The Growth-Inhibitory Effect of Increased Planting Density Can Be Reduced by Abscisic Acid-Degrading Bacteria.

High-density planting can increase crop productivity per unit area of cultivated land. However, the application of this technology is limited by the inhibition of plant growth in the presence of neighbors, which is not only due to their competition for resources but is also caused by growth regulators. Specifically, the abscisic acid (ABA) accumulated in plants under increased density of planting has been shown to inhibit their growth. The goal of the present study was to test the hypothesis that bacteria capable of degrading ABA can reduce the growth inhibitory effect of competition among plants by reducing concentration of this hormone in plants and their environment. Lettuce plants were grown both individually and three per pot; the rhizosphere was inoculated with a strain of Pseudomonas plecoglossicida 2.4-D capable of degrading ABA. Plant growth was recorded in parallel with immunoassaying ABA concentration in the pots and plants. The presence of neighbors indeed inhibited the growth of non-inoculated lettuce plants. Bacterial inoculation positively affected the growth of grouped plants, reducing the negative effects of competition. The bacteria-induced increase in the mass of competing plants was greater than that in the single ones. ABA concentration was increased by the presence of neighbors both in soil and plant shoots associated with the inhibition of plant growth, but accumulation of this hormone as well as inhibition of the growth of grouped plants was prevented by bacteria. The results confirm the role of ABA in the response of plants to the presence of competitors as well as the possibility of reducing the negative effect of competition on plant productivity with the help of bacteria capable of degrading this hormone.

Influence of Plant Growth-Promoting Rhizobacteria on the Formation of Apoplastic Barriers and Uptake of Water and Potassium by Wheat Plants.

The formation of apoplastic barriers is important for controlling the uptake of water and ions by plants, thereby influencing plant growth. However, the effects of plant growth-promoting bacteria on the formation of apoplastic barriers, and the relationship between these effects and the ability of bacteria to influence the content of hormones in plants, have not been sufficiently studied. The content of cytokinins, auxins and potassium, characteristics of water relations, deposition of lignin and suberin and the formation of Casparian bands in the root endodermis of durum wheat (Triticum durum Desf.) plants were evaluated after the introduction of the cytokinin-producing bacterium Bacillus subtilis IB-22 or the auxin-producing bacterium Pseudomonas mandelii IB-Ki14 into their rhizosphere. The experiments were carried out in laboratory conditions in pots with agrochernozem at an optimal level of illumination and watering. Both strains increased shoot biomass, leaf area and chlorophyll content in leaves. Bacteria enhanced the formation of apoplastic barriers, which were most pronounced when plants were treated with P. mandelii IB-Ki14. At the same time, P. mandelii IB-Ki14 caused no decrease in the hydraulic conductivity, while inoculation with B. subtilis IB-22, increased hydraulic conductivity. Cell wall lignification reduced the potassium content in the roots, but did not affect its content in the shoots of plants inoculated with P. mandelii IB-Ki14. Inoculation with B. subtilis IB-22 did not change the potassium content in the roots, but increased it in the shoots.

Effects of a Pseudomonas Strain on the Lipid Transfer Proteins, Appoplast Barriers and Activity of Aquaporins Associated with Hydraulic Conductance of Pea Plants.

Lipid transfer proteins (LTPs) are known to be involved in suberin deposition in the Casparian bands of pea roots, thereby reinforcing apoplast barriers. Moreover, the Pseudomonas mandelii IB-Ki14 strain accelerated formation of the Casparian bands in wheat plants, although involvement of LTPs in the process was not studied. Here, we investigated the effects of P. mandelii IB-Ki14 on LTPs, formation of the Casparian bands, hydraulic conductance and activity of aquaporins (AQPs) in pea plants. RT PCR showed a 1.6-1.9-fold up-regulation of the PsLTP-coding genes and an increase in the abundance of LTP proteins in the phloem of pea roots induced by the treatment with P. mandelii IB-Ki14. The treatment was accompanied with increased deposition of suberin in the Casparian bands. Hydraulic conductance did not decrease in association with the bacterial treatment despite strengthening of the apoplast barriers. At the same time, the Fenton reagent, serving as an AQPs inhibitor, decreased hydraulic conductance to a greater extent in treated plants relative to the control group, indicating an increase in the AQP activity by the bacteria. We hypothesize that P. mandelii IB-Ki14 stimulates deposition of suberin, in the biosynthesis of which LTPs are involved, and increases aquaporin activity, which in turn prevents a decrease in hydraulic conductance due to formation of the apoplast barriers in pea roots.

The Effects of Rhizosphere Inoculation with Pseudomonas mandelii on Formation of Apoplast Barriers, HvPIP2 Aquaporins and Hydraulic Conductance of Barley.

Pseudomonas mandelii strain IB-Ki14 has recently been shown to strengthen the apoplastic barriers of salt-stressed plants, which prevents the entry of toxic sodium. It was of interest to find out whether the same effect manifests itself in the absence of salinity and how this affects the hydraulic conductivity of barley plants. Berberine staining confirmed that the bacterial treatment enhanced the deposition of lignin and suberin and formation of Casparian bands in the roots of barley plants. The calculation of hydraulic conductance by relating transpiration to leaf water potential showed that it did not decrease in bacteria-treated plants. We hypothesized that reduced apoplastic conductivity could be compensated by the higher conductivity of the water pathway across the membranes. This assumption was confirmed by the results of the immunolocalization of HvPIP2;5 aquaporins with specific antibodies, showing their increased abundance around the areas of the endodermis and exodermis of bacteria-treated plants. The immunolocalization with antibodies against auxins and abscisic acid revealed elevated levels of these hormones in the roots of plants treated with bacteria. This root accumulation of hormones is likely to be associated with the ability of Pseudomonas mandelii IB-Ki14 to synthesize these hormones. The involvement of abscisic acid in the control of aquaporin abundance and auxins-in the regulation of and formation of apoplast barriers-is discussed.

Effects of Phytohormone-Producing Rhizobacteria on Casparian Band Formation, Ion Homeostasis and Salt Tolerance of Durum Wheat.

Inoculation with plant growth-promoting rhizobacteria can increase plant salt resistance. We aimed to reveal bacterial effects on the formation of apoplastic barriers and hormone concentration in relation to maintaining ion homeostasis and growth of salt-stressed plants. The rhizosphere of a durum wheat variety was inoculated with cytokinin-producing Bacillus subtilis and auxin-producing Pseudomonas mandelii strains. Plant growth, deposition of lignin and suberin and concentrations of sodium, potassium, phosphorus and hormones were studied in the plants exposed to salinity. Accumulation of sodium inhibited plant growth accompanied by a decline in potassium in roots and phosphorus in shoots of the salt-stressed plants. Inoculation with both bacterial strains resulted in faster appearance of Casparian bands in root endodermis and an increased growth of salt-stressed plants. B. subtilis prevented the decline in both potassium and phosphorus concentrations and increased concentration of cytokinins in salt-stressed plants. P. mandelii decreased the level of sodium accumulation and increased the concentration of auxin. Growth promotion was greater in plants inoculated with B. subtilis. Increased ion homeostasis may be related to the capacity of bacteria to accelerate the formation of Casparian bands preventing uncontrolled diffusion of solutes through the apoplast. We discuss the relative impacts of the decline in Na accumulation and maintenance of K and P content for growth improvement of salt-stressed plants and their possible relation to the changes in hormone concentration in plants.

Growth-Promoting Effect of Rhizobacterium (Bacillus subtilis IB22) in Salt-Stressed Barley Depends on Abscisic Acid Accumulation in the Roots.

An ABA-deficient barley mutant (Az34) and its parental cultivar (Steptoe) were compared. Plants of salt-stressed Az34 (100 mmol m-3 NaCl for 10 days) grown in sand were 40% smaller than those of "Steptoe", exhibited a lower leaf relative water content and lower ABA concentrations. Rhizosphere inoculation with IB22 increased plant growth of both genotypes. IB22 inoculation raised ABA in roots of salt-stressed plants by supplying ABA exogenously and by up-regulating ABA synthesis gene HvNCED2 and down-regulating ABA catabolic gene HvCYP707A1. Inoculation partially compensated for the inherent ABA deficiency of the mutant. Transcript abundance of HvNCED2 and related HvNCED1 in the absence of inoculation was 10 times higher in roots than in shoots of both mutant and parent, indicating that ABA was mainly synthesized in roots. Under salt stress, accumulation of ABA in the roots of bacteria-treated plants was accompanied by a decline in shoot ABA suggesting bacterial inhibition of ABA transport from roots to shoots. ABA accumulation in the roots of bacteria-treated Az34 was accompanied by increased leaf hydration, the probable outcome of increased root hydraulic conductance. Thereby, we tested the hypothesis that the ability of rhizobacterium (Bacillus subtilis IB22) to modify responses of plants to salt stress depends on abscisic acid (ABA) accumulating in roots.

Effects of Plant Growth Promoting Rhizobacteria on the Content of Abscisic Acid and Salt Resistance of Wheat Plants.

Although salinity inhibits plant growth, application of appropriate rhizosphere bacteria can diminish this negative effect. We studied one possible mechanism that may underlie this beneficial response. Wheat plants were inoculated with Bacillus subtilis IB-22 and Pseudomonas mandelii IB-Ki14 and their consequences for growth, water relations, and concentrations of the hormone abscisic acid (ABA) were followed in the presence of soil salinity. Salinity alone increased ABA concentration in wheat leaves and roots and this was associated with decreased stomatal conductance, but also with chlorophyll loss. Bacterial treatment raised ABA concentrations in roots, suppressed accumulation of leaf ABA, decreased chlorophyll loss, and promoted leaf area and transpiration. However, water balance was maintained due to increased water uptake by inoculated plants, brought about in part by a larger root system. The effect may be the outcome of ABA action since the hormone is known to maintain root extension in stressed plants. Root ABA concentration was highest in salt-stressed plants inoculated with B. subtilis and this contributed to greater root hydraulic conductivity. We conclude that bacteria can raise salt resistance in wheat by increasing root ABA, resulting in larger root systems that can also possess enhanced hydraulic conductivity thereby supporting better-hydrated leaves.

Cytokinin producing bacteria stimulate amino acid deposition by wheat roots.

Phytohormone production is one mechanism by which rhizobacteria can stimulate plant growth, but it is not clear whether the bacteria gain from this mechanism. The hypothesis that microbial-derived cytokinin phytohormones stimulate root exudation of amino acids was tested. The rhizosphere of wheat plants was drenched with the synthetic cytokinin trans-zeatin or inoculated with Bacillus subtilis IB-22 (which produces zeatin type cytokinins) or B. subtilis IB-21 (which failed to accumulate cytokinins). Growing plants in a split root system allowed spatial separation of zeatin application or rhizobacterial inoculation to one compartment and analyses of amino acid release from roots (rhizodeposition) into the other compartment (without either microbial inoculation or treatment with exogenous hormone). Supplying B. subtilis IB-22 or zeatin to either the whole root system or half of the roots increased concentrations of amino acids in the soil solution although the magnitude of the increase was greater when whole roots were treated. There was some similarity in amino acid concentrations induced by either bacterial or zeatin treatment. Thus B. subtilis IB-22 increased amino acid rhizodeposition, likely due to its ability to produce cytokinins. Furthermore, B. subtilis strain IB-21, which failed to accumulate cytokinins in culture media, did not significantly affect amino acid concentrations in the wheat rhizosphere. The ability of rhizobacteria to produce cytokinins and thereby stimulate rhizodeposition may be important in enhancing rhizobacterial colonization of the rhizoplane.