Investigating the potential of unsaturated fatty acids as antifungal crop protective agents
Abstract
Pathogenic fungi cause significant yield losses and quality reductions to many crops including canola, wheat, and barley. Toxic metabolites produced by fungal pathogens, along with excessive application of synthetic fungicides, can also pose risks to human and livestock health. Hydroxy unsaturated fatty acids (HUFAs) are novel alternatives to commonly used fungicides. Here, the antifungal activities of two HUFAs, coriolic acid (CA) and ricinoleic acid (RA), were assessed in vitro and in planta for their activity against the important phytopathogens Fusarium graminearum Schwabe, Pyrenophora tritici-repentis (Died.) Drechsler, Pyrenophora teres f. teres Drechsler, Sclerotinia sclerotiorum (Lib.) de Bary, Leptosphaeria maculans Ces. & De Not., and Aspergillus niger Tiegh. on selected media, monocots or dicots. The results in vitro indicated that both CA and RA showed the strongest inhibitory activity against L. maculans and A. niger, but their activities varied with different fungi. On wheat and barley, CA but not RA reduced disease severity caused by Pyrenophora spp.; on canola, treatment with high levels of CA and RA led to oxidative damage of the plant tissues, and treatment with low concentrations of CA and RA did not reduce disease severity caused by L. maculans or S. sclerotiorum on canola. Our findings suggest that the utility of HUFAs in reducing disease severity caused by pathogenic fungi depends on the dosage and the plant and fungus targeted. High concentrations of HUFA can be phytotoxic on certain plants. In addition to their direct antifungal properties, additional mechanisms may be involved in the disease reduction of CA, suggesting the need for further evaluation of its potential use in crop protection.
Résumé
Les champignons pathogènes entraînent une perte de rendement et une diminution de la qualité importantes pour de nombreuses cultures, dont le canola, le blé et l’orge. Les métabolites toxiques que libèrent les cryptogames et l’application d’une quantité excessive de fongicides synthétiques peuvent aussi poser des risques pour la santé humaine et animale. Les acides gras hautement insaturés (HUFAs — « hydroxy unsaturated fatty acids ») viennent de faire leur entrée parmi les fongicides. Les auteurs ont évalué l’effet de l’acide coriolique (CA — « coriolic acid ») et de l’acide ricinoléique (RA — « ricinoleic acid »), deux HUFA, in vitro et in planta sur les phytopathogènes Fusarium graminearum Schwabe, Pyrenophora tritici-repentis (Died.) Drechsler, Pyrenophora teres f. teres Drechsler, Sclerotinia sclerotiorum (Lib.) de Bary, Leptosphaeria maculans Ces. & De Not., et Aspergillus niger Tiegh., dans divers milieux de culture et sur des monocotylédones et des dicotylédones. Les résultats des essais in vitro indiquent que l’CA et l’RA inhibent le mieux L. maculans et A. niger, mais leur efficacité varie avec le cryptogame. Si l’CA atténue la gravité de la maladie attribuée à Pyrenophora spp. chez le blé et l’orge, tel n’est pas le cas de l’RA. En ce qui concerne le canola, l’application d’un taux élevé d’CA et d’RA entraîne des dommages aux tissus végétaux par oxydation, mais à faible concentration, aucun des deux acides n’atténue la gravité de la maladie causée par L. maculans ou S. sclerotiorum. Ces résultats laissent croire que l’utilité des HUFA dans la lutte contre les champignons pathogènes dépend du dosage, de la plante et du cryptogame. À forte concentration, les HUFA peuvent s’avérer toxique pour certains végétaux. Outre leurs propriétés antifongiques, il se peut que d’autres mécanismes interviennent dans la réduction de la maladie par l’CA, ce qui exigerait une analyse plus approfondie de son utilité éventuelle pour la protection des cultures. [Traduit par la Rédaction]
Introduction
Pathogenic fungi pose significant challenges to the growth and development of plants and threaten crop production worldwide (Montesinos et al. 2002; Strange and Scott 2005). Disease outbreaks cause severe yield losses and quality reductions to major crops including wheat (Triticum aestivum L.) (Shabeer and Bockus 1988; Savary et al. 2012), canola (Brassica napus L.) (Pageau et al. 2006; del Río et al. 2007; Hwang et al. 2016), and barley (Hordeum vulgare L.) (Jayasena et al. 2007; Murray and Brennan 2010). Moreover, toxic metabolites produced by some phytopathogenic fungi also pose significant risks to animal and human health (Fajardo et al. 1995; Bottalico and Perrone 2002; Yoshida et al. 2008). Current management practices include the application of synthetic fungicides; however, their extensive usage may pose risks to the environment and human and (or) animal (Alavanja et al. 2014). Furthermore, repeated application of fungicides can result in the development of insensitivity or resistance in the target organisms (Brown et al. 2004; Gossen et al. 2014). Therefore, it is important to develop alternate crop protection strategies to mitigate the negative effects of fungal plant pathogens.
Unsaturated fatty acids (UFAs) and hydroxy unsaturated fatty acids (HUFAs) showed antifungal activities that depend on specific structural characteristics (Prost et al. 2005; Cantrell et al. 2008; Pohl et al. 2011; Black et al. 2013). These compounds may offer an alternative for crop protection. For instance, coriolic acid (CA; 13-hydroxy-9,11-octadecadienoic acid), that was first extracted from the seed oil of Coriaria nepalensis Wall. (Tallent et al. 1966, 1968), inhibited spore germination and germ tube elongation of the rice blast fungus (Namai et al. 1993). CA also inhibited the in vitro growth of fungal pathogens including Cladosporium herbarum (Pers.) Link, Botrytis cinerea Pers., Phytophthora infestans (Mont.) de Bary, Phytophthora nicotianae Breda de Haan (syn. Phytophthora parasitica var. nicotianae Tucker), Aspergillus niger Tiegh., and Penicillium roqueforti Thom (Prost et al. 2005; Chen et al. 2016; Liang et al. 2017). Ricinoleic acid (RA; 12-hydroxy-9-cis-octadecenoic acid), constituting more than 80% of the seed oil of castor plant (Ricinus communis L.) (Bafor et al. 1991), also has growth-inhibiting activity against pathogenic fungi including A. niger and P. roqueforti in vitro (Chen et al. 2016; Liang et al. 2017). The HUFA mixture, including mono-, di-, and tri-OH fatty acids was also accumulated in the supernatant of liquid cultures of Pseudomonas aeruginosa (Schroeter 1872) Migula 1900 42A2 when supplemented with linoleic acid, and this HUFA mixture exhibited antifungal activity against the pathogenic fungi Verticillium dahliae Kleb., Macrophomina phaseolina (Tassi) Goid., Trichophyton ajelloi (Vanbreus.) Ajello (syn. Arthroderma uncinatum C.O. Dawson & Gentles), Trichophyton mentagrophytes (C.P. Robin) Sabour., and Talaromyces funiculosus (Thom) Samson, N. Yilmaz, Frisvad & Seifert (syn. Penicillium funiculosum Thom) (Martin-Arjol et al. 2010). Similarly, oleic acid (OA), a non-hydroxylated UFA, or the 9-cis-octadecenoic acid derivatives, methyl- and ethyl-oleate, also showed activity against powdery mildew of barley caused by Blumeria graminis f. sp. hordei (Choi et al. 2010). In addition to their activities against fungal phytopathogens, in planta antiviral activity of OA has also been demonstrated against tobacco mosaic virus (Zhao et al. 2017).
While several studies have evaluated the efficacy of UFAs and HUFAs in plant disease control (Granér et al. 2003; Prost et al. 2005; Pohl et al. 2011), to our knowledge the exogenous application of these fatty acids for the control of pathogenic fungi of important Canadian crops has not been reported. In this study, we tested the efficacy of two HUFAs, CA and RA, as potential crop protection agents against phytopathogenic fungi. Minimum inhibitory concentrations (MICs) for CA and RA against selected, relevant phytopathogenic fungi were determined, as well as their activity to reduce the disease severity of wheat, barley, and canola against their common pathogens.
Materials and Methods
Preparation of UFAs
CA was produced either through enzymatic oxidation of linoleic acid or extracted from seed oil of C. nepalensis (XinTai Seed Production and Wholesale Company, Jiangsu, People’s Republic of China), followed by purification using high-speed counter current chromatography as previously described (Liang et al. 2017). RA and OA, each with a purity >99%, were purchased from Nu-Check Prep, Inc. (Elysian, MN, USA).
Preparation of fungal isolates and inoculum
The fungal isolates Pyrenophora tritici-repentis (Died.) Drechsler AB 7-2 (Aboukhaddour et al. 2013), Pyrenophora teres f. teres Drechsler AB 06 and AB 34 (Akhavan et al. 2016, 2017), Sclerotinia sclerotiorum (Lib.) de Bary SS-01 (Navabi et al. 2010), Fusarium graminearum Schwabe G-1, Leptosphaeria maculans Ces. & De Not. RL-60, and A. niger FUA5001 were used in the experiments. Growth conditions for each isolate are provided in Table 1. Following sporulation on specific growth media, approximately 5 mL of sterile distilled water (SDW) was added to each culture and gently scraped with a sterile inoculation loop. The spore suspensions were collected and filtered through four layers of cheesecloth under sterile conditions to eliminate mycelial fragments. Spores were counted using a Neubauer counting chamber (Fein-Optik, Jena, Germany), and spore concentrations were adjusted to specific amounts for the subsequent experiments. In the case of S. sclerotiorum, mycelial plugs were used for inoculation of plants rather than spore suspensions.
Table 1.
Determination of MIC
The antifungal activities of fatty acids were determined using the broth microdilution method as described previously (Magnusson and Schnürer 2001) with some modifications. Briefly, the fatty acids were dissolved in 100% ethanol at a concentration of 53.33 g·L−1. The fatty-acid stock solution (100 μL) was mixed with 100 μL of potato dextrose broth or V8-juice broth (for L. maculans) or mMRS broth (for A. niger) and serially diluted 2-fold to obtain final concentrations of 20, 10, 5, 2.5, 1.25. 0.625, 0.312, 0.156, 0.078, and 0.039 g·L−1 in microtiter plates. Ethanol was evaporated by placing the uncovered microtiter plates in a laminar flow hood. Subsequently, the wells were inoculated with 33.3 μL of fungal spore suspensions containing about 104 spores·mL−1. MIC was defined as the lowest concentration of the analyte required to completely inhibit the visible growth of each fungus. MICs were determined visually 1 d after fungal growth was visible in the positive control. MIC values were calculated from the average of three independent experiments using replicate preparations of the spores. The whole experiment was repeated twice for each fungal isolate.
Plant materials
Canola (B. napus ‘Westar’ and DH12075 line) were used as susceptible genotypes for experiments with L. maculans and S. sclerotiorum, respectively (Sharma et al. 2010; Joshi et al. 2016). Their seeds were sown in plastic inserts (5 cm × 5 cm; one seed per insert) filled with Sunshine potting mix (W.R. Grace and Co., Fogelsville, PA, USA) and grown in growth cabinets under 22 °C day/18 °C night with a light intensity of ∼800 μmol·m−2·s−1 and a 16 h photoperiod for 3 wk. Seedlings were watered regularly and were fertilized by Peters® NPK (20–20–20) solution on 2-wk-old seedlings at a concentration of 200 ppm (Sharma et al. 2010). Wheat (T. aestivum ‘Katepwa’) and barley (H. vulgare ‘Xena’) were treated as susceptible genotypes for experiments with the pathogens P. tritici-repentis and P. teres f. teres, respectively. The seeds were sown in plastic square pots (12.7 cm; six seeds per pot) filled with Sunshine potting mix and grown in growth cabinets under 20 °C day/18 °C night with 16 h photoperiod at ∼800 μmol·m−2·s−1, then watered regularly without the application of any fertilizer for 10–13 d until plants reached the 2–3 leaf stage (Aboukhaddour et al. 2013; Akhavan et al. 2016).
Foliar treatments and in planta assessments
For all plant experiments, SDW containing 0.05% Tween-20 (Polysorbate 20, pH 7.5) was used as the solvent for UFAs to obtain concentrations ranging from 0.12 to 2 g·L−1 (Bajpai et al. 2009). Seedlings treated with SDW containing 0.05% Tween-20 (pH 7.5) and later inoculated with fungal spore suspension served as positive controls. Negative controls were seedlings treated with SDW containing Tween-20 but without subsequent inoculation with fungal suspension. The fungicide Bumper 418 EC (propiconazole 418 g·L−1; ADAMA Agricultural Solutions, Winnipeg, MB, Canada) was also used as a control at a concentration of 1.5 mL·L−1; it was applied 36 h before inoculation with each pathogen. In addition, OA was applied on wheat and barley plants to determine whether the absence of a hydroxyl group in fatty acid chains would alter ability of UFAs to reduce the severity of disease. Experiments were arranged in a randomized complete block design with three biological replicates for each treatment and were repeated twice for each crop. Six plastic inserts, each with one seedling, represented one biological replicate in case of canola ‘Westar’ cultivar and DH12075 line. A single pot with six seedlings represented one biological replicate in case of the wheat ‘Katepwa’ and barley ‘Xena’ cultivars. In total, there were 18 replicates (6 technical replications × 3 biological replicates) for each treatment.
Evaluation of disease responses in canola: L. maculans
To evaluate the response of canola ‘Westar’ to L. maculans after treatment with fatty acids, 3-wk-old seedlings were placed in a misting chamber 24 h prior to inoculation with a relative humidity of >95%. Seedlings were air-dried in the greenhouse for about 2 h and treated manually with approximately 10 mL of CA or RA per seedling at concentrations of 0.12, 0.25, 0.5, 1, and 2 g·L−1 using a plastic-bottle sprayer. After 4 h, two true leaves of each seedling were wounded on both sides of the mid-rib with a sterile needle and inoculated with 10 μL of an L. maculans spore suspension (107 spores·mL−1). After 2 h, the seedlings were placed back in the humidity chamber for 48 h and then transferred to a growth cabinet maintained as described above. Disease severity was evaluated 14 d post inoculation as described previously (Sharma et al. 2010). Briefly, leaves were scanned using an EPSON Perfection V19 Scanner (Epson America Inc., USA), and the images analyzed using the APS Assess version 2.0 software (Lamari 2008). Mean disease severity was calculated as the percentage of lesion area divided by leaf area for each leaf.
Evaluation of disease responses in canola: S. sclerotiorum
To evaluate the effect of CA and RA treatments on infection by S. sclerotiorum, we inoculated canola line DH12075 following Joshi et al. (2016). Briefly, 21-d-old seedlings were placed in a humidity chamber for 24 h and air-dried in the greenhouse for 2 h. They were then treated with solutions of CA and RA as described above for ‘Westar’. After 4 h, two true leaves of each seedling were wounded on either side of the mid-rib using a sterile needle and inoculated with 3-d-old mycelial plugs (5 mm) containing actively growing S. sclerotiorum excised from potato dextrose agar (PDA) plates. Leaves inoculated with PDA plugs without fungal cultures served as negative controls. After inoculation, the plants were left in the humidity chamber for 24 h and then transferred to a growth cabinet. Disease severity was assessed 48 h post inoculation using APS Assess version 2.0 software as described above for L. maculans (Lamari 2008).
Evaluation of disease responses in wheat and barley
To evaluate the effects of fatty acids on the ability of pathogens to infect wheat and barley, seedlings of the wheat ‘Katepwa’ and barley ‘Xena’ (at the 2–3 leaf stage) were treated manually with approximately 8 mL of CA, RA, and OA at five different concentrations (0.12, 0.25, 0.5, 1, and 2 g·L−1) with a plastic-bottle sprayer 4 h before inoculation. Inoculation of the wheat cultivars with P. tritici-repentis and barley cultivars with P. teres f. teres was performed as described by previous studies Aboukhaddour et al. (2013) and Akhavan et al. (2016), respectively, with some modifications. Briefly, the seedlings were sprayed with a conidial suspension at a concentration of 4 × 103 spores·mL−1 in the case of P. tritici-repentis and 1 × 104 for P. teres f. teres using a pressurized sprayer connected to a laboratory air source. The inoculated seedlings were covered with moist plastic bags and kept for 24 h on laboratory benches to maintain the relative humidity at almost 100%. Subsequently, the bags were removed, plants were returned to the previous growth conditions, and disease severity was assessed 6 d post inoculation as described above for canola.
Determination of reactive oxygen species (ROS) levels
The accumulation of superoxide (O2−) and hydrogen peroxide (H2O2) in the canola ‘Westar’ after treatment with CA, RA, and OA was determined by histochemical staining (Daudi and O’Brien 2012). Two different methods, foliar and wounding treatments, were used to determine the effects of treatments at 1 and 2 g·L−1 of each of CA, RA, and OA. In one instance, the second leaves of 3-wk-old ‘Westar’ seedlings were wounded at two sides of the mid-rib using a sterile needle. Then, 10 μL of CA, RA, and OA at 1 or 2 g·L−1 was applied on each wounded site. For the foliar treatments, 3-wk-old ‘Westar’ seedlings were sprayed with 10 mL of CA, RA, and OA at the same concentrations. After 6 h, the second leaf was excised from the seedlings (three seedlings for each treatment) and placed in 50 mL Falcon tubes separately. Untreated seedlings and leaves treated with the solvent used to prepare the fatty acid solutions (water-tween 0.05%) served as controls. Staining of the leaves with Nitroblue Tetrazolium (NBT) and 3,3′-Diaminobenzidine (DAB) was performed following prior literature (Daudi and O’Brien 2012; Kumar et al. 2014).
Statistical analysis
Disease scores were analyzed using the MIXED procedure of SAS version 9.4 (SAS Institute Inc., Cary, NC, USA). The model for the experiment included different treatments [negative control, positive control, CA 0.12, CA 0.25, CA 0.5, CA 1, CA 2, OA 0.12, OA 0.25, OA 0.5, OA 1, OA 2, RA 0.12, RA 0.25, RA 0.5, RA 1, RA 2 g·L−1, and fungicide (Bumper 418 EC at concentration of 1.5 mL·L−1)] as the fixed independent variables and disease severity as the dependent variable. Data from all experiments were pooled together for statistical analysis, and time (experimental blocks) were considered as random effects. If the data for certain treatments were not normally distributed, the Box-Cox transformation within PROC TRANSREG was used to find the most appropriate data transformation. Differences between means were analyzed using a Tukey’s multiple comparison test with a 95% confidence level and were reported as a mean ± the standard error of the mean. Differences were considered significant at P < 0.05.
Results
MICs of CA and RA against pathogenic fungi
To determine the antifungal activity of CA and RA in vitro, the broth microdilution method was used (Table 2). Highest sensitivity to CA was observed in the case of L. maculans and A. niger with MIC of 0.73 (±0.10) and 0.78 (±0.10) g·L−1, respectively. Similarly, in the case of RA, L. maculans and A. niger showed highest sensitivity with MIC values of 0.83 (±0.10) and 0.88 (±0.34) g·L−1. A weaker inhibitory activity of CA and RA was observed against the phytopathogens S. sclerotiorum, P. teres f. teres, and P. tritici-repentis with higher MIC values (Table 2). The weakest inhibitory activity of CA and RA was observed for F. graminearum with MIC of over 6 g·L−1 (Table 2). In general, CA and RA showed comparable antifungal activity on each of the fungi tested, but their antifungal activities varied on different fungi.
Table 2.
Note: SEM, standard error of the mean; ND, not determined. Data are shown as means ± of triplicate independent experiments analyzed in duplicate.
Foliar application of fatty acids on canola and its disease severity reduction
The antifungal effects in situ of CA and RA were investigated first through foliar application on seedlings of canola. The ability of CA and RA to inhibit fungal growth in planta was evaluated in two genotypes of canola, ‘Westar’ and DH12075, which were inoculated with L. maculans (blackleg disease) and S. sclerotiorum, respectively. ‘Westar’ is more susceptible to L. maculans than the DH12075 line. Our results indicated that blackleg disease in ‘Westar’ was not significantly reduced by treatments with HUFAs at 0.12–1 g·L−1 (Fig. 1). Similar observations were obtained with CA- and RA-treated DH12075 canola when challenged with S. sclerotiorum (Fig. 2); however, a higher concentration of HUFAs could not be tested against phytopathogens in situ in this case, because at 2 g·L−1 both CA and RA caused extensive necrotic lesions on the leaves of both canola lines, ultimately resulting in plant death (Fig. 3).
Fig. 1.
Fig. 2.
Fig. 3.
Effects of CA, RA, and OA on ROS accumulation in canola
To understand whether CA and RA triggered oxidative stress responses through a disturbance of the steady-state level of ROS in canola, resulting in the observed lesions on the leaves, the accumulation of H2O2 and O2− was evaluated in the canola ‘Westar’ after treatment with 1 and 2 g·L−1 of CA or RA. Additionally, to investigate the roles of hydroxy group of fatty acids in the lesion-causing process, treatments with OA, which is the non-hydroxy analogue of RA (1 and 2 g·L−1), was also tested for comparison. Our results from NBT histochemical staining for O2− indicated that the OA-treated leaves stained heavily (Fig. 4A); however, compared with controls, no staining for O2− was observed in the CA and RA treatments (Fig. 4A). H2O2 accumulation was also detected by histochemical staining with DAB (Fig. 4B). Treatment of leaves with CA, RA, and OA resulted in intense staining compared with the controls, indicating the accumulation of H2O2 (Fig. 4B).
Fig. 4.
The effects of CA, RA, and OA treatments on disease severity in wheat and barley
To investigate the antifungal performance of HUFAs in situ on monocotyledons, treatments of HUFA were applied on wheat and barley and their resistance was evaluated. Different from the dicotyledonous canola, CA treatment of wheat prior to inoculation with P. tritici-repentis (isolate AB 7-2) resulted in a reduction of visual symptoms (Fig. 5). A significant (P = 0.0001) reduction in disease severity was caused by CA of 0.5–2 g·L−1 in comparison with the positive control, in which a disease severity of 21% was observed (Fig. 6). Its activity to reduce disease severity on wheat was comparable to the fungicide control, fungicide bumper 418 EC (3.9%), at a concentration of 1.5 mL·L−1. In addition, this activity of CA to reduce disease severity on wheat was a dose-dependent response, as the 0.12%–0.25% CA did not show a protective effect. In contrast, the disease severity was not reduced by the treatments with RA and OA (Fig. 6). The difference between the disease severity reduction of CA and RA was surprisingly inconsistent with the similarity of the MIC of CA and RA against P. tritici-repentis (Table 2).
Fig. 5.
Fig. 6.
When barley seedlings were treated with CA at 0.5–2 g·L−1, a significant reduction in disease symptoms caused by P. teres f. teres (isolate AB 34) was also observed relative to the positive control (Fig. 7); however, the reduction in disease severity was not as strong as treatment with the fungicide bumper 418 EC (2.5%). RA and OA at any tested concentration did not significantly reduce the disease severity caused by P. teres f. teres in barley (Fig. 7). The overall ability of RA and OA to control these disease symptoms was significantly lower compared with CA (Fig. 7), which was inconsistent with their antifungal activity in vitro (Table 2).
Fig. 7.
Discussion
This study determined the antifungal activities of two HUFAs, CA and RA, against phytopathogenic fungi in vitro and in situ in dicot and monocot plants. Results here expand on prior studies with HUFA that were conducted with cucumbers, tobacco, and Arabidopsis; moreover, the comparison of different HUFA species and different host species provides important clues on possible mechanisms of their activity.
Antifungal tests in vitro showed that antifungal activities of HUFA depends on fungal species, which is consistent with the studies on HUFA against other phytopathogens and food spoilage and (or) fermentation fungi (Granér et al. 2003; Prost et al. 2005; Liang et al. 2017). In particular, comparison of the MIC measured here for the phytopathogens with MIC available in the literature for other fungi indicated that phytopathogens were more resistant to HUFA compared with food spoilage and (or) fermentation filamentous mold but not yeast (Liang et al. 2017). The underlying mechanisms of this variation of fungal sensitivity to HUFA remains to be elucidated, although the composition of fungal membranes such as sterol content has been linked to their sensitivity towards non-hydroxy fatty acids (Avis and Bélanger 2001). In addition, HUFA may affect fungi differently at various stages of development, such as asexual spore germination, hyphal growth, and sexual production (Mazur et al. 1991; Granér et al. 2003; Scala et al. 2014). How these additional roles of HUFA on fungal physiology contribute to their antifungal properties is not well understood, especially under field conditions that may involve more than one fungus.
In contrast with the antifungal activities in vitro, CA, but not its analogues RA and OA, reduced disease severity caused by P. tritici-repentis on wheat. Meanwhile, the active concentration of CA in situ against this pathogen (0.5 g·L−1) is even lower than its in vitro MIC (1.64 ± 0.46 g·L−1). The disease severity caused by P. teres f. teres on barley was also reduced by CA (0.5 g·L−1 in planta vs. MIC = 1.66 ± 0.20 g·L−1 in vitro), but not by the other tested fatty acids. This mismatch between MIC in vitro and effective concentration in planta suggests the possible activation of other plant defense mechanisms by HUFA. Similarly, a few previous studies have demonstrated that the MIC of HUFA in vitro cannot always entirely explain their antimicrobial performance in plants. For example, 2-OH linolenic acid (2-OH C18:3) exhibited a tissue-protective effect on tobacco leaves against phytopathogenic bacterial infection (Hamberg et al. 2003), but this protective effect was not attributed to its direct bacteriostatic or bactericidal effects against Pseudomonas syringae subsp. syringae van Hall and P. syringae pv. tabaci (Hamberg et al. 2003; Prost et al. 2005). A series of Arabidopsis mutants that do not respond to 9-lipoxygenase mediated signaling to increase the defense against microbial pathogens was also more susceptible to infection with P. syringae (Vellosillo et al. 2013). While 9-OH C18:3 is not inhibitory to P. syringae (Prost et al. 2005), it may serve as a signal for plant defense pathways that involve brassinosteroid-signaled cell wall synthesis, contributing to physical protection against P. syringae (Marcos et al. 2015). The mechanisms of plant protection mediated by CA were not examined in this study. Possibly, it can be attributed to their function as signaling compounds to trigger further pathogenic-related gene expression that regulates further pathogenic response (Weichert et al. 1999; Weber 2002). For example, an exogenously applied 10 μmol·L−1 solution of 13-OH C18:3, (13S, 9Z,11E,15Z)-13-hydroxy-9,11,15-octadecatrienoic acid can induce pathogenic-related gene PR1b expression in barley leaf segments (Weichert et al. 1999), and the PR1b proteins are antifungals (minimal concentration of 100 μg·mL−1 to inhibit the germination of zoospores by more than 90%) (Niderman et al. 1995).
The plant protection and phytotoxic effects of HUFA varied among the host species evaluated, noticeably between the dicots and monocots. Disease reduction by CA was observed only on the monocots, wheat, and barley. In addition, a reduction in disease severity was observed only on the whole plants but not seed treatments, indicating the effect of plant physiological stage on the efficacy of HUFA (data not shown). In contrast, in the dicot, foliar-sprayed CA or RA did not decrease disease severity on canola caused by L. maculans or S. sclerotiorum. This is distinct from results observed with the application of HUFA on tobacco leaves (Hamberg et al. 2003) and Arabidopsis (Vellosillo et al. 2013), which appeared to be effective. Furthermore, a high concentration (2 g·L−1) of CA or RA caused wilting and formed necrotic lesions on canola leaves but had no effect on seed germination (data not shown). The cause of this plant- and physiological stage–specific toxicity is not clear, but it has been observed on other important defense molecules such as salicylic acid (SA) (Hayat et al. 2010), indicating the delicate requirement for concentration, time, location, and plant on applying these molecules as antipathogenic agents.
In addition, our results suggest the phytotoxicity of HUFAs is associated with the generation of H2O2. Our results suggest that while the application of CA, RA, and OA resulted in the accumulation of H2O2, these treatments did not affect the accumulation of O2−; the accumulation of ROS may account partially for the death of canola plants treated with high concentration of HUFA. In plants, H2O2 triggers programmed hypersensitive cell death, which plays critical roles in plant defense (Levine et al. 1994). This hypersensitive response (HR) indicates the successful recognition of pathogens, and the triggered localized cell death is usually linked to restricting the infection spread (Morel and Dangl 1997; Zurbriggen et al. 2010). More importantly, HR is usually associated with the development of systemic acquired resistance (SAR), a broad-spectrum systemic mechanism of resistance to pathogenic infection (Balint-Kurti 2019). This process is mediated by the hormone SA, and the production of H2O2 is both upstream and downstream of SA synthesis (Herrera-Vasquez et al. 2015). Whether or not CA, RA, and their induction of H2O2 production directly contributed to the activation of HR and SAR, and whether it will support the development of a local application of HUFA on plant protection, requires further investigation. Contrary to the potential of CA and RA to activate HR, 2-OH linoleic acid protects the plant from cell death during HR induced by bacterial infection (Hamberg et al. 2003), although this effect is also specific to plant species (Vicente et al. 2012). A further study on the phytotoxicity or plant disease reduction of various HUFA structures, the induction of HR by HUFA, and their antifungal application on both dicotyledonous and monocotyledonous plants, is desirable.
In conclusion, this study identified HUFAs as promising candidates for plant protection, especially with an active concentration of CA to reduce disease caused by Pyrenophora spp. on wheat and barley; however, the efficacy of HUFA in planta depends on the concentration and molecular species of HUFA used, the plant host species, as well as the target pathogen. Based on our observations, additional studies and the further development of HUFAs may be worthwhile to further explore their utility as plant disease management tools. Efforts in developing alternative or complementary antifungal methods to the currently used agricultural fungicide or fungistatic agents will contribute to the reduction of food and agriculture waste caused by pathogenic or spoilage fungi.
Acknowledgements
Western Grains Research Foundation and Alberta Wheat Commission are acknowledged for their funding support for this project. Nuanyi Liang acknowledges funding of Globalink Graduate Fellowship from Mitacs. Michael G. Gänzle acknowledges funding from the Canada Research Chairs program.
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Canadian Journal of Plant Science
Volume 101 • Number 1 • February 2021
Pages: 73 - 85
Editor: Brian Beres
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Received: 30 April 2020
Accepted: 27 July 2020
Version of record online: 31 July 2020
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© 2021.
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AzadehYasari, NuanyiLiang, AidinForoutan, Michael G.Gänzle, Stephen E.Strelkov, and Nat N.V.Kav. 2020. Investigating the potential of unsaturated fatty acids as antifungal crop protective agents. Canadian Journal of Plant Science.
101(1): 73-85. https://doi.org/10.1139/cjps-2020-0113
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