Search

The strains of nodule bacteria differ greatly in their symbiotic properties and efficiency in nitrogen fixation and therefore, the selection of highly effective strains is of great importance when attempting to achieve a successful pre-sowing inoculation of common bean. The aim of this study was to determine the nodulation ability of indigenous rhizobial strains and to evaluate their compatibility and symbiotic efficacy with two common bean cultivars. A two-factorial pot experiment (12 indigenous rhizobial strains and two common bean cultivars) was set up in the greenhouse. In the full blossom phase, dry nodule biomass, shoot dry weight and N content in dry shoot was determined. The research revealed significant differences depending on the strain of nodule bacteria and common bean cultivars. The compatibility of indigenous and reference strains on the examined common bean cultivars was also observed. The cultivar Tresnjevac was significantly better than Slavonski zeleni in all measured parameters. The results revealed that the indigenous common bean rhizobial strains considerable differ in their symbiotic efficiency. The highest dry nodule biomass was determined when strain HO12 was applied, and the highest shoot dry weight with strains MB1 and DE7. The application of strain DE7 revealed the highest N content in dry shoot. The results indicate that these indigenous strains of common bean nodulating bacteria are characterized by the highest symbiotic efficiency and could be used for the pre-sowing inoculation of common bean after testing in field scale experiment.

Modern agriculture is based on mineral fertilizers and use of synthetic chemicals in plant protection. Plant growth promoting bacteria (PGPB) represent a promising alternative to chemicals employed in agricultural practice. The aim of this study was to isolate and characterize PGP strains with the ability to promote plant growth from three different locations in the city of Zagreb. Phenotypic characterization of isolates included testing the tolerance to unfavorable soil conditions as well as intrinsic antibiotic resistance, biochemical characterization and screening for plant growth promoting properties. Twenty-four isolates were isolated. 28 % isolates were positive for the phosphate solubilization test, while IAA was produced by all isolates. Half of all isolates had the ability to synthesize lytic enzymes and exopolysaccharides. Some differences were found between isolates regarding their phenotypic characteristic, especially resistance to low pH and high temperatures.

Probiotic fermented milks are a group of functional food that, in addition to their nutritional value, contain certain components considered beneficial to human health. For their production, besides probiotic bacterial strains, yeasts - most commonly Saccharomyces boulardii (S. boulardii), are used too in the form of an secondary culture. The biotherapeutic effect of this yeast on the human body has been most studied. S. boulardii is also considered a potential natural preservative due to its antagonistic effect against other microorganisms. Its survivability and its effects on organoleptic properties have been studied in the production of yogurt from cow’s and goat’s milk, kefir, and acidophilic fermented milk. Based on all these factors, these products can be considered as new functional dairy products.

Symbiotic associations between legumes and specific group of soil bacteria enable considerable entries of biologically fixed nitrogen into the soil. The rhizobia inoculation of soybean is a sustainable practice to induce atmospheric nitrogen fixation and subsequently improve crop productivity and soil fertility. The main advantage of indigenous rhizobial strains relates to their high competitiveness and adaptation ability to specific environmental conditions. Drought is one of the most important factors limiting nitrogen fixation, growth and yield of soybean. In this study, 60 indigenous rhizobia isolated from 29 different regions of Croatia were subjected to in vitro investigations of different water conditions. Based on the results obtained by the RAPD method, sequencing of the 16S rRNA gene and in vitro drought test representative strains were selected for further analysis of sequences rpoB, glnII, gyrB, nodC and nifH gene. Phenotypic characterization of isolates included testing the tolerance to unfavorable soil conditions intrinsic antibiotic resistance, biochemical characterization, assimilation of different carbon sources, generational time and screening for plant growth promoting properties (PGPR). Greenhouse experiment was setup using two factors on the basis of a completely randomized block design. The 16S rRNA results showed that 75% strains from this study belong to genus Bradyrhizobium. Phylogenetic analysis of the three housekeeping genes rpoB, gyrB and glnII showed that the strains were identified as B. diazoefficiens and B. ottawaense species for the first time in Croatian soils and species B. japonicum identified earlier. The results of the nodC gene analysis of indigenous rhizobial strains isolated from Croatian soils showed that all of them belong to the symbiovar glycinearum. The phylogeny of most nifH genes coincides with the phylogeny of the nodC gene indicating the co- evolution of these two genes. PGPR characterization of isolates showed their significant variability. Results of greenhouse experiment showed that the hightest nodule number was obtained when soybean was inoculated with indigenous strain S1/5 (B. japonicum). Nodule dry weight is parameter indicating nodulation capacity of strain. Strains S3/5 (B. japonicum) S25/2 (B. diazoefficiens) S32 (B. ottawaense) and S37 (B. japonicum) showed significant tolerance to unfavourable conditions and PGPR characterization.

Nowadays, climate changes are considered as one of the biggest challenges for the society overall, with a special highlight on vulnerable agriculture production as human activity which produce food. According to predictions As extreme climate events is gonig to be more often, and fl ood is one of them as a result of extreme heavy rain in short period of time a project “Potential of the rhizosphere microbiome in the adaptation of agriculture to climate change (PERSPIRE)”, funded by the EU Regional Development Fund, focused on the eff ects of this kind of fl ood. Aim of the research was to determine the eff ect of the short fl ood on model plant in seedling phenophase. Experiment in carried out in the controlled conditions (16 hours day/8 hours night ; 25 ˚C per day/20 ˚C per night ; 60-70% relative humidity), with the cabbage (Brassica oleracea var. capitata f. alba) used as a model plant. The experiment lasted, from seeding to the full end, for 57 days. Plants (triplicate trials) were subjected to either one (72 h duration) or two short-term fl ooding events (72 h duration, 10 days recovery between fl oods) at diff erent stages of development. At diff erent time points (day 0, after fl ooding and after recovery period) soil samples were taken and Nmin was determined.Result showed that fl ood treatment signifi cantly decreased Nmin content in the soil (around 50%), both after fi rst and second fl ood. During the recovery time Nmin content increased, but after second fl ood decreased again.

The use of beneficial microorganisms is of great importance in modern agriculture development. The concept of biodiversity based agriculture (BBA) relies on sustainable diversification of all biological components to increase ecosystem service provision and reduce the external input in farming systems. In the focus of many researchers as well as many biotech companies is a very diverse group of soil bacteria called plant growth-promoting rhizobacteria (PGPR) because of its beneficial effect on the overall well-being of the plant. PGPR include various nitrogen-fixing bacteria that enable the conversion of molecular nitrogen into available form for plant nutrition. Selection of high-quality rhizobial strains is a powerful tool for improving beneficial effect of legume inoculation particularly under adverse environmental conditions. Although for many years rhizobia were thought to be the only nitrogen-fixing inhabitants of legume nodules, recent studies revealed the existence of very diverse bacterial community within the nodules. The aim of the present study was to reveal the biodiversity within chickpea/lentil nodules, primarily to detect rhizobial strains with improved characteristics. Soil samples were taken from different locations along the coastal part of Croatia as well as from the southern part of Herzegovina. Isolation of indigenous chickpea and lentil rhizobia was performed by trap-host method. Around 185 isolates were obtained that have to be verified, identified and characterized. Most isolates showed unusual phenotypic properties for rhizobia indicating wide diversity within nodule microbiome. Several authentication tests of selected isolates were performed under controlled conditions in order to test their nodulation ability. In two experiments, 37 chickpea rhizobial strains were tested and nodulation was determined on plants inoculated with 19 isolates. The results of rrs gene sequencing revealed considerable diversity within nodule microbiome which includes Alpha-, Beta-, Gamma-proteobacteria and actinobacteria as well. Isolates that caused nodulation on chickpea roots, were clustered in very different groups of alfaproteobacteria. PGPR traits of selected isolates were tested and the results revealed that some of them have a beneficial effect on plant growth. Further analyzes are needed in order to determine the presence of nod and nif genes in isolates and to perform their more precise molecular and symbiotic characterization

Because of its negative impact on all aspects of the environment, society, and the economy, climate change is no longer seen as a global threat of the 21st century, but as such. Agricultural production is classified as one of the activities most likely to be affected by climate change. According to the predictions, extreme climate events will occur more frequently, which will certainly have a detrimental effect on the growth and development of crops and, consequently, on food production. One of the extreme climate events predicted for Croatia is heavy rainfall in spring, followed by floods. Therefore, the aim of this work was to determine the effects of short-term flooding on the nitrate content in cabbage leaves in the period after planting as part of the project "Potential of the Rhizosphere Microbiome in the Adaptation of Agriculture to Climate Change (PERSPIRE)" funded by the EU Regional Development Fund. The experiment was conducted with cabbage (Brassica oleracea var. capitata f. alba) under controlled conditions (16 hours day/8 hours night ; 25 ˚C day/20 ˚C night ; 60-70% relative humidity). The total duration of the experiment was 57 days (sowing to the end of the experiment). Plants were flooded twice for short periods (duration 72 hours, 10 days recovery time between floods), and leaves were sampled five times at different stages of development. There were also control plants with optimal irrigation. The results showed no significant differences in leaf nitrate content between flooded and non-flooded plants, except at the end of the first recovery period after the first flooding. The nitrate content in the leaves decreases with time. It can be assumed that the nitrate content in cabbage is not so affected by short-term flooding.

The study presents the results of genotypic and phenotypic characterization of plant growth promoting (PGP) bacteria isolated from various soybean cultivars. A total of 18 isolates were isolated from two different soybean cultivars (AFZG Ana and Gabriela). Morphological characterisation has shown that all of the isolates were rod-shaped gram negative bacteria. The sequencing has shown that 12 out of 18 isolates belong to the genus Pseudomonas, four of which belong to P. fluorescens species. Isolates which belong to the Pseudomonas genus have shown the highest ability of indole-3-acetic acid synthesis, phosphorous solubilization, and along with isolates SGN6, SGN7 and SGS4 (Sphingomonas sanguinis) potassium solubilisation too. All the isolates from Pseudomonas genus as well as those belonging to B. japonicum species have shown the protease synthesis abilities while amylase synthesis abilities was observed only in the isolate SAK2 (P. chlororaphis). The most efficient strains in in vitro biological nitrogen fixation assay belong to Pseudomonas and Rhizobium genera. Most of the isolates were positive to the acetic acid production while all of them have shown the ability of the exopolysaccharide production.

Climate change is one of the biggest challenges of our times, with agricultural sector predicted to beamong those mostly affected. It is projected that climate variability will cause a substantial effect on agroecosystem functioning with measurable consequences on agricultural production and food security. Project “Exploring adaptation potential of rhizosphere microbiota to climate change: towards sustainable agriculture in the future (PERSPIRE)”, funded by the European Regional Development Fund(ERDF), aims to study the effects of the extreme weather events, already being experienced as a consequence of climate variability, on the complex interaction between rhizosphere microbiota and plant holobiont. Rhizosphere microbiota accomplish various functions that contribute a wide range of essential services by acting as the primary driving agents of nutrient cycles, regulating dynamics of soil organic matter, soil carbon sequestration and greenhouse gas emission, degrading pollutants, modifying soil physical properties and water regimes therefore indirectly being responsible for the health, growth, development and productivity of the plants. In the same time, due to unpredictable nature of the extreme weather events, studies of their impacts on the climate-ecosystem feedbacks are limited, with almost nothing known of the way in which extreme weather affects the rhizosphere microbiota. In view of a changing climate, not only crop yields, but also agricultural practices, including crop protection, is expected to be deeply affected. Combination of increased volatilization and accelerated degradation under climate change scenario, both strongly affected by a high moisture content, elevated temperatures and direct exposure to sunlight, is expected to reduce environmental concentrations of both synthetic and natural active ingredients. In such “new” condition pesticides are presumed to be used in higher amounts, doses, frequencies and different varieties or types of productsapplied. Climate change will therefore surely change what we currently know about the behaviour of pesticides in soils as well as their effect on microbiota. As intense rainfall events will in many cases result in soil saturation/hypoxic or anoxic conditions both microbiota, plant as well as pesticide behaviour is expected to be greatly affected. To tackle the uncertainty of the climate change effects on the agroecosystems, within the project PERSPIRE, we will settle a microcosm experiment in which selected model plant Brassica oleracea var. viridis (collards) will be grown in conditions mimicking expected climate change scenario: (1) exposing soil and holobiont to repetitive short-term flooding events (48h) and (2) additions of high amounts of herbicide pendimethaline [N-(1- ethylpropyl)-3, 4-dimethyl-2, 6- dinitrobenzenamine] to soil. Complex interactions between rhizosphere microbiota and plant holobiont in these “new” climate-change driven conditions will be studied on different levels: (i) within the rhizosphere microbiota (changes in the structure, function and activity), (ii) within the plant (changes in physiology, productivity and growth), (iii) within soil compartment (changes in plant- nutrient capacity and pesticide behaviour). Studying the responses of the rhizosphere microbiota and plants in experiments mimicking expected climate change scenarios is necessary not only to better understand effects of climate change on the agricultural sector but also to explore resilience and recovery capacity of the plants under expected climate change.

Drought is one of the most important factors limiting nitrogen fixation, growth and yield of soybean. In this study, fifteen indigenous Bradyrhizobium japonicum strains isolated from different regions of Croatia were subjected to in vitro investigations of different drought conditions (simulated by polyethilene glycol (PEG) 6000 and increased NaCl concentrations). ERIC-PCR method was employed in order to determine genetic variability of strains. At the concentration of 15 % PEG 6000 indigenous strain B. japonicum IS1 isolated from eastern Slavonia region was most tolerant to the lack of water. At the concentraction of 30 % PEG 6000, B. japonicum IS2, was distinctively resistant to osmotic pressure, and the least tolerant was B. japonicum IS4, both isolated from eastern Slavonia. For all tested strains, good growth was observed at the concentration of 1 % NaCl and moderate growth was recorded for the strains from Koprivnica region. Indigenous strans isolated from Baranja, Istria and two from eastern Slavonia were very tolerant to high concentration of NaCl (4%). According to results of ERIC-PCR method genetic similarity was not related to location of the isolation. ERIC-PCR fingerprints revealed significant diversity between rhizobial strains regardless their sampling sites.

Glavni cilj ovog istraživanja bio je utvrditi genotipske, fenotipske i simbiozne značajke autohtonih sojeva kvržičnih bakterija izoliranih iz 27 uzoraka tla sakupljenih na području sjeverozapadne Hrvatske. Identifikacija je izvršena pomoću metode MALDI- TOF MS, RAPD-PCR i filogenetske analize rrs, recA, atpD i nodC gena. Fenotipska karakterizacija uključila je testiranje otpornosti na visoke koncentracije NaCl-a, različitih vrijednosti pH i temperatura, otpornost na antibiotike i iskorištavanje različitih izvora ugljika. U cilju utvrđivanja simbioznih svojstava odabranih 15 sojeva te njihove kompatibilnosti sa dvije tradicijske sorte graha postavljen je vegetacijski pokus u stakleniku. Rezultati identifikacije pokuzuju da većina sojeva iz ovog istraživanja pripada vrstama R. leguminosarum i R. hidalgonense, koja je nedavno otkrivena u Americi. Dva soja su identificirana kao R. pisi dok nekoliko sojeva predstavlja potencijalnu novu vrstu. U vegetacijskom pokusu, najučinkovitija su bila dva soja (23T i 16T) koja pripadaju vrsti R.leguminosarum. Osim što su bili visoko učinkoviti u fiksaciji dušika, sojevi 1AT i 2Z (R. leguminosarum) pokazali su značajnu otpornost na nepovoljne uvjete kao što su visoke koncentracije NaCl i visoke temperature. Stoga bi ove sojeve trebalo uključiti u buduća istraživanja s ciljem odabira visokokvalitetnih sojeva za primjenu u održivoj proizvodnji graha.

Kukci predstavljaju izvrstan izvor visokovrijednog i dobro probavljivog proteina za monogastrične domaće životinje. Protein kukaca u odnosu na konvencionalna proteinska krmiva odlikuje se višim sadržajem i probavljivosti aminokiselina te nižom cijenom proizvodnje što ga čini atraktivnim zamjenskim krmivom. Međutim, temeljem važećeg zakonodavstva, kukci i prerađeni proizvodi od kukaca trenutno nemaju znatan udio u proizvodnji hrane za životinje u Europskoj uniji. Navedeno stanje će se u skorijoj budućnosti najvjerojatnije promijeniti. Tako je od 2017. godine prerađeni životinjski protein podrijetlom iz kukaca dopušteno koristiti za akvakulturu, dok se korištenje u ostalim kategorijama monogastričnih domaćih životinja razmatra. U ovom će radu biti opisane nutritivne karakteristike kukaca, njihova uporabljivost i najnovija zakonska regulativa vezana uz uporabu ovog tipa životinjskog proteina.

Putem simbiozne fiksacije dušika mahunarke mogu zadovoljiti velik dio svojih potreba za dušikom na učinkovit i ekološki prihvatljiv način. Rizobije su skupina bakterija tla sa jedinstvenom sposobnošću tvorbe kvržica na korijenu mahunarki u kojima pomoću enzima nitrogenaze konvertiraju inertan atmosferski dušik u biljkama pristupačan amonijačni oblik. Zbog svog vrlo vrijednog hranidbenog sastava, grah se smatra jednom od najvažnijih mahunarki u ljudskoj prehrani. Unatoč vrlo povoljnim agroekološkim uvjetima, uzgoj graha u Republici Hrvatskoj je zanemaren i u stalnom padu. Brojne vrste rizobija mogu tvoriti simbiozne odnose sa grahom, međutim u tlu su često prisutni kompetitivni autohtoni sojevi tih bakterija koji su dobro prilagođeni na nepovoljne uvjete, no njihova simbiozna učinkovitost je uglavnom nepoznata. Glavne pretpostavke ovih istraživanja su da u tlima sjeverozapadne Hrvatske postoje autohtoni sojevi rizobija koji noduliraju grah te da se oni značajno razlikuju po genotipskim i fenotipskim svojstvima te po učinkovitosti u simbioznoj fiksaciji dušika. U cilju odabira najkvalitetnijih sojeva rizobija sakupljeni su uzorci tla iz područja sjeverozapadne Hrvatske u kojem postoji dugogodišnja tradicija uzgoja graha. Iz 27 uzoraka tala izolirano je 45 autohtonih sojeva rizobija koji su zatim identificirani molekularnim metodama. Izolati su najprije identificirani MALDI-TOF (Matrix-assisted Laser Desorption/Ionization Time-of-Flight) masenom spektrometrijom koja se bazira na analizi ukupnih ribosomalnih proteina. U dosadašnjim istraživanjima potvrđena je pouzdanost ove brze i osjetljive metode za identifikaciju brzorastućih i spororastućih rizobija. Pomoću RAPD (engl. random amplified polymorphic DNA) metode dobiven je uvid u genetsku raznolikost izolata te izvršeno grupiranje u 15 skupina iz kojih su odabrani reprezentativni izolati za daljnja istraživanja. Ona su uključila sekvenciranje 16S rRNA, atpD, recA i nodC gena što danas predstavlja standard u identifikaciji rizobija. Fenotipska karakterizacija uključila je utvrđivanje rasta na različitim temperaturama, pH vrijednostima, koncentracijama NaCl-a, sposobnosti iskorištavanja različitih izvora ugljika te otpornosti na antibiotike. U vegetacijskom pokusu ispitana je simbiozna učinkovitost 15 odabranih sojeva koja je uspoređena sa referentnim sojem, nebakteriziranom kontrolom te nebakteriziranom kontrolom uz dodatak mineralnog dušika. Drugi faktor pokusa bile su tradicionalne hrvatske sorte graha, Trešnjevac i Slavonski zeleni. Rezultati identifikacije autohtonih sojeva ukazuju na vrlo veliku raznolikost rizobija koji noduliraju grah u tlima sjeverozapadne Hrvatske. Rezultati MALDI- TOF MS analize pokazuju da se izolati iz ovog istraživanja podudaraju s vrstama Rhizobium leguminosarum, Rhizobium sophoriradicis, Rhizobium phaseoli, Rhizobium ecuadorense, Rhizobium acidisoli, Rhizobium indigoferae i Rhizobium pisi. Neke od ovih vrsta imaju gotovo identične 16S rRNA gene zbog čega je bilo neophodno analizirati sekvence njihovih konstitucijskih gena atpD i recA. Prema tim analizama, najzastupljenije vrste iz uzorkovanih tala su R. leguminosarum i R. hidalgonense koja je tek nedavno otkrivena u američkim tlima. Sekvence atpD i recA gena velikog broja sojeva (35 %) značajno se razlikuju od najbližih srodnih vrsta zbog čega se može pretpostaviti da predstavljaju potencijalno novu vrstu unutar roda Rhizobium. Analiza nodC gena pokazala je da svi izolati pripadaju simbiovaru phaseoli, a u ovom istraživanju prvi put je utvrđeno postojanje simbiovara phaseoli kod vrste R. pisi. Dokazana je različita otpornost sojeva na stresne uvjete u tlu od čega je najznačajnija karakteristika otpornost na nizak pH jer su brojna tla u Hrvatskoj kisela. Rezultati vegetacijskog pokusa pokazuju da je najveća masa suhe tvari kvržica utvrđena primjenom sojeva 26T (R. hidalgonense) i 22AT (R. pisi). Na biljkama bakteriziranim sojevima vrste R. leguminosarum (16T, 23T, 1AT i 2Z) utvrđena je najveća masa suhe tvari kao i najveća količina dušika u suhoj tvari biljke što upućuje na njihovu veću simbioznu učinkovitost. Zbog toga bi ove sojeve bilo korisno uključiti u daljnje programe selekcije.

Putem simbiozne fiksacije dušika mahunarke mogu zadovoljiti velik dio svojih potreba za dušikom na učinkovit i ekološki prihvatljiv način. Rizobije su skupina bakterija tla sa jedinstvenom sposobnošću tvorbe kvržica na korijenu mahunarki u kojima pomoću enzima nitrogenaze konvertiraju inertan atmosferski dušik u biljkama pristupačan amonijačni oblik. Zbog svog vrlo vrijednog hranidbenog sastava, grah se smatra jednom od najvažnijih mahunarki u ljudskoj prehrani. Unatoč vrlo povoljnim agroekološkim uvjetima, uzgoj graha u Republici Hrvatskoj je zanemaren i u stalnom padu. Brojne vrste rizobija mogu tvoriti simbiozne odnose sa grahom, međutim u tlu su često prisutni kompetitivni autohtoni sojevi tih bakterija koji su dobro prilagođeni na nepovoljne uvjete, no njihova simbiozna učinkovitost je uglavnom nepoznata. Glavne pretpostavke ovih istraživanja su da u tlima sjeverozapadne Hrvatske postoje autohtoni sojevi rizobija koji noduliraju grah te da se oni značajno razlikuju po genotipskim i fenotipskim svojstvima te po učinkovitosti u simbioznoj fiksaciji dušika. U cilju odabira najkvalitetnijih sojeva rizobija sakupljeni su uzorci tla iz područja sjeverozapadne Hrvatske u kojem postoji dugogodišnja tradicija uzgoja graha. Iz 27 uzoraka tala izolirano je 45 autohtonih sojeva rizobija koji su zatim identificirani molekularnim metodama. Izolati su najprije identificirani MALDI-TOF (Matrix-assisted Laser Desorption/Ionization Time-of-Flight) masenom spektrometrijom koja se bazira na analizi ukupnih ribosomalnih proteina. U dosadašnjim istraživanjima potvrđena je pouzdanost ove brze i osjetljive metode za identifikaciju brzorastućih i spororastućih rizobija. Pomoću RAPD (engl. random amplified polymorphic DNA) metode dobiven je uvid u genetsku raznolikost izolata te izvršeno grupiranje u 15 skupina iz kojih su odabrani reprezentativni izolati za daljnja istraživanja. Ona su uključila sekvenciranje 16S rRNA, atpD, recA i nodC gena što danas predstavlja standard u identifikaciji rizobija. Fenotipska karakterizacija uključila je utvrđivanje rasta na različitim temperaturama, pH vrijednostima, koncentracijama NaCl-a, sposobnosti iskorištavanja različitih izvora ugljika te otpornosti na antibiotike. U vegetacijskom pokusu ispitana je simbiozna učinkovitost 15 odabranih sojeva koja je uspoređena sa referentnim sojem, nebakteriziranom kontrolom te nebakteriziranom kontrolom uz dodatak mineralnog dušika. Drugi faktor pokusa bile su tradicionalne hrvatske sorte graha, Trešnjevac i Slavonski zeleni. Rezultati identifikacije autohtonih sojeva ukazuju na vrlo veliku raznolikost rizobija koji noduliraju grah u tlima sjeverozapadne Hrvatske. Rezultati MALDI-TOF MS analize pokazuju da se izolati iz ovog istraživanja podudaraju s vrstama Rhizobium leguminosarum, Rhizobium sophoriradicis, Rhizobium phaseoli, Rhizobium ecuadorense, Rhizobium acidisoli, Rhizobium indigoferae i Rhizobium pisi. Neke od ovih vrsta imaju gotovo identične 16S rRNA gene zbog čega je bilo neophodno analizirati sekvence njihovih konstitucijskih gena atpD i recA. Prema tim analizama, najzastupljenije vrste iz uzorkovanih tala su R. leguminosarum i R. hidalgonense koja je tek nedavno otkrivena u američkim tlima. Sekvence atpD i recA gena velikog broja sojeva (35 %) značajno se razlikuju od najbližih srodnih vrsta zbog čega se može pretpostaviti da predstavljaju potencijalno novu vrstu unutar roda Rhizobium. Analiza nodC gena pokazala je da svi izolati pripadaju simbiovaru phaseoli, a u ovom istraživanju prvi put je utvrđeno postojanje simbiovara phaseoli kod vrste R. pisi. Dokazana je različita otpornost sojeva na stresne uvjete u tlu od čega je najznačajnija karakteristika otpornost na nizak pH jer su brojna tla u Hrvatskoj kisela. Rezultati vegetacijskog pokusa pokazuju da je najveća masa suhe tvari kvržica utvrđena primjenom sojeva 26T (R. hidalgonense) i 22AT (R. pisi). Na biljkama bakteriziranim sojevima vrste R. leguminosarum (16T, 23T, 1AT i 2Z) utvrđena je najveća masa suhe tvari kao i najveća količina dušika u suhoj tvari biljke što upućuje na njihovu veću simbioznu učinkovitost. Zbog toga bi ove sojeve bilo korisno uključiti u daljnje programe selekcije. Symbiotic nitrogen fixation enables legumes to supply most of their needs for nitrogen in efficient and ecological manner. Rhizobia are group of prokaryotic organisms with the unique ability to form symbiotic relationships with legume roots. In this relationship a new organ called nodule is formed, in which conversion of atmospheric nitrogen into ammonia, which is available to plants, occurs. Besides the significant quantities of nitrogen fixed in a symbiotic relationship, utilization of this process in legume production enables improvement of soil fertility and reduction of mineral fertilizer application costs. Bean (Phaseolus vulgaris L.) is very valuable and economically important culture. It is rich in proteins, fiber, potassium, phosphorus and B vitamin and recently, it has been discovered that bean consumption can contribute to health improvement. However, bean production in Croatia is limited to small-scale farmers and generally neglected despite favourable environmental conditions. Numerous rhizobial species can nodulate bean. Competitive indigenous strains of these bacteria which are well adapted to soil stress conditions can often be present in the soil. Their symbiotic efficiency is usually unknown. It is assumed that native rhizobial populations are also present in Croatian soils and that they contain strains resistant to stressful soil conditions and hidden potential for efficient nitrogen fixation. The aim of this study was to assess genotypic, phenotypic and symbiotic features of indigenous rhizobia isolated from soils of Northwestern Croatia. Soil samples were collected from 27 different locations in Northwestern region of Croatia. Trapping host method was performed to obtain 45 isolates of indigenous bean symbionts. The identification of isolates was first performed by Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) which is based on the characteristic protein profiles for each microorganism. Protein profiles obtained from isolates were compared to those contained in database created in Salamanca University, containing the type strains of all currently described species from the Family Rhizobiaceae. The results of identification by MALDI-TOF MS were further compared to those based on gene analysis. Random Amplified Polymorphic DNA (RAPD) method was employed to assess rhizobial genetic diversity and to choose representative strains for further analysys of gene sequences. Since the proposal for using the 16S rRNA (rrs) gene as universal tool for classification and identification of bacteria, this gene is the basis for rhizobial classification. Therefore, the sequence analysis of this gene was performed in this research to classify isolates obtained from bean nodules. In many studies it was shown that the analysis of the rrs gene is not sufficient for accurate differentiation among rhizobial species since several species with identical rrs gene sequences have been described untill now. These species are distinguishable on the basis of the housekeeping recA and atpD genes analysis, which are the most useful phylogenetic markers for the identification of rhizobial isolates. Sequence analysis of those two genes were further step in identification of rhizobia at species level. Bean is a legume nodulated by several fast growing species of family Rhizobiaceae, mainly belonging to genus Rhizobium. Some of those species contained several symbiovars being the symbiovar phaseoli most usually present. NodC gene is the commonly used phylogenetic marker to define symbiovars within species of genus Rhizobium and its sequences were analysed to study simbiovars within species of indigenous rhizobia isolated from Croatian soils. Phenotypic characterization of isolates included testing the tolerance to unfavorable soil conditions such as high levels of NaCl, different pH values and temperatures as well as intristic antibiotic resistance and assimilation of different carbon sources. Greenhouse experiment was setup using two factors (2 x 18) on the basis of a completely randomized block design with three replications. The first factor in the experiment were two traditional Croatian landraces of bean, Trešnjevac and Slavonski zeleni. The second factor were 15 indigenous rhizobial strains selected from different RAPD groups, reference strain used to inoculate plants, negative control (uninoculated plants) and positive control (uninoculated plants with addition of 0,05 % KNO3). At the flowering stage plants were collected and afterwords nodule dry mass, dry aerial biomass and total N in each plant were measured in order to estimate symbiotic efficiency of indigenous strains. Analysis of variance was performed with the statistical package SAS 9.4 for Windows. SAS Institute Inc. According to our hypothesis, indigenous rhizobial strains nodulating bean have been found in the soils of Northwestern Croatia. The results obtained with MALDI-TOF MS methodology showed that the strains from this study matched with different species from the Rhizobium leguminosarum phylogenetic group with score values higher than 2.0. Using this criterion investigated isolates were identified as Rhizobium leguminosarum, Rhizobium sophoriradicis, Rhizobium phaseoli, Rhizobium ecuadorense, Rhizobium acidisoli, Rhizobium indigoferae i Rhizobium pisi. The results of the RAPD analysis showed a high diversity among the isolated strains and the existence of 15 groups with similarity lower than 70 % from which representative strains were selected for core and symbiotic gene analyses. The rrs gene sequences were firstly compared to those available in Genbank and the results showed that all strains from this study belong to genus Rhizobium being their closest related species those of the phylogenetic group of R. leguminosarum. Similarity values of 100 % were found between 11 strains with R. leguminosarum and R. hidalgonense and between 1 strain (22BZ) with R. pisi. The rest of the strains, namely 8Z, 9T and 13T presented similarity values higher than 99 %, but lower than 100 % with R. leguminosarum, R. pisi and R. etli respectively. Nine out of 11 strains that were closely related to the R. leguminosarum phylogenetic group were identified as this species after the analysis of the atpD and recA housekeeping genes (similarity value higher than 98 %). Other two strains (25T and 26T) were identified as R. hidalgonense, a species recently isolated in America from P. vulgaris nodules. The strain 8Z formed an independent lineage more closely related to the species R. ecuadorense, but the similarity values were lower than 97 % in recA and atpD genes indicating that the strains represented by 8Z probably belong to a new species within genus Rhizobium. Analysis of recA and atpD gene sequence of strain 22BZ indicates that this strain belongs to the species R. pisi. Strains 9T and 13T formed an independent branch in the phylogenetic tree of housekeeping genes which was most closely related to R. sophoriradicis, but with similarity values lower than 98 %. Further research is needed to assign this strain to this species or to new phylogenetic lineage. The results of the nodC gene analysis of indigenous rhizobial strains isolated from Croatian soils showed that all of them belong to the symbiovar phaseoli which has previously been found in different Rhizobium species. Nevertheless we have found for the first time this symbiovar in the species R. pisi. Some differences were found between isolates regarding their phenotypic characteristic. Resistance to low pH is one of the most important traits of isolates since many Croatian soils are acid. Results of greenhouse experiment showed that both Croatian landraces were compatible with indigenous rhizobial strains. Significant differences were found between strains in their symbiotic performance. Nodule dry weight is parameter indicating nodulation capacity of strain. The highest nodule dry weight was obtained when Slavonski zeleni was inoculated with indigenous strain 26T (R. hidalgonense). No significant differences were found between this strain and strain 22AT (R. pisi). Symbiotic efficiency was estimated by comparison of dry aerial biomass of plants inoculated with indigenous rhizobial strains with dry aerial biomass of uninoculated plants. In this sence, the most efficient were strains 23T and 16T, both belonging to R. leguminosarum, species. The highest total N content was obtained in plants inoculated with strain 16T indicating this strain was most efficient in nitrogen fixation while similar values were obtained with strains 2Z, 23T i 1AT. Six out of 15 strains showed higher symbiotic fixation efficiency than positive control, while similar values for this parameter were obtained for five more strains. Least efficient were strains of species R. hidalgonense (20Z and 26T). Besides being highly efficient in symbiotic nitrogen fixation, strains 1AT and 2Z (R. leguminosarum) showed significant tolerance to unfavourable conditions such as high NaCl concentrations and high temperatures. Therefore, these strains should be included in further investigation in order to select high quality strain for application as biofertilizer for sustainable bean production. Given that the knowledge about the natural population of rhizobia that nodulate bean in Croatia is very limited, these study provided an insight into the composition and characteristic of this group of microorganisms.

Soil bacterium, Sinorhizobium meliloti is of enormous agricultural value because of its ability to fix atmospheric nitrogen in symbiosis with an important forage crops such as alfalfa. The main aim of this study was to evaluate the stress tolerance of indigenous S. meliloti strains to adverse environmental conditions. Twenty rhizobial strains, isolated from different regions in Croatia, were genotipically characterized to assess diversity amongst natural field population. Stress tolerance assays were performed in order to select indigenous rhizobia with possible higer tolerance to unfavorable soil conditions. The growth of the strains was studied at different pH values, temperatures, carbohydrate sources and different concentrations of NaCl. Biochemical characteristics of rhizobial isolates and the susceptibility to different antibiotics were determined as well. The results showed that most of the strains can grow at temperatures higher than optimal and that strains tolerate both acidic and alkaline environment. It was found that indigenous strains can tolerate extremely high concentrations of NaCl. Lactose agar assay indicates that three isolates probably belong to the genus Agrobacterium. Urease activity was determined in most strains while only a few strains expressed catalase activity. The results showed that all indigenous rhizobial strains belong to the group of fast-growing rhizobia. In comparison to the reference strain, higher resistance of indigenous S. meliloti strains was determined. The results of 16S rDNA genotyping using PCR/RFLP analysis showed that 14 isolates could be assigned to S. meliloti while RAPD and ERIC-PCR fingerprints revealed significant genetic diversity among indigenous rhizobial strains. Better understanding of rhizobial response to adverse environmental conditions is of potential value for improving rhizobial inoculants and efficiency of symbiotic nitrogen fixation.Book

Symbiotic nitrogen fixation has important role in sustainable soybean production because of utilization of atmospheric nitrogen for soybean nutrition. Pre-sowing soybean seed inoculation with selected rhizobial strains is used to improve the amount of symbiotically fixed nitrogen. Besides strain selection, suitable inoculant formulation is important for the success of inoculant application. The aim of this research is the evaluation of symbiotic efficiency and compatibility of Bradyrhizobium japonicum strains with soybean cultivar as well as possibility of using different inoculant formulation in soybean production. During two years of field trials in eastern Slavonia, nodule dry weight, nitrogen content in plant, seed yield, weight of 1000 seeds, protein and oil content in seed were determined. Results of this study indicate that inoculant formulation as well as the use of selected strains affect nodulation, symbiotic and agronomic properties of soybean. Despite the differences in results in both experimental years, it can be concluded that the strains used as well as inoculant formulations are suitable for soybean inoculation in agroecological conditions of eastern Slavonia.

An important feature of sustainable agriculture is the effective management of nitrogen in the environment. Consequently, the natural process of symbiotic nitrogen fixation has a unique role in sustainable common bean production. The application of rhizobial inoculants enables the considerable reduction of mineral nitrogen what is of great ecological and economic importance. Nitrogen fixing capacity in agricultural systems depends on rhizobial strain, host plant and environmental conditions. It is well known that rhizobial strains strongly differ in their properties like symbiotic efficiency, competitiveness with other rhizobia in soil, compatibility with host plant, and adaptive ability to stress conditions in soil. Therefore, the selection of high quality rhizobial strains is one of the main presumptions for successfull inoculation. Furthermore, in order to maximize the contribution of biologically fixed nitrogen in soybean production, it is also very important to select the most effective symbiotic associations between common bean cultivar and rhizobial strain. However, indigenous rhizobial strains can be present in some soils. In specific agroecological conditions, these strains, usually show higher competitivity and adaptive ability in comparison with commercial strains while their symbiotic efficiency is usually unkonwn. Therefore, in order to improve the beneficial effect of common bean inoculation, it is important to determine the actual composition and the characteristics of rhizobial field population. The main aim of the present study was to identify and characterize the indigenous common bean rhizobia isolated from different field sites in central Croatia, to determine genetic diversity within rhizobial field population and to evaluate symbiotic properties of indigenous common bean rhizobia.

Simbiozna fiksacija dušika kod uzgoja mahunarki nastoji se što više koristiti u održivoj poljoprivrednoj proizvodnji. Jedna od značajnijih krmnih mahunarki je stočni ili krmni grašak (Pisum sativum L.). U zajednici s kvržičnim bakterijama vrste Rhizobium leguminosarum sv. viciae i Rizobium pisi, stočni grašak osigurava znatno korištenje atmosferskog dušika, inače ograničavajućeg čimbenika u intenzivnoj poljoprivrednoj proizvodnji. U svrhu veće iskoristivosti procesa biološke fiksacije dušika provodi se predsjetvena bakterizacija sjemena graška. Pretpostavlja se da uporaba herbicida predstavlja potencijalnu opasnost za aktivnost kvržičnih bakterija u tlu, zbog čega je potrebno istražiti njihov utjecaj na rast i razvoj kvržičnih bakterija. Cilj istraživanja je utvrditi utjecaj različitih koncentracija herbicida imazamoksa, pendimetalina i klomazona na rast deset sojeva vrste R. leguminosarum sv. viciae i R.pisi. Utvrđen je značajan utjecaj visokih doza herbicida klomazona na većinu ispitivanih sojeva. Inhibirao je rast sojeva R. l. sv. viciae 1001, Kž 16 i R.pisi 30132, a usporio rast sojeva Kž 23 i Kž 24. Inhibitorni učinak klomazona utvrđen je samo primjenom najviše istraživane koncentracije (10.000 ppm). Negativan učinak imazamoksa i pendimetalina na rast ispitivanih sojeva nije utvrđen.

Microorganisms play a key role in the soil ecosystem functioning and their role is essential in numerous soil processes. The aim of the present study was to determine the effect of soil covering with different materials and three nitrogen fertilizer rates on total number of bacteria, abundance of microorganisms involved in nitrogen cycling, abundance of cellulolytic microorganisms and dehydrogenase activity. Stationary field trial was set up on hydromeliorated terra rossa soil in Istria. Nine trial plots were formed, with three replications, which differed from one another in the amounts of nitrogen fertilizers applied as well as in soil surface covering. Sampling was carried out four times during the growing season. Effect of sampling season on total number of bacteria as well as on the abundance of nitrifiers and denitrifiers was determined. In both years, statistically significant effect of soil covering upon the cellulolytic bacteria was found. Nitrifiers were the least abundant of all examined groups of microorganisms in both years, and in 2010 significantly highest number of nitrifiers was obtained with 120 kg of nitrogen applied (in May). In June and October significantly highest number of denitrifiers was recorded in plots covered with polyethylene film. Dehydrogenase activity was highest in uncovered plots in three sampling seasons in 2010.This study revealed that soil covering and different nitrogen fertilizer rates had very different impact on total number of microorganisms and on microbiological processes involved in the nitrogen cycle.

The studying of rhizobial biodiversity opens up the possibility to preserve and maybe to exploit some indigenous strains with hidden symbiotic or ecological potential. In order to improve the beneficial effect of alfalfa inoculation it is important to characterize the indigenous strains and to obtain information about actual composition of rhizobial field population. The main aim of the present study was to characterize indigenous rhizobia associated with alfalfa (Medicago sativa L.) in different regions of Croatia. Over 250 isolates were obtained from root nodules of alfalfa. To study their diversity and characterize them in relation to environmental conditions of their soils of origin, a polyphasic approach was used. Stress tolerance assays revealed significant variations in pH tolerance while almost all isolates showed similar tolerance to elevated salt concentrations and growth temperatures. PCR-RFLP of 16S rDNA revealed most of isolates to be closely related to S. meliloti. However, several isolates produced slightly different RFLP pattern from S. meliloti type strain and other isolates. Dendrogram derived from AFLP profiles revealed considerable genetic diversity among S. meliloti isolates. Only a few strains were identical or nearly identical to each other. The strains originated from the same region were mostly grouped within the same cluster. Better understanding of rhizobial ecology and selection of locally adapted and genetically defined strains as well as their targeted application is one among many approaches to improve the nitrogen fixation efficiency and crop productivity.

The studying of rhizobial biodiversity opens up the possibility to preserve and maybe to exploit some indigenous strains with hidden symbiotic or ecological potential, particularly under unfavourable conditions. In order to improve the beneficial effect of alfalfa inoculation it is important to characterize the indigenous strains and to obtain information about actual composition of rhizobial field population. The main aim of the present study was to characterize indigenous rhizobia associated with alfalfa (Medicago sativa L.) in different regions of Croatia. The soil samples for rhizobial isolation were collected from three different regions in Croatia (Istrian peninsula, north-west Croatia and central Croatia). Greenhouse pot experiment was established in order to obtain nodules for isolation procedure. Due to better description of field sites, main physical and chemical characteristics were determined in all soil samples. Over 250 isolates were obtained from root nodules of alfalfa. To study their diversity and characterize them in relation to environmental conditions of their soils of origin, a polyphasic approach was used. Stress tolerance assays revealed significant variations in pH tolerance while almost all isolates showed similar tolerance to elevated salt concentrations and growth temperatures. Among all isolates recovered from different regions, significant number of isolates showed tolerance to acidic pH values with dominance of strains originating from Istrian region where 38% of isolates tolerated pH 4.5. The actual composition and genetic diversity of natural field population was studied by two PCR fingerprinting methods. PCR-RFLP of 16S rDNA revealed most of isolates to be closely related to S. meliloti. Cluster analysis of RFLP patterns obtained with RsaI, showed that none of the isolate was identical with S. medicae type strain. However, several isolates produced slightly different RFLP pattern from S. meliloti type strain and other isolates. Dendrogram derived from AFLP profiles revealed considerable genetic diversity among S. meliloti isolates. Only a few strains were identical or nearly identical to each other. The strains originated from the same region were mostly grouped within the same cluster. These results were confirmed by further analysis. Analysis of molecular variance (AMOVA) allowed relating the genetic structure of the symbiotic population to various factors, including location, soil pH and the type of vegetation. Majority of the factors considered were significant as source of genetic variation, indicating that these populations were structured according to their geographical location and environmental conditions. The certain number of isolates, showing unspecific rhizobial characteristics, were sequenced. The results confirmed the presence of other bacterial species within the nodules. Therefore, further investigations are needed for the assessment of hidden diversity within the nodules. Better understanding of rhizobial ecology and selection of locally adapted and genetically defined strains as well as their targeted application regarding the environmental conditions of certain production area is one among many approaches to improve the nitrogen fixation efficiency and crop productivity.

Prirodni proces simbiozne fiksacije dušika nastoji se što više intenzivirati predsjetvenom bakterizacijom sjemena soje sa učinkovitim sojevima Bradyrhizobium japonicum. Pristustvo adaptiranih i visoko kompetitivnih sojeva B. japonicum u tlu može ograničiti uspjeh predsjetvene bakterizacije čak i sa visoko učinkovitim komercijalnim sojevima. Glavni cilj ovih istraživanja je izolacija i identifikacija autohtonih sojeva B. japonicum iz različitih tipova tala istočne Slavonije te utvrđivanje genetske varijabilnosti unutar prirodne populacije kvržičnih bakterija. Istraživanjem je obuhvaćeno 19 različitih izolata B. japonicum. Identifikacija prirodne populacije B. japonicum te procjena genetičke raznolikosti provedena je na osnovi razlika u 16S genskoj regiji putem PCR-RFLP metode te na osnovu razlika unutar ukupne genomske DNA primjenom RAPD metode. Rezultati svake od ovih metoda prevedeni su u dvodimenzionalnu binarnu matricu i analizirani primjenom biostatističkog programa NTSYS kojim je dobiven dendrogram relativne sličnosti istraživanih sojeva. Dobivenim rezultatima utvrđeno je postojanje autohtone populacije B. japonicum u svim istraživanim tipovima tla. Na osnovi rezultata 16S rDNA PCR RFLP utvrđeno je da 15 izolata pripada vrsti B. japonicum dok ih se 4 značajno razlikuje. Rezultati RAPD metode pokazuju da postoji značajna genetska varijabilnost između ispitivanih sojeva. Istom metodom utvrđeno je postojanje dviju srodnih skupina unutar autohtone populacije dok se 4 izolata značajno razlikuju od svih istraživanih sojeva. Najveća sličnost utvrđena je između sojeva 52 i 59 izoliranih iz različitih uzoraka tla. Rezultati pedoloških analiza pokazuju da kemijske karakteristike ne utječu značajno na genetsku varijabilnost populacije.