Journal of Plant Physiology & Pathology ISSN: 2329-955X

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Research Article, J Plant Physiol Pathol Vol: 4 Issue: 3

Effects of Calcium Fertilization on the Susceptibility of Dioscorea Species to the Yam Storage Pathogens Aspergillus Niger van Tiegh and Botryodiplodia Theobromae Pat

Otusanya MO1*, Enikuomehin O1, Popoola A1, Adetunji M2, Kehinde O3, Okeleye K4, Latunde-Dada O1 and Amusa O5
1Department of Crop Protection, College of Plant Science and Crop Production, Federal University of Agriculture, Abeokuta, Nigeria
2Deoartment of Soil Science and Land Management, College of Plant Science and Crop Production, Federal University of Agriculture, Abeokuta, Nigeria
3Department of Biological Sciences, College of Natural Sciences, Federal University of Agriculture, Abeokuta, Nigeria
4Department of Plant Physiology and Crop Prod., Plant Science and Crop Production, Federal University of Agriculture, Abeokuta, Nigeria
5Department of Crop Protection, Olabisi Onabanjo University, Ago-Iwoye, Ijebu- Ode, Nigeria
Corresponding author : Otusanya M. Oluleke
Department of Crop Protection, College of Plant Science and Crop Production, Federal University of Agriculture, Abeokuta, Nigeria
E-mail: [email protected]
Received: May 03, 2016 Accepted: August 05, 2016 Published: August 10, 2016
Citation: Otusanya MO, Enikuomehin O, Popoola A, Adetunji M, Kehinde O, et al. (2016) Effects of Calcium Fertilization on the Susceptibility of Dioscorea Species to the Yam Storage Pathogens Aspergillus Niger van Tiegh and Botryodiplodia Theobromae Pat. J Plant Physiol Pathol 4:3. doi: 10.4172/2329-955X.1000155

Abstract

The effects of soil amendment with basal dose of nitrogen (60 kg ha- 1), phosphorus (30 kg ha-1) and potassium (75 kg ha-1) for Yams Dioscorea species in South West Nigeria and calcium carbonate were assessed on yield and susceptibility to two storage rot pathogens, in Dioscorea alata TDa 92-2, D. rotundata TDr 131 and D. esculenta TDe 89-1. The study site at the Teaching and Research Farms of the Federal University of Agriculture, Abeokuta, had been monocropped to yams without fertilizer for three years before the study. A 3 by 5 factorial desgn, with the Dioscorea species as main plots and 0 kg ha-1, 2 kg ha-1, 4 kg ha-1, 6 kg ha-1 and 8 kg ha-1 calcium carbonate as subplot treatments, was used in each of two years. Aspergillus niger van Tiegh. and Botryodiplodia theobromae Pat. were the two yam storage pathogens used. Pre-planting soil analysis in the two years revealed calcium, magnesium and potassium below the critical levels required for Dioscorea species in South Western Nigeria. Calcium fertilizer enhanced resistance and reduced weight loss in yam ‘’sections;’’ of D. rotundata TDr 131 and Dioscorea alata TDa 92-2 to the pathogens after long-terms storage of over four months in both years. Response of whole tubers incubated for an additional (after the four months) period of ten weeks and six weeks in years one and two respectively, was not significantiy (P=0.05) different from the control. Futher work to determine endogenous or physiological reasons for this is necessary. D. esculenta TDe 89-1 was more susceptible to the rot pathogens than the other two species. D. esculenta TDe 89-1 was also not significantly different in infection or weight loss than the control in the two years, necessitating a need to ascertain optimum fertilizer placement methods for this species in Nigeria. There were no differences in tuber number or tuber weight per plant at the treatment levels.

Keywords: Calcium fertilization; Monocropped; Aspergillus niger; Botryodiplodia theobromae

Keywords

Calcium fertilization; Monocropped; Aspergillus niger; Botryodiplodia theobromae

Introduction

Yams, Dioscorea species have a world-wide distribution from Asia to the Pacific Islands, Africa and West Indies [1]. White guinea yam, Dioscorea rotundata Pior and yellow guinea yam, Dioscorea cayenensis Lamk are native to the West African savannah where they are a staple food and are the two most cultivated species [2]. The most widespread in terms of cultivation is the water yam, D. alata [1]. Dioscorea opposita Thumb is native to China having been introduced into Europe with the Potato famine caused by the bacterial blight (induced by Phytophthora infestans) disease in the 1800’s [3]. Other species such as Dioscorea esculenta (Lour.), Burk called the lesser yam is native to South- East Asia, but currently has spread to Africa. Several of the cultivated species such as D. opposita are becoming popular in countries such as Japan and Korea and for the Asian food market in France and South America [3].
Storage losses in yams (Dioscorea spp.), which may be between 10 to 15% in the first three months and up to 60% or above after six months is a major constraint in production [4]. Fungi constitute a major problem in stored yams than any other single cause [5]. Methods that have been utilized in the control of storage losses by fungi in yams include fungicides especially benzimidazole derivatives such as benomyl and thiabendazole [6-8]. Others such as imazalil, limewash, borax, bordeaux mixture [7-9], and benzoic acid [10,11] have also been used. Chemical residues however are a threat to the environment and their high costs discourage farmers [12]. Biological control with a bacterium namely Bacillus subtilis had been used to prevent advancement of rot pathogens in infected yams and to protect tubers in the barn [12,13]. Ethanolic plant extracts of Aframomum meleguata and Ocimum gratissimum had inhibited rot pathogens such as A. niger and B. theobromae in vitro [14]. Other more effective means of control such as cool temperature storage and irradiation are however, very expensive, highly technical and unapplicable in field storage for peasant farmers who are responsible for about 70% of yam production in Nigeria [15].
A means of control of rots (both storage and field) in potato tubers and some fruits, as well as some field diseases of agricultural crops which has gained widespread use is that of calcium infiltration of the fruits and tubers as well as calcium fertilization of the growing plant. This use of calcium is based on one of its functional properties in the plant cell namely, the improvement of the structural integrity of both the plasmalemma and cell wall materials [16]. This reduces maceration of the cell wall by pectolytic enzymes produced by fungi as well as the multiplication and intercellular spread of the rot pathogen [17]. Apart from the structural roles in the cell wall and membrane, calcium is a counter-cation for inorganic and organic anions in the cell vacuole and cytosolic Ca2+ concentration, is an obligate intracellular messenger coordinating responses to numerous developmental cues and environmental challenges [18].
Vacuum infiltration of apples with calcium before storage at 0°C for six months, was found to reduce decay by Penicillium expansum and the resulting storage loss [19]. Soybean plants produced fewer and lower quality seeds when grown in decreased calcium concentrations [20]. Increase in calcium uptake strengthened cells of broccoli (Brassica oleracea L. var. Italica) against infection by the clubroot pathogen Plasmodiophora brassicae [21]. Elad et al. also reported good control in cucumber, eggplant and pepper crops against Botrytis cinerea infection with calcium fertilization [22]. Bacterial soft rot caused by Erwinia carotovora pv. atroseptica was greatly reduced in potato tubers by infiltration with calcium before storage and calcium fertilization of the growing plant [23,24]. Even in infected plant tissues, increased calcium has been associated with a decrease in the activity of pectolytic enzymes and an increase in the resistance of pectic materials in cell walls to degradation [25,26]. In yams (Dioscorea spp.) the role of calcium infiltration and fertilization in the control of and the conferring of resistance on tubers against storage rot pathogens, had been identified [27].
The objective of this study was to assess the effect of calcium fertiliser application on the susceptibility of Dioscorea species to the yam storage pathogens Aspergillus niger and Botryodiplodia theobromae.

Materials and Methods

Planting material
Three varieties of yam namely D rotundata TDr 131, D. alata TDa 92-2 and D. esculenta TDe 89-1 collected from the Root and Tuber Improvement Programme, International Institute of Tropical Agriculture in Ibadan, Oyo State, Nigeria were sown in each of two growing seasons.
Field plot and field lay-out
A field that had been monocropped to yam without fertilizer for three consecutive years was used for the study, at the permanent site of the Federal University of Agriculture Abeokuta, Teaching and Research Farms. The same plot was used in the second year. The experiment in both years was a 3 × 5 factorial, arranged in a split- plot with three replications. The three varieties of yam were the main-plot treatments and five CaCO3 levels the sub-plot treatments, randomly arranged per main-plot. Three rows of five 1 m by 1 m mounds and a total of 15 plants (on 15 mounds) constituted a sub-plot. Mounds were separated by 0.5 m and 0.25 m spaces respectively in the first and second year and each sub-plot separated from the other by a 0.5 m ridge on all sides. Main plots were separated by a distance of 1 m and a clean field border of 5 m radius was maintained.
Soil sampling and analysis
Soil samples were collected on the field plot before planting in each of the two growing seasons. Collection, drying and sieving was according to a standard procedure [28]. Soil pH was determined using a glass electrode pH metre in a 1:2, soil: distilled water ratio. Determination of exchangeable cations such as calcium, magnesium and potassium was done using standard methods [28,29].
Fertiliser
Calcium carbonate Analar grade was used for calcium fertilization in both years. Also NPK fertilizer at the required dosage of 60 kg nitrogen, 30 kg phosphorus, and 75 kg potassium per hectare was applied at 3 months after sowing [30].
Planting and fertilization
Similar weight (500 to 600 g) of seed yams of the three varieties were planted in the two years. Each sub-plot consisted of fifteen plants in three rows of five plants each with a total of 75 plants per main plot, 225 plants per replicate and a total of 675 plants. The required sub-plot treatments of calcium carbonate namely 0 kg ha-1, 2 kg ha-1, 4 kg ha-1, 6 kg ha-1 and 8 kg ha-1 and 60 kg N, 30 kg P and 75 kg K per hectare were applied to the yam plants in a circular form at 20 cm radius around each plant 3 months after sowing [30]. Plants were mulched with dry grass held down with a ball of soil after planting and stakes established one per mound/plant. Manual weeding was carried out as at when due, during both seasons.
Storage barn and structures
A storage barn of about 3.5 m by 3.5 m area was built at the mini-campus of the Federal University of Agriculture Isale-Igbein Abeokuta. It had no windows but was made of concrete from the floor to half of its height. The remaining half to the roof was of metal wiremesh supported with a closely-knit wire netting to ward off reptiles, rodents and insects. Its roof was of metal sheets secured with bars of wood. It had a coarse-sand floor and a wooden door secured with padlocks. Two storage structures were placed inside the barn to store the tubers as well as being a platform for storage experiments. They were fabricated of wood, metal wire mesh and wire net. Each was a rectangular wooden frame of three metres (3 m) length, one hundred centimetre (100 cm) width and two metres (2 m) height, covered along the top half of its height with metal wire mesh, supported with a closely-knit wire net also supported at the corners with bars of wood. The second half of its height was of wood on which it stood .The wire mesh with the supporting wire net was to ward off reptiles, rodents and insects. The front of the structures were made to open and close and secured with locks.
Harvest and storage of tubers
At maturity (9 months after planting), indicated by senescent leaves, number of tubers and weight (kg) of tubers produced per plant were recorded in both years. Harvested tubers were allowed to dry, were labelled and stored carefully on slabs of wood inside the two storage structures for the storage period of one hundred and forty-five days (4 months and 24 days) in the first season. Routine inspection was done during the storage period to secure or replace paper labels, and to dislodge sprouts. After this period the storage structures were evacuated of tubers, fumigated with baygon insecticide, washed with 80% sodium hypochlorite solution and left to dry for the first year storage experiments. In the second season, harvested tubers were stored for a period of one hundred and twenty-eight days (4 months and 8 days). Routine inspection was done within this period as in the first season. Thereafter storage structures were again fumigated washed with 80% sodium hypochlorite solution and left to dry for the second season storage experiments.
Preparation of Fungal culture medium
Aspergillus niger and Botryodiplodia theobromae isolated from and found to be pathogenic on tubers of yam (Dioscorea rotundata) were used for the experiment. Potato dextrose agar (PDA) was used as growth medium for the fungal isolates. It was prepared by dissolving 39 grams of PDA powder in 1000 ml of distilled water. This was then homogenized and sterilized in an autoclave at 1.05 kg cm-2 pressure and 121°C temperatures for 15 minutes [31]. Seven-day old pure cultures of the isolates were used for the storage experiments. Stocks were maintained on PDA in universal bottles at 28 ± 2°C
Inoculation of yam sections and yam tubers
The technique for the inoculation of yam sections was that of Otusanya [32] as shown in Figures 1 and 2. Tubers which had no abrations, holes, or lacerations were selected. They were dusted free of soil with a clean cloth. Where sections were used, the tubers were cut into the number of sections appropriate for the experimental design after the tail and head tip had been cut off. Sectioning was along the tuber axis with a sterile steel knife. Sections were then placed carefully with their periderm resting on the surface-sterilized wooden slabs in the storage structure for 24 hours. After the natural drying of the cut surfaces, the upper portion of each section was swabbed with cotton wool dipped in 96% ethanol. A hole about 15 mm deep was bored through the swabbed area with a 6 mm sterile cork borer, and tissue within cut free with sterile forceps. A 5 mm agar disc of the appropriate seven-day old fungal isolate was placed in the hole with another sterile cork borer. The cut tissue was replaced and incision sealed with petroleum jelly. The same inoculation technique was used for the whole tubers but without the cutting away of the head and tail tips (Figure 3). The yam sections and whole tubers were labelled and weighed before inoculation. They were then placed as before on the slabs in the storage structure. At the end of the period the yam sections and yam tubers were again weighed. The petroleum jelly on each site was cleared away with a clean spatula. Thereafter the yam was cut through the inoculation site with a swabbed (with methylated spirit) steel knife. Then the infected tissue was pared away carefully with a sterile scalpel on to aluminium foil (pre-weighed) and weighed.
Figure 1: Uninoculated yam sections.
Figure 2: Inoculated yam sections (Arrowed; Inoculation plug).
Figure 3: Inoculated yam tuber (Arrowed; Inoculation plug).
Storage experiments
The first storage experiment in the first year was a 2×5 factorial one. Whole tubers of D. rotundata TDr 131 and D. alata TDA 92-2 picked randomly from the harvest of the sub-plots in the field were maintained in the storage structure as the sub-plot treatments. In each of three replicates, the two yam species were the main-plots. The second experiment was with cut sections of the two yam species, while the factorial arrangement/combinations were as in the first storage experiment. Whole tubers and cut sections were inoculated as described above with the yam storage pathogen Aspergillus niger. Whole tubers and sections were incubated in the storage barn in the first year, for 10 weeks and 2 weeks respectively. The third experiment and the fourth followed the same pattern as the first and second but with the pathogen Botryodiplodia theobromae. The storage temperature and relative humidity during the incubation/storage period were 26°C-28°C and 76%-80% respectively. The difference in weights of the experimental units taken before inoculation and at the end of the storage experiment were calculated as a percentage of the initial weight to give percentage weight loss as in the formula
((A-B)/A)) 100 = Y (% weight loss)
Where A = initial weight of experimental unit (section/tuber) in grams
And B = final weight of experimental unit (section/tuber) in grams
Also percentage infection was assessed after amount of infected tissue “X” had been weighed and corrected for weight loss as in the formula: (100 X) /(100-Y)=C (corrected weight of infected tissue) Percentage infection was then calculated as in the formula:
( (A-C)/A) 100 = % Infection
Second year storage experiments followed the same pattern as in the first year. However the duration of the whole tubers storage experiment was six weeks only not ten weeks as in the first year. Cut sections storage experiment period was two weeks as in the first year. Also seed yams of D. alata TDA 92-2 were tested for their susceptibility along with the smaller D. esculenta TDE 89-1 tubers in a 2 x 5 factorial experiment in a split-plot design as with the larger tubers. For this latter experiment range of tuber weight was between 0.02 kg and 0.50 kg.
Data analysis
Data in percentage were transformed (arcsine) and then subjected to analysis of variance using Statistical Analysis System [33]. Means separation was according to Duncan’s multiple range test (DMRT).

Results

Soil analysis summary
Table 1 gives a summary of the soil analysis. In the first year range of pH in the main plots (of the field plot) was over 5 to over 6, while the mean was 6.4. The pre-planting mean values of the cations Calcium (Ca), Potassium (K) and Magnesium (Mg) in the plot were 0.20 cmol kg-1, 0.16 cmol kg-1 and 3.00 × 10-5 cmol kg-1 respectively, all of which are below the critical for yams in South West Nigeria [34]. In the second year, the range of pH of the soil in the mainplots was 5.42 to 7.18 and the mean 6.16 as shown in Table 1. The pre-planting mean values for cations Calcium (Ca), Potassium (K) and Magnesium (Mg) were 0.19 cmol kg-1, 0.17 cmol kg-1 and 2.55 × 10-5 cmol kg-1 respectively. These values are also below the critical levels required for yams in the South West of Nigeria [34].
Table 1: Soil analysis summary of exchangeable cations (cmol kg-1) in calcium-fertilised field plot sown to Dioscorea species.
Infection and weight loss in calcium-fertilised whole tubers and cut sections of D. alata TDa 92-2 and D. rotundata TDr 131 by A. niger after ten weeks and two weeks incubation (first year)
For the inoculation with A. niger, in the first year whole tubers of TDr 131 and TDa 92-2
lost weight about equally in ten weeks, that is over 18% (Table 2). There were no significant differences in infection or weight loss in the two cultivars or in infection and weight loss in the treatment means (Table 2). The cut sections of the two cultivars lost weight in the same magnitude in only two weeks as the whole tubers did in ten weeks (Table 2 and 3). Infection was higher (4.02%) in D. rotundata TDr 131 than in D. alata TDa 92-2 (2.40%) while weight loss was not significantly different in the two cultivars.
Table 2: Infection and weight loss in calcium-fertilised whole tubers of D. rotundata TDr 131 and D. alata TDa 92-2 by A. niger after ten weeks incubation (first year).
Table 3: Infection and weight loss in calcium-fertilised cut sections of D. rotundata TDr 131 and D. alata TDa 92-2 by A. niger after two weeks incubation (first year).
In the treatment category infection in treatments 2 kg ha-1 , 4 kg ha-1 and 8 kd ha-1 calcium carbonate were significantly less than the control while weight loss in the trearment categories were not significantly different from the control (Table 3). There was a species x calcium carbonate interaction in weight loss (Table 3).
Infection and weight loss in calcium –fertilized whole tubers and cut sections of D. alata TDa 92-2 and D. rotundata TDr 131 by Botryodiplodia theobromae after ten weeks and two weeks incubation (first year)
In the whole tubers infection and weight loss were not significantly different in D. alata TDa 92.2 and D. rotundata TDr 131 (Table 4). Infection and weight loss in the treatment means were not significantly different (Table 4). In the cut sections infection was over 1% while weight loss was over 12% in both TDa 92–2 and TDr 131 (Table 5). Weight loss in all treatment levels was significantly lower than the control (Table 5).
Table 4: Infection and weight loss in calcium-fertilized whole tubers of D. rotundata TDr 131 and D. alata TDa 92-2 by B. theobromae after ten weeks incubation (first year).
Table 5: Infection and weught loss in calcium-fertilised cut sections of D. alata TDa 92-2 and D. rotundata TDr 131 by B. theobromae after two weeks incubation (first year).
Treatment level 8 kg CaCO3 per hectare had the lowest (6.94%). The control was 21.59%. There was no significant difference in infection in the treatment means. There was a species × CaCO3 interaction both in infection and weight loss as shown in Table 5.
Infection and weight loss in calcium-fertilised whole tubers and cut sections of D. alata TDa 92-2 and D.rotundata TDr 131 by A. niger after six weeks and two weeks incubation (second year)
D. alata TDa 92-2 whole tubers were infected in the same magnitude as D. rotundata (Table 6). Loss in weight over the six week period was also similar, in TDa 92-2 and TDr 131 (Table 6). Treatments were similar in terms of weight loss also compared with the control. A marked significantly high level of infection occurred in the 2 kg ha-1 level over others, evidenced also by a higher infection in TDr 131 also in the 2 kg ha-1 level over the control (Table 6). In the cut sections infection in D. alata TDa 92-2 was similar to that of D. rotundata TDr 131, while weight losss was significantly higher in D. alata TDa 92-2 than in D. rotundata TDr 131 (Table 7). All calcium carbonate treatment levels had significantly lower weight loss than the control (Table 7). Category 2 kg ha-1 and 4 kg ha-1 each had over 20% weight loss while categories 6 kg ha-1 and 8 kg ha-1 had the lowest of 12.75% and 15.52% respectively. Infection in category 6 kg ha-1 was significantly higher than the control. This was buttressed further by species × calcium carbonate interaction in D. alata TDa 92-2 where infection in treatment level 4 kg ha-1 and 6 kg ha-1 were higher than the control . There were no differences in infection at the treatment levels in D. rotundata TDr 131 (Table 7).
Table 6: Infection and weught loss in calcium-fertilised whole tubers of D. alata TDa 92-2 and D. rotundata TDr 131 by A. niger after six weeks incubation (second year).
Table 7: Infection and weight loss in calcium-fertilised cut sections of D.alata TDa 92-2 and D. rotundata TDr 131 by A. niger after two weeks incubation (second year).
Infection and weight loss in calcium-fertilized whole tubers and cut sections of D. alata TDa 92-2 and D. rotundata TDr 131 by B. theobromae after six weeks and two weeks incubation (second year)
In the whole tubers loss in weight were 13.70% for TDa 92-2 and 10.91% for TDr 131 (Table 8). Overall infection was not significantly different in the two species.
Table 8: Infection and weight loss in calcium-fertilised whole tubers of D. alata TDa 92-2 and D. rotundata TDr 131 by B. theobromae after six weeks incubation (second year).
Weight loss and infection were not different from one another in the treatment categories. In the cut sections infection in D. alata TDa 92.2 and in D. rotundata TDr 131were not significantly different, neither was there any significant difference in weight loss in both cultivars (Table 9). Treatment category 6 kg ha-1 calcium carbonate had significantly lower loss in weight than the control (Table 9). Treatment levels 2kg ha-1 and 4kg ha-1 calcium carbonate had lower infection than the control (Table 9). There was a species × CaCO3 interact on both in weight loss and infection (Table 9). In D. rotundata TDr 131 there was lower weight loss in the 2 kg ha-1, 6 kg ha-1and 8 kg ha-1categories than in the control. Also there was a lower weight loss in the 6 kg ha-1 level in D. alata TDa 92-2. Infection however in D. alata TDa 92-2 were not significantly different at the treatment levels. In D. rotundata TDr 131 infection which was 2.74%, 8.73%, 4.83%, and 8.59% in the 2 kg ha-1, 4 kg ha-1, 6 kg ha-1 and 8 kg ha-1 calcium carbonate categories were all significantly lower than in the control which was 38.23% (Table 9).
Table 9: Infection and weight koss in calcium-fertilised cut sections of D. alata TDa 92-2 and D. rotundata TDr 131 by B. theobromae after two weeks incubation (second year).
Infection and weight loss in calcium-fertilized Dioscorea esculenta TDe 89-1 and Seed yam D. alata TDa 92-2 by A. niger and B. theobromae after two weeks incubation (second year)
Also in the second year, infection by A. niger was significantly higher in D. esculenta TDe 89-1 (25.03%) than in seed yam D. alata TDa 92-2 (10.46%), (Table 10). Weight loss was also higher in D. esculenta TDe 89-1. Treatment means were not significantly different from the control either in weight loss or infection, but in seed yam TDa 92-2 infection of 3.82% and 4.66% in the 6 kg ha-1 and 8 kg ha-1 calcium carbonate categories were significantly lower than the control of 16.24% (Table 10). But in the same treatment categories 6 kg ha-1 and 8 kg ha-1 in D. esculenta TDe 89-1, significantly higher infection of 43.13% and 44.99% than the control (17.68%) were recorded (Table 10).
Table 10: Infection and weight loss in calcium- fertilized seed yams of D. alata TDa 92-2 and tubers of D. sculenta TDe 89-1by A. niger after two weeks incubation.
Infection with B. theobromae was significantly higher (79.85%) in TDe 89-1 than in SYTDa 92-2 where it was only 10.28% as shown in Table 11. A higher weight loss of over 9% in the two weeks occurred in D. esculenta TDe 89-1 than in seed yam D. alata TDa 92-2 where 3.76% was recorded. There were no differences in treatment means in infection and weight loss. However, weight loss in treatment level 4 kg ha-1 CaCO3 was significantly lower (7.06%) in D. esculenta TDe 89-1 than in the control (11.34%).
Table 11: Infection and weight loss in calcium-fertilised seed yams of D. alata TDa 92-2 (SYTDa 92-2) and tubers of D. esculenta TDe 89-1 by B. theobromae after two weeks incubation.
Mean tuber number and weight per plant in calcium-fertilized Dioscorea species (first year)
For the harvest assessment in the first year, a significant difference was recorded in mean tuber number per plant (Table 12). The number was highest, that is 36.99 in Dioscorea esculenta TDe 89-1 and significantly lower 2.79 and 1.09 in D. alata TDa 92-2 and D. rotundata TDr 131 respectively. Tuber weight was lowest in D. esculenta TDe 89-1 (1.02 kg) followed by D. rotundata TDr 131 (2.87 kg) and highest in D. alata TDa 92-2 (4.08 kg), as shown in Table 12.
Table 12: Tuber number and tuber weight (kg) in calcium-fertilised Dioscorea species (first year).
There were no significant differences in the calcium carbonate levels for either tuber number or tuber weight.However treatment levels 2 kg ha-1 and 4 kg ha-1 recorded lower tuber weight per plant in D. alata TDa 92-2 than in the control. The values were 3.17 kg per plant and 2.77 kg per plant respectively while the control was 4.92 kg.
Mean tuber number and weight (kg) per plant in calcium-fertilised Dioscorea species (second year)
In the second year, tuber number was not significantly different in D. alata TDa 92-2 or D. rotundata TDr 131 while mean tuber weight was higher in the former (Table 13). There were no significant differences in tuber weight or tuber number in the calcium carbonate categories or species × CaCO3 interactions.
Table 13: Tuber number and tuber weight (kg) per plant in calcium-fertilized Dioscorea species (second year).

Discussion

In ten weeks of incubation of inoculated tubers of white yam and water yam with A. niger, loss in weight of whole tubers were about the same. Low infection in the two yam species is due to the fact that infection is generally low (below 10%) in 8 weeks and only rises sharply afterwards [35]. However infection was higher with the ubiquitous B. theobromae. In 6 weeks only in the second year, incubation with Aspergillus niger resulted in a higher infection in the two yam types, probably because of less favourable conditions of the environment [36]. These two fungi have been rated to be amongst the four most important storage pathogens of yams globally [32,37-40]. Yam sections also lost weight in the same percentage range in two weeks only because of the more rapid weight loss from the cut but dry surfaces [31]. Mean infection in the yam sections was about 500% higher in the same storage barn in the second season harvest than the first probably because of higher relative humidity and ambient temperatures [41]
There was a rapid spread of A.niger rot in the smaller and softer yam D. esculenta in the first season, such that they could not be used in the storage experiments. In the second year, the same species D. esculenta was 200% more susceptible to A. niger and 700% more susceptible to B. theobromae than D. rotundata or D. alata. A similar infection level was reported with A.niger on D. esculenta in a previous study by Otusanya and Jeger [11] while Ikotun [42] rated A.niger as one of the three species of Aspergillus to which D. esculenta was highly vulnerable. Weight loss in two weeks in D. esculenta was as high as in a study of rot development in D. rotundata, D. alata and D. esculenta under long-term storage conditions [11].
No significant changes (infection/weight loss) were recorded in the whole tubers compared with the control CaCO3 fertiliser had been administered so far in literature to improve yield in the Dioscorea [41]. Otusanya and Jeger [11], reported that after storage of tubers for ten weeks, benzoic acid inhibited A. niger rot compared to benlate or sodium hypochlorite solution in three yam species. In the report, days of storage of the different species before storage experiments were carried out was not specified. In this study, the tubers were stored for one hundred and forty five days (20 weeks and 5 days) and one hundred and twenty eight days (18 weeks and 2 days) in the first and second year respectively, before storage experiments were conducted for 10 weeks or 6 weeks. There is need for further work to establish endogenous/environmental factors responsible for the insignificance in infection/weight loss in whole tubers. The smaller tubers and seed yams were quite responsive in just two weeks (14 days) as they are too small to be left inoculated/infected for more than this period [32]. Yam sections inoculated for two weeks only, of D. rotundata and D. alata as well as seed yams of D. alata from the calcium-fertilised plots responded with lower infection with A. niger in year one and two respectively. Weight loss in the calcium carbonate-fertilised yam sections were also lower than in control tubers in both years. Otusanya and Jeger [35] reported that Brazilian D. alata yams (cut sections) responded significantly after 12 days only to infection by A. niger. Exceptions to the reduced infection in the calcium carbonatefertilised plots occurred in the lesser yam D. esculenta inoculated with A. niger and D. alata inoculated with A. niger both in the second year. Obigbesan and Agboola [31] reported 2.8 kg ha-1 uptake of calcium for an unspecified cultivar of D.alatta. Treatment levels 6 kg ha-1 CaCO3 and 8 kg ha-1 CaCO3 in this study are slightly below and above this amount respectively. Cultivars of the same species may differ in their calcium requirement as D. rotundata cvs ‘Aro’ and D. rotundata cvs ‘Efuru’ required 2.1 kg ha-1 and 3.9 kg ha-1 respectively in the report [30]. Kabeerathumma et al. [43] reported uptake of 19.8 kg ha-1 of calcium oxide by D. esculenta in India. Melteras [44] reported from studies on Australian cultivars of D. esculenta, that their roots were quite extensive increasing from about 10 cm after planting to more than 4 metres at tuber initiation. This was adduced as the reason for inconsistent responses of this species to fertilizer in the field. Therefore there would be need for further studies to ascertain optimal fertilizer placement method for D. esculenta. Fertiliser usage in yam (Dioscorea spp.) had been mainly to improve either dry matter production or tuber yield [34,45,46]. Recently, tillage systems (zero, ploughing, ridge, and mound), soil microclimate (temperature, and moisture), crop physiology and crop environment/ecology as well as use of organic fertilizer versus conventional ones have been monitored for yam improvement [47-49]. Fertiliser or mineral (calcium) nutrition to enhance disease resistance in yam storage had only been identified [27]. In this study, the site on which the experiment was done had calcium, potassium and magnesium nutrients less than the critical required for yam in South western Nigeria [34]. Adeleye et al. [46] supplemented organic (poultry) manure in two sites in Ondo state, in the South West of Nigeria, which had been left fallow (bush) for two years before planting yam. The result of the pre-planting soil analysis indicated nitrogen, phosphorus, potassium and calcium as being below the critical level for yam production. Use of NPK fertilizer supplemented with CaCO3 in this study is justified. Furthermore, responses of lower infection by A. niger and B. theobromae after longterm storage is an indication that calcium fertilization (mineral or organic) would improve resistance of yams to the storage pathogens. Enhanced resistance to disease with calcium fertilisation as in this study had been reported in vegetable crops such as pepper, tomato, eggplant and cucumber [22,50]. Among the tuber crops, diseases such as internal brown spot and sub-apical necrosis in potato have been controlled by calcium fertilization [24,51]. Tissue integrity in apple fruits and potato tubers was increased also with spraying as well as immersion in calcium solution [52-61]. Responses of low infection and weight loss in calcium carbonate-fertilised yams in this study are an indication that a balanced nutrition of calcium would improve storability of yam tubers against rots by A. niger and B. theobromae.

Acknowledgments

The authors wish to thank RESDEC (Research and Developement Centre), of the Federal University of Agriculture, Abeokura, for the part-funding of the field study. They also appreciate Dr Robert ASIEDU of the Root And Tuber Improvement Program, International Institute of Tropical Agriculture, Ibadan, Oyo State, Nigeria for the supply of the three improved clones or varieties Dioscorea alata TDa 92-2, Dioscorea rotundata TDr 131 and Dioscorea esculenta TDe 89-1 in the two years. We also thank Dr O. Oduwaye for technical assistance.

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