In vitro P - solubilization activity of halophilic fungi in salt - affected soils and their potential as bio - inoculants

Article information

Environmental Engineering Research. 2024;29(6)

Publication date (electronic) : 2024 May 6

doi : https://doi.org/10.4491/eer.2023.760

Priyanka Chandra, Arvind Kumar Rai

, Nirmalendu Basak

, Parul Sundha, Kailash Prajapat, Awtar Singh, Rajender Kumar Yadav

ICAR Central Soil Salinity Research Institute, Karnal132 001, Haryana, India

^†Corresponding author: E-mail: nirmalendubasak@rediffmail.com (N.B.), ak.rai@icar.gov.in (A.K.R.), rk.yadav@icar.gov.in (R.K.Y.), Tel: +91–9416145187 (N.B.), +91–8814878225 (N.K.R.), +91–9416333045 (R.K.Y.)

Received 2023 December 18; Revised 2024 April 29; Accepted 2024 May 5.

Abstract

Some fungi have a unique ability to adapt and establish beneficial association with crop in salt–affected soils. Therefore, present study was conducted to characterize the salinity–tolerance, P–solubilization and growth promotion ability of the fungal isolates from the rhizosphere of salt–tolerant crops. The rhizospheric soil showed an assemblage of fifteen phosphate–solubilizing fungal (PSF) isolates tolerant to salinity (5% NaCl w/v), alkalinity (pH 8.0), and thermal stress (up to 40°C). Soil pH, EC, organic carbon, KMnO₄–N, Olsen’s P, and NH₄OAc–K explained about 51.6% variability in the rhizosphere assemblage of the microorganisms. The genera Aspergillus and Penicillium were abundant in rhizosphere. The association of Penicillium spp. with crops was greater in soil of higher salinity than Aspergillus. The salt–tolerant fungi demonstrated variable effects on the germination of different crops. Siderophore and fungal biomass of the isolates cause 23% variability in plant growth parameters. The PSF strains PP3 and SA1 of the Penicillium and Aspergillus, respectively, showed high P solubilization without any appreciable change at high salinity. This study concluded that the PSF producing siderophores and phytohormones has significant importance for promoting growth and survival of crops in salt–affected soils.

Abstract

Graphical Abstract

Keywords: Fungi; Organic acid; Phosphatase activity; Phosphorus solubilization; Salinity

1. Introduction

Excessive soluble salts and exchangeable sodium cause osmotic and matric stress for crops growing in salt–affected soils (SAS) [1, 2] and lead to less productive or uninhabitable wastelands. The SAS are affected by limited water availability, specific ion toxicity, and nutrient deficiency [3, 4]. Plants adapt to such extreme conditions by developing mutualistic associations with specific microorganisms to offset these stresses [5–7]. A wide range of intricate interactions between the partners in the rhizosphere may directly or indirectly impact plant nutrition and health [8]. The inoculation of the Fusarium equiseti, Bipolaris sp. and arbuscular mycorrhizal fungi (AMF) had shown enhanced salt–tolerance and plant growth in ryegrass, maize, and soybean by regulating ion homeostasis and rhizospheric community [9–12]. The colonization by the endophytic fungi modulates the ion accumulation in leave to maintain low cytosolic Na: K ratio in plants [13]. The endophytic fungi upregulate the genes associated with high affinity potassium transporter to regulate the Na: K homeostasis. Increased photosynthesis rate, stomatal conductance and synthesis of carotenoid, chlorophyll and protein after inoculation also contributed to increased salinity tolerance in many crop plants [14, 15].

The dynamic process of interactions between plants and microbes provides plants with a variety of advantages, including improved resilience to biotic and abiotic challenges, biological disease management, improved nutrient uptake, and growth promotion [16]. Alterations and modifications by plants themselves within their rhizosphere encourage the growth of particular microorganisms. These interactions between plants and the associated microbes increases the plant's ability to adapt to harsh environments [17, 18]. Despite the detrimental qualities, the saline soils have distinctive ecological niches that inhabit extremophilic microorganisms with specialized adaptation mechanisms [5, 12, 19, 20] enabling plants to respond the salt stressors by generating phytohormones and solubilizing nutrients [21]. These microorganisms also help plants in these situations by reversing the harmful effects of salinity [22].

The fungus is prevalent in severe environments and has a significant impact on ecosystem processes by active participation in nutrient cycling, disease biocontrol, plant growth promotion, autoimmune improvement, and protective barrier against invasion of phytopathogens [23, 24]. It also had the capability of differential activation of the plant signaling pathways to reprogram the plant gene expression [25]. In saline soils, plants forge the mutualistic association with different endophytic and rhizospheric fungi [26–28]. Plants exert significant control over these rhizosphere fungi by synthesizing carbon– and energy–rich molecules and bioactive phytochemicals. Several plant growth–promoting fungi (PGPF) belonging to Gliocladium, Aspergillus, Phoma, Mortierella, Talaromyces, Fusarium, Penicillium, and Trichoderma genera had been reported from the rhizosphere of several plants [24–29]. Fungi from the Penicillium genus had been reported extensively to grow in extreme saline environments with appreciable PGP potential [26, 30, 31]. The P. funiculosum P1, Penicillium sp. NAUSF2 and P. chryosgenum were reported to increase the yield of the quinoa, Vigna radiata, and tomato, respectively, by increased phosphorus solubilizing potential (PSP), organic acid (OA), siderophore, and phytohormones production under saline soil conditions [32–34].

Although several reports highlighted the beneficial effect of the PGPF in stressed soil ecology, the information on the rhizospheric assemblage of the PGPF of the crops of variable salinity tolerance is limited. Hence, we hypothesize that the crop plants adapted to saline soil conditions might have evolved to forge the mutualistic association with different PGPF as per the specific requirement of adaptation in these soils. Hence, the PGPF inhabiting the rhizosphere may vary with crop, nutrient deficiency status and level of soil salinity stress. Wheat (Triticum aestivum), mustard (Brassica juncea L), sorghum (Sorghum bicolour L), and pearl millet (Pennisetum glaucum L.) are the common crops grown in saline soils. These crops possessing moderate to high tolerance to salinity were selected; and their rhizosphere soils were explored to harness salt–tolerant bio–inoculants with P solubilization and other plant growth promotion attributes. The P solubilization and production of other plant growth promotion substances in the plant rhizosphere are significantly influenced by filamentous fungi. Therefore, such fungi adapted to saline soil conditions can be employed as an alternative technique to improve P nutrition, growth promotion, and salt–tolerance of the crop plants. Keeping these points in view, this study was conducted to (i) characterize the rhizospheric assemblage of P–solubilizers in crops of varying salinity–tolerance; and (ii) assess the in-vitro P–solubilization and other plant growth promotion activities of the PGPF in high saline conditions.

2. Material and Methods

2.1. Soil Sampling

The soil samples were collected from the experimental farm of ICAR–Central Soil Salinity Research Institute situated in the western Indo—Gangetic Alluvial Plains, Panipat, Haryana, India (29°19'7.09'' to 29°19'10.0'' N latitude and 76°47'30.0'' to 76°48' 0.0'' E longitude, elevation of 230 to 231 m above mean sea level). The site has complex saline and saline—sodic soil with sandy loam soil texture and ustic soil moisture regime. The soil of the experimental farm is classified as Hasplustepts. The EC_e of the sites ranged from <4 to >30 dS m⁻¹ and pH_1:2 <8.2 to 9.0. A total of 15 rhizospheric soils of four crops (wheat cv. KRL 210 (Triticum aestivum L.), Pearl millet cv. HHB 197 (Pennisetum glaucum L.), sorghum cv. Hybrid Mayur (Sorghum bicolour L), and mustard cv. CS 56 (Brassica juncea L)) were collected at the flower initiation stage. The three adjacent plants of each crop were uprooted carefully to collect rhizosphere soil [21]. The soils adhering on the roots were collected aseptically and stored at <4°C for microbial isolations and other biochemical analysis. For physiochemical properties, bulk soil samples were collected adjacent to the plants collected for rhizosphere soils. The bulk soil samples were air–dried and sieved through a two mm sieve before analysis.

2.2. Soil Properties

The pH and electrical conductivity (EC) were estimated using a digital pH meter (Systronics ^μpH system 362), and EC meter (Systronics conductivity meter 306^μ^c), respectively in 1:2 (w/v) aqueous soil suspension. Electrical conductivity of saturation extract (EC_e) was determined by removal of aqueous extract of soil paste under suction at 0.88 kg cm⁻² [3]. Walkley and Black oxidizable organic C, KMnO₄ oxidizable–N, Olsen’s P and NH₄OAc extractable K were estimated following standard procedures [35]. The soil texture and saturation extract parameters were determined for the composite soil sample taken from all 15 sites (Table S1). The Shannon–Weiner diversity index (SDI) is an index of measuring the diversity of species in a habitat. The bacteria, actinobacteria and fungi population [20] were estimated in rhizosphere soil samples for computing the rhizosphere SDI [20] as described in Eq. (1).

(1) SDI=Σi=1S pi×ln pi

where pi is proportion of the species i in population, and S is the total number of species.

Pikovskaya agar medium supplemented with 5% NaCl was used for enumerating phosphate solubilizing fungi (PSF) and bacteria. Streptomycin (30 μg L⁻¹) and amphotericin B (50 μg L⁻¹) were supplemented to the medium to control bacteria and fungi growth for isolation of PSF and PSB, respectively. The isolates on Pikovskaya agar medium demonstrating halo zones around their colonies after 5 days were counted (Fig. S1a, b). Fungi with the highest zone of solubilization in each rhizospheric sample were picked on potato dextrose agar (PD) slants for further study after purification. A total of 15 distinct salt–tolerant phosphate solubilizing fungal isolates were selected for further study.

2.3. Fungal Characterization

The culture condition variation alters the expression of the biosynthestic gene expression responsible for structural diversity and production of the metabolites [36]. Therefore, the isolated fungi were incubated at 25°C for seven days on the agar medium such as Czapek–Dox (CZ), Potato–Dextrose (PD), Malt Extract (ME), and Yeast Extract Sucrose (YES), to identify the fungal–media combinations having the ability to metabolically utilize various substrates such as carbohydrates, and compounds as sole sources of carbon. Fungi have greater variability in the growth rate, mechanisms of sporulation, and nutrient requirements on different growth media, hence colony growth, colony obverse and reverse colors were recorded following standard procedure [37, 38]. The microscopic study was carried out on slides prepared from the fragment of a fungal colony (approximately 1–2 mm from the periphery) grown on MEA medium stained with lactophenol cotton blue (Himedia®) [39]. Microscopic characteristics focusing on conidiophore branching patterns were examined to identify the genus. The physiological characterization was carried out by monitoring fungal growth on Czapek Dox agar medium at pH 5 and 8. Fungal growth was also observed at different temperatures (25–40°C) [40–42].

2.4. Fungal Biomass and Salt–Tolerance Index

Fungal biomass under saline conditions was estimated in Pikoskaya’s broth containing 1 and 5% NaCl upto 15^th day. The salt–tolerance index (STI) was determined on PDA supplemented with 5% NaCl [43]. The STI was calculated by following formulae. (2):

(2) STI=diameter (cm) of fungi colony on NaCl-medium (cm)diameter of fungi on NaCl-free medium (cm)

2.5. Phosphorus Solubilizing Potential and Organic Acid Production

The PSP was confirmed by inoculating isolated fungal strains on Pikovskaya’s agar medium supplemented with 1 and 5% NaCl. Fungal culture disc was inoculated on Pikovskaya’s agar medium under aseptic condition and incubated at 28±2°C for 5 days. The halo zones developed around the colonies showed PSP. The results were represented as the diameter of zone in mm as phosphorus solubilization potential. The quantitative phosphorus solubilization by fungal isolates were studied under stationary conditions in Pikovskaya’s medium [44]. About twenty ml of growth medium supplemented with 1 and 5% NaCl (w/v) and tricalcium phosphate (TCP) (0.1% w/v) were inoculated with the 7 days old grown two disc (diameter 6 mm) of mycelia of fungi and incubated for 15 days at 28±2°C. The amount of the P released in media was monitored up to 15 days by the ascorbic acid blue color method [45]. The phosphorus solubilization ratio (PSR) was calculated by the following formulae. (3):

(3) sPSR=PS5NPS1N

where PS_5N is the phosphorus solubilized at 5% NaCl salinity level, and PS_1N is the phosphorus solubilized at 1% NaCl salinity level.

The organic acid (OA) production was determined by titrating culture broth with 0.05 N NaOH and was expressed as equivalent g tartaric acid L⁻¹ [46]. Change in pH was estimated by a digital pH meter.

2.6. Enzyme Assays

Amylase, cellulose [47], and protease [48] activities of fungal isolates were screened on medium containing carboxy methyl cellulose, soluble starch, and skimmed milk power. Enzymatic index (EI) was calculated as described [49] earlier using following eq. (4):

(4) EI=Rr

where, R = diameter halo zone, and r = diameter of fungal colony.

For the estimation of acid and alkaline phosphatase enzymes [50], 100 ml Erlenmeyer flask containing sterilized 25 ml Czapek Dox medium was inoculated with 2 mm disc of 5 day–old grown culture for 5 days at 25°C. The fungal mat and culture broth were centrifuged for 10 min at 12000 g. The cell–free culture filtrate was used to determine the extracellular acid and alkaline phosphatase. The biomass obtained was homogenized in 25 ml phosphate–buffered saline (PBS) aseptically and filtered with Whatmann 42 filter. The filtrate obtained was used to estimate the intracellular acid and alkaline phosphatases.

2.7. Plant Growth-Promoting Attributes

Indole acetic acid (IAA) [51] and hydrogen cyanide (HCN) [52] estimation was carried out in tryptophan and glycine–supplemented medium, respectively. Chrome azurol S dye was used to detect siderophores production [53]. The ammonia production was determined by incubating the isolates in peptone water [54]. Pikovskaya agar medium supplemented with 0.1% zinc oxide has been used to determine zinc solubilization [55].

2.8. In Vitro Germination Assay

The bioinoculant potential of fungi was evaluated on four different crops (wheat cv. KRL 210 (Triticum aestivum L.), pearl millet cv. HHB 197 (Pennisetum glaucum L.), sorghum cv. Hybrid Mayur (Sorghum bicolour L), and mustard cv. CS 56 (Brassica juncea L)) through in vitro germination assay. After surface sterilization, seeds of crops were treated with culture filtrate of each fungus. The culture filtrate of fungi was harvested at the log stage obtained after the fifth day of incubation in potato dextrose broth. The 5 ml of 1% NaCl in sterilized distilled water (w/v) was applied to seeds to maintain salinity. Germination was considered when the radicles were half of the seed length. The germination percentage, and root and shoot length were measured [6]. The germination rate and vigour index were calculated with the formulae [5]:

(5) Germination rate (%)=no.of seeds germinatedtotal no.of seeds×100

(6) Vigour index=Germination rate (%)×total plant length (cm)

2.9. Statistical Analysis

The SPSS software was used for all the statistical analysis. Shapiro Wilk’s and Bartlett’s Tests were applied for evaluating the normality and heterogeneity of the variance, respectively. The Kruskal-Wallis test was performed for analysis of variance (ANOVA) to characterize the effects of the crop rhizosphere, and fungal isolates. Similarly, the Mann Whitney U test was carried out to test the effect of fungus genera. Post–hoc analyses pertaining to treatments means were performed using the p values adjusted for Benferroni correction for multiple tests. The cluster analysis was carried out using R. The relationship between different soil variables with microbial population and diversity index in the study was established using Redundancy analysis (RDA). The Monte Carlo permutation test was carried out to verify the significance of RDA models. Contributions of the fungal properties, including plant growth potential and salt tolerance to the total variation in germination and vigor of four crops (wheat (Triticum aestivum L.), pearl millet (Pennisetum glaucum L.), sorghum (Sorghum bicolor L), mustard (Brassica juncea L) were determined by partial redundancy analysis (pRDA) using the Vegan package in R [56].

3. Results

3.1. Soil Properties and Rhizospheric Microbial Assemblage

The cultural and morphological characterization of the fungi was carried out by observing colony features under the two or more growth conditions. Different colony size of fungal isolates was observed on four growth media (Table 1). The morphological features of the fungal growth on PD, YE and CZ media and microscopic features on the ME media indicated that the fungal isolates belonged to the genus Penicillium and Aspergillus (Table 1; Fig. 1). Out of 15 fungal isolates, eleven were from Penicillium while the other four were related to Aspergillus. The rhizospheric soil samples collected for determining the microbial assemblage had different EC, pH, SOC, and available N, P, and K content (Table 2). The soil under the pearl millet had greater EC, SOC, and NH₄OAc–K, while KMnO₄–N was greater under the wheat crop. The EC was lowest under the wheat, while soil pH was similar in rhizosphere of all the crops. The highest rhizosphere microbial population was observed for pearl millet and lowest for wheat. However, the relative abundance of the P solubilizing bacteria (PSB) and fungi (PSF) were lowest for pearl millet compared to sorghum, mustard, and wheat. The rhizospheric PSF assemblage was greater than PSB in all the crops. The Shannon–Weiner diversity index ranged from 0.20–0.21 in all rhizospheric soils (Table 2). The association of Penicillium spp. with crops was greater in soils of higher salinity than Aspergillus. Penicillium was also a preferred partner in different crops under nutrient deprivation compared to Aspergillus. Sorghum inhabiting Penicillium as dominant P solubilizers had a greater population of the actinobacteria, bacteria, fungi, and P solubilizers but the relative proportion of the PSF and PSB were greater in sorghum with rhizospheric dominance of Aspergillus. Different soil parameters like EC_e, EC, pH, N, P, K, and SOC explained about 51.6% variability (Monte Carlo permutation test, p≤ 0.001) in the rhizospheric assemblage of the bacteria, fungi, actinomycetes, Shannon–Weiner diversity index and percentage of phosphorus solubilizing microbes (Fig. 2). RDA1 and RDA2 explained about 48.7 and 2.4% variability and EC, pH and KMnO₄–N had a significant marginal effect (p≤ 0.05; Table S2).

Table 1

Characterization of salt tolerant phosphorus solubilizing fungal isolates from rhizosphere of crops of different salinity tolerance on the basis of their morphological and microscopic characteristics; ^#First letter M: Mustard; P: Pearl millet; S: Sorghum; W: Wheat; second letter P: Penicillium; A: Aspergillus; number (1, 2, 3, 4): strain number; numbers followed by different letters (a–c) in the column arc significantly different (p≤ 0.05); CZ: Czapek-Dox Agar ; PD: Potato-Dextrose Agar; ME: Malt Extract Agar; YE: Yeast Extract Sucrose Agar

Fig. 1

Microscopic view of conidiophores and conidia of fungal isolates; C: conidia; P: phialide; M: metulae; V: vesicle; S: stalk; CP: conidiophore (depicted with white arrow); first letter M: mustard; P: pearl millet; S: sorghum; W: wheat; second letter P: Penicillium; A: Aspergillus; number (1, 2, 3, 4): strain number.

Table 2

Soil properties affecting microbial assemblages of rhizospheric soils of crops grown under saline soils; numbers followed by different letters a–c, A–B, and a–d in the column are significantly different for crop, PSF and crop × PSF, respectively (p ≤ 0.05); ^#First letter M; Mustard; P; Pearl millet; S: Sorghum; W: Wheat; second letter P: Penicillium; A: Aspergillus;

Fig. 2

Redundancy analyses (RDA) depicting soil parameter (blue arrows) shaping microbial composition (red arrows) of rhizospheres of different crops in salt affected soils;* (p< 0.05), ** (p< 0.01 and *** (p< 0.001) denotes most important variables of the most parsimonious RDA model (adj. R² =0.65). EC: EC_1:2; PH: pH_1:2; N: KMnO₄ oxidizable–N; P: Olsen–P; K: NH₄OAc–K; SOC: Organic carbon; BACT: bacteria; FUN: Fungi; ACT: actinobacteria; PSF: phosphate solubilizing fungi; PSB: phosphate solubilizing bacteria; PSFP: phosphate solubilizing fungi percentage; PSBP: phosphate solubilizing bacteria percentage; SDI: Shannon–Weiner diversity index.

3.2. Characterization of the Fungal Isolates

Variations in the growth–pattern and size of fungal isolates were observed under different growth conditions. All the fungal isolates demonstrated temperature tolerance upto 40°C and pH tolerance upto 8.0 (Fig. S2, 3). Aspergillus and Penicillium demonstrated comparable temperature and pH–tolerance. Both the fungi showed an increase in the colony diameter with an increase in temperature (Fig. S2). At each temperature, fungal strains from sorghum rhizosphere had greater colony diameter while from pearl millet had smaller colony diameter. The SA1 and SP4 had maximum colony diameter. Rhizosphere of sorghum and mustard has highly tolerant fungal isolates than pearl millet and wheat. The colony diameter of the fungi was lower in acidic pH compared to alkaline pH (Fig. S3). At both the pH, the colonies of the Aspergillus and Penicillium fungal strains from pearl millet were smaller compared to the sorghum, wheat and mustard.

All the fungi isolated from saline soils demonstrated a high–salt tolerance index (STI) of 0.86–0.95. The strain’s STIs were almost identical across the crops except for P2 (mustard). The salt tolerance index of P2 (mustard) was 0.86 compared to P1 (0.94) with an increase in the salinity at 5% NaCl in the growth media (Fig. 3a). Similarly, Aspergillus and Penicillium demonstrated indistinguishable STI. These salt–tolerant fungi exhibited variable zone of PS on Pikovskaya’s medium (Fig. S1c, d). The P solubilization zone varied among the fungal isolates and ranged from 2.20–5.07 mm, and isolate PP3 demonstrated the greater solubilization zone (Fig. 3b). Aspergillus showed better solubilization potential than Penicillium. The P solubilization zones of pearl millet, sorghum, and wheat rhizospheric fungi were similar but greater than the mustard.

Fig. 3

Effect of fungal isolates (F), Crops (C) and their interaction (F×C) on (a) salt tolerance index, (b) phosphorus solubilization zone, and (c) ratio of the biomass production on Pikovskaya’s Agar supplemented with 5% NaCl to the biomass production at 1% NaCl; P: Penicillium; A: Aspergillus; number (1, 2, 3, 4): strain number; ± data followed by numbers and capped lines on bars are standard deviation; n = 6, 12, 15, 12, 12, 33 and 3 for mustard, pearl millet, sorghum, wheat, Aspergillus, Penicillium and crop × PSF, respectively; numbers and bars followed by different letters (a–c) are significantly different (p≤ 0.05).

3.3. Fungal Biomass and Quantitative P–Solubilization

All the fungal isolates demonstrated salt–tolerance, and the ratio of biomass produced in media supplemented with 5% NaCl compared to 1% NaCl was similar except for a few isolates from pearl millet and sorghum rhizosphere (Fig. 3c). Although the fungal biomass produced in 5% NaCl media were greater for SA1, PP3, PP2 and SP2 (12.3–13.5 dry wt g⁻¹) (Table S3) but WP2 exhibited the least decline in P solubilization at high salt–concentration (Fig. 3c). The salinity of the growth media affected the P–solubilization potential of different isolates. The ratio of the P solubilization in 5% NaCl enriched media compared to the 1% NaCl media was < 1 for all the fungal isolates. All the crops had PSF with differential response to salinity. The WP2, SP2, PA1 and MP2 showed greater P solubilization ratio (p≤ 0.05) (Fig. 4a). But the P solubilization in 5% NaCl media was greater for PP3, SA1, WP1 and SP2 (27.1–28.9 mg ml⁻¹) (Table S3). The organic acid (OA) secretion in the growth media slightly increased with an increase in the salinity. The ratio of the OA released at 5% NaCl concentration compared to 1% NaCl media was >1 for mustard, pearl millet and wheat (Fig. 4b). Aspergillus and Penicillium showed similar response to salinity for OA production in growth media (Table S3). These fungal isolates declined the pH of the growth media with the increase in the incubation period (Table S4). The decline in pH was less at 5% NaCl concentration compared to 1%. The pH decline was greater for the pearl millet isolates than for other crops. The pH reduction was greater for the isolates PP2, PP3, SP2, SA1 and WP1 at both salinity levels.

Fig. 4

Effect of fungal isolates (F), Crops (C) and their interaction (F×C) on the ratio of (A) P solubilization, and (B) organic acid production by different fungal isolates after 15 days of incubation in Pikovskaya’s broth supplemented with 5% NaCl to the 1% NaCl concentration. P: Penicillium; A: Aspergillus; number (1, 2, 3, 4): strain number; ± data followed by numbers and capped lines on bars are standard deviation; n = 6, 12, 15, 12, 12, 33 and 3 for mustard, pearl millet, sorghum, wheat, Aspergillus, Penicillium and crop × PSF, respectively; numbers and bars followed by different letters (a–c) are significantly different (p≤ 0.05).

3.4. Enzymatic and Growth–Promoting Potential

The fungal isolates varied in production of phosphatase enzymes. Except for extracellular acid phosphatase, Aspergillus demonstrated higher phosphatase activity than Penicillium (Table 3). The PSF from the mustard rhizosphere showed lower activities of phosphatases compared to the pearl millet, sorghum and wheat. Isolates from Aspergillus genus also showed higher phosphatase compared to Penicillium. The fungal isolates PP3 and WA1 recorded greater acidic and alkaline phosphatase activity, respectively. Isolates from the mustard and wheat rhizosphere showed greater enzyme index (EI) for cellulase, amylase, and protease than sorghum and pearl millet (Table 3). The EI was almost similar for both the genera. Among the isolates, WA1 showed maximum EI for cellulase, amylase, and protease. Although the PSF from the Penicillium genus showed greater Zn solubilization, siderophore (Fig. S1e, f), ammonia, and IAA production, these isolates did not vary from one crop to another. Among the isolates, SP1 showed greater Zn solubilization, siderophore, IAA, and ammonia production ability than other PSF. In contrast to other growth–promoting attributes, the HCN secretion was greater in Aspergillus compared to Penicillium. The PSF from sorghum rhizosphere produced the maximum HCN, while pearl millet produced the lowest HCN.

Table 3

Extracellular enzymes production and plant growth promoting potential profile of salt tolerant fungal isolates from rhizosphere of crops of different salinity tolerance; ^#first letter M: Mustard; P: Pearl millet; S: Sorghum; W: Wheat; second letter P: Penicillium; A: Aspergillus; number (1, 2, 3, 4): strain number; numbers followed by different letters a–c, A–B, and a–i in the column are significantly different (p ≤ 0.05) for crop, PSF and crop × PSF, respectively.

3.5. Bio–Inoculant Potential

In vitro, germination and vigor including shoot and root length of different crops were substantially influenced by the application of the salt–tolerant fungi under saline conditions (Table S5). The PSF from the Penicillium genus had better plant growth promotion in wheat, whereas the sorghum and pearl millet’s root length and vigor index were higher with PSF from Aspergillus. The SA1 and SP1 were the most effective PSF for sorghum and wheat, respectively. Similarly, PP2 was the most effective PSF for promoting plant growth parameters of mustard crops. The redundancy analysis showed that siderophore, IAA, phosphatase, HCN and biomass production affected the variability 33% variability (Monte Carlo permutation test, p≤ 0.001) in the germination and vigor of sorghum, pearl millet, mustard, and wheat (Fig. 5; Table S6). Siderophore, HCN, and biomass production ability of the culture in saline condition were the most important parameters affecting the plant performance and showed a strong positive correlation with different growth parameters of the culture. These parameters caused 85% variation in the germination and vigor (Monte Carlo permutation test, p≤ 0.001; Fig. 5) (Table S6).

Fig. 5

Redundancy analyses (RDA) showing the fungal properties including its biomass (BioSAL), IAA (indole acetic acid), HCN (hydrogen cyanide), siderophore (Sid), extracellular alkaline phosphatase production (ElkP), extracellular acidic phosphatase production (EAP), phosphorus solubilization (PS) and zinc solubilization (ZS) (blue arrows) on germination and vigour (red arrows) parameters (shoot length (SL), root length (RL), no. of roots (NR), germination percentage (P), and vigour index (VI)) of the crops wheat (W), pearl millet (P), sorghum (S), mustard (M) treated with PSF; ** (p< 0.01) and ** (p≤ 0.001) denotes most important variables of the most parsimonious RDA model (adj. R²= 0.85).

4. Discussion

4.1. Soil Properties and Rhizospheric Microbial Assemblage

The rhizosphere, the junction where plant, soil, and microbial interactions converge in soil ecology, is home to an extensive array of microorganisms [57]. Plant exerts some selectivity in shaping the microbial composition of the rhizosphere by altering the rhizosphere soil environment through the secretion of low molecular weight organic acids and energy–rich exudates [58, 59]. Crop, specific variation in the microbial population and relative abundance of the PSB and PSF in different crops were mainly because of the selectivity of the host plants enabled by the exudation of metabolites to host the microbes capable of alleviating nutrient deprivation, and water and salt stresses [60, 61]. The numerous stressors present in the SAS threaten the emergence of crops. Plants acclimatize by forming a mutualistic interaction with particular beneficial microorganisms in such stressed soil settings to develop tolerance to the prevalent stresses [62, 63]. The greater assemblage of the PSF compared to the PSB in the saline soils highlights the plasticity and ability of the fungi to adapt in unfavorable environment [64]. Aspergillus and Penicillium were the most common genera among the salt–tolerant fungi that could survive in the severe salinity of the soils used in the current study. They are frequently found in soil and are crucial to the disintegration of organic carbon and the cycling of nutrients [24, 26, 31]. The relatively greater population of the Penicillium at higher soil salinity also corroborates the earlier studies highlighting the contribution of this genera in P solubilization and plant growth promotion in extreme saline soils [32–34]. High salinity, nutrient deprivation, and water stress favor the selective assemblage of the PSF with greater adaptability. The present study also established the importance of soil parameters like EC_e, EC, pH, N, P, K, and SOC explained about 52% variability in the rhizosphere assemblage of different crops in the saline soils.

Fungal isolates are ecological generalists about salinity, as shown by their wide range of tolerances [65]. Specialists are limited in scope; generalists are frequently omnipresent and highly adaptable in harsh circumstances. The PSF isolated from different rhizosphere also showed a high salt, temperature, and alkalinity tolerance. This suggested the greater plasticity in adaptation and the presence of mechanisms enabling resistance to sodium toxicity and water loss [66]. Several researchers had previously established the ability of Penicillium spp. to survive in severe circumstances [67]. Although these PSF could survive in high salt environments, results from the present study pointed out that the fungal biomass declined with increased salinity stress. The decline in fungal biomass and hyphal density with increased salinity was also observed earlier [68]. The reduction in growth with increasing NaCl concentrations might be because of altered cytoplasmic water activities and loss of intracellular water. Reports also highlighted the reduced successful colonization of PSF in saline soils because of the disruption in cell walls, reduced metabolic activities, increased ionic concentration in cell sap and disorganization of nucleic acid structure under osmotic stress [69, 70]. In the stressed environment, microbes also spend some energy producing the osmolytes such as ectoine, betaine, and proline, to balance the osmotic pressure in and outside the cell mass [71]. Likewise, for mycorrhizal fungi, the increased colonization of the AMF in salt–affected soil was associated with increased secretion of the glomalin [8]. Variation in fungal growth on growth media used in the study indicated the differential ability of these isolates to utilize variable nutrient source. Different media supported luxuriant growth of fungi which indicates the diverse medium can be utilized for the development of fungi as bio–inoculants for their bulk production.

4.2. P Solubilization and Plant Growth Promoting Potential of PSF

Cellulases, amylases, and proteases are three important ex tracellular non–pectinolytic hydrolases produced by fungi to maintain the proper balance of carbon and nutrients in the soil [72]. These enzymes also impact rhizosphere colonization by regulating physiological processes directly affecting the signaling, protein synthesis, and gene regulation [73]. Soil fungi can be categorized as ecosystem regulators involved in compound transformations and organic matter decomposition based on their functional characteristics [74–77]. In the present study, the P solubilization and pH decline was lower in–spite of increased organic acid secretion by different isolates at increased salinity level. The observed conundrum in P solubilization and organic acid secretion at high salinity might be because of the salinity–induced changes in the composition of organic acid secreted by the fungi as an adaptive response. These organic acids vary in their pKa value had varied effects on the pH of the media. The hydrolysis of organic acids with lower pKa produced pH lower than the acids with higher pKa [78, 79]. But all PSF isolates in high saline media exhibited about 75% potential of P solubilization in non–saline conditions. The intrinsic ability to solubilize phosphates under substantially high saline conditions appeared to exist in the strains as they were isolated from SAS. PSF was able to convert insoluble Pi into soluble orthophosphate forms (PO₄³⁻_, HPO₄²⁻, and H₂PO₄⁻) as they possess the potential to emanate various organic or inorganic acids that release H⁺ and reduce the medium's pH. The organic acids (OA) have carboxyl groups that bind to P by substituting for cations or by competing with them for P adsorption sites, increasing Pi solubilization and improving soil absorption of PO₄³⁻ [80]. The PSF isolates display notable levels of OA synthesis and Pi solubilization efficiency coupled with the pH decline of the growth media (r= 0.4–0.6; p≤ 0.05).

The study also noted a variation in the pH decline and PSI, highlighting the variability of the PSF associated with rhizosphere of different crops. Fungi that solubilize phosphate, can create extracellular phosphatase, an enzyme that can convert organic phosphate to inorganic phosphate. Siderophores are another organic low molecular weight molecule secreted by the PSF to facilitate the chelation of the Ca and other metal cations [81]. The fungal isolates examined in the current study could release IAA, a hormone found in plants. The observed variation in the rhizospheric assemblage and P solubilization index of the PSF from different crop rhizosphere was probably driven by the combined adaptive response of the plant–PSF interaction to a given salinity. The salinity–induced biochemical response of different PSF provided two distinct clusters (Fig. S4). The cluster analysis also indicates the nearest Euclidean distance of OA secretion, and PSP, for cluster I, while STI and fungal enzymatic profiles for cluster II. Thus, any of these two groups of variables can be used for segmenting different isolates for selecting the PSF with suitable characteristics for P solubilization and plant growth promotion. The SP1, SP2, PP2, SA1 and WA1 isolates were almost similar with respect to P solubilization and salinity–tolerance while different from the other isolates clustered in second group. Any of these isolates can be utilized for P solubilization at a targeted salinity condition. The PSF’s extracellular secretion of the siderophore and phytohormones enhances plant growth and development of roots. This was the reason for identifying the siderophore as the most important parameter, controlling about 14% variability in the plant growth promotion. The maximum vigor index of crops was shown by fungi producing large concentrations of siderophore and IAA.

5. Conclusions

The study concludes that the phosphorus–solubilizing fungi from the rhizosphere of the crops adapted to different salinity had appreciable variation in the assemblage of the P–solubilizing microorganisms. Variability in the rhizospheric assemblage was explained by the variation in electrical conductivity, pH, KMnO₄–N, Olsen–P, NH₄OAc–K, and soil organic C content of the soil. The PSF strains PP3 and SA1 of the Penicillium and Aspergillus genera, respectively, showed high P solubilization without appreciable change in P solubilization at high salinity. The PSF from different rhizosphere can tolerate appreciable salinity, temperature, and alkalinity tolerance. The siderophore, IAA, extracellular enzymes, and biomass production of the PSF were closely associated with seedlings growth promotion of wheat, mustard, sorghum, and pearl millet. These fungi can produce phosphatase and solubilize phosphate; they may be used as bio–inoculants to boost soil fertility while reducing fertilizer consumption. Furthermore, having the ability to solubilize phosphorus and produce siderophore and phytohormones in growth media highlights the significance of such fungus in promoting the growth and survival of crops in such harsh environments.

Supplementary Information

eer-2023-760-Supplementary.pdf

Acknowledgment

The authors would like to acknowledge the Director, ICAR–Central Soil Salinity Research Institute (CSSRI), Karnal (Haryana) for providing technical and laboratory assistance. The authors are thankful to Sh. Devender Yadav, Technical assistant for his technical assistance in soil sampling. This research article was approved by Prioritization, Monitoring and Evaluation Cell (PME), ICAR–CSSRI (Research Article No. 9/2023 dated 10.04.2023). For funding the authors are grateful to Indian Council of Agricultural Research, New Delhi (India) “Project: Development of endo-rhizospheric fungal consortia to increase salt tolerance in crops”; and Science and Engineering Research Board, Department of Science and Technology, Government of India (India) (EEQ/2021/000369); and Project (NRMACSSRISIL201700600930): “Development of Arbuscular Mycorrhizal Fungi (AMF) based plant biostimulant to enhance the productivity of salt–affected soils”.

Notes

Author Contributions

P.C. (Scientist) conceptualized and investigated the experiment and wrote the manuscript. A.K.R. (Principal Scientist and Head) did the formal analysis and wrote the manuscript and assisted to review and editing during revision. N.B. (Senior Scientist) did the formal analysis and wrote the manuscript and assisted to review and editing during revision. K.P. (Scientist) assisted in data curation and formal analysis. P.S. (Scientist) conducted data curation and formal analysis. A.S. (Scientist) conducted data curation and formal analysis. R.K.Y. (Principal Scientist and Director) supervised, reviewed and edited the manuscript.

Conflict-of-Interest Statement

The authors declare that they have no conflict of interest.

References

1. Rai AK, Basak N, Sundha P. Saline and sodic ecosystems in the changing world. Book: Soil science: fundamentals to recent advances In : Rakshit A, Singh SK, Abhilash PC, Biswas A, eds. Springer. Singapore: 2021. p. 175–190. https://doi:10.1007/978-981-16-0917-6_9.

2. Basak N, Barman A, Sundha P, Rai AK. Recent trends in soil salinity appraisal and management. Book: Soil analysis: recent trends and applications In : Rakshit A, Ghosh S, Chakraborty S, Philip V, Datta A, eds. Springer. Singapore: 2020. p. 143–161. https://doi:10.1007/978-981-15-2039-6_9.

3. Soni P, Basak N, Rai AK, et al. Deficit saline water irrigation under reduced tillage and residue mulch improves soil health in sorghum-wheat cropping system in semi–arid region. Sci. Rep 2021;11:1880. https://doi.org/10.1038/s41598-020-80364-4.

4. Rai AK, Basak N, Sundha P, Gobinath R, Dixit AK. Problem soils and their management for crop production. Book A text book on Recent advances in agronomy In : Kumar S, Tripathi AK, Palsaniya DR, Ghosh PK, eds. Kalyani Publishers; 2022. p. 4.1–4.14.

5. Singh A, Mishra S, Choudhary M, et al. Rhizobacteria improve rice zinc nutrition in deficient soils. Rhizosphere 2023;25:00646. https://doi.org/10.1016/j.rhisph.2022.100646.

6. Chandra P, Khobra R, Sundha P, Jasrotia P, Chandra P, Sharma R. Plant growth promoting Bacillus–based bio formulations improve wheat rhizosphere biological activity, nutrient uptake and growth of the plant. Acta Physiol. Plant 2021;43:139. https://doi:10.1007/s11738-021-03310-5.

7. Verma P, Chandra P, Rai AK, et al. Native rhizobacteria suppresses spot blotch disease, improves growth and yield of wheat under salt–affected soils. Plant Stress 2023;10:100234. https://doi.org/10.1016/j.stress.2023.100234.

8. Chandra P, Rai AK, Sundha P, Basak N, Kaur H. Rhizospheric soil–plant–microbial interactions for abiotic stress mitigation and enhancing crop performance. Book: Soil Health and Environmental Sustainability: Application of Geospatial Technology In : Shit PK, Adhikary PP, Bhunia GS, Sengupta D, eds. Springer International Publishing; 2022. p. 593–614. https://doi:10.1007/978-3-031-09270-1_26.

9. Feng Q, Cao S, Liao S, et al. Fusarium equiseti–inoculation altered rhizosphere soil microbial community, potentially driving perennial ryegrass growth and salt tolerance. Sci. Total Environ 2023;871:162153. https://doi:10.1016/j.scitotenv.2023.162153.

10. Krishnamoorthy R, Kim K, Subramanian P, Senthilkumar M, Anandham R, Sa T. Arbuscular mycorrhizal fungi and associated bacteria isolated from salt-affected soil enhances the tolerance of maize to salinity in coastal reclamation soil. Agric. Ecosyst. Environ 2016;231:233–239. https://doi.org/10.1016/j.agee.2016.05.037.

11. Lubna MAK, Asaf S, Jan R, et al. Endophytic fungus Bipolaris sp. CSL–1 induces salt tolerance in Glycine max. L via modulating its endogenous hormones, antioxidative system and gene expression. J. Plant Interact 2022;17:319–332. https://doi.org/10.1080/17429145.2022.2036836.

12. Chandra P, Singh A, Prajapat K, Rai AK, Yadav RK. Native arbuscular mycorrhizal fungi improve growth, biomass yield, and phosphorus nutrition of sorghum in saline and sodic soils of the semi–arid region. Environ. Exp. Bot 2022;201:104982. https://doi.org/10.1016/j.envexpbot.2022.104982.

13. Gupta S, Schillaci M, Walker R, Smith P, Watt M, Roessner U. Alleviation of salinity stress in plants by endophytic plant-fungal symbiosis: Current knowledge, perspectives and future directions. Plant Soil 2021;461:219–244. https://doi.org/10.1007/s11104-020-04618-w.

14. Zhang C, Wang W, Hu Y, et al. A novel salt–tolerant strain Trichoderma atroviride HN082102.1 isolated from marine habitat alleviates salt stress and diminishes cucumber root rot caused by Fusarium oxysporum . BMC Microbiol 2022;22:67. https://doi.org/10.1186/s12866-022-02479-0.

15. Abdelaziz ME, Kim D, Ali S, Fedoroff NV, Al–Babili S. The endophytic fungus Piriformospora indica enhances Arabidopsis thaliana growth and modulates Na(+)/K(+) homeostasis under salt–stress conditions. Plant Sci 2017;263:107–115. https://doi:10.1016/j.plantsci.2017.07.006.

16. Chandra P, Wunnava A, Verma P, Chandra A, Sharma RK. Strategies to mitigate the adverse effect of drought stress on crop plants—influences of soil bacteria: A review. Pedosphere 2021;31:496–509. https://doi.org/10.1016/S1002-0160(20)60092-3.

17. Mendes R, Garbeva P, Raaijmakers JM. The rhizosphere microbiome: significance of plant beneficial, plant pathogenic, and human pathogenic microorganisms. FEMS Microbiol. Rev 2013;37:634–663. https://doi:10.1111/1574-6976.12028.

18. Shahzad T, Chenu C, Genet P, et al. Contribution of exudates, arbuscular mycorrhizal fungi and litter depositions to the rhizosphere priming effect induced by grassland species. Soil Biol. Biochem 2015;80:146–155. https://doi:10.1016/j.soilbio.2014.09.023.

19. Rai AK, Johri S, Kaur H, Basak N, Sundha P. Salinity and sodicity influence mutualistic association of beneficial microorganism in arid soils. J. Soil Salin. Water Qual 2020;12:15–21.

20. Chandra P, Dhuli P, Verma P, et al. Culturable microbial diversity in the rhizosphere of different biotypes under variable salinity. Trop. Ecol 2020;61:291–300. https://doi:10.1007/s42965-020-00089-3.

21. Rai AK, Dinkar A, Basak N, et al. Phosphorus nutrition of oats genotypes in acidic soils: Exploiting responsive plant-microbe partnership. Appl. Soil Ecol 2021;167:104094. https://doi:10.1016/j.apsoil.2021.104094.

22. Upadhyay SK, Singh DP. Effect of salt–tolerant plant growth–promoting rhizobacteria on wheat plants and soil health in a saline environment. Plant Biol 2015;17:288–293. https://doi:10.1111/plb.12173.

23. Paul S, Rakshit A. Effect of seed bio-priming with Trichoderma viride strain bhu–2953 for enhancing soil phosphorus solubilization and uptake in soybean (Glycine max). Soil Sci . Plant Nutr 2021;21:1041–1052. https://doi.org/10.1007/s42729-021-00420-4J.

24. Adedayo AA, Babalola OO. Fungi that promote plant growth in the rhizosphere boost Crop Growth. J. Fungi 2023;9(2):239. https://doi.org/10.3390/jof9020239.

25. Hossain Md, Sultana F, Islam S. Plant Growth–Promoting Fungi (PGPF): Phytostimulation and induced systemic resistance. Book: Plant–Microbe Interactions in Agro–Ecological Perspectives: Volume 2: Microbial Interactions and Agro-Ecological Impacts In : Singh DP, Singh HB, Prabha R, eds. Springer; Singapore: 2017. 135–191. https://doi:10.1007/978-981-10-6593-4_6.

26. Galeano RMS, Silva SM, Yonekawa MKA, et al. Penicillium chrysogenum strain 34–P promotes plant growth and improves initial development of maize under saline conditions. Rhizosphere 2023;26:100710. https://doi.org/10.1016/j.rhisph.2023.100710.

27. Bibi N, Jan G, Jan FG, et al. Cochliobolus sp. acts as a biochemical modulator to alleviate salinity stress in okra plants. Plant Physiol. Biochem 2019;139:459–469. https://doi.org/10.1016/j.plaphy.2019.04.019.

28. Gul SL, Moon YS, Hamayun M, et al. Porostereum spadiceum–AGH786 Regulates the Growth and Metabolites Production in Triticum aestivum L. under salt stress. Curr. Microbiol 2022;79:159. https://doi.org/10.1007/s00284-022-02853-1.

29. Elgharably A, Nafady NA. Inoculation with Arbuscular mycorrhizae, Penicillium funiculosum and Fusarium oxysporum enhanced wheat growth and nutrient uptake in the saline soil. Rhizosphere 2021;18:100345. https://doi.org/10.1016/j.rhisph.2021.100345.

30. Bilal S, Shahzad R, Khan AL, Al-Harrasi A, Kim CK, Lee I. Phytohormones enabled endophytic Penicillium funiculosum LHL06 protects Glycine max L. from synergistic toxicity of heavy metals by hormonal and stress–responsive proteins modulation. J. Hazard. Mater 2019;379:120824. https://doi:10.1016/j.jhazmat.2019.120824.

31. Leitão AL, Enguita FJ. Gibberellins in Penicillium strains: Challenges for endophyte–plant host interactions under salinity stress. Microbiol. Res 2016;183:8–18. https://doi.org/10.1016/j.micres.2015.11.004.

32. Jin F, Hu Q, Zhao Y, Lin X, Zhang J, Zhang J. Enhancing quinoa growth under severe saline-alkali stress by phosphate solubilizing microorganism Penicillium funicuiosum P1. PLoS One 2022;17:e0273459. https://doi.org/10.1371/journal.pone.0273459.

33. Patel S, Parekh V, Patel K, Jha S. Plant growth–promoting activities of Penicillium sp. NAUSF2 ameliorate Vigna radiata salinity stress in phosphate–deficient saline soil. Appl. Biochem. Microbiol 2021;57:500–507. https://doi.org/10.1134/S000368382104013X.

34. Morsy M, Cleckler B, Armuelles–Millican H. Fungal endophytes promote tomato growth and enhance drought and salt tolerance. Plants 2020;9(7):877. https://doi.org/10.3390/plants9070877.

35. Sundha P, Rai AK, Basak N, Yadav RK, Sharma PC. P solubility and release kinetics in the leachate of saline–sodic soil: Effect of reclamation strategies and water quality. Soil Tillage Res 2022;222:105440. https://doi.org/10.1016/j.still.2022.105440.

36. Bills GF, Platas G, Fillola A, et al. Enhancement of antibiotic and secondary metabolite detection from filamentous fungi by growth on nutritional arrays. J. Appl. Microbiol 2008;104:1644–1658. https://doi:10.1111/j.1365-2672.2008.03735.x.

37. Frisvad JC. Media and growth conditions for induction of secondary metabolite production. Methods Mol. Biol 2012;944:47–58. https://doi.org/doi:10.1007/978-1-62703-122-6_3.

38. Raper KB, Thom C. Manual of the Penicillia Williams and Wilkins. Baltimore MD USA: 8751949. p. 875.

39. Gunasinghe N, Barbetti MJ, You MP, Dehigaspitiya P, Neate S. Dimorphism in Neopseudocercosporella capsellae, an emerging pathogen causing white leaf spot disease of Brassicas. Front. Cell Infect. Microbiol 2021;11:678231. https://doi.org/doi:10.3389/fcimb.2021.678231.

40. Pitt JI. The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces Academic Press Inc Ltd; 1979. p. 634.

41. Frisvad JC, Samson RA. Polyphasic taxonomy of Penicillium subgenus Penicillium. A guide to identification of food and air–borne terverticillate Penicillia and their mycotoxins. Stud. Mycol 2004;49:1–174.

42. Samson RA, Pitt JI. Integration of modern taxonomic methods for Penicillium and Aspergillus classification 1st Editionth ed. CRC Press; 2000. p. 524. https://doi.org/10.1201/9781482284188.

43. Al–Garni SMS, Khan MMA, Bahieldin A. Plant growth–promoting bacteria and silicon fertilizer enhance plant growth and salinity tolerance in Coriandrum sativum . J. Plant Interact 2019;14:386–396. https://doi:10.1080/17429145.2019.1641635.

44. Pikovskaya RI. Mobilization of phosphorus in soil in connection with the vital activity of some microbial species. Microbiology 1948;17:362–370.

45. Barton CJ. Photometric Analysis of Phosphate Rock. Anal Chem 201948;:1068–1073.

46. Panda B, Rahman H, Panda J. Phosphate solubilizing bacteria from the acidic soils of Eastern Himalayan region and their antagonistic effect on fungal pathogens. Rhizosphere 2016;2:62–71. https://doi:10.1016/j.rhisph.2016.08.001.

47. Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem 1959;31(3):426–428. https://doi.org/10.1021/ac60147a030.

48. Chandra P, Vij S. Molecular characterization and identification of bioactive peptides producing Lactobacillus sps. Based on 16S rRNA Gene Sequencing. Food Biotechnol 2018;32:1–14. https://doi:10.1080/08905436.2017.1413657.

49. Saroj P, Manasa P, Narasimhulu K. Characterization of thermophilic fungi producing extracellular lignocellulolytic enzymes for lignocellulosic hydrolysis under solid–state fermentation. Bioresour. Bioprocess 2018;5:31. https://doi.org/10.1186/s40643-018-0216-6.

50. Tabatabai MA, Bremner JM. Use of p–nitrophenyl phosphate for assay of soil phosphatase activity. Soil Biol. Biochem 1969;1:301–307. https://doi.org/10.1016/0038-0717(69)90012-1.

51. Bric JM, Bostock RM, Silverstone SE. Rapid in situ assay for indoleacetic acid production by bacteria immobilized on a nitrocellulose membrane. Appl. Environ. Microbiol 1991;57:535–538. https://doi:10.1128/aem.57.2.535-538.

52. Bakker AW, Schippers B. Microbial cyanide production in the rhizosphere in relation to potato yield reduction and Pseudomonas SPP–mediated plant growth–stimulation. Soil Biol Biochem 1987;19:451–457. https://doi.org/10.1016/0038-0717(87)90037-X.

53. Schwyn B, Neilands JB. Universal chemical assay for the detection and determination of siderophores. Anal. Biochem 1987;160:47–56. https://doi.org/10.1016/0003-2697(87)90612-9.

54. Ahmad F, Ahmad I, Khan MS. Screening of free–living rhizospheric bacteria for their multiple plant growth promoting activities. Microbiol. Res 2008;163:173–181. https://doi.org/10.1016/j.micres.2006.04.001.

55. Di Simine CD, Sayer J, Gadd G. Solubilization of zinc phosphate by a strain of Pseudomonas fluorescens isolated from a forest soil. Biol. Fertil. Soils 1998;28:87–94. https://doi.org/10.1007/s003740050467.

56. Dixon P. VEGAN, a package of R functions for community ecology. J. Veg. Sci 2003;14:927–930. https://doi.org/10.1111/j.1654-1103.2003.tb02228.x.

57. Raaijmakers JM, Paulitz TC, Steinberg C, Alabouvette C, Moënne-Loccoz . The rhizosphere: a playground and battlefield for soilborne pathogens and beneficial microorganisms. Plant Soil 2009;321:341–361. https://doi.org/10.1007/s11104-008-9568-6.

58. Jacoby RP, Koprivova A, Kopriva S. Pinpointing secondary metabolites that shape the composition and function of the plant microbiome. J. Exp. Bot 2021;72:57–69. https://doi.org/10.1093/jxb/eraa424.

59. Jacoby R, Peukert M, Succurro A, Koprivova A, Kopriva S. The role of soil microorganisms in plant mineral nutrition-current knowledge and future directions. Front. Plant Sci 2017;8:1617. https://doi.org/10.3389/fpls.2017.01617.

60. Pantigoso HA, Newberger D, Vivanco JM. The rhizosphere microbiome: Plant–microbial interactions for resource acquisition. J. Appl. Microbiol 2022;133:2864–2876. https://doi.org/10.1111/jam.15686. 36648151.

61. Chandra P, Tripathi P, Chandra A. Isolation and molecular characterization of plant growth-promoting Bacillus spp. and their impact on sugarcane (Saccharum spp. hybrids) growth and tolerance towards drought stress. Acta Physiol. Plant 2018;40:199. https://doi.org/10.1007/s11738-018-2770-0.

62. Sunita K, Mishra I, Mishra J, Prakash J, Arora NK. Secondary metabolites from halotolerant plant growth promoting rhizobacteria for ameliorating salinity stress in plants. Front. Microbiol 2020;11:567768. https://doi.org/10.3389/fmicb.2020.567768.

63. Otlewska A, Migliore M, Dybka–Stępień K, et al. When salt meddles between plant, soil, and microorganisms. Front. Plant Sci 2020;11:553087. https://doi.org/10.3389/fpls.2020.553087.

64. Wille L, Messmer MM, Studer B, Hohmann P. Insights to plant–microbe interactions provide opportunities to improve resistance breeding against root diseases in grain legumes. Plant Cell Environ 2019;42:20–40. https://doi.org/10.1111/pce.13214.

65. Gostinčar C, Lenassi M, Gunde-Cimerman N, Plemenitaš A. Fungal adaptation to extremely high salt concentrations. Adv. Appl. Microbiol 2011;77:71–96. https://doi.org/10.1016/B978-0-12-387044-5.00003-0.

66. Qiao Q, Zhang J, Ma C, et al. Characterization and variation of the rhizosphere fungal community structure of cultivated tetraploid cotton. PLoS ONE 2019;14(10):e0207903. https://doi.org/10.1371/journal.pone.0207903.

67. Evans S, Hansen RW, Schneegurt MA. Isolation and characterization of halotolerant soil fungi from the great salt plains of oklahoma. Cryptogam. Mycol 2013;34:329–341. https://doi.org/10.7872/crym.v34.iss4.2013.329.

68. Bencherif K, Boutekrabt A, Fontaine J, Laruelle F, Dalpè Y, Sahraoui AL. Impact of soil salinity on Arbuscular Mycorrhizal fungi biodiversity and microflora biomass associated with Tamarix Articulata Vahll rhizosphere in arid and semi–arid Algerian areas. Sci. Total Environ 2015;533:488–494. https://doi.org/10.1016/j.scitotenv.2015.07.007.

69. Bois G, Bertrand A, Piché Y, Fung M, Khasa DP. Growth, compatible solute and salt accumulation of five mycorrhizal fungal species grown over a range of NaCl concentrations. Mycorrhiza 2006;16:99–109. https://doi.org/10.1007/s00572-005-0020-y.

70. Hrynkiewicz K, Szymańska S, Piernik A, Thiem D. Ectomycorrhizal community structure of Salix and Betula spp. at a saline site in central poland in relation to the seasons and soil parameters. Water Air Soil Pollut 2015;226:99. https://doi.org/10.1007/s11270-015-2308-7.

71. Bowers KJ, Mesbah NM, Wiegel J. Biodiversity of poly–extremophilic Bacteria: does combining the extremes of high salt, alkaline pH and elevated temperature approach a physic–chemical boundary for life? Aquat. Biosyst 2009;5:9. https://doi.org/10.1186/1746-1448-5-9.

72. Frąc M, Hannula SE, Bełka M, Jędryczka M. Fungal biodiversity and their role in soil health. Front. Microbiol 2018;9:00707. https://doi.org/10.3389/fmicb.2018.00707.

73. Bond JS. Proteases: history, discovery, and roles in health and disease. J. Biol. Chem 2019;294:1643–1651. https://doi.org/10.1074/jbc.TM118.004156.

74. Tedersoo L, Bahram M, Pölme S, et al. Fungal biogeography. Global diversity and geography of soil fungi. Science 2014;346:1256688. https://doi.org/10.1126/science.1256688.

75. Gardi C, Jeffery S. Soil biodiversity 2009. Luxembourg: Joint Research Center, European Commission. p. 27.

76. Treseder KK, Lennon JT. Fungal traits that drive ecosystem dynamics on land. Microbiol Mol. Biol. Rev 2015;79:243–262. https://doi.org/10.1128/mmbr.00001-15.

77. Yang T, Adams JM, Shi Y, He Js, Jing X, Chen L, et al. Soil fungal diversity in natural grasslands of the Tibetan Plateau: associations with plant diversity and productivity. New Phytol 2017;215:756–765. https://doi.org/10.1111/nph.14606.

78. Li Z, Bai T, Dai L, et al. A study of organic acid production in contrasts between two phosphate solubilizing fungi: Penicillium oxalicum and Aspergillus niger . Sci. Rep 2016;6(1):1–8. https://doi.org/10.1038/srep25313.

79. Zúñiga–Silgado D, Rivera–Leyva JC, Coleman JJ, et al. Soil type affects organic acid production and phosphorus solubilization efficiency mediated by several native fungal strains from Mexico. Microorganisms 2020;8(9):1337. https://doi.org/10.3390/microorganisms8091337.

80. Kpomblekou-A K, Tabatabai MA. Effect of organic acids on release of phosphorus from phosphate rocks. Soil Sci 1994;158(6):442–453.

81. Renshaw JC, Robson G, Trinci AP, et al. Fungal siderophores: structures, functions and applications. Mycol. Res 2002;106:1123–1142. https://doi.org/10.1017/S0953756202006548.

Article information Continued

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Isolate Name^#	Morphological							Microscopic	Identification
									Identification
	Colony obverse color	Colony reverse color	CZ	PD	ME	YE	Phialide shape	Conidia shape

			Colony diameter (mm)
MP1	Greenish white with pigments	Dark brown	7.4^ab	7.3^b	7.9^ab	7.1^b	Ampulliform	Oval	Penicillium
MP2	Green with white margins	Brownish yellow	7.3^ab	7.9^b	8.1^ab	7.9^ab	Flask-shaped	Subglobose to ellipsoidal	Penicillium
PA1	Brown color with white margins	Brown-yellow	5.5^bc	6.7^bc	6.3^b	6.0^bc	Ampulliform	Oval	Aspergillus
PP1	Velvety colony yellowish orange	Light yellow to light orange	4.3^c	5.4^c	4.9^bc	5.2^c	Flask-Shaped	Oval	Penicillium
PP2	Greenish yellow	Bright yellow	5.4^bc	5.7^c	5.5^bc	5.1^c	Ampulliform	Globose to subglobose	Penicillium
PP3	Brown pigment on green mat	Greenish yellow	6^b	6.4^bc	5.9^bc	5.9^bc	Ampulliform	Globose, spherical,	Penicillium
SA1	Black and yellow edges	White	8.4^a	8.9^a	8.7^a	8.6^a	Flask-shaped	Globose	Aspergillus
SP1	Green with white margins	Brownish yellow	7.9^a	8.1^a	8.2^ab	8.1^ab	Flask-Shaped	Oval	Penicillium
SP2	Velvety colony yellowish orange	Light yellow to light orange	4.2^c	5.2^c	4.2^c	5.1^c	Ampulliform	Oval	Penicillium
SP3	Brilliant Green with white margins	Pale yellow	7.5^a	7.9^b	7.7^ab	7.6^ab	Ampulliform	Globose to subglobose	Penicillium
SP4	Greenish yellow	Yellow	8.4^a	8.8^a	8.6^a	8.3^ab	Flask-shaped	Globose to subglobose	Penicillium
WA1	Floccose, pale yellow to dark brown	Yellow	7.1^ab	8.1^a	8.0^ab	8.0^ab	Flask-shaped	Globose to subglobose	Aspergillus
WA2	Velvety colony yellowish orange	Light yellow to orange	4.5^c	5.5^c	4.3^c	5.0^c	Flask-shaped	Oval	Aspergillus
WP1	Greyish yellow	Orange-brownish	5.6^bc	6.2^c	6.2^bc	6.4^bc	Ampulliform	Ellipsoidal	Penicillium
WP2	Green serrated edges	Dull brown	5.5^bc	6.7b^c	6.1^bc	5.9^bc	Flask-shaped	Ellipsoidal	Penicillium

		EC_e	EC_1:2	pH_1:2	KMnO₄oxidizable^–N	Olsen^–P	NH₄O Ac^–K	Organic carbon	Bacteria (×10⁵)	Fungi (×10³)	Actino mycetes (×10⁴)	Phosphate solubilizing fungi (×10²)	phosphate solubilizing bacteria (×10²)	Phosphate solubilizing fungi percentage	Phosphate solubilizing bacteria percentage	Shannon-Weiner diversity index

		(dS m⁻¹)			(kg ha⁻¹)			g kg⁻¹	Colony Forming Unit					%
Crop	n

Mustard	6	1.9^bc	6.2^bc	8.2^a	124.3^bc	13.6^a	221.1^b	0.37^b	55.5^bc	18.7^bc	25.5^bc	2.5^b	1.9^a	1.39^a	0.04^a	0.20^a
Pearl millet	12	2.0^b	8.9^a	8.0^a	126.7^b	13.3^a	229.2^a	0.42^a	61.3^a	21.2^a	28.7^a	2.6^a	1.9^a	1.24^b	0.03^b	0.20^a
Sorghum	15	2.3^a	7.6^b	8.1^a	120.6^c	12.8^b	226.9^ab	0.35^b	58.3^b	19.7^b	27.1^b	2.5^b	1.9^a	1.35^a	0.04^a	0.21^a
Wheat	12	1.7^c	5.8^c	8.2^a	130.9^a	13.1^a	223.5^ab	0.40^ab	53.5^c	18.1^c	24.8^c	2.4^c	1.8^b	1.39^a	0.04^a	0.20^a

PSF

Aspergillus	12	2.1^B	6.9^B	8.1^A	126.3^A	13.2^A	230.4^A	0.40^A	48.5^B	17.1^B	23.5^B	2.4^B	1.8^B	1.43^A	0.04^A	0.21^A
Penicillium	33	2.2^A	7.4^A	8.1^A	125.2^A	13.1^A	224.2^B	0.38^B	60.7^A	20.4^A	27.8^A	2.5^A	1.9^A	1.31^B	0.03^B	0.20^B

Crop × PSF^#

MP	6	1.9^bc	6.2^bc	8.2^a	124.3^b	13.6^a	221.1^b	0.37^bc	55.5^bc	18.7^bc	25.5^b	2.5^b	1.9^a	1.39^b	0.04^b	0.20^b
PA	3	3.0^a	9.9^a	8.2^a	128.4^ab	13.7^a	232.5^a	0.47^a	57.6^b	20.6^b	27.8^b	2.5^b	1.9^a	1.25^c	0.03^c	0.21^ab
PP	9	2.6^b	8.5^ab	8.0^b	126.2^b	13.2^a	228.0^ab	0.41^a	62.5^b	21.4^b	29.1^ab	2.6^ab	1.9^a	1.24^c	0.03^c	0.20^b
SA	3	1.6^c	5.2^c	8.3^a	121.9^bc	13.0^a	232.0^a	0.35^b	37.5^d	13.3^d	18.8^d	2.1^c	1.7^b	1.63^a	0.05^a	0.22^a
SP	12	2.8^a	9.1^a	8.0^b	119.6^c	12.7^b	223.5^b	0.35^c	72.2a	23.1^a	32.6^a	2.7^a	2.0^a	1.17^c	0.03^c	0.20^b
WA	6	2.2^b	7.4^b	7.8^b	132.8^a	13.3^a	225.0^b	0.42^b	61.6b	21.3^b	28.8^b	2.6^ab	1.9a	1.23^c	0.03^c	0.20^b
WP	6	1.6^c	5.3^c	8.3^a	130.2^a	13.1^a	223.0^b	0.40^b	50.8c	17.1^c	23.5^c	2.4^b	1.8^ab	1.45^b	0.04^b	0.20^b

Treatment	n	Acidic phosphatase		Alkaline phosphatase		Cellulase	Amylase	Protease	Plant Growth Promoting potential

		Intracellular	Extracellular	Intracellular	Extracellular				Zinc solubilization	Siderophore	Ammonia production	Indole acetic acid	HCN

Crop		mg g⁻¹ dry wt pNP ml⁻h⁻¹				Enzymatic index			mm		(mgL⁻¹)		Absorbance
Mustard	6	113.4^b	81.0^c	86.93c	94.67^b	1.8^a	1.68^a	1.75^a	5.3^a	5.3^a	32.8^a	20.0^a	0.037^b
Pearl millet	12	187.8^a	300.1^a	203.0^b	199.9^a	1.6^b	1.59^b	1.77^a	5.2^a	5.0^a	35.9^a	21.9^a	0.025^c
Sorghum	15	177.2^a	205.3^b	162.4^b	197.0^a	1.7^ab	1.71^a	1.69^b	5.2^a	5.0^a	35.5^a	20.0^a	0.051^a
Wheat	12	168.4^a	192.3^b	318.1^a	179.8^ab	1.8^a	1.70^a	1.80^a	5.0^a	4.8^a	31.2^a	19.7^a	0.030^ab

PSF

Aspergillus	12	174.1^A	203.1^A	327.6^A	185.6^A	1.7^A	1.69^A	1.74^A	4.4^B	4.2^B	29.0^B	19.6^B	0.041^A
Penicillium	33	167.4^B	213.0^A	160.04^B	177.3^B	1.7^A	1.66^A	1.75^A	5.5^A	5.3^A	36.2^A	20.8^A	0.038^B

Crops × PSF^#

MP1	3	85.62^c	45.2ⁱ	71.7^h	73.4^c	1.8^a	1.67^b	1.70^bc	7.5^a	7.1^a	46.7^ab	22.4^a	0.032^b
MP2	3	141.2^b	116.8^h	102.1^g	115.9^b	1.8^a	1.69^b	1.80^ab	3.1^bc	3.4^bc	18.8^b	17.6^b	0.039^a
PA1	3	157.7^b	151.4^f	140.4^e	125.7^b	1.8^a	1.62^b	1.77^b	4.5^b	4.4^b	35.0^ab	21.6^ab	0.038^ab
PP1	3	154.1^b	136.5^g	113.8^f	119.5^b	1.7^a	1.63^b	1.86^a	8.0^a	7.4^a	55.2^ab	23.6^a	0.042^a
PP2	3	211.5^a	274.8^c	211.4c	268.4^a	1.5^b	1.65^b	1.77^b	4.3^b	4.2^bc	27.5^b	21.3^ab	0.031^b
PP3	3	228.0^a	637.8^a	346.6^b	286.0^a	1.6^b	1.45^b	1.68^bc	4.2^bc	4.0^bc	26.1^b	21.0^ab	0.027^b
SA1	3	207.2^a	254.5^cd	204.9^c	250.1^a	1.5^b	1.61^b	1.55^c	3.4^bc	3.5^bc	20.1^b	19.1^b	0.040^a
SP1	3	148.8^b	130.5^g	112.0^f	118.1^b	1.9^a	1.64^b	1.87^a	8.4^a	7.8^a	66.1^a	26.2^a	0.033^b
SP2	3	216.1^a	343.9^b	214.9^c	272.0^a	1.6b	1.65^b	1.54^c	7.8^a	7.3^a	50.9^ab	22.9^a	0.034^b
SP3	3	125.5^b	66.8ⁱ	79.2^h	102.0^b	1.7^a	1.80^a	1.75^b	4.0^bc	3.8^bc	25.3^b	20.7^ab	0.042^a
SP4	3	188.6^ab	230.8^d	201.3^c	242.8^a	1.7^a	1.88^a	1.74^b	2.6^c	2.4^c	15.4^b	11.0^b	0.04^a
WA1	3	171.9^ab	217.8^de	802.5^a	219.1^a	2.0^a	1.93^a	1.92^a	6.4^ab	5.6^ab	41.4^ab	21.8^ab	0.035^b
WA2	3	159.3^b	191.3^e	162.5^d	147.5^b	1.7^a	1.61b	1.73^b	2.9^c	3.1^c	17.0^b	15.0^b	0.037^ab
WP1	3	203.3^a	251.9^cd	205.4^c	247.6^a	1.6^b	1.62b	1.78^b	7.1^a	6.9^a	43.3^ab	22.0^a	0.026^b
WP2	3	139.4^b	108.2^h	102^g	105.0^b	1.7^a	1.63b	1.77^b	3.5^bc	3.7^bc	23.3^b	20.0^ab	0.035^b