Endophyte-induced Verticillium protection in tomato is range-restricted

Endophytes, bacterial, fungal or viral, colonize plants often without causing visible symptoms. More important, they may benefit host plants in many ways, most notably by preventing diseases caused by normally virulent pathogens. Previous studies have shown that an isolate of V. dahliae from eggplant, Dvd-E6, can colonize tomato endophytically, producing taller and more robust tomato plants while providing protection against a virulent V. dahliae, race 1 (Vd1) isolate. Expression analyses suggest this requires interplay between Dvd-E6 and the plant that involves resistance and defense genes. To examine the possibility of a broader effect, dual interactions have been further examined with a more distantly related pathogen, Verticillium albo-atrum (Vaa). The results indicate Dvd-E6 colonization selectively modifies the expression of specific tomato genes to be detrimental to Vd1 but not Vaa, providing evidence that Verticillium-induced protection is range-restricted.Key words: cross-protection, endophyte, lycopersicon, tolerance, VerticilliumAn “endophyte” commonly is defined as a “fungus or bacterium living within plants without causing visible symptoms of disease”; a “pathogen” is regarded as “a disease causing biological agent”. Historically, plant biologists have tended to consider these as two very different and distinct classes of organisms but accumulating evidence now suggests that the boundaries between mutualism and parasitism are not as defined as previously thought. Many organisms can occupy both ecological niches¹ depending on the genotype of the host, the genotype of the organism itself and interaction with the environment. Indeed, this “dual life style” may be a significant factor in the evolutionary dynamics of pathogen resistance, tolerance and susceptibility.²One of the more recent additions to the growing list of dual life style (endophyte/pathogen) fungi is Verticillium dahliae,³ a causal agent of vascular wilt disease or early dying syndrome in a broad range of the plant species.⁴ When Verticillium infects a plant three different host responses can occur: resistance, susceptibility or tolerance. The phenomenon of tolerance has been associated with Verticillium spp. for decades but research on mechanisms governing the development of the plant/Verticillium interaction has focused on the compatible and incompatible interactions and little is known about the tolerant state (reviewed in ref. ⁵). In a recent series of papers we have identified an isolate of Verticillium dahliae, known as Dvd-E6, that colonizes tomatoes, cv Craigella, resulting in a stable tolerant condition, that we have used as a model system to investigate the biological and molecular bases of plant tolerance to Verticillium spp. The Craigella/Dvd E6 interaction has a number of interesting but unanticipated properties. Host plants tend to be taller and more robust than their uninfected counterparts⁶ and colonization of Craigella by Dvd-E6 provides protection against its virulent Verticillium dahliae race 1 (Vd1) cousin, limiting both Vd1 colonization and symptom development during dual infections.⁷ Such attributes normally are associated with plant/endophyte partnerships, strongly suggesting that under some conditions, Verticillium dahliae can assume an endophytic role. Apparently interplay between Dvd-E6 and the plant establishes protection against the virulent Vd1.⁷Many endophytic infections provide protection for the host against a broad range of pathogens.¹ To test the ability of Dvd-E6 infection to protect Craigella against a more distantly related Verticillium pathogen we have now examined dual interactions with Verticillium albo-atrum (Vaa). Susceptible Craigella⁸ seedlings again were inoculated at the 4-leaf stage by dipping the roots in Vd1, Dvd-E6 or Vaa conidial suspensions (1 × 10⁷ spores/ml in 0.5% gelatin solution) to establish homogeneous interactions (reviewed in ref. ⁷). For dual interactions, seedlings were inoculated with Dvd-E6 spores at the 3-leaf stage and reinoculated at the 4-leaf stage with either Vd1 or Vaa spore suspension to establish the mixed infections. Control seedlings were root dipped in gelatin solution alone. Plants were scored for symptom expression on a 0 (no symptoms) to 5 (plant dead) scale as described by Shittu and co-workers (2009) and the top two-thirds of the stems harvested at 5 and 10 days post inoculation (d.p.i.) for extraction and fungal DNA assays.^7,9All experimental results are summarized in Figure 1. At 10 d.p.i., the disease scores for Dvd-E6-infected plants were approximately one-third that of the Vd1-or Vaa-infected plants. More interesting were the symptoms in the dual interactions, Dvd-E6/Vd1 mimicking the tolerant plants with low disease and Dvd-E6/Vaa-infected plants exhibiting the highest disease scores, similar to Vd1-or Vaa-infected plants. When the amount of fungal DNA in the stems was assessed, the total fungal biomass in both dual infections as well as the Dvd-E6 and Vd1-infected plants at 5 and 10 dpi was similar (light and dark gray bars), the amount of Vaa being somewhat lower. In the mixed infections, however, most of the DNA (>90%) was of Dvd-E6 origin (white bars). More important, the Vd1 DNA level in Dvd-E6/Vd1 plants was substantially reduced relative to plants infected with Vd1 alone while the Vaa DNA levels stayed about the same in the single and double infections. This indicates that Vd1 colonization is restricted⁷ by the presence of Dvd-E6 while Vaa is not.Open in a separate windowFigure 1Comparison of symptoms and levels of V. dahliae 1 or V. albo-atrum DNA in susceptible Craigella tomato simultaneously infected with V. dahliae Dvd-E6. Individual (E6, Vd1 and Vaa) and mixed (E6/Vd1 and E6/Vaa) infections were established as previously described.⁷ Plants were scored (upper) at 5 (light gray bars) and 10 (dark gray bars) dpi for symptoms (i.e., disease scores ± SD) or assayed⁷ for total levels of Verticillium DNA (i.e., ng/g plant tissue ± SD) in the stems (lower). In mixed infections levels for each fungus also were determined (black and white bars, respectively). Results summarize the data for 9–12 plants per time point for each interaction.These results demonstrate that Dvd-E6 infection protects Craigella tomatoes against colonization by and symptom development from Vd1 but not Vaa. Past studies often have suggested that the protective effect stems from an endophyte-induced activation of systemic acquired resistance (SAR) in the host providing protection against a broad range of pathogens.¹⁰ However, in the tolerant CS//Dvd-E6 interaction the protective effect appears to be targeted more directly, allowing Dvd-E6 to effectively restrict it''s virulent cousin, Vd1. In this context, it may be important that both of the Verticillium dahliae isolates from tomato are endemic to Ontario^7,11 and potentially in direct competition, while Verticillium albo-atrum from tomato is not. Previous molecular analyses suggested that the protective effect provided by Dvd-E6 colonization requires a genetic interplay with the host, selectively modifying the expression of specific tomato genes to be detrimental to Vd1. The experimental results presented here provide evidence that Verticillium-induced protection is restricted in range.