Effects of nitrogen fertilizer on phenolic acids of leaves of pear tree
Nine phenolic acids were separated and identified from the leaves of pear seedlings, including chlorogenic acid, epicatechin, ferulic acid, vanillin, gallic, caffeic acid, syringic acid, p-coumaric acid, and protocatechuic acid. chlorogenic acid, epicatechin, ferulic acid of them were the main phenolic acid from pear tree (Fig.S 1-4).
The content of ferulic acid from pear leaves treated with 500mg/kg and 1000mg/kg nitrogen fertilizers showed no significant different from that of the control, however the content of ferulic acid from pear leaves was significantly higher than that from the control pear leaves under the concentration of 1500mg/kg, (F3,20= 4.649, P= 0.013) (Fig. 1A). The content of chlorogenic acid and epicatechin from pear leaves treated with nitrogen fertilizers showed no significant difference from that of the control (chlorogenic acid: F3,20= 1.063, P= 0.387; Fig. 1B ; epicatechin: F3,20= 0.104, P= 0.0957; Fig. 1C).
Effects of ferulic acid on developmental duration of nymphs of C. chinensis
The developmental duration of the 5th instar and the 3rd -5th instar of C. chinensis nymphs decreased significantly (F2,57= 4.888, P= 0.011) in both two concentrations (0.24 ppm and 0.36 ppm) of ferulic acid treatment comparing with control treatment (Fig. 2). No significant differences were found among the developmental duration of the 3rd and 4th instar of C. chinensis in control and ferulic acid treatments (Fig. 2).
Effect of nitrogen fertilizer on honeydew secretion of C. chinensis
We found that nitrogen fertilizer could affect the secretion of C. chinensis honeydew, the secretion of honeydew gradually increased with the increase of nitrogen fertilizer. The secretion of honeydew from C. chinensis fed on pear leaves treated with nitrogen fertilizer was significantly higher than that of the control by testing with the direct measurement (F3, 80=17.512, P<0.001) (Fig. 3A) or the water-sensitive paper method (F3, 116=24.852, p< 0.001) (Fig. 3B).
Effect of nitrogen fertilizer on content of amino acids from pear leaves
A total of 19 amino acids were detected in the pear leaves treated with different concentrations of nitrogen fertilizer, including 9 non-essential amino acids and 10 essential amino acids for insects. The three main non-essential amino acids in leaves were glutamate, aspartate and asparagine, respectively. Arginine and threonine were the two most abundant essential amino acids in leaves. The result indicated that the content of amino acids in leaves showed an increase trend with the increase of nitrogen fertilizer. Most of the amino acids in leaves showed significantly difference under different concentrations of nitrogen fertilizer: (1) Non-essential amino acids: Aspartate F3, 8=16.950, p = 0.001, asparagine F3, 8=12.585, p = 0.002, glutamine F3, 8=201.814, p < 0.001, alanine F3, 8=33.428, p <0.001, glycine F3, 8= 16.413, p = 0.001, serine F3, 8=11.261, p = 0.003, proline F3, 8=140.439, p < 0.001. (2) Essential amino acids: Arginine F3, 8=595.35, p < 0.001,valine F3, 8= 13.099, p = 0.002, phenylalanine F3, 8= 5.042, p = 0.03, threonine F3, 8= 8.856, p = 0.06, tryptophan F3,8= 11.478, p = 0.03, histidine F3, 8= 14.307, p = 0.001 (Fig. 4).
Effect of nitrogen fertilizer on content of amino acids from honeydew of C. chinensis
Four essential amino acids (Fig. 5B) and eight non-essential amino acids (Fig. 5D) were identified in the honeydew secreted by C. chinensis. The results showed that the nitrogen fertilizer significantly affected the total content of amino acid (Fig. 5A & 5C) in the honeydew of C. chinensis, and the content of amino acids increased with the increase of nitrogen fertilizer, arginine ( F3, 8=5.166, p = 0.028), threonine (F3, 8=52.898, p < 0.001), glutamine (F3, 8=11.896, p = 0.003), asparagine (F3, 8=4.534, p =0.039), glycine (F3, 8=4.306, p = 0.044), serine (F3, 8=9.455, p = 0.005) were significantly higher than that of the control respectively.
The amino acids contained in the pear leaves, such as tyrosine, leucine, isoleucine, methionine, phenylalanine, lysine, and tryptophan weren’t detected from the honeydew of C. chinensis. The contents of amino acids in the honeydew of C. chinensis were significantly lower than that of pear leaves under non-treatment with nitrogen fertilizer, such as the non-essential amino acid glutamate (t4=3.91. p = 0.017), essential amino acids arginine (t4 = 7.74, p = 0.001), valine (t4 = 8.89, p = 0.001), phenylalanine (t4 = 12.03, p = 0.007), lysine (t4 = 4.81, p = 0.009), threonine (t4 =21.85, p< 0.001) (Fig. 5E). The content of amino acids in the honeydew of C. chinensis under the 1500 mg/kg of nitrogen fertilizer treatment, such as the non-essential amino acids alanine (t4 = 8.42. p = 0.001), proline (t4 = 4.28, p = 0.013), serine (t4 = 5.78, p = 0.004), were also significantly lower than that in pear leaves (Fig. 5F).
Effect of nitrogen fertilizer on the gene expression of amino acid synthesis of C. chinensis
A total of 116 differentially expressed genes (DEGs) were obtained between C. chinensis fed on pear seedlings with and without nitrogen fertilization, of which 2 were up-regulated and 114 were down-regulated (Fig. 6). Two up-regulated genes failed to match the protein sequence after comparison with the database through Blastx. 107 out of the 116 differential genes were annotated.
The down-regulated differential genes were enriched in several pathways in the KEGG metabolic pathway analysis, mainly including amino acid anabolism, pentose phosphate pathway and peroxidase (Fig. 7). KEGG pathway analysis showed that several DEGs were related to amino acid metabolism pathways, and all of them were down-regulated after treating with nitrogen fertilizer, including alanine, aspartic and glutamate metabolic pathways, tryptophan metabolic pathways, glycine, serine, and threonine metabolic pathways, etc. The results showed that the expression levels of the serine synthetic gene phosphoserine phosphatase (PSP), glycine synthetic gene alanine-glyoxylate aminotransferase, genes catalyzing the synthesis of glycine (serine hydroxymethyltransferase, glyA), asparagine (asparagine synthase) and glutamate (aspartate aminotransferase, AAT), peroxiredoxin and glutathione S-transferase, catalase (CAT), and sod-2 (superoxide dismutase) in C. chinensis were all down-regulated in the nitrogen-treated group. The expression level of the gene cystathionine gama-lyase that synthesized cysteine was not significantly changed after treatment with nitrogen fertilizer.
Verified by qPCR, the expression levels of genes phosphoserine phosphatase (t2 = 11.02, p = 0.008) and serine hydroxymethyltransferase (t2 = 10.068, p = 0.010) that synthesize serine and glycine were found significantly decreased in C. chinensis fed on pear seedlings treated with nitrogen fertilizer (Fig. 8). Meanwhile, the expression levels of asparagine synthesis gene asparagine synthase (t2 = -9.19, p = 0.012) and glutamate synthesis gene aspartate aminotransferase (t2 = -7.953. p = 0.015) were also significantly down-regulated in C. chinensis under nitrogen fertilizer treatment (Fig. 8).