In-Vitro Efficacy of Trichoderma viride Against Sclerotium rolfsii and Macrophomina phaseolina

The fungal pathogen causes serious widespread losses to agricultural crops worldwide. Therefore, economy of countries may worsen especially of developing countries. In addition, harmful chemical pesticides which are being used today for increasing crop production creates very serious health hazardous problems to human beings and ecosystem as a whole. The antagonistic potential of Trichoderma species which has been long known to control various soil-borne fungal pathogens in biological way may be utilized. The faster growth rates with which it competes with fungal pathogen mainly brings upon their antagonistic characteristics. An investigation was carried out in laboratory condition towards biological efficacy of T. viride on potato dextrose agar (PDA) medium for the bio-control of soilborne plant pathogens Sclerotium rolfsii and Macrophomina phaseolina in in-vitro condition. The dual culture technique was followed in which T. viride showed significant antifungal activities towards both the pathogens. T. viride significantly inhibited the mycelial radial growth of S. rolfsii by 75% and M. phaseolina by 71.42%. The results showed variable mycelial growth rate for all fungal isolates which was determined after 6 days of incubation in which T. viride showed minimum of 4.00 days to completely cover the petri-plates and S. rolfsii showed 4.33 days whereas M. phaseolina showed 6.33 days. Thus, T. viride showed encouraging results regarding their biopesticidal and biofungicidal potential against plant pathogens which may be endorsed to substitute harmful chemical supplements that exists in modern day agricultural practices.


Introduction
Sclerotium rolfsii (Sacc.) is considered to be ruinous against several crops worldwide with an extensive host range, which includes more than 500 species in 100 families worldwide (Agrios, 2005;Cilliers et al., 2000).The pathogen S. rolfsii may cause damping-off or stem rot on host plant with the help of sclerotial germination, which may measure 1-3 mm with mustard-like appearance formed on surfaces of the affected plant parts (Koike, 2004).The pathogen may exploit host plant internally as well as externally for their growth with white cottony-like mycelium formation.When this fungus first starts demonstrating its symptoms in soil then eradication becomes nearly impossible even with the help of chemical fungicides.Besides, unavailing use of chemicals it may prove ineffective and costly to growers.
Nowadays unfortunately there are hundreds of chemical substitutes available for present day agriculture systems to protect several species of crops from various kinds of diseases.In view of this growers are even opting for broad spectrum chemicals for faster results and some are using air service for spraying their fields in the notion of confirmed production and to gain certain monetary benefits.By contrast these chemical substitutes are far more devastating to environment and economy than we all are anticipating it today.So, biological control which, comprises of employment of various micro-organisms to control plant pathogens is seemed to be very beneficial as it may be economically as well as environmentally utile and safer option for modern agriculture practice today, which is nearly dependent on several harmful chemical pesticides.Amongst various biological antagonists Trichoderma species since long have been extensively used and investigated for their biocontrol ability as they suppresses pathogens by various mechanism such as competition (Baker and Cook, 1974;Harman et al., 2004;Reino et al., 2008).Henis et al. (1982) reported mycoparasitism (penetration and infection) of

Potato Dextrose Agar (PDA) medium
For preparation of potato dextrose agar (PDA) medium potatoes (220 g) were washed, peeled and diced.It was placed in casserole after addition of 1000 ml distilled water.Potatoes were boiled until they became soft enough to be eaten but not overcooked.Strain the decoction through cheese cloth (or thin layer of cotton) in the funnel; collect the liquid in the beaker.Restore the volume of decoction to 1000 ml by adding fresh distilled water and put back in the casserole.Potatoes were discarded.Dextrose (20 g) and agar (18 g) powder were added to the casserole.The medium was boiled with occasional stirring until the agar completely dissolved.The medium was transferred into test tubes and/or petri plates and/or into 250 ml Erlenmeyer flasks (plugged with cotton wool).Again aluminum foil was used to loosely cover.The medium was used to maintain cultures of pathogens (S. rolfsii and M. phaseolina), antagonistic isolates (T.viride) and produce sclerotia of S. rolfsii.

Sterilization
The whole volume of media was sterilized before inoculation.Sterilization was accomplished by heating, chemicals or ultraviolet radiation, also with steam.
Water and culture media were sterilized by heating in autoclave (steam pressure sterilizer), the pressure most commonly used was 15 pounds per square inch in excess of the atmospheric pressure, which gave a temperature of 121°C, usually maintained for 15-20 minutes.After sterilization, the tubes or bottles filled with proper amount of medium were still hot.Therefore, they were placed in an inclined position to make the slants.When the medium solidified on cooling, the test tubes were put in an upright position in an incubator and kept at 34-35°C for one day.The medium, in which bacteria, molds or yeast developed were discarded, since such organisms deprives the mycelium of food and oxygen and may also produce toxic substances that damage the mycelium and retards its growth.

Mycelial growth rate
Mycelial growth rate of Trichoderma viride was determined (Elad et al., 1981) as follows: Petri dishes (90 mm diameter) containing potato dextrose agar (PDA) was centrally inoculated with 5 mm of agar plugs from 7-day-old PDA culture of T. viride to determine the average linear growth.The plates were incubated at 28°C in oven under continuous light and inspected for three successive days.Observation of radial mycelia growth at right angles was recorded every 24 hours during that period.The colony diameter was measured as the mean of two perpendicular diameters measured at the third day minus the diameter at first day.The average linear growth rate (ALG) of assay was recorded as per (Elad et al., 1981) daily by measuring colony diameter calculated by using formula: Average Linear Growth (ALG) (mm/day) = [C3-C1]/T where: Trichoderma spp.against Sclerotium rolfsii, where chlamydospores were abundantly produced in contrast to conidia within the infected fungal sclerotia.Even Majumdar et al. (1996) showed antagonistic property of Trichoderma isolates towards against M. phaseolina.Among Trichoderma species as fungal antagonists, Trichoderma viride is considered to be one of the important antagonists that are being considered to eradicate widest range pathogens biologically.Thus, appropriate formulation of fungal antagonist seems to be an alternative way to decrease burden of excessive and harmful use of chemical pesticides.
Hence, the present study was undertaken to investigate biocontrol efficacy of T. viride in in-vitro condition against pathogen S. rolfsii and M. phaseolina by dual culture technique.

Collection of fungal isolates and culture maintenance
For initial screening of Trichoderma isolate (in vitro) for using its capacity as bio-controller, the fungi S. rolfsii was isolated from roots of groundnut plants showing yellowing and stems rot symptoms from the fields of Pune district, Maharashtra, India (18°32'N; 73°51'E).The infected plant material was brought to laboratory and was washed, cut into pieces of 3 mm segments and were surface sterilized for two minutes with 0.05% sodium hypochlorite solution and rinsed with three changes of sterile water.The segments were dried with tissue paper fungus was maintained in potato dextrose agar plates for seven days (28°C) impregnated with streptomycin.Plates of PDA (90 mm) were inoculated with mycelia plugs of S. rolfsii and incubated at 28°C for 2-3 weeks until mature sclerotia formed.The sclerotia were collected until needed and stored in test tubes at room temperature.Pure culture was obtained by sub-culturing with the help of single hyphal tip method and was maintained on potato dextrose agar slants in incubator at 28°C temperature.Later on the fungus was identified as Sclerotium rolfsii (KD-1) species, identified by Dr. Sanjay K. Singh (Scientist), Division of Mycology and Plant Pathology, ARI, 77, Pune, Maharashtra, India.The fungi Macrophomina phaseolina was kindly provided by Agharkar Research Institute, Pune, Maharashtra, India.Plugs of one week old M. phaseolina were inoculated in PDA and incubated at 28°C for 2-3 weeks until needed.Pure cultures of M. phaseolina were maintained on PDA slants in test tubes and kept in the refrigerator until required.

Antagonists
T. viride isolates were obtained from Department of Microbiology, Agricultural College of Pune, Maharashtra, India.Single colonies of the isolate were sub-cultured in PDA and stored in refrigerators to maintain their genetic purity.
C1: colony diameter in mm after one day of incubation; C3: colony diameter in mm after three days of incubation; T: the difference in time (day).The used experimental design was a completely randomized block with three replicates (plates) for each treatment.

Percent growth inhibition
The percent growth inhibition was calculated using the formula as per Edington, (1971): I (%) = CT/C×100 where: I: Percent growth inhibition; C: radial growth in control; T: radial growth in treatment.
In vitro antifungal activities of Trichoderma isolates against major pathogens (S. rolfsii and M. phaseolina) of groundnut plant in dual culture interaction.
The Trichoderma isolates were evaluated for their in vitro antifungal activities against two fungal pathogens of groundnut in dual culture.The antifungal activity against the facultative soil-borne fungi S. rolfsii, which was isolated locally from groundnut plants and M. phaseolina, obtained from Agharkar Research Institute, was determined in dual culture method.A 5 mm diameter mycelia block 7-day-old, removed from margin of young culture of Trichoderma isolate and of pathogen colonies, placed 3 cm apart near the edge on the surface of PDA medium.Three replicates of petri plates were incubated at 28°C, under continuous light, and inspected daily for approximately 7-8 consecutive days for mycoparatisim.The fungal colony was arranged in a manner by which it would meet 2-3 days after inoculation.The area of interaction of mycoparasite and the host was measured every 24 hours after contact (Dennis andWebster, 1971 a, 1971 b).A similar dual culture interaction was carried out for Trichoderma against M. phaseolina.The experiment was conducted as completely randomized design with three replicates in Petri plates.

Statistical analysis
The experiment was laid in complete randomized block design (CRBD) with three replicates.Data were expressed as mean value of three replicates.The values are mean ± SD.All the calculations were made by using a Microsoft Excel 2007 for data analysis.

The antifungal activities of Trichoderma isolate
The antifungal activity of T. viride was determined by testing their effect on radial growth rate.

Inhibition of radial growth
The antifungal activity of T. viride was determined by testing their effect on radial growth with dual culture method.The Trichoderma isolate inhibited the radial growth of S. rolfsii by 75% and M. phaseolina by 71.42% (Tab.1).Trichoderma viride showed significant inhibition of the test fungi S. rolfsii and M. phaseolina.

Mycelial growth rate (Average Linear Growth)
The average mycelial growth rate for three isolates and growth of isolate in dual culture are presented in (Tab.1).The highest colony diameter was 85.33 mm/day for Trichoderma viride followed by Sclerotium rolfsii 73.33 mm/day and Macrophomina phaseolina 70.33 mm/day.The efficacies of Trichoderma isolate to inhibit the growth of S. rolfsii and M. phaseolina in dual culture method was determined on PDA medium.Results are presented in (Tab.1).The percent inhibition by T. viride for S. rolfsii was 75% while for M. phaseolina the value was 71.42%.

The growth rate of fungal isolates in vitro
The results showed statistically significant difference in growth rate between the different isolates of the T. viride, S. rolfsii and M. phaseolina.T. viride showed a minimum of 4.00 days for the complete coverage of the medium, whereas S. rolfsii took 4.33 days and M. phaseolina 6.33 days, respectively.The T. viride's minimum time to complete coverage gives an edge over both S. rolfsii and M. phaseolina in the completion for food and space.
The fungal isolates T. viride, S. rolfsii and M. phaseolina exhibited colony diameter of about 24.5%, 20.5% and 11.5% respectively (Fig. 1).The bioagent T. viride inhibited growth of S. rolfsii by 75% and M. phaseolina by 71.42% in dual culture method over control (Tab.1).The maximum inhibition was observed by bioagent T. viride, It is well documented that Trichoderma spp. is mycoparasite (Elad and Chet, 1983) and has been widely used as antagonistic fungal agents.Several Trichoderma species are considered to be stoutly antagonistic to other phytopathogenic fungi (Coley-Smith, 1991;Harman, 1996).Their different mechanism of parasitism to other phytopathogenic fungi occurs by various mechanisms such as competition, antibiosis, mycoparasitism, induced resistance and inactivation of pathogen's enzyme.The direct mycoparasitic activity of Trichoderma species has been proposed as one of the major mechanism for their antagonistic activity against phytopathogenic fungi (Chet, 1990;Cruz, 1995).Results obtains in present investigation suggests competition by T. viride against pathogen being one of the main reason for biological control.Already the Trichoderma species are known to produce a number of antibiotics such as trichodermin, trichodermol, harzianum A and harzianolide (Claydon, 1991;Dickinson, 1995), which may help in reducing ill effects of harmful pathogens.Thus, T. virde was evaluated for their efficacy against pathogens S. rolfsii and M. phaseolina.
The present investigation reveals the rate of growth being faster by T. viride singly or in combination with pathogens in artificially controlled environment.This quick rate of growth may be helpful and beneficial to Trichoderma isolate as compared to plant pathogenic fungi in vitro in the competition for space and nutrients.
In our investigation we have showed that T. viride prevented infestation supposed to be incurred by pathogens S. rolfsii (75%) and M. phaseolina (71.42%) in controlled condition.In the present experiment Trichoderma isolate grew towards S. rolfsii and M. phaseolina colony and in case of T. viride + S. rolfsii as compared to T. viride + M. phaseolina.The antagonistic activity of T. viride against S. rolfsii and M. phaseolina was most likely due to mechanism of antibiosis and/or competition.
The growth rate of T. viride, S. rolfsii and M. phaseolina after 6 days of in vitro incubation showed 4 days for T. viride and S. rolfsii, whereas for M. phaseolina it took 3.66 days (Tab.2, Fig. 2).The present study observed almost similar findings to those of Inbar et al., (1996) who also reported a successful inhibition of Sclerotinia sclerotiorum by Trichoderma harzianum.

Discussion
Sclerotium rolfsii is the causal fungal pathogen that causes stem-rot on groundnut plants.The pathogen was collected from infected root and stem of groundnut from the fields of Pune district, Maharashtra, India.The pathogen Macrophomina phaseolina that causes charcoal rootrot was obtained from Agharkar Research Institute, Pune, Maharashtra, India.Trichoderma isolates were obtained from Department of Microbiology, Agricultural College of Pune, Maharashtra, India.The information in Tab. 1 illustrates the growth of fungal pathogen, individually and in combination with Trichoderma isolate.All three fungal

Tab. 1 .
Growth rate and dual culture technique using T. viride, S. rolfsii and M. phaseolina after 6 days of incubation in vitro value is mean of triplicate; ± = Standard deviation Abbreviations used: Tv = Trichoderma viride; Sr = Sclerotium rolfsii; Mp = Macrophomina phaseolina; PDA = Potato dextrose agar isolates showed variance in their growth on potato dextrose medium (PDA).The antagonistic growths of Trichoderma isolate on both S. rolfsii and M. phaseolina have been illustrated in Tab. 1.