| The aim of this study was mainly to isolate and identify high efficient plantgrowth-promoting rhizobacteria (PGPR) with specific-affintity in wheat plant roots as wellas research the dynamic and adsorption of PGPR in wheat roots. In total,32high efficientisolates were obtained by wheat germ agglutinin (WGA) labeled with fluoresceinisothiocyanate (FITC). Out of the32strains,17ones were obtained from the modifiedAshby medium,11ones from the potassium-releasing selective medium, and4strainsfrom the modified Pikovskaya medium. Based on phenotypic characterization, mostbacterial communities were white and milk-white, etc. Seventeen isolates were gramnegative whereas fifteen ones were gram positive. The oxidase test was positive for31isolates. The starch test was positive for27isolates, and the M.R. test was positive for3bacterial strains while the V.P. test was positive for4bacterial ones. Nine strains couldproduce H2S and eight strains could utilize glucose to produce acid. Most isolates couldutilize monosaccharides and a disaccharide as carbon sources. Based on the phylogeneticanalysis of the16SrRNA partial sequences, the results showed that the whole phylogenetictree was consisted by seven genuses, such as Bacillus, Pseudomonas and Arthrobacter etc.The main genus in phylogenetic tree concluded Bacillus sp. and Arthrobacter sp.It is well known that plant growth-promoting rhizobacteria play important roles inplant growth processes, so it is necessary to identification of plant growth promoting (PGP)traits, such as production of indole-3-acetic acid (IAA) and siderophores, phosphorussolubilization as well as inhibition to fungi pathogens. The results showed that out of32tested isolates,21(65.6%) strains were found to produce IAA. Three isolates producedmore than80mg/l IAA, and strain WS12showed the highest IAA production (103.3mg/l).Petri plate and liquid cultivation methods were employed to detect production ofsiderophores. The results showed that few isolated could produce siderophores on petriplates, which showed yellow-halo around the communities, this might attribute to the toxinof medium used. However, the medium could change to light-yellow when bacteriaproduced siderophores. Nineteen (59.3%) isolates produced siderophores by using CASreaction, with12strains producing them at high amounts5+. This indicates that detectionof siderophores by liquid cultivation is more efficient. In detection of phosphorussolubilization,31(96.8%) strains exhibited the ability to solubilize phosphate, arrangingfrom9.38-57.50μg/ml. Strains WS31showed the largest amount of solubilized phosphate,which was57.7μg/ml. The detection of phosphorus solubilization indicates that although isolates are obtained from different media, they have the same ability to solubilizephosphate. Most isolates showed nearly no inhibition to four fungi pathogens. However,strain WS07showed satisfied ability to inhibit four tested fungi. The largest inhibition wasdetected toward wheat scab (Fusarium Graminearum), the inhibition efficiency was51.40%, and the next were cotton fusarium wilt (Fusarium. oxysporum) and Maize(Bipolaria maydis), with inhibition efficiency of41.03%and39.32%, respectively. Thelowest inhibition was toward rice sheath blight fungi (Rhizoctonia solani), which inhibitionefficiency was37.7%. Overall, strain WS07showed satisfied inhibiting effect. It isimportant to determine growth-promoting properties of tested strains as it can be used inthe application of microbial fertilizers.Large tube cultivation method was used to determine the promoting effect of32strains on the growth in wheat seedlings. The results showed that the concentration ofbacterial cells had great impact on the wheat growth. Low concentration showed nopromotion whereas high concentration showed inhibition of wheat plant growth. Out of32tested strains,21isolates increased the lateral roots,22strains promoted the plant heightwhile23increased the root length. Inoculation with32isolates,11strains increased theshoot fresh weight and17strains promoted the root fresh weight. By determination ofwheat dry weights, the results showed that10strains increased shoot dry weight,19isolates increased the root dry weight. The proportion of promotion on wheat primary root,lateral root number, plant height, root length, total fresh weight, root fresh weight, shootdry weight and root dry weight were15.62,65.62,68.75,71.87,34.37,53.12,31.25and59.37%, respectively. The all promotions mentioned above reached significant differenceat p=0.05level. The results showed that tested strains did not show promotions in all testeditems, but in several indicators, such as number of lateral root, plant height, root length androot dry weight. After comparion of promotion items with bacterial PGP traits, the resultsshowed that most of bacteria (65.6%) possessing two or three PGP traits, the number inpromotion items which achieved significant difference were more larger, this indicates thatbacterial growth-promoting properties paly important role in plant growth. Theseexperimental results provide concrete data to facilitate research on biofertilizers.Electroporation was employed to transfer the plasmid pTR102to strain WS32in orderto make easier to study the adsorption in wheat root surface. The results showed that thelabeled strains had luminescence and the resistances to three kinds of antibiotics (tet, kmand chl). The transfer frequency was3.55×10-5~4.14×10-5while the lost frequency was19.7%, this indicate that luxAB gene shows well genetic stability. Therefore, the strain, which had resistance and was easier to detect, could be obtained by labeled with luxABgene and made more efficient in research the adsorption of microbial strains in plantrhizosphere.Attachment of bacteria to plant roots is a necessary and vital step when bacteriacolonize the wheat root surface. Strain WS32-L was chosen to study the adsorptionkinetics in wheat roots. The results showed that attachment of strains WS32-L to wheatroots reached the highest and stable amount (7.85±0.084lgCFU/g) in first60min. Theadsorption of strain WS32-L matched well with isothermal adsorption curve. The highestamount was6.47×108CFU/g and the adsorption coefficient α=4.5×10-9after calculation instandard curve. Herein, the attachment was significantly increased by treated the wheatroot with WGA when compared to the control treatments at p=0.01level. However,heat-treated WGA failed to increase the adsorption amount. Thus, WGA play an importantrole in attachment of bacteria in plant root. These findings expand the understanding of themechanisms which WGA is involved. In addition, the research on adsorption of bacteriacan give us a better understanding in colonization process of bacteria to plant roots.Fluorescence in situ hybridization (FISH) is a powerful tool to detect the rhizospheremicrobes. The colonization of plant growth promoting strain (Bacillus sp. WS24andPseudomonas sp. WS32) was investigated by FISH technique. Species-specific probeswere designed. The results showed that probes had specific toward the target strains andcould detected the tested strains. The detecting efficiency of Bacillus sp. could be greatlyimproved by pretreated strains with lysozyme. Under the mixture cultivation conditions,different specie-probes could well detect the target strains. By using FSIH technique, theresults showed that most bacterial cells adsorbed on wheat root surface and root hairs.Bacterial cells were distributed along longitudinal gap of plant root cells and formed in thepattern of dense biofilm. Moreover, the bacterial density in the root hair was the biggestand majority of bacterial cells were located on root hair surfaces. By using laser scanningconfocal microscopy, it also showed that bacteria were distributed in the peripheral surfacegap of plant root cells in wheat. This study also found that, regardless of different bacterialspecies, their colonization in wheat root position were most similar, this may indicate thatbacteria in wheat root colonization process possesses great competion of adhering sites.Herein, by using the FISH technique, we can visually observe the adsorption andcolonization of bacteria in wheat root surface. All the work performed here provides morespecific experimental data in the research of rhizosphere microorganisms. |
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