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

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

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

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

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

Effect of different fungicides on the growth of yeast from Saccharomyces sensu stricto group

Cilj ovog istraživanja bio je utvrditi djelovanje četiri različita fungicidna pripravka na bazi djelatnih tvari mankozeb, kaptan, iprodion i zoksamid na kvasce iz skupine Saccharomyces sensu stricto u laboratorijskim uvjetima. Kvasci iz ove skupine, posebice vrste Saccharomyces cerevisiae i Saccharomyces paradoxus od iznimne su važnosti za proizvodnju vina jer svojim metabolizmom utječu na sam proces fermentacije kao i na stvaranje brojnih spojeva važnih za aromu vina. Filter-disk metodom ispitivan je utjecaj fungicidnih pripravaka koji se primjenjuju za suzbijanje bolesti vinove loze u koncentracijama preporučenima od strane proizvođača kao i u nekim umanjenim i uvećanim koncentracijama. Dokazan je utjecaj pripravaka Cadillac 80WP®, Electis WG® i Stoper® na rast ispitivanih sojeva S. cerevisiae i S. paradoxus dok Kidan® nije imao utjecaj na rast kvasaca ni pri jednoj od ispitivanih koncentracija. Najveći negativan utjecaj imao je Cadillac 80WP® koji je inhibirao rast ispitivanih sojeva čak i pri upola nižim koncentracijama od propisanih. Nije utvrđena razlika u osjetljivosti između vrsta S. cerevisiae i S. paradoxus, dok se istovremeno može zaključiti da su referentni sojevi bili osjetljiviji na Cadillac 80WP®, Electis WG® i Stoper® u usporedbi s autohtonim izolatima.The aim of this study was to determine the effect of four different fungicides based on the active substances mancozeb, captan, iprodion and zoxamide on yeasts from Saccharomyces sensu stricto groups in laboratory scale. Yeasts from these group, particulary Saccharomyces cerevisiae and Saccharomyces paradoxus species, are of great importance for wine production, because their metabolism contributes to formation of important compounds but they also affects the fermentation process itself. Filter-paper disc method was employed in order to test the effect of fungicides which are usually applied for the control of grapevine diseases at doses/concentrations recommended by the manufacturers, as well as some lower and higher concentrations. The effect of Cadillac 80WP, Electis WG and Stoper on the tested strains S. cerevisiae and S. paradox species was observed, while Kidan did not affect the growth of yeasts at any of the concentrations tested. The greatest negative impact was observed for Cadillac 80WP, which inhibited the growth of tested strains even when the applied concentrations were lower than prescribed. No differences were found between S. cerevisiae and S. paradoxus strains regarding their susceptibility to tested fungicides, while it can be concluded that the reference strains were more sensitive to Cadillac 80WP, Electis WG, and Stoper compared to indigenous isolates

Obilježavanje stote godišnjice prve primjene inzulina

The year 2022 marked the one-hundredth anniversary of the first application of insulin. November 14th, the birth date of one of its main discoverers, Frederick Banting, was designated as World Diabetes Day. This paper comprises a narrative review of the history of the discovery of diabetes and insulin, progress in insulin development, important breakthroughs in insulin production and delivery, and a short commentary regarding potential future developments in insulin treatment. Diabetes, as one of the earliest recorded illnesses in medical writings, has been a focus of research for almost the entire written human history. Groundbreaking discoveries during the early 20th century have resulted in type 1 diabetes mellitus becoming a treatable, chronic condition. The relationship between good glycemic control and reduced occurrence of diabetes complications was established, which has enticed further development and refinements in insulin treatment, ranging from the purification and increased quality of insulin itself, as well as various inventions in its administration. Despite great achievements in insulin therapy so far, future research aims to avoid the need for subcutaneous administration and to create non-invasive means of insulin application.Godine 2022. obilježena je stota godišnjica prve primjene inzulina. Četrnaesti studenog, datum rođenja Fredericka Bantinga, jednog od izumitelja inzulina, proglašen je Svjetskim danom dijabetesa. U ovom radu pružamo narativni pregled povijesti otkrića šećerne bolesti i inzulina, napretka u razvoju inzulina, važnih otkrića u proizvodnji i isporuci inzulina te kratak komentar o potencijalnom budućem razvoju u inzulinskoj terapiji. Šećerna bolest, kao jedna od najranijih zabilježenih bolesti u medicinskim spisima, bila je tema istraživanja tijekom gotovo cijele pisane ljudske povijesti. Revolucionarna otkrića ranog 20. stoljeća dovela su do toga da šećerna bolest tip 1 postane lječivo, kronično stanje. Kada je utvrđen odnos između dobre kontrole glikemije i smanjene pojave komplikacija šećerne bolesti, potaknuti su daljnji razvoj i usavršavanje inzulinske terapije, uključujući pročišćavanje i unapređenje kvalitete samog inzulina, kao i razne izume u njegovoj primjeni. Unatoč velikim postignućima u inzulinskoj terapiji do sada, budućim istraživanjima cilj je izbjeći potrebu za potkožnom primjenom i stvoriti neinvazivne načine primjene inzulina

Obilježavanje stote godišnjice prve primjene inzulina

The year 2022 marked the one-hundredth anniversary of the first application of insulin. November 14th, the birth date of one of its main discoverers, Frederick Banting, was designated as World Diabetes Day. This paper comprises a narrative review of the history of the discovery of diabetes and insulin, progress in insulin development, important breakthroughs in insulin production and delivery, and a short commentary regarding potential future developments in insulin treatment. Diabetes, as one of the earliest recorded illnesses in medical writings, has been a focus of research for almost the entire written human history. Groundbreaking discoveries during the early 20th century have resulted in type 1 diabetes mellitus becoming a treatable, chronic condition. The relationship between good glycemic control and reduced occurrence of diabetes complications was established, which has enticed further development and refinements in insulin treatment, ranging from the purification and increased quality of insulin itself, as well as various inventions in its administration. Despite great achievements in insulin therapy so far, future research aims to avoid the need for subcutaneous administration and to create non-invasive means of insulin application.Godine 2022. obilježena je stota godišnjica prve primjene inzulina. Četrnaesti studenog, datum rođenja Fredericka Bantinga, jednog od izumitelja inzulina, proglašen je Svjetskim danom dijabetesa. U ovom radu pružamo narativni pregled povijesti otkrića šećerne bolesti i inzulina, napretka u razvoju inzulina, važnih otkrića u proizvodnji i isporuci inzulina te kratak komentar o potencijalnom budućem razvoju u inzulinskoj terapiji. Šećerna bolest, kao jedna od najranijih zabilježenih bolesti u medicinskim spisima, bila je tema istraživanja tijekom gotovo cijele pisane ljudske povijesti. Revolucionarna otkrića ranog 20. stoljeća dovela su do toga da šećerna bolest tip 1 postane lječivo, kronično stanje. Kada je utvrđen odnos između dobre kontrole glikemije i smanjene pojave komplikacija šećerne bolesti, potaknuti su daljnji razvoj i usavršavanje inzulinske terapije, uključujući pročišćavanje i unapređenje kvalitete samog inzulina, kao i razne izume u njegovoj primjeni. Unatoč velikim postignućima u inzulinskoj terapiji do sada, budućim istraživanjima cilj je izbjeći potrebu za potkožnom primjenom i stvoriti neinvazivne načine primjene inzulina

Isolation and phenotypic characterization of soybean (Glycine max L.) microsymbionts in continental Croatia

Simbioznom fiksacijom dušika leguminoze mogu zadovoljiti veliki dio svojih potreba za dušikom na učinkovit i ekološki prihvatljiv način. Rizobije su skupine bakterija tla sa jedinstvenom sposobnošću tvorbe kvržica na korijenu leguminoza u kojima pomoću enzima nitrogenaze konvertiraju inertan atmosferski dušik u biljkama pristupačan amonijačni oblik. Pretpostavlja se da su u tlima kontinentalne Hrvatske prisutni autohtoni sojevi rizobija koji noduliraju soju te da se sojevi međusobno razlikuju po fenotipskim i genotipskim svojstvima. U cilju odabira najkvalitetnijih sojeva rizobija sakupljeni su uzorci tla iz tri područja kontinentalne Hrvatske u kojem postoji dugogodišnja tradicija uzgoja soje. Fenotipska karakterizacija uključila je utvrđivanje rasta na različitim temperaturama, pH vrijednostima i rast na različitim koncentracijama soli, biokemijsku karakterizaciju, ispitivanje otpornosti na antibiotike. Fenotipska karakterizacija pokazala je značajnu otpornost na stresne uvjete okoliša, posebno otpornost na niski pH što je izuzetno važno jer Hrvatska ima jako puno kiselih tala. Kao najbolji sojevi pokazali su se sojevi SR1, SR2 te SR3 zbog čega bi ih bilo korisno uključiti u daljnji program 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 at the root of leguminosis in which they inert atmospheric nitrogen in plants is converted by nitrogenase enzymes. It is assumed that native rhizobial populations are also present in the soils of continental Croatia and that the strains differ from each other in phenotypic and genotypic properties. In order to select the best rhizobia strains, soil samples were collected from three areas of continental Croatia where there is a long tradition of soybean cultivation. Phenotypic characterization included the determination of growth at different temperatures, pH values and growth at different salt concentrations, biochemical characterization, antibiotic resistance testing. Phenotypic characterization has shown significant resistance to environmental stress conditions, particularly resistance to low pH, which is extremely important because Croatia has a lot of acid soils. Strains SR1, SR2 and SR3 have proven to be the best strains, therefore it would be useful to include them in a further selection programme