Peptide-Based Identification of Phytophthora Isolates and Phytophthora Detection in Planta.

Phytophthora is arguably one of the most damaging genera of plant pathogens. This pathogen is well suited to transmission via the international plant trade, and globalization has been promoting its spread since the 19th century. Early detection is essential for reducing its economic and ecological impact. Here, a shotgun proteomics approach was utilized for Phytophthora analysis. The collection of 37 Phytophthora isolates representing 12 different species was screened for species-specific peptide patterns. Next, Phytophthora proteins were detected in planta, employing model plants Solanum tuberosum and Hordeum vulgare. Although the evolutionarily conserved sequences represented more than 10% of the host proteome and limited the pathogen detection, the comparison between qPCR and protein data highlighted more than 300 protein markers, which correlated positively with the amount of P. infestans DNA. Finally, the analysis of P. palmivora response in barley revealed significant alterations in plant metabolism. These changes included enzymes of cell wall metabolism, ROS production, and proteins involved in trafficking. The observed root-specific attenuation in stress-response mechanisms, including the biosynthesis of jasmonates, ethylene and polyamines, and an accumulation of serotonin, provided the first insight into molecular mechanisms behind this particular biotic interaction.

Novel organ-specific genetic factors for quantitative resistance to late blight in potato.

Potato, Solanum tuberosum, is one of the major consumed food in the world, being the basis of the diet of millions of people. The main limiting and destructive disease of potato is late blight, caused by Phytophtora infestans. Here, we present a multi-environmental analysis of the response to P. infestans using an association panel of 150 accessions of S. tuberosum Group Phureja, evaluated in two localities in Colombia. Disease resistance data were merged with a genotyping matrix of 83,862 SNPs obtained by 2b-restriction site-associated DNA and Genotyping by sequencing approaches into a Genome-wide association study. We are reporting 16 organ-specific QTL conferring resistance to late blight. These QTL explain between 13.7% and 50.9% of the phenotypic variance. Six and ten QTL were detected for resistance response in leaves and stem, respectively. In silico analysis revealed 15 candidate genes for resistance to late blight. Four of them have no functional genome annotation, while eleven candidate genes code for diverse proteins, including a leucine-rich repeat kinase.

A substrate of the ABC transporter PEN3 stimulates bacterial flagellin (flg22)-induced callose deposition in Arabidopsis thaliana.

Nonhost resistance of Arabidopsis thaliana against Phytophthora infestans, a filamentous eukaryotic microbe and the causal agent of potato late blight, is based on a multilayered defense system. Arabidopsis thaliana controls pathogen entry through the penetration-resistance genes PEN2 and PEN3, encoding an atypical myrosinase and an ABC transporter, respectively, required for synthesis and export of unknown indole compounds. To identify pathogen-elicited leaf surface metabolites and further unravel nonhost resistance in Arabidopsis, we performed untargeted metabolite profiling by incubating a P. infestans zoospore suspension on leaves of WT or pen3 mutant Arabidopsis plants. Among the plant-secreted metabolites, 4-methoxyindol-3-yl-methanol and S-(4-methoxy-indol-3-yl-methyl) cysteine were detected in spore suspensions recollected from WT plants, but at reduced levels from the pen3 mutant plants. In both whole-cell and microsome-based assays, 4-methoxyindol-3-yl-methanol was transported in a PEN3-dependent manner, suggesting that this compound is a PEN3 substrate. The syntheses of both compounds were dependent on functional PEN2 and phytochelatin synthase 1. None of these compounds inhibited mycelial growth of P. infestans in vitro Of note, exogenous application of 4-methoxyindol-3-yl methanol slightly elevated cytosolic Ca2+ levels and enhanced callose deposition in hydathodes of seedlings treated with a bacterial pathogen-associated molecular pattern (PAMP), flagellin (flg22). Loss of flg22-induced callose deposition in leaves of pen3 seedlings was partially reverted by the addition of 4-methoxyindol-3-yl methanol. In conclusion, we have identified a specific indole compound that is a substrate for PEN3 and contributes to the plant defense response against microbial pathogens.

Insights into evolving global populations of Phytophthora infestans via new complementary mtDNA haplotype markers and nuclear SSRs.

In many parts of the world the damaging potato late blight pathogen, Phytophthora infestans, is spread as a succession of clonal lineages. The discrimination of genetic diversity within such evolving populations provides insights into the processes generating novel lineages and the pathways and drivers of pathogen evolution and dissemination at local and global scales. This knowledge, in turn, helps optimise management practices. Here we combine two key methods for dissecting mitochondrial and nuclear diversity and resolve intra and inter-lineage diversity of over 100 P. infestans isolates representative of key clonal lineages found globally. A novel set of PCR primers that amplify five target regions are provided for mitochondrial DNA sequence analysis. These five loci increased the number of mtDNA haplotypes resolved from four with the PCR RFLP method to 37 (17, 6, 8 and 4 for Ia, Ib, IIa, and IIb haplotypes, respectively, plus 2 Herb-1 haplotypes). As with the PCR RFLP method, two main lineages, I and II were defined. Group I contained 25 mtDNA haplotypes that grouped broadly according to the Ia and Ib types and resolved several sub-clades amongst the global sample. Group II comprised two distinct clusters with four haplotypes corresponding to the RFLP type IIb and eight haplotypes resolved within type IIa. The 12-plex SSR assay revealed 90 multilocus genotypes providing accurate discrimination of dominant clonal lineages and other genetically diverse isolates. Some association of genetic diversity and geographic region of contemporary isolates was observed; US and Mexican isolates formed a loose grouping, distinct from isolates from Europe, South America and other regions. Diversity within clonal lineages was observed that varied according to the age of the clone. In combination, these fine-scale nuclear and maternally inherited mitochondrial markers enabled a greater level of discrimination among isolates than previously available and provided complementary perspectives on evolutionary questions relating to the diversity, phylogeography and the origins and spread of clonal lineages of P. infestans.

A gold nanoparticle-based lateral flow biosensor for sensitive visual detection of the potato late blight pathogen, Phytophthora infestans.

Phytophthora infestans, the causal agent of late blight in potatoes and tomatoes, is the most important and ongoing pathogenic threat to agricultural production worldwide. Rapid and early identification of P. infestans is an essential prerequisite for countering the further spread of infection. In this study, a novel method for visual detection of P. infestans has been developed by integrating universal primer mediated asymmetric PCR with gold nanoparticle (AuNP)-based lateral flow biosensor. We employed asymmetric PCR to generate large amounts of single-stranded DNA (ssDNA) by amplifying a region of P. infestans-specific repetitive DNA sequence. The ssDNA products were then applied to the lateral flow biosensor to perform a visual detection using sandwich-type hybridization assays. In the presence of target DNA, sandwich-type hybridization reactions among the AuNP-probe, target DNA and capture probe were performed on the test line of the biosensor, and then a characteristic red band was produced for the accumulation of AuNPs. Quantitative analysis obtained by recording the optical intensity of the red band demonstrated that this biosensor could detect as little as 0.1 pg µL-1 genomic DNA. Furthermore, the specificity of the biosensor was confirmed by detecting three other Phytophthora species and two pathogenic fungi. We believe this method has potential application in early prediction of potato late blight disease and instigation of management actions to reduce the risk of epidemic development.

Simple and inexpensive long-term preservation methods for Phytophthora infestans.

Phytophthora infestans is one of the most notorious pathogen among Phytophthora species causing potato late blight disease. Stable and long-term preservation of this pathogen is essential for biological research and fungicide screening. The aim of this study was to find a suitable long-term preservation method for P. infestans. We adjusted the storage temperature, made a slight modification to the rye seed method, and compared the influence of four preservation methods (the mineral oil method, the sterile water method, the rye seed method, and the modified rye seed method) on survival, growth and virulence of four isolates of P. infestans. The results showed that all four methods maintained high viability of the tested P. infestans isolates, but the two rye seed methods were the best ways to maintain 100% viability of the P. infestans isolates without contamination. The four preservation methods did not significantly influence growth or morphological characteristics of the P. infestans isolates. The impacts of the four methods on the virulence of the four P. infestans isolates were isolate-specific. For isolates YF3 and 64093, all four methods were suitable for maintaining their virulence. Whilst for isolate HQK8-3, the rye seed and sterile water methods were more suitable to maintain its virulence than the other two methods. For isolate 32835, storage under mineral oil was the best method for maintaining its virulence. In view of these results, it is recommended P. infestans should be stored by several different storage methods to ensure the safety and stability of the isolates.

Matrix approach to the simultaneous detection of multiple potato pathogens by real-time PCR.

AIM: Create a method for highly sensitive, selective, rapid and easy-to-use detection and identification of economically significant potato pathogens, including viruses, bacteria and oomycetes, be it single pathogen, or a range of various pathogens occurring simultaneously. METHODS AND RESULTS: Test-systems for real-time PCR, operating in the unified amplification regime, have been developed for Phytophthora infestans, Pectobacterium atrosepticum, Dickeya dianthicola, Dickeya solani, Ralstonia solanacearum, Pectobacterium carotovorum, Clavibacter michiganensis subsp. sepedonicus, potato viruses Y (ordinary and necrotic forms as well as indiscriminative test system, detecting all forms), A, X, S, M, potato leaf roll virus, potato mop top virus and potato spindle tuber viroid. The test-systems (including polymerase and revertase) were immobilized and lyophilized in miniature microreactors (1·2 µl) on silicon DNA/RNA microarrays (micromatrices) to be used with a mobile AriaDNA® amplifier. CONCLUSIONS: Preloaded 30-reaction micromatrices having shelf life of 3 and 6 months (for RNA- and DNA-based pathogens, respectively) at room temperature with no special conditions were successfully tested on both reference and field samples in comparison with traditional ELISA and microbiological methods, showing perfect performance and sensitivity (1 pg). SIGNIFICANCE AND IMPACT OF THE STUDY: The accurate, rapid and user-friendly diagnostic system in a micromatrix format may significantly contribute to pathogen screening and phytopathological studies.

Environmental risk assessment of blight-resistant potato: use of a crop model to quantify nitrogen cycling at scales of the field and cropping system.

Environmental risk assessment of GM crops in Europe proceeds by step-wise estimation of effect, first in the plant, then the field plot (e.g. 10-100 m-2), field (1000-10,000 m-2) and lastly in the environment in which the crop would be grown (100-10,000 km2). Processes that operate at large scales, such as cycling of carbon (C) and nitrogen (N), are difficult to predict from plot scales. Here, a procedure is illustrated in which plot scale data on yield (offtake) and N inputs for blight resistant (both GM and non-GM) and blight-susceptible potato are upscaled by a model of crop resource use to give a set of indicators and metrics defining N uptake and release in realistic crop sequences. The greatest potential damage to environment is due to loss of N from the field after potato harvest, mainly because of the large quantity of mineral and plant matter, high in N, that may die or be left in the field. Blight infection intensifies this loss, since less fertiliser N is taken up by plants and more (as a proportion of plant mass) is returned to the soil. In a simulation based on actual crop sequences, N returns at harvest of potato were raised from 100 kg ha-1 in resistant to 150 kg ha-1 in susceptible varieties subject to a 40% yield loss. Based on estimates that blight-resistant types would require ~20% of the fungicide applied to susceptible types, introduction of resistant types into a realistic 6-year cropping sequence would reduce overall fungicide use to between 72 and 54% depending on the inputs to other crops in the sequence.

Characterization of Phytophthora infestans populations in northwestern Algeria during 2008-2014.

A total of 161 Phytophthora infestans isolates, collected from infected potato and tomato plants during 2008-2014, were characterized based on mating type, metalaxyl sensitivity and polymorphism at 12 simple sequence repeat (SSR) loci, in order to investigate the population of P. infestans in the north-west of Algeria, an emerging potato production region. The majority of isolates were of A2 mating type (112 isolates). A high percentage (89 %) of resistance to metalaxyl among isolates was detected. The metalaxyl resistant phenotype was present in both mating types with a higher percentage in A2 mating type isolates. SSR-based genotypic analysis of P. infestans population showed a low diversity. Genotype 13_A2 was the predominant in the population with a frequency of 67 % followed by 2_A1 (21 %) and 23_A1 (5 %). Genotype 23_A1 was detected only in tomato and potato isolates collected in 2013 and 2014.

Genetic Variation within Clonal Lineages of Phytophthora infestans Revealed through Genotyping-By-Sequencing, and Implications for Late Blight Epidemiology.

Genotyping-by-sequencing (GBS) was performed on 257 Phytophthora infestans isolates belonging to four clonal lineages to study within-lineage diversity. The four lineages used in the study were US-8 (n = 28), US-11 (n = 27), US-23 (n = 166), and US-24 (n = 36), with isolates originating from 23 of the United States and Ontario, Canada. The majority of isolates were collected between 2010 and 2014 (94%), with the remaining isolates collected from 1994 to 2009, and 2015. Between 3,774 and 5,070 single-nucleotide polymorphisms (SNPs) were identified within each lineage and were used to investigate relationships among individuals. K-means hierarchical clustering revealed three clusters within lineage US-23, with US-23 isolates clustering more by collection year than by geographic origin. K-means hierarchical clustering did not reveal significant clustering within the smaller US-8, US-11, and US-24 data sets. Neighbor-joining (NJ) trees were also constructed for each lineage. All four NJ trees revealed evidence for pathogen dispersal and overwintering within regions, as well as long-distance pathogen transport across regions. In the US-23 NJ tree, grouping by year was more prominent than grouping by region, which indicates the importance of long-distance pathogen transport as a source of initial late blight inoculum. Our results support previous studies that found significant genetic diversity within clonal lineages of P. infestans and show that GBS offers sufficiently high resolution to detect sub-structuring within clonal populations.

The cell biology of late blight disease.

Late blight, caused by the oomycete Phytophthora infestans, is a major global disease of potato and tomato. Cell biology is teaching us much about the developmental stages associated with infection, especially the haustorium, which is a site of intimate interaction and molecular exchange between pathogen and host. Recent observations suggest a role for the plant endocytic cycle in specific recruitment of host proteins to the Extra-Haustorial Membrane, emphasising the unique nature of this membrane compartment. In addition, there has been a strong focus on the activities of RXLR effectors, which are delivered into plant cells to modulate and manipulate host processes. RXLR effectors interact directly with diverse plant proteins at a range of subcellular locations to promote disease.

Genomic Analyses of Dominant U.S. Clonal Lineages of Phytophthora infestans Reveals a Shared Common Ancestry for Clonal Lineages US11 and US18 and a Lack of Recently Shared Ancestry Among All Other U.S. Lineages.

Populations of the potato and tomato late-blight pathogen Phytophthora infestans are well known for emerging as novel clonal lineages. These successions of dominant clones have historically been named US1 through US24, in order of appearance, since their first characterization using molecular markers. Hypothetically, these lineages can emerge through divergence from other U.S. lineages, recombination among lineages, or as novel, independent lineages originating outside the United States. We tested for the presence of phylogenetic relationships among U.S. lineages using a population of 31 whole-genome sequences, including dominant U.S. clonal lineages as well as available samples from global populations. We analyzed ancestry of the whole mitochondrial genome and samples of nuclear loci, including supercontigs 1.1 and 1.5 as well as several previously characterized coding regions. We found support for a shared ancestry among lineages US11 and US18 from the mitochondrial genome as well as from one nuclear haplotype on each supercontig analyzed. The other nuclear haplotype from each sample assorted independently, indicating an independent ancestry. We found no support for emergence of any other of the U.S. lineages from a common ancestor shared with the other U.S. lineages. Each of the U.S. clonal lineages fit a model where populations of new clonal lineages emerge via migration from a source population that is sexual in nature and potentially located in central Mexico or elsewhere. This work provides novel insights into patterns of emergence of clonal lineages in plant pathogen genomes.

Mitochondrial DNA assessment of Phytophthora infestans isolates from potato and tomato in Ethiopia reveals unexpected diversity.

Mitochondrial DNA (mtDNA) haplotypes were determined using restriction fragment length polymorphism (RFLP) for P. infestans sampled from 513 foliar lesions of late blight found on potato and tomato in different regions of Ethiopia. Among the four reported mitochondrial haplotypes of Phytophthora infestans, Ia, Ib and IIb were detected in 93 % of the samples analyzed but the vast majority of these were Ia. The remaining 7 % represented a previously unreported haplotype. DNA sequencing of this new haplotype also confirmed a single base nucleotide substitution that resulted in loss of EcoRI restriction site and gain of two additional MspI sites in cox1 and atp1 genes, respectively. There were 28 polymorphic sites among all nucleotide sequences including five reference isolates. Sites with alignment gaps were observed in P4 with one nucleotide deletion in 11 Ethiopian isolates. None of the reference sequence produced frame-shifts, with the exception of the 3-nucleotide deletion in the P4 region by Phytophthora andina, a feature that can be used to distinguish the new Ethiopian isolates from P. andina. While a distinguishing molecular data presented here clearly separated them from P. infestans, 7 % of the isolates that share this feature formed an important component of the late blight pathogen causing disease on Solanum tuberosum in Ethiopia. Thus, these Ethiopian isolates could represent a novel Phytophthora species reported for the first time here.

High genotypic diversity found among population of Phytophthora infestans collected in Estonia.

Potato late blight, caused by the oomycete pathogen Phytophthora infestans, is one of the most important diseases of potato worldwide. This is the first study characterising Estonian P. infestans population using the SSR marker genotyping method. 70 P. infestans isolates collected during the growing season in 2004 from eight potato fields in three different regions of Estonia were characterised with nine polymorphic SSR markers. A1 and A2 mating type isolates were detected from every studied field indicating the high potential for sexual reproduction, which raises the genotypic diversity in P. infestans population. Results revealed highly diverse P. infestans population in Estonia resembling the Northern European populations. Most of the multilocus genotypes were detected only once among the collected isolates. Subpopulations collected from different geographical regions of Estonia showed no differentiation from each other but instead formed one highly diverse group.

Loop-mediated isothermal amplification for detection of the tomato and potato late blight pathogen, Phytophthora infestans.

AIMS: To design and validate a colorimetric loop-mediated isothermal amplification assay for rapid detection of Phytophthora infestans DNA. METHODS AND RESULTS: Two sets of loop-mediated isothermal amplification (LAMP) primers were designed and evaluated for their sensitivity and specificity for P. infestans. ITSII primers targeted a portion of the internal transcribed spacer region of ribosomal DNA. These primers had a limit of detection of 2 pg P. infestans DNA and cross-reacted with the closely related species Phytophthora nicotianae. Rgn86_2 primers, designed to improve assay specificity, targeted a portion of a conserved hypothetical protein. These primers had a limit of detection of 200 pg P. infestans DNA and did not cross-react with P. nicotianae. The specificity of the Rgn86_2 assay was tested further using the closely related species P. andina, P. ipomoeae, P. mirabilis and P. phaseoli. Cross-reactions occurred with P. andina and P. mirabilis, but neither species occurs on tomato or potato. Both primer sets were able to detect P. infestans DNA extracted from tomato late blight leaf lesions. CONCLUSIONS: Two colorimetric LAMP assays detected P. infestans DNA from pure cultures as well as infected leaf tissue. The ITSII primers had higher sensitivity, and the Rgn86_2 primers had higher specificity. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of a LAMP assay for the detection of P. infestans, the causal organism of potato and tomato late blight. These assays have potential for immediate utility in plant disease research and diagnostic laboratories.

Characterization of Phytophthora infestans resistance to mefenoxam using FTIR spectroscopy.

Phytophthora infestans (P. infestans) is the causal agent of late blight in potato and tomato. This pathogen devastated the potato crops in Ireland more than a century years ago and is still causing great losses worldwide. Although fungicides controlling P. infestans have been used successfully for almost 100 years, some isolates have developed resistance to most common fungicides. Identification and characterization of these resistant isolates is required for better control of the disease. Current methods that are based on microbiological and molecular techniques are both expensive and time consuming. Fourier Transform Infra-Red spectroscopy (FTIR) is an inexpensive and reagent-free technique that provides accurate results in only a few minutes. In this study the infrared absorption spectra of the sporangia of P. infestans were measured to evaluate the potential of FTIR spectroscopy in tandem with multivariate analysis in order to classify those sporangia into those that were resistant and those that were non-resistant to the phenylamide fungicide mefenoxam. Based on individual measurements, our results show that FTIR spectroscopy enables classification of P. infestans isolates into mefenoxam resistant and mefenoxam non-resistant types with specificity of 81.9% and sensitivity of 75.5%. Using average spectra per leaf, it was possible to improve the classification results to 88% sensitivity and 95% specificity.

Detection of the virulent form of AVR3a from Phytophthora infestans following artificial evolution of potato resistance gene R3a.

Engineering resistance genes to gain effector recognition is emerging as an important step in attaining broad, durable resistance. We engineered potato resistance gene R3a to gain recognition of the virulent AVR3aEM effector form of Phytophthora infestans. Random mutagenesis, gene shuffling and site-directed mutagenesis of R3a were conducted to produce R3a* variants with gain of recognition towards AVR3aEM. Programmed cell death following gain of recognition was enhanced in iterative rounds of artificial evolution and neared levels observed for recognition of AVR3aKI by R3a. We demonstrated that R3a*-mediated recognition responses, like for R3a, are dependent on SGT1 and HSP90. In addition, this gain of response is associated with re-localisation of R3a* variants from the cytoplasm to late endosomes when co-expressed with either AVR3aKI or AVR3aEM a mechanism that was previously only seen for R3a upon co-infiltration with AVR3aKI. Similarly, AVR3aEM specifically re-localised to the same vesicles upon recognition by R3a* variants, but not with R3a. R3a and R3a* provide resistance to P. infestans isolates expressing AVR3aKI but not those homozygous for AVR3aEM.

Persistence of the mitochondrial lineage responsible for the Irish potato famine in extant new world phytophthora infestans.

The plant pathogen Phytophthora infestans emerged in Europe in 1845, triggering the Irish potato famine and massive European potato crop losses that continued until effective fungicides were widely employed in the 20th century. Today the pathogen is ubiquitous, with more aggressive and virulent strains surfacing in recent decades. Recently, complete P. infestans mitogenome sequences from 19th-century herbarium specimens were shown to belong to a unique lineage (HERB-1) predicted to be rare or extinct in modern times. We report 44 additional P. infestans mitogenomes: four from 19th-century Europe, three from 1950s UK, and 37 from modern populations across the New World. We use phylogenetic analyses to identify the HERB-1 lineage in modern populations from both Mexico and South America, and to demonstrate distinct mitochondrial haplotypes were present in 19th-century Europe, with this lineage initially diversifying 75 years before the first reports of potato late blight.

Effect of farming systems on the yield, quality parameters and sensory properties of conventionally and organically grown potato (Solanum tuberosum L.) tubers.

The objectives of this two-year research were to study the impact of two different farming types, conventional and organic, on the yield and sensory properties of five Lithuanian varieties of potato tuber. The parameters and properties examined were: phenolic acids; dry matter and starch content; and the spread and intensity of Phytophthora infestans growth. It was determined that potato yield fluctuates with the variety, but for conventional farming it is significantly (p<0.05) higher than that obtained by organic farming. The farming type has no significant effect (p>0.05) on the content of phenolic acids. No significant effect (p>0.05) of farming type on dry matter and starch content, or sensory properties was found. No significant relation (p>0.05) was found between the content of phenolic acids and P. infestans spread. The spread of P. infestans was faster and infection was heavier in organically grown potatoes.

Genotypic diversity and migration patterns of Phytophthora infestans in the Nordic countries.

In this study we investigated the genotypic diversity and the migration patterns of Phytophthora infestans in the Nordic countries. Isolates of P. infestans from outbreaks in 43 fields sampled in 2008 were collected using stratified sampling with country, field, and disease foci as the different strata. Microsatellites were used as markers to determine the genotypic variation in the sampled material. The results show a high genotypic variation of P. infestans in the Nordic countries with most of the genotypes found only once among the collected isolates. The major part of the genotypic variation was observed within the fields, with low differentiation between the fields. The observed low association of alleles among loci is consistent with frequent sexual reproduction of P. infestans in the Nordic countries. Coalescence analyses did not support a single common population for the four countries, thus indicating some degree of geographic differentiation. The analyses of migration patterns showed differing levels of gene flow among the Nordic countries. No correlation between migration rates and geographical distance could be seen. This could be explained by different degrees of genetic similarity between the pathogen populations in the different countries.