**3. Results and Discussion**

Total amino acid content in rice grains was significantly influenced by both the level of earthworm castings and cultivar (Table 1). There was no significant interaction effect between the level of earthworm castings and cultivar on total amino acid content in the grain of rice. Averaged across the four cultivars, total amino acid content in the grain was 8% higher under EC17 than under EC0. Huanghuazhan had the highest total amino acid content in grains, more than 10% higher than that of the other three cultivars.



EC0 and EC17 represent 0 and 17 kg earthworm castings m−2, respectively; Values in parentheses are SE (*n* = 4); \*, significance at the 0.05 probability level; \*\*, significance at 0.01 probability level; ns, non-significance at the 0.05 probability level.

N is a principal constitute element of amino acids. It is generally considered that amino acid content is positively linearly related to N content in the rice grain [8]. Some findings of this study are consistent with those of previous studies: Huanghuazhan, the cultivar with highest grain amino acid content, also had higher grain N content than the other three cultivars (Table 1). However, unexpectedly, no significant difference was recorded in the grain N content between the EC17 and EC0 treatments. Thus, increasing the grain N content was not the mechanism for the positive impact of applying earthworm castings on amino acid content in rice grains. Our study also showed that the EC17 treatment had a significantly higher ratio of amino acid to N than EC0 (Table 1). This result suggests that the increased grain amino acid content in rice induced by applying earthworm castings might be mostly attributable to the improved efficiency of converting N to amino acid. This could be further supported and informed by that the linear regression between grain amino acid and N contents was different under EC17 and EC0 (Figure 1). These results suggest that further investigations are required to determine the effects of earthworm castings on the metabolism of amino acid in rice grains.

**Figure 1.** Linear regression between total amino acid and N contents in grains of four rice cultivars under two levels of earthworm castings. EC0 and EC17 represent 0 and 17 kg earthworm castings m<sup>−</sup>2, respectively. Each data point is the mean for one cultivar under one level of earthworm castings. Error bars are SE (*n* = 4).

Application of earthworm castings can affect plant metabolism including protein synthetic activity [16,17]. In this regard, it is well documented that the castings and body secretions of earthworms contain a certain amount of plant hormones and hormone-like substances [18–20]. Earthworms can also enhance microbial biomass and activity due to their castings and mucus, and the byproducts of this microbial activity include plant hormones such as abscisic acids, auxins, cytokinins, ethylene, and gibberellins [21]. Although there is limited information available on the influences of plant hormones or plant hormone-like substances included in earthworm castings on amino acid metabolism in the rice crop, some relevant reports have been documented in other crops. For example, Muscolo et al. [22] found that earthworm-worked humic substances had an auxin-like effect on nitrogen metabolism in wild carrot, and Singh et al. [23] observed that gibberellic acid-like activity of vermicompost leachate resulted in an increase in amino acids in the common bean. Further study is required to determine if similar effects of plant hormones and hormone-like substances in earthworm castings are observed on amino acid metabolism in rice grains.

Application of earthworm castings has the potential for increasing amino acid content in rice grains, although the practicality of any approach must be taken into account. High labor costs are the key factor limiting the adoption of organic manures. In this study, the tested earthworm castings were not specially produced but were collected from no-tillage rice-oilseed rape rotation fields, where earthworms are known to be abundant (pers. obs.). Reduced or absent soil tillage in these fields can provide earthworms with an undisturbed biotope and hence may be favorable to increasing their populations, while oilseed rape plants can provide abundant food for earthworms by producing large volumes of residues. In addition, our previous study documented that adoption of no-tillage rice-oilseed rape cropping systems can maintain crop yields while saving labor [24]. Therefore, development of similar no-tillage systems may be an indirect but feasible way to increase grain amino acid content in rice by increasing earthworm activity.

**Author Contributions:** Conceptualization, M.H. and Y.Z.; Investigation, C.Z.; Writing, M.H.; Funding Acquisition, M.H. and Y.Z.

**Funding:** This research was funded by [the National Key R&D Program of China] grant number [2016YFD0300509] and [the Earmarked Fund for China Agriculture Research System] grant number [CARS-01].

**Conflicts of Interest:** The authors declare no conflict of interest.
