Root infection of potato by Spongospora subterranea: knowledge review and evidence for decreased plant productivity

Information is reviewed on root infection of potato by the plasmodiophorid Spongospora subterranea f. sp. subterranea. This pathogen has long been recognized as the cause of root galls (hyperplasia) and the economically important disease powdery scab on tubers (modified stolons). The significance for plant productivity of the zoosporangium stages of the pathogen in potato roots has only recently begun to be documented. Two experiments are described that assessed effects of S. subterranea root infection on potato plant root function and productivity. A greenhouse experiment measured root function and plant parameters for eight potato cultivars with markedly different susceptibilities to tuber powdery scab. Water uptake and plant growth were reduced by S. subterranea inoculation in all eight cultivars. The magnitudes of these negative effects, and intensities of root hyperplasia, differed among the cultivars, but were not related to respective susceptibilities to tuber powdery scab. A field trial assessed root function and plant productivity for a cultivar (Iwa) that is very susceptible to Spongospora tuber and root diseases. Soil water content beneath uninoculated plants was consistently less than for inoculated plants, indicating that inoculation reduced water uptake (root function). Inoculation reduced shoot and root dry weights, and reduced weight of tubers per plant by 42%. Spongospora subterranea causes three diseases of potato: root membrane dysfunction, root hyperplasia and tuber powdery scab. The root diseases caused by the pathogen are likely to be important both for powdery scab management and for deleterious effects on potato crop yields.     

Relationship between Spongospora subterranea f. sp. subterranea soil inoculum level,host resistance and powdery scab on potato tubers in the field

The relationship between initial soil inoculum level of Spongospora subterranea f. sp. subterranea (Sss) and the incidence and severity of powdery scab on potato tubers at harvest was investigated. In all experiments soil inoculum level of Sss (sporeballs/g soil) was measured using a quantitative real‐time PCR assay. Of 113 commercial potato fields across the UK, soil inoculum was detected in 75%, ranging from 0 to 148 Sss sporeballs/g soil. When arbitrary soil inoculum threshold values of 0, <10 and >10 sporeballs/g soil were set, it was observed that the number of progeny crops developing powdery scab increased with the level of inoculum quantified in the field soil preplanting. In four field trials carried out to investigate the link between the amount of inoculum added to the soil and disease development, disease incidence and severity on progeny tubers was found to be significantly (P < 0·01) greater in plots with increasing levels of inoculum incorporated. There was a cultivar effect in all years, with disease incidence and severity scores being significantly greater in cvs Agria and Estima than in Nicola (P < 0·01).     

Spongospora subterranea root infection assessed in two potato cultivars differing in susceptibility to tuber powdery scab

Infection by Spongospora subterranea of roots of two potato (Solanum tuberosum) cultivars, either very resistant or very susceptible to powdery scab on their tubers, was studied in a glasshouse experiment. Plants grown in sand/nutrient solution culture were inoculated with S. subterranea sporosori 2 weeks after planting. Plant parameters, the intensity of zoosporangium infection in roots, numbers of Spongospora root galls and amounts of Spongospora DNA in roots, measured using quantitative PCR (qPCR), were assessed at sequential harvests. Inoculation with S. subterranea reduced water use (56 days after planting) by 26% in the tuber resistant cultivar compared with uninoculated plants, and by 60% in the susceptible cultivar. Inoculation did not affect growth of the resistant cultivar, nor shoot mass of the susceptible cultivar, but caused a 38% reduction in root mass of the susceptible cultivar. The intensities of zoosporangium development in both cultivars were similar. The susceptible cultivar had approximately four times more Spongospora root galls g^?1 root mass than the resistant cultivar. Quantitative PCR detected S. subterranea DNA in roots 1 week after inoculation, and indicated a twofold greater amount of pathogen DNA in roots of the susceptible than the resistant cultivar. This study suggests that the S. subterranea zoosporangium stage in host roots is affected differently by host resistance factors than the sporosorus (root gall and tuber scab) stages. The study has also demonstrated the usefulness of qPCR for sensitive and consistent detection of S. subterranea across the duration of potato root infection.     

<Emphasis Type="Italic">Spongospora subterranea</Emphasis> soil contamination and its relationship to severity of powdery scab on potatoes

We investigated soil contamination by Spongospora subterranea f. sp. subterranea (Sss) and disease severity of powdery scab in 29 potato fields in Hokkaido, Japan, using a hydroponic culture method with tomato seedlings as bait plants. The quantity of Sss infection on the roots of bait plants was evaluated using the polymerase chain reaction (PCR) and expressed in terms of the infection potential in the soil. The infection potential was positively correlated with the disease severity of harvested tubers, whereas the spore ball density determined using PCR had an indistinct relationship with disease severity. The infection potential can be useful in evaluating soil contamination and in applying countermeasures against powdery scab.     

Powdery scab resistance in Solanum tuberosum: an assessment of cultivar × environment effect

Powdery scab of potato caused by Spongospora subterranea is one of the main disease problems in many potato production regions of the world. However, no efficient and economically sound control method is currently available. Host resistance will be a key component of the integrated management of powdery scab, but there are discrepancies in published powdery scab resistance ratings of cultivars between countries. In order to identify the main factors causing such discrepancies, 10 reference cultivars thought to have a range of susceptibility to powdery scab and potato mop‐top virus were cropped over 4 years in four to six locations across Europe and disease levels on roots and tubers were assessed using standardized scoring scales. Soil contamination was tested using real‐time PCR and ELISA. The cultivars performed as expected according to previous characterization, with one exception. No relationship was found between tuber and root susceptibility. Assessment of powdery scab symptoms 1 month before harvest gave results comparable to those assessed 2 months after harvest. Neither real‐time PCR nor ELISA soil test results were closely related to disease index data. The field trial results indicate that different scoring methods are the main factor for the discrepancy in resistance ratings, and that environmental conditions and/or soil inoculum level play a minor role. Furthermore, there was either no difference between the pathogen populations in each location or the resistance of most of the cultivars is polygenic.     

Prevalence and distribution of Potato mop‐top virus in Scotland

This study was undertaken to determine the current occurrence in Scottish seed potato crops of Potato mop‐top virus (PMTV), which is transmitted by Spongospora subterranea and causes spraing (brown arcs and lines) in the flesh of potato tubers, rendering them unsaleable. In 2004, a stratified survey of four commonly grown cultivars was conducted, while in 2007 and 2008, only samples from powdery scab‐affected crops were collected. The incidence of crops in which infection by PMTV was present was 37·5% in the stratified survey in 2004, but was greater in surveys in which tubers with powdery scab were tested (47·2% in 2007 and 44·6% in 2008). Similarly, the frequency of crops with incidences of more than 10% tuber infection was lower (9·4%) in 2004 than in 2007 (25·4%) and in 2008 (26·2%). Significant differences in crop infection were found amongst the four major seed‐producing regions and the counties within these regions. The incidence of crop and tuber infection was least for class Pre‐basic seed potatoes and greatest for class Super Elite 3 and Elite seed potatoes. The results indicate that the prevalence of PMTV has not increased since surveys in the early 1970s.     

The potential for the Rapid Screening of Potato Cultivars (Solanum tuberosum) for Resistance to Powdery Scab (Spongospora subterranea) using a Laboratory Bioassay

Powdery scab of potato, once established in a field, is difficult to control because of the longevity of the resting spores (cystosori) of the causal organism, Spongospora subterranea f.sp. subterranea. Host resistance is likely to be the most efficient in a long-term control strategy for preventing build-up of field inoculum and spread of the disease. Resistance screening of potato cultivars is mostly done in laborious field trials where disease development is likely to be unpredictable. A bioassay with potato tissue cultured plantlets and cystosori as inoculum is described and was tested for its potential to screen potato cultivars at an early stage for their relative susceptibility to powdery scab by comparing the lab results with field data. With cystosori inoculum of Swiss origin, the laboratory test showed clear differences between the potato cultivars in the severity of zoosporangial root infection which correlated better with ranked tuber infection data, compared to root galling. There are apparent differences in the relative trends in susceptibility between roots and tubers of five selected cultivars when using naturally infested soil instead of prepared cystosori as inoculum in the lab bioassay. Furthermore, differences in the severity of zoosporangial root infection of two selected cultivars were found when cystosori from different countries where used as inoculum. A possible host genotype × pathogen interaction is discussed. The bioassay has the potential to screen and select for resistant material at an early breeding stage thus making field trials not unnecessary but more economical. It will allow the use of a standard set of pathogen collections and facilitate testing for inoculum virulence in infested soils.     

Biocontrol of powdery scab of potato by seed tuber application of an antagonistic fungus, <Emphasis Type="Italic">Aspergillus versicolor</Emphasis>, isolated from potato roots

An environmentally friendly measure to control potato powdery scab caused by a protozoan pathogen Spongospora subterranea f.sp. subterranea (Sss) was developed by focusing on antagonistic microorganisms that were considered compatible with potato root. Five hundred and eight soil fungi, isolated from potato root cultivated in soil suspensions from four potato fields in Hokkaido, were screened for suppressiveness of root infection by Sss in a hydroponic culture system and for powdery scab severity in greenhouse and field experiments. Antagonistic isolate Im6-50, identified as Aspergillus versicolor, was selected as a potent biological control agent. In a 3-year field test, A. versicolor Im6-50 suppressed powdery scab with a protection value of 54–70 (100?=?complete protection) when applied directly on seed tubers compared with a protection value of 77–93 by the synthetic fungicide fluazinam. A. versicolor Im6-50 was detected from the surface of daughter tubers and from the soil in which the inoculated seed tubers were cultivated by PCR using species-specific primers. The establishment of A. versicolor Im6-50 on the stolon of inoculated potato plants and in the rhizosphere is considered to contribute to the mechanism for disease suppression.     

Detection and Quantification of Spongospora subterranea f. sp. subterranea in Soils and on Tubers Using Specific PCR Primers

PCR-based methods were developed for the detection and quantification of the potato pathogen Spongospora subterranea f. sp. subterranea (S. subterranea) in peel, tuber washings and soil. A partial sequence was obtained for S. subterranea ribosomal DNA and specific PCR primers (Sps1 and Sps2) were chosen from the internal transcribed spacer regions. These primers amplified a 391bp product from S. subterranea DNA but did not amplify DNA from potato or a range of soil-borne microbes, including related species. Diluted S. subterranea DNA was detected at a concentration equivalent to 25×10^–5 cystosori or 1 zoospore per PCR. Amplification was detected from peel and washings of infected and apparently healthy tubers, but not from peel of Scottish classified seed potatoes or axenically micropropagated potatoes. A rapid method for extracting S. subterranea DNA from soils was developed. This yielded DNA pure enough for PCR within 3h and facilitated the detection of 1–5 cystosori per gram of soil. A PCR quantification technique was developed involving comparison of product ratios obtained after co-amplification of S. subterranea DNA along with an internal standard (competitor DNA fragment). This quantitative technique was also adapted for use in soil. PCR detection of S. subterranea in soil was considerably more sensitive than previously reported immunoassays and was quicker and easier than conventional bait plant bioassays. Such an assay could be useful for developing disease risk assessments for field soils and seed potato stocks and for future studies on the ecology and control of S. subterranea.     

Resistance of potato genotypes to common and netted scab-causing species of Streptomyces

Common and netted scabs are two disfiguring bacterial diseases of potato tubers, caused by various groups of Streptomyces species. Common scab, caused primarily by Streptomyces scabies and Streptomyces europaeiscabiei , is characterized by more or less deep pustules on the tuber surface, while symptoms of netted scab, caused mainly by Streptomyces reticuliscabiei , are superficial, corky alterations of the tuber periderm. Some isolates of S. europaeiscabiei are able to induce both common and netted scab symptoms, and therefore constitute a third pathogenicity group. Like most bacterial diseases, potato scabs would be best controlled by using resistant cultivars. Repeated experiments with soil artificially infested with isolates of three species representative of the three pathogenicity groups showed the level and stability of cultivar resistance, as well as the existence of a range of aggressiveness among different isolates. The distribution of scab severity indexes recorded on a collection of 16 potato cultivars and 27 breeding clones grown in soil infested with common scab-inducing isolates was continuous, suggesting isolate nonspecific quantitative resistance. Least susceptible cultivars were Nicola, BF15, Sirtéma, and Charlotte, while Urgenta, Désirée, Ondine and Bintje were very susceptible. The same genotypes proved either highly susceptible (e.g. cvs Bintje, Désirée or Carmine) or highly resistant (e.g. cvs Charlotte, Sirtéma, Monalisa, BF15 or Belle de Fontenay) to isolates forming netted scab symptoms, suggesting isolate-specific qualitative resistance. The ability was confirmed of some isolates of S. europaeiscabiei to induce one or the other type of symptoms depending on cultivar and soil temperature.     

Improved immunological detection of S<Emphasis Type="Italic">pongospora subterranea</Emphasis>

The genus Spongospora has two members which are important pathogens of vegetables, S. subterranea f.sp. subterranea (Sss) and S. subterranea f.sp. nasturtii (Ssn). The close taxonomic relationship of these formae speciales is based on similar cystosori morphology. The potato disease powdery scab, caused by Sss, is difficult to control. The key control measure is avoidance, aimed at planting clean seed in clean soil. For the development of routine tests for the presence of the pathogen on tubers and in soil, a monoclonal antibody (MAb) was developed using Sss cystosori as immunogen. It detected less than one Sss cystosorus and recognised Sss material from many parts of the world. No cross-reactions with other Plasmodiophoromycetes including Plasmodiophora brassicae, Polymyxa betae, Polymyxa graminis and different Streptomyces species causing common and netted scab of potatoes were observed. A novel tuber sample test method was developed using a kitchen peeling machine. This detected two tubers with one powdery scab lesion each in a sample including eighteen uninfected tubers. When soil samples spiked with cystosori were tested with the MAb, different Sss infestation levels could be discriminated. Ssn cystosori gave absorbance values in ELISA as high as Sss cystosori, whereas fresh crook roots of watercress containing Ssnzoosporangia and plasmodia or mud from an Ssninfected watercress bed gave low absorbance values or no reaction. The potential of these findings for the development of a disease control management are discussed.     

Somaclonal selection in potato for resistance to common scab provides concurrent resistance to powdery scab

Variant somaclones of potato cultivar Russet Burbank, selected for resistance to common scab using in vitro cell selection techniques, were tested for resistance to powdery scab, another important disease of potato caused by Spongospora subterranea. This pathogen also invades roots, producing root galls. Most variants consistently showed increased resistance to powdery scab, both in field and glasshouse challenge, when compared to the parental cultivar, several significantly so. On average, the best variant reduced powdery scab incidence by 51% and severity (tuber surface coverage) by 64%. In contrast, no improvement in the extent of root infection and root galling was seen. These results suggest host interactions during root and tuber infection are distinct. Correlation analyses of disease indices amongst the selected variants showed no association between Sp. subterranea root infection and gall scores, nor between root infection and tuber disease severity. However, a weak positive association was found between root gall score and tuber disease, and a strong correlation between tuber disease incidence and severity scores. Interestingly, positive correlations were also found between the extent of powdery and common scab resistance expressed and both incidence and severity of these diseases within the variants, suggesting a common defence mechanism. The role of thaxtomin A in selecting for concurrent resistance to both diseases is discussed.     

Characterization of weeds and rotational crops as alternative hosts of Spongospora subterranea,the causal agent of powdery scab in Israel

Alternative hosts of Spongospora subterranea may allow multiplication and survival of the pathogen over time; thus, host range is important from an epidemiological aspect. Weeds and rotational crops, such as wheat and barley, were sampled from potato fields with a history of powdery scab (PS) and examined for the presence of S. subterranea by root staining followed by microscopic observations and by qPCR analysis after DNA extraction. The pathogen was detected in plants of 16 weed species from eight families and in volunteer plants of potato and wheat. The ability of the pathogen to infect weeds and rotational crops was further examined by artificial inoculations with sporosori in pot experiments. Successful inoculations occurred with 13 weed species from eight families and with 12 rotational crops from five families. The findings of this study indicate a wide host range in Israel; the families Malvaceae and Zygophyllaceae and the following species are reported for the first time as S. subterranea hosts: Solanum elaeagnifolium, Triticum aestivum, Cynodon dactylon, Phalaris paradoxa, Phalaris minor, Setaria verticillata, Rostaria cristata, Sinapis nigra, Arachis hypogaea, Medicago sativa, Astragalus hauraensis, Amaranthus albus, Chenopodium murale, Chenopodium opulifolium, Salsola soda, Malva nicaeensis, Chrysanthemum segetum, Verbesina encelioides, Ammi majus and Tribulus terrestris. Controlling weeds and avoiding the relevant rotational crops observed to be S. subterranea-positive and thus potential hosts, should be taken into consideration in the management of PS, to reduce pathogen inoculum build-up.     

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

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.     

Evaluation of the <Emphasis Type="BoldItalic">Sss</Emphasis> AgriStrip rapid diagnostic test for the detection of <Emphasis Type="BoldItalic">Spongospora subterranea</Emphasis> on potato tubers

Spongospora subterranea, f.sp. subterranea (Sss), which causes powdery scab, is mainly spread through infected seed tubers and survives in contaminated soil for many years. The visual assessment of tuber lots by inspectors carries the risk of misidentification due to the difficulty of distinguishing lesions caused by either Sss or Streptomyces spp.. To avoid this, the “Sss AgriStrip”, a rapid and lab-independent test tool based on a lateral flow immunoassay has been developed, and we assessed its accuracy and sensitivity for detecting Sss. The Sss AgriStrip performed as well as other lab-based identification methods. The Sss AgriStrip, microscopy, ELISA, PCR, and real-time PCR techniques identified infection with S. subterranea in all tubers with typical powdery scab lesions. When lots with tubers showing a mixture of typical and atypical (suspicious) symptoms were tested, the presence of S. subterranea was confirmed in all lesions by all methods. The DNA content was generally lower in atypical than in typical lesions. Diverse and suspicious symptoms, which were difficult to assign to either powdery or common scab, tested negative with Sss AgriStrip and the other methods. This was despite microscopic observation of sporosori-like structures in some samples. Isolation and molecular identification confirmed that these lesions were mostly caused by Streptomyces spp. The Sss AgriStrip is as sensitive as DAS-ELISA with a detection limit between 1 and 10 sporosori per ml buffer. It is ideal for rapid and selective detection of Sss on farms and border inspection points to prevent spread of the pathogen.     

Increased abundance of patatins,lipoxygenase and miraculins in a thaxtomin A-habituated potato Russet Burbank somaclone with enhanced resistance to common scab

Potato common scab caused by the actinobacterium Streptomyces scabiei is characterized by the formation of corky lesions on tubers that reduce their marketability. Management of common scab is very complex and often ineffective under various environmental conditions. Using potato varieties that are more resistant to common scab remains one of the most efficient strategies to control this disease. However, very little is known about the factors associated with resistance to common scab. Somaclone RB9 regenerated from thaxtomin A-habituated potato Russet Burbank calli produced tubers more resistant to common scab than the original variety. Comparison of the RB9 tuber proteome with that of Russet Burbank using label-free quantitative proteomic analysis revealed changes in the accumulation of defence-related proteins from the patatin and lipoxygenase (LOX) families, which are involved in the metabolism of lipids, and of two miraculins of the Kunitz-type protease inhibitors family. The implication of LOX during common scab infection was studied using synchronized minitubers developed from leaf-bud cuttings. S. scabiei infection stimulated the accumulation of LOX in both Russet Burbank and RB9 minitubers, but this accumulation was intensified in RB9 minitubers. Infection also increased LOX activity in Russet Burbank and RB9 minitubers. However, LOX activity measured in noninfected RB9 minitubers was similar to that of infected Russet Burbank minitubers, indicating endogenous activation of LOX activity in RB9 minitubers. We discuss how increased LOX abundance and activity in the somaclone RB9 may contribute to improving tuber defence against common scab.     

Detection and Quantification of Spongospora subterranea in Soil, Water and Plant Tissue Samples Using Real-Time PCR

A sensitive real-time polymerase chain reaction (PCR) assay was developed for the quantification of Spongospora subterranea, the cause of powdery scab and root galling in potato, and the vector of Potato mop top virus. A specific primer pair and a fluorogenic TaqMan® probe were designed to perform a quantitative assay for the detection of S. subterranea in soil, water and plant tissue samples. The assay was tested using DNA from cystosori, zoospores, plasmodia and zoosporangia of the pathogen. DNA was extracted directly from cystosori suspended in water and from clay soil with varying levels of added cystosori. DNA obtained from zoospores released into nutrient solution by cystosori in the presence of tomato bait plants was also tested, as was DNA from plasmodia and zoosporangia in infected tomato roots. In many cases, detection was successful even at low inoculum levels. This specific quantitative assay could therefore be a useful tool for studying the biology of S. subterranea, and for the optimisation of disease avoidance and control measures.     

Detection of the root lesion nematode Pratylenchus penetrans in potato tubers

The root lesion nematode Pratylenchus penetrans parasitizes a wide range of economically important crops, including potato (Solanum tuberosum). Damage by P. penetrans impacts not only the potato yield but can also reduce the tuber quality. Detailed information on tuber infection by P. penetrans is scarce for most cultivars and molecular detection of nematodes from infected tubers is needed. The objective of this study was to assess tuber symptomatology due to P. penetrans infection in 10 potato cultivars and to provide an accurate molecular methodology for nematode detection using tuber peels. Sprouts of certified potato seed from cultivars Agata, Agria, Camel, Désirée, Dirosso, Kennebec, Laura, Picasso, Royata, and Stemster were planted in 2 L pots, and soil was inoculated with 4 P. penetrans/g of soil. Sixty days after inoculation, tubers were harvested, inspected for lesions, and the number of nematodes/g of potato peel assessed. Observations of tubers with symptoms showed the presence of P. penetrans in superficial layers of peels around the lenticels and injured necrotic tissue. Different nematode stages were detected in tubers of all inoculated cultivars, varying from 4 to 46 nematodes/g of potato peel. Species-specific primers showed suitable sensitivity and reproducibility for the detection of P. penetrans in tuber potato peel samples. The molecular detection of P. penetrans directly from tuber peels can facilitate routine nematode inspections of potato seed tubers or cull potatoes for nematode detection, and prevent further dissemination of this species.     

Temporal association of potato tuber development with susceptibility to common scab and Streptomyces scabiei‐induced responses in the potato periderm

Using hydroponics and novel non‐destructive pot culture systems which enable inoculation at specific tuber development stages, the dynamics of common scab infection patterns in potato were studied in order to provide more precise identification of tuber physiological factors associated with susceptibility. At the whole‐tuber level, infection percentages were greatest when Streptomyces scabiei inoculation occurred early; at 2 weeks after tuberization (WAT) 68% of tubers became infected, contrasting with late inoculation (8 WAT), when only 4% infection occurred. The first‐formed internodes were most susceptible to infection, whilst later‐forming and slower‐expanding internodes were less susceptible. Detailed tuber physiological examination of internode 2 showed that pathogen‐induced changes, including increased phellem (periderm) thickness, cell layers and phellem suberization (key physiological features believed critical to S. scabiei infection) were promoted through S. scabiei inoculation. Sequential harvesting showed enhanced phellem suberization (28% greater than the control) within 7 days of pathogen exposure, while phellem thickness and layer responses were also initiated early in the infection process (10–14 days after pathogen exposure) and these responses were independent of symptom expression. Differences in cultivar response were observed, with greater phellem suberization observed 10 days after tuberization (DAT) in the common‐scab‐tolerant cv. Russet Burbank than in the susceptible cv. Desiree. Likewise, Russet Burbank had thicker and more numerous cell layers in the phellem (up to eight cell layers) during early tuber growth (20–30 DAT) than Desiree (up to six cell layers).