The Effect of Inorganic and Microbial Fertilizer Applications on the Grain Yield and Some Agronomic Characters of the bean (Phaseolus vulgaris L.)^##

The application of inorganic fertilisers is regarded as a prerequisite for enhancing the efficiency of agricultural production. However, the variation in the types and application amounts of inorganic fertilizers applied and the lack of knowledge about fertilization negatively affect the health of living beings and the environment. The overuse of chemical fertilizers hardens the soil, reduces soil fertility, pollutes air, water and soil and lessens important nutrients of soil and minerals, thereby bringing hazards to environment. (Phalvi et al., 2021). Despite high yield and quality in the short term, the use of synthetic fertilizers leads to a decrease in the biodiversity of agricultural soils (Tripathi et al., 2020). It causes the deterioration of soil aggregate structure and carbon sequestration (Gupta et al., 2020). As a result, it reduces soil fertility and contributes to desertification (Huebner, 2023). Therefore, the use of biological fertilizers has become a phenomenon that not only reduces the damage caused by synthetic fertilizers to the soil and the environment, but also provides the nutrients necessary for plant growth (Fasusi et al., 2023). The use of biofertilizers can provide quality products for human consumption by reducing chemical residues and at the same time reduce the risk of environmental pollution. However, the increase in chemical fertilizer prices and the addition of organic fertilizers, especially microbial biofertilizers, to the soil in order to protect the soil ecosystem have made it necessary to minimize the use of chemical fertilizers (Saikia et al., 2018).
       
Biofertilizers mostly consist of N fixing, P solubilizing and plant growth promoting microorganisms. These bacteria support root development with the N they fix and increase resistance to stress conditions. They promote plant development and growth through plant hormone and vitamin synthesis by increasing mineral uptake (Ajaykumar et al., 2022).
       
Microbial fertilizers containing rhizosphere bacteria that promote plant growth increase plant development and yield by providing plant nutrient uptake to the plant or by increasing the the uptake of plant nutrient elements, producing some plant hormones or by inhibiting pathogen growth (Javorekova et al., 2015).
       
Studies have shown that biofertilizers may not completely replace synthetic fertilizers, but are quite effective in facilitating reduced fertilization. Some research findings indicate that biofertiliser strains may potentially yield higher crop yields and improved quality when compared to the use of synthetic fertilisers. In such circumstances, the application of biofertilisers may offer a solution, providing additional benefits. From a broader perspective, biofertilizer strains have many advantages over synthetic fertilizers and significantly overlap with the sustainability vision. The aim of this study was to determine the effects of biofertilizer strains on yield and some agronomic characters of bean by comparing their performance with plants subjected to synthetic fertilizer and rhizobium inoculation in the conditions of Van Province in the Eastern Anatolia Region of Turkey.

Location and characterization of experimental area
 
Van province, where the research was conducted, is located in the Turkey’s Eastern Anatolia Region, between 37^o-39^o north latitude and 43o-45o east longitude and its altitude is 1.726 m. First year of the study (2020) was conducted in Van-Tuşba, within the Field Crops Department at Van Yüzüncü Yýl University’s Faculty of Agriculture. The subsequent phase of the study, conducted in 2022, carried out during the summer under irrigated conditions in the farmer’s field in Atalan village, located Gevaş district of Van. The 2021 trial was cancelled due to the lack of data due to the negativities experienced. In accordance with the long-term average, precipitation in Van-Tuşba reached a total of 94.3 mm, while Van-Gevaş experienced an average precipitation level of 119.2 mm. Additionally, the average temperature and relative humidity in the area were recorded at a temperature range of 19.2-17.3^oC and a humidity range of 45.8-56.5%, respectively. In the first year of the research, average precipitation during the growing season was recorded as 74.7 mm. In the same period, temperature was 19.7°C and the average relative humidity was 46.1%. While the relative humidity and average temperature data for the growing season were higher than the long-term average data, total precipitation remained below the long-term average. In the second year, average temperature and precipitation were below the long-term averages.
 
Experimental design
 
Akman 98 bean variety was used as seed material in the study. The experiment was carried out in the split plots in randomized blocks trial design with three replications in a 7 x 3 scheme. The treatments were composed of inoculations and reduced doses of NP during seeding and coverage fertilization. Microbial inoculations (N= inoculation with B. atrophaeus (Nitrogen fixing bacterial strain, TV 126C) (2.2-8.8 x 10⁶ cfu mL^-1); R= inoculation with R. gallicum (1 kg bacteria to 100 kg seeds); P= inoculation with Bacillus-GC group (Phosphate-solubilizing bacterial strain, TV119E) (2.2-8.8 x 10⁶ cfu mL^-1); NR= co-inoculation with B. atrophaeus + R. gallicum; PR= inoculation with Bacillus-GC group + R. gallicum; RN= inoculation with Bacillus-GC group + B. atrophaeus; NPR= inoculation with B. atrophaeus + Bacillus-GC group + R. gallicum) and reduced doses of NP (Ammonium sulfate (21%) and triple super phosphate (TSP) (42%) during seeding and coverage fertilization (unfertilized=control; NP50=50% of the recommended dose, 20 kg N/ha^-1,30 kg/ha^-1 P₂O₅); NP100=100% of the recommended dose of two parts, 40 kg N/ha^-1,60 kg/ha^-1 P₂O₅).
 
Installation of field research
 
The study was conducted for 2 years under irrigated conditions in the 2020 and 2022 growing seasons. Since data could not be obtained in 2021, it was excluded from evaluation. The trial plots were 3 m long, 2.5 m wide and consisted of five rows of plants. The planting density was arranged to be 100 kg seeds per hectare. In the trial, inorganic fertilizers were applied to the main plots, while microbial fertilizer combinations were applied to the subplots. Before planting, all of the P and half of the N from the inorganic fertilizers applied to the trial plots and incorporated with a rake. The remaining half of the nitrogenous fertilizer was applied at 30 days after planting.    

The field trial, which was repeated for two years, started on May 15, 2020 in the first year and May 20, 2022 in the second year. In the trial, weed control was carried out by hand plucking twice in the first trial year and three times in the second trial year until the flowering period. Thinning was done with the first hoe and throat filling was done with the second hoe. Irrigation was done regularly at certain intervals with the drip irrigation system. Since no disease or pest was detected, chemical control was not needed. Harvesting was done manually in a 3.0 m2 area in each plot between August 28 and September 03, 2020 for the first trial year and between August 30, 2022 and September 06, 2022 for the second trial year, once the plants turned yellow and the pods began drying.
 
Statistical analysis
 
Variance analyses of the data obtained from the study were performed according to the Split Plots in Randomized Blocks Trial Design with three replications. The results of the analysis of variance were evaluated according to the F test and the significant difference were compared and grouped according to the LSD multiple comparison test (IBM SPSS 22.0).

Inorganic and microbial fertilizer applications had an effect on plant height, but this effect was found to be statistically insignificant (P>0.05) in both years. According to inorganic fertilizer averages, the highest plant height value was obtained from NP100 (40 kg N/ha^-1,60 kg/ha^-1 P₂O₅) in both years, while the lowest was obtained from control application. In microbial fertilizer averages, the highest values were determined in NR (B. atrophaeus + R. gallicum) in the first year, PN (Bacillus-GC group + B. atrophaeus) in the second year and the lowest values were determined in R (R. gallicum) application. In both years of the study, the shortest plant height was obtained from the Control + R
(R. gallicum) fertilizer combinations and the longest plant height was obtained from the first year NP100 (40 kg N/ha^-1,60 kg/ha^-1 P₂O₅ )+NR (B. atrophaeus+R. gallicum) and the second year NP100 (40 kg N/ha^-1,60 kg/ha^-1 P₂O₅ )+PR (Bacillus-GC group+R. gallicum) fertilizer combinations (Table 1). Plant height is a hereditary trait, can be affected by the environmental conditions in which it is grows. According to Sozen  et al. (2021), variation in plant height can can ocur when beans are grown with different applications in the same environmental conditions, leading to differences plant heights across plant subjected to different fertilizer combinations.


       
Researchers who obtained similar results with the study, Doğan and Çýğ (2023), reported that plants achieved greater heights with microbial, inorganic and organic fertilizers, while the shortest plant height was detected in control application and that fertilizers had a positive effect on plant height. Soysal and Erman (2020) and Yolci and Tunçtürk (2022) reported that the effect of microbiological fertilizer applications was significant. Özsoy Altunkaynak and Ceyhan (2018) reported that different N and bacteria applications led to increased plant height compared to the control and a certain increase was obtained whwn N was combined with bacterial application. Additionally, Çağlaret_al(2024) reported that the highest plant height was obtained from chemical fertilizer applications with P-solubilizing and N-fixing bacteria and the lowest from control application.
       
In microbial fertilizer applications, the highest first pod height was obtained from NPR (B. atrophaeus+Bacillus-GC group+R. gallicum) in both years, the lowest from N (B. atrophaeus) in the first year and P (Bacillus-GC group) in the second year. In inorganic fertilizer applications, the highest first pod height was obtained from NP50 (20 kg N/ha^-1, 30 kg/ha^-1 P₂O₅ ) in both years and the lowest from control applications (Table 1). First pod height is especially important under conditions where harvesting will be done by machine. Therefore, harvest losses in beans are suitable for mechanized harvesting and where the first pods form at high altitudes are expected to be minimal (Elkoca and Çýnar, 2015). Similar to the study, there are researchers who stated that the difference between bacterial applications is statistically significant in terms of first pod height (Bulut, 2013; Ozaktan et al.,  2020; Soysal and Erman 2020), while other researchers found no significant effect (Akman, 2017). As seen in Table 1, statistically significant (P≤0.01) differences were found between microbial and inorganic fertilizer applications in the second year. The highest first pod height value (10.87 cm) was obtained with the NP100 (40 kg N/ha^-1, 60 kg/ha^-1 P₂O₅)+NPR (B.atrophaeus+Bacillus-GC group+R. gallicum) application, while the lowest (8.40 cm) was obtained in the control+PR (Bacillus-GC group+R. gallicum) application (Fig 1a).
       
According to microbial fertilizer averages, the lowest number of main branches in 2020 was observed with PR (Bacillus-GC group+R.gallicum) application and in 2022 it was with R (R. gallicum) applications and in inorganic fertilizer averages, the lowest was in control and the highest was in NP100 (40 kg N/ha^-1,60 kg/ha^-1 P₂O₅) applications in both years of the study. According to the application combinations, the lowest number of main branch were determined in control+R in the first year, the highest in the NP50 (20 kg N/ha^-1,30 kg/ha^-1 P₂O₅)+N (B.atrophaeus); the lowest in the NP₅₀ (20 kg N/ha^-1,30 kg/ha^-1 P₂O₅)+NPR (B.atrophaeus + Bacillus-GC group+R. gallicum) and the highest in the NP100 (40 kg N/ha^-1,60 kg/ha^-1 P₂O₅)+PR (Bacillus-GC group + R.gallicum) applications in the second year (Table 1). Bulut (2013) and other researchers (Akman, 2017; Gautam et al., 2024) reported that bacteria application had a significant effect on the number of branches in  plants.
       
According to microbial fertilizer averages, the highest number of pods per plant was detected from N (B. atrophaeus) in the first year and in the second year from PN (Bacillus-GC group+B. atrophaeus); and in inorganic fertilizer averages, the highest number of pods per plant was detected from NP100 (40 kg N/ha^-1, 60 kg/ha^-1 P₂O₅) applications in both years (Table 1). In terms of inorganic and microbial fertilizer combinations, the highest number of pods per plant was detected in NP100 (40 kg N/ha^-1,60 kg/ha^-1 P₂O₅)+NR (B. atrophaeus+R. gallicum) application in both years, while the lowest was in control+R (R. gallicum) applications in the first year and in control+N (B. atrophaeus) applications in the second year (Table 1). As seen in Table 1, there were significant (Pd≤0.01) differences between microbial and inorganic fertilizer applications in 2020. The highest value was obtained in NP100 (40 kg N/ha^-1, 60 kg/ha^-1 P₂O₅) + NR (B. atrophaeus + R. gallicum) and the lowest in control+N (B. atrophaeus) applications (Fig 1b). In bean cultivation, the number of pods per plant is one of the important criteria that always positively affects yield (Sozen et al., 2021). Doğan and Çýğ (2023) and other researchers (Soysal and Erman, 2020; Akman, 2017 and Aldemir et al., 2019) reported that microbial and inorganic fertilizer applications significantly effected in the number of pods per plant compared to the control. Sonkarlay et al., (2020) reported that when P was applied alone or in combination with organic fertilizer and biofertilizer, the number of pods per plant increased significantly compared to the control. When the application combinations were examined, the minimum number of grains per pod occurred in NP₅₀ (20 kg N/ha^-1,30 kg/ha^-1 P₂O₅) +PR (Bacillus-GC group + R. gallicum) in 2020, the maximum in NP₅₀ (20 kg N/ha^-1,30 kg/ha^-1 P₂O₅)+N (B. atrophaeus) and the minimum in NP₅₀ (20 kg N/ha^-1,30 kg/ha^-1 P₂O₅) +NPR (B. atrophaeus+ Bacillus-GC group + R. gallicum) and the maximum in control+PR (Bacillus-GC group+R. gallicum) applications in 2022. The difference in terms of application averages and combinations was found to be insignificant (Table 2). Researchers who stated that PGPBs increased the number of grains in pods (Galindo et al., 2022; Ma et al., 2019; Bulut, 2013; Akman, 2017) reported that inorganic fertilizers did not significantly increase the number of grains in pods (Şahin, 2018).


       
According to microbial fertilizer averages, the maximum 100-grain weight was obtained from the N (B. atrophaeus) application, while the minimum was from the R (R. gallicum) application in the first year of the study. In the second year the maximum 100-grain weight was obtained from the R (R. gallicum) and the minimum from the N (B. atrophaeus) applications. The difference between microbial applications in the first year was statistically significant (P≤0.01), while in the second year, the difference was insignificant (P≤0.01) both microbial and inorganic applications. According to the inorganic fertilizer applications average, the 100-grain weight was lowest in control treatment in the first year and in the NP₅₀ (20 kg N/ha^-1,30 kg/ha^-1 P₂O₅) second year while it was in the NP50 (20 kg N/ha^-1,30 kg/ha^-1 P₂O₅) in the first year and in the NP100 (40 kg N/ha^-1,60 kg/ha^-1 P₂O₅) second year (Table 2). In the application combinations, the highest 100-grain weight (25.15 g) was obtained from NP100 (40 kg N/ha^-1,60 kg/ha^-1 P₂O₅)+N (B. atrophaeus) and the lowest (20.83 g) from control+R (R. gallicum) (Fig 1c). The 100-grain weight, a critical factor affecting the yield and market quality in beans, is influenced by the hereditary characteristics of the plant and the environmental conditions under which it grows. In some researchers (Bulut, 2013; Akman, 2017) reported that microbial fertilizers positively impacts 100-grain weight, while Doğan and Çýğ (2023) reported that they did not. However, Şahin (2018) reported that inorganic fertilizers increased did not affect 100-grain weight significantly, while Doğan and Çýğ (2023) reported that they was affect significantly. The researchers’ findings support our findings.
       
According to the 2020 microbial fertilizer averages, the highest grain yield (2996.7 kg/ha) was observed in the NR (B. atrophaeus+R. gallicum) treatment, while the lowest (1925.3 kg/ha) was in the PR (Bacillus-GC group+R. gallicum) treatment. In the second year, the highest yield (3759.4 kg/ha) was in the PN (Bacillus-GC group+B. atrophaeus) application and the lowest (2450.6 kg/ha) in the N (B. atrophaeus) application. For inorganic fertilizers averages, the highest grain yield in both years (2719.0 and 3653.0 kg/ha, respectively) was obtained from the NP100 (40 kg N/ha^-1,60 kg/ha^-1 P₂O₅) and the least (2390.3 kg/ha) was obtained from control in the first year and NP50 (20 kg N/ha-1,30 kg/ha^-1 P₂O₅) (2923.8 kg/ha) application in the second year (Table 2). As seen in Table 2, microbial and inorganic fertilizer interactions were significant in both years. The highest value was obtained from NP100 (40 kg N/ha^-1,60 kg/ha^-1 P₂O₅) +NR (B.atrophaeus +R.gallicum) (3482 kg/ha in 2020 and 4497 kg/ha in 2022,) while the lowest value (1737.3 kg/ha in 2020) was obtained from NP50 (20 kg N/ha^-1,30 kg/ha^-1 P₂O₅) +PR (Bacillus-GC group +R.gallicum) and (2124.7 kg/ha in 2022) from NP₅₀ (20 kg N/ha^-1,30 kg/ha^-1 P₂O₅)+N (B.atrophaeus) (Fig 1d). When Table 2 is analyzed, according to microbial fertilizer averages in 2020, the highest biological yield (10769.4 kg/ha) was determined in N (B. atrophaeus) and the lowest (7249.8 kg/ha) in PR (Bacillus-GC group+R.gallicum) treatments. In 2022, NR (B. atrophaeus+R. gallicum) produced the highest yield (16512.7 kg/ha) and N (B. atrophaeus) the lowest (10329.4 kg/ha). In inorganic fertilizer applications, the highest value was obtained in NP100 (40 kg N/ha^-1,60 kg/ha^-1 P₂O₅) application in both years, followed by NP₅₀ (20 kg N/ha^-1,30 kg/ha^-1 P₂O₅) and control. Microbial and inorganic fertilizer interactions were statistically very significant (Pd£0.01) in both years. The highest value (12668.7 kg/ha) was obtained from the combination of NP100 (40 kg N/ha^-1,60 kg/ha^-1 P₂O₅)+NR (B. atrophaeus+R.gallicum) in 2020 and (17818.7 kg/ha) from NP50 (20 kg N/ha^-1,30 kg/ha^-1 P₂O₅)+NR (B. atrophaeus +R.gallicum) in 2022 (Fig 1e).


  
The increased availability of plant nutrients with the application of chemical fertilizers and microbial fertilizers has led to an increase in plant growth parameters. The activities of nitrogen fixation and P-solubilizing bacteria support plants starting within a few weeks from seed germination until the grain filling period. However, they provide slower and more continuous support compared to chemical fertilizers. Studies show that some of the N applied to the soil by chemical fertilization is lost by leaching, or denitrificatied due to evaporation processess (Geng et al., 2022; Zhang et al., 2023). In contrast, biologically sourced N is directly fixed in the the root zone of the plant reducing loses and enhancing nutrient availability (Guo et al., 2023). Therefore, it is believed that plants receiving N supple mentation until the late stages have higher biomass and higher yields. Healthy growing plants had a higher number of pods and 100-seed weight, resulting in higher grain and biological yield in beans. Various researchers have reported that a combination of PGPB strains can give superior results compared to the individual performances of the strains (Çýğ et al.,  2021; Timofeeva et al., 2023; Ceritoğlu et al., 2024). Many researchers have obtained similar results to the findings obtained as a result of our study (Dela et al., 2023; Singh et al., 2023; Doğan and Çýğ, 2023; Ahmad et al., 2022). Sonkarlay et al., 2020 and some other researchers (Singh et al., 2021; Yadav et al., 2021; Lanjewar et al., 2023; Namdeo and Bhatnagar, 2023) have suggested using these two fertilizer types together, since higher yields were obtained from areas where microbial and inorganic fertilizers were applied together. Sadeghipour (2017), in a study involving the application of vermicompost and chemical fertilizers, reported that the lowest biological productivity was obtained from control plots, followed by NPK fertilizers.

The research results revealed that the grain yield and yield components of beans responded differently to microbial and inorganic fertilizer applications. It was determined that NP100 (40 kg N/ha-1,60 kg/ha^-1 P₂O₅) inorganic fertilizer application showed the best results in terms of many agronomic characteristics. However, especially when applied as a consortium, microbial fertilizers showed a very close performance to synthetic fertilization. In almost all combinations, yield components increased compared to control applications. Most of the inoculations applied together with NP50 (20 kg N/ha^-1,30 kg/ha^-1 P₂O₅) dose caused an increase in bean grain yield. However, the increase was greater in inoculations made together with NP100 (40 kg N/ha^-1,60 kg/ha^-1 P₂O₅). The highest grain yield was obtained in NP100 (40 kg N/ha^-1,60 kg/ha^-1 P₂O₅) + NR (B. atrophaeus+ R. gallicum) application. Therefore, we recommend that NP100 (40 kg N/ha^-1,60 kg/ha^-1 P₂O₅) +NR (B. atrophaeus+ R. gallicum) applications be applied together in order to increase grain yield and yield components in bean farming.

This research was supported by Van YYU Scientific Research Projects Directorate within the scope of the doctoral project numbered FDK-2021-9567. I would like to thank Van YYU Scientific Research Projects Directorate for providing financial support for the doctoral thesis study.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.