Micronutrients Affect Expression of Induced Resistance Genes in Hydroponically Grown Watermelon against Fusarium oxysporum f. sp. niveum and Meloidogyne incognita

Abstract

The soil-borne pathogens, particularly Fusarium oxysporum f. sp. niveum (FON) and southern root-knot nematode (RKN, Meloidogyne incognita) are the major threats to watermelon production in the southeastern United States. The role of soil micronutrients on induced resistance (IR) to plant diseases is well-documented in soil-based media. However, soil-based media do not allow us to determine the contribution of individual micronutrients in the induction of IR. In this manuscript, we utilized hydroponics-medium to assess the effect of controlled application of micronutrients, including iron (Fe), manganese (Mn), and zinc (Zn) on the expression of important IR genes (PR1, PR5, and NPR1 from salicylic acid (SA) pathway, and VSP, PDF, and LOX genes from jasmonic acid (JA) pathway) in watermelon seedlings upon inoculation with either FON or RKN or both. A subset of micronutrient-treated plants was inoculated (on the eighth day of micronutrient application) with FON and RKN (single or mixed inoculation). The expression of the IR genes in treated and control samples was evaluated using qRT-PCR. Although, significant phenotypic differences were not observed with respect to the severity of wilt symptoms or RKN galling with any of the micronutrient treatments within the 30-day experimental period, differences in the induction of IR genes were considerably noticeable. However, the level of gene expression varied with sampling period, type and concentration of micronutrients applied, and pathogen inoculation. In the absence of pathogens, micronutrient applications on the seventh day, in general, downregulated the expression of the majority of the IR genes. However, pathogen inoculation preferentially either up- or down-regulated the expression levels of the IR genes at three days post-inoculation depending on the type and concentration of micronutrients. The results demonstrated here indicate that micronutrients in watermelon may potentially make watermelon plants susceptible to infection by FON and RKN. However, upon infection the IR genes are significantly up-regulated that they may potentially aid the prevention of further infection via SA- and JA-pathways. This is the first demonstration of the impact of micronutrients affecting IR in watermelon against FON and RKN infection.

Keywords: Fusarium; induced resistance; micronutrients; root-knot nematode; watermelon.

Figure 1

Schematic representation of the timeline (in days) of the greenhouse experiment and associated sampling to evaluate the effects of micronutrients (Fe or Mn or Zn) on induced resistance in watermelon seedlings when challenged with Fusarium oxysporum f. sp. niveum (FON) and Meloidogyne incognita (RKN) or both.

Figure 2

Relative expression of non-expressor pathogenesis-related gene 1 (NPR1), pathogenesis-related protein 1 (PR1), pathogenesis-related protein 5 (PR5), lipoxygenase (LOX), plant defensin (PDF), and vegetative storage protein (VSP) genes by qRT-PCR in watermelon leaves at 7 days post treatment with micronutrients (A) Fe, (B) Mn, and (C) Zn. Watermelon seedlings (cv. Sugar Baby; 3 weeks old) were either treated with Fe or Mn or Zn at high (3X), low (0.5X), or standard concentration (X, Steiner) for 7 days. ß-Actin was used as the reference gene. Plants in the Steiner treatment were considered as non-treated control. Data are the mean fold changes ± SE in gene transcript levels in tissues from micronutrient treated plants relative to tissues from non-treated control plants in Steiner. Letters indicate a significant difference between treatments with the Tukey–Kramer test (p < 0.05).

Figure 3

Relative expression of non-expressor pathogenesis-related gene 1 (NPR1), pathogenesis-related gene 1 (PR1), pathogenesis-related gene 5 (PR5), lipoxygenase (LOX), Plant defensin (PDF), and vegetative storage proteins (VSP) genes by qRT-PCR in leaves of watermelon plants at 11 days post-treatment with micronutrients Fe (A,D,G), Mn (B,E,H), and Zn (C,F,I) at high (3X), low (0.5X), and standard concentration (X) Steiner via hydroponics system and 3 days post-inoculation with 1 ml of 5 × 10⁵ microconidia of Fusarium oxysporum f. sp. niveum (FON) (A–C) or 1 mL of 6000 active J2s of Meloidogyne incognita (RKN) (D–F), or both (G–I). ß-Actin was used as the reference gene. Plants in the Steiner treatment were considered as non-treated control. Data are the mean fold changes ± SE in genes transcript levels of tissues from inoculated plants in micronutrient treatments relative to tissues from non-inoculated control plants in Steiner. Letters indicate a significant difference between treatments with the Tukey–Kramer test (p < 0.05). Primer sequences and PCR conditions for test and reference genes are given in Table 3.