Bioprospecting Beneficial Endophytic Bacterial Communities Associated With Rosmarinus Officinalis for Alleviating Plant Productivity


 The present study was aimed to isolate and identify root endophytic bacteria with multifunctional plant growth promoting (PGP) traits from medicinal plant Rosemarinus officinalis grown in the North-Western Himalayas. A total of 42 strains were isolated, exhibiting variable degrees of PGP traits, including P-solubilization (10-375 µg/ml), IAA (6-66 µg/ml), siderophore (32.37-301.48 %SU) production and antifungal activity in terms of percent growth inhibition (%GI) against Fusarium oxysporum (44.44-77.77 %GI), Fusarium graminiarum (48.88-71.42 %GI) and Rhizoctonia solani (44.44-77.7 %GI). 16S rDNA sequencing results showed lineage of these strains to 15 genera viz., Aneurinibacillus, Bacillus, Beijerinckia, Cedecea, Ensifer, Enterobacter, Kosakonia, Lactobacillus, Lysobacter, Oxynema, Pseudomonas, Pantoea, Paenibacillus, Pseudoxanthomonas and Serratia. The effect of 11 potential strains was selected for in vivo growth studies of R. officinalis. The results showed that the inoculation of Bacillus subtilis KU21, Pseudomonas aeruginosa SI12, and Cedecea lapagei KU14 significantly increased the physical growth parameters of plant over uninoculated control viz., number of lateral of branches (43.95-46.39 %), stem height (29.04-38.57 %), root length (32.31-37.14 %), shoot (34.76-40.91 %) and root biomass (62.89-70.70 %). Physiological characteristics such as total chlorophyll (30.41-30.96 %), phenol (14.43-24.55 %) and carotenoids (34.26-39.87 %) content, also showed a relative increase as compared to uninoculated control; furthermore, the macronutrients (NPK) contents of the plant as well as soil also showed an increase. The developed module may be recommended for sustainable production of R. officinalis in the North-Western Himalayan region without hampering the soil health and fertility.


Introduction
Plants develop an intricate association with a variety of microorganisms including rhizospheric, and endophytic, which have attracted the attention of scienti c community due to their veri ed bene ts (Cordero et al. 2017;Liotti et al. 2018). These microorganisms are natural inhabitants of host plant and can also develop in an endogenous fashion. This is true for endophytic microorganisms, which colonize plant tissues for at least a part of their life-cycle without any visible disease symptoms (Silva et al. 2019). Bacteria of endophyte community are diverse and able to disperse throughout plant tissues systemically (Bacon and White 2016). Such colonization provides many advantages as this community, independent of its environmental circumstances, can interact effectively with the plant (Santoyo et al. 2016). This improves plant growth and a defense against plant pathogens, which further enhances stress tolerance, and can promote the synthesis or development of bioactive compounds of interest (Khamwan et al. 2018;McMullin et al. 2018).
Various studies have shown that roots associated bacterial strains can promote plant growth by mechanisms such as phytostimulation, biofertilization, and biocontrol (Gaiero et al. 2013;Abbamondi et al. 2016). Such association has direct physiological effects on plant growth and production, such as: nitrogen xation, phosphate solubilization, production of ammonia, siderophores, phytohormones and hydrolytic enzymes (Silva et al. 2019). These effects provide for the need of plant nutrition in a sustainable manner, thereby reducing the use of chemical fertilizers and pesticides. This in turn preserves the soil biological diversity, thus providing an alternative to the conventional methods of cultivation and compelling the researchers to look for eco-friendly and sustainable agriculture production methods. To this end, the use of plant growth promoting rhizobacteria (PGPR) has emerged as an attractive approach in rosemary production. They are often used as plant growth enhancers under both normal and stressful conditions.
Medicinal plants are selective, while forming endophytic associations, since this choice may be based on the production of secondary metabolites and composition of root exudates; hence, a diverse group of bacterial communities exists based on their nutritional requirements, environment and soil type in which they are found (Maggini et al. 2018).
R. o cinalis (rosemary) is a member of Labiatae family and is one of the most popular medicinal herbs rich in polyphenols (carnosic acid and rosamarinic acid) and avanoids. It is native to Mediterranean region and cultivated worldwide in cool regions at elevations of 1000-3000 meter above sea level (masl) (semiarid and sub humid bioclimatic regions). In Himachal Pradesh, rosemary is grown at an elevation range of 1050-2100 masl falling in the mid-hills sub humid and high hills temperate wet zone. Owing to its numerous biological activities (antibacterial, antiproliferative, anti-in ammatory, and antioxidant), this plant has gained more interest from commercial point of view (Bourhia et al. 2019 Table S1. A total of twenty-four (3 samples×8 sites) composite root samples were obtained from all the locations and stored in plastic bags at 4 ºC for further assaying of bacterial community structure.

Isolation of bacterial endophytes
Surface sterilization of roots was performed for isolation of bacterial endophytes following the standard method (de Favaro et al. 2012) with slight modi cation as follows: root samples were washed under running water, sterilized with 70 % ethanol for 45 sec, and 2 % sodium hypochlorite for 5 min followed by washing 4-5 times with sterile distilled water. The surface sterility of roots was cross-checked by plating 100 µl of the nal wash and incubated overnight at 30 ºC. No growth on plate indicated complete sterilization of roots. Furthermore, root endophytic bacteria were isolated using serial dilution spread plate technique. An aliquot of 100µl of suspension (10 − 2 -10 − 4 dilutions) was spread on nutrient and tryptic soy agar medium and incubated at 30 ºC till the appearance of bacterial colonies (upto 5 days). Isolated bacteria were enumerated as colony forming units per gram of roots (cfu g − 1 root).
A total of 42 distinct morphotypes were selected and puri ed on respective medium. The pure culture of these strains was preserved on petri plates at 4 ºC for further analysis.

Morpho-biochemical characterization of endophytic bacteria
Microscopic examination of endophytic bacteria was done together with biochemical characterization according to Bergey's Mannual of Determinative Bacteriology (Holt et al. 1994). Antibiotic sensitivity test of isolates to standard antibiotics was evaluated using antibiotic sensitivity kits (Hexa universal 1, 2 and Hexa Pseudo 1,2) (Himedia, India).
Identi cation of endophytic bacteria 16S rDNA sequence analysis was employed for molecular identi cation of isolated bacteria. Genomic DNA was extracted using the conventional method (Sambrook et al. 1989) followed by PCR-mediated ampli cation with a set of universal primers (16SF: 5'-AGAGTTTGATCCTGGCTCAG-3'and16SF: 5'-AAGGAGGTGATCCAGCCGCA-3´). PCR reaction mix of 25 µl was prepared with 50 ng of template DNA, 20 pmol of each primer, 0.2 mM dNTPs, and 1U Taq polymerase in 1X PCR buffer. The reaction was cycled 35 times as denaturation at 94ºC for the 30s, annealing at 55ºC for 30 s and extension at 72ºC for 1 min 30 sec followed by a nal extension at 72ºC for 10 min. The PCR product was analyzed by gel electrophoresis on 1.2 % (w/v) agarose gel. A band of ~ 1400bp was excised from the gel and puri ed using a gel extraction kit (RBCs Real Genomics, New Taipei City, Taiwan) and was sequenced by Genei labs (Bangaluru, India). Based on 16S rDNA sequences, phylogenetically related bacteria were aligned using a BLASTn search (Altschul et al. 1997). Multiple alignments with sequences of related taxa were implemented using CLUSTAL W (Higgins et al. 1994). A neighbor-joining phylogenetic tree was constructed with other 16S rDNA sequences of the related taxa retrieved from the GenBank database using MEGA X software.
In vitro screening for traits involved in plant growth promotion Isolated bacteria were further authenticated by subsequent in vitro experiments to see whether they exhibited qualities which identi ed them as possible plant growth promoting bacterial endophytes (PGPBEs). Each in vitro screening test was conducted in triplicates. P-solubilization activity was determined by the method of Pikovskaya (1948). Quantitative production of IAA was estimated using the colorimetric method described by Gorden and Palleg (1957). To test the e cacy of endophytic bacteria as nitrogen xers, loop full of 24 h old culture of each isolate were streaked on nitrogen free agar medium and incubated for 72 h at 30 º C. Colonies showing growth on inoculated medium after being transferred ten times in the same medium were potent nitrogen xers (Jensen 1987).
The ability of isolates to produce siderophore and hydrocyanic acid (HCN) was also assessed by Schwyn and Neilands (1987) and Bakker and Schipper (1987) methods, respectively. For lytic enzyme activity, spot inoculation was done on minimal agar medium amended with 0.3% colloidal chitin for chitinase (Robert and Selitrennikoff 1988), starch agar medium for amylase (Shaw et al. 1995) and skim milk agar plates for protease activity (Fleming et al. 1975). Ammonia production was observed according to the method of Cappuccino and Sherman (1992).
The antagonistic activity of the bacterial isolates against test fungal pathogen viz., F. oxysporum (ITCC 7337), F. graminiarum (ITCC 5334) and R. solani (ITCC 5308), was done by agar dual plate method on malt extract agar (MEA) medium and the percent growth inhibition (%GI) was calculated as described by Vincent (1947).

Stimulation of plant growth
To test the e cacy of endophytic strains for stimulating plant growth, a pot experiment was conducted. Eleven isolates with best ex situ PGP traits were selected. The potting mixture was prepared by mixing sand, soil and farm yard manure (FYM) in a ratio of 1:2:1. The following mixture was then subjected to intermittent sterilization i.e. three successive autoclave cycles of 1 h each at 100 ºC with 24 h of incubation between each cycle (tyndallization). The pH of potting mixture was determined in 1:2.5 (soil:water) suspension and the electrical conductivity (E.C.) of the supernatant liquid was recorded and expressed in dSm − 1 (Jackson 1973). Furthermore, organic carbon (O.C.) was determined by chromic acid titration method of Walkley and Black (1934). Available N, P, and K contents of soil were determined following standard procedures (Tandon 2009 . Bacteria were subsequently pelleted by centrifugation at 6,000 rpm for 10 min. The pellets were washed with sterile distilled water three times, and the concentration of cells adjusted to 1× 10 8 cfu ml − 1 by dilution. This liquid formulation was used as inoculum (Mortensen 1997). Roots of seedlings were immersed in prepared inoculum for about 1 h before plantation. Two seedlings per pot were planted and allowed to grow for four months. Booster doses of liquid bacterial cultures of the same cell density were applied at the rate of 20 ml plant − 1 with 15 days interval after planting. Seedlings were watered daily during the rst two weeks of planting followed by irrigation once every two days. The following 12 treatments were arranged in a completely randomized block design (CRD) with three replications for each treatment: T1, control (uninoculated); Observations on plant growth parameters such as shoot and root length (cm), biomass (g), and the number of lateral branches per plant were recorded by following standard methods. Oven-dried plant samples were ground and sieved for the estimation of macronutrients (NPK). The total concentration of N in plant samples was determined using micro-Kjeldhal's method (Helrich 1990). Plant samples were digested in a diacid mixture of HNO 3 :HCLO 4 (4:1) for P and K analysis (Jackson 1973). P concentration was tested in the digested sample (Jackson 1973). K in the digest was analyzed using the ame photometer (Biogen, Microcontroller Flame Photometer) (Jackson 1967). Total chlorophyll, carotenoids, and total phenol content (TPC) of leaf samples were determined using methods of Withem et al. (1971), Brezeanu et al. (2005), and Faust and Mikulewics (1967), respectively.

Endophytic nature of bacterial strains
To test whether the bacterial strains were capable of colonizing plant tissues, enumeration of the total endophytic bacterial population was done using standard serial dilution spread plate method after termination of the experiment. Colonies which showed morphological characteristics similar to treated strain were selected and the dominance of inoculated strain was calculated according to Simpson's index of dominance (D) as: Where, Pi is the relative abundance of isolates calculated according to the following equation Pi = ni/N n i , is the number of inoculated strain colonies and N, is the total number of endophyte colonies

Statistical analysis
All the experiments were conducted under the statistical framework with three biological replications along with equal number of appropriate controls. The data obtained from the laboratory experiments and the net house was 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). The means and standard deviation of data were also calculated. Comparisons of treatment means were performed by the Fisher's Projected LSD (least signi cant differences) test at P ≤ 0.05 level of signi cance. PCA (principal component analysis) was performed on pot experiment, to evaluate the relationship between effects of endophyte inoculation on several plant growth parameters. PCA was performed using PAST 3.0 software.

Isolation and identi cation of endophytic bacteria
A total of 42 strains were isolated based on unique colony morphologies on medium used for isolation. Amongst these, 17 were Gram's positive while 25 were Gram's negative, varied from rods, cocci to coccobacilli. Endospore formation was observed in 16 isolates. Biochemically, low number of isolates were positive for indole (28.57 %) and hydrogen sul de (H 2 S) (9.52 %) production. Greater numbers of bacterial isolates were positive nitrate reduction (80.95 %) and oxidase production (61.90 %). All the isolates were positive for catalase except KU20 and KU25. 59.52 % were positive for methyl red, 40.47 % showed positive test for voges proskauer (VP); only 21.43 % bacterial isolates were able to hydrolyse gelatin. Almost all the isolates were able to ferment dextrose and sucrose, whereas a few isolates showed positive lactose fermentation test (Table S2).

Bene cial Plant traits of bacterial strains
In vitro screening revealed that most of the strains exhibited multiple PGP activities. All the strains substantially solubilized mineral P in PVK broth (70-375 µg ml − 1 ). Majority of isolates (85.71 %) synthesized IAA (10-66 µg ml − 1 ) and produced siderophore (92.85 %) that ranged from 35.71-301.48 %SU. The ability to x nitrogen on Jensen's nitrogen-free medium was recorded in 66.66% of strains. Strains exhibited potential bene cial traits belonging to genera Bacillus, Cedecea, Enterobacter, Pantoea and Pseudomonas (Fig. 2). All the bacterial strains were able to produce one or more cell wall degrading enzymes. In total, 73.81 % strains exhibited chitinase activity, and 80.95 % were protease and amylase producers, thereby conferring antagonistic and endophytic properties to the selected strains. Majority of bacterial strains (88.09 %) displayed ammonia production, whereas; only 40.47 % were HCN producers. Endophytes belonging to genera Aneurinibacillus, Bacillus, Cedecea, Pseudomonas and Paenibacillus possessed e cient antagonistic traits of plant growth promotion (Fig. 2) Effect of endophytic bacteria on plant growth promotion A pot experiment was conducted to validate the in vitro PGP activities of selected strains on the growth of R. o cinalis. The in vitro PGP traits of selected strains have been depicted in Table 1.

Effect on physiological characteristics
Photosynthetic pigments of R. o cinalis leaves were determined to evaluate the impact of endophytic strains on photosynthetic e ciency of host plant. Data corresponding to biochemical parameters revealed that treatment T5 (P. aeruginosa SI12) had maximum signi cant (P ≤ 0.05) increase in total chlorophyll (33.09%), carotenoids (39.87%) and total phenol content (24.55%) of R. o cinalis leaves over uninoculated control, which was at par with T8, T12 and T12 for chlorophyll and treatments T3, T7, T8, T11 and T12 in case of carotenoids (Fig. 3).
Principal component analysis of growth characteristics of R. o cinalis in response to inoculation with endophytic bacteria Principal component analysis of growth parameters of R. o cinalis revealed that PC1 and PC2 accounted for 94.67 % and 3.93 % of the total data variation, respectively (Fig. 4). PC1 comprised treatments with T3 (P. oryzihabitans KU5), T6 (P. japonensis KU13), T7 (P. putida KU2), and T9 (Pantoea agglomerans KA14) showed a strong relationship with several lateral branches and stem height. While in PC2, treatments with T4 (Bacillus exus KA10), T7 (P. putida KU2), and T9 (Pantoea agglomerans KA14) reported more in uence on root biomass, total chlorophyll, and phenol content. This analysis showed that inoculation with potential plant growth-promoting bacterial endophytes had a signi cant effect on growth and productivity of R. o cinalis.

Effect on plant nutrient concentration
The data appended in Fig. 5 illustrate that treatment (T12) receiving B. subtilis KU21 inoculation had maximum signi cant increase in N, P and K content by 33.33%, 61.54% and 54.54%, respectively over untreated control and was at par with T5 (P. aeruginosa SI12) and T8 (Cedecea lapagei KU14) for N content and with T8 (Cedecea lapagei KU14) for P content.

Effect on soil properties and endophytic population
None of the treatments in uenced the soil pH, E.C. and O.C. signi cantly in comparison with the initial soil test values (data not shown) recorded before the trial whereas the contents of available nutrients (NPK) increased signi cantly by the sole application of endophytic strains (Table 3). Maximum signi cant increase in available NPK content (16.79 %, 36.26 % and 7.56 %) were recorded in treatment T12 (B. subtilis KU21) which was statistically at par with T8 (C. lapagei KU14) for N, T5 (P. aeruginosa SI12) for P and T11 (B. simplex KA2) for K content. None of the treatments in uenced the soil pH, EC, OC signi cantly over soil initial test value (data not shown). Total endophytic bacterial count varied from 31.00 to 54×10 2 cfu g − 1 root with the maximum count (54×10 2 cfu g − 1 root) in Bacillus subtilis KU21 inoculated plants. Also, in the root endosphere of B. subtilis KU21 inoculated plants accounted for a maximum number (48×10 2 cfu g − 1 root) of bacterial colonies matching B. subtilis KU21 with maximum Simpson's index of dominance (0.79).
Intrinsic antibiotic resistance of potential endophytic bacteria B. subtilis KU21, C. lapagei KU14 and P. aeruginosa SI12 were evaluated for antibiotic sensitivity against combination of antimicrobial sensitivity discs (Table 4). It was observed that the tested strains were resistant to most of the antibiotics. But polymyxin B (300 µg) and colistin (10 µg) inhibited the growth of Pseudomonas aeruginosa SI12. C. lapagei KU21 was susceptible to neomycin (30 µg) and Co-trimoxazole (25 µg). Amikacin (30 µg) inhibited the growth of all three tested strains.

Discussion
Despite the great interest in plants used for the purpose of traditional medicine, little is known about the symbiotic associations of these plants with endophytic microorganisms (Silva et al. 2019). The present study is the rst of its kind to evaluate the multifunctional potential of bacterial strains isolated from the roots of R. o cinalis native to the North-Western Himalayan region of Himachal Pradesh, India. In this study, a collection of 42 root endophytic bacteria of R. o cinalis was obtained from 4 different rosemary cultivating locations of Himachal Pradesh. These strains were identi ed using 16S rDNA sequencing and phylogenetic analysis. The isolates belonged to 15 genera and 32 species, mainly belonging to Proteobacteria and Firmicutes. These results con rmed rich endophytic pool in medicinal plants, in agreement with the previous studies (Elmagzob et al. 2019;Silva et al. 2019;Abdelshafy Mohamad et al. 2020). The predominant reported genera were Pseudomonas and Bacillus. Our ndings are in line with the earlier studies which con rmed that Bacillus and Pseudomonas as PGPBEs have been widely found in medicinal plants such as Pinellia ternate, Lycium Chinese, Digitalis pupurae, (Miller et al. 2012); Lonicera japonica (Zhao et al. 2015); Ginkgo biloba (Yuan et al. 2012); Clerodendrum colebrookianum Walp. (Passari et al. 2016); Thyme vulgaris (Abdelshafy Mohamad et al. 2020).
The main purpose of the current study was to understand the interaction of endophytes with the host plant which involves mobilization of nutrients, production of phytohormones, siderophores, and antagonistic compounds. That is why bacterial strains were screened for in vitro traits of plant growth promotion with an aim to obtain potential candidates. These strains exhibited multifaceted PGP traits. Similar investigations reported that endophytic bacteria exhibited multiple traits of PGP (Egamberdieva et al. 2017). Among all, the strains of Bacillus, Pseudomonas and Cedecea exhibited the highest amounts of Psolubilization, siderophore, IAA, HCN, ammonia and lytic enzymes (chitinase, protease, and amylase). Earlier studies have also indicated that several Bacillus species isolated from medicinal plants produced phytohormones, solubilized P and improved growth of tomato (Abdelshafy Mohamad et al. 2020) and Pseudowintera colorata (Purushotham et al. 2020). Similarly, Pseudomonas species produced IAA and increased plant biomass of medicinal plant Astragalus mongholicus (Sun et al. 2019). In contrast, few studies had reported PGP potential of Cedecea. For example Beniassa et al. (2019) reported the potential of Cedecea as P-solubilizer, nitrogen xer and HCN producers under in vitro conditions. In vitro screening for antifungal activity showed that six strains inhibited the growth of all tested fungal pathogens (F. oxysporum, F. graminiarum, and R. solani). These antagonistic strains exhibited various antifungal properties viz., siderophore, chitinase, protease, amylase, HCN, and ammonia production, etc. Thus, these endophytes can protect the plant from phytopathogenic fungi either by degrading the cell wall or by stimulating systemic resistance in plants (Mohamad 2018). Similar work has been carried out by Egamberdieva et al. (2017), and Liu et al. (2017) who have reported that endophytic strains associated with medicinal plants Ziziphora capital and Ferula songorica respectively, exhibited antifungal activity by producing several lytic enzymes.
To authenticate the results of in vitro studies, we selected endophytic isolates possessing multifarious PGPTs for in vivo experiments. In pot experiment, the application of endophytic strains signi cantly increased the physical growth parameters of R. o cinalis seedlings over untreated control, especially Pseudomonas aeruginosa SI12, Cedecea lapagei KU14 and Bacillus subtilis KU21. The increased shoot/ root parameters in the inoculated plants is attributed to the release of a variety of plant growth regulators in the rhizosphere, resulting in an altered root architecture that may have prompted an expansion in the total root surface area and consequently, improved the water and nutrient uptake, especially N and P, with positive effects on plant growth as a whole (Montano et al. 2014). Similar results were documented by Gupta et al. (2016) with the isolates LS.B11 (Pseudomonas sp.) and EF.B3 (Burkholderia sp.) isolated from medicinal plants Echinacea purpurea and Lonicera japonica which showed an increase in various physical growth parameters of pea seedlings.
The inoculation of R. o cinalis seedlings with indigenous endophytes was also reported to have affected several physiological properties of plants. The signi cantly increased contents of total chlorophyll were observed in plants inoculated with Bacillis subtilis KU21, followed by Pseudomonas aeruginosa SI12, Cedecea lapagei KU14. The results of the present study corresponded with those of Zhang et al. (2008), who reported that PGPR B. subtilis GB03 improved photosynthetic capability by augmenting photosynthetic e ciency and chlorophyll levels in Arabidopsis. Plants possess a variety of antioxidant molecules predominantly phenols that alleviate the reactive oxygen species and defend host cells against adverse conditions. The results of the present study suggested that there were certain elicitors in the microbial cultures which played a vital role in enhancing the phenolic content of R. o cinalis leaves. Several studies have likewise reported the promising effect of endophytic bacteria in boosting phenolics and avanoid contents in Withania somnifera (L.), Dunal, and sweet basil (Gupta and Pandey 2015;Singh et al. 2016).
In case of nutrient acquisition, an improved NPK concentration in plants inoculated with Bacillis subtilis KU21 followed by Pseudomonas aeruginosa SI12, Cedecea lapagei KU14 was observed. This enhanced capacity of the plant to attain and utilize more nutrients could be attributed to the bioinoculation effect on the stimulated root system (Egamberdieva et al. 2017). Moreover, these microbes are also capable of solubilizing mineral nutrients, resulting in increased levels of available NPK in the soil, thereby facilitating their availability to plants (Setiawati and Mutmainnah 2016). For example, Bacillus and Pseudomonas, possessing mineral solubilizing and nitrogen-xing ability, signi cantly increased NP uptake in Zea mays L (Zahid et al. 2015). The endophytic strains of the current investigation were found to be capable of solubilizing P and xing N under in vitro conditions, thus providing more NP to R. o cinalis seedlings.
Each value within columns represents mean of three replicates Each value within columns represents mean of three replicates. Table 3 Effect of inoculation with endophytic bacteria on soil characteristics (post trial). Soil chemical characteristics (Kg ha -1 ) Total viable endophytic bacterial population (10 2 ×cfu g -1 root) Population of inoculated endophytic bacteria (10 2 ×cfu g -1 root) Within columns, means followed by same letter are not significantly different (least signi cant difference (LSD) at P ≤ 0.05).
Each value within columns represents mean of three replicates. Table 4 Antibiotic sensitivity test of endophytic bacteria Antibiotics Concentration (µg disc -1 ) Endophytic strains B. subtilis KU21 P. aeruginosa S112 C. lapagei KU14