Studies on the Compound Separation and Its Antifungal Potentiality of Fungi Isolated From Nagapattinam District, East Coast of India

 

Madhanraj P.1* and Panneerselvam A.2

1Dept of Microbiology, Thanthai  Hans  Roever  College  of  Arts  and  Science, Elambalur,  Perambalur - 621 212, Tamil Nadu, India.

2Dept of Botany and Microbiology, A.V.V.M Sri Pushpam College (Autonomous), Poondi, Thanjavur (Dt) – 613 503, Tamil Nadu, India.

 

ABSTRACT:

Altogether 37 species belonged to 10 genera were recorded. A preliminary screening of all the species isolated from soils were used for antifungal (antagonistic) activity against Fusarium semitectum, a known soil borne fungal pathogen. Among the species tested, Trichoderma koeningii   showed promising activity, inhibited the pathogenic fungus for the maximum both in   dual culture and in food poisoning technique. When HPLC fractionation of the mycelial extract of T. koeningii grown in PDA broth yielded three fractions. They were named as peak 1, peak 2 and peak 3.Among the three fractions, peak 2 alone showed the antifungal activity with the inhibition zone of 13 mm diameter against Fusarium semitectum. The functional groups present in peak 1 and 3 are almost same, but in the peak 2 some extra functional groups are present. These groups may be responsible for the antifungal activity of this fraction.

 

KEYWORDS: antifungal activity, dual culture and  food poisoning technique,       HPLC, compounds, biocontrol.

 

INTRODUCTION:

The ocean covers about 70 per cent of the earth’s surface and contains an extraordinary diversity of life. Interest in understanding of the marine organisms has been accelerated in recent years with growing recognition of their importance in human life as food, fine chemicals, pharmaceuticals, etc. Starting from Barghoorn and Linder (1944) till date, marine fungi have been extensively studied from different substrata. Soil borne plant pathogenic fungi create a major economical loss on many important crops. The management of the pathogen is a major problem among the agricultural community. Now-a-days the diseases are managed with the application of chemical pesticides. Use of chemical pesticides causes serious environmental problems, as they don’t undergo biodegradation. So, minimizing the application of pesticides has become the order of the day. To achieve this goal, the biological control methods can be effectively be used along with other methods of disease control. Antibiotic effect of cell free culture filtrates have been used to demonstrate the role of antibiosis in biological control (Khara and Hadwan, 1990; Naik and Sen, 1992; Tu, 1992; Panneerselvam and  Saravanamuthu, 1994, 1999; Muthukumar et al., 2006; Madhanraj et al.,2009).

 

Trichoderma sp. are filamentous soil fungi known to be effective biocontrol agents against a range of important soil borne pathogens. They are the most widely studied biocontrol agents (BCAs) against plant pathogens. Weindling and Emerson (1936) stated that they could excrete extracellular compound called gliotoxin. Since then, many antibiotics and extracellular enzymes were isolated and characterized. The biocontrol mechanisms were also established (Haran et al., 1996; Zhihe et al., 1998).


The marine fungi are believed to be the potential sources for a variety of secondary materials as they lead their life in a peculiar environmental conditions. Interest on this aspect could result in the discovery of production of various enzymes, antibiotics, etc. However, the outcome is very meagre in the estimated potential available in the nature. Thus, exploration of the marine fungal resources for antagonistic properties against different pathogens and identification of bioactive compounds from them would help in identifying evolving new biocontrol organisms and potential antibiotic substances.

 

MATERIALS AND METHOD:

Fungi isolates:

About 37 species were isolated from Nagapattinam coastal soil, 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. semitectum with all the fungi isolated. Based on this, three species of Trichoderma were selected for the study of antagonistic activity. Colony interaction between the test-organism and the soil fungi namely Trichoderma viride, T. koeningii and T. harzianum was studied in vitro in dual culture experiments. In the dual culture experiments T. koeningii inhibited the growth of the pathogen to the maximum extent. Hence, T. koeningii was taken for further studies.

 

Extraction and separation of compounds from mycelium:

Preparation of mycelial extract:

The mycelial discs of T. koeningii were inoculated into liquid potato dextrose medium and incubated in darkness for three weeks. After   incubation the fungal mycelial mat was harvested by filteration, pressed between folds of filter paper and then a weighed amount (1 g) of the fungal mycelium was extracted in 70% acetonitrite (10 ml). The extract was then filtered using Whatman No.1 filter paper. The filtrate was dried in vacuum. The residue was re-dissolved in HPLC solvent, i.e., acetonitrile : water : acetic acid (65:35:1,v/v/v) for further analysis.

 

Separation of components using High Performance Liquid:

Chromatography (HPLC):

The mycelial extract (0.5 ml) was injected into the RP-18 octadecylsilyl  silica (DDS) column (25 x 1 cm, i.e.) with LC-UV detector (Bioanalytical System, U.S.A.) and monitored at 254 nm. The flow rate was adjusted to 1.5 ml
min-1. The fractionated samples were collected in vials

 

Assay of Antifungal activity:

The HPLC fractions (purified compounds) from mycelial extract of  T. koeningii was tested for their antifungal activity against F. semitectum. Sterile paper discs (5 mm in diameter) were soaked with each fraction (70 ml) and dried completely. The discs were then placed on PDA in which the test fungal pathogen (F. semitectum) had been plated previously. Inhibition zones (mm) formed around paper discs were measured to find out the antifungal activity against the pathogen tested.

 

Structure analysis of the fermentation metabolite (Yong et al., 2003):

The compound structure was identified by the method of ultraviolet scanning, infrared scanning and nuclear magnetic resonance separation analysis as follows:

 

Ultraviolet scanning:

The fractionated sample was dissolved in acetonitrite and then detected its UV absorption values with Lambda 35 Ultraviolet scanner.

 

Infrared scanning:

A small quantity of solid sample product was collected, grinded adequately and was pressed to tablet with KBr method. Infrared spectrum of sample product was recorded in the range of 4000-400 cm-1 using Perking Elmer Rx1 infrared scanner.

 

Nuclear magnetic resonance:

The sample product was dissolved in deuterium and using TMS as inside marker the 1H-NMR spectrum was recorded with Bruker Nuclear Magnetic Resonance Scanner.

 

RESULT AND DISCUSSION:

The mycelial contents of T. koeningii were capable of inhibiting the growth of the pathogenic fungus, F. semitectum. The mycelial extract, when subjected to HPLC, yielded three fractions namely peak1, peak2 and peak3 (Table.1). Of which, the fraction with peak 2 showed strong activity, while rest of them did not show any such activity as they were minor peaks(Fig.1). It was about 38% in the total mycelial extract. Similarly, Watts et al. (1988) have isolated separated a fraction with antimicrobial property from mycelial extract of Trichoderma reesei and identified the compound as trichodermin, which inhibited the growth of Aspergillus flavus, A. niger, Cladosporium cucumerium, Fusarium solani, F. udum, Rhizoctonia solani, R. bataticola, Rhizopus stolonifer, Pythium sp., which is in agreement with the present findings that the mycelial compound of T. koeningii also could inhibit the growth of F. semitectum(Plate.1). The UV spectrum of all the peaks derived through the HPLC of mycelial extract revealed the presence of carboxyl group. The NMR and FTIR spectrum of peaks 1 and 3(Fig.2 and 4) showed the presence of phenolic group, alkene non-conjugated, primary alcohol, chloride, cyanide groups and CN groups and peak 2 (Fig.3)showed the aromatic hydroxyl (or) phenolic acid, cyanide group, carboxyl group, carbon-carbon double bond (or) alkene group, primary alcohol, chloride and CN groups. Of which the presence of CN group may be due to the impurities of the solvent acetonitrile used. The functional groups that are found only in peak 2 which were not recorded the peaks 1 and 3 such as aromatic hydroxyl (or) phenolic group, carboxyl group, carbon-carbon double bone (or) alkene group and CN groups would be responsible for the antifungal activity against F. semitectum. Thus the coastal soil isolate of T. koeningii could better serve as a biocontrol agent to suppress the growth of F. semitectum, a known pathogen. In the coastal soils that are influenced by seawater, T. koeningii represented all the stations with appreciable per cent contribution, and also grow well in broad range of salinity and pH under in vitro condition. Further, as the mycelial extract showed activity against the F. semitectum, it can be explored further for production of antifungal compounds, commercially.

 

Table 1.           HPLC fractions of mycelial extract, their RT and height

S. No.

RT

Name of the compound

Height (volts)

1.

2.21

Peak 1

0.453

2.

2.56

Peak 2

178

3.

2.61

Peak 3

0.242

 

 

Fig.1. HPLC of mycelial extract of Trichoderma koeningii

 

Fig.2(a-c) UV, FT-IR and H-NMR spectrum of Peak 1 derived through HPLC of the mycelial extract of T. koeningii

 

Fig.3(a-c) UV, FT-IR and H-NMR spectrum of Peak 2 derived through HPLC of the mycelial extract of T. koeningii

 

 

Fig.4(a-c) UV, FT-IR and H-NMR spectrum of Peak 3 derived through HPLC of the mycelial extract of T. koeningii

ACKNOWLEDGEMENT:

The authors are grateful to Secretary and Correspondent A.V.V.M. Sri Pushpam College, Poondi – 615 503, Thanjavur Dt. for providing laboratory facilities.

 

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Received on 15.03.2011

Accepted on 24.05.2011        

© A&V Publication all right reserved

Research Journal of Pharmaceutical Dosage Forms and Technology. 3(3): May-June 2011, 100-104