Image_1_The PGPR Bacillus aryabhattai promotes soybean growth via nutrient and chlorophyll maintenance and the production of butanoic acid.tif

Plant growth-promoting rhizobacteria (PGPR) colonize plant roots, establish a mutualistic relationship with the plants and help them grow better. This study reports novel findings on the plant growth-promoting effects of the PGPR Bacillus aryabhattai. Soil was collected from a soybean field, PGPR were isolated, identified, and characterized for their ability to promote plant growth and development. The bacterium was isolated from the soybean rhizosphere and identified as B. aryabhattai strain SRB02 via 16s rRNA sequencing. As shown by SEM, the bacterium successfully colonized rice and soybean roots within 2 days and significantly promoted the growth of the GA-deficient rice cultivar Waito-C within 10 days, as well as the growth of soybean plants with at least six times longer shoots, roots, higher chlorophyll content, fresh, and dry weight after 10 days of inoculation. ICP analysis showed up to a 100% increase in the quantity of 18 different amino acids in the SRB02-treated soybean plants. Furthermore, the 2-DE gel assay indicated the presence of several differentially expressed proteins in soybean leaves after 24 hrs of SRB02 application. MALDI-TOF-MS identified β-conglycinin and glycinin along with several other proteins that were traced back to their respective genes. Analysis of bacterial culture filtrates via GCMS recorded significantly higher quantities of butanoic acid which was approximately 42% of all the metabolites found in the filtrates. The application of 100 ppm butanoic acid had significantly positive effects on plant growth via chlorophyll maintenance. These results establish the suitability of B. aryabhattai as a promising PGPR for field application in various crops.</p

Table_1_The PGPR Bacillus aryabhattai promotes soybean growth via nutrient and chlorophyll maintenance and the production of butanoic acid.docx

Plant growth-promoting rhizobacteria (PGPR) colonize plant roots, establish a mutualistic relationship with the plants and help them grow better. This study reports novel findings on the plant growth-promoting effects of the PGPR Bacillus aryabhattai. Soil was collected from a soybean field, PGPR were isolated, identified, and characterized for their ability to promote plant growth and development. The bacterium was isolated from the soybean rhizosphere and identified as B. aryabhattai strain SRB02 via 16s rRNA sequencing. As shown by SEM, the bacterium successfully colonized rice and soybean roots within 2 days and significantly promoted the growth of the GA-deficient rice cultivar Waito-C within 10 days, as well as the growth of soybean plants with at least six times longer shoots, roots, higher chlorophyll content, fresh, and dry weight after 10 days of inoculation. ICP analysis showed up to a 100% increase in the quantity of 18 different amino acids in the SRB02-treated soybean plants. Furthermore, the 2-DE gel assay indicated the presence of several differentially expressed proteins in soybean leaves after 24 hrs of SRB02 application. MALDI-TOF-MS identified β-conglycinin and glycinin along with several other proteins that were traced back to their respective genes. Analysis of bacterial culture filtrates via GCMS recorded significantly higher quantities of butanoic acid which was approximately 42% of all the metabolites found in the filtrates. The application of 100 ppm butanoic acid had significantly positive effects on plant growth via chlorophyll maintenance. These results establish the suitability of B. aryabhattai as a promising PGPR for field application in various crops.</p

Development of necrosis (HR) on leaves of NT (L<i>er</i>) and P6-transgenic (A7 & B6) Arabidopsis.

<p>(A) Effect of SA-treatment. Panels show from top to bottom: untreated leaves and treated with 1.0 mM SA. (B) Effect of inoculation with virulent or avirulent <i>P</i>st. Panels show from top to bottom, uninfiltrated controls, leaves infiltrated with <i>Ps</i>t (DC3000) and leaves infiltrated with <i>Ps</i>t (<i>AvrB</i>). Black arrows indicate areas of leaf infiltrated with <i>Ps</i>t <i>AvrB</i> (bottom row). Necrosis was visualized by staining leaves with Trypan Blue 24 h after SA-treatment or infiltration with <i>Ps</i>t.</p

The effects of expression of P6 on NPR1.

<p>(A) Western blots of protein extracted from <i>npr1</i> mutant, P6-transgenic (A7, B6) and NT (L<i>er</i>) plants, separated by polyacrylamide gel electrophoresis. Tissue was harvested from plants either before (4 lanes on left) or 12 hours after (4 lanes on right) treatment with 1.0 mM SA. Upper panel shows blots probed with antibody to NPR1 and bands visualized by chemiluminescence. Bars on left indicate mobility of molecular weight markers; arrow indicates expected mobility of NPR1. Lower panel shows Ponceau-stained loading control; arrow indicates mobility of Rubisco Large Subunit (RBCL). (B) Western blots showing NPR1 accumulating in <i>N. benthamiana</i> leaves following agroinfiltration with a binary vector expressing NPR1 (HA-tagged at the N-terminus) under the control of a 35S promoter. Upper panel shows blots probed with anti-HA antibody and bands visualized by chemiluminescence. Lower panel shows Ponceau-stained loading control – arrow indicates mobility of Rubisco Large Subunit (RBCL). (Lane 1) HA:NPR1 co-infiltrated with empty binary vector pGWB17; (lane 2) HA:NPR1 co-infiltrated with P6 expressing binary vector pGWB-P6myc. (C) NPR1 transcript levels (in arbitrary units) determined by qPCR in P6-transgenic (A7 and B6) and NT (L<i>er</i>) plants. Bars represent mean levels of 3 independent biological samples each comprising pooled tissue from 3 plants. Error bars show standard deviations. (D) Confocal microscope images of representative pairs of guard cells from transgenic plants expressing an NPR1:GFP fusion. Panels a–c are in a NT background; panels d-f are in a P6-transgenic background (the progeny of a typical cross between the NPR1:GFP transgenic and B6). Panels a & d show samples from uninduced seedlings (infiltrated with water). Panels b & e and c & f show samples from seedlings following 5 min and 40 min (respectively) infiltration with 1.0 mM SA. Arrows indicate the nucleus.</p

Shoot length and fresh weight of rice seedlings in response to SA application (0.5 and 1.0 mM) in the presence and absence of salt stress (100 mM).

<p>Shoot length and fresh weight of rice seedlings in response to SA application (0.5 and 1.0 mM) in the presence and absence of salt stress (100 mM).</p

Effects of single application of SA or NaCl, and combination treatment of SA and NaCl on <i>OsCATA</i> expression in rice plants.

<p>Lowercase letters indicate a significant difference among treatments. Vertical bars with error bars indicate the average ± standard error (n = 3), and different letters indicate a significance difference at P < 0.05. All data were analyzed by DMRT.</p

Quantification of <i>AtPR-1</i> and <i>NbPR-1a</i> transcripts in response to pathogens.

<p>(A) <i>PR-1</i> transcripts in mock-inoculated and CaMV-infected NT (L<i>er</i>) and P6-transgenic (A7) Arabidopsis, determined by qPCR. Bars show mean levels (in arbitrary units) of 3 independent biological samples each comprising pooled tissue from 3 plants. Samples were harvested 14 dpi. Error bars show standard deviations. (B) <i>PR-1</i> transcripts in uninoculated controls and <i>Ps</i>t (<i>AvrB</i>) inoculated NT (L<i>er</i>) and P6-transgenic (A7) Arabidopsis, determined by qPCR. Bars show mean levels (in arbitrary units) of 3 independent biological samples each comprising pooled inoculated leaves from 3 plants. Samples were harvested 48 h after infiltration. Error bars show standard deviations. (C) <i>PR-1a</i> transcripts, determined by qPCR, in <i>N. benthamiana</i> leaves harvested 48 h after agroinfiltration. Samples were (U) uninfiltrated leaves, and leaves infiltrated with Agrobacterium carrying the following vectors (EV) pJO530, (pJO-BJI) pJO-BJI, (P6myc) pGWB-P6myc, (P6Y305P) pGWB-P6Y305P. Bars show mean levels (in arbitrary units) of 3 independent biological samples each comprising 3 pooled infiltrated leaf sections. Error bars show standard deviations.</p

Changes in chlorophyll fluorescence and the electric conductivity ratio after combination treatment of rice plants with SA and NaCl, and single treatment with SA or NaCl.

<p>A and B indicate changes in chlorophyll fluorescence and the EC ratio during different treatment times, respectively. Each symbol indicates the average, and the bar indicates ± standard error (n = 3).</p

Levels of free and conjugated SA in P6-transgenic and non-transgenic Arabidopsis following inoculation with <i>Ps</i>t (<i>AvrB</i>).

<p>(A) free SA, (B) SA-conjugates (SA-β-glucoside) in leaves of NT (L<i>er</i>) and P6-transgenic (A7 and B6) plants inoculated on a single leaf with 1.2×10³ cfu of bacteria. Leaves from uninoculated plants were used as controls. Each sample comprised the pooled tissue from 10 leaves harvested 48 h after inoculation. Bars show mean values from 3 samples, error bars indicate standard error.</p

Effects of single application of SA or NaCl, and combination treatment of SA and NaCl on <i>OsAPX1</i> expression in rice plants.

<p>Lowercase letters indicate a significant difference among treatments. Vertical bars with error bars indicate the average ± standard error (n = 3), and different letters indicate significant differences at P < 0.05. All data were analyzed by DMRT.</p