SOME VOLATILE METABOLITES PRODUCED BY THE ANTIFUNGAL-TRICHODERMA ASPERELLUM UZ-A4 MICROMYCETE

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INTRODUCTION
According to many scientists, more than 1.5 million microscopic fungal species are spread worldwide. However, approximately 10% of the fungi and 1% of them were studied on the spectra of secondary metabolites (Weber et al., 2007). It is known that the largest amount of natural preparation, 45% of antibiotics obtained based on secondary metabolites are produced by fungi. In this case, the share of asidialmacromycetes is 11%, while the share of micromycetes belonging to the Penicilium, Aspergillus, Trichoderma and Tolypocladium is 33%. The representatives of make up almost 99% of metabolites used in medicine and agriculture (Zhu et al., 2011). Genus Trichoderma the secondary metabolites formed are important for agriculture some of which are noteworthy for their antifungal properties against phytopathogenic fungi (Daoubi et al., 2009). Trichoderma metabolites are chemically diverse natural compounds of relatively low molecular weight produced primarily by microorganisms and plants. The secondary metabolites are biosynthezed pathways from primary metabolites (i.e., polycetides or mevalonate pathways derived from acetyl coenzyme A or amino acids) and are synthesized by certain genes. The expression of these genes is controlled by one or more global regulators (Herbert, 1989). The secondary metabolites exhibit several biological activities related to the body's survival functions, such as competing against other micro and macro-organisms, symbiosis, and ion exchange. Trichoderma the production of the secondary metabolites in fungi is often interrelated with specific stages of morphological dynamics, during the active growth phase the metabolites increase and the number of species increases. The secondary metabolites exhibit several biological functions and play an important role in regulating interactions between organisms. The are phytotoxins (secondary metabolites produced by plants phytopathogenic microorganisms), mycotoxins (secondary metabolites produced by fungi that cause disease and death in humans and animals), pigments (metabolites that form color compounds with antioxidant activity), and antibiotics (natural microbiological resistance or secondary metabolites capable of destruction) (Keller et al., 2005;Chiang et al., 2009). Among microorganisms, fungi of the Trichoderma are one of the most powerful biological agents in use today because they produce various metabolites against pathogenic microbes (Khan et al., 2020;Ming et al., 2012). Trichoderma lives in the soil and grows saprophytically on many substrates, such as tree bark, and plant roots and affects animals (a source of protein and enzyme-rich nutrients when added to feed) and plants (growth, development, microbiological protection) (Atanasova et al., 2013;Holzlechner et al., 2017). Some of the secondary metabolites produced by Trichoderma are important as drugs and a single compound (6-pentyl-a-piron) as a food flavor. Since the discovery of gliotoxin in the early 1930s, the extraction and study of metabolites from fungi of the Trichoderma genus have begun (Weindling and Emerson, 1936). Over the years, analytical studies have isolated more than 120 secondary metabolites from Trichoderma and determined their structures as well (Sivasithamparam and Ghisalberti, 1998;Reino et al., 2008). However, it is difficult to determine the exact amount of secondary metabolites produced by Trichoderma, which can form more than 1000 compounds, depending on the characteristics of the strain, environmental conditions and the sensitivity of the detection method. In recent years, genetic and genomic studies have revealed that Trichoderma secondary metabolites form new types of metabolites, taking into account biosynthetic pathways, fungal metabolism and environmental interactions (Reithner et al., 2007). These micromycetes produce several pharmaceutical and biotechnologically important secondary metabolites, including non-ribosomal peptides, terpenoids, pyrons, indolyl compounds, peptaibols, polycetides, sideophores, volatile and non-volatile terpenes (Contreras-Cornejo et al., 2016;Vinale et al., 2008;Velázquez-Robledo et al., 2011;Müller et al., 2013). One of them is a steroidal metabolite viridin, an antifungal compound isolated from various Trichoderma sp (T. koningii, T. viride, T. virens) (Golder and Watson, 1980;Singh et al., 2005). This antibiotic secondary metabolite exhibits potent antagonism to microorganisms such as Botrytis allii, Colletotrichum lini, Fusarium caeruleum, Penicillium expansum, Aspergillus niger va Stachybotrys atra (Reino et al., 2008). After reviewing these, we set the goal of our study is to analyze the secondary volatile metabolites that form the fungus Trichoderma and their properties.

MATERIALS AND METHODS Study area and Material Selection
The Trichoderma sp. 4 strain was selected from the soils of a cotton field infected with phytopathogenic diseases in the Bukhara region of the Republic of Uzbekistan in October 2019.

Fungi Identification
The soil samples were dried in air for 4 hours and the isolation of microorganisms was carried out by the method of serial dilutions. The inoculum was incubated at 28 +30°C for 5 days. Observation of the appearance of colonies was recorded for 3 to 5 days. Colonies with symptoms of Trichoderma in Petri dishes were isolated and kept clean for further study. Isolated strains were identified by classical methods based on morphology using the relevant literature (Park et al., 2005). The isolated strains were deposited at the Institute of Microbiology of the Academy of Sciences of the Republic of Uzbekistan, where they were kept at a low temperature (4-5 °C).

DNA Isolation and Purification
A 200 µl of the Trichoderma sp. 4 fungus sample was taken in a 1.5 ml plastic tube. Then 200 μl of 200 mM LiOAc, 1% SDS buffer was added, vortexed thoroughly and incubated at 70 o C for 5 minutes (Arnold et al., 2011). The samples were then centrifuged at 15,000 g for 5 min. The liquid portion was taken to a new plastic tube, an equal volume of 96% ethanol was added and vortexed. For DNA precipitation, the sample was stored at -20 o C for 1 h and centrifuged at 15,000 g for 5 min. The liquid in the tube was discarded and washed in 70% ethanol. The precipitate was dissolved in 100 μl of TE buffer and detected on a 0.8% agarose gel. Add another 100 μl of TE to the DNA dissolved in 100 μl of TE. 1 μl of RNAase was added, vortexed and incubated at 37 °C for 30 min. Then, 0.1 volume (20 μl) of NaAc and 1 volume of isopropanol (220 μl) were added. The sample was stored at -20°C for 1 hour and centrifuged at maximum speed for 10 minutes.The liquid in the tube was discarded and washed in 70% ethanol. The precipitate was dissolved in 50 μl of TE buffer and detected on a 0.8% agarose gel.

PCR Amplification of the ITS Fragment
Universal oligonucleotide primers of the internal transcribed spacer (ITS) gene were used for PCR amplification: ITS1-(TCCGTAGGTGAACCTGCGG), and ITS4-(TCCTCCGCTTATTGATATGC) (White et al., 1990). PCR amplification of DNA samples isolated from bacterial strains was conducted in the GenPak® PCR MasterMix kit. In this case, the reaction was prepared in a total volume of 20 μl, consisting of 10 μl of Dilution, 8.2 μl of double-distilled water, 0.4 μl of primer (ITS1 and ITS4) and 1 μl of DNA samples. PCR amplification optimization initial denaturation at 94 °C for 3 minutes, denaturation at 94 °C for 40 seconds, primer annealing at 55 °C for 40 seconds, elongation at 70 °C for 90 seconds, final elongation at 70 °C for 7 minutes, + ∞ at 4 o C, repeated for 35 cycles. Amplicons were detected by electrophoresis on a 2% agarose gel stained with ethidium bromide.

PCR Product Purification and Sequencing
For sequencing, PCR products were cut from 2% agarose gel and purified using the QIAquick® Gel Extraction Kit manual. The amount of purified PCR products was measured in a NanoDrop device. Sequencing of the samples was performed using BigDye Terminator v.3.1 cycle sequencing kit and Applied Biosystems® Genetic Analyzers, 3130 series sequence (Thermo Fischer Scientific, USA). The sequence result of this Trichoderma sp 4 strain was aligned with the species in NCBI BLAST (http://www.ncbi.nlm.nih.gov/BLAST). A phylogenetic tree for ITSof Trichoderma sp 4 was built via MEGA-X (versión10.1.8) software (Tamura et al., 2007).

Dual Culture Analysis
A dual culture analysis. To determine their antagonism, the strains of Alternaria alternata, Fusarium solani and Aspergillus niger, which have a phytopathogenic effect, were studied against the strain Trichoderma asperellum Uz-A4. A block (6 mm in diameter) was taken from the antagonist and phytopathogens and placed in the CDA nutrient medium in the same way on both sides of the Petri dish (5.5 mm) oppositely. The experiment was repeated 3 times. Placed in a thermostat with a temperature of 25 °C. The radius of colony antagonism on the 7th day was measured and calculated by the following formula (Mao et al., 2020).

Growth Inhibition Rate Control Colony Radius Treatment Colony Radius Fermentation
T. asperellum Uz-A4 strain was grown on Mandel's agar medium (in a test tube) for 6 days, and its suspension at a concentration of 106-7 spores/ml was used as an equivalent material. Microscopic fungus modified by Mandels (Mandels et al., 1962) 3; sucrose -20; (pH 5.5)) were grown on nutrient medium in 500 ml Erlenmeyer flasks. in 250 ml of nutrient medium, on an orbital shaker (shakers IKA® KS 130) at a speed of 180 rpm, at a temperature of 28-30 °C for 14 days.

Filtration
On the 14th day of growth, the biomass of the T. asperellum Uz-A4 strain was isolated from the culture liquid by double filtration through filter paper (Whatman #1). The culture fluid extracted from the biomass was stored at 4 °C.

Extraction
The separated culture liquid was extracted 3 times in a separating funnel 3:1 in ethyl acetate (EtOAc). The extraction was repeated every 2 hours. The aqueous layer at the bottom of the separating funnel was removed. The extract with ethyl acetate was dried at a temperature of 40°C under a vacuum (in a rotary evaporator) (Stracquadanio et al., 2020).

Analysis of VOCs
Unknown volatiles was detected in a YL 6900 GX/MS gas chromatography-mass spectrometric detector using a YL 6900 GX/MS (Young In Chromass, Korea) equipped with a DB-5MS column (30 m × 0.25 mm inner diameter, 0.25 μm film thickness) substances were identified. Oven temperature -initial -80°C/3.0 min, heating rate -15 °C/min to 250 °C, hold -3.0 min, Helium was used as carrier gas at a flow rate of 1.0 ml/min. Evaporator temperature -280 °C, flow section -1/20, analysis time -17 min. organized the Liquid samples were injected into disparities using a 1 μl microsyringe. The temperature of the transmission line was 300 °C, the ionization voltage was -70 eV, and the ion source temperature was 230 °C. Scanning range -30-350 a.m.u. The components were identified from the mass spectra of each component after comparison with the available spectral data in the MS library NIST 2017 (Guarrasi et al., 2017).

RESULTS
The PCR product of the ITS part of Trichoderma asperellum Uz-A4 strain (ON534075) is about 600 bp (Figure 1), and when we compared it with the data in the NCBI BLAST database, it showed that this strain is 100% similar to about 100 species of Trichoderma asperellum. We analyzed the phylogenetic tree with the species T.hamatum, T.atroviride, T.viride, T.harzianum and Protocrea pallida as an outgroup. We named this strain (ON534075) Trichoderma asperellum Uz-A4 strain based on the molecular identification of the ITS part. Phylogenetic tree of the ITS portion of Trichoderma asperellum strain Uz-A4 (blue) (Figure 2) and related species and Protocrea pallida as an outgroup. Antagonistic properties of Trichoderma asperellum Uz-A4 strain against phytopathogenic strains of A. alternata isolated from infected wheat (Triticum aestivum), A.
niger isolated from infected cucumber (Cucumis sativus L.) and F. solani isolated from bean (Phaseolus L.) were studied during the research. T. asperellum Uz-A4 strain showed 77% antagonism against the A. alternata strain in 7 days, stopped the growth of the phytopathogenic fungus, multiplied on its colonies, changed the color of the colony, and acted as a super parasite. As A. niger strain is a relatively strong phytopathogen, T. asperellum Uz-A4 strain produced 55% antagonism, 97% antagonistic zones were formed compared to F. solani strain, surrounded F. solani hyphae and growth was observed in the hyphae (Figure 3).   To detect volatiles in the secondary metabolites, the T. asperellum Uz-A4 strain was first grown in liquid culture and was filtered and separated from the biomass. Mass spectral gas chromatography (GC-MS) analysis was performed on the extracted liquid culture (Figure 4). Phenylethyl alcohol, 5-Hydroxymethylfurfural, dehydroacetic acid, 1-Dodecanol, 2,4-di-tertbutyl phenol, diethyl suberate, n-hexadecanoic acid, 1hexadecanol, 2-methyl-, phthalic acid, ethyl pentadactyl ester, mono(2-ethylhexyl) phthalate, octadecanoic acid were identified compared to GC-MS library base depending on the absorption rate of some spectra of the chromatogram (Table 1). When the liquid culture of the fungus T. asperellum Uz-A4 was extracted and volatile substances were detected, the presence of the substance-phenyl ethyl alcohol was demonstrated. The chromatogram showed that the absorption rate of this substance was 6,393 minutes ( Figure 5). Although phenyl ethyl alcohol is a volatile substance by nature, it further enhances the antagonistic ability of fungi of the Trichoderma genus. Phenylethyl alcohol have been shown in studies to inhibit the growth of F. Incarnatumfrom 21,68% to 74.29% (Intana et al., 2021). 5-Hydroxymethylfurfural secondary metabolite showed an absorption rate of 7,672 minutes ( Figure 6). It is known from the literature that 5-Hydroxymethylfurfural is a furfural sugar compound and is involved in the formation of enzymes (β-glucanase, cellulose) of fungi of the Trichoderma genus. Figure 4. T. asperellum Uz-A4 fungal strain culture extract GC chromatogram; 1) Phenylethylalcohol; 2)5-Hydroxymethylfurfural; 3) Dehydroaceticacid; 4) 1-Dodecanol; 5) 2,4-Di-tert-butylphenol; 6) Diethyl suberate; 7) n-Hexadecanoic acid; 8) 1-Hexadecanol, 2-methyl; 9) Phthalic acid, ethyl pentadecyl ester; 10) Mono(2-ethylhexyl) phthalate; 11) Octadecanoic acid.   Dehydroacetic acid showed an absorption rate of 9,155 minutes on the GC chromatogram (Figure 7). This substance is currently used in the storage and packaging of fruit and vegetables as well as cosmetics (Saravanakumar et al., 2018). The volatility of 1-Dodecanol was relatively high, resulting in an absorption rate of 10,094 minutes on the chromatogram (Figure 8). It is known from the literature that 1-Dodecanolis an organic product by nature and is a fatty alcohol. As metabolites of the microorganism, the fungi Aspergillus niger, Rhizopus oryzae, Aspergillus terreus, Trichoderma viridе, Aspergillus flavus micromycetes were also extracted from ethyl acetate and found to be metabolites in GC-MS (Shaikh and Mokat, 2017). 2,4-Di-tert-butylphenolGC showed an absorption rate of 10,388 minutes when analyzed( Figure 9). 2,4-Di-tertbutylphenolis a metabolite with cytotoxicity, insecticidal, nematidic activity, antagonistic properties (Zhao et al., 2020;Shobha et al., 2020;Chen and Dai, 2015). In a study on T. asperellum Uz-A4 extraction, diethylsuberate GC produced an absorption rate of 11,001 minutes in the analysis ( Figure 10).    n-Hexadecanoic acidGC showed an absorption rate of 13,764 minutes when analyzed ( Figure 11).The presence of n-hexadecanoic acid (6.17%) in the analysis of the metabolite T. atroviridi GC-MS. Due to the fatty acid content, its antioxidant and antibacterial properties have been known (Saravanakumar et al., 2018). 1-Hexadecanol produced a 14,007-minute absorption rate on a 2-methyl GC chromatogram ( Figure 12).
Among the secondary metabolites of Trichoderma, the antioxidant content of this substance is high (Ali, 2021). Phthalic acid, ethyl pentadecyl ester showed an absorption time of 14,588 minutes ( Figure 13). This substance is characterized by the fact that it is an active enzymatic and bioactive substance among secondary metabolites (Bahaa et al., 2019).   Mono(2-ethylhexyl) phthalate volatile metabolite showed an absorption rate of 14,817 minutes when T. asperellum Uz-A4 liquid culture was extracted with ethyl acetate (Figure 14). It is known from the literature that this substance is also actively involved for antifungal properties (Yang et al., 2013). Octadecanoic acid showed an absorption rate of 15,118 minutes in GC analysis ( Figure 15). Octadecanoic acid metabolite is a metabolite involved in the formation of carbohydrate containing substances (Kaushik et al., 2020).

DISCUSSION
Biopreparations based on Trichoderma are used in vegetable growing, melon growing, greenhouses, horticulture, viticulture and growing various ornamental plants and trees. This micromycete protects plants from phytopathogens. Increases seed germination capacity, enhances plant growth, increases metabolism, expands leaf plate surface, and improves soil structure. In recent years, when we got acquainted with the volatile metabolites identified in Trichoderma species, it was proposed as a biological protection agent that reduces the effects of plant diseases (Sunpapao et al., 2018;Andriamialisoa et al., 2004;Wonglom et al., 2019). The biological activity of Trichoderma fungi provides an important advantage in the competition with pathogens in terms of growth and development speed, competition for habitat and food. Trichoderma develops in the hyphae of phytopathogenic fungi, and wraps and destroys the cell walls with the help of lytic enzymes. As a result, it continues its life form as a hyperparasite. In addition, along with mycoparasitism, it also produces antibiotics. The limits the activity of pathogenic microorganisms (Harman, 1996). In figure 3, we can observe the development of the T. asperellum Uz-A4 strain in the hyphae of pathogenic microorganisms after antagonistic development against the F.solani strain.
The authors also noted the production of volatile and non-volatile antibiotics by Trichoderma species in the biocontrol of plants, the formation of antagonistic properties against phytopathogenic microorganisms (Ubalua and Oti, 2007). Volatile metabolites identified in our studies are metabolites that affect the antagonistic properties of the strain. In recent years, in connection with the rapid development of biotechnology, interest in microscopic fungi of the genus Trichoderma, which attract the attention of researchers in connection with their practical significance for obtaining biologically active substances, plant protection products and as an active destructor of plant polysaccharides. It is known that Trichoderma emits various metabolites: growth factors (auxins, cytokines and ethylene), organic acids, intracellular amino acids, vitamins and over 100 antibiotics (Benítez et al., 2004). "TopShield" (New York), "Trichodex" (Israel), "Sternifag SP", "Trichodermin", "SellovirdineV-G20x", "Gliokladin", "Viridin" (Russia), "Fungilex J", "Fekord-2012-S" (Belarus) and other biological drugs were developed by international scientists based on fungi belonging to the Trichoderma (Reithner et al., 2007).