2.1 Plant materials, soil and chemicals
Seven rice (Oryza sativa ) cultivars, Huagan-3, Huagan-8, Huafeng, Lingyou-6173, Lingyou-6365, Lingyou-3826 and Liaojing, were used for this study. Among them, allelopathic Huagan-3 and Lingyou-6173 have the ability for kin recognition (Yang, Li, Xu, & Kong, 2018), and other cultivars are non-allelopathic. ‘Huagan-3’ and ‘Huagan-8’ or ‘Lingyou-6173’ and ‘Lingyou-6365’ were bred or hybridized from reciprocal parents or identical male sterile, resulting in their relatedness. ‘Huafeng’ or ‘Lingyou-3826’ was obtained from other parents or male sterile that do not have consanguinity ties with the ‘Huagan-3’ and ‘Huagan-8’ or ‘Lingyou-6173’ and ‘Lingyou-6365’. These cultivars with different relatedness were divided into two sets of genotypes, indica-inbred and indica-hybrid. In the indica-inbred rice set, ‘Huagan-3’ was the focal rice cultivar (same cultivar, self). ‘Huagan-8’ and ‘Huafeng’ were the closely related (kin) and distantly related (non-kin) cultivars, respectively. In indica-hybrid rice set, ‘Lingyou-6173’ was the focal rice cultivar (same cultivar, self). ‘Lingyou-6365’ and ‘Lingyou-3826’ were the closely and distantly related cultivars, respectively. In addition, a japonica-inbred cultivar ‘Liaojing’ was used for comparative evaluation as a more distant non-kin for both sets. Finally, the relatedness of cultivars in both sets was determined by genetic distances relative to the focal cultivar (Yang, Li, Xu, & Kong, 2018). The genetic distance, allelopathic potential and grain biomass of these cultivars were presented in Supplementary Table 1.
Weeds seeds and soil were collected at Nanling experimental station of rice research, Anhui Province, China (30°56′52′′ N, 118°23′15′′ E), located in the low mountains on the southern side of the Yangtze River. Soil was collected randomly from the surface (0-10 cm) of a paddy field. The soil is typical fluvaquent, Etisol (US taxonomy) with pH 5.6, organic matter of 24.5 g·kg-1 and a fertility for total nitrogen of 1.4 g·kg-1, available phosphourus of 29.3 mg·kg-1 and available potassium of 56.8 mg·kg-1. Soil samples were air-dried, sieved (2 mm mesh) to remove plant tissues, and used in the series of greenhouse experiments described below.
The rice allelochemicals tricin and momilactone B were isolated from allelopathic rice PI312777 seedlings following Kong et al. (2004). Allantoin, related to kin recognition in rice lines (Yang, Li, Xu, & Kong, 2018), and other chemicals were purchased from China National Chemical Corporation (Beijing, China) and were of the highest purity available.