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Environ Eng Res > Volume 29(6); 2024 > Article
Chandra, Rai, Basak, Sundha, Prajapat, Singh, and Yadav: In vitro P - solubilization activity of halophilic fungi in salt - affected soils and their potential as bio - inoculants

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.

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 [57]. 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 [912]. 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 [2628]. 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 [2429]. 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 [3234].
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 ECe of the sites ranged from <4 to >30 dS m−1 and pH1:2 <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 306μc), respectively in 1:2 (w/v) aqueous soil suspension. Electrical conductivity of saturation extract (ECe) 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=1Spi×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) [4042].

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 15th 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 PS5N is the phosphorus solubilized at 5% NaCl salinity level, and PS1N 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)
Germinationrate(%)=no.ofseedsgerminatedtotalno.ofseeds×100
(6)
Vigourindex=Germinationrate(%)×totalplantlength(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 ECe, 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).

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.

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.

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.

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).

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 [3234]. 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 ECe, 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 [7477]. 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

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

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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.
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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: EC1:2; PH: pH1:2; 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.
/upload/thumbnails/eer-2023-760f2.gif
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).
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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).
/upload/thumbnails/eer-2023-760f4.gif
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).
/upload/thumbnails/eer-2023-760f5.gif
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
Isolate Name# Morphological Microscopic 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 7.4ab 7.3b 7.9ab 7.1b Ampulliform Oval Penicillium
MP2 Green with white margins Brownish yellow 7.3ab 7.9b 8.1ab 7.9ab Flask-shaped Subglobose to ellipsoidal Penicillium
PA1 Brown color with white margins Brown-yellow 5.5bc 6.7bc 6.3b 6.0bc Ampulliform Oval Aspergillus
PP1 Velvety colony yellowish orange Light yellow to light orange 4.3c 5.4c 4.9bc 5.2c Flask-Shaped Oval Penicillium
PP2 Greenish yellow Bright yellow 5.4bc 5.7c 5.5bc 5.1c Ampulliform Globose to subglobose Penicillium
PP3 Brown pigment on green mat Greenish yellow 6b 6.4bc 5.9bc 5.9bc Ampulliform Globose, spherical, Penicillium
SA1 Black and yellow edges White 8.4a 8.9a 8.7a 8.6a Flask-shaped Globose Aspergillus
SP1 Green with white margins Brownish yellow 7.9a 8.1a 8.2ab 8.1ab Flask-Shaped Oval Penicillium
SP2 Velvety colony yellowish orange Light yellow to light orange 4.2c 5.2c 4.2c 5.1c Ampulliform Oval Penicillium
SP3 Brilliant Green with white margins Pale yellow 7.5a 7.9b 7.7ab 7.6ab Ampulliform Globose to subglobose Penicillium
SP4 Greenish yellow Yellow 8.4a 8.8a 8.6a 8.3ab Flask-shaped Globose to subglobose Penicillium
WA1 Floccose, pale yellow to dark brown Yellow 7.1ab 8.1a 8.0ab 8.0ab Flask-shaped Globose to subglobose Aspergillus
WA2 Velvety colony yellowish orange Light yellow to orange 4.5c 5.5c 4.3c 5.0c Flask-shaped Oval Aspergillus
WP1 Greyish yellow Orange-brownish 5.6bc 6.2c 6.2bc 6.4bc Ampulliform Ellipsoidal Penicillium
WP2 Green serrated edges Dull brown 5.5bc 6.7bc 6.1bc 5.9bc Flask-shaped Ellipsoidal Penicillium
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;
ECe EC1:2 pH1:2 KMnO4oxidizableN OlsenP NH4O
AcK
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 1.9bc 6.2bc 8.2a 124.3bc 13.6a 221.1b 0.37b 55.5bc 18.7bc 25.5bc 2.5b 1.9a 1.39a 0.04a 0.20a
Pearl millet 12 2.0b 8.9a 8.0a 126.7b 13.3a 229.2a 0.42a 61.3a 21.2a 28.7a 2.6a 1.9a 1.24b 0.03b 0.20a
Sorghum 15 2.3a 7.6b 8.1a 120.6c 12.8b 226.9ab 0.35b 58.3b 19.7b 27.1b 2.5b 1.9a 1.35a 0.04a 0.21a
Wheat 12 1.7c 5.8c 8.2a 130.9a 13.1a 223.5ab 0.40ab 53.5c 18.1c 24.8c 2.4c 1.8b 1.39a 0.04a 0.20a

PSF

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

Crop × PSF#

MP 6 1.9bc 6.2bc 8.2a 124.3b 13.6a 221.1b 0.37bc 55.5bc 18.7bc 25.5b 2.5b 1.9a 1.39b 0.04b 0.20b
PA 3 3.0a 9.9a 8.2a 128.4ab 13.7a 232.5a 0.47a 57.6b 20.6b 27.8b 2.5b 1.9a 1.25c 0.03c 0.21ab
PP 9 2.6b 8.5ab 8.0b 126.2b 13.2a 228.0ab 0.41a 62.5b 21.4b 29.1ab 2.6ab 1.9a 1.24c 0.03c 0.20b
SA 3 1.6c 5.2c 8.3a 121.9bc 13.0a 232.0a 0.35b 37.5d 13.3d 18.8d 2.1c 1.7b 1.63a 0.05a 0.22a
SP 12 2.8a 9.1a 8.0b 119.6c 12.7b 223.5b 0.35c 72.2a 23.1a 32.6a 2.7a 2.0a 1.17c 0.03c 0.20b
WA 6 2.2b 7.4b 7.8b 132.8a 13.3a 225.0b 0.42b 61.6b 21.3b 28.8b 2.6ab 1.9a 1.23c 0.03c 0.20b
WP 6 1.6c 5.3c 8.3a 130.2a 13.1a 223.0b 0.40b 50.8c 17.1c 23.5c 2.4b 1.8ab 1.45b 0.04b 0.20b
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.
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 mlh−1 Enzymatic index mm (mgL−1) Absorbance
Mustard 6 113.4b 81.0c 86.93c 94.67b 1.8a 1.68a 1.75a 5.3a 5.3a 32.8a 20.0a 0.037b
Pearl millet 12 187.8a 300.1a 203.0b 199.9a 1.6b 1.59b 1.77a 5.2a 5.0a 35.9a 21.9a 0.025c
Sorghum 15 177.2a 205.3b 162.4b 197.0a 1.7ab 1.71a 1.69b 5.2a 5.0a 35.5a 20.0a 0.051a
Wheat 12 168.4a 192.3b 318.1a 179.8ab 1.8a 1.70a 1.80a 5.0a 4.8a 31.2a 19.7a 0.030ab

PSF

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

Crops × PSF#

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