Effect of Storage Methods and Management of Sweet Potato on the Incidence of Tuber Rot Induced by Rhizopus stolonifer in Kano, Nigeria

Different storage methods and management practices of sweet potatoes in Kano state, northwestern Nigeria, were investigated to fi nd out how they may affect the incidence of tuber rot. Three local government areas were selected for the sample collection. In each local government area, two sweet potato farming communities were selected, infected and uninfected tubers were sampled and taken to the laboratory for further studies. Pathogenicity test confi rmed Rhizopus stolonifer as the causal pathogen responsible for tuber rot and this fungus was used for the inoculation of fresh sweet potato tubers in all the storage and management methods used in the experiment. Tubers preserved using ash + sand and fungicide + sand showed the least incidence of tuber rots. Similarly, it was also observed that storage of tubers with sand alone showed a reduction in the severity of the disease across the storage weeks. There was no signifi cant difference in the incidence and severity of tuber rot between the two inoculation methods with both the treatment combinations. Our result showed that the incubation period of R. stolonifer to infect sweet potato tubers started at two days after inoculation with about 25% infection. The result also indicated that none of the tubers treated with fungicide, ash, and eucalyptus sprouted at both the storage weeks, this may be attributed to the possibility of having some inhibitory properties against sprouting.


Introduction
Sweet potato [Ipomea batatas (L.) Lam] is presently ranked the seventh most important crop in the world with a total of about 103 million tonnes in 2013 (FAO, 2015;Sugri et al., 2017). Asia accounts for close to 76% of total world production, followed by Africa (19.5%). The top fi ve sweet potato producing countries in the world are China, Nigeria, Uganda, Indonesia, and Tanzania with China being the leading producer with about 75.6 million tonnes, followed by Tanzania and Nigeria that produce around 3.57 and 2.73 million tonnes, respectively (Sugri et al., 2017). In Nigeria, sweet potato is one of the six most important root and tuber crops, other root crops are cassava, yam, Irish potato, cocoyam, and ginger. Although the sweet potato leaves are edible, the starchy tubers are by far the most important product (Bidzakin et al., 2014). The roots are mostly boiled, fried, roasted or baked for their rich sources of dietary energy (Oduola et al., 2018;Olaitan, 2012), quite essential for their beta carotene and vitamin C (Motsa et al., 2015;Naico and Lusk, 2010), and it is an imperative source of antioxidants and anthocyanidins (Oladoye et al., 2013). However, sweet potato is a highly perishable food source that is susceptible to destruction my microorganisms, metabolic spoilage, physical destruction and pest infestation after harvesting. The production of perishable products in Nigeria can be improved by increasing productivity and avoiding losses caused by storage rots (Echerenwa and Umechuruba, 2004). soften the tuber textures which make them susceptible to damage and microbial decay (Munish et al., 2006). Postharvest quality deterioration emanates from weight loss, weevil damage, sprouting and attack by microorganisms (Singh et al., 2016) with postharvest rots being predominantly reported as the main cause of post-harvest loss (da Silva and Clark, 2013;Olaitan, 2012). A wide variety of microorganisms, mostly moulds, have been implicated in tuber spoilage but few are implicated as primary pathogens (da Silva and Clark, 2013). The fungi associated with sweet potato rots comprises; Aspergillus niger, Fusarium oxysporum, Rhizopus stolonifer, Botryodiplodia theobroma and Penicillium sp. (Olaitan, 2012).
Rhizopus stolonifer (Zygomycetes), commonly known as the black bread mould, is a fungus which survives in soils and debris in the farm or around houses. Postharvest infection by this secondary pathogen commonly occurs in fruits or tubers pierced by insects or affected by physiological disorders or mechanical injuries (Agrios, 2005). Once a spore lands on a wounded tissue, it germinates and starts growing on the surface, producing a thick mycelium which at the same time produces cell degrading enzymes (amylases and pectinases) that denature the tissue, thus results in infection (Bautista-Baños et al., 2008).
Currently, both the traditional and modifi ed traditional methods of storage are practiced in most of sub-Saharan Africa and some developing countries (Abidin et al., 2016;Sowley et al., 2015). These include in-ground storage, heap storage, platform storage, pit storage, and under shade covering with grass amongst others (Dandago and Gungula, 2011). In some instances, sawdust and ash are added to improve the shelf-life (Amoah et al., 2011). In addition, fungicides such as Dichloronitroanline are used to protect tubers against Rhizopus soft rot. However, the use of synthetic fungicides, apart from their potential danger to both the farmer and the environment, are unaffordable by most farmers (Obagwu et al., 1997). Based on these notions this study was conducted in order to evaluate the effi cacy of using locally available materials and fungicide (used as a control) in the management of tuber rots on sweet potato.
The main objectives of this study were to identify the fungus associated with post-harvest deterioration in sweet potato in selected areas of Kano state, Nigeria, and to evaluate the effi cacy of locally available materials in the storage and the management of tuber rot of sweet potato.

Experimental Site
The experiment was conducted between November 2014 and February 2015 at the Pathology Laboratory of Crop Protection Department, Faculty of Agriculture, Bayero University, Kano, located at latitude 11˚ 58ˈ N and longitude 8˚ 25ˈE situated at 475 m above sea level in Sudan savannah ecological zone of Nigeria.

Collection of Sweet Potato Tubers
Tubers were collected from the six sweet potato producing areas of Kano state. Three local government areas were selected for the sampling, with two locations selected from each local government; namely Kumbotso L.

Collection of Materials for Tuber Rot Treatments
Fresh leaves of Eucalyptus camaldulensis, coarse sand and wood-ash were collected from the Faculty of Agriculture environs, Bayero University, Kano, while fungicide (Tricyclazole 75% WP) was purchased from Sabon Gari market, in Kano metropolis.

Isolation and Identifi cation of Fungal Pathogens Associated with Potato Tuber Rot
Infected potato tubers were cut toward the advance margin between the healthy and infected portions of the tubers with a surface sterilized knife; then were further cut into smaller portions of about 5 mm with a sterilized scalpel. The sections were surface sterilized in 1% sodium hypochlorite and rinsed with several changes of sterile distilled water. They were then plated on petri dishes containing potato dextrose agar (PDA) incorporated with streptomycin. The culture plates were incubated at room temperature (28˚ ± 2˚C) for seven days and observed daily for fungal development. The developing fungus was sub-cultured and pure cultures were obtained and kept for further use. The fungus was viewed under a microscope and identifi ed according to the morphology using an illustrated genera of imperfect fungi, fourth edition identifi cation manual as a guide (Barnett and Hunter, 1998). Based on the morphology, Rhizopus stolonifer was identifi ed to be associated with the deteriorating sweet potato tubers.

Pathogenicity and Determination of Incubation Period of Rhizopus stolonifera
To determine the pathogenicity of Rhizopus stolonifer fresh, healthy sweet potato tubers were inoculated with the fungus. This was done by washing the tubers with tap water, rinsed with distilled water and surface sterilized with 60% ethanol before a 5mm cylindrical disc was removed from the tuber using a cork borer. A disc of a seven days old culture of the isolated fungus was transferred into the holes created in the tubers. Cello tape was used to completely seal the opening of the hole created. The inoculated tubers were then incubated at room temperature (28˚C ± 2˚C) for seven days, and examined for infection and disease development.

Preparation of Materials for the Root Rot Treatments
The collected leaves of Eucalyptus camaldulensis were thoroughly washed with tap water and rinsed in sterile water. These leaves were then dried and grounded using a household blender (Binaton model). Twenty grams (20g) of each sample (Eucalyptus leaves, wood ash, and fungicide) were added to one litre of distilled water separately. These suspensions were vigorously stirred and later used in treating the tubers.

Evaluation of Storage Methods
In this experiment, two storage methods were used; treated tubers and stored in sand, untreated tubers and stored in sand, and control (untreated tubers and not stored in sand).

Treated Tubers and Sand Storage
Two hundred grams (200 g) of sweet potato tubers replicated four times were immersed into the suspensions/solutions of Eucalyptus leaves, wood ash or fungicide then allowed to stay for 10 minutes, removed and dried under shade. These tubers were then placed in plastic containers either containing 2/3 fi lled coarse sand or left in the container without any treatment and not covered with sand (control). These treatments were left for three weeks before observation. The treatment combinations are Treatment 1: sand + tuber treated with fungicide Treatment 2: sand + tuber treated with eucalyptus Treatment 3: sand + tuber treated with ash Treatment 4: sand alone Treatment 5: control (untreated tubers stored in container without sand)

Effi cacy of the Materials Used in Storage on Injured and Uninjured Tubers
One-hundred grams of sweet potato tubers were washed with tap water and rinsed with sterile water, dried under shade and surface sterilized with 10% ethanol. These tubers were treated with the suspension treatments prepared from Eucalyptus, wood ash and fungicide respectively. Injury on the tubers were infl icted artifi cially using a sterile cork borer, the fungus Rhizopus stolonifer was then introduced into the tuber and fi nally sealed with cello tape to avoid external contaminants. Another set of tubers were treated also with the suspensions but the fungus was smeared on the surfaces of the tubers. Data was collected at two days interval for ten days. The experimental units were arranged using a Complete Randomized Design (CRD) and the treatments were replicated four times.

Determination of Incidence and Severity on Sweet Potato Inoculated Tubers
The disease incidence (expressed as a percentage) and severity of the tubers infected with rot were recorded. Disease incidence was calculated using the formula: Disease incidence = Number of visible infected tubers × 100% Total number of tubers assessed A scale of 1 to 5 employed by Eze and Mauewesi (1990) was used with slight modifi cations to determine the disease severity as indicated below: 1: No disease 2: 1% area affected 3: 10% area affected 4: 25% area affected 5: >50% area affected

Data Analysis
Data collected were subjected to analysis of variance (ANOVA) using an SAS software version 9.1.3 and means were separated using Fisher's protected least signifi cant difference test at 5% level of signifi cant (P ≤ 0.05) and standard error (SE) where necessary.

Identifi cation of the Fungus and Pathogenicity Test
Based on the fungal morphology by Barnett and Hunter (1998) we identifi ed Rhizopus stolonifer as the fungus associated for the tuber rot of sweet potato. The pathogenicity test confi rmed the fungus as the causal agent of the tuber rot.

Effect of Storage Methods on the Incidence of Tuber Rot
As shown in Table 1, there was no symptoms of tuber rots observed in week one. However, treating the tubers with Eucalyptus + Sand showed a signifi cantly high disease incidence (100%) as compared to the control which showed no disease incidence (0%). In week three, ash + sand, fungicide + sand showed the least incidence of tuber rot (0%) however, they were statistically similar to sand alone. Sand alone and control are also statistically similar in week three.

Severity of Tuber Rots after Inoculation
The result of the severity of sweet potato tuber rot (Table 2) showed that there was no signifi cant difference between the treatments in week one. In week two, all the treatments were statistically similar except for eucalyptus + sand. In week three, sand + ash had the lowest severity of tuber rot however it was not signifi cantly different from fungicide + sand and sand alone.

Incidence on Tuber Rots on Inoculated Tubers
There was no signifi cant difference between the two inoculations methods in the incidence of tuber rot at both the treatments combinations (Table 3).

Severity of Tuber Rots on Inoculated Tubers
There was no signifi cant difference in the severity of the treatments in the two inoculation methods ( Table  4).

Determination of Incubation and Infection Period of Sweet Potato Tuber Rot
We evaluated the incubation period of the sweet potato rot on the inoculated tubers. The result indicated that there was no infection shown one day after inoculation (Figure 1). However, after two days of inoculation 25% of the tubers were infected and by the fourth day, 50% of tubers inoculated showed sign of rot. By day fi ve, 70% of the tubers were infected and were completely rotten.

Effect of Preservation and Storage Method
The result in Table 5 indicated that in the fi rst week some of the tubers treated with sand alone and the control started to sprout and this continued on in the third week. It was also observed that none of the tubers treated with fungicide, wood ash and eucalyptus sprouted.

Effect of Preservation and Storage Methods on Tuber Weight Loss
The tubers treated with Eucalyptus lost most of their weight (4.64%) as a result of rotting and drying; and also the same with fungicide treated tubers, even though the rate of rotten varies ( Figure 2); the least weight was from the sand alone 1.86 g (0.93%) followed by the control 3.66 g (1.80%) as a result of sprouting from the tubers. Fungicide and ash seem to  have inhibited sprouting; therefore the weight loss is only by drying which is less than 1% as that of sand and the control.
In this study, we focused on the different methods of storing sweet potato tubers as well as identifying the best possible control practices to limit postharvest loss due to tuber rots. Our result indicated that Rhizopus stolonifer was the causal agent for the sweet potato tuber rot. This is in line with several reports showing tuber rot of sweet potato caused by Rhizopus stolonifer during storage (Olaitan, 2012;Onuegbu, 2002;Oyewale, 2006), and they also suggest that the pathogen has the potential to cause severe damage to sweet potato tubers during postharvest handling when the condition is favourable. The effect of the preservation methods and the storage methods used on the incidence of tuber rot showed no signifi cant difference at week one in all the treatments (Table 1)    are statistically the same except Eucalyptus + sand which showed a high incidence of the disease. The best preservation method was observed in ash +sand, fungicide + sand at three weeks after storage. For the effects of these storage methods on the severity of tuber rot, the same trend was observed as in incidence at week one and two respectively ( Table 2). On the third week, sand + ash had the lowest severity of the disease, even though it did not differ from fungicide + sand and preserving on sand alone. The result of this study confi rms the fi nding of Amoah et al. (2011) who reported the effectiveness of wood ash and sawdust in the storage performance of sweet potatoes. Similarly, storing sweet potatoes in fresh river sand showed good storage potential for up to 5 months without been damaged or infected (Dandago and Gungula, 2011). Our result showed that treatment with wood-ash + sand or sand alone is capable of preventing tuber rot, signifying that ash and or sand may have fungi toxic effects which probably explain why it was able to prevent microbial growth than any other treatment combination.
No signifi cant difference was observed between the severity and incidence of tuber rot in all the treatments combination in both smears and injured inoculation methods across the post-inoculation days (Table  3 and 4). The mechanism behind non-signifi cant differences on these two inoculation methods is not clearly known. It may be attributed to the short duration of the experiment coupled with treatments combination that may have likely supressed the activities of the microbes. However, differences were observed between the means as compared by the standard error.
Our result showed the incubation period of Rhizopus stolonifer to infect sweet potato started at two days after inoculation with 25% infection (Figure 1). Infected tubers were found to be completely rotten at fi ve days after inoculation with about 70% level of infection. A similar pattern of disease infection with increasing days of storage was observed on sweet potato tubers stored after treatments for over a time (Oladoye et al., 2016). We suggested that Rhizopus stolonifer can infect within the shortest possible time as fast as 48hours, therefore management strategies should be applied even when tubers are to be stored for a short period of time. The result also indicated that none of the tubers treated with fungicide, ash, and eucalyptus sprouted at both the storage week (Table 5), this may be attributed to the possibility of these treatments having some inhibitory properties against sprouting. This is in line with Okigbo and Nmeka, (2005) who reported the effi cacy of Xylopia aethiopica and Zingiber offi cinale leaf extracts in controlling tuber rot of yam. Tuber treated with Eucalyptus lost most of their weight as a result of rotting and drying and the same scenario was also observed with the fungicide treated tubers. Sand treated tubers recorded the least weight loss of less than 1% (Figure 2). This is similar to the fi nding of Dandago and Gungula (2011) that fresh river sand is a good storage method used to keep sweet potato tubers for a long time without losing weight or reduction in quality or taste.

Conclusion
The results of this study revealed that Rhizopus stolonifer was found to be responsible for the sweet potato tuber rot. Based on the study, wood-ash + sand, fungicide + sand and sand alone are the more effective methods in suppressing Rhizopus stolonifer during storage of sweet potato. Results of this study can be useful to farmers in the postharvest storage of sweet potato so as to minimize losses caused by Rhizopus stolonifer. It was also found that the Rhizopus stolonifer can infect tubers as low as two days post inoculation period, therefore management strategies should be applied when tubers are to be stored even for a short period of time to avoid damage or infection. A mixture of fungicide with ash and eucalyptus leaf extract was found to suppress the sweet potato tubers from sprouting, suggesting that these two treatments are effective in managing the tuber rots successfully.