Multiplication of English isolates of potato pale cyst- nematode, Globodera pallida, on potatoes of differing nematode resistance and the identity of potato cyst- nematode populations

In pots, 25 populations of potato pale cyst-nematode, Globodera pallida Stone, differed significantly in their ability to multiply on potato clones P55/7 and ZC83/ 6, both fully resistant to G. pallida pathotype Pal. Neither clone was fully resistant to any of the populations. For 21 populations common to this and an earlier experiment, increase on the more resistant potatoes (P55/7, ZC83/6, cvs Sante, Paladin and Glenna) was correlated with their increase on less resistant potatoes (cvs Morag, Fingal and Valiant). Variation in virulence on these partially resistant potatoes was not matched by differences in the electrophoretic patterns of the nematodes' proteins. The identification of populations of G. rostochiensis (Woll.) Skarbilovich used in these experiments was confirmed by electrophoresis. All populations of G. pallida Stone, appeared to contain very small numbers of G. rostochiensis after subculture on susceptible potatoes (cv. Désirée).     

The effect of Solanum tuberosum×S. vernei hybrids on hatching of the potato cyst nematode Globodera pallida

The hatching activity of partially resistant Solanum tuberosum×S. vernei clones and susceptible cultivars was investigated in a series of tests with G. pallida conducted in pots in the glasshouse. Differences in hatching were apparent, with the partially resistant clones generally stimulating a lower total hatch than the S. tuberosum cultivars. It is concluded that reduced hatching activity can make a small contribution to overall resistance and perhaps also to the tolerance of a clone to G. pallida.     

Origin, distribution and 3D-modeling of Gr-EXPB1, an expansin from the potato cyst nematode Globodera rostochiensis

Southern analysis showed that Gr-EXPB1, a functional expansin from the potato cyst nematode Globodera rostochiensis, is member of a multigene family, and EST data suggest expansins to be present in other plant parasitic nematodes as well. Homology modeling predicted that Gr-EXPB1 domain 1 (D1) has a flat beta-barrel structure with surface-exposed aromatic rings, whereas the 3D structure of Gr-EXPB1-D2 was remarkably similar to plant expansins. Gr-EXPB1 shows highest sequence similarity to two extracellular proteins from saprophytic soil-inhabiting Actinobacteria, and includes a bacterial type II carbohydrate-binding module. These results support the hypothesis that a number of pathogenicity factors of cyst nematodes is of procaryotic origin and were acquired by horizontal gene transfer.     

The effect of partially resistant potato clones and aldicarb on the multiplication of the potato cyst nematode Globodera pallida in field trials

Potato cultivars with different degrees of resistance to Globodera pallida (PCN) were trialled at three infested sites in 1983, 1984 and 1985. These trials were primarily intended to assess the effects of PCN damage on the yield of tubers and consequently the sites chosen had high initial population densities of G. pallida. The population density of G. pallida was determined from samples taken both pre-planting and post-harvest and results showed that there were significant differences between the clones in the nematode multiplication rates. Although actual rates of multiplication varied between sites the relative differences between clones were maintained across sites. Under the susceptible control cultivars the populations generally increased even in the presence of nematicide. The partially resistant clones gave best control, as measured by nematode multiplication, at the sites with the highest initial population densities but gave good control at all sites when treated with nematicide irrespective of initial population density. The role of partial resistance in integrated control systems and the effectiveness of nematicides against G. pallida is discussed.     

Nematicidal effect of Lepidium sativum on activity and reproduction of potato cyst nematode Globodera rostochiensis in soil

Abstract

In Iran, potato cyst nematode (Globodera rostochiensis) jeopardizes the traditionally high yields of potatoes in Hamadan Province in the west of Iran. Biofumigation is an eco-friendly method for integrated management of plant parasitic nematodes. In the laboratory, water extracts of water cress, fenugreek and dill similarly reduced viability of second stage juveniles after 3?h of exposure, and decreased hatching of encysted eggs to less than 1%. Pre-treatment and combined tests similarly decreased hatch. The nematicidal efficiency of top green manure of Lepidium sativum on the survival of nematode was tested on a susceptible cv in microplots. The weights of biofumigated plants increased. Anti-hatching properties of water cress applied as a biofumigant reduced hatch by average of 56%. Reproduction rates were lowered to below one, and final populations of cysts and their egg contents were reduced by nearly 60% in treated soil. Biofumigation at a 1% amendment rate was sufficient to bring about these results, which were comparable with those achieved with 2 and 3% rates. Nematicidal isothiocyanates released after incorporating glucosinolate-containing brassica plants are fully biodegradable and less toxic than their synthetic equivalents, and their use is considered a safer alternative to soil fumigants such as methyl bromide.     

The effect of pyramiding two potato cyst nematode resistance loci to Globodera pallida Pa2/3 in potato

Globodera pallida is a major nematode pest causing severe constraints in many potato production regions worldwide. The most prevalent G. pallida pathotypes are Pa2 and Pa3, which exist in mixed populations referred to as Pa2/3. Due to heterogeneity for avirulence genes within these nematode populations, so far, breeders have failed to identify a single resistance gene source which offers complete resistance. Currently, there are two quantitative trait loci (QTL) available, $ GpaIV_{adg}^{s} $ and Gpa5, conferring partial levels of resistance to G. pallida pathotype Pa2/3. The objective of this research was to pyramid $ GpaIV_{adg}^{s} $ and Gpa5 through marker-assisted selection to investigate whether or not pyramiding provides increased resistance to G. pallida Pa2/3. We developed a population segregating for both resistance QTLs and, using diagnostic genetic markers (Contig237 and HC), we identified groups of individuals from this population containing each individual QTL, both QTLs simultaneously, and neither QTL. These individuals were assessed for their resistance levels against G. pallida Pa2/3 population Chavornay. We demonstrated that individuals carrying both QTLs showed a significant reduction in the number of cysts formed in comparison to genotypes carrying $ GpaIV_{adg}^{s} $ or Gpa5, indicating an additive effect. Overall these results show that MAS-based pyramiding of these QTLs is an effective strategy for breeding cultivars exhibiting very high levels of resistance to G. pallida pathotype Pa2/3.     

The effect of the potato cyst nematode Globodera pallida on in vitro root growth of potato genotypes, differing in tolerance

Roots of eighteen potato genotypes, differing in tolerance of G. pallida, were grown from tuberpieces on agar in Petri dishes. Juveniles of G. pallida were inoculated directly onto root tips. Root length was measured at various times after inoculation. Inoculation reduced root growth within one day. At later stages, genotypes differed strongly in growth of inoculated roots. Between four and seven days after inoculation, growth of inoculated roots was not significantly correlated with growth of untreated roots, and was only poorly correlated with tolerance assessed in the greenhouse or in the field. However, multiple regression analysis revealed that the tolerance of the tested genotypes was associated with both the rate at which they induced hatching and the growth of roots after inoculation. The combination of these two variables accounted for high percentages explained variance.     

The relationship between neutral lipid reserves and infectivity for hatched and dormant juveniles of Globodera spp.

A quantitative histochemical technique based on scanning microdensitometry has been used to investigate neutral lipid utilisation by hatched and dormant juveniles of Globodera rostochiensis and Globodera pallida. During storage in water at 20°C, hatched juveniles of the two species showed no statistical difference in their rate of lipid utilisation. A semi-logarithmic curve fitted to the pooled data suggests that 50% relative utilisation occurred after 36 ± 4 days of storage. Measurement of the neutral lipid reserves for unhatched juveniles of G. pallida dormant in field soils in Northern England for a maximum of 14 yr showed a reduced rate of metabolism with a 50% loss of neutral lipid reserves after 7·5 ± 1·1 yr. The use of neutral lipid reserves by the hatched, starved juveniles was correlated with their survival and with a loss of both locomotor activity and an ability to infect tomato seedlings. Furthermore, results suggest that a loss of mobility by juveniles from field populations of G. pallida immediately after hatching was more closely related to their neutral lipid reserves than the duration of their dormancy. Infection of plants was also reduced for one population. This suggests that measurement of the neutral lipid reserves of a population may be of value for modifying the economic threshold to take account of changes in infectivity during prolonged rotations.     

The nematicidal potential of local Bacillus species against the root-knot nematode infecting greenhouse tomatoes

Bacillus spp. were isolated from Iranian tomato fields and evaluated for their efficacy against root-knot nematodes, Meloidogyne javanica. The 52 spore-forming bacterium isolates were obtained from tomato rhizosphere following heat treatment. Eight isolates were chosen based on their potency in prevention of M. javanica egg hatch and juvenile mortality and production of proteolytic enzymes in Petri plates. Their ability to form biofilms were also determined in pot experiments. According to phenotypic traits and 16s rDNA sequencing, all selected isolates belonged to Bacillus spp. including B. cereus and B. pumilus. Treatment with bacterial culture filtrates in vitro caused juvenile mortality of 72 to 99% after 48 h. After four days, the percentage of egg hatch ranged from 1.6 to 59% depending on the isolate. Bacillus pumilus (ToIr-MA) and Bacillus sp. (ToIr-10) were found to have significant ability to produce extracellular proteases and to form maximum biofilm, considerably reducing the number of egg masses and root gall index (P = 0.05) in comparison to untreated plants. Application of ToIr-MA and ToIr-10 enhanced the fresh and dry weights of shoot and root systems. There was significant enhancement in dry root weight (45 and 50%) and shoot weight (67 and 75%). Results suggest that these two Bacillus spp. have potential as biocontrol agents against root-knot disease in tomato production.     

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.     

Purification and characterization of alpha-amylase from the infective juveniles of the nematode Heterorhabditis bacteriophora

Glycogen content and alpha-amylase activity were estimated in the infective juveniles (IJs) of Heterorhabditis bacteriophora at different times of storage. The glycogen content declined from 5.8 to 2.5 ng/IJ during storage for 40 days at 27 degrees C. The change in glycogen content coincided with the change of alpha-amylase activity during storage. alpha-Amylase was purified from IJs at zero time of storage by ion exchange chromatography and gel filtration. Ion exchange chromatography resolved alpha-amylase into three isoenzymes. The major isoenzyme alpha-amylase I had the highest specific activity and was purified to homogeneity. A molecular mass of 46-47 kDa was estimated for both the native and denatured enzyme, suggesting that the enzyme is monomeric. The Km values were 6.5 and 9.6 mg/ml using starch and glycogen as substrates, respectively. alpha-Amylase I showed optimum activity at pH 7.0 and had an optimum temperature of 40 degrees C. The enzyme was unstable at temperatures above 40 degrees C. The enzyme activity was severely inhibited by EDTA, p-CMB and iodoacetic acid, but potentiated by CaCl2 and NaCl. These results are discussed and compared with previously reported alpha-amylases in the insect hosts of the parasite.     

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 effect of ICAD-S on the formation and intracellular distribution of a nucleolytically active caspase-activated DNase

We show here that co-expression of murine CAD with either ICAD-L or ICAD-S in Escherichia coli as well as mammalian cells leads to a functional DFF complex, which after caspase-3 activation releases a nucleolytically active DNase. The chaperone activity of ICAD-S is between one and two orders of magnitude less effective than that of ICAD-L, as deduced from cleavage experiments with different activated recombinant DFF complexes produced in E.coli. With nucleolytically active EGFP fusion proteins of CAD it is demonstrated that co-expression of ICAD-S, which lacks the C-terminal domain of ICAD-L, including the NLS, leads to a homogeneous intracellular distribution of the DNase in transfected cells, whereas co-expression of human or murine ICAD-L variants lacking the NLS leads to exclusion of EGFP–CAD from the nuclei in ~50% of cells. These results attribute a particular importance of the NLS in the long isoform of the inhibitor of CAD for nuclear accumulation of the DFF complex in living cells. It is concluded that ICAD-L and ICAD-S in vivo might function as tissue-specific modulators in the regulation of apoptotic DNA degradation by controlling not only the enzymatic activity but also the amount of CAD available in the nuclei of mammalian cells.     

The white potato cyst nematode (Globodera pallida) – a critical analysis of the threat in Britain

Over the last 30 years, there has been an epidemic of the white potato cyst nematode (wPCN, Globodera pallida). It has progressively replaced the yellow species (yPCN, G. rostochiensis) throughout most of England and Wales and is now a widespread problem. As damaging populations of wPCN are enormous (>10⁹ eggs ha^?1), several crops of potato cultivars resistant only to yPCN were required to produce this change. The threat it poses is reflected in an increase in the numbers of soil samples being tested and in nematicide use, which has increased to > 25 000 ha of potatoes being treated annually. Computer modelling shows that current management of wPCN is mostly ineffective and populations will continue to increase. The multiplication rate of wPCN is inversely related to its population density at planting and, because of this, modelling shows that sufficient eggs are likely to survive to enable large populations of wPCN to “rebound” following nematicide treatment. This is supported by recent trial results showing that wPCN population increase was almost as great in nematicides‐treated plots as in the untreated. Modelling also showed that current rotations (typically potatoes once every 5 or 6 years) are too short to prevent wPCN populations from progressively increasing, even when used in conjunction with a nematicide. Similarly, except with avirulent populations, the partially resistant cultivars currently available will not prevent wPCN from increasing. However, as the effectiveness of partially resistant cultivars is independent of population density, they can be very effective when integrated with a nematicide. Unfortunately, only c. 8% of the potato area is planted with partially resistant cultivars, and much of that is in land not known to be infested with wPCN. Consequently, the current epidemic of wPCN is likely to become progressively more serious. However, many farmers are failing to recognise and respond to this threat until it is too late because of the slow rate of increase of wPCN, the difficulties of detecting small populations and the costs of nematicides. To respond to the current epidemic of wPCN, the greatest priority is to have available an increased number of commercially‐attractive partially resistant cultivars.     

The Influence of Potato Cultivar on Lipid Content and Fecundity of Bolivian and British Populations of Globodera rostochiensis

The influence of host cultivar on the lipid levels provided by a female to her progeny was investigated with Oil Red O stain and a quantitative image analyzer. A population of Globodera rostochiensis was multiplied at Toralapa Field Station in Bolivia on 25 different potato cultivars grown in that country. The mean neutral lipid content of newly formed second-stage juveniles varied significantly with cultivar over a 200% range. The corresponding range was only 18% and 28% for the same Bolivian and a UK population of G. rostochiensis, respectively, when both completed reproduction concurrently on 10 pot-grown European cultivars in the United Kingdom. Egg numbers per female varied with host for Bolivian cultivars that lack known partial resistance to Globodera spp. There was a 15-fold range between the most and least fecund nematode-host combinations (Kosi and Gendarme). The Bolivian G. rostochiensis population showed only a 2-fold range in mean eggs per cyst when grown on European cultivars in the UK. The fatty acid profiles of lipids from Bolivian G. rostochiensis cysts reared on Bolivian potato cultivars were dominated by C₂₀ (37-64%) and C₁₈ (28-46%) fatty acids and ranged from C₁₄ to C₂₂. The three major fatty acids detected were C_20:4:, C_20:1, and C_18:1. Few differences between cultivars were observed. For a UK population of G. rostochiensis reared on ssp. tuberosum, higher relative percentages of C₁₈ and monounsaturated fatty acids and lower relative percentages of C₂₀ and polyunsaturated fatty acids were found.     

Early responses of resistant and susceptible potato roots during invasion by the potato cyst nematode Globodera rostochiensis

Signals from roots of resistant (cv. Maris Piper) and susceptible (cv. Désirée) potato cultivars during invasion by second stage juveniles (J2s) of the potato cyst nematode, Globodera rostochiensis, were investigated. Novel experimental chambers enabled the recording of electrophysiological responses from roots during nematode invasion. The root cell membrane potentials were maintained throughout the 3 d required to assess invasion and feeding site development. The steady-state resting membrane potentials of Désirée were more negative than those of Maris Piper on day 1, but the reverse on day 3. After 5 d there was no difference between the two cultivars. Intracellular microelectrodes detected marked spike activity in roots after the application of J2s and there were distinct and reproducible differences between the two cultivars, with the response from Désirée being much greater than that from Maris Piper. The responses to mechanical stimulation of roots by blunt micropipettes and sharp electrodes were consistent and similar in both cultivars to the responses in Maris Piper obtained after nematode invasion, but could not account for the marked response found in Désirée. Exogenous application of exoenzymes, used to mimic nematode chemical secretions, resulted in a distinct depolarization pattern that, although similar in both cultivars, was different from patterns obtained during nematode invasion or mechanical stimulation. The pH of homogenates prepared from roots of both cultivars was measured and a Ca2+ channel blocker was used to assess the role of Ca2+ in nematode invasion. The results indicated a role for Ca2+ in the signalling events that occur during nematode invasion.