Objectives Metabolites are the basis of biological phenotypes, and their species and contents are significantly correlated with the nutritional quality of crops. We studied the metabolites of garlic bulbs using the metabolomics technology, to provide a theoretical base for scientific nutrient management of garlic production.

Methods A localized field garlic experiment was started since 2018 in Xuzhou City, Jiangsu Province. Four treatments were used: no nitrogen fertilizer control (CK), conventional chemical fertilizer (T1), 1/3 manure N combined with 2/3 chemical fertilizer N (T2), and pure manure application (T3). The total N input amount was equal in T1 and T2, and the nitrogen input in T3 was half of that in T1. At harvesting of garlic bulbs in May 2023, the appearance traits, yield and nutritional quality of garlic bulbs were investigated, and the metabolites were identified quantitatively using GC-MS, and the differential expressed metabolites were screened between treatments in pairs, based on metabolomics analysis, and their enrichment pathways were analyzed.

Results Compared with CK, T1, T2, and T3 treatments significantly increased the horizontal diameter, vertical diameter, bulb weight and first grade bulb proportion of garlic bulbs, resulting in significantly higher yield and economic effect, and T2 was recorded the best effect among the treatments. The three fertilization treatments did not show significant effects on vitamin C (Vc) content of garlic bulbs, but reduced the soluble sugar content in bulb, and T1 and T2 treatments were recorded significantly lower soluble sugar content than T3 treatment. T1 treatment reduced, while T2 increased, the bulb soluble protein content. Both T2 and T3 treatments significantly increased the allicin content. Applying nitrogen fertilizer significantly increased the free amino acid content of bulbs, with the highest under T2 treatment, followed by T1. All the PCA, cluster heat map and OPLS-DA model analysis showed significant differences in garlic bulb metabolite species and levels among the fertilization treatments. Based on the criteria of VIP>1.0, FC>2.0 or FC<0.5, and P<0.05, the deferentially expressed metabolites (DEM) were screened. Compared to CK, T1, T2, and T3 were screened 44, 41, and 20 DEMs, and among them, there were 15 commonly screened DEMs in the three treatments, mainly involving amino acid metabolism, secondary metabolism biosynthesis and aminoacyl-tRNA synthesis. Compared with T1, T2 was screened out 8 DEMs, four of them were enriched in cysteine and methionine, and zeatin biosynthesis and metabolism.

Conclusions The application of nitrogen fertilizer significantly influences the nutritional and economic traits of garlic bulbs. Adequate nitrogen supply enhances the presence of over 40 metabolites, thereby improving the overall quality of garlic bulbs. Under the same nitrogen application rate, inputting 1/3 of manure nitrogen produces four deferentially expressed metabolites which directly participate in biochemical pathways such as cysteine and methionine metabolism, as well as zeatin biosynthesis, thus further enhancing both the quality and yield of garlic.