The chemical components analysis of different extracts of A. argyi
In our preliminary study, we accidentally found that A. argyi powder significantly inhibited the germination and reduced the varieties and biomass of weeds in the field, when it was applied as a fertilizer originally. Therefore, we speculated that certain allelochemicals present in A. argyi might inhibit the growth of weeds. To investigated the possible allelochemicals in A. argyi, three solvents (water, 50% ethanol and pure ethanol) were used to extract the metabolites in A. argyi leaves. The three type of extracts were analysed by UPLC-Q-TOF-MS and the components were confirmed by comparison with synthetic standards and MS data in literatures9,10,11. As shown in Table 1 and supplement Fig. 1, we have identified a total of 29 components in A. argyi. Six main compound mass signals were identified in the water extract: caffeic acid, schaftoside, 4-caffeoylquinic acid, 5-caffeoylquinic acid, 3,5-dicaffeoylquinic acid and 3-caffeoylquinic acid. The main compounds of the 50% ethanol extract were 4,5-dicaffeoylquinic acid, 3-caffeoylquinic acid, schaftoside, rutin, kaempferol 3-rutinoside, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 3-caffeoy,1-p-coumaroylquinic acid, 1,3,4-tri-caffeoylquinic acid and eupatilin. The metabolites with higher contents in the pure ethanol extract were eupatilin, jaceosidin and casticin. Among these compounds, caffeic acid is very unique in water extract. Higher contents of schaftoside, 4-caffeoylquinic acid and 3-caffeoylquinic acid were observed in water extract and 50% ethanol extract, but very low concentrations were detected in the pure ethanol extract. 3,4-dicaffeoylquinic acid, jaceosidin, eupatilin and casticin were present at higher concentrations in the 50% ethanol extract and pure ethanol extract, but were detected at very low concentrations or were absent in the water-soluble extract. In a word, we have preliminarily identified the chemical components of different extracts.
Comparison of the allelopathic effects of different extracts of A. argyi
To explore the allelopathic effects of three different extracts of A. argyi, seed germination and seedling growth of B. pekinensis, L. sativa and O. sativa were investigated after treatment of A. argyi powder extracts. The results showed that the allelopathic inhibition increased in a concentration dependent manner. When seeds were incubated with extracts in a range of concentrations, the water-soluble extract of A. argyi powder exerted an extremely significant inhibitory effect on the germination index of all the three plants (Fig. 1a,b). While the 50% ethanol extract also showed striking allelopathic inhibitory effects on the germination index of B. pekinensis and L. sativa, but moderately inhibitory effects on O. sativa (Fig. 1c,d). Similarly, the pure ethanol extract only showed powerful inhibitory effects on the germination index of B. pekinensis and L. sativa, but no effects on O. sativa (Fig. 1e,f). Additionally, the water-soluble extract of A. argyi powder displayed extremely inhibition of the biomass of the three plants (Fig. 2a), while the 50% ethanol extract also exerted extremely significant allelopathic inhibitory effects on the biomass of B. pekinensis and L. sativa but inhibited O. sativa moderately (Fig. 2b). However, the pure ethanol extract exerted inhibitory effects on the biomass of these three plants only in high concentrations (Fig. 2c). Based on these results, the allelopathic intensity of the three different extracts of A. argyi was in the order of water-soluble extract > 50% ethanol extract > pure ethanol extract.
The different solvent exracts of A. argyi: (a,b) the water-soluble extract, (c,d) the 50% ethanol extract,and (e,f) the pure ethanol extract exert allelopathic effects on germination index of different plants. (n = 3,*P < 0.05, **P < 0.01, ***P < 0.001).
Allelopathic effects of the water-soluble extract (a), the 50% ethanol extract (b),and the pure ethanol extract(c) from A. argyi with different concentrations on biomass of three plants were compared. (n = 3,*P < 0.05, **P < 0.01, ***P < 0.001).
The water-soluble extract of A. argyi inhibited the germination and growth of different plants
From the above results, water-soluble extract of A. argyi powder exhibited the strongest allelopathic effects on plant growth. To evaluate systematacially, the germination rate, germination rate index, germination index, root length, stem length and biomass were selected as the allelopathic response indexes as previously reported12,13. As shown in Fig. 3 and Table 2, the germination rate, germination speed index, germination index, root length, stem length and biomass of B. pekinensis could be significantly inhibited by a low concentration extract (50 mg/ml), and the order of inhibition efficiency was: germination index > germination speed index > root length > germination rate > stem length > biomass. When the treatment concentration up to 100 mg/ml, all the allelopathic indexes were -1.00 which indicated that no seeds could germinate under this treatment concentration. For L. sativa, the order of inhibition efficiency was germination speed index > germination index > biomass > germination rate > root length > stem length. All allelopathy response indexes reached -1.00 when plants were treated with 150 mg/ml extract. For O. sativa, the six physiological indexes also could be inhibited by a low concentration of extract (50 mg/ml), but the changes were not as obvious as the changes in B. pekinensis and L. sativa. The intensity of inhibition on the six indexes was root length > stem length > biomass > germination index > germination speed index > germination rate. When O. sativa seeds treated with 100 mg/ml of extract, the allelopathic response index of root length and stem length were -1.00. The germination rate, germination speed index, germination index and biomass were -0.79, -0.91, -0.91 and -0.84, respectively, under the treatment with 150 mg/ml of extract. In brief, according to the comprehensive allelopathy index of the 6 indicators , the order in which they were sensitive to water-soluble extract of A. argyi were B. pekinensis (Cruciferae) > L. sativa (Compositae) > O. sativa (Gramineae).
The water-soluble extract of A. argyi inhibits the germination and growth of Brassica pekinensis, Lactuca sativa and Oryza sativa. Specific performance is in a series of indicators: (a) the germination rate, (b) the germination speed index, (c) the root length, (d) the stem length. (n = 3,*P < 0.05, **P < 0.01, ***P < 0.001).
A.argyi inhibited the germination and growth of different plants in pot experiment
Further, the allelopathic effects of A. argyi were evaluated in pot experiments via soil mixed with a certain proportion of A. argyi powder. Firstly, seeds of B. pekinensis, L. sativa, O. sativa, P. oleracea , O. corniculata and S. viridis were sown into the mixed soil to observe the effects of A. argyi on seeds germination and plant growth. As shown in Fig. 4, when the proportion of soil: A. argyi powder was 100:2, the germination rate of B. pekinensis and L. sativa was significantly inhibited, and the plant height of B. pekinensis, L. sativa, O. sativa and P. oleracea was also inhibited. As the proportion of A. argyi powder gradually enhanced, the level of inhibition of the germination rate and plant height of these six tested plants was gradually increased. When the ratio reached 100:8, the germination inhibition rates of B. pekinensis, L. sativa, O. sativa, P. oleracea, O. corniculata and S. viridis were 71.82%, 93.20%, 31.75%, 65.47%, 63.60% and 60.78%, respectively. The plant height inhibition rates were 51.76%, 71.39%, 64.99%, 65.70%, 40.94%, and 36.53%, respectively, and the leaves turn yellow gradually in many plants. Therefore, the results from the laboratory were remarkable, it is urgent to verify the A.argyi powder as a weed herbicide in the field.
Mean germination index and plants height plus standard deviation for the six species with different proportions of A. argyi treatment in pot experiment: Brassica pekinensis (a), Lactuca sativa (b), Oryza sativa (c), Portulaca oleracea (d), Oxalis corniculata (e), Setaria viridis (f). (n = 3,*P < 0.05, **P < 0.01, ***P < 0.001).
A.argyi inhibited the germination and growth of weeds in Chrysanthemum morifolium field
Then, A. argyi powder was applied into the C. morifolium field to explore the inhibition of weeds and evaluate the adverse effect on crops. After the application of A. argyi powder for one month, six species of weeds appeared in the control group, while 3 species of weeds appeared in the group treated with 0.1 kg/m2 powder, and only 1 species of weeds appeared in the group treated with 0.2 kg/m2 powder (Fig. 5). Our previous report14 showed that the inhibition rate of weeds species were 50% and 83.33%, respectively, in the low dose group and the high dose group. Furthermore, the quantity and biomass of weeds were inhibited by 46.61% and 60.98% in the 0.1 kg/m2 treatment group compared with control group14. The weed quantity and biomass were inhibited by 60.90% and 82.11%, respectively, in the 0.2 kg/m2 treatment group14. In addition, C. morifolium grew very well in the field with no growth inhibition by A. argyi powder. After C. morifolium was harvested in the autumn, there were no significant differences in the number of flowers and the weight of flowers between the experimental group and the blank control group. Therefore, A. argyi powder did not inhibit the growth of the existing crops in the field, but only exerted an obvious effect on the ungerminated weed seeds in the field. Importantly, A. argyi powder may be a potential raw material for developing safe and environmentally friendly plant-sourced herbicides.
Actual growth of weeds in Chrysanthemum morifolium field with A. argyi treatment.
A.argyi inhibited the germination and growth of weed via the suppression of chlorophyll synthesis and photosynthesis
The inhibitory effect of weeds has been well documented, but the mechanism is urgently to be revealed. As reported15, transcriptomic analysis is the rapid and objective method to explore the mechanism. For the convenience of transcriptome analysis, we choosed the accepted model plant Oryza sativa as the research object, based on the BGISEQ500 platform. Each sample produced an average of 6.76G of data, with a total of 87.42% of the reads were mapped to the reference genome. Further, genome-wide gene expression profiles were compared between A.argyi– and water-treated wild-type plants. In total, 311 differentially expressed genes (DEGs) with (log2|FC (ratio of treated/control)|≥ 1, p-value < 0.05) were selected for further investigation. Among them, 245 genes were up-regulated and 66 genes were down-regulated in the A.argyi-treated plants (Fig. 6a,b).
RNA-Seq Analysis of the O. sativa under two treatment models. (a) The volcano plot of DEGs. Different colors represent different gene expression trends. (b) Hierarchical cluster analysis of the significantly changed DEGs. The color key represents FPKM-normalized log2-transformed counts. (c) KEGG pathway classification analysis of DEGs. (d) Top 20 KEGG pathway enrichment analysis of up-regulated DEGs. The colors are shaded according to the Q-values level, as shown in the color bars gradually from low(red) to high(blue); the size of the circle indicates the number of DEGs from small (less) to big (more); the same of next figure. (e)Top 20 KEGG pathway enrichment analysis of down-regulated DEGs. (f) The expression of key DEGs in key pathways. The color key represents FPKM-normalized log2-transformed counts.
To elucidate the potential biological functions of the DEGs, Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used for pathway classification and enrichment analysis. The results showed that a maximum number of genes were classified in the metabolism category, followed by organismal systems, environmental information processing, cellular processes and genetic information processing (Fig. 6c). Furthermore, KEGG pathways of up- and down-regulated DEGs were investigated separately. For up-regulated DEGs, most genes were enriched in “Metabolic pathways” and “Biosynthesis of secondary metabolites”. In detail, “Drug metabolism—cytochrome P450″(11 unigenes), “Metabolism of xenobiotics by cytochrome P450” (10 unigenes), “Drug metabolism—other enzymes” (10 unigenes), and “Glutathione metabolism” (10 unigenes) , were the most significant enriched pathways(Fig. 6d). For down-regulated DEGs, Photosynthesis, Nitrogen metabolism and Porphyrin and chlorophyll metabolism pathways were the most significant enriched pathways (Fig. 6e). Specifically, 4 unigenes involved in Photosynthesis, 2 unigenes involved Nitrogen metabolism and 2 unigenes involved in Porphyrin and chlorophyll metabolism were down regulated upon A.argyi treatment (Fig. 6f). In summary, these results suggest that A.argyi inhibited the germination and growth of weed via multi-targets and multi-pathways, especially, inhibiting the photosynthesis and chlorophyll synthesis pathways.
Photosynthesis is one of the most important physiological activities of plants. Our transcriptome analysis showed that photosynthesis might be the targets that inhibited by A.argyi treatment. Meanwhile, we also observed that A.argyi treatment caused the leaves of weeds turned yellow gradually in the pot experiment. The down-regulated genes of photosynthesis were involved in “photosystem II”, “photosystem I” and “cytochrome b6/f complex” (Fig. 7a)16. And the DEGs of porphyrin and chlorophyll metabolism were mainly involved in the preceding part of chlorophyll synthesis pathway (Fig. 7b)17. Therefore, we speculated that suppression of chlorophyll synthesis and photosynthesis was one of the key mechanism of A.argyi to inhibit weeds.
The down-regulated DEGs of “ photosynthesis” pathway (a) and “chlorophyll biosynthesis” pathway (b).
The key genes involved in photosynthesis pathways were verified by RT-qPCR, and the results were in consistent with our transcriptome analysis. As shown in Fig. 8, nine genes (HEMA “encoding glutamyl-tRNA reductase”, HEML “encoding glutamate-1-semialdehyde aminotransferase”, CHLD “encoding Mg chelatase D subunit”, CHLH “encoding Mg chelatase H subunit”, CRD “encoding Mg-protoporphyrinogen IX monomethylester cyclase”, CHLG “encoding chlorophyll synthase”, PsbY “encoding a polyprotein of photosystem II”, PetC “encoding the polypeptide binding the Rieske FeS center”, Os04g38410 “encoding subunits of the LHCII complex”) of photosynthesis were significantly suppressed by A.argyi treatment in a concentration dependent manner. Our transcriptome data and RT-qPCR verification showed that the suppression of chlorophyll synthesis and photosynthesis was one of the key mechanism of A.argyi ’s inhibitory effect on weeds.
Expression of photosynthetic genes in O. sativa treated with different concentrations water-souble extact of A.argyi.
Source: Ecology - nature.com