Physiological and Biochemical Changes in Cucurbita moschata Duch. Ex. Poir Inoculated with a Nigerian Strain of Moroccan Watermelon Mosaic Virus (MWMV): Lagenaria breviflora Isolate

MATERIALS AND METHODS

Seed collection and planting: Seeds of Cucurbita moschata used in this study were obtained from Akparabong in Ikom Local Government Area of Cross River State Nigeria, located on latitude 5°57’N and longitude 8°44’E. The seeds were sown in steam-sterilized soil in 16 cm diameter polyethylene bags and maintained in the greenhouse at 25±3°C.

Virus source and preparation of inoculum: The virus used in the study is a strain of Moroccan Watermelon Mosaic Virus (MWMV) isolated from Lagenaria breviflorus. The virus inoculums was prepared by obtaining young symptomatic leaves of Lagenaria breviflorus. These leaves were titrated in a pre-sterilized cold mortar and pestle in disodium phosphate (Na_zHPO₄) butter 0.03 M, PH 8.0. The inoculum was applied on leaves dusted with carborundum (800 mesh), rinsed and then left for symptoms development.

Experimental design: Fourty five seedlings of inoculated and healthy plants of C. moschata were arranged in three groups each containing fifteen plants each prior to inoculation, the seedling growing in polyethylene bags were arranged in a randomized block design. Within each group nine were inoculated with the virus isolate and the remaining six were inoculated only with the buffer to serve as healthy controls.

Sample preparation for analysis: Two months post-inoculation, inoculated and healthy leaves were harvested randomly from fifteen plants (i.e., from each of the three groups). These leaves were dried in the oven at 65°C and milled into powdered form with the aid of an electric mill (National Food Grinder, Model MK 308, Japan). The powdered samples were then used for the phytochemical, proximate, mineral and antinutrient determination.

Standard procedures were used in the determination of phytochemicals. Alkaloids were determined using, Harbone (1973) method, tannins (Van Buren and Robinson, 1969). Vitamins and minerals were determined by atomic absorption spectrophotometer (Pye Uniam SP8-190, Spec. UK) as outline in AOAC (1995). Phytate was determined according to AOAC (1995), while oxalate content was determined by the method of Dye (1956) as described by Abara et al. (2000).

Effect of mwmv infection on growth parameters: Leaf area determination of inoculated (MWMV) and healthy plants of C. moschata was carried out at 7 days interval for a period of 56 Post-Inoculation Days (PID). Here inoculated and healthy leaf each was placed on a 1 mm² graph paper. The leaf size was traced on the paper and the total area calculated based on the number of squares covered within the traced region.

The formula for leaf area estimation is given as:

A = KLB (cm²), thus:

Effect of mwmv infection on shoot height and number of leaves: The effect of viral inoculated on short height was determined by measuring stem length in centimeters from soil level to the tip of the stem with the aid of a measuring tape. Measurements were taken as described above. The leaves of both inoculated and healthy plants were counted to determine the effect of the virus infection on the number of produced leaves over the same period of time.

Effect of MWMV infection on total chlorophyll content: Two gram leaf sample obtained from inoculated and healthy plants were used in the estimation of chlorophyll. Measurements were taken at 14 PID intervals. The leaves were grounded using mortar and pestle in 10 mL of 80% acetone. The homogenate were poured into test tubes and centrifuged for 3 min at 4,500 rpm. The supernatants were decanted and then used for chlorophyll estimation. Acetone 3 mL was used to set the blank at zero. To this acetone was added 0.1 mL each of the supernatants from inoculated and healthy samples and absorbance readings taken at wavelength 663 and 643 nm for chlorophyll a and b respectively with the aid of a spectrophotometer (Model 722S, England). Readings were taken from three replicates of each sample and chlorophyll contents were estimated using the formula of Strickland and Parsons (1972):

Chl a = (11.6 A663-1.3 A643) VX^–1
Chl b = (19.6 A643-4.7 A663) VX^–1
Chl a+b = (mg g^–1 FW)

where, A663 and A643 represent absorbance at 663 and 643 nm, respectively. A is absorbance, V^–1 is volume of 80% acetone, X^–1 is sample fresh weight, mg is milligram, g is gram and FW is fresh weight.

Data analysis: Data obtained in this study were analyzed using the independent t-test. Results were also expressed as percentage difference and difference between mean values were determined at 5% probability.

RESULTS

Results in Table 1 and Fig. 1a, b revealed significant (p≤0.05) decrease in shoot height, leaf area and number of leaves produced in C. moschata infected with Moroccan watermelon mosaic virus. The virus caused progressive decrease in the growth parameters studied with increasing period of development. Shoot height had a range in mean values for inoculated and healthy plants from 9.80±0.62-20.90±0.6 and 9.98±0.62-33.55±0.6 cm corresponding values for leaf area in inoculated and healthy plants ranged from 3.80±-0.3, 25.55±1.8 cm² and 3.90±0.3-56.05±1.2 cm². While mean values for number of leaves produced in inoculated and health plants ranged from 12.10±0.6-45.02±0.03 and 12.10±0.6-56.05±0.1. Infection of MWMV was insignificant at early period of growth 7 (PID).

Table 1:	Effect of MWMV infection on the growth parameters of Cucurbita moschata
p≤0.05, values are Mean±SD, N = 3 replicates, Percentage difference was obtained by expressing the difference between the values for inoculated and healthy as a percentage of the healthy

Fig. 1(a-b):	Symptoms induced by MWMV on C. moschata included mosaic, green vein-banding, rugosity and leaf malformation, (a) Healthy leaf and (b) Inoculated leaf

Table 2:	Effect of MWMV infection on chlorophyll content of Cucurbita moschata
Values are means of three replicates, Percentage difference was obtained by expressing the difference between the values for inoculated and healthy as a percentage of the healthy

Table 3:	Qualitative determination of phytochemicals in Cucurbita moschata inoculated with MWMV
+: Present, -: Absent

Infection of Cucurbita moschata with MWMV resulted in significant (p≤0.05) reduction in chlorophyll a, b and total chlorophyll contents. Higher reduction in chlorophyll content was recorded for chl a when compared with chl b. Lowest and highest mean values for chl a, b and total chl in inoculated plants were 30.8±0.02 and 42.8±0.03, 42.1±0.02 and 69.3±0.03 and 72.9 and 112.1 mg g^–1 FW, respectively. Corresponding mean values for healthy plants were 46.3±0.5 and 86.1±0.04, 65.6±0.03 and 113.4±0.01 and 111.9 and 199.5 mg g^–1 FW (Table 2).

In both inoculated and healthy plants, chlorophyll content attained a peak at 42 PID and declined. The result showed that MWMV infection caused significant (p≤0.05) decrease in the contents of chlorophyll when compared with healthy plants.

Results of qualitative determination of phytochemicals revealed that of the twelve phytochemicals screened in leaves samples of C. moschata infected with MWMV, nine (alkaloids, glycosides, saponins, tannins, flavonoids, terpenoids, steroids, polyphenols and reducing sugars) were present and three (phlobatanins, anthraquinones and hydroxymethyl anthraquinones) were absent, glycoside, flavonoids, steroids and reducing sugars were more in ethanolic extract when compared to aqueous, while polyphenols were more in aqueous extract than in ethanolic extract (Table 3).

Effect of MWMV infections on crude phytochemicals (Table 4) resulted in significant (p≤0.05) reductions in saponins, alkaloids, terpenoids, reducing sugars and steroids with percentage difference of 33.6, 32.1, 25.0, 42.0 and 15.0%, respectively.

Table 4:	Quantitative determination of phytochemicals in Cucurbita moschata Inoculation with MWMV
p≤0.05, values are Mean±SD, N = 3 replicates, Percentage difference was obtained by expressing the difference between the value for inoculated and healthy as a percentage of the healthy

Table 5:	Effect of MWMV Infection on Proximate Composition of Cucurbita moschata
Values are Mean±SD, N = 3 replicates, percentage difference was obtained by expressing the difference between the values for inoculated and healthy as a percentage of the healthy

Table 6:	Effect of MWMV Infection on Mineral Composition of Cucurbita moschata
Values are Mean±SD, N = 3 replicates, percentage difference was obtained by expressing the difference between the values for inoculated and healthy as a percentage of the healthy

Marginal reduction were recorded for tannins (3.2%) and polyphenol (2.8%). Glycosides and flavonoids were noise in inoculated sample when compared to the healthy.

Moroccan watermelon virus infection on the proximate composition of C. moschata (Table 5) indicated significant (p≤0.05) reductions in lipid, ash and carbohydrate contents with percentage difference of 25.0, 14.6 and 10.7%, respectively. Protein and fibre contents were significantly (p≥0.05) higher in inoculated samples than in healthy ones. Mean values of protein and fibre in healthy samples were 4.70±0.1 and 1.90±0.1 mg/100 g while inoculated samples had values of 5.80±0.1 and 2.10±0.1 mg/100 g. Marginal increases in inoculated samples were recorded for vitamin A, β-carotene and vitamin C.

Results obtained for effect of MWMV on mineral composition showed significant (p≤0.05) reductions in potassium, iron, zinc, magnesium and sodium with percentage reduction values of 48, 28, 25, 22 and 20%, respectively. Hundred percentage increase in inoculated sample was recorded for cupper while phosphorus and calcium depicted insignificant increases (Table 6).

Antioxidants (hydrocyanic acid, total oxalate, soluble oxalate and phytic acid) were significantly (p≥0.05) higher in inoculated samples the healthy with percentage difference of 22.1% for Hydrocyanic acid.

Table 7:	Effect of MWMV infection on antioxidant in Cucurbita moschata
Values are Mean±SD, N = 3 replicates, percentage difference was obtained by expressing the difference between the values for inoculated and healthy as a percentage of the healthy

DISCUSSION

This study was conducted to determine physiological and biochemical changes induced by MWMV in C. moschata. Results of the study show that MWMV caused considerable changes in the parameters investigated as systematic host of the virus (Table 7). It was well expressed in the reduction in the phytochemcial contents of saponins, alkaloids, terpenoids, reducing sugars and steroids. Reductions engendered by MWMV infection are in agreement with previous reports by El-Dougdoug et al. (2007), Duarte et al. (2008) and Mofunanya and Nta (2011). Significant increases in glycosides and flavonoids and insignificant increase in tannins in infected samples reported in this study are in line with findings by Uegaki et al. (1998), who reported on the accumulation of stress compounds due to virus infection. The accumulation of these chemical constituent in inoculated plants suggest that their synthesis is stimulated by stress caused by virus infection. While reduction in phytochemicals could be due to reduction in plant total fresh weight of leaves and demand for viral components synthesis which could cause the host cells to be in competition with normal biosynthetic pathways for essential precursors.

Significant (p≤0.05) reductions in proximate and mineral constituents are in the line with reports by Mofunanya et al. (2008). Antioxidants content were considerable higher in inoculated samples compared with healthy ones.

The reductions depicted in phytochemicals and mineral nutrients in this study require keen attention because the medicinal values of plants and vegetables are revealed by their phytochemical and other constituents as reported by Huseini et al. (2005). Edeoga et al. (2005) also documented that the presence of these chemical constituents that produce a definite physiological action in the body reveal it’s medicinal value. Leafy vegetables are of great importance to the health of individuals as well as communities. The urban and rural poor depend of vegetables as their main and cheap source of mineral elements (Ajakaiye et al., 1995). Vegetables are good sources of both macro and micro nutrients.

Phytochemicals are important to both human and animal as they have been linked with positive effect in health including cancer, high blood pressure, coronary heart disease, diabetic, ulcer, muscular degradation, psychotic disease with multiple actions on human health.

Infection of C. moschata by MWMV led to significant reduction in growth characteristics. It is not uncommon for virus infection to have a negative impact on plant especially by limiting their growth characteristics (Miteva et al., 2005). One explanation for leaf area decrease in inoculated plants could be due to the reduction in the amount of assimilate availability (Ayres, 1992). There thus appear to be a link between assimilate reduction and plant growths.

Reduction in chlorophyll contents agrees with previous studies by Funayama-Noguchi and Terashima (2006), Pineda et al. (2008), Singh and Shuka (2009), Pazarlar et al. (2013), Sinha and Srivastava (2010) and Mofunanya et al. (2014), who have all reported on reduction in chlorophyll content in many plants infected with different viruses.

The differences in chlorophyll content in inoculated and healthy plants is worthy of note as the leaves of inoculated plants appeared to be mosaic. Leaf chlorophyll content can be directly linked to stress physiology (Hooks et al., 2008). Chlorophyll a and b are the two most important pigments used in plant photosynthetic processes and low levels of these chlorophylls can directly limit photosynthetic activity (Taiz and Zeiger, 1991).

Loss of chlorophyll in infected plants has been attributed to impaired chlorophyll synthesis (Funayama-Noguchi and Terashima, 2006). Goncalves et al. (2005) attributed loss in chlorophyll to structural alternations in the photosynthetic apparatus while, Guo et al. (2005) attributed loss to reduction in the activities of some enzymes associated with electron transport and photosynthesis. From the above, various factors are associated with loss in chlorophyll in infected leaves. Moroccan watermelon mosaic virus is a potyvirus. Potyviruses have been known to cause chloroplast aggregation and resulting in a reduction in their number (Wei et al., 2010, 2013) and inhibition of carbon assimilation enzymes (Guo et al., 2005) chlorophyll reduction in C. moschata caused by MWMV infection in this study could be said to be due to a reduction in the number of chloroplast in the inoculated leaf tissues. Results of our study underline the fact that plant viruses unfavorably influence crops physiological processes.

Reduction in chlorophyll content led to a corresponding decrease in carbohydrate. Goodman et al. (2008) and Singh and Shuka (2009) also reported reduction in carbohydrate in other host plant virus combination. It has been established in many host virus infection that there is an increased demand of abnormal protein production required for the rapid synthesis of virus particle and for this purpose there has been increase in the diversion of assimilated carbon compounds towards protein synthesis eventually resulting in the decreased production of carbohydrate level in the leaves (Singh and Shuka, 2009). The decrease in the content of carbohydrate is due to increased respiration resulting from virus infection and the conversion of carbohydrate into amino acids used in protein synthesis of the viral coat. This reduction might also be due to the duration of virus infection (Muqit et al., 2007; Sinha and Srivastava, 2010).

CONCLUSION

From the results obtained it is clear that infection of C. moschata by a Nigerian strain of MWMV caused decreases in physiological and biochemical parameters. These reductions are of great concern both to the plant and to man who depend on this vegetable as a cheap source of mineral elements. Resources should be geared towards prevention of MWMV on C. moschata in order to ensure quality and guarantee-nutritive value by searching for resistant varieties of the vegetable and their cultivation are recommended.

ACKNOWLEDGMENT

The authors are grateful to Mr. S. M. Ekpo the Chief Technologist of the Department of Biochemistry, Faculty of Basic Medical Sciences, University of Calabar, Nigeria for Technical Assistance.