Expression Analysis of Host-Induced Genes and Proteins of Potato Pathogen Pectobacterium atrosepticum

Pectobacterium atrosepticum on Gram-negatiivinen bakteeri, joka aiheuttaa perunan tyvi- ja märkämätää. P. atrosepticum bakteerin optimilämpötila on melko alhainen ja se on yleinen lauhkeilla alueilla. Tyvimätä leviää pääasiassa siemenperunan välityksellä ja siksi se on ongelma erityisesti siemenperunan tuotannossa. P. atrosepticum kannan SCRI1043 genomi on julkaistu ja sitä tutkitaan malliorganismina märkä- ja tyvimädän taudinaiheuttamisen ymmärtämiseksi. Tämä opportunistinen taudinaiheuttaja voi elää isäntäkasvissa kuukausia piilevänä, aiheuttamatta näkyviä oireita. Suotuisissa olosuhteissa bakteerit alkavat jakautua ja tuottaa kasvin kudoksia hajottavia entsyymejä. Mädäntyvä kasvimassa tarjoaa ravinteita bakteerien kasvuun ja mahdollistaa isäntäkasvin asuttamisen. Soluseiniä hajottavien entsyymien merkitys taudinaiheuttamisessa on hyvin tunnettu, mutta oireettomasta jaksosta ja taudin alkuvaiheista tiedätään vain vähän. Bakteerin genomi sisältää monia toksiineja, adhesiineja, hemolysiineja ja muita proteiineja, joilla saattaa olla merkitys taudinaiheuttamisessa. Tässä työssä käytettiin proteomiikkaa ja mikrosiruanalysiä P. atrosepticum bakteerin erittyvien proteiinien ja geeniekspression tutkimiseen. Proteiinit, jotka eritetään ulos bakteerista, toimivat todennäköisesti taudinaiheuttamisessa, koska ne ovat suorassa kontaktissa isäntäkasvin kanssa. Analyysit suoritettiin olosuhteissa, jotka muistuttavat kasvin soluvälitilaa: matala pH, vähän ravinteita ja matala lämpötila. Isäntäkasvin läsnäolon vaikutusta proteiinien tuottoon ja geeniekspressioon tutkittiin lisäämällä perunauutetta kasvatusalustaan. Tutkimuksessa tunnistettiin P. atrosepticum bakteerin monia jo tunnettuja ja mahdollisesti taudinaiheuttamiseen liittyviä proteiineja. Perunauute lisäsi hiljattain tunnistetun, proteiinien eritysreittiä (tyyppi VI sekreetio, T6SS) koodaavien geenien ilmentymistä. Lisäksi bakteerin havaittiin erittävän useita T6SS:n liittyviä proteiineja kasvualustaan, johon oli lisätty perunauutetta. T6SS:n merkitys bakteereille on vielä epäselvä ja sen vaikutuksesta taudinaiheuttamiseen on julkaistu ristiriitaisia tuloksia. Märkä- ja tyvimädän ymmärtäminen molekulaarisella tasolla luo pohjan tautien kontrollointiin tähtäävään soveltavaan tutkimukseen. Tämä tutkimus lisää tietoa kasvi-patogeeni- interaktiosta ja sitä voidaan tulevaisuudessa käyttää hyväksi esimerkiksi diagnostiikassa, resistenttien perunalajikkeiden jalostuksessa tai viljely- ja varastointiolosuhteiden parantamisessa.

Concentrate feeding strategies for growing and finishing dairy bulls offered grass silage-based diets

The first aim of this thesis was to produce data for evaluating, developing and recommending biologically and economically efficient energy and protein feeding strategies for growing and finishing dairy bulls offered grass silage-based diets. The second aim was to calculate the energy and protein supplies of the dairy bulls fed different grass silage-cereal-based diets and, based on this, to estimate the possible need to revise the current Finnish energy and protein recommendations for growing dairy bulls. The third aim was to demonstrate the phosphorus supply of dairy bulls fed grass silage-cereal-based diets with or without protein supplementation in relation to current feeding recommendations for phosphorus. The results indicate that protein supplement is not needed for finishing dairy bulls (live weight more than 250 kg) when they are fed good-quality grass silage (digestible organic matter more than 650 g/kg dry matter, restricted fermentation with low concentrations of fermentation acids and ammonia N) and grain-based concentrate with a moderate (300-700 g/kg dry matter) concentrate level. The results also suggest that with total mixed ration feeding it is possible to use rather high concentrate proportions (700 g/kg dry matter) in feeding dairy bulls. According to this study, barley fibre is a suitable energy supplement with good-quality silage for growing dairy bulls. The results suggest that 50% of barley grain can be replaced with barley fibre without affecting growth. Also oats is a suitable energy supplement for dairy bulls. However, as a consequence of decreased energy intake, the gain and feed conversion of the bulls were slightly reduced in this study when barley grain was replaced by oats in the diet. Ultimately, the rationality of the use of barley fibre and oats in the future will depend on the price in relation to other concentrates. During the feeding experiments the calculated supply of energy was 10% higher than in the Finnish feeding recommendations for the present growth rate. This indicates that there is a need to update the Finnish feeding recommendations for dairy-breed growing bulls, and further calculations are needed for the energy supply of growing dairy bulls. The calculated supply of AAT (amino acids absorbed from the small intestine) was 38% higher than in the Finnish feeding recommendations for the present growth. Possibly, the present AAT-PBV system is not an optimal protein evaluation system for growing dairy bulls more than 250 kg live weight. The calculations based on the feeding experiments and the Finnish feeding recommendations indicate that in most cases the dairy bulls (live weight more than 250 kg) received enough P from the basic grass silage cereal-based diets without additional mineral feeds. Therefore there is no need to add P in the form of mineral mixtures.

Rhizoctonia solani as a potato pathogen : Variation of isolates in Finland and host response

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.

Winter feeding strategies for suckler cows in cold climatic conditions

In Finland, suckler cow production is carried out in circumstances characterized by a long winter period and a short grazing period. The traditional winter housing system for suckler cows has been insulated or uninsulated buildings, but there is a demand for developing less expensive housing systems. In addition, more information is needed on new winter feeding strategies, carried out in inexpensive winter facilities with conventional (hay, grass silage, straw) or alternative (treated straw, industrial by-product, whole-crop silage) feeds. The new feeding techniques should not have any detrimental effects on animal welfare in order to be acceptable to both farmers and consumers. Furthermore, no official feeding recommendations for suckler cows are available in Finland and, thus, recommendations for dairy cows have been used. However, this may lead to over- or underfeeding of suckler cows and, finally, to decreased economic output. In Experiment I, second-calf beef-dairy suckler cows were used to compare the effects of diets based on hay (H) or urea-treated straw (US) at two feeding levels (Moderate; M vs. Low; L) on the performance of cows and calves. Live weight (LW) gain during the indoor feeding was lower for cows on level L than on level M. Cows on diet US lost more LW indoors than those on diet H. The cows replenished the LW losses on good pasture. Calf LW gain and cow milk production were unaffected by the treatments. Conception rate was unaffected by the treatments but was only 69%. Urea-treated straw proved to be a suitable winter feed for spring-calving suckler cows. Experiment II studied the effects of feeding accuracy on the performance of first- and second-calf beef-dairy cows and calves. In II-1, the day-to-day variation in the roughage offered ranged up to ± 40%. In II-2, the same variation was used in two-week periods. Variation of the roughages offered had minor effects on cow performance. Reproduction was unaffected by the feeding accuracy. Accurate feeding is not necessary for young beef-dairy crosses, if the total amount of energy offered over a period of a few weeks fulfills the energy requirements. Effects of feeding strategies with alternative feeds on the performance of mature beef-dairy and beef cows and calves were evaluated in Experiment III. Two studies consisted of two feeding strategies (Step-up vs. Flat-rate) and two diets (Control vs. Alternative). There were no differences between treatments in the cow LW, body condition score (BCS), calf pre-weaning LW gain and cow reproduction. A flat-rate strategy can be practised in the nutrition of mature suckler cows. Oat hull based flour-mill by product can partly replace grass silage and straw in the winter diet. Whole-crop barley silage can be offered as a sole feed to suckler cows. Experiment IV evaluated during the winter feeding period the effects of replacing grass silage with whole-crop barley or oat silage on mature beef cow and calf performance. Both whole-crop silages were suitable winter feeds for suckler cows in cold outdoor winter conditions. Experiment V aimed at assessing the effects of daily feeding vs. feeding every third day on the performance of mature beef cows and calves. No differences between the treatments were observed in cow LW, BCS, milk production and calf LW. The serum concentrations of urea and long-chain fatty acids were increased on the third day after feeding in the cows fed every third day. Despite of that the feeding every third day is an acceptable feeding strategy for mature suckler cows. Experiment VI studied the effects of feeding levels and long-term cold climatic conditions on mature beef cows and calves. The cows were overwintered in outdoor facilities or in an uninsulated indoor facility. Whole-crop barley silage was offered either ad libitum or restricted. All the facilities offered adequate shelter for the cows. The restricted offering of whole-crop barley silage provided enough energy for the cows. The Finnish energy recommendations for dairy cows were too high for mature beef breed suckler cows in good body condition at housing, even in cold conditions. Therefore, there is need to determine feeding recommendations for suckler cows in Finland. The results showed that the required amount of energy can be offered to the cows using conventional or alternative feeds provided at a lower feeding level, with an inaccurate feeding, flat-rate feeding or feeding every third day strategy. The cows must have an opportunity to replenish the LW and BCS losses at pasture before the next winter. Production in cold conditions can be practised in inexpensive facilities when shelter against rain and wind, a dry resting place, adequate amounts of feed suitable for cold conditions and water are provided for the animals as was done in the present study.

A dynamic programming model for optimising feeding and slaughter decisions regarding fattening pigs

Costs of purchasing new piglets and of feeding them until slaughter are the main variable expenditures in pig fattening. They both depend on slaughter intensity, the nature of feeding patterns and the technological constraints of pig fattening, such as genotype. Therefore, it is of interest to examine the effect of production technology and changes in input and output prices on feeding and slaughter decisions. This study examines the problem by using a dynamic programming model that links genetic characteristics of a pig to feeding decisions and the timing of slaughter and takes into account how these jointly affect the quality-adjusted value of a carcass. The model simulates the growth mechanism of a pig under optional feeding and slaughter patterns and then solves the optimal feeding and slaughter decisions recursively. The state of nature and the genotype of a pig are known in the analysis. The main contribution of this study is the dynamic approach that explicitly takes into account carcass quality while simultaneously optimising feeding and slaughter decisions. The method maximises the internal rate of return to the capacity unit. Hence, the results can have vital impact on competitiveness of pig production, which is known to be quite capital-intensive. The results suggest that producer can significantly benefit from improvements in the pig's genotype, because they improve efficiency of pig production. The annual benefits from obtaining pigs of improved genotype can be more than €20 per capacity unit. The annual net benefits of animal breeding to pig farms can also be considerable. Animals of improved genotype can reach optimal slaughter maturity quicker and produce leaner meat than animals of poor genotype. In order to fully utilise the benefits of animal breeding, the producer must adjust feeding and slaughter patterns on the basis of genotype. The results suggest that the producer can benefit from flexible feeding technology. The flexible feeding technology segregates pigs into groups according to their weight, carcass leanness, genotype and sex and thereafter optimises feeding and slaughter decisions separately for these groups. Typically, such a technology provides incentives to feed piglets with protein-rich feed such that the genetic potential to produce leaner meat is fully utilised. When the pig approaches slaughter maturity, the share of protein-rich feed in the diet gradually decreases and the amount of energy-rich feed increases. Generally, the optimal slaughter weight is within the weight range that pays the highest price per kilogram of pig meat. The optimal feeding pattern and the optimal timing of slaughter depend on price ratios. Particularly, an increase in the price of pig meat provides incentives to increase the growth rates up to the pig's biological maximum by increasing the amount of energy in the feed. Price changes and changes in slaughter premium can also have large income effects. Key words: barley, carcass composition, dynamic programming, feeding, genotypes, lean, pig fattening, precision agriculture, productivity, slaughter weight, soybeans

Host Rock Classification (HRC) system for nuclear waste disposal in crystalline bedrock

A new rock mass classification scheme, the Host Rock Classification system (HRC-system) has been developed for evaluating the suitability of volumes of rock mass for the disposal of high-level nuclear waste in Precambrian crystalline bedrock. To support the development of the system, the requirements of host rock to be used for disposal have been studied in detail and the significance of the various rock mass properties have been examined. The HRC-system considers both the long-term safety of the repository and the constructability in the rock mass. The system is specific to the KBS-3V disposal concept and can be used only at sites that have been evaluated to be suitable at the site scale. By using the HRC-system, it is possible to identify potentially suitable volumes within the site at several different scales (repository, tunnel and canister scales). The selection of the classification parameters to be included in the HRC-system is based on an extensive study on the rock mass properties and their various influences on the long-term safety, the constructability and the layout and location of the repository. The parameters proposed for the classification at the repository scale include fracture zones, strength/stress ratio, hydraulic conductivity and the Groundwater Chemistry Index. The parameters proposed for the classification at the tunnel scale include hydraulic conductivity, Q´ and fracture zones and the parameters proposed for the classification at the canister scale include hydraulic conductivity, Q´, fracture zones, fracture width (aperture + filling) and fracture trace length. The parameter values will be used to determine the suitability classes for the volumes of rock to be classified. The HRC-system includes four suitability classes at the repository and tunnel scales and three suitability classes at the canister scale and the classification process is linked to several important decisions regarding the location and acceptability of many components of the repository at all three scales. The HRC-system is, thereby, one possible design tool that aids in locating the different repository components into volumes of host rock that are more suitable than others and that are considered to fulfil the fundamental requirements set for the repository host rock. The generic HRC-system, which is the main result of this work, is also adjusted to the site-specific properties of the Olkiluoto site in Finland and the classification procedure is demonstrated by a test classification using data from Olkiluoto. Keywords: host rock, classification, HRC-system, nuclear waste disposal, long-term safety, constructability, KBS-3V, crystalline bedrock, Olkiluoto

Rhizoctonia -Scots pine interactions: detection, impact on seedling performance and host defence gene response

Rhizoctonia spp. are ubiquitous soil inhabiting fungi that enter into pathogenic or symbiotic associations with plants. In general Rhizoctonia spp. are regarded as plant pathogenic fungi and many cause root rot and other plant diseases which results in considerable economic losses both in agriculture and forestry. Many Rhizoctonia strains enter into symbiotic mycorrhizal associations with orchids and some hypovirulent strains are promising biocontrol candidates in preventing host plant infection by pathogenic Rhizoctonia strains. This work focuses on uni- and binucleate Rhizoctonia (respectively UNR and BNR) strains belonging to the teleomorphic genus Ceratobasidium, but multinucleate Rhizoctonia (MNR) belonging to teleomorphic genus Thanatephorus and ectomycorrhizal fungal species, such as Suillus bovinus, were also included in DNA probe development work. Strain specific probes were developed to target rDNA ITS (internal transcribed spacer) sequences (ITS1, 5.8S and ITS2) and applied in Southern dot blot and liquid hybridization assays. Liquid hybridization was more sensitive and the size of the hybridized PCR products could be detected simultaneously, but the advantage in Southern hybridization was that sample DNA could be used without additional PCR amplification. The impacts of four Finnish BNR Ceratorhiza sp. strains 251, 266, 268 and 269 were investigated on Scot pine (Pinus sylvestris) seedling growth, and the infection biology and infection levels were microscopically examined following tryphan blue staining of infected roots. All BNR strains enhanced early seedling growth and affected the root architecture, while the infection levels remained low. The fungal infection was restricted to the outer cortical regions of long roots and typical monilioid cells detected with strain 268. The interactions of pathogenic UNR Ceratobasidium bicorne strain 1983-111/1N, and endophytic BNR Ceratorhiza sp. strain 268 were studied in single or dual inoculated Scots pine roots. The fungal infection levels and host defence-gene activity of nine transcripts [phenylalanine ammonia lyase (pal1), silbene synthase (STS), chalcone synthase (CHS), short-root specific peroxidase (Psyp1), antimicrobial peptide gene (Sp-AMP), rapidly elicited defence-related gene (PsACRE), germin-like protein (PsGER1), CuZn- superoxide dismutase (SOD), and dehydrin-like protein (dhy-like)] were measured from differentially treated and un-treated control roots by quantitative real time PCR (qRT-PCR). The infection level of pathogenic UNR was restricted in BNR- pre-inoculated Scots pine roots, while UNR was more competitive in simultaneous dual infection. The STS transcript was highly up-regulated in all treated roots, while CHS, pal1, and Psyp1 transcripts were more moderately activated. No significant activity of Sp-AMP, PsACRE, PsGER1, SOD, or dhy-like transcripts were detected compared to control roots. The integrated experiments presented, provide tools to assist in the future detection of these fungi in the environment and to understand the host infection biology and defence, and relationships between these interacting fungi in roots and soils. This study further confirms the complexity of the Rhizoctonia group both phylogenetically and in their infection biology and plant host specificity. The knowledge obtained could be applied in integrated forestry nursery management programmes.

DNA Packaging and Host Cell Lysis: Late Events in Bacteriophage PRD1 Infection

The object of this study is a tailless internal membrane-containing bacteriophage PRD1. It has a dsDNA genome with covalently bound terminal proteins required for replication. The uniqueness of the structure makes this phage a desirable object of research. PRD1 has been studied for some 30 years during which time a lot of information has accumulated on its structure and life-cycle. The two least characterised steps of the PRD1 life-cycle, the genome packaging and virus release are investigated here. PRD1 shares the main principles of virion assembly (DNA packaging in particular) and host cell lysis with other dsDNA bacteriophages. However, this phage has some fascinating individual peculiarities, such as DNA packaging into a membrane vesicle inside the capsid, absence of apparent portal protein, holin inhibitor and procapsid expansion. In the course of this study we have identified the components of the DNA packaging vertex of the capsid, and determined the function of protein P6 in packaging. We managed to purify the procapsids for an in vitro packaging system, optimise the reaction and significantly increase its efficiency. We developed a new method to determine DNA translocation and were able to quantify the efficiency and the rate of packaging. A model for PRD1 DNA packaging was also proposed. Another part of this study covers the lysis of the host cell. As other dsDNA bacteriophages PRD1 has been proposed to utilise a two-component lysis system. The existence of this lysis system in PRD1 has been proven by experiments using recombinant proteins and the multi-step nature of the lysis process has been established.

Outer Membrane Protease/Adhesin PgtE of Salmonella enterica: Role in Salmonella-Host Interaction

Salmonella enterica serovar Typhimurium is a common cause of gastroenteritis in humans and, occasionally, also causes systemic infection. During systemic infection an important characteristic of Salmonella is its ability to survive and replicate within macrophages. The outer membrane protease PgtE of S. enterica is a member of the omptin family of outer membrane aspartate proteases, which are beta-barrel proteins with five surface-exposed loops. The main goals of this study were to characterize biological substrates and pathogenesis-associated functions of PgtE and to determine the conditions where PgtE is fully active. In this study we found that PgtE requires rough lipopolysaccharide (LPS) to be functional but is sterically inhibited by the long O-antigen side chain in smooth LPS. Salmonella isolates normally are smooth with a long oligosaccharide O-antigen, and PgtE remains functionally cryptic in wild-type Salmonella cultivated in vitro. Interestingly, our results showed that due to increased expression of PgtE and to reduced length of the LPS O-antigen chains, the wild-type Salmonella expresses highly functional PgtE when isolated from mouse macrophage-like J774A.1 cells. Salmonella is thought to be continuously released from macrophages to infect new ones, and our results suggest that PgtE is functional during these transient extracellular growth phases. Six novel host protein substrates were identified for PgtE in this work. PgtE was previously known to activate human plasminogen (Plg) to plasmin, a broad-spectrum serine protease, and in this study PgtE was shown to interfere with the Plg system by inactivating the main inhibitor of plasmin, alpha2-antiplasmin. PgtE also interferes with another important proteolytic system of mammals by activating pro-matrix metalloproteinase-9 to an active gelatinase. PgtE also directly degrades gelatin, a component of extracellular matrices. PgtE also increases bacterial resistance against complement-mediated killing in human serum and enhances survival of Salmonella within murine macrophages as well as in the liver and spleen of intraperitoneally infected mice. Taken together, the results in this study suggest that PgtE is a virulence factor of Salmonella that has adapted to interfere with host proteolytic systems and to modify extracellular matrix; these features likely assist the migration of Salmonella during systemic salmonellosis.