Association between late blight resistance and foliage maturity type in potato. Physiological and genetic studies

Transcription

1 Association between late blight resistance and foliage maturity type in potato Physiological and genetic studies

2 Promotoren: Prof. dr. ir. P.C. Struik Hoogleraar in de Gewasfysiologie, Wageningen Universiteit Prof. dr. ir. L.C. van Loon Hoogleraar in de Fytopathologie, Universiteit Utrecht Co-promotor: Dr. ir. L.T. Colon Senior onderzoeker, Plant Research International, Wageningen Promotiecommissie: Prof. dr. ir. C.M.J. Pieterse, Universiteit Utrecht Dr. ir. J.J.H.M. Allefs, Agrico Research, Emmeloord Prof. dr. ir. E. Jacobsen, Wageningen Universiteit Prof. dr. L.H.W. van der Plas, Wageningen Universiteit Dit onderzoek is uitgevoerd binnen de onderzoekschool Production Ecology & Resource Conservation.

3 Association between late blight resistance and foliage maturity type in potato Physiological and genetic studies Marleen Visker Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, Prof. dr. ir. L. Speelman, in het openbaar te verdedigen op vrijdag 11 maart 2005 des namiddags te vier uur in de Aula.

4 Visker MHPW (2005) Association between late blight resistance and foliage maturity type in potato - Physiological and genetic studies Ph.D. thesis, Wageningen University, Wageningen, the Netherlands With references with summary in Dutch ISBN: x

5 ABSTRACT Potato (Solanum tuberosum) is grown throughout the world and is among the most important crops for global food supply. Late blight, caused by Phytophthora infestans, is the most important and destructive disease wherever potato is grown. Potato varieties with resistance to late blight can be used to contest the disease. The application of race-specific resistance has turned out not to be durable, because P. infestans is able to overcome this type of resistance. Race-non-specific resistance is expected to be more durable but, unfortunately, this type of resistance against P. infestans is consistently associated with late foliage maturity. The purpose of the research described in this thesis was to unravel the nature of the association between race-non-specific resistance to late blight and foliage maturity type in potato. The main goal was to determine whether the association between the two traits is genetic or physiological. As part of the physiological studies, single-node cuttings of potato were tested for their potential to serve as a model to predict foliage maturity type at any time during the cropping season. Single-node cuttings did not reflect the stage of tuber development or tuber induction of the plants from which they were taken, and they do not appear to reflect the physiological state of the whole plant adequately. Consequently, single-node cuttings cannot be used to predict foliage maturity type. A possible physiological association between late blight resistance and foliage maturity type was studied by relating the effects of plant age, leaf age, and leaf position on resistance. Leaf position had the largest and most significant effect: apical leaves were far more resistant to late blight than basal leaves. Plant age and leaf age had only minor effects. Thus, the resistance of a specific leaf remained about the same during its entire lifetime. Genetic studies were performed with seven different diploid potato progenies. Recombinant genotypes with early foliage maturity and resistance against P. infestans were not identified. Therefore, it cannot be concluded that the association between late blight resistance and foliage maturity type is due to closely linked genes. QTL analyses revealed loci for resistance to late blight on chromosomes 3, 5, and 10, and for foliage maturity type on chromosome 5. The locus for foliage maturity type could not be distinguished from the most important QTL for resistance: the allele of molecular marker GP21 that is associated with late blight resistance is also associated with late foliage maturity. Keywords: Earliness, Foliage maturity type, Late blight, Phytophthora infestans, Potato, QTL analysis, Resistance, Solanum tuberosum

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7 CONTENTS Chapter 1 General introduction 9 Chapter 2 Are cuttings suitable for assessing maturity type 23 in potato (Solanum tuberosum)? Chapter 3 Leaf position prevails over plant age and leaf age 39 in reflecting resistance to late blight in potato Chapter 4 Can the QTL for late blight resistance on potato chromosome 5 63 be attributed to foliage maturity type? Chapter 5 Correlation between late blight resistance 85 and foliage maturity type in potato Chapter 6 Genetic linkage of QTLs for late blight resistance and 109 foliage maturity type in six related potato progenies Chapter 7 General discussion 129 Summary 147 Samenvatting 151 Nawoord 157 Curriculum vitae 159

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9 Chapter 1 General introduction

10 Chapter 1 Potato The cultivated potato (Solanum tuberosum L.) originates from the Andean region of South America, where it is known as an ancient crop (Hawkes, 1978). It was introduced into Europe by the Spanish explorers about the year This first European potato was of the short-day andigena subspecies and, thus, did not tuberize at these Northern latitudes until very late in the season. Saving seed tubers of early-yielding plants (unintentionally) resulted in selection for earliness and, consequently, in potato that was adapted to tuberize under long-day conditions. This potato of the tuberosum subspecies (Figure 1.1) became accepted as a food crop in the mid-eighteenth century (Hawkes, 1978). The long-day adaptation has greatly contributed to the success of the potato that has spread from Europe into nearly every part of the world. Nowadays potato is grown throughout the world and provides an important part of the global food supply (Table 1.1). The crop thrives in the temperate regions of the Figure 1.1. Stylised drawing of the potato (Solanum tuberosum) plant (Hegi, 1975). 10

11 General introduction Table 1.1. Food quantities in metric tons (Mt) available for human consumption of the top five of globally important food crops. Data from the year 2001 (FAOSTAT data, February 2004). Food World Europe Netherlands Wheat Rice (milled) Potato Maize Cassava Northern Hemisphere (Table 1.2) and is the staple food in several European countries (Burton, 1989). In the last decennia, cultivated areas have gradually decreased in traditional potato growing countries such as Poland, Belarus and Germany (FAOSTAT data, February 2004), whereas developing countries have increased their share of world potato production to about one-third (CIP, 1997). The Dutch climate is particularly suited for growing potato, which has contributed to the Netherlands success in potato breeding, its leading position in seed potato production, and its well-established potato starch industry. Potato is the cash crop in arable farming in the Netherlands. Large areas of ware, starch and seed potato are cultivated, and yields are among the highest in the world (Tables 1.2 and 1.3). Table 1.2. Total potato production and potato yield per hectare of the global top ten countries, Europe and world. Data from the year 2001 (FAOSTAT data, February 2004). Note that the potato yield per hectare in the Netherlands is biased by the relatively large proportion of seed potato, grown for export (Table 1.3). Total potato production Potato yield per hectare Mt Mt/ha China New Zealand 44.6 Russian Federation Netherlands 43.4 India Germany 42.2 USA Kuwait 41.3 Poland Belgium 41.2 Ukraine Denmark 40.4 Germany United Kingdom 40.3 Belarus USA 40.2 Netherlands Switzerland 37.6 United Kingdom France 37.5 Europe Europe 15.5 World World

12 Chapter 1 Table 1.3. Potato production data from the Netherlands for the year 2001 (CBS, 2004). Ware potato Starch potato Seed potato Total Harvested area (ha) Yield (Mt/ha) Production (Mt) Potato late blight The most important and destructive disease wherever potato is grown is late blight (Fry and Goodwin, 1997; Fry et al., 2001), which reduces global potato production by 15% annually (CIP, 1997). Late blight is caused by the oomycete Phytophthora infestans (Mont.) de Bary. The pathogen is thought to originate from the central highlands of Mexico (Reddick, 1939; Andrivon, 1996), where it has evolved on wild relatives of the cultivated potato. P. infestans probably migrated into South America in ancient times, and subsequently into North America and Europe in the early 1840s (Bourke, 1964; Andrivon, 1996), where potato crops were practically wiped out in 1843 and 1845/1846, respectively. Crop losses were most severe in Ireland, where the notorious Potato Famine caused poverty, the death of 1 million people and the emigration of another 1.5 million (Large, 1940). The pathogen was transported into the rest of the world with infected European seed tubers (Fry et al., 1993). P. infestans is a hemibiotroph (Lucas, 1998) that can affect most parts of the potato plant: leaves, stems, flowers, fruits, stolons, and tubers. Damage is caused by destruction of the foliage and rot of the tubers. Infection usually starts with the deposition of a sporangium on a leaf or stem. The sporangium germinates either directly (18-24 C) or indirectly via zoospores (8-18 C). The germ tube penetrates the host and mycelium grows intercellularly, sending haustoria into adjoining cells (Figure 1.2). After a few days the infection becomes visible as a lesion, the appearance of which depends on its age and weather conditions. Lesions on leaves typically have a necrotic centre surrounded by pale green or chlorotic tissue (Figure 1.3A), while stem lesions are water-soaked and dark green to black. When conditions are optimal (18-24 C), the asexual reproduction cycle can be completed in four to five days with the formation of large numbers of new sporangia at the edge of the lesion. Formation of sporangia occurs only under wet conditions, and appears as white fluff (Figure 1.3B). Sporangia are viable for up to several hours, during which they are dispersed by wind or splashing water to start new infections (Fry et al., 2001). This asexual cycle can be completed over 25 times in a single cropping season and result in a significant loss of photosynthetic capacity of the potato plant. Spores that are washed off the leaves and stems can infect tubers, allowing 12

13 General introduction invasion by secondary parasites such as bacteria and fungi, and result in rot of the tubers (Fry et al., 2001). Infected tubers enable hibernation of the pathogen and are an important source of inoculum for new epidemics in the next cropping season (Andrivon, 1995; Fry et al., 2001). Dissemination can be local when infected tubers end on cull piles or produce volunteer plants, but also long-distance when they are transported as seed tubers. The asexual reproduction cycle can be complemented by the sexual cycle when both the A1 and A2 mating type are present, and sexual oospores are produced (Fry et al., 2001). Such oospores can endure adverse conditions, survive in the soil for several years, and serve as an additional source of primary inoculum (Andrivon, 1995). The worldwide population of P. infestans has long been dominated by a single clonal lineage, US-1, of the A1 mating type and reproducing asexually. Genetic variation in US-1 could result from mitotic recombination and mutation only (Goodwin et al., 1994). A new worldwide migration of P. infestans started in the late 1970s. The new population has displaced the US-1 clonal lineage rapidly in Europe and the United States, and displacements may also be occurring on other Figure 1.2. Disease cycle of late blight caused by Phytophthora infestans (Agrios, 1997). 13

14 Chapter 1 continents (Spielman et al., 1991; Fry et al., 1993; Fry and Goodwin, 1997). The new population appears more diverse and more aggressive than the previous one, and consists of both mating types A1 and A2. Reproduction in the new population is predominantly asexual, but sexual reproduction also occurs and can enhance the quick adaptation of the pathogen through meiotic recombination. Socio-economic impact of potato late blight Potato late blight is among the most costly biotic constraints on global food production. Resource-poor farmers suffer crop losses that account for US$ 2.75 billion per annum, while another US$ 100 million are spent on fungicides (CIP, 1997). Disease control is extremely difficult in the highland tropics, because inoculum is continuously present due to year-round potato cultivation. Late blight epidemics in industrialized countries are controlled by enormous amounts of fungicides. In the Netherlands over 50% of all chemical pesticides applied in agriculture are used for late blight control (CBS, 2004). Dutch farmers spray their potato crops 7 to 20 times per cropping season (Schepers, 2002), which adds up to about 1.7 million kilograms of active ingredients annually (CBS, 2004). This practice is expensive and does not contribute to sustainable agriculture. In addition, the development of P. infestans populations that are resistant to the therapeutic fungicide metalaxyl (Goodwin et al., 1996) reduces the reliability of chemical control A B Figure 1.3. Lesions on potato leaves caused by infection with Phytophthora infestans. A. Newly formed lesions on a compound leaf of variety Irene in the field. B. Sporulating lesion on a leaflet of variety Eersteling in the laboratory. 14

15 General introduction of late blight. Potato production without the use of fungicides, as in organic farming, is hardly economically feasible. The threat that late blight poses to potato production worldwide is best contested by an integrated approach that combines sanitation, resistant varieties, cultivation practices, and judicious use of appropriate fungicides (Fry et al., 1993; 2001). The global fight against P. infestans is taken on by GILB: the Global Initiative on Late Blight. This network was launched in 1996 with the goal to reduce the devastating effects of potato late blight all over the world. In the Netherlands, forces have been joined in the Masterplan Phytophthora ( ), which has resulted in an integrated control strategy and in a substantial increase of the awareness of the late blight problem among potato growers, researchers, and policy makers. Nowadays, all activities are joined in NILB: the Netherlands Initiative on Late Blight (Parapluplan Phytophthora). This initiative started in 2003 and aims to reduce the use of fungicides to control P. infestans in potato by 75% in Breeding for resistance against Phytophthora infestans in potato The potato varieties that were grown when late blight caused its first heavy attacks in the 1840s were all more or less susceptible to the disease (Müller and Black, 1952; Van der Planck, 1957). This initiated the search for late blight resistance in wild relatives of the potato in the second half of the nineteenth century (Hawkes, 1990). Resistance against P. infestans was found in the hexaploid Mexican species S. demissum and was introduced into potato breeding programmes in the beginning of the twentieth century (Müller and Black, 1952; Ross, 1986). S. demissum was the source of (at least) 11 race-specific resistance (R) genes, most of which provided complete resistance to late blight (Ross, 1986; Turkensteen, 1989). The dominant nature and monogenic inheritance of these R genes facilitated their introgression into the tetraploid potato crop (Umaerus, 1970). R gene-mediated resistance against P. infestans in potato is associated with a hypersensitive response (HR; Müller and Black, 1952; Ross, 1986): a rapid, localized cell death, which prevents further colonization of the plant tissue (Mittler and Lam, 1996). The HR is the result of a specific interaction between the plant and the pathogen: plant receptors that are encoded by the dominant R genes recognize pathogen elicitors that are encoded by dominant avirulence (Avr) genes (Heath, 1998). A mutation in an Avr gene may result in the loss of recognition by the cognate R gene product and, consequently, in the loss of resistance. Races of P. infestans that were no longer resisted by the R genes of S. demissum appeared soon after, or even before introgression of the resistance into commercial potato 15

16 Chapter 1 varieties, and are now present for (combinations of) all 11 known R genes (Turkensteen, 1993). Current potato varieties carry subsets of these 11 R genes (Ross, 1986; Turkensteen, 1989), but the effectiveness of this race-specific resistance depends on the unpredictable racial composition of the P. infestans population in the area of cultivation. The advantage of potato varieties with racespecific resistance to late blight disappears when such varieties become widely cultivated, because virulent races of the pathogen soon become the prevalent ones (Van der Plank, 1957). More recently discovered R genes of S. berthaultii (Ewing et al., 2000), S. bulbocastanum (Helgeson et al., 1998; Van der Vossen et al., 2003), and S. pinnatisectum (Kuhl et al., 2001) are now being investigated for their applicability in potato breeding. Race-non-specific resistance against P. infestans in potato is believed to be more durable, but can provide only partial protection. This resistance is also known as partial, horizontal, general, quantitative, or field resistance, and levels are similar for all races of P. infestans (Thurston, 1971). Race-non-specific resistance to late blight consists of components that affect infection efficiency, lesion growth rate, latent period, and sporulation capacity of the pathogen (Van der Zaag, 1959; Thurston, 1971). The resistance is characterized by a continuous variation in phenotypic appearance and a polygenic inheritance that make breeding for this trait rather complicated (Umaerus, 1970; Wastie, 1991). An additional difficulty is the uncertainty about the number of genes involved: molecular marker studies have identified Quantitative Trait Loci (QTLs) for race-non-specific resistance against P. infestans on all 12 potato chromosomes (Gebhardt and Valkonen, 2001; Simko, 2002). Race-non-specific resistance to late blight has been derived from wild species such as S. demissum, S. stoloniferum, S. vernei and S. verrucosum (Ross, 1986), but high levels of resistance have also been identified in numerous other wild Solanum species (Ross, 1986; Hawkes, 1990; Wastie, 1991). Unfortunately, most of the race-non-specific resistance fades away during the necessary back-crosses with S. tuberosum (Tazelaar, 1981). Although aimed at, true race-non-specificity is impossible to prove, while true durability can only be concluded after cultivation of a particular variety on a large acreage for a long period of time (Johnson, 1979). Illustrative is that potato varieties with late blight resistance that was thought to be race-non-specific also display hypersensitive necrosis, though at a slower rate (Vleeshouwers et al., 2000), and appear rather susceptible when exposed to certain aggressive races of P. infestans (Flier et al., 2003). The existence of R genes from S. demissum that do not provide complete resistance against P. infestans (R2, R4, R10, R11; Turkensteen, 1989), 16

17 General introduction and residual resistance conferred by defeated R genes (Stewart et al., 2003) also add to the experience that differences between race-specific and race-non-specific or durable resistance are not that clear. The development of effective fungicides during the twentieth century initially diminished the need for late blight resistant potato (Turkensteen, 1993). The current emphasis on sustainable agriculture, as well as the appearance of a new, more aggressive and fungicide-resistant P. infestans population has restored the need for resistant potato varieties (Duncan, 1999). Breeding for resistance to late blight is on the top of the priority list of GILB (CIP, 1997), and characterization of, and employing genes for resistance against P. infestans are important goals in largescale plant genome research projects such as the German GABI (Genome Analysis of the plant BIological system) and the Dutch CBSG (Centre for BioSystems Genomics). Foliage maturity type in potato and its relation with late blight resistance Race-non-specific resistance against P. infestans in potato is found only in late maturing genotypes (Toxopeus, 1958). All potato plants start with the build-up of foliage. In time, assimilates are also allocated to tuber development. In early maturing genotypes this allocation of assimilates is soon directed in favour of tuberization, the foliage is no longer maintained and plants become senescent. Late maturing genotypes invest more in foliage development. This results in a prolonged above-ground development and, thus, a larger canopy. Tuberization is slower, but due to the larger photosynthetic capacity and longer life span of the foliage a higher tuber yield is achieved (Kooman, 1995). However, very late maturity in potato varieties is unwelcome, because harvest at the very end of the cropping season can be difficult due to unfavourable (rainy) weather conditions, and the long period of growth that is required is inefficient in year-round production systems. The variation in maturity that is available in the current range of potato varieties allows cultivation of potato in different areas of the world, enables year-round supply of fresh potato, and satisfies marketing purposes. Assessing maturity is rather difficult, because tuber production occurs below-ground and, thus, can only be evaluated destructively. Fortunately, the trait of interest correlates well with maturity of the foliage (Celis-Gamboa, 2002), which can be evaluated more easily. Therefore, maturity of potato is generally assessed as maturity of the foliage, called foliage maturity type in this thesis. Foliage maturity type is composed of a syndrome of features, of which termination of apical growth, sagging of the plants, and yellowing of the leaves are the most important components. 17

18 Chapter 1 The association between race-non-specific resistance against P. infestans and late foliage maturity has been mentioned by several authors (e.g. Müller and Black, 1952; Ross, 1986; Wastie, 1991), but only a few provide original data (e.g. Van der Plank, 1957; Toxopeus, 1958; Swiezynski, 1990). The consequence of this association is the virtual non-existence of early maturing potato varieties with satisfactory levels of resistance to late blight (Figure 1.4). The underlying mechanism of this association is not known and could be either genetic or physiological (Toxopeus, 1958). In case of a genetic association, this can be the result of either closely linked, but different genes, or of a single, pleiotropic gene that affects both traits separately. In case of a physiological association, one or more genes affect the physiological state of the plant, which in turn influences both traits: late blight resistance and foliage maturity type. A physiological association is supported by observations that environmental conditions affect both traits. Illustrative is the effect of photoperiod: short photoperiods promote early foliage maturity (Pohjakallio et al., 1957) and simultaneously reduce resistance to late blight (Umaerus, 1959). The presence of separable genes for the two traits seems improbable, as potato breeders have fruitlessly tried to combine late blight resistance with early foliage maturity for decades (Muskens and Allefs, 2002). Breeding programmes worldwide involve screening of hundreds of thousands of genotypes each year, a number most likely sufficiently large to reveal even the rarest recombinant if proper identification is feasible within the selection procedures applied. Although these data seem to support a physiological association between race-non-specific resistance against P. infestans and foliage maturity type, the resistance is polygenic and the association with foliage maturity type may be different (genetic, physiological, or absent) on each of the different loci for resistance. Scope of this thesis The research described in this thesis was started to unravel the nature of the association between late blight resistance and foliage maturity type in potato. The main goal was to determine whether the association between the two traits is genetic or physiological. An integrated approach of genetic and physiological experiments has been pursued, with on the one hand a search for separable loci for the two traits, and on the other hand a search for proof of physiological association. Foliage maturity type can only be assessed relatively late in the cropping season, whereas resistance to late blight is detectable after the first infection with P. infestans, which generally occurs quite early in the season. Chapter 2 describes 18

19 General introduction Late blight resistance Resistant Susceptible Late Early Foliage maturity type Figure 1.4. Late blight resistance and foliage maturity type of all potato varieties on the Dutch list of varieties of field crops (Anonymous, 2004). attempts to enable evaluation of both traits concurrently, at an early stage of plant growth, by using single-node cuttings of potato. This approach turned out to be not applicable, because single-node-cuttings do not reflect the physiological state of the whole plant adequately. A possible physiological association between late blight resistance and foliage maturity type was studied by relating the effects of plant age, leaf age, and leaf position on resistance. Results are described in Chapter 3, and led to the conclusion that the effect of leaf position prevails over effects of plant age and leaf age in reflecting resistance against P. infestans. Simultaneously, the association between late blight resistance and foliage maturity type was studied genetically. At the start of this research, the association between the two traits had just been confirmed by means of molecular markers: a locus on chromosome 5 near marker GP21 appeared to have a major effect on resistance to late blight as well as on foliage maturity type (Van Eck and Jacobsen, 1996). Chapter 4 describes a detailed study of this locus, with analyses that were focused on determining whether this locus harbours a single QTL that affects both traits, or different QTLs that coincide on the same chromosomal region. Because the results were not conclusive, a more comprehensive and thorough search for recombinant genotypes and QTLs for both traits was undertaken that is described in Chapters 5 and 6. Recombinant genotypes were not retrieved, but QTLs for late blight resistance and foliage maturity type were identified, with some QTLs for resistance appearing to be independent of foliage maturity type. All results are summarized and discussed in Chapter 7, and the consequences for further breeding for resistance against P. infestans in early maturing genotypes are considered. 19

20 Chapter 1 REFERENCES Agrios GN (1997) Plant pathology, 4 th edition. Academic Press, London, p Andrivon D (1995) Biology, ecology, and epidemiology of the potato late blight pathogen Phytophthora infestans in soil. Phytopathology 85: Andrivon D (1996) The origin of Phytophthora infestans populations present in Europe in the 1840s: a critical review of historical and scientific evidence. Plant Pathol 45: Anonymous (2004) Potatoes. In: 79 th List of varieties of field crops. Stichting DLO, Wageningen, The Netherlands, p Bourke PMA (1964) Emergence of potato blight, Nature 203: Burton WG (1989) The potato. Longman, New York, pp 742. Celis-Gamboa WC (2002) The life cycle of the potato (Solanum tuberosum L.): from crop physiology to genetics. PhD Thesis, Wageningen University, Wageningen, pp 191. CIP (1997) CIP in The International Potato Center Annual Report. International Potato Center, Lima, pp 59. Duncan JM (1999) Phytophthora-an abiding threat to our crops. Microbiol Today 26: Ewing EE, Simko I, Smart CD, Bonierbale MW, Mizubuti ESG, May GD, Fry WE (2000) Genetic mapping from field tests of qualitative and quantitative resistance to Phytophthora infestans in a population derived from Solanum tuberosum and Solanum berthaultii. Mol Breed 6: Flier WG, Van den Bosch GBM, Turkensteen LJ (2003) Stability of partial resistance in potato cultivars exposed to aggressive strains of Phytophthora infestans. Plant Pathol 52: Fry WE, Goodwin SB, Dyer AT, Matuszak JM, Drenth A, Tooley PW, Sujkowski LS, Koh YJ, Cohen BA, Spielman LJ, Deahl KL, Inglis DA, Sandlan KP (1993) Historical and recent migrations of Phytophthora infestans: chronology, pathways, and implications. Plant Dis 77: Fry WE, Goodwin SB (1997) Resurgence of the Irisch potato famine fungus. BioScience 47: Fry WE, Thurston HD, Stevenson WR (2001) Late blight. In: Stevenson WR, Loria R, Franc GD, Weingartner DP (eds) Compendium of potato diseases, 2 nd edition. American Phytopathological Society, St. Paul, p Gebhardt C, Valkonen JPT (2001) Organization of genes controlling disease resistance in the potato genome. Annu Rev Phytopathol 39: Goodwin SB, Cohen BA, Fry WE (1994) Panglobal distribution of a single clonal lineage of the Irish potato famine fungus. Proc Natl Acad Sci USA 91: Goodwin SB, Sujkowski LS, Fry WE (1996) Widespread distribution and probable origin of resistance to metalaxyl in clonal genotypes of Phytophthora infestans in the United States and Western Canada. Phytopathology 86: Hawkes JG (1978) History of the potato. In: Harris PM (ed) The potato crop: the scientific basis for improvement. Chapman and Hall, London, p Hawkes JG (1990) The potato: evolution, biodiversity and genetic resources. Belhaven Press, London, pp

21 General introduction Heath MC (1998) Apoptosis, programmed cell death and the hypersensitive response. Eur J Plant Pathol 104: Hegi G (1975) Illustrierte Flora von Mitteleuropa, V. Band, 4. Teil, Dicotyledones, 3. Teil, Verlag Paul Parey, Berlin und Hamburg, p Helgeson JP, Pohlman JD, Austin S, Haberlach GT, Wielgus SM, Ronis D, Zambolim L, Tooley P, McGarth JM, James RV, Stevenson WR (1998) Somatic hybrids between Solanum bulbocastanum and potato: a new source of resistance to late blight. Theor Appl Genet 96: Johnson R (1979) The concept of durable resistance. Phytopathology 69: Kooman PL (1995) Yielding ability of potato crops as influenced by temperature and daylength. PhD Thesis, Wageningen Agricultural University, Wageningen, pp 155. Kuhl JC, Hanneman Jr. RE, Havey MJ (2001) Characterization and mapping of Rpi1, a late-blight resistance locus from diploid (1EBN) Mexican Solanum pinnatisectum. Mol Gen Genet 265: Large EC (1940) The advance of the fungi. Jonathan Cape, London, pp 488. Lucas JA (1998) Plant pathology and plant pathogens, 3 rd edition. Blackwell, Oxford, pp 274. Mittler R, Lam E (1996) Sacrifice in the face of foes: pathogen-induced programmed cell death in plants. Trends Microbiol 4: Müller KO, Black W (1952) Potato breeding for resistance to blight and virus diseases during the last hundred years. Z Pflanzenzüchtg 31: Muskens MWM, Allefs JJHM (2002) Breeding for late blight resistance; views from practice. In: Wenzel G, Wulfert I (eds) Potatoes today and tomorrow. Abstracts of papers and posters of the 15 th triennial conference of the EAPR. European Association for Potato Research, Bonn, p 85. Pohjakallio O, Salonen A, Antila S (1957) Analysis of earliness in the potato. Acta Agric Scand 7: Reddick D (1939) Where came Phytophthora infestans? Chronica Botanica 5: Ross H (1986) Late blight, Phytophthora infestans (Mont.) de Bary. In: Ross H (ed) Advances in Plant Breeding, vol 13. Potato breeding: problems and perspectives. Verlag Paul Parey, Berlin, p Schepers HTAM (2002) The development and control of Phytophthora infestans in Europe in In: PPO-special report no 8: Sixth workshop of an European Network for development of an integrated control strategy of potato late blight. Edingburgh, Scotland, september 2001, p Simko I (2002) Comparative analysis of quantitative trait loci for foliage resistance to Phytophthora infestans in tuber-bearing Solanum species. Am J Potato Res 79: Spielman LJ, Drenth A, Davidse LC, Sujkowski LJ, Gu W, Tooley PW, Fry WE (1991) A second world-wide migration and population displacement of Phytophthora infestans? Plant Pathol 40: Stewart HE, Bradshaw JE, Pande B (2003) The effect of the presence of R-genes for resistance to late blight (Phytophthora infestans) of potato (Solanum tuberosum) on the underlying level of field resistance. Plant Pathol 52:

22 Chapter 1 Swiezynski KM (1990) Resistance to Phytophthora infestans in potato cultivars and its relation to maturity. Genet Pol 31: Tazelaar MF (1981) The screening of Solanum species for horizontal resistance against late blight (Phytophthora infestans) and its use for breeding programmes. In: Abstracts of conference papers of the 8 th triennial conference of the EAPR. European Association for Potato Research, München, p Thurston HD (1971) Relationship of general resistance: late blight of potato. Phytopathology 61: Toxopeus HJ (1958) Some notes on the relations between field resistance to Phytophthora infestans in leaves and tubers and ripening time in Solanum tuberosum subsp. tuberosum. Euphytica 7: Turkensteen LJ (1989) Interaction of R-genes in breeding for resistance of potatoes against Phytophthora infestans. In: Fungal diseases of the potato. International Potato Center, Lima, p Turkensteen LJ (1993) Durable resistance of potatoes against Phytophthora infestans. In: Jacobs Th, Parlevliet JE (eds) Durability of disease resistance. Kluwer Academic Publishers, Dordrecht, p Umaerus V (1959) The relationship between peroxidase activity in potato leaves and resistance to Phytophthora infestans. Am Potato J 36: Umaerus V (1970) Studies on field resistance to Phytophthora infestans 5. Mechanisms of resistance and applications to potato breeding. Z Pflanzenzüchtg 63: Van der Plank JE (1957) A note on three sorts of resistance to late blight. Am Potato J 34: Van der Vossen E, Sikkema A, Te Lintel Hekkert B, Gros J, Stevens P, Muskens M, Wouters D, Pereira A, Stiekema W, Allefs S (2003) An ancient R gene from the wild potato species Solanum bulbocastanum confers broad-sprectrum resistance to Phytophthora infestans in cultivated potato and tomato. Plant J 36: Van der Zaag DE (1959) Some observations on breeding for resistance to Phytophthora infestans. Eur Potato J 2: Van Eck HJ, Jacobsen E (1996) Application of molecular markers in the genetic analysis of quantitative traits. In: Struik PC, Hoogendoorn J, Kouwenhoven JK, Mastenbroek LJ, Turkensteen LJ, Veerman A, Vos J (eds) Abstracts of conference papers, posters and demonstrations of the 13 th triennial conference of the EAPR. European Association for Potato Research, Wageningen, p Vleeshouwers VGAA, Van Dooijeweert W, Govers F, Kamoun S, Colon LT (2000) The hypersensitive response is associated with host and nonhost resistance to Phytophthora infestans. Planta 210: Wastie RL (1991) Breeding for resistance. In: Ingram DS, Williams PH (eds) Advances in Plant Pathology, vol 7. Phytophthora infestans: the cause of late blight of potato. Academic Press, London, p

23 Chapter 2 Are cuttings suitable for assessing maturity type in potato (Solanum tuberosum)? P.C. Struik, M.H.P.W. Visker, J.B. Pauwels, L.T. Colon Submitted

24 Chapter 2 ABSTRACT Two experiments were carried out to evaluate the potential of single-node cuttings of potato (Solanum tuberosum) as a tool to assess genotypic differences in maturity type. Plants were exposed to different photoperiodic treatments (different photoperiods, different numbers of photoperiodic cycles) and cuttings were taken at different plant ages. Cuttings from early (and to a lesser extent also late) maturing varieties exposed to short photoperiods showed strong induction to tuberize irrespective of plant age; the induction increased with an increase in the number of short photoperiodic cycles. The response of cuttings taken from early maturing varieties exposed to long photoperiods depended on plant age: cuttings showed stronger induction when mother plants were older; cuttings from late maturing varieties hardly tuberized after exposure to long photoperiods. The tuberization of the cuttings did not depend on the length of the long photoperiods (18 or 24 h), or on the number of cycles of a photoperiod of 18 h. Tuberization on cuttings did not properly reflect the tuber formation on the mother plants, although within varieties significant correlations between tuberization on cuttings and tuber yield per plant nine weeks after planting were found with different numbers of photoperiodic cycles of 12 h. Our experiments show that the cutting technique cannot be used on older plants to assess the maturity type of potato varieties, as there are interactions between photoperiod, genotype, plant age, and number of photoperiodic cycles, in the reflection of the degree of induction to tuberize on single-node cuttings. INTRODUCTION Tuberization is an essential developmental process in the life cycle of the potato (Solanum tuberosum) plant. It includes tuber induction, tuber initiation, and the initial phases of tuber formation. It is the result of a well-orchestrated sequence of highly complex physiological events and marks the onset of the production phase of the potato crop, often called the tuber bulking phase. Tuberization also triggers other changes, including changes in plant morphology (Ewing, 1985), a shift in the rate of photosynthesis, and a change in dry-matter partitioning (Ewing and Struik, 1992; Van Dam et al., 1996). Tuber induction is influenced by photoperiod (Gregory, 1965; Ewing and Struik, 1992). Tuber induction is advanced and tuberization is promoted by short photoperiods (or long nights). The same is true for other physiological processes, 24

25 Single-node cuttings such as flowering (Almekinders and Struik, 1994; 1996): the shorter the photoperiod, the faster the progress to flowering. The abundance of flowering is often enhanced by long photoperiods, which is associated with a delay of tuber bulking. Long photoperiods allow the formation of more sympodial branches (Almekinders and Struik, 1996), thus prolonging the period of formation of new inflorescences and increasing the numbers of flowers per inflorescence. The parallel progression to flowering and tuberization induced by short photoperiods, followed by the photoperiod-controlled competition between sympodial growth and flowering on the one hand, and tuber bulking on the other hand, illustrates the complexity of the whole-plant physiology (cf. Celis-Gamboa et al., 2003). Moreover, both the tuberization and the flowering response to photoperiod show significant interaction with many other environmental factors (such as temperature and light intensity), cultural practices (nitrogen supply, plant density), and intrinsic factors (such as genotype and the physiological age of the mother plant). While photoperiod affects tuberization, flowering, and dry-matter partitioning of the plant, it also influences the expression of resistance to late blight (Colon, 1994): resistance is lost at short photoperiods, a phenomenon associated with increased infection efficiency and increased lesion growth rate. This is especially interesting as it is well established that genotypes with early foliage maturity types are much more susceptible to late blight than genotypes with late maturity types (Chapter 5). There might be a direct link between the induction to tuberize and the susceptibility to late blight. The definition of maturity type as a genotypic trait in potato is usually based on either the timing of tuber formation (or the timing of the rapid increase in harvest index), or on the timing of leaf senescence, combined with sagging of plants and completion of apical canopy growth. To study the genetic-physiological linkage between maturity type and susceptibility to late blight we wanted to create a manageable definition of the concept of (foliage) maturity type and develop a simple and reproducible tool to assess maturity type at or before the time genotypic differences in late blight susceptibility are expressed. This tool should be reliable and allow the handling of large numbers of genotypes. According to Ewing (1985), the changes taking place in the whole plant after induction to tuberize are accurately simulated by the behaviour of single-node cuttings (or leaf-bud cuttings) taken from these plants. Single-node cuttings may therefore be used as simple and convenient models to study the physiology of the entire potato plant. Because their structure is simple and their physiological behaviour after cutting becomes independent of the mother tuber from which the 25

26 Chapter 2 mother plant is grown, cuttings might even show clearer responses than the whole plant from which they are taken. They are also easily manipulated and can be checked for developmental progress frequently and non-destructively (Struik, 1999). Earlier research showed the robustness of the method and illustrated its use for many diverse studies on various potato plant phenomena, including tuber initiation (Duncan and Ewing, 1984), secondary growth (Van den Berg et al., 1990), maturation and leaf senescence (McGrady and Ewing, 1990), and response to exogenously applied hormones (McGrady et al., 1986). Cuttings are especially suitable to test how environmental conditions, genetic differences, the application of growth substances, and other variables affect the degree to which leaves have been induced to cause stolon or tuber formation (ad verbatim citation from Ewing and Struik, 1992). The cutting technique extensively described by Ewing (1985) was an obvious candidate to use in our research, as this technique can be used to assess the induction to tuberize throughout the cropping season in field experiments (Ewing and Wareing, 1978). The cutting technique was also very attractive as we could then carry out assessments of maturity type and susceptibility to late blight on the same part of the plant, thus allowing us to relate these two phenomena directly and on an individual leaf basis. This paper illustrates that the cutting technique cannot be used to assess the maturity type of potato varieties, certainly not at later stages of growth, as there is a strong photoperiod-by-genotype (by plant age) interaction in the reflection of the degree of induction to tuberize by single-node cuttings. We illustrate this by describing the response of cuttings taken from a diverse set of varieties exposed to various photoperiods, in interaction with differential numbers of cycles of short and long photoperiods and different plant ages. MATERIALS AND METHODS Starting material Two large-scale experiments were carried out. Experiment 1 was carried out using plants obtained from pre-sprouted minitubers of similar physiological age and produced and stored under controlled conditions. For Experiment 2, in vitro plantlets were obtained from a commercial source. We chose to start with in vitro plantlets in the second experiment to avoid confounding effects of differences in physiological age of mother tubers between varieties used. 26

27 Single-node cuttings Varieties Experiment 1 included eight varieties varying in foliage maturity type. Foliage maturity type is expressed on a scale from 1 (very late) to 9.5 (very early) and these foliage maturity type data were taken from the appropriate Dutch lists of varieties of field crops. The early maturing varieties of Experiment 1 included Spunta (maturity type 7), Apollonia (8), Eersteling (8.5), and Minerva (8.5). Late maturing varieties were Robijn (maturity type 3), Pimpernel (3.5), Alpha (4), and Mondial (4.5). Experiment 2 included the early maturing variety Rode Eersteling (maturity type 9.5), the mid-early variety Nicola (6), and the late variety Astarte (3). Growing conditions, treatments, and experimental design In Experiment 1 plants were grown in two growth chambers, each set at 19 ± 0.5 C (photophase) and 11 ± 0.5 C (dark phase), with a period of either 10 or 16 h of photosynthetically active radiation (obtained from 50% Philips 400 W/HPI-T and 50% Philips 400 W/SON-T lamps at a density of three lamps per m² at 1.80 m above plant level). For each variety x photoperiod combination five plants were grown until 20, 27, 34, 41, 48, 55, 62, or 69 days after planting, at which plant ages cuttings were taken. Plants were grown in a potting soil in containers of 2.2 litre (plants grown until 20 or 27 days after planting) or of 5 litre (plants grown until 34 days after planting or longer). In Experiment 2 plants were grown in three greenhouses, set to maintain a temperature of 18 ± 2 C during the day (12 h) and 12 ± 1 C at night (12 h). Photoperiods were extended to 24 h using incandescent bulbs until treatments started. Treatments included three photoperiods (12, 18 or 24 h) maintained during the last 7, 14, 21, or 28 cycles before cuttings were taken. Natural light was supplemented using Philips 400 W/SON-T lamps (0.8 lamps per m 2 at 2 m above plant level). Per variety x photoperiod x number of photoperiodic cycles, plants were grown in a potting medium in containers of 5 litre. Two thirds of the plants were allocated to treatments with 7, 14, 21, or 28 cycles of a photoperiod of 12 or 18 h, one third remained at photoperiods of 24 h for the entire growth phase. The very reliable estimates obtained for the 24 h photoperiod treatment made it possible to use this treatment not only as the control but also as a treatment with 0 cycles of a photoperiod of either 12 or 18 h. All plants were used for cutting on the same date, i.e. 28 days after onset of first treatments (or nine weeks after planting). 27

28 Chapter 2 Cuttings Single-node cuttings or leaf-bud cuttings consist of a leaf and its subtended bud together with a small piece of stem. The stem piece, the base of the petiole and the bud are inserted into a well-moistened soil with the leaf upright. The subtended bud should not have shown too much growth as this hampers the expression of the level of induction of the leaf. Cuttings in Experiment 1 were taken using two stems per plant and for each stem taking the leaf numbers four, five, and six counting from the top, leaf one being the youngest leaf longer than 3.5 cm. This provided 30 cuttings per variety x plant age x photoperiod treatment combination. The cuttings of Experiment 1 were placed in trays with a transparent cover, containing a moist potting medium, in climate rooms under the same conditions as to which the plants from which the cuttings were taken had been exposed. The 3840 cuttings were examined 14 days after cutting. The cuttings of Experiment 2 were taken from the main stem only, using the leaf numbers four, five, and six. If the number of plants proved to be insufficient for three replicates of ten cuttings each (replicates corresponding to the greenhouses during the first five weeks of growth), cuttings were also taken from a lower branch. The latter were always allocated to the third replicate. This provided 90 cuttings per variety x photoperiod x number of photoperiodic cycles combination. The cuttings of Experiment 2 were placed in covered, transparent trays, filled with a moist potting medium and put in greenhouses set at a day/night temperature of 18 (12 h) / 12 (12 h) C and photoperiods of 12 h. The relative humidity was kept as high as possible. All 3240 cuttings of Experiment 2 were examined after 14 days. Scoring In Experiment 1, the evaluation of the structures formed was based on the diagram of Ewing and Struik (1992). As this diagram is very detailed, also discriminates on the basis of the development of the ancillary buds, and suggests equidistance between different structures, we used a less detailed scale of 0 (no bud growth, lowest level of induction), 1 (orthotropic shoot from the central bud irrespective of development of ancillary bud; score 2 in the Ewing and Struik scale), 5 (stolons present on either the central or the ancillary buds; scores 3-6/7 in the Ewing and Struik scale), and 9 (tuber on the central bud, highest level of induction; scores 7/8-9 in the Ewing and Struik scale). In Experiment 2, the evaluation of the structures was based on the total percentage of tuberization per experimental unit (including all structures on the main 28

29 Single-node cuttings bud with a tuber) followed by a correction for calculating percentages based on low numbers plus an arcsin sqrt(x) transformation of the data (cf. Struik et al., 1987). Tuber formation on plants In both experiments, mother plants were checked for the presence of tubers when cuttings were taken. In Experiment 1, all five plants per variety x photoperiod x plant age combination were harvested. In Experiment 2, ten plants per variety x photoperiodic treatment were harvested (16 for the treatments with photoperiods of 24 h). These plants were randomly selected. Number of tubers present and their total fresh and dry weights were recorded. Tuber yields reflect the tuber growth during the first ten weeks (Experiment 1) or nine weeks (Experiment 2) after planting. Statistical analysis Data recorded were analysed using GenStat 6 (GenStat, 2002). For Experiment 1, we carried out an ANOVA. Factors included in the fixed model comprised photoperiod, plant age, and variety (plus the contrast between early and late maturing varieties), and the three two-way interactions and one three-way interaction between these factors. Low absolute values of residuals called for some caution in the analysis, but this lack of variance was mainly associated with missing values. Results were verified with a REML (REstricted Maximum Likelihood) analysis of variance components. In both the statistical analysis and our presentation data were pooled over stem and leaf number. Data of Experiment 2 were analysed by an ANOVA. Factors included in this analysis of variance were photoperiod, number of photoperiodic cycles, variety, replicate (combining the effects of greenhouse during initial growth, plant, and stem), leaf number, and all possible interactions. RESULTS Experiment 1 We expected that the developmental score of the buds on the single-node cuttings would increase with plant age, and that this increase would be influenced both by foliage maturity type (variety type) and photoperiod. The ANOVA showed variance ratios that were especially large for the main effects of photoperiod and the contrast between early and late maturing varieties, 29