INTRODUCTION:

Fungi, being the most important saprophytic microbes in the soils, play a vital role in the bio-geochemical cycling of matter. This process improves the fertility of any environment and thereby supports productivity and biodiversity. On the other hand soil borne plant pathogenic fungi create a major economical loss on many important crops and the management of the pathogens area major problems 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 and the biological control methods has become the order of the day. (Khara and Hadwan, 1990; Naik and Sen, 1992; Tu, 1992; Panneerselvam and Saravanamuthu, 1994, 1999)

The existence of fungi in the marine habitat is known from early times and their significance as active participants in marine ecological processes has been proved well (Kohlmeyer and Kohlmeyer, 1979). However, abundant and accurate data on distribution are prerequisite for any bio-geographical analyses; such listings of fungi from particular geographic localities do not themselves from the complete part of the bio-geographical studies and they become part of it only when the distributions are analyzed either from a historical perspective or in terms of the ecological parameters. Further, the fungi in marine habitats are believed to be the potential sources for a variety of secondary metabolites as they lead their life in peculiar environmental conditions. Interest on this aspect could result in the discovery of production of various enzymes, antibiotics, etc. However, the outcome is very meager in the estimated potential available in the nature.

Thus, exploration of the fungal resources from marine habitats for antagonistic properties against different pathogens would help in identifying new biocontrol organisms and the present investigation is an attempt in this aspect.

MATERIALS AND METHODS:

Sampling:

The present investigation was carried out by collections and examination of soil samples from different sites in the backwater environment at Vellapallam (Nagapattinam District, Tamil Nadu) along south east coast of India in the month of May 2013. The soil samples were collected at a depth within 10 cm using alcohol sterilized metal spatula, kept in new polythene bags, sealed and transported to the laboratory immediately for the mycological examination. For the analysis of soil nutrients, one kg of soil was separately collected in polythene bags from each station.

Analysis of fungal flora:

Dilution plating technique described by Warcup (1950) was used to isolate the fungi from soils. For the plating, the Potato Dextrose Agar (PDA) Medium was prepared and serial dilutions were made using 1:1ratio of Distilled and Seawater.

The poured plates, in replicates, were incubated for a week long period at room temperature (24+2^oC) and the colonies growing on PDA plates with different morphology were counted separately and identified comparing with the standard works of Von Arx (1974), Ainsworth et al. (1973); Raper and Fennell (1965) and Ellis (1976). Axenic cultures of all the fungal species were also raised in PDA slants by transferring the growing edge of the colonies from the plates. Mean value of Total number colonies encountered in replicate plates is referred as Population Density and it is expressed in terms of colony forming unit (CFU) per gram of soil with dilution factor. Number of species is referred as species diversity. Percentage contribution is worked out as follows to find out the share of individual fungal species in the total fungal flora.

Percentage

Contribution

Total number of colonies of individual species

´ 100

Total number of colonies of all the species

Analysis of physico-chemical characteristics:

Moisture content was estimated by finding the weight difference of known quantity of soil before and after drying in a hot air oven at 60°C for 6 hours. The shade dried samples, after removing the debris, were suspended in distilled water (1:2 w/v) and allowed to settle down the sand particles. The pH of the suspension was read using pH meter (Systronics, India), to find out the soil pH. Electrical conductivity of soil was determined in the filtrate of the water extract using conductivity bridge as described by Jackson (1973), Cation exchange capacity (CEC) of the soil was determined by using 1 N ammonium acetate solution as described by Jackson (1973).

Organic carbon content was determined by adopting chromic acid wet digestion method as described by Walkley and Black (1934), available nitrogen was estimated by alkaline permanganate method as described by Subbiah and Asija (1956) and available phosphorus by Brayl method as described by Bray and Kutz (1945). Available potassium was extracted from soil with neutral 1 N ammonium acetate (1:5) and the potassium content in the extract was determined by using flame photometer (Standfold and English, 1949), calcium (Neutral 1 N NH₄ OAC extractable 1:5) was extracted with neutral 1 N ammonium acetate and the available calcium in the extract was determined by Versenate method (Jackson, 1973). Available micronutrients such as Zn, Cu and Mn were determined in the diethylene triamine pentaacetic extract of soil using Perkin-Elmer model 2280 Atomic Absorption Spectrophotometer (Lindsay and Norvell, 1978). Other nutrients such as magnesium, sodium and available iron were analysed following the method of Barnes (1959) and Muthuvel and Udayasoorian (1999).

To assess the relationship between physico-chemical parameters and total fungal population Pearson’s correlation analysis was employed using Statistical Package for Social Sciences (SPSS) software.

Antibiotic interactions assay:

Fusarium semitectum Berkeley and Ravenel, one of the soil borne broad spectrum fungal pathogen that cause a major disease in Cotton (Ciegler et al., 1982), was isolated from the coastal soils and used as a test organism. Three species of Trichoderma viz. Trichoderma viride, T. koeningii and T. harzianum that were isolated from the brackish water environment in the present study were screened as biocontrol agent their efficiency was determined in vitro in dual culture and food poisoning methods.

Dual culture method:

Dual culture interaction between the pathogen and test organisms was studied by inoculating over PDA plates with 6 mm mycelial discs of Fusarium semitectum and Trichoderma harzianum, T. viride and T. koeningi separately at a distance of 3 cm from each other. Three replicates were maintained for each set. Control was set in single and dual inoculated cultures of the fungus. The position of the colony margin on the back of the disc was recorded daily. The colony interactions between the pathogen and the test fungi were assessed following the model proposed by Porter (1924) and Dickinson and Broadman (1971). Five type of interaction grades as proposed by Skidmore and Dickinson (1976) have been used.

They are as follows:

Grade 1: Mutual intermingling growth without any microscopic sights of interaction.

Grade 2: Mutual intermingling growths where the growth of the fungus is ceased, and is being over grown by the opposed fungus.

Grade 3: Intermingling growth where the fungus under observation is growing into the opposed fungus either above (or) below.

Grade 4: Slight inhibition of both the interacting fungi with a narrow demarcation line (1-2 mm).

Grade 5: Mutual inhibition of growth at a distance of >2 mm.

Assessments were made when the fungi had achieved an equilibrium after which there was no further attraction in the growth. Since both of the organisms were mutually inhibited, the assessment was made for both organisms. The percentage inhibition of growth was calculated as follows.

Percentage inhibition of growth

r	=	growth of the fungus that was measured from the center of the colony towards the center of the plate in the absence of antagonistic fungus.
r¢	=	growth of the fungus that was measured from the center of the colony towards the antagonistic fungus.

Food poisoning method:

Agar blocks of equal size (5 mm dia) cut from the actively growing margin of the pathogenic fungus of F. semitectum, and the antagonists of Trichoderma harzianum, T. koeningi and T. viride were inoculated separately into 250 ml conical flasks containing 100 ml of sterilized potato dextrose broth medium. The flasks were incubated at 25 ± 2°C for 15 days. After 15 days of incubation the staling substances were filtered first through Whatman No.1 filter paper and then through Seitz filter (GS). The filtrates were transferred aseptically into sterile conical flask; condensed and stored at 4°C for further use.

The culture filtrates prepared in such a way were added separately to the cooled potato dextrose agar medium to give the concentrations of 5, 10, 15 and 20 per cent and allowed to solidify. After solidification 5 mm agar blocks cut from the actively growing margin of the test fungus (F. semitectum) was inoculated at the center of each plate. The plates were incubated at 25 ± 2°C for five days. The radial growth was measured periodically and the mean growth rate was calculated. Control was also maintained.

The percentage of inhibition of growth was calculated as follows:

Percentage of inhibition of growth	=	Growth in control – Growth in treatment	´ 100
		Growth in control

In order to assess the dominance of individual species in each site percentage contribution was worked out as follows.

% contribution	=	No. of colonies of fungus in a sample	x 100
		Total number all colonies of all the species in a sample

RESULTS:

Fungal flora:

The population density of fungi showed variations from 6.98 to 106 ´ 10² CFU/g and Species diversity varied from 5 to 9, in different sites. Altogether 14 species belonging to 4 genera were identified in the present study. Of them, 13 species under 3 genera were assignable to Deuteromycetes and one species to Zygomycetes. Species composition structure revealed the genus Aspergillus to represent maximum of 9 species followed by Trichoderma (3 species) and the rest by one species each, as follows:

	Zygomycetes
1.	Absidia glauca Hagem
	Deuteromycetes
2.	Aspergillus albicans
3.	A. awamori Kawachi
4.	A. candidus Link
5.	A. conicus Blochwitz
6.	A. flavipes Bainier and Sartory
7.	A. fumigatus Fresenius
8.	A. granulosus Raper and Thom
9.	A. ochraceous Wilhelm
10.	A. oryzae (Ahlburg in Korschelt) Cohn
11.	F. semitectum Berkeley and Ravenel
12.	Trichoderma koeningii Oudemans
13.	T. harzianum Rifai
14.	T. viride AA.Gams

Percentage contribution of the individual species to the total fungal population in each site showed variation. However, collective results of all the sites showed the maximum percentage contribution of 15.1% by A. granulosis. This was followed by A.awamori and A.fumigatus (11.3% each), T.harzianum (10.3%), A.flavipes (8.4%), T.viride and A.albicans (7.5%), Aspergillus ochraceous (6.6%), Absidia glauca and Fusarium semitectum (4.7% each), A. oryzae and Trichoderma koeningii (2.8% each), A. ustus (3.85%) (Table 1).

Physico–chemical characteristics:

Physico–chemical characteristics of the soil samples at these sites showed the following features: Moisture content of the soil was in the range from 00 to 00 % ; pH was in the range from 7.9 to 8.9; electrical conductivity from 0.04 to 1.31 dSm^-1; cation exchange capacity from 8.01 to 10.11 c.mol proton⁺/kg; organic carbon from 0.04 to 0.21%; available nitrogen from 0.011 to 0.22%; available phosphorus from 0.001 to 0.006%; available potassium from 0.011 to 0.057 ppm; available zinc from 0.36 to 0.57 ppm; available iron from 2.01 to 2.91 ppm; available copper from 0.14 to 0.34 ppm; available manganese from 1.22 to 1.53 ppm; calcium from 3.1 to 5.7 mg/kg; magnesium from 3.1 to 4.2 mg/kg; sodium from 0.29 to 1.73 mg/kg; and potassium 0.01 to 0.08 mg/kg (Table 2).

Correlation coefficient (r) values between physico-chemical parameters of soil and population density of fungi:

The Correlation analysis between population density of fungi and physic-chemical characteristics soil revealed a significant positive correlation with electrical conductivity (r = 0.981; p< 0.05) and organic matter content (r = 0.973; p< 0.05) (Table 3).

Antifungal activity:

Colony interactions between Trichoderma spp. and F. semitectum in dual culture:

The type of interactions between the pathogen and the Trichoderma spp. observed was of Group 4, i.e., slight inhibition of both the interacting fungi with a narrow demarcation line (1-2 mm) and the maximum percentage of inhibition of F. semitectum was due to

T. koeningii (61.3%) followed by T. harzianum (58.1%) and T. viride (55.5%) (Table 4).

Effect of culture filtrate of Trichoderma spp. on the growth of F. semitectum:

Culture filtrates of T. koeningii, T. harzianum and T. viride showed inhibitory effect on the growth of F. semitectum. The inhibitory effects of the fungi were measured as 22.7, 13.6 and 13.6 mm at 5% concentrations, 41.7,27.3 and 22.7 from at 10% concentrations, 63.6, 50.0 and 45.4 mm at 15% and 77.3, 68.2 and 63.6 mm at 20% concentrations (Table 5).

Table 1. Population density (CFU/g) and percentage contribution of fungi in different sites.

S. No	Fungi	Site 1		Site 2		Site 3		Site 4		Total No. of colonies	% Contribution
S. No	Fungi	PD	PC	PD	PC	PD	PC	PD	PC	Total No. of colonies	% Contribution
1.	Absidia glauca	-	-	2	0.66	3	1	-	-	5	4.7
3.	Aspergillus albicans	3	1	-	-	2	0.66	3	1	8	7.5
2.	A. awamori	4	1.33	4	1.33	4	1.33	-	-	12	11.3
4.	A. candidus	-	-	2	0.66	-	-	-	-	2	1.8
5.	A. conicus	-	-	-	-	5	1.66	-	-	5	4.7
6.	A. flavipes	2	0.66	-	-	-	-	7	2.33	9	8.4
7.	A. fumigatus	7	2.33	5	1.66	-	-	-	-	12	11.3
8.	A. granulosus	5	1.66	-	-	7	2.33	4	1.33	16	15.1
9.	A. ochraceous	2	0.66	-	-	-	-	5	1.66	7	6.6
10.	A. oryzae	-	-	3	1	-	-	-	-	3	2.8
11.	F. semitectum	1	0.33	4	1.33	-	-	-	-	5	4.7
12.	Trichoderma harzianum	4	1.33	-	-	-	-	7	2.33	11	10.3
13.	T. koeningii	3	1	-	-	-	-	-	-	3	2.8
14.	T.viride	-	-	8	2.66	-	-	-	-	8	7.5
	Total	31	10.3	28	9.3	21	6.98	26	8.65	106

PD - Population Density; PC- Percentage Contribution

Table 2. Physico-chemical character of the sampling soils

Sl. No.	Name of the parameter	Sample details
Sl. No.	Name of the parameter	I	II	III	IV
1.	Moisture content (%)
2.	pH	7.82	7.86	7.46	7.25
3.	Electrical conductivity (dsm^-1)	0.49	0.51	0.38	0.34
4.	Organic carbon (%)	0.32	0.25	0.29	0.24
5.	Organic matter (%)	0.64	0.50	0.58	0.48
6.	Available nitrogen (Kg/ac)	115.6	112.0	123.6	106.8
7.	Available phosphorus (Kg/ac)	4.85	4.25	3.75	3.89
8.	Available potassium(Kg/ac)	112.6	126.5	119.2	124.6
9.	Available zinc (ppm)	0.89	0.84	0.74	0.76
10.	Available copper (ppm)	0.49	0.42	0.46	0.49
11.	Available iron (ppm)	4.26	4.13	4.23	4.56
12.	Available manganese (ppm)	2.89	2.36	2.16	2.16

Table 3. Correlation coefficient (r) vales between physico-chemical parameters and population density of fungi

	pH	EC	OC	OM	AN	AP	AK	AZ	ACu	AFe	AMn	PDF
PDF	-0.894	0.981*	-0.706	0.973*	0.000	-0.238	0.206	-0.771	0.083	-0.301	-0.665	1

* Significant at 5% level (P< 0.05).

Table 4. Colony interaction between Fusarium semitectum (pathogen) and Trichoderma spp. (antagonists) in dual culture experiment

S.No.	Growth response of the antagonistic and test fungi	*T. koeningii*	*T. viride*	*T. harzianum*
1.	Colony growth of the pathogen towards antagonist (mm)	7	8	7
2.	Colony growth of the pathogen away from the antagonist (mm)	11	12	13
3.	% growth inhibition of the pathogen in the zone of the interaction	68.2	63.6	68.1
4.	Colony growth of the antagonist in control (i.e.) growth towards the centre of the plate in the absence of the pathogen	75	72	74
5.	Colony growth of the antagonist towards the pathogen (mm)	29	32	31
6.	Colony growth of the antagonist away from the pathogen (mm)	25	23	24
7.	% of growth inhibition in the zone of interaction	61.3	55.5	58.1

Growth of F. semitectum towards the centre of the plates in the absence of any antagonistic fungus (control) was 22 mm measurement was taken into 96 hours

Table 5. Effect of culture filtrates of Trichoderma spp. on the growth of F. semitectum

S.No.	Name of the fungi used in culture filtrates	Concentration (%)	Growth rate (mm) After 72 hours	Percentage of inhibition
	Control		22
1.	Trichoderma0harzianum	5	19	13.6
		10	16	27.3
		15	11	50.0
		20	7	68.2
2.	T. koeningii	5	17	22.7
		10	14	41.7
		15	8	63.6
		20	5	77.3
3.	T. viride	5	19	13.6
		10	17	22.7
		15	12	45.4
		20	8	63.6

DISCUSSION:

The present investigation revealed the existence of 14 species of fungi belonging to 4 genera in total with the population density ranged from 6.98 to 106 ´ 10² CFU/g and the species diversity from 5 to 9. All these fungal species were reported earlier from soils and a variety of substrates in the terrestrial environment (Gilman, 1995), from oceans and estuaries (Johnson and Sparrow, 1961) and coastal and marine environs (Subramanian and Raghukumar, 1974) as invasive fungi. Though the coastal soils are considered to be the transitional areas between the land and sea, it exhibit the occurrence of only terrestrial species, often described as facultative fungi based on their ability in growth and reproduction, but not the obligate fungi. Introduction of terrestrial species into this is facilitated through various sources such as plant litter, other organic materials and run off from soil.

Edaphic characteristics are believed to be responsible for the establishment of biotic community of any of the soil ecosystem. Hence, the soil characteristics were analyzed along with the fungal Population density. Among the different parameters analyzed EC values and OM content have showed significant positive correlation. EC value is an indirect measure for the salinity. Its positive relationship was interesting and contradictory to other observations. However, the EC value recorded in the present study comparatively was lower than the marine and brackish water sediments of Madras coast (Subramanian and Ragukumar, 1974) and mangroves of Andaman (Chandhuri et al., 2009). Organic Matter is considered to be the factor responsible to influence the population of any of the heterotrophic microorganisms (Nadimuthu, 1998) and but it showed variations in the narrow range of 0.24 to 0.32 per cent in different sites. But, rather than the edaphic factors, fungal community structure is believed to be responsible for the fungal population in the brackish water environment at Vellapallam. Aspergilli formed the bulk in terms of species diversity (9 species) and percentage contribution (69.5%) in the sampling sites and they are reported to possess tolerance and adaptive mechanisms to the varying marine environmental characteristics, by Pawar and Thirumalachar (1966), Upadhyay et al. (1978) and Nadimuthu (1998).

Trichoderma spp. are common saprophytic fungi found in almost all types of soil. Efficiency of terrestrial isolates of this genus as biocontrol against different pathogenic organisms are documented well (Papavizas, 1985; Sivan and Chet, 1986; Calvet et al., 1990; Elad et al., 1993; Spiegel and Chet, 1998; Mathur and Sarbhoy, 1978; Muthukumar et al., 2006; Madhanraj et al., 2009). Among the three species isolated and screened during the present investigation adopting dual culture technique, T. koeningii showed the maximum inhibitory effect against F. semitectum (61.3%) followed by T. viride and T. harzianum (58.1 and 55.5%). Food poisoning technique also confirmed the same order of inhibitory effect on the pathogen, which further confirm the activity. The activity recorded in the present study is promising and thus they could also be utilized as biocontrol agent to control F. semitectum diseases in the crops of coastal areas.

ACKNOWLEDGEMENTS:

The authors thank the Principals of the respective Colleges for their encouragement and the Indian Biotrack Research Institute, Thanjavur 613 005 for extending the facilities to carry out the work

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Received on 13.12.2014 Accepted on 22.02.2015

Asian J. Pharm. Res. 5(1): Jan.-Mar. 2015; Page 24-30

DOI: 10.5958/2231-5691.2015.00004.0

Ecology and Biocontrol Potential of Soil Fungi of a Backwater Environment along the East Coast of India

¹Department of Microbiology, Srimad Andavan Arts and Science College, Tiruchirappalli - 620 005, Tamil Nadu.

²Indian Biotrack Research Institute, Thanjavur, Tamil Nadu.

³P.G. Department of Plant Science, Avvaiyar Government College for Women, Karaikal 609 602, Puducherry U.T. India

*Corresponding Author E-mail: ibrilab@gmail.com

Ecology and Biocontrol Potential of Soil Fungi of a Backwater Environment along the East Coast of India

1Department of Microbiology, Srimad Andavan Arts and Science College, Tiruchirappalli - 620 005, Tamil Nadu.

2Indian Biotrack Research Institute, Thanjavur, Tamil Nadu.

3P.G. Department of Plant Science, Avvaiyar Government College for Women, Karaikal 609 602, Puducherry U.T. India

*Corresponding Author E-mail: ibrilab@gmail.com

¹Department of Microbiology, Srimad Andavan Arts and Science College, Tiruchirappalli - 620 005, Tamil Nadu.

²Indian Biotrack Research Institute, Thanjavur, Tamil Nadu.

³P.G. Department of Plant Science, Avvaiyar Government College for Women, Karaikal 609 602, Puducherry U.T. India