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Antimicrobial and Cytotoxic Activity of Macrophomina Phaseolina Isolated from Gummosis Infected Citrus Reticulata.

RUBAL C. DAS
Department of Biochemistry and Molecular Biology, University of Chittagong, Chittagong-4331, Bangladesh

ROBIUL HASAN BHUIYAN
Department of Biochemistry and Molecular Biology, University of Chittagong, Chittagong-4331, Bangladesh

MD. GOLAM KABIR
Department of Biochemistry and Molecular Biology, University of Chittagong, Chittagong-4331, Bangladesh

RAJIB BANIK
Department of Biochemistry and Molecular Biology, University of Chittagong, Chittagong-4331, Bangladesh

Abstract/ Executive Summary:

Macrophomina phaseolina is one of the pathogenic organisms of gummosis disease of orange tree (Citrus reticulata). The pathogen was identified from the observation of their colony size, shape, colour, mycelium, conidiophore, conidia, hyaline, spore, and appressoria in the PDA culture. The crude chloroform extracts from the organism showed antibacterial activity against a number of Gram positive and Gram-negative bacteria. The crude chloroform extract also showed promising antifungal activity against three species of the genus Aspergillus. The minimum inhibitory concentration (MIC) of the crude chloroform extract from M. phaseolina against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Shigella sonnie were 128 μgm, 256 μgm, 128 μgm and 64 μgm/ml respectively. The LD₅₀ (lethal dose) values of the cytotoxicity assay over brine shrimp of the crude chloroform extract from M. phaseolina was found to be 51.79 μgm/ml.

Keywords: Gummosis disease, Macrophomina phaseolina , Antibiotic activity, MIC, Cytotoxicity

Location: Department of Biochemistry and Molecular Biology, University of Chittagong, Chittagong

Start Date:

End Date:

Program Area: Pest Management

Commodity: Fruit

Objectives:

1. The study was designed to evaluate the antimicrobial activity and cytotoxicity of the chloroform extract of Macrophomina phaseolinsa.

Methodology:

The fungal materials were isolated from the ‘gummosis’ gum exudes of blister containing gum pockets, usually located on the trunk. The wood beneath the blister shows a pink-orange color collected from different orange fields of Agricultural Research Institute (BARI), Pahartali, Chittagong, Bangladesh. The specimens were collected in November-December. After collection, the samples were brought to the laboratory and detail observations on the symptoms of the gummosis disease infected orange fruits were made. After proper sterilization, 5-6 diseased orange fruits were incubated in humid chamber for 7-10 days at 28±2⁰C. The stock cultures were maintained on Potato Dextrose Agar (PDA). Sub-cultures were made at intervals of every 15 days. Examinations were made within 10-15 days under stereoscopic binocular microscope. Culture study was carried on with the culture obtained from the single spore. Isolation of pathogen - Isolation of pathogen from the different host fruits was made following direct method, plating method and single spore were then isolated. A suitable portion of culture of pure organism from PDA plate was subjected to microscopic examination under a stereoscopic binocular microscope. Isolation of the organism from artificially infected fruits with the organism - Previously surface sterilized and wounded by sterilized needle fruits were subjected to spraying by the conidial suspension of the purified organism with automizer. The inoculated fruits were transferred into germ-free full humidly desiccators and incubated at 28±2⁰C for 5-7 days. Observation was made after 5-7 days when fruits had developed characteristic lesions and compared with the naturally developed previously recorded symptoms. Re-isolation of the pathogen was made from the artificially infected fruits following the usual procedures described before. The morphological characters of the re-isolated organism were compared with the original isolates by which the fruits were inoculated. Isolation of antibiotic from the culture medium - Potato Dextrose (PD) Broth was used for antibiotic production. PDA slants were inoculated with spores of the organism and were allowed to grow until sporulation occurred. Mature spores were transferred to conical flasks containing 25 ml of sterilized PD broth medium to make spore suspension and again aseptically transferred to large culture flasks (500 ml) each containing 300 ml of sterile PD broth medium. These were incubated at 28±2⁰C for 10 days. After 10 days of incubation, the medium in the flasks turned into yellow colour with thick, uniform mat on the surface. The liquid was then separated from its mycelial mat and filtered through a fresh piece of cotton, and then Whatman filter paper 2. The filtrate was preserved at +4⁰C by adding 2/3 drops of toluene as preservative for the extraction of the antibiotics (metabolites). 100 ml of the culture filtrate was taken in a separating funnel. This was shaken for 30 minutes with 30 ml of chloroform for the first time. Then the lower layer having the chloroform extract was separated and kept in a suitable beaker. The remaining medium in the separating funnel was treated in the same manner and collected. The chloroform fraction thus obtained was evaporated under reduced pressure in a Rota-evaporator at 45⁰C, until a yellowish solid mass was obtained and the weight of the solid extract was measured. Determination of antibacterial activity - The in vitro sensitivity of the crude chloroform extract was determined by disc diffusion method against eight pathogenic bacteria (three Gram positive and five Gram negative). The test organisms used were Bacillus cereus BTCC 19, B. subtilis BTCC 17, B. megaterium BTCC 18, Staphylococcus aureus ATCC 6539, Escherichia coli ATCC 25922 , Salmonella typhi, AE 14613, Vibrio cholerae and Shigella sonnie ATCC 25931. Determination of antifungal activity - For fungal inoculums PDA pour plates were prepared. At the center of these 5 days old test fungi were transferred and incubated at (25±2) ºC. After 5 days of incubation they were ready for use. The poisoned food technique was used to screen for anti-fungal activity. Determination of Minimum Inhibitory Concentration (MIC) of crude chloroform extract - ‘Serial dilution technique’ was followed using nutrient broth medium to determine the MIC value of the compound against Bacillus subtilis , Shigella sonnie, Staphylococcus aureus and E. coli. Cytotoxicity Test (Brine shrimp lethality assay) - Cytotoxicity was performed by using shrimp lethality assay. The percentage lethality was determined by comparing the mean surviving larvae of the test and control tubes. LD₅₀ value was obtained from the best-fit line plotted concentration versus percentage of lethality.

Source of Information: The Chittagong Univ. J. B. Sci.,Vol. 5(1 &2): 125-133 , 2010

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

In the brine shrimp lethality bioassay, the chloroform extract of organism showed positive results, indicating that the extracts are biologically active. The mortality rate of brine shrimp nauplii was found to be increase with the increase of concentration of the sample and a plot of concentration versus percent mortality on graph paper gave an almost linear correlation between them. They had a notable antagonistic activity over both Gram positive and Gram-negative bacteria and the pathogen may play a vital role in combating against antibiotic resistant bacteria and fungi.

Knowledge Management: Journal