Structural characteristics and plant-beneficial effects of bacteria colonizing the shoots of field grown conventional and genetically modified T4-lysozyme producing potatoes

Genetically modified potatoes expressing antibacterial protein T4 lysozyme may offer effective control strategies for bacterial pathogens causing severe potato diseases. Apart from this beneficial effect, it is very important to investigate such engineered potatoes carefully for potential adverse effects on potato-associated bacteria which frequently exhibit plant beneficial functions such as plant growth promotion and antagonism towards pathogens invading the plant. Two field experiments were carried out in Spain to analyze the potential effects of conventional and genetically modified T4-lysozyme producing potatoes on shoot-associated bacteria. The first baseline field trial 2002 was performed in Meliana in which three conventional potato lines, Achirana Inta, Desirée, and Merkur, were cultivated and sampled at flowering. The second field trial was conducted in Cella in 2003 in order to compare the effects of a senescent transgenic, T4 lysozyme expressing potato trait, Desirée DL 12, with its isogenic, non-transformed parental line Desirée. Structural characteristics of potato shoot-associated bacteria was assayed by 16S rRNA-based terminal restriction fragment length polymorphism (T-RFLP) analysis and dominant community members within T-RFLP profiles were identified by sequence analysis of generated 16S rRNA gene libraries. Cultivable bacteria isolated from shoots of potatoes grown in the Meliana field trial were monitored for antibiosis against Ralstonia solanacearum, whereas isolates derived from shoots of potatoes cultivated in the Cella trial were screened for antagonism against Ralstonia solanacearum and Rhizoctonia solani, and for 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase production. Determined antagonists were identified by 16S rRNA gene analysis. All potato traits hosted a cultivar-specific community of bacteria with antagonism against the pathogens and/or potential to produce ACC deaminase. Several antagonists obtained from the Cella field potatoes were also observed as ACC deaminase producers. Community profiling revealed a greater diversity differentiation between the senescent T4 lysozyme expressing and parental Desirée lines grown in the Cella field as compared to the variations between the three flowering conventional lines cultivated in the Meliana field trial. Effects of the two varying field sites and different vegetation stages were greater than those of T4 lysozyme when investigating the community composition of bacteria colonizing the shoots of the Desirée line cultivated in both field trials.     

Effect of bacterial inoculation,plant genotype and developmental stage on root-associated and endophytic bacterial communities in potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis>)

Beneficial bacteria interact with plants by colonizing the rhizosphere and roots followed by further spread through the inner tissues, resulting in endophytic colonization. The major factors contributing to these interactions are not always well understood for most bacterial and plant species. It is believed that specific bacterial functions are required for plant colonization, but also from the plant side specific features are needed, such as plant genotype (cultivar) and developmental stage. Via multivariate analysis we present a quantification of the roles of these components on the composition of root-associated and endophytic bacterial communities in potato plants, by weighing the effects of bacterial inoculation, plant genotype and developmental stage. Spontaneous rifampicin resistant mutants of two bacterial endophytes, Paenibacillus sp. strain E119 and Methylobacterium mesophilicum strain SR1.6/6, were introduced into potato plants of three different cultivars (Eersteling, Robijn and Karnico). Densities of both strains in, or attached to potato plants were measured by selective plating, while the effects of bacterial inoculation, plant genotype and developmental stage on the composition of bacterial, Alphaproteobacterial and Paenibacillus species were determined by PCR-denaturing gradient gel-electrophoresis (DGGE). Multivariate analyses revealed that the composition of bacterial communities was mainly driven by cultivar type and plant developmental stage, while Alphaproteobacterial and Paenibacillus communities were mainly influenced by bacterial inoculation. These results are important for better understanding the effects of bacterial inoculations to plants and their possible effects on the indigenous bacterial communities in relation with other plant factors such as genotype and growth stage.     

RNAi-mediated silencing of endogenous <Emphasis Type="Italic">Vlnv</Emphasis> gene confers stable reduction of cold-induced sweetening in potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L. cv. Désirée)

Potato tubers must be cold-stored to extend their shelf life and maintain an uninterrupted supply chain for food processors. However, a side-effect of low-temperature storage is manifested in terms of cold-induced sweetening (CIS) of potato tubers, which reduces the processing quality and the commercial value of the end-products. RNA interference (RNAi) technology, whereby transgene-derived small interfering RNAs can trigger the homology-based knockdown of cognate host genes and can initiate gene silencing, has been successfully applied in crop improvement through targeted gene knockout in host plants. In the current study, transgenic potato plants (Solanum tuberosum cv. Désirée) were generated, expressing a 300 bp hairpin loop nucleotide sequence targeting the potato vacuolar invertase gene (VInv), under the constitutive Cauliflower mosaic virus 35S promoter. Tubers collected from transgenic lines showed a significant reduction in reducing sugar content after 180 days of cold storage, without showing any measurable off-target effects on plant morphology and tuberization compared to non-transformed control plants. The cold-stored tubers were further assayed for chip color, which showed a fairly light colored quality in the samples originating from RNAi lines. Together with similar effects seen in previously published experiments involving other potato varieties, the Désirée results described here establish the efficacy of using RNAi for the successful reduction of CIS in potato tubers.     

Modified light spectral conditions prior to cryopreservation alter growth characteristics and cryopreservation success of potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) shoot tips in vitro

Light is one of the most important factors affecting growth and morphogenesis of plants. Light intensity, photoperiod and spectral composition greatly affect morphogenetic responses of in vitro plants. Modification of light spectra during recovery after cryopreservation improves survival and regeneration, but the effect of modified light conditions prior to cryopreservation are not known. Therefore, the aim of the present study was to follow the photomorphogenetic response of potato plants (Solanum tuberosum L.) under different light qualities i.e. cool white fluorescent (CW) used as control, warm white (HQI), white LEDs (W), blue LEDs (B), red LEDs (R) and a combination of red with 10?% of blue LEDs (RB) prior to cryopreservation, affecting recovery of cultivars Agrie Dzeltenie, Bintje, Maret, Anti and Désirée in vitro. Light spectral quality had a significant effect on growth characteristics of potato plants in vitro. Red light (R) promoted elongation growth but biomass accumulation remained low under monochromatic light treatments. Some of the pre-cryopreservation light treatments significantly affected post-cryopreservation success. Under blue LEDs, high early recovery was observed for all cultivars tested, whereas under red (R) or (HQI), lowest survival percentages were obtained 2–4 weeks after thawing. Specifically, during early recovery, blue light increased survival from 26 to 66?%, 4 to 31?% and 16 to 48?% for cultivars Agrie Dzeltenie, Anti, and Désirée, compared to illumination by red LEDs. Therefore, light spectral quality prior to cryopreservation can significantly affect the cryopreservation success of potato shoot tips.     

Early responses of resistant and susceptible potato roots during invasion by the potato cyst nematode Globodera rostochiensis

Signals from roots of resistant (cv. Maris Piper) and susceptible (cv. Désirée) potato cultivars during invasion by second stage juveniles (J2s) of the potato cyst nematode, Globodera rostochiensis, were investigated. Novel experimental chambers enabled the recording of electrophysiological responses from roots during nematode invasion. The root cell membrane potentials were maintained throughout the 3 d required to assess invasion and feeding site development. The steady-state resting membrane potentials of Désirée were more negative than those of Maris Piper on day 1, but the reverse on day 3. After 5 d there was no difference between the two cultivars. Intracellular microelectrodes detected marked spike activity in roots after the application of J2s and there were distinct and reproducible differences between the two cultivars, with the response from Désirée being much greater than that from Maris Piper. The responses to mechanical stimulation of roots by blunt micropipettes and sharp electrodes were consistent and similar in both cultivars to the responses in Maris Piper obtained after nematode invasion, but could not account for the marked response found in Désirée. Exogenous application of exoenzymes, used to mimic nematode chemical secretions, resulted in a distinct depolarization pattern that, although similar in both cultivars, was different from patterns obtained during nematode invasion or mechanical stimulation. The pH of homogenates prepared from roots of both cultivars was measured and a Ca2+ channel blocker was used to assess the role of Ca2+ in nematode invasion. The results indicated a role for Ca2+ in the signalling events that occur during nematode invasion.     

Development of black dot disease (Colletotrichum coccodes (Wallr.) Hughes) and its effects on the growth and yield of potato plants

Seed tubers of cvs Désirée and Pentland Crown with different severities of black dot were planted in 1988 and 1989 at Rothamsted in fields in 4– or 7-course rotations, respectively. Tubers treated with prochloraz (1988) or imazalil (1989) were planted in some plots, and in others Colletotrichum coccodes inoculum was added to the soil at planting. In further experiments at Mepal, Cambridgeshire in 1989 and 1990 and at Rothamsted in 1990 on sites where potatoes had not been grown for more than 15 years, large amounts of inoculum were added to the soil around disease-free seed tubers of two (1989) or three (1990) cultivars at planting. In all experiments plants were sampled during the season and the effects of treatments on disease development, growth and yield were recorded. Disease on roots, stem bases and tubers was found early in the season and was more severe on Désirée than on Pentland Crown plants from fields in 4– or 7-course rotations. Severity increased throughout the season and with increasing amounts of disease on the seed tubers, especially with Desiree. Disease was also found on plants from disease-free tubers and was more severe in 1988 than 1989. At harvest, black dot on tubers was significantly more severe from severely affected than from disease-free seed, and was most severe where inoculum, especially large amounts, had been added at planting. Fungicide treatment decreased disease early in the season but had no effect on tuber infection at harvest. In 1989 the weight loss of seed tubers during sprouting increased with increasing amounts of black dot, but the disease had little effect on plant size through the season. At harvest the yield of ware tubers (>50 mm) decreased with severe disease but total tuber yields were not significantly affected. However, at harvest in 1988 severely affected seed yielded significantly less than healthy seed. Plants grown from mini-tubers were free from disease on sites where potatoes had not been grown for at least 15 years. Inoculum applied at planting caused severe disease on all cultivars in both years, whereas disease was slight on uninoculated plants. Inoculated plants senesced early at Mepal in 1990, but there were no significant differences in total tuber yield in any experiment. However, yields of ware tubers (>50 mm) were sometimes decreased and the total tuber number per plant increased.     

Increased killing of Bacillus subtilis on the hair roots of transgenic T4 lysozyme-producing potatoes

Transgenic potato plants expressing the phage T4 lysozyme gene which are resistant to the plant-pathogenic enterobacterium Erwinia carotovora subsp. carotovora have been constructed. The agricultural growth of these potatoes might have harmful effects on soil microbiota as a result of T4 lysozyme release into the rhizosphere. To assess the bactericidal effect of roots, we have developed a novel method to associate the cells of Bacillus subtilis with hair roots of plants and to quantify the survival of cells directly on the root surface by appropriate staining and fluorescence microscopy. With this technique, we found that the roots of potato plants (Désirée and transgenic control lines) without T4 lysozyme gene display measurable killing activity on root-adsorbed B. subtilis cells. Killing was largely independent of the plant age and growth of plants in greenhouse or field plots. Roots from potato lines expressing the T4 lysozyme gene always showed significantly (1.5- to 3.5-fold) higher killing. It is concluded that T4 lysozyme is released from the root epidermis cells and is active in the fluid film on the root surface. We discuss why strong negative effects of T4 lysozyme-producing potatoes on soil bacteria in field trials may not be observed. We propose that the novel method presented here to study interactions of bacteria with roots can be applied not only to bacterial killing but also to interactions leading to growth-sustaining effects of plants on bacteria.     

Field trials using single potato plants as whole plots, with and without nematicide treatment, to assess tolerance of attack by Globodera rostochiensis

It is important to assess the tolerance of new potato cultivars with partial resistance to potato cyst nematode attack. The experiments described here are intended to quantify tolerance adequately using a minimum of resources. When test plants were spaced widely in the field trials it was also possible to estimate their resistance levels but standard plant spacing allowed roots of neighbouring plants to encroach on a test plant's root space and to interfere with attempts to assay resistance. Standard plant spacing did, however, give more realistic yields per plant and competition from neighbouring plants was standardised by surrounding each test plant by four identical guard plants (cv. Désirée). Regressions of yield on severity of nematode attack were drawn when nematode infestation varied naturally across the trial site or when different levels of attack were created by applying nematicide at a range of dose rates. Cara was the most tolerant cultivar tested, with a complete spectrum of tolerance shown by other cultivars/clones. Some clones with a high degree of resistance were intolerant and it is precisely these that this type of test seeks to identify.     

β-1,3-glucanase class III promotes spread of PVYNTN and improves in planta protein production

Glucanases are enzymes regulating the size exclusion limit and permeability of plasmodesmata and play a role in biotic stress. In plant genomes, they are encoded as relatively large gene families divided into four classes. Most studies of plant virus interactions have focused on glucanases from classes I and II. In our study, we have evaluated the role of the β-1,3-glucanase class III (Glu-III) gene in the potato–potato virus Y^NTN (PVY^NTN) interaction and implemented the findings to plant biotechnology application. Potato cultivars Désirée and Santé, which are tolerant and extremely resistant to PVY^NTN, respectively, were stably transformed with Agrobacterium tumefaciens harbouring constructs for Glu-III overexpression. Localization of Glu-III protein in patches within the cell wall was determined by tagging the Glu-III protein with green fluorescent protein. Transgenic and non-transgenic plants were challenged with PVY^NTN and its multiplication and spreading was followed. Differences in viral spread were observed between transgenic lines overexpressing Glu-III and non-transgenic lines, with stronger and faster viral spread in transgenic Désirée, and some multiplication in transgenic Santé. In addition, the ability of Glu-III to improve in planta protein production after agroinfiltration was tested. The results have shown that Glu-III overexpression enables faster spreading of vectors between cells and better protein production, which could be beneficial in improving in planta protein production system using viral vectors.     

Expression of an abscisic acid-binding single-chain antibody influences the subcellular distribution of abscisic acid and leads to developmental changes in transgenic potato plants

Potato (Solanum tuberosum L. cv. Désirée) plants were transformed to express a single-chain variable-fragment antibody against abscisic acid (ABA), and present in the endoplasmic reticulum at to up to 0.24% of the soluble leaf protein. The resulting transgenic plants were only able to grow normally at 95% humidity and moderate light. Four-week-old plants accumulated ABA to high extent, were retarded in growth and their leaves were smaller than those of control plants. Leaf stomatal conductivity was increased due to larger stomates. The subcellular concentrations of ABA in the chloroplast, cytoplasm and vacuole, and the apoplastic space of leaves were determined. In the 4-week-old transgenic plants the concentration of ABA not bound to the antibody was identical to that of control plants and the stomates were able to close in response to lower humidity of the atmosphere. A detailed analysis of age-dependent changes in plant metabolism showed that leaves of young transformed plants developed in ABA deficiency and leaves of older plants in ABA excess. Phenotypic changes developed in ABA deficiency partly disappeared in older plants.     

Characteristics of oxidative stress in potato plants with modified carbohydrate metabolism

Effects of sugars on the development of hypothermia-induced oxidative stress were studied in leaves of two potato genotypes (Solanum tuberosum L., cv. Désirée): with normal carbohydrate metabolism and a genotype with increased sugar content modified by insertion of yeast-derived invertase gene. It was found that generation of proceeds more actively in transformed plants than in control plants. On the contrary H₂O₂ concentration and the catalese and peroxidase activities were lower. At the same time, the activities of superoxide dismutase were similar in plants of both genotypes. A short-term incubation of plants at ?7°C confirmed that a higher freezing tolerance of transformed plants was due to low-molecular-weight components of antioxidant protection system rather than to enzymatic component. Literature data and experimental results suggest that the protective effect of sugars is caused by their ability to scavenge ROS nonspecifically under stress conditions     

Production,storability, and regeneration of shoot tips of potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) encapsulated in calcium alginate hollow beads

Summary Shoot tips, of four potato cultivars (Désirée, Genet, Tigoni, and Tomensa), 3–4 mm in size, were precultured for 2 d on Murashige and Skoog (MS) solid medium, then encapsulated in calcium alginate to produce hollow bead synthetic seed capsules averaging 0.78 cm in diameter. Regeneration and ‘regrowth’ were tested on MS solid medium and on soil in the greenhouse, respectively. The encapsulated shoot tips were stored at 4 and 10°C for up to 390 d. For all cultivars, the encapsulated shoot tips stored at both temperatures for 180 d and at 4°C,for 270 d, 100% regeneration on MS solid medium was recorded. After 360 d in storage at 4°C, 70.8% (Tigoni), 66.7% (Genet), 58.3% (Désirée), and 51.5% (Tomensa) regeneration was recorded on MS medium, reducing to 15% (Tigoni), 25% (Genet), 10% (Désirée), and 0% (Tomensa) regeneration after 390 d in storage. ‘Regrowth’ of 93–100% was recorded for non-stored encapsulated shoot tips, directly transferred on soil in the greenhouse after a 2 wk preculture on MS solid medium with an added fungicide (carbendazim) in the encapsulating gel. The ‘regrown’ shoot tips produced plants showing normal development. The results presented here demonstrate that hollow bead synthetic seed capsules are an alternative propagating method for potato seed production.     

Salicylic acid is important for basal defense of Solanum tuberosum against Phytophthora infestans

The importance of the signaling compound salicylic acid for basal defense of potato (Solanum tuberosum L. cv. Désirée) against Phytophthora infestans, the causal agent of late blight disease, was assessed using transgenic NahG potato plants which are unable to accumulate salicylic acid. Although the size of lesions caused by P. infestans was not significantly different in wild-type and transgenic NahG plants, real-time polymerase chain reaction analyses revealed a drastic enhancement of pathogen growth in potato plants depleted of salicylic acid. Increased susceptibility of NahG plants correlated with compromised callose formation and reduced early defense gene expression. NahG plants pretreated with the salicylic acid analog 2,6-dichloro-isonicotinic acid allowed pathogen growth to a similar extent as did wild-type plants, indicating that salicylic acid is an important compound required for basal defense of potato against P. infestans.     

The effect of Erwinia carotovora subsp. atroseptica (blackleg) on potato plants. II. Compensatory growth

In field experiments in 1981 and 1982, uninoculated seed tubers (cv. Désirée) and those inoculated with Erwinia carotovora subsp. atroseptica at the rose (apical) or heel (stolon attachment) ends were planted at normal (35 cm) or double spacing; in additional plots, inoculated and uninoculated tubers were planted alternately. Inoculation, especially at the rose end, decreased plant height and sometimes resulted in blackleg symptoms. Individual plant yields were recorded at the end of the season. In plots of uniform seed type at normal spacing, inoculation decreased total yield compared with uninoculated by 12–13% (heel-end inoculation) or 26–40% (rose-end inoculation). At double spacing, yields increased compared with normal spacing by 44–58% (uninoculated or heel-end inoculation) or 30–39% (rose-end inoculation). When rose-end-inoculated and uninoculated seed tubers were planted alternately, inoculated plants yielded less and uninoculated plants more than in plots planted throughout with the same seed treatment. The abilities of inoculated and uninoculated plants to compensate for weak or missing neighbours were combined using equations to predict the yields of crops with different proportions of diseased or missing plants.     

The effects of deep cultivation and oxamyl on control of potato cyst-nematode, Globodera rostochiensis

Soil compaction associated with frequent cultivation of potatoes was partly removed with a deep winged-tine coulter. This increased the yield of tubers of cv. Cara in 1987 and 1988 and of cv. Désirée in 1987 in soil which was heavily infested with potato cyst-nematode, Globodera rostochiensis (Woll.), and which was treated with oxamyl at 5.6 kg ha^-1. In 1988, in soil not treated with oxamyl, deep cultivation significantly decreased the yield of cv. Cara. In both years, oxamyl decreased numbers of G. rostochiensis eggs in the soil following cv. Désirée potatoes but not following cv. Cara which were resistant to the nematode.     

Elicitation of Ethylene by Verticillium albo-atrum Phytotoxins in Potato

Petioles from a susceptible cultivar (Désirèe) of Solanum tuberosum treated with a low‐molecular mass toxin, separated from culture fluid of Verticillium albo‐atrum, produced greater quantities of ethylene than did petioles of a tolerant cultivar (Home Guard). Pretreatment of leaflets from cv. Désirèe with silver thiosulphite, which inhibits perception of ethylene, prevented the chlorosis and necrosis normally associated with exposure to the toxin. Similarly, application of aminoethoxyvinylglycine (AVG) an inhibitor of aminocyclopropane‐1‐carboxylic acid (ACC) synthase, to petioles of cv. Désirèe reduced toxin‐induced ethylene synthesis and symptom development. The data indicate that, in part, Verticillium‐toxin acts through induction of ethylene biosynthesis in the host tissues, and different responses of susceptible and tolerant potato cultivars to V. albo‐atrum are the result of differential production of ethylene.