Tomato Cultivars Resistant or Susceptible to Spider Mites Differ in Their Biosynthesis and Metabolic Profile of the Monoterpenoid Pathway

Abstract

The two-spotted spider mite (TSSM; Tetranychus urticae) is a ubiquitous polyphagous arthropod pest that has a major economic impact on the tomato (Solanum lycopersicum) industry. Tomato plants have evolved broad defense mechanisms regulated by the expression of defense genes, phytohormones, and secondary metabolites present constitutively and/or induced upon infestation. Although tomato defense mechanisms have been studied for more than three decades, only a few studies have compared domesticated cultivars' natural mite resistance at the molecular level. The main goal of our research was to reveal the molecular differences between two tomato cultivars with similar physical (trichome morphology and density) and agronomic traits (fruit size, shape, color, cluster architecture), but with contrasting TSSM susceptibility. A net house experiment indicated a mite-resistance difference between the cultivars, and a climate-controlled performance and oviposition bioassay supported these findings. A transcriptome analysis of the two cultivars after 3 days of TSSM infestation, revealed changes in the genes associated with primary and secondary metabolism, including salicylic acid and volatile biosynthesis (volatile benzenoid ester and monoterpenes). The Terpene synthase genes, TPS5, TPS7, and TPS19/20, encoding enzymes that synthesize the monoterpenes linalool, β-myrcene, limonene, and β-phellandrene were highly expressed in the resistant cultivar. The volatile profile of these cultivars upon mite infestation for 1, 3, 5, and 7 days, revealed substantial differences in monoterpenoid and phenylpropanoid volatiles, results consistent with the transcriptomic data. Comparing the metabolic changes that occurred in each cultivar and upon mite-infestation indicated that monoterpenes are the main metabolites that differ between cultivars (constitutive levels), while only minor changes occurred upon TSSM attack. To test the effect of these volatile variations on mites, we subjected both the TSSM and its corresponding predator, Phytoseiulus persimilis, to an olfactory choice bioassay. The predator mites were only significantly attracted to the TSSM pre-infested resistant cultivar and not to the susceptible cultivar, while the TSSM itself showed no preference. Overall, our findings revealed the contribution of constitutive and inducible levels of volatiles on mite performance. This study highlights monoterpenoids' function in plant resistance to pests and may inform the development of new resistant tomato cultivars.

Keywords: Phytoseiulus persimilis; Solanum lycopersicum; Terpene synthase; Tetranychus urticae (Koch); salicylic acid; volatile organic compounds.

Figure 1

The total number of adult and juvenile TSSMs per plant on the two tomato cultivars grown in a net house. Mite population was counted 5 and 8 weeks after TSSM infestation. At each time point, nine leaves from a plant were selected, twenty plants of each cultivar were sampled (Student's t-test; *p < 0.05; n = 20; mean ± SE). The time points that are presented are the ones in which the peak of the mite population was observed.

Figure 2

Plant damage rate of the tomato cultivars grown in a net house. (A) Photos from the tomato plants. (B) Plant damage rate of whole tomato plants. Plant severity symptoms were scored on three damage levels: (1) slight, (2) mild, and (3) severe. n = 24–26.

Figure 3

Evaluation of TSSM performance and oviposition rate on the two tomato cultivars Ofir and Shiran. (A) The mites were counted 13 days after infestation, 4-week-old tomato plants were infested with 10 TSSMs per plant. Asterisks indicate significant differences between cultivars (Student's t-test; *p < 0.01; n = 6; mean ± SE. (B) Oviposition rate was determined from 30 sets of leaf disks infested with one adult female mite per leaf disk. The figure shows the average total egg production per day. There was no significant difference between the number of eggs produced in the cultivars (ANOVA with df = 1; F = 1.1; p-value = 0.29). There was a significant difference in the number of eggs between sampling days in each cultivar (ANOVA with df = 4; F = 6.3; p < 0.0001). Different letters indicate significant differences determined by one-way ANOVA (p < 0.05; HSD).

Figure 4

Transcriptomic overview of two tomato cultivars infested with TSSMs for 3 days. (A) PCA plot was generated using 22,592 genes (transcripts with only zero values were excluded). (B) Venn diagram illustrates the number of genes that were differentially expressed (DEGs) between the two cultivars (Shiran/Ofir) and/or treatments (TSSM-infested/control). Absolute fold change >|2|, p < 0.05 FDR (n = 5–6 biological replicates).

Figure 5

Volatile compound contents of tomato plants infested with TSSM or untreated (control) for 1, 3, 5, or 7 days. The graphs show the average relative production at each day for (A) monoterpenes, including the sum of 3,7,7-trimethyl-1,3,5-cycloheptatriene, 4-carene, α-terpinene, β-myrcene, β-phellandrene, p-cymene, p-cymenene, limonene, and terpinolene. (B) β-pinene, (C) sequiterpenes represent the sum of α-humulene, β-elemene, β-trans-caryophyllene, and guaiazulene. (D) Irregular terpenes represent the sum of β-cyclocitral, β-homocyclocitral β-ionone, β-ionone epoxide, and crypton. (E) Methyl salicylate (MeSA) and (F) methyl benzoate. Data represent mean ± SE; n = 4–5. Asterisks indicate significant differences between the control and the infested in each cultivar (Student's t-test, *p < 0.05, **p < 0.01, and ***p < 0.001).

Figure 6

Olfactory response of Tetranychus urticae (TSSM) and Phytoseiulus persimilis to different tomato cultivars infested with TSSMs and untreated (control). (A) Percentage of TSSMs that chose the odors of infested plants (striped bars) for control plants (solid bars) or Ofir cultivar (light blue bars) for Shiran cultivar (light beige bars). (B) Percentage of P. persimilis that chose the odors of infested plants (striped bars) for control plants (solid bars) or Ofir cultivar (blue bars) for Shiran cultivar (beige bars). Experiments were repeated twice. In each experiment, 40 individual adult mites were tested (χ²-test, *p < 0.05).

Figure 7

Summary of the significant differences in gene expression of the terpene biosynthesis in the leaves of two tomato cultivars. Genes are shown in italics, red-colored expressed higher in the resistant cultivar (Ofir) than in the susceptible cultivar (Shiran). Volatiles with red background produced more in Ofir, and volatiles with blue background produced more in Shiran. Gene and volatiles identified in this work are shown in bold. CPT, cis-prenyltransferase; DMAPP, dimethylallyl diphosphate; E,E-FPP, trans-farnesyl diphosphate; FPPS, E,E-FPP synthase; GPP, geranyl diphosphate; GGPP, geranylgeranyl diphosphate; GGPPS, GGPP synthase; IPP, isopentenyl diphosphate; NPP, neryl diphosphate; NNPP, nerylneryl diphosphate; SSU, small subunit of GGPPS; TPS, terpene synthase; Z,Z-FPP, cis-farnesyl diphosphate.