
==== Front
BMC Plant Biol
BMC Plant Biol
BMC Plant Biology
1471-2229
BioMed Central London

38862926
5229
10.1186/s12870-024-05229-8
Research
Combining transcriptome and metabolome analysis to understand the response of sorghum to Melanaphis sacchari
Zhao Xin-Rui 12
Zhao Dong-Ting 12
Zhang Ling-Yu 12
Chang Jin-Hua jhchang2006@126.com

12
Cui Jiang-Hui cjianghui521@126.com

12
1 https://ror.org/009fw8j44 grid.274504.0 0000 0001 2291 4530 College of Agronomy, Hebei Agricultural University, Baoding, 071000 China
2 North China Key Laboratory for Crop Germplasm Resources of Education Ministry, Baoding, 071000 China
11 6 2024
11 6 2024
2024
24 52915 11 2023
31 5 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by/4.0/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Background

The sorghum aphid Melanaphis sacchari (Zehntner) (Homoptera: Aphididae) is an important insect in the late growth phase of sorghum (Sorghum bicolor L.). However, the mechanisms of sorghum response to aphid infestation are unclear.

Results

In this paper, the mechanisms of aphid resistance in different types of sorghum varieties were revealed by studying the epidermal cell structure and performing a transcriptome and metabolome association analysis of aphid-resistant and aphid-susceptible varieties. The epidermal cell results showed that the resistance of sorghum to aphids was positively correlated with epidermal cell regularity and negatively correlated with the intercellular space and leaf thickness. Transcriptome and metabolomic analyses showed that differentially expressed genes in the resistant variety HN16 and susceptible variety BTX623 were mainly enriched in the flavonoid biosynthesis pathway and differentially expressed metabolites were mainly related to isoflavonoid biosynthesis and flavonoid biosynthesis. The q-PCR results of key genes were consistent with the transcriptome expression results. Meanwhile, the metabolome test results showed that after aphidinfestation, naringenin and genistein were significantly upregulated in the aphid-resistant variety HN16 and aphid-susceptible variety BTX623 while luteolin was only significantly upregulated in BTX623. These results show that naringenin, genistein, and luteolin play important roles in plant resistance to aphid infestation. The results of exogenous spraying tests showed that a 1‰ concentration of naringenin and genistein is optimal for improving sorghum resistance to aphid feeding.

Conclusions

In summary, the physical properties of the sorghum leaf structure related to aphid resistance were studied to provide a reference for the breeding of aphid-resistant varieties. The flavonoid biosynthesis pathway plays an important role in the response of sorghum aphids and represents an important basis for the biological control of these pests. The results of the spraying experiment provide insights for developing anti-aphid substances in the future.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-024-05229-8.

Keywords

Sorghum aphid
Sorghum
Transcriptome
Metabolome
Flavonoid metabolic pathway
issue-copyright-statement© BioMed Central Ltd., part of Springer Nature 2024
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pmcIntroduction

Sorghum (Sorghum bicolor L.) represents the fifth most grown crop worldwide after maize, wheat, and rice [1–3] and is a C4 crop with high nitrogen and water use efficiency [4]. It has a strong environmental adaptability and wide distribution and is extensively grown in arid and semi-arid regions worldwide, thus playing a vital role in the world’s agroecosystems [4].The sorghum aphid (Melanaphis sacchari (Zehntner) (Homoptera: Aphididae) is a major pest of sorghum that seriously harms the yield and quality of sorghum under field conditions [5]. Sorghum aphids occurr on sugarcane and sorghum in India, China, South Africa, Japan, and the United States, mainly during late sorghum growth [6–8]. Sorghum aphids are widely distributed in sorghum and sugarcane growing areas [8], and their population growth is influenced by temperature and rainfall patterns [9], with populations increasing rapidly in warm and dry climates [8]. Moreover, the timing and severity of infestation vary with location and year [10–12]. Sorghum aphids cause increased water stress in plants as they feed on the underside of sorghum leaves and stalks and suck plant sap. This feeding process causes the direct loss of plant nutrients and sugars. [6, 7, 13]. If sorghum is infested with aphids during panicle initiation and flowering, it can lead to delayed plant development, reduction in the number of spikes, and reduction in the accumulation of photosynthates, which overall reduces sorghum yield. Sorghum aphids also important pests of forage sorghum because they secrete large amounts of honeydew during host survival, which can cause sorghum leaves to stick together. Moreover, the excessive accumulation of honeydew can lead to mechanical problems during cutting and baling and reduce harvesting efficiency and quality because of the associated growth of mold [14]. A cosmopolitan pest, the sorghum aphid has high dispersal and reproductive potential and can rapidly increase in number. A single female can produce up to 75 nymphs in 12 days [6], whereas 50 sorghum aphids can produce up to 500 nymphs within a week [6]. This significantly increases the risk of plant infestation. Once infested, sorghum aphids can grow exponentially and reach 30,000 aphids on a single plant [8]. This can cause direct as well as indirect economic losses. In large-scale sorghum cultivation with many sensitive varieties, sorghum aphids remain the most damaging pests. In this context, the promotion of biological control of aphids has gradually increased to improve the environmental sustainability of sorghum. Chemical spraying is the main method of sorghum aphid control; however, this method not only pollutes the environment and leads to insect resistance but also kills other beneficial insects, such as predators, parasites, and pollinators. Therefore, alternative methods of effective aphid control are required.

During the co-evolution of plants and insects, both have acquired strategies to avoid each other’s defense systems [1, 2]. The interaction between plants and insects has led to the development of complex plant defense systems that can recognize infectious molecules or signals in damaged cells and activate the plant’s immune response to herbivorous insects [1, 2, 14–16]. Plants respond to herbivore attacks through a complex and dynamic defense system that includes structural barriers, toxic chemicals, and natural enemies of target pests [15, 17, 18]. Plant-induced responses are important components of agricultural pest control and have been widely used to regulate populations of herbivorous insects [6, 19, 20]. The substances that induce defense are primarily secondary metabolites, including phenolic and flavonoid compounds. Secondary metabolites are compounds that do not affect normal plant growth and development but reduce the palatability of the plant tissues in which they are produced [2, 21]. Defensive (secondary) metabolites can be constitutive, stored in an inactive form, or induced upon insect or microbial attack. Secondary metabolites protect plants from different stresses and enhance their adaptive capacity [22, 23]. Aphid infestation of aphid-sensitive sorghum has resulted in the altered expression of functional genes involved in cell wall modification, photosynthesis, and phytohormone biosynthesis [24]. Dhurrin is a cyanogenic glycoside found in sorghum that likely plays an important role in plant–herbivore defense [25, 26]. Jasmonic acid mediated responses may play a key role in promoting herbivore resistance [25, 26]. However, the mechanisms underlying aphid resistance in sorghum require further investigation.

To further elucidate the aphid resistance mechanisms of sorghum, aphid stress experiments were conducted on the aphid-susceptible variety BTX623 and aphid-resistant variety HN16, which are widely grown in China. The microstructures of the leaves of aphid-susceptible and aphid-resistant varieties of sorghum were observed, and the transcriptome and metabolic changes in their leaves before and after aphid infestation were analyzed. Candidate genes, metabolites, and key pathways were then identified. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to validate candidate differentially expressed genes (DEGs). We conducted exogenous spraying experiments to determine whether metabolites affect aphid resistance in sorghum, identify efficient aphid control methods, and provide a reference for the development of aphid-resistant substances in sorghum.

Materials and methods

Microstructure observation of different resistant varieties

Aphid-resistant varieties HN16, Silimei, and BTX428 and aphid-sensitive varieties BTX623, QianSan, and 3197B were selected for sampling at the heading stage. This was followed by fixation with FAA fixation, paraffin embedding, sectioning, and staining saffron solid green to observe the epidermal arrangement of different aphid-resistant varieties under a microscope (Nikon, Japan, Nikon Eclipse E100). The epidermal cell parameters and relative thickness were determined using CaseViewer software.

Transcriptome determination

The representative high-resistance variety HN16 and the sensitive variety BTX623 were selected for the aphid infestation test. At the sorghum heading stage, aphids were lightly brushed on the lower surface of the 4th and 5th leaf with a brush. At 0, 1, 2, 3, and 4 days, aphid-infested parts of the leaves were collected to form mixed samples, and 10 samples were sequenced using a transcriptome. According to the sampling days, HN16 was numbered CKh (0d), Th1 (1d), Th2 (2d), Th3 (3d), Th4 (4d); and BTX623 was numbered CKb (0d), Tb1 (1d), Tb2 (2d), Tb3 (3d), Tb4 (4d) (Additional file 1). RNA concentration and purity were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). RNA integrity was assessed using an RNA Nano 6000 Assay Kit on an Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA) [27]. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparation [27]. Sequencing libraries were generated using the NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA), following the manufacturer’s recommendations. Index codes were added to attribute sequences for each sample. Hisat2 tools soft were used to map with reference genome [28]. The reference genome was downloaded from https://www.ncbi.nlm.nih.gov/genome/term=sorghum. Differential expression analysis of the two samples was performed using edgeR. FDR < 0.01 and fold change ≥ 2 were set as the thresholds for significantly differential expression [29].

Metabolome determination

Based on the results of the transcriptome assessment, a metabolome assessment was performed on HN16 and BTX623 cells at 0 and 3 days afterinfestation, respectively. Each treatment was repeated three times for a total of 12 samples. The samples and transcriptome materials belonged to different biological repeats of the same batch. Sample extraction, chromatomass spectrometry collection, and metabolite characterization and quantification were conducted by NetWare (Wuhan, China) in accordance with standard procedures and previous studies [30]. The software Analyst 1.6.3 was used to process the mass spectrum data. Significantly regulated metabolites between groups were determined by VIP ≥ 1 and absolute log2FC (fold change) ≥ 1 [29].

Quantitative real-time PCR

HN16 and BTX623 plants infested with aphids for 0 and 3 days were used for quantitative real-time PCR (qPCR). Transcriptome sequencing was performed using the same batch of biological replicates. RNA was extracted with reference to the Eastep® Super Total RNA Extraction Kit (SHANGHAI PROMEGA) kit [31]. cDNA synthesis was performed using HiFiScript gDNA Removal RT MasterMix (CWBIO) and cDNA was synthesized with reference to the AugeGreenTM qPCR Master Mix (US EVERBRIGHT, UE) kit for the step-by-step method using cDNA as the template and actin as the internal reference gene [27]. The qRT-PCR system was configured with 20ul, and the relative expression of genes was calculated using the 2-ΔΔCt method with an ABI7500 PCR instrument from Applied Biosystems for real-time quantification of fluorescence [32]. qRT-PCR was performed in triplicate for each gene. The qPCR temperature cycler was set to 95 °C for 2 min, 95 °C for 5 s, 60 °C for 30 s, and 45 cycles. Primer design was performed using Primer 5 software (Additional file 2).

Exogenous spraying test

The test varieties were the resistant variety HN16 and susceptible variety BTX623, while qiansan naringenin, luteolin, and henistein (95% purity, purchased on August 5, 2022, San Land, USA) were chosen as metabolites. Aphids were extracted from other plants that had been infested earlier in other fields.The field spraying concentrations were set to 0, 0.2‰, and 0.1‰ (Additional file 3). The metabolites were sprayed on the front and back of the sorghum leaves, and three plants were sprayed in each treatment, with each treatment replicated three times.

Results

Microstructure of sorghum leaves of different resistant varieties

The epidermal structure of the leaves differed significantly between the aphid-susceptible varieties (Fig. 1A, Additional file 4: L4-6) and aphid-resistant varieties (Fig. 1B, Additional file 4: L7-9). The epidermal cells of the aphid-resistant varieties (HN16, Silimei, and BTX428) were neatly and tightly arrange while those of the aphid-sensitive varieties (BTX623, QianSan, and 3197 B) were irregular in shape, uneven in cell size, and unevenly arranged, and had large cell gaps. Evaluation of the epidermal cell characteristics and leaf thickness and analysis of significant differences showed that bose inand aphid-resistant varieties and aphid-susceptible varieties (Additional file 4), the longitudinal and transverse diameters of upper epidermal cells differed insignificantly. In aphid-resistant varieties the longitudinal and transverse diameters of lower epidermal cells differed more and were closer to a rectangular shape compared with that in aphid-susceptible varieties, which were closer to a round shape. In aphid-resistant varieties, the cell gap was smaller and the cells were more tightly arranged, in aphid-susceptible varieties, which presented a larger cell gap and loosely arranged cells, the differences were significant. The difference in blade thickness was highly significant, with aphid-resistant varieties presenting thinner blades than aphid-susceptible varieties. In summary, the resistance of sorghum to aphids was positively correlated with the regularity of epidermal cells and negatively correlated with intercellular space and leaf thickness.Fig. 1 Microstructure of sorghum leaves and changes in aphid population after three days of inoculation. A Microstructure of leaves of sensitive sorghum varieties and their aphid population after three days of inoculation with aphids. B Microstructure of leaves of resistant sorghum varieties and their aphid population after three days of inoculation with aphids. Note: LDEC, longitudinal diameter of upper epidermal cells; TDUEC, transverse diameter of upper epidermal cells; LDLEC, Longitudinal diameter of lower epidermal cell; LECTD, lower epidermal cell transverse diameter; LEC L/T: lower epidermal cells longitudinal/transverse; CG, cell gap; BT, blade thickness; TECW, thickness of epidermal cell wall

Transcriptomic analysis of sorghum leaves

After sequencing quality control, a total of 78.68 GB of clean data were obtained. The clean data from each sample reached 7.12 GB, and the distribution of Q30 bases in each sample ranged from 94.67% to 94.95%. The GC content ranged from 53.09% to 55.06%. Clean reads were subjected to sequence alignment with the specified reference genome, with alignment efficiencies ranging from 90.17% to 94.46% (Additional file 5).

A total of 3,462 new genes were identified and 1,572 genes were annotated throughout the project. HN16 and BTX623 produced the highest number of differentially expressed genes three days after aphid infestation (Fig. 2A). Therefore, we further analyzed the differentially expressed genes in HN16 and BTX623 infested with aphids for 0 and 3 days. Among them, 7526 differential genes were identified in HN16, including 4113 upregulated genes, which were mainly enriched in the plant–pathogen interaction and MAPK signaling pathway, that is, plant, starch, and sucrose metabolism, and 3413 downregulated genes, which were mainly enriched in photosynthesis, that is, antenna proteins, photosynthesis, circadian rhythm, plant, and other pathways. A total of 4958 differential genes were identified in BTX623, including 3260 upregulated genes, mainly enriched in flavonoid biosynthesis, phenylpropanoid biosynthesis, and glutathione metabolism pathways. There were 1698 downregulated genes, mainly in photosynthesis-antenna proteins, photosynthesis, circadian rhythm, plant, and other pathways (Additional file 6, Sheets 1–2).Fig. 2 Transcriptome and metabolome analysis of different comparison groups after sorghum aphid infestation. A Number of DEGs in sorghum infested with aphids at different days after infestation compared to the control. B DEG Veen plot of different varieties of sorghum infested with aphids compared to the control. C Heat map of expression clustering of DEGs for each treatment. D DEG GO enrichment map after sorghum infestation by aphids compared to the control. E Histogram of DEG KEGG enrichment after sorghum infestation by aphids compared to the control. F Taxonomic map of metabolites detected through metabolomics of sorghum leaves. G KEGG enrichment map of DAMs after sorghum infestation by aphids compared to the control. H DAMs Veen diagram of different varieties of sorghum infested with aphids compared to the control. I Number of DAMs after sorghum infestation by aphids compared to the control. J Sorghum infestation by aphids after joint analysis with control compared to KEGG. Note: In panels A, D, E, G, and J, both left plots are HN16 and both right plots are BTX623; and in panels B and H, both left plots are upregulated and both right plots are downregulated

In the comparison with the Venn diagram of CKh vs. Th3 and CKb vs. Tb3 (Fig. 2B), 1741 differential genes were co-upregulated and 889 differential genes were co-downregulated. There were 1519 and 809 DEGs that were only downregulated in BTX623, and 2372 and 2524 DEGs that were only downregulated in HN16. The results showed that some genes changed at the transcriptional level after sorghum leaves were invaded by aphids. The clustering heat map shows the changes in the expression of each differential gene (Fig. 2C). GO analysis showed that the DEGs were enriched in three major functional categories, molecular function (MF), cellular component (CC), and biological process (BP) (Additional file 7, Sheets 1–6). DEGs in CKh compared with Th3 were enriched in all three GO classes. In the CC class, the integral components of the membrane, intracellular membrane-bounded organelles, and plasma membranes were the most enriched. In the BP and MF classes, RNA modification and protein serine/threonine kinase activity were the most abundant. DEGs of CKb relative to Tb3 were most abundant in CC and MF. The integral component of the membrane was most abundant in the CC class, and iron-ion binding was most abundant in the MF class (Fig. 2D).

To further analyze the biological functions of the DEGs, we performed KEGG enrichment analysis (Fig. 2E). Differential genes were upregulated in the comparison between CKh and Th3 and mainly related to plant–pathogen interactions, MAPK signaling pathway-plants, and flavonoid biosynthesis. In the comparison between CKb and Tb3, the upregulated genes were associated with flavonoid biosynthesis, phenylpropanoid biosynthesis, and glutathione metabolism. The downregulated genes in the two comparison groups were related to photosynthesis–antenna proteins, photosynthesis, and circadian rhythm-plants. These results show that aphid infestation inhibited the expression of genes related to photosynthesis antenna proteins, circadian rhythm, and decreased photosynthesis in sorghum. However, porphyrin and chlorophyll metabolism, benzoxazinoid biosynthesis, glyoxylate and dicarboxylate metabolism, and carotenoid biosynthesis were only downregulated in HN16 after aphid infestation. Plant hormone signal transduction was only upregulated in HN16 but not in aphid-infested BTX623. The upregulated metabolic pathways specific to BTX623 were phenylpropanoid biosynthesis, glutathione metabolism, biosynthesis of amino acids, and alpha-linolenic acid metabolism. This suggests that there may be differences in the mechanisms of different resistant varieties in response to aphid stress.

Metabolomic analysis of sorghum leaves

Using the UPLC-MS/MS detection platform, we detected 1106 metabolites. Subsequently, all the metabolites were classified into 11 major groups, the most diverse of which comprised 358 flavonoids, 171 phenolic acids, and 121 lipids (Fig. 2F). Many differential metabolites were identified in different comparisons, including 247 upregulated and 81 downregulated metabolites in the comparison of CKh with Th3, and 108 upregulated and 225 downregulated metabolites in the comparison of CKb with Tb3 (Fig. 2I, Additional file 8, Sheet 1).

The Venn diagram showed that there were 110 common upregulated metabolites in the comparison of CKh vs. Th3 and CKb vs. Tb3. Among these, flavonoids were the most abundant, comprising 74 types, accounting for 67.27% of the total upregulated metabolites. This was followed by lipids, comprising 14 types, accounting for 12.73% of the total upregulated metabolites, five types of common downward adjustment, one type of amino acid and their derivatives, one type of organic acid, two types of nucleotides and their derivatives, and one type of phenolic acid (Fig. 2h, Additional file 8 Sheet 1). The expression of the three flavonoids 2'-hydroxygenistein, naringenin, and 6-hydroxyluteolin was upregulated after aphid infestation of HN16 and BTX623. The expression of a nucleotide and its derivative, Inosine 5'-monophosphate, was downregulated after aphid infestation. In addition, 45 differential metabolites were regulated based on a comparison of CKh vs. Th3 and CKb vs. Tb3 (Additional file 8).

To further highlight the pre-and post-infestation profiles of the two sorghum varieties, we created a clustering heat map for the 160 DAMs mentioned above (Additional file 9). These metabolites were divided into nine species, that is, flavonoids, nucleotides and derivatives, and lipids. Most of the differential metabolites increased after BTX623 was infested with sorghum aphid compared to HN16 infestation, with a significant trend in the expression of flavonoid substances in BTX623 and HN16. For example, pme2960 Naringenin chalcone and Lmmp004504 2'-hydroxygenistein showed a significantly higher upward trend in expression in BTX623 than in HN16. Meanwhile, pmp000587 luteolin-7-O-(6''-malonyl) glucoside showed a significant upward trend in expression in HN16. In summary, sorghum aphid infestation had a significant effect on BTX623 and a moderate effect on HN16, particularly on flavonoid metabolites.

Based on the KEGG annotation, differential metabolites in different comparisons were enriched in many pathways, including flavonoid, amino acid, and secondary metabolite biosynthesis. Among the top 20 KEGG-enriched pathways in the different comparisons, those in the CKh vs. Th3 and CKb vs. Tb3 comparisons were the most significantly enriched in isoflavonoid biosynthesis, followed by flavonoid biosynthesis (Fig. 2G). This shows that metabolites enriched in flavonoid and isoflavonoid biosynthesis may play a role in direct or indirect defense against aphids.

Association analysis of metabolome and transcriptome

To more effectively visualize the co-enrichment of differential genes and metabolites, transcriptomic metabolomic KEGG enrichment analysis was performed (p < 0.05), and the pathway of the top 20 differential genes and metabolites for each group were enriched(Fig. 2J).The two varieties were enriched in some common functional pathways, such as flavonoid biosynthesis and isoflavonoid biosynthesis. DEGs and DAMs were enriched in both varieties, suggesting that these metabolic pathways may play a crucial role in sorghum resistance against aphids(Fig. 3A). Therefore, we analyzed the main DEGs and DAMs involved in these pathways. By comparing CKb vs. Tb3 and CKh vs. Th3, we identified 40 flavonoid biosynthesis-related genes, including chalcone synthase (CHS), isoflavone 3'-hydroxylase (I3’H), cytochrome P450 CYP73A100(P450), hydroxycinnamoyltransferase (HCT), and isoflavone 2'-hydroxylase (I2’H) (Additional file 10, Sheet 1). Among these 40 genes, the expression of 36 genes was significantly upregulated while that of 4 genes was significantly downregulated between CKb and Tb3. In addition, the expression of 33 genes was significantly upregulated while that of 7 genes was significantly downregulated between CKh and Th3. In the metabolomic analysis, flavonoid biosynthesis involved 18 differential metabolites such as naringenin, genistein, and apigenin. These metabolites were significantly upregulated in BTX623 and HN16 (Additional file 10, Sheet 2).Fig. 3 Flavonoid metabolic pathway and exogenous spraying (A) Flavonoid metabolic pathway, key genes, and key metabolites in red, DEG expression heat map CKb vs. Tb3 on the left, and CKh vs. Th3 on the right. B Transcriptome of different genes with real-time quantitative bar graphs. C Statistical plot of the aphid population after exogenous spraying of metabolites on sorghum leaves; * indicates a significant difference (p < 0.05) and n.s. indicates no significant difference. Note: The copyright license has been applied for

qRT-PCR

To validate the differential expression results obtained from the transcriptome analysis, we examined the relative expression levels of the nine selected regulatory genes using qRT-PCR. These genes are involved in flavonoid biosynthesis, including eugenol O-methyltransferase-like, C4H, CHS, and cytochrome P450 CYP73A100. The expression patterns of DEGs obtained using RNA-seq and qRT-PCR were highly consistent, highlighting the reliability of the RNA-seq results (Fig. 3B).

Validation of flavonoid metabolite spraying

To verify whether the screened metabolites would affect aphid resistance in sorghum, three sorghum varieties with different aphid resistance were selected for exogenous spraying experiments, they were the aphid-resistant varieties HN16 and aphid-sensitive varieties BTX623 and Qiansan. Among the metabolites involved in the flavonoid biosynthesis pathways, three metabolites, that is, naringenin, luteolin, and genistein, were used for foliar spraying using more mature extraction techniques, more widespread plant sources, and lower application costs. The results showed that all three metabolites had a greater control effect on aphids. Aphid reduction in the different resistant varieties was higher than those without the metabolites sprayed (Fig. 3C). However, the control effect of spraying different concentrations of metabolic substances was different, and the different treatments showed that the number of aphids sprayed with 0.2‰ concentration was significantly higher than that of 0.1‰ concentration. This indicates that spraying 0.1‰ concentration of metabolic substances could play a more effective role in controlling the aphid population. Three days after spraying, genistein at 0.1‰ concentration was more effective against aphids in BTX623, followed by naringenin at 0.1‰ concentration; naringenin at 0.1‰ concentration was more effective against aphids in QianSan, followed by genistein at 0.1‰ concentration, at HN16, the aphid population in all groups showed a decreasing trend, but spraying with 1‰ genistein showed the fastest decreasing trend, followed by spraying with 1‰ naringenin. This shows that spraying 1‰ concentration naringenin and 1‰ concentration genistein was more effective than spraying luteolin for aphid control.

Discussion

Physical defenses are the first line of defense against herbivorous insects and are mainly morphological and anatomical features that can directly prevent feeding by herbivorous insects and provide plants with a fitness advantage [33]. This includes prominent protrusions on the plant and microscopic changes in cell wall thickness from lignification and sub lignification [33–35]. The present study has shown that sorghum aphid resistance was positively correlated with epidermal cell arrangement tightness and negatively correlated with leaf thickness, which is in line with the results of Chang et al. [36]. In contrast, Luo et al. [37]. showed that cotton leaf thickness was not related to resistance to the Apolygus lucorum. Zhong et al. [38]showed that aphid resistance in tea trees was positively correlated with leaf thickness. These results indicate the differential activity of aphids in different crops.

Transcriptomic and metabolomic analyses have become common tools for evaluating interactions between plants and herbivorous insects [39]. In this study, we used a combined transcriptomic and metabolomic approach to comparatively analyze the genetic and metabolic changes involved in different genotypes of sorghum subjected to aphid infestation. This has increased our understanding of the potential mechanisms by which sorghum responds to aphid infestation. Studies have shown that in sorghum, rice and maize plants infested by sorghum aphids, rice stem borers (Chilo suppressalis), and corn aphids, the number of upregulated DEGs is higher than the number of downregulated DEG [40–42], indicating that insect feeding triggers host transcriptome recombination. Similarly, our DEG analysis showed that there were more upregulated DEGs than downregulated DEGs in response to aphid infestation. However, Wang et al. [43] showed that more DEGs were downregulated than upregulated when rice was infested with brown flies. Li et al. [44] showed that the number of upregulated and downregulated DEGs was similar when cotton was infested with sooty flies. These diverse results may be related to differences in aphid resistance between sorghum and other plants, differences in feeding between sorghum aphids and other insects, differences in the number of leaves infested by sorghum aphids compared to the number of plants stimulated by other herbivorous insects, or differences in the techniques used to detect gene expression.

The infestation of sorghum aphids can initiate a direct defense response of sorghum, induce flavonoid biosynthesis gene expression, and promote flavonoid production. The flavonoid biosynthesis is the most abundant pathway in sorghum after aphid infestation. In this pathway, aphid feeding increased gene expression of enzymes involved in sorghum flavonoid biosynthesis, including CHS, I3 'H, and HCT. This is consistent with previous research on transferase activities upregulated in later stages of infestation [45]. Activation of signal transduction pathways after an insect attack leads to concomitant changes in plant secondary metabolism [46]. Metabolomic analysis showed that the generalized upregulation of flavonoid biosynthesis and isoflavonoid biosynthesis under insect feeding seems to be a common strategy for sorghum to resist aphid feeding. Some metabolic substances in the pathway, such as genistein, naringenin, and naringenin chalcones, were significantly increased in abundance. This result further suggests that flavonoids may play a key role in their defense against aphids.

In a previous study by our experimental group, the sorghum aphid resistance gene RMES1 was localized and cloned in HN16 [47]. The results showed that sorghum material carrying the RMES1 gene was significantly more resistant to aphids than material that did not carry the RMES1 gene or material that had a mutation in the RMES1 gene. For example, HN16, which carries the RMES1 gene, is more resistant to aphids than BTX623, which does not carry the RMES1 gene. The differential genes screened in this study were mainly enriched in the flavonoid bioconjugate completion pathway. This is a major class of phytochemicals accounting for 5–10% of the known plant secondary metabolites [48]. Most of these have strong antioxidant activity and protect plants from insect pests by influencing the behavior, growth, and development of insects [49]. Depending on the dose applied, this group of metabolites can have different effect on the insect's feeding behavior, survival, and development [50–52]. The results have shown that naringenin, genistein, and luteolin reduced the number of aphids in sensitive plants while naringenin and genistein significantly increased aphid resistance in sensitive sorghum. This is consistent with the results of previous studies that demonstrated that naringenin, luteolin, and its derivatives have antibacterial and insecticidal activities [53–59]. The reduction in aphid numbers was significantly higher at 0.1‰ than at the other two concentrations. The results show that aphid feeding resistance does not necessarily increase with a higher concentration of metabolites. Previous studies have confirmed that exogenous JA has dual effects on aphid resistance of sorghum and will promote the growth and reproduction of aphids when the concentration exceeds the sensitivity of aphids [60]. The flavotoxin compounds extracted from Fusarium petiolate showed strong insecticidal activity at low concentrations [61]. Therefore, we concluded that sprayed flavonoids have dual effects on sorghum's resistance to aphid feeding. The reason for this phenomenon may be that the plant absorbs excessive flavonoids, resulting in excessive production and accumulation of ROS, which destroys the balance between oxidants and antioxidants and affects the ability of sorghum to resist aphids [62].

Conclusion

We observed the microstructure of the leaves of sorghum varieties with different levels of aphid resistance and found that the aphid resistance of sorghum is related to the leaf cell morphology, cell arrangement closeness, and blade thickness. A joint analysis of the transcriptome and metabolome was performed to obtain a large dataset related to sorghum response to aphid infestation. The results of the transcriptome–metabolome association analysis indicated that flavonoid biosynthesis plays an important role in the response of sorghum to aphids. To verify whether flavonoids affected sorghum resistance to aphids, we designed an exogenous spray experiment in which three concentrations (0, 0.1‰, and 0.2%) were selected for foliar application on the three sorghum varieties. The exogenous spraying of flavonoids showed that naringenin and genistein effectively increased the resistance of aphid-sensitive plants. The genes and metabolites identified in this study have provided new insights into the mechanisms underlying the response of sorghum to aphid infestation, and exogenous spraying of flavonoids may represent a potential approach for the biological control of sorghum aphids. However, the optimal application concentrations and mechanism of action in sorghum aphid resistance need to be further explored.

Supplementary Information

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Acknowledgements

Thanks to Biomarker, Metware Cloud and majorbio for their technical support in this study.

Authors’ contributions

XZ writing manuscript, data investigation and analysis. DZ and LZ assisted investigation, JCu and JCh designed the methodology and framework of this study, project management, manuscript review and revision. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by the Hebei Key Research & Development Program (19226370D and 21326305D).

Availability of data and materials

The dataset provided in this study has been uploaded to NCBI, The names of the repository/repositories and accession number(s) can be found below: BioProject, PRJNA1009505 (https://www.ncbi.nlm.nih.gov/sra/PRJNA1009505).

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Jinhua Chang as common communication. 

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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References

1. Chamarthi SK. Biochemical mechanisms of resistance to shoot fly, Atherigona soccata (rondani) in sorghum, sorghum bicolor (L.). Moench. J Appl Entomol. 2008.10.1111/j.1439-0418.2010.01564.x.
2. War AR Paulraj MG Ahmad T Buhroo AA Hussain B Ignacimuthu S Sharma HC Mechanisms of plant defense against insect herbivores Plant Signal Behav 2012 7 10 1306 1320 10.4161/psb.21663 22895106
War AR, Paulraj MG, Ahmad T, Buhroo AA, Hussain B, Ignacimuthu S, Sharma HC. Mechanisms of plant defense against insect herbivores. Plant Signal Behav. 2012;7(10):1306–20. 10.4161/psb.21663. 22895106 10.4161/psb.21663
3. Xin Z Wang M Cuevas HE Chen J Harrison M Pugh NA Morris G Sorghum genetic, genomic, and breeding resources Planta 2021 254 6 114 10.1007/s00425-021-03742-w 34739592
Xin Z, Wang M, Cuevas HE, Chen J, Harrison M, Pugh NA, Morris G. Sorghum genetic, genomic, and breeding resources. Planta. 2021;254(6):114. 10.1007/s00425-021-03742-w.34739592 10.1007/s00425-021-03742-w
4. Wang X Gowik U Tang H Bowers JE Westhoff P Paterson AH Comparative genomic analysis of C4 photosynthetic pathway evolution in grasses Genome Biol 2009 10 6 R68 10.1186/gb-2009-10-6-r68 19549309
Wang X, Gowik U, Tang H, Bowers JE, Westhoff P, Paterson AH. Comparative genomic analysis of C4 photosynthetic pathway evolution in grasses. Genome Biol. 2009;10(6):R68. 10.1186/gb-2009-10-6-r68.19549309 10.1186/gb-2009-10-6-r68
5. Armstrong JS Rooney WL Peterson GC Villenueva RT Brewer MJ Sekula-Ortiz D Sugarcane Aphid (Hemiptera: Aphididae): host range and sorghum resistance including cross-resistance from greenbug sources J Econ Entomol 2015 108 2 576 582 10.1093/jee/tou065 26470168
Armstrong JS, Rooney WL, Peterson GC, Villenueva RT, Brewer MJ, Sekula-Ortiz D. Sugarcane Aphid (Hemiptera: Aphididae): Host Range and Sorghum Resistance Including Cross-Resistance From Greenbug Sources. J Econ Entomol. 2015;108(2):576–82. 10.1093/jee/tou065. (Epub 2015 Jan 30 PMID: 26470168). 26470168 10.1093/jee/tou065
6. Bowling, R. D., Brewer, M. J., Kerns, D. L., Gordy, J., Seiter, N., Elliott, N. E., Buntin, G. D., Way, M. O., Royer, T. A., Biles, S., Maxson, E.(2016). Sugarcane aphid (Hemiptera: Aphididae): A new pest of sorghum in North America. Pest Management Science. 7(1). 10.1093/jipm/pmw011.
7. Paudyal S Armstrong JS Giles KL Hoback W Aiken R Payton ME Differential responses of sorghum genotypes to sugarcane aphid feeding Planta 2020 252 1 14 10.1007/s00425-020-03419-w 32621008
Paudyal S, Armstrong JS, Giles KL, Hoback W, Aiken R, Payton ME. Differential responses of sorghum genotypes to sugarcane aphid feeding. Planta. 2020;252(1):14. 10.1007/s00425-020-03419-w. PMID: 32621008.32621008 10.1007/s00425-020-03419-w
8. Singh BU Padmaja PG Seetharama N Biology and management of the sugarcane aphid, Melanaphis sacchari (Zehntner) (Homoptera: Aphididae), in sorghum: A review Crop Protection 2004 23 739 755 10.1016/j.cropro.2004.01.004
Singh BU, Padmaja PG, Seetharama N. Biology and management of the sugarcane aphid, Melanaphis sacchari (Zehntner) (Homoptera: Aphididae), in sorghum: A review. Crop Protection. 2004;23:739–55. 10.1016/j.cropro.2004.01.004.10.1016/j.cropro.2004.01.004
9. Chang CP Fang MN Tseng HY Studies on the life history and varietal resistance in grain sorghum aphid, Melanaphis sacchari Zehntner in central Taiwan Chin J Entomology 1982 2 70 81
Chang CP, Fang MN, Tseng HY. Studies on the life history and varietal resistance in grain sorghum aphid, Melanaphis sacchari Zehntner in central Taiwan. Chinese Journal of Entomology. 1982;2:70–81.
10. Brewer MJ Gordy JW Kerns DL Woolley JB Rooney WL Bowling RD Sugarcane aphid population growth, plant injury, and natural enemies on selected grain sorghum hybrids in Texas and Louisiana J Econ Entomol 2017 110 5 2109 2118 10.1093/jee/tox204 28962004
Brewer MJ, Gordy JW, Kerns DL, Woolley JB, Rooney WL, Bowling RD. Sugarcane Aphid Population Growth, Plant Injury, and Natural Enemies on Selected Grain Sorghum Hybrids in Texas and Louisiana. J Econ Entomol. 2017;110(5):2109–18. 10.1093/jee/tox204. 28962004 10.1093/jee/tox204
11. Szczepaniec A Assessment of a density-based action threshold for suppression of sugarcane aphids, (Hemiptera: Aphididae), in the Southern high plains J Econ Entomol 2018 111 5 2201 2207 10.1093/jee/toy176 29955822
Szczepaniec A. Assessment of a density-based action threshold for suppression of sugarcane aphids, (Hemiptera: Aphididae), in the Southern high plains. J Econ Entomol. 2018;111(5):2201–7. 10.1093/jee/toy176.29955822 10.1093/jee/toy176
12. Zapata SD Dudensing R Sekula D Esparza-Díaz G Villanueva R Economic impact of the sugarcane aphid outbreak in South Texas. Agricultural and Applied Economics Association Agricultural and Applied Economics Association 2016 50 104 128
Zapata SD, Dudensing R, Sekula D, Esparza-Díaz G, Villanueva R. Economic Impact of the Sugarcane Aphid Outbreak in South Texas. Agricultural and Applied Economics Association. Agricultural and Applied Economics Association. 2016;50:104–28.
13. Farquharson KL A phloem protein contributes to aphid resistance and heat stress tolerance Plant Cell 2017 29 10 2309 2310 10.1105/tpc.17.00781 28978759
Farquharson KL. A phloem protein contributes to aphid resistance and heat stress tolerance. Plant Cell. 2017;29(10):2309–10. 10.1105/tpc.17.00781. Epub 2017 Oct 4. PMID: 28978759; PMCID: PMC5774563.28978759 10.1105/tpc.17.00781
14. Hare JD Ecological role of volatiles produced by plants in response to damage by herbivorous insects Annu Rev Entomol 2011 56 161 180 10.1146/annurev-ento-120709-144753 21133760
Hare JD. Ecological role of volatiles produced by plants in response to damage by herbivorous insects. Annu Rev Entomol. 2011;56:161–80. 10.1146/annurev-ento-120709-144753.21133760 10.1146/annurev-ento-120709-144753
15. Howe GA Jander G Plant immunity to insect herbivores Annu Rev Plant Biol 2008 59 41 66 10.1146/annurev.arplant.59.032607.092825 18031220
Howe GA, Jander G. Plant immunity to insect herbivores. Annu Rev Plant Biol. 2008;59:41–66. 10.1146/annurev.arplant.59.032607.092825. PMID: 18031220.18031220 10.1146/annurev.arplant.59.032607.092825
16. Verhage A van Wees SC Pieterse CM Plant immunity: it's the hormones talking, but what do they say? Plant Physiol 2010 154 2 536 540 10.1104/pp.110.161570 20921180
Verhage A, van Wees SC, Pieterse CM. Plant immunity: it’s the hormones talking, but what do they say? Plant Physiol. 2010;154(2):536–40. 10.1104/pp.110.161570.20921180 10.1104/pp.110.161570
17. Hanley ME Lamont BB Fairbanks MM Rafferty CM Plant structural traits and their role in antiherbivore defense PPEES 2007 8 157 178 10.1016/j.ppees.2007.01.001
Hanley ME, Lamont BB, Fairbanks MM, Rafferty CM. Plant structural traits and their role in antiherbivore defense. PPEES. 2007;8:157–78. 10.1016/j.ppees.2007.01.001.10.1016/j.ppees.2007.01.001
18. Karban R The ecology and evolution of induced resistance against herbivores Funct Ecol 2011 25 339 347 10.1111/een.12771
Karban R. The ecology and evolution of induced resistance against herbivores. Funct Ecol. 2011;25:339–47. 10.1111/een.12771. 10.1111/een.12771
19. Agrawal AA Current trends in the evolutionary ecology of plant defense Funct Ecol 2011 25 420 432 10.1111/j.1365-2435.2010.01796.x
Agrawal AA. Current trends in the evolutionary ecology of plant defense. Funct Ecol. 2011;25:420–32. 10.1111/j.1365-2435.2010.01796.x.10.1111/j.1365-2435.2010.01796.x
20. Sharma HC Biotechnological approaches for pest management and ecological sustainability 2009 New York, USA.526 CRC Press/Taylor and Francis
Sharma HC. Biotechnological approaches for pest management and ecological sustainability. New York, USA.526: CRC Press/Taylor and Francis; 2009.
21. Handley R Ekbom B Agren J Variation in trichome density and resistance against a specialist insect herbivore in natural populations of Arabidopsis thaliana Ecological Entomology 2005 30 284 292 10.1111/j.0307-6946.2005.00699.x
Handley R, Ekbom B, Agren J. Variation in trichome density and resistance against a specialist insect herbivore in natural populations of Arabidopsis thaliana. Ecological Entomology. 2005;30:284–92. 10.1111/j.0307-6946.2005.00699.x. 10.1111/j.0307-6946.2005.00699.x
22. Morant AV Jørgensen K Jørgensen C Paquette SM Sánchez-Pérez R Møller BL Bak S Beta-glucosidases as detonators of plant chemical defense Phytochemistry 2008 69 9 1795 1813 10.1016/j.phytochem.2008.03.006 18472115
Morant AV, Jørgensen K, Jørgensen C, Paquette SM, Sánchez-Pérez R, Møller BL, Bak S. Beta-glucosidases as detonators of plant chemical defense. Phytochemistry. 2008;69(9):1795–813. 10.1016/j.phytochem.2008.03.006.18472115 10.1016/j.phytochem.2008.03.006
23. Walling LL The myriad plant responses to herbivores Plant Growth Regul 2000 19 195 216 10.1007/s003440000026
Walling LL. The myriad plant responses to herbivores. Plant Growth Regul. 2000;19:195–216. 10.1007/s003440000026.10.1007/s003440000026
24. Tetreault HM Grover S Scully ED Gries T Palmer NA Sarath G Louis J Sattler SE Global Responses of Resistant and Susceptible Sorghum (Sorghum bicolor) to Sugarcane Aphid (Melanaphis sacchari) Front Plant Sci 2019 22 10 145 10.3389/fpls.2019.00145
Tetreault HM, Grover S, Scully ED, Gries T, Palmer NA, Sarath G, Louis J, Sattler SE. Global Responses of Resistant and Susceptible Sorghum (Sorghum bicolor) to Sugarcane Aphid (Melanaphis sacchari). Front Plant Sci. 2019;22(10):145. 10.3389/fpls.2019.00145. PMID: 30853964; PMCID: PMC6396740.10.3389/fpls.2019.00145
25. Cheng WN Lei JX Rooney WL Liu TX Zhu-Salzman K High basal defense gene expression determines sorghum resistance to the whorl-feeding insect southwestern corn borer Insect Sci 2013 20 3 307 17 10.1111/1744-7917.12002 23955883
Cheng WN, Lei JX, Rooney WL, Liu TX, Zhu-Salzman K. High basal defense gene expression determines sorghum resistance to the whorl-feeding insect southwestern corn borer. Insect Sci. 2013;20(3):307–17. 10.1111/1744-7917.12002. Epub 2013 Jan 14. PMID: 23955883.23955883 10.1111/1744-7917.12002
26. Padmaja PG, Aruna C. Advances in Sorghum Insect Pest Resistance. Tonapi, In Woodhead Publishing Series in Food Science, Technology and Nutrition, Breeding Sorghum for Diverse End Uses, Woodhead Publishing. 293–312, ISBN 9780081018798. 2019.10.1016/B978-0-08-101879-8.00018-8.
27. Zhang H Wei Y Jia H Chen D Tang X Wang J Chen M Guo Y Immune activation of characteristic gut mycobiota Kazachstania pintolopesii on IL-23/IL-17R signaling in ankylosing spondylitis Front Cell Infect Microbiol 2022 20 12 1035366 10.3389/fcimb.2022.1035366
Zhang H, Wei Y, Jia H, Chen D, Tang X, Wang J, Chen M, Guo Y. Immune activation of characteristic gut mycobiota Kazachstania pintolopesii on IL-23/IL-17R signaling in ankylosing spondylitis. Front Cell Infect Microbiol. 2022;20(12):1035366. 10.3389/fcimb.2022.1035366. PMID: 36605130; PMCID: PMC9808786.10.3389/fcimb.2022.1035366
28. Li S Qi L Tan X Li S Fang J Ji R Small brown planthopper nymph infestation regulates plant defenses by affecting secondary metabolite biosynthesis in rice Int J Mol Sci 2023 24 5 4764 10.3390/ijms24054764 36902211
Li S, Qi L, Tan X, Li S, Fang J, Ji R. Small brown planthopper nymph infestation regulates plant defenses by affecting secondary metabolite biosynthesis in rice. Int J Mol Sci. 2023;24(5):4764. 10.3390/ijms24054764.36902211 10.3390/ijms24054764
29. Li H Yu X Meng F Zhao Z Guan S Wang L Ferulic acid supplementation increases lifespan and stress resistance via insulin/IGF-1 signaling pathway in C. elegans Int J Mol Sci 2021 22 4279 10.3390/ijms22084279 33924155
Li H, Yu X, Meng F, Zhao Z, Guan S, Wang L. Ferulic acid supplementation increases lifespan and stress resistance via insulin/IGF-1 signaling pathway in C. elegans. Int J Mol Sci. 2021;22:4279. 10.3390/ijms22084279. 33924155 10.3390/ijms22084279
30. Wang F Chen L Chen S Chen H Liu Y Characterization of two closely related citrus cultivars using UPLC-ESI-MS/MS-based widely targeted metabolomics PLoS One 2021 16 7 e0254759 10.1371/journal.pone.0254759 34283861
Wang F, Chen L, Chen S, Chen H, Liu Y. Characterization of two closely related citrus cultivars using UPLC-ESI-MS/MS-based widely targeted metabolomics. PLoS One. 2021;16(7):e0254759. 10.1371/journal.pone.0254759. PMID: 34283861; PMCID: PMC8291699.34283861 10.1371/journal.pone.0254759
31. Yang L Qi S Touqeer A Li H Zhang X Liu X Wu S SlGT11 controls floral organ patterning and floral determinacy in tomato BMC Plant Biol 2020 20 1 562 10.1186/s12870-020-02760-2 33317459
Yang L, Qi S, Touqeer A, Li H, Zhang X, Liu X, Wu S. SlGT11 controls floral organ patterning and floral determinacy in tomato. BMC Plant Biol. 2020;20(1):562. 10.1186/s12870-020-02760-2. PMID: 33317459; PMCID: PMC7734826.33317459 10.1186/s12870-020-02760-2
32. Livak KJ Schmittgen TD Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method Methods 2001 25 402 408 10.1006/meth.2001.1262 11846609
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 2001;25:402–8.11846609 10.1006/meth.2001.1262
33. Agrawal AA Fishbein M Jetter R Salminen JP Goldstein JB Freitag AE Sparks JP Phylogenetic ecology of leaf surface traits in the milkweeds (Asclepias spp.): chemistry, ecophysiology, and insect behavior New Phytol 2009 183 3 848 867 10.1111/j.1469-8137.2009.02897.x 19522840
Agrawal AA, Fishbein M, Jetter R, Salminen JP, Goldstein JB, Freitag AE, Sparks JP. Phylogenetic ecology of leaf surface traits in the milkweeds (Asclepias spp.): chemistry, ecophysiology, and insect behavior. New Phytol. 2009;183(3):848–67. 10.1111/j.1469-8137.2009.02897.x.19522840 10.1111/j.1469-8137.2009.02897.x
34. Arimura G Matsui K Takabayashi J Chemical and molecular ecology of herbivore-induced plant volatiles: proximate factors and their ultimate functions Plant Cell Physiol 2009 50 5 911 923 10.1093/pcp/pcp030 19246460
Arimura G, Matsui K, Takabayashi J. Chemical and molecular ecology of herbivore-induced plant volatiles: proximate factors and their ultimate functions. Plant Cell Physiol. 2009;50(5):911–23. 10.1093/pcp/pcp030.19246460 10.1093/pcp/pcp030
35. He J Chen F Chen S Lv G Deng Y Fang W Liu Z Guan Z He C Chrysanthemum leaf epidermal surface morphology and antioxidant and defense enzyme activity in response to aphid infestation J Plant Physiol 2011 168 7 687 693 10.1016/j.jplph.2010.10.009 21145126
He J, Chen F, Chen S, Lv G, Deng Y, Fang W, Liu Z, Guan Z, He C. Chrysanthemum leaf epidermal surface morphology and antioxidant and defense enzyme activity in response to aphid infestation. J Plant Physiol. 2011;168(7):687–93. 10.1016/j.jplph.2010.10.009. 21145126 10.1016/j.jplph.2010.10.009
36. Chang JH Zhang L Xia XY Li RR Luo YW Liu GQ Relationship between physical traits and aphid resistance of sorghum plants with different genotypes Journal of Agricultural University of Hebei. 2004 24 2 5 7
Chang, J. H., Zhang, L., Xia, X. Y., Li, R. R., Luo, Y. W., Liu, G. Q. (2004) . Relationship between Physical Traits and aphid resistance of sorghum plants with different genotypes. Journal of Agricultural University of Hebei. 24(2), 5–7. (in Chinese).
37. Luo JY Cui JJ Huang Q Relationship between chlorophyll, wax content and leaf thickness of cotton leaf and resistance to Chlorophorus viridis J Plant Prot 2011 38 04 320 326
Luo, J. Y., Cui, J. J., Huang, Q. (2011) . Relationship between chlorophyll, wax content and leaf thickness of cotton leaf and resistance to Chlorophorus viridis. Journal of Plant Protection. 38(04), 320–326. (in Chinese).
38. Zhong MY Study on resistance of different tea cultivars to tea aphid 2018 Guiyang Guizhou University
Zhong, M. Y. (2018) . Study on resistance of different tea cultivars to tea aphid. Guiyang: Guizhou University. (in Chinese).
39. Wang Q Liu X Liu H Fu Y Cheng Y Zhang L Shi W Zhang Y Chen J Transcriptomic and metabolomic analysis of wheat kernels in response to the feeding of orange wheat blossom midges (Sitodiplosis mosellana) in the Field J Agric Food Chem 2022 70 5 1477 1493 10.1021/acs.jafc.1c06239 35090120
Wang Q, Liu X, Liu H, Fu Y, Cheng Y, Zhang L, Shi W, Zhang Y, Chen J. Transcriptomic and Metabolomic Analysis of Wheat Kernels in Response to the Feeding of Orange Wheat Blossom Midges (Sitodiplosis mosellana) in the Field. J Agric Food Chem. 2022;70(5):1477–93. 10.1021/acs.jafc.1c06239. 35090120 10.1021/acs.jafc.1c06239
40. Puri H Grover S Pingault L Sattler SE Louis J Temporal transcriptomic profiling elucidates sorghum defense mechanisms against sugarcane aphids BMC Genomics 2023 24 1 441 10.1186/s12864-023-09529-5 37543569
Puri H, Grover S, Pingault L, Sattler SE, Louis J. Temporal transcriptomic profiling elucidates sorghum defense mechanisms against sugarcane aphids. BMC Genomics. 2023;24(1):441. 37543569 10.1186/s12864-023-09529-5
41. Liu Q Wang X Tzin V Romeis J Peng Y Li Y Combined transcriptome and metabolome analyses to understand the dynamic responses of rice plants to attack by the rice stem borer Chilo suppressalis (Lepidoptera: Crambidae) BMC Plant Biol 2016 16 1 259 10.1186/s12870-016-0946-6 27923345
Liu Q, Wang X, Tzin V, Romeis J, Peng Y, Li Y. Combined transcriptome and metabolome analyses to understand the dynamic responses of rice plants to attack by the rice stem borer Chilo suppressalis (Lepidoptera: Crambidae). BMC Plant Biol. 2016;16(1):259. 10.1186/s12870-016-0946-6. 27923345 10.1186/s12870-016-0946-6
42. Tzin V Fernandez-Pozo N Richter A Schmelz EA Schoettner M Schäfer M Ahern KR Meihls LN Kaur H Huffaker A Mori N Degenhardt J Mueller LA Jander G Dynamic maize responses to aphid feeding are revealed by a time series of transcriptomic and metabolomic assays Plant Physiol 2015 169 3 1727 1743 10.1104/pp.15.01039 26378100
Tzin V, Fernandez-Pozo N, Richter A, Schmelz EA, Schoettner M, Schäfer M, Ahern KR, Meihls LN, Kaur H, Huffaker A, Mori N, Degenhardt J, Mueller LA, Jander G. Dynamic Maize Responses to Aphid Feeding Are Revealed by a Time Series of Transcriptomic and Metabolomic Assays. Plant Physiol. 2015;169(3):1727–43. https://doi.org/10.1104/pp.15.01039. 26378100 10.1104/pp.15.01039
43. Wang F Ning D Chen Y Dang C Han NS Liu Y Ye GY Comparing gene expression profiles between Bt and non-Bt rice in response to brown planthopper infestation Front Plant Sci 2015 6 1181 10.3389/fpls.2015.01181 26734057
Wang F, Ning D, Chen Y, Dang C, Han NS, Liu Y, Ye GY. Comparing gene expression profiles between Bt and non-Bt rice in response to brown planthopper infestation. Front Plant Sci. 2015;6:1181. 10.3389/fpls.2015.01181.26734057 10.3389/fpls.2015.01181
44. Li J Zhu L Hull JJ Liang S Daniell H Jin S Zhang X Transcriptome analysis reveals a comprehensive insect resistance response mechanism in cotton to infestation by the phloem feeding insect Bemisia tabaci (whitefly) Plant Biotechnol J 2016 14 10 1956 1975 10.1111/pbi.12554 26923339
Li J, Zhu L, Hull JJ, Liang S, Daniell H, Jin S, Zhang X. Transcriptome analysis reveals a comprehensive insect resistance response mechanism in cotton to infestation by the phloem feeding insect Bemisia tabaci (whitefly). Plant Biotechnol J. 2016;14(10):1956–75. 10.1111/pbi.12554. 26923339 10.1111/pbi.12554
45. Serba DD Meng X Schnable J Bashir E Michaud JP Vara Prasad PV Perumal R Comparative transcriptome analysis reveals genetic mechanisms of sugarcane aphid resistance in grain sorghum Int J Mol Sci 2021 22 13 7129 10.3390/ijms22137129 34281180
Serba DD, Meng X, Schnable J, Bashir E, Michaud JP, Vara Prasad PV, Perumal R. Comparative transcriptome analysis reveals genetic mechanisms of sugarcane aphid resistance in grain sorghum. Int J Mol Sci. 2021;22(13):7129. 10.3390/ijms22137129. 34281180 10.3390/ijms22137129
46. Iqrar I Alam A Numan M Shinwari ZK Secondary metabolites: rapid evolving weapon in the plant-microbe warfare 2021
Iqrar I, Alam A, Numan M, Shinwari ZK. Secondary metabolites: rapid evolving weapon in the plant-microbe warfare. 2021.
47. Wang F Zhao S Han Y Shao YT Dong ZY Gao Y Zhang KP Liu X Li DW Chang JH Wang DW Efficient and fine mapping of RMES1 conferring resistance to sorghum aphid Melanaphis sacchari Molecular Breeding 2013 31 4 777 784 10.1007/s11032-012-9832-6
Wang F, Zhao S, Han Y, Shao YT, Dong ZY, Gao Y, Zhang KP, Liu X, Li DW, Chang JH, Wang DW. Efficient and fine mapping of RMES1 conferring resistance to sorghum aphid Melanaphis sacchari. Molecular Breeding. 2013;31(4):777–84. 10.1007/s11032-012-9832-6.10.1007/s11032-012-9832-6
48. Upasani SM Kotkar HM Mendki PS Maheshwari VL Partial characterization and insecticidal properties of Ricinus communis L foliage flavonoids Pest Manag Sci 2003 59 12 1349 1354 10.1002/ps.767 14667057
Upasani SM, Kotkar HM, Mendki PS, Maheshwari VL. Partial characterization and insecticidal properties of Ricinus communis L foliage flavonoids. Pest Manag Sci. 2003;59(12):1349–54. 10.1002/ps.767. 14667057 10.1002/ps.767
49. Tian F Woo SY Lee SY Park SB Im JH Chun HS Plant-based natural flavonoids show strong inhibition of aflatoxin production and related gene expressions correlated with chemical structure Food Microbiol 2003 109 104141 10.1016/j.fm.2022.104141
Tian F, Woo SY, Lee SY, Park SB, Im JH, Chun HS. Plant-based natural flavonoids show strong inhibition of aflatoxin production and related gene expressions correlated with chemical structure. Food Microbiol. 2003;109:104141.10.1016/j.fm.2022.104141
50. Simmonds MSJ Flavonoid-insect interactions: recent advances in our knowledge Phytochemistry 2003 64 21 30 10.1016/S0031-9422(03)00293-0 12946403
Simmonds MSJ. Flavonoid-insect interactions: recent advances in our knowledge. Phytochemistry. 2003;64:21–30. 12946403 10.1016/S0031-9422(03)00293-0
51. Arimura G Ozawa R Maffei ME Recent advances in plant early signaling in response to herbivory Int J Mol Sci 2011 12 6 3723 3739 10.3390/ijms12063723 21747702
Arimura G, Ozawa R, Maffei ME. Recent advances in plant early signaling in response to herbivory. Int J Mol Sci. 2011;12(6):3723–39. 10.3390/ijms12063723. 21747702 10.3390/ijms12063723
52. Zhao X Chen S Wang S Shan W Wang X Lin Y Su F Yang Z Yu X Defensive responses of tea plants (Camellia sinensis) against tea green leafhopper Attack: a multi-omics study Front Plant Sci 2020 10 1705 10.3389/fpls.2019.01705 32010173
Zhao X, Chen S, Wang S, Shan W, Wang X, Lin Y, Su F, Yang Z, Yu X. Defensive responses of tea plants (Camellia sinensis) against tea green leafhopper Attack: a multi-omics study. Front Plant Sci. 2020;10:1705. 10.3389/fpls.2019.01705.32010173 10.3389/fpls.2019.01705
53. Kotkar HM Mendki PS Sadan SV Jha SR Upasani SM Maheshwari VL Antimicrobial and pesticidal activity of partially purified flavonoids of Annona squamosa Pest Manag Sci 2022 58 1 33 37 10.1002/ps.414
Kotkar HM, Mendki PS, Sadan SV, Jha SR, Upasani SM, Maheshwari VL. Antimicrobial and pesticidal activity of partially purified flavonoids of Annona squamosa. Pest Manag Sci. 2022;58(1):33–7. 10.1002/ps.414.10.1002/ps.414
54. Padmavati M Reddy AR Flavonoid biosynthetic pathway and cereal defence response: an emerging trend in crop biotechnoloy J Plant Biochem Biotechnol 1999 8 1 15 20 10.1007/BF03263051
Padmavati M, Reddy AR. Flavonoid biosynthetic pathway and cereal defence response: an emerging trend in crop biotechnoloy. J Plant Biochem Biotechnol. 1999;8(1):15–20.10.1007/BF03263051. 10.1007/BF03263051
55. Li Y Zhou H Feng N Zheng D Ma G Feng S Liu M Yu M Huang X Huang A Physiological and transcriptome analysis reveals that prohexadione-calcium promotes rice seedling's development under salt stress by regulating antioxidant processes and photosynthesis PLoS One 2023 18 6 e0286505 10.1371/journal.pone.0286505 37315011
Li Y, Zhou H, Feng N, Zheng D, Ma G, Feng S, Liu M, Yu M, Huang X, Huang A. Physiological and transcriptome analysis reveals that prohexadione-calcium promotes rice seedling’s development under salt stress by regulating antioxidant processes and photosynthesis. PLoS One. 2023;18(6):e0286505. 10.1371/journal.pone.0286505. PMID: 37315011; PMCID: PMC10266641.37315011 10.1371/journal.pone.0286505
56. Nicholson R Wood K Phytoalexins and secondary products, where are they and how can we measure them? Physiol Mol Plant Pathol 2001 59 63 69 10.1006/pmpp.2001.0344
Nicholson R, Wood K. Phytoalexins and secondary products, where are they and how can we measure them? Physiol Mol Plant Pathol. 2001;59:63–9.10.1006/pmpp.2001.0344
57. Kariyat RR Gaffoor I Sattar S Dixon CW Frock N Moen J De Moraes CM Mescher MC Thompson GA Chopra S Sorghum 3-deoxyanthocyanidin flavonoids confer resistance against corn leaf aphid J Chem Ecol 2019 45 5–6 502 514 10.1007/s10886-019-01062-8 30911880
Kariyat RR, Gaffoor I, Sattar S, Dixon CW, Frock N, Moen J, De Moraes CM, Mescher MC, Thompson GA, Chopra S. Sorghum 3-deoxyanthocyanidin flavonoids confer resistance against corn leaf aphid. J Chem Ecol. 2019;45(5–6):502–14. 10.1007/s10886-019-01062-8.30911880 10.1007/s10886-019-01062-8
58. Mendk PS Patil SB Patil SV Patil MG Kothari RM Pesticidal activity of certain plant extracts to control stored grain pest Callosobruchus chinensis Pestology 1999 23 64 67
Mendk, P. S., Patil, S. B., Patil, S. V., Patil, M. G., Kothari, R. M. (1999) .Pesticidal activity of certain plant extracts to control stored grain pest Callosobruchus chinensis. 23, 64–67.
59. Wharton PS Nicholson RL Temporal synthesis and radiolabelling of the sorghum 3-deoxyanthocyanidin phytoale Nijveldt s and the anthocyanin, cyanidin 3-dimalonyl glucoside New Phytol 2000 145 3 457 69 10.1046/j.1469-8137.2000.00600.x 33862912
Wharton PS, Nicholson RL. Temporal synthesis and radiolabelling of the sorghum 3-deoxyanthocyanidin phytoale Nijveldt s and the anthocyanin, cyanidin 3-dimalonyl glucoside. New Phytol. 2000;145(3):457–69. 10.1046/j.1469-8137.2000.00600.x.33862912 10.1046/j.1469-8137.2000.00600.x
60. Grover S Puri H Xin Z Sattler SE Louis J Dichotomous role of jasmonic acid in modulating sorghum defense against aphids Mol Plant-microbe Interact 2022 35 9 755 67 10.1094/MPMI-01-22-0005-R 35394339
Grover S, Puri H, Xin Z, Sattler SE, Louis J. Dichotomous role of jasmonic acid in modulating sorghum defense against aphids. Mol Plant-microbe Interact. 2022;35(9):755–67. 10.1094/MPMI-01-22-0005-R.35394339 10.1094/MPMI-01-22-0005-R
61. Sotelo-Leyva C Avilés-Montes D Manuel Rivas-González J Figueroa-Brito R Abarca-Vargas R Toledo-Hernández E Salinas-Sánchez DO Xanthotoxin: an aphicidal coumarin from ficus petiolaris against Melanaphis sacchari Zehntner (Hemiptera: Aphididae) J Food Prot 2023 86 6 100084 10.1016/j.jfp.2023.100084 37019182
Sotelo-Leyva C, Avilés-Montes D, Manuel Rivas-González J, Figueroa-Brito R, Abarca-Vargas R, Toledo-Hernández E, Salinas-Sánchez DO. Xanthotoxin: an aphicidal coumarin from ficus petiolaris against Melanaphis sacchari Zehntner (Hemiptera: Aphididae). J Food Prot. 2023;86(6):100084. 10.1016/j.jfp.2023.100084.37019182 10.1016/j.jfp.2023.100084
62. Ku YS Ng MS Cheng SS Lo AW Xiao Z Shin TS Chung G Lam HM Understanding the composition, biosynthesis, accumulation and transport of flavonoids in crops for the promotion of crops as healthy sources of flavonoids for human consumption Nutrients 2020 12 6 1717 10.3390/nu12061717 32521660
Ku YS, Ng MS, Cheng SS, Lo AW, Xiao Z, Shin TS, Chung G, Lam HM. Understanding the composition, biosynthesis, accumulation and transport of flavonoids in crops for the promotion of crops as healthy sources of flavonoids for human consumption. Nutrients. 2020;12(6):1717. 10.3390/nu12061717.32521660 10.3390/nu12061717
