Phosphite Integrated in Late Blight Treatment Strategies in Starch Potato Does Not Cause Residues in the Starch Product.

Currently available fungicides against potato late blight are effective but there are concerns about the sustainability of frequent applications and the risks of fungicide resistance. Therefore, we investigated how potassium phosphite can be integrated into late blight control programs with reduced fungicides in field trials. Phosphite was somewhat less effective than the conventional fungicides at suppressing late blight in the foliage, and the tubers contained less starch. However, when we reduced the amount of phosphite and combined it with reduced amounts of conventional fungicides, we observed no differences in disease suppression, total yields, and tuber starch contents compared with the full treatments with conventional fungicides. The amount of phosphite detected in the harvested tubers was linearly associated with the amount of phosphite applied to the foliage. Our analyses indicate that phosphite could replace some fungicides without exceeding the current European Union standards for the maximum residue levels in potato tubers. No phosphite was detected in the starch from the tubers. In 1 of 2 years, early blight (caused by Alternaria solani) was less severe in the phosphite treatments than in the treatments without phosphite. The integration of phosphite into current treatment strategies would reduce the dependence on conventional fungicides.

Effector-driven marker development and cloning of resistance genes against Phytophthora infestans in potato breeding clone SW93-1015.

KEY MESSAGE: We show the usefulness of integrating effector screening in a breeding program and in resistance gene cloning, with Phytophthora resistance in the Swedish potato breeding clone SW93-1015 as an example. Phytophthora infestans is one of the most devastating plant pathogens worldwide. We have earlier found that the SW93-1015 potato breeding clone has an efficient resistance against P. infestans under field conditions in Sweden, which has an unusually high local diversity of the pathogen. This potato clone has characteristics that are different from classical R-gene-mediated resistance such as elevated levels of hydrogen peroxide (H2O2) under controlled conditions. Analysis of 76 F1 potato progenies from two individual crosses resulted in nearly 50% resistant clones, from both crosses. This result suggests that the SW93-1015 clone has a simplex genotype for this trait. Screening with over 50 different P. infestans effectors, containing the conserved motif RXLR (for Arg, any amino acid, Leu, Arg), revealed a specific response to Avr2, which suggests that SW93-1015 might contain a functional homolog of the R2 resistance gene. We cloned eight R2 gene homologs from SW93-1015, whereof seven have not been described before and one gene encoded a protein identical to Rpi-ABPT. Expression of this gene in potato cultivar Désirée provided R2-specific resistance, whereas other homologues did not. Using RNAseq analyses we designed a new DNA marker for the R2 resistance in SW93-1015. In summary, we have demonstrated the use of effector screening in practical breeding material and revealed the key resistance mechanism for SW93-1015.

Assessment of diversity and genetic relationships of Neonectria ditissima: the causal agent of fruit tree canker.

BACKGROUND: Neonectria ditissima is one of the most important fungal pathogens of apple trees, where it causes fruit tree canker. Information about the amount and partitioning of genetic variation of this fungus could be helpful for improving orchard management strategies and for breeding apple cultivars with high levels of genetically determined resistance. In this study single-spore Neonectria isolates originating from both the same and from different perithecia, apple cultivars and apple orchards in Sweden and Belgium, were evaluated for AFLP- and SSR-based genetic similarity and for mating system. RESULTS: Seven SSR loci produced a total of 31 alleles with an average of 4 alleles per locus, while 11 AFLP primer combinations produced an average of 35 fragments per primer combination and 71 % polymorphic fragments. An AFLP-based analysis of molecular variance (AMOVA) revealed that 89 % of the variation was found within orchards and 11 % between orchards. Genetic similarity among the studied isolates was illustrated with a principal coordinate analyseis (PCoA) and a dendrogram. AFLP-based Jaccard's similarity coefficients were the highest when single-ascospore isolates obtained from the same perithecium were compared, medium-high for isolates from different perithecia on the same tree, and lowest when isolates from different trees were compared. CONCLUSIONS: Based on the results of PCoA and AMOVA analysis, isolates from the same or geographically close orchards did not group together. Since AFLP profiles differed also when single-ascospore isolates from the same perithecium were compared, the mating system of N. ditissima is most likely heterothallic.

Genetic diversity and occurrence of the F129L substitutions among isolates of Alternaria solani in south-eastern Sweden.

BACKGROUND: Early blight, caused by the fungus Alternaria solani, occurs on potato mainly in the south-eastern part of Sweden, but also in other parts of the country. The aim of this study was to investigate the genetic diversity of A. solani populations from different potato growing regions in south-eastern Sweden using AFLP marker analysis. In addition, the cultured isolates were examined for substitutions in the gene encoding cytochrome b, associated with loss of sensitivity against QoI fungicides. RESULTS: Nei's gene diversity index for the Swedish populations of A. solani revealed a gene diversity of up to 0.20. Also genetic differentiation was observed among populations of A. solani from different locations in south-eastern Sweden. The mitochondrial genotype of the isolates of A. solani was determined and both known genotypes, GI (genotype 1) and GII (genotype 2), were found among the isolates. The occurrence of the F129L substitution associated with a loss of sensitivity to strobilurins was confirmed among the GII isolates. In vitro conidial germination tests verified that isolates containing the F129L substitution had reduced sensitivity to azoxystrobin and, at a lower extent, to pyraclostrobin. CONCLUSIONS: Genetic diversity was relatively high among isolates of A. solani in south-eastern part of Sweden. F129L substitutions, leading to reduced sensitivity to strobilurins, have been established in field populations, which may have implications for the future efficacy of QoI fungicides.

Plant Resistance Inducers against Pathogens in Solanaceae Species-From Molecular Mechanisms to Field Application.

This review provides a current summary of plant resistance inducers (PRIs) that have been successfully used in the Solanaceae plant family to protect against pathogens by activating the plant's own defence. Solanaceous species include many important crops such as potato and tomato. We also present findings regarding the molecular processes after application of PRIs, even if the number of such studies still remains limited in this plant family. In general, there is a lack of patterns regarding the efficiency of induced resistance (IR) both between and within solanaceous species. In many cases, a hypersensitivity-like reaction needs to form in order for the PRI to be efficient. "-Omics" studies have already given insight in the complexity of responses, and can explain some of the differences seen in efficacy of PRIs between and within species as well as towards different pathogens. Finally, examples of field applications of PRIs for solanaceous crops are presented and discussed. We predict that PRIs will play a role in future plant protection strategies in Solanaceae crops if they are combined with other means of disease control in different spatial and temporal combinations.

Proteomics and transcriptomics of the BABA-induced resistance response in potato using a novel functional annotation approach.

BACKGROUND: Induced resistance (IR) can be part of a sustainable plant protection strategy against important plant diseases. ß-aminobutyric acid (BABA) can induce resistance in a wide range of plants against several types of pathogens, including potato infected with Phytophthora infestans. However, the molecular mechanisms behind this are unclear and seem to be dependent on the system studied. To elucidate the defence responses activated by BABA in potato, a genome-wide transcript microarray analysis in combination with label-free quantitative proteomics analysis of the apoplast secretome were performed two days after treatment of the leaf canopy with BABA at two concentrations, 1 and 10 mM. RESULTS: Over 5000 transcripts were differentially expressed and over 90 secretome proteins changed in abundance indicating a massive activation of defence mechanisms with 10 mM BABA, the concentration effective against late blight disease. To aid analysis, we present a more comprehensive functional annotation of the microarray probes and gene models by retrieving information from orthologous gene families across 26 sequenced plant genomes. The new annotation provided GO terms to 8616 previously un-annotated probes. CONCLUSIONS: BABA at 10 mM affected several processes related to plant hormones and amino acid metabolism. A major accumulation of PR proteins was also evident, and in the mevalonate pathway, genes involved in sterol biosynthesis were down-regulated, whereas several enzymes involved in the sesquiterpene phytoalexin biosynthesis were up-regulated. Interestingly, abscisic acid (ABA) responsive genes were not as clearly regulated by BABA in potato as previously reported in Arabidopsis. Together these findings provide candidates and markers for improved resistance in potato, one of the most important crops in the world.

Phosphite-induced changes of the transcriptome and secretome in Solanum tuberosum leading to resistance against Phytophthora infestans.

BACKGROUND: Potato late blight caused by the oomycete pathogen Phytophthora infestans can lead to immense yield loss. We investigated the transcriptome of Solanum tubersoum (cv. Desiree) and characterized the secretome by quantitative proteomics after foliar application of the protective agent phosphite. We also studied the distribution of phosphite in planta after application and tested transgenic potato lines with impaired in salicylic and jasmonic acid signaling. RESULTS: Phosphite had a rapid and transient effect on the transcriptome, with a clear response 3 h after treatment. Strikingly this effect lasted less than 24 h, whereas protection was observed throughout all time points tested. In contrast, 67 secretome proteins predominantly associated with cell-wall processes and defense changed in abundance at 48 h after treatment. Transcripts associated with defense, wounding, and oxidative stress constituted the core of the phosphite response. We also observed changes in primary metabolism and cell wall-related processes. These changes were shown not to be due to phosphate depletion or acidification caused by phosphite treatment. Of the phosphite-regulated transcripts 40% also changed with ß-aminobutyric acid (BABA) as an elicitor, while the defence gene PR1 was only up-regulated by BABA. Although phosphite was shown to be distributed in planta to parts not directly exposed to phosphite, no protection in leaves without direct foliar application was observed. Furthermore, the analysis of transgenic potato lines indicated that the phosphite-mediated resistance was independent of the plant hormones salicylic and jasmonic acid. CONCLUSIONS: Our study suggests that a rapid phosphite-triggered response is important to confer long-lasting resistance against P. infestans and gives molecular understanding of its successful field applications.

Whole-genome sequencing elucidates the species-wide diversity and evolution of fungicide resistance in the early blight pathogen Alternaria solani.

Early blight of potato is caused by the fungal pathogen Alternaria solani and is an increasing problem worldwide. The primary strategy to control the disease is applying fungicides such as succinate dehydrogenase inhibitors (SDHI). SDHI-resistant strains, showing reduced sensitivity to treatments, appeared in Germany in 2013, shortly after the introduction of SDHIs. Two primary mutations in the SDH complex (SdhB-H278Y and SdhC-H134R) have been frequently found throughout Europe. How these resistances arose and spread, and whether they are linked to other genomic features, remains unknown. For this project, we performed whole-genome sequencing for 48 A. solani isolates from potato fields across Europe to better characterize the pathogen's genetic diversity in general and understand the development and spread of the genetic mutations that lead to SDHI resistance. The isolates can be grouped into seven genotypes. These genotypes do not show a geographical pattern but appear spread throughout Europe. We found clear evidence for recombination on the genome, and the observed admixtures might indicate a higher adaptive potential of the fungus than previously thought. Yet, we cannot link the observed recombination events to different Sdh mutations. The same Sdh mutations appear in different, non-admixed genetic backgrounds; therefore, we conclude they arose independently. Our research gives insights into the genetic diversity of A. solani on a genome level. The mixed occurrence of different genotypes, apparent admixture in the populations, and evidence for recombination indicate higher genomic complexity than anticipated. The conclusion that SDHI tolerance arose multiple times independently has important implications for future fungicide resistance management strategies. These should not solely focus on preventing the spread of isolates between locations but also on limiting population size and the selective pressure posed by fungicides in a given field to avoid the rise of new mutations in other genetic backgrounds.

Transcriptome Analysis of Potato Infected with the Necrotrophic Pathogen Alternaria solani.

Potato early blight is caused by the necrotrophic fungus Alternaria solani and can result in yield losses of up to 50% if left uncontrolled. At present, the disease is controlled by chemical fungicides, yet rapid development of fungicide resistance renders current control strategies unsustainable. On top of that, a lack of understanding of potato defences and the quantitative nature of resistance mechanisms against early blight hinders the development of more sustainable control methods. Necrotrophic pathogens, compared to biotrophs, pose an extra challenge to the plant, since common defence strategies to biotic stresses such as the hypersensitive response and programmed cell death are often beneficial for necrotrophs. With the aim of unravelling plant responses to both the early infection stages (i.e., before necrosis), such as appressorium formation and penetration, as well as to later responses to the onset of necrosis, we present here a transcriptome analysis of potato interactions with A. solani from 1 h after inoculation when the conidia have just commenced germination, to 48 h post inoculation when multiple cell necrosis has begun. Potato transcripts with putative functions related to biotic stress tolerance and defence against pathogens were upregulated, including a putative Nudix hydrolase that may play a role in defence against oxidative stress. A. solani transcripts encoding putative pathogenicity factors, such as cell wall degrading enzymes and metabolic processes that may be important for infection. We therefore identified the differential expression of several potato and A. solani transcripts that present a group of valuable candidates for further studies into their roles in immunity or disease development.

Powdery mildew resistance in 155 Nordic bread wheat cultivars and landraces.

The occurrence and distribution of seedling resistance genes and the presence of adult plant resistance to powdery mildew, was investigated in a collection of 155 Nordic bread wheat landraces and cultivars by inoculation with 11 powdery mildew isolates. Eighty-nine accessions were susceptible in the seedling stage, while 66 accessions showed some resistance. Comparisons of response patterns allowed postulation of combinations of genes Pm1a, Pm2, Pm4b, Pm5, Pm6, Pm8 and Pm9 in 21 lines. Seedling resistance was three times more frequent in spring wheat than in winter wheat. The most commonly postulated genes were Pm1a+Pm2+Pm9 in Sweden, Pm5 in Denmark and Norway, and Pm4b in Finland. Forty-five accessions were postulated to carry only unidentified genes or a combination of identified and unidentified genes that could not be resolved by the 11 isolates. Complete resistance to all 11 isolates was present in 18 cultivars. Adult plant resistance was assessed for 109 accessions after natural infection with a mixture of races. In all, 92% of the accessions developed less than 3-5% pathogen coverage while nine lines showed 10-15% infected leaf surface. The characterization of powdery mildew resistance in Nordic wheat germplasm could facilitate the combination of resistance genes in plant breeding programmes to promote durability of resistance and disease management.

Earlier occurrence and increased explanatory power of climate for the first incidence of potato late blight caused by Phytophthora infestans in Fennoscandia.

BACKGROUND: Late blight (caused by Phytophthora infestans) is a devastating potato disease that has been found to occur earlier in the season over the last decades in Fennoscandia. Up until now the reasons for this change have not been investigated. Possible explanations for this change are climate alterations, changes in potato production or changes in pathogen biology, such as increased fitness or changes in gene flow within P. infestans populations. The first incidence of late blight is of high economic importance since fungicidal applications should be typically applied two weeks before the first signs of late blight and are repeated on average once a week. METHODS: We use field observations of first incidence of late blight in experimental potato fields from five sites in Sweden and Finland covering a total of 30 years and investigate whether the earlier incidence of late blight can be related to the climate. RESULTS: We linked the field data to meteorological data and found that the previous assumption, used in common late blight models, that the disease only develops at relative humidity levels above 90% had to be rejected. Rather than the typically assumed threshold relationship between late blight disease development and relative humidity we found a linear relationship. Our model furthermore showed two distinct responses of late blight to climate. At the beginning of the observation time (in Sweden until the early 90s and in Finland until the 2000s) the link between climate and first incidence was very weak. However, for the remainder of the time period the link was highly significant, indicating a change in the biological properties of the pathogen which could for example be a change in the dominating reproduction mode or a physiological change in the response of the pathogen to climate. CONCLUSIONS: The study shows that models used in decision support systems need to be checked and re-parametrized regularly to be able to capture changes in pathogen biology. While this study was performed with data from Fennoscandia this new pathogen biology and late blight might spread to (or already be present at) other parts of the world as well. The strong link between climate and first incidence together with the presented model offers a tool to assess late blight incidence in future climates.

Leaf rust (Puccinia triticina) resistance in wheat (Triticum aestivum) cultivars grown in Northern Europe 1992-2002.

Diversity of resistance to leaf rust caused by Puccinia triticina can be enhanced in wheat (Triticum aestivum) cultivars through a better knowledge of resistance genes that are present in important cultivars and germplasm. Multi-pathotype tests on 84 wheat cultivars grown in Denmark, Finland, Norway and Sweden during 1992-2002 and 39 differential testers enabled the postulation of nine known genes for seedling resistance to leaf rust. Genes Lr1, Lr2a, Lr3, Lr10, Lr13, Lr14a, Lr17, Lr23 and Lr26 were found singly or in combination in 47 of the cultivars (55.9%). The most frequently occurring genes in cultivars grown in Sweden were Lr13 (20.4%), Lr14a (14.8%) and Lr26 (14.8%). Lr14a was the most common gene in cultivars grown in Norway (18.7%), Lr13 in Denmark (35.5%) and Lr10 in Finland (20.0%). Although 28 cultivars (33.3%) exhibited a response pattern that could not be assigned to resistance genes or combinations present in the tester lines, several pathotypes carried virulence and hence these genes or combinations are of limited use. Nine cultivars (10.7%) lacked detectable seedling resistance. One cultivar was resistant to all pathotypes used in the study.

Comparisons of early root cortical senescence between barley cultivars, Triticum species and other cereals.

The natural rate of root cortical death (RCD) in seminal roots was investigated in different cereals by staining the roots with acridine orange. In all cereals investigated the part of the cortex having stainable nuclei gradually decreased with increased age of the root. Wheat differed from barley, oats and rye in having a much faster rate of RCD In 15-d-old root regions of wheat grown in soil, only 10-20% of the radius of the root had stainable nuclei. In barley, oats and rye the amount of cortex with stainable nuclei varied between 65 and 80% Minor differences in RCD were found between different barley cultivars or accessions. Large variation in RCD was found between different Triticum species: T. monococcum, T. dicoccum and different Aegilops species had much slower RCD than did hexaploid wheat. These results are discussed in relation to microbial colonization of roots and susceptibility to root pathogens.

DNA fragmentation in cereal roots indicative of programmed root cortical cell death.

In cereals, a progressively increasing root cortical cell death (RCD) occurs from the root tip and upwards when measured with vital staining methods. In this study, nuclear DNA fragmentation was studied in seminal root segments of wheat and barley in order to investigate if the cell death resembled apoptosis. The fraction of cells with TUNEL-positive nuclei increased gradually with increasing root age in both the cortex and the stele. Southern analysis showed a typical ladder pattern, indicating nucleosomal fragmentation already in 2-day-old root segments, and this became more pronounced in older root segments. DNA fragmentation appeared to be more extensive in wheat than in barley roots. These results confirm earlier studies, where RCD has been found to be earlier initiated and to proceed at a faster rate in wheat. The characteristic DNA fragmentation found in the roots indicates programmed cell death with mechanistic similarities to apoptosis. Ultrastructural examination of nuclei in cortex cells with transmission electron microscopy revealed an increased chromatin condensation in older roots, particularly in wheat. In addition, we found nucleosomal DNA ladders in young leaf tissue from wheat but not from barley.

Paranoid potato: phytophthora-resistant genotype shows constitutively activated defense.

Phytophthora is the most devastating pathogen of dicot plants. There is a need for resistance sources with different modes of action to counteract the fast evolution of this pathogen. In order to better understand mechanisms of defense against P. infestans, we analyzed several clones of potato. Two of the genotypes tested, Sarpo Mira and SW93-1015, exhibited strong resistance against P. infestans in field trials, whole plant assays and detached leaf assays. The resistant genotypes developed different sizes of hypersensitive response (HR)-related lesions. HR lesions in SW93-1015 were restricted to very small areas, whereas those in Sarpo Mira were similar to those in Solanum demissum, the main source of classical resistance genes. SW93-1015 can be characterized as a cpr (constitutive expressor of PR genes) genotype without spontaneous microscopic or macroscopic HR lesions. This is indicated by constitutive hydrogen peroxide (H2O2) production and PR1 (pathogenesis-related protein 1) secretion. SW93-1015 is one of the first plants identified as having classical protein-based induced defense expressed constitutively without any obvious metabolic costs or spontaneous cell death lesions.

Stability of transgene integration and expression in subsequent generations of doubled haploid oilseed rape transformed with chitinase and beta-1,3-glucanase genes in a double-gene construct.

A double-gene construct with one chitinase and one beta-1,3-glucanase gene from barley, both driven by enhanced 35S promoters, was transformed into oilseed rape. From six primary transformants expressing both transgenes 10 doubled haploid lines were produced and studied for five generations. The number of inserted copies for both the genes was determined by Southern blotting and real-time PCR with full agreement between the two methods. When copy numbers were analysed in different generations, discrepancies were found, indicating that at least part of the inserted sequences were lost in one of the alleles of some doubled haploids. Chitinase and beta-1,3-glucanase expression was analysed by Western blotting in all five doubled haploid generations. Despite that both the genes were present on the same T-DNA and directed by the same promoter their expression pattern between generations was different. The beta-1,3-glucanase was expressed at high and stable levels in all generations, while the chitinase displayed lower expression that varied between generations. The transgenic plants did not show any major impact on fungal resistance when assayed in greenhouse, although purified beta-1,3-glucanase and chitinase caused retardment of fungal growth in vitro.