Molecular Biomonitoring during Rhizoremediation of Oil-Contaminated Soil

Rhizoremediation is the use of microbial populations present in the rhizosphere of plants for environmental cleanup. The idea of this work was that bacteria living in the rhizosphere of a nitrogen-fixing leguminous plant, goat's rue (Galega orientalis), could take part in the degradation of harmful monoaromatic hydrocarbons, such as benzene, toluene and xylene (BTEX), from oil-contaminated soils. In addition to chemical (e.g. pollutant concentration) and physical (e.g. soil structure) information, the knowledge of biological aspects (e.g. bacteria and their catabolic genes) is essential when developing the rhizoremediation into controlled and effective bioremediation practice. Therefore, the need for reliable biomonitoring methods is obvious. The main aims of this thesis were to evaluate the symbiotic G. orientalis - Rhizobium galegae system for rhizoremediation of oil-contaminated soils, to develop molecular methods for biomonitoring, and to apply these methods for studying the microbiology of rhizoremediation. In vitro, Galega plants and rhizobia remained viable in m-toluate concentrations up to 3000 mg/l. Plant growth and nodulation were inhibited in 500 mg/l m-toluate, but were restored when plants were transferred to clean medium. In the greenhouse, Galega showed good growth, nodulation and nitrogen fixation, and developed a strong rhizosphere in soils contaminated with oil or spiked with 2000 mg/l m-toluate. The high aromatic tolerance of R. galegae and the viability of Galega plants in oil-polluted soils proved this legume system to be a promising method for the rhizoremediation of oil-contaminated soils. Molecular biomonitoring methods were designed and/or developed further for bacteria and their degradation genes. A combination of genomic fingerprinting ((GTG)5-PCR), taxonomic ribotyping of 16S rRNA genes and partial 16S rRNA gene sequencing were chosen for molecular grouping of culturable, heterogeneous rhizosphere bacteria. PCR primers specific for the xylE gene were designed for TOL plasmid detection. Amplified enzyme-coding DNA restriction analysis (AEDRA) with AluI was used to profile both TOL plasmids (xylE primers) and, in general, aromatics-degrading plasmids (C230 primers). The sensitivity of the direct monitoring of TOL plasmids in soil was enhanced by nested C23O-xylE-PCR. Rhizosphere bacteria were isolated from the greenhouse and field lysimeter experiments. High genetic diversity was observed among the 50 isolated, m-toluate tolerating rhizosphere bacteria in the form of five major lineages of the Bacteria domain. Gram-positive Rhodococcus, Bacillus and Arthrobacter and gram-negative Pseudomonas were the most abundant genera. The inoculum Pseudomonas putida PaW85/pWW0 was not found in the rhizosphere samples. Even if there were no ecological niches available for the bioaugmentation bacterium itself, its conjugative catabolic plasmid might have had some additional value for other bacterial species and thus, for rhizoremediation. Only 10 to 20% of the isolated, m-toluate tolerating bacterial strains were also able to degrade m-toluate. TOL plasmids were a major group of catabolic plasmids among these bacteria. The ability to degrade m-toluate by using enzymes encoded by a TOL plasmid was detected only in species of the genus Pseudomonas, and the best m-toluate degraders were these Pseudomonas species. Strain-specific differences in degradation abilities were found for P.oryzihabitans and P. migulae: some of these strains harbored a TOL plasmid - a new finding observed in this work, indicating putative horizontal plasmid transfer in the rhizosphere. One P. oryzihabitans strain harbored the pWW0 plasmid that had probably conjugated from the bioaugmentation Pseudomonas. Some P. migulae and P. oryzihabitans strains seemed to harbor both the pWW0- and the pDK1-type TOL plasmid. Alternatively, they might have harbored a TOL plasmid with both the pWW0- and the pDK1-type xylE gene. The breakdown of m-toluate by gram-negative bacteria was not restricted to the TOL pathway. Also some gram-positive Rhodococcus erythropolis and Arthrobacter aurescens strains were able to degrade m-toluate in the absence of a TOL plasmid. Three aspects of the rhizosphere effect of G. orientalis were manifested in oil-contaminated soil in the field: 1) G. orientalis and Pseudomonas bioaugmentation increased the amount of rhizosphere bacteria. G. orientalis especially together with Pseudomonas bioaugmentation increased the numbers of m-toluate utilizing and catechol positive bacteria indicating an increase in degradation potential. 2) Also the bacterial diversity, when measured as the amount of ribotypes, was increased in the Galega rhizosphere with or without Pseudomonas bioaugmentation. However, the diversity of m-toluate utilizing bacteria did not significantly increase. At the community level, by using the 16S rRNA gene PCR-DGGE method, the highest diversity of species was also observed in vegetated soils compared with non-vegetated soils. Diversified communities may best guarantee the overall success in rhizoremediation by offering various genetic machineries for catabolic processes. 3) At the end of the experiment, no TOL plasmid could be detected by direct DNA analysis in soil treated with both G. orientalis and Pseudomonas. The detection limit for TOL plasmids was encountered indicating decreased amount of degradation plasmids and thus, the success of rhizoremediation. The use of G. orientalis for rhizoremediation is unique. In this thesis new information was obtained about the rhizosphere effect of Galega orientalis in BTEX contaminated soils. The molecular biomonitoring methods can be applied for several purposes within environmental biotechnology, such as for evaluating the intrinsic biodegradation potential, monitoring the enhanced bioremediation, and estimating the success of bioremediation. Environmental protection by using nature's own resources and thus, acting according to the principle of sustainable development, would be both economically and environmentally beneficial for society. Keywords: molecular biomonitoring, genetic fingerprinting, soil bacteria, bacterial diversity, TOL plasmid, catabolic genes, horizontal gene transfer, rhizoremediation, rhizosphere effect, Galega orientalis, aerobic biodegradation, petroleum hydrocarbons, BTEX

Agroforestry systems for sustainable livelihoods and improved land management in the East Usambara Mountains, Tanzania

Forest destruction for agriculture continues to be a major threat to the rich biological diversity in the East Usambara Mountains in the north-eastern corner of Tanzania. The highest ratio of endemic plant and animal species found on 100 km2 anywhere in the world is depending on the remaining natural forests. Forests are vitally important for the local population in many different ways, and nationally they are an important source of water and hydroelectricity. The soils, of low fertility and mostly acidic Ferrasols, mainly have the nutrients in the topsoil. After clear-cutting, the soils soon become poor when the topsoil is eroded. High-value cardamom is nowadays unsustainably cultivated in the natural forests of the East Usambaras. The general aim was to study the possibilities to develop new profitable and sustainable agroforestry systems for the benefit of the local people that could contribute to relieving the pressure on the remaining natural forests in the East Usambara Mountains. Results from a spice crop agroforestry trial, established in cooperation with a local farmer, showed a clear advantage of intercropping cardamom (Elettaria cardamomum) and black pepper (Piper nigrum) with trees, especially with Grevillea robusta. The nitrogen fixing tree species Gliricidia sepium also improved the nitrogen and organic matter content of the soil over levels found in the natural forest. With improved agroforestry methods for spice production the households generated as much as13 times the net income obtained with traditional forest cultivation practices. There are thus sustainable and profitable ways to cultivate spices as cash crops in well-managed homegardens. However, the farmers need stable markets, access to credit and comprehensive extension services. The soil fertility depletion should be reversed with organic manure application and an enabling policy environment for the smallholder-farming sector. Strong farmers organisations and equal rights to resources and decision-making are needed. Organic spices have an increasing demand, and their export would be profitable for these farmers. What is, however, most needed for a change is a political will of a government that understands the importance of agricultural and forestry development for poverty reduction.

The role of phosphorus as a regulator of bloom-forming diazotrophic cyanobacteria in the Baltic Sea

Eutrophication favours harmful algal blooms worldwide. The blooms cause toxic outbreaks and deteriorated recreational and aesthetic values, causing both economic loss and illness or death of humans and animals. The Baltic Sea is the world s only large brackish water habitat with recurrent blooms of toxic cyanobacteria capable of biological fixation of atmospheric nitrogen gas. Phosphorus is assumed to be the main limiting factor, along with temperature and light, for the growth of these cyanobacteria. This thesis evaluated the role of phosphorus nutrition as a regulating factor for the occurrence of nitrogen-fixing cyanobacteria blooms in the Baltic Sea, utilising experimental laboratory and field studies and surveys on varying spatial scales. Cellular phosphorus sources were found to be able to support substantial growth of the two main bloom forming species Aphanizomenon sp. and Nodularia spumigena. However, N. spumigena growth seemed independent of phosphorus source, whereas, Aphanizomenon sp. grew best in a phosphate enriched environment. Apparent discrepancies with field observations and experiments are explained by the typical seasonal temperature dependent development of Aphanizomenon sp. and N. spumigena biomass allowing the two species to store ambient pre-bloom excess phosphorus in different ways. Field experiments revealed natural cyanobacteria bloom communities to be predominantly phosphorus deficient during blooms. Phosphate additions were found to increase the accumulation of phosphorus relatively most in the planktonic size fraction dominated by the nitrogen-fixing cyanobacteria. Aphanizomenon sp. responded to phosphate additions whereas the phosphorus nutritive status of N. spumigena seemed independent of phosphate addition. The seasonal development of phosphorus deficiency is different for the two species with N. spumigena showing indications of phosphorus deficiency during a longer time period in the open sea. Coastal upwelling introduces phosphorus to the surface layer during nutrient deficient conditions in summer. The species-specific ability of Aphanizomenon sp. and N. spumigena to utilise phosphate enrichment of the surface layer caused by coastal upwelling was clarified. Typical bloom time vertical distributions of biomass maxima were found to render N. spumigena more susceptible to advection by surface currents caused by coastal upwellings. Aphanizomenon sp. populations residing in the seasonal thermocline were observed to be able to utilise the phosphate enrichment and a bloom was produced with a two to three week time lag subsequent to the relaxation of upwelling. Consistent high concentrations of dissolved inorganic phosphorus, caused by persistent internal loading of phosphorus, was found to be the main source of phosphorus for large-scale pelagic blooms. External loads were estimated to contribute with only a fraction of available phosphorus for open sea blooms. Remineralization of organic forms of phosphorus along with vertical mixing to the permanent halocline during winter set the level of available phosphorus for the next growth season. Events such as upwelling are important in replenishing phosphate concentrations during the nutrient deplete growth season. Autecological characteristics of the two main bloom forming species favour Aphanizomenon sp. populations in utilising the abundant excess phosphate concentrations and phosphate pulses mediated through upwelling. Whilst, N. spumigena displays predominant phosphorus limited growth mode and relies on more scarce cellular phosphorus stores and presumably dissolved organic phosphorus compounds for growth. The Baltic Sea is hypothesised to be in an inhibited state of recovery due to the extensive historical external nutrient loading, extensive internal phosphorus loading and the substantial nitrogen load caused by cyanobacteria nitrogen fixation. This state of the sea is characterised as a vicious circle .

Hiekkatympösen (Hebeloma cylindrosporum) L-aminohappo-oksidaasi -geenin eristäminen

Pohjoisella havumetsävyöhykkeellä typpi on usein kasvien kasvua rajoittava tekijä. Metsämaan typpivarannot koostuvat pääasiassa orgaaniseen ainekseen sitoutuneista typpiyhdisteistä, erityisesti aminohapoista. Ektomykorritsasienet osallistuvat metsämaassa tapahtuvaan typenkiertoon hajottamalla orgaanisia typpiyhdisteitä ja kuljettamalla niitä kasvien käytettäväksi. Sienisolun sisällä tapahtuvasta aminohappojen mineralisaatiosta tiedetään toistaiseksi melko vähän. Aminohappo-oksidaasit katalysoivat aminohappojen mineralisaatiota. Eräissä ektomykorritsaa muodostavien kantasienten suvuissa on osoitettu L-aminohappo-oksidaaseja (LAO). Toistaiseksi LAO-geeniä ei tunneta kantasienistä. Työssä kuvattiin ensimmäistä kertaa LAO-geeni kantasienistä. Hiekkatympösen LAO1- geenin cDNA:n 5´ ja 3´ päiden emäsjärjestykset määritettiin RACE-PCR -menetelmällä, josta saatujen sekvenssien perusteella suunniteltiin alukkeet koko geenin cDNA:n ja genomisen DNA:n monistamiseksi. Genomisen DNA ja cDNA -sekvenssien perusteella määritettiin hiekkatympösen LAO1-geenin rakenne. Hiekkatympösen LAO1-geeni koostuu viidestä eksonista ja neljästä intronista. Hiekkatympösen LAO1-geenin yläpuoliselta alueelta löydettiin typpimetabolian säätelyyn osallistuvan proteiinin sitoutumiskohta. LAO1-geeniä edeltävä geenin osittainen genominen DNA-sekvenssi määritettiin. Kangaslohisienen genomissa LAO1-geeniä edeltävä geeni oli ennustettu pyruvaattidekarboksylaasiksi. Lisäksi työssä määritettiin hiekkatympösen toisen LAOhomologin cDNA:n osittainen emäsjärjestys. Työssä tunnistettiin myös toisen kantasienen, kangaslohisienen, LAO-geeni. LAO-geeniksi tunnistettu kangaslohisienen geenimalli oli aiemmin ennustettu NCBI:n tietokannassa toiminnaltaan tuntemattomaksi proteiiniksi. Proteiinien sukupuun perusteella hiekkatympösen ja kangaslohisienen LAO:n kantamuoto on kahdentunut. Työstä saatu tutkimustulos tuo täysin uutta tietoa molekyylibiologian tasolla ektomykorritsasienten aminohappojen katabolisista reaktioista. Aminohappojen mineralisaation seurauksen muodostuneet ammoniumionit saattavat olla merkittävä typen lähde myös maan muille mikrobeille ja kasveille. On mahdollista, että ektomykorritsasienten LAO-entsyymi on yksi merkittävä tekijä metsämaan typenkierrossa.

Short-term plant-decomposer feedbacks in grassland plants

Plant species differ in their effects on ecosystem productivity and it is recognised that these effects are partly due to plant species-specific influences on soil processes. Until recently, however, not much attention was given to the potential role played by soil biota in these species-specific effects. While soil decomposers are responsible for governing the availability of nutrients for plant production, they simultaneously depend on the amount of carbon provided by plants. Litter and rhizodeposition constitute the two basal resources that plants provide to soil decomposer food webs. While it has been shown that both of these can have effects on soil decomposer communities that differ among plant species, the putative significance of these effects for plant nitrogen (N) acquisition is currently understudied. My PhD work aimed at clarifying whether the species-specific influences of three temperate grassland plants on the soil microfood-web, through rhizodeposition and litter, can feed back to plant N uptake. The methods and approach used (15N labelling of plant litter in microcosm experiments) revealed to be an effective combination of tools in studying these feedbacks. Plant effects on soil organisms were shown to differ significantly between plant species and the effects could be followed across several trophic levels. The labelling of litter further permitted the evaluation of plant acquisition of N derived from soil organic matter. The results show that the structure of the soil microfood-web can have a significant role in plant N acquisition when the structure is experimentally manipulated, such as when comparing systems consisting of microbes to those consisting of microbes and their grazers. However, despite this, the results indicate that differences in N uptake from soil organic matter between different plant species are not related to the effects these species exert on the structure of the soil microfood-web. Rather, these differences in N uptake seem to be determined by other species-specific traits of live plants and their litter. My results thus indicate that different resources provided by different plant species may not induce species-specific decomposer feedbacks on plant N uptake from soil organic matter. This further suggests that the species-specific plant effects on soil decomposer communities may not, at least in the short term, have significant consequences on plant production.

Lebanese Plants and Plant-Derived Compounds Against Colon Cancer

Colorectal cancer (CRC) is a major health concern and demands long-term efforts in developing strategies for screening and prevention. CRC has become a preventable disease as a consequence of a better understanding of colorectal carcinogenesis. However, current therapy is unsatisfactory and necessitates the exploration of other approaches for the prevention and treatment of cancer. Plant based products have been recognized as preventive with regard to the development of colon cancer. Therefore, the potential chemopreventive use and mechanism of action of Lebanese natural product were evaluated. Towards this aim the antitumor activity of Onopordum cynarocephalum and Centaurea ainetensis has been studied using in vitro and in vivo models. In vitro, both crude extracts were non cytotoxic to normal intestinal cells and inhibited the proliferation of colon cancer cells in a dose-dependent manner. In vivo, both crude extracts reduced the number of tumors by an average of 65% at weeks 20 (adenomas stage) and 30 (adenocarcinomas stage). The activity of the C. ainetensis extract was attributed to Salograviolide A, a guaianolide-type sesquiterpene lactone, which was isolated and identified through bio-guided fractionation. The mechanism of action of thymoquinone (TQ), the active component of Nigella sativa, was established in colon cancer cells using in vitro models. By the use of N-acetyl cysteine, a radical scavenger, the direct involvement of reactive oxygen species in TQ-induced apoptotic cells was established. The analytical detection of TQ from spiked serum and its protein binding were evaluated. The average recovery of TQ from spiked serum subjected to several extraction procedures was 2.5% proving the inability of conventional methods to analyze TQ from serum. This has been explained by the extensive binding (>98%) of TQ to serum and major serum components such as bovine serum albumin (BSA) and alpha-1-acid glycoprotein (AGP). Using mass spectrometry analysis, TQ was confirmed to bind covalently to the free cysteine in position 34 and 147 of the amino acid sequence of BSA and AGP, respectively. The results of this work put at the disposal for future development new plants with anti-cancer activities and enhance the understanding of the pharmaceutical properties of TQ, a prerequisite for its future clinical development.

Tropical dryland agroforestry on clay soils: : Analysis of systems based on Acacia senegal in the Blue Nile region, Sudan

Acacia senegal, the gum arabic producing tree, is the most important component in traditional dryland agroforestry systems in the Blue Nile region, Sudan. The aim of the present study was to provide new knowledge on the potential use of A. senegal in dryland agroforestry systems on clay soils, as well as information on tree/crop interaction, and on silvicultural and management tools, with consideration on system productivity, nutrient cycling and sustainability. Moreover, the aim was also to clarify the intra-specific variation in the performance of A. senegal and, specifically, the adaptation of trees of different origin to the clay soils of the Blue Nile region. In agroforestry systems established at the beginning of the study, tree and crop growth, water use, gum and crop yields, nutrient cycling and system performance were investigated for a period of four years (1999 to 2002). Trees were grown at 5 x 5 m and 10 x 10 m spacing alone or in mixture with sorghum or sesame; crops were also grown in sole culture. The symbiotic biological N2 fixation by A. senegal was estimated using the 15N natural abundance (δ15N) procedure in eight provenances collected from different environments and soil types of the gum arabic belt and grown in clay soil in the Blue Nile region. Balanites aegyptiaca (a non-legume) was used as a non-N-fixing reference tree species, so as to allow 15N-based estimates of the proportion of the nitrogen in trees derived from the atmosphere. In the planted acacia trees, measurements were made on shoot growth, water-use efficiency (as assessed by the δ13C method) and (starting from the third year) gum production. Carbon isotope ratios were obtained from the leaves and branch wood samples. The agroforestry system design caused no statistically significant variation in water use, but the variation was highly significant between years, and the highest water use occurred in the years with high rainfall. No statistically significant differences were found in sorghum or sesame yields when intercropping and sole crop systems were compared (yield averages were 1.54 and 1.54 ha-1 for sorghum and 0.36 and 0.42 t ha-1 for sesame in the intercropped and mono-crop plots, respectively). Thus, at an early stage of agroforestry system management, A. senegal had no detrimental effect on crop yield, but the pattern of resource capture by trees and crops may change as the system matures. Intercropping resulted in taller trees and larger basal and crown diameters as compared to the development of sole trees. It also resulted in a higher land equivalent ratio. When gum yields were analysed it was found that a significant positive relationship existed between the second gum picking and the total gum yield. The second gum picking seems to be a decisive factor in gum production and could be used as an indicator for the total gum yield in a particular year. In trees, the concentrations of N and P were higher in leaves and roots, whereas the levels of K were higher in stems, branches and roots. Soil organic matter, N, P and K contents were highest in the upper soil stratum. There was some indication that the P content slightly increased in the topsoil as the agroforestry plantations aged. At a stocking of 400 trees ha-1 (5 x 5 m spacing), A. senegal accumulated in the biomass a total of 18, 1.21, 7.8 and 972 kg ha-1of N, P, K and OC, respectively. Trees contributed ca. 217 and 1500 kg ha-1 of K and OC, respectively, to the top 25-cm of soil over the first four years of intercropping. Acacia provenances of clay plain origin showed considerable variation in seed weight. They also had the lowest average seed weight as compared to the sandy soil (western) provenances. At the experimental site in the clay soil region, the clay provenances were distinctly superior to the sand provenances in all traits studied but especially in basal diameter and crown width, thus reflecting their adaptation to the environment. Values of δ13C, indicating water use efficiency, were higher in the sand soil group as compared to the clay one, both in leaves and in branch wood. This suggests that the sand provenances (with an average value of -28.07 ) displayed conservative water use and high drought tolerance. Of the clay provenances, the local one (Bout) displayed a highly negative (-29.31 ) value, which indicates less conservative water use that resulted in high productivity at this particular clay-soil site. Water use thus appeared to correspond to the environmental conditions prevailing at the original locations for these provenances. Results suggest that A. senegal provenances from the clay part of the gum belt are adapted for a faster growth rate and higher biomass and gum productivity as compared to provenances from sand regions. A strong negative relationship was found between the per-tree gum yield and water use efficiency, as indicated by δ13C. The differences in water use and gum production were greater among provenance groups than within them, suggesting that selection among rather than within provenances would result in distinct genetic gain in gum yield. The relative δ15N values ( ) were higher in B. aegyptiaca than in the N2-fixing acacia provenances. The amount of Ndfa increased significantly with age in all provenances, indicating that A. senegal is a potentially efficient nitrogen fixer and has an important role in t agroforestry development. The total above-ground contribution of fixed N to foliage growth in 4-year-old A. senegal trees was highest in the Rahad sand-soil provenance (46.7 kg N ha-1) and lowest in the Mazmoom clay-soil provenance (28.7 kg N ha-1). This study represents the first use of the δ15N method for estimating the N input by A. senegal in the gum belt of Sudan. Key words: Acacia senegal, agroforestry, clay plain, δ13C, δ15N, gum arabic, nutrient cycling, Ndfa, Sorghum bicolor, Sesamum indicum

UV-induced NOy emissions in gas-exchange chambers enclosing Scots pine shoots: an analysis on their origin and significance

It is essential to have a thorough understanding of the sources and sinks of oxidized nitrogen (NOy) in the atmosphere, since it has a strong influence on the tropospheric chemistry and the eutrophication of ecosystems. One unknown component in the balance of gaseous oxidized nitrogen is vegetation. Plants absorb nitrogenous species from the air via the stomata, but it is not clear whether plants can also emit them at low ambient concentrations. The possible emissions are small and difficult to measure. The aim of this thesis was to analyse an observation made in southern Finland at the SMEAR II station: solar ultraviolet radiation (UV) induced NOy emissions in chambers measuring the gas exchange of Scots pine (Pinus sylvestris L.) shoots. Both measuring and modelling approaches were used in the study. The measurements were performed under noncontrolled field conditions at low ambient NOy concentrations. The chamber blank i.e. artefact NOy emissions from the chamber walls, was dependent on the UV irradiance and increased with time after renewing the Teflon film on chamber surfaces. The contribution of each pine shoot to the total NOy emissions in the chambers was determined by testing whether the emissions decrease when the shoots are removed from their chambers. Emissions did decrease, but only when the chamber interior was exposed to UV radiation. It was concluded that also the pine shoots emit NOy. The possible effects of transpiration on the chamber blank are discussed in the summary part of the thesis, based on previously unpublished data. The possible processes underlying the UV-induced NOy emissions were reviewed. Surface reactions were more likely than metabolic processes. Photolysis of nitrate deposited on the needles may have generated the NOy emissions; the measurements supported this hypothesis. In that case, the emissions apparently would consist mainly of nitrogen dioxide (NO2), nitric oxide (NO) and nitrous acid (HONO). Within studies on NOy exchange of plants, the gases most frequently studied are NO2 and NO (=NOx). In the present work, the implications of the emissions for the NOx exchange of pine were analysed with a model including both NOy emissions and NOy absorption. The model suggested that if the emissions exist, pines can act as an NOx source rather than a sink, even under relatively high ambient concentrations.

Potato virus A as a heterologous protein expression tool in plants

Viruksien käyttö tuotekehityksen ja tutkimuksen vaatimien proteiinien tuottamiseen, syötävien rokotteiden kehittämiseen ja geeniterapiaan edustavat kasvavia biotekniikan sovellusalueita. Perunan A-virus (PVA) kuuluu potyviruksiin, joiden proteiinit tuotetaan aluksi yhtenä suurena molekyylinä, joka pilkotaan yksittäisiksi proteiineiksi viruksen itsensä tuottamilla entsyymeillä. Siten virusgenomiin lisätty vieras geeni käännetään proteiiniksi virusproteiinien mukana. Lopputuloksena kaikkia proteiineja tuotetaan kasvisoluissa samansuuruinen määrä. Lisäksi, viruksen proteiinikuoren koontimekanismi sallii perintöaineksen merkittävän lisäyksen ilman että viruksen tartutuskyky merkittävästi heikkenee. Koska virus monistuu ja leviää koko kasviin, jo melko pieni määrä kasveja riittää huomattavan proteiinimäärän tuottamiseen esimerkiksi säännösten mukaisessa kasvihuoneessa. Tämän työn tarkoituksena oli muuntaa PVA:n genomia siten, että virus soveltuisi yhden vieraan proteiinin tai useiden erilaisten proteiinien samanaikaiseen tuottamiseen kasveissa. Aluksi kokeiltiin viruksen replikaasia ja kuoriproteiinia koodaavien genomialueiden välistä kohtaa ja ihmisestä peräisi olevaa geeniä, joka tuotti S-COMT-entsyymiä (katekoli-O-metyylitransferaasi). Sen aktiivisuuden rajoittaminen auttaa Parkinsonintaudin hoidossa. Kasvissa tuotettua S-COMT:ia voitaisiin käyttää lääkekehityksessä estolääkkeiden testaukseen. Kahden viikon kuluttua tartutuksesta tupakan lehdissä oli entsymaattisesti aktiivista S-COMT:ia n. 1 % lehden liukoisista proteiineista. PVA:n P1-proteiinia koodaavalta alueelta oli paikannettu kohta, johon ehkä voitaisiin siirtää vieras geeni. Asia varmistettiin siirtämällä tähän kohtaan meduusan geeni, joka tuottaa UV-valossa vihreänä fluoresoivaa proteiinia (GFP). GFP-geeniä kantava PVA levisi kasvissa ja lisääntyi n. 30-50 %:iin viruksen normaalista pitoisuudesta. Koko kasvi fluoresoi vihreänä UV-valossa. Vieras geeni voidaan sijoittaa myös potyviruksen P1- ja HCpro-proteiineja koodaavien alueiden väliin. Samaan PVA-genomiin siirrettiin kolme geeniä, yksi kuhunkin kolmesta kloonauskohdasta: GFP-geeni P1:n sisälle, merivuokon lusiferaasigeeni P1/HCpro-kohtaan ja bakteerin beta-glukuronidaasigeeni (GUS) replikaasi/kuoriproteiini-kohtaan. Virusgenomin ja itse viruksen pituudet kasvoivat 38 %, mutta virus säilytti tartutuskykynsä. Se levisi kasveissa saavuttaen n. 15 % viruksen normaalista pitoisuudesta. Kaikki kolme vierasta proteiinia esiintyivät lehdissä aktiivisina.

Microbial quality of hemp (Cannabis sativa L.) and flax (Linum usitatissimum L.) from plants to thermal insulation

Flax and hemp have traditionally been used mainly for textiles, but recently interest has also been focused on non-textile applications. Microbial quality throughout the whole processing chain of bast fibres has not previously been studied. This study concentrates on the microbial quality and possible microbial risks in the production chain of hemp and flax fibres and fibrous thermal insulations. In order to be able to utilize hemp and flax fibres, the bast fibres must be separated from the rest of the plant. Non-cellulosic components can be removed with various pretreatment processes, which are associated with a certain risk of microbial contamination. In this study enzymatic retting and steam explosion (STEX) were examined as pretreatment processes. On the basis of the results obtained in this study, the microbial contents on stalks of both plants studied increased at the end of the growing season and during the winter. However, by processing and mechanical separation it is possible to produce fibres containing less moulds and bacteria than the whole stem. Enzymatic treatment encouraged the growth of moulds in fibres. Steam explosion reduced the amount of moulds in fibres. Dry thermal treatment used in this study did not markedly reduce the amount of microbes. In this project an emission measurement chamber was developed which was suitable for measurements of emissions from both mat type and loose fill type insulations, and capable of interdisciplinary sampling. In this study, the highest amounts of fungal emissions were in the range of 10^3 10^5 cfu/m^3 from the flax and hemp insulations at 90% RH of air. The fungal emissions from stone wool, glass wool and recycled paper insulations were below 10^2 cfu/m^3 even at 90% RH. Equally low values were obtained from bast fibrous materials in lower humidities (at 30% and 80% RH of air). After drying of moulded insulations at 30% RH, the amounts of emitted moulds were in all cases higher compared to the emissions at 90% RH before drying. The most common fungi in bast fibres were Penicillium and Rhizopus. The widest variety of different fungi was in the untreated hemp and linseed fibres and in the commercial loose-fill flax insulation. Penicillium, Rhizopus and Paecilomyces were the most tolerant to steam explosion. According to the literature, the most common fungi in building materials and indoor air are Penicillium, Aspergillus and Cladosporium, which were all found in some of the bast fibre materials in this study. As organic materials, hemp and flax fibres contain high levels of nutrients for microbial growth. The amount of microbes can be controlled and somewhat decreased by the processing methods presented.

The Significance of Wild Plants in the Evolutionary Ecology of Three Major Viruses Infecting Cultivated Sweetpotato in Uganda

The studies presented in this thesis contribute to the understanding of evolutionary ecology of three major viruses threatening cultivated sweetpotato (Ipomoea batatas Lam) in East Africa: Sweet potato feathery mottle virus (SPFMV; genus Potyvirus; Potyviridae), Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus; Closteroviridae) and Sweet potato mild mottle virus (SPMMV; genus Ipomovirus; Potyviridae). The viruses were serologically detected and the positive results confirmed by RT-PCR and sequencing. SPFMV was detected in 24 wild plant species of family Convolvulacea (genera Ipomoea, Lepistemon and Hewittia), of which 19 species were new natural hosts for SPFMV. SPMMV and SPCSV were detected in wild plants belonging to 21 and 12 species (genera Ipomoea, Lepistemon and Hewittia), respectively, all of which were previously unknown to be natural hosts of these viruses. SPFMV was the most abundant virus being detected in 17% of the plants, while SPMMV and SPCSV were detected in 9.8% and 5.4% of the assessed plants, respectively. Wild plants in Uganda were infected with the East African (EA), common (C), and the ordinary (O) strains, or co-infected with the EA and the C strain of SPFMV. The viruses and virus-like diseases were more frequent in the eastern agro-ecological zone than the western and central zones, which contrasted with known incidences of these viruses in sweetpotato crops, except for northern zone where incidences were lowest in wild plants as in sweetpotato. The NIb/CP junction in SPMMV was determined experimentally which facilitated CP-based phylogenetic and evolutionary analyses of SPMMV. Isolates of all the three viruses from wild plants were genetically similar to those found in cultivated sweetpotatoes in East Africa. There was no evidence of host-driven population genetic structures suggesting frequent transmission of these viruses between their wild and cultivated hosts. The p22 RNA silencing suppressor-encoding sequence was absent in a few SPCSV isolates, but regardless of this, SPCSV isolates incited sweet potato virus disease (SPVD) in sweetpotato plants co-infected with SPFMV, indicating that p22 is redundant for synergism between SCSV and SPFMV. Molecular evolutionary analysis revealed that isolates of strain EA of SPFMV that is largely restricted geographically in East Africa experience frequent recombination in comparison to isolates of strain C that is globally distributed. Moreover, non-homologous recombination events between strains EA and C were rare, despite frequent co-infections of these strains in wild plants, suggesting purifying selection against non-homologous recombinants between these strains or that such recombinants are mostly not infectious. Recombination was detected also in the 5 - and 3 -proximal regions of the SPMMV genome providing the first evidence of recombination in genus Ipomovirus, but no recombination events were detected in the characterized genomic regions of SPCSV. Strong purifying selection was implicated on evolution of majority of amino acids of the proteins encoded by the analyzed genomic regions of SPFMV, SPMMV and SPCSV. However, positive selection was predicted on 17 amino acids distributed over the whole the coat protein (CP) in the globally distributed strain C, as compared to only 4 amino acids in the multifunctional CP N-terminus (CP-NT) of strain EA largely restricted geographically to East Africa. A few amino acid sites in the N-terminus of SPMMV P1, the p7 protein and RNA silencing suppressor proteins p22 and RNase3 of SPCSV were also submitted to positive selection. Positively selected amino acids may constitute ligand-binding domains that determine interactions with plant host and/or insect vector factors. The P1 proteinase of SPMMV (genus Ipomovirus) seems to respond to needs of adaptation, which was not observed with the helper component proteinase (HC-Pro) of SPMMV, although the HC-Pro is responsible for many important molecular interactions in genus Potyvirus. Because the centre of origin of cultivated sweetpotato is in the Americas from where the crop was dispersed to other continents in recent history (except for the Australasia and South Pacific region), it would be expected that identical viruses and their strains occur worldwide, presuming virus dispersal with the host. Apparently, this seems not to be the case with SPMMV, the strain EA of SPFMV and the strain EA of SPCSV that are largely geographically confined in East Africa where they are predominant and occur both in natural and agro-ecosystems. The geographical distribution of plant viruses is constrained more by virus-vector relations than by virus-host interactions, which in accordance of the wide range of natural host species and the geographical confinement to East Africa suggest that these viruses existed in East African wild plants before the introduction of sweetpotato. Subsequently, these studies provide compelling evidence that East Africa constitutes a cradle of SPFMV strain EA, SPCSV strain EA, and SPMMV. Therefore, sweet potato virus disease (SPVD) in East Africa may be one of the examples of damaging virus diseases resulting from exchange of viruses between introduced crops and indigenous wild plant species. Keywords: Convolvulaceae, East Africa, epidemiology, evolution, genetic variability, Ipomoea, recombination, SPCSV, SPFMV, SPMMV, selection pressure, sweetpotato, wild plant species Author s Address: Arthur K. Tugume, Department of Agricultural Sciences, Faculty of Agriculture and Forestry, University of Helsinki, Latokartanonkaari 7, P.O Box 27, FIN-00014, Helsinki, Finland. Email: tugume.arthur@helsinki.fi Author s Present Address: Arthur K. Tugume, Department of Botany, Faculty of Science, Makerere University, P.O. Box 7062, Kampala, Uganda. Email: aktugume@botany.mak.ac.ug, tugumeka@yahoo.com