Preliminary investigations on the control of yam (Dioscorea rotundata Poir) tuber rot through nanoscience

1 Introduction

Yam, a starchy tuber of the genus Dioscorea, is a staple food in the tropics and some parts of Europe and America. The southern part of Nigeria is known for high-volume production of yam where a high premium is attached to yam-based foods [1]. Over the last decade, yam became an export crop in most parts of the yam belt of West Africa, following high demand from other countries of the world. The post-harvest losses of yam, which occur shortly after harvest, have been a major challenge to the production and marketing of yams [2]. These losses are usually associated with microbial attack by rot-causing micro-organisms whose activities were exacerbated by mechanical injuries inflicted on yam tubers, which probably occurred during harvesting [3]. Research has shown that most of the post-harvest losses in yams result majorly from microbial attacks [4]. In south-eastern Nigeria, yams are stored in bans, carefully separated from each other under the shade until the next planting season. Storage or preservation of fresh yam tuber in low-temperature facilities such as refrigerators has been shown to keep the yam but causes dehydration, which induces deterioration when the yam is returned to room temperature [4]. Application of other preservation methods such as irradiation [4] has been reported to influence the physiological activities such as dormancy extension and slow rate of sprouting, which causes serious weight loss of stored yam tubers. Losses through microbial attack or pathogenic organisms are not readily controlled by the aforementioned technologies.

Besides, a number of micro-organisms, such as Botryodiplodia theobromae, Fusarium moniliforme, Penicillim sclerotigenum, Rosellina bunnode, Aspergillus niger, Hendersonula forulvidae, Macrophomina phaseoli, and Rhizopus nodosus, have been implicated as the major causes of yam rot in storage [5]. The use of synthetic chemicals of fungicide and bactericide in the control of microbial attacks of crops and crop products has been reported; however, these chemicals have been shown to have toxic effects on humans and the environment, in addition to declining potency of the chemicals following resistance by the target organisms [5]. Recently, botanical extracts (organic chemicals) with anti-fungal and anti-bacterial properties were reported to be effective in the control of some pathogenic organisms especially in the post-harvest management of crop and crop products [6]. These organic chemicals are non-toxic, readily available, cost-effective, and highly degradable [6]. The notable plants from where the organic chemicals are extracted include but are not limited to Azadirachta indica, Elaeis guineensis, Cassia alata, and Moringa oleifera [7]. Application of chemical treatments such as gibberellic acid, thiabendazole, and deltamethine on stored yam has been reported to be effective; however, they are imported products and may not even be affordable for the rural farmers that produce the yam [8]. There is, therefore, the need to develop a technology that is non-toxic to the environment, cost-effective, and could easily be adopted by rural farmers.

The application of low-cost nanobiotechnology – a combination of silver nitrate and neem Azadirachta indica leaf extract, otherwise termed AgNeemnano – might provide a viable solution to the post-harvest problems of yam. Each of the constituent elements has variously been shown to have anti-bacterial and anti-fungal properties [9, 10]. Nanobiotechnology is a combination of organic and metallic nanoparticles whose chemical property is far different from each constituent element [11]. Nanoparticles hold great potentials in various contexts. This study, therefore, sets to investigate the potential of nanobiotechnology (AgNeemnano) in the control of rots of fresh yam tubers in storage.

The objectives of this study were as follows:

1. To test the bioactivities of neem (Azadirachta indica) leaf extracts and silver nitrate (AgNeemnano) solution on rot-causing organisms of yam (Dioscorea spp.); and

2. To ascertain the minimum inhibitory concentration (MIC) of the AgNeemnano solution that inhibits the growth of rot-causing organisms in stored yam.

2 Materials and methods

Two experiments were conducted in the Department of Crop Science, University of Nigeria, Nsukka. Nsukka is located on a plateau by latitude 6°52′N and longitude 7°23′E on an altitude of 400 m above sea level. The experiments involved in vitro and in vivo trials conducted with a decaying yam tuber suspected to have been infested with rot-causing micro-organisms. The tuber had soft rot that covered about 25% of the entire yam tuber. Prior to the trials, the yam was kept in a humid environment so as to create a conducive ground for micro-organism infestation. Other materials were silver nitrate salt and neem (Azadirachta indica) leaf extracts. The fresh leaves of neem (A. indica) were harvested from the botanical garden of the Department of Plant Science and Biotechnology, University of Nigeria, Nsukka.

3 Results

The particle size distributions of the neem broth, silver nitrate, and the combination of neem and silver nitrate are shown in Figures 1–3, respectively. Neem has variable particle sizes, which were larger than the silver nitrate particles. A combination of the two substances (AgNeemnano) resulted in a colour change different from either the neem or silver nitrate, and the particle sizes were in between the sizes of neem and silver nitrate.

Figure 1:

Particle size distribution of neem broth.

Figure 2:

Particle size distribution of silver nitrate.

Figure 3:

Particle size distribution of AgNeemnano.

The rot-causing organisms found in yam tuber in the study area at the time of this study were Fusarium moniliforme and Lasiodiplodia theobromae. The bioactivities of AgNeemnano on F. moniliforme varied with the varying concentration of the solution (Table 1). At the concentration of 28.40 mg/ml, the IZD was 13.0 mm but increasing the concentration to 227.27 mg/ml resulted in an IZD of 20.0 mm. Similarly, varying the concentration of AgNeemnano affected its activities on L. theobromae (Table 2). The MIC of the AgNeemnano that inhibited the mycelial growth of F. moniliforme was about 0.9 mg/ml as shown in Figure 4, while the MIC of AgNeemnano for L. theobromae was <0.5 mg/ml (Figure 5). Doubling the concentration did not show a corresponding increase in the IZD. When the concentration was increased from 56.81 to 113.63 mg/ml for Fusarium moniliforme, the CUE was only 3 mm. When this concentration was doubled or increased to 227.27 mg/ml, the IZD increased in a decreasing trend with reduction in CUE. The cut surfaces of the untreated yam tuber appeared white or colourless, while the cut surface of tuber treated with AgNeemnano appeared dark brown (Figure 6). The isolated micro-organisms that were found in the yam tubers as captured by the Moticam camera had large particle sizes (Figure 7).

Figure 4:

Graph of the log concentration and IZD showing the point of MIC (in mg/ml) of AgNeemnano solution against Fusarium moniliforme.

Figure 5:

Graph of the log concentration and IZD showing the point of MIC (in mg/ml) of AgNeemnano solution against Lasiodiplodia theobromae.

Figure 6:

Photograph of untreated tuber (A) and tuber treated with AgNeemnano solution (B) against Fusarium moniliforme and Lasiodiplodia theobromae.

Figure 7:

Microphotographs of the organisms associated with soft rot in yam tuber (Dioscorea rotundata).

4 Discussion

The individual substances (neem broth and silver nitrate) used in this study had a peculiar colour and different particle sizes; however, when they were mixed together to generate AgNeemnano, a different colour and a different particle size were obtained. This is evidently a complete chemical reaction with a new product that has properties different from the individual component reactants.

This preliminary investigation of nanobiotechnology for its effectiveness as an anti-fungal agent of soft rot in yam gave encouraging and interesting results. The MIC of AgNeemnano that inhibited the mycelial growth of Fusarium moniliforme was about 0.9 mg/ml, while the MIC of AgNeemnano for Lasiodiplodia theobromae was <0.45 mg/ml. These micro-organisms were sensitive to the AgNeemnano solutions at very low concentrations compared to the MIC of Cassia alata leaf extracts (48.78 and 87.50 mg/ml) on Rhodotorula mucilaginosa and Scopulariopsis brevicaulis, respectively, as reported by Eze and Eze [8].

The MIC of any chemical is as important as the optimum inhibition concentration (OIC) for social, environmental, and economic reasons. It is evident in this study that while the MIC of AgNeemnano for Fusarium moniliforme is 24.40 mg/ml, the OIC is 113.63 mg/ml, beyond which the IZD decreased. Given that silver is non-toxic to humans and the environment, it is not economical to increase the concentration beyond this point as additional increases in concentration resulted in a very low increase in IZD, and therefore, poor CUE of the silverneem. The MIC of AgNeemnano for Lasiodiplodia theobromae was also the same with that of F. moniliforme but the OIC varied. The CUE of AgNeemnano decreased at the concentration of 113.63 mg/ml and increased again at the concentration of 227.27 mg/ml. Although it is not clear what caused the fall and rise in the value of the CUE of the silverneem on the L. theobromae, it is likely that the organism resisted the bioactivity of this chemical at that concentration but became susceptible when the concentration was further increased. The optimum inhibition concentration of silverneem for L. theobromae was not attained in this study; therefore, further research is recommended.

Interestingly, silver is metallic in nature; therefore, the combination of silver and neem (AgNeemnano) solution in this study is not likely to be degradable as in the case of Cassia alata leaf extracts or neem leaf extracts alone. This means that AgNeemnano could represent a lead source of novel antimicrobial agents capable of controlling rots of yam in storage if properly utilised. In rational drug therapy, the concurrent administration of two or more antimicrobial agents is often essential and sometimes mandatory in order to achieve the desired therapeutic effects [13].

The mycelial growth of the pathogenic organisms was noticeable even with unaided eyes. The dark brown colour of the treated tuber could be due to the drying up of yam exudates that sealed the cut surface and changed in colour, while the colourless or white surface of the untreated tuber suggests well-covered mycelial growth of the pathogenic organisms. The morphological features of the micro-organisms as captured by the Moticam camera suggest that these two organisms that cause yam rot in the study area have big particle sizes and might be virulent to other micro-organisms. Again, few numbers of pathogenic organisms of yam identified in this study could be attributed to the prevalent weather conditions, which probably were not conducive to other organisms. It has been reported that micro-organisms thrive better in a high-humidity environment with relative humidity of 70–90% than in low-humidity regions [14].

In this study, the sensitivity of the micro-organisms to AgNeemnano varied with the concentration, suggesting that this new metallic nanoparticle has multiple anti-microbial properties that could be applied to other fields of study, and not only in food crop preservation. This result also corroborates the report by Furno et al. [10] who had stated earlier that nanoparticles hold great potential in various contexts. A combination of silver nitrate and neem leaf extracts is a reaction of metal and a non-metallic substance resulting in a product that could maintain the durable property of metal with antimicrobial (antibacterial and antifungal) spectrum that deals with two or more organisms with different sensitivity patterns [14]. The most interesting aspect of this study is that the green metallic nanoparticles have no toxic chemicals and no effects on food [10].

5 Conclusion

This new method of biosynthesis of green metallic nanoparticles has different levels of bioactivities on the rot causing micro-organisms. Again, many micro-organisms were not found in this study. It is likely that location, type of yam, and time of storage could be responsible for the non-abundance of the micro-organisms in the study area. However, it is evident in this study that the use of metallic nanoparticles for storage of yam has the potential to keep yam free from all manners of microbial attack, and will enable yam to be stored for a period longer than the traditional 2–4 months after harvest. Further research is recommended to substantiate our findings, especially on the optimum concentration of the solution and different application methods. The technology is cheap, easy to apply, and with non-toxic effects on humans and the environment, and can be applied correctly in the farm field.