Factors affecting the detection of potato leafroll virus in potato foliage by enzyme-linked immunosorbent assay

Using antiserum globulins that reacted only weakly with plant materials, potato leafroll virus (PLRV) at 10 ng/ml was detected consistently by enzyme-linked immunosorbent assay (ELISA). The reaction with PLRV particles was slightly impaired in potato leaf extracts that were diluted less than 10^-1 but not at greater dilutions. Antiserum globulins that reacted more strongly with plant materials could be used satisfactorily for coating microtitre plates but were unsuitable for conjugating with enzyme. The detection end-point of PLRV, in leaf sap of potato cv. Cara plants grown from infected tubers in the glasshouse, was about 10^-2 and the virus was reliably detected in extracts of composite samples of one infected and 15 virus-free leaves. PLRV concentration was much less in extracts of roots or stolons than in leaf extracts. The virus was detected in infected leaves of all 27 cultivars tested. PLRV was readily detectable 2 wk before symptoms of secondary infection developed in field-grown plants of cv. Cara and Maris Piper and remained so for at least 5 wk. Its concentration was slightly greater in old than in young leaves and was similar to that in glasshouse-grown plants. In field-grown plants of cv. Maris Piper with primary infection, PLRV was detected in tip leaves 21–42 days after lower leaves were inoculated by aphids; in some shoots it later reached a concentration, in tip leaves, similar to that in leaves with secondary infection. Symptoms of primary infection developed in the young leaves of some infected shoots but were inconspicuous and were not observed until at least a week after PLRV was detected by ELISA.     

Effects of potato leafroll virus on the crop processes leading to tuber yield in potato cultivars which differ in tolerance of infection

The effects of infection with potato leafroll virus (PLRV) on the four crop processes leading to tuber fresh weight yield were examined in field plots of four cultivars (Montana, Pentland Crown, Maris Piper and King Edward) differing in tolerance to infection. Averaged across cultivars, infection decreased yield by 50%. This decrease was equally due to less light (total solar radiation) being intercepted, a lower efficiency with which intercepted light was converted into dry weight, and a smaller proportion of dry weight being partitioned to tubers. Dry matter content of the tubers was also diminished but to a lesser extent. The main difference between the cultivars in their response to infection was in the partitioning of dry weight. In Montana and Pentland Crown, harvest index was decreased by 15% in infected plants, whereas in the less tolerant cultivars, Maris Piper and King Edward, it was decreased by 25%. The decline in photosynthetic performance of Montana, a cultivar with slightly earlier maturity than the other three, was delayed in PLRV-infected plants. Effects on number of daughter tubers essentially reflected those on yield to the extent that average tuber weight did not change in Maris Piper, was one third less in King Edward, and the change was intermediate in Montana and Pentland Crown.     

Spread of potato leafroll virus is decreased from plants of potato clones in which virus accumulation is restricted

Tubers of eight potato clones infected with potato leafroll luteovirus (PLRV) were planted as ‘infectors’ in a field crop grown, at Invergowrie, of virus-free potato cv. Maris Piper in 1989. The mean PLRV contents of the infector clones, determined by enzyme-linked immunosorbent assay (ELISA) of leaf tissue, ranged from c. 65 to 2400 ng/g leaf. Myzus persicae colonised the crop shortly after shoot emergence in late May and established large populations on all plants, exceeding 2000/plant by 27 June. Aphid infestations were controlled on 30 June by insecticide sprays. Aphid-borne spread of PLRV from plants of the infector clones was assessed in August by ELISA of foliage samples from the neighbouring Maris Piper ‘receptors’. Up to 89% infection occurred in receptor plots containing infector clones with high concentrations of PLRV. Spread was least (as little as 6%) in plots containing infectors in which PLRV concentrations were low. Primary PLRV infection in guard areas of the crop away from infectors was 4%. Some receptor plants became infected where no leaf contact was established with the infectors, suggesting that some virus spread may have been initiated by aphids walking across the soil.     

Application of enzyme-linked immunosorbent assay to the detection of potato leafroll virus in potato tubers

Factors affecting the detection of potato leafroll virus (PLRV) by enzyme-linked immunosorbent assay (ELISA) in tubers of field-grown potato plants with primary or secondary infection were studied. The reactions of extracts of virus-free potato tubers were minimised by pre-incubating the extracts at room temperature and by careful choice of the dilution of enzyme-conjugated globulin. PLRV was reliably detected in tubers produced by secondarily infected plants of all six cultivars tested. PLRV concentration was greater in heel-end than in rose-end vascular tissue of recently harvested tubers but increased in rose-end tissue when tubers stored at 4°C for at least 5 months were placed at 15–24°C for 2 wk. PLRV occurred at greater concentration in tubers from plants of cv. Maris Piper with natural or experimentally induced primary infection than in tubers from secondarily infected plants; again PLRV concentration was greater in heel-end than in rose-end vascular tissue. Plants whose shoots were infected earliest in the growing season were invaded systemically and produced the greatest proportion of infected tubers; plants infected late in the season also produced infected tubers but PLRV was not detected in their shoot tops. PLRV concentration in tubers from the earliest-infected plants was less than in tubers from later-infected plants. PLRV was detected reliably by ELISA in tubers from progenies that were totally infected but was not detected in all infected tubers from partially infected progenies. ELISA is suitable as a routine method of indexing tubers for PLRV, although the virus will not be detected in all infected tubers produced by plants to which it is transmitted late in the growing season.     

Evidence of simple genetic control in potato of ability to restrict potato leaf roll virus concentration in leaves

Summary The concentration of potato leaf roll virus (PLRV), measured by quantitative enzyme-linked immunosorbent assay, in foliage of plants of cv Maris Piper and clone G7445(1) with secondary infection was 2,700 ng/g leaf and 120 ng/g leaf, respectively. In experiments to examine the genetic control of their ability to restrict the multiplication of PLRV, reciprocal crosses were made between these two clones. Among 40 genotypes from the progeny of the crosses, about half had a low PLRV concentration in plants with secondary infection and the other half had a high concentration. The possibility of monogenic control of the character that restricts PLRV multiplication in such clones of Solanum tuberosum is discussed.     

Restricted multiplication of potato leafroll virus in resistant potato genotypes

Plants of a range of potato genotypes differing in rating for field resistance to potato leafroll virus (PLRV) were inoculated with the virus by grafting or by aphids (Myzus persicae). Plants of all genotypes tested became infected by each inoculation method and PLRV was detected by ELISA in the upper leaves of all genotypes within 26 days after grafting. Most genotypes with high resistance ratings developed only mild primary and secondary symptoms whereas those with low resistance ratings developed more pronounced symptoms. However, one genotype (G7461(4)) with a high resistance rating was very severely affected. The concentrations attained by PLRV in genotypes with high resistance ratings were only 1–10% of those in genotypes with low resistance ratings. These differences in virus concentration were found in young leaves of plants with primary or secondary infection, whether inoculated by grafting or by aphids and whether grown in the glasshouse or the field. In older leaves, differences in virus concentration between genotypes were at least as pronounced as those in younger leaves. In contrast, PLRV concentration in vascular tissue at the heel end of tubers of plants with primary infection was similar for all the genotypes tested. Although low PLRV concentration was consistently associated with high resistance rating it is not the only form of resistance to PLRV occurring in potato.     

The effects of temperature on the availability and acquisition of potato leafroll luteovirus by Myzus persicae

The concentration of potato leafroll luteovirus (PLRV) did not differ in potato plants with secondary infections grown at 15°C or 27°C. Detached leaves of plants grown at 15°C or 27°C were used as sources of PLRV for peach-potato aphids (Myzus persicae Sulz.) both at 15°C and 27°C. At comparable temperature during virus acquisition, aphids which fed on leaves of plants kept previously at 15°C contained more viral antigen detected by ELISA than aphids which fed on leaves of plants grown at 27°C. The aphids which acquired PLRV at 27°C contained evidently more viral antigen than those which acquired PLRV at 15°C. The greatest amount of PLRV was found in the aphids which acquired the virus at 27°C from the leaves of plants kept at 15°C. The ability of M. persicae to transmit PLRV to Physalis ftoridana Rydb. generally decreased with decrease in the amount of PLRV in vectors.     

Multiple components of the resistance of potatoes to potato leafroll virus

In glasshouse experiments the ranking of potato genotypes for resistance to infection with potato leafroll virus (PLRV) using three concentrations of aphid-borne inoculum was the same as their field resistance ratings. In field-grown plants this resistance to infection increased in all genotypes as the plants aged but its rate of increase differed between genotypes. In tests on field-grown plants infected by aphid- or graft-inoculation, the proportion of virus-free progeny tubers increased the later the date of inoculation but was greater in resistant than in susceptible genotypes. This trend was most pronounced in the resistant clone G7445(1), in which the virus failed to move from the foliage to the tubers of some plants infected in glasshouse tests. The spread of PLRV will thus be minimised in crops of resistant compared with susceptible genotypes for three reasons: plants have greater resistance to infection, systemic spread of virus from their foliage to tubers is less likely and, as shown previously, the low concentration of virus particles in leaf tissue makes infected plants less potent sources of inoculum for aphids.     

The restricted distribution of potato leafroll luteovirus antigen in potato plants with transgenic resistance resembles that in clones with one type of host genemediated resistance

The distribution of virus-infected cells was examined, by fluorescence microscopy, within plants of a range of potato clones infected with potato leafroll luteovirus (PLRV). This range included nine PLRV-resistant clones, of which four were transgenic lines carrying the PLRV coat protein gene and five were conventionally bred. Plants of these clones were resistant to PLRV multiplication and accumulated less virus antigen in leaf tissue than did susceptible clones. Indirect fluorescent antibody staining of thin sections from carbodiimide-fixed petiole tissue revealed that in plants of PLRV-susceptible clones, virus-infected cells were abundant within both external (abaxial) and internal (adaxial) phloem bundles. In plants of the PLRV-resistant conventionally bred clones and in resistant transgenic lines of cv. Pentland Squire, virus-infected cells were much fewer in number and largely restricted to internal phloem bundles. In resistant transgenic lines of cv. Désirée, this restricted distribution of PLRV antigen was only detected in petioles of young leaves. The results suggest that the transgenic and a host-mediated type of resistance that restricts virtis multiplication have underlying similarities.     

Effects of potato genotype, oxamyl and the numbers of potato cyst-nematodes, Globodera rostochiensis or G. pallida on tuber yields and nematode increase

A new technique is described for establishing different numbers of the potato cyst-nematode Globodera rostochiensis in field soil, which leaves the soil homogeneous in nutrient status. Field plots established in this way were used to compare yield losses in four potato cultivars (Maris Piper, Pentland Crown, Pentland Dell and Désirée) associated with different numbers of G. rostochiensis. Over the range of 7.4 to 148.4 eggs g^-1 soil at planting, yield losses were 18.7% (Maris Piper), 53.2% (Désirée), 55.7% (Pentland Crown) and 63.5% (Pentland Dell). Similar results were obtained in another experiment on the same field in a different year using only lightly and heavily infested plots. Treating the seedbed soil with oxamyl before planting prevented significant injury to potatoes by G. rostochiensis but increased the yield of Pentland Dell and perhaps Désirée (but not Maris Piper or Pentland Crown) more than expected from nematode control alone. Treating heavily infested soil with such a nematicide cannot therefore be recommended as part of a valid procedure for establishing lightly and heavily infested plots for comparing tolerances of attack by potato cyst-nematodes in a range of potato genotypes. In peaty loam soils moderately or heavily infested with G. pallida, oxamyl at 5.6 kg a.i. ha^-1 incorporated into the seedbed before potatoes were planted generally increased tuber yields, though the effects varied considerably with the cultivar grown. Increase of G. pallida in these soils was controlled better by growing potatoes bred for resistance to it (ZB 35 – 29, Caxton, Santé, Morag, 11233 ab 22, Fingal, A27/23, Cromwell). Increase of G. pallida on susceptible cultivars varied greatly and Romano increased G. pallida no more than the resistant Morag. G. pallida is probably controlled best in peaty loam by growing a resistant cultivar in soil treated with a granular (non-fumigant) nematicide.     

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.     

Quantitative studies on the uptake and retention of potato leafroll virus by aphids in laboratory and field conditions

Enzyme-linked immunosorbent assay (ELISA) was adapted for the efficient detection and assay of potato leafroll virus (PLRV) in aphids. Best results were obtained when aphids were extracted in 0.05 M phosphate buffer, pH 7.0, and the extracts incubated at 37 °C for 1 h before starting the assay. Using batches of 20 green peach aphids (Myzus persicae), about 0.01 ng PLRV/aphid could be detected. The virus could also be detected in single aphids allowed a 1-day acquisition access period on infected potato leaves. The PLRV content of aphids depended on the age of potato source-plants and the position of source leaves on them. It increased with increase in acquisition access period up to 7 days but differed considerably between individual aphids. A maximum of 7 ng PLRV/aphid was recorded but aphids more usually accumulated about 0.2 ng PLRV per day. When aphids were allowed acquisition access periods of 1–3 days, and then caged singly on Physalis floridana seedlings for 3 days, the PLRV content of each aphid, measured subsequently, was not strongly correlated with the infection of P. floridana. The concentration of PLRV in leaf extracts differed only slightly when potato plants were kept at 15, 20, 25 or 30 °C for 1 or 2 wk, but the virus content of aphids kept on leaves at the different temperatures decreased with increase of temperature. PLRV was transmitted readily to P. floridana at all temperatures, but by a slightly smaller proportion of aphids, and after a longer latent period, at 15 °C than at 30 °C. The PLRV content of M. persicae fed on infected potato leaves decreased with increasing time after transfer to turnip (immune to PLRV). The decrease occurred in two phases, the first rapid and the second very slow. In the first phase the decrease was faster, briefer and greater at 25 and 30 °C than at 15 and 20 °C. No evidence was obtained that PLRV multiplies in M. persicae. These results are compatible with a model in which much of the PLRV in aphids during the second phase is in the haemocoele, and transmission is mainly limited by the rate of passage of virus particles from haemolymph to saliva. The potato aphid, Macrosiphum euphorbiae, transmitted PLRV much less efficiently than M. persicae. Its inefficiency as a vector could not be ascribed to failure to acquire or retain PLRV, or to the degradation of virus particles in the aphid. Probably only few PLRV particles pass from the haemolymph to saliva in this species. The virus content of M. euphorbiae collected from PLRV-infected potato plants in the field increased from early June to early July, and then decreased. PLRV was detected both in spring migrants collected from the plants and in summer migrants caught in yellow water-traps. PLRV was also detected in M. persicae collected from infected plants in July and August, and in trapped summer migrants, but their PLRV content was less than that of M. euphorbiae, and in some instances was too small for unequivocal detection.     

Infection of potato plants with potato leafroll virus changes attraction and feeding behaviour of Myzus persicae

Potato leafroll virus (PLRV; genus Polerovirus, family Luteoviridae) is a persistently transmitted circulative virus that depends on aphids for spreading. The primary vector of PLRV is the aphid Myzus persicae (Sulzer) (Homoptera: Aphididae). Solanum tuberosum L. potato cv. Kardal (Solanaceae) has a certain degree of resistance to M. persicae: young leaves seem to be resistant, whereas senescent leaves are susceptible. In this study, we investigated whether PLRV‐infection of potato plants affected aphid behaviour. We found that M. persicae's ability to differentiate headspace volatiles emitted from PLRV‐infected and non‐infected potato plants depends on the age of the leaf. In young apical leaves, no difference in aphid attraction was found between PLRV‐infected and non‐infected leaves. In fact, hardly any aphids were attracted. On the contrary, in mature leaves, headspace volatiles from virus infected leaves attracted the aphids. We also studied the effect of PLRV‐infection on probing and feeding behaviour (plant penetration) of M. persicae using the electrical penetration graph technique (DC system). Several differences were observed between plant penetration in PLRV‐infected and non‐infected plants, but only after infected plants showed visual symptoms of PLRV infection. The effects of PLRV‐infection in plants on the behaviour of M. persicae, the vector of the virus, and the implications of these effects on the transmission of the virus are thoroughly discussed.     

New approaches for restricting spread of potato leafroll virus by different methods of eradicating infected plants from potato crops

Experiments were made at Invergowrie in 1984 and 1985 to compare the spread of potato leafroll virus (PLRV) after removing infected plants by three different methods; conventional roguing, desiccation with diquat, or incineration for 45–60s using a propane gas flame. Potato leaf roll 'infector' plants, grown in plots of virus-free Maris Piper seed potatoes, were artificially infested in June with aphids (Myzus persicae) from a laboratory culture, and removed from the plots after 2 or 3 wk. In both years, natural infestations of potato aphids were scarce during this period. There was no significant difference in the proportion of tubers infected with PLRV in adjacent plants after the neighbouring infector plants had been rogued by hand or desiccated with diquat, but the proportion was considerably reduced following incineration of the infector plants. In 1984, the spread of PLRV in conventionally rogued plots was also significantly reduced by a mixture of deltamethrin plus heptenophos, applied four times from 80% crop emergence, and was almost eliminated by a treatment with aldicarb granules, either at planting, or as a side-dressing 5 wk later. In 1985, delaying infector removal by 8 days in early July significantly increased the spread of PLRV to neighbouring plants from 2.3% (1 July) to 8.3% (9 July). A single application of deltamethrin plus heptenophos to infectors 1 wk before removal did not significantly decrease spread. Although incineration was quick and effective, the value of this method of eradicating infector plants in seed potato crops is limited because it failed to destroy infected tubers.     

The susceptibility of stems of different potato cultivars to blackleg caused by Erwinia carotovora subsp. atroseptica

The susceptibility of stems of six potato cultivars to Erwinia carotovora subsp. atroseptica was assessed in two years (1981 and 1982) either by direct inoculation in the field or by inoculation of detached stems in the laboratory. These six and a further 22 cultivars were also assessed in three years (1982-84) by inoculating stems of glasshouse-grown plants. Different methods of inoculation and types of inocula were tested. In the field, wooden toothpicks rubbed in bacterial slime were more successful in establishing infection than when dipped in a bacterial suspension, but injection of bacterial suspension with a hypodermic needle was reliable in establishing infection over a range of concentrations. Detached stems were more readily infected and gave more consistent results compared with inoculation in the field. The range of reaction of the six cultivars was similar in both detached stem and glasshouse tests. The early cultivars Pentland Javelin and Ulster Sceptre were most susceptible and of the maincrop cultivars, Maris Piper was intermediate and Desiree and King Edward least susceptible whereas Pentland Crown showed greater resistance in the glasshouse than in the field. Glasshouse tests using hypodermic inoculation indicated a range of susceptibilities; the early cultivars Manna, Maris Bard and Estima were most susceptible and the maincrop Pentland cultivars Crown, Dell, Hawk, Ivory and Squire least susceptible.     

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.     

Transfer of resistance to potato leafroll virus, potato virus Y and potato virus X from Solarium brevidens to S. tuberosum through symmetric and designed asymmetric somatic hybridisation

Resistance to potato leafroll virus (PLRV), potato virus Y (PVY^o) and potato virus X (PVX) was studied in symmetric and asymmetric somatic hybrids produced by electrofusion between Solanum brevidens (2n=2×=24) and dihaploid S. tuberosum (2n=2×=24), and also in regenerants (B-hybrids) derived through protoplast culture from a single somatic hybrid (chromosome number 48). All of the somatic hybrids between 5. brevidens and the two dihaploid lines of potato cv. Pito were extremely resistant to PLRV and PVY^oand moderately resistant to PVX, irrespective of their chromosome number and ploidy level (tetraploid or hexaploid). Most (56%) of the asymmetric hybrids of irradiated S. brevidens and the dihaploid line of potato cv. Pentland Crown (PDH40) had high titres of PVY^osimilar to those of PDH40, whereas the rest of the hybrids had PVY^otitres less than a tenth of those in PDH40. Three B-hybrids had a highly reduced chromosome number (27, 30 and 34), but were however as resistant to PLRV, PVY^oand PVX as 5. brevidens. Two asymmetric hybrids and one B-hybrid were extremely resistant to PLRV but susceptible to both PVY and PVX. The results suggested that resistance to PLRV in 5. brevidens is controlled by a gene or genes different from those controlling resistance to PVY and PVX, and the gene(s) for resistance to PVY and PVX are linked in S. brevidens.     

Resistance to potato leaf roll virus multiplication in potato is under major gene control

The concentration of potato leaf roll virus (PLRV), as measured by a quantitative enzyme-linked immunosorbent assay, in the foliage of potato plants (Solanum tuberosum) of cv Maris Piper with secondary infection was 2900 ng/g leaf, whereas in clones G7445(1) and G7032(5) it was 180 ng/g leaf and 120 ng/g leaf, respectively. To examine the genetic control of resistance to PLRV multiplication, reciprocal crosses were made between the susceptible cultivar Maris Piper and the two resistant clones, and the three parents were selfed. Seedling progenies of these families were grown to generate tubers of individual genotypes (clones). Clonally propagated plants were graft-inoculated, and their daughter tubers were collected and used to grow plants with secondary infection in which PLRV concentration was estimated. The expression of resistance to PLRV multiplication had a bimodal distribution in progenies from crosses between Maris Piper and either resistant clone, and also in progeny from selfing the resistant parents, with genotypes segregating into high and low virus titre groups. Only the progeny obtained from selfing Maris Piper did not segregate, all genotypes being susceptible to PLRV multiplication. The pattern of segregation obtained from these progenies fits more closely with the genetical hypothesis that resistance to PLRV multiplication is controlled by two unlinked dominant complementary genes, both of which are required for resistance, than with the simpler hypothesis that resistance is conferred by a single dominant gene, as published previously.