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.     

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.     

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.     

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.     

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.     

Detection of potato leafroll and potato mop-top viruses by immunosorbent electron microscopy

Attachment of virus particles to antiserum-coated electron microscope grids (immunosorbent electron microscopy) provided a test that was at least a thousand times more sensitive than conventional electron microscopy for detecting potato leafroll (PLRV) and potato mop-top (PMTV) viruses. The identity of the attached virus particles was confirmed by exposing them to additional virus antibody, which coated the particles.
PLRV particles (up to 50/μm² of grid area) were detected in extracts of infected potato leaves and tubers, infected Physalis floridana leaves, and single virus-carrying aphids. On average, Myzus persicae yielded 10–30 times more PLRV particles than did Macrosiphum euphorbiae .
PMTV particles (up to 10/μm² of grid area) were detected in extracts of inoculated tobacco leaves, and of infected Arran Pilot potato tubers with symptoms of primary infection. Particles from tobacco leaves were of two predominant lengths, about 125 nm or about 290 nm, and fewer particles of other lengths were found than in previous work, in which partially purified or purified preparations of virus particles were examined, using grids not coated with antiserum.     

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.     

Aspects of resistance to sweet potato virus disease in sweet potato

In field trials during the first and the second rainy season of 1996 in Uganda, whiteflies were similarly abundant and aphids were absent on three clones of sweet potato (NIS-93–63, cv. Tanzania and cv. New Kawogo) although the three clones differed considerably in their resistance to sweet potato virus disease (SPVD), a complex disease resulting from infection by both the aphid-borne sweet potato feathery mottle virus (SPFMV) and the whitefly-borne sweet potato chlorotic stunt virus (SPCSV). This suggests that vector resistance does not determine the relative SPVD resistance of these genotypes. SPFMV alone had only a low virus titre in sweet potato cvs Tanzania and New Kawogo, became increasingly difficult to detect in plants of these cultivars and was seldom acquired by aphids. However, this resistance to SPFMV was not apparent in plants which were also infected with SPCSV. Plants then had a high SPFMV titre, appeared unable to eliminate SPFMV and provided good sources for aphids to acquire it.     

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.     

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.     

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.     

A major quantitative trait locus for resistance to Potato leafroll virus is located in a resistance hotspot on potato chromosome XI and is tightly linked to N-gene-like markers.

Potato leafroll virus (PLRV) causes one of the most widespread and important virus diseases in potato. Resistance to PLRV is controlled by genetic factors that limit plant infection by viruliferous aphids or virus multiplication and accumulation. Quantitative trait locus (QTL) analysis of resistance to virus accumulation revealed one major and two minor QTL. The major QTL, PLRV.1, mapped to potato chromosome XI in a resistance hotspot containing several genes for qualitative and quantitative resistance to viruses and other potato pathogens. This QTL explained between 50 and 60% of the phenotypic variance. The two minor QTL mapped to chromosomes V and VI. Genes with sequence similarity to the tobacco N gene for resistance to Tobacco mosaic virus were tightly linked to PLRV.1. The cDNA sequence of an N-like gene was used to develop the sequence characterized amplified region (SCAR) marker N127(1164) that can assist in the selection of potatoes with resistance to PLRV.     

Location of resistance factors in the leaves of potato and wild tuber-bearing Solanum species to the aphid Myzus persicae

Analysis of electrically recorded feeding behaviour of aphids was combined with colony‐development tests to search for sources of resistance to Myzus persicae (Sulzer) (Homoptera: Aphididae) in tuber‐bearing Solanum species (Solanaceae), aiming at a reduction of potato leaf roll virus (PLRV) transmission. Twenty genotypes, originating from 14 gene bank accessions, representing 13 wild tuber‐bearing Solanum spp., three Solanum tuberosum L. (potato) cultivars, and one S. tuberosum breeding line, were selected. Colony‐development tests were carried out in no‐choice experiments by placing adult aphids on plants of each genotype and counting numbers of nymphs and adults on young plants after 8 and 15 days, and on flowering plants after 14 and 30 days. Large differences were observed among genotypes: some developed small colonies and others developed large ones. Also, in a few genotypes, resistance in mature plants was different for leaves of different ages; young leaves were resistant to aphids whereas old senescent leaves were susceptible. The electrical penetration graph (DC‐EPG system) technique was used to study aphid feeding behaviour on each Solanum genotype for 6 h. Electrical penetration graph (EPG) results also showed large differences among the genotypes, indicating resistance at the leaf surface and at three different levels of plant tissue (epidermis, mesophyll, and phloem). Therefore, it was concluded that different mechanisms of resistance to M. persicae exist among the genotypes analysed. EPGs recorded from aphids on Solanum berthaultii Hawkes and Solanum tarijense Hawkes with and without glandular trichomes showed that strong surface resistance can bias EPG parameters associated with resistance located in deeper tissues. Experimental evidence is presented that the resistance to aphids in the genotypes with glandular trichomes strongly depends on these morphological structures.     

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.     

Replicase mediated resistance against potato leafroll virus in potato Desirée plants

Potato leafroll virus (PLRV) is a major menace for the potato production all over the world. PLRV is transmitted by aphids, and until now, the only strategy available to control this pest has been to use large amounts of insecticides. Transgenic approaches involving the expression of viral replicases are being developed to provide protection for plants against viral diseases. The purpose of this study was to compare the protection afforded by the differential expression of PLRV replicate transgene in potato plants cv. Desirée, Plants were genetically modified to express the complete sense PLRV replicase gene. Two constructions were used, one containing the constitutive 35SCaMV promoter and the other the phloem-specific RolA promoter from Agrobacterium rhizogenes. Transgenic plants were infected with PLRV in vitro, using infested aphids. In plants in which 35SCaMV controlled the expression of the PLRV replicase gene, signs of infection were initially detected, although most plants later developed a recovery phenotype showing undetectable virus levels 40 days after infection. In turn, those plants with the RolA promoter displayed an initial resistance that was later overcome. Different molecular mechanisms are likely to participate in the response to PLRV infection of these two types of transgenic plants.     

Transmission of potato leafroll virus in relation to the honeydew excretion of Myzus persicae

Honeydew excretion of single Myzus persicae nymphs on potato leafroll virus (PLVR)-infected Physalis floridana was studied during the acquisition access period (AAP) in relation to the efficiency of virus transmission.
With increasing length of the AAP, the percentage of nymphs that transmitted the virus increased. These nymphs produced significantly more honeydew droplets during the AAP on PLRV-infected P. floridana plants than nymphs which failed to transmit the virus. However, the number of honeydew droplets excreted during the AAP by transmitting nymphs did not affect the length of the latency period. Nymphs which infected the first test plant after a short latency period produced a similar amount of honeydew during the AAP to those with a longer latency period.
Honeydew excretion recorded on plants of varied age, showed that nymphs feeding on bottom leaves of infected plants produced more honeydew droplets than on comparable leaves of healthy plants. On infected plants, nymphs produced more honeydew droplets on bottom leaves with pronounced symptoms than on top leaves that hardly showed any symptom of PLRV infection.
The concentration of viral antigen measured by ELISA was lower in top leaves than in bottom leaves of infected plants. Nevertheless, nymphs feeding on top leaves transmitted the virus more efficiently than those which used bottom leaves as virus source. When bottom leaves were used as a virus source, the percentage of viruliferous nymphs decreased with plant age. These results indicate that the availability of virus for acquisition by aphids declines with increasing plant age and symptom severity.     

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.     

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.