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Bio-control of Lentil Wilt Disease by Trichoderma Harzianum

Ferdous Akter
Department of Agronomy and Agricultural Extension, University of Rajshahi, Rajshahi-6205, Bangladesh

Md. Giush Uddin Ahmed
Department of Agronomy and Agricultural Extension, University of Rajshahi, Rajshahi-6205, Bangladesh

M. Firoz Alam
Department of Botany, University of Rajshahi, Rajshahi- 6205

Md. Jahanggir Alam
Assistant Professor, Department of Botany, Lalmonirhat Govt. College, Lalmonirhat, Bangladesh

Nazma Begum
Assistant Professor, Dept. of Botany, New Degree Govt. College, Rajshahi, Bangladesh

Abstract/ Executive Summary:

Fusarium caused vascular wilt of lentil is a serious biotic threat, sometimes lowered the yield severely as pathogen resides the field always for their wide mode of nutrition. Trichoderma initiated bio-control was well accepted for their beneficial arsenal rather chemical hazard. Assessment of antagonism of T. harzianum against F. oxysporum by in-vitro and ex-vitro to reduce lentil wilt was the goal of this study. In vitro antagonistic study shows that T. harzianum IMI 392432 restricted the growth of F. oxysporum most in both method of dual culture but inhibition was highest (mean PIRG- 73%) in method -2 wherein test fungi paired in two opposite poles on PDA plate at 28±2°C temperature. 100% Trichoderma metabolites containing culture filtrate in ThFDA media totally prevent pathogen’s growth. It is assumed that the antibiotic compound produced by the T. harzianum turns poison substrate for the respective pathogen. Tricho- compost treated lentil plant showed a maximum reduction of wilt incidence compared to T. harzianum spore suspension and other combinations in ex-vitro pot study. Wilt disease appeared most (63%) in combining treatment of conventional compost + Fusarium spore suspension. Population density of both test fungi in conventional compost is high and the maximum population of T. harzianum found in conventional compost +Tricho-compost application

Keywords: Biological control, Antagonism, Filtrate, Tricho-compost, Biomass

Location: Agronomy and Agricultural Extension, University of Rajshahi.

Start Date:

End Date:

Program Area: Pest Management

Commodity: Lentil

Objectives:

Understanding the efficacy of T. harzianum and its culture filtrate to prevent the growth of pathogen in vitro was the prime initiative. T. harzianum was evaluated here against F. oxysporum as a bio- control tool to check devastating lentil wilt in pot cultivation.

Methodology:

Wilted lentil plants were collected from an infested field to laboratory. After washing and sterilization, the infected roots were transferred to a sterile Potato Dextrose Agar plate supplemented with streptomycin sulfate to prevent bacterial growth and incubated at 28±2°C for 7 days. Appeared fungal mycelium surrounding on root pieces transferred to the fresh plate soon and perpetuated the culture to obtain pure culture. The disease-causing agent then allowed to sporulate at 12 h photoperiod regime need for identification. The pathogen was primarily identified based on morphological characteristics such as type, shape and color of sexual or asexual spore assisted by ultra-microscope. The pathogenicity of the fungus F. oxysporum f. sp. lentis was confirmed using lentil cv. BARI Masur-7, under pot culture conditions in net house at the field of Agronomy and Agricultural extension, in the University of Rajshahi. Three Isolated strains of T. harzianum IMI 392432, IMI 392433, IMI 392434 (Rahman et al., 2011) were collected from the Microbiology & Biotechnology laboratory in the Department of Botany, University of Rajshahi. T. harzianum isolates cultured on Potato Dextrose Agar medium prepared following Anonymous standard procedure. Single spore culture of T. harzianum continued to get pure culture and stored for further use. Pure stock cultures were used to make conidial suspensions for soil inoculation of antagonist and plant pathogen were prepared following the procedure of Erskine and Bayaa (1996), El-Hassan (2004). Two techniques were followed to implement dual culture technique (Dhingra and Sinclair, 1995) of test fungi. In Method-1, 5 mm diam. mycelial disc of test antagonist (T. harzianum IMI 392432, 392433 & 392434) taken from 3 days old culture and was placed to pair against the same sized mycelial disc of F. oxysporum about 30mm from periphery on PDA plate of 90mm diam. The pathogen and antagonist disc placed at equal distances from the periphery of the Petri plate. The plates were incubated at 25±2°C in light condition for the days need to overlap the pathogen colony. In Method-2, 5mm- diam. mycelial plug of antagonist fungi was placed and paired with F. oxysporum at the opposite end in PDA plate or in the peripheral boundary. The plates were incubated at 28 ±2°C in light condition for the days need to overlap the pathogen colony. A single plug of same-sized antagonistic and test fungi alone was placed on PDA plate that served as control in both methods.

The presence and role of antifungal metabolites secreted by T. harzianum (IMI 392432) in liquid culture filtrate and their effect on pathogen (F. oxysporum) for reduction of biomass weight and mycelial growth was tested here. In this regards T. harzianum cultured primarily in 250ml culture flask containing 100ml Potato Dextrose Broth (PDB) medium and incubation was done in rotary shaker at 150 rpm with 25⁰C temperature by 24 hours rotation for 30 days. Fungal culture was filtered through Whatman No.1 filter paper and collect the broth. The fine Fungal mats were then harvested by centrifugation of culture broth at 4100×g for 10 minutes. Discarding the pellet, supernatant broth further filtered using a sterile What man micro GD/X syringe filter (Whatman International Ltd., New Jersey, USA) with a pore size of 0.22 µm. Fungus free filtrate confirmed finally by culturing them on PDA media and store in the refrigerator at 4°C temperature for use. Two types of filtrate media were prepared for antifungal study (i) ThFDA (T. harzianum Filtrate Dextrose Agar) medium were prepared using four concentrations of filtrates (25%, 50%, 75%, 100%) supplemented with 2% dextrose and 2% agar (wt/vol). (ii) ThFDB (T. harzianum Filtrate Dextrose Broth) was prepared using the same concentration of filtrates supplemented with only 2% dextrose (wt/vol). Both filtrate media were autoclaved at 121°C for 10 min. ThFDA media dispense in Petri-plates and 5mm diam. disc of three days old F. oxysporum culture place at the center of medium. PDA plates with no filtrate seeded with a plug from old F. oxysporum served as the control. The plates were incubated in a growth cabinet at 25±2°C temperature. After 7 days, the growth inhibition was analyzed by measuring the radial growth of the F. oxysporum colony following Edington et al (1971) formula mentioned before. ThFDB were dispense in 250ml conical flask at the rate of 100ml and 2 plugs of 5mm diam. mycelial disc of F. oxysporum seeded in flask which inoculated with rotary shaker (150 rpm) at 25±2°C temperature for 10 days. Sterilized PDB media seeded with the same plug of F. oxysporum culture similarly as a control to compare the filtrating effect. Biomass growth of the pathogen in ThFDB and control media, assessed by harvesting with centrifugation at 4100×g for 10 minutes. Fresh weight of test pathogen measured after 12-hour air drying at room temperature and dried in the oven at 50°c for 6 hours to get dry weight for each. Completely Randomized Design (CRD) with five replications were followed as experimental design in growth measurement of fungus from culture media as well as pot grown plant and data were analyzed statistically using MSTAT-C computer program and means were compared following Duncan’s Multiple Range Test (DMRT).

Source of Information: International Journal of Agriculture, Environment and Bioresearch, Vol. 3, No. 06; 2018

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

T. harzianum is quite capable to hinder the growth of F. oxysporum in agar plates as well as soil inoculation. Therefore, treating pathogen inoculation is the cause of the highest seedling wilt which minimized by T. harzianum at Tricho-compost application in this study.

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