Multifarious functional traits of free-living rhizospheric fungi, with special reference to Aspergillus spp. isolated from North Indian soil, and their inoculation effect on plant growth

Rhizospheric soil fungi are critical for plant and soil health. However, their multiple functional traits and impact on plant growth have not been systematically explored. During this study, biochemical traits of 73 indigenous soil fungal isolates and 15 unidentified isolates related to plant growth promotion and production of extracellular enzymes were studied. Forty four (65.67%) of the total isolates produced indole acetic acid (IAA) followed by siderophore (52.23%), phosphate solubilization (37.31%), and antibiotic (11.93%). 91.04% of the studied isolates produced ammonia whereas 28.35% produced organic acid. Extracellular enzyme activities of lipase, amylase, chitinase, and cellulase were detected among 95.52%, 61.11%, 35.82%, and 41.79% isolates, respectively. Based on these activities, 73 fungal isolates were categorized into different biotypes. Quantitative analysis of IAA production and phosphate solubilization was carried out for Aspergillus, Penicillium, and Rhizopus isolates. Aspergillus isolates exhibited varying activities of IAA production and phosphate solubilization. Most of the Aspergillus isolates and some other fungi demonstrated multiple activities. Based on the multiple traits of selected fungal isolates, Aspergillus sp-07, Penicillium sp-03, and Rhizopus sp-02 were further evaluated in different combinations for their inoculation effect on the growth and yield of wheat under field conditions. The results indicated that these isolates could be developed into bio-inoculants to enhance plant growth. The consortium of these three isolates was also found to be compatible and beneficial for plant growth.


Background
Fungi are an integral part of the terrestrial ecosystems and are known for their wide applications in the food, pharmaceutical, agricultural, and health care industries. Soil fungi play an important role in the biogeochemical cycling of the elements, maintaining soil fertility, and humus formation by decomposing organic materials (Frąc et al. 2018;Imran et al. 2020). Phosphate solubilization, nutrient mobilization, metal solubilization, and production of organic acid and extracellular enzymes by fungi have also been reported from different soil niches (Newbound et al. 2010;Frąc et al. 2018). Free-living filamentous soil fungi are diverse, adaptable, and beneficial for plants. They possess the extraordinary ability to degrade complex natural and synthetic organic compounds by producing a wide range of extracellular enzymes (Khalil et al. 2013;Zhao et al. 2014;El-Morsy et al. 2017). Indigenous soil fungi produce different enzymes including amylases, proteases, lipases, pectinases, and cellulases, which are important for industrial utilization (Park et al. 2017;McKelvey and Murphy 2018). These enzymes play vital roles in the soil ecosystem via nutrient turnover and maintaining soil fertility; however, their direct role in promoting plant growth is less explored.
The rhizosphere is a nutrient-rich zone for microorganisms and contains more microbial population density and diversity as compared with bulk soil. Extensive research data have been documented about the relation of mycorrhizal fungi in enhancing plant growth, effect on crop yield, and their significance to the environment (Bona et al. 2017;Carrara et al. 2018;Mohamed et al. 2019). However, such data about free-living rhizospheric soil fungi are limited. Soil bacteria have gained much attention as biofertilizers and biocontrol agents during the last few decades (Hafeez et al. 2006;Bhardwaj et al. 2014;Osman et al. 2015;Singh et al. 2019). The freeliving fungi have yet not been systematically screened for their potential in promoting plant growth. Different species of filamentous fungi have been reported with only one major character such as phosphate solubilization by Aspergillus and Penicillium (Elias et al. 2016). Trichoderma spp. are also known for phosphate-solubilizing properties and have been developed as phosphate solubilizer bioinoculant and biocontrol agent (Li et al. 2015;Shaw et al. 2016).
During this study, we hypothesized that multiple beneficial activities of fungal isolates are distributed among common free-living rhizospheric fungi, and the selection of fungi might enhance plant growth under field conditions. Considering the huge diversity of free-living rhizospheric filamentous fungi and the lack of concerted efforts to screen and utilize the rich diversity of indigenous free-living rhizospheric fungi in promoting plant growth, we used previously isolated rhizospheric fungi from the agricultural fields of Aligarh, Northern India (Imran et al. 2020). During this study, these fungi were further screened for plant growth-promoting traits and extracellular enzyme activities. The isolates with desired traits were also tested in different combinations to assess their potential in promoting plant growth under field conditions.

Rhizospheric free-living fungi used in this study
Seventy-three (73) rhizospheric fungi previously isolated from the agricultural fields adjacent to Aligarh city (Northern India) and tentatively identified based on the cultural, morphological, and molecular methods were used in this study. The details of these fungal isolates are available in a recent publication by Imran et al. (2020).

Cultivation of fungi
The fungal isolates were maintained at 4°C in the refrigerator by subculturing on Sabouroud Dextrose Agar (SDA) (Hi-Media Lab. Pvt., Mumbai, India). Fresh fungal cultures for the experiments were prepared by transferring a loop/fragment of mycelium/spore. The purity of freshly grown cultures (28°C for 3 to 7 days) was assessed by subsequent streak plate method followed by microscopic examination (Gilman 2001;Zafar and Ahmad 2005;Imran et al. 2020).

Screening of isolates for extracellular enzymes
All fungal isolates (73) were screened for the production of extracellular enzymes using conventional agar plate assays as follows:

Amylase activity
Starch-degrading activity of the cultures was estimated by the hydrolysis of starch on a medium (g·l -1 ) containing 5.0 g peptone, 3.0 g beef extract, 2.0 g starch (soluble), and 20 g agar. Freshly grown fungal spores of 3-day-old culture were spot inoculated in the center of the plate and incubated at 28°C for 7 days. Seven-day-old fungal cultures were flooded with an iodine solution (Yarrow 1998;Singh et al. 2014). The development of a pale yellow zone around a colony in the blue medium indicated the starchdegrading activity, and isolates were considered positive for amylase production.

Lipase activity
Fungal isolates were inoculated on tributyrin agar medium (g·l -1 ) containing 5.0 g peptone, 3.0 g beef extract, 10 tributyrin, and 15 g agar at pH 6.5. The development of a clear halo zone around the colony indicated the lipase activity after incubation at 28°C for 7 days (Cappuccino and Welsh 2018).

Chitinase activity
The production of chitinases was determined on the plates pre-poured with a layer of 15 g·l -1 agar and successively poured with a 3.0-ml overlayer of the test medium containing 25 g·l -1 chitin powder (Hi-Media) (Campbell and Williams 1951;Krithika and Chellaram 2016). After incubation at 28°C for 7 days, the formation of a clear zone around the colony in an opaque medium indicated the presence of extracellular chitinase.
In vitro detection of plant growth-promoting (PGP) traits of soil fungi Plant growth-promoting activities of isolated soil fungi were detected and quantified in vitro. PGP activities such as the production of indole acetic acid, siderophore, antibiotics, ammonia, organic acid, and phosphate solubilization were determined according to the standard methods as follows: Production and quantification of indole acetic acid (IAA) Fungal cultures were grown in Sabouroud Dextrose broth amended with 500 μg·ml -1 tryptophan at 28°C for 7 days. The fungal growth was separated from 7-day-old culture by filtration followed by centrifugation at 10,000 rpm for 20 min. Two milliliters of supernatant was taken from each tube and mixed with 4.0 ml of Salkowski's reagent (50 ml of 35% perchloric acid, and 1.0 ml of 0.5M FeCl 3 ), and 2-3 drops of orthophosphoric acid were added to each aliquot. The samples were incubated at room temperature for 25 min. The development of the rose pink color indicated the production of IAA (Ahmad et al. 2006). IAA was quantified by reading the Optical Density (OD) at 530 nm using a Spectronic 20D + spectrophotometer, whereas its concentration was measured through a standard IAA graph ranging from 10 to 100 mg/ml.

Phosphate solubilization and quantification
Phosphate solubilization was detected on Pikovskaya agar plates containing (g·l -1 ) 10 g glucose, 5.0 g tricalcium phosphate, 0.5 g yeast extract, 0.5 ammonium sulfate, 0.2 g MgSO 4 , 0.1g NaCl, and a trace amount of manganese sulfate and ferrous sulfate according to Ahmad et al. (2006). The fungal cultures were grown on the Pikovskaya agar plates at 28°C for 7 days. After the incubation period, a clear halo zone of tricalcium phosphate solubilization around the fungal colony indicated a positive result. The solubilization index was evaluated as the ratio of the total diameter (colony + halo zone) and colony diameter of the test fungi (Premono et al. 1996).
The release of soluble phosphate in the culture medium was measured to quantify the phosphate solubilization according to Gaur (1990). Briefly, the fungal cultures were separately inoculated (2.5 × 10 6 spor-e·ml -1 ) in 25 ml of Pikovskaya broth in a 100-ml flask and incubated at 28°C and 125 rpm for 7 days. The uninoculated medium with tricalcium phosphate served as the control. The culture was filtered and centrifuged at 10,000 rpm for 30 min, and the pH of the supernatant was determined. An aliquot of 10 ml supernatant and 10 ml of chloromolybdic acid were mixed in a separate tube, and 5-6 drops of chlorostannous acid were added. The volume was adjusted to 50 ml with double distilled water and the developed blue color was read at 600 nm. The amount of solubilized phosphate was calculated using the calibration curve of KH 2 PO 4 . Biomass was separately processed to determine its dry weight.

Production of siderophores
Siderophore production was detected by the FeCl 3 test (Jalal and van der Heim 1990). Briefly, 7-day-old culture grown in Sabouroud dextrose broth was filtered through Whatman filter paper (No. 42). Culture filtrate was centrifuged at 10,000 rpm for 20 min. Then, an aliquot of 1.0 ml was taken and 1.0 ml of 2% aqueous ferric chloride solution was added. The formation of reddish brown or red color indicated the siderophore production. Uninoculated SD broth was used as a negative control.

Production of organic acid
The Sabouroud dextrose agar media amended with 2% alizarin red S dye was used to examine the production of organic acid. Both media and Alizarin red S were separately autoclaved and mixed in molten media (50°C) before plates were poured. Spore suspension of different fungal isolates was spot inoculated in the center and incubated at 28 ± 2°C for 7 days. Medium acidification was visualized by the presence of pH indicator Alizarin red S, whereas the diameter of the yellow (acid) zone or change in color from red to yellow around the fungal colony indicated the production of organic acid (Prusky et al. 2001).

Production of ammonia
Ammonia production was detected in peptone water (Hi-Media). Fungal cultures were inoculated and incubated at 28°C for 7 days. After incubation, 1.0 ml of Nessler's reagent was added to each tube, and the development of brown to the yellow color indicated the positive test (Cappuccino and Welsh 2018).

Antibiotic activity of fungal culture filtrates
A modified agar well diffusion method was adopted to determine the antibacterial activity of fungal culture filtrates (Ahmad and Beg 2001). An aliquot of 0.1 ml was grown overnight at 37°C and appropriately diluted cultures (10 5 CFU·ml -1 ) of Escherichia coli and Staphylococcus aureus were spread on Muller-Hinton Agar (Hi-Media) plates. The wells of 8.0 mm diameter were punched into agar medium and filled with 100 μl of fungal culture filtrates obtained from 7-day-old culture. The plates were incubated at 37°C for 18-24 hrs. Antibiotic (chloramphenicol) concentration of 100 μg·ml -1 was used as the positive control. The zones of inhibition for bacterial growth were measured in millimeters around each well.
The role of soil fungi in promoting Wheat (Triticum aestivum) plant growth under field conditions The field experiments were carried out on wheat (Triticum aestivum) var. PBW 343, at the Agricultural fields of Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh, India. Wheat seeds were sown in 12 plots by following a completely randomized design. Crops were irrigated with tube-well water by drip irrigation.

Inoculum and plant culture
The plant growth-promoting fungal isolates (Aspergillus niger, Asp-07), Penicillium sp-03, and Rhizopus oryzae, Rsp-02) were cultured on Sabouraud dextrose agar plates at 30°C. The spores were scraped from the plates with a pasture pipette by using 2.0 ml sterile normal saline solution and transferred to a sterile test tube. The concentration of the spores was adjusted to 1 × 10 6 spore·ml -1 . The consortium of Aspergillus niger, Penicillium sp-03, and Rhizopus oryzae (Table 1) were prepared by adding equal volumes of spore suspensions of all the strains. Then, the surface-sterilized seeds were either soaked in the individual suspensions or consortium suspension for 2 h at 28°C on a shaker. The seeds were soaked in 0.85% normal saline solution for minimum dose treatment of inorganic fertilizers (NPK and DAP) and controls (Table 1). The seeds of each treatment were kept in 10 ml sterile NSS to assess the CFU/seed of the inoculated fungi on agar media. Control seeds (uninoculated) were surface sterilized by treating with mercuric chloride (0.1%). Then, control seeds were soaked in 0.85% saline followed by three-time washing with sterile distilled water before sowing. The experimental scheme is summarized in Table 1.
Inoculum density on the seeds was determined by agitating ten seeds from each treatment in 10 ml sterile NSS that was plated on agar plates after serial dilution. Mean fungal spore density per seed was determined after 4 days of incubation at 28 ± 2°C.

Harvesting and data collection
The wheat sampling was carried out at 50 DAS (vegetative), 70 DAS (flowering), and harvesting. The plants were randomly removed from each treatment during the sampling. Plants were extended and pressed for biometric analysis. The following growth and yield parameters were determined using standard methods.

Vegetative growth parameters
The wheat plant growth parameters such as shoot weight, shoot length, leaf numbers, Spikelet numbers, and other parameters were taken into account at three stages (50, 70, and 130 DAS).

Estimation of nitrogen content
Nitrogen content was estimated according to Lindner and Harley (1942). Dry plant material (1.0 g) was digested in concentrated H 2 SO 4 and 30% H 2 O 2 . A 10-ml aliquot of the digested material was added to a 50-ml flask. Two milliliters of 2.5 N sodium hydroxide was added into the flask to neutralize the excess acid, and 2.0 ml of sodium salicylate was added to prevent the turbidity. The volume of the solution was made up to the mark. An aliquot of 5.0 ml of this solution was taken, and 0.5 ml Nessler's reagent was added to it. The volume was made up to 10 ml with distilled water and allowed to stand for 5 min. Absorbance was read at 525 nm, whereas the standard curve was prepared by using ammonium sulfate solution.

Estimation of protein content
The protein content of wheat seeds was estimated according to Lowry et al. (1951). Briefly, 500 mg of the seeds soaked in PBS were finely ground in 5-10 ml PBS. The resultant sample extract was centrifuged, and the supernatant was used for protein estimation. Aliquots of 0.1 and 0.2 ml were taken from the sample extract, and the volume was made up to 1.0 ml. A tube containing 1.0 ml of water served as a blank. Five milliliters of copper solution was mixed into each tube including the blank. The contents were mixed well and allowed to stand for 10 min. A 0.5-ml aliquot of Folin's reagent was added to the mixture and incubated at room temperature for 30 min. The OD was read at 660 nm after the development of blue color. The standard curve of bovine serum albumin (BSA) solution was used to calculate the protein concentration as milligram per gram (mg·g -1 ) of seed.

Yield parameters
The yield parameters were studied at the time of harvesting including the weight of seeds per plant and weight of thousand seeds.

Statistical analysis
The experimental data were subjected to statistical analysis using Duncan's new multiple range test (MRT). The differences among means were calculated at P < 0.05.

Results
During this study, previously isolated fungal isolates (73) belonging to various genera including Aspergillus, Curvularia, Penicillium, Rhizopus, and Mucor (Imran et al. 2020) were screened for their potential in producing extracellular enzymes and plant growth-promoting activities. The study included 20 distinct isolates of Aspergillus followed by Curvularia (09), Mycelia Sterillia (05), and Penicillium (03), whereas the remaining 14 genera were represented by one or two isolates. Fifteen unidentified isolates were also included in the primary screening.
Screening of fungal isolates for extracellular enzyme and plant growth-promoting (PGP) activities The production of extracellular enzymes by 73 fungal isolates is shown in Table 2. Lipase was the most frequently produced enzyme by 70 fungal isolates (95.89%) followed by amylase which was produced by 45 isolates (61.64%). The production of chitinase and cellulose was observed in 27 (36.98%), and 30 (41.09%) fungal isolates, respectively ( Table 2). The majority of the isolates produced more than one enzyme, while Mucor spp. and Trichothecium could not produce any enzyme ( Table 2). The production of lipase and amylase was observed in the majority of the Aspergillus isolates [20 (100%, n = 20), and 16 (80%, n = 20) isolates, respectively]. However, only a few isolates were able to produce both chitinase and cellulose (40%, n =20) enzymes (Table 2). Free-living rhizospheric soil fungal isolates exhibited varying plant growth-promoting activities (Table 3). Sixty-one (83.56%) isolates out of the total 73 were found positive for ammonia production, the most frequently detected trait followed by the production of indole acetic acid (IAA) in 44 (60.27%) isolates. Siderophore production was observed in 35 (47.94%) isolates followed by the detection of phosphate solubilization in 25 (34.25%) isolates. 26.02% (19) isolates produced organic acid, whereas only 10.95% (8) isolates could produce antibiotics. The production of ammonia (60%), IAA (50%), and siderophore (40%) were the most frequently detected plant growth-promoting traits in Aspergillus isolates (n = 20) ( Table 3). The overall enzyme and PGP activities of Aspergillus isolates are presented in Table 4.

Assessment of plant growth-promoting activities of soil fungi on solid media
Phosphate solubilization and production of organic acid The phosphate-solubilizing activity of fungal isolates was determined on Pikovskaya agar plates, and efficient isolates were identified. The maximum phosphate solubilization activity among Aspergillus isolates was observed in Aspergillus sp-01 (SI = 2.40) followed by Aspergillus sp-03 (SI = 2.29). A few isolates belonging to other fungal genera also exhibited efficient phosphate solubilization on solid agar medium including Mucor sp-01 (SI = 2.57), Penicillium sp-03 (SI = 2.53), Alternaria sp-01 (SI = 2.33), Curvularia sp-02 (SI = 2.36), and other species (Table 5).
The production of organic acid was tested on SDA agar plates amended with Alizarin dye. The halo yellow zones around the fungal colonies were measured for the production of organic acid. Fungal isolates such as Aspergillus sp-07 (SI = 2.30), Alternaria sp-02 (SI = 2.35), Mycelia sterilia sp-01(SI = 2.66), and Unidentified sp-07 (SI = 2.33) efficiently produced organic acid. However, the Unidentified sp-03 (SI = 2.69) isolate remained the best producer of organic acid among all tested isolates ( Table 5).
The fungal isolates were grouped into 10 PGP groups/ types based on the presence or absence of one or more PGP traits such as phosphate solubilization and production of IAA, ammonia, and siderophore. Group I consisting of 12 isolates (Aspergillus sp-01, Aspergillus sp-02 .,Alternaria, Curvularia, Fusarium, Mucor, Penicillium, Rhizopus, Trichoderma, and three unidentified spp (sp-02, sp-10, and sp-14)) possessed all four PGP traits as shown in Table 6.

Production of indole acetic acid and biomass
The production of indole acetic acid was estimated in the presence of a fixed concentration of tryptophan (500 μg·ml -1 ) in liquid media. Aspergillus sp-05 produced the maximum amount (31.80 μg·ml -1 ) of IAA among the isolates of Aspergillus genera after 1 week of growth (Table 8). The isolates of other fungal genera also produced a significant amount of  IAA. Fusarium sp-01 produced a maximum (42.41 μg·ml -1 ) amount of IAA followed by Penicillium sp-03 (37.26 μg·ml -1 ), and Rhizopus sp-02 (35.27 μg·ml -1 ) ( Table 8).

Inoculation effects of selected fungal isolates on wheat growth and yield
Shoot length Wheat seeds inoculated with plant growth-promoting fungi (Aspergillus niger, Penicillium sp-03, and Rhizopus oryzae) demonstrated a variable plant shoot growth when grown in unfertilized and fertilized clay loam soil. The composite inoculation of microbial cultures resulted in significantly different shoot lengths of wheat at 50, 70, and 145 DAS (Tables 9 and  10). The single inoculation of Aspergillus niger significantly increased the shoot length by 32.8, 44.2, and 86.7 cm at 50, 70, and 145 DAS, respectively as compared with the uninoculated and unfertilized control. A similar trend was also observed in single inoculations of Penicillium sp-03 and Rhizopus oryzae . Among dual culture inoculations, the combination of Aspergillus niger + Penicillium sp-03 significantly (P < 0.05) increased the shoot growth by 35.7, 52.2, and 87.9 cm at 50, 70, and 145 DAS, respectively as compared with the control. Generally, the shoot length consistently increased in all treatments up to the harvesting stage. The combination of NPK+ Aspergillus niger showed a better response in comparison to NPK+ Penicillium sp-03 and NPK+ Rhizopus oryzae inoculants. Triple inoculation treatment (Aspergillus niger + Penicillium sp-03 + Rhizopus oryzae) produced better results than single   (Tables 9 and 10). The coinoculation of Aspergillus niger + Penicillium sp-03 +Rhizopus oryzae remained superior to all other treatments and significantly enhanced the shoot dry biomass by 27.3, 29.4, and 10.7 g at 50, 70, and 145 DAS, respectively relative to the control. In comparison, the dual inoculation treatment (Aspergillus niger + Penicillium sp-03) significantly (P < 0.05) increased the dry matter accumulation in shoots by 18.7, 21.5, and 8.8 g at the three stages (50, 70, and 145 DAS) of plant growth, respectively as compared with the control. In general, this dual combination performed better than other dual inoculation treatments. The dual inoculation of NPK with Aspergillus niger significantly increased the shoot biomass by 24.8, 24.7, and 9.4 g at 50, 70, and 145 DAS as compared with NPK + Penicillium sp-03 and NPK + Rhizopus oryzae inoculations. The single inoculation treatments of Aspergillus niger also significantly increased the shoot dry biomass by 14.8, 16.5, and 8.0 g as compared with the unfertilized control treatment at 50, 70, and 145 DAS respectively.
Leaf number Generally, the single inoculation treatments of Aspergillus niger, Penicillium sp-03, and Rhizopus oryzae significantly (P <0.05) increased the leaf number by 29.62%, 22.22%, 18.51%, respectively than controls at 145 DAS in 2007. The combination of NPK + Aspergillus niger significantly (P < 0.05) increased the leaf number by 80%, 50%, 40.74%/Ear at 50, 70, and 145 DAS, respectively than the control treatment (Tables 9  and 10). The results indicated that the inoculation of Aspergillus niger in combination with plant growth-promoting Penicillium sp-03 significantly (P < 0.05) increased the leaf number by 58.33% and 37.03% at 70 and 145 DAS, respectively as compared with the inoculation with unfertilized treatment or control. The combination of three cultures (Aspergillus niger + Penicillium sp-03 + Rhizopus oryzae) produced better results than dual and single inoculation treatments (Aspergillus niger + Penicillium sp-03, NPK + Aspergillus niger, Aspergillus niger, and Penicillium sp-03) and significantly (P < 0.05) increased the leaf numbers of wheat plants (Tables 9 and 10).

Spikelet number
The results presented in Table 10 indicate that the inoculation with a combination of Aspergillus niger and plant growth-promoting Penicillium sp-03 significantly (P < 0.05) increased the spikelet numbers as compared with the inoculation with unfertilized treatment or control. Generally, the combination of three cultures (Aspergillus niger + Penicillium sp-03 + Rhizopus oryzae) produced better results than dual and single inoculation treatments (Aspergillus niger + Penicillium sp-03, NPK + Aspergillus niger, Aspergillus niger, and Penicillium sp-03) and significantly (P < 0.05) increased the spikelet numbers of wheat plants. The combination of NPK + Aspergillus niger significantly (P < 0.05) increased the number of spikelets by 51.02%/Ear at harvesting time as compared with all other treatments. The single inoculation treatment of Aspergillus niger, Penicillium sp-03, and Rhizopus oryzae also significantly (P < 0.05) increased the spikelet numbers by 32.65%, 24.48%, and 20.40%, respectively in comparison with the control.
Ear number The treatments of NPK and Aspergillus niger, Penicillium sp-03 and Rhizopus oryzae, Penicillium sp-03 + Rhizopus oryzae, and Aspergillus niger + Penicillium sp-03 + Rhizopus oryzae depicted a significant (P < 0.05) 50% increase as compared with the unfertilized and uninoculated control. The combination of Penicillium sp-03 + Rhizopus oryzae also showed a similar effect on the ear numbers of the wheat plant. A single inoculation of Penicillium sp-03 and Rhizopus oryzae significantly (P < 0.05) increased the ear numbers (25%) than control (Table 10).
Grain number The results presented in Table 10 indicate that inoculation of Aspergillus niger in combination with plant growth-promoting Penicillium sp-03 significantly (P < 0.05) increased the grain number by 75%/Ear as compared with inoculation with unfertilized treatment or control. The combination of three cultures (Aspergillus niger + Penicillium sp-03 + Rhizopus oryzae) proved significantly (P < 0.05) better than dual and single inoculation treatments (Aspergillus niger + Penicillium sp-03, NPK + Aspergillus niger, Aspergillus niger, and Penicillium sp-03) and increased the grain numbers by 106%/Ear of the wheat plants. The combination of NPK + Aspergillus niger increased the number of grains by 84.37%/Ear at harvesting time as compared with the control. The single inoculation treatment of Aspergillus niger, Penicillium sp-03, and Rhizopus oryzae also significantly (P < 0.05) increased the number of the grains by 62.5%, 46.87%, and 43.75%/Ear, respectively, in comparison to the control (Table 10).
Seed yield The seed yield was expressed in terms of both weight of 1000 seeds (g) and seed weight (g·plant -1 ) after single and combined applications of beneficial rhizosphere microorganisms, as presented in Table 10. All treatments significantly increased the grain production during the wheat crop experiments. Single inoculation with Aspergillus niger significantly (P < 0.05) increased the grain yield (weight of 1000 seeds) by 57.01% as compared with the control. The dual inoculation treatments comparatively performed better than the single inoculation, fertilizer treatment, and control. However, inoculation with Aspergillus niger + Penicillium sp-03 was found to be the most effective than other single and dual inoculation treatments. The highest grain yield of 64.3 g·1000 seeds -1 was obtained in Aspergillus niger + Penicillium sp-03 + Rhizopus oryzae treatment, which was even greater than the best performing combination of NPK + Aspergillus niger (Table 10). The single inoculation of Aspergillus niger significantly (P < 0.05) increased the seed yield by 61.53 % than control. The co-inoculation of Aspergillus niger + Penicillium sp-03 was found better than other dual culture treatments. Triple inoculation of Aspergillus niger + Penicillium sp-03 + Rhizopus oryzae enhanced the seed yield by 19.7 g·plant -1 as compared with the control. The triple inoculation factor had a significantly greater effect on seed yield as compared with dual culture treatments. The application of NPK + Aspergillus niger was noted to be significantly superior to other combined treatments of fertilizers and cultures and increased the seed yield by 17.4 g·plant -1 as compared with the control (7.80 g·plant -1 ).
Seed protein The effect of microbial inoculation on seed protein (SP) was variable (Table 10). The maximum average seed protein (17.9%) was observed with the inoculation of Aspergillus niger + Penicillium sp-03 + Rhizopus oryzae in 2007. Among the dual inoculation treatments, the combination of Aspergillus niger + Penicillium sp-03 significantly enhanced the SP (5.10%) as compared with the control. The single inoculation of Aspergillus niger produced 14.1% SP as compared with the uninoculated and unfertilized control (10.80%). The application of NPK + Aspergillus niger was significantly superior to other combined treatments with fertilizers and demonstrated 16.8% seed protein as compared with 10.80% in the control. The comparison of SP mean values revealed that it decreased in the following order: triple < dual < single inoculation.

Discussion
Resident soil fungi and bacteria are crucial for maintaining soil fertility and crop productivity. The role of plant growth-promoting rhizobacteria and mycorrhizal fungi is well known. However, the rich diversity of free-living rhizospheric fungi has not been systematically screened and evaluated as plant growth-promoting fungi (PGPF). Since the majority of free-living soil fungi is not hostspecific; therefore, promising isolates can be developed as broad host range bio-inoculants for crops. During this study, common free-living soil fungi were isolated and evaluated as potential PGPF bio-inoculants. The screening was carried out in vitro and soil-plant system.
The extracellular enzymes of fungal origin have various industrial and biotechnological applications in food processing, manufacturing of detergents, textiles, pharmaceutical products, medical therapy, and molecular biology (Wösten 2019). Different extracellular fungal enzymes are known to degrade complex carbonaceous and nitrogenous substances of soil to synthesize humus that enhances soil fertility and nutrient availability for other organisms including plants (Frąc et al. 2018). These enzymes have been comprehensively studied and utilized in industrial applications. The fungal extracellular enzymes can effectively degrade organic matter and facilitate nutrient recycling. The isolates with efficient enzyme production can better survive in the rhizosphere and may also be indirectly beneficial for plant growth through nutrient turnover. Therefore, fungal extracellular enzymes with potential PGP traits were evaluated.
Lipase activity was noted to be the highest (95.52%) followed by amylase (61.11%), cellulase (41.79%), and chitinase (35.82%) among extracellular enzymes isolated from fungal strains. The production of these enzymes varied among different genera and even among the isolates of the same genus such as Aspergillus, Curvularia, and Mycelia Sterillia group. The differences in the genetic makeup, nature and regulatory mechanisms involved in enzyme synthesis might be the reasons behind varying enzyme production (Crueger et al. 1990). Lipases act upon the organic substrates in the rhizosphere to release the minerals, which are available for other microorganisms and plants (Costa and Peralta 1999;Wadia and Jain 2017). Amylase activity was next to lipase in most of the genera except for some isolates such as Fusarium, Chlamydospora, Mucor, Rhizopus, and Verticillium. The production of amylases is widely distributed among filamentous fungi (Rekha et al. 2013;Ogbonna et al. 2014;Verma and Verma 2018). Such fungi solubilize and utilize starch to co-mineralize nitrogenous compounds for better availability to plants in the rhizosphere. Similarly, cellulolytic activities were observed among certain groups of fungi like Aspergillus, Curvularia, Fusarium, Monotospora, Tricophyton, Trichoderma, Mycelia sterillia, Monillia, and some other isolates belonging to unidentified fungal strains. Fungi with cellulolytic activity play an important role in the enrichment and acceleration of the composting process leading to increased mineralization in the rhizosphere and might also modulate the microbial activity in the rhizosphere (Ram et al. 2014;Reddy et al. 2014;Saxena et al. 2015). Chitin is one of the most important structural polysaccharides that provide mechanical strength to organisms. Chitin is synthesized by plants, animals, and certain microbes except for bacteria and actinomycetes. Chitin-degrading activity is predominant among soil actinomycetes, bacteria, and fungi (Nampally et al. 2015;Veliz et al. 2017). The chitinase production might also have a lytic effect on phytopathogens containing chitin in their cell wall constituents. However, the actual mechanisms of phytopathogen suppression by a biocontrol organism through chitinase production have not been well illustrated. It was hypothesized during a study that chitinase may directly break down the fungal cell walls (Veliz et al. 2017). However, the role of chitinases in the carbon cycle of the biosphere is well known (Rathore and Gupta 2015).
Among the tested fungi, 37.31% fungal isolates belonging to Aspergillus, Curvularia, Alternaria, Trichophyton, Hormodendrum, Monotospora, Mucor, Microsporum, Penicillium, Trichoderma, and Verticillium solubilized phosphate. Several unidentified isolates (15) also served as phosphate solubilizers. The production of organic acid was detected among 28.35% fungal isolates belonging to different genera such as Aspergillus. Alternaria, Penicillium, Mucor, Fusarium, Trichoderma, Verticillium, and many unidentified isolates. Our findings of phosphate solubilization by Aspergillus, Curvularia, Alternaria, Trichophyton, Hormodendrum, Monotospora, Mucor, Microsporum, Penicillium, Trichoderma, and Verticillium are in agreement with previous studies (Gaur 1990, Scervino et al. 2010Altaf et al. 2018;Elias et al. 2016). The phosphate-solubilizing capacity of Trichophyton, Hormodendrum, Monotospora, and Microsporum species from Northern Indian soil is probably the first report. Similarly, our findings of organic acid production by certain fungi (Aspergillus, Rhizopus, Alternaria, Penicillium, Monotospora, and Verticillium) are in line with previous reports (Scervino et al. 2010;Liaud et al. 2014). The production of plant growth regulators is considered an important PGP trait of plant-associated microbes. The rhizospheric fungi capable of producing plant growth hormones (indole acetic acid (IAA)) might directly influence plant growth promotion (Chanclud and Morel 2016;Altaf et al. 2018). Forty-four (65.67%) isolates out of a total 67 produced detectable amounts of IAA and IAA-like compounds in vitro in the presence of tryptophan. These findings are supported by the previous reports on IAA production by Aspergillus, Curvularia, Alternaria, Trichophyton, and Penicillium (Abri et al. 2015;Fu et al. 2015;Altaf et al. 2018;Shah et al. 2019). Siderophore production is considered an indirect mechanism of plant growth promotion by rhizospheric microorganisms (Altaf et al. 2018). To solubilize ferric iron, many microbes utilize an efficient system consisting of low molecular weight (≥ 1000 da) compounds with high iron affinity, known as siderophores. Siderophores have recently received much attention in agriculture as a mechanism of promoting plant health and protection of plants from other fungal pathogens by depriving them of iron (Altaf et al. 2018;Kumar et al. 2018). Organic acid produced by Aspergillus spp. (citric acid and oxalic acid) might serve as siderophores (Haas 2014). Aspergillus spp. is known to produce hydroxamate-like siderophores (Haas 2014; Patel et al. 2017). During this study, siderophore production was noted in eight Aspergillus isolates followed by the isolates of Curvularia, Alternaria, Monotospora, Penicillium, Trichoderma, Chlamydospora, Mucor, and Trichothecium. Certain isolates of Mycelia sterilia and unidentified fungi could also produce siderophores. However, such activity was not detected among Rhizopus, Trichophyton, and Verticillium. Hydroxamate-type siderophore production in fungi belonging to zygomycetes, basidiomycetes, and ascomycetes has been reported, whereas the members of Mucorales produced carboxylates (Comensoli et al. 2017). Similarly, the production of ammonia by 91.04% fungi demonstrated their role in ammonification, which is the key compound for further cycling of nitrogen elements (Espenberg et al. 2018). The role of ammonia production in plant growth promotion is well known. The production of antibiotics by eight isolates of test fungi inhibited or suppressed pathogenic bacteria. However, such activity should be thoroughly investigated as it can also inhibit useful bacteria (Rani and Jain 2017). The antibiotic production by tested fungi signifies their ecological role for better survival and occupation of ecological niche in the rhizosphere.
The rhizospheric soil is rich in fungal diversity; therefore, inoculant strain must be a competitive and effective rhizosphere colonizer. These fungi have a high level of metal tolerance (data not shown) and are expected to have competitive advantages for survival and effective colonization in the rhizosphere. The consortium used in this study was based on the compatibility of the isolates and expressed multiple biochemical and PGP traits. The three most promising fungal isolates were selected and used as inoculants in single and combined treatments to assess their inoculation effect on the growth and yield of wheat under field conditions.
Inoculation response of single-tested fungi and in combinations of two or three species showed stimulatory effect on plant growth and yield attributes of wheat crop like shoot length as well as weight, spikelet, ear and grain number, seed yield, and protein content. Generally, the inoculation of three cultures (Aspergillus niger + Penicillium sp-03 + Rhizopus oryzae) together was observed most effective as compared with dual and single inoculation treatments and showed a significant increase in yield attributes of the wheat plants. Triple inoculation of Aspergillus niger + Penicillium sp-03 + Rhizopus oryzae enhanced the seed as compared with control. This triple inoculation factor had significantly greater effect on seed yield as compared with dual culture treatments. The combined effects of PFPG inoculation is expected due to the probability of expression of desired plant growth-promoting traits of these fungi which might have influenced the plant growth both by direct and indirect mechanisms. Inoculation of Aspergillus niger combination with plant growth-promoting Penicillium sp-03 significantly increased the grain number 75%/Ear as compared with the inoculation with unfertilized treatment or control. The phosphate-solubilizing capabilities of filamentous fungi and bacteria have been studied. Wahid and Mehana (2000) have reported the consortium effect of three phosphate-solubilizing fungal isolates (Aspergillus niger, A. fumigatus, and Penicillium pinophilum) on the faba bean and wheat. They observed that the yield of wheat and faba bean plants increased in response to the soil inoculation with three fungal species, and Penicillium pinophilum played the most effective role. The yield of wheat grains increased by 28.9 and 32.8% in the soil treated with rock phosphate and superphosphate, respectively. Similarly, the same treatments increased the production of faba bean seeds by 14.7 and 29.4%. The phosphorus uptake of both crops significantly increased after the inoculation of soil with tested fungi.
The bacterial isolate Pseudomonas sp. BR2, a mycorrhiza helper bacterium, significantly enhanced the early emergence of wheat seedlings (5 days after planting) under field conditions and increased the root dry matter yield by 128%. The two TPR-solubilizing fungal isolates Aspergillus awamori Nakazawa C1 and Penicillium chrysogenum Thom C13 also increased the root dry matter yields by 60 and 44%, respectively (Babana and Antoun 2006). Wakelin et al. (2007) tested three phosphatesolubilizing fungi (Penicillium radicum, Penicillium bilalae, and Penicillium sp. KC6-W2) to assess their ability in increasing the growth and phosphorus (P) nutrition of wheat, medic, and lentil. Penicillium sp. KC6-W2 was found to be the best plant growth-promoting (PGP) strain and significantly increased the shoot growth and dry mass in seven of the nine experiments.
The effect of three phosphate-solubilizing fungi including Penicillium expansum, Mucor ramosissimus, and Candida krissii on the seedling growth of wheat, has been reported by Xiao et al. (2009). All the isolates promoted growth, availability of soil phosphorus, and nitrogen uptake of wheat seedlings in the soil containing rock phosphate under pot cultivation conditions. In recent years, researchers are focusing on indigenous soil fungi as PGPF. For instance, Hossain et al. (2014) have demonstrated the ability of soil Penicillium sp. G15-1 in promoting plant growth and providing protection against plant pests and pathogens in cucumber plants. Recently, the plant growth-promoting activities of Alternaria sp. A13 have been reported in the Chinese herbal plant Salvia miltiorrhizae through promoting root growth and accumulation of active ingredients (Zhou et al. 2018).

Conclusion
Based on the extensive screening and evaluations, this study concludes that free-living soil fungi have great potential in promoting plant growth through diverse metabolic activities. These fungi require further detailed investigations to develop bio-inoculants with broadspectrum fitness to wheat and other crops under normal and stress conditions.