Biodiversity and symbiotic efficiency of indigenous rhizobial populations nodulating bean (Phaseolus vulgaris L.)

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

Biodiversity and symbiotic efficiency of indigenous rhizobial populations nodulating bean (Phaseolus vulgaris L.)

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

Biodiversity and symbiotic efficiency of indigenous rhizobial populations nodulating bean (Phaseolus vulgaris L.)

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

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

Improvement of Rhizobial Inoculants: A Key Process in Sustainable Soybean Production

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, 1000 seeds weight, protein and oil content in seed were determined. Results of this study indicate that inoculant formulation as well as the use of selected strains affects 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

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

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

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

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