Involvement of phytohormones in the development of interaction between wheat seedlings and endophytic <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain 11BM

Wheat (Triticum aestivum L., cv. Kazakhstanskaya 10) seedling growth activation by the endophytic 11BM strain of the bacterium Bacillus subtilis Cohn and the involvement of phytohormones in this process were studied. At the concentration used, bacteria enhanced wheat root and shoot growth as compared to control. Biotests demonstrated auxin-, cytokinin-, and gibberellin-like activities of the endophyte, but the result depended on the way of experiment performing. When wheat seeds were treated with the spores of B. subtilis 11BM strain, the concentrations of IAA and ABA increased transiently in the roots and shoots of the seedlings. The involvement of IAA oxidase in the plant response to inoculation with bacteria was shown; it was accompanied by a decrease in enzyme activity, which occurred later than auxin accumulation. It was concluded that observed changes in the hormonal status of wheat plants under the influence of the endophytic bacterial strain may be one of the mechanisms for seedling growth stimulation.     

Participation of Auxin Transport in the Early Response of the <i>Arabidopsis</i> Root System to Inoculation with <i>Azospirillum brasilense</i>

The potential of Plant Growth Promoting Rhizobacteria (PGPR) has been demonstrated in the case of plant inoculation with bacteria of the genus Azospirillum which improves yield. A. brasilense produces a wide variety of molecules, including the natural auxin indole-3-acetic acid (IAA), as well as other phytoregulators. However, several studies have suggested that auxin induces changes in plant development during their interaction with the bacteria. The effects of A. brasilense Sp245 on the development of Arabidopsis thaliana root were investigated to help explain the molecular basis of the interaction. The results obtained showed a decrease in primary root length from the first day and remained so throughout the exposure, accompanied by a stimulation of initiation and maturation of lateral root primordia and an increase of lateral roots. An enhanced auxin response was evident in the vascular tissue and lateral root meristems of inoculated plants. However, after five days of bacterization, the response disappeared in the primary root meristems. The role of polar auxin transport (PAT) in auxins relocation involved the PGP1, AXR4-1, and BEN2 proteins, which apparently mediated A. brasilense-induced root branching of Arabidopsis seedlings.     

Characterization of Novel Plant Growth Promoting Endophytic Bacterium <Emphasis Type="Italic">Achromobacter xylosoxidans</Emphasis> from Wheat Plant

Nine diazotrophic bacteria were isolated from surface-sterilized roots and culms of wheat variety Malviya-234, which is grown with very low or no inputs of nitrogen fertilizer. Out of the nine bacteria, four showed indole acetic acid (IAA) production, and five were positive for P solubilization. One isolate, WM234C-3, showed appreciable level of nitrogenase activity, IAA production, and P solubilization ability, and was further characterized with a view to exploiting its plant growth promoting activity. Based on 16S rDNA sequence analysis, this isolate was identified as Achromobacter xylosoxidans. Diazotrophic nature of this particular isolate was confirmed by Western blot analysis of dinitrogenase reductase and amplification of nifH. Analysis of the nifH sequence showed close homology with typical diazotrophic bacteria. Endophytic nature and cross-infection ability of WM234C-3 were tested by molecular tagging with gusA fused to a constitutive promoter followed by inoculation onto rice seedlings in axenic conditions. At 21 days after inoculation, the roots showed blue staining, the most intense color being at the emergence of lateral roots and root tips. Microscopic observation confirmed colonization of gus-tagged WM234C-3 in the intercellular spaces of cortical as well as vascular zones of roots. Inoculation of gus-tagged WM234C-3 to rice plants resulted in significant increase in root/shoot length, fresh weight, and chlorophyll a content. Plant growth promoting features coupled with cross-infection ability suggest that this endophytic bacterium may be exploited as agricultural agent for various crops after a thorough and critical pathogenicity test.     

Effect of phytohormones synthesized by rhizosphere bacteria on plants

New strains of rhizosphere microorganisms Azotobacter chroococcum Az d10, Bacillus megaterium Pl-04, and Bacillus mucilaginosus B-1574 were found to be able to synthesize cytokinins (CKs) and indolylacetic acid (IAA). Three forms of CKs—dihydrozeatin riboside, isopentenyl adenosine, and trans-zeatin riboside—were identified, whose ratio was different in the three bacterial cultures. Inoculation of cucumber (Cucumis sativus L.) plants increased the content of CKs and IAA in them by 35.6 and 21.3%, respectively, and also stimulated seed germination and increased the growth rate, the biomass of shoots, the number of lateral roots, and the root hair area, which ensured better plant nutrition. The IAA/CKs ratio shifted during bacterization towards CKs due to increase in the content of riboside forms, which apparently caused growth stimulation.     

Inhibition of Auxin Movement from the Shoot into the Root Inhibits Lateral Root Development in Arabidopsis

In roots two distinct polar movements of auxin have been reported that may control different developmental and growth events. To test the hypothesis that auxin derived from the shoot and transported toward the root controls lateral root development, the two polarities of auxin transport were uncoupled in Arabidopsis. Local application of the auxin-transport inhibitor naphthylphthalamic acid (NPA) at the root-shoot junction decreased the number and density of lateral roots and reduced the free indoleacetic acid (IAA) levels in the root and [³H]IAA transport into the root. Application of NPA to the basal half of or at several positions along the root only reduced lateral root density in regions that were in contact with NPA or in regions apical to the site of application. Lateral root development was restored by application of IAA apical to NPA application. Lateral root development in Arabidopsis roots was also inhibited by excision of the shoot or dark growth and this inhibition was reversible by IAA. Together, these results are consistent with auxin transport from the shoot into the root controlling lateral root development.     

Quantitative assessments of the host range and strain specificity of endophytic colonization by Klebsiella pneumoniae 342

Enteric bacteria, particularly Klebsiella, are common endophytes of plants. Endophytic colonization is important as these bacteria may be beneficial, either by providing fixed N or growth hormones to the host plant. In this work, we assessed the host range and strain specificity for endophytic colonization with Klebsiella pneumoniae 342 (Kp342) on five host plants. This strain was inoculated onto seedlings of Medicago sativa, Medicago truncatula, Arabidopsis thaliana, Triticum aestivum, and Oryza sativa. The type strain of K. pneumoniae, ATCC13883, was also inoculated on all of these hosts except M. truncatula. Both strains were labeled with GFP. Eight inoculum levels were used from 1 CFU to 10⁷ CFU per plant plus uninoculated controls. Six days after inoculation, the number of cells colonizing the rhizosphere and interior were determined. Inoculation with about one CFU of Kp342 was adequate to obtain high colonization levels on the rhizosphere and roots of all host plants. The type strain could colonize the interior of the host plant but the highest colonization levels were generally 100-fold lower than those obtained from Kp342 and those levels required at least 1000 cells in the inoculum. Thus, Kp342 was a more efficient colonizer of the plant apoplast. In addition, the monocots inoculated in this work were colonized endophytically in much higher numbers than were the dicots. Cells of Kp342 congregate at lateral root junctions suggesting the cells enter the plant through cracks created by lateral root extensions. The strain and host effects observed here suggest that endophytic colonization is an active process controlled by genetic determinants from both partners.     

Investigation of Arbuscular mycorrhizal Fungus and EDTA Efficiencies on Lead Phytoremediation by Sunflower in a Calcareous Soil

Lead (Pb) contamination of soils is a widespread problem. Mycorrhizal inoculation and synthetic chelators such as ethylenediaminetetraacetic acid (EDTA) may be useful for improving phytoremediation efficiency in Pb-contaminated soils. A greenhouse experiment was performed to study the influence of inoculation with arbuscular mycorrhizal fungus (AMF), Glomus mosseae, and addition of EDTA on phytoremediation of Pb by sunflowers (Helianthus annuus) in a calcareous soil. The experiment was a completely randomized design in a factorial arrangement with five levels of Pb, two levels of mycorrhizal treatments, and two levels of EDTA. Inoculation increased root colonization as Pb levels increased, but the addition of EDTA decreased it. Shoot and root dry matter yields increased by inoculation; however, they decreased with EDTA and Pb levels in co-application treatments. Pb concentration in shoots was significantly higher than that in roots, indicating a translocation factor greater than 1. Inoculation or addition of EDTA significantly increased Pb in roots and its translocation to shoots. The uptake index (UI) value increased in co-application of EDTA and AMF and the individual application of them; it is, therefore, concluded that both AMF and EDTA are effective in phytoremediation of Pb by sunflowers in the studied soil.     

Involvement of IAA in the interaction betweenAzospirillum brasilense andPanicum miliaceum roots

The possible involvement of IAA in the effect thatAzospirillum brasilense has on the elongation and morphology ofPanicum miliaceum roots was examined by comparing in a Petri dish system the effects of inoculation with a wild strain (Cd) with those of an IAA-overproducing mutant (FT-326). Both bacterial strains produced IAA in culture in the absence of tryptophan. At the stationary growth phase, production of IAA by FT-326 wasca. 12 times greater than that of Cd. When inoculation was made with bacterial concentrations higher than, 10⁶ colony forming units ml^–1 (CFU ml^–1), both strains inhibited root elongation to the same extent. At lower concentrations Cd enhanced elongation, by 15–20%, while FT-326 was ineffective. Both strains promoted root-hair development, and root-hairs were produced nearer the root tip the higher the bacterial concentration (e. g. root elongation region was reduced). Effects of FT-326 on root-hair development were greater than those of Cd. Acidified ether extracts of Cd and FT-326 cultures had inhibitory or promoting effects on root elongation depending on the dilution applied. At low dilutions, extracts from FT-326 were more inhibitory for elongation than those from Cd. At higher dilutions root elongation was promoted, but FT-326 extracts had to be more diluted than those from Cd. Dilutions that promoted root elongation contained supra-optimal concentrations of IAA, 1–3 orders of magnitude higher than those required for optimal enhancement by synthetic IAA. It is suggested that the bacteria produce in culture an IAA-antagonist or growth inhibitor that decreases the effectiveness of IAA action. The large variability reported for the effects ofAzospirillum on root elongation could be the result of the opposite effects on root elongation of IAA and other compounds, produced by the bacteria.     

Effects of the Inoculation of <Emphasis Type="Italic">Burkholderia vietnamensis</Emphasis> and Related Endophytic Diazotrophic Bacteria on Grain Yield of Rice

During a survey of endophytic diazotrophic bacteria associated with different rice varieties in Tamilnadu, some “endophytes” were obtained. Thirteen bacterial isolates from surface-sterilized roots and shoots were obtained in pure culture, which produced indole acetic acid (IAA) and reduced acetylene to ethylene. Polymerase chain reaction (PCR) amplification confirmed the presence of nif-H gene in all the isolates. Morphological, biochemical, and molecular characteristics indicated that all of them belonged to the genus Burkholderia One of them, MGK3, was consistently more active in reducing acetylene, and 16S rDNA sequences of isolate MGK3 confirmed its identification as Burkholderia vietnamiensis. Colonization of rice root was confirmed by strain MGK3 marked with gusA gene. The inoculated roots showed a blue color, which was most intense at the points of lateral root emergence and at the root tip. Transverse sections of roots, 15 days after inoculation, revealed beta-glucuronidase (GUS) activity within many of the cortical intercellular spaces next to the stele and within the aerenchyma. Nitrogen fixation was quantified by using ¹⁵N isotope dilution method with two different cultivars grown in pot and field experiments. Higher nitrogen fixation was observed in variety Ponni than in ADT-43, where nearly 42% (field) and 40% (pot) of the nitrogen was derived from the atmosphere (% Ndfa). Isolate MGK3 was used to inoculate rice seedlings in a comparison with four other diazotrophs, viz., Gluconacetobacter diazotrophicus LMG7603, Herbaspirillum seropedicae LMG6513, Azospirillum lipoferum 4B LMG4348, and B. vietnamiensis LMG10929. They were used to conduct two pot and four field inoculation experiments. MGK3 alone, and combined with other diazotrophs, performed best under both pot and field conditions: combined inoculation produced yield increases between 9.5 and 23.6%, while MGK3 alone increased yield by 5.6 to 12.16% over the uninoculated control treatment.     

The Rhizobium sp. strain NGR234 systemically suppresses arbuscular mycorrhizal root colonization in a split‐root system of barley (Hordeum vulgare)

Nitrogen‐fixing bacteria (rhizobia) form a nodule symbiosis with legumes, but also induce certain effects on non‐host plants. Here, we used a split‐root system of barley to examine whether inoculation with Rhizobium sp. strain NGR234 on one side of a split‐root system systemically affects arbuscular mycorrhizal (AM) root colonization on the other side. Mutant strains of NGR234 deficient in Nod factor production (strain NGRΔnodABC), perception of flavonoids (strain NGRΔnodD1) and secretion of type 3 effector proteins (strain NGRΩrhcN) were included in this study. Inoculation resulted in a systemic reduction of AM root colonization with all tested strains. However, the suppressive effect of strain NGRΩrhcN was less pronounced. Moreover, levels of salicylic acid, an endogenous molecule related to plant defense, were increased in roots challenged with rhizobia. These data indicate that barley roots perceived NGR234 and that a systemic regulatory mechanism of AM root colonization was activated. The suppressive effect appears to be Nod factor independent, but enhanced by type 3 effector proteins of NGR234.     

Nitrate-dependent control of root architecture and N nutrition are altered by a plant growth-promoting Phyllobacterium sp

Both root architecture and plant N nutrition are altered by inoculation with the plant growth-promoting rhizobacteria (PGPR) Phyllobacterium strain STM196. It is known that NO₃⁻ and N metabolites can act as regulatory signals on root development and N transporters. In this study, we investigate the possible interrelated effects on root development and N transport. We show that the inhibition of Arabidopsis lateral root growth by high external NO₃⁻ is overridden by Phyllobacterium inoculation. However, the leaf NO₃⁻ pool remained unchanged in inoculated plants. By contrast, the Gln root pool was reduced in inoculated plants. Unexpectedly, NO₃⁻ influx and the expression levels of AtNRT1.1 and AtNRT2.1 genes coding for root NO₃⁻ transporters were also decreased after 8 days of Phyllobacterium inoculation. Although the mechanisms by which PGPR exert their positive effects remain unknown, our data show that they can optimize plant development independently from N supply, thus alleviating the regulatory mechanisms that operate in axenic conditions. In addition, we found that Phyllobacterium sp. elicited a very strong induction of AtNRT2.5 and AtNRT2.6, both genes preferentially expressed in the shoots whose functions are unknown.     

Inhibition of primary roots and stimulation of lateral root development in Arabidopsis thaliana by the rhizobacterium Serratia marcescens 90–166 is through both auxin-dependent and -independent signaling pathways

The rhizobacterium Serratia marcescens strain 90–166 was previously reported to promote plant growth and induce resistance in Arabidopsis thaliana. In this study, the influence of strain 90-166 on root development was studied in vitro. We observed inhibition of primary root elongation, enhanced lateral root emergence, and early emergence of second order lateral roots after inoculation with strain 90–166 at a certain distance from the root. Using the DR5::GUS transgenic A. thaliana plant and an auxin transport inhibitor, N-1-naphthylphthalamic acid, the altered root development was still elicited by strain 90–166, indicating that this was not a result of changes in plant auxin levels. Intriguingly, indole-3-acetic acid, a major auxin chemical, was only identified just above the detection limit in liquid culture of strain 90–166 using liquid chromatography-mass spectrometry. Focusing on bacterial determinants of the root alterations, we found that primary root elongation was inhibited in seedlings treated with cell supernatant (secreted compounds), while lateral root formation was induced in seedlings treated with lysate supernatant (intracellular compounds). Further study revealed that the alteration of root development elicited by strain 90–166 involved the jasmonate, ethylene, and salicylic acid signaling pathways. Collectively, our results suggest that strain 90–166 can contribute to plant root development via multiple signaling pathways.     

The responsiveness of the <Emphasis Type="Italic">IAA2</Emphasis> promoter to IAA and IBA is differentially affected in <Emphasis Type="Italic">Arabidopsis</Emphasis> roots and shoots by flavonoids

The structural features of flavonoids which are involved in the modulation of auxin distribution in Arabidopsis thaliana were evaluated. An auxin-inducible promoter IAA2 fused to a reporter gene (GUS) was used to monitor the tissue responsiveness to auxins. The following aspects were investigated: 1) the influence of flavonoids (quercetin, naringenin, kaempferol, myricetin and isorhamnetin) on the distribution of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) in roots and leaves, 2) differences in flavonoid uptake into roots and shoots depending on flavonoid concentration in the medium, and 3) influence of structurally different flavonoids on the gravitropic response and growth of roots. The same flavonoids differently affected IAA and IBA distribution in leaves and roots. There were several structural requirements for the flavonoids which resulted in the changes of auxin response/distribution. Great differences between the ability of shoots and roots to take up quercetin were showed. Also, flavonoids influenced gravitropism and root growth of Arabidopsis seedlings in a structure-dependent manner.     

Differential IAA dose response relations of the axr1 and axr2 mutants of Arabidopsis

In comparison to wild type Arabidopsis thaliana, the auxin resistant mutants axr1 and axr2 exhibit reduced inhibition of root elongation in response to auxins. Several auxin-regulated physiological processes are also altered in the mutant plants. When wild-type, axr1 and axr2 seedlings were grown in darkness on media containing indoleacetic acid (IAA), promotion of root growth was observed at low concentrations of IAA (10^?11 to 10^?7M) in 5-day-old axr2 seedlings, but not in axr1 or wild-type seedlings. In axr1 there was little or no measurable root growth response over the same concentration range. In wild type, root growth was inhibited at concentrations greater than 10^?10M and no detectable root growth response was observed at lower concentrations. In addition, production of lateral roots in response to IAA increased in axr2 seedlings and decreased in axr1 seedlings relative to wild type. Promotion of root elongation and initiation of lateral roots in axr2 seedlings in response to auxin indicate that axr2 seedlings are able to perceive and respond to IAA.     

Azospirillum brasilense ameliorates the response of Arabidopsis thaliana to drought mainly via enhancement of ABA levels

Production of phytohormones is one of the main mechanisms to explain the beneficial effects of plant growth‐promoting rhizobacteria (PGPR) such as Azospirillum sp. The PGPRs induce plant growth and development, and reduce stress susceptibility. However, little is known regarding the stress‐related phytohormone abscisic acid (ABA) produced by bacteria. We investigated the effects of Azospirillum brasilense Sp 245 strain on Arabidopsis thaliana Col‐0 and aba2‐1 mutant plants, evaluating the morphophysiological and biochemical responses when watered and in drought. We used an in vitro‐grown system to study changes in the root volume and architecture after inoculation with Azospirillum in Arabidopsis wild‐type Col‐0 and on the mutant aba2‐1, during early growth. To examine Arabidopsis development and reproductive success as affected by the bacteria, ABA and drought, a pot experiment using Arabidopsis Col‐0 plants was also carried out. Azospirillum brasilense augmented plant biomass, altered root architecture by increasing lateral roots number, stimulated photosynthetic and photoprotective pigments and retarded water loss in correlation with incremented ABA levels. As well, inoculation improved plants seed yield, plants survival, proline levels and relative leaf water content; it also decreased stomatal conductance, malondialdehyde and relative soil water content in plants submitted to drought. Arabidopsis inoculation with A. brasilense improved plants performance, especially in drought.     

Wheat inoculation with Azospirillum brasilense Sp6 and some mutants altered in nitrogen fixation and indole-3-acetic acid production

Abstract Inoculation of wheat seedlings with Azospirillum brasilense Sp6 produced an increase in the number and length of the lateral roots as a plant response. Inoculation with a Nif⁻ mutant, A. brasilense SpF103, which is producer of indole-3-acetic acid (IAA), yielded a very similar plant response. However, inoculation with a Nif⁻ mutant, A. brasilense SpF57, which is a low producer of IAA, did not elitic any response from the plant. The data suggest that the root system response of wheat seedlings to bacterial inoculation is due mainly to production of auxin-type substances by the microorganism.     

Effect of time of inoculation on in vitro ectomycorrhizal colonization and nodule initiation in Acacia holosericea seedlings

The complex interactions that occur in systems with more than one type of symbiosis were studied using one isolate of Bradyrhizobium sp. and the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker and Couch inoculated on to the roots of Acacia holosericea A. Cunn. ex G. Don in vitro. After a single inoculation with Bradyrhizobium sp., bacteria typically entered the roots by forming infection threads in the root hair cells via the curling point of the root hair and/ or after intercellular penetration. Sheath formation and intercellular penetration were observed on Acacia roots after a single inoculation with Pisolithus tinctorius but no radial elongation of epidermal cells. Simultaneous inoculation with both microorganisms resulted in nodules and ectomycorrhiza on the root system, occasionally on the same lateral root. On lateral roots bearing nodules and ectomycorrhiza, the nodulation site was characterized by the presence of a nodule meristem and the absence of an infection thread; sheath formation and Hartig net development occurred regularly in the region of the roots adjacent to nodules. Prior inoculation with Bradyrhizobium sp. did not inhibit ectomycorrhizal colonization in root segments adjacent to nodules in which nodule meristems and infection threads were clearly present. Conversely, in ectomycorrhizae inoculated by bacteria, the nodule meristem and the infection thread were typically absent. These results show that simultaneous inoculation with both microorganisms inhibits infection thread development, thus conferring an advantage on fungal hyphae in the competition for infection sites. This suggests that fungal hyphae can modify directly and/or indirectly the recognition factors leading to nodule meristem initiation and infection thread development.     

Different Behaviour of Indole-3-Acetic Acid and Indole-3-Butyric Acid in Stimulating Lateral Root Development in Rice (Oryza sativa L.)

The plant hormone auxin has been shown to be involved in lateral root development and application of auxins, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA), increases the number of lateral roots in several plants. We found that the effects of two auxins on lateral root development in the indica rice (Oryza sativa L. cv. IR8) were totally different from each other depending on the application method. When the roots were incubated with an auxin solution, IAA inhibited lateral root development, while IBA was stimulatory. In contrast, when auxin was applied to the shoot, IAA promoted lateral root formation, while IBA did not. The transport of [³H]IAA from shoot to root occurred efficiently (% transported compared to supplied) but that of [³H]IBA did not, which is consistent with the stimulatory effect of IAA on lateral root production when applied to the shoot. The auxin action of IBA has been suggested to be due to its conversion to IAA. However, in rice IAA competitively inhibited the stimulatory effect of IBA on lateral root formation when they were applied to the incubation solution, suggesting that the stimulatory effect of IBA on lateral root development is not through its conversion to IAA.     

Effect of inoculation ofAzospirillum lipoferum on nitrogen fixation in rhizosphere soil,their association with root,yield and nitrogen uptake by mustard (Brassica juncea)

Summary A microplot field experiment was conducted in the presence or absence of P and N application to evaluate the influence of the seed inoculation of mustard (cv. Baruna T₅₉) withAzospirillum lipoferum on N₂-fixation in rhizosphere, association of the bacteria with the roots and grain yield and N uptake. Inoculation significantly increased the N content in rhizosphere soil particularly at early stage (40 days) of plant growth, which was accompanied by the increased association of the bacteria (A. lipoferum) in rhizosphere soil, root surface washing and surface-sterilized macerated root. A significant increase in grain yield and N uptake was also observed due to inoculation. Application of P particularly at the 20 kg. ha^–1 level further enhanced the beneficial effect ofAzospirillum lipoferum inoculation, while N addition markedly reduced such an effect.