Published on International Journal of Biology, Physics & Matematics
ISSN: 2721-3757, Volume 2, Issue 2, page 103 – 111
Publication Date: March 13, 2019
C. O. Ahonsi, A. G. Ogofure & A. O. Emoghene
Department of Microbiology, Faculty of Life Sciences, University of Benin, Ugbowo Campus
Nigerian Institute of Oil Palm Research (Nifor), Benin-Akure Road
Benin City, Edo State, Nigeria
The antimicrobial activity of neem leaf extract (Azadirachta indica) was investigated using agar welldiffusion methods on pathogens isolated from yam rot. The isolated bacterial pathogens; Pseudomonas species and Corynebacterium species and the implicated fungi pathogens; Aspergillus niger, Penicillum, Rhizopus and Trichoderma species were all susceptible to both the aqueous and ethanolic leaf extract of Azadirachta indica. The minimum inhibitory concentration (MIC) for Corynebacterium species was 0.25mg/ml for ethanolic extract and 1mg/ml for aqueous extract. Pseudomonas and Penicillium were more susceptible to the extract than other organisms isolated. Aspergillus niger, Penicillum and Trichoderma had similar MIC value of 0.25mg/ml for ethanolic extract and aqueous extract respectively. The effect of the extract varied with the type of solvent used, concentration of the leaf extract and the organisms on which it was tested on. Statistical analysis showed that there was no significant difference observed in aqueous and ethanol as extraction solvent p>0.05. The tremendous potential of the extract of neem (Azadirachta indica) in agriculture and health care was also discussed in details.The result of the pathogenicity test showed The intrinsic ability of some isolated fungal pathogens at causing significant rot of exposed yam tubers. The isolated fungal pathogens include Aspergillusniger PenicillumSpp RhizopusSpp TrichodermaSpp PseudomonasSpp corynebacteriumSpp The three plant material reduced the rot caused by the four organisms namely Aspergillusniger PenicillumSpp RhizopusSpp and TrichodermaSpp.
Keywords: Yam rot, bio-control, antimicrobial, bacteria, fungi, Azadirachta indica & neem.
In developing countries, the loss of crops due to pest, plant diseases and competitions from weeds is great. Pesticides/insecticides produced over the years aim to kill these pests in order to prevent these damage also tend to have adverse effects on humans in various ways most especially those manufactured from synthetic materials (Boadu.et al 2001)
It has been investigated by Mondali et al (2009) that large numbers of chemicals have been developed for the control of plant diseases. But due to growing awareness of the hazardous side effects of these chemicals, more and more emphasis is being given to the use of biocontrol agents. To this effect, major challenge is felt in the field of plant pathology to introduce some ecofriendly and safe alternative control strategies for agriculture which lead researchers to turn their attentions to plants and microorganisms as source of bio-control agents. As the sources of bio-control agent, ‘neem’ has already emerged at the top of the list of plants with the highest potential. This is no mistake when the National Research Council (1992) of USA gave an excellent account of this tree in their publication “Neem, tree for solving global problem”.
During the last three decades, neem has attracted the brains of the scientific community and has attained a place of pride in national and international scenario Bansal et al (2010). Not only neem is universally acknowledged as a ‘miracle tree’ but also ‘arista’ in Sanskrit – a word that means ‘perfect, complete and imperishable Muñoz-Valenzuela et al 2007
Neem that belongs to Mahogany family, Meliacea, harbors lots of species that have been the subject of botanical bio-control research. However, numerous studies have identified compounds within the neem plants as a reason for its extensive use in Asia and Africa subcontinent. Neem has been found to contain a vast array of biologically active compounds which are chemically diverse and have got an enormous therapeutic potential Bansal et al (2010).as well as medicinal and even more agrochemical properties since ancient times (AFD 2009)
The major bioactive compounds in neem leaves are salanin, nimbin, azadirone and azadirachtin Sadeghian et al (2007). (Indian neem tree), contains at least 35 potent biologically active principals of which nimbin and azadirachtin are the most active insecticidal ingredients and are present predominantly in seeds, leaves and other parts of the neem tree. Neem plant is known for its pesticide activity against more than 400 insect pests Siddiqui et al(2003) and a wide range of pathogens Amadioha (2000).The aim of this research is to investigate the in-vitro antimicrobial activity of neem (Azadirachta indica) leaf extract on plant pathogens using yam rot as a case study.
2. MATERIALS AND METHODS
2.1 Collection of materials
Leaves were collected from the neem (Azadirachta indica) tree at the Faculty of Education in the University of Benin, Benin City, Nigeria. The collected leaves were identified and authenticated by Dr. Akinnibosun of Department of Plant Biology and Biotechnology. The leaves were washed under running tap water to eliminate dust and other foreign particles as well as to clean the leaves thoroughly. The leaves were later allowed to dry. The chemical reagents for extraction were obtained from the Department of Chemistry, Faculty of Physical Sciences, University of Benin, Benin City, Nigeria. All glass wares were made from Pyrex in England.
2.2 Leaf extracts preparation
The completely shade (air) dried leaves were crushed and coarsely powdered by the appropriate machine. 20 g portion of the powdered plant leaves were soaked in solvents (80 ml of ethanol and aqueous water respectively) at ambient temperature for 24 hours under shaking condition. The extract was then filtered using Whatman filter and re-filtered. The filterate was kept in the freezer at -20 °C. The filtrate extracts can also be stored at 4 °C in air tight bottle (Maragathavalli et al., 2012).
2.3 Isolation of microorganism from yam rots
A rotten white yam tuber obtained from Oba market was taken as a sample and stored in a cool dry place. Serial dilution was carried out using six (6) serial test tubes. 1 g from different part of a rotten yam was weighed and pulverized properly using motar and pestle. Little quantity of sterile (distilled) water was added to make a stock culture and properly mixed. 1 ml of aliquot obtained from stock culture was added to tube 1 containing 9 ml and mixed. The same is repeated for tube 2-6. Pour plate method was employed for culturing on nutrient agar (NA) and potato dextrose agar (PDA) for bacteria and fungi isolation respectively. Petri dishes containing NA were incubated at 37 °C for about 24 hours while PDA plates were incubated at room temperature (28 ± 2 °C) for 2-3 days.
2.4 Characterization of Bacteria Isolate
Gram staining technique as well as biochemical tests which include catalase, oxidase, citrate, sugar fermentation test and coagulase test. Motility test was also carried out.
2.5 Antimicrobial assay for fungi and bacteria
Agar well diffusion method was adopted for the assay of bacteria and fungi. The technique of (Okigbo et al., 2006) was adopted. Media used for the test remain nutrient agar for former and potato dextrose agar for later. Ethanol extract filtrate was allowed to concentrate using an oven at a regulated temperature. Warm distilled water was used to reconstitute the extract to ensure proper mixing. Ethanol was not used to reconstitute the concentrate because of its effect on microorganism which could interfere with the result. Water extract filtrate was concentrated to a certain level and used for the sensitivity test. Six sterile test tubes were prepared each containing 1 ml of distilled water except tube 0 which serves as stock for the extract. 1 mg of the aliquot extract was taken from stock extract in tube 0 and dispensed to tube 1 that already has 1 ml of distilled water and mixed properly. 1 mg of the extract from the tube 1 was dispensed into tube 2 and mixed. The same was repeated for tube 3-6.
The inoculum was introduced aseptically to the petri dishes using cotton board. 2-3 drops of the extract of the same dilution as well as the same solvent was introduced into the three dug holes. For plates containing the same set of inoculum, flood the holes with extract of the same solvent but varying extract dilution in a step wise manner. The plates were then incubated in petri dishes containing an extract at about 37 °C for 24 hours.
2.6 Minimum inhibitory concentration
The minimum inhibitory concentration values were determined according to methods of (Maragathavalli et al., 2012) involving broth dilution assay of microdilution assay. Varying concentrations of the extracts (1.000 mg/ml, 0.500 mg/ml, 0.250 mg/ml, 0.125 mg/ml, 0.06250 mg/ml and 0.03125 mg/ml) were prepared. Controls were equally set up by using distilled water. The tube with least concentration of extract without growth after incubation was taken and recorded as the minimum inhibitory concentration.
2.7 Pathogenicity test
The yam tubers were prepared for pathogenicity tests following techniques described by(Okigbo et al., 2006 and Okigbo et al., 2005)Ten fresh healthy tubers of yam were washed with tap water and distilled water respectively and thereafter sterilized with 70 % ethanol. Cylindrical discs (4 mm) were removed from the tubers with a sterile 4 mm cork borer. The 4 mm discs of 5 days old cultures of the isolates were used to plug the holes created in the tubers respectively. The discs of the tuber in the cork borer was replaced and then sealed with Vaseline jelly to make it air tight. Sterile PDA disc used in place of the culture discs served as the control. This was done for all the isolates obtained in pure culture. Triplicates were made for each organism. The inoculated tubers and the control were each enclosed in a sterile polyethylene bag and incubated for eight days at room temperature (25-37 °C). A micro-humid environment was provided by enclosing sterile water soaked aseptic cotton wool in each set up.