Cisgenesis Does Not Solve the Late Blight Problem of Organic Potato Production: Alternative Breeding Strategies

Average potato yields in Dutch organic farming systems vary from 15 to 29 Mg/ha and are limited by low input of nitrogen and severe late blight attacks caused by the oomycete Phytophthora infestans. Under Dutch late blight regulations it is mandatory to kill the haulm at 7% infestation. The late blight attacks have been so early in the organic potato production of the past few years that its acreage is now gradually decreasing whereas consumer demand is increasing. Agronomic control strategies have limited success. First priority lies in breeding for highly resistant varieties to safeguard organic potato production in the Netherlands. Cisgenesis, however, is not an option for the organic sector. Although the product of cisgenesis does not contain genes from non-crossable species it is a result of a genetic engineering process which is excluded from use in organic agriculture. As the principles and standards of organic agriculture are process-based, cisgenesis does not comply with the norms and standards of organic agriculture. The arguments of the organic sector go well beyond the alleged risks of the gene technology and relate to the technology itself. Breeding at DNA-level, instead of at whole-plant level, violates the integrity of life as described in the concept of naturalness. The Dutch organic sector is now aiming at increasing the traditional breeding activities including the participation of farmer-breeders in close cooperation with the formal breeding companies. Additional selection methods need to be developed to include required traits other than late blight resistance, such as nutrient efficiency. Recently two varieties have been released with high resistance against late blight based on introgressing genes from Solanum bulbocastanum. Organic agriculture can benefit from marker assisted breeding to achieve adequate pyramiding of different, new sources of resistance.     

Impact of Agronomic Strategies (Seed Tuber Pre-sprouting,Cultivar Choice) to Control Late Blight (<Emphasis Type="Italic">Phytophthora infestans</Emphasis>) on Tuber Growth and Yield in Organic Potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) Crops

In order to optimize potato crop management in organic farming systems, knowledge of crop growth processes in relation to limitations and reductions by nitrogen (N) and disease is necessary. This paper examines the effect of different prevention measures (seed tuber pre-sprouting, choice of cultivars: resistance to Phytophthora infestans; earliness of tuber initiation) against disease-related constraints on yields, depending on the N-mediated growing conditions of organic potato crops. Under conditions of a relatively high N supply, accelerating the early development and tuber initiation of potato crops by seed tuber pre-sprouting (yield increase of c. 18–23%) or the selective choice of cultivars with an earlier tuber initiation (yield increase of c. 0–21%) are the most effective strategies in combating late blight. They bring forward the crop development c. 7–10 days, escaping the negative impact of the fungus on tuber growth. Under conditions of a relatively low N supply, these strategies do not affect final tuber yield. The reason for the differences of the effect depending on N supply of the crops is, that the lower the N supply, the shorter the period of time over which tuber growth takes place, independent of whether P. infestans becomes severe or not. Tuber growth of organic crops low in N has mostly ceased by the time late blight becomes potentially important for limiting yield, with the consequence that preventative measures against P. infestans are meaningless. Surprisingly, the level of cultivar haulm resistance to late blight did not affect tuber yields in years with an early and strong late blight development. Probably, the positive effect of a longer resistance to the fungus (c. 1 week) was counterbalanced by a later tuber initiation (also c. 1 week). In years with a moderate late blight incidence, less susceptible cultivars were able to produce higher tuber yields on fields with a lower N availability (c. 17–20%), probably due to higher N use efficiency and a better match of N mineralization and N uptake. Under conditions of a relatively high N supply, the tested cultivars with a higher haulm resistance to late blight were not able to improve tuber yields.     

Impact and Interaction of Nitrogen and Phytophthora infestans as Yield-limiting and Yield-reducing Factors in Organic Potato (Solanum tuberosum L.) Crops

For organic potato producers, the two main challenges are disease and nutrient management. Both are limited by regulations that on the one hand prohibit the use of chemical fertilizers, especially nitrogen, and on the other hand prohibit most synthetic pesticides. Late blight caused by Phytophthora infestans is commonly thought to be the most yield-reducing factor. However, because there is no really effective fungicide available to control late blight, there are virtually no yield loss data available for organic farming conditions. In this paper, the state of the art of organic potato tuber growth under on-farm conditions with respect to disease and nutrient management is summarized by field trials and on-farm surveys on commercial organic crops carried out in the years 1995–1998. Soil nitrogen (N) levels, plant N uptake, disease development of P. infestans and potato yield were measured. Results indicated that N availability was most important in limiting yields in organic potato crops. From on-farm data, a model including disease development, growth duration of the crops until foliage decay and different parameters related to N status of the crop could explain 73% of the observed variation in yield. Only 25% of this variation in yield could be attributed to the influence of late blight. Differences in N availability explained 48%. In conclusion, several points emerged from the results. In organic farming, yields are mainly limited by nutrient availability in spring and early summer. The effects of late blight on yields may often be overestimated and cannot be deduced from results in conventional farming because of the strong interaction with nutrient status. Depending on N availability, tubers stop growing between mid-July (70–90 kg N ha⁻¹ uptake), the end of July (110–140 kg N ha⁻¹ uptake) and mid-August (140–180 kg N ha⁻¹ uptake) due to N limitations. The higher the N status of a potato crop, the longer the growing period needed to achieve the attainable yield and the higher the probability that late blight stops further tuber growth and becomes the key tuber-yield-limiting factor. In the second part of this paper, the interactive effects of soil N availability and the impact of P. infestans on yield in the presence and absence of fungicides from 1996 to 1998 for mid-early main crops are reported. An empirical schematic model of disease impact depending on N availability was developed.     

Synergy of cymoxanil and mancozeb when used to control potato late blight

Summary Synergism between fungicides could be used to reduce the application dosage. Synergism between cymoxanil and mancozeb was studied using potted potato plants under field conditions. In three experiments the preventive efficacy of the fungicides and fungicide mixtures in the treatments were studied using a bio-assay. Potato plants were sprayed with the fungicides or fungicide mixtures. Leaves were picked from the potato plants with regular intervals after fungicide application. The efficacy of the fungicides was studied in a bioassay by inoculating the leaves withPhytophthora infestans zoosporangia. The Abbot method was used to show synergistic effects of the cymoxanil/mancozeb mixtures tested. In two of three experiments synergy of the fungicide mixtures was found.     

The economic impact of potato late blight on US growers

Summary Potato growers have been able to control the fungus,Phytophthora infestans, that causes late blight with fungicides, but at an increasingly higher cost. A Delphi survey was conducted with thirteen experts to estimate the impact of late blight on potato yields, storage losses and fungicide use. It was estimated late-blight fungicides cost $77.1 million and lost revenue for US growers was an additional $210.7 million. These total costs, which average $507 per hectare, do not include non-fungicide control practices.     

Exploring the Natural Biodiversity of Potato for Late Blight Resistance

This paper gives an overview of the activities in Sub-project 4 of the BIOEXPLOIT programme. The European genebank collections of potato need to be narrowed down into (customized) core collections by focusing on loci associated with disease resistance. Therefore, new and more efficient molecular methods are being developed to identify these loci, extensive genotyping of resistance to Phytophthora infestans and signalling loci is being performed, and genotyped selections of accessions are phenotypically analysed for their resistance specificities. The results of these activities are being assembled in an integrated database.     

Correlation between resistance to late blight in foliage and tubers in potato clones from parents of contrasting resistance

Summary The relationship between resistance to late blight in foliage and tuber was examined on 50 clones of each of five progenies from crosses where one parent was resistant in both foliage and tuber and the other susceptible. Foliage resistance was assessed in a field trial and tuber resistance in a laboratory test on glasshouse-grown tubers. The genetical and environmental components of variance and the phenotypic and genetical correlations between foliage and tuber blight scores were determined for each progeny. Foliage and tuber resistance were correlated, indicating either that both are determined by the same genes or by different linked genes. The environmental component of variation was greater for tuber blight than foliage blight. We suggest that the most effective way of selecting for resistance to both aspects of the disease in a breeding programme is to select those resistant in the foliage and then screen them for tuber resistance.     

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.     

Improving Potato Production in Kenya, Uganda and Ethiopia: A System Diagnosis

Increased productivity of potatoes can improve the livelihood of smallholder potato farmers in Kenya, Uganda and Ethiopia and is required to meet the growing demand. This paper investigates the opportunities for potato system improvement that could result in improved productivity. Through a diagnosis of the potato systems in the three countries on the basis of surveys and stakeholder workshops, seed potato quality management, bacterial wilt control, late blight control and soil fertility management were identified as key technical intervention topics. For effective problem solving in these areas, the functioning of the potato innovation system requires improvement to better deliver the functions of potato marketing as well as knowledge development and information exchange. With use of a ‘system failure framework’ the shortcomings of the potato innovation system are identified and discussed and options for improvement are suggested.     

The effect of the moisture level of potting compost on the susceptibility of glasshouse-grown tubers to late blight

Summary The effect of the moisture level of potting compost at harvest on the susceptibility of tubers of three cultivars to late blight was investigated in the glasshouse in three successive years. Three moisture regimes (dry, moist, wet) were imposed at flowering time, the tubers harvested 3 weeks later and immediately dipped in a zoospore suspension ofPhytophthora infestans. Tubers from dry compost were significantly more susceptible than those from wet or moist compost, which showed similar levels of susceptibility.     

Differential resistance to tuber late blight in potato cultivars without R-genes

Summary In 1983 and 1984 there were reports in Norway of more severe late blight infection than usual on tubers of cv. Pimpernel, a cultivar without R-genes known to be somewhat resistant to both haulm and tuber infection. Results from routine tests confirmed earlier experience that cv. Pimpernel shows stable resistance to tuber late blight, although the ranking of cultivars may vary between years. In experiments during three harvest seasons tubers of different cultivars were inoculated with isolates ofP. infestans obtained from stocks of cv. Pimpernel with severe tuber late blight and with other isolates. In these experiments the isolates from cv. Pimpernel caused more rot on tubers of Pimpernel than did other isolates.     

Testing the resistance of potato genotypes to tuber late blight

Summary The method hitherto used in Norway for testing for tuber resistance to late blight, inoculating newly harvested unwounded tubers, is described. Superficial wounding of the tubers prior to inoculation increased the level of infection. Tests on wounded and unwounded tubers ranged the cultivars in the same order, but the significance level was higher for wounded tubers. When the tubers were harvested 16 days before inoculation instead of the same day, unwounded tubers of susceptible genotypes were less infected, but using the mean for wounded and unwounded tubers, the distinction between susceptible and resistant genotypes was still clear. Tubers inoculated 7 days after harvest were most infected when wounded shortly before inoculation, but tubers wounded two days before inoculation were infected more than unwounded tubers, and the separation between susceptible and resistant genotypes was more distinct than for unwounded tubers.

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Zusammenfassung Die derzeit in Norwegen verwendete Methode zur Prüfung der Knollenresistenz gegen Braunf?ule wird beschrieben. Die Knollen werden vorsichtig geerntet und auf feuchten Filterpapier in Styropor-Boxen (‘Fischboxen’) gelegt. Am gleichen Tag werden die Knollen mit einer Zoosporen/Sporangien-Suspension vonPhytophthora infestans besprüht. Nach Inokulation werden die Boxen bei 15°C gelagert und dabei 20 Stunden lang zwecks Erhaltung hoher Feuchtigkeit gestapelt. Danach k?nnen die Knollen abtrocknen. In Untersuchungen erh?hte sich die Zahl der Infektionen nach Rollen der Knollen über 10 Nagelspitzen, 2 mm hoch und von einer h?lzernen Basis aufw?rts weisend (Tabellen 2 und 3). Bei verwundeten und nichtverwundeten Knollen waren die Rangfolgen der Sorten nicht signifikant unterschiedlich. Für die getesteten Sorten jedoch war die Unterscheidung zwischen resistent und anf?llig, wie derF-Test aus der Varianzanalyse zeigte, bei verwundeten Knollen besser (Tabelle 3). Das Vorkommen von Braunf?ule in unverwundeten Knollen anf?lliger Sorten wurde reduziert, wenn die Knollen 16 Stunden vor der Inokulation anstatt am gleichen Tag geerntet wurden (Tabellen 1 und 2). Bei Verwendung des Durchschnitts bei verwundeten und nichtverwundeten Knollen war die Unterscheidung resistenter Sorten von anf?lligen Sorten noch klar m?glich (Tabelle 1). 7 Tage nach der Ernte inokulierte Knollen wurden am st?rksten infiziert, wenn sie kurz vor Inokulation verwundet wurden (Tabelle 4). Zwei Tage vor Inokulation verwundete Knollen zeigten zudem noch st?rkere Infektion als unverwundete Knollen. Für die in dieser Studie verwendeten Knollen war die Unterscheidung der Sorten etwas besser bei einem Tag vor Inokulation verwundete Knollen m?glich als bei Knollen, die am gleichen Tag oder zwei Tage vor Inokulation verwundet worden waren. Nichtverwundete Knollen von. Pflanzen aus 3 verschiedenen Pflanzterminen waren nahezu gleich anf?llig (Tabelle 5). Es liess sich kein signifikanter Effekt der Knollenreife auf die Resistenz feststellen.

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Résumé La méthode utilisée jusqu'ici en Norvège pour apprécier la résistance des tubercules au mildiou est décrite. Les tubercules sont récoltés soigneusement et placés sur papier filtre dans des bacs à poisson en polystyrène. Le jour même, les tubercules sont soumis à la pulvérisation d'une suspension de zoospores et de sporanges dePhytophthora infestans. Après l'inoculation, les bo?tes sont placées à 15°C et empilées pendant 20 heures afin de conserver un niveau élevé d'humidité. Après ce temps, les tubercules peuvent sécher. Lors d'expériences de blessures superficielles des tubercules en les faisant rouler sur 10 pointes (de 2 mm de haut) de clous fixés sur un support de bois, on augmente le niveau de l'infection (tableaux 2 et 3). Le classement des cultivars n'est pas significativement différent entre les tubercules blessés et non blessés mais chez les cultivars éprouvés, la distinction entre les résistants et les sensibles est meilleure chez les tubercules blessés, comme le montre la valeur duF des analyses de variance (tableau 3). L'incidence de la maladie chez les tubercules non blessés de cultivars sensibles est réduite quand les tubercules sont récoltés 16 jours avant l'inoculation, comparée à une récolte réalisée le jour même (tableaux 1 et 2). En utilisant la moyenne des valeurs obtenues avec des tubercules blessés et non blessés, la distinction entre cultivars sensibles et résistants est encore claire (tableau 1). Les tubercules inoculés 7 jours après la récolte sont plus infectés quand ils sont blessés peu de temps avant l'inoculation (tableau 4). De plus, les tubercules blessés deux jours avant l'inoculation sont encore plus infectés que les tubercules non blessés. Avec les cultivars utilisés dans l'étude, la distinction entre cultivars est nettement meilleure chez les tubercules blessés un jour avant l'inoculation que chez ceux blessés le jour même ou deux jours avant. Les tubercules non blessés de récoltes issues de plantations différées toutes les 6 semaines possèdent quasiment la même sensibilité (tableau 5). Aucun effet de la maturité des tubercules sur la résistance ne peut être déterminé.

     

Applied Biotechnology to Combat Late Blight in Potato Caused by <Emphasis Type="Italic">Phytophthora Infestans</Emphasis>

Potato is an important crop, grown worldwide. It suffers from many pests and diseases among which late blight, caused by the oomycete Phytophthora infestans, is the worst. The disease is still causing major damage in many potato production areas and control is only possible by applying fungicides frequently. The knowledge on the molecular biology and genetics of the interaction between the plant and the oomycete is developing rapidly. These are relevant fields of study, currently dominated by the discovery of many resistance genes and numerous effector proteins and the analysis of their specific mode of action. These studies may yield essential information needed for the development of durable resistance. The long-term and worldwide effort to breed for resistance so far has had little effect. A novel breeding approach may change this. It is based on cisgenic modification (CM) consisting of marker-free pyramiding of several resistance genes and their spatial and temporal deployment yielding dynamic varieties that contain potato genes only. It is envisioned that this CM approach with potato’s own genes will not only prove societally acceptable but may also result in simplifications in the legislation on use of the CM approach. Various parties in the potato research arena intend to cooperate in this novel approach in a number of developing countries where potato substantially contributes to food security. The use of resources such as land, water and energy improves when the effect of late blight is markedly reduced.     

Transgenic potato plants expressing oxalate oxidase have increased resistance to oomycete and bacterial pathogens

Summary Potato (cv. Bintje) was transformed with a gene encoding an oxalate oxidase from wheat under the control of the CaMV35S promoter. Transgenic potato plants produced high constitutive levels of H₂O₂ as visualized by 4-chloro-l-naphtol staining. The resistance of these plants was tested againstPhytophthora infestans. An increased level of resistance to the disease was marked by a reduced number of lesions as well as by a decreased number of sporangia formed per lesion. In addition, oxalate oxidase overexpressing plants also exhibited improved resistance toStreptomyces reticuliscabiei, the causal agent of netted scab. Increased expression of oxalate oxidase had no effect on the interaction withErwinia carotovora. These experiments show that overexpression of oxalate oxidase represents a potentially interesting approach for protection of potato to pathogens.     

Effects of Mulch and Potato Hilling on Development of Foliar Blight (<Emphasis Type="Italic">Phytophthora infestans</Emphasis>) and the Control of Tuber Blight Infection

Foliar and tuber blight caused by Phytophthora infestans accounts for significant losses in potatoes in field and storage. Nevertheless, limited research has been published on the effects of cultural practices on late blight control. Field experiments were conducted in two years on Howard gravely loam soil in New York State to evaluate the effectiveness of mulching using oat straw and hilling in preventing tuber blight infection for cvs Allegany and Katahdin. Potato hilling and mulching had little effect on foliar blight development. The cultivar affected the disease development in the foliage, with cv. Allegany showing lower foliar late blight than cv. Katahdin. Tuber blight incidence averaged 25% for cv. Allegany and 3% for cv. Katahdin in hilled plots, while in the mulched plots the incidence of tuber blight averaged 33% for cv. Allegany and 10% for cv. Katahdin. The straw hay mulch was ineffective in tuber blight control. Tubers set at a soil depth of more than 7 cm had lower tuber blight incidence than shallow tubers set at a depth of less than 7 cm. In both years, hilling provided partial protection of tubers but its effectiveness was limited in the presence of favourable conditions for late blight development. Even though large hills had proportionally a lower tuber blight incidence than medium-sized hills, the difference between the different hill sizes was not significant. These studies suggest that the use of cultivars with foliage resistance to late blight in combination with cultural practices may partially reduce the incidence of tuber blight. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation for endorsement by Cornell University or the US Department of Agriculture.     

Societal Costs of Late Blight in Potato and Prospects of Durable Resistance Through Cisgenic Modification

In the European Union almost 6 Mha of potatoes are grown representing a value of close to €6,000,000,000. Late blight caused by Phytophthora infestans causes annual losses (costs of control and damage) estimated at more than €1,000,000,000. Chemical control is under pressure as late blight becomes increasingly aggressive and there is societal resistance against the use of environmentally unfriendly chemicals. Breeding programmes have not been able to markedly increase the level of resistance of current potato varieties. New scientific approaches may yield genetically modified marker-free potato varieties (either trans- and/or cisgenic, the latter signifying the use of indigenous resistance genes) as improved variants of currently used varieties showing far greater levels of resistance. There are strong scientific investments needed to develop such improved varieties but these varieties will have great economic and environmental impact. Here we present an approach, based on (cisgenic) resistance genes that will enhance the impact. It consists of five themes: the detection of R-genes in the wild potato gene pool and their function related to the various aspects in the infection route and reproduction of the late blight causing pathogen; cloning of natural R-genes and transforming cassettes of single or multiple (cisgenic) R-genes into existing varieties with proven adaptation to improve their value for consumers; selection of true to the wild type and resistant genotypes with similar qualities as the original variety; spatial and temporal resistance management research of late blight of the cisgenic genetically modified (GM) varieties that contain different cassettes of R-genes to avoid breaking of resistance and reduce build-up of epidemics; communication and interaction with all relevant stakeholders in society and transparency in what research is doing. One of the main challenges is to explain the different nature and possible biological improvement and legislative repercussions of cisgenic GM-crops in comparison with transgenic GM-crops. It is important to realize that the present EU Directive 2001/18/EC on GM crops does not make a difference between trans- and cisgenes. These rules were developed when only transgenic GM plants were around. We present a case arguing for an updating and refinement of these rules in order to place cisgenic GM-crops in another class of GM-plants as has been done in the past with (induced) mutation breeding and the use of protoplast fusion between crossable species.     

Biochemical and morphological characterization of potato clones differing in their resistance to late blight

Summary Morphological and biochemical parameters that could be involved in resistance to late blight were studied in non-infected and in infected potato hybrids resulting from a cross betweenSolanum phureja, resistant toPhytophthora infestans, and a susceptibleSolanum tuberosum. Some morphological differences between resistant and susceptible hybrids, indicating a positive correlation between stem diameter and phloem thickness in the stem and resistance toP. infestans, were observed. The lignin content in the leaves of the resistant hybrid rose upon infection byP. infestans. In the leaves of the susceptible hybrid, a diminution of the lignin content could be observed upon infection byP. infestans. In the same context, peroxidasic activity raised upon infection byP. infestans in both resistant and susceptible hybrids. Further characterization of the hybrid clones based on the polymorphism of peroxidases was attempted using isoelectric focusing.     

Inheritance of resistance to late blight in foliage and tubers of progenies from parents differing in resistance

Summary TenSolanum tuberosum genotypes differing in resistance to late blight (Phytophthora infestans (Mont.) de Bary) in foliage and tubers were intercrossed to determine the inheritance of disease resistance in their progenies. Plots of 10–15 clones per progeny were established in each of 2 years and resistance assessed by field or laboratory tests. The parental genotypes were similarly tested each year. The parents differed in general combining ability (GCA) for both foliage blight (FB) and tuber blight (TB). The parental and GCA scores were significantly correlated for both aspects of the disease, but the correlations between foliage and tuber scores for parents and for GCAs were not significant. Three parental genotypes were highly resistant in both foliage and tubers, and the genotype with the highest GCA for resistance to both FB and TB (cv. stirling) is recommended as the best parent. There was no evidence of strong genetic correlation between both aspects of resistance, and it is suggested that both be selected for in a breeding programme.     

A simple non-destructive technique for healthy and diseased leaf area assessment of potato plants

Summary A method for assessing the leaf area of potato plants was based on the number of leaves coinciding with imaginary vertical lines descending from a horizontal grid of points above the plant or crop canopy. Leaves seen through each vertically aligned pair of holes in a perforated double table, covering the midpoint of the exposed area, were counted and moved aside one by one, until the ground was seen. For both green and diseased surfaces the leaf area, or its horizontal component, was calculated as the product of sum of records and the grid cell size. Tested against a destructive method, the technique worked equally well for healthy and diseased leaf area of potato plants infected byPhytophthora infestans. The theoretic estimate of the standard error for a single measurement was derived as the geometric mean of the leaf area and the grid cell size. The method is also suitable forLAI of crop stands.