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Reaction of Amaranthus hybridus L. (Green) to Telfairia Mosaic Virus (TeMV) Infection

The reaction of Amaranthus hybridus to Telfairia mosaic virus (TeMV) infection was investigated. Infected and healthy (control) leaf samples were obtained, pulverized and analyzed to determine the nutritional quality of the vegetable due to TeMV infection. Results obtained revealed that the virus caused significant (p<0.05) reductions in fibre (50.9%), fat (49.7%) and protein (32.5%) in infected samples compared to healthy ones. The virus also engendered significant reductions in the contents of Na, P, Fe, Mg, Cu and Ca with marginal reduction for K in infected samples when compared to the healthy. The TeMV caused significant reduction in phytic acid while infection led to increases in total oxalate, soluble oxalate and hydrocyanide acid. The virus caused significant reductions (p<0.05) in vitamin A (55.43%), C (43.1%), B1 (34.6%), B2 (26.6%), B6 (17.8%) and B3 (13.1%), respectively while, reduction in vitamin E was insignificant. The reaction of amino acids profile of Amaranthus hybridus to TeMV infection revealed significance decrease in methionine (35.3%), valine (34.1%), cysteine (31.9%), arginine (26.1%), isoleucine (25.9%), glycine (22.8%), lysine (17.3%), threonine (15.0%), phenylalanine (12.1%) and leucine (11.8%). Increases in infected samples were obtained for glutamic acid (19.8%), aspartic acid (9.5%) and proline (8.9%). Amaranthus hybridus reaction to TeMV infection revealed marked reductions in the nutritional quality of the vegetable that is a major source of nutrient for both rural and urban dwellers.


INTRODUCTION
Amaranthus hybridus popularly (called smooth Amaranthus or amaranth or pig weed) is cultivated in several areas of the world including south America, Africa, India, China and the United States (He and Corke, 2010). Amaranthus hybridus is an annual herbaceous plant. It is a common species in waste places, cultivated fields and barnyard. The commercial production of Amaranthus hybridus is increasing throughout the world as an important alternative food source (Kauffmann and Weber, 1990;Rawate, 1983). Amaranthus hybridus is cultivated on a commercial scale in southern Nigeria. It constitutes a major part of the diet of the people in middle and Southern parts of Nigeria, where they are mostly used in soups preparation (Oke, 1983;Mepba et al., 2007). In Congo, their leaves are eaten as spinach or green vegetables (Dhellot et al., 2006). In Mozambique and West Africa the leaves are boiled and mixed with a groundnut sauce are eaten as salad (Oliveira and de Carvalho, 1975;Martin and Telek, 1979). This vegetable has also been reported to contain squalene which has beneficial effects on cancers and industry (Rao et al., 1998;Smith, 2000;He et al., 2003). Int. J. Virol.,11 (2): [87][88][89][90][91][92][93][94][95]2015 Developing countries of the world depend on starch-based foods as the main staple food for both energy and protein supply. Protein deficiency which is prevalent among the poor populace as documented by FAO is accounted for in part. In Nigeria, where the daily diet of people is dominated by highly starchy foods. Vegetables serve as indispensable constituent in the diet (Akubugwo et al., 2007).
The production of A. hybridus, a highly nutritious leafy vegetables of tropical West Africa is reported to be limited by a number of diseases such as wet rot of leaves and young stalks caused by Choanephora cucurbitarum , damaging of the flowering head caused by lygus bug Lygus lineolarius and curly top diseases most of which are of viral etiology (Maboko, 1998). Worldwide, diseases caused by virus have been recognized to constitute one of the major factors limiting vegetable production (Grogan, 1980). Amaranthus Mosaic Virus (AMV) was also reported on Amaranthus in Nigeria (Thottappily, 1988). Amaranthus species serve as experimental host to a number of viruses (Nyamupingidza and Machakaire, 2003). Amaranthus species have been reported as susceptible host of TeMV (Shoyinka et al., 1987). The virus was reported to be seedborne in Telfairia occidentalis (Anno-Nyako, 1988). Infected leaves exhibited mosaic severe leaf malformation and distortion and reduced leaf size.
Studies by Akubugwo et al. (2007) and Maiyo et al. (2010) centered on phytochemical, mineral and antimicrobial activity of leaves of A. hybridus. Reports also exist on the performance of A. hybridus as affected by enrichment of compost with urea during composting (John and Udoinyang, 2007) and effect of AMV on the growth characters of A. hybridus (Ehinmore and Kareem, 2010). However, information is lacking on the effect of TeMV on the nutritional quality of this all important vegetable. This work is therefore aimed at documenting the reaction of Amaranthus hybridus to infection by TeMV.

MATERIALS AND METHODS
Collection of seeds and planting: Seeds of A. hybridus were obtained from Akparabong market in Ikom Local Government Area of Cross River State (CRS), Nigeria. They were sun-dried for two days to enhance germinability and thereafter scattered on a steam-sterilized fertile garden soil in 16 cm diameter polyethylene bags. The seeds germinated and were inoculated with TeMV at the four leaf stage. The inoculum was prepared from diseased leaves of T. occidentalis and applied on A. hybridus by the conventional leaf-rub method (mechanical or sap inoculation) after application of carborundum (800 mesh). The inoculated leaves were then rinsed with water and left for symptom development while the control plants were only inoculated with the buffer.

Experimental design and inoculation of experimental plants:
Test plants were arranged in two groups each containing 30 plants each which were set out in three rows of ten plants. Prior to inoculation, plants to be inoculated with the virus and those of controls in both groups were arranged in a randomized complete block design in the greenhouse with average temperature of 25±3°C. The inoculation of the test plants was as described above. Within each plant groups, fifteen were inoculated with the virus and the remaining fifteen inoculated with buffer only to serve as controls. The set up was monitored for symptom expression (8-10 days post-inoculation), which included mosaic, severe leaf malformation and distortion characteristic of TeMV infected A. hybridus. At three months period of development, infected and healthy leaves were harvested, oven-dried, grinded into fine powder and used to determine infection of TeMV on the nutritional quality of Amaranthus hybridus. Int. J. Virol., 11 (2): 87-95, 2015 Sample analysis: The proximate compositions of (Moisture, crude protein, ash, lipid and fibre) of the samples were determined by the method of the Association of Official Analytical Chemists (AOAC., 1984). Mineral contents (Ca, Fe, Mg, Zn, Cu and P) were determined using atomic absorption spectrophotometer as outlined in AOAC (1984). Sodium and K were estimated by flame photometry. The antinutrients: hydrocyanate was determined according to AOAC (1984), phytate (Abara et al., 2000) and oxalate (Dye, 1956) as described by Abara et al. (2000). Vitamin A content was determined spectrophotometrically using the hexane method. Vitamin C by modified method of Bessey (1944), vitamin B 1 (Thiamine) and vitamin B 2 (riboflavin and nicotinamide by AOAC (1984). While, amino acids were determined using methods described by Spackman et al. (1958).

Statistical analysis:
The data obtained in this study was analyzed using the Student t-test. Results were also expressed as percentage difference and differences between mean values were determined at 5% probability.

RESULTS
The results of this study revealed that TeMV infection of A. hybridus caused significant reductions (p<0.05) in fibre, fat and protein contents when compared to the values obtained for the healthy controls (Table 1). The mean values of 4.23±0.2, 2.34±0.2 and 12.10±0.1 (g/100 g) were recorded for fibre, fat and protein respectively in infected sampled with corresponding mean values of 8.61±0.1, 4.65±0.01 and 17.92±0.2 (g/100 g) for control samples. However, results obtained for ash, moisture and carbohydrate showed marginal reductions when compared to the healthy controls. The percentage reduction in the content of these proximate composition engendered by TeMV ranged from 2.15% for carbohydrate to 49.68 for fat.
Results as presented in Table 2 highlight the effects of TeMV on mineral composition of A. hybridus infection of A. hybridus of resulted in significant decreases in all the elements with the exception of potassium. Significant decreases obtained for Na, P, Fe, Zn, Mg, Cu and Ca in infected samples were 0.07±0.02, 12.11±0.1, 6.21±0.02, 2.79±0.01, 0.02±0.01 and 29.21±0.1 mg/100 g,   Effects of TeMV on antinutrient contents of A. hybridus were found to be significantly higher in infected samples compared to the healthy controls with the exception of phytic acid. The mean values obtained for total oxalate, soluble oxalate and hydrocyanide acid in infected samples were 54.79±0.01, 48.51±0.01 and 3.13±0.1 (mg/100 g), respectively while, corresponding values for the healthy controls were 38.82±0.02, 31.79±0.01 and 1.57±0.01. Infection of TeMV on A. hybridus caused significant reduction (p<0.05) in phytic acid with mean value for infected sample of 80.10±0.02 and values for healthy sample of 120.30±0.3 (mg/100 g), respectively. Values for percentage difference in anti-nutrient ranged from 33.4% for phytic acid to 52.6% for soluble oxalate (Table 3).
Results as presented in Table 5 show significant (p< 0.05) decline in sulphur containing amino acids: cysteine, isoleucine, leucine, lysine and methionine with a range in percentage decline from 11.8% (leucine) to 35.3% (methionine). Phenylalanine and tyrosine (aromatic amino acids) also showed decline due to TeMV infection. Nonessential amino acids (glutamic acid, aspartic acid and proline) were significantly higher in infected samples. Percentage reductions due to infection of TeMV on Amaranthus hybridus for essential and nonessential amino acids ranged from 5.0% (tyrosine) to 35.3% (methionine). The significant decline in essential amino acids (histidine, lysine, isoleucine, leucine, methionine, phenylalanine, threonine, tryptophan and valine) and nonessential amino acids (alanine, serine, arginine, cysteine, tyrosine and glycine) and increase in infected samples of glutamic acid, aspartic acid and proline.

DISCUSSION
The reaction of A. hybridus to TeMV infection resulted in caused significant (p<0.05) reductions in fibre, fat and protein contents, all the mineral elements with exception of K, vitamins A, C, B 1 , B 6 and B 3 with significant increases in anti nutrients with the exception of phytic acid in infected samples when compared to healthy ones. Previous reports of virus infection on protein contents are varied and seem depended on host-virus combination. Muqit et al. (2007) reported a decrease in the protein content of Benincasa hispidia infected with Bottle gourd mosaic virus (BgMV) and Watermelon mosaic virus-2 (WMV-2) singly. Mofunanya et al. (2008) have also reported a decrease in the protein content to T. occidentalis inoculated with TeMV. Owolabi et al. (2010) observed a decrease in protein content in Ivy gourd infected by a Nigeria strain of Moroccan Watermelon Mosaic Virus (MWMV). A decrease in carbon, nitrogen and protein in Daucus carota due to infection of Cucumber mosaic virus and increased P in diseased plant has been reported by Afreen et al. (2011). Soya bean mosaic virus (SoyMV) and Cowpea Chlorotic Mottle Virus (CCMV) (Demski and Jellum, 1975), Potato Virus X (PVX) and Potato virus Y (Gommaa et al., 2004) and Tobacco Mosaic Virus (TMV) (Barka and El-Maaty, 2008) have been reported to cause decreases in the protein contents in soybean, Nicotiana tabacum and tomato, respectively. Results of this study however, contrasts with those of White and Blakke (1992), who reported increased level of protein in barley infected with WSMV and BSMV separately. Milavec et al. (2001) and Hemida (2005) have also documented increases in the protein content in potato-PVY NTN and Vicia faba-BYMV combination. Significant reductions in fibre and fat observed in this study is consistents with those of Mofunanya et al. (2008) in T. occidentalis-TeMV combination and Owolabi et al. (2010) in Ivy gourd-MWMV combination.
Reductions in mineral elements recorded for Na, P, Fe, Mg, Cu and Ca in this study correspond to previous reports by Mofunanya et al. (2008) and Owolabi et al. (2010) but differs from that of Nogueira et al. (1996) who reported increased level of Fe, while the level of Mg was similar to that of the control. Salama et al. (1975) and Shattuck (1987) have reported increased levels of P in infected leaf tissues of broad bean and Brassica napus spp. Rapifera infected with Broad bean mosaic virus (BBMV). Owolabi et al. (2010) recorded significant reductions in Mg, Fe and Ca with increases in P, Mn and K in Ivy gourd leaves (Coccinia barteri) infected with a Nigerian strain of MWMV.
Significant increase in the contents of total oxalate, soluble oxalate and hydrocyanide acid obtained in infected samples in this study agrees with previous reports by Mofunanya et al. (2008). The reductions in vitamins in TeMV infected A. hybridus correspond to report of reductions in vitamin A and C in T. occidentalis infected by TeMV. Mofunanya et al. (2008), Owolabi et al. (2010, Raj et al. (2005) and Prakash et al. (2006) and Kapinga et al. (2009) also reported losses in these vitamins in other plant-virus combination.
Reductions in amino acids profile due to TeMV infection of A. hybridus in this study are in consonance with results of previous reductions resulting from TeMV infection in T. occidentalis ecotypes (Mofunanya et al., 2009). Mofunanya et al. (2009) reported a decrease in infected samples of essential and nonessential amino acids with increase in infected samples of glutamic acid, aspartic acid, valine and proline. Reductions by BYMV in Vicia faba and Phaseolus vulgaris (Hemida, 2005) and MWMV in leaves of Ivy gourd (Owolabi et al., 2010). The decrease in amino acids may be due to their utilization by the virus and enhanced enzyme activities in leaves while increase in these amino acids which are protein constituents may be due the fact that the coat protein of a virus in this case TeMV may come to represent about half the protein in disease leaf (Hull, 2002).
Accumulation of proline and other amino acids is a common metabolic response of higher plants to both abiotic and biotic stress, many plants accumulate high amount of proline in their tissues (Drossopoulos et al., 1985;Mazid et al., 2011).
The presence of these amino acids in A. hybridus agrees with results of Akubugwo et al. (2007). Seventeen instead of twenty amino acids were determined. This was due the conversion of asparagines and glutamine to glutamic acid and aspartic acid respectively and the destruction of tryptophan that occur during hydrolysis (Wathelet, 1999). The reductions in these minerals could be due to the possible adverse effects and alteration in plant metabolism induced by viral infection.
Plant nutrients are vital to keep the body healthy and alive. They are needed to build and repair cells and tissues of the body as well as maintenance of the organs and bone in working optimally and to provide warmth, energy and fuel. Eating food rich in nutrients can help in preventing common ailments and life threatening disease and illness (Premier, 2002). Adewale and Olorunju (2013) reported that the cheapest and readily available sources of proteins, vitamins, minerals and amino acids are vegetables.
The significant reductions in proximate, mineral elements, vitamins and amino acids profile caused by the reaction of A. hybridus to TeMV infection reduced the nutritional value of this all important vegetable where large quantities are consumed daily by the Nigerian populace. These reductions should be looked into jealously because consumption of infected leaves is of least significance as it has just lower amount of the required nutrients when compared to healthy leaves. The use of resistant variety as a practical means of controlling the virus and biotechnological research that will result in the production and deregulation of virus-resistant A. hybridus through coat protein gene transfer should be intensified.