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.
|Abstract (english)|| |
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
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
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.