Restricted distribution of potato leafroll virus antigen in resistant potato genotypes and its effect on transmission of the virus by aphids

Enzyme-linked immunosorbent assay was used to measure the concentration of potato leafroll virus (PLRV) antigen in different parts of field-grown secondarily infected plants of three potato genotypes known to differ in resistance to infection. The antigen concentration in leaves of cv. Maris Piper (susceptible) was 10–30 times greater than that in cv. Pentland Crown or G 7445(1), a breeder's line (both resistant). Differences between genotypes in antigen concentration were smaller in petioles and tubers (5–10-fold) and in above-ground stems (about 4-fold), and were least in below-ground stems, stolons and roots (about 2-fold). PLRV antigen, detected by fluorescent antibody staining of tissue sections, was confined to phloem companion cells. In Pentland Crown, the decrease in PLRV antigen concentration in leaf mid-veins and petioles, relative to that in Maris Piper, was proportional to the decrease in number of PLRV-containing companion cells; this decrease was greater in the external phloem than in the internal phloem. The spread of PLRV infection within the phloem system seems to be impaired in the resistant genotypes. Green peach aphids (Myzuspersicae) acquired < 2800 pg PLRV/aphid when fed for 4 days on infected field-grown Maris Piper plants and < 58% of such aphids transmitted the virus to Physalis floridana test plants. In contrast, aphids fed on infected Pentland Crown plants acquired <120 pg PLRV/aphid and <3% transmitted the virus to P. floridana. The ease with which M. persicae acquired and transmitted PLRV from field-grown Maris Piper plants decreased greatly after the end of June without a proportionate drop in PLRV concentration. Spread of PLRV in potato crops should be substantially decreased by growing cultivars in which the virus multiplies to only a limited extent.     

Inhibited Long-Distance Movement of Potato Leafroll Virus to Tubers in Potato Genotypes Expressing Combined Resistance to Infection, Virus Multiplication and Accumulation

Plants of two potato clones which, in preliminary greenhouse assessments, showed resistance to multiplication and accumulation of potato leafroll virus (PLRV) were graft or aphid inoculated with the virus and grown in the greenhouse; plants of a moderately susceptible cultivar were used for comparison in all experiments. A high concentration of aphid‐borne inoculum was used to ensure strong infection pressure. Clone M62759 appeared to be highly resistant to PLRV infection, whereas clone PS1706 was more susceptible. Both clones expressed a high level of resistance to virus multiplication, when primary or secondary infection was assayed by enzyme‐linked immunosorbent assay. Moreover, PLRV was detected in only few or none of the progeny plants of clone M62759, which thus strongly inhibited virus transport to tubers. The study on PLRV translocation from aphid‐inoculated shoots to uninoculated shoots sprouted from the same tubers showed that no specific mechanisms are likely to impair PLRV movement through the tubers of the resistant genotypes. These results indicate that three valuable components of the resistance to PLRV are probably closely linked in the genotype, a combination that seems to occur rather rarely in potato clones. Nevertheless, selecting potato genotypes for the complex resistance to PLRV may prove to be a worthwhile part of breeding programmes, provided that the genetic mechanisms governing particular types of resistance are better recognized.     

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.     

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.     

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.     

Resistance to potato leafroll luteovirus can be greatly improved by combining two independent types of heritable resistance

Twelve potato clones were exposed to infection by aphids with potato leafroll luteovirus (PLRV) in three field trials in order to assess their resistance to infection. Up to 92% of the plants of some clones became infected, although other clones were relatively resistant to infection and one clone remained virus-free in all three trials. The resistance of the same 12 clones to PLRV multiplication was assessed in glasshouse-grown plant: lants were graft-inoculated and their daughter tubers were used to grow plants with secondary infection. High concentrations of PLRV were found in some clones (c. 1700 ng/g leaf) while in others much less virus accumulated (as little as 60 ng/g leaf). However, clones in which little virus accumulated were not necessarily those which were most resistant to infection in the field, and there was no association between the two types of resistance. Nevertheless, both types of resistance were found in some clones. The clone G8107(1), which remained virus-free in all the field exposure trials, was also the most resistant to PLRV multiplication. The combination of these two types of resistance in cultivars should help to eliminate the spread of PLRV in crops.     

Characterization of the expression and inheritance of potato leafroll virus (PLRV) and potato virus Y (PVY) resistance in three generations of germplasm derived from Solanum etuberosum

Potato virus Y (PVY) and potato leafroll virus (PLRV) are two of the most important viral pathogens of potato. Infection of potato by these viruses results in losses of yield and quality in commercial production and in the rejection of seed in certification programs. Host plant resistance to these two viruses was identified in the backcross progeny of a Solanum etuberosum Lindl. somatic hybrid. Multiple years of field evaluations with high-virus inoculum and aphid populations have shown the PVY and PLRV resistances of S. etuberosum to be stably expressed in two generations of progeny. However, while PLRV resistance was transmitted and expressed in the third generation of backcrossing to cultivated potato (Solanum tuberosum L. subsp. tuberosum), PVY resistance was lost. PLRV resistance appears to be monogenic based on the inheritance of resistance in a BC₃ population. Data from a previous evaluation of the BC₂ progeny used in this study provides evidence that PLRV resistance was partly conferred by reduced PLRV accumulation in foliage. The field and grafting data presented in this study suggests that resistance to the systemic spread of PLRV from infected foliage to tubers also contributes to the observed resistance from S. etuberosum. The PLRV resistance contributed by S. etuberosum is stably transmitted and expressed through sexual generations and therefore would be useful to potato breeders for the development of PLRV resistant potato cultivars.     

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.     

Experiments on the Spread of Potato Virus X Between Plants in Contact

Experiments on the spread of five strains of potato virus X were made with seven potato varieties and with tomato plants both under glass and in the field. Spread by leaf contact between healthy and infected plants was confirmed, and it was also found that spread could occur between plants whose only contact was below ground.
The rate of spread was much greater in tomato than in potato plants, and virulent strains of the virus, which achieve a high concentration in infected plants, spread more rapidly than avirulent strains. In only one experiment with potatoes did more than 10% of the healthy potato plants exposed to infection become infected during one season.
Datura stramonium and tomato plants became infected when growing in soil containing sap or residues from X -infected plants.
It was common in the field for potato plants whose foliage gave no reaction for virus X at the end of the season to yield a mixed progeny of healthy and infected tubers. Such infections are thought to result from underground spread.
Attempts to transmit virus X from infected to healthy potatoes by means of Rhizoctonia solani failed. No examples of infection were found except when healthy plants came into direct contact with sources of the virus.     

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.     

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.     

Factors affecting the field infection of potato tubers of different cultivars by blight (Phytophthora infestans)

The relationships between rain and blight (Phytophthora infestans) were studied in unsprayed crops of cultivars differing widely in foliage and tuber susceptibility. The occasions when tubers were infected depended on rain and not cultivar, but numbers of tubers infected after rain was affected by the blight susceptibility of the cultivar. Infected tubers were first found when less than 5 % (BMS key) of the potato foliage was infected but few fresh infections occurred when 50–75% of the foliage had been destroyed. Some tubers were infected after 8 mm rain (tubers near the surface with even less) but large increases in numbers of tubers infected usually occurred only after 25 mm or more had increased soil moisture to above ‘field capacity’ around the tuber for at least 24 h. The most susceptible cultivars Ulster Ensign and Arran Banner had all plants with some tuber blight, and some plants with all tubers affected and often many lesions per tuber. Cultivars of intermediate susceptibility, King Edward and Up-to-Date, had some plants without blighted tubers, many with a few and very few with all. The more resistant cultivars Majestic and Arran Viking had many plants without infected tubers and many lesions that aborted while still necrotic threads, so that the fungus did not spread. Most infections occurred through tuber eyes, lenticels or sometimes growth cracks. The distribution of blight lesions on tubers differed in the different seasons, for example, lenticels were most commonly infected on Arran Banner and Ulster Ensign and eyes on King Edward, Majestic and Arran Viking. In late or slowly developing attacks, lesions on stems became more numerous and larger than in fast, early attacks and were prolific sources of spores on King Edward and Up-to-Date but not on Majestic and Arran Viking. Because much rain water runs down the stems of Up-to-Date and King Edward, stem lesions can provide an important source of inoculum for tubers.     

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.     

Increased sensitivity of detection of plant viruses obtained by using a fluorogenic substrate in enzyme-linked immunosorbent assay

The fluorogenic substrate 4-methylumbelliferyl phosphate (MUP) of alkaline phosphatase was compared with the chromogenic substrate p-nitrophenyl phosphate (NPP) in tests for plant viruses by enzyme-linked immunosorbent assay (ELISA). In tests on leaf extracts of squash infected with prune dwarf virus, Chenopodium quinoa and apple infected with apple mosaic virus (ApMV), and potato infected with potato leafroll virus (PLRV), MUP increased sensitivity 2–16 times, the smallest and greatest increases being obtained with ApMV (in apple) and PLRV respectively. In similar tests on 21 dormant PLRV-infected potato tubers, sensitivity was increased 2–4 times with 13 tubers, but the two substrates gave the same detection end-points with eight tubers. When individual seeds of potato plants infected with the Andean potato calico strain of tobacco ringspot virus were tested, the virus was detected in virtually all seeds by MUP-ELISA, but detection by NPP-ELISA was inefficient unless absorbance values were measured after overnight incubation at 4 °C, instead of after 2 h at room temperature. In tests on Myzus persicae carrying PLRV and Sitobion avenae carrying barley yellow dwarf virus (BYDV), both viruses were consistently detected in a greater proportion of individual aphids by MUP-ELISA than NPP-ELISA irrespective of whether incubation was for 2 h at room temperature or overnight at 4 °C. The effeciency of detection of virus in single viruliferous aphids by MUP-ELISA was not decreased by grouping with one or four non-viruliferous aphids but was decreased (PLRV) or greatly decreased (BYDV) by grouping with nine. MUP-ELISA and transmission tests to Physalis floridana seedlings (2–3 day inoculation access periods) both detected PLRV in most individual M. persicae, but the results obtained with the two methods did not correlate completely. In similar tests for BYDV in individual S. avenae, virtually all aphids transmitted BYDV to oat seedlings during a 3-day inoculation access period but it was subsequently detected by MUP-ELISA in less than half of them. By contrast, MUP-ELISA detected PLRV in most viruliferous M. persicae even after they had fed for 3 days on Chinese cabbage, a non-host for this virus.     

Two allelic or tightly linked genetic factors at the <Emphasis Type="Italic">PLRV.4</Emphasis> locus on potato chromosome XI control resistance to potato leafroll virus accumulation

A novel locus for potato resistance to potato leafroll virus (PLRV) was characterized by inheritance studies and molecular mapping. The diploid parental clone DW 91-1187 was resistant to PLRV accumulation in both inoculated plants and their tuber progeny. The resistance to PLRV accumulation present in DW 91-1187 was not transmitted to any F₁ offspring when crossed with a PLRV susceptible clone. Instead, one half of the F₁ individuals exhibited undetectable amounts of PLRV as determined by ELISA during the primary infection assay, but accumulated PLRV in their tuber progeny plants. The other half was clearly infected both in the inoculated and tuber-born plants. The inheritance of resistance to PLRV accumulation may be explained by a model of two complementary alleles of a single gene (PLRV.4) or by two complementary genes that are closely linked in repulsion phase. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers linked to the PLRV.4 locus were selected. The two complementary factors were closely linked in coupling phase to the alternative alleles UBC864₆₀₀ and UBC864₈₀₀ of DNA marker UBC864. These markers may be used for marker-assisted selection of genotypes having both factors for resistance to PLRV accumulation. The PLRV.4 locus was mapped to a central position on linkage group XI of the potato molecular map, where no resistance locus has been mapped previously.     

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.     

Strong resistance to potato tuber necrotic ringspot disease in potato induced by transformation with coat protein gene sequences from an NTN isolate of Potato virus Y

The potato cv. Igor is susceptible to infection with Potato virus Y (PVY) and in Slovenia it has been so severely affected with NTN isolates of PVY causing potato tuber necrotic ringspot disease (PTNRD) that its cultivation has ceased. Plants of cv. Igor were transformed with two transgenes that contained coat protein gene sequence of PVY^NTN. Both transgenes used PVY sequence in a sense (+) orientation, one in native translational context (N‐CP), and one with a frame‐shift mutation (FS‐CP). Although most transgenic lines were susceptible to infection with PVY^NTN and PVY^O, several lines showed resistance that could be classified into two types. Following manual or graft inoculation, plants of partially resistant lines developed some symptoms in foliage and tubers, and virus titre in the foliage, estimated by ELISA, was low or undetectable. In highly resistant (R) lines, symptoms did not develop in foliage and on tubers, and virus could not be detected in foliage by ELISA or infectivity assay. Four lines from 34 tested (two N‐CP and two FS‐CP) were R to PVY^NTN and PVY^O and one additional line was R to PVY^O. When cv. Spey was transformed with the same constructs, they did not confer strong resistance to PVY^O.