Nutritional Qualities Evaluation of Ten Accessions of Cowpea ( Vigna unguiculata L. Walp) Infected by Cercospora Leaf Spot Disease

Background and Objective: Despite the importance of cowpea ( Vigna unguiculata L. Walp) in Nigeria, its production in commercial quantity is constrained majorly by fungal diseases, especially Cercospora Leaf Spot (CLS). Therefore, a study was conducted to evaluate the nutritional qualities of ten cowpea accessions infected by CLS planted in the screen house Materials and Methods: The experiment was laid out in Completely Randomized Design with three replications. Data collected were on stomatal conductance, leaf chlorophyll content, disease evaluation and proximate analysis. The data collected were subjected to mixed model Analysis of Variance (ANOVA). Means of significant treatments were separated using Fisher Protected Least Significant Difference (LSD) at p<0.05. Results: The effect of CLS on stomatal conductance, leaf chlorophyll content, disease evaluation and proximate analysis of the cowpea accessions differed significantly with TVU-9182 having the highest value of ash, crude fibre, moisture and fat at 3.92, 3.13, 15.67 and 4.48%, respectively with the value of carbohydrate at 51.99%. The TVU-9175 had the lowest value of ash, protein, moisture, crude fibre and fat at 2.17, 16.55, 8.67, 1.73 and 2.48%, respectively with the highest value of carbohydrate at 68.41%. The ten accessions were significantly susceptible (p<0.5) to CLS with high susceptibility at 10 Weeks After Planting (WAP). Conclusion: The results showed that TVU-9174 and TVU-9182 had better nutritional qualities after CLS infection than the other infected cowpea accessions. Hence, TVU-9174 and TVU-9182 could be used as breeding materials based on the nutritional contents after CLS infection.


INTRODUCTION
Cowpea is one of the important staple food in Africa and a major food crop in Nigeria. The cowpea crop has an average essential protein content of 25% and is rich in vitamins and minerals. A major food of the tropics with low fat and a major delicacy to humans and livestock, highly beneficial to human and animal health 1,2 . It is estimated that 22% of the world's 141 Mha of cowpea is planted in rain-fed fields in West Africa, especially Nigeria 3,4 , where Cercospora Leaf Spot Disease (CLSD) is a major cowpea disease that affects the balance in the synthesis of photosynthetic pigments resulting in poor quality and yield reduction 3 . These changes in the amounts of photosynthetic pigments in the cowpea plant due to the CLS infection are closely associated with plant foliage lesion and death at the reproductive stage [5][6][7] . Hence, the ability to maintain the photosynthetic rate in the cowpea plant determines the disease tolerance 8 . Optimization of this physiological response to CLS to attain reproductive growth is a prerequisite for stable nutrients in cowpea seed development. This ability helps the plant to adapt to such limiting disease conditions 8,9 . The physiological responses of the cowpea plant under disease conditions can affect the nutritional qualities of cowpea seeds produced. Hence, the objectives of this study were to investigate the effect of CLSD on the nutritional qualities of ten cowpea accessions.

MATERIALS AND METHODS Study area:
Screen house experiments were conducted in a Completely Randomized Design (CRD) with three replications at the Institute of Agriculture Research and Training (I.A.R and T) Obafemi Awolowo University, Ibadan, (longitude 3°15'N and latitude 7°23'E) at an altitude of 650 m above sea level. The annual rainfall of 1,320 mm with minimum and maximum annual temperatures of 20.1-23.6 and 27.1-34.5°C, respectively. The cowpea materials were obtained from the IITA gene bank and were selected based on various characteristics such as ease of tolerance, resistance, susceptibility to disease and yield. The cowpea materials used were ten different cowpea accessions in Table 1.
Research protocol: Pure Cercospora fungi culture isolated on Potato Dextrose Agar (PDA) from cowpea plants grown under natural field conditions was vortexed with 100 mL of sterilized distilled water. The concentration of the conidia suspension of the fungi was adjusted with a hemocytometer to 3×10 5 spore mLG 1 and later incubated for 3 days before it was inoculated onto the planted cowpea seedlings in the screen house damped with transparent cellophane cover for 24 hrs to provide a humid environment for the Cercospora fungus to thrive. A control experiment was also set up without any fungi inoculation to elucidate the standard nutritional content of the ten cowpea accessions.
The following physiological variables were taken after infection between the hrs of 9 am to 12 pm noon at 10 Weeks After Planting (WAP) with the control experiment.
Leaf chlorophyll was measured using SPAD meter 11 (Model 2900PDL) made in Italy. Harvesting and post-harvest operation: Harvesting was carried out when the pods of the cowpea plants turned to dry yellow from green colour. The pods were later threshed to obtain the seed grains and recorded as yield.
Proximate and nutrients quality determination: Moisture content, crude fibre, fat and ash content were determined using the method described by Njoku et al. 13 while carbohydrate and crude protein content were determined 14 . Proximate analysis was determined on the infected harvested seeds and the control experiment with no Cercospora leaf spot disease to ascertain the cowpea nutritional quality.
Statistical analysis: Data collected were subjected To mixed model Analysis of Variance (ANOVA). Means of significant treatments were separated using Fisher Protected Least Significant Difference (LSD) at a 5% probability level.

RESULTS AND DISCUSSION
Effect of Cercospora Leaf Spot Disease (CLSD) at the reproductive growth stage (10 WAP) was evidentin the stomatal conductance and leaf chlorophyll content in Table 2. A significant difference in cowpea accessions×disease severity was observed in the stomatal conductance, leaf chlorophyll content and the 100 g seed weight. The interaction effect between the cowpea accession and disease severity was not statistically significant. The mean total stomatal conductance (80.7 mmol mG 2 secG 1 ) was observed in the CLSD infected cowpea accessions while the total mean for the control was 274.38 mmol mG 2 secG 1 . Thus, the highest stomatal conductance for the CLSD infected cowpea accessions was on TVU-9174 (115.9 mmol mG 2 secG 1 ) accession while TVU-13664 (32.8 mmol mG 2 secG 1 ) had the least significant stomata conductance. The leaf chlorophyll content of TVU-9174 (48.33) under optimum disease severity was significantly higher than those established under non-disease infection at the reproductive growth stage ( Table 2). The cowpea accession: TVU-13664 had the least leaf chlorophyll content (37.00) when compared statistically under this disease condition as the highest leaf chlorophyll content (53.73) was also observed in TVu-9174 in the control experiment, which was significantly different from that of TVU-13664 (37.01) in the non-infected condition. A similar significant pattern of stomatal conductance and chlorophyll decline was observed across the cowpea accessions with disease incidence and severity.   The result of the proximate composition of the ten cowpea accessions harvested after CLSD infection is shown in Table 4.  Tannin: The tannin content recorded was significantly low for all the ten cowpea accessions. The TVU-9195 (0.06%) had the highest tannin content closely followed by TVU-9175 and TVU-9106 with 0.05%, while the least tannin content was detected in TVU-9182 (0.00) it was however not detected in TVU-9179, TVU-9185 and TVU-13664. The presence of tannin in the cowpea act as a suppressor against CLS infection.

Oxalate:
The oxalate content of the ten cowpea accession infected by CLSD ranged between 0.11-0.06%. where TVU-13664 (0.11%) had the highest percentage and the least percent was recorded in Tvu-9185 (0.06%). Oxalate occurs naturally in cowpea legume plants and assists in defence response but they have little or no useful effect on human health through high level causes flatulence and constipation in man.

Saponin:
The low saponin content of the ten cowpea accessions affected by CLSD varied from 0.02-0.00%. The TVU-9172 and TVU-9185 cowpea accessions had the highest level of saponin (0.02%) while it was rarely found in TVU-13664 (ND). The low presence of saponin accounted for the susceptibility of the cowpea to Cercospora leaf spot infection. This is because Saponin possesses anti-microbial activities that control fungal infections 14,15 .
The result in Table 3 and 4 (the infected cowpea and the control) when compared. Table 4 Table 3). The result was in-line with what was observed based on the susceptibility of the ten different accessions of cowpea to the Cercospora leaf spot disease.

DISCUSSION
The lack of empirical data for the response of cowpea accessions to infection by Cercospora Leaf Spot Disease (CLSD) and its effects on nutritional qualityis one of the factors limiting the progress of breeding of cowpea for nutritional improvement in Nigeria and the Sub-Sahara African States. From this study, the results showed that TVU-9185 and TVU-9172 had significantly the highest leaf photosynthetic rate, stomatal conductance which was evident in the plant physiological response to CLSD on the harvested seeds after CLSD infection. This gave a significant difference (p<0.05) in the cowpea accessionsused while the least value was observed in TVU-9175. The stomatal conductance generally decreased with the disease severity and incidence, the tendency of reduction of stomatal conductance under the disease incidence and severity is consistent with observations made on photoinhibition on plant 16,17 . A reduction in the photosynthetic rate was more pronounced in the CLSD infected cowpea plant than in the control. This result agrees with the observations 17,18 that the reduction in the leaf photosynthetic absorption was a result of the infection of CLSD through its circumvention of free oxygen needed by the cowpea plant to carry out photosynthesis this was evidenced by the clear downward record of the stomata conductance that created a metabolic bottleneck on the plant ability to use chlorophyll and sunlight 6,19 . Thus, significant cowpea accessional difference in the net photosynthesis was observed in the infected cowpea accessions. Thus, TVU-9174 and TVU-9182 showed higher photosynthetic rates compared to TVU-13664. This implied that TVU-9174 and TVU-9182 accessions are fairly more tolerant to the CLSD infection than Tvu-13664 and can photosynthesize under certain levels of disease response as similar observations were emphasized [20][21][22][23][24][25] . The findings from this study showed that the exposure to CLSD affected the photosynthetic rate and in turn affected the nutrient uptake heavily resulting in a decline in growth rate and seed formation at the reproductive stage of the affected cowpea accessions evidenced in the nutritional content [20][21][22][23][24][25] . The change in the nutritional quality was a result of the infection caused by CLS disease by the inhibition of the photosynthetic activity of the cowpea plant as the plants managed to attain reproductive growth for seed production, thus, affecting the nutrient quality and yields.
From this study, the result of disease incidence and severity showed that the Cercospora leaf spot disease affected the proximate composition differently which could be a result of the differences in their level of inherent genetic resistance and tolerance which corroborated the findings 26 which also had the same results on disease incidence and severity of Cercospora leaf spot on the groundnutplant. The proximate analysis result of the CLSD infected cowpea accessions and the control showed that CLS has a significant effect on the nutritional qualities and anti-nutrition composition of the ten cowpea accessions tested on proximate analysis such as protein, ash, moisture, crude fibre fat carbohydrate, phytate tannin, oxalate and saponin as compared in Table 3 and 4.

CONCLUSION
From this study, Cercospora Leaf Spot (CLS) inhibited the physiological functions of the reproductive growth of the cowpea plants as evidenced in the low stomatal conductance, decreased photosynthesis and changes in the nutritional qualities of the ten cowpea accessions infected by Cercospora Leaf Spot Disease (CLSD).
This study also revealed that TVU-9174, TVU-9182 and TVU-9185 cowpea accessions had a better adaptive response to physiological changes and the reduction in their nutritional qualities was induced by CLSD. This is an advantage to farmers and breeders as these accessions could be recommended as breeding materials for their physiological tolerance to CLS infection and nutritional qualities. Hence, more research is still recommended to evaluate the response of cowpea plants to CLSD dynamics and its influence on the nutritional quality of cowpea accessions.

SIGNIFICANCE STATEMENT
This study discovers the influence of Cercospora leaf spot disease on the nutritional quality of cowpea (Vigna unguiculata) that can be beneficial for both cowpea breeders, farmers and consumers. Hence, this study will help the researcher to compare the nutritional qualities of susceptible cowpea infected by CLSD and recommend them as breeding materials for farmers to ensure food security and improved cowpea germplasm which is critical to cowpea production that many breeders have ignored and have not been explored. Thus, the best cowpea biotechnology practice is to improve the nutritional quality and palatability of cowpea accessions affected by CLSD to boost their nutritional content, subsequent yield increase and resistant accessions to the disease may be arrived at.