Detection of hatching inhibitors and hatching factor stimulants for golden potato cyst nematode, Globodera rostochiensis, in potato root leachate

In a comparison of four potato varieties, in-soil hatch of the golden potato cyst nematode (Globodera rostochiensis) was positively correlated to in vitro hatch in response to potato root leachate (PRL). The in-soil hatch of cysts of G. rostochiensis to two of the four varieties was significantly less than that of the control (cysts in gravel without potato plants) in the first 2 wk after plant emergence, suggesting the production of hatching inhibitors (HIs) by young potato plants. The hatching factor: hatching inhibitor ratio of PRL was positively associated with the net hatching activity of the PRL. Four zones of HI activity were resolved following gel permeation chromatography of PRL on Sephadex G-10. Hatch-inactive chemicals, which stimulated the activity of hatching factors (HFs) in PRL (hatching factor stimulants, HSs), were also isolated from PRL, hatch levels induced by individual HFs responding differently to the same HS preparation. The complex interactions between individual HFs and other hatching chemicals in PRL was illustrated when addition of the hatch-active potato glycoalkaloid α-solanine caused both inhibition and stimulation of PRL-induced hatch, depending on the α-solanine concentration.     

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.     

Response of Globodera rostochiensis to exogenously applied hatching factors in soil

The exogenous application of hatching factors (HFs) to soil in the field gave an approximately 50% reduction in the population size of Globodera rostochiensis. This control was found to be due to a combination of suicide hatch and increased in‐egg mortality caused presumably by incomplete hatch stimulation. The rates of hatch and emergence of J2s from cysts of G. rostochiensis were found to be dependent on hatching factor concentration in vitro and in the field. Encysted eggs of G. rostochiensis in the field during the first year of rotation after the previous year's potato crop were found to be more sensitive to HF concentration than those in the second year of rotation.     

Hatching of Globodera pallida juveniles by diffusate of potato genotypes, differing in tolerance to G. pallida

Hatching induced by root diffusate, obtained from various potato genotypes, and by standard potato root diffusate, was determined in vitro. The used potato genotypes differed considerably in tolerance to Globodera pallida. A three parameter logistic model was used to describe the numbers of hatched juveniles in relation to time of exposure to root diffusate. Clear differences in hatching characteristics between genotypes were found. Some tolerant genotypes induced hatching of G. pallida juveniles relatively slowly, compared to intolerant genotypes. Other tolerant genotypes, however, induced hatching as fast as intolerant genotypes, and no significant correlation between hatching parameters and tolerance was found.     

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.     

The suitability of some natural and artificial substrates as oviposition sites for the insidious flower bug, Orius insidiosus

A variety of natural and artificial substrates were tested for their suitability as oviposition sites for the predatory anthocorid bug Orius insidiosus. The bugs oviposited into models of beans constructed from Parafilm, but none of the eggs laid into these models hatched. In choice tests, beans stems were preferred to both bean pods and potato sprouts. Hatching rates at 100% r.h. were highest (59%) for bean pods and significantly lower for both potato sprouts (39%) and bean stems (29%). At 60% r.h., hatching rates for bean pods (39%), bean stems (34%) and potato sprouts (33%) were similar. At 30% r.h. hatching rates for all three substrates were less than 15%. The results suggest that bean stems can be used successfully as an alternative to bean pods for the mass rearing of these biological control agents.     

Plant–parasite coevolution: A weak signature of local adaptation between Peruvian Globodera pallida populations and wild potatoes

Plant–parasite coevolution has generated much interest and studies to understand and manage diseases in agriculture. Such a reciprocal evolutionary process could lead to a pattern of local adaptation between plants and parasites. Based on the phylogeography of each partner, the present study tested the hypothesis of local adaptation between the potato cyst nematode Globodera pallida and wild potatoes in Peru. The measured fitness trait was the hatching of cysts which is induced by host root exudates. Using a cross‐hatching assay between 13 populations of G. pallida and root exudates from 12 wild potatoes, our results did not show a strong pattern of local adaptation of the parasite but the sympatric combinations induced better hatching of cysts than allopatric combinations, and there was a negative relationship between the hatching percentage and the geographical distance between nematode populations and wild potatoes. Moreover, a strong effect of the geographic origin of root exudates was found, with root exudates from south of Peru inducing better hatching than root exudates from north of Peru. These results could be useful to develop new biocontrol products or potato cultivars to limit damages caused by G. pallida.     

The response of eggs of the white potato cyst nematode Globodera pallida to diffusate from potato and mustard roots

Brief exposure of eggs of Globodera pallida to potato root diffusate not only initiated hatching but also caused the majority of unhatched juveniles to respond more rapidly to subsequent treatment with diffusate. Eggs previously exposed to diffusate had a peak hatch after 1 or 2 days compared with 4 days for untreated eggs. Mustard root diffusate prevented hatch, and further stimulation with potato root diffusate was necessary to re-initiate it. Eggs previously treated with potato root diffusate for 24 h were much more sensitive to drought than untreated eggs. These results are discussed in relation to the theory that potato root diffusate alters the permeability of the eggshell as an initial step in the hatching process.     

Screening of non-tuber bearing Solanaceae for resistance to and induction of juvenile hatch of potato cyst nematodes and their potential for trap cropping

Ninety accessions of non‐tuber bearing Solanaceae were screened for (i) resistance to and (ii) stimulatory effect on juvenile hatch of potato cyst nematodes, and (iii) their growth under temperate climatic conditions. All plant species belonging to the genus Solanum tested induced hatching but this effect was most pronounced for plant species of the Solanum nigrum complex. Hatching of juveniles was hardly or not stimulated by other plant genera of the Solanaceae. Solanum sisymbriifolium combined a high hatching effect with complete resistance to both Globodera rosiochiensis and G pallida. Two S. nigrum varieties showed full resistance to G rostochiensis and a high level of resistance to G pallida. Moreover, S. sisymbriifolium and the two varieties of S. nigrum performed very well under Dutch field conditions and, therefore, they are suggested as candidate trap crops for the control of potato cyst nematodes.     

Behavior of newly hatched potato tuber moth larvae,Phthorimaea operculella Zell. (Lepidoptera: Gelechiidae), in relation to their host plants

A study was made of neonate larvae of Phthorimaea operculella.Host finding from soil-laid eggs and dispersal from hosts and nonhosts were first examined. Of first-instar larvae hatching from soil-laid eggs, 80% found the potato plant while roughly 50% found each of the other three plants (datura, tobacco, and tomato). Dispersal from potato, datura, and tobacco was very low, while on tomato it was higher and a high mortality was observed in the 24 h period. Dispersal from nonhost plants was high. Behavior on leaves of hosts and non-hosts is described and some host-plant specific behaviors are identified. Effects on behavior of some of the physical and chemical factors are described. Methylene dichloride extracts of potato leaf wax reduced locomotion rates and the number of turns during locomotion. Also methylene dichloride extract and, to a lesser extent, methanol extract caused biting behavior.     

Effects of Host Variety,Photoperiod and Chemical Treatments on Hatching of Globodera rostochiensis

Golden nematode cysts were collected after they had completed one generation on an early, a mid-season, or a late-maturing potato variety (''Superior,'' ''Katahdin,'' and ''Sebago,'' respectively). The plants were grown in 8.5- or 16-h photoperiods and treated with gibberellic acid (GA) or xylocaine or both. When treated subsequently with potato root-diffusate few (13.3%) juveniles hatched from cysts collected from Sehago, more (22.3%) from cysts collected from Katahdin, and most (36.0%) from cysts collected from Superior. Hatching was greater from cysts collected from plants treated with GA than from untreated plants. Short photoperiods decreased hatching from cysts collected from Katahdin but increased hatching from cysts collected from Superior and Sebago. Gibberellic acid had no detectable effect on hatching, but EDTA had some.     

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.     

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.     

Growth duration and root length density of Solanum sisymbriifolium (Lam.) as determinants of hatching of Globodera pallida (Stone)

Solanum sisymbriifolium is a trap crop for potato cyst nematodes (PCN). In this study, we quantified the effect of different periods of growth of S. sisymbriifolium and root length density on hatching of Globodera pallida, using potato and fallow treatments as references. One‐year‐old and 2‐year‐old G. pallida cysts were used in greenhouse experiments carried out in containers over 2 years. Two methods were used in study hatching. In the first method, 7.5‐cm‐diameter soil cores were removed and backfilled with infested soil. In the second method, cysts were buried in nylon bags. The soil cores infested with cysts used in the first method had very poor root colonisation as compared to surrounding bulk soil. Therefore, the effect of S. sisymbriifolium was strongly underestimated by the soil core method. Hatching of PCN juveniles from cysts, measured with the nylon bag method, increased with the duration of growth of S. sisymbriifolium from 47% after 6 weeks of crop growth up to 75% after 21 weeks of crop growth. Reductions per depth layer were also correlated with root length density and varied between 42.6% at 0.26 cm cm^?3 and 85.3% at 5.8 cm cm^?3. Based on a single exponential decay function, a general method is presented to estimate for any PCN management measure the average reduction in the number of years needed to ensure that the PCN population falls below a given density. Calculated reductions in the number of years ranged from 2.3 years for 59% hatching (equivalent to 90 days of S. sisymbriifolium) to 4.4 years for 75% of hatching (equivalent to 150 days of S. sisymbriifolium). These reductions were independent of initial and final population density. Our results corroborate the hatch‐inducing effect of S. sisymbriifolium, underline the importance of growth duration and root length density as determinants of the reduction in PCN population that can be achieved and draw attention to the pitfall in methodology that can arise in the study of hatch stimulation.     

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.     

Comparison of the life cycle of potato cyst nematode (<Emphasis Type="Italic">Globodera rostochiensis</Emphasis>) pathotype Ro1 on selected potato cultivars

The life cycle of Globodera rostochiensis pathotype Ro1 was studied under experimental conditions on selected potato cultivars (Korela, Albina, Vivaldi, Veronika, Vera, Monalisa, Victoria, Maranca, Désirée) in Slovakia during two growing seasons. Two peaks of second stage juveniles (J2) were found in the soil; the first peak three and four weeks after planting in the first and second year, respectively. The last J2 were found on 23 September. The number of J2 found in the second peak was much higher. First J2 associated with roots were observed 18 days, on middle early and seritonous cultivars 34 days after planting, but fourth stage juveniles (J4) were observed 40 days after planting in both cultivar groups. First adult males were found in soil 43 and 46 days after planting, respectively, and the last males two weeks later. White females filled with eggs were observed on roots 61 days after planting. The cycle from hatching of J2 in the soil to the hatching of J2 from brown cysts required 68 days in the first year and 60 days in the second year.     

Inorganic ions and the hatching of Heterodera spp

Of various inorganic ions tested for their ability to stimulate hatching of eggs of the cyst nematodes of cereals (Heterodera avenae Woll), carrot (H. carotae Jones), cabbage (H. cruciferae Franklin), soybean (H. glycines Ichinohe), pea (H. goettingiana Liebs.), potato (H. rostochiensis Woll.), beet (H. schachtii Schm.), tobacco (H. tabacum Lownsbery & Lownsbery) and clover (H. trifolii Goffart), some were active. Zn²⁺ hatched many eggs of seven species and some of H. goettingiana, but inhibited hatch of H. avenae to below that in water. Zinc salts are the first recorded very active hatching stimulants for H. glycines in vitro. Many other metal ions stimulated hatching of H. schachtii eggs to varying extents; these also hatched some but not all of the other species. Vanadate ions were particularly effective for H. rostochiensis, more so than Zn²⁺. No ion increased the hatch of H. avenae to above that in water. The most active ions were not those most abundant in soil. The behaviour of different ions with different species did not suggest any obvious affinities between species, but the differences between the hatching of H. tabacum and H. rostochiensis add weight to the view that, despite morphological similarities and overlapping host ranges, they are distinct species rather than pathotypes of a single species. Ions and other hatching agents may be absorbed by materials within the egg or larva and alter the structure and function of these materials. The lack of correlation between the hatching of H. schachtii by ions and the known stability sequences of various biological metal-binding systems suggests that there may be several sites of action that differ in their response.     

Field performance of Solanum sisymbriifolium, a trap crop for potato cyst nematodes. II. Root characteristics

Hatching of potato cyst nematodes is induced by root exudates of Solanaceae, such as Solanum sisymbriifolium, and is therefore related to root length distribution of this crop. A mathematical model was derived to relate the hatching potential to root length density (RLD). A series of field experiments was carried out to study actual root length distribution of S. sisymbriifolium in relation to shoot properties and to provide input into the model. Using a modified Poisson distribution formula for the three‐dimensional distribution of roots in a volume of soil, the relation between the zone of influence of hatching agents and the RLD could be derived. On this basis, the minimal RLD was estimated, which is needed to expose 75%, 90% or 95% of cysts to root exudates, as a function of the length of the zone of influence of hatching agents on cysts. The logarithm of the total root length showed a linear relation with the logarithms of above‐ground biomass and with leaf area index. Root diameter distribution was the same for all crops examined and independent of soil depth. Fine roots (<0.4 mm in diameter) constituted around 50% of total root length. Using a zone of influence of 1.00, 0.75 and 0.50 cm around the centre of each root, a minimal RLD for sufficient soil exploration (75%) was estimated. Depth at which that minimal RLD was exceeded was linearly related to total root length (km m^?2) and to above‐ground crop biomass, enabling estimations being made of the potential hatching efficacy as related to measurable properties of S. sisymbriifolium crops. The proposed approach to derive potential hatching effects from crop properties needs further validation; particularly, the distance of influence of root exudates is a critical factor.     

Changes in a Heterodera rostochiensis population through the growing season

Changes in the numbers of all stages of Heterodera rostochiensis in plants and soil were studied in a potato crop growing on infected land. Hatching and invasion occurred early in the season, when plants were very young and temperatures were below those considered necessary for hatching and invasion in Long Island, U.S.A. More larvae hatched from cysts nearer than away from potato roots. Males were abundant during June and July, when fertilization probably occurs. Many new cysts appeared during late June and early July. In May/June, there were many larvae in the roots of young plants but later there were few; a slight increase during August probably indicated a small second generation. The larvae for this second invasion may have come from the most recently formed cysts, which may have been stimulated to hatch either by root exudates or by substances from other soil organisms.     

Environmental Fate of Soil Applied Neonicotinoid Insecticides in an Irrigated Potato Agroecosystem

Since 1995, neonicotinoid insecticides have been a critical component of arthropod management in potato, Solanum tuberosum L. Recent detections of neonicotinoids in groundwater have generated questions about the sources of these contaminants and the relative contribution from commodities in U.S. agriculture. Delivery of neonicotinoids to crops typically occurs as a seed or in-furrow treatment to manage early season insect herbivores. Applied in this way, these insecticides become systemically mobile in the plant and provide control of key pest species. An outcome of this project links these soil insecticide application strategies in crop plants with neonicotinoid contamination of water leaching from the application zone. In 2011 and 2012, our objectives were to document the temporal patterns of neonicotinoid leachate below the planting furrow following common insecticide delivery methods in potato. Leaching loss of thiamethoxam from potato was measured using pan lysimeters from three at-plant treatments and one foliar application treatment. Insecticide concentration in leachate was assessed for six consecutive months using liquid chromatography-tandem mass spectrometry. Findings from this study suggest leaching of neonicotinoids from potato may be greater following crop harvest in comparison to other times during the growing season. Furthermore, this study documented recycling of neonicotinoid insecticides from contaminated groundwater back onto the crop via high capacity irrigation wells. These results document interactions between cultivated potato, different neonicotinoid delivery methods, and the potential for subsurface water contamination via leaching.