Screening of bacterial isolates for IAA production
In general, all the isolates produced IAA indicating the ubiquitous occurrence of IAA-producing potentiality in bacteria, particularly from the rhizosphere of chickpeas. Out of 80 bacteria, 67 bacteria were found to produce IAA indicating the IAA production is common function of almost all bacteria. The bacteria produced IAA through tryptophan-dependent pathway in the range of 3.8 to 70.0 µg ml− 1 whereas produced in the range of 1.13 to 53.33 µg ml− 1 through tryptophan independent pathway indicating efficient functionality of tryptophan dependent pathway for IAA production by bacteria (Supplementary Table 2). The prominent bacterial isolates producing substantial amount of IAA were KS-14, BEMS-9-1, BS-2, BKS-2, RS-6, IS-4, IS-7, BS-4, GS-5, RS-3, IS-11 etc (Supplementary Table 2).
IAA production in Tris-minimal (TM) broth supplemented with casein enzymic hydrolysate and Acicase, Vitamin Free (acid hydrolysis of milk protein)
Eight potential high IAA-producing bacterial isolates along with the 5 reference bacterial strains were evaluated to observe the operation of tryptophan-dependent or - independent pathways for IAA biosynthesis. Out of these eight bacterial isolates, KS-14, BEMS-9-1 and BS-2 produced significantly higher amounts of IAA i.e, 24, 21 and 18 µgml− 1 in TM broth supplemented with casein enzymic hydrolysate whereas produced the lower amounts, i.e., 12, 08 and 07 µg ml− 1 in TM broth with Acicase (casein acid hydrolysate), respectively (Fig. 1).
Identification of IAA-producing bacterial isolates
All the eight isolates had medium to large creamy white colour colonies of irregular and undulated or lobate shape with smooth surfaces in the early growth stages, which dried in the later growth stages. (Supplementary Table 3). In particular, the small cells of isolate KS-14 and the long rod-shaped cells of isolate BS-2 were arranged either single or in chains. All the isolates showed Gram’s positive reaction and formed endospores except B. tequilensis (BS-2) which did not show clear endospore formation despite repeated staining with malachite green. (Fig. 2). The sequencing of 16S rRNA genes of these isolates revealed that the isolates KS-14, BEMS-9-1 and BS-2 were closely related to Brevibacillus formosus DSM 9885T (99.93%), Bacillus paramycoides NH24A2T (99.93%) and Bacillus tequilensis KCTC 13622T (99.86%), respectively. Further, the phylogenetic analysis grouped these isolates into Brevibacillus and Bacillus clades. (Fig. 3).
IAA-producing bacteria with PGP traits
Brevibacillus formosus KS-14 was positive for siderophore and ammonium production but negative for phosphate and zinc solubilization. Bacillus paramycoides BEMS9-1 and Bacillus tequilensis BS-2 were positive for siderophore and ammonium production as well as phosphate and zinc solubilization. The PGP traits of Bacillus tequilensis BS-2 were comparatively elevated than the other two strains (Table 1).
Biocompatibility among bacteria
B. formosus KS-14 was compatible with B. paramycoides BEMS-9-1 but not compatible with B. tequilensis BS-2. B. paramycoides BEMS-9-1 was found compatible with B. tequilensis BS-2 (Supplementary Fig. 3).
Evaluation of IAA producing B. formosus KS-14, B. paramycoides BEMS-9-1 and B. tequilensis BS-2 on wheat crop growth under pot conditions
Effects on seed germination and vigour indices After 30 days of sowing, maximum germination of 88% was recorded when seeds were inoculated with 50% RDF + B. formosus KS-14 + B. paramycoides BEMS-9-1 followed by when inoculated with 50% RDF + B. formosus KS-14 alone. Percent germination of 77% was recorded with the absolute control after 30 days of sowing. Similarly, a significant increment of 4813 and 98 in vigour indices I and II, respectively, was recorded in plants inoculated with 50% RDF + B. formosus KS-14 + B. paramycoides BEMS-9-1 followed by 3813 and 81 in plants inoculated with 50% RDF + B. paramycoides BEMS-9-1 + B. tequilensis BS-2. The other treatments and absolute control had vigour indices I of 2034 and vigour indices II of 41 compared to vigour indices I of 2432 and vigour indices II of 58 in plants treated with the RDF (Table 2).
Table 2
Effects of IAA bacteria alone and their consortia on seed germination and vigour index-I and vigour-II of wheat
Treatments | Seed germination (%) | vigor index-I | vigor index-II |
Absolute control | 74.34 ± 2.70 | 2034.41 ± 21 | 41.34 ± 0.99 |
RDF* | 80.14 ± 5.61 | 2431.56 ± 51 | 58.32 ± 1.53 |
50% RDF + Brevibacillus formosus KS-14 | 83.14 ± 1.97 | 3561.5 ± 324 | 70.42 ± 0.75 |
50% RDF + Bacillus paramycoides BEMS-9-1 | 82.14 ± 1.38 | 3661.5 ± 35 | 70.42 ± 1.42 |
50% RDF + Bacillus tequilensis BS-2 | 80.12 ± 3.12 | 2513.16 ± 294 | 51.34 ± 1.22 |
50% RDF + Brevibacillus formosus KS-14 & Bacillus paramycoides BEMS-9-1 | 88.43 ± 3.15 | 4813.12 ± 468 | 98.43 ± 0.67 |
50% RDF + Brevibacillus formosus KS-14& Bacillus tequilensis BS-2 | 81.43 ± 3.26 | 2661.21 ± 381 | 71.00 ± 1.18 |
50% RDF + Bacillus paramycoides BEMS-9-1& Bacillus tequilensis BS-2 | 82.15 ± 0.45 | 3812.53 ± 467 | 72.23 ± 0.50 |
50% RDF + Brevibacillus formosus KS-14, Bacillus paramycoides BEMS-9-1& Bacillus tequilensis BS-2 | 80.3 ± 2.41 | 2571.31 ± 289 | 74.32 ± 1.11 |
LSD (P = 0.05) | 9.25 | 867.61 | 3.17 |
Plant growth and physiological parameters
It was observed that the treatment with 50% RDF + B. formosus KS-14 + B. paramycoides BEMS-9-1 significantly increased the plant height up to 81% and 26% over absolute control and RDF control, respectively. Shoot and root dry weight, chlorophyll content, root volume and root surface area had increased to 235.51%, 221.21%, 266.66%, 150.42% and 171.80% respectively, over absolute control and 128.66%, 156.79%, 81%, 88.45% and 108.50% respectively, over RDF control. On the contrary, the consortium treatments of 50% RDF + B. formosus KS-14, B. paramycoides BEMS-9-1 + B. tequilensis BS-2 and 50% RDF + B. formosus KS-14 + B. tequilensis BS-2 drastically reduced all growth parameters (Table 3).
Table 3
Effects of IAA bacteria alone and their consortia on plant growth parameters
Treatments | Plant height (cm) | Dry wt. of shoot (gm) | Dry wt. of root (gm) | Chlorophyll content (mg/g) | Root volume (cm3) | Root surface area (cm3) |
Absolute control | 35.12 ± 1.67 | 2.15 ± 0.578 | 0.66 ± 0.0557 | 1.56 ± 0.272 | 40.83 ± 1.33 | 98.54 ± 1.38 |
RDF* | 50.32 ± 1.56 | 3.14 ± 0.601 | 0.81 ± 0.0681 | 3.12 ± 0.587 | 54.31 ± 2.15 | 128.46 ± 4.91 |
50% RDF + Brevibacillus formosus KS-14 | 57.12 ± 1.85 | 5.21 ± 1.132 | 1.82 ± 0.0721 | 4.85 ± 0.547 | 87.43 ± 1.31 | 205.55 ± 6.02 |
50% RDF + Bacillus paramycoides BEMS-9-1 | 52.02 ± 1.65 | 4.92 ± 1.172 | 0.92 ± 0.0265 | 3.94 ± 0.576 | 61.42 ± 0.74 | 151.88 ± 6.40 |
50% RDF + Bacillus tequilensisBS-2 | 45.34 ± 0.27 | 3.13 ± 0.289 | 0.82 ± 0.0839 | 3.11 ± 0.302 | 46.31 ± 2.01 | 104.23 ± 8.27 |
50% RDF Brevibacillus formosus KS-14&Bacillus paramycoides BEMS-9-1 | 63.43 ± 1.64 | 7.18 ± 0.601 | 2.08 ± 0.0841 | 5.65 ± 0.469 | 102.35 ± 2.30 | 267.84 ± 10.53 |
50% RDF + Brevibacillus formosus KS-14& Bacillus tequilensis BS-2 | 51.34 ± 1.59 | 4.34 ± 0.702 | 0.82 ± 0.0306 | 3.45 ± 0.020 | 53.12 ± 1.27 | 132.70 ± 4.16 |
50% RDF + Bacillus paramycoides BEMS-9-1& Bacillus tequilensis BS-2 | 59.32 ± 2.54 | 6.81 ± 0.589 | 1.75 ± 0.3332 | 5.10 ± 0.508 | 80.46 ± 1.54 | 186.07 ± 8.99 |
50% RDF + Brevibacillus formosus KS-14, Bacillus paramycoides BEMS-9-1& Bacillus tequilensis BS-2 | 48.41 ± 2.12 | 5.12 ± 0.607 | 0.72 ± 0.0569 | 3.07 ± 0.330 | 71.37 ± 5.69 | 166.31 ± 0.25 |
LSD (P = 0.05) | 5.41 | 2.37 | 0.36 | 1.19 | 7.21 | 17.23 |
Soil enzymes activities and IAA production
In general, the dehydrogenase activity (DHA) and acid phosphatase activity (ACP) and alkaline phosphatase activity (ALP) were significantly increased after inoculation of consortium (50% RDF + B.formosus KS-14 + B. paramycoides BEMS-9-1). At 40 DAS, the treatments of 50% RDF + B. formosus KS-14 + B. paramycoides BEMS-9-1 and 50% RDF + B. formosus KS-14 increased the DHA to 97% and 75%, respectively over absolute control (33%) and RDF (18%). However, at 110 DAS, this increase was 191% over the absolute control and 35% over the RDF. At 40 DAS and 110 DAS, the treatment of a consortium (50% RDF + B. formosus KS-14 + B. paramycoides BEMS-9-1) increased ACP to 91% and 94%, respectively over absolute control (37%) and RDF (20%). Whereas, the treatment of another consortium (50% RDF + B. formosus KS-14) at 40 DAS and 110 DAS increased ACP to 67% and 91%, respectively, over absolute control. In particular, at 110 DAS, the pattern of ACP was disturbed and no pattern recorded. Similarly, at 40 DAS, ALP increased to 61% with the treatment of 50% RDF + B. formosus KS-14 + B. paramycoides BEMS-9-1; whereas, at 110 DAS the ALP increased up to 113%, over absolute control (27%) and RDF (25%). Further, at 40 DAS the amount of IAA in soil was significantly higher i.e., 73% over absolute control and 15% over RDF, after the treatment of 50% RDF + B. formosus KS-14 + B. paramycoides BEMS-9-1; however, at 110 DAS this increase was only 61.77% over the absolute control and 6.48% over RDF. Voidly, B. tequilensis BS-2 alone or in consortia did not influence the soil enzymatic activities and soil IAA production (Table 4).
Table 4
Effects of IAA bacteria alone and their consortia on soil enzyme activity and soil IAA production
Treatments | Dehydrogenase activity (pKat g-1 soil) | Acid phosphatase activity (nKat 100 g-1 soil) | Alkaline phosphatase activity (nKat 100g-1 soil) | Soil IAA (µg IAA g− 1) |
40 days | 110 Days | 40 days | 110 Days | 40 days | 110 Days | 40 days | 110 Days |
Absolute control | 3.80 ± 0.59 | 1.80 ± 0.11 | 25.6 ± 1.41 | 20.15 ± 1.15 | 48.43 ± 1.44 | 30.38 ± 1.84 | 42.34 ± 1.41 | 25.40 ± 1.70 |
RDF* | 5.65 ± 0.66 | 3.88 ± 0.57 | 35.78 ± 1.81 | 28.76 ± 1.68 | 61.56 ± 1.40 | 49.63 ± 2.27 | 63.50 ± 1.96 | 40.78 ± 1.32 |
50% RDF + Brevibacillus formosus KS-14 | 6.65 ± 0.59 | 4.68 ± 0.59 | 42.78 ± 1.24 | 38.50 ± 1.38 | 71.43 ± 1.67 | 64.67 ± 2.42 | 56.08 ± 3.43 | 39.67 ± 2.32 |
50% RDF + Bacillus paramycoides BEMS-9-1 | 5.90 ± 0.54 | 3.67 ± 0.60 | 38.88 ± 1.86 | 31.70 ± 1.49 | 62.98 ± 1.73 | 56.38 ± 2.46 | 51.83 ± 1.82 | 35.09 ± 0.03 |
50% RDF + Bacillus tequilensis BS-2 | 3.67 ± 0.38 | 2.13 ± 0.18 | 31.09 ± 1.39 | 22.98 ± 1.27 | 54.87 ± 2.39 | 45.89 ± 2.27 | 44.33 ± 2.18 | 30.0 ± 1.38 |
50% RDF + Brevibacillus formosus KS-14& Bacillus paramycoides BEMS-9-1 | 7.50 ± 0.83 | 5.24 ± 0.57 | 48.87 ± 1.84 | 39.07 ± 0.62 | 78.09 ± 4.38 | 61.97 ± 1.82 | 73.16 ± 1.85 | 41.09 ± 1.38 |
50% RDF + Brevibacillus formosus KS-14& Bacillus tequilensis BS-2 | 5.14 ± 0.58 | 3.23 ± 0.57 | 35.71 ± 1.23 | 25.54 ± 1.28 | 57.12 ± 3.58 | 48.93 ± 1.79 | 52.79 ± 1.98 | 32.45 ± 1.40 |
50% RDF + Bacillus paramycoides BEMS-9-1& Bacillus tequilensis BS-2 | 5.80 ± 0.56 | 4.89 ± 0.65 | 38.09 ± 1.03 | 29.87 ± 0.77 | 65.4 ± 3.19 | 56.54 ± 2.50 | 44.20 ± 2.38 | 28.90 ± 2.08 |
50% RDF + Brevibacillus formosus KS-14, Bacillus paramycoides BEMS-9-1& Bacillus tequilensisBS-2 | 5.75 ± 0.15 | 4.87 ± 0.48 | 31.45 ± 2.37 | 26.8 ± 0.19 | 62.06 ± 4.12 | 58.98 ± 2.50 | 50.54 ± 3.20 | 37.9 ± 4.47 |
LSD (P = 0.05) | 1.721 | 1.574 | 5.191 | 2.635 | 8.928 | 7.76 | 7.16 | 6.19 |
Micronutrient content in shoot, grains and rhizosphere soil of wheat
Nutrient assimilation by the shoot of wheat increased significantly upon inoculation of the high IAA-producing bacterial isolates. Inoculation of 50% RDF + B. formosus KS-14 + B. paramycoides BEMS-9-1 significantly increased the Zn, Fe, Cu and Mn contents up to 195%, 82%, 104% and 97% respectively, as compared to 82%, 24%, 68% and 27%, respectively in absolute control and RDF in the shoots of wheat plants. The other treatments did not show a significant variation for Zn content in shoots when compared to RDF but the content of other micronutrients in shoots (Fe, Cu and Mn) were significantly influenced by all the treatments over absolute control and RDF. Similarly, the consortium (50% RDF + B. formosus KS-14 + B. paramycoides BEMS-9-1) increased Zn, Fe, Cu and Mn concentrations up to 40%, 50%, 167% and 126%, respectively as compared to 27%, 18%, 87% and 34%, respectively over absolute control and RDF in wheat grains. The Cu concentration was significantly high after the treatment of 50% RDF + B. paramycoides BEMS-9-1 + B. tequilensis BS-2 and it was up to 182.84% over absolute control. Besides, the pattern of zinc content in grains under all the treatments was same as in the case of zinc content in shoots. The inoculation of 50% RDF + B. formosus KS-14 + B. paramycoides BEMS-9-1 significantly mobilized Zn, Fe, Cu and Mn content and increased their availability by 35%, 71%, 59% and 69% respectively, as compared to 27%, 18%, 87% and 34%, respectively over absolute control and RDF (Table 5).
Table 5
Effects of IAA bacteria alone and their consortia on macro- and micro-nutrient content in grain of wheat
Treatments | Macronutrients (%) | Macronutrients (µg g− 1 ) |
N | P | K | S | Zn | Fe | Cu | Mn |
Absolute control | 1.01 ± 0.08 | 0.14 ± 0.04 | 1.15 ± 0.03 | 0.11 ± 0.01 | 37.46 ± 2.30 | 47.19 ± 2.52 | 6.13 ± 0.58 | 9.14 ± 0.62 |
RDF* | 1.56 ± 0.28 | 0.18 ± 0.01 | 1.68 ± 0.06 | 0.18 ± 0.61 | 41.40 ± 1.49 | 61.44 ± 3.24 | 8.76 ± 0.58 | 13.39 ± 0.50 |
50% RDF + Brevibacillus formosus KS-14 | 2.16 ± 0.17 | 0.33 ± 0.05 | 2.35 ± 0.16 | 0.39 ± 0.10 | 43.27 ± 1.79 | 66.71 ± 3.26 | 13.35 ± 0.59 | 14.38 ± 0.60 |
50% RDF + Bacillus paramycoides BEMS-9-1 | 2.00 ± 0.11 | 0.26 ± 0.06 | 2.26 ± 0.60 | 0.23 ± 0.04 | 40.28 ± 1.09 | 61.34 ± 1.72 | 13.09 ± 0.55 | 14.23 ± 1.06 |
50% RDF + Bacillus tequilensis BS-2 | 1.41 ± 0.19 | 0.23 ± 0.04 | 1.92 ± 0.11 | 0.15 ± 0.03 | 35.69 ± 1.32 | 54.17 ± 2.85 | 10.27 ± 0.48 | 13.09 ± 0.56 |
50% RDF + Brevibacillus formosus KS-14& Bacillus paramycoides BEMS-9-1 | 3.20 ± 0.06 | 0.37 ± 0.05 | 3.27 ± 0.44 | 0.31 ± 0.01 | 52.40 ± 2.40 | 72.31 ± 3.91 | 16.34 ± 0.59 | 18.00 ± 0.85 |
50% RDF + Brevibacillus formosus KS-14& Bacillus tequilensis BS-2 | 2.10 ± 0.06 | 0.35 ± 0.06 | 3.51 ± 0.60 | 0.20 ± 0.01 | 38.10 ± 1.65 | 56.30 ± 3.35 | 13.07 ± 0.59 | 13.47 ± 0.41 |
50% RDF + Bacillus paramycoides BEMS-9-1& Bacillus tequilensis BS-2 | 3.01 ± 0.06 | 0.24 ± 0.04 | 3.23 ± 0.58 | 0.23 ± 0.04 | 42.5 ± 2.03 | 64.30 ± 3.15 | 17.25 ± 0.56 | 17.31 ± 0.64 |
50% RDF + Brevibacillus formosus KS-14, Bacillus paramycoides BEMS-9-1& Bacillus tequilensis BS-2 | 2.90 ± 0.49 | 0.25 ± 0.03 | 3.13 ± 0.58 | 0.14 ± 0.02 | 38.41 ± 1.45 | 66.40 ± 3.66 | 11.12 ± 0.89 | 14.31 ± 0.69 |
LSD (P = 0.05) | 0.68 | 0.12 | 1.27 | 0.61 | 5.38 | 8.59 | 1.90 | 2.16 |
Shoot weight and grain yield
At maturity, inoculation of 50% RDF + B. formosus KS-14 + B. paramycoides BEMS-9-1 significantly increased shoot and grain yield to 78% and 111% respectively, over the absolute control followed by the treatment of 50% RDF + B. paramycoides BEMS-9-1 + B. tequilensis BS-2 that increased shoot and grain yield to 66% and 108% respectively. Further, it was observed that shoot yield was significantly increased but not grains yield when compared with RDF. In the case of both shoot yield and grain yield, except for 50% RDF + B. formosus KS-14 + B. paramycoides BEMS-9-1, all other treatments did not significantly increased these over RDF. The percentage increase in grain yield was only 15%; whereas, shoot yield was 35% over the RDF. In this case, also, the detrimental effect of B. tequilensis BS-2 was noted on shoots and grain yield (Table 6). This might be due to either suppressing the activity of B. paramycoides BEMS-9-1 or by reducing the population of B. paramycoides BEMS-9-1 or both.
Table 6
Effects of IAA bacteria alone and their consortia on yield parameters of wheat
Treatments | Shoot (g/plant) | Grain (g/plant) |
Absolute control | 6.82 ± 0.423 | 2.65 ± 0.350 |
RDF* | 8.95 ± 0.890 | 4.84 ± 0.448 |
50% RDF + Brevibacillus formosus KS-14 | 10.12 ± 0.500 | 5.11 ± 0.105 |
50% RDF + Bacillus paramycoides BEMS-9-1 | 8.42 ± 0.748 | 4.85 ± 0.453 |
50% RDF + Bacillus tequilensis BS-2 | 7.23 ± 0.783 | 4.01 ± 0.313 |
50% RDF + Brevibacillus formosus KS-14& Bacillus paramycoides BEMS-9-1 | 12.12 ± 0.569 | 5.58 ± 0.465 |
50% RDF + Brevibacillus formosus KS-14& Bacillus tequilensis BS-2 | 8.12 ± 1.011 | 4.38 ± 0.254 |
50% RDF + Bacillus paramycoides BEMS-9-1& Bacillus tequilensis BS-2 | 11.34 ± 0.667 | 5.50 ± 0.298 |
50% RDF + Brevibacillus formosus KS-14, Bacillus paramycoides BEMS-9-1& Bacillus tequilensis BS-2 | 9.50 ± 1.094 | 4.50 ± 0.401 |
LSD (P = 0.05) | 2.193 | 1.044 |
Principal Component Analysis (PCA)
To differentiate between treatments and to assess the relationships between the variables, principal component analysis (PCA) was executed using mean values data of soil enzymes, plant physiological parameters and macro- and micro-nutrients associated with soil, shoot and grains. We compared the performance of various treatments based on the above-mentioned parameters with the scores resulting from PCA. Principal Components (PCs) with eigenvalues > 1.0 were selected as per the Kaiser concept, which explained maximum variance. The first PCA (Fig. 4a) separated the treatments based on the activity of soil enzymes and soil IAA represented most of the parameters towards RDF (T2), 50% RDF + Brevibacillus formosus KS-14 + Bacillus paramycoides BEMS-9-1 (T6), 50% RDF + Bacillus paramycoides BEMS-9-1 (T4), 50% RDF + Brevibacillus formosus KS-14 (T3) and 50% RDF + Brevibacillus formosus KS-14 + Bacillus paramycoides BEMS-9-1 + Bacillus tequilensis BS-2 (T9). PC1 and PC2 accounted for 83.52 and 8.4% variance, respectively, constituting total variance 91.9%. The factor loadings were highest for DHA I (0.98), ACP II (0.96) ALP I (0.96%), IAA II (0.85%) of soil in PC 1 (Supplementary Table 4A). The second PCA illustrates soil macro- and micro-nutrients (Fig. 4b). Two principal components were extracted, and accounted for 63.7% (PC1 38.4%; PC2 25.3%) total variance of the initial dataset. The PCA separated the treatments based on the status of soil nutrients represented most of the nutrients towards 50% RDF + Bacillus paramycoides BEMS-9-1 + Bacillus tequilensis BS-2 (T8), 50% RDF + Brevibacillus formosus KS-14 + Bacillus paramycoides BEMS-9-1 (T6), RDF (T2), 50% RDF + Brevibacillus formosus KS-14 (T3) and 50% RDF + Brevibacillus formosus KS-14 + Bacillus paramycoides BEMS-9-1 + Bacillus tequilensis BS-2 (T9). The highest positive loadings were displayed by N (0.92), Mn (0.87) and P (0.65) of soil in PC1 (Supplementary Table 4B). The third PCA (Fig. 4c) displayed plant physiological parameters. The two principal components explained total 92.8% variation with PC1 accounting for 88.9% and PC2 for 3.9% and separated treatments towards 50% RDF + Bacillus paramycoides BEMS-9-1 (T4), 50% RDF + Brevibacillus formosus KS-14 + Bacillus paramycoides BEMS-9-1 (T6), 50% RDF + Bacillus paramycoides BEMS-9-1 + Bacillus tequilensis BS-2 (T8) and 50% RDF + Brevibacillus formosus KS-14 (T3). The highest positive loadings were observed for plant height (0.97), chlorophyll content (0.97), root volume (0.96) and root surface area (0.96) along PC1 (Supplementary Table 4C). In the fourth PCA (Fig. 4c), PC1 and PC2 together accounted for total variance of 87.7% (79.46% PC1 & 8.23 PC 2). The shoot macro- and micro-nutrients significantly enhanced under 50% RDF + Brevibacillus formosus KS-14 (T3), 50% RDF + Bacillus paramycoides BEMS-9-1 (T4), 50% RDF + Bacillus paramycoides BEMS-9-1 + Bacillus tequilensis BS-2 (T8) and 50% RDF + Brevibacillus formosus KS-14 + Bacillus paramycoides BEMS-9-1 (T6) along PC1. The highest positive loadings were observed for Fe (0.95), N (0.82) and Mn (0.96) of shoot in PC1 (Supplementary Table 4D). In the fifth PCA, PC1 (73.96%) and PC2 (11.55%) explained 85.5% of the total variance (Fig. 4d). The grain macro- and micro-nutrients significantly enhanced under 50% RDF + Brevibacillus formosus KS-14 (T3), 50% RDF + Brevibacillus formosus KS-14 + Bacillus paramycoides BEMS-9-1 (T6), 50% RDF + Bacillus paramycoides BEMS-9-1 + Bacillus tequilensis BS-2 (T8) and 50% RDF + Brevibacillus formosus KS-14 + Bacillus tequilensis BS-2 (T7) with highest positive loadings for Mn (0.94), N (0.92), Cu (0.90) and Fe (0.90) (Supplementary Table 4E). In general, treatment 50% RDF + Brevibacillus formosus KS-14 + Bacillus paramycoides BEMS-9-1 (T6) and 50% RDF + Brevibacillus formosus KS-14 (T3) significantly influenced the variances such as DHA I (0.98), ACP II (0.96) ALP I (0.98) and IAA II (0.85) of soil, Zn (0.98), Mn (0.96) and S (0.94) of shoot, plant height (0.97), chlorophyll content (0.97), root volume (0.96) and root surface area (0.96), Zn (0.96),Mn (0.96) and S (0.94) of shoot and grain Mn (0.94), N (0.92), Cu (0.93), Fe (0.90) of grain of that might have contributed to overall growth and yield of wheat plant.