Seed biopriming with P- and K-solubilizing Enterobacter hormaechei sp. improves the early vegetative growth and the P and K uptake of okra (Abelmoschus esculentus) seedling

Limited information is available that seed biopriming with plant growth-promoting Enterobacter spp. play a prominent role to enhance vegetative growth of plants. Contrary to Enterobacter cloacae, Enterobacter hormaechei is a less-studied counterpart despite its vast potential in plant growth-promotion mainly through the inorganic phosphorus (P) and potassium (K) solubilization abilities. To this end, 18 locally isolated bacterial pure cultures were screened and three strains showed high P- and K-solubilizing capabilities. Light microscopy, biochemical tests and 16S rRNA gene sequencing revealed that strains 15a1 and 40a were closely related to Enterobacter hormaechei while strain 38 was closely related to Enterobacter cloacae (Accession number: MN294583; MN294585; MN294584). All Enterobacter spp. shared common plant growth-promoting traits, namely nitrogen (N2) fixation, indole-3-acetic acid production and siderophore production. The strains 38 and 40a were able to produce gibberellic acid, while only strain 38 was able to secrete exopolysaccharide on agar. Under in vitro germination assay of okra (Abelmoschus esculentus) seeds, Enterobacter spp. significantly improved overall germination parameters and vigor index (19.6%) of seedlings. The efficacy of root colonization of Enterobacter spp. on the pre-treated seedling root tips was confirmed using Scanning Electron Microscopy (SEM). The pot experiment of bioprimed seeds of okra seedling showed significant improvement of the plant growth (> 28%) which corresponded to the increase of P and K uptakes (> 89%) as compared to the uninoculated control plants. The leaf surface area and the SPAD chlorophyll index of bioprimed plants were increased by up to 29% and 9% respectively. This report revealed that the under-explored species of P- and K-solubilizing Enterobacter hormaechei sp. with multiple plant beneficial traits presents a great potential sustainable approach for enhancement of soil fertility and P and K uptakes of plants.

120 [23]. A fresh 24 h colony culture was spot on the solidified agar using a sterile inoculating 121 needle and the plate was incubated at 30°C for 5 days in triplicate. The halo zone formation 122 was interpreted as solubility index (SI) which from now onwards was termed as P solubility 123 index (PSI) and K solubility index (KSI) respectively and determined using the the ratio of the 124 total halo diameter to the colony diameter [6]. 125 126 Antagonistic interaction assay 127 Antagonistic interaction assay between selected isolates was characterized by pour plate 128 method as described by Grossart et al., (2004). A molten nutrient 1% agar (2.5 mL) was mixed 129 with 50 µL target isolate suspension (1×10 8 CFU/mL). The cell suspension agar was poured 130 onto a nutrient agar plate (Sigma) and left for 10 min until it was completely solidified. An 131 aliquot (10 µL) of test strain (1×10 8 CFU/mL) was applied onto the lawn and thereafter 132 incubated at 30°C for 3 days. The strains were tested in triplicate against each other and 133 observed daily for inhibition zones. Inhibitory activity was recorded when the inhibition zone 134 was equal to or more than 4 mm of the diameter of the applied colony in both parallels.
Note: P and K solubilization were categorized as low activity when the halo zone is < 5 mm 313 (denoted as +) or high activity when the halo zone > 5 mm (denoted as ++).

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Based on in vitro plate screening (Fig 1) The evolutionary relationship was reconstructed using the neighbour-joining method 406 [33] to generate the optimal phylogenetic tree with the sum of branch length of 0.14724404 407 (Fig 4). 422 Enterobacter spp. were also examined on the ability to demonstrate multi-putative plant growth 423 promoting activities such as N 2 fixation, IAA production, GA3 production, zinc solubilization, 424 siderophore and exopolysaccharide (EPS) production (   Table).  499 Data are mean value of six biological replicates with ± symbol denoting standard deviation. Means followed by 500 different letters in the same column indicate significant differences at P<0.05. 501 502 Leaf surface area and SPAD chlorophyll index of bioprimed seedlings were increased 504 significantly (P<0.05) as compared to uninoculated seedlings (Table 5). Treatment using E.
505 hormaechei 40a showed the highest improvement in leaf surface area (18.5 cm 2 ) followed by 506 E. hormaechei 15a1 (18.5 cm 2 ) and E. cloacae 38 (15.7 cm 2 ). Likewise, the SPAD chlorophyll 507 index of the leaves of the bioprimed seedlings was increased up to 37.9. Soil viable cell of 508 bioprimed seedlings recorded a higher CFU ranged from 6.0 × 10 7 to 9.5 × 10 7 CFU/g soil as 509 compared to uninoculated seedlings (3.5 × 10 7 ). 510 511 564 Synergistic effect maybe more meaningful to be assessed when one strain with a desired 565 specific activity is paired with another strain with distinctive beneficial traits as to complement 566 a collective purpose [1].

567
The most common mechanism adopted by bacteria during P solubilization was the 568 secretion of multiple organic acids [6]. In this study, the organic acid production of 569 Enterobacter spp. was analysed using HPLC where the most notable amount of gluconic acid 570 was detected, followed by malic, citric, succinic, acetic and GA3 acid. The intense amount of 571 gluconic acid present in the growth media was an indicator of the active conversion from its