Simbiozne zajednice između većine leguminoza i posebnih skupina bakterija tla (rizobija) omogućuju značajan unos biološki vezanog dušika u tlo. Selekcija visoko učinkovitih sojeva rizobija koje noduliraju soju jedan je od preduvjeta uspješne primjene bakterizacije sjemena soje. Pretpostavlja se da su u tlima različitih regija Hrvatske prisutni autohtoni sojevi rizobija koji noduliraju soju, da postoji visoki stupanj varijabilnosti u prirodnoj populaciji te da se sojevi međusobno razlikuju po fenotipskim, genotipskim i simbioznim svojstvima. U cilju odabira najkvalitetnijih sojeva rizobija koji noduliraju soju sakupljeni su uzorci tla iz 29 regija kontinentalnog i mediteranskog dijela RH. Ukupno je izolirano 60 autohtonih sojeva rizobija koji su identificirani RAPD (engl. random amplified polymorphic DNA) metodom kojom je dobiven uvid u genetsku raznolikost izolata na osnovu koje je izvršeno grupiranje u 24 skupine, dok se identifikacija na razini vrste provela sekvenciranjem 16S rRNA gena. In vitro ispitivanjem tolerantnosti izolata na sušu utvrđena je najveća otpornost sojeva koji pripadaju rodu Ensifer i Bradyrhizobium. Na osnovu tri provedene metode odabrano je 12 reprezentativnih izolata za daljnu analizu koja je uključivala sekvenciranje rpoB, gyrB, glnII gena u cilju točnije identifikacije reprezentativnih izolata, kao i simbiozni nodC gen i gen za fiksaciju dušika nifH. Fenotipska karakterizacija uključila je utvrđivanje rasta na različitim temperaturama, pH vrijednostima, koncentracijama NaCl-a, sposobnosti iskorištavanja različitih izvora ugljika, biokemijsku karakterizaciju izolata, otpornost na antibiotike, utvrđivanje generacijskog vremena izolata kao i PGRP (engl. plant growth promoting rhizobactria) karakterizaciju. U vegetacijskom pokusu ispitana je simbiozna učinkovitost i tolerantnost na sušu 12 odabranih sojeva koja je uspoređena sa referentnim sojem i nebakteriziranom kontrolom. Rezultati filogenetske analize 16S rRNA gena pokazali su da 75 % izolata pripada rodu Bradyrhizobium, dok je preostali dio izolata svrstan u rodove Ensifer, Microbacterium, Rhizobium i Agrobacterium. Filogenetskom analizom tri konstitucijska gena rpoB, gyrB i glnII po prvi puta u tlima RH identificirane su vrste B. diazoefficiens i B. ottawaense kao i vrsta B. japonicum koja je ranije identificirana. Analiza nodC gena pokazala je da svi izolati osim S27 pripadaju simbiovaru glycinearum, dok se filogenija većine nifH gena podudara se sa filogenijom nodC gena što ukazuje na koevoluciju ova dva simbiozna gena. Dokazana je različita otpornost sojeva na stresne uvjete posebno otpornost na niski pH što je od izuzetne važnosti budući da Hrvatska ima jako puno kiselih tala, dok je PGPR karakterizacijom utvrđeno da soj S32 (B. ottawaense) producira IAA, egzopolisaharide, litičke enzima i otapa fosfate čime direktno promovira rast i razvitak soje. Rezultati vegetacijskog pokusa pokazuju da je najveći broj kvržica po biljci utvrđen primjenom soja S1/5 (B. japonicum), najveća masa suhe tvari kvržica utvrđena primjenom sojeva S37 (B. japonicum), S32 (B. ottawaense) i S25/2 (B. diazoefficiens). Na biljkama bakteriziranim sojevima vrste B. diazoefficiens (S25/2) i B. japonicum (S1/5) utvrđena je najveća masa suhe tvari kao i najveća količina dušika u suhoj tvari biljke što upućuje na njihovu veću simbioznu učinkovitost. Najveći indeks količine klorofila u biljci utvrđen je primjenom sojeva S3/5 (B. japonicum) i S25/2 (B. diazoefficiens). Sojevi S1/5 (B. japonicum) i S25/2 (B. diazoefficiens) odlikuju se najvećom simbioznom učinkovitošću u uvjetima suše zbog čega bi ih bilo korisno uključiti u daljnji program selekcije.
Symbiotic associations between legumes and specific group of soil bactera (rhizobia) enable considerable entries of biologically fixed nitrogen into the soil. Efforts are therefore made to intensify the natural process of symbiotic nitrogen fixation by legume inoculation. Effective symbiotic nitrogen fixation can significantly reduce the need for mineral nitrogen fertilizers. Rhizobia form a symbiotic association with leguminous plants, which results in the formation of an organ called a nodule on the root of the host plant. Inside the root nodule, rhizobia convert atmospheric nitrogen to ammonia, which may then be utilized for plant growth. Approximately 120 rhizobial species in 15 genera of α-proteobacteria and β-proteobacteria have been documented for diverse legumes. Soybean is the most important legume in the world and its seeds contain 40% protein and 20% oil. The northeastern region of China, the Korean peninsula, and Japan are the origin of soybean. China, USA, Brazil, and Argentina are the largest producers in the world. In recent years, the demand for soybean has increased in Europe due to increasing consumer demand for soybean as a healthy alternative to meat as well as to reduce the import of genetically modified soybean products from USA and South America. However, soybean cultivation is still relatively new in central Europe even though areas of soybean cultivation increased two-fold between 2013 and 2018. There is also large potential to grow soybean under the cooler and less favorable environments in northern parts of Europe. The rhizobia inoculation of soybean is a sustainable practice to induce atmospheric nitrogen fixation and subsequently improve crop productivity and soil fertility. Different environmental factors affect legume and rhizobia symbiosis, such as temperature, pH, salinity, drought, the origin of cultivars and the amount of nitrogen in soil. The main advantage of indigenous rhizobial strains relates to their high competitiveness and adaptation ability to specific environmental conditions. It is assumed that native rhizobial populations are also present in Croatian soils and that they contain strains resistant to stress soil conditions and with hidden potential for efficient nitrogen fixation. Soil samples were collected from 29 different region of Croatia. Trapping host method was performed to obtain 60 isolates of indigenous soybean symbionts. The identification of isolates was first performed by Random Amplified Polymorphic DNA (RAPD) method was employed to assess rhizobial genetic diversity. Since the sequencing of 16S rRNA gene is considered as universal tool for identification of bacteria, it is also widespread used for rhizobia as well. 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. In rhizobia, the variation in 16S rRNA gene sequences was shown to be unreliable to resolve species below the genus level. In many of the rhizobial groups, this region has slight divergence between species. Particularly in the genus Bradyrhizobium, phylogenetic classification at species level using this gene known to be difficult because the16S rRNA gene is highly conserved in the genus Bradyrhizobium and the sequence similarity is relatively high among closely related species.
Drought is one of the most important factors limiting nitrogen fixation, growth and yield of soybean. Recent advances in current understanding of the effects of drought on soybean growth have predominantly been based on evaluation of above-ground (shoot) traits, with flowering and seed stages particularly sensitive to drought stress. In contrast, drought effects on soybean roots, and specifically root nodules, has been less studied. In this study, 60 indigenous rhizobia isolated from different regions of Croatia were subjected to in vitro investigations of different water conditions (simulated by polyethilene glycol (PEG) 6000).
Based on the results obtained by the RAPD method, sequencing of the 16S rRNA gene and in vitro drought test representative strains were selected for further analysis of sequences rpoB, glnII, gyrB, nodC and nifH gene. Housekeeping protein coding genes are chromosomal in origin and are constitutively expressed in all cells under normal and patho-physiological conditions in order to maintain basic cellular functions of an organism. To overcome the limitations of rRNA genes, the sequence analysis of multiple protein coding genes known as multilocus sequence analysis (MLSA), has been recently considered as a preferred method to study closely related species and to discriminate strains of the same species. In general, they have higher level of sequence divergence compared to the 16S rRNA gene but are conserved enough to retain genetic information and therefore their sequences show better discrimination than 16S rRNA gene sequences. In rhizobia, the symbiotic genes that are required for nodule formation (nod) and for nitrogen-fixation (fix and nif genes) in symbiosis with the host legume are located either on plasmids or symbiosis islands in the chromosomes of the bacteria.
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 intrinsic antibiotic resistance, biochemical characterization, assimilation of different carbon sources, generational time and screening for plant growth promoting properties (PGPR).
Greenhouse experiment was setup using two factors (2 x 14) on the basis of a completely randomized block design with three replications. The first factor in the experiment were 12 indigenous rhizobial strains selected from different RAPD groups 16S rRNA sequencing results and in vitro drought test, reference strain B. japonicum 344 used to inoculate plants, negative control (uninoculated plants). The second factor was moisture (two irrigation regimes- drought and optimal conditions). At the flowering stage plants were collected and afterwords nodule number, nodule dry mass, aboveground dry biomass, total N and chlorophyll content index in each plant were measured in order to estimate symbiotic efficiency and drought tolerance of indigenous strains. Analysis of variance was performed with the statistical package SAS 9.4 for Windows. SAS Institute Inc.
The 16S rRNA gene sequences were firstly compared to those available in Genbank and the results showed that 75 % strains from this study belong to genus Bradyrhizobium, while the remaining strains belong to the genus Ensifer, Microbacterium, Rhizobium and Agrobacterium. Phylogenetic analysis of the three housekeeping genes rpoB, gyrB and glnII showed that the strains were identified as B. diazoefficiens and B. ottawaense (similarity value higher than 99 %) species for the first time in Croatian soils and species B. japonicum identified earlier. Six isolates were identified as B. japonicum, four as B. diazoefficiens and two as B. ottawaense. The strain S27 formed an independent lineage and they certainly represents a new species. 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 glycinearum except S27 which formed an independent lineage. The phylogeny of most nifH genes coincides with the phylogeny of the nodC gene indicating the co-evolution of these two genes. PGPR characterization of isolates showed their significant variability. The best strain in this study was S32 which was syntetsized of indole-3-acetic acid, produced exopolysaccharides, phosphate solubilizing activity and lytic enzymes priduction. This strain might have great potential to improve soybean productivity under different stress conditions.
Results of greenhouse experiment showed that the hightest nodule number was obtained when soybean was inoculated with indigenous strain S1/5 (B. japonicum). Nodule dry weight is parameter indicating nodulation capacity of strain. The highest nodule dry weight was obtained when soybean was inoculated with indigenous strains S37 (B. japonicum), S32 (B. ottawaense) and S25/2 (B. diazoefficiens). The highest dry aerial biomass was
obtained when soybean was inoculated indigenous rhizobial strains S25/2 (B. diazoefficiens) and S1/5 (B. japonicum). The highest total N content was obtained in plants inoculated with strains indicating this strains were most efficient in nitrogen fixation S25/2 (B. diazoefficiens) and S1/5 (B. japonicum). The highest chlorophyll content index was obtained in plants inoculated with strains S3/5 (B. japonicum) and S25/2 (B. diazoefficiens).
Besides being highly efficient in symbiotic nitrogen fixation, strains S3/5 (B. japonicum) S25/2 (B. diazoefficiens) S32 (B. ottawaense) and S37 (B. japonicum) showed significant tolerance to unfavourable conditions such as high NaCl concentrations, high temperatures and PGPR characterization especially strain S32 (B. japonicum). Therefore, these strains should be included in further investigation in order to select high quality strain for application as biofertilizer for sustainable soybean production.