Tomato progeny inherit resistance to the nematode Meloidogyne javanica linked to plant growth induced by the biocontrol fungus Trichoderma atroviride

Abstract

Root-knot nematodes (RKN) are major crop pathogens worldwide. Trichoderma genus fungi are recognized biocontrol agents and a direct activity of Trichoderma atroviride (Ta) against the RKN Meloidogyne javanica (Mj), in terms of 42% reduction of number of galls (NG), 60% of number of egg masses and 90% of number of adult nematodes inside the roots, has been observed in tomato grown under greenhouse conditions. An in vivo split-root designed experiment served to demonstrate that Ta induces systemic resistance towards Mj, without the need for the organisms to be in direct contact, and significantly reduces NG (20%) and adult nematodes inside tomato roots (87%). The first generation (F1) of Ta-primed tomato plants inherited resistance to RKN; although, the induction of defenses occurred through different mechanisms, and in varying degrees, depending on the Ta-Mj interaction. Plant growth promotion induced by Ta was inherited without compromising the level of resistance to Mj, as the progeny of Ta-primed plants displayed increased size and resistance to Mj without fitness costs. Gene expression results from the defense inductions in the offspring of Ta-primed plants, suggested that an auxin-induced reactive oxygen species production promoted by Ta may act as a major defense strategy during plant growth.

Figure 1. Experimental procedure used for in vivo analysis of systemic defense in tomato plants.

Five-week-old tomato plants (F0) were transplanted following a split-root system designed experiment and treated with Trichoderma atroviride T11 (Ta) and/or Meloidogyne javanica (Mj) in the following way: i) (w/w) the two root system halves treated with sterile water (control); ii) (w/Ta) one root system half treated with Ta; iii) (w/Mj) one root system half treated with Mj; iv) (Ta/Mj) one root system half treated with Ta and the other half with Mj; and v) (w/Ta + Mj) one root system half with treated with Ta plus Mj. F1 plants, derived from the corresponding F0 treatment, were inoculated with Mj when they were 21-days old. See Methods for more details.

Figure 2. In vivo biocontrol ability of Trichoderma atroviride T11 (Ta) against Meloidogyne javanica (Mj).

(A) Number of galls (NG), (B) Number of egg masses (NEM), and (C) NEM/NG ratio calculated at 30 days after infection with Mj when tomato plants were six-weeks old. The split-root treatments were as indicated in the legend of Fig. 1. Data of three plants per treatment from two independent experiments (n = 6) were submitted to ANOVA and the comparison of means by Tukey test (P < 0.05) carried out. Bars with different letters indicate statistically significant differences between the different treatments.

Figure 3. Resistance induced by Trichoderma atroviride T11 (Ta) against Meloidogyne javanica (Mj) in tomato plants.

(A) Number of total nematodes in roots (NTNR) and (B) Number of nematodes corresponding to different developmental stages (J2, second stage juvenile; J3, third stage juvenile; J4, fourth stage juvenile; and adult). NTNR and number of nematodes calculated at 15 days after inoculation with Mj when tomato plants were six-week old. The split-root treatments were as indicated in the legend of Fig. 1. Data of four plants per treatment from two independent experiments (n = 8) were submitted to ANOVA and the comparison of means by Tukey test (P < 0.05) was carried out. Bars with different letters indicate statistically significant difference between the different treatments.

Figure 4. Defense marker gene expression patterns in tomato roots in response to Trichoderma atroviride T11 (Ta) and/or Meloidogyne javanica (Mj).

Total root cDNA was subjected to qPCR to quantify LOX1, PR1, TPX1, LERBOH1 and LECHS2 gene expression related to different plant defense responses at 1, 4 and 6 days after inoculation with Mj. The actin gene was used as a control. Values correspond to relative measurements against the respective control conditions (w/w) (2^−ΔΔCT = 1) for each time point, and are expressed as log₁₀. Error bars represent standard deviations of the mean values for three technical replicates of cDNA from two independent experiments. Each cDNA was obtained from a root-pool of three different F0 plants for each treatment and time point. The levels of expression were tested using one-way analysis of variance (ANOVA) followed by Tukey’s test. For each gene and time, different letters represent significative differences (P < 0.05).

Figure 5. Defense gene expression in Trichoderma atroviride-primed tomato plant progeny.

Total RNA extracted from two-week-old F1 plants, derived from split-root tomato plants subjected to the five treatments indicated in Fig. 1. cDNA was subjected to qPCR to quantify LOX1, PR1, TPX1, LERBOH1, LECHS2, MYC2, NPR1, ERF1 and ARF1 gene expression. The actin gene was used as a control. Values correspond to relative measurements against the respective control condition (w/w) (2^−ΔΔCT = 1) and are expressed as log₁₀. Error bars represent standard deviations of the mean values for three technical replicates of cDNA from two independent experiments. Each cDNA was obtained from ten different F1 plants for each treatment. The levels of expression were tested using one-way analysis of variance (ANOVA) followed by Tukey’s test. For each gene different letters represent significative differences (P < 0.05).

Figure 6. Phenotypical differences among Trichoderma atroviride-primed tomato plant progeny.

(A) Frontal view of two-week-old F1 plants, derived from seeds collected from split-root tomato plants subjected to the five treatments indicated in the legend of Fig. 1. (B) The same plants viewed from above. Green mass percentages are referred to that of the control plants (w/w = 100%) and obtained from five plants per treatment of two independent experiments.

Figure 7. Transgenerational biocontrol against Meloidogyne javanica (Mj) in Trichoderma atroviride-primed tomato plant progeny.

(A) Number of galls (NG), (B) Number of egg masses (NEM), and (C) NEM/NG ratio calculated in 51-day-old F1 plants at 30 days after infection with Mj, when plants were 21-days old. These plants were derived from seeds collected from split-root tomato plants subjected to the five treatments indicated in the legend of Fig. 1. Data from five plants per treatment of two independent experiments (n = 10) were submitted to ANOVA, and the comparison of means by Tukey test (P < 0.05) was carried out. Bars with different letters indicate statistically significant differences among treatments.