Volatile organic compounds emitted from endophytic fungus Trichoderma asperellum T1 mediate antifungal activity, defense response and promote plant growth in lettuce (Lactuca sativa)

Fungal Ecology 43 (2020) 100867

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Volatile organic compounds emitted from endophytic fungus Trichoderma asperellum T1 mediate antifungal activity, defense response and promote plant growth in lettuce (Lactuca sativa) Prisana Wonglom a, Shin-ichi Ito b, c, Anurag Sunpapao d, e, * a

Faculty of Technology and Community Development, Thaksin University, Phatthalung Campus, 222 Moo 2, Ban Phrao Sub-District, Pa Payom District, Phattalung, 93110, Thailand Department of Biological and Environmental Sciences, Graduate School of Science and Technology for Innovation, Yamaguchi University, Yamaguchi, 7538515, Japan c Research Center for Thermotolerant Microbial Resources (RCTMR), Yamaguchi University, Yamaguchi, 753-8515, Japan d Pest Management Biotechnology and Plant Physiology, Prince of Songkla University, Hatyai, Songkhla, 90112, Thailand e Department of Pest Management, Faculty of Natural Resources, Prince of Songkla University, Hatyai, Songkhla, 90112, Thailand b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 November 2018 Received in revised form 24 August 2019 Accepted 26 August 2019 Available online xxx

Trichoderma species are applied as biological control agents and biofertilizers to control plant diseases and enhance crop yields. The ability to inhibit pathogens, induce defense responses, and promote plant growth can result from the production of volatile organic compounds (VOCs). In this study, we evaluated the effects of VOCs from Trichoderma asperellum T1 on those multifaceted actions in lettuce. The VOCs released by T. asperellum T1 inhibited fungal growth of two leaf spot fungal pathogens, Corynespora cassiicola and Curvularia aeria. Lettuces responded to VOCs by increasing activities of the cell-wall degrading enzymes chitinase and b-1,3-glucanase to 1.26 U mL
1 and 4.45 U mL
1, respectively, above those in the control. Accumulation of cell-wall degrading enzymes in lettuce that had been treated with VOCs resulted in morphological changes to fungal cell-walls. Exposure to the VOCs emitted by T. asperellum T1 significantly increased numbers of leaves and roots, plant biomass and total chlorophyll content in lettuce. Furthermore, GC/MS analysis revealed that T. asperellum T1 emitted 22 volatile compounds, which are involved in antifungal activity, inducing defense responses, and promoting growth in lettuce. © 2019 Elsevier Ltd and British Mycological Society. All rights reserved.

Corresponding Editor: Prof. L. Boddy Keywords: Antifungal activity Growth promotion Defense response GC/MS Lettuce Volatile organic compound

1. Introduction Trichoderma species are generally known as biological control agents (BCAs) against several plant diseases. Several Trichoderma species have multifaceted actions including parasitism (Matroudi et al., 2009; Monteiro et al., 2010), antibiosis (Howell, 1998), emission of volatile antifungal compounds (Dennis and Webster, 1971; Vinodkumar et al., 2017; Sunpapao et al., 2018), production of defense-related enzymes (Matroudi et al., 2009; Ting and Chai, 2015; Asad et al., 2015), induction of defense response (Singh et al., 2013; Sunpapao et al., 2018) and systemic resistance in plants (Hoitink et al., 2006; Mathys et al., 2012), as well as

* Corresponding author. Pest Management Biotechnology and Plant Physiology, Prince of Songkla University, Hatyai, Songkhla, 90112, Thailand. E-mail address: [email protected] (A. Sunpapao). https://doi.org/10.1016/j.funeco.2019.100867 1754-5048/© 2019 Elsevier Ltd and British Mycological Society. All rights reserved.

promoting plant growth (Conteras-Conrnejo et al., 2009; Vinodkumar et al., 2017). In agriculture, Trichoderma species are often added to soil or to hydroponic systems to increase biomass and control some soil-borne pathogens. Trichoderma species are known to produce several metabolites of agricultural significance (Mathivanan et al., 2008). The metabolites exhibit antimicrobial activities, as well as induction of resistance to plant pathogens (Engelberth et al., 2001; Mukherjee et al., 2012). For instance, harzianolide produced by Trichoderma koningii and T. harzianum exhibits antifungal activity and is a plant growth regulator (Cutler et al., 1991; Ghisalberti and Rowland, 1993). The trichokonin produced by T. koningii has the same attributes, i.e. antifungal and plant defense inducer (Xiao-Yan et al., 2006). Volatile metabolites or volatile organic compounds (VOCs) emitted by Trichoderma species are of high interest in the context of these effects. VOCs have low boiling points, high vapor pressure, and low molecular mass (Insam and Seewald, 2010). They are

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produced by plants (Dudareva et al., 2006) and microorganisms (Campos et al., 2010). They are chemically diverse and include aliphatic hydrocarbons, terpenes and fatty acids (Vinodkumar et al., 2017). It has been reported that the VOCs released by Trichoderma play an important role in its antifungal activity (Vinodkumar et al., 2017; Sunpapao et al., 2018); they can induce systemic resistance (Kishimoto et al., 2005; Yi et al., 2009) and promote plant growth (Vinale et al., 2008). Our recent publication has shown that T. spirale T76-1 displays antifungal activity against lettuce leaf spot fungi, Corynespora cassiicola and Curvularia aeria by several mechanisms including production of volatile antifungal compounds (Baiyee et al., 2019). Another species of Trichoderma, T. asperellum, has been used as a biocontrol agent to control Pythium myriotylum, which causes root rot in cocoyam (Mbarga et al., 2012), and Sclerotium sclerotiorum, which causes stem rot in carnation (Vinodkumar et al., 2017). However, the ability of VOCs emitted by T. asperellum T1 to contribute to antifungal activity, induce defense response, and promote plant growth of lettuce has not yet been clarified. The aim of this study was to determine whether the VOCs of T. asperellum T1 are the major factor in (i) antifungal activity against leaf spot fungi of lettuce C. cassiicola and C. aeria, (ii) inducing defense response in lettuce, and (iii) promoting growth in lettuce. We also characterized the volatile profiles emitted by T. asperellum T1 using gas chromatography-mass spectrometry (GC/MS) to determine which of the metabolites was responsible for the effects.

2. Materials and methods 2.1. Sources of Trichoderma and pathogens

Lettuce seeds (green oak cultivar) were surface disinfected with 70% ethyl alcohol followed by 10% sodium hypochloride (NaOCl) and rinsed with sterile distilled water (DW) to remove excess NaOCl. The seeds were then air dried in laminar flow air cabinet for 1 h on sterile Whatman filter paper. The seeds were placed on Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) containing 1 ppm indole acetic acid (IAA) in cell culture plates. The culture plates were incubated in growth chamber with humidity of 60 ± 2%, temperature 25 ± 2  C and photoperiod of 16:8 for 14 d. 2.4. Plant and Trichoderma volatile exposure bioassay Exposure of lettuce plants to Trichoderma VOCs was performed using 6-well cell culture plates 3.3 cm in diameter (SPL Life Sciences, Gyeonggi-do, Korea). The wells on one side of the culture plate contained PDA for culturing T. asperellum T1, the other side of the culture plate contained sterile lettuce seeds. They were grown in a shared atmosphere on the culture plate and incubated in a growth chamber. For control treatments, the control wells without Trichoderma inoculation (PDA alone) were handled in the same way. Lettuce seeds (4 seeds per well) were germinated in the absence or presence of VOCs released by T. asperellum T1 to the shared atmosphere in a growth chamber with 16-h photoperiod at 25  C for 14 d (Jalali et al., 2017). The experiments were set up in triplicate and repeated twice. At the end of VOC exposure periods, the lettuce plants were removed from the exposure conditions and subjected to enzyme assay, phenotypic characterization, and determination of chlorophyll content. 2.5. Enzyme assay

Trichoderma asperellum T1 and the pathogens causing leaf spot on lettuces, namely C. cassiicola (Chairin et al., 2017) and C. aeria (Pornsuriya et al., 2018), were obtained from the Culture Collection of Pest Management, Faculty of Natural Resources, Prince of Songkla University, Thailand. T. asperellum T1 and pathogens were cultured on potato dextrose agar (PDA) (HiMedia, Mumbai, India) for 5 d at 28 ± 2  C before use in this study.

2.2. Volatile antifungal bioassay To test the activity of the volatiles emitted by T. asperellum T1 in inhibiting mycelial growth of lettuce pathogens, a volatiles antifungal bioassay was used as previously described by Dennis and Webster (1971) with some modification. T. asperellum T1 was grown on PDA in 9 cm Petri dishes (10 mL medium per dish) (Citotest, Jiangsu, China) for 5 d. The plates had agar plugs (0.5 cm in diameter) cut from stock cultures inserted centrally, and then the lid of each dish was replaced by a Petri dish base containing PDA inoculated with test fungi. The two plates were taped together with Parafilm and incubated at 28 ± 2  C for 7 d. For control cases, the lids of control plates without Trichoderma inoculation were treated the same way. The experiments were set up in triplicate and repeated twice. Colony diameters of the tested fungi were measured and the percentage of inhibition was calculated:

Percentage inhibition of fungal diameter ¼ ð%Þ

2.3. Lettuce plants and growth conditions

Dc
Dt  100 Dc

where Dc is the colony diameter of pathogen in the untreated control and Dt is the colony diameter of pathogen in the treatment (Prapagdee et al., 2008).

To examine the induction of defense response in lettuce plants, enzyme activities of lettuces treated with VOCs were compared with untreated lettuces (control). Protein extraction from treated and untreated lettuce was conducted with 0.1 M potassium phosphate buffer (KPB) pH 7.0 for chitinase and b-1,3-glucanase assays (Chairin and Petcharat, 2017). The extracted lettuces were then centrifuged at 14,000 g for 20 min at 4  C, and the supernatants were collected and analyzed immediately. Enzyme activities of chitinase and b-1,3-glucanase were determined with the 3,5dinitrosalicylic acid (DNS) method (Miller, 1959). Colloidal chitin and laminarin (Sigma-Aldrich, Missouri, USA) were used as the substrates in chitinase and b-1,3-glucanase assay, respectively. Reducing sugar released in the reaction mixtures was determined from absorbance in a UV/VIS spectrophotometer UV5300 (METASH, Shanghai, China) at 550 nm and at 575 nm for b-1,3-glucanase and for chitinase, respectively. Each enzyme activity determination had three replicates and was assayed twice (i.e., three experimental replicates and two technical replicates). 2.6. SEM analysis To confirm that the crude extract from lettuce contained cellwall degrading enzymes and caused abnormal fungal cell-walls, three agar plugs of each of the pathogens were submerged in crude extract from lettuce treated with VOCs and from control lettuce, incubated at 37  C for 1 h as previously described by Wonglom et al. (2019). Next the samples were incubated in 3% glutaraldehyde at 4  C for 24 h. The samples were then dehydrated in a series of alcohol dilutions (30, 50, 60, 70, 80, 90 and 100%). After the samples were dried, they were coated with gold and observed with a scanning electron microscope (JSM-580 LV, JEOL, USA) at the Science Equipment Center, Prince of Songkla University.

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2.7. Plant chlorophyll measurements Total chlorophyll content of lettuces was determined using a UV/VIS spectrophotometer (UV5300, METASH) according to the method previously described by Arnon, 1949. Lettuce shoots (0.5 g) were ground in a small mortar with a pestle, and then submerged overnight in 1 mL of 80% acetone in the dark at 4  C. The total chlorophyll content was calculated as: Chlorophyll a (mg mL
1) ¼ 12.7 A663 e 2.69 A645 Chlorophyll b (mg mL
1) ¼ 22.9 A645 e 4.68 A663 Total chlorophyll (mg mL
1) ¼ Chlorophyll a þ Chlorophyll b Here A645 ¼ absorbance at 645 nm, and A663 ¼ absorbance at 663 nm. The total chlorophyll content was converted to mg g
1 fresh weight (FW). 2.8. Histological staining To detect the possible growth of endophytic Trichoderma or possible pathogenic fungi contaminating the culture plants, histological staining with trypan blue was conducted. Whole lettuce plants treated with VOCs of Trichoderma were submerged in trypan blue solution 12 h (van Wees, 2010). Chlorophyll was removed by soaking the lettuces in 95% ethanol overnight (12 h). Lettuce plants without VOCs served as a negative control. Stained plant tissues were examined under a stereomicroscope S8AP0 (Leica microsystems, Wetzlar, Germany). 2.9. GC/MS analysis T. asperellum T1 was cultured on PDA in a chromatography vial 20 mm in diameter, 20 mL capacity (PerkinElmer, Waltham, USA) and incubated at 28 ± 2  C for 14 d. VOCs of T. asperellum T1 were extracted by solid-phase microextraction (SPME) according to the method previously described by Arthur et al. (1992). SPME fiber (DVB/CAR/PDMS fiber) was exposed to the vapor phase above the fungal culture for 30 min. The adsorbent fiber was inserted into the injection port of the gas chromatograph SQ8 (PerkinElmer Co., Ltd. Thailand). The capillary column used in this study was AT-5MS (5% phenylmethylpolysiloxane) with dimensions of 30 m  0.25 mm i.d. and 0.25 mm film thickness. The column temperature was programmed for an initial 45  C, and was then increased at a rate of 7  C min
1 to a final temperature of 230  C. Purified helium was used as the carrier gas at a flow rate of 1 mL min
1. Electron impact (EI) mass spectra were collected at 70 eV ionization voltage over the m/z range 29e550. The ion source and quadrupole temperatures were both set at 200  C. Computer searches of the National Institute of Standards and Technology (NIST, v17, 2014) Mass Spectral Library Search Chromatogram were done to identify the VOCs emitted by T. asperellum T1. 2.10. Effect of VOCs on antifungal activity, enhancing enzyme activity and plant growth development To test effect of each compound putatively involved in antifungal activity against both C. cassiicola and C. aeria, antifungal bioassay of the volatiles was conducted as described in 2.2. The compounds 2-ethylhexanol (Sigma-Aldrich, Missouri, USA), 1nonanol (Sigma-Aldrich, Missouri, USA) and 6-pentyl-2H-pyran2-one (6-PP) (Sigma-Aldrich, Missouri, USA) were used in this study to compare with the total VOCs of T. asperellum T1. The 2ethylhexanol and 1-nonanol and 6-PP were dissolved in ethanol

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and adjusted to 75 mM concentration (Garnica-Vergara et al., 2015), and 20 ml of each was then applied to a sterile cotton pad and subjected to the method described in 2.2. The tested plates were incubated at 28 ± 2  C for 7 d. The experiments were set up in triplicate and repeated twice. Colony diameters of the tested fungi were measured and the percentage of inhibition was calculated as described in section 2.2. To test the effect of 6-PP in inducing a defense response in lettuce, the experiments described in 2.4 and 2.5 were carried out. 6PP at a concentration of 25 mL mL
1 (Parker et al., 1997) was applied onto sterile cotton pads to compare the effects of this VOC with the total VOCs of T. asperellum T1. Lettuce seeds were germinated in the absence/presence of 6-PP in shared atmosphere in a growth chamber with 16-h photoperiod at 25  C for 14 d. The experiments were set up in triplicate and repeated twice. The chitinase and b1,3-glucanase assays used were described in 2.5. To test the effect of 6-PP and total VOCs on plant growth, experiments described in 2.4 were performed. 6-PP, at a concentration 25 mL mL
1 (Parker et al., 1997), was applied onto a sterile cotton pad and compared with total VOCs of T. asperellum T1. Lettuce seeds were germinated in the absence/presence of individual VOCs in shared atmosphere in a growth chamber with 16-h photoperiod at 25  C for 14 d (Jalali et al., 2017). The experiments were set up in triplicate and repeated twice. At the end of VOCs exposure periods, the lettuce plants were removed from the exposure conditions and subjected to phenotypic characterization as described in 2.4. 2.11. Statistical analysis Significant differences among the various treatments (the effect of volatile(s) on fungal growth, enzyme activities, biomass and chlorophyll content) were subjected to one-way analysis of variance (ANOVA). A Student's t-test and Duncan's multiple range test (DMRT) was used to determine statistically significant differences between treated samples and the untreated control (Gomez and Gomez, 1984; Naznin et al., 2014). 3. Results 3.1. VOCs emitted from T. asperellum T1 inhibit fungal growth To determine the effects of VOCs emitted by T. asperellum T1 on fungal growth of lettuce leaf spot fungi, C. cassiicola and C. aeria, a volatile antifungal assay was conducted. We found that the colony diameters of control fungi were larger than those of treated fungi (Fig. 1A). Percentage inhibitions of C. cassiicola and C. aeria by the volatiles from T. asperellum T1 were 61.31% and 41.46%, respectively (Fig. 1B). 3.2. Induction of defense-related enzymes in lettuce by VOCs emitted from T. asperellum T1 After 2 weeks of exposure, the plant defense-related enzymes of lettuce, induced by T. asperellum T1 VOCs, were assessed by measuring activities of the cell-wall degrading enzymes b-1,3glucanase and chitinase. The activities of these enzymes were enhanced by exposure to T. asperellum T1 (Fig. 2). The activity of chitinase was 0.010 ± 0.01 U mL
1 for control lettuce and 1.26 ± 0.01 U mL
1 for treated lettuces. The b-1,3-glucanase activity was 1.83 ± 0.01 U mL
1 for control lettuce and 4.45 ± 0.02 U mL
1 for treated lettuce (Fig. 2). The activities of cell-wall degrading enzymes in treated lettuce were significantly higher than in control lettuces (p < 0.05). Furthermore, a crude extract from lettuce treated with VOCs caused morphological changes (wilt and

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Fig. 1. Antifungal activity of volatile organic compounds emitted by Trichodrma asperellum T1 on fungal growth (A), and percentage of inhibition (B).

Fig. 2. Enzyme activities of chitinase (A) and b-1,3-glucanase (B) in control lettuce and those treated with volatile organic compounds emitted by Trichoderma asperellum T1. Asterisks indicate statistically significant differences by Student's t-test, p < 0.05.

abnormalities such as irregular and rough cell-wall surface) in both fungal pathogens, whereas in the control treatment they remained normal (Fig. 3). 3.3. VOCs emitted by T. asperellum T1 display plant growth promoting ability Following 14 d of exposure, lettuce plants were collected for measurement of biomass, phenotypic characteristics and total chlorophyll content. We defined growth promotion of T. asperellum T1 to be the case when the majority of plant biomass, phenotypic

characteristics (number of shoots and roots, fresh and dry weight) and chlorophyll content were significantly higher than in the control (p < 0.05). In the presence of VOCs from T. asperellum T1, there were significant increases in average numbers of shoots and roots, and biomass and chlorophyll content (Figs. 4 and 5). The average number of leaves was 2.8 ± 0.8 for the control and 4.4 ± 0.5 following treatment, whereas the average number of roots was 4.0 ± 1.2 for the control and 6.2 ± 1.3 for the treatment plants (Fig. 4A). The VOCs increased plant biomass (Fig. 4B), as fresh weights of control and treated lettuce were 27.8 ± 7.6 mg and 81.2 ± 19.8 mg, respectively. Regarding the dry weights of lettuce

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Fig. 3. Crude extract of lettuce treated with VOCs caused changes in fungal morphology: (A) untreated C. cassiicola, (B) VOCs treated C. cassiicola, (C) untreated C. aeria, and (D) VOCs treated C. aeria.

(Fig. 4C), control lettuce and treated lettuce weighed 4.4 ± 1.0 mg and 8.3 ± 2.2 mg, respectively. VOCs also increased total chlorophyll content (Fig. 6). Chlorophyll a content was 4.15 ± 0.01 mg g
1 FW for control lettuce and 4.42 ± 0.02 mg g
1 FW for treated lettuces. Chlorophyll b content was 1.58 ± 0.04 mg g
1 FW for control lettuces and 2.21 ± 0.02 mg g
1 FW for treated lettuce. 3.4. Histology study Trypan blue staining showed no endophytic Trichoderma, or other fungi, in lettuce tissues throughout the 2 weeks of exposure to Trichoderma VOCs (Fig. 7). 3.5. GC/MS profiling of T. asperellum T1 VOCs The results are summarized in Table 1 from a GC/MS profiling of the natural compounds produced by T. asperellum T1 that were responsible for inhibiting C. cassiicola and C. aeria, for inducing defense response, and for promoting growth in lettuce. A total of 22 compounds were identified using the NIST library (Fig. 8). Most compounds were tentatively identified with a greater than 50% probability. The compounds’ carbon counts (C) ranged from C2 (ethanol) to C18 (ethyl palmitate). The SPME collection showed that VOCs emitted by T. asperellum T1 were identified as acids, alcohols, aldehydes, alkanes, pyran and fatty acid groups (Table 1). The 6pentyl-2H-pyran-2-one (6-PP) was dominant, making up 14.20% of peak area (Table 1). Fig. 8 shows the mass spectrum of the compounds, including those identified as 2-ethyl-1-hexanol, 1nonanol and 6-pentyl-2H-pyran-2-one. 3.6. Effect of each volatile compound on antifungal activity, defense response and phenotypic characterization of lettuce The percentage inhibition of C. cassiicola by 2-ethylhexanol, 1nonanol, 6-PP and total VOCs were 25.20 ± 5.80, 18.33 ± 6.29,

11.67 ± 2.60 and 48.75 ± 2.16% respectively, whereas inhibition of C. aeria was 24.78 ± 5.78, 28.33 ± 13.76, 13.75 ± 5.22 and 39.16 ± 3.81%, respectively (Fig. 9). The activities of cell wall degrading enzymes were increased by exposure to 6-PP and total VOCs of T. asperellum T1. The activity of chitinase was 0.009 ± 0.037 U mL
1 for control lettuce, and 0.022 ± 0.033 U mL
1 and 0.001 ± 0.0007 U mL
1 for total VOCs and 6-PP treated lettuce, respectively (Fig. 10). The b-1,3-glucanase activity was 0.193 ± 0.050 U mL
1 for control lettuce, and 0.473 ± 0.022 U mL
1 and 0.343 ± 0.005 U mL
1 for total VOCs and 6-PP treated lettuce, respectively (Fig. 10). Phenotypic characteristics of lettuce, including fresh weight, dry weight, number of root and number of leaves treated with 6-PP and total VOCs were significantly higher than those of control (Table 2). Fresh weight of control lettuce was 18.67 ± 4.04 mg, whereas for 6-PP and total VOCs treated lettuce it was 35.00 ± 10.00 and 76.67 ± 7.64 mg, respectively. Dry weight of control lettuce was 1.83 ± 0.76 mg, and for 6-PP and total VOCs treated lettuce it was 4.17 ± 1.04 and 8.00 ± 2.29 mg, respectively. Number of roots in control lettuce was 4.33 ± 0.58, and 6-PP and total VOCs treated lettuce had 6.33 ± 0.58 and 6.67 ± 1.53 roots, respectively. Moreover, number of leaves in control lettuce was 2.67 ± 0.58, and in 6-PP and total VOCs treated lettuce it was 4.67 ± 0.58 and 4.33 ± 0.58 leaves, respectively. 4. Discussion VOCs from microorganisms have recently been proposed as BCAs that can reduce the effects of several plant diseases (Andrimialisoa et al., 2010; Sunpapao et al., 2018; Baiyee et al., 2019; Wonglom et al., 2019), induce defense responses (Ryu et al., 2004; Kishimoto et al., 2005; Yi et al., 2009) and enhance plant growth (Ryu et al., 2003; Lugtenberg and Kamilova, 2009; CortesBarco et al., 2010; Blom et al., 2011; Garnica-Vergara et al., 2015). The antifungal compounds detected in culture extracts of T. asperellum T1 are members of the following compound classes:

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Fig. 4. Phenotypic characteristics of lettuce: numbers of shoots and roots (A), fresh weight (B), and dry weight (C), when treated with volatile organic compounds emitted by Trichoderma asperellum T1. Asterisks indicate statistically significant differences by Student's t-test, p < 0.05.

alcohols, aldehydes, pyrans and fatty acids. Ethanol and 2ethylhexanol from the alcohol group were found in VOCs at 3.85% and 4.70%, respectively (Table 1), and contribute to antifungal activity (Fernado et al., 2005). 1-Nonanol is in an aldehyde represented in the VOCs by about 2.85% (Table 1), having various biological activities such as antimicrobial and antifungal activities (Fernado et al., 2005). 6-PP is a compound of the pyran class, found as a major compound at 14.2% (Table 1). Biological properties of 6PP include antifungal activity (Parker et al., 1997: Andrimialisoa et al., 2010; Baiyee et al., 2019). Ethyl palmitate, a fatty acid, was detected in VOCs at 1.94% (Table 1) with antifungal activity (Choi et al., 2010). However, although fatty acids are less effective than other compounds and chemical fungicides, they have been reported to exhibit antimicrobial activities (Pohl et al., 2011). It has been shown that VOCs from microorganisms can be involved in inducing defense responses against pathogen infections and can cause systemic resistance (Naznin et al., 2014). However, prior literature does not indicate the 22 compounds tentatively identified in this study (Table 1) as potential inducers of defense response in lettuce. Our current results demonstrate that lettuce plants exposed to VOCs emitted by T. asperellum T1 had elevated activities of the cell-wall degrading enzymes chitinase and b-1,3glucanase (Fig. 2). Direct SEM imaging showed degradation of fungal pathogen cell walls (Fig. 3), probably by these enzymes. Furthermore, VOCs of T. asperellum T1 improved the resistance of lettuce against leaf spot pathogens (Data not shown). VOCs emitted by several microbes have been shown to be plant

growth promoters (Ryu et al., 2003; Lugtenberg and Kamilova, 2009; Blom et al., 2011). Exposed lettuce plants also displayed significant increases in plant biomass, numbers of leaves and roots, and total chlorophyll content (Figs. 4e6). This agrees with a prior report by Lee et al. (2016). The GC/MS analysis identified several compounds, some of which have been reported as plant growth promoters. It has been shown that 6-PP produced by Trichoderma spp. improves or inhibits plant growth depending on the concentration (Harman et al., 2004; Vinale et al., 2008). Prior studies have shown that plants exposed to volatile 2-methyl-1-butanol have altered plant size and chlorophyll concentration compared with controls (Ryu et al., 2003; Hung et al., 2015), and we found that T. asperellum T1 in our study produced 2-methyl-1-butanol. Baiyee et al. (2019) showed that T. spirale T76-1 emitted five dominant VOCs including 2-amylfuran, ethanol, ethyl 2methylbutyrate, phenylethyl alcohol and 6-PP. The volatile compounds produced by T. spirale T76-1 revealed several compounds involved in antifungal activity, especially ethanol, phenylethyl alcohol and 6-PP (Baiyee et al., 2019). However, the authors did not clarify the effect of each compound involved in antifungal activity. Based on the result of our study, T. asperellum T1 produced 6-PP, as was also the case for T. spirale T76-1. We also showed that 6-PP displayed antifungal activity against fungal disease pathogens (Fig. 9). Based on GC/MS analysis, volatiles emitted by T. asperellum T1 had 22 identified compounds (Table 1). Several of these compounds such as ethanol, 2-ethylhexanol, 1-nonanol, 6-PP and ethyl

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Fig. 5. Growth of lettuce in a shared atmosphere with Trichoderma asperellum T1 for 14 days: control plates exposed to plain PDA (A), lettuce exposed to T. asperellum T1 VOCs (B), lettuce exposed to T. asperellum T1 VOCs (lower panel) are larger than without VOCs (upper panel) (C).

Fig. 7. Trypan blue staining of control plant (A), and plant exposed to T. asperellum T1 VOCs for 2 weeks (B). Fig. 6. Total chlorophyll content of lettuces with and without exposure to T. asperellum T1 VOCs. Asterisks indicate statistically significant differences by Student's t-test, p < 0.05.

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Table 1 GC/MS profiling of volatile organic suggested compounds released by Trichoderma asperellum T1. No.

Compound

Molecular formula

RT (min)

% Match

% Area

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Ethanol O-butylhydroxylamine 2-Methyl-1-butanol 2-Amylfuran 2-Ethyl-1-hexanol Cycloheptane 2,4-Dihydroxybenzaldehyde 1-Nonanol Phosphonoacetic Acid 2-Ethylhexyl hexyl sulfite Cyclomethicone 6 N1,N1,N4-Tris(tert-butyldimethylsilyl) succinamide 6-Pentyl-2H-pyran-2-one 2,5-Cyclohexadiene-1,4-dione, 2,6-bis(1,1-dimethylethyl)6-Undecylamine 10-Epi-gamma-Eudesmol Succinic acid, 2-(2-chlorophenoxy)ethyl ester 6-Undecylamine 2,5-diisopropylphenol, trifluoroacetate ester 6-Undecylamine 2,6-Bis(1,1-dimethylethyl)-4-(1-oxopropyl)phenol Ethyl palmitate

CH3CH2OH C4H11NO C5H12O C9H14O C8H18O C7H14 C7H6O3 C9H20O C2H5O5P C14H30O3S C12H36O6Si6 C22H50N2O2Si3 C10H14O2 C14H20O2 C11H25N C15H26O C14H17ClO5 C11H25N C14H17F3O2 C11H25N C17H26O2 C18H36O2

1.45 2.21 3.04 4.71 5.19 5.63 6.02 6.66 7.93 8.66 8.96 9.84 11.10 11.19 11.23 13.21 13.28 13.69 15.70 15.90 15.94 16.91

87.20 80.80 77.70 85.30 91.10 77.60 68.30 80.50 56.60 78.80 83.90 57.50 84.40 77.90 84.70 74.80 60.1 73.10 55.40 86.20 77.10 80.00

3.85 0.74 0.81 1.16 4.70 1.02 0.68 2.85 0.83 0.64 2.25 0.96 14.20 4.06 2.18 2.41 13.05 4.23 1.83 3.86 1.97 1.94

Fig. 8. A chromatogram of volatile organic compounds from T. asperellum T1. The peaks at 5.19, 6.66, and 11.10 min are tentatively attributed to 2-ethyl-1-hexanol, 1-nonanol and 6pentyl-2H-pyran-2-one, respectively.

palmitate have been shown to inhibit pathogenic fungi (Loewenthal, 1961; Fernado et al., 2005; Parker et al., 1997: Andrimialisoa et al., 2010; Choi et al., 2010), whereas 6-PP and 2methyl-1-butanol act as plant growth promoters (Ryu et al., 2003; Harman et al., 2004; Vinale et al., 2008; Hung et al., 2015). 6-PP was the dominant compound from some Trichoderma species and has been shown to induce growth and reduce disease symptoms (Vinale et al., 2008). However, we still do not know which components in the volatiles emitted by Trichoderma species induce defense responses in lettuce. As the VOCs are complex mixtures and their production is induced by the environmental conditions, it is difficult to attribute the effects to individual volatile compounds or their mechanisms (McNeal and Herbert, 2009; Insam and Seewald, 2010; Polizzi et al., 2011; Bailly and Weisskopf, 2012; Lee et al., 2016). In the present study we found that 2-ethylhexanol, 1-nonanol and 6-PP inhibit both tested fungal pathogens in viro (Fig. 9). This result is in agreement with previous studies (Fernado et al., 2005;

Parker et al., 1997). However, the inhibition by each compound was below that of the total VOCs. T. asperellum T1 emitted VOCs as a complex mixture of several compounds (Table 1), and some compounds might work synergistically to inhibit fungal growth, with more effective inhibition than for each compound singly. The ability of 6-PP and total VOCs to induce defense responses was confirmed by assaying cell-wall degrading enzymes. Our results showed that chitinase and b-1,3-glucanase activities in 6-PP and total VOCs treated plants were higher than in the control (Fig. 10). Furthermore, treatment with 6-PP and total VOCs improved phenotypic characteristics in comparison to the control (Table 2). That 6-PP can promote plant growth is in agreement with Lee et al. (2016). Based on results presented in this study, 6-PP is one of several compounds emitted by T. asperellum T1 that showed ability to suppress fungal growth, induce a defense response and promote plant growth in lettuces. The results from our study revealed that VOCs emitted by T. asperellum T1 mediated (i) antifungal activity against leaf spot

P. Wonglom et al. / Fungal Ecology 43 (2020) 100867

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Table 2 Phenotypic characteristics of lettuces when treated with 6-PP and total VOCs. Treatment

Fresh weight (mg)

Dry weight (mg)

No. of roots

No. of leaves

control 6-PP VOCs

18.67a ± 4.04c 35.00 ± 10.00b 76.67 ± 7.64a

1.83 ± 0.76c 4.17 ± 1.04b 8.00 ± 2.29a

4.33 ± 0.58b 6.33 ± 0.58a 6.67 ± 1.53a

2.67 ± 0.58b 4.67 ± 0.58a 4.33 ± 0.58a

a Mean from four replications ± SD. Values in same column followed by the same letter are not significantly different, according to Duncan's multiple range test (DMRT) p ˃ 0.05.

Acknowledgements This work was supported by the Prince of Songkla University (grant no. NAT6202117-0), the Center of Excellence in Agricultural and Natural Resources Biotechnology (CoE-ANRB) phase 2, the Japan Society for the Promotion of Science (JSPS) and the National Researcher Council of Thailand (NRCT) Core-to-Core Program. The authors would like to thank PerkinElmer Co., Ltd. Thailand for GC/ MS analysis. The copy-editing service of RDO/PSU and the helpful comments of Dr. Seppo Karrila are gratefully acknowledged. References Fig. 9. Antifungal activity of 2-ethylhexanol, 1-nonanol, 6-PP and total VOCs emitted by Trichodrma asperellum T1 against growth of Corynespora cassiicola (A), and Curvularia aeria (B).

Fig. 10. Activities of chitinase and b-1,3-glucanase enzymes in control lettuce, and in 6-PP and total VOCs treated lettuce. The letters indicate statistically significant differences according to Duncan's multiple range test, p < 0.05.

fungal pathogens of lettuces (C. cassiicola and C. aeria), (ii) defense responses, including enhanced cell-wall degrading enzyme activities, (iii) improved plant growth and (iv) increased chlorophyll content in lettuces.

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