Evaluation of Cowpea Genotypes for Virus Resistance Under Natural Conditions in Uganda

Cowpea (Vigna unguiculata L. Walp) is an important grain legume in most parts of Sub Saharan Africa. However, viral diseases are a major limiting production factor causing significant yield losses. An experiment was conducted to evaluate the reaction of 105 different cowpea genotypes to viral infection in different agro-ecological zones of Uganda. The aim was to identify genotypes that could serve as sources of resistance to virus infection. Virus infection in these experiments occurred naturally through insect vectors. Results showed that there were significant differences in disease reaction among genotypes within and among agro-ecological zones in terms of Area Under Disease Progress Curve (AUDPC) and incidence. Interactions of genotype by season (GXS), genotype by location (GXL) and genotype by location by season (GXLXS) also significantly affected reaction to viral infection among genotypes. Introduced cowpea genotypes exhibited a more susceptible viral disease reaction compared to the landraces over the two seasons in the three locations. A number of landraces such as WC32, WC18, NE43, NE15, WC35B consistently showed resistance to virus infection in the three locations and therefore could be good sources of resistance. Low disease pressure (AUDPC) was also recorded on SECOW2W (released variety) as reported by previous studies. The landraces also gave consistently higher grain yield values compared to the introduced genotypes. Overall, data from this study showed that locally adapted cowpea genotypes offer resistance to virus infection and may be desirable germplasm for Ugandan cowpea breeding programs.


Introduction
Cowpea (Vigna unguiculata L. Walp) is a grain legume crop with high protein, minerals and vitamins and is utilized as a fresh vegetable (pods and leaves), and as fodder.Cowpea is an early maturing crop and therefore helps reduce the "hunger period" that often occurs prior to harvest in farming communities (Singh et al., 2002;Timko et al., 2007a).It is an important source of income especially to the rural households after selling the grain and the fresh leaves (Isubikalu et al., 1999;Singh et al., 2002;Timko et al., 2007a).Cowpea curbs erosion through its rapid ground coverage and fixes atmospheric nitrogen thus improving soil fertility (Singh et al., 2002;Tarawali et al., 2002;Casky et al., 2002).In Uganda, cowpea is an important food security crop especially in the eastern and northern regions where up to 90% of the crop is grown (Adipala et al., 1999).
Wherever cowpea is grown, viral diseases are a major constraint to production and yield (Bashir & Hampton, 1996).More than 20 viruses affect cowpea production worldwide (Thottappilly & Rossel, 1985).Yield losses of almost 90% or even total crop failure have been reported (Kaiser & Mossahebi, 1975;Raheja & Leleji, 1974).In Uganda, the main viruses infecting cowpea are; Cowpea aphid borne mosaic (CABMV), Cucumber mosaic virus (CMV), Cowpea mild mottle virus (CPMMV) and Cowpea severe mosaic virus (CPSMV) (Amayo et al., 2012;Orawu et al., 2005;Edema et al., 1997).The seed-borne nature of these viruses renders them very destructive to emerging seedlings and insect vectors can spread these further (Ndiaye et al., 1993;Bashir et al., 2002).These viruses are transmitted by several insect species in a non persistent manner and therefore use of insecticides is not an effective method of control (Umaharan et al., 1997).Genetic resistance, therefore, is the best alternative in reducing crop losses due to these diseases.
To identify host resistance, it is important to evaluate different genotypes under field conditions in different environments (Goenaga et al., 2011;Maphosa et al., 2013;Oloka et al., 2008).This study was therefore undertaken to study the reaction of 105 cowpea genotypes to natural virus infection to identify sources of resistance for breeding.

Experimental Sites and Their Characteristics
The study was conducted in 2012 during two seasons, herein referred as 2012A and 2012B, at three locations in Uganda that are known to grow cowpeas extensively (Table 1).These locations have differing edaphic characteristics, land use types and cropping systems, and climatic conditions that influence crop growth, vector populations and virus disease development.and Eledu (1999).

Genotypes and Experimental Design
A total of 105 cowpea genotypes that included landraces (82), commercially released varieties (1) and introductions (22).The introductions were provided by the International Institute of Tropical Agriculture (IITA) (Table 2).The landraces were collected from the northern and eastern regions (NE), western and central regions (WC) of Uganda.
At all sites, the fields were ploughed twice before harrowing to prepare a fine seedbed before planting.Each genotype was planted in two rows measuring 4 m long with spacing of 60 cm between rows and 30 cm between plants within rows.The experimental design was α-design (incomplete block design) as described by Patterson and Williams (1976a)

Data Collection Method
For each trial, data were collected at 14-days interval starting three weeks after planting (WAP) until the appearance of the first ripe pods.Viral disease incidence was recorded on all plants per plot while severity was recorded on ten randomly selected plants in a plot.Disease severity was based on visual estimation of the diseased plants as manifested by the different symptoms on a modified scale of 1-5 where 1 = no symptoms on all leaves, 2 = slight symptoms (1 to 25% of the leaves infected), 3 = moderate symptoms (26 to 50 % leaves infected), 4 = prominent symptoms with stunting (51 to 75% of leaves infected), 5 = highly severe symptoms with stunting (> 75% of leaves infected) (Gumedzoe et al., 1997).

Statistical Analysis
Disease severity data were used to compute area under disease progress curve (AUDPC) as described by Campbell and Madden (1990).
where; n = number of successive readings, Y i = disease severity at time i, t i = number of days after the first observation on assessment date i.Data were also recorded on number of days to 50% flowering (midbloom), number of days to maturity and yield.Data was subjected to analysis of variance (ANOVA) using the linear mixed models (REML) in Genstat discovery Edition 4 (http://discovery.genstat.co.uk) and R version 3.1.0(R core team, 2014).Mean AUDPC and incidence per genotype were computed per location as well as per season and genotype.

Results
Area under disease progress and disease incidence were high in Budaka and lowest in Tororo over the two seasons (Table 3).In the first season (2012A), lowerAUDPC and disease incidence were recorded than in the second season (2012B).Overall, mean AUDPC was lower in 2012A than in 2012B, with Budaka 12A recording the highest values followed by Tororo12A and then Serere12A.In 2012B, Budaka12B had the highest disease severity followed by Serere 12B and lastly Tororo12B.Based on genotype source, cowpea genotypes from IITA had higher AUDPC and disease incidence values than genotypes from Uganda (Table 4).Among the Ugandan genotypes, accessions from the western and central regions of the country had lower disease levels (Table 4).Analysis of variance showed significant differences among genotypes, seasons and locations for AUDPC.The site x season, site x genotype, season x genotype and site x season x genotype interactions were also highly significant (Table 5).Among locations, analysis of variance indicated that there were significant genotype, season and genotype x season interaction for AUDPC and disease incidence (Table 6).However, seasonal differences had the strongest effects on both AUDPC and disease incidence.The effects of genotype and genotype x season on AUDPC were greater in Budaka than for Serere and Tororo.*** = significant at 0.001 level, ** = significant at 0.05 level.
Mean AUDPC for the 105 genotypes screened for reaction under natural infestation are shown in Table 7.Average AUDPC across the three locations over the two seasons, ranged from 39.4 (on genotype WC48) to 94.9 (genotype NE46).It is evident from the table that none of the genotypes evaluated were immune.The introduced germplasm from IITA exhibited more susceptible reaction than the local accessions (Table 7 and Figure 1).Among the local genotypes, the accessions WC48, MU19 and NE43 from the western and central region, Makerere University and Northern and Eastern Uganda respectively had the lower AUDPC.A range of typical virus symptoms were also observed on most genotypes during evaluation such as; leaf mosaic, necrosis, chlorosis, vein clearing, vein banding, purpling, leaf curling, leaf deformation, and blotching.Number of days to 50% flowering over the two seasons ranged from 45 days to 73 days in Budaka, 39 days to 63 days in Serere and 42 days to 67 days in Tororo.The average number of days to 50% flowering in 2012A was 57 days, 44 days and 53 days for Budaka12A, Serere12A and Tororo12A respectively.In 2012B, the average number of days to 50% flowering was 52 days, 54days and 52 days in Budaka12B, Serere12B and Tororo12B respectively (Table 8).Number of days to maturity across the two seasons ranged from 69 days to 101 days in Budaka, 71 days to 88 days in Serere and 67 days to 99 days in Tororo.Average number of days to maturity in 2012A was 87 days in Budaka, 81 days in Serere and 85 days in Tororo while in 2012B, the average number of days to maturity was 74 days, 81 days and 89 days respectively in Budaka, Serere and Tororo respectively (Table 8).

Discussion
There was variation in disease pressure across sites and seasons with higher AUDPC recorded in 2012B than in 2012A.These differences can be attributed to the influence of environmental conditions such as rainfall, temperature and relative humidity (Kaisser & Mossahebi, 1975;Edema et al., 1997).Edema et al. (1997) however reported higher disease incidence in the first (wet season) than in the dry season.In our study, this could be attributed to rapid symptom development that normally occurs in dry conditions (Schuerger & Hammer, 1995).Also under dry conditions, cowpea plants do not show considerable plasticity and recovery growth and hence are severely affected by viral infections (Booker et al., 2005).
During the trials at the three sites, the genotypes expressed a range of symptoms some appearing on the primary leaves shortly after germination.The symptoms ranged from leaf mosaic, necrosis, chlorosis, vein clearing, vein banding, purpling, leaf curling, leaf deformation, and blotching.It has been reported that symptom expression depends on the virus type or strain, genotype, species and plant age, time of the year and environmental conditions and some viruses cause similar or related symptoms (Shoyinka et al., 1997).Some symptoms such as mosaic, necrosis, chlorosis, vein banding, stunted growth, leaf deformation, and mottling are associated with viruses such as Cowpea aphid borne mosaic virus (CABMV), Blackeye cowpea mosaic virus (BICMV), Cowpea yellow mosaic virus (CYMV) (Aliyu et al., 2012;Orawu, 2007).
Results from this study also show that with the exception of genotype NE46, most of the landraces and the released variety (SECOW2W) were either resistant or moderately resistant compared to the introduced genotypes.However, Orawu et al. (2012) found Ebelat (landrace) to be susceptible especially to Cowpea aphid borne mosaic virus (CABMV).In this study, the cowpea genotypes were screened for resistance to field viruses and therefore, genotypes such as WC32, WC18, NE43, NE15, WC35B and SECOW2W with low disease severity may offer multiple virus resistance.Breeding for disease resistance in cowpea is a complex problem because of the occurrence of multiple virus infections in a single field/plant (Amayo et al., 2012;Orawu et al., 2012;Shoyinka et al., 1997).However, most of these viruses are transmitted by the same vectors which offers an opportunity to utilize horizontal resistance to vector transmission in breeding programmes (Shoyinka et al., 1997).The development of resistant varieties would be the most effective and environmentally friendly means of controlling these viral diseases.In the course of developing varieties that are resistant to a particular disease, the breeder has to select a resistant individual as a parent.The gene(s) conferring resistance can then be transferred to a cultivated variety in order to obtain improved lines (Ogundiwin et al., 2002).There is no ecological restriction to viruses affecting cowpea and therefore screening of genotypes under natural field conditions in different agro-ecological conditions is critical for identifying resistant genotypes (Shoyinka et al., 1997).The introduced genotypes were more susceptible compared to the local genotypes.Differential response of genotypes is common in disease resistance screening and can be attributed to differences in environmental conditions, pathogen variability and virulence (Gremillion et al., 2011).

Conclusion
Overall, results from this study identified a number of local landraces in addition to the released variety (SECOW2W) as useful sources of resistance to cowpea viruses that can be used in the local breeding program.These include; WC32, WC18, NE43, NE15, WC35B.Follow up studies both in the field and green house involving high virus innoculum levels (by artificial inoculation) need to be undertaken to confirm the levels of resistance in these genotypes.This study also showed consistency among genotypes in reaction to virus infection in the three locations over the two seasons.These study sites are therefore ideal for future screening of genotypes for resistance or susceptibility to virus infection.

Figure 1 .
Figure 1.Relationship of mean yield (kg/ha) by genotypes across 2012A and 2012B

Figure 2 .
Figure 2. Variation of AUDPC by genotypes across study sites

Figure 3 .
Figure 3. Relationship of mean yield (kg/ha) by genotypes across study sites

Table 1 .
Altitude and climatic data at three experimental sites in Uganda * Agroecological zone, 1 Northern Moist Farmlands, 2 Southern and Eastern Lake Kyoga Basin, 3 Lake Victoria Crescent and Mbale Farmlands.Source: 4 Meteorological stations at the experimental sites in 2012 and * AEZ delineation according to Wortmann

Table 2 .
Description of cowpea genotypes used in the study NE = Northern and Eastern, Uganda; WC = Western and Central, Uganda; MAK = Makerere University, Uganda; IITA = International Institute of Tropical Agriculture, Nigeria.

Table 3 .
Mean AUDPC and mean disease incidence across locations

Table 4 .
Mean AUDPC and mean disease incidence according to source of planting material NE = Northern and Eastern; WC = Western and Central; MAK = Makerere University collection; IITA = International Institute of Tropical Agriculture, Nigeria.

Table 5 .
Analysis of variance for area under disease progress curve among cowpea genotypes evaluated for two seasons *** = significant at 0.001 and 0.003 respectively.

Table 6 .
Variation of AUDPC and incidence across experimental locations

Table 7 .
Overall Cowpea genotype reaction based on AUDPC

Table 8 .
Average number of days to 50%flowering, maturity and average yield across study sites in 2012A and 2012B