Identification of volatile organic compounds of some Trichoderma species using static headspace gas chromatography-mass spectrometry

 INTRODUCTION

Fungi produce various volatile organic compounds (VOCs) that due to their small sizes and high vapor pressure are readily able to diffuse through the atmosphere and soils at normal temperature and pressure. VOCs generally have low to medium water solubility and often have a distinctive odor (Hung et al. 2015). Up to now, approximately 500 VOCs have been detected in fungal metabolites. From more than 100,000 species of described fungi, only about 100 species have been studied for VOC production (Korpi et al. 2009; Hung et al. 2015). VOCs play important signaling roles in fungal natural environments. Many ecological interactions are mediated by VOCs, between fungi, plants and bacteria (Morath et al. 2012). They appear as intermediate and final products of different metabolic pathways and principallydevoted to mono- and sesquiterpenes, alcohols, ketones, lactones, esters, fatty acids, sulfur-containing compounds, simple pyranes and benzene derivatives (Korpi et al. 2009). These metabolites are involved in different biological processes such as biocontrol or communication between microorganisms and their living environment (Bitas et al. 2013). They can mediate defense against predators, parasites and diseases, and may be produced for competition between species (Stoppcher et al. 2010).

Fungal strains of the genus Trichoderma are well-known producers of volatile compounds. The VOCs profile of a known species or strain will vary depending on the substrate, duration of incubation, type of nutrients, temperature, and other environmental parameters (Siddiquee et al. 2012; Tait et al. 2013). VOCs of the filamentous biocontrol fungi like Trichoderma spp., act antibiotically against range of plant pathogenic moulds and can confer plant growth promoting effects as well as systemic resistance to plants, thus rendering plants less susceptible to the fungal pathogens (Vinale et al., 2008). The ability of Trichoderma spp. to produce a significant number of volatile (e.g. pyrones, sesquiter-
penes) and nonvolatile secondary metabolites (e.g. peptaibols) has been reviewed recently (Reino et al. 2008).

The Trichoderma strains have been used as biocontrol agents with different mechanisms, such as, mycoparasitism, antibiosis, competition for nutrients, cell wall-lytic enzyme activity, and induction of systemic resistance to pathogens in planta (vinal et al. 2006; Norouzi et al. 2014; Habibi et al. 2015). Determination of volatile fungal metabolites usually is determined by gas chromatography (GC) methods and has been detected for different fungal genera such as Aspergillus, Fusarium, Mucor, Penicillium and Trichoderma. After culture of the fungi in liquid (Pinches and Apps 2007) or on solid growth medium (Nemcovic et al. 2008), volatiles can be extracted in different ways, such as with organic solvents (Reithner et al. 2005), solid phase extraction using C18 or silica gel columns (Keszler et al. 2000), online gas enrichment on adsorption tubes or various headspace (HS) techniques: e.g. static headspace, dynamic headspace (purge and trap) and solid phase microextraction (Stoppcher et al. 2010). In static headspace analysis, the volatiles in the sample are allowed to equilibrate with the air in an airtight container. After equilibration, a known volume of air is collected from the sample, frequently in a gas-tight syringe, and injected directly into the gas chromatograph (GC). After GC separation on nonpolar stationary phases, the constituents of complex mixtures of VOCs can be identified by mass spectro-
metry (MS) (Siddiquee 2014). Mass spectrometric detection can detect individual volatiles from complex mixtures. Structure characterization and confirmation of identity is usually achieved by comparison of mass spectra with library spectra (Jeleń, 2003; Stoppcher et al. 2010).

The objective of this research was to detect volatile organic compounds from the headspace of Trichoderma cultures by using static headspace gas chromatography-mass spectrometry. This is a powerful approach for the direct profiling of VOCs, because fungi are cultured directly in headspace vials and HS-GC-MS measurement is realized in a fully automated method (Guler et al. 2015).

MATERIALS AND METHODS

Fungal isolates and growth conditions

In this study, three native biocontrol Trichoderma species were used. Trichoderma harzianum (NCBI GeneBank accession No. JX173852.1(, T. virens (6011) accession No. KP671477.1, and T. atroviridae

(1-3) were obtained from the Mycology Laboratory, Department of Plant Protection, Gorgan University of Agricultural Sciences and Natural Resources.  Morphological identification of the last isolate has been confirmed by Dr. Zafari (Bu-Ali Sina University). It was compatible with type specimen from Mashhad collection, too (Zafari et al. 2002). They were isolated from the soil of canola and cucurbits farms in Gorgan and were successful in biological control of different phytopathogens (Abdolahian et al. 2012; Norouzi et al. 2014; Habibi et al. 2015). All the fungal strains were maintained on potato dextrose agar (PDA) (Merck, Germany) slants at room temperature and subcultured bimonthly. From actively growing margins of PDA cultures, a
5 mm diameter plug of each Trichoderma species, was placed on the centre of slants consisting of 5 mL of sterile PDA in 20 mL headspace vials. The control vials were consisted of only sterile PDA culture (without Trichoderma plug). Three replicates were considered for each treatment. The vials were sealed with screw-caps containing gas-tight silicone/teflon septa and incubated at 22 °C for 5 days. A single GC-MS measurement was carried out, for all fungal cultures and control vials.

HS-GC/MS conditions

After 10 min of equilibration at 90 °C, extraction of volatiles from the headspace of the fungal cultures was carried out by the aid of a COMBI PAL autosampler (CTC ANALYTICS, Switzerland). For the detection of fungal VOCs, a GC Agilent 7890A equipped with an Agilent 5975C mass selective detector was used. GC–MS analyses were performed with ionization energy of 70ev. Identification of volatile metabolites was conducted using a nonpolar capillary colum (DB-5): 60 m, 0.25 mm, 0.25 µm. Oven program: 40 °C (hold 2 min), 10 °C/min to
200 °C, 25 °C/min to 260 °C (hold 25 min).

Injector temperature was hold at 250 °C (splitless mode) and detector temperature was set at 280 °C.  The carrier gas was helium (He) at the flow-rate of 1 ml/min. The scan range was 45-550 m/z. Fungal metabolites were identified by comparison of the obtained mass spectrum with mass spectral libraries (NIST08.L).

RESULTS

According to NIST08.L mass spectra library of the GC–MS analysis, 30 volatile compounds were identified in the headspace of cultures. The retention time and abundance of these compounds are shown in  (Tables1-3). The detected VOCs in the culture samples, included cycloalkene, alcohol, ketone, ester, organic acid, monoterpene, sesquiterpene,sulphur and nitrogen compounds. Most of detected compounds by this method were related to monoterpenes and sesquiterpenes. Chemical structures of some

identified VOCs are illustrated in Fig. 1 (https://pubchem.ncbi.nlm.nih.gov). In all three species, limonene is the common compound.

Table 1. Volatile metabolites of the biocontrol fungus Trichoderma harzianum identified by HS-GC-MS.

RT: Retention Time

DISCUSSION

Volatile organic compounds have been shown to be involved in interactions between filamentous fungi and their living environment. Thus, analytical methods for the identification of volatile compounds are the key to considering their formation and functions in the biological interactions.

Some identified VOCs were previously reported in various standard laboratories as shown in references list in Tables 1-3. Isoamyl alcohol, limonene and 2, 2-dimethoxy-1,2-diphenyl-ethanone have been identif-
ied in all three species in this study (Tables 1-3). Limonene had the most frequency in these three species. This compound is biosynthesised from acetyl-CoA via the intermediate mevalonate. It has been shown antitumor activities in animal models and in cell culture experiments (Wagner et al. 2003). Khethr et al. (2008) investigated the antibacterial and antifungal activities of limonene against five pathogenic bacterial and fungal strains, and reported that this compound has antibacterial effect, without any antifungal activity.

Table 2. Volatile metabolites of the biocontrol fungus T. virens (6011) identified by HS-GC-MS.

RT: Retention Time

They also declared that this compound was the major component in the Trichoderma extract. Tajick et al. (2014) has been detected limonene in secondary metabolites of Penicillium purpurogenum.

The following VOCs, just detected in T. atroviridae (1–3): ethanol, beta–bisabolene, epizonarene, farnesol, beta–guaiene, alpha–gurjunene, beta–himachalene, beta–sesquiphellandrene, widdrene, zingiberene, diethylac–etylene, benzoic acid–4nitroso–ethyl ester and propanoic acid. In this isolate, ethanol and isoamyl alcohol had major amounts after limonene (Table 3). Based on antifungal effects of these compounds, its biocontrol activity can be related to them.

Four compounds have been recognized only in
T. harzianum: cembrene, beta–elemene, alpha–muurolene and 6–methyl–5–nonen–4–one (Table 1). Unique metabolites were identified in T. virens (6011) include cadinene, calamenene, caryophyllene, beta-eudesmol, alpha–farnesene, 1,2,3,4,5-pentamethyl-1,3-cyclopentadiene and 2–amino–5,7–dimethyl thiazolo[4,5–b]pyridine (Table 2).

Sivasithamparam and Ghisalberti (1998) declared that different species of one family and different isolates of one species, can often produce significantly different compounds. It means that secondary metabolites express the individuality of species in chemical terms. They also stated that, widely separate species could produce the same class of the secondary metabolite and sometimes even the same secondary metabolites.

Zeringue et al. (1993) identified also alpha-gurjunene, caryophyllene, cadinene, alpha-muurolene in aflatoxigenic strains of Aspergillus flavus. Ethanol, beta-bisabolene, alpha-farnesene, beta-himachalene, dl-limonene, beta-sesquiphellandrene, caryophyllene and zingiberene have been detected in T. atroviride and T. viride (Stoppacher et al. 2010; Polizzi et al. 2011; Polizzi et al. 2012; Hung et al. 2013). The bisabolenes are a large group of sesquiterpenes that various biological activities (nematicidal and antimicrobial activities) have been reported for them (Wu et al. 2011). Sesquiterpenes share the same metabolic precursor mevalonate as the monoterpenes and are converted to the final structures by the action of sesquiterpene synthases. They presented a structurally complex compound class that showed antimicrobial and antiviral activities (Fraga 2012; Stoppacher et al. 2010).

Kundu et al. (2013) demonstrated significant antifungal activity of cadinenederivatives that makes them as a source of antifungal agent for the development of a natural fungicide.

Table 3. Volatile metabolites of the biocontrol fungus T. atroviridae (1-3) identified by HS-GC-MS.

RT: Retention Time

Matasyoh et al. (2013) presented antifungal activity of cadinene and beta–bisabolene against mycotoxigenic Aspergillus, Fusarium and Penicillium species. Dahham et al. (2015) demonstrated antimicr-
obial activities of caryophyllene against pathogenic bacterial and fungal strains. Caryophyllene could enhance plant growth and increase stress resistance (Morath et al. 2012; Bitas et al. 2013). Caryophyllene oxide, an oxygenated terpenoid, well known as preservative in food, drugs and cosmetics, has been shown in vitro antifungal effect against dermatophytes (Yang et al. 1999). Azevedo et al. (2013) reported

 that 7-hydroxycalamenene-rich oils presented high antimicrobial activity. Siddiqui et al. (2013) reported M. scandens extract had a remarkable antifungal effect against Rhizoctonia solani, Pythium graminicola and Fusarium oxysporum. They clarified that the key role for their antifungal activities was related to the presence of phenolic compounds, oxygenated monoterpenes and sesquiterpene hydrocarbons such as beta–caryophyllene, d-cadinene, alpha–cubebene, caryophyllene oxide, beta-himachalene and beta–farnesene. These compounds have already been detected in this study.

Fig. 1. Chemical structures of some identified VOCs in Trichoderma species (pubchem.ncbi.nlm.nih.gov).