Plant Pathology & Microbiology Antifungal Activity of Some Plant Extracts against (Colletotrichum Musae) the Cause of Postharvest Banana Anthracnose

The present study was conducted to determine the efficacy of extracts of some plant species against Colletotrichum musae . Methanolic extracts of 21 plant species were screened for their inhibitory effect against C. musae using paper disc method and spore germination assay. Among them, extract of Prosopis juliflora exhibited superior antifungal activity (30.7 mm), followed by Acacia albida (19 mm) as compared to nill in the control. On the other hand, carbendazim, which was used as a standard chemical check, demonstrated by far the highest inhibition zone of 51.7 mm diameter. Extracts from A. albida, Dovalis abyssinica and P. juliflora reduced conidial germination to 0.2, 0.5 and 0.3%, respectively, which didn’t vary statistically from 1.2% in Carbendazim. Six plant species, viz., A. albida, Azadirachta indica, Argemone mexicana, D. abyssinica, P. juliflora and Vernonia amygdalina , that showed high to moderate antifungal activity in the preliminary screening, were further tested for their thermal stability at 60°C and for the efficacy of their aqueous extracts against C. musae. Extracts of the tested plant species were found to be heat stable and aqueous extracts of A. albida showed the highest antifungal activity (18 mm), followed by P. juliflora (12.3 mm). Further studies need to be undertaken to isolate the active compounds from those extracts with fungicidal potential.


Introduction
Anthracnose caused by the fungus Colletotrichum musae, is the most important postharvest disease of banana that can result in 30-40% losses of marketable fruit [1]. Recently, plant products have attracted high attention to search for some phytochemicals for their exploitation as antimicrobials; such plant products would be easily degradable less phyto-toxic and safe to human health [2]. However, no efforts have been made in Ethiopia to screen plants that have potential antimicrobial properties against C. musae. Several investigations have evaluated spices and medicinal plants with the aim of using them for plant disease management [3][4][5]. However, although many such plants have shown antimicrobial activity, their practical use for disease management has been limited because of the high price they fetch for other purposes. Thus, attention should be focused on plants with limited known economic values.
It has been suggested that combining plant extracts with nonchemical postharvest treatments, such as heat treatment, might control postharvest anthracnose diseases effectively [6]. However, the antifungal substances that are present in the plant extracts may be unstable at higher temperatures [7]. Accordingly, there is a need for investigating a heat stable plant extract that could be integrated with hot water treatments. This study was undertaken to determine the inhibitory effects of extracts of plants with limited economic value against C. musae.

Isolation of the pathogen and inoculum preparation
Banana fruits showing anthracnose disease symptoms were collected and used for isolation of Colletotrichum musae. Pure culture was maintained on PDA medium. Inoculum was prepared by dislodging the surface of 10 days old culture with sterile distilled water and adjusted to 105 ml -1 using a hemacytometer.

Extraction of plant samples
Extraction was done using methanol and water. The methanolic extracts were used for preliminary screening of the plant specimens for their potency against the fungal pathogen, while aqueous extracts were used for further testing. Fifty gram of powdered samples of each of the plant specimens was extracted with 250 ml solvent (methanol or water). The methanolic and aqueous extracts were filtered and reduced to dryness on rotary evaporator under vacuum at 40°C and 60°C water bath temperatures, respectively [8,9]. The extract of each plant was weighed, re-dissolved and then tested for antifungal activities.
Briefly, sterile filter paper discs (6 mm in diameter) were impregnated with 10 μl plant extract (at a concentration of 100 μg/μl). Five milliliter of a culture suspension of C. musae with a concentration of 1×10 6 conidia ml -1 was added to 250 ml of PDA medium which had been previously cooled down to ca. 50°C. The medium was then poured into Petriplate and allowed to solidify. The impregnated discs were dried and placed with forceps aseptically on the surface of the pre-inoculated PDA medium. Paper discs that were similarly handled but without plant extract served as untreated control and paper discs impregnated with commercial fungicide carbendazim (at the concentration of 2 μg/ μl) served as a positive control. Four days of after incubation (at 28°C), the diameter of inhibition zone around the test discs was measured and the degree of inhibition of fungal growth expressed on a 0-4 scale was recorded [9], where: 0=no inhibition zone visible, 1=inhibition zone barely distinct, fungal growth and sporulation only slightly inhibited, 2=inhibition zone well distinct fungal growth ca. 50% of the control, slight sporulation, 3=inhibition zone with sparse (ca. 25% of the control) fungal growth, 4=inhibition zone free of visible fungal growth.

Spore germination assay:
In this assay, about 0.5 ml of methanolic plant extract was pipetted into sterile test tubes and the solvent was allowed to evaporate at room temperature. About 4.5 ml volume of potato dextrose broth was pipetted into the test tubes and mixed with extracts. At the same time an aliquot (100 μl) of spore suspension (adjusted to 10 6 conidia ml -1 ) was added into each tube. After 24 hr of incubation at 28°C on a rotary shaker, a drop of the mixture from each tube were placed in a microscope slide and slides were fixed in lactophenol cotton blue and observed microscopically for spore germination. The same volume of methanol was added in place of plant extracts in the control samples and carbendazim at the concentration of 2.5 mg/ml served as a positive control. A conidium was considered as germinated if the length of the germ tube was at least half the length of the conidium. The number of germinated conidia was counted out of 100 randomly selected conidia in three replicate slides. Percentage spore germination was calculated according to the following formula: pore Germination (%)=(Germinated Spores (No.)/(Total Spores (No.))×100 Thermal inactivation of plant extracts: Plant extracts showing potential antifungal activity in the preliminary evaluation were further tested for thermal stability. About 1 ml of methanolic extracts in glass tubes were exposed to 60°C in a water bath for 10 min and cooled to room temperature. Afterwards, the paper disc method was employed to assess their effect on antifungal activity against C. musae as described under Section 2.4.1.

Statistical data analysis:
Percentage data on spore germination inhibition was arcsine transformed before statistical analysis. The data were subjected to analysis of variance (ANOVA) using SAS software version 9.2 [10]. Means were separated using Duncan's Multiple Range test (DMRT) at P ≤ 0.05.

Antifungal activity of methanolic extracts
Preliminary screening of 21 plant species was conducted for antifungal activity against Colletotrichum musae using mycelial inhibition test and spore germination assay.

Mycelial inhibition test:
Out of the 21 plant species tested, Prosopis juliflora exhibited the highest antifungal activity with inhibition zone of 30.7 mm diameter against C. musae. The methanolic extract of A. albida showed the second best antifungal activity (19 mm), followed by D. The present study has demonstrated the antifungal potential of methanolic extract of P. juliflora that corresponded with the work done by [11] who reported methanolic extract of P. juliflora leaves to have excellent in vitro antifungal activity against soil mycoflora i.e. Fusarium oxysporum, Rhizoctonia solani, Alternaria alternata and Curvularia lunata. Extracts from A. albida showed potent activity and this result is in agreement with the previous finding of [12]. Similarly, antifungal effect of A. mexicana was previously reported by [13]. The antifungal activity of methanolic extract of D. abyssinica has also been reported [14].
Spore germination assay: Methanolic extract from A. albida, D. abyssinica and P. juliflora showed potent antifungal activity in reducing the spore germination to 0.2, 0.5 and 0.3%, respectively. Carbendazim, used as a standard chemical check, markedly reduced spore germination percentage to 1.2% which is not significantly different (P>0.05) from the above most effective plant extracts (Tables 1 and 2). The extracts   In the present study, spore germination was reduced to below 1% by methanolic extracts of A. albida, D. abyssinica and P. juliflora. Previous study conducted by [15] revealed that extracts of P. juliflora completely inhibited the germination of conidia of Phaeoisariopsis personata.

Thermal stability and antifungal activity of aqueous extracts:
Extracts of A. albida, A. indica, A. mexicana, D. abyssinica, P. juliflora, and V. amygdalina that showed high activity against C. muase in the preliminary screening were further tested for their thermal stability and for the potency of their aqueous extracts. The results showed that there was no significant difference (P>0.05) between heated (at 60°C) and unheated plant extracts in their efficacy against the test pathogen (Table 3). This indicates that temperature slightly above the hot water treatment (HWT) range had no effect on the antifungal activity of the plant extracts. The result of the study is supported by previous works [16,17] that natural extract components are heat stable under moderate (50-80°C) heat treatment. It is worthy to note that heat treatment slightly but insignificantly increased inhibition zone diameter (Table  3). In addition, the degree of inhibition of fungal growth was increased from 3.5 to 4 scales in the case of A. indica. This is in agreement with the earlier findings [18,19] that heat treatments increased the activity of plant extracts. This may be due to an increase in the release of active compounds and free radicals [20].
Similarly, among the six plant species extracts tested, the highest zone of inhibition was exhibited by A. albida (18 mm) followed by P. juliflora (12.3 mm) ( Table 4). Extracts of A. mexicana and D. abyssinica showed low antifungal activity with an inhibition zone of 7 and 6.7 mm diameter, respectively. On the other hand, aqueous extracts of A. indica and V. amygdalina showed no antifungal activity and the pathogen grew even on the paper discs. Distilled water, used as negative control, showed no zone of inhibition as expected. The fungi-toxic effect of aqueous extracts of P. juliflora against Aspergillus species [21] and Fusarium species [22] have been reported, which is in line with present result.
It is important to note that the activity of methanolic extracts of P. juliflora, A. indica, A. mexicana and D. abyssinica showed greater activity than their aqueous extracts (Tables 1 and 4). This is in harmony with the work of [11] that showed methanolic extracts have greater activity than the aqueous extract. This might be due to better solubility of the active components in organic solvents [23]. Furthermore, [24] indicated that such observations can be rationalized in terms of the polarity of the compounds being extracted by each solvent and, in addition to their intrinsic bioactivity, by their ability to dissolve or diffuse in the media.