alexa Growth Inhibition Potentials of Leaf Extracts from Four Selected Euphorbiaceae against Fruit Rot Fungi of African Star Apple (Chrysophyllum albidum G. Don)
ISSN: 2157-7471
Journal of Plant Pathology & Microbiology

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Growth Inhibition Potentials of Leaf Extracts from Four Selected Euphorbiaceae against Fruit Rot Fungi of African Star Apple (Chrysophyllum albidum G. Don)

Ilondu EM1* and Bosah BO2

1Department of Botany, Faculty of Science, Delta State University, Abraka, Nigeria

2Department of Agronomy, Faculty of Agriculture, Delta State University, Asaba Campus, Nigeria

*Corresponding Author:
Ilondu EM
Department of Botany
Faculty of Science
Delta State University, Abraka, Nigeria
Tel: +2348036758249
E-mail: [email protected]

Received date: September 07, 2015; Accepted date: October 07, 2015; Published date: October 11, 2015

Citation: Ilondu EM, Bosah BO (2015) Growth inhibition potentials of Leaf Extracts from Four Selected Euphorbiaceae against Fruit Rot Fungi of African Star Apple (Chrysophyllum albidum G. Don). J Plant Pathol Microb 6:306. doi:10.4172/2157-7471.1000306

Copyright: © 2015 Ilondu EM, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

The efficacy of ethanolic leaf extracts from Phyllanthus amarus, Euphorbia hirta, Euphorbia heterophylla and Acalypha fimbriata in inhibiting the growth of post-harvest fruit rot fungi of Chrysophyllum albidum was investigated at the concentrations of 100, 80, 60 40 and 20 mg/ml in-vitro. The fungi isolated from rotted fruits and their frequency of occurrence includes Aspergillus niger (69.6%) and Fusarium solani (30.4%). These fungal isolates were cultured on different leaf extracts agar and their radial mycelia growth was observed. The antifungal activities increased with increase in concentrations of the plant extracts with E. heterophylla extract most effective in inhibiting the growth of A. niger while A. fimbriata extract was more effective in the inhibition of F. solani than other extracts. Phytochemical screening of the plant extracts revealed the presence of saponins, alkaloids, glycosides, terpenes, steroids, flavonoids, tannins and phenols. Gas Chromatography Mass Spectrometry (GC-MS) analysis revealed the presence a complex mixture of constituents ranging from 7 compounds in E. hirta, 10 compounds in A. fimbriata, 11 compounds in E. heterophylla and 14 compounds in P. amarus. The result of this study is an indication that these Euphorbiaceae could be a potential source of antifungal agents.

Keywords

Growth inhibition; Leaf extracts; Euphorbiaceae; Rot fungi; Chrysophyllum albidum

Introduction

Chrysophyllum albidum G. Don commonly called African star apple and locally called udara (Igbo), agbalumo (Yoruba) belongs to the family Sapotaceae [1]. It features prominently in the compound agro forestry system for fruit, food, cash income and other auxiliary uses including environmental purposes. It is also a tree that is common throughout the Tropical Central, East and West Africa regions for its sweet edible fruit and various ethnomedical uses [2].

C. albidum fruits are widely eaten in Southern Nigeria. The fruit is seasonal (December-March), when ripe. It is flattened seeds or sometimes fewer by abortion. The fruit is ovoid to sub-globose pointed at the apex and up to 6 cm long and 5 cm in diameter. The skin or peel is grey when immature turning orange red, pinkish or light yellow within the pulp having three to five seeds arranged as a star [3].

The fruit has been found to have the highest content of ascorbic acid with 1000 to 3330 μg of ascorbic acid per 100 gm of edible fruit or about 100 times that of oranges and 10 times of that of guava or cashew. It is also an excellent source of vitamins B and D as well as iron [4]. Umoh [5] and Ureigho [6] reported on the proximate composition, minerals and vitamins content of Chrysophyllum albidum.

The fruit has immense economic potential, especially following the report that jams that could compete with rasp berry jams and jellies could be made from it and it is eaten especially as snack by both young and old [2]. The fruits contain 90% anacadic acid, which is used industrially in protecting wood and as a source of resin. The fruits can be used in the preparation of wine, soft drink, jams and jellies [3,6].

The seed are used for local games; it is also a source of oil, which used for diverse purposes [7]. The seeds along with those of other Sapotaceae are used as anklets in dancing. It was also discovered in the removal of Ni2+ ions from synthetic wastewater [8]. The cotyledons are useful in the preparation of medicine for the treatment of infertility problems in both male and female; infertility due to the presence of abnormalities within the uterus and female tubes, abdominal pains in dysmenorrheal, secondary ammenorrhae in women (loss or absence of menstrual cycle). The seed cotyledon has been reported to possess antihyperglycemic and hypolipidemic effects [9].

Fungi have been reported to be associated with post harvest deterioration of agricultural products in Nigeria. However, F. solani, L. theobromae, Rhizopus spp and A. flavus have been reported to be associated with C. albidum [10]. Since most microbial spores are small in size and light, they could settle on the surface of African Star Apple fruits resulting in the range of microbial group isolated from them.

Preserving the freshness of these fruits for many days or months is therefore the problem, which most farmers and the traders seek to solve. Control of fruit rot by employing the use of local preservatives (plant extracts) like Afromomum danielli, Afromomum melegueta and chemical disinfectants like (parazone), sodium chloride and sodium benzoate at mild form has been suggested to reduce the losses due to storage moulds [10].

The objective of this study is therefore to isolate and identify fungi associated with C. albidum fruits rot in storage as well as to determine the effects of various concentrations of ethanolic extracts of Phyllanthus amarus, Euphorbia hirta, Euphorbia heterophylla and Acalypha fimbriata on the identified fungi.

Materials and Methods

Collection of plant materials for the study

Mature healthy and rotted C. albidum fruits were purchased at Abraka Main Market, Delta State. Fresh and healthy leaves of Euphorbia hirta, Euphorbia heterophylla Phyllantus amarus and Acalypha fimbriata free from insect and pathogen attack were collected from different areas within Abraka community. Abraka (Ethiope East Local Government Area of Delta State lies within latitude 05° 47ËŁN and longitude 06° 06ËŁE of the Equator with an annual rainfall of 3,097.8 mm, annual relative humidity of 83% and annual mean temperature of 30.6°C [11]. The plants were identified using Akobundu and Agyakwa [12].

Isolation and identification of fungi

Isolation and identification of fungi from diseased C. albidum fruits was carried out using the method adopted from Ilondu [13]. Sections, 4 mm long, excised from the margins of diseased spot with sterile razor blade were surface-sterilized for 2 min in 2% aqueous solution of commercial bleach (sodium hypochlorite solution), rinsed in two changes of sterile distilled water. The disinfected tissue pieces were blotted between sterile Whatman No. 1 filter paper and aseptically plated on potato dextrose agar (PDA) plates (3 pieces per plate). The plates were then incubated at room temperature (32 ± 2°C) for five days. Any observed mycelial growth was repeatedly transferred to fresh PDA plates until pure cultures of isolates were obtained.

The frequency of isolations of the different types of fungi associated with C. albidum fruit rot diseases was determined. The number of times each fungus was encountered was recorded. The percentage frequency of occurrence was calculated with the formula below:

Equation

Plant sample preparation and extraction procedures

The plants were collected into polyethylene bags and taken to the laboratory for processing. The leaves were separately plucked and rinsed in flowing tap water, shade dried on the bench in a ventilated section of the Department of Botany herbarium at ambient temperature (30°C ± 2) for two weeks [14]. Dried leaves were separately ground into powder using an electric blender before extraction. For extraction procedures, one hundred gram of each pulverized sample was put into Soxhlet extractor and three hundred milliliter of absolute ethanol (HPLC grade) was added and extracted for 8hrs for each batch of sample. The extracts were evaporated on a rotary evaporator at 40°C to remove excess alcohol. The solvent free extracts were stored at 4°C till needed.

Phytochemical tests

One gram of powdered sample was subjected to phytochemical test for alkaloid (Myers reagent), Flavonoids were determined by magnesium rebbon test, Sapoins by chloroform and H2SO4 tests, Tannins, by Ferric salt test, Sterol by Chloroform-acetic anhydride, Terpenes and phenols by following the procedures of Oyewale and Audu [15].

Extract analysis

GC-MS analysis was done at National Research Institute for Chemical Technology (NARICT) Zaria, Kaduna state, Nigeria. A SHIMADZU GCMS-QP 2010 Plus system was used. The GC-MS was operated under the following conditions: Column oven temperature: 70°C; Injection temperature: 250°C; Injection mode: split; Pressure: 104.1 kPa; Total flow: 6.2 ml/min; Column flow: 1.59 ml/min; Linear velocity: 46.3 cm/sec; Purge flow: 3.0 mL/min; and Split ratio: 1.0. The generated chromatogram was recorded. The identification of the components was carried out using the peak enrichment technique of reference compounds and computer matching with those of NIST.05 library mass spectrum [14,16].

Effect of extracts on fungal growth

Different concentrations (100, 80, 60 40 and 20 mg/ml) were prepared from each of the extracts. One millilitre of each level of concentration was aseptically incorporated into 20 ml of cool molten PDA in sterile test tube. Each medium was homogenized by gentle agitation before dispensing into sterile 9 cm Petri dishes. The control was set up using extract free PDA plates. The plates were allowed to set for 3 hr. The effect of the extracts on fungal growth was determined using the method of Chohan et al. [17]. This was done by inoculating at the Centre of 90 cm Petri plates with a mycelia disc (4 mm) obtained from the colony edge of 7-day old culture of the test fungi. Three replicates of both the control and PDA-extract plates per isolate were incubated at room temperature (28 ± 2°C) and radial growth was measured with a metric ruler daily for seven days. Colony diameter was taken as the means along two directions on two perpendicular lines drawn on the reverse of the plates. The percentage inhibition was calculated by the method of Ayodele et al. [18].

Data analysis

Data obtained were subjected to Analysis of Variance (ANOVA) using Statistical Package for Social Science (SPSS) version 17.0 and means were separated according to Duncan’s Multiple Range Test (DMRT) at 5% probability level.

Results

The fungi isolated from the diseased Chrysophyllum albidum fruits were Aspergillus niger and Fusarium solani. A. niger occurred more frequently with 69.6% followed by F. solani with 30.4% (Table 1). The classes of natural products present in the plant investigated are shown in Table 2. Tannins, saponins, steroids and phenols were present in all the plants. Alkaloids were present in E. hirta, E. heterophylla and A. fimbriata except P. amarus. Flavoinoids was only present in E. hirta. Glycosides and terpenes were present only in E. hirta and A. fimbriata.

Fungal isolate No of times isolated Percentage frequency (%) Pathogenicity of isolates
Aspergillus niger 80 69.6 +
Fusarium solani 35 30.4 +

Table 1: Percentage occurrence of fungi associated with Chrysophyllum albidum.

Phytochemicals P. amarus E. hirta E. heterophylla A. fimbriata
Saponins + + + +
Alkaloids - + + +
Tannins + + + +
Flavonoids - + - -
Steroids + + + +
Glycosides - + - +
Terpenes - + - +
Phenols + + + +

Table 2: Phytochemical Screening of Plants used in the study.

The gas chromatography profiles of the plants extracts used in the study were shown in Figures 1-4. The analysis of the extract revealed complex mixture of constituents ranging from 7-14 compounds in the samples (Table 3).

plant-pathology-microbiology-GC-MS-Chromatogram

Figure 1: GC-MS Chromatogram for P. amarus.

plant-pathology-microbiology-GC-MS-Chromatogram-hirta

Figure 2: GC-MS Chromatogram for E. hirta.

plant-pathology-microbiology-GC-MS-Chromatogram-heterophylla

Figure 3: GC-MS Chromatogram for E. heterophylla.

plant-pathology-microbiology-GC-MS-Chromatogram-fimbriata

Figure 4: GC-MS Chromatogram for A. fimbriata.

Plant Extracts Peak No Retention time (min) % peak Compound formula Name of compound
P. amarus 1 25.381 1.09 C14H22O Phenol3,5-bis(1,1-dimethylethyl)
2 30.543 9.45 C17H34O2 Hexadecanoic acid, methyl ester
3 30.868 5.51 C16H36O2 n-Hexadecanoic acid
4 31.066 9.17 C18H36O2 Hexadecanoic acid, ethyl ester
5 31.745 9.50 C19H34O2 11,14-octadecadienoc acid, methyl ester
6 31.802 22.92 C19H34O2 10-octadecenoic acid, methyl ester
7 31.978 8.69 C19H38O2 Octadecanoic acid, methyl ester
8 32.223 19.33 C20H34O2 9,12,15-octadealrienoic acid, ethyl ester (z,z,z)
9 32.405 3.85 C20H40O2 Octadecanoic acid, ethyl ester
10 33.205 1.99 C21H42O2 Eicosanoic acid, methyl ester
11 34.017 2.31 C18H31C10 9,12-octadecadienoyl chloride (z,z)
12 37.501 2.71 C22H28O7 Carissanol dimethyl ether
13 38.330 1.20 C34H22 Dibenz(a,h) anthracene, 12-diphenyl
14 39.463 2.29 C10H14O2 Benzene, 4-ethyl-2-dimethoxy-
E. hirta 1 25.383 1.94 C14H22O Phenol, 3,5-bis(1,1-dimethylethyl)
2 30.542 12.68 C17H34O2 Hexadecanoic acid, methyl ester
3 30.869 8.85 C16H32O2 n-hexadecanoic acid
4 31.067 6.33 C18H36O2 Hexadecanoic acid, ethyl ester
5 31.747 16.27 C19H34O2 9,12-octadecadienoic acid methyl ester
6 31.806 44.9 C19H36O2 9-octadecenoic acid (z)-, methyl ester
7 31.98 9.33 C19H38O2 Octadecanoic acid, methyl ester
E. heterophylla 1 5.496 4.78 C8H10 O-xylene
2 25.386 3.47 C14H22O Phenol3,5-bis(1,1-dimethylethyl)
3 30.542 3.94 C17H34O2 Hexadecanoic acid, methyl ester
4 30.868 14.71 C16H32O2 n-Hexadecanoic acid, methyl ester
5 31.061 7.84 C18H36O2 Hexadecanoic acid, ethyl ester
6 31.798 9.22 C19H36O2 10-Octadecenoic acid, methyl ester
7 32.093 22.22 C22H42O2 Erucic acid
8 32.224 21.39 C18H32O2 9,12-Octadecadienoica cid (z,z)-
9 32.944 2.97 C37H74NO8P Hexadecanoic acid
10 34.020 8.33 C18H34O 13-Octadecenal
11 37.644 1.13 C10H14O2 1,4-Benzenedimethanol, alpha, alpha, dimethyl
A. fimbriata 1 3.070 2.42 C5H12O 1-Butanol,3-methyl-/isopentyl alcohol
2 5.43 10.52 C8H10 Xylene/benzene1,2-dimethyl
3 25.372 1.70 C14H32O Phenol3,5-bis(1,1-dimethyl ethyl)
4 30.537 5.83 C17H34O2 Pentadecanoic acid
5 30.867 17.42 C16H32O2 n-Hexadecanoic acid
6 31.057 7.95 C18H36O2 Hexadecanoic acid ethyl ester
7 31.797 18.47 C19H36O2 10-Octadecenoic acid, methyl ester
8 32.093 28.88 C22H42O2 Erucic acid
9 32.401 3.25 C20H40O2 Octadecanoic acid, ethyl ester
10 34.015 3.56 C16H30O Cis-9-Hexadecenal

Table 3: Major identified constituents of the plant extracts.

Phenol 3,5-bis (1,1-dimethylethyl) were recorded in all plant. Hexadecanoid acid, methyl ester were recorded in all the plant except A. fimbriata. 10-Otadecenoic acid, methyl ester was recorded to be the most abundant of all the (14) compounds identified in P. amarus, 9-Octadecenoic acid (Z)-methyl ester was most abundant among the (7) compounds in E. hirta, Erucic acid was most abundant in E. heterophylla and A. fimbriata.

The two fungi were very sensitive to various concentrations of the plant extracts tested since the extracts significantly reduced the mycelia growth of the fungi at all concentrations (Table 4). However, the effectiveness of the plant extracts increased with increased in concentration and this was significantly different (p<0.05) when compared to the control. Similarly, percentage growth inhibition generally increased with increase in concentration of the leaf extracts when compared to the control. Although, the plant extracts could not give complete inhibition at the highest concentration tested, their effectiveness increased with increase concentrations.

Extract conc. (mg/ml) P. amarus E. hirta E. heterophyta A. Fimbriata
A. niger F. solani A. niger F. solani A. niger F. solani A. niger F. solani
0 4.30a 4.30a 4.30a 4.30a 4.30a 4.30a 4.30a 4.30a
20 2.07b 3.1b 2.83b 2.83b 1.53b 1.90b 1.87b 1.53b
40 1.95c 2.23c 2.40c 1.93c 0.95c 1.73b 1.40c 0.58c
60 1.13c 1.85d 1.97d 1.63c 0.72c 0.85c 0.92d 0.42c
80 0.92d 1.27e 1.50e 1.03d 0.62c 0.62c 0.75d 0.30c
100 0.84d 0.92f 1.03f 0.82d 0.47c 0.42c 0.60d 0.22c

Table 4: Radial mycelia growth (cm) of fungi isolated from Chrysophyllum albidum fruits when exposed to various concentrations of plant leaf extracts.

There was no significant difference in the inhibitory effect of P. amarus on A. niger at the concentrations of 20 and 40 mg/ml, 60 and 80 mg/ml concentrations with E. heterophylla as well as 80 and 100 mg/ml concentrations with A. fimbriata. Similarly, there was no significant difference in the inhibitory effect of A. fimbriata extract on F. solani from 60-100 mg/ml concentrations (Table 5). A. niger was most sensitive to E. heterophylla followed by A. fimbriata, P. amarus and E. hirta respectively. Similarly, F. solani was most sensitive to A. fimbriata followed by E. heterophylla, E. hirta and P. amarus.

Conc. (%) P. amarus E. hirta E. heterophylla A. fimbriata
  F. Solani A. niger F. Solani A. niger F. Solani A. niger F. Solani A. niger
0 0f 0d 0f 0f 0f 0e 0d 0d
20 27.91e 51.86d 45.81e 34.19e 55.51e 64.42d 64.42c 56.51c
40 48.14d 54.65c 55.43d 44.18d 59.77d 77.91c 86.49b 67.44b
60 56.98c 73.72b 62.09c 54.19c 80.37c 83.43b 90.23a 78.75ab
80 70.47b 78.61a 76.21b 65.12b 85.58b 85.58b 93.02a 82.68a
100 78.61a 80.47a 80.93a 76.05a 90.31a 89.07a 94.89a 86.05a

Table 5: Percentage growth inhibition of fungal isolates from Chrysophyllum albidum fruits after exposure to varying concentrations of leaf extract of various plants.

Discussion

The present study showed that two fungi were associated with post harvest fruit rot disease of Chrysophyllum albidum, which include Aspergillus niger and Fusarium solani. These fungi have previously been reported as fruit rot pathogens [13,19,20].

Aspergillus niger has the highest percentage occurrence of 69.6% followed by F. solani which is 30.4%. This was enhanced by the light spores, which are easily dispersed by wind. Similarly Aspergillus species are capable of utilizing an enormous variety of substrates as the result of large number of enzymes they produce [21].

Phytochemical screening of the plants revealed the presence of saponin, alkaloid, tanin, steroids, Phenols, terpenes, glycosides, and flavonoids. The presence of these secondary metabolites could be responsible for their antifungal activity. Egwin et al. have earlier demonstrated the presence of tannins in Euphorbia hirta and opined that it may account for its antimicrobial activity. Tannins have been reported to be toxic to bacteria, filamentous fungal and yeast [22]. Ogbo and Oyibo [19] reported that the presence of alkaloids, saponins and terpenoids in the extract of Ocimum gratissimum may have accounted for the broad spectrum of activities on the fungal isolate tested.

The analysis of the plant extract of the leaves in this study showed a complex mixture of constituents. The total number of compounds identified varied from 7-14 in all the plant samples. It is possible that these compounds identified in the plant extracts were responsible for the observed fungi-toxic effects in the study. Sunderham [23] reported that the toxic action of the plant extract of E. heterophylla is due to the combined action of its constituents this is similar to the observations of Ilondu [14,16].

Erucic acid was the highest constituent found in E. heterophylla (22.22%) and A. fambiata (28.88%) extracts. Antimicrobial activity of Eruca sativa seed oil has been reported to be due to higher concentration of erucic acid present in the oil [24,25]. Varied concentrations of fatty acid including their ethyl and methyl esters were found abundant in all the plant extracts. Several researchers have reported the antifungal activity of fatty acid and their ethyl and methyl esters against pathogenic fungi [14,16,26].

The percentage inhibition of the mycelia growth of the tested fungi was found to increase as concentration of the plant extracts increased. This may be as a result of the presence of the biologically active antimicrobial compounds of the extracts in higher quantity at lower dilutions, this findings is in consonance with the work of Fernadex et al. [27] who suggested that with increasing concentrations the antagonistic property of the extract increased.

The above result clearly confirms that the test fungi varied widely in the degree of their susceptibility to the extracts. The extract of Euphorbia heterophylla was the most effective of all the extracts in inhibiting the growth of Aspergillus niger followed by Acalypha fimbriata, Phyllanthus amaraus and Euphorbia hirta. While ethanolic extracts of Acalypha fimbriata was the most effective in inhibiting the growth of Fusarium solani followed by Ephorbia heterophylla, Euphorbia hirta and Phyllanthus amarus. Previous studies have shown that ethanolic leaf extracts of E. hirta, E. heterophylla, A. fimbriata, P. amarus and other species of these genera were capable of inhibiting the growth of bacteria, and fungi [13,23,28-32].

Conclusion

The result of this study is an indication that these Euphorbiaceae could be a potential source of antifungal agents. Knowledge of chemical constituents of non-economic plants is desirable because such information could be valuable in discovering new source of economic materials, which may be precursors for the synthesis of complex chemical substances. Such screening of various natural organic compounds and identification of active agents is the need of the century for the formulation of plant biofungicide and improvement of food security for the timing world population.

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