1 Introduction

Plants have always been faced with potential deleterious organisms such as fungi, bacteria, viruses and nematodes responsible for crop losses worldwide. Synthetic fungicides are primary means to control postharvest diseases. They are used alone, combined in mixtures or applied separately in sequence (Ismail and Zhang, 2004). Nevertheless, several fungicides have been removed from the market due to possible toxicological risks. Over recent decades, there has increasing public pressure to reduce the use of synthetic fungicides in agricultural products and their presence in the environment (Cowan, 1999). The use of botanicals has hence been used in recent times as an alternative treatment to inhibit the growth of fungi on postharvest fruits. The ultimate aim of recent research in postharvest control has been the development of alternative control strategies to reduce dependency on synthetic fungicides. Plants have ability to synthesize aromatic secondary metabolites, like phenols, phenolic acids, quinones, flavones, flavonoids flavonols, tannins and coumarins (Cowan, 1999). The components with phenolic structures, like carvacrol, eugenol, and thymol, were highly active against the pathogen. These groups of compounds show antimicrobial effect and serves as plant defence mechanisms against pathogenic microorganisms (Das et al., 2010). Botanicals are considered for their sustainable solutions in agriculture, reduction of crop losses, eco-friendly, easily bio-degradable, cheaper and their integrated diseases management. They have been shown to possess good efficacy against fungi, bacteria and viruses. This research work therefore focuses on the activities of leaf extracts of cashew and pawpaw against mycelia growth of Aspergillus niger and Aspergillus flavus isolated from decayed cashew fruits. The result obtained is expected to form a prospect for the in vivo control of spoilage pathogens on cashew fruits using these botanicals.

2 Results

2.1 Activities of aqueous leaf extracts of cashew (Anarcadium occidentale) and pawpaw (Carica papaya) against mycelia of Aspergillus niger

On day three of incubation, only pawpaw leaf of 2% concentration among the tested extracts and concentrations had mean zone of 0.00 ± 0.00 cm as its mean zone of inhibition which implied that there was no zone of inhibition (Table 1). However, 2% and 6% cashew leaf extracts had their respective zones of inhibition measured as 0.50 ± 0.388 cm and 0.30 ± 0.388 cm which were not significantly different from each other, Mancozeb with inhibition zone of 0.20 ± 0.549 cm and 6% pawpaw extract with mean zone of inhibition of 0.60 ± 0.388 cm (Table 1). Hence, they were all significantly different from the control with the mean zone of inhibition of 0.00 ± 0.00 cm (Table 1). Hence, the order of the zones of inhibition of A. niger with the tested extracts and concentrations was 6% pawpaw leaf extract > 2% cashew leaf extract > 6% cashew leaf extract > Mancozeb > 2% pawpaw leaf extract.

2.2 Activities of aqueous leaf extracts of cashew (Anarcadium occidentale) and pawpaw (Carica papaya) against mycelia of Aspergillus flavus

Similarly, on day 3 of incubation of Aspergillus flavus, among the tested extracts and concentrations, only 2% pawpaw leaf extract had no mean zone of inhibition (0.00 ± 0.00 cm) (Table 2). Nevertheless, 6% cashew leaf extract had its mean zone of inhibition (1.55 ± 0.096 cm) significantly different from 6% pawpaw leaf extract with inhibition zone of 1.15 ± 0.096 cm, 2% cashew leaf extract with mean zone of inhibition of 1.25 ± 0.096 cm and control plate with 0.00 ± 0.00 cm but significantly not different from Mancozeb with mean zone of inhibition 1.40 ± 0.135 cm (Table 2). Hence, the order of antifungal activities of the tested extracts and concentrations against mycelia of A. flavus was 6% cashew leaf extract > Mancozeb > 2% cashew leaf extract > 6% pawpaw leaf extract > 2% pawpaw leaf extract (Table 2).

2.3 Phytochemical constituents of aqueous leaf extracts of cashew (Anarcadium occidentale) and pawpaw (Carica papaya)

Results of qualitative phytochemical constituents of aqueous leaf extracts of cashew and pawpaw showed that tannin, alkaloid and cardiac glycosides were present in both extracts (Table 3). However, phlobatannin was absent in both cashew and pawpaw leaf extracts. Saponin and flavonoid were present in cashew leaf extract but absent in pawpaw leaf extract (Table 3).

3 Discussion

Aqueous extracts of cashew and pawpaw retarded or inhibited mycelia growth of the species of Aspergillus in vitro. Findings from the research work showed that cashew (Anacardium occidentale) leaf extract had the highest antifungal activity against Aspergillus flavus and the least antifungal activity against Aspergillus niger. The presence of bioactive ingredients in the cashew leaves like tannins, saponins, alkaloids and flavanoids add to their antimicrobial activities. This was supported by the work of Hutchings et al. (2003) and Okorondu et al. (2010) that antimicrobial properties of plant extracts had been attributed to the presence of alkaloids and flavonoids.Phytochemicals with bitter taste such as alkaloids and flavonoids have been found to possess antimicrobial properties and interestingly, results of qualitative analysis of the cashew leaf extract revealed the presence of high concentration of tannins, saponins, flavonoids and alkaloids in the aqueous extract.

The pawpaw (Carica papaya) leaf extracts had no inhibitory zone against the mycelia of Aspergillus flavus but was able to inhibit the mycelia growth of Aspergillus niger using agar well diffusion method. This was supported by the works of Amadioha (1998); Owolade and Osikanlu (1999) and Adejumo et al. (2000) who reported the efficacy of extracts from Carica papaya and Acalypha ciliata in reducing the mycelia growth of Erysiphe cichoracearum, Collectotrichum capsici and Protomycopsis phaseoli. The aqueous extracts investigated revealed the presence of alkaloids, flavonoids, tannins, cardiac glycosides, and reducing sugars. Alabi et al. (1990) had done comparative studies on antimicrobial and anti-fungal property of extracts of fresh and dried leaves of Carica papaya. Their phytochemical analysis indicated presence of alkaloids, tannins, saponoins, glycosides and phenols in the leaf extract. This conformed with preliminary phytochemical screening of the extract as obtained in this work. This agrees with the results of Snowdon (1990) that the crude extracts of this plant possessed some inhibitory components which cause significant reduction in mycelia growth of fungi such as Aspergillus niger and Fusarium solani.

4 Materials and Methods

4.1 Isolation from decayed cashew fruits

Isolation of mycelia of Aspergillus species from decayed cashew fruit was made by cutting out the interface between the healthy and the disease issue and placing pieces of the affected fruit rind without surface sterilization on the plates of solidified Malt Extract Agar (MEA). The plates were then incubated at 28ºC ± 2ºC for 3 days. Subcultures of the isolates were prepared by transferring agar cut with distinct mycelium to sterilize Petri dishes containing solidified MEA and then incubated at 28ºC ± 2ºC until pure culture were obtained. The resulting pure culture was then used for the morphological characterization of each isolate.

4.2 Morphological identification of fungal isolates

A drop of cotton -in- blue lactophenol solution was put on slide. Each isolate was put on a slide and was covered with a cover slip. Excess liquid was drained with a filter paper and the isolate was examined under microscope. Examination was done with x40 objective lens for the presence and type of hyphae, mycelium either dark or clear and spore morphology and each isolate was identified using the text of Alexopoulous et al. (1996).

4.3 Collection and drying of selected plant leaves

Cashew (Anacardium occidentale) and pawpaw (Carica papaya) leaves were obtained from Federal University of Technology Akure environ in Ondo state, Nigeria and brought to the Department of Biology Laboratory, Federal University of Technology Akure. The leaves were spread out and air dried at 28ºC ± 2ºC for a period of 5 days after which they were grinded using a mechanized blender.

4.4 Phytochemical screening of leaf extracts of cashew and pawpaw

Extracts were prepared from the powered sample according to the method of Harbourne (1998) but with slight modification. Water was used for the extraction. For aqueous extraction, exactly 50 g of each powered sample was soaked into 500 mls of water. The solution was allowed to stand for 24 hours after which it was first sieved with a clean muslin cloth and filtered with Whatman No.1 filter paper. The filtrate was collected in a sterile clean beaker and concentrated in vacuo using rotary evaporator (Resona, Germany). The phytochemical screening of each extract was done according to the method described by Trease and Evans (2004). The phytochemicals screened for were tannin, saponin, phlobatannin, flavonoid, alkaloid and cardiac glycosides.

4.5 Preparations of different concentrations of cashew and pawpaw leaf extracts used for antifungal assay

Cashew leaf extract of 2% concentration was prepared by adding 2g of the leaf powder into 8 mls of distilled water. The procedure was also repeated for 2% pawpaw leaf extract but with pawpaw leaf powder. Similarly, 6% cashew leaf extract was prepared by adding 6g of the leaf powder into 4 mls of distilled water. This was also repeated for 6% pawpaw leaf extract but with pawpaw leaf powder. After 24 hours, the mixtures were filtered and the aqueous extracts obtained were then tested for antifungal activities.

4.6 Testing for antifungal action of aqueous leaf extracts of cashew and pawpaw

The antimicrobial activity of each of the leaf extracts was assayed using agar well diffusion method described by Madigan et al. (2002) with slight modification. The mycelium of Aspergillus niger and Aspergillus flavus cut with 6mm diameter cork borer was inoculated separately and aseptically on plate containining solidified MEA. This was done in triplicate for each isolate. Wells of 6 mm were bored on the MEA plates with sterile cork borer, 2 cm away from the inoculum and 2% concentration of each extract was then introduced into the wells on the agar plates. This was also repeated for 6% concentration. The plates were then incubated at 28ºC and observed daily for clear zones which are indicative of the zones of inhibition of the isolate by the extract. Agar well without any leaf extract served as control plate. Mancozeb (a fungicide) was also tested against the mycelia growth of Aspergillus species. This was also done in triplicate.

4.7 Statistical analysis

The data obtained for antifungal activity was subjected to analysis of variance and where significant, the means were compared at 5% level of probability using New Duncan’s Multiple Range Test (SPSS version 20.0).

5 Conclusion

Aqueous extracts used for the study retarded or inhibited mycelia growth of the two species of Aspergillus in vitro. The water – soluble antifungal principles in the plants are responsible for the antifungal activities. The facts that these plants used in this study are easily available, with easy method of extraction, they can be exploited in the control of decay on postharvest fruits. Hence, cashew leaf and pawpaw leaf extracts may serve as potential antifungal alternatives for treatments of cashew fruits against Aspergillus flavus and A. niger respectively.

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