# Increased Grain Amino Acid Content in Rice with Earthworm Castings
## 2 Materials and Methods  
A micro-plot field experiment was done in Changsha (28\({}^{\circ}\)11\({}^{\prime}\) N, 113\({}^{\circ}\)04\({}^{\prime}\) E), Hunan Province, China in 2017. The soil in the experimental field was a Fluvisol (FAO taxonomy) with the following chemical properties at the upper 20 cm layer: pH 5.75, 34.2 g organic matter kg\({}^{-1}\), 81.6 mg available N kg\({}^{-1}\), 34.4 mg available P kg\({}^{-1}\), and 56.7 mg available K kg\({}^{-1}\).  
Thirty-two bottomless plastic boxes (length \(\times\) width \(\times\) height = 40 cm \(\times\) 40 cm \(\times\) 30 cm) were pushed into the soil of the experimental field to a depth of 20 cm to establish micro-plots. Four rice cultivars (Huanghuazhan, Liangyoupejiu, Longliangyou 97, and Xiangliangyou 396) were grown and each micro-plot was treated with either earthworm castings (17 kg m\({}^{-2}\), EC17) or no castings (0 kg m\({}^{-2}\), EC0). The level of earthworm castings for EC17 was chosen according to the product of duration of oilseed rape-growing season (218 days) and daily production rate of earthworm castings (78 g m\({}^{-2}\) day\({}^{-1}\)) in a no-tillage rice-oilseed rape rotation field in Nanxian (29\({}^{\circ}\)21\({}^{\prime}\) N, 112\({}^{\circ}\)25\({}^{\prime}\) E), Hunan Province in 2015. The daily production rate of earthworm castings was obtained from an investigation conducted on 10 randomly selected 1-m\({}^{2}\) areas in the field on the first day after oilseed rape harvest. The earthworm castings (pH 7.89, 61.4 g organic matter kg\({}^{-1}\), 128 mg available N kg\({}^{-1}\), 44.2 mg available P kg\({}^{-1}\), and 254 mg available K kg\({}^{-1}\)) used in the experiment were collected from several no-tillage rice-oilseed rape rotation fields in Nanxian after oilseed rape harvest in 2016. The micro-plots were arranged in a split-plot design with four replicates, using the level of earthworm castings as the main plots and cultivars as the subplots.  
Rice seeds were pre-germinated and then sown in a seedbed on 10 May. Rice seedlings were transplanted on 5 June with one seedling per hill and four hills per micro-plot. Earthworm castings were applied and incorporated in the upper 20 cm of the soil on the day before transplanting. Urea was split in three applications: 6.0 g N m\({}^{-2}\) at 1 day before transplanting (basal), 3.6 g N m\({}^{-2}\) at 7 days after transplanting, and 2.4 g N m\({}^{-2}\) at panicle initiation. Superphosphate was applied as basal at a rate of 4.8 g P\({}_{2}\)O\({}_{5}\) m\({}^{-2}\). Potassium chloride (8.4 g K\({}_{2}\)O m\({}^{-2}\)) was split equally in two applications: one at basal and the other at panicle initiation. The micro-plots were flooded with a water depth of about 5 cm until 7 days before maturity. Agrochemicals were used to control diseases, insects, and weeds.  
At maturity, rice grains were sampled from each micro-plot. The sampled grains were oven-dried at 70 \({}^{\circ}\)C to a constant weight and then hulled, ground, and sieved (100 mesh). About 1.0 g of the sieved sample was weighed, hydrolyzed, and derivatized to determine total amino acid content with high-performance liquid chromatography method [15]. Approximately 0.5 g of the sieved sample was digested by H\({}_{2}\)SO\({}_{4}\)-H\({}_{2}\)O\({}_{2}\) method to determine the total N content. The N analysis was carried out using a segmented flow analyzer (SAN Plus, Skalar Inc., Breda, The Netherlands). The ratio of amino acid to N (total amino acid content/total N content) was calculated.  
Data analysis including analysis of variance and linear regression analysis was performed using Statistix 8.0 (Analytical Software, Tallahassee, FL, USA).