Evaluation of Antifungal Activity of Plant Extracts against Papaya Anthracnose (Colletotrichum gloeosporioides)

Papaya (Carica papaya L.) is a popular and economically important fruit tree of tropical and subtropical countries [1]. The leading global producers of papaya are Brazil, Colombia, Democratic Republic of Congo, Ethiopia, Guatemala, India, Indonesia, Mexico, Nigeria and Philippines [2]. Papaya is known as “common man’s fruits’’. It is rich sources of vitamin A, C and calcium. The ripe fruit is prone to many diseases, among which anthracnose caused by Colletotrichum gloeosporioides (Penz.) Penz & Sacc. is an economically important disease during transit, storage and market [3-5]. In general, the fungus initiates infection as soon as flowering starts and stays latent until the postharvest environment conditions favor colonization of fruit tissue [3,6]. According to Coursey [7], postharvest losses of approximately 40100% have been generally reported in papaya in developing countries.


Introduction
Papaya (Carica papaya L.) is a popular and economically important fruit tree of tropical and subtropical countries [1]. The leading global producers of papaya are Brazil, Colombia, Democratic Republic of Congo, Ethiopia, Guatemala, India, Indonesia, Mexico, Nigeria and Philippines [2]. Papaya is known as "common man's fruits''. It is rich sources of vitamin A, C and calcium. The ripe fruit is prone to many diseases, among which anthracnose caused by Colletotrichum gloeosporioides (Penz.) Penz & Sacc. is an economically important disease during transit, storage and market [3][4][5]. In general, the fungus initiates infection as soon as flowering starts and stays latent until the postharvest environment conditions favor colonization of fruit tissue [3,6]. According to Coursey [7], postharvest losses of approximately 40-100% have been generally reported in papaya in developing countries.
Synthetic fungicides are currently used as the primary means for the control of plant diseases. However, the alternative control methods are needed because of the negative public perceptions about the use of synthetic chemicals, resistance to fungicides among fungal pathogens and high development cost of new chemicals [8,9]. Application of higher concentrations of chemicals in an attempt to overcome anthracnose disease increases the risk of high levels of toxic residues, which is, particularly serious, since papaya fruit is consumed in relatively short time after harvest [10].
Bioactive products of plants are less persistent in environment and are safe for mammals, other non target organisms [11][12][13], and for the control of postharvest disease than synthetics [14]. A number of plant species have been reported to possess natural substances that are toxic to many fungi causing plant diseases [15,16]. Ranaware et al. [17] indicated the efficacy of aqueous plant extracts as potential inhibitors of Alternaria carthami. Similarly, Dwivedi and Shukla [18] reported the effectiveness of aqueous extracts of different species of plants against Fusarium oxysporum.
Papaya anthracnose is one of the major diseases of the crop in Ethiopia [19]. Hence, this study was conducted with objective of determining the in vitro effect of plant extracts on conidial germination, mycelial growth of Colletotrichum gloeosporioides and their efficacy against the development of postharvest papaya anthracnose.

Isolation of target pathogen
Colletotrichum gloeosporioides was isolated from papaya fruits showing anthracnose lesions. An isolate of the pathogen grown in pure culture was maintained in PDA culture tubes at 4°C, and used as stock culture throughout the study [20,21].

In vitro evaluation of botanicals
Sample collection and extraction: The potential extracts were selected from a screening of nineteen plant species. The plants were collected from Haramaya and Ambo areas of Ethiopia, in 2010. The experiment was conducted at the Plant Pathology Laboratory of the School of Plant Sciences at Haramaya University. The plant specimens (leaves) were shade dried at a room temperature and milled into a fine powder. Following the procedures employed by Amare [22], 50 gram of the pulverized plant specimens were extracted with 250 ml ethyl acetate by stirring for 2 hrs on magnetic stirrer. The extract was filtered through folded filter paper into a 500 ml round bottom flask and reduced to dryness on a rotary evaporator at 40°C water bath temperature. About 50 mg of the ethyl acetate extracts of each plant was weighed, redissolved in 1 ml of the extraction solvent and then tested for antifungal activities. Paper disc assay: Filter paper discs, 6 mm diameter, were sterilized by dry heat for 1 h at 160°C oven temperature and impregnated with each of the test extracts by applying 10 µL of the extract solution using a capillary pipette. The culture media containing spore suspension of Colletotrichum gloeosporioides was poured into 14.5 cm in diameter petri plate and allowed to solidify. After the carrier solvent evaporated from the paper discs, they were placed on the surface of the medium; the plates were incubated for 4 days. The diameter of inhibition zone was measured in mm and the degree of inhibition of the fungal growth expressed on a 0-4 scale was recorded, 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, and 4=inhibition zone free of visible fungal growth [22] (Figure 1a and 1b).

Conidial germination test:
Conidia of C. gloeosporioides were adjusted using hemacytometer to a concentration of 10 5 condia/ml. Ten µL of plant extracts and 90 µL of the conidial suspension were mixed and the mixtures were added to the surface of dried depression slides. The slides were then placed on a glass rod in petri dish under moistened conditions and incubated at 25°C for 24 h. Control conidia received an equivalent amount of the solvent. After incubation, slides were fixed in lactophenol cotton blue and observed microscopically for spore germination. The experiment was laid out in CRD with three replications. The number of conidia germinated was scored to calculate the percentage inhibition of conidial germination.

In vivo antifungal assay of plant extracts
Aqueous extracts were tested for their effect on the papaya anthracnose development on harvested fruit. "Solo" papaya was obtained from Yilma State Farm in Dire Dawa, Ethiopia. For this purpose, undamaged, matured fruits of comparable size, color class and free from any pesticide were used. Aqueous solutions of selected plant species were evaluated at a concentration of 10 and 25%. Conidial suspension of C. gloeosporioides was prepared and adjusted to 10 5 conidia/ml Papaya fruits were surface-sterilized by dipping in 1% sodium hypochlorite solution for 10 min, rinsed in sterile distilled water and inoculated by dipping into spore suspension of C. gloeosporioides. After incubation for 15 h in plastic bag, fruits were dipped into extracts, while the control fruits were dipped into sterile distilled water. Five fruits (i.e. replications) for each concentration of extracts were used and arranged in CRD [21]. As of first symptom appearance, data on incidence and severity of anthracnose was recorded. Disease incidence was expressed as the percentage of fruits showing symptom. Disease severity was rated on 1 to 5 scale, where 1=0% of surface fruit rotten, 2=1-25%, 3=26-50%, 4=51-75%, and 5=76-100% [23]. Overall quality was assessed according to the following score: 1-2= fruit not marketable; 3=poor quality, limited marketability; 4-5=fair quality, marketable; 6-7=good quality, marketable; 8-9=excellent quality [24]. Percentage of marketability was assessed as a ratio of the number of fruits with scores 6, 7 for overall quality against the total number of fruits [25].

Statistical analysis
Analysis of variance (ANOVA) was carried out with the statistical software SAS v. 9.0 and Least Significant Difference (LSD) at 5% probability level were used for mean comparison. Severity was square root transformed, while spore germination was arcsine transformed before statistical analysis.

In vitro effect of botanicals on mycelial growth and spore germination of Colletotrichum gloeosporioides
There was a highly significant difference (P<0.0001) among the antifungal effects of ethyl acetate extracts on inhibition zone and degrees of inhibition against the test fungus. Extracts of Echinops sp. and Lantana camara more strongly inhibited growth of the pathogen than the remaining extracts. On the other hand, the inhibition resulting from other extracts of both solvents ranged from weak to moderately active. Ethyl acetate extracts of Lantana camara had the highest inhibition zone among plants. This was followed by Artemisia afara, Echinops sp., Lantana viburnoides, Ruta chalepensis and Vernonia amygdalina ( Table 1). Lantana camara were found to be superior in mycelial growth reduction among different botanical tested against anthracnose of papaya [5]. The preservative nature of some plant extracts has been known for centuries, and there has been renewed interest in the antimicrobial properties of extracts from aromatic plants [12,26]. Numerous investigations on the genus Echinops have resulted in the isolation of thiophenes. Thiophenes from Echinops have been reported to possess many biological activities, including insecticidal and fungicidal [13]. Bautista-Banos et al. [23] tested leaf and stem

extracts of various plant species against Colletotrichum gloeosporioides.
In in vitro experiment, leaf extracts of Citrus limon were found to be inhibitive the in vitro radial growth of C. gloeosporioides. In general, the presence of antimicrobial substances in the different extracts which caused the inhibition of radial growth in vitro agrees with reports of other studies [27][28][29].
The result of the in vitro screening tested against C. gloeosporioides revealed that there was a highly significant difference (P<0.0001) in effects among ethyl acetate extracts of plants on spore germination ( Table 1). From tested extracts, Lantana camara gave the lowest spore germination (10.1%), followed by Lantana viburnoides (12.6%), Echinops sp. (13.3%) and Ruta chalepensis (13.7%), with no significant difference among them. The remaining ethyl acetate extracts showed also varying degrees of inhibition of spore germination ranging from 84.6% in Ruta chalepensis to 48.3% in Citrus limon (Table 1). Commercial essential oils of Ruta chalepensis and Thymus vulgaris and extracts of Ocimum basilicum and Vernonia amygdalina were found to be effective in reducing conidial germination of C. gloeosporioides [14,30]. Antifungal activities of 13 plant extracts were tested against conidial germination of C. gloeosporioides and Zingiber officinales were reported to be effective in minimizing conidial germination [31].

Effect of extracts on anthracnose development and quality of papaya
Extracts of botanicals evaluated for their efficacy against papaya anthracnose on papaya fruit that had been artificially inoculated by C. gloeosporioides showed a highly significant difference (P<0.0001) among the treatments in the incidence and severity of the disease. The incidence and severity of anthracnose was lowest in fruits treated with Echinops sp. extract, which were statistically at par with the positive control. Within the aqueous extract concentrations, fruits treated with 25% had relatively lower incidence and severity of anthracnose than those treated with 10% aqueous extracts, with the exception of Ruta chalepensis and Thymus serrultus. Overall, fruits treated with aqueous plant extracts had lower severity than the untreated control (Figure 2a  and 2b). Water is a universal solvent, used to extract plant products with antimicrobial activity. Nearly all of the identified antimicrobial compounds from plants are aromatic or saturated organic compounds, thus water is among other solvents that are most commonly used for preliminary investigation of antimicrobial activity in plants [32]. The presences of Furoquinolines and coumarins are reported in Rutaceae family to exhibit antifungal activity [33,34]. The antimicrobial properties of extracts from various species have been proven to affect fungal development in vivo [24,30,35].
In this study, there was a highly (P<0.0001) significant difference in marketability of fruits treated with extracts. The results showed that extracts of different plant species substantially varied in their antifungal potentials and the difference might accrue from the variability in chemical constituents of the plants. The highest marketability was achieved in fruits treated with Echinops sp. extracts at a concentration of 25% (Figure 2c). Bhaskara et al. [36] reported the antifungal activity of thyme oil against B. cinerea and R. stolonifer. Similarly, the inhibitory effects of thyme oil against C. gloeosporioides growth in in vitro evaluations and over development of storage rots in papaya fruit [37].
Postharvest diseases like C. gloeosporioides greatly reduce the storage life papayas. However, dipping fruit in plant extracts inhibited rot development during storage [35,38]. Navel orange fruits treated with aqueous extract kept on quality of navel orange under cold storage condition, and reduced the incidence and severity of green rot disease comparing with the control treatment [27]. The report by Anthony et al. [39] also showed that extracts was effective in controlling postharvest diseases, while maintaining the fruit quality. Plants are known to contain a number of secondary substances like phenols, flavonoids, quinines, essential oils, alkaloids, saponins and steroids. Some of these plant-based metabolites have antimicrobial properties, and are toxic to phytopathogens [40].

Conclusions
Examination of plant extracts on C. gloeosporioides in this study showed promising prospects for the utilization of plant extracts in postharvest disease control. In vivo experiments showed that Echinops sp. (25%) extract reduced postharvest diseases on papaya caused by C. gloeosporioides, while maintaining overall quality of the fruit. Further studies on isolation and characterization of the active (antifungal) compound are needed.