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.     

蚜虫中携带马铃薯卷叶病毒检测方法的改进

马铃薯卷叶病毒( Potato leafroll virus,PLRV)对马铃薯生产的危害极大,是一种极为重要的马铃薯病毒病。 RT－PCR是马铃薯卷叶病毒检测较为常用的方法,该方法检测准确率高、成本低、适用范围广。但在实际生产中其检测对象多为染病植株,对PLRV传播的主要介体桃蚜( Myzus persicae)的检测,则由于蚜虫体积小、RNA提取难度大、成本高、且不能复检,因而在生产中不能被广泛使用。该研究以马铃薯感病植株和带毒蚜虫为材料,利用改进的RNA提取方法从它们中提取到PLRV的RNA,并以CP 基因设计特异性引物,进行PCR检测。结果表明：该方法提取的RNA完整性好,可用于蚜虫中PLRV检测,且同样适用于对马铃薯感病植株的检测。另外,通过对田间有翅蚜和无翅蚜携带 PLRV 情况进行检测发现,无翅蚜 PLRV 检出率为100％,有翅蚜PLRV检出率也高达60％,证明该体系在生产中的实用性。该研究使用改进的RNA提取方法,提取蚜虫中RNA,并利用RT－PCR进行了PLRV检测,与以前的方法相比简单实用,可被应用于生产检测中。该研究结果为马铃薯生产中PLRV的防控提供了一种新的手段。     

Effects of sub-lethal imidacloprid levels on potato leafroll virus transmission by Myzus persicae

The effects of sub-lethal imidacloprid concentrations on acquisition and inoculation of potato leafroll virus (PLRV) by Myzus persicae (Sulzer) (Hemiptera: Aphididae) were investigated. In experiments using two aphid clones to acquire PLRV from infected potatoes, virus transmission declined significantly with increasing concentrations of imidacloprid. The same was true in experiments using imidacloprid-treated Physalis floridana Rydb. as acquisition sources. When viruliferous M. persicae were placed on uninfected, imidacloprid-treated P. floridana, there were significant declines in PLRV transmission. Sub-lethal concentrations of imidacloprid clearly inhibited both acquisition and inoculation of PLRV by M. persicae, either through poisoning, temporary intoxication, and/or antifeedant effects.     

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.     

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.     

Rates of growth and increase ofMyzus persicae on virus-infected potatoes according to type of virus-vector relationship

Growth, reproduction and survival (=performance) of the aphidMyzus persicae Sulzer was measured on virus-free and virus-infected potato plants The principle objective was to evaluate if various viral infections affected aphid performance differently, and if so, whether any order in the performance response of the aphid was discernible according to the type of virus-vector relationship. Three viruses varying in their dependency onM. persicae as a vector were used. Plants infected with potato leafroll virus (PLRV), a circulative virus highly dependent uponM. persicae for dispersal and transmission, were superior hosts as determined by the significantly greater mean relative growth rate (MRGR) and intrinsic rate of increase (r_m) ofM. persicae compared with those of aphids reared on other plants. Plants infected with potato virus Y, a noncirculative virus less dependent uponM. persicae for dispersal than PLRV, were intermediate in their quality based upon intermediate MRGR and r_m values. Plants infected with potato virus X, a nonvectored virus independent ofM. persicae, were least suitable hosts along with the group of virus-free plants according to the lower MRGR and r_m values.     

Comparison of the potential rate of population increase of brown and green color morphs of Sitobion avenae (Homoptera: Aphididae) on barley infected and uninfected with Barley yellow dwarf virus

Life tables of brown and green color morphs of the English grain aphid, Sitobion avenae (Fabricius) reared on barley under laboratory conditions at 20 ± 1°C, 65% ± 5% relative humidity and a photoperiod of 16 : 8 h (L : D) were compared. The plants were either: (i) infected with the Barley yellow dwarf virus (BYDV); (ii) not infected with virus but previously infested with aphids; or (iii) healthy barley plants, which were not previously infested with aphids. Generally, both color morphs of S. avenae performed significantly better when fed on BYDV‐infected plants than on plants that were virus free but had either not been or had been previously infested with aphids. Furthermore, when fed on BYDV‐infected plants, green S. avenae developed significantly faster and had a significantly shorter reproductive period than the brown color morph. There were no significant differences in this respect between the two color morphs of S. avenae when they were reared on virus‐free plants that either had been or not been previously infested with aphids. These results indicate that barley infected with BYDV is a more favorable host plant than uninfected barley for both the color morphs of S. avenae tested, particularly the green color morph.     

Insecticidal control of aphids and the spread of potato leafroll virus in potato crops in eastern Scotland

Field experiments were carried out in eastern Scotland in 1976-78 to test the ability of granular insecticides, applied to soil at planting, and of insecticide sprays applied to the foliage, to control aphids and spread of potato leafroll virus (PLRV) in potatoes. The three years provided contrasting opportunities for virus spread. In 1976, the main vector of PLRV, Myzus persicae, arrived in early June and multiplied rapidly in untreated plots, and PLRV spread extensively. In 1977, M. persicae arrived 4–6 wk later than in 1976 and most spread of PLRV, which was less than in 1976, occurred after the end of July. In 1978, few M. persicae were recorded but the potato aphid, Macrosiphum euphorbiae, arrived early and very large populations developed in untreated plots. However, little spread of PLRV occurred in 1978, supporting other evidence that M. euphorbiae is an inefficient vector of PLRV in field conditions. In each year, granular insecticides decreased PLRV spread to a quarter or less of that in control plots. Thiofanox gave somewhat better and longer-lasting control of aphid populations than disulfoton, especially of M. persicae, but did not give greater control of PLRV spread. Application of three (1976) or five (1977) sprays of demeton-S-methyl to plots treated with granular insecticides further improved the control of M. euphorbiae but had less or no effect on M. persicae, especially where organophosphorus resistant aphids (R1 strain) were found. These supplementary sprays of insecticide did not further improve the control of PLRV but, in 1978, four sprays of demephion or pirimicarb to plots not treated with granular insecticide decreased PLRV spread. These data, together with previous findings, indicate that the amount of virus spread depends on the date of arrival and rate of multiplication of M. persicae in relation to the timing and effectiveness of removal of PLRV sources in crops. It is concluded that in Scotland insecticide granules should be used routinely only in crops of the highest grade of seed potato. Their use for other grades need be considered only in years following mild winters, when aphids can be expected to enter crops earlier and in larger numbers.     

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.     

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.     

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.     

Some effects of spraying with thiabendazole on the susceptibility of sugar beet to yellowing viruses and on their vector, Myzus persicae (Sulz.)

In a field experiment fewer sugar-beet plants became infected with aphid-transmitted yellowing viruses in plots that had been sprayed with solutions of thiabendazole lactate than in water-sprayed plots, after exposure to natural infestation with aphids. Subsequent glasshouse tests showed that foliar sprays of o·o1 % thiabendazole lactate in water significantly reduced the proportion of inoculated sugar-beet plants which became infected with beet yellows virus (BYV) or beet mild yellowing virus (BMYV) after inoculation with viruliferous Myzus persicae (Sulz.). This effect on virus transmission was not apparently due to a direct insecticidal action of thiabendazole, because adult aphids usually survived equally well on sprayed and unsprayed plants. Treatment of test plants with thiabendazole did not affect the transmission of beet mosaic virus to them by M. persicae. The fecundity of M. persicae was greatly reduced by transferring them to plants which had been sprayed with thiabendazole or by spraying them with thiabendazole before transfer to unsprayed plants. The fertility of adult Aphis fabae Scop, was also reduced by spraying with thiabendazole. The mechanisms whereby thiabendazole affected fecundity of aphids and transmission of viruses are not understood.     

Spread of barley yellow dwarf virus and relative importance of local aphid vectors in central Washington

Data from bioassays of field collected aphids, barley indicator plants exposed to natural conditions, and various types of aphid traps were used to describe the spread of barley yellow dwarf virus (BYDV) in wheat and barley near Prosser, Washington. Bioassays were also used to assess the relative importance of local vector species. Of alate aphids collected from grain in the 1982 and 1983 fall migration seasons, 3.4–14–5% transmitted BYDV. Data from concurrent and post-migration assays of resident aphids (apterae and nymphs) reflected an increase in the proportion of infected plants in the field. Maximum increase in the percentage of viruliferous aphids occurred in late November and December of 1982 and November of 1983. The 1982 increase occurred after aphid flights had ceased for the year, suggesting active secondary spread. Collections in pitfall traps and infected trap plants from November to February confirmed aphid activity and virus spread. Rhopalosiphum padi was the most important vector in central Washington in 1982 and 1983 because of its abundance and relative BYDV transmission efficiency. Metopolophium dirhodum was more winter-hardy than R. padi and equal to R. padi in its efficiency as a vector; however, it was not as abundant as R. padi except during the mild winter of 1982–83, when it was a major contributor to secondary spread. Sitobion avenae may be important in years when it is abundant, but it was only a quarter as efficient as R. padi. Rhopalosiphum maidis was a much less efficient vector than R. padi and it only reached high populations in late autumn barley.     

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.     

Barley yellow dwarf virus in aphids caught in suction traps, 1969-73

Suction traps operating at low level (1 5 m) were used to catch live alate Rhopalosiphum padi, Macrosiphum (Sitobion) avenae and Metopolophium dirhodum which were tested for transmission of barley yellow dwarf virus (BYDV). The first species caught and infective was R. padi, followed by M. (S.) avenae infective some 2–3 wk later and M. dirhodum 3–4 wk later still. Never more than 11-5% of the annual catch of any species transmitted BYDV and the proportion fluctuated from week to week and between seasons in different years. The relative abundance of infective vectors of ths three species varied; annual numbers of infective M. (S.) avenae and M. dirhodum varied inversely with infective R. padi, the latter also usually transmitted severer virus. The results of the infectivity tests have been compared with the catches of these aphids by the Rothamsted Insect Survey and show that numbers of alate aphids do not necessarily indicate the likely incidence of BYDV.     

The effect of barley yellow dwarf virus on honeydew production by the cereal aphids, Sitobion avenae and Metopolophium dirhodum

Individual S. avenae and M. dirhodum excreted significantly fewer droplets of honeydew on plants infected with BYDV than on healthy plants. S. avenae excreted less honeydew on the ears than on the leaves of wheat. M. dirhodum excreted less than S. avenae on the leaves. The size of honeydew droplets increased with the age of aphids but was not affected by BYDV infection. Possible reasons for the observed effects of BYDV on honeydew excretion are discussed.