Poultry manure influenced growth , yield and nutritional quality of containerized aromatic pepper ( Capsicum annuum L . , var ‘ Nsukka Yellow ’ )

‘Nsukka Yellow’ pepper is an aromatic pepper (Capsicum annuum) grown in specific rural locales in southeastern Nigeria. Ripe fruits are predominantly yellow and are exported to Nigerian cities where they command premium price. The need to develop appropriate technologies for adaptation and cultivation of ‘Nsukka Yellow’ pepper in cities where there are scarcity of agricultural lands motivated evaluation of the pepper as a potherb using three poultry manure (PM) rates (0, 5 and 10 t/ha). Threeweek old seedlings were transplanted into 11 L containers and laid out in a completely randomized design replicated 10 times. Growth, yield and nutritional quality of fruits (at different ripening stages) were determined in response to applied manure. Plant height, number of leaves and number of branches increased with increasing level of PM, which translated to increased number of fruits and fruit weight per plant. Plants that received 10 t/ha of PM produced the highest fruit yield. Mature green, half ripe and fully ripe fruits were analyzed for ash, carbohydrate, fat, crude fiber, moisture, protein, alkaloid, flavonoid, tannin and volatile oil contents using standard methods. Both yellow fruits and red fruits (offtype from mutated branches) were harvested and analyzed for nutritional quality. Fruits harvested from 10 t/ha PM had the highest percent fat, crude fiber, moisture content, alkaloid, flavonoid, tannin and volatile oil. However, 5 t/ha produced fruits with the highest ash and protein contents; fruits from plants without manure had the highest percent carbohydrate. Fully ripe fruits had the highest percent carbohydrate, crude fiber, alkaloid, flavonoid, tannin and volatile oil, but green fruits had higher values for moisture and protein. Red fruits had the highest values for fat, carbohydrate, protein, alkaloid, flavonoid and volatile oil while the yellow type had the highest percent values for ash, crude fiber and tannin. Data on growth, yield and nutritional quality had significant positive response to the PM rates, suggesting that 10 t/ha is recommendable for growing the pepper as a potherb.


INTRODUCTION
Pepper (Capsicum annuum) originated in South America and spread into Asia and Africa (George, 1985).It is among the most commonly grown crops throughout Africa because of its utilization in soup, stews and salads (Heiser, 1995).There are five cultivated species of Capsicum, which are Capsicum annuum, Capsicum Plate 1. Fruits of ripe 'Nsukka Yellow' pepper.
Nigeria is the largest producer of pepper in Africa covering about 50% of total Africa production (Adetula and Olakojo, 2006).Nigeria was named 12 th among top producing countries of chillies and peppers in the world (Hays, 2009).Idowu-Agida et al. (2012) reported that Nigeria has more than 200 selections of pepper.Ten genotypes of aromatic pepper types have also been characterized in the derived savanna ecology of southeastern Nigeria (Abu and Uguru, 2006;Abu et al., 2011).
In almost every tropical country, pepper has become the most popular condiment used to add zest and flavor to otherwise dull foods (Kochhar, 1986;Alabi, 2006).It has extensive culinary uses.It is used in pickles, ketchup and sauce seasoning dishes and in sausages (Purseglove, 1968).Pepper is rich in vitamins especially ascorbic acid and vitamin A. Bosland and Votava (2000) reported that pepper contains more vitamin C than tomato.Uzo (1982) reported that pepper is the major source of capsaicin, an alkaloid, which is used in medicine as a digestive stimulant.Peppers also contain various phenolics, flavonoids and carotenoids (Amakura et al., 2002;Materska and Perucka, 2005).Carotenoids are important colorants in vegetables, which often impart orange and red colors (Delgado-Vargas and Paredes-Lopez, 2003).Carotenoids in peppers include capsanthin, β-carotene and capsorubin (Howard et al., 2000;Guzman et al., 2010).The yellow-orange color of peppers is formed by αand β-carotene, zeaxanthin, lutein and β-cryptoxanthin and these compounds are antioxidants and can reduce harmful oxidation reactions in human body; thus consumption of peppers may prevent various diseases associated with free radical oxidation, such as cardiovascular disease, cancer and neurological disorders (Howard et al., 2000).
The group of pungent components peculiar to the fruits of Capsicum plants is called capsaicinoids.The environment, especially the climate, light intensity, soil type, moisture level, fertilization and temperature during plant growth, is considered to have an impact on capsaicinoid levels, as does the age of the fruit (Estrada et al., 2002).Asiegbu and Uzo (1984) reported that 'Nsukka yellow' pepper of the species C. annuum is a flavor pepper with a distinctive aroma and predominantly yellow color (Plate 1), which enhances its acceptability, thereby making it to attract higher prices than other peppers in groceries.Ripe 'Nsukka Yellow' pepper fruits are predominantly yellow (Plate 1) and are usually exported to Nigerian cities where they command premium price.It is widely speculated that the use of inorganic manure has resulted in dis-flavor of the 'Nsukka yellow' pepper, and it has also been argued that the use of organic manure is the best option in the cultivation of the pepper (Onugha, 1999).
Optimum organic manure needs for containerized 'Nsukka Yellow' pepper has not been reported in literature.The main bulk of pepper production in southeastern Nigeria is in the hands of small-scale farmers.They have limited resources to buy inorganic fertilizer because of the high cost.Organic manure that is more available as homestead wastes is a popular alternative to inorganic fertilizer and may be more affordable (Ndukwe et al., 2010(Ndukwe et al., , 2012;;Ndubuaku et al., 2014).There is a need to work out the optimum manure rate for container-grown pepper, and that was addressed in this paper.*Corresponding author.E-mail: paul.baiyeri@unn.edu.ng.Tel: +234(0)8039281834.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License

MATERIALS AND METHODS
The field experiment was conducted in the research farm of the Department of Crop Science, University of Nigeria, Nsukka, Nigeria located at latitude 06° 25 1 N, longitude 07° 24 1 S and at an altitude of 477.2 m above sea level.

Nursery operation
A ground nursery measuring 1.8 x 3 m was used for raising the seedlings.The nursery site was cleared of grasses and hoed to a fine tilt.Cured poultry manure was thoroughly mixed with the topsoil and watered to field capacity.Seeds were drilled in rows spaced at about 10 cm apart to a planting depth of about 2 cm, a day after nursery bed preparation.The seeds were covered with fine sand, watered lightly and mulched; furthermore, routine nursery maintenance practices were performed until seedlings were transplanted with ball of earth to the experimental pots.

Field experimentation
Thirty 11-L calibrated plastic pots were perforated at three points at the bottom to ensure drainage and were filled with 13.5 kg (dry weight) of soil each.The thirty pots were randomly allotted to the three experimental treatments, that is, 0, 5 and 10 t/ha cured poultry manure, giving 10 replications per treatment laid out in completely randomized design.Seedlings were watered, mulched and capped immediately after transplanting to provide temporary shade, reduce excessive moisture loss through evapo-transpiration, and to ensure ease of seedling establishment.Application of manure as top-dressing was done two weeks after transplanting the seedlings.

Plant growth and yield
Data were collected on plant height (cm), number of leaves and branches per plant over time.Fruits were harvested as ripening sets in and cumulative harvest per week was recorded as yield (grams) per plant.Harvest lasted for about three months; cumulative number of fruits harvested and the total yield (grams) per pot were recorded.Fruits weight loss under ambient laboratory condition was determined over time.

Soil and manure analyses
Soil samples were collected from representative spots on the experimental site from where soil was collected for potting using soil auger to a depth of 20 cm, the samples were made into a composite sample.A sub-sample was taken, air-dried, crushed and sieved with 2-mm mesh sieve after which physical and chemical analyses were carried out.A sample of the poultry manure used for experiment was also collected and analyzed for percentages of NPK.Standard laboratory procedures (Bouyoucos 1951;AOAC, 2005) were followed for the determination of physicochemical properties of the soil and manure used.Phosphorus was determined using phospho-molybedate method as described by Pearson (1976).5 ml of the dissolved ash sample was pipetted into a test tube and 5 ml of molybdate solution was added.The standard calibration curve was prepared using phosphate salt.The absorbance was read at 470 nm in a spectrophotometer (Spectrolam 21) and the concentration calculated from the standard curve.
Analyses were carried out at the laboratory of the Department of Soil Science, University of Nigeria, Nsukka, Enugu State, Nigeria.

Laboratory experimentation on nutritional quality of fruits
Nutrient contents of 'Nsukka yellow' pepper as influenced by poultry manure rate and ripening stages were evaluated at the Department of Crop Science Analytical Laboratory.This was a follow-up study to the field experimentation outlined above.Fruits were harvested at three different ripening stages and notable sport mutations were utilized.Thus, the experiment was a 3 x 3 x 2 factorial in completely randomized design, comprising the initial three levels of poultry manure (0, 5 and 10 t/ha), three ripening stages of fruits (Green, half-ripe and fully ripe) and two colour types of the pepper (the conventional yellow type, and off-type from spot mutations of branches, colored red).Mature fruit samples were washed, dried with paper towel, ground and finely diced for subsequent analyses.All analyses were prepared in triplicate and percent ash, carbohydrate, fat, crude fiber, moisture, protein, alkaloid, flavonoid, tannin and volatile oil contents were determined using standard methods.Moisture was determined by gravimetric method.

Nutritional and phytochemical analyses
Proximate composition of respective treatment samples was performed to determine percent ash, crude fiber, protein, fats, carbohydrate and moisture contents of fruits using the standard methods by association of official analytical chemist (AOAC, 2005).Similarly, total alkaloids, total flavonoids, tannin and volatile oil were determined.Two grams of the ground sample put in silica dish was heated in a muffle furnace at 600°C for 3 h and allowed to cool in a desiccator and weighed to determine percent ash.Crude protein was determined by the micro-Kjeldahl method while percent crude fat was by Soxhlet extraction method.Following AOAC (2005) standard method, crude fiber was determined, however moisture content of samples were determined using 2 g of samples dried to constant weight at 60°C in a hot air circulating oven for 24 h.
Determination of total alkaloids, total flavonoids and tannin was by spectrophotometric method but volatile oil was determined using direct steam distillation with petroleum spirit at 40°C (AOAC, 2005).

Statistical analysis
All data were subjected to analysis of variance (ANOVA) using Genstat release 7.22 DE (GENSTAT, 2009).Test of significance of treatments means was by Fisher's least significant difference (F-LSD) at 5% probability level, as outlined by Obi (2002).Second order interaction involving manure rate, ripening stage and pepper type was further modeled with genotype-genotype-environment (GGE) biplot analysis (Yan, 2001).

RESULTS
The textural class of the potted soil was sandy clay loam; the soil was acidic and generally low in essential plant nutrients.Meanwhile, the poultry manure utilized was high in pH, organic carbon and organic matter; besides, the exchangeable minerals were relatively high (Table 1).
Significance tests of variance components shown in Table 2 revealed that manure rate (MR), fruit type (FT) and ripening stage (RS) were very highly significant (p ≤ 0.001) for most traits, although FT did not significantly influence percent fat.First and second order interactions of MR, FT and RS were significant for most traits except flavonoids, tannins and volatile oils.

Growth and yield data
Table 3 shows that physiologically timed events such as (time to first, 50 and 100%) anthesis, fruiting and harvesting responded to the rate of manure.In all cases, there were inverse responses of days to specific event as manure rate increased from zero to 10 t/ha.As expected, plant growth traits, that is, plant height, number of branches and number of leaves per plant, increased as plant age and rate of poultry manure increased (Table 4).Significant (p ≤ 0.05) treatment effect was obtained for plant height from the 10 th week after transplanting, however, number of branches and leaves per plant varied significantly as from the sixth week.These growth characters increased progressively with increased poultry manure.Poultry manure significantly (p ≤ 0.05) influenced the cumulative number of fruits and weight of fruits harvested per plant (Table 5).The number and weight of fruits harvested when plants received poultry manure were several multiples of the number and weight harvested from plants not fertilized.Application of 10 t/ha of poultry manure outperformed the 5 t/ha application rate.Frequency of fruit harvest was not statistically significant (Table 5).Evaluation of fruits weight loss under tropical ambient laboratory condition revealed that the quantity of poultry manure applied significantly (p ≤ 0.05) affected the percent moisture loss over the period of study (Table 6).
In all cases, no application of manure increased the percent moisture loss while the least loss of moisture was from fruits that received the highest dosage of manure.Correlative responses of yield components to plant growth were determined to ascertain the strength of relationships between growth (plant height, number of branches and number of leaves) at different growth ages and the eventual fruit yield (Table 7).Plant height, number of branches and number of leaves at 6, 10 and 14 weeks after transplanting (WAT) had significant (p ≤ 0.05) and positive relationships with number of fruits harvested and fruit weight.In some cases, growth traits of older plants had higher correlation coefficient with yield components (Table 7).

Data on proximate and phytochemical qualities
The proximate quality traits were significantly affected by ripening stage (RS) across manure rates (MR) (Table 8).
Irrespective of MR, ripe fruits had higher percent crude fat, fiber and carbohydrate, meanwhile unripe (green) fruits harvested from plants fertilized with 10 t/ha contained the highest percent protein.All the phytochemicals were higher in ripe fruits regardless of the manure rate.
Combined effects of MR and FT showed that yellow fruits contained higher ash, crude fiber and moisture across the MR, but the red fruit type had higher carbohydrate and slightly higher protein (Table 9).Red fruits contained more alkaloids, flavonoids and volatile oils irrespective of the quantity of manure earlier applied to the crop.However, tannin was slightly higher in yellow fruits.It is noteworthy that increasing manure rate increased the quantity of phytochemicals in both the red and yellow fruits.Table 10 presents variations in fruits biochemical qualities due to ripening stages of the yellow and red fruit types.Fully ripe fruits of both types contained higher proportion of ash, fat, carbohydrate and crude fiber, but protein and moisture were higher in unripe (green) fruits.Regardless of fruit types fully ripe fruits had higher alkaloids, flavonoids, tannin and volatile oils.The second order interaction involving manure rate (MR), fruit type (FT) and ripening stage (RS) showed how the combined effects of the three factors affected the phytochemicals in pepper fruit (Figure 1).
The biplot explained 95.1% of the total existing variation suggesting that deductions from it are reliable.Ripe fruits of the red type that were harvested from 10 t/ha poultry manure (M10rfr) contained higher quantity of phytochemicals especially flavonoid and volatile oils.It was evident from the biplot that application of manure tended to increase quantity of phytochemicals in the fruits especially when the fruits were fully ripe or half ripe before harvesting (Figure 1).Proximate quality traits responded significantly to the combined effects of MR, FT, and RS as shown in the biplot in Figure 2, which explained 81.8% of the total variation.Yellow pepper fruits grown under 5 t/ha or 10 t/ha manure regimes and harvested green contained more protein and moisture.Percent carbohydrate was highest in ripe red fruits that were not fertilized (M0rfr).Similarly, quantity of fat was higher in ripe yellow fruit type that manure was not applied (M0yfr).There were higher values of crude fiber and ash in ripe yellow fruits grown under either 5-or 10-t/ha manure (M5yfr or M10yfr).

DISCUSSION
The physicochemical analysis of the soil sample showed that the soil was low in essential plant nutrient elements and was acidic following Ibedu et al. (1988) classification of soils in the zone.However, the poultry manure applied was sufficiently high in pH and essential elements to bridge putative nutrient deficiency gap of the soil used.
The quality of growth of potted plants is influenced to a large extent by the quality of the growth substrate provided for growing the plants.
The adequacy of the essential nutrient elements in the container certainly will influence growth performance of the plants.Thus, it was appropriate that the quantity (rate) of the poultry manure used, which invariably influenced the adequacy of essential nutrient elements in the container, influenced growth, yield and nutrient quality of 'Nsukka Yellow' pepper.
Plant growth characters increased as the organic manure level increased, which is attributable to greater supply of plant nutrients with incremental application of poultry manure since the un-amended soil was low in nutrient content.Asiegbu and Uzo (1984) reported a high response of eggplant and onion to incremental application of organic manure in a similar soil at Nsukka.Similarly, these authors reported that farmyard manure (FYM) benefitted fruit set in eggplant and generally enhanced size characters in both onion and eggplant.
The positive response of physiological-time events to manure rates suggested that phenological changes (anthesis, fruiting and fruit maturity) in 'Nsukka Yellow' pepper could be influenced by the quantity of nutrient elements (determined by the quantity of manure applied) available for growth.This is corroborated by the significant increase in growth traits (plant height, number of branches and number of leaves) as manure rate increased.
For most crops there is a direct relationship between growth and yield.This was evident in this study as plant height, number of branches and number of leaves were positively and significantly correlated with number of fruits and weight of fruits harvested.Abu et al. (2013) reported a similar finding for field grown aromatic peppers.
In their study, fruit yield could be predicted from the combined effects of number of nodes, number of leaves and fruits per plant, with above 80% predictive accuracy.In the current study, it was notable from the correlative responses that yield could be predicted from 6-week old plants although 10-week old plants were stronger estimator), the positive relationship suggest that the better the crop performance at this stage, the more the yield, thus allowing for effective nutrient management in the case of early season poor crop performance.The result (suggested that, providing adequate growth environment for  containerized plants will enhance its yield capacity. The use of organic manure especially from poultry source has become increasingly important in southeastern Nigeria because of its role in increasing the nutritive value of most fruit crops and leafy vegetables (Ndukwe et al., 2010(Ndukwe et al., , 2012;;Ndubuaku et al., 2014).In the current study, application of poultry manure increased most of the nutritional properties of the 'Nsukka Yellow' pepper.Significant effect of poultry manure rate on the nutrient quality of pepper is also supported by an earlier study on passion fruits (in the same location), which showed that nutritional quality of the juice varied with poultry manure rate (Ani and Baiyeri, 2008).
Nutritional quality of pepper fruits varied significantly with ripening stage, suggesting that ripening may have enhanced biosynthesis of these nutrients.Noteworthy is that green (unripe) pepper fruits had higher percent protein, although alkaloids, flavonoids and volatile oils increased with ripening.Earlier studies on plantain fruits reported that ripening increased the quantity of most of the nutrient elements assayed for (Baiyeri and Unadike, 2001;Baiyeri et al., 2011).

Conclusion
Growth and yield performances and nutritional quality of pepper could be manipulated positively by the application of poultry manure.'Nsukka Yellow' pepper could be cultivated in pots without compromising both yield and quality attributes of the pepper.Application of 10 t/ha poultry manure is recommended for growing the crop.

Figure 1 .
Figure 1.Biplot analysis of combine effects of poultry manure rate, different ripening stages and pepper type on the phytochemical qualities of 'Nsukka pepper' (yellow and red).

PC2Figure 2 .
Figure 2. Biplot analysis of combined effects of poultry manure rate, different ripening stages and pepper type on the proximate qualities of 'Nsukka pepper' (yellow and red).

Table 1 .
Physical and chemical characteristics of soil and poultry manure used.

Table 2 .
Test of significance of variance components showing degree of freedom and probability of significance.

Table 3 .
Days to first, 50 and 100% anthesis, fruiting and harvesting as influenced by rate of poultry manure.

Table 4 .
Effect of manure rates on plant height (cm), number of branches and number of leaves per plant over time.

Table 5 .
Effect of manure rate on components of yield of 'Nsukka Yellow' pepper.

Table 6 .
Percent fruit weight loss of 'Nsukka Yellow' pepper over time (days) as influenced by poultry manure rates.
DAS: Days after storage.

Table 7 .
Relationship between plant growth parameters and yield components.

Table 8 .
Combined effects of manure rate and ripening stage of fruits on nutritional quality of Nsukka Yellow pepper.

Table 9 .
Combined effects of manure rate and pepper type (yellow and red) on nutritional quality.

Table 10 .
Combined effects of pepper type (yellow and red) and ripening stage on nutritional quality.