ASSESSMENT OF THE ANTIFUNGAL ACTIVITY OF Cymbopogan citratus (Lemon grass) LEAF EXTRACT AGAINST SOME PHYTOPATHOGENS

ince the beginning of mankind, nature has been a source of medicinal agent for thousands of years. A plant pathogen is an infectious organism that is capable of causing disease to plants. This research work was aimed at assessing the antifungal activity of Cymbopogan citratus leaf extract on some phytopathogenic fungi isolated from agricultural soils. Aspergillus niger , Aspergillus tamari and Mucor circinelloides were isolated from an agricultural soil. Aspergillus tamari displayed highest susceptibility to the 50% ethanolic extract of the plant with a mycelial radial growth inhibition measurement of 18.25 ± 0.25 mm while Mucor circinelloides displayed the least susceptibility to the 100% ethanolic extract with a mycelial radial growth inhibition of 45.25 ± 0.25 mm. The results were significantly different from the controls (63.25 ± 0.75 mm, Aspergillus niger ; 43.25 ± 1.25 mm, Aspergillus tamari and 63.75 ± 0.75 mm, Mucor circinelloides ). In the same vein, the aqueous extract also displayed appreciable antifungal activity. The radial growth inhibition of the aqueous extract ranged from 24.25 ± 0.25 mm (25%, Mucor circinelloides ) to 34.50 ± 0.50mm (100%, Aspergillus tamari ). These were significantly different from the controls. This Study has shown that lemon grass leaf extract possesses antifungal activities to control phytopathogens, from the result obtained, it can be stated that lemon grass can be exploited as an alternative to chemical fungicides in combating resistance amongst phytopathogens.


INTRODUCTION
Cymbopogan citratus (lemon grass) of the Poaceae family is a monocotyledonous aromatic perennial germinal plant which is tall with slender sharp-edged green leaves pointed at the apex, now cultivated widely in other tropical regions and warm temperate but native to tropical Asia (Bleasel et al., 2002).In Nigeria, the use of Lemon grass cannot be overemphasized.It is used for malaria therapy, stomach discomfort and when combined with two or more plants are more effective for malaria therapy.Research carried out has revealed that Cymbopogan citratus (lemon grass) has both antibacterial and antifungal properties.Benefits of the oil which is also used as an antifungal agent are its use as pesticides and preservatives (Tzortzakis and Economakis, 2007).Others include making of soaps, creams, candles, detergents and candles.Continuous evolution of microbial resistance has led to the search for novel and antimicrobial compounds.Plant extracts are employed globally for their antiviral, antibacterial and antifungal activities (Odugbemi, 2006).Secondary metabolites which are produced as by-products, activation of phytochemical constituents as well as provision of food, fuel, shelter are other reasons for the use of plant extracts.Benefits of medicinal plants are verification of pharmacological effects and anti-infectious agents (Ushimaru et al., 2007).
Plant pathogens such as Mucor circinelloides, Aspergillus niger, Aspergillus tamari are have been implicated severally in plant diseases (Webber et al., 2014).They cause rot disease in fruits, vegetables and medicinal plants (Snowdon, 1990).Fungicides have been used in the control in the management and control of post-harvest fungal rots.However, new pathogen races which are harmful to the environment and human health.As an alternative to synthetic fungicides plant extract has been proposed due to their acceptability because they are believed to be less hazardous than their chemical counterparts (Shah et al., 2011).The study was thus aimed at determining the antifungal activity of lemon grass leaf extracts (aqueous and ethanolic) against Mucor circinelloides, Aspergillus niger and Aspergillus tamari.

METHODOLOGY COLLECTION OF PLANT MATERIALS
Cymbopogan citratus (lemon grass) leaves was obtained from a Garden in the University of Benin, Ugbowo Campus, Edo State, Nigeria, authenticated at the Department of Plant Biology and Biotechnology.They wereassigned a voucher number (UBH-C451).The leaves were air dried, macerated using a laboratory pestle and mortar and sieved with a 1 mm sieve.The powdered leaves were kept in bottle for further analysis.

COLLECTION OF SOIL SAMPLE
Agricultural soil samples, from which the test isolates were to be isolated from, were collected from three different farmlands at Ekosodin community, Edo State, Nigeria.At each location soil sample was taken at a depth of 2-15cm and homogenized.The samples were then taken to the laboratory using a sterile polyethylene bags (Rauha et al., 2000).

PREPARATION OF CULTURE MEDIUM
The culture medium used (Potato Dextrose Agar) in this study were prepared aseptically according to the manufacturer's instructions (Cheesbrough, 2000).

ISOLATION OF FUNGI
Serial dilution plate method was used for the isolation process.Soil dilutions were made by suspending 1g of soil of each sample in 10ml of sterile distilled water.Dilutions of 10 3 , 10 4 and 10 5 were used to isolate fungi in order to avoid over-crowding of the fungal colonies.1ml of the suspension of each concentration was added to sterile petri dishes, in triplicates of each dilution, containing sterile potato dextrose agar medium.Chloramphenicol (250mg) in solution was added to the medium for preventing bacteria growth, before pouring to Petri plates.The plates were then incubated at 28 ± 2 0 C for 4-7 days.Organisms were easily isolated because they form surface colonies that were well dispersed particularly at higher dilutions.

IDENTIFICATION OF FUNGAL ISOLATES
The colony growth parameters which included length and width of the colony, the presence of absence of aerial mycelium, the colour, wrinkles, furrows and any other pigment production were the macro morphological characters evaluated.The fungi were identified with the help of standard procedure and relevant literature (Nagamani et al., 2006).

PREPARATION OF AQUEOUS EXTRACT
The plant's aqueous extract was made by weighing 200g of the powder on a scale and soaking it in 500ml of distilled water in a 1L conical flask.After being agitated for 30 minutes, it was allowed to stand for 48 hours.The extract was filtered twice: first through a fine cloth and once more using Whatman filter paper.Until it was needed, the extract was kept in a container with a label and a tight seal, and it was kept refrigerated at 4 o C according to the methods of Rauha et al. (2000).

PREPARATION OF ETHANOLIC EXTRACT
10g of lemon grass leaf powder that had been air dried was soaked in a container containing 1000 ml of ethanol solvent.The mixture was well mixed and left at room temperature for 24 hours.The solution was first filtered with a muslin cloth and then again using Whatman filter paper.The filtrate obtained was concentrated by complete evaporation of solvent in a water bath at 50 o C. Solution was stored at 4 o C after collecting in sterilized bottles until required.This was in consonance with the methods of Rauha et al., (2000).

ANTIFUNGAL ASSAY
Two (2) ml of the various extract concentration was poured into Petri dishes and then 18ml of PDA medium was added to it.A 5mm diameter agar disc bearing the hyphae of the fungus from a 7-day old colony grown on potato dextrose agar medium was transferred to the center of each Petri dish.This process was repeated for all the fungus and was replicated.Inoculated Petri dishes were incubated at 28 ± 2 0 C. Media inoculated with pathogen alone served as negative control.The average diameters of the fungal mycelia were measured on the 7th day of incubation and percentage of mycelial radial growth inhibition was calculated according to the formula below.

Percentage mycelial radial growth inhibition
Where; R= Linear growth of fungus on control plates r= Linear growth of fungus on petri dishes with lemon grass extract (Bahkali et al, 2016).

DATA ANALYSIS
All data were analyzed using the ANOVA statistical method with Statistical Package for Social Scientists (SPSS, version 21.0) and were reported as means ± standard errors of replicate measurements.Significant was defined as a P˂0.05 value.The means were further analyzed using Duncan test (Ogbeibu, 2005).

RESULTS
From the results of this study, lemon grass leaf extract was able to inhibit the growth of the tested fungi.Aspergillus niger displayed a higher susceptibility to the ethanolic extract than aqueous extract while for the aqueous extract, the susceptibility of the organisms varied with concentration.Cultural and microscopic characteristics of the fungi isolated from the agricultural soil used in this study are shown in Plate 1A, 1B, 2A, 2B, 3A and 3B.
Table 1 shows the cultural and morphological characteristics of fungal isolate.The result reveal the presence of Aspergillus niger on PDA as black mycelia growth with yellow growing edges plate while the microscopic view revealed that the conidia shape is globules and the texture of conidia is pigmented, Mucor circinelloides displayed white cottonlike colony on Potato Dextrose Agar plates.Colonies of Mucor circinelloides showed non-septate hyphae, the shape of the conidia is globules having an ellipsoidal texture and Aspergillus tamari appeared as yellow mycelia growth with white growing edges on potato dextrose agar plate while the microscopic view revealed that the conidia was globule shaped and pigmented.

DISCUSSION
In order to manage plant diseases, synthetic fungicides are presently the main tool employed.The public's misperception about the usage of synthetic pesticides, fungal disease resistance to fungicides, and the expensive expense of developing new compounds need alternate control strategies.Since they frequently have low mammalian toxicity, minimal environmental impact, and widespread acceptability among the general public, the applications of plant-derived compounds as disease control agents have been researched (El-Mougyet al., 2004).In this study, the antifungal activities of Lemon grass against Aspergillus niger, Aspergillus tamarii and Mucor circinelloides was investigated.The aqueous extract of Lemon grass had a higher antifungal activity against Aspergillus niger and A. tamarii, probably because the phytochemicals are water soluble or some phytochemicals with antifungal properties may have been destroyed by the extracting solvent, in agreement with the study conducted by Diba et al. (2007).It is also worthy of note that the antifungal activities of the lemon grass extract used in the study were not concentration dependent in line with the studies of Hadi and Kashefi (2013).The aqueous extract was also observed to show better effect against the test organisms compared to the ethanolic extracts.
Furthermore, it was observed that the ethanolic extract of Lemon grass demonstrated antifungal effect against the test isolates, though not significantly different from the inhibitory effect of the aqueous extract, but were significantly different from the controls especially at 100% concentration.The antifungal activity of lemon grass extracts against the test fungi in this study, maybe due to the presence of bioactive compounds such as tanins, flavonoids, saponins and acidic PH as opined by Hameed et al. (2017).

AJHSE-CONF-2023 4(1)
ORIBHABOR & IYEKEKPOLOR | 07 The study reveals and confirms the fungi toxic potentials of ethanol and aqueous water extracts of lemon grass at different concentrations, the plant extract could be further developed to produce natural fungicides.

CONCLUSION
The results obtained from this study indicate that Lemon grass extract possess antifungal activities against Aspergillus niger, Aspergillus tamarii and Mucor circinelloides in vitro.Lemon grass extract can serve as an eco-friendly alternative to combat the growing resistance development in pest.Further studies should however be conducted to ascertain the effectiveness of these natural fungicides in vivo.
Cultural and microscopic characteristic of Aspergillus niger A: 7-day old pure culture of Aspergillus niger B: Photomicrographs of Aspergillus niger.The black arrow shows the mycellia and red arrow shows globose phialides 2A 2B Plate 2A and B: Cultural and microscopic characteristic of Aspergillus tamarii A: 7-day old pure culture of Aspergillus tamarii B: Photomicrographs of Aspergillus tamarii.The black arrow shows the globose conidia and red arrow shows mycelia.and B: Cultural and microscopic characteristic of Mucor circinelloides A. 7-day old pure culture of Mucor circinelloides B. Photomicrographs of Mucor circinelloides

Table 1 :
Cultural and microscopic characterization of fungal isolate

Table 2 :
Antifungal activity of the Lemon grass ethanolic extract demonstrated by the mycelia radial growth inhibition (mm)

Table 3 :
Antimicrobial activity of the Lemon grass aqueous extract demonstrated by the mycelia radial growth inhibition (mm)