In Vitro and In Vivo Biological Evaluation of Crude Extracts of Dioscorea dumetorum and Psidium guajava Leaves in the Control of Storage Rot of Onion (Allium cepa L.) Bulbs

The antifungal activity of the ethanolic crude leaf extracts of Dioseorea dumetorum and Psidium quajava on the fungal pathogens isolated from infected onion bulbs were investigated in vitro and in vivo. The pathogens were Aspergillus niger and Rhizopus stolonifer. For the in vitro antifungal assay, various concentrations of the extracts ranging from 10g/200ml, 20g/200ml, 30g/200ml, 40g/200ml and 50g/200ml were separately added to PDA media. The fungal pathogens were separately inoculated into the media and incubated for seven days. For the in vivo assay, healthy onion bulbs were properly surface sterilized using 99% ethanol and sodium hypochlorite (bleach). The sterile onion bulbs were then sprayed with the extracts at different concentrations two hours prior to inoculation with the fungal pathogens. Each onion bulb after being treated with different concentration of the extracts of 10g/200ml, 20g/200ml, 30g/200ml, 40g/200ml and 50g/200ml were dipped into beakers containing dissolved spore of each pathogen and incubated for seven days. Results of the in vitro antifungal assay shows that at 10g/200ml – 50g/200ml concentration, ethanolic leaf extracts of D. dumetorum and P. guajava completely inhibited the radial growth of A. niger and R. stolonifer after seven days observation period. For the in vivo antifungal assay, results shows that the extracts had a significant effect (P<0.05) on the mycelial growth of the fungal pathogens at all the different concentrations tested. Pathogen growth inhibition was most effective at 30g/200ml, 40g/200ml and 50g/200ml concentrations. The inhibitory action of the extracts increased/decreased with a corresponding increase/decrease in the concentration of the plant extracts. Ethanolic extracts of D. dumetorum was found to be more effective in inhibiting the growth of the fungal pathogens than P. guajava extracts.


INTRODUCTION
The onion (Allium cepa L.), also known as bulb (Fritsch and Friesen, 2002), or common onion, is one of the most widely cultivated species of the genus Allium. The species is probably native to South-western Asia but is now commercially grown throughout the world, chiefly in the temperate zones (Ozer and Koycu, 2004). The onion often called as "queen of kitchen" is commonly used for cooking by almost all the people. It ensures excellent taste to dishes and also exhibits a number of therapeutic properties such as antibacterial, antifungal, antihelmentic, anti-inflammatory, antiseptic and antispasmodic. Due to its enormous commercial and medicinal value, the plant is now cultivated in almost all countries of the world and consumed across the globe (Rai and Yadar, 2006).
Onion belongs to the Lily family, Alliaceae, however, some classification place it in the family Liliaceae (Fritsch and Friesen, 2002). Modern varieties of onion typically grow to a height of 15 to 45cm (6 to 18inch) with real stem very short, formed at the base of the plant in the form of a disk with adventitious root at the base, the leaves are yellowish-green and grow alternately in a flattened, fan-shaped varying from globule to ovoid to oblate up to 20cm in diameter (Brickell, 2002). The species is biennial but usually grown as annuals in the temperate climates. In Nigeria, it is mostly cultivated during the dry season (October to April) and as such are easily propagated, transported and stored (Zohary and Hopf, 2000).
Common onions are normally available in three colour varieties ranging from yellow or brown onions (called red in some European countries), are full flavour red and are the onions of choice for everyday use. With its pungent aroma and strong flavour, it is a good all-round onion. Their varieties are grown in countries like Australia, Nigeria and they vary from cream gold and muray brown. Other varieties of onion crop also include Red onion; these are sometimes called Spanish onions, which have purplish red skin and white flesh tinged with red. This onion tends to be medium to large size and can have a mild to sweet flavour but after being stored for a short time can become quite pungent. They are often consumed raw, grilled or lightly cooked with other foods or added as colour to salads, these variety of onion plant can be stored three to four months under ideal condition, and their varieties include red shine, red wing and red emperor. Although white onions are considered to be the strongest in flavour after brown onions, their varieties vary in size, skin characteristics and flavour; they include Bianca, Gladalan white and white Spanish (Mower, 2013).
Onion is a valuable ingredient in diet due to its content of sugars, vitamins and minerals (Ole et al., 2004). Most species of onion contain low amount of essential nutrient, are low in fats and have an energy value of 166KJ (40 cal) per 100g. The onion cultivars are about 89% water, 4% sugar, 1% protein, 2% fiber and 0.1% fat (Fern, 2011). Furthermore, onion contain bioactive compound such as phenol that are under basic research to determine their possible properties in human (Slimestad et al., 2004). Yellow and Red onions have considerable content of anthocyanin pigments with 25 different compounds representing 10% of total flavonoid content (Yang et al., 2004). As a result of the bioactive compounds contained in onion, some people suffer from allergic reaction after handling them. Some of the allergic reactions include eye irritation, bronchial asthma, blurred vision, intense itching etc. This stinging sensation can be avoided by cutting onions under running water or submerged in a basin of water (Williamson et al., 1997).
Onion as an important vegetable crop is eaten all over the world. They play the role of being antioxidant and cardio-protective in nature as a result is rich in vitamin B, C, E and other trace elements (Conant, 2006). Recent report suggest that onion plays an important role in preventing cancer, diabetes and even common cold and are also reputed to increase fertility in men and milk in nursing mothers, more so to cure headache when mixed with milk and vinegar (Rai and Yadar, 2006). In addition, Allium Species are used as traditional medicine in countries like Europe, North Asia and Africa to treat intestinal worms, gastroenteritis, heart diseases and other ailments (Brewster,1999) planting them closely to other vulnerable crops such as carrot discourages insect attack on those crops (Wane and Taskeen, 2011).
Onions have greater water and sugar content that gives them their sweeter-and milder-tasting but reduces their shelf life (Olsson et al., 2010). Onion are better stored at room temperature, optimally in a single layer, in mesh bags in a dry cool, dark , well-ventilated location, storing them in a cool dry place with plenty of circulating air can help prolong their shelf life approximately one to five months irrespective of type (Jauron, 2009). For propagation, the onion plant are monocotyledons, they sometimes behave like the annuals, by setting seed-bolting in their first year. They grow from the base of their leaves pushing their leaves up and out from the base, by contrast, grow outward from the edges of the leaves (Savon et al., 2006), may also be grown from seed or seed sets ranging from 3.5 to 4.5kg/ha when transplanted, 4 to 6kg/ha when direct seeded with spacing usually 25-40cm between rows.
Onions are adapted to moist soils where nutrients are in sufficient quantity; viability falls rapidly in hot humid conditions and this may be 0% in less than a year especially in their F 1 hybrid (klaas and Friesen, 1998).
Fungi especially moulds are important pathogens of fruit and vegetables particularly under tropical and subtropical conditions (Adebayo and Diyaulo, 2003). The effect of storage rots includes reduction in the quantity and quality of onion which affects the markets value (Adebayo and Diyaulo, 2003). Black mould disease caused by Aspergillus niger Van Tieghem is a limiting factor in onion production worldwide (Ozer and Koycu, 2004). Aspergillus niger Van Tieghem is a 82 IJPPE Volume 1 high temperature fungus, with an optimum temperature range from growth 28-34 o C, are ubiquitous in occurrence, it attacks/infect bulbs of onion in field/storage, whenever they find injured tissues by producing various toxins (Srinivasan and Shanmugam, 2006). Other fungal pathogens in association with post-harvest rot of onion include Neck rot of onion caused by Botrytis allii, white rot and a host of other pathogens (Amienyo and Ataga, 2007). According to Okigbo (2009) and Okigbo and Nmeka (2005), in Nigeria, for instance, about 98% of the food consumed is produced by the local farmers. Most of these farmers are not educated and therefore in most cases cannot practice the various control measures recommended for disease control. In addition to these inadequacies, most of the chemicals are not available and where they are, they are very expensive. These problems have necessitated the search for alternatives to synthetic fungicides. Among the various alternatives, natural plant products that are biodegradable, ecologically, friendly and readily available have therefore been sort by scientists worldwide. Diseases and deterioration by fungal pathogens constitute a menace in the production and storage of onion bulb thereby decreasing their food and market value. In view of this, the aim and objectives of this research work was to isolate and identify the fungal pathogens associated with storage rot of onion bulb as well as evaluate the antifungal potentials of crude extracts of Psidium guajava and Dioscora dumetorum on fungal pathogens associated with postharvest storage rot of onion bulb both in vitro and in vivo.

Sources of materials
Infected and uninfected onion bulbs were obtained from three different markets (Watt, Akim and Marian) in Calabar Metropolis, Cross River State, Nigeria and wrapped in sterile cellophane bags and transported to the Laboratory for further studies. Dioscorea dumetorum and Psidium guajava leaves were obtained from the Botanic Garden of the Department of Botany, University of Calabar, Calabar, Cross River state, Nigeria. The research was carried out in the Laboratory of the Department of Botany, University of Calabar, Calabar, Cross River State, Nigeria.

Isolation of fungal pathogens and morphological identification
Cut sections of the diseased onion bulbs were surface sterilized with 70% sodium hypochlorite (bleach) solution for 1min and rinsed quickly in 3 changes of sterile distilled water, blotted dry on Whatman's No.1 filter paper and placed on Potato Dextrose Agar (PDA) in Petri dishes. Four (4) sections were inoculated per Petri dish. The plates were incubated at 28 ± 1°C until fungal growth was noticed. After 7 days, the different isolates were subcultured on freshly prepared PDA to obtain their pure culture. Isolated fungi were microscopically (Olympus optical, Phillipines) identified as far as possible using the identification guides of the International Mycological Institute, Kew, Barnett and Hunter, (1998), Dugan, (2006).

Pathogenicity test and Koch's postulates
Pathogenicity test was carried out using the method of Amienyo and Ataga (2006). Healthy onion bulbs were washed in distilled water and surface sterilized with 70% Sodium hypochlorite solution. A 5mm diameter cork borer was used to cut disc from the bulbs (three discs per bulb) and cultures of the isolates (four days old) were introduced into bulbss and replaced with the discs. The discs portions were sealed with Vaseline to prevent entry by other pathogens. They were kept for 24-48 hours wrapped in sterilized cellophane bags. A sterile cotton wool was soaked with distilled water and dropped inside the cellophane bags to enable the organism to breath (respire). The

International Journal of Pharmacology, Phytochemistry and Ethnomedicine
Vol. 1 inoculated fruits established symptoms after 24 hours, tissue segment from the healthy and infected bulbs was excised, surface sterilized and plated into freshly prepared PDA and incubated at 28+1 o C for four days. Pure cultures of the isolate were identified.

Preparation of plant extracts
Leaves of Psidium guajava and Dioscorea dumetorum obtained were washed with distilled water and oven dried at a temperature of 80 o C for 24 hours grounded into fine powder and extracted separately using 200ml of 99% concentration of ethanol.
In vitro antifungal assay 5ml of each concentration was first poured into different Petri dishes using sterile syringe. The sterile Potato Dextrose Agar (PDA) was also poured into the plates containing the solvent extracts after which the plates containing the solvent extracts were gently swirled to ensure mixing. The media was allowed to solidify and with a sterilized No.2 cork borer of 5.5mm in diameter, a disc of the matured culture was punched out and inoculated at the centre of plates and incubated at room temperature of (28+1 o C) for 7days. As a control, the dishes were inoculated on PDA instead of extracts-agar mix. Three (3) control plates were prepared for each extract. Measurement of the mycelia radial growth was done daily for seven days (Umana et al., 2014).

In vivo antifungal assay
In vivo antifungal assay of the plant extracts was carried out using the method described by Poppe et al., (2003) with some modifications. Whole healthy onion bulbs were properly surfaced sterilized using 99% ethanol rinsed with distilled water and allowed to dry for 2-3minutes. The sterile bulbs were then sprayed with the extracts at different concentrations, two hours prior to inoculation with the fungal pathogens. Each bulb after being treated with different concentrations of the extracts of 10g/200ml, 20g/200ml, 30g/200ml, 40g/200ml and 50g/200ml were dipped into beaker containing dissolved spores of each pathogen. The bulbs were then allowed to dry for 2-5minutes, after which each bulb was incubated at room temperature (28+1 o C) for twenty-eight days. The control which was also dipped in beakers containing the dissolved spores of each pathogen was not treated with any of the extracts. Growth rate were measured after one week of inoculation and was repeated after every four days for four times.

Identified fungal pathogens
The fungal pathogens isolated and identified as the causative agents of storage rot of onion bulbs from this study and used were Aspergillus niger and Rhizopus stolonifer.

Pathogenicity test and Koch's postulates
Result from the pathogenicity confirmed that the two fungal isolates Aspergillus niger and Rhizopus stolonifer were responsible for the storage rot of onion bulbs obtained from three major markets in Calabar Metropolis of Cross River State, Nigeria. Pathogenicity was established 24 -48 hours after inoculation (Plate 1).

In vitro effect of the plant extracts on the radial growth of A. niger and R. stolonifer at the different concentrations.
The in vitro effect of the crude plant extracts at the different concentrations on the radial growth of the fungal isolates is presented in (Tables 1 -4). Results showed that, at 10g/200ml, 20g/200ml, 30g/200ml, 40g/200ml and 50g/200ml D. dumetorum and P. guajava extracts completely inhibited the radial growth of Aspergillus niger and Rhizopus stolonifer at the different concentrations tested when compared with the control (Plate 2).

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In vivo effect of the crude plant extracts on the growth and sporulation of A. niger and R. stolonifer at the different concentrations.
The in vivo effect of the plant extracts at different concentrations on the radial growth of the fungal isolates is presented in (Tables 5-8). Investigations from the study showed that extracts of Dioscorea dumetorum and Psidium guajava had a significant effect on the isolated pathogens at all the concentration levels (10g/200ml, 20g/200ml, 20g/200ml, 40g/200ml and 50g/200ml) tested when compared with the control. Results (Table 5) showed that at 30g/200ml, 40g/200ml and 50g/200ml concentrations, Dioscorea dumetorum extract completely inhibited the radial growth of Aspergillus niger on the first and second day of incubation. However, at 50g/200ml concentration, extract of D. dumetorum completely inhibited the radial growth of A. niger in the first four days when compared with the control. While results ( Table 6) showed that D. dumetorum extract completely inhibited the radial growth of Rhizopus stolonifer at 40g/200ml and 50g/200ml concentrations on the first and second day and at 50g/200ml on the third day of incubation respectively. Results (Table 5-8) showed that there was a corresponding increase in the antifungal activity of the plant extracts as the concentration of the extracts increased from 10g/200ml to 50g/200ml. Results (Table 7 and 8) showed that Psiduim guajava extract completely inhibited Aspergillus niger at 50g/200ml concentration on the first and second day of incubation. While at 50g/200ml Psidium guajava extract inhibited the radial growth of R. stolonifer at 50g/200ml concentration only on the first day of incubation. Results therefore, showed that Dioscorea dumetorum leaf extracts was more effective inhibiting the growth of Aspergillus niger and Rhizopus stolonifer than those of Psidium guajava. Results also showed that the extracts were more effective in inhibiting the growth of Aspergillus niger than that of Rhizopus stolonifer.

Concentration
Incubation period (Days) and radial growth (cm 2 )

DISCUSSION
The efficacy of two plant extracts was tested in vitro and in vivo against the radial growth of two pathogenic fungi associated with post harvest storage rot of onion bulb. The fungal pathogens isolated were identified as Aspergillus niger and Rhizopus Stolonifer. The results of this study revealed that the fungi were responsible for the post harvest rot of onion bulbs in Calabar Metropolis as evidenced by the pathogenicity tests. The fungal spores of these pathogens could be air-borne and therefore spread by wind and may land on susceptible host like onion bulbs, fruits and other plants. The pathogens (A. niger and R. stolonifer) were reported by Shehu and Muhamed (2011) to cause storage rot fungi of onion bulb. Also Adebayo and Diyaulo (2003) reported (A. niger and R. stolonifer) as pathogens of post harvest rot of onion, which is in agreement with the finding of this study. The in vitro and in vivo inhibitory effects of D. dumetorum and P. guajava extracts on the isolated fungal pathogens are presented in (Tables 1-8). The results showed that, the extracts significantly (P<0.05) inhibited the radial growth of the fungal pathogens at the different concentrations tested and the rate of inhibition differed from one extract to the other. However, results, (Table 1-4) showed that in the invitro study, the plant extracts completely inhibited the radial growth of the fungal isolates at all the different concentration levels tested, while in the in vivo, increase in antifungal activity was observed with the corresponding increase in the concentrations of all the plant extracts (Table 5-

CONCLUSION
The fungal pathogens isolated and identified from this study as the causative agents of postharvest storage rot of onion bulb obtained from different markets in Calabar Metropolis of Cross River State were Aspergillus niger and Rhizopus stolonifer. Pathogenicity test confirmed that these pathogens actually caused storage rot of onion bulb. The efficacy of the crude plant extracts (Dioscorea dumetorum and Psidium guajava) against storage rot fungi was tested in vitro and in vivo. The in vitro results showed that the extracts significantly inhibited the mycelia growth of the fungal pathogens at all the concentrations tested when compared with the control. However, in the in vivo assay, the crude plant extracts significantly (p≤ 0.05) inhibited the mycelia growth of the fungal isolates at the higher different concentrations tested and the rate of inhibition differed from each other. The extracts showed significant effect on the growth and sporulation of A. niger and R. stolonifer both in vitro and in vivo at all the concentration levels tested and as such can be used in the control of postharvest storage rot of onion bulb.