Decoding the Plant Growth Promotion and Antagonistic Potential of Bacterial Endophytes From Ocimum sanctum Linn. Against Root Rot Pathogen Fusarium oxysporum in Pisum sativum

Abstract

The present study demonstrates plant growth promotion and induction of systemic resistance in pea (Pisum sativum) plant against Fusarium oxysporum f.sp. pisi by two bacterial endophytes, Pseudomonas aeruginosa OS_12 and Aneurinibacillus aneurinilyticus OS_25 isolated from leaves of Ocimum sanctum Linn. The endophytes were evaluated for their antagonistic potential against three phytopathogens Rhizoctonia solani, F. oxysporum f. sp. pisi, and Pythium aphanidermatum by dual culture assay. Maximum inhibition of F. oxysporum f. sp. pisi was observed by strains OS_12 and OS_25 among all root rot pathogens. Scanning electron microscopy of dual culture indicated hyphal distortion and destruction in the case of F. oxysporum f. sp. pisi. Further, volatile organic compounds (VOCs) were identified by gas chromatography-mass spectrometry (GC-MS). The GC-MS detected eight bioactive compounds from hexane extracts for instance, Dodecanoic acid, Tetra decanoic acid, L-ascorbic acid, Trans-13-Octadecanoic acid, Octadecanoic acid. Both the endophytes exhibited multifarious plant growth promoting traits such as indole acetic production (30-33 μg IAA ml^-1), phosphate solubilization, and siderophore and ammonia production. Pot trials were conducted to assess the efficacy of endophytes in field conditions. A significant reduction in disease mortality rate and enhancement of growth parameters was observed in pea plants treated with consortium of endophytes OS_12 and OS_25 challenged with F. oxysporum f.sp. pisi infection. The endophytic strains elicited induced systemic resistance (ISR) in pathogen challenged pea plants by enhancing activities of Phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO), ascorbate oxidase (AO), catalase (CAT) and total phenolic content. The endophytes reduced the oxidative stress as revealed by decrease in malondialdehyde (MDA) content and subsequently, lipid peroxidation in host plant leaves. Robust root colonization of pea seedlings by endophytes was observed by scanning electron microscopy (SEM) and fluorescence microscopy. Thus, plant growth promoting endophytic P. aeruginosa and A. aneurinilyticus can be further exploited through bio-formulations for sustainable protection of crops against root rot diseases as bio-control agents.

Keywords: Fusarium root rot; biological control; endophytes; induced systemic resistance; plant growth promotion; volatile compounds.

FIGURE 1

Dual culture assay for investigating in vitro antagonistic potential of bacterial endophytes inhibiting the mycelial growth of fungal pathogens on PDA medium after 7 days of incubation. The endophytes antagonists OS_12 (D–F) and OS_25 (G–I) have limited the growth of hyphae of pathogens with respect to control plates which showed dense hyphal growth of fungal pathogens. (A) Fusarium oxysporum f. sp. pisi, (B) Rhizoctonia solani, and (C). Pythium aphanidermatum.

FIGURE 2

Scanning electron micrographs of mycelia of F. oxysporum f. sp. pisi: in the absence of antagonistic endophytes (A). In the presence of strain OS_12 only showing dense proliferation of bacteria over the mycelial surface and hyphal destruction (B). In the presence of strain OS_25 only showing adhesion of bacteria to fungus mycelial structure (C). SEM micrograph of consortium (OS_12 + OS_25) treated mycelia of F. oxysporum f. sp. pisi exhibiting disaggregated hyphal structures with bacterial adhesion and proliferation on mycelial surface (D). Scale bar equals 10 μm (A,B,D) and 5 μm (C).

FIGURE 3

Gas chromatography–mass spectrometry (GC-MS) ion chromatogram of bioactive volatile compounds identified from hexane extract sample of bacterial isolate OS_12 (A) and OS_25 (B) with evidenced peaks (shown with blue dot) of compounds presented in Supplementary Table 1.

FIGURE 4

Assessment of multiple plant growth promoting traits of endophytic strains OS_12 and OS_25. (A,B) Zone of phosphate solubilization on NBRIP medium amended with Tricalcium phosphate. (C,D) Production of siderophore on CAS agar medium.

FIGURE 5

Phylogenetic tree based on a partial 16S rRNA nucleotide sequences showing the position of strains OS_12 and OS_25 with respect to other related taxa constructed with Neighbor joining method using version 10.1 of MEGA software (https://www.megasoftware.net/). The 16S rRNA gene sequences of closely related species were retrieved from NCBI GenBank databases. Evolutionary distance was computed using Maximum Composite Likelihood method. Bootstrap values (percentage of 1,000 replicates) higher than 40% are shown at node points. Bar, 0.020 substitutions per nucleotide position.

FIGURE 6

Effect of antagonistic bacterial endophytes strains OS_01 and OS_03 inoculation, single and dual combination on the root rot disease incidence after 7 days in F. oxysporum challenged pea plants. Severity disease symptoms in the roots system of pea plants (A) grown in soil infested with F. oxysporum only, (B) grown in sterile soil without infestation with F. oxysporum, (C) primed with strain OS_12 and grown in infected soil, (D) primed with strain OS_25 and grown in infected soil, (E) primed with bacterial consortia of OS_12 and OS_25 grown in infected soil.

FIGURE 7

Effect of seed biopriming with endophytic biostimulant strains OS_12 and OS_25 plants on length parameter of root and shoot biomass of pea plants sown in plastic pots under normal (dark blue and dark yellow bars) and pathogenic F. oxysporum stress (red and gray bars) conditions. Columns represent Mean values ± standard deviation (n = 3 replicates per treatment). Different letters indicate statistical difference between treatments (Turkey’s posttest, P < 0.05) in root length and shoot length under normal and biotic stress conditions.

FIGURE 8

Effect of seed biopriming with endophytic biostimulant strains OS_12 and OS_25 plant on fresh weight of root (A) and shoot (B) biomass of pea plants sown in plastic pots under normal (green bar) and pathogenic F. oxysporum stress (purple bar) conditions. Columns represent Mean values ± standard deviation (n = 3 replicates per treatment). Different letters indicate statistical difference between treatments (Turkey’s posttest, P < 0.05) under normal and biotic stress conditions.

FIGURE 9

Effect of endophytic biostimulant strains OS_12 and OS_25 plant on photosynthesis associated pigments- total chlorophyl and carotenoid content of pea plants under normal (dark gray bar) and pathogenic F. oxysporum stress (dark red bar) conditions. Columns represent mean values ± standard deviation (n = 3 replicates per treatment). Different letters indicate statistical difference between treatments (Turkey’s posttest, P < 0.05) in enhancing chlorophyll and carotenoid content under normal and biotic stress conditions.

FIGURE 10

Effect of pathogen and endophytic bacterial inoculation on activities of different defense related antioxidative enzymes (A) phenylalanine ammonia lyase (PAL), (B) polyphenol oxidase (PPO), (C) peroxidase (PO), (D) ascorbate oxidase (AO), and (E) catalase (CAT) under different treatment conditions. Columns represent Mean values ± Standard deviation (n = 3). Dark yellow bar, normal conditions; blue bar, pathogen challenged conditions; Different letters indicate statistical difference between treatments (Turkey’s post test P < 0.05).

FIGURE 11

Effect of pathogen and endophytic bacterial inoculation on activities of different defense related antioxidative enzymes (A) total phenolic content, (B) malondialdehyde (MDA) content. Columns represent Mean values ± Standard deviation (n = 3). Blue bar, normal conditions; yellow bar, pathogen challenged conditions. Different letters indicate statistical difference between treatments (Turkey’s post test P < 0.05).

FIGURE 12

Scanning electron microscopic analysis of pea plant roots inoculated by consortia of Pseudomonas aeruginosa OS_12 and Aneurinibacillus aneurinilyticus OS_25 (A–C). (A) Roots of non-inoculated pea plants, (B) bacterial cells (arrows) swarming in the vicinity of rhizoplane, (C) bacterial cells (arrows) in the intercellular space between epidermis and cortex of root system. Scale bar equals 3 μm (A), 1 μm (B), and 2 μm (C). Fluorescent microscopy images of root sections stained with acridine orange (D,E). (D) Control pea root sample without any inoculation, (E) root section of consortium inoculated pea plant with arrows pointing bacterial cells inside the epidermis (bar equals 10 μm).

FIGURE 13

Principal component analysis of effect of different treatment (shown in circles with blue dot) with respect to control (uninoculated) and bacterial inoculation either single (OS_12 or OS_25) or in combination (OS_12 + OS_25) in the presence and absence of F. oxysporum f. sp. pisi (FOP) on growth parameters, defense enzymes and mortality rate of pea plants (shown in red dots). The X-axis and Y-axis of PCA plot represent first and second principal components (PCs). Symbols: RL, root length; SL, shoot length; SFW, shoot fresh weight; RFW, root Fresh weight; CHL, chlorophyll content; CART, carotenoid content.