Late Blight Pathogen Phytophthora Infestans Research Articles

CRISPR/Cas9 genome editing of potato StDMR6-1 results in plants less affected by different stress conditions.

Potato is the third most important food crop, but cultivation is challenged by numerous diseases and adverse abiotic conditions. To combat diseases, frequent fungicide application is common. Knocking out susceptibility genes by genome editing could be a durable option to increase resistance. DMR6 has been described as a susceptibility gene in several crops, based on data that indicates increased resistance upon interruption of the gene function. In potato, Stdmr6-1 mutants have been described to have increased resistance against the late blight pathogen Phytophthora infestans in controlled conditions. Here, we present field evaluations of CRISPR/Cas9 mutants, in a location with a complex population of P. infestans, during four consecutive years that indicate increased resistance to late blight without any trade-off in terms of yield penalty or tuber quality. Furthermore, studies of potato tubers from the field trials indicated increased resistance to common scab, and the mutant lines exhibit increased resistance to early blight pathogen Alternaria solani in controlled conditions. Early blight and common scab are problematic targets in potato resistance breeding, as resistance genes are very scarce. The described broad-spectrum resistance of Stdmr6-1 mutants may further extend to some abiotic stress conditions. In controlled experiments of either drought simulation or salinity, Stdmr6-1 mutant plants are less affected than the background cultivar. Together, these results demonstrate the prospect of the Stdmr6-1 mutants as a useful tool in future sustainable potato cultivation without any apparent trade-offs.

Оценка устойчивости перспективных гибридов цветного картофеля к фитофторозу

Abstract. Potatoes with pigmented tubers contain anthocyanins and other phenolic compounds that demonstrate antioxidant activity and have a beneficial effect on human health. The nutritional value and other consumer qualities of available potato varieties must be combined with resistance to various infections, primarily late blight, which leads to significant yield losses and considerable costs for chemical protection. The purpose of this work was to assess late blight resistance of 46 promising potato hybrids with different pigmentation patterns, both in the field and by laboratory methods, including molecular screening. The objectives of this study included obtaining promising hybrids of colored potatoes characterized by a set of economically importatnt traits, laboratory and field assessment of the resistance of leaves and tubers to the late blight pathogen Phytophthora infestans, as well as molecular screening of late blight resistance gene markers (Rpi genes). The production, cultivation and evaluation of the hybrid material was carried out using standard methods. Field and laboratory resistance of leaves and tubers was assessed on a 9-point scale. Molecular screening was carried out using PCR analysis. The scientific novelty of the work lies in the fact that for the first time a comprehensive assessment of the resistance to late blight of hybrid material of colored potatoes obtained on the territory of Russian Federation was carried out. According to the results obtained, the studied samples demonstrated a high level of resistance of tubers and leaves in field conditions and laboratory tests. However, the results of the study did not reveal a relationship between laboratory or field resistance and the presence of Rpi gene markers, which may be due to the influence of many independent factors that determine horizontal resistance.

Anti-Oomycete Effect and Mechanism of Salicylic Acid on Phytophthora infestans.

Pathogenic oomycetes infect a wide variety of organisms, including plants, animals, and humans, and cause massive economic losses in global agriculture, aquaculture, and human health. Salicylic acid (SA), an endogenous phytohormone, is regarded as an inducer of plant immunity. Here, the potato late blight pathogen Phytophthora infestans was used as a model system to uncover the inhibitory mechanisms of SA on pathogenic oomycetes. In this research, SA significantly inhibited the mycelial growth, sporulation, sporangium germination, and virulence of P. infestans. Inhibition was closely related to enhanced autophagy, suppression of translation initiation, and ribosomal biogenesis in P. infestans, as shown by multiomics analysis (transcriptomics, proteomics, and phosphorylated proteomics). Monodansylcadaverine (MDC) staining and Western blotting analysis showed that SA promoted autophagy in P. infestans by probably targeting the TOR signaling pathway. These observations suggest that SA has the potential to control late blight caused by P. infestans.

Bio-synthesized ZnO NPs using cow urine in vitro & in vivo efficacy against late blight pathogen

Biosynthesis of nanoparticles and their use to harvest agricultural products are a brilliant approach for vegetables and crops with large cultivation areas. Phytophthora, an oomycete, is one of the most serious challenges to tomato crop productivity. In this study, ZnO nanoparticles (ZnO NPs) have been synthesized using cow urine. The X-ray diffraction pattern confirms the hexagonal structure, and symmetry of ZnO NPs have a space group (P63mc). SEM and FTIR identified surface morphology and formation of metal oxide functional groups. A prominent absorption peak executes the electronic transitions from the valence band to the conduction band. In vitro and in vivo efficacy of ZnO NPs, cow urine, and the combination of (CU + ZnONPs) were checked against late blight pathogen (Phytophthora infestans). The role of ROS in ZnO NPs represents by a reduction in the growth inhibition of Phytophthora infestans and the cytotoxic effect of ZnO NPs mediated through ROS.

Breeding of Genetically Engineered Indonesian Potato, Bio Granola, Resistant to Late Blight Pathogen Phytophthora infestans

Breeding of Genetically Engineered Indonesian Potato, Bio Granola, Resistant to Late Blight Pathogen Phytophthora infestans

Comparative transcriptome profiling of potato cultivars infected by late blight pathogen Phytophthora infestans: Diversity of quantitative and qualitative responses

The Estonia potato cultivar Ando has shown elevated field resistance to Phytophthora infestans, even after being widely grown for over 40 years. A comprehensive transcriptional analysis was performed using RNA-seq from plant leaf tissues to gain insight into the mechanisms activated for the defense after infection. Pathogen infection in Ando resulted in about 5927 differentially expressed genes (DEGs) compared to 1161 DEGs in the susceptible cultivar Arielle. The expression levels of genes related to plant disease resistance such as serine/threonine kinase activity, signal transduction, plant-pathogen interaction, endocytosis, autophagy, mitogen-activated protein kinase (MAPK), and others were significantly enriched in the upregulated DEGs in Ando, whereas in the susceptible cultivar, only the pathway related to phenylpropanoid biosynthesis was enriched in the upregulated DEGs. However, in response to infection, photosynthesis was deregulated in Ando. Multi-signaling pathways of the salicylic-jasmonic-ethylene biosynthesis pathway were also activated in response to Phytophthora infestans infection.

Uptake of oomycete RXLR effectors into host cells by clathrin-mediated endocytosis.

Filamentous (oomycete and fungal) plant pathogens deliver cytoplasmic effector proteins into host cells to facilitate disease. How RXLR effectors from the potato late blight pathogen Phytophthora infestans enter host cells is unknown. One possible route involves clathrin-mediated endocytosis (CME). Transient silencing of NbCHC, encoding clathrin heavy chain, or the endosome marker gene NbAra6 encoding a Rab GTPase in the model host Nicotiana benthamiana, attenuated P. infestans infection and reduced translocation of RXLR effector fusions from transgenic pathogen strains into host cells. By contrast, silencing PP1c isoforms, susceptibility factors not required for endocytosis, reduced infection but did not attenuate RXLR effector uptake. Endosome enrichment by ultracentrifugation and sucrose gradient fractionation revealed co-localization of RXLR effector Pi04314-RFP with clathrin-coated vesicles. Immunopurification of clathrin- and NbAra6-associated vesicles during infection showed that RXLR effectors Pi04314-RFP and AvrBlb1-RFP, but not apoplastic effector PiSCR74-RFP, were co-immunoprecipitated during infection with pathogen strains secreting these effectors. Tandem mass spectrometry analyses of proteins co-immunoprecipitated with NbAra6-GFP during infection revealed enrichment of host proteins associated with endocytic vesicles alongside multiple pathogen RXLR effectors, but not apoplastic effectors, including PiSCR74, which do not enter host cells. Our data show that the uptake of P. infestans RXLR effectors into plant cells occurs via CME.

Nucleotide-binding leucine-rich repeat network underlies nonhost resistance of pepper against the Irish potato famine pathogen Phytophthora infestans.

Nonhost resistance (NHR) is a robust plant immune response against non-adapted pathogens. A number of nucleotide-binding leucine-rich repeat (NLR) proteins that recognize non-adapted pathogens have been identified, although the underlying molecular mechanisms driving robustness of NHR are still unknown. Here, we screened 57 effectors of the potato late blight pathogen Phytophthora infestans in nonhost pepper (Capsicum annuum) to identify avirulence effector candidates. Selected effectors were tested against 436 genome-wide cloned pepper NLRs, and we identified multiple functional NLRs that recognize P. infestans effectors and confer disease resistance in the Nicotiana benthamiana as a surrogate system. The identified NLRs were homologous to known NLRs derived from wild potatoes that recognize P. infestans effectors such as Avr2, Avrblb1, Avrblb2, and Avrvnt1. The identified CaRpi-blb2 is a homologue of Rpi-blb2, recognizes Avrblb2 family effectors, exhibits feature of lineage-specifically evolved gene in microsynteny and phylogenetic analyses, and requires pepper-specific NRC (NLR required for cell death)-type helper NLR for proper function. Moreover, CaRpi-blb2-mediated hypersensitive response and blight resistance were more tolerant to suppression by the PITG_15 278 than those mediated by Rpi-blb2. Combined results indicate that pepper has stacked multiple NLRs recognizing effectors of non-adapted P. infestans, and these NLRs could be more tolerant to pathogen-mediated immune suppression than NLRs derived from the host plants. Our study suggests that NLRs derived from nonhost plants have potential as untapped resources to develop crops with durable resistance against fast-evolving pathogens by stacking the network of nonhost NLRs into susceptible host plants.

Exploring the Role of Salicylic Acid in Regulating the Colonization Ability of Bacillus subtilis 26D in Potato Plants and Defense against Phytophthora infestans

Plant colonization by endophytic bacteria is mediated by different biomolecules that cause dynamic changes in gene expression of both bacteria and plant. Phytohormones, in particular, salicylic acid, play a key role in the regulation of endophytic colonization and diversity of bacteria in methaphytobiome. For the first time it was found that salicylic acid influenced motility in biofilms and transcription of the surfactin synthetase gene of the endophytic strain Bacillus subtilis 26D in vitro. Treatment of Solanum tuberosum plants with salicylic acid, along with B. subtilis 26D, increased the number of endophytic cells of bacteria in potato internal tissues and level of transcripts of bacterial surfactin synthetase gene and decreased transcription of plant PR genes on the stage of colonisation with endophytes. Thus, the production of surfactin plays an important role in endophytic colonization of plants, and salicylic acid has an ability to influence this mechanism. Here we firstly show that plants treated with salicylic acid and B. subtilis 26D showed enhanced resistance to the late blight pathogen Phytophthora infestans, which was accompanied by increase in transcriptional activity of plant PR-genes and bacterial surfactin synthetase gene after pathogen inoculation. Therefore, it is suggested that salicylic acid can modulate physiological status of the whole plant–endophyte system and improve biocontrol potential of endophytic strains.

SWATH-MS based quantitative proteomics analysis reveals novel proteins involved in PAMP triggered immunity against potato late blight pathogen Phytophthora infestans.

Potato is the most important non-grain food in the world, while late blight caused by Phytophthora infestans seriously threatens the production of potato. Since pathogen-associated molecular patterns (PAMPs) are relatively conserved, PAMP-triggered immunity (PTI) can provide durable resistance to late blight for potato. However, knowledge of the regulatory mechanisms of PTI against oomycete pathogens at protein levels remains limited due to the small number of identified proteins. In the present work, changes in the proteome profile of Nicotiana benthamiana leaves upon P. infestans PAMP induction were examined using the SWATH-MS (sequential windowed acquisition of all theoretical mass spectra) approach, which provides quantification of protein abundances and large-scale identification of PTI-related proteins. A total of 4401 proteins have been identified, of which 1429 proteins were differentially expressed at least at one time point of 8, 12, 24 and 48h after PAMP induction, compared with the expression at 0h when immediately after PAMP induction. They were further analyzed by expression clustering and gene ontology (GO) enrichment analysis. Through functional verification, six novel DEPs of 19 candidates were proved to be involved in PTI responses, including mitochondrial phosphate carrier protein (MPT) 3, vesicle-associated membrane protein (VAMP) 714, lysophospholipase (LysoPL) 2, ascorbate peroxidase (APX) 1, heat shock 70 kDa protein (HSP) 2 and peptidyl-prolyl cis-trans isomerase FKBP (FKBP) 15-1. Taken together, the time course approach and the resulting large-scale proteomic analyses have enlarged our understanding of PTI mechanisms and provided a valuable resource for the discovery of complex protein networks involved in the resistance response of potato to late blight.

Transcriptomic and metabolomic profiling revealed the role of succinoglycan Riclin octaose in eliciting the defense response of Solanum tuberosum.

Activating the defense response of plants by elicitors provides a promising method for biocontrol of pathogens. The homogeneous octaose (RiOc) which was depolymerized from the succinoglycan Riclin was investigated as a novel elicitor to activate the immune system of potato (Solanum tuberosum L.). After foliar spray, RiOc quickly induced accumulation of reactive oxygen species in potato leaves in a dose-dependent manner. Transcriptomic analysis revealed that 2712 out of 30,863 genes were differentially expressed at the early stage (24h), while 367 of them were changed later (72h). Results from the transcriptome and quantitative RT-PCR suggested that RiOc was probably perceived by the receptor LYK3 and it activated the MKK2/3/9/-MPK6/7 signaling cascade and promoted the salicylic acid-mediated defense response. Meanwhile, RiOc changed the metabolome profile of potato leaves over time as demonstrated by the 1H NMR-based metabolomic analysis. Homeostasis of amino acids was affected at the early stage while the secondary metabolism was strengthened later. More importantly, RiOc significantly reduced the severity of potato leaf lesions caused by the late blight pathogen Phytophthora infestans. In conclusion, RiOc effectively improved the resistance of potato to P. infestans by eliciting the salicylic acid-mediated defense response. RiOc becomes a promising carbohydrate-based elicitor for biocontrol of plant pathogens. KEY POINTS: • Homogeneous Riclin octaose was a novel elicitor for biocontrol of plant pathogens. • Riclin octaose primed the salicylic acid-mediated defense response of potato plants. • Riclin octaose changed the metabolome profile of potato leaves over time.

In-vitro Evaluation of Fungicide Sensitivity of Tomato Leaf Blight Pathogens

Tomato early and late blight diseases caused by Alternaria solani and Phytophthora infestans respectively, are constraints to tomato production globally. Conventional use of commercial synthetic fungicides in management of tomato blight disease has become a key input for tomato production among farmers in Tharaka Nithi County, Kenya. Indiscriminate use and application of sub-lethal doses of fungicides negate disease management efforts, and may lead to gradual fungicide resistance. Yet, local tomato farmers have continued to use the fungicides without periodic evaluation of their effectiveness. This study was carried out in-vitro to evaluate the efficacy of six synthetic commercial fungicides used by farmers around River Ruguti, against two tomato leaf blight pathogens; Alternaria solani and Phytophthora infestans. The poison food method was used to evaluate fungicides known by trade names and application levels; Mancozeb (640 g kg-1) + Metalaxyl (40 g kg-1), Mancozeb 640 g/kg + Metalaxyl 80 g/kg, Mancozeb, Propineb700 g/kg + Cymoxanil 60 g/kg, Carbendazim and Triticonazole at different concentration (25%, 50% and 75%). The in-vitro plate experiment was laid out in a Complete Randomized Design with 3 replicates, and data on mycelia growth inhibition analyzed through General Linear Model (α=.05) and significant means separated using Least significant difference (LSD) using Scientific Analysis System version 9.4. All the tested fungicides significantly (p≤ 0.05) inhibited mycelial growth of tested pathogen. Percentage inhibition for early blight pathogen (Alternaria solani) was 80.42% compared to late blight pathogen Phytophthora infestans at 69.51%. Mancozeb (640 g kg-1) + Metalaxyl (40 g kg-1) and Propineb700 g/kg + Cymoxanil 60 g/kg recorded higher per cent inhibition of mycelia growth of 92.4% and 89.71% respectively. Carbendazim recorded lower per cent inhibition of 39.15%. Mycelia growth inhibition increased with an increase in fungicide concentration. Lower inhibition of 71.78% was observed at 25% concentration as compared to 50% and 75% with 76.77% and 76.36% respectively. Fungicides screened varied in mycelia inhibition against P. infestans and A. solani isolates with Mancozeb (640 g kg-1) + Metalaxyl (40 g kg-1) and Propineb700g/kg +Cymoxanil 60g/kg giving significantly (p≤ 0.05) better inhibition while Carbendazim had the lowest inhibition effect. Increased fungicide concentration effectively inhibited mycelia growth. Thus higher concentration of fungicide application is recommended in cases where there is low efficacy of fungicides.

Natural Genetic Diversity in the Potato Resistance Gene RB Confers Suppression Avoidance from Phytophthora Effector IPI-O4.

RB is a potato gene that provides resistance to a broad spectrum of genotypes of the late blight pathogen Phytophthora infestans. RB belongs to the CC-NB-LRR (coiled-coil, nucleotide-binding, leucine-rich repeat) class of resistance (R) genes, a major component of the plant immune system. The RB protein detects the presence of class I and II IPI-O effectors from P. infestans to initiate a hypersensitive resistance response, but this activity is suppressed in the presence of the Class III effector IPI-O4. Using natural genetic variation of RB within potato wild relatives, we identified two amino acids in the CC domain that alter interactions needed for suppression of resistance by IPI-O4. We have found that separate modification of these amino acids in RB can diminish or expand the resistance capability of this protein against P. infestans in both Nicotiana benthamiana and potato. Our results demonstrate that increased knowledge of the molecular mechanisms that determine resistance activation and R protein suppression by effectors can be utilized to tailor-engineer genes with the potential to provide increased durability.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

New Honorary Member of the BSPP

Jonathan D. G. Jones grew up in London and graduated in Botany from Cambridge University followed by graduate work at the PBI, Cambridge. His career in plant–microbe interactions began with Ausubel at Harvard, studying Rhizobium–legume associations. He then worked at Advanced Genetic Sciences Inc. where his expertise using broad host-range plasmids underpinned the development of plant genetic engineering tools using Agrobacterium. Since 1988, he has led a research team at The Sainsbury Lab (TSL), Norwich, where he isolated the tomato Cf-9 gene for resistance to the fungus Cladosporium fulvum. Cf-9 encodes a pioneer cell surface “receptor-like protein” immune receptor, with extracellular leucine-rich repeats (LRRs). He isolated six such genes, each recognizing different fungal virulence factors. These preceded the Nobel prize-winning discoveries of similar Toll-like receptors in animal innate immunity. He went on to characterize complex immune receptor-encoding haplotypes with multiple paralogs and infer their evolutionary trajectories. He discovered that solvent-exposed amino acids in plant immune receptor LRRs are hypervariable and under diversifying selection for specific recognition of distinct pathogen molecules, and that unequal crossing over between paralogous genes at complex loci also contributes to receptor diversification. In a Cf-2 resistance gene suppressor screen, he discovered Rcr3, a cysteine protease required for Cf-2 to recognize the Avr2. In collaboration with the de Wit laboratory in Wageningen, he showed that Cf-2 “guards” Rcr3, activating defence when Rcr3 is inhibited by the Avr2 protease inhibitor. The resulting guard hypothesis was the subject of an influential paper with Dangl in 2001. He also identified several protein kinases and E3 ubiquitin ligases involved in Cf-dependent immunity, verifying their requirement for defence activation, and was first to show the role of receptor-like cytoplasmic kinases in cell surface receptor-mediated immunity. Most plant resistance genes encode intracellular nucleotide-binding LRR (NLR) immune receptors. He cloned one of the earliest (RPP5) and has defined >12 more. He was first to reveal the extreme haplotype diversity at the RPP5 locus between Arabidopsis accessions, again verifying extreme diversity in solvent-exposed amino acids of the LRRs of different paralogs, and unequal crossovers between paralogs in their evolution. In an influential 2006 paper with Dangl, he articulated the roles of cell surface “pattern-triggered immunity” and intracellular “effector-triggered immunity” in plant defence, integrating them for the first time. He has led discoveries on cross-talk between various hormone signalling pathways, in pathogen effector mechanisms, and in the role of variation in detection capacity in population resistance to disease. Some NLR receptors carry integrated domains (IDs) and are encoded adjacent in the genome to another NLR gene. Arabidopsis RPS4 and RRS1 are a paradigmatic example; RRS1 carries a WRKY transcription factor domain and the pair creates an immune receptor that detects pathogen effectors that act on WRKY domains. His work provided the first demonstration that NLRs can evolve to be immune receptors that contain IDs that mimic authentic pathogen targets. His fascination with genetic variation in host–pathogen interactions led him to develop sequence capture-based methods to interrogate plant immune receptor diversity. Resistance gene enrichment sequencing (RenSeq) has greatly accelerated our understanding of plant immune receptor repertoires. He also developed capture-based methods to investigate the genetic diversity of obligate parasites growing on hosts (PenSeq). He used RenSeq to address nonhost resistance (NHR) and defined the genetic basis of Arabidopsis NHR to Brassica-infecting races of white rust, revealing the crucial role of NLR receptors. Solanum americanum shows NHR to the potato late blight pathogen Phytophthora infestans. By isolating two NLR immune receptors, Rpi-amr1 and Rpi-amr3, from S. americanum, defining their recognized effectors from P. infestans, and verifying the efficacy of Rpi-amr1, Rpi-amr3, and Rpi-vnt1 in transgenic potato field experiments, he showed that their deployment might enable NHR to control blight in the field. He has played a key role in making the case that this approach to controlling crop disease is useful, safe, and sustainable. His discoveries in plant molecular genetics earned him election as Professor at UEA (1996), EMBO member (1998), Royal Society Fellow (2003), and International Member of US National Academy of Sciences (2015). He served in Royal Society working groups on food security and coauthored the Royal Society’s 2009 “Reaping the Benefits” report.

Cleavage of a pathogen apoplastic protein by plant subtilases activates host immunity.

The plant apoplast is a harsh environment in which hydrolytic enzymes, especially proteases, accumulate during pathogen infection. However, the defense functions of most apoplastic proteases remain largely elusive. We show here that a newly identified small cysteine-rich secreted protein PC2 from the potato late blight pathogen Phytophthora infestans induces immunity in Solanum plants only after cleavage by plant apoplastic subtilisin-like proteases, such as tomato P69B. A minimal 61amino acid core peptide carrying two key cysteines, conserved widely in most oomycete species, is sufficient for PC2-induced cell death. Furthermore, we showed that Kazal-like protease inhibitors, such as EPI1, produced by P.infestans prevent PC2 cleavage and dampen PC2 elicited host immunity. This study reveals that cleavage of pathogen proteins to release immunogenic peptides is an important function of plant apoplastic proteases.

Characterization of a Late Blight Resistance Gene Homologous to R2 in Potato Variety Payette Russet

Breeding for late blight resistance has traditionally relied on phenotypic selection, but as the number of characterized resistance (R) genes has grown, so have the possibilities for genotypic selection. One challenge for breeding russet varieties is the lack of information about the genetic basis of resistance in this germplasm group. Based on observations of strong resistance by ‘Payette Russet’ to genotype US-23 of the late blight pathogen Phytophthora infestans in inoculated experiments, we deduced the variety must contain at least one major R gene. To identify the gene(s), 79 F1 progeny were screened using a detached leaf assay and classified resistant vs. susceptible. Linkage mapping using markers from the potato SNP array revealed a single resistant haplotype on the short arm of chromosome group 4, which coincides with the R2/Rpi-abpt/Rpi-blb3 locus. PCR amplification and sequencing of the gene in Payette revealed it is homologous to R2, and transient expression experiments in Nicotiana benthamiana confirmed its recognition of the Avr2 effector. Sequencing of a small diversity panel revealed a SNP unique to resistant haplotypes at the R2 locus, which was converted to a KASP marker that showed perfect prediction accuracy in the F1 population and diversity panel. Although many genotypes of P. infestans are virulent against R2, even when defeated this gene may be valuable as one component of a multi-genic approach to quantitative resistance.

An Improved PCR Method for Rapid and Accurate Identification of Mating Types in the Late Blight Pathogen Phytophthora infestans

Mating type is a critical trait in heterothallic organisms. In plant pathogenic oomycetes, like the late blight pathogen Phytophthora infestans, it is usually identified through pairing between tester and candidate isolates, a method that is both laborious and applicable to live isolates only. Therefore, developing simple and fast PCR tests to reliably identify P. infestans mating types is of great interest for population genetic studies. A multiplex PCR assay combining the amplification of a locus diagnostic for P. infestans and of one diagnostic for the A1 mating type was developed and validated on a collection of 1,441 samples, covering the current and past diversity of European P. infestans populations. These samples were obtained from either freeze-dried mycelium or FTA cards on which diseased leaflets had been pressed. The multiplex assay correctly identified mating types in 97.4% of these samples. The main source of incorrect assignment was the lack of amplification of the A1 diagnostic allele, due to insufficient DNA quality and/or quantity in the reaction mix. This multiplex PCR, applicable to both live and stored material, thus constitutes a useful addition to the set of molecular tools available for population typing in P. infestans.

Screening of Wild Potatoes Identifies New Sources of Late Blight Resistance.

Late blight (LB) of potato is considered one of the most devastating plant diseases in the world. Most cultivated potatoes are susceptible to this disease. However, wild relatives of potatoes are an excellent source of LB resistance. We screened 384 accessions of 72 different wild potato species available from the U.S. Potato GeneBank against the LB pathogen Phytophthora infestans in a detached leaf assay (DLA). P. infestans isolates US-23 and NL13316 were used in the DLA to screen the accessions. Although all plants in 273 accessions were susceptible, all screened plants in 39 accessions were resistant. Resistant and susceptible plants were found in 33 accessions. All tested plants showed a partial resistance phenotype in two accessions, segregation of resistant and partial resistant plants in nine accessions, segregation of partially resistant and susceptible plants in four accessions, and segregation of resistant, partially resistant, and susceptible individuals in 24 accessions. We found several species that were never before reported to be resistant to LB: Solanum albornozii, S. agrimoniifolium, S. chomatophilum, S. ehrenbergii, S. hypacrarthrum, S. iopetalum, S. palustre, S. piurae, S. morelliforme, S. neocardenasii, S. trifidum, and S. stipuloideum. These new species could provide novel sources of LB resistance. P. infestans clonal lineage-specific screening of selected species was conducted to identify the presence of RB resistance. We found LB resistant accessions in Solanum verrucosum, Solanum stoloniferum, and S. morelliforme that were susceptible to the RB overcoming isolate NL13316, indicating the presence of RB-like resistance in these species.

Genome-wide identification of Argonautes in Solanaceae with emphasis on potato

Regulatory small RNAs (sRNAs) play important roles in many fundamental processes in plant biology such as development, fertilization and stress responses. The AGO protein family has here a central importance in gene regulation based on their capacity to associate with sRNAs followed by mRNA targeting in a sequence-complementary manner. The present study explored Argonautes (AGOs) in the Solanaceae family, with emphasis on potato, Solanum tuberosum (St). A genome-wide monitoring was performed to provide a deeper insight into gene families, genomic localization, gene structure and expression profile against the potato late blight pathogen Phytophthora infestans. Among 15 species in the Solanaceae family we found a variation from ten AGOs in Nicotiana obtusifolia to 17 in N. tabacum. Comprehensive analyses of AGO phylogeny revealed duplication of AGO1, AGO10 and AGO4 paralogs during early radiation of Solanaceae. Fourteen AGOs were identified in potato. Orthologs of AGO8 and AGO9 were missing in the potato genome. However, AGO15 earlier annotated in tomato was identified. StAGO15 differs from the other paralogs having residues of different physico-chemical properties at functionally important amino acid positions. Upon pathogen challenge StAGO15 was significantly activated and hence may play a prominent role in sRNA-based regulation of potato defense.

Trichoderma Volatile Organic Compounds as a Biofumigation Tool against Late Blight Pathogen Phytophthora infestans in Postharvest Potato Tubers.

We tested the ability of 14 strains of Trichoderma to emit volatile compounds that decreased or stopped the growth of Phytophthora infestans. Volatile organic compounds (VOCs) emitted from Trichoderma strains designated T41 and T45 inhibited the mycelial growth of P. infestans grown on a laboratory medium by 80 and 81.4%, respectively, and on potato tubers by 93.1 and 94.1%, respectively. Using the DNA sequence analysis of the translation elongation factor region, both Trichoderma strains were identified as Trichoderma atroviride. VOCs emitted by the strains were analyzed, and 39 compounds were identified. The most abundant compounds were 3-methyl-1-butanol, 6-pentyl-2-pyrone, 2-methyl-1-propanol, and acetoin. Electron microscopy of the hyphae treated with T. atroviride VOCs revealed serious morphological and ultrastructural damages, including cell deformation, collapse, and degradation of cytoplasmic organelles. To our knowledge, this is the first report describing the ability of Trichoderma VOCs to suppress the growth of the late blight potato pathogen.