Agriculture lands have been significantly depleted of fertility as important plant nutrients (mainly nitrogen and phosphorus) are not easily available under dry and semi-arid conditions. Nutritional nitrogen and phosphorus are each significant macronutrients in plant growth (Hag Husein et al., 2021). Applying mineral fertilizers to improve soil characteristics faces many challenges, especially in Iraqi soils due to high carbonate concentration. This carbonate causes phosphorus to precipitate, converting it into calcium phosphate, which is not available for plants and thus reduces its availability. Nitrogen is subjected to leaching or conversion into gaseous forms including denitrification and ammonia volatilization, diminishing the fertilizer effectiveness of N (Anitha et al., 2022).
       
The fungus Gigaspora sp. is an important root-beneficial fungus, forming a mutualistic relationship with plant roots. During symbiosis, the fungus receives carbohydrates and energy from the host plant. The fungal hyphae track network increases the surface area for nutrient absorption from soil (Alkobaisy et al., 2020). Fungi are primary mediators of increasing phosphorus accessible by converting insoluble phosphorus to available forms due to production of acids and phosphate-soluble organic compounds within the rhizosphere (Al-Baldawy et al., 2019).
       
Organic fertilizers are defined as substances derived from the microbial degradation of organic matter and animal manure at various stages. They are among the most significant natural resources for enhancing soil fertility and maintaining its production Dey et al., (2024). They enhance the physical, chemical and biological characteristics of the soil by augmenting its organic matter content and improving its structure and capacity to retain water and nutrients. Moreover, they stimulate advantageous bacteria and directly provide nutrients to the plant, enhancing the biological processes that transform nutrients into forms readily assimilated by the plant (Havlin et al., 2016).
       
Subathra and Devika (2025) conducted a comparative study on the effect of biochar produced via hydrothermal liquefaction of multiple waste streams (including sewage sludge/wastewater) on the growth and productivity of tomato, pepper and okra. The compared its performance with chemical fertilization and commercial biofertilizers and found that biochar produced by this method can function as an effective biofertilizer for enhancing crop yield.
       
Research indicates that incorporating phosphate fertilizer into the soil enhances soil chemical composition and fertility, thereby augmenting the uptake of vital nutrients such as nitrogen, phosphorus and potassium, particularly at elevated application rates (Raj and Kumar, 2025). Lettuce (Lactuca sativa L.) is a leafy vegetable of significant nutritional and commercial value. It is used for human and animal consumption. Historically, farmers have utilized sewage sludge as a fertilizer for this crop because of its elevated levels of macro and micronutrients.

In this study, a field experiment was conducted in Al-Daghara. The field was disked twice and leveled, followed by an amendment to the soil. The space was divided into 3 sections, consisting of 18 experimental units. From the field, a 0-30 cm depth composite soil sample was obtained and thoroughly blended to ensure homogeneity. The material was then air-dried and processed through a 2 mm sieve for subsequently physical, chemical and biological investigations (Table 1).


       
The experiment consisted of three variables: Biofertilizer [Inoculated with Gigaspora sp., 30 (G₁) and no inoculation (G₀)], sewage sludge [at 0 (W₀), 2 t ha^-1 (W₁) and 4 t ha^-1 (W₂) and mineral fertilizer [at 0 (D₀), 50% (D₁) and 100% (D₂)]. 45 days after planting, 3^rd was during flowering. The NPK fertilizer applied had composition of 0-46-18 (N-P-K). The field was established in accordance with the Randomized Complete Block Design (RCBD) (Bozóki et al., 2025). Least significant difference (LSD) was used at a significance level of 0.05. The studied traits are:
 
Mycorrhizal infection rate
 
The infection rate was evaluated during the flowering and end-of-season stages. Plant root systems were randomly selected from each experimental unit and washed. The roots were cleaned with a narrow stream of water to remove any associated soil. After that, roots with a length of 1 cm were placed in test tubes. Roots were treated with a 10% KOH solution in the tubes. The tubes were then placed in a 90°C water bath for 30 to 60 min. After that, they washed with distilled water and soaked in the 10% alkaline hydrogen peroxide (H₂O₂) solution for 15-60 minutes. Ten per cent hydrochloric acid (HCl) was given for a duration of 3 minutes. So, the staining process used formalin aceto alcohol (FAA) solution to kept microorganism structures from morphometric changes. Then, the stained hairs were subjected to staining in the acid fuchsin solution during 15 to 20 minutes at a temperature of 90°C. The materials were then extracted with the addition of lactic acid. Each of the 10 dyed root slices (1 cm long) on a glass slide were examined microscopically. The percentage of root hair infection was then calculated as follows:

 
The ratio was calculated using the methodology established by Kormanik et al. (1980).
 
Number of spores
 
The spore count was determined by weighing 10 g of soil containing root hairs (the root zone) at two intervals: blossoming and the season’s conclusion. Subsequently, 100 ml of distilled water was added to the test tubes, which were then centrifuged at 4000 rpm for 5 minutes. Subsequently, the contents of the test tubes were disposed of and the precipitate was retrieved. A 50% sugar solution was created by adding 100 ml of distilled water to 100 g of the sugar and distributing it evenly among the tubes. The solution was subsequently centrifuged at 4000 rpm. The filtrate was collected and filtered in series through a sieve of (25, 35, 100 and 175) µm pore size sieves to remove residual contaminants. Each sieve was separately collected and placed into glass dishes. The sugar was eliminated by washing with distilled water and this process was performed three times. Afterwards, one milliliter of the purified solution was placed on a glass slide to count spores in 10 g soil, according to Gerdemann and Nicolsin (1963).
 
Dry weight of vegetative components
 
Vegetative components dry weight was based on cutting respective vegetative parts. We estimated the dry weight of the foliage.
 
Total yield
 
The overall yield was determined by the formula:

Fungal infection rate
 
The perforation in the Gigaspora sp. significantly reduced the incidence during the blossoming and end-of-season stages by 64.019% and 56.221%, respectively. The control treatment calculated averages of 40.161% and 36.504%. This is in line with Al-Anzi (2024). Fungal infection achieved significantly higher levels when treated with organic fertilizer as depicted in table.
       
The application of organic fertilizer at 2 t ha^-1 (W₁) produced average values of 54.873% and 49.711% during the blossoming and end-of-season stages, respectively. The control treatment exhibited average values of 47.173% for flowering and 40.457% for the end-of-season stages, respectively This is consistent with Al-Shabani (2005).
       
Table 2 indicates that mineral fertilization significantly influenced the results, with values rising under the treatment using 100% of the fertilizer guideline (D₂), achieving 58.176% and 52.187% during the flowering and end-of-season phases, respectively. The control treatment exhibited average values of 46.927% for the flowering stage and 41.349% for the end-of-season stage. This is consistent with the findings of Sylvia and Neal (1990).


       
The data indicate that the interaction between Gigaspora sp. and mineral fertilizer exerts a considerable effect. Treatment G₂D₂ had the highest infection rates during the flowering and end-of-season phases, with 73.761% and 62.801%, respectively. The control treatment exhibited average rates of 37.618% at the flowering stage and 31.804% at the end-of-season stage this is consistent with Garmendia et al. (2005).
       
The table indicates that the interaction between organic and mineral fertilizers has an impact. The fungal infection values rose due to treatment (W₂D₂) to 64.615 and 56.260, respectively, during the flowering and end-of-season stages. Conversely, the control treatment had average values of 43.112 and 35.017 for the flowering and end-of-season stages, respectively this is consistent with Al-Nuaimi (1987).
       
The three-way interaction exerted a significant influence, with treatment (G₁W₂D₂) demonstrating the most pronounced effect, with average values of 84.463 and 69.573 for the flowering and end-of-season stages, respectively. The control treatment exhibited average values of 32.570 and 25.243.
 
Number of spores
 
The inoculated plants with Gigaspora sp. exerted a considerable influence. This application increased the number of spores to 45,539 and 49,120 spores per 100 g of soil for flowering and end-of-season stages. The control treatment yielded 31.860 and 34.633 spores per 100 g of soil, respectively. This is consistent with (Ahamad et al., 2023).
       
Table 3 indicates that organic fertilization influenced spore count. Treatment W₂ markedly elevated the spore count at both the flowering and end-of-season phases, yielding averages of 45.712 and 42.511 spores per 100 g of soil, respectively. The control treatment produced 36.410 and 33.053 spores per 100 g of soil. This is due to the enhancement of soil moisture and fertility by the application of organic fertilizers. This subsequently amplifies the activity of beneficial bacteria and enhances their efficacy in performing essential functions, resulting in a higher spore count. This is consistent with Al-Shabani (2005).


       
The (Table 4) indicates that mineral fertilization markedly elevated spore count. Treatment (D₂) markedly elevated the spore count at both the flowering and end-of-season phases, resulting in 43.044 and 46.850 spores per 100 g of soil, respectively. The control treatment exhibited average values of 37.831 and 34.951 spores per 100 g of soil at the flowering and end-of-season stages, respectively. This aligns with Tanwar and Aggarwal (2012).        

                                                    
       
Our data demonstrate an effect of the interaction between Gigaspora sp. and mineral fertilizer is very important for the increase of number spores. Treatment G₁D₂ achieved a significant increase in spore amount, with an average of 56.537 spores and 51.863 per 100 g soil, respectively for the flowering and end-of-season stages (Table 3). The average values of the control treatment for flowering and end-of-season stages were 32.206 spores per 100 g of soil and 29.480 spores per 100 g of soil, respectively.
       
As can be seen with (Table 5) the interaction between organic and mineral fertilizers (W₂D₂) exercised a highly significant influence on all observations, producing 55.082 and 49.600 spores per 100 g of soil for flowering phase and end-season phase treatments, respectively. The averages for the control treatment are 33.893 and 30.315 spores per 100 g of soil in flowering and end-of-season soil samples, respectively consistent with the findings of Turk et al., (2006).


       
The triple treatment interaction demonstrated a substantial impact, with treatment G₁W₂D₂ exhibiting a markedly elevated average spore count of 67.633 and 59.563 spores  100 g of soil, compared with the control treatment, which averaged 28.440 and 25.367 spores  100 g of soil, during both the blooming and end-of-season phases, respectively.
 
Dry weight of the vegetative parts
 
Inoculation with the fungus Gigaspora sp. significantly increased the dry weight of vegetative parts. The average dry weight of the vegetative parts increased significantly with G₁ treatment, with values equal to 62.514 g per plant compared to 47.142 g per plant in the control supported by the observations of Utobo et al., (2011).
       
The results in (Table 6) showed that organic fertilization significantly increased the vegetative mass of the plant. The W₂ was particularly remarkable and had an average vegetative biomass of 62.121 g per plant over the course of the study. The control treatment produced an average of 48.661 g plant^-1, according to Sumner (2000).


       
The results showed that the application of mineral fertilizers significantly increased dry weight of vegetative mass. Highest values were noted during the treatment D₂, with an average 65.308 g per plant. Results indicate an average yield of 43.388 g per plant for the control treatment, according to the Nora et al. (2017). The data show that the fungus Gigaspora sp. and the amount of mineral fertilizer applied has a great impact on the vegetative dry weight. In the same way, G₁D₂ (interaction treatment) was 76.343 g and the control treatment was 39.747 g. This concurs with important implications of (Hag Husein et al., 2021).
       
The interaction between organic and mineral fertilizers using analysis of variance significantly affects, as follows: 22 bulek treated W₂D₂ indicates an average plant weight of 76.855 g per plant than the dry weight control leaves (36.177 g). That is in line with the findings of Koodi et al., (2017).
       
There was a significant increase in vegetative dry weight (Table 2) from the triadic interaction among treatments. Treatment G₁W₂D₂ produced a mean plant weight increase of 91.000 g compared to the control treatment at 34.630 g.
 
Total yield
 
The (Table 7) shows strong effects of inoculation with the fungus Gigaspora sp. on yield enhancement. The yield at the control treatment was 55.070 t ha^-1 while treatment G₁ provided a significant increase of production with 64.810 t ha^-1. This finding is in line with the findings by Dey et al., (2024). This tabular summary(7) shows that the yield increase was significantly affected by organic fertilizer. Based on comparisons with the control-identified as 42.500 t ha^-1 the yield obtained with treatment W₂ was greater by 70.760 t ha^-1. The production was significantly increased due to the mineral fertilization. This treatment was equivalent to 83.630 t ha^-1 higher in yield than that of the control, which averaged 40.840 t ha^-1 as also observed by Jassim et al., (2014). The data show that Gigaspora sp. fungus interaction results in such cells and mineral fertilizer significantly increases yield. Yield over time It has been shown that G₂D₂ (interaction treatment) yield of 83.940 t ha^-1 was higher than the control treatment (G₁D₁ which produced yield 33.470 t ha^-1). That goes along with similar fact, showing up in the same work of Al-Samarrai and Al-Tamimi (2018).


       
The interaction between organic and mineral fertilizers leads to significant increases in production. Under the interaction treatment W₂D₂, the average yield was 97.650 t ha^-1 and the one from control treatment was 22.840 t ha^-1 as has been noted by Al-Sahaf and Ati (2007). G₁W₂D₂ triple interaction had a significant 98.050 t ha^-1 increase in production over control treatment with an average of 10.620 t ha^-1.

The effects of Gigaspora sp. visitation, sewage sludge applications and NPK fertilization are reported with respect to mycorrhizal infection level, spore density, vegetative dry weight in lettuce plants obtained from soil-transplant system and yield. Results were even better for the combination of all factors, with the triple interaction (G₁W₂D₂) producing the highest values, showing that integrated fertilization is an important strategy to promote sustainable lettuce production.

We, the authors of the manuscript submitted for publication in the Agricultural Science Digest, hereby declare that we have no conflict of interest to disclose. We confirm that there are no financial, personal, professional, academic, institutional, or other relationships that could be perceived to influence the research, interpretation of data, manuscript preparation, or publication decision related to this work. We further confirm that we have received no external funding for this research.