Effect of soil inoculum level and environmental factors on potato powdery scab caused by Spongospora subterranea
E-mail: [email protected]
Present address: Scottish Agricultural Science Agency, 82 Craigs Road, East Craigs, Edinburgh EH12 8NJ, UK.
Abstract
The effects of soil inoculum level and three environmental factors (soil type, soil moisture regime and temperature) on the incidence and severity of powdery scab caused by Spongospora subterranea were investigated in potato plants grown under controlled environmental conditions. Symptoms of powdery scab on tubers were assessed visually, after which DNA was extracted from tuber peelings and quantified in a real-time polymerase chain reaction assay using primers and a TaqMan® probe specific to S. subterranea to establish tuber infection levels. Soil inoculum concentration of S. subterranea did not significantly affect the incidence and severity of either tuber infection or powdery scab symptoms at maturity. No significant differences in disease incidence and severity were found between sandy, loamy and clay soils, although the two lighter soils yielded more powdery scab than clay soil. Constant dampness of the soil resulted in significantly more disease than a fluctuating moisture regime. Infection and disease levels were high at all three temperatures tested (9, 12 and 17°C), but symptoms were most severe at 12°C. The percentage of plants with infected tubers did not increase after tuber initiation, although the amount of S. subterranea DNA detected in tubers and the severity of powdery scab symptoms increased in mature plants. Latent tuber infections were found to be common, especially under conditions suboptimal for disease development. This new information may be important for the prevention of powdery scab in potato-growing areas around the world.
Introduction
Powdery scab of potato is an important disease throughout the world and may cause extensive losses in seed and ware potato crops (Wale, 2000). The powdery scab pathogen Spongospora subterranea is also the vector of potato mop-top virus (PMTV), one of the causes of spraing (Jones & Harrison, 1969). Spongospora subterranea belongs to the plasmodiophorids and forms persistent resting structures called cystosori, or sporeballs, containing primary zoospores that can infect all underground structures of the host. Secondary zoospores are released from zoosporangia formed in the roots of suitable host plants. Apart from potato and related species, the host range of S. subterranea comprises other crop plants, such as oilseed rape, sugarbeet and spinach, and a large number of common weeds, including chickweed, poppy, nettle and fat hen. In potato and a few other hosts, infected root systems may develop galls containing cystosori (Würzer, 1964; Janke, 1965; Jones & Harrison, 1969, 1972; Andersen et al., 2002). Infected potato tubers can form cankers and scabs that eventually attain a powdery appearance through the formation of masses of cystosori (Hims & Preece, 1975).
The role of nonsolanaceous hosts in the incidence and severity of powdery scab in potato remains unclear (de Boer, 2000), as does the relative importance of other inoculum sources that can be either soil- or seedborne (Wale, 2000). Powdery scab is especially prevalent in cool, wet climates (Wale, 2000), but the exact effects of temperature and moisture on disease incidence is unknown. Other areas of the biology of the pathogen, such as the effect of soil type on disease incidence and severity, and the occurrence of latent tuber infections, are also poorly understood. Spraing caused by PMTV is regularly found in plants without powdery scab symptoms, which suggests that latent tuber infections by S. subterranea could be common (Jones & Harrison, 1969; Wale, 2000).
A real-time PCR assay specific to S. subterranea was recently developed and is a quick and reliable method for detecting and quantifying S. subterranea DNA in host plant tissues (van de Graaf et al., 2002, 2003). Use of this method in studies on S. subterranea could greatly increase the knowledge of the biology of this pathogen.
The objective of this work was to study the effect of soil inoculum level and several environmental factors on infection of potato tubers by S. subterranea and on subsequent symptom development using real-time PCR in combination with conventional techniques. A better understanding of the pathogen will eventually lead to improved disease prevention and control.
Materials and methods
Preparation of inoculum and soils
Potato tubers with powdery scab symptoms were harvested in Aberdeenshire, Scotland, in 2000 and stored in the dark at 4°C. Tubers were washed with tap water and left to dry overnight at room temperature. Scabs containing cystosori were then scraped off the tuber surface with a scalpel and ground with a pestle and mortar. The resulting powder was passed through a 53-µm-mesh sieve and suspended in sterile distilled water. The concentration of cystosori was established microscopically using a haemocytometer.
Three different types of soil from Aberdeenshire, loamy sand, sandy loam and silty clay (from now on referred to as sand, loam and clay, respectively), were collected, air-dried, passed through a 1-cm-mesh sieve and stored at room temperature. Soil moisture curves were established for the sieved soils using standard water-column and pressure-plate techniques.
Disease trial
The three different types of soil were moistened with distilled water to bring the moisture level to −1 bar (damp). The water added contained different amounts of cystosori, resulting in inoculum concentrations of five, 15 and 50 cystosori per g soil. Control treatments were moistened with water that did not contain inoculum. The water and soil were mixed thoroughly by hand and the mixture distributed into clean 3 L plastic plant pots. One ‘nongerminated’ seed potato tuber of cv. Estima was planted in each pot at a depth of 3 cm below soil level and grown in a growth room with a 16-h light regime at a constant temperature of 12°C. The pots were watered regularly with distilled water to maintain constant dampness in the soil, but the humidity of the air was not regulated.
An additional soil moisture regime in which soil was saturated at the start of the experiment, left to dry for 10 days and then saturated again was applied to the loam soil only. This procedure was repeated until the end of the experiment. All loam-soil treatments were incubated at three separate temperatures (9, 12 and 17°C).
Plants were lifted at two growth stages: tuber initiation and maturity. Plants at tuber-initiation stage were lifted 2 (17°C), 3 (12°C) or 4 months (9°C) after planting. Mature plants were lifted when the foliage had died. There were five replicates per treatment for each growth stage, but the trial itself was carried out only once. An overview of the different treatments and number of replicates used in this trial is given in Table 1.
| Soil type | Temperature (°C) | Moisture regime | Soil inoculum levels (cystosori per g soil) | Growth stages | Total no. of tubers planted |
|---|---|---|---|---|---|
| Sand | 12 | Constant | 0, 5, 15, 50 | Tuber initiation, maturity | 1 × 1 × 1 × 4 × 2 × 5 = 40 |
| Clay | 12 | Constant | 0, 5, 15, 50 | Tuber initiation, maturity | 1 × 1 × 1 × 4 × 2 × 5 = 40 |
| Loam | 9, 12, 17 | Constant, fluctuating | 0, 5, 15, 50 | Tuber initiation, maturity | 1 × 3 × 2 × 4 × 2 × 5 = 240 |
Visual assessment
The tubers or swollen stolon ends (≥ 0·5 cm) of each plant were harvested, washed, visually assessed for powdery scab symptoms and peeled completely. Peelings from tubers of the same plant were combined, air-dried at room temperature, and then stored in the freezer before DNA extraction. Powdery scab disease symptom severity was determined on the basis of percentage tuber surface area covered with powdery scab using the scale published by Merz (2000), which ranges from 1 (no symptoms) to 7 (> 75% covered with powdery scab). Scores were determined for each individual tuber and then averaged per plant.
DNA extraction
DNA from the potato tuber peelings was extracted using a Nucleon® PhytoPure plant DNA extraction kit (Tepnel Life Sciences, Manchester, UK). In each case, the plant tissue samples were cut into small pieces and 0·1 g samples taken at random. For samples where the total weight was less than 0·1 g, all available material was used.
Each peeling sample was ground in 600 µL of Reagent 1 of the PhytoPure plant DNA extraction kit in a 1·5 mL tube. To each tube, 0·2 g sterile 0·1-mm zirconia/silica beads and 0·2 g sterile 1·0-mm glass beads were added, after which the samples were shaken at 5000 rpm using a Biospec® Mini Bead Beater (Biospec Products, Bartlesville, USA) for 1 min to rupture the cystosori. The tubes were centrifuged at 3000 g for 10 min and the supernatant was transferred to new microtubes. The DNA-extraction-kit protocol was then followed.
Real-time PCR amplification
Real-time quantitative PCR was performed as described by van de Graaf et al. (2003) using the DNA extracted from the potato tuber peelings. A range of standards with known amounts of S. subterranea DNA was included in the real-time PCR assay, for which DNA was extracted from a known amount of cystosori following the method of Bell et al. (1999) and diluted. The standards were used to create a standard curve of the critical threshold (Ct) value against the logarithm of the amount of cystosori. Using this standard curve, the amount of S. subterranea DNA in each unknown sample was expressed in cystosorus equivalents (hereafter referred to as units) on the basis of their Ct values (van de Graaf et al., 2003). A nontemplate control with 1 µL TE buffer instead of DNA was included in every assay and all samples were tested in duplicate and then averaged. Scores were adjusted for the amount of tuber peeling (mg dry weight) used for DNA extraction. Plants were assumed to have tubers infected with S. subterranea if the tuber DNA sample tested positive (≥ 3 units) in the real-time PCR assay.
Data analysis
Results were tested using Friedman two-way anova, Mann–Whitney U-tests, chi-square tests and Fisher's exact tests (α = 0·05).
Results
Effect of soil inoculum level
The number of cystosori added to the soil was found to have no significant effect on the level of tuber infection (DNA) or disease (powdery scab symptoms). At tuber initiation, significant differences were observed in the incidence and severity of infection caused by inoculum between the levels of five and 50 cystosori per g soil, when the average values from all growing conditions were taken. At maturity, no significant differences in infection rates or disease symptoms were found between the three levels of added inoculum (Table 2). On average, the amount of DNA detected in tubers of mature plants was much higher when 50 cystosori per g soil were used, compared with the other inoculum levels, but this was mainly the result of one plant having a very high level of S. subterranea DNA (38 729 units per mg tuber peeling) and was therefore not significant.
| Growth stage | Number of cystosori added per g soil | n | Average no. of tubers per plant | Percentage of plants with infected tubersa | Average amount of DNA in units per mg tuber skin tissueb (± SE)a | Percentage of plants with powdery scaba | Average powdery scab severityc (± SE)a | Percentage of plants with latently infected tubersa |
|---|---|---|---|---|---|---|---|---|
| Tuber initiation | ||||||||
| 0 | 38 | 3·1 | 50 a | 1 ± 0·5 a | 0 a | 1·00 ± 0 a | 50 ab | |
| 5 | 36 | 3·4 | 69 ab | 42 ± 25 b | 8 ab | 1·09 ± 0·05 b | 64 bc | |
| 15 | 34 | 2·9 | 82 bc | 284 ± 161 c | 6 ab | 1·04 ± 0·03 b | 77 c | |
| 50 | 25 | 2·9 | 92 c | 282 ± 215 c | 12 ab | 1·13 ± 0·08 b | 80 c | |
| Maturity | ||||||||
| 0 | 37 | 3·0 | 54 a | 252 ± 205 c | 16 b | 1·17 ± 0·08 b | 46 abe | |
| 5 | 33 | 2·9 | 73 abc | 1205 ± 780 c | 52 c | 1·59 ± 0·15 c | 27 ef | |
| 15 | 32 | 2·9 | 78 bc | 1197 ± 584 c | 66 c | 1·95 ± 0·19 c | 22 f | |
| 50 | 26 | 2·9 | 73 abc | 3548 ± 1794 c | 65 c | 1·99 ± 0·25 c | 27 af | |
- a Results in the same column not sharing a common letter are significantly different (a = 0·05).
- b One unit is equivalent to the average amount of DNA extracted from one cystosorus of S. subterranea quantified in a real-time PCR assay.
- c On a scale of 1 (no symptoms) to 7 (> 75% tuber surface area covered with powdery scab) (Merz, 2000).
The average powdery scab severity in the control treatment was significantly lower than in any of the inoculated treatments. Tuber infection was found in 52% of all noninoculated controls, but the amount of DNA detected in these plants was much lower than in the inoculated treatments and mostly occurred without the development of powdery scab symptoms (Table 2).
At tuber initiation, latent infections were significantly more common in the treatments with higher inoculum concentrations. However, at maturity, the situation was reversed and most latent infections were found in the noninoculated control plants. At both growth stages, the percentage of latently infected plants from soil inoculated with five cystosori per g was not significantly different from the percentage in noninoculated soil (Table 2).
At all inoculum levels except the highest, the average amount of S. subterranea DNA detected in tubers was significantly higher in infected mature plants with powdery scab than in those without symptoms. The percentage of latently infected plants having very low levels of S. subterranea DNA (< 1 unit per mg skin) was significantly higher in the noninoculated control (58%) than in any of the three inoculated treatments, which did not differ significantly from each other (39, 27 and 19% latent infection with < 1 unit per mg for five, 15 and 50 cystosori per g, respectively) [data not shown].
Some observations were missing, mainly because of nonemergence resulting from fusarium dry rot of the seed tubers in the soil, which was especially common at the highest soil inoculum level (Table 2). Other reasons for missing observations included the fact that some plants formed no or only very small daughter tubers, and the presence of other tuber diseases which occasionally prevented successful scoring for powdery scab.
Since all three inoculum levels resulted in high levels of infection and disease at maturity, and few significant differences were found between them, the results of the treatments with added inoculum were averaged in the analysis of the effect of the environmental factors given below.
Effect of soil type
No significant effect of soil type on tuber infection or incidence and severity of powdery scab symptoms was found for inoculated plants grown at 12°C. At both growth stages, tuber infection and symptom development were generally more severe in sand and loam soils than in clay soil, but the differences between the three soil types were not significant (Table 3). At tuber initiation, powdery scab symptoms were observed only on plants grown in sandy soil, but in mature plants, powdery scab was no more common or severe on plants from sandy soil than those grown in loam soil. The average amount of DNA extracted from tuber skins was relatively high in sand-grown plants at both growth stages (Table 3), but the variation between samples was large and the differences between the soil types were therefore not statistically significant.
| Growth stage | Soil type | n | Percentage of plants with infected tubersa | Average amount of DNA in units per mg tuber skin tissueb (± SE)a | Percentage of plants with powdery scaba | Average powdery scab severityc (± SE)a | Percentage of plants with latently infected tubersa |
|---|---|---|---|---|---|---|---|
| Tuber initiation | |||||||
| Sand | 14 | 79 a | 693 ± 415 a | 21 ab | 1·24 ± 0·14 a | 64 ab | |
| Loam | 15 | 93 a | 55 ± 35 a | 0 a | 1·00 ± 0 a | 93 a | |
| Clay | 15 | 67 a | 16 ± 14 a | 0 a | 1·00 ± 0 a | 67 a | |
| Maturity | |||||||
| Sand | 14 | 86 a | 5218 ± 3198 a | 71 c | 2·29 ± 0·33 b | 29 bc | |
| Loam | 15 | 80 a | 1662 ± 985 a | 80 c | 2·43 ± 0·34 b | 13 c | |
| Clay | 14 | 71 a | 678 ± 369 a | 57 bc | 1·59 ± 0·18 ab | 21 c | |
- a Results in the same column not sharing a common letter are significantly different (a = 0·05)
- b One unit is equivalent to the average amount of DNA extracted from one cystosorus of S. subterranea quantified in a real-time PCR assay.
- c On a scale of 1 (no symptoms) to 7 (> 75% tuber surface area covered with powdery scab) (Merz, 2000).
At tuber initiation, most latent infections were found in plants grown in loam soil, but at maturity, plants grown in loam soil had less latent infections than those grown in sand or clay soil. Differences in the percentage of plants with latent infections between the three soil types were not significant at either growth stage.
Effect of soil moisture level
In loam soil, no significant differences in tuber infection and disease levels were found between the two moisture regimes when assessed at tuber initiation. However, in mature plants, tuber infection and powdery scab were significantly more common and severe on plants grown at constant dampness than those receiving fluctuating moisture levels. Only the percentage of plants with latent tuber infections did not differ significantly between the two soil moisture regimes (Fig. 1). Fluctuating soil moisture levels promoted the development of fusarium dry rot in some of the mother tubers and prevented germination. There were therefore fewer observations for soil with fluctuating moisture than for damp soil (20 and 43, respectively).
Effect of soil moisture regime on incidence and severity of tuber infection and powdery scab by Spongospora subterranea in inoculated mature potato cv. Estima plants grown in loam soil. The results for different inoculum levels and temperatures are combined. Differences between paired columns with different letters are significant (α = 0·05) (n = 43 and 20, respectively).
Effect of temperature
Tuber infection rates were high (up to 100%) in inoculated damp loam soil at all three temperatures at both growth stages, but powdery scab symptoms were present only at maturity (Fig. 2). At maturity, there was no significant effect of temperature on the percentage of plants with infected tubers or powdery scab symptoms. However, the average amount of DNA per mg tuber skin was significantly higher in plants grown at 9°C than in those grown at 17°C. In addition, powdery scab severity was significantly higher at 12°C than at 9 or 17°C. Disease severity at 12°C was relatively high, with an average disease score of 2·4 (Fig. 2).
Effect of temperature on incidence and severity of tuber infection and powdery scab by Spongospora subterranea in inoculated mature potato cv. Estima plants grown in damp loam soil. The results for different inoculum levels are combined. Differences between grouped columns with different letters are significant (α = 0·05) (n = 13, 15 and 15, respectively).
DNA of S. subterranea was detected in symptomless tubers at all three temperatures. More than twice as many latent infections occurred in mature plants grown at 9 or 17°C than in those grown at 12°C, but these differences were not significant (Fig. 2).
The effect of temperature on infection and disease development was different for plants grown in soil with fluctuating moisture levels compared with those grown in constantly damp soil. At 9°C, no powdery scab symptoms were observed on tubers grown in soil with fluctuating moisture levels, whereas ∼70% of plants developed powdery scab when grown in damp soil. This difference in disease incidence between moisture regimes was not observed at 12 or 17°C, even though the amount of S. subterranea DNA detected in tubers originating from plants grown in soil with fluctuating wetness was very low at all three temperatures (0·1, 4 and 64 units per mg tuber skin, respectively) [data not shown].
Disease development in time
A comparison was made of the incidence and severity of infection and disease, averaged for all inoculated treatments, between tuber initiation and maturity (Fig. 3). The majority of plants harvested at tuber initiation had tubers infected by S. subterranea and, although the average amount of pathogen DNA recovered from tubers increased during the growing season, no additional plants were infected after tuber initiation. Powdery scab symptoms were found on some very young tubers, but both the percentage of plants with powdery scab and the average disease severity increased significantly with time. The percentage of plants with latently infected tubers was very high at tuber initiation (73%), but decreased to just over 25% in mature plants (Fig. 3).
Incidence and severity of tuber infection and powdery scab by Spongospora subterranea in inoculated potato cv. Estima plants at two different growth stages. The results are the averages of different growing conditions. Differences between paired columns with different letters are significant (α = 0·05) (n = 95 and 91, respectively).
Discussion
This study provides new information on the biology of S. subterranea. The results show that, within the range of relatively low inoculum levels tested, the concentration of cystosorus inoculum in soil had little effect on the incidence and severity of infection and disease. It appeared that very low levels of soil inoculum (< five cystosori per g soil) were enough to cause high levels of disease. This lack of a significant effect of soil inoculum level on disease agrees with earlier findings. Burnett (1991) tested a range of soil inoculum densities between 0·03 and 333 cystosori per g soil and found that the initial inoculum level in soil was not important if environmental conditions were conducive to disease development. In Burnett's experiments, infection levels no longer increased when soil inoculum levels were raised above three cystosori per g soil. Similarly, for potato plants in hydroculture, the maximum effective inoculum level was found to be only one cystosorus mL−1. Burnett's (1991) findings can be explained by the fact that each cystosorus contains hundreds of zoospores, and it was on the basis of these findings that relatively low inoculum levels were chosen for the present experiments. Also, once infection has been established in the potato roots, secondary zoospores released from zoosporangia greatly reduce the importance of soil inoculum.
In the present trials, tuber infection levels were high for most treatments, even the noninoculated control. Disease in noninoculated soils was most likely the result of natural contamination of the soil or the seed tubers used.
No significant differences in infection and disease levels were found between the three soil types tested. Nevertheless, powdery scab was generally more severe in the two lighter soils than in the clay soil. In some countries, powdery scab has been reported to be a particular problem in sandy and, to a lesser extent, loamy soils (de Boer, 2000; Wale, 2000). It is known that acid soils can prevent disease by inducing the release of zinc and manganese, which are toxic to S. subterranea zoospores (Burnett, 1991). However, no significant differences in pH or heavy metal contamination (e.g. zinc) were present between the three soil types used in the experiments described above, and are therefore unlikely to have played a role in the differences observed. Soil pore size is smaller in clay soil than in sandy and loamy soils and could have restricted zoospore movement or caused low oxygen levels unfavourable to S. subterranea, resulting in reduced infection and disease.
Burnett (1991) found no significant differences in powdery scab between soils with constant and fluctuating wetness, and de Boer (1991) reported no differences in disease incidence between poorly and well-drained soils. However, in the present study, plants grown in soil at constant dampness showed higher levels of infection and disease than plants grown under a fluctuating moisture regime. Very low disease levels were observed in soils with fluctuating wetness, which could have been caused by a combination of the relatively low inoculum levels, the increased compactness of the soils as a result of the moisture regime, and a reduction in plant vigour resulting from dry periods. It is well known that in practice, powdery scab is often more prevalent in wet years or areas (Wale, 2000), when strong fluctuations in soil moisture level are less common and periods of soil dryness do not occur. The results of the soil-moisture-regime treatments concur with the idea that the moisture level of the soil does not need to be high to obtain high infection and disease levels. Zink et al. (2004) reported that even in well-drained soils, conditions in the root zone remained adequate for extensive powdery scab development. This can be explained by the fact that zoospores may be able to be released and infect plants at moisture levels close to wilting point (Burnett, 1991).
For decades, it has been clear that powdery scab is most severe in cooler regions (Janke, 1963). In warmer countries, the disease has mostly been a problem when potatoes are grown at a high altitude or irrigated with cold water (Wale, 2000; Zink et al., 2004). The optimum temperature for powdery scab symptoms found in the present studies (12°C) agrees with earlier findings by de Boer et al. (1985), who also observed powdery scab at higher temperatures (17 and 20°C), but not at 10°C. Harrison et al. (1997) suggested that zoospores of S. subterranea could be active at temperatures much lower than 12°C. The high levels of disease found at 9°C in the present studies support this idea and explain why powdery scab can be a problem in regions with low average temperatures.
Latent infections of potato tubers by S. subterranea were first mentioned by Jones & Harrison (1969) in their study of PMTV. Diriwächter & Parbery (1991) described symptomless infections of potato around the tuber lenticels, whilst Bell et al. (1999) reported amplification of DNA from peelings taken from symptomless potato tubers in a conventional PCR assay with primers specific to S. subterranea. It is possible that some positive reactions in the present real-time PCR assay of symptomless tuber peelings were a result of cystosori stuck to the tuber surface or in lenticels (de Boer et al., 1982). However, the tubers were washed rigorously and the fact that the detection of S. subterranea DNA in symptomless tubers did not increase with soil inoculum level suggests that most latent infections were caused by the presence of the pathogen in tuber tissues rather than by cystosori on the surface.
The results confirm that tuber initiation is a critical phase for infection, as previously reported by Burnett (1991) and Diriwächter & Parbery (1991), perhaps because the lenticels, through which S. subterranea zoospores enter the potato tubers, suberize as the tubers are maturing (Diriwächter & Parbery, 1991). The incidence of latent infection decreased after tuber initiation because more tubers formed powdery scab symptoms. It has been suggested that infected tubers which have not formed mature scabs at the time of harvest may develop necrotic lesions during storage, which can contain cystosori without showing typical powdery scab symptoms (Kole, 1954). However, little is known about the importance of latent and immature infections as a source of contamination of seed tubers. The present experiments suggest that latent infections might be associated with conditions unfavourable for disease development, such as suboptimal temperature and low inoculum concentration, but further studies are needed in this area.
Despite the fact that the trial was not repeated, its results give new insight into several aspects of the biology of S. subterranea. The observed effects of soil type, soil moisture regime and temperature agree with observations from the field and will help to optimize further studies on disease prevention and control. The effect of soil inoculum at low levels on the incidence and severity of disease and the high frequency of latent tuber infections are discoveries which could have important consequences for powdery scab management around the world.
Acknowledgements
Funding of this work by the Scottish Executive Environment & Rural Affairs Department and the British Potato Council is gratefully acknowledged. Many thanks are due to Dr Danny Cullen (SCRI) for designing the primers and TaqMan® probe. The authors would like to thank Louise Sullivan, Sandie Linton and Alison Ward for technical assistance, Dr Paul Hallett (SCRI) for assistance with soil-moisture-curve measurements, and Dr Alex Hilton (SAC) for the supply of soil.
