Monitoring current season potato leafroll virus movement with an immunosorbent direct tissue blotting assay

A direct tissue blotting assay (DTBA) was used to track the movement of potato leafroll virus (PLRV) from newly infected foliage to the tubers. Plant and tuber characteristics were recorded to assess plant growth stage at inoculation and PLRV effect on yield. Russet Burbank potatoes were planted at different times in 1991 and 1992 to provide plants of different maturities which were then inoculated using PLRV carrying aphids. Aphids were allowed to feed two to three days after which an insecticide was applied. Stems and tubers were tested periodically for PLRV with DTBA after inoculation. Indexed tubers were grown out and ELISA tested in the greenhouse the following winter to confirm results of summer serological tests. Plant age affected percentages more consistently than did inoculation date. When plants approximately 43 days from planting were inoculated at different dates, early inoculation produced a higher percentage of infected plants. Conversely, when plants approximately 62 days from planting were inoculated at different dates, late inoculation resulted in a higher percentage of infected plants. However, early inoculation of young plants resulted in the highest infection percentages. Tuber size and yield were negatively affected by higher percentages of leafroll regardless of the stage of growth at inoculation. DTBA is best used for detecting PLRV in foliage of plants grown from infected tubers (i.e. secondary PLRV). DTBA is less accurate for detecting primary PLRV.     

Potato virus X infection of Netted Gem potato

When Netted Gem potato plants were inoculated with potato virus X 7 to 31 days before defoliation the tubers were not uniformly infected. Testing of a single germinating eye chosen at random from each tuber detected fewer than half of the tubers from which stem-end, mid, or apical-end sections produced infected plants. The number of completely and partially infected tubers increased as the length of time between inoculation and defoliation was increased. Infection was not correlated with tuber size. Partially infected tubers produced equal numbers of infected plants from stem-end and apical-end sections.     

Transmission of potato leafroll virus to or from tuber slices by aphids feeding through a membrane

Summary Eye-bearing slices, cut from healthy potato tubers and placed between Parafilm membranes, were inoculated with potato leafroll virus (PLRV) byMyzus persicae. PLRV was detected by ELISA and by transmission tests in tuber slices and in plants grown from the slices of the susceptible cv. Désirée, but not in those of the resistant cv. Arkula. These results suggest that PLRV replication and transport within tuber phloem is controlled by specific mechanisms of resistance.M. persicae was also able to acquire and transmit PLRV toPl floridana from slices cut from tubers of infected plants. The aphids effectively transmitted PLRV from slices cut from the sprouting rose end but they failed to transmit it from slices cut from the heel end of tubers.     

The effect of potato virus X on susceptibility of potato tubers toFusarium roseum ‘Avenaceum’

Potato virus X (PVX)-free and mild PVX-infected tuber lines originating from the same Katahdin clone were compared for susceptibility to tuber rot incited byFusarium roseum (Lk.) Synd. and Hans. ‘Avenaceum’. The effect of interval between top-kill and harvest on development ofFusarium tuber rot was also determined. A series of tubers from both PVX-free and PVX-infected tuber lines was either (i) mechanically bruised and inoculated by immersion in a spore suspension ofF. roseum ‘Avenaceum’, (ii) mechanically bruised but not inoculated, (iii) inoculated only or (iv) neither bruised nor inoculated. Tubers from all treatments were then stored at temperatures of 18–21 C (65–70 F) for a 3-week period prior to being placed at 4 C (39 F) for the remainder of the storage period. The amount of rot was determined after a 5–6 month storage period at the latter temperature. In a 4-year study PVX-free tubers were significantly (1% level) more susceptible to infection byF. roseum ‘Avenaceum’ than tubers infected with mild PVX. PVX-free tubers left in the ground for 2 or 3 weeks after top-kill were significantly (1% level) more susceptible toFusarium dry rot than those infected with the virus; however, there was no significant difference in disease incidence between PVX-free and PVX-infected tubers when harvested 4–5 weeks after top-kill. Tubers, both PVX-free and PVX-infected, remaining in the ground for a 2 week period following top-kill were significantly (1% level) more susceptible toFusarium tuber rot than those harvested 3 weeks after top-kill; similarly, both PVX-free and PVX-infected tubers harvested 3 weeks after top-kill were significantly (1% level) more susceptible than those left in the ground for a 4–5 week interval.     

Multiplication ofPseudomonas solanacearum in resistant potato plants and the establishment of latent infections

When 1-mo-old plants of a wilt-resistant clone ofSolanum phureja (1386.15) were stem-inoculated with three strains ofPseudomonas solanacearum (K60, S123, and S206), the bacteria multiplied rapidly at the point of inoculation and then moved in the vascular system to other parts of the stem. Resistant plants showed a remarkable ability to support relatively high populations of the bacterium in the absence of disease symptoms. Although multiplication in this resistant clone was substantially less than in susceptible Russet Burbank potato plants, large numbers of bacteria (up to 624 × 10⁴ cells of K60 per 5-cm stem segment) reached the base of the stem of plants maintained at high temperature (28°C) for 20 days after stem inoculation. From the base of the stem, the bacteria moved rapidly into the roots and tubers. Strains ofP. solanacearum differed in their ability to cause latent tuber infection in different resistant potato clones. When 11S.phureja ×S. tuberosum hybrids were stem-inoculated, maintained at 28°C for 3 wk and then grown to maturity at 20°C., most of the clones yielded tubers infected by one or more strains. The race 1 strain (K60) was the most infectious; 53.8% of all tubers harvested from all plants inoculated with this isolate carried latent infections. Because one clone (BR 53.1) never yielded infected tubers, there appear to be genetic factors which may be useful in breeding programs aimed at eliminating latent tuber infection.     

Effect of time of inoculation with potato leafroll virus on development of net necrosis and stem-end browning in potato tubers

The Green Mountain cultivar was used in field tests to determine the effects of inoculating potato plants at various times with the potato leafroll virus (PLRV) on development of internal necrosis of tuber tissue. Viruliferous apterae of the green peach aphid,Myzus persicae (Sulz.), were placed on each stem in all hills to be inoculated in each 3.0 m single-row plot. Planting and inoculation dates were varied in all field experiments and, in one, several vine-killing dates were also included. All harvested tubers were stored for approximately four months at 10°C to enhance development of internal necrosis prior to examination. Similar but smaller greenhouse studies involving both apterous and alate green peach aphids were also conducted using Green Mountain, Irish Cobbler, and Russet Burbank cultivars. All results showed that as inoculation was delayed relative to plant development, more net necrosis (NN) occurred. Conversely, when plants were inoculated early, stem-end browning (SEB) rather than NN predominated. A high percentage of naturally occurring SEB tubers (cv. Russet Burbank) were found by ELISA to contain PLRV. Plants produced by these tubers only rarely developed leafroll symptoms. These findings suggest a previously unsuspected causal relationship between SEB and PLRV. Implications of this apparent relationship on the epidemiology of potato leafroll in Maine are discussed.     

Latent infection of potato tubers byPseudomonas solanacearum

Strains ofPseudomonas solanacearum differed in their ability to infect tubers of different resistant potato clones grown in infested soil. When eight resistant clones (Solanum phureja orS.phureja ×S. tuberosum hybrids) were grown at 24–28°C in soil infested with a race 1 or a race 3 strain of the bacterium, relatively few plants had wilt symptoms at harvest, but 26.7% and 9.2% of the tubers harvested from plants infected with the race 1 and race 3 strains, respectively, carried latent infections. Some infected clones never yielded diseased tubers, however. The development of symptoms above ground was not correlated with the incidence of tuber infection in any particular clone. No tuber infection occurred in tolerant or resistant clones grown in infested soil at cool (12–22°C) temperatures. Tubers were inoculated directly in an attempt to evaluate the ability of bacteria to multiply in these tissues at different temperatures. Highly virulent strains ofP. solanacearum survived in susceptible tubers in higher numbers and for longer periods than in resistant ones. Low temperature (4°C) had a deleterious effect on survival of the bacterium in tubers, but did not completely eliminate the pathogen even after 40 days.     

Effect of time and date of inoculation,plant age,and temperature on translocation of potato leafroll virus into potato tubers

Movement of potato leafroll virus (PLRV) to tubers following placement of viruliferous aphids on foliage was studied on the medium-maturing cultivar, Katahdin and the late-maturing cultivar, Russet Burbank. Inoculation was begun on August 20 and continued at three-day intervals until mid-September. There was no trend of increasing or decreasing numbers of leafroll-infected tubers from early to late inoculation. Several variables were examined to determine their effects on the incidence of PLRV-infected tubers. Multiple regression analyses showed that inoculation date, maximum daily temperature, minimum daily temperature, plant age, and length of time from inoculation to harvest explained 38% of PLR incidence in tubers of cv. Katahdin. Length of time from inoculation to harvest and minimum daily temperature explained 40% of PLR incidence in tubers of the cv. Russet Burbank.     

Resistance toPseudomonas solanacearum in potato: Infectivity titrations in relation to multiplication and spread of the pathogen

When several wilt-resistant potato clones were tested against a highly virulent strain from Mexico (No. 276) by standard stem inoculation methods, only one clone ofSolanum phureja was resistant. When different inoculum concentrations were introduced quantitatively for infectivity titrations, however, different levels of resistance were clearly defined among clones previously rated as susceptible. Linear regression was used to estimate the ED₅₀ values (dosage required to wilt 50% of the population) for each of seven clones. These values ranged from 3 and 100 colony-forming units (CFU) for Katahdin and Russet Burbank, respectively, to 2.1 x 10⁶ CFU for S.phureja clone 1386.15. The distribution of bacteria in stems of Russet Burbank plants inoculated with the compatible strains 276 and K-60 and the incompatible strains B1 and S-210 was very different. In all cases, incompatible bacteria were not detected 10 cm above the inoculation point in the stem, even by 12 days after inoculation, whereas compatible strains multiplied rapidly at this site by 6 days. At the inoculation site and at sites immediately adjacent to it, however, incompatible bacteria attained populations that only differed by one order of magnitude from those of compatible bacteria. Similar results were obtained when a resistant clone (MS 118.24) was inoculated with the virulent strain K-60. Thus, resistance was characterized both by reduced acropetal spread and by tolerance to large numbers of the bacterium.     

Use of enzyme-linked immunosorbent assay to detect potato leafroll virus in field grown potato,cv. Russet Burbank

Use of enzyme-linked immunosorbent assay (ELISA) in detecting potato leafroll infections in field grown potato, cv. Russet Burbank, was studied from 1986 to 1988 at Rosemount, Minnesota. The objective was to determine relative reliability of current season foliage ELISA, tuber tissue ELISA, and tuber progeny foliage ELISA. Serological tests were most accurate when foliage of tuber progenies was tested. ELISA underestimated total leafroll infection when current season foliage from the inoculated plant was used, in those plants inoculated during late tuber bulking stage. Current season foliage ELISA tests using newly expanded terminal leaflets were more reliable than were tests using older leaflets. Leafroll infection was detected in the current season foliage and tuber progenies (tuber tissue as well as tuber progeny foliage) of some plants seven days after inoculation. Most current season foliage infections were detected by day 14–28 depending on year. Differences among years were most likely caused by variation in quality of virus source plants and numbers of vectors used in inoculation. ELISA tests on tuber tissue were almost as effective as ELISA tests on tuber progeny foliage in detecting potato leafroll 20 days after inoculation, but ELISA on tuber tissue substantially underestimated infection if plants were sampled earlier. Maximum percent tuber infection occurred 20 days or more after inoculation. Movement of the virus from the inoculated stem to other stems decreased with increased plant age at inoculation. Percent infected tubers declined with increased plant age at inoculation. Action thresholds developed for aphids in managing potato leafroll virus should take into account the temporal change in percent infected tubers.     

Improved efficiency of detection of potato mop-top furovirus in potato tubers and in the roots and leaves of soil-bait plants

Summary A reverse trancription-polymerase chain reaction (RT-PCR) assay was devised and shown to be sensitive and reliable for the detection of potato mop-top virus (PMTV) RNA sequences in the flesh of virus infected potato tubers, and in the roots and leaves of soil-bait plants. This assay was compared with an enzyme-linked immunosorbent assay incorporating PMTV specific monoclonal antibodies (TAS-ELISA). The tests were devised to improve the efficiency of detection of viruliferousSpongospora subterranea in agricultural soils, and PMTV in potato tubers. RT-PCR detected PMTV RNA sequences in the roots and leaves of bait plants after three weeks growth in viruliferous soil, three weeks before the bait plants themselves developed symptoms, and two weeks before the virus was detected by TAS-ELISA. Both RT-PCR and TAS-ELISA detected PMTV in the tubers of primary-infected potatoes. RT-PCR and TAS-ELISA were shown to be more sensitive and reliable than conventional baittests and sap inoculation methods for the detection and diagnosis of PMTV.     

Position and date of seed tuber inoculation byErwinia carotovora ssp.atroseptica and appearance of potato blackleg

Summary The date of inoculation and the part of the potato seed tuber inoculated both influenced the number of blackleg plants appearing in June, July and August. In winter, inoculation of the heel end was more effective than inoculation of the rose end of seed tubers, but there were no significant differences following inoculation earlier, in autumn, or later, in spring.     

Systemic induced resistance to late blight in potato by treatment with salicylic acid andPhytophthora cryptogea

Summary Tests for systemic induced resistance (SIR) to late blight were performed with different potato cultivars in field trials and in growth chamber experiments. The non-pathogenPhytophthora cryptogea (Pc) and salicylic acid (Sa) were used as inducer agents. In the field, plants were naturally infected byPhytophthora infestans while the indoor plants were inoculated. The degree of SIR obtained varied with the cultivar used and also with the type of inducer. For example in the field the susceptible cv. St. Cecilia had enhanced resistance with Pc whereas Sa caused increased susceptibility. Inbred resistant cvs acquired increased resistance after treatment with Sa. In a separate pilot experiment in a growth chamber, Sa at two different concentrations was injected into potato seed tubers before planting. The higher Sa concentration increased resistance in genetically resistant cultivars while the lower Sa concentration brought about increased susceptibility.     

马铃薯植株感染Y病毒后生理指标变化与抗病性的关系

本研究分别以对PVY感病、过敏和极端抗性的品种脱毒试管苗为材料,比较了接种PVY后不同时间内超氧化物歧化酶(SOD)和游离脯氨酸含量2个指标的变化。从整体上看,未接种PVY条件下,感病品种(Superior)植株的SOD活性较极端抗性品种(IVP35)和过敏型品种(Desiree)低。接种后,IVP35的SOD活性下降;Desiree在接种前期0.5～1 d SOD的活性下降,2～7 d后SOD活性上升,再后7～14 d又下降;Superior接种PVY后,SOD活性有上升趋势,且在接种2～3 d变化率出现最大值。另外,IVP35和Desiree在接种PVY后SOD变化率小,变化范围分别为-13.4%～4.6%和-17.8%～8.7%;Superior接种PVY后,SOD活性变幅较大,范围为5.1%～79.2%。被测的3个品种无论接种与否,极端抗性品种和过敏型品种其植株内部游离脯氨酸含量均明显高于感病品种。正常情况下,IVP35和Desiree的游离脯氨酸含量为41.39～48.77μg.g-1干重和33.42～39.11μg.g-1干重,而Superior仅为8.91～10.72μg.g-1干重。接种PVY后,Superior的游离脯氨酸含量明显上升,达到11.23～21.54μg.g-1干重;虽然IVP35和Desiree的游离脯氨酸含量也呈上升趋势,但上升幅度小,含量分别为48.92～72.13μg.g-1干重和42.46～71.34μg.g-1干重。     

Potato leafroll virus concentration in the vascular region of potato tubers examined by enzyme-linked immunosorbent assay (ELISA)

Summary Enzyme-linked immunosorbent assay (ELISA), used in conjunction with a new rapid extraction method, showed that potato leafroll virus (PLRV) concentration in the vascular region of infected potato tubers decreases from the heel to the rose end. Lower virus concentration at the rose than at the heel end was found not only in dormant tubers but also in tubers three weeks after breaking dormancy although the difference was then less pronounced. These results were obtained from plants with both primary and secondary infection by one of two French virus isolates which behave differently in respect of either accumulation in the ants or in their serological properties or both.     

Production of phytoalexins, glycoalkaloids and phenolics in leaves and tubers of potato cultivars with different degrees of field resistance after infection withPhytophthora infestans

Summary The kinetics of accumulation of phytoalexins, glycoalkaloids and phenolics was studied in two potato cultivars differing in their degrees of field resistance when infected withPhytophthora infestans. Tuber slices and leaves of cvs Pampeana INTA (high degree of field resistance, free of R genes) and Bintje (susceptible) were infected with race C (complex race 1, 3, 5, 7, 11) ofPhytophthora infestans. Phytoalexins and phenolics accumulated in tuber and leaf tissues which had been inoculated. The levels of these compounds in the susceptible cv. Bintje were relatively low and similar to those found before inoculation. Leaves of cv. Pampeana INTA had a very high glycoalkaloid content, suggesting that glycoalkaloids may play a role in protection of leaves against the fungus. However, we could find no correlation between resistance and glycoalkaloid content of tubers. Our results suggest a major role of phytoalexins, phenolics and glycoalkaloids in the complex mechanisms of field resistance.     

Assessing six Austrian potato cultivars for resistance toErwinia carotovora subsp.atroseptica

Summary The susceptibility of six new Austrian cultivars to soft rot, stem rot and blackleg caused byErwinia carotovora subsp.atroseptica was assessed over 2 years in two locations. Soft rot susceptibility was assessed by whole tuber and half tuber tests, stem rot by stem inoculation and blackleg by planting inoculated tubers in the glasshouse. The susceptibility of the cultivars tested with the tuber and blackleg test methods were in general agreement. The half tuber test was more precise in differentiating intermediate levels of susceptibility than the whole tuber test. The reaction of the cultivars to soft rot could be classified as susceptible (Gina and Romina), moderately susceptible (Ditta, Sonja and Bintje), or slightly susceptible (Treff, Komet and Désirée). Results from the stem rot test did not agree with this classification and also depended upon the physiological state of plants as influenced by growing season and glasshouse conditions.     

Interactions betweenPotato leafroll virus and potato plants and their implications for assessment of infection resistance

The effects of Potato leafroll virus (PLRV) isolates and inoculation method on the resistance to PLRV in potato clones and cultivars were investigated. In the season of inoculation, aphid inoculation resulted in higher proportions of infected plants and higher virus concentration in infected plants, as compared to graft inoculation. In these respects, grafting showed superiority over aphid inoculation in the tuber progeny of inoculated plants, though distinct dominance of aphid inoculation was observed for some tested clones. This leads to the conclusion that both methods should be applied as complementary tests for the efficient selection of highly resistant potato clones. For inoculations, two isolates of PLRV were applied, and one of them, isolate L7, was routinely used in screening breeding materials for resistance to the virus. The second isolate originated from plants of the highly resistant clone DW84-1457, which were incidentally infected with PLRV. The virus isolated from these plants was able to infect only specific clones, which were resistant to isolate L7. At the same time, potato clones with resistance derived from a source different from that present in DW84-1457, and standard susceptible and resistant cultivars, showed generally lower infection ratings after inoculation with the new isolate. This result suggests that the isolates may be different strains of PLRV. It is possible that under the strong selection pressure of resistance genes present in clone DW84-1457, a new isolate I-1457 could evolve the ability to overcome resistance to infection controlled by these genes.     

Enzymatic discoloration and phenolic content of potato tubers from cultivars resistant and susceptible to the golden nematode

Phenolic content and discoloration were determined for 13 commercial potato cultivars and 6 potato clones grown at 2 locations over a period of 5 successive years. Tubers from 6 potato cultivars and 5 potato clones resistant to the golden nematode were compared with tubers from 7 potato cultivars and 1 clone known to be susceptible to the golden nematode. Tubers from resistant plants were lower in phenols and discolored less than tubers from susceptible plants.