Nusrat Jahan
Ex-student
Agrotechnology discipline, Khulna University
Sabiha Sultana
Assistant Professor
Agrotechnology discipline, Khulna University
S. K Adhikary
Professor
Agrotechnology discipline, Khulna University
Sanzida Rahman
Ex-student
Agrotechnology discipline, Khulna University
Suraiya Yasmin
Ex-student
Agrotechnology discipline, Khulna University
Growth performance, Trichoderma harzianum, Different culture media
Plant Protection Laboratory of Agrotechnology Discipline, Khulna University
Pest Management
An experiment was conducted in the Plant Protection Laboratory of Agrotechnology Discipline, Khulna University, to evaluate the performance of different media for growth of T. harzianum. An isolate of T. harzianum was collected from the preserved isolates of Bangladesh Agricultural research Institute (BARI), Joydebpur, Gazipur. PDA was prepared following the standard procedure (Anonymous, 1968). After preparation of media, pH of the medium was adjusted to 6.00 by adding 1% HCl using pH meter. After melting the prepared medium was sterilized in an autoclave at 121°C temperature for 20 minutes.
For the Multiplication of T. harzianum PDA medium was poured in sterilized petridishes, 20ml in each. A 5mm block of the 3 days old pure culture of T. harzianum was placed upside down at the center of each plate. The block was cut with the help of a flame sterilized cork borer (5mm diameter). The inoculated petridishes were kept in the growth chamber at 27±2ºC temperature. All the works were done under aseptic condition.
For the Preservation of T. harzianum Sterilized PDA medium were poured into sterilized test tubes, 10ml in each. Then the test tubes were sterilized in an autoclave at 121°C temperature for 20 minutes. After autoclaving, slanting of test tube was done at 45° angles to increase the surface area of the medium in the test tube. 7 days old fungal hyphae with the help of a inoculation niddle were placed in the test tube. After inoculation test tubes were kept in the growth chamber at 27±2ºC temperature. Preparation of Different Media Potato Dextrose Agar (PDA) PDA was prepared following the standard procedure (Anonymous, 1968) and the pH was adjusted to 6.00 using pH meter with the help of 1% HCl. The medium was then sterilized in an autoclave at 121°C temperature for 20 minutes. Modified Potato Dextrose Agar (MPDA) Modified PDA was prepared by using 125gm potato and 15gm dextrose instead of 200g potato and 20g dextrose respectively. The pH of the medium was adjusted to 6.00 using pH meter with the help of 1% HCl. The medium was then sterilized in an autoclave at 121°C temperature for 20 minutes. Water Agar (WA) Water agar was prepared following the standard procedure (Anonymous, 1968). 1000ml distilled water was boiled at first. After boiling it was poured into a beaker. The beaker is then placed on a heater with magnetic stirrer to mix 15g agar and then adjusted to 1000ml by distilled water. The pH of the medium was adjusted to 6.00 using pH meter with the help of 1% HCl. The medium was then sterilized in an autoclave at 121°C temperature for 20 minutes.
Carrot agar (CA) 400g carrot was cut into slice and boiled in 1000ml distilled water. After boiling it was sieved into a beaker. The beaker is then placed on a heater with magnetic stirrer to mix 20g agar and then adjusted to 1000ml by distilled water. The pH of the medium was adjusted to 6.00 using pH meter with the help of 1% HCl. The medium was then sterilized in an autoclave at 121°C temperature for 20 minutes. Cornmeal agar (CMA) 20g cornmeal was weighted on an electric balance and mixed with 1000ml boiling water slowly. After mixing, it was sieved into a beaker. The beaker is then placed on a heater with magnetic stirrer to mix 15g agar and then adjusted to 1000ml by distilled water. The pH of the medium was adjusted to 6.00 using pH meter with the help of 1% HCl. The medium was then sterilized in an autoclave at 121°C temperature for 20 minutes. Pouring of Media In all cases of media 20ml medium was poured in each petridish. where as five plates for each medium. Inoculation and Incubation Advanced hyphae of 3 days old culture was used for inoculation. A 5mm block of the mycelium was cut with flame sterilized cork borer (5mm). The mycelial blocks were taken from the edge of the colony. Each mycelial block was placed upside down at the centre of each plate. Five replicated plates were used for each medium. The inoculated petridishes were kept in the growth chamber at room temperature (27±2°C) and 90% relative humidity (RH) until the mycelia touch the edge of petridishes. Measurement of Average Linear Growth Rate (ALGR) of T. harzianum on Different Growth Media After 3 days of incubation, linear growth (mm) of T. harzianum was recorded. Linear growth measured by averaging three diameters taken from each colony. Average linear growth rate was measured by the following formula (Aneja, 1993 and Elad et al., 1981). ALGR (mm/day) = (C3-C0)/3 Where C3= Colony diameter after 3 days of inoculation C0= Initial colony diameter of inoculation.
Colony Characteristics of T. harzianum After 4 days of incubation, different colony characters such as surface, color, margin, texture and hyphal thickness was observed visually in each different media. Measurement of Fresh Weight and Dry Weight of T. harzianum on different media For the measurement of fresh weight and dry weight of T. harzianum was grown in 100ml of potato dextrose broth (PDB), modified potato dextrose broth (MPDB), water broth (WB), carrot broth (CB), cornmeal broth (CMB) in 250ml conical flask. These media were inoculated with a 5mm block of 3 days old culture of T. harzianum grown on PDA. The inoculated conical flasks were kept in the growth chamber at 27±2°C temperature and 90% RH. The conical flasks were shaked at 24 hours of interval till 14 days of inoculation to inhibit colony formation on the surface of growth. After 14 days inoculation the fungal mass in each conical flask was separated by filtration using Whatman paper No. 1. The filtrates were discarded and the fungal biomass on the filter paper was air dried at room temperature for 24 hours. Then the fresh weight (mg/100ml) was measured with electric balance. The fungal biomass of all treatments was oven dried at 70°C temperature for 72 hours. Then the oven dried fungal biomass was weighted (mg/100ml) to have the dry weight. Experimental Design and Data Analysis Experimental Design was Completely Randomized Design (CRD) with five replications and data was analyzed statistically using MSTAT-C computer program and means were compared following Duncan’s Multiple Range Test (DMRT).
IOSR Journal of Agriculture and Veterinary Science (IOSR-JAVS) e-ISSN: 2319-2380, p-ISSN: 2319-2372.Volume 3, Issue 4 (May. - Jun. 2013), PP 44-50 www.iosrjournals.org
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