Morphological and molecular identification of indigenous arbuscular mycorrhizal fungi in the rhizosphere of chickpea (Cicer arietinum) and their role in nutrient uptake

Study sites and sample collection

Several studies were conducted to collect Cicer arietinum L. at the flowering stage, along with rhizospheric soils, from the districts of Bhakkar (31°37′30.90″N, 71°03′56.66″E) and Khushab (32°17′48.01″N, 72°21′9.00″E) in Punjab, Pakistan. These districts are located in lowland semi-arid plains at an elevation of 168 m above sea level, with an average temperature of 35°C (Fig. 1). C. arietinum and rhizospheric soils were collected in sterilised bags from different sites during the spring season (2019–2020). A total of 44 soil and root samples (20 from Bhakkar and 24 from Khushab) were gathered from 20 selected sites in these districts, at a depth of 10–15 cm. The samples were stored for a week at 4°C, while the root samples were kept at −20°C for molecular analysis.

Fig. 1.

Location of study sites in Districts (Khushab and Bhakkar) Punjab, Pakistan.

Physiochemical soil analysis

We analysed physiochemical features with the isolation of different AMF spores using the method described by (Trejo-Aguilar and Banuelos 2020).

Soil organic matter: we used the procedure of Schulte and Hoskins (1995):

Soil pH:  this was calculated as the negative log of hydrogen ion activity, determined by a pH meter (McLean 1983).

Soil moisture: we used filter paper to clean all of the Petri dishes for the soil moisture analysis. Weight of empty Petri dishes (without lid) (W₁) as well as 10 g soil sample (W₂) in Petri dishes were noted and measured initial weight before oven (W₃ = W₁ + W₂). This was placed in an oven at 106°C for 4 h and measured the weight (W₄ = after drying) (Sparks et al. 2020):

Soil texture: we used hydrometer values used to compute the percentage of sand, silt, and clay, which was used to determine the soil’s textural class according to the USDA soil textural triangle (Gee and Bauder 1986).

Electrical conductivity (EC): we measured the quantity of soluble inorganic salts in each amount of soil is referred to as salinity. An EC bridge is used to test salinity and to determine soil EC (Blake 1965).

Total soil nitrogen: nitrogen determination was made in three steps of digestion, distillation, and titration. In digestion, a mixture of 1 g CuSO₄ and 7 g K₂SO₄ in the ratio of 1:7 was used with a 0.2-g test sample. In distillation, 60 mL of 40% NaOH was dispensed into digested sample along with the 5-mL indicator of boric acid and 200 mL of 0.2% bromocresol for 10 min. Finally, the distillate was analysed for ammonium by titrating against 0.005 N HCl (Bremner and Mulvaney 1982):

AB-DTPA extractable P: we added 10 g of soil and 20 mL of the AB-DTPA solution, filtered the suspension and added 5 mL of the mixed ascorbic reagent. The phosphorus content was then measured at 880 nm using a spectrophotometer (Madurapperuma and Kumaragamage 1999).

AB-DTPA extractable K, Na, Ca, Mg; and micronutrients (Zn, Fe, Mn): 10 g of soil sample was suspended in  20 mL of the AB-DTPA solution. Flame photometer was used to calculate Na and K concentration while atomic absorption spectrophotometer was used to assess the extract’s micronutrient contents with Ca and Mg (McLean 1983):

Root assessment, spores population, isolation, and identification

Chickpea samples were assessed for root colonisation using the procedure described by Phillips and Hayman (1970). The roots were washed to remove soil particles, cut into 1 cm segments, and preserved in 70% formalin acetic acid (FAA). For measuring colonisation, the roots were cleared in 15% (w/v) KOH and placed in a water bath at 90°C for 25–30 min. After cooling, the roots were rinsed with water and stained with 0.5% (w/v) acid fuchsin. Finally, the roots from the collected samples were examined under a compound microscope to observe AMF colonisation. Percentage of root colonisation was calculated by the equation:

Wet sieving and decanting were used to separate coarse soil particles, while organic particles and AMF spores were retained on sieves with various diameters (40 µm, 100 µm, and 150 µm) (Gerdemann and Nicolson 1963). AMF diversity was identified based on spore size, shape, and colour (Perez and Schenck 1989). Single spores were observed and focused under a compound microscope for enumeration, with spore density calculated per 10 g of soil. The INVAM (International Culture Collection of Vesicular Arbuscular Mycorrhizal Fungi) reference species description was used for identifying AMF spore diversity (Gerdemann and Nicolson 1963; Giovannetti and Mosse 1980).

Plant analysis

A total of 1 g of weighed plant powder (WS) was recorded for its initial weight before oven drying (WPS = WP + WS). For ash determination in chickpea., a 1-g sample was placed in a crucible (Wcs) and heated in a furnace at 550°C for 4 h. The crucible with ash was then weighed (WCA), and the ash content was calculated as described by (Bremner and Mulvaney (1982).

Protein percentage in chickpea shoots was determined using the method described by Madurapperuma and Kumaragamage (1999), with protein measured by multiplying the percentage of nitrogen by a factor of 0.25.

For fat determination, 1 g of the sample was wrapped in filter paper and placed in a thimble. The Soxhlet extraction unit was set up with the sample flask containing 70% petroleum ether. The flask was placed in an oven to remove moisture and petroleum ether, with the drying temperature set at 110°C for 30 min (Bremner and Mulvaney 1982). The percentage of fat was determined (Eqn 6).

For fibre analysis, a 2-g sample was treated with 2% HCl solution (4 mL HCl + 196 mL distilled water) and heated in a water bath for 3 h. The mixture was filtered through muslin cloth to separate the residues, which were then treated with 2% NaOH solution for an additional 3 h in a water bath. The residues were transferred to a crucible and placed in an electric oven at 105°C for 3 h, followed by heating in a muffle furnace at 650°C for 3 h, according to the methods described by AOAC (2005).

Elemental analysis

For the analysis of macronutrient (P, K, Na, Ca, Mg) and micronutrient (Zn, Fe, Mn) concentrations, 1 g of the sample was placed in a flask with 10 mL of concentrated HNO3 and allowed to sit for 24 h. After this period, 4 mL of perchloric acid was added, and the mixture was heated until white fumes appeared. The digest was then diluted by adding 100 mL of distilled water and filtered using Whatman 42 filter paper.

A volume of 1 mL of the filtrate was used to measure phosphorus using a photometer, with phosphorus determined by a spectrophotometer at 880 nm. Potassium and sodium concentrations were assessed using a flame photometer, and micronutrient concentrations were measured by atomic absorption spectrophotometer, following the method described in Madurapperuma and Kumaragamage (1999).

Molecular analysis

PCR parameters

For primers AML1-AML2 and NS1-NS2, the PCR was run for 35 cycles with the following conditions: 95°C for 5 min, 94°C for 30 s, 55°C for 35 s, 72°C for 1 min, and a final extension at 72°C for 10 min. The PCR products were separated using 1.5% agarose gel for AML1-AML2 and 2% agarose gel for NS1-NS2, with 100 mL of 1 × TBE buffer and 3 μL of ethidium bromide. A 100 bp DNA ladder was used as a control. These parameters were applied to all primers except for the annealing temperature. The annealing temperature for Glomus1310-GLOM5.8 and GLOM5.8R-ITS1F was 55°C, while it was 59.5°C for GLOM5.8-ITS1F and 60.7°C for both ARCH1311F-ITS4R and ITS1F-ITS4R (Goswami et al. 2018). Technical replicates use gradient PCR to perform multiple PCR reactions from the same DNA sample, whereas biological replicates use DNA from particular samples that were previously identified visually (Ganguly et al. 2024). Multivariate ANOVA and s.e. at P < 0.05 were used for statistical analysis. Details of the different parameters studied in this research are in Fig. 2.

Fig. 2.

Schematic representation showing different parameters studied.

Physiochemical soil analysis

Chickpea samples were collected from various sites in the districts of Bhakkar and Khushab, Pakistan. Significant physicochemical soil analysis mean values from these sites are in Tables 1 and 2. Mean soil organic matter (SOM) values were 0.65% for Bhakkar and 0.52% for Khushab. Average pH of the 44 samples ranged from 6.12 to 7.88, indicating that the soil was neutral in nature. Observed pH values were statistically significant at P < 0.05, which was a key indicator of AMF spore diversity.

The soil textural class in both districts was loamy. In Bhakkar, the highest EC values recorded were 2.4 dS/m, 1.7 dS/m, and 1.5 dS/m from Tinda Thal, Umar Wali, and Zamey Wala, respectively. In Jandan Wala and Kallurkot, EC values were 1.5 dS/m and 2.7 dS/m, respectively. The concentration of soil nitrogen was high, with values of 5472 mg/kg in Bhakkar and 4510 mg/kg in Khushab, which were not significant (7.72) at P < 0.05.

Table 1 shows the significant relationships among the physicochemical properties of the soil samples from Bhakkar and Khushab. Pphosphorus concentration in Bhakkar soil samples was higher than Khushab, with a notable phosphorus concentration of 4.7 mg/kg measured at Umar Wali in Bhakkar, which had a high organic matter content of 1.12%. Low rhizospheric phosphorus standard is 15 mg/kg; thus both districts indicated phosphorus deficiency (Nair and Sollenberger 2022). Potassium concentrations were reasonable, with 134.6 mg/kg in Bhakkar and 188 mg/kg in Khushab. Other elements such as Ca (0.039), Fe (0.284), and Mg (0.080) showed significantly different results (P < 0.05) (Table 2).

AMF diversity

Diversity of AMF depends on spore sizes (AOAC 2005). For example, the size average range across replicates for Glomus spores was 10−40 μm, Acaulospora ranged from 125 μm to 149 μm, Sclerocystis ranged from 110 μm to 155 μm, and Gigaspora ranged from 65 μm to 112 μm. Among the collected samples, Glomus was the smallest in size but the most abundant genus (Fig. 3a–f).

Fig. 3.

Genera of AMF detected. (a–d, f) Glomus, (e) Acaulospora, (g) Gigaspora, (h) Scutellospora, (i) Sclerocystis, and (j) Sclerocystis.

Root colonisation

The intensity of root colonisation of chickpea by AMF varied among sampled sites due to differences in the physicochemical properties of the soil (Bhakkar, s.d. 14.31; Khushab, s.d. 17.50) (Perez and Schenck 1989). The Tinda Thal site in Bhakkar had a high root colonisation rate of 75.9%, while the Rakhutra site in Khushab showed a rate of 72.16%, which positively influenced the plant nutrients concentration (Table 3). Kallurkot samples exhibited similar rates of 18.41% and 16.34%, respectively. AMF structures, including arbuscules and vesicles, were identified in the collected samples. Levels of root colonisation varied randomly in both districts of Bhakkar and Khushabin (Figs 4, 5).

Fig. 4.

Root colonisation of chickpea by AMF in Khushab and Bhakkar districts, Pakistan.

Fig. 5.

Root colonisation structures of AMF in chickpeas showing external and internal hyphae, vesicles, and arbuscules in Bhakkar and Khushab, districts, Pakistan.

Spore densities

Spore densities of different genera of AMF were enumerated in soil samples collected from the districts of Bhakkar and Khushab (Fig. 6). Although spore abundances varied among sampling sites, the mean spore densities from both districts were nearly the same. The Kallurkot site in Bhakkar had a high spore density of 36 spores/10 g of soil, while a high spore count of 29 at the Quaidabad site in Khushab. Glomus spores were the most abundant in rhizopshere soils of chickpea.

Fig. 6.

AMF spore density in the rhizosphere soil of chickpea in Bhakkar and Khushab districts, Pakistan.

Plant analysis

Moisture content of chickpea samples varied slightly among sites in Bhakkar, with a mean value of 8.62%; the highest being 9.33% from the Jandan Wala site. In Khushab, mean moisture content was 9.20%, ranging from 9.33% (Shah Wala site) to 7.55% (Rakhutra site). Moisture values in Khushab were generally higher than Bhakkar.

Mean ash content of chickpea samples from Bhakkar was 1.72%; the highest was 4.31% at Zamey Wala and the lowest was 0.57% at Jandan Wala. There were differences in ash content among various sites in Bhakkar. In Khushab, the ash content ranged from 3.15% (Shah Wala) to 0.25% (Quaidabad), showing variations among the selected sites. The mean fat content in chickpea samples from Bhakkar was 1.16%. The Umar Wali site had a high fat value of 1.8%. Fat values varied less among Bhakkar sites, while a high value of 2.1% was obtained from the Rakhutra site in Khushab. In Bhakkar distract, mean fibre content was 3.78%, with highest at Jandan Wala (7.45%) and the lowest at Umar Wali (1.05%). In Khushab district, mean fibre content was 3.24%, with the highest at Rakhutra (5.05%) (Table 4). Similarities in ash, fat, and fibre content are illustrated in the standard error graph. Proteins and carbohydrates were also found to be significantly abundant (Fig. 7).

Protein content showed less variation among samples from Bhakkar, with the highest protein value of 22.75% obtained from Tinda Thal. In Khushab, protein concentrations were 17.5% at Adhikot and 10.5% at Shah Wala. The highest carbohydrate values were observed in samples from both Bhakkar and Khushab, with mean concentrations of 71.72% and 72.86%, respectively (Table 5 and Fig. 7).

Fig. 7.

Plant moisture, ash, fat, fibre, protein, carbohydrate content of chickpeas in Bhakkar and Khushab districts, Pakistan.

Elemental analysis

Mean nitrogen value was 2.98%, with a high of 3.64% recorded from Tinda Thal in Bhakkar. Kallurkot samples showed a similar nitrogen percentage of 1.54%, which is relatively low for Bhakkar. Mean nitrogen value in Khushab was lower than Bhakkar, with less variation observed among Khushab samples. The Adhikot site in Khushab had a high nitrogen value of 2.8%.

Mean phosphorus values were 32.98 mg/kg for Bhakkar and 0.58 mg/kg for Khushab, indicating that phosphorus concentration is low in Khushab. The Zamey Wala site in Bhakkar had a high phosphorus concentration of 68.20 mg/kg, while the Rakhutra site in Khushab had the highest concentration in samples from that district. Phosphorus content in shoots from Bhakkar was higher than Khushab.

Mean potassium value for Khushab samples was 43.06 mg/kg, higher than Bhakkar (33.32 mg/kg). Zamey Wala site had the lowest potassium content of 4.49 mg/kg in Bhakkar. The highest and lowest shoot potassium values, 89.22 mg/kg and 15.40 mg/kg were observed at the Noorpur and Adhikot sites, respectively, in Khushab.

Mean calcium value was 22.48 mg/kg in Bhakkar, with the highest shoot calcium concentration of 52.00 mg/kg at Zamey Wala. Mean magnesium values were 0.24 mg/kg in Bhakkar and 32.10 mg/kg in Khushab. Magnesium content showed little variation among Bhakkar sites, reflecting similar AMF root colonisation frequencies.

Mean sodium value was 24.50 mg/kg, with a high shoot sodium content of 66.22 mg/kg from Umar Wali and a low value of 2 mg/kg from Kallurkot in Bhakkar. Sodium content showed minimal variation among Khushab sites, with a mean of 15.95 mg/kg and similar values of 17.2 mg/kg observed at Adhikot, Noorpur, and Rakhutra. Rakhutra recorded a high sodium value of 15.8 mg/kg in Khushab.

Mean zinc concentrations were 15.8 mg/kg in Bhakkar and 11.8 mg/kg in Khushab. Umar Wali had the highest zinc value of 33 mg/kg among Bhakkar sites, while Noorpur had the highest zinc concentration of 15 mg/kg in Khushab. Noorpur and Quaidabad both had similar zinc contents of 14 mg/kg in their samples. Zinc content showed little variation among Khushab samples.

Mean iron values were 7.25 mg/kg for Bhakkar and 7.22 mg/kg for Khushab. Jandan Wala samples showed both high and low iron values of 11.9 mg/kg and 3.29 mg/kg, respectively. Iron content varied among sites in both districts.

Mean manganese value was 33.00 mg/kg for Bhakkar, with a high value of 57 mg/kg, while Zamey Wala had a low content of 13 mg/kg. Noorpur had a high manganese content of 64.40 mg/kg. Standard errors for nitrogen and phosphorus showed significant differences, while similar significant values were found for potassium, calcium, and manganese.

Detailed elemental analysis is in Table 3. Elemental significance levels were found to be similar for P, K and Mn (Table 6).

The relationship between soil spore densities collected from sites in Bhakkar and Khushab districts and plant elemental properties is in Table 7. Spore diversity positively affected the concentrations of plant elements. However, due to minimal differences in spore densities, there were not significant fluctuations in the plant elemental properties.

Molecular analysis of arbuscular mycorrhizae fungi in Cicer arietinum L

The ladder DNA (L) along primer sets AML1-AML2 generated bands at 700–900 bp, NS1-NS4 at 900–1100 bp, GLOM5.8-ITS1F at 700–1000 bp, Glomus 1310-GLOM 5.8 at 500–700 bp, and ITS1F-ITS4R at 1000–1100 bp (Fig. 8). AML1-AML2 confirmed the presence of AMF diversity in the roots of chickpea collected from both Bhakkar and Khushab districts. The phylogenetic tree illustrating the clades and distances among the AMF genera associated with the primers is in Fig. 9. The evolutionary history was inferred using the Neighbor-Joining method, and evolutionary distances were calculated with the Maximum Composite Likelihood method, expressed in terms of the number of base substitutions per site. The analysis included 24 nucleotide sequences, with ambiguous positions removed for each sequence pair (pairwise deletion option). The final dataset contained 1242 positions. Evolutionary analyses were conducted in MEGA11, producing a cladogram that shows the distances and different traits among the AMF genera Glomus, Archaeospora, Diversispora, Funneliformis, Acaulospora, and Scutellaspora.

Fig. 8.

AMF PCR bands (AML1 and AML2), AMF diversity PCR bands in different combinations of (GLOM5.8, ITS1F4, GOM1310, NS1 and NS4) primers.

Fig. 9.

Phylogenetic analysis of AMF species using AMF specific primers.

Physicochemical soil analysis and AMF

In chickpea farming, the application of biofertilisers based on AMF improves crop production, stress tolerance, and nutrient uptake. By lowering reliance on chemical inputs, AMF promotes sustainable farming while simultaneously increasing production and nutritional quality. Including regional AMF strains can increase efficacy even more, providing chickpea production systems with an economical and sustainable option (Pellegrino and Bedini 2014; Saurabh Jha and Songachan 2023). Evidence of increased nutrient absorption induced by AMF symbiosis has been documented (Wang et al. 2023). To explore the potential role of AMF in chickpea in its natural environment, our investigated physicochemical soil properties, AMF spore abundance, diversity, colonisation, and nutrient distribution within the plants. SOM concentrations and EC magnitudes in the districts of Bhakkar and Khushab were similar (Tables 1–2), consistent with previous reports (Krishnamoorthy et al. 2015; Khalid Chaudhry et al. 2016; El Hazzat et al. 2018; Makkar et al. 2018; Saeed et al. 2020). The pH of the investigated sites was neutral, which influenced the abundance of AMF with significance at P < 0.05 (Krishnamoorthy et al. 2015; Saeed et al. 2020). The study results support the view that AMF play a pivotal role in phosphorus acquisition, as the mean phosphorus concentration in Bhakkar soil (4.38 mg/kg) was higher than in Khushab (1.68 mg/kg), attributed to differences in the strength of AMF root colonisation. However, soil nitrogen levels in both districts were not significantly affected by AMF colonisation (Bhatt and Singh 2020; Saeed et al. 2020; Alimi et al. 2021) (Tables 1, 2).

Morphological analysis, root colonisation, and spore density of AMF in rhizospheric soil of chickpeas

Our morphological investigation showed variations in spore sizes among AMF taxa (Glomus, Acaulospora, Gigaspora, Sclerocystis) (Fig. 3), similar to the findings of El Hazzat et al. (2018) andDroh et al. (2023). The study identified Glomus as the dominant and smallest genus, consistent with previous results (Alguacil et al. 2003; Sasvári et al. 2011; Jacquemyn et al. 2011; Hipólito-Piedras et al. 2024) (Fig. 3). Differences in AMF chickpea root colonisation levels at various AMF sampling sites were attributed to the uneven distribution of soil physicochemical properties across the districts (Pepe et al. 2016; Ndeko et al. 2024). Our results highlighted the importance of AMF root colonisation for nutrient acquisition, particularly phosphorus. Variations in AMF root colonisation levels were observed with 40.56% in Khushab compared with 35.45% in Bhakkar (Houngnandan et al. 2009; Marizal and Syariyah 2017) (Figs 4, 5). Average AMF spore densities per 100 g of soil were 19.2 in Bhakkar and 15.6 in Khushab. The density of AMF spores at Bhakkar positively influenced the concentrations of N, P, Na, and Zn in plants of C. arietinum, which was also reported previoulsy (Diagne et al. 2020; Mulyadi and Jiang 2023) (Fig. 6, Table 7).

Plant and elemental analyses of chickpeas

Moisture, fat, and carbohydrate were positively influenced by AMF colonisation in Khushab compared with Bhakkar. Variations in plant ash content observed in this study differed from the findings of Mehrvarz and Chaichi (2008). Mean percentages of moisture, fat, and carbohydrate were 9.10%, 1.25%, and 72.8%, respectively, in Khushab compared with 8.62%, 1.16%, and 71.7%, respectively, in Bhakkar (Table 4). The impact of AMF on these plant components was statistically significant (P < 0.05), aligning with previous studies (Wu et al. 2006; Manoharan et al. 2008; Dobo 2022; Amir and Crossay 2024). Both districts showed similar effects of AMF on protein and fibre concentrations, as reported by (Lester 2009; Egberongbe et al. 2010; Sarah and Burni 2013; Samanhudi et al. 2014) (Tables 4–5). Results from elemental analysis were consistent with previous findings (Xia et al. 2007; Xie et al. 2022), highlighting the role of AMF in nutrient absorption.

Plant potassium levels varied between districts due to differences in AMF colonisation, with Bhakkar showing 33.02 mg/kg and Khushab 43.06 mg/kg (Bai et al. 2008; Subramanian et al. 2008; Naderi et al. 2010; Sajedi and Rejali 2011) (Table 3). N and P concentrations were not significantly affected by AMF in either district (Sajedi and Rejali 2011). P content in Khushab plant samples was notably lower compared to studies on Zea mays (Tian et al. 2004), Gossypium (Sharifi et al. 2007), and Glycine max (Yaseen et al. 2016). Higher calcium concentrations in Khushab compared with Bhakkar further indicated the influence of AMF frequency, as also reported by Ci et al. (2021) and Liu et al. (2019) for Arachis hypogea. Magnesium and sodium concentrations were positively influenced by indigenous AMF taxa, supported by findings from Arias et al. (2015) and Ramakrishnan and Selvakumar (2012), though this contradicted the work of Naderi et al. (2010). Zinc levels were 15.8 mg/kg in Bhakkar and 11.8 mg/kg in Khushab, differing from the study of Maffo et al. (2022) due to difference in AMF colonisation. Iron (Fe) concentrations in plant shoots across both districts under AMF influence have been previously reported (Arias et al. 2015). The standard errors for phosphorus showed significant differences, while potassium, calcium, and manganese also showed significant values. The interactions of AMF with magnesium, zinc, and iron were not significant in either Bhakkar or Khushab (Tables 3, 6). Because of current agronomic techniques, the potential of AMF as biofertilisers has not been completely utilised despite their accepted benefits (Beleri 2023). Increased crop yields and sustainability can result from combining several legume species with AMF, especially in low-input agricultural systems (Zhao et al. 2022).

Molecular analysis of AMF in the roots of chickpeas

Molecular and morphological analyses indicated that Glomus was the predominant AMF genus sporulating with chickpeas in both districts of Punjab. The amplification of the 18S rDNA gene of the small subunit from the chickpea root samples was performed using specific primers (Fig. 8). The goal of the gradient PCR was to isolate different fragments of AMF rDNA based on their size. The primers used enabled the amplification of gene fragments approximately 1100 bp in length. Our results showed that the root samples contained AMF sizes ranging 250–1100 bp (Lee et al. 2008). There was consistency between molecular and morphological investigations in identifying the genera. Primers AML1 and AML2 efficiently amplified AMF sequences from chickpea  roots, unlike primers NS1-NS4, GLOM5.8, ITS1F4, and GLOM1310 (Ogoma et al. 2021; Noreen et al. 2023). Phylogenetic analysis indicated that all sequences belonged to the phylum Glomeromycota. The resulting cladogram (Fig. 9) included 10 taxa and 21 species. This included one taxa each from Scutellospora, Gigaspora, Entrophospora, Dentiscutata, Diversispora, and Sclerocystis, along with three from Archaeospora and six each from Acaulospora and Glomus (Noreen et al. (2023).

Conclusions

Plant analysis of chickpea showed significant variations in nutrient composition across sites in Bhakkar and Khushab districts. Moisture content was slightly higher in Khushab, averaging 9.20%, compared with 8.62% in Bhakkar. Ash content varied significantly, ranging from 0.57% to 4.31% in Bhakkar and from 0.25% to 3.15% in Khushab. Fat content was relatively consistent in Bhakkar with a mean of 1.16%, while the highest value of 2.1% was recorded in Khushab. Fibre content showed more variation in Bhakkar with the hightest at 7.45%, whereas Khushab had a slightly lower mean value of 3.24%. Protein and carbohydrates were also found to be significantly abundant, highlighting the nutritional richness of the samples. These findings suggest site-specific differences that may be influenced by environmental conditions and AMF activity. Elemental analysis of chickpeas showed higher nitrogen (up to 3.64%) and phosphorus (32.98 mg/kg) in Bhakkar, while Khushab had more potassium (43.06 mg/kg) and magnesium (32.10 mg/kg). Calcium, sodium, and zinc were higher in Bhakkar; iron levels were similar in both districts (~7.2 mg/kg). Manganese was high in both, reaching 64.40 mg/kg in Khushab. Nutrient levels varied notably across sites. Molecular analysis of the AMF association with soil from Bhakkar and Khushab revealed the presence of 21 AMF taxa, with two genera, Glomus and Acaulospora, identified as the most dominant. This is the first detailed study of AMF and chickpea in these districts of Punjab, Pakistan. Our findings showed the strength of indeginous AMF association with chickpea in natural fields of Punjab, Pakistan with a sign of future work. Our results indicated that mineral concentrations were the primary factor influencing AMF diversity and distribution patterns. The amplification of the 18S gene of AMF rDNA confirmed that chickpeas hosted AMF. The choice of sampling sites was crucial for promoting the establishment of mycorrhizal symbiosis, which is beneficial for chickpea cultivation. AMF colonisation in chickpeas accelaerated the intake of nutrients, particularly P, K and Ca. It is recommended that developing AMF inoculums on a commercial scale could be a transformative step in biofertiliser production, potentially enhancing agricultural productivity. Farmers should also supply commercial-grade AMF biofertiliser inoculum that is cast-free.