Functional characterization of endophytic bacteria isolated from feather grass (<i>Chloris virgata</i> Sw.)

Rusi Lata; Sandip Chowdhury; Surendra Kumar Gond; Rusi Lata; Sandip Chowdhury; Surendra Kumar Gond

doi:10.48130/grares-0024-0005

Figures (5) Tables (6)

Figure 1.
Venn diagram showing biochemical characterization of bacterial endophytes.
Figure 2.
Venn diagram showing extracellular enzyme assay exhibited by bacterial endophytes.
Figure 3.
% Bacterial endophytes of C. virgata with PGP activity.
Figure 4.
Heatmap for plant growth promoting traits of the bacterial endophytes was prepared in terms of color intensity by normalisation of mean values of the results.
Figure 5.
Neighbor-joining phylogenetic tree based on the partial sequence of the 16S rRNA gene of bacterial isolates from C. virgata and their related type strains. The evolutionary distances were computed using the p-distance. Bootstrap values are given at branch nodes and are based on 1,000 replicates.

Isolates	Colour	Surface	Shape	Edge/margin	Elevation	Opacity
CHVLB1	Off-white	Shiny	Circular	Smooth	Raised	Opaque
CHVRB1	Off-white	Irregular	−	Lobate	Flat	Opaque
CHVRB2	Off-white to pink	Irregular	−	Undulate	Raised	Opaque
CHVRB3	Off-white to pink	Shiny	Circular	Lobate	Raised	Opaque
CHVRB4	Off-white to pink	Mucoidal, glistening	−	Entire	Raised	Opaque
CHVRB5	Off-white	−	−	Lobate	Flat	Opaque
CHVRTB1	Off-white	Dull	Circular	Entire	Raised	Opaque
CHVRTB2	Yellow	−	Irregular	Undulate	Raised	Opaque
CHVRTB3	Off-white	−	Irregular	Undulate	Raised	Opaque
CHVRTB4	Off-white	−	−	−	Flat	Opaque
CHVRTB5	Off-white	Irregular	−	Lobate	Flat	Opaque
CHVRTB6	Off-white	Irregular	−	Lobate	Raised	Opaque
CHVRTB7	Off-white	Slimy, glistening	Circular	Entire	Raised	Opaque
* CHVLB: Chloris virgata Leaf bacteria; CHVRB: Chloris virgata Rhizome bacteria; CHVRTB: Chloris virgata Root bacteria.

Table 1.

Morphological characteristic of endophytic bacterial isolates from Chloris virgata.

Isolates	Indole production	Catalase	Oxidase	Methyl Red test	Voges-Proskauer test	Citrate utillisation	Oxidative fermentation		NO₃⁻
Isolates	Indole production	Catalase	Oxidase	Methyl Red test	Voges-Proskauer test	Citrate utillisation	With oil	Without oil	NO₃⁻
CHVLB1	−	+	−	−	+	+	+	+	+
CHVRB1	+	+	−	−	+	+	+	+	−
CHVRB2	+	+	−	−	+	+	+	+	+
CHVRB3	+	+	−	−	+	+	−	+	+
CHVRB4	+	+	−	−	+	+	+	+	+
CHVRB5	+	+	−	−	+	+	+	+	+
CHVRTB1	+	+	−	−	+	+	+	+	+
CHVRTB2	+	+	−	−	+	+	+	+	−
CHVRTB3	+	+	−	−	+	+	+	+	+
CHVRTB4	+	+	−	+	+	+	+	+	+
CHVRTB5	+	−	−	−	+	+	+	+	−
CHVRTB6	+	−	−	+	−	−	+	+	+
CHVRTB7	+	+	−	−	+	+	+	+	+
(+) = presence of activity; (−) = absence of activity.

Table 2.

Biochemical characterization of endophytic bacteria.

Isolates	Enzyme activity				Phosphate utillisation		Siderophore production	Ammonia production	HCN production	Acc deaminase	Nitrogen free growth
Isolates	Amylase	Cellulase	Protease	Lipase	TCP	DCP	Siderophore production	Ammonia production	HCN production	Acc deaminase	Nitrogen free growth
CHVLB1	−	+++	+++	−	−	+	+++	++	−	+	+++
CHVRB1	+++	++	−	+++	−	+	+	+++	−	+	+
CHVRB2	+++	+++	++	+++	+	+	++	+++	−	+	+
CHVRB3	+++	+++	+++	+++	+	+	+	+++	−	+	+
CHVRB4	+++	+++	+++	+++	+	+	+++	+++	−	+	+
CHVRB5	+++	+++	++	+++	−	+	−	+++	−	+	+
CHVRTB1	+++	+++	+++	+++	−	+	+++	++	−	+	+++
CHVRTB2	+++	+++	+++	+++	+	+	+++	++	−	+	+++
CHVRTB3	−	+++	+++	−	+	+	+++	++	−	+	+++
CHVRTB4	+++	−	−	+++	+	−	+++	++	−	−	−
CHVRTB5	+++	+	−	+++	−	+	−	+++	−	−	+
CHVRTB6	+++	+	−	+++	−	−	+	+++	−	−	+
CHVRTB7	+	−	+++	+	+	−	−	+++	−	+	+
* HCN: Hydrogen cyanide; TCP: Tricalcium phosphate, DCP: Dicalcium phosphate; Number of (+) sign indicates strength of activity, (−) indicates absence of activity.

Table 3.

Qualitative observation of PGP traits of the endophytic isolates.

Isolates	Indole acetic acid production (µg·mL⁻¹)		Gibberellic acid production (µg·mL⁻¹) (540 nm)	Siderophore production (CAS ASSAY) % PSU	Ammonia production (µg·mL⁻¹)
Isolates	Without trp	With trp	Gibberellic acid production (µg·mL⁻¹) (540 nm)	Siderophore production (CAS ASSAY) % PSU	Ammonia production (µg·mL⁻¹)
CHVLB1	0.033 ± 0.006	13.50 ± 0.004	22.22 ± 0.003	32.71 ± 0.008	35.45 ± 0.004
CHVRB1	0.036 ± 0.006	16.91 ± 0.008	146.72 ± 0.004	41.05 ± 0.012	71.28 ± 0.031
CHVRB2	0.039 ± 0.003	17.62 ± 0.006	70.64 ± 0.009	49.40 ± 0.003	95.27 ± 0.009
CHVRB3	0.038 ± 0.005	20.76 ± 0.003	74.92 ± 0.007	49.30 ± 0.003	98.33 ± 0.006
CHVRB4	0.044 ± 0.004	17.84 ± 0.004	73.93 ± 0.009	48.96 ± 0.006	94.86 ± 0.005
CHVRB5	0.028 ± 0.009	23.95 ± 0.005	97.64 ± 0.008	8.16 ± 0.003	73.64 ± 0.032
CHVRTB1	0.038 ± 0.004	12.19 ± 0.007	^*c149.03 ± 0.012	49.08 ± 0.002	69.05 ± 0.003
CHVRTB2	0.035 ± 0.014	18.14 ± 0.004	136.84 ± 0.006	27.50 ± 0.025	97.29 ± 0.031
CHVRTB3	0.037 ± 0.01	11.53 ± 0.002	103.57 ± 0.005	48.83 ± 0.002	74.89 ± 0.015
CHVRTB4	0.032 ± 0.007	^*b29.53 ± 0.008	96.33 ± 0.005	^*d69.77 ± 0.063	^*e276.13 ± 0.13
CHVRTB5	^*a0.054 ± 0.012	16.42 ± 0.008	92.05 ± 0.003	36.55 ± 0.10	79.97 ± 0.01
CHVRTB6	0.033 ± 0.016	22.96 ± 0.004	102.26 ± 0.005	32.04 ± 0.004	70.09 ± 0.007
CHVRTB7	0.037 ± 0.02	24.25 ± 0.005	57.79 ± 0.003	11.01 ± 0.002	66.54 ± 0.003
a = highest IAA produced without trp supplement; b = highest IAA produced with trp supplement; c = highest GA3 concentration produced; d = highest PSU released; *e = highest concentration of ammonia released.

Table 4.

Quantitative observation of PGP traits of the endophytic isolates.

Isolates	5% NaCl	10% NaCl	15% NaCl	20% NaCl	0.15 MPA	0.49 MPA	1.03 MPA
CHLB1	+	+	−	−	+	+	+
CHRB1	+	+	−	−	+	+	+
CHRB2	+	+	−	−	+	+	+
CHRB3	+	+	−	−	+	+	+
CHRB4	+	+	−	−	+	+	+
CHRB5	+	−	−	−	+	+	+++
CHRTB1	+	+	−	−	+	+	+
CHRTB2	+	+	−	−	+	+	+++
CHRTB3	+	+	−	−	+	+	++
CHRTB4	+	+	−	−	+	+	+++
CHRTB5	+	+	−	−	+	+	++
CHRTB6	+	−	−	−	+	+	++
CHRTB7	+	−	−	−	+	+	+++
Number of (+) sign indicates the increased appearance of colonies on media, (−) indicates absence of activity.

Table 5.

Screening for salinity and osmotic stress tolerance on minimal DF (Dworkin and Foster) media.

Endophytic isolates	Accession number	Bacterial genus with highest similarity	Accession number of closest match	Identity %
CHVLB1	MW680956	Bacillus sp.	NR_116240.1	99.45
CHVRB1	MW680957	Bacillus sp.	NR_148786.1	98.97
CHVRB2	MW680958	Bacillus sp.	NR_112686.1	98.64
CHVRB3	MW680959	Bacillus sp	NR_112686.1	99.75
CHVRB4	MW680960	Bacillus sp.	NR_112686.1	99.84
CHVRTB1	MW680961	Glutamicibacter sp.	NR_026190.1	99.21
CHVRTB2	MW680962	Bacillus sp.	NR_113265.1	99.84
CHVRTB3	MW680963	Bacillus sp.	NR_112686.1	99.92
CHVRTB4	MW680964	Bacillus sp.	NR_157609.1	99.25

Table 6.

Taxonomic identification and sequence homology of isolated bacterial endophytes with genus or species affiliation and corresponding identity percentage in the NCBI database.