Late blight (LB) caused by the oomycete Phytophthora infestans, is a major disease of potato and tomato worldwide and can cause up to 100% yield losses. The devastating economic impact of this disease intensified the related pathology and genetics research since the occurrence of Irish famine in 1840s, with a side gain of major scientific discoveries. For example, many of the crucial steps involved in LB defense response in host plants have been elucidated through the use of modern cytological and molecular biology techniques. Also, genetic and biochemical studies have revealed differences between oomycetes and pathogenic fungi, which has led to more selective use of chemicals for LB control. Furthermore, the discovery of P. infestans two mating types and the resultant generation of more aggressive lineages by sexual recombination stresses the need for an integrated and sustainable approach to LB control. These measures would include the use of cultural practices, selective fungicide applications, and genetic resistance. In potato at least a dozen major resistance genes and several quantitative trait loci (QTLs) for LB resistance have been identified, and most modern cultivars have been bred with one or more resistance genes. In tomato, though most commercial cultivars are susceptible to LB, a few major resistance genes and several QTLs have been identified and several breeding programs around the world are developing breeding lines and commercial cultivars with LB resistance. Most recently, a few fresh-market tomato hybrid cultivars with LB resistance were released by the North Carolina State University Tomato Breeding Program in the United States. There is, however, an insufficient number of potato and tomato cultivars with LB resistance, resulting in continued expensive as well as the hazardous and increasingly ineffective use of chemicals for disease control. In an era when both host plants and P. infestans genomes are sequenced and considerable genomic information is available, it is not unexpected that a more sustainable solution to controlling LB is on the horizon. In this review, we summarize the recent achievements in better understanding of the P. infestans pathogenesis, host-pathogen interactions, and the progress made in developing genetic resistance in potato and tomato.

The cultivated tomato, Solanum lycopersicum L., is the second most consumed vegetable crop after potato and unquestionably the most popular garden crop in the world. There are more varieties of tomato sold worldwide than any other vegetable crop. Most of the commercial cultivars of tomato have been developed through phenotypic selection and traditional breeding. However, with the advent of molecular markers and marker-assisted selection (MAS) technology, tomato genetics and breeding research has entered into a new and exciting era. Molecular markers have been used extensively for genetic mapping as well as identification and characterization of genes and QTLs for many agriculturally important traits in tomato, including disease and insect resistance, abiotic stress tolerance, and flower- and fruit-related characteristics. The technology also has been utilized for marker-assisted breeding for several economically important traits, in particular disease resistance. However, the extent to which MAS has been employed in public and private tomato breeding programs has not been clearly determined. The objectives of this study were to review the publically-available molecular markers for major disease resistance traits in tomato and assess their current and potential use in public and private tomato breeding programs. A review of the literature indicated that although markers have been identified for most disease resistance traits in tomato, not all of them have been verified or are readily applicable in breeding programs. For example, many markers are not validated across tomato genotypes or are not polymorphic within tomato breeding populations, thus greatly reducing their utility in crop improvement programs. However, there seems to be a considerable use of markers, particularly in the private sector, for various purposes, including testing hybrid purity, screening breeding populations for disease resistance, and marker assisted backcross breeding. Here we provide a summary of molecular markers available for major disease resistance traits in tomato and discuss their actual use in tomato breeding programs. It appears that many of the available markers may need to be further refined or examined for trait association and presence of polymorphism in breeding populations. However, with the recent advances in tomato genome and transcriptome sequencing, it is becoming increasingly possible to develop new and more informative PCR-based markers, including single nucleotide polymorphisms (SNPs), to further facilitate the use of markers in tomato breeding. It is also expected that more markers will become available via the emerging technology of genotyping by sequencing (GBS).

The cultivated potato, Solanum tuberosum is affected by a variety of diseases with late blight (LB) caused by Phytophthora infestans being the most severe. The disease is mostly controlled by the application of large quantities of fungicides, which represent a financial burden on farmers indeveloping countries and pose risks to both human health and the environment. A more effective and environmentally friendly strategy to prevent damages caused by P. infestans is to use resistant potato cultivars. In the early days of breeding for LB resistance, a small number of resistance (R) genes from the wild Mexican species, Solanum demissum, were introgressed into modern potato varieties. These genes conferred race-specific resistance, which was rapidly overcome by new isolates of the pathogen. Recently, a number of new R genes have been identified and cloned from several wild potato species .Taking advantage of genetic engineering, our strategy is to use three of these new R genes (RB, Rpiblb2 and Rpi-vnt1.1) in a triple gene construct, p CIP 99, based on the hypothesis that simultaneous mutation for pathogenicity against all three genes is unlikely, and therefore the resistance conferred by the construct should be durable. We plan to deploy the three stacked R genes into the potato variety “Victoria” (known in Kenya and Uganda as “Asante”). To date, we have produced more than 100 transgenic events which are currently being characterized. The pathogen population will be characterized concurrently to assess the expected durability of this resistance.

Sclerotinia stem rot caused by the fungus Sclerotinia sclerotiorum is one of the most damaging and difficult-to-manage diseases of oilseed rape (Brassica napus) and mustard (B. juncea). Identifying oilseed Brassica genotypes with effective resistance offers the best long-term prospect for improved management of the disease. Despite some significant interactions between oilseed Brassica genotypes and S. sclerotiorum isolates reported in earlier studies, mostly a single pathogen isolate has been used to identify resistant genotypes. This paper reports the results of studies involving 53 isolates of S. sclerotiorum from the northern and southern agricultural regions of Western Australia where Sclerotinia stem rot is a serious disease of oilseed rape. Colony characteristics of isolates on potato dextrose agar were determined, and two field virulence (i.e., levels of disease severity) studies conducted. The first field study included 14 Brassica genotypes against three S. sclerotinia isolates and the second had eight Brassica genotypes against 50 S. sclerotinia isolates. Only colony diameter of isolates growing on potato dextrose agar was correlated with stem lesion length in the field. In both field experiments, there were significant effects of isolates and host genotypes, as well as a significant interaction between isolates and genotypes in relation to stem lesion length. In the first field experiment, mean stem lesion length ranged from 1cm in the highly resistant B. napus ZY006 to at least 9cm for susceptible genotypes Brassica juncea #2 and B. juncea Montara. The latter genotype was the most susceptible with a mean stem lesion length of 11.1. Expression of high-level resistance in B. napus ZY006 was largely independent of S. sclerotiorum isolate. In contrast, responses in B. napus genotypes, Zhongyou 821, 06-6-3792, RT108 and Charlton were much more isolate-dependent. In the first field experiment, with a greater number of host genotypes than S. sclerotiorum isolates, a higher variance ratio occurred for isolates (VR=158.4) than for host genotypes (VR=10.7). In the second field experiment, with a larger number of S. sclerotinia isolates than hosts, host genotypes had a higher variance ratio (VR=458.9) compared to the isolates (VR=71.2). Increasing the number of isolates greatly improved the capacity to differentiate levels of resistance among test genotypes. In this experiment, some genotypes showed more consistent resistant reactions (e.g., B. napus Mystic and B. juncea Xinyou 9) across different isolates and these are ideal targets for commercial exploitation of this resistance in oilseed Brassica breeding programs. From this experiment, a standardized set of three B. napus and three B. juncea genotypes were shown to be suitable for use as universal differentials to characterize pathotypes of S. sclerotiorum using octal nomenclature. Eight distinct pathotypes of S. sclerotiorum were delineated and characterized and these six host differential genotypes can now be utilized to identify and monitor the incidence and distribution of current and future pathotypes of S. sclerotiorum. Further, by providing a reliable means to characterize pathotypes of S. sclerotiorum, for the first time not only allows identification of resistance(s) against the predominant pathotype(s) of S. sclerotiorum prevailing in a particular region, but also now allows oilseed rape breeding programs to combine host resistances against several specific pathotypes of S. sclerotiorum into future cultivars.

A two-year survey was conducted to investigate the level of genetic variability occurring across growing seasons within natural populations of Alternaria solani, the cause of early blight in potato. Genetic diversity among 151 isolates, taken from a disease resistance breeding trial, was assessed using seven random amplified polymorphic DNA (RAPD) primers and sequence analyses of portions of the internal transcriber spacer (ITS) region and Alt a1 gene. A. solani isolates were grouped into 19 RAPD profiles to examine the distribution patterns of genetically distinct isolates within and between years. Seven RAPD profiles were found spanning both years with profiles 6 and 13 being the most prevalent. Five unique profiles were found only in 2008 and seven were found only in 2009. No variation was observed among isolates of A. solani based on ITS and Alt a1 sequence analyses, but a distinction between A. solani and A. dauci, a close relative outgroup was identified. Pathogenicity was also assessed using a tissue culture plantlet assay on four isolates and two reference cultures. Differences in virulence were observed among the isolates examined.

Potato is the most important non-cereal crop in the world. Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating disease of potato. In the mid-19th century, P. infestans attacked the European potato fields and this resulted in a widespread famine in Ireland and other parts of Europe. Late blight remains the most important pathogen to potato and causes a yearly multi-billion US dollar loss globally. In Europe and North America, late blight control heavily relies on the use of chemicals, which is hardly affordable to farmers in developing countries and also raises considerable environmental concerns in the developed countries. The structure of P. infestans populations can change quickly by migration, sexual recombination and sub-clonal variation. Migration and the reconvening of the two mating types considerably raised the level of genetic diversity in the global P. infestans population, leading to a more variable population with a presumed higher level of adaptability as compared to the previously, purely asexually, reproducing population. How can the P. infestans population efficiently be monitored with such diverse genotypes? A high-throughput, high-resolution and easy-handled set of markers would be favorable for this purpose. Few genetic markers, if any, have found such widespread use as SSRs. Sequencing allows the identification of large numbers of microsatellites by bioinformatics. So far, however, only a limited number of informative microsatellite loci had been described for P. infestans and none have been mapped. This thesis first describes the development and mapping of SSR markers in P. infestans and integration with other SSRs to generate a multiplex SSR set and its application in the population analysis of P. infestans from four countries are described with the developed multiplex SSRs. Finally, the use of this knowledge in resistance breeding of potato is shortly indicated and discussed. Chapter 1 describes the historic population changes of P. infestans at the global level and the current population trends. It summarizes microsatellite as favorite molecular markers for studying pathogen population diversity and assesses monitoring of population dynamics in more detail for resistance breeding in potato. The selection and identification of new SSR markers is presented in Chapter 2. From EST and genomic sequences from P. infestans we identified 300 non-redundant SSR loci by a bioinformatic screening pipeline. Based on the robustness, level of polymorphism and map position eight SSR markers were selected, which were assembled in two multiplex PCR sets and labeled with two different fluorescent dyes to allow scoring after single capillary electrophoresis. This successful multiplex SSR approach encouraged the development of fast, accurate and high-throughput genotyping, in an one-step multiplex PCR method to facilitate worldwide screening of P. infestans populations. Published SSRs and the 8 new SSRs were integrated. All these SSR markers were re-evaluated and the 12 most informative SSRs were selected to set up a standard set for global application (Chapter 3). The 12-plex SSRs are distributed over different chromosomes, significantly increasing the resolution of genotyping compared to the previous set of 8 SSRs. The 12-plex SSRs were integrated to one-step fluorescence-based multiplex reaction, which plays a key role to facilitate highly paralleled genotyping and efficient dissection of the more complex P. infestans populations. This multiplex PCR for P. infestans populations is (i) simple, as only one PCR is needed to perform multi-locus typing with twelve markers; (ii) rapid, as the genotyping results can be available in 1 day; and (iii) reproducible and adapted to different laboratories. The genotyping data from different geographic populations were submitted to the Euroblight database. With the same SSR set and the bin set, a comparable global database can easily be achieved. As indicated earlier, more recent analyses of P. infestans populations highlight the appearance of many new genotypes via migration and/or sexual recombination. To practice the newly developed 12-plex SSR set and dissect the current population structure, several P. infestans populations from 4 different continents were selected for analysis. These include Chinese (Chapter 4), Dutch (Chapter 5), Ecuadorian (Chapter 6) and Tunisian (Chapter 7) populations. China has become the largest potato producing country not only for potato cultivation area but also in Megaton potato production. Interprovincial trade of consumption and seed potatoes is very important and frequent in China. Although both, the A1 and A2 mating types are found in China, to this date, no evidence of an active sexual cycle based on changes in allele frequency was found. With the ten SSRs, a large genotypic survey of in nation-wide collection of 228 P. infestans isolates was performed (Chapter 4). One of the three dominant clonal lineages CN-04 (A2) in this Chinese population was genetically similar to a major clonal lineage identified in Europe, called “Blue_13” with A2 mating type. It was not possible to critically assess the origin of this clonal lineage. This study is the first report of “Blue_13” outside Europe. The virulence spectrum of selected Chinese P. infestans isolates showed seven different virulence spectra varying from 3 to 10 differentials. The CN04 genotypes were identified as more aggressive and more virulent genotypes, one of whom had the full virulence pattern after using the potato differential set. Within the Chinese P. infestans population, the genotypes strongly clustered according to their six sampling provinces, which seems not to be influenced by the frequent interprovincial trading activities of seed potatoes. The mating type ratio and the SSR allele frequencies indicate that in China the contribution of the sexual cycle to P. infestans on population dynamics is minimal. It was concluded that the migration through asexual propagules and the generation of sub-clonal variation are the dominant driving factors behind the Chinese P. infestans population structure. The Netherlands has a long history of population studies on local P. infestans isolates and a substantial amount of commercial potato varieties growing in the field. One decade (2000-2009) of isolate sampling in 5 different regions provided the basis for a good understanding of the population dynamics in the Netherlands (Chapter 5). The surveyed population revealed the presence of several clonal lineages and a group of sexual progenies. The major clonal lineage with A2 mating type is known as “Blue_13”, but also two distinct clonal lineages with A1 mating type in this study have been identified. This survey witnesses that the Dutch population was undergoing dramatic changes in the ten years under study. The most notable change was the emergence and spread of A2 mating type strain “Blue_13”. The results emphasize the importance of the sexual cycle in generating genetic diversity and the importance of the asexual cycle as the propagation- and dispersal mechanism for successful genotypes. In addition to the neutral SSR markers a molecular marker for the virulence of isolates on potato lines that contain the Rpi-blb1 R-gene has been developed. Using this Avr-blb1 marker and the corresponding virulence assay we report, for the first time, the presence of Rpi-blb1 breaker isolates in the Netherlands even before a Rpi-blb1 containing resistant variety was introduced. The 12 breaker isolates only occurred in sexual progeny. So far the asexual spread of such virulent isolates has been limited because of the absence of Rpi-blb1 containing varieties in the field. Remarkably, on the other end of the world in the Andes, the region of potato origin, the situation is far less complex as far as P. infestans is concerned. There are more than 400 potato landraces in Ecuador and the planting habit by local farmers by traditional cultivation at small scale in the highlands is different from potato cultivation in other potato countries in North America or Europe (Chapter 6). Phytophthora isolates in Ecuador belong to two closely related species, P. infestans (on potato and tomato) and P. andina (on non-tuber bearing host), but SSR analysis of 66 isolates indicated that the two species are separated in two clearly distinguished genetic groups. Two ancient clonal lineages of P. infestans appeared to be dominant in Ecuador one is found only on tomato the other one only on potato. Within the potato isolates, but not in the tomato isolates, there is a large sub-clonal variation caused by (partial) polyploidization and loss of alleles. In Tunisia, potato is cultivated in three to four partly overlapping seasons while tomato is grown either in greenhouses or as aerial crop in most potato producing regions. Chapter 7 revealed, among 165 isolates of five regions, the presence of a major clonal lineage (NA-01, A1 mating type, Ia mtDNA haplotype) that seems to consist of races that are relatively simple. Another highly genetic diverse group of isolates was found containing more complex races and isolates with both mating types. Season clustering indicated that at least some of the new genotypes generated by sexual reproduction overlapped between seasons and such a sexual progeny may play an important role in the next season epidemics. On tomato, mostly asexual progeny was identified with two mtDNA haplotypes but less nuclear genotypes, compared to potato. This study shows that the P. infestans population is currently changing, and the old clonal lineage is being replaced by a more complex, genetically diverse and sexually propagating population in two sub-regions in Tunisia. Despite the massive import of potato seeds from Europe, the P. infestans population in Tunisia is still clearly distinct from the European population. Chapter 8 discusses the application of microsatellites in monitoring genetic diversity of late blight and the potential use in resistance breeding. Monitoring of the local P. infestans population for new virulent genotypes with the differential potato set in combination with screening for effector variation, allows early detection of adaptation of certain genotypes within the P. infestans population to particular resistance genes in a specific region. This provides the possibilities to determine which broad spectrum R-genes are still useful in order to adapt the control strategy by resistance breeding to the new situation. One way of doing that is to replace the existing varieties by other varieties with stacked non-broken R-genes obtained by marker assisted selection or to add additional R-genes to existing (R-gene containing) varieties by transformation. In a transgenic or cisgenic approach, additional broad spectrum R-genes could be added by re-transformation. As we have shown, the right R-gene management strategy in potato breeding, but also in potato production, should include the direct monitoring of local pathogen populations by using the differential set and the 12-plex SSR set.

Potato is the most important non-cereal crop in the world. Late blight, caused by the oomycete pathogen Phytophthora infestans, is the most devastating disease of potato. In the mid-19th century, P. infestans attacked the European potato fields and this resulted in a widespread famine in Ireland and other parts of Europe. Late blight remains the most important pathogen to potato and causes a yearly multi-billion US dollar loss globally. In Europe and North America, late blight control heavily relies on the use of chemicals, which is hardly affordable to farmers in developing countries and also raises considerable environmental concerns in the developed countries. The structure of P. infestans populations can change quickly by migration, sexual recombination and sub-clonal variation. Migration and the reconvening of the two mating types considerably raised the level of genetic diversity in the global P. infestans population, leading to a more variable population with a presumed higher level of adaptability as compared to the previously, purely asexually, reproducing population. How can the P. infestans population efficiently be monitored with such diverse genotypes? A high-throughput, high-resolution and easy-handled set of markers would be favorable for this purpose. Few genetic markers, if any, have found such widespread use as SSRs. Sequencing allows the identification of large numbers of microsatellites by bioinformatics. So far, however, only a limited number of informative microsatellite loci had been described for P. infestans and none have been mapped. This thesis first describes the development and mapping of SSR markers in P. infestans and integration with other SSRs to generate a multiplex SSR set and its application in the population analysis of P. infestans from four countries are described with the developed multiplex SSRs. Finally, the use of this knowledge in resistance breeding of potato is shortly indicated and discussed. Chapter 1 describes the historic population changes of P. infestans at the global level and the current population trends. It summarizes microsatellite as favorite molecular markers for studying pathogen population diversity and assesses monitoring of population dynamics in more detail for resistance breeding in potato. The selection and identification of new SSR markers is presented in Chapter 2. From EST and genomic sequences from P. infestans we identified 300 non-redundant SSR loci by a bioinformatic screening pipeline. Based on the robustness, level of polymorphism and map position eight SSR markers were selected, which were assembled in two multiplex PCR sets and labeled with two different fluorescent dyes to allow scoring after single capillary electrophoresis. This successful multiplex SSR approach encouraged the development of fast, accurate and high-throughput genotyping, in an one-step multiplex PCR method to facilitate worldwide screening of P. infestans populations. Published SSRs and the 8 new SSRs were integrated. All these SSR markers were re-evaluated and the 12 most informative SSRs were selected to set up a standard set for global application (Chapter 3). The 12-plex SSRs are distributed over different chromosomes, significantly increasing the resolution of genotyping compared to the previous set of 8 SSRs. The 12-plex SSRs were integrated to one-step fluorescence-based multiplex reaction, which plays a key role to facilitate highly paralleled genotyping and efficient dissection of the more complex P. infestans populations. This multiplex PCR for P. infestans populations is (i) simple, as only one PCR is needed to perform multi-locus typing with twelve markers; (ii) rapid, as the genotyping results can be available in 1 day; and (iii) reproducible and adapted to different laboratories. The genotyping data from different geographic populations were submitted to the Euroblight database. With the same SSR set and the bin set, a comparable global database can easily be achieved. As indicated earlier, more recent analyses of P. infestans populations highlight the appearance of many new genotypes via migration and/or sexual recombination. To practice the newly developed 12-plex SSR set and dissect the current population structure, several P. infestans populations from 4 different continents were selected for analysis. These include Chinese (Chapter 4), Dutch (Chapter 5), Ecuadorian (Chapter 6) and Tunisian (Chapter 7) populations. China has become the largest potato producing country not only for potato cultivation area but also in Megaton potato production. Interprovincial trade of consumption and seed potatoes is very important and frequent in China. Although both, the A1 and A2 mating types are found in China, to this date, no evidence of an active sexual cycle based on changes in allele frequency was found. With the ten SSRs, a large genotypic survey of in nation-wide collection of 228 P. infestans isolates was performed (Chapter 4). One of the three dominant clonal lineages CN-04 (A2) in this Chinese population was genetically similar to a major clonal lineage identified in Europe, called “Blue_13” with A2 mating type. It was not possible to critically assess the origin of this clonal lineage. This study is the first report of “Blue_13” outside Europe. The virulence spectrum of selected Chinese P. infestans isolates showed seven different virulence spectra varying from 3 to 10 differentials. The CN04 genotypes were identified as more aggressive and more virulent genotypes, one of whom had the full virulence pattern after using the potato differential set. Within the Chinese P. infestans population, the genotypes strongly clustered according to their six sampling provinces, which seems not to be influenced by the frequent interprovincial trading activities of seed potatoes. The mating type ratio and the SSR allele frequencies indicate that in China the contribution of the sexual cycle to P. infestans on population dynamics is minimal. It was concluded that the migration through asexual propagules and the generation of sub-clonal variation are the dominant driving factors behind the Chinese P. infestans population structure. The Netherlands has a long history of population studies on local P. infestans isolates and a substantial amount of commercial potato varieties growing in the field. One decade (2000-2009) of isolate sampling in 5 different regions provided the basis for a good understanding of the population dynamics in the Netherlands (Chapter 5). The surveyed population revealed the presence of several clonal lineages and a group of sexual progenies. The major clonal lineage with A2 mating type is known as “Blue_13”, but also two distinct clonal lineages with A1 mating type in this study have been identified. This survey witnesses that the Dutch population was undergoing dramatic changes in the ten years under study. The most notable change was the emergence and spread of A2 mating type strain “Blue_13”. The results emphasize the importance of the sexual cycle in generating genetic diversity and the importance of the asexual cycle as the propagation- and dispersal mechanism for successful genotypes. In addition to the neutral SSR markers a molecular marker for the virulence of isolates on potato lines that contain the Rpi-blb1 R-gene has been developed. Using this Avr-blb1 marker and the corresponding virulence assay we report, for the first time, the presence of Rpi-blb1 breaker isolates in the Netherlands even before a Rpi-blb1 containing resistant variety was introduced. The 12 breaker isolates only occurred in sexual progeny. So far the asexual spread of such virulent isolates has been limited because of the absence of Rpi-blb1 containing varieties in the field. Remarkably, on the other end of the world in the Andes, the region of potato origin, the situation is far less complex as far as P. infestans is concerned. There are more than 400 potato landraces in Ecuador and the planting habit by local farmers by traditional cultivation at small scale in the highlands is different from potato cultivation in other potato countries in North America or Europe (Chapter 6). Phytophthora isolates in Ecuador belong to two closely related species, P. infestans (on potato and tomato) and P. andina (on non-tuber bearing host), but SSR analysis of 66 isolates indicated that the two species are separated in two clearly distinguished genetic groups. Two ancient clonal lineages of P. infestans appeared to be dominant in Ecuador one is found only on tomato the other one only on potato. Within the potato isolates, but not in the tomato isolates, there is a large sub-clonal variation caused by (partial) polyploidization and loss of alleles. In Tunisia, potato is cultivated in three to four partly overlapping seasons while tomato is grown either in greenhouses or as aerial crop in most potato producing regions. Chapter 7 revealed, among 165 isolates of five regions, the presence of a major clonal lineage (NA-01, A1 mating type, Ia mtDNA haplotype) that seems to consist of races that are relatively simple. Another highly genetic diverse group of isolates was found containing more complex races and isolates with both mating types. Season clustering indicated that at least some of the new genotypes generated by sexual reproduction overlapped between seasons and such a sexual progeny may play an important role in the next season epidemics. On tomato, mostly asexual progeny was identified with two mtDNA haplotypes but less nuclear genotypes, compared to potato. This study shows that the P. infestans population is currently changing, and the old clonal lineage is being replaced by a more complex, genetically diverse and sexually propagating population in two sub-regions in Tunisia. Despite the massive import of potato seeds from Europe, the P. infestans population in Tunisia is still clearly distinct from the European population. Chapter 8 discusses the application of microsatellites in monitoring genetic diversity of late blight and the potential use in resistance breeding. Monitoring of the local P. infestans population for new virulent genotypes with the differential potato set in combination with screening for effector variation, allows early detection of adaptation of certain genotypes within the P. infestans population to particular resistance genes in a specific region. This provides the possibilities to determine which broad spectrum R-genes are still useful in order to adapt the control strategy by resistance breeding to the new situation. One way of doing that is to replace the existing varieties by other varieties with stacked non-broken R-genes obtained by marker assisted selection or to add additional R-genes to existing (R-gene containing) varieties by transformation. In a transgenic or cisgenic approach, additional broad spectrum R-genes could be added by re-transformation. As we have shown, the right R-gene management strategy in potato breeding, but also in potato production, should include the direct monitoring of local pathogen populations by using the differential set and the 12-plex SSR set.

Breeding method: The variety was bred at the Plant Breeding Station Úhřetice within the programme aimed at development of short-straw wheat varieties with breadmaking quality. Because it appeared promising under the conditions of Central Europe to introduce Rht1 gene in order to obtain both high ear productivity and good breadmaking quality (Czech J. Genet. Plant Breed., 34, 1998: 81–85), selection for F2 plants showing insensitivity to applied gibberellic acid (carrying the dwarfing gene Rht1 of the parental variety Vlada) was performed in cooperation with RICP Prague-Ruzyně. Afterwards the standard pedigree selection was applied, including line testing, reselection of spikes and repeated yield testing since F6. The line 6237B (SG-RU-24) was tested in Czech Official Trials from 1999 to 2001 and registered in the generation F13. Disease resistance: The variety is resistant to yellow rust (8), moderately resistant to brown rust and brown leaf spot diseases (6–7) and medium resistant to stem rust (5). It shows good resistance to Septoria nodorum (8) and Fusarium (7) head blights. The resistance to powdery mildew is medium on leaves (5–6) and higher than average (7) on spikes. In the trials of RICP Prague-Ruzyně this variety proved to have moderate resistance to barley yellow dwarf virus. Grain quality: According to the results of Official Tests it is a bread wheat of quality B. It showed high falling number (300 s), high sprouting resistance, medium to high protein content (13%) and relatively lower loaf volume (523 ml) and SDS sedimentation volume (50 ml). It has very good flour properties (high water binding capacity: 61%) and high test weight (810 kg/hl). Frost resistance is medium to high, with lethal temperature (LT 50) around –18°C (tests of RICP Prague-Ruzyně by P. Prášilová). Grain yield: In three year official trials this variety outyielded standards in maize, sugar beet, cereal and potato growing regions by 11.0, 7.0, 4.0 and 5.0%, respectively. The latest comparisons with modern varieties in trials of BS Úhřetice showed the presence of effective response to different types of environment (combinations of treatments occurring in agricultural practice). Other characters: It is a medium late variety, with medium length of straw (90–95 cm) and good resistance to lodging (Rht1 gene). The spike is white, with short scurs and of medium density and tapered shape. Thousand grain weight is high (50–55 g) and stable in different environments. Electrophoretic characteristics of HMW GLU subunits is the following: 1, 6+8, 5+10.

马铃薯晚疫病是由致病疫霉[Phytophthora infestans (Mont.) de Bary]引起的第一大作物病害，目前已严重限制了全球马铃薯的生产和发展。马铃薯晚疫病抗性分子机理的研究一直是抗病育种中的热点问题。本研究利用病毒诱导的基因沉默 (virus-induced gene silencing, VIGS)技术，在本氏烟草(Nicotina benthamiana)中沉默两个马铃薯晚疫病水平抗性相关的基因片段EL732276和EL732318。目的基因沉默后的烟草接种晚疫病菌P. infestans，根据病斑生长速率LGR(length grow rate)来了解这两个基因是否参与烟草对晚疫病的抗性反应。结果表明，目的基因片段EL732276和EL732318被成功地插入烟草脆裂病毒(Tobacco rattle virus, TRV)载体中。利用携带八氢番茄红素脱氢酶基因(phytoene desaturae, PDS)的TRV重组载体病毒接种烟草，感染病毒的烟草植株表现光漂白现象，说明VIGS体系有效。用携带目的片段的重组载体病毒TRV∶EL732276和TRV∶EL732318感染烟草植株，一个月后，烟草叶片接种晚疫病菌P. infestans，从接种P. infestans的烟草叶片上可以看出，感染TRV空载体的对照烟草叶片上病斑扩展速率非常缓慢，而目的片段EL732276和EL732318沉默后的烟草叶片可见明显的水浸状病斑和少量白色的晚疫病菌菌丝。进一步的分析表明TRV∶EL732276重组载体病毒侵染的烟草接种P. infestans后LGR值极显著上升，TRV:EL732318病毒侵染的烟草接种P. infestans后LGR值显著上升，表明EL732276和EL732318同源基因在烟草中沉默后，烟草对P. infestans的抗性显著降低。研究结果认为，病毒诱导的基因沉默技术可在本氏烟草中初步快速鉴定马铃薯抗病相关基因的功能。 | Potato late blight, ranks as world agriculture's most destructive disease, which are caused by Phytophthora infestans (Mont.) de Bary, and potato production has been seriously limited by this disease. Research on molecular mechanism of resistance against P. infestans is a very important issue in potato breeding program. To date, many ESTs involved in potato late blight resistance have been isolated by the molecular biology methods such as suppression subtractive hybridization, DNA microarray and cDNA-AFLP. To screen and analysis of key candidate genes of potato against P. infestans, functional studies of these ESTs are the most urgent thing. In the present study, two potatoes’ ESTs EL732276 and EL732318 which involved in late blight resistance were silenced in Nicotiana benthamiana using virus-induced gene silencing (VIGS). The gene silenced N.benthamiana leaves were inoculated with P. infestans and length grow rate (LGR) of lesions were measured. The RT-PCR results showed that the fragments of candidate genes EL732276 and EL732318 have been successfully cloned into Tobacco rattle virus (TRV) vector. The N.benthamiana leaves infected with TRV carrying the phytoene desaturase gene (PDS) were bleached, indicating that the VIGS system was successful. One month late, the N.benthamiana leaves infected with TRV carrying the genes EL732276 and EL732318 fragment individually were inoculated with P. infestans. The results showed that, compared to the control, LGR values were significantly increased in the EL7322276 and EL732318 silenced N.benthamiana plants, suggesting that P. infestans resistance was dramatically decreased after these two genes silenced in N.benthamiana. This founding was consistent with the results that water soaked lesions and white mycelium obviously observed on EL732276 or EL732318 silenced N.benthamiana leaves after P. infestans inoculation, whereas the lesions developed very slowly on the control leaves which infected with TRV empty vector. The results suggest that functions of potato genes involved in late blight resistance can be rapidly confirmed in N.benthamiana by VIGS technique.

A produção de batata (Solanum tuberosum L.) é limitada por vários fatores, dentre estes, as doenças de plantas. A requeima causada por Phytophthora infestans e a pinta preta causada por Alternaria grandis são consideradas de grande importância. Estudos sobre a epidemiologia e resistência da requeima e pinta preta da batateira são muito importantes para melhor entender estes patossistemas. Para avaliar a resistência de cultivares à requeima em folhas de batata e verificar como essa resistência a requeima relaciona como o tipo de maturação foliar e o tipo de pele em cultivares de batata plantadas no Brasil, 2 experimentos de campo foram conduzidos em diferentes condições ambientais na Universidade Federal de Viçosa (UFV). Para o primeiro experimento foi usado 34 cultivares (tratamentos) e para o segundo experimento foi usado 17 cultivares. A maioria dos cultivares de batata foram suscetíveis a requeima. Os cultivares com os maiores níveis de resistência a requeima (resistente e moderadamente resistente) são de ciclo mais tardios (meio tardio e tardio). A maioria dos cultivares que foram classificados como moderadamente suscetível e suscetível foram mais precoces (precoce e meio precoce). No geral, cultivares que foram mais resistentes a requeima possuem pele áspera, enquanto que a pele das cultivares mais suscetíveis foram lisas. Para estimar a severidade da requeima e da pinta preta da batateira em folhas e em plantas de parcelas experimentais é necessário o uso de uma escala diagramática (EDG) e uma escala descritiva (EDS), respectivamente. Atualmente, existem essas duas escalas somente para a requeima. Para a pinta preta há apenas a EDG, que compreende diagramas de folhas de batata com a severidade da pinta preta variando de 0 a 50%, a qual possui limitações. Sendo assim, foi desenvolvida e validada uma EDG e uma EDS. A EDG proposta contem ilustrações de folhas com doze níveis de severidade da doença (0,05; 0,5; 2; 4; 8; 16; 32; 48; 62; 82; 96 e 100%). A EDS foi uma adaptação da EDS desenvolvida para avaliar a severidade da requeima da batateira. Ambas EDG e EDS melhoraram a acurácia, precisão e reprodutibilidade das estimativas de severidade da pinta preta da batateira. Estas escalas podem ser utilizadas para avaliar a severidade para o melhoramento de plantas visando à resistência, screening de fungicida e caracterização de patótipo. Para avaliar a resistência de cultivares à pinta preta em folhas de batata e verificar como essa resistência a requeima relaciona como o tipo de maturação foliar e o tipo de pele em cultivares de batata plantadas no Brasil, 3 experimentos de campo foram conduzidos em diferentes condições ambientais na UFV. Para o primeiro e o segundo experimento foram usadas 26 cultivares (tratamentos) e para o terceiro experimento foi usado 24 cultivares. A maioria dos cultivares de batata foram suscetíveis e moderadamente suscetíveis à pinta preta da batateira. O nível de resistência de alguns cultivares a pinta preta mudou de acordo com as condições ambientais. Os cultivares resistentes a pinta preta são de meia estação, meio tardio e tardio. Não foi observado nenhum cultivar suscetível a pinta preta com ciclo mais tardio (meio tardio e tardio). Na maioria dos casos, os cultivares suscetíveis a pinta preta são de ciclo mais precoce (precoce e meio precoce). Cultivares resistentes a pinta preta tiveram pele áspera, meio áspera e lisa. Cultivares suscetíveis à pinta preta com pele áspera não foram observados. Na maioria dos casos, os cultivares que foram suscetíveis e moderadamente suscetíveis à pinta preta possuem pele lisa. Objetivando comparar epidemias de requeima e pinta preta da batateira sobre diferentes condições ambientais e programas de aplicação de fungicidas, três experimentos foram conduzidos usando a cultivar Ágata. Cada experimento consistiu de dois ensaios lado a lado e cada ensaio foi conduzido no delineamento em blocos casualizados com 5 tratamentos e 5 repetições. Aos 30 dias após o plantio, as plantas de batata do ensaio 1 foram inoculadas artificialmente com um isolado do grupo de compatibilidade A2 de P. infestans (200 esporângios/mL) e as plantas do ensaio 2 foram inoculadas com isolados de A. grandis (200 conídios/mL). As aplicações de fungicidas iniciaram aos 7 dias após a inoculação. A severidade de ambas doenças foram quantificadas a cada dois dias e os dados de severidade foram usados para calcular a área abaixo da curva de progresso (AACPD). A produtividade e os danos foram também obtidos. A requeima foi mais agressiva do que a pinta preta sobre as diferentes condições climáticas, e isso refletiu na produtividade. A requeima causou danos de 82,4%, enquanto que a pinta preta causou danos de 44,9%. A eficiência dos fungicidas mudou sobre as diferentes condições ambientais, mostrando a importância do uso de ferramentas que monitoram o clima, como o uso de sistema de previsão, para o controle eficiente das doenças. | Conselho Nacional de Desenvolvimento Científico e Tecnológico | Potato (Solanum tuberosum L.) production is limited by several factors, including diseases. Among the diseases that affect potatoes, late blight caused by Phytophthora infestans and early blight caused by Alternaria grandis are considered of great importance. Studies about epidemiology and resistance of potato to late blight and early blight are very important to better understand these pathosystems. To assess the resistance of potato cultivars to foliar late blight and examine how resistance to foliar late blight relates to foliage maturity type and skin type in potato cultivars in Brazil, two field experiments were carried out under different environmental conditions at the Universidade Federal de Viçosa (UFV) in Viçosa- MG. The first experiment used 34 cultivars (treatments). The second experiment used 17 cultivars. The majority of potato cultivars were susceptible to foliar late blight. The cultivars with the highest levels of resistance to foliar late blight (resistant and moderately resistant) were later maturity (mid-late and late maturity). Most cultivars that have been classified as moderately susceptible or susceptible were earlier maturity (early or mid-early maturity). In general, cultivars that are more resistant to foliar late blight had a rougher skin, whereas the skins of the more susceptible cultivars had smoother. To estimate the severity of late blight and early blight of the potato on the leaves and experimental plots, it is necessary to use a standard area diagram (SAD) and a field key (FK), respectively. Currently, there are these two scales to late blight only. For early blight, there is only a SAD, comprising diagrams of potato leaves with early blight severity ranging from 0 to 50%. However, this scale has limitations. For this, were developed and validated a SAD and FK. The proposed SAD contains illustrations of leaves with twelve disease severity levels (0.05, 0.5, 2, 4, 8, 16, 32, 48, 62, 82, 96 and 100%). The proposed FK was an adaptation of a FK developed to assess the severity of potato late blight. Both the SAD and FK improved raters ability to accurately, precisely and reliably estimate potato early blight severity, and as such these scale can be used for assessing severity for many purposes, including plant breeding for resistance, fungicide screening, and pathotype characterization. To assess the resistance of potato cultivars to foliar early blight and examine how resistance to foliar early blight relates to foliage maturity type and skin type in potato cultivars in Brazil, three field experiments were carried out under different environmental conditions at the UFV in Viçosa-MG. The first and second experiments used 26 cultivars (treatments). The third experiment used 24 cultivars. The majority of potato cultivars were susceptible and moderately susceptible to foliar early blight. The resistance levels of some cultivars to foliar early blight changed according to the environmental conditions. The resistant cultivars to foliar early blight had mid-season, mid-late or late maturity. No cultivars susceptible to foliar early blight were observed to have later maturity (mid-late or late maturity). In most cases, the cultivars found to be susceptible to foliar early blight had earlier maturity (early or mid-early maturity). The cultivars found to be resistant had a rough, mid-rough or smooth skin. Cultivars that were susceptible to foliar early blight and had rough skin were not observed. In most cases, the cultivars that were found to be susceptible or moderately susceptible to foliar early blight had smooth skin. Aiming to compare late blight and early blight potato epidemics under different environmental conditions and fungicide applications programs, three experiments were carried out using the Ágata cultivart. Each experiment consisted of two side-by-side trials, and each trial was set in a randomised blo ck design with 5 treatments and 5 replications. At 30 days after inoculation, the potato plants of trial 1 were artificially inoculated with the A2 mating type isolate (200 sporangia/mL) of P. infestans and the potato plants of trial 2 were artificially inoculated with isolates (200 conidia/mL) of A. grandis. Fungicide applications started seven days after inoculation. The late blight severity and the early blight severity were quantified every two days, and the disease severity data were used to calculate the area under the disease progress curve (AUDPC). The yield and its loss were also measured. The late blight was more aggressive than the early blight under different environmental conditions, and this was reflected in the yields. The late blight reached yield losses of 82.4%, while the early blight reached yield losses of 44.9%. Fungicide efficiency changed under the different environmental conditions, showing the importance of using tools that monitor the weather, such as forecast systems, for effective disease control.