Novel synergism of Cedecea lapagei KU14 and Bacillus subtilis KU21for sustainable productivity of Rosmarinus officinalis in Northwest Himalayas

doi:10.21203/rs.3.rs-1501235/v1

Download PDF

Research Article

Novel synergism of Cedecea lapagei KU14 and Bacillus subtilis KU21for sustainable productivity of Rosmarinus officinalis in Northwest Himalayas

https://doi.org/10.21203/rs.3.rs-1501235/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Aims

Rosmarinus officinalis (rosemary) is most extensively used culinary herbs rich in antioxidants and several other secondary metabolites. Soil quality affects the biosynthesis of secondary metabolites thereby reducing their quality. An approach to improve soil quality and boosting plant productivity in a sustainable manner is the use of plant growth promoting (PGP) endophytic bacteria. Previously three endophytic bacteria associated with roots of R. officinalis were characterized for in vitro PGP traits. The purpose of this study was to assess (1) the compatibility of isolated potential indigenous endophytic bacteria for PGP traits to develop potential consortium biofertilizer, and (ii) how the coordinated interaction among these strains mediates the response for productivity and nutrient acquisition of R. officinalis.

Methods

In vitro synergistic interactions among isolated strains (Bacillus subtilis KU21, Pseudomonas aeruginosa SI12 and Cedecea lapagei KU14) were examined for multiple PGP traits. In vivo studies were carried out to assess synergistic effects of theses strains on productivity and soil quality of R. officinalis.

Results

The consortia of strains exhibited higher production of IAA, siderophore and phosphorus solubilization. Further, assessment of consortia of these strains significantly improved rosemary growth parameters, nutrient acquisition and soil quality over single inoculation and uninoculated control.

Conclusions

This is the first field report that describes the efficacy of novel endophytic strains i.e., B. subtilis KU21+ C. lapagei KU14 for improving rosemary production and soil health simultaneously, may be recommended for sustainable production of R. officinalis in Northwest Himalayan region.

Rosmarinus officinalis

endophytic bacteria

growth attributes

Cedecea lapagei

Bacillus subtilis

Pseudomonas aeruginosa

Aromatic and medicinal plants have recently attracted a lot of interest in the food industry. R. officinalis (Rosemary) is one of the most extensively used spices in food processing from the Lamiaceae family of herbs (Gharib et al. 2016). Rosemary is a perennial evergreen shrub (upto 3 meters height), which has also been named as the “Herb of the Year” in 2001 by the International Herb Association (Indian mirror). It has numerous biological activities such as antibacterial, antiproliferative, anti-inflammatory, and antioxidant (Bourhia et al. 2019). Rosemary grows in a wide range of climates i.e., 1000–3000 metres above mean sea level (masl) and is native to Europe, Asia, and Africa, primarily in the Mediterranean Sea region (Pintore et al. 2002). Rosemary has been successfully cultivated practically everywhere in the world in recent years, largely for its aromatic leaves and essential oil extraction. In India, rosemary has been planted extensively in cooler areas, one of which being Himachal Pradesh, where it is grown in Kangra, Kullu, and Mandi districts. Owing to its biological activities it is widely used by tribal communities of the state, thereby leading to its increased demand and production in the recent years (Sharma et al. 2021).

Rosemary and its compounds were the foremost natural antioxidants to be commercialized (Yanishlieva et al. 2006). Although secondary metabolites in therapeutic and aromatic plants are traditionally governed by their genotypes, environmental conditions also have a substantial influence on their biosynthesis. The soil quality is one of the most crucial environmental elements. Improved soil quality boosts plant productivity, concurrently mitigating climate change impacts through carbon sequestration (Shrestha et al. 2013). The management of soil quality relies heavily on maintaining an optimum quantity of soil organic matter and biological nutrient cycling (Ghosh et al. 2015).

Two types of soil bacteria have been shown to improve soil health and quality by increasing nutrient availability in the rhizosphere: rhizospheric bacteria, which are often found around plant roots, and endophytic bacteria, which are found within the plant's tissues (even though endophytic bacteria can also be found in the soil as free-living bacteria) (Lacava and Azevedo 2013). To promote host growth, rhizospheric and endophytic PGP bacteria use similar, if not identical, methods. The fundamental distinction is that once PGP endophytic bacteria have established themselves within the host plant's tissues, they are no longer exposed to the vagaries of shifting soil physico-chemical conditions (Glick 2012).

Pseudomonas and Bacillus are the prominent genera in endophyte community of medicinal plants (Sharma et al. 2021). Several studies have also registered their yield enhancement and growth promoting potential of host plants under different agro-climatic conditions (Bacillus and Brevibacillus; ALKahtani et al. 2020, B. subtilis; Jabborova et al. 2021, B. subtilis; Sharma et al. 2021, P. aeruginoa; Chandra et al. 2020, P. segetis; Rodriguez et al. 2020). Another interesting genus that has been reported as PGP endophyte is Cedecea, of family Enterobacteriaceae. The in vitro traits of plant growth promotion are well documented for Cedecea (Benaissa et al. 2019; Sharma et al. 2021).

Without causing any symptoms, endophytic bacteria inhabit plant's internal tissues (Schulz and Boyle 2006). They have a strong relationship with their host plant in order to obtain easily available nutrients and to establish a friendly environment within the apoplastic space (Weyens et al. 2009). A stronger interaction between plant and endophytic bacteria can be built to enhance growth, pathogen resistance and yield (Stajkovic et al. 2009). Endophytes induce resistance against phytopathogens in host plant by releasing variety of secondary metabolites such as siderophores, antibiotics, and lytic enzymes etc. (Silva et al. 2019). They also aid in degradation of pollutants, solubilization of minerals (phosphate and zinc), or assimilation of nitrogen, as well as the release of phytohormones (auxin or cytokinin) and vitamins (Silva et al. 2019). Therefore, PGP bacterial endophytes are of particular interest to rosemary cultivation in a sustainable manner, as they stand out amongst the most acceptable and eco-friendly approaches to be applied as biofertilizers or as biopesticides (Sharma et al. 2021; Sadegh Kasmaei et al. 2019).

In our recent work, the growth stimulating capacity of native PGP strains of Cedecea lapagei KU21, B. subtilis KU14, and P. aeruginosa SI12 for R. officinalis was well examined under protected conditions (Sharma et al. 2021). Continuing the studies further, as till date, no published study has linked the efficacy of indigenous PGP bacterial endophytes and their consortia on the productivity and soil health of R. officinalis under in vivo conditions in the North-Western Himalayas. Keeping this in mind, the current study aims to assess (i) the compatibility of potential indigenous endophytic bacteria B. subtilis KU21, P. aeruginosa SI12, and C. lapagei KU14 for PGP traits to develop as consortium biofertilizer, and (ii) how the coordinated interaction among these strains mediates the response for growth and nutrient acquisition in R. officinalis.

Bacterial strains and their maintenance

Endophytic strains of B. subtilis KU21, P. aeruginosa SI12 and C. lapagei KU14 previously isolated from root tissues of R. officinalis (Sharma et al. 2021) were used in the present study. These strains were preserved on nutrient agar (NA) plates at 4 ºC for further experiments.

Biocompatibility Interaction Assay And Characterization For Pgp Traits

Potential endophytic strains were screened for inter-compatibility with each other on nutrient agar using cross streak method (Monthon and Sawangnop 2011; Nazmul and Rahman 2014). Each of the three strains was streaked as straight line on NA plate followed by single streak with similar strains at 90^° angle to the previously streaked strains and incubated at 30°C for 24–48 h. No zone of inhibition around the crosses between streaked lines of strains indicated protocooperation among the tested strains. Protocooperation between B. subtilis KU21, P. aeruginosa SI12 and C. lapagei KU14 was also assessed for growth spectro-photometrically (OD₆₀₀ nm) at 24, 48 and 72 h of incubation in nutrient broth as single and co-inoculants (Kumawat et al. 2021).

Each of three selected strains (individual and combinations) were further evaluated for in vitro PGP traits i.e., P-solubilization (Pikovskaya 1948), IAA (Gorden and Palleg 1957) and siderophore production (Schwyn and Neilands 1987) as per standard protocols.

Plant Growth Stimulation

Preparation of inoculum

The selected endophytic strains (B. subtilis KU21, P. aeruginosa SI12 and C. lapagei KU14) were grown in nutrient broth at 30 ºC in a rotary incubator shaker at 120 rpm for 48 h (Thermo Scientific™ Sorvall™ ST 8). The bacterial cells were then harvested by centrifugation at 3,000 rpm for 15 min. The obtained pellets were then washed using autoclaved distilled water and the cells were suspended in autoclaved distilled water at a concentration of 1× 10⁸ cfu/mL by dilution. The following formulations were used for in vivo growth promotion studies (Mortensen 1997).

Procurement of seedlings

Two months old healthy (disease and infection free) seedlings of R. officinalis procured from Council of Scientific and Industrial Research-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh (India) were used for plant growth promotion studies under both net house and field conditions.

Net house studies

Potential endophytic strains (single and combinations) were evaluated for growth promotion of R. officinalis under net house conditions. Pot experiment was conducted for a period of three months (July-September 2018) at Department of Basic Sciences, College of Horticulture, Dr Yashwant Singh Parmar University of Horticulture and Forestry, Nauni-Solan, Himachal Pradesh (India).

Sand, soil, and farmyard manure (FYM) were mixed in a 1:2:1 ratio to make the potting mixture. The following mixture was then subjected to intermittent sterilization, which consisted of three consecutive autoclave cycles of one hour each at 100 ^oC, with a 24 h incubation period in between (tyndallization). The pH of the above mixture was examined using a soil:water (1:2.5) suspension and the electrical conductivity (E.C.) of the supernatant liquid was recorded and expressed in dSm^− 1 (Jackson 1973). Furthermore, chromic acid titration method of Walkley and Black (1934) was used to determine organic carbon (O.C.). Standard procedures were used to determine the available N, P, and K contents of soil (Tandon 2009).

The chemical properties of the potting mixture were: E.C. (0.70 dSm^− 1); O.C. (1.22%); available nitrogen (N), phosphorus (P) and potassium (K) (292.03, 22.70 and 312.21 kg/ha, respectively) with pH 7.04. The soil used for the pot (20.00 cm diameter and 16.00 cm deep) had a loamy texture that belonged to the order Entisols as per United States department of Agriculture Soil Taxonomy (USDA). Each pot was filled with 4kg of potting mixture. R.officinalis seedlings were washed thoroughly under running tap water in order to remove soil from roots. Seedlings bacterization was carried out as per Sharma et al. (2021) for single strains and for different combinations, 1:1 ratio of respective strains was mixed before seedlings treatment. The following eight treatments (3 replications per treatment) were subjected to one-way analysis of variance (ANOVA) using completely randomized design: T1, control (untreated); T2, P. aeruginosa SI12; T3, B. subtilis KU21; T4, C. lapagei KU14; T5, B. subtilis KU21 + P. aeruginosa SI12; T6, P. aeruginosa SI12 + C. lapagei KU14; T7, B. subtilis KU21 + C. lapagei KU14; T8, B. subtilis KU21 + C. lapagei KU14 + P. aeruginosa SI12. Two seedlings were planted in each pot and left to grow for three months. All seedlings cultivation practices and inoculum applications were carried out according to Sharma et al. (2021). Observations on physical growth parameters of plants viz., number of lateral branches per plant, plant height and biomass were recorded at the termination of experiment. Estimation of macronutrient contents i.e., N, P and K in oven dried plant samples was also performed using methods of Helrich 1990, Jackson 1973 and Jackson 1967, respectively. The physiological parameters of leaf samples (total chlorophyll, carotenoids, and total phenol content) were determined using standard protocols (Witham et al. 1971, Yang et al. 1998 and Faust and Mikulewics 1967 respectively).

Field studies

Based on outcome of the net house studies, two consortia (B. subtilis KU21 + C. lapagei KU14 and B. subtilis KU21 + C. lapagei KU14 + P. aeruginosa SI12) were selected (best performers) to study their synergistic effects on R. officinalis under open field conditions. The experiment was conducted during the year 2018–2019 at Research Farm of Department of Forest products, Dr Yashwant Singh Parmar University of Horticulture and Forestry, Nauni, Solan (H.P.) located at an altitude of 1270 masl with latitude of 30.15 ºN and longitude of 77.11 ºE fall under mid hill conditions.

The field was thoroughly ploughed by tractor followed by planking about 15 days prior to the date of planting. Field was levelled and sufficient provisions for drainage were made. Raised beds (1 metre square at 90×45 cm spacing) were prepared and 2.5 kg of farmyard manure and sand each per bed was applied at the time of final field preparation before planting of seedlings. The physico-chemical status of the soil on experimental field was sandy loam in texture with nearly neutral pH (7.01), E.C. (0.51 dSm^− 1) was in safe range and high in O.C. (1.71%). The available N (260.00 Kg/ha), P (26.70 Kg/ha) and K (298.23 Kg/ha) were in the medium range. Roots of R. officinalis seedlings were washed under running tap water to remove the adhered soil and then dipped for 1 h in respective cell suspensions. The treatments were as follows: T1; control (un-inoculated), T2; B. subtilis KU21 + C. lapagei KU14 and T3; B subtilis KU21 + C. lapagei KU14 + P. aeruginosa SI12. The seedlings were then planted (4 no. per bed). The following treatments were arranged in randomized block design with seven replications per treatment. Booster doses in liquid bacterial cultures of the same cell density were applied at the rate of 20 ml/plant with 15 days interval in between and after planting. Hand weeding was done regularly during the plant growth period, to keep the beds free from weeds. Irrigation was done at alternate days.

Following observations on various plant parameters (physical and physiological), nutrient concentration, soil nutrient analysis was recorded at 120 (vegetative stage) and 240 (flowering stage) days after planting (DAP) as per the methods described in above section (net house studies). Plant nutrient uptake (kg/ha) was calculated by multiplying total NPK concentration of whole plant with plant biomass. Soil health parameters in terms of dehydrogenase, phytase and phosphatse enzymes activity was investigated using the methods of Casida (1977), Boyce et al. (2004) and Tabatabai and Bremner (1969), respectively.

Statistical analysis

All experiments were carried out within a statistical framework, with the appropriate number of replications per treatment and controls. The data from the laboratory and pot experiments (3 replications per treatment) were subjected to one-way analysis of variance (ANOVA) using SPSS version 16 (SPSS Inc., Chicago, IL, USA) and Microsoft Excel 7.0. (Microsoft, Redmond, WA, USA). Field studies, on the other hand, were subjected to a two-way analysis of variance with seven replications per treatment. The Fisher's Projected LSD (least significant differences) test was used to compare treatment means at P ≤ 0.05. To assess the relationship between effects of endophyte inoculation on several growth parameters of R. officinalis, principal component analysis (PCA) was performed on using R 4.1.3 software.

Bio-Compatibility Interaction Assay and Screening for in vitro plant growth promoting traits

PGP endophytic strains of B. subtilis KU21, P. aeruginosa SI12 and C. lapagei KU14 did not showed any inhibitory effect for growth toward each other on NA in cross streak method. Mutual protocooperation was detected between these three strains in terms of optical density (OD₆₀₀ nm) at different intervals of time (Fig. S1). Statistically highest optical density was recorded for B. subtilis KU21 + C. lapagei KU14 (1.32) followed by B. subtilis KU21 + C. lapagei KU14 + P. aeruginosa SI12 (1.02) after 72 h of incubation (Fig S1c). In contrast, single inoculants registered minimum optical density.

In vitro screening revealed that mutual protocooperation of different strains exhibited elevated level of different PGP traits over single inoculants (Table S1). All the strains (single and consortia) showed P-solubilization ranged from 17.60–42.00 mg/ml. Significantly (P ≤ 0.05) higher P-solubilization was shown by B. subtilis KU21 + C. lapagei KU14 consortium (42.00 mg/ml), which was statistically at par with consortium of B. subtilis KU21 + C. lapagei KU14 + P. aeruginosa SI12 (40.00 mg/ml).

Single as well as combinations of strains gave positive test for IAA production in the presence of tryptophan (ranged from 3.00–6.00 mg/ml). Significantly highest amount of IAA production was registered for B. subtilis KU21 + C. lapagei KU14 + P. aeruginosa SI12 (6.00 mg/ml) which was statistically at par with the consortia of B. subtilis KU21 + P. aeruginosa SI12 (5.80 mg/ml) and B. subtilis KU21 + C. lapagei KU14 (5.60 g/ml). Similarly, all the tested inoculants were also positive for siderophore production (ranged from 35.71-305.56%SU) with significantly maximum production for consortia of B. subtilis KU21 + C. lapagei KU14 + P. aeruginosa SI12and B. subtilis KU21 + P. aeruginosa SI12 i.e., 305.56%SU.

In vivo studies of plant growth promotion

A pot experiment was conducted to validate the in vitro PGP activities of single strains and their combinations (P. aeruginosa SI12, B. subtilis KU21, C. lapagei KU14, B. subtilis KU21 + P. aeruginosa SI12, P. aeruginosa SI12 + C. lapagei KU14, B. subtilis KU21 + C. lapagei KU14 and B. subtilis KU21 + C. lapagei KU14 + P. aeruginosa SI12) on the growth of R. officinalis under in vivo conditions.

All the treatments significantly (P ≤ 0.05) increased growth parameters of R. officinalis over untreated control. Plants receiving combined inoculation of B. subtilis KU21 + C. lapagei KU14 (T7) showed maximum stimulatory effects for overall growth parameters over uninoculated control, which was statistically at par with the treatment receiving consortium of B. subtilis KU21 + C. lapagei KU14 + P. aeruginosa SI12 (T8) inoculation (Table 1). The incremental effects for T7 and T8 were 31.01% and 28.94% for number of lateral branches, 35.99% and 34.46% for stem height, 36.73% and 35.69% for root length, 34.78% and 33.33% for shoot biomass, 39.67% and 36.85% for root biomass, respectively over untreated control.

Table 1

Effect of endophyte inoculation (single and consortia) on growth parameters of *R. officinalis* (pot experiment).
Treatment	Number of lateral branches	Stem height (cm)	Root length (cm)	Root biomass (g/plant)	Shoot biomass (g/plant)
T1 (uninoculated control)	15.01 ± 0.03^h	32.67 ± 0.12^h	15.38 ± 0.11^h	0.69 ± 0.02^g	4.26 ± 0.07^h
T2 (P. aeruginosa SI12)	18.00 ± 0.188^cdefg	41.71 ± 0.18^def	18.98 ± 0.15^defg	0.84 ± 0.01^cdef	5.42 ± 0.12^efg
T3 (B. subtilis KU21)	18.17 ± 0.06^cde	41.83 ± 0.17^de	19.25 ± 0.07^cdef	0.86 ± 0.01^abcde	5.48 ± 0.08^def
T4 (C. lapagei KU14)	18.07 ± 0.05^cdef	40.31 ± 0.05^g	19.3 ± 0.19^cde	0.88 ± 0.02^abcd	5.51 ± 0.18^de
T5 (B. subtilis KU21 + P. aeruginosa SI12)	18.21 ± 0.03^cd	42.34 ± 0.06^c	19.75 ± 0.12^cd	0.9 ± 0.02^abc	5.59 ± 0.16^cd
T6 (P. aeruginosa SI12 + C. lapagei KU14)	18.37 ± 0.04^c	42.12 ± 0.04^cd	19.92 ± 0.26^c	0.92 ± 0.02^ab	5.73 ± 0.03^bc
T7 (B. subtilis KU21 + C. lapagei KU14)	19.66 ± 0.12^a	44.43 ± 0.08^a	21.03 ± 0.09^a	0.93 ± 0.01^a	5.95 ± 0.04^a
T8 (B. subtilis KU21 + C. lapagei KU14 + P. aeruginosa SI12)	19.36 ± 0.12^ab	43.93 ± 0.07^ab	20.87 ± 0.27^ab	0.92 ± 0.01^ab	5.83 ± 0.14^ab
C.D. (P ≤ 0.05)	0.46	0.52	0.81	0.08	0.18
^{The data represent mean of 3 replicates ± standard error (SE). Within columns, means followed by same letter are not significantly different (P ≤ 0.05)}.

Photosynthetic pigments of R. officinalis leaves were determined to evaluate the impact of endophytic strains on photosynthetic efficiency of host plant. Data corresponding to biochemical parameters revealed that highest significant (P ≤ 0.05) increase in total chlorophyll (32.23%) and phenol content (24.72%) was registered for treatment receiving consortium of B. subtilis KU21 + C. lapagei KU14 (T7) over uninoculated control (T1) and was statistically at par with all the inoculated treatments for chlorophyll and treatments T5 and T8 for phenol content (Fig. S2). Whereas treatment T8 (B. subtilis KU21 + C. lapagei KU14 + P. aeruginosa SI12) had maximum significant increase in total carotenoid (32.69%) content of R. officinalis leaves over untreated control and was at par with treatments T2, T3, T6 and T7 (Fig. S2).

To evaluate the effect of endophyte inoculation on growth parameters of R. officinalis, score and loading plots of PCA were made (Fig. S3). All the treatments were successfully dispersed by first two principal components (Fig. S3a). The treatment T1 (control) was segregated from the other treatments, suggesting that these treatments were different than control. PC1 accounted for 96.60% while PC2 comprised of 2.30% of the total data variation (Fig. S3b). In the present study, almost all the growth parameters of rosemary were grouped, and increased in response to endophytic strains inoculation. This analysis showed that inoculation with potential PGP bacterial endophytes had a significant effect on growth and productivity of R. officinalis.

The data appended in Fig. 1 illustrate that, maximum significant enhancement in plant total N and P content was registered with the consortium of B. subtilis KU21 + C. lapagei KU14 (21.05 and 38.09%, respectively) over the control treatment and was statistically at par (P ≤ 0.05) with treatments T3, T5, T6 and T8 for N and T3, T5 and T8 for P content. Inoculation with consortium of B. subtilis KU21 + C. lapagei KU14 + P. aeruginosa SI12 (T8) tended to enhance maximum plant K content by 11.70% in comparison to uninoculated control which was statistically at par with treatments T5, T6 and T7.

In case of soil properties, none of the treatments significantly influenced the soil pH, E.C. and O.C. over the initial soil parameters (data not shown) documented before the trial, however treatment with endophytic strains significantly enhanced the levels of available NPK contents over untreated control (Fig. 1). Maximum significant increase in available N and K contents (13.45 and 8.37%, respectively) were recorded in plants receiving B. subtilis KU21 + C. lapagei KU14 (T7) inoculation, was statistically at par with treatment T8. While treatment T8 (B. subtilis KU21 + C. lapagei KU14 + P. aeruginosa SI12) showed maximum significant increase in soil available P (32.07%) which was statistically at par with treatment T7.

Synergistic Effects Of Compatible Consortia In Rosemary Under Field Conditions

On the basis outcome of pot experiment, best performing treatments (B. subtilis KU21 + C. lapagei KU14; T7 and B. subtilis KU21 + C. lapagei KU14 + P. aeruginosa SI12; T8) were used for field experimentation.

The response of rosemary seedlings for different consortia of endophytic strains under field conditions at vegetative (120 DAP) and flowering stage (240 DAP) revealed improvement in various plant parameters over uninoculated control (Table 2).

Table 2

Effect of selected consortia on growth parameters of *R. officinalis* (field conditions).
Endophytic strains	Number of lateral branches		Mean	Stem height (cm)		Mean	Root length (cm)		Mean	Root biomass (g/plant)		Mean	Shoot biomass (g/plant)		Mean
	Days of planting			Days of planting			Days of planting			Days of planting			Days of planting
	120 days	240 days		120 days	240 days		120 days	240 days		120 days	240 days		120 days	240 days
T1 (uninoculated control)	12.32 ± 0.08	17.75 ± 0.13	15.03	27.00 ± 1.63	31.00 ± 1.22	29.00	24.00 ± 2.18	30.00 ± 1.63	27.00	0.69 ± 0.14	1.00 ± 0.16	0.84	29.31 ± 0.70	31.50 ±1.58	30.40
T2 (B. subtilis KU21 + C. lapagei KU14)	15.34 ± 0.12	24.25 ± 0.11	19.79	34.56 ± 1.96	43.00 ± 2.69	38.78	29.00 ± 1.46	40.20 ± 0.94	34.60	0.88 ± 0.23	1.38 ± 0.31	1.13	36.78 ± 1.71	42.20 ±1.79	39.49
T3 (B subtilis KU21 + C. lapagei KU14 + P. aeruginosa SI12)	14.75 ± 0.11	23.45 ± 0.25	19.10	33.21 ± 2.14	41.95 ± 2.14	37.58	28.00 ± 1.63	39.00 ± 0.81	33.50	0.86 ± 0.17	1.31 ± 0.14	1.08	34.95 ± 1.04	40.50 ±1.10	37.72
Mean	14.14	21.82		31.59	38.65		27.00	36.40		0.810	1.23		33.68	38.06
C.D. (P ≤ 0.05) Treatment Year Treatment×Year	0.44 0.36 0.62			0.60 0.49 0.85			1.03 0.84 1.46			0.13 0.10 NA			0.88 0.72 1.24
^{The data represent mean of 7 replicates ± standard error (SE). Within columns, means followed by same letter are not significantly different (P ≤ 0.05)}.

Numeric enhancement in lateral branches at 120 DAP was registered for seedlings of treatment T2 (B. subtilis KU21 + C. lapagei KU14) followed by treatment T3 (B subtilis KU21 + C. lapagei KU14 + P. aeruginosa SI12) i.e., 24.51% and 19.72%, respectively over untreated control (T1). Treatment of seedlings with dual inoculant (B. subtilis KU21 + C. lapagei KU14) showed statistically significant increase in stem height (28.00%), root length (20.83%), root biomass (27.53%) and shoot biomass (25.48%), respectively over un-inoculated control at vegetative state (120 DAP). Similarly at 240 DAP, maximum per cent increase in number of lateral branches (36.61%), stem height and shoot biomass (38.71 and 33.96%), root length and biomass (34.00 and 38.00%) were also recorded for seedling inoculated with consortia of B. subtilis KU21 + C. lapagei KU14 (T2).

The inoculation of rosemary seedlings with the different combinations of potential endophytic strains positively affected several physiological properties of the plants over untreated control (Fig. S4). During vegetative growth (120 DAP), seedlings receiving treatment T2 (B. subtilis KU21 + C. lapagei KU14) showed highest increase in total chlorophyll (21.09%), and carotenoids (19.54%) content of leaves in comparison to uninoculated control and was statistically at par with T3 for carotenoid content. Maximum increase in total phenol content (20.18%) of plant leaves was recorded for treatment T3 (B subtilis KU21 + C. lapagei KU14 + P. aeruginosa SI12) and was statistically at par with treatment T2.

Similar trend was noticed for these biochemical parameters at flowering stage (240 DAP) over untreated control. Difference among dual (T2) and triple inoculants (T3) treatment for physiological parameters were not significant (Fig. S4). However, inoculation with B. subtilis KU21 + C. lapagei KU14 (T2) showed maximum rise in total chlorophyll (37.95%), phenol (34.40%) and carotenoids (27.27%) content of plants over T1 (un-inoculated control).

The data appended in Fig. 2 revealed that both the treatments (T2 and T3) significantly enhanced NPK uptake of plants over untreated control (T1) during vegetative (120 DAP) as well as flowering stage (240 DAP). Maximum enhancement in plant NPK uptake at 120 and 240 DAP was registered for treatment T2 (B. subtilis KU21 + C. lapagei KU14). These incremental effects were 29.92% and 45.67% for N, 28.03%, 40.71% for P and 28.41% and 41.76% for K at 120 DAP and 240 DAP, respectively.

In case of soil physico-chemical parameters, none of the tried treatments influenced the soil pH, E.C. and O.C. significantly over initial soil test values (data not shown) recorded before the trial as well as un-inoculated control, whereas consortia of potential endophytic strains increased soil available macronutrients contents (NPK) significantly at both vegetative and flowering stage (Fig. 2). Consortia of B. subtilis KU21 + C. lapagei KU14 (T2) supremely increased soil available N (5.4% and 9.37%), P (4.81% and 17.11%) and K (4.10% and 16.46%) contents at 120 and 240 DAP, respectively. However no significant difference was observed among treatments T2 and T3 for soil available NPK content during both vegetative (120 DAP) and flowering stage (240 DAP).

Conjoint application of potential endophytic isolates showed statistically significant increase in soil enzymes activity i.e., dehydrogenase, phytase and phosphatase over uninoculated control. Significantly high increase in dehydrogenase activity (27.05% and 28.25%) was recorded for treatment T2 (B. subtilis KU21 + C. lapagei KU14) at 120 and 240 DAP, respectively over T1 (uninoculated control). Likewise highest increase in soil phytase (8.74% and 10.06%) and phosphatase (12.87% and 13.09%) activity were also registered for soil receiving B. subtilis KU21 + C. lapagei KU14 treatment during both vegetative and flowering stage over un-inoculated control (Table S2).

The use of bacterial inoculants is a widely established method in intensive agriculture systems for increasing agricultural production while maintaining long-term ecological balance in the agro-ecosystem (Berg et al. 2013). In our previous investigation, three potential strains (B. subtilis KU21, C. lapagei KU14 and P. aeruginosa SI12) were proven to significantly enhance the growth parameters of R. officinalis seedlings under protected conditions (Sharma et al. 2021). Therefore, were further evaluated for growth promotion of R. officinalis under open field conditions in order to enhance plant productivity in a sustainable manner.

In the present study, selected endophytic strains (B. subtilis KU21, C. lapagei KU14 and P. aeruginosa SI12) were found to be compatible with each other in cross streak plate assay. This could be because compatible strains occupy different rhizosphere niches and are thus non-inhibitory to each other in a natural ecosystem, thereby synergizing their effect as a whole (Kumawat et al. 2019a). Moreover, spectrophotometric growth interaction studies revealed the establishment of protocooperation between B. subtilis KU21, C. lapagei KU14 and P. aeruginosa SI12 during combined inoculation in single medium due to release of non-reactive metabolites. In a similar study by Angelina et al. (2020) reported positive interaction of B. subtilis and Pseudomonas fluorescens under both in vivo and in vitro conditions in Lactuca sativa L.

Several studies have shown that endophytes can enhance plant growth either directly via P-solubilization, IAA and siderophore production or indirectly through antimicrobial activities against phytopathogens via, hydrolytic enzymes and ammonia production (Gaiero et al. 2013; Abbamondi et al. 2016; Hassan 2017; Sharma et al. 2021). Therefore, in vitro PGP traits were assessed for single strains and combination of these strains. The results showed maximum production of IAA, siderophore and P-solubilization in case of combined inoculation of endophytic strains (B. subtilis KU21 + C. lapagei KU14 and B. subtilis KU21 + C. lapagei KU14 + P. aeruginosa SI12) in comparison to single inoculants. The results are consistent with Kumawat et al. (2019a) who documented combined application of P-solubilizing Bradyrhizobium sp. (LSBR-3) + P. aeruginosa (LSE-2) in soybean improved P uptake in soil as well as in plants and enhanced plant growth as a whole. In a similar study, significantly high amount of IAA and siderophore production was obtained with co-inoculation of Leclercia adecarboxylata (LSE1) and Bradyrhizobium sp. (LSBR3) as compared to single inoculants in soybean (Kumawat et al. 2019b).

To validate the results of in vitro screening of enhancement in PGP traits by consortium over single inoculants, in vivo studies were carried out. In pot experiment, consortia of endophytic strains especially B. subtilis KU21 + C. lapagei KU14 and B. subtilis KU21 + C. lapagei KU14 + P. aeruginosa SI12 significantly increased physical growth parameters of R. officinalis seedlings in comparison to single inoculants and un-inoculated control. Significant improvements in the number of lateral branches, root and shoot parameters of R. officinalis with combined inoculation of B. subtilis KU21 + C. lapagei KU14 and B. subtilis KU21 + C. lapagei KU14 + P. aeruginosa SI12 in comparison to single inoculants can be attributed to synergism observed in in vitro studies. Jha et al. (2012) had reported the effect of combined application of Pseudomonas fluorescens, Burkholderia cepacia and Aeromonas vaga on increased plant growth parameters over un-inoculated control of mungbean. Subramanian et al. (2015) also reported an increase in root and shoot parameters in soybean receiving consortium of Bradyrhizobium japonicum MN110 and Bacillus megaterium LNL6 in comparison to single inoculants treatment and un-inoculated control.

The inoculation of R. officinalis seedlings with indigenous endophytes (single and consortia) positively affected several physiological properties of plants. Significant increase in total chlorophyll, carotenoids and phenol content were observed in plants inoculated with B. subtilis KU21 + C. lapagei KU14 followed by B. subtilis KU21 + C. lapagei KU14 + P. aeruginosa SI12. The results of the present study are consistent with Zhang et al. (2019), who reported that consortia of Bacillus cereus AR156, Bacillus subtilis SM21, and Serratia sp. XY21 significantly improved photosynthetic capability by augmenting photosynthetic efficiency and chlorophyll levels in sweet pepper. Plants possess a variety of antioxidant molecules predominantly phenols that alleviate the reactive oxygen species and defend host cells against adverse conditions. The current study's findings revealed that certain elicitors were present in the bacterial cultures that had fundamental role in increasing phenolic content of rosemary leaves. Various reports have also indicated a positive effect of co-inoculation with a mixture of PGPRs in increasing phenolics and flavanoid contents in Lycopersicon esculentum Mill and Allium cepa L. (Kannan and Sureendar 2009; Pellegrini et al. 2021)

The combined inoculation with endophytic strains (B. subtilis KU21, C. lapagei KU14 and P. aeruginosa SI12) significantly increased plant macronutrient contents (NPK) in comparison to single inoculants and untreated control. The plant’s increased capability for getting and utilizing more nutrients is accredited to the teaming effects of inoculated strains for increasing surface area of roots (by Phyto stimulants secretion) and promoting absorption of nutrients from soil as a result of altered root (Egamberdieva et al. 2017). Furthermore, these bacteria were mineral solubilizers, hence resulting in higher quantities of accessible minerals (NPK) in the soil and so increasing plant availability and uptake (Setiawati and Mutmainnah 2016; Adhikari and Pandey 2020). For example, consortium of B. subtilis and P. fluorescens having P-solubilizing and nitrogen-fixing ability, significantly enhanced NP content in lettuce (Angelina et al. 2020). The consortia of inoculants of the current investigation were found to be capable of solubilizing P and fixing N more than single inoculants under in vitro conditions, thus providing more NP to R. officinalis seedlings.

Field experiment studies illustrated significant growth benefits of synergistic association of rosemary with combined application of endophytic strains (B. subtilis KU21, C. lapagei KU14 and P. aeruginosa SI12). Co-inoculation with B. subtilis KU21, C. lapagei KU14 significantly increased physical (number of lateral branches and root, shoot parameters) as well as physiological growth (total chlorophyll, phenol and carotenoids content) characteristics of R. officinalis. This improvement can be attributed to better colonization of PGP strains due to protocooperation, more secretion of phytohormones thereby alteration in root architecture and improved nutrient availability (Gupta and Pandey 2019; Samaddar et al. 2019). Present results are conferred with Kumar et al. (2016) who confirmed synergistic effects of Pseudomonas putida and Bacillus amyloliquefaciens in growth improvement of mungbean.

The combined inoculation of B. subtilis KU21and C. lapagei KU14 was known to show high N, P, K uptake in rosemary as compared to un-inoculated control treatment. These results are in agreement with He et al. (2019), inoculation with Bacillus spp. and P. putida in combination significantly increased the N, P and K concentration in plant biomass of tomato. This increase is attributed to more availability of NPK in soil as a result of inoculation with endophytic bacterial strains that resulted in the increase of root length and root hair formation. Bais et al. (2004) had also reported an increase in nutrient uptake due to enhancement in ions flux at root plane in the presence of PGP bacteria.

Soil enzymes signify the microbial diversity in plant’s vicinity in terms of their activity and are well known to catalyze several reactions such as decomposition of biomass and mineralization of organic compounds (phosphorus, potassium). Soil enzyme activities also increases uptake of plant nutrients from soil by means of plant microbe interactions (El-Nohrawy and Omara 2017; Bargaz et al. 2018; Kumawat et al. 2019a). In our study, significantly maximum soil enzyme activities (dehydrogenase, phosphatase and phytase) were registered for treatment T2 (B. subtilis KU21 + C. lapagei KU14) over untreated treatment T1 (control). The present findings are in line with Kumawat et al. (2019a) who reported co-inoculation of Bradyrhizobium sp. LSBR-3 + P. aeruginosa LSE-2 resulted in significant increase of soil dehydrogenase and phosphatase enzyme in soyabean.

In essence, this is the first report on in vivo growth promoting potential of endophytic strain C. lapagei KU14 in combination with B. subtilis KU21 and P. aeruginosa SI12 of R. officinalis. After field assessment, our results unequivocally proved that strain C. lapagei KU14 in co-ordination build beneficial association with the host and stimulated plant growth. Thus, the findings of this exploration could be utilized for development of an eco-friendly and cost-effective technology for biofertilization of R. officinalis which diminish the utilization of synthetic manures and save non-renewable energy source utilization.

Acknowledgments

The authors are thankful to the Indian Council of Agricultural Research, New Delhi, India, for providing financial assistance through the All-India Network Project on Soil Biodiversity and Biofertilizer.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

Conceptualization: Anjali Chauhan; Methodology: Minakshi Sharma; Formal analysis and investigation: Gaurav Sood; Writing - original draft preparation: Minakshi Sharma; Writing - review and editing: Gaurav Sood; Resources: Bhupender Dutt; Supervision: Anjali Chauhan.

Data Availability

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

Code availability

Not available.

Abbamondi GR, Tommonaro G, Weyens N, Thijs S, Sillen W, Gkorezis P (2016) Plant growth-promoting effects of rhizospheric and endophytic bacteria associated with different tomato cultivars and new tomato hybrids. Chem Biol Technol Agric. http://doi.org/10.1186/s40538-015-0051-3

Adhikari P, Pandey A (2020) Bioprospecting plant growth promoting endophytic bacteria isolated from Himalayan yew (Taxus wallichiana Zucc.). Microbiol Res http://doi.org/10.1016/j.micres.2020.126536

ALKahtani MDF, Fouda A, Attia KA et al (2020) Isolation and characterization of plant growth promoting endophytic bacteria from desert plants and their appli cation as bioinoculants for sustainable agriculture. Agronomy 10:1325. http://doi.org/10.3390/agronomy10091325

Angelina E, Papatheodorou Efimia M, Demirtzoglou T, Monokrousos N (2020) Effects of Bacillus subtilis and Pseudomonas fluorescens inoculation on attributes of the Lettuce (Lactuca sativa L.) soil rhizosphere microbial community: The role of the management system. Agronomy 10:1428. doi:10.3390/agronomy10091428

Bais HP, Fall R, Vivanco JM (2004) Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production. Plant Physiol 134:307-319.

Bargaz A, Lyamlouli K, Chtouki M, Zeroual Y, Dhiba D (2018) Soil microbial resources for improving fertilizers efficiency in an integrated plant nutrient management system. Front Microbiol. 9:1606. 10.3389/fmicb.2018.01606

Benaissa A, Djebbar R, Abderrahmani A (2019) Diversity of plant growth-promoting rhizobacteria of Rhus tripartitus in the arid soil of Algeria (Ahaggar) and their physiological properties under abiotic stresses. Adv Hortic Sci 32:525-534.

Berg G, Alavi M, Schmidt CS, Zachow C, Egamberdieva D, Kamilova F, Lugtenberg B (2013) Bio-control and osmoprotection for plants under saline conditions. In: de Bruijn Frans J (ed) Molecular microbial ecology of the Rhizosphere. Wiley, New York. https://doi.org/10.1002/9781118297674

Bourhia M, Laasri FE, Aourik H et al (2019) Antioxidant and antiproliferative activities of bioactive compounds contained in Rosmarinus officinalis used in the Mediterranean Diet. Evid.-based complementary altern. Med https://doi.org/10.1155/2019/7623830

Boyce A, Casey A, Walsh G (2004) A phytase enzyme-based biochemistrypractical particularly suited to students undertaking courses inbiotechnology and environmental science. Biochem Mol Biol Educ 32: 336-340. doi:10.1002/bmb.2004.494032050392

Casida LE (1977) Microbial metabolic activity in soil as measured bydehydrogenase determinations. Appl Environ Microbiol 34:630-636.
Chandra H, Kumari P, Bisht R et al (2020) Plant growth promoting Pseudomonas aeruginosa from Valeriana wallichii displays antagonistic potential against three phytopathogenic fungi. Mol Biol Rep 47:6015-6026. https://doi.org/10.1007/s11033-020-05676-0

Egamberdieva D, Wirth S, Behrendt U, Ahmad P, Berg G (2017) Antimicrobial activity of medicinal plants correlates with the proportion of antagonistic endophytes. Front Microbiol 8:199.

El-Nohrawy S, Omara A (2017) Effectiveness of coinoculation with Pseudomonas koreensis and rhizobia on growth, nodulation and yield of common bean (Phaseolus vulgaris L.). Microbiol Res J Int 21 1-15.

Faust SD, Mikulewicz EW (1967) Factors influencing the condensation of 4 aminoantipyrine with derivatives of hydroxybenzene – II. Influence of hydronium ion concentration on absorptivity. Water Res 1:509-522.

Gaiero JR, McCall CA, Thompson KA, Day NJ, Best AS, Dunfield KE (2013) Inside the root microbiome: Bacterial root endophytes and plant growth promotion. Am J Bot 100: 1738–1750.

Gharib F, Ghazi S, Aly H, El-Araby M, Moustafa S (2016) Effect of soil type and water content on rosemary growth and essential oil yield. Int J Sci Eng Res 7:183–89.

Ghosh S, Ow LF, Wilson B (2015) Influence of biochar and compost on soil properties and tree growth in a tropical urban environment. Int J Environ Sci Technol 12:1303-10. https://doi.org/10.1007/s13762-014-0508-0.

Glick BR. 2012. Plant growth promoting rhizobacteria: Mechanisms and Applications. Scientificia doi: 10.6064/2012/963401.

Gordon SA, Palleg LG (1957) Quantitative measurement of IAA. Plant Physiology 10:37-38.

Gupta S, Pandey S (2019) ACC deaminase producing bacteria with multifarious plant growth promoting traits alleviates salinity stress in French bean (Phaseolus vulgaris) plants. Front. Microbiol 10:1506. http://doi.org/10.3389/fmicb.2019.01506

Hassan SED (2017) Plant growth-promoting activities for bacterial and fungal endophytes isolated from medicinal plant of Teucrium polium L. J Adv Res 8:687-695.

He Y, Pantigoso HA, Wu Z, Vivanco JM (2019) Co-inoculation of Bacillus sp. and Pseudomonas putida at different development stages acts as a biostimulant to promote growth, yield and nutrient uptake of tomato. J Appl Microbiol 127:196-207.

Helrich K (1990) Official and tentative methods of analysis. Association of Official Analytical Chemists. Washington:William Star Wetglad http://www.indianmirror.com/ayurveda/rosemary.html.

Jabborova D, Enakiev Y, Sulaymanov K, Kadirova D, Ali A, Annapurna K (2021) Plant growth promoting bacteria Bacillus subtilis promote growth and physiological parameters of Zingiber officinale Roscoe. Plant Sci. Today 8:66-71.

Jackson ML (1967) Soil chemical analysis. Bombay: Oxford and IBH Publishing House

Jackson ML (1973) Soil chemical analysis. New Delhi: Prentice-Hall of India.

Jha CK, Saraf M (2012) Evaluation of multispecies plant growth promoting consortia for the growth promotion of Jatropha curcas L. J Plant Growth Regul 31:588-598.

Kannan V, Sureendar R (2009) Synergistic effect of beneficial rhizosphere microflora in biocontrol and plant growth promotion. J Basic Microbiol 49:158-64. doi: 10.1002/jobm.200800011

Kumar P, Pandey P, Dubeya RC, Maheshwari DK (2016) Bacteria consortium optimization improves nutrient uptake, nodulation, disease suppression and growth of the common bean (Phaseolus vulgaris) in both pot and field studies. Rhizosphere 2:13-23.

Kumawat KC, Sharma P, Nagpal S, Gupta RK, Sirari A, Nair RM, Bindumadhava H, Singh S (2021) Dual microbial inoculation, a game changer? – bacterial biostimulants with multifunctional growth promoting traits to mitigate salinity stress in spring mungbean. Front Microbiol. https://doi.org/10.3389/fmicb.2020.600576

Kumawat KC, Sharma P, Sirari A, Singh I, Gill BS, Singh U, Saharan K (2019 a) Synergism of Pseudomonas aeruginosa (LSE-2) nodule endophyte with Bradyrhizobium sp. (LSBR-3) for improving plant growth, nutrient acquisition and soil health in soybean. World J Microbiol Biotechnol 35:47. https://doi.org/10.1007/s11274-019-2622-0

Kumawat KC, Sharma P, Singh I, Sirari A, Gill BS (2019 b) Co‑existence of Leclercia adecarboxylata (LSE‑1) and Bradyrhizobium sp. (LSBR‑3) in nodule niche for multifaceted efects and proftability in soybean production. World J Microbiol Biotechnol 35:172. https://doi.org/10.1007/s11274-019-2752-4

Lacava PT and Azevedo JL. 2013. Endophytic Bacteria: A Biotechnological Potential in Agrobiology System. Bacteriain Agrobiology: Crop Productivity DOI: 10.1007/978-3-642-37241-4_1

Monthon L, Sawangnop S (2011) A comparison of two methods used for measuring the antagonistic activity of Bacillus species. Walailak J Sci Technol 5:161-171.

Mortensen CN (1997) Seed bacteriology laboratory guide. Copenhagen (Denmark): Danish Government Institute of Seed Pathology for Developing Countries.

Nazmul H, Rahman M (2014) Antagonistic activity of antibiotic producing Streptomyces sp. against fish and human pathogenic bacteria. Braz Arch Biol Technol 57:233-237.

Pellegrini M, Spera DM, Ercole C, Del Gallo M (2021) Allium cepa L. inoculation with a consortium of plant growth-promoting bacteria: effects on plants, soil, and the autochthonous microbial community. Microorganisms 9:639. https://doi.org/10.3390/ microorganisms9030639

Pikovskaya RI (1948) Mobilization of phosphorus in soil in connection with the vital activity of some microbial species. J Microbio 7:362-370.

Pintore G, Usai M, Bradesi P, Juliano C, Boatto G, Tomi F, Chessa M, Cerri R, Casanova J (2002). Chemical composition and antimicrobial activity of Rosmarinus officinalis L. oils from Sardinia and Corsica. Flavour Fragr J 17:15-19. https://doi.org/10.1002/ffj.1022

Rodriguez M, Torres M, Blanco L et al (2020) Plant growth-promoting activity and quorum quenching-mediated biocontrol of bacterial phytopathogens by Pseudomonas segetis strain P6. Sci Rep 10:4121. https://doi.org/10.1038/s41598-020-61084-1

Sadegh Kasmaei L, Yasrebi J, Zarei M et al (2019) Influence of plant growth promoting rhizobacteria, compost, and biochar of Azolla on Rosemary (Rosmarinus Officinalis L.) growth and some soil quality indicators in a calcareous soil. Commun Soil Sci Plant Anal 50: 119-131.

Samaddar S, Chatterjee P, Choudhury AR, Ahmed S, Sa T (2019). Interactions between Pseudomonas spp. and their role in improving the red pepper plant growth under salinity stress. Microbiol. Res 219:66-73. http://doi.org/10.1016/j.micres.2018.11.005

Schulz B, Boyle C (2006) What are endophytes? In: Schulz BJE, Boyle CJC, Sieber TN (eds) Microbial root endophytes. Springer-Verlag, Berlin, pp 1–13

Schwyn B, Neilands JB (1987) Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160:47-56.

Setiawati C, Mutmainnah L (2016) Solubilization of potassium-containing mineral by microorganisms from the sugarcane rhizosphere. Agric Sci Procedia 9:108-117.

Sharma M, Sood G, Chauhan A (2021) Bioprospecting beneficial endophytic bacterial communities associated with Rosmarinus officinalis for sustaining plant health and productivity. World J Microbiol Biotechnol 37:135. doi: 10.1007/s11274-021-03101-7

Shrestha RK, Lal R, Rimal B (2013) Soil carbon fluxes and balances and soil properties of organically amended no-till corn production systems. Geoderma 197:177-85. https://doi.org/10.1016/j.geoderma.2013.01.005.

Silva CF, Vitorino LC, Mendonça MAC, Araujo WL, Dourado MN, Albuquerque LC (2019) Screening of plant growth-promoting endophytic bacteria from the roots of the medicinal plant Aloe vera. S A J. http:// doi.org/10.1016/j.sajb.2019.09.019

Stajkovic O, De Meyer S, Milicic B, Willems A, Delic D (2009) Isolation and characterization of endophytic non-rhizobial bacteria from root nodules of alfalfa (Medicago sativa L.). Bot Serb 33:107-114.

Subramanian P, Kim K, Krishnamoorthy R, Sundaram S, Sa T (2015) Endophytic bacteria improve nodule function and plant nitrogen in soybean on co-inoculation with Bradyrhizobium japonicum MN110. Plant Growth Reg 76:327-332.

Tabatabai MA, Bremner JM (1969) Use of p-nitrophenyl phosphate forassay of soil phosphatase activity. Soil Biol Biochem 1:301-307. doi:10.1016/0038-0717(69)90012-1
Tandon HLS (2009) Methods of analysis of soil, plant, water, fertilizers, and organic manures. (Fertiliser Development and Consultation Organisation: New Delhi).

Walkley A, Black TA (1934) An estimation of soil organic matter and purposed modification of chromic acid titration method. Soil Sci 37:29-38.

Weyens N, Vsn DD, Taghavi S, Vangronsveld J (2009) Phtyoremediation: plant endophyte partnerships take the challenge. Curr Opin Biotechnol 20:248-254.

Witham FH, Bladydes DF, Delvins RM (1971) Experiments in plant physiology. New York: Van Nostrand Reinhold; p. 245

Yang CM, Chang KW, Yin MH, Huang HM (1998) Methods for the determination of the chlorophylls and their derivatives. Taiwania 43(2):116-122.

Yanishlieva NV, Marinova E, Pokorny J (2006) Natural antioxidants from herbs and spices. Eur J Lipid Sci Technol 108:776-93. https://doi.org/10.1002/ejlt.200600127

Zhang LN, Wang DC, Hu Q, Dai XQ, Xie YS et al (2019) Consortium of plant growth-promoting rhizobacteria strains suppresses sweet pepper disease by altering the rhizosphere microbiota. Front Microbiol 10:1668. https://doi.org/10.3389/fmicb.2019.01668

Download PDF

Version 1

posted

You are reading this latest preprint version

Novel synergism of Cedecea lapagei KU14 and Bacillus subtilis KU21for sustainable productivity of Rosmarinus officinalis in Northwest Himalayas

Status:

Version 1

Abstract

Figures

Introduction

Material And Methods

Bacterial strains and their maintenance

Biocompatibility Interaction Assay And Characterization For Pgp Traits

Plant Growth Stimulation

Preparation of inoculum

Procurement of seedlings

Net house studies

Field studies

Statistical analysis

Results

Synergistic Effects Of Compatible Consortia In Rosemary Under Field Conditions

Discussion

Declarations

Data Availability

References

Supplementary Files

Status:

Version 1