Response of Nine Cultivars of Pepper ( Capsicum spp . ) to Infection by Four Viruses under Natural Field Conditions in the Coastal Savanna Zone of Ghana 1

A study was conducted to evaluate the reaction of two local and seven exotic pepper cultivars to four viral diseases in the coastal savanna zone of Ghana. The objective was to evaluate the response of the pepper cultivars to the viruses under natural field conditions. The plants were assessed for symptom severity as well as disease incidence. Symptom expression varied among the cultivars, with severity ranging from 0.5% at 4 Weeks after Transplanting (WAT) to 47.2% at 14 WAT. Disease incidence also varied significantly among the cultivars and ranged from 1.1% at 4 WAT to 61.3% at 14 WAT. Enzyme-Linked Immune Sorbent Assay (ELISA) was performed against PVMV, TMV, CMV and PMMV for each cultivar. The test showed the presence of all four viruses in the leaf extracts, with each cultivar infected by at least one of the four viruses. PVMV and CMV were the predominant viruses identified. Mixed infections of two or more viruses were observed in all the cultivars. TMV was detected in all cultivars except the two hybrids, Sunny F1 and Forever F1, while PMMV was detected in only cv. Legon 18 which had a mixed infection of all four viruses.


INTRODUCTION
Pepper belongs to the genus Capsicum and family Solanaceae (Ochoa-Alejo and Ramírez-Malagón, 2001).It is an indispensable ingredient for food preparation throughout the world (Green and Kim, 1991) and is also utilized in the pharmaceutical, cosmetic and poultry industries (Jin et al., 2009).
In Ghana, pepper is widely produced for both local consumption and export to the European market.Currently, Ghana is the fifth largest exporter of peppers to the European Union, where the demand has been growing by about 17% annually since 2000 (Millennium Development Authority, 2010).However, the incidence of viral diseases has become a major constraint to production.Some 35 viruses have been reported to infect peppers worldwide causing considerable economic losses in terms of reduced fruit quality and marketable yield (Jin et al., 2009).In Africa, 11 viruses have been found to infect pepper crops (Gafalla, 2003).The most important of them include Pepper Veinal Mottle Virus (PVMV) and Cucumber Mosaic Virus (CMV).Planting of resistant cultivars offers the best control measure as the use of chemical or cultural means has not been very effective.A number of sources of virus resistance have been reported in wild and cultivated lines of pepper and several virus-resistant lines have already been released.However, some viruses, particularly Cucumber Mosaic Virus (CMV) and the leaf curl virus complex, continue to pose a challenge to breeding programmes (Green and Kim, 1994).
In Ghana, seeds of some exotic pepper varieties under various trade names are being sold to farmers by commercial seed companies for planting.Adaptability of these cultivars to the local environment especially to viruses is not yet known.There is, therefore, the need to assess the response of these cultivars to viruses present locally.This will allow the identification of resistant or tolerant cultivars for recommendation to farmers in the short term and in the long term utilized in breeding programmes to improve well-adapted local cultivars.Furthermore, it will allow for the development and evaluation of better control measures, thereby, reducing production losses and increasing the quality of the crop.This project, therefore, sought to evaluate the response of seven exotic as well as two local cultivars of pepper to four pepper viruses in the Coastal Savanna agroecological zone under natural field conditions.

MATERIALS AND METHODS
Source of seeds: Seeds of the exotic varieties used for the study namely, California Wonder (CW), Big Sun (BS), Archard (AR), Bombadier (BO), Forever F1 (FF), Sunny F1 (SF) and Antillas (AT) were obtained from Technisem, Accra.The local cultivars; Legon 18 (LE) and Anloga (AN) which were used as checks were obtained from Agrimat, Accra and a farmer's field respectively.

Nursery establishment and field planting:
hundred and fifty seeds each of the nine cultivars were sown and raised in plastic trays in a sterilized soil mixture composed of sandy loam and decomposed coconut fibre in a ratio of 2:1.The trays were kept in insect proof cages.At four weeks after emergence, the seedlings were transplanted in the field in a Randomized Complete Block Design (RCBD) with three replications.Thirty seedlings per cultivar were planted per plot at a spacing of 0.75 fertilizer (19-19-19) was applied as a starter solution at transplanting.Eighty grams (80 g) of fertilizer material was dissolved in 4.5 L of water and applied at 15 per seedling.No insecticide was used throughout the study.
Host response in the field: Phenotypic data on host reaction were recorded in terms of symptom expression following a six-point scoring scale modified after Piper et al. (1996).Using the symptom severity scores, percentage severity was estimated as: { { {4I <?F @ HG ?=B >?AF??{ 4I <?F @ HG ?J I H?> BCAB?GH >?AF??
Disease incidence, which measures the extent of propagation of a disease within a given field (Agrios, 2005), was also estimated using the formula: According to González-Pérez et al. (2011).

Virus detection:
The technique of Antibody Sandwich ELISA (DAS performed following the method of Clark and Adams (1977) for the detection of Cucumber Mosaic Virus (CMV), Pepper Veinal Mottle Virus (PVMV), Tobacco Mosaic Virus (TMV) and Pepper Mild Mottle Virus (PMMV).The ELISA kits used for the virus detection were obtained from DSMZ Plant Virus Collection, Braunchweig, Germany.
Leaf extract was prepared by grinding 0.5 young leaves in 5 mL of General ELISA

MATERIALS AND METHODS
tic varieties used for the study namely, California Wonder (CW), Big Sun (BS), Archard (AR), Bombadier (BO), Forever F1 (FF), Sunny F1 (SF) and Antillas (AT) were obtained from Technisem, Accra.The local cultivars; Legon 18 (LE) used as checks were obtained from Agrimat, Accra and a farmer's field Nursery establishment and field planting: One the nine cultivars were sown and raised in plastic trays in a sterilized soil sed of sandy loam and decomposed coconut fibre in a ratio of 2:1.The trays were kept in insect proof cages.At four weeks after emergence, the seedlings were transplanted in the field in a Randomized Complete Block Design (RCBD) with hirty seedlings per cultivar were per plot at a spacing of 0.75×0.65 cm.NPK 19) was applied as a starter solution at transplanting.Eighty grams (80 g) of fertilizer material was dissolved in 4.5 L of water and applied at 15 mL er seedling.No insecticide was used throughout the Phenotypic data on host reaction were recorded in terms of symptom expression point scoring scale modified after Piper .(1996).Using the symptom severity scores, >?AF?? × 100 Disease incidence, which measures the extent of within a given field (Agrios, 2005), was also estimated using the formula: direct Double-ELISA (DAS-ELISA) was performed following the method of Clark and Adams (1977) for the detection of Cucumber Mosaic Virus (CMV), Pepper Veinal Mottle Virus (PVMV), Tobacco Mosaic Virus (TMV) and Pepper Mild Mottle Virus ELISA kits used for the virus detection were obtained from DSMZ Plant Virus Collection, Leaf extract was prepared by grinding 0.5 g of ELISA extraction buffer containing 20 g of Poly Vinyl Pyrrolidone (PVP), 2 g of ovalbumin, 1.3 g of sodium sulpthite (anhydrous), 0.2 g of sodium azide, 0.5 mL of tween 20, 8 g of sodium chloride and 0.2 g of potassium dihydrogen orthophosphate, 1.15 g of disodium hydrogen orthophosphate and 0.2 g of potassium chloride.Positive and negative controls were included on each ELISA microtiter plate for validation of the test results.Absorbance values were measured at 405 using spectrophotometer (Multiskan Ascent VI.25 Version 1.3.1)and a reaction was considered positive when the absorbance value was at least two times higher than that for the healthy control.
Statistical analysis: Disease incidence was obtained making counts of infected plants for each cultivar and expressing it as a percentage of the total number of plants.The disease incidence values were arcsine transformed before Analysis of Variance Similarly, percentage symptom severity values also transformed by Arcsine before ANOVA.Data were analyzed using GENSTAT statistical software and the treatment means were separated using Tukey's pair wise comparison.

RESULTS AND DISCUSSION
The nine pepper cultivars exhibited various degrees of symptoms of viral infection.These included mosaic, yellowing, vein clearing, puckering, leaf curling, leaf distortion and stunting (Fig. 1).The  However, symptoms alone are insufficient in virus identification since their expression may be influenced by the host cultivar, strain of virus, age of the host, environmental conditions as well as possible coinfection with other viruses (Sherwood et al., 1986).Furthermore, different viruses may induce similar symptoms, while insect damage, particularly by thrips and mites, may also produce symptoms similar to virus infection (Green and Kim, 1994).Generally, symptom expression among the different cultivars increased in severity with time and ranged from 0.5% at 4 Weeks after Transplanting (WAT) to 47.2% at 14 WAT (Fig. 2).Anloga recorded the highest symptom severity of 42.7 and 47.2% at 12 and 14 WAT respectively.On the other hand, cv.Sunny F1 recorded the least symptom severity throughout the whole period of study except at 14 WAT when cv. Arhcard had the least.At 6 WAT, there was no significant difference in symptom severity between cvs.Archard and Sunny F1 but both were significantly different from cv.California Wonder.However, at 8 WAT, a significant difference was observed between cv.Sunny F1 and California Wonder.Symptom severity among the cultivars was not significantly different at 4, 10, 12 and 14 WAT.Differences in the pattern and degree of symptom severity among the cultivars could be attributed to differences in genetic make-up of the cultivars as well as the strain of the virus and possible co-infection with other viruses (Sherwood et al., 1986).
Using antisera to the different viruses in a DAS-ELISA test, CMV, PVMV, PMMoV and TMV were all detected among the cultivars, with each cultivar infected by at least one of the four viruses (Table 1).CMV and PVMV were the most common viruses detected.All symptomatic samples of cvs.Archard and Anloga were found to be infected by PVMV.This virus has been reported in several West African countries where disease incidences as high as 100% have frequently been observed (Atiri and Dele, 1985).Similarly, CMV was detected in all symptomatic samples of Anloga, followed by Legon 18 (83.3%)and Forever F1 (75%).The high incidence of PVMV and CMV observed in this study is in agreement with Arogundade et al. (2012) who recorded an incidence in the range of 55.0 to 87.50% for PVMV and CMV in single as well as mixed infections on pepper during a survey in Nigeria.The high incidence of these two viruses in this study might be due to a number of factors such as proximity of the pepper plants to alternative hosts, climatic conditions, low quality seed and vector activity as reported by Shah et al. (2009).
Although TMV was found to be present in seven out of the nine cultivars, it was detected in few samples in each cultivar, with Legon 18 recording the highest infection (33.3%).Interestingly, cv.California Wonder, which has been labeled as resistant to TMV, was found to be infected by the virus (25%).It is possible that the plants may have been infected by a different strain of the virus that is found locally.PMMoV was detected only in symptomatic plants of cv.Legon 18 (33.3%).
Cases of mixed infections of two or more viruses were also observed.The most common was the coinfection by PVMV and CMV which occurred in 100% of samples of cv.Anloga, 75% of cv.Legon 18, 66.6% of cv.Archard, 58.3% of cv.Sunny F1 and 50% of cv.
California wonder with cv.Big Sun recording the least (16.7%)Co-infection of pepper by PVMV and CMV has also been reported elsewhere (Hameed et al., 1995).Mixed infection of all four viruses was found in cv.Legon 18.This variety was bred in Ghana and has been cultivated as far back as 1981 (Amoatey, 1981).The high rate of infection observed in this cultivar could be due to recycling of seeds which may have resulted in the accumulation of viruses.

CONCLUSION
Viral diseases are considered to be a major limiting factor in pepper production in Ghana, causing serious losses.In the present study, both local and exotic pepper cultivars were found to be susceptible, with each cultivar infected by at least one of the four viruses tested.Mixed infections involving two or more viruses were also recorded.CMV and PVMV were identified as the most common viruses, occurring in all the cultivars These findings have revealed the need for a nationwide survey to monitor disease pattern of these viruses, assess the crop condition as well as collecting information regarding their prevalence, distribution and occurrence.The outcome of such a study would serve as a guide in management strategies to minimize crop losses and ensure sustainable production of pepper.Furthermore, the findings have necessitated the need for the production and use of virus-free seed by pepper farmers, breeding for virus resistance and adoption of efficient seed certification systems as well as strict quarantine measures to prevent the introduction of viruses especially PMMV to other areas both within and outside the country where this virus is not known to occur.

Fig. 2 :
Fig. 2: Symptom severity among nine local and exotic cultivars of pepper infected by four viruses (p≤0.05)

Table 1 :
DAS-ELISA detection of CMV, PVMV, PMMV and TMV in leaf extracts of nine pepper cultivars