GROWTH AND YIELD VARIABILITY OF GROUNDNUT (Arachis hypogaea L.) CULTIVARS INFECTED WITH COWPEA APHID-BORNE MOSAIC VIRUS DISEASE

Groundnut (Arachis hypogaea) is a major legume crop with diverse uses in Nigeria. Its productivity is however threatened by Cowpea aphid-borne mosaic virus (CABMV) in most groundnut producing areas, resulting in huge losses. This study was conducted to determine the resistance of some commercial groundnut cultivars under CABMV disease. The experiment was conducted in Minna, Southern Guinea Savanna agro-ecological zone of Nigeria. Twenty groundnut cultivars were evaluated separately as healthy and CABMV infected trials. The experiment was laid out in randomised complete block design with three replications. Seedlings were infected with the virus by mechanical inoculation at 10 days after sowing. The plants were observed for disease incidence, growth and yield attributes. The data collected were subjected to analysis of variance, principal component and cluster analyses at p≤0.05. One hundred percent infection was found regardless of the cultivar. The healthy plants exhibited significantly (p<0.05) higher morphological and yield parameters than infected plants. Principal component analysis revealed that 100-seed weight accounted for the greatest variability in healthy (eigen vector = 0.6239) and CABMV infected (eigen vector = 0.6005) plants. Cluster analysis showed that 18 (90 %) cultivars formed cluster 1, whereas one cultivar each was found in cluster 2 and 3. The top three cultivars for 100-seed weight: “SAMNUT 23” (56.0 g), “SAMNUT 25” (50.5 g) and “SAMNUT 26” (50.9 g) were the most tolerant to CABMV, whereas “ICG92267” was identified as the best cultivar for dry matter production (24.0 g/plant). Planting of these tolerant cultivars is recommended in order to mitigate the stresses imposed by CABMV.


INTRODUCTION
Groundnut (Arachis hypogaea L.) is an important grain legume in tropical cropping systems of Africa which is typically grown in subsistence nature by resource poor farmers (Haussmann et al., 2012).Cultivated groundnut originated from South America (Scott and Vikas, 2014).It is an annual oilseed and food legume crop grown in numerous environments all over the world between 40 o N and 40 o S. Groundnut is ranked fifth position among oilseed crops worldwide after oil palm, soyabean, rapeseed and sunflower (FAO, 2014).It serves as an excellent source of plant-based protein and contains high contents of various minerals, vitamins and plant compounds (Muhammad et al., 2013).Its haulms are essential animal feed containing 8-15 % protein, 1-3 % lipids, 9-17 % minerals, and 38-45 % carbohydrates (Janila et al., 2016).Groundnut is used as therapeutic food in Africa and many developing nations of the world to combat malnutrition in children (UNICEF, 2007).It has been found to produce more oil per hectare than any other food crop hence possibilities are being considered to explore it for bio-diesel production (Prasad et al., 2011).Groundnut is commonly grown in intercrops due to its nitrogen fixing ability thereby enriching the soil for other crops (Konlan et al., 2013).Studies have shown that groundnut could fix between 40 and 80 kg N per hectare in one year (Janila et al., 2013).
In sub Saharan Africa, 32 viruses induce yield losses in groundnut and the major ones are Cowpea aphid-borne mosaic virus (CABMV), Cucumber mosaic virus (CMV), Indian peanut clump virus (IPCV), Peanut clump virus (PCV), Peanut mottle virus (PeMoV), Peanut streak virus (PStV) and Cowpea mild mottle virus (CPMMV-severe strain) (Sastry and Zitter, 2014).All these viruses are transmitted through groundnut seed and are regarded as important quarantine pests.Cowpea aphid-borne mosaic virus has been known in Nigeria since 1976 as a devastating virus disease of legume in all the agro-ecological zones (Shoyinka et al., 1997).The virus has flexuous filamentous particles raging from 727 to 765 nm in length and 11 nm wide (Damiri et al., 2013).It is a potyvirus whose particles are not affected by Mg ions (Luis et al., 2013).Most often, the fecundity of CABMV soars under field conditions through increased aphid populations, availability of susceptible host plants and the presence of initial infection foci (Luis et al., 2013).Infection due to CABMV has resulted in yield losses that ranged from 13 to 87 % and sometimes total crop loss could be encountered depending on the level of susceptibility of the cultivars (Alegbejo, 2015).Besides sole infection, CABMV can co-exist with other viruses like Cowpea mottle virus (CPMoV) or CMV (Arogundade et al., 2010), causing a mixed or combined infections with more damaging effect on farmers' field.Although a lot of studies have been conducted to determine the pathogenicity of CABMV disease on cowpea there is scanty information on the effect of the virus on groundnut in Nigeria.Groundnut cultivars with in-built characteristics of CABMV disease resistance have the potential to produce higher yields on farmers' fields.Knowledge of the resistance status of commercial groundnut cultivars would contribute to Nigeria's food security.Therefore, the objective of this study was to evaluate 20 commercial cultivars of groundnut for CABMV resistance in Southern Guinea Savanna agro-ecological zone.

Study Location
The study was conducted at the Teaching and Research Farm, Federal University of Technology, Minna during the 2016 cropping season.The coordinates of the site were 9 O 51ʹN, 6 O 44ʹE and 212 m above sea level.The site of the experiment is located in the Southern Guinea Savannah ecological zone of Nigeria.The mean annual rainfall is 1200 mm and is distributed between April and early October of each year.Temperature ranges between 35.0 o C and 37.5 o C while relative humidity varies from 40 to 80 %.According to Adeboye et al. (2011), soils in Minna are generally classified as Alfisols.

Treatments, Experimental Design and Field Layout
The twenty groundnut cultivars served as the treatments.Each cultivar was evaluated separately as infected and control (healthy).The experiment was laid out in randomised complete block design with three replications.The overall plot size was 40 m × 12 m (480 m 2 ).The plots containing the infected plants had a total size of 15 m × 2 m (30 m 2 ) containing 20 rows of 2 m long each with an alley of 3 m between the replicates.Uninfected (control) plots of each cultivar also had a total size of 15 m × 2 m (30 m 2 ) containing 20 rows of 2 m long each with an alley of 3 m between the replicates.The infected and control plots were established side by side with an isolation distance of 10 m in order to prevent virus contaminations.

Source and Multiplication of Virus Inoculum
The CABMV isolate used was obtained from the stock in the Department of Crop Production, Federal University of Technology, Minna.The inoculum was multiplied in the screen house to obtain enough quantity required for the field work.This was accomplished by planting susceptible cowpea cultivar (Ife Brown) into 20 pots at 5 plants per pot.The plants were infected with CABMV extract at 10 days after sowing.Inoculation of the cowpea seedlings was carried out after grinding the inoculum (1 g of CABMV infected leaf) in inoculation buffer (0.1 M sodium phosphate dibasic, 0.1 M potassium phosphate monobasic, 0.01 M ethylene diamine tetra acetic acid and 0.001 M L-cystine per litre of distilled water, adjusted to pH 7.2) using a pre-cooled sterilized mortar and pestle (Adamu et al., 2015).Two microlitres of βmercapto-ethanol was added to the extract prior to application.The upper leaf surface of the target plants were dusted with 600-mesh carborundum (Fisher Scientific, Fair Lawn, NJ) powder before the inoculum extract was applied.The inoculated leaves were then rinsed with cold distilled water and observed for disease symptoms.The infected leaves were harvested at three weeks after inoculation (WAI).Leaf tissues infected with CABMV were preserved in vials (1 g of the leaf/vial) at room temperature.Each vial contained 5 g silica gel as moisture absorber covered with 1 g of non-absorbent cotton wool.

Field Establishment, Inoculation and Crop Management
The experimental site was ploughed with tractor in the first week of May, 2016.Manual ridging was then carried out at one week after ploughing.Groundnut seeds were sown in the second week of

Growth and Yield Variability of Groundnut (Arachis hypogaea L.) Cultivars
May, 2016 on the ridges at the rate of two seeds per hole, using 75 cm × 25 cm inter-and intra row spacing.Seedlings were thinned to one plant per stand at one week after emergence.Manual weeding was carried out at 3, 5 and 8 weeks after planting.The earlier preserved virus isolates (CABMV infected leaves) were used to inoculate the treatment plots on the field.Inoculation was carried out following the same procedure as described above.Lambda cyhalothrin 30 % EC insecticide was sprayed (Badii et al., 2013) at the rate of 2.5 litres/ha in three applications to control aphids and other insects.Insecticide was applied weekly starting from 2 weeks after sowing (WAS) until the fourth week.

Data Collection and Statistical Analysis
Disease incidence was recorded at 1 and 2 weeks after inoculation (WAI).It was taken as percentage of the total plants that exhibited symptoms of CABMV disease.Disease symptoms were scored using a scale of 1-5 (Adamu et al., 2015) as follows: 1 = no symptoms (apparently healthy plant) 2 = mild mosaic (10-30 % infection) 3 = moderate mosaic (31-50 % infection) 4 = severe mosaic, chlorosis and stunting (51-70 % infection) 5 = very severe mosaic, chlorosis, stunting and plant dead (>70 % infection) The growth and yield parameters observed were plant height (cm), leaf diameter (cm), number of branches per plant, number of days to 50% flowering, number of pod per plant, pod weight per plant (g), fresh haulm weight per plant (g), dry haulm weight per plant (g), 100-seed weight (g) and pod yield (kg ha -1 ).The data were subjected to analysis of variance, principal component and cluster analyses, using Statistical Analysis System (SAS, 2008).The cultivars were classified into three clusters based on the reductions in their growth and yield parameters using Unweighted Pair Group Method with Arithmetic (UPGMA) mean (Adama et al., 2015).
X 1 where X 1 is mean of healthy plants and X 2 is mean of infected plants.

Principal Component Analysis Based on Growth and Yield Traits
The first five principal components of healthy plants accounted for 83.8 % of the total variation (Table 5).The PC 1 was strongly correlated with dry haulm weight per plant (eigen vector = 0.4388), plant height (eigen vector = 0.4386) and fresh haulm weight per plant (eigen vector = 0.4089).The PC 2 was loaded with pod yield (eigen vector = 0.5899) and pod weight per plant (eigen vector = 0.5894).The third principal component was mainly loaded with number of days to 50 % flowering (eigen vector = 0.6094) while variability in PC 4 was mainly due to the number of branches per plant (eigen vector = 0.5861) and leaf diameter (eigen vector = 0.5599.The fifth principal component was positively correlated with 100-seed weight (eigen vector = 0.6329).Leaf diameter, number of pods per plant, pod weight per plant and pod yield contributed positively to the variability among "ICG-01276", "ICG-92267", "ICG-IS-13986", "SAMNUT 25" and "SAMNUT 26" (Figure 1 A).

DISCUSSION
All cultivars expressed significant variability in morphological and yield traits between the healthy and CABMV-infected plants.This observation showed that CABMV reduced the potential performance of the entire groundnut cultivars evaluated.This supported the findings of Kaitisha (2001) who identified CABMV as a major biotic constraint to groundnut productivity in sub Saharan Africa.Over the years, researchers have intensified efforts aiming at providing adequate protection for plants and obtaining maximum yield.The fact that 100 % disease incidence was found on the inoculated plants indicated that none of the groundnut cultivars exhibited immunity to the virus.Cultivation of tolerant varieties is an alternative and effective control strategy in the absence of immune varieties.The cultivars "ICG-5195" and "ICG-6654" which exhibited less than 10 % reduction in 100-seed weight probably contained CABMV tolerant genes.
Seed weight is an important trait because of its direct relationship with seed size (Biçer, 2009), overall yield and economic return.For instance, Upadhyaya et al. (2006) reported seed size as an important trait for trade and component of yield and adaptation in chickpea (Cicer arietinum).In developing countries, including Nigeria most farmers rely on the seeds from previous harvest for field establishment at the next cropping season.Farmers generally prefer varieties with large seeds because such seeds would contain more food for early germination and seedling emergence, vigorous plants and are more likely to produce higher yields.However, since none of the cultivars attained its potential seed weight under infected condition, the top three cultivars "SAMNUT 23", "SAMNUT 25" and "SAMNUT 26" which gave high 100-seed weight among the infected plants could be described as the most tolerant to CABMV disease.Dry matter was least affected in "ICG-01276" (less than 20 % reduction) among the infected plants, which revealed some level of tolerance to infection.However, in absolute term, "ICG-92267" which exhibited the highest dry matter among the infected plants could be a choice cultivar for the production of hay in CABMV prone areas.The result of principal components which showed that 100-seed weight was responsible for the greatest variability observed in the growth and yield parameters of both healthy and infected plants.This revealed the importance of seed weight in groundnut breeding.
Cultivars' potential for this parameter was not realized in the CABMV infected plants, thereby revealing the deleterious impact of the virus in susceptible cultivars.The result of cluster analysis was an indication of the variability among the evaluated groundnut cultivars.Cluster 1 consisted of cultivars which could be explored for various purposes.For instance, the cultivars in cluster 1 could be utilized for fresh forage production; "ICG-IS-13003" which is the only member of cluster 2 would be suitable for hay (dry haulm), whereas the cultivar FDRF7-67 that formed cluster 3 would be best for seed production.The inconsistent responses of the cultivars under CABMV disease was partly due to the fact that growth and yield characters were under the influence of different genes.Therefore, it was possibly because the genes conferring disease resistance did not interact synergistically with those controlling growth and yield attributes.This supported the view of Vanhaeren et al. (2016) who reported that growth processes are governed by complex genetic networks.Similarly, studies have shown that in diseased plant, plant-virus interactions interfere with a broad range of cellular Growth and Yield Variability of Groundnut (Arachis hypogaea L.) Cultivars processes, such as hormonal regulation, cell cycle control and endogenous transport of macromolecules, among others (Pallas and Garcia, 2011).Therefore, the physiology of infected plants was destabilized, resulting in lack of co-ordination among the various organs.

CONCLUSION AND RECOMMENDATIONS
This study has revealed the vulnerability of some commercial groundnut cultivars and variability in their response to CABMV disease.The infected groundnut cultivars expressed significant reduction for most of the growth and yield traits due to their susceptibility to the pathogen.Although none of the groundnut cultivars exhibited immunity to CABMV disease, some cultivars showed certain levels of tolerance and consequently gave appreciable yield."SAMNUT 23", "SAMNUT 25" and "SAMNUT 26" were the top three cultivars for 100-seed weight while "ICG-92267" was identified as the best for dry matter production.Planting of these tolerant cultivars is recommended in order to mitigate the stresses imposed by CABMV disease.

Figure 1 :
Figure 1: Biplot of the first two principal components from (A) Healthy and (B) Cowpea aphid-borne mosaic virus infected groundnut plants

Figure 2 :
Figure 2: Dendogram of the reduction in growth and yield attributes of groundnut cultivars infected with Cowpea aphid-borne mosaic virus, according to Unweighted Pair Group Method with Arithmetic (UPGMA) mean

Table 1 :
Mean plant height (cm), leaf diameter (cm) and number of branches per plant in Cowpea aphid-borne mosaic virus infected compared with healthy groundnut plants

Table 3 :
Mean fresh and dry haulm weight per plant (g) of Cowpea aphid-borne mosaic virus infected compared with healthy groundnut plants

Table 2 :
Mean number of days to 50 % flowering, pods per plant (no.), and pod weight per plant (g) in Cowpea aphid-borne mosaic virus infected compared with healthy groundnut plants

Table 4 :
Mean 100-seed weight per plant and pod yield (kg ha -1 ) of Cowpea aphid-borne mosaic virus infected compared with healthy groundnut plants

Table 5 :
Eigen vectors from the principal components (PCs) of growth and yield attributes in healthy and Cowpea aphid-borne mosaic virus infected groundnut plants