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Volatile organic compounds of some Trichoderma spp. increase growth and induce salt tolerance in Arabidopsis thaliana
Fungal Ecology 29 (2017) 67e75
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Volatile organic compounds of some Trichoderma spp. increase growth and induce salt tolerance in Arabidopsis thaliana Farnaz Jalali a, Doustmorad Zafari a, *, Hooman Salari b a b
Bu-Ali Sina University, Department of Plant Protection, College of Agriculture, Hamedan, Iran Razi University, Department of Agronomy and Plant Breeding, College of Agriculture and Natural Resources, Kermanshah, Iran
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 July 2016 Received in revised form 12 June 2017 Accepted 27 June 2017
Many beneficial effects of Trichoderma spp. on plant growth and/or resistance to biotic/abiotic stresses can result from the production of bioactive compounds including volatile organic compounds (VOCs). We evaluated the effects of the volatile mixtures from 13 strains of different Trichoderma species on induction of tolerance to salt stress (100 mM NaCl) as well as growth promotion of Arabidopsis thaliana. Plants responded differently due to the presence of VOCs from various Trichoderma species ranging from both growth promotion and induction of salt tolerance to no significant changes under any of the conditions tested. In plants exposed for 2 weeks to VOCs of the selected strain, i.e. Trichoderma koningii, there was less H2O2 accumulation under salt stress compared to that in control plants. This result may reflect the possible role of VOCs of this strain in plant protection against oxidative damage under salt stress. Together, induction of salt tolerance using VOCs should be added to the known mechanisms of plant vigor enhancement by Trichoderma spp. © 2017 Elsevier Ltd and British Mycological Society. All rights reserved.
Corresponding Editor: Barbara Joan Schulz Keywords: Volatile organic compounds (VOCs) Induced systemic tolerance (IST) Abiotic stress Growth promotion Salt tolerance Arabidopsis thaliana Trichoderma spp. Bioactive compounds 3, 30 -Diaminobenzidine Reactive oxygen species (ROS)
1. Introduction Trichoderma spp. (teleomorph Hypocrea) are saprotrophic fungal inhabitants of soil that also may function as opportunistic, avirulent plant symbionts (Harman et al., 2004; Hermosa et al., 2012; Zaidi et al., 2014). Beneficial roles of Trichoderma spp. for plants comprise indirect and direct interactions. Indirect interaction with plants occurs via mycoparasitism and competition with, or antagonism of plant pathogens, mainly fungi and nematodes (Harman et al., 2004; Shoresh et al., 2010; Zaidi et al., 2014). Direct interaction of the fungus with plants can lead to an increase in plant growth, especially of roots, and particularly under stress (Harman, 2000; Shoresh et al., 2010; Hermosa et al., 2012), systemic resistance to disease (Bae et al., 2011; Mathys et al., 2012), systemic tolerance to abiotic stress, including water deficit (drought) (Bae
* Corresponding author. E-mail addresses: [email protected] (F. Jalali), [email protected] (D. Zafari), [email protected] (H. Salari). http://dx.doi.org/10.1016/j.funeco.2017.06.007 1754-5048/© 2017 Elsevier Ltd and British Mycological Society. All rights reserved.
et al., 2009; Mastouri et al., 2010; Zaidi et al., 2014), salt (Yildirim et al., 2006; Mastouri et al., 2010; Brotman et al., 2013; Zaidi et al., 2014), chilling stress and heat stress (Mastouri et al., 2010; Shoresh et al., 2010); enhancement of the vigor of poor quality seeds (Mastouri et al., 2010; Zaidi et al., 2014); bioremediation of contaminated soil (Adams and De-Lij, 2007; Shoresh et al., 2010); solubilization of phosphorus and increased availability of micronutrients and improved nitrogen use efficiency (NUE) by plants (Shoresh et al., 2010; Harman, 2011a, 2011b). Various compounds released by Trichoderma spp. into the zone of interaction with plants are associated with these advantageous effects and include proteins, peptides, secondary metabolites, and volatile organic compounds (VOCs) (Shoresh et al., 2010; Hermosa et al., 2012). VOCs are low molecular mass, usually hydrophobic, compounds with high vapor pressure which due to their small size can diffuse through the atmosphere and soils (Morath et al., 2012; Hung et al., 2015). Their physical properties make them valuable for interspecies communication as ‘infochemicals’ or ‘semiochemicals’, especially in non-aqueous environments (Hung et al., 2013).
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VOCs of different Trichoderma species inhibit growth of other fungi, in particular wood decay basidiomycetes and plant pathogens, thereby improving plant health (Dennis and Webster, 1970; Wheatley et al., 1997; Humphris et al., 2001). The role of microbial VOCs in direct interaction with plants, including growth promotion and induction of systemic resistance (ISR) to disease, was first reported by Ryu et al. (2003, 2004) for bacterial VOCs. There has also been growing awareness regarding the important role of Trichoderma VOCs in direct interactions with plants. For example, the volatile 6-n-pentyl-6H-pyran-2-one (6PP) produced by some strains of Trichoderma harzianum and Trichoderma atroviride induces defense mechanisms against Botrytis cinerea and Leptosphaeria maculans in tomato and canola, respectively (Vinale et al., 2008). The enhancement of growth of Arabidopsis thaliana by volatile mixtures emitted from the biocontrol fungus Trichoderma viride was reported by Hung et al. (2013). ContrerasCornejo et al. (2014) reported that Trichoderma virens VOCs elicit both development and defense programs in Arabidopsis. Moreover, co-cultivation of Arabidopsis with Trichoderma asperellum IsmT5 leads to increased numbers of trichomes, accumulation of defense-related compounds and expression of defense-related genes (Kottb et al., 2015). More recently, Lee et al. (2016) screened 20 strains, representing 11 different Trichoderma species, and identified nine Trichoderma strains that produced plant growth promoting VOCs. Although abiotic stresses are the most harmful factors that lower productivity of crops worldwide, no investigations have been reported regarding the role of Trichoderma or any other fungal VOCs in improving abiotic stresses in plants. However, involvement of Bacillus subtilis GB03 VOCs in induction of systemic tolerance (IST) to salt stress has been reported (Zhang et al., 2008). Salt stress is one of the most commonly observed abiotic stresses that significantly reduce yield and affect almost every aspect of the physiology and biochemistry of plants (Josine et al., 2011). Considering the fact that, “A single treatment of plants that could simultaneously promote growth and confer resistance to stresses would be of importance to agricultural plant production.” (Mastouri et al., 2010), the goal of our study was to identify potential strains among several species of Trichoderma which could induce salt tolerance as well as growth promotion in Arabidopsis via VOC production. After selecting one strain on the basis of its growth promoting and salt tolerance inducing effects, we tested whether the VOCs from this strain could improve salt tolerance by reducing hydrogen peroxide (H2O2) accumulation as the most stable reactive oxygen species (ROS) to be accumulated in plants under stress (Bose et al., 2014).
2. Materials and methods 2.1. Sources of Trichoderma strains and Arabidopsis thaliana seeds Trichoderma strains were obtained from the Mycological Laboratory of Plant Protection Department of Bu-Ali Sina University, Hamedan, Iran. These strains have been identified on the basis of morphological characteristics and sequences of two genes (tef and ITS region of rRNA), but due to some commercial purpose they could not be deposited in a public database, yet (Nazmi, unpublished data). The strains screened were: an anamorph of Hypocrea orientalis, T. asperellum, T. atroviride (2 strains), Trichoderma brevicompactum, Trichoderma citrinoviride, Trichoderma crassum, T. harzianum, Trichoderma koningii, Trichoderma koningiopsis, Trichoderma longibrachiatum, T. viride and Trichoderma viridescens. A. thaliana seeds (Ecotype Columbia-0) were obtained from the seed bank of Razi University, Kermanshah, Iran.
2.2. Sterilization and growth conditions for A. thaliana Seeds were surface-sterilized by treating them sequentially in a 70% ethanol, 5% NaOCl (bleach solution), rinsing in sterile water and stratifying at 4 C for 3 d on 0.1% (w/v) agarose to synchronize germination (Weigel and Glazebrook, 2002). Seeds were plated after stratification with an appropriate density in 90 15 mm Petri plates containing 25 ml of full strength Murashige and Skoog (MS) medium including vitamins (MO222.0025, Duchefa Biochemie, Haarlem, The Netherlands) supplemented with 3% sucrose and 0.4% (w/v) purified agar. The plates were transferred to a growth chamber at 21 C ± 2 C with a 16 h photoperiod. Plates were incubated vertically to allow seedlings to grow over the agar surface and to facilitate the transfer of germinated seedlings after 4 d. 2.3. Plant exposure experiments Five seedlings were transferred onto one part of a 100 15 mm partitioned Petri dish (split- or I-plate) containing either 15 ml of MS medium or 15 ml of MS supplemented with NaCl (100 mM) for salt stress. In each case, Trichoderma spp. were inoculated as discs, 5 mm in diameter, cut from the growing edge of the fungal mycelium on culture plates, onto a Petri plate (35 10 mm) containing 4 ml of malt extract agar (MEA, 105398, Merck, Darmstadt, Germany). Trichoderma spp. plates were placed into the empty region of the split plate and allowed to grow with Arabidopsis. For controls, the same plate-within-a-plate system was used with sterile media (MEA) without fungal inoculation (Lee et al., 2015). Plants and fungi were grown for 14 d at 21 C ± 2 C with a 16 h photoperiod. At the end of the exposure period, individual plants were removed from the testing condition; fresh weight and total chlorophyll content were assessed (Hung et al., 2013). 2.4. Plant chlorophyll measurements Total chlorophyll content of plants was determined using a spectrophotometer (Varian Technologies, Cary 100 scan, Pal Alto, California, US) according to the method described by Hung et al. (2013). Shoot tissue was submerged overnight in 1 ml of 80% acetone in the dark at 4 C. The total chlorophyll content (chlorophyll a and b) was calculated from the equation [(8.02) (A663) þ (20.2) (A645)] V/1000 W, where V is volume and W is plant shoot fresh weight (Palta, 1990). 2.5. CO2 assay To separate the VOC-based plant growth promotion from the effects of CO2 accumulation that might occur in enclosed dualculture systems (Piechulla and Schnitzler, 2016), CO2 trapping was performed in a way similar to that of Lee et al. (2016) for the strain selected from the screening experiment, i.e. T. koningii. With this method, KOH is used to trap CO2 as K2CO3, leading to lower CO2 concentrations while microbial volatile organic compounds (mVOCs) become enriched relative to CO2 (Lee et al., 2016). Briefly, a sterile cotton ball containing 3 ml of 0.1 M KOH was placed onto a sterile aluminum cap. Then the cap was placed into an empty region of the three-partitioned Petri dishes (100 15 mm) containing the fungal culture and plants in other parts, separately. Plates were kept under the same condition as described above. At the end of the exposure period, individual plants were sampled to assess fresh weight and total chlorophyll content. 2.6. Histochemical stainings Histochemical staining was performed on plants exposed to
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VOCs of the selected strain (T. koningii) and control plants as described by Hung et al. (2013). In summary, to detect the possible growth of endophytic Trichoderma or possible pathogenic fungi that may have contaminated the cultured plants, trypan blue staining was used. Trypan blue also stains dead plant cells, so its absence is an indicator of plant health. Whole plants were submerged in a trypan blue solution (BI1014-100, Bioidea Company, Tehran, Iran) overnight. To stain for hydrogen peroxide (H2O2), the whole plant was submerged in a 3, 30 -diaminobenzidine (DAB, D12384, Sigma Aldrich, St Louis, Mo, US) solution (1 mg ml_1, pH 3.8) for 5 h. In each case, chlorophyll was removed by soaking the plant in 95% ethanol overnight. Stained plant tissues were examined macroscopically and microscopically. 2.7. Measuring H2O2 content Hydrogen peroxide content of Arabidopsis seedlings that had or had not been exposed to VOCs from T. koningii was measured spectrophotometrically according to Alexieva et al. (2001). The reaction mixture consisted of 0.5 ml supernatant of 0.1% trichloroacetic acid (TCA) seedling extract, 0.5 ml of 100 mM Kphosphate buffer (pH 7), and 2 ml reagent (1 M KI, w/v in fresh double-distilled water). The blank consisted of 0.5 ml of 0.1% TCA without seedling extract. After 1 h of incubation in darkness at room temperature, the absorbance was measured at 390 nm. The amount of hydrogen peroxide was calculated using a standard curve prepared with known concentrations of H2O2. 2.8. Experimental design and data analysis A fully factorial completely randomized design was used. For the screening test, there were two factors including fungal VOC mixtures at 14 levels (emitted from control medium and 13
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Trichoderma isolates) and salt at two levels (0 and 100 mM NaCl). For the CO2 and H2O2 assays, the factors were VOC mixtures at two levels (emitted from control medium and T. koningii) and salt at two levels (0 and 100 mM NaCl). Analyses were performed using the average of five seedlings in each Petri dish as one treatment. Each treatment had three technical replications and experiments were repeated independently two times, resulting in a total number of 6 replicates. The normality test of the data at 5% probability level, analysis of variance and Duncan's multiple range tests were performed for all quantitative data using SPSS (ver. 16.0) and SAS (ver. 9.1), respectively (for chlorophyll content the data were transformed and analyzed based on a logarithm, according to the normality test results). We also created a scatter plot to show percentage of difference from the control for each trait (shoot and root fresh weight and chlorophyll content) in plants exposed to VOCs of different Trichoderma strains using XLSTAT 2017 software. Confidence ellipses were based on chi-square, at 95% confidence interval. 3. Results After 2 weeks of exposure, the morphological and physiological responses of Arabidopsis to Trichoderma spp. VOCs were assessed by measuring phenotypic characteristics including shoot fresh weight, root fresh weight, and total chlorophyll content. Plants exhibited different responses in the presence of VOCs from various Trichoderma strains ranging from growth promotion and induction of salt tolerance to no significant changes in any of the conditions tested (Fig. 1). In the absence of salt, there was a significant (p 0.05) increase in the shoot and root fresh weight and chlorophyll content of Arabidopsis exposed to volatile mixtures emitted from T. viride, T. atroviride 2, T. longibrachiatum, T. citrinoviride, T. harzianum,
Fig. 1. Arabidopsis seedlings exposed to Trichoderma spp. VOCs and control plants in the absence and presence of salt (100 mM NaCl) for 14 d. Hypocrea orientalis (A), T. asperellum (B), T. atroviride 1 (C), T. atroviride 2 (D), T. brevicompactum (E), T. citrinoviride (F), T. crassum (G), T. harzianum (H), T. koningii (I), T. koningiopsis (J), T. longibrachiatum (K), T. viride (L), T. viridescens (M). Equivalent to controls (¼), Plant growth promotion (þ).
70 F. Jalali et al. / Fungal Ecology 29 (2017) 67e75 Fig. 2. Arabidopsis seedlings exposed to Trichoderma VOCs in the absence and presence of salt (100 mM NaCl). Mean of shoot fresh weight (mg) (A), root fresh weight (mg) (B), and chlorophyll content (mg g1) (C) of seedlings exposed to VOCs for 2 weeks compared to control plants (Number of replicates, N ¼ 6). Error bars represent the standard deviation of the mean. * indicates significance compare to the control using Duncan's multiple range test (p < 0.05).
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T. koningii, T. koningiopsis, H. orientalis and T. viridescence compared to control plants (Fig. 2). In the presence of VOCs from these strains, plants were taller with more leaf surface area, more lateral roots and leaves with a darker hue of green than control plants (Data not shown). Plants co-cultivated with VOCs from T. asperellum, T. atroviride 1, T. brevicompactum and T. crassum did not show any significant changes (Figs. 1 and 2). Under salt stress, plants exposed to VOCs emitted from T. koningii, T. viridescence and H. orientalis increased significantly (p 0.05) in shoot fresh weight compared to control plants (Fig. 2A); those exposed to VOCs from T. koningii and H. orientalis also exhibited a significant increase in root fresh weight compared with control plants (Fig. 2B). Total chlorophyll content decreased under salt stress (100 mM) in control plants as well as plants exposed to VOCs as compared to those without salt stress. However, under salt stress as compared to control plants, exposure to VOCs of T. viridescence, T. koningii, T. asperellum, T. harzianum, T. atroviride 2, T. koningiopsis, T. atroviride 1, H. orientalis and T. viride led to a
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significant increase (p 0.05) in chlorophyll content (Fig. 2C). Using a scatter plot and statistical analysis, we were able to cluster strains on the basis of their VOCs' effects on the plant shoot fresh weight, plant root fresh weight, and total chlorophyll content with and without salt stress (Fig. 3). Concerning shoot fresh weight, three groups were distinguished: (1) strains which improved this trait under both conditions including T. koningii, T. viridescence and H. orientalis; (2) strains which improved this trait only in the absence of salt, i.e. T. atroviride 2, T. viride, T. longibrachiatum, T. citrinoviride, T. harzianum, T. koningiopsis; and (3) strains which did not induce any significant changes in either of the conditions, i.e. T. asperellum, T. atroviride 1, T. brevicompactum and T. crassum (Fig. 3A). In the case of root fresh weight, the categories were the same with the exception of T. viridescence, whose VOCs did not improve this trait under salt stress (Fig. 3B). Strains were categorized into four groups according to the effect of their VOCs on increasing chlorophyll content of the plants as follow: (1) strains which improved this trait under both conditions
Fig. 3. Scatter plots showing the effects of Trichoderma strains' VOCs on plant growth and induction of salt tolerance. X and Y axes represent percentage difference to control plants of plants exposed to VOCs of Trichoderma strains in the absence and presence of salt with respect to shoot fresh weight (A), root fresh weight (B) and chlorophyll content (C). Confidence ellipses were based on chi-square, at 95% confidence interval. Hypocrea orientalis (A), T. asperellum (B), T. atroviride 1 (C), T. atroviride 2 (D), T. brevicompactum (E), T. citrinoviride (F), T. crassum (G), T. harzianum (H), T. koningii (I), T. koningiopsis (J), T. longibrachiatum (K), T. viride (L), T. viridescens (M).
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Fig. 4. A. thaliana plants grown with or without KOH solution and with or without exposure to VOCs of T. koningii for 2 weeks in the absence and presence of salt (100 mM NaCl) (N ¼ 6). Control (no VOC exposure and no salt) (A), exposure to T. koningii VOCs without salt (B), control (salt but no VOC exposure) (C) and exposure to T. koningii VOCs with salt (D).
included T. viride, T. atroviride 2, T. harzianum, T. koningii, T. koningiopsis, H. orientalis and T. viridescens; (2) strains which improved this trait only in the absence of salt stress, i.e. T. longibrachiatum and T. citrinoviride; (3) strains which improved this trait only under salt
stress, i. e T. asperellum and T. atroviride 1; and (4) strains which did not induce any significant changes in chlorophyll content under either of the conditions i.e. T. brevicompactum and T. crassum (Fig. 3C). Considering all the above aspects, T. koningii was the most successful species tested with respect to growth promotion, as well as induced salt tolerance via production of VOCs, and was thus chosen for further studies. This strain increased shoot and root fresh weight and chlorophyll content of plants in the absence of salt by 101%, 152% and 240%, respectively, and 62%, 128% and 383%, respectively under salt stress (Fig. 3). Trapping possible CO2 accumulation with 0.1 M KOH solution did not eliminate the observed beneficial effects of T. koningii VOCs under any of the conditions tested (Fig. 4). Without salt stress, there was still a 97% increase in shoot fresh weight, a 151% increase in root fresh weight and a 244% increase in chlorophyll content for plants exposed to T. koningii VOCs and KOH solution compared to the negative control grown only in KOH that was significantly different from the control without KOH (p 0.05) (Fig. 5). There was also a 69% increase in shoot fresh weight, a 111% increase in root fresh weight and a 390% increase in chlorophyll content for plants exposed to Trichoderma VOCs and KOH solution compared to the negative control grown only with KOH under salt stress that was significantly different from the control with neither KOH or NaCl (p 0.05) (Fig. 5). Trypan blue staining revealed no endophytic Trichoderma or other contaminating fungi in plant tissues throughout the 2 weeks of exposure to Trichoderma VOCs. In addition, under salt stress there seemed to be less cell death in plants exposed to VOCs of T. koningii than in control plants (Fig. 6).
Fig. 5. A. thaliana exposed to KOH solution and plants exposed to T. koningii VOCs and KOH solution for 2 weeks in the presence and absence of salt (100 mM NaCl). Mean of shoot fresh weight (mg) (A), root fresh weight (mg) (B), and chlorophyll content (mg g1) (C) (N ¼ 6). Error bars represent the standard deviation of the mean.* indicates significance compare to control using Duncan's multiple range test (p < 0.05).
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Fig. 6. Trypan blue staining of control plants and plants exposed to T. koningii VOCs for 2 weeks. Control (no VOC exposure and no salt) (A), plant exposed to T. koningii VOCs without salt (B), control (salt but no VOC exposure) (C), and plant exposed to T. koningii VOCs with salt (D).
The near lack of DAB staining in tissues of both exposed and control plants in the absence of salt, indicated an absence of stress responses. However after 2 weeks of exposure, lower amounts of H2O2 accumulation were observed in the plants exposed to T. koningii VOCs under salt stress compared to controls (Fig. 7), suggesting that VOCs of T. koningii may be involved in plant protection against oxidative damage under this condition. Data from spectrophotometrical determination of H2O2 content in plants also confirmed DAB staining results. The level of H2O2 was not significantly different between exposed and control plants after 14 d of co-cultivation under no salt stress. H2O2 content increased significantly (p 0.05) in exposed and unexposed plants under salt stress. However, this level was significantly (p 0.05) lower in exposed plants compared to controls (Fig. 8).
4. Discussion
Fig. 7. DAB staining of control plants and plants exposed to T. koningii VOCs for 2 weeks. Control (no VOC exposure and no salt) (A), plant exposed to T. koningii VOCs without salt (B), control (salt but no VOC exposure) (C) and plant exposed to T. koningii VOCs with salt (D).
Our screening experiment enabled us to identify nine out of 13 Trichoderma strains that produced plant growth promoting VOCs, thereby supporting the report of Hung et al. (2013) and Lee et al. (2016) regarding growth promoting effects of T. harzianum, T. longibrachiatum and T. viride VOCs on Arabidopsis. In addition to growth promotion, VOCs of some strains increased salt tolerance of Arabidopsis seedlings without physical contact, a phenomenon which was called “aromatic therapy for plant stresses” by Ryu
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Fig. 8. H2O2 content in control plants and plants exposed to T. koningii VOCs for 2 weeks in the presence and absence of salt (100 mM NaCl) (N ¼ 6). Error bars represent the standard deviation of the mean. * indicates significance compared to control using Duncan's multiple range test (p < 0.05).
(2015) for bacterial VOCs. There were significant differences amongst Trichoderma spp. regarding effects of their VOCs on Arabidopsis under the different growth conditions we tested. For instance, VOCs of some species of Trichoderma, i.e. T. longibrachiatum and T. citrinoviride, which promoted plant growth in the absence of salt, did not do so when the medium contained salt. On the other hand, VOCs of some species, i.e. T. asperellum and T. atroviride1, which increased chlorophyll content of plants under salt stress, did not do so in the absence of salt. It was also notable that plants exposed to VOCs of the different strains of T. atroviride also induced varied responses. In the absence of salt, exposure to T. atroviride2 had a growth promoting effect on Arabidopsis, whereas growth of plants exposed to T. atroviride1 VOCs did not significantly differ from that of the controls. These results show that there are strain and species differences among Trichoderma isolates regarding the affects of their VOC emissions on Arabidopsis. The volatile profiles of these Trichoderma isolates should be studied, as well as the cognate pathways in Arabidopsis that are affected by these different VOCs mixtures. A single treatment of plants that could simultaneously promote growth and confer resistance to stresses would be of importance to agricultural plant production (Mastouri et al., 2010). On the basis of our screening experiment, we selected T. koningii as a promising biocontrol agent which can induce growth promotion as well as salt tolerance via its VOC emissions. In other studies, it was shown that the volatile metabolites of one strain of T. koningii had an inhibitory effect on the growth of the plant pathogen B. cinerea (Alizadeh et al., 2007). Our observations add to knowledge about the direct beneficial effects of T. koningii VOCs on plants. It is known that accumulation of microbial respiratory CO2 in experimental setups that are sealed to be gas-tight, or otherwise prevent unrestricted gas exchange, can lead to plant growth promotion, starch accumulation, and stress and pathogen resistance (Kai and Piechulla, 2009; Jin et al., 2015). Thus, it is paramount to assure that the VOC-based plant growth promotion is not due to possible co-occurring effects of CO2 fertilization in the system (Kai and Piechulla, 2009). However, since trapping of CO2 produced by T. koningii using KOH did not reduce the growth promotion observed in our experimental system, we can assume that growth promotion was not due to CO2. Lee et al. (2016) used the same system and found no significant differences in the concentration of CO2 between microhabitats containing Trichoderma and ambient air. The causal link between reactive oxygen species (ROS) production and stress tolerance is not as straightforward as one may expect. Molecules such as H2O2 play a crucial signalling role for plants in many biological processes including plant growth,
development, hormonal action, biotic resistance, abiotic tolerance, and many other physiological phenomena when produced in a controlled manner (Bose et al., 2014). However, under different stresses, ROS production can exceed the scavenging capacity of the plant and accumulate to toxic levels that can damage cell components (Shoresh et al., 2010). Mastouri et al. (2010) reported that inoculation of tomato seeds by T. harzianum T22 led to a decrease in the adverse effects of different stresses. They suggested that T22 inoculation reduced damage resulting from accumulation of ROS in stressed plants. Our observation from DAB staining and H2O2 content measurement, i.e. a reduced level of H2O2 in plants exposed to T. koningii VOCs after 2 weeks, may support the mechanism proposed by Mastouri et al. (2010). This reduction in H2O2 level was also accompanied by the presence of fewer necrotic cells in plants exposed to T. koningii VOCs under salt stress as revealed by trypan blue staining. Thus, our data seem to support the model that treatment with T. koningii VOCs ameliorates salt stress by inducing plant physiological protection against oxidative damage. Accumulation of more H2O2 in Arabidopsis exposed to VOCs of T. virens (for 5 d) and exposed to VOCs of T. asperellum (for 9 d) than in control plants was reported by Contreras-Cornejo et al. (2014) and Kottb et al. (2015), respectively. They also showed that Arabidopsis plants exposed to the VOCs were more robust against B. cinerea infection, showing that VOCs had affected ROS homeostasis under stress. On the other hand, Hung et al. (2013) did not find any difference between control and T. viride VOCs exposed plants in accumulation of H2O2 after 2 weeks in non-stressed plants. The difference between our results and those of Contreras-Cornejo and coworkers and Kottb and coworkers is probably due to different time points selected for assays, as well as different mixtures of Trichoderma VOCs emitted by the species and their subsequent effects on different plant pathways. In the future, more detailed experiments should be performed to reveal the exact role of Trichoderma VOCs in ROS homeostasis in plant stress responses. To our knowledge, this is the first report of a screening of Trichoderma spp. with regard to effects of their VOCs on salt tolerance. We hypothesize that volatile metabolites emitted by Trichoderma act as signalling molecules that induce pathways related to plant growth and salt tolerance. Experiments should be done to identify the specific Trichoderma VOCs involved in eliciting the growth promotion and salt tolerance, as well as to identify the specific genetic pathways in Arabidopsis plants which are activated by VOCs. In addition, future experiments should be performed to determine the efficiency of naturally produced VOCs by Trichoderma spp. in soil under greenhouse conditions. These experiments are presently being conducted by our research group.
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The direct use of Trichoderma metabolites, or alternatively in combination with the living fungus or other beneficial microbes or their products, is an important future target for research and development (R&D) programs in the public and private sectors worldwide (Lorito and Woo, 2015). Screening of the Trichoderma strains with respect to their beneficial metabolites is a first step towards attaining these goals. Induction of salt tolerance by Trichoderma VOCs as signalling compounds should be added to the known mechanisms that enhance plant vigor. Acknowledgments We appreciate Professor Joan W. Bennett (Rutgers University) for her reading over the manuscript and her professional comments and Dr. Samantha Lee (Rutgers University) for her intellectual inputs. Also, we thank Dr. Sohbat Bahraminezhad (Razi University) for his assistance with the statistical analysis and Dr. Saeid Abbasi and Dr. Rouhallah Sharifi (Razi University) for their suggestions and comments. References Adams, P., De-Lij, A.A.M., 2007. Trichoderma harzianum Rifai 1295-22 mediates growth promotion of crack willow (Salix fragilis) saplings in both clean and metal-contaminated soil. Microb. Ecol. 54, 306e313. Alexieva, V., Sergiev, I., Mapelli, S., Karanov, E., 2001. The effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat. Plant, Cell & Environ. 24, 1337e1344. Alizadeh, H.R., Sharifi-Tehrani, A., Hedjaroude, G.A., 2007. Evaluation of the effects of chemical versus biological control on Botrytis cinerea agent of gray mould disease of strawberry. Commun. Agric. Appl. Biol. Sci. 72, 795e800. Bae, H., Sicher, R.C., Kim, M.S., Kim, S.H., Strem, M.D., Melnick, R.L., Bailey, B.A., 2009. The beneficial endophyte Trichoderma hamatum strain DIS 219b promotes growth and delays the onset of the drought response in Theobroma cacao. J. Exp. Bot. 60, 3279e3295. Bae, H., Roberts, D.P., Lim, H.S., Strem, M., Park, S.C., Ryu, C.M., Melnick, R.L., Bailey, B.A., 2011. Endophytic Trichoderma strains from tropical environments delay disease and induce resistance against Phytophthora capsici in hot pepper using multiple mechanisms. MPMI 24, 336e351. Bose, J., Rodrigo-Moreno, A., Shabala, S., 2014. ROS homeostasis in halophytes in the context of salinity stress tolerance. J. Exp. Bot. 65, 1241e1257. Takayuki, T., Fernie, A.R., Chet, I., Brotman, Y., Landau, U., Cuadros-Inostroza, A., Viterbo, A., Willmitzer, L., 2013. Trichoderma-plant root colonization: escaping early plant defense responses and activation of the antioxidant machinery for saline stress tolerance. PLoS Pathog. 9 (3) http://dx.doi.org/10.1371/journal ppat.1003221. Contreras-Cornejo, H.A., Macias-Rodriguez, L., Herrera-Estrella, A., Lopez-Bucio, J., 2014. The 4-phosphopantetheinyl transferase of Trichoderma virens plays a role in plant protection against Botrytis cinerea through volatile organic compound emission. Plant Soil 379, 261e274. Dennis, C., Webster, J., 1970. Antagonistic properties of species groups of Trichoderma: II. Production of volatile antibiotics. Trans. Br. Mycol. Soc. 57, 41e48. Harman, G.E., 2000. Myths and dogmas of biocontrol. Changes in perceptions derived from research on Trichoderma harzianum T-22. Plant Dis. 84, 377e393. Harman, G.E., Howell, C.R., Viterbo, A., Chet, I., Lorito, M., 2004. Trichoderma speciesdopportunistic, avirulent plant symbionts. Nat. Rev. Microbiol. 2, 43e56. Harman, G.E., 2011a. Trichoderma-not just for biocontrol anymore. Phytoparasitica 39, 103e108. Harman, G.E., 2011b. Multifunctional fungal plant symbionts: new tools to enhance plant growth and productivity. New Phytol. 189, 647e649. Hermosa, R., Viterbo, A., Chet, I., Monte, E., 2012. Plant-beneficial effects of Trichoderma and of its genes. Microbiology 158, 17e25. Humphris, S.N., Wheatley, R.E., Bruce, A., 2001. The effect of specific volatiles
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Volatile organic compounds of some Trichoderma spp. increase growth and induce salt tolerance in Arabidopsis thaliana Farnaz Jalali a, Doustmorad Zafari a, *, Hooman Salari b a b
Bu-Ali Sina University, Department of Plant Protection, College of Agriculture, Hamedan, Iran Razi University, Department of Agronomy and Plant Breeding, College of Agriculture and Natural Resources, Kermanshah, Iran
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 July 2016 Received in revised form 12 June 2017 Accepted 27 June 2017
Many beneficial effects of Trichoderma spp. on plant growth and/or resistance to biotic/abiotic stresses can result from the production of bioactive compounds including volatile organic compounds (VOCs). We evaluated the effects of the volatile mixtures from 13 strains of different Trichoderma species on induction of tolerance to salt stress (100 mM NaCl) as well as growth promotion of Arabidopsis thaliana. Plants responded differently due to the presence of VOCs from various Trichoderma species ranging from both growth promotion and induction of salt tolerance to no significant changes under any of the conditions tested. In plants exposed for 2 weeks to VOCs of the selected strain, i.e. Trichoderma koningii, there was less H2O2 accumulation under salt stress compared to that in control plants. This result may reflect the possible role of VOCs of this strain in plant protection against oxidative damage under salt stress. Together, induction of salt tolerance using VOCs should be added to the known mechanisms of plant vigor enhancement by Trichoderma spp. © 2017 Elsevier Ltd and British Mycological Society. All rights reserved.
Corresponding Editor: Barbara Joan Schulz Keywords: Volatile organic compounds (VOCs) Induced systemic tolerance (IST) Abiotic stress Growth promotion Salt tolerance Arabidopsis thaliana Trichoderma spp. Bioactive compounds 3, 30 -Diaminobenzidine Reactive oxygen species (ROS)
1. Introduction Trichoderma spp. (teleomorph Hypocrea) are saprotrophic fungal inhabitants of soil that also may function as opportunistic, avirulent plant symbionts (Harman et al., 2004; Hermosa et al., 2012; Zaidi et al., 2014). Beneficial roles of Trichoderma spp. for plants comprise indirect and direct interactions. Indirect interaction with plants occurs via mycoparasitism and competition with, or antagonism of plant pathogens, mainly fungi and nematodes (Harman et al., 2004; Shoresh et al., 2010; Zaidi et al., 2014). Direct interaction of the fungus with plants can lead to an increase in plant growth, especially of roots, and particularly under stress (Harman, 2000; Shoresh et al., 2010; Hermosa et al., 2012), systemic resistance to disease (Bae et al., 2011; Mathys et al., 2012), systemic tolerance to abiotic stress, including water deficit (drought) (Bae
* Corresponding author. E-mail addresses: [email protected] (F. Jalali), [email protected] (D. Zafari), [email protected] (H. Salari). http://dx.doi.org/10.1016/j.funeco.2017.06.007 1754-5048/© 2017 Elsevier Ltd and British Mycological Society. All rights reserved.
et al., 2009; Mastouri et al., 2010; Zaidi et al., 2014), salt (Yildirim et al., 2006; Mastouri et al., 2010; Brotman et al., 2013; Zaidi et al., 2014), chilling stress and heat stress (Mastouri et al., 2010; Shoresh et al., 2010); enhancement of the vigor of poor quality seeds (Mastouri et al., 2010; Zaidi et al., 2014); bioremediation of contaminated soil (Adams and De-Lij, 2007; Shoresh et al., 2010); solubilization of phosphorus and increased availability of micronutrients and improved nitrogen use efficiency (NUE) by plants (Shoresh et al., 2010; Harman, 2011a, 2011b). Various compounds released by Trichoderma spp. into the zone of interaction with plants are associated with these advantageous effects and include proteins, peptides, secondary metabolites, and volatile organic compounds (VOCs) (Shoresh et al., 2010; Hermosa et al., 2012). VOCs are low molecular mass, usually hydrophobic, compounds with high vapor pressure which due to their small size can diffuse through the atmosphere and soils (Morath et al., 2012; Hung et al., 2015). Their physical properties make them valuable for interspecies communication as ‘infochemicals’ or ‘semiochemicals’, especially in non-aqueous environments (Hung et al., 2013).
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VOCs of different Trichoderma species inhibit growth of other fungi, in particular wood decay basidiomycetes and plant pathogens, thereby improving plant health (Dennis and Webster, 1970; Wheatley et al., 1997; Humphris et al., 2001). The role of microbial VOCs in direct interaction with plants, including growth promotion and induction of systemic resistance (ISR) to disease, was first reported by Ryu et al. (2003, 2004) for bacterial VOCs. There has also been growing awareness regarding the important role of Trichoderma VOCs in direct interactions with plants. For example, the volatile 6-n-pentyl-6H-pyran-2-one (6PP) produced by some strains of Trichoderma harzianum and Trichoderma atroviride induces defense mechanisms against Botrytis cinerea and Leptosphaeria maculans in tomato and canola, respectively (Vinale et al., 2008). The enhancement of growth of Arabidopsis thaliana by volatile mixtures emitted from the biocontrol fungus Trichoderma viride was reported by Hung et al. (2013). ContrerasCornejo et al. (2014) reported that Trichoderma virens VOCs elicit both development and defense programs in Arabidopsis. Moreover, co-cultivation of Arabidopsis with Trichoderma asperellum IsmT5 leads to increased numbers of trichomes, accumulation of defense-related compounds and expression of defense-related genes (Kottb et al., 2015). More recently, Lee et al. (2016) screened 20 strains, representing 11 different Trichoderma species, and identified nine Trichoderma strains that produced plant growth promoting VOCs. Although abiotic stresses are the most harmful factors that lower productivity of crops worldwide, no investigations have been reported regarding the role of Trichoderma or any other fungal VOCs in improving abiotic stresses in plants. However, involvement of Bacillus subtilis GB03 VOCs in induction of systemic tolerance (IST) to salt stress has been reported (Zhang et al., 2008). Salt stress is one of the most commonly observed abiotic stresses that significantly reduce yield and affect almost every aspect of the physiology and biochemistry of plants (Josine et al., 2011). Considering the fact that, “A single treatment of plants that could simultaneously promote growth and confer resistance to stresses would be of importance to agricultural plant production.” (Mastouri et al., 2010), the goal of our study was to identify potential strains among several species of Trichoderma which could induce salt tolerance as well as growth promotion in Arabidopsis via VOC production. After selecting one strain on the basis of its growth promoting and salt tolerance inducing effects, we tested whether the VOCs from this strain could improve salt tolerance by reducing hydrogen peroxide (H2O2) accumulation as the most stable reactive oxygen species (ROS) to be accumulated in plants under stress (Bose et al., 2014).
2. Materials and methods 2.1. Sources of Trichoderma strains and Arabidopsis thaliana seeds Trichoderma strains were obtained from the Mycological Laboratory of Plant Protection Department of Bu-Ali Sina University, Hamedan, Iran. These strains have been identified on the basis of morphological characteristics and sequences of two genes (tef and ITS region of rRNA), but due to some commercial purpose they could not be deposited in a public database, yet (Nazmi, unpublished data). The strains screened were: an anamorph of Hypocrea orientalis, T. asperellum, T. atroviride (2 strains), Trichoderma brevicompactum, Trichoderma citrinoviride, Trichoderma crassum, T. harzianum, Trichoderma koningii, Trichoderma koningiopsis, Trichoderma longibrachiatum, T. viride and Trichoderma viridescens. A. thaliana seeds (Ecotype Columbia-0) were obtained from the seed bank of Razi University, Kermanshah, Iran.
2.2. Sterilization and growth conditions for A. thaliana Seeds were surface-sterilized by treating them sequentially in a 70% ethanol, 5% NaOCl (bleach solution), rinsing in sterile water and stratifying at 4 C for 3 d on 0.1% (w/v) agarose to synchronize germination (Weigel and Glazebrook, 2002). Seeds were plated after stratification with an appropriate density in 90 15 mm Petri plates containing 25 ml of full strength Murashige and Skoog (MS) medium including vitamins (MO222.0025, Duchefa Biochemie, Haarlem, The Netherlands) supplemented with 3% sucrose and 0.4% (w/v) purified agar. The plates were transferred to a growth chamber at 21 C ± 2 C with a 16 h photoperiod. Plates were incubated vertically to allow seedlings to grow over the agar surface and to facilitate the transfer of germinated seedlings after 4 d. 2.3. Plant exposure experiments Five seedlings were transferred onto one part of a 100 15 mm partitioned Petri dish (split- or I-plate) containing either 15 ml of MS medium or 15 ml of MS supplemented with NaCl (100 mM) for salt stress. In each case, Trichoderma spp. were inoculated as discs, 5 mm in diameter, cut from the growing edge of the fungal mycelium on culture plates, onto a Petri plate (35 10 mm) containing 4 ml of malt extract agar (MEA, 105398, Merck, Darmstadt, Germany). Trichoderma spp. plates were placed into the empty region of the split plate and allowed to grow with Arabidopsis. For controls, the same plate-within-a-plate system was used with sterile media (MEA) without fungal inoculation (Lee et al., 2015). Plants and fungi were grown for 14 d at 21 C ± 2 C with a 16 h photoperiod. At the end of the exposure period, individual plants were removed from the testing condition; fresh weight and total chlorophyll content were assessed (Hung et al., 2013). 2.4. Plant chlorophyll measurements Total chlorophyll content of plants was determined using a spectrophotometer (Varian Technologies, Cary 100 scan, Pal Alto, California, US) according to the method described by Hung et al. (2013). Shoot tissue was submerged overnight in 1 ml of 80% acetone in the dark at 4 C. The total chlorophyll content (chlorophyll a and b) was calculated from the equation [(8.02) (A663) þ (20.2) (A645)] V/1000 W, where V is volume and W is plant shoot fresh weight (Palta, 1990). 2.5. CO2 assay To separate the VOC-based plant growth promotion from the effects of CO2 accumulation that might occur in enclosed dualculture systems (Piechulla and Schnitzler, 2016), CO2 trapping was performed in a way similar to that of Lee et al. (2016) for the strain selected from the screening experiment, i.e. T. koningii. With this method, KOH is used to trap CO2 as K2CO3, leading to lower CO2 concentrations while microbial volatile organic compounds (mVOCs) become enriched relative to CO2 (Lee et al., 2016). Briefly, a sterile cotton ball containing 3 ml of 0.1 M KOH was placed onto a sterile aluminum cap. Then the cap was placed into an empty region of the three-partitioned Petri dishes (100 15 mm) containing the fungal culture and plants in other parts, separately. Plates were kept under the same condition as described above. At the end of the exposure period, individual plants were sampled to assess fresh weight and total chlorophyll content. 2.6. Histochemical stainings Histochemical staining was performed on plants exposed to
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VOCs of the selected strain (T. koningii) and control plants as described by Hung et al. (2013). In summary, to detect the possible growth of endophytic Trichoderma or possible pathogenic fungi that may have contaminated the cultured plants, trypan blue staining was used. Trypan blue also stains dead plant cells, so its absence is an indicator of plant health. Whole plants were submerged in a trypan blue solution (BI1014-100, Bioidea Company, Tehran, Iran) overnight. To stain for hydrogen peroxide (H2O2), the whole plant was submerged in a 3, 30 -diaminobenzidine (DAB, D12384, Sigma Aldrich, St Louis, Mo, US) solution (1 mg ml_1, pH 3.8) for 5 h. In each case, chlorophyll was removed by soaking the plant in 95% ethanol overnight. Stained plant tissues were examined macroscopically and microscopically. 2.7. Measuring H2O2 content Hydrogen peroxide content of Arabidopsis seedlings that had or had not been exposed to VOCs from T. koningii was measured spectrophotometrically according to Alexieva et al. (2001). The reaction mixture consisted of 0.5 ml supernatant of 0.1% trichloroacetic acid (TCA) seedling extract, 0.5 ml of 100 mM Kphosphate buffer (pH 7), and 2 ml reagent (1 M KI, w/v in fresh double-distilled water). The blank consisted of 0.5 ml of 0.1% TCA without seedling extract. After 1 h of incubation in darkness at room temperature, the absorbance was measured at 390 nm. The amount of hydrogen peroxide was calculated using a standard curve prepared with known concentrations of H2O2. 2.8. Experimental design and data analysis A fully factorial completely randomized design was used. For the screening test, there were two factors including fungal VOC mixtures at 14 levels (emitted from control medium and 13
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Trichoderma isolates) and salt at two levels (0 and 100 mM NaCl). For the CO2 and H2O2 assays, the factors were VOC mixtures at two levels (emitted from control medium and T. koningii) and salt at two levels (0 and 100 mM NaCl). Analyses were performed using the average of five seedlings in each Petri dish as one treatment. Each treatment had three technical replications and experiments were repeated independently two times, resulting in a total number of 6 replicates. The normality test of the data at 5% probability level, analysis of variance and Duncan's multiple range tests were performed for all quantitative data using SPSS (ver. 16.0) and SAS (ver. 9.1), respectively (for chlorophyll content the data were transformed and analyzed based on a logarithm, according to the normality test results). We also created a scatter plot to show percentage of difference from the control for each trait (shoot and root fresh weight and chlorophyll content) in plants exposed to VOCs of different Trichoderma strains using XLSTAT 2017 software. Confidence ellipses were based on chi-square, at 95% confidence interval. 3. Results After 2 weeks of exposure, the morphological and physiological responses of Arabidopsis to Trichoderma spp. VOCs were assessed by measuring phenotypic characteristics including shoot fresh weight, root fresh weight, and total chlorophyll content. Plants exhibited different responses in the presence of VOCs from various Trichoderma strains ranging from growth promotion and induction of salt tolerance to no significant changes in any of the conditions tested (Fig. 1). In the absence of salt, there was a significant (p 0.05) increase in the shoot and root fresh weight and chlorophyll content of Arabidopsis exposed to volatile mixtures emitted from T. viride, T. atroviride 2, T. longibrachiatum, T. citrinoviride, T. harzianum,
Fig. 1. Arabidopsis seedlings exposed to Trichoderma spp. VOCs and control plants in the absence and presence of salt (100 mM NaCl) for 14 d. Hypocrea orientalis (A), T. asperellum (B), T. atroviride 1 (C), T. atroviride 2 (D), T. brevicompactum (E), T. citrinoviride (F), T. crassum (G), T. harzianum (H), T. koningii (I), T. koningiopsis (J), T. longibrachiatum (K), T. viride (L), T. viridescens (M). Equivalent to controls (¼), Plant growth promotion (þ).
70 F. Jalali et al. / Fungal Ecology 29 (2017) 67e75 Fig. 2. Arabidopsis seedlings exposed to Trichoderma VOCs in the absence and presence of salt (100 mM NaCl). Mean of shoot fresh weight (mg) (A), root fresh weight (mg) (B), and chlorophyll content (mg g1) (C) of seedlings exposed to VOCs for 2 weeks compared to control plants (Number of replicates, N ¼ 6). Error bars represent the standard deviation of the mean. * indicates significance compare to the control using Duncan's multiple range test (p < 0.05).
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T. koningii, T. koningiopsis, H. orientalis and T. viridescence compared to control plants (Fig. 2). In the presence of VOCs from these strains, plants were taller with more leaf surface area, more lateral roots and leaves with a darker hue of green than control plants (Data not shown). Plants co-cultivated with VOCs from T. asperellum, T. atroviride 1, T. brevicompactum and T. crassum did not show any significant changes (Figs. 1 and 2). Under salt stress, plants exposed to VOCs emitted from T. koningii, T. viridescence and H. orientalis increased significantly (p 0.05) in shoot fresh weight compared to control plants (Fig. 2A); those exposed to VOCs from T. koningii and H. orientalis also exhibited a significant increase in root fresh weight compared with control plants (Fig. 2B). Total chlorophyll content decreased under salt stress (100 mM) in control plants as well as plants exposed to VOCs as compared to those without salt stress. However, under salt stress as compared to control plants, exposure to VOCs of T. viridescence, T. koningii, T. asperellum, T. harzianum, T. atroviride 2, T. koningiopsis, T. atroviride 1, H. orientalis and T. viride led to a
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significant increase (p 0.05) in chlorophyll content (Fig. 2C). Using a scatter plot and statistical analysis, we were able to cluster strains on the basis of their VOCs' effects on the plant shoot fresh weight, plant root fresh weight, and total chlorophyll content with and without salt stress (Fig. 3). Concerning shoot fresh weight, three groups were distinguished: (1) strains which improved this trait under both conditions including T. koningii, T. viridescence and H. orientalis; (2) strains which improved this trait only in the absence of salt, i.e. T. atroviride 2, T. viride, T. longibrachiatum, T. citrinoviride, T. harzianum, T. koningiopsis; and (3) strains which did not induce any significant changes in either of the conditions, i.e. T. asperellum, T. atroviride 1, T. brevicompactum and T. crassum (Fig. 3A). In the case of root fresh weight, the categories were the same with the exception of T. viridescence, whose VOCs did not improve this trait under salt stress (Fig. 3B). Strains were categorized into four groups according to the effect of their VOCs on increasing chlorophyll content of the plants as follow: (1) strains which improved this trait under both conditions
Fig. 3. Scatter plots showing the effects of Trichoderma strains' VOCs on plant growth and induction of salt tolerance. X and Y axes represent percentage difference to control plants of plants exposed to VOCs of Trichoderma strains in the absence and presence of salt with respect to shoot fresh weight (A), root fresh weight (B) and chlorophyll content (C). Confidence ellipses were based on chi-square, at 95% confidence interval. Hypocrea orientalis (A), T. asperellum (B), T. atroviride 1 (C), T. atroviride 2 (D), T. brevicompactum (E), T. citrinoviride (F), T. crassum (G), T. harzianum (H), T. koningii (I), T. koningiopsis (J), T. longibrachiatum (K), T. viride (L), T. viridescens (M).
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Fig. 4. A. thaliana plants grown with or without KOH solution and with or without exposure to VOCs of T. koningii for 2 weeks in the absence and presence of salt (100 mM NaCl) (N ¼ 6). Control (no VOC exposure and no salt) (A), exposure to T. koningii VOCs without salt (B), control (salt but no VOC exposure) (C) and exposure to T. koningii VOCs with salt (D).
included T. viride, T. atroviride 2, T. harzianum, T. koningii, T. koningiopsis, H. orientalis and T. viridescens; (2) strains which improved this trait only in the absence of salt stress, i.e. T. longibrachiatum and T. citrinoviride; (3) strains which improved this trait only under salt
stress, i. e T. asperellum and T. atroviride 1; and (4) strains which did not induce any significant changes in chlorophyll content under either of the conditions i.e. T. brevicompactum and T. crassum (Fig. 3C). Considering all the above aspects, T. koningii was the most successful species tested with respect to growth promotion, as well as induced salt tolerance via production of VOCs, and was thus chosen for further studies. This strain increased shoot and root fresh weight and chlorophyll content of plants in the absence of salt by 101%, 152% and 240%, respectively, and 62%, 128% and 383%, respectively under salt stress (Fig. 3). Trapping possible CO2 accumulation with 0.1 M KOH solution did not eliminate the observed beneficial effects of T. koningii VOCs under any of the conditions tested (Fig. 4). Without salt stress, there was still a 97% increase in shoot fresh weight, a 151% increase in root fresh weight and a 244% increase in chlorophyll content for plants exposed to T. koningii VOCs and KOH solution compared to the negative control grown only in KOH that was significantly different from the control without KOH (p 0.05) (Fig. 5). There was also a 69% increase in shoot fresh weight, a 111% increase in root fresh weight and a 390% increase in chlorophyll content for plants exposed to Trichoderma VOCs and KOH solution compared to the negative control grown only with KOH under salt stress that was significantly different from the control with neither KOH or NaCl (p 0.05) (Fig. 5). Trypan blue staining revealed no endophytic Trichoderma or other contaminating fungi in plant tissues throughout the 2 weeks of exposure to Trichoderma VOCs. In addition, under salt stress there seemed to be less cell death in plants exposed to VOCs of T. koningii than in control plants (Fig. 6).
Fig. 5. A. thaliana exposed to KOH solution and plants exposed to T. koningii VOCs and KOH solution for 2 weeks in the presence and absence of salt (100 mM NaCl). Mean of shoot fresh weight (mg) (A), root fresh weight (mg) (B), and chlorophyll content (mg g1) (C) (N ¼ 6). Error bars represent the standard deviation of the mean.* indicates significance compare to control using Duncan's multiple range test (p < 0.05).
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Fig. 6. Trypan blue staining of control plants and plants exposed to T. koningii VOCs for 2 weeks. Control (no VOC exposure and no salt) (A), plant exposed to T. koningii VOCs without salt (B), control (salt but no VOC exposure) (C), and plant exposed to T. koningii VOCs with salt (D).
The near lack of DAB staining in tissues of both exposed and control plants in the absence of salt, indicated an absence of stress responses. However after 2 weeks of exposure, lower amounts of H2O2 accumulation were observed in the plants exposed to T. koningii VOCs under salt stress compared to controls (Fig. 7), suggesting that VOCs of T. koningii may be involved in plant protection against oxidative damage under this condition. Data from spectrophotometrical determination of H2O2 content in plants also confirmed DAB staining results. The level of H2O2 was not significantly different between exposed and control plants after 14 d of co-cultivation under no salt stress. H2O2 content increased significantly (p 0.05) in exposed and unexposed plants under salt stress. However, this level was significantly (p 0.05) lower in exposed plants compared to controls (Fig. 8).
4. Discussion
Fig. 7. DAB staining of control plants and plants exposed to T. koningii VOCs for 2 weeks. Control (no VOC exposure and no salt) (A), plant exposed to T. koningii VOCs without salt (B), control (salt but no VOC exposure) (C) and plant exposed to T. koningii VOCs with salt (D).
Our screening experiment enabled us to identify nine out of 13 Trichoderma strains that produced plant growth promoting VOCs, thereby supporting the report of Hung et al. (2013) and Lee et al. (2016) regarding growth promoting effects of T. harzianum, T. longibrachiatum and T. viride VOCs on Arabidopsis. In addition to growth promotion, VOCs of some strains increased salt tolerance of Arabidopsis seedlings without physical contact, a phenomenon which was called “aromatic therapy for plant stresses” by Ryu
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Fig. 8. H2O2 content in control plants and plants exposed to T. koningii VOCs for 2 weeks in the presence and absence of salt (100 mM NaCl) (N ¼ 6). Error bars represent the standard deviation of the mean. * indicates significance compared to control using Duncan's multiple range test (p < 0.05).
(2015) for bacterial VOCs. There were significant differences amongst Trichoderma spp. regarding effects of their VOCs on Arabidopsis under the different growth conditions we tested. For instance, VOCs of some species of Trichoderma, i.e. T. longibrachiatum and T. citrinoviride, which promoted plant growth in the absence of salt, did not do so when the medium contained salt. On the other hand, VOCs of some species, i.e. T. asperellum and T. atroviride1, which increased chlorophyll content of plants under salt stress, did not do so in the absence of salt. It was also notable that plants exposed to VOCs of the different strains of T. atroviride also induced varied responses. In the absence of salt, exposure to T. atroviride2 had a growth promoting effect on Arabidopsis, whereas growth of plants exposed to T. atroviride1 VOCs did not significantly differ from that of the controls. These results show that there are strain and species differences among Trichoderma isolates regarding the affects of their VOC emissions on Arabidopsis. The volatile profiles of these Trichoderma isolates should be studied, as well as the cognate pathways in Arabidopsis that are affected by these different VOCs mixtures. A single treatment of plants that could simultaneously promote growth and confer resistance to stresses would be of importance to agricultural plant production (Mastouri et al., 2010). On the basis of our screening experiment, we selected T. koningii as a promising biocontrol agent which can induce growth promotion as well as salt tolerance via its VOC emissions. In other studies, it was shown that the volatile metabolites of one strain of T. koningii had an inhibitory effect on the growth of the plant pathogen B. cinerea (Alizadeh et al., 2007). Our observations add to knowledge about the direct beneficial effects of T. koningii VOCs on plants. It is known that accumulation of microbial respiratory CO2 in experimental setups that are sealed to be gas-tight, or otherwise prevent unrestricted gas exchange, can lead to plant growth promotion, starch accumulation, and stress and pathogen resistance (Kai and Piechulla, 2009; Jin et al., 2015). Thus, it is paramount to assure that the VOC-based plant growth promotion is not due to possible co-occurring effects of CO2 fertilization in the system (Kai and Piechulla, 2009). However, since trapping of CO2 produced by T. koningii using KOH did not reduce the growth promotion observed in our experimental system, we can assume that growth promotion was not due to CO2. Lee et al. (2016) used the same system and found no significant differences in the concentration of CO2 between microhabitats containing Trichoderma and ambient air. The causal link between reactive oxygen species (ROS) production and stress tolerance is not as straightforward as one may expect. Molecules such as H2O2 play a crucial signalling role for plants in many biological processes including plant growth,
development, hormonal action, biotic resistance, abiotic tolerance, and many other physiological phenomena when produced in a controlled manner (Bose et al., 2014). However, under different stresses, ROS production can exceed the scavenging capacity of the plant and accumulate to toxic levels that can damage cell components (Shoresh et al., 2010). Mastouri et al. (2010) reported that inoculation of tomato seeds by T. harzianum T22 led to a decrease in the adverse effects of different stresses. They suggested that T22 inoculation reduced damage resulting from accumulation of ROS in stressed plants. Our observation from DAB staining and H2O2 content measurement, i.e. a reduced level of H2O2 in plants exposed to T. koningii VOCs after 2 weeks, may support the mechanism proposed by Mastouri et al. (2010). This reduction in H2O2 level was also accompanied by the presence of fewer necrotic cells in plants exposed to T. koningii VOCs under salt stress as revealed by trypan blue staining. Thus, our data seem to support the model that treatment with T. koningii VOCs ameliorates salt stress by inducing plant physiological protection against oxidative damage. Accumulation of more H2O2 in Arabidopsis exposed to VOCs of T. virens (for 5 d) and exposed to VOCs of T. asperellum (for 9 d) than in control plants was reported by Contreras-Cornejo et al. (2014) and Kottb et al. (2015), respectively. They also showed that Arabidopsis plants exposed to the VOCs were more robust against B. cinerea infection, showing that VOCs had affected ROS homeostasis under stress. On the other hand, Hung et al. (2013) did not find any difference between control and T. viride VOCs exposed plants in accumulation of H2O2 after 2 weeks in non-stressed plants. The difference between our results and those of Contreras-Cornejo and coworkers and Kottb and coworkers is probably due to different time points selected for assays, as well as different mixtures of Trichoderma VOCs emitted by the species and their subsequent effects on different plant pathways. In the future, more detailed experiments should be performed to reveal the exact role of Trichoderma VOCs in ROS homeostasis in plant stress responses. To our knowledge, this is the first report of a screening of Trichoderma spp. with regard to effects of their VOCs on salt tolerance. We hypothesize that volatile metabolites emitted by Trichoderma act as signalling molecules that induce pathways related to plant growth and salt tolerance. Experiments should be done to identify the specific Trichoderma VOCs involved in eliciting the growth promotion and salt tolerance, as well as to identify the specific genetic pathways in Arabidopsis plants which are activated by VOCs. In addition, future experiments should be performed to determine the efficiency of naturally produced VOCs by Trichoderma spp. in soil under greenhouse conditions. These experiments are presently being conducted by our research group.
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The direct use of Trichoderma metabolites, or alternatively in combination with the living fungus or other beneficial microbes or their products, is an important future target for research and development (R&D) programs in the public and private sectors worldwide (Lorito and Woo, 2015). Screening of the Trichoderma strains with respect to their beneficial metabolites is a first step towards attaining these goals. Induction of salt tolerance by Trichoderma VOCs as signalling compounds should be added to the known mechanisms that enhance plant vigor. Acknowledgments We appreciate Professor Joan W. Bennett (Rutgers University) for her reading over the manuscript and her professional comments and Dr. Samantha Lee (Rutgers University) for her intellectual inputs. Also, we thank Dr. Sohbat Bahraminezhad (Razi University) for his assistance with the statistical analysis and Dr. Saeid Abbasi and Dr. Rouhallah Sharifi (Razi University) for their suggestions and comments. References Adams, P., De-Lij, A.A.M., 2007. Trichoderma harzianum Rifai 1295-22 mediates growth promotion of crack willow (Salix fragilis) saplings in both clean and metal-contaminated soil. Microb. Ecol. 54, 306e313. Alexieva, V., Sergiev, I., Mapelli, S., Karanov, E., 2001. The effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat. Plant, Cell & Environ. 24, 1337e1344. Alizadeh, H.R., Sharifi-Tehrani, A., Hedjaroude, G.A., 2007. Evaluation of the effects of chemical versus biological control on Botrytis cinerea agent of gray mould disease of strawberry. Commun. Agric. Appl. Biol. Sci. 72, 795e800. Bae, H., Sicher, R.C., Kim, M.S., Kim, S.H., Strem, M.D., Melnick, R.L., Bailey, B.A., 2009. The beneficial endophyte Trichoderma hamatum strain DIS 219b promotes growth and delays the onset of the drought response in Theobroma cacao. J. Exp. Bot. 60, 3279e3295. Bae, H., Roberts, D.P., Lim, H.S., Strem, M., Park, S.C., Ryu, C.M., Melnick, R.L., Bailey, B.A., 2011. Endophytic Trichoderma strains from tropical environments delay disease and induce resistance against Phytophthora capsici in hot pepper using multiple mechanisms. MPMI 24, 336e351. Bose, J., Rodrigo-Moreno, A., Shabala, S., 2014. ROS homeostasis in halophytes in the context of salinity stress tolerance. J. Exp. Bot. 65, 1241e1257. Takayuki, T., Fernie, A.R., Chet, I., Brotman, Y., Landau, U., Cuadros-Inostroza, A., Viterbo, A., Willmitzer, L., 2013. Trichoderma-plant root colonization: escaping early plant defense responses and activation of the antioxidant machinery for saline stress tolerance. PLoS Pathog. 9 (3) http://dx.doi.org/10.1371/journal ppat.1003221. Contreras-Cornejo, H.A., Macias-Rodriguez, L., Herrera-Estrella, A., Lopez-Bucio, J., 2014. The 4-phosphopantetheinyl transferase of Trichoderma virens plays a role in plant protection against Botrytis cinerea through volatile organic compound emission. Plant Soil 379, 261e274. Dennis, C., Webster, J., 1970. Antagonistic properties of species groups of Trichoderma: II. Production of volatile antibiotics. Trans. Br. Mycol. Soc. 57, 41e48. Harman, G.E., 2000. Myths and dogmas of biocontrol. Changes in perceptions derived from research on Trichoderma harzianum T-22. Plant Dis. 84, 377e393. Harman, G.E., Howell, C.R., Viterbo, A., Chet, I., Lorito, M., 2004. Trichoderma speciesdopportunistic, avirulent plant symbionts. Nat. Rev. Microbiol. 2, 43e56. Harman, G.E., 2011a. Trichoderma-not just for biocontrol anymore. Phytoparasitica 39, 103e108. Harman, G.E., 2011b. Multifunctional fungal plant symbionts: new tools to enhance plant growth and productivity. New Phytol. 189, 647e649. Hermosa, R., Viterbo, A., Chet, I., Monte, E., 2012. Plant-beneficial effects of Trichoderma and of its genes. Microbiology 158, 17e25. Humphris, S.N., Wheatley, R.E., Bruce, A., 2001. The effect of specific volatiles
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