Change In Allelopathic Potential Of Rice And Its Molecular Mechanism In The Responses To Barnyardgrass (Echinochloa Crus-galli L.) Stress

In order to elucidate the change in allelopathic potential of rice and its molecular mechanism in the responses to barnyardgrass (BYG) stress, allelopathic rice accession PI312777 (PI) and non-allelopathic rice accession Lemont (LE) were chosen as the donor plants and BYG (Echinochloa crus-galli L. Beauv.) as the receiver plant in a hydroponic rice/weed mixed culture system. The different rice:BYG ratios were as follows:20 rice plants:5 BYG plants (4:1) per pot,20 rice plants:10 BYG plants (2:1) per pot, and 20 rice plants:20 BYG plants (1:1) per pot. The rice control comprised 20 rice plants with no BYG plants and BYG control comprised 20 BYG plants with no rice plants. Firstly, some indexes were investigated to evaluate the change of two rice accessions and BYG in rice/weed mixed culture system, that are (1) Morphological parameters including root length, plant height, and plant dry weight of the plants; (2) Physiological parameters including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), the contents of soluble carbohydrate and soluble protein of the plants; (3) Photosynthetic parameters, such as net photosynthetic rate, stomatal conductance, transpiration rate, intercellular CO2 concentration, stomatal limitation, and chlorophyll content of the plants; (4) Nutrient uptake and utilization efficiency of N, P, K of the plants. Secondly, suppression subtractive hybridization (SSH) was used to detect the differential expression genes between the two rice accessions under BYG stress at the rice:BYG ratio of 1:1. Thirdly, quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the differential expression of 7 relevant genes encoding Phenylalanine ammonia-lyase, Cinnamate-4-hydroxylase, Hydroxylase, CoA-ligases, O-Methyltransferases, Cinnamoyl-CoA, and Cinnamoyl alcohol dehydrogenases which involved in phenolic acid metabolism, and 7 relevant genes encoding 3-Hydroxy-3-methylglutaryl-CoA reductase, Mevalonate kinase, Monoterpenes cyclase, Sesquiterpene cyclase, Diterpene cyclase, Phytoene synthase, and Squalene synthase which involved in terpene metabolism in rice root and its leaf under BYG stress. The phenolics contents in rice root, leaf, and root exudates, and the terpenoid contents in rice root exudates were determined by HPLC and GC/MS, respectively. Finally,5 high-bioactive oxygenic terpenoids, namely (-)-carveol, (+)-carvone, (-)-menthone, (-)-carvyl acetate, and (+)-cedrol were chosen as allelochemical candidates. The optimum combination of the five terpenoids was statically estimated by orthogonal rotatable central composite design with five variables and five concentration levels. Further, the effect of the optimal combination mixture of the five oxygenic terpenoids on BYG and paddy weeds was validated in laboratory bioassay, greenhouse experiment, and field test, respectively.The results could be summarized as follows:(1) Two rice accessions showed the inhibitory effect on accompanying weed BYG in the mixed culture system and the inhibitory rate was increased with increasing weed density. However, PI showed higher inhibitory rate than LE. For example, at the ricc.BYG ratio of 1:1, the inhibitory rate of PI on root length, plant height, and plant dry weight of BYG were 63.66%,54.47%, and 46.71% respectively, but they were 31.56%,20.19%, and 24.34% respectively when mixed culture with LE. Analysis of allelopathy-competition separation in rice/weed mixture has shown that PI contributed to more allelopathic effect than its resource competition, showing the inhibitory rates coming from allelopathic effect were 39.06% (4:1),40.88% (2:1), and 49.39%(1:1). However, the inhibitory rates of LE from allelopathic effect were only 9.57% (4:1), 8.36%(2:1), and -3.19%(1:1). The competition indices showed that the competitive inhibitory effect of PI on BYG was more than two times higher than that of LE on BYG. The inter-specific interaction of BYG to PI was facilitation and that of BYG to LE was inhibition. This finding suggested that there existed different survival strategies in different allelopathic potential rice accessions in rice/weed mixed culture system. PI showed better growth by enhancing its allelopathic potential, and LE was a struggle for existence by strengthening its predatory ability to resource inversely. PI has an obvious competitive advantage in rice/weed mixed culture system.(2) The activities of SOD, CAT and POD, the content of soluble carbohydrate and soluble protein in leaf and root of BYG were significant declined in rice/weed mixed culture system. The inhibitory rates of abovementioned parameters were increased with increasing weed density. However, PI showed higher inhibitory effect on abovementioned parameters than LE. For example, at the rice:weed ratio of 1:1, the inhibitory rate of PI on above indexes of BYG were 36.39%,50.93%,48.29%,33.14% and 51.74% respectively, but those were 7.33%,22.30%,12.82%,6.18% and 10.24% respectively by LE. The inhibitory rate of PI on the chlorophyll content of BYG leaf was 26.52% (4:1),31.69% (2:1), respectively and 41.29%(1:1), but the data of LE on BYG were 4.14% (4:1),6.49%(2:1), and 11.11% (1:1), respectively. The N, P, K uptake and utilization efficiencies of BYG were decreased with higher weed density ratio in PI/BYG mixed culture. In LE/BYG mixed culture, the N, P, K uptake efficiencies of BYG was decreased, but utilization efficiency was increased with higher weed density ratio. This result suggested that the allelochemicals exudated from PI not only interfere with the absorption of the target plant, but also with the internal metabolism.(3) The results of SSH analysis showed that under BYG stress, the enhanced expression genes in PI root were genes that associated with allelochemical metabolism, plant growth and cell cycle relative, stress resistance and signal transduction, and protein metabolism. One of them was the gene encoding 4-coumarate-CoA ligase, a key enzyme in phenylpropanoid pathway, which was responsible for the synthesis of phenolic secondary metabolic compounds to suppress the accompanying BYG. Another is cytochrome P450 monooxygenase, which is involved in a wide range of biosynthetic reactions, leading to various fatty acid conjugates, plant hormones, defensive compounds, impressing terpenoids, which are the substrates for the synthesis of plant cytochrome, will be more synthesized to supply plant cytochrome synthesis under BYG stress.(4) The results of qRT-PCR showed that the 7 relevant genes involved in phenolics metabolism in PI leaf and root were up-regulated. The fold of up-regulation was increased with increasing weed density. However, six out of seven genes were down-regulated in LE, except the gene encoding Phenylalanine ammonia-lyase. By HPLC determination, the phenolics contents in leaf, root and root exudates of the two rice accessions were increased with increasing weed density. However, the phenolics contents were higher in PI than that in LE. For example, at the rice:weed ratio of 1:1, the phenolics contents in leaf, root and root exudates of PI were 3.67,3.27, and 4.59 times more than that in LE, respectively. At the same time, the 7 relevant genes involved in terpene metabolism were up-regulated in two rice accessions, but the up-regulation folds were higher in PI than in LE. By GC/MS determination, the terpenoids contents in root exudates was about 1.5 times higher in PI than that in LE. The relatively high component is oxygenic terpenoids, accounting for more than 85% of the terpenoids. (5) On the basis of a single terpenoid bioassay, five high-bioactive oxygenic terpenoids, namely (-)-carvcol, (+)-carvonc, (-)-mcnthonc, (-)-carvyl acetate, and (+)-cedrol were chosen as candidates. By the result of orthogonal rotatable central composite design, the optimal combination mixture of the five oxygenic terpenoids was as follow:0.033mmol·L-1 (-)-carveol,0.03 mmol·L-1 (+)-carvone, 0.08mmol·L-1 (-)-menthone,0.02mmol·L-1 (-)-carvyl acetate, and 0.001mmol·L-1 (+)-cedrol. Theoretically, the maximum inhibitory rate of the optimal combination mixture was 93.69%. In Petri dish bioassay, the pre-germination seeds of BYG did not regrown. In hydroponic, the inhibitory rates of the optimal combination mixture on the root length, plant height, and plant dry weight of BYG were 69.60%,23.93%, and 58.24 respectively. The inhibitory rates of SOD, POD and CAT activity were 76.79%,78.54% and 74.23% respectively in BYG root, and were 59.49%,58.05% and 74.86% respectively in leaf of the target weed. In paddy soil and in monoculture experiments, the inhibitory rates of the optimal combination mixture on the root length, plant height, and plant dry weight of BYG were 48.58%,28.11%, and 39.88 respectively. However, the root length, plant height, and plant dry weight of the two normal rice accessions, MinHui63 and ShanYou63, were not significantly affected. The effects of the optimal combination mixture on BYG when grown with rices were similar to those in the monoculture experiment. The mixture was not harmful to the two normal rice accessions. In field potted test,12 paddy weeds were found in the control pots. However, after treated by the optimal combination mixture of the five oxygenic terpenoids,11 of the 12 weeds were significantly suppressed except Common Duckweed (Lemna minor L.) showing stimulative effect. The inhibitory rate on aboveground dry weight of 11 weeds was 90.34%.In summary, under BYG stress, the relevant genes involved in phenolics and terpenoid metabolisms in PI were up-regulated significantly, accelerating the synthesis of allelochemicals such as phenolics and terpenoids. These allelochemicals destroyed effectively the protective enzyme system in BYG, inhibited its photosynthetic parameters, decreased the contents of chlorophyll, soluble carbohydrate and soluble protein of BYG, and suppressed the nutrient uptake and utilization efficiency of BYG. These effects ultimately led to disturb the growth of BYG. The combination mixture of oxygenic terpenoids, showing the higher bioactive at lower concentrations, was recommended as the most potential candidate of natural herbicides.