The importance of the infected seed tuber and soil inoculum in transmitting Potato mop-top virus to potato plants

Authors


Abstract

Potato mop-top virus (PMTV), the cause of spraing in potato tubers, is transmitted by Spongospora subterranea, the cause of powdery scab, and by planting infected seed tubers. This study was undertaken to determine the relative importance of these sources of infection in seed potato production in Scotland. The transmission of PMTV from tested seed tubers to daughter plants was examined over 2 years and six cultivars. The development of foliar symptoms varied with year and cultivar. Infection of daughter tubers derived from PMTV-infected seed tubers was more prevalent on plants affected by foliar symptoms than those without symptoms. The rate of transmission of PMTV from infected seed tubers to daughter tubers ranged from 18 to 54%. Transmission was affected by cultivar and by origin of seed tubers used for a cultivar, but not by a cultivar's sensitivity to PMTV infection. The incidence of PMTV in daughter tubers of cv. Cara grown from seed potatoes from one source (common origin) by more than 25 seed producers was examined over two successive generations. The incidence of PMTV in daughter tubers was not correlated with that in the seed tubers but appeared to be strongly associated with soil inoculum. The incidence of PMTV was correlated with powdery scab in those crops in which both were present. There was some evidence from soil tests conducted in 2006 using a tomato bait plant and real-time RT-PCR that planting PMTV-infected seed potatoes could increase the risk of introducing the virus into land not infested by PMTV.

Introduction

Potato mop-top virus (PMTV) is a rod-shaped virus of the genus Pomovirus (Torrance & Mayo, 1997) and has a tripartite genome consisting of three single-stranded, positive sense RNA molecules (Torrance et al., 1999). In tubers of potato (Solanum tuberosum), PMTV infection can induce symptoms known as spraing which are slightly raised lines and rings on the tuber surface and/or brown arcs and lines in the flesh of sensitive cultivars (Calvert & Harrison, 1966; Harrison & Jones, 1971). These symptoms render tubers unsuitable for consumption or processing. Misshapen and cracked tubers may also be produced by plants derived from PMTV-infected seed tubers (Calvert, 1968; Tenorio et al., 2006). The virus can also produce symptoms on the foliage (Calvert, 1968), although often only a proportion of stems on a plant are affected. However, the development of foliar symptoms is strongly affected by environmental conditions (Calvert, 1968; Carnegie et al., 2010a) and thus their prevalence can vary considerably amongst the countries in which PMTV is present. For example, foliar symptoms are relatively common in the cool climate of Scotland (Carnegie et al., 2010b) but rare in Scandinavian countries with the exception of Norway (Santala et al., 2010).

The virus can spread from infected seed tubers to the foliage and tubers of the daughter plant but its systemic spread occurs more slowly and erratically than with other viruses such as Potato leafroll virus (Jones, 1988; Carnegie et al., 2010b). Cooper & Harrison (1976) found that tubers from plants of cv. Red Craigs Royal with foliar symptoms produced only a proportion (c. 50%) of daughter plants with foliar symptoms in the following year. Similarly, in a small study assessing the incidence of PMTV in daughter tubers of cv. Atlantic, Carnegie et al. (2010b) reported a rate of transmission of 19–42% and suggested that this was affected by the presence and severity of foliar symptoms. Symptomless infection by PMTV can occur in foliage (Browning et al., 2002) and in tubers (Latva-Kilby et al., 2009) and thus evidence of transmission based on symptoms may underestimate the true rate of virus transmission.

Spongospora subterranea, the cause of powdery scab, was considered by Jones & Harrison (1969) to be the vector for the transmission of PMTV and this was proved conclusively by Arif et al. (1995) who demonstrated that PMTV could be acquired by S. subterranea from roots of infected plants and then transmitted to healthy bait plants. PMTV has often been found to occur in Scotland in the absence of powdery scab on individual tubers (Jones & Harrison, 1969) or on tubers within crops (Carnegie et al., 2012). In the Peruvian Andes, Tenorio et al. (2006) also reported the absence of a correlation between infection by PMTV and the incidence or severity of powdery scab. However, S. subterranea can also infect roots, resulting in the development of root galls and this infection occurs at temperatures greater than the optimum range of 12–15°C for powdery scab (van de Graaf et al., 2007). Carnegie et al. (2010a) demonstrated that PMTV infection from PMTV-infested soil occurred at similar rates between 12 and 20°C and concluded that infection by PMTV could occur through roots or tubers infected by S. subterranea without necessarily resulting in powdery scab.

Soil infestations by PMTV have been detected using sensitive bait plants such as Nicotiana benthamiana in which diagnostic symptoms of systemic infection by PMTV can be produced, although sometimes not reliably (Arif et al., 1994). Virus-specific tools, e.g. antibodies and PCR primers, have been developed more recently enabling the virus to be detected in the tissue of bait plants (Santala et al., 2010). Davey (2009a) described a test using tomato as a bait plant and real-time, reverse-transcription polymerase chain reaction (RT-PCR) to detect the virus in roots after 2–3 weeks in test soil. However, the effectiveness of such tests has not been evaluated in the field or related to disease outbreaks in crops.

The objective of this study was to determine experimentally the transmission of PMTV from tested PMTV-infected seed tubers to foliage and daughter tubers in the field. The development of PMTV was monitored in seed potato crops grown from common origin seed potatoes during two consecutive multiplications of commercial seed potatoes at a large range of locations in Scotland. The effectiveness of a tomato and real-time RT-PCR soil assay in detecting PMTV in soil samples from the production fields was also assessed.

Materials and methods

Infected seed tuber experiments

Four experiments were conducted over 2 years at SASA's Gogarbank Farm to examine the transmission of PMTV from infected seed tubers to daughter plants of a range of cultivars. Samples of seed potatoes were obtained from commercial crops identified as being considerably infected by PMTV. Tubers from each identified crop were tested by enzyme-linked immunosorbent assay (ELISA) to obtain PMTV-infected and PMTV-free tubers. Cultivars, numbers of PMTV-infected and -free tubers planted in each experiment and the number of replicates in randomized blocks are shown in Table 1. In both years, because only a few suitable crops were identified, two samples of seed potatoes of the same cultivar grown by different producers were chosen for inclusion in the experiments and were identified as different seed sources (Table 1). These were cv. Nicola in 2004 and cv. Cara in 2005. In addition, in 2004, the infected tubers of cv. Cara were divided into lots of 120 with spraing symptoms and 120 without symptoms. Each plot consisted of four drills, each of 10 tubers, except for Experiment 2 with cv. Slaney in which there were two drills. Tubers were spaced 0·38 m apart along the drills that were 0·7 m apart. Plots were separated by planting tubers of the blue skinned cv. Edzell Blue in the adjacent drills and along the end of each drill. A compound fertilizer was applied just before planting at the rate of 330 units N, 660 units P and 660 units K per hectare. Weeds were controlled by applying a mixture of linuron and paraquat at 25% plant emergence. Pesticides were applied according to local practice to control pests and diseases. Haulm was killed by applying diquat dibromide (Reglone, Syngenta) at the manufacturer's recommended rate.

Table 1. Details of cultivars, number of Potato mop-top virus (PMTV)-free and -infected tubers planted and number of replicates included in randomized block experiments in 2004 and 2005 at SASA's Gogarbank Farm
YearExperimentCultivarNo. of seed tubers for plantingNo. of replicates
PMTV-freePMTV-infected
  1. a

    Two seed sources A and B.

  2. b

    Infected seed divided: tubers with spraing and symptomless tubers.

  3. c

    Two seed sources C and D.

20041Maris Piper, Nicolaa1201203
2Slaney 80 804
3Cara120120/120b3
20054Carac, Nicola, Rooster, Winston1201203

Foliage was inspected for visual symptoms characteristic of PMTV infection during the growing season and affected plants marked with canes. Leaflets from plants with and without symptoms were sampled and tested for PMTV by double-antibody sandwich ELISA (DAS-ELISA; Fox et al., 2005). Plants of a different cultivar and plants affected by other viruses or blackleg were marked separately and their produce discarded at harvest.

At harvest, first the daughter tubers from plants with symptoms in each plot were lifted by fork and placed in separate new polypropylene bags and then the produce of symptomless plants was harvested into bags using a single-row digger. Tubers were stored in the dark at 18 ± 2°C for 2 weeks followed by at least 1 week at 4 ± 2°C. For each plot, all daughter tubers from plants with symptoms and 150 randomly sampled tubers from symptomless plants were tested for PMTV by DAS-ELISA before conducting an assessment for spraing.

Experiments in 2004

The cultivars were Maris Piper and Nicola (in the case of the latter, seed potatoes were sourced from two separate growers, A and B) in Experiment 1, cv. Slaney in Experiment 2 and cv. Cara in Experiment 3. Tubers were planted on 18 May, haulm desiccant was applied on 2 September and daughter tubers were harvested 3 weeks later.

Experiment in 2005

In Experiment 4, the cultivars were Cara (seed sources C and D), Nicola, Rooster and Winston. Tubers were planted on 10 May, haulm desiccant was applied on 18 August and daughter tubers were harvested in early October.

PMTV transmission during commercial seed potato multiplication

During the 2000s, seed potatoes of cv. Cara were being multiplied by a unique system. The initial seed potatoes were multiplied by a Pre-basic seed potato grower in Ross & Cromarty in the north of Scotland, to produce class SE1 or SE2 seed potatoes that were then distributed to over 25 producers of basic category seed potatoes, who grew the material for a further 1 or 2 years before marketing, largely for end use production. The incidence of PMTV during this multiplication process was assessed between 2004 and 2006 to gain information on the development of PMTV in crops at a large range of locations in Scotland when grown initially from common origin seed potatoes.

In 2004, seed potatoes from one crop of class SE1 and another of class SE2 were distributed to 31 growers of which 19 were in Angus, seven in Perthshire, two in Fife, and one in each of Moray, Banffshire and Aberdeenshire. In 2005, all common origin seed potatoes were of class SE2 and were used to produce 27 crops by 26 growers of which 14 were in Angus, six in Perthshire, two in each of Fife and Aberdeenshire and one in each of Moray and Banffshire. In 2005, 57 crops were produced from the first generation seed potatoes and in 2006, there were 28 crops.

Each year, before the common origin seed potatoes were despatched to the growers, a sample of 150 tubers was collected from each crop by a seed potato inspector and sent to SASA for PMTV testing. Random samples of 200 (2004) or 150 (2005 and 2006) daughter tubers were collected from each crop as soon as possible after harvest. After all the samples were received, they were stored as described above for the infected seed tuber experiments to induce spraing symptoms.

Virus and disease assessment

All tubers were washed before conducting the assessments.

Leaflet and tuber testing for PMTV by DAS-ELISA

For plants with symptoms, one leaflet was collected from each of four compound leaves on the affected stems. For symptomless plants, one leaflet was sampled from four compound leaves on separate stems of the plant. The four leaflets were placed into the rear of a BIOREBA homogenization bag (BIOREBA AG). Five millilitres of leaf extraction buffer were added to the bag and leaflets were ground using a Homex 5 homogenizer (BIOREBA) after which a further 5 mL of buffer were added to the bag.

In order to determine if a tuber was infected by PMTV, cores of tuber tissue, 25–30 mm in length and 5 mm in diameter, were taken with a cork borer from the rose- and stolon-end of each tuber and placed together in a homogenization bag to which 5 mL of tuber extraction buffer were added before grinding, as for leaflets.

ELISA tests were conducted as described by Carnegie et al. (2012) using a PMTV-specific monoclonal antibody (http://www.sasa.gov.uk/diagnostics/antibody-unit).

Disease assessment

The number of tubers affected by powdery scab was determined by visual examination. Tubers were cut lengthwise to expose six surfaces that were examined for spraing symptoms.

Soil testing for PMTV

Two probe and primer pairs for the detection of each of the RNA 1 and RNA 2 molecules in the PMTV genome were designed using Primer Express v. 1.5 (Applied Biosystems) and synthesized by MWG-Biotech AG. One primer and probe combination was also designed to amplify the RNA 3 molecule and these newly designed primer and probe combinations were evaluated in comparison with the RNA 3 primer set designed by Mumford et al. (2000). After assessing their effectiveness in detecting PMTV in naturally infested soil samples using tomato as the bait plant and real-time RT-PCR assay (Davey, 2009a), one RNA 2 primer/probe combination (forward primer 5′-AGAATTGRCATCGAAACAGCA-3′, reverse primer 5′-GTCGCGCTCCAATTTCGTT-3′ and dual-labelled probe 5′FAM-CCACAAACAGACAGGTATGGTCCGGAA-TAMRA3′) was selected for further comparative testing with the RNA 3 set of Mumford et al. (2000) using soil samples collected from fields used to produce the seed potato crops of cv. Cara.

In 2004, samples were collected from 31 fields in early to mid-August and from 28 fields in 2006 prior to planting and again in the spring of the following year. Sampling units within fields were confined to 4 ha or less. Cores, 5 × 1 cm, were taken at various points over each unit during a W-shaped walk to obtain c. 600 mL soil. On receipt at SASA, each sample was spread out on a 30 × 40 cm plastic tray and placed in a drying cabinet at room temperature for at least 1 week. Where there was more than one sample for a field, these were bulked and mixed. Three replicate lots, each of 20 g, were taken at random from each field sample for testing. Tests for PMTV were then conducted using the tomato and real-time RT-PCR assay as set out by Davey (2009a). Samples were deemed to be positive if two out of the three replicates analysed were positive.

Statistical analysis

Prior to analysis, the percentage of tubers infected by PMTV and affected by spraing in each replicate plot in the experiments was calculated in proportion to the relative incidence of plants with foliar symptoms and those without. A binomial logistic regression model (GenStat v. 8.1, Lawes Agricultural Trust, 2005) was used to analyse data from the experiments. Chi-squared (χ2) one-tailed test of significance was also used in these experiments to compare the frequency of PMTV tuber infection in plants with foliar symptoms with those without symptoms where sample sizes were unequal. In the field study with cv. Cara, the relationships between the incidences of PMTV, spraing and powdery scab in daughter tubers were tested using correlation and linear regression analysis.

Results

Infected seed tuber experiments

Symptoms and PMTV detection in foliage

The expression of symptoms did not differ between plants derived from PMTV-infected seed tubers and those from PMTV-free seed tubers, except that the few severe symptoms that did develop were confined to plants from PMTV-infected seed tubers. Foliar symptoms on plants of cvs Slaney and Cara consisted of distortion of the leaf and/or a reduction in size of leaflets but were confined to only one or two stems on a plant with some limited shortening of the stem. Symptoms on plants of cv. Nicola were generally more severe than those on the other cultivars with the occasional ‘mop-top’ plant being observed. Cultivars Rooster and Winston developed no foliar symptoms and only one plant of cv. Maris Piper developed symptoms, which were very mild.

On plants derived from PMTV-free seed tubers, foliar symptoms characteristic of PMTV infection were observed on a relatively small proportion of plants of four stocks (cvs Nicola (A and B) and Slaney in 2004; cv. Cara (D) in 2005; Table 2). However, PMTV was detected by ELISA in the foliage of plants of only 3 stocks (cvs Nicola (B) and Slaney in 2004; cv. Nicola in 2005).

Table 2. Mean incidence of foliar symptoms attributable to Potato mop-top virus (PMTV) and foliar infection by PMTV, as determined by ELISA, on plants of six cultivars derived from PMTV-free and PMTV-infected seed tubers and mean incidence of PMTV and spraing in daughter tubers over 2 years
YearHealth status of seed tuberExperimentCultivar% plants with PMTV foliar symptoms% plants PMTV detected% daughter tubers
Infected by PMTVAffected by spraing
  1. NS, not significant.

  2. a

    Letter indicates seed potatoes obtained from different producers.

  3. b

    Probability derived from regression analysis for infected seed only.

  4. c

    < 0·05.

  5. d

    < 0·01.

  6. e

    < 0·001.

2004PMTV-free1Maris Piper0000
Nicola (A)a5010
Nicola (B)2200
2Slaney1110·2
3Cara0030
PMTV-infected1Maris Piper4db33c34e0e
Nicola (A)23d23c33e2e
Nicola (B)10d12c18e1e
2Slaney5721425
No spraing3Cara44NS18NS54e3e
Spraing3Cara30NS31NS45e12e
2005PMTV-free4Cara (C)0010
Cara (D)100·20·2
Nicola0210
Rooster000·20
Winston000·20
PMTV-infected Cara (C)13e32NS34e11e
Cara (D)9e36NS41e5e
Nicola15e32NS32e1e
Rooster0e27NS23e1e
Winston0e40NS38e0·2e

The incidence of foliar symptoms on plants derived from PMTV-infected seed tubers was generally greater in 2004 than in 2005 and varied amongst varieties (Table 2). In 2004, symptoms were most prevalent on plants of cv. Slaney, followed by cv. Cara, then cv. Nicola. The frequency of symptoms on cvs Cara and Nicola was broadly similar in 2005 (Table 2). Over the 2 years, the incidence of foliar symptoms was nil or very low in cvs Maris Piper, Rooster and Winston although the incidence of PMTV detection in leaves by ELISA was not different to that for the other cultivars. In 2005, the incidence of PMTV in foliage did not differ significantly amongst the cultivars but in 2004 it was least for plants of cv. Nicola from seed source B and greatest for plants of cv. Maris Piper. In 2004, the incidence of foliar symptoms and foliar infection by PMTV did not differ significantly between plants derived from infected seed tubers with spraing symptoms and those from tubers with no symptoms.

Tuber infection by PMTV and spraing

PMTV was detected in daughter tubers of 8 out of 10 stocks derived from PMTV-free seed tubers but generally at very low incidences, 1% or less (Table 2). On plants derived from PMTV-infected seed tubers, the incidence of PMTV in daughter tubers varied between 18 and 54% (Table 2) and was significantly affected by cultivar and source of seed tubers used for a cultivar. In 2005, PMTV was least prevalent in daughter tubers of cv. Rooster and most prevalent in those of cv. Cara from seed source D. However, the incidence of PMTV in tubers of cv. Cara was greater for those derived from seed tubers from origin D than for origin C. A difference between tubers from different seed origins was also recorded in 2004 with cv. Nicola.

In both years, the incidence of spraing was much lower than that for PMTV tuber infection. Spraing was most frequent in tubers of cv. Cara in both years. However, in 2005 there was also a difference between the two origins of seed tubers of cv. Cara used for planting. Seed tubers with spraing symptoms produced daughter tubers with significantly more spraing than those from seed tubers without symptoms but this was the reverse of the pattern for PMTV tuber infection. The incidence of PMTV in daughter tubers was greater for plants with foliar symptoms than for those with no foliar symptoms in all 6 comparisons, although statistically significant for only three (Table 3). The difference was greatest for the two Nicola (A and B) stocks in 2004.

Table 3. Percentage of daughter tubers infected by Potato mop-top virus (PMTV) for plants with or without PMTV foliar symptoms derived from PMTV-infected tubers
YearCultivarPMTV foliar symptomsNo PMTV foliar symptoms math formula
  1. a

    Figure in parenthesis is number of tubers tested.

  2. b

    Letter indicates seed potatoes obtained from different producers.

  3. c

    < 0·05.

  4. d

    < 0·001.

2004Maris Piper38 (37)a33 (610) 0·4
Nicola (A)b50 (224)25 (458)29·1c
Nicola (B)42 (88)14 (450)17·6c
2005Cara (C)37 (221)34 (450) 0·5
Cara (D)47 (183)40 (450) 2·5
Nicola37 (212)31 (450) 4·2d

PMTV transmission during commercial seed potato multiplication

Common origin seed potatoes

PMTV was detected in 0·5% of tubers of both class SE seed potato crops produced in 2003 for planting in 2004. The seed tubers produced in 2004 contained 0·7% PMTV.

First year of multiplication from common origin seed potatoes

In 2004, PMTV was not detected in daughter tubers of 29% of crops produced from common origin seed potatoes but only one crop was free from powdery scab (Table 4). However, PMTV was less common in 2005 with 41% of crops being free of the virus. Powdery scab was also less common in 2005 with 19% of crops having no diseased tubers. In addition, the incidence of tuber infection by PMTV in PMTV-infected crops differed considerably from that for powdery scab. In 2004, the incidence of PMTV in daughter tubers exceeded 15% in only two crops (6%) compared with 55% of the crops having more than 20% of their tubers affected by powdery scab. The difference was much less in 2005 with 7% of crops exceeding 15% tuber infection by PMTV compared with 26% of crops with more than 20% of tubers affected by powdery scab.

Table 4. Incidence of Potato mop-top virus (PMTV) and powdery scab on daughter tubers of 31 crops (2004) and 27 crops (2005) of cv. Cara derived from seed tubers of a common origin with 0·5 and 0·7% tuber infection by PMTV in 2004 and 2005 respectively
Tubers infected by PMTV (%)No. of cropsTubers affected by powdery scab (%)No. of crops
2004200520042005
0911015
>0–51911>0–10910
>5–1001>10–2045
>10–1512>20–3023
>15–2000>30–4023
>20–2500>40–5030
>25–3001>50–6040
>30–3501>60–7030
>35–4010>70–8021
>40–4500>80–9010
>45–5000   
>50–5510   

In 2004, eight of nine daughter crops in which PMTV was not detected were affected by powdery scab and in 2005, five out of 11 were affected. In these crops the incidence of powdery scab varied from 3·5 to 76·1% in 2004 and from 3·3 to 77·3% in 2005. In 2004, one crop contained 0·5% tuber infection by PMTV but no powdery scab was seen in the sampled tubers. For those crops in which both PMTV and powdery scab were present, there was a significant (< 0·05) linear correlation between the incidence of tuber infection by PMTV and that of tubers affected by powdery scab in both years (2004, r = 0·48, 20 d.f.; 2005, r = 0·61, 14 d.f.).

Second year of multiplication from common origin seed potatoes

When crops were grown from seed tubers with varying amounts of PMTV infection in 2005, the incidence of PMTV in daughter tubers was not correlated (r = –0·09, 55 d.f.) with that in the seed tubers. The incidence of PMTV in daughter tubers was 10% or lower in 88% of the 57 crops with the greatest amount of tuber infection being 47% (Table 5). This latter crop was produced from seed potatoes in which PMTV had been found in 1·4% of sampled tubers. Nine other sister crops were also produced from these seed potatoes. PMTV was not detected in the daughter tubers of two crops but another contained 28% PMTV tuber infection. The greatest amount of tuber infection by PMTV in seed potatoes used for planting in 2005 was 34·9%. Three crops produced from these seed potatoes yielded one crop with no detectable infection by PMTV and two crops with 2·0 and 1·3% of tubers infected by PMTV.

Table 5. Incidence of Potato mop-top virus (PMTV) and powdery scab on second generation daughter tubers of 57 crops (2005) and 28 crops (2006) of cv. Cara derived from seed tubers of a common origin
Tubers infected by PMTV (%)No. of cropsTubers affected by powdery scab (%)No. of crops
2005200620052006
01910053
>0–52713>0–101717
>5–1043>10–2091
>10–1520>20–30123
>15–2000>30–4030
>20–2510>40–5040
>25–3010>50–6041
>30–3511>60–7021
>35–4010>70–8001
>40–4500>80–9010
>45–5011   
>50–5500   

Similarly, the greatest incidence of PMTV in the 18 crops of seed potatoes used for planting in 2006 was 13·3% and that in 28 daughter crops was 45·3%. As in the previous year, the incidence of PMTV in seed tubers was not correlated with that in daughter tubers (r = 0·06, 26 d.f.) The two crops with the greatest amounts of infection of daughter tubers by PMTV, 45·3 and 30·4%, were derived from seed potatoes in which PMTV had not been detected in the test sample. In contrast, seed potatoes with 13·3% of tubers infected by PMTV produced two daughter crops with 4·7 and 0·7% tuber infection.

The number of second generation crops with no powdery scab on their daughter tubers was 5 out of 57 (9%) crops in 2005 and 3 out of 27 (11%) crops in 2006 (Table 5). The greatest incidence of powdery scab on daughter tubers was 84·7% in 2005 and 71·3% in 2006. In each year, one crop was found to contain 0·7% tuber infection by PMTV but no powdery scab. There was also a strong significant correlation (< 0·001) between the incidence of PMTV in daughter tubers and that of powdery scab for those crops in which both PMTV and powdery scab occurred (Fig. 1).

Figure 1.

Linear relationship ((a) 2005, y = –3·12 + 0·41x, r = 0·64, 36 d.f., < 0·001; (b) 2006, y = –1 + 0·42x, r = 0·82, 16 d.f., < 0·001) between the incidence of tuber infection by Potato mop-top virus (PMTV) and the incidence of powdery scab on tubers of seed potato crops of cv. Cara containing both virus and disease which were grown in Scotland as second generation basic category seed potatoes derived from common origin seed potatoes.

Over the 3 years, the incidence of spraing was strongly correlated with the incidence of PMTV in the daughter tubers of those crops in which both virus and symptoms were present (Fig. 2). However, the estimated incidence of spraing was lower than that for PMTV. In addition, spraing was also recorded in some tubers of 10 PMTV-free crops, at incidences varying from 0·7 to 20%.

Figure 2.

Linear relationship (y = 0·37 + 0·73x, r = 0·92, 144 d.f., < 0·001) between the incidence of tuber infection by Potato mop-top virus (PMTV) and the incidence of spraing in tubers of Scottish seed potato crops of cv. Cara in which both virus and disease were present between 2004 and 2006.

Soil testing for PMTV

PMTV was detected in 144 out of 425 triplicate soil samples in all the tests conducted in 2004 and 2006. However, the RNA 3 primer set did not detect PMTV in 23·6% of triplicates in which the virus was found using the RNA 2 primer set; therefore, only the results for RNA 2 primer set were used in analysing and presenting the field soil results. In 2004 and 2006, PMTV was detected in all fields soils which produced crops with >10% tuber infection by PMTV (Table 6). For crops in which the amount of tuber infection by PMTV ranged from >0–5%, PMTV was found in only seven out of 19 field soils sampled in August in 2004 and in nine out of 13 soils sampled before planting in 2006. PMTV was also detected in some soils producing crops in which no infection of daughter tubers by PMTV was detected. In 2004, infestation by PMTV was found in one out of nine soils producing PMTV-free crops and in five out of 10 soils in 2006. Of the 18 field soils in which PMTV was detected before planting in 2006, only 15 were found to be infested in the post-harvest samples. In contrast, six out of the 10 fields free of PMTV before planting were found to be infested by PMTV after harvest. PMTV-infected seed tubers had been planted in only four of the six field soils.

Table 6. Proportion of field soils in which Potato mop-top virus (PMTV) was detected when sampled in early to mid-August in 2004 and before planting in 2006 using an RNA 2 primer set in relation to the incidence of PMTV tuber infection in daughter tubers of seed potato crops of cv. Cara
Tubers infected by PMTV (%)Number of soils PMTV detected/total tested
20042006
  1. –, no crops within this range of daughter tuber infection.

01/95/10
>0–57/199/13
>5–102/3
>103/32/2

Discussion

Foliar symptoms in the seed tuber transmission experiments were generally mild but did vary in frequency amongst the cultivars and between years. The latter may be a reflection of growing conditions in the two test years because the development of foliar symptoms is favoured by cool conditions, particularly in spring (Calvert, 1968; Carnegie et al., 2009). The symptoms on cvs Cara and Nicola were, however, similar to those reported by Carnegie et al. (2010b) for those cultivars in field crops. The incidence of symptomless foliar infection on plants of the susceptible cultivars Cara and Nicola derived from PMTV-infected seed tubers was greater in 2005 when symptoms were less prevalent than in 2004 (Table 2). This suggests that the movement of PMTV into foliage was unaffected by year, but the extent of symptom expression was. The incidence of PMTV infection in daughter tubers derived from PMTV-infected seed tubers was greater for plants showing foliar symptoms than for symptomless plants (Table 3). This is in agreement with the results of Carnegie et al. (2010b) who similarly tested plants of cv. Atlantic in field crops. Removing plants with symptoms from infected crops would, therefore, reduce the overall incidence of tuber infection in the harvested crop, but this strategy may be impractical if plants with symptoms are too prevalent and ineffective if the amount of symptomless infection is too high.

Testing for PMTV in the seed transmission experiments confirmed the relatively low rate of movement that occurs for this virus between seed and daughter tubers. The rate varied from 54% for cv. Cara to 18% for cv. Nicola (seed source B) in Experiment 1 (Table 2). This range is similar to the rates of 19–41% reported by Carnegie et al. (2010b) when testing daughter tubers of cv. Atlantic derived from seed tubers from plants with foliar symptoms of PMTV infection. While the rate of transmission appeared to be affected by cultivar, more surprisingly it also differed as much when seed tubers were sourced from different farms. One possible explanation could be that tubers from the different producers carried different amounts of spore balls, which could provide an additional contribution to PMTV infection of the plant during the growing season through root infection (Santala et al., 2010). However, this does emphasize the need to treat individual comparisons between cultivars or treatments with some caution. It is also clear that the rate of PMTV transmission was not affected by the sensitivity of a cultivar to foliar or tuber symptoms, supporting the findings of Carnegie et al. (2009). For example, in 2005, the incidence of daughter tuber infection by PMTV was similar for the insensitive cvs Rooster and Winston and the sensitive cvs Cara and Nicola. In addition, cultivars that were insensitive to foliar infection also appeared to be insensitive to tuber infection e.g. cvs Maris Piper, Rooster and Winston (Table 2). Infected seed tubers of insensitive cultivars can, therefore, act as carriers of PMTV from one field to another, where the virus could be picked up by a PMTV-free population of S. subterranea.

In the seed tuber transmission experiments, testing seed tubers from PMTV-infected stocks to obtain PMTV-free seed tubers for planting revealed that a very low proportion of infected tubers may be missed by an ELISA test using two bulked cores. Overall, this proportion was around 0·8% based on the incidence of foliar symptoms and 0·6% based on foliar infection detected by ELISA (Table 2). I. Browning (SASA, UK, personal communication) found that testing rose and middle cores detected at least 99·5% of the infected tubers found by testing three cores: rose, middle and heel. Use of molecular tests may increase the effectiveness of tuber testing (Latva-Kilby et al., 2009) although the additional cost may outweigh the practical benefit.

The results of the study of crops of cv. Cara indicate that soil inoculum is considerably more important than seed inoculum in causing significant amounts of infection of daughter tubers by PMTV, and hence potentially disease, during one growing season. This was illustrated by the high incidences of tuber infection that occurred in a few crops in both years when grown from common origin seed potatoes containing very small amounts of PMTV infection (Tables 45). The relative importance of soil inoculum was confirmed by the absence of a relationship between the incidence of PMTV in seed and daughter tubers in the second generation of multiplication from common origin seed potatoes. The source of this infection will have been spore balls of S. subterranea carrying PMTV. The occurrence of high incidences of tuber infection by PMTV was considerably less frequent than those of tuber infection by S. subterranea as expressed by powdery scab symptoms (Table 4). However, a statistically significant correlation between the incidence of PMTV and powdery scab was recorded in all 3 years in Scotland when only the data from PMTV-infected crops with powdery scab were included in the analysis. Applying this criterion meant that crops affected by spraing from other causes and powdery scab affected crops in which PMTV was not present were excluded from the analysis and allowed the relationship between infection by S. subterranea and PMTV to be revealed. The correlation was stronger in 2005 and 2006 than in 2004 but relatively weak overall, indicating that PMTV infection can be associated with infections by S. subterranea other than those causing powdery scab, as reported previously by Jones & Harrison (1969) and Carnegie et al. (2012). In addition, such an association may not occur in other environments, such as Peru (Tenorio et al., 2006). Carnegie et al. (2010a) showed that PMTV infection could occur in the absence of powdery scab through root and stolon infection, particularly at temperatures greater than 16°C. Carnegie et al. (2012) demonstrated that PMTV was present in only 46% of crops containing daughter tubers affected by powdery scab and this may account for those crops of cv. Cara with powdery scab but no detectable tuber infection by PMTV.

Testing field soils before planting for the presence of PMTV could, therefore, be a useful management tool in determining the risk of PMTV infection of sensitive cultivars. In the study using the seed production system for cv. Cara, PMTV was detected in seven out of eight fields that produced crops with more than 5% tuber infection, although it was also found in the soil from six fields producing crops with no detectable PMTV in the sampled daughter tubers (Table 6). This latter finding may have been the result of unfavourable local conditions for PMTV infection or of limitations in the sampling and test methodology. Davey (2009b) found PMTV to be unevenly distributed in two infested fields when sampling was conducted in 1 ha units so useful benefits might be achieved by a more extensive study of the distribution of PMTV in fields using smaller sampling units in order to provide more detailed information to guide future sampling strategies. A limitation of the tomato and real-time RT-PCR method is that it does not allow the amount of PMTV infestation in a sample to be measured. While some workers (Qu et al., 2006; Nakayama et al., 2007; Brierley et al., 2012) have recorded a relationship between the concentration of spore balls in soil and the amount of powdery scab on daughter tubers, others (Parker, 1984; Christ, 1989; van de Graaf et al., 2005) found none. No studies have been done on this aspect for PMTV.

Although seed inoculum does not appear to be important in causing economically serious occurrences of PMTV disease in a growing year, it is likely to be critical in introducing the virus into PMTV-free soils. Santala et al. (2010) considered that seed potatoes had been the main source of infestation in the island of Lolland in Denmark after large-scale potato production started there in 2000. Introductions may occur either through seed tubers carrying viruliferous spore balls of S. subterranea which are deposited in soil after planting or by a soil population of S. subterranea acquiring the virus from infected plants, as demonstrated by Arif et al. (1995). The infestation rate in a field was considered by Santala et al. (2010) to be more closely related to the amount of PMTV in adhering soil than to the incidence of PMTV in tubers. The results in the current study with soil testing before and after cropping with cv. Cara in 2006 provided some limited evidence that planting PMTV-infected seed potatoes can increase the risk of introducing PMTV into clean land.

Although the incidence of spraing following the fluctuating temperature treatment was strongly related to the incidence of PMTV in the tubers of cv. Cara in the 3 years of commercial seed production (Fig. 2), overall only 73% of infected tubers developed spraing. Most of this infection appeared to result from soil inoculum. This contrasts with the mean of 32% for infected daughter tubers of cv. Cara in the seed tuber transmission experiments, suggesting that the risk of spraing developing in PMTV-infected tubers is greater when infection is derived from soil inoculum than from seed inoculum. Calvert (1968) did not list spraing as a symptom of secondary infection, i.e. resulting from infected seed tubers. Nevertheless, these results and those of Carnegie et al. (2009) show that spraing can develop in daughter tubers derived from PMTV-infected seed tubers, albeit at a lower rate than for primary infected tubers. In trials with 10 cultivars, Carnegie et al. (2009) found that the proportion of infected tubers developing spraing was generally much greater for primary infection than for secondary infection. Overall, these results confirm that the controlled temperature treatment of tubers after harvest can help to provide a good indication of potential disease risk for spraing, although it is also clear that spraing symptoms can develop which are not attributable to infection by PMTV and thus could result in false estimations of PMTV infection.

These results confirm that PMTV could be eliminated from potato crops in only a few generations of seed multiplication because the rate of transmission from seed to daughter tuber was relatively low. However, the rate of transmission was greater for plants with foliar symptoms than for symptomless plants, indicating that their removal could reduce overall crop infection more quickly. Although transmission was not affected by cultivar, the prevalence of foliar and tuber symptoms varied amongst cultivars; some were identified as being relatively insensitive. The field study of PMTV infection in seed crops of cv. Cara multiplied over two successive generations at a large range of Scottish locations showed that the incidence of PMTV in daughter tubers was not correlated with the incidence of virus in seed tubers, but appeared to be associated with soil inoculum. Analysis also revealed an underlying linear relationship between the incidence of powdery scab and PMTV. Some limited soil testing indicated that planting PMTV-infected seed potatoes could result in the infestation of clean land.

Acknowledgements

The authors thank R. Holmes of Virology and Zoology Section, SASA for conducting the ELISA tests, the financial contribution of Potato Council Ltd for TD and the inspectors of Rural Payments and Inspections Division who collected the samples. The authors would also like to thank Adrian Roberts of Biomathematics & Statistics Scotland for his advice.

Ancillary