Introduction

The heavy reliance on chemical N-P-K fertilizers in intensive vegetable farming has raised significant environmental and health concerns. Nitrate accumulation in leafy greens, especially spinach, is a major food safety challenge. Nitrate is directly linked to nitrogen metabolism and accumulates in plant tissue when its uptake exceeds the plant's capacity to use it for growth and protein synthesis. Plant growth-promoting bacteria (PGPB) offer a sustainable alternative by enhancing nutrient bioavailability through mechanisms such as phosphate solubilization, nitrogen fixation, and siderophore production. Growth-promoting bacteria prevent nitrate accumulation in leaves by improving nitrogen uptake and transport. While the efficacy of PGPB is well-documented in cereal crops, their potential to mitigate the negative effects of reduced fertilization in high-demand vegetables like spinach remains under-explored.

Materials and Methods

This study aimed to evaluate the efficiency of PGPB in improving spinach characteristics under greenhouse conditions using a factorial experiment based on a completely randomized design. The treatments consisted of two levels of N-P-K fertilizer (100% of recommended rate and 70% of the recommended rate) and four levels of microbial inoculation (no bacteria, Pseudomonas putida, Phytobacter diazotrophicus, and a mix of both). The experiment was conducted in 3 kg pots. Six seeds were sown 1 cm below the soil surface and one milliliter of each strain was inoculated under each seed. The soil moisture of the pots was maintained at approximately 75% of field capacity moisture by weight during the experiment. After two weeks, the plants were thinned to three plants. The plants were harvested 50 days after planting. Parameters including leaf nitrogen (N), phosphorus (P), potassium (K), iron (Fe), chlorophyll content, nitrate concentration, and nitrate reductase enzyme activity were measured.



Results

The results showed that the interaction effect of fertilizer level and bacterial inoculation was significant for all measured traits. Microbial inoculation, particularly the mix of bacteria, effectively compensated for the yield loss caused by the 30% reduction in chemical fertilizer. The highest leaf fresh weight was obtained in both the 100% and 70% recommended NPK fertilizer treatments when plants were inoculated with the combined bacterial consortium, representing 1.7- and 2.2-fold increases, respectively, compared with the corresponding non-inoculated treatments. Application of 100% of the recommended NPK fertilizer in combination with the mixed bacterial inoculum increased leaf nitrogen (62%), phosphorus (23%), potassium (51%), and iron (32%) contents compared with the corresponding non-inoculated treatment. The second-highest contents of these nutrients were observed in the treatment receiving 70% of the recommended fertilizer rate together with the mixed bacterial inoculum. Total chlorophyll content declined with reduced fertilizer application; however, bacterial inoculation compensated for this reduction, such that no significant difference was observed between the inoculated treatments and the treatment receiving 100% of the recommended fertilizer rate. The highest total chlorophyll content was measured in the treatment receiving 100% of the recommended fertilizer rate together with the mixed bacterial inoculum,

which was 34% higher than the corresponding non-inoculated treatment. The highest leaf nitrate content (6123 mg kg⁻¹) was recorded in the treatment receiving 100% of the recommended fertilizer rate without bacterial inoculation. The lowest leaf nitrate content was measured in the treatment receiving 70% of the recommended fertilizer rate combined with the mixed bacterial inoculum, representing a 25% reduction compared with the corresponding non-inoculated treatment. The highest nitrate reductase activity was measured in the treatment receiving 70% of the recommended fertilizer rate together with the mixed bacterial inoculum, showing a 47% increase compared with the corresponding non-inoculated treatment. There was a negative correlation between nitrate content in the leaf and nitrate reductase activity. The results indicated that chlorophyll content was positively correlated with the contents of nitrogen, phosphorus, potassium, and iron, as well as with nitrate reductase activity. Plant fresh weight was also positively correlated with chlorophyll content, the contents of nitrogen, phosphorus, potassium, and iron, and the activity of nitrate reductase.





Conclusion

Overall, these findings suggest that the combined application of Pseudomonas putida and Phytobacter diazotrophicus with 70% of the recommended NPK fertilizer rate may represent a promising strategy to reduce chemical fertilizer use by 30% without substantial yield loss, while enhancing the nutritional quality of spinach. Nevertheless, further validation under field conditions and diverse environmental settings is required before this approach can be recommended for widespread agricultural use.