In Vitro and In Vivo Activity of Selected Plant Extracts against Papaya (Carica papaya L.) Anthracnose (Colletotrichum gloeosporioides)

Copyright: © 2014 Ademe A, 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. In Vitro and In Vivo Activity of Selected Plant Extracts against Papaya (Carica papaya L.) Anthracnose (Colletotrichum gloeosporioides)


Introduction
Anthracnose, caused by Colletotrichum gloeosporioides, is one of the most widespread and devastating diseases of papaya, especially during storage [1,2]. It is a major constraint to papaya production as well as to export of the fruit to bigger overseas markets [3]. Anthracnose infections are usually initiated in the field at early stages of fruit development, but the pathogen remains quiescent until the fruit reaches the climacteric phase [4].
Anthracnose of papaya can be controlled by fungicide treatments but application of higher concentrations of chemicals in an attempt to overcome anthracnose increases the risk of high levels of toxic residues, which is particularly serious since papaya fruit is consumed in relatively short time after harvest [5]. Plant extracts are emerging as safer alternatives to conventional fungicides for the control of plant diseases [6]. Natural product-based fungicides have the ability to decompose rapidly, thereby reducing their risk to the environment [7]. The antifungal activities of different plant species and the importance of plants as possible sources of natural fungicides are well established. Numerous studies have demonstrated the fungicidal potentials of plant extracts against postharvest fungi. Studies on the inhibitory effects of a diversity of extracts to control fungi such as Botrytis cinerea, C. gloeosporioides, Glomerella cingulata, Penicillium expansum, Pestalotia psidii, Phomposis mangiferae, Rhizopus stolonifer and others have proved the fungicidal potentials of extracts [8,9].
The fungicidal potentials of plant extracts against postharvest fungi and specifically towards C. gloeosporioides were reported [9][10][11]. There is a need for continued research efforts to developing effective and economical alternative methods for the management of papaya anthracnose. Most plant specious used in this experiment had medicinal value and are known to contain a number of secondary substances with antimicrobial properties and are toxic to phytopathogens [6,[12][13][14][15][16][17].
Papaya anthracnose is one of the major diseases of the crop in Ethiopia [18]. However, research on postharvest diseases of perishable fruits, particularly papaya anthracnose in Ethiopia, is limited. This paper reports on the in vitro effect of extracts of medicinal and other plant species from Ethiopia against C. gloeosporioides as well as their in vivo effect on anthracnose development on artificially inoculated papaya fruit.

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 potato dextrose agar culture tubes at 4°C and used as stock culture throughout the study.

In vitro screening of plant extracts for fungicidal activity
Plant specimen collection and extraction: Specimens of leaves and twigs of full grown plant species were collected from Ambo and Haramaya, Ethiopia with latitude and longitude of 8°59'N 37°51'E and 9°24'N 42°01'E, respectively. Leaves of selected plant specimens were dried under shade, milled using laboratory mill, and extracted with methanol. Fifty grams of the milled plant specimens were extracted with 250 mL methanol by stirring for 2 h 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 MeOH extracts of each plant were weighed, redissolved in 1 mL of the extraction solvent and then tested for antifungal activities.

Abstract
Anthracnose is the major postharvest disease of papaya wherever the fruit crop is grown. The present investigation was conducted with the objectives of evaluating plant extracts for their activity against Colletotrichum gloeosporioides in vitro and for controlling anthracnose on artificially inoculated 'solo' papaya fruit. Plant specimens were collected from Ambo and Haramaya, Ethiopia, dried under shade and extracted using methanol. Out of 18 plant extracts tested, nine of them showed activity against C. gloeosporioides. Methanol extract of Echinops sp. of 10 µL from the concentration of 50 mg/ml resulted in the highest inhibition zone of 13.5 mm against mycelial growth of C. gloeosporioides. Spore germination of C. gloeosporioides was reduced by 98.7%, 97.7% and 97.3% over the control by extracts of Echinops sp., Thymus serrulatus and Ocimum lamifolium, respectively. Among four botanicals evaluated in vivo as 10% and 25% aqueous extracts, Echinops sp. at 25% concentration kept disease severity score at 1.3 out of 5 (i.e. less than 1% fruit surface affected) and maintained quality of papaya fruit during 14 days experimental period. Further study is necessary on sensory analysis and developing botanicals as natural fungicides.
Paper disc assay of antifungal activity: Filter paper discs, 6 mm diameter, were sterilized for 1 h and impregnated with the test extracts by applying 10 µL of the extract solution using a capillary pipette. Autoclaved malt extract agar which was cooled to about 45°C in a water bath was seeded with 10 5 conidia/mL of spore suspension of C. gloeosporioides, poured into 14.5 cm diameter petri dish and allowed to solidify. After the carrier solvent evaporated from the paper discs in a laminar flow cabinet, ten discs were placed nearly equidistantly on the surface of the medium; the plates were then incubated for 4 days. The experiment was laid out in Completely Randomized Design (CRD) with three replications. The diameter of inhibition zone was measured in mm, and the degree of inhibition of fungal growth was recorded on a 0-4 scale, 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 [13].

Conidial germination test:
Spore suspension of C. gloeosporioides was adjusted to a concentration of 10 5 conidia/mL using a hemacytometer. 10 µL of plant extracts and 90 µL of the conidial suspension were mixed and the mixtures were added to the surface of depression slides. The slides were then placed on a glass rod in petri dish layered with moistened filter paper and incubated at 25°C for 24h. Conidial suspension mixed with an equivalent amount of the solvent served as control. The experiment was laid out in CRD with three replications. After incubation, a drop of lactophenol was added to the depression slide and the mount was observed microscopically for spore germination. A conidium was considered as germinated when the length of the germ tube exceeded its diameter. The number of conidia germinated was counted and expressed as percentage of germination.
In vivo assay of plant extracts: Extracts that showed antifungal activity was further tested for their effect against papaya anthracnose on harvested fruit. "Solo" papaya was obtained from Yilma State Farm in Dire Dawa, Ethiopia. Undamaged, matured fruits of comparable size and color class and free from any pesticide were used. Aqueous extracts of Echinops sp., Thymus serrulatus, Vernonia amygdalina and Ruta chalepensis were evaluated at concentrations of 10 and 25% (w/v). Conidial suspension of C. gloeosporioides was prepared from 10-day old culture and adjusted to 10 5 conidia/mL using hemacytometer. 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 covered by plastic sheet until conidia germinated, fruits were dipped into aqueous extracts, while the control fruits were dipped into sterile distilled water [18,19]. Carbendazim was used as positive control. Five fruits (i.e. replications) were used for each of the treatments. The experiment was laid out in CRD.
To determine titratable acidity fruit pulp tissues (10 g) were homogenized with distilled water (40 mL) using blender. Using one to two drops of phenolphthalein (1%) as indicator, 5 mL of the filtrate was titrated using 0.1 N NaOH to an endpoint pink. The results were expressed as percentage of citric acid per 100 g fresh weight. Ascorbic acid was determined using the dye method and expressed as mg 100 g -1 of fresh fruits [22].

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

In vitro effect of extracts on mycelial growth and spore germination of C. gloeosporioides
Mycelial growth of C. gloeosporioides was significantly (P<0.05) inhibited by methanol extracts of nine plant species (Table 1). The effect of the extracts ranged from weak to strong (shown on 0-4 scale). Strong antifungal activity was exhibited by both leaves and twings methanol extracts of Echinops sp. and Thymus serrulatus. Growth inhibition score of four was recorded for extracts of these plants, indicating complete inhibition of growth and sporulation of the fungus. Echinops sp. had the highest inhibition zone diameter of 13.5 mm, which was then followed by that of Ruta chalepensis, Thymus serrulatus, Vernonia amygdalina and Zingiber officinale (Table 1). Similarly to mycelial growth, there were significant differences among treatments on germination of spores (P<0.05). Among the nine methanol extracts, Echinops sp., Thymus serrultus and Ocimum lamifolium showed strong inhibition with only 1.1%, 2.0% and 2.3% spores germinated, accounting for 98.7, 97.7 and 97.3% inhibition of spore germination over the control, respectively ( Table 1).

Effect of plant extracts on anthracnose development and quality of papaya fruit
All the four aqueous extracts tested significantly reduced anthracnose severity on papaya fruit that had been artificially inoculated with C. gloeosporioides ( Table 2). The severity of anthracnose on a 1-5 scale was 1.3 (which is equivalent to less than 1% fruit area affected) in fruits treated with Echinops sp. extract at a concentration of 25% that was statistically at par with that of the positive control (carbendazim) after 14 days of incubation.   Fruits treated with 25% aqueous extract of Echinops sp. had pH and TSS values of 5.57 and 7.8, respectively, which are statistically at par with that of carbendazim treated fruit. The highest TSS was recorded from the untreated control. There was no difference in terms of TA among fruits treated with different concentrations of V. amygdalina, R. chalepensis and Thymus serrultus. However, Echinops sp. at a concentration of 25% resulted in TA value comparable to that of fruits treated with carbendazim. In general, fruits treated with aqueous extracts of plants had higher titrable acidity and ascorbic acid content than the untreated control (Table 2). Fruits in the untreated control ripened quickly and this led to the reduction of titrable acidity and ascorbic acid content and increase in the pH and total soluble solid contents of papaya fruits.

Discussion
The result demonstrated that compounds extracted from plants vary in their efficacy in inhibiting C. gloeosporioides growth which is likely to be due to variability on the availability and solubility of active compounds. The findings of this study are in agreement with previous reports on the antifungal activity of Echinops sp., Ruta chalepensis, Thymus serrulatus and Artemisia genus [13,14,16,23]. Previous phytochemical investigation of Ruta chalepensis resulted in isolation of a number of alkaloids and coumarins and the active ingredients of this plant have antifungal properties that could prove beneficial to agriculture [12,24].
Crude extracts of Vernonia amygdalina exhibited antifungal activity and the groups of compounds present that were likely to be responsible for the fungitoxicity were identified to be glycosides, saponins and tannins [15]. In addition, extract of Thymus vulgaris and Zingiber officinale oil are reported to inhibit mycelial growth of phytopathogenic fungi [25,26]. Similarly, complete inhibition of Helminthosporium solani, Aspergillu niger, Penicillium digitatum and Mucor piriformis was reported by extract Z. officinale at 25% concentration. The phytochemical analysis of extracts revealed the presence of tannins, phlobatannins, steroids, tarpenes, saponins, flavonoids and alkaloids [17].
The extracts of tested plants showed high inhibition on spore germination percentage compared to untreated control. This agrees with the report of Barrera-Necha et al. [27] which reported the inhibition of C. gloeosporioides spores with essential oil of Ruta chalepensis. Similarly, Anand and Bhaskaran [28] indicated that in Zingiber officinale extract only 39.3 and 29.4% of spores of C. capsici and Alternaria alternata, respectively, germinated. It is noteworthy that inhibition of spore germination by the extracts is desirable towards the management of papaya anthracnose [4].
Increased in incidence and severity of the disease resulted in softening and rotting of fruit tissue which in turn leading to reduction in the marketability of the fruits [29]. A similar trend was observed in present study. The organic acids in papaya are known to be largely citric and malic acids, and the increase in pH during ripening and storage could be due to the metabolic processes of the fruit that result in decrease of these organic acids [30,21]. The reason for increased TSS content during storage is mainly due to conversion of starch into soluble sugar with advances in ripening [21]. Likewise, decrease in acidity during storage demonstrated fruit ripening [31]. Earlier, Selvaraj et al. [30] and Mahmud et al. [21] demonstrated that the ascorbic acid and titrable acidity of papaya first increases then decrease, while pH and the TSS values increase during senescence. In conclusion, extract of Echinops sp. inhibited growth and spore germination of C. gloeosporioides as well as anthracnose development on artificially inoculated papaya fruits and could be used for practical management of papaya anthracnose.