Mucor sp. HELF2 was isolated as an endophyte from the flower of an ethnomedicinally valuable plant Helicteres isora collected from forests in the East Singbhum district, Jharkhand, West Bengal, India, and stored, maintained on PDA slants and Petri plates at 4 ± 2°C and 25 ± 2°C, respectively. In brief, plant parts were thoroughly washed by running tap water for 5 min, sodium hypochlorite (2–10%) for 2 min, and hydrogen peroxide (2%) for 1 min, respectively, and explants were incubated on water agar plates at 27°C on biological oxygen demand incubator for endophyte isolation. Water agar plates were supplemented with antibiotics (streptomycin and tetracycline- 50 mg L⁻¹) to avoid bacterial endophytes. The effectiveness of this sterilisation and isolation process was cross-checked by the explant imprintation technique described by Schulz et al. (1993). In brief, the aliquots used for explant sterilisation were spread on a water agar medium and incubated under the same conditions. After, 3–5 days of incubation, fungal hyphae emerged from the tissues and they were transferred to PDA plates for optimum growth (Schulz et al., 1993). Emerging fungal hyphal tips were transferred to PDA (Potato Dextrose Agar) medium and morphology (both macroscopic and microscopic) of the fungal isolate was recorded using light (Primo Star, Zeiss, Germany) and stereo microscope (Stemi 508, Zeiss, Germany).

The organism was identified by rDNA-based molecular technique as there was no reproductive structure produced by the endophytic fungi even in a medium with carnated leaves. In brief, genomic DNA of the fungal isolate was obtained (using DNeasy Plant Minikit-Qiagen, Germany) and a polymerase chain reaction was performed using the two universal primers named ITS1 (5’-TCCGTAGGTGAACCTTGCGG-3′) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3′; Laich and Andrade, 2016; Landum et al., 2016). The PCR products were separated using 1% agarose gel in 1X TAE buffer (90 mM Tris-acetate and 2 nM EDTA, pH 8.0), stained with ethidium bromide (0.5 μg mL⁻¹), and documented using BIO-RAD Gel Doc EZ imager version 5.1 (United States). PCR products were sent for direct bi-directional sequencing using ABI 3730xl Genetic Analyzer (Applied Biosystems, United States) to Bioserve Biotechnologies (India) Pvt. Ltd., A Repro Cell Company, Hyderabad, India. The obtained consensus sequence of 620 bp was used for further study. Sequences were submitted to GenBank and were compared to the GenBank database using BLAST. Fifteen sequences along with HELF2 were selected and aligned using the multiple alignment software program Clustal W and the phylogenetic tree was prepared using MEGA 11 (Tamura et al., 2021).

Endophytic fungi were grown in different 250 mL Erlenmeyer flasks with 50 mL potato dextrose broth in a shaker incubator at 120 rpm for 8 days. An initial medium pH of 6 and a medium temperature of 28°C were maintained.

To detect the optimum culture conditions for the maximum production of GRH, fungi were grown in varying fermentation times (4–10 days), then in different medium pH (5.5–7.9), and then at varying incubation temperatures (20–30°C) in separate Erlenmeyer flasks with separate PDB medium. To find out the requirement of additional nutrients for maximum GRH production and mycelial growth, various carbon sources (5 g%, w/v of fructose, glucose, maltose, starch, rhamnose, raffinose, glycerol), various organic and inorganic nitrogen sources (0.4 g% w/v of peptone, ammonium nitrate, urea, ammonium chloride, glycine and yeast extract) in different Erlenmeyer flasks were used with PDB as the basal medium. After the finalisation of the additional carbon and nitrogen sources, their optimum concentration was confirmed by using different concentrations of these products on a PDB medium and the respective biomass and GRH amounts were calculated. A variety of ionic salts (0.1 g%, w/v of MgCl₂, FeCl₃, KCl, NaCl) and phosphate sources (0.1 g%, w/v including NaH₂PO₄, K₂HPO₄, KH₂PO₄) were analysed separately to detect their role in fungal biomass and GRH production (Mahapatra and Banerjee, 2013, 2016).

To detect the O₂ requirement, fungi were grown with different medium volumes in 250 mL Erlenmeyer flasks. Headspace volume, medium volume, total volume, and medium depth in flask culture were measured for the indirect measurement of the organism’s O₂ requirement (Wonglumsom et al., 2000).

Further optimisation was performed with the RSM (Response surface methodology). The investigational design was a Box–Behnken experimental setup with the four most important factors obtained from the OVAT system. The four independent factors had three different levels (−1, 0, and + 1) each for the experiment. GRH production was set to a second-order polynomial equation by the means of multiple regression techniques. The model involving the most significant factors was derived. The system performance follows the subsequent second-order polynomial equation: Y = β₀+ Σβ_iX_i + Σβ_ijX_iX_j + Σβ_iiX²_i, where Y is the predicted response or dependent variable, x_i and x_j are independent factors, β₀ is the intercept of the regression equation, β_i is the linear coefficient, β_ii is the quadratic coefficient and β_ij is the interaction coefficient (Mahapatra and Banerjee, 2013, 2016).

Fungal biomass was separated from the culture extract by centrifugation at 10,000 rpm. Mycelial biomass was dried at 55°C for 24 h and weighed. The supernatant was concentrated in a rotary evaporator under low pressure at 40°C. Chilled absolute ethanol was added to the concentrated supernatant (5:1 v/v), mixed thoroughly, and kept for 24 h at freezing conditions finally, the recovery of viscous precipitate was done by centrifugation at 10,000 rpm for 10 min. The recovered polysaccharide was dialyzed in a cellulose membrane (MW cut off 10,000) against distilled water for 24 h. It was tested for sugar and protein contents following the methods of Dubois et al. (1956) and Lowry (1951) with glucose and bovine serum albumin as the standard. Obtained EPS solution was concentrated in a rotary evaporator under low pressure at 40°C for characterisation.

GRH was purified by gel chromatographic technique using a Sepharose-6B gel filtration column (65 × 2 cm) and average molecular weight was determined following the methods of Mahapatra and Banerjee (2013). The dried polysaccharide was subjected to characterisation using GC–MS with some pre-treatments (Proestos et al., 2006). Using a water bath at 70°C for 15 min, 100 mg of dried exopolysaccharide was combined with 1 mL MeOH, 20 μl ribitol (which serves as an internal standard), and 20 μl nor-leucine. The entire mixture was then centrifuged for 5 min at 10,000 rpm, and the supernatant was immediately dried and dissolved in 20 μl of methoxy-amine HCL for 120 min at 37°C. The final 1 μl of the derivatized EPS sample was loaded onto the GC–MS for analysis of monosaccharide composition after 40 μl of TMS (Trimethyl siloxane) had been added. The instrument was set up with a 30 m × 0.25 mm DB-5 Ultra Inert column. With a split ratio of 25:1, the inlet temperature was 230°C, and the MS transfer line temperature was 250°C. A constant flow mode was used for the column’s flow, which was 1.3 mL min⁻¹ with an average linear starting velocity of 39 cm sec⁻¹. Helium was used as the carrier gas, and ZB − 1701 served as the guard column. The program was isothermal, holding at 70°C for 5 min, increasing by 10°C per minute to 180°C, holding for 2 min, increasing by 10°C per minute again to 220°C, holding for 1 min, and finally ramping up by 2.5°C per minute. Up to 265°C with a 1-min hold, then a ramp up to 285°C with a 1-min hold, and finally a climb of 1°C per min up to 290°C with a 0.6-min hold. The mass spectrum was obtained in scan mode from 40 to 650 amu with a detection threshold of 100 ion counts while the detector was in positive ion mode. Appropriate configurations (D-dextrorotatory and L-laevorotatory form) of the sugars were identified by matching them with the NIST library. To detect the sugar linkages, procedures of Ciucanu and Kerek (1984) and Das et al. (2009) were adopted using the GC–MS equipment.

Initially, healthy and disease-free rice seeds (O. sativa ssp. indica MTU-7093 Swarna) were surface sterilised with a series of surface disinfectants: sodium hypochlorite (2.5%) for 20 min, deionised double-distilled water (3–5 times thoroughly) and then soaked in water for germination followed by storing at 22°C for 72–96 h. Uniformly seeds were transferred to a hydroponics box supplemented with Hoagland solution and were replaced at an interval of 3–4 days (Chen et al., 2011). Seedlings reaching an age of 15–20 days were divided into four separate groups with the control group (un-inoculated water), 20 ppm, 50 ppm, and 100 ppm GRH application, respectively, at a frequency of 3 times a day (morning, noon, and afternoon) for 45 days. Simultaneously, 20% polyethylene glycol (PEG)-6,000 (for 7 days) was mixed with a hydroponic solution as a drought-inducing component, and all the biochemical tests were performed from drought-induced seedlings. The fresh weight of rice seedlings was measured and leaves were stored at −20°C for further biochemical estimations.

The relative water content (RWC) of the treated and control plants were calculated in percentage following the method of Arndt et al. (2015). Fresh leaves were plucked and fresh tissue weight (FW) was measured, then immersed in a 50 mL tube with distilled water, and placed in the dark at 4°C for 20 h. Further, the leaves were dried with filter paper and again weighed for turgid weight (TW) calculation. Lastly, the same leaves were incubated at 80°C for a period of 72 h and dry weight (DW) was measured immediately. Relative water content was calculated by the formula RWC (%) = (FW−DW)/ (TW−DW).

The chlorophyll content (mg g⁻¹ of fresh weight) of the fresh leaves was measured according to the modified formula of Lichtenthaler and Wellburn (1983). Firstly, fresh leaves (0.5 g fresh weight) were split into small pieces and immediately dissolved in 50 mL methanol (80% v/v), covered with black paper, or kept in dark conditions for 24–36 h at 28–30°C. Centrifugation was performed and the supernatant was estimated (645 nm and 653 nm) for chlorophyll contents. Chlorophyll content (mg L⁻¹ FW) = 8.05 A₆₅₃ + 20.29 A₆₄₅.

To calculate the proline contents of the seedling methods proposed by Bates et al. (1973) were adopted. Leaves (0.5 g fresh weight) were split into small pieces and put in a test tube. Further treatment was done by mixing with 5 mL of 3% sulfosalicylic acid, incubated in a water bath for 10 min and 2 mL of the supernatant was mixed thoroughly with 2 mL of acetic acid, and 3 mL of 2.5% ninhydrin. Finally, the mixture was incubated in the water bath for a time period of 40 min⁻¹ h and extracted using 4 mL methylbenzene, optical density was measured at 520 nm and compared with a proline standard curve.

Soluble sugar contents (in terms of mg g⁻¹ fresh weight) were measured by the method of Watanabe et al. (2000). Fresh leaves (0.2 g fresh weight) were crushed in 80% v/v ethanol (10 mL) and centrifuged at 8000 g for 10 min at 4°C. We mixed 1 mL of supernatant thoroughly with 3 mL of anthrone reagent followed by heating at 100°C for 10–12 min, which was stopped by rapid cooling them on the ice. Finally, at 620 nm absorbance was estimated using glucose as a standard.

Malondialdehyde content (nmol g⁻¹ fresh weight) was reported according to the method of Del Buono et al. (2011) 0.5 gm of fresh leaves were homogenised in 5% (w/v) trichloroacetic acid (TCA), centrifuged at 12000 g for a time period of approximately 15–20 min and then the supernatant was mixed with 5 mL of 0.5% thiobarbituric acid (TBA)-prepared with 20% TCA followed by incubation for 25 min and cooling at 100°C and room temperature, respectively. Finally, after centrifugation (7,500 g for 5 min), the supernatant was measured for its absorbance at 450, 532, and 600 nm. The amount of MDA was calculated by the following formula MDA content (nmol g⁻¹) = 6.45 (A₅₃₂-A₆₀₀)−0.56A₄₅₀.

The methods of Lei et al. (2015) were followed for the measurement of peroxidase activity. The system contained multiple chemicals; 2.9 mL of 0.05 M phosphate buffer, 0.5 mL of 2% H₂O₂, 0.1 mL of 2% guaiacol, and also 0.1 mL of crude enzyme extract followed by the absorbance measurement at 470 nm. Lastly, POD activity was calculated as an amount of guaiacol oxidised per minute in nanomoles per minute per mg of protein. At the end of the reaction, the absorbance was measured at 470 nm. POD activity was defined as the amount of guaiacol oxidised per minute, and was expressed as nanomoles per minute per mg of protein.

Catalase activity was calculated following the protocols of Lei et al. (2015). O.1 mL H₂O₂ (2%) and 2 mL phosphate buffer (50 mM-pH 7.0) were mixed and the whole reaction was initiated by the addition of 0.1 mL of crude enzyme extract. Finally, the catalase activity was measured (at 240 nm) in terms of the decrease of values of H₂O₂ per minute, as nanomoles/min/gm of protein.

Superoxide dismutase activity was assayed following the protocols of Lei et al. (2015). The whole system contained a series of valuable freshly prepared reagents; 1.5 mL of 0.05 M phosphate buffer, 0.3 mL of 130 mM methionine solution, 0.3 mL of 750 μM nitroblue tetrazolium solution, 0.3 mL of 100 μM EDTA -Na₂ solution, 0.3 mL of 20 μM lactochrome solution, 0.5 mL of distilled water and finally 0.1 mL of crude enzyme extract. The complete reaction was initiated at 4000 Lx of illumination for a constant 20 min with no interruption. The control set comprises the same set of reagents and illumination but with no crude enzyme extract, rather replaced with a phosphate buffer. The third setup of control contains only phosphate buffer followed by incubation in dark conditions for the same time period of 20 min. Finally, after the completion of the reaction, the absorbance was estimated at 560 nm of wavelength. One unit of SOD activity was defined as the amount of enzyme which inhibits NBT reduction by 50%, also the results were expressed as unit/mg protein.

All experiments were performed in triplicate and the results are presented as means ± standard errors (SE). Data were analysed by Prism GraphPad version 9.2.0 (332) software (San Diego, California, United States). BBD experiments were done in Minitab (version 20.2).

The organism had enormously intertwining hyphae and septate hyaline whitish mycelium, and it lacked any sexual or asexual reproductive structures (Figures 1A–C). rDNA sequence data of the isolate was deposited in GenBank (ON146358). A BLAST search of the earlier existing database indicates a close genetic connection with other species of Mucor and the evolutionary history of the endophytic fungal isolate HELF2 was included using the neighbour-joining method (Saitou and Nei, 1987). The most appropriate phylogenetic tree with a total branch length of 0.00649585 is represented in Figure 1D. The tree was constructed to scale with branch lengths in similar units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances between the species were analysed using the maximum composite likelihood method (Tamura et al., 2021) and were in the units of the number of base substitutions per site. Gaps and missing data were removed from the dataset. There was a total of 620 nucleotides in the final dataset.

Mucor sp. HELF2 was grown on a 250 mL Erlenmeyer flask in submerged condition for 10 days and the highest production of fungal GRH and biomass was detected after 8 days of fermentation (Table 1). A temperature of 26°C and a medium pH of 7.1 was found to be the most suitable one for GRH production. Glucose and peptone at a concentration of 11 g L⁻¹ and 5.5 g L⁻¹ were found to be the most appropriate ones for maximum GRH yield (Table 1). NaCl at a concentration of 0.1 g L⁻¹ was the most effective salt (or source of metal ions) for GRH production. The detailed effect of different parameters on biomass and GRH production is summarised in Table 1.

The amount of dissolved oxygen in the fermentation medium affects EPS production. It depends on the medium volume, the headspace of the medium, and the medium depth. A medium volume of 75 mL in a 320 mL Erlenmeyer flask with 245 mL of headspace volume and 2.1 cm of medium depth and 2.53 cm of surface area was found to be the criteria for optimum GRH production (Table 2).

After OVAT optimisation RSM was adopted using a three-level Box Behnken Design. The most important four factors (glucose concentration, peptone concentration, medium pH, and fermentation time) with five replicates at the center points were established as a model for analysis of GRH production. The experimental design with variable predicted and measured values of GRH was presented in Table 3. Maximum GRH production was noted at the five replicated center points. The predicted response Y for GRH production by Mucor sp. HELF2 was described as coded factors in the following equation Y_GRH = 5.9810–0.168024x₁–0.39671x₂-0.09103x₃-0.37144x₄-0.25686x₁x₂–0.03737x₁x₃ + 0.09369x₁x₄–0.0367 x₂x₃ + 0.32736 x₂x₄–0.0337 x₃x₄₋0.65663 x²₁-0.6395 x²₂₋0.50414 x²₃–0.85426 x²₄. Here Y_GRH is the predicted GRH yield and x₁, x₂, x₃, and x₄ are the four coded factors of glucose concentration, peptone concentration, medium pH, and fermentation time (day) respectively. A regression analysis with detailed statistical data related to the experiment is presented in Table 4. The F-test data of 1994.082 proved that the model was significant. The adjusted determinant coefficient (R² Adj) was found to be 0.9998 which represents that there is a high degree of correlation between the experimental and predicted values and there is more than 99% variation in response that could be predicted by second-order polynomial prediction equation. Adeq precision was reported to be 111.901 which indicates that the model is appropriate. The lack of fit F-value of 5.782 and p-value (p < 0.0001) was not at all valuable to the pure error and the fitness of the model was perfect. The high degree of precision and uniformity of the investigational outcomes were proved by the p value of lack of fit- 0.2830 (> 0.05) and a p value of probability (>F less than 0.05). The other linear and quadratic effects of glucose concentration, urea concentration, M-pH, and fermentation time were also significant (p < 0.0001). Finally, three-dimensional response surface plots and contour plots were constructed by Minitab (20.2) for a clear understanding of the effects of the parameters on GRH production (Figure 2).

The model predicted a maximum response of 20.10 g L⁻¹ GRH yield when the necessary components are 5.2 g L⁻¹ of peptone, 10.5 g L⁻¹ of glucose, 7.05 MpH, and 180 (7.5 days) h of fermentation time. These predictions were authenticated by performing laboratory experiments in flask culture by triplicate with an outcome of 19.951 ± 0.091 g L⁻¹ of GRH.

Exopolysaccharide produced by Endophytic Mucor sp. HELF2 was precipitated by applying chilled ethanol and crude EPS was then dialysed, and purified by gel filtration chromatography with a Sepharose-6B column. One major fraction obtained was eluted between 29 and 42 tubes (Figure 3C) and the colorimetric test confirms the absence of proteins in those fractions. The fraction was further investigated for monosaccharide analysis. The molecular weight of the homogeneous EPS was calculated from a calibration curve of standard dextran as ~2.98 × 10⁵ Da (Figure 3D). Monosaccharide analysis of the derivatised EPS samples showed the occurrence of galactose, fucose, and glucose in a 13:2:1 ratio with D, L, and D configuration, respectively (Table 5). Each repeating unit of the fraction contained 13 galactose, two fucoses, and one glucose, which indicates that the studied EPS contained approximately 104 repeating units. We, therefore, considered that the Galactose Rich Heteropolysaccharide (GRH) could have been produced by endophytic Mucor sp. HELF2. FT-IR analysis of the EPS sample revealed the occurrence of strong absorption peaks at particular wavelengths of 3400.71, 2950.89, 1651.56, 1489.73 which represents C-H, O-H, C-O asymmetric stretching respectively, which indicates the basic characteristics and purity of the carbohydrate moiety. Figures 3A,B represent the FT-IR spectrum and GC–MS spectrum of the crude and derivatised EPS, respectively.

GRH-sprayed rice seedlings were found to be much healthier, and more vigorous in terms of their fresh weight and relative water contents in comparison to the control (only drought-inducing agent-PEG treated). The control plants were characterised by low growth, chlorosis, and wilting of leaves. The rice seedlings exhibited maximum growth promotion after continuous 14 days of GRH treatment. The relative water content and fresh weight of the 50 ppm GRH treated plants were found to be higher than the plants of the control set, and plants treated with 20 ppm and 100 ppm GRH dosage. There was a 1.31, 2.38, and 1.74-time improvement in the fresh weight of seedlings in the 20, 50, and 100 ppm GRH treated plants compared to the control. The relative water contents were also increased by 1.14, 1.58, and 1.26 times in 20, 50, and 100 ppm GRH treated plants than the control one. There was a 3, and 4 times increase in root length and shoot length of the treated (50 ppm GRH) plant, respectively, compared to the control. The fresh weight of the seedlings were found to be improved after the GRH treatment and the 50-ppm GRH application was also found to be the most effective in comparison to the control. Table 6 represents the improved physical characteristics of the treated seedlings. Figure 4 represents the treated (20, 50, and 100 ppm) and control rice seedlings, showing their physical changes.

Not only physical but also biochemical characteristics were improved in the case of GRH-treated plants even after severe drought situations. The chlorophyll contents of the treated plants were found to be more elevated (2.32 times higher for 50 ppm GRH treated one) than the untreated control (Figure 5A). The proline is a potent indicator of plant stress and higher proline contents indicate higher resistance towards stress and better adaptation to that stressful situation. Here the GRH-treated plant shows higher accumulations (approximately 3.89 times higher for 50 ppm GRH treatment) of proline contents than the control ones (Figure 5B). The presence of increased soluble sugar content in the plant tissues also indicates the higher survival ability of plants in drought-stress situations. In this experiment, we found almost 3.5 times higher accumulation of soluble sugar contents in 50 ppm GRH-treated plants in comparison to the only PEG-treated one (Figure 5C). On the other hand, the MDA (Malondialdehyde) content is found to be correlated with lipid peroxidation and membrane damage. Higher MDA content in the plant tissues indicates the detrimental situation induced by the stress factors. In the case of treated seedlings, there was a sharp six time decrease in MDA contents compared to the control (Figure 6A). Other enzymatic antioxidative parameters were found to be also elevated after GRH treatment even in extreme drought situations. SOD, CAT, and POD activities increased up to 1.44, 2.09, and 1.79 times, respectively, in the case of GRH-treated rice seedlings compared to those treated only with PEG (Figures 6B–D).