Exploring New Sources of Viral Resistance in Cowpea Germplasm and Its Elaboration through Multivariate Analysis

Sixty-eight genotypes were acquired from PGRI, NARC, Islamabad, Pakistan. Collections representing Pakistan were 62, while 4 were obtained from International Institute of Tropical Agriculture (IITA) and one from China (Iqbal et al., 2003). These genotypes were grown under field conditions at PGRI, NARC, Islamabad (33°44’ N and 73°08’ E, 540 m) in an augmented design with three replications. One local check was repeated after every 10 rows. One row of 4 meters length was planted for each genotype with row to row and plant to plant spacing as 75 cm and 20 cm, respectively. Recommended cultural practices were followed throughout the crop season (Iqbal et al., 2017).
 
Qualitative traits
 
Field data on diverse qualitative traits (leaf shape, seed surface, twinning tendency, anthocynin pigment, plant type, fodder type and cream color) were recorded. Data were taken of individual plant and then their mean values were compared and analyzed using summary statistics with the help of MS Excel Windows 2010.
 
Serology and Direct antigen coating-enzyme linked immunosorbent assay (DAC-ELISA)
 
Leaves were collected from ten plants of each genotype from the field to screen against four viral antisera to cowpea seed borne virus viz., CMV, CABMV, BlCMV and BCMV for DAC-ELISA. As majority of the virus antisera are produced in rabbit, therefore, the rabbit immunoglobulin attached to antigens are probed with enzyme labeled anti-rabbit immunoglobulin produced in goat. In place of immuno- globulins, the use of Protein-A conjugate is equally good as that of anti-rabbit conjugate. The fresh and healthy samples were collected at 50% flowering stage for each genotype in polyethylene bags and stored at 4°C. ELISA buffer was used to ground leaves before loading in the ELISA plates. Antigens were prepared in 0.02 M carbonate buffer (pH 9.6) and used for coating ELISA plates followed by the addition of crude antiserum (not purified IgG as in case of DAS-ELISA) with its optimum concentration. 200 mL extract of each sample was loaded in each well of ELISA plate. Two wells each for healthy and positive control were used for each virus. The loaded plates were incubated at 37°C for 2 hours, then washed with washing buffer three times at interval of one minute and dried on paper towel (Bashir et al., 2000).
 
Cross absorption of crude antiserum was made with healthy plant extract in conjugate buffer (antibody buffer) to have a 1:100 dilution, then filtering through cheese cloth. Two hundred µl cross-absorbed solution was added into each well of ELISA plate and incubated the plate at 37°C for 1 hr prior to adding it to micro titer plate. The solution of alkaline phosphatase (ALP) conjugate (Sigma-3687) was prepared in conjugate solution (pH 7.4) at a dilution of 1:1000 for all the viruses to be screened against specific antiserum. Two hundred µl of this solution was added in each well and incubated at 37°C for 2 hr. The substrate solution was prepared by dissolving P-Nitrophenyl Phosphate (PNPP) tablets in substrate buffer (pH 9.8). One tablet (5 mg) was used for 30 ml buffer. Substrate solution (200 µl) was added into each well and ELISA reading was taken using BIORAD Multiscan (Model Atto 550 nm Yamato Scientific Co. Ltd, Japan) 30 minutes after adding substrate buffer. Reading of the healthy wells was considered as positive. The data on disease rating were recorded as 0 (resistant), where no reaction and 1 (susceptible), where reaction was observed. Although, variation in reaction intensity was observed but even weak reaction (even slightly higher than control that was considered at ELISA reading < 0.200 and the values greater than this were considered as susceptible). Genetic linkage for disease reaction was constructed on the basis of Unweighted Pair Group Mean Averages (UPGMA) and scattered diagram using computer software STATISTICA version 6.0 and SPSS version 10.01 for windows.

Cowpea genetic diversity existed in Pakistan comprising 39 genotypes from Punjab, 21 from KPK, 1 each from Baluchistan and Gilgit Baltistan. These areas are known as centers of biodiversity (Iqbal et al., 2017).  Qualitative traits presented in (Table 1) showed wide range of variability present in these traits. Leaf shape (LS) was divided into four descriptors. Forty two genotypes were ovate, 12 were lanceolate narrow and 14 were lanceolate broad whereas no line was observed as rhombic. Plants with ovate leaf shape were found to be best for moisture absorption and synthesis of food proteins while lanceolate broad and narrow leaf shape is also a distinguishing trait in cowpea which could have the ability to resist drought, this trait was prominent in exotic germplasm obtained from IITA, Nigeria and hence the plants with these characters could be utilized for breeding and hybridization of local and exotic cowpea in rainfed conditions. All plants were found to be glabrous i.e. without hairs (100%) which help in harvest and trample. Erect plant type was recorded in 49 lines was dominant trait as compared to prostrate in (13), pronounced (2) and spreading (4). Erect plant type is preferred for fodder use and during humid conditions get more light while prostrate type is preferred for planting under rainfed conditions as it facilitates moisture uptake and its efficient conservation. On the other hand genetic variation was recorded low in twinning tendency and anthocynin pigment. Further evaluation and exploitation of these traits with other economic traits may help to improve this trait (Al-Saady et al., 2018). Thirty two genotypes were identified and selected as fodder type, rest 36 were non-fodder type. Fodder type can be utilized as dual purpose (Iqbal et al., 2021). Cream color in 29 lines was prominent which is commercially valued and needs to be multiplied.
 

 
Resistant sources are reported for one or two viruses but no investigation is available on multiple resistances (Bashir et al., 2002). ELISA (Clark and Adams, 1977) generally detects concentration of virus lower than 0.01 µg/ml as compared to immunodiffusion test (1 µg/ml), micro precipitin test (0.5 µg/ml), electron microscopy (0.1 µg/ml) and infectivity assay (0.05 µg/ml) (Van Dan Heuvel and Peters, 1989; Bar-Joseph and Garnsay, 1981).

As six antisera (CMV, CABMV, BICMV, CSMV, BCMV and SBMV) were used for detection, identification of cowpea seed borne viruses, two antisera (CSMV and SBMV) were not properly functioned as they gave high back ground reaction. Results presented in Table 1 generated by DAC-ELISA revealed that genotypes of diverse origin (27005, 27041, 27075, 27141, 27145, 27146, 27147, 27158, 27160, 27167, 27172, IT85F-1380 and IT86D-719) were found highly resistant to four viral antisera (CMV, CABMV, BICMV and BCMV). Resistant genotypes may serve as resistant source for developing new varieties. Only one genotype 27008 of local origin was found highly susceptible to all the viral antisera. Genotype number F0214 obtained from China was found resistant to CMV although susceptible to other three viruses. Seven genotypes (27001, 27006, 27012, 27025, 27027, 27052 and 27064) were found only resistant to black eye cowpea mosaic virus but susceptible to other three antisera. Twenty seven genotypes were observed resistant to cowpea mosaic virus, whereas susceptible to other three antisera. As a whole twelve different disease reactions of resistant and susceptible to different antisera were recorded through DAC-ELISA (RRRR, SSSS, SSRS, RSSS, SRRS, RSSR, RRSS, SSRR, SRRR, RRSR, RSRR and RRRS).
 
Previously CMV was not detected in cowpea germplasm collected from Pakistan (Bashir and Hampton, 1996). Genotypes viz., 27008, 27001, 27006, 27012, 27025, 27027, 27052, 27064, 27003, 27009, 27011, 27017, 27018, 27022 and 27144, all were observed susceptible to CMV. The CMV symptoms were more severe in plants infected with mixed infection of BICMV than with single infection. This may be due to synergistic effect of two viruses (Pio-Ribeiro et al., 1978). These lines may serve as resistant sources for breeding program provided their resistance is confirmed by artificial virus inoculation. There are several control strategies for viral diseases including the use of virus free seed or vegetative propagules, prevention of infection by breaks in cropping or control of weed hosts, prevention of transmission by vectors and breeding for resistance (Fraser, 1992).

Two types of viral symptoms were observed under field condition. Some plants showed bright yellowing with mosaic pattern and such types of symptoms were more common. Second type of symptoms appeared in the form of molting, vein banding and vein clearing, mosaic and yellowing. Among 16 genotypes which were observed resistant under field conditions due to no visible virus like symptoms, either single or mixed infection of two to three viruses gave reaction in three genotypes (27052, 27099 and F0214) by ELISA and these were considered as susceptible. The genotypes resistant to antisera were CMV (53), CABMV (21), BICMV (28) and BCMV (22), whereas, for susceptible ones were for CMV (15), CABMV (47), BICMV (40) and BCMV (46). Under field conditions, the viral disease increased with the passage of time and was attributed to favorable climatic factors, abundance of inoculum and vector for viruses or due to synergistic effect of two or more viruses. Overall viral disease incidence ranged from 0-66% based on ELISA results. Thirteen genotypes (27005, 27041, 27075, 27141, 27145, 27146, 27147, 27158, 27160, 27167, 27172, IT85F-1380 and IT86D-719) were resistant to all the four viral diseases (Fig 1).
 

 
In response to disease severity, twelve clusters were obtained from unpaired group mean averages (UPGMA). Cluster I, III, VI, VII, IX consisted of one genotype, cluster II (3), cluster IV (7), cluster V (27), cluster VIII (13), cluster X (4), Cluster XI (2) and cluster XII counted 7 genotypes in each case. Genotype 27008 originated from Pakistan was present in cluster VI and was susceptible to all antisera CMV, CABMV, BICMV and BCMV, whereas 13 genotypes were present in cluster VIII which were found resistant to all the four antisera applied. Out of these two were from IITA, Nigeria and 11 others were from Pakistan. The identified genotypes screened against seed borne viruses can be safely used in any breeding program. Clusters were diverse in nature and so were their responses to different antisera. This may prove their worth in future breeding programs or in combination with resistant, susceptible and highly susceptible lines. Cluster diagram indicated that resistant genotypes were placed in separate cluster, whereas susceptible to CMV, CABMV, BICMV and BCMV were grouped in different clusters. Genotypes resistant under field condition were from all the three sources, i.e., China, Nigeria and Pakistan. PCA revealed first two factors with eigen-values greater than unity contributed more than 80 per cent variability among 68 genotypes for four viral diseases, hence a scattered diagram was constructed (Fig 2). Twelve distinct groups were observed and these were in coordination with cluster analysis. The genotypes resistant to all the four viruses were grouped in the upper right box along with other three genotypes which were resistant to three different viruses. On the contrary, the genotypes susceptible to all the viruses were in the lower left box.
 

Thirteen genotypes (27005, 27041, 27075, 27141, 27145, 27146, 27147, 27158, 27160, 27167, 27172, IT85F-1380 and IT86D-719) were found resistant to all above viral diseases therefore, suggested for breeding programs aiming development of resistant cultivars with better yield potential.

Authors are grateful to IITA, Nigeria for cowpea germplasm and Dr. M. Bashir, Ex-PL (CDRP) for providing antisera against CMV, CABMV, SBMV and BICMV.