Assessing the efficacy of multifunctional and ACC- deaminase producing Trichoderma harzianum and plant growth-promoting bacteria in the alleviation of salt-stress in Ocimum sanctum

doi:10.21203/rs.3.rs-1596262/v1

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Assessing the efficacy of multifunctional and ACC- deaminase producing Trichoderma harzianum and plant growth-promoting bacteria in the alleviation of salt-stress in Ocimum sanctum

https://doi.org/10.21203/rs.3.rs-1596262/v1

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Salinity is a significant concern in crop production, causing severe losses in agricultural yields. Ocimum sanctum, also known as Holy Basil, is an important ancient medicinal plant used in the Indian traditional system of medicine. The present study explores the use of ACC deaminase-producing strains of plant-growth-promoting bacteria (PGPB) namely Str-8 (Halomonas desiderata), Fd-2 (Achromobacter xylosoxidans), Sd-6 (Brevibacterium halotolerans), Art-7 (Burkholderia cepacia), and Ldr-2 (Bacillus subtilis), and T. harzianum (Th), possessing multi-functional properties like growth promotion, stress alleviation, and root-inducing properties, for using them as bioinoculants for enhancing O. sanctum yield and tolerance to salt stress. The results showed that co-inoculation of Th and PGPBs enhanced plant height and fresh herb weight by 3.78–17.65% and 7.86–58.76% respectively; maximum being in Th + Fd-2 and Th + Art-7 compared to untreated salinized plants. Doubly inoculated plants increased pigments, phenol, flavonoids, protein, sugar, relative water content, and nutrient uptake (Nitrogen and Phosphorous) as compared to monocultures and untreated salinized plants. In addition, combined inoculations in plants resulted in lower Na⁺, MDA, H₂O₂, CAT, APX activities, and also lower ACC accumulation (49.75 to 72.38% over non-treated salinized plant) in O. sanctum, which probably played a significant role in minimizing the deleterious effects of salinity. Overall multifactorial analysis showed that co-inoculation of Th and PGPBs improved O. sanctum growth, its physiological activities, and alleviated salt stress compared to single inoculated and untreated salinized plants.

Plant growth-promoting bacteria

Trichoderma harzianum

Ocimum sanctum

Salinity

ACC accumulation

Ocimum sanctum (Family: Lamiaceae), also called the queen of all medicinal herbs, is grown throughout the tropical Asian region, including India, for the last 3000 years (Gupta et al. 2002; Mondal et al. 2009). Various chemical constituents have been identified in O. sanctum essential oil including eugenol, cinnamyl acetate, β-elemene, rosmarinic acid, flavonoids, ursolic acid, linolenic acid (Kelm et al. 2000; Kothari et al. 2004; Wakchaure et al. 2017). Different plant parts of O. sanctum, like stem, root, seeds, flower, and leaves contain some phytochemicals which have therapeutic potential and are being used in anticancer, antiasthmatic, antidiabetic, hepatoprotective, hypolipidaemic and antistress drugs (Gupta et al. 2002; Wakchaure et al. 2017). Among the variety of abiotic stresses, salinity is the primary threat to agriculture, causing the most significant loss in crop production worldwide (Yadav, 1993). The major components affected by salt stress are plant yield and secondary metabolite content (Nikolova et al. 2005; Jamil et al. 2006). Several researchers have previously reported that alkalinity and salinity have a negative impact on the growth and yield of various crop plants (Bhatnagar and Yadav 1980; Abrol, 1986; Chodhary et al. 1996; Dharmendra and Bijendra 1997). According to the latest statistics, saline lands affect about 10% of the world’s land surfaces (Szabolcs 1989). The use of chemical fertilizers and irrigation with salt-affected water in agricultural practices is the main cause of soil salinity (Woodward et al. 2002). Ideal soil considered for the cultivation of Ocimum spp. is well-drained sandy loam to loam soils (Hussain et al. 1988). The resultant main effects of salinity are ionic imbalance and hyperosmotic stress. The effect of ionic imbalance causes molecular damage resulting in impairment in plant growth. Under severe salinity conditions, the amount of potassium, calcium, and magnesium in the tissues of many plant species gets reduced (Greenway and Munns, 1980). Consequently, this leads to cell death and plant death (Zhu et al. 1997; Xiong and Zhu 2002). Barbieri et al. (2012) have reported that basil showed a reduction in stomatal density, delay in abscisic acid, and proline accumulation under salt stress conditions. Treating the plants with PGPB (plant growth-promoting bacteria) is known to induce growth and defense systems in plants against various stresses modulating their secondary metabolites (Ali et al. 2014; Barnwal et al. 2017; Ahmad et al. 2015; Cho et al. 2015). Trichoderma spp. has been formerly used as biofertilizer and biocontrol agent to enhance crop production and tolerance to environmental stresses (Mastouri et al. 2010; Shoresh et al. 2010). Likewise, various PGPBs are found useful in enhancing crop growth, nutrient uptake, symbiotic properties, and tolerance to environmental stresses in various plants via various mechanisms including synthesis and secretion of growth-enhancing compounds like phytohormones, siderophores, certain enzymes, and activities such as fixation of atmospheric nitrogen and solubilization of phosphates (Barnawal et al. 2012; 2013; 2014; 2017; Abd Allah et al. 2018; Saleh et al. 2019; Sapre et al. 2018). Some physiological changes that could be induced by certain enzymes in plants can carry out these functions. Among these enzymes, ACC (1-aminocyclopropane-1-carboxylate) deaminase is essential for ethylene regulation (Valluru et al., 2016). ACC can be metabolized by some PGPBs possessing ACC- deaminase activity into ammonia and α-ketobutyrate; or else, ACC can be transformed into ethylene (Barnwal et al. 2012; Madhaiyan et al. 2006). The ethylene produced in plants, when combined with auxins, can promote plant growth as well its development, especially the development of roots (Gschwendtner et al., 2016). The strains of bacteria having ACC deaminase activity can mitigate harmful effects on plants induced by stress-induced ethylene. Previous studies have demonstrated that inoculating bacteria with ACC deaminase can lessen the effects of salt-stress in a variety of plants (Barnawal et al. 2013; Himadri and Tapan 2021; Subhan et al. 2020). Although there are many reports on the application of T. harzianum and PGPB alone to increase salinity tolerance in various plants; no or limited attempts on co-inoculation of T. harzianum and PGPB to enhance salt stress tolerance in O. sanctum have been made. Therefore, the present study focuses on studying the effect of combined inoculations of ACC deaminase producing T. harzianum and PGPBs in reducing salt stress in O. sanctum.

Culture Selections and Compatibility study

Five PGPB strains Str-8 (Halomonas desiderata; GenBank accession no. JQ436849), Ldr-2 (Bacillus subtilis; GenBank accession no. JX996197), Fd-2 (Achromobacter xylosoxidans; GenBank accession no. JQ 975414), Art-7 (Burkholderia cepacia; GenBank accession no. KP 198544), Sd-6 (Brevibacterium halotolerans; GenBank accession no. NR042638) were obtained from Crop Protection Division, CSIR- Central Institute of Medicinal and Aromatic Plants, Lucknow, India. Plant growth-promoting activities of these selected strains have been previously evaluated on various aromatic and medicinal crops (Barnawal et al. 2012; 2013; Maji et al. 2013; Bharti et al. 2015). These selected strains were further characterized for various plant growth-promoting activities such as ACC deaminase production, nitrogen fixation, solubilization of phosphates, IAA, and siderophore production. All the strains were checked for their compatibility with T. harzianum (US Patent No. 6475772).

In vitro testing was performed to determine the compatibility of all PGPBs with T. harzianum by following the method of Singh et al. (2019). Freshly prepared bacterial cultures (24 h old) were streaked at the periphery of a Petri plate, poured with {NA+PDA (1:1)} growth media, and incubated for 24 h at 26±2°C. After one day, 5 mm disc of T. harzianum (5 days old culture) was placed in the centre of the plate and further incubated at 26±2°C for 5 days. The absence of an inhibition zone indicated the compatibility between the bacterial and fungal strains. Each test was independently replicated four times.

Biofilm study

For in vitro biofilm study, compatible microbes were used for the crystal violet assay (Triveni et al.2012; Singh et al. 2019). Three ml of T. harzianum suspension (5 days old) and 5 ml of bacterial culture (2 days old) were mixed in 250 ml broth and incubated for 16 days at 30°C. Fungal-based bacterial biofilm was observed under an LCD microscope US3 model (BR Biochem Life Sciences Pvt. Ltd.) as per the procedure of Alexander (2009).

Preparation of bacterial and T. harzianum inoculums

PGPB strains and T. harzianum were grown separately in nutrient broth (24 h) and potato dextrose broth (7 days), respectively. Bacterial cells were harvested by centrifugation (8,000 x g at 4° C for 10 min), washed thoroughly, and re-suspended in sterilized NaCl solution. The bacterial suspension’s CFU (colony forming unit) count was maintained at 3×10⁸ml^-1 (Bharti et al. 2013). T. harzianum culture was filtered through cheeseclothinto a sterile glass bottle and suspended in sterile saline solution. The spore count has adjusted to approximately 3.0 x 10⁸ spores ml^–1.

Glasshouse experiment

The experiments in pots were conducted with O. sanctum cv CIM “Ayu” to assess the effect of selected PGPBs and T. harzianum, either alone or in various combinations, on the growth and secondary metabolite status of plants under saline conditions. Under glasshouse conditions, experiments were conducted in 20 cm tall and 20 cm internal diameter earthen pots filled with a mixture of soil: vermicompost {(3:1); 3 kg pot^-1} in a completely randomized block design (CRBD) with 4 replications. Surface sterilization of roots of O. sanctum was done by dipping the roots in NaOCl (4%) for 1 minute, preceded by several washes with sterilized distilled water (SDW) several times. Before planting, roots were dipped for 30 min in microbial cultures, and the remaining culture (5 ml pot^-1) was poured into the same pot (Maji et al. 2013). The potting mixture without inoculation served as a control (Bharti et al. 2013; Singh et al. 2019). Plantlets were inoculated with the same microbial cell suspension (3×10⁸ CFU/ml) after 15 days. For pots in the glasshouse that were watered on a regular basis, a temperature of 28 ±2°C and a humidity of 50% were maintained.

Treatments applied were negative control (untreated non-salinized); Positive control (untreated salinized), T. harzianum (Th); Halomonas desiderata (Str-8); Bacillus subtilis (Ldr-2); Achromobacter xylosoxidans (Fd-2); Burkholderia cepacia (Art-7); Brevibacterium halotolerans (Sd-6); Th +Str-8; Th +Ldr-2; Th +Fd-2; Th+Sd-6; Th +Art-7. All the pots were watered three times a week with SDW for the first 15 days after inoculation of cultures and then periodically supplemented with NaCl (salt) solution at concentrations steadily increasing from 50 mM to 500 mM NaCl solution till harvesting. Plants were harvested after 90 days of planting. Negative control plants were not supplemented with saline solution (EC=0.587 mS at 20.5 °C).

Plant growth measurement, electrolyte leakage, and relative water content

Growth parameters like the height of the plant and herb weight (fresh), were recorded after the harvest. Electrolyte leakage and relative water content were also determined by following the methods of Hnilickova et al. (2019) and Medici et al. (2003), respectively.

Photosynthetic pigments

Chlorophyll and carotenoid contents were determined by previously described methods of Lichtenthaler and Buschmann (2001) and Singh et al. (2019). A UV-VIS Spectrophotometer (Spectra Max plus 384) was used to measure absorbance at 665.2, 662.4, and 470 nm.

Proline, Malondialdehyde (MDA), hydrogen peroxide, and sugar content

Proline content in the leaf was determined, as illustrated by Bates et al. (1973). The concentration of MDA, a lipid peroxidation product, was analyzed by the TBA method in leaf samples (Hodges 1999). To determine hydrogen peroxide content, fresh leaf tissues were extracted in TCA (0.5ml; 0.1%), and then centrifuged. H₂O₂ content was calculated as illustrated by Velikova et al. (2000). The total soluble sugar in leaf samples was determined as described previously by Irigoyen et al. (1992).

Antioxidant enzymatic activities

Leaf tissues were extracted in100 mM phosphate buffer (4 ml; pH: 7.2) using mortar and pestle, as Aroca et al. (2003) reported. After centrifugation (18000xg for 10 min at 4°C), the supernatant was used for evaluating enzyme activity.

Ascorbate Peroxidase (APX): The enzyme’s activity was assessed in a 1ml mixture of potassium phosphate buffer (100 mM; pH: 7.0), hydrogen peroxide (0.5 mM), Ascorbate (5 mM), and enzyme extract. To start the reaction, H₂O₂ was added, and the reduction in the absorbance was recorded at 290 nm for 1 min (Nakano and Asada 1981).

Catalase (CAT): For assessing the catalase activity method of Aebi (1984) was followed. The activity of CAT was determined by recording the decline in absorbance at 240nm in a reaction mixture containing 10 mM phosphate buffer, 150 mM hydrogen peroxide, and enzyme extract and calculating the rate of decomposition H₂O_2.

Total protein content: It was calculated using the Lowry et al. (1951) method, and Bovine Serum Albumin was used as a standard.

Total flavonoid and phenol content

Fresh leaves were extracted in 0.5 ml ethanol (80%) thrice and centrifuged. Supernatants were then pooled, and the total phenolic content was calculated using the Folin–Ciocalteu method, as previously reported by Zhang et al. (2006), using gallic acid as a standard. The total flavonoid content was measured using the aluminum chloride colorimetric method described by Chang et al. (2002) and quercetin as the standard.

Measurement of ACC level in the plant tissues

ACC concentrations in root samples were estimated by following Barnawal et al. (2012). Frozen leaf samples were extracted in 80% methanol (5ml) containing butylated hydroxytoluene (2 mg l⁻¹) thrice and samples were pooled. After centrifugation (12000x g for 15 min at 4°C), the supernatants were combined and evaporated using a rotary evaporator. ACC level was estimated by gas chromatography as reported by Barnawal et al. (2012). The supernatant was mixed with 0.1 ml HgCl₂ (80 mM) in vials and air tightened with rubber septa. Afterward, sodium hypochlorite solution (0.2 ml) was injected in the vials through septa, shaken well, and incubated for 8 minutes, and analyzed by gas chromatography for ethylene determination (Barnawal et al. 2012; 2013; 2014).

Foliar nutrient uptake

In air-dried samples, sodium, nitrogen, and phosphorous content were estimated by wet digestion with HNO₃ + H₂O₂. The Kjeldahl method was used for nitrogen estimation (Singh et al. 2009). Flow Inject Analyzer (Foss FI- ASTAR 5000) was used for phosphorus and ﬂame photometer for sodium estimation (Singh et al. 2009; 2019).

Statistical analysis

Statistical analysis was performed by ANOVA method with IBM SPSS Statistics 20 software. Means and standard errors were determined for 4 replicate values, and means were compared using DMRT’s signiﬁcance level under 0.05. The principal component analysis was applied using XLSTAT 2021.1.1 Addinsoft to reveal the correlation among treatments and parameters.

Compatibility study

In vitro cross streak assay, biofilm formation, and the microscopic study showed that all the five strains Str-8, Ldr-2, Fd-2, Sd-6, and Art-7 were compatible with T. harzianum.

Effect on plant growth, electrolyte leakage, and relative water content

No effect of single and dual inoculations was noticed on plant height over positive control (untreated salinized) plants. Of the monocultures, Sd-6 inoculated plants showed increased fresh herb weight over positive control. On the other hand, in doubly inoculated plants, Th+Art-7, Th+Ldr-2, and Th+Fd-2 inoculations significantly increased fresh herb weight compared to monocultures and positive control (Table1). Maximum enhancement in plant growth was noticed in Th +Art-7, (58.77%over control), while in doubly inoculated plants Th+Ldr-2 showed maximum increase in fresh herb weight over its respective monoculture i.e., 38.05%.

The plants inoculated with monocultures exhibited a significant decrease in electrolyte leakage, which was reduced further with dual inoculation of Th+Art-7 over monocultures and positive control. Str-8, Fd-2, and Sd-6 inoculated plants significantly enhanced relative water content over positive control in monocultures. Th+Ldr-2 followed by Th+Sd-6 and Th+Fd-2 increased the relative water content of inoculated plants further over monocultures and positive control (Table 1).

Table 1: Effect of bio inoculants on height, fresh herb weight, electrolyte leakage and relative water content in O. sanctum under salt stress

Treatments	Height (cm)	Fresh herb weight (g)	Electrolyte Leakage (dS/m)	Relative Water content (%)
Positive Control	59.5±2.90^ab	36.67±1.44^d	80.94±0.54^a	69.12±1.13^g
Th	64.25±5.72^ab	42.61±5.54^cd	69.28±1.16^d	74.72±0.42^f
Str-8	55.75±2.25^b	42.11±3.50^cd	63.79±1.09^e	78.34±0.81^e
Ldr-2	57.25±1.89^ab	39.55±3.38^d	65.66±0.63^e	74.60±1.13^f
Fd-2	67.5±5.58^ab	42.59±6.54^cd	41.38±1.59^g	82.45±0.42^cd
Sd-6	70±0.71^a	52.00±3.88^abc	65.60±1.22^e	80.03±0.78^de
Art-7	61.75±2.59^ab	45.68±1.36^bcd	60.25±0.84^f	72.75±0.57^f
Th+ Str-8	62.75±5.91^ab	42.17±0.99^cd	72.34±0.69^c	80.43±1.28^de
Th+ Ldr-2	67.25±1.31^ab	54.60±2.37^ab	71.56±0.50^cd	88.44±0.57^a
Th+ Fd-2	70±2.86^a	53.81±2.51^ab	69.98±1.02^cd	84.11±0.92^bc
Th+ Sd-6	67.75±2.29^ab	52.04±3.12^abc	78.14±0.70^b	86.43±1.02^ab
Th+ Art-7	68±5.12^ab	58.22±2.95^a	39.14±0.42^g	81.22±1.00^d
Negative Control	60±4.71^ab	52.56±2.32^abc	28.04±0.75^h	87.00±0.43^a

Th: Trichoderma harzianum; Sd-6: Brevibacterium halotolerans; Art-7: Burkholderia cepacia; Ldr-2: Bacillus subtilis; Fd-2: Achromobacter xylosoxidans; Values represents means ±SE (n=4); positive control: untreated salinized plants; negative control: untreated non salinized plants; Different letters denote significant difference among treatments according to Duncan’s Multiple Range Test (P<0.05)

Effect on total chlorophyll and carotenoids

All the monoculture treatments, except Sd-6, increased total chlorophyll content over positive control, while among dual inoculations, Th+Fd-2 and Th+Art-7 treated plants recorded a significant increase in total chlorophyll content over positive control. Inoculation of four monocultures out of five resulted in enhanced carotenoid content in plants. Th+Fd-2 showed a maximum increase in carotenoid content; however, the increase was not significant over monocultures (Figure 1).

Effect on antioxidant enzymes

The positive control plants recorded significantly higher CAT and APX activities over the negative control (untreated non-salinized) plants. However, all the single and dual inoculated plants reduced CAT and APX activities compared to positive control plants. In the case of dual inoculations, Th+Ldr-2, Th+Fd-2, and Th+Sd-6 inoculated plants recorded a decrease in CAT but not in APX activity as compared to monocultures. However, it was significantly lower than positive control plants (Figure 2).

Effect on Protein, Sugar, H₂O₂, MDA, and proline content

All monocultures recorded an increase in protein content over positive control except Ldr-2 inoculated plants. Application of Th+Sd-6 and Th+Ldr-2, in case of dual inoculations, recorded a significant increase in protein content (i.e., 142.57% and 137.63%, respectively) compared to positive control plants; however, no dual treatment was found significantly superior over monocultures. Likewise, the sugar content was enhanced by all single and dual inoculated plants as compared to positive control plants. Again, no dual inoculated plants could significantly enhance sugar content in comparison to the monocultures (Table 2). Proline content was not significantly increased by any monoculture, while in dual inoculations, it was significantly higher in Th+Ldr-2 inoculated plants (i.e., 10.53% over the positive control plants). All the monocultures recorded a reduction in MDA content, and in dual inoculated plants, it was significantly reduced by Th+Ldr-2 inoculated plants (i.e., 48.05% over the positive control plants) followed by Th+Fd-2 and Th+Art-7 as compared to monocultures and positive control. Similarly, all the monocultures recorded a reduction in H₂O₂ content when compared with positive control. In the case of dual inoculations, namely Th+Ldr-2, Th+Sd-6 and Th+Art-7, plants showed reduction in H₂O₂ content over monocultures and positive control. The highest reduction was recorded in Th+Sd-6, Th+Fd-2, and Th+Art-7 inoculated plants; a reduction of 71.38%, 64.51%, and 73.54%, respectively, as compared to positive control plants (Table 2).

Table 2: Effect of bioinoculants on biochemical parameters in O. sanctum under salt stress

Treatments	Protein (mg g^-1 FW)	Sugar (µg g^-1 FW)	Hydrogen peroxide (mg g^-1 FW)	MDA (nmol g^-1FW)	Proline (mg g^-1 FW)
Positive Control	225.16±4.47^g	191.35±14.15^d	9.75±0.04^a	4.87±0.04^a	12.54±0.08^b
Th	530.48±17.39^b	498.22±15.46^ab	4.50±0.17^d	3.31±0.01^de	8.02±0.21^d
Str-8	427.53±6.93^de	512.85±4.87^ab	7.88±0.15^b	3.41±0.06^cd	4.79±0.02^f
Ldr-2	250.24±1.32^g	347.12±6.76^c	3.60±0.06^fg	3.27±0.04^de	3.74±0.04^gh
Fd-2	410.47±11.31^de	336.00±43.95^c	6.14±0.35^c	3.44±0.08^cd	6.17±0.44^e
Sd-6	381.95±3.84^def	515.32±0.60^ab	4.38±0.02^d	4.26±0.02^b	5.73±0.06^e
Art-7	343.37±5.06^f	523.18±2.00^ab	8.17±0.10^b	3.59±0.06^c	2.82±0.05ⁱ
Th+ Str-8	436.02±2.30^cd	520.87±1.97^ab	4.06±0.02^de	3.30±0.02^de	7.75±0.04^d
Th+ Ldr-2	535.05±26.67^b	470.16±13.17^b	3.24±0.09^g	2.53±0.02^h	13.86±0.17^a
Th+ Fd-2	493.17±61.08^bc	348.34±10.03^c	3.46±0.30^fg	3.18±0.05^ef	12.43±0.05^b
Th+ Sd-6	546.16±3.69^b	527.12±1.22^ab	2.79±0.03^h	3.42±0.06^cd	8.90±0.32^c
Th+ Art-7	370.16±8.58^ef	538.90±2.21^a	2.58±0.02^h	2.81±0.18^g	4.31±0.20^fg
Negative Control	838.54±8.89^a	491.60±48.21^ab	3.72±0.04^ef	3.05±0.04^f	3.29±0.07^hi

Th: Trichoderma harzianum; Sd-6: Brevibacterium halotolerans; Art-7: Burkholderia cepacia; Ldr-2: Bacillus subtilis; Fd-2: Achromobacter xylosoxidans; positive control: untreated salinized plants; negative control: untreated non salinized plants; Values represents means ±SE (n=4); Different letters denote significant difference among treatments according to Duncan’s Multiple Range Test (P<0.05)

Effect of various bioinoculants on nutrient uptake

In salinized plants, sodium concentration was reduced by all single and dual inoculated plants over the positive control. All the monocultures recorded a reduction in sodium content, while in the case of dual inoculations, all inoculated plants showed significantly reduced sodium content compared to monocultures and positive control plants. A slight increase in nitrogen content was recorded by all monocultures, except Sd-6, over positive control. In contrast, dual inoculation of Th+Sd-6 recorded a maximum increase in nitrogen content over monocultures and positive control plants. Th and Str-8 significantly increased phosphorous content among all monocultures over the positive control. Also, in plants with dual inoculation of Th+Str-8 and Th+Art-7, significantly higher phosphorous content was recorded over monocultures and positive control (Figure 3).

Effect on ACC content

All treatments significantly decreased ACC content, with the highest reduction in T. harzianum inoculated plants over the untreated salinized plants. However, all the dual inoculated plants showed reduction in ACC content, significant reduction was found in Th+Fd-2(61.88%), Th+Sd-6 (56.99%) and Th+Ldr-2 (38.95%) compared to theirrespective bacterial monocultures (Figure 4).

Effect on total phenol and flavonoid content

All monocultures recorded an increase in total phenol and flavonoid contents over positive control. In dual inoculations, total phenol content was significantly increased by all the doubly inoculated plants as compared to monocultures and positive control. Flavonoid content was also significantly enhanced by all the dual inoculations over monocultures and positive control plants (Figure 5).

Microbial consortia are gaining strong acceptance by agriculturists around the globe as a possible alternative to chemical fertilizers and are thus being incorporated into important nutrient and disease management programs (Kaushal et al. 2019). However, the combination of strains/organisms with multifunctional activities like T. harzianum and selected PGPBs in plant growth promotion and abiotic stress alleviation has not been focussed much, especially in the case of medicinal and aromatic plants. Therefore, efforts were made to enhance the plant growth under stress with combined inoculation of organisms that can reduce plant stress, improve the nutrient uptake and increase the yields under salinity stress in O. sanctum. Previous reports of the association of bacteria and fungi residing in roots, leaves, and seeds of Withania somnifera have been reported to improve plant growth and photosynthetic efficacy (Pandey et al. 2018). In the present study, inoculation of T. harzianum and PGPBs, both possessing ACC deaminase activities, considerably increased the fresh herb weight, total chlorophyll, proline, total sugar, protein, phenol, and flavonoids in O. sanctum as compared to positive control plants under salt stress. The present work also showed more accumulation of phenols and flavonoids in dual inoculated plants over monocultures and positive control supporting better growth and radical scavenging property (Cheynier 2012; Singh et al. 2013). It is hypothesized that the enhancement in growth, biochemical parameters indicating stress alleviation might be possibly due to the synergistic effects of microbial partners T. harzianum and PGPBs. It has been previously reported that Trichoderma having ACC deaminase activity could enhance growth, minimized ionic toxicity, and tolerance to salt stress in wheat (Zhang et al. 2019). Likewise, ACC deaminase-producing bacteria have been shown in studies to improve plant growth during environmental stresses that increase ethylene production (Glick et al. 2007; Belimov et al. 2009; Barnawal et al. 2014; 2017). In the present study, ACC deaminase producing T. harzianum and PGPBs effectively reduced electrolytic concentrations than positive control plant. The lesser permeability of the plasma membrane chiefly pointed towards the integrity and constancy of cellular tissues by the microbial consortia interaction over the positive control (Garg and Manchanda 2009). In addition, the RWC of dual inoculated plants was recorded higher than the positive control plants in this study, indicating the beneficial role of microbial consortia in reducing salt stress by fetching more water from sources inaccessible to control plants (Singh et al. 2013). Another study found that consortium consisting of B. japonicum and P. putida improved salt tolerance in soybean, facilitating better nutrient uptake in the plants (Egamberdieva et al. 2017). Our results were also on a similar line as a slight increase in nitrogen and phosphorus content was observed over positive control in microbe-treated plants. In addition, numerous studies have reported that salt stress can increase the toxic levels of cellular Na⁺ concentration in plants and restrained K⁺ absorption, disturbing the intracellular ionic homeostasis in plants. Thus, a reduction in Na⁺ accumulation in plants provides tolerance to salt stress and induces plant growth (Deng et al. 2016; Chen et al. 2007). In the current investigation, the results revealed that Na + ion concentration was reduced in leaves of all single and dual inoculated plants; most significantly in Th + Ldr-2 treated plants over positive control. Other parameters i.e., MDA, hydrogen peroxide, antioxidant enzymes (CAT, APX), and ACC accumulation, were reduced by different single and dual inoculated plants; results were more encouraging in dual inoculated O. sanctum plants under salt stress. Salt-induced oxidative damage in terms of MDA and H₂O₂ was alleviated by B. cereus inoculation in Vigna radiata (Islam et al. 2016). The decreased MDA and H₂O₂ content in dual inoculated plants in our experiment confirm the previous studies where plants were protected from salt-induced oxidative damage by various plant beneficial microbes (Bharti et al. 2014; Barnawal et al. 2014). The present work recorded the highest increase in content of proline by Th + Ldr-2 and sugar content by Th + Art-7 treated plants compared to positive control plants. Under salt stress, osmolytes such as proline, sugar, and amino acids play a key role in maintaining the water balance of cells by regulating cellular metabolism (AbdAllah et al. 2016; Agami et al. 2016). A study reported that PGPR strains, Serratia sp. and Rhizobium sp., alleviated soil salinity by affecting antioxidant enzymes, photosynthesis, nutrient uptake, and growth of Lactuca sativa. Our results showed that soil salinity increased the enzyme activity in positive control plants, while PGPBs and Th inoculated decreased the CAT and APX enzyme activity. In earlier studies, reduction in CAT and APX enzymes as a result of microbial consortia treatment in various plants under stress conditions have been reported (Upadhyay et al. 2012; Han and Lee, 2005; Kumar et al. 2018). On the contrary, in some studies, inoculation with PGPR in plants increased enzymatic activities under saline conditions (Singh et al. 2017). These studies, although valuable, are rather inexplicable. It has been observed that in plant defense mechanism, ROS does not depend merely on a few enzymatic activities but on the total antioxidant defence system (Liang et al, 2003). It may be concluded that each PGPR strain utilizes its own distinct mechanism to affect different plant genotypes for alleviation of abiotic stress (Akbari et al, 2020). Another study showed a decrease in antioxidant enzyme activities in PGPR inoculated paddy, signifying a slightly lower ROS scavenging and dissimulating plant's capability, suggesting the role of PGPR in salt tolerance (Jha and Subramanian, 2014). For reducing the stress, another major mechanism used by plant growth-promoting microbes consists of minimizing ethylene levels through hydrolyzing the ACC through an enzyme ACC deaminase. It is known that certain PGPB containing ACC deaminase enzymes can convert ACC (immediate precursor of ethylene) to ammonia and α-ketobutyrate (Singh et al. 2015; Gamalero and Glick 2015; Raghuwanshi and Prasad 2018). We used selected PGPBs and a T. harzianum strain possessing ACC deaminase activity in the present study. Nitrogen and phosphorous are essential macronutrients for plant growth as well as development and their levels increase the plant tolerance to stress (Averina et al. 2014). Furthermore, elevated levels of NO₃ in the rooting medium have been shown to stimulate ACC-oxidase activity, resulting in enhanced ethylene production. The inoculation of these strains in the Ocimum plant could effectively reduce salinity-induced damage due to high nutrient uptake by dual inoculated plants over single inoculations, which is similar to the finding of Zhou et al, 2011 and Guo et al, 2010. This probably happened because of the substantial reduction in the content of the ACC (direct substrate for ethylene biosynthesis), thereby reducing salt-induced damage mediated by ethylene. ACC content in the individual application of Th was reduced and further decreased by all dual inoculations except Th + Art-7 compared to bacterial monocultures, which may represent the basal cellular ethylene level needs to be present for cellular homeostasis. This also suggests that other growth-promoting activities of selected microbes (like phosphate solubilization, IAA production, siderophore production, and fixation of nitrogen) play a major role in the observed effect in this study. Hence, it is apparent that PGPB that produce ACC deaminase have the potential to reduce abiotic stress-induced ethylene levels and its damaging effect on plants (Glick, 2014). Another study supporting our results showed that consortium of Aneurinibacillus aneurinilyticus and Paenibacillus spp. inoculation in French beans significantly reduced ethylene levels by 60% induced by salt stress and improved the biomass and total chlorophyll content (Gupta and Pandey, 2019). The current findings of the dual culture compatibility assay, crystal violet assay, and pot study showed that all the PGPBs were compatible with Th and showed signs of their cumulative growth promotion activity in dual culture. It has been observed that microbial consortia having a syntrophic relationship put forth favourable and improved results in plant growth and secondary metabolite content (Morris et al. 2013).

Multifactorial Analysis

The PCA (principal component analysis) and their correlation analysis specified the relationship between measured parameters and their treatments (Fig. 6). The 1st factor showed 46.80%, whereas the 2nd factor represented 13.71% of the variance of data. In view of this PCA, three clusters were created in the biplot, which included chlorophyll, carotenoids, protein, flavonoids as one cluster. Phenols, sugars, fresh weight, nitrogen, phosphorous, relative water content, height formed the second cluster, while electrolyte leakage, hydrogen peroxide, catalase, ascorbate peroxidase, ACC content, sodium, proline fell in the third cluster. The parameters in each cluster were closely related to each other. Similarly, treatments also had 2 clusters: cluster 1, which had Th + Ldr-2, Th + Art-7, Th + Str-8, Th + Fd-2, and Th + Sd-6, while the 2nd cluster had Ldr-2, Art-7, Str-8, Fd-2, Th, and Sd-6. However, negative and positive controls were located far-away from the other treatments showing different responses from other clusters. Based on the factorial analysis, cluster 1 and cluster 2 parameters were closely related to all the dual inoculations, while cluster 3 parameters were closely related to the monocultures.

In the current study, it has been revealed that a compatible combination of T. harzianum and PGPBs sustained better growth and abiotic stress tolerance compared to single strains or non-inoculated salinized plants. Exploring T. harzianum and PGPBs possessing plant growth-promoting and stress alleviation and biocontrol potential may thus play a significant role in developing bioformulations useful in integrated nutrient management and stress management programs. Therefore, this study strengthens the viewpoint that the application of microbial consortia-based bio formulations can be successfully used for increasing agricultural production especially in salt-affected areas.

Acknowledgments

The author (SS) is thankful to the Department of science and technology (Ref no. DST/DISHA/SoRf-PM-016/2013) for financial support and Director, CSIR-Central Institute of Medicinal and Aromatic Plants for providing necessary facilities for the present work. The author would also like to thank Dr. Vikas Kumar Patel (Art-7) and Dr. Deepti Barnawal (Fd-2 and Ldr-2) for supplying bacterial strains for this study.

Authors contribution

Designing the research SS and AK, performing the experiments SS, ACC analysis CSC, Analyzing the data SS, Writing the manuscript SS, AS, Supervision PV, Supervision and Writing AK

Compliance with Ethical Standards

Conflict of interest: The authors declare that they have no conflict of interest regarding the publication of this paper.

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No competing interests reported.

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Assessing the efficacy of multifunctional and ACC- deaminase producing Trichoderma harzianum and plant growth-promoting bacteria in the alleviation of salt-stress in Ocimum sanctum

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