Extraction and Identification of Secondary Metabolites Produced by Trichoderma atroviridae (6022) and Evaluating of their Antifungal Effects

Fungi release wide spectrum of secondary metabolites that belong to several chemical groups with different biochemical origins. These materials produce as intermediate and end products of diverse metabolic pathways. The profile of the secondary metabolites for a known species or strain will vary depending on the substrate, the duration of incubation, the type of nutrients, temperature and other environmental parameters. Trichoderma spp. are well-known producers of secondary metabolites with different biological activities. The secondary metabolites with antibiotic activity can be classified into two main types. Low molecular weight and volatile metabolites which are involved in complex Trichoderma plant-pathogen interactions. They belong to various structure classes such as alcohols, ketones, alkanes, furans, simple aromatic compounds, isocyanate compounds, volatile terpenes, some polyketides, butenolides, and pyrones. All of them are relatively nonpolar compounds with a significant vapor pressure. These volatile organic compounds (VOCs) in the soil environment could be traveled over distance and affect the physiology of the pathogens. They also enhance growth and systemic resistance in plants. These VOCs have been evaluated for T. atroviride, T. harzianum, T. viride, T. longibrachiatum, T. pseudokoningii and T. aureoviride. High molecular weight metabolites (like peptaibols) are polar metabolites which act directly by contact between Trichoderma species and competitor organisms. Due to potent separation and highly sensitive detection, gas chromatography-mass spectrometry (GC-MS) is the main method for detection of the fungal VOCs. Mass spectrometric detection offers the possibility to identify individual volatiles from complex mixtures. Structure characterization and confirmation of identity are usually achieved by comparison of mass spectra with library spectra and the determination of chromatographic retention indices. Due to the capacity of Trichoderma species in crop protection and promoting vegetative growth, they are marketed as biopesticides, biofungicides and biofertilizers. The identification of molecules with such biological activities can support the development of new biopesticides and biofertilizers based on Trichoderma metabolites. The aim of this study was to investigate antifungal effects and chemical composition of secondary metabolites produced by Trichoderma atroviridae(6022) against Macrophomina phaseolina and Sclerotinia sclerotiorum.
Antifungal effects of isolate 6022 against M. phaseolina and S. sclerotiorum were evaluated under invitro condition by dual culture technique, volatile (Dennis & Webster 1971) and non-volatile (Vinal 2006) metabolites. Volatile metabolites tests were done in 4 cases: Co-culture, 24, 48 and 72 hour cultures. For considering non-volatile metabolites of this isolate, different concentrations of culture filtrate and mycelial mass have been prepared in (autoclaved) potato dextrose agar (PDA), individually. Secondary metabolites were extracted via 4 processes by using of organic solvents (Siddiquee 2012), Headspace technique (Stoppacher 2010) and soxhlet water bath distillation methods for mycelial mass (Dubey 2011) and identified by using the GC-MS device with nonpolar column (DB-5).
In dual culture test, isolate 6022 inhibited the mycelial growth of the pathogen, then over ran and sporulated on the mycelia. The related results for the volatile test in 24, 48, 72h and Co-cultures, indicated that the antagonist inhibited the mycelial growth of the pathogen and production of sclerotia in culture media (PDA). Results of the non-volatile test (in different concentrations) showed significant inhibitory effects on mycelial growth and production of sclerotia. After extraction and GC separation, the constituents of mixtures can be detected via mass spectrometry (MS) by comparison of mass spectra with library spectra searching. According to mass spectra library, 61 compounds such as some alkanes, alkenes, phenolic, alcoholic, terpenoid and aromatic compounds have been detected by 4 processes that among them, alkanes had the highest frequency. Many important compounds with the antifungal effect such as iso-amyl-alcohol, 2-ethyl-1-hexanol, 1-pentanol, stearic acid, palmitic acid, bis (2-ethylhexyl) phthalate, di-n-octyl phthalate have been identified. So, inhibitory effects of isolate 6022 are related to these compounds.
Secondary metabolites of the biocontrol fungus Trichoderma have been involved in different biological processes such as biocontrol or communication between fungi and their living environment. These fungi have antibiotic activity against plant pathogenic fungi, so the requirement for monitoring Trichoderma VOCs profiles has been offered. The results showed that isolate 6022 has the potential for controlling above mentioned pathogens so it can be the suitable alternative for chemical toxins. This method is simple for extraction of the secondary metabolites, under laboratory conditions and studied in Iran for the first time.