Bioprospecting of endophytic bacteria and their role in plant growth promotion -

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Bioprospecting of endophytic bacteria and their role in plant growth promotion

A total of 67 putative endophytic bacteria were isolated using different nutrient growth media from three diverse maize genotypes. Out of the 67, 10 endophytic bacterial isolates were selected for further characterization on the basis of plant growth-promoting (PGP) attributes. Phosphorus (P) and potassium (K) solubilization was observed in about 25% of the bacterial isolates. Additionally, bacterial endophytes’ ability to undergo mechanisms like nitrogenase activity, production of indole acetic acids, and siderophores was also studied. Among the 10 selected bacterial strains, three efficient endophytic PGP strains EU-A2SK1, EU-M4ARAct, and EU-E1RT3-1 were identified as Pseudomonas brenneri, Ewingella americana, and Pantoea agglomerans, respectively. These three efficient endophytic bacterial strains were tested on the maize seeds. The isolates efficiently increased the shoot length and enhanced anthocyanin, chlorophyll content, physiological available iron, and total protein content when compared to untreated control maize plants at 60 days of maize plant growth. These bacterial strains, as single or in a consortium, could be useful as bioinoculants for sustainable agriculture.

Materials and methods

Isolation and Enumeration of Bacterial Endophytes

Healthy genotypes of maize as local red, local white, and anthocyanin rich were collected from different fields of Baru Sahib, Sirmaur, Himachal Pradesh, India, for the isolation and enumeration of endophytic bacteria. Each of the three replicated samples of each genotype of maize was collected, labeled, and transported to the laboratory. The plant roots were washed properly for the removal of adhering soil under running tap water. The bacterial endophytes were isolated using the standard methods and diverse growth medium were utilized, such as Burks media, tryptic soy agar, Jensen’s agar, Kings B agar, yeast mannitol agar, nutrient agar, Azotobacter media, T3A media, Actinomycete isolation agar, and Luria Bertani agar for isolation followed by incubation at 30°C–35°C. The purified colonies were maintained at 4°C in slants and (25%) glycerol stock at −80°C.

Screening of Bacterial Endophytes for PGP- Attributes

The bacterial endophytes were initially screened for direct PGP attributes, including solubilization of phosphorus on Pikovskaya agar, potassium on Alexandrov agar medium and zinc on nutrient agar supplemented with insoluble compounds of zinc, respectively. Screening of nitrogen-fixing ability of endophytic microbes was tested by the acetylene reduction assay. The other PGP attributes, indole-3-acetic-acid, siderophores, and ammonia production were carried out using the standard method.

Phosphorus solubilization

The quantitative estimation of selected bacterial endophytes for solubilization of phosphorus was carried out. In 25 ml of the phosphate medium broth (supplemented with 0.5% tri-calcium phosphate), 1 ml of bacterial suspension was inoculated and incubated for 1 week at a temperature of 30°C. A week later, incubated bacterial suspension was centrifuged and the supernatant was collected for phosphorus estimation. To 1 ml of supernatant, 10 ml of ammonium molybdate solution was added and the volume was made to 45 ml by addition of distilled water which was followed by the addition of 4–5 drops of chlorostannous acid and optical density (OD) at 600 nm. The concentration of P was expressed in mg/L.

Indole-3-Acetic Acid Production

The quantitative estimation of indole acetic acids (IAA) production was carried out by inoculating 1 ml bacterial suspension in 25 ml of Luria-Bertani (LB) broth. Quantitatively, the IAA production was carried out in two sets, one set of LB broth containing 0.25M L-tryptophan and the other set without tryptophan incubated at 30°C for 3 days. In both sets, Salkowski’s reagent was added and observed for the development of pink colorations and IAA concentration was estimated by taking the OD at 530 nm.

Biological Nitrogen Fixation

The ability of putative endophytic bacterial culture for nitrogen-fixing attributes was carried out using the acetylene reduction assay technique. The nitrogenase activity was expressed as the amount of ethylene produced (in moles) per unit time and cell number. Protein concentration was estimated by Bradford’s method.

In-vitro Antagonistic Activity

The bacterial endophytes property of antagonism was evaluated against Fusarium graminerum.

Evaluation of Endophytic Bacteria on the Growth Promotion of Maize

Based on the different PGP attributes of bacterial endophytes, strains EU-A2SK1, EU-M4ARAct, and EU-E1RT3-1 were selected for exploring their ability to promote seedling growth of F1 pioneer hybrid maize under greenhouse conditions. Bacterial endophytes were grown overnight in nutrient broth on a rotary shaker at an incubation temperature of 30°C. All the pots were placed in a completely randomized design in a greenhouse for the avoidance of contamination. For each treatment, there were three replicates. After 1 week of germination, thinning was conducted for the maintenance of two plants per pot. The seedlings were watered properly for maintaining moisture content. At regular periods of 30, 60, and 90 days, the shoot length, number of tillers, and number of spikes were measured. The treatment in the present study consisted of EU-A2SK1, EU-M4ARAct, and EU-E1RT3-1, 50% P (half dose of di-ammonium phosphate), 100% P (full dose of di-ammonium phosphate), and control, respectively.

Analysis of Biochemical Levels

The content of chlorophyll and anthocyanin in maize leaves was calculated according to standard method. For chlorophyll content, 100 mg of fresh maize leaves was placed in a test tube containing 10 ml of methanol: H2O:Conc. HCl (80:20:1 v/v) and placed on a shaker in dark at 4°C, followed by filtration through Whatman No. 1 filter paper after 72 hours and OD at 530 and 657 nm, respectively. Fresh 100 mg chopped maize leaves were incubated with 25 ml of 1.5% 1–10 O-phenanthroline solution at 25°C. At 510 nm, the absorbance of the solution was read by the atomic absorption spectrometer. The content of total protein in maize leaves was calculated according to standard method described by Bradford.

Molecular Characterization of Endophytic Bacteria

The PCR amplification of 16S rRNA was conducted using the primers pA (5′-AGAGTTTGATCCTGGCTCAG-3′) and pH (5′-AAGGAGGTGATCCAGCCGCA-3′). The amplified 16S rRNA PCR product was selected for the phylogenetic analysis. Furthermore, the Polymerase Chain Reaction (PCR) product of the partial 16S rRNA gene was sequenced at Science of the Genome (SciGenome) (Chennai, India). Using the BLASTn program available at GenBank, the sequences of bacterial endophytes were aligned to those which were already available. A phylogenetic tree was constructed using the neighbor-joining (NJ) method, implemented in the program MEGA 4.0.2. The partial 16S rRNA sequences were submitted to National Center for Biotechnology Information (NCBI).

Screening of Bacterial Endophytes for PGP-Attributes

Out of the 67 endophytic bacteria, 10 bacterial isolates were selected on the basis of PGP attributes. Out of the 10 endophytic bacteria, all bacterial isolates exhibited PGP attributes of phosphorus and potassium solubilization, whereas only two isolates was positive for zinc solubilization. The production of siderophores, ammonia, and IAA were positive for 7, 5, and 2 bacterial strains, respectively. The nitrogenase activity was demonstrated by two bacterial isolates. Isolate EU-E1RT3-1 solubilized the highest amount of phosphorus (326.7 ± 0.08 mg l−1) followed by EU-M4ARAct (155.0 ± 0.20 mg l−1). Isolate EU-E1RT3-1 demonstrated highest production of IAA (21.00 ± 0.00 mg l−1) without the addition of tryptophan in screening media; furthermore, the strain EU-A2SK1 produced similar amounts of IAA (21.20 ± 0.01 mg/lt−1) with the addition of tryptophan during the quantitative estimation of IAA production in LB broth.

Evaluation of Endophytic Bacteria on Growth Promotion of Maize

The maize seedlings were bacterized with endophytic bacterial strains EU-A2SK1, EU-M4ARAct, and EU-E1RT3-1. The inoculation of endophytic bacteria and chemical fertilizer (di-ammonium phosphate) resulted in higher shoot length when compared to the uninoculated control. The treatment of maize seedlings with Pantoea agglomerans EU-E1RT3-1 and Ewingella americana EU-M4ARAct resulted in a significantly higher shoot length when compared to the un-inoculated control and other treatments at 30, 60, and 90 days. Although both these treatments were statistically analogous, treatment with P. agglomerans EU-E1RT3-1 resulted in an increased shoot length (127.5 cm) when compared with E. americana EU-M4ARAct (123.3 cm) at 90 days, respectively. The treatment of maize seedlings with a full dose (100%) of diammonium phosphate (DAP) resulted in an increased shoot length (122.5 cm) when compared to the half dose (50%) of DAP (119.5 cm) after 90 days.

The total chlorophyll content was maximum in maize seedlings treated with P. agglomerans EU-E1RT3.1 [127.6 μg/g fresh weight leaf (FWL),], followed by E. americana EU-M4ARAct (111.5 μg/g FWL), when compared to the un-inoculated control (66.8 μg/g FWL) after 60 days. In the present study, bacterized maize seedlings after 60 days of plant growth revealed a maximum increase in the anthocyanin content compared to the un-inoculated.

In conclusion, it was found that endophytes served as a valuable pool of bacteria with PGP- abilities. According to the results obtained in this study, strain P. agglomerans EU-E1RT3-1, possessing the capacity for P and K solubilization, and IAA production successfully improved plant growth. The strain with multiple PGP activities reported in this investigation seems to be an ideal candidate as a bioinoculant. P. agglomerans EU-E1RT3-1 in future may be used as an efficient biofertilizer for improving the health and productivity of crops. However, further studies must be conducted for the commercial scale production of NPK–based biofertilizers.

 

Reference:

Rana, K.L., Kour, D., Kaur, T., Devi, R., Yadav, A. and Yadav, A.N., 2021. Bioprospecting of endophytic bacteria from the Indian Himalayas and their role in plant growth promotion of maize (Zea mays L.). Journal of Applied Biology and Biotechnology, 9(3), pp.4-0.