GC-MS metabolite profiling of <Emphasis Type="Italic">Phytophthora infestans</Emphasis> resistant to metalaxyl

Phytophthora infestans is the most important potato pathogen worldwide. Various alternatives have been used to control the pathogen, including continuous applications of phenylamide fungicides which has caused a rapid development of resistance in populations of P. infestans. Despite the importance of the disease, metabolite profiling of fungicide-resistant P. infestans has not been reported. In vitro resistance of Phytophthora infestans isolates to metalaxyl was characterized and metabolic changes in resistant isolates were evaluated at low (0.5 mg/L) and high (100 mg/L) concentrations of the fungicide. About 70% of the isolates tested showed resistance to metalaxyl and a total of 49 metabolites were differently expressed in resistant isolates growing in the presence of the fungicide. Principal components analysis revealed a distinct metabolite profile of resistant isolates exposed to both low and high levels of metalaxyl. The main metabolites responsible for the clustering in both fungicide concentrations included fatty acids such as hexadecanoic and octadecanoic acids, sugars such as glucose and fructose, aminoacids such as proline and valine, and organic acids such as butanedioic and propanoic acids. Potential resistance-related metabolic pathways are mostly involved in the regulation of the pathogen’s membrane fluidity and included the fatty acid biosynthesis as well as the glycerophospholipid metabolism pathways. This is the first metabolomic-based characterization of fungicide resistance in plant pathogens.     

Outcome of sexual reproduction in the <Emphasis Type="Italic">Phytophthora infestans</Emphasis> population in Estonian potato fields

In this study, the Estonian population of Phytophthora infestans was characterized with mating type, sensitivity to metalaxyl, virulence on 11 potato R-gene differentials and 12 SSR markers to show the outcome of potential sexual reproduction in the population. During the three years 2010–2012, 141 P. infestans isolates, collected from 23 potato fields, showed quite a high and stable frequency of the A2 mating type, 48% of the total population. In 87% of all sampled potato fields, both mating types were recorded, suggesting continuous sexual reproduction of P. infestans and possible oospore production. Metalaxyl-sensitive isolates prevailed in all three years (68 out of 99 isolates). Amongst the 95 isolates tested, 51 virulence races were found. The race structure was diverse, and most pathotypes were unique, appearing only once; the two most common pathotypes, 1.2.3.4.6.7.10.11 and 1.2.3.4.7.10.11, comprised 35% of the population. The P. infestans population was genetically highly diverse and most of the multilocus genotypes (MLGs) appeared only once. Furthermore, all of the MLGs appeared in only one of the three sampling years. Our results confirm that the high diversity in the Estonian P. infestans population is most likely the result of frequent sexual reproduction, which benefits the survival, adaptability and diversity of the pathogen in the climate of North-Eastern Europe.     

Reduced efficacy of fluazinam against <Emphasis FontCategory="SansSerif" Type="Italic">Phytophthora infestans</Emphasis> in the Netherlands

Phytophthora infestans is the causal organism of potato late blight, the most important disease in potato, the second most important arable crop in Europe. The P. infestans population in Europe is well known for its sudden changes in composition. Currently it is composed of a wide variety of genotypes, some of which are dominant clonal lines while others are rare or even unique to a year or location. Fungicides play a crucial role in the integrated control of late blight. Since its introduction in the Netherlands in 1992, fluazinam has been used in late blight control strategies in ware and starch potatoes. It has a broad spectrum of activity and is effective against a range of diseases including potato late blight. Fluazinam interrupts the pathogen cell’s energy production process by an uncoupling effect on oxidative phosphorylation. It is considered to have a low resistance risk. Until recently, reduced efficacy against fluazinam was not detected in P. infestans surveys in Europe. In this paper we present the finding of a new clonal lineage (EU_33_A2) of P. infestans in the Netherlands and the reduced efficacy of fluazinam to control one of the EU_33_A2 isolates in field experiments carried out in 2011 and 2015 under high disease pressure. The potential effects of this finding on practical late blight control strategies are discussed.     

Characterization of Egyptian <Emphasis Type="Italic">Phytophthora infestans</Emphasis> population using simple sequence repeat markers

The plant pathogenic oomycete, Phytophthora infestans, is the causal agent of late blight disease in tomato and potato. For characterizing Egyptian P. infestans isolates by DNA marker analysis, 40 isolates of P. infestans were collected from different locations in Egypt during two growing seasons (2012/2013 and 2013/2014). The 40 isolates were grouped into seven genotypes, in which 24 alleles were detected. The identified genotypes were not completely associated with geographic location and sample collection years. These results provide genetic and geographical information for developing a program to manage late blight disease.     

Races of <Emphasis Type="Italic">Phytophthora infestans</Emphasis> isolated from potato in Hokkaido,Japan

The virulence of 29 isolates of Phytophthora infestans collected in potato fields in Hokkaido, Japan, in 2013 and 2014, was tested for race identification. Thirteen different races were identified, each of which had five to eight virulence factors. All of the isolates caused a virulent reaction against plants with R1 and R7, and most of the isolates caused a virulent reaction against plants with R3, R4, R10, and R11. On the other hand, no isolate was virulent against plants with R9. These results demonstrate that the current Japanese P. infestans population is more complex than the population in the 1990s from the viewpoint of race.     

Characterization of <Emphasis Type="Italic">Dickeya</Emphasis> and <Emphasis Type="Italic">Pectobacterium</Emphasis> strains obtained from diseased potato plants in different climatic conditions of Norway and Poland

Soft rot and blackleg of potato caused by pectinolytic bacteria lead to severe economic losses in potato production worldwide. To investigate the species composition of bacteria causing soft rot and black leg of potato in Norway and Poland, bacteria were isolated from potato tubers and stems. Forty-one Norwegian strains and 42 Polish strains that formed cavities on pectate medium were selected for potato tuber maceration assays and sequencing of three housekeeping genes (dnaX, icdA and mdh) for species identification and phylogenetic analysis. The distribution of the species causing soft rot and blackleg in Norway and Poland differed: we have demonstrated that mainly P. atrosepticum and P. c. subsp. carotovorum are the causal agents of soft rot and blackleg of potatoes in Norway, while P. wasabiae was identified as one of the most important soft rot pathogens in Poland. In contrast to the other European countries, D. solani seem not to be a major pathogen of potato in Norway and Poland. The Norwegian and Polish P. c. subsp. carotovorum and P. wasabiae strains did not cluster with type strains of the respective species in the phylogenetic analysis, which underlines the taxonomic complexity of the genus Pectobacterium. No correlation between the country of origin and clustering of the strains was observed. All strains tested in this study were able to macerate potato tissue. The ability to macerate potato tissue was significantly greater for the P. c. subsp. carotovorum and Dickeya spp., compared to P. atrosepticum and P. wasabiae.     

Molecular aspects of the potato — Phytophthora infestans interaction

The fungal pathogenPhytophthora infestans is the causal organism of late blight, one of the most devastating diseases of potato. In the past, various aspects of the potato-P. infestans interaction have been studied extensively. In this paper we briefly review the current knowledge of the molecular events associated with the interaction and in addition we discuss a new approach for analyzing the molecular basis of pathogenicity ofP. infestans.     

Rice <Emphasis Type="Italic">OsPBL1</Emphasis> (ORYZA SATIVA ARABIDOPSIS PBS1-LIKE 1) enhanced defense of <Emphasis Type="Italic">Arabidopsis</Emphasis> against <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> DC3000

The receptor-like cytoplasmic kinases (RLCK family VII) are required for plant defense against various pathogens. Previously, OsPBL1 (ORYZA SATIVA ARABIDOPSIS PBS1-LIKE 1) was isolated from rice as a potential RSV (rice stripe virus) resistant factor, but its physiological roles in plant defense are yet to be investigated. In this study, we demonstrated that OsPBL1increased defense against P. syringae in transgenic Arabidopsis. To ascertain the role of OsPBL1 gene in plant defense, OsPBL1 tagged with HA (i.e. Hemagglutinin) was overexpressed in Arabidopsis and examined for the resistance against Pseudomonas syringae pv. tomato DC3000 (i.e. Pst DC3000). At 3 dpi of Pst DC3000, transgenic Arabidopsis lines exhibited the reduced chlorotic lesion and propagation of P. syringae, compared to wild type. Elevated pathogen resistance of transgenic lines was correlated with increased H₂O₂ accumulation and callose deposition on the infected leaves. It was also revealed that expression levels of salicylic acid dependent genes such as PR1, PR2, and PR5, were induced higher in transgenic lines than wild type. Taken together, our data suggested that OsPBL1 exerted the role in defense against pathogen attacks in plant via mainly facilitating salicylic acid dependent pathway.     

Chitin elicitor receptor kinase 1 (CERK1) is required for the non-host defense response of <Emphasis Type="Italic">Arabidopsis</Emphasis> to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. Sp. <Emphasis Type="Italic">cubense</Emphasis>

Banana wilt disease is a typical vascular disease caused by the fungal pathogen Fusarium oxysporum f. sp. cubense 4 (Foc 4). Pattern recognition receptors in the plant cell membrane can recognize pathogen-associated molecular patterns (PAMPs) to activate multi-layer defense responses, including defense gene expression, stomatal closure, reactive oxygen species (ROS) burst and callose deposition, to limit pathogen growth. In the present study, we found that chitin elicitor receptor kinase 1 (CERK1) was required for the non-host resistance of Arabidopsis thaliana to Foc B2 (a strain of Foc 4). The cerk1 mutant had weaker defense responses after Foc B2 treatment, including lower expression of PAMP- and salicylic acid-responsive genes, no stomatal closure, lower ROS level and less callose deposition, than that of the wild-type plant. Consistent with this, the cerk1 mutant plants exhibited higher susceptibility to non-host pathogen Foc B2. These results suggest the crucial importance of CERK1 in Foc B2-triggered non-host resistance.     

Recent and future developments in potato fungal pathology

An overview is given on the world-wide occurrence and frequency of mating type A2 ofPhytophthora infestans, on ploidy levels, increased virulence and fitness of the pathogen population in Germany and the Netherlands. Experiments on the survival of oospores in the open showed that these may survive in Europe until the next growing season. The resistance reaction of the differential set of R-genes used to determine virulence of races ofP. infestans is found to vary by differential clone and test method. Work has been started to come to a more homogenous differential set. The impact of oospores and the new population ofP. infestans on epidemiology and hence on disease forecasting is unknown.During the past 40 years, considerable research efforts have been made to reveal the biochemical pathways of pathogenesis, compatibility and incompatibility of the host-pathogen reaction ofP. infestans. However, the questions about host-pathogen recognition, the genes involved and which chemical compounds cause hypersensitivity or durable resistance, have not been answered yet. It is expected that the modern molecular biological approaches will more rapidly lead to such answers. One of such approaches, RFLP fingerprinting, indicated a total replacement of the old European population ofP. infestans by a new one, and revealed the origin of Mendelian aberrations found to be due to selfing when crossing isolates.The world-wide occurrence ofPhoma exigua var.foveata, the causal organism of gangrene, is discussed and a clue on its possible origin and time of introduction in Europe is given. Soil infestation is a minor but persistent source of infection and impedes elimination of the pathogen. Green-cropharvesting, a new vine killing method, considerably reduced the level of black scurf caused byRhizoctonia solani and of gangrene as compared to other vine killing methods. Promising results have been obtained combining green-crop-harvesting and the application of antagonistic organisms in controlling black scurf andErwinia spp. Integrated control of dry rot and gangrene seems to have a better future than the use of systemic fungicides.     

AlgU contributes to the virulence of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tomato</Emphasis> DC3000 by regulating production of the phytotoxin coronatine

Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), which causes bacterial speck disease of tomato, has been used as a model pathogen to investigate the molecular basis of plant–pathogen interactions. The function of many potential virulence factors encoded in the Pst DC3000 genome and their modes of action are not fully understood. P. syringae is known to produce the exopolysaccharide alginate. Although AlgU, a sigma factor, is known to regulate the expression of genes such as algD related to alginate biosynthesis, the molecular mechanisms of AlgU in the virulence of Pst DC3000 is still unclear. To investigate the function of AlgU and alginate in plant–bacterial pathogen interactions, we generated ΔalgU and ΔalgD mutants. After inoculation with ΔalgU but not ΔalgD, host plants of Pst DC3000 including tomato and Arabidopsis had milder disease symptoms and reduced bacterial populations. Expression profiles of Pst DC3000 genes revealed that AlgU can regulate not only the expression of genes encoding alginate biosynthesis, but also the expression of genes related to type III effectors and the phytotoxin coronatine (COR). We also demonstrated that the ΔalgU mutant showed full virulence in the Arabidopsis fls2 efr1 double mutant, which is compromised in the recognition of PAMPs. Further, the application of COR was able to restore the phenotype of the ΔalgU mutant in the stomatal response. These results suggest that AlgU has an important role in the virulence of Pst DC3000 by regulating COR production.     

SGT1 and HSP90 are essential for age-related non-host resistance of Nicotiana benthamiana against the oomycete pathogen Phytophthora infestans

The oomycete pathogen, Phytophthora infestans, is the causal agent of potato late blight, which is one of the most destructive and economically important plant diseases. We investigated the interaction between P. infestans and Solanaceous model plant Nicotiana benthamiana. Mature N. benthamiana plants were resistant to 8 isolates of P. infestans, whereas relatively young plants were susceptible to all isolates. Analysis with virus-induced gene silencing (VIGS) indicated that NbSGT1 and NbHSP90, genes essential for the function of R proteins, are required for the resistance of N. benthamiana to P. infestans. NbSGT1 was also required for the production of reactive oxygen species (ROS), hypersensitive cell death and expression of NbEAS, a gene for phytoalexin biosynthesis, induced by INF1, a secretory protein derived from P. infestans. These results suggested that N. benthamiana possibly possesses a broad-spectrum R protein against P. infestans, which requires an SGT1/HSP90-dependent mechanism, for the recognition of a conserved molecular pattern of P. infestans.     

Phytotoxin produced by the netted scab pathogen, <Emphasis Type="Italic">Streptomyces turgidiscabies</Emphasis> strain 65, isolated in Sweden

Streptomyces spp. are a highly diverse group of bacteria most of which are soil-inhabiting saprophytes. A few are plant pathogens that produce a family of phytotoxins called thaxtomins and cause significant economic losses, e.g., by reducing the marketability of potato tubers (Solanum tuberosum). In northern Europe, S. scabies, S. turgidiscabies and S. europaeiscabiei are the most common plant pathogenic species. In this study, a Streptomyces strain isolated from a netted scab lesion on a tuber of potato cv. Bintje in northern Sweden was identified as S. turgidiscabies but was found to differ in the genomic region carrying genes required for thaxtomin biosynthesis. Our results showed that the strain did not produce thaxtomin but rather phytotoxin fridamycin E, which is an anthraquinone novel to plant pathogenic Streptomyces spp. Fridamycin E was shown to reduce or inhibit sprouting of potato microtubers in vitro. While fridamycin E is known to have antibiotic activity against Gram-positive bacteria, the inhibitory activity of fridamycin E on plant growth is a novel finding.     

The occurrence of the A2 mating type of Phytophthora infestans in the Netherlands; significance and consequences

Phytophthora infestans (Mont.) de Bary, the causal agent of potato late blight, was first discovered in Europe in 1845. Until 1980, only A1 mating type isolates were known to occur in Europe. The absence of A2 mating type isolates restrained the fungus from sexual reproduction. In the early 1980s, A2 mating type isolates were discovered in Europe. Presumably, a new introduction ofP. infestans isolates originating from Mexico had taken place. In this paper, the significance of the presence of A1 and A2 mating type isolates in the Netherlands is reviewed. Now that both mating types are present, sexual reproduction can occur and its consequences for the control of potato late blight are discussed.     

The <Emphasis Type="Italic">Foc</Emphasis> gene governs resistance to race 3 of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">cucumerinum</Emphasis> in the cucumber cv. SMR-18

Bois noir (BN) is the most widespread European grapevine yellows disease caused by ‘Candidatus Phytoplasma solani’. Although our knowledge of the mechanisms of interactions of this pathogenic bacteria with host is largely unknown, the plant-pathogen system of BN is commonly used as a model system for studying grapevine yellows diseases. We applied here a conceptual model of general plant pathology – a disease triangle for describing interactions among the host plant, the pathogen and the environment. We generated a proof-of-concept statistical model for disease triangle using original experimental data and different statistical and data mining approaches for a selected system of ‘Ca. P. solani’ infection of cv. ‘Chardonnay’ grapevine plants. We monitored individual plants from a single vineyard over a period of six years. Phytoplasma content, the expression of 21 selected grapevine genes and environmental conditions were recorded and related to disease severity. Our model predicts that in described conditions BN is a function of the expression of grapevine gene VvDMR6, summer rainfall and abundance of ‘Ca. P. solani’. The greatest impact among elements of the disease triangle is attributed to the pathogen, and is independent of the pathogen titer. We showed that this first de facto representation of the disease triangle is useful for showing disease dynamics over several years and could be applied to other plant-pathogen systems. The overall results of this study will contribute to understanding of ‘Ca. P. solani’ biology and its interactions with grapevine host.     

Antagonistic effects of biofilm-forming bacterial strains co-inoculated with blood disease pathogenic strain, <Emphasis Type="Italic">Ralstonia syzygii</Emphasis> subspecies <Emphasis Type="Italic">celebesensis</Emphasis> in banana plants

A blood disease pathogenic strain, Ralstonia syzygii subspecies celebesensis was used to study the possible association of biofilm-forming bacteria with the development and severity of blood disease in banana plants. Therefore, the objective of this study was to determine the effects of mono-culture and co-culture inoculation of isolated biofilm-forming bacteria with the blood disease pathogen in banana pseudostems in glasshouse conditions. Putative biofilm-forming bacteria were isolated from an infected banana plant and were further identified using 16SrRNA sequencing. Four isolates, identified as Enterobacter hormaechei, Enterobacter cloacae, Kosakonia radicincitans and Klebsiella pneumoniae, were inoculated as a mono- and co-culture with R. syzygii subsp. celebesensis into 2 months old banana plants. The observation after the 8 weeks of post inoculation showed that plants which were co-inoculated with the pathogen and K. radicincitans, a biofilm-forming bacterium, were the most susceptible towards the infection. In contrast, plants under two treatments (which were co-inoculated with the pathogen and E. cloacae and the pathogen with E. hormaechei) were less susceptible towards the infection. This study revealed the antagonistic effects of two biofilm-forming strains which reduced the severity of infection caused by the pathogenic agent. Scanning electron micrographs of the cross section of plant rhizomes indicated the dissimilarity of adhesion and host colonization conditions of the pathogen in each infected plant from different treatments.     

Evaluation of PCR markers for <Emphasis Type="Italic">Phytophthora infestans</Emphasis> mating type determination

Late blight is a devastating potato disease caused by Phytophthora infestans. This organism can reproduce asexually and sexually between the strains of two mating types named A1 and A2. Mating type is an important strain characteristic affecting the pathogen’s population structure. We validate different PCR markers for P. infestans mating type determination by comparison of the results obtained with the markers (W16, S1, PHYB) with the pairing test results for 26 isolates collected worldwide and for a group of 146 Polish isolates. This study identifies an interesting feature of the isolates of genotype US-1. For all these A1 mating type isolates, the product specific for A2 isolates is amplified using the marker W16. Analysis of sequences of W16 PCR products indicates high similarity of the US-1 isolates with modern A2 mating type isolates. When US-1 isolates are excluded from analysis, 95 and 96% of isolates are correctly assigned by markers W16 and S1, respectively, when compared with the pairing test results. Marker PHYB produces 14% of discrepant results with the pairing test. Our results show that molecular markers can be useful tools for P. infestans mating type determination, but their application should be preceded by validation in each local population since their efficiency may vary depending on a pathogen’s genotype.     

Interaction of root colonizing biocontrol agents demonstrates the antagonistic effect against <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis> on tomato

Plant growth promoting Bacillus subtilis MSS9 and Bacillus licheniformis MSS14 were isolated from the tomato rhizosphere. These isolates were capable of inhibiting the fungal pathogen, Fusarium oxysporum f. sp. lycopersici causing fusarium wilt in tomato, tested by dual culture method and by mycolytic enzyme production. The isolates have the capacity to form biofilm on the microtitre plate. Scanning electron microscopy revealed good colonization capacity of Bacillus licheniformis MSS14 on tomato plant root as compared to Bacillus subtilis MSS9, pot experiments were also analyzed to study the effects of both rhizobacterial cultures on pathogen development and plant growth. It was observed that MSS14 reduces the incidence of Fusarium oxysporum f. sp. lycopersici in tomato and there was significant increase in vegetative parameters like root length, shoot length, plant wet weight, dry weight and chlorophyll content after which indicates that the root colonization property of the culture MSS14 helps in enhancing the biocontrol capacity against pathogen than that of MSS9.     

Comparison of leaf proteomes of potato (<Emphasis Type="BoldItalic">Solanum tuberosum</Emphasis> L.) genotypes with ER- and HR-mediated resistance to PVY infection

The Ny-1 and Ry-fsto genes of potato (Solanum tuberosum L.) confer hypersensitive response (HR) and extreme resistance (ER), respectively, to Potato virus Y (PVY). ER-type resistance was also observed in potato plants with both alleles (Ny-1 and Ry-fsto). Using two-dimensional electrophoresis (2-DE), quantitative differences between PVY-infected and non-infected control plants were found for 35, 32 and 15 protein spots identified in leaves of potato cultivar Rywal (possessing Ny-1), and potato tetraploid clones PW 363 (with Ry-fsto) and PB 08–137 (with Ny-1 + Ry-fsto), respectively. We recognized 29, 12 and 21 PVY-induced protein spots involved in qualitative changes in Rywal, PW 363 and PB 08–137 plants, respectively, which were processed and analysed using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. A database search indicated that these 62 proteins belong to various functional categories. Their expression was genotype-specific. In the case of cultivar Rywal with HR-mediated resistance, proteins involved in photosynthesis and primary metabolism were the most abundant. For PW 363 and PB 08–137, both with ER–mediated resistance, stress-responsive proteins were the most numerous. Only two proteins – glutamate–glyoxylate aminotransferase 2 (GGAT2) and monodehydroascorbate reductase 5 (MDHAR5) – were identified in all three genotypes. These two proteins are components of the reactive oxygen species (ROS) defence mechanism against pathogens in plants. The present study showed that the differences in PVY-induced proteins in the leaves of Ny-1, Ry-fsto and Ny-1 + R-fsto genotypes do not correspond to the type of gene conferring the resistance or to the observed phenotype.