Surveys of virus diseases on pepper ( Capsicum spp . ) in South-west Nigeria

Surveys to determine the incidence, diversity and distribution of viruses infecting pepper (Capsicum spp.) were conducted in six states (Oyo, Ondo, Osun, Ogun, Ekiti and Lagos) of South-west Nigeria in 2010 and 2011. Leaf samples from symptomatic and asymptomatic plants were collected at random from farmers’ fields and were analyzed for viruses using antigen coated plate enzyme-linked immunosorbent assay (ACP-ELISA). Symptoms observed on infected plants included mosaic, mottling, puckering, reduction in leaf size, vein yellowing, leaf and fruit deformation and stunting. The average disease incidence was 79% in 2010 and 76% in 2011; the average disease severity score was 2.9 in both years. This study identified eight viruses in the leaf samples as follows: Potato virus Y (PVY), Potato virus X (PVX), Pepper veinal mottle virus (PVMV), Pepper mild mottle virus (PMMV), Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Tobacco etch virus (TEV) and Tomato mosaic virus (ToMV). Incidence of PVY was the highest (79%), followed by TEV (67%), CMV (61%), and PVMV (58%); lowest in ToMV (23%). Mixed infections were common in the farmers' fields and high incidence suggests the cultivars are highly susceptible to viral infections.


INTRODUCTION
Chilli (Capsicum spp.) is one of the world's most popular vegetables; it is consumed, fresh or processed and used mainly as a spice and condiment (Sonago, 2003).Hot peppers, fresh, dried or processed, are an important item in all local markets in Africa.Production of green chilli and peppers in Africa is estimated to be 2.88 million t on *Corresponding author.E-mail: arogundade_olawale@yahoo.co.uk.Tel: +2348055360425/+2347038216421.Abbreviations: DMRT, Duncan's multiple range test; ACP-ELISA, antigen coated plate enzyme-linked immunosorbent assay; PVY, potato virus Y; PVX, potato virus X; PVMV, pepper veinal mottle virus; PMMV, pepper mild mottle virus TMV, tobacco mosaic virus; CMV, cucumber mosaic virus; TEV, tobacco etch virus; ToMV, tomato mosaic virus.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 363,937 ha of land (FAO, 2014); in West Africa the total production is 754,260 ton 108,452 ha with Nigeria contributing 500,000 ton 60,000 ha (FAO, 2014).Ogun State is the largest producer of Capsicum spp., in the south-west Nigeria with a production of 51.753t from 29,800 ha followed by Ekiti state with 48.85 t from 38,130 (APS, 2009).The two States are responsible for more than 50% of the total pepper production in the region (APS, 2009).The crop is cultivated mostly during the rainy season but also in the dry season in places where irrigation is available.It is mainly produced under subsistence conditions by smallholder farmers and is a significant source of household income.Peppers are used in stew and some local dishes all over the country although the types and quantities used vary in different areas.Four main varieties are grown in Nigeria: Bird peppers-atawere (Capsicum frutescens), Cayenne pepper or red pepper-Sombo (C.frutescens), Hot pepper-atarodo (Capsicum chinense), Sweet peppertatase (Capsicum annum).The fruits of these varieties vary in size, color, shape, and pungency but they are all hot.Both bird pepper and cayenne pepper are used either fresh or dried; atarodo and tatase are mostly used fresh (Bosland and Votata, 2000).
Pepper is susceptible to over 40 viruses (Kim et al., 2009).In Nigeria, most of the pepper fields exhibit complex symptoms of mosaic, mottle, leaf distortion, vein chlorosis and stunting that cause considerable losses in yield and plant vigor.Viral diseases caused in pepper are one of the most severe constraints in the production of the crop (Arogundade et al., 2012).Previous surveys showed high incidence of Pepper veinal mottle virus (PVMV) and Cucumber mosaic virus (CMV) in Nigeria (Arogundade et al., 2012).However, most viral diseases said to be infecting pepper were identified based on the basis of symptoms expression.Identifying the specific common viral diseases of pepper would facilitate understanding and knowledge of how best to manage them to prevent production losses.Hence, this study aimed at determining the incidence and severity of viral diseases of pepper growing areas of south-west Nigeria.

Survey and sample collection
Surveys were conducted in pepper farms for viral disease incidence and severity during the 2010 and 2011 planting seasons in six states: Oyo, Ogun, Osun, Ondo, Ekiti and Lagos.Thirty four farms were surveyed in 2010 and 25 in 2011.For each location within a state, farms not less than 0.5 acre (2000 m 2 ) were selected for evaluation.Observation was taken on 30 plants per site by walking along a W-shaped path in a field with plants on each side spaced at equal distance from one another.Five plants were randomly sampled from the population of plant showing virus symptoms.In each local government area (LGA), 625 leaf samples from as many plants showing symptoms of mosaic, chlorosis, yellowing, stunting, mottle, necrosis, leaf deformation and leaf bunching (Figure 1) were collected and stored in zip-lock plastic sampling bags.Percentage incidence of viral diseases was calculated and the severity score of the plants counted for disease incidence was calculated using the Mean of these scores ≥2 was expressed to determine the average severity of viral diseases in the field.

Antigen coated plate-enzyme-linked immunosorbent assay (ACP-ELISA)
Leaf samples were bagged separately and kept on dry ice while in the field until they were brought into the laboratory for further analysis.Leaf samples collected were stored at 4°C.These samples were tested using ACP-ELISA for the presence of Potato virus Y (PVY), Potato virus X (PVX), PVMV, Pepper mild mottle virus (PMMV), Tobacco mosaic virus (TMV), CMV, Tobacco Etch virus (TEV) and Tomato mosaic virus (ToMV) using homologous rabbit polyclonal antiserum available in the Virology Unit at IITA-Ibadan.About 0.1 g of the leaf sample was ground in 1 ml of carbonate coating buffer (0.015 M Na2CO3 and 0.0349 M NaHCO3); 100 µl was dispensed into each well of the ELISA plate.The plate was incubated at 37°C for 1 h and later washed three times with phosphate buffered saline (PBS) containing 0.05% (v/v) Tween-20 (PBS-T) with a 3 min interval between each wash.Polyclonal antiserum was cross-adsorbed in healthy pepper leaf sap extract (1:20 w/v) diluted in the conjugate buffer (PBS-T containing 0.02% (w/v) egg albumin and 0.2% (w/v) PVP-40,000).All the antisera used were diluted at 1:1000 (v/v) in conjugate buffer except CMV that was diluted at 1:3000 (v/v), and 100 μl polyclonal antisera were used for virus detection.After incubating the ELISA plate for 1 h at 37°C, the plate was washed three times with PBS-T.One hundred (100) μl of alkaline phosphatase conjugated anti-rabbit antibody diluted at 1:15000 (v/v) in conjugate buffer was used as secondary antibody and the plate was incubated at 37°C for 1 h.The plate was washed three times with PBS-T, and 100 μl of 0.001 g/ml of pnitrophenyl phosphate in 10% (v/v) diethanolamine buffer (pH 9.8) was added per well and incubated at room temperature for 1 h.Healthy pepper plants (Capsicum spp.) were used as the negative control.After 1 h the absorbance was measured at 405 nm in a BIO-RAD multiscan ELISA reader (ELx 800, Universal Microplate Reader).The samples were considered as virus positive when the ELISA reading was at least twice that of the healthy pepper leaf sap control.
Individual virus incidence was determined as a percentage of the total number of infected samples over the total leaf samples analyzed.Statistical analysis system (SAS) v2008 was used to analyze incidence and severity.Means were separated using Duncan's Multiple Range Test (DMRT) at 5% level of probability.

Disease incidence
The average disease incidence recorded was 79% in 2010 (Table 1) and 76% in 2011 (Table 2).The incidence and severity of infection varied significantly depending on the location.In general, the disease incidence was highest in Ekiti state (96.67%) in 2010, while Oyo had the least (66.90%) (Table 1).Ondo had the highest incidence in 2011 with 91.67%; followed by Oyo state with 80.17%, Osun had the least with 65.17% (Table 2).The southwest agro-ecological zone of Nigeria had an average disease severity score of 2.9 during the 2010 surveys and 2.8 in 2011.The disease severity was highest in Osun (average severity score of 3.1), closely followed by Ekiti (3.0).The lowest level of disease severity was recorded in Lagos state (2.6) during the 2010 surveys (Table 1).As for incidence, disease severity was highest in Ondo (3.3), followed by Oyo (2.9) during the 2011 surveys (Table 2).

Prevalence, distribution and relative occurrence of pepper viruses
Data generated from the survey revealed the relative occurence of viruses as follows: PVY 78.67%, PVX 57.17%, PVMV 57.5%, PMMV 43.0%, TMV 38.17%, CMV 61.17%, TEV 66.5% and ToMV 22.5% in the zone during the periods under survey (Table 3).The relative occurrence of PVY was highest in across all states compared to the other seven viruses.ToMV was not detected in Ekiti and Lagos (Table 3).The viruses detected were found to be distributed throughout the states surveyed.However, single infection were not detected in any of the states surveyed as all area had more than one viral infection in the infected plants (Table 4) and the viruses were distributed throughout south-west Nigeria (Figure 2).

DISCUSSION
The surveys showed that the incidence and severity of viral diseases of Capsicum spp., were generally high across the zone.A relatively high average of 78% incidence and an average severity index of 2.8 were observed in the two years surveys that were conducted.The serological method used in this study revealed the occurrence of eight viruses in pepper fields across the states studied.The PVY is the most commonly occurring plant virus in pepper followed by TEV, CMV, PVMV, PVX, PMMV, TMV and ToMV.Previous studies also identified PVY and PVMV as the most common viruses infecting pepper (Green and Kim, 1991;Atiri, 1992;Fajinmi, 2010) in the same zone.The high occurrence of the viruses especially PVY, TEV, CMV, PVMV and PVX in the zone   might be connected with their ability to remain infective for many months in alternative weed hosts together with a good breeding environment for the vectors of the virus that aids effective transmission (Agrios, 2005).Moreover, the major means for PMMV, TMV and ToMV to spread is through the use of infected pepper seeds as planting material as only a few seedlings need to be infected for the viruses to spread rapidly by vectors (AVRDC, 2014).
The rapid spread within crops could occur due to mechanical transmission by workers (from contaminated hands, clothing, and tools) during routine farm operations such as transplanting, pruning, grafting, and other farm activities.
Management necessitates good agronomic practices such as the control of weeds within and around pepper farms, which can reduce virus inoculum and breeding of

Figure 2 .
Figure 2. Distribution of eight viruses infecting pepper in south-west Nigeria.

Table 1 .
Incidence and severity of virus diseases of pepper in south-west Nigeria in 2010.

Table 2 .
Incidence and severity of virus diseases of pepper in south-west Nigeria in 2011.

Table 3 .
Frequency of pepper viruses detected in leaf samples collected from farmers' fields in south-west Nigeria.