Megalurothrips usitatus Directly Causes the Black-Heads and Black-Tail Symptoms of Cowpea along with the Production of Insect-Resistance Flavonoids

Abstract

The thrip (Megalurothrips usitatus) damages the flowers and pods of the cowpea, causing "black-heads and black-tails" (BHBT) symptoms and negatively affecting its economic value. However, the mechanism by which BHBT symptoms develop is still unknown. Our results showed that the microstructure of the pod epidermis was altered and the content of the plant's resistance-related compounds increased after a thrip infestation. However, the contents of protein and free amino acids did not change significantly, suggesting that the nutritional value was not altered. Pathogens were found not to be involved in the formation of BHBT symptoms, as fungi and pathogenic bacteria were not enriched in damaged pods. Two herbivory-induced flavonoids-7,4'-dihydroxyflavone and coumestrol-were found to exert insecticidal activity. Our study clarified that BHBT symptoms are directly caused by the thrip. Thresholds for pest control need to be reconsidered as thrip herbivory did not degrade cowpea nutrition.

Keywords: Megalurothrips usitatus; cowpea; flavonoid; nutrition; pathogen; thrip.

Figure 1

Morphology structure of healthy pods and BHBT pods. (A): Healthy pods, CK. (B): BHBT pods, T. (C): M. usitatus nymphs attacking pods, scale bar = 500 µm. (D): Cowpea pod of BHBT symptoms, scale bar = 100 µm. (E): Fresh section of CK cross section, scale bar = 500 µm. (F): Fresh section of BHBT, scale bar = 100 µm. (G): Fresh section of CK longitudinal section, scale bar = 100 µm. (H): Fresh section of BHBT, scale bar = 100 µm. (I): Paraffin section of CK cross section, scale bar = 250 µm. (J): Paraffin section of cross section of BHBT, scale bar = 250 µm. (K): paraffin section of longitudinal section of CK, scale bar = 250 µm. (L): paraffin section of longitudinal section of BHBT, scale bar = 250 µm. (M): SEM cross section of CK, scale bar = 300 µm. (N): SEM of cross section of BHBT, scale bar = 300 µm. (O): SEM of longitudinal section of CK, scale bar = 100 µm. (P): SEM of longitudinal section of BHBT, scale bar = 100 µm. EP: Epidermis. PA: Parenchyma cell. VB: Vascular bundle. R: Ridges. S: Stomatal pore. SP: Stomatal pore. Blue arrows indicate M. usitatus nymphs. Black arrows indicate stomatal locations. White arrows indicate damaged tissues. Red arrows indicate epidermal cells. Red boxes indicate the morphology of epidermal cells. CK: (A,E,G,I,K,M,O). T: (B–D,F,H,J,L,N,P).

Figure 2

Contents of physiological and biochemical indexes in cowpea pods. (A): Cellulose content. (B): Lignin content. (C): Pectin content. (D): Callose content. (E): Reactive oxygen species (ROS) content. (F): Abscisic acid (ABA) content. (G): Jasmonic acid (JA) content. (H): Jasmonoyl–isoleucine (JA-Ile) content. (I): Salicylic acid (SA) content. Each value represents the mean ± SE of five replicates. Bars with different lower-case letters indicate significant differences based on a t-test at the p ≤ 0.05 level.

Figure 3

Protein content of healthy pods and BHBT pods. T: BHBT pods, CK: healthy pods. Each value represents the mean ± SE of five replicates. Bars with different lowercase letters indicate significant differences based on a t-test at the p ≤ 0.05 level.

Figure 4

Analysis of bacterial community in cowpea pod. (A): Principal component analysis of OUT-level of epidermal bacterial communities in healthy pods (CK) and BHBT pods (T). (B,C): alpha diversity analysis at OUT-level of epidermal bacterial communities in healthy pods and BHBT pods. (D): The relative abundance analysis of epidermal bacterial communities in healthy pods and BHBT pods at the genus level. (E): The significant difference of epidermal bacterial communities at genus level between healthy pods and BHBT pods. The differences were considered significant based on a t-test at * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 5

Specialized metabolites analysis of cowpea pods. (A): Composition of secondary metabolites in BHBT pod (T). (B): Venn diagram of secondary metabolites in healthy pods (CK) and BHBT pods. (C): Principal component analysis of secondary metabolites in healthy and BHBT pods. (D): Cluster analysis of secondary metabolites in healthy and BHBT pods. (E): Analysis of the enrichment pathway of secondary metabolites in healthy and BHBT pods. (F): KEGG biosynthesis pathway map of secondary metabolites in healthy and BHBT pods. Red indicates upregulation of specialized metabolites, green indicates downregulation of specialized metabolites, and black indicates that specialized metabolites are not detected. 7,4′-Dihydroxyflavone and coumestrol were highly upregulated in the flavonoid biosynthesis pathway and the isoflavonoid biosynthesis pathway. (G): bioassay of 7,4′-Dihydroxyflavone and coumestrol. T: The concentration is 4 mg/mL, CK: 4% DMSO. The differences were considered significant based on a t-test at * p ≤ 0.05.