Studies on the Compounds and Its Antifungal Potentiality of Fungi Isolated From Paddy Field Soils of Jenbagapuram Village, Thanjavur District, and South India

Senthilkumar G., *Madhanraj P. and Panneerselvam A.

Dept of Botany and Microbiology, A.V.V.M Sri Pushpam College (Autonomous), Poondi, Thanjavur (Dt) – 613 503, Tamil Nadu, India

*Corresponding Author E-mail: micromadhan@sify.com

ABSTRACT:

Totally 42 species belonged to 20 genera were recorded. A preliminary screening of all the species isolated from soils were made for antifungal (antagonistic) activity against Fusarium oxysporum, a known soil borne fungal pathogen. Among the species tested the Trichoderma harzianum inhibited the pathogenic fungus to the maximum both in dual culture and in food poisoning technique. Gas chromatography mass spectrum analysis of acetonitrile extract of the filtrate of T. harzianum revealed the presence of six compounds represents six major peaks. The peaks correspond with diethyl phthalate, tetradecanoic acid 9,12 – octadecadienoic acid (z, z), oleic acid, 1, 2 –benzene di oxylic acid, diisooctyl ester and squalene.

KEYWORDS: Fungal extract, Compounds, GC-MS

INTRODUCTION:

Now a days the diseases are managed with the application of chemical pesticides. Use of chemical pesticides causes environmental problem, as they don’t undergo biodegradation. So minimizing the application of pesticides has become order of the day. To achieve this goal the biological control methods can be effectively used along with other methods of disease control. Trichoderma sp. is filamentous soil fungus known to be effective biocontrol agents (BCAS) against plant pathogens. Weindling and Emerson (1936) started that they could excrete extra cellular compound called gliotoxin. Since then many antibiotics and extra cellular enzymes were isolated and characterized. Their biocontrol mechanisms were also established (Haran et al., 1996. Zhihe et al., 1998).

Inventory of biologically active compounds has gained importance in recent years. This involves the process such as extraction, separation, purification and characterization. The compound resulted in the process are proved to interesting in their structure and effective activity against various pathogens. Moreover the compounds (both extra and intra-cellular) are considered as a key factor to identify the organisms.

MATERIALS AND METHODS:

Fungal isolates:

About 42 species were isolated from Jenbagapuram paddy soil,Thanjavur Dt., Tamil nadu, India. All these strains were screened for their antifungal activity against pathogenic fungi.

Antibiotic interactions assay:

A preliminary screening was conducted against F. oxysporum with all the fungi isolated. Based on this, ten species were selected for the study of antagonistic activity. Colony interaction between the test-organism and the soil fungi namely A. niger, A. terreus, A. nidulans, A. sydowi, A. fumigatus, A. flavus, P. janthinellum, P. chrysogenum, T. viride and T. harzianum was studied in vitro in dual culture experiments. In the dual culture experiments T. harzianum inhibited the growth of the pathogen to the maximum extent. Hence, T. harzianum was taken for further studies.

Gas chromatography – Mass Spectrum analysis of the culture filtrate (Liu et al., 2007):

Extraction of antifungal compounds:

The fungus which showed promising activity against the pathogen was cultured in liquid potato dextrose medium at 24^°C in darkness for three weeks. After incubation, the culture was filtered twice through Whatman No.1 filter paper and Seitz filter (G.5). To 100 ml of culture filtrate, 10 ml of ethyl acetate was added in a separation funnel (250 ml), shaken well for 3 min. and the solvent and aqueous layer were separated. The acetonitrile layer of the culture filtrate was used for further analysis.

Gas chromatography – Mass Spectrometry (GC-MS):

Volatile components were identified by GC-MS using a coloumn Elite-1 (100% Dimethyl poly siloxane), 30 x 0.25 mm x 1µm df equipped with GC clarus 500 Perkin Elmer. The turbo mass-gold-perkin-Elmer detector was used.

The carrier gas flow rate was 1 ml per min, split 10:1, and injected volumes were 2 µl. The column temperature was maintained initially at 110^°C for 2 min (hold) followed by increases up to 200^°C at the rate of 5^°/min-9 min (hold). The injector temperature was 250^°C and this temperature was held constant for 36 min. The electron impact energy was 70 eV, Julet line temperature was set at 200⁰C and the source temperature was set at 200^°. Electron impact (EI) mass scan (m/z) was recorded in the 45-450 aMU range.

Using computer searches on the NIST Ver.2.1 MS data library and comparing the spectrum obtained through GC-MS the compounds present in the crude sample were identified.

RESULTS AND DISCUSSION:

Understanding the mechanisms involved in the antagonistic effect of Trichodema spp. against plant pathogen are important in selection of suitable biocontrol agent for effective and safe utilization. Different isolates of Trichoderma have various effects of fungal antagonism and on the plant health. The possible mechanism of antagonism employed by Trichoderma spp. realized so far include competitions, antibiosis by producing non volatile volatile antibiotics and exploitation (Harman and Hadar, 1983).

Table 1. Characteristic features of antifungal compound isolated from T. harzianum BY GC-MS

S. No	RT	Name of the compound	Molecular Formula	MW	Peak area %	Compound nature**	Activity**
1.	16.59	Diethyl phthalate	C₁₂H₁₄O₄	222	31.14	Plasticizer compound	Antimicrobial Antifouling
2.	24.39	Tetradecanoic acid	C₁₄H₂₈O₂	228	22.24	Linoleic acid	Anti inflammatory, hypocholesterolemic, cancer preventive, hepatoprotective, nematicide, insectifuge, antihistaminic, antieczemic, antiacne, 5-alpha reductase inhibitor antiandrogenic, antiarthritic, anticoronary and insectifuge
3.	27.59	9, 12 – Octadecadienoic acid (Z, Z)-	C₁₈H₃₂O₂	280	17.52	Oleic acid	Antiinflammatory, antiandrogenic, cancer preventive, dermatitigenic, hypocholesterolemic, 5-alpha reductase inhibitor, anemiagenic, insectifuge and flavor
4.	28.00	Oleic acid	C₁₈H₃₄O₂	282	4.69	Myristic acid	Antioxidant, cancer preventive, nematicide, lubricant hypocholesterolemic
5.	33.97	1, 2 Benzenedi carboxylic acid, diisooctyl ester	C₂₄H₃₈O₄	390	14.01	Triterpene	Antibacterial, antioxidant, antitumor, cancer preventive, immunostimulant, chemo preventive, lipoxygenase-inhibitor and pesticide
6.	38.39	Squalene	C₃₀H₅₀	410	10.39	Plasticizer compound	Antimicrobial and antifouling

**Source: Dr. Duke's Phytochemical and Ethnobotanical Databases RT- Retention Time MW- Molecular weight

Fig.1. GC – Mass Spectrum of the culture filtrate of Trichoderma harzianum

When the extract of acetonitrile culture filtrate of T. harzianum was subjected to GC-MS analysis to find out the components produced by the fungus, it yielded six prominent peaks with retention time 16.59, 24.39, 27.59, 28.00, 33.97 and 38.39 min. The peaks with reaction time 16.59 min. corresponds to diethyl phthalate with 31.14% of peak area; 24.39 min. corresponds to the tetradecanoic acid with 22.24% of peak area; 27.59 corresponds to 9, 12–octadecadienoic acid (z,z) - with 17.52% of peak area; 28.00 min. corresponds to the oleic acid with 4.69% of peak area; 33.97 min. corresponds to 1,2 – benzenedicarboxylic acid, diisooctyl ester with 14.01% of peak area; 38.39 min. corresponds squalene with 10.39% of peak area and biological activity and chemical structure of phytocompound were identified(Table 1 and Fig 1).

This proved that T. harzianum is capable of producing many compounds that are produced by many other fungal species. The antimicrobial activity of the 1, 2- benzenedicarboxylic acid and diisooctyl ester have already been reported by Ushadevi (2008) from the marine isolates of P. lividum and T. lignorum. Thus, these compounds were also isolated in the present investigation, individually and in combination with other compounds such as diethyl phthalate, tetradecanoic acid, 9, 12 – octadecadienoic acid Z, Z), oleic acid and squalene. Thus the present investigation concludes that 1,2-dicarboxylic acid and diisooctyl ester along with other compounds would have suppressed the growth of F. oxysporum.

Likewise there are reports on the occurrence of tetradecanoic acid, dodecanoic acid and n-hexadecanoic acid in the extract of heads space of Aspergillus versicolar, dodecanoic acid and tetradecanoic acid from P. chrysogenum (Griffith et al., 2007), pentadecanoic acid and oleic acid from Mortierella alpine (Wang et al., 2005), and oleic acid from Phytophthora cinnamomi (Zaki et al., 1983).

Squalene isolated from Rhizoctonia solani, A. flavus, A. fimigatus, Penicillium afrovenetum, Phytophthora cinnamomi, P. cactorum, Pythium graminicola and P. ultimum has been reported by Gottlieb (1978) and 1,2 benzene dicarboxylic acid, di iso octyl ester from Penicillium lividum (Ushadevi, 2008).

The phytochemical analysis of T. harzianum was also studied using thin layer chromatography. The results revealed the presence of saponin, flavonoids, sterol, tannin and phenol. In the present study, it was found that saponin showed antimicrobial activity. Hence, relatively high antimicrobial activity of T. harzianum could be attributed to the presence of these compounds.

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Received on 13.03.2011 Modified on 20.03.2011

Asian J. Pharm. Res. 1(1): Jan.-Mar. 2011; Page 19-21