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Therefore the carried out study aimed at determination of the effect of high-calcium brown coal ash and compost being produced from municipal sewage sludge on the content and bioaccumulation of heavy metals in potato tubers, wheat grains and rapeseeds during a three-year period. Rapeseeds contained most Cd whereas wheat rains less. Potato tubers, wheat grains and rapeseeds contained more Mn, Ni and Zn in the fertilization objects with municipal sewage sludge with or without coal ash and compared to those where calcium carbonate or coal ash had been introduced into the soil at a dose corresponding to 1.5 Mg CaO · ha–1 at the beginning of this study. Differences in the Mn, Ni and Zn contents in test plants between the fertilization objects with sewage sludge of with and without addition of ash were not significant.

Исследования проведены на трех видах грунтов, загрязненных тяжелыми металлами (ТМ). Первый – нативная почва, отобранная в городском сквере (НП)), второй – компост на основе листового опада (К), третий – почвогрунт (ПГ), состоящий на 40% из полученного компоста и на 60% из привезенной почвообразующей породы, торфа, строительного песка. В качестве тест объектов растений выбраны растения петрушки (Petroselinum Mill.) и кресс-салата (Lepidium sativum). Тест-культуры характеризовались наибольшим абсолютным накоплением цинка, свинца и меди. Накопление кобальта, никеля, хрома и кадмия за исследованный период незначительно. Выявлена тесная положительная корреляция между содержанием свинца в зелени кресс-салата и петрушки (r = 0,72-0,82) и содержанием его в грунтах. Установлена положительная корреляция между величиной коэффициента биологического накопления и содержанием ТМ в тест-растениях (r = 0,48-0,99) за исключением кобальта. Наиболее благоприятным для выращивания растений показал себя ПГ, где величина коэффициента биологического накопления ТМ имела значения равные или значительно ниже, чем у НП и К. В условиях эксперимента на биодоступность металла из почвы в растение влиял вид грунта и вид тест-растения. Двухфакторный дисперсионный анализ показал преимущественное положительное влияние вида грунта на накопление в тест-растениях Cu, Pb, Ni (F > Fкрит (75,79 > 19 при р < 0,01; 55,81 > 19 при р < 0,01; 302,14 > 19 при р < 0,003)). На накопление Zn в зелени тест-растений в большей степени повлиял вид выращиваемого растения (F > Fкрит; 121,89 > 18,51 при р < 0,008)., The studies were carried out on soils contaminated with heavy metals (HM). The first is the native soil sampled in the city square (NS)), the second is compost based on leaf litter (C), the third is substrate (S), consisting of 40% of the compost obtained and 60% of the imported soil-forming rock, peat, construction sand. Parsley (Petroselinum Mill.) And watercress (Lepidium sativum) plants were selected as test objects of plants. Test cultures were characterized by the highest absolute accumulation of zinc, lead and copper. The accumulation of cobalt, nickel, chromium and cadmium over the studied period is insignificant. A close positive correlation was revealed between the content of lead in watercress and parsley greens (r = 0.72-0.82) and its content in soils. A positive correlation was revealed between the value of the coefficient of biological accumulation and the HM content in test plants (r = 0.48-0.99), with the exception of cobalt. The most favorable for growing plants proved to be S, where the value of the coefficient of biological accumulation of HM, had values equal or significantly lower than that of NS and C. Under the experimental conditions, the type of soil and the type of test plant influenced the bioavailability of metal from the soil to the plant. Two-way analysis of variance revealed the predominant positive effect of the type of soil on the accumulation of Cu, Pb, Ni in test plants (F > Fcrit (75.79 > 19 at p < 0.01; 55.81 > 19 at p < 0.01; 302.14 > 19 at p < 0.003)). The accumulation of Zn in the greens of test plants was more influenced by the type of the grown plant (F > Fcrit; 121.89 > 18.51 at p < 0.008).

The selection of bioindicator species for ethoxysulfuron can enable the study of the behavior of this herbicide in the soil through the bioassay technique. Thus, this study aimed to select species with potential for use as bioindicators of the presence of this herbicide in the soil. A completely randomized design with four replications was used in which four plant species (sorghum, cucumber, beet and tomato) were cultivated in soil submitted to the application of six doses of ethoxysulfuron (0, 18.75, 37.5, 75, 112.5 and 150 g ha-1). Phytotoxicity, plant height (PH), shoot dry matter (SDM), root dry matter (RDM) and total dry matter (TDM) were evaluated. Different levels of tolerance of the species to ethoxysulfuron were observed: the increased doses of ethoxysulfuron reduced PH, SDM, RDM and TDM, as well as increased phytotoxicity for cucumber, beet and tomato. Sorghum showed low sensitivity to the herbicide. The highest doses inhibited the growth in height and the accumulation of dry matter of cucumber. Beet behaved as an extremely sensitive species, with a reduction of 100% in PH and dry matter in the second lowest dose (37.5 g ha-1). Among the evaluated species, the cucumber has greater potential for use as bioindicator in studies with ethoxysulfuron. A seleção de espécies bioindicadoras do ethoxysulfuron pode viabilizar o estudo do comportamento deste herbicida no solo pela técnica de bioensaio, constituindo assim o objetivo deste trabalho. Utilizou-se o delineamento inteiramente casualizado, com quatro repetições, em que quatro espécies vegetais (sorgo, pepino, beterraba e tomate) foram cultivadas em solo submetido à aplicação de seis doses de ethoxysulfuron (0; 18,75; 37,5; 75; 112,5 e 150 g ha-1). Avaliou-se a fitointoxicação, altura de plantas (ALT) e matéria seca da parte aérea (MSPA), da raiz (MSR) e total (MST). Foram observados diferentes níveis de tolerância das espécies ao herbicida: o aumento das doses de ethoxysulfuron reduziu as variáveis ALT, MSPA, MSR e MST e aumentou a fitointoxicação para pepino, beterraba e tomate. O sorgo apresentou baixa sensibilidade ao herbicida. As maiores doses avaliadas inibiram o crescimento em altura e o acúmulo de matéria seca do pepino. A beterraba se comportou como espécie extremamente sensível, com redução de 100% em ALT e matéria seca a partir da segunda menor dose (37,5 g ha-1). Entre as espécies avaliadas, o pepino apresenta maior potencial para uso como bioindicadora em estudos com ethoxysulfuron.

The chemical and biological trait approach have been applied to sediment pollution assessment of the Veliki Bački canal (Serbia) –VBC. For this assessment, pseudo-total metal contents, pollution load index (PLI), contamination factor (CF), enrichment factor (EF), sequential extraction procedure (SEP), pore water metal concentrations, geoaccumulation factor (Igeo), sediment quality guidelines (SQGs) and risk assessment code (RAC) have been used. Furthermore, the phytotoxic effects of the contaminated sediment have been assessed according to the physiological and morphological trait changes of the test plant species (maize, white mustard, barley and cucumber) in a bioassay. The chemical analysis has shown that an assessed sediment from VBC is extremely polluted with Cd, Zn, Ni whereas pore water is polluted with Cu, Zn, Ni. The results which have been acquired in plant bioassays indicate the different sensitivity of the plants. While pore water from the contaminated sediment had no effect on maize traits, it considerably inhibited the physiological and morphological traits of barley, cucumber and white mustard. These species are potentially good indicators of impaired sediment quality.

Pepino mosaic virus (PepMV) has caused great concern in the greenhouse tomato industry after it was found causing a new disease in tomato in 1999. The objective of this paper is to investigate alternative hosts and compare important biological characteristics of the three PepMV strains occurring in Europe whentested under different environmental conditions. To this end we compared the infectivity and symptom Development of three, well characterized isolates belonging to three different PepMV strains, EU-tom, Ch2 and US1, by inoculating them on tomato, possible alternative host plants in the family Solanaceae and selected test plants. The inoculation experiments were done in 10 countriesfrom south to north in Europe. The importance of alternative hosts among the solanaceous crops and the usefulness of test plants in the biological characterization of PepMV isolates are discussed. Our data for the three strains tested at 10 different European locations with both international and local cultivars showed that eggplant is an alternative host of PepMV. Sweet pepper is not an important host of PepMV, but potato can be infected when the right isolate is matched with a specific cultivar. Nicotiana occidentalis 37B is a useful indicator plant for PepMV studies, since it reacts with a different symptomatology to each one of the PepMV strains.

The aim of this study was to evaluate the impact of low internal exposure to 137Cs on L. sativum meristem cells and Tradescantia stamen hair cells. It also compared the impact of 137Cs internal and external irradiation of similar level on the plant seed germination and root growth. Compared to control, the tested internal (0.0007 mGy to 0.7 mGy) and external (0.04 mGy to 5.5 mGy) 137Cs ionising radiation doses stimulated the elongation of L. sativum roots by 11 % to 12 % and 24 % to 33 %, respectively. Internal 137Cs exposure (0.0003 mGy to 0.5 mGy) for 14 days caused 1.2 % to 1.6 % of somatic mutations and 19 % to 87 % of non-viable stamen hair in Tradescantia., Radom je istražen učinak izlaganja čitavih biljaka ionizirajućem zračenju izvora 137Cs na stanice meristema vegetativnog tkiva (korijen) i generativnog tkiva (stanice dlaka filamenata prašnika). Istraživanjem su se, također, pokušale utvrditi razlike u učinku zračenja na klijavost sjemena i rast korijena biljaka u ovisnosti o tome je li izvor zračenja u samoj biljci ili izvan nje. Značajan toksičan učinak zračenja utvrđen je samo u biljaka vrste Lepidium sativum L., i to na rast korijena. Neovisno o položaju izvora zračenja, doze od 0.0007 mGy do 0.7 mGy povećale su izduživanje korijena za 11 % do 12 %, a doze od 0.04 do 0.5 mGy za 24 % do 33 % u odnosu na kontrolu. Interne doze zračenja izvora 137Cs od 0.0003 mGy do 0.5 mGy tijekom 14-dnevnog izlaganja dovele su do pojave somatskih mutacija u 1.2 % do 1.6 % stanica dlaka prašničkih filamenata roda Tradescantia. Ujedno, 19 % do 87 % stanica izgubilo je vijabilnost, što upućuje na inhibiciju reproduktivne sposobnosti biljaka djelovanjem ionizirajućeg zračenja.

Biological N2 fixation is carried out exclusively by prokaryotes, either in the free-living form or in mutualistic symbioses with green algae, legumes and actinorhizal plants. The most agronomica1ly relevant symbiosis is, by fàr, that formed between soil rhizobia and legume roots. In addition, the input of fixed N2 by this symbiosis into agricultural systems represents an economical and environmentally- friendly altemative to chemical fertilization. Therefore, the improvement of the efficiency of symbiotic N2 fixation, both under physiological and stressful conditions, is highly desirable.The process of N2 fixation occurs in specialized organs, called nodules, that are formed as a result of a complex exchange of molecular signals (flavonoids, Nod factors) between the rhizobia and the host plant. During nodule formation, bacteria differentiate into bacteroids, which express the nitrogenase enzymatic complex. This catalyzes; the reduction of N2 to ammonium, requires ATP, Mg2+ and an electron donor, and is irreversibly inactivated by O2. During the last few years, the very early stages of nodule formation are being dissected at the molecular level. It is clear that the signal transduction pathway, which is triggered after binding of compatible Nod factors to receptors, involves oscillatory changes in cytoplasmic calcium, ethylene and protein kinases. Several lines of evidence indicate that reactive oxygen species (ROS) are also involved in signal transduction during nodule formation as well as in natural and stress- induced nodule senescence. However, critical questions remain to be answered: what specific ROS and at what specific step(s) of nodulation do they participate, how can Nod factors suppress the plant's defensive response, or how ROS production escapes control during nodule senescence.In this thesis we have devised three strategies that may be of value for the cellular and molecu1ar study of symbiosis and in particular ROS metabolism in nodules. First, we report a novel method that allows the isolation of root hairs, with high yield and purity, from the model legume M. truncatula. We also found in M. truncatula a protein, MtRH2, homologous to pea PsRH2, that is specifically localized in the root epidermis. This feature was exploited to monitor the root hair isolation procedure and can also be used for specific expression of antioxidant enzymes in root hairs. A second strategy that may be helpful to study the function of ROS is gene inactivation. We show that A. rhizogenes-mediated RNA interference (RNAi) is a fast and effective tool for this purpose. The introduction of RNAi in legumes and Arabidopsis using Agrobacterium rhizogenes based transformation was set up with genes with a known knockout phenotype. The efficiency of the technique was demonstrated by using RNAi to knock-down the KOJAK gene of Arabidopsis, which is involved in root hair development. We also show that RNAi silencing is restricted to the epidermal cells where RNAi constructs are expressed. A third strategy to study ROS metabolism is the overexpression of important antioxidant enzymes, such as superoxide dismutases (SODs), in transgenic legumes. Three lines of transgenic alfalfa (M. sativa) were designed to overexpress, respectively, MnSODs in chloropIasts, MnSOD in mitochondria and FeSODs in chloropIasts. Analysis of SOD composition in these plants led us to discover a novel compensatory effect in the activities of MnSOD and FeSOD in the leaves, which was not due to changes in mRNA levels. We also provide evidence that SOD activity in plants is regulated at least partially at the post-translational level and that FeSOD performs important antioxidant functions other than the scavenging of superoxide radicals generated in photosynthesis. Finally, in this thesis we discuss the significance, applications and prospects of the work done in the light of current research on nodule formation and senescence.

126 Pag., Tabls., Figs. Thesis Wageningen University, The Netherlands, Biological N2 fixation is carried out exclusively by prokaryotes, either in the free-living form or in mutualistic symbioses with green algae, legumes and actinorhizal plants. The most agronomically relevant symbiosis is, by far, that formed between soil rhizobia and legume roots. In addition, the input of fixed N2 by this symbiosis into agricultural systems represents an economical and environmentally-friendly alternative to chemical fertilization. Therefore, the improvement of the efficiency of symbiotic N2 fixation, both under physiological and stressful conditions, is highly desirable. The process of N2 fixation occurs in specialized organs, called nodules, that are formed as a result of a complex exchange of molecular signals (flavonoids, Nod factors) between the rhizobia and the host plant. During nodule formation, bacteria differentiate into bacteroids, which express the nitrogenase enzymatic complex. This catalyzes the reduction of N2 to ammonium, requires ATP, Mg2+ and an electron donor, and is irreversibly inactivated by O2. During the last few years, the very early stages of nodule formation are being dissected at the molecular level. It is clear that the signal transduction pathway, which is triggered after binding of compatible Nod factors to receptors, involves oscillatory changes in cytoplasmic calcium, ethylene and protein kinases. Several lines of evidence indicate that reactive oxygen species (ROS) are also involved in signal transduction during nodule formation as well as in natural and stress-induced nodule senescence. However, critical questions remain to be answered: what specific ROS and at what specific step(s) of nodulation do they participate, how can Nod factors suppress the plant's defensive response, or how ROS production escapes control during nodule senescence. In this thesis we have devised three strategies that may be of value for the cellular and molecular study of symbiosis and in particular ROS metabolism in nodules. First, we report a novel method that allows the isolation of root hairs, with high yield and purity, from the model legume M. truncatula. We also found in M. truncatula a protein, MtRH2, homologous to pea PsRH2, that is specifically localized in the root epidermis. This feature was exploited to monitor the root hair isolation procedure and can also be used for specific expression of antioxidant enzymes in root hairs. A second strategy that may be helpful to study the function of ROS is gene inactivation. We show that A. rhizogenes-mediated RNA interference (RNAi) is a fast and effective tool for this purpose. The introduction of RNAi in legumes and Arabidopsis using Agrobacterium rhizogenes based transformation was set up with genes with a known knockout phenotype. The efficiency of the technique was demonstrated by using RNAi to knock-down the KOJAK gene of Arabidopsis, which is involved in root hair development. We also show that RNAi silencing is restricted to the epidermal cells where RNAi constructs are expressed. A third strategy to study ROS metabolism is the overexpression of important antioxidant enzymes, such as superoxide dismutases (SODs), in transgenic legumes. Three lines of transgenic alfalfa (M. sativa) were designed to overexpress, respectively, MnSODs in chloroplasts, MnSOD in mitochondria and FeSODs in chloroplasts. Analysis of SOD composition in these plants led us to discover a novel compensatory effect in the activities of MnSOD and FeSOD in the leaves, which was not due to changes in mRNA levels. We also provide evidence that SOD activity in plants is regulated at least partially at the post-translational level and that FeSOD performs important antioxidant functions other than the scavenging of superoxide radicals generated in photosynthesis. Finally, in this thesis we discuss the significance, applications and prospects of the work done in the light of current research on nodule formation and senescence.

A brief account of the history of the development of artificial diets for phytophagous insects is given. Some conceptions with regard to terminology are discussed (chapter 3).Artificial diets for the larvae of the Colorado potato beetle, Leptinotarsa decemlineata Say, were developed and in addition some factors determining the feeding and rate of growth of the larvae have been brought to light.Larvae of the four consecutive instars reared on a diet were compared concerning their feeding and growth response (chapter 4). From the increased feeding activity of the larvae of the successive instars it appeared that an advance in tolerance towards the diet was observed as the larvae grew older. In addition it was noticed that as being in an advanced instar, the larvae were better able to accomplish their development on the diet.Because of the above mentioned phenomenon diets for the third instar larvae were based on the results of tests on the effects of various substances on larval growth (chapter 5). The nature of the substances tested as well as the concentration ratios of the dietary constituents appeared to be the most critical factors. Macro-molecular substances had to be included in the diet in view of the physico-chemical requirements for feeding and growth.First instar larvae reared on the best diets formulated so far for the third instar, could only attain the third instar.Non-aseptic rearing of newly hatched larvae on an autoclaved diet revealed that although palatability remained unchanged, the nutritive value of the diet was increased remarkably by heat treatment (chapter 6, section 6.5). Further experiments, however, are required to determine the factors causing this positive effect of autoclaving.In many cases during subsequent experiments 50 % or more of the individuals reared on an autoclaved diet from the time of hatching attained maturity. Addition of potato leaf powder appeared to promote feeding; however, it has no effect on growth. Inclusion of fresh potato leaves in the diet at a level of 10 % led to an increased feeding activity as well as an improved growth rate. However, growth ceased after two generations on diets with or without crude material from the host plant.In the present study it has been shown that ascorbic acid is required for larval growth. Commercial β-sitosterol, stigmasterol, cholesterol and ergosterol proved to be equally effective as sterol sources.An aseptic rearing technique for studies of the nutritional requirements was also developed, however, without satisfactory results.The improved quality of the diet by means of autoclaving enabled a mass rearing of larvae under non-aseptic conditions.In the general discussion (chapter 7) it was concluded that in principle the Colorado potato beetle could be reared on an artificial diet without addition of specific substances from the host plant. The results of the present study support the general opinion that the nutritional requirements for phytophagous insects are qualitatively more or less similar, but differ to a great extent quantitatively between species.

A brief account of the history of the development of artificial diets for phytophagous insects is given. Some conceptions with regard to terminology are discussed (chapter 3).Artificial diets for the larvae of the Colorado potato beetle, Leptinotarsa decemlineata Say, were developed and in addition some factors determining the feeding and rate of growth of the larvae have been brought to light.Larvae of the four consecutive instars reared on a diet were compared concerning their feeding and growth response (chapter 4). From the increased feeding activity of the larvae of the successive instars it appeared that an advance in tolerance towards the diet was observed as the larvae grew older. In addition it was noticed that as being in an advanced instar, the larvae were better able to accomplish their development on the diet.Because of the above mentioned phenomenon diets for the third instar larvae were based on the results of tests on the effects of various substances on larval growth (chapter 5). The nature of the substances tested as well as the concentration ratios of the dietary constituents appeared to be the most critical factors. Macro-molecular substances had to be included in the diet in view of the physico-chemical requirements for feeding and growth.First instar larvae reared on the best diets formulated so far for the third instar, could only attain the third instar.Non-aseptic rearing of newly hatched larvae on an autoclaved diet revealed that although palatability remained unchanged, the nutritive value of the diet was increased remarkably by heat treatment (chapter 6, section 6.5). Further experiments, however, are required to determine the factors causing this positive effect of autoclaving.In many cases during subsequent experiments 50 % or more of the individuals reared on an autoclaved diet from the time of hatching attained maturity. Addition of potato leaf powder appeared to promote feeding; however, it has no effect on growth. Inclusion of fresh potato leaves in the diet at a level of 10 % led to an increased feeding activity as well as an improved growth rate. However, growth ceased after two generations on diets with or without crude material from the host plant.In the present study it has been shown that ascorbic acid is required for larval growth. Commercial β-sitosterol, stigmasterol, cholesterol and ergosterol proved to be equally effective as sterol sources.An aseptic rearing technique for studies of the nutritional requirements was also developed, however, without satisfactory results.The improved quality of the diet by means of autoclaving enabled a mass rearing of larvae under non-aseptic conditions.In the general discussion (chapter 7) it was concluded that in principle the Colorado potato beetle could be reared on an artificial diet without addition of specific substances from the host plant. The results of the present study support the general opinion that the nutritional requirements for phytophagous insects are qualitatively more or less similar, but differ to a great extent quantitatively between species.