Izolacija i karakterizacija endofitskih bakterija iz soje (Glycine max L.)

Endophytic bacteria colonize the internal tissue of the plant usually forming beneficial association with their host. The main aim of this study was to genotypically and phenotypically characterize endophytic bacteria isolated from roots, stems and nodules of two soybean cultivars (AFZG Ana and Gabriela). A total of 29 endophytes were isolated from three different tissues of soybean. Genotypic characterization included rrs genes sequencing. Among soybean endophytes five different genera of bacteria were identified: Pseudomonas spp., Sphingomonas spp., Bradyrhizobium spp., Rhizobium spp., and Agrobacterium spp. Most of the isolates were Gram negative, rod-shaped, without capability of capsule production. The variability of the phenotypic characteristics of isolates was demonstrated, as well as the resistance to adverse environmental conditions such as high temperature, increased salt content, and low and high pH values. Most strains showed tolerance to the specific antibiotics. Most of the catalase positive isolates belong to Bradyrhizobium or Rhizobium spp., while the catalase negative isolates belong to the Pseudomonas, Sphingomonas and Agrobacterium spp. It was also shown that 85% of the tested isolates were oxidase negative while those that were positive belong to the Pseudomonas spp. Further characterization of endophytes is needed to determine their influence on plant growth.Endofitske bakterije koloniziraju unutrašnjost biljnih tkiva stvarajući pozitivne interakcije sa svojim domaćinom. Glavni cilj ovog istraživanja bio je genotipska i fenotipska karakterizacija endofitskih bakterija izoliranih iz korijena, stabiljke i nodula dva različita kultivara soje (AFZG Ana and Gabriela). Izolirano je ukupno 29 endofita iz tri različita tkiva soje. Genotipska karakterizacija uključivala je sekvenciranje rrs gena. Identificirano je pet skupina bakterija različitih rodova: Pseudomonas spp., Sphingomonas spp., Bradyrhizobium spp., Rhizobium spp. i Agrobacterium spp. Većina izolata je Gram negativna, štapićastog oblika, bez mogućnosti stvaranja kapsule. Dokazana je varijabilnost fenotipskih karakteristika izolata, ali i otpornost prema nepovoljnim uvjetima poput povišene temperature, povećanog sadržaja soli te niske i visoke pH vrijednosti. Većina sojeva pokazala je tolerantnost na istraživane antibiotike. Većina katalaza pozitivnih izolata pripadaju rodovima Bradyrhizobium ili Rhizobium, dok su katalaza negativni izolati identificirani kao Pseudomonas, Sphingomonas i Agrobacterium spp. Dokazano je da 85% ispitanih izolata ne reagira na enzim oksidazu, dok oni koji reagiraju pripadaju vrsti Pseudomonas spp. Potrebna je daljnja karakterizacija endofita kako bi se utvrdio njihov utjecaj na razvoj biljke

Fenotipska i genotipska raznolikost sojeva Sinorhizobium meliloti izoliranih iz tala Zadarske županije

Soil bacteria, Sinorhizobium melilo i is of great agricultural importance because of its ability to fix atmospheric nitrogen in symbiosis with alfalfa, a very valuable forage crop. The main aim of this study was to evaluate tolerance of indigenous S. melilo i strains to stress environmental conditions. Twenty rhizobial strains, isolated from different regions in Croatia, were genotipically characterized to assess diversity amongst natural population. Stress tolerance assays were performed in order to select indigenous rhizobia with tolerance to unfavorable soil conditions. The growth of the strains was studied at different pH values, temperatures, NaCl and heavy metals concentrations. The results of 16S rDNA genotyping using PCR/RFLP analysis showed that 17 isolates could be assigned to S. melilo i while RAPD and ERIC-PCR fingerprints revealed significant genetic diversity among indigenous rhizobial strains. In comparison to the reference strain, higher resistance to adverse growth conditions of indigenous S. melilo i strains was determined. Most of the strains grew at temperatures higher than optimal and tolerated both acidic and alkaline environment. It was found that indigenous strains tolerate extremely high NaCl concentrations. Better understanding of rhizobial response to adverse environmental conditions is of potential value for improving rhizobial inoculants and efficiency of symbiotic nitrogen fixationBakterija tla, Sinorhizobium meliloti važna je u poljoprivrednoj proizvodnji zbog sposobnosti fiksacije atmosferskog dušika u simbiozi s lucernom koja je vrlo vrijedna krmna kultura. Glavni cilj ovog istraživanja bio je procijeniti otpornost autohtonih sojeva S. meliloti na stresne uvjete okoliša. Najprije je genotipski karakterizirano dvadeset sojeva rizobija, izoliranih iz različitih regija u Hrvatskoj, kako bi se procijenila raznolikost unutar prirodne populacije. Ispitivanje otpornosti na stres provedeno je u cilju odabira autohtonih sojeva rizobija otpornih na nepovoljne uvjete u tlu. Ispitivan je rast sojeva pri različitim pH vrijednostima, temperaturama, koncentracijama NaCl-a i teškim metalima. Rezultati genotipizacije 16S rDNA PCR / RFLP analizom pokazali su da 17 izolata pripada vrsti S. meliloti, dok je primjenom RAPD i ERIC-PCR metode utvrđena značajna genetska raznolikost među autohtonim sojevima rizobija. Utvrđena je veća otpornost autohtonih sojeva S. meliloti na nepovoljne uvjete rasta u usporedbi s referentnim sojem. Većina sojeva rasla je na temperaturama višim od optimalnih i toleriralo je i kiselo i alkalno okruženje. Utvrđeno je da autohtoni sojevi podnose ekstremno visoke koncentracije NaCl. Bolje razumijevanje prilagodbe rizobija na nepovoljne uvjete okoliša važna je u svrhu poboljšanja inokuluma i učinkovitost simbiozne fiksacije dušik

Identification of Indigenous Bradyrhizobium japonicum Strains Isolated from Different Soil Types in Western Slavonia

Proučavanje prirodne populacije Bradyrhizobium japonicum kao i selekcija najučinkovitijih sojeva za komercijalnu poizvodnju, nameće potrebu za brzim i pouzdanim metodama identifikacije. Glavni cilj ovih istraživanja je komparacija različitih metoda za identifikaciju autohtonih sojeva B. japonicum izoliranih iz različitih tipova tala zapadne Slavonije. Fiziološki testovi, ispitivanje otpornosti na antibiotike, SDS-PAGE (sodium dodecil sulfat poliakrilamid gel elektroforeza) ukupnih staničnih proteina i RAPD (random amplified polymorphic DNA) analiza, korišteni su za identifikaciju sojeva i utvrđivanje varijabilnosti unutar prirodne populacije B. japonicum. Rezultati svake navedene metode prevedeni su u dvodimenzionalnu binarnu matricu i nakon analize podataka primjenom biostatističkog NTSYS programa, dobiven je dendrogram koji pokazuje relativnu sličnost između istraživanih sojeva. Na osnovi rezultata svih primjenjenih metoda utvrđeno je da je RAPD analiza najosjetljivija i najpouzdanija za identifikaciju sojeva. Najmanje razlike između B. japonicum sojeva utvrđene su ispitivanjem otpornosti na antibiotike. Na osnovi rezultata svih metoda, sa iznimkom prirodne otpornosti na antibiotike, utvrđeno je da se izolati bitno razlikuju od referentnih sojeva iz komercijalnog inokuluma te se mogu smatrati autohtonim za to područje. Proteinski i RAPD profili jasno su ukazali na postojanje dvaju bitno različitih grupa sojeva unutar prirodne populacije.Fast and reliable strain identification methods are needed for studying Bradyrhizobium japonicum field population as well as for the selection the most efficient strains for commercial inoculant production. The main aim of the present investigation was to compare different methods for identification of indigenous B. japonicum strains isolated from different soil types in western Slavonia. Physiological tests, intrinsic antibiotic resistance, SDS-PAGE (sodium dodecil sulphate polyacrilamide gel electrophoresis) of total cell proteins and RAPD (random amplified polymorphic DNA) analysis were used for strain identification and the assessment of variability within natural B. japonicum population. The results of each method were converted to two-dimensional binary matrix and dendrograms, showing relative similarity among B. japonicum strains, were obtained by using biostatistical NTSYS programe. The results showed that among all the methods used in this work, that RAPD analysis was the most sensitive and reliable for strain identification. The lowest level of differentiation among B. japonicum strains were determined by using intrinsic antibiotic resistance. All the methods used in this work, with the exception of intrinsic antibiotic resistance, revealed that all field isolates essentially differed from commercial strains and so can be regarded as indigenous strains for that area. Protein and RAPD profiles very clearly indicated that among natural field population two highly divergent group of strains can be determined

Karakteristike i uloga mezofilne kulture bakterija mliječne kiseline

The use of lactococci is widespread and has the longest tradition in industrial mesophilic lactic cultures technology. The principal concern of the dairy industry is the reliability and stability of these cultures. This paper describes the major technologically important properties of mesophilic lactic cultures when they are used for fermentation of milk and the production of cheese. This paper includes some of the specific properties of lactococci strains which can help in making the right choice of strains, depending on the product being produced. For example, most dairy fermentation require rapid acid production, ability to produce desired changes in milk and lack of off-flavour production. Certain strains of lactococci have the ability to produce exopolysaccharides or bacteriocines thus contributing to sensory properties of dairy products. In addition, these bacteria make important contributionLaktokoki u sastavu mezofilne kulture bakterija mliječne kiseline odavno se koriste u mljekarskoj industriji u proizvodnji različitih mliječnih proizvoda. Uspjeh fermentacije primarno je ovisan o upotrebljenoj kulturi od koje se zahtjeva osiguraje poželjnih svojstva na učinkovit i ponovljiv način. U radu su opisana glavna tehnološka svojstva mezofilne kulture laktokoka u fermentaciji mlijeka i proizvodnji sira. Navedena su i specifična svojstva određenih sojeva laktokoka koja mogu poslužiti kao kriterij u odabiru sojeva, za točno određeni mliječni proizvod. Općenito, od sojeva se zahtjeva brzo stvaranje mliječne kiseline i sposobnost stvaranja isključivo željenih promjena u mlijeku. Određeni sojevi laktokoka imaju sposobnost stvaranja egzopolisaharida ili bakteriocina i na taj način povoljno djeluju na senzorna svojstva proizvoda. Osim toga sojevi mezofilne kulture laktokoka imaju sposobnost proteolize nužne za tijek zrenja i formiranje okusa sira. analyses

Phenotypic characterization of indigenous soybean rhizobia

Kvržične bakterije u simbioznim zajednicama s mahunarkama omogućuju značajan unos biološki vezanog dušika u tlo. Veliku važnost ima odabir sojeva koji su otporni na stresne uvjete u tlu. Danas se sve više susrećemo s ekstremnim vremenskim uvjetima te je od iznimne važnosti selekcija visoko učinkovitih sojeva kvržičnih bakterija (rizobija) kako bi predsjetvena bakterizacija sjemena mahunarki bila uspješna. Cilj ovog istraživanja bio je utvrditi fenotipske karakteristike sojeva kvržičnih bakterija izoliranih iz kvržica soje u uvjetima koji su izvan optimalnih vrijednosti za njihov rast. Uzorci tla za izolaciju sojeva prikupljeni su s područja Koprivničko-križevačke, Bjelovarsko-bilogorske i Vukovarsko- srijemske županije. Sposobnost rasta sojeva ispitivana je na različitim pH vrijednostima, temperaturama, koncentracijama soli, izvorima ugljikohidrata te je testirana osjetljivost na antibiotike. U svrhu utvrđivanja biokemijskog statusa svakog pojedinog izolata provedena su tri biokemijska testa (oksidaza, ureaza i katalaza) te su istražene morfološke karakteristike izolata. Utvrđeno je da je većina autohtonih sojeva rizobija iz ovog istraživanja u in vitro uvjetima pokazala značajnu otpornost na ispitivane uvjete, ali i da se međusobno značajno razlikuju po određenim svojstvima. Izolati K2 i K3 iz Koprivničko-križevačke županije pokazali su najveću tolerantnost na povišene koncentracije soli. Od ispitivanih antibiotika, ampicilin je imao najslabiji utjecaj na izolate, dok su na streptomicin izolati bili najosjetljiviji. Dobiveni rezultati ukazuju na značaj selekcije autohtonih sojeva rizobija.Nodule bacteria in symbiotic communities with legumes allow a significant uptake of biologically fixed nitrogen into the soil. The selection of strains that are resistant to stressful soil conditions is of great importance. Selection of highly efficient strains of nodule bacteria (rhizobia) for successful pre-sowing bacterization of legume seed is especially important today, when we are increasingly faced with extreme weather conditions. The aim of this study was to determine the phenotypic characteristics of strains of nodule bacteria isolated from soybean nodules under conditions that are beyond the optimal values for their growth. Soil samples for strain isolation were collected from Koprivnica-Križevci, Bjelovar- Bilogora and Vukovar-Srijem counties. The ability of strains to grow was tested at different pH values, temperatures, salt concentrations and carbohydrate sources. Antibiotic susceptibility was tested as well. In order to determine the biochemical status of each individual isolate, three biochemical tests (oxidase, urease and catalase) were performed, and the morphological characteristics of the isolates were investigated. It was found that most indigenous strains of rhizobia from this study in vitro showed significant resistance to testing conditions, but they also differ significantly from each other in certain properties. Isolates K2 and K3 from Koprivnica-Križevci county showed the highest tolerance to elevated salt concentrations. From tested antibiotics, ampicillin showed to have the weakest effect on the selected isolates, while the isolates were most sensitive to streptomycin. The obtained results indicate the importance of selection of autochthonous strains of rhizobia

Resistance of indigenous Bradyrhizobium japonicum strains on moisture deficiency stress

Suša je jedan od najvažnijih faktora koji ograničava fiksaciju dušika, rast i prinos soje. U ovom radu petnaest autohtonih sojeva Bradyrhizobium japonicum izoliranih iz različitih regija Hrvatske podvrgnuto je in vitro ispitivanjima otpornosti na sušu simuliranu polietilen glikolom (PEG) 6000, te na povišen sadržaj soli. ERIC-PCR metoda korištena je za utvrđivanje genetske varijabilnosti autohtonih bakterija. Pri koncentraciji 15% PEG 6000 autohtoni soj izoliran iz područja istočne Slavonije B. japonicum IS1 bio je najtolerantniji na nedostatak vode. Pri koncentraciji PEG 6000 od 30%, autohtoni soj B. japonicum IS2 bio je izrazito otporan na osmotski stres, dok je najmanje tolerantan bio soj B. japonicum IS4. Oba soja izolirana su iz područja istočne Slavonije. Kod svih ispitivanih sojeva uočen je dobar rast pri koncentraciji od 1% NaCl, a umjeren rast je utvrđen kod autohtonog soja iz područja Koprivnice. Autohtoni soj iz Baranje, Istre i dva iz istočne Slavonije bili su vrlo tolerantni na izrazito visoke koncentracije od 4% NaCl. Prema rezultatima ERIC-PCR metode utvrđeno je da genetska sličnost nije uvjetovana mjestom izolacije. Najveća genetska sličnost bila je utvrđena kod sojeva izoliranih iz područja Baranje, istočne Slavonije, Koprivnice i zapadne Slavonije.Drought is one of the most important factors limiting N 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 east 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 from Baranja, Istria and two from eastern Slavonia were very tolerant to high concentration of 4% NaCl. According to results of ERIC-PCR method genetic similarity was not related to location of the isolation. The greatest genetic similarity was observed for strains isolated from Baranja region, eastern Slavonia, Koprivnica and western Slavonia

Selection of Stress Tolerant Indigenous Rhizobia Nodulating Alfalfa (Medicago sativa L.)

Alfalfa (Medicago sativa L.) is a very important forage crop, which forms a symbiotic relationship with nodule bacterium Sinorhizobum meliloti. 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 phenotypically characterized to assess diversity amongst natural field population. The growth of the strains was studied at different pH values, temperatures, carbohydrate sources and different concentrations of NaCl. 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. Most strains possess the enzyme urease while only a small number of them possess the enzyme catalase. The results showed that all indigenous strains belonged to the group of fast-growing rhizobia and that they were more tolerant to the antibiotics tested in comparison to the reference S. meliloti strain. Better understanding of rhizobial response to adverse environmental conditions is of important value for improving rhizobial inoculants and efficiency of symbiotic nitrogen fixation

Influence of co-inoculation of Bradyrhizobium japonicum and Pseudomonas fluorescens on growth of soybean (Glycine max L.)

Soybean (Glycine max L.) is a very important legume in the human diet as well as fodder crop. Previous studies have reported that co-inoculation with PGPR (plant growth-promoting bacteria) and rhizobia promotes symbiotic nitrogen fixation efficiency, increases plant nitrogen content and reduces the occurrence of soybean diseases, thus reducing pesticide use, and protecting the environment. The aim of this study was to determine the impact of co-inoculation of soybean seeds with different strains of nodule bacteria Bradyrhizobium japonicum as well as PGPR (Pseudomonas fluorescens). Indigenous strains of beneficial bacteria used in this research were isolated from different locations in Croatia. Isolates were identified by performing 16S rRNA gene sequencing. The application of different strains of nodule bacteria resulted in a statistically justified effect on all investigated properties, with the exception of root length. A positive influence of P. fluorescens on the increase in shoot dry weight was observed with the co-inoculation with the reference strain B. japonicum and two indigenous strains. The application of P. fluorescens did not show statistically justified differences in any of the investigated properties. Co-inoculation of nodules and PGPR bacteria is a very important biotechnological tool in agriculture, which is why research in this area should definitely be continued and expanded to a larger number of P. fluorescens strains

Genotypic Characterisation of Indigenous Rhizobium leguminosarum bv. viciae Field Population in Croatia

Genetic diversity of thirteen Rhizobium legumnosarum bv. viciae strains isolated from different field sites in continental part of Croatia was investigated. All rhizobial isolates were obtained either from plants grown in pots containing soil samples or from field grown plants. The strains were analyzed for DNA polymorphism using two DNA fingerprinting methods - randomly amplified polymorphic DNA (RAPD-PCR) and repetitive extragenomic palindromic- PCR (rep-PCR). Both methods resulted in very similar grouping of strains. Cluster analysis of rep- and RAPD-PCR profi les showed significant differences among Rh. leguminosarum bv. viciae isolates. The highest differences were detected among reference strains and all field isolates revealing considerable genetic diversity of rhizobial field populations. These results suggest the presence of adapted indigenous Rhizobium leguminosarum bv. viciae strains, probably with higher competitive ability, whose symbiotic properties have to be evaluated in further investigations