As sessile organisms, plants are presented with numerous biotic challenges such as herbivory and pathogen attack. Plants initiate responses to these challenges by harnessing tightly regulated phytohormone networks. Salicylic acid levels increase in plants following pathogen infection and SA is critical for the development of systemic acquired resistance . There are two enzymatic pathways for the generation of SA: one via phenylalanine ammonia lyase and the other via isochorismate synthase . In tomato , Arabidopsis and Nicotiana benthamiana, most pathogen-induced SA appears to be synthesized via the ICS pathway . Plants with compromised SA synthesis or signaling have greatly diminished defenses against pathogens, as is the case with SA-deficient transgenic plants expressing a bacterial salicylate hydroxylase or ICS mutants like sid2 , and mutants in downstream targets of SA such as npr1 . SAR induction by biotic agents coincides with increases in SA levels and a systemic transcriptional reprograming that primes the plant to respond rapidly to minimize the spread or severity of further infections . This transcriptional reprograming includes the expression of pathogenesis-related genes and deployment of peroxidases and other defense factors. In addition to induction by biotic agents, SAR responses are induced by exogenous application of SA to the foliage or roots . Plant activators are chemicals that have no direct antimicrobial activity but induce disease resistance . A number of synthetic compounds have been developed that induce SAR by increasing SA accumulation and/or by acting on downstream targets of SA . For example, the plant activator, probenazole, effective against bacterial, fungal, and oomycete diseases, stimulates SAR by increasing SA levels . 1,2,3-Benzothiadiazole-7-thiocarboxylic acid-S-methyl-ester , sold under the trade name, Actigard,grow bucket stimulates SAR in many plant species without inducing SA accumulation . Tiadinil [TDL; N–4-methyl-1,2,3-thiadiazole-5-carboxamide] is a plant activator that was registered in Japan in 2003 under the trade name, V-GET. TDL was developed for disease management in rice where it is applied to nursery-grown seedlings for transplanting to production fields . TDL is very effective for control of rice blast disease caused by Magnaporthe oryzae and appears to induce resistance in a manner similar to BTH by acting on downstream targets of SA .
The TDL metabolite,4-methyl-1,2,3-thiadiazole-5-carboxylic acid, is responsible for the SAR activation .The effect of brief episodes of root stress such as salinity and water deficit at levels that commonly occur in agriculture is well documented in plant–oomycete interactions, wherein stress events predispose plants to levels of inoculum they would normally resist . The phytohormone abscisic acid accumulates rapidly in roots and shoots as an adaptive response to these abiotic stresses, but also contributes to the increased disease proneness of the plants . Antagonism between SA and ABA is well documented in relation to plant defense responses to pathogens . Previously, ABA was found to have an antagonistic effect on SAR which was induced by 1,2-benzisothiazol-3-one1,1-dioxide and BTH in Arabidopsis and tobacco . However, it is not known if plant activators that target SA signaling impact the ABA-mediated susceptibility to root pathogens that occurs following predisposing root stress in tomato. Because of the potential for unwanted trade offs and signaling conflicts in plants exposed to different stresses, as can occur in the field, we investigated how predisposing root stress impacts chemically induced resistance in tomato. The objective of this study was to determine the effect of pretreatment of tomato seedlings with TDL and BTH on salt-induced predisposition to the foliar bacterial pathogen Pseudomonas syringae pv. tomato and to the soil borne oomycete pathogen Phytophthora capsici. TDL is of particular interest in the context of soil borne pathogens such as Phytophthora capsici because it is often applied to plants as a root dip. We also determined the impact of SA, TDL and BTH on ABA accumulation during a predisposing episode of salt stress. The results show that TDL applied to roots strongly protects the leaves from disease caused by Pst in both non-stressed and salt-stressed plants. In contrast, neither TDL nor BTH protects roots from Phytophthora capsici.
The protection induced by plant activators against Pst does not result from reduced ABA accumulation and, although overall disease is less in both non-stressed and salt-stressed plants by chemically induced SAR, plant activators do not reverse the salt-induced increment in disease severity.To determine the effect of SA on ABA accumulation during salt stress, ABA levels were measured in WT plants pre-treated with SA, TDL, or BTH. Following salt stress treatment for 18 h, roots and shoots were collected and immediately frozen in liquid N2.The tissues were lyophilized and placed at −20◦C until extraction. The lyophilized tissue was ground in liquid N2 to a fine powder with a mortar and pestle, 50–100 mg samples were collected, and each sample transferred to a micro-fuge tube. Cold 80% methanol containing butylated hydroxytoluene at 10 μg ml−1 was added to each tube, which was then vortexed. The extracts were placed on ice and agitated occasionally for 30 min. The tubes were centrifuged for 5 min at 10,000 × g, and the supernatants collected. The pellet was extracted with 0.5 ml of 80% methanol and centrifuged to collect the supernatant. This step was repeated, all three supernatants were combined, and the methanol concentration of the extract adjusted to 70%. The extracts were applied to pre-wetted Sep-pak C18 columns and eluted with 5 ml of 70% methanol. The eluate containing ABA was concentrated to near dryness at 37◦C under vacuum and the volume adjusted to 300 μl with deionized water. The samples were analyzed by competitive immuno assay with an ABA immuno assay kit according to the manufacturer’s directions. Results are expressed as nanomoles of -ABA per gram dry weight of tissue. To determine the effect of the nahG transgene on ABA levels, roots and shoots from WT and NahG plants were processed using the same procedure as above.To determine if plant activators induce resistance to Pst under different stress regimes in our experimental format, roots of hydroponically grown seedlings of cv. “New Yorker” were treated with TDL and then either not salt-stressed or exposed to 0.2 M NaCl for 18 h prior to inoculation.
In preliminary experiments, several concentrations of TDL were evaluated for phytotoxicity and for efficacy against bacterial speck disease with 10 ppm TDL selected as this concentration provided an optimal response. Concentrations higher than 10 ppm of TDL caused a slight bronzing of the roots and depressed growth of the seedlings, suggesting a mild phytotoxicity of the chemical in our experimental format at these higher levels. Inoculated salt-stressed seedlings had more severe disease symptoms and a significantly higher titer of pathogen than non-stressed, inoculated plants. Pretreatment with TDL at 10 ppm significantly reduced Pst colonization and symptom severity in “New Yorker” plants in both non-stressed and salt-treated seedlings . However, TDL did not prevent the proportional increase in Pst colonization observed in salt-stressed plants relative to the non-stressed controls.Since TDL harnesses SA-mediated defenses, we treated SA deficient NahG plants to see if TDL induces resistance under the different stress regimes in this highly susceptible background. As expected, NahG plants were more susceptible to Pst and accumulated significantly less SA following Pst infection than the WT background “New Yorker.” However, TDL provided strong protection in the NahG plants and mitigated the predisposing effect of salt-stress on bacterial speck disease.In a previous study we showed that ABA-deficient tomato mutants displayed a much reduced predisposition phenotype to salt stress . To determine if the protective effect of TDL is altered within an ABA-deficient tomato mutant,dutch bucket for tomatoes seedlings of WT and an ABA-deficient mutant within this background, sitiens, were treated in the same format and stress regimes as above. TDL significantly reduced Pst symptoms and colonization in both non-stressed and salt-treated plants of “Rheinlands Ruhm.” However, 3.6- and 5.4-fold increases in pathogen titer as a result of salt-stress were observed in both the control and TDL-treated plants, respectively, indicating that TDL did not prevent the proportional increase in Pst colonization in salt-stressed plants, similar to the results with “New Yorker” and NahG plants. In contrast, the sitiens mutant was not predisposed to Pst by salt stress and had significantly reduced symptoms and colonization by the pathogen than the background “Rheinlands Ruhm” . Nonetheless, TDL pretreatment of sitiens provided further protection against Pst .To determine if plant activators protect tomato roots and crowns against the oomycete pathogen, Phytophthora capsici, and predisposing root stress, tomato seedlings were treated with TDL or BTH , not stressed or salt-stressed as above, and then inoculated. There was no protection provided by the plant activators against disease caused by Phytophthora capsici in either the control or salt-treated plants, as reflected in symptom severity and pathogen colonization .Because elevated levels of ABA in tomato can enhance susceptibility to Pst and Phytophthora capsici, the effect of SA, TDL, and BTH on ABA levels was determined in roots and shoots. ABA concentrations in either shoots or roots at the time selected for inoculation in our treatment sequence were not altered by SA . However, a trend of increasing ABA accumulation was observed in TDL- and BTH treated “New Yorker” plants relative to the corresponding control plants . Although the increase in ABA accumulation in the plants treated with these plant activators is not statistically significant at P ≤ 0.05, it can be said that SA, TDL, and BTH do not reduce ABA content relative to untreated plants . In addition, salt stress did not further increase the levels of ABA in plants that had been pretreated with TDL or BTH, which were similar to the salt stressed controls.In a previous study, we demonstrated the predisposing effect of salt stress and a role for ABA as a determinative factor in predisposition in the tomato–Phytophthora capsici interaction .
The present study is the first report of salt-induced predisposition to the bacterial speck pathogen, Pst, in tomato. Furthermore, the results with the ABA-deficient sitiens mutant are consistent with the salt-induced susceptibility to Pst being mediated by ABA . These results conform to studies in Arabidopsis where ABA has been reported to promote susceptibility to Pst .Because SA has been shown to protect tomato against salt stress, possibly by an ABA-dependent mechanism , plant activators that operate via the SA pathway were evaluated for effect on salt-induced predisposition. Protection of tomato against bacterial speck disease by BTH is well documented , and TDL has previously been shown to reduce the severity of bacterial and fungal infections without inducing SA accumulation . Here, TDL was shown to protect against Pst in both non-stressed and salt-stressed tomato plants. TDL pretreatment strongly reduced disease and colonization by Pst in both “New Yorker” and SA-deficient NahG plants. TDL, or more likely its biologically active metabolite, SV-03, presumably allows the NahG plants to mount an SAR response to Pst infection in the absence of SA accumulation . TDL provided protection in both non-stressed and salt-stressed plants, but did not reverse the predisposing effect of salt stress. An increase in Pst colonization was observed in the salt-stressed, TDL-pretreated plants of both genotypes, with comparable percentage increases relative to the corresponding non-stressed controls in “New Yorker” and NahG plants. This indicates that TDL does not reverse the salt-stress effect on disease, per se, and likely targets stress network signaling independently of an ABA-mediated process that conditions the salt-induced susceptibility observed in this system . “Rheinlands Ruhm” also displayed salt-induced predisposition to Pst. Pretreatment with TDL significantly reduced Pst colonization in both “Rheinlands Ruhm” and sitiens . Similarly, TDL provided protection in both non-stressed and saltstressed plants, but did not reverse the predisposing effect of salt stress in “Rheinlands Ruhm” plants. The salt-induced increment in colonization by the pathogen was comparable in both the untreated and TDL-treated plants . The ABA-deficient mutant, sitiens, is considerably less susceptible to Pst than its background “Rheinlands Ruhm,” and does not exhibit salt-induced predisposition .Protection by plant activators against foliar pathogens is well established . However, relatively few studies have examined these compounds against soilborne pathogens and so TDL and BTH were evaluated for protection against root infection by Phytophthora capsici. Neither TDL nor BTH induced resistance or impacted salt-induced predisposition to Phytophthora capsici . Phytophthora capsici is an aggressive root and crown pathogen with a hemibiotrophic parasitic habit that triggers both SA- and jasmonic acid-mediated responses during infection of tomato .