Antagonistic and growth-promoting potential of multifarious bacterial endophytes against

Background: Chickpea is the most important legume crop in India, and its productivity is significantly lowered due to biotic constraints such as wilt caused by Fusarium oxysporum f. sp. ciceris. Endophytes help plants to thrive better under stress conditions by regulating plant hormones and inducing systemic resistance. The aim of the present study was to identify chickpea root bacterial endophytes having antagonistic ability for the management of the Fusarium wilt. Results: Twenty endophytic bacterial strains were isolated from the susceptible and resistant chickpea cultivar, amongst which 35% bacterial endophytes gave positive results for siderophore and 15% isolates were HCN producers, whereas 55% showed good growth on ACC-supplemented medium. Based upon 16S rRNA phylogenetic analysis, efficient endophytic bacterial isolates CRBE1, CRBE3 and CRBE7 were identified as Priestia megaterium, Brucella haematophila and Microbacterium paraoxydans, respectively. Bacterial endophyte CRBE7 and CRBE3 showed pronounced antagonistic activity against Fusarium oxysporum f. sp. ciceris under in vitro conditions. Inoculation of chickpea with isolated bacterial endophytes alone and in combination through seed priming resulted in significant biocontrol activity against Fusarium wilt of chickpea under screenhouse conditions. Biopriming of chickpea seeds alone with CRBE3 and as consortium of CRBE3 + CRBE7 upsurged the phenol content in susceptible and resistant chickpea varieties JG 62 and HC 3, which indicated the role of promising endophytes as potential bio-agents under in vivo conditions. Conclusions: Chickpea root bacterial endophytes Priestia megaterium (CRBE1), Brucella haematophila (CRBE3) and Microbacterium paraoxydans (CRBE7) exhibiting antagonistic activity could be incorporated in integrated disease management module against Fusarium wilt of chickpea.


Background
Chickpea (Cicer arietinum L.) is an important leguminous crop. India is the largest chickpea producer in the world with production volume amounting to nearly ten million metric tonnes (https:// www. stati sta. com/ stati stics/ 722203/ chick peas-produ ction-volume-by-count ry-world wide/). Chickpea productivity is significantly lowered due to biotic stresses, namely wilt caused by Fusarium oxysporum f. sp. ciceris in various regions of the world including India (Jiménez-Díaz 2015). In India, the disease was reported to reduce chickpea production annually by 10-15% (Dubey et al. 2007), reaching up to 70% in years of severe outbreak (Halila and Strange 1996).
Endophytic bacteria are known to promote the plant growth by producing siderophores, auxins, phytohormones and enzymes, nitrogen fixation, solubilizing phosphate, releasing ammonia or by supplying essential vitamins to plants (Saini et al. 2015). These microbes provide protection against phyto-pathogens mediated by volatile metabolites including hydrogen cyanide and ammonia, iron acquisition by siderophores production of antibiotic, exopolysaccharides, production of cell wall degrading enzymes and antioxidant enzymes like superoxide dismutase, catalase, peroxidase and phenols (Nagpal et al. 2021).
Identification and use of PGPR along with antagonistic properties against plant pathogens were essential and promoted throughout the world for sustainable crop production. Kumari and Khanna (2018) reported 74.5% and 70.3% reduction in Fusarium wilt incidence in chickpea genotypes GPF-2 and JG-41, respectively, on bacterial inoculation along with production of siderophore, HCN and plant growth hormones. Bacillus megaterium significantly reduced the fungal growth of F. graminearum in wheat (Pan et al. 2015) and triggers salicyclic acid-dependent resistance in rice to mitigate the disease caused by F. proliferatum (Cheng et al. 2020). The biological control of Ralstonia solanacearum, incitant of potato wilt disease through endophytic bacteria Bacillus, Pseudomonas and Microbacterium was observed by Bahmani et al. (2021). Despite adequate knowledge on potential growth-promoting bacterial endophytes in chickpea, the studies on their bio-control potential against F. oxysporum f. sp. ciceris are scanty. Moreover, use of indigenous endophytic bacteria possessing plant growth traits along with bio-control potential is the best eco-friendly and ecologically efficient approach within the framework of integrated disease management. Therefore, the present investigation aimed to exploit the bacterial symbionts that possessed both plant growth-promoting and antagonistic ability, as potential biocontrol agents.

Isolation of endophytic bacteria
Endophytic bacteria were isolated from the roots of chickpea cultivars, viz. HC 5 (resistant) and JG 62 (susceptible) grown in Fusarium wilt infected plot at Pulses Section, Department of Genetics and Plant Breeding, CCS Haryana Agricultural University, Hisar, India (Latitude: 29°14′92'' N, Longitude: 75°72′17'' E and Altitude: 215 m AMSL). The standard protocol for isolation of endophytic bacteria given by Sangwan et al. (2021) was followed. Roots were washed with running tap water and surface sterilized sequentially in 75% (v/v) ethanol for 2 min, 2.6% (w/v) sodium hypochlorite solution for 5 min and 75% (v/v) ethanol for 1 min followed by thorough five to six times washing with sterile distilled water. For isolation of bacterial endophytes, 1 g root was crushed in pestle and mortar with 10 ml sterile distilled water to get uniform paste and allowed to settle down for 15-20 min.
The supernatant was further diluted five times in 10 ml water, and approximately 10 µl was used for streaking on nutrient agar plates and incubated at 28 ± 1 °C for 3 days. The colonies of bacterial endophytes were characterized based on visual observations, purified using streak plate technique and maintained on nutrient agar slants at 4 °C for further study.

Plant growth-promoting abilities of bacterial endophytes
Isolated bacterial endophytes were tested for their plant growth-promoting abilities, namely siderophore production, hydrogen cyanide (HCN) production, 1-aminocyclopropane 1-carboxylic acid (ACC) utilization. Siderophore production of isolated bacterial endophytes was identified using Chrome azurol S assay (Schwyn and Neilands 1987). From each log phase grown culture, 10 µl was spotted on the siderophore plates and incubated at 28 ± 1 °C for 3-4 days. The presence of siderophore was indicated by the formation of yellow halo zone around the colonies. The production of hydrogen cyanide was detected after 72 h of incubation of bacterial isolates in King's B broth at 28 ± 1 °C, using alkaline picrate soaked filter paper fixed inside the test tube. A colour change from yellow to reddish brown was noted as indication of weak, moderate or strong cyanogenic potential. (Alstrom and Burns 1989).
For ACC utilization, the minimal medium (Dworkin and Foster 1958) supplemented with 2 mM ACC was prepared (Penrose and Glick 2003). A loopful of bacterial endophytes from 48 h old culture was spotted on ACC supplemented minimal medium plates. Growth of the isolates on ACC and ammonium sulphate amended medium plates was observed after 5 days of incubation at 28 ± 1 °C. The minimal medium plates having ammonium sulphate were used as control for growth comparison of different isolates.

Isolation of pathogen
The Fusarium wilt-infected chickpea plants were collected from the wilt infected plot at Pulses Section, CCS Haryana Agricultural University, Hisar, India. The pathogen was isolated from the infected roots of chickpea plants, and culture was identified at Department of Plant Pathology, CCS Haryana Agricultural University, Hisar, India.

In vitro efficacy of bacterial endophytes
The bacterial endophytes were evaluated for their inhibitory potential against Fusarium identified strain under in vitro following dual culture technique on potato dextrose agar (HIMEDIA M096) plates with four replications maintained for each bacterial isolate. A 5-mm mycelial disk from a one-week-old fungus culture was placed in the center of a 90-mm-diameter Petri dish containing PDA medium. The bacterial strain was applied in two opposite streaks at approximately 30 mm from the fungal plug. The medium plates were incubated at 28 ± 1 °C, and split growth of pathogen was observed when the control plates were completely filled with test pathogen. The percentage growth inhibition was calculated following the formula of Vincent (1947).

Morphological, biochemical and molecular characterization
Three bacterial endophytes that exhibited prominent antagonistic activities were morphologically and biochemically characterized as per procedure described in Bergey's Manual of Determinative Bacteriology (Holt et al. 1994). For molecular identification, the 16S rRNA region of different isolates was amplified using forward primer (F:5′AGA GTT TGA TCC TGG CTC AG 3′) and reverse primer (R:5′AAG GAG GTG ATC CAG CCG CA 3′) (Weisburg et al. 1991). The amplified product was sequenced at Biotechnology Laboratory, University of LUVAS, Hisar, Haryana (India). The sequence for all the endophytes was obtained and analysed using BLAST. Identification up to species level was ascertained as maximum homology (≥99 %) to a type strain sequence in the GenBank. Phylogenetic tree for the available data sets was deduced using the neighbour joining program, MEGA version 11.0.8 (Kumar et al. 2018).

In vivo efficacy of bacterial endophytes and their impact on total phenol content in chickpea plants
The identified Fusarium strain was multiplied on potato dextrose broth, while the bacterial endophytes, CRBE1, CRBE3 and CRBE7 were multiplied in nutrient broth for 2 days at 28 ± 1 °C for artificial inoculation. The experiment was conducted with seven treatments, viz. CRBE1 alone, CRBE3 alone, CRBE7 alone, CRBE1 + CRBE3, CRBE3 + CRBE7, CRBE1 + CRBE7 and CRBE1 + CRBE3 + CRBE7, in earthen pots under screen house conditions during crop season 2019-2020 in completely randomized design with three replications. Each pot was filled with five kg of sterilized sandy soil inoculated with test pathogen for each replication; three pots were maintained. The pathogen was inoculated as soil drench with conidial suspension adjusted to 10 6 conidia/ ml. Seeds of chickpea cultivars, viz. JG 62 and HC 3, were treated separately with 1 ml inoculum of each of the bacterial endophyte independently and in combination. The bacterial suspension was adjusted to 10 8 cells/ ml, and for co-inoculation, the cell suspension of both endophytic bacterial strains was mixed in 1:1 ratio. Uninoculated surface sterilized seeds served as control and four seeds were sown in each pot. The observations on disease incidence were recorded by examining the healthy and disease plants.
Total phenols in root samples at different time intervals were estimated by following the standard procedure (Thimmaiah 2006). A 100 mg of dried root sample was taken, and 10 ml of 80% methanol was added to it. It was kept in water bath at 80 °C for 30 min. Five ml of 80% methanol was added to the supernatant and incubated in water bath for half an hour. The above step was performed again. The supernatant collected was centrifuged at 6000 rpm for 15 min and transferred to other clean test tube. After drying the samples completely by evaporation in water bath at 80 °C, 5 ml water was added in each test tube and test tubes were vortexed. One ml of this solution was mixed with 4.0 ml water. This solution (0.5 ml) was transferred to a test tube, and 0.5 ml Folin-Ciocalteu reagent was added to it. It was left for 3 min followed by addition of 2 ml of 20% sodium carbonate solution. The absorbance was measured at 650 nm. The amount of phenol expressed (µg/g dry weight) in the sample was estimated from the standard curve prepared gradually by taking catechol as the standard phenol content.

Statistical analysis
Data were analysed statistically after angular transformation using appropriate design (Panse et al. 1978), and the treatments were compared at p = 0.05.

Isolation of pathogen
The Fusarium wilt-infected chickpea plants were collected from the wilt infected plot at Pulses Section, CCS Haryana Agricultural University, Hisar, India. The isolated pathogen from the infected roots of chickpea plants was identified as Fusarium oxysporum f. sp. ciceris, by comparing the morphological and conidial characteristics of the standard culture.

In vitro efficacy of bacterial endophytes against Fusarium oxysporum f. sp. ciceris
The twenty endophytic bacteria isolated from chickpea roots were evaluated for their antagonistic activity Page 4 of 9 Khanna et al. Egyptian Journal of Biological Pest Control (2022) 32:17 against F. oxysporum f. sp. ciceris under in vitro conditions. All the endophytic bacterial isolates were found to inhibit the fungal growth ranging from 14.34 to 69.38%. Endophytes, CRBE3 and CRBE7 (statistically at par) exhibited growth inhibition of 63.95 and 69.38%, respectively, followed by CRBE1, CRBE19 and CRBE5 which suppressed the fungal growth by 53.10, 51.16 and 48.06%, respectively, while the isolates CRBE4, CRBE6, CRBE11 and CRBE12 were the least effective in inhibiting mycelial growth of the test pathogen (Table 1).

Morphological, biochemical and molecular characterization of bacterial endophytes
Based on colony morphology and Gram reaction, isolate CRBE1 was found as a Gram-positive bacterium having flat, smooth and shiny colony surface, while isolate CRBE3 was a Gram-negative bacterium, showing shiny white colour colonies on nutrient agar medium with smooth and flat surface. Isolate CRBE7 was a Gram positive bacterium, and its colonies appeared yellowish on nutrient agar medium. Based on more than 98% sequence homology of 16S rRNA gene with the gene sequences available in the NCBI nucleotide database, potential endophytic bacterial isolates (CRBE1, CRBE3 and CRBE7) were identified as Priestia megaterium (Accession no. OK482778), Brucella haematophila (Accession no. OK482777), and Microbacterium paraoxydans (Accession no. OK482779), respectively (Figs. 2, 3).

In vivo efficacy of endophytic bacterial isolates against Fusarium oxysporum f. sp. ciceris and effect on total phenol content
Based on in vitro assay and plant growth-promoting traits, three antagonistic endophytic bacteria CRBE1, CRBE3 and CRBE7 were tested alone and in combination under screen house conditions for their antifungal activity against F. oxysporum f. sp. ciceris. Seed priming with cultures of endophytes significantly reduced the disease incidence over control in chickpea varieties, JG 62 and

Fig. 1 Venn diagram showing plant growth-promoting traits of chickpea root bacterial endophytes
Page 5 of 9 Khanna et al. Egyptian Journal of Biological Pest Control (2022) 32:17 HC 3. Seed priming with CRBE3 (Brucella haematophila) in chickpea variety JG 62 was found to be the most effective in suppressing the wilt incidence from 69.44 to 11.11%, while the other treatments were found to be statistically at par. In resistant variety HC 3, seed treatment with endophyte CRBE3 curtailed the disease incidence from 22.22 to 2.78% (Table 2). However, when the consortium of endophytes used, their potential antagonistic activity against Fusarium wilt hampered as compared to their alone application in both the varieties. Plant phenolic compounds play a vital role in plant defence and fluctuation in concentration is observed in response to invading pathogen. In resistant chickpea variety HC 3, maximum mean phenol content, i.e. 366.49 µg/g, was recorded on seed treatment with isolate CRBE3 which was significantly higher than the other treatments, while minimum phenol content was recorded in plants after seed treatment with CRBE1. Seed treatment with CRBE1 + CRBE7 and consortium of CRBE1 + CRBE3 + CRBE7 was found to be statistically at par with a phenol content of 223.60 and 228.47 µg/g, respectively. There was significant increase in phenol content of chickpea plants of resistant variety HC 3 with the increase in duration of sampling. The maximum phenol content was recorded in the 3rd week of sampling (299.62 µg/g) followed by the 2nd one, while it was a minimum in the 1st week of sampling. The interaction between bacterial endophytes and duration of sampling was significant. The highest phenol content (412.70 µg/g) was recorded in the 3rd week of sampling in plants of resistant chickpea variety HC 3 raised from seeds primed with CRBE3 followed by CRBE3 + CRBE7 and CRBE7 alone which were statistically at par (Table 3).
Similar trend with respect to phenol content was recorded in susceptible variety JG 62. Maximum phenol content was recorded in plants treated with CRBE3 (201.56 µg/g) followed by CRBE3 + CRBE7 (168.64 µg/g). However, it was significantly lower in chickpea variety JG 62 in comparison with resistant variety HC 3 irrespective of duration of sampling.

Discussion
Endophytes facilitate plant growth by protecting plants from plant pathogens and increasing its tolerance against various biotic and abiotic stresses (Khanna et al. 2019). In the present study, 20 morphologically distinct endophytic bacteria were isolated from the roots of susceptible and resistant chickpea varieties. Several researchers reported a wide variation in genera of endophytic bacteria from the roots of chickpea (Batra et al. 2020   Page 7 of 9 Khanna et al. Egyptian Journal of Biological Pest Control (2022) 32:17 and humans (Hu et al. 2020). The results of the present study were in accordance with those of Lee et al. (2020) who reported plant growth-promoting activity of Brucella anthropi strain T16R-87 and Saini et al. (2017) who observed the ability of Ochrobactrum intermedium CP-2 chickpea isolate to produce siderophore. Endophytic bacterial isolate CRBE1, identified as Priestia megaterium in the present study, was previously known as Bacillus megaterium (Biedendieck et al. 2021), produced HCN along with ACC utilization, while isolate CRBE7 (Microbactrum haematophilum) showed positive reaction for siderophore and HCN production. Under in vitro conditions, CRBE3 (Brucella haematophila) showed maximum mycelial inhibition (63.95%) of F. oxysporum f. sp. ciceris. Similarly, Lee et al. (2020) reported that B. anthropi strain T16R-87 conferred resistance against bacterial wilt pathogen, Ralstonia solanacearum. Shoaib et al. (2020) reported antagonistic activity of Ochrobactrum ciceri in contending charcoal rot disease of mung bean. The other two isolates CBRE7 and CRBE1, also, showed pronounced antifungal activity against the test pathogen. Antagonistic activity of Priestia sp. against Fusarium wilt was also reported by Bashir et al. (2021) in sunflower. Rangeshwaran et al. 2002 also reported that under in vitro conditions, B. subtilis isolate PDBCEN 3 and Rb29 inhibited the mycelial growth of F. oxysporum f. sp. ciceris, while isolate Rb29 reduced the wilt incidence both in pot and field conditions. Kumari and Khanna (2018) recorded maximum control of mycelial proliferation of the pathogen with Ps16b (45.7%) and Ps45 (87.3%) in dual culture plate and liquid broth assay amongst 61 chickpea rhizosphere isolates, while Mukherjee et al. (2020) reported that amongst the isolates retrieved from chickpea seed, Pseudomonas aeruginosa BHUJPCS-7 showed significant antifungal activity against F. oxysporum f. sp. ciceris.
In the present study, seed priming with endophytic bacterial isolate CRBE3 curtailed the Fusarium disease severity in both the chickpea varieties and found to be superior as individual treatment rather combination or consortium. Maximum phenol content (412.70 µg/g) was observed in plants of chickpea variety HC 3 raised from seed treated with CRBE3. Similarly, Maheshwari et al. (2019) reported that seed treatment with B. subtilis strain (CPHR3) was the most effective in promoting chickpea growth in pots under screen house conditions. Datta and Lal (2012) also correlated Fusarium wilt resistance with phenol content in chickpea varieties and observed maximum phenol content (21.8 mg gdw −1 ) in wilt-resistant cultivars and minimum (16.5 mg gdw −1 ) in susceptible lines of chickpea.
The results of the present study highlighted the significance of sustainable agronomic practices for improving the crop yield, while reduction in the adverse effect of chemical pesticides through seed biopriming of chickpea with root endophytic bacteria was demonstrated.

Conclusions
It was concluded that the comprehensive diversity of endophytic bacteria from C. arietinum plants along with their plant growth promoted potential both in vitro and in vivo. Three effective multi-traits endophytes exhibited plant growth-promoting traits and bio-control potential against a devastating pathogen F. oxysporum f. sp. ciceris inciting Fusarium wilt. Such bio-control agents can be used as a component in integrated disease management Week × Treatment 12.78 19.37