Browsing by Subject "kasvipatologia"

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  • Pasanen, Miia (Helsingin yliopisto, 2020)
    The aim of this dissertation was to characterize Pectobacterium strains isolated in Finland. Pectobacterium species cause soft rot and blackleg on wide range of plants in cultivated areas worldwide. Potato is an important food crop and source of food cultivated all over the world. Pectobacterium species interfere with potato production at all stages of cultivation. Pectobacterium species belong to the Pectobacteriaceae family with the soft rot bacteria Dickeya genus. In this study, two Pectobacterium model strains, SCC3193 and SCC1, isolated in Finland during 80s and initially defined as belonging to P. carotovorum species were further examined. Biochemical tests of the strains were conducted to understand their characteristics of the bacterial strains and their differences to closely related bacterial strains. The strain SCC3193, originally determined as belonging to P. carotovorum, was redefined as P. wasabiae in the present study. However, it did not fully share the same biochemical profile with the P. wasabiae type strain and based on genome comparisons it was later placed into a novel species P. parmentieri. Furthermore, phylogenetic position of the Pectobacterium strain SCC1 was determined. Also, the strain SCC1 was originally defined as P. carotovorum, but it was observed in the phylogenetic analysis that it clustered apart from P. carotovorum type strain, and thus its taxonomical status could not be confirmed at the time of the analysis. It was later included into a novel species called Pectobacterium versatile. In addition, Pectobacterium strains isolated from diseased potato tubers in 2004, and initially classified as P. carotovorum, were characterized in this study. According to biochemical analyzes, these bacteria isolated from potato stems resembled P. carotovorum but had a low virulence on potato tuber and citrate-negative phenotype. Two genomes of these atypical Finnish stem isolates were produced to study their genome content and phylogenetic position in Pectobacterium genus. Average nucleotide identity (ANI) analysis showed that these isolates were similar to Pectobacterium polaris, a highly virulent new species recently identified in Norway. However, the Finnish isolates were most similar to atypical P. polaris isolates in ANI analysis and biochemical tests. Genome comparisons showed that the all the atypical isolates harbored similar genomic islands not present in P. polaris type strain. Alltogether, taxonomic and genomic studies placed the atypical P. polaris strains into a new subspecies, here called P. polaris subsp. parvum. One of the P. polaris subsp. parvum strains had been isolated in the Netherlands already in 1970s, but originally misidentified as P. carotovotum, which suggests that similar isolates were present in Europe also before. This study provides novel information about the taxonomy and ecology of Pectobacterium species existing in Finland. Taxonomic status of P. carotovorum isolates redefined in this and other studies show that Pectobacterium strains previously included into P. carotovorum species could be divided into several novel species with genome-based methods. The precise identification of bacterial species poses challenges for plant protection. The information from the study can be used for potato production and plant protection in the future.
  • Mattinen, Laura (Helsingfors universitet, 2008)
    The plant pathogen Pectobacterium atrosepticum is a Gram-negative bacterium that causes the diseases soft rot and blackleg in potato. Of the soft rotting bacteria, it has the lowest temperature optimum and it is prevalent in temperate climates. Blackleg spreads mainly through contaminated seed tubers and therefore the disease is a problem especially in the production of seed potatoes. The genome of the P. atrosepticum strain SCRI1043 has been published and it has been studied as a model organism to understand the pathogenesis of soft rot and blackleg. It is an opportunistic pathogen that can live as a biotroph in the host plant for months, without causing symptoms. As the environmental conditions turn favourable, the bacteria start to multiply and produce large amounts of plant cell wall degrading enzymes that macerate the plant tissues. The degraded plant tissue provides nutrients for the bacterial growth and enables colonization of the host. The role of the tissue degrading enzymes in pathogenesis is well known, but the latent phase and beginning of the disease are poorly understood. The genome of the bacterium revealed many putative toxins, adhesins, hemolysins and proteins that might have a function in pathogenesis. In this work, proteomics and microarray analysis were used to study the secreted proteins (secretome) and gene expression of P. atrosepticum. The proteins secreted by the bacterium are likely to have a role in virulence, because they are in direct contact with the host cells. The analyses were performed in conditions resembling plant apoplastic space: low pH, minimal nutrients and low temperature. To reveal the host induced genes and proteins, potato extract was added to the cultivation medium. Many known and putative virulence factors were identified in this study. Potato extracts induced the expression of a recently described secretion pathway, type VI secretion system (T6SS). This conserved system was first described in 2006 and since then it has been the subject of intense scrutiny in many laboratories. In this study, the T6SS of P. atrosepticum was characterized. P. atrosepticum was found to secrete hemolysin-coregulated proteins (Hcp) in a T6SS-dependent manner, and the secretion was dependent on VasK, a putative membrane component of the apparatus. A putative sigma-54 dependent regulator VasH was demonstrated to be a positive regulator of T6SS. Previous evidence indicated that T6SS was a virulence mechanism but the results of this study do not support that hypothesis, since the secretion mutant and regulatory mutant of P. atrosepticum T6SS had increased virulence in the potato maceration assay.
  • Hannukkala, Asko (MTT Agrifood Research Finland, 2012)
    Potato late blight (Phytophthora infestan) is one of the most destructive diseases of potato. The history of late blight occurrence in Finland 1845 1981 is described based on newspaper articles and scientific reports. The disease occurrence and severity in 1982 2011 was monitored in variety and fungicide efficacy trials carried out in MTT Agrifood Research Finland and Potato Research Institute. The effect of climatic factors on late blight outbreaks was modelled for the period 1993 2002. In total 4927 P. infestans isolates were collected in 1990 2010to determine mating type, R-gene virulence race, mitochondrial haplotype and response to fungicides metalaxyl and propamocarb-HCl . Development in fungicide use from 1953 to 2010 is described based on statistics for fungicide sales. Disease symptoms caused by the potato late blight pathogen were probably first described in 1845 in Finland. In 1847 and 1848 the disease was widespread, occurring in various parts of the country. From 1849 to the 1980s one to five severe late blight epidemics were reported per decade. During this period late blight appeared in the fields at the end of the growing season. During 1990s there was a rapid shift towards early outbreaks of late blight and since then the first late blight outbreaks have been reported at midsummer. The shift towards early epidemics has led to significant increase in fungicide applications in the 1990s and 2000s. The mating type A2 was first found in 1992 at low proportion in Finland. Since then the proportion of A1:A2 has stabilised close to 50:50%. This change in mating type ratio coincides with the shift towards early outbreaks of late blight, suggesting an increasing role of oospores as a source of primary inoculum. The Finnish P. infestans population is also very diverse based on the spectrum of R-gene virulence races and genetic marker studies indicating sexual reproduction in population. Metalaxyl-resistant isolates dominated the population in the early 1990s. After adapting anti-resistance strategies resistance has almost disappeared from the population. Potato late blight is currently under control in conventional potato production as long as effective fungicides are available. Due to public demand for decreasing use of pesticides in agriculture more resistant potato cultivars should be developed and cultural practices to reduce primary inoculum sources should be developed.
  • Gabrenaite-Verkhovskaya, Rasa (Helsingin yliopisto, 2007)
    The particles of Potato virus A (PVA; genus Potyvirus) are helically constructed filaments that contain multiple copies of a single type of coat-protein (CP) subunit and a single copy of genome-linked protein (VPg), attached to one end of the virion. Examination of negatively-stained virions by electron microscopy revealed flexuous, rod-shaped particles with no obvious terminal structures. It is known that particles of several filamentous plant viruses incorporate additional minor protein components, forming stable complexes that mediate particle disassembly, movement or transmission by insect vectors. The first objective of this work was to study the interaction of PVA movement-associated proteins with virus particles and how these interactions contribute to the morphology and function of the virus particles. Purified particles of PVA were examined by atomic force microscopy (AFM) and immuno-gold electron microscopy. A protrusion was found at one end of some of the potyvirus particles, associated with the 5' end of the viral RNA. The tip contained two virus-encoded proteins, the genome-linked protein (VPg) and the helper-component proteinase (HC-Pro). Both are required for cell-to-cell movement of the virus. Biochemical and electron microscopy studies of purified PVA samples also revealed the presence of another protein required for cell-to-cell movement the cylindrical inclusion protein (CI), which is also an RNA helicase/ATPase. Centrifugation through a 5-40% sucrose gradient separated virus particles with no detectable CI to a fraction that remained in the gradient, from the CI-associated particles that went to the pellet. Both types of particles were infectious. AFM and translation experiments demonstrated that when the viral CI was not present in the sample, PVA virions had a beads-on-a-string phenotype, and RNA within the virus particles was more accessible to translation. The second objective of this work was to study phosphorylation of PVA movement-associated and structural proteins (CP and VPg) in vitro and, if possible, in vivo. PVA virion structural protein CP is necessary for virus cell-to-cell movement. The tobacco protein kinase CK2 was identified as a kinase phosphorylating PVA CP. A major site of CK2 phosphorylation in PVA CP was identified as a single threonine within a CK2 consensus sequence. Amino acid substitutions affecting the CK2 consensus sequence in CP resulted in viruses that were defective in cell-to-cell and long-distance movement. The CK2 regulation of virion assembly and cell-to-cell movement by phosphorylation of CP was possibly due to the inhibition of CP binding to viral RNA. Four putative phosphorylation sites were identified from an in vitro phosphorylated recombinant VPg. All four were mutated and the spread of mutant viruses in two different host plants was studied. Two putative phosphorylation site mutants (Thr45 and Thr49) had phenotypes identical to that of a wild type (WT) virus infection in both Nicotiana benthamiana and N. tabacum plants. The other two mutant viruses (Thr132/Ser133 and Thr168) showed different phenotypes with increased or decreased accumulation rates, respectively, in inoculated and the first two systemically infected leaves of N. benthamiana. The same mutants were occasionally restricted to single cells in N. tabacum plants, suggesting the importance of these amino acids in the PVA infection cycle in N. tabacum.
  • Marttinen, Eeva (Helsingin yliopisto, 2020)
    Knowledge of the defense responses of mosses against pathogens has gained less attention than has our knowledge of the pathogens of vascular plants. Recently, the use of mosses has gained attention because mosses are used for greenhouse production as well as landscaping. In particular, the landscaping and greening of buildings have become popular because these initiatives offer one solution for mitigating urban problems such as heat islands and flooding. Mosses are an easy and lightweight solution for greening purposes as they can survive the harsh rooftop environment and have great stormwater retention. However, the health of plants is fundamental to achieving the benefits of greening. Like vascular plants, mosses also are susceptible to plant diseases. Many fungi damage mosses by causing brown patches of greenish moss. Brown patches on mosses are a characteristic sign of fungal infection. However, plants have various defense mechanisms, the first of which consists of preexisting structural and chemical defenses. Second, the plant immune system uses specific receptors with which to recognize the molecular structures of microbes that are not present on the surface of the plant's own cells. Receptor-mediated sensing of these structures can trigger early defense responses of plant, which can make the plant resistant to the attacking microbe. The model moss Physcomitrella patens, like the vascular plants, senses the molecular structures of microbes. For example, exposure of P. patens to chitosan—a component of the fungal cell wall—significantly increases peroxidase activity and oxygen radical formation. Oxygen radicals in turn affect many biological events; they can directly damage the pathogen or stimulate the plant's defenses. Currently, little is known about the peroxidase-based defense as well as chitosan-induced signaling pathways of P. patens. The aim of the research presented in this thesis was to study the pathogens of green roof mosses and establishes the host range of the isolated pathogens; the study also utilized a Physcomitrella mutant collection to identify genes involved in chitosan-induced signaling pathway. Fungal species that naturally inhabit mosses at a moss farm in Japan and on green roof environments in southern Finland were isolated and the ability of these fungi to infect and cause disease symptoms on the model moss P. patens was tested. In addition, the pathogenicity of fungus species towards vascular plants was also assessed. To elucidate the genes involved in chitosan-induced peroxidase activity, part of the Physcomitrella mutant collection was screened using the oxidation of 2,2´-azino-bis(3-ethylbenzothiazoine-6-sulfonic acid) as an indicator of peroxidase activity. Genome walking analyses were used to identify which genes were mutated within each moss line of interest. The work described in this thesis demonstrates that mosses used for greening are colonized by many different fungal isolates. These studies reveal that several fungal genera such as Fusarium, Trichoderma, Phoma and Alternaria cause severe symptoms in P. patens. Moreover, our results demonstrate that mosses and vascular plants have common pathogens. The fungal isolates Fusarium avenaceum and Cladosporium oxysporum obtained from moss panels caused disease symptoms on barley and carrots, respectively, and also on two different moss species. The results also demonstrate that the Physcomitrella mutant collection is a valuable source for identifying genes involved in the chitosan-induced signaling pathway. Screening of part of the mutant collection and further analyses revealed that Rossmann fold protein is a significant part of the signaling chain leading to upregulated peroxidase activity induced by chitosan. In addition, this Rossmann fold protein is an important factor for normal lipoxygenase (LOX) expression and might contribute to defense against fungal pathogens. The results from this doctoral thesis provide new insights into the pathogens of green roofs, the host range of the pathogens and the molecular mechanisms involved in disease control and defense responses in moss. The knowledge gained concerning the pathogenicity of Trichoderma isolates and the host range of pathogenic fungi should be considered when planting moss farms and cultivating crops in close proximity to each other or when applying biological control agents containing Trichoderma species to green roofs. Furthermore, these results may encourage the use of the Physcomitrella mutant collection to identify candidate genes for signaling pathways to elucidate the molecular mechanisms underlying the defense responses of mosses.
  • Hautsalo, Juho (Helsingin yliopisto, 2020)
    Fusarium head blight (FHB) disease and the mycotoxins produced by its causal agents such as Fusarium graminearum Schwabe, F. culmorum (Wg. G. Sm.) and F. langsethiae Torp and Nirenberg have become a growing problem for oat (Avena sativa L.) production in the northern countries over the last decades. Since Nordic oats and oat products are branded as high quality and healthy food, FHB has to be managed. Controlling FHB by agricultural or manufacturing practices can be a cumbersome task. Therefore development of resistant cultivars would offer a highly needed and economical solution to the problem. The primary aim of this thesis was to improve the tools for FHB resistance breeding in oats. Resistance among genebank accessions and Nordic oat breeding material, inoculation methods and potential traits to measure resistance were evaluated in several field and greenhouse experiments in Finland. The results obtained from the field experiments had more practical relevance than greenhouse experiments in identification of cultivars that accumulate less DON. In the field conditions, the escape mechanisms such as early flowering, height or high rate of anther extrusion through open flowering can all affect to DON accumulation and Fusarium infection within an oat plant. Large variation in several Fusarium-associated traits among the breeding material indicates that FHB resistance in Finnish oats can be improved. However, the results also underline that there are several components of resistance that need to be considered, since the estimates for DON accumulation, germination capacity and proportion of Fusarium infected kernels did not give similar rankings for all of the genotypes. Further research is recommended in order to gain better understand the genetics of FHB resistance in oats. In addition, more efficient methods for the phenotyping of large progenies are needed. In future breeding, the amount of phenotyping can be reduced when readiness for genomic selection is on place. Also other Fusarium species and mycotoxins need to be considered in future resistance studies.
  • Lehtonen, Mikko (Helsingin yliopisto, 2013)
    Mosses (Bryophyta) are among the basal embryophytes, which constitute a large part of the photosynthesizing biomass and carbon storage on Earth. Albeit inhabited by a number of fungal species, little is known about how this important portion of flora maintains its health status. Vascular plants (tracheophytes) respond to micro-organisms by the production of reactive oxygen species (ROS), the activation of signaling cascades leading to altered gene expression, protein synthesis and the secretion of extracellular proteins and metabolites. But how do mosses respond to pathogens and pathogen elicitors? The aim of this study was to understand interactions between mosses and fungal pathogens by finding out how the moss Physcomitrella patens responds to a fungal cell wall -derived compound chitosan, which induces defense in vascular plants, by identifying the genes involved in the response and determining their significance against fungal pathogens by creating knockout plants. Chitosan treatment was used to study the production of ROS, extracellular enzyme activity, the expression of defense gene homologs of vascular plants and the changes in the extracellular proteome of P. patens. The resulting information was utilized to generate knock-out moss plants, and since natural pathogens of P. patens are not known, the responses of the knock-out moss plants were tested using fungi isolated from commercially grown Sunagoke moss Racomitrium japonicum. The pathogenicity of these fungi was tested also on selected vascular plants. Chitosan caused the generation of superoxide and a rapid increase in peroxidase activity resulting from a single peroxidase (Prx34) of P. patens. The induction of genes related to oxidative stress and defense signaling was observed. Proteomics studies of extracellular proteins revealed secretion of a large number of proteins in response to chitosan treatment. Fungal infection in P. patens induced the formation of papillae, cell wall reinforcement, chloroplast repositioning, spreading necrosis and a reaction reminiscent of a hypersensitive response. The ΔPrx34 mutants of P. patens were susceptible not only to pathogenic but also to saprophytic fungi. The previously described salt sensitive ΔPpTSPO1 mutants of P. patens, lacking the PpTSPO1 gene involved in mitochondrial tetrapyrrole transport and abiotic (salt) stress tolerance, also showed enhanced susceptibility to fungal infection. Certain fungi including Cladosporium oxysporum and Fusarium avenaceum isolated from disease lesions in Sunagoke moss could also infect P. patens and vascular crop plants. The studies pointed out Prx34 as the first specific host factor that is pivotal for pathogen defense in a bryophyte. PpTSPO1 appears as a new link between abiotic and biotic stress signaling and illustrates the importance of redox homeostasis required for efficient pathogen defense. Analysis of extracellular proteins of P. patens indicates an active basal defense towards fungi and expands the current knowledge of bryophyte secretory proteins. Furthermore, results suggest that mosses and vascular plants may be linked by a common microbial interface consisting of pathogenic fungi. These findings have implications that have gained little previous attention.
  • Serenius, Marjo (Helsingin yliopisto, 2006)
    Spring barley is the most important crop in Finland based on cultivated land area. Net blotch, a disease caused by Pyrenophora teres Drech., is the most damaging disease of barley in Finland. The pressure to improve the economics and efficiency of agriculture has increased the need for more efficient plant protection methods. Development of durable host-plant resistance to net blotch is a promising possibility. However, deployment of disease resistant crops could initiate selection pressure on the pathogen (P. teres) population. The aim of this study was to understand the population biology of P. teres and to estimate the evolutionary potential of P. teres under selective pressure following deployment of resistance genes and application of fungicides. The study included mainly Finnish P. teres isolates. Population samples from Russia and Australia were also included. Using AFLP markers substantial genotypic variation in P. teres populations was identified. Differences among isolates were least within Finnish fields and significantly higher in Krasnodar, Russia. Genetic differentiation was identified among populations from northern Europe and from Australia, and between the two forms P. teres f. teres (PTT, net form of net blotch) and P. teres f. maculata (PTM, spot form of net blotch) in Australia. Differentiation among populations was also identified based on virulence between Finnish and Russian populations, and based on prochloraz (fungicide) tolerance in the Häme region in Finland. Surprisingly only PTT was recovered from Finland and Russia although both forms were earlier equally common in Finland. The reason for the shift in occurrence of forms in Finland remained uncertain. Both forms were found within several fields in Australia. Sexual reproduction of P. teres was supported by recover of both mating types in equal ratio in those areas although the prevalence of sexual mating seems to be less in Finland than in Australia. Population from Krasnodar was an exception since only one mating type was found in there. Based on the substantial high genotypic variation in Krasnodar it was suggested go represent an old P. teres population, whereas the Australian samples were suggested to represent newer populations. In conclusion, P. teres populations are differentiated at several levels. Human assistance in dispersal of P. teres on infected barley seed is obvious and decreases the differentiation among populations. This can increase the plant protection problems caused by this pathogen. P. teres is capable of sexual reproduction in several areas but the prevalence varies. Based on these findings it is apparent that P. teres has the potential to pose more serious problems in barley cultivation if plant protection is neglected. Therefore, good agricultural practices, including crop rotation and the use of healthy seed, are recommended.
  • Vuorinen, Anssi (Helsingin yliopisto, 2011)
    Peruna kestää A-virusta estämällä sen leviämistä Peruna on maissin ohella maailman kolmanneksi tärkein ravintokasvi vehnän ja riisin jälkeen. Perunaa lisätään kasvullisesti mukuloita istuttamalla, jolloin virukset siirtyvät sairaiden siemenmukuloiden välityksellä kasvukaudesta toiseen. Virustauteja voi torjua ainoastaan terveen siemenperunan ja kestävien lajikkeiden avulla. Kestävyys perustuu usein siihen, että kasvi estää viruksen leviämisen tartuntakohdasta välttyäkseen virustaudilta. Tässä työssä tutkittiin kolmea perunan A-viruksen (PVA) liikkumista estävää kestävyysmekanismia perunassa. Lisäksi työn kokeelliseen osaan oleellisesti kuuluvaa virustartutusta varten kehitettiin uusi paranneltu versio geenipyssystä. Tämä itse rakennettu laite optimoitiin PVA:n tartuttamiseen mahdollisimman helposti ja pienin käyttökustannuksin. Tutkimuksen kohteena olleessa perunan risteytysjälkeläistössä oli PVA:ta kestäviä kasveja (ryhmä nnr), jotka estivät viruksen liikkumisen aiheuttamatta oireita tartutuskohdassa, sekä kasveja, joissa PVA aiheutti kuolioläikkinä näkyvän yliherkkyysvasteen (ryhmä HR). Molemmissa kestävyystyypeissä virus pystyi monistumaan ja leviämään solusta soluun paikallisesti, mutta liikkuminen muihin kasvinosiin nilan kautta estyi. Ryhmän nnr kasveissa PVA-tartunta ei aiheuttanut tilastollisesti merkitsevää muutosta useimpien geenien ilmenemiseen tartuntakohdassa. Ainoastaan geeniperhe, joka ilmentää tiettyä proteinaasi-inhibiittoria (PI), reagoi PVA:han 24 tuntia tartutuksesta. Kun tämän PVA:han reagoivan geeniperheen jäsenet hiljennettiin nnr- perunalinjoissa, ne muuttuivat alttiiksi PVA:lle ja virus levisi tartuntakohdasta muihin kasvinosiin. Tulos osoittaa, että PI on viruskestävyystekijä. Lisäksi muut tutkimuksessa saadut tulokset tukevat mahdollisuutta, että PI estää PVA:n P1-proteinaasin toimintaa. HR-linjoissa todettiin erilaisiin puolustusvasteisiin liittyvien PR-geenien aktivoitumista PVA-tartunnan seurauksena, mutta myös ilman sitä kasvien kasvettua mullassa noin neljä viikkoa. Sen sijaan solukkoviljelyssä tai vasta kaksi viikkoa mullassa kasvaneissa kasveissa vastaavaa ei vielä todettu. Tulos viittaa siihen, että HR-perunat reagoivat herkemmin ympäristöön ja/tai kasvin kehitysasteeseen laukaisten puolustusvasteita, jotka saattavat parantaa kestävyyttä taudinaiheuttajia vastaan. Kolmas tutkittu kestävyystyyppi havaittiin Pito-perunalajikkeessa. Se muistutti nnr-kestävyyttä siten, että myös siinä viruksen liikkuminen nilassa muihin kasvinosiin estyi. PVA:n todettiin pysähtyvän vasta lehtiruodin tyvelle muodostuvaan irtoamisvyöhykkeeseen, mitä havainnollistettiin käyttämällä muunnettua PVA-rotua, joka tuotti UV-valossa fluoresoivaa vihreää valoa. Tulos viittaa siihen, että virus ei pääse kulkemaan vyöhykkeeseen kuuluvan suojaavan kerroksen läpi, jollei sillä ole pääsyä nilaan. Tällainen kestävyys on tarpeen, jotta virus ei voi korvata nilakuljetusta solusta soluun leviämisellä. Tulokset tuovat uusia näkökulmia kasvien viruskestävyyteen ja auttavat selittämään viruksen nilakuljetuksen estymistä sekä solusta soluun leviämisen pysähtymistä kestävissä kasveissa.
  • Lehtonen, Mari (Helsingin yliopisto, 2009)
    Rhizoctonia solani is a soil inhabiting basidiomycetous fungus able to induce a wide range of symptoms in many plant species. This genetically complex species is divided to 13 anastomosis groups (AG), of which AG-3 is specialized to infect potato. However, also a few other AGs are able to infect or live in close contact with potato. On potato, R. solani infection causes two main types of diseases including stem canker observed as a dark brown lesions on developing stems and stolons, and black scurf that develops on new tubers close to the time of harvest. These disease symptoms are collectively called a ‘Rhizoctonia disease complex’. Between the growing seasons R. solani survives in soil and plant debri as sclerotia or as the sclerotia called black scurf on potato tubers which when used as seed offer the main route for dispersal of the fungus to new areas. The reasons for the dominance of AG-3 on potato seem to be attributable to its highly specialization to potato and its ability to infect and form sclerotia efficiently at low temperatures. In this study, a large nationwide survey of R. solani isolates was made in potato crops in Finland. Almost all characterized isolates belonged to AG-3. Additionally, three other AGs (AG-2-1, AG-4 and AG-5) were found associated with symptoms on potato plants but they were weaker pathogens on potato than AG-3 as less prone to form black scurf. According to phylogenetic analysis of the internal transcribed sequences (ITS) of the ribosomal RNA genes the Finnish AG-3 isolates are closely related to each other even though a wide variation of physiological features was observed between them. Detailed analysis of the ITS regions revealed single nucleotide polymorphism in 14 nucleotide positions of ITS-1 and ITS-2. Additionally, compensatory base changes on ITS-2 were detected which suggests that potato-infecting R. solani AG-3 could be considered as a separate species instead of an AG of R. solani. For the first time, molecular defence responses were studied and detected during the early phases of interaction between R. solani AG-3 and potato. Extensive systemic signalling for defence exploiting several known defence pathways was activated as soon as R. solani came into close contact with the base of a sprout. The defence response was strong enough to protect vulnerable sprout tips from new attacks by the pathogen. These results at least partly explain why potato emergence is eventually successful even under heavy infection pressure by R. solani.
  • Karjalainen, Eveliina (Helsingin yliopisto, 2019)
    Yeasts are significant plant pathogens, yet plant-yeast interactions are poorly understood. Mammals have immune receptors for yeast-specific MAMPs (microbe-associated molecular patterns), suggesting that plants might have similar receptors as well. Detection of MAMPs by PRRs (pattern recognition receptors) triggers the plant immune system, leading to the first phase of PTI (pattern-triggered immunity). The yeast cell wall is layered, so only some of the potential MAMP molecules, like mannans, are easily available for the plant receptors. In order to utilize yeasts and PRR-based yeast resistance in agriculture and forestry, it is crucial to identify these receptors and gain a better understanding of their functions in plant-yeast interactions. PRRs can be divided into two groups, RLPs (receptor-like proteins) and RLKs (receptor-like kinases). Some of the L-type lectin receptor kinases have already been shown to participate in the interactions between plants and fungi or fungal like pathogens, making them promising candidates for yeast receptors. G-type lectins remain less studied but have promising genes as well. In this thesis, we use forward and reverse genetics methods to show that lectin receptor kinases might include putative yeast pattern recognition receptors. We also establish the first protocol and genetic screen for the identification of plant PRRs participating in the recognition of yeast cell wall MAMPs. Only genes present in both silver birch (Betula pendula) and arabidopsis (Arabidopsis thaliana) were examined to enable following studies with an Arabidopsis model system of the interactions between silver birch and the dimorphic birch pathogen Taphrina betulina. This information can later be used in plant production with other plants and their yeasts as well.
  • Jalli, Marja (2010)
    In Finland, barley, Hordeum vulgare L., covers 50 % of the total acreage devoted to cereal cultivation. The most common disease of barley in Finland is net blotch, a foliar disease caused by the ascomycete Pyrenophora teres Drechsler. Disease resistance based on plant genes is an environmentally friendly and economical way to manage plant diseases caused by biotic stresses. Development of a disease resistance breeding programme is dependent on knowledge of the pathogen. In addition to information on the epidemiology and virulence of a pathogen, knowledge on how the pathogen evolves and the nature of the risks that might arise in the future are essential issues that need to be taken into account to achieve the final breeding aims. The main objectives of this study were to establish reliable and efficient testing methods for Pyrenophora teres f. teres virulence screening, and to understand the role of virulence of P. teres f. teres in Finland from a disease resistance breeding point of view. The virulence of P. teres was studied by testing 239 Finnish P. teres f. teres isolates collected between 1994 2007 originating from 19 locations, and 200 P. teres progeny isolates originating from artificially produced P. teres matings. According to the results of this study, screening for P. teres f. teres isolates on barley seedlings under greenhouse conditions is a feasible and cost efficient method to describe the virulence spectrum of the pathogen. Inoculum concentration and the seedling leaf used to gauge virulence had significant effects. Barley grain size, morphological traits of P. teres isolates, spore production and growth rate on agar did not affect the expression of virulence. A common barley differential set to characterize the P. teres virulence was developed and is recommended to be used globally. The virulence spectrum of Finnish P. teres f. teres isolates collected in 1994-2007 was constant both within and between the years. The results indicated differences in the pathogen s aggressiveness and in barley genotypes resistance. However, differences in virulence were rarely significant. Unlike in laboratory conditions, no indications of changes in virulence caused by the sexual reproduction have been observed in Finnish barley fields. In Finland, durable net blotch resistance has been achieved by introducing resistance from other barley varieties using traditional crossing methods, including wide crossing, and testing the breeding material at early generations at several sites under natural infection pressure. Novel resistance is available, which is recommended to minimize the risk of selection of virulent isolates and breakdown of currently deployed resistance.
  • Nykyri, Johanna (Helsingin yliopisto, 2013)
    The aim of this study was to characterize novel virulence or fitness genes of Pectobacterium wasabiae SCC3193 and Pectobacterium atrosepticum SCRI1043, which are potato pathogenic model strains. Members of the Pectobacterium genus cause plant diseases called soft rot, blackleg and aerial stem rot. Potato is one of the most economically important crops affected by these diseases. The Pectobacterium genus belongs to the bacterial family Enterobacteriaceae and is closely related to many common animal pathogens, such as Escherichia coli and Salmonella. Together, the bacteria belonging to genus Pectobacterium and to the other soft-rot genus Dickeya are called soft-rot enterobacteria. In this study, novel virulence genes were identified and characterized using genomic and post-genomic approaches. The study began by examining the genomic sequences of the model strains; functional studies of the genes were then conducted based on function predictions of the genes of interest. The genome of P. atrosepticum SCRI1043 was sequenced in 2004, and the genomes of P. wasabiae SCC3193 and the P. wasabiae type strain, which were used for comparative genomics, were sequenced by a collaborator of this study. The genome analyses revised the species designation of the model strain SCC3193 and the genome analyses together with transcriptional studies revealed candidates for novel virulence genes for post-genomic studies. In the post-genomic studies, the genes of interest were mutated and complemented by expression of the respective gene from a plasmid in the mutant background in several phenotypic experiments. The phenotypes of the novel mutant strains were evaluated in several virulence assays in potato and tobacco, motility assays, attachment assays, growth assays in several media, gene expression assays in several media and plant cell wall-degrading enzyme production measuring assays, all in comparison to the wild-type strain and the complemented strain. Plant cell wall-degrading enzymes are the main pathogenicity factors in genus Pectobacterium; the other tested functions are known to be related to virulence or may be related to virulence. Novel virulence determinants were suggested in this study, including the type VI secretion system, the Flp/Tad pilus and a two component system regulating the pilus, the SirB protein of unknown function and a genomic island containing a predicted lipoprotein transporter and a HopL1-like protein that is similar to the type III secretion system effector HopL1 in Pseudomonas syringae. This study provides novel information about the phylogeny and virulence of devastating plant pathogens that are present in Finland and worldwide. This information could be utilized in subsequent applied studies that could enhance plant health as an economically important part of potato production and industry.