated plots in the lightly infested field, there were more nematode eggs following a crop of Pentland Crown than before. In contrast, Maris Piper markedly decreased post-cropping populations and except at one site, where dazomet further decreased nematode numbers, combining nematicides with the resistant cultivar failed to decrease nematode numbers further. Nematicides decreased the numbers of larvae invading potato roots by up to 95%, oxamyl at 5–6 kg/ha being consistently the best treatment.     

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.     

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.     

Differences in the tolerance of potato cultivars to potato cyst nematodes (Globodera rostochiensis and G. pallida) in field trials with and without nematicides

In field trials Cara, Brio, Maris Piper and Pentland Javelin were consistently more tolerant of damage by Globodera rostochiensis yielding more than Corsair, Pentland Dell, Maris Anchor and Maris Peer, in untreated, heavily infested soil and giving the smallest increases to nematicide treatment. No yield or growth differences were found between plants in untreated and nematicide treated plots at a nematode-free site. The most tolerant cultivars all had a gene (H₁) for resistance to G. rostochiensis derived from Solanum tuberosum ssp. andigena and in soil infested with G. pallida the tolerance of at least one resistant cultivar (Maris Piper) appeared to be lessened. However, some resistant cultivars were comparatively intolerant, even to G. rostochiensis. Early cultivars were generally less tolerant than late maturing cultivars but there were exceptions. Amongst cultivars with resistance derived from Solanum vernei the early cultivar Guardian was more tolerant than the main crop cultivar Corsair. The effect on the yield of several cultivars of a range of densities of G. rostochiensis, produced either by applying different rates of a nematicide or by cropping in the previous year, was examined at two sites. The results indicated that the slope of the regression for yield in relation to nematode density was less for tolerant than intolerant cultivars. At sites infested with G. rostochiensis Maris Piper was found to be consistently more tolerant than Pentland Crown.     

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.     

An efficient cDNA-AFLP-based strategy for the identification of putative pathogenicity factors from the potato cyst nematode Globodera rostochiensis

A new strategy has been designed to identify putative pathogenicity factors from the dorsal or subventral esophageal glands of the potato cyst nematode Globodera rostochiensis. Three independent criteria were used for selection. First, genes of interest should predominantly be expressed in infective second-stage juveniles, and not, or to a far lesser extent, in younger developmental stages. For this, gene expression profiles from five different developmental stages were generated with cDNA-AFLP (amplified fragment length polymorphism). Secondly, the mRNA corresponding to such a putative pathogenicity factor should predominantly be present in the esophageal glands of pre-parasitic juveniles. This was checked by in situ hybridization. As a third criterion, these proteinaceous factors should be preceded by a signal peptide for secretion. Expression profiles of more than 4,000 genes were generated and three up-regulated, dorsal gland-specific proteins preceded by signal peptide for secretion were identified. No dorsal gland genes have been cloned before from plant-parasitic nematodes. The partial sequence of these three factors, A4, A18, and A41, showed no significant homology to any known gene. Their presence in the dorsal glands of infective juveniles suggests that these proteins could be involved in feeding cell initiation, and not in migration in the plant root or in protection against plant defense responses. Finally, the applicability of this new strategy in other plant-microbe interactions is discussed.